The Biological Effect of Platelet-Rich Plasma on Rotator Cuff Tears: A Prospective Randomized In Vivo Study.

The positive effect of platelet-rich plasma (PRP) on tendon metabolism has been extensively investigated and proven in vitro. Additionally, in vivo animal studies have correlated the application of PRP with the enhancement of tenocyte anabolic activity in the setting of tendon degeneration. However, less is known about its in vivo effect on human tendon biology. The purpose of the current prospective randomized comparative study was to evaluate the effect of PRP on torn human supraspinatus tendon. Twenty consecutive eligible patients with painful and magnetic resonance imaging (MRI)-confirmed degenerative supraspinatus tendon tears were randomized in a one-to-one ratio into two groups. The patients in the experimental group (n = 10) underwent an ultrasound-guided autologous PRP injection in the subacromial space 6 weeks before the scheduled operation. In the control group (n = 10), no injection was made prior to surgery. Supraspinatus tendon specimens were harvested from the lateral end of the torn tendon during shoulder arthroscopy and were evaluated under optical and electron microscopy. In the control group, a mixed cell population of oval and rounded tenocytes within disorganized collagen and sites of accumulated inflammatory cells was detected. In contrast, the experimental group yielded abundant oval-shaped cells with multiple cytoplasmic processes within mainly parallel collagen fibers and less marked inflammation, simulating the intact tendon structure. These findings indicate that PRP can induce microscopic changes in the ruptured tendon by stimulating the healing process and can facilitate a more effective recovery.

Effect of magnetic microbeads on sustained and targeted delivery of transforming growth factor-beta-1 for rotator cuff healing in a rat rotator cuff repair model.

Structural failure is a well-established complication of rotator cuff repair procedures. To evaluate the effect of magnetic microbeads, designed for precise drug delivery via magnetic force, on sustained transforming growth factor-beta-1 (TGF-ß1) release and rotator cuff healing in a rat rotator cuff repair model. TGF-ß1 laden microbeads were prepared, and baseline in vitro experiments included the magnetization of the microbeads and TGF-ß1 release tests. In an in vivo experiment using a rat rotator cuff repair model on both shoulders, 72 rats were randomly assigned to three groups (24 per group): group A, conventional repair; group B, repair with and simple TGF-ß1 injection; and group C, repair with magnet insertion into the humeral head and TGF-ß1 laden microbead injection. Delivery of TGF-ß1 was evaluated at 1 and 7 days after the intervention using PCR, Western blot, and immunohistochemistry. At 6 weeks post-intervention, rotator cuff healing was assessed using biomechanical and histological analysis. The in vitro experiments confirmed the magnetization property of the microbeads and sustained delivery of TGF-ß1 for up to 10 days. No difference in the TGF-ß1 expression was found at day 1 in vivo. However, at day 7, group C exhibited a significantly elevated expression of TGF-ß1 in both PCR and Western blot analyses compared to groups A and B (all P < 0.05). Immunohistochemical analysis revealed a higher expression of TGF-ß1 at the repair site in group C on day 7. At 6 weeks, biomechanical analysis demonstrated a significantly higher ultimate failure load in group C than in groups A and B (P < 0.05) and greater stiffness than in group A (P = 0.045). In addition, histological analysis showed denser and more regular collagen fibers with complete continuity to the bone in group C than in groups A and B, a statistically significant difference according to the semi-quantitative scoring system (all P < 0.05). The use of the TGF-ß1 laden magnetic microbeads demonstrated sustained delivery of TGF-ß1 to the repair site, improving rotator cuff healing.

Evaluation of the metabolic activity of the torn rotator cuff muscles by 18 F-2-deoxy- d -glucose PET-computed tomography scan.

OBJECTIVES: Fatty atrophy and fatty infiltration have been considered as limiting factors for rotator cuff repair. The metabolic activity of the muscle can be measured noninvasively by PET. In our study, we aim to compare the metabolic activity between the shoulders with rotator cuff tears and normal shoulders. METHODS: All the patients with unilateral full-thickness rotator cuff tears were included. The patients were divided into two groups based on fatty atrophy and the metabolic activities of the rotator cuff muscles, trapezius, and deltoid were calculated using an 18 F-2-deoxy- d -glucose PET-computed tomography scan for comparison. RESULTS: A total of 17 patients were included. The standardized uptake values were compared between the affected shoulder and the normal shoulders. There was a significant increase in uptake in the insertion sites and musculotendinous junctions in the rotator cuff torn group. The standardized uptake values showed no significant difference between the low-grade and high-grade groups. CONCLUSION: Our first hypothesis was also proven wrong; when we found that there was no statistically significant difference in the metabolic activity in muscle bellies of normal shoulders and those with rotator cuff tears. Our second hypothesis was proven wrong when found that there was no statistically significant difference in the metabolic activities of rotator cuff muscles between high-grade and low-grade fatty atrophy groups. The metabolic activities of the middle deltoid and trapezius are inversely related. Based on the findings of our study, fatty atrophy or fatty infiltration alone cannot be considered a limiting factor for rotator cuff repair.

Transcriptomics reveals dynamic changes in the "gene profiles" of rat supraspinatus tendon at three different time points after diabetes induction.

OBJECTIVE: There is increasing evidence that type 2 diabetes mellitus (T2DM) is an independent risk factor for the occur of tendinopathy. Therefore, this study is the first to explore the dynamic changes of the "gene profile" of supraspinatus tendon in rats at different time points after T2DM induction through transcriptomics, providing potential molecular markers for exploring the pathogenesis of diabetic tendinopathy. METHODS: A total of 40 Sprague-Dawley rats were randomly divided into normal (NG, n = 10) and T2DM groups (T2DM, n = 30) and subdivided into three groups according to the duration of diabetes: T2DM-4w, T2DM-8w, and T2DM-12w groups; the duration was calculated from the time point of T2DM rat model establishment. The three comparison groups were set up in this study, T2DM-4w group vs. NG, T2DM-8w group vs. NG, and T2DM-12w group vs. NG. Differentially expressed genes (DEGs) in 3 comparison groups were screened. The intersection of the three comparison groups' DEGs was defined as key genes that changed consistently in the supraspinatus tendon after diabetes induction. Cluster analysis, gene ontology (GO) functional annotation analysis and Kyoto encyclopedia of genes and genomes (KEGG) functional annotation and enrichment analysis were performed for DEGs. RESULTS: T2DM-4w group vs. NG, T2DM-8w group vs. NG, and T2DM-12w group vs. NG detected 519 (251 up-regulated and 268 down-regulated), 459 (342 up-regulated and 117 down-regulated) and 328 (255 up-regulated and 73 down-regulated) DEGs, respectively. 103 key genes of sustained changes in the supraspinatus tendon following induction of diabetes, which are the first identified biomarkers of the supraspinatus tendon as it progresses through the course of diabetes.The GO analysis results showed that the most significant enrichment in biological processes was calcium ion transmembrane import into cytosol (3 DEGs). The most significant enrichment in cellular component was extracellular matrix (9 DEGs). The most significant enrichment in molecular function was glutamate-gated calcium ion channel activity (3 DEGs). The results of KEGG pathway enrichment analysis showed that there were 17 major pathways (p < 0.05) that diabetes affected supratinusculus tendinopathy, including cAMP signaling pathway and Calcium signaling pathway. CONCLUSIONS: Transcriptomics reveals dynamic changes in the"gene profiles"of rat supraspinatus tendon at three different time points after diabetes induction. The 103 DEGs identified in this study may provide potential molecular markers for exploring the pathogenesis of diabetic tendinopathy, and the 17 major pathways enriched in KEGG may provide new ideas for exploring the pathogenesis of diabetic tendinopathy.

Rotator Cuff Tendinopathy: Pathways of Apoptosis.

Rotator cuff repair is usually successful, but retear is not uncommon. It has been previously identified that there is a higher incidence of apoptosis in the edges of the torn supraspinatus tendon. A prospective cohort study was conducted with 28 patients-14 rotator cuff tear patients, 5 instability patients, and 9 Anterior cruciate ligament reconstruction patients to determine whether there was any increase in several genes implicated in apoptosis, including Fas receptor (FasR), Fas ligand, Aifm-1, Bcl-2, Fadd, Bax, and caspase-3. There was a significant expression of Bax (P=0.2) and FasR (P=0.005) in the edges of torn supraspinatus tendons, and in intact subscapularis tendons, there was a significant expression of caspase-3 (P=0.02) compared with samples from the torn supraspinatus tendon (P=0.04). The cytochrome c pathway, with its subsequent activation of caspase-3, as well as the TRAIL-receptor signaling pathway involving FasR have both been implicated. The elevated expression of Bax supported the model that the Bax to Bcl-2 expression ratio represents a cell death switch. The elevated expression of Bax in the intact subscapularis tissue from rotator cuff tear patients also may confirm that tendinopathy is an ongoing molecular process.

Collagen degradation assessment with an in vitro rotator cuff tendinopathy model using multiparametric ultrashort-TE magnetization transfer (UTE-MT) imaging.

PURPOSE: This study aims to assess ultrashort-TE magnetization transfer (UTE-MT) imaging of collagen degradation using an in vitro model of rotator cuff tendinopathy. METHODS: Thirty-six supraspinatus tendon specimens were divided into three groups and treated with 600 U collagenase (Group 1), 150 U collagenase (Group 2), and phosphate buffer saline (Group 3). UTE-MT imaging was performed to assess changes in macromolecular fraction (MMF), macromolecule transverse relaxation time (T2m), water longitudinal relaxation rate constant (R1m), the magnetization exchange rate from the macromolecular to water pool (Rm0 w) and from water to the macromolecular pool (Rm0 m), and magnetization transfer ratio (MTR) at baseline and following digestion and their differences between groups. Biochemical and histological studies were conducted to determine the extent of collagen degradation. Correlation analyses were performed with MMF, T2m, R1m, Rm0 w, Rm0 m, and MTR, respectively. Univariate and multivariate linear regression analyses were performed to evaluate combinations of UTE-MT parameters to predict collagen degradation. RESULTS: MMF, T2m, R1m, Rm0 m, and MTR decreased after digestion. MMF (r = -0.842, p < 0.001), MTR (r = -0.78, p < 0.001), and Rm0 m (r = -0.662, p < 0.001) were strongly negatively correlated with collagen degradation. The linear regression model of differences in MMF and Rm0 m before and after digestion explained 68.9% of collagen degradation variation in the tendon. The model of postdigestion in MMF and T2m and the model of MTR explained 54.2% and 52.3% of collagen degradation variation, respectively. CONCLUSION: This study highlighted the potential of UTE-MT parameters for evaluation of supraspinatus tendinopathy.

Lineage tracing reveals a novel PDGFRß+ satellite cell subset that contributes to myo-regeneration of chronically injured rotator cuff muscle.

Massive rotator cuff (RC) tendon tears are associated with progressive fibro-adipogenesis and muscle atrophy that altogether cause shoulder muscle wasting. Platelet derived growth factor ß (PDGFRß) lineage cells, that co-express PDGFRα have previously been shown to directly contribute to scar formation and fat accumulation in a mouse model of irreversible tendon and nerve transection (TTDN). Conversely, PDGFRß+ lineage cells have also been  shown to be myogenic in cultures and in other models of skeletal muscle injury. We therefore hypothesized that PDGFRß demarcates two distinct RC residing subpopulations, fibro-adipogenic and myogenic, and aimed to elucidate the identity of the PDGFRß myogenic precursors and evaluate their contribution, if any, to RC myo-regeneration. Lineage tracing revealed increasing contribution of PDGFRß+ myo-progenitors to the formation of GFP+ myofibers, which were the most abundant myofiber type in regenerated muscle at 2 weeks post-TTDN. Muscle regeneration preceded muscle atrophy and both advanced from the lateral site of tendon transection to the farthest medial region. GFP+/PDGFRß+Sca-1-lin-CXCR4+Integrin-ß1+ marked a novel subset of satellite cells with confirmed myogenic properties. Further studies are warranted to identify the existence of PDGFRß+ satellite cells in human and other mouse muscles and to define their myo-regenerative potential following acute and chronic muscle injury.

Histological and biochemical changes in a rat rotator cuff tear model with or without the subacromial bursa.

The subacromial bursa (SAB) plays an important role in the tendon healing process. Based on previous reports, co-culture of the rotator cuff (RC) and SAB have been shown to increase the tendon-related gene expressions, inflammatory cytokines, and tensile strength. However, the nature of the specific biochemical alterations during the inflammatory and repair phases of tendon healing with or without the SAB remain unknown. Using a full-thickness RC tear rat model, we determined how the presence or absence of the SAB alters the histological characteristics and gene expressions. After 3 and 6 weeks, tissues were collected for histological and real-time quantitative polymerase chain reaction (RT-qPCR) evaluations. Results showed greater cell density at 3 weeks, neovascularization and tendon thickening at 6 weeks with SAB preservation. Immunostaining revealed significant increases in type 3 collagen (COL3) expression at 6 weeks with SAB preservation. The RT-qPCR results showed that SAB preservation induced significant increases in the expression of scleraxis, matrix metalloproteinase-13 (MMP-13), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase (iNOS) at 3 weeks and significant increases in COL3, IL-10, and arginase-1 (Arg-1) at 6 weeks. An RC tear undergoes more appropriate inflammatory and repair phases during the tendon healing process when the SAB is retained.

Optimized design of an enthesis-mimicking suture anchor-tendon hybrid graft for mechanically robust bone-tendon repair.

Repair of functionally graded biological interfaces requires joining dissimilar materials such as hard bone to soft tendon/ligament, with re-injuries/re-tears expected to be minimized by incorporating biomimicking, stress-reducing features within grafts. At bone-tendon interfaces (entheses), stress can be reduced via angled insertion, geometric flaring, mechanical gradation, and interdigitation of tissues. Here, we incorporated enthesis attributes into 3D in silico and physical models of a unique suture anchor-tendon hybrid graft (SATHG) and investigated their effects on stress reduction via finite element analyses (FEA) studies. Over 20 different simulations altering SATHG angulation, flaring, mechanical gradation, and interdigitation identified an optimal design, which included 90° angulation, 25° flaring, and a compliant (ascending then descending) mechanical gradient in SATHG's bone-to-tendon-like transitional region. This design reduced peak stress concentration factor (SCF) by 43.6 % relative to an ascending-only mechanical gradient typically used in hard-to-soft tissue engineering. To verify FEA results, SATHG models were fabricated using a photocrosslinkable bone-tendon-like polyurethane (QHM polymer) for ex vivo tensile assessment. Tensile testing showed that ultimate load (132.9 N), displacement-at-failure (1.78 mm), stiffness (135.4 N/mm), and total work-to-failure (422.1 × 10-3 J) were highest in the optimized design. Furthermore, to assess envisioned usage, SATHG pull-out testing and 6-week in vivo implantation into large, 0.5-cm segmental supraspinatus tendon defects was performed. SATHG pull-out testing showed secure bone attachment while histological assessment such as hematoxylin and eosin (H&E) together with Safranin-O staining showed biocompatibility including enthesis regeneration. This work demonstrates that engineering biomaterials with FEA-optimized, enthesis-like attributes shows potential for enhancing hard-to-soft tissue repair. STATEMENT OF SIGNIFICANCE: Successful repair of hard-to-soft tissue injuries is challenging due to high stress concentrations within bone-tendon/ligament grafts that mechanically compromise repair strength. While stress-reducing gradient biomaterials have been reported, little-to-no attention has focused on other bone-tendon/ligament interface (enthesis) features. To this end, a unique bone-tendon graft (SATHG) was developed by combining two common orthopaedic devices along with biomimetic incorporation of four enthesis-like features to reduce stress and encourage widespread clinician adoption. Notably, utilizing designs based on natural stress dissipation principles such as anchor insertion angle, geometric flaring, and mechanical gradation reduced stress by 43.6 % in silico, which was confirmed ex vivo, while in vivo studies showed SATHG's ability to support native enthesis regeneration. Thus, SATHG shows promise for hard-to-soft tissue repairs.

Analysis of differentially expressed genes in torn rotator cuff tendon tissues in diabetic patients through RNA-sequencing.

BACKGROUND: Rotator cuff tears (RCT) is a common musculoskeletal disorder in the shoulder which cause pain and functional disability. Diabetes mellitus (DM) is characterized by impaired ability of producing or responding to insulin and has been reported to act as a risk factor of the progression of rotator cuff tendinopathy and tear. Long non-coding RNAs (lncRNAs) are involved in the development of various diseases, but little is known about their potential roles involved in RCT of diabetic patients. METHODS: RNA-Sequencing (RNA-Seq) was used in this study to profile differentially expressed lncRNAs and mRNAs in RCT samples between 3 diabetic and 3 nondiabetic patients. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were performed to annotate the function of the differentially expressed genes (DEGs). LncRNA-mRNA co-expression network and competing endogenous RNA (ceRNA) network were constructed to elucidate the potential molecular mechanisms of DM affecting RCT. RESULTS: In total, 505 lncRNAs and 388 mRNAs were detected to be differentially expressed in RCT samples between diabetic and nondiabetic patients. GO functional analysis indicated that related lncRNAs and mRNAs were involved in metabolic process, immune system process and others. KEGG pathway analysis indicated that related mRNAs were involved in ferroptosis, PI3K-Akt signaling pathway, Wnt signaling pathway, JAK-STAT signaling pathway and IL-17 signaling pathway and others. LncRNA-mRNA co-expression network was constructed, and ceRNA network showed the interaction of differentially expressed RNAs, comprising 5 lncRNAs, 2 mRNAs, and 142 miRNAs. TF regulation analysis revealed that STAT affected the progression of RCT by regulating the apoptosis pathway in diabetic patients. CONCLUSIONS: We preliminarily dissected the differential expression profile of lncRNAs and mRNAs in torn rotator cuff tendon between diabetic and nondiabetic patients. And the bioinformatic analysis suggested some important RNAs and signaling pathways regarding inflammation and apoptosis were involved in diabetic RCT. Our findings offer a new perspective on the association between DM and progression of RCT.

Untargeted metabolomics reveals dynamic changes in metabolic profiles of rat supraspinatus tendon at three different time points after diabetes induction.

Objective: To investigate the dynamic changes of metabolite composition in rat supraspinatus tendons at different stages of diabetes by untargeted metabolomics analysis. Methods: A total of 80 Sprague-Dawley rats were randomly divided into normal (NG, n = 20) and type 2 diabetes mellitus groups (T2DM, n = 60) and subdivided into three groups according to the duration of diabetes: T2DM-4w, T2DM-12w, and T2DM-24w groups; the duration was calculated from the time point of T2DM rat model establishment. The three comparison groups were set up in this study, T2DM-4w group vs. NG, T2DM-12w group vs. T2DM-4w group, and T2DM-24w group vs. T2DM-12w group. The metabolite profiles of supraspinatus tendon were obtained using tandem mass spectrometry. Metabolomics multivariate statistics were used for metabolic data analysis and differential metabolite (DEM) determination. The intersection of the three comparison groups' DEMs was defined as key metabolites that changed consistently in the supraspinatus tendon after diabetes induction; then, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed. Results: T2DM-4w group vs. NG, T2DM-12w group vs. T2DM-4w group, and T2DM-24w group vs. T2DM-12w group detected 94 (86 up-regulated and 8 down-regulated), 36 (13 up-regulated and 23 down-regulated) and 86 (24 up-regulated and 62 down-regulated) DEMs, respectively. Seven key metabolites of sustained changes in the supraspinatus tendon following induction of diabetes include D-Lactic acid, xanthine, O-acetyl-L-carnitine, isoleucylproline, propoxycarbazone, uric acid, and cytidine, which are the first identified biomarkers of the supraspinatus tendon as it progresses through the course of diabetes. The results of KEGG pathway enrichment analysis showed that the main pathway of supraspinatus metabolism affected by diabetes (p < 0.05) was purine metabolism. The results of the KEGG metabolic pathway vs. DEMs correlation network graph revealed that uric acid and xanthine play a role in more metabolic pathways. Conclusion: Untargeted metabolomics revealed the dynamic changes of metabolite composition in rat supraspinatus tendons at different stages of diabetes, and the newly discovered seven metabolites, especially uric acid and xanthine, may provide novel research to elucidate the mechanism of diabetes-induced tendinopathy.

Intra-articular site-specific distribution of advanced glycation end products in the shoulder of patients with diabetes mellitus having rotator cuff tears.

BACKGROUND: Advanced glycation end products (AGEs) are compounds formed due to aging and diabetes mellitus (DM). They activate NADPH oxidase (NOX) by binding to their receptors, thereby increasing the production of reactive oxygen species (ROS), which cause oxidative stress. In this study, we investigated the effects of AGEs on the tissues of the shoulder joint (such as rotator cuff synovium, and capsule) in patients with DM having rotator cuff tears. METHODS: This study included eight patients with DM who underwent surgical treatment for rotator cuff tears with contracture. The rotator cuff, synovium, and joint capsule were harvested at the time of surgery and evaluated by hematoxylin-eosin staining. Furthermore, immunostaining was used for evaluating AGEs and receptor for AGEs (RAGE), cell activity, ROS, and apoptosis. Quantitative real-time polymerase chain reaction (qPCR) was employed for the cellular evaluation of NOX, interleukins, RAGE, and collagen. RESULTS: The AGEs and RAGE staining as well as the ratio of ROS and apoptosis were in the following order: rotator cuff > joint capsule > synovium. In contrast, the cellular activity was significantly higher in the synovium than in the other regions. The type I collagen expression (as shown by qPCR) as well as the RAGE and NOX expressions were as follows: rotator cuff > joint capsule > synovium. Conversely, the expression of inflammatory cytokines (i.e., IL-6 and IL-1ß) was higher in the synovium than in the other regions. CONCLUSIONS: Our study is among the first to evaluate the effects of AGEs on each tissue of the shoulder joint in patients with DM having rotator cuff tears and contractures. The accumulation of AGEs in each tissue of the shoulder joint could reveal the locations affected by DM, which can lead to a better understanding of the pathophysiology of DM-related shoulder diseases.

Potential function of Scx+/Sox9+ cells as progenitor cells in rotator cuff tear repair in rats.

Tendons and their attachment sites to bone, fibrocartilaginous tissues, have poor self-repair capacity when they rupture, and have risks of retear even after surgical repair. Thus, defining mechanisms underlying their repair is required in order to stimulate tendon repairing capacity. Here we used a rat surgical rotator cuff tear repair model and identified cells expressing the transcription factors Scleraxis (Scx) and SRY-box 9 (Sox9) as playing a crucial role in rotator cuff tendon-to-bone repair. Given the challenges of establishing stably reproducible models of surgical rotator cuff tear repair in mice, we newly established Scx-GFP transgenic rats in which Scx expression can be monitored by GFP. We observed tissue-specific GFP expression along tendons in developing ScxGFP transgenic rats and were able to successfully monitor tissue-specific Scx expression based on GFP signals. Among 3-, 6-, and 12-week-old ScxGFP rats, Scx+/Sox9+ cells were most abundant in 3-week-old rats near the site of humerus bone attachment to the rotator cuff tendon, while we observed significantly fewer cells in the same area in 6- or 12-week-old rats. We then applied a rotator cuff repair model using ScxGFP rats and observed the largest number of Scx+/Sox9+ cells at postoperative repair sites of 3-week-old relative to 6- or 12-week-old rats. Tendons attach to bone via fibrocartilaginous tissue, and cartilage-like tissue was seen at repair sites of 3-week-old but not 6- or 12-week-old rats during postoperative evaluation. Our findings suggest that Scx+/Sox9+ cells may function in rotator cuff repair, and that ScxGFP rats could serve as useful tools to develop therapies to promote rotator cuff repair by enabling analysis of these activities.

Mechanisms of ferroptosis with immune infiltration and inflammatory response in rotator cuff injury.

The processes driving ferroptosis and rotator cuff (RC) inflammation are yet unknown. The mechanism of ferroptosis and inflammation involved in the development of RC tears was investigated. The Gene Expression Omnibus database was used to obtain the microarray data relevant to the RC tears for further investigation. In this study, we created an RC tears rat model for in vivo experimental validation. For the additional function enrichment analysis, 10 hub ferroptosis-related genes were chosen to construct the correlation regulation network. In RC tears, it was discovered that genes related to hub ferroptosis and hub inflammatory response were strongly correlated. The outcomes of in vivo tests showed that RC tears were related to Cd68-Cxcl13, Acsl4-Sat1, Acsl3-Eno3, Acsl3-Ccr7, and Ccr7-Eno3 pairings in regulating ferroptosis and inflammatory response. Thus, our results show an association between ferroptosis and inflammation, providing a new avenue to explore the clinical treatment of RC tears.

Multi-Proteomic Analysis Reveals the Effect of Protein Lactylation on Matrix and Cholesterol Metabolism in Tendinopathy.

Tendinopathy is a disease with surging prevalence. Lacking understanding of molecular mechanisms impedes the development of therapeutic approaches and agents. Lysine lactylation (Kla) is a newly discovered post-translational modification related to glycolysis. It has long been noted that manipulation of glycolysis metabolism could affect tendon cell function, tendon homeostasis, and healing process of tendon. However, protein lactylation sites in tendinopathy remain unexplored. Here, we conducted the first proteome-wide Kla analysis in tendon samples harvested from patients with rotator cuff tendinopathy (RCT), which identified 872 Kla sites across 284 proteins. Compared with normal counterparts, 136 Kla sites on 77 proteins were identified as upregulated in the pathological tendon, while 56 sites on 32 proteins were downregulated. Function enrichment analysis demonstrated that the majority of proteins with upregulated Kla levels functioned in organization of the tendon matrix and cholesterol metabolism, accompanied by lower expression levels which meant impaired cholesterol metabolism and degeneration of the tendon matrix, indicating potential cross-talk between protein lactylation and expression levels. At last, by western blotting and immunofluorescence, we verified the correlation between high lactylation and the downregulation of matrix and cholesterol-related proteins including BGN, MYL3, TPM3, and APOC3. ProteomeXchange: PXD033146.

Characterization of Histone Modifications in Late-Stage Rotator Cuff Tendinopathy.

The development and progression of rotator cuff tendinopathy (RCT) is multifactorial and likely to manifest through a combination of extrinsic, intrinsic, and environmental factors, including genetics and epigenetics. However, the role of epigenetics in RCT, including the role of histone modification, is not well established. Using chromatin immunoprecipitation sequencing, differences in the trimethylation status of H3K4 and H3K27 histones in late-stage RCT compared to control were investigated in this study. For H3K4, 24 genomic loci were found to be significantly more trimethylated in RCT compared to control (p < 0.05), implicating genes such as DKK2, JAG2, and SMOC2 in RCT. For H3K27, 31 loci were shown to be more trimethylated (p < 0.05) in RCT compared to control, inferring a role for EPHA3, ROCK1, and DEFß115. Furthermore, 14 loci were significantly less trimethylated (p < 0.05) in control compared to RCT, implicating EFNA5, GDF6, and GDF7. Finally, the TGFß signaling, axon guidance, and regulation of focal adhesion assembly pathways were found to be enriched in RCT. These findings suggest that the development and progression of RCT is, at least in part, under epigenetic control, highlighting the influence of histone modifications in this disorder and paving the way to further understand the role of epigenome in RCT.

Pathological alterations in the expression status of rotator cuff tendon matrix components in hyperlipidemia.

Hyperlipidemia is an important risk factor in the development and progression of tendon pathology, however its role in aggravating rotator cuff tendon injury (RCTI) is largely unknown. We aimed to assess the expression status of key extracellular matrix (ECM) components in the tendon tissues and tenocytes under hyperlipidemia. Shoulder rotator cuff (RC) tendon tissues harvested from the swine model of hyperlipidemia displayed alterations in histomorphometry and the expression status of major ECM component proteins including COL-I, COL-III, COL-IV, COL-V, COL-VI, MMP2, and MMP9. Similarly, the LDL- and oxLDL-challenged tenocytes displayed altered expression of the same proteins at both transcriptional and translational levels. In addition, the lipid uptake and cellular reactive oxygen radicals predominated in the lipid-challenged tenocytes compared to the control. Overall, the LDL-treated cells displayed predominant pathological alterations compared to the ox-LDL-treated cells. Further understanding regarding the underlying molecular mechanisms driving the tendon matrisome alteration and subsequent aggravated RCTI pathology in hyperlipidemia could open novel translational avenues in the management of RCTI.

Bioprinted living tissue constructs with layer-specific, growth factor-loaded microspheres for improved enthesis healing of a rotator cuff.

Substantial challenges remain in constructing the native tendon-to-bone interface for rotator cuff healing owing to the enthesis tissues' highly organized structural and compositional gradients. Herein, we propose to bioprint living tissue constructs with layer-specific growth factors (GFs) to promote enthesis regeneration by guiding the zonal differentiation of the loaded stem cells in situ. The sustained release of tenogenic, chondrogenic, and osteogenic GFs was achieved via microsphere-based delivery carriers embedded in the bioprinted constructs. Compared to the basal construct without GFs, the layer-specific tissue analogs realized region-specific differentiation of stem cells in vitro. More importantly, bioprinted living tissue constructs with layer-specific GFs rapidly enhanced the enthesis regeneration in a rabbit rotator cuff tear model in terms of biomechanical restoration, collagen deposition, and alignment, showing gradient interface of fibrocartilage structures with aligned collagen fibrils and an ultimate load failure of 154.3 ± 9.5 N resembling those of native enthesis tissues in 12 weeks. This exploration provides a feasible strategy to engineer living tissue constructions with region-specific differentiation potentials for the functional repair of gradient enthesis tissues. STATEMENT OF SIGNIFICANCE: Previous studies that employed acellular layer-specific scaffolds or stem cells for the reconstruction of the rotator cuff faced challenges due to their insufficient capability to rebuild the anisotropic compositional and structural gradients of native enthesis tissues. This manuscript proposed a living tissue construct with layer-specific, GFs-loaded µS, which can direct in situ and region-specific differentiation of the embedded stem cells to tenogenic, chondrogenic, and osteogenic lineages for functional regeneration of the enthesis tissues. This bioprinted living tissue construct with the unique capability to reduce fibrovascular scar tissue formation and simultaneously facilitate enthesis tissue remodeling might provide a promising strategy to repair complex and gradient tissues in the future.

Evaluating the role of type 2 diabetes mellitus in rotator cuff tendinopathy: Development and analysis of a novel rat model.

Objective: To establish and validate an intact rotator cuff rat model for exploring the pathophysiological effects of type 2 diabetes on the rotator cuff tendon in vivo. Methods: A total of 45 adult male rats were randomly divided into a control group (n = 9) and type 2 diabetes group (n=36). The rats were sacrificed at 2 weeks (T2DM-2w group, n=9), 4 weeks (T2DM-4w group, n=9), 8 weeks (T2DM-8w group, n=9), and 12 weeks (T2DM-12w group, n=9) after successful modeling of type 2 diabetes. Bilateral shoulder samples were collected for gross observation and measurement, protein expression(enzyme-linked immunosorbent assay,ELISA), histological evaluation, biomechanical testing, and gene expression (real-time quantitative polymerase chain reaction, qRT-PCR). Results: Protein expression showed that the expression of IL-6 and Advanced glycation end products (AGEs)in serum increased in type 2 diabetic group compared with the non-diabetic group. Histologically, collagen fibers in rotator cuff tendons of type 2 diabetic rats were disorganized, ruptured, and with scar hyperplasia, neovascularization, and extracellular matrix disturbances, while Bonar score showed significant and continuously aggravated tendinopathy over 12 weeks. The biomechanical evaluation showed that the ultimate load of rotator cuff tendons in type 2 diabetic rats gradually decreased, and the ultimate load was negatively correlated with AGEs content. Gene expression analysis showed increased expression of genes associated with matrix remodeling (COL-1A1), tendon development (TNC), and fatty infiltration (FABP4) in tendon specimens from the type 2 diabetic group. Conclusion: Persistent type 2 diabetes is associated with the rupture of collagen fiber structure, disturbance in the extracellular matrix, and biomechanical decline of the rotator cuff tendon. The establishment of this new rat model of rotator cuff tendinopathy provides a valuable research basis for studying the cellular and molecular mechanisms of diabetes-induced rotator cuff tendinopathy.

Spatial transcriptomics tools allow for regional exploration of heterogeneous muscle pathology in the pre-clinical rabbit model of rotator cuff tear.

BACKGROUND: Conditions affecting skeletal muscle, such as chronic rotator cuff tears, low back pain, dystrophies, and many others, often share changes in muscle phenotype: intramuscular adipose and fibrotic tissue increase while contractile tissue is lost. The underlying changes in cell populations and cell ratios observed with these phenotypic changes complicate the interpretation of tissue-level transcriptional data. Novel single-cell transcriptomics has limited capacity to address this problem because muscle fibers are too long to be engulfed in single-cell droplets and single nuclei transcriptomics are complicated by muscle fibers' multinucleation. Therefore, the goal of this project was to evaluate the potential and challenges of a spatial transcriptomics technology to add dimensionality to transcriptional data in an attempt to better understand regional cellular activity in heterogeneous skeletal muscle tissue. METHODS: The 3' Visium spatial transcriptomics technology was applied to muscle tissue of a rabbit model of rotator cuff tear. Healthy control and tissue collected at 2 and 16 weeks after tenotomy was utilized and freshly snap frozen tissue was compared with tissue stored for over 6 years to evaluate whether this technology is retrospectively useful in previously acquired tissues. Transcriptional information was overlayed with standard hematoxylin and eosin (H&E) stains of the exact same histological sections. RESULTS: Sequencing saturation and number of genes detected was not affected by sample storage duration. Unbiased clustering matched the underlying tissue type-based on H&E assessment. Connective-tissue-rich areas presented with lower unique molecular identifier counts are compared with muscle fibers even though tissue permeabilization was standardized across the section. A qualitative analysis of resulting datasets revealed heterogeneous fiber degeneration-regeneration after tenotomy based on (neonatal) myosin heavy chain 8 detection and associated differentially expressed gene analysis. CONCLUSIONS: This protocol can be used in skeletal muscle to explore spatial transcriptional patterns and confidently relate them to the underlying histology, even for tissues that have been stored for up to 6 years. Using this protocol, there is potential for novel transcriptional pathway discovery in longitudinal studies since the transcriptional information is unbiased by muscle composition and cell type changes.