Blocking BMP Inhibitors Enhances HMSCs Differentiation on Microstructured Titanium Surfaces

Objective: Human mesenchymal stem cells (HMSCs) differentiate into osteoblasts on microstructured titanium (Ti) surfaces, increasing bone formation and subsequent osseointegration. Bone morphogenic proteins (BMP) regulate osteoblastic differentiation and bone formation, and are associated with implant osseointegration. However, addition of BMP had only a moderate effect on osseointegration with many side effects, possibly due to increased levels of several inhibitors to BMP, including noggin, cerberus, gremlin, and chordin induced by surface microstructure. Here, we examine if blocking BMP inhibitors can enhance HMSC differentiation on microstructured Ti surfaces.

Method: HMSCs were cultured on tissue culture polystyrene or Ti [PT (Ra<0.4μm); sandblasted/acid-etched (SLA, Ra=3.2μm); hydrophilic-SLA (modSLA)]. Expression of BMP pathway inhibitors was measured by real-time qPCR. HMSCs were treated with control IgG or with blocking antibodies to cerberus, chordin, gremlin1, or noggin every day for 7 days. HMSCs or noggin-silenced HMSCs (shNOG) were cultured on the same substrates and for 7 days. In both experiments, cell number, alkaline phosphatase specific activity (ALP), osteocalcin (OCN), OPG, TGF-β1, and VEGF were measured (n=6/condition, analyzed with ANOVA/Bonferroni’s modified Student’s t-test).

Result: Cells on SLA and modSLA increased NOG, GREM1, CER1, and CHOR gene expression. Antibody treatment against cerberus, gremlin, and noggin increased ALP, OCN, OPG, VEGF, and TGF-β1 on all surfaces, but the effect was strongest on microstructured Ti. The effect was more robust in noggin-antibody treated cells. shNOG cells produced more ALP, OCN, OPG, TGF-β1, BMP-2, and BMP-4 than HMSCs on Ti, with the greatest effect on rough surfaces, confirming the antibody blocking experiments.

Conclusion: The present study indicates that molecules associated with the BMP signaling pathway regulate HMSC differentiation on microstructured Ti surfaces. Regulating BMP inhibitors enhances HMSCs differentiation and this approach seems to be a better therapy than exogenous BMP to enhance osseointegration and peri-implant bone formation.