Tenocytes from several mammal species have been shown to be prone to phenotypic drift at early sub-culture passages. In the present study we compared allogenic and xenogenic serum supplementation suitability as a supplement for the in vitro expansion of equine tenocytes (eTCs), in combination with the presence or absence of crowding conditions. eTCs were isolated from superficial digital flexor tendon and expanded in normal growth medium containing DMEM, 10% appropriate serum, 1% penicillin/streptomycin solution. Isolation was performed by migration method in growth medium containing the selected serum. Silver staining, densitometry, zymography, immunofluorescence, metabolic activity, proliferation, viability and morphology were performed after 3, 5 and 7 days in culture with a seeding density of 10,000 cells/cm2. Treatment conditions were equine serum (ES) or foetal bovine serum (FBS), with or without 75 μg/mL of crowding agent carrageenan (CR). Viability and metabolic activity of eTCs were affected by FBS. eTCs in ES reached higher cell density than in FBS in day 7, especially with CR. Morphology of eTCs was maintained under different sera. Silver staining on pepsin digested cell layers shows that collagen type I deposition rate is remarkably enhanced in the presence of CR in all conditions. Immunofluorescence showed increased expression for collagen I, III, V and VI in both sera in the presence of CR. Deposition of all collagen types but type VI was increased by ES supplementation. We conclude that ES in combination with CR can represent a reliable choice for the ex vivo expansion of eTCs.

RNA-Seq or whole transcriptome shotgun sequencing has been adopted in the last years as a reference technique to determine the presence and the quantity of different species of RNA in determined biological samples, thanks to it allows the identification every single RNA species transcribed from a reference genome. Meta-profiling takes advantage of the public availability of an increasing set of RNA-Seq data produced by different laboratories to summarize the expression levels of the different RNA species of many samples according to their biological context, giving the opportunity to perform comparisons on the gene expression profiles of different tissues by integrating data derived from a high number of studies. By using Genevestigator™; a platform which integrates RNA-Seq data into meta-profiles, we have performed a comparison between the gene expression profiles of bone, cartilage, muscle tendon and skin by means of interrogating its database with different gene sets and families with relevance to the function of the tissues of the musculoskeletal system. The collagen gene family and genes coding for proteoglycans, matrix metalloproteinases and tissue inhibitors of metalloproteinases, mechanotransduction-related proteins and signalling pathways involved in tissue development and differentiation have been analysed. Hierarchical clustering for every gene set was performed for the understanding the differences and similarities between the different tissues included in the analyses. The results of this study will help to improve our understanding of the musculoskeletal system, and will help to identify new biomarkers and signalling pathways of specific relevance for the bone, cartilage, muscle and tendon.

Porcine and fish by-products in particular are rich sources for collagen, which is the main component of the extracellular matrix (ECM). Although there are studies investigating different collagen derived from various tissue sources for the purpose of creating biomaterials, the comparison of biophysical, biochemical and biological properties of type II collagen isolated from cartilaginous tissues has yet to be assessed. In addition, it has been shown from previous studies that sex steroid hormones affect the collagen content in male and female animals, herein, type II collagens from male and female porcine cartilage were assessed in order to investigate gender effects on the property of collagen scaffolds. Moreover, type II collagen has a supportive role in articular cartilage in the knee joint. Therefore, the aim is to assess the properties of type II collagen scaffolds as a function of species, tissue and gender for cartilage regeneration. Type II collagen was extracted from male and female porcine trachea, auricular, articular cartilage and cartilaginous fish through acid-pepsin digestion at 4°C. SDS-PAGE was conducted to confirm the purity of extracted collagen. Collagen sponges were created via freeze-drying. Scaffold structure and pore size were evaluated by scanning electron microscopy (SEM). Thermal stability was assessed by differential scanning calorimetry (DSC). Sponges were seeded with human adipose derived stem cells to assess chondro-inductive potential of collagen sponges after 7, 14 and 21 days of culture. In conclusion, collagen sponges support the proliferation and differentiation of human adipose derived stem cells to different extents.

Cell-based scaffold-free tissue equivalents present a limited clinical translation as consequence of the delayed extracellular matrix (ECM) deposition due to the prolonged production time in vitro. Different combinations of media supplements such as ascorbic acid, growth factors, oxygen tension among others can modulate the cell fate or the ECM synthesis. New research lines are focusing on the use of macromolecular crowders (MMCs) as media supplement for cell sheet production due to their ability to increase ECM deposition by volume exclusion effect, pro-collagenases alosteric regulation, matrix self-assembly by confinement and diffusion limitation (most probably, modulating the interaction between the ECM, MMPs and TIMPs). Herein, different molecular weights and concentrations of a natural potential MMC (Crowder-A) have been tested in equine adipose-derived stem cell (eADSC) and human dermal fibroblast (hADF) cultures in comparison with other commonly used crowders such as carrageenan and the Ficoll™ cocktail 70 KDa and 400 KDa. The eADSCs were characterized according to the current criteria for horse MSCs. Tri-lineage and FACS analysis showed eADSC osteogenic and adipogenic potentials and the presence of the markers CD29, CD44, CD90. The screening of the aforementioned Crowder-A was performed in cultures of 15,000 cells / cm² for the eADSCs and 25,000 cells / cm² for the hADFs during 3, 5, and 7 days. Non-MMC conditions were used as negative controls. Collagen type I was analysed by SDS-PAGE. Other collagen types were studied by immunocytochemistry assays. Significant increase of some ECM components was observed in some concentrations and molecular weights of the Crowder-A.

Introduction

Collagen is the predominant component of extracellular matrix in various connective tissues and makes up to 25% to 35% of the whole protein content in animal bodies. Type II collagen was first introduced from chicken sternal cartilage and presents supportive function in cartilaginous tissue. Since type II collagen is the major component of cartilage in joint, this study is aiming to determine an optimal type II collagen material for the development of medical devices for articular cartilage regeneration. In order to make more effective use of underutilized food waste, type II collagens from mammalian tissue sources (porcine tracheal cartilage; auricular cartilage; articular cartilage) and marine tissue sources (cuckoo ray, blonde ray, thorn back ray, lesser spotted dogfish) were isolated through acid-pepsin digestion under 4°C and characterized by various biological, biochemical and biophysical analysis. Pepsin cleaves the telopeptide region of the collagen molecule and pepsin treated collagen extraction ensures higher collagen yield. Telopeptide-free collagen reveals cytocompatibility, biodegradability and lower toxicity. The number and size of collagen chains were revealed by SDS-polyacrylamide gel electrophoresis. Intermolecular crosslinking density was quantified by Ninhydrin assay. Thermal stability was tested by differential scanning calorimetry (DSC) and enzymatic degradation was assessed by collagenase assay. Human chondrocytes were seeded on to collagen sponges at a density of 30,000 cells per sponge. Cell morphology (DAPI/ Rhodamine Phalloidin), viability(LIVE/DEAD®), proliferation(PicoGreen®) and metabolic activity (alamarBlue®) were analysed. Quantitative morphometric analysis was carried out using ImageJ software.