Poster N16 - Investigating Titanium Mediated Surface Effects on Osseointegration via Semaphorin 3C

Introduction: The semaphorin family is a group of nerve-derived factors involved in various processes such as cell motility, angiogenesis, cardiogenesis, tumor growth, and immune response, and acts as a coupling factor between osteoblasts and osteoclasts. Semaphorins provide a connection between neurogenesis and osteogenesis as Human bone marrow stromal cells (hBMSCs) produce these nerve factors, suggesting that semaphorins provide a connection between neurogenesis and osteogenesis during peri-implant bone formation. Osseointegration is the structural and functional connection between bone tissue and implant surface, which is highly regulated by primary bone remodeling and depends on crosstalk between osteoblasts and osteoclasts. Semaphorin 3A has shown significant effects on osteoblast differentiation while regulating osteoclasts. Semaphorin 3C (sema3C) is from the same class as semaphorin 3A, suggesting that it may also be involved. Previous research demonstrated that there may be cleavage of the sema3C protein, causing different lengths to be available and having various functions, including storage in the extracellular matrix. Therefore, the aim of this study is to evaluate the role of sema3C on osseointegration and to localize its variants.

Materials and

Methods: To determine the titanium surface mediated effect, hBMSC (osteoblast progenitor cells) and MG63 cells (preosteoblasts) were grown on tissue culture polystyrene (TCPS), smooth hydrophobic titanium (PT), or micro- roughened hydrophilic titanium (mSLA) and harvested for DNA, osteocalcin (OCN), and sema3C production. To investigate the autocrine effect of sema3C on osteoblasts, 95kD sema3C treated hBMSCs and MG63 cells were grown on TCPS, PT, mSLA, and microroughened hydrophobic titanium (SLA) and assessed for DNA and osteogenic markers via ELISA. The paracrine effect of sema3C was investigated by determining sema3C’s effects on osteoclasts. RAW264.7 (preosteoclast) cells were stimulated with RANKL and treated with 95kD sema3C or conditioned media from hBMSCs grown on TCPS, PT, SLA, or mSLA and assessed for osteoclastogenic markers via RT-PCR. To assess the localization of sema3C variants, western blots were performed on combined cell and matrix, isolated cells, and conditioned media from hBMSCs, MG63 cells, and MCF7 breast cancer cells (positive control) grown on TCPS and on SLA surfaces.

Results, Conclusions, and Discussions: hBMSCs and MG63 decreased DNA and increased OCN and sema3C on mSLA compared to TCPS and PT. 95kD sema3C treatment did not affect surface-mediated osteoblastic activity markers in hBMSCs and MG63 cells measured via OCN, bone morphogenetic protein 2 (BMP2), osteopontin (OPN), osteoprotegerin (OPG) and vascular endothelial growth factor 165 (VEGF) ELISAs. 0.2ng/mL of 95kD sema3C reduced RANKL-stimulated osteoclast activation markers, nuclear factor of activated T-cells cytoplasmic 1 (NFATC1), tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), chloride voltage-gated channel 7 (CLCN7), osteoclast-associated receptor (OSCAR), and triggering receptor expressed on myeloid cells 2 (TREM2). Conditioned media from hBMSCs grown on SLA reduced RAW264.7 expression of osteoclast activation markers, NFATc1, TRAP, and CLCN7, while conditioned media from mSLA grown hBMSCs reduced NFATc1, TRAP, and OSCAR compared to TCPS. Conditioned media from hBMSCs grown on PT showed no differences in RAW264.7 expression of osteoclast activity markers compared to TCPS. Western blots of hBMSCs, MG63 cells, and MCF7 cells on TCPS showed 95kD and 65kD variants of sema3C, found within the combined cell and matrix as well as the isolated cell, while only the 65kD variant was secreted to the conditioned media. Similar results were found on SLA surfaces where 95kD was found in the cells and matrix, while 65kD was found in the cells and the conditioned media.

These data show the production of sema3C is upregulated only on microroughened hydrophilic mSLA titanium surface. Sema3C treatment showed no surface-mediated autocrine effect but rather works via a paracrine mechanism to downregulate osteoclast activity, shown by a reduction of osteoclastogenesis markers when preosteoclast cells were treated with recombinant 95kD sema3C or conditioned media from microroughened titanium surfaces. Western blot analysis demonstrated two variants of sema3C, including full-length 95kD bound to the matrix, and the variant 65kD found in the conditioned media and cells, both on TCPS and titanium surfaces. The role of 65kD sema3C on osseointegration has yet to be fully elucidated.

Sema3C acts as a regulator of osseointegration through two variants in which the function of the full-length variant is now known, but the shorter 65kD length function will be examined in future studies.

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