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Osteoblast adhesion on poly(L-lactic acid)/polystyrene demixed thin film blends: effect of nanotopography, surface chemistry, and wettability
Lim, J. Y., J. C. Hansen, et al. (2005), Biomacromolecules 6(6): 3319-27.
Abstract: Biomaterial surface characteristics are critical cues that regulate cell function. We produced a novel series of poly(l-lactic acid) (PLLA) and polystyrene demixed nanotopographic films to provide nonbiological cell-stimulating cues. The increase in PLLA weight fraction (phi) in blend solutions resulted in topography changes in spin-cast films from pit-dominant to island-dominant morphologies having nanoscale depth or height (3-29 nm). Lower molecular weight PLLA segregated to the top surface of demixed films, as observed by X-ray photoelectron spectroscopy and secondary ion mass spectroscopy (SIMS). For phi > or = 0.5, the topmost film layer was predominantly filled with PLLA (>96% by SIMS at 20-A depth). Nanotextured substrata stimulated osteoblastic cell adhesion to a greater degree than did flat PLLA (phi = 1), and this effect was more pronounced for nanoisland (phi = 0.7 and 0.9) relative to nanopit topographies (phi = 0.5). Demixed films having relatively lower water contact angles generally enhanced cell adhesion and spreading. Our results reveal that cell adhesion is affected by surface chemistry, topography, and wettability simultaneously and that nanotextured surfaces may be utilized in regulating cell adhesion.

Osteoblast cell adhesion on a laser modified zirconia based bioceramic
Hao, L., J. Lawrence, et al. (2005), J Mater Sci Mater Med 16(8): 719-26.
Abstract: Due to their attractive mechanical properties, bioinert zirconia bioceramics are frequently used in the high load-bearing sites such as orthopaedic and dental implants, but they are chemically inert and do not naturally form a direct bond with bone and thus do not provide osseointegration. A CO2 laser was used to modify the surface properties with the aim to achieve osseointegration between bioinert zirconia and bone. The surface characterisation revealed that the surface roughness decreased and solidified microstructure occurred after laser treatment. Higher wettability characteristics generated by the CO2 laser treatment was primarily due to the enhancement of the surface energy, particularly the polar component, determined by microstructural changes. An in vitro test using human fetal osteoblast cells (hFOB) revealed that osteoblast cells adhere better on the laser treated sample than the untreated sample. The change in the wettability characteristics could be the main mechanism governing the osteoblast cell adhesion on the YPSZ.

Osteoblast differentiation with titania and titania-silica-coated titanium fiber meshes
Meretoja, V. V., A. E. De Ruijter, et al. (2005), Tissue Eng 11(9-10): 1489-97.
Abstract: Two surface-reactive sol-gel coatings, namely titania (TiO2) and a mixture of titania and silica (TiSi), were applied to titanium fiber meshes. Differentiation of rat bone marrow stromal cells toward an osteogenic phenotype with coated and uncoated (cpTi) substrates was compared. The amount of DNA in cpTi and TiSi matrices did not increase after day 3, but with TiO2 matrices the amount increased for 7 days. The prolonged period of proliferation with TiO2 scaffolds resulted in a delay in alkaline phosphatase induction. However, osteocalcin incorporation into extracellular matrix by day 14 was greater with TiO2 scaffolds than with cpTi scaffolds. Calcium deposition was also greater with TiO2-coated substrates than with uncoated substrates. With the TiSi scaffolds osteocalcin production and mineralization were lower than with the cpTi scaffolds. The current study confirms our previous findings that titanium fiber mesh supports attachment, growth, and differentiation of rat bone marrow stromal cells. Furthermore, the osteogenic capacities of cell-scaffold constructs under cell culture conditions were increased with a sol-gel-derived titania coating, but not with a titania-silica coating.

Osteoblast growth promotion by protein electrostatic self-assembly on biodegradable poly(lactide)
Zhu, H., J. Ji, et al. (2005), J Biomater Sci Polym Ed 16(6): 761-74.
Abstract: Extracellular matrix (ECM)-like coating was developed on biodegradable biomaterials based on the electrostatic self-assembly (ESA) technique to promote osteoblast growth. Poly(ethylenimine) (PEI) was first employed to obtain a stable positively charged surface on poly (DL-lactide) (PDL-LA) substrate. Gelatin was selected as ECM-like biomacromolecule to deposit on the activated PDL-LA substrate using the ESA technique. zeta-Potential results showed alternating charge of polyelectrolytes (PEI/gelatin) layering on PDL-LA microspheres. Quartz crystal microbalance (QCM) measurement further verified the gradual deposition of PEI/gelatin on PDL-LA thin film. Osteoblast cells (MC3T3) were chosen to test the cell behavior on modified PDL-LA substrates. The osteoblast test about cell activity, intracellular total DNA content, total protein content and cell morphology by SEM investigation on ECM-like multilayer-modified PDL-LA substrate showed to promote osteoblast growth. Comparing conventional coating methods, polyelectrolyte multilayers are easy and stable to prepare. It may be a good choice for the surface modification of complex biomedical devices. These very flexible systems allow broad medical applications for drug delivery and tissue engineering.

Osteoblast interactions with calcium phosphate ceramics modified by coating with type I collagen
Brodie, J. C., E. Goldie, et al. (2005), J Biomed Mater Res A 73(4): 409-21.
Abstract: Complications associated with the use of autogenous bone in the repair or replacement of tissue lost through injury or disease have driven the search for alternative sources of graft material. Bioceramics containing hydroxyapatite (HA), tricalcium phosphate (TCP), or composites that combine the best properties of both of these materials are among the principal candidates. In this study, we have investigated the in vitro proliferation, morphology, and viability of an immortalized rat osteoblast cell line cultured on HA, TCP, and composites of the two in the ratios 75:25 (H75), 50:50 (H50), and 25:75 (H25) for 28 days. The biocompatibility of each material was examined in the presence and absence of a collagen coating. With the exception of H50, cell proliferation, quantified by carboxyfluorescein fluorescence, was enhanced by collagen coating of all materials for the first 14 days, although at later time points cell numbers were unaffected. It is notable that the collagen coating was least stable on H50, the only material not to show enhancement of cell growth on coating. Confocal laser scanning microscopy confirmed that cell growth was more extensive on coated materials over the first 7-14 days in culture, and the development of cell extensions and bridges across the pores in the materials was observed. Results indicate that collagen coating of calcium phosphate ceramics may also increase their compatibility and osseointegration in vivo.

Osteoblast responses one hour after load-induced fluid flow in a three-dimensional porous matrix
Tanaka, S. M., H. B. Sun, et al. (2005), Calcif Tissue Int 76(4): 261-71.
Abstract: When bone is loaded, substrate strain is generated by the external force and this strain induces fluid flow that creates fluid shear stress on bone cells. Our current understanding of load-driven gene regulation of osteoblasts is based primarily on in vitro studies on planer two-dimensional tissue culture substrates. However, differences between a flat layer of cells and cells in 3-dimensional (3D) ECM are being recognized for signal transduction. Proliferation and differentiation of osteoblasts are affected by substrate geometry. Here we developed a novel 3D culture system that would mimic physiologically relevant substrate strain as well as strain-induced fluid flow in a 3D porous collagen matrix. The system allowed us to evaluate the responses of osteoblasts in a 3D stress-strain environment similar to a mechanical field to which bone is exposed. Using MC3T3-E1 osteoblasts grown in the 3D collagen matrix with and without hydroxyapatite deposition, we tested the role of strain and the strain-induced fluid flow in the expression of the load-responsive genes such as c-fos, egr1, cox2, osteopontin, and mmp1B involved in transcriptional regulation, osteogenesis, and rearrangement of ECM. Strain-induced fluid flow was visualized with a microspheres approximately 3 microm in diameter in real time, and three viscoelastic parameters were determined. The results obtained by semi-quantitative PCR, immunoblot assay, enzymatic activity assays for collagenase and gelatinase, and mechanical characterization of collagen matrices supported the dominant role of strain-induced fluid flow in expression of the selected genes one hour after the mechanical treatment.

Osteocalcin enhances bone remodeling around hydroxyapatite/collagen composites
Rammelt, S., M. Neumann, et al. (2005), J Biomed Mater Res A 73(3): 284-94.
Abstract: The effect of osteocalcin (OC), an extracellular bone matrix protein, on bone healing around hydroxyapatite/collagen composites was investigated. Cylindrical nanocrystalline hydroxyapatite implants of 2.5-mm diameter containing 2.5% biomimetically mineralized collagen type I were inserted press-fit into the tibial head of adult Wistar rats. To one implant group, 10 mug/g OC was added. Six specimens per group were analyzed at 2, 7, 14, 28, and 56 days. After 14 days, newly formed woven bone had reached the implant surface of the OC implants whereas a broad fibrous interface could still be observed around controls. Woven bone was formed directly around both implant groups after 28 days and had been replaced partially by lamellar bone around the OC implants only. No significant differences in total bone contact were seen between both groups after 56 days. The higher number of phagocytosing cells and osteoclasts characterized immunohistochemically with ED1, cathepsin D, and tartate-resistant alkaline phosphatase around the OC implants at the early stages of bone healing suggests an earlier onset of bone remodeling. The earlier and increased expression of bone-specific matrix proteins and multifunctional adhesion proteins (osteopontin, bone sialoprotein, CD44) at the interface around the OC implants indicates that OC may accelerate bone formation and regeneration. This study supports the observations from in vitro studies that OC activates both osteoclasts and osteoblasts during early bone formation.

Osteochondral repair in the rabbit model utilizing bilayered, degradable oligo(poly(ethylene glycol) fumarate) hydrogel scaffolds
Holland, T. A., E. W. Bodde, et al. (2005), J Biomed Mater Res A 75(1): 156-67.
Abstract: In this study, hydrogel scaffolds, based on the polymer oligo(poly(ethylene glycol) fumarate) (OPF), were implanted into osteochondral defects in the rabbit model. Scaffolds consisted of two layers-a bottom, bone forming layer and a top, cartilage forming layer. Three scaffold formulations were implanted to assess how material composition and transforming growth factor-beta1 (TGF-beta1) loading affected osteochondral repair. Critical histological evaluation and scoring of the quantity and quality of tissue in the chondral and subchondral regions of defects was performed at 4 and 14 weeks. At both time points, no evidence of prolonged inflammation was observed, and healthy tissue was seen to infiltrate the defect area. The quality of this tissue improved over time with hyaline cartilage filling the chondral region and a mixture of trabecular and compact bone filling the subchondral region at 14 weeks. A promising degree of Safranin O staining and chondrocyte organization was observed in the newly formed surface tissue, while the underlying subchondral bone was completely integrated with the surrounding bone at 14 weeks. Material composition within the bottom, bone-forming layer did not appear to affect the rate of scaffold degradation or tissue filling. However, no bone upgrowth into the chondral region was observed with any scaffold formulation. TGF-beta1 loading in the top layer of scaffolds appeared to exert some therapeutic affect on tissue quality, but further studies are necessary for scaffold optimization. Yet, the excellent tissue filling and integration resulting from osteochondral implantation of these OPF-based scaffolds demonstrates their potential in cartilage repair strategies.

Osteoclastic differentiation by mononuclear phagocytes containing biomaterial particles
Sabokbar, A., R. Pandey, et al. (1998), Arch Orthop Trauma Surg 117(3): 136-40.
Abstract: Aseptic loosening of implant components is a common and important complication of both cemented and uncemented prosthetic joint replacements. Wear particles derived from organic polymer and metal implant biomaterials are commonly found within macrophages and macrophage polykaryons in the fibrous membrane between loose implant components and the host bone undergoing resorption. In order to determine whether biomaterial particle-containing, foreign-body macrophages may contribute to periprosthetic bone resorption, we cultured murine monocytes that had phagocytosed particles of biomaterials commonly employed in bone implant surgery [polymethylmethacrylate (PMMA), ultra-high molecular weight polyethylene (PE), titanium and chromium-cobalt] on bone slices and glass coverslips with UMR 106 osteoblast-like stromal cells in the presence of 1,25-dihydroxy-vitamin D3. Under these conditions, all biomaterial particle-containing, foreign-body macrophages differentiated into osteoclastic cells, i.e. tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells capable of extensive lacunar bone resorption. This study shows that particle phagocytosis by macrophages does not abrogate the ability of these cells to undergo osteoclast differentiation. These findings emphasise the importance of the foreign-body macrophage response to biomaterial wear particles in the pathogenesis of aseptic loosening.

Osteoclastic resorption of calcium phosphate coatings applied with electrostatic spray deposition (ESD), in vitro
Siebers, M. C., K. Matsuzaka, et al. (2005), J Biomed Mater Res A 74(4): 570-80.
Abstract: Calcium phosphate (CaP) coatings have been applied on titanium implants to improve the bioactivity in order to favor the initial bone healing response. Recently, a new technique has been developed to apply CaP coatings: electrostatic spray deposition (ESD). Although ESD-derived coatings have several benefits, it is not known whether they are degradable. This study was designed to examine the cell-mediated degradation of two ESD-derived coatings with different chemical compositions, that is, beta-tricalcium phosphate (beta-TCP) and carbonate apatite (CA). First, coatings were deposited and analyzed physiochemically. Subsequently, rat bone marrow-derived osteoclastlike cells were seeded on the coatings, and analyzed with osteoclast-specific markers, scanning electron microscopy, and transmission electron microscopy. Results showed that both coatings exhibited porous morphologies, with an average pore size of less than 1 microm (beta-TCP), or larger than 1 microm (CA). After heat treatment, both coatings were crystalline in structure. The Ca/P ratios were 1.4 to 1.5 for the beta-TCP coating, and 1.8 to 2.0 for the CA coating. After 8 and 12 days of culture, multinucleated osteoclastlike cells were observed on both coatings. The osteoclast phenotype was confirmed by tartrate resistant acid phosphatase (TRAP) staining, and immunostaining against the calcitonin receptor. Using scanning electron microscopy, numerous resorption lacunae were observed in both coatings. Finally, transmission electron microscopy of TRAP-positive cells confirmed the osteoclastlike aspect of the cells revealing multiple nuclei and a ruffled border. In conclusion, CaP coatings produced with the ESD process can be degraded by osteoclasts.

Osteoclastic resorption of Ca-P biomaterials implanted in rabbit bone
Basle, M. F., D. Chappard, et al. (1993), Calcif Tissue Int 53(5): 348-56.
Abstract: The nature of the multinucleated cells involved in the resorption processes occurring inside macroporous calcium-phosphate biomaterials grafted into rabbit bone was studied using light microscopy, histomorphometric analysis, enzymatic detection of tartrate-resistant acid phosphatase (TRAP) activity, scanning, and electron microscopy. Samples were taken at days 7, 14, and 21 after implantation. As early as day 7, osteogenesis and resorption were observed at the surface of the biomaterials, inside the macropores. Resorption of both newly formed bone and calcium-phosphate biomaterials was associated with two types of multinucleated cells. Giant multinucleated cells were found only at the surface of the biomaterials; they showed a large number of nuclei, were TRAP negative, developed no ruffled border, and contained numerous vacuoles with large accumulation of mineral crystals from the biomaterials. Osteoclasts exhibited TRAP positivity and well-defined ruffled border. They were observed at the surface of both newly formed bone and biomaterials, around the implant, and inside the macropores. In contract with the biomaterials, infoldings of their ruffled border were observed between the mineral crystals, deeply inside the microporosity. The microporosity of the biomaterials (i.e., the noncrystalline spaces inside the biomaterials) increased underneath this type of cell as compared with underneath giant cells or to the depth of the biomaterials. These observations demonstrate that macroporous calcium-phosphate biomaterials implanted in bone elicit osteogenesis and the recruitment of a double multinucleated cell population having resorbing activity: giant multinucleated cells that resorb biomaterials and osteoclasts that resorb newly formed bone and biomaterials.

Osteoclasts resorb protein-free mineral (Osteologic discs) efficiently in the absence of osteopontin
Contractor, T., B. Babiarz, et al. (2005), In Vivo 19(2): 335-41.
Abstract: Osteopontin (OPN) is both a matrix protein in mineralized tissues and a cytokine, and it has a pivotal role in osteoclast-mediated bone resorption. Here, using a proprietary hydroxyapatite substitute for bone mineral (Osteologic discs), we investigated the requirement for OPN in mineral resorption. Resorption pits formed in the Osteologic discs, revealed by staining with silver nitrite (Von Kossa stain), were analyzed using the NIH Image J program, which can determine the number of pits formed per unit area, their average size, and the fractional area resorbed. After a preincubation of bone marrow cells from OPN -/- and OPN +/+ mice with M-CSF to allow the multiplication of osteoclast precursors on cell culture plastic, osteoclast formation on both Osteologic discs and standard cell culture plates was induced with soluble receptor activator of NFkappaB ligand, sRANKL. We did not detect a dramatic difference in osteoclast formation between OPN +/+ and OPN -/- cells, as judged by staining for tartrate-resistant acid phosphatase in osteoclasts formed on cell culture plastic, nor was there a significant difference in the ability of the osteoclasts to form resorption pits in the Osteologic discs. Additionally, none of six different anti-OPN monoclonal antibodies had a significant and reproducible effect on the formation or subsequent functioning of the OPN+/+ osteoclasts. These studies suggest that, in contrast to what has been found for normal bone, the efficiency of dissolution of a ceramic, protein-free (excepting protein adsorbed from the culture medium) hydroxyapatite/tri-calcium phosphate substrate by osteoclasts is not substantially enhanced by endogenous or exogenous OPN.

Osteogenic cell contact with biomaterials influences phenotype expression
Kudelska-Mazur, D., M. Lewandowska-Szumiel, et al. (2005), Cell Tissue Bank 6(1): 55-64.
Abstract: Relationship between (1) osteoblast adhesion and spreading, and (2) phenotype expression was investigated. Cellular adhesion and spreading were estimated after short time (24 h), whereas proliferation and other osteoblast functions--after 7 days. Primary human osteogenic cells were seeded on the samples of titanium (T), surgical steel (S) and tissue culture polystyrene (PS), and incubated at 37 degrees C. After 24 h a number of samples were stained with crystal violet and Hoechst; the average single cell area (spreading) and adhering cell number was measured on each sample. The remaining cultures were supplemented with dexamethasone (10 nM) and beta-glycerophosphate (5 mM), and incubation was continued for 7 days. The cells on each sample were counted and the following tests were performed: XTT mitochondrial activity assay, total protein content, alkaline phosphatase activity (ALP), Sirius Red test for collagen, osteocalcin and calcium concentration. After 24 h significantly greater cell spreading (p < 0.05) and number (p < 0.05) were on T than on S. After 7 days significantly higher on T than on S were: ALP activity (p < 0.000001), collagen (p < 0.0015) and calcium concentration (p < 0.03). XTT results were bigger on S than on T. In control - XTT results were higher than on the metals; collagen and ALP were lower than on T, and calcium level was significantly lower than on T and S (p < 0.025). After 7 days there were no differences in cell number between T and S. Cell number (24 h) correlated with ALP activity (7 days) on steel (coefficient of correlation, CC = 0.866) and titanium (CC = 0.742). The spreading correlated on steel and on titanium with calcium concentration (CC = 0.645 on S, CC = 0.696 on T) and collagen level (CC = -0.638 on S, CC = -0.69 on T). CONCLUSIONS: Better conditions for osteoblast phenotype expression on T after 7 days of culture coincided with greater adhesion and spreading of cells after 24 h on T, as compared with S. The initial contact of cells with underlying surface may influence osteoblast functions and possibly, bone regeneration and implant osteointegration in vivo. Early cell spreading may be an indicator of further expression of osteoblast phenotype and may be important for application of osteogenic cells in reconstructive surgery.

Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds
Jarrahy, R., W. Huang, et al. (2005), Am J Physiol Cell Physiol 289(2): C408-14.
Abstract: 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.

Osteogenic differentiation within intact human embryoid bodies result in a marked increase in osteocalcin secretion after 12 days of in vitro culture, and formation of morphologically distinct nodule-like structures
Cao, T., B. C. Heng, et al. (2005), Tissue Cell 37(4): 325-34.
Abstract: Osteogenic lineages derived from human embryonic stem cells hold much promise for clinical application in bone regeneration, in addition to providing a useful research model in developmental biology, and for pharmacological and cytotoxicity screening of bone-related biomaterials and drugs in vitro. Previously, osteogenic differentiation of human embryonic stem cells was achieved through dissociation of embryoid bodies by trypsinization, prior to culture with osteogenesis-promoting medium. This study therefore attempted a new approach: that is to achieve osteogenesis within intact human embryoid bodies. After 22 days of culture in osteogenesis-promoting medium comprising a cocktail of ascorbic acid, beta-glycerophosphate and dexamethasone, the attached embryoid bodies exhibited much cellular outgrowth and migration, and formed morphologically distinct nodule-like structures. These were somewhat similar to osteogenic nodules formed by mesenchymal stem cells, as reported by previous studies. Immunohistochemical staining and RT-PCR analysis confirmed the presence of osteogenic cells within these nodule-like structures. Additionally, the quantitative assay of osteocalcin secretion demonstrated a rapid sharp increase in osteocalcin expression on day 12 of in vitro culture, which could suggest the appearance of differentiated osteoblasts from day 12 onwards. Future work will attempt to investigate whether other cytokines, growth factors and chemical compounds could further enhance osteogenesis within intact human embryoid bodies.

Osteogenic protein-1 is most effective in stimulating nucleus pulposus and annulus fibrosus cells to repair their matrix after chondroitinase ABC-induced in vitro chemonucleolysis
Takegami, K., H. S. An, et al. (2005), Spine J 5(3): 231-8.
Abstract: BACKGROUND CONTEXT: Chondroitinase ABC (C-ABC) is used in chemonucleolysis to degrade, with great specificity, the chondroitin sulfate and dermatan sulfate chains of proteoglycans (PGs). A recent study showed that osteogenic protein-1 (OP-1) is very effective in stimulating the production and formation of the extracellular matrix by rabbit intervertebral disc cells. PURPOSE: To test the hypothesis that the repair of the extracellular matrix of the intervertebral disc after chemonucleolysis by C-ABC can be stimulated by exposure to a low dose of a growth factor, OP-1. STUDY DESIGN: An alginate bead cell culture system was used to monitor the effects of OP-1 on the repair of damaged matrices after in vitro chemonucleolysis with C-ABC. METHODS: Rabbit nucleus pulposus (NP) or annulus fibrosus (AF) cells cultured for 2 weeks in alginate gel were briefly exposed to low concentrations of C-ABC and then cultured in the presence or absence of OP-1. The control group was cultured without enzyme treatment for the same period in the absence of OP-1. At each time point, the contents of DNA and proteoglycan accumulation and proteoglycan synthesis were measured. RESULTS: NP or AF cells cultured in alginate beads, which were digested with C-ABC and then treated with OP-1, recover PG content more rapidly than those cultured in the absence of OP-1. The major contributor to the superior matrix repair in the cells treated with OP-1 was an up-regulation of proteoglycan synthesis. CONCLUSIONS: OP-1 was effective in stimulating matrix repair by NP and AF cells after their matrices were nearly totally depleted of sulfated glycosaminoglycans. The use of OP-1 after chemonucleolysis might help the disc to regain biomechanical strength, weakened by enzyme digestion, by stimulating matrix metabolism.

Osteoinduction by bone morphogenetic protein 2-expressing adenoviral vector: application of biomaterial to mask the host immune response
Sonobe, J., Y. Okubo, et al. (2004), Hum Gene Ther 15(7): 659-68.
Abstract: We constructed a human bone morphogenetic protein 2 (BMP-2)-expressing adenoviral vector, AxCABMP-2, which showed osteoinduction in immunosuppressed rats. In immunocompetent rats, new bone was not induced, because of the rapid elimination of transduced cells. Biomaterials such as collagen can be used as carriers for the delivery of DNA vectors, allowing prolonged expression of plasmid DNA in normal animals. We evaluated osteoinduction with AxCABMP-2 and atelopeptide type I collagen in immunocompetent rats. Collagen plus AxCABMP-2 (BMP group), collagen plus AxCALacZ (LacZ group), or collagen alone (CL group) was implanted into calf muscle pouches in immunocompetent rats, or AxCABMP-2 alone (injection group) was injected into the calf muscle. On days 3, 7, 14, and 21 after treatment, osteoinduction was evaluated. In the BMP group, bone formation was not observed on days 3 and 7. On day 14, radiographic formation was seen, but little bone formation was detected histologically. On day 21, new bone formation was observed both radiologically and histologically. In the other groups, osteoinduction was not found at any time. Immunohistochemical analysis on days 3 and 7 revealed decreased immunogenicity in the BMP group compared with the injection group. These findings suggested that collagen was an effective masking material for our vector.

Osteoinduction by calcium phosphate biomaterials
Yuan, H., Z. Yang, et al. (1998), J Mater Sci Mater Med 9(12): 723-6.
Abstract: Different materials were implanted in muscles of dogs to study the osteoinduction of calcium phosphate biomaterials. Bone formation was only seen in calcium phosphate biomaterials with micropores, and could be found in hydroxyapatite (HA) ceramic, tricalcium phosphate/hydroxyapatite ceramic (BCP), beta-TCP ceramic and calcium phosphate cement. The osteoinductive potential was different in different materials. The results indicate that osteoinduction can be a property of calcium phosphate biomaterials when they exhibit specific chemical and structural characteristics.

Osteoinductive biomaterials for medical implantation
Toriumi, D. M., C. A. East, et al. (1991), J Long Term Eff Med Implants 1(1): 53-77.
Abstract: The search for the ideal implant material continues since presently available implants all have significant drawbacks. This paper reviews three studies that we recently completed with bone-inducing implants. Long-term clinical follow-up of 75 allogeneic demineralized bone implants showed an average degree of resorption of 49%. Implants used for dorsal nasal augmentation showed an average degree of resorption of 50.7%, increasing to 82.5% after a 24-month follow-up. The efficacy of demineralized bone implants is dependent on many factors including site of implantation, method of preparation, etc. Transforming growth factor-beta 1 (TGF-beta 1) is a regulator of bone formation. We combined recombinant TGF-beta 1 with demineralized bone powder in a rabbit facial augmentation model. At 6 weeks, there was evidence of increased bone formation in the implants containing TGF-beta 1. Even though TGF-beta 1 can increase bone formation in demineralized bone implants, the overall bone-inducing activity in these implants seems to be suboptimal. Osteoinductive factor extract (OFE) is a partially purified bone factor preparation that has been shown to form bone when implanted into rats. Using a collagen/ceramic carrier, we implanted OFE into a rabbit facial augmentation model. At 21 d, histomorphometry revealed numerous osteoblasts and bone formation in the OFE implants. The bone-inducing activity of many partially purified osteoinductive preparations such as OFE is probably due to Bone Morphogenetic Protein-2A (BMP-2A), which has been shown to induce bone formation in its recombinant form. Recombinant DNA methodology provides the technology necessary to produce these molecules in their homogeneous form, permitting evaluation of bone-inducing activity in a preparation free of contaminants. Finally, the ideal carrier must be devised to permit safe and effective delivery of recombinant bone-inducing factors.

Osteopontin and bone metabolism: a histology and scintigraphy study in rats
Gordjestani, M., L. Dermaut, et al. (2005), Int J Oral Maxillofac Surg 34(7): 794-9.
Abstract: Osteopontin (OPN) is one of the major non-collagen proteins in extracellular bone matrix. To elucidate the function of OPN in bone metabolism, a cellular defect was created in parietal bone and tibia of 12 rats. In Group 1, the left defects were filled with OPN-coated hydroxyapatite (OPN-H). In Group 2, the right defects were filled with non-coated hydroxyapatite (N-H). In both groups, the contra lateral defects were used as control defects. In Group 3, OPN-H was inserted in the left defects and N-H in the right defects. Bone metabolism was measured by (45)Ca and technetium-99m methylene diphosphonate scintigraphy for 4 weeks. Scintigraphy did not show any significant differences in bone metabolism between the defects filled with OPN-H and N-H. A higher bone metabolism was measured between the parietal defects filled with OPN-H or N-H in comparison with the parietal control defects. This difference, however, was not significant and was less for tibia defects. Histological observation (7th week) shows less inflammatory cells at the tibia defects filled with OPN-H compared to the tibia defects filled with N-H. This study did not show any acceleration or inhibition of bone metabolism in parietal or tibia bone in rats, but there is some evidence that OPN might influence inflammatory cells in bone matrix.

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Last Modified: 8 February 2006