200. Effect of implant surface roughness and porosity on osteoblastic genes expression in bone marrow Mesenchymal stem cells

BACKGROUND CONTEXT 3D printing can allow for the manufacturing of sophisticated open-lattice interbody fixation devices with surface roughness which is the biproduct of the manufacturing process. Such characteristics can lead to better osteointegration and faster fusion rates to PEEK or smoothed machined titanium implants. PURPOSE We compared P3D (Rough Ti),SmTi (Smooth),and PEEK surfaces for bone differentiation using fluorescing bone marrow stem cells. Gene expression of a comprehensive panel of bone-related genes was evaluated to assess osteogenesis rate and magnitude, including a bone formation regulator and early/late bone differentiation markers. STUDY DESIGN/SETTING Lab experiment. OUTCOME MEASURES Osteogenic gene expression. METHODS BSP-GFP/DMP-RFP transgenic mouse bone marrow stem cells were used to monitor bone mineralization with fluorescently-tagged genes. Bone marrow was harvested and stem cells were cultured and expanded. The BSP and DMP proteins, located in bone tissue and involved in mineralization, were evaluated for gene activation in newly formed osteoblasts and mineral-embedded osteocytes. Low-density, first passaged cells from the primary bone marrow stromal culture (BMSCs) were seeded at 1x106 cells/ mL onto three replicate plates: P3D, Ti with smooth surface (SmTi) or PEEK (ultra-low cell attachment plates, Corning Fisher Scientific). From days 7 through 22, cell constructs were placed under osteogenic conditions (alpha MEM (ThermoFisher Cat# 12571063) supplemented with 10% FBS, 50 µg/ml ascorbic acid, and 4 mM ß-glycerophosphate). Media was replenished every 2-3 days. The plates were harvested on days 3, 7, 14, and 22 and evaluated for fluorescence and RNA expression. Cell entry into osteogenesis was determined by fluorescence intensity (GFPtopaz, mCherry, or double positive) at day 22. Negative fluorescence was defined as no osteogenesis initiation. RESULTS Significantly greater percentage of BMSC-derived progenitor cells were osteogenic on the P3D Ti surface at day 22 (p<0.05) as compared to SmTi and PEEK. Osteogenic gene expression was evaluated over time on each surface. On each surface, almost all osteogenic markers increased over time with maximum osteogenic gene expression generally occurring at approximately day 14. The magnitude of gene expression was substantially higher for most markers on the P3D surface as compared to PEEK or SmTi. At each time point, the fold-increase for osteogenic expression between P3D and PEEK or SmTi was evaluated. BMSC-derived progenitor cells cultured on P3D resulted in significantly (P<0.005) greater increases in the late osteogenic markers Bglap and Phex (indicative of osteocyte formation) by more than 1,000- and 100-fold, respectively, at day 14. At day 22, stem cells cultured on P3D showed significant increases (P<0.005) in early osteogenic markers Alp and Col1A1, by over 15- and 35-fold respectively. CONCLUSIONS 3D-printed rough Ti surface enabled a greater proportion of stem cells entering the osteogenic lineage relative to PEEK and SmTi surfaces. Stem cells on the P3D surface resulted in greater gene expression master regulators of bone differentiation, early bone markers, and markers associated with bony mineralization. Faster and more robust late-stage osteogenic differentiation was demonstrated by over a 100-fold increase in osteocalcin and Phex for P3D relative to PEEK and SmTi. These attributes may facilitate a more rapid and stable fixation of the bone-implant interface during spinal fusion. FDA Device/Drug Status This abstract does not discuss or include any applicable devices or drugs. 3D printing can allow for the manufacturing of sophisticated open-lattice interbody fixation devices with surface roughness which is the biproduct of the manufacturing process. Such characteristics can lead to better osteointegration and faster fusion rates to PEEK or smoothed machined titanium implants. We compared P3D (Rough Ti),SmTi (Smooth),and PEEK surfaces for bone differentiation using fluorescing bone marrow stem cells. Gene expression of a comprehensive panel of bone-related genes was evaluated to assess osteogenesis rate and magnitude, including a bone formation regulator and early/late bone differentiation markers. Lab experiment. Osteogenic gene expression. BSP-GFP/DMP-RFP transgenic mouse bone marrow stem cells were used to monitor bone mineralization with fluorescently-tagged genes. Bone marrow was harvested and stem cells were cultured and expanded. The BSP and DMP proteins, located in bone tissue and involved in mineralization, were evaluated for gene activation in newly formed osteoblasts and mineral-embedded osteocytes. Low-density, first passaged cells from the primary bone marrow stromal culture (BMSCs) were seeded at 1x106 cells/ mL onto three replicate plates: P3D, Ti with smooth surface (SmTi) or PEEK (ultra-low cell attachment plates, Corning Fisher Scientific). From days 7 through 22, cell constructs were placed under osteogenic conditions (alpha MEM (ThermoFisher Cat# 12571063) supplemented with 10% FBS, 50 µg/ml ascorbic acid, and 4 mM ß-glycerophosphate). Media was replenished every 2-3 days. The plates were harvested on days 3, 7, 14, and 22 and evaluated for fluorescence and RNA expression. Cell entry into osteogenesis was determined by fluorescence intensity (GFPtopaz, mCherry, or double positive) at day 22. Negative fluorescence was defined as no osteogenesis initiation. Significantly greater percentage of BMSC-derived progenitor cells were osteogenic on the P3D Ti surface at day 22 (p<0.05) as compared to SmTi and PEEK. Osteogenic gene expression was evaluated over time on each surface. On each surface, almost all osteogenic markers increased over time with maximum osteogenic gene expression generally occurring at approximately day 14. The magnitude of gene expression was substantially higher for most markers on the P3D surface as compared to PEEK or SmTi. At each time point, the fold-increase for osteogenic expression between P3D and PEEK or SmTi was evaluated. BMSC-derived progenitor cells cultured on P3D resulted in significantly (P<0.005) greater increases in the late osteogenic markers Bglap and Phex (indicative of osteocyte formation) by more than 1,000- and 100-fold, respectively, at day 14. At day 22, stem cells cultured on P3D showed significant increases (P<0.005) in early osteogenic markers Alp and Col1A1, by over 15- and 35-fold respectively. 3D-printed rough Ti surface enabled a greater proportion of stem cells entering the osteogenic lineage relative to PEEK and SmTi surfaces. Stem cells on the P3D surface resulted in greater gene expression master regulators of bone differentiation, early bone markers, and markers associated with bony mineralization. Faster and more robust late-stage osteogenic differentiation was demonstrated by over a 100-fold increase in osteocalcin and Phex for P3D relative to PEEK and SmTi. These attributes may facilitate a more rapid and stable fixation of the bone-implant interface during spinal fusion.