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Fibroblast growth factors (FGF) are osteoblast mitogens, but their effects on bone formation are not clearly understood. Most in vitro studies examining the effects of FGFs on osteoblasts have been performed only during the initial proliferative stage of osteoblast culture. In these studies, we examined the consequential effect of acidic FGF in cultures of rat fetal diploid osteoblasts that undergo a developmental differentiation program producing a mineralized bone-like matrix. During the initial growth period (days 1-10), addition of acidic FGF (100 micrograms/ml) to actively proliferating cells increased (P < 0.05) 3H-thymidine uptake (2,515 +/- 137, mean +/- SEM vs. 5,884 +/- 818 cpm/10(4) cells). During the second stage of maturation (days 10-15), osteoblasts form multilayered nodules of cells and accumulate matrix, followed by mineralization (stage 3, days 16-29). Addition of acidic FGF to the osteoblast cultures from days 7 to 15 completely blocked nodule formation. Furthermore, addition of acidic FGF after nodule formation (days 14-29) inhibited matrix mineralization, which was associated with a marked increase in collagenase gene expression, and resulted in a progressive change in the morphology of the nodules, with only a few remnants of nonmineralized nodules present by day 29. Histochemical and biochemical analyses revealed a decrease in alkaline phosphatase and mineral content, confirming the acidic FGF-induced inhibition of nodule and matrix formation. To identify mechanisms contributing to these changes, we examined expression of cell growth and bone phenotypic markers. Addition of acidic FGF during the proliferative phase (days 7-8) enhanced histone H4, osteopontin, type I collagen, and TGF-beta mRNA levels, which are coupled to proliferating osteoblasts, and blocked the normal developmental increase in alkaline phosphatase and osteocalcin gene expression and calcium accumulation. Addition of acidic FGF to the cultures during matrix maturation (days 14-15) reactivated H4, osteopontin, type I collagen, and TGF-beta gene expression, and decreased alkaline phosphatase and osteocalcin gene expression. In an in vivo experiment, rats were treated with up to 60 micrograms/kg/day acidic FGF intravenously for 30 days. Proliferation of osteoblasts and deposition of bone occurred in the marrow space of the diaphysis of the femur in a dose-related fashion. The metaphyseal areas were unaffected by treatment. In conclusion, our data suggest that acidic FGF is a potent mitogen for early stage osteoblasts which leads to modifications in the formation of the extracellular matrix; increases in TGF-beta and collagenase are functionally implicated in abrogating competency for nodule formation. Persistence of proliferation prevented expression of alkaline phosphatase and osteocalcin, also contributing to the block in the progression of the osteoblast developmental sequence.
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PMID:Acidic fibroblast growth factor inhibits osteoblast differentiation in vitro: altered expression of collagenase, cell growth-related, and mineralization-associated genes. 872 64

In a recent in vitro study, chemical microroughening of a bioactive glass surface was shown to enhance attachment of MG-63 osteoblastic cells to glass. The current study was designed to delineate the effects of microroughening on the gene expression patterns of bone markers during osteogenesis and new bone remodeling on bioactive glass surface in vivo. With the use of a rat model of paired comparison, a portion of the medullary canal in the proximal tibia was evacuated through cortical windows and filled with microroughened or smooth bioactive glass microspheres. The primary bone-healing response and subsequent remodeling were analyzed at 1, 2, and 8 weeks, respectively, by radiography, pQCT, histomorphometry, BEI-SEM, and molecular biologic analyses. The expression of various genes for bone matrix components (type I collagen, osteocalcin, osteopontin, osteonectin) and proteolytic enzymes (cathepsin K, MMP-9) were determined by Northern analysis of the respective mRNAs. Paired comparison showed significant differences in the mRNAs levels for specific bone matrix components at 2 weeks: osteopontin was significantly higher (p =.01) and osteonectin significantly lower (p =.05) in bones filled with microroughened microspheres than in those filled with smooth microspheres. Bones filled with microrough microspheres also showed significantly increased ratios of cathepsin K and MMP-9 (both markers of osteoclastic resorption) to type I collagen (p =.02 and p =.02, respectively) at 2 weeks and a significantly increased expression of MMP-9 at 8 weeks (p =.05). The pQCT, histomorphometric, and BEI-SEM analyses revealed no significant differences in the pattern of bone-healing response. Based on these results, microroughening of a bioactive glass surface could trigger temporal changes in the expression of specific genes especially by promoting the resorption part of new bone-remodeling processes. Future studies are needed to evaluate if the observed changes of gene expression are directly related to the microrough surface of any biomaterial or are biomaterial specific.
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PMID:Molecular biologic comparison of new bone formation and resorption on microrough and smooth bioactive glass microspheres. 1263 86

Alkaline-heat-treated titanium self-forms an apatite surface layer in vivo. The aim of the present study was to materialistically characterize the surface of alkaline-heat-treated titanium immersed in simulated body fluid (AHS-TI) and to examine the differentiation behavior of osteoblasts on AHS-TI. SEM, thin-film XRD, FTIR, and XPS analyses revealed that AHS-TI contained a 1.0- micro m-thick, low-crystalline, and [002] direction-oriented carbonate apatite surface. Human osteoblast-like SaOS-2 cells were cultured on polystyrene, titanium, and AHS-TI, and RT-PCR analyses of osteogenic differentiation-related mRNAs were conducted. On AHS-TI, the expression of bone sialoprotein mRNA was up-regulated as compared with that on polystyrene and titanium (p < 0.05). On AHS-TI, the expression of osteopontin and osteocalcin mRNAs was up-regulated as compared with that on polystyrene (p<0.05). The results indicate that the apatite was bone-like and accelerated the osteogenic differentiation of SaOS-2, suggesting that alkaline-heat treatment might facilitate better integration of titanium implants with bone.
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PMID:Characterization of apatite formed on alkaline-heat-treated Ti. 1515 53

Biomimetic apatites have been reported to promote osteogenic activities in numerous in vivo and in vitro models, but the precise mechanism by which the apatite microenvironment promotes such activities is not well understood. Such mechanistic studies require reproducible model systems that are relevant to tissue engineering practices. Although two-dimensional (2D) apatite-coated polystyrene culture dishes provide practicality and reproducibility, they do not simulate the effects of the three-dimensional (3D) microenvironment and degrading polymeric substrates. A simple 3D model system to address these relevant effects, and its utilization in the investigation of apatite-promoted osteoblastic differentiation in vitro is reported in this paper. Apatite coating was achieved by sequentially immersing poly(lactide-co-glycolide) (PLGA) scaffolds into different simulated body fluids (SBF). SEM, EDX, FTIR, TEM electron diffraction confirmed the apatite coating to comprise of calcium-deficient carbonated hydroxyapatite crystals. While both apatite-coated and non-coated PLGA scaffolds supported MC3T3-E1 attachment, spreading, and proliferation, significant differences in osteoblastic differentiation were observed. Relative to non-coated controls, quantitative real-time PCR revealed significant apatite-associated suppression of alkaline phosphatase (ALP), early upregulation of osteopontin (OPN) at 3 days, and upregulation of osteocalcin (OCN) and bone sialoprotein (BSP) at 4 weeks. In summary, apatite-promoted osteoblastic differentiation can be observed in a 3D model system that is relevant to tissue engineering.
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PMID:In vitro response of MC3T3-E1 pre-osteoblasts within three-dimensional apatite-coated PLGA scaffolds. 1600 21

Many osteoporotic fracture patients are candidates for concurrent treatment with bisphosphonates and bioceramic bone graft substitutes. Osteopromotive silica-based bioactive glasses are known to induce accelerated local bone turnover and adjunct antiresorptive agents, such as zoledronic acid, may affect the process. The current study examined the effect of adjunct zoledronic acid therapy on bioactive glass incorporation. In Harlan Sprague-Dawley rats (n = 80), a standardized region of the proximal tibia was subjected to ablation of local bone marrow and filled with bioactive glass (BG) microspheres. Experimental animals received zoledronic acid (1.5 mug/kg, s.c., once a week, started 1 week before surgery) or doxycycline (a metalloproteinase inhibitor) (33 mg/kg, daily gavage) as a control agent. BG incorporation and geometric bone properties were followed by sequential pQCT imaging. The final outcome at 8 weeks was analyzed by digital radiography, histomorphometry, BEI-SEM, EDXA and muCT. The mRNA levels of markers for bone resorption (cathepsin K, TRACP, MMP-9, MMP-13) and synthesis (type I, II, III collagens, osteocalcin, osteonectin, osteopontin) were measured for determination of local bone turnover. Bones filled with BG microspheres produced 2.5-fold more intramedullary new bone than controls with bone marrow ablation only, but the BG filling delayed the recovery of pQCT strength strain index (SSI) of the bones. Adjunct therapy with zoledronic acid enhanced new bone formation on BG microspheres and particularly improved the SSI values of the BG-filled bones (P < 0.05). The zoledronic acid therapy alone (without BG filling) produced the highest amount of intramedullary new bone (6-fold more than in unfilled controls, P < 0.001) but did not show a similar benefit in SSI. The analyses of mRNA expression confirmed high local bone turnover in all bones with BG filling. At the 9th week of zoledronic acid treatment, bones with and without BG filling showed increased mRNA levels of bone resorption markers and decreased mRNA levels of markers for synthesis, indicating that a corrective resorption process was already in progress in response to massive accumulation of medullary new bone at earlier stages of the therapy. Adjunct antiresorptive therapy seems to be beneficial for incorporation of bioactive glass microspheres and does not block local natural remodeling processes. In the current model, the therapy even resulted in favorable remodeling of the tubular bone structure.
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PMID:Effect of zoledronic acid on incorporation of a bioceramic bone graft substitute. 1633 90

The aims of the present study were to fabricate a novel porous polylactic acid (PLLA) composite scaffold and evaluate the capacity of the scaffold in carrying recombinant bone morphogenetic protein 2 (rhBMP2) for engineering bone formation. The structures of the PLLA scaffolds were evaluated by SEM and the controlled release of rhBMP2 from the composite scaffolds was assayed by ELISA. Bone induction by the scaffolds loaded with or without rhBMP2 was performed in the calf muscle of twenty Wistar rats for 3, 7, 10, 14, and 28 days. Tissue specimens were examined by Masson's trichrome and von Kossa stainings, and immunohistochemistry of bone proteins. Our results indicated that a moderate foreign body reaction was found in control scaffolds, which lasted for 4 weeks. The addition of rhBMP2 to this novel scaffold dramatically alleviated the adverse responses to PLLA. Enhanced deposition of collagen matrix and endochondral formation were observed in rhBMP2-PLLA scaffolds at 7-10 days, compatible with an early release of rhBMP2 in the composite scaffolds. Bone sialoprotein and osteopontin were demonstrated simultaneously. Von Kossa staining was observed in the test group at 10-14 days. In conclusion, the PLLA scaffolds exhibited the capability of carrying rhBMP2 for inducing bone formation within 2 weeks. These results suggest that rhBMP2-PLLA scaffold may be applicable in tissue engineering.
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PMID:Bone tissue engineering with novel rhBMP2-PLLA composite scaffolds. 1722 6

Low temperature setting calcium phosphate cements (CPC) formed from reactive calcium phosphate precursors are receiving great attention in the fields of orthopaedics and tissue engineering. The purpose of this study was to evaluate the mechanical properties and osteocompatibility of a novel calcium deficient hydroxyapatite (CDSHA) with a Ca/P ratio of 1.6 developed in our laboratories and compare it to a previously developed calcium deficient hydroxyapatite (CDHA) with a Ca/P ratio of 1.5. The results demonstrated that the calcium-deficient hydroxyapatites (HA) formed from the CPCs were similar to biological HA at physiological temperature and the elastic moduli of CDHA and CDSHA were found to be 174.42 +/- 20.41 MPa (p < 0.05) and 115.86 +/- 24.8 MPa (p < 0.05), respectively. The surface morphologies of the two calcium deficient HA's formed were identical with a micro/nano porous structure as evidenced from SEM. The cellular proliferation on CDHA, and CDSHA, was comparable to the control, tissue culture polystyrene (TCPS) (p < 0.05). Alkaline phosphatase activity was significantly elevated on CDHA and CDSHA matrices at early time points when compared with the control (TCPS) (p < 0.05). Osteoblast cells gene expression on CDHA, and CDSHA showed type I collagen, alkaline phosphatase, osteocalcin, and osteopontin activity at both 7 and 14 days of culture. Thus, novel calcium-deficient HAs, CDHA, and CDSHA formed at low temperature are promising candidates for orthopaedic applications based on their ability to promote osteoblast cell adhesion and gene expression in vitro.
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PMID:Novel low temperature setting nanocrystalline calcium phosphate cements for bone repair: osteoblast cellular response and gene expression studies. 1733 35

During mineralization of the avian eggshell, there is a sequential and orderly deposition of both matrix and mineral phases. Therefore, the eggshell is an excellent model for studying matrix-mineral relationships and the regulation of mineralization. Osteopontin, as an inhibitor of crystal growth, potently influences the formation of calcium phosphate and calcium carbonate biominerals. The purpose of this study was to characterize matrix-mineral relationships, specifically for osteopontin, in the avian eggshell using high-resolution transmission (TEM) and scanning (SEM) electron microscopy to gain insight into how calcite crystal growth is structured and compartmentalized during eggshell mineralization. Osteopontin was localized at the ultrastructural level by colloidal-gold immunocytochemistry. In EDTA-decalcified eggshell, an extensive matrix network was observed by TEM and SEM throughout all regions and included interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries that compartmentalized different eggshell structural units. In fractured and undecalcified eggshell, osteopontin was immunolocalized on the {104} crystallographic faces of calcite-its natural cleavage plane. The specific occlusion of osteopontin into calcite during mineralization may influence eggshell structure to modify its fracture resistance.
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PMID:Colloidal-gold immunocytochemical localization of osteopontin in avian eggshell gland and eggshell. 1825 19

A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.
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PMID:3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells. 1845 45

Periodontal tissue engineering is expected to overcome the limitations associated with the existing regenerative techniques for the treatment of periodontal defects involving alveolar bone, cementum, and periodontal ligament. Cell-based tissue engineering approaches involve the utilization of in vitro expanded cells with regenerative capacity and their delivery to the appropriate sites via biomaterial scaffolds. The aim of this study was to establish living periodontal ligament cell-containing structures on electrospun poly(DL-lactic-co-glycolic acid) (PLGA) nanofiber membrane scaffolds, assess their viability and characteristics, and engineer multilayered structures amenable to easy handling. Human periodontal ligament (hPDL) cells were expanded in explant culture and then characterized morphologically and immunohistochemically. PLGA nanofiber membranes were prepared by the electrospinning process; mechanical tensile properties were determined, surface topography, nanofiber size, and porosity status were investigated with SEM. Cells were seeded on the membranes at approximately 50,000 cell/cm(2) and cultured for 21 days either in expansion or in osteogenic induction medium. Cell adhesion and viability were demonstrated using SEM and MTT, respectively, and osteogenic differentiation was determined with IHC and immunohistomorphometric evaluation of osteopontin, osteocalcin, and bone sialoprotein marker expression. At days 3, 6, 9, and 12 additional cell/membrane layers were deposited on the existing ones and multilayered hybrid structures were established. Results indicate the feasibility of periodontal ligament cell-containing tissue-like structures engineering with PDL cells and electrospun nanofiber PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures amenable to macroscopic handling.
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PMID:Periodontal ligament cellular structures engineered with electrospun poly(DL-lactide-co-glycolide) nanofibrous membrane scaffolds. 1849 92


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