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Over 240 million people missing teeth worldwide experience lingering problems such as difficulty speaking and eating, undesirable aesthetics, and resorption of bone supporting neighboring teeth. The gold standard of treatment utilizes grafts to attach a function-restoring implant to supporting bone. Current graft materials suffer from problems including autologous donor site morbidity, long resorption time, incomplete integration with the maxillae or mandible, and structural weakness. Patient-specific, cellularized bone grafts may be a solution to these issues by accelerating and improving the quality of regenerated bone. Recently, encapsulation of mesenchymal stem cells within self-assembling type I collagen oligomer matrices has been shown to support rapid mineralization of small-scale bone constructs (cylinders with diameter and height of 6mm and 1mm, respectively) in vitro. However, this method’s volume and geometric constraints for nutrient transport and cell viability are still unknown. In this study, the effects of construct size and medium formulation on mineralization were investigated using conventional static culture methods. To create constructs, human adipose stem cells (hASCs) were embedded in oligomer matrices, allowed to polymerize, and compressed to final cell and fibril densities of 3x107 cells/mL and 50 mg/mL, respectively. Varying construct sizes (maximum diameter and thickness of 11 mm and 0.81 mm) were cultured for 1 week in growth medium or osteogenic medium with varying calcium concentrations. Alizarin red staining was used to detect calcium deposits indicative of cell-induced mineralization. Preliminary data suggests that culture in osteogenic medium supplemented with both 8 mM and 16 mM calcium may induce rapid, uniform mineralization across all sizes tested, and 16 mM calcium supplementation induces greater mineralization. However, additional validation by direct measurement of cell viability and osteogenic differentiation will be needed to better compare bone regeneration as a function of scale.

Millions of craniofacial bone defects occur annually as a result of trauma, congenital defects, disease, or tooth extraction. When present in the oral cavity, these defects are associated with adverse impacts on speech, mastication, and aesthetics. Thus, there is a clinical need for interventional strategies to restore and preserve alveolar bone mass to improve the success of future treatment options intended to reestablish functionality and aesthetics. Guided bone regeneration using bone grafts and a membrane represent the current standard of care for repairing alveolar bone defects, but face a number of limitations related to resorption time and structural integrity. Improvements may be directed toward the development of bone graft substitutes that are patient-specific, provide structural support to the defect, and promote rapid bone regeneration. Here, we describe the design of a densified, oligomer collagen-fibril bone regeneration template inspired by the natural progression of bone defect healing. As the dominant component of the bone organic matrix and the soft callus formed first during bone fracture healing, type I collagen provides a physiologically-relevant material for promoting bone regeneration. The polymerizable oligomer collagen formulation preserves fibrillar and suprafibrillar microstructures necessary for directing mineral deposition and also facilitates cellular encapsulation with minimal shape distortion. The densified oligomer collagen-fibril materials promote accelerated and enhanced mineralization by encapsulated human adipose-derived stem cells in vitro when compared to conventional collagen-based bone regeneration strategies. This works supports the continued development of oligomer collagen-fibril bone regeneration templates to restore and preserve alveolar bone mass in vivo.