Jellyfish Collagen: A Biocompatible Collagen Source for 3D Scaffold Fabrication and Enhanced Chondrogenicity.

Osteoarthritis (OA) is a multifactorial disease leading to degeneration of articular cartilage, causing morbidity in approximately 8.5 million of the UK population. As the dense extracellular matrix of articular cartilage is primarily composed of collagen, cartilage repair strategies have exploited the biocompatibility and mechanical strength of bovine and porcine collagen to produce robust scaffolds for procedures such as matrix-induced chondrocyte implantation (MACI). However, mammalian sourced collagens pose safety risks such as bovine spongiform encephalopathy, transmissible spongiform encephalopathy and possible transmission of viral vectors. This study characterised a non-mammalian jellyfish (Rhizostoma pulmo) collagen as an alternative, safer source in scaffold production for clinical use. Jellyfish collagen demonstrated comparable scaffold structural properties and stability when compared to mammalian collagen. Jellyfish collagen also displayed comparable immunogenic responses (platelet and leukocyte activation/cell death) and cytokine release profile in comparison to mammalian collagen in vitro. Further histological analysis of jellyfish collagen revealed bovine chondroprogenitor cell invasion and proliferation in the scaffold structures, where the scaffold supported enhanced chondrogenesis in the presence of TGFß1. This study highlights the potential of jellyfish collagen as a safe and biocompatible biomaterial for both OA repair and further regenerative medicine applications.

Acid sphingomyelinase promotes SGK1-dependent vascular calcification.

In chronic kidney disease (CKD), hyperphosphatemia is a key factor promoting medial vascular calcification, a common complication associated with cardiovascular events and high mortality. Vascular calcification involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs), but the complex signaling events inducing pro-calcific pathways are incompletely understood. The present study investigated the role of acid sphingomyelinase (ASM)/ceramide as regulator of VSMC calcification. In vitro, both, bacterial sphingomyelinase and phosphate increased ceramide levels in VSMCs. Bacterial sphingomyelinase as well as ceramide supplementation stimulated osteo-/chondrogenic transdifferentiation during control and high phosphate conditions and augmented phosphate-induced calcification of VSMCs. Silencing of serum- and glucocorticoid-inducible kinase 1 (SGK1) blunted the pro-calcific effects of bacterial sphingomyelinase or ceramide. Asm deficiency blunted vascular calcification in a cholecalciferol-overload mouse model and ex vivo isolated-perfused arteries. In addition, Asm deficiency suppressed phosphate-induced osteo-/chondrogenic signaling and calcification of cultured VSMCs. Treatment with the functional ASM inhibitors amitriptyline or fendiline strongly blunted pro-calcific signaling pathways in vitro and in vivo. In conclusion, ASM/ceramide is a critical upstream regulator of vascular calcification, at least partly, through SGK1-dependent signaling. Thus, ASM inhibition by repurposing functional ASM inhibitors to reduce the progression of vascular calcification during CKD warrants further study.

Protective effects of Phlomis umbrosa extract on a monosodium iodoacetate-induced osteoarthritis model and prediction of molecular mechanisms using transcriptomics.

BACKGROUND: Phlomis umbrosa Turczaninow root has been traditionally used to treat fractures, rheumatoid arthritis, and arthralgia. However, the effects and mechanisms of P. umbrosa on osteoarthritis (OA) remain poorly understood and a functional genomic approach has not been investigated. AIM: The purpose of this study was to investigate the effects and mechanisms of P. umbrosa extract (PUE) on OA using transcriptomic analysis. METHODS: We performed joint diameter measurements, micro computed tomography, and histopathological analysis of monosodium iodoacetate (MIA)-induced OA rats treated with PUE (200 mg/kg) for 3 weeks. Gene expression profiling in articular cartilage tissue was then performed using RNA sequencing (RNA-seq) followed by signaling pathway analysis of regulatory genes. RESULTS: PUE treatment improved OA based on decreased joint diameter, increased joint morphological parameters, and histopathological features. Many genes involved in multiple signal transduction pathway and collagen activation in OA were differentially regulated by PUE. These included genes related to Wnt/ß-catenin, OA pathway, and sonic hedgehog signaling activity. Furthermore, PUE treatment downregulated cartilage damage factors (MMP-9, MMP-13, ADAMTs4, and ADMATs5) and upregulated chondrogenesis (COL2A1 and SOX-9) by regulating the transcription factors SOX-9, Ctnnb1, and Epas1. CONCLUSION: Based on the results of gene expression profiling, this study highlighted the molecular mechanisms underlying the effects of PUE in MIA-induced OA rats. The findings provide novel insight into the mechanisms by which PUE treatment-induced gene expression changes may influence OA disease progression. Taken together, the results suggest that PUE may be used as a source of therapeutic agents for OA.

Effect of quercetin on chondrocyte phenotype and extracellular matrix expression.

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.

Biosafety evaluation of culture-expanded human chondrocytes with growth factor cocktail: a preclinical study.

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor ß2 (TGF ß2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes' expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.

Chondrogenic differentiation of Wharton's Jelly mesenchymal stem cells on silk spidroin-fibroin mix scaffold supplemented with L-ascorbic acid and platelet rich plasma.

In this research, hWJ-MSCs were grown on silk scaffolds and induced towards chondrogenesis by supplementation with L-ascorbic acid (LAA) or platelet rich plasma (PRP). Silk scaffolds were fabricated with salt leaching method by mixing silk fibroin (SF) with silk spidroin (SS). The silk fibroin was obtained from Bombyx mori cocoon that had been degummed, and the silk spidroin was obtained from wild-type spider Argiope appensa. The effect of scaffold composition and inducer on cell proliferation was observed through MTT assay. The most optimal treatment then continued to be used to induce hWJ-MSC towards chondrogenic differentiation for 7 and 21 days. Scaffolds characterization showed that the scaffolds produced had 3D structure with interconnected pores, and all were biocompatible with hWJ-MSCs. Scaffold with the addition of 10% SS + 90% SF showed higher compressive strength and better pore interconnectivity in comparison to 100% silk fibroin scaffold. After 48 h, cells seeded on scaffold with spidroin and fibroin mix had flattened morphology in comparison to silk fibroin scaffold which appeared to be more rounded on the scaffold surface. Scaffold with 10% (w/w) of silk spidroin (SS) + 90% (w/w) of silk fibroin (SF) was the most optimal composition for cell proliferation. Immunocytochemistry of integrin ß1 and RGD sequence, showed that scaffold with SS 10% provide better cell attachment with the presence of RGD sequence from the spidroin silk which could explain the higher cell proliferation than SF100% scaffold. Based on Alcian Blue staining and Collagen Type II immunocytochemistry (ICC), cells grown on 10% SS + 90% SF scaffold with 10% PRP supplementation were the most optimal to support chondrogenesis of hWJ-MSCs. These results showed that the addition of spidroin silk from A. appensa. had impact on scaffold compressive strength and chondrogenic differentiation of hWJ-MSC and had the potential for further development of bio-based material scaffold in cartilage tissue engineering.

Aqueous extract of Arctium lappa L. root (burdock) enhances chondrogenesis in human bone marrow-derived mesenchymal stem cells.

BACKGROUND: Arctium lappa L. root (burdock root) has long been recommended for the treatment of different diseases in traditional Chinese medicine. Burdock root possesses anti-oxidative, anti-inflammatory, anti-cancer, and anti-microbial activities. The aim of the study was to elucidate whether aqueous extract of burdock root regulates mesenchymal stem cell proliferation and differentiation. METHODS: Human bone marrow-derived mesenchymal stem cells in 2D high density culture and in 3D micromass pellets were treated with chondrogenic induction medium and chondral basal medium in the absence or presence of aqueous extract of burdock root. The chondrogenic differentiation was accessed by staining glucosaminoglycans, immunostaining SOX9 and type II collagen and immuonblotting of SOX9, aggrecan and type II collagen. RESULTS: Treatment of aqueous extract of burdock root increased the cell proliferation of hMSCs. It did not have significant effect on osteogenic and adipogenic differentiation, but significantly enhanced chondrogenic induction medium-induced chondrogenesis. The increment was dose dependent, as examined by staining glucosaminoglycans, SOX9, and type II collagen and immunobloting of SOX9, aggrecan and type II collagen in 2D and 3D cultures. In the presence of supplemental materials, burdock root aqueous extract showed equivalent chondrogenic induction capability to that of TGF-ß. CONCLUSIONS: The results demonstrate that aqueous extract of Arctium lappa L. root promotes chondrogenic medium-induced chondrogenic differentiation. The aqueous extract of burdock root can even be used alone to stimulate chondrogenic differentiation. The study suggests that the aqueous extract of burdock root can be used as an alternative strategy for treatment purposes.

Silver Fir (Abies alba L.) Polyphenolic Extract Shows Beneficial Influence on Chondrogenesis In Vitro under Normal and Inflammatory Conditions.

Several plant polyphenols have been shown to reduce osteoarthritis symptoms due to their antioxidant, anti-inflammatory and immunomodulatory properties. We investigated the effects of two different polyphenolic extracts (Belinal, Pycnogenol) and two different polyphenols (resveratrol, quercetin) on the chondrogenic potential of bone-derived mesenchymal stem/stromal cells (MSCs) from healthy donors and patients with osteoarthritis. Our main aim was to determine whether Belinal, a commercially available polyphenolic extract from silver fir (Abies alba L.) branches, has comparable chondrogenic potential with the other tested extract and the polyphenols under inflammatory and non-inflammatory conditions. In our study, Belinal promoted significantly greater chondrogenesis compared to the untreated (p = 0.0289) and resveratrol-treated (p = 0.0468) MSCs from patients with hip osteoarthritis under non-inflammatory conditions. Under inflammatory conditions, chondrogenesis was significantly enhanced for MSCs treated with Belinal compared to the control (p = 0.0483). The other extract and the polyphenols did not show any significant effects on chondrogenesis under non-inflammatory or inflammatory conditions. None of the tested extracts and polyphenols showed significant effects on chondrogenesis in healthy donors, under either non-inflammatory or inflammatory conditions. Our data show that Belinal can boost the chondrogenesis of MSCs derived from patients with osteoarthritis, under both non-inflammatory and inflammatory conditions.

Broccoli extract increases drug-mediated cytotoxicity towards cancer stem cells of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCC) are malignant neoplasms with poor prognosis. Treatment-resistant cancer stem cell (CSC) is one reason for treatment failure. Considerable attention has been focused on sulforaphane (SF), a phytochemical from broccoli possessing anticancer properties. We investigated whether SF could enhance the chemotherapeutic effects of cisplatin (CIS) and 5-fluorouracil (5-FU) against HNSCC-CSCs, and its mechanisms of action. METHODS: CD44+/CD271+ FACS-isolated CSCs from SCC12 and SCC38 human cell lines were treated with SF alone or combined with CIS or 5-FU. Cell viability, colony- and sphere-forming ability, apoptosis, CSC-related gene and protein expression and in vivo tumour growth were assessed. Safety of SF was tested on non-cancerous stem cells and in vivo. RESULTS: SF reduced HNSCC-CSC viability in a time- and dose-dependent manner. Combining SF increased the cytotoxicity of CIS twofold and 5-FU tenfold, with no effects on non-cancerous stem cell viability and functions. SF-combined treatments inhibited CSC colony and sphere formation, and tumour progression in vivo. Potential mechanisms of action included the stimulation of caspase-dependent apoptotic pathway, inhibition of SHH pathway and decreased expression of SOX2 and OCT4. CONCLUSIONS: Combining SF allowed lower doses of CIS or 5-FU while enhancing these drug cytotoxicities against HNSCC-CSCs, with minimal effects on healthy cells.

Folic acid rescues corticosteroid-induced vertebral malformations in chick embryos through targeting TGF-ß signaling.

Folic acid (FA) is routinely supplemented in the food of pregnant women or women planning a pregnancy, but whether FA exerts a positive effect on preventing fetal bone malformation remains obscure. In this study, we first exposed chick embryos with different concentrations of FA (1-10,000 pmol/egg) and studied vertebral mineralization and ossification through alcian blue and alizarin red as well as hematoxylin and eosin staining. Morphological measurements of the thoracic vertebral bodies demonstrated that 100 pmol/egg FA exhibited the tendency of shortening the growth plate, extended the ossification center, and increased the amount of Type I collagen. Second, we suggested that FA treatment promotes osteogenesis by demonstrating increased RUNX family transcription factor 2 (Runx2) and Osterix expressions in MC3T3-E1 and ATDC5 cells. Transforming growth factor-ß (TGF-ß) signaling was also upregulated by FA exposure, and addition of smad2/3 small interfering RNA knocks down FA-induced increased p-smad2/3, Runx2, and Osterix expression in vitro during chondrogenesis induction. Third, we employed dexamethasone (Dex), exposed chick embryos as an animal model of skeletal developmental retardation, to explore whether FA could rescue the loss of embryonic bone mass. Micro-computed tomography imaging showed that the addition of FA improved the reduction of bone mass in our model. Histological analysis of the vertebral bodies revealed that FA dramatically improved the delayed turnover of the zones of growth plate caused by Dex exposure. Immunofluorescence on the chick embryonic vertebrae and chondrocytes showed that FA supplementation upregulated the expression of TGF-ß1, p-smad2/3, and improved Runx2 as well as Osterix expression in the Dex + FA group compared with the Dex group. Lastly, we found that supplementation with TGF-ß1 (1 ng/egg) rescued bone mass loss caused by Dex as was also seen in FA exposure. Taken together these results, our data revealed that FA supplementation was able to rescue Dex exposure-induced inhibitive osteogenesis through targeting on the TGF-ß signaling pathway.

Chondrogenic differentiation of mesenchymal stem/stromal cells on 3D porous poly (ε-caprolactone) scaffolds: Effects of material alkaline treatment and chondroitin sulfate supplementation.

Cartilage defects resultant from trauma or degenerative diseases (e.g., osteoarthritis) can potentially be repaired using tissue engineering (TE) strategies combining progenitor cells, biomaterial scaffolds and bio-physical/chemical cues. This work examines promoting chondrogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs) by combining the effects of modified poly (ε-caprolactone) (PCL) scaffolds hydrophilicity and chondroitin sulfate (CS) supplementation in a hypoxic 5% oxygen atmosphere. 3D-extruded PCL scaffolds, characterized by µCT, featured a 21 mm-1 surface area to volume ratio, 390 µm pore size and approximately 100% pore interconnectivity. Scaffold immersion in sodium hydroxide solutions for different periods of time had major effects in scaffold surface morphology, wettability and mechanical properties, but without improvements on cell adhesion. In-situ chondrogenic differentiation of BM-MSC seeded in 3D-extruded PCL scaffolds resulted in higher cell populations and ECM deposition along all scaffold structure, when chondrogenesis was preceded by an expansion phase. Additionally, CS supplementation during BM-MSC expansion was crucial to enhance aggrecan gene expression, known as a hallmark of chondrogenesis. Overall, this study presents an approach to tailor the wettability and mechanical properties of PCL scaffolds and supports the use of CS-supplementation as a biochemical cue in integrated TE strategies for cartilage regeneration.

Impact of Prevalence Ratios of Chondroitin Sulfate (CS)- 4 and -6 Isomers Derived from Marine Sources in Cell Proliferation and Chondrogenic Differentiation Processes.

Osteoarthritis is the most prevalent rheumatic disease. During disease progression, differences have been described in the prevalence of chondroitin sulfate (CS) isomers. Marine derived-CS present a higher proportion of the 6S isomer, offering therapeutic potential. Accordingly, we evaluated the effect of exogenous supplementation of CS, derived from the small spotted catshark (Scyliorhinus canicula), blue shark (Prionace glauca), thornback skate (Raja clavata) and bovine CS (reference), on the proliferation of osteochondral cell lines (MG-63 and T/C-28a2) and the chondrogenic differentiation of mesenchymal stromal cells (MSCs). MG-G3 proliferation was comparable between R. clavata (CS-6 intermediate ratio) and bovine CS (CS-4 enrichment), for concentrations below 0.5 mg/mL, defined as a toxicity threshold. T/C-28a2 proliferation was significantly improved by intermediate ratios of CS-6 and -4 isomers (S. canicula and R. clavata). A dose-dependent response was observed for S. canicula (200 µg/mL vs 50 and 10 µg/mL) and bovine CS (200 and 100 µg/mL vs 10 µg/mL). CS sulfation patterns discretely affected MSCs chondrogenesis; even though S. canicula and R. clavata CS up-regulated chondrogenic markers expression (aggrecan and collagen type II) these were not statistically significant. We demonstrate that intermediate values of CS-4 and -6 isomers improve cell proliferation and offer potential for chondrogenic promotion, although more studies are needed to elucidate its mechanism of action.

Yam-Containing Serum Promotes Proliferation and Chondrogenic Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells and Synthesis of Glycosaminoglycan.

The chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is affected by a variety of factors, including environmental, physical, and chemical factors and growth factors, and traditional Chinese medicine (TCM) preparations can further influence this process. In this study, the effects of different concentrations of Yam-containing serum of rabbits on BMSC proliferation and chondrogenic differentiation were investigated, as were the underlying molecular mechanisms. The growth and proliferation of BMSCs were significantly enhanced upon treatment with Yam-containing serum. Under both monolayer and micromass culture conditions, Yam-containing serum promoted the differentiation of BMSCs into chondrocytes. Toluidine blue staining results revealed that chondrocyte differentiation in the group treated by Yam-containing serum was significantly more pronounced than in the control group. Glycosaminoglycan levels, as measured by 1,2-dimethylmethylene blue (DMMB) detection, were significantly higher in cells of the Yam-containing group relative to the control group. This is the first study to our knowledge that demonstrates that Yam-containing serum can promote BMSC proliferation and chondrogenic differentiation. This study therefore lays an experimental groundwork for further application of TCM as a means of treating degenerative cartilage diseases and provides an experimental and theoretical basis for the combination of TCM and stem cells for the treatment of such diseases.

Effect of quercetin on chondrocyte phenotype and extracellular matrix expression / 中国天然药物

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.

Cladophora glomerata methanolic extract promotes chondrogenic gene expression and cartilage phenotype differentiation in equine adipose-derived mesenchymal stromal stem cells affected by metabolic syndrome.

BACKGROUND: Chondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential. OBJECTIVES: In this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome. METHODS: Cultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis. RESULTS: The different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCsEMS) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation. CONCLUSION: Chondrogenesis in EqASCsEMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.

Icariin-conditioned serum engineered with hyaluronic acid promote repair of articular cartilage defects in rabbit knees.

BACKGROUND: Osteochondral defects mostly occur as a result of trauma or articular degeneration. The poor regenerative ability of articular cartilage remains osteochondral defects are a tricky problem to deal with. The modern treatment strategies mainly focus on cartilage tissue engineering with bioactive materials. In this study, we aimed to develop icariin conditioned serum (ICS) together with hyaluronic acid (HA) and determine their ability in reparing osteochondral tissue in a critical-sized defect in rabbit knees. METHODS: Primary chondrocytes were incubated with serum conditioned with icariin at different concentrations, then cell proliferation rates and glycosaminoglycan (GAG) secretion were detected. Rabbits were treated with intra-articular injection of 0.5 mL normal saline (NS), ICS, HA and ICS + HA in the right knee joint, respectively. ICRS scores were used to assess the macroscopic cartilage regeneration. Histological and immunohistochemical analysis including H&E, Safranin O, toluidine blue and collagen II staining were used to determine the repair of cartilage and the regeneration of chondrocytes. RESULTS: Icariin at a low dose of 0.94 g/kg was identified to have significantly promoted the proliferation of chondrocytes and enhance the secretion of GAG. Femoral condyle from rabbits treated by ICS together with HA was observed to be integrated with native cartilage and more subchondral bone regeneration. ICS together with HA could promote repair of the cartilage defect and increase the neoformation of cartilage. CONCLUSIONS: These results demonstrated the potential of ICS combined with HA to promote reparative response in cartilage defects and the possible application in bioactive material based cartilage regeneration therapies.

Nutritional supplementation with myo-inositol in growing mice specifically augments mandibular endochondral growth.

INTRODUCTION: The purpose of this study was to examine growth-promoting effects of myo-inositol nutritional supplementation on the mandible in experimental animals. METHODS: Mice were fed on diets that contained various concentration of myo-inositol for 3 to 12 weeks. The length of the mandible, maxilla, and femur were measured on µCT images. The mandible and tibia were examined histologically and immunohistochemically. The effects of myo-inositol on cell proliferation and chondrocytic differentiation were examined using ATDC5 cells. RESULTS: Myo-inositol supplementation had no effects on body weight, length, and maxilla and femur lengths. However, the length of mandible and the thickness of the mandibular condylar cartilage (MCC) were increased by myo-inositol supplement. Microarray analysis revealed that Pik3cd was highly expressed in MCC as compared to that in the cartilage of the tibial growth plate, which was confirmed by real-time RT-PCR and immunohistochemistry. ATDC5 cells also highly expressed Pik3CD. Myoinositol induced increases in cell proliferation and chondrocytic differentiation in ATDC5 cells. The addition of a PIK3CD inhibitor blocked the induction of cell proliferation by myo-inositol in ATDC5 cells. CONCLUSIONS: Nutritional supplementation with myo-inositol in growing mice augmented mandibular endochondral growth without any systemic effects. The specific promotion of mandibular growth by myoinositol is primarily dependent on the specific intensive expression of PIK3CD in the MCC.

Simvastatin With PRP Promotes Chondrogenesis of Bone Marrow Stem Cells In Vitro and Wounded Rat Achilles Tendon-Bone Interface Healing In Vivo.

BACKGROUND: Tendons and ligaments are joined to bone in a specialized interface that transmits force from muscle to bone and permits body movement. Tendon/ligament injuries always occur in the interface areas, and injured tendons/ligaments have a limited healing response because the insertion site is composed of a fibrocartilaginous zone. PURPOSE: To study the effect of simvastatin with platelet-rich plasma (PRP) on chondrogenesis of rat bone marrow stem cells (BMSCs) in vitro and wounded rat Achilles tendon-bone interface healing in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: The in vitro model was performed by the culture of rat BMSCs with various concentrations of simvastatin (0, 10, 50, 100 nM) for 2 weeks. The effect of simvastatin on the chondrogenic differentiation of the BMSCs was examined by histochemical analysis and real-time quantitative reverse transcription polymerase chain reaction. The in vivo model was carried out by testing the healing effect of simvastatin with PRP on 12 wounded rat Achilles tendon-bone interfaces. RESULTS: Simvastatin induced chondrogenic differentiation of rat BMSCs in a concentration-dependent manner as evidenced by histological staining and real-time quantitative reverse transcription polymerase chain reaction. The wounds treated with simvastatin alone or with simvastatin-containing PRP gel healed much faster than the wounds treated with saline alone or PRP alone. Histological analysis showed that higher percentages of healed tissues were positively stained with safranin O and fast green in wounds treated with simvastatin-containing PRP gel than in the other 3 groups. Immunohistochemical analysis further demonstrated these findings, as evidenced by more positively stained healed tissues with collagen I and II antibodies in the wound areas treated with simvastatin-containing PRP gel than the other 3 groups. CONCLUSION: The combination of simvastatin with PRP induced chondrogenesis of BMSCs in vitro and enhanced fibrocartilage formation in vivo. The simvastatin-PRP gel treatment promotes wounded tendon-bone interface healing in clinical treatment. CLINICAL RELEVANCE: The combination of simvastatin with PRP may be a good clinical treatment for wounded tendon/ligament junction healing, especially for acute sports-related tendon/ligament injuries.

Proteomic analysis of the effects of antler extract on chondrocyte proliferation, differentiation and apoptosis.

Deer antlers are unique cranial appendages capable of regeneration and rapid growth. In addition, deer antlers have been widely used in traditional Chinese medicine to promote the function of the kidneys, reproductive system, bones and nervous system. It has been shown that water-soluble substances are the major bioactive components within the deer antlers. In this study, we prepared aqueous extracts from deer antlers during a rapid growth stage. We investigated the effects of antler extracts on primary chondrocytes by analyzing their protein expression patterns using isobaric tags for relative and absolute quantitation technology. We demonstrated that antler extracts promote chondrocyte proliferation and prevent chondrocyte differentiation and apoptosis by controlling multiple cellular processes involved in genomic stability, epigenetic alterations, ribosome biogenesis, protein synthesis and cytoskeletal reorganization. Antler extracts significantly increased the expression levels of proliferation markers Mki67 and Stmn1 and differentiation inhibitor Acp5 as well as cellular apoptosis inhibitors Ndufa4l2 and Rcn1. Thus, this study has greatly expanded our current knowledge of the molecular effects of antler extracts on chondrocytes. It has also shed new light on possible strategies to prevent damage to and to treat cartilage and its related diseases by using aqueous extracts from growing Sika deer antlers.

Effects of malvidin, cyanidin and delphinidin on human adipose mesenchymal stem cell differentiation into adipocytes, chondrocytes and osteocytes.

BACKGROUND: Anthocyanidins are plant phytochemicals found at high concentrations in berries, vegetables and flowers. Anthocyanidins have been extensively investigated due to their antioxidative, antidiabetic and anti-inflammatory effects. Few studies show that anthocyanidins decrease obesity and improve bone density. However, the effects of anthocyanidins on tissue regeneration have not been sufficiently clarified. Human mesenchymal stem cells (MSCs) are multipotent adult stem cells responsible for the regeneration of fat, bone and cartilage. Although MSCs are often used for screening of biologically active compounds, so far, the effect of anthocyanidins on MSC differentiation has not been addressed. PURPOSE: The aim of this study was to analyse the effect of anthocyanidins malvidin, cyanidin and delphinidin on adipose tissue-derived MSC differentiation into adipocytes, osteocytes and chondrocytes. STUDY DESIGN AND METHODS: Differentiation into adipocytes, osteocytes and chondrocytes was carried out in the defined cell culture conditions in the presence or absence of malvidin, cyanidin and delphinidin. The differentiation was confirmed by cytochemical staining and tissue-specific gene and protein expression. Antiobesity and anti-diabetes drug liraglutide was used as a reference drug in this study. RESULTS: Delphinidin inhibited MSC adipogenesis and downregulated FABP4 and adiponectin genes. Malvidin induced a significantly higher accumulation of calcium deposits in MSCs comparing to untreated MSCs, as well as upregulated the osteocyte-specific gene BMP-2 and Runx-2 expression and induced BMP-2 secretion. Cyanidin and delphinidin demonstrated a chondrogenesis stimulating effect by upregulation of Col2a1 and aggrecan. CONCLUSION: Altogether, our data show that anthocyanidins malvidin, cyanidin and delphinidin exert favourable effects on MSC osteogenesis and chondrogenesis whereas delphinidin inhibits adipogenesis. These results suggest that anthocyanidin effects on tissue regeneration could be further analysed in depth in vivo.