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Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity with sufficient mechanical properties. This study tailored the phosphoric acid-based binder solution concentration to 8.75 wt% to maximize cytocompatibility and mechanical strength, with a supplementation of Tween 80 to improve printing. To further enhance the formulation, collagen was dissolved into the binder solution to fabricate collagen-calcium phosphate composites. Reducing the viscosity and surface tension through a physiologic heat treatment and Tween 80, respectively, enabled reliable thermal inkjet printing of the collagen solutions. Supplementing the binder solution with 1–2 wt% collagen significantly improved maximum flexural strength and cell viability. To assess the bone healing performance, we implanted 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks. The implants were confirmed to be osteoconductive, with new bone growth incorporating the degrading scaffold materials. In conclusion, this study demonstrates optimization of material parameters for 3D printed calcium phosphate scaffolds and enhancement of material properties by volumetric collagen incorporation via inkjet printing.

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Collagen gels were seeded with rabbit bone marrow-derived mesenchymal stem cells (MSCs) and contracted onto sutures at initial cell densities of I , 4, and 8 million cellslml. These MSC-collagen composites were then implanted into full thickness, full length, central defects created in the patellar tendons of the animals providing the cells. These autologous repairs were compared to natural repair of identical defects on the contralateral side. Biomechanical, histological, and morphometric analyses were performed on both repair tissue types at 6, 12, and 26 weeks after surgery. Repair tissues containing the MSC-collagen composites showed significantly higher maximum stresses and moduli than natural repair tissues at 12 and 26 weeks postsurgery. However, no significant differences were observed in any dimensional or mechanical properties of the repair tissues across seeding densities at each evaluation time. By 26 weeks, the repairs grafted with MSC-collagen composites were one-fourth of the maximum stress of the normal central portion of the patellar tendon with bone ends. The modulus and maximum stress of the repair tissues grafted with MSCLcollagen composites increased at significantly faster rates than did natural repairs over time. Unexpectedly, 28% of the MSC ~ collagen grafted tendons formed bone in the regenerating repair site. Except for increased repair tissue volume, no significant differences in cellular organization or histological appearance were observed between the natural repairs and MSC-collagen grafted repairs. Overall, these results show that surgically implanting tissue engineered MSC-collagen composites significantly improves the biomechanical properties of tendon repair tissues, although greater MSC concentrations produced no additional significant histological or biomechanical improvement.

Mesenchymal stem cells (MSCs) were isolated from bone marrow of 18 adult New Zealand White rabbits. These cells were culture expanded, suspended in type I collagen gel, and implanted into a surgically induced defect in the donor s right patellar tendon. A cell-free collagen gel was implanted into an identical control defect in the left patellar tendon. Repair tissues were evaluated biomechanically (n = 13) and histomorphometrically (n = 5) at 4 weeks after surgery. Compared to their matched controls, the MSC-mediated repair tissue demonstrated significant increases of 26% (p < 0.001), 18% (p < 0. 01), and 33% (p < 0.02) in maximum stress, modulus, and strain energy density, respectively. Qualitatively, there appeared to be minor improvements in the histological appearance of some of the MSC- mediated repairs, including increased number of tenocytes and larger and more mature-looking collagen fiber bundles. Morphometrically, however, there were no significant left-right differences in nuclear aspect ratio (shape) or nuclear alignment (orientation). Therefore, delivering a large number of mesenchymal stem cells to a wound site can significantly improve its biomechanical properties by only 4 weeks but produce no visible improvement in its microstructure.

A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that ∼70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP-2-producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene-enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair.Introduction: The loss of cellular activity in a structural bone allograft markedly reduces its healing potential compared with a live autograft. To further understand the cellular mechanisms for structural bone graft healing and repair and to devise a therapeutic strategy aimed at enhancing the performance of allograft, we established a segmental femoral structural bone graft model in mice that permits qualitative and quantitative analyses of graft healing and neovascularization. Materials and Methods: Using this segmental femoral bone graft model, we transplanted live isografts harvested from Rosa 26A mice that constitutively express ␤-galactosidase into their wildtype control mice. In an attempt to emulate the osteogenic and angiogenic properties of periosteum, we applied a cell-based, adenovirus-mediated gene therapy approach to engraft BMP-2-producing bone marrow stromal cells onto devitalized allografts. Results: X-gal staining for donor cells allowed monitoring the progression of periosteal progenitor cell fate and showed that 70% of osteogenesis was attributed to cellular proliferation and differentiation of donor progenitor cells on the surface of the live bone graft. Quantitative CT analyses showed a 3-fold increase in new bone callus formation and a 6.8-fold increase in neovascularization for BMP-2/stromal cell-treated allograft compared with control acellular allografts. Histologic analyses showed the key features of autograft healing in the BMP-2/stromal cell-treated allografts, including the formation of a mineralized bone callus completely bridging the segmental defects, abundant neovascularization, and extensive resorption of bone graft. Conclusions:The marked improvement of healing in these cellularized allografts suggests a clinical strategy for engineering a functional periosteum to improve the osteogenic and angiogenic properties of processed allografts.

Pools of human adipose-derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell-based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population. The goal of this study was to test the hypothesis that single hADAS are multipotent at a clonal level. hADAS cells were isolated from liposuction waste, and ring cloning was performed to select cells derived from a single progenitor cell. Forty-five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage-specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histological and biochemical analyses. Eighty one percent of the hADAS cell clones differentiated into at least one of the lineages. In addition, 52% of the hADAS cell clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron-like cells (52%) than of adipocytes (12%), possibly due to the loss of adipogenic ability after repeated subcultures. The findings are consistent with the hypothesis that hADAS cells are a type of multipotent adult stem cell and not solely a mixed population of unipotent progenitor cells. However, it is important to exercise caution in interpreting these results until they are validated using functional in vivo assays.

Over the past 8 years, our group has been continuously improving tendon repair using a functional tissue engineering (FTE) paradigm. This paradigm was motivated by inconsistent clinical results after tendon repair and reconstruction, and the modest biomechanical improvements we observed after repair of rabbit central patellar tendon defects using mesenchymal stem cell-gelsuture constructs. Although possessing a significantly higher stiffness and failure force than for natural healing, these first generation constructs were quite weak compared to normal tendon. Fundamental to the new FTE paradigm was the need to determine in vivo forces to which the repair tissue might be exposed. We first recorded these force patterns in two normal tendon models and then compared these peak forces to those for repairs of central defects in the rabbit patellar tendon model (PT). Replacing the suture with end-posts in culture and lowering the mesenchymal stem cell (MSC) concentration of these constructs resulted in failure forces greater than peak in vivo forces that were measured for all the studied activities. Augmenting the gel with a type I collagen sponge further increased repair stiffness and maximum force, and resulted in the repair tangent stiffness matching normal stiffness up to peak in vivo forces. Mechanically stimulating these constructs in bioreactors further enhanced repair biomechanics compared to normal. We are now optimizing components of the mechanical signal that is delivered in culture to further improve construct and repair outcome. Our contributions in the area of tendon functional tissue engineering have the potential to create functional load-bearing repairs that will revolutionize surgical reconstruction after tendon and ligament injury. ß