Chemical Compounds Released from Specific Osteoinductive Bioactive Materials Stimulate Human Bone Marrow Mesenchymal Stem Cell Migration.

In this work, a poly(L-lactide-co-glycolide) (PLGA)-based composite was enriched with one of the following sol-gel bioactive glasses (SBG) at 50 wt.%: A1-40 mol% SiO2, 60 mol% CaO, CaO/SiO2 ratio of 1.50; S1-80 mol% SiO2, 20 mol% CaO, CaO/SiO2 ratio of 0.25; A2-40 mol% SiO2, 54 mol% CaO, 6 mol% P2O5, CaO/SiO2 ratio of 1.35; S2-80 mol% SiO2,16 mol% CaO, 4 mol% P2O5, CaO/SiO2 ratio of 0.20. The composites and PLGA control sheets were then soaked for 24 h in culture media, and the obtained condition media (CM) were used to treat human bone marrow stromal cells (hBMSCs) for 72 h. All CMs from the composites increased ERK 1/2 activity vs. the control PLGA CM. However, expressions of cell migration-related c-Fos, osteopontin, matrix metalloproteinase-2, C-X-C chemokine receptor type 4, vascular endothelial growth factor, and bone morphogenetic protein 2 were significantly increased only in cells treated with the CM from the A1/PLGA composite. This CM also significantly increased the rate of human BMSC migration but did not affect cell metabolic activity. These results indicate important biological markers that are upregulated by products released from the bioactive composites of a specific chemical composition, which may eventually prompt osteoprogenitor cells to colonize the bioactive material and accelerate the process of tissue regeneration.

Molecular Indicators of Biomaterials Osteoinductivity - Cell Migration, BMP Production and Signalling Turns a Key.

In this work we dissected the osteoinductive properties of selected, PLGA-based scaffolds enriched with gel-derived bioactive glasses (SBGs) of either binary SiO2-CaO or ternary SiO2-CaO-P2O5 system, differing in CaO/SiO2 ratio (i.e. high -or low-calcium SBGs). To assess the inherent ability of the scaffolds to induce osteogenesis of human bone marrow stromal cells (BMSC), the study was designed to avoid any osteogenic stimuli beyond the putative osteogenic SBG component of the studied scaffolds. The bioactivity and porosity of scaffolds were confirmed by SBF test and porosimetry. Condition media (CM) from BMSC-loaded scaffolds exhibited increased Ca and decreased P content corresponding to SBGs CaO/SiO2 ratio, whereas Si content was relatively stable and overall lower in CM from scaffolds containing binary SBGs. CM from cell-loaded scaffolds containing high-calcium, binary SBGs promoted migration of BMSC and BMP-response in reporter osteoblast cell line. BMSC culture on these scaffolds or the ones containing ternary, low-calcium SBGs resulted in the activation of BMP-related signaling and expression of several osteogenic markers. Ectopic bone formation was induced by scaffolds containing binary SBGs, but high-calcium ones produced significantly more osteoid. Scaffolds containing ternary SBGs negatively influenced the expression of osteogenic transcription factors and Cx43, involved in cell-cell interactions. High-calcium scaffolds stimulated overall higher Cx43 expression. We believe the initial cell-cell communication may be crucial to induce and maintain osteogenesis and high BMP signaling on the studied scaffolds. The presented scaffolds' biological properties may also constitute new helpful markers to predict osteoinductive potential of other bioactive implant materials.

Gellan gum hydrogels cross-linked with carbodiimide stimulates vacuolation of human tooth-derived stem cells in vitro.

The natural polysaccharides are promising compounds for applications in regenerative medicine. Gellan gum (GG) is the bacteria-derived polysaccharide widely used in food industry. Simple modifications of its chemical properties make GG superior for the development of biocompatible hydrogels. Beside reversible cationic integration of GG chains, more efficient binding is accomplished with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). However, the side-products of polymer cross-linking might affect viability and differentiation of stem cells introduced into the hydrogels. We found that O-acylisourea (EDU) stimulates autophagy-based vacuolation in both periodontal ligament and dental pulp stem cells. 24-h treatment of cells with GG extracts cross-linked with 15 mM EDC developed large cytoplasmic vacuoles. Freshly prepared EDU (2-6 mM) but not 15 mM EDC solutions initiated vacuole development with concomitant reduction of cell viability/metabolism. Most of the vacuoles stained with acridine orange displayed highly acidic environment further confirmed by flow cytometric analysis. Western blot of the LC3 autophagy marker followed by a transmission electron microscopy indicated the process is autophagy-dependent. We propose that the high reactivity of EDU with intracellular components initiates autophagy, although the targets of EDU remain unknown. Nevertheless, a burst release of EDU from GG hydrogels might modulate negatively cellular processes and final effectiveness of tissue regeneration.

Development of tannic acid-enriched materials modified by poly(ethylene glycol) for potential applications as wound dressing.

The interests in the biomedical impact of tannic acid (TA) targeting production of various types of biomaterials, such as digital microfluids, chemical sensors, wound dressings, or bioimplants constantly increase. Despite the significant disadvantage of materials obtained from natural-based compounds and their low stability and fragility, therefore, there is an imperative need to improve materials properties by addition of stabilizing formulas. In this study, we performed assessments of thin films over TA proposed as a cross-linker to be used in combination with polymeric matrix based on chitosan (CTS), i.e. CTS/TA at 80:20 or CTS/TA at 50:50 and poly(ethylene glycol) (PEG) at the concentration of 10% or 20%. We evaluated their mechanical parameters as well as the cytotoxicity assay for human bone marrow mesenchymal stem cells, human melanotic melanoma (MNT-1), and human osteosarcoma (Saos-2). The results revealed significant differences in dose-dependent of PEG regarding the maximum tensile strength (σmax) or impact on the metabolic activity of tissue culture plastic. We observed that PEG improved mechanical parameters prominently, decreased the hemolysis rate, and did not affect cell viability negatively. Enclosed data, confirmed also by our previous reports, will undoubtedly pave the path for the future application of tannic acid-based biomaterials to treat wound healing.

Properties of scaffolds based on chitosan and collagen with bioglass 45S5.

Scaffolds based on chitosan (CTS), collagen (Coll) and glycosaminoglycans (GAG) mixtures cross-linked by tannic acid (TA) with bioglass 45S5 addition were obtained with the use of the freeze-drying method. The prepared scaffolds were characterised for morphology, mechanical strength and degradation rate. Moreover, cell viability on the obtained scaffolds was measured with and without the presence of ascorbic acid and dexamethasone. The main purpose of the research was to compare the effectiveness of bioglass 45S5 influence on the physicochemical and biological properties of scaffolds. The results demonstrated that the scaffolds based on the blends of biopolymers cross-linked by TA are stable in an aqueous environment. Scanning electron microscope images allowed the observation of a porous scaffold structure with interconnected pores. The addition of bioglass nanoparticles improved the mechanical properties and decreased the degradation rate of composite materials. The biological properties were improved for 20% tannic acid addition compared to 5%. However, the addition of bioglass 45S5 did not change to cells response significantly.

Normal and cancer cells response on the thin films based on chitosan and tannic acid.

A novel aspect of tissue engineering is the material selective influence on the different cell types. The cell viability is a parameter which determine the cell ability to proliferate in the contact with material. In the experimental study the thin films based on chitosan and tannic acid mixture in ratio 80/20, 50/50 and 20/80 were tested. The surface roughness decreases with increasing tannic acid content. The cell culture was established on the proposed films. In vitro tests were carried out for the different cell lines as MNT-1, SK-MEL-28, Saos-2, HaCaT and BMSC. The result showed the dependence on the material influence on the various cell lines.

The Effect of Surface Modification of Ti13Zr13Nb Alloy on Adhesion of Antibiotic and Nanosilver-Loaded Bone Cement Coatings Dedicated for Application as Spacers.

Spacers, in terms of instruments used in revision surgery for the local treatment of postoperative infection, are usually made of metal rod covered by antibiotic-loaded bone cement. One of the main limitations of this temporary implant is the debonding effect of metal-bone cement interface, leading to aseptic loosening. Material selection, as well as surface treatment, should be evaluated in order to minimize the risk of fraction and improve the implant-cement fixation the appropriate manufacturing. In this study, Ti13Zr13Nb alloys that were prepared by Selective Laser Melting and surface treated were coated with bone cement loaded with either gentamicin or nanosilver, and the effects of such alloy modifications were investigated. The SLM-made specimens of Ti13Zr13Nb were surface treated by sandblasting, etching, or grounding. For each treatment, Scanning Electron Microscope (SEM), contact profilometer, optical tensiometer, and nano-test technique carried out microstructure characterization and surface analysis. The three types of bone cement i.e., pure, containing gentamicin and doped with nanosilver were applied to alloy surfaces and assessed for cement cohesion and its adhesion to the surface by nanoscratch test and pull-off. Next, the inhibition of bacterial growth and cytocompatibility of specimens were investigated by the Bauer-Kirby test and MTS assay respectively. The results of each test were compared to the two control groups, consisting of commercially available Ti13Zr13Nb and untreated SLM-made specimens. The highest adhesion bone cement to the titanium alloy was obtained for specimens with high nanohardness and roughness. However, no explicit relation of adhesion strength with wettability and surface energy of alloy was observed. Sandblasting or etching were the best alloys treatments in terms of the adhesion of either pure or modified bone cements. Antibacterial additives for bone cement affected its properties. Gentamicin and nanosilver allowed for adequate anti-bacterial protection while maintaining the overall biocompatibility of obtained spacers. However, they had different effects on the cement's adhesive capacity or its own cohesion. Furthermore, the addition of silver nanoparticles improved the nanomechanical properties of bone cements. Surface treatment and method of fabrication of titanium affected surface parameters that had a significant impact on cement-titanium fixation.

Incorporation of Ca ions into anodic oxide coatings on the Ti-13Nb-13Zr alloy by plasma electrolytic oxidation.

The present work concerns the surface modification of The Ti-13Nb-13Zr alloy by electropolishing and plasma electrolytic oxidation (PEO) process in Ca-containing electrolytes: calcium formate and calcium lactate solutions (0.1-1.0 mol dm-3) under voltages of 200 and 400 V. As a result of the PEO process, a porous oxide layer containing incorporated calcium compounds was formed on the Ti-13Nb-13Zr alloy surface. The morphology and chemical composition of the modified Ti-13Nb-13Zr alloy were investigated using scanning electron microscopy (SEM + EDS), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). An increase in the applied voltage caused an increase in the number of pores and an increase in the amount of calcium incorporated in the oxide layer. Analysis showed that all samples were covered by titanium oxide, which was present in the form of anatase and/or rutile. In course of the experiments, it was showed that the proposed procedure has a positive effect on the overall bioactivity of the Ti-13Nb-13Zr alloy. Bioactivity investigations using simulated body fluid (SBF) confirmed the formation of apatite on the anodized surfaces. The cell adhesion results obtained by the use of human bone marrow mesenchymal stem cells (hBMSC) demonstrated that the PEO coatings on the Ti-13Nb-13Zr alloy remarkably enhanced the cytocompatibility of the substrate, indicating a potential application in orthopedic surgeries. The incorporation of Ca into the oxide layer proceeded to a higher extent when the PEO treatment was performed in the calcium lactate bath. The oxide layers formed in the calcium lactate solution exhibited also superior biological behavior towards hBMSC. This can be ascribed to the presence of carboxylic groups onto coatings' surface (as identified by XPS), which facilitated the anchoring of cells and tissues.

Ectopic bone formation by gel-derived bioactive glass-poly-L-lactide-co-glycolide composites in a rabbit muscle model.

In this study we aimed to assess the in vivo osteoinductive properties of two composite scaffolds made of PLGA (poly-L-lactide-co-glycolide) and two types of gel-derived bioactive glasses, namely a high silica S2 bioactive glass (S2-PLGA composites) or high lime A2 bioactive glass (A2-PLGA composites). To achieve that, the potential of the composites to induce ectopic bone formation in a rabbit muscle has been examined along with the control PLGA scaffold. Cylinder-like scaffolds of 7 × 3 mm (width × height) were implanted into pouches created in the latissimus dorsi muscle of 18 New Zealand rabbits. The tissue sections were obtained at 6, 12 or 24 weeks post-surgery (six rabbits per each time point) and stained with hematoxylin-eosin. The process of wound healing, the formation of collagen-rich connective tissue and its transition to cartilage were examined by Sirius red and Alcian blue histological stainings. We also performed immunohistochemical verification of the presence of osteoblast- and osteoclast- like cells in the vicinity of the scaffolds. A typical foreign body reaction and wound healing process was observed for all implanted scaffolds. Osteoblast- and osteoclast-like cells were observed in the vicinity of the scaffolds as determined by the immunohistochemical staining for Osteocalcin, BMP-2 and Cathepsin K. Compared to plain PLGA scaffolds, numerous osteoblast-like cells were observed 12 weeks post implantation near the composites and the scaffolds gradually degraded as bone formation proceeded. S2-PLGA and A2-PLGA composites display osteoinductive properties in vivo. Furthermore, they are more effective at inducing ectopic bone formation in a rabbit muscle compared to plain PLGA. Thus these SBG-PLGA composite scaffolds have potential for clinical applications in dental and/or orthopedic-bone tissue engineering.

A single short session of media perfusion induces osteogenesis in hBMSCs cultured in porous scaffolds, dependent on cell differentiation stage.

Perfusing culture media through porous cell-seeded scaffolds is now a common approach within many tissue engineering strategies. Human bone-marrow derived mesenchymal stem cells (hBMSC) are a clinically valuable source of osteoprogenitors that respond to mechanical stimuli. However, the optimal mechanical conditions for their osteogenic stimulation in vitro have not been defined. Whereas the effects of short durations of media fluid flow have been studied in monolayers of osteoblastic cells, in 3D culture continuous or repeated perfusion is usually applied. Here, we investigated whether a short, single perfusion session applied to hBMSCs cultured in 3D would enhance their osteogenesis in vitro. We cultured hBMSCs on gelatine-coated, porous polyurethane scaffolds with osteogenic supplements and stimulated them with a single 2-h session of unidirectional, steady, 2.5 mL/min media perfusion, at either early or late stages of culture in 3D. Some cells were pre-treated in monolayer with osteogenic supplements to advance cell differentiation, followed by 3D culture also with the osteogenic supplements. We report that this single, short session of media perfusion can markedly enhance the expression of bone-related transcription and growth factors, and matrix components, by hBMSCs but that it is more effective when cells reach the pre-osteoblast or osteoblast differentiation stage. These findings could aid in the optimization of 3D culture protocols for efficient bone tissue engineering. Biotechnol. Bioeng. 2016;113: 1814-1824. © 2016 Wiley Periodicals, Inc.

In vitro bioactivity investigations of Ti-15Mo alloy after electrochemical surface modification.

Titanium and its aluminum and vanadium-free alloys have especially great potential for medical applications. Electrochemical surface modification improves their surface bioactivity and stimulates osseointegration process. In this work, the effect of plasma electrolytic oxidation of the ß-type alloy Ti-15Mo surface on its bioactivity is presented. Bioactivity of the modified alloy was investigated by immersion in simulated body fluid (SBF). Biocompatibility of the modified alloys were tested using human bone marrow stromal cells (hBMSC) and wild intestinal strains (DV/A, DV/B, DV/I/1) of Desulfovibrio desulfuricans bacteria. The particles of apatite were formed on the anodized samples. Human BMSC cells adhered well on all the examined surfaces and expressed ALP, collagen, and produced mineralized matrix as determined after 10 and 21 days of culture. When the samples were inoculated with D. desulfuricans bacteria, only single bacteria were visible on selected samples. There were no obvious changes in surface morphology among samples. Colonization and bacterial biofilm formation was observed on as-ground sample. In conclusion, the surface modification improved the Ti-15Mo alloy bioactivity and biocompatibility and protected surface against colonization of the bacteria. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 903-913, 2016.

Bioactivity of coatings formed on Ti-13Nb-13Zr alloy using plasma electrolytic oxidation.

In this work, we investigated the bioactivity of anodic oxide coatings on Ti-13Nb-13Zr alloy by plasma electrolytic oxidation (PEO) in solutions containing Ca and P. The bioactive properties of the films were determined by immersion in simulated body fluid (SBF), and their biocompatibility was examined using adult human bone marrow derived mesenchymal stem cells (hBMSCs). The oxide layers were characterised based on their surface morphology (SEM, AFM, profilometry) as well as on their chemical and phase compositions (EDX, XRF, XRD, XPS). We report that anodic oxidation of Ti-13Nb-13Zr led to the development of relatively thick anodic oxide films that were enriched in Ca and P in the form of phosphate compounds. Furthermore, the treatment generated rough surfaces with a significant amount of open pores. The surfaces were essentially amorphous, with small amounts of crystalline phases (anatase and rutile) being observed, depending on the PEO process parameters. SBF soaking led to the precipitation of small crystals after one week of experiment. During culturing of hBMSCs on the bioactive Ti-13Nb-13Zr surfaces the differentiation of human mesenchymal stem cells toward osteoblasts was promoted, which indicated a potential of the modified materials to improve implant osseointegration.

Osteoinductive activity of insulin-functionalized cell culture surfaces obtained using diazonium chemistry.

Polymeric surfaces suitable for cell culture (DR/Pec) were constructed from diazoresin (DR) and pectin (Pec) in a form of ultrathin films using the layer-by-layer (LbL) technique. The surfaces were functionalized with insulin using diazonium chemistry. Such functionalized surfaces were used to culture human mesenchymal stem cells (hMSCs) to assess their suitability for bone tissue engineering and regeneration. The activity of insulin immobilized on the surfaces (DR/Pec/Ins) was compared to that of insulin dissolved in the culture medium. Human MSC grown on insulin-immobilized DR/Pec surfaces displayed increased proliferation and higher osteogenic activity. The latter was determined by means of alkaline phosphatase (ALP) activity, which increases at early stages of osteoblasts differentiation. Insulin dissolved in the culture medium did not stimulate cell proliferation and its osteogenic activity was significantly lower. Addition of recombinant human bone morphogenetic protein 2 (rhBMP-2) to the culture medium further increased ALP activity in hMSCs indicating additive osteogenic action of immobilized insulin and rhBMP-2.

Biopolymer-based hydrogels as injectable materials for tissue repair scaffolds.

The progress in tissue regeneration is strongly dependent on the development of biocompatible materials with properties resembling those of a native tissue. Also, the application of noninvasive methods of delivering the scaffold into the tissue defect is of great importance. In this study we present a group of biopolymer-based materials as potential injectable scaffolds. In contrast to other studies involving collagen neutralization or additional incubation of gel in genipin solution, we propose collagen and collagen-chitosan gels crosslinked in situ with genipin. Since some parameters of the cells should be considered in the microscale, the steady-state fluorescence anisotropy was applied to study the microenvironment of the gels. To our knowledge we are the first to report on microrheological properties, such as gel time and microviscosity, for this group of hydrogels. Rapid gelation at physiological temperatures found makes these materials of special interest in applications requiring gel injectability. Physico-chemical investigation showed the influence of the crosslinking agent concentration and chitosan addition on the crosslinking degree, swelling ratio, gel microviscosity, and the degradation rate. Strong correlation was revealed between the surface wettability and the viability of cultured mesenchymal stem cells. Cytotoxicity studies indicated that the collagen-chitosan hydrogels showed the best biocompatibility.

Degradation, bioactivity, and osteogenic potential of composites made of PLGA and two different sol-gel bioactive glasses.

We have developed poly(L: -lactide-co-glycolide) (PLGA) based composites using sol-gel derived bioactive glasses (S-BG), previously described by our group, as composite components. Two different composite types were manufactured that contained either S2-high content silica S-BG, or A2-high content lime S-BG. The composites were evaluated in the form of sheets and 3D scaffolds. Sheets containing 12, 21, and 33 vol.% of each bioactive glass were characterized for mechanical properties, wettability, hydrolytic degradation, and surface bioactivity. Sheets containing A2 S-BG rapidly formed a hydroxyapatite surface layer after incubation in simulated body fluid. The incorporation of either S-BG increased the tensile strength and Young's modulus of the composites and tailored their degradation rates compared to starting compounds. Sheets and 3D scaffolds were evaluated for their ability to support growth of human bone marrow cells (BMC) and MG-63 cells, respectively. Cells were grown in non-differentiating, osteogenic or osteoclast-inducing conditions. Osteogenesis was induced with either recombinant human BMP-2 or dexamethasone, and osteoclast formation with M-CSF. BMC viability was lower at higher S-BG content, though specific ALP/cell was significantly higher on PLGA/A2-33 composites. Composites containing S2 S-BG enhanced calcification of extracellular matrix by BMC, whereas incorporation of A2 S-BG in the composites promoted osteoclast formation from BMC. MG-63 osteoblast-like cells seeded in porous scaffolds containing S2 maintained viability and secreted collagen and calcium throughout the scaffolds. Overall, the presented data show functional versatility of the composites studied and indicate their potential to design a wide variety of implant materials differing in physico-chemical properties and biological applications. We propose these sol-gel derived bioactive glass-PLGA composites may prove excellent potential orthopedic and dental biomaterials supporting bone formation and remodeling.

Modification of Smad1 linker modulates BMP-mediated osteogenesis of adult human MSC.

We examined whether bone morphogenetic protein (BMP)-mediated osteogenesis of adult human mesenchymal stem cells (MSCs) is regulated by extracellular signal-regulated kinase phosphorylation of Smad1. Adenoviral constructs carrying either unmodified human Smad1 or Smad1 mutated in the linker region to preclude extracellular signal-regulated kinase phosphorylation were expressed in human and rodent cells. Unlike unmodified Smad1, expression of mutated Smad1 facilitated BMP-stimulated expression of osteoblast markers in human MSC but had no effect on either rat MSC or mouse pre-osteoblastic MC3T3-E1 cells. Expression of mutated Smad1 in adult human MSC cultures also resulted in increased nuclear accumulation of BMP-activated Smads and elevated gene transcripts characteristic of differentiating osteoblasts. These results may partly explain the poor efficacy of BMP in some human bone therapies and indicate an important mechanism regulating BMP-mediated bone formation in adults.

Gel-derived bioglass as a compound of hydroxyapatite composites.

Despite the excellent biocompatibility of hydroxyapatite and bioglass, their clinical applications are limited to non-load-bearing implants and implant coatings due to their low mechanical properties. We have developed two different composites made of hydroxyapatite (HA) and gel-derived bioglasses designated S2 (80 mol% SiO(2)-16 mol% CaO-4 mol% P(2)O(5)) or A2 (40 mol% SiO(2)-54 mol% CaO-6 mol% P(2)O(5)). We show that the combination of hydroxyapatite with either bioglass results in better composite bioactivity and biocompatibility compared to HA alone. We used a commercially available hydroxyapatite that was sintered with varying additions (10%, 50%) of A2 or S2 bioglass. Scanning electron microscopy and x-ray diffraction were used to characterize the microstructure and phases of the composites. The elastic properties of bioglass/HA composites were analyzed with the use of the pulse ultrasonic technique. The bioactivity (surface activity) of the composites was assessed by determining the changes of surface morphology and composition after soaking in simulated body fluid (SBF) for 7 and 14 days. The biocompatibility of the obtained composites was then assessed in vitro using adult human bone marrow stromal cells. Cells were seeded on the material surfaces at a density of 10(4) cells cm(-2) and cultured for 7 days in non-differentiating and osteogenic conditions. The number of live cells was estimated in both standard and osteogenic cultures, followed by alkaline phosphatase (ALP) activity assay in osteogenic cultures. We determined that 10 wt% addition of A2 (E = 12.24 GPa) and 50 wt% addition of S2 (E = 16.96 GPa) to the HA base results in higher Young's modulus of the composites compared to pure hydroxyapatite (E = 9.03 GPa). The rate of Ca-P rich layer formation is higher for bioglass/HA composites containing A2 bioglass compared to the composites containing S2 bioglass. Evaluation of cell growth on the bioglass/HA composites showed that the incorporation of either 50 wt% S2 or 50 wt% A2 into the hydroxyapatite base significantly improves cell viability when compared to cells grown on pure HA. Also the cellular activity of ALP, an early marker of osteoblasts, increases with the amount of bioglass addition to the composites.

Sol-gel bioactive glasses support both osteoblast and osteoclast formation from human bone marrow cells.

We have examined the ability of bioactive sol-gel glass ceramics to support both osteoblast and osteoclast differentiation from human bone marrow cells (HBMC). Nucleated cells from human bone marrow were cultured on tissue culture plastic and on two sol-gel coatings: A2 glass-ceramic containing 54 mol % CaO/40 mol % SiO(2) and S2 glass-ceramic containing 16 mol % CaO/80 mol % SiO(2). Osteoblast differentiation was followed by measuring alkaline phosphatase (ALP) activity, mRNA levels for ALP, osteopontin, RANK ligand (RANKL), and immunofluorescent co-localization of ALP and RANKL. Osteoclasts were identified by morphology and positive staining for tartrate-resistant acid phosphatase (TRAP). ALP activity and mRNA levels were similar for cells on A2 coatings and on tissue culture plastic, but mRNA levels of osteopontin and RANKL were tenfold higher on A2 than on plastic. Cultures on A2 coatings also contained multinucleated osteoclasts staining positively for TRAP. In contrast, cells cultured on S2 coatings had the characteristics of more differentiated osteoblasts as measured by higher ALP expression. However, the levels of osteopontin and RANKL mRNA on S2 glass were lower than on A2 glass and there were fewer, weakly staining TRAP-positive multinucleate cells. Thus, sol-gel glass-ceramic materials differing in CaO/SiO(2) ratios can produce markedly different effects on the osteoblast and osteoclast differentiation from HBMC.

Bone morphogenetic protein regulation of early osteoblast genes in human marrow stromal cells is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling.

Bone marrow stromal cells (MSC) are the major source of osteoblasts for bone remodeling and repair in postnatal animals. Rodent MSC cultured with bone morphogenetic proteins (BMPs) differentiate into osteoblasts, but most human MSC show a poor osteogenic response to BMPs. In this study we demonstrate that BMP-induced osteogenesis in poorly responsive human MSC requires modulation of ERK and phosphatidylinositol 3-kinase (PI3-K) pathways. Either treating human MSC cultures with the MAPK/ERK kinase inhibitor PD98059 or transferring them to serum-free medium with insulin or IGF-I permits BMP-dependent increases in the expression of the early osteoblast-associated genes, alkaline phosphatase and osteopontin. Increased expression of these genes in BMP-treated, serum-free cultures correlates with increased nuclear levels of activated Smads, whereas serum-free cultures of human MSC expressing constitutively active MAPK/ERK kinase show decreased expression of early osteoblast genes and decreased nuclear translocation of BMP-activated Smads. Inhibiting ERK activity in human MSC also elevates the expression of Msx2, a transcription factor that is directly regulated by Smad-binding elements in its promoter. Therefore, growth factor stimulation leading to high levels of ERK activity in human MSC results in suppressed BMP-induced transcription of several early osteoblast genes, probably because levels of BMP-activated nuclear Smads are decreased. In contrast, inhibiting the insulin/IGF-I-activated PI3-K/AKT pathway decreases BMP-induced alkaline phosphatase and osteopontin expression in serum-free cultures of human MSC, but increases BMP activation of Smads; thus, PI3-K signaling is required for BMP-induced expression of early osteoblast genes in human MSC either downstream or independent of the BMP-activated Smad signaling pathway.

Different effects of BMP-2 on marrow stromal cells from human and rat bone.

Bone morphogenetic proteins (BMPs) promote the differentiation of osteoprogenitor cells, and also induce osteogenesis in bone marrow stromal cells (MSC) from rats and mice. However, compared to results with animal models, BMPs are relatively inefficient in inducing human MSC to undergo osteogenesis, and are much less effective in promoting bone formation in human clinical trials. Previous studies indicated that, while human MSC respond to dexamethasone with elevated levels of the osteoblast marker alkaline phosphatase, most isolates of human MSC fail to show alkaline phosphatase induction in response to BMP-2, BMP-4, or BMP-7. Several other genes known to be induced by BMPs are appropriately regulated; thus, human MSC are capable of some BMP-activated signaling. Analysis of the BMP receptors ALK-3 and ALK-6 indicated that, although ALK-6 mRNA was not expressed in human MSC, overexpressing a constitutively active ALK-6 receptor did not induce elevated alkaline phosphatase. Real-time RT-PCR was used to investigate expression of several osteoblast-related transcription factors in MSC after 6 days' exposure to BMP2 or dexamethasone. Msx-2, a transcription factor that has been reported to inhibit differentiation of osteoprogenitor cells, showed 10-fold elevation in BMP-2-treated human MSC, but not in BMP-2-treated rat MSC. Overexpression of Msx-2 in human and rat MSC, however, did not alter alkaline phosphatase levels, which suggests that absence of BMP-stimulated alkaline phosphatase was not caused by the BMP-2-induced increase in Msx-2. Although Runx2 isoforms have been implicated in control of osteoblast differentiation, levels of this transcription factor were unaffected by BMP treatment. Expression of the FKHR transcription factor, which has been reported to regulate alkaline phosphatase transcription in mouse cells, showed a modest increase in response to BMP-2, but a much greater increase in dexamethasone-treated cells. We propose that BMP regulation of the bone/liver/kidney alkaline phosphatase gene is indirect, requiring expression of new transcription factor(s) that behave differently in rodent and human MSC.