Cartilage Extracellular Matrix Formation Research Articles

3D bioprinting of ultrashort self-assembling peptides to engineer scaffolds with different matrix stiffness for chondrogenesis.

62Articular cartilage is a nonvascularized and poorly cellularized tissue with a low self-repair capacity. Therefore, damage to this tissue due to trauma or degenerative joint diseases such as osteoarthritis needs a high-end medical intervention. However, such interventions are costly, have limited healing capacity, and could impair patients' quality of life. In this regard, tissue engineering and three-dimensional (3D) bioprinting hold great potential. However, identifying suitable bioinks that are biocompatible, with the desired mechanical stiffness, and can be used under physiological conditions is still a challenge. In this study, we developed two tetrameric self-assembling ultrashort peptide bioinks that are chemically well-defined and can spontaneously form nanofibrous hydrogels under physiological conditions. The printability of the two ultrashort peptides was demonstrated; different shape constructs were printed with high shape fidelity and stability. Furthermore, the developed ultrashort peptide bioinks gave rise to constructs with different mechanical properties that could be used to guide stem cell differentiation toward specific lineages. Both ultrashort peptide bioinks demonstrated high biocompatibility and supported the chondrogenic differentiation of human mesenchymal stem cells. Additionally, the gene expression analysis of differentiated stem cells with the ultrashort peptide bioinks revealed articular cartilage extracellular matrix formation preference. Based on the different mechanical stiffness of the two ultrashort peptide bioinks, they can be used to fabricate cartilage tissue with different cartilaginous zones, including the articular and calcified cartilage zones, which are essential for engineered tissue integration.

Polycaprolactone Triol-Citrate Scaffolds Enriched with Human Platelet Releasates Promote Chondrogenic Phenotype and Cartilage Extracellular Matrix Formation.

In this paper we report the differentiating properties of platelet-rich plasma releasates (PRPr) on human chondrocytes within elastomeric polycaprolactone triol-citrate (PCLT-CA) porous scaffold. Human-derived chondrocyte cellular content of glycosaminoglycans (GAGs) and total collagen were determined after seeding into PCLT-CA scaffold enriched with PRPr cells. Immunostaining and real time PCR was applied to evaluate the expression levels of chondrogenic and extracellular gene markers. Seeding of chondrocytes into PCLT-CA scaffold enriched with PRPr showed significant increase in total collagen and GAGs production compared with chondrocytes grown within control scaffold without PRPr cells. The mRNA levels of collagen II and SOX9 increased significantly while the upregulation in Cartilage Oligomeric Matrix Protein (COMP) expression was statistically insignificant. We also report the reduction of the expression levels of collagen I and III in chondrocytes as a consequence of proximity to PRPr cells within the scaffold. Interestingly, the pre-loading of PRPr caused an increase of expression levels of following extracellular matrix (ECM) proteins: fibronectin, laminin and integrin β over the period of 3days. Overall, our results introduce the PCLT-CA elastomeric scaffold as a new system for cartilage tissue engineering. The method of PRPr cells loading prior to chondrocyte culture could be considered as a potential environment for cartilage tissue engineering as the differentiation and ECM formation is enhanced significantly.

Effects of musculoskeletal related growth factors on cartilage extracellular matrix formation

Effects of musculoskeletal related growth factors on cartilage extracellular matrix formation

Expression of xylosyltransferase-1 is modulated by fibronectin fragment in human articular chondrocytes

Purpose: Xylosyltransferase-1 (XT-1), encoded by xylt1 gene, is an essential anabolic enzyme to catalyze the initial and rate-determining step in glycosaminoglycan chain synthesis. The effect of fibronectin fragments (FN-fs), generated by proteolytic cleavage of FN and known as damage-associated molecular pattern (DAMP) molecules, on cartilage metabolism was poorly characterized. In this study we examined 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f)-mediated XT-1 expression mechanism and its signaling pathway, determining the role of 29-kDa FN-f in cartilage matrix synthesis. Methods: Human articular chondrocytes were enzymatically isolated from articular cartilage and cultured in monolayer. In 29-kDa FN-f-stimulated chondrocytes, the relative levels of mRNA and protein for XT-1 were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. In order to investigate the effects of 29-kDa FN-f on XT-1, human chondrocytes were transfected with small interfering RNAs (siRNAs) targeting TLR-2. Results: The level of aggrecan and XT-1 in human osteoarthritis cartilage was significantly decreased compared to normal cartilage. XT-1 expression in cultured primary articluar chondrocytes showed a periodic oscillation in both mRNA and protein level. 29-kDa FN-f significantly suppressed the mRNA and protein levels of XT-1 at 14 h and 24 h, respectively. Inhibition of mitogen activated protein kinase and nuclear factor-κB signaling pathway restored 29-kDa FN-f-inhibited XT-1 expression. Knockdown of toll like receptor-2 (TLR-2) using small interference RNA revealed that the decrease of XT-1 expression by 29-kDa FN-f is mediated by TLR-2 signaling pathway. In addition, Sp3, a repressor of XT-1 promoter, was up-regulated by 29-kDa FN-f. Knockdown and overexpression experiments revealed that XT-1 expression was modulated by 29-kDa FN-f-stimulated Sp3 in primary articular chondrocytes. XT- expression was promoted by AP-1 inhibitor and suppressed by AP-1 activator. Both c-jun and c-fos were activated by 29-kDa FN-f. Conclusions: These results demonstrated that 29-kDa FN-f plays a detrimental role in the regulation of cartilage extracellular matrix formation including XT-1 expression.

Comparative Proteomic Analysis of Normal and Collagen IX Null Mouse Cartilage Reveals Altered Extracellular Matrix Composition and Novel Components of the Collagen IX Interactome

Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.

Changes in the Chondrocyte and Extracellular Matrix Proteome during Post-natal Mouse Cartilage Development

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.

Inhibition of β‐catenin signaling in chondrocytes induces delayed fracture healing in mice

Appropriate and controlled chondrogenesis and endochondral ossification play fundamental roles in the fracture healing cascade, a regenerative process involved in highly coordinated biological events, including the Wnt/β-catenin signaling pathway. To examine the role and importance of this pathway in chondrocytes, we studied bone repair of closed tibias fractures in Col2a1-ICAT transgenic mice, in which the Wnt/β-catenin signaling pathway is specially inhibited in chondrocytes. Radiological, histological, and histomorphometric analyses at 7, 9, 12, 14, 21, and 28 days after fracture demonstrated the bone repairs were retarded in Col2a1-ICAT transgenic mice, due to reduced and delayed cartilage formation, chondrocyte hypertrophy, and bone generation. In addition, at 5 weeks, Col2a1-ICAT transgenic mice exhibited a weak mechanical tolerance to four-point bending. Furthermore, quantitative-PCR analysis revealed that the expression of genes associated specifically with cartilage extracellular matrix formation (collagen II, collagen X, and mmp13), bone remodeling (alp, collagen I, and osteocalcin), and vascular extravagation (vegf), and transcriptional activators involved in cartilage generation and ossification (sox9 and runx2) was decreased and delayed in the fracture sites of Col2a1-ICAT transgenic mice during healing. Collectively, these results suggest that Wnt/β-catenin signaling is critical for fracture healing, especially with respect to chondrogenesis and endochondral ossification. Thus, our study provides insight into the possible mechanisms of and therapeutic targets for improving normal facture repair and the healing of non-union fractures.

Comprehensive Profiling of Cartilage Extracellular Matrix Formation and Maturation Using Sequential Extraction and Label-free Quantitative Proteomics

Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n = 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n = 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo.