Regenerative Potential of Platelet Concentrate Lysate in Mechanically Injured Cartilage and Matrix-Associated Chondrocyte Implantation In Vitro

Adjuvant therapy in autologous chondrocyte implantation (ACI) can control the post-traumatic environment and guide graft maturation to support cartilage repair. To investigate both aspects, we examined potential chondro-regenerative effects of lysed platelet concentrate (PC) and supplementary interleukin 10 (IL-10) on mechanically injured cartilage and on clinically used ACI scaffolds. ACI remnants and human cartilage explants, which were applied to an uniaxial unconfined compression as injury model, were treated with human IL-10 and/or PC from thrombocyte concentrates. We analyzed nuclear blebbing/TUNEL, sGAG content, immunohistochemistry, and the expression of COL1A1, COL2A1, COL10A1, SOX9, and ACAN. Post-injuriously, PC was associated with less cell death, increased COL2A1 expression, and decreased COL10A1 expression and, interestingly, the combination with Il-10 or Il-10 alone had no additional effects, except on COL10A1, which was most effectively decreased by the combination of PC and Il-10. The expression of COL2A1 or SOX9 was statistically not modulated by these substances. In contrast, in chondrocytes in ACI grafts the combination of PC and IL-10 had the most pronounced effects on all parameters except ACAN. Thus, using adjuvants such as PC and IL-10, preferably in combination, is a promising strategy for enhancing repair and graft maturation of autologous transplanted chondrocytes after cartilage injury.

Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast–cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p < 0.0001) except when co-cultured on dentine, when osteoclast formation was inhibited (p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their ‘native’ tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.

Strategies to Modulate the Redifferentiation of Chondrocytes

Because of the low self-healing capacity of articular cartilage, cartilage injuries and degenerations triggered by various diseases are almost irreversible. Previous studies have suggested that human chondrocytes cultured in vitro tend to dedifferentiate during the cell-amplification phase and lose the physiological properties and functions of the cartilage itself, which is currently a critical limitation in the cultivation of cartilage for tissue engineering. Recently, numerous studies have focused on the modulation of chondrocyte redifferentiation. Researchers discovered the effect of various conditions (extracellular environment, cell sources, growth factors and redifferentiation inducers, and gene silencing and overexpression) on the redifferentiation of chondrocytes during the in vitro expansion of cells, and obtained cartilage tissue cultured in vitro that exhibited physiological characteristics and functions that were similar to those of human cartilage tissue. Encouragingly, several studies reported positive results regarding the modulation of the redifferentiation of chondrocytes in specific conditions. Here, the various factors and conditions that modulate the redifferentiation of chondrocytes, as well as their limitations and potential applications and challenges are reviewed. We expect to inspire research in the field of cartilage repair toward the future treatment of arthropathy.

Progenitor Cells in Healthy and Osteoarthritic Human Cartilage Have Extensive Culture Expansion Capacity while Retaining Chondrogenic Properties

Objective Articular cartilage-derived progenitor cells (ACPCs) are a potential new cell source for cartilage repair. This study aims to characterize endogenous ACPCs from healthy and osteoarthritic (OA) cartilage, evaluate their potential for cartilage regeneration, and compare this to cartilage formation by chondrocytes. Design ACPCs were isolated from full-thickness healthy and OA human cartilage and separated from the total cell population by clonal growth after differential adhesion to fibronectin. ACPCs were characterized by growth kinetics, multilineage differentiation, and surface marker expression. Chondrogenic redifferentiation of ACPCs was compared with chondrocytes in pellet cultures. Pellets were assessed for cartilage-like matrix production by (immuno)histochemistry, quantitative analyses for glycosaminoglycans and DNA content, and expression of chondrogenic and hypertrophic genes. Results Healthy and OA ACPCs were successfully differentiated toward the adipogenic and chondrogenic lineage, but failed to produce calcified matrix when exposed to osteogenic induction media. Both ACPC populations met the criteria for cell surface marker expression of mesenchymal stromal cells (MSCs). Healthy ACPCs cultured in pellets deposited extracellular matrix containing proteoglycans and type II collagen, devoid of type I collagen. Gene expression of hypertrophic marker type X collagen was lower in healthy ACPC pellets compared with OA pellets. Conclusions This study provides further insight into the ACPC population in healthy and OA human articular cartilage. ACPCs show similarities to MSCs, yet do not produce calcified matrix under well-established osteogenic culture conditions. Due to extensive proliferative potential and chondrogenic capacity, ACPCs show potential for cartilage regeneration and possibly for clinical application, as a promising alternative to MSCs or chondrocytes.

Microbial Transglutaminase Improves ex vivo Adhesion of Gelatin Methacryloyl Hydrogels to Human Cartilage

Current surgical techniques to treat articular cartilage defects fail to produce a satisfactory long-term repair of the tissue. Regenerative approaches show promise in their ability to generate hyaline cartilage using biomaterials in combination with stem cells. However, the difficulty of seamlessly integrating the newly generated cartilage with the surrounding tissue remains a likely cause of long-term failure. To begin to address this integration issue, our strategy exploits a biological enzyme (microbial transglutaminase) to effect bioadhesion of a gelatin methacryloyl implant to host tissue. Mechanical characterization of the bioadhesive material shows that enzymatic crosslinking is compatible with photocrosslinking, allowing for a dual-crosslinked system with improved mechanical properties, and a slower degradation rate. Biocompatibility is illustrated with a 3D study of the metabolic activity of encapsulated human adipose derived stem cells. Furthermore, enzymatic crosslinking induced by transglutaminase is not prevented by the presence of cells, as measured by the bulk modulus of the material. Adhesion to human cartilage is demonstrated ex vivo with a significant increase in adhesive strength (5.82 ± 1.4 kPa as compared to 2.87 ± 0.9 kPa, p &lt; 0.01) due to the addition of transglutaminase. For the first time, we have characterized a bioadhesive material composed of microbial transglutaminase and GelMA that can encapsulate cells, be photo crosslinked, and bond to host cartilage, taking a step toward the integration of regenerative implants.

Effects of long-term deep freezing on human femoral cartilage: differential scanning calorimetric (DSC) analysis and histopathological evaluations

Results from our previous study proved that differential scanning calorimetry (DSC) is a reliable method for qualitative evaluation of morphological changes in human cartilage samples caused by cryopreservation and storage time correlates with graft quality [1, 2]. Our current aim was to specify/characterise the effects of deep-freezing on hyaline cartilage in conjunction with length of storage time. Detailed analysis included DSC and histological measurements which showed distinctive differences. Based on our histological examinations, we could not confirm significant association between cartilage destruction and long-time storage, but the DSC results exhibited a definite change in thermal parameters after the 6th week cryopreservation. A modified and more detailed analysis would be useful to determine the effects of deep-freezing.

SPRY4 acts as an indicator of osteoarthritis severity and regulates chondrocyte hypertrophy and ECM protease expression

Osteoarthritis (OA) causes serious changes in the metabolic and signaling pathways of chondrocytes, including the mitogen-activated protein kinase (MAPK) pathway. However, the role of sprouty RTK signaling antagonist 4 (SPRY4), an inhibitor of MAPK, in the human cartilage tissues and chondrocytes remains to be understood. Here, using SPRY4 gene delivery into healthy and degenerated chondrocytes, we elucidated the role of SPRY4 in preventing chondrocyte hypertrophy. In addition to using the human cartilage tissues with the destabilization of the medial meniscus (DMM) model in Sprague-Dawley (SD) rats, the role of SPRY4 in cartilage tissues and chondrocytes was explored through their molecular and histological analyses. In order to determine the effects of SPRY4 on healthy human chondrocyte hypertrophy, small interfering RNA (siRNA) was used to knock down SPRY4. Lentiviral transduction of SPRY4 into degenerated human chondrocytes allowed us to investigate its ability to prevent hypertrophy. SPRY4 expression levels were higher in healthy human cartilage tissue and chondrocytes than in degenerated human cartilage tissues and hypertrophy-induced chondrocytes. The knockdown of SPRY4 in healthy chondrocytes caused an increase in hypertrophy, senescence, reactive oxygen species (ROS) production, and extracellular matrix (ECM) protease expression. However, all these factors decreased upon overexpression of SPRY4 in degenerated chondrocytes via regulation of the MAPK signaling pathway. We conclude that SPRY4 is a crucial indicator of osteoarthritis (OA) severity and could play an important role in preventing OA in the cartilage by inhibiting chondrocyte hypertrophy.

Zone-Dependent Architecture and Biochemical Composition of Decellularized Porcine Nasal Cartilage Modulate the Activity of Adipose Tissue-Derived Stem Cells in Cartilage Regeneration

Previous anatomical studies have shown different functional zones in human nasal septal cartilage (NC). These zones differ in respect to histological architecture and biochemical composition. The aim of this study was to investigate the influence of these zones on the fate of stem cells from a regenerative perspective. Therefore, decellularized porcine septal cartilage was prepared and subjected to histological assessment to demonstrate its equivalence to human cartilage. Decellularized porcine NC (DPNC) exposed distinct surfaces depending on two different histological zones: the outer surface (OS), which is equivalent to the superficial zone, and the inner surface (IS), which is equivalent to the central zone. Human adipose tissue-derived stem cells (ASCs) were isolated from the abdominal fat tissue of five female patients and were seeded on the IS and OS of DPNC, respectively. Cell seeding efficiency (CSE), vitality, proliferation, migration, the production of sulfated glycosaminoglycans (sGAG) and chondrogenic differentiation capacity were evaluated by histological staining (DAPI, Phalloidin, Live-Dead), biochemical assays (alamarBlue®, PicoGreen®, DMMB) and the quantification of gene expression (qPCR). Results show that cell vitality and CSE were not influenced by DPNC zones. ASCs, however, showed a significantly higher proliferation and elevated expression of early chondrogenic differentiation, as well as fibrocartilage markers, on the OS. On the contrary, there was a significantly higher upregulation of hypertrophy marker MMP13 (p < 0.0001) and GAG production (p = 0.0105) on the IS, whereas cell invasion into the three-dimensional DPNC was higher in comparison to the OS. We conclude that the zonal-dependent distinct architecture and composition of NC modulates activities of ASCs seeded on DPNC. These findings might be used for engineering of cartilage substitutes needed in facial reconstructive surgery that yield an equivalent histological and functional structure, such as native NC.