Comparison of knee joint and temporomandibular joint development in pig embryos.

Although the knee joint (KNJ) and temporomandibular joint (TMJ) all belong to the synovial joint, there are many differences in developmental origin, joint structure and articular cartilage type. Studies of joint development in embryos have been performed, mainly using poultry and rodents. However, KNJ and TMJ in poultry and rodents differ from those in humans in several ways. Very little work has been done on the embryonic development of KNJ and TMJ in large mammals. Several studies have shown that pigs are ideal animals for embryonic development research. Embryonic day 30 (E30), E35, E45, E55, E75, E90, Postnatal day 0 (P0) and Postnatal day 30 (P30) embryos/fetuses from the pigs were used for this study. The results showed that KNJ develops earlier than TMJ. Only one mesenchymal condensate of KNJ is formed on E30, while two mesenchymal condensates of TMJ are present on E35. All structures of KNJ and TMJ were formed on E45. The growth plate of KNJ begins to develop on E45 and becomes more pronounced from E55 to P30. From E75 to E90, more and more vascular-rich cartilage canals form in the cartilage regions of both joints. The cartilaginous canal of the TMJ divides the condyle into sections along the longitudinal axis of the condyle. This arrangement of cartilaginous canal was not found in the KNJ. The chondrification of KNJ precedes that of TMJ. Ossification of the knee condyle occurs gradually from the middle to the periphery, while that of the TMJ occurs gradually from the base of the mandibular condyle. In the KNJ, the ossification of the articular condyle is evident from P0 to P30, and the growth plate is completely formed on P30. In the TMJ, the cartilage layer of condyle becomes thinner from P0 to P30. There is no growth plate formation in TMJ during its entire development. There is no growth plate formation in the TMJ throughout its development. The condyle may be the developmental center of the TMJ. The chondrocytes and hypertrophic chondrocytes of the growth plate are densely arranged. The condylar chondrocytes of TMJ are scattered, while the hypertrophic chondrocytes are arranged. Embryonic development of KNJ and TMJ in pigs is an important bridge for translating the results of rodent studies to medical applications.

Stromal-vascular fraction and adipose-derived stem cell therapies improve cartilage regeneration in osteoarthritis-induced rats.

This study aimed to evaluate the effects of the stromal vascular fraction (SVF) and adipose-derived stem cells (ADSCs) on cartilage injury in an osteoarthritis (OA) rat model. Sodium iodoacetate (3 mg/50 µL) was used to induce OA in the left knee joint of rats. On day 14 after OA induction, 50 µL of SVF (5 × 106cells), ADSCs (1 × 106 cells), or 0.9% normal saline (NS) was injected into the left knee-joint cavity of each group. The macroscopic view and histological sections revealed that the articular cartilage in the NS group was damaged, inflamed, uneven and thin, and had hyperchromatic cell infiltration. Notably, the cartilage surface had recovered to nearly normal and appeared smooth and bright on day 14 in the SVF and ADSC groups. Additionally, the white blood cell counts in the SVF and ADSC groups were higher than those in the NS group on day 14. Plasma IL-1ß levels on days 7 and 14 were reduced in the SVF and ADSC groups. These results indicated that both SVF and ADSC treatments may assist in articular cartilage regeneration after cartilage injury. Cell therapy may benefit patients with OA. However, clinical trials with humans are required before the application of SVF and ADSC treatments in patients with OA.

Role of Ciliary Protein Intraflagellar Transport Protein 88 in the Regulation of Cartilage Thickness and Osteoarthritis Development in Mice.

OBJECTIVE: Mechanical and biologic cues drive cellular signaling in cartilage development, health, and disease. Primary cilia proteins, which are implicated in the transduction of biologic and physiochemical signals, control cartilage formation during skeletal development. This study was undertaken to assess the influence of the ciliary protein intraflagellar transport protein 88 (IFT88) on postnatal cartilage from mice with conditional knockout of the Ift88 gene (Ift88-KO). METHODS: Ift88fl/fl and aggrecanCreERT2 mice were crossed to create a strain of cartilage-specific Ift88-KO mice (aggrecanCreERT2 ;Ift88fl/fl ). In these Ift88-KO mice and Ift88fl/fl control mice, tibial articular cartilage thickness was assessed by histomorphometry, and the integrity of the cartilage was assessed using Osteoarthritis Research Society International (OARSI) damage scores, from adolescence through adulthood. In situ mechanisms of cartilage damage were investigated in the microdissected cartilage sections using immunohistochemistry, RNAScope analysis, and quantitative polymerase chain reaction. Osteoarthritis (OA) was induced in aggrecanCreERT2 ;Ift88fl/fl mice and Ift88fl/fl control mice using surgical destabilization of the medial meniscus (DMM). Following tamoxifen injection and DMM surgery, the mice were given free access to exercise on a wheel. RESULTS: Deletion of Ift88 resulted in progressive reduction in the thickness of the medial tibial cartilage in adolescent mice, as well as marked atrophy of the cartilage in mice during adulthood. In aggrecanCreERT2 ;Ift88fl/fl mice at age 34 weeks, the median thickness of the medial tibial cartilage was 89.42 µm (95% confidence interval [95% CI] 84.00-93.49), whereas in Ift88fl/fl controls at the same age, the median cartilage thickness was 104.00 µm (95% CI 100.30-110.50; P < 0.0001). At all time points, the median thickness of the calcified cartilage was reduced. In some mice, atrophy of the medial tibial cartilage was associated with complete, spontaneous degradation of the cartilage. Following DMM, aggrecanCreERT2 ;Ift88fl/fl mice were found to have increased OARSI scores of cartilage damage. In articular cartilage from maturing mice, atrophy was not associated with obvious increases in aggrecanase-mediated destruction or chondrocyte hypertrophy. Of the 44 candidate genes analyzed, only Tcf7l2 expression levels correlated with Ift88 expression levels in the microdissected cartilage. However, RNAScope analysis revealed that increased hedgehog (Hh) signaling (as indicated by increased expression of Gli1) was associated with the reductions in Ift88 expression in the tibial cartilage from Ift88-deficient mice. Wheel exercise restored both the articular cartilage thickness and levels of Hh signaling in these mice. CONCLUSION: Our results in a mouse model of OA demonstrate that IFT88 performs a chondroprotective role in articular cartilage by controlling the calcification of cartilage via maintenance of a threshold of Hh signaling during physiologic loading.

Fetal Immunomodulatory Environment Following Cartilage Injury-The Key to CARTILAGE Regeneration?

Fetal cartilage fully regenerates following injury, while in adult mammals cartilage injury leads to osteoarthritis (OA). Thus, in this study, we compared the in vivo injury response of fetal and adult ovine articular cartilage histologically and proteomically to identify key factors of fetal regeneration. In addition, we compared the secretome of fetal ovine mesenchymal stem cells (MSCs) in vitro with injured fetal cartilage to identify potential MSC-derived therapeutic factors. Cartilage injury caused massive cellular changes in the synovial membrane, with macrophages dominating the fetal, and neutrophils the adult, synovial cellular infiltrate. Correspondingly, proteomics revealed differential regulation of pro- and anti-inflammatory mediators and growth-factors between adult and fetal joints. Neutrophil-related proteins and acute phase proteins were the two major upregulated protein groups in adult compared to fetal cartilage following injury. In contrast, several immunomodulating proteins and growth factors were expressed significantly higher in the fetus than the adult. Comparison of the in vitro MSCs proteome with the in vivo fetal regenerative signature revealed shared upregulation of 17 proteins, suggesting their therapeutic potential. Biomimicry of the fetal paracrine signature to reprogram macrophages and modulate inflammation could be an important future research direction for developing novel therapeutics.

A scaffold-free approach to cartilage tissue generation using human embryonic stem cells.

Articular cartilage functions as a shock absorber and facilitates the free movement of joints. Currently, there are no therapeutic drugs that promote the healing of damaged articular cartilage. Limitations associated with the two clinically relevant cell populations, human articular chondrocytes and mesenchymal stem cells, necessitate finding an alternative cell source for cartilage repair. Human embryonic stem cells (hESCs) provide a readily accessible population of self-renewing, pluripotent cells with perceived immunoprivileged properties for cartilage generation. We have developed a robust method to generate 3D, scaffold-free, hyaline cartilage tissue constructs from hESCs that are composed of numerous chondrocytes in lacunae, embedded in an extracellular matrix containing Type II collagen, sulphated glycosaminoglycans and Aggrecan. The elastic (Young's) modulus of the hESC-derived cartilage tissue constructs (0.91 ± 0.08 MPa) was comparable to full-thickness human articular cartilage (0.87 ± 0.09 MPa). Moreover, we have successfully scaled up the size of the scaffold-free, 3D hESC-derived cartilage tissue constructs to between 4.5 mm and 6 mm, thus enhancing their suitability for clinical application.

The Interaction between microRNAs and the Wnt/ß-Catenin Signaling Pathway in Osteoarthritis.

Osteoarthritis (OA) is a chronic disease affecting the whole joint, which still lacks a disease-modifying treatment. This suggests an incomplete understanding of underlying molecular mechanisms. The Wnt/ß-catenin pathway is involved in different pathophysiological processes of OA. Interestingly, both excessive stimulation and suppression of this pathway can contribute to the pathogenesis of OA. microRNAs have been shown to regulate different cellular processes in different diseases, including the metabolic activity of chondrocytes and osteocytes. To bridge these findings, here we attempt to give a conclusive overview of microRNA regulation of the Wnt/ß-catenin pathway in bone and cartilage, which may provide insights to advance the development of miRNA-based therapeutics for OA treatment.

Chondro-inductive hyaluronic acid/chitosan coacervate-based scaffolds for cartilage tissue engineering.

Injuries related to articular cartilage are among the most challenging musculoskeletal problems because of poor repair capacity of this tissue. The lack of efficient treatments for chondral defects has stimulated research on cartilage tissue engineering applications combining porous biocompatible scaffolds with stem cells in the presence of external stimuli. This work presents the role of rat bone marrow mesenchymal stem cell (BMSC) encapsulated-novel three-dimensional (3D) coacervate scaffolds prepared through complex coacervation between different chitosan salts (CHI) and sodium hyaluronate (HA). The 3D architecture of BMSC encapsulated scaffolds (HA/CHI) was shown by scanning electron microscopy (SEM) to have an interconnected structure to allow cell-cell and cell-matrix interactions. Chondrogenic induction of encapsulated BMSCs within HA/CHI coacervates demonstrated remarkable cellular viability in addition to the elevated expression levels of chondrogenic markers such as sex determining region Y-box 9 protein (SOX9), aggrecan (ACAN), cartilage oligomeric matrix protein (COMP) and collagen type II (COL2A1) by immunofluorescence staining, qPCR and ELISA test. Collectively, HA/CHI coacervates are promising candidates for future use of these scaffolds in cartilage tissue engineering applications.

3D-bioprinted gradient-structured scaffold generates anisotropic cartilage with vascularization by pore-size-dependent activation of HIF1α/FAK signaling axis.

Articular cartilage injury is one of the most common diseases in orthopedics, which seriously affects patients' life quality, the development of a biomimetic scaffold that mimics the multi-layered gradient structure of native cartilage is a new cartilage repair strategy. It has been shown that scaffold topography affects cell attachment, proliferation, and differentiation; the underlying molecular mechanism of cell-scaffold interaction is still unclear. In the present study, we construct an anisotropic gradient-structured cartilage scaffold by three-dimensional (3D) bioprinting, in which bone marrow stromal cell (BMSC)-laden anisotropic hydrogels micropatterns were used for heterogeneous chondrogenic differentiation and physically gradient synthetic poly (ε-caprolactone) (PCL) to impart mechanical strength. In vitro and in vivo, we demonstrated that gradient-structured cartilage scaffold displayed better cartilage repair effect. The heterogeneous cartilage tissue maturation and blood vessel ingrowth were mediated by a pore-size-dependent mechanism and HIF1α/FAK axis activation. In summary, our results provided a theoretical basis for employing 3D bioprinting gradient-structured constructs for anisotropic cartilage regeneration and revealed HIF1α/FAK axis as a crucial regulator for cell-material interactions, so as to provide a new perspective for cartilage regeneration and repair.

Enhanced matrix production by cocultivated human stem cells and chondrocytes under concurrent mechanical strain.

Conventional treatments of osteoarthritis have failed to re-build functional articular cartilage. Tissue engineering clinical treatments for osteoarthritis, including autologous chondrocyte implantation, provides an alternative approach by injecting a cell suspension to fill lesions within the cartilage in osteoarthritic knees. The success of chondrocyte implantation relies on the availability of chondrogenic cell lines, and their resilience to high mechanical loading. We hypothesize we can reduce the numbers of human articular chondrocytes necessary for a treatment by supplementing cultures with human adipose-derived stem cells, in which stem cells will have protective and stimulatory effects on mixed cultures when exposed to high mechanical loads, and in which coculture will enhance production of requisite extracellular matrix proteins over those produced by stretched chondrocytes alone. In this work, adipose-derived stem cells and articular chondrocytes were cultured separately or cocultivated at ratios of 3:1, 1:1, and 1:3 in static plates or under excessive cyclic tensile strain of 10% and results were compared to culturing of both cell types alone with and without cyclic strain. Results indicate 75% of chondrocytes in engineered articular cartilage can be replaced with stem cells with enhanced collagen over all culture conditions and glycosaminoglycan content over stretched cultures of chondrocytes. This can be done without observing adverse effects on cell viability. Collagen and glycosaminoglycan secretion, when compared to chondrocyte alone under 10% strain, was enhanced 6.1- and 2-fold, respectively, by chondrocytes cocultivated with stem cells at a ratio of 1:3.

Pseudolaric acid B ameliorates synovial inflammation and vessel formation by stabilizing PPARγ to inhibit NF-κB signalling pathway.

Synovial macrophage polarization and inflammation are essential for osteoarthritis (OA) development, yet the molecular mechanisms and regulation responsible for the pathogenesis are still poorly understood. Here, we report that pseudolaric acid B (PAB) attenuated articular cartilage degeneration and synovitis during OA. PAB, a diterpene acid, specifically inhibited NF-κB signalling and reduced the production of pro-inflammatory cytokines, which further decreased M1 polarization and vessel formation. We further provide in vivo and in vitro evidences that PAB suppressed NF-κB signalling by stabilizing PPARγ. Using PPARγ antagonist could abolish anti-inflammatory effect of PAB and rescue the activation of NF-κB signalling during OA. Our findings identify a previously unrecognized role of PAB in the regulation of OA and provide mechanisms by which PAB regulates NF-κB signalling through PPARγ, which further suggest targeting synovial inflammation or inhibiting vessel formation at early stage could be an effective preventive strategy for OA.

Versican contributes to ligament formation of knee joints.

Versican is a large proteoglycan in the extracellular matrix. During embryonic stages, it plays a crucial role in the development of cartilage, heart, and dermis. Previously, we reported that Prx1-Vcan conditional knockout mice, lacking Vcan expression in mesenchymal condensation areas of the limb bud, show the impaired joint formation and delayed cartilage development. Here, we investigated their phenotype in adults and found that they develop swelling of the knee joint. Histologically, their newborn joint exhibited impaired formation of both anterior and posterior cruciate ligaments. Immunostaining revealed a decrease in scleraxis-positive cells in both articular cartilage and ligament of Prx1-Vcan knee joint, spotty patterns of type I collagen, and the presence of type II collagen concomitant with the absence of versican expression. These results suggest that versican expression during the perinatal period is required for cruciate ligaments' formation and that its depletion affects joint function in later ages.

Overexpression of long non-coding RNA AP001505.9 inhibits human hyaline chondrocyte dedifferentiation.

Autologous chondrocyte implantation (ACI) is an effective method for treating chronic articular cartilage injury and degeneration; however, it requires large numbers of hyaline chondrocytes, and human hyaline chondrocytes often undergo dedifferentiation in vitro. Moreover, although long non-coding RNAs (lncRNAs) regulate gene expression in many pathological and physiological processes, their role in human hyaline chondrocyte dedifferentiation remains unclear. Here, we examined lncRNA and mRNA expression profiles in human hyaline chondrocyte dedifferentiation using microarray analysis. Among the many lncRNAs and mRNAs that showed differential expression, lncRNA AP001505.9 (ENST00000569966) was significantly downregulated in chondrocytes after dedifferentiation. We next performed gene ontology, pathway, and CNC (coding-non-coding gene co-expression) analyses to investigate potential regulatory mechanisms for AP001505.9. Pellet cultures were then used to redifferentiate dedifferentiated chondrocytes, and AP001505.9 expression was upregulated after redifferentiation. Finally, both in vitro and in vivo experiments demonstrated that AP001505.9 overexpression inhibited dedifferentiation of chondrocytes. This study characterizes lncRNA expression profiles in human hyaline chondrocyte dedifferentiation, thereby identifying new potential mechanisms of chondrocyte dedifferentiation worthy of further investigation.

Molecular and Cellular Effects of Chemical Chaperone-TUDCA on ER-Stressed NHAC-kn Human Articular Chondrocytes Cultured in Normoxic and Hypoxic Conditions.

Osteoarthritis (OA) is considered one of the most common arthritic diseases characterized by progressive degradation and abnormal remodeling of articular cartilage. Potential therapeutics for OA aim at restoring proper chondrocyte functioning and inhibiting apoptosis. Previous studies have demonstrated that tauroursodeoxycholic acid (TUDCA) showed anti-inflammatory and anti-apoptotic activity in many models of various diseases, acting mainly via alleviation of endoplasmic reticulum (ER) stress. However, little is known about cytoprotective effects of TUDCA on chondrocyte cells. The present study was designed to evaluate potential effects of TUDCA on interleukin-1ß (IL-1ß) and tunicamycin (TNC)-stimulated NHAC-kn chondrocytes cultured in normoxic and hypoxic conditions. Our results showed that TUDCA alleviated ER stress in TNC-treated chondrocytes, as demonstrated by reduced CHOP expression; however, it was not effective enough to prevent apoptosis of NHAC-kn cells in either normoxia nor hypoxia. However, co-treatment with TUDCA alleviated inflammatory response induced by IL-1ß, as shown by down regulation of Il-1ß, Il-6, Il-8 and Cox2, and increased the expression of antioxidant enzyme Sod2. Additionally, TUDCA enhanced Col IIα expression in IL-1ß- and TNC-stimulated cells, but only in normoxic conditions. Altogether, these results suggest that although TUDCA may display chondoprotective potential in ER-stressed cells, further analyses are still necessary to fully confirm its possible recommendation as potential candidate in OA therapy.

Chondrogenesis of human amniotic fluid stem cells in Chitosan-Xanthan scaffold for cartilage tissue engineering.

Articular chondral lesions, caused either by trauma or chronic cartilage diseases such as osteoarthritis, present very low ability to self-regenerate. Thus, their current management is basically symptomatic, progressing very often to invasive procedures or even arthroplasties. The use of amniotic fluid stem cells (AFSCs), due to their multipotentiality and plasticity, associated with scaffolds, is a promising alternative for the reconstruction of articular cartilage. Therefore, this study aimed to investigate the chondrogenic potential of AFSCs in a micromass system (high-density cell culture) under insulin-like growth factor 1 (IGF-1) stimuli, as well as to look at their potential to differentiate directly when cultured in a porous chitosan-xanthan (CX) scaffold. The experiments were performed with a CD117 positive cell population, with expression of markers (CD117, SSEA-4, Oct-4 and NANOG), selected from AFSCs, after immunomagnetic separation. The cells were cultured in both a micromass system and directly in the scaffold, in the presence of IGF-1. Differentiation to chondrocytes was confirmed by histology and by using immunohistochemistry. The construct cell-scaffold was also analyzed by scanning electron microscopy (SEM). The results demonstrated the chondrogenic potential of AFSCs cultivated directly in CX scaffolds and also in the micromass system. Such findings support and stimulate future studies using these constructs in osteoarthritic animal models.

An alginate-poly(acrylamide) hydrogel with TGF-ß3 loaded nanoparticles for cartilage repair: Biodegradability, biocompatibility and protein adsorption.

Current implantable materials are limited in terms of function as native tissue, and there is still no effective clinical treatment to restore articular impairments. Hereby, a functionalized polyacrylamide (PAAm)-alginate (Alg) Double Network (DN) hydrogel acting as an articular-like tissue is developed. These hydrogels sustain their mechanical stability under different temperature (+4 °C, 25 °C, 40 °C) and humidity conditions (60% and 75%) over 3 months. As for the functionalization, transforming growth factor beta-3 (TGF-ß3) encapsulated (NPTGF-ß3) and empty poly(lactide-co-glycolide) (PLGA) nanoparticles (PLGA NPs) are synthesized by using microfluidic platform, wherein the mean particle sizes are determined as 81.44 ± 9.2 nm and 126 ± 4.52 nm with very low polydispersity indexes (PDI) of 0.194 and 0.137, respectively. Functionalization process of PAAm-Alg hydrogels with ester-end PLGA NPs is confirmed by FTIR analysis, and higher viscoelasticity is obtained for functionalized hydrogels. Moreover, cartilage regeneration capability of these hydrogels is evaluated with in vitro and in vivo experiments. Compared with the PAAm-Alg hydrogels, functionalized formulations exhibit a better cell viability. Histological staining, and score distribution confirmed that proposed hydrogels significantly enhance regeneration of cartilage in rats due to stable hydrogel matrix and controlled release of TGF-ß3. These findings demonstrated that PAAm-Alg hydrogels showed potential for cartilage repair and clinical application.

Acetabular Coverage Decreases at the End of Skeletal Growth: A 3DCT Study of Healthy Hips.

BACKGROUND: Abnormalities in size and position of the acetabulum have been linked to both developmental dysplasia of the hip and femoroacetabular impingement. Owing to its 3-dimensional (3D) complexity, plain radiography and cross-sectional studies [computed tomography (CT) and magnetic resonance imaging] have limitations in their ability to capture the complexity of the acetabular 3D anatomy. The goal of the study was to use 3D computed tomography reconstructions to identify the acetabular lunate cartilage and measure its size at varying ages of development and between sexes. METHODS: Patients aged 10 to 18 years with asymptomatic hips and a CT pelvis for appendicitis were reviewed. Patients were stratified by sex and age: preadolescent (10 to 12), young adolescent (13 to 15), and old adolescent (16 to 18) in equal proportions. Materialise 3-matic was used to generate a 3D pelvic model, and the acetabular lunate cartilage surface area was calculated. The lunate cartilage was divided into anatomic segments: superior (11:00 to 1:00), anterior (1:00 to 4:00), and posterior (8:00 to 11:00). The femoral head surface area was calculated to control for patient size. Mixed effects models were generated predicting segment size where side was treated as a repeated measure. Absolute and relative (lunate cartilage to femoral head) models were generated. RESULTS: Sixty-two patients (124 hips) were included. Females showed a significant decrease in femoral head coverage as age increased overall and in the 3 subsegments. The majority of changes occurred between the preadolescent and young adolescent groups. Males did not show an overall change, but the superior and anterior anatomic subgroups showed a significant decrease in coverage between the young and old adolescent groups. Male lunate cartilages were absolutely, but not relatively, larger than females. No clinically significant side-to-side differences were noted. CONCLUSIONS: The relative femoral head coverage by the acetabular lunate cartilage reduced with increasing age, suggesting the growth of the femoral head outpaces the acetabular lunate cartilage's growth. This was more prominent in females. This study has important implications for expected acetabular coverage changes in the latter aspects of pediatric and adolescent development. LEVEL OF EVIDENCE: Level III-diagnostic study.

Marine Collagen Hydrolysates Downregulate the Synthesis of Pro-Catabolic and Pro-Inflammatory Markers of Osteoarthritis and Favor Collagen Production and Metabolic Activity in Equine Articular Chondrocyte Organoids.

Articular cartilage experiences mechanical constraints leading to chondral defects that inevitably evolve into osteoarthritis (OA), because cartilage has poor intrinsic repair capacity. Although OA is an incurable degenerative disease, several dietary supplements may help improve OA outcomes. In this study, we investigated the effects of Dielen® hydrolyzed fish collagens from skin (Promerim®30 and Promerim®60) and cartilage (Promerim®40) to analyze the phenotype and metabolism of equine articular chondrocytes (eACs) cultured as organoids. Here, our findings demonstrated the absence of cytotoxicity and the beneficial effect of Promerim® hydrolysates on eAC metabolic activity under physioxia; further, Promerim®30 also delayed eAC senescence. To assess the effect of Promerim® in a cartilage-like tissue, eACs were cultured as organoids under hypoxia with or without BMP-2 and/or IL-1ß. In some instances, alone or in the presence of IL-1ß, Promerim®30 and Promerim®40 increased protein synthesis of collagen types I and II, while decreasing transcript levels of proteases involved in OA pathogenesis, namely Htra1, and the metalloproteinases Mmp1-3, Adamts5, and Cox2. Both Promerim® hydrolysates also decreased Htra1 protein amounts, particularly in inflammatory conditions. The effect of Promerim® was enhanced under inflammatory conditions, possibly due to a decrease in the synthesis of inflammation-associated molecules. Finally, Promerim® favored in vitro repair in a scratch wound assay through an increase in cell proliferation or migration. Altogether, these data show that Promerim®30 and 40 hold promise as dietary supplements to relieve OA symptoms in patients and to delay OA progression.

Cell-free scaffolds functionalized with bionic cartilage acellular matrix microspheres to enhance the microfracture treatment of articular cartilage defects.

Microfracture surgery remains the most popular treatment for articular cartilage lesions in the clinic, but often leads to the formation of inferior fibrocartilage tissue and damage to subchondral bone. To overcome these problems, extracellular matrix (ECM) scaffolds derived from decellularized natural cartilaginous tissues were introduced and showed excellent biological properties to direct the differentiation of bone marrow stem cells. However, besides the limited allogenic/allogenic supply and the risk of disease transfer from xenogeneic tissues, the effectiveness of ECM scaffolds always varied with a high variability of natural tissue quality. In this study, we developed composite scaffolds functionalized with a cell-derived ECM source, namely, bionic cartilage acellular matrix microspheres (BCAMMs), that support the chondrogenic differentiation of bone marrow cells released from microfracture. The scaffolds with BCAMMs at different developmental stages were investigated in articular cartilage regeneration and subchondral bone repair. Compared to microfracture, the addition of cell-free BCAMM scaffolds has demonstrated a great improvement of regenerated cartilage tissue quality in a rabbit model as characterized by a semi-quantitative analysis of cells, histology and biochemical assays as well as micro-CT images. Moreover, the variation in ECM properties was found to significantly affect the cartilage regeneration, highlighting the challenges of homogenous scaffolds in working with microfracture. Together, our results demonstrate that the biofunctionalized BCAMM scaffold with cell-derived ECM shows great potential to combine with microfracture for clinical translation to repair cartilage defects.

Changes in DNA methylation accompany changes in gene expression during chondrocyte hypertrophic differentiation in vitro.

During osteoarthritis (OA), articular chondrocytes undergo phenotypic changes that resemble developmental patterns characteristic of growth plate chondrocytes. These phenotypic alterations lead to a hypertrophy-like phenotype characterized by altered production of extracellular matrix constituents and increased collagenase activity, which, in turn, results in cartilage destruction in OA disease. Recent studies have shown that the phenotypic instability and dysregulated gene expression in OA are associated with changes in DNA methylation patterns. Subsequent efforts have aimed to identify changes in DNA methylation with functional impact in OA disease, to potentially uncover therapeutic targets. Here, we paired an in vitro 3D/pellet culture system that mimics chondrocyte hypertrophy with RNA sequencing (RNA-Seq) and enhanced reduced representation of bisulfite sequencing (ERRBS) to identify transcriptomic and epigenomic changes in murine primary articular chondrocytes undergoing hypertrophy-like differentiation. We identified hypertrophy-associated changes in DNA methylation patterns in vitro. Integration of RNA-Seq and ERRBS datasets identified associations between changes in methylation and gene expression. Our integrative analyses showed that hypertrophic differentiation of articular chondrocytes is accompanied by transcriptomic and epigenomic changes in vitro. We believe that our integrative approaches have the potential to uncover new targets for therapeutic intervention.

Surgical Induction of Posttraumatic Osteoarthritis in the Mouse.

Given the prevalence and the scope of the personal and societal burden of osteoarthritis (OA), investigators continue to be deeply interested in understanding the pathogenic basis of disease and developing novel disease modifying OA therapies. Because joint trauma/injury is considered a leading predisposing factor in the development of OA, and since posttraumatic OA is one of the most common forms of OA in general, large animal and rodent models of knee injury that accurately recapitulate the OA disease process have become increasingly widespread over the past decade. To enable study in the context of defined genetic backgrounds, investigative teams have developed standardized protocols for injuring the mouse knee that aim to induce a reproducible degenerative process both in terms of severity and temporal pacing of disease progression. The destabilization of the medial meniscus (DMM) is one of the most commonly employed surgical procedure in rodents that reproducibly models posttraumatic OA and allows for the study of disease progression from initiation to end-stage disease. The description provided here sets the stage for both inexperienced and established investigators to employ the DMM procedure, or other similar surgical destabilization methods, to initiate the development of posttraumatic OA in the mouse. Successful application of this method provides a preclinical platform to study the mechanisms driving the pathogenesis of posttraumatic OA and for testing therapeutic strategies to treat it.