Quantification and 3D visualization of new vessel networks in vivo remains a major unresolved issue in tissue engineering constructs. This study has examined the potential of combining the use of a radio opaque dye and micro-CT to visualize and quantify microvascular networks in 3D in vivo. We have applied this technique to the study of neoangiogenesis in a bone impaction graft model in vivo as proof of concept. Tissue engineered constructs were created with natural (morsellised allograft) and synthetic grafts (Poly Lactic Acid, PLA) Culture expanded human bone marrow stromal cells (HBMSC) labeled with a fluorescent probe (Cell Tracker Green, CTG) to measure cell viability, were seeded onto prepared scaffolds (morsellised allograft or PLA) and impacted with a force equivalent to a standard femoral impaction (474J/m2). The impacted HBMSC / scaffolds and scaffolds alone were contained within capsules and implanted subcutaneously into severely compromised immunodeficient mice. Radiopaque dye was infused into all vessels via cardiac cannulation prior to removal of implants. Micro CT imaging and immunohistochemistry was performed in all samples. Cell survival was evident by abundant fluorescent staining. The average number of blood vessels penetrating the capsules were 16.33 in the allograft / HBMSC constructs compared to 3.5 (p=0.001) in the allograft alone samples and 32.67 in the PLA / HBMSC constructs compared to 7.67 (p=0.001) in the PLA alone samples. The average total vessel volume within the capsules was 0.43mm3 in the allograft / HBMSC constructs compared to 0.04mm3 (p=0.05) in the allograft alone samples and 1.19mm3 in the PLA / HBMSC constructs compared to 0.12mm3 (p=0.004) in the PLA alone samples. Extensive staining for Type 1 Collagen, new matrix and Von Willebrand factor in living tissue engineered constructs confirmed osteogenic cell phenotype, and new blood vessel formation respectively. In summary, these studies demonstrate, HBMSC combined with either morsellised allograft or PLA can survive the forces of femoral impaction, differentiate along the osteogenic lineage and promote neovascularisation in vivo. Successful combined neovascularisation and bone formation in impacted tissue engineered constructs in vivo augers well for their potential use in IBG. This novel technique utilising contrast enhanced 3D reconstructions in combination with immunohistochemistry enables quantification of neovascularisation and new bone formation in impacted tissue engineered constructs with widespread experimental application in regenerative medicine and tissue engineering analysis.
Impaction bone grafting with morsellised allograft is a recognized technique to reconstitute loss of bone stock often encountered during revision hip surgery. Concerns over disease transmission, high costs and limited supply has led to interest in synthetic grafts. Poly (lactic acid) (PLA) grafts are attractive to the tissue engineering community as a consequence of their biocompatibility, ease of processing into three-dimensional structures, their established safety as suture materials and the versatility that they offer for producing chemically defined substrates for bone graft matrices. This study set out to examine the potential of PLA scaffolds augmented with human bone marrow stromal cells in impaction bone grafting (IBG).
The use of fresh morsellised allograft in impaction bone grafting for revision hip surgery remains the gold standard. Bone marrow contains osteogenic progenitor cells that arise from multipotent mesenchymal stem cells and we propose that in combination with allograft will produce a living composite with biological and mechanical potential. This study aimed to determine if human bone marrow stromal cells (HBMSC) seeded onto highly washed morsellised allograft could survive the impaction process, differentiate and proliferate along the osteogenic lineage and confer biomechanical advantage in comparison to impacted allograft alone. Future work into the development of a bioreactor is planned for the potential safe translation of such a technique into clinical practice.
The mean hallux valgus angle was reduced from 39.6 degrees to 31.8 degrees and the second MTP angle from 28.3 degrees to 19.4 degrees. Pre-operatively 28% of the lesser toe MTP joints were aligned compared with 83% post-operatively. All of the WMOs fused. Two first MTP joint fusions resulted in non-unions and required successful revision surgery. In five cases metalwork was removed from the hallux because of discomfort. In two cases, metalwork was removed because of superficial wound infection. Infection subsequently resolved after a course of oral antibiotics. Nine patients stated they would recommend the procedure.