Statement of Clinical Significance: There remains the need to develop materials and surfaces that can increase the rate of implant osseointegration. Though osteoanabolic agents, like bone morphogenetic protein (BMP), can provide signaling for osteogenesis, the appropriate design of implants can also produce an innate cellular response that may reduce or eliminate the need to use additional agents to stimulate bone formation. Studies show that titanium implant surfaces that mimic the physical properties of osteoclast resorption pits regulate cellular responses of bone marrow stromal cells (MSCs) by altering cell morphology, transcriptomes, and local factor production to increase their differentiation into osteoblasts without osteogenic media supplements required for differentiation of MSCs on tissue culture polystyrene (TCPS). The goal of this study was to determine how cells in contact with biomimetic implant surfaces regulate the microenvironment around these surfaces in vitro. Two different approaches were used. First, unidirectional signaling was assessed by treating human MSCs grown on TCPS with conditioned media from MSC cultures grown on Ti6Al4V biomimetic surfaces. In the second set of studies, bidirectional signaling was assessed by coculturing MSCs grown on mesh inserts that were placed into culture wells in which MSCs were grown on the biomimetic Ti6Al4V substrates. The results show that biomimetic Ti6Al4V surface properties induce MSCs to produce factors within 7 days of culture that stimulate MSCs not in contact with the surface to exhibit an osteoblast phenotype via endogenous BMP2 acting in a paracrine signaling manner.

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The aim of this study was to examine if soluble factors produced by MSCs in contact with titanium-aluminum-vanadium implants possessing a complex biomimetic topography can stimulate osteogenesis ectopically. Ti6Al4V disks were grit-blasted and acid-etched to create surfaces possessing macroscale and microscale roughness (MM), microscale and nanoscale topography (MN), and macro-/micro-/nano-scale topography (MMN). Polyether-ether-ketone (PEEK) disks were fabricated by machining to medical-grade specifications. Cellular response of MSCs was quantified by proteins and local factors present in the conditioned media (CM) of MSCs grown in growth media (GM) for 14 days. Subsequently, MSCs were cultured on tissue culture polystyrene (TCPS) and on MMN disks and the culture-conditioned media (CM) were collected, lyophilized, and used to examine ectopic bone formation in the gastrocnemius of athymic nude mice (ANMs), based on a modified ASTM2529-13 standard guide for assessing osteoinductivity of demineralized bone matrix (DBM). We then compared osteoinduction by iDBM, iDBM+rhBMP2 (R&D Systems) at a concentration similar to in vitro production of MSCs on MMNTM surfaces (MMN-CM concentration), and iDBM+MMN-CM. MMN-CM was collected from MSCs grown on MMNTM surfaces for D5-D10, pooled, and lyophilized. Bone formation was assessed by micro-CT and histomorphometry (H&E staining) with the histology scored according to ASTM 2529-13. MSCs cultured on MMN surfaces differentiate and produce an increase in local factors, including, but not limited to, BMP2. Assessed with a gel-capsule carrier in a mouse muscle pouch model, a combination of paracrine factors in the CM of MMN cultures can induce ectopic bone formation compared to control groups. These results indicate that the increased bone formation arises from the local response by MSCs to a biomimetic, multi scale surface topography. Furthermore, by producing an osteoinductive microenvironment, the MSCs may support better osseointegration due to the local effects of surface-dependent osteogenic differentiation.