Transforming growth factor β1 (TGF-β1) has been suggested to be a candidate cytokine in the field of bone tissue engineering. Cytokines serve important roles in tissue engineering, particularly in the repair of bone damage; however, the underlying molecular mechanisms remain unclear. In the present study, the effects of TGF-β1 on the osteogenesis and motility of hFOB1.19 human osteoblasts were demonstrated via the phenotype and gene expression of cells. Additionally, the role of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin/S6 kinase 1 (PI3K/AKT/mTOR/S6K1) signalling pathway in the effects of TGF-β1 on osteoblasts was investigated. It was demonstrated using Cell Counting Kit-8 and flow cytometry assays that the proliferation of human osteoblasts was promoted by 1 ng/ml TGF-β1. In addition, alkaline phosphatase activity, Alizarin red staining, scratch-wound and Transwell assays were conducted. It was revealed that osteogenesis and the migration of cells were regulated by TGF-β1 via the upregulation of osteogenic and migration-associated genes. Alterations in the expression of osteogenesis- and migration-associated genes were evaluated following pre-treatment with a PI3K/AKT inhibitor (LY294002) and an mTOR/S6K1 inhibitor (rapamycin), with or without TGF-β1. The results indicated that TGF-β1 affected the osteogenesis and mineralisation of osteoblasts via the PI3K/AKT signalling pathway. Furthermore, TGF-β1 exhibited effects on mTOR/S6K1 downstream of PI3K/AKT. The present study demonstrated that TGF-β1 promoted the proliferation, differentiation and migration of human hFOB1.19 osteoblasts, and revealed that TGF-β1 affected the biological activity of osteoblasts via the PI3K/AKT/mTOR/S6K1 signalling pathway. Our findings may provide novel insight to aid the development of bone tissue engineering methods for the treatment of bone injury.

Background The body is unable to repair and regenerate large area bone defects. Moreover, the repair capacity of articular cartilage is very limited. There has long been a lack of effective treatments for osteochondral lesions. The engineered tissue with biphase synthetic for osteochondral repair has become one of the hot research fields over the past few years. In this study, an integrated biomanufacturing platform was constructed with bone marrow mesenchymal stem cells (BMSCs)/porous tantalum (pTa) associated with chondrocytes/collagen membranes (CM) to repair large osteochondral defects in load-bearing areas of goats. Methods Twenty-four goats with a large osteochondral defect in the femoral heads of the left hind legs were randomly divided into three groups: eight were treated with chondrocytes/CM-BMSCs/pTa, eight were treated with pure CM-pTa composite, and the other eight goats were untreated. The repair effect was assessed by X-ray, gross observation, and histomorphology for 16 weeks after the operation. In addition, the biocompatibility of chondrocytes/CM-BMSCs/pTa was observed by flow cytometry, CCK8, immunocytochemistry, and Q-PCR. The characteristics of the chondrocytes/CM-BMSCs/pTa were evaluated using both scanning electron microscopy and mechanical testing machine. Results The integrated repair material consists of pTa, injectable fibrin sealant, and CM promoted adhesion and growth of BMSCs and chondrocytes. pTa played an important role in promoting the differentiation of BMSCs into osteoblasts. Three-dimensional CM maintained the phenotype of chondrocytes successfully and expressed chondrogenic genes highly. The in vivo study showed that after 16 weeks from implantation, osteochondral defects in almost half of the femoral heads had been successfully repaired by BMSC-loaded pTa associated with biomimetic 3D collagen-based scaffold. Conclusions The chondrocytes/CM-BMSCs/pTa demonstrated significant therapeutic efficacy in goat models of large osteochondral defect. This provides a novel therapeutic strategy for large osteochondral lesions in load-bearing areas caused by severe injury, necrosis, infection, degeneration, and tumor resection with a high profile of safety, effectiveness, and simplicity.

Alginate/chitosan/alginate (ACA) hydrogel microcapsules were modified with methoxy poly(ethylene glycol) (MPEG) to improve protein repellency and biocompatibility. Increased MPEG surface graft density (n(S)) on hydrogel microcapsules was achieved by controlling the grafting parameters including the buffer layer substrate, membrane thickness, and grafting method. X-ray photoelectron spectroscopy (XPS) model was employed to quantitatively analyze n(S) on this three-dimensional (3D) hydrogel network structure. Our results indicated that neutralizing with alginate, increasing membrane thickness, and in situ covalent grafting could increase n(S) effectively. ACAC(PEG) was more promising than ACC(PEG) in protein repellency because alginate supplied more -COO(-) negative binding sites and prevented MPEG from diffusing. The n(S) increased with membrane thickness, showing better protein repellency. Moreover, the in situ covalent grafting provided an effective way to enhance n(S), and 1.00 ± 0.03 chains/nm(2) was achieved, exhibiting almost complete immunity to protein adsorption. This antifouling hydrogel biomaterial is expected to be useful in transplantation in vivo.

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 mater...

Cell encapsulation technology holds promise for the cell-based therapy. But poor mechanical strength and biocompatibility of microcapsule membrane are still obstacles for the clinical applications. A novel strategy is presented to prepare AC₁ C₂ A microcapsules with bi-functional membrane (that is, both desirable biocompatibility and membrane stability) by sequentially complexing chitosans with higher deacetylation degree (C₁) and lower deacetylation degree (C₂) on alginate (A) gel beads. Both in vitro and in vivo evaluation of AC₁C₂ A microcapsules demonstrate higher membrane stability and less cell adhesion, because the introduction of C₂ increases membrane strength and decreases surface roughness. Moreover, diffusion test of AC₁C₂ A microcapsules displays no inward permeation of IgG protein suggesting good immunoisolation function. The results demonstrate that AC₁C₂ A microcapsules with bi-functional membrane could be a promising candidate for microencapsulated cell implantation with cost effective usage of naturally biocompatible polysaccharides.