Osteogenic effects of bioactive glass on bone marrow stromal cells

Bioactive glass (BG) is an effective synthetic bone graft material. BG granules of narrow size range (300-355 mum) have the ability to form new bone tissue inside excavations produced by in vivo resorption. Previously, we demonstrated that BG stimulates the differentiation of cultured osteoblast precursors if the glass surface was biomimetically modified by the formation of bone-like apatite and adsorption of serum proteins. We now report that modified BG can also increase the rate at which multipotential rat bone marrow stromal cells (rMSC) will undergo osteogenesis. BG promoted rMSC osteogenesis both when cells were plated in contact with BG and when cells were not directly in contact with the BG. Alkaline phosphatase activity, a marker of bone cell differentiation, was used as an indicator for osteogenesis. Alkaline phosphatase activity of rMSCs exposed to osteoinducers such as ascorbate, dexamethasone, and BMP-2 was enhanced in the presence of BG. The stimulatory effect of BG was more pronounced in rMSC cultures with low basal alkaline phosphatase activity than in those with higher activity. The enhanced differentiation of rMSCs was associated with both a change in rMSC morphology and altered chemical composition of the cell culture media. rMSCs cultured on BG in the presence of BMP or dexamethasone exhibited a more rounded osteoblast-like appearance as compared with cells grown on tissue culture plastic. In the presence of BG, elevated levels of calcium and silicon in the culture medium were observed throughout the 7-day culture period, suggesting a continuous dissolution of surface-modified BG and resulting release of BG dissolution products. The data suggest that both surface- and solution-mediated events play a role in the osteogenic effect of BG.     

Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells

The growth and differentiation of mesenchymal stem cells (MSCs) is controlled by various growth factors, the activities of which can be modulated by heparan sulfates (HSs). We have previously noted the necessity of sulfated glycosaminoglycans for the fibroblast growth factor type 2 (FGF-2)-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of HS to cultures of primary rat MSCs stimulates their proliferation, leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation, and osteogenic differentiation of rat bone marrow stem cells (rMSCs) when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. We show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth, resulting in the cells being unable to reach the critical density necessary to induce differentiation. Blocking FGFR1 signaling in postconfluent osteogenic cultures significantly increased calcium deposition. Taken together our data suggest that FGFR1 signaling plays an important role during osteogenic differentiation, first by stimulating cell growth that is closely followed by an inhibitory effect once the cells have reached confluence. It also confirms the importance of HS as a coreceptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.     

The effect of decellularized bone/bone marrow produced by high-hydrostatic pressurization on the osteogenic differentiation of mesenchymal stem cells

Decellularized bone/bone marrow was prepared to provide a microenvironment mimicking that of the bone marrow for three-dimensional culture in vitro. Bone/bone marrows were hydrostatically pressed at 980 MPa at 30 °C for 10 min to dismantle the cells. Then, they were washed with EGM-2 and further treated in an 80% EtOH to remove the cell debris and lipid, respectively. After being rinsed and shaken with PBS again, treated bone/bone marrows were stained with hematoxylin and eosin (H-E) to assess the efficacy of decellularization. Cells were determined to have been completely removed through H-E staining of their sections and DNA quantification. Rat mesenchymal stem cells (rMSCs) were seeded on the decellularized bone/bone marrows and cultured for 21 days. The adhesion of rMSCs on or into decellularized bone/bone marrows was confirmed and proliferated over time in culture. The osteogenic differentiation effect of decellularized bone/bone marrows on rMSCs in the presence or absence of dexamethasone was investigated. Decellularized bone/bone marrows without dexamethasone significantly increased alkaline phosphatase (ALP) activity, indicating promoted osteogenic differentiation of rMSCs. In an animal study, when decellularized bone/bone marrows were implanted into the rat subcutaneous, no immune reaction occurred and clusters of the hematopoietic cells could be observed, suggesting the decellularized bone/bone marrows can provide a microenvironment in vivo.     

Natural coral exoskeleton as a bone graft substitute: a review

Natural coral graft substitutes are derived from the exoskeleton of marine madreporic corals. Researchers first started evaluating corals as potential bone graft substitutes in the early 1970s in animals and in 1979 in humans. The structure of the commonly used coral, Porites, is similar to that of cancellous bone and its initial mechanical properties resemble those of bone. The exoskeleton of these high content calcium carbonate scaffolds has since been shown to be biocompatible, osteoconductive, and biodegradable at variable rates depending on the exoskeleton porosity, the implantation site and the species. Although not osteoinductive or osteogenic, coral grafts act as an adequate carrier for growth factors and allow cell attachment, growth, spreading and differentiation. When applied appropriately and when selected to match the resorption rate with the bone formation rate of the implantation site, natural coral exoskeletons have been found to be impressive bone graft substitutes. The purpose of this article is to review and summarize all the pertinent work that has been published on natural coral as a bone graft including in vitro, animal and clinical human studies. Preliminary report of our own experiments as well as our recommendations on the use of coral are also included.     

Is there a relation between vitamin D,interleukin-17, and bone mineral density in patients with inflammatory bowel disease?

IntroductionIn inflammatory bowel diseases (IBD), osteopenia and osteoporosis constitute a significant medical problem. Cytokines, especially IL-17, play an important role in the pathogenesis of IBD and osteoporosis. Vitamin D is a regulator of bone metabolism, and helps maintain immune system homeostasis.Material and methodsThe research sample consisted of 208 persons: 83 patients (age 35 ±11.99 years) with Crohn’s disease (CD); 86 patients (age 39.58 ±14.74 years) with ulcerative colitis (UC); and 39 persons (age 30.74 ±8.63 years) in the control group (CG). Clinical data on bone mineral density of the lumbar spine (L2-L4), bone mineral density of the femoral neck (FN), and body mass index (BMI) were collected. 25OHD and IL-17 serum concentrations were also measured.ResultsBody mass index (kg/m²) results: in CD, 21.51 ±3.68; in UC, 23.31 ±4.38; and in CG, 24.57 ±3.45 (p < 0.01). Densitometry results for L2–L4 T-score SD: in CD –0.83 ±1.45; in UC –0.47 ±1.15; in CG 0.09 ±0.70. Densitometry results for FN T-score SD: in CD –0.62 ±1.26; in UC –0.29±1.17; in CG 0.41 ±1.03 25OHD (ng/ml) serum concentrations: in CD, 21.33±12.50; in UC, 22.04±9.56; in CG, 21.56±9.11 (ns). IL-17 (pg/ml) serum concentrations: in CD, 8.55±10.99; in UC, 11.67±12.97; in CG, 5.16±9.11 (ns).ConclusionsInflammatory bowel diseases patients and persons from the CG did not differ in vitamin D or IL-17 levels. Patients with a mild course of the disease had a higher vitamin D concentration and bone mineral density. In UC, higher vitamin D concentrations were associated with lower IL-17 concentrations. The IBD patients with a severe course of the disease had a lower body mass than those in the CG and the patients with a mild course of the disease.     

Graft materials and bone marrow stromal cells in bone tissue engineering

Bone augmentation procedures rely on osteogenic/osteoconductive properties of bone graft material (BGM). A further improvement is represented by use of autologous bone marrow stromal cells (BMSC), expanded in vitro and seeded on BGM before implantation in the bone defect. The effect of different BGMs on BMSC osteogenic differentiation was evaluated. BMSC were cultured in vitro in the presence of different BGM (natural, synthetic, and mixed origins). Cellular morphology was analyzed with scanning electron microscopy. The capability of BMSC to differentiate was determined in vitro by alkaline phosphatase gene expression and enzyme activity at different time points (7, 14, and 28 days) and in vivo by ectopic bone formation of implanted tissue constructs in an immunodeficient murine model. BGM supports the cell adhesion and osteogenic differentiation of BMSC developing a useful tool in the bone tissue engineering.     

The use of injectable,thermosensitive poly(organophosphazene)–RGD conjugates for the enhancement of mesenchymal stem cell osteogenic differentiation

An injectable and thermosensitive poly(organophosphazene)–RGD conjugate to enhance functionality was synthesized by a covalent amide linkage between a cell adhesion peptide, GRGDS and carboxylic acid-terminated poly(organophosphazene). The aqueous solutions of synthesized poly(organophosphazene)–GRGDS conjugates existed in an injectable fluid state at room temperature and immediately formed a hydrogel at body temperature. The rabbit mesenchymal stem cells (rMSCs) on the polymer–GRGDS conjugate (conjugate 1-2, 0.05 mol fraction as GRGDS) hydrogel constructs using an injection method into a nude mouse were proved to express markers at mRNA level for all stages towards osteogenesis and mainly a sharp increase of osteocalcin (OCN, a typical late osteogenic differentiation marker) levels at 4th week post-induction indicated that the maturation process has started within this period. By histological and immunohistochemical evaluations, significantly high mineralization level by calcium contents was detected qualitatively and collagen type I (Col I), a major characteristic marker protein, was mainly and highly expressed by the rMSCs cultivated in the polymer–GRGDS conjugate hydrogel constructs formed into the nude mouse. The results suggest that the poly(organophosphazene)–GRGDS conjugate to enhance biofunctionality hold a promise for cell delivery material to induce osteogenic differentiation of MSC for enhancing ectopic bone formation.     

Bioactive membranes for bone regeneration applications: Effect of physical and biomolecular signals on mesenchymal stem cell behavior

This study focuses on the in vitro characterization of bioactive elastin-like recombinamer (ELR) membranes for bone regeneration applications. Four bioactive ELRs exhibiting epitopes designed to promote mesenchymal stem cell adhesion (RGDS), endothelial cell adhesion (REDV), mineralization (HAP), and both cell adhesion and mineralization (HAP-RGDS) were synthesized using standard recombinant protein techniques. The materials were then used to fabricate ELR membranes incorporating a variety of topographical micropatterns including channels, holes and posts. Primary rat mesenchymal stem cells (rMSCs) were cultured on the different membranes and the effects of biomolecular and physical signals on cell adhesion, morphology, proliferation, and differentiation were evaluated. All results were analyzed using a custom-made MATLAB program for high throughput image analysis. Effects on cell morphology were mostly dependent on surface topography, while cell proliferation and cell differentiation were largely dependent on the biomolecular signaling from the ELR membranes. In particular, osteogenic differentiation (evaluated by staining for the osteoblastic marker osterix) was significantly enhanced on cells cultured on HAP membranes. Remarkably, cells growing on membranes containing the HAP sequence in non-osteogenic differentiation media exhibited significant up-regulation of the osteogenic marker as early as day 5, while those growing on fibronectin-coated glass in osteogenic differentiation media did not. These results are part of our ongoing effort to develop an optimized molecularly designed periosteal graft.     

A Novel Semisynthetic Molecule Icaritin Stimulates Osteogenic Differentiation and Inhibits Adipogenesis of Mesenchymal Stem Cells

Background We previously reported that the constitutional flavonoid glycosides derived from herb Epimedium (EF, composed of seven flavonoid compounds with common nuclear stem) exerted beneficial effects on the bone, including promoting bone formation and inhibiting bone marrow fat deposition. Recent in vivo study showed that Icaritin was a common metabolite of these constitutional flavonoid glycosides, indicating that Icaritin is a bioactive compound. The present study was designed to investigate whether Icaritin could promote osteogenic differentiation and suppress adipogenic differentiation of marrow mesenchymal stem cells (MSCs).Methods Primary MSCs were harvested from adult mice and exposed to Icaritin to evaluate whether it could promote osteogenesis and suppress adipogenesis using the following assays: determination of alkaline phosphatase (ALP) activity and mineralization; mRNA expression of osteogenic differentiation marker Runx2; osteocalcin and bone sialoprotein (BSP) by RT-PCR; quantification of adipocyte-like cells by Oil Red O staining assay and mRNA expression for adipogenic differentiation markers peroxisome proliferator-activated receptor gamma (PPARγ); adipocyte fatty acid binding protein (aP2) and lipoprotein lipase (LPL) by RT-PCR. For the underlying mechanism, glycogen synthase kinase-3beta (GSK3β) and β-catenin were also explored by western blotting.Results Icaritin promoted osteogenic differentiation and maturation of MSCs as indicated by increased mRNA expression for Runx2, osteocalcin and BSP, and enhanced ALP activity and mineralization; Icaritin inhibited adipogenic differentiation, as indicated by decreased mRNA expression for PPARγ, LPL, aP2, and suppressed formation of adipocyte-like cells; Icaritin inactivated GSK3β and suppressed PPARγ expression when promoting osteogenesis and suppressing adipogenesis of MSCs.Conclusion This was the first study demonstrating that the novel semisynthetic molecule Icaritin could stimulate osteogenic differentiation and inhibit adipogenesis of MSCs, which was associated with the suppression of GSK3β and PPARγ.     

The effect of bioactive glass content on synthesis and bioactivity of composite poly (lactic-co-glycolic acid)/bioactive glass substrate for tissue engineering

Tissue engineering offers a promising new approach to bone tissue grafting. One material that has received attention in this regard is the polymer poly (lactic-co-glycolic acid) (PLGA). It has the advantage of controllable bioresorption and ease of processing. Another material of interest is bioactive glass (BG), which shows the ability to stimulate osteoblastic differentiation of osteoprogenitor cells. In this study, we reported on the optimal synthesis parameters and the kinetics of formation of calcium phosphate (Ca-P) phase at the surface of PLGA/BG composites. The formation of calcium phosphate layer was confirmed using scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDXA). PLGA-30%BG microspheres based porous scaffolds for bone tissue engineering were examined for their ability to promote osteogenesis of marrow stromal cells (MSC). This porous scaffold supported both MSC proliferation and promoted MSC differentiation into cells expressing the osteoblast phenotype. It therefore demonstrates significant potential as a bone replacement material.     

Effects of hydroxyapatite in 3-D chitosan-gelatin polymer network on human mesenchymal stem cell construct development

Human mesenchymal stem cells (hMSCs) have great potential in bone tissue engineering, and hydroxyapatite (HA), a natural component of human hard tissues, is believed to support hMSC growth and osteogenic differentiation. In this study, two types of biomimetic composite materials, chitosan-gelatin (CG) and hydroxyapatite/chitosan-gelatin (HCG), were fabricated and compared to examine the effects of HA on hMSC adhesion and 3-D construct development. The 2-D membranes were prepared to examine the influence of HA on adhesion efficiency of hMSCs, while 3-D porous scaffolds were produced to investigate the effects of HA on material adsorption properties and 3-D hMSC construct development. HA was found to promote protein and calcium ion adsorption of the 3-D porous scaffolds in the complete tissue culture media. HMSCs exhibited higher initial cell adhesion efficiency to 2-D HCG membranes, and maintained higher proliferation rates in the 3-D porous HCG than CG scaffolds with 3.3 times higher final DNA amount in HCG scaffolds over a 35-day period. Colony forming unit-fibroblast (CFU-F) assays showed that higher percentages of cells maintained their progenicity in the 3-D porous HCG scaffolds over the 35-day culture period. Differentiation assays indicated that the multi-lineage differentiation potential of the hMSCs was preserved in both 3-D porous scaffolds. However, higher alkaline phosphate activity was detected in the 3-D porous HCG scaffolds upon osteogenic induction indicating improved osteogenic differentiation potential. The results demonstrate that enhanced protein and calcium ion adsorption properties of HA in the CG polymer network improve initial cell adhesion and long-term growth, favor osteogenic differentiation upon induction, as well as maintain the progenicity of the 3-D hMSC constructs.     

Repair of cranial bone defects with adipose derived stem cells and coral scaffold in a canine model

Adipose derived stem cells (ASCs) with osteogenic differentiation potential have been documented as an alternative cell source for bone regeneration. However, most of previous in vivo studies were carried out on small animals along with relatively short-term follow-up. In this study, we investigated the feasibility of using ASCs and coral scaffolds to repair a cranial bone defect in a canine model, and followed up the outcome for up to 6 month. Autologous ASCs isolated from canine subcutaneous fat were expanded, osteogenically induced, and seeded on coral scaffolds. Bilateral full-thickness defects (20 mm×20 mm) of parietal bone were created. The defects were either repaired with ASC-coral constructs (experimental group) or with coral alone (control group). Three-dimensional CT scan showed that new bones were formed in the experimental group at 12 weeks post-implantation, while coral scaffolds were partially degraded in the control group. By radiographic analysis at 24 weeks post-transplantation, it was shown that an average of 84.19±6.45% of each defect volume had been repaired in experimental side, while the control side had only 25.04±18.82% of its volume filled. Histological examination revealed that the defect was repaired by typical bone tissue in experimental side, while only minimal bone formation with fibrous connection was observed in the control group. The successful repair of critical-sized bone defects via the current approach substantiates the potentiality of using ASCs with coral scaffolds for bone regeneration.     

miR-34b对骨髓间充质干细胞成骨分化的影响及相关机制

目的：探讨miRNA-34b在骨髓间充质干细胞成骨分化过程中的表达及其可能的作用靶点和作用机制。方法：采用密度梯度离心和全骨髓贴壁相结合的方法分离培养人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hBMSCs),并在体外诱导成骨分化。采用实时荧光定量PCR技术,检测hBMSCs成骨分化过程中的miR-34b的表达水平;然后过表达miR-34b,进一步观察其对hBMSCs成骨分化的影响。同时检测过表达miR-34b对成骨分化的关键信号通路之一Notch信号通路的活性影响,初步探讨其可能涉及的作用机制。结果：成功分离出hBMSCs,并构建了hBMSCs体外诱导成骨分化模型;且随着成骨诱导培养时间的延长,miRNA-34b表达水平逐渐降低。ALP活性检测、茜素红染色检测结果显示过表达miRNA-34b后,ALP活性显著降低,且茜素红染色的钙盐结节明显减少;同时Western blot实验结果显示过表达miRNA-34b后,成骨特异性标记分子Runx2的蛋白表达水平显著下降(P<0.05)。此外,过表达miRNA-34b后,Notch信号通路的活性显著降低。结论：miRNA-34b能够负向调控人骨髓间充质干细胞成骨分化;其作用机制可能与抑制Notch信号通路的活性有关,提示miRNA-34b可以作为诊断和靶向治疗慢性炎症性骨疾病的潜在作用靶点。     

Synergistic effects of dimethyloxalylglycine and butyrate incorporated into α-calcium sulfate on bone regeneration

Osteogenesis is closely related to angiogenesis, and the combined delivery of angiogenic and osteogenic factors has been suggested to enhance bone regeneration. Small molecules have been explored as alternatives to growth factors for tissue regeneration applications. In this study, we examined the effects of the combined application of angiogenic and osteogenic small molecules on bone regeneration using a prolyl hydroxylase, dimethyloxalylglycine (DMOG), and a histone deacetylase inhibitor, butyrate. In a critical size bone defect model in rats, DMOG and butyrate, which were incorporated into α calcium sulfate (αCS), resulted in synergistic enhancements in bone and blood vessel formation, eventually leading to bone healing, as confirmed by micro-CT and histological analyses. In MC4 pre-osteoblast cultures, DMOG and butyrate enhanced the pro-angiogenic responses and osteoblast differentiation, respectively, which were evaluated based on the levels of hypoxia inducible factor (HIF)-1α protein and the expression of pro-angiogenic molecules (VEGF, home oxidase-1, glucose transporter-1) and by alkaline phosphatase (ALP) activity and the expression of osteoblast phenotype marker molecules (ALP, α1(I)col, osteocalcin, and bone sialoprotein). DMOG combined with butyrate synergistically improved osteoblast differentiation and pro-angiogenic responses, the levels of which were drastically increased in the cultures on αCS disks. Furthermore, it was demonstrated that αCS increased the level of HIF-1α and as a consequence VEGF expression, and supported osteoblast differentiation through the release of calcium ions from the αCS. Altogether, the results of this study provide evidence that a combination treatment with the small molecules DMOG and butyrate can expedite the process of bone regeneration and that αCS can be an efficient delivery vehicle for the small molecules for bone regeneration.     

Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSC) are a promising cell source with pluripotency and self-renewal properties. Design of simple and robust biomaterials with an innate ability to induce lineage-specificity of hiPSC is desirable to realize their application in regenerative medicine. In this study, the potential of biomaterials containing calcium phosphate minerals to induce osteogenic differentiation of hiPSC was investigated. hiPSC cultured using mineralized gelatin methacrylate-based matrices underwent osteogenic differentiation ex vivo, in both two-dimensional and three-dimensional cultures, in growth medium devoid of any osteogenic-inducing chemical components or growth factors. The findings that osteogenic differentiation of hiPSC can be achieved through biomaterial-based cues alone present new avenues for personalized regenerative medicine. Such biomaterials that could not only act as structural scaffolds, but could also provide tissue-specific functions such as directing stem cell differentiation commitment, have great potential in bone tissue engineering.