Calcium Mineral Deposition Research Articles

Simvastatin delivery on PEEK for bioactivity and osteogenesis enhancements

A strategy developed for obtaining positive cellular responses remains to be focused in the filed of functional biomimetics. In this study, a hydrogel covered simvastatin-loaded polyetheretherketone (PEEK) bio-composites was constructed with the purpose of bone tissue regeneration therapy. Briefly, a three-dimensional (3D) porous structure was fabricated on PEEK surface; then the substrate was functionalized with the poly(L-lactic acid)/simvastatin porous film and hyaluronic acid hydrogel subsequently. In vitro cell attachment, proliferation, and cytoskeletal observation experiments reveal that our scaffolds show better bio-affinity due to the layer of hyaluronic acid hydrogel compared with control. Furthermore, the alkaline phosphatase activity, calcium mineral deposition evaluation, and gene expression for osteogenic potential all exhibit that the superior osteogenic differentiation of MC3T3-E1 pre-osteoblasts on our scaffolds. Therefore, our PEEK samples loaded with simvastatin and covered with hyaluronic acid hydrogel hold great potential in clinical applications for bone repair.

Osteogenic differentiation of preconditioned bone marrow mesenchymal stem cells with lipopolysaccharide on modified poly-l-lactic-acid nanofibers.

Tissue engineering is an interdisciplinary expertise that involves the use of nanoscaffolds for repairing, modifying, and removing tissue defects and formation of new tissues. Mesenchymal stem cells (MSCs) can differentiate into a variety of cell types, and they are attractive candidates for tissue engineering. In the current study, the electrospinning process was used for nanofiber preparation, based on a poly-l-lactic-acid (PLLA) polymer. The surface was treated with O 2 plasma to enhance hydrophilicity, cell attachment, growth, and differentiation potential. The nanoscaffolds were preconditioned with lipopolysaccharide (LPS) to enhance induction of differentiation. The nanoscaffolds were categorized by contact angle measurements and scanning electron microscopy. The MTT assay was used to analyze the rate of growth and proliferation of cells. Osteogenic differentiation of cultured MSCs was evaluated on nanofibers using common osteogenic markers, such as alkaline phosphatase activity, calcium mineral deposition, quantitative real-time polymerase chain reaction, and immunocytochemical analysis. Based on the in vitro results, primed MSCs with LPS on the PLLA nanoscaffold significantly enhanced the proliferation and osteogenesis of MSCs. Also, the combination of LPS and electrospun nanofibers can provide a new and suitable matrix to support stem cells' differentiation for bone tissue engineering.

Graphene-Oxide-Decorated Microporous Polyetheretherketone with Superior Antibacterial Capability and In Vitro Osteogenesis for Orthopedic Implant.

Due to its similar elastic modulus of human bones, polyetheretherketone (PEEK) has been considered as an excellent cytocompatible material. However, the bioinertness, poor osteoconduction, and weak antibacterial activity of PEEK limit its wide applications in clinics. In this study, a facile strategy is developed to prepare graphene oxide (GO) modified sulfonated polyetheretherketone (SPEEK) (GO-SPEEK) through a simple dip-coating method. After detailed characterization, it is found that the GO closely deposits on the surface of PEEK, which is attributed to the π-π stacking interaction between PEEK and GO. Antibacterial tests reveal that the GO-SPEEK exhibits excellent suppression toward Escherichia coli. In vitro cell attachment, growth, differentiation, alkaline phosphatase activity, quantitative real-time polymerase chain reaction analyses, and calcium mineral deposition all illustrate that the GO-SPEEK substrate can significantly accelerate the proliferation and osteogenic differentiation of osteoblast-like MG-63 cells compared with those on PEEK and SPEEK groups. These results suggest that the GO-SPEEK has an improved antibacterial activity and cytocompatibility in vitro, showing that the developed GO-SPEEK has a great potential as the bioactive implant material in bone tissue engineering.

Evaluation of long-term biocompatibility and osteogenic differentiation of graphene nanosheet doped calcium phosphate-chitosan AZ91D composites

To improve the biodegradable performance and osteoinductivity of magnesium (Mg) alloy, a novel graphene nanosheet (GNS) doped micro-arc oxidized-AZ91D (MAO-AZ91D) based calcium phosphate (CaP)-chitosan (CS) (GNS-CaP-CS/AZ91D) composite was fabricated. Their long-term in vitro biocompatibility, osteoinductivity and the related signaling pathway were evaluated. The electrochemical test, Mg concentration and SEM results showed that the corrosion rate of GNS-CaP-CS/AZ91D became much slower, whose corrosion degree with immersion time being 90 d was similar to that of AZ91D being 16 d. Good biocompatibility and non-cytotoxicity were observed during the whole immersion process of GNS-CaP-CS/AZ91D. Extracts from GNS-CaP-CS/AZ91D especially in the early stage (16 days and before) could significantly enhance alkaline phosphatase (ALP) activity, calcium mineral deposition and osteoblast-related genes expression of human bone marrow mesenchymal stem cells (hBMSCs). GNS released from GNS-CaP-CS/AZ91D composites might serve a critical role to induce osteogenic differentiation through ERK/MAPK pathway in the long-term immersion process, while, PI3K/Akt signaling only significantly in the early stage. Moreover, Mg element could promote osteogenic differentiation through ERK/MAPK pathway and the maximum concentration was about the range between 200 and 500 ppm.

Endocytic mechanisms and osteoinductive profile of hydroxyapatite nanoparticles in human umbilical cord Wharton's jelly-derived mesenchymal stem cells.

BackgroundAs a potentially bioactive material, the widespread application of nanosized hydroxyapatite (nano-HAP) in the field of bone regeneration has increased the risk of human exposure. However, our understanding of the interaction between nano-HAP and stem cells implicated in bone repair remains incomplete.MethodsHere, we characterized the adhesion and cellular internalization of HAP nanoparticles (HANPs) with different sizes (20 nm np20 and 80 nm np80) and highlighted the involved pathway in their uptake using human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs). In addition, the effects of HANPs on cell viability, apoptosis response, osteogenic differentiation, and underlying related mechanisms were explored.ResultsIt was shown that both types of HANPs readily adhered to the cellular membrane and were transported into the cells compared to micro-sized HAP particles (m-HAP; 12 μm). Interestingly, the endocytic routes of np20 and np80 differed, although they exhibited similar kinetics of adhesion and uptake. Our study revealed involvement of clathrin- and caveolin-mediated endocytosis as well as macropinocytosis in the np20 uptake. However, for np80, clathrin-mediated endocytosis and some as-yet-unidentified important uptake routes play central roles in their internalization. HANPs displayed a higher preference to accumulate in the cytoplasm compared to m-HAP, and HANPs were not detected in the nucleolus. Exposure to np20 for 24 h caused a decrease in cell viability, while cells completely recovered with an exposure time of 72 h. Furthermore, HANPs did not influence apoptosis and necrosis of hWJ-MSCs. Strikingly, HANPs enhanced mRNA levels of osteoblast-related genes and stimulated calcium mineral deposition, and this directly correlated with the activation in c-Jun N-terminal kinases and p38 pathways.ConclusionOur data provide additional insight about the interactions of HANPs with MSCs and suggest their application potential in hard tissue regeneration.

Biocompatibility and Osteogenic/Calcification Potential of Casein Phosphopeptide-amorphous Calcium Phosphate Fluoride

IntroductionCasein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and CPP-ACP with fluoride (CPP-ACFP) have been shown to provide bioavailable ions to promote mineralization. Hence, the aim of this study was to evaluate the materials’ biocompatibility and osteogenic/calcification potential for endodontic applications. MethodsHuman and mouse osteoblast-like and fibroblast-like cell lines were incubated with 0.05%–3.0% w/v CPP-ACP and CPP-ACFP, and toxicity, proliferation, alkaline phosphatase, interleukin (IL)-1α, and IL-6 production, collagen type I, osteocalcin, and osteopontin production, and mineralization/calcification were determined. ResultsCPP-ACP and CPP-ACFP were non-toxic and had no significant effect on proliferation or production of the inflammatory cytokine IL-1α. Alkaline phosphatase activity of the osteoblast-like cells was significantly increased (P < .05) by CPP-ACP and CPP-ACFP, as was the production of the osteotropic cytokine IL-6, the formation of calcium mineral deposits, and the secretion of mineralization-related proteins (collagen type I and osteocalcin). ConclusionsCPP-ACP and CPP-ACFP are biocompatible and have the potential to induce osteoblastic differentiation and mineralization. Potential applications include apexification, perforation repair, vital pulp therapy, and regenerative endodontic procedures.

Osteoblast Behavior on Silicon and Porous-Silicon Substrates

Osteoblast viability, proliferation, protein expression and mineralization were studied on bare, micro- and nanoporous silicon (Si) substrates. Micro- and nano-porous-Si substrates were prepared by anodic etching of silicon in ethanolic hydrofluoric acid and characterized using scanning electron and atomic force microscopies. Mouse osteoblasts were cultured on these substrates and cellular response to these surfaces was assessed using the Live/Dead Cell Viability assay and the MTT assay for cell proliferation. Osteoblast functionality was assessed using immunohistochemistry for bone protein specific markers. Osteoblasts grew well on micro- and nanoporous silicon substrates over the twenty-one day experimental period supporting the assessment that these are suitable cell supportive surfaces. Cell proliferation rates on bare and nanoporous silicon were similar initially, however, nanoporous silicon displayed enhanced cell proliferation, in comparison to bare silicon, after 14 days in culture. Immunocytochemical assays, using bone specific markers, showed positive reactions for osteonectin and osteopontin expression on all substrates with staining intensity increasing over the 21-day experimental period. Calcium mineral deposits were quantified using the Alizarin Red histochemical assay and nanoporous silicon induced the highest level of calcium mineral production in comparison to bare and microporous silicon. The data supports the potential use of nanoporous silicon as a surface implant coating for dental and orthopedic applications. The ability to dope (and then release) drugs or growth factors from the silicon nanopores offers the potential for a multi-functional implant surface.

Gelatin-Derived Graphene-Silicate Hybrid Materials Are Biocompatible and Synergistically Promote BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells.

Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased the ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPARγ was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms.

Toward mimicking the bone structure: design of novel hierarchical scaffolds with a tailored radial porosity gradient

Guiding bone regeneration poses still unmet challenges due to several drawbacks of current standard treatments in the clinics. A possible solution may rely on the use of three-dimensional scaffolds with optimized structural properties in combination with human mesenchymal stem cells (hMSCs). Bone presents a radial gradient structure from the outside, where the cortical bone is more compact (porosity ranging from 5% to 10%), toward the inner part, where the cancellous bone is more porous (porosity ranging from 50% to 90%). Here, we present a new scaffold design with a built-in gradient in porosity, which approximate the radial bone structure. The pores of the outer ring were 500 μm, the ones in the middle zone were 750 μm and the inner part presented pores of 1000 μm. The porosity of each scaffold region resembled the gradient present in bone, with the outer ring having a porosity of 29.6% ± 5%, the middle and inner regions a porosity of 50.8% ± 8.1% and 77.6% ± 3.2% respectively. hMSCs behavior was analyzed in terms of growth, extracellular matrix deposition and differentiation toward the osteogenic lineage. A trend was displayed by the hMSCs residing in different zones of the gradient scaffolds after 7, 14 and 28 days of culture in mineralization medium. Osteogenic differentiation was influenced by pore size and location in scaffolds displaying a radial porosity gradient. Cell differentiation was confirmed by gene expression with upregulation of Runx2 and bone sialoprotein markers. Mineralization staining further confirmed the maturation of cell differentiation, as indicated by the presence of calcium and phosphate mineral deposits.

Osteoblast differentiation of mesenchymal stem cells on modified PES-PEG electrospun fibrous composites loaded with Zn2SiO4 bioceramic nanoparticles

Tissue engineering has attracted a great deal of interest by combining fibrous scaffolds and stem cells regarding bone regeneration applications. In the present study, polymeric fibrous polyethersulphone-polyethylene glycol (PES-PEG) was fabricated by electrospinning. It was then treated with NH3 plasma to enhance surface hydrophilicity, cell attachment, growth and differentiation potential. X-ray photoelectron spectroscopy (XPS) measurements were used to evaluate the modification of the scaffold's surface chemistry. Electrospun scaffolds were coated with willemite (Zn2SiO4) bioceramic nanoparticles. Scaffold characterization was done by scanning electron microscope (SEM), differential scanning calorimetry (DSC), contact angle measurements and tensile analysis. MTT assay was used to assess the biocompatibility of fibrous scaffolds loaded with Zn2SiO4 regarding proliferation support. Osteogenic differentiation of cultured human mesenchymal stem cells (hMSCs) on fibers was evaluated using common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium mineral deposition, quantitative real-time PCR (qPCR) and immunocytochemical analysis (ICC). According to the results, proliferation and osteogenic differentiation of hMSCs were significantly enhanced after coating Zn2SiO4 on fibrous scaffolds. These results were detected by higher ALP activity, biomineralization and expression of osteogenic related genes and proteins in differentiated hMSCs. In conclusion, our results indicated that the combination of Zn2SiO4 nanoparticles and electrospun fibers is able to provide a new, suitable and more efficient matrix to support stem cells differentiation for bone tissue engineering applications.

Mechanical and Vascular Cues Synergistically Enhance Osteogenesis in Human Mesenchymal Stem Cells.

Development and maintenance of a vascular network are critical for bone growth and homeostasis; strategies that promote vascular function are critical for clinical success of tissue-engineered bone constructs. Co-culture of endothelial cells (ECs) with mesenchymal stem cells (MSCs) and exposure to 10% cyclic tensile strain have both been shown to regulate osteogenesis in isolation, but potential synergistic effects have yet to be explored. The objective of this study was to expose an MSC-EC co-culture to 10% cyclic tensile strain to examine the role of this mechanical stimulus on MSC-EC behavior. We hypothesized that paracrine signaling from ECs would stimulate osteogenesis of MSCs, and exposure to 10% cyclic tensile strain would enhance this anabolic signal. Human umbilical vein ECs and human bone marrow-derived MSCs were either monocultured or co-cultured at a 1:1 ratio in a mixed osteo/angiogenic medium, exposed to 10% cyclic tensile strain at 1 Hz for 4 h/day for 2 weeks, and biochemically and histologically analyzed for endothelial and osteogenic markers. While neither 10% cyclic tensile strain nor co-culture alone had a significant effect on osteogenesis, the concurrent application of strain to an MSC-EC co-culture resulted in a significant increase in calcium accretion and mineral deposition, suggesting that co-culture and strain synergistically enhance osteogenesis. Neither co-culture, 10% cyclic tensile strain, nor a combination of these stimuli affected endothelial markers, indicating that the endothelial phenotype remained stable, but unresponsive to the stimuli evaluated in this study. This study is the first to investigate the role of cyclic tensile strain on the complex interplay between ECs and MSCs in co-culture. The results of this study provide key insights into the synergistic effects of 10% cyclic tensile strain and co-culture on osteogenesis. Understanding mechanobiological factors affecting MSC-EC crosstalk will help enhance strategies for creating vascularized tissues in tissue engineering and regenerative medicine.

Biomimetic scaffolds containing nanofibers coated with willemite nanoparticles for improvement of stem cell osteogenesis

Nowadays, discovering osteogenesis stimulating effectors is one of the major topics in bone tissue engineering and regenerative medicine. In this study, the proliferation rate and osteogenic differentiation potency of adipose-derived mesenchymal stem cells (AT-MSCs) cultured on poly (l-lactide acid) (PLLA) and willemite-coated PLLA were investigated by MTT assay and common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium mineral deposition and bone-related genes expression.Willemite-coated PLLA showed a higher proliferation support to AT-MSCs in comparison to PLLA and TCPS. During the period of study, AT-MSCs cultured on willemite-coated PLLA scaffolds exhibited the greatest ALP activity and mineralization. Gene expression analysis demonstrated that the highest expression of four important osteogenic-related genes, osteonectin, Runx2, collagen type 1 and osteocalcin was observed in stem cells cultured on willemite-coated PLLA nanofibrous scaffolds. According to the results, willemite-coated PLLA could be a suitable substrate to support the proliferation and osteogenic differentiation of stem cells and holds promising potential for bone tissue engineering and regenerative medicine applications.

A carboxymethyl chitosan and peptide-decorated polyetheretherketone ternary biocomposite with enhanced antibacterial activity and osseointegration as orthopedic/dental implants.

Carbon fiber-reinforced polyetheretherketone (CFRPEEK) possesses biomechanical properties such as elastic modulus similar to human bones and is becoming a dominant alternative to replace the traditional metallic implants. The defective osseointegration and bacterial infection risk of CFRPEEK, however, impede its clinical adoption. In the current study, a newly-developed carbon fiber-reinforced polyetheretherketone/nanohydroxyapatite (CFRPEEK/n-HA) ternary biocomposite was functionalized by covalently grafting carboxymethyl chitosan (CMC) followed by the decoration of a bone-forming peptide (BFP) assisted via the polydopamine tag strategy. Antibacterial test with Staphylococcus aureus (S. aureus) indicated that the CMC and peptide-conjugated substrates (pep-CMC-CFRPEEK/n-HA) significantly suppressed bacterial adhesion. In vitro cell attachment/growth, spreading assay, alkaline phosphatase activity, real-time PCR analysis, osteogenesis-related protein expression and calcium mineral deposition all disclosed greatly accelerated adhesion, proliferation and osteo-differentiation of human mesenchymal stem cells (hMSCs) on the pep-CMC-CFRPEEK/n-HA biocomposite due to the additive effect of the CMC polysaccharide and the small osteoinductive peptide. More importantly, in vivo evaluation of the beagle tibia model by means of micro-CT, histological analysis, SEM observation and fluorescent labeling confirmed the remarkably boosted bioactivity and osteointegration. The CFRPEEK/n-HA ternary composite with the dual functions of bacterial adhesion reduction and osteointegration promotion holds great potential as a bioactive implant material in orthopedic/dental applications based on this scheme.

Enhanced osteoconductivity of polyethersulphone nanofibres loaded with bioactive glass nanoparticles in in vitro and in vivo models.

There is growing need for new scaffold constructions for synthetic bone graft substitutes to repair large bone lesions. A very promising and important class of new implants for tissue engineering is based on three-dimensional scaffolds and bioceramics. In this study, after investigation of mechanical properties of polyethersulphone (PES) nanofibres, fabricated by electrospinning methodology and coated with bioactive glass (BG), cells of the MG-63 line were cultured on surfaces of these scaffolds. Their capacity to support MG-63 proliferation was also investigated in vitro by MTT assay. Osteoconductivity on these scaffolds was investigated by the common osteogenic markers alkaline phosphatase (ALP) activity, calcium mineral deposition and bone-related gene activation. Next, a bone reconstruction of rat critical-size defects model was evaluated using radiographic imaging analysis (digital mammography), computed tomography and histological examination. In vitro results indicated that biocompatibility and osteogenic markers of MG-63 cells were significantly enhanced after coating PES with BG. Based on in vivo results, new bone formation in the defect site was enhanced in implanted rats in comparison with a control group. The highest reconstruction was observed in animals implanted with BG-coated nanofibres. Osteoconductivity of PES nanofibres was markedly enhanced after coating them with BG, and introduction of this construct as new bone-graft substitute for bone loss and defects is indicated.

Peptide-laden mesoporous silica nanoparticles with promoted bioactivity and osteo-differentiation ability for bone tissue engineering

Combination of mesoporous silica materials and bioactive factors is a promising niche-mimetic solution as a hybrid bone substitution for bone tissue engineering. In this work, we have synthesized biocompatible silica-based nanoparticles with abundant mesoporous structure, and incorporated bone-forming peptide (BFP) derived from bone morphogenetic protein-7 (BMP-7) into the mesoporous silica nanoparticles (MSNs) to obtain a slow-release system for osteogenic factor delivery. The chemical characterization demonstrates that the small osteogenic peptide is encapsulated in the mesoporous successfully, and the nitrogen adsorption–desorption isotherms suggest that the peptide encapsulation has no influence on mesoporous structure of MSNs. In the cell experiment, the peptide-laden MSNs (p-MSNs) show higher MG-63 cell proliferation, spreading and alkaline phosphatase (ALP) activity than the bare MSNs, indicating good in vitro cytocompatibility. Simultaneously, the osteogenesis-related proteins expression and calcium mineral deposition disclose enhanced osteo-differentiation of human mesenchymal stem cells (hMSCs) under the stimulation of the p-MSNs, confirming that BFP released from MSNs could significantly promote the osteogenic differentiation of hMSCs, especially at 500μg/mL of p-MSNs concentration. The peptide-modified MSNs with better bioactivity and osteogenic differentiation make it a potential candidate as bioactive material for bone repairing, bone regeneration, and bio-implant coating applications.

Enhanced healing of rat calvarial defects with MSCs loaded on BMP-2 releasing chitosan/alginate/hydroxyapatite scaffolds.

In this study, we designed a chitosan/alginate/hydroxyapatite scaffold as a carrier for recombinant BMP-2 (CAH/B2), and evaluated the release kinetics of BMP-2. We evaluated the effect of the CAH/B2 scaffold on the viability and differentiation of bone marrow mesenchymal stem cells (MSCs) by scanning electron microscopy, MTS, ALP assay, alizarin-red staining and qRT-PCR. Moreover, MSCs were seeded on scaffolds and used in a 8 mm rat calvarial defect model. New bone formation was assessed by radiology, hematoxylin and eosin staining 12 weeks postoperatively. We found the release kinetics of BMP-2 from the CAH/B2 scaffold were delayed compared with those from collagen gel, which is widely used for BMP-2 delivery. The BMP-2 released from the scaffold increased MSC differentiation and did not show any cytotoxicity. MSCs exhibited greater ALP activity as well as stronger calcium mineral deposition, and the bone-related markers Col1α, osteopontin, and osteocalcin were upregulated. Analysis of in vivo bone formation showed that the CAH/B2 scaffold induced more bone formation than other groups. This study demonstrates that CAH/B2 scaffolds might be useful for delivering osteogenic BMP-2 protein and present a promising bone regeneration strategy.

Fibroblast-like synoviocytes induce calcium mineral formation and deposition.

Calcium crystals are present in the synovial fluid of 65%–100% patients with osteoarthritis (OA) and 20%–39% patients with rheumatoid arthritis (RA). This study sought to investigate the role of fibroblast-like synoviocytes (FLSs) in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla) protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s). The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

Initial stages of calcium uptake and mineral deposition in sea urchin embryos.

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. We reconstructed various stages of the formation pathway of calcium carbonate from calcium ions in sea water to mineral deposition and integration into the forming spicules. Monitoring calcium uptake with the fluorescent dye calcein shows that calcium ions first penetrate the embryo and later are deposited intracellularly. Surprisingly, calcium carbonate deposits are distributed widely all over the embryo, including in the primary mesenchyme cells and in the surface epithelial cells. Using cryo-SEM, we show that the intracellular calcium carbonate deposits are contained in vesicles of diameter 0.5-1.5 μm. Using the newly developed airSEM, which allows direct correlation between fluorescence and energy dispersive spectroscopy, we confirmed the presence of solid calcium carbonate in the vesicles. This mineral phase appears as aggregates of 20-30-nm nanospheres, consistent with amorphous calcium carbonate. The aggregates finally are introduced into the spicule compartment, where they integrate into the growing spicule.

The effects of functionalized titanium with alendronate and bone morphogenic protein-2 for improving osteoblast activity

The aim of this study was to investigate the effect of functionalized titanium (Ti) with alendronate (Aln) and bone morphogenic protein-2 (BMP-2) for enhancement of osteoblast activity in vitro. Aln and/or BMP-2 were sequentially immobilized to the heparinized-Ti (Hep-Ti) surface. The compositions of pristine Ti and Hep-Ti with or without Aln and/or BMP-2 were characterized by scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS). Aln and/or BMP-2 onto Hep-Ti surface were released in a sustained manner. Osteoblast activities on all Ti substrates were investigated by cell proliferation assays, alkaline phosphate (ALP) activity, calcium deposition, gene expressions of osteocalcin and osteopontin. Aln/BMP-2/Hep-Ti significantly enhanced ALP activity, calcium mineral deposition, and gene expressions of osteoblast cells when compared with pristine Ti, Aln/Hep-Ti, and BMP-2/Hep-Ti. From these results, functionalized Ti substrates with alendronate and BMP-2 such as Aln/BMP-2/Hep-Ti are a promising material for the enhanced osteoblast activities in orthopedic and dental fields.

Enhanced reconstruction of rat calvarial defects achieved by plasma-treated electrospun scaffolds and induced pluripotent stem cells

Tissue engineering with a combination of stem cells and nanofibrous scaffolds has attracted interest with regard to bone regeneration applications. In the present study, human induced pluripotent stem cells (iPSCs) were cultured on polymeric nanofibrous polyethersulfone (PES) with and without plasma treatment. The capacity of PES and plasma-treated PES (Plasma-PES) scaffolds to support the proliferation and osteogenic differentiation of iPSCs was investigated by MTT assay and for common osteogenic markers such as alkaline phosphatase activity, calcium mineral deposition and bone-related genes. Plasma-PES scaffolds with or without iPSCs were subsequently used to evaluate bone regeneration of critical-size defects in the rat by digital mammography, multislice spiral-computed tomography imaging and histological analysis. The results of in vitro analysis showed that plasma treatment significantly enhanced iPSC proliferation and osteogenesis. After 8 weeks of iPSC-loaded Plasma-PES implantation, no mortality or complication was observed in animals or at the site of surgery. Imaging analysis revealed more extensive bone reconstruction in rats receiving nanofibers compared with untreated control groups. Moreover, Plasma-PES seeded with iPSCs induced the highest regeneration of bone defects among all groups. These findings were confirmed by histological staining. Affective osseointegration was observed in implanted scaffolds. Thus, plasma-treated nanofibrous scaffolds are suitable tissue-engineered matrices for supporting the proliferation and osteogenic differentiation of iPSCs and might also be appropriate for the reconstruction of bone defects.