Cranial bone defects: current and future strategies

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  • Institute of Reconstructive Plastic Surgery, New York University Langone Medical Center, New York, New York
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Bony defects in the craniomaxillofacial skeleton remain a major and challenging health concern. Surgeons have been trying for centuries to restore functionality and aesthetic appearance using autografts, allografts, and even xenografts without entirely satisfactory results. As a result, physicians, scientists, and engineers have been trying for the past few decades to develop new techniques to improve bone growth and bone healing. In this review, the authors summarize the advantages and limitations of current animal models; describe current materials used as scaffolds, cell-based, and protein-based therapies; and lastly highlight areas for future investigation. The purpose of this review is to highlight the major scaffold-, cell-, and protein-based preclinical tools that are currently being developed to repair cranial defects.

Abbreviations used in this paper: ADMSC = adipose-derived mesenchymal stem cells; BCP = biphasic calcium phosphate; BMMSC = bone marrow–derived mesenchymal stem cell; BMP = bone morphogenetic protein; HA = hydroxyapatite; OCP = octacalcium phosphate; rhBMP = recombinant human BMP; TCP = tricalcium phosphate; VEGF = vascular endothelial growth factor.

Bony defects in the craniomaxillofacial skeleton remain a major and challenging health concern. Surgeons have been trying for centuries to restore functionality and aesthetic appearance using autografts, allografts, and even xenografts without entirely satisfactory results. As a result, physicians, scientists, and engineers have been trying for the past few decades to develop new techniques to improve bone growth and bone healing. In this review, the authors summarize the advantages and limitations of current animal models; describe current materials used as scaffolds, cell-based, and protein-based therapies; and lastly highlight areas for future investigation. The purpose of this review is to highlight the major scaffold-, cell-, and protein-based preclinical tools that are currently being developed to repair cranial defects.

Abbreviations used in this paper: ADMSC = adipose-derived mesenchymal stem cells; BCP = biphasic calcium phosphate; BMMSC = bone marrow–derived mesenchymal stem cell; BMP = bone morphogenetic protein; HA = hydroxyapatite; OCP = octacalcium phosphate; rhBMP = recombinant human BMP; TCP = tricalcium phosphate; VEGF = vascular endothelial growth factor.

Bony defects in the craniomaxillofacial skeleton can occur as a result of congenital defects (for example, in 2001, 37,732 children underwent surgery to repair birth defects) or acquired injuries (for example, in 2001, 24,298 patients required maxillofacial surgery for injuries to the face and jaw) (www.surgeryencyclopedia.com). Regardless of their cause, bony defects are functionally debilitating, socially incapacitating, and biomedically and economically burdensome.

The war on terrorism has brought a myriad of new challenges to maxillofacial surgeons, plastic surgeons, and neurosurgeons: combat-associated craniomaxillofacial injuries. Wars in Iraq and Afghanistan have resulted in the greatest incidence of head trauma since the Vietnam conflict. Increased survival because of body armor and advanced battlefield medicine, as well as the increased use of explosive devices, has contributed to the increased incidence of craniomaxillofacial combat injuries. Patients once not considered amenable to reconstructive surgery are now being aggressively treated and are surviving devastating head trauma. This unique patient population ultimately requires reconstruction of the cranial skeleton for protection of the brain as well as aesthetic and functional restoration of the calvaria or the bones of the face. These patients require countless procedures and are often left with poor aesthetic and functional results.

It is now well known that successful spontaneous calvarial reossification only occurs in infants younger than 2 years of age.27 Thus, a variety of materials and methods have been proposed to restore such defects including autogenous bone grafts and allogeneic banked bone, demineralized matrix pastes, ceramic scaffolds, and even synthetic materials and bone substitutes such as calcium ceramics. More recently, cell-based alternatives and BMPs have also been used.26 The multitude of methods reflects both the inadequacy of each technique, as well as the pressing need to adequately reconstruct the skeleton. While each method may achieve craniofacial reconstruction, each possesses inherent limitations, such as donor-site morbidity, an obligatory graft resorption phase, contour irregularities, insufficient autogenous resources, disease transmission, graft-versus-host disease, immunosuppression, structural failure, and foreign body infection. These limitations preclude most large defects from being repaired with these materials. Therefore, the need for new and improved treatment options is urgent.

To solve these issues, scientists, physicians, and engineers are collaborating to design new tissues to repair cranial defects. Because bone formation is an intricate and dynamic process, an interdisciplinary team effort is a requirement for the generation of functional tissue. Many strategies have been employed including cell-based therapies, cellular and acellular scaffolds, recombinant gene therapy, and topical small molecule therapies among others. Although bone-tissue engineering for the treatment of cranial defects is a multistep and multicomponent process, for clarity we will separately highlight every major tool that is currently being developed to solve common craniofacial problems.

In Vivo Animal Models Of Cranial Defects

The in vivo regeneration of bone for healing critical-size calvarial defects is an ongoing area of active research. Physicians and scientists are generating novel methods and combination treatments for addressing cranial defects, and these strategies abound in the literature. Animal models have the limitations of recreating only limited cranial defects but are our best option currently available. We will first focus on describing small-animal models and then shortly describe larger-animal models that are used less frequently (Table 1).

TABLE 1:

Characteristics of currently used animal models

Animal ModelSize of Critical DefectsProsCons
mouse5 mmsmall size, easy maintenance, low costsmall size, poor surgical precision
rat8 mmslightly larger than mouse: still relatively affordable w/ more surgical precisionsimilar to mouse model but more expensive
rabbit15 mmnot much larger than rat but has larger critical defect so more surgical controlmore expensive than rats; few benefits other than larger defect size
monkey15 mmclosely related to humans so most translational modelexpensive, ethical concerns
canine20 mmlarger defect size for more surgical precision & controlexpensive, ethical concerns
sheep22 mmlarger defect size for more surgical precision & controlexpensive, ethical concerns

Small-Animal Models

Mouse

The mouse is currently the most commonly used animal model in basic science research owing to its ease of maintenance and relatively low cost. Additionally, sophisticated molecular and cellular biology analytical tools are readily available and yield highly reproducible results.1 In the mouse, a critical-size cranial defect is defined as a bony deficit greater than or equal to 5 mm.63,67,80 Such a defect, if left untreated, will not heal in the life of an animal.102 Generally, a critical-size defect in the mouse calvaria is created by using a trephine that makes a circular defect in the cranial skeleton16 (Fig. 1). The challenge in using this particular model is the small size of the mouse. Because of the intimate relationship of the cerebral cortex, the dura mater, and the calvaria, a durasparing craniotomy is technically challenging. Sparing the dura mater is in fact critically important since several studies have shown that the dura is instrumental in bony regeneration of the skull.1,35,117

Fig. 1.
Fig. 1.

Mice calvarial defect model. A: A 3-mm-diameter round dental bur was used to create a 3-mm (noncritical) calvarial defect on the left parietal bone in a wild-type mouse. The underlying dura mater was kept intact. B: A 3D micro-CT scan of a calvarial defect immediately following wound creation, showing the perfect bone defect.

As previously noted, humans up to 2 years of age have the capacity to spontaneously heal cranial defects that would be “critical” in an adult human. In 2003, Aalami et al.1 reported similar findings in a mouse model of juvenile and adult cranial defect healing: in the juvenile mice (6 days old) a significantly greater portion of critical-size calvarial defects was healed than in the adults (60 days old). The authors were particularly careful in sparing the dura mater in these procedures. Prior studies have shown that although juvenile animals have the capacity to spontaneously heal critical-size defects, this ability is negated if the dura mater is compromised.17 Further support of the observation that the dura mater is influential in bony healing is the finding that critical-size bony defects in adult guinea pigs can be rescued by allotransplantation of juvenile guinea pig dura mater to the adult defect.41 To preserve the dura mater, craniotomies in mice should to be performed with the aid of a dissecting microscope. The other option, of course, is to use a larger-animal model such as the rat.

Rat

The rat is another commonly used animal model for studying critical-size defects. In the rat, a critical-size cranial defect is defined as a bony deficit greater than or equal to 8 mm.103 In 1982, Takagi and Urist110 determined that 8-mm-diameter cranial defects in Sprague-Dawley rats healed to 5 mm by 4 weeks and by 12 weeks no further healing was noted; however, the first to examine critical-sized defects in the rat were Freeman and Turnbull30 10 years earlier.113 Benefits of the rat over a smaller animal to study cranial defects are its larger size and the consequence that one is thereby permitted to perform technically sound craniotomies.

Larger-Animal Models

Larger-animal models have also been used, and there are pros and cons to using these animals. On the one hand, the primary benefit is the greater control and precision that the larger size allows intraoperatively. Larger animals such as the rabbit, guinea pig, dog, sheep, and monkey provide the opportunity for more control in the surgical area. On the other hand, these animals are more expensive to purchase and maintain, and they also take up a lot of space.

The first studies of critical-size cranial defects were conducted in 1899 by Berezowsky,8 who reported that the presence of the dura mater in rabbits is required for calvarial bony healing to occur. In rabbits, the critical-size defect is a bony deficit greater than or equal to 15 mm.28,29 Other larger animals have been described in the literature as models for studying critical-size defects. Dogs, for example, have been frequently used to study calvarial defects.31,97,115 Canine critical-size calvarial defects are 20 mm in diameter.19 More recently, sheep have been used to study critical-size defects (22 mm)116 as have other nonhuman primates such as monkeys (15 mm).43 Clearly, many different animals have been used as model systems for studying critical-size defects of the cranial skeleton. The animal chosen by a research team must suit their needs and be both ethically acceptable and economically feasible to purchase and maintain.

Materials Used for Defect Repair

Ever since critical-size defects were first described and the limitations of endogenous healing were understood, physicians have come to the conclusion that novel strategies should be developed to enhance bony healing. Hence, scaffolds have been one of the main targets of ongoing research. The idea has been that a biocompatible and biodegradable matrix inserted into a cranial defect will support, guide, and enhance bone healing. After the failed use of empty scaffolds,94 people have been trying to improve the efficacy of the scaffold by using it as a delivery system or a carrier for cells and/or growth factors. To facilitate the delivery of cells, drugs, and extracellular matrix material to a localized bony defect, an appropriate carrier device is often required, and several different materials have been proposed (Table 2). The ideal scaffold will have to be biocompatible and osteocompatible and allow for osteogenesis, osteoinduction, and osteoconduction (Fig. 2). In addition, the scaffold must demonstrate mechanical strength and resilience, and its degradation products must not trigger any inflammatory reaction.

TABLE 2:

Characteristics of preferred scaffolds for bone-tissue engineering purposes

ScaffoldCompositionProsConsResorption RateRelease MineralsStiff or Moldable
HACa10(PO4)6(OH)2native inorganic bone matrixbrittle & resorbs slowlyvery slow (takes yrs)yesstiff
β-TCPCa3(PO4)2native bone calcification mineralssignificantly less bone laid down than β-TCP resorbedvery slow (>3 yrs)yesstiff
BCPvariablecombination of native bone matrix & mineralsmust vary amount of HA & β-TCP to generate useful scaffoldrapid (wks)yesstiff
calcium sulfateCaSO4·2H2Ohighly moldable & easily shapednot native bone materialrapid (4–12 wks)yesmoldable
OCPCa8H2(PO4)6.5H2Ocomposed of native bone minerals, osteoinductivenot as much known about material as traditional calcium phosphateslow (6 mos to over 1 yr)yesstiff
advanced nanomaterialsvariablecustomizable & can be generated w/ desired characteristicsnew & underdeveloped technologyvariablenomoldable
Fig. 2.
Fig. 2.

Bone-tissue engineering: properties and characteristics to consider for the design of a scaffold. The ideal scaffold will have to be biocompatible and osteocompatible (to be inserted into a cranial defect to support, guide, and enhance bone healing) and allow for osteogenesis, osteoinduction, and osteoconduction. In addition, the scaffold must demonstrate mechanical strength and resilience and its degradation products must not trigger an inflammatory reaction. The surrounding environment and the seeding of cells onto the scaffold play a major role for bone growth as well.

Many new polymers and synthetic materials have been developed in recent years and are being tested as scaffolds. However, all current scaffolds have in common the properties of being both biocompatible and resorbable.111 In addition to the multitude of new compounds and polymers currently developed, for many years people have been using calcium phosphate–or calcium carbonate–based scaffolds because of their similar composition to bone.

Calcium Phosphate Scaffolds

Beta-Tricalcium Phosphate

Beta-tricalcium phosphate is one of the earliest compounds to be used as a scaffold for osseous regeneration.81 It has a compressive and tensile strength that is nearly equivalent to cancellous bone,49 and this makes it an attractive compound for use as a scaffold material. As early as 1920, there were reports that β-TCP, when injected into the gap of a segmental bony defect, increased bone union.5 In research models, β-TCP has been used as an empty scaffold to promote bone healing in rats40 and has also been shown to promote bone regeneration in cranial defects of canines when the scaffold is seeded with bone marrow–derived stromal cells.114 Additionally, investigators have used a composite scaffold composed of β-TCP, collagen, and autologous bone fragments fixated with fibrin glue to correct cranial defects in canines.60 Although β-TCP is replaced by bone in vivo, replacement does not occur in a predictable 1:1 ratio. In other words, as β-TCP is resorbed, less new bone is laid down than β-TCP is resorbed.42 This has limited its clinical applicability up to this point.

Hydroxyapatite

Intuitively, the ideal biomaterial should be a biomimetic reproduction of the matrix of native bone. The inorganic element of the natural bone matrix consists of mainly crystalline mineral salts in the form of HA. In addition to providing the raw materials for mineralized matrix formation, HA has a nanoscale topography that promotes cellular adhesion, differentiation, growth, and proliferation. For these reasons, many groups have advocated the use of HA scaffolds in regenerating bone both in vivo and in vitro. To prevent ischemia and allow for vascularization of the scaffold and proper seeding of cells within it, the scaffold needs to have pores that have a relative diameter of about 100–150 μm.46 The geometry and porosity of HA scaffolds have also been shown to be important in the capacity of these scaffolds to heal cranial defects in rabbits.25 One downside to this scaffold material is that it tends to be brittle, not easily molded, and its resorption rate is also very slow. To enhance its usability as a scaffold material, people have added polymers such as Pluronic F-127 to HA to increase its moldability.15,125 In 1997, Lew et al.69 demonstrated that empty HA scaffolds could promote osteoconductive bone healing in canine calvarial defects. Additionally, by coating a zirconium porous scaffold with an outer layer of highly porous HA, calvarial defects in rabbits have been demonstrated to exhibit up to 50% new bone formation at 12 weeks.58

Biphasic Calcium Phosphates

Biphasic calcium phosphates are mixtures of β-TCP and HA of varying amounts.95 As a BCP scaffold is resorbed in vivo, it releases calcium and phosphate ions into the microenvironment of the implant, and these ions can then be used to build bone de novo.104 Although there have not been much published data on using BCP scaffolds to heal critical-size cranial defects, the scaffolds have been used to successfully aid in bone regeneration in mandibular defects in canines48,105 and iliac wing defects in goats.64 Additionally, a BCP scaffold (60% HA plus 40% β-TCP) seeded with rhBMP-2 was used to demonstrate complete spinal fusion in a nonhuman primate model.101 In our laboratory, we can fabricate HA and TCP scaffolds using a custom-designed 3D microprinting process (Fig. 3). We have demonstrated that these BCP scaffolds, when implanted into a critical-size alveolar defect in rats either empty or seeded with rhBMP-2, are capable of inducing new bone formation.84 Further experiments using HA-TCP scaffolds are being pursued in our laboratory.

Fig. 3.
Fig. 3.

Scaffold printing system. The 3D microprinting process is fully customized (diameter, pore size, strut size, and porosity) and can produce novel scaffolds in less than 3 hours. A: The 3D microprinting processor. B: Several HA-TCP sample scaffolds. C: The microarchitecture of a customized scaffold.

Octacalcium Phosphate

Octacalcium phosphate is a biological precursor of apatite crystals11 and has been shown to promote differentiation and maturation of osteoblasts in vitro.6,72,108 Studies have also shown that OCP, compared with HA or β-TCP, has the capacity to increase attachment of osteoblasts to the scaffold.32 Furthermore, OCP/collagen scaffolds have been demonstrated to direct improved bone regeneration in rat calvarial defects compared with HA or β-TCP alone or HA/collagen or β-TCP/collagen scaffolds.54 An additional benefit of OCP scaffolds is that they are osteoinductive, which is not an attribute of HA or β-TCP.37,42

Calcium Sulfate Scaffolds

Calcium sulfate, also known as plaster of Paris, is thought to act as an osteoconductive substrate for the invasion of blood vessels and associated osteogenic cells; for this to occur, it is imperative that the calcium sulfate scaffold be in intimate contact with viable endosteum or periosteum.16 Additionally, calcium sulfate scaffolds serve as an excellent site of adherence for osteoblasts106 and cause no appreciable inflammation on implantation.93 In vivo studies have been conducted in rat calvarial defect models and demonstrate that calcium sulfate, when combined with allogeneic bone matrix, promotes bone formation.98 For more information about calcium sulfate scaffolds please see review article by Pietrzak and Ronk.96

Advanced Scaffold Materials

The recent advent of nanotechnology and advances in polymer chemistry have led to the development of many novel and ingenious scaffold materials. Osathanon et al. recently described the use of a nanofibrous fibrin scaffold for bony regeneration in a mouse calvarial defect model.89 This scaffold served to immobilize enzymatically active alkaline phosphatase, which promoted bone formation by increasing the amount of biologically available inorganic phosphate. In another recently published article the authors used PuraMatrix (a self-assembling peptide nanomaterial) seeded with either mesenchymal stem cells or platelet-rich plasma to aid in bone regeneration.123 This scaffold has the added benefit of being easily molded and readily shaped to conform to any bony defect.61 Electrospun silk fibrin scaffolds can also be used for bone regeneration because the nanofibers of the scaffold function like the native bone extracellular matrix.70,120 This electrospun silk fibrin scaffold promotes osteoblast proliferation and increases osteoblastic alkaline phosphatase activity.90 Many scaffold materials have been used as well: polyethylene glycol,53,78,111 nano-HA membranes,124 poly β-amino esters,10 injectable chitosan gel, minimally invasive tissue-engineered bone,107 and others. An exhaustive evaluation of current and future scaffold materials is available in the literature and is beyond the scope of this article.

Materials for Pediatric Use

Importantly, titanium implants, alloplastic, and inorganic scaffold materials such as those mentioned above are not the most appropriate options for the pediatric or adolescent patient.51 Because these patients require further ossification of the cranial vault, autogenous and allogeneic banked bone are better options because of their capacity for osteointegration and growth with the pediatric skull. Despite this general consensus among neurosurgeons, plastic surgeons, and maxillofacial surgeons, successful treatment of pediatric patients using hybrid alloplastic materials has been achieved.7

Cell-Based Strategies

In addition to a proper scaffold for recruitment of cells that promote bone growth, it may be necessary to deliver cells to a bony defect to achieve proper regeneration. While tremendous progress has been made in the design, engineering, and manufacturing of construct technology, scientists are still struggling to find appropriate cell sources. Those cell-based strategies that use a scaffold material may come in 2 varieties: 1) scaffolds preseeded with cells and then implanted or 2) acellular scaffolds that require in vivo recruitment of autologous cells.122 Acellular scaffolds may also be designed to recruit cells from nearby or distant sources.39 Moreover cells may be used in bony regeneration without the use of a scaffold.

Many different cell types may be used. In this section we will discuss the variety of cell sources as well as their current use and success in generating neovascularized craniofacial constructs.

Bone Marrow–Derived Mesenchymal Stem Cells

Bone marrow–derived mesenchymal stem cells are multipotent cells that have the capacity to differentiate into many mesodermally derived cell types.21 They can be easily differentiated in vitro into osteoblasts. Bone marrow–derived mesenchymal stem cells have mainly been used in 2 different ways: either seeded on a scaffold after in vitro osteogenic differentiation or seeded directly as uninduced BMMSCs on a scaffold. In the latter case, to make BMMSCs more potent promoters of bone growth, they may be coadministered with osteogenic cytokines such as BMP-2 and FGF-2.76 Treatment of BMMSCs with BMPs has been demonstrated to help heal critical-size defects in the rat44,45 (77.45% healed in 8 weeks)21 as well as in the rabbit14 (“near complete repair” at 3 months). Furthermore, when treating cranial defects in rats, naive BMMSCs are more potent inducers of osteogenesis than platelet-rich plasma.57 One of the limitations of using BMMSCs to promote bone growth is that they have a limited life span. Nakahara et al. demonstrated that bone repair is enhanced by delivering BMMSCs to the defect that has been transfected with human telomerase reverse transcriptase, thus immortalizing the BMMSC cell population.83 That being said, BMMSC populations are of limited quantity, and therefore it would be beneficial to have a source of adult stem cells that is more extensive. The recent discovery of ADMSCs provides a promising contender.

Adipose-Derived Mesenchymal Stem Cells

Adipose-derived mesenchymal stem cells are multipotent cells that can differentiate into numerous cell types including osteogenic cell types19 (Fig. 4); the apparent availability of these cells makes them a potent cell source for bone-tissue engineering. This cell type, with its multipotent differentiating capacity, was characterized in 2001 by Zuk et al.,126 and the use of ADMSCs to treat calvarial defects was first studied by Cowan et al.18 3 years later. Cowan et al. showed that implanted poly(lactic-coglycolic acid) scaffolds seeded with ADMSCs promoted complete bone bridging in 12 weeks in a rat model of calvarial defects and also that the ADMSCs contributed 84–99% of the newly formed bone as determined by chromosomal detection. Although naive ADMSCs may have some therapeutic benefit when transplanted into a bony defect,50 preimplantation osteoinduction seems to provide more benefit.22,24,123 When critical-size cranial defects were made in canines, Liu et al.71 demonstrated that osteoinduced ADMSCs successfully repaired the defect when seeded on coral scaffolds, and Cui et al.19 confirmed that this reossification remains present at 6 months postimplantation.57 Osteoinduced ADMSCs have also been shown to repair critical-size defects in rabbits when seeded on polylactic acid scaffolds.50 Future research into the applicability of ADMSCs for regeneration of osseous structures will undoubtedly yield important clinical insights.

Fig. 4.
Fig. 4.

Photomicrographs showing human mesenchymal stem cell multilineage differentiation. Under the appropriate stimuli, mesenchymal stem cells can differentiate into various cell types: undifferentiated (A; alkaline phosphatase, original magnification × 400), bone nodule (B; von Kossa, original magnification × 400), endothelial tubule (C; fluorescent GFAP, original magnification × 200), cartilage (D; Alcian blue, original magnification × 400), adipocyte (E; Oil Red O, original magnification × 200), neuron (F; GFAP, original magnification × 400).

Chondrocytes

Most of the bony skeleton forms by endochondral ossification. Briefly, this involves the proliferation and hypertrophy of chondrocytes followed by apoptosis and replacement with osteoblasts. Although calvarial bone is formed by intramembranous bone formation that is independent of chondrocytes, it appears that chondrocytes may actually be able to help heal critical-size defects of the calvaria.23,52,79,87,88 Just this year, Doan et al.23 demonstrated that chondrocytes, when implanted directly into a critical-size cranial defect in mice, heal the defect by 6 weeks postimplantation. Montufar-Solis et al.79 had first made this observation about chondrocytes in healing mouse cranial defects 6 years earlier.

Recombinant Protein–Based Strategies

Recombinant protein–based strategies for improving bone healing are based on the premise that augmenting endogenously produced cytokines will enhance bone regeneration.27 Although the use of many different recombinant proteins such as fibroblast growth factor, activin, BMP, and VEGF for improved healing of bony defects in vivo has been demonstrated,55,56,100,118,121 we will focus on the osteogenic BMPs and angiogenic VEGF. These 2 morphogens can be delivered to a calvarial defect by preseeding one of the previously discussed scaffolds with the recombinant protein. This allows for concentrated local delivery of the protein and avoids systemic administration.

Bone Morphogenetic Proteins

Bone morphogenetic proteins come in many different isoforms that play many dynamic roles in patterning of the embryo, bone growth and formation, and other processes (Table 3). Endogenous BMPs are typically found in the human body at a concentration of 2 mg/kg of cortical bone.109 Thanks to the isolation and cloning of the BMPs in 1981,99 recombinant human proteins can be made in high yield by transfecting Escherichia coli cells with human genes and utilizing the bacterial cellular machinery to produce the protein of interest in large quantities.66 Bone morphogenetic protein–2, –4, and –7 have all been shown to induce bone formation in vivo, and Hyun et al.47 have demonstrated this in a model of rat calvarial defects. Bone morphogenetic proteins have been shown to be strongly osteoinductive and can be delivered to a wound bed in numberous ways. Murine stromal cells were transfected by a retrovirus carrying a BMP-4 transgene, and these cells were then embedded in a gelatinous matrix and placed into a critical-size defect in a rat model;36 this treatment completely healed the defect by 4 weeks. Additionally, a chitosan gel matrix loaded with BMP-2 and placed into a rat cranial defect was shown to promote bone regeneration.13 Finally, rats with critical-size calvarial defects have been completely healed by utilizing collagen or β-TCP scaffolds embedded with rhBMP-4.3 It is important to note that the use of BMPs in bone regeneration has been demonstrated in noncranial spinal defects in humans. Two studies have demonstrated that bilateral treatment of the posterolateral spine with rhBMP-2 resulted in complete spinal fusion in 100% of patients receiving the treatment.9,73 The action of BMPs in vivo can even be enhanced by coadministration of NELL-1,2 COX-2 inhibitors,65 treatment with simvastatin,86 or a combination of simvastatin and α-TCP.85 Last, it is important to clarify that in Europe, rhBMP-2 is commercially available for the treatment of acute open tibial fractures (InductOS implant kit, Medtronic Sofamor Danek and Wyeth Pharmaceuticals) as a lyophilized powder, dissolved in sterile water, and applied to an absorbable collagen matrix made of Type 1 bovine collagen. A similar kit (InFuse bone graft, Medtronic Sofamor Danek) is available in Europe and in the US for the treatment of degenerative lumbar disc disease.110

TABLE 3:

Bone morphogenetic proteins grouped by amino acid sequence homology*

Group 1Group 2Group 3Group 4Group 5Group 6
BMP-2BMP-5GDF-5BMP-9GDF-10 (BMP-3b)BMP-15
BMP-4BMP-6GDF-6 (BMP-13)BMP-10BMP-3
BMP-7 (OP-1)GDF-7 (BMP-12)
BMP-8 (OP-2)

* The human genome codes for more then 20 BMPs and with the exception of BMP-1, which was mistakenly classified as a BMP, these low-molecular-weight glycoproteins belong to the transforming growth factor–beta family. The BMP family has been grouped into subsets based on amino acid sequence homology. Abbreviations: GDF = growth differentiation factor; OP = osteogenic protein.

† BMP-2, BMP-4, and BMP-7 have all been shown to induce bone formation in vivo.

Neovascularization Strategies

Numerous groups have shown that the combination of artificial scaffolds and osteoprogenitor cells can lead to the formation of new bone,12,33,75 but the clinical applicability of this technique is still questionable. The limited clinical success may be explained by a lack of vascularization. Vascularization is vital for the survival of the implanted cells on a carrier material after implantation, and apart from that, vascularization is a critical process during bone growth and repair. Studies have shown that fracture healing and ectopic bone formation can be blocked by the administration of angiogenesis inhibitors.38,82 Other investigators have demonstrated that new bone formation in porous scaffolds was significantly increased by the insertion of a vascular pedicle in the scaffold4,59 and that endothelial cells form vascular structures in vitro,20,34,102 which can anastomose to the vasculature of the host after implantation.68,112

Thus, promoting vasculogenesis is an important aspect of promoting bone growth when attempting to correct calvarial defects. Briefly, this can be accomplished in 1 of 3 ways: 1) impregnating a scaffold with VEGF or another angiogenesis-promoting substance as previously discussed, 2) seeding endothelial cells or other vasculogenic cells onto a scaffold, or 3) implanting scaffolds into highly vascular tissues, waiting for vessel ingrowth to occur, and then transplanting the vascularized scaffold to the bony defect. Vascular endothelial growth factor is a very potent angiogenic morphogen.122 Incorporation of VEGF into a scaffold material has been shown to induce angiogenesis and promote bone formation.62 In mice, this is especially true when the VEGF is released slowly over time through the use of a calcium phosphate scaffold (in one study, bone healing occurred at a rate 1.56 times as fast119). Additionally, a combination therapy of VEGF and BMPs seems to have a synergistic effect on bone formation during the first few weeks of treatment: critical-size defects made in rat calvaria have been rescued by coadministration of VEGF/BMP-2 embedded in a gelatinous matrix. The 2 compounds acted synergistically to promote bone formation at 4 weeks postimplantation but exhibited little more effect than BMP-2 alone at 12 weeks.92 As for the implantation of a scaffold into a highly vascularized tissue, a site of choice may be the abdominal mesentery117 or any tissue with a rich arterial supply and venous drainage.74 Briefly, this technique involves implantation of a scaffold into a highly vascularized tissue, waiting for the scaffold to become vascularized, removing the scaffold, and replanting the scaffold into the cranial defect. Future research into promoting neovascularization in bony defects will yield important methods by which we can improve osteogenesis and bone regeneration.

Discussion

After many years of research on bone-tissue engineering and after numerous attempts to develop an ideal scaffold including the appropriate combination of bone growth factors, we have yet to find the answer. A major limitation is the current availability of cranial defect models. While large models may seem more intuitive, technically, economically, and even ethically, smaller models such as the mouse or the rat are currently more feasible and are thus used more commonly. Nevertheless, there will always be a translational issue concerning the application of results seen in an animal model to the human body.

There is widespread agreement that the ideal solution would be to find a way to enhance endogenous bone healing in a patient without the use of an external device like a scaffold. Because our technology has not gotten to that point yet, however, scientists have focused their research on developing biocompatible and osteocompatible scaffolds capable of osteoinduction and osteoconduction. These scaffolds have ranged broadly in their composition from synthetic to natural, and all of them have had different properties, benefits, and flaws. Because its structure is so close to actual bone, HA-TCP has been one of the most frequently investigated materials. It has undeniable advantages such as its composition and its capacity to be both osteoconductive and osteoinductive when in the presence of osteogenic cells (www.surgeryencyclopedia.com);77 however, it is rarely osteoinductive when implanted on its own.91 The major downside to using HA-TCP scaffolds is that they are quite brittle and resorb rather slowly. As previously mentioned, OCP scaffolds are being generated as a potential scaffold with similar characteristics to HA-TCP, but they have the added benefits of being both osteoconductive and osteoinductive. It seems that OCP scaffolds are superior to HA-TCP in their capacity to serve as an attachment site for osteoblasts and their ability to help direct bone formation in vivo. Octacalcium phosphate scaffolds may have a bright future in bone regeneration.

Due to the progress of cell-based therapy, the idea of bringing osteogenic cells to a defect has been actively investigated. Many cells types are being studied, and the ability of BMMSCs and ADMSCs to differentiate into osteoblasts and chondrocytes has led researchers to focus on them. Seeded on the appropriate scaffold and placed in the appropriate environment, these two cell types have shown their ability to create bone. However, a major issue has always been, and still remains, that the quantity of cells available is limited; this is especially true when considering mesenchymal stem cells.

Since their discovery in the 1960s, BMPs have been providing us great hope but disappointment as well. They were thought to be the missing link for ex vivo and in vivo bone growth stimulation but proved to be less successful than expected. That said, recent advances in rhBMP delivery vehicles, as well as the application of BMPs in orthopedic surgery, have renewed expectations in their capacity to regenerate bone in humans.

Research led to the observation that vascularization may be a key missing factor for bone-tissue engineering. Every single tissue needs a patent blood supply to survive and bone is no exception to the rule. Thus, the use of vasculogenic factors and cells became obvious. Both have shown great results, enhancing bone formation and bone healing.

The research on cranial bone defects is promising. There have been leaps and bounds in the past 2 decades, and certainly the next few decades will show even more progress. For those people born with craniomaxillofacial defects or those in whom they are acquired due to trauma, this research is an integral part of their future livelihood. It has been demonstrated that cranial bone defects can be healed in animal models, and this provides hope for applications in humans. It is only a matter of time before we are able to give patients with craniomaxillofacial defects a second chance.

Conclusions

This review highlights most of the current translational bone tissue–engineering strategies. Although numerous different materials and techniques have been and are under investigation all around the world, we have yet to find the perfect solution to bone reconstruction; that said, we have made tremendous progress. We are getting closer every day, and ultimately, we may find the gene-modified, cell-based, tissue-engineering strategies that will almost perfectly imitate nature and succeed today's reconstructive strategies.

Disclosure

The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

Author contributions to the study and manuscript preparation include the following. Analysis and interpretation of data: Barr. Drafting the article: Szpalski. Critically revising the article: Warren, Szpalski, Wetterau, Saadeh.

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Contributor Notes

Address correspondence to: Stephen M. Warren, M.D., Institute of Reconstructive Plastic Surgery, New York University School of Medicine, 560 First Avenue, TH-169, New York, New York 10016. email: stephen.warren.md@gmail.com.
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    Mice calvarial defect model. A: A 3-mm-diameter round dental bur was used to create a 3-mm (noncritical) calvarial defect on the left parietal bone in a wild-type mouse. The underlying dura mater was kept intact. B: A 3D micro-CT scan of a calvarial defect immediately following wound creation, showing the perfect bone defect.

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    Bone-tissue engineering: properties and characteristics to consider for the design of a scaffold. The ideal scaffold will have to be biocompatible and osteocompatible (to be inserted into a cranial defect to support, guide, and enhance bone healing) and allow for osteogenesis, osteoinduction, and osteoconduction. In addition, the scaffold must demonstrate mechanical strength and resilience and its degradation products must not trigger an inflammatory reaction. The surrounding environment and the seeding of cells onto the scaffold play a major role for bone growth as well.

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    Scaffold printing system. The 3D microprinting process is fully customized (diameter, pore size, strut size, and porosity) and can produce novel scaffolds in less than 3 hours. A: The 3D microprinting processor. B: Several HA-TCP sample scaffolds. C: The microarchitecture of a customized scaffold.

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    Photomicrographs showing human mesenchymal stem cell multilineage differentiation. Under the appropriate stimuli, mesenchymal stem cells can differentiate into various cell types: undifferentiated (A; alkaline phosphatase, original magnification × 400), bone nodule (B; von Kossa, original magnification × 400), endothelial tubule (C; fluorescent GFAP, original magnification × 200), cartilage (D; Alcian blue, original magnification × 400), adipocyte (E; Oil Red O, original magnification × 200), neuron (F; GFAP, original magnification × 400).

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