The Extracellular Matrix of Articular Cartilage Controls the Bioavailability of Pericellular Matrix-Bound Growth Factors to Drive Tissue Homeostasis and Repair

The extracellular matrix (ECM) has long been regarded as a packing material; supporting cells within the tissue and providing tensile strength and protection from mechanical stress. There is little surprise when one considers the dynamic nature of many of the individual proteins that contribute to the ECM, that we are beginning to appreciate a more nuanced role for the ECM in tissue homeostasis and disease. Articular cartilage is adapted to be able to perceive and respond to mechanical load. Indeed, physiological loads are essential to maintain cartilage thickness in a healthy joint and excessive mechanical stress is associated with the breakdown of the matrix that is seen in osteoarthritis (OA). Although the trigger by which increased mechanical stress drives catabolic pathways remains unknown, one mechanism by which cartilage responds to increased compressive load is by the release of growth factors that are sequestered in the pericellular matrix. These are heparan sulfate-bound growth factors that appear to be largely chondroprotective and displaced by an aggrecan-dependent sodium flux. Emerging evidence suggests that the released growth factors act in a coordinated fashion to drive cartilage repair. Thus, we are beginning to appreciate that the ECM is the key mechano-sensor and mechano-effector in cartilage, responsible for directing subsequent cellular events of relevance to joint health and disease.     

COMP and TSP-4: Functional Roles in Articular Cartilage and Relevance in Osteoarthritis

Osteoarthritis (OA) is a slow-progressing joint disease, leading to the degradation and remodeling of the cartilage extracellular matrix (ECM). The usually quiescent chondrocytes become reactivated and accumulate in cell clusters, become hypertrophic, and intensively produce not only degrading enzymes, but also ECM proteins, like the cartilage oligomeric matrix protein (COMP) and thrombospondin-4 (TSP-4). To date, the functional roles of these newly synthesized proteins in articular cartilage are still elusive. Therefore, we analyzed the involvement of both proteins in OA specific processes in in vitro studies, using porcine chondrocytes, isolated from femoral condyles. The effect of COMP and TSP-4 on chondrocyte migration was investigated in transwell assays and their potential to modulate the chondrocyte phenotype, protein synthesis and matrix formation by immunofluorescence staining and immunoblot. Our results demonstrate that COMP could attract chondrocytes and may contribute to a repopulation of damaged cartilage areas, while TSP-4 did not affect this process. In contrast, both proteins similarly promoted the synthesis and matrix formation of collagen II, IX, XII and proteoglycans, but inhibited that of collagen I and X, resulting in a stabilized chondrocyte phenotype. These data suggest that COMP and TSP-4 activate mechanisms to protect and repair the ECM in articular cartilage.     

Structural Variations in Articular Cartilage Matrix Are Associated with Early-Onset Osteoarthritis in the Spondyloepiphyseal Dysplasia Congenita (Sedc) Mouse

Heterozgyous spondyloepiphyseal dysplasia congenita (sedc/+) mice expressing a missense mutation in col2a1 exhibit a normal skeletal morphology but early-onset osteoarthritis (OA). We have recently examined knee articular cartilage obtained from homozygous (sedc/sedc) mice, which express a Stickler-like phenotype including dwarfism. We examined sedc/sedc mice at various levels to better understand the mechanistic process resulting in OA. Mutant sedc/sedc, and control (+/+) cartilages were compared at two, six and nine months of age. Tissues were fixed, decalcified, processed to paraffin sections, and stained with hematoxylin/eosin and safranin O/fast green. Samples were analyzed under the light microscope and the modified Mankin and OARSI scoring system was used to quantify the OA-like changes. Knees were stained with 1C10 antibody to detect the presence and distribution of type II collagen. Electron microscopy was used to study chondrocyte morphology and collagen fibril diameter. Compared with controls, mutant articular cartilage displayed decreased fibril diameter concomitant with increases in size of the pericellular space, Mankin and OARSI scores, cartilage thickness, chondrocyte clustering, proteoglycan staining and horizontal fissuring. In conclusion, homozygous sedc mice are subject to early-onset knee OA. We conclude that collagen in the mutant’s articular cartilage (both heterozygote and homozygote) fails to provide the normal meshwork required for matrix integrity and overall cartilage stability.     

The Use of Infrapatellar Fat Pad-Derived Mesenchymal Stem Cells in Articular Cartilage Regeneration: A Review

Cartilage is frequently damaged with a limited capacity for repair. Current treatment strategies are insufficient as they form fibrocartilage as opposed to hyaline cartilage, and do not prevent the progression of degenerative changes. There is increasing interest in the use of autologous mesenchymal stem cells (MSC) for tissue regeneration. MSCs that are used to treat articular cartilage defects must not only present a robust cartilaginous production capacity, but they also must not cause morbidity at the harvest site. In addition, they should be easy to isolate from the tissue and expand in culture without terminal differentiation. The source of MSCs is one of the most important factors that may affect treatment. The infrapatellar fat pad (IPFP) acts as an important reservoir for MSC and is located in the anterior compartment of the knee joint in the extra-synovial area. The IPFP is a rich source of MSCs, and in this review, we discuss studies that demonstrate that these cells have shown many advantages over other tissues in terms of ease of isolation, expansion, and chondrogenic differentiation. Future studies in articular cartilage repair strategies and suitable extraction as well as cell culture methods will extend the therapeutical application of IPFP-derived MSCs into additional orthopedic fields, such as osteoarthritis. This review provides the latest research concerning the use of IPFP-derived MSCs in the treatment of articular cartilage damage, providing critical information for the field to grow.     

Mechanophysical Cues in Extracellular Matrix Regulation of Cell Behavior

The extracellular matrix (ECM) is a macromolecular network that can provide biochemical and structural support for cell adhesion and formation. It regulates cell behavior by influencing biochemical and physical cues. It is a dynamic structure whose components are modified, degraded, or deposited during connective tissue development, giving tissues strength and structural integrity. The physical properties of the natural ECM environment control the design of naturally or synthetically derived biomaterials to guide cell function in tissue engineering. Tissue engineering is an important field that explores physical cues of the ECM to produce new viable tissue for medical applications, such as in organ transplant and organ recovery. Understanding how the ECM exerts physical effects on cell behavior, when cells are seeded in synthetic ECM scaffolds, is of utmost importance. Herein we review recent findings in this area that report on cell behaviors in a variety of ECMs with different physical properties, i.e., topology, geometry, dimensionality, stiffness, and tension.     

Cartilage Oligomeric Matrix Protein,Diseases, and Therapeutic Opportunities

Cartilage oligomeric matrix protein (COMP) is an extracellular matrix (ECM) glycoprotein that is critical for collagen assembly and ECM stability. Mutations of COMP cause endoplasmic reticulum stress and chondrocyte apoptosis, resulting in rare skeleton diseases. The bouquet-like structure of COMP allows it to act as a bridging molecule that regulates cellular phenotype and function. COMP is able to interact with many other ECM components and binds directly to a variety of cellular receptors and growth factors. The roles of COMP in other skeleton diseases, such as osteoarthritis, have been implied. As a well-established biochemical marker, COMP indicates cartilage turnover associated with destruction. Recent exciting achievements indicate its involvement in other diseases, such as malignancy, cardiovascular diseases, and tissue fibrosis. Here, we review the basic concepts of COMP and summarize its novel functions in the regulation of signaling events. These findings renew our understanding that COMP has a notable function in cell behavior and disease progression as a signaling regulator. Interestingly, COMP shows distinct functions in different diseases. Targeting COMP in malignancy may withdraw its beneficial effects on the vascular system and induce or aggravate cardiovascular diseases. COMP supplementation is a promising treatment for OA and aortic aneurysms while it may induce tissue fibrosis or cancer metastasis.     

Evaluation of Three-Dimensional Bioprinted Human Cartilage Powder Combined with Micronized Subcutaneous Adipose Tissues for the Repair of Osteochondral Defects in Beagle Dogs

Cartilage lesions are difficult to repair due to low vascular distribution and may progress into osteoarthritis. Despite numerous attempts in the past, there is no proven method to regenerate hyaline cartilage. The purpose of this study was to investigate the ability to use a 3D printed biomatrix to repair a critical size femoral chondral defect using a canine weight-bearing model. The biomatrix was comprised of human costal-derived cartilage powder, micronized adipose tissue, and fibrin glue. Bilateral femoral condyle defects were treated on 12 mature beagles staged 12 weeks apart. Four groups, one control and three experimental, were used. Animals were euthanized at 32 weeks to collect samples. Significant differences between control and experimental groups were found in both regeneration pattern and tissue composition. In results, we observed that the experimental group with the treatment with cartilage powder and adipose tissue alleviated the inflammatory response. Moreover, it was found that the MOCART score was higher, and cartilage repair was more organized than in the other groups, suggesting that a combination of cartilage powder and adipose tissue has the potential to repair cartilage with a similarity to normal cartilage. Microscopically, there was a well-defined cartilage-like structure in which the mid junction below the surface layer was surrounded by a matrix composed of collagen type I, II, and proteoglycans. MRI examination revealed significant reduction of the inflammation level and progression of a cartilage-like growth in the experimental group. This canine study suggests a promising new surgical treatment for cartilage lesions.     

Histone Extraction from Human Articular Cartilage for the Study of Epigenetic Regulation in Osteoarthritis

Osteoarthritis (OA) is a chronic disease that affects articular cartilage, causing its degeneration. Although OA is one of the most prevalent pathologies globally, there are no definitive treatments available. Recently, research has focused on elucidating the complex interplay that takes place between inflammatory processes and epigenetic regulation, showing that histone post-translational modifications (PTMs) can exert a pronounced effect on the expression of OA-related genes. OA chondrocytes enhance the production of interleukin 1β (IL-1β) and interleukin 8 (IL-8), which are epigenetically regulated. These cytokines upregulate the synthesis of matrix metalloproteinases (MMPs) and aggrecanases, which promote the extracellular matrix (ECM) destruction. This motivates the study of histone PTMs to investigate the epigenetic regulation of proinflammatory molecules, but the absence of specific protocols to extract histones from human articular cartilage has complicated this task. The lack of effective methods can be explained by the structural complexity and low cellularity of this tissue, which are responsible for the biomechanical properties that allow the movement of the joint but also complicate histone isolation. Here, we provide a histone extraction procedure specifically adapted for cryopreserved human articular cartilage that can be useful to understand epigenetic regulation in OA and accelerate the search for novel strategies.     

Main and Minor Types of Collagens in the Articular Cartilage: The Role of Collagens in Repair Tissue Evaluation in Chondral Defects

Several collagen subtypes have been identified in hyaline articular cartilage. The main and most abundant collagens are type II, IX and XI collagens. The minor and less abundant collagens are type III, IV, V, VI, X, XII, XIV, XVI, XXII, and XXVII collagens. All these collagens have been found to play a key role in healthy cartilage, regardless of whether they are more or less abundant. Additionally, an exhaustive evaluation of collagen fibrils in a repaired cartilage tissue after a chondral lesion is necessary to determine the quality of the repaired tissue and even whether or not this repaired tissue is considered hyaline cartilage. Therefore, this review aims to describe in depth all the collagen types found in the normal articular cartilage structure, and based on this, establish the parameters that allow one to consider a repaired cartilage tissue as a hyaline cartilage.     

High Density Infill in Cracks and Protrusions from the Articular Calcified Cartilage in Osteoarthritis in Standardbred Horse Carpal Bones

We studied changes in articular calcified cartilage (ACC) and subchondral bone (SCB) in the third carpal bones (C3) of Standardbred racehorses with naturally-occurring repetitive loading-induced osteoarthritis (OA). Two osteochondral cores were harvested from dorsal sites from each of 15 post-mortem C3 and classified as control or as showing early or advanced OA changes from visual inspection. We re-examined X-ray micro-computed tomography (µCT) image sets for the presence of high-density mineral infill (HDMI) in ACC cracks and possible high-density mineralized protrusions (HDMP) from the ACC mineralizing (tidemark) front (MF) into hyaline articular cartilage (HAC). We hypothesized and we show that 20-µm µCT resolution in 10-mm diameter samples is sufficient to detect HDMI and HDMP: these are lost upon tissue decalcification for routine paraffin wax histology owing to their predominant mineral content. The findings show that µCT is sufficient to discover HDMI and HDMP, which were seen in 2/10 controls, 6/9 early OA and 8/10 advanced OA cases. This is the first report of HDMI and HDMP in the equine carpus and in the Standardbred breed and the first to rely solely on µCT. HDMP are a candidate cause for mechanical tissue destruction in OA.     

Extracellular Vesicles: Interplay with the Extracellular Matrix and Modulated Cell Responses

The discovery that cells secrete extracellular vesicles (EVs), which carry a variety of regulatory proteins, nucleic acids, and lipids, has shed light on the sophisticated manner by which cells can communicate and accordingly function. The bioactivity of EVs is not only defined by their internal content, but also through their surface associated molecules, and the linked downstream signaling effects they elicit in target cells. The extracellular matrix (ECM) contains signaling and structural molecules that are central to tissue maintenance and repair. Recently, a subset of EVs residing within the extracellular matrix has been identified. Although some roles have been proposed for matrix-bound vesicles, their role as signaling molecules within the ECM is yet to be explored. Given the close association of EVs and the ECM, it is not surprising that EVs partly mediate repair and regeneration by modulating matrix deposition and degradation through their cellular targets. This review addresses unique EV features that allow them to interact with and navigate through the ECM, describes how their release and content is influenced by the ECM, and emphasizes the emerging role of stem-cell derived EVs in tissue repair and regeneration through their matrix-modulating properties.     

An Update on the Role of Leptin in the Immuno-Metabolism of Cartilage

Since its discovery in 1994, leptin has been considered as an adipokine with pleiotropic effects. In this review, we summarize the actual information about the impact of this hormone on cartilage metabolism and pathology. Leptin signalling depends on the interaction with leptin receptor LEPR, being the long isoform of the receptor (LEPRb) the one with more efficient intracellular signalling. Chondrocytes express the long isoform of the leptin receptor and in these cells, leptin signalling, alone or in combination with other molecules, induces the expression of pro-inflammatory molecules and cartilage degenerative enzymes. Leptin has been shown to increase the proliferation and activation of immune cells, increasing the severity of immune degenerative cartilage diseases. Leptin expression in serum and synovial fluid are related to degenerative diseases such as osteoarthritis (OA), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Inhibition of leptin signalling showed to have protective effects in these diseases showing the key role of leptin in cartilage degeneration.     

Expression and Localization of Thrombospondins,Plastin 3, and STIM1 in Different Cartilage Compartments of the Osteoarthritic Varus Knee

Osteoarthritis (OA) is a multifactorial disease which is characterized by a change in the homeostasis of the extracellular matrix (ECM). The ECM is essential for the function of the articular cartilage and plays an important role in cartilage mechanotransduction. To provide a better understanding of the interaction between the ECM and the actin cytoskeleton, we investigated the localization and expression of the Ca²⁺-dependent proteins cartilage oligomeric matrix protein (COMP), thrombospondin-1 (TSP-1), plastin 3 (PLS3) and stromal interaction molecule 1 (STIM1). We investigated 16 patients who suffered from varus knee OA and performed a topographical analysis of the cartilage from the medial and lateral compartment of the proximal tibial plateau. In a varus knee, OA is more pronounced in the medial compared to the lateral compartment as a result of an overloading due to the malalignment. We detected a location-dependent staining of PLS3 and STIM1 in the articular cartilage tissue. The staining intensity for both proteins correlated with the degree of cartilage degeneration. The staining intensity of TSP-1 was clearly reduced in the cartilage of the more affected medial compartment, an observation that was confirmed in cartilage extracts by immunoblotting. The total amount of COMP was unchanged; however, slight changes were detected in the localization of the protein. Our results provide novel information on alterations in OA cartilage suggesting that Ca²⁺-dependent mechanotransduction between the ECM and the actin cytoskeleton might play an essential role in the pathomechanism of OA.     

Chaenomeles Fructus (CF), the Fruit of Chaenomeles sinensis Alleviates IL-1β Induced Cartilage Degradation in Rat Articular Chondrocytes

Osteoarthritis (OA) causes persistent pain, joint dysfunction, and physical disability. It is the most prevalent type of degenerative arthritis, affecting millions of people worldwide. OA is currently treated with a focus on pain relief, inflammation control, and artificial joint surgery. Hence, a therapeutic agent capable of preventing or delaying the progression of OA is needed. OA is strongly associated with the degeneration of the articular cartilage and changes in the ECM, which are primarily associated with a decrease in proteoglycan and collagen. In the progress of articular cartilage degradation, catabolic enzymes, such as matrix metalloproteinases (MMPs), are activated by IL-1β stimulation. Given the tight relationship between IL-1β and ECM (extra-cellular matrix) degradation, this study examined the effects of Chaenomeles Fructus (CF) on IL-1β-induced OA in rat chondrocytes. The CF treatment reduced IL-1β-induced MMP3/13 and ADAMTS-5 production at the mRNA and protein levels. Similarly, CF enhanced col2a and aggrecan accumulation and chondrocyte proliferation. CF inhibited NF-κB (nuclear factor kappa B) activation, nuclear translocation induced by IL-1β, reactive oxygen species (ROS) production, and ERK phosphorylation. CF demonstrated anti-OA and articular regeneration effects on rat chondrocytes, thus, suggesting that CF is a viable and fundamental therapeutic option for OA.     

The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes

Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint.     

Baseline Stiffness Modulates the Non-Linear Response to Stretch of the Extracellular Matrix in Pulmonary Fibrosis

Pulmonary fibrosis (PF) is a progressive disease that disrupts the mechanical homeostasis of the lung extracellular matrix (ECM). These effects are particularly relevant in the lung context, given the dynamic nature of cyclic stretch that the ECM is continuously subjected to during breathing. This work uses an in vivo model of pulmonary fibrosis to characterize the macro- and micromechanical properties of lung ECM subjected to stretch. To that aim, we have compared the micromechanical properties of fibrotic ECM in baseline and under stretch conditions, using a novel combination of Atomic Force Microscopy (AFM) and a stretchable membrane-based chip. At the macroscale, fibrotic ECM displayed strain-hardening, with a stiffness one order of magnitude higher than its healthy counterpart. Conversely, at the microscale, we found a switch in the stretch-induced mechanical behaviour of the lung ECM from strain-hardening at physiological ECM stiffnesses to strain-softening at fibrotic ECM stiffnesses. Similarly, we observed solidification of healthy ECM versus fluidization of fibrotic ECM in response to stretch. Our results suggest that the mechanical behaviour of fibrotic ECM under stretch involves a potential built-in mechanotransduction mechanism that may slow down the progression of PF by steering resident fibroblasts away from a pro-fibrotic profile.     

Molecular Mechanisms of Changes in Homeostasis of the Dermal Extracellular Matrix: Both Involutional and Mediated by Ultraviolet Radiation

Skin aging is a multi-factorial process that affects nearly every aspect of skin biology and function. With age, an impairment of structures, quality characteristics, and functions of the dermal extracellular matrix (ECM) occurs in the skin, which leads to disrupted functioning of dermal fibroblasts (DFs), the main cells supporting morphofunctional organization of the skin. The DF functioning directly depends on the state of the surrounding collagen matrix (CM). The intact collagen matrix ensures proper adhesion and mechanical tension in DFs, which allows these cells to maintain collagen homeostasis while ECM correctly regulates cellular processes. When the integrity of CM is destroyed, mechanotransduction is disrupted, which is accompanied by impairment of DF functioning and destruction of collagen homeostasis, thereby contributing to the progression of aging processes in skin tissues. This article considers in detail the processes of skin aging and associated changes in the skin layers, as well as the mechanisms of these processes at the molecular level.     

Age Dependent Changes in Cartilage Matrix,Subchondral Bone Mass,and Estradiol Levels in Blood Serum,in Naturally Occurring Osteoarthritis in Guinea Pigs

The Dunkin Hartley (DH) guinea pig is a widely used naturally occurring osteoarthritis model. The aim of this study was to provide detailed evidence of age-related changes in articular cartilage, subchondral bone mineral density, and estradiol levels. We studied the female Dunkin Hartley guinea pigs at 1, 3, 6, 9, and 12 months of age (eight animals in each group). Histological analysis were used to identify degenerative cartilage and electron microscopy was performed to further observe the ultrastructure. Estradiol expression levels in serum were assessed, and matrix metalloproteinase 3 and glycosaminoglycan expression in cartilage was performed by immunohistochemistry. Bone mineral density of the tibia subchondral bone was measured using dual X-ray absorptiometry. Histological analysis showed that the degeneration of articular cartilage grew more severe with increasing age starting at 3 months, coupled with the loss of normal cells and an increase in degenerated cells. Serum estradiol levels increased with age from 1 to 6 months and thereafter remained stable from 6 to 12 months. Matrix metalloproteinase 3 expression in cartilage increased with age, but no significant difference was found in glycosaminoglycan expression between 1- and 3-month old animals. The bone mineral density of the tibia subchondral bone increased with age before reaching a stable value at 9 months of age. Age-related articular cartilage degeneration occurred in Dunkin Hartley guinea pigs beginning at 3 months of age, while no directly positive or negative correlation between osteoarthritis progression and estradiol serum level or subchondral bone mineral density was discovered.