Collagenase Preparations Research Articles

Dupuytren’s contracture treated with collagenase Clostridium histolyticum

Treatment of Dupuytren’s contracture improves the quality of life of patients, but standard open fasciectomy surgery is traumatic and requires long-term rehabilitation. The study aimed to determine whether injectable treatment with collagenase preparations was effective in comparison with open fasciectomy. Forty patients were examined. Of these, 15 were treated with Clostridium histolyticum collagenase preparations (the main group), and 25 underwent limited fasciectomy (the control group). In 11 (73.3%) patients of the main group, restoration of mobility of the affected joints was achieved after the first injection, in 3 (20%) – after the second, and in 1 (6.7%) – after the third. Patients in the main group remained able to work after treatment and did not require rehabilitation; the function of the upper limb was fully restored on the day of intervention. During the observation period, 12 (80%) patients in the main group were concerned about dry skin at the intervention site, and 5 (33.3%) patients were concerned about itching and discolouration of the skin at the intervention site. In 25 (100%) patients of the control group, joint mobility was fully restored. The ability to use the hand after surgery in patients of the control group was limited for 6 (4.5; 10) days. All patients in the control group required rehabilitation to relieve stiffness and restore the functional capabilities of the hand. The period of disability in these patients lasted 16 (12; 24.5) days. 11 (44%) patients had complaints of pain or discomfort in the intervention area during the follow-up, and 24 (96%) patients were bothered by itching. Satisfaction with the choice of treatment method in the main group was more frequent compared to the control group. Relapses during the observation period were absent in both groups. Thus, the efficacy of Clostridium histolyticum collagenase preparations are comparable to the results of surgical treatment. Minimally invasive treatment is optimal for patients with mild disease, as well as for those who have a low risk of contracture recurrence. The results of this study can be useful for surgeons when choosing the optimal method of treating Dupuytren’s contracture

CHARACTERISATION OF COLLAGENOLITYC ACTIVITY OF Coprinus spp.

In this research the screening of several higher fungi - basidiomycetes cultures for the presence of collagenolytic activity was carried out. The highest collagenase activity was detected in submerged culture of higher fungus Coprinus lagopides. The enzyme preparation of collagenase was isolated from culture liquid of this producer. The optimum temperature and pH of the collagenolytic activity of the enzyme preparation were determined. The selection of nitrogen and carbon sources and ratios of carbon and nitrogen sources in the composition of nutrient mediums was carried out in order to increase the collagenase activity of fungus submerged culture. In addition to the collagenase activity of producers culture the amount of accumulated biomass, pH and protein concentration in the culture liquid of basidiomycete were also determined.

IR-SPECTROSCOPIC STUDY OF IMMOBILIZATION OF SELENIUM COMPOUNDS ON BIOMODIFIED COLLAGEN

The aim of the work was to research the spectral characteristics of collagen substances bioengineered by using complex collagenolytic proteases preparation obtained before and after the immobilization of selenium compounds in acidic and alkaline media and to comparate the degree of interaction of selenium preparations with collagen matrix upon immobilization and its influence on the conformation of protein molecules. The results of the immobilization of selenium preparations on biomodified collagen by the IR spectroscopy method are presented, analyzed, and discussed. Biomodified collagen was obtained from beef trimming waste (veins and tendons) by sequential peroxide-alkaline and enzymatic hydrolysis with the food collagenase preparation. The following sources of selenium were used as compounds with bioprotective properties for subsequent immobilization on biomodified collagen proteins: 4,4-di[3(5-methyldiprazolil)]selenide (DMDPS) with the content of 0,657g DMDMS in 100 cm3 and sodium selenite. Spectrograms are carried out for products of biomodification of collagen before sorption of compounds of selenium, (at the rate of 1.2 g-6 of selenium on 1 g of collagen) in acid (рН =5) and alkaline (рН =10) environments at a research of influence of compounds of selenium on IR spectrums of products of biomodification of collagen. It was established, that the immobilization takes place by a chemical reaction of selenium preparations with functional groups of the side chains of protein molecules, and its degree varies in the range Na2SeO3 (pH=5) > 4,4-di[3(5-methyldiprazolil)]selenide (DMDPS) > Na2SeO3 (pH=10). It is shown that under the interaction of selenium products with collagen, there is no change in the conformations of its molecules occurred.

EXPERIENCE WITH TRANSVAGINAL APPLICATION OF ELECTROPHORESIS IN THE COMPLEX THERAPY OF PATIENTS WITH CHRONIC SALPINGOOPHORITIS

Chronic salpingooophoritis occupies one of the leading places in the structure of gynecological diseases (60-70%) with a tendency to generalization and chronicization of the inflammatory process, the development of serious pathophysiological and pathomorphological changes in affected tissues, involvement of the reproductive system in the pathological process and a significant negative effect on the quality of life. In clinical conditions, 25 patients (mean age 29.9 ± 3.6 years) with a verified diagnosis of chronic salpingooophoritis were examined and treated. Treatment included transvaginal electrophoresis of the complex of collagenases (15 procedures for a course, 3-4 times a week). Efficacy was assessed taking into account the indices of the scale of symptoms, degree of pain syndrome and quality of life. Transvaginal electrophoresis with collagenase preparations has a high efficiency for all clinical symptoms of chronic salpingooophoritis, which is confirmed by the dynamics of the index of the symptom scale, the reduction of which was 74.9%, while the global therapeutic effectiveness of the method was 80%. The developed method is highly effective in terms of relief of pain, which significantly contributes to improving the quality of life of this category of patients.

Comparison of non-surgical methods for the treatment of deep partial thickness skin burns of the hand

This paper describes a randomized, controlled, parallel-group, single-center clinical trial designed to compare non surgical treatment methods of deep partial thickness skin burns of the hand.All patients were scanned with the Laser Doppler Imaging device to determine the depth of the burn wound. Viable keratinocytes sites were determined according to the established Perfusion Units (PU) measurement system. The trial enrolled 87 patients with hand burn wounds in the section of 260–600PU.Hand burn patients were divided into the following four groups: treated with hydrocolloid dressings; treated with mechanical debridement of monofilament polyester fibers pad and then applying silver sulfadiazine; treated with gauze dressings containing enzymatic collagenase preparation. The fourth group of patients was treated with silver sulfadiazine and gauze dressings. This group was considered as the control group. The wound healing status was assessed after 3, 7, 14 and 21 days. Burn scars and injured extremity function were assessed after six months according to the Vancouver Scar Scale and Disabilities of the Arm, Shoulder and Hand Outcome Measure.The fastest epithelialization of hand burn wounds was observed in the patients group treated with hydrocolloid dressings (15, 7 days, p<0,05). The patients of this group also had less scars and a better hand function.

An Optimized and Validated Method for Isolation and Characterization of Lymphocytes from HIV+ Human Gut Biopsies.

The gastrointestinal (GI) tract harbors most of the body's immune cells and is also a major HIV reservoir in ART-treated patients. To achieve a cure, most HIV-infected cells must be identified and eliminated. While obtaining gut biopsies is a relatively noninvasive method of sampling relevant tissue for monitoring HIV activity, immune cell isolation from these limited tissue samples has proven to be challenging. Enzymatic tissue digestion is required for maximal immune cell isolation from gut biopsies. However, these enzymatic digestions can also be detrimental for preservation of cellular surface markers that are required for accurate identification of various subsets of leukocytes. In this study, we describe an optimized protocol for isolation of lymphocytes from human gut biopsies. We also discuss our validation results, which show that compared with several other collagenase preparations, the use of CSLPA maintains high lymphocyte recovery while preserving the integrity of most cellular surface antigens that we tested. Importantly, chemokine receptors that are used to characterize various subsets of T cells, which are notorious for being digested during a typical enzymatic tissue digestion, are highly preserved using this protocol.

Substantiation of conditions for biomodification of starfish for production of fodder additives

Biotechnological stage in technology of fodder products of starfish is substantiated. The starfishes Evasterias echinosoma and Patiria pectinifera caught in the Severnaya Bay (Primorye coast of the Japan Sea) are used in the experiments on enzymic hydrolysis for production of the fodder additive with high biological value. The hydrolysis provides the more accessible forms of nutrients for animals and birds. Impacts of the enzyme preparation concentration, temperature, pH, time of hydrolysis, and water duty on efficiency of enzymic hydrolysis are tested in the experiments; these parameters influence on the amount of accumulated amino nitrogen and on the hydrolysis degree is evaluated. The following rational values are determined for the parameters in the process of protein-mineral fodder additive production: concentration of enzyme collagenase preparation - 0.25-1.00 %; time of treatment - 2-4 hours; temperature - 45-55 o C; pH - 6.0; water duty - 0.2.

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.

Effect of enzymatic debridement with two different collagenases versus mechanical debridement on chronic hard-to-heal wounds.

A chronic ulcer is usually defined as an injury that does not spontaneously evolve towards healing and does not progress through normal healing stages such as inflammation, proliferation and remodelling. This study was designed in order to compare two types of collagenases with mechanical debridement alone. It was thus possible to evaluate their differences in terms of pain and debridement efficacy. Patients were divided into three groups: 30 patients were daily dressed using an ointment based on collagenase produced by Vibrio alginolyticus (B group), 30 patients were daily dressed using an ointment based on a collagenase preparation derived from Clostridium histolyticum (N group) and 30 patients underwent classical mechanical debridement (M group). Complete wound healing over a period of 8 weeks occurred in 24 patients (27%) out of 90;10 patients belonging to the B group, 8 patients to the N group and 6 patients to the M group. This study was performed in order to highlight the differences between two commercially available collagenase-based ointments in comparison with mechanical debridement alone. At the final time point of week, the difference in the percentage of debridement was not statistically significant in all groups, but at 4 weeks, the debrided area in the B group was larger with respect to the N and M groups, suggesting a more rapid wound bed cleansing process. On the basis of our experience, collagenase derived from V. alginolyticus with hyaluronic acid showed chemical and physical properties that make it a product of great manageability and ensure the protection of peri-wound skin. Moreover, less pain was experienced by the patients.

Collagenase Followed by Compression for the Treatment of Earlobe Keloids

Many therapeutic options are available for treating keloids, but success rates vary widely, and the keloids often recur. The Food and Drug Administration has recently approved intralesional collagenase for the treatment of Dupuytren's contracture. This medication has not been explored for the treatment of earlobe keloids, a common problem. To evaluate the safety and clinical efficacy of intralesional collagenase followed by compression for the treatment of earlobe keloids. Six earlobe keloids in six patients were injected with a commercial collagenase preparation. Study participants were asked to use compression earrings daily thereafter. Patients were examined and photographed 1day, 2weeks, 4weeks, 10months, and 12months after injection. Adverse events were assessed at each visit, and the keloids were measured and photographed. All patients had a decrease in the size of their earlobe keloid by an average of 50% (p=.02). Three of the six participants chose to have their earlobe keloids surgically excised for cosmetic reasons 6, 8, and 11months after enrollment, so measurements for data analysis for these patients were taken after only 1, 1 and 10months. All participants returned for follow-up at the last study visit 1year after study commencement. The three patients who completed the study were pleased with the improvement of their earlobe keloid, although complete clearance was not achieved. Side effects included injection site swelling, tenderness, and one ulceration that spontaneously resolved within 2weeks. Intralesional collagenase followed by compression appears to be a safe and modestly effective treatment for earlobe keloids. This approach warrants further investigation in larger studies with longer follow-up in motivated patients who decline surgical excision.

Characterizing and quantifying leukocyte populations in human adipose tissue: Impact of enzymatic tissue processing

Adipose tissue inflammation is a major mechanistic link between obesity and chronic disease. To isolate and characterize specific leukocyte populations, e.g. by flow cytometry, tissue needs to be processed to digest the extracellular matrix. We have systematically compared the impact of different commonly used collagenase preparations, digestion times, and normalization strategies on the reproducibility of flow cytometric phenotyping of adipose tissue leukocyte populations. Subcutaneous adipose tissue was obtained from 11 anonymous donors undergoing elective procedures at a plastic surgery clinic in Seattle, WA. We found that collagenase alone consistently produced better cell yields (p=0.007) than when combined with additional proteases such as the commercially available liberases. Moreover, liberase significantly degraded the cell surface expression of CD4 (p<0.001) on T cells and to a lesser extent CD16 (p=0.058) on neutrophils. Extension of the digestion interval from 30 to 120min did not significantly impact cell viability (p=0.319) or yield (p=0.247). Normalization by either ‘live-gate’ or percentage of CD45pos leukocytes exhibited the lowest coefficient of variation for tissue digests between 60 and 75min, compared to normalization per gram of tissue, which consistently exhibited the greatest variability. Our data suggest that digestion of adipose tissue using pure collagenase for 60–75min provides the best cell yield and viability, with minimal degradation of cell surface markers used to identify immune cell subpopulations, and best reproducibility independent of the normalization strategy.

Improved Method of Human Islet Isolation for Young Donors

Background Although islet transplantation using young donors is more effective than older donors, islet isolation from young donor is notoriously difficult. This may relate to islet ontogeny and collagen composition in the young pancreas. Therefore, we examined whether a high concentration of collagenase could improve the separation of islets from exocrine tissues resulting in an high islet yield. Methods We used six human pancreata from brain-dead donors of less than 30 years old. Islet isolation was performed based on the Edmonton protocol with modifications. All pancreata were digested with Collagenase NB1 Premium Grade (Serva). The pancreas was expanded by injecting either 200 mL of cold collagenase solution (2.5 mg/mL, standard group, n = 3) or 100 mL of solution (5 mg/mL, new group, n = 3) in a controlled manner under low pressure for 5 minutes. Then the pressure was raised for another 5 minutes. The following procedure and evaluation were performed based on the Edmonton protocol. Results Phase II time in the new group was significantly shorter than the standard group. The ratio of embedded islets in the new group was significantly lower than the standard group. The postpurification islet equivalents per pancreas weight (IEQ/g) and the recovery rate in the new group were higher than the standard group, but not significantly. There was no significant difference in the postpurification purity, viability, and final tissue volume. Conclusion Our simple modification with an initially concentrated collagenase preparation using a syringe significantly improved the ratio of embedded islets, resulting in a higher yield from young donors.

Tissue Dissociation Enzyme Neutral Protease Assessment

Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate–conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures.

Reply to Burgdorf et al.: The mannose receptor is not involved in antigen cross-presentation by steady-state dendritic cells

We thank Burgdorf et al. for suggesting potential explanations for the few discrepancies between our studies (1, 2). Our collagenase treatment did not prevent us from detecting the mannose receptor (MR) on splenic macrophages and inflammatory dendritic cells (DC), or from confirming a role for MR in OVA uptake by the latter (1). We have analyzed MR expression on cells purified with or without collagenase digestion with identical results: macrophages express MR, but steady-state DC do not (Fig. 1). Furthermore, OVA uptake was not affected by the use of collagenase (Fig. 1). Some commercial preparations of collagenase contain trypsin-like activity that might affect MR detection or OVA uptake; we used collagenase tested for the absence of this activity. Previous studies also failed to detect MR in steady-state DC (3, 4). These studies included in situ staining of tissue sections, which would … 1To whom correspondence should be addressed. E-mail: villadangos{at}wehi.edu.au.

Human Placenta Is a Potent Hematopoietic Niche Containing Hematopoietic Stem and Progenitor Cells throughout Development

Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.

High-level expression of his-tagged clostridial collagenase in Clostridium perfringens

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.

Comparative Analysis of Highly Defined Proteases for the Isolation of Adipose Tissue-Derived Stem Cells

Before the potential of adipose tissue-derived stem cells can fully be exploited for a broad scope of tissue-engineering and cell-based therapeutical applications, an effective and reproducible method for isolation is needed. To comparatively analyze five highly defined protease formulations, Blendzyme 1-4, liberase H1 and a crude collagenase mixture in the course of digestion that consisted of three 1-h intervals. The resulting digests of human adipose tissue aspirates were evaluated for the yield of nucleated cells, viability and frequency of specific lineages, in particular CD90, CD34 and CD45, by flow cytometry. The functionality of the cells was assessed as to the colony-forming capacity in limiting dilution assays. Based on all evaluation criteria, Blendzymes 1 and 2 and liberase H1 demonstrated a superior performance and highest consistency. Blendzyme 3 clearly underperformed compared with all other enzymes, and the performance of the rest of enzymes appeared erratic. As for the length of digestion, a 2-h interval appeared optimal when weighing both the yield and functionality of the cells in the stromal vascular fractions obtained from different adipose tissue samples. Our results demonstrate that the highly purified proteases provide a valuable alternative to crude collagenase preparations, especially in scenarios where a high definition and reproducibility of the digestion process is of importance.

Comparative evaluation of the depth of collagen and hyaluronic acid hydrolysis in vitro by collagenase and hyaluronidase preparations

The molecular weights of collagen and hyaluronic acid solutions after their incubation with collagenase and hyaluronidase were evaluated by capillary viscosimetry. The results indicate high amylolytic activity of collagenase and the absence of proteolytic activity in hyaluronidase in an in vitro system.

Dynamics of hydration of cicatricial tissue during local therapy with collagenase preparations

The components of complex dielectric permeability of cicatricial tissue at the frequency of 55 GHz were studied during local therapy of cicatrices with Fermenkol (a complex of collagenolytic proteases). Electrical characteristics of tissues in the microwave range were interpreted in terms of hydration parameters (total content of water in tissues and the ratio of structured and free water). The rightfulness of interpretations is discussed on the basis of the results of measurements of cell suspension models (native blood and its fractions brought to a standard hematocrit). The results attest to a relationship between hydration of the cicatricial tissue and its morphology and function.

A comparative study of collagenase complex and new homogeneous collagenase preparations for scar treatment

The unique feature of collagenase is the ability to cleave the native triple helix of intact collagen molecules. This ability makes it particularly effective in the removal of detritus but not in scar treatment. Three crude collagenase preparations containing Cl. histolyticum collagenase, collagenases PC from the hepatopancreas Paralithodes camtshatica and collagenase D from larvae Dermestes frischii were observed as potential drugs for scar treatment. Activity of collagenase D is significantly superior to that of king crab collagenase PC. The observed effect is superior to that of Cl. histolyticum collagenase. It was established that collagenase D may be used for treatment of keloids and hypertrophic scars. Liniment Morikrol containing collagenase PC was effectively applied for scar treatment and in other cases where skin may regenerate.