CD201+ fascia progenitors choreograph injury repair.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

Organ dependency on fascia connective tissue.

Fascia is a specialized connective tissue system that encapsulates and interconnects between tissues and organs throughout the body. The fascia system regulates pain sensation, organ inflammation, trauma, and fibrotic diseases. This mini-review summarizes recent findings from animal models, which reveal the inter-dependency between tissues/organs and the fascia system. Special mechanisms are explored of fascia response to skin inflammatory processes and fibrotic microenvironments in trauma models. We highlight the functionally diverse communities of its fascia-born fibroblasts and the significance of their stage-specific differentiation and communication to disease progression. Understanding the molecular mechanisms and cellular processes within the fascia microenvironment may serve as a basis for future clinical translation.

The Impact of Sciatic Nerve Injury on Extracellular Matrix of Lower Limb Muscle and Thoracolumbar Fascia: An Observational Study.

Peripheral nerve injury (PNI) is a complex clinical challenge resulting in functional disability. Neurological recovery does not always ensure functional recovery, as extracellular matrix (ECM) alterations affect muscle function. This study evaluates hyaluronan (HA) and collagen concentration in the gastrocnemius muscle and thoracolumbar fascia (TLF) in unilateral lower limb PNI rats to explore systemic ECM alterations following PNI and their impacts on functional recovery. Eighteen 8-week-old male Sprague-Dawley rats were divided into experimental (n = 12 left sciatic nerve injury) and control (n = 6) groups. After six weeks, motor function was evaluated. Muscle and TLF samples were analysed for HA and collagen distribution and concentrations. SFI and gait analysis confirmed a functional deficit in PNI rats 6 weeks after surgery. HA concentration in both sides of the muscles decreased by approximately one-third; both sides showed significantly higher collagen concentration than healthy rats (12.74 ± 4.83 µg/g), with the left (32.92 ± 11.34 µg/g) significantly higher than the right (20.15 ± 7.03 µg/g). PNI rats also showed significantly lower HA (left: 66.95 ± 20.08 µg/g; right: 112.66 ± 30.53 µg/g) and higher collagen (left: 115.89 ± 28.18 µg/g; right: 90.43 ± 20.83 µg/g) concentrations in both TLF samples compared to healthy rats (HA: 167.18 ± 31.13 µg/g; collagen: 47.51 ± 7.82 µg/g), with the left TLF more affected. Unilateral lower limb PNI induced HA reduction and collagen accumulation in both the lower limb muscles and the TLF, potentially exacerbating motor function impairment and increasing the risk of low back dysfunctions.

Effect of nanoparticle-mediated delivery of SFRP4 siRNA for treating Dupuytren disease.

Dupuytren disease (DD) is a progressive fibrous proliferative disease. It invades the palmar aponeurosis and extends to the finger fascia, eventually leading to flexion contracture of the metacarpophalangeal or interphalangeal joint. At present, surgical resection and the local injection of collagenase are the main methods for the treatment of DD, but postoperative complications and high recurrence rates often occur. Bioinformatics analysis showed that the increased expression of SFRP4 protein was closely related to the incidence of DD. Persistent and effective inhibition of SFRP4 expression may be a promising treatment for DD. We prepared SFRP4 siRNA/nanoparticle complexes (si-SFRP4) and negative siRNA/nanoparticle complexes (NC) and applied them in vitro and in vivo. Flow cytometry analysis showed that si-SFRP4 could be successfully transfected into DD cells. MTT and EdU staining assays showed that the OD values and percentage of EdU-positive cells in the si-SFRP4 group were significantly lower than those in the NC group. Scratch tests showed that the wound healing rate of the si-SFRP4 group was lower than that of the NC group, and the difference was statistically significant. The expression of SFRP4 and α-SMA protein in the si-SFRP4 group significantly decreased in both DD cells and xenografts. Compared with the NC group, the xenograft quality of the si-SFRP4 group was significantly reduced. Masson's trichrome staining showed that the collagen and fibrous cells in the si-SFRP4 group were more uniform, slender, parallel and regular. The above experimental results suggest that the proliferation and metabolism of palmar aponeurosis cells and the quality of metacarpal fascia xenografts were both significantly decreased. We speculated that nanoparticle-mediated SFRP4 siRNA can be used as a potential new method for the treatment of DD.

Proteomic Analysis of Dupuytren's Contracture-Derived Sweat Glands Revealed the Synthesis of Connective Tissue Growth Factor and Initiation of Epithelial-Mesenchymal Transition as Major Pathogenetic Events.

Dupuytren's contracture (DC) is a chronic and progressive fibroproliferative disorder restricted to the palmar fascia of the hands. Previously, we discovered the presence of high levels of connective tissue growth factor in sweat glands in the vicinity of DC nodules and hypothesized that sweat glands have an important role in the formation of DC lesions. Here, we shed light on the role of sweat glands in the DC pathogenesis by proteomic analysis and immunofluorescence microscopy. We demonstrated that a fraction of sweat gland epithelium underwent epithelial-mesenchymal transition illustrated by negative regulation of E-cadherin. We hypothesized that the increase in connective tissue growth factor expression in DC sweat glands has both autocrine and paracrine effects in sustaining the DC formation and inducing pathological changes in DC-associated sweat glands.

Pathophysiological and Therapeutic Roles of Fascial Hyaluronan in Obesity-Related Myofascial Disease.

Myofascial disease is an important complication associated with obesity and one of the leading causes of physical disability globally. In the face of limited treatment options, the burden of myofascial disorders is predicted to increase along with the escalating prevalence of obesity. Several pathological processes in obesity contribute to modifications in fascial extracellular matrix mechanical and biological properties and functions. Changes in adipose tissue metabolism, chronic inflammatory phenotype, oxidative stress, and other mechanisms in obesity may alter the physiochemical and biomechanical properties of fascial hyaluronan. Understanding the pathophysiological importance of hyaluronan and other components of the fascial connective tissue matrix in obesity may shed light on the etiology of associated myofascial disorders and inform treatment strategies. Given its unique and favorable pharmacological properties, hyaluronan has found a broad range of clinical applications, notably in orthopedic conditions such as osteoarthritis and tendinopathies, which share important pathophysiological mechanisms implicated in myofascial diseases. However, while existing clinical studies uniformly affirm the therapeutic value of hyaluronan in myofascial disorders, more extensive studies in broader pharmacological and clinical contexts are needed to firmly validate its therapeutic adaptation.

Fascial Innervation: A Systematic Review of the Literature.

Currently, myofascial pain has become one of the main problems in healthcare systems. Research into its causes and the structures related to it may help to improve its management. Until some years ago, all the studies were focused on muscle alterations, as trigger points, but recently, fasciae are starting to be considered a new, possible source of pain. This systematic review has been conducted for the purpose of analyze the current evidence of the muscular/deep fasciae innervation from a histological and/or immunohistochemical point of view. A literature search published between 2000 and 2021 was made in PubMed and Google Scholar. Search terms included a combination of fascia, innervation, immunohistochemical, and different immunohistochemical markers. Of the 23 total studies included in the review, five studies were performed in rats, four in mice, two in horses, ten in humans, and two in both humans and rats. There were a great variety of immunohistochemical markers used to detect the innervation of the fasciae; the most used were Protein Gene Marker 9.5 (used in twelve studies), Calcitonin Gene-Related Peptide (ten studies), S100 (ten studies), substance P (seven studies), and tyrosine hydroxylase (six studies). Various areas have been studied, with the thoracolumbar fascia being the most observed. Besides, the papers highlighted diversity in the density and type of innervation in the various fasciae, going from free nerve endings to Pacini and Ruffini corpuscles. Finally, it has been observed that the innervation is increased in the pathological fasciae. From this review, it is evident that fasciae are well innerved, their innervation have a particular distribution and precise localization and is composed especially by proprioceptors and nociceptors, the latter being more numerous in pathological situations. This could contribute to a better comprehension and management of pain.

Osteogenic Differentiation of Human Adipose-Derived Stem Cells Seeded on a Biomimetic Spongiosa-like Scaffold: Bone Morphogenetic Protein-2 Delivery by Overexpressing Fascia.

Human adipose-derived stem cells (hADSCs) have the capacity for osteogenic differentiation and, in combination with suitable biomaterials and growth factors, the regeneration of bone defects. In order to differentiate hADSCs into the osteogenic lineage, bone morphogenetic proteins (BMPs) have been proven to be highly effective, especially when expressed locally by route of gene transfer, providing a constant stimulus over an extended period of time. However, the creation of genetically modified hADSCs is laborious and time-consuming, which hinders clinical translation of the approach. Instead, expedited single-surgery gene therapy strategies must be developed. Therefore, in an in vitro experiment, we evaluated a novel growth factor delivery system, comprising adenoviral BMP-2 transduced fascia tissue in terms of BMP-2 release kinetics and osteogenic effects, on hADSCs seeded on an innovative biomimetic spongiosa-like scaffold. As compared to direct BMP-2 transduction of hADSCs or addition of recombinant BMP-2, overexpressing fascia provided a more uniform, constant level of BMP-2 over 30 days. Despite considerably higher BMP-2 peak levels in the comparison groups, delivery by overexpressing fascia led to a strong osteogenic response of hADSCs. The use of BMP-2 transduced fascia in combination with hADSCs may evolve into an expedited single-surgery gene transfer approach to bone repair.

Hyaluronan and the Fascial Frontier.

The buzz about hyaluronan (HA) is real. Whether found in face cream to increase water volume loss and viscoelasticity or injected into the knee to restore the properties of synovial fluid, the impact of HA can be recognized in many disciplines from dermatology to orthopedics. HA is the most abundant polysaccharide of the extracellular matrix of connective tissues. HA can impact cell behavior in specific ways by binding cellular HA receptors, which can influence signals that facilitate cell survival, proliferation, adhesion, as well as migration. Characteristics of HA, such as its abundance in a variety of tissues and its responsiveness to chemical, mechanical and hormonal modifications, has made HA an attractive molecule for a wide range of applications. Despite being discovered over 80 years ago, its properties within the world of fascia have only recently received attention. Our fascial system penetrates and envelopes all organs, muscles, bones and nerve fibers, providing the body with a functional structure and an environment that enables all bodily systems to operate in an integrated manner. Recognized interactions between cells and their HA-rich extracellular microenvironment support the importance of studying the relationship between HA and the body's fascial system. From fasciacytes to chronic pain, this review aims to highlight the connections between HA and fascial health.

Sensitivity of the Fasciae to the Endocannabinoid System: Production of Hyaluronan-Rich Vesicles and Potential Peripheral Effects of Cannabinoids in Fascial Tissue.

The demonstrated expression of endocannabinoid receptors in myofascial tissue suggested the role of fascia as a source and modulator of pain. Fibroblasts can modulate the production of the various components of the extracellular matrix, according to type of stimuli: physical, mechanical, hormonal, and pharmacological. In this work, fascial fibroblasts were isolated from small samples of human fascia lata of the thigh, collected from three volunteer patients (two men, one woman) during orthopedic surgery. This text demonstrates for the first time that the agonist of cannabinoid receptor 2, HU-308, can lead to in vitro production of hyaluronan-rich vesicles only 3-4 h after treatment, being rapidly released into the extracellular environment. We demonstrated that these vesicles are rich in hyaluronan after Alcian blue and Toluidine blue stainings, immunocytochemistry, and transmission electron microscopy. In addition, incubation with the antagonist AM630 blocked vesicles production by cells, confirming that release of hyaluronan is a cannabinoid-mediated effect. These results may show how fascial cells respond to the endocannabinoid system by regulating and remodeling the formation of the extracellular matrix. This is a first step in our understanding of how therapeutic applications of cannabinoids to treat pain may also have a peripheral effect, altering the biosynthesis of the extracellular matrix in fasciae and, consequently, remodeling the tissue and its properties.

Osteoinduction within BMP-2 transduced muscle tissue fragments with and without a fascia layer: implications for bone tissue engineering.

Bone can be engineered in vivo by implantation of gene-activated muscle tissue fragments. This expedited approach may be further improved by use of muscle tissue with attached fascia. The aim of this in vitro study was to provide an in depth comparison of the osteogenic differentiation capacity of muscle alone and muscle with fascia after BMP-2 transduction. Skeletal muscle tissue from rats was cut into pieces with and without a fascia layer on the surface. Adenoviral BMP-2 or GFP vectors were used for transduction. Osteogenic differentiation within the tissue fragments was evaluated and compared by qRT-PCR, alizarin red S staining, histomorphometry and immunohistology. Transduction efficiency and level of transgene expression were higher for muscle with fascia than muscle alone. Transduction with BMP-2 led to a significant upregulation of bone marker genes, proteins, and calcium deposition in both groups. Interestingly, histological evaluation revealed that osteoinduction did not occur within the fascia layer itself. The upregulation of bone marker genes in muscle with fascia was significantly lower after 2 weeks but similar after 4 weeks of in vitro culture in comparison to muscle alone. The fascia layer led to higher transduction efficiency and enhanced BMP-2 expression. Despite fascia's lower capacity for osteogenic differentiation, muscle implants may benefit from the fascia layer by the improved ability to deliver BMP-2. The presented data may contribute to the development of a novel, cost-effective, single-surgery bone engineering technology and encourage the evaluation of the osteoregenerative potential of muscle with fascia in an animal model.

The effect of the volume of supra-inguinal injected solution on the spread of the injectate under the fascia iliaca: a preliminary study.

The fascia iliaca compartment is the compartment confined by the fascia iliaca (FI) and a muscular layer formed by the iliac- and psoas muscle. This compartment creates a virtual tunnel that contains the femoral nerve (FN), the obturator nerve (ON), and the lateral femoral cutaneous nerve (LFCN) of the lumbar plexus. In this pilot study, we aimed to determine the suggested volume needed to reach the three target nerves of the lumbar plexus (FN, ON, and LFCN) with a single-injection ultrasound-guided supra-inguinal fascia iliaca compartment (S-FICB). A computer tomography (CT scan)-guided step-up/step-down sequence was used to determine the suggested injection volume to target all three nerves. Subsequently, an anatomist blinded for the injected volume and CT findings, dissected the cadavers, and evaluated the spread of dye underneath the fascia iliaca. In total, seven pelvic areas of four cadavers were evaluated on CT scan and dissected. Distribution of dye underneath the FI in relation to the FN, ON, and the LFCN was recorded in all dissected cadavers. Combining CT and dissection findings, the suggested volume to reach the FN, ON, and LFCN with an S-FICB was 40 mL.

Doxycycline shows dose-dependent changes in hernia repair strength after mesh repair.

BACKGROUND: Ventral hernia is a commonly occurring surgical problem. Our earlier studies have shown that a 30 mg/kg dose of doxycycline can significantly impact the strength of polypropylene (PP) mesh in a rat hernia repair model at 6 and 12 weeks. The objective of the present study was to investigate the dose dependence of doxycycline treatment on hernia repair strengths in rats. STUDY DESIGN: Fifty-six Sprague-Dawley rats underwent hernia repair with either PP mesh (n = 28) or sutures only (primary; n = 28); both groups were further divided into four doxycycline groups of seven animals each: control (0 mg/kg), low (3 mg/kg), medium (10 mg/kg), and high (30 mg/kg). One day before hernia repair surgery, animals received doxycycline doses by gavage and continued receiving daily until euthanasia. After 8 weeks, rats were euthanized and tissue samples from hernia repaired area were collected and analyzed for tensile strength using a tensiometer (Instron, Canton, MA, USA), while MMPs 2, 3, and 9, and collagen type 1 and 3 were analyzed by western blotting. RESULTS: In mesh-repaired animals, medium and high doxycycline dose repaired mesh fascia interface (MFI) showed significant increase in tensile strength when compared to control. In the primary repaired animals, there was no significant difference in MFI tensile strength in any dose group. In medium-dose MFI, there was a significant reduction in MMPs 2, 3, and 9. In this animal group, MFI showed significant increase in collagen 1 and significant reduction in collagen type 3 when compared to control. CONCLUSION: It is possible to improve the strength of mesh-repaired tissue by administering a significantly lower dose of the drug, which has implications for translation of the findings.

An analysis of the expression of collagen I and III genes in the fascia of obese patients.

BACKGROUND: The etiology of incisional hernias in the population of morbidly obese patients remains unclear. Most likely, factors other than purely mechanical are at play; it has been ascertained that nonobese patients suffering from inguinal and incisional hernias display alterations in the architecture of the connective tissue. The goal of this study has been to evaluate and compare the relative expression of collagen type I and III genes in the rectus abdominis muscle sheath (RMS) of obese and nonobese individuals to investigate their possible influence on the quality of the connective tissue. MATERIALS AND METHODS: RMS specimens were harvested in the early stages of either bariatric or non-bariatric laparotomies; total RNA was isolated and enzymatically purified from the tissue samples. The resulting material was subjected to a quantitative and qualitative analysis; reverse transcription reactions were then performed and the resulting complementary DNA was used in real-time reverse transcription polymerase chain reactions. The biopsy specimens were also examined by scanning electron microscopy. RESULTS: The real-time reverse transcription polymerase chain reactions, performed on complementary DNA, provided specific amplicons for individual genes. The efficacy of the reactions was rather low. An almost twofold decrease of the relative expression level for type I and III collagen was observed between the two patient groups; the results did not reach statistical significance. Scanning electron microscope photographs have documented a marked difference in the ultrastructure of the RMS in both groups. CONCLUSIONS: The authors have shown that changes in messenger RNA levels for collagen type I and III genes may be related to the pathogenesis of incisional hernia through alterations in the ultrastructure of the RMS fascia. Our report should be considered preliminary; the results should be verified on a larger group of patients.

[Metabolism of collagen in patients with Dupuitren's contracture].

Results of investigation of collagen metabolism in Dupuitren's contracture (DC) were summarized. The patients were operated for calculous cholecystitis and DC stages II - III. The changes revealed witnessed about more expressed degradation of collagen and affection of the elastin components of connective tissue. On background of the pathological process progress in palmar aponeurosis in patients, suffering DC, a content of oxyproline have enhanced trustworthy in urine and reduced in tissue of a changed palmar aponeurosis.

A transcriptomic analysis of incisional hernia based on high-throughput sequencing technology.

PURPOSE: Incisional hernia is a common postoperative complication; however, few transcriptomic studies have been conducted on it. In this study, we used second-generation high-throughput sequencing to explore the pathogenesis and potential therapeutic targets of incisional hernias. METHODS: Superficial fasciae were collected from 15 patients without hernia and 21 patients with an incisional hernia. High-throughput sequencing of the fascia was performed to generate an expression matrix. We analyzed the matrix to identify differentially expressed genes (DEGs) and performed gene ontology and enrichment analyses of these DEGs. Additionally, an external dataset was utilized to identify key DEGs. RESULTS: We identified 1,823 DEGs closely associated with extracellular matrix (ECM) imbalance, bacterial inflammatory response, and fibrillar collagen trimerization. TNNT3, CMAY5, ATP1B4, ASB5, CILP, SIX4, FBN1 and FNDC5 were identified as key DEGs at the intersection of the two expression matrices. Moreover, non-alcoholic fatty liver disease-related, TNF, and IL-17 signaling pathways were identified as key enrichment pathways. CONCLUSIONS: We identified eight key DEGs and three pathways associated with incisional hernias. Our findings offer new insights into the pathogenesis of incisional hernias and highlight potential targets for their prevention and treatment.

Proteomics analysis of deep fascia in acute compartment syndrome.

Acute compartment syndrome (ACS) is a syndrome in which local circulation is affected due to increased pressure within the compartment. We previously found in patients with calf fractures, the pressure of fascial compartment could be sharply reduced upon the appearance of tension blisters. Deep fascia, as the important structure for compartment, might play key role in this process. Therefore, the aim of the present study was to examine the differences in gene profile in deep fascia tissue in fracture patients of the calf with or without tension blisters, and to explore the role of fascia in pressure improvement in ACS. Patients with lower leg fracture were enrolled and divided into control group (CON group, n = 10) without tension blister, and tension blister group (TB group, n = 10). Deep fascia tissues were collected and LC-MS/MS label-free quantitative proteomics were performed. Genes involved in fascia structure and fibroblast function were further validated by Western blot. The differentially expressed proteins were found to be mainly enriched in pathways related to protein synthesis and processing, stress fiber assembly, cell-substrate adhesion, leukocyte mediated cytotoxicity, and cellular response to stress. Compared with the CON group, the expression of Peroxidasin homolog (PXDN), which promotes the function of fibroblasts, and Leukocyte differentiation antigen 74 (CD74), which enhances the proliferation of fibroblasts, were significantly upregulated (p all <0.05), while the expression of Matrix metalloproteinase-9 (MMP9), which is involved in collagen hydrolysis, and Neutrophil elastase (ELANE), which is involved in elastin hydrolysis, were significantly reduced in the TB group (p all <0.05), indicating fascia tissue underwent microenvironment reconstruction during ACS. In summary, the ACS accompanied by blisters is associated with the enhanced function and proliferation of fibroblasts and reduced hydrolysis of collagen and elastin. The adaptive alterations in the stiffness and elasticity of the deep fascia might be crucial for pressure release of ACS.

Widespread Vascularization and Correlation of Glycosaminoglycan Accumulation to Tendon Pain in Human Plantar Fascia Tendinopathy.

BACKGROUND: Plantar fasciitis is a painful tendinous condition (tendinopathy) with a high prevalence in athletes. While a healthy tendon has limited blood flow, ultrasound has indicated elevated blood flow in tendinopathy, but it is unknown if this is related to a de facto increase in the tendon vasculature. Likewise, an accumulation of glycosaminoglycans (GAGs) is observed in tendinopathy, but its relationship to clinical pain is unknown. PURPOSE: To explore to what extent vascularization, inflammation, and fat infiltration were present in patients with plantar fasciitis and if they were related to clinical symptoms. STUDY DESIGN: Descriptive laboratory study. METHODS: Biopsy specimens from tendinopathic plantar fascia tissue were obtained per-operatively from both the primary site of tendon pain and tissue swelling ("proximal") and a region that appeared macroscopically healthy at 1 to 2 cm away from the primary site ("distal") in 22 patients. Biopsy specimens were examined with immunofluorescence for markers of blood vessels, tissue cell density, fat infiltration, and macrophage level. In addition, pain during the first step in the morning (registered during an earlier study) was correlated with the content of collagen and GAGs in tissue. RESULTS: High vascularization (and cellularity) was present in both the proximal (0.89%) and the distal (0.96%) plantar fascia samples, whereas inconsistent but not significantly different fat infiltration and macrophage levels were observed. The collagen content was similar in the 2 plantar fascia regions, whereas the GAG content was higher in the proximal region (3.2% in proximal and 2.8% in distal; P = .027). The GAG content in the proximal region was positively correlated with the subjective morning pain score in the patients with tendinopathy (n = 17). CONCLUSION: In patients with plantar fasciitis, marked tissue vascularization was present in both the painful focal region and a neighboring nonsymptomatic area. In contrast, the accumulation of hydrophilic GAGs was greater in the symptomatic region and was positively correlated with increased clinical pain levels in daily life. CLINICAL RELEVANCE: The accumulation of GAGs in tissue rather than the extent of vascularization appears to be linked with the clinical degree of pain symptoms of the disease.

MMP-14 and MMP-2 are key metalloproteases in Dupuytren's disease fibroblast-mediated contraction.

Dupuytren's disease (DD) is a common fibrotic condition of the palmar fascia, leading to deposition of collagen-rich cords and progressive flexion of the fingers. The molecular mechanisms underlying the disease are poorly understood. We have previously shown altered expression of extracellular matrix-degrading proteases (matrix metalloproteases, MMPs, and 'a disintegrin and metalloprotease domain with thrombospondin motifs', ADAMTS, proteases) in palmar fascia from DD patients compared to control and shown that the expression of a sub-set of these genes correlates with post-operative outcome. In the current study we used an in vitro model of collagen contraction to identify the specific proteases which mediate this effect. We measured the expression of all MMPs, ADAMTSs and their inhibitors in fibroblasts derived from the palmar fascia of DD patients, both in monolayer culture and in the fibroblast-populated collagen lattice (FPCL) model of cell-mediated contraction. Key proteases, previously identified in our tissue studies, were expressed in vitro and regulated by tension in the FPCL, including MMP1, 2, 3, 13 and 14. Knockdown of MMP2 and MMP14 (but not MMP1, 3 and 13) inhibited cell-mediated contraction, and knockdown of MMP14 inhibited proMMP-2 activation. Interestingly, whilst collagen is degraded during the FPCL assay, this is not altered upon knockdown of any of the proteases examined. We conclude that MMP-14 (via its ability to activate proMMP-2) and MMP-2 are key proteases in collagen contraction mediated by fibroblasts in DD patients. These proteases may be drug targets or act as biomarkers for disease progression.

Cyclic loading of tendon fascicles using a novel fatigue loading system increases interleukin-6 expression by tenocytes.

Repetitive strain or 'overuse' is thought to be a major factor contributing to the development of tendinopathy. The aims of our study were to develop a novel cyclic loading system, and use it to investigate the effect of defined loading conditions on the mechanical properties and gene expression of isolated tendon fascicles. Tendon fascicles were dissected from bovine-foot extensors and subjected to cyclic tensile strain (1 Hz) at 30% or 60% of the strain at failure, for 0 h (control), 15 min, 30 min, 1 h, or 5 h. Post loading, a quasi-static test to failure assessed damage. Gene expression at a selected loading regime (1 h at 30% failure strain) was analyzed 6 h post loading by quantitative real-time polymerase chain reaction. Compared with unloaded controls, loading at 30% failure strain took 5 h to lead to a significant decrease in failure stress, whereas loading to 60% led to a significant reduction after 15 min. Loading for 1 h at 30% failure strain did not create significant structural damage, but increased Collagen-1-alpha-chain-1 and interleukin-6 (IL6) expression, suggesting a role of IL6 in tendon adaptation to exercise. Correlating failure properties with fatigue damage provides a method by which changes in gene expression can be associated with different degrees of fatigue damage.