Is cultured tendon fibroblast a good model to study tendon healing?

Cultured tendon fibroblasts (CTFs) from intact explants are widely used to study tendon healing in vitro. The significance of these findings may rely on similarities between CTFs and healing tendon fibroblasts in situ. Our purpose was to compare CTFs with fibroblasts cultured from healing tendons. We cultured CTFs from intact and healing tendons at day 7 and day 14 postinjury in a rat model of patellar donor site injury. The mRNA expression of COL1A1, COL3A1, decorin, and biglycan, with or without supplementation of 1 ng/mL TGF‐β1, was compared by quantitative real‐time RT‐PCR. The expression of proliferation cell nuclear antigen (PCNA) and α‐smooth muscle actin (α‐SMA) was determined by immunostain. COL3A1 and decorin mRNA in CTFs was lower as compared to day 7 healing fibroblasts, but its biglycan mRNA level was higher than day 14 healing fibroblasts. TGF‐β1 increased COL1A1 and decorin mRNA in CTFs, but decreased the mRNA of all four genes in day 7 healing tendon fibroblasts. CTFs exhibited lower PCNA immunopositivity as compared to day 7 and day 14 healing fibroblasts, but a higher α‐SMA immunopositivity than cultured day 14 healing fibroblasts. These findings showed that CTFs did not resemble healing tendon cells with respect to major cellular activities related to tendon healing. Thus, fibroblasts from healing tendon may be a more appropriate model for studying cellular activities in tendon healing. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:374–383, 2008     

The role of the macrophage in tendinopathy and tendon healing

The role of the macrophage is an area of emerging interest in tendinopathy and tendon healing. The macrophage has been found to play a key role in regulating the healing process of the healing tendon. The specific function of the macrophage depends on its functional phenotype. While the M1 macrophage phenotype exhibits a phagocytic and proinflammatory function, the M2 macrophage phenotype is associated with the resolution of inflammation and tissue deposition. Several studies have been conducted on animal models looking at enhancing or suppressing macrophage function, targeting specific phenotypes. These studies include the use of exogenous biological and pharmacological substances and more recently the use of transgenic and genetically modified animals. The outcomes of these studies have been promising. In particular, enhancement of M2 macrophage activity in the healing tendon of animal models have shown decreased scar formation, accelerated healing, decreased inflammation and even enhanced biomechanical strength. Currently our understanding of the role of the macrophage in tendinopathy and tendon healing is limited. Furthermore, the roles of therapies targeting macrophages to enhance tendon healing is unclear. Clinical Significance: An increased understanding of the significance of the macrophage and its functional phenotypes in the healing tendon may be the key to enhancing tendon healing. This review will present the current literature on the function of macrophages in tendinopathy and tendon healing and the potential of therapies targeting macrophages to enhance tendon healing.     

Temporal expression of nitric oxide synthase isoforms in healing Achilles tendon.

We investigated the temporal expressions of the three nitric oxide synthase (NOS) isoforms by semi-quantitative polymerase chain reaction (PCR) assays and by immunoblot analysis, following Achilles tendon transection in rats. Four days after injury, there were increases in the steady-state levels of mRNA for all three NOS isoforms, with peaks for the inducible isoform (iNOS) (23-fold increase) at day 4, the endothelial isoform (eNOS) (24-fold increase) at day 7 and the neuronal isoform (bNOS) (seven-fold increase) at day 21. The temporal expression of NOS isoforms at a protein level was consistent with the results at the mRNA level. We have previously shown a five-fold increase in the NOS activity, as detected by 3H-arginine to 3H-citrulline conversion, at day 7 postinjury. These findings indicate that all three NOS isoforms are expressed during tendon healing with differential expression patterns during the various phases of tendon healing. These findings may prove clinically relevant with respect to strategies for regulating tendon healing.     

Current progress in growth factors and extracellular vesicles in tendon healing

Tendon injury healing is a complex process that involves the participation of a significant number of molecules and cells, including growth factors molecules in a key role. Numerous studies have demonstrated the function of growth factors in tendon healing, and the recent emergence of EV has also provided a new visual field for promoting tendon healing. This review examines the tendon structure, growth, and development, as well as the physiological process of its healing after injury. The review assesses the role of six substances in tendon healing: insulin-like growth factor-I (IGF-I), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and EV. Different growth factors are active at various stages of healing and exhibit separate physiological activities. IGF-1 is expressed immediately after injury and stimulates the mitosis of various cells while suppressing the response to inflammation. VEGF, which is also active immediately after injury, accelerates local metabolism by promoting vascular network formation and positively impacts the activities of other growth factors. However, VEGF's protracted action could be harmful to tendon healing. PDGF, the earliest discovered cytokine to influence tendon healing, has a powerful cell chemotaxis and promotes cell proliferation, but it can equally accelerate the response to inflammation and relieve local adhesions. Also useful for relieving tendon adhesion is TGF- β, which is active almost during the entire phase of tendon healing. As a powerful active substance, in addition to its participation in the field of cardiovascular and cerebrovascular vessels, tumour and chronic wounds, TGF- β reportedly plays a role in promoting cell proliferation, activating growth factors, and inhibiting inflammatory response during tendon healing.     

The early inflammatory response after flexor tendon healing: A gene expression and histological analysis

Despite advances in surgical techniques over the past three decades, tendon repairs remain prone to poor clinical outcomes. Previous attempts to improve tendon healing have focused on the later stages of healing (i.e., proliferation and matrix synthesis). The early inflammatory phase of tendon healing, however, is not fully understood and its modulation during healing has not yet been studied. Therefore, the purpose of this work was to characterize the early inflammatory phase of flexor tendon healing with the goal of identifying inflammation‐related targets for future treatments. Canine flexor tendons were transected and repaired using techniques identical to those used clinically. The inflammatory response was monitored for 9 days. Temporal changes in immune cell populations and gene expression of inflammation‐, matrix degradation‐, and extracellular matrix‐related factors were examined. Gene expression patterns paralleled changes in repair‐site cell populations. Of the observed changes, the most dramatic effect was a greater than 4,000‐fold up‐regulation in the expression of the pro‐inflammatory factor IL‐1β. While an inflammatory response is likely necessary for healing to occur, high levels of pro‐inflammatory cytokines may result in collateral tissue damage and impaired tendon healing. These findings suggest that future tendon treatment approaches consider modulation of the inflammatory phase of healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:645–652, 2014.     

Localized delivery of ibuprofen via a bilayer delivery system (BiLDS) for supraspinatus tendon healing in a rat model

The high prevalence of tendon retear following rotator cuff repair motivates the development of new therapeutics to promote improved tendon healing. Controlled delivery of non-steroidal anti-inflammatory drugs to the repair site via an implanted scaffold is a promising option for modulating inflammation in the healing environment. Furthermore, biodegradable nanofibrous delivery systems offer an optimized architecture and surface area for cellular attachment, proliferation, and infiltration while releasing soluble factors to promote tendon regeneration. To this end, we developed a bilayer delivery system (BiLDS) for localized and controlled release of ibuprofen (IBP) to temporally mitigate inflammation and enhance tendon remodeling following surgical repair by promoting organized tissue formation. In vitro evaluation confirmed the delayed and sustained release of IBP from Labrafil-modified poly(lactic-co-glycolic) acid microspheres within sintered poly(ε-caprolactone) electrospun scaffolds. Biocompatibility of the BiLDS was demonstrated with primary Achilles tendon cells in vitro. Implantation of the IBP-releasing BiLDS at the repair site in a rat rotator cuff injury and repair model led to decreased expression of proinflammatory cytokine, tumor necrotic factor-α, and increased anti-inflammatory cytokine, transforming growth factor-β1. The BiLDS remained intact for mechanical reinforcement and recovered the tendon structural properties by 8 weeks. These results suggest the therapeutic potential of a novel biocompatible nanofibrous BiLDS for localized and tailored delivery of IBP to mitigate tendon inflammation and improve repair outcomes. Future studies are required to define the mechanical implications of an optimized BiLDS in a rat model beyond 8 weeks or in a larger animal model.     

Aging does not alter tendon mechanical properties during homeostasis,but does impair flexor tendon healing

Aging is an important factor in disrupted homeostasis of many tissues. While an increased incidence of tendinopathy and tendon rupture are observed with aging, it is unclear whether this is due to progressive changes in tendon cell function and mechanics over time, or an impaired repair reaction from aged tendons in response to insult or injury. In the present study, we examined changes in the mechanical properties of Flexor Digitorum Longus (FDL), Flexor Carpi Ulnaris (FCU), and tail fascicles in both male and female C57Bl/6 mice between 3 and 27 months of age to better understand the effects of sex and age on tendon homeostasis. No change in max load at failure was observed in any group over the course of aging, although there were significant decreases in toe and linear stiffness in female mice from 3 to 15 months, and 3 to 27 months. No changes in cell proliferation were observed with aging, although an observable decrease in cellularity occurred in 31‐month old tendons. Given that aging did not dramatically alter tendon mechanical homeostasis we hypothesized that a disruption in tendon homeostasis, via acute injury would result in an impaired healing response. Significant decreases in max load, stiffness, and yield load were observed in repairs of 22‐month old mice, relative to 4‐month old mice. No changes in cell proliferation were observed between young and aged, however, a dramatic loss of bridging collagen extracellular matrix was observed in aged repairs suggest that matrix production, but not cell proliferation leads to impaired tendon healing with aging. Results © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2716–2724, 2017.

     

Current aspects of tendon healing

This review describes structure, function and healing of tendinous tissue and discusses new biologically based treatment options to modulate tendon healing. The repair process after tendon rupture results in a morphologically different and biomechanically inferior structure compared to a normal tendon. The collagen fibril diameters are decreased months after the traumatic lesion and show also different phenotypes. We know that cytokines and growth factors are key components for normal tissue development and regulate wound healing processes. Some growth factors have been detected to influence tenocytes by promoting cell proliferation and matrix synthesis. Application of the adequate growth factors at certain periods during the repair process might improve the healing result after tendon rupture. However, most of these growth factors are proteins which are rapidly metabolized by the organism. Transfer of growth factor genes into tenocytes might eliminate this problem by a continuous local release of growth factors at the healing site.     

Treatment with rhBMP12 or rhBMP13 increase the rate and the quality of rat Achilles tendon repair

Tendon injuries that result in partial or complete tears often come from chronic, repetitive use, or from sudden trauma. In some cases, torn tendons can be repaired, but such repairs often fail to completely restore tendon function. We used global gene expression profiling and histological examination to study tendon repair to elucidate key molecular processes that regulate the rate and quality of tissue restoration. Using a rat Achilles tendon transection model, tissue was collected at 3, 7, 10, and 15 days postinjury. The pattern of gene expression in the repairing tissue paralleled the healing phases of inflammation, matrix formation, and matrix reorganization. Newly formed repaired tissue is characterized by cells expressing many genes associated with tendon formation, thereby potentially distinguishing this repair tissue from other types of repair or scar tissue. Addition of recombinant human bone morphogenic protein (rhBMP)12 or rhBMP13, also known as growth and differentiation factors (GDFs) 6 and 7, 1 day after injury yielded increases in tissue volume, rate of cellular infiltration, and in changes in levels of key mRNAs involved in tendon repair. Altogether, our results indicate that rhBMP12 or rhBMP13 enhance the rate of tendon repair. A better understanding of the key molecular regulators of tendon repair could lead to the development of new therapies for tendon injuries and the identification of diagnostic markers that indicate the status of tendon repair after injury. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1604–1612, 2011     

Comparison of the effects of laser, ultrasound, and combined laser + ultrasound treatments in experimental tendon healing

BACKGROUND AND OBJECTIVE: Therapeutic ultrasound (US) and laser (L) treatments accelerate and facilitate wound healing, and also have beneficial effects on tendon healing. This randomized control study was designed to evaluate the effects of low-intensity US and low-level laser therapy (LLLT) on tendon healing in rats. STUDY DESIGN/MATERIALS AND METHODS: Eighty-four healthy male Swiss-Albino rats were divided into three groups consisting of 28 rats, the left Achilles tendons were used as treatment and the right Achilles tendons as controls. The right and left Achilles tendons of rats were traumatized longitudinally. The treatment was started on postinjury day one. We applied the treatment protocols including low-intensity US treatment in Group I (US Group), Sham US in Group II (SUS Group), LLLT in Group III (L Group), Sham L in Group IV (SL Group), US and LLLT in Group V (US + L Group), and Sham US and Sham L in Group VI (SUS + SL Group). The US treatment was applied with a power of 0.5 W/cm2, a frequency of 1 MHz, continuously, 5 minutes daily. A low-level Ga-As laser was applied with a 904 nm wavelength, 6 mW average power, 1 J/ cm2 dosage, 16 Hz frequency, for 1 minute duration, continuously. In the control groups, the similar procedures as in the corresponding treatment groups were applied with no current (Sham method). The treatment duration was planned for 9 days (sessions) in all groups, except the rats used for biochemical evaluation on the 4th day of treatment, which were treated for 4 days. We measured the levels of the tissue hydroxyproline for biochemical evaluation on the 4th, 10th, and 21st days following the beginning of treatment and the tendon breaking strength on the 21st day following the beginning of treatment for biomechanical evaluation. Seven rats in each group were killed on the 4th, 10th, and 21st days for biochemical evaluation and on the 21st day for biomechanical evaluation. RESULTS: The hydroxyproline levels were found to be significantly increased in the treatment groups on the 10th and 21st days compared to their control groups (P < 0.05). In comparison of the treatment groups on the 4th, 10th, and 21st days of the treatment, the levels of tissue hydroxyproline were found to be more increased in combined US+L Group compared with US Group and L Group, but the difference was not significant (P > 0.05). In comparison of the tendon breaking strengths, it was found as significantly increased in the treatment groups compared with their control groups (P < 0.05), although there was no significant difference between the treatment groups. CONCLUSIONS: Although US, L, and combined US + L treatments increased tendon healing biochemically and biomechanically more than the control groups, no statistically significant difference was found between them. Also we did not find significantly more cumulative positive effects of combined treatment. As a result, both of these physical modalities can be used successfully in the treatment of tendon healing.     

Joint immobilization reduces the expression of sensory neuropeptide receptors and impairs healing after tendon rupture in a rat model

Healing after mobilization versus immobilization was assessed in a model of rat Achilles tendon rupture, by RT‐PCR at 8 and 17 days and by histological analyses at 14 and 28 days postrupture. The expression of mRNA for extracellular matrix (ECM) molecules (collagen type I and type III, versican, decorin, and biglycan), and the subjective histological maturation of the healing area were analyzed. Effects of immobilization on healing were related to changes in the peripheral expression of substance P (NK₁)‐ and calcitonin gene‐related peptide (CRLR and RAMP‐1)‐ receptors. At 8 days postinjury, mRNA levels for ECM molecules were equal in both groups. However, by day 17, the ECM mRNA expression in the mobilized group had increased up to ～14× that of the immobilized group, which were comparable to intact tendon values. Histological analysis confirmed a higher regenerating activity in the mobilized group, with an increased amount of blood vessels, fibroblasts, and new collagen. The expression of sensory neuropeptide receptors in the mobilized group exhibited a significant increase from 8 to 17 days postinjury similar to the increased ECM mRNA expression, whereas the immobilized group at 17 days exhibited levels comparable to the intact tendon values. Therefore, immobilization postrupture appears to hamper tendon healing, a process which may prove to be directly linked to a downregulated peripheral sensitivity to sensory neuropeptide stimulation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:274–280, 2009     

Development of a mouse model of supraspinatus tendon insertion site healing

Supraspinatus (SS) tendon tears are common musculoskeletal injuries whose surgical repair exhibits the highest incidence of re‐tear of any tendon. Development of therapeutics for improving SS tendon healing is impaired by the lack of a model that allows biological perturbations to identify mechanisms that underlie ineffective healing. The objective of this study was to develop a mouse model of supraspinatus insertion site healing by creating a reproducible SS tendon detachment and surgical repair which can be applied to a wide array of inbred mouse strains and genetic mutants. Anatomical and structural analyses confirmed that the rotator cuff of the mouse is similar to that of human, including the presence of a coracoacromial (CA) arch and an insertion site that exhibits a fibrocartilagenous transition zone. The surgical repair was successfully conducted on seven strains of mice that are commonly used in Orthopaedic Research suggesting that the procedure can be applied to most inbred strains and genetic mutants. The quality of the repair was confirmed with histology through 14 days after surgery in two mouse strains that represent the variation in mouse strains evaluated. The developed mouse model will allow us to investigate mechanisms involved in insertion site healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:25–32, 2015.     

Growth factor and protease expression during different phases of healing after rabbit deep flexor tendon repair

The purpose of the study was to contribute to the mapping of molecular events during flexor tendon healing, in particular the growth factors insulin‐like growth factor‐1 (IGF‐1), vascular endothelial growth factor (VEGF) and nerve growth factor (NGF), matrix metalloproteinases (MMP‐3 and MMP‐13) and their inhibitors (tissue inhibitors of metalloproteinases, TIMP‐1 and TIMP‐3, and the protease cathepsin K. In a rabbit model of flexor tendon injury, the mRNA expression for the growth factors, MMPs and TIMPs were measured in tendon and tendon sheath tissue at several time points (3, 6, 21, and 42 days) representing different phases of the healing process. We found that MMP‐13 remained increased during the study period, whereas MMP‐3 returned to normal levels within the first week after injury. TIMP‐3 was down‐regulated in the tendon sheaths. Cathepsin K was up‐regulated in tendons and sheaths after injury. NGF was present in both tendons and sheaths, but unaltered. IGF‐1 exhibited a late increase in the tendons, while VEGF was down‐regulated at the later time points. In conclusion, we have demonstrated the presence of NGF in flexor tendons. MMP‐13 expression appears to play a more protracted role in flexor tendon healing than MMP‐3. The relatively low levels of endogenous IGF‐1 and VEGF mRNA following injury support their potential beneficial role as exogenous modulators to optimize tendon healing and strength without increasing adhesion formation. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:886–892     

Recent progress in flexor tendon healing

 Although advances in the treatment of flexor tendon injuries have led to improved clinical outcomes during the past several decades, a subset of patients continue to experience a loss of function. Using a canine model of sharp transection of the flexor digitorum profundus tendon followed by repair and rehabilitation using clinically relevant techniques, we have examined the influence of multistrand suture and postoperative rehabilitation variables on digital function and tendon strength. Our findings highlight the critical role of repair technique in providing a stiff and strong repair and indicate that continued refinement of suture techniques is warranted in order to minimize repair-site elongation (gap). Gap formation continues to occur at a high frequency, and the formation of gaps greater than 3 mm delays the accrual of repair-site strength that occurs with time. Furthermore, our results indicate that passive-motion rehabilitation that produces a moderate amount of tendon excursion (2 mm) at low levels of tendon force (5 N) is sufficient to inhibit adhesion formation and to promote healing. Increases in excursion or force beyond these levels do not accelerate the healing process. These findings suggest that we are approaching the limit of the extent to which we can modulate healing by manipulating rehabilitation variables such as tendon excursion and force. Future advances will probably require manipulation of the biological factors that promote healing. Received: December 20, 2001     

CD44 deficiency improves healing tendon mechanics and increases matrix and cytokine expression in a mouse patellar tendon injury model

CD44 plays an important role in inflammation and healing. Previous studies investigated its role in inflammatory diseases and skin wounds; however, the role of CD44 in tendon healing is unknown. Therefore, we investigated the effect of CD44 in the healing of the patellar tendon in a knockout mouse model. We hypothesized that in comparison to wild‐type counterparts, CD44 knockout mice would have decreased material parameters, increased organization, decreased expression of proinflammatory cytokines, and increased expression of matrix components during healing. These hypotheses were tested through an in vivo surgical model and mechanical, organizational, and gene expression analyses. Material strength and tissue organization were significantly improved in the CD44 knockout mouse. This could be attributed to increased expression of cytokines and matrix components that are also elevated in regenerative healing. Our study showed that the absence of CD44 in a mouse patellar tendon injury creates an environment that is conducive to regenerative healing through altered gene expression, resulting in superior material properties and reduced cross‐sectional area. Therefore, limiting the role of CD44 may improve healing parameters in adult tendon injury. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1386–1391, 2009     

Effects of montelukast sodium on tendon healing: An experimental study

Introduction:

Montelukast sodium (MS) a selective leukotriene antagonist of the cysteinyl leukotriene receptor, has been used in the treatment of asthma and allergic rhinitis. In this study, we evaluated the effect of MS on the early inflammatory phase (histological) of nonsynovial tendon healing.

Materials and Methods:

Rats were divided randomly into two groups (n = 6 each). MS (Singulair) was administered to one group at 10 mg/kg/day [250 g/day intraperitoneally (i.p.)]. The control group was administered 250 g/day of 0.9% saline i.p. This nonsynovial tendon was longitudinally divided at the midportion, cut transversely and then sutured. In both groups, the rats were sacrificed by decapitation 10 days later.

Results:

Decreased inflammatory cell infiltration and more properly oriented collagen fibres were observed in the MS group''s histopathological specimens as compared to the control group''s (P < 0.05). Additionally, vascularity was decreased in the MS group.

Conclusion:

MS decreased tendon healing, apparently by inhibiting the early inflammatory phase of nonsynovial tendon healing.