Enhanced ex vivo expansion of adult mesenchymal stem cells by fetal mesenchymal stem cell ECM

Large-scale expansion of highly functional adult human mesenchymal stem cells (aMSCs) remains technologically challenging as aMSCs lose self renewal capacity and multipotency during traditional long-term culture and their quality/quantity declines with donor age and disease. Identification of culture conditions enabling prolonged expansion and rejuvenation would have dramatic impact in regenerative medicine. aMSC-derived decellularized extracellular matrix (ECM) has been shown to provide such microenvironment which promotes MSC self renewal and “stemness”. Since previous studies have demonstrated superior proliferation and osteogenic potential of human fetal MSCs (fMSCs), we hypothesize that their ECM may promote expansion of clinically relevant aMSCs. We demonstrated that aMSCs were more proliferative (∼1.6×) on fMSC-derived ECM than aMSC-derived ECMs and traditional tissue culture wares (TCPS). These aMSCs were smaller and more uniform in size (median ± interquartile range: 15.5 ± 4.1 μm versus 17.2 ± 5.0 μm and 15.5 ± 4.1 μm for aMSC ECM and TCPS respectively), exhibited the necessary biomarker signatures, and stained positive for osteogenic, adipogenic and chondrogenic expressions; indications that they maintained multipotency during culture. Furthermore, fMSC ECM improved the proliferation (∼2.2×), size (19.6 ± 11.9 μm vs 30.2 ± 14.5 μm) and differentiation potential in late-passaged aMSCs compared to TCPS. In conclusion, we have established fMSC ECM as a promising cell culture platform for ex vivo expansion of aMSCs.     

The potential of anisotropic matrices as substrate for heart valve engineering

Cells environment is increasingly recognized as an important function regulator through cell–matrix interactions. Extracellular matrix (ECM) anisotropy being a key component of heart valves properties, we have devised a method to create highly porous anisotropic nanofibrillar scaffolds and studied their suitability as cell-support and interactions with human adipose derived stem cells (hADSCs) and human valve interstitial cells (hVICs). Anisotropic nanofibrillar scaffolds were produced by a modified jet-spraying method that allows the formation of aligned nanofibres (600 nm) through air-stream diffraction of a polymer solution (poly (ε-caprolactone, PCL) and collection onto a variably rotating drum. The resulting matrices of high porosity (99%) mimicked valve mechanical anisotropy. Dynamically seeded hADSC and hVIC cultured on scaffolds up to 20 days revealed that hADSC and hVIC penetration within the matrices was improved by anisotropic organization. Within 10 days, cells populated the entire scaffolds thickness and produced ECM (collagen I, III and elastin). As a result, mechanical properties of the constructs were improved over culture, while remaining anisotropic. In contrast to isotropic matrices, anisotropy induced elongated hADSCs and hVICs morphology that followed nanofibres orientation. Interestingly, these morphological changes did not induce hADSC differentiation towards the mesoderm lineages while hVIC recovered a physiological phenotype over culture in the biomimetic matrices. Overall, this study indicates that highly porous anisotropic jet-sprayed matrices are interesting candidates for valve tissue engineering, through anisotropic mechanical properties, efficient cell population, conservation of stem cells phenotype and recovery of hVIC physiological phenotype.     

Induction of epithelial-to-mesenchymal transition in proximal tubular epithelial cells on microfluidic devices

In proteinuric nephropathy, epithelial-to-mesenchymal transition (EMT) is an important mechanism that causes renal interstitial fibrosis. The precise role of EMT in the pathogenesis of fibrosis remains controversial, partly due to the absence of suitable in vitro or in vivo models. We developed two microfluidic and compartmental chips that reproduced the fluidic and three-dimensional microenvironment of proximal tubular epithelial cells in vivo. Using one microfluidic device, we stimulated epithelial cells with a flow of healthy human serum, heat-inactivated serum and complement C3a, which mimicked the flow of urine within the proximal tubule. We observed that epithelial cells exposed to serum proteins became apoptotic or developed a mesenchymal phenotype. Incubating cells with C3a induced similar features. However, cells exposed to heat-inactivated serum did not adopt the mesenchymal phenotype. Furthermore, we successfully recorded the cellular morphological changes and the process of transmigration into basement membrane extract during EMT in real-time using another three-dimensional microdevice. In conclusion, we have established a cell-culture system that mimics the native microenvironment of the proximal tubule to a certain extent. Our data indicates that EMT did occur in epithelial cells that were exposed to serum proteins, and C3a plays an essential role in this pathological process.     

Preparation of silica–poly(methyl methacrylate) composite with a nanoscale dual-network structure and hardness comparable to human enamel

ObjectiveThis study aims to prepare an organic–inorganic composite with a nanoscale dual-network structure composed of a ceramic skeleton and infiltrated resin to mimic the mechanical properties of human enamel.MethodsA porous silica block was obtained by sintering a green body composed of SiO₂ nanoparticles and poly(vinyl alcohol) organic binder. Methyl methacrylate monomers were infiltrated into the porous silica block and thermally polymerized to form poly(methyl methacrylate) (PMMA). A monolithic SiO₂–PMMA composite was obtained, and its nanoscale structure was investigated. Its mechanical properties were characterized by Vickers hardness, elastic modulus, and flexural strength tests.ResultsThe SiO₂–PMMA composite had a nanoscale dual-network structure composed of a silica–ceramic skeleton with PMMA-filled continuous 10–20 nm sized pores. The mechanical properties of the composite depended on the SiO₂ content, which could be adjusted by modifying sintering time of the porous silica block. The mechanical properties of the composite exhibited wide variations with Vickers hardness values of 54–756, elastic moduli of 7–54 GPa, and flexural strengths of 75–120 MPa.SignificanceThe preparation of a SiO₂–PMMA composite with a dual-network structure at the nanoscale was demonstrated, and the composite was characterized with respect to its hardness compatibility with human enamel.     

猫眼草素的结构和立体化学

根据生源学说、光谱分析及化学合成,将猫眼草素的原定结构A式修改为B₁式。分子中氧杂环上的两个取代基互为反式,并以外消旋体(R,R+S,S)的形式存在。     

A New Absorbable Synthetic Substitute With Biomimetic Design for Dural Tissue Repair

Dural repair products are evolving from animal tissue–derived materials to synthetic materials as well as from inert to absorbable features; most of them lack functional and structural characteristics compared with the natural dura mater. In the present study, we evaluated the properties and tissue repair performance of a new dural repair product with biomimetic design. The biomimetic patch exhibits unique three‐dimensional nonwoven microfiber structure with good mechanical strength and biocompatibility. The animal study showed that the biomimetic patch and commercially synthetic material group presented new subdural regeneration at 90 days, with low level inflammatory response and minimal to no adhesion formation detected at each stage. In the biological material group, no new subdural regeneration was observed and severe adhesion between the implant and the cortex occurred at each stage. In clinical case study, there was no cerebrospinal fluid leakage, and all the postoperation observations were normal. The biomimetic structure and proper rate of degradation of the new absorbable dura substitute can guide the meaningful reconstruction of the dura mater, which may provide a novel approach for dural defect repair.     

Characteristic and in vitro bioactivity of a microarc-oxidized TiO(2)-based coating after chemical treatment

Microarc oxidation (MAO) was used to prepare a TiO(2)-based coating containing Ca and P on titanium alloy. An alkali treatment was developed to modify the surface of the MAO coating to improve the apatite-forming ability of the coating. The chemically treated MAO coating exhibits a modified layer, with the main constituents being O, Ti, Ca and Na, showing anatase. The modified MAO coating shows a rough and porous morphology containing numerous nanoflakes of approximately 100nm thickness. During the alkali treatment process, P on the surface of the MAO coating shows a main dynamic process of dissolution; however, Ca exhibits a re-deposition process as well as dissolution. The formation of the modified layer could be explained by this mechanism: negatively charged HTiO(3)(-) ions are formed on the MAO coating due to the attack of OH(-) ions on the TiO(2) phase. The HTiO(3)(-) ions could incorporate sodium from the alkali solution and calcium from the alkali solution and MAO coating. The apatite-forming ability of the MAO coating is improved remarkably by the simple chemical treatment, since the surface of the alkali-treated MAO coating could provide abundant Ti-OH groups probably formed by ionic exchanges between (Ca2+, Na+) ions of the alkali-treated MAO coating and H3O+ ions of a simulated body fluid (SBF). Moreover, Ca released from the alkali-treated MAO coating increases the degree of supersaturation of SBF, promoting the formation of apatite. The apatite induced by the alkali-treated MAO coating possesses carbonated structure and pore networks on the nanometer scale.     

Nanorod diameter modulated osteogenic activity of hierarchical micropore/nanorod-patterned coatings via a Wnt/β-catenin pathway

Hierarchical micropore/nanorod-patterned strontium doped hydroxyapatite (Ca₉Sr₁(PO₄)₆(OH)₂, Sr₁-HA) structures (MNRs) with different nanorod diameters of about 30, 70 and 150 nm were coated on titanium, to investigate the effect of nanorod diameter on osteogenesis and the involved mechanism. Compared to micropore/nanogranule-patterned Sr₁-HA coating (MNG), MNRs gave rise to dramatically enhanced in vitro mesenchymal stem cell functions including osteogenic differentiation in the absence of osteogenic supplements and in vivo osseointegration related to the nanorod diameter with about 70 nm displaying the best effects. MNRs activated the cellular Wnt/β-catenin pathway by increasing the expression of Wnt3a and LRP6 and decreasing the expression of Wnt/β-catenin pathway antagonists (sFRP1, sFRP2, Dkk1 and Dkk2). The exogenous Wnt3a significantly enhanced the β-catenin signaling activation and cell differentiation on MNG, and the exogenous Dkk1 attenuated the enhancing effect of MNRs on them. The data demonstrate that MNRs favor osseointegration via a Wnt/β-catenin pathway.     

Effectiveness of an acellular synthetic matrix in the treatment of hard‐to‐heal leg ulcers

Hard‐to‐heal leg ulcers are a major cause of morbidity in the elderly population. Despite improvements in wound care, some wounds will not heal and they present a significant challenge for patients and health care providers. A multi‐centre cohort study was conducted to evaluate the effectiveness and safety of a synthetic, extracellular matrix protein as an adjunct to standard care in the treatment of hard‐to‐heal venous or mixed leg ulcers. Primary effectiveness criteria were (i) reduction in wound size evaluated by percentage change in wound area and (ii) healing assessed by number of patients healed by end of the 12 week study. Pain reduction was assessed as a secondary effectiveness criteria using VAS. A total of 45 patients completed the study and no difference was observed between cohorts for treatment frequency. Healing was achieved in 35·6% and wound size decreased in 93·3% of patients. Median wound area percentage reduction was 70·8%. Over 50% of patients reported pain on first visit and 87·0% of these reported no pain at the end of the study. Median time to first reporting of no pain was 14 days after treatment initiation. The authors consider the extracellular synthetic matrix protein an effective and safe adjunct to standard care in the treatment of hard‐to‐heal leg ulcers.