MicroRNAs as Prognostic Markers for Chondrogenic Differentiation Potential of Equine Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are a promising cell source for cartilage tissue regeneration in animals and humans but with large interdonor variation in their in vitro chondrogenic differentiation potential. Underlying molecular mechanisms responsible for culture-expanded MSC heterogeneity remain poorly understood. In this study, we sought to identify variations in microRNA (miRNA) signatures associated with cultured equine MSC chondrogenic differentiation potential from different donors. Neocartilage tissue generated from equine cord blood-derived MSCs was categorized as having either high or low chondrogenic potential (LCP) based on their histological appearance and quantification of glycosaminoglycan deposition. Using next-generation sequencing, we identified 30 differentially expressed miRNAs among undifferentiated MSC cultures that corresponded with their chondrogenic potential. Of note, MSCs with LCP upregulated miR-146a and miR-487b-3p, which was also observed by quantitative real-time polymerase chain reaction. Our findings suggest that miRNA profiling of equine MSC cultures may have prognostic value in selecting MSC donors with regard to their chondrogenic differentiation potential.

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Unique and overlapping GLI1 and GLI2 transcriptional targets in neoplastic chondrocytes.

Excessive Hedgehog (Hh) signaling in chondrocytes is sufficient to cause formation of enchondroma-like lesions which can progress to chondrosarcoma. To elucidate potential underlying mechanisms, we identified GLI1 and GLI2 target genes in human chondrosarcoma. Using chromatin immunoprecipitation (ChIP) sequencing and microarray data, in silico analyses were conducted to identify and characterize unique and overlapping GLI1 and GLI2 binding regions in neoplastic chondrocytes. After overlaying microarray data from human chondrosarcoma, 204 upregulated and 106 downregulated genes were identified as Hh-responsive Gli binding targets. After overlaying published Gli ChIP-on-chip data from mouse, 48 genes were identified as potential direct downstream targets of Hedgehog signaling with shared GLI binding regions in evolutionarily conserved DNA elements. Among these was BMP2, pointing to potential cross-talk between TGF beta signaling and Hh signaling. Our identification of potential target genes that are unique and common to GLI1 and GLI2 in neoplastic chondrocytes contributes to elucidating potential pathways through which Hh signaling impacts cartilage tumor biology.

Cultural Factors Influencing Osteoarthritis Care in Asian Communities: A Review of the Evidence.

With the prevalence of osteoarthritis (OA) increasing internationally, there is a need to study the impact of this disease on culturally diverse populations. Individuals of Asian descent make up more than 60% of the world population, yet comprehensive information on the cultural factors that impact OA care is not available. Scoping review methodology using directed content analysis was employed to identify and analyze existing research on OA care for Asians. A categorization matrix was developed using the six care areas from the OA clinical practice guidelines along with an additional three non-clinical areas (cross-cultural adaptation of clinical tools; psychological well-being; family systems and informal care) identified in an initial scan resulting in a total of nine OA care areas to guide initial coding. A full scoping review was conducted across five databases resulting in 656 abstracts screened. All text was coded using the categorization matrix and resulting subthemes were identified. A total of 74 articles were analyzed with 23 subthemes identified across the nine categories. Four new perspectives emerged to support OA care for Asian populations: (1) the importance of family and peer assistance, (2) the importance of culturally specific activities, (3) distrust in western medicine, and (4) impact of positive coping mechanisms on health appraisals. While Asians are more susceptible to knee and hand OA because of their cultural lifestyle factors (e.g. squatting for chores, hygiene and religious activities), and traditional beliefs on OA management (e.g. traditional diet, topical oils, physical therapy), many do not present themselves for conventional treatments (e.g. surgery) until all traditional treatments are exhausted. The results suggest that cultural factors influence the uptake of OA management practices among Asians. Greater awareness of these cultural factors may improve diagnosis, treatment, and management of OA among Asian patients.