Apatite/amelogenin coating on titanium promotes osteogenic gene expression.

Osteoblast differentiation and extracellular matrix production are pivotal processes for implant osseointegration or bone tissue engineering. We hypothesized that a biomimetic coating on titanium surfaces, consisting of apatite and amelogenin, would promote such processes. Human Embryonic Palatal Mesenchymal pre-osteoblasts were used as a model for the evaluation of cell adhesion and spreading patterns, as well as mRNA expression of certain osteoblastic gene products. Real-time PCR showed significant (p < 0.05) increase in expression of type I collagen, alkaline phosphatase, and osteocalcin from cells grown on titanium with an apatite/amelogenin composite, as compared with that from cells grown on a pure titanium or apatite coating only. Osteocalcin expression was specifically stimulated by amelogenin added to the culture media. Enhanced attachment and cell spreading were also observed. The biomimetic coating promoting cell adhesion and osteoblast differentiation may have great potential for future dental and biomedical applications.

Rotary culture enhances pre-osteoblast aggregation and mineralization.

Three-dimensional environments have been shown to enhance cell aggregation and osteoblast differentiation. Thus, we hypothesized that three-dimensional (3D) growth environments would enhance the mineralization rate of human embryonic palatal mesenchymal (HEPM) pre-osteoblasts. The objective of this study was to investigate the potential use of rotary cell culture systems (RCCS) as a means to enhance the osteogenic potential of pre-osteoblast cells. HEPM cells were cultured in a RCCS to create 3D enviroments. Tissue culture plastic (2D) cultures served as our control. 3D environments promoted three-dimensional aggregate formations. Increased calcium and phosphorus deposition was significantly enhanced three- to 18-fold (P < 0.001) in 3D cultures as compared with 2D environments. 3D cultures mineralized in 1 wk as compared with the 2D cultures, which took 4 wks, a decrease in time of nearly 75%. In conclusion, our studies demonstrated that 3D environments enhanced osteoblast cell aggregation and mineralization.

Altered Cbfa1 expression and biomineralization in an osteosarcoma cell line.

Osteoblast differentiation and expression are regulated by Cbfa1 transcription factors. Recent evidence suggests that Cbfa1 may also regulate bone mineralization. The purpose of this study was to characterize Cbfa1 expression in relation to mineralization in rat UMR106-01 osteoblastic cell cultures. UMR106-01 BSP cultures consistently form bone-like mineral, whereas the UI subclone mineralize gradually. BSP and UI cultures were grown for 48 h and then treated with beta-glycerophosphate. BSP cultures had alizarin red stained calcifications and mineral-like deposits within 24 h of phosphate. Atomic absorption spectroscopy measured significantly (P<0.0001) more calcium in the phosphate-treated BSP cultures than in the UI. Cbfa1 message was detected in the BSP and UI cultures, but the Cbfa1 N-terminal isoform was deficient in UI and appeared to be up-regulated in the phosphate-treated BSP cultures. Cbfa1 protein levels were also reduced in the UI. DNA sequence from the RT-PCR products was utilized to design Taqman Real-time RT-PCR reagents. Quantitative Real-time RT-PCR analysis showed that Cbfa1 mRNA levels relative to endogenous 18 s rRNA were lower in the slower mineralizing UI cultures. Furthermore, the Cbfa1 N-terminal isoform mRNA levels were significantly (P<0.001) lower in the slower mineralizing cultures. Transfection with Cbfa1 or isoform antisense caused a significant (P<0.001) reduction in mineralization. Therefore, Cbfa1 expression may be associated with bone-like mineral formation in rat UMR106-01 osteoblastic cell cultures.

Implant surface roughness affects osteoblast gene expression.

The transcription factor Cbfa1 regulates osteoblast differentiation and expression of genes necessary for the development of a mineralized phenotype. The purpose of this study was to determine if Cbfa1 and BSPII gene expression are influenced by implant surface microtopography. Osteoblasts were cultured on 600-grit (grooved) or sandblasted (roughened) cpTi implant discs. Mineralization was evaluated by Alizarin-Red-S staining. Real Time PCR was used for quantitative analysis of Cbfa1 and BSPII gene expression. Enhanced mineralization was seen in osteoblasts grown on roughened implant surfaces relative to tissue culture plastic. Real Time PCR showed significant (P < 0.05) increases in Cbfa1 gene expression in cells grown on roughened, as compared with grooved, implant surfaces. BSPII gene expression was also increased on rough surfaces in the UMR cells, but was reduced in the rat calvarial osteoblast cultures. These results suggest that osteoblast gene expression and mineralization are affected by roughened implant surface microtopographies during osseointegration of dental implants.

Osteoblast integrin adhesion and signaling regulate mineralization.

Integrin adhesion and signaling events may contribute to the progressive differentiation of the osteoblast and to the initiation of a mineralized matrix. The purpose of our study was to begin to analyze the role of integrin receptors, in particular alpha2beta1, alpha5beta1, and alphaVbeta3, regarding mediation of the initiation of a mineralized matrix. Integrin-perturbation assays were conducted in microdot cultures of UMR-106-01 Bone Sialoprotein (BSP) osteoblast cells. For phenotypic analysis, we performed bright-field microscopy and Aliziran Red S staining to analyze effects on mineralization initiation. Mineralization was reduced significantly (P < 0.001) following the addition of alpha5- or beta1-integrin subunit antibody by approximately 20% and 45%, respectively--alphaVbeta3 integrin by nearly 65%, and alpha2beta1 integrin by nearly 95%. This effect was reversible following the removal of the antiintegrin antibody. These results suggest that integrin adhesion and signaling events may contribute to the ability of this cell line to mediate the initiation of the mineralization phenotype biologically.

Early cell adhesion events differ between osteoporotic and non-osteoporotic osteoblasts.

In osteoporosis, the regenerative capacity of bone is compromised, which may involve altered osteoblast (OB) activity. This could be attributed to an inappropriate synthesis and assembly of an extracellular matrix (ECM), altered cell adhesion to the ECM, or be due to inappropriate downstream activation of adhesion-mediated signaling cascades through proteins such as focal adhesion kinase (FAK). The purpose of our study was to compare early adhesion-mediated events using previously described and characterized clinically derived OBs obtained from human patients undergoing major joint arthroplasty for osteoporosis or osteoarthritis. The presence or absence of osteoporosis was established with a radiographic index. Using light microscopy and crystal violet staining, we show that OB cells derived from sites of osteoporosis do not attach and spread as well as non-osteoporotic (OP) OB cells. OP cells initially have a more rounded morphology, and show significantly less (P < 0.001) attachment to serum-coated tissue culture plastic over a 24 h time period. Immunofluorescent labeling after 24 h of attachment showed that OP OB focal adhesions (FAs) and stress fibers were less defined, and that the OP cells were smaller and had a more motile phenotype. When normalized protein lysates were Western blotted for phosphotyrosine (PY) a band corresponding to pp125FAK was identified. FAK tyrosine phosphorylation was evident at 6 h in both the OP and non-OP OBs, but decreased or was absent through 24 h in OP OBs. These results suggest early adhesion-mediated events, such as cell adhesion, attachment, and FAK signaling via PY may be altered in OP OBs.

Primary human bone cultures from older patients do not respond at continuum levels of in vivo strain magnitudes.

Osteoporosis is characterized by excessive loss of bone mass, while exercise is believed to maintain or enhance bone mass. Since exercise marginally affects osteoporosis, we wondered whether bone cells from osteoporotic patients would fail to respond to strain. Primary human bone-like cultures were obtained from females over age 60 with hip arthroplasty procedures performed for either osteoporotic fracture (n = 8) or non-osteoporotic osteoarthrosis (n = 5). Cultures (96,000 cell/cm2) were strained in rectangular optically clear silastic wells. Three periods of uniaxial substratum strain (1000 micro-strain, 1 Hz, 10,000 cycles, sine wave) were provided every 24 h using a four-point bending, computer-controlled device. Results at a frequency of 1 Hz were compared to cultures exposed to 20 Hz with bone cells derived from one osteoarthritic subject. Alterations in protein level expression of bone-related proteins were determined using a semi-quantitative confocal approach along with enzyme (alkaline phosphatase) activity and enzyme mRNA copy number using cRNA RT-PCR. Strain did not alter levels of bone-related protein levels, enzyme activity, or steady state copy number per cell in response to strain in either group. Strained cultures from osteoporotic patients exhibited little variation from unstrained controls, while individual cultures from osteoarthritic patients exhibited increases in one protein or the other. The results suggest that bone cells from older individuals may not be responsive to continuum levels of strain anticipated with vigorous activity.

The effects of scaling procedures on epithelial cell growth on titanium surfaces.

This study examined in vitro epithelial cell growth on titanium surfaces which had been instrumented by 3 types of periodontal curets. Commercially pure titanium discs were uniformly scaled using plastic, stainless steel, or gold-coated curets and then seeded with a microdot (50,000 cells per 10 microliters) of rat gingival epithelial cells. After 5 days of growth, epithelial cell surface area (mm2) coverage was measured on photographed specimens using a computer digitizing system. Scanning electron microscopic evaluation showed a denser central core of cellular growth on the stainless steel-treated surfaces as compared to the other surface treatments. Epithelial surface area coverage on the stainless steel, plastic, and control groups did not vary significantly among groups. However the gold-coated curet exposed surfaces supported significantly less epithelial growth than the stainless steel and control surfaces. A better understanding of in vitro epithelial cell responses to different titanium surface characteristics will assist in designing implant scaling procedures which allow maintenance of the implant perimucosal seal.

Characterizations of titanium implant surfaces. III.

There are several reports in the literature concerning the similarities and the differences between the oxide on cpTi and Ti-6A1-4V alloy; however, their biological sequelae are not entirely known. In this work, a series of surface characterization techniques were used in conjunction with short term in vitro biological assays to assess the effects of materials selection (cpTi and Ti alloy) on osteoblast-like cell responses. Surface analysis indicated that with the exception of oxide thickness, there were no significant differences in surface characteristics between the two implant materials. These results were reflected in the biological studies, where the levels of cell attachment and adaptation of the attached cells to the titanium surfaces were similar. These results are in general agreement with previous in vivo studies and continue to indicate that cpTi and Ti alloy are suitable, biologically compatible materials for fabrication of dental implants.

Attachment of epithelial cells and fibroblasts to ceramic materials.

This study examined in vitro gingival epithelial and fibroblast cell attachment to ceramic materials made of tricalcium phosphate and/or magnesium aluminate spinel. The composite made of tricalcium phosphate and spinel is called 'osteoceramic'. These ceramics had various compositions and surface structures, which were initially characterized. Cell attachment assays were performed using both cell types to compare cellular response to the ceramic materials. Specimens were also prepared for scanning electron microscopy to investigate cellular morphology. The highest levels of cell attachment for gingival epithelial cells were observed on the rough osteoceramic surface, whereas gingival fibroblasts attached least to the rough osteoceramic surface.