Cartilage Extracellular Matrix Production Research Articles

Liquid platelet-rich fibrin produced via horizontal centrifugation decreases the inflammatory response and promotes chondrocyte regeneration in vitro.

Objective: Recently, liquid platelet-rich fibrin (PRF), a rich source of concentrated platelets and growth factors, has emerged as a promising agent for stimulating tissue regeneration. However, its specific efficacy in chondrocyte proliferation and cartilage regeneration remains underexplored. To address this question, we investigated liquid PRF obtained through horizontal centrifugation and compared its effects with hyaluronic acid (HA), a high molecular weight glucosamine supplement widely used in clinical practice to safeguard against chondral damage. Materials and Methods: Liquid PRF, produced using horizontal centrifugation (liquid H-PRF) at 500g for 8min, served as our experimental agent. We conducted cell viability and proliferation assays using PRF-conditioned medium. We assessed the chondrocyte phenotype of ATDC5 cells through toluidine blue and alcian blue staining, real-time polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence staining. Furthermore, we examined the expression of genes involved in inflammation through RT-PCR and Western blot analysis. Results: Liquid H-PRF exerted notable effects on chondrocytes, influencing proliferation, inflammatory responses, and chondrogenic differentiation. The H-PRF group displayed significantly higher expression of chondrogenic markers, including Col2a1, compared to HA-treated cells, whereas aggrecan expression was significantly higher in the HA group. PRF also demonstrated the ability to reduce inflammatory levels in chondrogenic ATDC5 cells, and this effect was further enhanced when PRF from the buffy coat zone was added. In comparison, chondrocytes cultured in the HA group produced significantly fewer inflammatory factors than those in the PRF group, as confirmed qualitatively by Western blot analysis. Conclusion: Liquid H-PRF emerged as a potent stimulator for chondrogenesis and a regulator of the inflammatory response, achieving levels similar to HA. Moreover, liquid H-PRF exhibited strong potential for enhancing the production of cartilage extracellular matrix and promoting chondrogenic regeneration with notably increased Col2a1 levels. Future research should encompass animal studies and human trials to further evaluate the comparative effectiveness of liquid PRF versus HA, potentially as an alternative or complementary strategy for future clinical applications.

Reinforcement of Hydrogels with a 3D-Printed Polycaprolactone (PCL) Structure Enhances Cell Numbers and Cartilage ECM Production under Compression

Hydrogels show promise in cartilage tissue engineering (CTE) by supporting chondrocytes and maintaining their phenotype and extracellular matrix (ECM) production. Under prolonged mechanical forces, however, hydrogels can be structurally unstable, leading to cell and ECM loss. Furthermore, long periods of mechanical loading might alter the production of cartilage ECM molecules, including glycosaminoglycans (GAGs) and collagen type 2 (Col2), specifically with the negative effect of stimulating fibrocartilage, typified by collagen type 1 (Col1) secretion. Reinforcing hydrogels with 3D-printed Polycaprolactone (PCL) structures offer a solution to enhance the structural integrity and mechanical response of impregnated chondrocytes. This study aimed to assess the impact of compression duration and PCL reinforcement on the performance of chondrocytes impregnated with hydrogel. Results showed that shorter loading periods did not significantly affect cell numbers and ECM production in 3D-bioprinted hydrogels, but longer periods tended to reduce cell numbers and ECM compared to unloaded conditions. PCL reinforcement enhanced cell numbers under mechanical compression compared to unreinforced hydrogels. However, the reinforced constructs seemed to produce more fibrocartilage-like, Col1-positive ECM. These findings suggest that reinforced hydrogel constructs hold potential for in vivo cartilage regeneration and defect treatment by retaining higher cell numbers and ECM content. To further enhance hyaline cartilage ECM formation, future studies should focus on adjusting the mechanical properties of reinforced constructs and exploring mechanotransduction pathways.

Platelet-Rich Plasma Does Not Inhibit Inflammation or Promote Regeneration in Human Osteoarthritic Chondrocytes In Vitro Despite Increased Proliferation.

ObjectiveThe aims of the study were to assess the anti-inflammatory properties of platelet-rich plasma (PRP) and investigate its regenerative potential in osteoarthritic (OA) human chondrocytes. We hypothesized that PRP can modulate the inflammatory response and stimulate cartilage regeneration.DesignPrimary human chondrocytes from OA knees were treated with manually prepared PRP, after which cell migration and proliferation were assessed. Next, tumor necrosis factor-α–stimulated chondrocytes were treated with a range of concentrations of PRP. Expression of genes involved in inflammation and chondrogenesis was determined by real-time polymerase chain reaction. In addition, chondrocytes were cultured in PRP gels and fibrin gels consisting of increasing concentrations of PRP. The production of cartilage extracellular matrix (ECM) was assessed. Deposition and release of glycosaminoglycans (GAG) and collagen was quantitatively determined and visualized by (immuno)histochemistry. Proliferation was assessed by quantitative measurement of DNA.ResultsBoth migration and the inflammatory response were altered by PRP, while proliferation was stimulated. Expression of chondrogenic markers COL2A1 and ACAN was downregulated by PRP, independent of PRP concentration. Chondrocytes cultured in PRP gel for 28 days proliferated significantly more when compared with chondrocytes cultured in fibrin gels. This effect was dose dependent. Significantly less GAGs and collagen were produced by chondrocytes cultured in PRP gels when compared with fibrin gels. This was qualitatively confirmed by histology.ConclusionsPRP stimulated chondrocyte proliferation, but not migration. Also, production of cartilage ECM was strongly downregulated by PRP. Furthermore, PRP did not act anti-inflammatory on chondrocytes in an in vitro inflammation model.

The Follistatin-like Protein 1 Pathway Is Important for Maintaining Healthy Articular Cartilage.

ObjectiveWe sought to determine whether follistatin‐like protein 1 (FSTL1), a protein produced by articular chondrocytes, promotes healthy articular cartilage and prevents chondrocytes from undergoing terminal differentiation to hypertrophic cells.MethodsIn vitro experiments were performed with immortalized human articular chondrocytes. The cells were transduced with a lentivirus encoding human FSTL1 small hairpin RNA or with an adenovirus encoding FSTL1. A quantitative polymerase chain reaction was used for gene expression analysis. Protein expression was assessed by Western blotting. Co‐immunoprecipitation was used to identify interacting partners of FSTL1. FSTL1 expression in human articular cartilage was analyzed using confocal microscopy.ResultsDownregulation of FSTL1 expression in transforming growth factor β (TGFβ)‐stimulated chondrocyte pellet cultures led to chondrocyte terminal differentiation characterized by poor production of cartilage extracellular matrix and altered expression of genes and proteins involved in cartilage homeostasis, including MMP13, COL10A1, RUNX2, COL2A1, ACAN, Sox9, and phospho‐Smad3. We also showed that FSTL1 interacts with TGFβ receptor proteins, Alk1 and endoglin, suggesting a potential mechanism for its effects on chondrocytes. Transduction of chondrocytes with an FSTL1 transgene increased COL2A1 expression, whereas it did not affect MMP13 expression. FSTL1 protein expression was decreased in human osteoarthritic cartilage in situ.ConclusionOur data suggest that FSTL1 plays an important role in maintaining healthy articular cartilage and the FSTL1 pathway may represent a therapeutic target for degenerative diseases of cartilage.

Platelet lysate is beneficial for chondrocyte expansion but ineffective in 3d culture

Purpose: The purpose of this study is to define the potential of platelet lysate as a xeno-free alternative in expansion of human chondrocytes for cell therapy purposes. Additionally, the effects of using platelet lysate for redifferentiation of chondrocytes in 3D pellets are assessed. Methods: Platelet lysate was prepared from human blood and platelet enrichment was measured. Chondrocytes were isolated from human osteoarthritic cartilage (age 55-84, mean 66 years) and subjected to a range of concentrations of platelet lysate in monolayer culture. Cell proliferation and morphology were assessed after 7 days. In addition, expression of chondrogenic genes was determined by quantitative real-time PCR. Next, platelet lysate-expanded chondrocytes (passage 2) were cultured in 3D cell pellets without any additional growth factors and cartilage matrix production was assessed after 28 days. Glycosaminoglycan (GAG) and collagen production in the pellets were quantified by biochemical analyses as well as (immuno)histochemistry. Moreover, platelet lysate was supplemented into redifferentiation medium of chondrocytes in 3D pellets. Cartilage matrix production was assessed with similar outcomes. Results: Platelet lysate had a dose-dependent effect on chondrocyte proliferation, but expression of chondrogenic markers was decreased in monolayers when compared to bovine serum-expanded monolayers. After 28 days of subsequent 3D pellet culture, GAG production was significantly higher in pellets consisting of chondrocytes expanded with 1% platelet lysate compared to controls (figure 1). When used for redifferentiation of chondrocyte pellets, platelet lysate significantly decreased the production of GAGs and collagen. This was confirmed by (immuno)histochemistry. Conclusions: In conclusion, low concentrations of platelet lysate stimulate chondrocyte proliferation in 2D monolayer and cartilage extracellular matrix production in subsequent 3D pellet culture. However, this does not work for high concentrations of platelet lysate. Furthermore, this study showed unfavourable effects on redifferentiation of 3D chondrocyte cultures in platelet lysate. Thus, platelet lysate can be seen as a promising xeno-free alternative to be used for in vitro expansion of chondrocytes for cartilage tissue engineering or cartilage repair therapies.

The matrix synthesis and anti-inflammatory effect of autologous leukocyte-poor platelet rich plasma in human cartilage explants.

To determine the effects of autologous leukocyte-poor platelet-rich plasma (LP-PRP) on the expression of markers involved in cartilage-extracellular matrix production and inflammation in cartilage explants bearing osteoarthritis. Cartilage explants and LP-PRP were obtained from 10 patients who underwent total knee arthroplasty. The explants were cultured in spinner flasks for 28 days in the presence of interleukin (IL)-1β and/or LP-PRP. The gene expression of catabolic (MMP13, ADAMTS5, and IL1β) and anabolic factors (COL2A1, ACAN, and SOX9) was quantified. A histological assessment was performed according to a modified Mankin score, and quantification of type II and I collagen deposition. The gene expression of catabolic factors and the Mankin score were lower in LP-PRP- and LP-PRP/IL-1β- than in IL-1β-treated explants, suggesting less matrix degradation in explants cultured in the presence of LP-PRP. Higher expression of genes involved in cartilage matrix restoration was observed in LP-PRP and LP-PRP/IL-1β- when compared to IL-1β-treated explants. The explants treated with LP-PRP and LP-PRP/IL-1β exhibited a higher deposition of type II collagen as well as a lower deposition of type I collagen and also better surface integrity and a significant increase in the number of chondrocytes. LP-PRP treatment favored restoration in early osteoarthritic cartilage and reduced the pro-inflammatory effect of IL-1β. LP-PRP is a promising therapy for early osteoarthritis, as it promotes extracellular matrix repair, reduces inflammation, and slows cartilage degeneration.

MMP3 cleaves agrin in cartilage and reduces its chondrogenic potency

Purpose: Osteoarthritis is a chronic disabling disease characterized by cartilage breakdown for which there is no cure. We recently discovered that the heparan sulphate proteoglycan Agrin is expressed in cartilage, where it is essential for the maintenance of the chondrocytic phenotype and for the production of cartilage extracellular matrix whilst exogenous Agrin enhances chondrocyte differentiation and cartilage formation in vitro and in vivo. Here we study the mechanism that leads to loss of Agrin in OA and the functional consequences of Agrin cleavage by metalloproteinase 3 (MMP3). Methods: Agrin expression was determined by immunohistochemistry. Osteoarthritis was induced in 8 week old 129sv mice by destabilisation of the medial meniscus (DMM) and N-Agrin and C-Agrin expression was evaluated by immunofluorescence at 7 days and 8 weeks post-surgery. Ex vivo human cartilage samples were treated with MMP3 and the cleavage of C-Agrin was compared by immunofluorescence. The effect of MMP3 on Agrin transfected chondrocytes was analysed by qPCR. Results: We discovered that detection of Agrin in osteoarthritic samples is lost in earlier stages of disease progression when using an antibody detecting the C-terminal portion of Agrin compared to when using an antibody recognizing the N-terminal domain. This could be explained by the well-described MMP3 mediated Agrin cleavage which separates the N-terminal domain (predicted to adhere to the basement membrane through interaction with laminin) from the C-terminal domain. In keeping with this hypothesis, treatment of chondrocyte cultures or cartilage explants with recombinant MMP3 resulted in the loss of Agrin detection when using the C-terminal, but not the N-terminal antibody. Using antibodies specific to the N and C-terminal portions of Agrin, we found that C-Agrin is lost in the articular cartilage as early as 2days post surgical-induction of OA (destabilization of the medial meniscus), whereas N-Agrin remains highly detectable for at least 7days post surgical-induction of OA. In functional terms, treatment with recombinant MMP3 reduced the capacity of Agrin to upregulate SOX9 mRNA. Conversely, overexpression of Agrin in chondrocytes strongly downregulated MMP3 mRNA expression, thereby suggesting a negative feedback loop. Conclusions: The cleavage of Agrin by MMP-3 in chondrocytes may contribute to the progression of OA by reducing SOX9 transcription.

Modulation of chondrogenic differentiation of human mesenchymal stem cells in jellyfish collagen scaffolds by cell density and culture medium.

Studies on tissue-engineering approaches for the regeneration of traumatized cartilage focus increasingly on multipotent human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes. The present study applied porous scaffolds made of collagen from the jellyfish Rhopilema esculentum for the in vitro chondrogenic differentiation of hMSCs. Culture conditions in those scaffolds differ from conditions in high-density pellet cultures, making a re-examination of these data necessary. We systematically investigated the influence of seeding density, basic culture media [Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM)] with varying glucose content and supplementation with fetal calf serum (FCS) or bovine serum albumin (BSA) on the chondrogenic differentiation of hMSCs. Gene expression analyses of selected markers for chondrogenic differentiation and hypertrophic development were conducted. Furthermore, the production of cartilage extracellular matrix (ECM) was analysed by quantification of sulphated glycosaminoglycan and collagen type II contents. The strongest upregulation of chondrogenic markers, along with the highest ECM deposition was observed in scaffolds seeded with 2.4 × 106 cells/cm3 after cultivation in high-glucose DMEM and 0.125% BSA. Lower seeding densities compared to high-density pellet cultures were sufficient to induce in vitro chondrogenic differentiation of hMSCs in collagen scaffolds, which reduces the amount of cells required for the seeding of scaffolds and thus the monolayer expansion period. Furthermore, examination of the impact of FCS and α-MEM on chondrogenic MSC differentiation is an important prerequisite for the development of an osteochondral medium for simultaneous osteogenic and chondrogenic differentiation in biphasic scaffolds for osteochondral tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.

Immobilized lentivirus vector on chondroitin sulfate-hyaluronate acid-silk fibroin hybrid scaffold for tissue-engineered ligament-bone junction.

The lack of a fibrocartilage layer between graft and bone remains the leading cause of graft failure after anterior cruciate ligament (ACL) reconstruction. The objective of this study was to develop a gene-modified silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS) hybrid scaffold for reconstructing the fibrocartilage layer. The scaffold was fabricated by lyophilizing the CHS mixture with braided silk cables. The scanning electronic microscopy (SEM) showed that microporous CHS sponges were formed around silk cables. Each end of scaffold was modified with lentiviral-mediated transforming growth factor-β3 (TGF-β3) gene. The cells on scaffold were transfected by bonded lentivirus. In vitro culture demonstrated that mesenchymal stem cells (MSCs) on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of cartilage-specific genes also increased with culture time. After 2 weeks, the MSCs were distributed uniformly throughout scaffold. Deposited collagen was also found to increase. The chondral differentiation of MSCs was verified by expressions of collagen II and TGF-β3 genes in mRNA and protein level. Histology also confirmed the production of cartilage extracellular matrix (ECM) components. The results demonstrated that gene-modified silk cable-reinforced CHS scaffold was capable of supporting cell proliferation and differentiation to reconstruct the cartilage layer of interface.

Effect of flow perfusion conditions in the chondrogenic differentiation of bone marrow stromal cells cultured onto starch based biodegradable scaffolds

Cartilage tissue engineering (TE) typically involves the combination of a 3-D biodegradable polymeric support material, with primary chondrocytes or other cell types able to differentiate into chondrocytes. The culture environment in which cell–material constructs are created and stored is an important factor. Various bioreactors have been introduced in TE approaches to provide specific culturing environments that might promote and accelerate cells’ potential for chondrogenic differentiation and enhance the production of cartilage extracellular matrix. The aim of the present study was to investigate the chondrogenic differentiation of goat bone marrow cells (GBMCs) under flow perfusion culture conditions. For that purpose, GBMCs were seeded into starch–polycaprolactone fiber mesh scaffolds and cultured in a flow perfusion bioreactor for up to 28days using culture medium supplemented with transforming growth factor-β1. The tissue-engineered constructs were characterized after several end points (7, 14, 21 and 28days) by histological staining and immunocytochemistry analysis, as well as by glycosaminoglycan and alkaline phosphatase quantification assays. In addition, the expression of typical chondrogenic markers was assessed by real-time reverse-transcription polymerase chain reaction analysis. In general, the results obtained suggest that a flow perfusion microenvironment favors the chondrogenic potential of GBMCs.

Targeting Growth Factors in Arthritis: A Rational for Restoring the IGF-I Response in Chondrocytes

The role of growth factors in the pathogenesis of arthritis has received considerably less attention as compared to proinflammatory cytokines in recent years. This is understandable as it is evident that current anti-tumor necrosis factor-α (TNFα) therapy is efficacious in at least in 60% of the rheumatoid arthritis (RA) patients and when given in combination with methotrexate stops radiological progression of bone erosion. Other proinflammatory cytokines, such as interleukin-1 (IL-1) and IL-17 have also been implicated in the dysregulation of bone and cartilage remodelling characteristic of rheumatoid arthritis (RA) and/or osteoarthritis (OA). The direct effects of anti-cytokine treatment on cartilage pathology are not that easy to determine in humans and it remains to be seen if amelioration of cartilage destruction or better cartilage repair has been accomplished with anti-TNF therapy. A complicating factor is that chondrocytes in RA and OA become non-responsive to insulin-like growth factor-I (IGF-I). IGF-I is the main growth factor for stimulation of chondrocyte proteoglycan and collagen type II synthesis in order to maintain the cartilage matrix integrity. This article reviews our current understanding of the role of IGF-I on cartilage extracellular matrix production and the mechanisms underlying the cause of IGF-I non-responsiveness, and in particular the inhibition of IGF-receptor signalling by suppressor of cytokine signalling (SOCS) proteins. The epigenetic and cytokine-dependent regulation of SOCS expression may give an explanation for the loss of IGF-I signalling in chondrocytes in ageing-related diseases such as OA and RA. Whether IGF-1 is replaceable by other growth factors to maintain the cartilage extracellular matrix will be discussed. It is concluded that in addition to the current use of anti-cytokine therapy in arthritis the IGF-I response of chondrocytes must be restored in order to achieve cartilage matrix repair.