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The translation of tissue engineering approaches to the clinic has been hampered by the inability to find suitable multipotent cell sources requiring minimal in vitro expansion. Enhanced bone marrow (eBM), which is obtained by reaming long bone medullary canals and isolating the solid marrow putty, has large quantities of stem cells and demonstrates significant potential to regenerate bone tissues. eBM, however, cannot impart immediate load-bearing mechanical integrity or maintain the gross anatomical structure to guide bone healing. Yet, its putty-like consistency creates a challenge for obtaining the uniform seeding necessary to effectively combine it with porous scaffolds. In this study, we examined the potential for combining eBM with mechanically strong, osteoinductive trabecular bone scaffolds for bone regeneration by creating channels into scaffolds for seeding the eBM. eBM was extracted from the femurs of adult Yorkshire pigs using a Synthes reamer-irrigator-aspirator device, analyzed histologically, and digested to extract cells and characterize their differentiation potential. To evaluate bone tissue formation, eBM was seeded into the channels in collagen-coated or noncoated scaffolds, cultured in osteogenic conditions for 4 weeks, harvested and assessed for tissue distribution and bone formation. Our data demonstrates that eBM is a heterogenous tissue containing multipotent cell populations. Furthermore, coating scaffolds with a collagen hydrogel significantly enhanced cellular migration, promoted uniform tissue development and increased bone mineral deposition. These findings suggest the potential for generating customized autologous bone grafts for treating critical-sized bone defects by combining a readily available eBM cell source with decellularized trabecular bone scaffolds.

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OBJECTIVE: To characterize and evaluate osteoarthritic (OA) chondrocytes, in comparison to normal chondrocytes, through a novel 3-dimensional (3-D) culture system, poly(ethylene-glycol) diacrylate (PEGDA). The cytokine interleukin 1ß (IL-1ß) was also used to simulate an in vitro OA model. METHODS: Normal and OA chondrocytes were cultured in monolayer and analyzed for changes in cartilage-specific gene expressions due to passage number. Then, cells were encapsulated in PEGDA to evaluate phenotype and matrix production capabilities through the in vitro culture system. Characterization was conducted with polymerase chain reaction (PCR), biochemical analyses, and histological staining. 3-D encapsulated chondrocytes (human and bovine) were also treated with IL-1ß to characterize how the cytokine affects gene transcription and extracellular matrix (ECM) content. RESULTS: In 2-dimensional monolayer, anabolic genes were down-regulated significantly in both normal and OA chondrocytes. In 3-D culture, OA chondrocytes demonstrated significantly higher expressions of catabolic genes when compared to normal cells. Differentiation medium resulted in significantly more matrix production than growth medium from OA chondrocytes, indicated through histological staining. In addition, normal chondrocytes responded more significantly to exogenous administration of IL-1ß than OA chondrocytes. Temporary initial stimulation of IL-1ß to OA chondrocytes resulted in comparable gene expressions to untreated cells after 3 weeks of in vitro culture. CONCLUSIONS: Our findings demonstrate the use of OA chondrocytes in tissue engineering and their significance for potential future cartilage regeneration research through their matrix production capabilities and the use of a hydrogel culture system.

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Tissue engineered bone grafts have the potential to be used to treat large bone defects due to congenital abnormalities, cancer resections, or traumatic incidents. Recent studies have shown that perfusion bioreactors can be used to generate grafts of clinically relevant sizes and shapes. Despite these scientific and technological successes, there is uncertainty regarding the translational utility of bioreactor-based approaches due to the perceived high costs associated with these procedures. In fact, experiences over the past two decades have demonstrated that the widespread application of cell-based therapies is heavily dependent on the commercial viability. In this article, we directly address the question of whether bioreactors used to create bone grafts have the potential to be implemented in clinical approaches to bone repair and regeneration. We provide a brief review of tissue engineering approaches to bone repair, clinical trials that have employed cell-based methods, and advances in bioreactor technologies over the past two decades. These analyses are combined to provide a perspective on what is missing from the scientific literature that would enable an objective baseline for weighing the benefit of extended in vitro cultivation of cells into functional bone grafts against the cost of additional cultivation. In our estimation, the cost of bioreactor-based bone grafts may range from $10,000 to $15,000, placing it within the range of other widely used cell-based therapies. Therefore, in situations where a clear advantage can be established for engineered grafts comprising patient-specific, autologous cells, engineered bone grafts may be a clinically feasible option.

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There is a significant need for improved therapy for bone regeneration. The delivery of recombinant bone morphogenetic proteins has been approved for clinical use to promote osteogenesis, but still has limitations such as expense, degradation of the proteins in vivo and difficulties retaining protein at the site of injury. Localized gene delivery is a promising alternative therapy, as it would allow sustained expression of specific osteoinductive growth factors by cells near the damaged site. We have engineered an injectable system for localized, sustained nonviral gene delivery from alginate hydrogels containing preosteoblastic cells and calcium phosphate-DNA nanoparticles. The nanoparticles utilized in this report are stable, on the order of 100 nm, and have a high DNA incorporation efficiency (>66%). When the nanoparticles were incorporated in alginate hydrogels, sustained release of DNA was observed. Furthermore, MC3T3-E1 preosteoblast cells exhibited the capacity to form bony tissue in as little as two and half weeks when mixed with DNA nanoparticles encoding for BMP-2 into the alginate hydrogels and injected subcutaneously in the backs of mice. This injectable, minimally invasive gene delivery system may be efficacious in bone regeneration applications.

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