principles of tissue engineering || bone regeneration

CHAPTER 55

Bone Regeneration
Arun R. Shrivats, Pedro Alvarez, Lyndsey Schutte and Jeffrey O. HollingerDepartment of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania
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INTRODUCTIONBone regeneration is a marvelous pas de deux of biology and biomechanics wrapped in discrete

temporal events nurtured by a lush vascular cascade. The outcome of the highly complex

process of regeneration is the restoration of form and function, ideally, enduring, to a boneinsufficiency. Broadly, such an insufficiency may be stated as any discontinuity in bone integrity,

spanning microfractures, through macrofractures to developmental-congenital defects to avulsive

trauma and surgical resection.

An epochal question: How to harness the intrinsic regenerative capacity of bone? Bone

regenerative tacticians have proposed a deconstructive approach, that is: What are the compo-sitional elemental components of bone beginning with cells, extracellular matrices (organic

and inorganic) and soluble signaling molecules? Further posing the questions: How do these

compositional elements inspire the multi-tiered temporal osteogenic regenerative cascade? Isthere a trigger factor? Are there co-factors?

The prototypic and conventional approach to bone regeneration for this chapter is to highlight

bone regeneration by exploiting contemporary, albeit pedestrian approaches of autografts,allografts and xenografts. First, we will define these modalities and emphasize what is good

about them and what is lacking. We will further offer consensus definitions of the biological

and biomechanical properties of regeneration, stating specific performance parameters thatdefine the temporal road map for the regenerative cascade. As such, we will exploit a fracture

healing model as a prototype for the regenerative process. We will introduce key directionalconcepts that must be acknowledged for the rational design and development of a regenerative

therapy. These concepts will include the osteogenic biohemodynamic cascade and for bone

regenerative therapies, the importance of a 4D environmental blastema matrix where embryo-genesis is recapitulated. And lastly, we will offer stringent performance criteria with objective

data to provide a framework for the rational design and fielding of a master tool kit for bone

regenerative therapeutics.

CURRENT CLINICAL PRACTICESWe will begin with an explanation of the most commonly employed techniques for boneregeneration. At the current time, this practice revolves around natural (as opposed to syn-

thetic) bone and its derivatives.

We will first establish a broad criterion for the generic term graft.We will carefully partition the

term graft and implant; then abandon this rigor in favor of clinical jargon.

Principles of Tissue Engineering. http://dx.doi.org/10.1016/B978-0-12-398358-9.00055-0

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PART 14Musculoskeletal System

Grafts contain cells; implants do not. Therefore, an autograft (recipient and donor being thesame individual), contains cells. An allograft (recipient and donor being genetically matched

individuals), contains cells. However, traditional tissue banking procedures effectively and

efficiently remove cells from allo-stock bone, rendering the allo-stock product acellular andnon-immunogenic. Consequently, such a product should be defined scientifically as an

alloimplant. Nevertheless, clinical jargon has made an exception to the stringent scientificdefinition, and while incorrect, allograft has evolved as a clinically acceptable term for an

acellular allo-stock product. Therefore, in this chapter, we will use the term allograft (despite the

fact that the term alloimplant is scientifically correct).

In order to understand the rationale for the use of bone-derived products for regeneration,

it is important to define the criteria by which we measure successful bone regeneration.

Bone healing and subsequent new bone formation after the implantation of a graft occurthrough the processes of osteogenesis, osteoinduction and/or osteoconduction [1].

Osteogenesis is the process by which osteoblasts at the defect site express osteoid that

subsequently mineralizes, yielding new bone. Osteoinduction is the induction of osteopro-genitor cells (or other non-differentiated cells) to differentiate down an osteoblast lineage.

Osteoconduction is the property by which a graft supports the attachment of new osteoblasts

and osteoprogenitor cells. In this situation, the graft must provide an interconnectedstructure through which new cells migrate and blood vessels form (i.e., angiogenesis).

An ideal bone graft is characterized by all three of these phenomena and clinically will

promote regeneration of bone that is physiologically and functionally indistinguishablefrom the pre-injury defect site.

Bone autografts are harvested from a donor site in a patient (usually the iliac crest) andimplanted in that same patient at the bone deficient locale (i.e., recipient site) [2]. Autografts

are considered to be the gold standard for bone grafting applications for several reasons.

Firstly, autografts satisfy all three of the previously mentioned bone regeneration criteriae thatis, they are osteoconductive, osteoinductive and promote osteogenesis. The autograft is

intrinsically vascularized and may be co-harvested with a vascular pedicle. Consequently, the

autograft provides an ideal combination of biological signals for integration of the new boneto the recipient site. Additionally, autograft is the patient’s own tissue, thus mitigating the risk

of immunological sequelae. However, there are drawbacks associated with the use of auto-

grafts: they are limited in quantity, shape restrictions often require extensive intra-operativemodifications, and donor-recipient procedures require a ‘harvesting’ site on the same patient

[3]. As with any surgery, surgical complications may include inflammation, infection, chronic

pain and donor site morbidity [4].

Allografts are tissues harvested from healthy, prescreened human donors and are processed and

preserved for implantation in a patient. The benefits of allografts include eliminating the needfor a second surgical site on the patient requiring the graft. However, allogeneic bone (i.e., the

allograft bone donated by the same species: human-to human) is not necessarily immuno-

priviledged and may activate an immune response in the new host [5]. Additionally, duringtissue bank processing, allografts go through freeze-drying, ‘washing’ (to render the product

cell-free), demineralization (partial or complete e residual mineral may be up to approx-

imately 4e6%), and gamma-irradiation or ethylene oxide sterilization. While these techniqueslessen the risk of disease transmission and immunological responses, they also reduce the

osteogenic potential of the graft.

An alternative to auto- and allografts are xenografts: bone from non-human species.Xenografts are processed to ensure sterility and biocompatibility and this processing must

mitigate disease transmission [6]. Xenogeneic bone is administered as a bone void filler.

Examples of xenogeneic bone products include BioOss [7], an inorganic matrix from cowsand XCM� (Synthes), porcine organic bone matrix. While xenografts may have their uses in

bone regeneration, the risks of disease transmission (however minimal they may be) and

CHAPTER 55Bone Regeneration

ethical issues have limited their clinical appeal and thus, they have not really ‘caught on’ inthe clinic.

Autografts and allografts have many clinical benefits and thus constitute a majority of bonegraft procedures. However, recognized limitations have necessitated the search for alternatives.

Therefore, laboratory inspired bone tissue-engineering approaches have been assiduously

pursued. We emphasize that a rational approach to regenerative bone tissue-engineeredproducts must be based on fundamental osteobiology. It is thus imperative to understand

mechanisms, concepts and the guiding principles of the osteogenic cascade.

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CONCEPTS AND DEFINITIONSOurbodies have evolved sophisticatedmechanisms to healwounds.When injured, there are two

distinct responses that can be initiated: repair and regeneration. Repair is the restoration of thecontinuity of tissues at the injury site, but not necessarily by the same cells and tissues that existed

prior to the injury. Regeneration, however, is a series of biologically- and biomechanically-

inspired events that produce restoration of form and function of the injured tissues to a state thatis biologically and functionally indistinguishable from the pre-injury wound site [8].

The molecular-level biological agents involved in wound healing and regeneration are slowly

being elucidated. Our collective understanding of these processes has increased. One area,however, that still causes confusion is the nomenclature in literature of biological factors,

specifically between growth factors and cytokines. In this chapter, growth factors are defined as

biologic agents whose actions are exerted on cells of mesenchymal lineage and cytokines aredefined as agents acting on cells of hematopoietic lineages, including immune cells.

Biological factors are crucial for regenerative processes and can be classified into three cat-egories e autocrine, paracrine and endocrine. Autocrine signaling refers to a cell releasing

an agent that acts on the same cell type. Paracrine signals affect those cells that are in the same

area as the cell from which the signal originated. Endocrine signals are those that must travelthrough the bloodstream in order to reach their target.

Biology is characterized by redundancy, which results in many similarities among responses oftissues to injury, particularly with respect to the biological agents [9,10]. One key difference

between osseous and non-osseous (aka ‘soft’) tissues is their respective regenerative potential.

Soft tissues heal exclusively by scar (i.e., fibroblast generated collagen) tissue formation,resulting in the restoration of tissue continuity at the injury site at the expense of the original

tissue function. Osseous tissues, however, have the potential to regenerate to a state that is

biologically and biomechanically indistinguishable from that derived from embryogenesis.

If bone would always spontaneously regenerate and overcome any manner of insufficiency/

defect, this chapter on bone tissue regeneration would not be necessary. There are limitations

to the regenerative potential of osseous tissue that renders defects (i.e., deficiencies: congenital,surgical, avulsive) greater than a certain size unlikely to regenerate [11]. This size has been

defined as a critical sized defect (CSD), which is:

‘the smallest size intraosseous wound in a particular bone and species of animal thatwill not heal spontaneously during the lifetime of the animal’ [12,13].

The central question is: how do we extend our regenerative capabilities for sub-critical sizedefects to bone insufficiencies larger than the critical size?

The answer lies in elucidating, defining and understanding the differences in each of thesepathways. Specifically, what is the osteogenic pathway for a critical sized defect? For a non-

critical sized defect? Consequently, a prototypic pathway is an obligatory first step. Conse-

quently, the next section introduces a fracture healing model. This model is defined by systematicprocesses of meticulously choreographed events that lead to bone regeneration. Bone tissue


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engineers must exploit and recapitulate the complex process of fracture healing, capture thebiological and biomechanical nuances in its evolution, and thus, define performance criteria

for achieving bone regeneration in critical size defects with tissue-engineered products.

FRACTURE HEALING MODELIn developing novel bone tissue-engineering tools, we seek to extend our bodies natural

regenerative capabilities to include bone defects larger than the critical size. Fracture healing is

the prototypic physiological model for bone regeneration. It is a multistage process that ischaracterized by complex, yet well-orchestrated, predictable steps in response to an injury.

Fracture healing begins immediately following the injury and ends following the remodeling ofthe newly formed bone into mature bone. This process has components that recapitulate the

processes of de novo bone formation during embryogenesis [14].

The process of fracture healing is a multi-phase, multi-tiered series of events segmented intofour main steps:

1) The formation of a hematoma,

2) The migration and mitosis of mesenchymal cells,3) Cartilage formation and substitution of cartilage by bone, and

4) Remodeling [15].

Immediately after a bone is fractured, the damage to local vasculature at the fracture site isresponsible for producing a hematoma, or a blood clot. A hematoma is a localized collection of

blood products including platelets, leukocytes, macrophages, fibrin and soluble biologicalgrowth factors and cytokines. This first phase of fracture healing, termed the destructive phase,

lasts for about three days and is characterized by inflammation and local hypoxia [9].

The constructive phase of regeneration follows the destructive phase (Fig. 55.2) and beginsroughly three days after the injury. It is characterized by new vasculature formation due to the

migration and subsequent capillary formation of endothelial cells. Local hypoxia during the

destructive phase is a stimulant for the formation of new blood vessels (i.e., angiogenesis).The formation of new vasculature allows for the recruitment of mesenchymal stem cells

FIGURE 55.1Bone will regenerate ‘gaps’ smaller than a critical size but when a ‘gap’ exceeds a certain volumetric dimension, theoutcome of the healing process is fibrosis rather than osteogenesis.

FIGURE 55.2The temporal activity of key cellphenotypes and events during fracturehealing.


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(or pericytes: fusiform cells of themesenchymal lineage that line blood vessels) that differentiate

into chondrocytes and osteoblasts as well as providing the conduits for monocytic-derived os-teoclasts [16,17]. The chondrocytes are responsible for cartilage matrix formation and the osteo-

blasts produce the new bone and the osteoclast-osteoblast coupling remodel that bone.

Granulation tissue condenses into a ‘soft’ callus that acts as a stabilizing buttress, supportingthe bone fragments at the distal and proximal bone fragments. The elegance of this soft callus

formation is that it provides the bone fracture site with a cartilaginous scaffold that acts as both

a fixation and stabilization structure and a template for subsequent mineralization [18].Chondrocytes in the soft callus undergo a programmed cell death (apoptosis); concurrently,

osteoblasts deposit bone matrix (osteoid) to replace the soft callus. At the fracture locale, over

the course of months to years, the bone is remodeled to a physiological status indis-tinguishable from the pre-fractured condition [19].

The processes driving the biology and biomechanics of bone regeneration at a fracture remainin large part, a mystery. There are many highly complex interactions among multiple cell types

and mediated by soluble and non-soluble signaling agents that have not been sufficiently

characterized and elucidated. The profoundly compelling challenge for biomedical tissue engineers isto deconstruct the regenerative process of fracture healing and subsequently stitch those components

together to produce osteo-regenerative therapies for applications in bone defects exceeding the

‘critical size’.

PERFORMANCE CRITERIA FOR BONE REGENERATIONWe establish a goal for bone regeneration: Recapitulate the Biohemodynamic Cascade of fracturehealing on a larger scale, one that may be applied to osseous regeneration of the critical sized

defect. We then ask the following: How can we mimic fracture healing to develop therapies

that consistently regenerate bone in gap defects? What must bone tissue engineering developas a therapy to regenerate bone defects that exceed the ability of the body to spontaneously

promote regeneration, that is, to regenerate bone in critically sized defects? The clinical re-

quirements for bone regeneration demand that we develop products that outperform theexisting standards set by autografts and allografts. We must develop a biologically- and

biomechanically-based therapy that is osteo-angiogenic, that is: fulfills the parameters of the

biohemodynamic cascade of fracture healing.

Our approach for achieving this is two-pronged: Since bones have an intrinsic ability for

regeneration, our first mission is to design therapeutics that do not interfere with the naturalregenerative processes. The body executes a network of biological and biomechanical


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events that are highly complex; as such, we must not interfere with the intrinsic processes butrather, supplement them. The second tactic to our approach is that bone regeneration thera-

peutics must work synergistically with endogenously-driven healing cues. Therefore, we must

carefully engineer therapeutics to match the biology and biomechanics of natural boneregenerative processes.

Safety

A fundamental requirement of any bone regeneration therapeutic is that it must do no harm to

the patient. This is a basic ethical principle for scientists, clinicians and the corporate sector.

Doing no harm to the patient includes ensuring the biocompatibility of bone regenerativetherapeutics. When a bone therapeutic is implanted at the site of a bone insufficiency, the first

interactions are with host blood and blood proteins. Therapeutics must not elicit either an

acute or a chronic immunological response [20].

An additional factor affecting biocompatibility, and therefore the safety of a therapy, is the

degradation properties of its materials. Therapeutics based on materials such as metals, ce-ramics and polymers may degrade in the body. Polymeric systems such as the polyalpha

hydroxy acids degrade by hydrolysis or enzymatic processes. While we have already stressed the

importance of biocompatibility upon implantation, of further importance is the biocompati-bility of degradation products. Polyalpha hydroxy acids such as polylactide and polyglycolide

degrade into lactic and glycolic acids, producing an acidotic environment. As such, it is crucial

that therapeutic bone grafts degrade in a fashion that does not hinder bone regenerativeactivities.

Mechanical properties

We have discussed the importance of avoiding immunologic responses, but we have not yetmentioned the biomechanical requirements for a bone regeneration therapeutic. Initially,

a bone defect site is in a state of dynamic instability, where structural integrity has been

compromised but may still be subjected to loads. Consequently, the biomaterial must providesufficient mechanical strength and be able to accommodate to tensile, compressive and shear

forces. Moreover, the material must degrade in a coordinated temporal manner with the

new bone formation as well as support the new vasculature. If a bone graft material is degradedtoo quickly (i.e., before the infiltration of bone begins e roughly four weeks after the injury),

the area will fail to provide the requisite support. Conversely, if the material remains at the

bone recipient site for longer than this four week window, it will impede regeneration.Furthermore, implanted materials in bonemust support physiological loads, for which there is

a biomechanical threshold. A material that exceeds this threshold will cause stress shielding of

the bone and promote bone resorption. Many of the terms and end-stage properties statedabove are well-known to bone tissue engineers. However, what is neither well-known nor

precisely defined, are the quantitative performance criteria that match to ‘sufficent strength’,

‘degradation’ and ‘how slowly or rapidly’ a bone regenerative material should either beretained or degraded. An inadequate product will be engineered when specific performance

criteria are lacking. Consequently, a focused effort must be made by bone tissue engineers toaccurately and precisely define, with rigorous science, performance criteria for bone tissue-

engineered product. Based on the timeline of fracture healing, an implanted material should

be fully degraded by the formation of the soft callus, providing a target of roughly two to fourweeks for full degradation [18].

Bone regenerative properties

General performance criteria for bone graft therapeutics must include properties of osteo-conductivity, osteoinductivity and osteogenicity.


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An osteoconductive graft will support the attachment and differentiation of the many differentcell types that migrate to the defect site. One method for influencing osteoconductivity in

synthetic grafts is by tailoring the hydrophilicity and hydrophobicity of the graft surfaces.

These surface properties enable cell attachment and protein adsorption; however, they aloneare not sufficient for a suitable therapeutic [21].

Another strategy for improving cellular adhesion to bone graft materials is to engineer peptide-based adhesion sequences, such as the arginine-glycine-aspartic acid (RGD) sequence and

extracellular matrix components such as collagen, fibronectin [22], and vitronectin. Moreover,

the inclusion of angiogenic growth factors in the composition, such as vascular endothelialgrowth factor and basic fibroblast growth factor, can contribute to the osteoinductive ability of

a bone biomaterial [4]. This approach is akin to compositional engineering. Compositional

engineering of bone regeneration therapies must comply with the temporal and dosing profileof the osteogenic cascade (Fig. 55.3). Specific biological factors are expressed at certain, desig-

nated time(s) during osteogenesis and bone regeneration. Moreover, there may be a waxing

and waning of biological agents. These agents evoke an outcome driven by cell phenotypesthat craft extracellular matrices resulting in tissue regeneration. We posit that bone ‘gaps’ less

than a certain size will regenerate as a consequence of sufficient biological cues and cellular

craftsmen; exceeding that certain size, that is, being critically sized, these ‘gaps’ will undergofibrogenesis rather than osteogenesis. Consequently, only by following the biological roadmap

with its meticulously timed and dosed boundaries that define the osteogenic cascade

(Fig. 55.3), will the bone tissue engineer be able to produce a predictable and safe clinicaloutcome: bone regeneration of a critically sized defect.

Another approach to bone regeneration materials exploits architectural engineering. Wedefine this as the physical properties that may be adjusted in a material to inspire bone

regeneration. An example of architectural engineering is pore structure (i.e., void volume) [23].

Macroporosity (pore size >50 mm) has been shown to promote cell ingrowth as well assupport angiogenesis. Microporosity (pore size <10 mm), on the other hand, plays a role in

cellular signaling via transport of biologically active signaling agents [24]. A combination of

the macro- and microporosity may sustain osteogenesis and angiogenesis [25] throughout thematerial, assuming those pores are interconnected. Further, pore size range, pore distribution

and volumetric porosity comprise the three parameters available to the bone tissue engineer

for modulating the design of a product that matches the intended clinical use. The designof a porous structure for bone regeneration is a delicate art as volumetric void volume is

inversely related to overall strength of the material.

FIGURE 55.3Relative levels of key biological factorsin the bone regeneration cascade,compiled from multiple sources[9,15,18,26e28].

TABLE 55.1 Recommended temporal windows (given as days postinjury) in which to deliver fracture healing biologicalsbased on extensive literature reviews [14,16,28e35]

Growth factor Proposed delivery period

PDGF 0 e 3 daysVEGF 14 e 21 days, though required earlier as wellBMP BMP-4: 3 e 5 days

BMP-2: 3 e 21 daysPRP 0 e 5 days


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Recapitulating osteogenesis within bone defect requires precise, dose-dependent external

contributions. Therefore, we are obligated to pay attention to the specific spatial, dosing and

temporal events that occur in fracture healing and embryogenic bone formation. By deliveringcells and biological factors to a bone defect site, we may overcome the limitations in bone

regenerative potential caused by an insufficiency. However, cells and biologicals must be

delivered at the appropriate dose, times and with release profiles mimicking the osteogeniccascade (see Fig. 55.3, above). For example, the delivery of pro-inflammatory cytokines

must occur within the first 72 hours after injury. If delivery of these cytokines is delayed,

constructive phase events of bone regeneration may be impeded. Table 55.1 summarizes ourrecommended delivery times for several growth factors based on their consensus appearance

in bone regeneration.

CLASSICAL RESEARCH APPROACHESNow that we have highlighted the performance criteria for bone regeneration approaches, we

will discuss current tactics in bone regeneration research. It is worth noting that in spite of the

criteria provided above, there are many approaches to bone tissue regeneration. Moreover,owing to the tremendous complexity (and diversity of opinions) about the process of bone

regeneration, it is naı̈ve to assume a single approach either will fit all clinical indications orprovide solutions in all circumstances. However, we underscore that contemporary efforts to

promote bone regeneration focus on cells, biological factors and materials.

Cell-based approaches for bone tissue engineering

The antecedents for the cell phenotypes in bone regeneration trace their pedigree to

hematopoietic and mesenchymal lineages. Cells that resorb bone, debris, and implant ma-terial e such as macrophages and osteoclasts e are derived from hematopoietic monocytes.

Monocytes may diverge to either macrophages or osteoclasts depending on the biological

environmental cues. Macrophages release cytokines to recruit mesenchymal cells for neovas-cularization, as well as orchestrate an anti-inflammatory, pro-healing environment [36].

Macrophages and monocytes, as well as the osteoclasts are marquis players during the

destructive phase of bone regeneration.

During the constructive phase, mesenchymal cells differentiate to pre-osteoblasts. The peri-

osteum and endosteum, as well as vascular lining pericytes [31], are the putative sources for the

preosteoblast lineage. Biological cues for the progression of the differentiation of the precursorcells to pre-osteoblasts are under the aegis of bone morphogenetic proteins.

The regeneration of bone in a bone deficient site is a time-sensitive process; as such, if boneformation by osteoblasts does not occur in register with soft callus remodeling, the outcome

will be fibrogenesis rather than osteogenesis. Moreover, mechano-transductive signals

matched to bone will not occur: fibrous tissue is not biofunctionally equivalent to osseoustissue. Consequently, a ‘pool’ of programmable osteoblastic precursors must be localized to

TABLE 55.2 The advantages and disadvantages of commonly used biomaterials for bone regeneration

Material Advantages Disadvantages References

Alginate Can be used for drug delivery, cellencapsulation, wound dressing,anti-adhesion applications;biodegradable

Anti-adhesive, mechanicallyweak

[86e89]

Bone Mineral Matrix Low immunogenicity, welldocumented success

Potential for diseasetransmission, decreasedmechanical strength, limitedsupply

[88,90,91]

Chitosan Sponge Can be used as a hemostat or todeliver soluble signaling molecules,biodegradable

Mechanically weak [88,92,93]

Collagen Sponge Biocompatible and osteocompatible,antigenicity can be weakened byremoving telopeptides,biodegradable, adhesive, highlyporous, can be incorporated withnew tissue matrix or combined withother materials

Lack of rigidity (mechanicallyweak), potentially antigenic

[88,94,95]

Collagen-Ceramiccomposite

Biocompatible, biodegradable,degradation profile better thancollagen alone, spatially adaptable,can control shape, higher stiffnessthan collagen sponge, increasedparticle and defect wall adhesion

Mechanically weak [88,96e98]

Hyaluronic Acid Can be used as a coating or to delivercells or signaling molecules, lowimmunogenicity, hydrophilic,injectable

Low cell adhesion,mechanically weak

[88,99,100]

Hydroxyapatite Moldable, can be used for coatings,high degree of tissue integration,creates undistinguishable unionswith bone, low immune response

Risk of disease transmission,requires increased time forbony restoration

[88,101,102]

Polycaprolactone(PCL)

Can be used as delivery system,mechanical properties can beadjusted

Hydrophobic, degrades slowly [88,103e105]

Poly(lactice-co-glycolic acid) (PLGA)

Can be used as a delivery system,biodegradable, mechanicalproperties and biodegradationkinetics are suited for boneapplications

Acidic Degradation products [88,106e108]

Polyphosphazenes Fine-tunable degradation profile,thermosensitive, injectable, supportscell attachment

Acidic degradation products [88,107,109]

Polyurethane Biostable, biocompatible, adjustablemechanical properties (durability,toughness), cost effective

Risk of toxic degradationproducts

[88,107,110]

Tyrosine-derivedPolycarbonates

Biodegradable, cell-impermeable,low immunogenicity, can be used asa delivery system, mechanicalproperties (stiffness and strength)suited for bone applications, costeffective

Hydrophobic [88,111e113]


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the healing bone regenerative domain (i.e., by chemoattraction), expanded in quantity(the process of mitogenesis) and modulated to an end-stage phenotype (i.e., undergo dif-

ferentiation) to produce the desired outcome: restoration of form and function. The timing

of this event is but one of many ‘key’ movements in the dynamics of osteogenesis. Sources forosteoblastic precursor cells include mesenchymal stem cells (MSCs), adipocytes and peri-

vascular pericytes populations contiguous to the bone wound. The tempo of regenerationmust be orchestrated by a meticulous sequence between these inducible cell populations and

the cueing biological signals that will produce osteoblasts. Exploiting osteoblast precursor

populations is the inspiration for dedicated cell-based therapies for bone regeneration.

The bone-marrow-derived mesenchymal stem cells (BMSCs) are commonly used considered

the ‘gold standard’ [37e39]. Bone-marrow-derivedmesenchymal stem cells have been used for

gap bone defect treatments and purportedly improve short-term and long-term healing[40,41]. However, there are limitations to BMSCs for bone regeneration that include the post-

operative painful sequelae from the ‘collection’ procedure, the uncertainty about the quantity

of cells necessary for a predictable outcome, variability of cell quantity that may be harvestedfrom patients and ambiguity regarding the actual quantity of viable cells that have been

implanted in the recipient site. Moreover, the lack of standardization and rigor associated with

the reports on bone-marrow-derived stem cell treatments have led to controversy and vocalopposition for the clinical merit of the procedure. Despite significant clinical and scientific

deficiencies of the procedure, aspirated BMSCs remain the ‘gold standard’, albeit somewhat

lusterless.

The research on induced pluripotent stem (iPS) cells has made progress over the last five

years. It is exciting that an individual’s epithelial cells could be secured, for example, andde-differentiated to stem cells and subsequently modulated to an osteogenic differentiation

pathway. Reports suggest iPS cells will differentiate into osteoblasts and produce bone

either by being cultured in differentiation media or by viral induction of the Runx2 gene[42,43]. With iPS cells, however, a significant drawback to their therapeutic potential is

their tendency to form teratomas, even after being differentiated down an osteogenic

pathway; in the future this may be solved by irradiation of the iPS cells or by a refinementof iPS production [44].

Adipose derived stem cells offer another source for autogenous MSCs. While the procedure tocollect the stem cells is more invasive than needed for iPS cells, the harvested tissue is one

that most patients would be more willing to endure. The adipose derived mesenchymal stem

cells (ADSCs) have the advantage of undergoing less processing steps before becoming oste-oblasts and e since they are already differentiated into a mesenchymal lineage e have less of

a risk of accidentally becoming non-osseous cells. Studies have reported that ADSCs can

undergo osteogenic differentiation, but do not produce mineralized matrix [45,46]. A moresophisticated understanding of ADSCs will be needed before this type of therapy advances into

the clinic.

Human umbilical cord derived MSCs (UCMSCs) are readily available, can be harvestedinexpensively with no donor site morbidity and can differentiate to osteoblasts [47].

UCMSCs may be harvested from the Wharton’s jelly post-partem. The UCMSCs expand easier

in culture than BMSCs [48] and have been increased 300-fold in culture without loss ofdifferentiation potential [49]. Furthermore, UCMSCs have the advantage of being immuno-

priviledged due to immunosuppressive isoforms of HLA, as well as the ability to suppress

splenocytes and T cells [50,51]. This biological phenomenon allows these UCMSCs to bematched with a wider range of tissue-types, providing allografts to patients with rare

immunotypes. Moreover, studies suggest hUCMSCs may differentiate into osteoblasts and

produce mineralized matrix, however, the outcome is less robust than that produced byhBMSCs [49,52,53].


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Biological therapies for bone tissue engineering

Earlier in this chapter we provided an overview of the fracture healing process that operated on

a macroscopic scale. The emphasis was placed on the ‘big-picture’ events at the bone defectsite. Therefore, we will now emphasize selective molecular cues that play a role in the osteo-

genic cascade. We will note the harmonized release of biologicals among multiple cell phe-

notypes that direct the osteogenic process and lead to bone regeneration

The dynamic fate of biological signaling molecules at a bone defect underscores the four-

dimensional nature of bone regeneration. Consensus abounds on the three spatial dimensions

(X, Y and Z) of volumetric bone regeneration’; however, there also exists the crucial temporalaspect. Consequently, we emphasize that bone regeneration must be viewed as a dynamic

four dimensional process. Biological signaling molecules function effectively for a limited

window of time to elicit an appropriate outcome on a dedicated target cell. Consequently, it isimportant to engineer the delivery of biological signaling agents in a bone regeneration

therapy with a precise understanding of their temporal pathways during natural bone regen-

eration [18].

The biological signaling agents we examine in this chapter can be broadly classified into the

following categories:

1) Pro-inflammatory cytokines,

2) Growth and differentiation factors, and

3) Angiogenic factors.

Pro-inflammatory cytokines are active following bone injury and establish and maintain the

initial destructive environment. Growth and differentiation factors function during the destructive

and constructive phases while angiogenic factors are focal points during the re-vascularizationof the injury site. The focus of this section will be on growth and differentiation factors along

with angiogenic factors. There are many growth factors that belong in this group and in an

effort to provide a more meaningful summary of the most likely factors that will beencountered in the clinic, we will down-select to specific factors based on known effects and

published data regarding the bone regenerative processes.

We begin with the destructive phase of fracture healing that is characterized by an acidotic,

hypoxic environment. The primary cell types in this environment are platelets, lymphocytes

and macrophages, which release fibroblast growth factor (FGF), transforming growth factor-b (TGF-b), and platelet-derived growth factor (PDGF).

Members of the FGF family are present in the wound site for up to three weeks, and as such,

have a broad range of activities [35]. Chief among these activities is the stimulation ofendothelial cell migration and subsequent angiogenesis. FGFs affect the migration and pro-

liferation of chondrocytes during the constructive phase of bone regeneration [54]. TGF-b growth factors functions as a mitogen for fibroblasts, marrow stem cells and pre-osteoblasts.

Both FGF and non-bone morphogenetic protein (BMP) members of the TGF-b super family of

proteins propel bone regeneration up to several weeks post injury.

PLATELET-DERIVED GROWTH FACTOR

PDGF is a dimeric growth factor composed of monomers linked by a disulphide linkage. Fourindividual chains (A, B, C, and D) combine to make five dimers (AA, AB, BB, CC and DD) of

which one dimer in particular e BB e plays a key role in bone regeneration [30]. During

embryogenesis, PDGF promotes formation and differentiation of somites, which are meso-dermal structures that eventually mature into precursors for bone, muscle and skin [55].

Following an injury to bone, PDGF is released from macrophages and the a-granules of

platelets and is a potent chemoattractant and mitogenic factor for cells of the mesenchymallineage. At the site of injury, PDGF will recruit fibroblasts, endothelial cells, osteoblasts as well


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as cells of the immune system. PDGF is active for the first 72 hours after injury and as such,is an exemplary candidate for delivery early in the regenerative process [35]. Additionally,

as a promoter of angiogenesis, PDGF plays a role in the re-vascularization of the bone defect

site [29].

PDGF is secreted by platelets, macrophages, osteoblasts and fibroblasts. In addition to its

mitogenic and chemoattractive properties, PDGF will increase osteoprotegerin expression invascular smooth muscle cells (an inhibitor of osteoclasts) and functions during embryonic

development [56]. As a modulator of early-stage wound and fracture healing, PDGF has

a significant impact on recruiting (i.e., functioning as a chemoattractant) cells to the fracturesite. As such, it is a strong candidate for delivery within 72 hours of bone injury.

PLATELET-RICH PLASMA

Platelet-rich plasma (PRP) provides an interesting alternative to the delivery of PDGF alone.

PRP consists of a centrifugated blood fraction that contains a concentration of platelets that isoften several fold greater than physiological platelet concentrations at wound sites [57]. This

concentrated solution is achieved through a multi-step centrifugation to separate plateletsfrom other cells found in blood. In the fracture healing model, the recruitment of immune

cells as well as cells of the mesenchymal lineage begin with the release of PDGF from platelets.

Consequently, the logic for the clinical application of PRP is that while one cannot preciselymimic the composition of the growth factors endogenously released by platelets in an

osseous wound, increasing the concentration of platelets at the site may augment the healing

process [58]. That is, there will be an increase in the presence of downstream growth factorssuch as Angiopoietin-2, endodermal growth factor (EGF), TGF-b1, FGF-2, and predominantly,

PDGF and Vascular endothelial growth factor (VEGF). It is generally accepted that many of

these factors are necessary for bone regeneration. Purportedly the administration of PRP willassure the appropriate healing milieu composition is available and suitable for the regener-

ative process [58]. Moreover, there is the argument that PRP is autogenous, patients, as their

own donor-recipient pair, consequently, will benefit. Regrettably, the notion of PRP effec-tiveness has been met with skepticism. An absence of rigorously designed scientific and clinical

studies and a proliferation of anecdotal ‘evidence’ have significantly diminished possible

benefits. Controversy over PRP has mitigated against clinical acceptance where successfuloutcomes seem to be segregated to localized clinical practices.

Nevertheless, given the strong biological rationale for the use of PRP in bone regeneration

therapies, it is somewhat surprising to see that its use has resulted in controversial results inliterature [57,58]. There are a few potential explanations for the range of reported results.

Firstly, the method of preparation of the PRP may differ in many of these studies, leading to

invalid comparisons among results. Another explanation for the discrepancies could be vari-ations in the administered dose of PRP. Like any ‘drug’, PRP functions optimally when present

within a certain therapeutic range [59] and studies report using different concentrations[60,61]. In spite of this controversy, PRP remains an intriguing option for growth factor-based

augmentation of bone regeneration, particularly when administered immediately following

injury.

VASCULAR ENDOTHELIAL GROWTH FACTOR

VEGF is angiogenic and includes agents of the angiopoietin pathway. Of these major angio-

genic regulators, VEGF is particularly important for bone regeneration [62]. As a major

regulator of both vasculogenesis (i.e., spontaneous formation of blood vessels) and angiogenesis(i.e., the sprouting of new blood vessels from existing vessels), VEGF plays an important role in

bone tissue regeneration [63]. Vasculogenesis, the de novo formation of vascular networks,

speaks to the similarities that exist between fracture healing and embryonic bone develop-ment. However, with respect to bone regeneration, the angiogenic capabilities of VEGF are of


1213

more interest in this application e that is, there is a heightened need for the creation of bloodvessels from pre-existing vasculature during bone regeneration.

VEGF is expressed in hypertrophic chondrocytes, but not in resting or proliferating ones [19].During osteogenic healing, the soft callus is invaded by blood vessels, resulting in the trans-

formation of a predominantly cartilaginous matrix into osseous tissue. This underscores

the importance of vascularization of the soft callus as it allows for it to be gradually replaced bywoven bone. With regards to temporal expression, VEGF is expressed predominantly 14 to

21 days following injury [35]. As such, it is a viable candidate for delivery to the bone wound

site during the early remodeling and mineralization phases of bone regeneration.

Upregulation and delivery of VEGF inhibitors prevent osteogenesis [64]. Additionally, the

inhibition of VEGF receptors impairs the healing of bone defects in mice [32]. VEGF has beenshown to function in a synergistic fashion with another group of proteins called the bone

morphogenetic proteins [31]. That is, VEGF alone does not promote bone regeneration but

rather, it acts in a coordinated way with BMPs to increase the recruitment of mesenchymalstem cells to the defect site and promote their differentiate into active osteoblasts. Studies

report that the dose of VEGF administered to the injury site needs to be finely tuned in

accordance with BMP delivery to avoid adverse effects [65]. With respect to promotingosteoinduction, VEGF is an excellent candidate for inclusion in porous scaffolds. Its local release

in a programmed profile titrated to bone regeneration may be an effective strategy in treating

gap bone defects [65].

BONE MORPHOGENETIC PROTEINS

No chapter on bone regeneration would be complete without inclusion of the BMP family.

Marshal Urist discovered bone morphogenetic proteins and determined that demineralizedbone matrix induced bone formation as a consequence of its endogenous BMP [66]. Urist’s

epochal work opened a therapeutic paradise; however, it is a paradise that includes peril for the

unwary. We will in this section, underscore the good, bad and ugly regarding the therapeuticapplications of recombinant human (rh)BMP.

While the actions of BMPs have been known for decades, its specific mechanisms of actionwere elucidated only just recently [67]. BMPs function by binding to type I and I serine/

threonine kinase receptors and activating intracellular signal transduction through SMAD

proteins 1, 5 and 8. These signals, when translocated into the nucleus, lead to an upregulationof osteoblast transcription factors such as RUNX2 (or core binding factor 1), COL1A1

(Collagen Type I), and OSX (Osterix) among others (see Fig. 55.4).

The most potent members of the BMP family involved in bone regeneration appear to be BMP-2, -4, and -7 homodimers [68,69]. BMPs are active ‘early’ in the osteogenic cascade. BMP-4 is

predominantly active from zero to five days following injury, with its peak closer to day 5.

BMP-2 is active throughout the entire bone regeneration process, culminating in the remod-eling of the woven bone to lamellar and Haversian bone and BMP-7 is active following day 14.

The cellular targets of BMPs include pluripotent mesenchymal cells, bone marrow cells, pre-osteoblasts, myoblasts, fibroblasts and neural cells. Key markers of osteoblast upregulation

include alkaline phosphatase, osteocalcin, osteopontin and osteonectin e the presence of

these markers can be used as indicators of cell differentiation from precursors to the osteo-blastic lineage [70]. The effects of BMPs on precursor cells are dose-dependent. It is well

documented that appropriate doses of BP can induce differentiation of precursor cells into

osteoblasts, as well as stimulate cartilage formation and alkaline phosphatase activity [33].However, ‘low’ BMP concentrations in vitro may promote differentiation into adipocytes. The

general effects of BMP on osteoblasts and periosteal cells, however, involve an increase in DNA

synthesis activity and the transcription of genes involved in the synthesis of bone matrixproteins.

FIGURE 55.4Key components of the BMP signaling pathway, ultimatelyleading to bone formation. The COL1A1 gene is just one of

many genes activated downstream of RUNX2. Other key

markers include Osterix, Alkaline Phosphatase, Osteocalcin,

Osteonectin and Bone Sialoprotein though the exact

mechanisms by which each of these becomes activated is not

yet understood.


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The evidence for the role BMPs in bone regeneration is overwhelming. There is a plethora ofexamples where BMP delivery to bone deficient sites promotes regeneration in a variety of

animal models [71e75]. Such overwhelming evidence inspired an industry to focus on clinicalapplications for BMP though perhaps the rush to the clinic affected judgment. Specifically, the

compelling clinical utility of BMP must be countered with caution for potential sinister out-

comes. For example, BMPs have been detected in osteosarcomas [76]. In osteosarcomas, thequestion is whether BMP expression is an effect or a cause of the tumor. Further, a concern with

rhBMP’s in the clinic has been the supraphysiological dose for clinical effect. The therapeutic

effectiveness of rhBMP requires milligram dosing. Consequently, off target effects have beenreported with supraphysiological dosing, including calcification of heart valves, heterotopic

bone formation, airway obstruction, neuropathies. These outcomes underscore the ineffec-

tiveness of the rhBMP delivery system as well as the potency of rhBMP to promote uncon-trolled clinical sequelae.

Biomaterial therapies for bone tissue engineering

One method for the precise, controlled delivery of biologicals and cells to a bone defect site is

through their sequestration within synthetic bone graft materials. Synthetic biomaterialsallow for the incorporation of cells and biological factors that will have release kinetic profiles

and dosing levels matching the physiological profiles of the osteogenic cascade. The following

is a focused summary of frequently used biomaterials for bone regeneration.

BIOACTIVE INORGANIC MATERIALS

Currently, there is a wide range of inorganic materials available, with a similar composition to

the inorganic matrix of bone, that have gained clinical interest. Among these materials arehydroxyapatite, tricalcium phosphate and bioactive glasses [77,78]. Bioactive inorganic ma-

terials, certain ceramics and bioglasses can undergo a time-dependent kinetic modification to

their surfaces [79,80]. However, the brittle nature of these materials cannot match bone and,therefore, they are not suitable for load-bearing applications.


1215

POLYMERS

Polymers, whether biological or synthetic, have also attracted clinical enthusiasm. The ability

to modify the physical, mechanical and compositional properties of polymers allows for

a design with specific clinical targets. Among the most commonly used biological polymericmaterials are collagen-based polymers and hyaluronic acid [81]. However, there are concerns

regarding the potential risk of disease transmission, immunogenicity, sourcing and relatively

weak mechanical properties of these materials [82]. Commonly used synthetic polymersinclude polycaprolactone (PCL), polyurethane, poly(lactic-co-glycolic acid) (PLGA), and

tyrosine-derived polycarbonates. These polymers may be processed using clever synthesis and

manufacturing techniques exploiting porogen leaching, phase separation, fiber meshingand microsphere sintering to make 3D-engineered constructs [82e85].

VISION FOR BONE REGENERATIONWhen designing practical therapeutics for bone regeneration, it is necessary to be cognizant of

the regulations set forth by the United States Food and Drug Administration (FDA). The design

and validation of therapeutics targeted for the clinic begins on a lab bench but ends withthe treatment of patients in hospital operating rooms. As such, we must anticipate regulatory

oversight.

However, this is a delicate art; components of the contemporary bone regenerative tool kit are

clearly not the answer, but radical new concepts often invoke vociferous and contrary re-

sponses from the FDA. In essence, we must strive to achieve a more effective treatment than iscurrently available but at the same time, be mindful of the eventual reaction from the FDA.

This section highlights some novel approaches to bone regeneration that we embrace as steps

in the right direction.

The approach for bone tissue regeneration should focus on the complex phenomenon of

wound healing, which requires a 4D structure (time, is the 4th element), competent bone-forming cells, and biological stimulants [114]. Contemporary approaches emphasize materials

that may deliver a signaling molecule in synchrony with the wound healing and bone

regeneration cascades [115]. The most commonly used medical device for recombinanthuman bone morphogenetic protein-2 (rhBMP-2) delivery is Medtronic’s InFuse�, which is

essentially a collagen sponge. InFuse� is currently FDA-approved for maxillary alveolar bone

augmentation (i.e., sinus lift procedures) and single level posterior lateral spinal fusion and isan example of a therapy that combines a growth factor-based approach with biomaterial

delivery. Another FDA-approved therapy is GEM21S�, which is a rhPDGF-BB and a beta-

tricalcium phosphate matrix. GEM21S� is FDA-approved for periodontal regenerativeprocedures. In contrast to a single growth factor approach, is a strategy to deliver multiple

biological cues to promote osteogenic differentiation as well as the incorporation of growth

factor binding peptides, proteins and glycosaminoglycans into scaffolds [82]. However, thedaunting challenge with this approach, in addition to achieving the appropriate temporal

release profiles, is a regulatory obstacle.

An alternative to the signaling molecule delivery approach is the acceleration of bone healingvia a construct made from extracellular matrix (ECM) components, which naturally deliver

osteoinductive proteins and signaling cues. Two promising strategies for accomplishing this

are to fabricate a scaffold from demineralized bone matrix [116] or to deliver in vitro syn-thesized ECM frommarrow stromal cells (MSCs), osteoblasts, or fibroblasts to the fracture site

[117]. Materials may be coated with ECM from MSCs or with an adhesive peptide such as

fibronectin to promote osteogenic differentiation upon implantation [117,118].

A unique approach to bone regeneration is the use of microparticles to deliver DNA plasmids

or siRNAs into cells to induce (or prevent) osteogenic differentiation. Just as upregulatingRUNX2 and OSX e two transcriptional regulators for bone formation emay incite osteoblast


1216

lineage progression and in turn, bone formation, silencing RUNX2 and OSX may have theopposite effect. Therefore, siRNAs have been used against these two transcription factors to

prevent ectopic ossification [119e121]. Amore relevant tactic for promoting bone regeneration

is delivering siRNAs against known inhibitors of osteogenic differentiation such as TWIST1.Alternative pathways for promoting bone regeneration are parathyroid hormone (PTH) 1e34

amino acid sequence and BMP-2 signaling. However the advantage of delivering siRNAsand plasmids over other signaling molecules is their stability and a lower risk of eliciting off

target effects.

Gene therapy, which was originally proposed for correction of genetic defects, has beenreported to induce the expression of molecules that can promote a regenerative response

[122]. The most common gene therapies use vectors to enhance the expression of a particular

gene [123]. Viruses have been explored as gene delivery vectors, with retroviruses, adenovirus,lentiviruses and adeno-associated viruses being the most promising vectors [124e128].

However, the immunogenic potential of these vectors, as well as the risk of dysregulating

normal gene function has kept them from advancing to the clinic [129,130]. Regardless, thenatural abilities of viruses to bypass cellular defenses and modify host DNA make them a very

powerful tool for guiding the fate of cells at the defect site.

Hydrogels, another bone regeneration therapy option, can be used to deliver cells with the

potential to differentiate into bone or vasculature. In order to prepare these implants, cells

are seeded in hydrogel constructs and cultured in bioreactors. After the appropriate matu-ration period, the construct is implanted into a bone defect. Natural hydrogels, such as

collagen and hyaluronic acid, have the intrinsic physical and biological characteristics of

ECM; they are able to direct cellular function and interact with osteoprogenitors at theimplant site [131]. However, the limitations of working with natural hydrogels include dif-

ficulty in processing and tailoring them for specific purposes. Additionally, hydrogel

degradation products may invoke an immune response. On the other hand, synthetic ma-terials, such as poly-N-isopropylacrylamide (poly-NIPAM), can be manufactured into

hydrogels with consistent, reproducible properties. The downside of synthetic hydrogels,

however, is minimal cell interaction, and the requirement of tailoring them to direct cellularfunction [131].

CONCLUSIONBone is complex tissue-organ system that presents many challenges in the quest to augment its

regenerative capabilities. In spite of its natural ability to regenerate, there are limitations to the

endogenous regenerative potential. We must harness the intrinsic regenerative capacity ofbone and extend it to bone insufficiencies that do not spontaneously regenerate. At this point,

we have begun to slowly elucidate the molecular-level biological signals involved in bone

regeneration. However, in order to advance bone regeneration therapeutics with twenty-firstcentury science, there is a crucial need to aim for more ambitious, radical lines of research. The

status quo of autografts, allografts and xenografts are no longer adequate and necessitate the

search for transforming alternatives. The majority of current research into bone therapeuticsfocuses on individual cell-, growth factor- and materials-based approaches that are rather

pedestrian and there is a need for radical, innovative strategies.

We offer some starting points: namely, to design therapeutics that work synergistically with

natural bone regenerative mechanisms. A key component of this approach involves mini-

mizing the interference with these processes that often occurs as a result of implants lackingbiocompatibility. Both the implant materials and its degradation products must not extend the

destructive phase of bone regeneration. At the same time, it must support the physiological

loads associated with bone tissue, though there is a fine threshold for achieving this. Theimplant must degrade in a coordinated temporal manner with the formation of new bone at


the defect site. Finally, the implant must be osteoconductive, osteoinductive and osteogenic toenhance bone regeneration at the defect site.

In conclusion, in this chapter we highlighted the advantages and disadvantages of contem-porary bone regenerative methods. We discussed the importance of the fundamentals of basic

osteobiology as a requisite road map for the design and development of bone therapies. We

provided our vision for bone regeneration and identified clinical performance criteria thatmust be defined and met. Finally, we shared some visionary approaches to bone regeneration.

It is our hope that this chapter provides some guidance towards improving the regeneration

capabilities of the structural tour de force that is bone.

AcknowledgmentsWe would like to acknowledge members of the Bone Tissue Engineering Center at Carnegie Mellon University,specifically April Watt, Eric Hsu, and Amy Donovan for contributing Figures 1, 2 and 3.

1217

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