Oral doxycycline prevents skin-associated adverse effects induced by injectable collagenase in a rodent model of capsular contracture around silicone implants.

BACKGROUND: The collagenase of the bacterium Clostridium histolyticum (CCH) is already an established treatment for fibroproliferative diseases like M. Dupuytren and M. Peyronie Although results are comparable to surgical intervention, skin laceration is a severe and relevant side effect. Doxycycline (DOX) recently rose interest as an inhibitor of matrix-metalloproteinases alongside its capabilities of skin accumulation. It therefore might be a potential skin protective agent in the use of CCH. METHODS: For simulation of a fibroproliferative disease adjacent to the skin, we utilized a rodent model of capsular fibrosis involving silicone implants and subsequent fibrotic capsule formation. For in-vitro studies, fibrotic capsules were excised and incubated with 0.9 mg/ml CCH and four different doses of DOX. For in-vivo experiments, animals received 0.0, 0.3 or 0.9 mg/ml CCH injections into the fibrotic capsules with or without prior oral DOX administration. Outcome analysis included histology, immunohistochemistry, gene expression analysis, chemical collagen and DOX concentration measurements as well as µCT imaging. RESULTS: In-vitro, DOX showed a dose-dependent inhibition of CCH activity associated with increasing capsule thickness and collagen density and content. In-vivo, oral DOX administration did neither interfere with capsule formation nor in effectiveness of CCH dissolving fibrotic capsule tissue. However, skin thickness and especially collagen density was significantly higher compared to control groups. This led to a reduced rate of clinical skin lacerations after DOX administration. CONCLUSION: DOX inhibits CCH and accumulates in the skin. Thereby, DOX can effectively reduce skin laceration after CCH treatment.

Extracellular matrix and Hippo signaling as therapeutic targets of antifibrotic compounds for uterine fibroids.

BACKGROUND: Uterine fibroids are highly prevalent, collagen-rich, mechanically stiff, fibrotic tumors for which new therapeutic options are needed. Increased extracellular matrix (ECM) stiffness activates mechanical signaling and Hippo/YAP promoting fibroid growth, but no prior studies have tested either as a therapeutic target. We tested the hypothesis that injection of a purified form of collagenase Clostridium histolyticum (CCH) that selectively digests type I and type III collagens would alter ECM stiffness, Hippo signaling, and selectively reduce fibroid cell growth. We also used two FDA-approved drugs, verteporfin and nintedanib, to elucidate the role of Hippo/YAP signaling in uterine fibroid and myometrial cells. METHODS: The clinical trial was registered (NCT02889848). Stiffness of samples was measured by rheometry. Protein expression in surgical samples was analyzed via immunofluorescence. Protein and gene expression in uterine fibroid or myometrial cell lines were measured by real time PCR and western blot, and immunofluorescence. RESULTS: Injection of CCH at high doses (0.1-0.2 mg/cm3 ) into fibroids resulted in a 46% reduction in stiffness in injected fibroids compared to controls after 60 days. Levels of the cell proliferation marker proliferative cell nuclear antigen (PCNA) were decreased in fibroids 60 days after injection at high doses of CCH. Key Hippo signaling factors, specifically the transcriptionally inactive phosphorylated YAP (p-YAP), was increased at high CCH doses, supporting the role of YAP in fibroid growth. Furthermore, inhibition of YAP via verteporfin (YAP inhibitor) decreased cell proliferation, gene and protein expression of key factors promoting fibrosis and mechanotransduction in fibroid cells. Additionally, the anti-fibrotic drug, nintedanib, inhibited YAP and showed anti-fibrotic effects. CONCLUSIONS: This is the first report that in vivo injection of collagenase into uterine fibroids led to a reduction in Hippo/YAP signaling and crucial genes and pathways involved in fibroid growth. These results indicate that targeting ECM stiffness and Hippo signaling might be an effective strategy for uterine fibroids.

Surgical outcomes after collagenase Clostridium histolyticum failure in patients with Peyronie's disease in a multicenter clinical study.

In the present study we aimed to investigate the surgical outcomes of patients with persistent penile curvature (PC) after Collagenase Clostridium histolyticum (CCH) intraplaque injections. Data from 90 patients with persistent PC after CCH in a multicentre study from 6 andrological centres were retrospectively reviewed. Three standardized surgical techniques were performed. Group 1: plaque incision grafting (PIG) with penile prosthesis implant (PPI); Group 2: PIG without PPI; Group 3: Nesbit technique. Hospital stay, operative time, postoperative complications and PC persistency/recurrence (> 20°) were evaluated. Overall satisfaction and functional outcomes were assessed through International Index of Erectile Function-Erectile Function (IIEF-EF), Peyronie's Disease Questionnaire (PDQ), Female Sexual Function Index (FSFI) administered pre and 3 months postoperatively. Of all, 25 (27.8%) patients received grafting procedure + PPI (Group 1), 18 (20.0%) patients belonged to Group 2, and 47 (52.2%) to Group 3. Bovine pericardium graft and collagen fleece have been used in in 22 (51.2%) and 21 (48.8%) patients, respectively. Median penile length after surgery was 13.0 cm (IQR 12.0-15.0). After surgery, Group 1 showed higher increase in penile length after surgery and better improvements in terms of PDQ-PS. In contrast, both IIEF-EF and FSFI scores did not differ among groups. Overall, 86 (95.6%) did not report any complication. 4 (4.4%) patients had PC recurrence; of those, 2 (8.0%), 1 (5.6%) and 1 (2.1%) cases were observed in Group 1, Group 2 and Group 3, respectively. In case of persistent PC after CCH, surgical correction by grafting with or without concomitant PPI or Nesbit technique emerged as a technically feasible, effective and safe procedure, with no significant postoperative complications.

Current role of the collagenase Clostridium histolyticum in Dupuytren's disease treatment.

BACKGROUND: Collagenase Clostridium histolyticum (CCH) is a recent treatment for Dupuytren disease, which is a fibroproliferative disorder that leads to progressive, persistent digital flexion contracture that interferes with basic daily activities. While CCH has changed the treatment of this hand disorder, numerous concerns have to be analyzed. AIMS: Our purpose is to assess the current status of this medical treatment. METHODS: Literary review based on a manual search on PubMed, Web of Science, and Google Academic. RESULTS: Pharmacoeconomic analyses support the use of CCH, but long-term studies showing that it should be favored over conventional surgery or other treatments are lacking. Treatment decisions, therefore, must be guided by current data, which include a 5-year recurrence rate of 47%. Complications following CCH treatment are also a controversial topic, as rates of over 90% have been reported, although most of the complications are mild and self-limiting. A definition and classification of CCH-related complications is sorely needed. If we exclude adverse effects that could be considered inherent to the mechanisms of action of CCH, then the complication rate would be similar to rates reported for other techniques. Although CCH is becoming an increasingly popular treatment for Dupuytren disease, the potential applications of this modality, are much higher than currently believed, for more disorders characterized by excessive fibrosis. CONCLUSION: Currently, the administration of this treatment is promising although long-term studies are necessary to see the real role that this drug can play in both Dupuytren's disease and other fibrotic disorders.

Altered long noncoding RNA profile after intracerebral hemorrhage.

OBJECTIVE: We investigated the expression pattern of long noncoding RNAs (lncRNA) and messenger RNAs (mRNA) from two different intracerebral hemorrhage (ICH) rat models, and performed gene ontology and gene/protein interaction analyses. METHODS: We harvested hemorrhagic brain 1, 3, and 7 days after ICH induction by stereotactic collagenase injection. We performed microarray analyses with Agilent array platform to compare the expression of lncRNA and mRNAs from hemorrhagic and normal brains. The RNA expression patterns were also examined from the autologous blood injection ICH model at days 1 and 3, and significantly altered lncRNAs from two ICH models were validated by quantitative reverse transcriptase-polymerase chain reaction. Gene ontology analysis and pathway analysis were performed with differentially expressed mRNAs after ICH. Gene and protein interaction analysis was performed to elucidate the functional role of upregulated lncRNA in neuronal damage. RESULTS: Among the 13,661 lncRNAs studied, 83, 289, and 401 lncRNAs were significantly elevated after 1, 3, and 7 days after collagenase-induced ICH, respectively. NR_027324, or H19, was the most upregulated lncRNA after 1 day from the two ICH models and its elevation persisted until the 7th day. Gene ontology analysis revealed that immune-related biological processes such as immune response, immune system process, and defense response were upregulated from both ICH models. Gene and protein interaction study demonstrated that NR_027324 was closely related to the type I interferon signaling pathway. INTERPRETATION: This study illustrates the dynamic expression pattern of the lncRNA profile following ICH, and that H19 is the most consistently upregulated lncRNA after ICH.

Minocycline Preserves the Integrity and Permeability of BBB by Altering the Activity of DKK1-Wnt Signaling in ICH Model.

Disruption of the blood-brain barrier (BBB) and subsequent neurological deficits are the most severe consequence of intracerebral hemorrhage (ICH). Minocycline has been wildly used clinically as a neurological protective agent in clinical practice. However, the underlying mechanisms by which minocycline functions remain unclear. Therefore, we assessed the influence of minocycline on BBB structure, neurological function, and inflammatory responses in a collagenase-induced ICH model, and elucidated underlying molecular mechanisms as well. Following a single injection of collagenase VII-S into the basal ganglia, BBB integrity was assessed by Evans blue extravasation while neurological function was assessed using an established neurologic function scoring system. Minocycline treatment significantly alleviated the severity of BBB disruption, brain edema, and neurological deficits in ICH model. Moreover, minocycline decreased the production of inflammatory mediators including TNF, IL-6, and MMP-9, by microglia. Minocycline treatment decreased DKK1 expression but increased Wnt1, ß-catenin and Occludin, a phenomenon mimicked by DKK1 silencing. These data suggest that minocycline improves the consequences of ICH by preserving BBB integrity and attenuating neurologic deficits in a DKK1-related manner that involves enhancement of the Wnt1-ß-catenin activity.

Collagen V Is a Potential Substrate for Clostridial Collagenase G in Pancreatic Islet Isolation.

The clostridial collagenases, H and G, play key roles in pancreatic islet isolation. Collagenases digest the peptide bond between Yaa and the subsequent Gly in Gly-Xaa-Yaa repeats. To fully understand the pancreatic islet isolation process, identification of the collagenase substrates in the tissue is very important. Although collagen types I and III were reported as possible substrates for collagenase H, the substrate for collagenase G remains unknown. In this study, collagen type V was focused upon as the target for collagenases. In vitro digestion experiments for collagen type V were performed and analyzed by SDS-PAGE and mass spectrometry. Porcine pancreatic tissues were digested in vitro under three conditions and observed during digestion. The results revealed that collagen type V was only digested by collagenase G and that the digestion was initiated from the N-terminal part. Tissue degradation during porcine islet isolation was only observed in the presence of both collagenases H and G. These findings suggest that collagen type V is one of the substrates for collagenase G. The enzymatic activity of collagenase G appears to be more important for pancreatic islet isolation in large mammals such as pigs and humans.

Loss of stiffness in collagen-rich uterine fibroids after digestion with purified collagenase Clostridium histolyticum.

BACKGROUND: Uterine fibroids are a significant health problem. These common benign tumors occur in 70-80% of women before age 50 years and often cause bleeding and pain and can interfere considerably with daily life. Current treatment options are limited. Fibroids contain substantial amounts of altered and disordered collagens, which contribute to their bulk. Targeting these collagens directly presents a novel treatment approach. OBJECTIVES: We sought to test the hypothesis that a highly purified collagenase Clostridium histolyticum will digest interstitial collagen in uterine fibroids and reduce their stiffness and thereby evaluate the feasibility that this collagenase C histolyticum can be developed into an alternative treatment for fibroids. A secondary objective was to describe the collagen content of the fibroid tissue. STUDY DESIGN: Fibroid tissue cubes (1 cm3; n = 154) were cut from 17 uterine fibroids that were obtained from 7 consented subjects undergoing scheduled hysterectomies. Tissue cubes were injected with diluent, placebo, or highly purified collagenase C histolyticum (0.05, 0.1, or 0.2 mg/cube) and incubated at 37°C for 24, 48, 72, or 96 hours. At each time point, 6 noninjected control cubes were also evaluated. Tissue cubes were photographed before and after incubation. Myometrial samples (n = 21) were also evaluated. Stiffness was quantified through rheometry by measuring complex shear moduli of the tissues. Percent fibrosis was determined by computerized analysis of Masson-trichrome-stained slides. Digestion of collagen fibrils was confirmed by transmission electron microscopy. RESULTS: Fibrosis in untreated fibroids ranged from 37% to 77%, reflecting the collagen-rich nature of these tumors. After treatment with collagenase for 96 hours, fibrosis ranged from 5.3% to 2.4%. Transmission electron microscopy confirmed complete digestion of collagen fibrils. Tissue stiffness was reduced with all 3 doses of collagenase treatment and at all 4 time points. Longer incubation times with collagenase caused greater reduction in stiffness, and treated cubes lost their cuboidal shape and had gelatinous/liquefied centers. At 96 hours the stiffness in tissues treated with the lowest dose was reduced to 966 ± 106 Pascal compared with the diluent-treated control at the same time (5323 ± 903 Pascal; P < .0001; by analysis of variance with Tukey-Kramer). CONCLUSION: Uterine fibroids have a high content of collagen that can be effectively digested by highly purified collagenase C histolyticum, resulting in reduced tissue stiffness. Loss of stiffness may decrease bulk symptoms in vivo and possibly lead to shrinkage of fibroids through changed mechanotransduction, leading ultimately to reduced fibroid symptoms of pain and bleeding. Clinical trials are necessary to evaluate the safety and efficacy of collagenase C histolyticum including the rate of regrowth of fibroids. The data of this study provide a strong rationale for using this purified collagenase in clinical trials as a local treatment for women with fibroids.

Efficacy and Safety of the Collagenase of the Bacterium Clostridium Histolyticum for the Treatment of Capsular Contracture after Silicone Implants: Ex-Vivo Study on Human Tissue.

BACKGROUND: The fibrotic capsule that surrounds silicone implants consists mainly of collagen. The FDA-approved collagenase of the bacterium clostridium histolyticum provides a reasonable treatment option. Safety and efficacy at the female breast site must be evaluated before clinical utilization. MATERIALS AND METHODS: We incubated 20 samples of fibrotic capsule as well as 12 full thickness skin grafts harvested from the female breast site for 24 hours with different doses of collagenase. Outcome measures involved histological assessment of thickness and density of the capsule tissue as well as the skin grafts. Furthermore, we performed a collagen assay and immunohistochemistry staining for collagen subtypes. RESULTS: Collagenase treatment was able to degrade human capsule contracture tissue ex-vivo. The remaining collagen subtype after degradation was type 4 only. 0.3 mg/ml of collagenase was most effective in reducing capsule thickness when compared with higher concentrations. Of note, effectiveness was inversely related to capsule density, such that there was less reduction in thickness with higher capsule densities and vice versa. Furthermore, the application of 0.3mg/ml collagenase did not lead to thinning or perforation of full thickness skin grafts. CONCLUSION: Adjustment of collagenase dose will depend on thickness and density of the contracted capsule. A concentration of 0.3mg/ml seems to be safe and effective in an ex-vivo setting. The remaining collagen subtype 4 is suitable to serve as a neo-capsule/acellular tissue matrix. Collagenase treatment for capsular contracture may soon become a clinical reality.

[Effects of exogenous enzymes on the degradation of adhesive-dentin interfaces].

OBJECTIVE: To compare the effects of exogenous enzymes on the degradation of adhesive-dentin interface. METHODS: Forty molars were sectioned to expose the middle-coronal dentin surface and randomly divided into two adhesive systems: an etch-and-rinse adhesive Adper Single Bond 2 and a self-etching adhesive G-Bond. After composite building up, the specimens were then randomly assigned to four groups(n=5 for each group)as follows: group 1, 24 h of water storage(the control group); group 2, six months of water storage; group 3, twelve weeks storage in artificial saliva containing clostridium histolyticum collagenase; group 4, twelve weeks storage in artificial saliva containing cholesterolesterase. The microtensile bond strengths(MTBS)were then tested. The failure modes and nanoleakage were analyzed. RESULTS: After aging treatments, the three aging groups showed significantly lower MTBS compared with the control group in both adhesive systems(P<0.05). For etch-and-rinse adhesive Adper Single Bond 2, the MTBS of group 3([19.6±3.5]MPa)was lower than that of group 2([23.4±4.2]MPa)and group 4([24.2±4.2]MPa)(P<0.05). For self-etching adhesive G-Bond, there was no difference on MTBS among different aging groups(P>0.05). SEM observation showed that, compared with the control group, water storage(group 2)and the exogenous enzymes(group 3 and 4)increased the nanoleakage expression(silver deposition)of both adhesive systems. Adhesive failure was the predominant fracture modes in all groups. CONCLUSIONS: Storage in artificial saliva containing clostridium histolyticum collagenase or cholesterol esterase could be used to accelerate the degradation process of adhesive-dentine interface.

[Effect of dopamine on the activity of matrix metalloproteinases and degradation of dentin collagen].

OBJECTIVE: To investigate the inhibition effect of dopamine on the activity of matrix metalloproteinases (MMP) and the effect of dopamine on degradation of dentin collagen for its potential use in caries treatment and dentin adhesive. METHODS: In the experiment of MMP activity test, 2.0 g/L dopamine + 1.0 g/L highly purified collagenase type VIII from Clostridium histolyticum served as the experimental group, and deionized water + 1.0 g/L highly purified collagenase type VIII from Clostridium histolyticum served as the negative control group, and 2% chlorhexidine + 1.0 g/L highly purified collagenase type VIII from Clostridium histolyticum served as the positive control group, and the mixture volume ratio of the two ingredients in every group was 1:9. After 15 minutes, the enzyme activity of each sample was tested by MMP activity colerimetric quantitative detection kits, and the test was repeated 5 times in each group. In the experiment of collagen degradation, the dentin slices were demineralized with 37% phosphoric acid for 1 min. In sequence, 2 dentin slices were used to observe the morphology, and the remaining 30 dentine slices were randomly divided into three groups (n = 10) according to random number table: the negative control ones were stored in 100 µl deionized water and 900 µl collagenase (7 days, 37 °C), the positive control ones were stored in 100 µl chlorhexidine and 900 µl collagenase (7 days, 37 °C) and the experimental specimens were stored in 100 µl dopamine and 900 µl collagenase (7 days, 37 °C). The degraded collagen was investigated by assaying hydroxyproline. The framework of collagen was evaluated with field emission scanning electron microscope (FE-SEM). RESULTS: The statistical results of completely random design ANOVA showed that the MMP activity and the amount of degraded collagen of the negative control group [(0.089 ± 0.011) µmol · min⁻¹ · mg⁻¹ and (2 837 ± 201) µg/cm²] were significantly higher than those of the positive control group [(0.038 ± 0.006) µmol · min⁻¹ · mg⁻¹ and (1 288 ± 172) µg/cm²] and the experimental group [(0.030 ± 0.009) µmol · min⁻¹ · mg⁻¹ and (1 389 ± 255) µg/cm²] (P < 0.05). SEM observation indicated that the structural integrity of the collagen network on dentin still existed in experiment samples and positive control groups, however, collagen fibrils were destructed and the structural integrity disappeared in the negative control groups. CONCLUSIONS: Dopamine may inhibit MMP activity and reduce the amount of degraded collagen.

Enzymatic debridement in necrotizing pancreatitis.

Multiple organ failure and pancreatic necrosis are the factors that determine prognosis in acute pancreatitis attacks. We investigated the effects of collagenase on the debridement of experimental pancreatic necrosis. The study covered 4 groups; each group had 10 rats. Group I was the necrotizing pancreatitis group. Group II was the collagenase group with pancreatic loge by isotonic irrigation following necrotizing pancreatitis. Group III was the collagenase group with pancreatic loge following necrotizing pancreatitis. Group IV was the intraperitoneal collagenase group following necrotizing pancreatitis. The progress of the groups was compared hematologically and histopathologically. There was no difference among the groups regarding the levels of leukocyte, hemogram, and urea. The differences in AST levels between Group I and II; and differences in glucose, calcium, LDH, AST, and amylase between Group II and III; between Group II and IV; between Group I and III; and between Group I and IV were statistically significant (P < 0.05). There were statistically significant differences between Group II and III, and Group II and IV regarding edema, acinar necrosis, inflammatory cell infiltration, hemorrhage, and fat necrosis (P < 0.05). In conclusion, the collagenase preparation used in this experimental pancreatitis model was found to be effective in the debridement of pancreatic necrosis.

Acetylated derivative of glaucine inhibits joint inflammation in collagenase-induced arthritis.

CONTEXT: Osteoarthritis (OA) has become by far the most common joint disorder. A number of studies using OA animal models have explored the effects of agents that can modulate bone metabolism. OBJECTIVE: In the present study, we investigated the effect of acetylated derivative of plant alkaloid glaucine (ADG) on experimental OA in mice. MATERIALS AND METHODS: Arthritis was induced by two intraarticular (i.a.) injections of collaganase. Histopathological changes were observed through hematoxylin and eosine (H&E), safranin O and toluidine blue staining. Differentiation of bone marrow (BM) cells was evaluated by tartarate-resistant acid phosphatase (TRAP) assay. The expression of phospho-Janus kinase 2 (pJAK2) and phospho signal transducer and activator of transcription3 (pSTAT3) expression in the joints was determined by immunohistochemistry. RESULTS: We established that ADG significantly decreased cell infiltration (2.32 ± 0.14 versus 1.62 ± 0.13), cartilage loss (2.42 ± 0.12 versus 1.12 ± 0.10) and bone erosion (1.76 ± 0.13 versus 1.04 ± 0.14) in arthritic mice. It appeared that the substance inhibited in a dose-dependent manner osteoclast differentiation in vitro. ADG suppressed the expression of pJAK2 in the joint and partially affected the expression of pSTAT3. CONCLUSION: Present results suggest that ADG is a suitable candidate for further development as an anti-arthritic agent.

The effect of mouthwashes containing biguanides on the progression of erosion in dentin.

BACKGROUND: Dental erosion is caused by frequent exposure to acids without the involvement of microorganism. This study analyzed the effect of biguanides (polyhexamethylene biguanide - PHMB and chlorhexidine - CHX) on dentin erosion due to their possible influence on the enzymatic degradation of the demineralized organic matrix. METHOD: Sixty bovine dentin specimens were prepared. On both sides of their surface, nail varnish was applied to maintain the reference surfaces for the determination of dentin loss. Samples were cyclically de- and remineralized for 6 days. Demineralization was performed with a 0.87 M citric acid solution (6×5 min daily). Thereafter, samples were treated with distilled water (negative control), 0.12% CHX (positive control), 0.07% PHMB, Sanifill Perio Premium™ (0.07% PHMB plus 0.05% NaF), or F solution (0.05% NaF) for 1 min and then subjected to enzymatic challenge for 10 min using a bacterial collagenase (Clostridium hystoliticum, 100 µg/ml). Dentin loss was assessed using profilometry (µm) daily. Data were analyzed using 2-way repeated measures-ANOVA and Bonferroni's test (p < 0.05). RESULTS: Dentin loss progressed significantly for all groups during the 6 days. After the 3rd day, Sanifill Premium™, CHX, and PHMB significantly reduced dentin erosion compared to control. On the 6th day, the lowest mean (±SD) dentin loss was observed for Sanifill Perio Premium™ (94.4 ± 3.9 µm). PHMB and CHX led to intermediate dentin loss (129.9 ± 41.2 and 135.3 ± 33.5 µm, respectively) that was significantly lower than those found for negative control (168.2 ± 6.2 µm). F (157.4 ± 6.1 µm) did not significantly differ from negative control. CONCLUSIONS: Sanifill Perio Premium™ mouthwash has a good potential to reduce dentin loss, which might be associated with the presence of PHMB.

Addition of Grape Seed Extract Renders Phosphoric Acid a Collagen-stabilizing Etchant.

Previous studies found that grape seed extract (GSE), which is rich in proanthocyanidins, could protect demineralized dentin collagen from collagenolytic activities following clinically relevant treatment. Because of proanthocyanidin's adverse interference to resin polymerization, it was believed that GSE should be applied and then rinsed off in a separate step, which in effect increases the complexity of the bonding procedure. The present study aimed to investigate the feasibility of combining GSE treatment with phosphoric acid etching to address the issue. It is also the first attempt to formulate collagen-cross-linking dental etchants. Based on Fourier-transformed infrared spectroscopy and digestion assay, it was established that in the presence of 20% to 5% phosphoric acid, 30 sec of GSE treatment rendered demineralized dentin collagen inert to bacterial collagenase digestion. Based on this positive result, the simultaneous dentin etching and collagen protecting of GSE-containing phosphoric acid was evaluated on the premise of a 30-second etching time. According to micro-Raman spectroscopy, the formulation containing 20% phosphoric acid was found to lead to overetching. Based on scanning and transmission electronic microscopy, this same formulation exhibited unsynchronized phosphoric acid and GSE penetration. Therefore, addition of GSE did render phosphoric acid a collagen-stabilizing etchant, but the preferable phosphoric acid concentration should be <20%.

Bioactive chitosan nanoparticles and photodynamic therapy inhibit collagen degradation in vitro.

INTRODUCTION: Collagen is the major structural protein of human dentin. Degradation of collagen by bacterial enzymes can facilitate microbial penetration, compromise structural/interfacial integrity, and lower resistance to fracture of dentin. We evaluated the ability of photodynamic therapy (PDT), bioactive chitosan nanoparticles (CSnp), or PDT in combination with CSnp to inhibit bacterial collagenase-mediated degradation of collagen. METHODS: Rat type 1 fibrillar collagen matrices were untreated or treated with 2.5% glutaraldehyde (GD), 2.5% GD followed by 1% CSnp, 1% CSnp, PDT (rose bengal activated with 540 nm light at 40 J/cm(2)), or 1% CSnp followed by PDT. Samples, except those used as untreated controls, were exposed to Clostridium histolyticum collagenase (125 CDU/mL) for 24 hours. The soluble digestion products were assessed by hydroxyproline assay, and the remaining adherent collagen was quantified by picrosirius red staining. Fourier transform infrared spectroscopy, immunoblotting, and scanning electron microscopy were used to study the interaction between CSnp/PDT with type 1 collagen. The data were analyzed by 1-way analysis of variance and post hoc Tukey test. RESULTS: As assessed by hydroxyproline release into the medium, collagen treated with CSnp, PDT, or a combination of CSnp and PDT exhibited less degradation than untreated controls (3.6-fold, 1.7-fold, and 7.9-fold reduction, respectively; P < .05). Compared with all other treatments, GD-treated collagen was the most resistant to collagenolytic degradation (239.6-fold reduction, P < .05). The abundance of post-treatment residual collagen, as measured by picrosirius red staining, was inversely related to the extent of collagen degradation. Analysis of collagen cross-links with Fourier transform infrared spectroscopy showed that PDT or GD treatments enhanced collagen cross-linking. Immunoblotting of sedimented CSnp indicated that CSnp and collagenase bound with low affinity. However, CSnp-bound collagenase showed a significant reduction in collagenolytic activity compared with controls (P < .05). CONCLUSIONS: Combined photochemical cross-linking of rat tail collagen by PDT and binding to CSnp inhibit collagenolytic activity.

Effectiveness of collagenase in preventing postoperative intra-abdominal adhesions.

INTRODUCTION: The purpose of this study is to investigate the effectiveness of Collagenase clastridiopeptidase an enzyme preparation used in enzymatic debridement in preventing adhesions brought about by peritoneal damage. METHODS: The study covers a total of 40 rats in 4 groups each having 10 rats. Group 1: The control group. Group 2: Normal saline group. Group 3: Sterile Novuxol group. Group 4: The group where the intraperitoneal and systemic effects of sterile Novuxol were investigated. Adhesion frequency and grades were scored on the post-op 11th day according to Granat. Blood work including hemoglobin, aspartate aminotransferase, alanine aminotransferase, urea, creatinine, and albumin level measurements were performed. Toxicity was investigated histopathologically through samples taken from the liver and the peritoneum from Group 4. RESULTS: Adhesion frequency was found to be 80% on the right and 90% on the left for Group 1, while it was 50% on both left and right for Group 2, and 30% on the right and 10% on the left for Group 3. Adhesion stages were found to be 1: 2.35 ± 1.42 for Group 1, 0.31 ± 1.15 for Group 2, and 0.20 ± 0.41 for Group 3. Adhesion stage of the Sterile Novuxol Group was lower than all the other groups (p < 0.05). Biochemical and hematological parameters were similar in all groups (p > 0.05). Histopathological analysis revealed no hepatotoxicity. CONCLUSIONS: According to the results of our study, we believe that Sterile Novuxol can be a good anti-adhesive agent considering its ease of use, non-toxicity, and effectiveness.

In vitro study of novel collagenase (XIAFLEX®) on Dupuytren's disease fibroblasts displays unique drug related properties.

Dupuytren's disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH)--Xiaflex®--is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex® against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex® exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex® did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex® significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex® enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex® on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.

pH in the bacteria-contaminated wound and its impact on clostridium histolyticum collagenase activity: implications for the use of collagenase wound debridement agents.

PURPOSE: We sought to understand the influence of pH on Clostridium collagenase activity, using both in vitro and in vivo, and to understand the influence of bacterial contamination on pH in vivo. DESIGN: Artificial wound eschar was used for the assessment of debridement efficacy in vitro and the wound fluid of a contaminated pig wound model was used for examining pH during healing using in vivo techniques. METHODS: We used a collagen-based artificial wound eschar to test collagenase activity in the collagenase product under various pH conditions. We evaluated bacterial contaminated wounds, using a pig wound model with a bacterial load including Pseudomonas aeruginosa, coagulase-negative staphylococci, and Fusobacterium sp to track the pH of wounds in relation to bacterial load. RESULTS: The pH levels in the wound fluid were all above neutral. They varied from 9.2 on day 1 to 8.3 on day 10. Collagenase achieved its highest activity around a pH of 8.5 when tested in Tris-buffered saline. Using artificial wound eschar, the optimal pH range for C collagenase was determined to be more than a pH of 6. The total initial microbial load (day 0) was higher than levels at any other time during the study. The levels of P aeruginosa began to decrease on day 1, but by day 4 the total pseudomonas population had rebounded to near day 0 levels. This was followed by a distinct decrease and by day 21 levels were lower than those on day 0. The coagulase negative staphylococci (CNS) population behaved differently than the pseudomonas population, decreasing and remaining decreased relative to day 0 through day 14. However, by day 21 the CNS population had increased to near day 0 levels. This greatly influenced the increase in total bioburden by day 21, indicating that it was primarily due to the increase in CNS. CONCLUSION: The data demonstrate that wound pH in a model of contaminated pig wounds is alkaline during the first 10 days of healing. As healing progresses, pH decreases with no significant change in the level of bacterial bioburden. C collagenase exhibited robust activity in the pH range found in this contaminated pig wound model, suggesting it can effectively debride necrotic tissue in the environment found in most chronic wounds in humans.

Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria.

Tissue dissociation enzymes are critical reagents that affect the yield and quality of human pancreatic islets required for islet transplantation. The United States Food and Drug Administration's oversight of this procedure recommends laboratories to set acceptance criteria for enzymes used in the manufacture of islet products for transplantation. Currently, many laboratories base this selection on personal experience because biochemical analysis is not predictive of success of the islet isolation procedure. This review identifies the challenges of correlating results from enzyme biochemical analysis to their effectiveness in human islet isolation and suggests a path forward to address these challenges to improve control of the islet manufacturing process.