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Background: The network of intermediate filament proteins underlying the inner nuclear membrane forms the nuclear lamin. A- and B-type lamins are the major components of the nuclear lamina. Lamins function in many nuclear activities. The role of lamin A and transcription factors (NF-kB) as anti-apoptotic is well documented. Recently, lamin A has also been considered as a mechanosensor protein that is able to maintain nuclear integrity from mechanical insults. We aimed to verify how lamin A expression varies in healthy cuff cells and in those with different-sized tears where various mechanical stresses are present., Methods: Forty-three patients with rotator cuff tear (RCT) [23M-20F, mean age (SD): 63.5 (6.1)] were enrolled. Tissue samples excised from the most medial point of tear margins were analyzed for lamin A expression by immunohistochemistry. Controls were represented by samples obtained by normal supraspinatus tendons excised from patients submitted to reverse shoulder prosthesis implant [8M-7F, mean age (SD): 67.9 (7.1)]. The intensity of staining was graded, and an H-score was assigned. Statistical analysis was performed., Results: Our study revealed a moderate intensity of lamin A in the healthy cuff tendons, a higher expression of this protein in the small tears, and a significant decrease of lamin A with increasing tear size (p < 0.0001)., Conclusions: Our study emphasizes the importance of early repair of small RCTs since nuclear stability is maintained, and the cellular function is protected by lamin A overexpression. High re-tear of massive cuff repair could be due to cellular apoptosis and nuclear modifications induced by lamin A lack., Level of Evidence: III.

Fatty infiltration (FI) of rotator cuff (RC) muscles is common in patients with RC tears. Studies have demonstrated that fibro-adipogenic progenitors (FAPs), a population of resident muscle stem cells, are the main contributors of FI, which adversely affects muscle quality and RC repair success. Although FI is common in RC injuries, it is not frequently reported after other musculotendinous injuries. Additionally, studies have shown the development of different pathology patterns across muscle groups suggestive of intrinsic differences in cellular composition and behavior. This study evaluates FAP distribution and differentiation properties across anatomic locations in mice. Muscles from seven different anatomic locations were harvested from PDGFRα-eGFP FAP reporter mice. FAPs were quantified using histology and FACS sorting with BD Aria II with CD31 - /CD45 - /Integrinα7 - /Sca-1 + and PDGFRα reporter signal (n = 3 per muscle). The cells were analyzed for adipogenesis using immunocytochemistry and for proliferation properties with Brdu-Ki67 staining. In a separate group of mice, RC and tibialis anterior muscles received glycerol injection and were harvested after 2 weeks for FI quantification (n = 4). One-way analysis of variance was used for statistical comparisons among groups, with significance at p < 0.05. FAPs from the RC, masseter, and paraspinal muscles were more numerous and demonstrated greater proliferative capacity and adipogenic potency than those from the tibialis anterior and gastrocnemius. The RC demonstrated significantly greater levels of FI than the tibialis anterior after glycerol-injection injury. Clinical Significance: This study suggests differences in FAP distribution and differentiation characteristics may account for the propensity to develop FI in RC tears as compared with other musculotendinous injuries. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1113-1121, 2020., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Background: Rotator cuff tendon tears are typically degenerative and usually affect the region of tendon insertion on bone. The remnant torn tendon is degenerative and may not be an ideal source for progenitor cells for cell-based therapies. Therefore, the aim of this study was to determine if musculotendinous junction (MTJ), which is adjacent to tendon would be a viable alternate source of progenitor stem cells. We also sought to study the gene expression profile MTJ progenitors and compare it with progenitors isolated from RC tendon, RC muscle and other existing tissue sources (bone marrow, adipose tissue, and Achilles tendon)., Methods: Rotator cuff tendon (RCT), muscle (RCM), and RCMTJ as well as Achilles tendon (AT) tissues were harvested from healthy male Lewis rats and progenitor cultures were established from these tissues and also from bone marrow and adipose tissue. Quantitative RT-PCR was performed on RNA extracts from intact tissues and progenitor cells using a custom array for the mesenchymal stem cell (MSC) differentiation marker genes. The gene expression profile of MSC differentiation markers within four tissues types, six progenitor cells, and between tissue and their corresponding progenitors were compared., Results: Progenitors cells can be isolated from rat rotator cuff musculotendinous tissue and their pattern of MSC gene expression was similar to the rotator cuff tendon progenitors for majority of the genes tested. However, there were significant differences between the MSC gene expression patterns of RCMTJ and RCM progenitors. Furthermore, there were differences in gene expression between the RCMTJ tissue and its progenitor cells with respect to MSC differentiation markers. The gene expression pattern of RCMTJ tissue was similar to RCM tissue with respect to markers of chondrogenesis, myogenesis, tenogenesis, and MSC specific markers., Conclusion: We demonstrate that the musculotendinous junction contains distinct set of progenitor cells and their MSC gene expression pattern is similar to rotator cuff tendon progenitors. RCMTJ progenitors will be an attractive option for cell-based regenerative treatment of chronic rotator cuff tears.

Background: Bone mineral density of the humeral head is an independent determining factor for postoperative rotator cuff tendon healing. Bisphosphonates, which are commonly used to treat osteoporosis, have raised concerns regarding their relationships to osteonecrosis of the jaw and to atypical fracture of the femur. In view of the prevalence of rotator cuff tear in osteoporotic elderly people, it is important to determine whether bisphosphonates affect rotator cuff tendon healing. However, no studies have investigated bisphosphonates' cytotoxicity to human rotator cuff tendon fibroblasts (HRFs) or bisphosphonates' effects on rotator cuff tendon healing. The purpose of this study was to evaluate the cytotoxicity of alendronate (Ald), a bisphosphonate, and its effects on HRF wound healing., Methods: HRFs were obtained from human supraspinatus tendons, using primary cell cultures. The experimental groups were control, 0.1 μM Ald, 1 μM Ald, 10 μM Ald, and 100 μM Ald. Alendronate exposure was for 48 h, except during a cell viability analysis with durations from 1 day to 6 days. The experimental groups were evaluated for cell viability, cell cycle and cell proliferation, type of cell death, caspase activity, and wound-healing ability., Results: The following findings regarding the 100 μM Ald group contrasted with those for all the other experimental groups: a significantly lower rate of live cells (p < 0.01), a higher rate of subG1 population, a lower rate of Ki-67 positive cells, higher rates of apoptosis and necrosis, a higher number of cells with DNA fragmentation, higher caspase-3/7 activity (p < 0.001), and a higher number of caspase-3 positive staining cells. In scratch-wound healing analyses of all the experimental groups, all the wounds healed within 48 h, except in the 100 μM Ald group (p < 0.001)., Conclusions: Low concentrations of alendronate appear to have little effect on HRF viability, proliferation, migration, and wound healing. However, high concentrations are significantly cytotoxic, impairing cellular proliferation, cellular migration, and wound healing in vitro.

Degenerative changes to rotator cuff tendons are often diagnosed in elderly patients. Spontaneous healing of degenerative tendons is rather inefficient as a result of the lack of a suitable microenvironment for tendon stem cell differentiation and vascularization. Herein, multilayer hyaluronic acid/chitosan (HA/CS) nanofilms were assembled by a layer-by-layer assembly method onto aligned poly(ε-caprolactone) (PCL) nanofibers for use in healing degenerative tendons. Materials testing showed that the number of layers of HA/CS nanofilms could adjust the hydrophilicity and wettability of the nanofibrous membranes. In vitro, the optimal 8-layer (termed as 8LP) membrane afforded aligned morphology of tendon stem/progenitor cells (TSPCs) and up-regulated mRNA expression of tenogenic markers (SCX, BGN). In a rabbit model with disorganized rotator cuff tendons, the 8LP group up-regulated mRNA levels of collagen I/III and tenascin (TNC) at 6 weeks, but not 12 weeks, post-surgery as compared to the native PCL group. Next, vascular endothelial growth factor-loaded 8LP (termed as 8LP-V) was prepared. Compared to 8LP, 8LP-V produced higher levels of angiogenesis in the tendons at 6 or 12 weeks post-surgery, thus supplying endogenous pre-tendon growth factors (TGF-β, IGF-1) to further enhance tenogenic transcriptional factors. As a result, 8LP-V yielded thicker collagen fibers and/or higher tendon stiffness as compared to the 8LP and clinical pericardial patch groups. This study highlights the rational design of LbL-assembled multilayer HA/CS films to upregulate tenogenesis for robust healing of degenerative rotator cuff tendons.

Background: Fat accumulation commonly occurs in chronically torn rotator cuff muscles, and increased fat within the rotator cuff is correlated with poor clinical outcomes. The extent of lipid deposition is particularly pronounced in injured rotator cuff muscles compared with other commonly injured muscles such as the gastrocnemius. Satellite cells, which are a tissue-resident muscle stem-cell population, can differentiate into fat cells. We hypothesized that satellite cells from the rotator cuff have greater intrinsic adipogenic differentiation potential than do gastrocnemius satellite cells, and this difference is due to variations in epigenetic imprinting between the cells., Methods: Satellite cells from gastrocnemius and rotator cuff muscles of mice were cultured in adipogenic media, and the capacity to differentiate into mature muscle cells and adipogenic cells was assessed (n ≥ 9 plates per muscle group). We also performed DNA methylation analysis of gastrocnemius and rotator cuff satellite cells to determine whether epigenetic differences were present between the 2 groups (n = 5 mice per group)., Results: Compared with the gastrocnemius, satellite cells from the rotator cuff had a 23% reduction in myogenic differentiation and an 87% decrease in the expression of the differentiated muscle cell marker MRF4 (myogenic regulatory factor 4). With respect to adipogenesis, rotator cuff satellite cells had a 4.3-fold increase in adipogenesis, a 12-fold increase in the adipogenic transcription factor PPARγ (peroxisome proliferator-activated receptor gamma), and a 65-fold increase in the adipogenic marker FABP4 (fatty-acid binding protein 4). Epigenetic analysis identified 355 differentially methylated regions of DNA between rotator cuff and gastrocnemius satellite cells, and pathway enrichment analysis suggested that these regions were involved with lipid metabolism and adipogenesis., Conclusions: Satellite cells from rotator cuff muscles have reduced myogenic and increased adipogenic differentiation potential compared with gastrocnemius muscles. There appears to be a cellular and genetic basis behind the generally poor rates of rotator cuff muscle healing., Clinical Relevance: The reduced myogenic and increased adipogenic capacity of rotator cuff satellite cells is consistent with the increased fat content and poor muscle healing rates often observed for chronically torn rotator cuff muscles. For patients undergoing rotator cuff repair, transplantation of autologous satellite cells from other muscles less prone to fatty infiltration may improve clinical outcomes.

Reducing rotator cuff failure after repair remains a challenge due to suboptimal tendon-to-bone healing. In this study we report a novel biomaterial with engineered tendon-fibrocartilage-bone composite (TFBC) and bone marrow-derived mesenchymal stem cell sheet (BMSCS); this construct was tested for augmentation of rotator cuff repair using a canine non-weight-bearing (NWB) model. A total of 42 mixed-breed dogs were randomly allocated to 3 groups (n = 14 each). Unilateral infraspinatus tendon underwent suture repair only (control); augmentation with engineered TFBC alone (TFBC), or augmentation with engineered TFBC and BMSCS (TFBC + BMSCS). Histomorphometric analysis and biomechanical testing were performed at 6 weeks after surgery. The TFBC + BMSCS augmented repairs demonstrated superior histological scores, greater new fibrocartilage formation and collagen fiber organization at the tendon-bone interface compared with the controls. The ultimate failure load and ultimate stress were 286.80 ± 45.02 N and 4.50 ± 1.11 MPa for TFBC + BMSCS group, 163.20 ± 61.21 N and 2.60 ± 0.97 MPa for control group (TFBC + BMSCS vs control, P = 1.12E-04 and 0.003, respectively), 206.10 ± 60.99 N and 3.20 ± 1.31 MPa for TFBC group (TFBC + BMSCS vs TFBC, P = 0.009 and 0.045, respectively). In conclusion, application of an engineered TFBC and BMSCS can enhance rotator cuff healing in terms of anatomic structure, collagen organization and biomechanical strength in a canine NWB model. Combined TFBC and BMSCS augmentation is a promising strategy for rotator cuff tears and has a high potential impact on clinical practice., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Platelet rich plasma (PRP) is widely used in rotator cuff repairs but its effect on the healing process is unclear. Several cell culture studies on the effect of allogenic PRP have reported promising results but are not transferable to clinical practice. The aim of the present study is to assess the possible effect of autologous PRP on rotator cuff tendon cells. The amount of growth factors involved with tendon-bone healing (PDGF-AB, IGF-1, TGF-β1, BMP-7 and -12) is quantified., Methods: Rotator cuff tissue samples were obtained from (n = 24) patients grouped by age (>/< 65 years) and sex into four groups and cells were isolated and characterized. Later, autologous PRP preparations were obtained and the effect was analyzed by means of cell proliferation, collagen I synthesis and expression of collagen I and III. Furthermore, the PRPs were quantified for growth factor content by means of platelet-derived growth factor (PDGF-AB), insulin-like growth factor (IGF-1), transforming growth factor (TGF-β1), as well as bone morphogenetic protein (BMP) -7 and - 12., Results: Cell proliferation and absolute synthesis of collagen I were positively affected by PRP exposure compared to controls (p < 0.05), but expression and relative synthesis of collagen I (normalized to cell proliferation) were significantly reduced. PRP contained high amounts of IGF-1 and lower levels of TGF-β1 and PDGF-AB. The amounts of BMP-7 and -12 were below the detection limits., Conclusions: PRP is a source of growth factors such involved with tendon-bone healing. PRP had an anabolic effect on the human rotator cuff tenocytes of the same individual in vitro by means of cell proliferation and absolute, but not relative collagen I synthesis. These results encourage further studies on clinical outcomes with more comparable standards in terms of preparation and application methods., Level of Evidence: Controlled laboratory study.

Purpose: Tendinopathy is a significant clinical problem thought to be associated with altered mechanical loading. Explant culture models allow researchers to alter mechanical loading in a controlled in vitro environment while maintaining tenocytes in their native matrix. However, current models do not accurately represent commonly injured tendons, ignoring contributions of associated musculature and bone, as well as regional collagen structure. This study details the characterization of amouse rotator cuff explant culture model, including bone, tendon, and muscle (BTM)., Materials and Methods: Following harvest, BTM explants were maintained in stress-deprived culture for one week and tendon was then assessed for changes in cell viability, metabolism, matrix structure and content., Results: Matrix turnover occurred throughout culture as manifested in both gene expression and biosynthesis, but this did not translate to net changes in total collagen or sulfated glycosaminoglycan content. Furthermore, tendon structure was not significantly altered throughout culture. However, we found significant cell death in BTM tendons after 3 days in culture, which we hypothesize is cytokine-induced. Using a targeted multiplex assay, we found high levels of pro-inflammatory cytokines released to the culture medium from muscle and bone, levels that did cause cell deathin tendon-alone controls., Conclusions: Overall, this model presents an innovative approach to understandingrotator cuff injury and tenocyte mechanobiology in a clinically-relevant tendon structure. Our model can be a powerful tool to investigate how mechanical and biological stimuli can alter normal tendon health and lead to tendon degeneration, and may provide a testbed for therapeutics for tendon repair.

Introduction: The purpose of this study was to evaluate the effect of allogenic leukocyte-reduced platelet-rich plasma on human tenocytes after treatment with prednisolone and to develop a standardization of its application for clinical practice., Methods: A leukocyte-reduced PRP was produced using the Arthrex Double Syringe (Arthrex, Inc., Naples, FL, USA), in a modified single-spin separation method. Human tenocytes were isolated from discarded rotator cuff segments. Tenocytes were cultured in the presence of PRP and prednisolone, both alone and in combination. Control samples were treated in media containing 2% FCS for 72 h. After 72 h of incubation, cell cycle kinetics of tenocytes were analyzed to assess proliferation., Results: Incubation of the tenocytes with PRP alone for 48 h led to high proliferation rate (10% PRP, 28.0 ± 10.5%; 20% PRP, 40.9 ± 3.3%). Incubation in the presence of prednisolone led to a significant decrease of the proliferation rate (5.2 ± 3.1%; p < 0.05). Treatment with PRP for 48 h significantly increased the proliferation of tenocytes in a dose-dependent manner (10% PRP, 28.0 ± 10.5%; 20% PRP, 40.9 ± 3.3%; p < 0.05). The presence of prednisolone resulted in a decreased tenocyte proliferation (5.2 ± 3.1%; p < 0.05), whereas addition of PRP for 24 and 48 h after prednisolone exposure did not show any compensating effect independent of PRPs concentration (10% PRP, 3.7 ± 3.0%; 20% PRP, 2.5 ± 2.5%). However, a significantly increased cell proliferation of tenocytes was evident when PRP was applied 24 h after prednisolone incubation for 48 h (31.0 ± 3.4 and 34.3 ± 4.7%)., Conclusion: The use of leukocyte-reduced PRP stimulates the proliferation of tenocytes and antagonizes the negative effect of prednisolone 24 h after treatment. Addition of PRP 48 h after treatment with prednisolone has no positive effect on the proliferation rate of tenocytes.

Purpose: To analyze the ability of ropivacaine, bupivacaine, and triamcinolone to induce apoptosis and necrosis in fibroblasts, tenocytes, and human mesenchymal stem cells., Methods: Human dermal fibroblasts, adipose-derived human mesenchymal stem cells (hMSCs), and tenocytes gained from the rotator cuff tendon were seeded with a cell density of 0.5 × 10 4 /cm 2 . One specimen of ropivacaine, bupivacaine, and triamcinolone was tested separately on the cells with separate concentrations of 0.5%, 0.25%, and 0.125% for each specimen. The negative control received no agent, only a change of medium. The incubation period for each agent was 30 minutes. After a change of medium and 1 hour, 24 hours, and 7 days of incubation, 10 4  cells were harvested and analyzed via fluorescence-activated cell sorting with double-staining with annexin V and propidium iodide. Statistical analysis to determine significant difference (P < .05) between the groups with SPSS statistics 23 through one-way analysis of variance with a univariate general linear model was performed., Results: Bupivacaine showed necrosis-inducing effects on fibroblasts and tenocytes, with the necrotic effect peaking at 0.5% and 0.25%. Ropivacaine and triamcinolone caused no significant necrosis. Compared with fibroblasts and tenocytes, hMSCs did not show significant necrotic or apoptotic effects after exposure to bupivacaine. Overall, no significant differences in apoptosis were detected between different cell lines, varying concentrations, or time measurements., Conclusions: Bupivacaine 0.5% and 0.25% have the most necrosis-inducing effects on fibroblasts and tenocytes. Ropivacaine caused less necrosis than bupivaine. Compared with fibroblasts and tenocytes, hMSCs were not affected by necrosis using any of the tested agents. A significant apoptosis-inducing effect could not be detected for the different cell lines., Clinical Relevance: Possible cell toxicity raises questions of concern for intra-articular injections using local anesthetics and corticosteroids. The present study demonstrates the necrotic and apoptotic effects of ropivacaine, bupivacaine, and triamcinolone and may give recommendations for intra-articular use of local anesthetics and corticosteroids., (Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

To achieve biological regeneration of tendon-bone junctions, cell sheets of human rotator-cuff derived cells were used in a rat rotator cuff injury model. Human rotator-cuff derived cells were isolated, and cell sheets were made using temperature-responsive culture plates. Infraspinatus tendons in immunodeficient rats were resected bilaterally at the enthesis. In right shoulders, infraspinatus tendons were repaired by the transosseous method and covered with the cell sheet (sheet group), whereas the left infraspinatus tendons were repaired in the same way without the cell sheet (control group). Histological examinations (safranin-O and fast green staining, isolectin B4, type II collagen, and human-specific CD31) and mRNA expression (vascular endothelial growth factor; VEGF, type II collagen; Col2, and tenomodulin; TeM) were analyzed 4 weeks after surgery. Biomechanical tests were performed at 8 weeks. In the sheet group, proteoglycan at the enthesis with more type II collagen and isolectin B4 positive cells were seen compared with in the control group. Human specific CD31-positive cells were detected only in the sheet group. VEGF and Col2 gene expressions were higher and TeM gene expression was lower in the sheet group than in the control group. In mechanical testing, the sheet group showed a significantly higher ultimate failure load than the control group at 8 weeks. Our results indicated that the rotator-cuff derived cell sheet could promote cartilage regeneration and angiogenesis at the enthesis, with superior mechanical strength compared with the control. Treatment for rotator cuff injury using cell sheets could be a promising strategy for enthesis of tendon tissue engineering. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:289-296, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Purpose: Mechanical stimulation plays an important role in the development and remodelling of tendons. The aim of the study was to evaluate the effects of mechanical stimulation on the expression of extracellular matrix proteins in human primary rotator cuff (RC) fibroblasts., Methods: RC fibroblasts were isolated from patients with degenerative RC tears and characterized using flow cytometry and immunohistochemistry. Cells were stimulated using the Flexcell FX5K™ Tension System. The stimulation regime was a uniaxial sinusoidal waveform with 10 % elongation and a frequency of 0.5 Hz, whereby each cycle consists of 10-s strain and 30-s relaxation. Data were normalized to mechanically unstimulated control groups for every experimental condition. RT-qPCR was performed to determine relative mRNA levels, and collagen production was measured by a colorimetric assay., Results: The positive expression of CD91 and CD10, and negativity for CD45 and CD4 confirmed the fibroblast phenotype of RC primary cells. RT-qPCR revealed that 10 % continuous cyclic strain for 7 and 14 days induced a significant increase in the mRNA expression both on the matrix metalloproteinases MMP1, MMP3, MMP13, and MMP14 and on the extracellular matrix proteins decorin, tenascin-C, and scleraxis. Furthermore, mechanically stimulated groups produced significantly higher amounts of total collagen., Conclusion: These results may contribute to a better understanding of strain-induced tendon remodelling and will form the basis for the correct choice of applied force in rehabilitation after orthopaedic surgery. These findings underline the fact that early passive motion of the joint in order to induce remodelling of the tendon should be included within a rehabilitation protocol for rotator cuff repair.

The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets, and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: (1) plasma (PPP), (2) plasma and platelets (PAP), (3) plasma and macrophages (PPPM), (4) plasma, platelets and macrophages (PAPM), (5) phosphate buffered saline (PBS), and (6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype, and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine whether these changes in cellular function will translate into improved tendon healing., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Purpose: The most frequent complications after rotator cuff repair (RCR) are non-healing and re-tear. Age and gender are both proven risk factors for faulty RCR. This study analyzed the effects of female sex steroids and calciotropic hormones on tendon-derived cell characteristics., Methods: Tendon-derived cells from rat supraspinatus were treated with estradiol-17β (E2); soy isoflavones (daidzein, genistein, biochainin A); raloxifene and estrogen receptors α and β agonists and antagonists; and less-calcemic vitamin-D analog, parathyroid hormone, and vehicle control for 24 h. Cell proliferation and mRNA expression of estrogen receptor α and β, vitamin-D receptor (VDR), scleraxis, and collagen-1 were assessed., Results: E2, Biochainin A, raloxifene, and vitamin-D significantly increased tendon-derived cell proliferation. Estrogen receptor α antagonists neutralized tendon-derived cells response to estradiol 17-β; however, estrogen receptor β antagonists did not have an effect. Scleraxis expression decreased following estradiol 17-β and vitamin-D treatments. Vitamin-D significantly reduced collagen-1 expression, while estradiol 17-β had no effect. Vitamin-D and estradiol 17-β upregulated VDR expression., Conclusions: Significant tendon-derived cell proliferation can be achieved with commonly prescribed female sex and calciotropic hormones. However, collagen-1 expression remained constant or decreased following the administration of these hormones. Female sex steroids and vitamin-D promoted tendon-derived cell proliferation via estrogen receptor α and VDR, not estrogen receptor β. Amplified cell proliferation was not associated with increased scleraxis and collagen-1 expression. These results have important implications to the properties of healing tendon and possible pharmaceutical therapies for patients with torn RC. Further research is warranted to expose the underling mechanisms of these effects.

An imbalance between matrix metalloproteases (MMPs) and the tissue inhibitors of metalloproteases (TIMPs) may have a negative impact on the healing of rotator cuff tears. The aim of the project was to assess a possible relationship between clinical and radiographic characteristics of patients such as the age, sex, as well as the degenerative status of the tendon and the MMPs and TIMPs in their tenocyte-like cells (TLCs). TLCs were isolated from ruptured supraspinatus tendons and quantitative Real-Time PCR and ELISA was performed to analyze the expression and secretion of MMPs and TIMPs. In the present study, MMPs, mostly gelatinases and collagenases such as MMP-2, -9 and -13 showed an increased expression and protein secretion in TLCs of donors with higher age or degenerative status of the tendon. Furthermore, the expression and secretion of TIMP-1, -2 and -3 was enhanced with age, muscle fatty infiltration and tear size. The interaction between MMPs and TIMPs is a complex process, since TIMPs are not only inhibitors, but also activators of MMPs. This study shows that MMPs and TIMPs might play an important role in degenerative tendon pathologies.

Introduction: The bursa subacromialis (BS) provides the gliding mechanism of the shoulder and regenerates itself after surgical removal. Therefore, we explored the presence of mesenchymal stem cells (MSCs) within the human adult BS tissue and characterized the BS cells compared to MSCs from bone marrow (BMSCs) on a molecular level., Methods: BS cells were isolated by collagenase digest from BS tissues derived from patients with degenerative rotator cuff tears, and BMSCs were recovered by adherent culture from bone-marrow of patients with osteoarthritis of the hip. BS cells and BMSCs were compared upon their potential to proliferate and differentiate along chondrogenic, osteogenic and adipogenic lineages under specific culture conditions. Expression profiles of markers associated with mesenchymal phenotypes were comparatively evaluated by flow cytometry, immunohistochemistry, and whole genome array analyses., Results: BS cells and BMSCs appeared mainly fibroblastic and revealed almost similar surface antigen expression profiles, which was CD44(+), CD73(+), CD90(+), CD105(+), CD106(+), STRO-1(+), CD14(-), CD31(-), CD34(-), CD45(-), CD144(-). Array analyses revealed 1969 genes upregulated and 1184 genes downregulated in BS cells vs. BMSCs, indicating a high level of transcriptome similarity. After 3 weeks of differentiation culture, BS cells and BMSCs showed a similar strong chondrogenic, adipogenic and osteogenic potential, as shown by histological, immunohistochemical and RT-PCR analyses in contrast to the respective negative controls., Conclusions: Our in vitro characterizations show that BS cells fulfill all characteristics of mesenchymal stem cells, and therefore merit further attention for the development of improved therapies for various shoulder pathologies.

Background: Many clinical and radiographic studies suggest that patient age and sex have an influence on rotator cuff (RC) repair outcomes. However, these findings result from retrospective statistical analyses and cannot provide a causal answer., Purpose: To analyze whether age and sex influence the biological potential at the time of RC repair or midterm clinical and radiographic outcomes. Also assessed was the effect of the biological potential on intraindividual clinical/radiographic results., Study Design: Cohort study; Level of evidence, 2., Methods: A total of 40 patients underwent arthroscopic RC repair. At the time of surgery (t = 0), supraspinatus tendon biopsy specimens were obtained, cultivated, and assessed for their biological potential, particularly (1) cell growth and (2) collagen type I production. After a follow-up at 24 months (t = 1), all patients were assessed by clinical scores (Constant score, subjective shoulder value, American Shoulder and Elbow Surgeons [ASES] score, and Western Ontario Rotator Cuff Index [WORC] score) and underwent magnetic resonance imaging to determine RC integrity. The data were examined for age- and sex-related differences and to identify the correlation between biological potential (t = 0) and clinical/radiographic outcome (t = 1)., Results: The follow-up rate for the imaging and clinical evaluation was 100%. Age, but not sex, influenced the biological tendon cell parameters at t = 0. However, there was no effect of age or sex on the clinical and radiographic results at t = 1. Furthermore, no correlation was observed between the initial biological parameters and later clinical outcomes or radiographic RC integrity. Finally, there was no significant difference between intact and nonhealed repairs in terms of the respective clinical scores., Conclusion: Age, but not sex, was found to have a negative effect on RC tendon cell biology. However, neither sex nor, in particular, a higher age influenced repair outcomes after 24 months., (© 2015 The Author(s).)

Background: Concern has recently arisen over the safety of local anesthetics used on human tissues., Hypothesis: Aminoamide local anesthetics have cytotoxic effects on human rotator cuff tenofibroblasts., Study Design: Controlled laboratory study., Methods: Cultured human rotator cuff tenofibroblasts were divided into control, phosphate buffered saline (PBS), and local anesthetic study groups; the PBS study group was further subdivided by pH level (pH 7.4, 6.0, and 4.4). The 6 local anesthetic subgroups (0.2% and 0.75% ropivacaine, 0.25% and 0.5% bupivacaine, and 1% and 2% lidocaine) were also studied at 10% dilutions of their original concentrations. Exposure times were 5, 10, 20, 40, or 60 minutes for the higher concentrations and 2, 6, 12, 24, 48, or 72 hours for the lower concentrations. Cell viability was evaluated through live, apoptotic, and necrotic cell rates using the annexin V-propidium iodide double-staining method. Intracellular reactive oxygen species (ROS) and the activity of mitogen-activated protein kinases (MAPKs) and caspase-3/7 were investigated., Results: The control and PBS groups showed no significant differences in cell viability (P > .999). In the local anesthetic study groups, cell viability decreased significantly with increases in anesthetic concentrations (P < .001) and exposure times (P < .001), with the exception of the lidocaine subgroups, where this effect was masked by the very high cytotoxicity of even low concentrations. Among the studied local anesthetic subgroups, 0.2% ropivacaine was the least toxic. The levels of intracellular ROS of each local anesthetic subgroup also increased significantly (P < .05). The studied local anesthetics showed increases in the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 as well as in levels of caspase-3/7 activity (P < .001)., Conclusion: The cytotoxicity of the anesthetics studied to tenofibroblasts is dependent on exposure time and concentration. Of the evaluated anesthetics, ropivacaine is the least toxic in the clinically used concentration. The studied anesthetics induce tenofibroblast cell death, mediated by the increased production of ROS, by the increased activation of ERK1/2, JNK, and p38 and by the activation of caspase-3/7., Clinical Relevance: This study identified the cytotoxic mechanisms of aminoamide local anesthetics acting on rotator cuff tenofibroblasts. The greatest margin of safety was found in lower anesthetic concentrations in general and more specifically in the use of ropivacaine., (© 2014 The Author(s).)

Full-thickness rotator cuff tears are one of the most common causes of shoulder pain in people over the age of 65. High retear rates and poor functional outcomes are common after surgical repair, and currently available extracellular matrix scaffold patches have limited abilities to enhance new tendon formation. In this regard, tissue-engineered scaffolds may provide a means to improve repair of rotator cuff tears. Electrospinning provides a versatile method for creating nanofibrous scaffolds with controlled architectures, but several challenges remain in its application to tissue engineering, such as cell infiltration through the full thickness of the scaffold as well as control of cell growth and differentiation. Previous studies have shown that ligament-derived extracellular matrix may enhance differentiation toward a tendon or ligament phenotype by human adipose stem cells (hASCs). In this study, we investigated the use of tendon-derived extracellular matrix (TDM)-coated electrospun multilayered scaffolds compared to fibronectin (FN) or phosphate-buffered saline (PBS) coating for use in rotator cuff tendon tissue engineering. Multilayered poly(ɛ-caprolactone) scaffolds were prepared by sequentially collecting electrospun layers onto the surface of a grounded saline solution into a single scaffold. Scaffolds were then coated with TDM, FN, or PBS and seeded with hASCs. Scaffolds were maintained without exogenous growth factors for 28 days in culture and evaluated for protein content (by immunofluorescence and biochemical assay), markers of tendon differentiation, and tensile mechanical properties. The collagen content was greatest by day 28 in TDM-scaffolds. Gene expression of type I collagen, decorin, and tenascin C increased over time, with no effect of scaffold coating. Sulfated glycosaminoglycan and dsDNA contents increased over time in culture, but there was no effect of scaffold coating. The Young's modulus did not change over time, but yield strain increased with time in culture. Histology demonstrated cell infiltration through the full thickness of all scaffolds and immunofluorescence demonstrated greater expression of type I, but not type III collagen through the full thickness of the scaffold in TDM-scaffolds compared to other treatment groups. Together, these data suggest that nonaligned multilayered electrospun scaffolds permit tenogenic differentiation by hASCs and that TDM may promote some aspects of this differentiation.

To assess the in vitro effect of platelet-rich plasma (PRP) on biological activity of the human rotator cuff fibroblasts and to describe the optimal dose-response to maximize cellular stimulation while reducing potential risk. Rotator cuff (RC) fibroblasts of n = 6 patients (mean age of 65.2 years) undergoing arthroscopic cuff tear reconstruction were cultured in vitro for 21 days and stimulated with PRP in three different concentrations (1-, 5-, and 10-fold). Samples were obtained for DNA and GAG measurement at 1, 7, 14, and 21 days. The biological outcomes were regressed on the PRP concentration. The application of PRP significantly influenced the fibroblast proliferation and activity of the human rotator cuff with elevated glycosaminoglycan (GAG) and DNA levels. The dosage of PRP had the significantly highest impact on this proliferation using a onefold or fivefold application. PRP has a significant effect on fibroblast proliferation of the human rotator cuff in vitro with an optimal benefit using a onefold or fivefold PRP concentration. This study justifies further in vivo investigations using PRP at the human rotator cuff., (Copyright © 2013 Orthopaedic Research Society.)

Purpose: The role of apoptosis in the progression of rotator cuff tendinopathy remains poorly understood. In particular, the extent of apoptosis in the partially torn supraspinatus tendon has not been well examined., Methods: Biopsies were obtained from nine partially torn supraspinatus tendons, from the matched intact subscapularis tendons, and from 10 reference subscapularis tendons. Immunohistochemistry was used to assess the density of apoptotic cells (activated caspase-3; Asp175), proliferation (Ki67), and p53 (M7001), a key protein involved in regulating cell death. The Bonar scale was used to evaluate tendon degeneration., Results: The density of apoptotic tendon cells and the density of cells expressing p53 were significantly increased in both the partially torn supraspinatus tendons and in the matched subscapularis tendons, compared with uninjured reference tendons. The Bonar score revealed significant tendon degeneration in the partially torn supraspinatus tendons compared with both matched and reference subscapularis tendons. Tendon cell proliferation was significantly increased in the partially torn supraspinatus tendons compared with reference subscapularis tendons., Conclusions: Partial-thickness tears of the supraspinatus tendon demonstrated an increased density of apoptotic, p53+ tendon cells. The fact that apoptosis was accompanied by increased tendon cell proliferation suggests that apoptosis may be related to an ongoing injury-repair process. Increased tenocyte apoptosis may be a relatively early feature in rotator cuff tendinopathy and could represent a possible target for therapeutic intervention.

Background: Stem cell therapy is expected to offer new alternatives to the traditional therapies of rotator cuff tendon tears. In particular, resident, tissue-specific, adult stem cells seem to have a higher regenerative potential for the tissue where they reside., Hypothesis: Rotator cuff tendon and long head of the biceps tendon possess a resident stem cell population that, when properly stimulated, may be induced to proliferate, thus being potentially usable for tendon regeneration., Study Design: Controlled laboratory study., Methods: Human tendon samples from the supraspinatus and the long head of the biceps were collected during rotator cuff tendon surgeries from 26 patients, washed with phosphate-buffered saline, cut into small pieces, and digested with collagenase type I and dispase. After centrifugation, cell pellets were resuspended in appropriate culture medium and plated. Adherent cells were cultured, phenotypically characterized, and then compared with human bone marrow stromal cells (BMSCs), as an example of adult stem cells, and human dermal fibroblasts, as normal proliferating cells with no stem cell properties., Results: Two new adult stem cell populations from the supraspinatus and long head of the biceps tendons were isolated, characterized, and cultured in vitro. Cells showed adult stem cell characteristics (ie, they were self-renewing in vitro, clonogenic, and multipotent), as they could be induced to differentiate into different cell types--namely, osteoblasts, adipocytes, and skeletal muscle cells., Conclusion: This work demonstrated that human rotator cuff tendon stem cells and human long head of the biceps tendon stem cells can be isolated and possess a high regenerative potential, which is comparable with that of BMSCs. Moreover, comparative analysis of the sphingolipid pattern of isolated cells with that of BMSCs and fibroblasts revealed the possibility of using this class of lipids as new possible markers of the cell differentiation status., Clinical Relevance: Rotator cuff and long head of the biceps tendons contain a stem cell population that can proliferate in vitro and could constitute an easily accessible stem cell source to develop novel therapies for tendon regeneration.

Tendon bone healing of the rotator cuff is often associated with non-healing or recurrent defects, which seems to be influenced by the patient's age and sex. The present study aims to examine cellular biological characteristics of tenocyte-like cells that may contribute to this impaired rotator cuff healing. Moreover, a therapeutic approach using growth factors could possibly stimulate tendon bone healing. Therefore, our second aim was to identify patient groups who would particularly benefit from growth factor stimulation. Tenocyte-like cells isolated from supraspinatus tendons of female donors younger and older than 65 years of age were characterized with respect to different cellular biological parameters, such as cell density, cell count, marker expression, collagen-I protein synthesis, and stem cell potential. Furthermore, cells of the donor groups were stimulated with BMP-2 and BMP-7 (200 and 1000 ng/ml) in 3D-culture and analyzed for cell count, marker expression and collagen-I protein synthesis. Female donors older than 65 years of age showed significantly decreased cell count and collagen-I protein synthesis compared to cells from donors younger than 65 years. Cellular biological parameters including cell count, collagen-I and -III expression, and collagen-I protein synthesis of cells from both donor groups were stimulated with BMP-2 and BMP-7. The cells from donors older than 65 years revealed a decreased stimulation potential for cell count compared to the younger group. Cells from female donors older than 65 years of age showed inferior cellular biological characteristics. This may be one reason for a weaker healing potential observed in older female patients and should be taken into consideration for tendon bone healing of the rotator cuff.

Triamcinolone acetonide (TA) injections are widely used to treat enthesopathy, but they may induce adverse effects such as tendon impairment and rupture. Platelet-rich plasma (PRP) is a blood fraction containing high platelet concentrations and various growth factors that play a role in tissue repair processes. The purpose of this study is to investigate whether TA has deleterious effects on human rotator cuff-derived cells, and if PRP can protect these cells from the effects of TA. Human rotator cuff-derived cells were cultured with and without TA and PRP, and the culture without any additive served as the control. Cell morphology was assessed at days 7 and 21. Cell viability was evaluated at days 1, 7, 14, and 21 by a water-soluble tetrazolium salt assay. Induction of apoptosis was measured by immunofluorescence staining and flow cytometry at day 7. Induction of cleaved caspase-3 was measured by immunofluorescence staining at day 7. The cells cultured with TA had a flattened and polygonal shape at day 7. The cells cultured with both TA and PRP were similar in appearance to control cells. Exposure to TA also significantly decreased cell viability, but cell viability did not decrease when PRP was added along with TA. The number of apoptotic cells increased with TA exposure, while addition of PRP prevented cell apoptosis. In conclusion, the deleterious effect of TA was prevented by PRP, which can be used as a protective agent for patients receiving local TA injections., (Copyright © 2012 Orthopaedic Research Society.)

Nicotine is one of the major chemical components of the cigarette smoke, which has been known as a risk factor for tendon ruptures including rotator cuff tears. This study investigated the effect of nicotine on tenocytes under cyclic-stretched condition. Particularly, we focused on the morphologic changes of tenocytes and their expression of MMPs. Primary porcine tenocytes were obtained from the infraspinatus tendon. The cells were cultured on elastic chambers under static or cyclic-stretched condition for 24 h in the existence of nicotine (0, 1, 10, and 100 µM). Cell shape, gene expression of collagen type I and III, MMPs (-1, -2, -3, -9, and -13) and TIMPs (-1, -2, and -3) and enzyme activity of MMP-9 were analyzed using immunohistochemistry, RT-PCR, and zymography. Tenocytes exposed to nicotine represented significantly decreased gene expressions in MMP-9 (p < 0.001) and TIMP-3 (p < 0.05) under the cyclic stretch. Enzymatic activity of MMP-9 was also reduced by nicotine exposure in a dose-dependent manner (p < 0.001). The down-regulation of MMP and TIMP expression by nicotine shown in our in vitro experiment might deteriorate normal metabolism of the tendon. These mechanisms might affect the mechanical properties of the extracellular matrix of the rotator cuff tendon., (Copyright © 2012 Orthopaedic Research Society.)

The self-healing potential of each tissue belongs to endogenous stem cells residing in the tissue; however, there are currently no reports mentioned for the isolation of human rotator cuff-derived mesenchymal stem cells (RC-MSCs) since. To isolate RC-MSCs, minced rotator cuff samples were first digested with enzymes and the single cell suspensions were seeded in plastic culture dishes. Twenty-four hours later, nonadherent cells were removed and the adherent cells were further cultured. The RC-MSCs had fibroblast-like morphology and were positive for the putative surface markers of MSCs, such as CD44, CD73, CD90, CD105, and CD166, and negative for the putative markers of hematopoietic cells, such as CD34, CD45, and CD133. Similar to BM-MSCs, RC-MSCs were demonstrated to have the potential to undergo osteogenic, adipogenic, and chondrogenic differentiation. Upon induction in the defined media, RC-MSCs also expressed lineage-specific genes, such as Runx 2 and osteocalcin in osteogenic induction, PPAR-γ and LPL in adipogenic differentiation, and aggrecan and Col2a1 in chondrogenic differentiation. The multipotent feature of RC-MSCs in the myogenic injury model was further strengthened by the increase in myogenic potential both in vitro and in vivo when compared with BM-MSCs. These results demonstrate the successful isolation of MSCs from human rotator cuffs and encourage the application of RC-MSCs in myogenic regeneration.

Background: Platelet-rich plasma (PRP) contains various growth factors and appears to have a potential to promote tendon healing, but evidence is lacking regarding its effect on human tenocytes from rotator cuff tendons with degenerative tears., Hypothesis: Platelet-rich plasma stimulates cell proliferation and enhances matrix gene expression and synthesis in tenocytes isolated from human rotator cuff tendons with degenerative tears., Study Design: Controlled laboratory study., Methods: Tenocytes were enzymatically isolated and cultured. To evaluate cell proliferation, tenocytes were cultured with 10% (vol/vol) platelet-poor plasma (PPP), PRP activated with calcium, and PRP activated with calcium and thrombin at platelet concentrations of 100, 200, 400, 800, 1000, 2000, 4000, 8000, and 16,000 × 10(3)/µL for 14 days. Cell number was measured at days 7 and 14. To investigate matrix gene expression and synthesis, cells were cultured with a PPP or PRP gel (10% vol/vol) at a platelet concentration of 1000 × 10(3)/µL for 14 days. Quantitative real-time reverse transcriptase polymerase chain reaction was performed to determine the expressions of type I and III collagen, decorin, tenascin-C, and scleraxis, and measurements of total collagen and glycosaminoglycan (GAG) synthesis were conducted at days 7 and 14., Results: Platelet-rich plasma significantly increased cell proliferation at days 7 and 14 in a dose-dependent manner, and the addition of thrombin moved up the plateau of proliferation. Platelet-rich plasma significantly induced the gene expression of type I collagen at day 7 but not at day 14, while it significantly promoted that of type III both at days 7 and 14. The ratio of type III/I collagens did not change at days 7 and 14. The expressions of decorin and scleraxis significantly increased at day 14, whereas that of tenascin-C significantly increased at days 7 and 14. Platelet-rich plasma significantly increased total collagen synthesis at days 7 and 14 and GAG synthesis at day 14., Conclusion: Platelet-rich plasma promoted cell proliferation and enhanced gene expression and the synthesis of tendon matrix in tenocytes from human rotator cuff tendons with degenerative tears., Clinical Relevance: These findings suggest that PRP might be used as a useful biological tool for regenerative healing of rotator cuff tears by enhancing the proliferation and matrix synthesis of tenocytes from tendons with degenerative tears.

Background: Rotator cuff repair is prone to incomplete regeneration. To explore biological improvements of tendon-bone healing, it was the aim of this study to investigate the influence of growth factors bone morphogenetic protein (BMP)-2 and BMP-7 on tenocyte cell activity and matrix gene expression and production. A beneficial effect of these factors would be promising to improve tendon-bone healing in vivo., Methods: Tenocyte-like cells were isolated from human rotator cuff tissue samples (supraspinatus and long head of biceps tendon) and incubated with BMP-2 (100-1000 ng/mL) and BMP-7 (100-2000 ng/mL), both alone and in combination. At days 0, 3, and 6, cell activity was assessed. At day 6, collagen type I production and the expression of several tendon-, bone-, and cartilage-related markers (collagen types I-III, osteocalcin, scleraxis) were evaluated., Results: Dose-dependent effects of both investigated growth factors on tenocyte-like cells were observed. Application of BMP-2 increased collagen type I production significantly but its expression only slightly. Cell activity was decreased in higher doses over time. For BMP-7, a significant increase in collagen type I production and expression, as well as increased cell activity, was observed. The addition of both factors resulted in decreased parameters when compared with BMP-7 alone. The expression of collagen types II and III, osteocalcin, and scleraxis was not significantly affected by application of BMPs., Conclusion: Besides the well-known effects of BMP-2 and BMP-7 on osteoblasts, this study describes further effects on rotator cuff tendon cell biology. Both tissue types potentially need to be addressed to improve tendon-bone healing of the rotator cuff., (Copyright © 2012 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.)

Degeneration of the rotator cuff tendon, which involves apoptosis of the tenofibroblasts, is one of the most common shoulder problems that can lead eventually to a full-thickness rotator cuff tendon tear. The current authors evaluated both the ability of anthocyanins, which are powerful antioxidants, to reduce apoptosis in oxidation-stressed rotator cuff tenofibroblasts, and the molecular mechanism for this antiapoptotic action. Anthocyanins demonstrated a dose-dependent ability to inhibit H(2)O(2)-induced apoptosis in cultured tenofibroblasts, as assessed by MTT assay and FACS analysis. H(2)O(2) increased the phosphorylation of extracellular regulated kinase1/2 (ERK1/2) and of c-Jun N-terminal kinase (JNK) and the production of reactive oxygen species (ROS). In contrast, treatment with anthocyanins decreased this activation of ERK1/2 and JNK, as confirmed by Western blot analysis, and reduced the production of ROS, as verified by fluorescent microscopic and FACS analyses. These findings suggest that anthocyanins, by suppressing JNK, ERK1/2, and intracellular ROS production, have a concentration-dependent antiapoptotic effect on rotator cuff tenofibroblasts exposed to an oxidative stressor, and may have therapeutic potential., ((c) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Rotator cuff tears are common soft tissue injuries of the musculoskeletal system that heal by formation of repair tissue and may lead to high retear rates and joint dysfunction. In particular, tissue from chronic, large tendon tears is of such degenerative nature that it may be prone to retear after surgical repair. Besides several biomechanical approaches, biologically based strategies such as application of growth factors may be promising for increasing cell activity and production of extracellular tendon matrix at the tendon-to-bone unit. As a precondition for subsequent experimental growth factor application, the aim of the present study was to establish and characterize a human rotator cuff tendon cell culture. Long head biceps (LHB)- and supraspinatus muscle (SSP)- tendon samples from donor patients undergoing shoulder surgery were cultivated and examined at the RNA level for expression of collagen type-I, -II and -III, biglycan, decorin, tenascin-C, aggrecan, osteocalcin, tenomodulin and scleraxis (by Real-time PCR). Finally, results were compared to chondrocytes and osteoblasts as control cells. An expression pattern was found which may reflect a human rotator cuff tenocyte-like cell culture. Both SSP and LHB tenocyte-like cells differed from chondrocyte cell cultures in terms of reduced expression of collagen type-II (p

The role of hormones in the pathogenesis of tendinopathy is not well recognised, even though the use of anabolic steroids is correlated with a higher incidence of spontaneous tendon ruptures. The aim of this study was to investigate the effects of dihydrotestosterone (DHT) on human tenocyte cultures from the intact supraspinatus tendon of male subjects. Cultured human tenocytes were seeded into culture plates at a density of 5 x 10(4) cells per well and incubated for 24 h. Then, 10(-9) M-10(-7) M DHT or Dulbecco's modified Eagle's medium (DMEM) only (control) was added to the culture plate wells. Cell morphology assessment and cell proliferation tests were performed 48, 72 and 96 h after DHT treatment. DHT-treated tenocytes showed an increased proliferation rate at DHT concentration higher than 10(-8) M. Differences in cell numbers between control and DHT-treated cells were statistically significant (P < 0.05) after 48 and 72 h of treatment with DHT concentrations of 10(-8) and 10(-7) M. The tenocytes treated with DHT (10(-8) and 10(-7) M) became more flattened and polygonal compared to control cells that maintained their fibroblast-like appearance during the experiment at each observation time. In conclusion, in vitro, progressive increasing concentration of DHT at doses greater than 10(-8) M had direct effects on male human tenocytes, increasing cell number after 48 and 72 h of treatment, and leading to a dedifferentiated phenotype after 48 h of treatment. This effect can be important during tendon-healing and repair, when active proliferation is required. Our results represent preliminary evidence for a possible correlation between testosterone abuse and shoulder tendinopathy.

Reparative strategies for the treatment of injuries to tendons, including those of the rotator cuff of the shoulder, need to address the formation of the cartilage which serves as the attachment apparatus to bone and which forms at regions undergoing compressive loading. Moreover, recent work indicates that cells employed for rotator cuff repair may need to synthesize a lubricating glycoprotein, lubricin, which has recently been found to play a role in tendon tribology. The objective of the present study was to investigate the chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells in monolayer and three-dimensional culture, and to compare the behavior with bone marrow-derived mesenchymal stem cells (MSCs). The results demonstrated that while tendon cells in various media, including chondrogenic medium, expressed lubricin, virtually none of the MSCs synthesized this important lubricating molecule. Also of interest was that the cartilage formation capacity of the tendon cells grown in pellet culture in chondrogenic medium was comparable with MSCs. These data inform the use of tendon cells for rotator cuff repair, including for fibrocartilaginous zones., ((c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Rotator cuff disease is becoming more prevalent and is a significant source of pain and disability. Current methods of treating rotator cuff dysfunction have inconsistent results and there is a high rate of recurrent tear and failure. This has prompted the search for novel solutions and the use of tissue engineered strategies and approaches. This article looks at the current state of tissue engineering methods for rotator cuff regeneration.

Background: Rotator cuff tears are a common cause of shoulder pain and impairment. Subacromial glucocorticoid injections are widely used for treatment of epiphenomenons of chronic impingement syndrome with the possible side effects of tendon rupture and impaired tendon healing., Methods: Using qRT-PCR, western blot, immunoflourescence, and measurement of 3H-thymidine uptake we investigated the effects of the crystalline glucocorticoid triamcinolone acetonide (TAA) when added to the culture medium of isolated human rotator cuff tendon cells., Results: After 2 weeks of incubation, the cells had lost their fibroblastic appearance and parallel orientation, which is characteristic of cellular degeneration in vivo. Moreover, expression and secretion of collagen I was strongly reduced, and there was a decrease in proliferation rate. Cell migration was blocked and the rate of expression of the matrix metalloproteinases MMP2, MMP8, MMP9, and MMP13 was reduced, but expression of TIMP1 (a tissue inhibitor of MMPs) was upregulated, indicating a reduction in the cellular capacity for tendon repair. In addition, changes in cellular differentiation were observed: the number of adipocytes increased and levels of the protein Sox9-a marker of differentiating and mature chondrocytes-were elevated in triamcinolone acetonide treated cells., Interpretation: These results may indicate that the use of TAA is one reason for weaker mechanical tendon properties and for the high rate of re-rupture after supraspinatus tendon repair.

Background: The effect of corticosteroids on tendon properties is poorly understood, and current data are contradictory and diverse. The biomechanical effect of steroids on rotator cuff tendon has not been studied, to our knowledge. The current study was undertaken to characterize the biomechanical effects of corticosteroid exposure on both uninjured and injured rat rotator cuff tendon., Methods: One hundred and twenty-three male Sprague-Dawley rats were randomly assigned to four groups: control (C), tendon injury (I), steroid exposure (S), and tendon injury plus steroid exposure (I+S). Unilateral tendon injuries consisting of a full-thickness defect across 50% of the total width of the infraspinatus tendon were created. Steroid treatment consisted of a single dose of methylprednisolone placed into the subacromial space. At one, three, and five weeks postoperatively, the shoulders were harvested and the infraspinatus tendon was subjected to biomechanical testing. Two specimens from each group were used for histological analysis., Results: At one week, maximum load, maximum stress, and stiffness were all significantly decreased in Group S compared with the values in Group C. Mean maximum load decreased from 37.9 N in Group C to 27.5 N in Group S (p < 0.0005). Mean maximum stress decreased from 18.1 MPa in Group C to 13.6 MPa in Group S (p < 0.0005). Mean stiffness decreased from 26.3 N/mm in Group C to 17.8 N/mm in Group S (p < 0.0005). At one week, mean maximum stress in Group I+S (17.0 MPa) was significantly decreased compared with the value in Group I (19.5 MPa) (p < 0.0005). At both the three-week and the five-week time point, there were no significant differences between Group C and Group S or between Group I and Group I+S with regard to mean maximum load, maximum stress, or stiffness. Histological analysis showed fat cells and collagen attenuation in Groups S and I+S. These changes appeared to be transient., Conclusions: A single dose of corticosteroids significantly weakens both intact and injured rat rotator cuff tendons at one week. This effect is transient as the biomechanical properties of the steroid-exposed groups returned to control levels by three weeks.

Outcomes obtained in patients with two-tendon rotator cuff tear submitted to repair reinforced with porcine small intestinal submucosa (SIS) have not been as encouraging as those observed in animal models. We verify the capacity of SIS to be used as a physical support for a culture of cuff cells. During arthroscopic repairs of large rotator cuff tears, we removed a fragment of supraspinatus tendon. Samples were treated for obtaining a cuff cell culture. Daily microscopic analysis, to observe adhesion to substrate, replication and cell shape was performed. A confluent monolayer was obtained in 1 week. Cells at the second passage were collected and seeded onto scaffold and cultured for 7-30 days. A morphological and immunohistochemical evaluation was performed. After 1 week, a monolayer of tendinous-like cells lay along the surface of the SIS. Within two weeks, a multicellular layer was observable in many foci of the scaffold. After a month, the cells completely invaded the numerous splits of the SIS and were positive to monoclonal anti-type I collagen antibody. Our experimental study has proved that a cuff cell culture can be performed using SIS as substrate. The culture covers the SIS surface, therefore it may reduce immune or non-specific inflammatory reactions.

The debilitating effects of rotator cuff tears and the high incidence of failure associated with current grafts underscore the clinical demand for functional solutions for tendon repair and augmentation. To address this challenge, we have designed a poly(lactide-co-glycolide) (PLGA) nanofiber-based scaffold for rotator cuff tendon tissue engineering. In addition to scaffold design and characterization, the objective of this study was to evaluate the attachment, alignment, gene expression, and matrix elaboration of human rotator cuff fibroblasts on aligned and unaligned PLGA nanofiber scaffolds. Additionally, the effects of in vitro culture on scaffold mechanical properties were determined over time. It has been hypothesized that nanofiber organization regulates cellular response and scaffold properties. It was observed that rotator cuff fibroblasts cultured on the aligned scaffolds attached along the nanofiber long axis, whereas the cells on the unaligned scaffold were polygonal and randomly oriented. Moreover, distinct integrin expression profiles on these two substrates were observed. Quantitative analysis revealed that cell alignment, distribution, and matrix deposition conformed to nanofiber organization and that the observed differences were maintained over time. Mechanical properties of the aligned nanofiber scaffolds were significantly higher than those of the unaligned, and although the scaffolds degraded in vitro, physiologically relevant mechanical properties were maintained. These observations demonstrate the potential of the PLGA nanofiber-based scaffold system for functional rotator cuff repair. Moreover, nanofiber organization has a profound effect on cellular response and matrix properties, and it is a critical parameter for scaffold design.

Nitric oxide (NO) has a variety of physiological roles, including acting as a key mediator in various phases of tendon healing, but its importance as a modulator of gene expression during tendon healing has not been well studied. The current study used microarray analysis to elucidate global gene expression after transfection with inducible nitric oxide synthase (iNOS) in tenocytes isolated from the injured rotator cuff tendons of human patients. We show that the expression of a wide range of genes is affected by NO, with many activated genes having known roles in healing. Of particular significance is that NOS overexpression stimulates the transcription and translation of a range of extracellular matrix genes important to the structure of connective tissues such as tendons, including collagen Ialpha1, collagen IIIalpha1, collagen IValpha5, biglycan, decorin, laminin, and matrix metalloproteinase 10 (MMP10). These genes were also shown to respond to stimulation by the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) in a dose-dependent manner. We further show that varying levels of NO significantly affect cellular adhesion in tenocytes, a critical process during tendon repair. These findings will be of use when optimizing the dose of NO delivery in further work investigating NO as potential treatment of tendon injuries.

Several studies have demonstrated that inflammation in the subacromial bursa is an important component in the pathogenesis of impingement syndrome. We have demonstrated in a previous study that many inflammatory cytokines, including stromal cell-derived factor 1 (SDF-1, CXCL12), are increased in the subacromial bursa [Blaine et al. 2005. J Shoulder Elbow Surg 14(Suppl 1):84S-89S]. SDF-1 is a potent chemotactic and angiogenic factor that stimulates recruitment of inflammatory cells. In the current study, we proposed that the resident cells in subacromial bursal tissue produce SDF-1, which can play a role in the inflammatory reponse of bursal tissue, and that this chemokine can be regulated by steroid (dexamethasone) and nonsteroidal anti-inflammatory medications (NSAIDs). Twenty-two subacromial bursa tissues (18 bursitis and 4 normal bursa) were obtained intraoperatively from patients during shoulder surgery and analyzed using the cDNA Array technique in accordance with an IRB approved protocol. cDNA array results were confirmed with real-time reverse transcription-polymerase chain reaction (RT-PCR). Bursal cells (from 4 normal bursa, 3 bursitis) and two normal bone marrow with whole tissue explants were cultured for one passage. Cell culture supernatants were collected and SDF-1 protein was detected with enzyme-linked immunosorbent assay (ELISA). Cultured bursal cells were treated with a COX-2 inhibitor and dexamethasone, and cells was harvested at 1-day and 4-day intervals. SDF-1 expression was evaluated by real-time RT-PCR and ELISA. cDNA Array analysis demonstrated that the gene expression of SDF-1 was increased in patients with subacromial bursitis compared to controls (p < 0.05). Real-time RT-PCR also revealed that the mRNA expression of SDF-1 in bursitis tissue is increased 10-fold over control tissue. While the normal bursal cells produced negligible amounts of SDF-1 protein, cultured cells derived from bursitis lesion released as much SDF-1 protein (235 pg/100,000 cells) as normal bone marrow stromal cells (283 pg/100,000 cells) as measured by ELISA. The addition of a COX-2 inhibitor and dexamethasone to bursitis cell lines led to decreased SDF-1 expression levels compared to untreated bursitis cell lines. These studies demonstrate that there is a significant elevation of SDF-1 expression in the subacromial bursa of patients with rotator cuff disease. Furthermore, this chemokine can be downregulated by COX-2 inhibitors and steroids. These results provide biologic evidence for the use of steroid and NSAIDs in the treatment of subacromial bursitis. In the future, targeted inhibition of molecules such as SDF-1 in the subacromial bursa may present a therapeutic strategy that may avoid the side effects of these other (steroid and NSAID) medications.

Collagen deposition is an important process that occurs during wound healing. We and others have shown that nitric oxide (NO) is important in tendon healing. The mechanisms whereby healing is enhanced are, however, undetermined. The aim of this study was to investigate whether NO could enhance collagen synthesis in cultured human tendon cells via exogenous NO and via an adenovirus containing the gene for inducible nitric oxide synthase (Ad-iNOS). Tendon cells from the torn edge of the tendons of patients undergoing rotator cuff repair surgery were cultured following collagenase digestion, and stimulated with exogenous NO (SNAP), transfected with Ad-iNOS, and treated with the NOS inhibitor, L-NMMA. Total protein and collagen synthesis were evaluated by (3)H-proline and collagenase sensitive (3)H-proline incorporation in human tendon cells. High doses of exogenous NO (SNAP) inhibited collagen synthesis. Lower doses enhanced total protein and collagen synthesis of the tendon cells. Ad-iNOS successfully transfected active iNOS into human tendon cells in vitro and also enhanced total protein and collagen synthesis of the tendon cells. The NOS inhibitor, L-NMMA, inhibited the effects of iNOS on the cells. Our studies show for first time that nitric oxide can enhance collagen synthesis in human tendon cells in vitro. These results may explain, in part, at least, the beneficial effects of NO donors in animal models and during the treatment of tendonopathies in human clinical trials. ., ((c) 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.)

Adenovirus-mediated gene transfer is a potential method for enhancing tendon healing. We investigated the transfection of Ad5CMVntLacZ, an adenovirus containing the reporter gene LacZ, in primary cultured human rotator cuff tendon cells and in a rat Achilles tendon healing model in vivo. Ad5CMVempty, the adenoviral vector containing no inserted gene, was used as a control for adenoviral transfection alone. Activity of beta-galactosidase,the protein expressed by LacZ gene, was measured using a beta-galactosidase assay and detected visually by X-gal staining. Cultured cells were successfully transfected without impairing cell viability. Maximal beta-galactosidase activity was detected when cells were transfected at the dose of 1000 PFU/cell. The duration of LacZ expression was six days with a peak value at 24 h post-transfection. A transfection rate of 100% was obtained at 5000 PFU/cell. Successful in vivo transfection by Ad5CMVntLacZ was obtained in healing rat Achilles tendon as confirmed by X-gal staining. 0.4% of tendon cells were transfected when Ad5CMVntLacZ was injected into the tendon at a dose of 10(6) PFU. The rate rose to 2% with 10(8) PFU and 3% with 10(9) PFU. The duration of LacZ expression in vivo was 17 days. Transfection efficiency was enhanced threefold and localization improved when a gelatin sponge was used to deliver the adenovirus. The results demonstrate that adenovirus can be used to deliver a gene of interest to cultured human rotator cuff tendon cells and healing tendon, with gelatin sponge implantation enhancing adenoviral transfection efficiency in vivo.

The rat shoulder animal model has been used previously to study the role of intrinsic injury (modeled as an acute insult to the tendon), extrinsic injury (modeled as external subacromial impingement), and overuse factors on rotator cuff tendinosis. These studies demonstrated that it is possible to produce rotator cuff tendinosis with any one of these factors in isolation. The current study uses the rat shoulder model to study the roles of extrinsic compression, overuse, and overuse in combination with extrinsic compression, on the development of rotator cuff tendinosis. The results of this study demonstrate that the injury created by overuse plus extrinsic compression is greater than the injuries created by overuse or extrinsic compression alone, particularly when important biomechanical variables are considered. While ineffective in causing a change in supraspinatus tendon properties in animals with normal cage activity, extrinsic compression had a significant and dramatic effect when it was combined with overuse activity. Without an additional factor, such as overhead activity, the extrinsic compression alone may be insufficient to cause tendinosis. The results of the present study support the role of multiple factors in the etiology of some rotator cuff injuries.

The topography of muscle spindles and Golgi tendon organs in the rotator cuff and surrounding shoulder muscles of a small laboratory marsupial (monodelphis domestica) were studied using light microscopy of serial sections. The shoulder joint of monodelphis has a large degree of freedom of movement allowing this animal to use the upper extremities for a wide range of activities like climbing and manipulating food. Thus, similar to the situation in man the shoulder joint is mainly secured by muscles. Silver stained serial paraffin sections were examined under the light microscope and the distribution of muscle spindles and Golgi tendon organs was reconstructed using three-dimensional image processing. In the two animals examined 113 and 131 muscle spindles respectively were found within the 4 rotator cuff muscles. In addition, 76 and 40 Golgi tendon organs respectively were seen at the musculo-tendinous junctions of these muscles preferentially close to the insertion at the humerus head. Also the surrounding shoulder muscles contain both muscle spindles and Golgi tendon organs in large numbers, but the ratio of Golgi tendon organs per muscle spindle appears to be lower. Number and localization of muscle spindles and Golgi tendon organs suggest, that these receptors are important for both reflex control of shoulder muscle tone as well as monitoring of static position and movement in the shoulder joint.