To evaluate the functional performance of the biomimetic cellular scaffold for bone graft healing, the following parameters should be considered: (1) osseointegration of allograft to host bone; (2) neovascularization of the graft; (3) remodeling of the graft; and (4) biomechanics of bone allograft. These parameters constitute important benchmarks for evaluation of efficacy for bone graft incorporation and repair.
Due to the lack of living cells on bone allograft, the osseointegration of allograft to host bone is slow and limited. In addition, the absence of osteogenic and angiogenic activity of the necrotic cortical bone often cause persistent fibrotic tissue formation. Thus, the primary goal of the cell-based therapy for bone allograft healing is to inhibit fibrotic tissue formation and accelerate osseointegration or incorporation of allograft to host bone. Osseointegration between donor graft and host bone is a challenging problem in graft repair. If not properly guided, newly formed bone could be poorly organized and therefore contribute little to the biomechanical function of the graft. A successful cellular replacement for periosteum should be designed to offer the directional bone growth, eg, to inhibit fibrotic tissue formation while promoting differentiation, proliferation, and directional migration of osteoblastic cells along bone allograft surface. Several innovative approaches have been used to add appropriate surface topographies to membranes in guided tissue regeneration for periodontal tissue regeneration [70
]. Conceivably, these techniques could also be used in our current design for regeneration of periosteal bone formation.
Experimental and clinical experience indicate vascularity of the graft bed is critical for bone graft healing and incorporation [86
]. Vascularity is essential for providing optimal blood supply to maintain survival of the osteogenic cells. The angiogenic response from the host is five to 10 times greater with fresh isograft compared to cryopreserved isograft in the subcutaneous bone graft transplantation model [52
]. A reciprocal role of angiogenesis and osteogenesis in bone repair has been illustrated in several bone repair and regeneration models [28
]. Bone formation can be enhanced by angiogenic growth factors such as vascular endothelial growth factor (VEGF), FGF, or platelet-derived growth factor (PDGF). VEGF further synergizes with BMP-2 stimulating bone formation [72
]. In contrast, suppression of angiogenesis blocks bone formation. On the other hand, osteogenic factors, including BMPs, are potent stimulators for angiogenesis, suggesting bone formation depends on angiogenesis.
To evaluate the neovascularization during bone graft healing, we have developed micro-CT image-based 3D stereologic techniques for quantifying the vasculature and new bone callus formation for structural graft healing [102
]. This approach allows analysis of bone and vasculature in the same sample. When applied to a direct model for the analysis of vasculature, we can perform histomorphometric analysis to determine the vascular volume, connectivity, vessel number, mean vessel thickness, vessel separation, and degree of anisotropy in a femoral bone-grafting model. This approach ensures accurate nondestructive evaluation of structural bone graft healing and vascularization [21
Due to the limited osteogenesis and poor vascularity of devitalized bone, remodeling of structural bone graft is limited. In some cases, decoupling of graft remodeling occurs due to insufficient osteoblast recruitment and activity, which results in loss of bone stock and failure of the grafted allograft bone [22
]. The only effective way to save the failing allograft is to place a vascularized autograft side by side with the allograft. The transplantation of an autograft improves vascularity and brings osteogenic periosteal cells to repair the failing allograft. The data obtained from retrieved large human bone allograft suggest the remodeling of the allograft is positively correlated with the extent of osteogenesis on the graft. The area near the host-graft junction is remodeled more extensively than the area at the midshaft of the allograft. Based on these findings, we presume enhanced osteogenesis and angiogenesis on bone allograft will facilitate bone allograft remodeling and curb early osteoclastic bone loss. In our subsequent studies, in which we implanted BMP-2-producing mesenchymal stem cells around bone allograft [98
], we observed that with improved vascularity and enhanced bone formation surrounding allografted bone the remodeling of necrotic bone allograft was improved compared to acellular allograft. However, the extent of resorption and remodeling was slower than autograft, in which the donor graft was rapidly replaced by new bone 9 weeks after transplantation [98
]. With reduced resorption and improved incorporation of the allograft, we found transplantation of stem cell treated allograft resulted in better biomechanics, outperforming live bone isograft in torsional testing [98
]. The delayed resorption of bone allograft could be due to the lack of living cells in bone, eg, osteocytes.
Evaluation of mechanical performance of the allografted bone is essential for determining the outcome of allograft healing. Mechanical analyses also provide the opportunity to study the structure-function relationship in bone graft healing if combined with micro-CT-based imaging analyses. Identification of micro-CT-based geometric parameters that correlate to the mechanical properties of the healing bone may be useful to interpret the effects of novel therapeutic interventions [78
]. Once established, these parameters can be used to evaluate and predict the functional performance of clinical bone graft using noninvasive imaging technology. Using a murine bone-grafting model, we demonstrated that although allograft incorporated into the host over time, the mechanical properties remain severely impaired compared to live autografts mainly due to the lack of new bone around donor bone graft. Unlike autografts that have a bridging callus by 6 weeks, allografts generate a callus that remains limited to the host side at the cortical bone junction at 6 weeks, and slowly creeps onto the allograft cortex and remodels down thereafter. An analysis of the connectivity of the graft to the host bones indicates that allografts are largely disconnected from the host especially at 6 weeks. From these studies, we identified three predictive correlations between combinations of the micro-CT parameters and the measured ultimate torque and torsional rigidity of the murine grafts, respectively [75
]: (1) amount of new bone formation around bone graft; (2) cross-sectional polar moment of inertia of the graft callus; and (3) connectivity of the bone allograft to the host bone.