Nuclear lamin A in rotator cuff tear margin tenocytes: an antiapoptotic and cell mechanostat factor.

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.

Gene expression profiling of progenitor cells isolated from rat rotator cuff musculotendinous junction.

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.

Bioactive LbL-assembled multilayer nanofilms upregulate tenogenesis and angiogenesis enabling robust healing of degenerative rotator cuff tendons in vivo.

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-ß, 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.

Reduced Myogenic and Increased Adipogenic Differentiation Capacity of Rotator Cuff Muscle Stem Cells.

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.

Engineered tendon-fibrocartilage-bone composite and bone marrow-derived mesenchymal stem cell sheet augmentation promotes rotator cuff healing in a non-weight-bearing canine model.

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.

The effect of autologous platelet rich plasma on tenocytes of the human rotator cuff.

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-ß1, 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-ß1), 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-ß1 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.

Leukocyte-reduced platelet-rich plasma increases proliferation of tenocytes treated with prednisolone: a cell cycle analysis.

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.

Cell Toxicity in Fibroblasts, Tenocytes, and Human Mesenchymal Stem Cells-A Comparison of Necrosis and Apoptosis-Inducing Ability in Ropivacaine, Bupivacaine, and Triamcinolone.

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 × 104/cm2. 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, 104 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.

Rotator cuff repair using cell sheets derived from human rotator cuff in a rat model.

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.

Impact of cyclic mechanical stimulation on the expression of extracellular matrix proteins in human primary rotator cuff fibroblasts.

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.

Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold.

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.

The response of cells derived from the supraspinatus tendon to estrogen and calciotropic hormone stimulations: in vitro study.

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-17ß (E2); soy isoflavones (daidzein, genistein, biochainin A); raloxifene and estrogen receptors α and ß 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 ß, 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-ß; however, estrogen receptor ß antagonists did not have an effect. Scleraxis expression decreased following estradiol 17-ß and vitamin-D treatments. Vitamin-D significantly reduced collagen-1 expression, while estradiol 17-ß had no effect. Vitamin-D and estradiol 17-ß 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 ß. 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.

Characterization of bursa subacromialis-derived mesenchymal stem cells.

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.

Do patient age and sex influence tendon cell biology and clinical/radiographic outcomes after rotator cuff repair?

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.

Isolation of mesenchymal stem cells from shoulder rotator cuff: a potential source for muscle and tendon repair.

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.

Nicotine reduced MMP-9 expression in the primary porcine tenocytes exposed to cyclic stretch.

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.

Increased levels of apoptosis and p53 in partial-thickness supraspinatus tendon tears.

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.

BMP-2 and BMP-7 affect human rotator cuff tendon cells in vitro.

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.

Characterization of tendon cell cultures of the human rotator cuff.

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

Effect of dihydrotestosterone on cultured human tenocytes from intact supraspinatus tendon.

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.