Incidence rates of bone fractures are on the rise due to an aging population, and nearly 10% of fractures fail to heal without exogenous treatment. With limitations on current clinical treatments, there is growing demand for the development of effective new therapies;​ bone tissue engineering (BTE) represents a promising approach to address the problem of healing bone defects.

Biomaterials are a critical component of tissue engineering strategies aimed at treating skeletal defects. In order to best facilitate or stimulate bone healing, biomaterials should demonstrate a number of key traits including biocompatibility, osteoinductivity, and osteoconductivity. Additionally, since bone is a load-bearing tissue, biomaterials used in bone defect applications should have sufficient mechanical properties to maintain structure and function when placed into a defect site. A range of materials have been used in BTE with varying degrees of success, although natural polymers – specifically the demineralized bone matrix (DBM) – exhibit osteoinductive properties and have been shown to contribute to bone healing. However, poor mechanical properties leave DBM primarily as filler or support material in clinical applications. Additionally, natural tissues must be decellularized and sterilized prior to clinical use, processes that can alter their efficacy. Therefore, we set out to modify the mechanical properties of DBM by incorporating a stiff mineral phase within the bulk of the collagenous matrix without compromising the structure and function of the native tissue.

The initial work in this dissertation was aimed at designing, building, and testing a unique automated system to reproducibly remineralize DBM. By exposing the DBM to solutions containing different mineral ions – a process we termed alternate solution immersion, or ASI – we demonstrated the ability to nucleate and grow mineral within the DBM, fabricating remineralized bone matrices (RBM). These RBM contained approximately 40% mineral by volume, and were significantly stiffer compared to the DBM starting material. We then set out to characterize the mineral formed via our ASI process. Multiple analysis techniques, including FTIR, identified the mineral as brushite, a biocompatible, FDA approved material used as a bone repair substrate. After confirming the formation of a stiff, biocompatible mineral, we set out to improve the safety of the material by reducing immunological concerns via an antigen removal (AR) step. However we noticed an unintended consequence:​ following AR treatment the ASI process resulted in more mineral and stiffer RBM compared to matrices that did not experience AR treatment. One potential mechanism of action identified was the removal of a number of non-collagenous proteins (NCPs) known to act as inhibitors of mineralization both in vivo and in vitro.

Finally, we designed an in vitro cell culture experiment to assess the ability of RBM to support the survival and differentiation of an important progenitor cell source, bone marrow-derived mesenchymal stromal cells (MSCs). We cultured MSCs on RBM, DBM, and PLG scaffolds. RBM demonstrated a tendency for improved cell adhesion and better seeding efficiency compared to the other scaffold materials. DNA content on RBM was increased nearly 10-fold compared to other groups after 3 weeks in culture. Additionally, MSCs cultured on both RBM and DBM had increased osteogenic gene expression after 1 and 2 weeks, exhibiting increases in both early and late osteogenic marker genes. These data, coupled with increased mechanical properties and demonstrated biocompatibility following ASI-remineralization, indicate RBM is an attractive substrate for further study regarding BTE applications.