Enzymatic Collagen Degradation Research Articles

Mechanisms of Degradation of Collagen or Gelatin Materials (Hemostatic Sponges) in Oral Surgery: A Systematic Review

Objective: The goal of this systematic review was to identify the mechanisms associated with the enzymatic degradation of collagen and gelatin biomaterials and the possible associated flaws. Methods: Four databases (PubMed, B-On, Cochrane Library, and ResearchGate) were used for the bibliographic search of articles. The research question was formulated using the PCC method, (P): collagen or gelatin sponges, hydrogels, and scaffolds; concept (C): enzymatic degradation of collagen or gelatin sponges, hydrogels, and scaffolds; and context (C): effect of enzymatic action on degradation time of collagen or gelatin sponges, hydrogels, and scaffolds. The search was contextualized according to PRISMA recommendations. The identification and exclusion of evidence followed the PRISMA criteria, with specific inclusion and exclusion factors being stipulated for the selection of articles. The risk of bias assessment was performed using the QUIN Scale. Results: The initial search was composed of 13,830 articles after removing duplicates; 56 articles followed for the full-text reading; 45 were excluded; then, 11 articles were obtained, constituting the results of this systematic review. All studies evaluated the materials using gravimetric analysis, and collagenases were the proteases used for the degradation solution. The materials tested were as follows: human-like collagen (HLC) hydrogel with microbial transglutaminase (MTGase), gelatin sponges subjected to different types of crosslinking, and collagen scaffolds with different types of crosslinking. The period of analysis varied between 0.25 h and 35 days. It was possible to highlight the lack of uniformity in the protocols used, which varied largely, thus influencing the degradation times. The risk of bias was low in nine studies and medium in two studies. Conclusions: This systematic review identified a gap in the literature, highlighting the absence of in vitro studies using human saliva and a collagenase concentration close to the physiological levels to simulate oral dynamics. However, based on existing literature, the mechanisms associated with collagen enzymatic degradation in collagen and gelatin biomaterials were comprehensively understood, answering the first research question postulated. In response to the second research question, the main shortcomings identified in the laboratory evaluation of mechanisms associated with collagen enzymatic degradation in collagen and gelatin biomaterials included the lack of standardization in degradation test protocols; this limited inter-study comparisons, which increased heterogeneity. Additionally, variations in collagenase concentrations and types influenced collagen degradation rates, and inappropriate evaluation intervals hindered the identification of total degradation time.

Modeling of mechanosensitive remodeling processes in collagen fibers

AbstractA large number of biochemical processes such as enzymatic collagen degradation are involved in the remodeling of biological soft tissue constituents. Experiments have found that moderate strain may stabilize fibrillar collagen to enzymatic degradation. This strain stabilization is crucial for the homeostatic balance during the tissue remodeling process in healthy soft tissue. This article studies a material model applicable to the modeling of collagenous soft tissue. The ground substance properties are taken to be time‐independent, while the mechanical behavior of the fibers is modeled in terms of the Helmholtz free energy of the underlying protofibers along with a survival kernel that accounts for how the fiber density is dependent upon the deformation of the overall material. The examples illustrate the consequences of a compressively induced mechanical disruption to the homeostatic fiber remodeling, and they show the differences in the re‐approach to homeostasis when either the stress or strain remains constant.

Modulation of the biophysical and biochemical properties of collagen by glycation for tissue engineering applications.

The structural and functional properties of collagen are modulated by the presence of intramolecular and intermolecular crosslinks. Advanced Glycation End-products (AGEs) can produce intermolecular crosslinks by bonding the free amino groups of neighbouring proteins. In this research, the following hypothesis is explored: The accumulation of AGEs in collagen decreases its proteolytic degradation rates while increasing its stiffness. Fluorescence Lifetime Imaging (FLIM) and Fourier-transform infrared spectroscopy (FTIR) detect biochemical changes in collagen scaffolds during the glycation process. The accumulation of AGEs increases exponentially in the collagen scaffolds as a function of Methylglyoxal (MGO) concentration by performing autofluorescence measurement and competitive ELISA. Glycated scaffolds absorb water at a much higher rate confirming the direct affinity between AGEs and interstitial water within collagen fibrils. In addition, the topology of collagen fibrils as observed by Atomic Force Microscopy (AFM) is a lot more defined following glycation. The elastic modulus of collagen fibrils decreases as a function of glycation, whereas the elastic modulus of collagen scaffolds increases. Finally, the enzymatic degradation of collagen by bacterial collagenase shows a sigmoidal pattern with a much slower degradation rate in the glycated scaffolds. This study identifies unique variations in the properties of collagen following the accumulation of AGEs. STATEMENT OF SIGNIFICANCE: In humans, Advanced Glycation End-products (AGEs) are naturally produced as a result of aging process. There is an evident lack of knowledge in the basic science literature explaining the biomechanical impact of AGE-mediated crosslinks on the functional and structural properties of collagen at both the nanoscale (single fibrils) and mesoscale (bundles of fibrils). This research, demonstrates how it is possible to harness this natural phenomenon in vitro to enhance the properties of engineered collagen fibrils and scaffolds. This study identifies unique variations in the properties of collagen at nanoscale and mesoscale following accumulation of AGEs. In their approach, they investigate the unique properties conferred to collagen, namely enhanced water sorption, differential elastic modulus, and finally sigmoidal proteolytic degradation behavior.

Photoinitiator‐Free Two‐Photon Polymerization of Biocompatible Materials for 3D Micro/Nanofabrication

AbstractTwo‐photon polymerization is expected to realize the fabrication of biological tissues for regenerative medicine and drug development because of its capability of 3D fabrication with submicrometer spatial resolution. However, photopolymerization initiators generally added to resins have low biocompatibility and water solubility, which pose a challenge for the biological application of micro‐ and nanostructures fabricated by photopolymerization. In this study, two‐photon polymerization is performed using biocompatible materials without photoinitiators and the preservation of the material properties of the fabricated 3D structures is evaluated. Poly(ethylene glycol) diacrylate (PEGda) and collagen are successfully fabricated using a femtosecond pulsed laser at a wavelength of 400 nm with a feature size of 100 nm without photoinitiator. The excitation intensities required for the polymerization of PEGda and collagen are about 450 and 150 kW cm−2, respectively, which are smaller than those reported with using photoinitiators. Raman spectroscopy is used to confirm that the water‐swelling polymer network of PEGda and the enzymatic degradation of collagen, which are important factors in the application with live cells, are maintained in the fabricated structures.

Maturation Process and Characterization of a Novel Thermostable and Halotolerant Subtilisin-Like Protease with High Collagenolytic Activity but Low Gelatinolytic Activity.

Enzymatic degradation of collagen is of great industrial and environmental significance; however, little is known about thermophile-derived collagenolytic proteases. Here, we report a novel collagenolytic protease (TSS) from thermophilic Brevibacillus sp. WF146. The TSS precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain, a β-jelly roll (βJR) domain, and a prepeptidase C-terminal (PPC) domain. The maturation of TSS involves a stepwise autoprocessing of the N-terminal propeptide and the PPC domain, and the βJR rather than the PPC domain is necessary for correct folding of the enzyme. Purified mature TSS displayed optimal activity at 70°C and pH 9.0, a half-life of 1.5 h at 75°C, and an increased thermostability as the NaCl concentration increased up to 4 M. TSS possesses an increased number of surface acidic residues and ion pairs, as well as four Ca2+-binding sites, which contribute to its high thermostability and halotolerance. At high temperatures, TSS exhibited high activity toward insoluble type I collagen and azocoll but showed a low gelatinolytic activity, with a strong preference for Arg and Gly at the P1 and P1' positions, respectively. Both the βJR and PPC domains could bind but not swell collagen, and thus facilitate TSS-mediated collagenolysis via improving the accessibility of the enzyme to the substrate. Additionally, TSS has the ability to efficiently degrade fish scale collagen at high temperatures. IMPORTANCE Proteolytic degradation of collagen at high temperatures has the advantages of increasing degradation efficiency and minimizing the risk of microbial contamination. Reports on thermostable collagenolytic proteases are limited, and their maturation and catalytic mechanisms remain to be elucidated. Our results demonstrate that the thermophile-derived TSS matures in an autocatalytic manner and represents one of the most thermostable collagenolytic proteases reported so far. At elevated temperatures, TSS prefers hydrolyzing insoluble heat-denatured collagen rather than gelatin, providing new insight into the mechanism of collagen degradation by thermostable collagenolytic proteases. Moreover, TSS has the potential to be used in recycling collagen-rich wastes such as fish scales.

Effects of zinc fluoride on inhibiting dentin demineralization and collagen degradation in vitro: A comparison of various topical fluoride agents.

Root caries is developed because of demineralization followed by enzymatic collagen degradation. This in vitro study aimed to examine the inhibitory efficacy of ZnF2 on dentin demineralization and collagen degradation. Bovine dentin specimens were treated either with ZnF2 or HCl-acidified ZnF2 (ZnF2/HCl) and then demineralized. Anti-demineralization efficacy was assessed by TMR as mineral loss (ΔZ). The efficacy was compared with silver diammine fluoride (SDF), KF, and acidulated phosphate fluoride (APF). For evaluating anti-collagen degradation, EDTA-demineralized dentin specimens were treated by one of four fluoride agents [SDF, APF, ZnF2/HCl, NaF] followed by collagenase challenge. The eroded depth of collagen layer in the lesion was assessed using optical microscope. ΔZ of SDF, KF, ZnF2/HCl, and APF were significantly lower compared with ZnF2 and Control (no treatment). Regarding anti-collagen degradation, SDF and ZnF2/HCl demonstrated a significant difference in the eroded depth compared with Control. Although SDF possessed higher efficacy, ZnF2/HCl might be beneficial as a staining-free agent.

Collagen containing microcapsules: Smart containers for disease controlled therapy

The protein collagen is the major component of connective tissue and it is involved in many biological functions. Its degradation is at the basis of different pathological processes. The up-regulated expression of matrix metalloproteinases and the down-regulated expression of their inhibitors are the causes for such degradation. The aim of this work was to evaluate the possibility to fabricate collagen based containers for drug encapsulation and release by cellular demand by the action of matrix metalloproteinases. In present work collagen type I based microcapsules were fabricated by means of the layer-by-layer assembly of oppositely charged collagen and poly (stirene sulfonate) onto colloidal particles, followed by removal of the cores to obtain hollow microcapsules. The process of shell growth on planar supports was monitored by quartz crystal microbalance. X-ray reflectivity measurements were carried out at the solid/water interface to study the interaction of matrix metalloproteinase 1 with LbL films of collagen. The morphology of hollow capsules was characterized by scanning electron microscopy, and compared to that of capsules exposed to the matrix metalloproteinase 1. Finally the matrix metalloproteinase 1 mediated permeability of capsules variation was studied by Confocal Laser Scanning Microscopy. The results demonstrated the possibility to fabricate a drug delivery system where the release of the drug is dependent on the biochemistry of the pathological state.

Grape seed proanthocyanidins increase collagen biodegradation resistance in the dentin/adhesive interface when included in an adhesive

Objectives Contemporary methods of dentin bonding could create hybrid layers (HLs) containing voids and exposed, demineralised collagen fibres. Proanthocyanidins (PA) have been shown to cross-link and strengthen demineralised dentin collagen, but their effects on collagen degradation within the HL have not been widely studied. The purpose of this study was to compare the morphological differences of HLs created by BisGMA/HEMA model adhesives with and without the addition of grape seed extract PA under conditions of enzymatic collagen degradation. Methods Model adhesives formulated with and without 5% PA were bonded to the acid etched dentin. 5-μm-thick sections cut from the bonded specimens were stained with Goldner's trichrome. The specimens were then exposed to 0.1% collagenase solution for 0, 1, or 6 days. Following collagenase treatment, the specimens were analysed with SEM/TEM. Results Staining did not reveal a difference in the HLs created with the two adhesives. SEM showed the presence of intact collagen fibrils in all collagenase treatment conditions for specimens bonded with adhesive containing PA. These integral collagen fibrils were not observed in the specimens bonded with adhesive without PA after the same collagenase treatment. TEM confirmed that the specimens containing PA still showed normal collagen fibril organisation and dimensions after treatment with collagenase solution. In contrast, disorganised collagen fibrils in the interfacial zone lacked the typical cross-banding of normal collagen after collagenase treatment for specimens without PA. Conclusions The presence of grape seed extract PA in dental adhesives may inhibit the biodegradation of unprotected collagen fibrils within the HL.

Collagen Organization during Cardiac Fibroblast‐mediated Collagen Gel Contraction

Collagen within the extracellular matrix (ECM) in the heart is responsible for connecting myocytes to one another and mechanically couples neighboring cells. In the developing heart, the collagen matrix accumulates rapidly during early post-natal life and develops an organization which is maintained in the adult heart. The mechanisms by which cardiac fibroblasts organize collagen within the cardiac ECM are unclear; although probably involve a combination of mechanical tension and enzymatic remodeling. To investigate this process, neonatal cardiac fibroblasts were dispersed within a collagen gel and cultured in either a mechanically loaded or unloaded environment. Using confocal reflection microscopy combined with circular statistical analysis, both cell distribution, cell morphology and collagen fiber distribution have been analyzed. While initially cells display a rounded morphology and are surrounded by randomly organized collagen, over time as the fibroblasts extend cell processes the collagen network develops a radial organization around the cell with some areas on the cell surface displaying numerous collagen attachments and others lacking any visible collagen interaction. In addition, areas devoid of collagen are also visible, suggesting enzymatic degradation of collagen. Fibroblasts appear to differentially orient within the gels, having a more circumferential orientation in the outer regions of the gels and a more random orientation in the center of the gel. These studies provide direct insight into the organizational changes which occur during collagen remodeling.

NEUTRAL AND ALKALINE MUSCLE PROTEASES OF MARINE FISH AND INVERTEBRATES A REVIEW

Muscle proteases active at neutral and alkaline pH's include calcium-activated neutral proteases, heat-activated thiol proteases, serine proteases, and metallo-proteases. They participate to different extents in postmortem degradation of fish muscle myofibrillar and scaffold proteins. Their activity in fish, the presence of endogenous activators and inhibitors, pH, and temperature. Recent studies indicate that neutral and alkaline proteases have more impact on the postmortem deterioration in quality of fish muscles than the cathepsins active at acid pH. Significant quality losses are caused by enzymatic collagen degradation in raw tissues and by heat-activated enzymes in fish gels.

Biochemical changes in human cervical connective tissue after intracervical application of prostaglandin E 2

Cervical biopsies were taken during the first trimester from primigravidae and plurigravidae at different time points after intracervical application of prostaglandin E 2-gel. Collagenase activity was determined by a highly specific technique using native, triple helical collagen as substrate. Elastase- α1-proteinase-inhibitor complex (elastase) was measured by a commercially available assay, and glycosaminoglycan (GAG) analyses were performed as described by Greiling et al. (5, 6). The maximum activity of collagenase was found 2 hours after PGE 2 application in plurigravidae and 4 hours after application in primigravidae. Elastase activity rose nearly 7-fold to maximum values 4 hours after PGE 2 application. The total GAG concentrations and the dermatan sulfate concentrations increased by approximately 10 %, while the hyaluronic acid concentrations were found to be elevated significantly by nearly 50 % in the PGE 2-primed cervices. We conclude that a time-dependent enzymatic collagen degradation by collagenases and other proteinases and an increase in hyaluronic acid concentrations are the significant biochemical events underlying PG-induced cervical ripening.

Grundlagen der physiologischen und medikamentös induzierten Zervixreifung - Neuere morphologische und biochemische Befunde*

Maturation of the cervix during pregnancy is an essential pre-requisite for an uncomplicated delivery at term. Physiological cervical ripening is characterised by a diffuse loosening of the collagenous connective tissue with widely scattered collagen fibrils and an increased amount of extracellular ground substance. These morphological changes are similar to those after prostaglandin (PG)-pre-treatment of the cervix. The local application of PG leads to a marked mulifocal loosening of the collagen fibre bundles with "activated" fibroblasts, characterised by a fine granular loosening of the cytoplasm, vacuolised enlarged mitochondria and an increased number of cytoplasmatic vesicles close to the cell surface. In the course of pregnancy the volume of the cervix increases by a significant rise in synthesis of collagen, protein, glycosaminoglycan and fibronectin. The change in consistency during late pregnancy corresponds to a significant decrease in dermatan sulphate coninciding with a marked increase in hyaluronic acid concentration associated with increased water uptake. Contrary to the already published literature, enzymatic collagen degradation does not play an important role in physiological cervical maturation. The action of catabolic enzymes (collagenases, glycosidases), liberated from polymorphonuclear leukocytes invading the extracellular matrix, is responsible for the rapid dilatation of the cervix at parturition. This process is limited by the immediate postpartum insudation of the cervix by plasma containing highly potent proteinase inhibitors (e.g. alpha 2-macroglobulin). PG-induced cervical ripening is associated with a time-limited enzymatic collagen degradation, an increased synthesis of non-collagenous proteins and a significant increase in hyaluronic acid concentration. Our basic biochemical findings in cervical ripening and dilatation during parturition may greatly contribute to the development of new concepts in the causal treatment of cervical pathology during pregnancy.

Enzymatic collagen degradation in the pregnant guinea pig cervix during physiological maturation of the cervix and after local application of prostaglandins

The role of enzymatic collagen degradation in prostaglandin-induced and physiological cervical ripening was studied in guinea pigs. The cervices were removed from (a) 8 non-pregnant guinea pigs, (b) 8 animals at day 45 of pregnancy, (c) 14 pregnant animals of comparable gestational age which had either an intracervical application of 0.2 ml 5% tylose or 10 μg sulprostone gel, and (d) 8 guinea pigs at day 63 to 65 of pregnancy. Collagenase activity was assayed in a highly specific and sensitive system using native collagen type I as substrate. Protease activity was measured by the method of Green and Shaw. Collagen fragments were identified by SDS-polyacrylamide electrophoresis (SDS-PAGE) of acetic-soluble fractions. Collagenase and protease activities were found in all extracts from the different groups. However, there were no differences in enzymatic activities between the non-pregnant, early-pregnant and late-pregnant cervical specimens. Prostaglandin pre-treatment of the cervix led to no significant increase in either collagenase or protease activity as compared to the control groups. The absence of typical collagen degradation products in the SDS-PAGE suggested that no significant collagen breakdown had taken place. In contrast to previously published literature, we conclude that enzymatic collagen degradation is unlikely to be a key factor in prostaglandin-induced and physiological cervical ripening.

The role of collagenases and proteases in prostaglandin-induced cervical ripening

Tissue samples were taken from the posterior lip of the cervix of 10 non-pregnant women, 10 women having a termination of pregnancy at 9 – 12 weeks' gestation and 16 women having a termination who had had an intracervical application of either 50 μg sulprostone gel or 2 ml 5% tylose 8 – 12 hours previously. The efficacy of cervical priming was demonstrated objectively by tonometric measurements. Collagenase activity was determined by a new highly specific technique using native, triple helical collagen. Protease activity was measured by a modified Anson-test. For identification of collagen fragments SDS-polyacrylamide electrophoresis was done on the acetic acid soluble fractions. — The sulprostone gel induced effective cervical ripening in all of the patients. Collagenase and protease activity were found in all extracts from the different groups, however, PG-pretreatment of the cervix led to no significant increase in enzymatic activities. In addition, the absence of typical collagen cleavage products in the SDS-electrophoresis suggested that no significant collagen breakdown had occurred. In contrast to previously published literature we conclude that enzymatic collagen degradation does not play a predominant role in PG-induced cervical ripening.

Collagen degradation in the pregnant human cervix at term and after prostaglandin-induced cervical ripening.

The role of enzymatic collagen degradation in prostaglandin-induced and physiological cervical ripening was studied by determining collagenase and other proteolytic activity in extracts of cervical biopsies. Collagenase activity was assayed in a highly specific and sensitive system using native collagen type I as substrate. The intracervical application of sulprostone gel prior to termination of 1st trimester pregnancy led to a marked improvement in cervical dilatability. Collagenase and proteolytic activity were found to be significantly higher in postpartum samples than in specimens obtained from the nonpregnant or early pregnant cervix. After sulprostone treatment enzymatic activities were only marginally elevated. Analysis of extractable peptides showed that sub partu collagen was degraded in preference to noncollagenous proteins into very small fragments, whereas in the process of prostaglandin-induced cervical ripening collagen degradation appears to be of minor importance.

Noninfected Corneal Ulceration

The pathogenesis of noninfected corneal ulcers consistently relates to persistent or extensive epithelial defects, concomitant with stromal inflammatory cell infiltration and enzymatic degradation of collagen and ground substance. The therapeutic approaches to these disorders may be considered on three levels: (1) determination of etiology and institution of primary therapy; (2) promotion of epithelial healing; and (3) limitation of ulceration and support of repair. The latter two aspects feature pharmacological management and limited surgical modalities, particularly the use of tissue adhesive.

Decision-making in the Therapy of External Eye Disease: Noninfected Corneal Ulcers

Despite diverse and multiple clinical etiologies, the pathogenesis of noninfected corneal ulcers consistently relates to persistent or extensive epithelial defects, concomitant with stromal inflammatory cell infiltration and enzymatic degradation of collagen and ground substance. The therapeutic approaches to these disorders may be considered on three levels: (1) the determination of etiology and initiation of primary therapy; (2) promotion of epithelial healing; and (3) limitation of ulceration and support of repair, the latter aspects currently featuring pharmacologic management and limited surgical modalities, particularly the use of tissue adhesive.

Immunopathological Mechanisms in Rheumatoid Arthritis at the Dual Interface of the Synovial Membrane: The Joint Cavity and the Pannus

The significance of immune responses is examined in relation to clinicopathological changes in the rheumatoid joint and the concept of a dual site of action is developed. In the joint cavity interaction between immune complexes and complement is central to the phenomena observed, which depend on the generation of chemotactic factors and the ingress of polymorphs. Other chemical mediators--e.g. prostaglandins and lymphokines--are envisaged as playing a secondary or augmentory role. At the pannus-cartilage (and bone) junction the possibility is considered that changes are due to activation of a variety of cell types which results in enzymatic degradation of collagen in cartilage, osteolastic activity in bone and prostaglandin-induced depletion of bone. The production of migration inhibition factors by rheumatoid membranes is examined and some evidence in support of their lymphokine nature is found by gel filtration; their role as cell activators is considered.

Mechanochemical studies of enzymatic degradation of insoluble collagen fibers.

A mechanochemical method was developed for studying the enzymatic degradation of insoluble collagen fibers. The method involves stretching the collagen fiber to a fixed extension in the presence of a solution of collagenase and measuring the rate of relaxation of the force induced in the fiber. In this work, bacterial collagenase was used for reasons of availability. We observed invariably an exponential decrease in force with respect to ttime. The slope of the linear plot of logarithm of the force versus time was taken as a measure of the rate of enzymatic degradation. This rate was found a) to vary linearly with collagenase concentration; b) to be maximal at pH 7-8; c) to vary with temperature according to the Arrhenius relationship in the range 10-56 degrees C; d) to be reduced to varying extent by addition of EDTA omicron-phenanthroline, 2,3-dimercaptopropanolol, and D,L-cysteine; e) to be minimal when the strain on the fiber was ca. 4%; f) to be increased dramatically by denaturation of the collagen fiber; and g) to be reduced by an increase in the crosslink density of the collagen fiber. Except for the effect of strain, which can not be conveniently studied by existing methods these results are consistent with those observed by other methods for the study of the enzymatic degradation of collagen. The mechanochemical method is, however, uniquely suited to monitor continuously the enzymatically induced decay in the stress-bearing ability of collagen fibers. It has also been found useful in the design of collagenous implants with specified resistance to enzymatic degradation in vivo.

Enzymatic Degradation of Collagena

SUMMARYFiein cannot hydrolyze native collagen. Collagen can he degraded by this enzyme only after it has been denatured by heat, low pH, or high salt concentrations. This appears to he true also for the action of bromelain, papain, and trypsin on collagen.