Engineering a dysbiotic biofilm model for testing root caries interventions through microbial modulation.

BACKGROUND: This study aimed to engineer and optimise a dysbiotic biofilm model to develop in vitro root caries for investigating microbial modulation strategies. The model involved growing complex biofilms from a saliva inoculum collected from four volunteers using two strategies. In the first strategy ("pre-treatment strategy"), bovine root slabs were used, and two natural compounds were incorporated at time 0 of the 10-day biofilm experiment, which included sucrose cycles mimicking the cariogenic environment. In the second strategy ("post-treatment strategy"), mature biofilms were grown in a modified Calgary biofilm device coated with collagen and hydroxyapatite for 7 days and then were exposed to the same natural compounds. The metatranscriptome of each biofilm was then determined and analysed. Collagenase activity was examined, and the biofilms and dentine were imaged using confocal and scanning electron microscopy (SEM). Mineral loss and lesion formation were confirmed through micro-computed tomography (µ-CT). RESULTS: The pH confirmed the cariogenic condition. In the metatranscriptome, we achieved a biofilm compositional complexity, showing a great diversity of the metabolically active microbiome in both pre- and post-treatment strategies, including reads mapped to microorganisms other than bacteria, such as archaea and viruses. Carbohydrate esterases had increased expression in the post-treated biofilms and in samples without sugar cycles, while glucosyltransferases were highly expressed in the presence of sucrose cycles. Enrichment for functions related to nitrogen compound metabolism and organic cyclic component metabolism in groups without sucrose compared to the sucrose-treated group. Pre-treatment of the roots with cranberry reduced microbial viability and gelatinase (but not collagenase) activity (p < 0.05). SEM images showed the complexity of biofilms was maintained, with a thick extracellular polysaccharides layer. CONCLUSIONS: This root caries model was optimized to produce complex cariogenic biofilms and root caries-like lesions, and could be used to test microbial modulation in vitro. Pre-treatments before biofilm development and cariogenic challenges were more effective than post-treatments. The clinical significance lies in the potential to apply the findings to develop varnish products for post-professional tooth prophylaxis, aiming at implementing a strategy for dysbiosis reversal in translational research. Video Abstract.

Enhancing uterine receptivity for embryo implantation through controlled collagenase intervention.

Ineffective endometrial matrix remodeling, a key factor in infertility, impedes embryo implantation in the uterine wall. Our study reveals the cellular and molecular impact of human collagenase-1 administration in mouse uteri, demonstrating enhanced embryo implantation rates. Collagenase-1 promotes remodeling of the endometrial ECM, degrading collagen fibers and proteoglycans. This process releases matrix-bound bioactive factors (e.g., VEGF, decorin), facilitating vascular permeability and angiogenesis. Collagenase-1 elevates embryo implantation regulators, including NK cell infiltration and the key cytokine LIF. Remarkably, uterine tissue maintains structural integrity despite reduced endometrial collagen fiber tension. In-utero collagenase-1 application rescues implantation in heat stress and embryo transfer models, known for low implantation rates. Importantly, ex vivo exposure of human uterine tissue to collagenase-1 induces collagen de-tensioning and VEGF release, mirroring remodeling observed in mice. Our research highlights the potential of collagenases to induce and orchestrate cellular and molecular processes enhancing uterine receptivity for effective embryo implantation. This innovative approach underscores ECM remodeling mechanisms critical for embryo implantation.

Effects of Pulsed Electromagnetic Field Treatment on Skeletal Muscle Tissue Recovery in a Rat Model of Collagenase-Induced Tendinopathy: Results from a Proteome Analysis.

Tendon disorders often result in decreased muscle function and atrophy. Pulsed Electromagnetic Fields (PEMFs) have shown potential in improving tendon fiber structure and muscle recovery. However, the molecular effects of PEMF therapy on skeletal muscle, beyond conventional metrics like MRI or markers of muscle decline, remain largely unexplored. This study investigates the metabolic and structural changes in PEMF-treated muscle tissue using proteomics in a rat model of Achilles tendinopathy induced by collagenase. Sprague Dawley rats were unilaterally induced for tendinopathy with type I collagenase injection and exposed to PEMFs for 8 h/day. Gastrocnemius extracts from untreated or PEMF-treated rats were analyzed with LC-MS/MS, and proteomics differential analysis was conducted through label-free quantitation. PEMF-treated animals exhibited decreased glycolysis and increased LDHB expression, enhancing NAD signaling and ATP production, which boosted respiratory chain activity and fatty acid beta-oxidation. Antioxidant protein levels increased, controlling ROS production. PEMF therapy restored PGC1alpha and YAP levels, decreased by tendinopathy. Additionally, myosins regulating slow-twitch fibers and proteins involved in fiber alignment and force transmission increased, supporting muscle recovery and contractile function. Our findings show that PEMF treatment modulates NAD signaling and oxidative phosphorylation, aiding muscle recovery through the upregulation of YAP and PGC1alpha and increasing slow myosin isoforms, thus speeding up physiological recovery.

Apo-Lactoferrin Inhibits the Proteolytic Activity of the 110 kDa Zn Metalloprotease Produced by Mannheimia haemolytica A2.

Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.

Pilot Study for Isolation of Stromal Vascular Fraction with Collagenase Using an Automated Processing System.

There are many potential therapeutic applications for autologous adipose-derived stromal cells. These cells are found in a heterogeneous population isolated from adipose tissue called the stromal vascular fraction (SVF). Closed automated systems are available to release cells from the adherent stroma. Here, we test one system to evaluate the heterogeneous output for yield, purity, cellular characterization, and stemness criteria. The SVF was isolated from three donors using the Automated Cell Station (ACS) from BSL Co., Ltd., Busan, Republic of Korea. The SVF cellular output was characterized for cell yield and viability, immunophenotyping analysis, pluripotent differentiation potential, adhesion to plastic, and colony-forming units. Additionally, the SVF was tested for endotoxin and collagenase residuals. The SVF yield from the ACS system was an average volume of 7.9 ± 0.5 mL containing an average of 19 × 106 nucleated cells with 85 ± 12% viability. Flow cytometry identified a variety of cells, including ASCs (23%), macrophages (24%), endothelial cells (5%), pericytes (4%), and transitional cells (0.5%). The final concentrated product contained cells capable of differentiating into adipogenic, chondrogenic, and osteogenic phenotypes. Furthermore, tests for SVF sterility and purity showed no evidence of endotoxin or collagenase residuals. The ACS system can efficiently process cells from adipose tissue within the timeframe of a single surgical procedure. The cellular characterization indicated that this system can yield a sterile and concentrated SVF output, providing a valuable source of ASCs within the heterogeneous cell population.

Aortic tissue stiffness and tensile strength are correlated with density changes following proteolytic treatment.

INTRODUCTION: Dissection or rupture of the aorta is accompanied by high mortality rates, and there is a pressing need for better prediction of these events for improved patient management and clinical outcomes. Biomechanically, these events represent a situation wherein the locally acting wall stress exceed the local tissue strength. Based on recent reports for polymers, we hypothesized that aortic tissue failure strength and stiffness are directly associated with tissue mass density. The objective of this work was to test this novel hypothesis for porcine thoracic aorta. METHODS: Three tissue specimens from freshly harvested porcine thoracic aorta were treated with either collagenase or elastase to selectively degrade structural proteins in the tissue, or with phosphate buffer saline (control). The tissue mass and volume of each specimen were measured before and after treatment to allow for density calculation, then mechanically tested to failure under uniaxial extension. RESULTS: Protease treatments resulted in statistically significant tissue density reduction (sham vs. collagenase p = 0.02 and sham vs elastase p = 0.003), which in turn was significantly and directly correlated with both ultimate tensile strength (sham vs. collagenase p = 0.02 and sham vs elastase p = 0.03) and tangent modulus (sham vs. collagenase p = 0.007 and sham vs elastase p = 0.03). CONCLUSIONS: This work demonstrates for the first time that tissue stiffness and tensile strength are directly correlated with tissue density in proteolytically-treated aorta. These findings constitute an important step towards understanding aortic tissue failure mechanisms and could potentially be leveraged for non-invasive aortic strength assessment through density measurements, which could have implications to clinical care.

Evaluation of Monomer-containing Isocyanate Groups on Stabilizing Demineralized Dentin Matrix Against Bond Interface Degradation.

PURPOSE: To evaluate the effect of urethane methacrylate precursor (UMP) on the enzymatic resistance of demineralized dentin (DD) matrices. MATERIALS AND METHODS: Experimental treatments containing 0 (control), 1, and 5 mmol/L UMP dissolved in an acetone (Ace) solution were formulated. Dentin matrix specimens were demineralized in vitro and immersed in the experimental treatments for 1 h. The treated specimens were then stored in 0.1 mg/mL collagenase solution for 24 h, after which their dry mass loss and hydroxyproline (HYP) release were assessed. The swelling ratios of specimens in each group were also evaluated. The interaction between UMP and the dentin matrix was observed using field-emission scanning electron microscopy (FE-SEM). Endogenous enzyme activity in dentin was evaluated using confocal laser scanning microscopy (CLSM). RESULTS: Compared with the other treatment groups, treatment with 1 mM and 5 mM UMP-Ace significantly decreased the dry mass loss, HYP release and swelling ratio of the DD matrix (p < 0.05). FE-SEM and CLSM observations showed that treatment with UMP-Ace protected the structure of the dentin matrix and decreased porosity within the dentin-collagen network. CONCLUSION: Treatment with 1 mM and 5 mM UMP-Ace protects DD matrix against collagenase degradation and may be clinically useful for improving the durability of the hybrid layer.

Validation of a rapid collagenase activity detection technique based on fluorescent quenched gelatin with synovial fluid samples.

BACKGROUND: Measuring collagenase activity is crucial in the field of joint health and disease management. Collagenases, enzymes responsible for collagen degradation, play a vital role in maintaining the balance between collagen synthesis and breakdown in joints. Dysregulation of collagenase activity leads to joint tissue degradation and diseases such as rheumatoid arthritis and osteoarthritis. The development of methods to measure collagenase activity is essential for diagnosis, disease severity assessment, treatment monitoring, and identification of therapeutic targets. RESULTS: This study aimed to validate a rapid collagenase activity detection technique using synovial fluid samples. Antibody microarray analysis was initially performed to quantify the levels of matrix metalloproteinase-9 (MMP-9), a major collagenase in joints. Subsequently, the developed gelatin-based test utilizing fluorescence measurement was used to determine collagenase activity. There was a significant correlation between the presence of MMP-9 and collagenase activity. In addition, Lower Limit of Detection and Upper Limit of Detection can be preliminary estimated as 8 ng/mL and 48 ng/mL respectively. CONCLUSIONS: The developed technique offers a potential point-of-care assessment of collagenase activity, providing real-time information for clinicians and researchers. By accurately quantifying collagenase activity, healthcare professionals can optimize patient care, improve treatment outcomes, and contribute to the understanding and management of joint-related disorders. Further research and validation are necessary to establish the full potential of this rapid collagenase activity detection method in clinical practice.

Enzymatic Digestion of the Intervertebral Disc Alters Intradiscal Injection and Leakage Mechanics.

Intradiscal injection is required to deliver therapeutic agents to the intervertebral disc (IVD) nucleus pulposus (NP). However, injectate leakage following needle retraction may result in decreased treatment efficacy and adverse side effects. While enzymatic digestion is a common research approach for simulating degeneration in healthy animal IVDs, contributions to the leakage phenomenon are unknown. In this study, bovine caudal discs were treated with injection into the NP of either a tris buffer control, collagenase (to primarily target collagen), or trypsin (to primarily target proteoglycans) and then injected with fluorescent saline using a through-puncture defect protocol. Pressure-volume records during injection were used to determine volume and pressure at leakage. Discs were then frozen, transected, and photographed to visualize injectate dispersion. Collagenase treatment resulted in a large increase in injectate dispersion, along with a decrease in injection pressure relative to control. Trypsin treatment resulted in a moderate increase in dispersion, with no associated effect on pressure. This study concludes that care should be taken when employing enzymatic digestion to simulate IVD degeneration, as NP tissue disruption may affect both retention and dispersion of subsequent therapeutic injections.

The Therapeutic Potential of Intra-Articular Injection of Synthetic Deer Antler Peptides in a Rat Model of Knee Osteoarthritis.

Synthetic deer antler peptides (TSKYR, TSK, and YR) stimulate the proliferation of human chondrocytes and osteoblasts and increase the chondrocyte content of collagen and glycosamino-glycan in vitro. This study investigated the peptide mixture's pain relief and chondroprotective effect in a rat model of collagenase-induced osteoarthritis. Thirty-six adult male Sprague-Dawley rats were divided into three groups: control (saline), positive control (hyaluronic acid), and ex-perimental (peptides). Intra-articular collagenase injections were administered on days 1 and 4 to induce osteoarthritis in the left knees of the rats. Two injections of saline, hyaluronic acid, or the peptides were injected into the same knees of each corresponding group at the beginning of week one and two, respectively. Joint swelling, arthritic pain, and histopathological changes were evaluated. Injection of the peptides significantly reduced arthritic pain compared to the control group, as evidenced by the closer-to-normal weight-bearing and paw withdrawal threshold test results. Histological analyses showed reduced cartilage matrix loss and improved total cartilage degeneration score in the experimental versus the control group. Our findings suggest that intra-articular injection of synthetic deer antler peptides is a promising treatment for osteoarthritis.

Immobilization of collagenase in inorganic hybrid nanoflowers with enhanced stability, proteolytic activity, and their anti-amyloid potential.

Organic-inorganic hybrid nanomaterials are considered as promising immobilization matrix for enzymes owing to their markedly enhanced stability and reusability. Herein, collagenase was chosen as a model enzyme to synthesize collagenase hybrid nanoflowers (Col-hNFs). Maximum collagenase activity (155.58 µmol min-1 L-1) and encapsulation yield (90 %) were observed in presence of Zn(II) ions at 0.05 mg/mL collagenase, 120 mM zinc chloride and PBS (pH 7.5). Synthesized Col-Zn-hNFs were extensively characterized by scanning electron microscopy (SEM), energy dispersive X-ray (EDX), X-ray diffraction (XRD), Fourier transform infrared (FTIR), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS) and zeta potential measurements. SEM images showed flower-like morphology with average size of 5.1 µm and zeta potential of -14.3 mV. Col-Zn-hNFs demonstrated superior relative activity across wide pH and temperature ranges, presence of organic solvents and surfactants as compared to its free form. Moreover, Col-Zn-hNFs exhibited excellent shelf life stability and favorable reusability. Col-Zn-hNFs showed the ability to suppress and eradicate fully developed insulin fibrils in vitro (IC50 = 2.8 and 6.2 µg/mL, respectively). This indicates a promising inhibitory potential of Col-Zn-hNFs against insulin amyloid fibrillation. The findings suggest that the utilization of Col-Zn-hNFs as a carrier matrix holds immense potential for immobilizing collagenase with improved catalytic properties and biomedical applications.

Comparing collagenase and silver sulfadiazine in deep second-degree burn treatment.

The indications for collagenase ointment (CO) and its efficacy are not clearly established in the treatment of second-degree burn wounds. To evaluate the efficacy of CO versus silver sulfadiazine ointment (SSD) in the treatment of second-degree burn wounds. A total of 170 eligible patients with deep second-degree burns, aged 18-65 years, with injuries occurring within 48-96 h, and having a total wound area of less than 30% of the total body surface area were included from 5 centers in China. The primary outcome was the wound healing time, and the secondary outcomes were the clearance time of wound necrotic tissues, wound healing rate, and wound inflammation. The study included 85 patients in SSD group and 84 in CO group in the modified intention-to-treat (mITT) population. The median time of wound healing was comparable in both groups (10 days vs. 10.5 days P = 0.16). The time for wound necrotic tissue removal was significantly shortened by CO compared with SSD (5 vs. 10 days P < 0.01). Wound inflammation, pain, wound healing rate, and scar were compared with SSD (all P-values > 0.05). No adverse events, such as infection or allergic reactions to the drugs and materials used, were reported. Both CO and SSD could heal the burn wounds at 10 days of treatment. However, CO significantly shortened the time of wound necrotic tissue removal by 5 days. Trial Registration: ChiCTR2100046971.

Thermostable Bacterial Collagenolytic Proteases: A Review.

Collagenolytic proteases are widely used in the food, medical, pharmaceutical, cosmetic, and textile industries. Mesophilic collagenases exhibit collagenolytic activity under physiological conditions, but have limitations in efficiently degrading collagen-rich wastes, such as collagen from fish scales, at high temperatures due to their poor thermostability. Bacterial collagenolytic proteases are members of various proteinase families, including the bacterial collagenolytic metalloproteinase M9 and the bacterial collagenolytic serine proteinase families S1, S8, and S53. Notably, the C-terminal domains of collagenolytic proteases, such as the pre-peptidase C-terminal domain, the polycystic kidney disease-like domain, the collagen-binding domain, the proprotein convertase domain, and the ß-jelly roll domain, exhibit collagen-binding or -swelling activity. These activities can induce conformational changes in collagen or the enzyme active sites, thereby enhancing the collagen-degrading efficiency. In addition, thermostable bacterial collagenolytic proteases can function at high temperatures, which increases their degradation efficiency since heat-denatured collagen is more susceptible to proteolysis and minimizes the risk of microbial contamination. To date, only a few thermophile-derived collagenolytic proteases have been characterized. TSS, a thermostable and halotolerant subtilisin-like serine collagenolytic protease, exhibits high collagenolytic activity at 60°C. In this review, we present and summarize the current research on A) the classification and nomenclature of thermostable and mesophilic collagenolytic proteases derived from diverse microorganisms, and B) the functional roles of their C-terminal domains. Furthermore, we analyze the cleavage specificity of the thermostable collagenolytic proteases within each family and comprehensively discuss the thermostable collagenolytic protease TSS.

Ultrasound Therapy With High-Pressure Pulses Is Effective to Reduce the Effects of Collagenase-Induced Tendinopathy in Rat's Achilles Tendon.

OBJECTIVE: We propose an ultrasonic treatment for collagenase-induced tendinopathy in rat's Achilles tendon using pulses with a low number of cycles, high acoustic pressure and very low duty cycle. METHODS: Twenty rats were used to perform the experiment. Four experimental groups of calcaneal tendons were studied: control (n = 6), sham (n = 4), collagenase-induced tendinopathy (n = 8) and ultrasound-treated collagenase-induced tendinopathy (n = 8). Surgical intervention was performed to expose the tendons prior to collagenase injection. A 1 MHz ultrasonic tansducer with a focusing lens was used. Ultrasonic treatments were used with an average total treatment time of 2.5 min, 20-cycle pulses, pressure amplitude p = 7 MPa, and 0.02% duty cycle. Histopathology of the samples was performed to evaluate nuclear density, acute inflammation, and signs of neovascularization. Collagen (types I and III), elastic fibers, and glycosaminoglycans were also analyzed. RESULTS: No tendon involvement was found by the surgical process. Ultrasonic treatment is safe, as it does not affect healthy tendons. When collagenase infiltrated animals were treated with US, a clear predominance of type I collagen fibers and a similar collagen ratio profile to that observed in the control and sham groups was observed, with a higher density of elastic fibers compared to the control and sham groups and a significant increase in the density of glycosaminoglycans. CONCLUSION: The ultrasound treatment proposed reduces the effects of the artificial collagenase lesion to reach the basal level after 45 d.

A multifunctional protein pre-coated metal-organic framework for targeted delivery with deep tissue penetration.

Targeted drug delivery using metal-organic frameworks (MOFs) has shown significant progress. However, the tumor microenvironment (TME) impedes efficient MOF particle transfer into tumor cells. To tackle this issue, we pre-coated nano-sized MOF-808 particles with multifunctional proteins: glutathione S-transferase (GST)-affibody (Afb) and collagenase, aiming to navigate the TME more effectively. The surface of MOF-808 particles is coated with GST-Afb-a fusion protein of GST and human epidermal growth factor receptor 2 (HER2) Afb or epidermal growth factor receptor (EGFR) Afb which has target affinity. We also added collagenase enzymes capable of breaking down collagen in the extracellular matrix (ECM) through supramolecular conjugation, all without chemical modification. By stabilizing these proteins on the surface, GST-Afb mitigate biomolecule absorption, facilitating specific tumor cell targeting. Simultaneously, collagenase degrades the ECM in the TME, enabling deep tissue penetration of MOF particles. Our resulting system, termed collagenase-GST-Afb-MOF-808 (Col-Afb-M808), minimizes undesired interactions between MOF particles and external biological proteins. It not only induces cell death through Afb-mediated cell-specific targeting, but also showcases advanced cellular internalization in 3D multicellular spheroid cancer models, with effective deep tissue penetration. The therapeutic efficacy of Col-Afb-M808 was further assessed via in vivo imaging and evaluation of tumor inhibition following injection of IR-780 loaded Col-Afb-M808 in 4T1tumor-bearing nude mice. This study offers key insights into the regulation of the multifunctional protein-adhesive surface of MOF particles, paving the way for the designing even more effective targeted drug delivery systems with nano-sized MOF particles.

Optimization and mechanisms of proteolytic enzyme immobilization onto large-pore mesoporous silica nanoparticles: Enhanced tumor penetration.

Immobilization of proteolytic enzymes onto nanocarriers is effective to improve drug diffusion in tumors through degrading the dense extracellular matrix (ECM). Herein, immobilization and release behaviors of hyaluronidase, bromelain, and collagenase (Coll) on mesoporous silica nanoparticles (MSNs) were explored. A series of cationic MSNs (CMSNs) with large and adjustable pore sizes were synthesized, and investigated together with two anionic MSNs of different pore sizes. CMSNs4.0 exhibited the highest enzyme loading capacity for hyaluronidase and bromelain, and CMSNs4.5 was the best for Coll. High electrostatic interaction, matched pore size, and large pore volume and surface area favor the immobilization. Changes of the enzyme conformations and surface charges with pH, existence of a space around the immobilized enzymes, and the depth of the pore structures, affect the release ratio and tunability. The optimal CMSNs-enzyme complexes exhibited deep and homogeneous penetration into pancreatic tumors, a tumor model with the densest ECM, with CMSNs4.5-Coll as the best. Upon loading with doxorubicin (DOX), the CMSNs-enzyme complexes induced high anti-tumor efficiencies. Conceivably, the DOX/CMSNs4.5-NH2-Coll nanodrug exhibited the most effective tumor therapy, with a tumor growth inhibition ratio of 86.1 %. The study provides excellent nanocarrier-enzyme complexes, and offers instructive theories for enhanced tumor penetration and therapy.

Collagenase motors in gelatine-based hydrogels.

Nano/micromotors outperform Brownian motion due to their self-propulsive capabilities and hold promise as carriers for drug delivery across biological barriers such as the extracellular matrix. This study employs poly(2-(diethylamino)ethyl methacrylate) polymer brushes to enhance the collagenase-loading capacity of silica particle-based motors with the aim to systematically investigate the impact of gelatine viscosity, motors' size, and morphology on their propulsion velocity. Notably, 500 nm and 1 µm motors achieve similar speeds as high as ∼15 µm s-1 in stiff gelatine-based hydrogels when triggered with calcium. Taken together, our findings highlight the potential of collagenase-based motors for navigating the extracellular matrix, positioning them as promising candidates for efficient drug delivery.

Collagen degradation assessment with an in vitro rotator cuff tendinopathy model using multiparametric ultrashort-TE magnetization transfer (UTE-MT) imaging.

PURPOSE: This study aims to assess ultrashort-TE magnetization transfer (UTE-MT) imaging of collagen degradation using an in vitro model of rotator cuff tendinopathy. METHODS: Thirty-six supraspinatus tendon specimens were divided into three groups and treated with 600 U collagenase (Group 1), 150 U collagenase (Group 2), and phosphate buffer saline (Group 3). UTE-MT imaging was performed to assess changes in macromolecular fraction (MMF), macromolecule transverse relaxation time (T2m), water longitudinal relaxation rate constant (R1m), the magnetization exchange rate from the macromolecular to water pool (Rm0 w) and from water to the macromolecular pool (Rm0 m), and magnetization transfer ratio (MTR) at baseline and following digestion and their differences between groups. Biochemical and histological studies were conducted to determine the extent of collagen degradation. Correlation analyses were performed with MMF, T2m, R1m, Rm0 w, Rm0 m, and MTR, respectively. Univariate and multivariate linear regression analyses were performed to evaluate combinations of UTE-MT parameters to predict collagen degradation. RESULTS: MMF, T2m, R1m, Rm0 m, and MTR decreased after digestion. MMF (r = -0.842, p < 0.001), MTR (r = -0.78, p < 0.001), and Rm0 m (r = -0.662, p < 0.001) were strongly negatively correlated with collagen degradation. The linear regression model of differences in MMF and Rm0 m before and after digestion explained 68.9% of collagen degradation variation in the tendon. The model of postdigestion in MMF and T2m and the model of MTR explained 54.2% and 52.3% of collagen degradation variation, respectively. CONCLUSION: This study highlighted the potential of UTE-MT parameters for evaluation of supraspinatus tendinopathy.

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.

Impact of different tissue dissociation protocols on endothelial cell recovery from developing mouse lungs.

Flow cytometry and fluorescence-activated cell sorting are widely used to study endothelial cells, for which the generation of viable single-cell suspensions is an essential first step. Two enzymatic approaches, collagenase A and dispase, are widely employed for endothelial cell isolation. In this study, the utility of both enzymatic approaches, alone and in combination, for endothelial cell isolation from juvenile and adult mouse lungs was assessed, considering the number, viability, and subtype composition of recovered endothelial cell pools. Collagenase A yielded an 8-12-fold superior recovery of viable endothelial cells from lung tissue from developing mouse pups, compared to dispase, although dispase proved superior in efficiency for epithelial cell recovery. Single-cell RNA-Seq revealed that the collagenase A approach yielded a diverse endothelial cell subtype composition of recovered endothelial cell pools, with broad representation of arterial, capillary, venous, and lymphatic lung endothelial cells; while the dispase approach yielded a recovered endothelial cell pool highly enriched for one subset of general capillary endothelial cells, but poor representation of other endothelial cells subtypes. These data indicate that tissue dissociation markedly influences the recovery of endothelial cells, and the endothelial subtype composition of recovered endothelial cell pools, as assessed by single-cell RNA-Seq.