Current cell-based tissue engineering strategies have limited clinical applicability due to the need for large cell numbers and prolonged culture periods that lead to phenotypic drift. In vitro microenvironmental modulators have been proposed to mimic the native tendon. Standard in vitro culture conditions result in delayed extracellular matrix (ECM) deposition, impairing the development of scaffold-free approaches. ECM deposition can be enhanced by macromolecular crowding (MMC), a biophysical phenomenon that governs the milieu of multicellular organisms. We assessed a multifactorial biophysical approach, using MMC and mechanical loading, on different cell sources to determine their suitability for in vitro fabrication of tendon-like tissue. Human dermal fibroblasts (DFs), tenocytes (TCs) and bone marrow mesenchymal stem cells (BMSCs) were cultured with MMC under static and uniaxial strain culture conditions. TCs and DFs exhibited alignment perpendicular to the load, whilst BMSCs did not show preferential alignment. When MMC was used, DFs and BMSCs showed increased deposition of collagen I, the main component in tendon ECM. DFs presented ECM composition similar to TCs with collagen types III, V and VI present. Gene expression analysis revealed upregulation of tenogenic markers by TCs and DFs, such as scleraxis and thrombospondin-4, under both loading and MMC. The combined use of MMC and mechanical stimulation is suitable for TCs phenotype maintenance and can modulate the phenotype of DFs and BMSCs differentially. This study provides insight into response of different cell sources to biophysical cues and contributes to further development of cell therapies for tendon repair and regeneration.

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.

The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones (tendon, fibrocartilage, mineralized fibrocartilage and bone). After injury, the native structure is often not re-established and a mechanically weaker fibrovascular scar is formed. Traditionally used monotherapies have failed to be effective, posing the need for multi-cargo localized delivery vehicles. We hypothesize that multilayer collagen-based scaffolds can serve as delivery vehicles for specific bioactive molecules with tenogenic, chondrogenic and osteogenic potential to enhance the functional regeneration of the enthesis. Three-layer scaffolds composed by a tendon-like layer of collagen type I, a cartilage-like layer of collagen type II and a bone-like layer of collagen type I and hydroxyapatite were fabricated by an iterative layering freeze-drying technique. The scaffolds were cross-linked with varying concentration of 4-arm polyethylene glycol (4s-PEG) and the biological and mechanical properties were assessed. Each layer was functionalized with platelet-derived growth factor, insulin growth factor, heparan sulfate or bone morphogenetic protein 7 and their tenogenic, chondrogenic and osteogenic potential on bone-marrow derived stem cells was investigated in vitro. Scaffolds cross-linked with 1 mM 4s-PEG showed 60% free amines reduction respect to non-cross-linked scaffolds, were stable in collagenase over 24 hours and had a compression modulus of 30 kPa. The bioactive molecules had a sustained release profile (approximately 50 ng/mL) over 5 days as a function of cross-linking. Preliminary in vitro studies confirmed the chondrogenic potential of heparin sulfate and insulin growth factor by the increase of proteoglycans.

Complex pathophysiologies involve different signalling mechanisms, with a multitude of often interconnected potential therapeutic targets. Therefore, there is a need for the development of multi-compartment delivery vehicles for combinatorial and synergistic therapeutic approaches. In this study it was hypothesized that multi-compartment crosslinked collagen type I systems can deliver multiple bioactive agents in a controlled manner in an in vitro model condition of skin fibrosis. Multi-compartment collagen-based systems were made using solutions of dialyzed type I collagen mixed with 10× PBS, after which they were neutralised and crosslinked with 1 and 2.0 mM 4 arm-succinimidyl glutarate ester PEG (4 arm-PEG-SG), respectively, followed by incubation at 37ºC. The systems were characterised through swelling assessment, collagenase degradation assay and compression tests. The release of encapsulated drugs from the hydrogels was studied by ELISA and the effect of the delivered bioactive agents was assessed through imaging and quantification for fibrotic markers in an in vitro model. A pilot study using FITC-dextran proved that the inner compartment was capable of promoting a sustained release over a long period of time (7 days), which was further confirmed with drug release assays using a TNF-α antagonist and recombinant decorin, fitting the intended therapeutic release profile. Protein expression studies showed a decrease of endogenous collagen type I and α-smooth muscle actin expression (p<0.05) indicating amelioration of fibrosis. In summary, this indicates that this system is suitable for dual delivery of multiple bioactive agents, resulting in a controlled release in vitro and illustrating its potential in therapy.

The formation of postoperative adhesions poses a major complication in surgery, especially in the treatment of tendon, where adhesions can result in an alteration of the biomechanical and gliding properties, impeding a proper functioning of the tendon. Current treatments to prevent adhesions in the tendon are mainly based on the use of mechanical barriers which isolate the tendon and prevent fibrin deposition. Despite the positive results in preclinical models, these results have not been translated to clinics. Thus, in this study we propose a porcine peritoneum xenograft as an alternative antiadhesion barrier which integrates a basal membrane, since the presence of a basal membrane together with an epithelium or mesothelium layer prevents the formation of adhesions in vivo. First results have shown the suitability of the porcine peritoneum xenograft as an antiadhesion barrier due to its lower crosslinking ratio (p<0.05) and faster degradation by MMPs in vitro than a commercially available tendon product, which suggest a faster remodelling in vivo. On the other hand, the porcine peritoneum showed higher mechanical properties (p<0.01) and a lower coefficient of friction (p<0.01), characteristics that make the porcine peritoneum an appropriate material for tendon regeneration. Furthermore, the presence in the xenograft of a collagen type IV and laminin network after decellularisation was confirmed with immunohistochemistry, which poses the potential of the porcine peritoneum as antiadhesion device due to the presence of a basal membrane. Preliminary cell assessment experiments showed different morphology of adult dermal fibroblast (ADFs) on the different sides of the material (basal membrane and connective tissue) due to the differences in composition of both layers. Furthermore, the culture of ADFs during 7 days in media conditioned with the porcine peritoneum resulted in higher proliferation and metabolic activity (p<0.05) than those observed in the control and the media conditioned with the commercial product, suggesting the presence of growth factors in the porcine peritoneum which promote the growth of cells. Although positive results have been observed regarding the potential of porcine peritoneum as antiadhesion barrier for tendon regeneration, further studies which assess the influence of the basal membrane on cell behaviour and confirm the potential of the xenograft as antiadhesion barrier are being carried out.