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Introduction Osteoarthritis is a highly prevalent, age-associated joint disease characterized by cartilage degeneration, joint dysfunction, and chronic pain. We previously developed a bone morphogenetic protein 7 derived peptide p[63–82], which may be a novel disease-modifying treatment option for OA. In this study we aimed to optimize the bioactivity and biostability of this peptide in the intra-articular environment to evaluate the therapeutic potential of these peptides to treat osteoarthritis. Methods 33 peptide modifications of p[63–82] were custom-designed and synthesized to optimize the bioactivity. Chondrocytes and synovial fluid were collected from end-stage osteoarthritic patients at total knee arthroplasty surgery. To validate improvements in bioactivity, gene expression analysis, glycosaminoglycan content, matrix metalloproteinase-13 protein levels and alkaline phosphatase activity was measured. Results Several biochemical approaches were used to explore optimization of the original p[63–82] peptide. One cyclized peptide (C2) was able to significantly increase the expression of collagen type 2 and decrease expression of collagen type 10 , matrix metalloproteinase-13 and prostaglandin-endoperoxide synthase 2 . The linear p[63–82] peptide and the cyclic peptide variant C2 in the same concentration were effective in suppressing the osteoartritic phenotype in SW1353 cells, despite the presence of interleukin-1β or osteoarthritic-synovial fluid. However, peptide variant C2 had a significantly more favorable bioactivity as compared to p[63–82] in reducing matrix metalloproteinase-13 protein levels in the osteoarthritic-synovial fluid exposed condition. At lower concentrations, the cyclic peptide C2 showed a higher bioactivity as compared to the linear p[63–82] peptide. When the activity of both peptides on primary human articular chondrocytes was evaluated, we found that the linear p[63–82] peptide as well as peptide C2 counteract the hypertrophic and inflammatory state of primary OA chondrocytes. Conclusion This study demonstrates that among various tested modifications of p[63–82], one cyclic variant (C2) showed similar results in bioactivity as compared to the linear peptide p[63–82], whilst the other modified peptide variants had inactive bioactive properties as compared to the original p[63–82] peptide. This highlights the challenge in enhancing peptide properties without compromising their biological activity and emphasises the need for a cautious approach in peptide modification for therapeutic use. This research underscores that while cyclization and other structural changes may offer benefits, they should be carefully evaluated on a case-by-case basis.

The fibrocartilage chondrocyte phenotype has been recognized to attribute to osteoarthritis (OA) development. These chondrocytes express genes related to unfavorable OA outcomes, emphasizing its importance in OA pathology. BMP7 is being explored as a potential disease-modifying molecule and attenuates the chondrocyte hypertrophic phenotype. On the other hand, BMP7 has been demonstrated to relieve organ fibrosis by counteracting the pro-fibrotic TGFβ-Smad3-PAI1 axis and increasing MMP2-mediated Collagen type I turnover. Whether BMP7 has anti-fibrotic properties in chondrocytes is unknown. Human OA articular chondrocytes (HACs) were isolated from end-stage OA femoral cartilage (total knee arthroplasty; n = 18 individual donors). SW1353 cells and OA HACs were exposed to 1 nM BMP7 for 24 h, after which gene expression of fibrosis-related genes and fibrosis-mediating factors was determined by RT-qPCR. In SW1353, Collagen type I protein levels were determined by immunocytochemistry and western blotting. PAI1 and MMP2 protein levels and activity were measured with an ELISA and activity assays, respectively. MMP2 activity was inhibited with the selective MMP-2 inhibitor OA-Hy. SMAD3 activity was determined by a (CAGA) 12 -reporter assay, and pSMAD2 levels by western blotting. Following BMP7 exposure, the expression of fibrosis-related genes was reduced in SW1353 cells and OA HACs. BMP7 reduced Collagen type I protein levels in SW1353 cells. Gene expression of MMP2 was increased in SW1353 cells following BMP7 treatment. BMP7 reduced PAI1 protein levels and -activity, while MMP2 protein levels and -activity were increased by BMP7. BMP7-dependent inhibition of Collagen type I protein levels in SW1353 cells was abrogated when MMP2 activity was inhibited. Finally, BMP7 reduced pSMAD2 levels determined by western blotting and reduced SMAD3 transcriptional activity as demonstrated by decreased (CAGA) 12 luciferase reporter activity. Our data demonstrate that short-term exposure to BMP7 decreases the fibrocartilage chondrocyte phenotype. The BMP7-dependent reduction of Collagen type I protein expression seems MMP2-dependent and inhibition of Smad2/3-PAI1 activity was identified as a potential pathway via which BMP7 exerts its anti-fibrotic action. This indicates that in chondrocytes BMP7 may have a double mode-of-action by targeting both the hypertrophic as well as the fibrotic chondrocyte phenotype, potentially adding to the clinical relevance of using BMP7 as an OA disease-modifying molecule.

BMP7 is a morphogen capable of counteracting the OA chondrocyte hypertrophic phenotype via NKX3-2. NKX3-2 represses expression of RUNX2, an important transcription factor for chondrocyte hypertrophy. Since RUNX2 has previously been described as an inhibitor for 47S pre-rRNA transcription, we hypothesized that BMP7 positively influences 47S pre-rRNA transcription through NKX3-2, resulting in increased protein translational capacity. Therefor SW1353 cells and human primary chondrocytes were exposed to BMP7 and rRNA (18S, 5.8S, 28S) expression was determined by RT-qPCR. NKX3-2 knockdown was achieved via transfection of a NKX3-2-specific siRNA duplex. Translational capacity was assessed by the SUNsET assay, and 47S pre-rRNA transcription was determined by transfection of a 47S gene promoter-reporter plasmid. BMP7 treatment increased protein translational capacity. This was associated by increased 18S and 5.8S rRNA and NKX3-2 mRNA expression, as well as increased 47S gene promotor activity. Knockdown of NKX3-2 led to increased expression of RUNX2, accompanied by decreased 47S gene promotor activity and rRNA expression, an effect BMP7 was unable to restore. Our data demonstrate that BMP7 positively influences protein translation capacity of SW1353 cells and chondrocytes. This is likely caused by an NKX3-2-dependent activation of 47S gene promotor activity. This finding connects morphogen-mediated changes in cellular differentiation to an aspect of ribosome biogenesis via key transcription factors central to determining the chondrocyte phenotype.

Osteoarthritis is the most common degenerative joint disorder. MicroRNAs are gene expression regulators that act post-transcriptionally to control tissue homeostasis. Microarray analysis was undertaken in osteoarthritic intact, lesioned and young intact cartilage. Principal component analysis showed that young intact cartilage samples were clustered together; osteoarthritic samples had a wider distribution; and osteoarthritic intact samples were separated into two subgroups, osteoarthritic-Intact-1 and osteoarthritic-Intact-2. We identified 318 differentially expressed microRNAs between young intact and osteoarthritic lesioned cartilage, 477 between young intact and osteoarthritic-Intact-1 cartilage and 332 between young intact and osteoarthritic-Intact-2 cartilage samples. For a selected list of differentially expressed microRNAs, results were verified in additional cartilage samples using qPCR. Of the validated DE microRNAs, four—miR-107, miR-143-3p, miR-361-5p and miR-379-5p—were selected for further experiments in human primary chondrocytes treated with IL-1β. Expression of these microRNAs decreased in human primary chondrocytes treated with IL-1β. For miR-107 and miR-143-3p, gain- and loss-of-function approaches were undertaken and associated target genes and molecular pathways were investigated using qPCR and mass spectrometry proteomics. Analyses showed that WNT4 and IHH, predicted targets of miR-107, had increased expression in osteoarthritic cartilage compared to young intact cartilage and in primary chondrocytes treated with miR-107 inhibitor, and decreased expression in primary chondrocytes treated with miR-107 mimic, suggesting a role of miR-107 in chondrocyte survival and proliferation. In addition, we identified an association between miR-143-3p and EIF2 signalling and cell survival. Our work supports the role of miR-107 and miR-143-3p in important chondrocyte mechanisms regulating proliferation, hypertrophy and protein translation.

Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ). The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.

NF-κB/p65 has been reported to be involved in regulation of chondrogenic differentiation. However, its function in relation to key chondrogenic factor Sox9 and onset of chondrogenesis during endochondral ossification is poorly understood. We hypothesized that the early onset of chondrogenic differentiation is initiated by transient NF-κB/p65 signaling. The role of NF-κB/p65 in early chondrogenesis was investigated in different in vitro, ex vivo and in vivo endochondral models: ATDC5 cells, hBMSCs, chicken periosteal explants and growth plates of 6 weeks old mice. NF-κB/p65 activation was manipulated using pharmacological inhibitors, RNAi and activating agents. Gene expression and protein expression analysis, and (immuno)histochemical stainings were employed to determine the role of NF-κB/p65 in the chondrogenic phase of endochondral development. Our data show that chondrogenic differentiation is facilitated by early transient activation of NF-κB/p65. NF-κB/p65-mediated signaling determines early expression of Sox9 and facilitates the subsequent chondrogenic differentiation programming by signaling through key chondrogenic pathways. The presented data demonstrate that NF-κB/p65 signaling, as well as its intensity and timing, represents one of the transcriptional regulatory mechanisms of the chondrogenic developmental program of chondroprogenitor cells during endochondral ossification. Importantly, these results provide novel possibilities to improve the success of cartilage and bone regenerative techniques.

The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by hypertrophic differentiation of the engineered cartilaginous tissue caused by endochondral ossification. Since a healthy cartilage matrix contains high amounts of Aggrecan and COMP, we hypothesized that their supplementation in the biogel used in the generation of subperiosteal cartilage mimics the composition of the cartilage extracellular matrix environment, with beneficial properties for the engineered cartilage. Supplementation of COMP or Aggrecan was studied in vitro during chondrogenic differentiation of rabbit periosteum cells and periosteum-derived chondrocytes. Low melting agarose was supplemented with bovine Aggrecan, human recombinant COMP or vehicle and was injected between the bone and periosteum at the upper medial side of the tibia of New Zealand white rabbits. Generated subperiosteal cartilage tissue was analyzed for weight, GAG and DNA content and ALP activity. Key markers of different phases of endochondral ossification were measured by RT-qPCR. For the in vitro experiments, no significant differences in chondrogenic marker expression were detected following COMP or Aggrecan supplementation, while in vivo favorable chondrogenic marker expression was detected. Gene expression levels of hypertrophic markers as well as ALP activity were significantly decreased in the Aggrecan and COMP supplemented conditions compared to controls. The wet weight and GAG content of the in vivo generated subperiosteal cartilage tissue was not significantly different between groups. Data demonstrate the potential of Aggrecan and COMP to favorably influence the subperiosteal microenvironment for the in vivo generation of cartilage for the optimization of cartilage regenerative approaches.

NSAIDs are used to relieve pain and decrease inflammation by inhibition of cyclooxygenase (COX)-catalyzed prostaglandin (PG) synthesis. PGs are fatty acid mediators involved in cartilage homeostasis, however the action of their synthesizing COX-enzymes in cartilage differentiation is not well understood. In this study we hypothesized that COX-1 and COX-2 have differential roles in chondrogenic differentiation. ATDC5 cells were differentiated in the presence of COX-1 (SC-560, Mofezolac) or COX-2 (NS398, Celecoxib) specific inhibitors. Specificity of the NSAIDs and inhibition of specific prostaglandin levels were determined by EIA. Prostaglandins were added during the differentiation process. Chondrogenic outcome was determined by gene- and protein expression analyses. Inhibition of COX-1 prevented Col2a1 and Col10a1 expression. Inhibition of COX-2 resulted in decreased Col10a1 expression, while Col2a1 remained unaffected. To explain this difference expression patterns of both COX-enzymes as well as specific prostaglandin concentrations were determined. Both COX-enzymes are upregulated during late chondrogenic differentiation, whereas only COX-2 is briefly expressed also early in differentiation. PGD2 and PGE2 followed the COX-2 expression pattern, whereas PGF2α and TXA2 levels remained low. Furthermore, COX inhibition resulted in decreased levels of all tested PGs, except for PGD2 and PGF2α in the COX-1 inhibited condition. Addition of PGE2 and PGF2α resulted in increased expression of chondrogenic markers, whereas TXA2 increased expression of hypertrophic markers. Our findings point towards a differential role for COX-enzymes and PG-production in chondrogenic differentiation of ATDC5 cells. Ongoing research is focusing on further elucidating the functional partition of cyclooxygenases and specific prostaglandin production.

Treatment of full-thickness damage to hyaline cartilage is hampered by the limited availability of autologous healthy cartilage and the lengthy, cost-prohibitive cell isolation and expansion steps associated with autologous cartilage implantation (ACI). Here we report a strategy for de novo engineering of ectopic autologous cartilage (EAC) within the subperiosteal space (in vivo bioreactor), through the mere introduction of a biocompatible gel that might promote hypoxia-mediated chondrogenesis, thereby effectively overcoming the aforementioned limitations. The EAC is obtained within 3 wk post injection of the gel, and can be press-fit into an osteochondral defect where it undergoes remodeling with good lateral and subchondral integration. The implanted EAC showed no calcification even after 9 mo and attained an average O'Driscoll score of 11 (versus 4 for controls). An \"on demand\" autologous source of autologous cartilage with remodeling capacity is expected to significantly impact the clinical options in repair of trauma to articular cartilage.

In addition to the well-known cartilage extracellular matrix-related expression of Sox9, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox9: an immediate early and a late (extracellular matrix associated) phase expression. In this study, we aimed to determine what biological processes are driven by Sox9 during this early phase of chondrogenic differentiation. Sox9 expression in ATDC5 cells was knocked down by siRNA transfection at the day before chondrogenic differentiation or at day 6 of differentiation. Samples were harvested at 2 h and 7 days of differentiation. The transcriptomes (RNA-seq approach) and proteomes (Label-free proteomics approach) were compared using pathway and network analyses. Total protein translational capacity was evaluated with the SuNSET assay, active ribosomes were evaluated with polysome profiling, and ribosome modus was evaluated with bicistronic reporter assays. Early Sox9 knockdown severely inhibited chondrogenic differentiation weeks later. Sox9 expression during the immediate early phase of ATDC5 chondrogenic differentiation regulated the expression of ribosome biogenesis factors and ribosomal protein subunits. This was accompanied by decreased translational capacity following Sox9 knockdown, and this correlated to lower amounts of active mono- and polysomes. Moreover, cap- versus IRES-mediated translation was altered by Sox9 knockdown. Sox9 overexpression was able to induce reciprocal effects to the Sox9 knockdown. Here, we identified an essential new function for Sox9 during early chondrogenic differentiation. A role for Sox9 in regulation of ribosome amount, activity, and/or composition may be crucial in preparation for the demanding proliferative phase and subsequent cartilage extracellular matrix production of chondroprogenitors in the growth plate .