Single nucleus and spatial transcriptomic profiling of healthy human hamstring tendon.

The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell-cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell-cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

RNA sequencing and immunofluorescence of the myotendinous junction of mature horses and humans.

The myotendinous junction (MTJ) is a specialized interface for transmitting high forces between the muscle and tendon and yet the MTJ is a common site of strain injury with a high recurrence rate. The aim of this study was to identify previously unknown MTJ components in mature animals and humans. Samples were obtained from the superficial digital flexor (SDF) muscle-tendon interface of 20 horses, and the tissue was separated through a sequential cryosectioning approach into muscle, MTJ (muscle tissue enriched in myofiber tips attached to the tendon), and tendon fractions. RT-PCR was performed for genes known to be expressed in the three tissue fractions and t-distributed stochastic neighbor embedding (t-SNE) plots were used to select the muscle, MTJ, and tendon samples from five horses for RNA sequencing. The expression of previously known and unknown genes identified through RNA sequencing was studied by immunofluorescence on human hamstring MTJ tissue. The main finding was that RNA sequencing identified the expression of a panel of 61 genes enriched at the MTJ. Of these, 48 genes were novel for the MTJ and 13 genes had been reported to be associated with the MTJ in earlier studies. The expression of known [COL22A1 (collagen XXII), NCAM (neural cell adhesion molecule), POSTN (periostin), NES (nestin), OSTN (musclin/osteocrin)] and previously undescribed [MNS1 (meiosis-specific nuclear structural protein 1), and LCT (lactase)] MTJ genes was confirmed at the protein level by immunofluorescence on tissue sections of human MTJ. In conclusion, in muscle-tendon interface tissue enriched with myofiber tips, we identified the expression of previously unknown MTJ genes representing diverse biological processes, which may be important in the maintenance of the specialized MTJ.

[A correlation study between the Mohawk expression level and the collagen fiber diameter of hamstring tendon graft after anterior cruciate ligament reconstruction].

OBJECTIVE: To evaluate the correlation between the Mohawk (MKX) expression level and the collagen fiber diameter of autologous hamstring tendon graft during the stable graft remodeling phase after anterior cruciate ligament (ACL) reconstruction. METHODS: Between January 2018 and August 2018, patients who underwent arth-roscopic single-bundle anatomical ACL reconstruction with autologous hamstring tendons for at least 48 months and also underwent second-look arthroscopy were enrolled in study. During the second-look arthroscopic procedures, ACL graft biopsies were performed from the surface of central part of the ligament. MKX expressions of ACL grafts were analysed by real-time fluorescent quantitative PCR (qRT-PCR). The ultrastructure of collagen fibers of grafts were evaluated by transmission electron microscopy, which included average diameter of collagen fibers (D c), average diameter of large-diameter collagen fibers (D L), average diameter of small-diameter collagen fibers (D S), and large-small collagen fibers ratio (R L/S). The correlation between MKX expression level and graft collagen fiber diameter was calculated. RESULTS: Twenty-six patients met the selection criteria and their ACL graft specimens were enrolled in the study. The interval between ACL reconstruction and second-look arthroscopy was 52-128 months, with an average of 78.6 months. Arthroscopic graft remodeling score was 3-6 (mean, 4.8). There were 17 cases of excellent remodeling and 9 cases of fair remodeling. All ACL grafts showed typical bimodal distributions of both large-diameter collagen fibers and small-diameter collagen fibers, but the ultrastructural characteristics of the graft collagen fibers were different according to different remodeling status under arthroscopy. The D C, D L, D S, and R L/S of the graft specimens were (65.2±9.3) nm, (91.6±10.5) nm, (45.7±8.6) nm, and 0.73±0.12, respectively. The relative expression level of MKX was 1.42±0.11, which was positively linearly correlated with D C, D L, and R L/S, and the correlation coefficient was statistically significant ( r=0.809, P=0.000; r=0.861, P=0.000; r=0.942, P=0.000), while there was no significant correlation between D S and relative expression level of MKX ( r=0.147, P=0.238). Regression analysis showed that the relative expression level of MKX could predict the D C, D L, and R L/S results of the ACL graft specimens ( P<0.05). CONCLUSION: After autologous hamstring tendon grafts stepped into stabilized remodeling phase, MKX expression level could predict the diameter measurement results of collagen fibers and be used as an important evaluation basis for graft collagen anabolic metabolism.

Morphological and molecular characterization of human hamstrings shows that tendon features are not influenced by donor age.

PURPOSE: Age-related modifications of tendons, such as reduced tenocyte proliferation and modified extracellular matrix (ECM) turnover, have been previously described, but results are often incomplete and discordant. The aim of this study was to investigate, using morphological and molecular methods, the effect of ageing on human tendons and tenocytes, especially focusing on the collagen turnover pathways, in order to understand how the ageing process could influence tendon biology and structure. METHODS: Morphological analysis was performed on fragments from human semitendinosus and gracilis tendons harvested from 10 adult (mean age 41.8 ± 13.3 years) and 6 aged healthy patients (mean age 72.7 ± 7.0 years) by haematoxylin and eosin, Sirius red and Alcian blue staining. The expression of genes and proteins involved in collagen turnover and focal adhesions was assessed by real-time PCR, slot blot and zymography in cultured tenocytes. Cytoskeleton arrangement was studied by immunofluorescence and cell migration by wound healing assay. RESULTS: The structure and composition of ECM in ageing tendons are preserved as well as the expression of genes and proteins involved in collagen turnover pathways. Although morphological analysis revealed that ageing tenocytes tended to an impaired migration potential and that actin filaments are occasionally shorter and randomly distributed, the expression of proteins involved in focal adhesion formation is preserved. CONCLUSION: Results of this study suggest that the structure of ageing tendons is preserved and that ageing tenocytes maintain their ability for ECM remodelling, supporting the hypothesis that ageing tendons maintain their biomechanical properties. The biological reliability of aged tendons has a clinical relevance, supporting the use of tendon autografts also in the elderly patients. Since the common and successful orthopaedic procedure of anterior cruciate ligament reconstruction using either autografts or allografts is becoming more common in older age groups, these findings suggest that the donor age would not significantly influence the clinical outcome.