a literature review on skeletal muscle tissue engineering, cell damage … · 2003-06-19 · 1 a...

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A literature review on

skeletal muscle tissue engineering,

cell damage and cell death,

damage markers and decubitus

Literature report of PhD Thesis

Debby GawlittaBMTE 03.27May, 2003

Promotor: F.P.T. Baaijens

Coaches: C.V.C. Bouten

C.W.J. Oomens

Eindhoven University of TechnologyDepartment of Biomedical EngineeringSection Materials TechnologyDivision Biomechanics and Tissue Engineering

Contents

Introduction 5

1 Overview of groups working on muscle tissue engineering 61.1 Technical University Eindhoven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.1.1 In-vitro models to study compressive strain-induced muscle cell damage . . . . . . 61.1.2 Internal reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.2 University of London . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.2.1 Compression induced damage in a muscle cell model in vitro . . . . . . . . . . . . 8

1.3 Brown University School of Medicine, Providence RI . . . . . . . . . . . . . . . . . . . . . 81.3.1 Organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.3.2 Human bioartificial muscles HBAMs . . . . . . . . . . . . . . . . . . . . . . . . . . 101.3.3 Current research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.4 University of Michigan, MI —– 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.4.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.4.2 Myooids from primary cultures and cell lines . . . . . . . . . . . . . . . . . . . . . 121.4.3 Protocols for Dennis’s and Kosnik’s myooids from cell lines . . . . . . . . . . . . . 131.4.4 Current and Future research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.5 University of Michigan, MI —– 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151.5.1 Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.6 Harvard Medical School, Boston MA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.6.1 Vascularized three-dimensional skeletal muscle tissue-engineering . . . . . . . . . . 16

1.7 Pittsburgh, PA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.8 Birmingham, Alabama . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171.9 Berkeley, California . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171.10 Tokyo, Japan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181.11 Osaka, Japan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

1.11.1 Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181.12 Groningen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

1.12.1 Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191.13 London and Stanmore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

1.13.1 Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191.14 Other interesting literature on muscle tissue engineering . . . . . . . . . . . . . . . . . . . 20

2 Cell damage, cell death and mechanotransduction 232.1 The healthy muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.1.1 Molecular muscle movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2

3

2.1.2 Quantification of muscle performance . . . . . . . . . . . . . . . . . . . . . . . . . 262.2 Cell damage and mechanotransduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

2.2.1 Second messengers and growth factors . . . . . . . . . . . . . . . . . . . . . . . . . 272.2.2 Cascade of muscle cell damage after eccentric muscle action . . . . . . . . . . . . . 282.2.3 Exercise-induced oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.2.4 Measurement of markers for cell damage . . . . . . . . . . . . . . . . . . . . . . . 29

2.3 Definitions of cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352.3.1 Apoptosis in muscle tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382.3.2 Markers for cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3 Pressure sores 40

4 Discussion 42

Bibliography 46

A Covering cells on cover glass with 3% agarose 57

B Protocol for blob formation from C2C12 cells 59

C Cell culture media 61

D Freezing cells 63

E Marker assays 64E.1 PI/CTG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64E.2 PI/Annexin V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65E.3 Creatine Kinase (CK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66E.4 Myoglobin (Mb) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69E.5 Nitric oxide (NO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70E.6 MalonDiAldehyde (MDA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

F Definitions 71

Introduction

Bedridden and immobilized patients as well as those wearing prostheses are sensitive to the developmentof pressure sores (decubitus or pressure ulcers). Pressure sores are sites of restricted tissue degenerationof the skin or underlying tissues, such as subcutaneous fat and muscles. They are believed to be causedby several factors. One of the hypothesized causes of decubitus is tissue compression. The tissue is thencompressed between the supporting surface (e.g. a matras) and a bony prominence at the other side.Especially the deep pressure sores that start from skeletal muscle tissue can evolve unnoticed (if visuallyinspected from the outside) towards the skin. The outer appearance of these wounds is treacherous as thesmall superficial hole may be the entrance to a large subcutaneous cavity. Therefore, these deep ulcersare the hardest to cure and usually surgical intervention is needed before wound healing starts. Moredetailed information on decubitus aetiology and prevalence will be presented in the third chapter.There are no clinical markers available up-to-date that give away the presence of these deep sores. Ifdeep sores are to be prevented, more insight is required in the development of such tissue degeneration.The several proposed hypotheses for the development of deep sores need to be tested. The most commontheory states that tissue compression causes occlusion of capillary beds, which induces local ischemia [49].Another hypothesis is occlusion of the lymphatic system, which leads to an accumulation of waste productsand cell death [70]. Several groups believe that reperfusion injury occurs after removal of the tissueload [57]. In our group it has been proposed that sustained cell deformation is an additional trigger forcell damage and can lead to pressure sores [16], [15]. All or some of these hypotheses combined or each oneby itself may cause pressure sores. It is unknown when which trigger induces muscle cell damage. Withinthis project, a first focus will be on the influence of sustained cell deformation on damage development.

In this review, the ingredients for an experiment to test the latter hypothesis are discussed. A skeletalmuscle model is necessary, as well as a way to compress the tissue, and a way to measure and quantifydamage of the tissue. In order to avoid experiments in animals, a representative skeletal muscle model hasto be applied. A currently popular method to produce artificial organs is tissue engineering because noanimals need to be sacrificed. An overview of the published methods for engineering a muscle is presentedin the first chapter. This tissue engineered muscle model will be compressed in a controlled environment.Damage development in the engineered tissue can be monitored by means of confocal microscopy, andsecretion measurement in the medium. To better define the damage, the composition of the healthymuscle is discussed ere damage development and different forms of cell death are explained in chapter 2.From the different steps in the damage cascade of tissue, markers are presented. In literature requirementsfor an ideal marker (apart from e.g. measurability) are proposed to facilitate the choice of the propermarker in a certain situation [82]:

• the marker’s concentration in skeletal muscle should be high

• the marker should not be present in other tissues

4

Introduction 5

• rapid and complete release of the marker after skeletal muscle injury

• homogeneous distribution of the marker throughout skeletal muscle tissue

• release in proportion to extent of the damage

• persistence in plasma; long enough for convenient diagnostics; short enough to be able to measure’new’ injury; e.g. several hours

• suitable to analyze as stat parameter

Finally, it will be discussed which tissue engineered muscle is chosen and which markers are the mostpromising for pressure sore diagnosis and may exhibit a damage threshold.

It is emphasized by the author that this review merely presents the top of some icebergs, rather than thebulk ice mass that is hidden under the surface. This may be parallelled by stage IV decubitus woundswhich exhibit this same characteristic.

Chapter 1

Overview of groups working on muscletissue engineering

In this chapter an overview is presented containing several groups that are or were working on tissueengineered skeletal muscle constructs. On the last page of this chapter, in table 1.1, the contents of thischapter are summarized.

1.1 Technical University Eindhoven

Professor: F.P.T. BaaijensC.V.C. Bouten, R.G.M. Breuls, E.A.G. Peeters, C.W.J. OomensDept. Biomedical Engineering and Dept. Mechanical Engineering,Eindhoven University of Technology,PO Box 513,5600MB Eindhoven,The NetherlandsPhone: +31 (0)40 247 3006Fax: +31 (0)40 244 7355

Correspondence: [email protected]

Main objective: to study the effects of sustained mechanical loading on soft tissues (focussed on muscletissue) by using experimental techniques and numerical modeling. Hypothesis: sustained inhomogeneousdeformation of (cells within the) muscle tissue is the primary trigger for tissue damage.

1.1.1 In-vitro models to study compressive strain-induced muscle cell damage

E. Peeters: single C2C12 myotubes (or if desired parallel myotube monlayers) are transversely loaded inunconfined compression under a glass tip. Forces and displacements can be measured. Cells are monitoredby CLSM and light microscopy.M. Daniels: biochemical and histology assays and statistical analyses of (1.105) C2C12 cell damage dueto compressive straining. Compression of multiple large myotube monolayers. Porous glass plungers ona monolayer covers with a 1 mm thick layer of 3% low gelling temperature agarose.D. Bader & R. Breuls: 1) Myoblasts are seeded in 3% low gelling temperature agarose at 2.106cells/ml

6

Section 1.1 7

into 5x5 mm cylindrical constructs. They are kept in growth medium for 4 days and cultured indifferentiation medium for 8 days. A mixture of myotubes and myoblasts at 1.106cells/ml is obtained andunconfined compression is performed under CLSM deformation monitoring.2) Parallel-aligned myotubes (seeding density 4.106cells/ml) embedded in a collagen/matrigel matrixare produced to mimic the architecture of muscle tissue, based on Vandenburgh et al. 1996. After 3days growth medium is replaced by differentiation medium. Specimens are then transversely loaded inunconfined compression. Cell deformation is assessed by CLSM.E.M.H. Bosboom: in vivo experiments using animal models.A.Stekelenburg: MRI assessment of in vivo damage using a rat model. Currently, the numerical modelis being improved to become more realistic by application of anisotropy and non-linear material behavioras well as refined contact between indentor and skin. Material properties of muscle under compression isbeing determined.Future: clinically relevant straining protocols, with magnitudes 0-40% and durations 0-24h.

1.1.2 Internal reports

L. Mulder, May 2001: ’Preparation and evaluation of tissue engineered muscle.’ Organoids areproduced in rectangular wells with grids for attachment and collagen coating, according to Vandenburgh.Results: no gelling and flake formation probably due to low pH. Later constructs (2) show a necrotic core(might be caused by diffusion). Min life span was 25 days. Recommendations: it takes two to performthe protocol with greater speed; produce smaller amounts than ten constructs at the same time; preparethe cell solution before preparing the gel solution. Highly passaged C2C12 myoblasts tend to differentiatetowards myocyte which makes it harder to get tubuli.R. Lems, Feb 2002: ’Chemically induced alignment of muscle fibers.’ C2C12 cells were culturedon substrates with gold lanes on glass that were covered with cell adhesive and cell resistantsurfacemonolayers. Muscle fibers will orient themselves parallel or perpendicular to ridge and groove structureswith groove depths in the order of micrometers. The gold-on-glass substrates investigated do not havea topological effect on cell growth and orientation, meaning that the myoblasts and myotubes have tobe chemically guided into parallel arrays. This was achieved after chemical coating (PMPS-b-POEGMApolymer film) and subsequent removal of the polymer. Myoblasts as well as myotubuli oriented themselvesin the direction of the lanes.K.v.d. Hout, Jan 2002: ’Chemical orientation of muscle cells in vitro.’ Chemical pretreatment ofgold-on-glass can change the degree of cell attachment.R. Sladek, Jul 2001: ’Modified substrates for anisotropic muscle cell growth.’ C2C12 cells are culturedon chemically modified gold-on-glass. Cell attachment is better if the surface is more hydrophilic. Theorientational response of the cells is dependent on the broadness of the gold lanes. Cells align paralleland perpendicular to the gold lanes, with a higher tendency to orient perpendicular to the lanes. Theorientation was clearer with the 100µm gold lanes than with the 200µm lanes.M. Janssen, May 2002: ’Release of biomarkers by cultured muscle cells as a result of cell damage.’A monolayer of predominantly myotubes is covered with agarose. Cells are subsequently compressed byweights for 0-24h. The markers, lactate dehydrogenase (irrev.), creatine kinase (irrev.) and nitric oxide(rev.) are measured during these 24 h. CK is a marker for myotubuli and is not present in myoblasts. Nocell deformation achieved, so no conclusions concerning the relationship of marker release and deformation.LDH seems not very appropriate as a marker.E. Peeters, Feb 1998: ’Deformation of myoblasts, mounted in agarose constructs.’ Myoblasts werecompressed (0-30%) using a three-dimensional in vitro agarose model and visualized with confocal microscopy.Cell deformation was quantified by cell diameter, surface area, volume strains and a deformation index

8 CHAPTER 1. OVERVIEW OF GROUPS WORKING ON MUSCLE TISSUE ENGINEERING

(ratio of x and y diameter). After imposing strain on the substrates a 10 minute stress relaxation periodwas allowed before the cells were scanned. Sub-saturation scanning was applied to limit the effects ofphoto-bleaching and to ensure that image brightness had no effect on dimensional measurements. A greatadvantage is that it allows calculation of strains of individual cells. Cells were scanned on days 3, 4 and9.

1.2 University of London

Professor: D.L. BaderY-N. Wang, D.A. Lee, M.M. KnightIRC in Biomedical Materials and Medical Engineering Division,Queen Mary,University of London,Mile End,London E1 4NS. UKPhone: +44(0)20 7882 5274Fax: +44(0)20 8983 1799


1.2.1 Compression induced damage in a muscle cell model in vitro

Goal: The system is employed to describe thresholds for cellular breakdown in terms of magnitude andduration of construct compression. Materials and Methods: C2C12 mouse myoblasts are embeddedin a homogeneous agarose gel to examine the damaging effects of prolonged applied pressures (static aswell as dynamic). Culturing: 2.106cells/ml were maintained in growth medium for 8 (or 4) days andthen transferred to differentiation medium in which they were cultured for 9 (or 8) days. 10% (eq.18mmHg) and 20% (eq. 32 mmHg) strains are imposed on cylinders cut from the agarose /cell suspensionfor periods ranging from 0.5 to 12 hours. Cell damage was assessed by either H-E staining, calcein AMor ethidium homodimer-1. The number of apoptotic cells is estimated using the DNA nick-translationmethod. Results: Significantly higher values of dead /damaged cells was found for specimens that weresubjected to the higher strain values. This was primarily due to apoptosis. Amount of damage wasdependent on the duration and magnitude of loading. Damage thresholds: tables......Future: structural, mechanical and biochemical changes in response to sustained cellular deformation.Incorporation of an ecm in the model to improve the invitro model to be more realistic (in vivo). Otherstudies: The same study with strains up to 40% was described in [15].

1.3 Brown University School of Medicine, Providence RI

Professor:H.H. Vandenburgh, P. Karlisch, L. Farr, S. Swasdison, C.A. PowellBrown University School of Medicine, Room 227,164 Summitt Avenue,Providence, RI 02906, USA;Phone: 401-793-4273Fax: 401-751-2398

Section 1.3 9

Correspondence: herman [email protected]

1.3.1 Organogenesis

Production in silicone wells Skeletal muscle development is regulated by factors, including nutrition,hormones, electrical activity, and tension.Wells are constructed from silicone rubber sheeting with a stainless steel mesh insert (figure 1.1). Thewells are subsequently cleaned with ’Pre-Zyme’ r© and washed. The wells are screwed to a stainless steelbar at each side. The wells are stretched 50% in their length direction. The stretched wells are coated withrat tail collagen I. The set-up is sterilized with ethylene oxide or autoclaved in a glass petri dish. Finally,before starting cell culture, the wells are preincubated with EBSS for several hours. Avain myoblastsproliferate and fuse into multinucleated myofibers beginning 48 hours after plating, align parallel to thedirection of substratum tension, and become striated and contractile by 96-120 hours. 3-5 days afterplating, the cell layer shifts off the bottom of the silicone rubber wells but remains attached to the screensat the bottom. The thus formed organoids contain organized and contractile myofibers. Mammalianmuscle organoids are produced with rat neonatal myoblasts. Primary cells were chilled in a 1:6 solutionof matrigel : collagen (col I, 1.6 mg/ml) prepared with GM. The cell suspension is kept on ice and pipettedinto the wells with precooled pipet tips at a concentration of 4.106 cells/0.75 ml in the wells. Gellingoccurs at 37 ◦C for 2-6 hours and then 1 ml GM is pipetted on top of the gel. Cultures are maintainedin GM for 3 days, FM for 3 days and MM for up to 4 weeks. Internal tensions in the gel align theforming myofibers. The organoids can then be transferred to other growth chambers without releasingthe tension. [88]

Production in silicone tubing Murine C2C12 rhGH cells were suspended in 1.6 mg/ml rat tailcollagen I in C2GM, NaOH to neutralize to pH 7.1 and 1:6 MATRIGEL was added. The suspensionwas immediately cast into molds from silicone rubber tubing containing either Velcro or stainless steelscreening for organoid attachment. The molds were glued to the bottom of a culture dish and incubatedfor 2-4h to form the gel. Then C2GM was added for 3-5 days; FM for 4-5 days, after 1-2 days in vitro,the gel detached from the silicone rubber mold and was held at its ends by the screening; 5-12 days inMM before implantation. [90]C2GM: DMEM with high glucose, 5% fetal calf serum, 15% defined supplemental calf serum, penicillin100 units/ml, streptomycin 0.1mg/ml.FM: DMEM with high glucose, 2% horse serum, penicillin 100 units/ml. Fusion efficiency is 75-80%.MM: DMEM with high glucose, 10% horse serum, 5% FCS, penicillin 100 units/ml, cytosine arabinoside(araC) 1 µg/ml.Tissue-engineered primary adult sheep muscle cells genetically engineered to express either rhVEGF orrhIGF-1 secreted the bioactive proteins locally in the sheep heart for at least 30 days. [51]


Figure 1.1: The forming organoid in the silicone well is shown on the left and the formed organoid isshown on the right. [78]

Mechanogenesis The utilization of more complex repetitive mechanical stimuli for extended periods oftime to induce the monolayer of oriented myotubes into an in vivo-like three-dimensional muscle organ,was described. The rectangular culture wells (collagen-coated??) were also used for this experiment.The stretched cells were embryonic mononucleated muscle cells. The mechanical stimulation consisted ofrepetitive stretching and unidirectional stretching. At days 7-8 after plating, cells were initially stretchedto 5% dynamical strain with 30 minute rest intervals. The repetitive stretching increased to 20% strainwith 5 minute rest intervals after two weeks in culture. The uniaxial elongation was set at 0.25-1.0 mm/24h. The cell monolayer detached from the elastic substratum after 6-10 days. The monolayer rolled upwardand inward to form a solid rodlike structure by 2 weeks of culture. After 3-4 weeks in culture in a crosssection, the folded monolayer has integrated into a solid tissue with a well-defined epimysium made upof mononucleated fibroblasts. Muscle fibers in cross section begin to display a polygonal checkerboardpattern similar to that seen in vivo, with both primary and secondary-like myofibers. [89]

1.3.2 Human bioartificial muscles HBAMs

Human artificial muscles were created from cells that were obtained after isolation from needle biopsies(60-80% myogenic based on desmin positive staining) and a collagen/matrigel mixture [68]. To improvethe morphological and mechanical properties of the constructs, mechanical stimulation of the constructswas performed. Within 24 hours after pouring the cell suspension into the molds, it had contracted,detached from the mold and was held in place at the attachment sites (two 2-mm diameter stainless steelpins). Unidirectional stretching was started at 24 hours. The HBAMs were stretched at approximately 3.5µm per 10 minutes. This equalled a total stretch of 2 mm after four days. Subsequently, they were heldat this 10% total stretch for three more days. At day 8 a repetitive stretch-relaxation pattern was started,consisting of 3 sets of 5 stretch-relaxations (within in 90 seconds) and 28 minutes of rest after the thirdset. The gradual stretching during 4 days was supposed to simulate bone growth during embryogenesis.Repetitive stretch-relaxation cycles were imposed on the constructs because the loading pattern wasknown to be similar to the one that caused skeletal muscle hypertrophy in monolayer avian skeletalmuscle cultures [92]. On days 8-10 the HBAMs were stretched 5% (1 mm), on days 10-12 10% (2 mm),and on days 12-16 15% (3 mm). Constructs were stained for sarcomeric tropomyosin and myosin heavychain and HBAM cross sectional area, myofiber diameter, and percent HBAM cross section occupied bymyofibers were calculated with a Zeiss image analysis software package. Repetitively stimulated constructsexhibited higher elasticity (i.e. lower E-modulus, and less stiff!!), 12% larger diameter, and 40% increasedpercentage of myofiber area. Nondestructive method to determine passive force and viscoelastic properties

Section 1.4 11

of constructs is presented.

1.3.3 Current research

BAMs (Bio-Artificial Muscles) generate, when electrically stimulated, 1-2% of the force generated bynormal skeletal muscle. This is due to the high extracellular matrix content, low myofiber packing density,and less than fully differentiated myofibers in the BAMs. Manipulation of the tissue- culture medium,growth factor supplements, perfusion conditions, electrical stimulation, and repetitive mechanical loadingshould eventually allow the production of stronger, more in vivo-like constructs. [91]

The mechanogenic second messengers involved in tension regulation of cell growth are currently underinvestigation. Our laboratory utilizes tissue cultured aneural skeletal and cardiac muscle cells grownon an elastic substratum. Computerized mechanical cell stimulators are used to examine hormonaland gene expression alterations associated with increased mechanical loads. The interaction of tensionand cellular sensitivity to insulin-like growth factors, glucocorticoids, and anabolic steroids are underinvestigation along with prostaglandins as mechanogenic second messengers. Understanding the basicmolecular mechanisms by which muscle tension stimulates growth could lead to new treatments forskeletal muscle atrophy under reduced tension conditions and pathological cardiac hypertrophy resultingfrom the increased tension associated with hypertension.Mechanical forces during embryonic and postnatal development also play an important role in organogenesis.We have utilized our computerized instrumentation and techniques to form individual skeletal muscleand cardiac muscle cells into in vivo-like three dimensional ”organoid” structures. The skeletal muscleorganoids have been used to study the effects of space travel on muscle atrophy in several NASA shuttleflight experiments. In addition, we are utilizing these tissue-engineered structures as growth factor deliverysystems by implantation in vivo of organoids formed from genetically modified muscle cells.

1.4 University of Michigan, MI —– 1

Professor:P.E. Kosnik, M.E. Gilbert, J.A. Faulkner, R.G. Dennis(Institute of Gerontology, and Department of Biomedical Engineering,)University of Michigan,300 N. Ingalls, Room 956,Ann Arbor, MI, 48109-2007, USA;


1.4.1 Background

Quote from Bob’s webpage [3]: The State of the Art in Functional Skeletal Muscle Tissue Engineeringcan be easily summarized by first defining the function of skeletal muscle. Though muscle tissue performsmany functions for the body, some arising from the emergent properties of muscle cells organized intowhole muscle organs, such as heat generation and protein synthesis, the most basic definition of muscletissue function is the generation of controlled force, work, and power. It is necessary to quantify thecontractility of the muscle tissue, to organize the tissue in such a way as to promote the generation ofdirected force, and exert control over the contractions for research in this area to be considered engineering,rather than cell biology. After all, spontaneous contractions in cultured skeletal muscle cells were first


reported in 1915 (Lewis), and this was not construed as ’engineering’. Defining Functional Skeletal MuscleTissue Engineering in this way, it is possible to assert that at this time there are only three research groupsin the world engineering functional skeletal muscle in vitro: Herman Vandenburgh and Paul Kosnik inProvidence, RI; myself and Hugh Herr at MIT, and the Muscle Mechanics Laboratory at the Universityof Michigan. The myooid was seen to spontaneously contract immediately after each feeding.No functional data were measured, as the instrumentation was not in place at that time.

1.4.2 Myooids from primary cultures and cell lines

First, myooids were cultured from primary cultures of adult rat myogenic precursor cells. Anchors wereeither acellularized muscle or silk sutures. The myooids were 12 mm in length and 0.1-1 mm in diameterand the formation process is shown in figure 1.2. Fibroblasts and myotubes generated their own ECM.Within 48 hours of formation the myooids began to contract spontaneously at approximately 1 Hz. Themyotubes in the myooids appeared to remain in an early developmental state due to the absence of signalsto promote expression of adult myosin isoforms, although this does not account for all the loss in P0.This indicates that major deficiencies arise in the force development of myooids due, at least in part, tothe inclusion of noncontractile material and the disordered structure of the sarcomeres. [27]

Figure 1.2: The rolling myooid is shown on the left and the formed myooid is shown on the right. [3]

Materials and Methods: Before myooids were prepared, culture dishes were pretreated with SYLGARDsubstrate to allow pinning of suture anchors and to which cells do not adhere. For cell attachment thesubstrate was subsequently coated with mouse laminin and presoaked with growth medium for 5-8 days.After cell isolation (fibroblasts, myoblasts, satellite cells), culture was started with differentiation medium.Results: After 2-3 weeks the monolayer detached from the substrate but stayed attached to the twoanchors. The detached (rolled) monolayer is then called the myooid. In the myooid, the fibroblasts forman annulus that surrounds a core of myotubes/muscle fibers. The annulus is relatively larger in neonatalmyooids than in adult myooids. Geometry: 1.2cm length, ±100-600 µm diameter. Within 48 hours offormation, the myooids began to contract spontaneously (0.8-1.5 Hz). [50]

Myooids were cultured from primary cultures, as described above, but also from cell lines. From primarycells adult mouse myooids, neonatal rat myooids and adult rat myooids were cultured and compared tomyooids that were cultured from C2C12 myoblasts and 10T1/2 fibroblasts. After approximately 30 daysof total culture, myooid cross-sectional area, rheobase, chronaxie, resting baseline force, twitch force,time to peak tension, one-half relaxation time and peak isometric force were measured. The specific force

Section 1.4 13

generated by the myooids was 2-8% of the force generated by skeletal muscles of control rodents. Thecell line based myooids exhibited a greater rheobase, time to peak tension and one-half relaxation timecompared to myooids cultured from adult rodent cultures. The cell based myooids as well as the neonatalrat cell myooids had greater resting baseline force than those from adult rodent cells. [28]

1.4.3 Protocols for Dennis’s and Kosnik’s myooids from cell lines

A more detailed protocol is described in appendix 4.Preparations and requirements:

• 10T1/2 fibroblasts

• C2C12 myoblasts (CRL-1772)

• DMEM (11995-065 GIBCO)

• FBS (10437-036 GIBCO)

• pencillin G

• silk sutures (size-0, Ethicon, metric size 3.5)

• natural mouse laminin (23017-015 GIBCO)

• Dulbecco’s PBS (14190-136 GIBCO)

• 35-mm diameter culture dishes (Falcon 1008)

• SYLGARD (Dow Chemical Corporation, type 184 elastomer)

• HS (16050-114 GIBCO)

• stainless steel pins, 0.10mm (Fine Science Tools, model 26002-10)

Planning:

• day 0: SYLGARD coating of petri dish; let elastomer develop for 1 week on level shelf

• day 7: store dish for de-toxication for another two weeks

• day 21: laminin coating of SYLGARD dish; allow to dry overnight

• day 22: pinning of the wet coated suture-ends to the coated substrate; allow to dry overnight

• day 23: add GM to the dish to presoak; sterilize set-up (60-90 min under UV) and incubate withGM for 5-8 days

• day 30: plate cells 1.105 cells/cm2 in a ratio of cell types between 30-80% C2C12; wait for cells toreach confluence, meanwhile refresh medium every 2(-3) days

• day 32: refresh GM

• day 33-38: if cells have reached confluence, change to DM

• day 39-60: check if monolayer detachment has occurred yet


1.4.4 Current and Future research

Future research will be directed toward integrating nerve and muscle tissue in vitro to increase theexcitability of the tissue and enhance the expression of adult phenotypes (figure 1.3). The purposeis to promote enhanced excitability and contractility of the engineered muscle constructs by providingnerve-derived trophic factors and synaptic stimulation at the neuromuscular junction. Other futurestudies will focus on the application of mechanical and electrical interventions to promote growth anddevelopment of the tissue, improved mechanical interface between the muscle and its attachment pointsusing tissue engineered tendons, and ultimately the addition of a vascular system with self-organizingvascular endothelium. [3]

Figure 1.3: In a collaboration with Marlene Calderon, M.D. Dennis and his group were able to develop anerve-muscle co-culture system in which axonal projections from a ventral horn explant projected to themuscle construct in vitro, as shown in the image. [3]

In collaboration with Herman H. Vandenburgh, Ph.D., Brown University and Paul Kosnik, Ph.D., CellBased Delivery, Inc. R. Dennis is working on the development of automated tissue culture systems tomonitor the contractility and excitability of engineered skeletal muscle constructs in culture (figure 1.4).Their objective is to provide a scalable robotic platform to automate the setup, culture, and contractilefunction of cultured muscle for use in drug testing and discovery. This research is closely linked to Pauland Herman’s work on the engineering of skeletal muscle for use in gene therapy and the cell baseddelivery of bioactive compounds.

Figure 1.4: A single Development Emulator unit is shown on the left. The units are modular, and aremaintained in organized stacks (shown on the right) in a cell culture incubator. The central feature

Section 1.6 15

of the developmental emulator units is the local control of the electromechanical interventions by anembedded microprocessor (the largest integrated circuit visible at the right). Visible to the extreme rightis a myooid (skeletal muscle construct), attached above by a linear servo motor (stepper motor, planetarygear head, and rack-and-pinion), and attached below to a force transducer. On the PC board (not shown)is the stimulation electronics for electrically stimulating the muscle tissue. All of the components of theDevelopment Emulator are designed specifically for use with this system. [3]

1.5 University of Michigan, MI —– 2

Professor:Rowley J.A., Madlambayan G., Sheridan M.H., Shea L.D., Peters M.C., Mooney D.J.Department of Biomedical Engineering,University of Michigan,Colleges of Engineering and Dentistry,Rm. 3074, H.H. Dow Bldg,2300 Hayward Avenue,Ann Arbor, MI 48109-2136, USATel: 001-313-763-4816Fax: 001-313-763-0459


1.5.1 Research

Alginates are naturally occurring polysaccharides that gel in the presence of divalent cations. Theyare copolymers containing mannuronic acid (M) and guluronic acid (G). These subunits differ in theirability to contribute to hydrogel cross-linking and thus alter the physiological properties of the resultanthydrogels. In this study the M:G ratio is varied as well as the RGD-ligand density on the gels to study theeffects on C2C12 cell proliferation and cell differentiation. Differentiation was measured qualitatively bymonitoring cell fusion and quantitatively by measuring the activity of muscle creatine kinase (colorimetricassay from Sigma, kit 520). RGD incorporation efficiency was comparable for different M:G ratios asassessed by radio active labeling. The rate of proliferation significantly increased as the G-content of thealginate increased. Also, the highest G-content substrates promoted extensive fusion of the myoblasts byday 3, with subsequently increasing levels of muscle creatine kinase activities. Furthermore, increasingRGD density to 30 fmol/cm2, led to an increase in cell proliferation (increasing more had no additionaleffect). This also increased fusion. However, peptide density did not cause myoblast fusion on theM-alginate gels. [76]

It is hypothesized that vascularization of engineered tissues can be enhanced by the local, controlleddelivery of molecules which induce capillary formation. The study is focussed on the delivery of vascularendothelial growth factor (VEGF) by different PLGs (PGA, 50:50 PLG, 75:25 PLG, 85:15 PLG, PLLA).These polymers were foamed with CO2, N2 or He. After that, in some samples salt leaching was performed.Significant porosity (95%) in 85:15 PLG resulted from foaming with CO2 gas as compared to N2 and He.For different compositions the copolymers all foamed to >90% porosity. For 85:15 PLG, appr. 84% ofthe incorporated growth factor is released within the first two days, whereas in 75:25 PLG a sustainedrelease for 70 days is observed. [79]Heavy-chain myosin is a differentiation marker for skeletal muscle. [75]


1.6 Harvard Medical School, Boston MA

Professor:A.K. Saxena, J. Marler, M. Benvenuto, G.H. Willital, J.P. VacantiDept. of Surgery,Children’s Hospital,Harvard Medical School,300 Longwood Avenue,Boston, MA 02114, USA


1.6.1 Vascularized three-dimensional skeletal muscle tissue-engineering

Rat neonatal myoblasts were seeded onto polyglycolic acid meshes to engineer skeletal muscle in vivo.After 30 days, 45 days and six weeks vascularized three-dimensional constructs were explanted with theability to generate neo-muscle-like tissue. Histology by hematoxylin-eosin staining, immunohistochemistryby alpha sarcomeric actin and desmin skeletal muscle marker. [77]

1.7 Pittsburgh, PA

Professor:C.A. DiEdwardo, P. Petrosko, T.O. Acarturk, P.A. DiMilla, W.A. LaFramboise, P.C. Johnson Divisionof Plastic and Maxillifacial Reconstructive Surgery,University of Pittsburgh Medical Center,676 Scaife Hall,3550 Terrace Street,Pittsburgh, PA 15261, USA

Skeletal muscle engineering depends on the unique regenerative properties of the satelite cells and theability to harness and direct the intrinsic cells programs associated with determination, proliferation, anddifferentiation. Human primary skeletal muscle cells can be harvested and successfully grown in vitrofrom either fetal muscle or from satellite cells of adult muscle. [29]Critical issues in functional muscle development in vitro include an understanding of the effects ofmechanical and electrical stimulation on cultured muscle cells and the role of the extracellular matrix(natural or synthetic) in the migration, attachment, and differentiation of myoblasts on biomaterialsurfaces. Mechanical forces have been shown to directly alter the transcription of myosin heavy chaingenes and can alter the flux rates of prostaglandins E2 and F2. Unidirectional mechanical stretch inducescell alignment parallel to the direction of force. Alignment occurs only if stretch is applied before cellsattach. In contrast, on a substratum undergoing continuous stretch-relaxation cycling, myotubes orientperpendicular to the direction of movement.Several biomaterials have been applied in muscle tissue engineering, such as DEAE-cellulose supports,collagen based biomaterial scaffolds, alginate hydrogels.

Section 1.10 17

1.8 Birmingham, Alabama

Professor:S. Swasdison, R. Mayne Department of Cell Biology,University of Alabama,Birmingham 35294

This group was originally interested in the attachment of muscle fibers to extracellular matrix, whichoccurs at the myotendinous junction. Two methods were developed in which long-term cocultures ofquail skeletal muscle cells and chicken tendon fibroblasts so that all of the fibers develop in a highlyoriented manner. In both methods the cells were grown on a scratched Bionique teflon membrane thatwas subsequently pinned into wax in a petri dish. The fibroblasts were seeded on the outside and myoblastsin between. After four weeks of culture the muscle fibers contract freely, detach from the membrane andform a muscle bundle-like structure. In the bundle both ends of the fibers remained attached to thetendon fibroblasts and stabilized fixed pins at each end of the culture. In the other method, instead offour weeks of static culture the cells were transferred into a collagen gel after two weeks of culture andgrown for three more weeks. Strong attachments to the collagen I gel were formed with the ends of eachmuscle fiber. In both methods cells contract spontaneously and vigorously and were not overgrown byfibroblastic cells. [85], [86]

1.9 Berkeley, California

Professor:Strohman RC, Bayne E, Spector D, Obinata T, Micou-Eastwood J, Maniotis A.Department of Molecular and Cell Biology,University of California,Berkeley, CA, 94720

Appropriate conditions can induce a complex organization of muscle fibers and connective tissue compartmentsspontaneously in culture. Materials and methods: Saran Wrap (Dow Chemical Co.) membranepieces were pinned to a Sylgard base. The base was prepared by polymerizing 5 ml of Sylgard #184(Dow Chemical Co.) in a 60mm Falcon tissue culture dish. The membrane and dish are flooded witha 0.1% collagen solution in water and allowed to dry under UV. Chicken myocytes and fibroblasts wereobtained from 12-day-old embryos. Subsequently, (6.5.103or5or6) cells were seeded in 5 ml medium. Noantibiotics or fungicides were used because they delay myogenesis and inhibit muscle specific proteins.No attempt was made to discourage fibroblast growth and the fibroblast network is necessary to stabilizethe muscle fiber array and to produce adhesion to the stainless steel pins. Note that the pins prevent themonolayer from rolling up completely. Results: Cell fusion occurs at day 3. In subsequent weeks, themyotubes develop cross striations and undergo extensive contractions. Meanwhile, the fibroblast growthcontinues and the muscle fibers become embedded in a 3D network of fibroblasts. Spontaneous twitchingof the fibers started at day 10-15 of culture. 10-20 second bursts of approximately 3-5 twitches/secondwere observed. While the total organization of the tissue lacks the detailed histology of that seen in theanimal, the three connective tissue compartments of muscle have been spontaneously generated in thetissue cultures. Results: Innervation does not appear to be required for orderly shifts in gene expressionfrom embryonic to adult patterns. [83]


1.10 Tokyo, Japan

Professor:Kishi K.; Satoh H.; Tanaka T.; Kubota Y.; Imanishi N.; Nakajima H.; Nakajima T.Department of Reconstructive Surgery,School of Medicine,Tokyo 160-8582, Japan

In their paper [46] the principle of skeletal muscle regeneration, and the challenges to achieve skeletalmuscle tissue engineering are described.

1.11 Osaka, Japan

Professor:Okano T., Matsuda T.Department of Bioengineering,National Cardiovascular Center Research Institute,5-7-1 Fujishirodai, Suita, Osaka 565, Japan

Correspondence: Takehisa Matsuda

1.11.1 Research

Three hybrid types C2C12 cells were incorporated in collagen I gels and molded in three ways: adisc-type, a polyester mesh-reinforced type and a tubular type. Culture occurred in GM (20% FBS) for 4days and in DM (2% horse serum). Periodic mechanical stress loading to the mesh-reinforced hybrid tissueaccelerated the cellular orientation along the axis of the stretch. In the tubular hybrid tissue, collagenand cells became oriented circumferentially with time. Future outlook: improving cellular density andcellular orientation and angiogenesis in hybrid tissue. [61]Increasing cell density and orientation Hybrid muscular tissues are prepared from C2C12 cells andcollagen I. A rod-shaped (centrifuged, 1000 rpm, 5 min) gel was cultured in an agarose gel coated dish (3dGM, 4d DM). Only a few necrotic cells were observed in the core. In another experiment a ring-shapedtissue was stretched at 60 rpm. Densely accumulated cells and collagen fibers were oriented in thestretching direction. From these two experiments it became clear that a centrifugal cell packing methodand mechanical loading make hybrid muscular tissues appear closer to the cell density and orientation innative tissues. [60]Vascularization of hybrid tissue Centrifugally packed collagen I gels seeded with C2C12 cells wereimplanted into nude mice. Grafts (when implanted: 0.3 mm diameter, 1 mm length) were explanted at 1,2 and 4 weeks to assess the formation of a capillary network. After four weeks, a dense capillary networkwas formed in the vicinities and on the surfaces of the grafts. [59]

1.12 Groningen

Professor:van Wachem P.B., van Luyn M.J.A., Ponte da Costa M.L., Brouwer L.A.Fac for Medical Sciences,

Section 1.13 19

Cell Biology and Biomaterials,University of Groningen,Bloemsingel 10,9712KZ Groningen

1.12.1 Research

The collagen network, evaluated in this study [94], showed non-cytotoxicity, a slow degradation, and highingrowth of collagen producing fibroblasts in previous work. However, muscle was not found to growinto the dermal sheep collagen (DSC), formed by modified cross-linking procedures. Therefore, mouseC2C12 myoblasts were seeded in the previously developed dermal sheep collagen. After culture of 7 days(at day 3 differentiation medium was added) myotube formation had taken place. Cells were distributedreasonably and cell survival was 45%, as determined from toluidine blue staining on 2 µm sections. Future:improving spreading facilities for the cells by pretreatment of the collagen bundles with fibronectin or anadditional fibrin network; use of immunocytochemical techniques to further identify extracellular matrixand follow differentiation by α actin staining.In 1999, again DSC was used as a cell carrier for implantation in the abdominal wall muscle. Foetal ratmyoblasts were seeded in DSC-discs and non-seeded discs served as controls. The discs were implantedup to six weeks. Ingrowth of host cells and tissue at the margins proceeded faster with the seeded discs.It was concluded that the chosen method of myoblast seeding did not result in the regeneration of muscleduring the observation period. [93]

1.13 London and Stanmore

Professor: R.A. BrownR.A. Brown, M. Eastwood, V. Mudera, R.T. Prajapati, D.A. McGrouther

1.13.1 Research

A culture force monitor (figure 1.5) has been developed by this group [32], [31].

Figure 1.5: The tensioning-Culture Force Monitor. Indicated are the force transducer (f), the culture well(w), and the micro-stepping motor system (m). [33]

This device enables precise uni-axial mechanical loads to be applied to fibroblast populated collagenlattices whilst simultaneously recording the total mechanical load across the lattice (i.e. including the


cellular contraction). Through finite element analysis the exact loading pattern in the lattice can bedefined [33]. The collagen and fibroblast orientations have been studied under mechanical loading patternswith this force monitor.More recently, the differences in force generation of skeletal myoblasts, dermal fibroblasts, and smoothmuscle cells were measured in a 3D culture model in which cells contract a collagen gel construct. Theforce transducer in the set-up was from the Measurements group, Basingstoke, England. One end of theconstruct is connected to a fixed point, the other to the force transducer. The output signal was processedwith a Labview program.Construct morphologies were studied after fixation and toluidine blue staining, FITC Phalloidin stainingof F-actin, rhodamine phalloidin. The total cellular RNA was isolated for RT-PCR and cDNA amplification.With this technique IGF-1 Ea and MGF mRNA expressions were quantified [20].

1.14 Other interesting literature on muscle tissue engineering

The role of stem cells in skeletal and cardiac muscle repair,Journal of Histochemistry and Cytochemistry, Volume 50, Issue 5, 2002, Pages 589-610Grounds M.D.; White J.D.; Rosenthal N.; Bogoyevitch M.A.Muscle injuries and repair: Current trends in research,Journal of Bone and Joint Surgery - Series A, Volume 84, Issue 5, 2002, Pages 822-832Huard J.; Li Y.; Fu F.H.Three dimensional culture system for myocyte growth and differentiation,American Society of Mechanical Engineers, Bioengineering Division (Publication) BED, Volume 29, 1995,Pages 91-92Mulder M.M., McElwain J.F., Tresco P.A.Abstract: An interdisciplinary approach has been taken in the development of a porous 3-D matrix for growing muscle cells in vitro. This system may be

useful as a muscle tissue surrogate for experimental and clinical applications such as in the delivery of genetically engineered cells and in the reconstruction

of damaged or diseased skeletal or cardiac muscle tissue.

Skeletal myogenesis on elastomeric substrates: implications for tissue engineering,Journal of Biomaterials Science. Polymer Edition, Volume 9, Issue 7, 1998, Pages 731-748Mulder, M M; Hitchcock, R W; Tresco, P AAbstract: Studies geared towards understanding the interaction between skeletal muscle and biomaterials may provide useful information for the development

of various emerging technologies, ranging from novel delivery vehicles for genetically modified cells to fully functional skeletal muscle tissue. To determine

the utility of elastomeric materials as substrates for such applications, we asked whether skeletal myogenesis would be supported on a commercially available

polyurethane, Tecoflex(TM) SG-80A. G8 skeletal myoblasts were cultured on Tecoflex(TM) two-dimensional solid thin films fabricated by a spin-casting

method. Myoblasts attached, proliferated, displayed migratory activity and differentiated into multinucleated myotubes which expressed myosin heavy

chain on solid thin films indicating that Tecoflex(TM) SG-80A was permissive for skeletal myogenesis. Porous three-dimensional (3-D) cell scaffolds were

fabricated in a variety of shapes, thicknesses, and porosities by an immersion precipitation method, and where subsequently characterized with microscopic

and mechanical methods. Mechanical analysis revealed that the constructs were elastomeric, recovering their original length following 100% elongation.

The 3-D substrates were seeded with muscle precursors to determine if muscle differentiation could be obtained within the porous network of the fabricated

constructs. Following several weeks in culture, histological studies revealed the presence of multinucleated myotubes within the elastomeric material. In

addition, immunohistochemical analysis indicated that the myotubes expressed the myosin heavy chain protein suggesting that the myotubes had reached

a state of terminal differentiation. Together the results of the study suggest that it is indeed feasible to engineer bioartificial systems consisting of skeletal

muscle cultivated on a 3-D elastomeric substrate.

A mathematical model that predicts the force-frequency relationship of human skeletal muscle, Muscle &Nerve, published online 15 Aug 2002Ding J., Wexler A.S., Binder-Macleod S.A.,

Section 1.14 21

Stem cell route to neuromuscular therapies, Muscle & Nerve, published online 16 Sep 2002Partridge T.A.Gene technology and tissue engineering,Minimally Invasive Therapy and Allied Technologies, Volume 11, Issue 3, 2002, Pages 93-99Andree C.; Kullmer M.; Wenger A.; Schaefer D.J.; Kneser U.; Stark G.B.Abstract: This review discusses principles and methods of delivering genes encoding growth factors into cells, together with their respective advantages and

disadvantages.


Table

1.1:O

verviewof

groupsworking

onengineered

skeletalm

uscletissue

Location

Con

structs

Nam

es#

cellsFocu

s/researchE

indhovenC

ollagen/Matrigel

Baaijens

Bouten

4.106

cells/ml

ε=

0-50%C

2C12

-m

urineB

reulsPeeters

Oom

ensLondon

Agarose

Bader

Wang

Lee

2.106

cells/ml

ε=

0-40%C

2C12

-m

urineK

nightP

rovidenceC

ollagen/Matrigel

Vandenburgh

Pow

ell5.33.10

6cells/m

lε

=0-20%

C2C

12-

murine

Swasdison

Karlisch

dynamic

loadingM

yoblasts-

avianM

yoblasts-

neonatalrat

Biopsies

-hum

anM

ichigan(I)

Biopsies

-neonatal

ratD

ennisK

osnik1.10

5cells/cm

2nerve

coculturesB

iopsies-

adultrat

FaulknerG

ilbertC

2C12+

10T1/2

-m

urineB

iopsies-

adultC

3Hm

iceM

ichigan(II)

Alginates

PLG

sM

ooneyR

owley

scaffoldsR

GD

sC

2C12

-m

urineSheridan

vascularizationB

ostonP

GA

Vacanti

Saxenain

vivovascularization

Myoblasts

-neonatal

ratP

ittsburghsilanized

glassA

carturkD

iMilla

review,m

yotubee.g.

ED

Aand

13FD

iedwardo

alignment,

myoblasts

C2C

12-

murine

onbiom

aterialsB

irmingham

Skeletalm

uscle+

Swasdison

Mayne

5.105

cells/ml(quail)

myotendinal

junctionFibroblasts

-chicken

1.105

cells/ml(chicken)

coculturesw

ith(out)collagen

gelB

erkeleyM

yocytes-

embryonic

chickenStrohm

anB

ayne0.23

cells/cm2

orientedm

yogenesisFibroblasts

-em

bryonicchicken

Obinata

Spectorno

antibiotics/fungicideson

mem

branesTokyo

Kishi

Nakajim

aJapanese

reviewTanaka

SatohO

sakaC

2C12

-m

urineO

kanoM

atsudahigh

celldensity

collagenI

gelsorientationin

vivovascularization

Groningen

C2C

12-

murine

vanW

achemvan

Luyn

4.106

cells/ml

muscle

regenerationM

yoblasts-

fetalrat

Collagen

London

andStanm

oreC

2C12

-m

urineB

rown

Eastw

ood4.10

6cells/m

l3D

muscle

organoidsFibroblasts

-derm

alM

uderam

easurement

offorce

Smooth

muscle

cellsgeneration

Chapter 2

Cell damage, cell death andmechanotransduction

In this chapter the appearance of the healthy muscle and its molecular contraction mechanism will bediscussed. The properties by which a muscle’s functional performance can be measured are defined.Furthermore, events that occur during and after cell damage and different mechanisms that lead to celldeath will be presented. Finally, markers for cell damage and cell death are proposed.

2.1 The healthy muscle

Figure 2.1: On the left the anatomy of the healthy muscle is depicted. On the right a more detailedoverview of a muscle fiber with its myofibrils is drawn and on the bottom a muscle fiber is shown with aclearer distinction of the bands and discs. (SR= sarcoplasmic reticulum)

Three different kinds of muscle are found in vertebrates: 1) heart or cardiac muscle make up the wall ofthe heart; 2) smooth muscle is found in the walls of all hollow organs except for the heart; 3) skeletal orstriated muscle is the muscle attached to the skeleton as its name implies. The remainder of this section

23

24 CHAPTER 2. CELL DAMAGE, CELL DEATH AND MECHANOTRANSDUCTION

will focus on the latter muscle which is under voluntary control.Before starting a description of the damaged and dead muscles and cells, it is good to take a quick lookat the anatomy of a healthy muscle. In figure 2.1 (top left) a part of a muscle is shown that is attachedto the bone through a tendon. The complete muscle bundle is embraced by a membrane that is calledthe epimysium. The largest structural bundles that can be found in a muscle are the fascicles which areenclosed by the perimysium. Among the fascicles some blood vessels run to supply oxygen and nutrientsto the muscle tissue and to remove its waste products. A fascicle in turn consists of muscle fibers (thecells). Each muscle fiber is surrounded by its endomysium. A muscle fiber contains myofibrils that arestacked lengthwise, mitochondria (or sarcosomes), endoplasmic reticulum (or sarcoplasmic reticulum),and many nuclei. The multiple nuclei arise from the fact that each muscle fiber develops from the fusionof muscle cells (myoblasts). The nuclei as well as the mitochondria are located just beneath the plasmamembrane (sarcolemma) while the sacroplasmic reticulum extends between the myofibrils (figure 2.1,right). The striated pattern of the muscle fiber is created by alternating dark A bands and light I bands.The A bands are bisected by the H zone and the I bands are bisected by the Z line (figure 2.1, bottomleft). Each myofibril is made up of arrays of parallel filaments. The thick filaments, composed of theprotein myosin, have a diameter of about 15 nm. The thin filaments, mainly composed of actin, troponinand tropomyosin, have a diameter of about 5 nm. The entire array of thick and thin filaments betweenthe Z lines is called a sarcomere.

Section 2.1 25

2.1.1 Molecular muscle movement

Calcium ions, Ca2+, link action potentials in a muscle fiber to contraction. In a resting muscle, Ca2+ isstored in the sarcoplasmic reticulum (SR). Spaced along the sarcolemma (plasma membrane) of the musclefiber are inpocketings of the membrane that form tubules of the so-called T system. These tubules plungerepeatedly into the interior of the fiber. The tubules terminate near calcium-filled sacs of the sarcoplasmicreticulum. [2]

Figure 2.2: Overview of muscle contraction on amolecular level.

Each action potential created at the neuromuscularjunction sweeps quickly along the sarcolemma and iscarried into the T system. The arrival of the actionpotential at the ends of the T system triggers therelease of Ca2+ from the SR among the filaments. Thechange in Ca2+ concentration in the cytosol followingexcitation is impressive; the resting concentration is10−7M, but following successive stimuli it rises to 10−5M(a 100-fold change) [55]. Two types of channels releasecalcium from the SR: the DHP channels and the ryanodinereceptors.Because of the speed of the action potential (7 cm/s[55]), the action potential arrives virtually simultaneouslyat the ends of all the tubules of the T system, ensuringthat all sarcomeres contract in unison. When theprocess is over, the calcium is pumped back into thesarcoplasmic reticulum using a Ca2+ ATPase.The calcium ions that are released among the filamentsbind there to troponin(-C subunit) on the thin filaments.This uncovers the attachment sites of the actin molecule.The myosin head attaches to the actin filament, causinga release of Pi (step 3, attachment, in figure 2.2).The release of Pi causes a conformational change ofthe myosin head that is associated with the relativemovement of the actin and myosin filaments (step 4,power stroke, in figure 2.2). During the power strokeADP is released. Upon subsequent binding of ATP tothe myosin head, the myosin head is uncoupled fromthe actin again (step 1, detachment, in figure 2.2). TheATP is hydrolysed and ADP and Pi remain on themyosin head while its conformation is changed backagain (step 2, resetting, in figure 2.2). The cycle is nowcompleted. By each cycle the sarcomere is shortenedby 1%. If the muscular contraction is studied on thelevel of the A and I bands, the Z lines come closertogether, the widths of the I bands and the H zones decrease and the width of the A bands remains thesame during muscle contraction.


2.1.2 Quantification of muscle performance

In this subsection some properties of muscle tissue will be defined concerning the excitability and contractilityof muscle tissue.

• Excitability is the capability of being activated by and reacting to stimuli.

• Contractility is a muscle’s capability to shorten.

• The process of contracting takes some 50 msec; relaxation of the fiber takes another 50-100 msec.In figure 2.3 shocks are given at 1/sec and the muscle responds with a single twitch. At 5/sec and10/sec, the individual twitches begin to fuse together, a phenomenon called clonus. At 50 shocksper second, the muscle goes into the smooth, sustained contraction of tetanus. Clonus and tetanusare possible because the refractory period is much briefer than the time needed to complete a cycleof contraction and relaxation. Note that the amount of contraction is greater in clonus and tetanusthan in a single twitch. [2]

Figure 2.3: The muscle fiber’s contractional response is shown as a function of pulse frequency. [2]

• At the critical fusion frequency (a pulse frequency) the Ca2+ concentration does not show aripple anymore. In figure 2.3 this the stimulation frequency at which the clonus fuses into tetanus;thus a pulse frequency exceeding the critical fusion frequency causes a tetanized state of the musclefiber.

Figure 2.4: Left: The relationship between stimulus strength and stimulus duration in terms ofmuscle excitability is plotted. If a stimulus has strength and duration combined above the line, themuscle is excited. Also, the determination of chronaxie and rheobase are shown [9]. Right: Twitchcharacteristics: Contraction time (Tc), Half relaxation time (Thr), Duration of the twitch (Ttw),Maximal force (Fmax).

Section 2.2 27

• Apart from an upper limit at which muscle tissue can be excited, the minimum stimulus strengththat will produce a response is called the rheobase. Not only the magnitude of a stimulusdetermines if a response is evoked. The duration of the stimulus also plays a role, as shown infigure 2.4. The duration that gives a response at twice the rheobase strength is called chronaxie.Lower values of both rheobase and chronaxie indicate greater excitability.

• If a muscle contracts and moves the attached weight according to its own shortening, the contractionis isotonic. If a muscle is prevented from shortening by e.g. fixing both ends, the contraction isisometric. A concentric contraction is when a muscle contracts while it is allowed to shorten,whereas during an eccentric contraction the muscle lengthens during contraction.

• Twitch contraction time is the interval between the onset of tension and the peak value (figure2.4).

• The isometric force is a muscle’s force that is exerted during isometric (constant length) contraction.The peak isometric force, P0, is the peak value of force that is reached during contraction. Thespecific force, sP0, is peak isometric force divided by the cross-sectional area (P0/A). The specificP0 of adult phenotype skeletal muscle is approximately 300 kN/m2 [35].

• The resting baseline force, Pb, is the force that is exerted by the muscle in a non-stimulated(resting) situation.

• A muscle twitch is an activation of a muscle fiber by a single firing of a motor neuron (twitch infigure 2.4). A twitch can also be created by applying a small electric shock across the membraneof a muscle cell. The result in either case is an increase in tension in the muscle fiber. A muscletwitch is defined as the contraction of a whole muscle in response to a stimulus that causes anaction potential in one or more muscle fibers. The twitch force should then be defined as the forceexerted by a muscle fiber during a single contraction.

• The time to peak tension, Pt, is called the contraction time (Tc in figure 2.4).

• One-half relaxation time RT1/2 is the time that has passed since the onset of the muscle twitchuntil the force has declined to half the maximum value (figure 2.4).

2.2 Cell damage and mechanotransduction

2.2.1 Second messengers and growth factors

From Peeters [64]: Second messengers are important to transfer the signal from the mechanosensor to thenucleus. Growth factors are signaling molecules that stimulate a cell to grow or proliferate. The inductionof cell growth by growth factors is similar to the load responding second messenger system for unicellularsystems. Skeletal muscle cells are reported to release not only growth factors upon mechanical stimulationbut also increase the amount of associated binding proteins. Elements and structures in the cell that areinvolved in converting mechanical loads into biochemical signals (mechanosensors) are summarized below:

• Stretch activated ion channels (SAC): The SACs can be specific and non-specific to certainions. The channel activity occurs in bursts whose frequency increases with tension, while thechannel current does not change with tension. Besides stretch activated channels, there existstretch-inactivated channels which decrease their permeability in response to stretch. SAC are


activated by membrane tension. It is assumed that SAC are attached to a tension bearing element(which may be part of the cytoskeleton).

• Integrins and the cytoskeleton: It is unclear whether the cytoskeleton responds directly tomechanical forces or if it is part of the signal transduction cascade. The actin cytoskeleton is coupledto components of the extracellular matrix by integrins. The existence of over 20 distinct integrinsraises the likelihood of individualization of signaling pathways. It is proposed that conformationalchanges within membrane proteins or cytoskeletal proteins start mechanotransduction.

• G-proteins: G-proteins are signal transducing proteins that couple a large number of membranebound receptors to a variety of intracellular effector systems. These effectors are ion channels orenzymes that generate regulatory proteins or second messengers. Second messengers can generatedramatic intracellular changes including protein phosphorylation, gene transcription, cytoskeletonreorganization, secretion and membrane polarization. It is not clear whether G-proteins are directlyactivated by mechanical forces or if they are just involved downstream of the mechanotransducers.

2.2.2 Cascade of muscle cell damage after eccentric muscle action

Eccentric muscle action is muscle elongation during contraction. The damage that can be caused bythis kind of action starts with cytoskeleton and sarcolemma disruption such that desmin is lost from theintracellular area (desmin plays a role in the mechanical integration of adjacent myofibrils by connectingthem at the Z-line). In the first 24 hours the connecting units between sarcomeres are damaged. Thisprocess is called streaming of Z-lines [55]. Type II fibers are damaged more frequently than type I fiberswhich is partly due to the wider Z-lines in type I fibers and a thicker connective tissue surrounding thefibers [4]. Following the initial exercise-induced muscle fiber damage the whole-body protein turnover isincreased for several days. In a murine model, protein degradation was elevated after 48 hours when alsophagocytes infiltrate. The protein synthesis was increased also after 48 hours. The phagocyte infiltrationand IL-1β secretion are part of an inflammatory response in the cascade following unaccustomed eccentricmuscle action [82]. However, the inflammatory response will not be discussed in this review.On days 10-15 necrosis and inflammation of the tissue can be observed [55]. The 2-3 weeks after that,much of the tissue is repaired and regenerated. The eccentric fiber damage is due to Ca2+ and freeradicals in the cytoplasm.

• The calcium elevation activates the nonlysosomal cysteine protease, calpain. Calpain cleaves cytoskeletalproteins such as desmin, α-actinin, vimentin and integrin and myofibrillar proteins such as troponinand tropomyosin and thus plays a key role in disassembling and remodeling of the cytoskeletalmatrix. Three major proteolytic processes are believed to be responsible for the breakdown ofproteins in skeletal muscle: the lysosomal pathway, Ca2+ activated pathway (with calpain) andthe ubiquitin-proteasome-dependent pathway [14]. The first two pathways contribute to 15-20% ofthe total protein breakdown. Other activated proteases break down proteins in the mitochondria.Circulating complement components also gain acces to the fiber through gaps in the plasmalemma,are deposited, and assist in the destruction of the myofibrils, organelles, and tubular systems.Myofibrils retract from a necrotic site and form a stump that becomes covered with newly formedmembrane. [55]The most sensitive marker for rise in plasma levels of muscle enzymes is creatine kinase, CK (afterheavy muscle exercise). A rise in CK can be observed 24 hours after loading and the value returns tonormal after another day or two. Cleaved troponin I (TnI) and myosin heavy chain (MHC) can bedetected in peripheral blood by specific assays and have also been described as markers of skeletalmuscle injury. [82]

Section 2.2 29

• The role of increased free radical production and HSP formation after skeletal muscle damagefollowing exercise are reviewed by McArdle [54]. The mechanisms by which skeletal muscle damageafter unaccustomed or excessive exercise occurs are poorly understood. Oxygen consumption isincreased during exercise as is the production of reactive oxygen species. A number of these speciesare released into the muscle extracellular space during contractile activity. The most damagingmuscle exercise is eccentric contraction. There is considerable evidence that supplementation withspecific antioxidants results in the reduction of some indicators of exercise-induced free radicalactivity. However, the role of free radical production in eccentric exercise-induced muscle damagehas been less well studied and data are conflicting. After eccentric contractions, small focal lesionswithin the muscle can be detected by electron microscopy. This direct trauma to the muscle maylead to a ’secondary’ increase in free radical activity although the site of free radical generationappears to be primarily from invading phagocytic cells. The effect of antioxidant supplementationremains a matter of dispute.Another cytoprotective response in skeletal muscle and other cells is the increased production ofstress or heat shock proteins (HSPs). HSPs are important components of the cellular protectiveresponse against reactive oxygen species. An increase in the muscle content of HSPs occurs followingexercise in humans. More on HSPs is explained in subsection 2.2.4.

2.2.3 Exercise-induced oxidative stress

Radical formation in the human body happens frequently to oxygen: e.g. O−2 . Exercise increases the

production of free radicals, as oxygen consumption is increased. Cell membranes contain unsaturated fattyacids that are very susceptible to radical attack. This process is called lipid peroxidation and increasespermeability. This results in a Ca2+ influx, loss of intracellular enzymes, and an influx of lysosomalenzymes. The primary defense of the cell is superoxide dismutase (SOD), which catalyzes the reaction:2O−

2 + 2H+ → H2O2 + O2

Though H2O2 is not a radical, it has been shown to damage nucleic acids. The reduction of H2O2 iscatalyzed by other antioxidants, glutathione peroxidase and catalase. Glutathione is a major nonenzymaticantioxidant and has several important functions including removal of H2O2 and recycling of vitamin E.Vitamin E can also be reduced by reaction with vitamin C (ascorbate). Another important vitamin isβcarotene (precursor to vitamin A), in that it quenches singlet oxygen (O−

2 ) and inhibits lipid peroxidation.[4]

2.2.4 Measurement of markers for cell damage

From Sorichter 1999, Hagisawa 1988, Peeters 2000, probes.com [82], [37], [64], [7]

Calcium markers

• Aequorin emits light in the presence of free Ca2+, microinjection into the cell.

• quin-2 for Ca2+ measurement, see fura-2.

• fura-2. The Ca2+ concentration is presented by different colors. In a study by Carroll et al [19], theCalcium concentrations were monitored using the fluorescent calcium indicator fura-2. To verify thecorrection for kinetic delay of the calcium fura-2 reaction, the calcium equilibrating time was assessedby a more rapidly equilibrating calcium indicator, mag-fura-2. It was concluded that the correctionmade for the kinetic delay, was allowed. Fura-2 was incorporated in the cells by exposure to 2µM


fura-2 AM (acetoxymethyl ester, cell permeant form) in a 1 mM Ca2+ mammalian Ringer solution(in agarose 45 minutes at 37 ◦C, before agarose for 10 minutes at 37 ◦C) Then the fibers were washedwith dye-free 1 mM Ca2+ Ringer solution (20 minutes at 37 ◦C). A 75W xenon lamp was used forepi-illumination. The dye was excited at 358nm (isosbestic) and 380nm (calcium). Emission wasrecorded at 510nm. The isosbestic wavelength is defined as the wavelength at which two molecularspecies show the same molar absorption coefficient. Furthermore, fura-2 is considered to be superiorto quin-2 as a marker for Ca2+, because of several reasons [10]: the use of the isosbestic point forquantitative analysis of the calcium concentration, 30x more intense fluorescence signal, less sensitiveto cross-coloration of Mg, Mn, Zn, or Fe, and last but not least fura-2 has a higher dissociationconstant (lower affinity) for Ca2+, which facilitates measurement of higher concentrations.

• Ca2+ concentrations can further be imaged with fluo-4 AM staining. Figure 2.5 contains imagesof changes in intracellular free Ca2+ in cells, monitored at 9-second intervals with fluo-4 AM. Inorder to induce an influx of Ca2+, the cells were depolarized with 50 mM KCl in frame 2 andexposed to 5 M ionomycin (a Ca2+ ionophore) in frame 8. The images are pseudocolored accordingto fluorescence intensity, with red representing high Ca2+ concentrations and blue representing lowCa2+ concentrations. The images were acquired using a fluorescence microscope equipped witha longpass filter set appropriate for fluorescein and a Photometrics Quantix cooled CCD camera.Fluo-4 exhibits a Kd for Ca2+ of 345 nm. The probe is excited at 488 nm and is available as acell-impermeant potassium salt or as its cell-permeant AM ester. [7], [47]

Figure 2.5: Calcium concentration imaging in time with fluo-4 AM staining. [7]

• Calcium ionophore A23187 (Sigma) is a marker for calcium fluxes. The Ca2+ ionophore (A-23187,Molecular probes) is commonly used for in situ calibrations of fluorescent Ca2+ indicators, toequilibrate intracellular and extracellular Ca2+ concentrations and to permit Mn2+ to enter thecell to quench intracellular dye fluorescence.

Metabolic markers

• Inorganic phosphatase reflects the status of cellular metabolism as it relates to oxygen and ATPavailability. In-phos is sensitive to tissue ischemia and it equilibrates after release of indentation,whereas tissue damage is ongoing. Inositol triphosphate is generated by a surface receptor after asignalling molecule has bound to it. The in-phos subsequently stimulates the release of Ca2+ fromthe ER into the cytosol.

• Lactate production. Lactate can be measured by an enzymatic method, LACT, by BoehringerMannheim. [81]. Another interesting way to measure lactate was reported by Ferguson-Pell and

Section 2.2 31

Hagisawa in 1988. They measured lactate concentrations in sweat during and after indentation ofsoft tissues. During the indentation elevated levels of lactate were registered [34] that returned tonormal after the load was removed.

Stress markers

• HSPs, heat shock proteins, are so called because they are induced in response to thermalstress (and other stresses such as mechanical loading), most notably after hyperthermia. Theyare usually named after their molecular weight and include the small HSPs such as αβcrystallin,mitochondrial HSP60 and HSP10, the HSP70 family, and the larger HSPs such as HSP90 andHSP110. HSPs are present at low levels in the unstressed cell, where they act as molecularchaperones in various compartments within the cell, associating with newly synthesized proteinsand ensuring it is folded and functions correctly. The increased content of HSPs following stressis thought to provide considerable protection against subsequent periods of damaging stress andto facilitate rapid recovery and remodeling if damage occurs. All HSPs act to preserve cellularintegrity, HSP70 has the capacity to refold a wide array of proteins and other HSPs have morespecific roles within the cell. HSP60 is mainly located in the mitochondria together with its HSP10chaperone. [54]So, HSPs repair denatured proteins and protect other proteins from damage. Their recovery timeis 10-24 hours and they can be measured in large quantities after 24 hours (MEMO C. Bouten).Measurement can be performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),Western [44] and Northern blotting. After nondamaging exercise (proved by absence of CK production)the muscle content of HSP60 and HSP70 in humans increased [44]. HSP70 levels did not changesignificantly until six days postexercise. Some subjects, however, showed a clear rise in HSP70content after 24-48h.

• Simultaneous measurements of intracellular Ca2+ with fura-2 and nitric oxide production withDAF-2 have been reported. PubMed D-23841 D-23842 D-23843 ”Fluorescent Indicators forImaging Nitric Oxide Production.” Kojima H, Urano Y, Kikuchi K, Higuchi T, Hirata Y, NaganoT. Angew Chem Int Ed Engl 38, 3209-3212 (1999) PN41280.PubMed C-7912 D-23843 ”In vivo imaging of an elicitor-induced nitric oxide burst in tobacco.”Foissner I, Wendehenne D, Langebartels C, Durner J. Plant J 23, 817-824 (2000) PN40889.

• Nitric oxide (NO) within few minutes through membrane upon stretching; nitrite: Griess assay(spectrophotometric). DAF-FM (D-23841), DAF-FM diacetate (D-23842, D-23844), DAA (D-23840),Luminol (L-8455). The nitric oxide (NO) radical is very unstable, and no equilibrium indicatorsfor NO are known. However, NO is readily oxidized to the nitrosonium cation (NO+), whichis moderately stable in aqueous solutions but highly reactive with nucleophiles, or other nitrogenoxides. Under aerobic conditions, these reactive nitrogen oxides can be trapped by various amines, inparticular by aromatic amines to form diazonium salts or aromatic 1,2-diamines to form benzotriazoles.Probably the most successful indicator for nitric oxide has been 4,5-diaminofluorescein diacetate(DAF-2 diacetate), which was developed by Kojima and collaborators. As with other fluoresceindiacetates, DAF-2 diacetate is membrane permeant and is deacetylated by intracellular esterasesto 4,5-diaminofluorescein (DAF-2). DAF-2, however, remains essentially nonfluorescent until itreacts with the nitrosonium cation (produced by spontaneous oxidation of nitric oxide) to forma fluorescent heterocycle, which becomes trapped in the cell’s cytoplasm. However, the DAF-FMreagent has some important advantages over DAF-2: 1) Spectra of the NO adduct of DAF-FM areessentially independent of pH above pH 5.5; 2) The NO adduct of DAF-FM is significantly more


photostable than that of DAF-2; 3) DAF-FM is a more sensitive reagent for NO than is DAF-2(NO detection limit for DAF-FM 3 nM versus 5 nM for DAF-2). DAF-2 and its diacetate arenot available from Molecular Probes. It is anticipated that DAF-FM should give equal or superiorperformance to DAF-2 in most applications. Under physiological conditions, NO is readily oxidizedto nitrite and nitrate or it is trapped by thiols as an S-nitroso adduct. The Griess reagent provides asimple and well characterized colorimetric assay for nitrites, and nitrates that have been reduced tonitrites, with a detection limit of about 100 nM. Nitrites react with sulfanilic acid in acidic solutionto form an intermediate diazonium salt that couples to N-(1-naphthyl)ethylenediamine to yield apurple azo derivative that can be monitored by absorbance at 548 nm. More information can befound in appendix E.5.

• MDA, malondialdehyde, is a marker for membrane integrity and a product of fatty acid oxidation(lipid peroxidation) as a result of protein damage. Only a little volume is required for measurementand it can be detected by HPLC with fluorometric detection [56] or UV-spectrophotometry. Cellmembranes contain unsaturated fatty acids that are very susceptible to radical attack. This processis called lipid peroxidation and increases permeability. This results in a Ca2+ influx, loss ofintracellular enzymes, and an influx of lysosomal enzymes. Chiaradia et al [21] determined MDAand gluthatione contents in horses. After blood collection, the plasma was immediately separatedfrom the blood by centrifugation. The plasma was mixed with either butylated hydroxytoluene(BHT) or 5,5%-dithio-bis-(2-nitrobenzoic acid) (Ellman’s reagent) and than stored at -30C untilthe MDA or the glutathione assay, respectively. Total glutathione, which included both reducedglutathione and glutathione disulphide, content of the plasma was measured using the glutathionereductase- Ellman’s reagent recirculating assay. Due to the very low amount of glutathione presentin the plasma, the above enzymatic cycling was used as it continuously reduces glutathione-Ellman’sadduct using NADPH. The MDA content in the blood, as a measure of lipid peroxidation end-products,was assayed by separating the MDA-thiobarbituric acid adduct by high-performance liquid chromatoraphyHPLC and using fluorescence detection [30]. The fluorescence method is described in appendix E.6.

Mitochondrial markers

• Mitochondria are found in eukaryotic cells, where they make up as much as 10% of the cellvolume. They are pleomorphic organelles with structural variations depending on cell type, cell-cyclestage and intracellular metabolic state. The key function of mitochondria is energy productionthrough oxidative phosphorylation (OxPhos) and lipid oxidation. Several other metabolic functionsare performed by mitochondria, including urea production and heme, non-heme iron and steroidbiogenesis, as well as intracellular Ca2+ homeostasis. Mitochondria also play a pivotal role inapoptosis a process by which unneeded cells are removed during development, and defective cellsare selectively destroyed without surrounding organelle damage in somatic tissues. For many ofthese mitochondrial functions, there is only a partial understanding of the components involved,with even less information on mechanism and regulation.Mitochondrial activity is a determinant for muscle cell metabolism (via Rhodamine 123 retentiontechnique). Although conventional fluorescent stains for mitochondria, such as rhodamine 123 andtetramethylrosamine, are readily sequestered by functioning mitochondria, they are subsequentlywashed out of the cells once the mitochondrion’s membrane potential is lost. This characteristiclimits their use in experiments in which cells must be treated with aldehyde-based fixatives or otheragents that affect the energetic state of the mitochondria. Therefore, MitoTracher probes weredeveloped by Molecular Probes. [7]

Section 2.2 33

• The green-fluorescent JC-1 probe (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanineiodide, T-3168) exists as a monomer at low concentrations or at low membrane potential. However,at higher concentrations (aqueous solutions above 0.1 M) or higher potentials, JC-1 forms red-fluorescent”J-aggregates” that exhibit a broad excitation spectrum and an emission maximum at 590 nm.Thus, the emission of this cyanine dye can be used as a sensitive measure of mitochondrial membranepotential. Also, a JC-9 exists.

Markers of membrane leakage

• CK is a marker for membrane leakage which is mostly associated with inevitable cell death.Therefore, more information on this marker can be found in the section ’Markers for cell death’ andin appendix E.3.

• Myoglobin is a marker for membrane damage via loss of this intracellular enzyme (17.8 kDa). Itfacilitates oxygen diffusion and serves as oxygen reservoir in striated muscle fibers. In skeletal musclemyoglobin is mainly found in the slow twitch fibers. Myoglobin from the skeletal and heart musclecan not be distinghuised. Myoglobin can be measured within minutes by automated assays, such asimmuno-nephelometry, immunoturbidimetry, rapid-enzyme immunoassays [81]. The commerciallyavailable enzyme immunoassay (Myoglobin-Access; Sanofi-Diagnostics-Pasteur, Marnes-la-Coquette,France) was used by Onuoha et al. [62] and Sorichter et al. [81] (see also H-FABP). It can be detectedwithin 0.5-2 hours in the circulation. The clearance from circulation is rapid; 3 hours half-life. Asmyoglobin is much smaller than CK and LDH, it is released into the serum earlier. More informationcan be found in appendix E.4.

• Heart fatty acid binding protein (H-FABP) concentrations in human skeletal muscle are muchlower than in heart. After skeletal muscle injury, H-FABP and myoglobin show a similar patternof release into and clearance from plasma. FABP increases rapidly after injury, usually 2-4 hoursin parallel with myoglobin and peak values occurring about 5 to 10 hours later. Lack of muscletype specificity of both markers may be addressed by calculating the myoglobin over H-FABP ratio:20-70 depending on fiber type composition and approximately 4-5 in case of heart muscle injury.

• Carbonic anhydrase (CA) isoenzyme III. CA is a soluble protein (29kD) that efficiently catalyzesthe hydration of CO2 to bicarbonate and a proton. The CA III isoform is mainly expressed inslow-twitch skeletal muscle fibers. As for H-FABP, calculation of the ration of myoglobin over CAIII is proposed for increasing the muscle type specificity.

Proteins lost from extracellular structures

• Cleaved troponin I (TnI) and myosin heavy chain (MHC) can be detected in peripheral bloodby specific assays and have been described as markers of skeletal muscle injury. [82] From Pinelli2002 [67]: Necrosis markers in cardiac muscles: troponin I, myoglobin, creatine kinase MB.TnI occurs in three forms: skeletal TnI (sTnI) exists as a slow twitch (sTnI-slow) and a fast twitch(sTnI-fast) isoform, and also a myocardial isoform (cTnI) exists. sTnI can be measured with animmunoenzymometric assay [62].

• Myosin is the primary component of the thick filaments and the predominant protein in musclefibers. Myosin is a hexameric structurally bound contractile protein containing 4 light and 2 heavychains (MHC, molecular weight 230,000). MHC can be cleaved into its subfragments by enzymes.Concentrations of MHC can be measured by an immunoradiometric assay (ERIA Diagnostics


Pasteur, Marnes la Coquette, France [81]). [52]. One MHC weighs approximately 23 kDa. Asall MHC of muscle fibers appears to be structurally bound, the protein is expected to specificallyindicate muscle fiber necrosis. [82]

Intracellular signaling

• cyclic adenosinemonophosphate (cAMP): In collaboration with Atto Bioscience, MolecularProbes offers Cyclic AMP Fluorosensor (FlCRhR, C-6660), the first fluorescent probe availablefor the nondestructive measurement of cyclic AMP (cAMP) in live cells by digital video imaging,confocal laser-scanning microscopy or microphotometry. This probe consists of cAMP-dependentprotein kinase A (PKA) in which its recombinant catalytic (C) and regulatory (R) subunits arelabeled with fluorescein and rhodamine, respectively. Fluorescence resonance energy transfer (FRET)from fluorescein to nearby rhodamine labels occurs readily in the holoenzyme configuration (C2R2)but is eliminated upon subunit dissociation in response to cAMP binding (4 molecules of cAMPper C2R2 holoenzyme). Consequently, the ratio of fluorescein ( 520 nm) and rhodamine ( 580nm) emission intensities excited at 488 nm can be quantitatively related to cAMP concentration.Cyclic AMP Fluorosensor is a protein complex with an aggregate molecular weight of 172,000daltons; therefore, it must be pressure microinjected into the cytoplasm for intracellular cAMPmeasurements. We offer a detailed protocol for this procedure (Cyclic AMP Fluorosensor (FlCRhR)).Complete cAMP response calibration of the probe is currently only possible in vitro. The affinityfor cAMP and the kinase activity of the probe are similar to those of the unlabeled protein kinase.

• protein kinase C: Protein kinase C (PKC) is a key player in many transmembrane signal transductionsystems. This Ca2+-dependent serine/threonine protein kinase is activated in the presence ofcertain membrane-derived lipids such as diacylglycerols and phosphatidyl serines, phosphorylatinga wide variety of substrates, including ion-channel proteins and cytoskeletal proteins. MolecularProbes offers a highly purified preparation of PKC holoenzyme (P-7432, Protein Kinase C), whichis prepared from rat brain using ion-exchange and affinity chromatography. These chromatographictechniques yield an enzyme preparation that is >95% pure by gel electrophoresis and contains theα, β and γ-isoforms (77,000 to 80,000 daltons). Purified PKC can be used to identify substrates,to determine the state of phosphorylation of PKC substrates and to serve as a positive control inPKC assays.

Other cell damage markers

• Aspartate aminotransferase (ASAT) was the first marker for laboratory diagnosis of muscle injury.However, it lacks muscle fiber specificity and by now has lost its diagnostic significance.

• A new imaging agent for skeletal muscle damage is Tc-99m-glucarate. It may become a usefulagent for clinical non-invasive monitoring of skeletal muscle damage after ischemia-reperfusion. [96]

• Leucine incorporation is an indicator of myofibril turnover.

• 3H-thymidine incorporation is an indicator of DNA synthesis that increases in loaded and hypertrophicmuscles.

• bFGF increases in the medium of cyclically loaded muscle cells and can be determined with ELISA.

• DNP enzyme loss indicates ultrastructural damage.

Section 2.3 35

• FDxLys is an indicator of myotube membrane wounding.

• Tetrodotoxin blocks stress-receptor sodium channels.

• Prostaglandins (F2α) are second messengers that mark protein turnover and can be determined byradioimmunoassays.

• Indomethacin blocks protein turnover in C2C12 cells.

• αactin (aa) ; muscle specific??

• inositol phosphatases

• c-fos

2.3 Definitions of cell death

It is necessary to be able to distinguish several ways in which a cell can die. Terms such as apoptosis,oncosis, necrosis and programmed cell death are used to designate the different pathways. However, thereis a lot of confusion on the exact definitions of the cell death pathways and the overlap in definitions. Inthis section an attempt has been made to clarify and define these terms.Cell death was first described by Virchow in 1858 and macroscopic observations led to the terms degeneration,mortification and necrosis. The first microscopic denominations for cell death were reported in 1879.Karyorhexis indicates the disintegration of the nucleus, karyolysis describes the disappearance of thenucleus, chromatolysis is combination of nucleus disintegration and subsequent vanishing and pyknosisconsists of contraction of nuclear contents to a deep-staining irregular mass. Cell death research wasgreatly enhanced with the introduction of the term apoptosis in 1972 by Kerr [42]. A biochemicaldistinction between apoptosis and necrosis lies in the fragmentation of the genome and cleavage ordegradation of several cellular proteins in apoptosis. Originally the phenomenon of decreasing cell volume,ruffled cell membranes (budding), condensed and marginated chromatin, increased cellular calcium,cell segregation with the formation of numerous vesicles (apoptotic bodies) containing intact organellesand/or nuclear material and the release of cytochrom c from the mitochondria [11], was called shrinkingnecrosis [24]. But after about a year it was renamed as apoptosis (the Greek word for falling of leaves). Themorphological features characterizing necrosis will be summarized later in this section and are illustratedin figure 2.6. Macrophages recognize the apoptotic cell fragments by their expression of phosphatidylserine on the outside of the plasma membrane (also vitronectin receptors and certain carbohydratescan evoke phagocytosis). Changes in the surface molecules prevent an inflammatory response that doesoccur in necrosis. If cells are not recognized by the phagocytes (or they are not present, e.g. in vitroculture) they may undergo secondary or apoptotic necrosis. In this terminology, necrosis is defined asthe final stage for any cell death process. During secondary necrosis no inflammatory reaction occurs [53].


Necrosis

Phagocytosis,

inflammation

Blebbing

Oncosis Apoptosis

Budding

or nearby cells

by macrophages

Apoptosis

Necrosis

Phagocytosis

Figure 2.6: On the left an illustration of apoptosis and necrosis according to Kerr et al. [43] is shown,while on the right apoptosis and oncosis, according to Majno and Joris [53], are illustrated.

Necrosis is marked by cellular swelling, often accompanied by chromatin condensation and eventuallyleading to cellular and nuclear lysis with subsequent inflammation. Apoptotic cells that undergo secondarynecrosis show several of these features, except for the inflammation. In contrast to necrosis, the plasmamembrane of apoptotic cells remains intact until very late in the process. Furthermore, the apoptoticprocess is coordinated and requires ATP as energy source, while that is not the case for necrosis.Another important term in cell death is oncosis. Oncosis, the Greek word for swelling, is a form of passiveor accidental cell death, also referred to as swelling necrosis. This type of lethal injury is characterizedby nuclear and cytoplasmic swelling, vacuolization of cytoplasm, and mitochondrial swelling. It is thusdifferent from apoptosis , which is not accidental and which is characterized by cell shrinkage. Cell deathcaused by ischemia is often associated with features of oncosis. The term oncosis is used frequently tomean necrosis or to refer to the early phase of primary necrosis. Both types of cell death (i.e. oncoticand apoptotic) are pre-mortal processes and lead to postmortem changes collectively termed necrosis (seefigure 2.6). The three terms mentioned before, pyknosis, karyorhexis and karyolysis, are not specific foreither apoptosis or oncosis.The term ’programmed cell death’ refers to the ’fixed’ pathway followed by dying cells, whether or notwith the characteristic morphology of apoptosis. Three mechanisms that are involved in apoptosis are areceptor-ligand mediated mechanism, a mitochondrial pathway and a pathway in which the endoplasmaticreticulum plays a central role; they all lead to caspase activation (figure 2.7). Once the caspases areactivated, the final apoptotic stage of CAD (caspase activated DNase) is started [11]. For more informationon caspase activity, the reader is referred to an evaluation by Khler et al (2002, [45]). According to

Section 2.3 37

Procaspase-8

tBid

Cell membrane

Caspase-8

Death receptor oligomer

APOPTOSIS

Smac/DIABLO XIAP Survivin

CrmABid

Floppase FADD

Death ligands (Fas and TNF )α

BaxBad Bcl-xL

Bcl-2

PSPS PS

PS

PS

AIF

?????????

CytosolMitochondrium

disruption

Inducing factor

Procaspase-9

Cytochrom c

????????

Membrane potential

Apoptosom

APAF-1+

NO

+

Procaspaseproteases

caspase-9

caspase-1caspase-3

Figure 2.7: A scheme of the apoptotic pathways, adapted from Huang [40] and Adams [11]. The bluearrows indicate a stimulatory influence, whereas the red arrows indicate inhibition. For a more detailedoverview the reader is referred to Cruchten and Broeck [24]

Huang [40], two major apoptotic pathways exist, both of which involve a family of cysteine proteases withaspartate specificity, the already mentioned caspases, as the executioners of apoptosis. The mitochondriaprovide one of the apoptotic pathways from which cytochrome c is released upon the stimulation by avariety of cell-death triggers. This leads to activation of Apaf-1 (apoptosis protease activating factor-1),caspase-9, and caspase-3. Another apoptotic pathway involves the ligation of death receptors such as Fasand activation of caspase-8 and subsequently caspase-3.The role of the mitochondria in apoptosis is further explained by Richter et al [73] in terms of the ATPproduction. The mitochondrial membrane potential is the driving force for mitochondrial ATP synthesis.It is proposed that the cellular ATP level is an important determinant for cell death, either by apoptosis ornecrosis. If the ATP drops below a certain level, apoptosis follows if enough ATP is available (rememberthat apoptosis is an active cell process). If the drop is too severe, necrosis is started instead of apoptosis.It is unknown if the ATP drop is the consequence or the cause of cell death. An ADP/ATP ratio of about


0.2 was the critical discriminator between survival and apoptosis in all cell types. All cellular processesinvolved in apoptosis are summarized in figure 2.7. Apoptosis can be prevented according to the depictedpathways. Bcl-2 for example stimulates an antioxidative response in cells and thereby prevents apoptosis.From Joza et al 2001 [41]: Our data provide genetic evidence for a caspase-independent pathway ofprogrammed cell death that controls early morphogenesis.From Hakem et al 1998 [38]: Comparison of the requirement for Casp9 and Casp3 in different apoptoticsettings indicates the existence of at least four different apoptotic pathways in mammalian cells.

2.3.1 Apoptosis in muscle tissue

However, apoptosis in differentiated tissues such as muscle fibers is less well-defined. Muscle fibers containmultiple nuclei of which some can be targeted for death while others are unaffected. Apoptosis can beactivated endogenously through a genetically defined program or by exogenous proteins, cytokines andhormones, as well as xenobiotic compounds (e.g. radiation, oxidative stress and hypoxia). Early inapoptosis a series of procaspase proteases is activated. Then, caspase-1, -3 and -9 are activated andapoptosis is detectable.Apoptotic nuclei are detectable for approximately 1-3 hours after the onset of apoptosis. In mononucleatedcells the complete apoptotic cell is removed by phagocytosis, whereas in multinucleated cells only theapoptotic nucleus is removed leaving the rest of the cell intact (nuclear death).It appears that no apoptosis occurs in skeletal muscle cells (in vivo) after ischemia-reperfusion.

2.3.2 Markers for cell death

Up to date, no gold standard is available for the proof of apoptosis. Common methods, such as TUNEL(terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) staining and DNA laddering,may not suffice to indicate apoptosis. The disadvantage of some immunological methods (such as Bcl-2,Bax, Bcl-Xl, caspase-induced PARP cleavage, mitochondrial release of cytochrom c and orthe cleavageof procaspase-3) is that they only provide indirect evidence of apoptosis. It is therefore important not torely on a single method to detect or even quantify apoptosis.Some of the assays available to detect cell death, for example the Annexin-V apoptosis assay, do notdiscriminate between apoptosis and oncosis. The cell surface receptor Porimin has been associated withcell death by oncosis [98].

• The concentration of CK correlates with the duration of ischemia and the degree of damage to thetissue [37]. When damage occurs, the intracellular CK enzyme leaks into the extracellular spacewith elevated levels of CK in blood serum resulting from increased efflux from tissues. CK wasbelieved to provide an early detection of muscle damage that is at least as sensitive as morphologicevaluation. Course of damage can be evaluated with CK. CK is a key enzyme in cellular energeticsand in muscular metabolism [82]. It catalyzes the reversible transfer of a phosphate residue in highenergy binding between ATP and creatine. CK exists in a cytosolic and a mitochondrial form. Thethree cytosolic isoenzymes are CKBB (CK-1), CKMB (CK-2) and CKMM (CK-3) (B is brain formand M is muscle form). The mitochondrial form consists of two identical subunits (CK-Mi). Thehighest concentration of CK in all organs is found in skeletal muscle. The CK activity in skeletalmuscle is accounted for by CKMM. In slow-twitch fibers CKMB content is 5-10% and fast-twitchfibers contain 1-3% CKMB. With endurance training, CKMB accumulates in skeletal muscle andmay then serve as indicator of skeletal muscle injury. In chronic muscle injury, such as Duchenne

Section 2.3 39

muscular dystrophy, the CKMB content may reach up to 10-50%. CKMM itself also appears in threeisoforms. An MM1/MM3 ratio in plasma >0.7 indicates skeletal muscle damage [52]. Additionalisoforms can be detected by more sophisticated methods that are not suitable for routine use inlaboratories.In an experiment [66], during which cells were loaded under an agarose layer of 1.4 mm, CKdiffusion through the layer took 4 hours, whereas it took the cells 5 hours to break down CK.Furthermore, it was observed that CK activity was not decreased by storing the medium in a fridge(CK concentration was determined by a CK-kit from Sigma). More information on CK assays canbe found in appendix E.3.

• Lactate dehydrogenase enzyme is released into the extracellular space and is a consequenceof increased permeability of the cellular membrane. It correlates closely with CK in response toischemic damage. LDH is found in almost every tissue. It is an important enzyme of glucosemetabolism. Similar to ASAT, LDH nowadays has only minor significance, if any, for muscle fiberinjury diagnosis.

• Annexin-V, detection by fluorescent probe annexin V FITC. The probe binds to phosphatidylserine when it flipflops from the membrane interior to the exterior. It then presents its binding sitefor the annexin. Marker for apoptosis, irreversible damage. Nuclear fragmentation, mitochondrialmembrane potential flux and caspase-3 activation apparently precede phosphatidylserine ”flipping”during apoptosis, while permeability to propidium iodide and cytoskeletal collapse occur later [7].This is contradicted by Denecker et al. [26] by the fact that exposure of phosphatidyl serine precedesor coincides with the decrease in mitochondrial transmembrane potential and release of cytochromc. The difference in fluorescence intensity between apoptotic and nonapoptotic cells stained byfluorescent annexin V conjugates from Molecular Probes, as measured by flow cytometry, is typicallyabout 100-fold. Fluorescein (FITC) annexin V (A-13199), is a green-fluorescent conjugate that hasbeen extensively used by a number of laboratories to detect apoptotic cells populations. Fluoresceinannexin V is frequently used in combination with propidium iodide to detect necrotic cells, as inthe Vybrant Apoptosis Assay Kit #3 (V-13242).From [71] Apoptotic cells staining with annexin V-FITC, but not PI, appear in the lower right (LR)quadrant of data plots. Necrotic cells appear in the upper right (UR) quadrant, staining with bothPI and annexin V-FITC. More information on the PI/Ann V duostaining can be found in appendixE.2

• MitoTracker probes have been used to mark early apoptosis. The MitoTracker Green FMprobe has the advantage that it is essentially nonfluorescent in aqueous solutions and only becomesfluorescent once it accumulates in the lipid environment of mitochondria. Hence, backgroundfluorescence is negligible, enabling researchers to clearly visualize mitochondria in live cells immediatelyfollowing addition of the stain, without a wash step.

• Propidium iodide can be applied to stain dead cells. It enters the cell nucleus after the cell hasdied. In combination with cell tracker green this stain is applied in viability assessment. A duostainwith annexin-V is also possible. Then, apoptotic cells stain with annexin-V and necrotic cells stainboth annexin-V and PI [72].

Chapter 3

Pressure sores

Every year 60,000 people die from a bedsore complication (in the states) [8].

”up to 80% of individuals with [spinal cord injury] will have a pressure sore during their lifetime, and30% will have more than one pressure sore.” (Lindsey, 2000) [1].

Pressure sores are defined as localized areas of tissue degeneration in skin and /or underlying tissues, suchas the subcutaneous connective tissue or the muscle tissue. Pressure sores develop either superficially orfrom within the deep tissue depending on the kind of external mechanical loading and the local geometriesof the tissue and underlying bone. Depending on the tissue layers that are damaged, the decubitus issubdivided into four stages. These stages can evolve from one another or develop directly. The first stageis characterized by a surface reddening of the skin (figure 3.1). The skin is unbroken and the wound issuperficial. This would be a light sunburn or a first degree burn as well as a beginning decubitus ulcer.The burn heals spontaneously or the decubitus ulcer quickly fades when pressure is relieved on the area.The second stage is characterized by a blister either broken or unbroken. A partial layer of the skin isnow injured. Involvement is no longer superficial. In the third stage, the wound extends through all ofthe layers of the skin. It is a primary site for a serious infection to occur. A stage IV wound extendsthrough the skin and involves underlying muscle, tendons and bone. The diameter of the wound is notas important as the depth. This is very serious and can produce a life threatening infection, especiallyif not aggressively treated. Another characteristic is the smell of these wounds that may cause socialisolation of the patient. All of the goals of protecting, cleaning and alleviation of pressure on the area stillapply. Nutrition and hydration is now critical. Without adequate nutrition, this wound will not heal.Stage V is an older classification. It appears in some older literature. A stage V wound is extremelydeep, having gone through the muscle layers and now involves underlying organs and bone. It is difficultto heal. Surgical removal of the necrotic or decayed tissue is the usual treatment. Amputation may benecessary in some situations. Death usually occurs from sepsis. [6]The onset of pressure sores is influenced by several risk factors, such as age, impaired mobility, andan increased environmental or body temperature. The sores result in diminished pain sensation and/ordiminished musculoskeletal activity. The most adhered hypothesis is that pressure sores are caused bylocal ischaemia of the soft tissues following occlusion or collapse of capillaries. It is true that capillaryperfusion decreases as mechanical loading increases. Within two hours of compressive loading tissuedegeneration leading to pressure sores can occur, whereas the consequences of intermittent loading areless severe. No controlled studies relating shear stress to the occurrence of tissue damage have beenperformed. However, application of shear stress reduced the time to develop visible tissue damage if

40

Section 3.0 41

compared to compression alone.Compression induced muscle breakdown starts at the cellular level with nuclear pyknosis (sign of celldeath) and disintegration of the contractile proteins and cell membrane, followed by inflammatory reactionsand tissue necrosis. Magnitude and duration of the compression both affect cellular breakdown. Theunderlying pathways are poorly understood. Most probably, the aetiology of pressure sores is multifactorial.

Figure 3.1: The four stages of decubitus wounds are illustrated.

What about the prevalence of decubitus? Prevalence studies have been conducted for the past five yearsin Dutch hospitals (general as well as academic), nursing homes, old people’s homes (rest homes), homecare, rehabilitation centers, and psychiatric hospitals. In hospitals the prevalence among the completepatient population is roughly between 15 and 20%; except for the nursing homes and psychiatric hospitals,where the prevalence lies between 30 and 33% (table 3.1).

Table 3.1: Prevalence of decubitus stages after institution in percentages (2002)

Stage 1 Stage 2 Stage 3 Stage 4 Total Total without first stageAcademic hospital 7.2 5.9 2.8 0.6 16.5 9.3General hospital 11.6 6.7 3.0 1.0 22.3 10.7Nursing home 19.2 8.0 4.2 1.6 33.0 13.8Rest home 11.2 3.6 1.2 0.3 16.2 5.0Home care 9.3 4.9 3.4 0.9 18.5 9.2Rehabilitation center 2.2 15.7 0.0 3.4 21.3 19.1Psychiatric hospital 21.2 0.0 3.0 6.1 30.3 9.1

From table 3.1 it is also clear that decubitus IV hardly occurs anymore, except for patients from therehabilitation centra and psychiatric hospitals.

Chapter 4

Discussion

The questions to be answered are: Why do muscle cells die in deep decubitus; is this due to ischemia,reperfusion injury, obstruction of the interstitial transport, or cell deformation? What kind of damageand cell death develop during muscle-involving decubitus? In the proposed project, attention will bedrawn mainly to the role of cell deformations, nutritional state, and oxygen availability. To find answersto these questions, three key players are necessary: the muscle model, devices to inflict the damage (e.g.compressional and hypoxic), and cell damage and cell death markers. These three points will be discussedbefore the experiments are proposed.

Muscle modelQuite a few groups were and are working on subjects related to muscle tissue engineering. This reviewfocussed on skeletal muscle tissue engineering. The two most important and promising groups (accordingto the currently available literature) were those from Michigan (Dennis) and Rhode Island (Vandenburgh).Most groups applied approaches based on addition of an artificial extracellular matrix to the engineeredmuscle construct; mostly a collagen network. The one exception was Dennis’s group (note that Vandenburghet al. reported the formation of muscle organs from a monolayer back in 1991 [89] after the publicationof rolling (co)cultures by Strohman et al. [83]). They added fibroblasts to their culture of muscle cells toserve as a source for extracellular matrix components. The advantage of this method is that complicationsthat are related to the gel (e.g. compaction) or another bio(artificial) material are not encountered.Furthermore, the resulting myooids, as they are called, resemble the natural tissue more than the gelconstructs because the additionally added ECM is omitted. Also, the myooid muscle model has anadditional advantage over the currently applied model in our lab, the blobs, in that it has paralleloriented myotubes instead of the randomly arranged muscle tubes in the blobs. If tissue responses andcharacteristics are tested, a model that better approximates the real tissue is, of course, preferred.Cells from different sources have been incorporated in myooids. Neonatal and adult cells have beenisolated from mice and rats. Also, myooids from murine cell lines have been assembled. A twice as highmyooid formation rate was reported for primary cell myooids (95% of the cases), opposed to cell linebased myooids (50% success rate). The primary cell myooids exhibit better functional (excitability andcontraction) properties. An advantage of cell line based myooids is their convenience of culture and easeof obtainment.An important limitation of all methods for construct production is the dimension of the produced tissues.The maximum construct diameter is restricted (to about 500 µm) because of diffusion of oxygen andnutrients to the inner tissue. Microvascularization may offer a solution to this problem. So far, thisprocess has only been achieved after implanting C2C12/ collagen-based constructs into mice [59]. After

42

Discussion 43

four weeks of implantation, a dense capillary network had formed on the vicinities and on the surface ofthe grafts. Furthermore, the inflammatory response of the tissue can not be studied in the present tissueengineered models.It is proposed to first culture the myooids from the mouse cell lines to assess the protocol obtainedfrom literature. A next step might be the isolation of cells from human or animal muscle biopsiesand subsequent culturing in myooids. Human myooids can give more insight in interspecies differencesand may also be the basis for implantable tissue engineered muscles and improved muscle models forresearch purposes. Up-to-date, the only human bioartificial muscles (HBAMs) have been produced byVandenburghs group [68] by incorporating cells from needle biopsies in a collagen/matrigel mixture. In amore ideal situation, no collagen or matrigel should be added to obtain a muscle equivalent as mentionedbefore. If the myooid culture does not result in satisfactory muscle models, tests may also be conductedon the collagen/matrigel blobs or on myotube monolayers.

Compression device The tissue engineered muscle constructs eventually have to serve as skeletalmuscle models for damage development. The response of muscle models to e.g. mechanical stimulationhas been evaluated by means of a viability assay by Breuls et al. [17]. In the same way, tests can bedesigned to investigate a damage threshold rather than a ’death-hold’. To establish thresholds for damagedevelopment after a ’desired’ stimulus (for instance mechanical), markers can be measured. The influenceof the duration and magnitude of a mechanical stimulus on the tissue response can be investigated.Likewise, the influence of dynamic compression, influence of contractional state of the muscle construct,and compression before and after reperfusion can be studied. Two systems, present in our lab, that areable to impose compressional forces on muscle constructs, have been described in literature [17], [65].Breuls’ system is a rather robust system with a climate control (temperature and humidity) for the tissueand glass indentors for the compression. During the experiment, the cells can be monitored by confocallaser microscopy and fluorescent dyes. The cell compressor by Peeters is more delicate and refined. Thesystem also has the environmental control and confocal imaging, but a much smaller glass indentor.An extra feature of this system is the ability of force registration and a contemporary measurement ofindentor displacement, and cell deformation. Moreover, tip displacement in all directions is computerized,compared to manual movements in Breuls’ system. No real choice has been made up to date concerningthe exact features of the compressional device. Also, a set-up has to be developed for testing the effectof anoxia or hypoxia on cells.

Cell damage and cell death markersIn the present review several markers for skeletal tissue damage are presented. Markers can be foundduring all events that follow damaging of muscle cells: nuclear fragmentation, mitochondrial membranepotential flux, caspase(-3) activity, phosphatidyl serin flipping, increased membrane permeability (to PI),and cytoskeletal collapse. Roughly, the markers can be divided into visible and biochemical markers. Byboth means, a quantitative view of the damage development can be obtained. Visible markers, such asfura-2 and fluo-4 AM, can give detailed information on calcium concentrations in the cells, which riseafter cell damage. Fura-2 can not be measured in our lab because the laser wavelength that is needed forexcitation is not available. Most markers for reversible damage, such as NO and HSPs, are released uponcell stress and are not specific for cell damage or cell type. Almost all other markers that are presentedin this review are released during the irreversible stage of damage. The cell may then as well be declareddead. For live/dead staining CTG/PI duostaining proved to be a useful tool [18]. Another combinationis Annexin V with PI to stain for apoptosis and necrosis (or better: cell death).As mentioned before, a lot of proteins are released into the circulation during damage to the cell membraneor extracellular matrix. A selection of markers that are applicable in the proposed muscle model is

44 CHAPTER 4. DISCUSSION

presented in table 4.1. Depending on their listed characteristics a choice can be made for a marker (ormarkers) that is best suited for the intended research. Note that in the proposed set-up with myooids,skeletal muscle specificity may also be a relevant characteristic, due to the presence of fibroblasts in themuscle construct.

Table 4.1: Possible markers for skeletal muscle damage

CK Mb sTnI LDH MHC NO MDAAssay ease + +- - + - + +Skeletal muscle specificity +- +- + - +- - -Peak time 6h 2h 6h - 2d <1h 1-6hTotal + +- -+ - - + +-

NO is a very early marker of all kinds of cell stress. Another muscle tissue non-specific marker is MDA.MDA can be measured if lipid peroxidation of the cell membrane occurs. Both of these early markerscan be measured relatively easily.Plasma CK activity is most frequently used as a marker for skeletal muscle damage. It must be kept inmind that CK is only present in fused myoblasts, the myotubes. Some attention has been devoted to thedifferent CKMM isoforms that can indicate muscle damage. However, the presence of the forms is notguaranteed in the chosen muscle model because the presence of the cleavage enzyme, carboxypeptidase,that is responsible for the different isoforms, is not certain. A disadvantage of the cytosolic CK proteinis that no difference between temporary membrane leakage (reversible damage) and leakage by cell death(irreversible damage) can be made. The same is the case for myoglobin, which is also a cytoplasmicprotein. Therefore, interest in research has switched to extracellular proteins such as myosin heavy chain(MHC) and sTnI. MHC is not suitable for early diagnosis as it peaks after two days. An early markerwith skeletal muscle specificity is sTnI. TnI peaks within 24 hours and remains elevated for at least 1 to2 days. However, these two proteins can not be measured by commercially available assays. Therefore,measurement of myoglobin (in combination with H-FABP) and CK in plasma are still the currentlyrecommended markers for monitoring eccentric muscle actions to control specific training sessions andavoid overtraining [82]. NO can be used in combination with one of the later markers to indicate earlydamage. A disadvantage of NO is that it is released by a cell upon a stressful situation and not necessarilyupon damage (therefore measurement in combination with late markers is required).The necessity for the development of a clinically relevant marker for deep pressure sores may be questionable.Although type three and four decubitus hardly occur anymore in hospitals, this disease still can be foundin rehabilitation centers and nursing homes for the elderly (table 3.1). And, as there is no quantitativeway to assess the extent of deep pressure sores, type IV wounds are still primarily diagnosed by theeye, which is not an optimal method, especially if the patient is wheelchairbound and insensitive to pain(paraplegia). (X-ray diagnosis is performed when the patient or medical staff have already noticed thedeep wound and want to check for osteomyelitis.) For this reason, the ’real’ prevalence of decubitus mightdiffer from the ’measured’ prevalence. Therefore, a clinically relevant marker is still necessary.

Apart from investigating the influence of mechanical stimulation on engineered skeletal muscle tissue,other factors that can be derived from the different hypotheses for decubitus development may play a role.These factors are nutritional supply, oxygen tension, or temperature. Knowledge gained by evaluatingthe effect of the aforementioned factors, can be applied to explain development of deep pressure sores.

Discussion 45

In summary, the following experiments are proposed:

• Different static loading of muscle models (i.e. differentiated monolayers and blobs if myooids can’tbe produced). Under the increasing deformations a ratio live/dead and apoptosis/necrosis has tobe determined. Also, the effect in time has to be studied on damage and death development.

• It has to be validated that ischemia is not the cause of cell death in the deformation experiments. Inischemic cell death necrosis occurs. With the PI/Annexin duostaining an apoptosis/necrosis ratiocan be determined. Ischemic conditions can be mimicked by a gas mixture of 95% hydrogen and5% carbon dioxide instead of normal air. Both ’gases’ will be humidified and heated for the cells.These different gas environments may also be employed during the static loading in order to checkfor a synergistic effect on cell damage. Other options to test the ischemic hypothesis of decubitusmay be measurement of HIF-1α (hypoxia inducible factor) (offline measurement), pH measurement(fluorophore) or GFP transfection of cells with a hypoxia responsive element in the promotor.

• A series of dynamic loading experiments with different frequencies and magnitudes, possibly underthe two gas conditions mentioned before.

• The hypothesis of an influence of the nutritional status of the cells may be tested with for examplethe glucose content of the medium.

• Reperfusion damage may be difficult to test in the proposed setting. An animal model may be moresuitable.

• Other experiments may focus on the role of the extracellular matrix, the cytoskeleton or electricalstimulation of the muscle construct in cell damage during loading.

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[66] R.T. Pijls. Ck as a biomarker for damaged muscle cells? Internal report BMTE 02.39, TechnicalUniversity Eindhoven, MaTe, September 2002.

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[76] J.A. Rowley and D.J. Mooney. Alginate type and rgd density control myoblast phenotype. Journalof Biomedical Materials Research, 60(2):217–223, 2002.

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[98] C. Zhang, Y. Xu, J. Gu, and S.F. Schlossman. A cell surface receptor defined by a mab mediates aunique type of cell death similar to oncosis. Proc Natl Acad Sci U S A, 95(11):6290–6295, 1998.

Protocols for Dennis’s and Kosnik’smyooids

Protocols were adapted from Dennis and Kosnik [27], [28], [50]. One culture dish is prepared for theformation of each myooid.

Culturing C3H10T1/2 clone 8 cells

• Growth medium:500 ml Basal Eagle’s medium (BME) with Earle’s basal salt solution (EBSS) (Sigma B1522)5 ml L-glutamine (2mM)50 ml FBS30.25 mg penicillin G (=100 U/ml, Biochrom A 321-42)

• Seed cells at 2000 per cm2. If a new vial is thawed, seed in T25, so 50,000 cells per flask.

GENERAL REMARK: Pipette very carefully at the edge of the dishes after laminin coatingbecause the laminin or the cells wash off easily!!!

Day 0: SYLGARD coating

The SYLGARD substrate allows anchor materials to be pinned in place. It provides a surface to whichcells do not adhere without a suitable coating.

Preparations and requirementsSYLGARD base substance and catalyst (Dow Corning, silicone elastomer type 184), petri dishes (35mm,Falcon 1008)

Procedure

• Prepare SYLGARD solution by mixing the base substance with the catalyst (10:1 weight-ratio)until the mixture is uniformly colored. Avoid air enclosure.

• Fill each 35-mm culture dish with 1.5 ml SYLGARD solution to yield a 2 mm deep layer.

• Cap the dishes subsequently and allow to sit on a level surface for 1 week (NB Although the materialis hard enough for further processing after 24 hours, the final mechanical and electrical propertiesare reached after 7 days).

53

54 Appendix

• Store for at least 2 weeks before use to reduce cell toxicity.

Day 20: Preparations day 21

Thaw the laminin coating solution (9.62µg/3ml) in the fridge at 4 ◦C overnight.

Day 21: Laminin coating of dishes

Laminin coating provides a surface to which cells can adhere. Two protocols are presented in this section.One according to Dennis [27] who uses laminin from Gibco. The first one contains the adjustments thatare needed if laminin from Sigma is used.

Preparations and requirementsPut 250 ml sterile PBS on ice in a LAF cabinet, 1 mg laminin (Laminin from Engelbreth-Holm-Swarmmurine sarcoma (basement membrane), Sigma L2020, storage at -20 ◦C) on ice, 10 (sterile!) eppendorfcups or Nalgene cryovials on ice, 5x 50 ml falcon tubes on ice, 4x 15 ml falcon tubes on ice. Place 3x 25ml pipette, 2x 10 ml pipette, 1 ml pipette tips in de -20◦C freezer. SYLGARD coated dishesPreparation information for the laminin coating solution: for each dish 3 ml sterile PBS solution with9.62µg laminin is needed. π*r2 = 9.62 cm2. The laminin is supplied as 1 mg dissolved in 1 ml.

Laminin (Sigma L2020) handling before actual coating

• Thaw the product slowly !!!! overnight at 2-8 ◦C. Keep in mind that if the product has beenwarmed up at room temperature and it gels, it can not be reactivated for use. (So you work on ice).

• For suture end immersion a concentration of 50 µg/ml is required. Therefore, first add the 1 mllaminin solution to 19 ml sterile PBS. (1000 µg in 20 ml = 50 µg/ml)

• Take 5 ml from the 20 ml and aliquot in amounts of 1 or 0.5 ml in the eppendorf cups. Thesealiquots will be used for the soaking of the suture ends.

• Add 218.86 ml PBS to the remaining 15 ml of laminin solution. The concentration of the resultingsolution is 750 µg/ 233.86 ml = 9.62 µg/ 3 ml = 3.207 µg/ml. This means practically (because nosterile flask for 233 ml is present):

Table 2: Distribution of the remaining 233.86 ml of PBS and laminin solution

Total 15 ml Laminin 218.86 ml PBSTubes 4x 50 ml 3x 9ml 1x 6mlEach tube (4x) 3.22 ml lam + 46.78 PBS (3x) 0.58 ml lam + 8.42 ml PBS 0.39 ml lam + 5.61 ml PBS

The actual laminin coating of the dishes

• Thaw slowly (overnight, in fridge!!) the laminin coating solution of the stock, which contains9.62µg laminin in 3 ml sterile Dulbecco’s PBS; this equals 1 µg/cm2 for each dish.

• Coat the SYLGARD-coated dishes with 3 ml laminin stock solution. Allow the dishes to dry in anopen biological safety hood with blower running overnight.

Appendix 55

• (On day 22) Remove salt cristals from the DPBS by adding 3 ml DPBS to each dish and thoroughaspiration.

• Thaw the concentrated laminin solution (50 µg/ml) in the fridge at 2-6 ◦C.

Day 22: Laminin coated silk suture anchors

Preparations and requirementsPBS, 6mm segments of size-0 (Ethicon, metric size 3.5)(Ethicon, Sommmerville, NJ) braided silk, scissors,forceps, needle-shaped dentist tool, 50 µg laminin/ ml PBS solution, cut 3 mm off pins (0.10 mm indiameter, 10 mm long, from stainless steel (Fine Science Tools, Foster City, CA, model 26002-10)) andpin them into a rubber stop until use

Procedure

• Remove salt cristals from the DPBS by adding 3 ml DPBS to each dish and thorough aspiration.

Within one day of completing the laminin-coating process the anchors should be placed. Meanwhile thedishes are stored on a shelf to dry.

• Cut 6mm-long segments of size-0 braided silk sutures.

• Hold each segment from one end to the center with a pair of forceps and fray the other end with aneedle-shaped dentist tool.

• Immerse in a concentrated solution of 50 µg natural mouse laminin/ ml DPBS (pH=7.2).

• Pin the wet suture ends into the laminin-coated dishes, separated by 12 mm (internal, frayed ends)and allow them to dry overnight (suture ends and pins are referred to as anchors).

Day 23: Presoaking with GM

To remove residual toxins from the anchors the whole dish is presoaked. It may also provide deposition ofadhesion molecules from the serum in the GM onto the substrate surface and thus improved cell adhesion.

Preparations and requirementsC2C12 GM, UV bulb for LAF cabinet

Procedure

• Add 3 ml growth medium (GM) to each dish

• Cover the dishes and expose them to UV (253.7 nm) in a hood for 60-90 minutes for sterilization.

• Then with GM and anchors, the dishes were presoaked in an incubator at 5%CO2, 37 ◦C for 5-8days before cells were plated.

With inadequate presoaking, the cells did not attach to the anchors, and the succes rate for myooidformation was less than 5%. Longer presoaking times also resulted in a reduced succes rate, presumablydue to degradation of the laminin coating on the SYLGARD substrate. Storage of the laminin-coateddishes at 4 ◦C for two weeks prior to presoaking also resulted in significant loss of cell monolayer adhesionto the SYLGARD substrate.

56 Appendix

Day 30: Cell seeding

Preparations and requirementsFor each dish to be seeded 0.5.106 C2C12 cells and 0.5.106 10T1/2 cells are needed, C2C12 GM, trypsin,trypan blue testing set

Procedure

• Plate 1.105 cells/cm2 in a cell type ratio between 30-80% C2C12. In total this comes down to±1.106 cells/dish. This implies that 5.105 cells of each type (C2C12 as well as 10T1/2) are neededfor each dish.

• Wait for the cells to reach confluence while refreshing the medium every 2-3 days.

• Within one day after reaching confluence, change to differentiation medium (DM) and feed threetimes a week.

Day 39-60: Monolayer detachment

• (± One week after the first addition of DM) Check if monolayer has detached yet.

• If detachment is incomplete, use a pair of sterile forceps to gently tease the myooid from thesubstrate.

• Measure myooid diameters with a calibrated eyepiece reticle while each myooid is viewed througha x10 objective lense on an inverted microscope.

Formed myooids often appeared inactive until electrically stimulated. In the cell line cocultures myooidsformed only ±50% of the time.

The culture media (CM) were GM and DM:CM-C2C12: 465 ml DMEM (11995-065) + 35 ml FBS + 100 U/ml penicillin GDM: 465 ml DMEM + 35 ml Horse Serum + 100 U/ml penicillin GGM: 400 ml HAM F-12 + 100 ml FBS + 100 U/ml penicillin G

My media:CM-10T1/2: 500 ml BME-EBSS(Sigma B1522) + 5 ml L-glutamine + 50 ml FBS + 5 ml non-essentialamino acids + 30.25 mg penicillin G (=100 U/ml, Biochrom A 321-42)GM-C2C12: 500 ml DMEM high glucose + 5 ml L-glutamine (end concentration 2 mM) + 75 ml FBS +5 ml non-essential amino acids + 10 ml HEPES + 2.5 ml gentamycinDM-C2C12: 500 ml DMEM (FG0415) + 10 ml Donor HS (S9133) + 5 ml gentamycin + 5 ml non-essentialamino acids (K0293) + 10 ml HEPES (1M, L1613)

Appendix A

Covering cells on cover glass with 3%agarose

Note that the amounts of agarose, medium and PBS can be adjusted in ratio to any desired amount.

Preparations and requirements:oven, pipettes (5 and 10 ml), LAF cabinet, burner, 15 ml falcon tube, waterbath, agarose powder, PBS(not necessarily sterile), autoclave, medium, cover glasses with cells (round), pair of tweezers, 15 ml falcontube, roller mixer, pipet boy

Procedure:

• Weigh 0.3 g agarose and dissolve in 5 ml PBS in a small autoclavable glass bottle.

• Autoclave solution in liquid cycle (±1-1.5h).

While autoclaving:

• Place roller mixer, 15 ml falcon tube and pipettes (5 and 10 ml) in oven at 45 ◦C.

• Place waterbath in LAF cabinet and set to 38-39 ◦C.

• Warm medium in waterbath, a volume equal to the volume of the agarose solution (5 ml).

After autoclaving:

• Place solution on roller mixer in the oven for 10 minutes to cool. Get a 5 ml pipet and the falcontube from the oven.

• Transfer the agarose solution into the 15 ml falcon tube to the waterbath and optionally wait foranother 5 minutes. Meanwhile get a 10 ml pipette from the oven.

• Add the medium to the agarose solution with the pre-heated 10 ml pipette. Mix gently and try toavoid formation of air bubbles. Place complete solution back into the waterbath (1 minute).

• Take the cells from the incubator.

• Sterilize a pair of tweezers in the burner flame.

57

58 Appendix A

• (Carefully!!!) Take a cover glass from the wells plate with the tweezers. NB Do not remove mediumbefore taking cover glass out of the wells plate.

• Pipet agarose solution from the tube and let a drop spread on the cover glass at the cell side.

• Allow the gel to ’solidify’ in the air and place the coated glasses subsequently into a set-up or 6-wellsplate and cover with medium.

Appendix B

Protocol for blob formation from C2C12cells

Culturing C2C12 cells

• Growth medium:500 ml high glucose DMEML-glutamine (2mM, or 5 ml)75 ml FBS 5 ml non-essential amino acids2.5 ml gentamycin10 ml HEPES

• Differentiation medium:500 ml DMEM (FG0415)10 ml Donor HS (S9133)5 ml gentamycin5 ml non-essential amino acids (K0293)10 ml HEPES (1M, L1613)Dennis-DM: 465 ml DMEM + 35 ml Horse Serum + 100 U/ml penicillin G

• Seed cells at approximately 10,000 per cm2, so 750,000 in a T75. Split cells 1:6 or 1:7.

Day -1: Preparations and stock solutions for blob formation

• Acetic acid solution 0.2%: 200 µl acetic acid in 100 ml distilled water. Autoclave.

• PBS: dissolve 1 tablet per 200 ml ultra pure water. Autoclave.

• NaOH 0.5M: dissolve 1 g NaOH in 100 ml distilled water. Autoclave.

• Prepare the collagen stock solution before making the blobs. Dissolve 10 mg (Sigma # C-7661) in3.125 ml ice cold acetic acid (0.2% sterile). This gains a solution with a collagen concentration of3.2 mg/ml.

• Prepare growth medium (GM) for C2C12 cells: 500 ml DMEM high glucose, 100 ml FCS (20%),10 ml Hepes (2%), 5 ml non-essential amino acids (1%), 5 ml gentamycin (1%) (recipe by Roel) or500 ml DMEM F9 0435, 75 ml FCS (15%), 10 ml Hepes (= 20 mM) (2%), 5 ml non-essential aminoacids (1%), 2.5 ml gentamycin (0.5%).

59

60 Appendix B

Day 0: Preparing the mixture and molding the blobs

• Put an empty tube in the -20 ◦C freezer for gel processing.

• Get the heating lamp from Marcel Wijlaars.

• Trypsinize the cells, resuspend in 1 ml and count with trypan blue. Enter the number of cells (ordesired number if you are not using all cells for blob formation) in the excel sheet ’blob.xls’. Therequired volumes for blob formation are calculated.

• Place a box with ice in the LAF cabinet. Put enough collagen stock solution (3.2 mg/ml), GM,matrigel and NaOH on ice with the prepared cell suspension in the LAF cabinet.

• Add the calculated amount (minus 1 ml that was already added for counting) of GM (may as wellbe cold medium) to obtain a cell suspension with 1.25 million cells/ml. Place in ice until furtherprocessing.

• Take the empty tube from the freezer and add the desired amount of collagen stock solution.

• Add the small amount of GM to serve as a pH indicator. Mix gently.

• Add the 0.5M NaOH until a light red color is indicating a pH of 7 (you have to look very closely tonotice a color at all...). Mix gently again and avoid bubble formation.

• Add the matrigel and mix very gently but thoroughly.

• Finally add the cell suspension to the mixture. If necessary use the green needles for mixing (Beaware of the risk of bubble formation!!!)

• Place the heating lamp but do not turn it on yet!

• Pipette 0.75 ml of the cell-gel mixture into each well. Handle the pipette in a vertical direction inthe center of the well to avoid adhesion to the well wall.

• Turn the heating lamp on. If the blobs have gelled enough to be transferred, carefully move theminto the incubator and avoid shaking for the next two hours.

• After two hours, gently overlay each gel with 1 ml of warm GM.

• Maintenance: refresh GM every 24 hours. Replace GM by differentiation medium after three days.On day 8 you should have obtained mature muscle fibers.

Appendix C

Cell culture media

Culturing C3H10T1/2 clone 8 cells (murine fibroblasts)

• Growth medium:500 ml Basal Eagle’s medium (BME) with Earle’s basal salt solution (EBSS) (Sigma B1522)5 ml L-glutamine (2mM)50 ml FBS30.25 mg penicillin G (=100 U/ml, Biochrom A 321-42)

• Seed cells at 2000 per cm2. If a new vial is thawed, seed in T25, so 50,000 cells per flask.

Culturing 3T3 murine fibroblasts

• Growth medium:500 ml DMEM low glucose500 ml HAMs F-125 ml L-glutamine (2mM)100 ml FBS10 ml penstrepif desired also add 10 ml amfotericine (anti-funghi and anti-yeast)

• Seed cells at ? per cm2.

Culturing C2C12 murine myoblasts

• Growth medium:500 ml high glucose DMEML-glutamine (2mM, or 5 ml)75 ml FBS 5 ml non-essential amino acids2.5 ml gentamycin10 ml HEPES

• Differentiation medium:500 ml DMEM (FG0415)10 ml Donor HS (S9133)5 ml gentamycin5 ml non-essential amino acids (K0293)10 ml HEPES (1M, L1613)(Dennis-DM: 465 ml DMEM + 35 ml Horse Serum + 100 U/ml penicillin G)

61

62 Appendix C

• Seed cells at approximately 10,000 per cm2, so 750,000 in a T75. Split cells 1:6 to 1:10.

Appendix D

Freezing cells

• Prepare freezing medium and medium without antibiotics or defrost aliquotsMake 10T1/2 medium without antibiotics. Take 45 ml and put it in a 50 ml falcon tube. Add 4,5ml (10%) DMSO. Be careful! DMSO is very toxic. The DMSO content is twice te concentrationthat is desired (i.e. 5%) as the medium will be diluted one more time before freezing. Also, storesome medium without antibiotics.

• Check if the container with isopropyl alcohol is available (250 ml isopropyl alcohol needs to bereplaced after having been in use for 5 times). It can fit 18 vials.

• Check for medium contamination (because they are without antibiotics)

• ProcedureWash cells twice with PBS

• Trypsinize cells and neutralize the trypsin by addition of twice its amount in normal medium

• Centrifuge at 1000 rpm for 5-10 minutes

• Remove supernatant, add 1 ml medium without antibiotics and count the cells with trypan blue

• Add ice cold culture medium without antibiotics to an amount of twice the desired cell concentration.In other words: add half of the total volume that is required to obtain the desired end concentration.Usually the desired cell concentration is 0.5-2 million cells/ml.

• Add an equal amount of freezing medium (with 10% DMSO, so final concentration is 5%)

• Transfer in 1 or 1.5 ml aliquots into cryovials (2 ml). Label the vial with the date, cell line, yourname and the cell passage.

• Place the vials in the cryocontainer and put it in the -80 ◦C freezer for at least 4 hours

• Remove vials from the freezer and store cells in the liquid nitrogen container. Clearly mark the viallocations in the log book

63

Appendix E

Marker assays

E.1 PI/CTG

Preparation and requirementsWork in a LAF cabinet.Attached C2C12 myoblasts or myotubes, PI, tube for preparation of CTG solution, 70% ethanol. Warmculture medium and PBS, thaw CTG, arrange vacuum suction in LAF cabinet (glass pipette in tube)

Procedure- CTG staining

• prepare the CTG solution by adding 10 µl CTG per required ml medium (10 µM CTG solution)

• remove the original culture medium from cells with glass pipette

• wash cells twice with PBS: add PBS and remove with glass pipette

• add ’pure’ medium to cells from a control group and add 10 µM CTG solutions to the cells thatshould be stained

• incubate for 15 minutes

• rinse twice with PBS, second time: 15 minutes on shaking table with PBS to remove as muchbackground CTG as possible

• remove PBS and add plain medium

• incubate 30 minutes to allow cells to produce fluorescent markers (NB although 5 minutes shouldbe sufficient as well)

Procedure- PI staining

• prepare a 10 µM PI solution by adding 10 µl PI per ml medium

• remove medium from cells

• rinse with PBS

64

Appendix E 65

• add 10 µM PI solutions to the cells

• (incubate 10 minutes)

Procedure- analysis

• check results under CLSM with appropriate settings for CTG (excite at 488 nm and emissionbetween 505-530 nm) and PI (excite at 543 nm and λ= 617 nm is reflected) and if possible reusesomeone’s old configurations

E.2 PI/Annexin V

Protocol adapted from Molecular Probes

Preparation and requirementsPrepare sterile annexin binding buffer (recipe for 100 ml). This step is not necessary for viable staining:

• 10 mM HEPES: get 1 ml from the 1M solution in cabinet;1 ml contains 1 mmol → 1 mmol/100 ml = 10 mmol/1000 ml = 10 mM.

• 140 mM NaCl: 0.818 g NaCl;Mw=58.44 u → 0.140*58.44= 8.18 g for 1 l, so you need 0.818 g for 100 ml solution.

• 2.5 mM CaCl2: ±0.035 g;Mw=110.98 u, Mw(dihydrate)=147 u → 110.98*0.0025= 0.28 g in 1 l, 147*0.0025= 0.37 g in 1 l,so ±0.035 g is needed for 100 ml solution.

• pH needs to be 7.4. Autoclave the solution.

ProcedureAfter performing several tests I have come to the following conclusions:From the Molecular Probes company no real concentration of the probe (Annexin V FITC, A-13199,500 µl) could be obtained. From a concentration assay the following concentration turned out to givesatisfactory staining: 2 µl probe/ 1 ml medium. The staining lasted for several days, although theintensity decreased with time. Staining was performed in either the aforementioned annexin bindingbuffer or medium. The binding buffer had no extra effects because calcium is present in the culturemedium. However, cells died in the buffer within one day. Therefore, if you use the probe as viable stain,proceed as follows:

• wash cells in PBS

• add 2 µl annexin solution/ml medium to the cells. The probe is expensive, so use the smallestamount of medium as possible. For more intensive staining a concentration of 10 µl annexin solution/1 ml medium may be used.

• add the PI dye in a concentration of 10 µl PI solution/ml medium

• to let the binding to the stains develop, place the cells in the incubator for approximately 10 minutes.If desirable, a positive apoptotic control can be included in the experiment by exposing the cells to1 µg staurosporine/ml medium.

66 Appendix E

• Results can be interpreted as follows: cells that do not stain are alive; cells nuclei that stain reddied from necrosis; cells that stain green (a circular membrane) died from apoptosis; and cells thatstain both red and green may have initially started an apoptotic pathway and eventually died fromnecrosis, or they may have died from necrosis. An example can be found in figure E.1.

Figure E.1: An example of cell staining with annexin (green channel, upper right) and PI (red channel,upper left). At the bottom left the transmission signal shows myotubes and the bottom right picturerepresents the three other channels combined.

E.3 Creatine Kinase (CK)

After eccentric exercise serum concentrations of creatine kinase, myoglobin and myosin heavy chain areelevated. They are indirect indicators of muscle damage. Creatine kinase activity was measured bymeans of an N-acetylcysteine activated, optimized UV test obtained from Merck. Myosin heavy chainconcentrations were measured by an immunoradiometric assay. [52]Creatine Kinase (CK) is an enzyme located in the cell. It is involved in cellular energy metabolism bycatalyzing the reversible transfer of a high energy phosphate group from adenosine triphosphate (ATP)to creatine, resulting in ADP and creatinephosphate (figure E.2). CK consists of 2 different subunitsM and B, each with a molecular weight of 41 kdalton. So, three compositions of CK might occur: theiso-enzymes CKMM (CK3), CKMB (CK2) and CKBB (CK1). Also a unique dimeric mitochondrial formexists.

Figure E.2: Reaction that is catalyzed by creatine kinase.

The CKMM isoenzyme can be fractionated into subtypes or ’isoforms’ by their isoelectric points. Theseisoforms are MM1, MM2, and MM3 with the respective isoelectric points of 6.50, 6.70, 7.05 [13]. CKMMis most abundant in skeletal muscle (97-99%) and myocardial muscle (about 80%). CKMB is part ofmyocardial muscle (nearly 20%) and of skeletal muscle (1-3%). CKBB is most abundant in brain tissueand tissues from the intestines. After necrosis of the cell, CK enters the circulation and is cleared by the

Appendix E 67

lymphatic system. In circulation the enzyme carboxypeptidase cleaves the carboxyterminal amino acidlysine from the M- and B-subunit (figure E.3).

Figure E.3: Cleavage of the carboxyterminal amino acid lysine from the M subunits. [5]

Wevers firstly described this phenomenon in 1977. As lysine is strongly positive, it is possible with highvoltage electrophoresis on agar gel to discriminate between M subunits with and without the presence oflysine. The CKMM-isoform with lysine at both subunits is called the tissue form (MM3), the isoform withlysine at one subunit the intermediate form (MM2) and the subform with lack of lysine at both subunitsis called the plasmaform (MM1). Isoforms are also detectable for the CKMB-isoenzyme. The isoformwith lysine at the end of the M-subunit is called the tissue form (MB2). The one without lysine at theend of the M-subunit is called the plasmaform (MB1). From experiments with column chromatographyand monoclonal antibodies directed against the M carboxyterminal lysine, it has become clear, that atleast three CKMB isoforms circulate in vivo: first, the already mentioned tissue form, second, a formwith a lysine on the M subunit but not on the B subunit, and third, a form with lysines absent fromboth carboxyterminal subunits. So, the cleavage of lysine from the B monomer seems to be favored.Comigration on electrophoresis of the tissue isoform and the isoform with a lysine removed from the Bmonomer appears to account for the separation of only two species. [5]The full potential of the diagnostic utility of MM and MB isoforms will not be realized until a reliable,sensitive, simple, and rapid quantitative assay becomes available (especially for myocardial infarction). [12]Skeletal muscle injury becomes apparent as a venous CPK elevation, after 2-3 hours of occlusion and canbe prevented by short periods of recirculation. [25]Analyses of CKMM isoforms in human plasma can provide useful information on the extent and relativetime course following an episode of (skeletal) muscle damage. [63]

Assays

Sample preparation

Ethylene glycol bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) (5 mmol/l) is immediatelyadded to the serum to prevent in vitro conversion of MM isoforms. [13]2-mercaptoethanol (final concentration 10 mmol/l) and EGTA (final concentration 30 mmol/l) were addedto the samples to inhibit carboxypeptidase N-mediated isoforms conversion after sample collection. [39]EGTA and 2-mercaptoethanol were added, EGTA to inhibit conversion. [36]

Total CK activity

• CK-kit from Sigma. [66]

68 Appendix E

• Determination of total CK with Roche CK NAC reagents. Total CKMB with ICON CK-MB kit(Hybritech Inc, San Diego, CA) and by Roche Isomune technologies. [22]

• Total CK activity is a composite of the activity of the various isoforms. [69]

• Total CK activities were measured by the method of the Scandinavian Committee on Enzymes onthe Centrifichem 600 analyzer at 30 ◦C. [80]

• Total CK and CKMB were determined using Ektachem analyser (Johnson and Johnson, Beerse,Belgium). [84]

• Plasma total CK activity was measured with CK NAC reagent at 37 ◦C in a Cobas Mira analyzer(Roche). Plasma total CKMB activity was measured immunochemically with the Roche Isomunekit and quantified with CK NAC reagent. [39]

• Total CK activity was determined by a modification of the Rosalki procedure. [13]

• CK activities were measured with an N-acetylcysteine activated, optimized UV test from Merck(Darmstadt, Germany); upper limit of the reference interval is 80 U/l for men. [52]

• Method of Rosalki for total CK. Total CKMB activity was determined with a sensitive and precisebatch adsorption assay (Lancer MB CK Isoenzyme separation System, Sherwood Medical, St Louis,Mo). This method is used for clinical CKMB determinations. Puleo1989 for more info... This systemmeasures both the absolute activity of the two subforms and the relative percent of each subform,so delay for independent MB assay is not necessary!! [36]

Electrophoresis studies - CKMM separation

• 1 µl of sample is applied to agarose gels, allowed to absorb for 1 minute and electrophoresed at 1400V for 15 minutes. Then each gel is overlaid with reagents optimized for coupling CK activity withreduction of NAD+ and incubated 45 ◦C for 270 seconds. The gels are subsequently dried and theisoforms are quantified by densitometry. [22]

• CK isoenzymes were separated by electrophoresing 1 µl of serum on 1% agarose gel plate (Specialfilm, Corning) at 90 V for 20 minutes in 0.05 M MOPSO buffer, pH 7.8, and the formation ofNADH was detected by overlaying 1 ml of Cardiotrac-CK isoenzyme reagent (coupled enzymaticRosalki reaction) in MES buffer (2-N-morpholino ethane sulfonic acid) with sucrose for 20 minutesat 37 ◦C and drying it at 60 ◦C in an oven for 25 minutes. Dried gels were scanned in amicrocomputer-controlled densitometer (Corning 780) using the fluorescent mode with excitationwavelength of 365 nm and emission wavelength of 460 nm. [80]

• CKMM isoforms were separated on Titan III cellulose acetate (Helena laboratories, Beaumont, TX,USA), and agarose gel (Corning) electrophoresis. Cellulose acetate electrophoresis was carried outby applying the sample on a Titan III plate and electrophoresing at 800 V for 12 minutes. Afterincubation with CK substrate (CKMB isoenzyme reagent kit from Helena) at 45 ◦C for 6 minutes,the plate was dried at 55 ◦C for 5 minutes and the fluorescence was scanned in the densitometer.For agarose gel electrophoresis, 1 µl sample, diluted to contain 150-1100 U/l of CKMM activity,was applied to the agarose gel and electrophoresed at 170 V for 45 minutes using ice-cooled buffer.The following procedure was identical to that of CK isoenzymes. [80]

Appendix E 69

• Electrophoresis on agarose gel (12 g/l)using Cardio RepTM (Helena Labs, USA) for CKMM andCKMB isoform patterns. 25 ◦C, 900 V and 40 mA, 6 minutes. Then standard reagent for CKanalysis is applied (3 minutes) and excess of reagent is removed. Incubation 5 min at 50 ◦C. Gel isdried at 55 ◦C for 4 minutes. Densitometric scanning. MM3/MM1 ratios were calculated from thearea under the curves of the densitometric scans. [84]

• The activity of the CKMM isoenzyme was calculated by mutiplying the percentage of CKMMcontained in the fraction (determined by electrophoresis, REP Helena labs) by the total CK activity.The activity of each MM isoform was calculated by mutiplying its percentage in the fraction bythe total activity of the isoenzym MM as measured by high resolution agarose gel electrophoresis.CKMB was determined by the Isomune kit; Tandem-E CKMB II assay system (Hybritech, CA) [39]

• Total CKMM concentrations can be determined by subtraction of CKMB concentration from totalCK content. Western blotting of MM isoforms was performed by Ramasamy [69]

Isoelectric focusing - CKMM separation

• CK-MM isoforms were separated by flatbed isoelectric focusing (IEF), using the ’Multiphor 2’system (LKB, Gaithersburg, MD) and precast Isogel agarose IEF gels (FMC BioProducts, Rockland,ME – www.cambrex.com part code 56015), pH 3.0-10.0. 2µl samples were directly applied onto thegel and focused for 20 minutes at 1500V, 20 mA, and 25 W constant power. CKMM isoformswere quantified by overlaying the gel with CK reagent (CKSVR Reagent; Calbiochem Behring, SanDiego, CA), incubated for 15 minutes at 37 ◦C, then dried for 15 minutes and scanning densitometrywas performed. Assay time was 70 minutes. Samples with activities > 1000 U/l were diluted withTris buffer (50 mmol/l, pH 7.2). CKMM isoforms were identified by their isoelectric points - MM37.05, MM2 6.70, MM1 6.50 - and expressed as a percentage of total CK. [13]

• Flatbed isoelectric focusing [23], also from Apple group.

• CKMM isoforms were separated using isoelectric focusing techniques. In most samples threeisoforms were detected. However, a few serum samples exhibited two more isoforms. The isoelectricpoints were determined as 7.26 for MM1, 6.85 for MM2, and 6.45 for MM3. The two additionalisoforms were seen at 7.12 and 6.65. [63]

E.4 Myoglobin (Mb)

Myoglobin is a relatively small protein that is abundant in the cytosol of striated muscle cells, bothskeletal and myocardial. Myoglobin is released rapidly after tissue injury and may be elevated as early as1 hour after myocardial injury. Myoglobin is also cleared rapidly by renal excretion, so abnormal levelsmay return to baseline values in six to twelve hours.The commercially available enzyme immunoassay (Myoglobin-Access; Sanofi-Diagnostics-Pasteur, Marnes-la-Coquette,France) was used by Onuoha et al. [62] and Sorichter et al. [81]. Commercially available analyzers areBayer Immuno I, Behring OPUS Series, Ciba Corning ACS:180, Dade International Systems, SanofiDiagnostics Access, Tosoh Medics System. Therefore, it might be interesting to try to send samples tohospitals for analysis.

70 Appendix E

E.5 Nitric oxide (NO)

For now, the reader has to be referred to the information available in the tissue engineering lab or toMark Daniels.

E.6 MalonDiAldehyde (MDA)

Malondialdehyde (MDA), a metabolite of lipid peroxides detactable in plasma, was used as an indicator oflipid peroxidation. Plasma MDA concentration were estimated as reactive substances by a thiobarbituricacid adduction (TBARS) method described by Yagi [97], and Wang et al. [95]: the assay indirectlyquantifies lipid hydroperoxides by measuring aldehyde breakdown products of lipid peroxidation. Insummary, 4 ml of 1/12 sulfuric acid and 0.5 ml of 10% phosphotungstic acid were added to 20 µl sampleand mixed thoroughly. After centrifugation at 3,000 g for 10 minutes, the liquid phase was decanted(removed). 4 ml of double-distilled water and 1.0 ml TBA reagent (0.67% 2-thiobarbituric acid/aceticacid, 1:1) were then added to each sample, mixed, and heated at 95 deg for 1 hour. Samples were cooledwith tap water. 5 ml of n-butyl-alcohol were added, and the samples were vigorously shaken for 1 minuteand centrifuged. The n-butyl-alcohol phase, which contained the lipid peroxides, was used for MDAanalysis with a fluorospectrophotometer with excitation at 515 nm and emission at 553 nm [48].Measurement of tissue MDA levels was performed by Topsakal et al. (2002) [87] in a way very similar to theone mentioned before according to the method of Ohkawa, 1979 [58]: Lipid peroxidation in injured spinalcord was estimated by the thiobarbituric acid reaction method for MDA (MDA defined as the productof lipid peroxidation) described by Ohkawa et al. to give a red species absorbing at 535 nm. The MDAresults were expressed as nmol/g wet tissue. 0.2 ml of 10% (weight/volume) tissue homogenate was addedto 0.2 ml of 8.1% sodium dodecyl sulfate and a 1:5 aqueous solution of thiobarbituric acid. The mixturewas diluted to 4.0 ml with distilled water heated in an oil bath at 95C for 60 min. After cooling with tapwater, 1.0 ml of distilled water and 5.0 ml of a mixture of N-butanol and pyridine (15:1 volume:volume)were added and the mixture was shaken. After centrifugation at 4000 rpm for 10 min, the organic layerwas taken and its absorbance at 532 nm was measured spectrophotometrically. Tetramethoxy propanewas measured as an external standard, and the level of lipid peroxides was expressed as nanomoles ofMDA per gram wet weight.An advantage of the TBARS method is the ease with which it can be performed. A drawback is the lowsensitivity, as well as the low specificity of the method. A more accurate and specific method is HPLC.A highly specific, simple and rapid method was described by Roman et al. (2002), using reverse phasecolumn MAC in a methanol/phosphate buffer mobile phase [74].

Appendix F

Definitions

Satellite cell: (1) sparse population of mononucleate cells found in close contact with muscle fibers invertebrate skeletal muscle. Seem normally to be inactive, but may be important in regeneration afterdamage. May be considered quiescent stem cell. (2) An alternative name for glial cell.Myooid: a three-dimensional skeletal muscle construct cultured from mammalian myoblasts and fibroblasts.Pyknosis: contraction of nuclear contents to a deep-staining irregular mass; sign of cell death.Glial cells: specialized cells that surround neurons, providing mechanical and physical support, andelectrical insulation between neurons.Myoblast: cell that by fusion with other myoblasts gives rise to myotubes that eventually develop intoskeletal muscle fibers. The term is sometimes used for all the cells recognisable as immediate precursorsof skeletal muscle fibers. Alternatively, the term is reserved for those post-mitotic cells capable of fusion,others being referred to as presumptive myoblasts.Myotube: elongated multinucleate cells (three or more nuclei) that contain some peripherally locatedmyofibrils. They are formed in vivo or in vitro by the fusion of myoblasts and eventually developinto mature muscle fibers that have peripherally located nuclei and most of their cytoplasm filled withmyofibrils. In fact, there is no very clear distinction between myotubes and muscle fibres proper.C2C12 cells: skeletal mouse muscle cells (myoblasts) from a commercially available cell line. This lineis subcloned from the C2 line that was originally isolated by Yaffe and Saxel (1977). The cells showdoubling times of 14 hours and have a density dependent growth rate. After fusion the myoblasts willdifferentiate into myotubes.ProNectin F: ProNectin F is a recombinant non-animal-source polymer that promotes attachment ofeven weakly adherent cell lines. Optimized for pharmaceutical applications, this bead performs well inserum-free and even in protein-free media. The coating consists of multiple copies of the human fibronectinRGD attachment domain.Epimysium: the external connective-tissue sheath of a muscle; Endomysium: Connective tissue sheathsurrounding individual muscle fibers.Isotonic contraction: the muscle shortens and lifts whatever load has been attached to its tendon.External work.Isometric contraction: muscle is prevented from shortening by fixing both ends. Tension at attachmentpoints is generated, rather than external work.Concentric contractions: muscles are allowed to shorten.Eccentric or pliometric (McArdle02) contractions: muscles lengthen during contraction.Twitch contraction time: the interval between the onset of tension and the peak value.Twitch: a single action potential (pulse) causes a brief rise in the interfibrillar Ca2+ concentration,

71

72 Appendix F

immediately followed by a fall in concentration. A longer lasting high Ca2+ concentration is obtained bysending more impulses to the sarcoplasmic reticulum.Critical fusion frequency: at this pulse frequency the Ca2+ concentration does not show a rippleanymore.Tetanized state: state if pulse frequency exceeds the critical fusion frequency.Rheobase: minimum stimulus strength that will produce a response (V). NB high values of C50 andR50 indicate low tissue excitability, which is undesirable.Chronaxie: duration that gives a response when the nerve is stimulated at twice the rheobase strength.Maintained stretch: induces the formation of new sarcomeres, including fresh myofilaments, at theextremities of the fiber (McComas, p.71) Severe intermittent stretch: as in forceful isometric oreccentric contractions results in the synthesis of new myofilaments around existing myofibrils, but thereis no increase in sarcomeres. (McComas)Northern blotting: mRNA expression is measured (MEMO C. Bouten).Western blotting: a blot that consists of a nitrocellulose sheet containing spots of protein for identificationby a suitable molecular probe and is used especially for the detection of antibodies.Southern blotting: a blot consisting of a nitrocellulose sheet containing spots of DNA for identificationby a suitable molecular probe.Apoptosis: a physiological, conserved program of cellular suicide, characterized by cell shrinkage,membrane blebbing, breaking up the cells in a number of membrane bound fragments (apoptotic bodies),cytoplasmic condensation, nuclear condensation, endonuclease-catalyzed DNA-fragmentation and releaseof mitochondrial cytochrom c into the cytoplasm. The apoptotic program is executed by a cascade ofhighly specific caspases.Necrosis:Bleb: Protrusion from the surface of a cell, usually approximately hemispherical; may be filled with fluidor supported by a meshwork of microfilaments.Apoptosome: a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks ofapoptosis.2-mercaptoethanol: BME is suitable for reducing protein disulfide bonds prior to polyacrylamidegel electrophoresis and is usually included in a sample buffer for SDS-PAGE at a concentration of5%. Cleaving intermolecular (between subunits) disulfide bonds allows the subunits of a protein toseparate independently on SDS-PAGE. Cleaving intramolecular (within subunit) disulfide bonds allowsthe subunits to become completely denatured so that each peptide migrates according to its chain lengthwith no influence due to secondary structure. (Sigma)Ethylene glycol bis (β-aminoethyl ether)-EGTA: EGTA inhibits in vitro carboxypeptidase-mediatedsubform conversion after sample collection.Gel electrophoresis: electrophoresis in which molecules (as proteins and nucleic acids) migrate througha gel and especially a polyacrylamide gel and separate into bands according to size.Isoelectric focusing (IEF): can be described as electrophoresis in a pH gradient set up between acathode and anode with the cathode at a higher pH than the anode. Because of the amino acids inproteins, they have amphoteric properties and will be positively charged at pH values below their IpHand negatively charged above. This means that proteins will migrate toward their IpH. Most proteinshave a IpH in the range of 5 to 8.5.Isoelectric focusing takes place in a pH gradient and is limited to molecules which can be either positivelyor negatively charged (amphoteric molecules), like proteins, enzymes and peptides. Separation happensin a pH gradient which is formed by special amphoteric buffers (ampholytes) having high buffer capacitiesat their pI (isoelectric point). The pH gradient is produced by an electric field. Before an electric fieldis applied the gel has a uniform pH-value and almost all the carrier ampholytes are charged. When

Appendix F 73

an electric field is applied, the negatively charged ampholytes move towards the anode, the positivelycharged ones to the cathode and their velocity depend on the magnitude of therir net charge. The carrierampholytes align themselves in between the cathode and the anode according to their pI, and determinethe pH of their environment. A stable gradually increasing pH gradient depending on the initial mixtureof ampholytes is formed.

a literature review on skeletal muscle tissue engineering, cell damage … · 2003-06-19 · 1 a...

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