Objective: Functional suitability and phenotypic stability of ectopic transplants are crucial factors in the clinical application of mesenchymal stem cells (MSCs) for articular cartilage repair, and might require a stringent control of chondrogenic differentiation. This study evaluated whether human bone marrow–derived MSCs adopt natural differentiation stages during induction of chondrogenesis in vitro, and whether they can form ectopic stable cartilage that is resistant to vascular invasion and calcification in vivo.Methods: During in vitro chondrogenesis of MSCs, the expression of 44 cartilage‐, stem cell–, and bone‐related genes and the deposition of aggrecan and types II and X collagen were determined. Similarly treated, expanded articular chondrocytes served as controls. MSC pellets were allowed to differentiate in chondrogenic medium for 3–7 weeks, after which the chondrocytes were implanted subcutaneously into SCID mice; after 4 weeks in vivo, samples were evaluated by histology.Results: The 3‐stage chondrogenic differentiation cascade initiated in MSCs was primarily characterized by sequential up‐regulation of common cartilage genes. Premature induction of hypertrophy‐related molecules (type X collagen and matrix metalloproteinase 13) occurred before production of type II collagen and was followed by up‐regulation of alkaline phosphatase activity. In contrast, hypertrophy‐associated genes were not induced in chondrocyte controls. Whereas control chondrocyte pellets resisted calcification and vascular invasion in vivo, most MSC pellets mineralized, in spite of persisting proteoglycan and type II collagen content.Conclusion: An unnatural pathway of differentiation to chondrocyte‐like cells was induced in MSCs by common in vitro protocols. MSC pellets transplanted to ectopic sites in SCID mice underwent alterations related to endochondral ossification rather than adopting a stable chondrogenic phenotype. Further studies are needed to evaluate whether a more stringent control of MSC differentiation to chondrocytes can be achieved during cartilage repair in a natural joint environment.

Healing of fractures is dependent on vascularization of bone, which is in turn promoted by VEGF. It was shown that 0.1 and 1 mg of pVEGF165-GAM led to a significant increase in vascularization and bone regeneration in defects that would otherwise have led to atrophic nonunions. Introduction: One reason for lack of bone healing in nonunions is the absence of vascularization. In skeletogenesis, which is tightly linked to angiogenesis, vascular endothelial growth factor (VEGF) promotes the vascularization of the growth plate and transformation of cartilage to bone. We postulate that a gene-activated matrix (GAM), created with a plasmid coding for human VEGF165, coated on a collagen sponge could efficiently accelerate bone healing in large segmental defects. Materials and Methods: Sixty New Zealand white rabbits received a 15-mm critical size defect on one radius, which was filled with either 0.1 or 1 mg plasmid-DNA as GAM. Radiographs were obtained every 3 weeks. After 6 or 12 weeks, animals were killed. New bone was measured by μCT scans. Vascularity was measured using anti-CD31 staining of endothelial cells in 18 regions of interest per implant. Results: Scaffold and control plasmid showed no defect healing, whereas most of the animals in the VEGF groups showed partial or total bone regeneration. Significantly more bone was found in the VEGF groups, with no significant differences between the 0.1- and 1-mg groups. Immunohistochemical staining of endothelial cells revealed that the VEGF groups showed two to three times the number of vessels and a significantly larger endothelial area after 6 weeks. Twelve weeks after surgery, the amount of vascularization decreased, whereas more new bone was detectable. Conclusions: The rabbit critical size defect was appropriate in size to produce atrophic nonunions. We showed that angiogenesis and osteogenesis can be promoted by a VEGF165-GAM that is an appropriate tool to induce bone healing in atrophic nonunions.