The exceptional mechanical properties of the load-bearing connection of tendon to bone rely on an intricate interplay of its biomolecular composition, microstructure and micromechanics. Here we identify that the Achilles tendon-bone insertion is characterized by an interface region of ∼500 μm with a distinct fibre organization and biomolecular composition. Within this region, we identify a heterogeneous mechanical response by micromechanical testing coupled with multiscale confocal microscopy. This leads to localized strains that can be larger than the remotely applied strain. The subset of fibres that sustain the majority of loading in the interface area changes with the angle of force application. Proteomic analysis detects enrichment of 22 proteins in the interfacial region that are predominantly involved in cartilage and skeletal development as well as proteoglycan metabolism. The presented mechanisms mark a guideline for further biomimetic strategies to rationally design hard-soft interfaces.

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.

artilage defects due to trauma, degenerative arthritis, or inflammatory arthritis affect approximately one out of five adults and represent a major cause of pain and disability (1-3). Because cartilage defects do not heal spontaneously, interventions are needed to induce repair. Bone marrow-derived autologous mesenchymal stromal cells (MSCs) can differentiate into chondrocytes and have been implanted into cartilage defects to restore joint health (4). However, cartilage repair outcomes of matrix-associated stem cell implants (MASIs) in patients have been highly variable: While some investigators reported full-thickness hyaline cartilage regeneration (5,6), others reported a failure rate of up to 50% for MASIs (7,8). Limited cell transplant survival was identified as the most important obstacle for successful cartilage repair (9). An imaging test that could help predict MASI outcomes would greatly enhance our ability to develop more successful cell transplant procedures. MRI is the primary modality for cartilage imaging (10,11). However, MRI within the 1st few weeks after MASI cannot help distinguish between grafts that will and grafts that will not repair the underlying cartilage defect (9). To date, successful cartilage repair is diagnosed many months after MASI, on the basis of a reduction in cartilage defect size at morphologic MRI (10,11). Unfortunately, failed cartilage repair and scar formation are difficult to correct at that time. Timely detection of an impending graft failure could enable rescue interventions

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.

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Abstract:The analysis of tissue network characteristics and cell distribution using histological methods is widely used. However, image analysis still relies on manual evaluation methods, known as semi-quantitative analysis, which are time-consuming and to a certain degree user-specific. For this reason, automated imaging processing methods have an enormous potential to increase sample processing and reduce the variation that is caused by a user-specific evaluation. This work demonstrates the feasibility of using a semi-automated image analysis process based on the open source software framework ImageJ and the plug-in Angiogenesis Analyzer to evaluate the quantitative degree of tissue damage within 3D cell constructs after mechanical loading. Within a proof-of-concept study, the semi-automated approach was applied to calculate the Node-to-Free Ratio (N/F-Ratio) and perform a strut analysis for histological evaluation of mechanically compressed samples of human osteoblast-derived 3D constructs. The N/F-Ratio revealed a median value of 1.29 for the control, whereas the values for the mechanically compressed samples decreased to 0.97 for 20% compression, 0.85 for 40%, and 0.86 for 60%. The strut analysis indicated a decrease of the connected branches with increasing compression rate. The newly developed and time-saving processing workflow was successfully established and can be carried out using available, open source software solutions.