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HFD leads to increased body weight and impairs glucose tolerance. A-Body weights from mice in both HFD (black bars) and LFD groups (white bars) were compared at the time of surgery and the time of harvest. At both points, the HFD group weighed significantly more than the LFD group. B-Elevated fasting blood glucose levels were observed in HFD (black bars), relative to LFD mice (white bars). C-Mice from the HFD group (black line) demonstrated an impaired ability to respond to glucose loading relative to LFD (red line), as shown by significantly elevated blood glucose levels at 30, 60, and 120 minutes after delivering a glucose bolus. (*) Indicates p<0.05. https://doi.org/10.1371/journal.pone.0181127.g001
Source publication
Type II Diabetes (T2DM) dramatically impairs the tendon healing response, resulting in decreased collagen organization and mechanics relative to non-diabetic tendons. Despite this burden, there remains a paucity of information regarding the mechanisms that govern impaired healing of diabetic tendons. Mice were placed on either a high fat diet (T2DM...
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Citations
... This altered macrophage polarization can prolong scar formation and impair biomechanical properties. Thus, fibrosis-induced macrophages have been identified as potential targets to improve tendon healing in patients with DM [3]. With the rapidly increasing number of patients with DM, potential therapeutic targets for preventing and treating tendon disorders in these patients are being reported. ...
... With the rapidly increasing number of patients with DM, potential therapeutic targets for preventing and treating tendon disorders in these patients are being reported. However, the effects of DM on tendon function and healing remain unclear, and no robust treatment is available [3]. Thus, these issues will be addressed once preclinical models of DM tendinopathy and tendon healing have been characterized and there is better understanding of the cell types and signaling pathways involved. ...
Rotator cuff tendon tears are a leading cause of shoulder pain. They are challenging to treat, and tendon–bone healing has a high failure rate despite successful surgery. Tendons connect the muscles and bones, which make them important for the body’s overall mobility and stability. Metabolic diseases, including diabetes or high blood pressure, can affect the healing process after repair of a damaged tendon. With a global incidence of 9.3%, diabetes is considered as a significant risk factor for rotator cuff tendon healing because it causes structural, inflammatory, and vascular changes in the tendon. However, the mechanisms of how diabetes affects tendon healing remain unknown. Several factors have been suggested, including glycation product accumulation, adipokine dysregulation, increased levels of reactive oxygen species, apoptosis, inflammatory cytokines, imbalanced matrix-metalloproteinase-to-tissue-inhibitor ratio, and impaired angiogenesis and differentiation of the tendon sheath. Despite the effects of diabetes on tendon function and healing, few treatments are available to improve recovery in these patients. This review summarizes the current literature on the pathophysiological changes of the tendon in diabetes and hyperlipidemia. Preclinical and clinical evidence regarding the association between diabetes and tendon healing is presented. Moreover, current approaches to improve tendon healing in patients with diabetes are reviewed.
... 9,10, 14 An imbalance in ECM synthesis and degradation from tissueresident cells and both tissue-resident cells and extrinsic immune cells, respectively, during healing are key reasons implicated in the development of fibrosis. 15,16 In both acute and chronic tendon injuries, inflammatory cytokines IL-1β, IL-6, TNF-α, and matrix metalloproteinases (MMPs) such as MMP-1, -3, and -13 are up-regulated. 15,16 However, there is minimal information on how macrophage-induced inflammatory cytokines impact the bioactivities of ECM remodeling enzymes, MMPs, and their inhibitors tissue inhibitor matrix metalloproteinases (TIMPs) in tendon-derived cells, which are vital for tendon ECM remodeling. ...
... 15,16 In both acute and chronic tendon injuries, inflammatory cytokines IL-1β, IL-6, TNF-α, and matrix metalloproteinases (MMPs) such as MMP-1, -3, and -13 are up-regulated. 15,16 However, there is minimal information on how macrophage-induced inflammatory cytokines impact the bioactivities of ECM remodeling enzymes, MMPs, and their inhibitors tissue inhibitor matrix metalloproteinases (TIMPs) in tendon-derived cells, which are vital for tendon ECM remodeling. In the context of equine DDFT injuries, determining the biological mechanisms controlling macrophages and DDF tenocytes may shed light on DDFT fibrosis that occurs during the healing process. ...
OBJECTIVE
To investigate matrix metalloproteinase (MMP) and their inhibitors tissue inhibitor matrix metalloproteinase (TIMP) gene expression and secretion during equine deep digital flexor tendon (DDFT) tenocyte and macrophage (undifferentiated, proinflammatory, and regulatory) co-culture.
SAMPLE
Third passage DDF tenocytes and donor-matched macrophages differentiated from peripheral blood CD14 ⁺ monocytes from 5 healthy horses ages 9–11 years, euthanized for reasons unrelated to musculoskeletal conditions.
METHODS
Passage 3 DDT tenocyte aggregate cultures were co-cultured with undifferentiated (control), proinflammatory (granulocyte-macrophage colony-stimulating factor; GM-CSF pretreated and lipopolysaccharide + interferon gamma-primed; LPS+IFN-γ) or regulatory (interleukin-4 and interleukin-10-primed; IL-4 + IL-10) macrophages in direct and transwell co-cultures for 72 hours. MMP-1, -2, -3, -9, -13, and TIMP -1, -2 mRNA were measured via real-time Polymerase Chain Reaction (rtPCR). Co-culture media MMP -3, -9, and TIMP -1, -2 concentrations were quantified via ELISA.
RESULTS
Direct co-culture of DDF tenocytes with proinflammatory macrophages for 72 hours increased MMP-1, -3, and -13 mRNA levels whereas, MMP-9 mRNA levels decreased. Direct and transwell co-culture with proinflammatory and regulatory macrophages resulted in increased MMP-3 and decreased MMP-9 media concentrations. While direct co-culture with regulatory macrophages significantly increased TIMP-1 mRNA, overall, TIMP mRNA and culture media concentrations were largely unchanged.
CLINICAL RELEVANCE
Cell-to-cell contact between DDF tenocytes and macrophages is not essential to induce MMP gene expression and secretion. Co-culture systems offer a viable in vitro platform to screen and evaluate immunomodulatory properties of therapies aimed at improving equine intrasynovial tendon healing.
... [40,41] Preclinical studies also demonstrated that the tendons of rats with obesity induced by high-fat-diet had compromised biomechanical and healing properties. [42,43] The cause might be the increased mechanical stress and low-grade, chronic inflammatory microenvironment. [44] The inflammatory status seems to be caused by adipocytes because they suffer from hypoxic, mechanical, and oxidative stress secondary to cell hypertrophy, ultimately leading to cellular apoptosis, release of the intracellular pro-inflammatory molecules and continues recalling of macrophages. ...
... Diabetes is a chronic metabolic disease that is complicated by delayed wound healing and dysregulation of the inflammatory phase wound repair [15][16][17][18][19], leading to chronic wounds and substantial morbidity. To identify secreted factors in the diabetic wound bed associated with delayed cutaneous wound closure as potential therapeutic targets, we used mice lacking the Leptin receptor (Lepr −/− ; db/db), which is a Type II diabetic model that is hyperglycemic and presents with a well-defined phenotype of delayed cutaneous wound closure [20][21][22][23]. We used sterile subcutaneous PVA sponge implants in mice as a source for EVs based on a recruitment of neutrophils, monocytes and macrophages similar to the inflammation phase of the wound healing [24][25][26]. ...
Chronic metabolic diseases such as diabetes are characterized by delayed wound healing and a dysregulation of the inflammatory phase of wound repair. Our study focuses on changes in the payload of extracellular vesicles (EVs) communicating between immune cells and stromal cells in the wound bed, which regulate the rate of wound closure. Adoptive transfer of EVs from genetically defined mouse models are used here to demonstrate a functional and molecular basis for differences in the pro-reparative biological activity of diabetic (db/db) vs. wildtype EVs in wound healing. We identify several members of the Serpin family of serine protease inhibitors that are absent in db/db EVs, then we overexpress Serpin A1, F2 and G1 in EVs to evaluate their effect on wound healing in db/db mice. Serpins have an important role in regulating levels of elastase, plasmin and complement factors that coordinate immune cell signaling in full thickness wounds in a diabetic model. Here, we establish a novel therapeutic approach by engineering the payload of EVs based on proteomic analysis. Serpin-loaded EVs were used to rescue the Serpin deficiency identified by proteomics and promote wound healing in db/db mice, as well as evaluated how EVs affected extracellular matrix remodeling and the resolution of tissue injury. Therefore, we propose that the identification of EV payloads that are downregulated in diabetic wounds can be systematically analyzed for their functional activity and potential as a therapeutic, based on whether their re-expression in engineered EVs restores normal kinetics of tissue repair in chronic wounds.
Graphical Abstract
... Melanocytes and fibroblasts may contribute to re-pigmentation [164]. Cellular interactions at wound sites promote fibroblast attachment and produce a new connective tissue matrix and double paracrine interaction between keratinocytes and fibroblasts [165]. These findings suggest that EVs from skin tissues play unique roles in cutaneous wound repair. ...
Wound healing refers to the healing process that occurs after the skin and other tissues are separated or damaged by internal or external forces. It is a complex combination of tissue regeneration, granulation tissue hyperplasia, and scar formation, and shows the synergistic effects of these processes. After skin damage, the environment around the wound and the cells at site of the damage respond immediately, and a range of cytokines and growth factors are released. In cutaneous injury, extracellular vesicle (EV) signaling plays a vital role in the healing process via paracrine and endocrine mechanisms. EVs are natural intercellular and inter-organ communication tools that carry various bioactive substances for message exchange. Stem cells and stem cell EVs facilitate tissue repair, showing promising potential in regenerative medicine. Nevertheless, EVs derived from specific skin tissue cells, such as epidermal cells, fibroblasts, vascular endothelial cells and inflammatory cells, also play important roles in cutaneous tissue repair. Here, we describe the characteristics of wound healing, concentrating on the production and functions of EVs derived from specific skin cells, and provide new ideas for wound therapy using EVs.
... Loss of ECM regulation, such as favoring degradation over synthesis, could alter the ECM responses to damage the tissue assembly. It is no surprise that the dysregulation of degenerative enzymes, such as MMPs, has been thought to play an essential role in developing tendon pathology in diabetes as overexpression of MMPs may favor ECM degradation [37]. Similarly, if inhibitory TIMPs are less expressed, the environment may also favor degradation by allowing MMPs to act on the ECM for a more extended period. ...
Background
Tendon pathologies affect a large portion of people with diabetes. This high rate of tendon pain, injury, and disease appears to manifest independent of well-controlled HbA1c and fasting blood glucose. Advanced glycation end products (AGEs) are elevated in the serum of those with diabetes. In vitro , AGEs severely impact tendon fibroblast proliferation and mitochondrial function. However, the extent that AGEs impact the tendon cell transcriptome has not been evaluated.
Objective
The purpose of this study was to investigate transcriptome-wide changes that occur to tendon-derived fibroblasts following treatment with AGEs. We propose to complete a descriptive approach to pathway profiling to broaden our mechanistic understanding of cell signaling events that may contribute to the development of tendon pathology.
Methods
Rat Achilles tendon fibroblasts were treated with glycolaldehyde-derived AGEs (200μg/ml) for 48 hours in normal glucose (5.5mM) conditions. In addition, total RNA was isolated, and the PolyA ⁺ library was sequenced.
Results
We demonstrate that tendon fibroblasts treated with 200μg/ml of AGEs differentially express 2,159 gene targets compared to fibroblasts treated with an equal amount of BSA-Control. Additionally, we report in a descriptive and ranked fashion 21 implicated cell-signaling pathways.
Conclusion
Our findings suggest that AGEs disrupt the tendon fibroblast transcriptome on a large scale and that these pathways may contribute to the development and progression of diabetic tendinopathy. Specifically, pathways related to cell cycle progression and extracellular matrix remodeling were affected in our data set and may play a contributing role in the development of diabetic tendon complications.
... Increasing attention is being given to the disruption of immune system activity in diabetes by hyperglycemia and/or chronic inflammation [6][7][8] particularly in adipose tissue in the case of obesity/type 2 diabetes [9]. These observations have profound implications for DPN, given the parallel emergence of the importance of neuroimmune crosstalk in neuronal dysfunction. ...
Background
The incidence of diabetes and diabetic peripheral neuropathy continues to rise, and studies have shown that macrophages play an important role in their pathogenesis. To date, macrophage tracking has largely been achieved using genetically-encoded fluorescent proteins. Here we present a novel two-color fluorescently labeled perfluorocarbon nanoemulsion (PFC-NE) designed to monitor phagocytic macrophages in diabetic neuropathy in vitro and in vivo using non-invasive near-infrared fluorescent (NIRF) imaging and fluorescence microscopy.
Methods
Presented PFC-NEs were formulated with perfluorocarbon oil surrounded by hydrocarbon shell carrying two fluorescent dyes and stabilized with non-ionic surfactants. In vitro assessment of nanoemulsions was performed by measuring fluorescent signal stability, colloidal stability, and macrophage uptake and subsequent viability. The two-color PFC-NE was administered to Lepr db/db and wild-type mice by tail vein injection, and in vivo tracking of the nanoemulsion was performed using both NIRF imaging and confocal microscopy to assess its biodistribution within phagocytic macrophages along the peripheral sensory apparatus of the hindlimb.
Results
In vitro experiments show two-color PFC-NE demonstrated high fluorescent and colloidal stability, and that it was readily incorporated into RAW 264.7 macrophages. In vivo tracking revealed distribution of the two-color nanoemulsion to macrophages within most tissues of Lepr db/db and wild-type mice which persisted for several weeks, however it did not cross the blood brain barrier. Reduced fluorescence was seen in sciatic nerves of both Lepr db/db and wild-type mice, implying that the nanoemulsion may also have difficulty crossing an intact blood nerve barrier. Additionally, distribution of the nanoemulsion in Lepr db/db mice was reduced in several tissues as compared to wild-type mice. This reduction in biodistribution appears to be caused by the increased number of adipose tissue macrophages in Lepr db/db mice.
Conclusions
The nanoemulsion in this study has the ability to identify phagocytic macrophages in the Lepr db/db model using both NIRF imaging and fluorescence microscopy. Presented nanoemulsions have the potential for carrying lipophilic drugs and/or fluorescent dyes, and target inflammatory macrophages in diabetes. Therefore, we foresee these agents becoming a useful tool in both imaging inflammation and providing potential treatment in diabetic peripheral neuropathy.
... However, Lin et al. [50] showed that an abnormally increased ratio of M1 to M2 correlated with pathological fibrosis during tendon healing. However, recent studies have demonstrated that M2 macrophages are also associated with fibrosis, and excessive M2 macrophage activation can lead to increased scar formation and impair normal tendon healing [51,52]. This suggests that the excellent outcome of tendon/ligament regeneration--reduced damage by mitigating inflammation, enhanced tissue regeneration and limited pathological fibrosis, may be achieved by the sequential transformation of macrophage phenotype instead of a specific phenotype of macrophages. ...
Musculoskeletal disorders are common in clinical practice. Repairing critical-sized defects in musculoskeletal systems remains a challenge for researchers and surgeons, requiring the application of tissue engineering biomaterials. Successful application depends on the response of the host tissue to the biomaterial and specific healing process of each anatomical structure. The commonly-held view is that biomaterials should be biocompatible to minimize local host immune response. However, a growing number of studies have shown that active modulation of the immune cells, particularly macrophages, via biomaterials is an effective way to control immune response and promote tissue regeneration as well as biomaterial integration. Therefore, we critically review the role of macrophages in the repair of injured musculoskeletal system soft tissues, which have relatively poor regenerative capacities, as well as discuss further enhancement of target tissue regeneration via modulation of macrophage polarization by biomaterial-mediated immunomodulation (biomaterial properties and delivery systems). This active regulation approach rather than passive-evade strategy maximizes the potential of biomaterials to promote musculoskeletal system soft tissue regeneration and provides alternative therapeutic options for repairing critical-sized defects.
... 24,37 Previous preclinical studies on the effects of either an HFD or a low-fat diet to impair the tendon healing response have used C57BL6/J mice, which carry a genetic predisposition to diet-induced type II diabetes. 3,19 These studies reported inferior biomechanical and healing properties at the digital flexor tendon healing site, with increased and prolonged M2 macrophage activity. Importantly, this animal model did not induce a diabetic or hypercholesterolemic phenotype, both of which have been shown to impair tendon healing and are often concurrent with obesity/metabolic syndrome. ...
... An HFD has been previously shown to induce tendinopathies in weightbearing and nonweightbearing tendons of rats observed this response in tendons to an HFD, which is possibly explained by inadequate dietary induction periods to disrupt tendon homeostasis. 3,19,44 In this study, there were differences in the body mass of the HFD and HF-CD groups in the first 12 weeks of the study period, with the HF-CD group appearing more responsive to the HFD and exhibiting a greater body mass. The variability among individual humans and animals in the extent to which they are predisposed to obesity in an obesogenic environment can be attributed to complex genetic and epigenetic mechanisms. ...
Background
Being overweight or obese is associated with poor outcomes and an increased risk of failure after rotator cuff (RC) surgery. However, the effect of obesity on enthesis healing has not been well characterized.
Hypotheses
Diet-induced obesity (DIO) would result in inferior enthesis healing in a rat model of RC repair, and a dietary intervention in the perioperative period would improve enthesis healing.
Study Design
Controlled laboratory study.
Methods
Male Sprague-Dawley rats were divided into 3 weight-matched groups (n = 26 per group): control diet (CD), high-fat diet (HFD), or HFD until surgery and then CD thereafter (HF-CD). After 12 weeks, the left supraspinatus tendon was detached, followed by immediate repair. Animals were sacrificed, and RCs were harvested at 2 and 12 weeks after surgery for biomechanical and histological evaluations. Metabolic end points were assessed using dual-energy X-ray absorptiometry and plasma analyses.
Results
DIO was established in the HFD and HF-CD groups before surgery and subsequently reversed in the HF-CD group after surgery. At 12 weeks after surgery, the body fat percentage ( P = .0021) and plasma leptin concentration ( P = .0025) were higher in the HFD group compared with the CD group. Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD groups compared with the CD group at 12 weeks after surgery, with semiquantitative scores of 6.20 ( P = .0078), 4.98 ( P = .0003), and 8.68 of 15, respectively. The repaired entheses in the HF-CD group had a significantly lower load to failure ( P = .0278) at 12 weeks after surgery compared with the CD group, while the load to failure in the HFD group was low but not significantly different ( P = .0960). There were no differences in the biomechanical and histological results between the groups at 2 weeks after surgery. Body mass at the time of surgery, plasma leptin concentration, and body fat percentage were negatively correlated with histology scores and plasma leptin concentration was correlated with load to failure at 12 weeks after surgery.
Conclusion
DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring a normal weight with dietary changes after surgery did not improve healing outcomes.
Clinical Relevance
Obesity is a potentially modifiable factor that impairs RC healing and increases the risk of failure after surgery. Exploring interventions that improve the metabolic state of obese patients and counseling patients appropriately about their modest expectations after repair should be considered.
... TNF-α was shown to affect wound healing by counteracting healing processes in keratinocytes and inducing polarization of macrophages to M1 phenotype [36]. A generally stronger polarization of monocytes into macrophages (both M1 and M2 type) with prolonged inflammation was reported in diabetic mice with impaired tendon healing, leading to increased scar formation [37]. ...
Diabetes mellitus is a main risk factor for delayed fracture healing and fracture non-unions. Successful fracture healing requires stimuli from different immune cells, known to be affected in diabetics. Especially, application of mononuclear cells has been proposed to promote wound and fracture healing. Thus, aim was to investigate the effect of pre-/diabetic conditions on mononuclear cell functions essential to promote osteoprogenitor cell function. We here show that pre-/diabetic conditions suppress the expression of chemokines, e.g., CCL2 and CCL8 in osteoprogenitor cells. The associated MCP-1 and MCP-2 were significantly reduced in serum of diabetics. Both MCPs chemoattract mononuclear THP-1 cells. Migration of these cells is suppressed under hyperglycemic conditions, proposing that less mononuclear cells invade the site of fracture in diabetics. Further, we show that the composition of cytokines secreted by mononuclear cells strongly differ between diabetics and controls. Similar is seen in THP-1 cells cultured under hyperinsulinemia or hyperglycemia. The altered secretome reduces the positive effect of the THP-1 cell conditioned medium on migration of osteoprogenitor cells. In summary, our data support that factors secreted by mononuclear cells may support fracture healing by promoting migration of osteoprogenitor cells but suggest that this effect might be reduced in diabetics.
