A conserved strategy to attack collagen: The activator domain in bacterial collagenases unwinds triple-helical collagen.

Bacterial collagenases are important virulence factors, secreted by several pathogenic Clostridium, Bacillus, Spirochaetes, and Vibrio species. Yet, the mechanism by which these enzymes cleave collagen is not well understood. Based on biochemical and mutational studies we reveal that collagenase G (ColG) from Hathewaya histolytica recognizes and processes collagen substrates differently depending on their nature (fibrillar vs. soluble collagen); distinct dynamic interactions between the activator and peptidase domain are required based on the substrate type. Using biochemical and circular dichroism studies, we identify the presumed noncatalytic activator domain as the single-domain triple helicase that unwinds collagen locally, transiently, and reversibly.

Enhancement of skin rejuvenation and hair growth through novel near-infrared light emitting diode (nNIR) lighting: in vitro and in vivo study.

The study aimed to explore the impact of a novel near-infrared LED (nNIR) with an extended spectrum on skin enhancement and hair growth. Various LED sources, including White and nNIRs, were compared across multiple parameters: cytotoxicity, adenosine triphosphate (ATP) synthesis, reactive oxygen species (ROS) reduction, skin thickness, collagen synthesis, collagenase expression, and hair follicle growth. Experiments were conducted on human skin cells and animal models. Cytotoxicity, ATP synthesis, and ROS reduction were evaluated in human skin cells exposed to nNIRs and Whites. LED irradiation effects were also studied on a UV-induced photoaging mouse model, analyzing skin thickness, collagen synthesis, and collagenase expression. Hair growth promotion was examined as well. Results revealed both White and nNIR were non-cytotoxic to human skin cells. nNIR enhanced ATP and collagen synthesis while reducing ROS levels, outperforming the commonly used 2chip LEDs. In the UV-induced photoaging mouse model, nNIR irradiation led to reduced skin thickness, increased collagen synthesis, and lowered collagenase expression. Additionally, nNIR irradiation stimulated hair growth, augmented skin thickness, and increased hair follicle count. In conclusion, the study highlighted positive effects of White and nNIR irradiation on skin and hair growth. However, nNIR exhibited superior outcomes compared to White. Its advancements in ATP content, collagen synthesis, collagenase inhibition, and hair growth promotion imply increased ATP synthesis activity. These findings underscore nNIR therapy's potential as an innovative and effective approach for enhancing skin and promoting hair growth.

The combination of hyaluronic acid and collagenase in the treatment of skin ulcers: an open, multicenter clinical study assessing safety and tolerability of Bionect Start®.

OBJECTIVE: Several clinical studies have shown that hyaluronic acid collagenase is well-tolerated and very effective in managing chronic venous ulcers. The aim of the present study is to confirm the safety and tolerability of daily application in patients suffering from cutaneous ulcers of different etiologies. The efficacy of the treatment and its impact on patients' quality of life are also assessed. PATIENTS AND METHODS: Patients with a clinical diagnosis of skin ulcer with devitalized/fibrinous/slough tissue that could delay the healing process were enrolled in the study. The hyaluronic acid/collagenase ointment was applied topically until wound closure or total debridement of non-viable tissue was achieved, however, with a limit of 30 days. Monitoring was performed weekly, either through outpatient visits or telephone surveys. Assessments included adverse events, local irritation reactions, pain at dressing changes, and wound bed status. Patients were also requested to complete a quality-of-life questionnaire. RESULTS: The study involved 96 patients with a mean age of 71 years. The patients suffered mainly from traumatic (21.9%), venous (15.6%), or pressure ulcers (12.5%); in 26% of cases, ulcers had mixed etiology. In approximately 32% of patients, the ulcer had been present for more than 6 months, and 18.1% of subjects had previously undergone surgical wound debridement. CONCLUSIONS: Daily application of hyaluronic acid-collagenase achieved the following results: i) absence of adverse events related to the use of the product; ii) significant reduction in the degree of localized irritation and pain at dressing changes; iii) significant support to wound bed preparation; iv) trend towards improvement in the quality of life and health status of the patients.

Application of the galactomannan gel from Cassia grandis seeds for biomedical purposes: Study of the incorporation of collagenases and their release profile.

The galactomannan-based gel from Cassia grandis seeds was used to incorporate Penicillium sp. UCP 1286 and commercial collagenases. Experiments were carried out according to a 23-full factorial design to identify the most significant parameters for the incorporation process. The pH of the incorporation solution (pHi), stirring time (t), and initial protein concentration in the crude extract (PCi) were selected as the three independent variables, and the efficiency of collagenase incorporation (E) and collagenolytic activity (CA) after 360 min as the responses. pHi and PCi showed positive statistically significant effects on E, while CA was positively influenced by pHi and t, but negatively by PCi. The fungi collagenase was released from the gel following a pseudo-Fickian behavior. Additionally, no <76 % of collagenase was efficiently incorporated into the gel retaining a high CA (32.5-69.8 U/mL). The obtained results for the commercial collagenase (E = 93.88 %, CA = 65.8 U/mL, and n = 0.10) demonstrated a pseudo-Fickian behavior similar to the fungi-collagenase. The results confirm the biotechnological potential of the gel as an efficient matrix for the incorporation of catalytic compounds; additionally, the incorporation of collagenases was achieved by retaining the proteases CA and releasing them in a controlled manner.

Miao medicine Gu Yan Xiao tincture inhibits mTOR to stimulate chondrocyte autophagy in a rabbit model of osteoarthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: The Gu Yan Xiao tincture, a blend of traditional Chinese herbs, is traditionally used for osteoarthritis and related pain. This study investigated its mechanism of action in order to rationalize and validate its therapeutic use. AIM OF THE STUDY: This study analyzed, in a rabbit model of knee osteoarthritis, whether and how Gu Yan Xiao tincture exerts therapeutic benefits by modulating chondrocyte autophagy. MATERIALS AND METHODS: The active constituents within the GYX tincture were identified using liquid chromatography-mass spectrometry. The rabbit model was established by injecting animals with type II collagenase intra-articularly, and the effects of topically applied tincture were examined on osteoarthritis lesions of the knee using histopathology, micro-computed tomography and x-ray imaging. Effects of the tincture were also evaluated on levels of inflammatory cytokines, matrix metalloproteases, and autophagy in chondrocytes. As a positive control, animals were treated with sodium diclofenac. RESULTS: The tincture mitigated the reduction in joint space, hyperplasia of the synovium and matrix metalloproteases in serum that occurred after injection of type II collagenase in rabbits. These therapeutic effects were associated with inhibition of mTOR and activation of autophagy in articular chondrocytes. Inhibiting mTOR with rapamycin potentiated the therapeutic effects of the tincture, while inhibiting autophagy with 3-methyladenine antagonized them. CONCLUSIONS: Gu Yan Xiao tincture mitigates tissue injury in a rabbit model of osteoarthritis, at least in part by inhibiting mTOR and thereby promoting autophagy in chondrocytes. These results rationalize the use of the tincture not only against osteoarthritis but also potentially other diseases involving inhibition of autophagy in bones and joints.

Evaluation of Collagenase Activity from Crab Hepatopancreas in Different Model Systems.

The effect of Kamchatka crab hepatopancreas containing three collagenolytic isoenzymes Collagenase KK and proteinases of Streptomyces lavendulae on metabolic activity and cell death were carried out on in vitro models. It was shown that changes in the protein structure under the influence of Collagenase KK occurred earlier than under the effect of bacterial proteinases. At the same time, activity of Collagenase KK was significantly higher than that of bacterial proteinases (p<0.01). Both preparations had a pronounced time- and dose-dependent effects on metabolic activity of cells. Collagenase KK had low cytotoxic effect, and cells mainly died by apoptosis. Thus, hepatopancreas collagenase has a high activity and proapoptotic effect on cells and can be used in low concentrations for enzymatic disaggregation of tissues.

Forward Light Scattering of the Vitreous Gel After Enzymatic Aging: An In Vitro Model to Study Vitreous Opacification.

Purpose: Symptomatic vitreous opacifications, so-called floaters, are difficult to objectively assess majorly limiting the possibility of in vitro studies. Forward light scattering was found previously to be increased in eyes with symptomatic floaters. Using an objective setup to measure forward light scattering, we studied the effects of enzymatically digesting the components of the vitreous body on straylight to develop an in vitro model of vitreous opacifications. Methods: Fifty-seven porcine vitreous bodies were digested using hyaluronidase, collagenase, trypsin, and bromelain, as well as using a combination of hyaluronidase + collagenase and hyaluronidase + bromelain. A modified C-Quant setup was used to objectively assess forward light scattering. Results: Depletion of hyaluronic acid majorly increased vitreous straylight (mean increase 34.4 deg2/sr; P = 0.01), whereas primarily digesting the vitreous gel with collagenase or trypsin did not significantly affect straylight. When collagenase or bromelain is applied in hyaluronic acid depleted vitreous gels, the increase in forward light scattering is reversed partially. Conclusions: The age-related loss of hyaluronic acid primarily drives the increase in vitreous gel straylight induced by conglomerates of collagen. This process can be reversed partially by digesting collagen. This in vitro model allows the objective quantification and statistical comparison of straylight burden caused by vitreous opacities and, thus, can serve as a first testing ground for pharmacological therapies, as demonstrated with bromelain.

An injectable thermosensitive hyaluronic acid/pluronic F-127 hydrogel for deep penetration and combination therapy of frozen shoulder.

Frozen shoulder (FS) is a common and progressive shoulder disorder that causes glenohumeral joint stiffness, characterized by inflammation and fibrosis. The treatment options are quite limited, and the therapeutic response is hindered by the fibrous membrane formed by excessive collagen and the rapid removal by synovial fluid. To address these challenges, we designed a hyaluronic acid/Pluronic F-127 (HP)-based injectable thermosensitive hydrogel as a drug carrier loaded with dexamethasone and collagenase (HPDC). We screened for an optimal HP hydrogel that can sustain drug release for approximately 10 days both in vitro and in vivo. In the meanwhile, we found that HP hydrogel could inhibit the proliferation and diminish the adhesion capacity of rat synovial cells induced by transforming growth factor-ß1. Furthermore, using an established immobilization rat model of FS, intra-articular injection of HPDC significantly improved joint range of motion compared to medication alone. Relying on sustained drug release, the accumulated collagen fibers were degraded by collagenase to promote the deep delivery of dexamethasone. These findings showed a positive combined treatment effect of HPDC, providing a novel idea for the comprehensive treatment of FS.

Anti-collagenase, Anti-elastase, Anti-urease, and Anti-cancer Potentials of Isokaempferide as Natural Compound: In vitro and in silico Study.

One of the main goals of medicinal chemistry in recent years has been the development of new enzyme inhibitors and anti-cancer medicines. The isokaempferide' ability to inhibit the enzymes urease, elastase, and collagenase were also studied. The results showed that isokaempferide was the most effective compound against the assigned enzymes, with IC 50 values of 23.05 µM for elastase, 12.83 µM for urease, and 33.62 µM for collagenase respectively. It should be emphasized that natural compound was more effective at inhibiting some enzymes. Additionally, the compound was tested for their anti-cancer properties using colon, lung, breast cancer cell lines. The chemical activities of isokaempferide against urease, collagenase, and elastase were investigated utilizing the molecular docking study. The anti-cancer activities of the compound were evaluated against lung cancer cells such as SPC-A-1, SK-LU-1, 95D, breast cancer cells like MCF7, Hs 578Bst, Hs 319.T, and UACC-3133 cell lines, and colon cancer cell lines like CL40, SW1417, LS1034, and SW480. The chemical activities of isokaempferide against some of the expressed surface receptor proteins (EGFR, estrogen receptor, CD47, progesterone receptor, folate receptor, CD44, HER2, CD155, CXCR4, CD97, and endothelin receptor) in the mentioned cell lines were assessed using the molecular docking calculations. The results showed the probable interactions and their characteristics at an atomic level. The docking scores revealed that isokaempferide has a strong binding affinity to the enzymes and proteins. In addition, the compound formed powerful contact with the enzymes and receptors. Thus, isokaempferide could be potential inhibitor for enzymes and cancer cells.

Differential Protease Specificity by Collagenase as a Novel Approach to Serum Proteomics That Includes Identification of Extracellular Matrix Proteins without Enrichment.

Circulating extracellular matrix (ECM) proteins are serological biomarkers of interest due to their association with pathologies involving disease processes such as fibrosis and cancers. In this study, we investigate the potential for serum biomarker research using differential protease specificity (DPS), leveraging alternate protease specificity as a targeting mechanism to selectively digest circulating ECM protein serum proteins. A proof-of-concept study is presented using serum from patients with cirrhotic liver or hepatocellular carcinoma. The approach uses collagenase DPS for digestion of deglycosylated serum and liquid-chromatography-trapped ion mobility-tandem mass spectrometry (LC-TIMS-MS/MS) to enhance the detection of ECM proteins in serum. It requires no sample enrichment and minimizes the albumin average precursor intensity readout to less than 1.2%. We further demonstrate the capabilities for using the method as a high-throughput matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS) assay coupled with reference library searching. A goal is to improve the depth and breadth of biofluid proteomics for noninvasive assays.

Early delivery of human umbilical cord mesenchymal stem cells improves healing in a rat model of Achilles tendinopathy.

Objective: This study aimed to explore the efficacy and optimal delivery time of human umbilical cord mesenchymal stem cells (hUC-MSCs) in treating collagenase-induced Achilles tendinopathy. Methods: Achilles tendinopathy in rats at early or advanced stages was induced by injecting collagenase I into bilateral Achilles tendons. A total of 28 injured rats were injected with a hUC-MSC solution or normal saline into bilateral tendons twice and sampled after 4 weeks for histological staining, gene expression analysis, transmission electron microscope assay and biomechanical testing analysis. Results: The results revealed better histological performance and a larger collagen fiber diameter in the MSC group. mRNA expression of TNF-α, IL-1ß and MMP-3 was lower after MSC transplantation. Early MSC delivery promoted collagen I and TIMP-3 synthesis, and strengthened tendon toughness. Conclusion: hUC-MSCs demonstrated a therapeutic effect in treating collagenase-induced Achilles tendinopathy, particularly in the early stage of tendinopathy.

Surgery, Needle Fasciotomy, or Collagenase Injection for Dupuytren Contracture : A Randomized Controlled Trial.

BACKGROUND: Surgery, needle fasciotomy, and collagenase injection are used to treat Dupuytren contracture. The treatment decision requires balancing initial morbidity and costs of surgery against its potential long-term benefits over needle fasciotomy and collagenase. OBJECTIVE: To compare the effectiveness of surgery, needle fasciotomy, and collagenase injection at 3 months and 2 years (secondary time points of the trial). DESIGN: A multicenter, randomized, outcome assessor-blinded, superiority trial. (ClinicalTrials.gov: NCT03192020). SETTING: 6 public hospitals in Finland. PARTICIPANTS: 302 persons with treatment-naive Dupuytren contracture (contracture angle <135°). INTERVENTION: Surgery (n = 101), needle fasciotomy (n = 101), or collagenase (n = 100). MEASUREMENTS: The primary outcome was the success rate, defined as greater than 50% contracture release and patients reaching the patient acceptable symptom state. Secondary outcomes included hand function, pain, quality of life, patient satisfaction, residual contracture angle, finger flexion, risk for retreatment, and serious adverse events. RESULTS: A total of 292 (97%) and 284 (94%) participants completed the 3-month and 2-year follow-ups. Success rates were similar at 3 months: 71% (95% CI, 62% to 80%) for surgery, 73% (CI, 64% to 82%) for needle fasciotomy, and 73% (CI, 64% to 82%) for collagenase. At 2 years, surgery had superior success rates compared with both needle fasciotomy (78% vs. 50%; adjusted risk difference [aRD], 0.30 [CI, 0.17 to 0.43]) and collagenase (78% vs. 65%; aRD, 0.13 [CI, 0.01 to 0.26]). Secondary analyses paralleled with the primary analysis. LIMITATION: Participants were not blinded. CONCLUSION: Initial outcomes are similar between the treatments, but at 2 years success rates were maintained in the surgery group but were lower with both needle fasciotomy and collagenase despite retreatments. PRIMARY FUNDING SOURCE: Research Council of Finland.

Revisiting rabbit models for keratoconus: A long-term study on collagenase-induced disease progression.

Keratoconus (KC) is a degenerative disorder resulting from the degradation of the stromal collagen fibril network in the cornea, leading to its thinning and conical deformation. Various studies have established animal models of KC by using the collagenase type II enzyme to gain a better understanding of the pathogenesis, however, long-term monitoring or follow-up of the models have not been reported so far. This study evaluates the long-term stability of collagenase type II-induced KC in a rabbit model. Six New Zealand rabbits were divided into 4 study groups with 3 eyes per group. The groups were control (group 1), 0.5% proparacaine + 5 min collagenase treatment on day 0 and day 30 (group 2), 0.5% proparacaine + 10 min collagenase treatment on day 0 (group 3) and, mechanical debridement + 2 min collagenase treatment on day 0 (group 4). Inflammation was observed in group 4 till week 10. Significant decrease in the central corneal thickness was observed in group 3 by week 4 (p < 0.001) however, the thickness was regained in the subsequent follow-ups in all the groups. Keratography results showed no changes in Km values but an increased astigmatic power in all groups. Scanning electron microscopy images revealed thinner collagen fibrils arranged in a mesh-like pattern above the uniform layer of the collagen lamellae in the central part of the treated corneas. Similarly, histological staining revealed loosely packed stromal fibrils in the anterior portion of the cornea which corroborates with the immunofluorescent staining results. This study revealed the remodeling of the corneal structure by eight weeks of collagenase treatment. Consequently, the possibility of creating a rabbit keratoconus model induced by collagenase may warrant further consideration.

Isolation and characterization of rabbit limbal niche cells.

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs. Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRß) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRß and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRß in rabbit-LNCs P4. In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRß+).

Enhanced molecular release from elderly bone samples using collagenase I: insights into fatty acid metabolism alterations.

BACKGROUND: Bone is a metabolically active tissue containing different cell types acting as endocrine targets and effectors. Further, bone is a dynamic depot for calcium, phosphorous and other essential minerals. The tissue matrix is subjected to a constant turnover in response to mechanical/endocrine stimuli. Bone turnover demands high energy levels, making fatty acids a crucial source for the bone cells. However, the current understanding of bone cell metabolism is poor. This is partly due to bone matrix complexity and difficulty in small molecules extraction from bone samples. This study aimed to evaluate the effect of metabolite sequestering from a protein-dominated matrix to increase the quality and amount of metabolomics data in discovering small molecule patterns in pathological conditions. METHODS: Human bone samples were collected from 65 to 85 years old (the elderly age span) patients who underwent hip replacement surgery. Separated cortical and trabecular bone powders were treated with decalcifying, enzymatic (collagenase I and proteinase K) and solvent-based metabolite extraction protocols. The extracted mixtures were analyzed with the high-resolution mass spectrometry (HRMS). Data analysis was performed with XCMS and MetaboAnalystR packages. RESULTS: Fast enzymatic treatment of bone samples before solvent addition led to a significantly higher yield of metabolite extraction. Collagenase I and proteinase K rapid digestion showed more effectiveness in cortical and trabecular bone samples, with a significantly higher rate (2.2 folds) for collagenase I. Further analysis showed significant enrichment in pathways like de novo fatty acid biosynthesis, glycosphingolipid metabolism and fatty acid oxidation-peroxisome. CONCLUSION: This work presents a novel approach for bone sample preparation for HRMS metabolomics. The disruption of bone matrix conformation at the molecular level helps the molecular release into the extracting solvent and, therefore, can lead to higher quality results and trustable biomarker discovery. Our results showed ß-oxidation alteration in the aged bone sample. Future work covering more patients is worthy to identify the effective therapeutics to achieve healthy aging.

Isolation of Primary Mouse Hepatocytes and Non-Parenchymal Cells from a Liver with Precancerous Lesions.

In the early stages of liver carcinogenesis, rare hepatocytes and cholangiocytes are transformed into preneoplastic cells, which can progressively acquire a neoplastic phenotype, favored by the failure of natural antitumor immunosurveillance. The detailed study of both hepatic parenchymal (e.g., hepatocytes) and non-parenchymal cells (NPCs), such as immune cells, could help understand the cellular microenvironment surrounding these pre-cancerous and neoplastic lesions.Cultures of primary hepatocytes are of interest in various biomedical research disciplines, serving as an ex vivo model for liver physiology. Obtaining high viability and yield of primary mouse hepatocytes and other liver cell populations is technically challenging, thus limiting their use. In the first section of the current chapter, we introduce a protocol based on the two-step collagenase perfusion technique (by inferior vena cava) to isolate hepatocytes and, to a lower extent, NPCs and detailed the different considerations to take into account for a successful perfusion. The liver is washed by perfusion, hepatocytes are dissociated with collagenase, and different cell populations are separated by centrifugation. Various techniques have been described for the isolation of healthy and malignant hepatocytes; however, the viability and purity of the isolated cells is frequently not satisfactory. Here, we significantly optimized this protocol to reach improved yield and viability of the hepatocytes and concomitantly obtain preserved NPC populations of the liver.Within NPCs, tissue-resident or recruited immune cells are essential actors regulating hepatocarcinogenesis. However, simultaneous isolation of hepatic leukocytes together with other cell types generally yields low immune cell numbers hindering downstream application with these cells. In the second section of this chapter, as opposed to the first section primarily aiming to isolate hepatocytes, we present a tissue dissociation protocol adapted to efficiently recover leukocytes from non-perfused bulk (pre-)cancerous livers. This protocol has been optimized to be operator-friendly and fast compared to other liver processing methods, allowing easy simultaneous sample processing to retrieve hepatic (tumor-infiltrating) immune cells.

A Novel Tendon Injury Model, Induced by Collagenase Administration Combined with a Thermo-Responsive Hydrogel in Rats, Reproduces the Pathogenesis of Human Degenerative Tendinopathy.

Patellar tendinopathy is a common clinical problem, but its underlying pathophysiology remains poorly understood, primarily due to the absence of a representative experimental model. The most widely used method to generate such a model is collagenase injection, although this method possesses limitations. We developed an optimized rat model of patellar tendinopathy via the ultrasound-guided injection of collagenase mixed with a thermo-responsive Pluronic hydrogel into the patellar tendon of sixty male Wistar rats. All analyses were carried out at 3, 7, 14, 30, and 60 days post-injury. We confirmed that our rat model reproduced the pathophysiology observed in human patients through analyses of ultrasonography, histology, immunofluorescence, and biomechanical parameters. Tendons that were injured by the injection of the collagenase-Pluronic mixture exhibited a significant increase in the cross-sectional area (p < 0.01), a high degree of tissue disorganization and hypercellularity, significantly strong neovascularization (p < 0.01), important changes in the levels of types I and III collagen expression, and the organization and presence of intra-tendinous calcifications. Decreases in the maximum rupture force and stiffness were also observed. These results demonstrate that our model replicates the key features observed in human patellar tendinopathy. Collagenase is evenly distributed, as the Pluronic hydrogel prevents its leakage and thus, damage to surrounding tissues. Therefore, this model is valuable for testing new treatments for patellar tendinopathy.

Collagenase-based wound debridement agent induces extracellular matrix supporting phenotype in macrophages.

Macrophages assume diverse phenotypes and functions in response to cues from the microenvironment. Earlier we reported an anti-inflammatory effect of Collagenase Santyl® Ointment (CSO) and the active constituent of CSO (CS-API) on wound macrophages in resolving wound inflammation indicating roles beyond debridement in wound healing. Building upon our prior finding, this study aimed to understand the phenotypes and subsets of macrophages following treatment with CS-API. scRNA-sequencing was performed on human blood monocyte-derived macrophages (MDM) following treatment with CS-API for 24 h. Unbiased data analysis resulted in the identification of discrete macrophage subsets based on their gene expression profiles. Following CS-API treatment, clusters 3 and 4 displayed enrichment of macrophages with high expression of genes supporting extracellular matrix (ECM) function. IPA analysis identified the TGFß-1 pathway as a key hub for the CS-API-mediated ECM-supportive phenotype of macrophages. Earlier we reported the physiological conversion of wound-site macrophages to fibroblasts in granulation tissue and impairment of such response in diabetic wounds, leading to compromised ECM and tensile strength. The findings that CSO can augment the physiological conversion of macrophages to fibroblast-like cells carry significant clinical implications. This existing clinical intervention, already employed for wound care, can be readily repurposed to improve the ECM response in chronic wounds.

[Anisotropy and viscoelasticity of different corneal regions in rabbit corneal ectasia model].

The mechanical properties of the cornea in corneal ectasia disease undergo a significant reduction, yet the alterations in mechanical properties within distinct corneal regions remain unclear. In this study, we established a rabbit corneal ectasia model by employing collagenase II to degrade the corneal matrix within a central diameter of 6 mm. Optical coherence tomography was employed for the in vivo assessment of corneal morphology (corneal thickness and corneal curvature) one month after operation. Anisotropy and viscoelastic characteristics of corneal tissue were evaluated through biaxial and uniaxial testing, respectively. The results demonstrated a marked decrease in central corneal thickness, with no significant changes observed in corneal curvature. Under different strains, the elastic modulus of the cornea exhibited no significant differences in the up-down and naso-temporal directions between the control and model groups. However, the cornea in the model group displayed a significantly lower elastic modulus compared to the control group. Specifically, the elastic modulus of the central region cornea in the model group was significantly lower than that of the entire cornea within the same group. Moreover, in comparison to the control group, the cornea in the model group exhibited a significant increase in both creep rate and overall deformation rate. The instantaneous modulus and equilibrium modulus were significantly reduced in the model cornea. No significant differences were observed between the entire cornea and the central cornea concerning these parameters. The results indicate that corneal anisotropy remains unchanged in collagenase-induced ectatic cornea. However, a significant reduction in viscoelastic properties is noticed. This study provides valuable insights for investigating changes in corneal mechanical properties within different regions of ectatic corneal disease.

Effect of accelerated high-fluence riboflavin and rose bengal-mediated corneal cross-linking on resistance to enzymatic digestion.

PURPOSE: This study evaluated the effect of high-fluence accelerated corneal cross-linking on the resistance to enzymatic digestion, assessing two chromophore/light combinations: riboflavin/UV-A light (RF/UV-A) and rose bengal/green light (RB/green). METHODS: Freshly prepared ex-vivo porcine corneas (n = 189) were divided into 8 groups groups. Group A corneas were unirradiated controls without chromophore soaking (A0), or soaked with riboflavin (A1) or rose bengal (A2). Group B corneas underwent accelerated epi-off RF/UV-A CXL at fluences of 5.4 J/cm² (B1), 10 J/cm² (B2), or 15 J/cm² (B3). Group C corneas underwent accelerated epi-off RB/green CXL at fluences of either 10 J/cm² (C1) or 15 J/cm² (C2). Following CXL, all corneas were digested in 0.3% collagenase-A solution, and the time until complete dissolution was measured. RESULTS: Non-irradiated controls exposed to RF and RB enhanced corneal resistance to collagenase digestion, with RB having a stronger effect than RF. RF/UV-A-treated corneas showed significantly increased digestion resistance with increasing fluence levels. RB/green-treated corneas displayed enhanced digestion resistance with each increase in fluence up to 10 J/cm²; a 15 J/cm² fluence yielded similar digestion resistance times to a 10 J/cm² fluence, suggesting a plateau effect in accelerated RB/green CXL protocols. CONCLUSIONS: When compared to standard-fluence treatments, high-fluence accelerated epi-off CXL using both riboflavin and rose bengal significantly increases resistance to enzymatic digestion. The optimal settings for clinical protocols might be 15 J/cm² (30 mW/cm² for 8 min 20 s) for RF/UV-A and 10 J/cm² (15 mW/cm² for 11 min 7 s) for RB/Green Light.