Project description:Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21?days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14?days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo. STATEMENT OF SIGNIFICANCE:Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration.
Project description:Human chondrocytes are expanded and used in autologous chondrocyte implantation techniques and are known to rapidly de-differentiate in culture. These chondrocytes, when cultured on tissue culture plastic (TCP), undergo both phenotypical and morphological changes and quickly lose the ability to re-differentiate to produce hyaline-like matrix. Growth on synoviocyte-derived extracellular matrix (SDECM) reduces this de-differentiation, allowing for more than twice the number of population doublings (PD) whilst retaining chondrogenic capacity. The goal of this study was to apply RNA sequencing (RNA-Seq) analysis to examine the differences between TCP-expanded and SDECM-expanded human chondrocytes. Human chondrocytes from three donors were thawed from primary stocks and cultured on TCP flasks or on SDECM-coated flasks at physiological oxygen tension (5%) for 4 passages. During log expansion, RNA was extracted from the cell layer (70?90% confluence) at passages 1 and 4. Total RNA was column-purified and DNAse-treated before quality control analysis and next-generation RNA sequencing. Significant effects on gene expression were observed due to both culture surface and passage number. These results offer insight into the mechanism of how SDECM provides a more chondrogenesis-preserving environment for cell expansion, the transcriptome-wide changes that occur with culture, and potential mechanisms for further enhancement of chondrogenesis-preserving growth.
Project description:Current tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential.Porcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically.Human chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions.Synoviocyte-derived matrix supports enhanced expansion of human chondrocytes such that the chondrocytes are maintained in a state from which they can re-differentiate into a cartilage phenotype after significantly more population doublings. Also, low oxygen tension supports GAG, but not collagen, accumulation. These findings are a step towards the production of a more functional, tissue engineered cartilage.
Project description:INTRODUCTION: Rheumatoid arthritis (RA) leads to progressive destruction of articular cartilage. This study aimed to disclose major mechanisms of antirheumatic drug action on human chondrocytes and to reveal marker and pharmacological target genes that are involved in cartilage dysfunction and regeneration. METHODS: An interactive in vitro cultivation system composed of human chondrocyte alginate cultures and conditioned supernatant of SV40 T-antigen immortalised human synovial fibroblasts was used. Chondrocyte alginate cultures were stimulated with supernatant of RA synovial fibroblasts, of healthy donor synovial fibroblasts, and of RA synovial fibroblasts that have been antirheumatically treated with disease-modifying antirheumatic drugs (DMARDs) (azathioprine, gold sodium thiomalate, chloroquine phosphate, and methotrexate), nonsteroidal anti-inflammatory drugs (NSAIDs) (piroxicam and diclofenac), or steroidal anti-inflammatory drugs (SAIDs) (methylprednisolone and prednisolone). Chondrocyte gene expression profile was analysed using microarrays. Real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay were performed for validation of microarray data. RESULTS: Genome-wide expression analysis revealed 110 RA-related genes in human chondrocytes: expression of catabolic mediators (inflammation, cytokines/chemokines, and matrix degradation) was induced, and expression of anabolic mediators (matrix synthesis and proliferation/differentiation) was repressed. Potential marker genes to define and influence cartilage/chondrocyte integrity and regeneration were determined and include already established genes (COX-2, CXCR-4, IL-1RN, IL-6/8, MMP-10/12, and TLR-2) and novel genes (ADORA2A, BCL2-A1, CTGF, CXCR-7, CYR-61, HSD11B-1, IL-23A, MARCKS, MXRA-5, NDUFA4L2, NR4A3, SMS, STS, TNFAIP-2, and TXNIP). Antirheumatic treatment with SAIDs showed complete and strong reversion of RA-related gene expression in human chondrocytes, whereas treatment with NSAIDs and the DMARD chloroquine phosphate had only moderate to minor effects. Treatment with the DMARDs azathioprine, gold sodium thiomalate, and methotrexate efficiently reverted chondrocyte RA-related gene expression toward the 'healthy' level. Pathways of cytokine-cytokine receptor interaction, transforming growth factor-beta/Toll-like receptor/Jak-STAT (signal transducer and activator of transcription) signalling and extracellular matrix receptor interaction were targeted by antirheumatics. CONCLUSIONS: Our findings indicate that RA-relevant stimuli result in the molecular activation of catabolic and inflammatory processes in human chondrocytes that are reverted by antirheumatic treatment. Candidate genes that evolved in this study for new therapeutic approaches include suppression of specific immune responses (COX-2, IL-23A, and IL-6) and activation of cartilage regeneration (CTGF and CYR-61).
Project description:Articular cartilage regeneration is insufficient to restore sports injuries or defects that can occur from trauma. Treatment options for cartilage repair include autologous chondrocyte implantation (ACI) by isolation, expansion, and reimplantation of healthy donor chondrocytes. Chondrocyte expansion onto 2D substrates leads to dedifferentiation and loss of the cellular phenotype. We aimed to overcome the state of dedifferentiation by biochemical stimuli with platelet derivatives such as platelet-rich plasma (PRP) and hyperacute serum (HAS) to achieve sufficient cell numbers in combination with variable oxygen tension. Human articular chondrocytes from osteoarthritic (OA) cartilage chondrocytes were switched from 10% FCS supplementation to either 10% PRP or 10% HAS after initial passaging for further experiments under normoxic (20% O2) or hypoxic (1% O2) conditions. An XTT assay measured the effect of PRP or HAS on the cell proliferation at 3, 6, and 9?days. The chondrogenic redifferentiation potential of dedifferentiated chondrocytes was determined with reverse transcriptase quantitative real-time PCR for markers of expression for type II collagen (COL2A1), type I collagen (COL1A1), and matrix metalloproteinases MMP3, matrix metalloproteinase 13 (MMP13) at 24 and 72?h. Measured protein levels of 100% PRP or HAS by multiplex quantification revealed basic fibroblast growth factor, G-CSF, and PDGF were significantly higher in PRP than in HAS (p?<?0.05) but LEPTIN levels did not differ. The quantified protein levels did not differ when isolated from same donors at a different time. Chondrocyte proliferation indicated that supplementation of 10% HAS enhanced the proliferation rate compared to 10% PRP or 10% FCS at 6 and 9?days significantly (p?<?0.05). mRNA levels for expression of COL1A1 were significantly downregulated (p?<?0.05) when cultured with 10% PRP than 10% HAS or 10% FCS under normoxic/hypoxic conditions. COL2A1 was significantly upregulated (p?<?0.05) in PRP than 10% HAS or 10% FCS. MMP3 expression was downregulated after 72?h under all conditions. MMP13 was upregulated with 10% PRP at both 24 and 72?h but significantly downregulated under hypoxia (1% O2) for all circumstances. While HAS has its effect on chondrocyte proliferation, PRP enhances both proliferation and redifferentiation of dedifferentiated chondrocytes. PRP can replace standard usage of FCS for chondrogenic priming and expansion as implications for clinical use such as ACI procedures.
Project description:Sialic acids frequently occur at the terminal positions of glycoprotein N-glycans present at chondrocyte surfaces or in the cartilage matrix. Sialic acids are transferred to glycoproteins in either alpha-2,3 or alpha-2,6 linkage by specific sialyltransferases (SiaTs) and can potentially affect cell functions and cell-matrix interactions. The present study aimed to assess the relationship between the expression of the human chondrocyte phenotype and the sialylation of chondrocyte glycoprotein N-glycans.The transcription of 5 SiaT was quantified using real-time Reverse transcription polymerase chain reaction (RT-PCR) assays. N-glycan analysis was performed using LC-ESI-MS. Primary human chondrocytes were cultured in monolayer or alginate beads and compared to the chondrocyte cell lines C-28/I2 and SW1353. In addition, effects of interleukin-1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) on primary cells were assessed.Primary human chondrocytes predominantly express alpha-2,6-specific SiaTs and accordingly, alpha-2,6-linked sialic acid residues in glycoprotein N-glycans. In contrast, the preponderance of alpha-2,3-linked sialyl residues and, correspondingly, reduced levels of alpha-2,6-specific SiaTs are associated with the altered chondrocyte phenotype of C-28/I2 and SW1353 cells. Importantly, a considerable shift towards alpha-2,3-linked sialic acids and alpha-2,3-specific SiaT mRNA levels occurred in primary chondrocytes treated with IL-1beta or tumour necrosis factor-alpha (TNF-alpha).The expression of the differentiated chondrocyte phenotype is linked to the ratio of alpha-2,6- to alpha-2,3-linked sialic acids in chondrocyte glycoprotein N-glycans. A shift towards altered sialylation might contribute to impaired cell-matrix interactions in disease conditions.
Project description:Osteoarthritis (OA) in articular joints is a prevalent disease. With increasing life expectancy, the need for therapies other than knee replacement arises. The intrinsic repair capacity of cartilage is limited, therefore alternative strategies for cartilage regeneration are being explored. The purpose of this study is first to investigate the potential of platelet lysate (PL) as a xeno-free alternative in expansion of human OA chondrocytes for cell therapy, and second to assess the effects of PL on redifferentiation of expanded chondrocytes in 3D pellet cultures. Chondrocytes were isolated from human OA cartilage and subjected to PL in monolayer culture. Cell proliferation, morphology, and expression of chondrogenic genes were assessed. Next, PL-expanded chondrocytes were cultured in 3D cell pellets and cartilage matrix production was assessed after 28 days. In addition, the supplementation of PL to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by quantitative biochemical analyses, as well as by (immuno)histochemistry. A dose-dependent effect of PL on chondrocyte proliferation was found, but expression of chondrogenic markers was decreased when compared to FBS-expanded cells. After 28 days of subsequent 3D pellet culture, GAG production was significantly higher in pellets consisting of chondrocytes expanded with PL compared to controls. However, when used to supplement redifferentiation medium for chondrocyte pellets, PL significantly decreased the production of GAGs and collagen. In conclusion, chondrocyte proliferation is stimulated by PL and cartilage production in subsequent 3D culture is maintained. Furthermore, the presences of PL during redifferentiation of 3D chondrocyte strongly inhibits GAG and collagen content. The data presented in the current study indicate that while the use of PL for expansion in cartilage cell therapies is possibly beneficial, intra-articular injection of the product in the treatment of OA might be questioned.
Project description:The Runt-related transcription factor 2 (RUNX2) is critical for bone formation as well as chondrocyte maturation. Matrix metalloproteinase (MMP)-13 is a major contributor to cartilage degradation in osteoarthritis (OA). We and others have shown that the abnormal MMP13 gene expression in OA chondrocytes is controlled by changes in the DNA methylation status of specific CpG sites of the proximal promoter, as well as by the actions of different transactivators, including RUNX2. The present study aimed to determine the influence of the methylation status of specific CpG sites in the RUNX2 promoter on RUNX2-driven MMP13 gene expression in OA chondrocytes. We observed a significant correlation between MMP13 mRNA levels and RUNX2 gene expression in human OA chondrocytes. RUNX2 overexpression enhanced MMP13 promoter activity, independent of the MMP13 promoter methylation status. A significant negative correlation was observed between RUNX2 mRNA levels in OA chondrocytes and the percentage methylation of the CpG sites in the RUNX2 P1 promoter. Accordingly, the activity of the wild type RUNX2 promoter was decreased upon methylation treatment in vitro. We conclude that RUNX2 gene transcription is regulated by the methylation status of specific CpG sites in the promoter and may determine RUNX2 availability in OA cartilage for transactivation of genes such as MMP13.
Project description:Dedifferentiation of chondrocytes during cell expansion is one of the barriers in tissue construction for cartilage repair. To understand chondrocyte behavior and improve cell expansion in monolayer culture, this study investigated the effects of morphological changes and cellular aggregation on the maintenance of chondrogenic capacity by observing the expression patterns of chondrogenic (collagen type II and aggrecan) and dedifferentiation (collagen type I) markers. Primary human chondrocytes were cultured on either a polystyrene surface (PS) or a polyamidoamine dendrimer surface with a fifth-generation (G5) dendron structure to create a one-step process of cell expansion and the maintenance of chondrogenic activities prior to the construction of cell sheets.During the first two passages (P0 - P2), the relative mRNA level of collagen type II decreased in all cultures, while that of collagen type I increased. Remarkably, the level of collagen type II was higher and aggrecan was retained in the chondrocytes, forming cell aggregates and showing some round-shaped cells with less production of stress fibers on the G5 surface compared to fibroblast-like chondrocytes with abundant stress fibers on the PS surface. The numbers of P2 chondrocytes on the G5 and PS surfaces were nearly the same and sufficient for construction of chondrocyte sheets using a temperature-responsive plate. Without a supporting material during cell sheet manipulation, chondrocyte sheets spontaneously detached and exhibited a honeycomb-like structure of stress fibers. Unlike the chondrocyte sheets constructed from cells on the PS surface, the chondrocyte sheets from cells on the G5 surface had higher chondrogenic activities, as evidenced by the high expression of chondrogenic markers and the low expression of dedifferentiation markers.The one-step process of cell expansion and maintenance of chondrogenic activity could be obtained using the G5 surface. Human chondrocyte sheets were successfully constructed with high chondrogenic activity. These findings may lead to an alternative cultivation technique for human chondrocytes that offers high clinical potential in autologous chondrocyte implantation.
Project description:Methacrylated hyaluronic acid (MeHA) and chondroitin sulfate (CS)-biofunctionalized MeHA (CS-MeHA), were crosslinked in the presence of a matrix metalloproteinase 7 (MMP7)-sensitive peptide. The synthesized hydrogels were embedded with either human mesenchymal stem cells (hMSCs) or chondrocytes, at low concentrations, and subsequently cultured in a stem cell medium (SCM) or chondrogenic induction medium (CiM). The pivotal role of the synthesized hydrogels in promoting the expression of cartilage-related genes and the formation of neocartilage tissue despite the low concentration of encapsulated cells was assessed. It was found that hMSC-laden MeHA hydrogels cultured in an expansion medium exhibited a significant increase in the expression of chondrogenic markers compared to hMSCs cultured on a tissue culture polystyrene plate (TCPS). This favorable outcome was further enhanced for hMSC-laden CS-MeHA hydrogels, indicating the positive effect of the glycosaminoglycan binding peptide on the differentiation of hMSCs towards a chondrogenic phenotype. However, it was shown that an induction medium is necessary to achieve full span chondrogenesis. Finally, the histological analysis of chondrocyte-laden MeHA hydrogels cultured on an ex vivo osteochondral platform revealed the deposition of glycosaminoglycans (GAGs) and the arrangement of chondrocyte clusters in isogenous groups, which is characteristic of hyaline cartilage morphology.