Towards 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 3D printing of living human bone in a single-step for in-situ bone regeneration. Human mesenchymal stem-cells (hBMSCs) were enriched with CD 117+ osteoprogenitor cells, dispersed in different collagen I, RAD 16I hydrogels mixes and applied onto 3-dimensional printed (3DP) beta-TCP/PLGA scaffolds, printed from ultra-high-resolution volumetric CT (VCT) images of a human thumb. Constructs were directly implanted subcutaneously into nude mice for 6 weeks. In-vivo radiographic VCT scanning and histological evaluations were performed at 1, 2, 4 and 6 weeks, 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-situ using a single-step procedure with constructs comprised of RAD 16I /collagen I hydrogel, CD 117+ enriched hBMSCs and porous beta-TCP/PLGA scaffolds.