Oxidative stress induces senescence in chondrocytes

Cellular senescence is a program activated during diverse situations of cell stress. Chondrocytes differ from other somatic cells as articular cartilage is an avascular tissue. The effects of oxidative stress on chondrocytes are still unknown. Our studies were to investigate into the proliferation potential, cytological features and the telomere linked stress response system of human osteoarthritic chondrocytes, subjected to acute or prolonged oxidant challenge with hydrogen peroxide. Telomere length was measured using the telomere restriction fragment assay, gene expression was determined by RT‐PCR. Sub‐lethal doses of oxidative stress induced cell‐cycle arrest, senescent‐morphological features and senescence‐associated β‐galactosidase positivity. Prolonged oxidative treatment had no effects on cell proliferation or morphology. Sub‐lethal and prolonged low doses of oxidative stress considerably accelerated telomere attrition. The effects of sub‐lethal oxidative stress regarding proliferation and telomere biology were more distinct in senescent cells. Acute oxidant insult caused up‐regulation of p21 expression to levels comparable to senescent cells. TRF2 protects telomere ends and showed elevated expression levels. SIRT1 and XRCC5 enable cells to cope with unfavorable growing conditions. Both were up‐regulated after oxidant insult, but expression levels decreased in aging cells. Taken together, oxidative stress considerably accelerated telomere shortening and cellular aging in chondrocytes. Senescent cells showed a reduced tolerance to oxidative stress. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1114–1120, 2011     

MGF enhances tenocyte invasion through MMP‐2 activity via the FAK‐ERK1/2 pathway

Tendon regeneration and healing requires tenocytes to move to the repair site followed by proliferation and synthesis of the extracellular matrix. A novel synthetic growth factor, mechano‐growth factor (MGF), has been discovered to have positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury‐induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. In this study, using a transwell system, we found that MGF‐C25E (a synthetic mechano‐growth factor E peptide) significantly promotes tenocyte invasion, which was accompanied by the increased phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2) as well as the increased activity of matrix metalloproteinases‐2 (MMP‐2). The MMP‐2 inhibitor OA‐Hy blocked MGF‐C25E‐promoted tenocyte invasion. Inhibitors of FAK or ERK1/2 blocked MGF‐C25E‐promoted tenocyte invasion and MMP‐2 activity as well. These results indicate that MGF‐C25E promotes tenocyte invasion by increasing MMP‐2 activity via the FAK‐ERK1/2 signaling pathway. Taken together, our findings provide the first evidence that MGF‐C25E enhances tenocyte invasion and indicate that it may serve as a potential repair material for promoting the healing and regeneration of injured tendons.     

Cellular senescence occurring in the rabbit medial collateral ligament during healing

Medial collateral ligament (MCL) healing proceeds in a temporally ordered fashion after injury. Despite the critical roles of fibroblasts during ligament repair, the phenotypic features of these healing fibroblasts have not been well characterized. Here, we show that healing MCL fibroblasts obtained from rabbits at 3‐week postinjury exhibited higher rates of senescent phenotypes and produced higher levels of TGF‐β1, collagens, α‐SMA, and matrix metalloproteinases (MMPs), than the corresponding fibroblasts from sham‐operated MCLs. Mechanical stretch further enhanced the cellular senescence and the expression of TGF‐β1, collagens, α‐SMA, and MMPs in both sham and healing MCL fibroblasts. In addition to MCL fibroblasts at 3‐week postinjury, the increased cellular senescence was also detected in healing MCL fibroblasts obtained at 4‐ and 6‐week postinjury. Most importantly, the association between the cellular senescence and ligament healing was confirmed in tissue sections by the senescence‐associated β‐galactosidase (SA‐β‐gal) staining. Using a recombinant TGF‐β1 and a neutralizing antibody, we found that those phenotypic changes, such as cellular senescence and the expression of collagens and MMPs, in MCL fibroblasts under mechanical loading conditions were regulated through TGF‐β1. Taken together, our results propose that cellular senescence and turnover of extracellular matrixes regulated by TGF‐β1 in MCL fibroblasts are critical for ligament healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:81–90, 2012     

Regenerative and protective effects of calcium silicate on senescent fibroblasts induced by high glucose

Diabetic wounds are a common complication of diabetes and therefore a pressing issue for clinicians. High‐glucose (HG)‐induced fibroblast senescence is mainly responsible for delayed wound healing. Calcium silicate (CS), a kind of bioceramic, is thought to have regenerative properties. The aim of this study was to determine the regenerative and protective effects of CS on senescent fibroblasts induced by HG. Fibroblasts were passaged five times and treated with HG and CS. Compared with the normal glucose (NG) group, the proliferation, migration, and differentiation capacity of HG‐induced fibroblasts significantly decreased (P < .05). After treatment with CS, the functions of HG‐induced senescent fibroblasts were partly restored (P < .05). The mechanism of the regenerative and protective effects of CS may be related to the decreased reactive oxygen species generation, improved senescent state (SA‐β‐gal expression decreased), up‐regulated expression of Smad2 and phosphorylated Smad2, and down‐regulated expression of p16, p21, and p53. An in vivo experiment also demonstrated that CS had a therapeutic effect on diabetic wounds via differentiation of fibroblasts into myofibroblasts and enhanced collagen deposition. These results indicate that CS may be a promising candidate for diabetic wound therapy.     

屈肌腱愈合过程中产生的相对分子质量为45 000的蛋白对腱细胞生长的作用

目的 了解肌腱愈合过程中产生的相对分子质量为45000的蛋白对腱细胞生长、增殖的作用。方法 按Handerson分步酶消化法分离刚孵化的Leghorn鸡的践屈肌腱腱细胞。选用F－12培养基，加入不同浓度的肌腱愈合过程中产生的相对分子质量为45000的蛋白，与腱细胞进行原代培养。通过绘制细胞生长曲线及MTT比色试验，观察该蛋白对腱细胞的作用。结果 屈肌腱愈合过程中产生的相对分子质量为45000的蛋白，可促进腱细胞的增殖，其增殖作用与该蛋白的浓度在一定范围内呈量效关系。结论 屈肌腱愈合过程中产生的相对分子质量为45000的蛋白，对腱细胞的增殖有显著的促进作用。     

Down‐regulation of mir‐27b promotes angiogenesis and fibroblast activation through activating PI3K/AKT signaling pathway

To study the effects of mir‐27b on angiogenesis and fibroblast activation and to explore its further mechanism. Humanmicrovascular endothelial cell (HMEC)‐1 and humannormal skin fibroblast (BJ) cells were treated with mir‐27b inhibitor negative control reagent, mir‐27b inhibitor, LY294002, and mir‐27b inhibitor + LY294002, respectively. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) was used to detect the T‐cell proliferation. The migration ability was detected by Scratch assays. The angiogenesis of HMEC‐1 cells was observed by in vitro tube formation assay. The mRNA and protein expression of vascular endothelial growth factor (VEGF) in HMEC‐1 cells and the mRNA and protein expression of collagen I, collagen III, α‐SMA, and MMP1 in BJ cells were detected by quantitativereal‐time polymerase chain reaction (qRT‐PCR) and Western blot, respectively. Meanwhile, the PI3K/protein kinase B (AKT) pathway‐related proteins were also detected by Western blot. The proliferation, migration, angiogenesis, the mRNA and protein expression of VEGF and the protein expression of p‐PI3K and p‐AKT in HMEC‐1 cells were increased after treated with mir‐27b inhibitor. Meanwhile, the proliferation, migration, and the protein expression of collagen I, collagen III, α‐SMA, MMP1, p‐PI3K, and p‐AKT in BJ cells were increased after treated with mir‐27b inhibitor. However, the angiogenesis and fibroblast activation of mir‐27b inhibitor was reversed by LY294002, and the activate effect to PI3K/AKT pathway was also inhibited. Down‐regulation of mir‐27b could promote angiogenesis and fibroblast activation, and its mechanism is related to activate PI3K/AKT signaling pathway.     

Low-level laser irradiation stimulates tenocyte proliferation in association with increased NO synthesis and upregulation of PCNA and cyclins

Low-level laser therapy is commonly used to treat tendinopathy or tendon injury. Tendon healing requires tenocyte migration to the repair site, followed by proliferation and synthesis of the extracellular matrix. There are few evidence to elucidate that low-level laser promote tenocyte proliferation. This study was designed to determine the effect of laser on tenocyte proliferation. Furthermore, the association of this effect with secretion of nitric oxide (NO) and the expressions of proliferating cell nuclear antigen (PCNA) and cyclins D1, E, A, and B1 was investigated. Tenocytes intrinsic to rat Achilles tendon were treated with low-level laser (660 nm). Tenocyte proliferation was evaluated by MTT assay and immunocytochemistry with Ki-67 stain. NO in the conditioned medium was measured by ELISA. Western blot analysis was used to evaluate the protein expressions of PCNA and cyclins D1, E, A, and B1. The results revealed that tenocytes proliferation was enhanced dose dependently by laser. NO secretion was increased after laser treatment. PCNA and cyclins E, A, and B1 were upregulated by laser. In conclusion, low-level laser irradiation stimulates tenocyte proliferation in a process that is mediated by upregulation of NO, PCNA, and cyclins E, A, and B1.     

Biologic behavior of an in vitro hydrated collagen gel-human tenocyte tendon model

An in vitro human tenocyte-collagen gel model was developed to study tenocyte-mediated Type I collagen fibril reorganization, proliferation, and Type I collagen gene expression. Human tenocytes, obtained from extrasynovial forearm flexor tendons from children 5 to 10 years of age were cultured on plastic or in a cylinder of hydrated Type I collagen gel. Collagen solution was seeded with human tenocytes at 5 x 10(5) cells/mL and gelled in cylinder molds; gel cylinders without human tenocytes served as controls. Gel cylinders were pinned to troughs to create noncompliance. The gel cylinders were analyzed for collagen birefringence and cell shape at 7 and 21 days and for proliferation and gene expression for Type I collagen at 7 days. Under conditions of noncompliance, human tenocytes reoriented Type I collagen into longitudinal bundles resembling the parallel organization of collagen in native tendons. Tenocyte shape became fusiform between the collagen bundles which mimics the morphologic features of a tenocyte in vivo. The structural changes in the tenocytes and matrix are accompanied by downregulation of human tenocyte proliferation and Type I collagen gene expression. When released from the gel cylinder and grown again on plastic, human tenocytes resume proliferation and Type I collagen gene expression. The human tenocytes in this in vitro gel cylinder model system control fibril reorganization and proliferation, resembling their behavior during the development and repair of native tendons.     

Mechanism of mast cell adhesion to human tenocytes in vitro

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell‐fibroblast co‐culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte‐conditioned media; the tenocyte‐derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte‐derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte‐derived ECM proteins, in particular fibronectin. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:9–16, 2015.

     

Extracellular matrix expression of human tenocytes in three‐dimensional air–liquid and PLGA cultures compared with tendon tissue: Implications for tendon tissue engineering

Tenocyte transplantation may prove to be an approach to support healing of tendon defects. Cell–cell and cell–matrix contacts within three‐dimensional (3D) cultures may prevent tenocyte dedifferentiation observed in monolayer (2D) culture. The present study compares both neotissue formation and tenocyte extracellular matrix (ECM) expression in 2D and 3D cultures directly with that of native tendon, in order to determine optimal conditions for tendon tissue engineering. Primary human tenocytes were embedded in poly[lactic‐co‐glycolic‐acid] (PLGA)‐scaffolds and high‐density cultures. Neotissue formation was examined by hematoxyline–eosine (H&E) and immunofluorescence staining. Gene expression of ECM proteins and vascular endothelial growth factor (VEGF) was compared at days 0 (2D), 14, and 28 in 3D cultures and tendon. Histomorphology of 3D culture showed tendon‐like tissue as tenocyte cell nuclei became more elongated and ECM accumulated. Type I collagen gene expression was higher in 2D culture than in tendon and decreased in 4‐week‐old 3D cultures, whereas type III collagen was only elevated in high‐density culture compared with tendon. Decorin and COMP were reduced in 2D and increased in 3D culture almost to ex vivo level. These results suggest that the 3D high‐density or biodegradable scaffolds cultures encourage the differentiation of expanded monolayer tenocytes in vitro to tendon‐like tissue. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1170–1177, 2010     

Identification of Senescent Cells in the Bone Microenvironment

Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16^Ink4a, profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence‐associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age‐related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16^Ink4a expression by real‐time quantitative polymerase chain reaction (rt‐qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence‐associated distension of satellites (SADS), ie, large‐scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction‐induced foci (TIFs) than osteocytes from young mice. Corresponding with the age‐associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age‐associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age‐related bone loss. © 2016 American Society for Bone and Mineral Research     

Modified house‐keeping gene expression in a rat tail compression loading‐induced disc degeneration model

House‐keeping genes (HKGs) are generally used as endogenous controls for molecular normalization in quantitative PCR analysis. However, whether all the so‐called HKGs are useful for intervertebral disc research is controversial. Our objective was, using a prevalidated rat tail static compression loading‐induced disc degeneration model, to clarify the feasibility of common HKGs for gene‐quantification in the nucleus pulposus cells. In real‐time RT‐PCR for five HKGs [β‐actin, β‐glucuronidase, β‐2 microglobulin, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), and lactate dehydrogenase A (LDHA)], static compression at 1.3 MPa for up to 56 days demonstrated messenger RNA (mRNA) expression levels of consistent β‐2 microglobulin and GAPDH, slightly up‐regulated β‐glucuronidase, and fairly down‐regulated β‐actin and LDHA. Especially, β‐actin had a drastic suppression to 0.15‐fold in the loaded relative to unloaded discs at 7 days. In immunofluorescence, β‐actin showed a significant down‐regulation to almost undetectable levels from 28 days, while GAPDH was constantly detected throughout. β‐Actin mRNA and protein‐distribution are thought to be affected by the loading treatment, however, GAPDH mRNA and protein‐distribution can retain relatively stable expressions. Under prolonged static compression, β‐actin and probably LDHA are inappropriate, and GAPDH is a feasible HKG as internal references in the disc cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1284–1290, 2011     

Role of pulsed electromagnetic fields (PEMF) on tenocytes and myoblasts—potential application for treating rotator cuff tears

The post‐surgery integrity of the tendons and muscle quality are the two major factors in success of rotator cuff (RC) repair. Though surgical techniques for rotator cuff repair have significantly improved in the past two decades, there are no effective treatments to improve tendon‐to‐bone healing and muscle quality after repair at this point in time. Pulsed electromagnetic fields (PEMF) have previously been used for promoting fracture healing. Previous studies have shown that PEMF has a positive role in promoting osteoblast precursors proliferation and differentiation. However, PEMFs effect on tenocytes and muscle cells has not been determined fully yet. The purpose of this study is to define the role of a commercially available PEMF on tenocytes and myoblasts growth and differentiation in vitro. Human rotator cuff tenocytes and C2C12 murine myoblasts were cultured and treated with PEMF for 2 weeks under regular and inflammatory conditions. Our results showed that 2 weeks treatment of PEMF enhanced gene expressions of growth factors in human rotator cuff tenocytes under inflammatory conditions. PEMF significantly enhanced C2C12 myotube formation under normal and inflammatory conditions. Results from this study suggest that PEMF has a positive role in promoting tenocyte gene expression and myoblast differentiation. Therefore, PEMF may potentially serve as a non‐operative treatment to improve clinical incomes rotator cuff tendon repairs. Results © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:956–964, 2017.

     

Three‐dimensional high‐density co‐culture with primary tenocytes induces tenogenic differentiation in mesenchymal stem cells

Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and tissue engineering and may represent an attractive option for tendon repair and regeneration. Thus far the ability of MSCs to differentiate into tenocytes in vitro has not been investigated. Experiments were performed with and without growth factors (IGF‐1, TGF‐β1, IGF‐1/TGF‐β1, PDGF‐BB, and BMP‐12), in co‐cultures of tenocytes and MSCs mixed in different ratios and by culturing MSCs with spent media obtained from primary tenocytes. Tenogenesis was induced in MSCs through a combination of treatment with IGF‐1 and TGF‐β1, in high‐density co‐cultures and through cultivation with the spent media from primary tenocytes. Electron microscopy and immunoblotting were used to demonstrate up‐regulation of collagen I/III, decorin, tenomodulin, β1‐Integrin, MAPKinase pathway (Shc, Erk1/2), and scleraxis in the co‐cultures and provide simultaneous evidence for the inhibition of apoptosis. In monolayer co‐cultures extensive intercellular contacts between MSCs and tenocytes were observed. Cells actively exchanged vesicles, which were labeled by using immunofluorescence and immunogold techniques, suggesting the uptake and interchange of soluble factors produced by the MSCs and/or tenocytes. We conclude that MSCs possess tenogenic differentiation potential when provided with relevant stimuli and a suitable microenvironment. This approach may prove to be of practical benefit in future tissue engineering and tendon regenerative medicine research. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1351–1360, 2011     

Paracrine anti‐fibrotic effects of neonatal cells and living cell constructs on young and senescent human dermal fibroblasts

Senescent cells observed in the area of chronic wounds have been proposed to affect wound healing. Therapeutic approaches against chronic wounds include, among others, the local application of living cell constructs (LCCs), containing fibroblasts and/or keratinocytes. Accordingly, the aim of the present work was to examine the effects of factors secreted by early passage neonatal fibroblasts and LCCs—in the form of a conditioned medium (CM)—on senescent adult dermal fibroblasts regarding functions related to the healing process, i.e., cell proliferation, alpha‐smooth muscle actin and metalloproteinase expression, and collagen synthesis. Target cells were fibroblasts senescent either due to subsequent divisions (replicative senescence) or due to an exogenous stress (stress‐induced premature senescence). No effect on the proliferation of senescent fibroblasts was observed, as expected. All CMs were found to inhibit overall collagen synthesis both in early passage and in senescent fibroblasts. The LCC‐derived CM was found to be more potent than fibroblast‐derived CMs and, furthermore, to inhibit alpha‐smooth muscle actin expression. In conclusion, these results may indicate anti‐contractile and anti‐fibrotic activities of factor(s) secreted by neonatal skin fibroblasts, and more intensely by LCCs on adult donor‐derived fibroblasts. These activities seem to persist during senescence of the target cells.     

Proliferation and differentiation of human tenocytes in response to platelet rich plasma: an in vitro and in vivo study

Platelet rich plasma (PRP) is the autologous plasma fraction with a platelet-rich cellular component which is enriched with a number of growth factors. Due to its availability and low cost, PRP has become an increasingly popular clinical tool as an alternative source of growth factors for various applications, for example, tendon regeneration but with limited success in clinical trials. The main objective of the current study was to determine whether activated PRP [i.e., platelet rich plasma-clot release (PRCR)] could be used to induce the proliferation and collagen synthesis in human tenocyte in vitro. The advantage of using PRCR is that the platelet-derived bioactive factors are more concentrated and could initiate a more rapid and accelerated healing response than PRP. Our results demonstrated that 10% PRCR treatment accelerated the extent of cell proliferation and collagen production by human tenocytes in vitro. The expression of specific tenocyte markers were similar to conventional fetal bovine serum (FBS)-treated tenocytes implanted in mice within 14 days of implantation in diffusion chambers. Moreover, relatively more collagen fibrils were evident in PRCR-treated tenocytes in vivo as compared to 10% FBS-treated cells. Overall, our feasibility study has indicated that PRCR can induce human tenocyte proliferation and collagen synthesis which could be implemented for future tendon regeneration in reconstructive surgeries.     

Restoration of Cellular Proliferation and Characteristics of Human Tenocytes by Vitamin D

Vitamin D (Vit D) increases calcium absorption in the intestine after binding to the Vit D receptor (VDR). The VDR has also been identified in muscle cells. Vit D supplementation resulted in improved muscle strength. However, there is a paucity of studies of the role of Vit D on tenocytes. We investigated the effects of Vit D on damaged tenocytes. Human tenocytes were treated with dexamethasone (Dex) to induce cell injury. Expression of the tenocyte‐related markers tenomodulin (Tnmd), tenascin C (Tnc), scleraxis (Scx), mohawk (Mkx), and collagen (Col) 1 and 3 were measured. Then, tenocytes were cotreated with Vit D. 1‐α‐Hydroxylase and VDR were explored in tenocytes. With 10 μM Dex, the growth of tenocytes was significantly inhibited, and the gene expression of Tnmd, Tnc, Scx, Mkx, Col 1 and 3 also decreased. When tenocytes were cotreated with Vit D, cell proliferation recovered in a dose‐dependent manner, and the expression of TNMD and Col 1 improved. When studying the mechanisms of the effects of Vit D on tenocytes, reactive oxygen species produced by Dex decreased with Vit D, and the phosphorylation of extracellular signal–regulated kinase and p38 was stimulated by Vit D cotreatment. 1‐α‐Hydroxylase and VDR were found in tenocytes, indicating that the cells have the ability to use an inactive form of Vit D and interact with it. Vit D is known to perform diverse actions and its protective effects on tenocytes suggest its beneficial role in tendon in addition to muscle and bone. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2241–2248, 2019