Effects of rhBMP-2/7 Heterodimer and RADA16 Hydrogel Scaffold on Bone Formation During Rabbit Mandibular Distraction.

PURPOSE: The effects of a recombinant human bone morphogenetic protein-2/7 (rhBMP-2/7) heterodimer and a RADA16 (Ac-RADARADARADARADA-CONH2) hydrogel scaffold on bone formation during distraction osteogenesis were evaluated. MATERIALS AND METHODS: Forty New Zealand white rabbits, which underwent mandibular lengthening, were randomly divided into 5 groups. One group served as the control group. The others received 2 µg of rhBMP-2 homodimer, 2 µg of rhBMP-2/7 heterodimer, 100 µL of RADA16, or 100 µL of RADA16 plus 2 µg of rhBMP-2/7 heterodimer in the mandibular distraction gap at the beginning of distraction. Fluorine-18-labeled fluoride positron emission tomography was used to assess osteogenesis both after distraction and at the end of consolidation. Dual-energy x-ray absorptiometry (DEXA) examination and bone histologic findings were also evaluated. RESULTS: At the end of distraction, the radioactivity concentration in the distracted area was significantly greater in the RADA16 plus rhBMP-2/7 heterodimer group than in the other groups (P < .01). The differences among the other 4 groups were also statistically significant in the following order: rhBMP-2/7 heterodimer group greater than the rhBMP-2 homodimer group, which was greater than the RADA16 group (or control group; P < .05). However, the radioactivity concentration of the RADA16 group was slightly greater than that of the control group with a nonsignificant difference (P > .05). By the end of consolidation, the activity in the control group, RADA16 group, rhBMP-2 homodimer group, and rhBMP-2/7 heterodimer group had significantly diminished (P < .05). However, the activity in the RADA16 plus rhBMP-2/7 heterodimer group remained at the same level (P > .05). The DEXA results and bone histologic findings indicated that more callus regeneration was noted in the RADA16 plus rhBMP-2/7 heterodimer group than in any other group. CONCLUSIONS: The use of rhBMP-2/7 heterodimer and RADA16 hydrogel scaffold significantly promoted mandibular distraction osteogenesis.

Enhanced attachment, proliferation, and differentiation of human gingival fibroblasts on titanium surface modified with biomolecules.

Surface modification of dental implants with biomolecules is of particularly interest recently. To mimic the structure and function of native extracellular matrix (ECM), a derivative of hyaluronic acid (HA), HA-GRGDSP, was synthesized, Arg-Gly-Asp (RGD)-containing collagen (Col)/HA multilayer polyelectrolyte films (MPFs) coating was fabricated on titanium (Ti) through alternate deposition of Col and HA-GRGDSP with 4.5 assembly cycles; moreover, bioactive molecule, basic fibroblast growth factor (bFGF), was also incorporated into such coating. This coating was then carefully characterized using scanning electronic microscope (SEM) and scanning force microscopy (SFM); bFGF release from the coating was also evaluated. (Col + bFGF)/HA-RGD coating was successfully deposited on Ti surface, and about 300 pg of bFGF could be slowly released from this coating for a week. This coating significantly promoted the initial cell attachment of human gingival fibroblasts (HGFs) compared with other groups (p < 0.05), and HGFs adhered and spread better on this coating than other groups (p < 0.05). Regarding cell proliferation and differentiation of HGFs, they were greatly stimulated when cultured on this coating (p < 0.05). These results indicated that surface modification of Ti using biomolecules might improve the sealing between the neck section of a dental implant and the soft tissue.

Effect of heat treatment on H2O2/HCl etched pure titanium dental implant: an in vitro study.

BACKGROUND: Surface chemistry of dental implant plays an important role in osseointegration. Heat treatment might alter surface chemistry and result in different biological response. The aim of this study was to investigate the roles of heat treatment of H2O2/HCl-treated Ti implants in cell attachment, proliferation and osteoblastic differentiation. MATERIAL/METHODS: Sandblasted, dual acid-etched and H2O2/HCl heat-treated discs were set as the control group and sandblasted, dual acid-etched H2O2/HCl-treated discs were the test group. Both groups' discs were sent for surface characterization. MC3T3-E1 cells were seeded on these 2 groups' discs for 3 hours to 14 days, and then cell attachment, cell proliferation and cell differentiation were evaluated. RESULTS: Scanning electron microscope analysis revealed that the titanium discs in the 2 groups shared the same surface topography, while x-ray diffraction examination showed an anatase layer in the control group and titanium hydride diffractions in the test group. The cell attachment of the test group was equivalent to that of the control group. Cell proliferation was slightly stimulated at all time points in the control group, but the alkaline phosphatase (ALP) activity and osteocalcin (OC) production increased significantly in the test group compared with those in the control group at every time point investigated (p<0.05 or p<0.01). Moreover, the osteoblastic differentiation-related genes AKP-2, osteopontin (OPN) and OC were greatly up-regulated in the test group (p<0.05 or p<0.01). CONCLUSIONS: The results implied that surface chemistry played an important role in cell response, and H2O2/HCl etched titanium surface without subsequent heat treatment might improve osseointegration response.

The optimal dose of recombinant human osteogenic protein-1 enhances differentiation of mouse osteoblast-like cells: an in vitro study.

OBJECTIVE: There is no certain conclusion on the effect of recombinant human Osteogenic Protein-1 (OP-1, BMP-7) on the proliferation of the osteoblast-like cell line, MC3T3-E1. Furthermore, the optimal dose of rhOP-1 on cell differentiation still needs to be elucidated. This investigation aims to delineate the biofunctional characteristics of rhOP-1 in inducing osteoblastogenesis of MC3T3-E1 through in vitro time-course and dose-response studies. DESIGN: MC3T3-E1 cells were cultured for 1, 4, 7 days with the addition of different rhOP-1 concentrations (0, 10, 20, 50, 100, 200, 400 ng/ml), and cell proliferation and cell differentiation were examined. RESULTS: MC3T3-E1 cell proliferation was stimulated by rhOP-1 in a dose-dependent manner (0-400 ng/ml) on day 1, whereas on day 4 and 7, it was still stimulated at low concentrations (10, 20, 50 ng/ml) but inhibited at high ones (200, 400 ng/ml). The alkaline phosphatase (ALP) activity, osteocalcin (OC) production, collagen deposition and extracellular matrix mineralization were dramatically elevated by rhOP-1 treatment, as a function of culture time and rhOP-1 concentration, and all of them reached a plateau at the concentration of 200 ng/ml. Real-time quantitative RT-PCR results showed Runx2, AKP-2, OC and Nog mRNA expressions increased in a dose- and time-dependent manner, and their expressions were significantly higher at high rhOP-1 concentrations than that of low ones. No significant differences were found between the effects of 200 ng/ml rhOP-1 and 400 ng/ml rhOP-1 on the differentiation of MC3T3-E1 cells, except the expression of Nog mRNA, whose expression level was much higher at 400 ng/ml than that at 200 ng/ml. CONCLUSIONS: These results suggest that cell proliferation of MC3T3-E1 is depended on culture time and rhOP-1 concentration, rhOP-1 could stimulate the differentiation of MC3T3-E1 cells and the optimal concentration could be 200 ng/ml.

H2O2/HCl and heat-treated Ti-6Al-4V stimulates pre-osteoblast proliferation and differentiation.

The purpose of the present study was to evaluate the bioactivity of chemical treatment of titanium alloy (Ti-6Al-4V) in vitro. Smooth-surface discs of Ti-6Al-4V were used in this study. Sandblasted, dual acid-etched and H(2)O(2)/HCl heat-treated discs were set as test group, and sandblasted, dual acid-etched discs as control group. SEM and XRD analysis revealed a porous anatase gel layer on rough surface in the test group and a rough surface in the control group. Mouse pre-osteoblasts (MC3T3-E1 cells) were cultured on these 2 group discs, and then cell proliferation and differentiation were examined 4 days, 7 days, and 14 days after cell seeding. Cell proliferation was greatly stimulated at all time points when cultured in test group (P < .05). The alkaline phosphatase (ALP) activity and osteocalcin (OC) production were much higher in the test group compared with the control group at every time point investigated (P < .05). Furthermore, in the test group, the expressions of alkaline phosphatase-2, osteocalcin, and collagen type I alpha 1 mRNAs were significantly up-regulated as compared with those in the control group (P < .05 or P < .01). The results suggested that H(2)O(2)/HCl and heat-treatment might facilitate better integration of Ti-6Al-4V implants with bone.

Designer self-assembling peptide scaffold stimulates pre-osteoblast attachment, spreading and proliferation.

A new peptide scaffold was made by mixing pure RADA16 (Ac-RADARADARADARADA-CONH2) and designer peptide RGDA16 (Ac-RADARGDARADARGDA-CONH2) solutions, and investigate any effect on attachment, spreading and proliferation of pre-osteoblast (MC3T3-E1). The peptides, RADA16 and RGDA16, were custom-synthesized. They were solubilized in deionized water at a concentration of 10 mg/ml (1% w/v), the RGDA16 peptide solution was mixed 1:1 with RADA16 solution and a new peptide solution RGDAmix was produced. The RGDAmix and RADA16 solution were directly loaded in 96-well plates and cover slips, and two different peptide scaffolds were formed with the addition of maintenance medium (alpha-MEM) in several minutes. About 1.0 x 10(4) MC3T3-E1 cells were seeded on each hydrogel scaffold, and then the cell morphological changes were observed using a fluorescence microscope at 1 h, 3 h and 24 h timepoint, respectively. Cell attachment was evaluated 1 h, 3 h and 24 h after cell seeding and cell proliferation was determined 4d, 7d and 14d after cell seeding. The RGDAmix scaffold significantly promoted the initial cell attachment compared with the RADA16 scaffold. MC3T3-E1 cells adhered and spread well on both scaffolds, however, cells spread better on the RGDAmix scaffold than on the RADA16 scaffold. Cell proliferation was greatly stimulated when cultured on RGDAmix scaffold. The RGD sequence contained peptide scaffold RGDAmix significantly enhances MC3T3-E1 cells attachment, spreading and proliferation.