Gene-Activated Titanium Surfaces Promote In Vitro Osteogenesis

Abstract

Commercially pure titanium (CpTi) and its alloys possess favorable mechanical and biological properties for use as implants in orthopedics and dentistry. However, failures in osseointegration still exist and are common in select individuals with risk factors such as smoking. Therefore, in this study, a proposal was made to enhance the potential of CpTi discs for osseointegration by coating their surfaces with nanoplexes comprising polyethyleneimine (PEI) and plasmid DNA encoding bone morphogenetic protein-2 (pBMP-2). The nanoplexes were characterized for size and surface charge at a range of N/P ratios. CpTi discs were surface characterized for morphology and composition before and after nanoplex coating using scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and X-ray powder diffraction (XRD). The cytotoxicity and transfection ability of CpTi discs coated with nanoplexes of varying N/P ratios in human bone marrow derived mesenchymal stem cells (BMSCs) was measured via MTS assays and flow cytometry, respectively. The CpTi discs coated with nanoplexes prepared at an N/P ratio of 10 (N/P-10) were considered optimal, resulting in 75% cell viability and 14% transfection efficiency. ELISA results demonstrated a significant enhancement in BMP-2 protein secretion by BMSCs 7 days post-treatment with CpTi discs coated with PEI/pBMP-2 nanoplexes (N/P-10), compared to the controls. Real time PCR data demonstrated that the BMSCs treated with PEI/pBMP-2 nanoplex coated CpTi discs resulted in an enhancement of runx-2, alkaline phosphatase and osteocalcin gene expressions on day 7, post-treatment. In addition, these BMSCs demonstrated enhanced calcium deposition on day 30 post-treatment as determined by qualitative (alizarin red staining) and quantitative (atomic absorption spectroscopy) assays. Thus, from all the above data it can be concluded that PEI/pBMP-2 nanoplex (N/P-10) coated CpTi discs have the potential to induce osteogenesis and enhance osseointegration.

Bone Regeneration Using Gene-Activated Matrices

“…Compared to controls, the transfection of BMSCs by PEI-pDNA (BMP-2) nanoplexes from the titanium surface resulted in enhanced expression of osteogenic markers including, runx-2, alkaline phosphatase and osteocalcin. (52) Compared to controls, enhanced calcium deposition as determined by qualitative (alizarin red staining) and quantitative (atomic absorption spectroscopy) assays was noted in transfected cells on day 30 post-treatment. (52) The results highlight the potential of this novel approach of using gene-activated titanium surfaces to enhance osseointegration.…”

“…(51) Our group developed a novel approach of coating the titanium surface with polymer-pDNA nanoplexes to enhance osseointegration. (52) We showed that discs coated with PEI-pDNA (BMP-2) nanoplexes prepared at an N/P ratio of 10 (N/P-10) resulted in 75% cell viability and 14% transfection efficiency in bone marrow stromal cells (BMSCs) in vitro . Compared to controls, the transfection of BMSCs by PEI-pDNA (BMP-2) nanoplexes from the titanium surface resulted in enhanced expression of osteogenic markers including, runx-2, alkaline phosphatase and osteocalcin.…”

“…(52) Compared to controls, enhanced calcium deposition as determined by qualitative (alizarin red staining) and quantitative (atomic absorption spectroscopy) assays was noted in transfected cells on day 30 post-treatment. (52) The results highlight the potential of this novel approach of using gene-activated titanium surfaces to enhance osseointegration.…”

A proof of concept gene‐activated titanium surface for oral implantology applications

“…Titanium disks made of commercially pure titanium were prepared as described previously (Atluri, Lee, Seabold, Elangovan, & Salem, ). Briefly, disks were sanded with a variable speed grinder–polisher (Ecomet 3, Buehler, Lake Bluff, IL, USA) using grinding papers (CarbiMet, Buehler) ascending to grit number 600.…”

Recent Advances in Musculoskeletal Tissue Regeneration

“…By implanting gene-activated matrices (GAMs) in animal models, we have shown that sustained gene expression and continuous osteogenic protein production in situ can be achieved in a way that stimulates osteogenesis and bone repair within osseous defects (1,2). We have also shown that critical parameters substantially affecting the therapeutic efficacy of gene therapy include the choice of osteogenic transgene(s), selection of vectors, the scaffold material, the wound environment, and the selection of delivery strategies (2)(3)(4)(5)(6)(7)(8). For example, we have developed a non-viral gene delivery system for bone regeneration utilizing a collagen scaffold to locally deliver complexes encoding for plateletderived growth factors (PDGF-B) (2).…”