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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.

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) 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.)

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.

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.