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A history of conventional means for filling critical size bone defects using grafting and bone regeneration techniques.
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An introduction as to what materials have so far been used for the creation of bone substitute and vascular augmentation substitutes for a composite bone and vascular void filler.
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Possible future directions that will need to be considered in the successful creation of composite vascularized bone void fillers for segmental defects.
Abstract
The management of large segmental bone defects caused by trauma or disease remains clinically challenging within orthopaedics. The major impediment to bone healing with current treatment options is insufficient vascularization and incorporation of graft material. Lack of rapid adequate vascularization leads to cellular necrosis within the inner regions of the implanted material and a failure of bone regeneration. Current treatment options for critical size bone defects include the continued “gold standard” autograft, allograft, synthetic bone graft substitutes, vascularized fibular graft, induced membrane technique, and distraction osteogenesis. Bone tissue engineering (BTE) remains an exciting prospect for the treatment of large segmental bone defects; however, current clinical integration of engineered scaffolds remains low. We believe that the barrier to clinical application of bone tissue engineering constructs lies in the lack of concomitant vascularization of these scaffolds. This mini-review outlines the progress made and the significant limitations remaining in successful clinical incorporation of engineered synthetic bone substitutes for segmental defects.
Treatment of segmental bone defects following traumatic injury, Cierny-Mader Stage III and IV osteomyelitis, and en bloc tumor resections remains clinically challenging. It is our opinion that the main obstacle to successful segmental long bone defect filling when using grafting techniques, be they autograft or synthetic, is the lack of adequate vascularization of the implanted materials or tissue engineered constructs. Without adequate vascularization, the inner regions of the graft either undergo cellular necrosis (can be the case with large segmental autografts) or failure of proper cellular infiltration and regeneration, leading to graft failure [
Bone is a complex organ with an intricate design. The inner matrix, cancellous bone, is a porous network formed by trabeculae, red marrow, and vasculature that acts as the site of hematopoiesis while providing strength, flexibility, and decreasing the weight of bone. The outer matrix, cortical bone, is a dense region with vascular canals that is capable of withstanding a high mechanical load and resisting bending and torsion forces. The outermost layer of bone, the periosteum, is a thin vascular membrane that houses osteoblasts and progenitor cells and is capable of forming new bone. The extracellular matrix provides a scaffold for bone deposition, strengthens the construct, and houses signaling factors that are crucial to bone formation, growth, remodeling, and resorption [
Together, these form the three components necessary for tissue regeneration: an osteoconductive scaffold for cell adhesion, osteoinductive signaling molecules to guide bone formation, and osteogenic cells themselves. It is important to note that at the cellular level, efficient exchange of nutrients and waste is possible because cells and vessels are at most 100 to 300 microns apart [
]. Despite advancements in manufacturing synthetic bone and soft tissue composites, their clinical integration remains low.
We believe that the barrier to clinical application of bone tissue engineering (BTE) constructs is the lack of concomitant vascularization techniques for these grafts [
]. At present, BTE efforts are not integrated with vascular tissue engineering innovations. As such, state of the art vascular substitutes are not designed for bone applications and vice versa [
]. It remains to be seen whether microvascular surgical techniques can aid in the revascularization of engineered bone substitutes. To date, this “Holy Grail” of bone tissue engineered constructs for treatment of critical sized bony defects has not been a reasonable alternative.
Current techniques of segmental long bone defect filling
The gold standard for bone repair remains autografting, or the transposition of a patient's bone from a distant region of the body to the site of the bony defect. This graft possesses all three components necessary for tissue regeneration: an osteoconductive scaffold, osteoinductive signaling molecules, and osteogenic cells [
]. Unfortunately, autograft is limited in supply and bone harvest is linked to pain and morbidity at the donor site.
Allografts are another option. These are comprised of decellularized bone that has been harvested from non-self, human donors. Allografts lack osteogenic cells and functioning inductive proteins; however, they possess osteoconductive properties as their extracellular matrix provides a scaffold for growth. Some surgeons have augmented allograft bone with autograft bone marrow aspirate concentrates to restore osteoinductivity to these scaffolds. This technique has shown promise. Both autograft and allograft also lack rapid vascularization into the body of the grafted material. Complications of allograft use include fracture, non-union, infection, and immunogenic response.
Synthetic bone graft substitutes such as hydroxyapatite, calcium phosphate, calcium sulfate, and tricalcium phosphate derivatives are osteoconductive [
]. There is an unlimited supply of synthetic graft. These materials also have a long shelf life and do not carry a risk of disease transfer; however, they lack osteoinductive and osteogenic properties and to some extent can lack cortical stability [
]. Outcomes of graft utilization for autograft, allograft, and synthetic bone substitutes are unpredictable, particularly when bone defect size is greater than 4 cm [
Several clinical approaches have been implemented for segmental long bone defect filling including distraction osteogenesis, induced membranes, and vascularized fibular graft. Distraction osteogenesis takes advantage of the inherent ability of bone to regenerate under tension [
]. An external fixator is applied with tensioned wires that allow for gradual distraction using the principles of Ilizarov, to promote bone formation utilizing the technique he described as “Bone Transport” [
]. Eventually, distraction across the corticotomy leads to new bone formation that replaces the critical size defect. When this transported bone meets with the segment of remaining bone at the “docking site,” grafting is performed. Compression is then applied across the site to gain bony union. Disadvantages include a lengthy duration of treatment, bulky apparatus surrounding the affected limb, pin site infection, and pain from surrounding structure stretching [
Complications associated with distraction osteogenesis for infected nonunion of the femoral shaft in the presence of a bone defect: a retrospective series.
]. Some of these disadvantages can now be ameliorated with new design internal bone transport nails.
Induced membrane technique involves a two-stage procedure wherein a temporary cement spacer is introduced into a bone defect and is later removed and replaced by autograft and possible allograft adjunct [
. This vascularized environment provides nourishment for bone repair once autograft is added. An obvious downside to this technique is the need for a second operation. Additional complications include infection, malunion, fracture, and nonunion. Infection can be decreased by impregnating the cement spacer with antibiotics [
]. This technique provides less reliable results than that obtained by experienced surgeons in the methods of bone transport; most results reported on induced membrane technique often achieve at best an implant-dependent union.
Vascularized fibular grafts can also be used to reconstruct skeletal defects [
]. They are harvested with an attached arteriovenous pedicle and can be grafted with surrounding muscle, fascia, or skin to meet the needs of the recipient tissue [
]. It is indicated for treatment of large segmental bone defects (greater than five or six centimeters in length) or for areas with a poorly vascularized soft tissue envelope [
]. Various osteotomies can be performed to the graft because of its dual vascular supply from both endosteal and periosteal vessels supplied by the peroneal artery and vein [
]. A transverse osteotomy allows creation of a “double barrel” construct to match the length and width of a defect, while a longitudinal osteotomy can be utilized to strengthen partial cortical defects [
]. This is unlike the creeping substitution experienced by avascular grafts wherein graft necrosis leads to resorption, and later, new bone formation [
]. As with other autografts, graft availability is limited. Complications are more prominent at the recipient site as compared to the donor site and include infection, nonunion, graft fatigue fracture, and vascular compromise [
Tissue engineering could provide a single-stage solution for surgical treatment of segmental long bone defects. The ideal engineered graft would be osteoconductive, osteoinductive, promote osteogenesis and angiogenesis, allow load transfer with weight-bearing activities, and be biocompatible with host tissue. Many promising constructs exist, but none meet all of the qualities outlined above. The list of materials and manufacturing methods that have been trialed in attempt to create a better engineered bone scaffold are quite extensive (Table 1).
Table 1Manufacturing methods and materials that have been utilized in the construction of synthetic rigid porous scaffolds to replace bone voids.
An injection molding process for manufacturing highly porous and interconnected biodegradable polymer matrices for use as tissue engineering scaffolds.
Tissue-engineered bone formation using periosteal-derived cells and polydioxanone/pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells.
]. Ceramics are commonly used. These include hydroxyapatite, bioactive glass, and tricalcium phosphate. They are biocompatible and osteoconductive with pores for cell ingrowth; however, they are also brittle [
]. Natural scaffolds such as hyaluronic acid, collagen, and alginate also provide pores for cell adherence and ingrowth, but lack the mechanical strength to be stand-alone scaffolds [
]. As such, ceramics are often coated in synthetic biodegradable polymers [e.g. polylactic acid (PLA), polyglycolic acid (PGA), or polylactic acid co-glycolic acid (PLGA)] to increase strength and osteoinductivity [
The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite-PCL composites.
]. Similarly, natural scaffolds can be strengthened through combination with other compounds. Small signaling molecules such as bone morphogenic proteins (BMPs), fibroblast growth factors (FGFs), and vascular endothelial growth factors (VEGFs) among others have been utilized to induce cell differentiation and vascular growth in scaffolds [
While bone tissue engineering has provided promising results, it has become increasingly clear that efforts should be directed towards hierarchical integration of bone scaffolds and vascular networks to create constructs that support both osteogenic and angiogenic growth. Exchange of nutrients and waste between individual cells and capillaries within bone is limited to between 100-300 micrometers [
]. This poses a limitation to graft integration from the cellular level to macroscale incorporation into host tissue. Vascular networks within a bone tissue engineered construct must consist of large vessels as well as microvascular beds to provide gradient driven blood of sufficient volume across the entire scaffold. This is necessary for the rapid and complete integration of the scaffold into the host, as well as to allow for viability of any cell line added to the composite (Fig. 1).
Fig. 1ASegmental defect of bone with constructed synthetic graft matrix and synthetic vascular tree with inflow and outflow conduits to allow for immediate perfusion of the segmental structural graft. 1B: Following inset and microvascular anastomoses of arterial inflow (red) and venous outflow (blue) graft vascular tree. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 1ASegmental defect of bone with constructed synthetic graft matrix and synthetic vascular tree with inflow and outflow conduits to allow for immediate perfusion of the segmental structural graft. 1B: Following inset and microvascular anastomoses of arterial inflow (red) and venous outflow (blue) graft vascular tree. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Clinical integration of tissue engineered vascular grafts has been challenging, in part due to limited hemocompatibility causing thrombogenic occlusion and stenosis from intimal hyperplasia [
]. The ideal vessel graft should be biocompatible with similar biochemical, permeability, and compliance properties to host tissue. Most importantly, because these grafts must be of small diameter and require microsurgical techniques to integrate them to the host vascular tree, these foreign graft materials must allow for endothelialization either in vitro or rapidly after anastomosis is completed to prevent thrombosis of the graft matrix.
Manufacturing
Elastomers are polymers that possess viscoelasticity, allowing them to withstand more repetitive strain and cyclic stress compared to other materials [
]. Elastomer grafts provide a surface for cell adhesion, cell integration, and proliferation within graft tissue, ultimately allowing substitution of graft with host vascular tissue. Several researchers have attempted to manufacture small synthetic hollow fibers that could serve as structural bridges for vascular conduits within a synthetic bone filling scaffold. Some have used elastomeric polyester urethane with varying success [
Fabrication and characterization of permeable degradable poly(dl-lactide-co-glycolide) (PLGA) hollow fiber phase inversion membranes for use as nerve tract guidance channels.
]. Although these grafts have been promising in large vessel applications, they have yet to demonstrate viability in microvascular applications. As no good in vivo data currently exists for these grafts, the integration of a vascular tree component to these bony scaffold models is limited.
Several authors have tried to incorporate vascularized tissue into ceramic scaffolds by adding vascular growth factors or directly placing vascularized tissue beds into the ceramic composite [
Capillary vessel network integration by inserting a vascular pedicle enhances bone formation in tissue-engineered bone formation using interconnected porous hydroxyapatite ceramics.
Preefabrication of vascularized bone graft using an interconnected porous calcium hydroxyapatite ceramic in presence of vascular endothelial growth factor and bone marrow mesenchymal stem cells: experimental study in rats.
]. To date, none have successfully incorporated a synthetic vessel into a structural component to achieve true immediate vascular reconstitution of the scaffold graft [
Capillary vessel network integration by inserting a vascular pedicle enhances bone formation in tissue-engineered bone formation using interconnected porous hydroxyapatite ceramics.
Preefabrication of vascularized bone graft using an interconnected porous calcium hydroxyapatite ceramic in presence of vascular endothelial growth factor and bone marrow mesenchymal stem cells: experimental study in rats.
]. One of the authors has engineered a vascular graft substitute that is compatible with both human endothelial and mesenchymal stem cell lines, allowing for cell adhesion to and proliferation within the synthetic vascular graft [
]. Using an animal model, they placed an autologous explanted microcirculatory bed (EMB) consisting of an afferent artery, capillary beds, efferent vein, and surrounding parenchymal tissue into a bioreactor and maintained tissue viability ex vivo for 24 hours [
]. Later, the EMBs were seeded with either multipotent adult progenitor cells, bone marrow derived mesenchymal stem cells, or adipose tissue derived mesenchymal stem cells [
]. They found that each of these cell lines could egress from the microcirculation into the parenchymal space and form proliferative clusters that maintained viability [
]. This may represent a more viable method to augment engineered scaffolds in the future.
Rigid porous scaffolds
The scaffold acts as an artificial extracellular matrix, a temporary structure that allows for cell adhesion, proliferation, and differentiation, ultimately with degradation of the graft and replacement with host cells. Ideally, the construct is biocompatible, strong, osteoconductive, and osteoinductive. Many scaffolds have been tried, but no clear evidence exists to suggest that one is particularly better than another [
The literature suggests that composite tissue bone scaffolds likely provide the best alternative to synthetic rigid porous scaffolds. These integrated scaffolds have commonly been made with hydroxyapatite nanoparticles (HA), biphasic calcium phosphate (BCP), beta tricalcium phosphate (β-TCP), and the synthetic biodegradable polymer polycaprolactone (PCL). PCL is simple to manufacture, easy to manipulate, and mixes well with other materials such as polymers and ceramics [
The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite-PCL composites.
]. Their composite scaffold had a higher compressive strength than other HA/β-TCP scaffolds (2.1 MPa versus 0.1-0.3 MPa) as well as enhanced elasticity, similar to the stress-strain characteristics of normal bone [
The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite-PCL composites.
]. These highly porous scaffolds supported osteoblast integration and differentiation in vitro with increased alkaline phosphatase activity and increased osteogenic gene expression. The coating greatly improved the bioactivity and mechanical properties of this scaffold, reducing the general brittleness compared to most ceramics.
Bruyas, et al. printed PCL/β-TCP scaffolds in an attempt to mimic a new bone matrix. By varying scaffold porosity and ceramic content, they too found that composition has an effect on surface properties, rate of degradation, and mechanical strength which further affects osteoconduction, proliferation, and differentiation [
Systematic characterization of 3D-printed PCL/β-TCP scaffolds for biomedical devices and bone tissue engineering: influence of composition and porosity.
]. Further, a PCL/β-TCP composite with higher ceramic content possessed a rougher surface, accelerated rate of degradation, and lower elasticity as compared to a composite with lower ceramic content [
Systematic characterization of 3D-printed PCL/β-TCP scaffolds for biomedical devices and bone tissue engineering: influence of composition and porosity.
Systematic characterization of 3D-printed PCL/β-TCP scaffolds for biomedical devices and bone tissue engineering: influence of composition and porosity.
Systematic characterization of 3D-printed PCL/β-TCP scaffolds for biomedical devices and bone tissue engineering: influence of composition and porosity.
]. Kang, et al. also found that the physical properties of a simple crystalline structure, in their case β-TCP, could be advantageously modified with the addition of another biocompatible polymer [
]. This composite was less brittle and had increased compressive strength, bending strength, and toughness as compared to the porous β-TCP alone, while still maintaining the interconnected porous microstructure [
]. These results can provide guidance in customizing the biological properties of rigid porous scaffolds for the future.
Combining of vascular and bone engineered entities into a composite
Again, it seems that the reason for the limited clinical success of synthetic bone scaffold substitutes has been the lack of vascular tree integration into the scaffold design [
]. This integration is necessary to provide immediate nutrition and waste disposal for any cell line acutely added to the matrix. Additionally, vascular networks within the scaffold and anastomoses between scaffold and host are essential for graft integration as a viable composite tissue. Unfortunately, microsurgical integration of tissue engineered vascular grafts has been limited by the inability to maintain flow and vascular patency within these grafts. This challenge is magnified by the length of microvascular synthetic conduit needed to perfuse a rigid porous scaffold and difficulty maintaining microvascular patency on the distal, low-pressure venous end. Until these can be overcome, the “Holy Grail” remains unattainable (Fig. 1).
Grayson, et al. described a technique of bioreactor generated bone of specific size and shape grown with an interstitial flow of culture medium seeded with human mesenchymal stem cells [
]. This is a promising technique for bone generation; however, they remain stunted by a lack of ability to maintain nutrient perfusion to the graft after removal from the bioreactor and placement into the host.
Conclusion and future directions
Segmental long bone defects remain one of the most challenging orthopaedic conditions to treat. These have classically been treated with bone grafting techniques, and more recently with clinical techniques such as distraction osteogenesis and induced membranes. Each technique has its limitations, ranging from donor site morbidity to infection and long duration of treatment. Bone tissue engineering remains an exciting prospect for the treatment of these defects. Traditional bone tissue engineering has focused on designing a scaffold that is osteoconductive, osteoinductive, and osteogenic; however, current efforts have not been integrated with cutting edge innovations in vascular tissue engineering. The translational future of vascularized bone tissue engineering scaffolds will be determined by our ability to integrate these two designs into one.
In the end, composite rigid scaffolds will not make or break the success of synthetic bone graft substitutes. Instead, this will depend on our ability to create an acute living tissue bed in the critical size bone defect to allow differentiation into vascularized bone. It is quite possible that the skillfully engineered rigid composite scaffolds may not even play a role in the final solution for this ongoing dilemma.
Declaration of Competing Interest
We wish to draw the attention of the Editor to the following facts which may be considered as potential conflicts of interest and to significant financial contributors to this work. All authors have no conflict of interest in regard to this work.
Complications associated with distraction osteogenesis for infected nonunion of the femoral shaft in the presence of a bone defect: a retrospective series.
An injection molding process for manufacturing highly porous and interconnected biodegradable polymer matrices for use as tissue engineering scaffolds.
Tissue-engineered bone formation using periosteal-derived cells and polydioxanone/pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells.
The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite-PCL composites.
Fabrication and characterization of permeable degradable poly(dl-lactide-co-glycolide) (PLGA) hollow fiber phase inversion membranes for use as nerve tract guidance channels.
Capillary vessel network integration by inserting a vascular pedicle enhances bone formation in tissue-engineered bone formation using interconnected porous hydroxyapatite ceramics.
Preefabrication of vascularized bone graft using an interconnected porous calcium hydroxyapatite ceramic in presence of vascular endothelial growth factor and bone marrow mesenchymal stem cells: experimental study in rats.
Systematic characterization of 3D-printed PCL/β-TCP scaffolds for biomedical devices and bone tissue engineering: influence of composition and porosity.
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