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Toward regenerating a human thumb in situ.

Regenerative technology promises to alleviate the problem of limited donor supply for bone or organ transplants. Most expensive and time consuming is cell expansion in laboratories. We propose a method of magnetically enriched osteoprogenitor stem cells, dispersed in self-assembling hydrogels and applied onto new ultra-high resolution, jet-based, three-dimensional printing of living human bone in a single-step for in situ bone regeneration. Human bone marrow-derived mesenchymal stem cells (hBMSCs) were enriched with CD 117+ cells, dispersed in different collagen I and RAD 16I hydrogel mixes, and applied onto three-dimensional printed btricalcium phosphate=poly(lactic-co-glycolic acid) scaffolds, printed from ultra-high-resolution volumetric CT images of a human thumb. Constructs were directly implanted subcutaneously into nude mice for 6 weeks. In vivo radiographic volumetric CT scanning and histological evaluations were performed at 1, 2, 4, and 6 weeks, and expression of bone-specific genes and biomechanical compression testing at 6 weeks endpoint. Time-dependant accumulation of bone-like extracellular matrix was most evident in CD 117+ hBMSCs using collagen I=RAD 16I hydrogel mix. This was shown histologically by Toluidine blue, von Kossa, and alkaline phosphatase staining, paralleled by increased radiological densities within implants approximating that of human bone, and confirmed by high expression of bone-specific osteonectin and biomechanical stiffness at 6 weeks. Human origin of newly formed tissue was established by expression of human GAPDH using RT-PCR. Statistical analysis confirmed high correlations between biomechanical stiffness, radiological densities, and bone markers. Bone tissue can be successfully regenerated in vivo using a single-step procedure with constructs composed of RAD 16I=collagen I hydrogel, CD 117+-enriched hBMSCs, and porous b-tricalcium phosphate=poly(lactic-co-glycolic acid) scaffolds.