1

Influence of modified alginate hydrogels on mesenchymal stem cells

and olfactory bulb-derived glial cells cultures.

Krzysztof Marycz1, Dariusz Szarek2, Jakub Grzesiak1*, Karol Wrzeszcz1

1 Electron Microscopy Laboratory, University of Environmental and Life Sciences, Kozuchowska 5b, Wroclaw;

tel. +4871 3205 888; krzysztofmarycz@interia.pl, grzesiak.kuba@gmail.com, wh44@wp.pl,

2 Department of Neurosurgery, Wroclaw University Hospital, szarekdariusz@gmail.com

* corresponding author

Abstract

BACKGROUND: Great potential of cellular therapies has generated extensive research in

the field of cells harvesting and culturing. Transplantation of cell cultures has been used in a

variety of therapeutic programs but in many cases it appeared that biomaterial scaffold or

sheath would enhance cells regenerative potential.

OBJECTIVE: Hydrogels composed of different proportions sodium and calcium alginates,

were undertaken to evaluate their influence on mesenchymal stem cells and olfactory bulb-

derived glial cells cultures. Additionally, these biomaterials were also enriched with fibrin

protein.

METHODS: The structure of materials was visualized by means of scanning electron

microscopy. After seeding with cells - hydrogels were observed with inverted and

fluorescence microscope. Cell’s morphology, behavior and phenotype were analyzed in

investigated materials by means of light, fluorescence and scanning electron microscopes.

Also, viability assay was performed with Alamar Blue cytotoxic test.

RESULTS: Our observations showed that basic alginate hydrogels had significant influence

on both cell types. Materials maintained cells alive, which is desired attribute, however none

2

of them kept cells in normal, flat form. Alginates with significant calcium component kept

cells alive for longer period of culture.

CONCLUSIONS: Addition of fibrin protein resulted in material’s biocompatibility

properties improvement, by creation of adhesion surface, which helps cells to keep proper

morphology and behavior. Our findings suggest that addition of fibrin protein to alginate

hydrogels improves them as cell carriers for regenerative medicine applications.

Keywords: hydrogel, fibrin, glial cells, mesenchymal stem cells.

1. Introduction

The main goal of regenerative medicine is to concentrate organism’s natural potential

with simultaneous amplification of tissues repair processes [1]. The most simple forms of

stem cell therapy are applied as cellular suspension injections directly into injured tissues [2,

3]. Adult mesenchymal stem cells, as multipotent, can differentiate into healthy tissue [4].

Also their immunomodulatory properties play crucial role in recovery processes [5, 6, 7].

Their beneficial properties have been showed by many researchers, but methods of

implantation usually don’t give satisfying results.

Ones of the biggest challenges for medicine are the nervous system injuries. In novel

therapeutic strategies, transplantation of the glial cells isolated from olfactory bulb are taken

under consideration in central nervous system disorders. It was showed that these cells can

promote elongation of axons and support their regenerative potential. Their usage in the

treatment of central nerve systems injuries could be of great importance. The mechanism by

which they do promote axon’s outgrowth is, among others, depended on neurotrophins

secretion, such as p75 protein and creating a gap according to pathway hypothesis [8, 9, 10].

3

Method of stem cell delivery is very important for the therapy efficiency. Direct

intravenous injection, as one of the first stem cells application methods, showed that cells

have the ability to find and settle the injured tissue site. However, the percentage of properly

localized cells is insufficient for reconstructing damaged tissue [11]. It was indicated that

most of them were trapped in lung’s vessels [12]. Direct applications of suspended cells into

the wounds have given better results, with limited cells loss. However, in this approach

significant percentage of cells are not immobilized in desired place and are flushed out by

organism’s fluids [13].To elaborate the most efficient way of stem cell therapy, they should

be concentrated in greater number and situated in destined tissues more stable. Cell carriers

with particular structure, besides cells embedding functions, are the physical fulfillment in

sites of tissue loss. With similar mechanical properties, such materials could mimic the

mechanical function of tissue. For this, particular forms of scaffolds have been elaborated

(biofilms, sponges, hydrogels, microcapsules), with various results in vitro and in vivo [14,

15, 16, 17, 18]. It is important that the cells carrier should be compatible with cells so they

could create normal connections, proliferate, and keep proper morphology and phenotype

[19]. Promising results could be obtained using modified alginate hydrogels as cells’ carriers.

Their advantages include biodegradability, immunological neutrality, bioabsorbability and

permeability for tissue fluids. Combinations of alginates with other substances could promote

specific differentiation of cells, allowing complete regeneration. For example, addition of type

II collagen, promotes chondrogenic differentiation of mesenchymal cells, even without

stimulation by inducing medium [20, 21, 22, 23]. Clinical experiments using alginate implants

also showed positive results. It was proven that bridging a gap in peripheral nerve with

alginates gives significant clinical improvement in comparison to auto nerve graft [24, 25].

In this experiment olfactory bulb-derived glial cells (OBGCs), as well as mesenchymal

stem cells (MSC) isolated from rat adipose tissue were cultured in various modifications of

4

alginate hydrogel. It was shown that alginates without additives could maintain cells alive;

however, investigated cells could not make intercellular connections and proliferated slowly.

We have found that addition of fibrin enables alginate gels to maintain cell web in areas

where fibers are present. It was also found that artificial environment of alginates influenced

cells; however, addition of fibrin makes their surrounding more natural, allowing them

keeping normal morphology and behavior.

2. Material and Methods

2.1. Cell isolation

Adipose tissue, olfactory bulbs and blood were collected from four adult Wistar rats, of

random gender, average weight 250gWistar bred.

2.1.1. OBGCs

Isolation and culture method was previously described [26], but it was modified for purposes

of this research. For cell isolation, olfactory bulbs were carefully dissected from forebrain and

placed in HBSS. Next, they were extensively washed, minced, digested in 0,2%

collagenase/10min, disrupted by syringe needles (18G, 20G and 22G) and washed in Hank’s

balanced salt solution (HBSS, Sigma Aldrich). Cells were then centrifuged for 5 minutes at

300xg, resuspended in fresh DMEM/F12:Ham with 10% FBS and 1% antibiotics. Cells were

plated and cultured in 25 cm2 T-flask, in 37°C/5%CO2/95% humidity incubator. After seven

days, cells were taken for experiments.

5

2.1.2. MSCs

Isolation and culture procedures were based on modified methods previously published [27].

Briefly, one gram of adipose tissue was collected per individual. Adipose tissue was then

washed, digested in 0,2% collagenase for 30minutes, centrifuged at 1200xg for 10minutes and

the pellet was resuspended in DMEM/F12:Ham supplemented with 10% FBS, with addition

of 1% antibiotic/antimycotic solution (all from Sigma Aldrich). Cell suspensions were seeded

in T-flasks and after first passage were undertaken to experiments.

2.2. Plasma isolation

Anticoagulated rat whole blood was centrifuged for 10 minutes at 300 x g to obtain clean

plasma layer; it was collected and stored in temperature below -80°C. Just before planned

application, when cells were ready to use, plasma was thawed in 4°C. Next, it was centrifuged

at 2000 x g for 15 minutes in 4°C. Upper half of plasma was discarded, and the pellet was

resuspended in half decreased volume of plasma.

2.3. Alginate hydrogels preparation

Sodium and calcium alginates were dissolved in 0,9% NaCl and the solutions were filtered

through 0,45 µm and 0,22 µm syringe filters. For experiments, hydrogels were composed of

2% pure sodium alginate acid solution with addition of 2% pure calcium alginate solution, in

6

ratios 1:4, 1:1 and 4:1 (sodium alginate per calcium alginate). Each solution was polymerized

by addition to sterile 102mM CaCl2 solution.

2.4. Fibrin alginate preparation

Independently prepared alginate solutions were mixed with isolated plasma fraction, in ratios

1:4, 1:1 and 4:1. Mixtures were polymerized with 102mM CaCl2 solution. Additionally, fibrin

in plasma was polymerized with calcium chloride and incubation in 37°C/5%CO2/95% to

obtain pure fibrin scaffolds.

2.5. Material examinations

Hydrogels were evaluated with scanning electron microscope (SEM), after fixation process in

4% paraformaldehyde and dehydration in ethanol, in critical point dry (Polaron, Quorum

Technologies). Detailed observations and documentations were made by means of scanning

electron microscope (EVO LS15, Zeiss).

2.6. Cell culture

Cultured cells (125 x 103 per well) were mixed with prepared alginate solutions, and the

solutions were polymerized with 102mM CaCl2. Prepared hydrogels were placed in high

glucose Dulbecco Modified Eagle Medium (for MSCs’ culture) or DMEM/F12:Ham’s (for

OBGCs’ culture), supplemented with 10% fetal bovine serum and 1% of antibiotics. Cultures

were kept in 37°C/5%CO2 in 95% humidity for 21 days, with medium change every second

day.

2.7. Evaluation of proliferation rate

7

The proliferation activity was assessed by Alamar Blue (TOX-8, Sigma Aldrich) assay kit.

Briefly, staining solution was added to the culture wells at concentration of 10% and

incubated for 2 hours, according to manufacturer’s procedure. Measurements of absorbance

were made (600nm wavelength, 690nm as a reference wavelength). Blank sample readings

were subtracted from results. Absorbance was converted to cell number using standard curve

made simultaneously by measurement of wells with increasing cell number.

2.8. Morphology and phenotype evaluation

Cell’s phenotype in elaborated materials was observed under fluorescence inverted

microscope (Axio Observer A1, Zeiss), with immunofluorescence staining applications. For

MSCs, CD44 and CD105 markers presence was evaluated, while in OBGCs, p75 and glial

fibrillary acidic protein (GFAP) markers presence was investigated. Additionally, nuclei and

f-actin fibers were visualized to evaluate cell’s morphology and grow pattern. Procedure

included washing in PBS, fixation in 4% paraformaldehyde, permeabilization and blocking

with 0,05% triton x-100/4% bovine serum albumin (15minutes in room temperature). After

that, primary antibodies were added for one hour in temperature room (concentration 1:500),

followed by secondary antibodies addition (concentration 1:400). Simultaneously, the

phalloidin (atto-488) and DAPI solutions were added. After rinsing in PBS, materials were

observed in fluorescence inverted microscope.

3. Results

3.1. The structure of materials

8

3.1.1. Pure alginates

Materials showed different structure that was dependent of preparation process. Pure sodium

alginate, polymerized with 102mM CaCl2, was tight and brittle, but homogenous and

transparent. Ultrastructural SEM examination revealed smooth surface without visible fibers.

Pure calcium alginate was unable to polymerize, however addition of just 5% sodium alginate

enabled the cross linking. Hydrogels composed of sodium/calcium alginate in ratio of 4:1

were softer and less brittle than pure sodium gel. Increase of calcium alginate concentration

ratio made hydrogels less stable and less transparent, with sol form in case of 5%Na/95%Ca

alginates ratio. It also occurred in ultrastructural changes, with greater roughness and visible

fibers (fig.1).

3.1.2. Fibrin alginates

Addition of fibrin protein made all combinations of alginate hydrogels more plastic and

compact. SEM examinations showed increased presence of fibers, connected with increase of

fibrin concentration. Finally, polymerized fibrin protein revealed the most compact and labile

structure, with prevalence of fiber component, in relation to previous materials (fig.1).

3.2. Influence on cells

3.2.1. Pure alginates

Mesenchymal cells suspended in elaborated hydrogels kept their viability. Pure sodium

alginate environment prevented adhesion of cells to polystyrene surface, as well as to the

material. Cells had rounded form and created cell aggregates in a suspension. Three-

dimensional cellular web was not detected. Similar cell’s morphology was seen in Na/Ca

9

alginate, but cells adhered to culture vessel’s surface, with typical morphology. However,

increase of calcium concentration madecells susceptible to creating agglomerates, which

decayed shortly after (fig. 2). The proliferative activity of cells differed depending on material

and cell type. After first day, sodium alginate increased the proliferation ratio of MSCs,

however, over the next few days their potential significantly decreased. For other investigated

alginates, increase of proliferation ratio was correlated with increase of calcium concentration

(tab. 1,2). Immunostaining analysis showed negative reaction, suggesting the absence of

characteristic markers for mesenchymal stem cell populations.

Analogous situation was noticed in case of glial cells. Only calcium-sodium alginates allowed

cells to adhere to well surface and kept normal morphology, with two- and three-spindle

shaped, elongated cells. In all pure alginates cells did not grow in a suspension, proliferated

slowly and did not create connections with each other (fig.3, tab.3,4). They also did not

express p75 protein or expressed it in limited scale. However, cells were staying alive which

was proven by metabolic activity tests.

3.2.2. Fibrin alginates

Addition of fibrin enabled emergence of three dimensional web of MSCs in hydrogel,

resulting mainly from fibers presence. Increase of fibrin concentration made, respectively,

increase of material’s uniformity and ability to maintain cultures in three dimensional state.

Pure fibrin allowed cells to create dense web, with regular dispersion and multiple cell

connections. Cells were properly elongated, spindle shaped and flattened. Additionally, no

agglomerates were seen in this kind of material. Addition of fibrin made alginates more

appropriate for maintaining proliferation. This also enabled the normal protein expression

(fig.3A). Finally, pure fibrin scaffold showed the best proliferative capacity for cells, with no

activity decreasing after the 7th day (tab. 1, 2).

10

Glial cellsalso kept normal proliferative activity and morphology in the presence of fibrin,

with typical bi- or tripolar cells. They also created long connections only in protein – enriched

hydrogels. Cells showed proliferative activity only with fibrin presence (tab. 3). Finally, cells

expressed p75 receptor and GFAP, but also in monolayer and in presence of fibrin (fig.3B).

Nevertheless, in case of cells growing as three dimensional webs in fibrin alginates, these

markers were unnoticed.

4. Discussion

Autotransplantations of stem cells can give significant benefits in treatment of many

disorders (eg. of locomotive system, of cardiovascular system), which was well documented

in various experiments [28, 29]. Autologous character of transplantations excludes the

possibility of draft – host adverse reactions [30]. Increased concentration of cells in wound

sites promotes regeneration by creating conductive environment, by paracrine signaling,

among others [31]. However, cells injected in injured tissues are easily rinsed by blood and

other fluids or are damaged by syringe pressure, which limit their regeneration capability

[32]. In case of neuroregeneration, OBGC should be placed in environment which could

maintain their growth and possess proper 3D structure, and even more mechanical and

chemical properties similar to grafted tissue. It is also important that cells must synthesize and

secrete the neural growth factors in normal, physiological way. Axon outgrowth depends on

these factors in environment [33].

OBGCs cultured in our tested pure alginate hydrogels were not able to keep

proper morphology and phenotype, however the material maintained cells still alive. Altered

11

metabolism of cells caused arrest of proliferation, what correlates with regenerative potential

decrease. In our experiment, pure alginate hydrogels indeed maintained cells alive, even with

no proliferative capacity. Though, the cell metabolism was decreased, and cells didn’t create

any three - dimensional connections within the gel. It could be explained by the lack of

attaching sites within hydrogels. Although alginates fulfilling the most of suitable properties,

they do not always allow cells to maintain proper form and functions. Maintaining cell’s

normal morphology, the possibility to adhere and create cell – cell connections is also

fundamental. In case of nerve regeneration, it is important to create dense, polarized web from

glial cells processes to allow axon outgrowth [33, 34, 35, 36]. Combination of alginate gel

with particular peptides or proteins makes gel suitable for maintaining three dimensional cell

web [37]. One of them is fibrin, which could be easily isolated from patients’ blood. It creates

natural fibers, compatible with organism’s cells and their actions [38, 39]. The fact that MSC

grow in fibrin clots easily [40, 41] prompt us to use this protein in our experiment.

Addition of proteins created a scaffold in which cells could adhere. After addition of

fibrin to hydrogels cells begun to adhere, made connections and proliferated in increased

ratio. SEM observations confirmed that cells adhered only to fibrin fibers, while the

material’s fragments without protein were unsettled (fig. 2). This fact suggests that optimal

hydrogel components should have, at least partially, fibrous and protein character, which

facilitates cell adhesion process and assures binding sites. In pure fibrin gels, MSC and

OBGC were distributed evenly and created dense interconnections web (cell to cell), and their

proliferative activity significantly increased. Yet fibrin itself causes scar tissue formation in

vivo, which is undesirable feature. Alginates are biocompatible, allow immobilized cells to

survive and block migration of fibroblasts. Particular combination of alginates with

appropriate concentration of fibrin could be the key for its pro-regenerative properties in vivo.

12

Mesenchymal stem cells, despite their significant proliferative activity, were limited in

this manner in alginates without biological additives. OBGCs were also inhibited in their

growth, but on the other hand they possess naturally limited proliferative capacity [26].

Addition of fibrin increased the number of cells divisions to level occurring in normal, two –

dimensional control cultures, with just 10% of protein concentration (tab.1, 2). Growth curves

showed different course, with increased proliferation in samples with more than 10% of fibrin

concentration. It could be explained by known, stimulating properties of this protein. Growth

inhibition of MSCs in alginate – fibrin gels was correlated with the lack of free, unsettled

fibers. That observation was not noticed in glial cells. The proliferation rate of OBGCs was

not sufficient enough to observe this process. Finally, differences seen on fluorescence

pictures suggested the influence of artificial environment on cells physiology. Their

phenotype reflects their function, so markers assigned to the particular type of cell take direct

participation in cellular processes. Presence of CD44 on mesenchymal cells is connected with

their adhesive abilities, whereas CD105 takes part in cell differentiation (as one of TGF

superfamily) [42, 43]. Glial fibrillary acidic protein, as well as p75 protein characterizes the

glial cells. Presence of p75 is strictly connected with stimulation the axon to elongation [26].

We showed, that cells expressed these proteins properly only in fibrin presence and only on

well bottom’s surface (fig. 3B). It could be explained by the influence of fibrin material on

MSCs that induced change of expression profile, eg. during differentiation. Negative staining

reactions in case of pure alginates suggest that in these hydrogels cells do not express these

particular proteins, probably due to cell’s hibernated-like state.

In summary, alginate hydrogels can be successfully used as cells carriers, but they

have to contain additives that provide suitable structure for cell to grow and maintain normal,

physiological functions. We presume that pure alginate hydrogels are good primary

biomaterial suitable for further improvements, depending on particular clinical requirement.

13

Our hydrogel modifications using fibrin suggest that other biological active components, eg.

biolipids or glycoproteins could be successfully enclosed within. Our results showed that

alginate hydrogels enriched with fibrin provide positive in vitro results in proliferation,

adhesion and phenotype of investigated cells, which suggest that these findings should be

confirmed by in vivo research. Combination of alginates enriched in fibrin is promising cell

carrier and can be useful in various clinical applications in the future.

5. References

[1] Caplan AI. Adult mesenchymal stem cells for tissue engineering versus regenerative

medicine. J. Cell. Physiol. 2007; 213(2): 341-347.

[2] Black LL, Gaynor J, Adams C, Dhupa S, Sams AE, Taylor R, Harman S, Gingerich DA,

Harman R. Effect of intraarticularinjections of autologous adipose-derived mesenchymal stem

and regenerative cells on clinical signs of chronic osteoarthritis of the elbow joint in dogs.

Vet. Therap. 2008; 9(3): 192-200.

[3] Stamegna JC, Felix MS, Roux-Peyronnet J, Rossi V, Feron F, Gauthier P, Matarazzo V.

Nasal OEC transplantation promotes respiratory recovery in a subchronic rat model of

cervical spinal cord contusion. Exp. Neurol. 2011; 229(1): 120-131.

[4] Strem BM, Hicok KC, Zhu M, Wulur I, Alfonso Z, Schreiber RE, Fraser JK, Hedrick MH.

Multipotential differentiation of adipose tissue-derived stem cells. Keio J. Med. 2005; 54(3):

132-141.

[5] Iyer SS, Rojas M. Anti-inflammatory effects of mesenchymal stem cells: novel concept

for future therapies. Expert Opin. Biol. Ther. 2008; 8(5): 569-581.

14

[6] Wan CD, Cheng R, Wang HB, Liu T. Immunomodulatory effects of mesenchymal stem

cells derived from adipose tissues in a rat orthotopic liver transplantation model.

Hepatobiliary Pancreat. Dis. Int. 2008; 7(1): 29-33.

[7] Hoogdujin MJ, Popp F, Verbeek R, Masoodi M, Nicolaou A, Baan C, Dahlke MH. The

immunomodulatory properties of mesenchymal stem cells and their use for immunotherapy.

Int. Immunopharmacol. 2010; 10(12): 1496-1500.

[8] Ramon-Cueto A, Munoz-Quiles C. Clinical application of adult olfactory bulb

ensheathing glia for nervous system repair. Exp. Neurol. 2011; 229(1): 181-194.

[9] Ziegler MD, Hsu D, Takeoka A, Zhong H, Ramon-Cueto A, Phelps PE, Roy RR,

Edgerton VR. Further evidence of olfactory ensheathing glia facilitating axonal regeneration

after a complete spinal cord transection. Exp. Neurol. 2011; 229(1): 109-119.

[10] Raisman G, Barnett SC, Ramón-Cueto A. Repair of central nervous system lesions by

transplantation of olfactory ensheathing cells. Handb Clin Neurol. 2012; 109:541-549.

[11] Devine SM, Bartholomew AM, Mahmud N, Nelson M, Patil S, Hardy W, Sturgeon C,

Hewett T, Chung T, Stock W, Sher D, Weissman S, Ferrer K, Mosca J, Deans R, Moseley A,

Hoffman R. Mesenchymal stem cells are capable of homing to the bone marrow of non-

human primates following systemic infusion. Exp. Hematol. 2001; 29(2): 244-255.

[12] Harting MT, Jimenez F, Xue H, Fischer UM, Baumgartner J, Dash PK, Cox CS.

Intravenous mesenchymal stem cell therapy for traumatic brain injury. J. Neurosurg. 2009;

110(6): 1189-1197.

[13] Sorrell JM, Caplan AI. Topical delivery of mesenchymal stem cells and their function in

wounds. Stem Cell Research and Therapy 2010; 1:30; doi:10.1186/scrt30.

[14] Levenberg S, Huang NF, Lavik E, Rogers AB, Itskovitz-Eldor J, Langer R.

Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds.

PNAS 2003; 100: 12741-12746.

15

[15] Medrado GCB, Machado CB, Valerio P, Sanches MD, Goes AM. The effect of a

chitosan-gelatin matrix and dexamethasone on the behavior of rabbit mesenchymal stem cells.

Biomed.Mater. 2006; 1(3): 155-161.

[16] Pan L, Ren Y, Cui F, Xu Q. Viability and differentiation of neural precursors on

hyaluronic acid hydrogel scaffold. J. Neurosci. Res. 2009; 87(14): 3207-3220.

[17] Li J, Sun H, Zhang R, Li R, Yin Y, Wang H, Liu Y, Yao F, Yao K. Modulation of

mesenchymal stem cells behaviors by chitosan/gelatin/pectin network films. J. Biomed.

Mater. Res. B: Appl. Biomater. 2010; 95B: 308-319.

[18] Ortinau S, Schmich J, Block S, Liedmann A, Jonas L, Weiss DG, Helm CA, Rolfs A,

Frech MJ. Effect of 3D-scaffold formation on differentiation and survival in human neural

progenitor cells. Biomedical Engineering OnLine 2010; 9:70; doi:10.1186/1475-925X-9-70.

[19] Discher DE, Mooney DJ, Zandstra PW. Growth factors, matrices, and forces combine

and control stem cells. Science 2009; 324(5935): 1673-1677.

[20] Mosahebi A, Wiberg M, Terenghi G. Addition of fibronectin to alginate matrix improves

peripheral nerve regeneration in tissue-engineered conduits. Tiss. Eng. 2003; 9(2): 209-218.

[21] Perets A., Baruch Y, Weisbuch F, Shoshany G, Neufeld G, Cohen S. Enhancing the

vascularization of three-dimensional porous alginate scaffolds by incorporating controlled

relase basic fibroblast growth factor microspheres. J. Biomed. Mater.Res. 2002; A 65: 489-

497.

[22] Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M, Fujinaga T. Chondrogenic

differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different

hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis.

Biotech.Bioeng. 2006; 93(6): 1152-1163.

16

[23] Yang C, Frei H, Rossi FM, Burt HM. The differential in vitro and in vivo responses of

bone marrow stromal cells on novel porous gelatin-alginate scaffolds. J. Tissue Eng. Regen.

Med. 2009; 3(8): 601-614.

[24] Hashimoto T, Suzuki Y, Kitada M, Kataoka K, Wu S, Suzuki K, Endo K, Nishimura Y,

Ide C. Peripheral nerve regeneration through alginate gel: analysis of early outgrowth and late

increase in diameter of regenerating axons. Exp Brain Res. 2002; 146:356-368.

[25] Matsuura S, Obara T, Tsuchiya N, Suzuki Y, Habuchi T. Cavernous nerve regeneration

by biodegradable alginate gel sponge sheet placement without sutures. Urology 2006;

68(6):1366-1371.

[26] Higginson JR, Barnett SC. The culture of olfactory ensheathing cells (OECs) – a distinct

glial cell type. Exp Neurol. 2011; 229(1):2-9.

[27] Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK,

Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol

Biol Cell 2002; 13:4279-4295.

[28] Ankrum J, Karp JM. Mesenchymal stem cell therapy: two steps forward, one step back.

Trends in Molecular Medicine 2010; 16(5):203-209.

[29] Prockop DJ, Gregory CA, Spees JL. One strategy for cell and gene therapy: harnessing

the power of adult stem cells to repair tissues. PNAS 2003; 100:11917-11923.

[30] Tosi P, Zamagni E, Ronconi S, Benni M, Motta MR, Rizzi S, Tura S, Cavo M. Safety of

autologous hematopoietic stem cell transplantation in patients with multiple myeloma and

chronic renal failure. Leukemia 2000; 14(7): 1310-1313.

[31] Ratajczak MZ, Kucia M, Jadczyk T, Greco NJ, Wojakowski W, Tendera M, Ratajczak J.

Pivotal role of paracrine effects in stem cell therapies in regenerative medicine: can we

translate stem cell-secreted paracrine factors and microvesicles into better therapeutic

strategies? Leukemia 2012; 26(6):1166-1173.

17

[32] Aguado BA, Mulyasasmita W, Su J, Lampe KJ, HeilshornSC. Improving viability of

stem cells during syringe needle flow through the design of hydrogel cell carriers. Tissue Eng.

Part A, 2012; 18(7-8):806-815.

[33] Jiao Y, Novozhilova E, Karlen A, Muhr J, Olivius P. Olfactory ensheathing cells

promote neurite outgrowth from co-cultured brain stem slice. Exp. Neurol. 2011; 229(1): 65-

71.

[34] Chuah MI, Hale DM, West AK. Interaction of olfactory ensheathing cells with other cell

types in vitro and after transplantation: Glial scars and inflammation. Exp. Neurol. 2011;

229(1): 46-53.

[35] Novikova LN, Lobov S, Wiberg M, Novikov LN. Efficacy of olfactory ensheathing cells

to support regeneration after spinal cord injury is influenced by method of culture preparation.

Exp. Neurol. 2011; 229(1): 132-142.

[36] Sasaki M, Lankford KL, Radtke C, Honmou O, Kocsis JD. Remyelination after olfactory

ensheathing cell transplantation into diverse demyelinating environments. Exp. Neurol. 2011;

229(1): 88-98.

[37] Dhoot NO, Tobias CA, Fischer I, Wheatley MA. Peptide-modified alginate surfaces as a

growth permissive substrate for neurite outgrowth. J. Biomed. Mater.Res. 2004; A 71: 191-

200.

[38] Kalbermatten DF, Kingham PJ, Mahay D, Mantovani C, Pettersson J, Raffoul W, Balcin

H, Pierer G, Terenghi G. Fibrin matrix for suspension of regenerative cells in an artificial

nerve conduit. J. Plast. Reconstr.Aesth. Surg. 2008; 61(6): 669-675.

[39] O’Cearbhaill ED, Murphy M, Barry F, McHugh PE, Barron V. Behavior of human

mesenchymal stem cells in fibrin-based vascular tissue engineering constructs. Ann. Biomed.

Eng. 2010; 38(3): 649-657.

18

[40] Ho W, Tawil B, Dunn JCY, Wu BM. The behavior of human mesenchymal stem cells in

3D fibrin clots: dependence on fibrinogen concentration and clot structure. Tissue Eng 2006;

12(6):1587-1595.

[41] Seybold D, Schildhauer TA, Gessman J, Muhr G, Koller M, Roetman B. Osteogenic

differentiation of human mesenchymal stromal cells is promoted by a leukocytes containing

fibrin matrix. Langenbecks Arch Surg. 2010; 395(6):719-726.

[42] Goodison S, Urquidi V, Tarin D. CD44 cell adhesion molecules. MP, Mol. Pathol. 1999;

52(4): 189-196.

[43] Rege TA, Hagood JS. Thy-1 as a regulator of cell-cell and cell-matrix interactions in

axon regeneration, apoptosis, adhesion, migration, cancer and fibrosis. FASEB J. 2006; 20(8):

1045-1054.

19

Table 1. Proliferation results of MSCs in various materials combinations. Cell quantity in

thousands, time in days; Na – sodium alginate, Ca – calcium alginate, F - fibrin.

20

Table 2. Differences in proliferation of MSCs depended on material’s composition. Cell

quantity in thousands, Na – sodium alginate, Ca – calcium alginate, F – fibrin.

21

Table 3. Proliferation results of OBGCs in various materials combinations. Na – sodium

alginate, Ca - calcium alginate, F – fibrin; cell quantity in thousands, time in days.

22

Figure 1. Appearance of obtained hydrogels: 1 – 100% sodium alginate; 2 – 1:4

sodium:calcium alginate; 3 – 1:4 sodium:calcium alginate with 25% fibrin content; 4 – 1:4

sodium:calcium alginate with 75% fibrin content; 5 – pure fibrin hydrogel. Macroscopic

pictures seen on upper row, ultrastructural pictures on bottom row.

Figure 2. Presence of cells in materials. 1 - 100% sodium alginate; 2 - 1:4

sodium:calcium alginate; 3 - 1:4 sodium:calcium alginate with 25% fibrin content; 4 - 1:4

sodium:calcium alginate with 50% fibrin content; 5 - 100% fibrin hydrogel. Left column -

SEM pictures; middle column – inverted, phase contrast microscope pictures; right column –

fluorescence pictures: actin showed in green, nuclei in yellow; magnification 100x.

Figure 3. A – CD44 molecule expression in MSCs in fibrin-containing alginates (mag. 200x);

B – p75 receptor (red) and GFAP (green) expression in OBGCs in 50% fibrin – containing

alginates (mag. 100x).

23

Figure 1.

24

Figure 2.

25

Figure 3.