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Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_II | Pages 287 - 287
1 May 2009
Bolland B Kanczler J Dunlop D Oreffo R
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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.


Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_II | Pages 287 - 287
1 May 2009
Bolland B Kanczler J Ginty P Shakesheff K Dunlop D Oreffo R
Full Access

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).

Methods: In vitro and in vivo studies were performed on impacted morsellised PLA seeded with human bone marrow stromal cells (HBMSC) and compared to PLA alone. In vitro samples were incubated under osteogenic conditions and in vivo samples were implanted subcutaneously into severely compromised immunodeficient mice, both for 4 weeks. In vitro samples were analysed for cell viability, DNA content, specific alkaline phosphatase activity, immunohistochemistry and mechanical shear testing using a cam shear tester. In vivo samples were analysed for cell viability, immunohistochemistry and evidence of neovascularisation and new bone formation using contrast enhance micro computer tomography.

Results: HBMSC survival post impaction, as evidenced by cell tracker green staining, was seen throughout the samples in vitro and in vivo. In vitro there was a significant increase in DNA content (P< 0.001) and specific alkaline phosphatase activity (P< 0.001) in PLA / HBMSC samples compared to impacted PLA alone. Mechanical shear testing of in vitro PLA / HBMSC samples demonstrated a significant increase in shear strength and interparticulate cohesion compared to PLA alone. Immunohistochemistry for type I collagen, osteocalcin, confirmed cell differentiation along the osteogenic lineage in vitro and in vivo. In vivo studies showed a significant increase in blood vessel number and volume penetrating the PLA / HBMSC constructs (32.6 vessels, 1.19mm3, p=0.02, p=0.004) compared to PLA alone (7.6vessels, 0.12mm3). There was a significant relative increase in new bone formation in the PLA / hBMSC constructs (0.47mm3) compared to PLA alone (0mm3), further confirmed with positive staining for osteoid using Goldners Trichrome.

Conclusion: HBMSC seeded onto PLA can withstand the forces of femoral impaction and continue to differentiate and proliferate along the osteogenic lineage. Furthermore PLA / hBMSC constructs in vitro offer a mechanical advantage over PLA alone and in vivo induce neovascularisation and new bone formation. From both a biological and mechanical perspective these studies have demonstrated that PLA is a suitable and beneficial bone graft extender for use in IBG.