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The tendon-bone interface (enthesis) is a highly sophisticated biomaterial junction that allows stress transfer between mechanically dissimilar materials. The enthesis encounters very high mechanical demands and the regenerative capacity is very low resulting in high rupture recurrence rates after surgery. Tissue engineering offers the potential to recover the functional integrity of entheses. However, recent enthesis tissue engineering approaches have been limited by the lack of knowledge about the cells present at this interface. Here we investigated the cellular differentiation of enthesis cells and compared the cellular pattern of enthesis cells to tendon and cartilage cells in a next generation sequencing transcriptome study. We integrated the transcriptome data with proteome data of a previous study to identify biomarkers of enthesis cell differentiation. Transcriptomics detected 34468 transcripts in total in enthesis, tendon, and cartilage. Transcriptome comparisons revealed 3980 differentially regulated candidates for enthesis and tendon, 395 for enthesis and cartilage, and 946 for cartilage and tendon. An asymmetric distribution of enriched genes was observed in enthesis and cartilage transcriptome comparison suggesting that enthesis cells are more chondrocyte-like than tenocyte-like. Integrative analysis of transcriptome and proteome data identified ten enthesis biomarkers and six tendon biomarkers. The observed gene expression characteristics and differentiation markers shed light into the nature of the cells present at the enthesis. The presented markers will foster enthesis tissue engineering approaches by setting a bench-mark for differentiation of seeded cells towards a physiologically relevant phenotype.

Interfaces between tendon/ligament and bone ("entheses") are highly specialized tissues that allow for stress transfer between mechanically dissimilar materials. Entheses show very low regenerative capacity resulting in high incidences of failure after surgical repair. Tissue engineering is a promising approach to recover functionality of entheses. Here, we established a protocol to decellularize porcine entheses as scaffolds for enthesis tissue engineering. Chemical detergents as well as physical treatments were investigated with regard to their efficiency to decellularize 2 mm thick porcine Achilles tendon entheses. A two-phase approach was employed: study 1 investigated the effect of various concentrations of sodium dodecyl sulfate (SDS) and t-octylphenoxypolyethoxy-ethanol (Triton X-100) as decellularization agents. The most efficient combination of SDS and Triton was then carried forward into study 2, where different physical methods, including freeze-thaw cycles, ultrasound, perfusion, and hydrostatic washing were used to enhance the decellularization effect. Cell counts, DNA quantification, and histology showed that washing with 0.5% SDS + 1% Triton X-100 for 72 h at room temperature could remove ~ 98% cells from the interface. Further investigation of physical methods proved that washing under 200 mmHg hydrostatic pressure shortened the detergent exposing time from 72 h to 48 h. Biomechanical tensile testing showed that the biomechanical features of treated samples were preserved. Washing under 200 mmHg hydrostatic pressure with 0.5% SDS + 1% Triton X-100 for 48 h efficiently decellularized entheses with preservation of matrix structure and biomechanical features. This protocol can be used to efficiently decellularize entheses as scaffolds for tissue engineering.

Recent advances in gene delivery into cells allow improved therapeutic effects in gene therapy trials. To increase the bioavailability of applied cells, it is of great interest that transfected cells remain at the application site and systemic spread is minimized. In this study, we tested clinically used biodegradable poly(lactic acid-co-glycolic acid) (PLGA) scaffolds (Vicryl & Ethisorb) as transient carriers for genetically modified cells. To this aim, we used human fibroblasts and examined attachment and proliferation of untransfected cells on the scaffolds in vitro, as well as the mechanical properties of the scaffolds at four time points (1, 3, 6 and 9 days) of cultivation. Furthermore, the adherence of cells transfected with green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF165) and also VEGF165 protein secretion were investigated. Our results show that human fibroblasts adhere on both types of PLGA scaffolds. However, proliferation and transgene expression capacity were higher on Ethisorb scaffolds most probably due to a different architecture of the scaffold. Additionally, cultivation of the cells on the scaffolds did not alter their biomechanical properties. The results of this investigation could be potentially exploited in therapeutic regiments with areal delivery of transiently transfected cells and may open the way for a variety of applications of cell-based gene therapy, tissue engineering and regenerative medicine.