Low Collagenase Research Articles

Enzyme-injected method of enzymatic dispersion at low temperature is effective for isolation of smooth muscle cells from human esophagogastric junction.

The present study was conducted to examine the feasibility of in vitro isolation and primary culture of smooth muscle cells (SMCs) from the esophagogastric junction (EGJ). Smooth muscles of EGJ were harvested from 23 patients with esophageal cancer during esophagostomy from January 2015 to December 2017. Enzymatic dispersion (ED) was performed for isolation. Collagenase II and Trypsin/EDTA were applied by enzyme injection (EI) into tissue fragments or immersion of tissue fragments into enzyme solution. Growth characteristics and proliferation [Cell Counting Kit-8 (CCK-8)] of cells were recorded for both smooth muscle cell medium (SMCM) and DMEM/F12 containing 10% newborn bovine serum (10%-F12). All ED methods could isolate primary cells; EI was the most effective method with low collagenase II concentration (0.5 mg/ml) at 4˚C for 14-24 h. Primary cells demonstrated mainly spindle- and long-spindle-shaped with ‘hills and valleys’ morphology. The CCK-8 assay in SMCM showed better proliferation results than in 10%-F12. After passaging for 4-8 generations in SMCM or 2-4 generations in 10%-F12, cells enlarged gradually with passages and lost spindle structures. mRNA and proteins of α-smooth muscle actin (α-SMA), smooth muscle 22 α (SM22α), vimentin, desmin, CD90 and proliferating cell nuclear antigen were detected in tissues and cells with different levels of expression. SMCs of esophageal circular muscle, esophageal longitudinal muscle, gastric circular muscle near sling in gastric bottom and gastric circular muscle near clasp in lesser gastric curvature, all cultured in 10%-F12, exhibited superior smooth muscle phenotypes compared with SMCs cultured in SMCM in terms of α-SMA, SM22α and vimentin expression. The EI method of ED at low temperature appears effective for isolation and primary culture of SMCs from human EGJ in vitro.

Morphine Promotes Colonization of Anastomotic Tissues with Collagenase - Producing Enterococcus faecalis and Causes Leak

BackgroundDespite ever more powerful antibiotics, newer surgical techniques, and enhanced recovery programs, anastomotic leaks remain a clear and present danger to patients. Previous work from our laboratory suggests that anastomotic leakage may be caused by Enterococcus faecalis strains that express a high collagenase phenotype (i.e., collagenolytic). Yet the mechanisms by which the practice of surgery shifts or selects for collagenolytic phenotypes to colonize anastomotic tissues remain unknown. MethodsHere, we hypothesized that morphine, an analgesic agent universally used in gastrointestinal surgery, promotes tissue colonization with collagenolytic E. faecalis and causes anastomotic leak. To test this, rats were administered morphine in a chronic release form as would occur during routine surgery or vehicle. Rats were observed for 6 days and then underwent exploratory laparotomy for anastomotic inspection and tissue harvest for microbial analysis. These results provide further rationale to enhanced recovery after surgery (i.e., ERAS) programs that suggest limiting or avoiding the use of opioids in gastrointestinal surgery. ResultsResults demonstrated that compared to placebo-treated rats, morphine-treated rats demonstrated markedly impaired anastomotic healing and gross leaks that correlated with the presence of high collagenase-producing E. faecalis adherent to anastomotic tissues. To determine the direct role of morphine on this response, various isolates of E. faecalis from the rats were exposed to morphine and their collagenase activity and adherence capacity determined in vitro. Morphine increased both the adhesiveness and collagenase production of four strains of E. faecalis harvested from anastomotic tissues, two that were low collagenase producers at baseline, and two that were high collagenase producers at baseline. ConclusionThese results provide further rationale to enhanced recovery after surgery (i.e., ERAS) programs that suggest limiting or avoiding the use of opioids in gastrointestinal surgery.

A metallo-keratinase from a newly isolated Acinetobacter sp. R-1 with low collagenase activity and its biotechnological application potential in leather industry.

Microbial keratinase is a well-recognized enzyme that can specifically degrade insoluble keratins. A keratinase-producing bacterium was isolated from a duck ranch soil and identified as Acinetobacter sp. R-1 based on the biochemical characteristics and 16S rDNA gene sequencing. It showed high keratinase activity and low collagenase activity. The keratinase was purified to electrophoretic homogeneity with 6.69% recovery, 2.68-fold purification and an estimated molecular weight of 25 kDa. Additionally, the keratinase showed optimal activity at 50 °C and pH11. Keratinase activity of Acinetobacter sp. significantly increased in the presence of Li(+), Na(+), and Ca(2+), while it was completely inhibited by EDTA, indicating it was a metallo-keratinase. Moreover, the crude keratinase from Acinetobacter sp. R-1 could thoroughly depilate goat skin and simultaneously modify the wool surface, which indicated its applicable potential in leather and textile industries.

Enzyme mediated beam house operations of leather industry: a needed step towards greener technology

Abstract Leather industries, due to pollution of water, careless disposal of solid wastes and gaseous emissions, are categorized as a red industry and are under deep pressure to develop environmentally efficient leather-making processes to comply with modern pollution and discharge legislation. In the present study, completely enzyme based beam house operations (dehairing & degreasing) of leather industry has been standardized for skins & hides using Bacillus licheniformis protease and Bacillus subtilis lipase. Owing to its immense importance and demand the productions of these enzymes were optimized and scaled up in a 300 L bioreactor resulting in a maximum yield of 4568 U/mL in 18 h for protease and 34.91 IU/mL of lipase in 36 h. 100% enzymatic dehairing & degreasing of skins and hides could be obtained at pH value of 8.0 and temperature 30–37 °C with the enzyme concentration of 2–5% (w/v) of protease for dehairing and 5–10% (w/v) of lipase for degreasing within 8–12 h. An interesting observation noted for enzyme based leather processing was that natural skin colour was preserved along with a drastic reduction of BOD and COD values of effluent. Crust leather formed using enzymatically processed skins exhibited similar physical and tactile properties as observed for conventional crust leather. On the bases of internally quenched fluorescent substrate specific method, this protease is subtilisin-like serine protease with low collagenase activity. High collagenase activity can damage the hide (leather) grain and the physical-mechanical characteristics of the hides. Successful demonstrations of large size (Industrial trials −100 kg skin/hide at Central Leather Research Institute, Chennai, India) 100% chemical free dehairing and degreasing further supports the authenticity of the work. Thus all this makes the beam house operations of leather tannery enzymatically feasible and environmentally benign.

Novel keratinase from Trichoderma harzianum MH-20 exhibiting remarkable dehairing capabilities

The production of protease blend preparations containing keratinase, collagenase, alkaline, neutral and acidic proteases was investigated employing five fungal and one bacterial isolates grown on chicken feathers waste as protein-rich substrate, intending for suitable application in hide unhairing. Of the fungal isolates tested, Trichoderma harzianum MH-20 proved to be the most potent and produced distinct protease blend after 7 days in shaken cultures. This preparation included mainly active constitutive α-keratinase and low collagenase level. Other minor protease activities (alkaline, neutral and acidic) proteases were also detected. The operational blend properties suggest potential uses of this enzyme preparation in many industrial applications, particularly the hide unhairing process without any lime and sulphide addition. All the analytical measurements and microscopical observations on the enzymatic unheared skins by this fungal preparation in comparison with the lime-sulphide treated skin are reported and discussed.

Regional Effects of Enzymatic Digestion on Knee Meniscus Cell Yield and Phenotype for Tissue Engineering

An abundant cell source is the cornerstone of most tissue engineering strategies, but extracting cells from the knee meniscus is hindered by its dense fibrocartilaginous matrix. Identifying a method to efficiently isolate meniscus cells is important, as it can reduce the cost and effort required to perform meniscus engineering research. In this study, six enzymatic digestion regimens used for cartilaginous cell isolation were used to isolate cells from the outer, middle, and inner regions of the bovine knee meniscus. Each regimen in each region was assessed in terms of cell yield, impact on cell phenotype, and cytotoxicity. All digestion regimens caused an overall upregulation of cartilage-specific genes Sox9, collagen type I (Col 1), collagen type II (Col 2), cartilage oligomeric matrix protein, and aggrecan (AGC) in cells from all meniscus regions, but was highest for cells isolated using 1075 U/mL of collagenase for 3 h (high collagenase). In response to isolation, outer meniscus cells showed highest upregulation of Sox9 and Col 1 genes, whereas greatest upregulation for middle meniscus cells was seen in Col 1 expression, and Col 2 expression for inner cells. Cell yield was highest in all regions when subjected to 45 min of 61 U/mL pronase followed by 3 h of 1075 U/mL collagenase (pronase/collagenase [P/C]) digestion regimen (outer: 6.57±0.37, middle: 12.77±1.41, inner: 22.17±1.47×10(6) cells/g tissue). The second highest cell yield was achieved using the low collagenase (LC) digestion regimen that applied 433 U/mL of collagenase for 18 h (outer: 1.95±0.54, middle: 3.3±4.4, inner: 6.06±2.44×10(6) cells/g tissue). Cytotoxicity analysis showed higher cell death in the LC group compared with the P/C group. Self-assembled constructs formed from LC-isolated cells were less dense than constructs formed from P/C-isolated cells, and P/C constructs showed higher glycosaminoglycan content and compressive moduli than LC constructs. All isolation methods tested resulted in similar phenotypic changes in meniscus cells from each region. These results indicate that, compared with other common isolation protocols, the P/C isolation method is able to more efficiently isolate meniscus cells from all regions that can produce tissue engineered constructs.

Effect of human umbilical cord mesenchymal stem cell-secretion on proliferation and apoptosis in hepatocytes

Objective To investigate the effect of human umbilical cord mesenchymal stem cell paracrine substance on proliferation and apoptosis of liver cells in vitro. Methods Mesenchymal stem cells (MSC)were separated from human umbilical cord with type Ⅳ collagenase and trypsogen digestion method and cultured in vitro. The human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) which contain paracrine substance of human umbilical cord mesenchymal stem cells (HUCMSC) was prepared. Hepatocytes were isolated from SD rats by low concentration collagenase perfusion procedure. There were three groups in the experiment, control group, 2% MSC-CM group and 8% MSC-CM group. The proliferation of normal hepatocytes were assayed with MTT method. We detected the urea and albumin level in culture supernatant to assay the hepatocyte function under different concentration MSC-CM. Hepatocytes were induced for apoptosis by Actinomycin D and tumor necrosis factor alpha (TNF-α),and the apoptosis effect of different concentration MSC-CM was assayed with LIVE/DEAD Viability/Cytotoxicity Kit. Results The MTT assay showed that the absorbance of 2% MSC-CM group was significantly increased (P＜0. 01), and the urea and albumin levels of 2 % MSC-CM group were also significantly increased when compared with control group(P＜0. 01).LIVE/DEAD Viability/Cytotoxicity Kit revealed that hepatocyte survival rate of 2 % MSC-CM group was increased when compared with control group(P＜0. 05), there were no significant differences in above-mentioned experiments when 8% MSC-CM group compared with control group. Conclusion The low concentration MSC-CM could stimulate normal hepatocyte proliferation, inhibit impaired hepatocyte apoptosis and improve hepatocyte function. Key words: Umbilical cord mesenchymal stem cell; Paracrine; Hepatocyte; Regeneration; Apoptosis

Role of Retinal Detachment Subretinal Fluid on Extracellular Matrix Metabolism

Purpose: To examine the in vitro effects of subretinal fluid (SRF) from patients with retinal detachment on extracellular matrix turnover. Methods: Matrix metalloproteinase (MMP) activity was explored by radiolabeled substrate degradation and zymography. Human lung fibroblasts were used to analyze SRF effects on collagen synthesis and cell proliferation. Transforming growth factor-β₂ (TGF-β₂) concentration was determined in SRF samples by a quantitative sandwich enzyme immunoassay. Results: MMP-2 and MMP-9 gelatinolytic activity was observed in all SRF samples. Collagenase activity was not detected in SRF from patients with holes and recurrent retinal detachment. All SRF samples stimulated fibroblast proliferation and collagen synthesis but SRF samples from recurrent retinal detachment patients with previous retinal tears had the largest effect with the largest TGF-β₂ concentration. Conclusions: The gelatinolytic activity found in all SRF samples might be associated with the retinal detachment process. Low collagenase activity with an increase in collagen synthesis could indicate a risk factor to the development of proliferative vitreoretinopathy.

Isolation of porcine pancreatic islets for xenotransplantation studies: Effects of low collagenase digestion temperatures

Abstract: Islets of Langerhans were isolated from porcine pancreata by a modification of our previously described method. The modification involved the use of a low temperature of collagenase digestion (30°C) during the process of islet isolation. The resulting islets were then evaluated in vitro and in vivo and compared to islets isolated at the regular 37°C temperature.The islets produced at the low temperature were more compact compared to the control islets. In the dextran density gradient these islets were deposited at the interface of the 1.060 and 1.068 g/ml density bands as compared to 1.050 and 1.060 g/ml for the control islets. In addition, the experimental islets contained a higher proportion of compact, unfragmented islets (68%) compared to the regular islets (55%), and their uptake of the dithizone stain was considerably slower than with the control islets. All ten batches of freshly isolated microencapsulated islets produced at both temperatures responded to the glucose stimulation. After 4 weeks of in vitro culture the islets of both groups microencapsulated in alginate‐polylysine‐alginate (APA) microcapsules still retained glucose responsiveness, with the experimental islets demonstrating significantly higher responsiveness to the high glucose (16.7 mM) and 0.1 mM IB MX stimulation. The morphology of unencapsulated islets in the experimental group following 4 weeks of in vitro culture indicates much firmer islet structure compared to the control islets. In addition, the unencapsulated experimental islets following the 4 week culture were still found to have secreted insulin when exposed to glucose. In transplantation studies both the experimental and the control islets normalized diabetic hyperglycemia in diabetic mice in a comparable fashion. In general, the low temperature digestion results in superior islets in terms of their morphology, viability, and physiological function.

Proteinases in subretinal fluid.

Degradation of the extracellular matrix by secreted proteases is connected to cell migration and proliferation in invasive growth and in scar tissue formation. In retinal detachment, retinal pigment epithelium (RPE) cells loosened from their monolayer are often seen in the subretinal fluid (SRF) and the vitreous, where they may participate in the scar tissue formation of proliferative vitreoretinopathy. To evaluate the role of SRF constituents on the release of RPE cells, we analyzed SRF in patients with retinal detachment for the presence of enzymes able to degrade extracellular matrix. SRF was collected altogether from 16 patients undergoing retinal reattachment surgery and analyzed for activities against some of the key enzymes in extracellular proteolysis, namely collagenases, gelatinases, elastase and cathepsin G. Seventy-two-kilodalton gelatinase was found in all SRF samples studied, whereas the neutrophil-type 92-kDa gelatinase could not be detected. Low collagenase, elastase and cathepsin G activities could also be detected in some samples. The predominant type of matrix metalloproteinase present in SRF is the 72-kDa MMP-2. The proteolytic activity in SRF may be connected to the release of RPE cells into SRF and to degradation of components of the vitreous exposed to SRF.

Effects of collagenase concentration on the purity and viability of isolated porcine pancreatic islets for use in xenotransplantation studies

Abstract: The use of lower concentrations of collagenase (0.8 mg/ml as compared to 1.25 and 1.5 mg/ml) resulted in isolation of porcine pancreatic islets retaining their original compact, wholesome appearance. In in vitro experiments, 100% of islet batches isolated with the low collagenase concentration responded to glucose stimulation, while only 63.1% of islet batches isolated with the high concentration of the enzyme did. The response to high glucose challenge of islets isolated with the low enzyme concentration was considerably higher compared to high collagenase concentration islets.In in vivo experiments, intraperitoneal transplants of microencapsulated islets isolated using 0.8 mg/ml of collagenase reversed diabetes in diabetic BALB‐c mice in a significantly larger proportion of recipients (87.5%) as compared to 33.3% of recipients grafted with 1.5 mg/ml collagenase islets.The results of this study demonstrate that the use of a low collagenase concentration is conducive to isolation of islets of a superior morphology, purity, viability, and functioning ability.

Critical remarks on the role of collagenolytic enzymes in carcinomas of the head and neck.

Collagenolytic activity of eight squamous cell carcinomas of the head and neck as well as xenografts of such tumors in nude mice was investigated by two different methods. High enzyme activities seem to be present in necrotic tumor areas, whereas the germinative zones tend to show lower collagenase levels. There is experimental evidence for the assumption of a collagen-stabilizing factor (CSF). A hypothetic concept including the results of other authors is developed concerning the role of collagenase in infiltrative growth of malignant tumors. The greater part of these questions being unanswered, further studies on collagenolytic activity in malignancy will be necessary.

Collagenase and proteinase induction in implants of bone matrix

Enzymes capable of digesting collagen and non-collagenous proteins are present in implants of bone matrix. In the early stages of bone morphogenesis, implants produce relatively large amounts of trypsin-labile proteins and have high non-collagenolytic neutral proteinase and low collagenase activities. Enzymatic activity is maximal three weeks after implantation. The results indicate that increased synthesis of non-collagenous proteins and non-collagenolytic proteinases precedes the induction of significant amounts of collagenases. The importance of these findings in bone morphogenesis is discussed.