Analysis of a Chinese pedigree affected with familial short stature due to 15q25.3q26.1 deletion involving the ACAN gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze the genetic etiology of a Chinese pedigree affected with short stature.@*METHODS@#A child with familial short stature (FSS) who had presented at the Ningbo Women and Children's Hospital in July 2020 and his parents and paternal and maternal grandparents were selected as the study subject. Clinical data of the pedigree was collected, and the proband was subjected to routine growth and development assessment. Peripheral blood samples were collected. The proband was subjected to whole exome sequencing (WES), and the proband, his parents and grandparents were subjected to chromosomal microarray analysis (CMA).@*RESULTS@#The height of the proband and his father was 87.7cm (-3 s) and 152 cm (-3.39 s) respectively. Both of them were found to harbor a 15q25.3-q26.1 microdeletion, which has encompassed the whole of the ACAN gene which is closely associated with short stature. The CMA results of his mother and grandparents were all negative, and above deletion has not been included in population database and related literature, and was rated as pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). After 14 months of rhGH treatment, the height of the proband has increased to 98.5 cm (-2.07 s).@*CONCLUSION@#The 15q25.3-q26.1 microdeletion probably underlay the FSS, in this pedigree. Short-term rhGH treatment can effectively improve the height of the affected individuals.

Kindlin-2 loss in condylar chondrocytes causes spontaneous osteoarthritic lesions in the temporomandibular joint in mice / 国际口腔科学杂志·英文版

The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint (TMJ) osteoarthritis (OA); however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4 (Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2 and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore, Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.

Clinical and genetic analysis of five Chinese pedigrees affected with short stature due to variants of ACAN gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical and genetic characteristics of five Chinese pedigrees affected with short stature.@*METHODS@#A retrospective analysis was carried out for the clinical data and results of genetic testing for the probands. A literature search was also conducted.@*RESULTS@#The five probands have all featured short stature with a family history. Genetic testing has revealed that they have harbored variants of the ACAN gene, including p.Val2042Argfs*6, p.Val1597del, c.630-1G>A, c.23delT and c.2026+1G>A(previously reported).@*CONCLUSION@#Except for short stature, children harboring heterozygous variants of the ACAN gene may have no involvement of other systems. Some of these children may response to short-term growth hormone treatment.

Hyaluronic acid has chondroprotective and joint-preserving effects on LPS-induced synovitis in horses

The intra-articular use of hyaluronic acid (HA) for the treatment of synovitis and osteoarthritis is still controversial. As a consequence, corticosteroids remain the most frequently employed therapeutic agents, despite their potential systemic and local deleterious effects. This study examined the anti-inflammatory, antioxidant, and chondroprotective activities of low and high molecular weight hyaluronic acid (LMW-HA and HMW-HA) on lipopolysaccharide (LPS)-induced synovitis in horses compared to triamcinolone acetonide (TA). LPS was injected in the metacarpophalangeal joints, which were treated intra-articularly with either TA (as control) or LMW-HA or HMW-HA. Joint clinical evaluation and synovial fluid (SF) analysis were performed at 0, 8, 24, and 48 h. The white blood cell counts (WBC), prostaglandin E2 (PGE2), interleukin (IL)-1, IL-6, IL-10, tumor necrosis factor-α, chondroitin sulfate (CS) and HA concentrations, oxidative burst, and HA molecular weights were measured. TA reduced the lameness, swelling, and PGE2 release but increased the SF CS concentrations enormously at 24h and 48h, and decreased the SF HA modal molecular weight. These results indicate the breakdown of articular cartilage aggrecan and SF HA. In contrast, LMW-HA and HMW-HA were less effective in reducing the inflammation symptoms, but preserved the joints because only a modest increase in CS occurred at 24 h, decreasing at 48 h, and the SF HA was maintained. The HA-treatment also had anti-inflammatory actions, and LMW-HA was the most effective in reducing the release of cytokine. In summary, the HA treatment inhibited efficiently the digestion of cartilage proteoglycans and SF HA breakdown.

Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure

BACKGROUND: Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro. METHODS: In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5 MPa with a frequency of 0.5 Hz and 4 h per day for 1 week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively. RESULTS: Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-β1 growth factor (10 ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-β1, respectively. CONCLUSION: The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.

LncRNA MALAT1/MiR-145 Adjusts IL-1β-Induced Chondrocytes Viability and Cartilage Matrix Degradation by Regulating ADAMTS5 in Human Osteoarthritis

PURPOSE: Accumulating evidence suggests that microRNA-145 (miR-145) plays an important role in osteoarthritis (OA), which is a chronic progressive joint disease. Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes metastasis in cancers and functions as a sponge for miR-145. However, the role of MALAT1/miR-145 in OA pathogenesis has not yet been elucidated. MATERIALS AND METHODS: The expression of MALAT1 and miR-145 was examined by quantitative real-time PCR; the interaction between miR-145, MALAT1 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5 was verified by luciferase reporter assay. Correlations among MALAT1, miR-145, and ADAMTS5 were analyzed by Spearman rank analysis. Chondrocytes viability and cartilage extracellular matrix (ECM) degradation were investigated with cell viability assay and Western blotting analyzing expression of ADAMTS5, collagen type 2 alpha 1 (COL2A1), aggrecan (ACAN), and cartilage oligomeric matrix protein (COMP). RESULTS: MALAT1 was upregulated, and miR-145 was downregulated in OA samples and IL-1β-induced chondrocytes. Mechanically, miR-145 could directly bind to MALAT1 and ADAMTS5. Moreover, miR-145 expression was negatively correlated with MALAT1 and ADAMTS5 expression in OA patients, whereas MALAT1 and ADAMTS5 expression was positively correlated. Functionally, overexpression of MALAT1 inhibited chondrocyte viability and promoted cartilage ECM degradation in IL-1β-induced chondrocytes. In support thereof, MALAT1 silencing and miR-145 upregulation exerted the opposite effect in IL-1β-induced chondrocytes. Moreover, the effect of MALAT1 was counteracted by miR-145 upregulation, and ADAMTS5 restoration could abate miR-145 effects. CONCLUSION: An MALAT1/miR-145 axis contributes to ECM degradation in IL-1β-induced chondrocytes through targeting ADAMTS5, suggesting that MALAT1/miR-145/ADAMTS5 signaling may underlie human OA pathogenesis.

Evaluation of Polycaprolactone-Associated Human Nasal Chondrocytes as a Therapeutic Agent for Cartilage Repair

BACKGROUND: In this study, we manufactured a complex of human nasal septal cartilage (hNC) with polycaprolactone (PCL) for transplantation into cartilaginous skeletal defects and evaluated their characteristics.METHODS: Nasal septum tissue was obtained from five patients aged ≥ 20 years who were undergoing septoplasty. hNCs were isolated and subcultured for three passages in vitro. To formulate the cell–PCL complex, we used type I collagen as an adhesive between chondrocyte and PCL. Immunofluorescence staining, cell viability and growth in the hNC–PCL complex, and mycoplasma contamination were assessed.RESULTS: hNCs in PCL showed viability ≥ 70% and remained at these levels for 9 h of incubation at 4 ℃. Immunostaining of the hNC–PCL complex also showed high expression levels of chondrocyte-specific protein, COL2A1, SOX9, and aggrecan during 24 h of clinically applicable conditions.CONCLUSION: The hNC–PCL complex may be a valuable therapeutic agent for implantation into injured cartilage tissue, and can be used clinically to repair cartilaginous skeletal defects. From a clinical perspective, it is important to set the short duration of the implantation process to achieve effective functional implantation.

Ginsenoside Rd inhibits IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1β, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1β by suppressing the increase in IL-1β, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1β-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1β to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Activation of β-catenin signaling in aggrecan-expressing cells in temporomandibular joint causes osteoarthritis-like defects / 国际口腔科学杂志·英文版

β-Catenin plays a critical role in cartilage formation and development. To further understand the role of β-catenin in osteoarthritis (OA) development in temporomandibular joint (TMJ), we have generated β-catenin conditional activation mice (β-cat(ex3) ) by breeding Agc1-CreER mice with β-catenin mice. Results of histologic analysis showed the progressive TMJ defects in 3- and 6-month-old β-cat(ex3) mice (tamoxifen induction was performed at 2 weeks of age), including decreased chondrocyte numbers in the superficial layer associated with less Alcian blue staining, increased numbers of hypertrophic chondrocytes in deep layers, and rough articular surface. Compared to the TMJ phenotype of β-cat(ex3) mice, β-cat(ex3) mice showed much severe morphological defects in the superficial layer of TMJ. This may reflect that Agc1-CreER mice could efficiently target cells in the superficial layer of TMJ. Results of immunostaining showed significantly increased expression of MMP13, Col-X, Adamts4, and Adamts5 in TMJ of β-cat(ex3) mice. Results of proliferating cell nuclear antigen (PCNA), Ki67, and terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) staining further demonstrated that cell proliferation was decreased and cell apoptosis was increased in condylar cartilage of β-cat(ex3) mice. Our findings indicate that abnormal upregulation of β-catenin in TMJ leads to defects assembling to OA-like phenotype, further demonstrating that β-catenin plays a critical role in TMJ pathogenesis.

In Vitro Anti-Inflammation and Chondrogenic Differentiation Effects of Inclusion Nanocomplexes of Hyaluronic Acid-Beta Cyclodextrin and Simvastatin

The aim of this study was to prepare inclusion nanocomplexes of hyaluronic acid-β-cyclodextrin and simvastatin (HA-β-CD/SIM) and evaluate in vitro anti-inflammation effects on lipopolysaccharide (LPS)-activated synoviocytes and chondrogenic differentiation effects on rat adipose-derived stem cells (rADSCs). The β-CD moieties in HA-β-CD could incorporate SIM to form HA-β-CD/SIM nanocomplexes with diameters of 297–350 nm. HA-β-CD/SIM resulted in long-term release of SIM from the nanocomplexes for up to 63 days in a sustained manner. In vitro studies revealed that HA-β-CD/SIM nanocomplexes were able to effectively and dose-dependently suppress the mRNA expression levels of proinflammatory markers such as matrix metallopeptidase-3 (MMP-3), MMP-13, cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) in LPS-stimulated synoviocytes. HA-β-CD/SIM-treated rADSCs significantly and dose-dependently enhanced mRNA expressions of aggrecan, collagen type II (COL2A1), and collagen type X (COL10A1), implying that HA-β-CD/SIM greatly induced the chondrogenic differentiation of rADSCs. Conclusively, HA-β-CD/SIM nanocomplexes will be a promising therapeutic material to alleviate inflammation as well as promote chondrogenesis.

Micromass cultures are effective for differentiation of human amniotic fluid stem cells into chondrocytes

OBJECTIVES: Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days. METHODS: Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee. RESULTS: SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected. CONCLUSION: We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells.

Identification of a novel heterozygous mutation of ACAN in a Korean family with proportionate short stature

Aggrecan is a proteoglycan in the extracellular matrix of growth plate and cartilaginous tissues. Aggrecanopathy has been reported as a genetic cause not only for severe skeletal dysplasia but also for autosomal dominant short stature with normal to advanced bone age. We report a novel heterozygous mutation of ACAN in a Korean family with proportionate short stature identified through targeted exome sequencing. We present a girl of 4 years and 9 months with a family history of short stature over three generations. The paternal grandmother is 143 cm tall (−3.8 as a Korean standard deviation score [SDS]), the father 155 cm (−3.4 SDS), and the index case 96.2 cm (−2.9 SDS). Evaluation for short stature showed normal growth hormone (GH) peaks in the GH provocation test and a mild delayed bone age for chronological age. This subject had clinical characteristics including a triangular face, flat nasal bridge, prognathia, blue sclerae, and brittle teeth. The targeted exome sequencing was applied to detect autosomal dominant growth palate disorder. The novel variant c.910G>A (p.Asp304Asn) in ACAN was identified and this variant was found in the subject's father using Sanger sequencing. This is the first case of Korean familial short stature due to ACAN mutation. ACAN should be considered for proportionate idiopathic short stature, especially in cases of familial short stature.

The minor collagens in articular cartilage

Articular cartilage is a connective tissue consisting of a specialized extracellular matrix (ECM) that dominates the bulk of its wet and dry weight. Type II collagen and aggrecan are the main ECM proteins in cartilage. However, little attention has been paid to less abundant molecular components, especially minor collagens, including type IV, VI, IX, X, XI, XII, XIII, and XIV, etc. Although accounting for only a small fraction of the mature matrix, these minor collagens not only play essential structural roles in the mechanical properties, organization, and shape of articular cartilage, but also fulfil specific biological functions. Genetic studies of these minor collagens have revealed that they are associated with multiple connective tissue diseases, especially degenerative joint disease. The progressive destruction of cartilage involves the degradation of matrix constituents including these minor collagens. The generation and release of fragmented molecules could generate novel biochemical markers with the capacity to monitor disease progression, facilitate drug development and add to the existing toolbox for in vitro studies, preclinical research and clinical trials.

Platelet Rich Plasma and Culture Configuration Affect the Matrix Forming Phenotype of Bone Marrow Stromal Cells

We aim to examine the influence of platelet rich plasma (PRP) and spatial cues in cartilage/bone matrix forming proteins, and to evaluate the mitotic and chemotactic potential of PRP on human mesenchymal stem cells (hMSCs). Directed cell migration towards PRP gradients was assessed in chemotactic chambers, and recorded by time-lapse microscopy. hMSCs cultured in three-dimensional (3D) scaffolds were visualized by scanning electron microscopy; Hoechst dye was used to confirm cell confluence in 3D-constructs and monolayers before experimental treatment. MSCs were treated with 10% PRP lysate or 10% PRP lysate supplemented with TGF-β-based differentiation medium. The expression of cartilage (COL2A1, Sox9, ACAN, COMP), and bone (COL1A1, VEGF, COL10A1, Runx2) fundamental genes was assessed by real time PCR in monolayers and 3D-constructs. PRP had mitotic (p <.001), and chemotactic effect on hMSCs, Ralyleigh test p = 1.02E - 10. Two and three-week exposure of MSCs to PRP secretome in 3Dconstructs or monolayers decreased Sox9 expression (p <0.001 and p = 0.050) and COL2A1, (p = 0.011 and p = 0.019). MSCs in monolayers exposed to PRP showed increased ACAN (p = 0.050) and COMP (p <0.001). Adding TGF-β-based differentiation medium to PRP increased COMP, and COL2A1 expression at gene and protein level, but merely in 3D-constructs, p <0.001. TGF-β addition to monolayers reduced Sox9 (p <0.001), aggrecan (p = 0.004), and VEGF (p = 0.004). Cells exposed to PRP showed no changes in hypertrophy associated genes in either monolayers or 3Dconstructs. Our study suggests hMSCs have high-degree of plasticity having the potential to change their matrix-forming phenotype when exposed to PRP and according to spatial configuration.

Effects of Trichostatin A on the Chondrogenesis from Human Mesenchymal Stem Cells

Histone deacetylase inhibitors (HDACi) are a class of compounds that suppress the function of histone deacetylases (HDACs). This study was performed to examine the effects of Trichostatin A (TSA), a typical HDACi, on chondrogenesis of human bone marrow mesenchymal stem cells (hBMMSCs) and related molecular pathways. After evaluating the concentration for cytotoxicity and HDAC activity, hBMMSCs underwent chondrogenic differentiation in pellet culture with or without TSA for 21 days. The weight of TSA-treated pellets was 25% lower than that of untreated pellets. DNA level was not significantly different, but glycosaminoglycan content per DNA level was lower in TSA-treated pellets than that of untreated pellets. Gene expression of the chondrogenic markers (SOX9, Aggrecan, and Col2A1) decreased by by 12.9-fold, 8.9-fold, and 7.6-fold respectively in TSA-treated pellets compared with that in TSA-untreated pellets. TSA-treated pellets had lower cell density and lower proteoglycan staining content compared with those of TSA-untreated pellets. A microarray analysis from TSA-treated pellets showed that 1,467 chondrogenic-related genes were downregulated and 1,524 were upregulated by more than 2-fold compared with TSA-untreated pellets. Col10A1, TGF-β3, and SOX9 decreased significantly by 10-fold, 2.1-fold, and 3.2-fold respectively in TSA-treated pellets compared with those in untreated pellets, whereas expression of BMP4 and FGFR3 increased significantly by 2.1-fold and 5.4-fold respectively. It is concluded that TSAinhibits chondrogenesis and does not seem to be useful for cartilage tissue engineering of hBMMSCs.

Effects of Type I Collagen Concentration in Hydrogel on the Growth and Phenotypic Expression of Rat Chondrocytes

It is controversial whether type I collagen itself can maintain and improve chondrogenic phenotype of chondrocytes in a three-dimensional (3D) environment. In this study, we examined the effect of type I collagen concentration in hydrogel (0.5, 1, and 2 mg/ml) on the growth and phenotype expression of rat chondrocytes in vitro. All collagen hydrogels showed substantial contractions during culture, in a concentration-dependent manner, which was due to the cell proliferation. The cell viability was shown to be the highest in 2 mg/ml collagen gel. The mRNA expression of chondrogenic phenotypes, including SOX9, type II collagen, and aggrecan, was significantly up-regulated, particularly in 1 mg/ml collagen gel. Furthermore, the production of type II collagen and glycosaminoglycan (GAG) content was also enhanced. The results suggest that type I collagen hydrogel is not detrimental to, but may be useful for, the chondrocyte culture for cartilage tissue engineering.

Porous Microcarrier-Enabled Three-Dimensional Culture of Chondrocytes for Cartilage Engineering: A Feasibility Study

Cartilage repair is substantially intractable due to poor self-healing ability. Porous microspheres can be a fascinating three-dimensional matrix for cell culture and injectable carrier in cartilage engineering. In this study, we assessed the feasible use of porous biopolymer microspheres for chondrocyte carriers. When seeded onto the blended biopolymer microspheres and followed by a dynamic spinner flask culture, the chondrocytes showed robust growth behaviors during the culture period. The gene expressions of SOX9, type II collagen, and aggrecan were significantly upregulated after 2-week of culture. Furthermore, immunolocalization of type II collagen and secretion of glycosaminolglycan became prominent. The results suggest the feasible usefulness of the porous microspheres as the cell culture matrix and the subsequent delivery into cartilage defects.

Alginate-Hyaluronic Acid-Collagen Composite Hydrogel Favorable for the Culture of Chondrocytes and Their Phenotype Maintenance

Articular cartilage has limited regeneration capacity, thus significant challenge has been made to restore the functions. The development of hydrogels that can encapsulate and multiply cells, and then effectively maintain the chondrocyte phenotype is a meaningful strategy to this cartilage repair. In this study, we prepared alginate-hyaluronic acid based hydrogel with type I collagen being incorporated, namely Alg-HA-Col composite hydrogel. The incorporation of Col enhanced the chemical interaction of molecules, and the thermal stability and dynamic mechanical properties of the resultant hydrogels. The primary chondrocytes isolated from rat cartilage were cultured within the composite hydrogel and the cell viability recorded revealed active proliferation over a period of 21 days. The mRNA levels of chondrocyte phenotypes, including SOX9, collagen type II, and aggrecan, were significantly up-regulated when the cells were cultured within the Alg-HA-Col gel than those cultured within the Alg-HA. Furthermore, the secretion of sulphated glycosaminoglycan, a cartilage-specific matrix molecule, was recorded higher in the collagen-added composite hydrogel. Although more in-depth studies are required such as the in vivo functions, the currently-prepared Alg-HA-Col composite hydrogel is considered to provide favorable 3-dimensional matrix conditions for the cultivation of chondrocytes. Moreover, the cell-cultured constructs may be useful for the cartilage repair and tissue engineering.

Inhibitory effects of SRT1720 on the apoptosis of rabbit chondrocytes by activating SIRT1 via p53/bax and NF-κB/PGC-1α pathways / 华中科技大学学报（医学）（英德文版）

SRT1720, a new discovered drug, was reported to activate silent information regulator 1 (SIRT1) and inhibit the chondrocyte apoptosis. However, the underlying mechanism remains elusive. In the present study, the chondrocytes were extracted from the cartilage tissues of New Zealand white rabbits, cultured in the presence of sodium nitroprusside (SNP) (2.5 mmol/L) and divided into five groups: 1, 5, 10, and 20 μmol/L SRT1720 groups and blank control group (0 μmol/L SRT1720). MTT assay was used to detect the chondrocyte viability and proliferation, and DAPI staining and flow cytometry to measure the chondrocyte apoptosis. The expression levels of SIRT1, p53, NF-κB/p65, Bax, and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) were detected by Western blotting and the expression levels of SIRT1, type II collagen, and aggrecan mRNA by RT-PCR. The results showed that in the SRT1720-treated groups, the nuclei of chondrocytes were morphologically intact and had uniform chromatin. In the blank control group, nuclear rupture into debris was observed in chondrocytes. With the SRT1720 concentration increasing, the chondrocyte viability increased, the apoptosis rate decreased, the protein expression levels of SIRT1 and PGC-1α and the mRNA expression levels of type II collagen and aggrecan increased ({ptP}<0.05), and the expression levels of p53, NF-κB and bax decreased (P<0.05). It was suggested that SRT1720 inhibits chondrocyte apoptosis by activating the expression of SIRT1 via p53/bax and NF-κB/PGC-1α pathways.

Effect of Hijikia fusiforme extracts on degenerative osteoarthritis in vitro and in vivo models

BACKGROUND/OBJECTIVES: The inhibitory effect of Hijikia fusiforme (HF) extracts on degenerative osteoarthritis was examined in primary cultured rat cartilage cells and a monosodium iodoacetate (MIA)-induced osteoarthritis rat model. MATERIALS/METHODS: In vitro, cell survival and the expression of matrix metalloproteinases (MMPs), collagen type I, collagen type II, aggrecan, and tissue inhibitor of metalloproteinases (TIMPs) was measured after H2O2 (800 µM, 2 hr) treatment in primary chondrocytes. In vivo animal study, osteoarthritis was induced by intra-articular injection of MIA into knee joints of rats, and then RH500, HFE250 and HFE500 were administered orally once a day for 28 days. To determine the anti-inflammatory effects of HFE, nitric oxide (NO), prostaglandin E2 (PGE2) expression were measured. In addition, real-time PCR was performed to measure the genetic expression of MMPs, collagen type I, collagen type II, aggrecan, and TIMPs. RESULTS: In the in vitro assay, cell survival after H2O2 treatment was increased by HFE extract (20% EtOH). In addition, anabolic factors (genetic expression of collagen type I, II, and aggrecan) were increased by HFE extract (20% EtOH). However, the genetic expression of MMP-3 and 7, known as catabolic factors were significantly inhibited by treatment with HFE extract (20% EtOH). In the in vivo assay, anabolic factors (genetic expression of collagen type I, II, aggrecan, and TIMPs) were increased by oral administration of HFE extract. However, the genetic expression of MMP-3 and 7, known as catabolic factors, and production of NO and PGE2 were significantly inhibited by treatment with oral administration of HFE extract. CONCLUSIONS: HFE extract inhibited articular cartilage degeneration through preventing extracellular matrix degradation and chondrocyte injury.