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Alginate-based microcapsule has becoming a promising carrier for probiotic encapsulation due to the improved stress resistant ability. Besides the physical protection of microcapsules, bacterial quorum sensing (QS) is another prominent factor affecting microbial stress resistance in microcapsules. In the present study, Vibrio harveyi cells were entrapped and proliferated into cell aggregates in alginate-based microcapsules. The microenvironment composed of cells and biomacromolecules was regulated by the diameter, alginate concentration and core state of microcapsule. Then the effect of microenvironment on bacterial QS capacity was investigated, including bioluminescence, autoinducers (AIs) production and QS related genes expression. The highest diameter of 1200 μm and highest alginate concentration of 2.0 % w/v under the investigation range presented strongest QS capacity, and the maintenance of hydrogel core could enhance bacterial QS. Moreover, the mechanism analysis revealed that the formed biofilm on the surface of cell aggregates hampered the outward transfer of AIs, and the local AIs inside the cell aggregates induced stronger bacteria QS by close-range interaction. As a whole, these findings are helpful to guide the technological development and optimization of microencapsulated probiotics with stronger stress resistance, and the potential application in food, dairy, wastewater treatment and biosensor.

Extrusible biomaterials have recently attracted increasing attention due to the desirable injectability and printability to allow minimally invasive administration and precise construction of tissue mimics. Specifically, self-healing colloidal gels are a novel class of candidate materials as injectables or printable inks considering their fascinating viscoelastic behavior and high degree of freedom on tailoring their compositional and mechanical properties. Herein, we developed a novel class of adaptable and osteogenic composite colloidal gels via electrostatic assembly of gelatin nanoparticles and nanoclay particles. These composite gels exhibited excellent injectability and printability, and remarkable mechanical properties reflected by the maximal elastic modulus reaching ∼150 kPa combined with high self-healing efficiency, outperforming most previously reported self-healing hydrogels. Moreover, the cytocompatibility and the osteogenic capacity of the colloidal gels were demonstrated by inductive culture of MC3T3 cells seeded on the three-dimensional (3D)-printed colloidal scaffolds. Besides, the biocompatibility and biodegradability of the colloidal gels was proved in vivo by subcutaneous implantation of the 3D-printed scaffolds. Furthermore, we investigated the therapeutic capacity of the colloidal gels, either in form of injectable gels or 3D-printed bone substitutes, using rat sinus bone augmentation model or critical-sized cranial defect model. The results confirmed that the composite gels were able to adapt to the local complexity including irregular or customized defect shapes and continuous on-site mechanical stimuli, but also to realize osteointegrity with the surrounding bone tissues and eventually be replaced by newly formed bones.

Ginkgo biloba L. extract (EGb) is one of the world's most extensively used herbal medicines. Due to the diverse pharmacological properties of EGb, it has been used in the treatment of neurological illnesses, as well as cardiovascular and cerebrovascular ailments. However, the effect and pharmacological mechanism of EGb on steroid-induced necrosis of the femoral head (SINFH) are still unclear.SINFH remains a challenging problem in orthopedics. Previous investigations have shown that EGb has the potential to reduce the occurrence of SINFH. The goal was to determine the effect and mechanism of EGb in preventing SINFH by inhibiting apoptosis and improving vascular endothelial cells (VECs) functions.CCK-8, nitric oxide (NO) production and flow cytometry were used to determine the cell apoptosis and function. The scratch and angiogenesis tests assessed migration and tube formation. Western blot analysis detected the expressions of apoptosis-related proteins and PI3K/AKT/eNOS pathway-related proteins. Apoptosis and angiogenesis were also detected treated with the inhibitors. A mouse model of SINFH was established. Paraffin section was used to determine the necrotic pathology and apoptosis. Vessels in the femoral heads were assessed by immunofluorescence staining.When stimulated by methylprednisolone (MPS), cell viability, NO generation and tube formation were decreased, the apoptotic rate increased. Simultaneously, MPS decreased the expression levels of p-PI3K, p-AKT, and p-eNOS. EGb increased the expression levels of these proteins, restrained apoptosis, and restored cell functions. The addition of the inhibitors decreased anti-apoptotic effect and angiogenesis. In addition, when compared to the model mice, there were fewer empty lacunae and normal trabecular arrangement after taking different doses of EGb. The protective effect was also confirmed by the vascular quantitative analysis in vivo.This study established that EGb increased endothelial cell activity and inhibited apoptosis and function loss induced by MPS, elucidating the effect and molecular mechanism of EGb on early SINFH.

Background As 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. Methods In 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. Results The 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. Conclusion Overall, 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.

Controlling the adhesion of mammalian cells at the interface between materials and biological environments is a real challenge when designing materials for tissue engineering applications. The surface properties of implanted materials are known to have a significant impact on cell adhesion. Herein, two types of polyelectrolyte multilayers (PEMs) hydrogel membrane based on marine-derived polysaccharides of chitosan (CHI) and alginate (ALG) biopolymers were fabricated by the layer-by-layer (LbL) technique using simple approach involving the change in assemble sequence of chitosan with different degree of deacetylation (DD). The effect of PEMs formation on the surface properties and their effects on the adhesion of mouse fibroblast cell (L929) of the two membranes were studied. The results showed that the formations of ALG/CHI membranes were related to the rigidity and conformations of chitosan molecules. The adhesion of L929 cell was strongly depended on the surface roughness rather than stiffness. The surface of PEMs can be strongly cytophilic (cell adhesive, terminated with chitosan (C1)) or strongly cytophobic (cell resistant, terminated with chitosan (C2)). The results obtained indicate that ALG/CHI PEMs with different surface morphology and roughness could be used in vitro to manipulate cell behaviors to improve upon the design of tissue-engineered membranes.

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.

In vivo microcapsule implantation suffers from varying degrees of inflammatory responses. Considering the complications of the implantation process and the individual variation in laboratory animals, a feasible model, constructed in vitro by culturing fibroblasts on the surfaces of different types of microcapsules, is established in the present study. A high consistency is shown between the cell adhesion on beads or microcapsules based on the developed model in vitro and their fibrosis formation after implantation in vivo, which can be used to evaluate the biocompatibility of the microcapsules. Moreover, the relationship between the surface properties of the microcapsules and the cell adhesion behavior was explored using this developed model. It was found that cell adhesion was aggravated with chitosan reaction, but was reduced with alginate neutralization. This was closely related to the variation trend of surface charges, but not surface roughness and the stiffness of alginate-based microcapsules during the process of chitosan coating and alginate neutralization. This model could therefore provide a rapid evaluation and guidance to tailor the optimal surface properties for long-term transplantation.

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 (AC) microcapsules with desired strength and biocompatibility are preferred in cell-based therapy. Sequential assembly of higher and lower deacetylated chitosans (C1 and C2 ) on alginate has produced AC1 C2 microcapsule with improved membrane strength and biocompatibility. In this article, the assembly and complexation processes of two cationic chitosans on anionic alginate were concerned, and the cause and influence of sequentially assembling chitosans on AC1 C2 microcapsules membrane formation were evaluated. It was found that C1 complexation was the key factor for deciding the membrane thickness of AC1 C2 microcapsule. Specifically, the binding amount of C2 positively related to the binding amount of C1 , which suggested the first layer by C1 complexation on alginate had no obvious resistance on the sequential cationic C2 complexation. Further analyses demonstrated that outward migration of alginate molecules and inward diffusion of both chitosans under electrostatic interaction contributed to the sequential coating of C2 on first C1 layer. Moreover, C2 complexation through the surface to inner layer of membrane helped smoothen the first layer by C1 complexation that displayed a synergy role on the formation of AC1 C2 microcapsule membrane. Therefore, the two chitosans played different roles and synergistically contributed to membrane properties that can be easily regulated with membrane complexation time.

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 AC(1)C(2)A microcapsules with bifunctional membrane (that is, both desirable biocompatibility and membrane stability) by sequentially complexing chitosans with higher deacetylation degree (C-1) and lower deacetylation degree (C-2) on alginate (A) gel beads. Both in vitro and in vivo evaluation of AC(1)C(2)A microcapsules demonstrate higher membrane stability and less cell adhesion, because the introduction of C-2 increases membrane strength and decreases surface roughness. Moreover, diffusion test of AC(1)C(2)A microcapsules displays no inward permeation of IgG protein suggesting good immunoisolation function. The results demonstrate that AC(1)C(2)A microcapsules with bi-functional membrane could be a promising candidate for microencapsulated cell implantation with cost effective usage of naturally biocompatible polysaccharides.

Alginate-chitosan (AC) microcapsule with polyelectrolyte complex (PEC) membrane has been attractive in biotechnological area such as cell immobilization fermentation. The PEC membrane has been known important to control microcapsule performance, but the knowledge was mainly obtained with empty or drug-loaded microcapsules. In this paper, the emphasis was to explore the influence of cell growth on PEC membrane characteristics, that is, to study the membrane characteristics in dynamic process. AC microcapsule entrapping yeast cells (Saccharomyces cerevisiae BY4741) was used as model, and the PEC membrane characteristics including membrane thickness, swelling behavior, permeability, and component were studied during cell culture process. It was found that both the membrane thickness and volume swelling degree of AC microcapsule increased, however, membrane permeability decreased with the growth of entrapped yeast cells. Fluorescence-labeling studies suggested the concentrated alginate layer squeezed by the proliferated cells and macromolecular metabolites layer secreted by proliferated cells contributed to above increment. The former PEC membrane, together with the new layers, constituted the 'apparent membrane', which increased the diffusion resistance of substances. (C) 2011 Elsevier B.V. All rights reserved.

In this study, an improved indirect cell coculture system was constructed by using a polyelectrolyte complex membrane generated by alginate (A) and chitosan (C). Methodologies of characterizing thickness and permeability of flat AC membrane were first established due to the importance of these two parameters in determining intercellular distance and degree of contact between cocultured cells. Compared to reaction time, both alginate concentration and molecular weight (Mw) of chitosan play more dominant roles in determining the membrane thickness and diffusion coefficients. Moreover, cells in the alginate gel and on the AC membrane could maintain high cell viability. Thus, an improved indirect cell coculture system constructed by flat AC membrane was fabricated and characterized, which provides a robust tool to study the effect of intercellular distance and degree of contact between cocultured cells on cell–cell interactions. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43100.

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.

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.