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Biomedical subjects

Timothy A Miller

Publications and source records attributed to Timothy A Miller.

11 recordsLinked to original sources
PubMed2005

Characterization of growth and osteogenic differentiation of rabbit bone marrow stromal cells.

BACKGROUND: The rabbit is recognized as an excellent model to study the repair of bony defects with tissue engineered constructs. However, the use of rabbit bone marrow stromal cells (RBMSCs) has been limited despite the proven benefits of autologous BMSCs in the formation of bone. The purpose of this study was to characterize the growth and differentiation pattern of RBMSCs and their response to growth factors. MATERIAL AND METHODS: BMSCs were isolated from New Zealand White rabbits and cultured. Serial cell counts of parallel cultures were taken daily to determine cell growth. Response of RBMSCs to varying doses of recombinant human BMP-2 (rhBMP-2) and their time course was gauged by alkaline phosphatase (ALP) activity. The osteoblastic differentiation potential of RBMSCs in response to rhBMP-2 treatment was determined by evaluating the expression pattern of various genes involved with osteogensis using northern analysis. Von Kossa staining was performed to determine the effect of rhBMP-2 on the mineralization capabilities of RBMSCs. RESULTS: The growth rate of RBMSCs severely declined after first passage and this rate was further suppressed by TGF-beta1. The optimal dose response of rhBMP-2 was determined to be 50 ng/ml and its time course displayed increasing alkaline phosphatase activity over time. Two osteogenic markers, collagen I and osteopontin, were up regulated by rhBMP-2 treatment. Finally, the mineralization capability of RBMSCs was determined to be enhanced by rhBMP-2 treatment. CONCLUSION: Our work indicates that RBMSCs possess strong osteogenic potential and can be successfully applied toward bone tissue engineering in a rabbit model.

Animals↗
PubMed2005

The effect of biomimetic apatite structure on osteoblast viability, proliferation, and gene expression.

The conventional biomimetic apatite coating process can be accelerated by immersing substrates into concentrated simulated body fluid (5 x SBF) at 37 degrees C to form an initial coating of apatite precursor spheres, and transform the precursors into plate-like apatite structures. Depending on processing parameters, different apatite structures can be created over the same substrate. The purpose of this study is to investigate the effects of the different apatite microenvironment on cell spreading, viability, proliferation, and gene expression. MC3T3-E1 preosteoblasts were cultured on five surfaces: conventional apatite (CA), precursor apatite spheres (PreA), large plate-like apatites (LgA), small plate-like apatites (SmA), and tissue culture grade polystyrene (TCPS). PreA induced significantly higher cell death during the first two weeks. TCPS supported more uniform spreading (1 day) and higher proliferation (2 weeks) than CA, LgA, and SmA. Apatites restricted spreading and promoted the extension of cellular projections along the textured surfaces under confocal microscopy observation. By 3 weeks, LgA induced highest expression of mature osteogenic markers osteocalcin (OCN) and bone sialoprotein (BSP) in both regular and osteogenic culture media based on quantitative real-time RT-PCR. The results of this study suggest differential cell responses to subtle changes in apatite microenvironment.

3T3 Cells↗
PubMed2005

Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds.

Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(l-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions.

Alkaline Phosphatase↗
PubMed2004

BMP-2 exerts differential effects on differentiation of rabbit bone marrow stromal cells grown in two-dimensional and three-dimensional systems and is required for in vitro bone formation in a PLGA scaffold.

Osteogenic differentiation of bone marrow stromal cells (BMSC) in a three-dimensional (3-D) scaffold has not been well studied. In this work, we studied expression of bone-related genes during differentiation of rabbit BMSCs in response to bone morphogenetic protein (BMP)-2 in both 2-D and 3-D culture systems. When BMSCs were cultured on films (2-D) of biodegradable poly(lactide-co-glycolide) (PLGA), increases in mRNA expression of type I collagen (Col I) and vascular endothelial growth factor (VEGF) became evident after 1 week. However, expression of both genes was only mildly stimulated by BMP-2. Expression of the osteopontin gene was highly stimulated by BMP-2 treatment. Expression of chordin, a BMP antagonist, increased significantly after 7 days. The increase was abrogated by BMP-2 treatment. BMP-2 was also able to stimulate mineralization of cultured BMSCs. After cells were switched to 3-D PLGA scaffolds for 24 h, expression of osteopontin and VEGF were markedly increased while expression of type I collagen and chordin remained unchanged. Expression of Col I did not increase with time in a 3-D culture as it did when cells were cultured on a 2-D film. We further explored the possibility of engineering bone tissue in vitro by seeding BMSCs into PLGA scaffolds. Cellular differentiation and bone formation in the scaffolds were analyzed histologically at 2 weeks and 2 months. Secretion of ECM by cells was evident at both 2-week and 2-month scaffolds, and was enhanced by rhBMP-2. Striking differences in 2-month scaffolds were observed between BMP-treated and untreated cells. A woven bone-like structure appeared in the scaffolds treated with BMP-2. The structure was verified to be bone-related by: (1) the presence of organized collagen fibrils; (2) the presence of mineral; and (3) morphological features of trabecular bone. Although collagen was abundant in the untreated 2-month scaffolds, it was disorganized. The untreated scaffolds also lacked mineral deposits, which were present in 2-D cultured cells even in the absence of BMP-2. Our results indicate that the requirement of osteo-inductive agents, such as BMP-2, is essential for bone tissue engineering.

Animals↗
PubMed2004

Osteopontin is a negative regulator of proliferation and differentiation in MC3T3-E1 pre-osteoblastic cells.

Osteopontin (OPN) is an important mediator of bone remodeling. However, the role of OPN in the process of bone formation is not fully understood. In previous studies, we have shown that MC3T3-E1 pre-osteoblastic cells at higher passage number exhibited weakened osteogenic capacity and elevated OPN mRNA expression. In this work, we investigated the role of OPN on proliferation and differentiation of low-passage MC3T3-E1 cells by studying stable cell lines overexpressing either OPN mRNA or its antisense RNA. Overexpression was verified by both Northern and Western blot analyses. Overexpression of OPN markedly inhibited proliferation as determined by daily cell counts, while overexpression of antisense RNA stimulated cellular proliferation. We also examined the effect of OPN level on BMP-2-induced alkaline phosphatase activity. Overexpression of OPN inhibited BMP-2 responsiveness while overexpression of antisense RNA enhanced the effect of BMP-2 on alkaline phosphatase activity. Increased OPN expression also caused decreases in expression of osteocalcin and bone sialoproteins while a reduction of OPN level caused the opposite. Furthermore, endogenous OPN expression in response to BMP-2 exhibited a biphasic pattern, that is, it was initially inhibited and then enhanced by the treatment of BMP-2, indicating that OPN might function as a negative feedback regulator for osteoblastic differentiation. Finally, overexpression of OPN inhibited mineral deposition. In contrast, overexpression of antisense RNA enhanced mineral deposition. These results indicate that OPN is a negative regulator of proliferation and differentiation in MC3T3-E1 cells.

3T3 Cells↗
PubMed2002

Overexpression of BMP-2 modulates morphology, growth, and gene expression in osteoblastic cells.

Bone morphogenetic proteins (BMP) play a pivotal role in growth and differentiation of osteoblastic lineage cells. BMPs are potent stimulators of bone formation in various animal models. To understand the mechanism of BMP action in bone cells, we have investigated the effects of overexpression of the BMP-2 gene on proliferation and differentiation of UMR-106 rat osteosarcoma cells. A stable UMR-106 cell line overexpressing the BMP-2 gene was established by transfection of cells using a mammalian expression vector harboring human BMP-2 cDNA followed by G418 selection. After introduction of the BMP-2 gene, UMR-106 cells appeared more spindle-shaped in morphology compared to the predominantly cuboidal appearance of the parental cells. Overexpression of BMP-2 markedly inhibited proliferation as measured by cell counting and [3H]thymidine incorporation assays. Extracellular matrix (ECM) derived from cells overexpressing BMP-2 exhibited a less supportive effect on proliferation of UMR cells than did ECM derived from parental cells. Furthermore, cell-cell communication through gap junctions was reduced more than 50% as determined by nondisruptive fluorescent dye transfer assays. Overexpression of BMP-2 significantly stimulated expression of osteocalcin and alkaline phosphatase genes, indicating its role in osteoblastic differentiation. There was little effect on osteopontin gene expression.

Alkaline Phosphatase↗
PubMed2002

Expression and localization of estrogen receptor-beta in annulus cells of the human intervertebral disc and the mitogenic effect of 17-beta-estradiol in vitro.

BACKGROUND: Recent evidence suggests that estrogens exert effects in different tissues throughout the body, and that the estrogen receptor beta (ERbeta) may be important for the action of estrogen (17-beta-estradiol) on the skeleton. The cellular localization of ERbeta in the human intervertebral disc, however, has not yet been explored. METHODS: Human disc tissue and cultured human disc cells were used for immunocytochemical localization of ERbeta. mRNA was isolated from cultured human disc cells, and RT-PCR amplification of ERbeta was employed to document molecular expression of this receptor. Cultured human disc cells were tested to determine if 17-beta-estradiol stimulated cell proliferation. RESULTS: In this report data are presented which provide evidence for ERbeta gene expression in human intervertebral disc cells in vivo and in vitro. Culture of annulus cells in the presence of 10-7 M 17-beta-estradiol significantly increased cell proliferation. CONCLUSIONS: These data provide new insight into the biology of cells in the annulus of the intervertebral disc.

Journal Article↗
PubMed2002

Retinoids inhibit squamous cell carcinoma growth and intercellular communication.

BACKGROUND: Retinoids have been shown to inhibit the growth of squamous cell carcinoma and other malignancies. They have also been shown to alter gap junctional intercellular communication (GJIC) and the expression of connexins, the protein subunits of gap junctions. We report in this study that the alteration of GJIC by retinoids may be directly related to inhibitory effects on cell growth. MATERIALS AND METHODS: SCC-13 cells were treated with all-trans retinoic acid (tRA) and 13-cis retinoic acid (cRA) at 10(-7) and 10(-6) M concentrations in culture. No treatment and ethanol vehicle controls were included for each experiment. Serial cell counts of parallel cultures were performed to determine cell growth. The parachute technique was performed in combination with fluorescence activated cell sorting (FACS) analysis to determine GJIC. Northern and Western blot analysis were performed to assess connexin mRNA and protein expression. RESULTS: The growth rate was inhibited for cells treated with tRA (10(-6) M) (P < 0.05) and cRA (10(-6) M) (P = 0.068) vs. vehicle control. GJIC was significantly inhibited with both tRA (10(-7) and 10(-6) M) (P < 0.001) and cRA (10(-7) and 10(-6) M) (P < 0.001) at 24, 48, and 96 h as determined by FACS analysis. To correlate GJIC with cell growth, we studied the effect of glycyrrhetinic acid, a known inhibitor of GJIC. Glycyrrhetinic acid also significantly inhibited cell growth (P < 0.05) vs. control. Connexin 26 and connexin 43 mRNA and protein expression were not significantly altered after retinoid treatment. CONCLUSION: Retinoic acids inhibit both cell growth and GJIC in SCC-13 cells. Retinoids may inhibit cell growth through alteration of GJIC in SCC-13 cells.

Blotting, Northern↗