Mature Cartilage Research Articles

Extensive chondroid differentiation in a Wilms tumor following chemotherapy: Clinical relevance and implications

Wilms tumor (WT) is the most common childhood tumor of the kidney, which histologically mimics various stages of nephrogenesis. Spectrum of chemotherapy induced histopathological changes has been described in literature, and is one of the most important predictors of disease outcome and survival rates. Extensive chondroid differentiation and absence of necrosis in a postchemotherapy nephrectomy specimen of a radiologically proven Wilms tumor is an unusual finding. Herein, we present the case of 6-year-old girl from South Africa who received a 10-week course of chemotherapy upon radiological diagnosis of WT, and post-surgery nephrectomy specimen revealed extensive mature cartilage island formation on histology raising diagnostic dilemma. Reporting of such cases is extremely important to recognize tumor morphological heterogeneity, particularly post- chemotherapy, and developing consensus for selecting further treatment and clinical follow-up.

The horse as a model for translational orthopedic research: examples of studies on the regeneration of cartilage and bone conducted in Costa Rica

The paper provides results published or to be published of long-term in vivo equine studies to evaluate techniques of possible regenerative matrices of cartilage and bone, by means of cell-free implants or stimulation of the bone marrow. From the fixation techniques analyzed, it can be concluded that the best alternatives are the pressure technique for subchondral defects and a novel hydrogel with self-adhesive capacity for chondral defects. The equine coxal tuberosity was used for the first time as a model for regeneration studies of bone defects, analyzing scaffolds based on tricalcium phosphate, polymers and nanoparticles, by means of 3-D printing. Osteoconductivity, osteoinductivity, and the importance of microporosity were documented.Given that decellularized materials do not always give significant desired results in the regeneration of cartilage, it is important to conduct long-term studies. The technique of nanofracture and a novel self-adhesive hydrogel in the knee of the equine showed promising preliminary results in the regeneration of cartilage.The knee and the coxal tuberosity of the horse represent models of studying cartilage and bone regeneration in a true translational sense as a source of highly valuable information for clinical studies, for both horses and humans.

Tissue-derived decellularized extracellular matrices toward cartilage repair and regeneration.

The inability of cartilage tissue to self-heal due to its avascular nature often leads to conditions such as osteoarthritis, traumatic rupture of cartilage, and osteochondrosis. The cartilage provides cushioning effects between the joints and avoids bone frictions. The extracellular matrix (ECM) of cartilage consists predominantly of collagens, elastin, proteoglycans and glycoproteins. A number of tissue engineered ECM derived biological scaffolds and matrices are available for cartilage regeneration. The decellularized tissues provide appropriate bioactive cues in the absence of cellular components, hence avoiding immunological issue. However, the decellularization process involves several cellular disruption techniques that may alter the ECM architecture affecting bioactivity. Therefore, development of cell-free cartilage biomaterials with unaltered ECM integrity and bioactivity is of paramount necessity by smart selection of modified techniques and agents. Herein, we described about various decellularization methods, agents, techniques, and their applications in tissue/cartilage decellularization. It also contemplates various difficulties and future perspectives to troubleshoot the existing obstructions in tissue-derived cartilage matrices and their applications.

N-methyl-D-aspartate (NMDA) receptor expression and function is required for early chondrogenesis

BackgroundIn vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated.MethodsWe studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting.ResultsHere we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein.ConclusionsWe propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts.

Gene expression markers in horse articular chondrocytes: Chondrogenic differentiaton IN VITRO depends on the proliferative potential and ageing. Implication for tissue engineering of cartilage

Chondrocyte dedifferentiation is a key limitation in therapies based on autologous chondrocyte implantation for cartilage repair. Articular chondrocytes, obtained from (metacarpophalangeal and metatarsophalangeal) joints of different aged horses, were cultured in monolayer for several passages (P0 to P8). Cumulative Populations Doublings Levels (PDL) and gene expression of relevant chondrocyte phenotypic markers were analysed during culturing. Overall data confirmed that, during proliferation in vitro, horse chondrocytes undergo marked morphological and phenotypic alterations of their differentiation status. Particularly, the dedifferentiation started early in culture (P0-P1) and was very marked at P3 subculture (PDL 4–6): proliferative phase after P3 could be critical for maintenance/loss of differentiation potential. In elderly animals, chondrocytes showed aspects of dedifferentiation shortly after their isolation, associated with reduced proliferative capacity. Regarding the gene expression of major cartilage markers (Col2, Aggrecan, SOX9) there was a very early reduction (P1) in proliferating chondrocytes independent of age. The chondrocytes from adult donors showed a more stable expression (up to P3) of some (Col6, Fibromodulin, SOX6, TGβ1) markers of mature cartilage; these markers could be tested as parameter to determine the dedifferentiation level. This study can provide parameters to identify up to which “culture step” chondrocytes for implantation with a conserved phenotypic potential can be obtained, and to test the efficiency of biomaterial scaffold or chondroinductive media/signals to maintain/recover the chondrocyte phenotype. Moreover, the determination of levels and time related expression of these markers can be useful during the chondroinduction of mesenchymal stem cells.

Type III collagen is a key regulator of the collagen fibrillar structure and biomechanics of articular cartilage and meniscus

Despite the fact that type III collagen is the second most abundant collagen type in the body, its contribution to the physiologic maintenance and repair of skeletal tissues remains poorly understood. This study queried the role of type III collagen in the structure and biomechanical functions of two structurally distinctive tissues in the knee joint, type II collagen-rich articular cartilage and type I collagen-dominated meniscus. Integrating outcomes from atomic force microscopy-based nanomechanical tests, collagen fibril nanostructural analysis, collagen cross-link analysis and histology, we elucidated the impact of type III collagen haplodeficiency on the morphology, nanostructure and biomechanical properties of articular cartilage and meniscus in Col3a1+/− mice. Reduction of type III collagen leads to increased heterogeneity and mean thickness of collagen fibril diameter, as well as reduced modulus in both tissues, and these effects became more pronounced with skeletal maturation. These data suggest a crucial role of type III collagen in mediating fibril assembly and biomechanical functions of both articular cartilage and meniscus during post-natal growth. In articular cartilage, type III collagen has a marked contribution to the micromechanics of the pericellular matrix, indicating a potential role in mediating the early stage of type II collagen fibrillogenesis and chondrocyte mechanotransduction. In both tissues, reduction of type III collagen leads to decrease in tissue modulus despite the increase in collagen cross-linking. This suggests that the disruption of matrix structure due to type III collagen deficiency outweighs the stiffening of collagen fibrils by increased cross-linking, leading to a net negative impact on tissue modulus. Collectively, this study is the first to highlight the crucial structural role of type III collagen in both articular cartilage and meniscus extracellular matrices. We expect these results to expand our understanding of type III collagen across various tissue types, and to uncover critical molecular components of the microniche for regenerative strategies targeting articular cartilage and meniscus repair.

Proliferation medium in three-dimensional culture of auricular chondrocytes promotes effective cartilage regeneration invivo.

IntroductionCartilage regeneration have been attracted attentions because of the poor ability of cartilage tissues to regenerate. Three-dimensional (3D) culture of chondrocytes is considered to be advantageous for cartilage regeneration. Although it is plausible that maturation of the constructs before transplantation positively affects the chondrogenesis, matured constructs after cultures for longer periods do not necessarily result in effective cartilage regeneration. In this study, we compared different types of culture media including growth factors which are clinically available. We prepared differentiation medium containing insulin-like growth factor-1 (IGF-1), proliferation medium containing fibroblast growth factor-2 (FGF-2) and insulin, and combination of them, and compared their efficacies on chondrogenesis when used in 3D culture of engineered cartilage constructs.MethodsCartilage constructs were fabricated by auricular chondrocytes and atelocollagen, and they were 3D-cultured with four types of media: control medium, differentiation medium, proliferation medium, and combination medium. After 3 weeks of culture, the constructs were analyzed for cell number, gene and protein expressions and mechanical properties. The constructs were also transplanted into nude mice. After 8 weeks, the degree of cartilage regeneration was evaluated. Constructs manufactured with canine auricular chondrocytes were subjected to autologous transplantation into beagles and examined for cartilage regeneration.ResultsDuring 3D culture, remarkably high gene expression of type II collagen was detected in the construct cultured with the differentiation medium whereas cell apoptosis were suppressed in the proliferation medium. When transplanted into nude mice, the constructs 3D-cultured in the proliferation medium produced abundant cartilage matrices. In autologous implantation model, the construct cultured in the proliferation medium again showed better chondrogenesis than those in other media.ConclusionsThe present study indicates that 3D culture with the proliferation medium maintains the cell viability to potentiate the subsequent cartilage regeneration. Here, we propose that not only differentiation but also high cell viability accompanied by proliferation factors should be taken into account to improve cartilage regeneration.

Understanding the Cellular and Molecular Mechanisms That Control Early Cell Fate Decisions During Appendicular Skeletogenesis.

The formation of the vertebrate skeleton is orchestrated in time and space by a number of gene regulatory networks that specify and position all skeletal tissues. During embryonic development, bones have two distinct origins: bone tissue differentiates directly from mesenchymal progenitors, whereas most long bones arise from cartilaginous templates through a process known as endochondral ossification. Before endochondral bone development takes place, chondrocytes form a cartilage analgen that will be sequentially segmented to form joints; thus, in the cartilage template, either the cartilage maturation programme or the joint formation programme is activated. Once the cartilage differentiation programme starts, the growth plate begins to form. In contrast, when the joint formation programme is activated, a capsule begins to form that contains special articular cartilage and synovium to generate a functional joint. In this review, we will discuss the mechanisms controlling the earliest molecular events that regulate cell fate during skeletogenesis in long bones. We will explore the initial processes that lead to the recruitment of mesenchymal stem/progenitor cells, the commitment of chondrocyte lineages, and the formation of skeletal elements during morphogenesis. Thereafter, we will review the process of joint specification and joint morphogenesis. We will discuss the links between transcription factor activity, cell–cell interactions, cell–extracellular matrix interactions, growth factor signalling, and other molecular interactions that control mesenchymal stem/progenitor cell fate during embryonic skeletogenesis.

PURE CARTILAGINOUS CHORISTOMA ON THE VENTRAL SURFACE OF TONGUE: A DOUBLE RARITY

Choristomas are tumor like mass that are aggregates of microscopically normal tissue in an abnormal location. Commonly reported choristomas are of Osseous, Cartilaginous, Glandular and Glial types. The oral cavity is an unusual site of presentation where the most common site is dorsum of the tongue. Ventral aspect is an extremely rare site with only four cases reported till date. We report here a case of a 24-year-old male who presented with a small swelling on the ventral surface of the tongue. The histopathological examination revealed features of a well-circumscribed mass composed of exclusively mature cartilage encased within a dense fibrous connective tissue. The case is presented for its double rarity of site which is a ventral surface of the tongue and the histopathology which revealed a pure form of cartilaginous choristoma.

Morphological and biomechanical characterization of immature and mature nasoseptal cartilage

Nasoseptal cartilage has been assumed to be isotropic, unlike the well-defined zonal organization of articular cartilage attributed to postnatal biomechanical loading. We know from clinical experience that malrotation of surgical nasoseptal cartilage grafts can lead to increased graft absorption. Other studies have also suggested directionally dependent compressive stiffness suggesting anisotropy, but morphological investigations are lacking. This study characterizes immature and mature native bovine nasoseptal cartilage using a combination of immunohistochemistry, biomechanical testing and structural imaging. Our findings indicate that there is extensive postnatal synthesis and reorganization of the extracellular matrix in bovine nasoseptal cartilage, independent of joint loading forces responsible for articular cartilage anisotropy. Immature nasoseptal cartilage is more cellular and homogenous compared to the zonal organization of cells and extracellular matrix of mature cartilage. Mature samples also exhibited greater glycosaminoglycan content and type II collagen fibre alignment compared to immature cartilage and this correlates with greater compressive stiffness. Engineered neocartilage often consists of immature, isotropic, homogenous tissue that is unable to meet the functional and mechanical demands when implanted into the native environment. This study demonstrates the importance of anisotropy on biomechanical tissue strength to guide future cartilage tissue engineering strategies for surgical reconstruction.

Depth-dependent ratcheting strains of young and adult articular cartilages by experiments and predictions

BackgroundRatcheting strain is produced due to the repeated accumulation of compressive strain in cartilage and may be a precursor to osteoarthritis. The aim of this study was to investigate the ratcheting behaviors of young and adult articular cartilages under cyclic compression by experiments and theoretical predictions.MethodsA series of uniaxial cyclic compression tests were conducted for young and adult cartilage, and the effects of different loading conditions on their ratcheting behaviors were probed. A theoretical ratcheting model was constructed and applied to predict the ratcheting strains of young and adult cartilages with different loading conditions.ResultsRatcheting strains of young and adult cartilages rapidly increased at the initial stage, followed by a slower increase in subsequent stages. The strain accumulation value and its rate for young cartilage were greater than them for adult cartilage. The ratcheting strains of the two groups of cartilage samples decreased with increasing stress rate, while they increased with increasing stress amplitude. As the stress amplitude increased, the gap between the ratcheting strains of young and adult cartilages increased gradually. The ratcheting strains of young and adult cartilages decreased along the cartilage depth from the surface to the deep layer. The ratcheting strains of different layers increased with the compressive cycle, and the difference among the three layers was noticeable. Additionally, the theoretical predictions agreed with the experimental data.ConclusionsOverall, the ratcheting behavior of articular cartilage is affected by the degree of articular cartilage maturation.

MiR-26a-5p is a Stable Reference Gene for miRNA Studies in Chondrocytes from Developing Human Cartilage.

miRNAs are emerging as key regulators of complex biological systems in several developmental processes. qRT-PCR is a powerful tool to quantitatively assess the profiles and modulation of miRNA expression. In the emerging field of cartilage maturation studies, from precursor to hypertrophic chondrocytes, few data about miRNA regulation are available, and no consensus on the best reference gene (RG) has been reached. This is a crucial pitfall since reliable outcomes depend on proper data normalization. The aim of this work was to identify reliable and stable miRNA RGs, basing the analysis on available high throughput qRT-PCR miRNA data (from the NCBI Gene Expression Omnibus database, GSE49152) obtained from human embryonic cartilage tissues enriched in the precursor, differentiated, and hypertrophic chondrocytes. Four normalization approaches were used, and the stability was quantified by combining BestKeeper, delta-Ct, geNorm, and NormFinder statistical tools. An integrated approach allowed to identify miR-26a-5p as the most stable RG and miR-212-3p as the worst one. RNU44, used in original dataset analysis, performed as second best RG. Applications of different normalization strategies significantly impacted the profiles and modulation of miRNA expression. Herein presented results point out the crucial need of a consensus on data normalization studies aimed at dissecting miRNA role in human cartilage development, to avoid the postulation of unreliable biological conclusions.

Development and Characterization of Acellular Caprine Choncal Cartilage Matrix for Tissue Engineering Applications.

Because of poor regenerative capabilities of cartilage, reconstruction of similar rigidity and flexibility is difficult, challenging, and restricted. The aim of the present investigation was to develop cost-effective acellular xenogeneic biomaterial as cartilage substitution. Two novel biometrics have been developed using different chemical processes (Na-deoxycholate + SDS and GndHCl + NaOH) to decellularize caprine (goat) ear cartilage and further extensively characterized before preclinical investigation. Complete cell removal was ascertained by hematoxylin and eosin staining followed by DNA estimation. No adverse effect on extracellular matrix (ECM) was found by quantifying collagen and sulfated glycosaminoglycans (sGAG) content as well as collagen, sGAG and elastin staining. Results showed no drastic changes in ECM structure apart from desired sGAG loss. Scanning electron microscopy images confirmed cellular loss and unaltered orientation. Nano-indentation study on cartilage matrices indicated interesting output showing better results among decellularized groups. Increased elastic modulus and hardness indicated better stiffness and more active energy dissipation mechanism due to decellularization. Fluid uptake and retention property remained unchanged after decellularization as analyzed by swelling behavior study. Additionally, acellular materials were confirmed to be nonreactive and nonhemolytic as assessed by in vitro hemocompatibility study. In vivo study (up to 3 months) on rabbits showed no symptoms of graft rejection/ tissue necrosis, established through postoperative histology and biochemical analyses of tissue explants. With regard to size, shape, biomechanics, source of origin and nonimmunogenic properties, these developed materials can play versatile role in biomedical/ clinical applications and pave a new insight as alternatives in cartilage reconstruction.

Cartilage tissue engineering combining microspheroid building blocks and microneedle arrays

ABSTRACTPurpose: Scaffold-free cartilage tissue engineering circumvents issues with scaffold seeding, potential toxicity response, and impaired host integration. However, precisely controlling and maintaining a scaffold-free construct shape have been challenging. We explored the feasibility of microneedle arrays to print tissue using cellular microspheroids as building blocks.Materials and Methods: Human embryonic-derived mesenchymal stem cells or infrapatellar fat pad mesenchymal stem cells were used to create microspheroids of 500 µm in diameter, which were assembled on microneedle arrays in a predefined arrangement using a robotic system under computer vision. Microspheroids on microneedles were cultured to permit fusion into a tissue construct. Infrapatellar fat pad mesenchymal stem cell constructs were either implanted into chondral defects created in human osteoarthritic cartilage explants or maintained on the microneedle array for 3 weeks. Embryonic-derived mesenchymal stem cell constructs were designed to be press-fit into 3 mm subchondral defects in New Zealand White rabbits and maintained for up to 8 weeks to assess retention, early tissue repair, and more mature cartilage regeneration.Results: Microspheroids of both cell types fused together in culture to form neotissues of predefined shape and size. Infrapatellar fat pad mesenchymal stem cell neotissues expressed high levels of chondrogenic genes and integrated with the surrounding osteoarthritic host cartilage. Embryonic-derived mesenchymal stem cell constructs generated chondrogenic neotissue in vivo as early as 2 weeks and more mature tissue by 8 weeks with increased glycosaminoglycan deposition.Conclusions: We constructed defined scaffold-free shapes by bioprinting and fusing microspheroids. Proof of concept was shown in the repair of ex vivo osteoarthritic human cartilage and in vivo rabbit osteochondral (OC) defects.

GC–MS method for metabolic profiling of mouse femoral head articular cartilage reveals distinct effects of tissue culture and development

The metabolic profile of cartilage is important to define as it relates to both normal and pathophysiological conditions. Our aim was to develop a precise, high-throughput method for gas/chromatography-mass/spectrometry (GC-MS) semi-targeted metabolic profiling of mouse cartilage. Femoral head (hip) cartilage was isolated from 5- and 15-week-old male C57BL/6J mice immediately after death for invivo analyses. In vitro conditions were evaluated in 5-week-old samples cultured ±10% fetal bovine serum (FBS). We optimized cartilage processing for GC-MS analysis and evaluated group-specific differences by multivariate and parametric statistical analyses. 55 metabolites were identified in pooled cartilage (4 animals per sample), with 29 metabolites shared between invivo and invitro conditions. Multivariate analysis of these common metabolites demonstrated that culturing explants was the strongest factor altering cartilage metabolism, followed by age and serum starvation. In vitro culture altered the relative abundance of specific metabolites; whereas, cartilage development between five and 15-weeks of age reduced the levels of 36 out of 43 metabolites >2-fold, especially in TCA cycle and alanine, aspartate, and glutamate pathways. In vitro serum starvation depleted six out of 41 metabolites. This study describes the first GC-MS method for mouse cartilage metabolite identification and quantification. We observed fundamental differences in femoral head cartilage metabolic profiles between invivo and in vitro conditions, suggesting opportunities to optimize in vitro conditions for studying cartilage metabolism. In addition, the reductions in TCA cycle and amino acid metabolites during cartilage maturation illustrate the plasticity of chondrocyte metabolism during development.

Biomimetic cartilage scaffold with orientated porous structure of two factors for cartilage repair of knee osteoarthritis

A dual-layer biomimetic cartilage scaffold was prepared by mimicking the structural design, chemical cues and mechanical characteristics of mature articular cartilage. The surface layer was made from collagen (COL), chitosan (CS) and hyaluronic acid sodium (HAS). The transitional layer with microtubule array structure was prepared with COL, CS and silk fibroin (SF). The PLAG microspheres containing kartogenin (KGN) and the polylysine-heparin sodium nanoparticles containing TGF-β1 (TPHNs) were constructed for the surface, transitional layer, respectively. The SEM result showed that the dual-layer composite scaffold had a double structure similar to natural cartilage. The vitro biocompatibility experiment showed that the biomimetic cartilage scaffold with orientated porous structure was more conducive to the proliferation and adhesion of BMSCs. A rabbit KOA cartilage defect model was established and biomimetic cartilage scaffolds were implanted in the defect area. Compared with the surface layer and transitional layer scaffolds group, the results of dual-layer biomimetic cartilage scaffold group showed that the defects had been completely filled, the boundary between new cartilage and surrounding tissue was difficult to identify, and the morphology of cells in repair tissue was almost in accordance with the normal cartilage after 16 weeks. All those results indicated that the biomimetic cartilage scaffold could effectively repair the defect of KOA, which is related to the fact that the scaffold could guide the morphology, orientation, and proliferation and differentiation of BMSCs. This work could potentially lead to the development of multilayer scaffolds mimicking the zonal organization of articular cartilage.

High-Temperature Requirement A1 Protease as a Rate-Limiting Factor in the Development of Osteoarthritis

Preserving the mature articular cartilage of joints is a critical focus in the prevention and treatment of osteoarthritis. We determined whether the genetic inactivation of high-temperature requirement A1 (HtrA1) can significantly attenuate the degradation of articular or condylar cartilage. Two types of mouse models of osteoarthritis were used, a spontaneous mutant mouse model [type XI collagen-haploinsufficient (Col11a1+/-) mice] and two post-traumatic mouse models [destabilization of the medial meniscus (DMM) on the knee and a partial discectomy (PDE) on the temporomandibular joint]. Three different groups of mice were generated: i) HtrA1 was genetically deleted from Col11a1+/- mice (HtrA1-/-;Col11a1+/-), ii) HtrA1-deficient mice (HtrA1-/-) were subjected to DMM, and iii) HtrA1-/- mice were subjected to PDE. Knee and temporomandibular joints from the mice were characterized for evidence of cartilage degeneration. The degradation of articular or condylar cartilage was significantly delayed in HtrA1-/-;Col11a1+/- mice and HtrA1-/- mice after DMM or PDE. The amount of collagen type VI was significantly higher in the articular cartilage in HtrA1-/-;Col11a1+/- mice, compared with that in Col11a1+/- mice. The genetic removal of HtrA1 may delay the degradation of articular or condylar cartilage in mice.

Mechanosensitive MiRs regulated by anabolic and catabolic loading of human cartilage

Elucidation of whether miRs are involved in mechanotransduction pathways by which cartilage is maintained or disturbed has a particular importance in our understanding of osteoarthritis (OA) pathophysiology. The aim was to investigate whether mechanical loading influences global miR-expression in human chondrocytes and to identify mechanosensitive miRs responding to beneficial and non-beneficial loading regimes as potential to obtain valuable diagnostic or therapeutic targets to advance OA-treatment. Mature tissue-engineered human cartilage was subjected to two distinct loading regimes either stimulating or suppressing proteoglycan-synthesis, before global miR microarray analysis. Promising candidate miRs were selected, re-evaluated by qRT-PCR and tested for expression in human healthy vs OA cartilage samples. After anabolic loading, miR microarray profiling revealed minor changes in miR-expression while catabolic stimulation produced a significant regulation of 80 miRs with a clear separation of control and compressed samples by hierarchical clustering. Cross-testing of selected miRs revealed that miR-221, miR-6872-3p, miR-6723-5p were upregulated by both loading conditions while others (miR-199b-5p, miR-1229-5p, miR-1275, miR-4459, miR-6891-5p, miR-7150) responded specifically after catabolic loading. Mechanosensitivity of miR-221 correlated with pERK1/2-activation induced by both loading conditions. The miR-response to loading was transient and a constitutive deregulation of mechano-miRs in OA vs healthy articular cartilage was not observed. MiRs with broader vs narrower mechanosensitivity were discovered and the first group of mechanosensitive miRs characteristic for non-beneficial loading was defined that may shape the proteome differentially when cartilage tissue is disturbed. The findings prompt future investigations into miR-relevance for mechano-responsive pathways and the corresponding miR-target molecules.

Maturation of the medial femoral condyle epiphyseal growth plate and osteochondral junction

Purpose: The osteochondral junction region has an important role in osteoarthritis and many early pathophysiological features (e.g. chondrocyte hypertrophy) of the disease recapitulate development events. Femorotibial osteoarthritis arises most commonly in the medial compartment in humans and horses. Furthermore, juvenile osteochondrosis (JOCD) also develops in this region, and if not treated successfully, the persisting incongruency can lead to secondary osteoarthritis. The maturation of the epiphyseal growth cartilage and the osteochondral junction of the medial femoral condyle (MFC) of the distal femur is poorly described in the literature. Horses are also increasingly recognized to be appropriate large animal models for cartilage repair investigations and information on maturation of the osteochondral junction is lacking. Our goal was to describe the normal development of the equine MFC osteochondral junction from birth to 2 years old. Methods: Distal femurs were collected from Thoroughbred horses (n=61, 0-2 years old), presented for post-mortem examination for reasons unrelated to this study. Radiographs and tomodensitometry were performed to screen for healthy specimens without focal osteochondral junction lesions. Micro-tomodensitometry (μ-CT), histology and immunohistochemistry were performed on the selected MFCs (n=29). μ-CT: A region of interest (ROI) was selected for study in a weight bearing area of the MFC that also incorporated developmental disease (JOCD and equine cysts/osteochondrosis) prone sites. Cartilage thickness (Cth) and trabecular bone volume were measured. 3D images were also constructed and the osteochondral junction morphology was assessed. Histology: MFC epiphyseal growth plate structure was studied on hematoxylin phloxine and saffron (HPS) stained sections. Collagen structure (picrosirius red) and proteoglycan content (Safranin O fast green) were also evaluated. Chondrocyte lacunae diameters were measured in predefined regions below the articular surface and near the osteochondral junction as an indicator of chondrocyte hypertrophy. Collagen X (Col X) immunohistochemical staining was also employed as a marker of endochondral ossification. Results: μ-CT: The Cthranged from 3.8mm in the youngest specimens to 1.2mm at 2 years of age. The Cthdecreased from birth to 7 months and remained constant thereafter (regression into 2 segments, p<0.0001). From birth to 6 months of age, the ossification front was porous on 3D images (fig 1.a) and later had a smooth appearance (fig 1. d). The trabecular bone volume did not change with age and was significantly higher at the periphery of the condyle, near the cartilage, compared to the center (p<0.0001). Histology: The structure of the epiphyseal growth cartilage differed from that reported for the physis as there was no columnar organization of the chondrocytes. The safranin O fast green and picrosirius stains showed a stratification of the extracellular matrix content that varied with age. The osteochondral unit was immature up to 6 months of age: Cartilage canals containing blood vessels crossed the ossification front (fig 1.b) and the chondrocytes were undergoing hypertrophy near the cartilage-bone junction. The chondrocyte lacunar volume increased 14-fold beside the osteochondral junction in the youngest specimens (fig 1.c). Expression of Col X was evident in the extracellular matrix surrounding hypertrophic chondrocytes in site matched specimens in the juvenile specimens alone. At six months of age, the absence of chondrocyte hypertrophy and Col X immunostaining indicated the conclusion of endochondral ossification. This coincided with the smallest Cth, similar to mature articular cartilage and the appearance of a narrow band of compact bone indicating formation of a subchondral cortical bone plate (fig 1.e). Conclusions: The equine MFC osteochondral junction is structurally immature and undergoes endochondral ossification up to 6 months of age. At this time, the histological structure of a subchondral cortical bone plate emerges. Cartilage canals and hypertrophic chondrocytes are no longer present and the cartilage thickness is stable and consistent with a mature articular cartilage. Although we have explored the structural and matrix maturation events, additional studies are now required to assess the biomechanical maturation of the region. New information is provided on MFC osteochondral junction development that may help towards the understanding of the remodeling events occurring in osteoarthritic joints.

TRPV5 and TRPV6 are expressed in placenta and bone tissues during pregnancy in mice

ABSTRACTWe investigated the dynamic expression of calcium transporters, TRPV5 and TRPV6, in placenta and bone to determine their role in maternal and fetal calcium balance during gestation. In placenta, TRPV5 was expressed predominantly in syncytiotrophoblasts of the labyrinthine zone, whereas TRPV6 was expressed in spongiotrophoblasts of the junction zone. In bone, the two transporters were found in osteoblasts, osteoclasts, cartilage and bone matrices. During the first half of gestation, TRPV5 and TRPV6 levels in bone were increased on pregnancy day (P) 0.5, then decreased on P3.5 followed by a slight increase on P6.5. During the second half of pregnancy, both the proteins and their mRNAs gradually increased from P9.5 to P15.5−P17.5 in both bone and placenta, followed at parturition by relatively high amounts in placenta, but markedly decreased amounts in bone. The expression pattern is likely related to the fetal and maternal calcium requirement during gestation, which may be regulated by estrogen and other hormones, because the fetal demand for calcium is greatest during the last few days of gestation for rats; maternal calcium metabolism is designed to meet the calcium needs of the fetus during this period. We found that TRPV5 and TRPV6 are involved in calcium transport in the placenta and bone, and therefore play a role in calcium homeostasis during embryonic and fetal development.