Osteogenic Protein-1 Research Articles

Effects of overexpression of Notch intracellular domain on proliferation and osteogenic differentiation of human periodontal ligament stem cells

Objective: To investigate the effect of overexpression of Notch intracellular domain (NICD) on proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSC). Methods: The third generation hPDLSC with stable overexpressing of NICD were assigned as experimental group, normal hPDLSC were as negative control group and hPDLSC transfected with empty vector were as blank control group. The effect of overexpressing NICD on proliferation ability of hPDLSC was detected by using cell counting kit-8 (CCK-8). Alizarin Red staining and real-time quantitative PCR (qPCR) were used to detect the effects of NICD on cementum attachment proteins (CAP), osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2) and Notch signal pathway receptor Notch1. The effect of overexpressing NICD on hPDLSC osteogenic protein RUNX2 and flag marker protein (used to label NICD) were detected by using Western blotting. Results: CCK-8 results showed that there were no significant differences in A values amongst the three groups for 1-2 days (P>0.05). The number of cells in the experimental group was significantly increase than that of the two control groups from the third to seventh days (A values were 0.203±0.016, 0.364±0.014, 0.449±0.020, 0.549±0.020 and 0.570±0.020, respectively) (P<0.05). Alizarin red staining showed that compared with the blank control group and negative control group, the mineralized nodules in the experimental group had smaller formation range and lighter color, and the differences were statistically significant (P<0.05). The expressions of CAP gene (0.751±0.058, 0.887±0.025), osteocalcin gene (0.592±0.051, 0.670±0.045) and RUNX2 gene (0.319±0.038, 0.684±0.055) at 14 and 21 days in the experimental group were significantly lower than those in the negative control group respectively (P<0.05). However, the expression levels of Notch1 gene at 14 and 21 days (2.507±0.047, 4.041±0.219) were significantly higher than those of negative and blank control groups (P<0.05). The results of Western blotting showed that the expressions of flag marker protein (0.167±0.007, 0.204±0.010) at 14 and 21 days in the experimental group were significantly higher than those in the negative and blank control groups (P<0.05). However, the expressions of RUNX2 protein (0.075±0.006, 0.074±0.013) at 14 and 21 days were significantly lower than that in the negative control group (0.092±0.003, 0.118±0.008) and blank control group (0.174±0.006, 0.212±0.008) (P<0.05). Conclusions: Overexpression of NICD can promote the proliferation capacity of hPDLSC and inhibit its osteogenic differentiation.

Evaluation of fibroblast growth factor-2 on the proliferation of osteogenic potential and protein expression of stem cell spheroids composed of stem cells derived from bone marrow.

Fibroblast growth factor-2 (FGF-2) is reported to have various functions and is considered a key human mesenchymal stem cell mitogen, often supplemented to increase human mesenchymal stem cell growth rates. The purpose of this study was to evaluate the effects of FGF-2 on cellular viability and osteogenic differentiation using three-dimensional cell spheroids of stem cells. Three-dimensional cell spheroids were fabricated using concave silicon elastomer-based microwells in the presence of FGF-2 at concentrations of 0, 30, 60 and 90 ng/ml. Qualitative cellular viability was determined with a confocal microscope, and quantitative cellular viability was evaluated using a Cell Counting Kit-8 assay. Alkaline phosphatase activity and Alizarin Red S staining were used to assess osteogenic differentiation. Spheroids were well formed in silicon elastomer-based concave microwells on Day 1. The average spheroid diameters at Day 1 for FGF-2 at 0, 30, 60 and 90 ng/ml were 202.2±3.0, 206.6±22.6, 208.8±6.8 and 196.6±26.7 µm, respectively (P>0.05). The majority of the cells in the cell spheroids emitted green fluorescence. The relative Cell Counting Kit-8 assay values for FGF-2 at 0, 30, 60 and 90 ng/ml at Day 1 were 100.0±5.5, 101.8±8.8, 99.2±4.8 and 103.4±9.6% (P>0.05). The addition of FGF-2 at 60 ng/ml concentration produced the highest value for alkaline phosphatase activity. Mineralized extracellular deposits were evenly observed in each group, and the highest value was identified for FGF-2 groups at 60 ng/ml concentration for Alizarin Red S staining. Based on these findings, it was concluded that FGF-2 may increase alkaline phosphatase activity or Alizarin Red S staining, and further studies are needed to fully elucidate the mechanisms of FGF-2.

Influence of epigallocatechin-3-gallate in promoting proliferation and osteogenic differentiation of human periodontal ligament cells

BackgroundEpigallocatechin-3-gallate (EGCG) was recently proposed to have the potential to regulate bone metabolism, however, its influence on osteogenesis remains controversial. The present study aimed to investigate the effects of EGCG on the proliferation and osteogenesis of human periodontal ligament cells (hPDLCs).MethodsCells were cultured in osteogenic medium and treated with EGCG at various concentrations. Cell proliferation was analyzed using a CCK-8 assay and acridine orange (AO)/ethidium bromide (EB) staining. Flow cytometry was used to measure the intracellular reactive oxygen species (ROS) potential of hPDLCs. The expression levels of osteogenic marker genes and proteins in hPDLCs, including type I collagen (COL1), runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osterix (OSX), were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. In addition, alkaline phosphatase (ALP) activity was monitored both quantitatively and qualitatively. Extracellular matrix mineralization was further analyzed by alizarin red S staining.ResultsThe results showed that EGCG concentrations from 6 to 10 μM increased the ROS level and inhibited the cell proliferation of hPDLCs. EGCG concentrations from 2 to 8 μM effectively increased extracellular matrix mineralization, in which 4 and 6 μM EGCG generated the most mineralizing nodules. The ALP activity and the mRNA and protein expression levels of the tested osteogenic markers were most strongly up-regulated by treatment with 4 and 6 μM EGCG.ConclusionsThe present study demonstrated that EGCG might promote the osteogenesis of hPDLCs in a dose-dependent manner, with concentrations of 4 and 6 μM EGCG showing the strongest osteogenic enhancement without cytotoxicity, indicating a promising role for EGCG in periodontal regeneration in patients with deficient alveolar bone in the future.

Fabrication and Evaluation of 3D Printed Poly(l-lactide) Scaffold Functionalized with Quercetin-Polydopamine for Bone Tissue Engineering.

Quercetin (Qu), a bioflavonoid, has been reported to positively affect bone metabolism. For the first time, Qu with different concentrations was utilized to functionalize 3D-printed poly(l-lactide) (PLLA) scaffold with the aid of a polydopamine (PDA) layer through a convenient and effective way in this study. Results revealed that the coexistence of PDA and Qu can capacitate the 3D-printed PLLA scaffold to possess rougher surface, as well as better hydrophilicity and compressive properties. The resulting PDA- and Qu-modified PLLA scaffolds (Qu/PD-PLLA) can sustainably release Qu to some extent, which is more beneficial to the proliferation and attachment of MC3T3-E1 cells, upregulating ALP activity and calcium nodules as well as promoting the expression of related osteogenic genes and proteins. More significantly, such a positive impact of the Qu on the cell affinity and osteogenic activity played in a dose-dependent manner. This study revealed the potential of the 3D-printed Qu/PD-PLLA scaffolds with a certain amount of Qu as bone-repair materials.

Biological responses of human bone mesenchymal stem cells to Ti and TiZr implant materials.

Titanium-zirconium alloy (TiZr1317) is a new material used for biological implants. There are several studies on the effects of TiZr implants on the biological characteristics of human bone mesenchymal stem cells (hBMSCs). The purpose of this study was to investigate the biological responses of hBMSCs to implant holes affected by the physicochemical properties of oral implants (TiSLA , TiSLActive , TiZrSLA , and TiZrSLActive ). Grade 4 Ti and TiZr (13-17% Zr) substrates were modified by sand-blasted large-grit acid-etched (SLA) or hydrophilic sand-blasted large-grit acid-etched (SLActive), resulting in four types of surface with complex microstructures corresponding to the commercially-available implants SLA, RoxolidSLA, SLActive, and RoxolidSLActive (Institute Straumann AG, Basel, Switzerland). Physicochemical properties were detected and the biological responses of hBMSCs were observed. Surface morphology characterization by scanning electron microscopy and atomic force microscopy revealed differences between the four groups. SLActive had higher surface energy/wettability than SLA, indicating that increased surface energy/wettability can promote the absorption of osteogenic proteins and enhance osseointegration. hBMSCs seeded on SLActive substrates exhibited better performance in terms of cell attachment, proliferation and osteoblastic differentiation than cells seeded on SLA. Because of their more suitable physicochemical properties, TiSLActive and TiZrSLActive materials demonstrated more pronounced effects on the biological responses of hBMSCs compared with TiSLA and TiZrSLA .

Effect of porous tantalum on promoting the osteogenic differentiation of bone marrow mesenchymal stem cells in vitro through the MAPK/ERK signal pathway

BackgroundAs an ideal new graft material, porous tantalum (pTa) has excellent mechanical properties and corrosion resistance and has received increased attention in the biomedical field because of its excellent cytocompatibility and ability to induce bone formation. However, the molecular mechanism of its potential to promote osteogenesis remains unclear, and very few reports have been published on this topic. MethodsIn this study, we first produced porous Ti6Al4V (pTi6Al4V) and pTa with the same pore size by three-dimensional printing combined with chemical vapour deposition. The number of adhesions between pTa and pTi6Al4V and bone marrow mesenchymal stem cells (BMSCs) after 1 day of culture was detected by the live/dead cell staining method. The proliferation activity of the two groups was determined after culture for 1, 3, 5 and 7 days by the cell counting kit-8 method. In addition, the osteogenic activity, mRNA expression levels of osteogenic genes alkaline phosphatase (ALP), osterix (OSX), collagen-I (Col-I), osteonectin (OSN) and osteocalcin (OCN) and protein expression levels of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signalling pathway marker p-ERK of the two groups cultured for 7, 14 and 21 days were determined by the ALP activity assay, real-time quantitative polymerase chain reaction (Q-PCR) and Western blotting, respectively. Subsequently, the two groups were treated with the MAPK/ERK–specific inhibitor U0126, and then, the mRNA expression levels of osteogenic genes and protein expression levels of p-ERK in the cultures were determined by Q-PCR and Western blotting, respectively. ResultsThe live/dead cell staining and cell counting kit-8 assays showed that the adhesion and proliferation activities of BMSCs on pTa were significantly better than those on pTi6Al4V. In addition, the ALP activity assay and Q-PCR showed that pTa harboured osteogenic activity and that the osteogenic genes ALP, OSX, Col-I, OSN and OCN were highly expressed, and by Western blotting, the expression of p-ERK protein in the pTa group was also significantly higher than that in the pTi6Al4V group. Subsequently, using the MAPK/ERK–specific inhibitor U0126, Western blotting showed that the expression of p-ERK protein was significantly inhibited and that there was no difference between the two groups. Furthermore, Q-PCR showed that osteogenic gene expression and ALP expression levels were significantly increased in the pTa group, and there were no differences in the OSX, Col-I, OSN and OCN mRNA expression levels between the two groups. ConclusionOverall, our research found that compared with the widely used titanium alloy materials, our pTa can promote the adhesion and proliferation of BMSCs, and the molecular mechanism of pTa may occur via activation of the MAPK/ERK signalling pathway to regulate the high expression of OSX, Col I, OSN and OCN osteogenic genes and promote the osteogenic differentiation of BMSCs in vitro.The translational potential of this article: Our self-developed pTa material produced by three-dimensional printing combined with the chemical vapour deposition method not only retains excellent biological activity and osteoinductive ability of the original tantalum metal but also saves considerably on material costs to achieve mass production of personalised orthopaedic implants with pTa as a stent and to accelerate the wide application of pTa implants in clinical practice, which have certain profound significance.

Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bone-like Matrix Deposition in Vitro.

Large bone defects represent a significant challenge for clinicians and surgeons. Tissue engineering for bone regeneration represents an innovative solution for this dilemma and may yield attractive alternate bone substitutes. Three-dimensional (3D) printing with inexpensive desktop printers shows promise in generating high-resolution structures mimicking native tissues using biocompatible, biodegradable, and cost-effective thermoplastics, which are already FDA-approved for food use, drug delivery, and many medical devices. Microporous 3D-printed polylactic acid scaffolds, with different pore sizes (500, 750, and 1000 μm), were designed and manufactured using an inexpensive desktop 3D printer, and the mechanical properties were assessed. The scaffolds were compared for cell growth, activity, and bone-like tissue formation using primary human osteoblasts. Osteoblasts showed high proliferation, metabolic activity, and osteogenic matrix protein production, in which 750 μm pore-size scaffolds showed superiority. Further experimentation using human mesenchymal stem cells on 750 μm pore scaffolds showed their ability in supporting osteogenic differentiation. These findings suggest that even in the absence of any surface modifications, low-cost 750 μm pore-size 3D-printed scaffolds may be suitable as a bone substitute for repair of large bone defects.

Cyclic tensile stress promotes osteogenic differentiation of adipose stem cells via ERK and p38 pathways

The present study aimed to elucidate whether extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinases pathways participate in the transduction of mechanical stretch exerted on adipose stem cells (ASCs) into intracellular osteogenic signals, and if so whether both pathways have time-dependent feature. Rat ASCs were cultured in osteogenic medium for 72 h and assigned into three sets, namely ERK1/2 inhibitor treated set, p38 inhibitor treated set, and the control set. After inhibitor treatment, all cells were subjected to cyclic stretch(2000 με, 1 Hz) on a four-point bending mechanical loading device. Protein and mRNA samples were acquired at six time points: 0, 15 min, 30 min, 1 h, 2 h and 6 h. Western blot showed phosphorylation level of ERK1/2 was elevated by cyclic tensile stress at all time points, while p38 at 15 min, 30 min and 1 h, and the elevation can be completely blocked by corresponding inhibitors. The treatment by ERK1/2 inhibitor was shown to antagonize the up-regulation of osteogenic genes bone morphogenetic protein 2 (BMP-2) and runt-related transcription factor 2 (Runx2) by mechanical stretch at 15 min and 6 h, whereas p38 inhibitor took effect at 15 min only. The results suggested both ERK and p38 could be positive mediators of stretch-induced osteogenic differentiation of ASCs, and ERK stimulate the stretch-induced osteogenic differentiation at both early and late stages while p38 responds to mechanical stretch in a more rapid fashion.

Strontium enhances BMP-2 mediated bone regeneration in a femoral murine bone defect model.

The application of strontium is one option for the clinical treatment of osteoporosis-a disease characterized by reduced bone density and quality-in order to reduce the risk of vertebral and nonvertebral fractures. Unlike other drugs used in osteoporosis therapy, strontium shows a dual effect on bone metabolism by attenuating cellular resorption and simultaneously enhancing new bone tissue formation. Current concerns regarding the systemic application of highly dosed strontium ranelate led to the development of strontium-modified scaffolds based on mineralized collagen (MCM) capable to release biologically active Sr2+ ions directly at the fracture site. In this study, we investigated the regenerative potential of these scaffolds. For in vitro investigations, human mesenchymal stromal cells were cultivated on the scaffolds for 21 days (w/ and w/o osteogenic supplements). Biochemical analysis revealed a significant promoting effect on proliferation rate and osteogenic differentiation on strontium-modified scaffolds. In vivo, scaffolds were implanted in a murine segmental bone defect model-partly additionally functionalized with the osteogenic growth factor bone morphogenetic protein 2 (BMP-2). After 6 weeks, bridging calluses were obtained in BMP-2 functionalized scaffolds; the quality of the newly formed bone tissue by means of morphological scores was clearly enhanced in strontium-modified scaffolds. Histological analysis revealed increased numbers of osteoblasts and blood vessels, decreased numbers of osteoclasts, and significantly enhanced mechanical properties. These results indicate that the combined release of Sr2+ ions and BMP-2 from the biomimetic scaffolds is a promising strategy to enhance bone regeneration, especially in patients suffering from osteoporosis. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:174-182, 2020.

Developing a Strontium-Releasing Graphene Oxide-/Collagen-Based Organic-Inorganic Nanobiocomposite for Large Bone Defect Regeneration via MAPK Signaling Pathway.

Significant efforts have been dedicated to fabricating favorable biomaterial-based bone substitutes for the repair of large bone defects. However, the development of bone biomaterials with suitable physiochemical and osteoinductive properties remains a challenge. Here, novel strontium-graphene oxide (Sr-GO) nanocomposites that allow long-term release of Sr ions are fabricated, which are used to reinforce collagen (Col) scaffolds through covalent cross-linking. The prepared Sr-GO-Col scaffold demonstrates significantly high water retention rates and excellent mechanical properties compared with unmodified Col scaffolds. The Sr-GO-modified Col scaffolds display a strong effect on adipose-derived stem cells by facilitating cell adhesion and osteogenic differentiation and by promoting the secretion of angiogenic factors to stimulate the in vitro tube formation of endothelial cells. Additionally, the secretion of angiogenic VEGF and osteogenic BMP-2 proteins is increased, which may be attributed to the synergistic effects of GO and Sr on the activation of the MAPK signaling pathway. The Sr-GO-Col constructs were then transplanted into rat critical-size calvarial bone defects, which showed the best bone regeneration and angiogenesis outcome at 12 weeks. Moreover, histological staining results show that the Sr-GO-Col group achieved complete defect bridging with the newly formed bone tissue and the residual Sr-GO nanoparticles are phagocytosed and degraded by multinucleated giant cells. These findings reveal that the incorporation of inorganic Sr-GO nanocomposites into biocompatible Col scaffolds is a viable strategy for fabricating favorable substitutes that enhance the regeneration of large bone defects.

Osteogenic protein-1 alleviates high glucose microenvironment-caused degenerative changes in nucleus pulposus cells.

Increasing evidence has indicated a close relationship between diabetes mellitus (DM) and disc degeneration. As a potential therapeutic growth factor, osteogenic protein-1 (OP-1) has lots of protective effects on the healthy disc cell’s biology. The present study was aimed to investigate the effects of OP-1 on degenerative changes of nucleus pulposus (NP) cells in a high glucose culture. Rat NP cells were cultured in the baseline medium or the high glucose (0.2 M) culture medium. OP-1 was added into the high glucose culture medium to investigate whether its has some protective effects against degenerative changes of NP cells in the high glucose culture. NP cell apoptosis ratio, caspase-3/9 activity, expression of apoptosis-related molecules (Bcl-2, Bax, and caspase-3), matrix macromolecules (aggrecan and collagen II), and matrix remodeling enzymes (MMP-3, MMP-13, and ADAMTS-4), and immuno-staining of NP matrix proteins (aggrecan and collagen II) were evaluated. Compared with the baseline culture, high glucose culture significantly increased NP cell apoptosis ratio, caspase-3/9 activity, up-regulated expression of Bax, caspase-3, MMP-3, MMP-13 and ADAMTS-4, down-regulated expression of Bcl-2, aggrecan and collagen II, and decreased staining intensity of aggrecan and collagen II. However, the results of these parameters were partly reversed by the addition of OP-1 in the high glucose culture. OP-1 can alleviate high glucose microenvironment-induced degenerative changes of NP cells. The present study provides that OP-1 may be promising in retarding disc degeneration in DM patients.

Abstract 90: L-Type Voltage Gated Calcium Channels and Sodium Phosphate Symporters are Required for Osteogenic Differentiation Induced by Nanoparticulate Mineralized Collagen Materials

PURPOSE: The increasing understanding that modulation of extracellular matrix (ECM) components can instruct differential cell fate determination has resulted in significant interest in the synthesis of ECM-inspired materials for regeneration.Previously we described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material that efficiently induced osteogenic differentiation of human mesenchymal stem cells (hMSCs) and calvarial bone healing without exogenous growth factors or progenitor cell expansion via an autogenous activation of the bone morphogenetic protein receptor (BMPR) signaling pathway. Such activities were not induced in non-mineralized collagen glycosaminoglycan (Col-GAG) materials, suggesting that the upstream trigger is related to the inorganic content of the material. In this work, we evaluated the contributions of inorganic ion signaling to MC-GAG-induced osteogenic differentiation. METHODS: Primary hMSCs were osteogenically differentiated on collagen glycosaminoglycan materials in the absence or presence of inhibitors to the L-type voltage gated calcium channels (L-VGCC), calcium sensing receptor (CaSR), and the sodium phosphate symporters (PiT-1/2) using nifedipine, NPS2143,and phosphonoformic acid (PFA), respectively. Western blots were performed for phosphorylated and total Smad1/5, ERK1/2, Akt, p38, JNK1/2, total Runx2, and actin. Expression of BMPs were assessed with immunofluorescent staining of scaffold sections. Mineralization was evaluated by Alizarin red staining and micro-computed tomographic analyses. Analyses of variance with posthoc comparisons were performed. RESULTS: PFA diminished phosphorylated Smad1/5, BMP-2, and Runx2 expression as well as mineralization for both Col-GAG and MC-GAG scaffolds. While NPS2143 diminished Runx2 and mineralization on Col-GAG, no effects on osteogenic protein expression, BMP-2 expression, or mineralization were evident on MC-GAG. In contrast, no effects were mediated by nifedipine on Col-GAG,whereas nifedipine inhibited phosphorylated Smad1/5, phosphorylated ERK1/2, Runx2, BMP-2 staining, and mineralization on MC-GAG. CONCLUSION: In combination, these data suggested that the autogenous activation of the BMPR signaling pathway induced by MC-GAGis first triggered by calcium and phosphate influxvia L-VGCCs and the sodium and phosphate symporters.

The physicochemical and biomechanical profile of forsterite and its osteogenic potential of mesenchymal stromal cells.

It has been demonstrated that nanocrystalline forsterite powder synthesised using urea as a fuel in sol-gel combustion method had produced a pure forsterite (FU) and possessed superior bioactive characteristics such as bone apatite formation and antibacterial properties. In the present study, 3D-scaffold was fabricated using nanocrystalline forsterite powder in polymer sponge method. The FU scaffold was used in investigating the physicochemical, biomechanics, cell attachment, in vitro biocompatibility and osteogenic differentiation properties. For physicochemical characterisation, Fourier-transform infrared spectroscopy (FTIR), Energy dispersive X-ray (EDX), X-ray diffraction (XRD), Raman spectroscopy, X-ray photoemission spectrometer (XPS) and Brunauer-Emmett-Teller (BET) were used. FTIR, EDX, XRD peaks and Raman spectroscopy demonstrated correlating to FU. The XPS confirmed the surface chemistry associating to FU. The BET revealed FU scaffold surface area of 12.67 m2/g and total pore size of 0.03 cm3/g. Compressive strength of the FU scaffold was found to be 27.18 ± 13.4 MPa. The human bone marrow derived mesenchymal stromal cells (hBMSCs) characterisation prior to perform seeding on FU scaffold verified the stromal cell phenotypic and lineage commitments. SEM, confocal images and presto blue viability assay suggested good cell attachment and proliferation of hBMSCs on FU scaffold and comparable to a commercial bone substitutes (cBS). Osteogenic proteins and gene expression from day 7 onward indicated FU scaffold had a significant osteogenic potential (p<0.05), when compared with day 1 as well as between FU and cBS. These findings suggest that FU scaffold has a greater potential for use in orthopaedic and/or orthodontic applications.

Effect of Hydroxyapatite Surface on BMP-2 Biological Properties by Docking and Molecular Simulation Approaches.

The interactions between osteogenic proteins and the biomaterial surface are crucial to the application of biomaterials, in which the conformational or orientational change of the adsorbed protein on the solid surfaces is one of the most important interactions other than the protein adsorption. Although some progress has been made in the mechanism of protein adsorption on the surface of hydroxyapatite (HAP) in recent years, there is still insufficient atomistic/molecular information about the conformation and orientation of proteins upon adsorbing on solid surfaces. In this study, different orientations and conformations of bone morphological protein-2 (BMP-2) adsorbed on the surface of HAP were calculated through the protein-solid surface docking approach; the relationship between optimal adsorption and biological activity of BMP-2 was investigated by applying a combination of molecular dynamic simulation (MD) and steered molecular dynamic simulation (SMD). Two optimal adsorption conformers were screened out according to the docking results on the basis of orientations of BMP-2 with different epitopes. Subsequent MD and SMD results showed that the knuckle epitope of BMP-2 was easier to adsorb on the surface of HAP(100) than the wrist epitope accompanying certain conformational changes. Such an absorption mode led to the wrist epitope of BMP-2 being exposed to the environment and then being identified/interacted with type I receptors on the stem cell membrane, which further induces the differentiation of stem cells into osteoblasts. Current simulation provided a theoretical high-throughput screening method for the protein-biomaterial adsorption states. It can be extended to more research on different protein adsorptions on the surface of different materials. The simulation results provided more information at the molecular and atomic levels to further interpret the mechanism of osteoinductivity from the perspective of growth factor adsorption. Meanwhile, we believe that it should be a meaningful attempt to screen biomaterial key factors by the high-throughput method, which might become a promising way to develop or optimize new biomaterials.

Macrophage inhibits the osteogenesis of fibroblasts in ultrahigh molecular weight polyethylene (UHMWPE) wear particle-induced osteolysis

BackgroundIn the ultrahigh molecular weight polyethylene (UHMWPE) prosthetic environment, fibroblasts affected by wear particles have the capacity of osteogenesis to reduce osteolysis. We aimed to assess the effects of macrophages on the osteogenic capability of fibroblasts treated with UHMWPE wear particles.MethodsThe effect of different concentrations of UHMWPE (0, 0.01, 0.1, and 1 mg/ml, respectively) on macrophage proliferation were validated by MTT assay to determine the optimum one. The fibroblasts viability was further determined in the co-culture system of UHMWPE particles and macrophage supernatants. The experiment was designed as seven groups: (A) fibroblasts only; (B) fibroblasts + 1 mg/ml UHMWPE particles; and (C1–C5) fibroblasts + 1/16, 1/8, 1/4, 1/2, and 1/1 supernatants of macrophage cultures stimulated by 1 mg/ml UHMWPE particles vs. fibroblast complete media, respectively. Alizarin red staining was used to detect calcium accumulation. The expression levels of osteogenic proteins were detected by Western blot and ELISA, including alkaline phosphatase (ALP) and osteocalcin (OCN).ResultsThe concentration of 0.1 mg/ml was considered as the optimum concentration for macrophage proliferation due to the survival rate and was highest among the four concentrations. Fibroblast viability was better in the group of fibroblasts + 1/16 ratio of macrophage supernatants stimulated by 1 mg/ml of UHMWPE particles than the other groups (1:8, 1:4, 1:2, 1:1). ALP and OCN expressions were significantly decreased in the group of fibroblasts + 1/4, 1/2, and 1/1 supernatants stimulated by 1 mg/ml of UHMWPE particles compared with other groups (1/8, 1/16) and the group of fibroblasts + 1 mg/ml UHMWPE (p < 0.5).ConclusionsMacrophages are potentially involved in the periprosthetic osteolysis by reducing the osteogenic capability of fibroblasts treated with wear particles generated from UHMWPE materials in total hip arthroplasty.

A Novel Non-invasive Effective Method for Potential Treatment of Degenerative Disc Disease: A Hypothesis

The pathophysiology of the intervertebral discs plays a significant role in the people's life quality. There is not adequate research done in the pathogenesis and treatment of intervertebral disc degeneration. Alternately, self-educated physiology offers a novel and noninvasive method to reverse the degenerated discs. In this single case study, report attempts have been made to highlight the effect of the self-educative physiology, on magnetic resonance imaging investigations, of progressive healing, on the degenerated intervertebral discs. Based on this novel method, an effort has been made to review literature on the degeneration of intervertebral discs and available mode of treatments and then to propose a hypothesis for the biochemical mechanisms of healing. The idea is that transforming growth factor-β1 from seminal plasma secretions may contribute to releasing the osteogenic protein- 1 which induces nucleus pulposus and annulus fibrosus cells in intervertebral discs for repairs. In addition, the patient's medical history is presented with background information.

Erythropoietin attenuates high glucose-induced oxidative stress and inhibition of osteogenic differentiation in periodontal ligament stem cell (PDLSCs)

Diabetes mellitus and periodontitis have long been considered to be biologically linked. Erythropoietin (EPO) has multiple biological functions, such as stimulating the proliferation and differentiation of erythroid progenitor cells and reducing glucose-induced oxidative stress via different mechanisms, acting as a direct antioxidant. The purposes of the study to examine the anti-oxidative effect of EPO on reducing high glucose-induced oxidative stress of hPDLSCs and provide a better understanding of the mechanism of these processes. PDLSCs were induced by highglucose (HG, 30 mM) in the presence or absence of EPO. Cell proliferation was measured by MTT assay. The reactiveoxygen species (ROS) level, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were detected to evaluate oxidative stress. qRT-PCR and western blot analysis were used to examine the expression of osteogenic related genes and protein (Runx2 and Osterix). Alizarin Red-S staining was used to detect mineralized nodule formation. The results showed that EPO promote the proliferation of PDLSCs, which was suppressed by high glucose (30 mM). Moreover, EPO attenuated high glucose (30 mM) induced oxidative stress by reducing the levels of ROS and MDA, and increasing the SOD activity. Furthermore,EPO alleviate high glucose(30 mM) induced suppression of osteogenic differentiation ability in PDLSCs, as evidenced by the up-regulated mRNA and protein expression of Runx2 and Osterix and increased ALP activity. In conclusion, EPO attenuates high glucose-induced oxidative stress, inhibitory effect of proliferation and inhibition of osteogenic differentiation in periodontal ligament stem cell (PDLSCs).

ERα and Wnt/β‑catenin signaling pathways are involved in angelicin‑dependent promotion of osteogenesis.

Reports of the ameliorative effect of angelicin on sex hormone deficiency-induced osteoporosis have highlighted this compound as a candidate for the treatment of osteoporosis. However, the molecular mechanisms of action of angelicin on osteoblast differentiation have not been thoroughly researched. The aim of the present study was to evaluate the effect of angelicin on the proliferation, differentiation and mineralization of rat calvarial osteoblasts using a Cell Counting Kit-8, alkaline phosphatase activity and the expression of osteogenic genes and proteins. Treatment with angelicin promoted the proliferation, matrix mineralization and upregulation of osteogenic marker genes including collagen type I α 1 and bone γ-carboxyglutamate in fetal rat calvarial osteoblasts. Furthermore, angelicin promoted the expression of β-catenin and runt related transcription factor 2, which serve a vital role in the Wnt/β-catenin signaling pathway. Consistently, the osteogenic effect of angelicin was attenuated by the use of a Wnt inhibitor. Moreover, angelicin increased the expression of estrogen receptor α (ERα), which also serves a key role in osteoblast differentiation. Taken together, these results demonstrated that angelicin may promote osteoblast differentiation through activation of ERα and the Wnt/β-catenin signaling pathway.

Age dependent effects of NELL-1 isoforms on bone marrow stromal cells

ObjectiveNELL-1 is an osteogenic protein first discovered to control ossification of the cranium. NELL-1 exists in at least two isoforms. The full-length NELL-1 contains 810 amino acid (aa) (NELL-1810), the N-terminal-truncated NELL-1 isoform contains 570 aa (NELL-1570). The differences in cellular effects between NELL-1 isoforms are not well understood. Methods: Here, BMSC were derived from adult or aged mice, followed by overexpression of NELL-1810 or NELL-1570. Cell morphology, proliferation, and gene expression were examined. Results/Conclusions: Overall, the proliferative effect of NELL-1570 was age dependent, showing prominent induction in adult but not aged mice.

Bone regeneration of multichannel biphasic calcium phosphate granules supplemented with hyaluronic acid

The present work is to investigate the efficiency of hyaluronic acid (HyA) supplemented biphasic calcium phosphate (BCP) injectable granule to promote the bone regeneration. The effect of adding HyA to the multichannel BCP granule (MCG-HyA) was studied in terms of morphology, chemical structure, porosity, in-vitro and in-vivo biocompatibility, RT-PCR, western blot and compared with MCG. The addition of HyA to MCG successfully made the granules injectable type. In-vivo studies in rabbit model showed an enhancement in bone formation after 4 weeks of implantation and better handling characteristics for MCG-HyA than MCG. RT-PCR and Western Blotting studies revealed that MCG-HyA significantly unregulated the osteogenic gene and protein expressions respectively. Our results indicated that MCG-HyA could be used as a promising injectable bone substitute in clinical applications.