Long non-coding RNA musculin antisense RNA 1 promotes proliferation and suppresses apoptosis in osteoarthritic chondrocytes via the microRNA-369-3p/Janus kinase-2/ signal transducers and activators of transcription 3 axis.

Increasing evidence indicates that long non-coding RNAs (lncRNAs) play critical roles in osteoarthritis (OA). The present study aimed to investigate the underlying molecular mechanism of lncRNA musculin antisense RNA 1 (MSC-AS1) in OA. RT-qPCR was used to detect MSC-AS1 levels in cartilage tissues from patients with OA. The effects of MSC-AS1 knockdown on the viability and apoptosis in OA were evaluated via CCK-8 and TUNEL assays. The StarBase database was used to predict the binding sites between microRNA (miR)-369-3p and MSC-AS1 or JAK2, which were confirmed via the dual-luciferase reporter assay. The results demonstrated that MSC-AS1 expression was downregulated in OA. Functional analysis indicated that the addition of MSC-AS1 promoted viability and inhibited inflammation and the apoptosis of chondrocytes. In addition, MSC-AS1 regulated the survival of OA chondrocytes by sponging miR-369-3p. JAK2 was confirmed as a direct target of miR-369-3p, and MSC-AS1 regulated JAK2/STAT3 signaling via miR-369-3p in OA chondrocytes. Taken together, our results suggest that MSC-AS1 may regulate the miR-369-3p/JAK2/STAT3 signaling pathway to accelerate the viability, and inhibit inflammation and cell apoptosis in OA chondrocytes.

Galectin network in osteoarthritis: galectin-4 programs a pathogenic signature of gene and effector expression in human chondrocytes in vitro.

Galectin-4 (Gal-4) is a member of the galectin family, which have been identified as galactose-binding proteins. Gal-4 possesses two tandem repeat carbohydrate recognition domains and acts as a cross-linking bridge in sulfatide-dependent glycoprotein routing. We herein document its upregulation in osteoarthritis (OA) in correlation with the extent of cartilage degradation in vivo. Primary human OA chondrocytes in vitro respond to carbohydrate-inhibitable Gal-4 binding with the upregulation of pro-degradative/-inflammatory proteins such as interleukin-1ß (IL-1ß) and matrix metalloproteinase-13 (MMP-13), as documented by RT-qPCR-based mRNA profiling and transcriptome data processing. Activation of p65 by phosphorylation of Ser536 within the NF-κB pathway and the effect of three p65 inhibitors on Gal-4 activity support downstream involvement of such signaling. In 3D (pellet) cultures, Gal-4 presence causes morphological and biochemical signs of degradation. Taken together, our findings strongly support the concept of galectins acting as a network in OA pathogenesis and suggest that blocking their activity in disease progression may become clinically relevant in the future.

Chondrosarcoma with Target-Like Chondrocytes: Update on Molecular Profiling and Specific Morphological Features.

This is the first histological and molecular analysis of two chondrosarcomas with target-like chondrocytes that were compared with a group of conventional chondrosarcomas and enchondromas. The unique histological feature of target-like chondrocytes is the presence of unusual hypertrophic eosinophilic APAS-positive perichondrocytic rings (baskets). In the sections stained with Safranin O/Fast green, the outer part of the ring was blue and the material in the lacunar space stained orange, similarly to intercellular regions. Immunohistochemical examination showed strong positivity for vimentin, factor XIIIa, cyclin D1, osteonectin, B-cell lymphoma 2 apoptosis regulator (Bcl-2), p53 and p16. The S-100 protein was positive in 25 % of neoplastic cells. Antibodies against GFAP, D2-40 (podoplanin), CD99, CKAE1.3 and CD10 exhibited weak focal positivity. Pericellular rings/baskets contained type VI collagen in their peripheral part, in contrast to the type II collagen in intercellular interterritorial spaces. Ultrastructural examination revealed that pericellular rings contained an intralacunar component composed of microfibrils with abundant admixture of aggregates of dense amorphous non-fibrillar material. The outer extralacunar zone was made up of a layer of condensed thin collagen fibrils with admixture of non-fibrillar dense material. NGS sequencing identified a fusion transcript involving fibronectin 1 (FN1) and fibroblast growth factor receptor 2 (FGFR2) at the RNA level. At the DNA level, no significant variant was revealed except for the presumably germline variant in the SPTA1 gene.

Bioprinting of human nasoseptal chondrocytes-laden collagen hydrogel for cartilage tissue engineering.

Skin cancer patients often have tumorigenic lesions on their noses. Surgical resection of the lesions often results in nasal cartilage removal. Cartilage grafts taken from other anatomical sites are used for the surgical reconstruction of the nasal cartilage, but donor-site morbidity is a common problem. Autologous tissue-engineered nasal cartilage grafts can mitigate the problem, but commercially available scaffolds define the shape and sizes of the engineered grafts during tissue fabrication. Moreover, the engineered grafts suffer from the inhomogeneous distribution of the functional matrix of cartilage. Advances in 3D bioprinting technology offer the opportunity to engineer cartilages with customizable dimensions and anatomically shaped configurations without the inhomogeneous distribution of cartilage matrix. Here, we report the fidelity of Freeform Reversible Embedding of Suspended Hydrogel (FRESH) bioprinting as a strategy to generate customizable and homogenously distributed functional cartilage matrix engineered nasal cartilage. Using FRESH and in vitro chondrogenesis, we have fabricated tissue-engineered nasal cartilage from combining bovine type I collagen hydrogel and human nasoseptal chondrocytes. The engineered nasal cartilage constructs displayed molecular, biochemical and histological characteristics akin to native human nasal cartilage.

Fibrin Gel Suspended Autologous Chondrocytes as Cell-based Material for long-term Injection Laryngoplasty.

OBJECTIVES/HYPOTHESIS: Injection laryngoplasty of materials for unilateral vocal-fold paralysis has shown various results regarding the long-term stability of the injected material. We evaluated a fibrin-gel based cell suspension with autologous chondrocytes in-vitro and in-vivo as long-term-stable vocal-fold augmentation material in an animal model. STUDY DESIGN: This study compises an in vitro cell-culture part as well as an in vivo animal study with New Zealand White Rabbits. METHODS: In in-vitro experiments, auricular chondrocytes harvested from 24 New Zealand White Rabbits cadavers were cultivated in pellet cultures to evaluate cartilage formation for 4 weeks using long-term-stable fibrin gel as carrier. Injectability and injection volume for the laryngoplasty was determined in-vitro using harvested cadaveric larynxes. In-vivo 24 Rabbits were biopsied for elastic cartilage of the ear and autologous P1 cells were injected lateral of one vocal cord into the paraglottic space suspended in a long-term-stable fibrin gel. Histologic evaluation was performed after 2, 4, 12, and 24 weeks. RESULTS: During 12-week pellet culture, we found extracellular matrix formation and weight-stable cartilage of mature appearance. In-vivo, mature cartilage was found in two larynxes (n = 6) at 4 weeks, in four (n = 6) at 12 weeks, and in five (n = 6) at 24 weeks mostly located in the paraglottic space and sometimes with spurs into the vocalis muscle. Surrounding tissue was often infiltrated with inflammatory cells. Material tended to dislocate through the cricothyroid space into the extraglottic surrounding tissue. CONCLUSIONS: A cell-based approach with chondrocytes for permanent vocal-fold augmentation has not previously been reported. We have achieved the formation of structurally mature cartilage in the paraglottic space, but this is accompanied by difficulties with dislocated material, deformation of the augmentation, and inflammation. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E1624-E1632, 2021.

MicroRNA-1 promotes cartilage matrix synthesis and regulates chondrocyte differentiation via post-transcriptional suppression of Ihh expression.

Indian hedgehog signaling molecule (Ihh) is known to play critical roles in chondrogenesis and cartilage development. However, it remains largely unknown how Ihh is regulated during the process. Previous studies suggest that Ihh plays an important regulatory role in the growth and development of articular cartilage, but whether it is regulated by miRNAs is unclear. The present study aimed to investigate the effects of miR­1 on chondrocyte differentiation and matrix synthesis, and to determine whether miR­1 can regulate the Ihh signaling pathway. In the present study, the expression level of miR­1 was altered via transfection of the miR­1 mimic or inhibitor in mouse thorax chondrocytes, and the impact on chondrocyte phenotypes and Ihh expression was examined. Overexpression of miR­1 promoted the expression of the matrix synthesis­associated molecules collagen (Col)­II and aggrecan, two key components in cartilage matrix. Conversely, overexpression of miR­1 significantly downregulated the expression of chondrocyte differentiation markers Col­X and matrix metallopeptidase 13. Moreover, overexpression of miR­1 dose­dependently inhibited endogenous Ihh expression, and an association was observed between miR­1 and Ihh expression. The 3' untranslated region (UTR) of Ihh from various species contains two miR­1 binding sites. Luciferase reporter assays indicated that miR­1 post­transcriptionally suppressed Ihh expression, which was dependent on the binding of miR­1 to one of the two putative binding sites of the Ihh 3'UTR. Furthermore, via inhibition of Ihh expression, miR­1 decreased the expression of molecules downstream of Ihh in the Hedgehog signaling pathway in mouse thorax chondrocytes. This study provided new insight into the molecular mechanisms of miR­1 in regulating chondrocyte phenotypes via targeting the Ihh pathway.

Optimization of culture media to enhance the growth of tissue engineered cartilage.

Tissue engineering is a promising option for cartilage repair. However, several hurdles still need to be overcome to develop functional tissue constructs suitable for implantation. One of the most common challenges is the general low capacity of chondrocytes to synthesize cartilage-specific extracellular matrix (ECM). While different approaches have been explored to improve the biosynthetic response of chondrocytes, several studies have demonstrated that the nutritional environment (e.g., glucose concentration and media volume) can have a profound effect on ECM synthesis. Thus, the purpose of this study was to optimize the formulation of cell culture media to upregulate the accumulation of cartilaginous ECM constituents (i.e., proteoglycans and collagen) by chondrocytes in 3D culture. Using response surface methodology, four different media factors (basal media, media volume, glucose, and glutamine) were first screened to determine optimal media formulations. Constructs were then cultured under candidate optimal media formulations for 4 weeks and analyzed for their biochemical and structural properties. Interestingly, the maximal accumulation of proteoglycans and collagen appeared to be elicited by different media formulations. Most notably, proteoglycan accumulation was favored by high volume, low glucose-containing DMEM/F12 (1:1) media whereas collagen accumulation was favored by high volume, high glucose-containing F12 media. While high glutamine-containing media elicited increased DNA content, glutamine concentration had no apparent effect on ECM accumulation. Therefore, optimizing the nutritional environment during chondrocyte culture appears to be a promising, straight-forward approach to improve cartilaginous tissue formation. Future work will investigate the combined effects of the nutritional environment and external stimuli.

Bionic composite hydrogel with a hybrid covalent/noncovalent network promoting phenotypic maintenance of hyaline cartilage.

The injectable composite hydrogel based on collagen and hyaluronic acid provided a bionic three-dimensional microenvironment and mimetic natural extracellular matrix (ECM) for the growth of cells in vivo and has been widely researched and developed for cartilage tissue engineering. Here, a novel injectable bionic hydrogel with hybrid covalent/noncovalent network derived from covalent conjugation of HA-SH and noncovalent supramolecular self-assembly of BPAA-AFF-OH short peptide was fabricated to overcome the collagen immunogenicity of animal origin and effectively maintain its biological function. Moreover, through optimizing the network structure and polymer composition, the bionic HS5FFAB5 hydrogel presented a reliable mechanical strength which depended on the highly integrated fiber structure between HA-SH and FFAB-AFF-OH molecules. The results in vitro and in vivo proved that HA-SH could provide a fundamental frame structure, while the supramolecular hydrogels could reinforce this structure via hydrogen bonds and hydrophilic/hydrophobic interactions, and endow bionic hydrogels with more abundant cell adhesion sites. The bionic composite hydrogel could improve the cell adhesion and proliferation when compared to HA-SH hydrogel, and enhanced chondrogenic related gene expression and matrix secretion by three-dimensional co-cultured in vitro and subcutaneous implantation in vivo, which further promoted phenotypic maintenance of hyaline cartilage. This bionic hydrogel with a hybrid covalent/noncovalent network is supposed to have potential application prospects in cartilage regeneration.

Spatiotemporal neocartilage growth in matrix-metalloproteinase-sensitive poly(ethylene glycol) hydrogels under dynamic compressive loading: an experimental and computational approach.

Enzyme-sensitive hydrogels containing encapsulated chondrocytes are a promising platform for cartilage tissue engineering. However, the growth of neotissue is closely coupled to the degradation of the hydrogel and is further complicated due to the encapsulated cells serving as the enzyme source for hydrogel degradation. To better understand these coupled processes, this study combined experimental and computational methods to analyze the transition from hydrogel to neotissue in a biomimetic MMP-sensitive poly(ethylene glycol) (PEG) hydrogel with encapsulated chondrocytes. A physics-based computational model that describes spatial heterogeneities in cell distribution was used. Experimentally, cell-laden hydrogels were cultured for six weeks under free swelling or subjected daily to one-hour of dynamic compressive loading. Extracellular matrix (ECM) synthesis rates were used as model inputs, and the model was fit to the experimentally determined construct modulus over time for the free swelling condition. Experimentally, ECM accumulation comprising collagen II and aggrecan increased over time concomitant with hydrogel degradation observed by a loss in PEG. Simulations demonstrated rapid degradation in regions of high cell density (i.e., cell clusters) reaching complete degradation by day 13, which facilitated localized ECM growth. Regions of low cell density degraded more slowly, had limited ECM, and led to the decrease in construct modulus during the first two weeks. The primary difference between the two culture environments was greater ECM accumulation in the clusters under free swelling, which facilitated a faster recovery in construct modulus. By 6 weeks the compressive modulus increased 2.5-fold to 107 kPa under free swelling, but dropped 1.6-fold to 26 kPa under loading. In summary, this biomimetic MMP-sensitive hydrogel supports neocartilage growth by facilitating rapid ECM growth within cell clusters, which was followed by slower growth in the rest of the hydrogel. Subtle temporal differences in hydrogel degradation and ECM accumulation, however, had a significant impact on the evolving mechanical properties.

Kartogenin enhances the therapeutic effect of bone marrow mesenchymal stem cells derived exosomes in cartilage repair.

The effectiveness of mesenchymal stem cells (MSC) in the treatment of cartilage diseases has been demonstrated to be attributed to the paracrine mechanisms, especially the mediation of exosomes. But the exosomes derived from unsynchronized MSCs may be nonhomogeneous and the therapeutic effect varies between samples. Aim: To produce homogeneous and more effective exosomes for the regeneration of cartilage. Materials & methods: In this study we produced specific exosomes from bone marrow MSCs (BMSC) through kartogenin (KGN) preconditioning and investigated their performance in either in vitro or in vivo experiments. Results & conclusion: The exosomes derived from KGN-preconditioned BMSCs (KGN-BMSC-Exos) performed more effectively than the exosomes derived from BMSCs (BMSC-Exos). KGN preconditioning endowed BMSC-Exos with stronger chondral matrix formation and less degradation.

Three-dimensional cartilage tissue regeneration system harnessing goblet-shaped microwells containing biocompatible hydrogel.

Differentiation of stem cells into chondrocytes has been studied for the engineering of cartilage tissue. However, stem cells cultured two-dimensionally have limited ability to differentiate into chondrocytes, which led to the development of three-dimensional culture systems. A recently developed microtechnological method uses microwells as a tool to form uniformly sized spheroids. In this study, we fabricated an array (10 × 10) of goblet-shaped microwells based on polydimethylsiloxane for spheroid culture. A central processing unit (CPU) was used to form holes, and metallic beads were used to form hemispherical microwell geometry. The holes were filled with Pluronic F-127 to prevent cells from sinking through the holes and allowing the cells to form spheroids. Viability and chondrogenic differentiation of human adipose-derived stem cells were assessed. The fabrication method using a micro-pin mold and metallic beads is easy and cost-effective. Our three-dimensional spheroid culture system optimizes the efficient differentiation of cells and has various applications, such as drug delivery, cell therapy, and tissue engineering.

MiR-9 facilitates cartilage regeneration of osteoarthritis in rabbits through regulating Notch signaling pathway.

OBJECTIVE: To explore the effect of micro ribonucleic acid (miR-9) on cartilage regeneration of osteoarthritis in rabbits through the regulation of the Notch signaling pathway. MATERIALS AND METHODS: A total of 30 specific pathogen-free Sprague-Dawley rabbits were randomly divided into control group (healthy rabbits, n=10), model group (osteoarthritis model, n=10) and miR-9 group (osteoarthritis model + miR-9 interference, n=10). The degeneration degree of rabbit knee articular cartilage in three groups was assessed through the Mankin's score. The morphology of cartilage tissues was observed under an optical microscope. Expressions of the Notch1, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) proteins, and collagen II (CII) in chondrocytes were detected via the immunohistochemical assay. RESULTS: In rabbits of control group, the articular cartilage had a smooth surface and complete structure, and the cells were arranged orderly with a clear tidal line. A large number of articular chondrocytes died in model group, while it was improved in miR-9 group. The Mankin's score was 3.52±0.79 points in control group, 6.73±0.95 points in model group, and 5.37±0.61 points in miR-9 group, showing significant differences among the three groups (p<0.05). Results of immunohistochemistry showed that the protein expressions of Notch1 and Bax were higher in model group, but lower in control group and miR-9 group (p<0.05). The protein expression of Bcl-2 was lower in model group but was upregulated in control group and miR-9 group (p<0.05). The results of Reverse Transcription-Polymerase Chain Reaction (RT-PCR) revealed that the expressions of Notch1 and Bax in control group were lower than those in model group and miR-9 group (p<0.05), while the expression of Bcl-2 in model group was lower than that in control group and miR-9 group (p<0.05). According to the results of immunohistochemistry, the CII optical density (OD) value was 0.18±0.013, 0.25±0.05 and 0.22±0.009 in control group, model group, and miR-9 group, respectively. It could be seen that the CII OD value was the highest in model group, indicating that the CII expression in articular cartilage in osteoarthritis was negatively correlated with the severity of osteoarthritis. CONCLUSIONS: MiR-9, through the down-regulation of the expressions of Notch and Bax, can activate the Bcl-2 to promote the differentiation and regeneration of chondrocytes. It can facilitate the cartilage regeneration of osteoarthritis in rabbits through the mediation of the CII expression.

Expression of IFT140 During Bone Development.

Primary cilia, hair-like organelles projecting from the surface of cells, are critical for sensing extracellular stimuli and transmitting molecular signals that regulate cell functions. During bone development, cell cilia are found in several types of cells, but their roles require further investigation. Intraflagellar transport (IFT) is essential for the formation and maintenance of most eukaryotic cilia. IFT140 is a core protein of the IFT-A complex. Mutations in IFT140 have been associated with cases of skeletal ciliopathies. In this study, we examined the expression of IFT140 during bone development. The results showed that, compared with many soft tissues, Ift140 (mRNA level) was highly expressed in bone. Moreover, its expression level was downregulated in the long bones of murine osteoporosis models. At the histological level, IFT140 was characteristically expressed in osteoblasts and chondrocytes at representative stages of bone development, and its expression level in these two types of cells was observed in two waves. These findings suggest that IFT140 may play an important role in the process of chondrogenic and osteogenic differentiation during bone development.

Analysis of Proteoglycan Synthesis and Secretion in Cell Culture Systems.

Experimental protocols for the synthesis and secretion of proteoglycans in cell culture models are important to study specific biosynthetic steps or disorders in which a defect in proteoglycans is expected. We describe a method using 35S-sulfate to metabolically label newly synthesized proteoglycans from cell cultures in order to measure proteoglycan synthesis and secretion. The method is set up for fibroblast and chondrocyte cultures, but can be extended to other cell types.

Light Absorptive Properties of Articular Cartilage, ECM Molecules, Synovial Fluid, and Photoinitiators as Potential Barriers to Light-Initiated Polymer Scaffolding Procedures.

OBJECTIVE: Many in vivo procedures to repair chondral defects use ultraviolet (UV)-photoinitiated in situ polymerization within the cartilage matrix. Chemical species that absorb UV light might reduce the effectiveness of these procedures by acting as light absorption barriers. This study evaluated whether any of the individual native biochemical components in cartilage and synovial fluid interfered with the absorption of light by common scaffolding photosensitizers. MATERIALS: UV-visible spectroscopy was performed on each major component of cartilage in solution, on bovine synovial fluid, and on four photosensitizers, riboflavin, Irgacure 2959, quinine, and riboflavin-5'-phosphate. Molar extinction and absorption coefficients were calculated at wavelengths of maximum absorbance and 365 nm. Intact articular cartilage was also examined. RESULTS: The individual major biochemical components of cartilage, Irgacure 2959, and quinine did not exhibit a significant absorption at 365 nm. Riboflavin and riboflavin-5'-phosphate were more effectual light absorbers at 365 nm, compared with the individual native species. Intact cartilage absorbed a significantly greater amount of UV light in comparison with the native species. CONCLUSION: Our results indicate that none of the individual native species in cartilage will interfere with the absorption of UV light at 365 nm by these commonly used photoinitiators. Intact cartilage slices exhibited significant light absorption at 365 nm, while also having distinct absorbance peaks at wavelengths less than 300 nm. Determining the UV absorptive properties of the biomolecules native to articular cartilage and synovial fluid will aid in optimizing scaffolding procedures to ensure sufficient scaffold polymerization at a minimum UV intensity.

Effect of gelatin source and photoinitiator type on chondrocyte redifferentiation in gelatin methacryloyl-based tissue-engineered cartilage constructs.

Gelatin methacryloyl (GelMA) hydrogels are a mechanically and biochemically tuneable biomaterial, facilitating chondrocyte culture for tissue engineering applications. However, a lack of characterisation and standardisation of fabrication methodologies for GelMA restricts its utilisation in surgical interventions for articular cartilage repair. The purpose of this study was to determine the effects of gelatin source and photoinitiator type on the redifferentiation capacity of monolayer-expanded human articular chondrocytes encapsulated in GelMA/hyaluronic acid methacrylate (HAMA) hydrogels. Chondrocyte-laden hydrogels reinforced with multiphasic melt-electrowritten (MEW) medical grade polycaprolactone (mPCL) microfibre scaffolds were prepared using bovine (B) or porcine-derived (P) GelMA, and photocrosslinked with either lithium acylphosphinate (LAP) and visible light (405 nm) or Irgacure 2959 (IC) and UV light (365 nm). Bulk physical properties, cell viability and biochemical features of hydrogel constructs were measured at day 1 and day 28 of chondrogenic cell culture. The compressive moduli of all groups increased after 28 days of cell culture, with B-IC displaying similar compressive strength to that of native articular cartilage (∼1.5 MPa). Compressive moduli correlated with an increase in total glycosaminoglycan (GAG) content for each group. Gene expression analysis revealed upregulation of chondrogenic marker genes in IC-crosslinked groups, whilst dedifferentiation gene markers were upregulated in LAP-crosslinked groups. mPCL reinforcement correlated with increased accumulation of collagen I and II in B-IC, B-LAP and P-IC groups compared to non-reinforced hydrogels. A reduction in cell viability was noted in all samples at day 28, potentially due to the generation of free radicals during photocrosslinking or cytotoxicity of the photoinitiators. In summary, hydrogel constructs prepared with bovine-derived GelMA and photocrosslinked with Irgacure 2959 and 365 nm light displayed properties most similar to native articular cartilage after 28 days of cell culture. The differences in biological response between investigated construct types emphasises the necessity to characterise and standardise biomaterials before translating in vitro tissue engineering research to preclinical applications for articular cartilage injuries.

A Complex Relationship between Visfatin and Resistin and microRNA: An In Vitro Study on Human Chondrocyte Cultures.

Growing evidence indicates the important role of adipokines and microRNA (miRNA) in osteoarthritis (OA) pathogenesis. The purpose of the present study was to investigate the effect of visfatin and resistin on some miRNA (34a, 140, 146a, 155, 181a, let-7e), metalloproteinases (MMPs), and collagen type II alpha 1 chain (Col2a1) in human OA chondrocytes and in the T/C-28a2 cell line. The implication of nuclear factor (NF)-κB in response to adipokines was also assessed. Chondrocytes were stimulated with visfatin (5 or 10 µg/mL) and resistin (50 or 100 ng/mL) with or without NF-κB inhibitor (BAY-11-7082, 1 µM) for 24 h. Viability and apoptosis were detected by MMT and cytometry, miRNA, MMP-1, MMP-13, and Col2a1 by qRT-PCR and NF-κB activation by immunofluorescence. Visfatin and resistin significantly reduced viability, induced apoptosis, increased miR-34a, miR-155, miR-181a, and miR-let7e, and reduced miR-140 and miR-146a gene expression in OA chondrocytes. MMP-1, MMP-13, and Col2a1 were significantly modulated by treatment of OA chondrocytes with adipokines. Visfatin and resistin significantly increased NF-κB activation, while the co-treatment with BAY11-7082 did not change MMPs or Col2a1 levels beyond that caused by single treatment. Visfatin and resistin regulate the expression levels of some miRNA involved in OA pathogenesis and exert catabolic functions in chondrocytes via the NF-κB pathway. These data confirm the complex relationship between adipokines and miRNA.

Exosomes derived from mature chondrocytes facilitate subcutaneous stable ectopic chondrogenesis of cartilage progenitor cells.

BACKGROUND: Developing cartilage constructed with the appropriate matrix composition and persistent chondrogenesis remains an enduring challenge in cartilage defects. Cartilage progenitor cell (CPC)-based tissue engineering has attracted recent attention because of its strong chondrogenic differentiation capacity. However, due to the lack of a suitable chondrogenic niche, the clinical application of CPC-regenerated cartilage in the subcutaneous environment remains a challenge. In this study, exosomes derived from chondrocytes (CC-Exos) were used to provide the CPC constructs with a cartilage signal in subcutaneous environments for efficient ectopic cartilage regeneration. METHODS: Rabbit CPC-alginate constructs were prepared and implanted subcutaneously in nude mice. CC-Exos were injected into the constructs at the same dose (30 µg exosomes per 100 µL injection) after surgery and thereafter weekly for a period of 12 weeks. Exosomes derived from bone mesenchymal stem cells (BMSC-Exos) were used as the positive control. The mice in the negative control were administered with the same volume of PBS. At 4 and 12 weeks after implantation, the potential of CC-Exos and BMSC-Exos to promote chondrogenesis and stability of cartilage tissue in a subcutaneous environment were analyzed by histology, immunostaining, and protein analysis. The influences of BMSC-Exos and CC-Exos on chondrogenesis and angiogenic characteristics in vitro were assessed via coculturing with CPCs and human umbilical vein endothelial cells. RESULTS: The CC-Exos injection increased collagen deposition and minimized vascular ingrowth in engineered constructs, which efficiently and reproducibly developed into cartilage. The generated cartilage was phenotypically stable with minimal hypertrophy and vessel ingrowth up to 12 weeks, while the cartilage formed with BMSC-Exos was characterized by hypertrophic differentiation accompanied by vascular ingrowth. In vitro experiments indicated that CC-Exos stimulated CPCs proliferation and increased expression of chondrogenesis markers while inhibiting angiogenesis. CONCLUSIONS: These findings suggest that the novel CC-Exos provides the preferable niche in directing stable ectopic chondrogenesis of CPCs. The use of CC-Exos may represent an off-the-shelf and cell-free therapeutic approach for promoting cartilage regeneration in the subcutaneous environment.

Cartilaginous extracellular matrix derived from decellularized chondrocyte sheets for the reconstruction of osteochondral defects in rabbits.

Cartilaginous extracellular matrix (ECM) materials derived from decellularized native articular cartilage are widely used in cartilage regeneration. However, it is difficult for endogenous cells to migrate into ECM derived from native cartilage owing to its nonporous structure and dense nature. Moreover, current decellularization approaches frequently lead to architectural breakdown and potential loss of surface composition of ECM. To solve this problem, we aimed to establish a novel biological ECM scaffold from chondrocyte sheets for cartilage regeneration. We cultured chondrocytes harvested from the auricular cartilage of 4-week-old New Zealand rabbits and enabled them to form cell sheets. These sheets were decellularized using sodium dodecyl sulfate (SDS) with three different concentrations, namely, 1%, 5%, and 10%, followed by 1% Triton X-100 and deoxyribonuclease enzyme solution. In vitro microstructural examination and mechanical tests demonstrated that 1% SDS not only removed chondrocytes completely but also maintained the native architecture and composition of ECM, thus avoiding the use of high-concentration SDS. Application of decellularized chondrocyte sheets for osteochondral defects in rabbits resulted in substantial host remodeling and variant regeneration of osteochondral tissues. One percent SDS-treated decellularized chondrocyte sheets contributed to the superior reconstruction of osteochondral defects as compared with 5% and 10% SDS groups, which includes vascularized subchondral bone, articular cartilage with adequate thickness, and integration with host tissues. Furthermore, ECM from 1% SDS significantly increased the migrating potential of bone marrow mesenchymal stem cells (BMSCs) in vitro. RT-PCR and western blot also revealed that ECM increased the expression of SOX-9 in BMSCs, whereas it decreased COL-X expression. In conclusion, our results suggested that the chondrocyte sheets decellularized with 1% SDS preserved the integrity and bioactivity, which favored cell recruitment and enabled osteochondral regeneration in the knee joints of rabbits, thus offering a promising approach for articular cartilage reconstruction without cell transplantation. STATEMENT OF SIGNIFICANCE: Although biological extracellular matrix (ECM) derived from decellularized native cartilage has been widely used in cartilage regeneration, it is difficult for endogenous cells to migrate into ECM owing to its dense nature. Moreover, current decellularization approaches lead to architectural breakdown of ECM. This study established a novel biological ECM from decellularized chondrocyte sheets for cartilage regeneration. Our results suggested that cartilaginous ECM favored cell recruitment and enabled osteochondral regeneration in rabbits, thus offering a promising approach for articular cartilage reconstruction without cell transplantation. SDS 1% adequately decellularized the chondrocytes in cell sheets, whereas it maintained the native architecture and composition of ECM, thereby avoiding the use of high-concentration SDS and providing a new way to acquire cartilaginous ECM.

The role of leptin in osteoarthritis.

BACKGROUND: The pathogenesis of osteoarthritis (OA) is not clear; leptin may be related to its pathogenesis. METHODS: We reviewed articles on leptin in OA, chondrocytes, and in vitro experiments. It is concluded that leptin may lead to OA via some signaling pathways. At the same time, the concentration of leptin in vitro experiments and OA/rheumatoid arthritis (RA) patients was summarized. RESULTS: Leptin levels in serum and synovial fluid of OA/RA patients were higher than normal person. In the condition of infection and immunity, serum leptin levels in the peripheral blood significantly increase. Because of the close relationship between obesity, leptin, and OA, it is crucial to study the effects of weight loss and exercise intervention on serum leptin levels to improve the symptoms of OA patients. CONCLUSION: Treatment for leptin-increased obesity may be a treatment for OA. The role of leptin in OA cannot be ignored and needs to be further studied.