Bacterial Collagenase Research Articles

Differential Biodegradation Kinetics of Collagen Membranes for Bone Regeneration.

Native collagen-based membranes are used to guide bone regeneration; but due to their rapid biodegradation, this treatment is often unpredictable. The purpose of this study was to investigate the biodegradability of natural collagen membranes. Three non-cross-linked resorbable collagen barrier membranes were tested: Derma Fina (porcine dermis), Evolution Standard (equine pericardium) and Duo-Teck (equine lyophilized collagen felt). 10 × 10 mm2 pieces of membranes were submitted to three different degradation procedures: (1) hydrolytic degradation in phosphate buffer solution, (2) enzyme resistance, using a 0.25% porcine trypsin solution, and (3) bacterial (Clostridium histolyticum) collagenase resistance test. Weight measurements were performed with an analytic microbalance. Thickness was measured with a digital caliper. Membranes were analyzed at different time-points, up to 21 d of immersion. A stereomicroscope was used to obtain membranes’ images. ANOVA and Student Newman Keuls were used for mean comparisons (p < 0.05), except when analyzing differences between time-points within the same membrane and solution where pair-wise comparisons were applied (p < 0.001). Derma Fina attained the highest resistance to all degradation challenges. Duo-Teck was the most susceptible membrane to degradation, complete degradation occurred as soon as 8 h. The bacterial collagenase solution performed as the most aggressive test as all membranes presented 100% degradation before 21 d.

TREM2 activation attenuates neuroinflammation and neuronal apoptosis via PI3K/Akt pathway after intracerebral hemorrhage in mice

BackgroundNeuroinflammation is an important host defense response to secondary brain injury after intracerebral hemorrhage (ICH). Triggering receptor expressed on myeloid cells 2 (TREM2) confers strong neuroprotective effects by attenuating neuroinflammation in experimental ischemic stroke. Recent studies suggest that apolipoprotein E (apoE) is a novel, high-affinity ligand of TREM2. This study aimed to investigate the effects of TREM2 activation on neuroinflammation and neuronal apoptosis in a mouse model of ICH.MethodsAdult male CD1 mice (n = 216) were subjected to intrastriatal injection of bacterial collagenase. The TREM2 ligand, apoE-mimetic peptide COG1410 was administered intranasally at 1 h after ICH induction. To elucidate the underlying mechanism, TREM2 small interfering RNA (siRNA) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were administered intracerebroventricularly prior to COG1410 treatment. Neurobehavioral tests, brain water content, immunofluorescence, western blotting, and Fluoro-Jade C- and terminal deoxynucleotidyl transferase dUTP nick end labeling staining were performed.ResultsEndogenous TREM2 expression was increased and peaked at 24 h after ICH. TREM2 was expressed on microglia, astrocytes, and neurons. COG1410 improved both short-term and long-term neurological functions, reduced brain edema, inhibited microglia/macrophage activation and neutrophil infiltration, and suppressed neuronal apoptotic cell death in perihematomal areas after ICH. Knockdown of endogenous TREM2 by TREM2 siRNA aggravated neurological deficits and decreased the expression of TREM2 in naïve and ICH mice. COG1410 was associated with upregulation of TREM2, PI3K, phosphorylated-Akt, and Bcl-2 and downregulation of TNF-α, IL-1β, and Bax after ICH. The neuroprotective effects of COG1410 were abolished by both TREM2 siRNA and PI3K inhibitor LY294002.ConclusionsOur finding demonstrated that TREM2 activation improved neurological functions and attenuated neuroinflammation and neuronal apoptosis after ICH, which was, at least in part, mediated by activation of PI3K/Akt signaling pathway. Therefore, activation of TREM2 may be a potential therapeutic strategy for the management of ICH patients.

NT-4 attenuates neuroinflammation via TrkB/PI3K/FoxO1 pathway after germinal matrix hemorrhage in neonatal rats

BackgroundNeuroinflammation plays an important role in pathogenesis of germinal matrix hemorrhage (GMH). Neurotrophin-4 (NT-4) is a member of the neurotrophin family and interacts with the tropomyosin receptor kinase B (TrkB). NT-4 has been shown to confer neuroprotective effects following cerebral ischemia. We aimed to investigate the neuroprotective function of NT-4-TrkB signaling, as well as its downstream signaling cascade phosphatidylinositol-3-kinases (PI3K)/protein kinase B (Akt)/forkhead box protein O1 (FoxO1), following GMH in neonatal rats.MethodsGMH was induced by intraparenchymal injection of bacterial collagenase (0.3 U) in P7 rat pups. A total of 163 pups were used in this study. Recombinant human NT-4 was administered intranasally at 1 h after the collagenase injection. The selective TrkB antagonist ANA-12, selective PI3K inhibitor LY294002, and FoxO1 activating CRISPR were administered intracerebroventricularly at 24 h prior to NT-4 treatment to investigate the underlying mechanism. Short-term and long-term neurobehavioral assessments, immunofluorescence staining, Nissl’s staining, and Western blot were performed.ResultsExpression of phosphorylated TrkB increased after GMH, reaching the peak level at day 3 after hemorrhage. TrkB receptors were observed on neurons, microglia, and astrocytes. The administration of rh-NT-4 induced phosphorylation of TrkB, expression of PI3K, and phosphorylation of Akt. Meanwhile, it decreased FoxO1 and IL-6 levels. Selective inhibition of TrkB/PI3K/Akt signaling in microglia increased the expression levels of FoxO1 and pro-inflammatory cytokines. FoxO1 activating CRISPR increased the expression of IL-6, suggesting that FoxO1 might be a potential inducer of pro-inflammatory factors. These results suggested that PI3K/Akt/FoxO1 signaling may be the downstream pathway of activation of TrkB. The rat pups treated with rh-NT-4 performed better than vehicle-treated animals in both short-term and long-term behavioral tests.ConclusionThese data showed that rh-NT-4 reduced the expression levels of pro-inflammatory cytokines, improved neurological function, attenuated neuroinflammation, and thereby mitigated post-hemorrhagic hydrocephalus after GMH by TrkB/PI3K/Akt/FoxO1 pathway. These results indicated that rh-NT-4 could be a promising therapeutic strategy to ameliorate neuroinflammation and hydrocephalus after GMH or other similar brain injuries.

Serelaxin activates eNOS, suppresses inflammation, attenuates developmental delay and improves cognitive functions of neonatal rats after germinal matrix hemorrhage

Germinal matrix hemorrhage (GMH) is a detrimental form of neonatal CNS injury. Following GMH-mediated eNOS inhibition, inflammation arises, contributing to GMH-induced brain injury. We investigated the beneficial effects of Serelaxin, a clinical tested recombinant Relaxin-2 protein, on brain injury after GMH in rats. We investigated whether effects of Serelaxin are mediated by its ability to activate the GMH-suppressed eNOS pathway resulting in attenuation of inflammatory marker overproduction. GMH was induced by intraparenchymal injection of bacterial collagenase (0.3U). Seven day old Sprague–Dawley rat pups (P7) were used (n = 63). GMH animals were divided in vehicle or serelaxin treated (3 µg once, 30 µg once, 30 µg multiple, i.p., starting 30 after GMH and then daily). Sham operated animals were used. We monitored the developmental profile working memory and spatial function (T-maze and open field test respectively). At day 28, all rats underwent MRI-scans for assessment of changes in cortical thickness and white matter loss. Effects of Serelaxin on eNOS pathway activation and post-GMH inflammation were evaluated. We demonstrated that Serelaxin dose-dependently attenuated GMH-induced developmental delay, protected brain and improved cognitive functions of rats after GMH. That was associated with the decreased post-GMH inflammation, mediated at least partly by amelioration of GMH-induced eNOS inhibition.

Pressure-induced collagen degradation in arterial tissue as a potential mechanism for degenerative arterial disease progression

Collagen fibre degradation is a strain-dependent process, whereby the magnitude of experienced strain dictates the rate of enzymatic cleavage. Studies have identified conflicting findings as to whether strain inhibits or enhances collagen degradation, which may be explained by the tissue type and tissue scale investigated, as well as the strain range considered. The aim of this study is to identify, for the first time, the strain-dependent degradation response of intact arterial vessels experiencing physiological pressures and apply these findings to a computational model to better understand degenerative arterial diseases, such as aneurysms.To achieve this, a series of quasi-static pressure inflation experiments were carried out on intact arteries in the presence of purified bacterial collagenase at physiologically relevant pressures to investigate collagen matrix degradation in the vascular wall. A complementary computational model was developed to explore the complex role of pressure, non-collagenous matrix contribution, and collagen fibre crimp in the ultimate degradation response of the vessel.Pressure induced inflation-degradation results identified an increased rate of vessel expansion and reduced time to failure with increasing pressure in the vessels. Interestingly, our computational model was able to capture this same response, including the elevated rates of degradation which occur at low pressures. These findings highlight the critical role of strain in collagen degradation, particularly in cases of arterial disease, such as aneurysm formation, whereby structural integrity may be compromised.

Targeting Cell Contractile Forces: A Novel Minimally Invasive Treatment Strategy for Fibrosis.

Fibrosis is a complication of tendon injury where excessive scar tissue accumulates in and around the injured tissue, leading to painful and restricted joint motion. Unfortunately, fibrosis tends to recur after surgery, creating a need for alternative approaches to disrupt scar tissue. We posited a strategy founded on mechanobiological principles that collagen under tension generated by fibroblasts is resistant to degradation by collagenases. In this study, we tested the hypothesis that blebbistatin, a drug that inhibits cellular contractile forces, would increase the susceptibility of scar tissue to collagenase degradation. Decellularized tendon scaffolds (DTS) were treated with bacterial collagenase with or without external or cell-mediated internal tension. External tension producing strains of 2-4% significantly reduced collagen degradation compared with non-tensioned controls. Internal tension exerted by human fibroblasts seeded on DTS significantly reduced the area of the scaffolds compared to acellular controls and inhibited collagen degradation compared to free-floating DTS. Treatment of cell-seeded DTS with 50mM blebbistatin restored susceptibility to collagenase degradation, which was significantly greater than in untreated controls (p < 0.01). These findings suggest that therapies combining collagenases with drugs that reduce cell force generation should be considered in cases of tendon fibrosis that do not respond to physiotherapy.

Analyzing Structural and Functional Characteristics of Collagenase from Bacillus cereus MH19 via in silico Approaches

Background: Collagenase is a type of proteolytic enzyme that specifically hydrolyzes native collagen with a three-dimensional helical structure. The structure and properties of collagenase vary with different sources and types. In addition to the well-characterized Clostridium collagenase, other collagenases are largely unknown. Various gene and protein databases have been widely used to mine novel functional genes in the genome. Gene mining and sequence analysis are effective methods for studying these enzymes. Objective: The present study aimed to understand the molecular, structural, and functional characteristics of collagenase from Bacillus cereus MH19 using a bioinformatics approach. Method: Based on the three-generation sequencing PacBio technique, Sequencing the Bacillus cereus MH19 genome. Function annotation is completed by blasting genes with different databases. Collagenases were investigated based on the physiochemical properties, phylogenetic relation, and domain architecture. The 3D structure model of the selected collagenase has been constructed and verified by SAVES. Results: There were 5 collagenases in Bacillus cereus MH19 with a molecular weight distribution ranging from 36-110 kDa. The analysis of evolutionary relationship between different collagenases indicating that the BCC000504 and BCC003388 collagenase gene sequences are closer to the typical collagenase genes ColG, ColA, and ColH, followed by BCC003615. The domain and function analysis showed that the collagenases BCC000504 and BCC003388 were similar to the collagenases ColG, ColA and ColH. BCC004271 was similar to BCC004272, and BCC003615 might be quite different from other collagenases. The secondary structure of collagenase was analyzed. The SAVES evaluation indicates that 3D structural modeling of the selected collagenase is acceptable. Conclusion: This study provides an overview of the molecular, functional, and structural characteristics of collagenase from Bacillus cereus MH19, which helps to understand the bacterial collagenase. The characterization of the collagenase will certainly expand the application range of collagenase.

Heterogeneously Strained Tissue Collagen Resists Collagenase Degradation Where Strains are High

Tissue homeostasis reflects a balance of synthesis and degradation, and past studies of homogeneously stressed tissues and collagen materials indicate collagen degradation is suppressed by strain. Such processes are studied here in isolated tendons and in beating embryonic hearts. Collagen fibrils in tendon fascicles orient along the tension axis, but heterogeneous strains and gradients were induced using three-point bending plus adhesion, with patterned photo-bleaching of a fluorescent fascicles used to measure strain. Tissue microstructure was simultaneously imaged using Second Harmonic Generation (SHG) signal while deformed fascicle samples were exposed to purified Matrix Metalloproteinase-1 (MMP-1) or Bacterial collagenase (BC). Within physiological strain limits (i.e. ∼5-8%), the decrease in degradation rate (relative to strain-free) was nearly independent of collagenase type despite different cleavage mechanisms, whereas tissue locations sustaining higher strains showed the degradation rate became almost independent of strain magnitudes. The dependence of degradation rate on mechanical strain suggests a sequestration of collagen's cleavage sites, which we posit because permeation and mobility of fluorescent collagenase and dextran are strain-independent up to ∼5-8% strains. Normal beating hearts are also subjected to ∼5% peak strain in a spatiotemporally coordinate contractile wave, and the hearts maintain collagen mass until strain is suppressed by inhibition of myosin-II. Endogenous MMPs then degrade the collagens in 30-60 min based on addition or not of MMP inhibitors and quantitation by calibrated mass spectrometry. Both tissue systems under heterogeneous strains indicate a degradative sculpting of tissue locations that sustain the lowest strains.

Abstract TP336: The Intestinal Barrier Dysfunction Induced by Intracerebral Hemorrhage Contributes to the Brain Edema

Background and Purpose: This study aims to investigate whether intracerebral hemorrhage (ICH) can lead to intestinal barrier dysfunction, and whether ICH-induced intestinal injury plays a role in brain edema. Methods: ICH mice model was prepared by an intrastriatal injection of bacterial collagenase. The following parameters were investigated at 3 h, 6 h, 12 h, 1 d, 2 d, 3 d, or 7 d after ICH preparation. Mice were given intragastrically with FITC-dextran and the intestinal permeability was evaluated by serum fluorescence measurement. Serum lipopolysaccharide (LPS) level was assayed with ELISA kits. Ileum and jejunum were stained with hematoxylin and eosin. Intestinal mucosal injuries were graded according to the scoring method (grade 0 to grade 4). Brain water content was evaluated via a wet/dry weight method. Correlations of intestinal injury, intestine permeability, LPS, and brain edema were analyzed using Pearson’s correlation analysis. Results: Compared with the Sham group, Ileum and jejunum damage occurred at 6 h after ICH, and the ICH-induced intestinal injury continued until 7 d. In line with the histopathological findings, the degree of ileum and jejunum injury was significantly increased at 6 h after ICH, showing mostly scores in Grade 1 to Grade 3 ( P &lt; 0.05 or P &lt; 0.01). After 6-h ICH, the intestinal permeability to FITC-dextran was higher compared to the Sham group, and the increase of intestinal permeability lasted 7 d ( P &lt; 0.01). From 6 h to 7 d, serum LPS was significantly augmented ( P &lt; 0.01). The brain content of the ipsilateral hemispheres was increased at 12 h, 1 d, 2 d, and 3 d after ICH ( P &lt; 0.05 or P &lt; 0.01), and the brain content of the contralateral hemispheres was also enhanced at 1 d, 2 d, and 3 d after ICH ( P &lt; 0.01). The ileum and jejunum injury were positively associated with intestine permeability (r = 0.625, P &lt; 0.01, r = 0.465, P &lt; 0.01, respectively). The intestine permeability was positively associated with the serum level of LPS (r =0.585, P &lt; 0.01). The LPS levels were positively associated with brain water content (r = 0.338, P &lt; 0.01). Conclusion: ICH can cause intestinal mucosal injury. Consequently, the increase of intestinal permeability results in the translocation of endotoxins, which contributes to ICH-induced brain edema.

Effect of tea root-derived proanthocyanidin fractions on protection of dentin collagen

ObjectivesProanthocyanidins (PAs) have been widely used as effective agents for dentin collagen cross-linking to enhance the biomechanics and biostability of dentin in vitro. However, the effects and protective mechanisms of various tea root-derived PA components on dentin remain undefined. This study evaluated the effects of these tea root-derived PA components on dentin biomechanics and biostability.MethodsIn this study, ethyl acetate and n-butyl alcohol were used to extract PAs with different degrees of polymerization from tea roots; the effects of these PA extracts on dentin were evaluated.ResultsDentin was treated with glutaraldehyde, ethyl acetate, n-butyl alcohol, or water. PAs with a high degree of polymerization, extracted using n-butyl alcohol, were able to more effectively improve dentin collagen cross-linking, increase resistance to bacterial collagenase digestion, and enhance dentin elasticity, relative to treatment with glutaraldehyde or PAs with a low degree of polymerization (extracted using ethyl acetate). Additionally, treatment with aqueous extract of tea roots was detrimental to dentin stability and function.ConclusionsPAs with a high degree of polymerization were effective for dentin protection and restoration in vitro, suggesting clinical treatment potential for tea root-derived PAs.

Systemic inflammatory response to the Radial Pressure Wave Therapy (RPWT) in collagenase-induced Achilles tendinopathy treated with Adipose Derived Stem Cells or Platelet Rich Plasma

Novel tendinopathy treatment protocols should be assessed for safety. The goal of this work was to compare differences in selected systemic inflammatory marker concentrations after two treatment protocols for collagenase induced Achilles tendinopathy in sheep. 14 sheep (aged 5 and 6 years, Polish Mountain Sheep breed, weight 60-70kg) were injected with bacterial collagenase type 1A-S (Clostridium histolyticum, C-5894, Sigma Aldrich, Poznań, Poland) bilaterally to Achilles tendons. Subsequently, the animals were injected with Platelet Rich Plasma (7 sheep) or Adipose Derived Stem Cells (7 sheep) to induced tendinopathy foci. Left limbs of all sheep were additionally treated with Radial Pressure Wave Therapy (RPWT) focused above the tendinopathy origins. Treatment progress was controlled by ultrasound scans, and tendon samples were taken on the 126th day of the experiment. Serum Amyloid A (SAA) concentration showed mild elevation before the experiment (2 sheep from group I, 4 sheep from group II) and two days after the intratendinous growth factors injection ( 4 sheep from group I, 3 sheep from group II) combined with RPWT (mean 22,63 mg/L and 53, 6 mg/L respectively). Haptoglobine (Hp) concentration increased from 0 to 0,01 g/L in 2 animals from group I two days after injection. These values declined to 0 during the course of the treatment. Fibrinogen (Fb) concentrations were within reference levels throughout the research, although mild elevation was observed before the treatment course in 6 sheep from group I and 1 sheep from group II. In conclusion, addition of RPWT to growth factors injections in the treatment of yatrogenic Achilles tendinopathy in sheep did not induce systemic inflammatory response.

In vitro histomechanical effects of enzymatic degradation in carotid arteries during inflation tests with pulsatile loading

In this paper, the objective is to assess the histomechanical effects of collagen proteolysis in arteries under loading conditions reproducing in vivo environment. Thirteen segments of common porcine carotid arteries (8 proximal and 5 distal) were immersed in a bath of bacterial collagenase and tested with a pulsatile tension/inflation machine. Diameter, pressure and axial load were monitored throughout the tests and used to derive the stress-stretch curves and to determine the secant circumferential stiffness. Results were analysed separately for proximal and distal segments, before and after 1, 2 and 3h of enzymatic degradation. A histological analysis was performed to relate the arterial microstructure to its mechanical behavior under collagen proteolysis. Control (before enzymatic degradation) and treated populations (after 1, 2 or 3h of enzymatic degradation) were found statistically incomparable, and histology confirmed the alteration of the fibrous structure of collagen bundles induced by the collagenase treatment. A decrease of the secant circumferential stiffness of the arterial wall was noticed mostly at the beginning of the treatment, and was less pronounced after 1h. These results constitute an important set of enzymatically damaged arteries that can be used to validate biomechanical computational models correlating structure and properties of blood vessels.

Verapamil and collagenase differentially affect collagen metabolism in experimental model of Peyronie's disease

ObjectivesPeyronie's disease (PD) is accompanied by remodelling of connective tissue into fibrotic plaque. Treatment of the inflammatory and fibrotic phases of the disease is not established. The aim of the study was to evaluate the effect of verapamil (VER) and bacterial collagenase (COLL) on collagen metabolism and cell migration in fibroblasts with experimental wound healing and inflammation as an in vitro model of PD. Materials and methodsIn vitro model of PD was designed using experimental model of inflammation induced by Interleukin-1 (IL-1) in cultured fibroblasts and mechanical damage of the cells. Cell viability, cell proliferation, collagen biosynthesis, prolidase activity and cell migration were studied in both models of the cells treated with VER and COLL. ResultsVER decreased cell viability, DNA and collagen biosynthesis and increased prolidase activity in control fibroblast, while in “wounded” fibroblasts it significantly decreased all the processes. COLL did not affect cell viability and DNA biosynthesis, while inhibited collagen biosynthesis and prolidase activity in both control and “wounded” fibroblasts. In IL-1-treated fibroblasts VER inhibited all studied processes except prolidase activity, while COLL inhibited only collagen biosynthesis and prolidase activity. COLL accelerated cell migration, while VER attenuated the process in fibroblast model of wound healing, compared to control cells. ConclusionVER and COLL attenuate collagen biosynthesis in both fibroblast models. The VER-dependent inhibition of collagen biosynthesis was accompanied by inhibition of DNA biosynthesis at high prolidase activity, while COLL affected this process through inhibition of prolidase activity at high rate of DNA biosynthesis. It shows that anti-fibrotic activity of VER/COLL and anti-inflammatory activity of VER may represent approach to establish standard treatment of PD.

Результаты нехирургического лечения витреомакулярной тракции с применением бактериальной коллагеназы

Purpose. To study the efficacy of a developed method of non-surgical treatment of vitreomacular traction (VMT) using the bacterial collagenase. Material and methods. The study group included 65 cases of VMT with a length from 20 to 3100 μm. The VMT was isolated in 22 cases, and it was complicated in 43 cases: by macular hole (29 eyes) or epiretinal fibrosis (14 eyes). Results. In 3 cases (4.6%) within three months after the diagnosis there was a spontaneous resolution of traction with the restoration of the macular profile. In 48 cases (73.9%) an elimination of VMT after the treatment was achieved. In 14 cases (21.5%) VMT could not be eliminated. In 15 cases it was possible to achieve not only the elimination of VMT, but also the closure of macular hole. In 9 cases, it was possible to achieve the resolution of VMT and epiretinal fibrosis with the improved visual acuity. Conclusion. The obtained results showed that the developed method of non-surgical treatment of VMT can be the method of choice in the treatment of both isolated and complicated VMT of any length.

An ex vivo tissue model of cartilage degradation suggests that cartilage state can be determined from secreted key protein patterns.

The pathophysiology of osteoarthritis (OA) involves dysregulation of anabolic and catabolic processes associated with a broad panel of proteins that ultimately lead to cartilage degradation. An increased understanding about these protein interactions with systematic in vitro analyses may give new ideas regarding candidates for treatment of OA related cartilage degradation. Therefore, an ex vivo tissue model of cartilage degradation was established by culturing tissue explants with bacterial collagenase II. Responses of healthy and degrading cartilage were analyzed through protein abundance in tissue supernatant with a 26-multiplex protein profiling assay, after exposing the samples to a panel of 55 protein stimulations present in synovial joints of OA patients. Multivariate data analysis including exhaustive pairwise variable subset selection identified the most outstanding changes in measured protein secretions. MMP9 response to stimulation was outstandingly low in degrading cartilage and there were several protein pairs like IFNG and MMP9 that can be used for successful discrimination between degrading and healthy samples. The discovered changes in protein responses seem promising for accurate detection of degrading cartilage. The ex vivo model seems interesting for drug discovery projects related to cartilage degradation, for example when trying to uncover the unknown interactions between secreted proteins in healthy and degrading tissues.

Activity And Kinetics of α-Glucosidase Inhibition by Collagen Hydrolysate From Thunnus albacares Bone

Controlling of postprandial hyperglycemia using α-glucosidase inhibitors is one of the therapeutic approaches for the treatment of type 2 diabetes. Collagen hydrolysate, as an α-glucosidase inhibitor has not been reported. Therefore, This study intends to evaluate the activity and kinetics of inhibition of collagen hydrolysate against the α-glucosidase enzyme. Collagen hydrolysate was prepared from Thunnus albacares bone enzymatically using bacterial collagenase at hydrolysis times (0; 0.5; 1; 2; 3; 4; 5 and 6 h). The type of inhibition was determined by enzyme kinetics analysis. Collagen hydrolysate obtained at hydrolysis time of 1 h has the highest inhibitory activity of 24.47 %. Based on enzyme kinetics analysis, collagen hydrolysate showed a show a type of competitive inhibition. These results revealed that collagen hydrolysate has the potential as a natural antidiabetic in controlling blood glucose levels in type 2 diabetes.

EARLY EXCISION AND GRAFTING VERSUS CONSERVATIVE TREATMENT IN THE MANAGEMENT OF MAJOR BURNS: A COMPARATIVE STUDY

Background : 1 Full thickness burns lose their eschar in 2-6 weeks through bacterial collagenase production and daily mechanical debridement . The practice of leaving these dead tissues only serves as a nidus for infection that can lead to the patient's death. Hence the standard procedure is surgical removal of eschar with grafting techniques. Methods:Atotal of 50 patients divided in 25 patients in each group were included first group was managed conservatively and second underwent th early excision and grafting within 5 day upto 10% TBSA. Results: Early excision and grafting patients required significantly (p=0.04) more blood transfusions than conservatively managed patients. The mean hospital stay was significantly (p=0.05) lower by 10 days in early excision and grafting than conservatively managed. Conclusion : Early Excision and Grafting group was found to have significantly shorter hospital stay with decresed painful debridement but required more blood transfusions than conservatively managed patients.

Siponimod (BAF-312) Attenuates Perihemorrhagic Edema And Improves Survival in Experimental Intracerebral Hemorrhage.

Background and Purpose- Perihemorrhagic edema (PHE) is associated with poor outcome after intracerebral hemorrhage (ICH). Infiltration of immune cells is considered a major contributor of PHE. Recent studies suggest that immunomodulation via S1PR (sphingosine-1-phosphate receptor) modulators improve outcome in ICH. Siponimod, a selective modulator of sphingosine 1-phosphate receptors type 1 and type 5, demonstrated an excellent safety profile in a large study of patients with multiple sclerosis. Here, we investigated the impact of siponimod treatment on perihemorrhagic edema, neurological deficits, and survival in a mouse model of ICH. Methods- ICH was induced by intracranial injection of 0.075 U of bacterial collagenase in 123 mice. Mice were randomly assigned to different treatment groups: vehicle, siponimod given as a single dosage 30 minutes after the operation or given 3× for 3 consecutive days starting 30 minutes after operation. The primary outcome of our study was evolution of PHE measured by magnetic resonance-imaging on T2-maps 72 hours after ICH, secondary outcomes included evolution of PHE 24 hours after ICH, survival and neurological deficits, as well as effects on circulating blood cells and body weight. Results- Siponimod significantly reduced PHE measured by magnetic resonance imaging (P=0.021) as well as wet-dry method (P=0.04) 72 hours after ICH. Evaluation of PHE 24 hours after ICH showed a tendency toward attenuated brain edema in the low-dosage group (P=0.08). Multiple treatments with siponimod significantly improved neurological deficits measured by Garcia Score (P=0.03). Survival at day 10 was improved in mice treated with multiple dosages of siponimod (P=0.037). Mice treated with siponimod showed a reduced weight loss after ICH (P=0.036). Conclusions- Siponimod (BAF-312) attenuated PHE after ICH, increased survival, and reduced ICH-induced sensorimotor deficits in our experimental ICH-model. Findings encourage further investigation of inflammatory modulators as well as the translation of BAF-312 to a human study of ICH patients.

Sustained Release of Phosphates From Hydrogel Nanoparticles Suppresses Bacterial Collagenase and Biofilm Formation in vitro.

Intestinal disease or surgical intervention results in local changes in tissue and host-derived factors triggering bacterial virulence. A key phenotype involved in impaired tissue healing is increased bacterial collagenase expression which degrades intestinal collagen. Antibiotic administration is ineffective in addressing this issue as it inadvertently eliminates normal flora while allowing pathogenic bacteria to “bloom” and acquire antibiotic resistance. Compounds that could attenuate collagenase production while allowing commensal bacteria to proliferate normally would offer major advantages without the risk of the emergence of resistance. We have previously shown that intestinal phosphate depletion in the surgically stressed host is a major cue that triggers P. aeruginosa virulence which is suppressed under phosphate abundant conditions. Recent findings indicate that orally administered polyphosphate, hexametaphosphate, (PPi) suppresses collagenase, and biofilm production of P. aeruginosa and S. marcescens in animal models of intestinal injury but does not attenuate E. faecalis induced collagenolytic activity (Hyoju et al., 2017). Systemic administration of phosphates, however, is susceptible to rapid clearance. Given the diversity of collagenase producing bacteria and the variation of phosphate metabolism among microbial species, a combination therapy involving different phosphate compounds may be required to attenuate pathogenic phenotypes. To address these barriers, we present a drug delivery approach for sustained release of phosphates from poly(ethylene) glycol (PEG) hydrogel nanoparticles. The efficacy of monophosphate (Pi)- and PPi-loaded NPs (NP-Pi and NP-PPi, respectively) and a combination treatment (NP-Pi + NP-PPi) in mitigating collagenase and biofilm production of gram-positive and gram-negative pathogens expressing high collagenolytic activity was investigated. NP-PPi was found to significantly decrease collagenase and biofilm production of S. marcescens and P. aeruginosa. Treatment with either NP-Pi or NP-Pi + NP-PPi resulted in more prominent decreases in E. faecalis collagenase compared to NP-PPi alone. The combination treatment was also found to significantly reduce P. aeruginosa collagenase production. Finally, significant attenuation in biofilm dispersal was observed with NP-PPi or NP-Pi + NP-PPi treatment across all test pathogens. These findings suggest that sustained release of different forms of phosphate confers protection against gram-positive and gram-negative pathogens, thereby providing a promising treatment to attenuate expression of tissue-disruptive bacterial phenotypes without eradicating protective flora over the course of intestinal healing.

Sixty years in the making: collagenase Clostridium histolyticum, from benchtop to FDA approval and beyond.

The introduction of collagenase Clostridium histolyticum (CCH) as the first and only FDA-approved non-surgical treatment for Peyronie's disease (PD) has been an important step in its management. Our aim is to provide an overview of the historical origins of CCH and its development through FDA approval and beyond for the treatment of PD. A PubMed search using the terms Peyronie OR Peyronie's AND collagenase and limited to clinical research studies resulted in 24 articles that were examined for the current review. PD is a connective tissue disorder of the penile tunica albuginea involving fibrotic penile plaques that cause abnormal curvature and, in many cases, erectile pain. Although the exact mechanism and underlying pathophysiology are not well characterized, the known lability of these plaques to exogenous bacterial collagenase combined with a lack of effective medical therapies led to the development of CCH as an evidence-based treatment of PD. The initial discovery of collagenase was followed by in vitro studies on PD plaque tissue and following the phase 3 IMPRESS trial culminated in FDA approval of CCH in 2013. Future directions in CCH therapy include improved patient selection, use in acute phase PD, adjuvant and combination therapies, and novel delivery mechanisms. CCH provides an effective non-surgical treatment option for men with PD. We have traced the development of CCH in the treatment of PD from the earliest in vitro investigations to comprehensive multi-study meta-analyses confirming its highly rated efficacy when compared to other historical non-surgical remedies.