Osteogenic Differentiation Of MG-63 Cells Research Articles

3D printed OCP bone scaffold with alginate enhancing osteogenic differentiation in MG-63 cells

3D printed OCP bone scaffold with alginate enhancing osteogenic differentiation in MG-63 cells

Bone marrow mesenchymal stem cell-derived exosomal microRNA-382 promotes osteogenesis in osteoblast via regulation of SLIT2

BackgroundOsteoporosis (OP) is a systemic skeletal disorder with increased bone fragility. Human bone marrow mesenchymal stem cells (hBMSCs) have multi-lineage differentiation ability, which may play important roles in osteoporosis. In this study, we aim to investigate the role of hBMSC-derived miR-382 in osteogenic differentiation.MethodsThe miRNA and mRNA expressions in peripheral blood monocytes between persons with high or low bone mineral density (BMD) were compared. Then we collected the hBMSC-secreted sEV and examined the dominant components. The over-expression of the miR-382 in MG63 cell and its progression of osteogenic differentiation were investigated by qRT-PCR, western blot and alizarin red staining. The interaction between miR-382 and SLIT2 was confirmed by dual-luciferase assay. The role of SLIT2 was also confirmed through up-regulation in MG63 cell, and the osteogenic differentiation-associated gene and protein were tested.ResultsAccording to bioinformatic analysis, a series of differential expressed genes between persons with high or low BMD were compared. After internalization of hBMSC-sEV in MG63 cells, we observed that the ability of osteogenic differentiation was significantly enhanced. Similarly, after up-regulation of miR-382 in MG63 cells, osteogenic differentiation was also promoted. According to the dual-luciferase assay, the targeting function of miR-382 in SLIT2 was demonstrated. Moreover, the benefits of hBMSC-sEV in osteogenesis were abrogated through up-regulation of SLIT2.ConclusionOur study provided evidence that miR-382-contained hBMSC-sEV held great promise in osteogenic differentiation in MG63 cells after internalization by targeting SLIT2, which can be served as molecular targets to develop effective therapy.

Generation of graphene oxide and nano-bioglass based scaffold for bone tissue regeneration

Graphene oxide (GO) offers a distinct opportunity in the field of biomedical engineering owing to its exceptionally high mechanical strength, excellent electrical conductivity, high optical transparency, and favorable biocompatibility. In this article, nanocomposite biocompatible GO-based scaffolds (chitosan/gelatin/nanobioglass/GO) Ch-G-NBG-GO were successfully fabricated through freeze drying technique (−40 °C) and evaluated for various physico-chemical and biological properties. The prepared Ch-G-NBG-GO composites have been investigated for their structural, physiochemical, and surface morphology via x-ray diffraction (XRD), high resolution scanning electron microscope, Fourier transform infrared spectroscopy, thermogravimetric analysis (TGA), energy-dispersive x-ray Spectroscopy and, differential scanning colorimetry (DSC) respectively. The morphological analysis showed the porous interconnected network of scaffold formed. Average pore size for the Ch-G-NBG-GO scaffolds were in between 90 and 120 μm, which was very close to the control scaffolds. XRD data revealed the successful incorporation of NBG and GO and distribution across the scaffolds. Porosity of the fabricated scaffolds were in the range between 75.3% and 77.3% which was very close to the control scaffold with 79% porosity. The studies also reveal that after GO incorporation, the weight loss reduced (0.11 ± 0.02–0.095 ± 0.03), scaffolds were firmly stable at room temperature even after a long duration of 28 d. The crystallinity added to the scaffolds due to addition of GO nanoparticles improved the mechanical strength of these scaffolds. The compressive modulus changed from (5.7 to 8.51) MPa after GO addition. Swelling ratio changed drastically especially in case of Ch-NBG-90%GO (4.9 ± 0.04–4 ± 0.01). DSC and TGA data revealed the thermal stability of GO incorporated scaffolds due to the proper interaction between GO/NBG with chitosan-gelatin blend. The scaffold’s potential for bone tissue engineering was evaluated by testing its cytocompatibility for MG-63 cell line. It revealed suitable cell attachment and proliferation of cells compared to the Ch-G-NBG scaffold. MTT assay showed that Ch-G-NBG-GO scaffold below 90% GO concentration possess best biocompatibility. But in case of Ch-G-NBG-90%GO scaffold, the cell proliferation was reduced when compared to control scaffolds. Alkaline phosphatase activity suggested improved osteogenic differentiation of MG-63 cells over GO based scaffolds and this was due to the osteogenic potential of NBG and GO present in the scaffolds. Based on these results, the nano-biocomposite scaffold appears to have the potential for utilization in bone tissue restoration, replacement and regeneration.

Multifunctional polyethylene imine hybrids decorated by silica bioactive glass with enhanced mechanical properties, antibacterial, and osteogenesis for bone repair

Inorganic/organic hybrids and bioactive glasses demonstrate promising potential as bone substitute biomaterials. A sol-gel hybrid consisting of silica bioactive glass and biodegradable polymer can combine the high bioactivity of a glass with the toughness of a polymer. In this study, multifunctional hybrids with a combination of organic-inorganic hybrid structure class II consisting of polyethyleneimine (PEI) generation 4 (G4) and bioactive glass with enhanced mechanical properties, mineralization, antibacterial, and osteogenesis activities were synthesized by the sol-gel method. Glycidoxypropyl) trimethoxysilane (GPTMS) with different concentrations was used as a covalent bonding agent between PEI polymer and bioactive glass. The effect of GPTMS content was assessed in the presence and absence of calcium in the hybrid structures in terms of morphology, wettability, mechanical properties, antibacterial activity, cell viability, and in vitro osteogenic differentiation properties. By increasing the amount of GPTMS, the compressive strength increased from 1.95 MPa to 2.34 MPa, which was comparable to human trabecular bone. All the hybrids presented antibacterial activity against Staphylococcus aureus, forming an inhibition zone of 13–16 mm. An increase in cell viability of 82.22% in PSCaG90 was obtained after 1 day of MG-63 cell culture. Alkaline phosphatase expression and mineralization of MG-63 cells increased in the PSCaG90 hybrid in the absence of an osteogenic medium compared to PSG60 and PSG90. The PSCaG90 hybrid indicated considerable in vitro osteogenic capacity in the absence of a differentiation medium, expressing high levels of bone-specific proteins including collagen I (COL1A1), Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osteocalcin (OCN), compared to calcium-free hybrids. Overall, our results suggest that the presence of calcium in the PSCaG90 leads to a significant increase in osteogenic differentiation of MG-63 cells even in the absence of differentiation medium, which suggests these hybrid structures with multifunctional properties as promising candidates for bone repair.

Osteogenic effects of exosomes derived from human chorion membrane extracts

ObjectiveHuman chorion membrane extracts (CME) are known to exhibit osteogenic effects when used for treating human osteoblast-like cells (MG63 cells), but the active compound in CME remains unknown. The aim of this study was to identify the presence of exosomes in CME and to determine the osteogenic effect of CME exosomes on MG63 cells.MethodsExosomes were isolated from human placenta CME using the ExoQuick-TC solution and were characterized. The activity and deposition of alkaline phosphatase (ALP) on MG63 cells cultured with or without exosomes in osteogenic induction medium (OIM) were determined. Human amniotic membrane extracts (AME) were used as controls as they had not affected the osteogenic differentiation of MG63 cells in our previous study.ResultsTransmission electron microscopy (TEM) revealed that exosomes isolated from CME and AME (CME-Exo and AME-Exo, respectively) had a cup-shaped structure. NanoSight™ particle tracking analysis (NTA) confirmed that the size of these exosomes was 100–150 nm. In vitro osteogenic experiments demonstrated that the exosomes from CME, but not those from AME, presented increased alkaline phosphatase (ALP) activity and resulted in the mineralization of MG63 cells in a dose-dependent manner.ConclusionExosomes were identified in CME and AME from the human placenta. Further, the exosomes from CME were found to be capable of promoting osteogenic differentiation, suggesting that exosomes are a key component of CME that stimulate the osteogenesis of human osteoblast-like cells. CME exosomes can be developed as promising therapeutic candidates for bone regeneration.

Simvastatin-Loaded Nanomicelles Enhance the Osteogenic Effect of Simvastatin

Objectives. The present study intended to further verify that simvastatin-loaded nanomicelles (SVNs) enhanced the role of simvastatin (SV) in promoting osteoblast differentiation in vitro and to evaluate the effect of SVNs on bone defect repair in vivo. Methods. SVNs were synthesized by dialysis. MG63 cells were subjected to intervention with 0.25 μmol/l of SVNs and SV. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit and flow cytometry were used to determine cell proliferation activity, cell cycle distribution, and apoptosis. The osteoblastic differentiation of MG 63 cells was evaluated by measuring alkaline phosphatase (ALP) activity, ALP staining, and the expression levels of the osterix (Osx) and osteocalcin (OC) proteins. In addition, 0.5 mg of SVNs or SV was applied to the skull defect area of rabbits. Micro-CT, hematoxylin and eosin (HE) staining, and Masson’s trichrome staining were used for qualitative and quantitative evaluation of new bone in three dimensions and two dimensions. Results. The SVNs had a mean diameter of 38.97 nm. The encapsulation and drug-loading efficiencies were 54.57 ± 3.15 % and 10.91 ± 0.63 % , respectively. In vitro, SVNs and SV can inhibit the proliferation activity and promote osteogenic differentiation of MG63 cells by arresting MG63 cells at the G0/G1 phase without increasing the apoptosis rate. In vivo quantitative results showed that the bone mineral density (BMD), bone volume (BV)/total volume (TV) ratio, and trabecular number (Tb.N) in the gelatin sponge with SVNs (SVNs-GS) group and gelatin sponge with SV (SV-GS) group were 362.1%, 292.0%; 181.3%, 158.0%; and 215.2%, 181.8% of those in the blank control (BC) group, respectively. Histological results identified the new bone tissue in each group as irregular fibrous bone, and the arrangement of trabecular bone was disordered. There were significantly more osteoblasts and new capillaries around the trabecular bone in the SVNs-GS group and SV-GS group than in both the BC and drug-free nanomicelle (DFNs) groups. Both in vitro and in vivo, SVNs exhibited greater osteogenic efficacy than SV. Conclusion. SVNs significantly improved the osteogenic efficacy of SV.

Cytocompatibility, stability and osteogenic activity of powder metallurgy Ta-xZr alloys as dental implant materials.

Tantalum (Ta) and zirconium (Zr) alloys were found to had low elastic modulus and similar biomechanical characteristics as the human bone. However, the biocompatibility and osteogenic potential of Ta-xZr alloyswith different proportions (20, 30, 40 and 50% Zr by atom) remains to be investigated. In this study, the biocompatibility of Ta-xZr alloys and commercially pure titanium (cpTi) was evaluated in vitro by cell counting kit-8 assay. The adhesion of MG63 osteoblasts to the surface of the alloys was observed by fluorescence microscopy, and their morphology was analyzed by scanning electron microscopy (SEM). The expressions of alkaline phosphatase (ALP), Ki67, osteocalcin (OC), collagen-I (Col-I) and Integrin β1 mRNA in the cultured cells were determined by RT-PCR. As a result, Ta-xZr (x = 20, 30, 40 and 50 at%) alloys were non-toxic and supported proliferation of the MG63 cells. The osteoblasts adhered to the Ta-xZr alloys, and subsequently spread and proliferated rapidly. Furthermore, the cells grown on Ta-20Zr and Ta-30Zr expressed high levels of ALP, Col I and OC, indicating that the Ta-xZr alloys can induce osteogenesis. In conclusion, Ta-xZr alloys promoted the adhesion, proliferation and osteogenic differentiation of MG63 cells. The Ta-xZr composites with a higher proportion of Ta exhibited superior osteogenic activity, and Ta-30Zr is therefore a promising alternative for Ti implants.

Enhanced bioactivity and osteogenic property of carbon fiber reinforced polyetheretherketone composites modified with amino groups

Polyetheretherketone (PEEK) is considered as a potential dental and orthopedic implant material owing to its favorable thermal and chemical stability, biocompatibility and mechanical properties. However, the inherent bio-inert and inferior osseointegration of PEEK have hampered its clinical application. In addition, carbon fiber is widely used as a filler to reinforce polymers for sturdy composites owing to its high strength, modulus, etc. In the study, carbon fiber reinforced PEEK (CPEEK) composites were fabricated and modified with amino groups by plasma-enhanced chemical vapor deposition surface modification technique. The surface characterization of composites was evaluated by FE-SEM, EDS, AFM, Water contact angle, XPS and FTIR, which revealed that amino groups were successfully incorporated on the modified CPEEK surface and significantly increased the hydrophilicity. In vitro study, cell adhesion, proliferation, ALP activity, ECM mineralization, real-time PCR analysis, and ELISA analysis showed the adhesion, proliferation and osteogenic differentiation of MG-63 cells on the amino group-modified CPEEK surface were higher than the CPEEK, equal to or better than pure titanium. Hence, the results indicated that the amino group-modified CPEEK possessed enhanced bioactivity and osteogenic property, which may be a potential candidate material for dental implants.

Micro/nano-hierarchical structured TiO2 coating on titanium by micro-arc oxidation enhances osteoblast adhesion and differentiation.

Nano-structured and micro/nano-hierarchical structured TiO2 coatings were produced on polished titanium by the micro-arc oxidation (MAO) technique. This study was conducted to screen a suitable structured TiO2 coating for osteoblast adhesion and differentiation in dental implants. The formulation was characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and wettability testing. Adhesion, proliferation and osteogenic differentiation of MG63 cells were analysed by SEM, Cell Counting Kit-8 (CCK-8) and quantitative real-time PCR. The micro/nano-hierarchical structured TiO2 coating with both slots and pores showed the best morphology and wettability. XRD analysis revealed that rutile predominated along with a minor amount of anatase in both TiO2 coatings. Adhesion and extension of MG63 cells on the micro/nano-hierarchical structured TiO2 coating were the most favourable. MG63 cells showed higher growth rates on the micro/nano-hierarchical structured TiO2 coating at 1 and 3 days. Osteogenic-related gene expression was markedly increased in the micro/nano-hierarchical structured TiO2 coating group compared with the polished titanium group at 7, 14 and 21 days. These results revealed the micro/nano-hierarchical structured TiO2 coating as a promising surface modification and suitable biomaterial for use with dental implants.

Nanostructured titanium surfaces fabricated by hydrothermal method: Influence of alkali conditions on the osteogenic performance of implants

Hydrothermal method is an easy-to-use approach for creating nanostructured surfaces on titanium (Ti). However, whether the alkali conditions of this method influence the osteogenic potential of the modified surfaces remains unknown. In this study, we fabricated nanostructured surfaces, termed the Ti-1, Ti-5, and Ti-10 groups, by using the hydrothermal method in 1 M, 5 M, and 10 M NaOH aqueous solutions, respectively. An untreated Ti surface served as a control. The osteogenic performance of modified surfaces was systemically investigated, including the proliferation and osteogenic differentiation of human osteoblast-like MG63 cells in vitro and the osteointegration of implants in a rabbit femoral condyle defect model. After hydrothermal treatment, the hydrophilicity of modified surfaces was greatly enhanced. The Ti-1 group showed a nanowire-like topography, while the Ti-5 and Ti-10 groups exhibited a nanopetal-like topography with different pore sizes. Compared with the untreated Ti surface, the modified surfaces showed good cytocompatibility and enhanced the osteogenic differentiation of MG-63 cells. Compared with the other modified surfaces, the Ti-5 group was the most favourable for the osteogenic differentiation of cells, showing higher levels of alkaline phosphatase activity, osteogenic gene expression, mineralization and osteoprotegerin secretion. Twelve weeks after implantation at the bone defects, the Ti-5 group showed superior peri-implant bone regeneration and higher peak push-out force than the other groups. Overall, this study revealed the crucial role of alkali conditions of hydrothermal method in modulating the material characteristics of modified surfaces and their osteogenic performance in vitro and in vivo, highlighting the need for optimizing the processing conditions of hydrothermal method for enhanced osteointegration.

Semaphorin3A enhances osteogenesis of MG63 cells through interaction with Schwann cells invitro.

Bone remodeling is under the control of various signals and systems in the body, including the nervous system. Semaphorin (Sema) 3A is a chemorepellent protein which regulates bone mass. Schwann cells, having a pivotal role following nerve injury, interact with Sema3A under numerous circumstances. The present study established a co‑culture system of MG63 and Schwann cells to investigate the role of the interaction between Sema3A and Schwann cells in osteogenesis. The results from the alkaline phosphatase assay, calcium nodule staining and the analysis of the osteogenic gene expression revealed that Sema3A inhibits osteogenic differentiation of MG63 cells in single‑cell culture and promotes osteogenic differentiation of MG63 cells in co‑culture with Schwann cells, in a concentration‑dependent manner. These findings suggest that the presence of Schwann cells induces Sema3A‑associated osteogenic differentiation in bone cells, and also reveals the pivotal role of Sema3A as a regulator in the skeletal and nervous systems, thus contributing to a better understanding of the interaction between these systems.

MiR-34a Improves the Proliferation and Osteogenic Differentiation of MG63 Cells Under Short-Term Mechanical Force Loading

miR-34a Improves the Proliferation and Osteogenic Differentiation of MG63 Cells Under Short-Term Mechanical Force Loading

Differential effects of amnion and chorion membrane extracts on osteoblast-like cells due to the different growth factor composition of the extracts.

Human amniotic membrane extracts contain numerous growth factors and bioactive substances. However, osteogenic effects of amnion and chorion membrane extracts (AME and CME, respectively) on osteoblasts are unclear. In this study, we explored the ability of AME and CME to promote the osteogenic differentiation of osteoblast-like MG-63 cells. MG-63 cells were cultured in osteogenic induction medium (OIM) with or without exogenous AME and CME. CME enhanced the osteogenic differentiation of MG-63 cells compared with AME, as indicated by increased mineralization; alkaline phosphatase activity; and mRNA expression of osteogenic marker genes encoding integrin-binding sialoprotein (IBSP), RUNX2, OSTERIX, and osteocalcin (OCN). Interestingly, AME and CME contained different combinations of osteogenesis-related growth factors, including basic fibroblast growth factor (bFGF), transforming growth factor beta-1 (TGFβ-1), and epidermal growth factor (EGF), which differentially regulated the osteogenic differentiation of MG-63 cells. bFGF and TGFβ-1 present in CME positively regulated the osteogenic differentiation of MG-63 cells, whereas EGF present in AME negatively regulated the differentiation of MG-63 cells. Moreover, exogenous treatment of EGF antagonized CME-induced mineralization of extracellular matrix on MG-63 cells. We compared the osteogenic efficacy of CME with that of BMP2, bFGF, and TGFβ-1 alone or their combinations. We observed that CME greatly enhanced osteogenesis by providing a conductive environment for the differentiation of MG-63 cells. Together, our results indicated that human AME and CME exerted differential effects on osteogenesis because of the presence of different compositions of growth factors. In addition, our results highlighted a new possible strategy of using CME as a biocompatible therapeutic material for bone regeneration.

Calcium Silicate Improved Bioactivity and Mechanical Properties of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Scaffolds.

The poor bioactivity and mechanical properties have restricted its biomedical application, although poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) had good biocompatibility and biodegradability. In this study, calcium silicate (CS) was incorporated into PHBV for improving its bioactivity and mechanical properties, and the porous PHBV/CS composite scaffolds were fabricated via selective laser sintering (SLS). Simulated body fluid (SBF) immersion tests indicated the composite scaffolds had good apatite-forming ability, which could be mainly attributed to the electrostatic attraction of negatively charged silanol groups derived from CS degradation to positively charged calcium ions in SBF. Moreover, the compressive properties of the composite scaffolds increased at first, and then decreased with increasing the CS content, which was ascribed to the fact that CS of a proper content could homogeneously disperse in PHBV matrix, while excessive CS would form continuous phase. The compressive strength and modulus of composite scaffolds with optimal CS content of 10 wt % were 3.55 MPa and 36.54 MPa, respectively, which were increased by 41.43% and 28.61%, respectively, as compared with PHBV scaffolds. Additionally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated MG63 cells had a higher proliferation rate on PHBV/CS composite scaffolds than that on PHBV. Alkaline phosphatase (ALP) staining assay demonstrated the incorporation of CS significantly promoted osteogenic differentiation of MG63 cells on the scaffolds. These results suggest that the PHBV/CS composite scaffolds have the potential in serving as a substitute in bone tissue engineering.

Induction of osteogenic differentiation of osteoblast-like cells MG-63 during cultivation on fibroin microcarriers

We have developed microcarriers made from silk fibroin. Microcarriers can be used as a substrate for cell cultivation and cell delivery during cell-based therapy and for the construction of bioengineered tissue. Fibroin microcarriers were mineralized, which led to the appearance of calcium phosphate crystals on their surface. The ability of mineralized and nonmineralized microcarriers to support osteogenic differentiation of the osteoblast-like cell line MG-63 was estimated by alkaline phosphatase activity, an early marker of bone formation. The experiment showed cells actively proliferating on the surface of both mineralized and nonmodified microcarriers. Culturing MG-63 on the surface of fibroin microcarriers resulted in an increase of alkaline phosphatase activity indicative of osteogenic differentiation of MG-63 cells in the absence of inductors. The level of alkaline phosphatase was higher when mineralized microcarriers were used. Alkaline phosphatase activity of MG-63 cells cultivated using traditional two-dimensional approaches were close to zero. As opposed to conventional monolayer culturing, microcarrier culture cells are in a three-dimensional environment that is closer to physiological conditions. This can have a significant impact on their morphology and functional properties. During this study, we also characterized mechanical properties of porous scaffolds used for microcarriers.

Promotion of osteogenic differentiation by amnion/chorion membrane extracts.

The amniotic membrane is a favorable biomaterial to apply in the field of tissue engineering because of its unique biological properties. Human amniotic membranes consist of 2-layered sheets containing numerous growth factors, cytokines and other bioactive substances. In this study, we explored the potential of amnion membrane extracts (AME) and amnion/chorion membrane extracts (A/CME) to promote osteogenic differentiation of osteoblast-like (MG-63) cells. MG-63 cells were cultured in osteogenic induction medium (OIM) with or without 100 µg/mL of AME or A/CME. To determine the early and late differentiation of osteogenesis, alkaline phosphatase (ALP) activity and calcium deposition were measured at 3, 7, 10 and 24 days. Expression of specific genes associated with osteogenic differentiation, including osteocalcin (OCN), osteopontin (OPN), runt domain-containing transcription factor (Runx2) and osterix (OSX) was also determined. In vitro experiments demonstrated that A/CME increased ALP activity, osteogenic gene expression and mineralization under osteogenic-inducing conditions. Notably, we found that A/CME contained growth factors related to osteogenesis, including fibroblast growth factors and transforming growth factors, which potentially promoted osteogenic differentiation of MG-63 cells to a greater extent than AME. These results indicate that A/CME is capable of providing growth factors and other substrates for osteogenic differentiation, which significantly increased the efficacy of osteogenesis in MG-63 cells. Taken together, the results of this study suggest that human A/CME is a promising biomaterial with therapeutic potential in bone regeneration applications.

Synthesis of Biocompatible Hydroxyapatite Using Chitosan Oligosaccharide as a Template.

In this study, a novel biocompatible hydroxyapatite (HA) was synthesized by using chitosan oligosaccharide (COS) as a template. These HA samples were studied by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). The biocompatibility of HA samples was evaluated via cell viability, cell morphology and alkaline phosphatase staining of MG-63 cell lines. The results show that HA synthesized in the presence of COS was favorable to proliferation and osteogenic differentiation of MG-63 cells. These hydroxyapatites are potentially attractive biomaterials for bone tissue engineering applications.

Improving Osteogenesis Activity on BMP-2-Immobilized PCL Fibers Modified by the γ-Ray Irradiation Technique.

The purpose of this study was to demonstrate the ability of BMP-2-immobilized polycaprolactone (PCL) fibers modified using the γ-ray irradiation technique to induce the osteogenic differentiation of MG-63 cells. Poly acrylic acid (AAc) was grafted onto the PCL fibers by the γ-ray irradiation technique. BMP-2 was then subsequently immobilized onto the AAc-PCL fibers (BMP-2/AAc-PCL). PCL and surface-modified PCL fibers was characterized by evaluation with a scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), and contact angle. The biological activity of the PCL and surface-modified PCL fibers were characterized by alkaline phosphatase (ALP) activity, calcium deposition, and the mRNA expression of osteocalcin and osteopontin in MG-63 cells. Successfully grafted AAc and PCL fibers with immobilized BMP-2 were confirmed by XPS results. The results of the contact angle showed that BMP-2/AAc-PCL fibers have more hydrophilic properties in comparison to PCL fibers. The ALP activity, calcium deposition, and gene expressions of MG-63 cells grown on BMP-2/AAc-PCL fibers showed greatly induced osteogenic differentiation in comparison to the PCL fibers. In conclusion, these results demonstrated that BMP-2/AAc-PCL fibers have the potential to effectively induce the osteogenic differentiation of MG-63 cells.

A spatial patternable macroporous hydrogel with cell-affinity domains to enhance cell spreading and differentiation

Cell adhesion and spreading are two essential factors for anchorage-dependent cells such as osteocytes. An adhesive macroporous hydrogel system, in which cell-affinitive domains and sufficient cytoskeleton reorganization space were simultaneously constructed, was proposed in this report to support cell adhesion and spreading, respectively, and facilitate cell differentiation and function establishment eventually. The adhesive macroporous alginate hydrogel was developed by RGD peptide graft and gelatin microspheres hybridization to generate cellular adhesion sites and highly interconnected macropores. The successful stretched morphology and enhanced osteogenic differentiation of MG-63 cells in this modified alginate hydrogel showed clearly the feasibility that cell function may be effectively facilitated. Besides, this hydrogel model can be further applied to construct complex micropatterned structure, such as individual microgels in shapes of circle, square, cross and ring, and osteon-like structure containing both osteogenic and vascularized area generated by a double-ring assembly. These results should provide this adhesive macroporous photocrosslinkable hydrogel system as potential three-dimensional scaffolds for guiding tissue formation, especially for the bioengineering of tissues that have multiple cell types and require precisely defined cell–cell and cell–substrate interactions.

RE: “The influence of RAMP1 overexpression on CGRP-induced osteogenic differentiation in MG-63 cells in vitro: An experimental study”

The aim of this study was to elucidate the influence of receptor activity modifying protein 1 (RAMP1) overexpression on the expression and distribution of calcitonin receptor-like receptor (CRLR) in MG-63 cells. Our research also focused on whether RAMP1 overexpression enhanced the promoting effect of exogenous CGRP on osteogenic differentiation in MG-63 cells. We first constructed a eukaryotic expression vector containing human RAMP1 and stably transfected it into MG-63 cells. Real-time PCR and Western blotting were used to determine the expression levels of RAMP1 and CRLR mRNA and protein, respectively. Immunofluorescence analysis was employed to compare the distribution of CRLR in transfected cells. After treatment with CGRP, the extent of osteogenic differentiation was evaluated by simultaneous monitoring of alkaline phosphatase activity, the expression patterns of osteoblastic markers and mineralisation staining. We found that RAMP1 was more highly expressed in the transfected group compared with the control groups (P < 0.01). The CRLR expression was significantly higher than that in the control groups (P < 0.05). In addition, after 7 days of CGRP treatment to induce osteogenic differentiation, the expression of collagen I mRNA was markedly increased in the transfected group (P < 0.05). The transfected group exhibited more granular precipitation in the cytoplasm with alkaline phosphatase staining after 7 and 14 days of differentiation. When stained with Alizarin Red, cells overexpressing RAMP1 were darker and formed many mineralised nodules with clear boundaries and calcium deposition typical of mineralised bone matrix structures at 28 days post-induction of differentiation. The CGRP-induced ALP activity in the RAMP1 overexpression group was significantly higher 3, 6 and 9 days after induction than that in the two control groups (P < 0.05). RAMP1 overexpression promotes CRLR expression, localisation on the cell membrane and enhanced CGRP-mediated differentiation of MG-63 cells. This study contributes to a better understanding of the molecular mechanisms governing CGRP-induced MG-63 differentiation.