A hybrid scaffold of poly(lactide-<Emphasis Type="Italic">co</Emphasis>-glycolide) sponge filled with fibrin gel for cartilage tissue engineering

The poly(lactide-co-glycolide)(PLGA) sponge fabricated by a gelatin porogen leaching method was filled with fibrin gel to obtain a hybrid scaffold for chondrocytes culture in vitro.The fibrin gel evenly distributed in the hybrid scaffold with visible fibrinogen fibers after drying.In vitro culture it was found that in the hybrid scaffold the chondrocytes distributed more evenly and kept a round morphology as that in the normal cartilage.Although the chondrocytes seeded in the control PLGA sponges showed similar proliferation behavior with that in the hybrid scaffolds,they were remarkably elongated,forming a fibroblast-like morphology.Moreover,a larger amount of glycosaminoglycans was secreted in the hybrid scaffolds than that in the PLGA sponges after in vitro culture of chondrocytes for 4 weeks.The results suggest that the fibrin/PLGA hybrid scaffold may be favorably applied for cartilage tissue engineering.     

Effects of adenovirus-mediated bFGF, IL-1Ra and IGF-1 gene transfer on human osteoarthritic chondrocytes and osteoarthritis in rabbits

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.     

A Convenient Approach to Stereoisomeric Iminocyclitols: Generation of Potent Brain‐Permeable OGA Inhibitors

Pyrrolidine‐based iminocyclitols are a promising class of glycosidase inhibitors. Reported herein is a convenient epimerization strategy that provides direct access to a range of stereoisomeric iminocyclitol inhibitors of O‐GlcNAcase (OGA), the enzyme responsible for catalyzing removal of O‐GlcNAc from nucleocytoplasmic proteins. Structural details regarding the binding of these inhibitors to a bacterial homologue of OGA reveal the basis for potency. These compounds are orally available and permeate into rodent brain to increase O‐GlcNAc, and should prove useful tools for studying the role of OGA in health and disease.     

Kaempferia parviflora Extract Alleviated Rat Arthritis,Exerted Chondroprotective Properties In Vitro,and Reduced Expression of Genes Associated with Inflammatory Arthritis

Kaempferia parviflora Wall. ex Baker (KP) has been reported to attenuate cartilage destruction in rat model of osteoarthritis. Previously, we demonstrated that KP rhizome extract and its active components effectively suppressed mechanisms associated with RA in SW982 cells. Here, we further evaluated the anti-arthritis potential of KP extract by using multi-level models, including a complete Freund’s adjuvant-induced arthritis and a cartilage explant culture model, and to investigate the effects of KP extract and its major components on related gene expressions and underlying mechanisms within cells. In arthritis rats, the KP extract reduced arthritis indexes, with no significant changes in biological parameters. In the cartilage explant model, the KP extract exerted chondroprotective potential by suppressing sulfated glycosaminoglycans release while preserving high accumulation of proteoglycans. In human chondrocyte cell line, a mixture of the major components equal to their amounts in KP extract showed strong suppression the expression of genes-associated inflammatory joint disease similar to that of the extract. Additionally, KP extract significantly suppressed NF-κB and MAPK signaling pathways. The suppressing expression of necroptosis genes and promoted anti-apoptosis were also found. Collectively, these results provided supportive evidence of the anti-arthritis properties of KP extract, which are associated with its three major components.     

HPLC identification of oversulphated chondroitin as the major glycosaminoglycan of human tonsils

The glycosaminoglycans of human nasopharyngeal and palatine tonsils, obtained after surgical dissection due to tonsillitis, were isolated and characterized by means of enzyme susceptibility and HPLC. Chondroitin/dermatan sulphate were the major glycosaminoglycans identified. A large proportion of this glycosaminoglycan was made up of oversulphated structures, namely DeltaDi-di(4,6)S, which were found mainly in invertebrate tissues and in mast cells.     

Thermal analysis of the osteoarthritic human hyaline cartilage

Summary Arthritis of major joints, especially osteoarthritis of the knee is a very frequent disease of human beings mainly in the developed countries. The pathology of osteoarthritis has been subject of many publications before, using a wide spectrum of different methods to evaluate degenerative changes of hyaline cartilage. The authors examined osteoarthritic human knee joint hyaline cartilage with differential scanning calorimetry. The different stages of cartilage degeneration have been verified by histological examinations. The research group demonstrated thermal differences between various stages of osteoarthritis. Besides explaining possible causes for experienced thermodynamic effects, the authors reflect upon future research ways and the possibilities of applying the method in practice.     

New thermogravimetric protocol for the investigation of normal and damaged human hyaline cartilage

Osteoarthritis, although classically conceived of as a degenerative consequence of aging, is a disease with an increasingly well-characterized molecular pathophysiology. Pathologic changes in cartilage composition and molecular organization, as well as elevated water content, alter the exquisite balance of biomechanical properties. Much of what is known about changes in the extracellular matrix in osteoarthritis comes from animal models. Previously, thermogravimetric methods have not been used for compositional thermoanalytical study of normal and degenerative human hyaline cartilage. For this reason the research group established a sufficient new thermogravimetric protocol, which proved water content elevation contributing to disease progression.     

Protein profile of osteoarthritic human articular cartilage using tandem mass spectrometry

Articular cartilage contains both chondrocyte cells and extracellular matrix (ECM) components. Currently, comprehensive information concerning the protein composition of human articular cartilage tissue is somewhat lacking. In this report we detail the use of tandem mass spectrometry (MS/MS) for a preliminary global identification of proteins from human articular knee cartilage tissue from patients diagnosed with osteoarthritis. Knee cartilage supernatant was fractionated using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), in-gel digested and peptide sequences were then determined by performing on-line nano-liquid chromatography (LC)/MS/MS experiments using an ion trap mass spectrometer. Altogether, over 100 different proteins from nearly 700 unique peptide sequences were detected by MS/MS. The majority of the proteins identified are involved in ECM organization (35%), signal transduction and cell communication (14%), immune response (11%) and metabolism and energy pathways (11%). Proteins observed included several well-known cartilage components as well as lower abundant lesser known ECM proteins. Possible degradation products in the cartilage sample, such as from cartilage link protein, could also be detected by our mass spectrometry methods. We show here that mass spectrometry can be utilized as a tool for a fast, accurate and sensitive analysis of a complex mixture of cartilage proteins. It is believed that this type of proteomic analysis will aid future work centered on investigating the pathology of this and other related joint diseases.     

Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis

Rheumatoid arthritis (RA) and osteoarthritis (OA), two common types of arthritis, affect the joints mainly by targeting the synovium and cartilage. Increasing evidence indicates that a significant network connects synovitis and cartilage destruction during the progression of arthritis. We recently demonstrated that hypoxia-inducible factor (HIF)-2α causes RA and OA by regulating the expression of catabolic factors in fibroblast-like synoviocytes (FLS) or chondrocytes. To address the reciprocal influences of HIF-2α on FLS and chondrocytes, we applied an in vitro co-culture system using a transwell apparatus. When co-cultured with HIF-2α-overexpressing chondrocytes, FLS exhibited increased expression of matrix metalloproteinases and inflammatory mediators, similar to the effects induced by tumor-necrosis factor (TNF)-α treatment of FLS. Moreover, chondrocytes co-cultured with HIF-2α-overexpressing FLS exhibited upregulation of Mmp3 and Mmp13, which is similar to the effects induced by interleukin (IL)-6 treatment of chondrocytes. We confirmed these differential HIF-2α-induced effects via distinct secretory mediators using Il6-knockout cells and a TNF-α-blocking antibody. The FLS-co-culture-induced gene expression changes in chondrocytes were significantly abrogated by IL-6 deficiency, whereas TNF-α neutralization blocked the alterations in gene expression associated with co-culture of FLS with chondrocytes. Our results further suggested that the observed changes might reflect the HIF-2α-induced upregulation of specific receptors for TNF-α (in FLS) and IL-6 (in chondrocytes). This study broadens our understanding of the possible regulatory mechanisms underlying the crosstalk between the synovium and cartilage in the presence of HIF-2α, and may suggest potential new anti-arthritis therapies.     

软骨细胞体外增殖去分化的拉曼光谱分析

基于显微拉曼光谱技术,对组织工程的软骨种子细胞在传代增殖过程中的去分化进行单细胞分析。首先,对体外单层培养的第1-4代(P1-P4)大鼠软骨细胞样本进行了单细胞拉曼光谱检测,由此识别出软骨细胞中各种碱基、糖基、氨基酸等主要物质分子结构的特征峰集合。随后,分析拉曼光谱中若干重点特征峰强度随细胞传代次数的变化,发现软骨细胞体外增殖过程中核酸(789、1094、1576 cm~(-1))含量降低、Ⅱ型胶原(特异组分为羟脯氨酸,1207 cm~(-1))和蛋白聚糖(特异组分为糖胺聚糖,1042、1063、1126、1160 cm~(-1))合成下降、脂质(1304 cm~(-1))及磷酸盐(957 cm~(-1))含量增加等分子水平变化,从而在活体单细胞层次初步揭示了去分化引起软骨细胞增殖变缓、分泌减弱、形态纤维化等现象的分子机制。     

微流控芯片流体剪切力和肿瘤坏死因子-α共同作用对大鼠软骨细胞表型的影响

流体剪切力是生物体内普遍存在的一种生物力学形式,是细胞微环境的重要组成部分,对细胞多种生物学行为有重要调节作用。该研究以微流控芯片技术为基础,建立了一种基于流阻原理能同时产生4个不同大小流体剪切力的微流控芯片平台,用以研究低流速的流体剪切力对大鼠原代软骨细胞表型维持的影响。结果表明,流体剪切力可促进软骨细胞的表型维持。还加入了肿瘤坏死因子-α(TNF-α),考察流体剪切力和TNF-α共同作用对软骨细胞表型的影响。结果表明,在剪切力和TNF-α共同作用下,软骨细胞的Ⅱ型胶原和蛋白多糖表达明显下调。该研究为软骨组织工程和骨性关节炎的疾病研究提供有力的研究平台,为骨关节疾病治疗和防治提供了理论依据。     

A Silk Fibroin and Peptide Amphiphile‐Based Co‐Culture Model for Osteochondral Tissue Engineering

New biomaterials with the properties of both bone and cartilage extracellular matrices (ECM) should be designed and used with co‐culture systems to address clinically applicable osteochondral constructs. Herein, a co‐culture model is described based on a trilayered silk fibroin‐peptide amphiphile (PA) scaffold cultured with human articular chondrocytes (hACs) and human bone marrow mesenchymal stem cells (hBMSCs) in an osteochondral cocktail medium for the cartilage and bone sides, respectively. The presence of hACs in the co‐cultures significantly increases the osteogenic differentiation potential of hBMSCs based on ALP activity, RT‐PCR for osteogenic markers, calcium analyses, and histological stainings, whereas hACs produces a significant amount of glycosaminoglycans (GAGs) for the cartilage region, even in the absence of growth factor TGF‐β family in the co‐culture medium. This trilayered scaffold with trophic effects offers a promising strategy for the study of osteochondral defects.

     

A study of magnetic field effects on fibroblast cultures part 3. The evaluation of the effects of static and extremely low frequency (ELF) magnetic fields on glycosaminoglycan metabolism in fibroblasts,cell coats and culture medium

Cultures of fibroblasts isolated from murine liver by the method of tissue trypsinization were exposed to a static magnetic field (0.49 T) and an extremely low frequency (ELF) magnetic field (50 Hz, 0.020 T). The cultures were exposed to magnetic fields for exposure periods of 2, 4, 8, 16, 32 and 64 min on four consecutive days. During the experiment we investigated the glycosaminoglycans isolated from the fibroblast, their coats and the culture medium. The investigations concerned heparan sulphate (DS) and chondroitin sulphates (CS). The changes observed in the fibroblast cultures exposed to ELF magnetic field suggest an increase in sulphate ion content in the glycosaminoglycans investigated, i.e. increased synthesis of the compounds. The ELF magnetic field also affects the degree of glycosaminoglycan sulphatization. Some changes in the quantitative relations between HS, DS and CS were also noted. The static magnetic field had no effect on glycosaminoglycan metabolism, i.e. there were no alterations in incorporation of labeled sulphur into sulphate glycosaminoglycans.     

Pigmentation Chemistry and Radical‐Based Collagen Degradation in Alkaptonuria and Osteoarthritic Cartilage

Alkaptonuria (AKU) is a rare disease characterized by high levels of homogentisic acid (HGA); patients suffer from tissue ochronosis: dark brown pigmentation, especially of joint cartilage, leading to severe early osteoarthropathy. No molecular mechanism links elevated HGA to ochronosis; the pigment's chemical identity is still not known, nor how it induces joint cartilage degradation. Here we give key insight on HGA‐derived pigment composition and collagen disruption in AKU cartilage. Synthetic pigment and pigmented human cartilage tissue both showed hydroquinone‐resembling NMR signals. EPR spectroscopy showed that the synthetic pigment contains radicals. Moreover, we observed intrastrand disruption of collagen triple helix in pigmented AKU human cartilage, and in cartilage from patients with osteoarthritis. We propose that collagen degradation can occur via transient glycyl radicals, the formation of which is enhanced in AKU due to the redox environment generated by pigmentation.     

糖胺多糖酶解产物—不饱和二糖的高效液相色谱分析

介绍了一种糖胺多糖的二糖组成成分分析方法－高效液相色谱法。该方法柱前处理简单。应用该方法对人体尿液中糖胺多糖的二糖组成进行了分析     

Thermo-Sensitive Dual-Functional Nanospheres with Enhanced Lubrication and Drug Delivery for the Treatment of Osteoarthritis

Osteoarthritis is a typical degenerative joint disease related to a lubrication deficiency of articular cartilage, which is characterized by increased friction at the joint surface and severe inflammation of the joint capsule. Consequently, therapies combining lubrication restoration and drug intervention are regarded as a promising strategy for the treatment of osteoarthritis. In the present study, thermo-sensitive dual-functional nanospheres, poly[N-isopropylacrylamide-2-methacryloyloxyethyl phosphorylcholine] (PNIPAM-PMPC), are developed through emulsion polymerization. The PNIPAM-PMPC nanospheres could enhance lubrication based on the hydration lubrication mechanism by forming a tenacious hydration layer surrounding the zwitterionic headgroups, and achieve local drug delivery by encapsulating the anti-inflammatory drug diclofenac sodium. The lubrication and drug release tests showed improved lubrication and thermo-sensitive drug release of the nanospheres. The in vitro test using cytokines-treated chondrocytes indicated that the PNIPAM-PMPC nanospheres were biocompatible and upregulated anabolic genes and simultaneously downregulated catabolic genes of the articular cartilage. In summary, the developed PNIPAM-PMPC nanospheres, with the property of enhanced lubrication and local drug delivery, can be an effective nanomedicine for the treatment of osteoarthritis.     

Surface modification with peptide for enhancing chondrocyte adhesion and cartilage regeneration in porous scaffolds

The present study presents the regeneration of cartilage in hybrid scaffolds comprising polyethylene oxide (PEO) and chitosan with surface CDPGYIGSR. This surface peptide was grafted via crosslinking onto the scaffolds. The pores in the scaffolds were interconnected and uniformly distributed with an average diameter about 200-250 μm. A high weight percentage of PEO in the matrix yielded a rugged topography of the pore surfaces. The adhesion of bovine knee chondrocytes (BKCs) in the peptide-grafted scaffolds was more efficient than that in the peptide-free scaffolds. In addition, the constructs with surface peptide could stimulate chondrogenesis with enhanced quantities of BKCs, glycosaminoglycans (GAGs), and collagen over cultivation. The histological staining of the proliferated BKCs and secreted GAGs indicated that the surface peptide favored the production of neocartilage in the constructs. Moreover, the immunochemical staining against type II collagen demonstrated the maintenance of phenotypic chondeocytes on the peptide-grafted surfaces. The peptide-grafted PEO/chitosan scaffolds can be applied to the treatment for injured cartilage in preclinical trials.