Effect Of Enzymatic Degradation Research Articles

Effect of enzymatic degradation and hydrolysis on 3D-printed resin-based composite material for temporary dental crowns and bridges

Abstract Additive manufacturing of dental prostheses e.g. composite materials is continuously finding broader application in dentistry. In the oral environment, the materials are subjected to hydrolysis and accelerated hydrolysis by enzymes. With the aim to compare an additively manufactured composite with a self-curing and a CAD/CAM material of the same composition, the effect of enzymatic degradation and hydrolysis on the materials were investigated. The experimental composites consisted of 50 wt. % inorganic filler and 50 wt. % monomer matrix (BisEMA, BisGMA, TEGDMA). The influence of hydrolysis via a buffer system was investigated in comparison to enzymatic degradation by cholesterol esterase over 22 days. The detection of degradation products over time was conducted by HPLC-DAD analysis. Degradation products and monomers calibrated were: TEGDMA, BisGMA, BisEMA, MA, TEGMA, Bis-HPPP and E-bis-PA. Surface roughness of polished specimens was measured by AFM and hardness with a Vickers micro hardness tester. After enzymatic degradation and hydrolysis, BisGMA was detected in the eluates of the additively manufactured composites. The samples fabricated by CAD/CAM behaved similar during hydrolysis, where additionally MA was detected. After enzymatic degradation, furthermore TEGMA and BisEMA were detected qualitatively. In contrast, the self-curing material exhibited traces of MA, TEGMA, TEGDMA and BisGMA after enzymatic degradation. Hereof detectable upon hydrolysis were MA, TEGMA and BisGMA. Surface roughness was comparable for all manufactured samples whereas the hardness was lowest of the 3D printed material. Enzymatic degradation and hydrolysis had no effect on surface roughness and hardness. The additively manufactured composite appeared to have low susceptibility to enzymatic degradation and hydrolysis. Due to the manufacturing process, the polishability and initial hardness are poorer.

Effect of collagenase\u2013gelatinase ratio on the mechanical properties of a collagen fibril: a combined Monte Carlo\u2013molecular dynamics study

Loading in cartilage is supported primarily by fibrillar collagen, and damage will impair the function of the tissue, leading to pathologies such as osteoarthritis. Damage is initiated by two types of matrix metalloproteinases, collagenase and gelatinase, that cleave and denature the collagen fibrils in the tissue. Experimental and modeling studies have revealed insights into the individual contributions of these two types of MMPs, as well as the mechanical response of intact fibrils and fibrils that have experienced random surface degradation. However, no research has comprehensively examined the combined influences of collagenases and gelatinases on collagen degradation nor studied the mechanical consequences of biological degradation of collagen fibrils. Such preclinical examinations are required to gain insights into understanding, treating, and preventing degradation-related cartilage pathology. To develop these insights, we use sequential Monte Carlo and molecular dynamics simulations to probe the effect of enzymatic degradation on the structure and mechanics of a single collagen fibril. We find that the mechanical response depends on the ratio of collagenase to gelatinase—not just the amount of lost fibril mass—and we provide a possible mechanism underlying this phenomenon. Overall, by characterizing the combined influences of collagenases and gelatinases on fibril degradation and mechanics at the preclinical research stage, we gain insights that may facilitate the development of targeted interventions to prevent the damage and loss of mechanical integrity that can lead to cartilage pathology.

Effects of enzymatic degradation on dynamic mechanical properties of the vitreous and intravitreal nanoparticle mobility

Intravitreal mobility of nanocarriers may have implications on the efficacy of the encapsulated drug in the treatment of vitreo-retinal diseases, with any changes in the integrity of the vitreous microstructure influencing nanoparticle biodistribution. This study investigated enzymatically digested vitreous models to mimic the aging eye. Collagenase, hyaluronidase, or trypsin was employed to selectively digest the structural components of the vitreous. Physical properties of digested bovine vitreous were initially assessed via texture analysis and oscillatory shear testing. Morphological changes in bovine vitreous microstructure were visualized by scanning electron microscopy and diffusion dynamics of hyaluronic acid coated nanoparticles through degraded porcine and bovine vitreous were examined using fluorescence spectroscopy and multiple particle tracking microscopy, respectively. After enzymatic treatment, the vitreous liquefied and its dynamic mechanical properties significantly changed with a decrease in stiffness and an increase in damping capacity. Micrographs confirmed specific digestion of each of the structural vitreous components. Furthermore, enzymatic degradation reduced steric hindrance and enhanced convective flow within the vitreous, resulting in increased intravitreal nanoparticle mobility which could alter the drug pharmacokinetics.

Effects of Enzymatic Degradation after Loading in Temporomandibular Joint

Synovial fluid of the joint decreases friction between the cartilage surfaces and reduces cartilage wear during articulation. Characteristic changes of synovial fluid have been shown in patients with osteoarthritis (OA) in the temporomandibular joint (TMJ). OA is generally considered to be induced by excessive mechanical stress. However, whether the changes in synovial fluid precede the mechanical overloading or vice versa remains unclear. In the present study, our purpose was to examine if the breakdown of joint lubrication affects the frictional properties of mandibular condylar cartilage and leads to subsequent degenerative changes in TMJ. We measured the frictional coefficient in porcine TMJ by a pendulum device after digestion with hyaluronidase (HAase) or trypsin. Gene expressions of interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), matrix metalloproteinases (MMPs), type II collagen, and histology were examined after prolonged cyclic loading by an active pendulum system. The results showed that the frictional coefficient increased significantly after HAase (35%) or trypsin (74%) treatment. Gene expression of IL-1β, COX-2, and MMPs-1, -3, and -9 increased significantly in enzyme-treated TMJs after cyclic loading. The increase in the trypsin-treated group was greater than that in the HAase-treated group. Type II collagen expression was reduced in both enzyme-treated groups. Histology revealed surface fibrillation and increased MMP-1 in the trypsin-treated group, as well as increased IL-1β in both enzyme-treated groups after cyclic loading. The findings demonstrated that the compromised lubrication in TMJ is associated with altered frictional properties and surface wear of condylar cartilage, accompanied by release of pro-inflammatory and matrix degradation mediators under mechanical loading.

Effect of enzymatic degradation on the mechanical properties of biological scaffold materials

Biological scaffolds must support a complex balance of resisting enzymatic degradation while promoting tissue remodeling. Thus, the purpose of this study was to evaluate the effects of in vitro enzymatic exposure on the mechanical properties of biological scaffolds. It was hypothesized that exposure to an enzyme solution would result in decreased tensile strength and that crosslinked scaffolds would resist enzymatic degradation more effectively than noncrosslinked scaffolds. Nine scaffolds were evaluated (four porcine dermis: Permacol™, CollaMend™, Strattice™, XenMatrix™; two human dermis: AlloMax™, FlexHD(®); two bovine pericardium: Veritas(®), PeriGuard(®); and one porcine small intestine submucosa: Surgisis™). Ten specimens (n = 10) were hydrated in saline at 37 °C and subjected to uniaxial testing to establish baseline properties. 50 specimens (n = 50) were incubated in collagenase solution at 37 °C for 2, 6, 12, 24, or 30 h (n = 10 each group) followed by uniaxial tensile testing. Tensile strength was significantly reduced after 30 h for CollaMend™, AlloMax™, Veritas(®), Strattice™, XenMatrix™, Permacol™, and FlexHD(®) (p < 0.01), while PeriGuard(®) demonstrated a slight increase in tensile strength (p = 0.0188). Crosslinked bovine pericardium (PeriGuard(®)) maintained greater tensile strength than noncrosslinked bovine pericardium (Veritas(®)) throughout all exposure periods (p < 0.0001). Similarly, crosslinked porcine dermis (Permacol™) maintained greater tensile strength than noncrosslinked porcine dermis (Strattice™ and XenMatrix™) throughout all exposure periods (p < 0.0001). Materials that deteriorate rapidly after in vitro enzymatic exposure may also deteriorate rapidly in vivo, particularly when exposed to a wound environment with elevated levels of matrix metalloproteinases. Permacol™, CollaMend™, Strattice™, FlexHD(®), and PeriGuard(®) survived the longest incubation period (30 h) and withstood mechanical testing. XenMatrix™, AlloMax™, Veritas(®), and Surgisis™ degraded more quickly and did not survive the longer exposure periods. Scaffolds that maintain strength characteristics after in vitro collagenase exposure may be advantageous for long-term hernia repair scenarios where elevated enzyme levels are expected.

Alginate grafted with poly(ε‐caprolactone): effect of enzymatic degradation on physicochemical properties

AbstractThe paper discusses the enzymatic behaviour of a series of copolymers composed of alginate grafted with poly(ε‐caprolactone) (PCL) of various lengths and degrees of substitution. The study is focused on viscosity measurements and pyrene probe fluorescence with or without two enzymes: alginate lyase, which breaks the alginate backbone; and esterase, which breaks PCL pendent groups. Alginate lyase is inactive at pH = 3.8 and degrades quickly all copolymers at pH = 6.3. The degradation is not complete and is slowed down by the presence of PCL. Esterase degrades only copolymers with long pendent PCL groups. It has no effect on copolymers when PCL has a length of 530 g mol−1. These systems are good candidates for controlled release of drugs using an enzymatic method. Copyright © 2012 Society of Chemical Industry

Influence of Enzymatic Degradation on Physical Properties of Poly(ε‐caprolactone) Films and Sponges

AbstractThe effect of enzymatic degradation on poly(ε‐caprolactone) (PCL) films and sponges was investigated at 37 °C using Pseudomonas lipase. Film samples were prepared by the solution casting method, while sponges were obtained by the freeze extraction method. The porosity was 17 and 60% respectively. Weight loss, morphology, crystallinity and mechanical properties were studied. The kinetic study on the enzymatic degradation of PCL porous samples depends on porosity, suggesting that degradation took place on the surface, not suffering bulk degradation. The non‐dependence on crystallinity indicates that degradation occurred in both phases, amorphous and crystalline, at the same time.

Characterisation of a collagen membrane for its potential use in cardiovascular tissue engineering applications

In this study, Biomend, a collagen membrane conventionally used in the regeneration of periodontal tissue, is investigated for its possible use in the field of cardiovascular tissue engineering. A key requirement of most potential tissue engineering scaffolds is that degradation occurs in tandem with tissue regeneration and extra cellular matrix remodelling. To this end, it is crucial to understand the degradation mechanics and mechanisms of the material and to investigate the practicability of using Biomend as a possible scaffold material. With this in mind, methodologies for the initial characterisation of the scaffold material were determined. The mechanical properties of Biomend samples, subjected to various degrees of hydration and enzymatic degradation, were examined primarily through tensile testing experiments. The effects of enzymatic degradation were monitored quantitatively, by observing weight loss, and visually, by studying micrographs. Cell adhesion and viability were of primary concern. Confocal laser scanning microscopy was employed to illustrate endothelialisation on the surface of this collagen membrane. Fluorescence microscopy was used to visualise cell viability on the membrane surface. These images, coupled with assays to measure cell activity, suggest that Biomend is not a suitable substrate to allow endothelialisation. In summary, this collagen membrane has suitable mechanical properties with the potential to control its degradation rate. However, since poor endothelial cell viability was observed on the membrane, it may not be suitable for use in cardiovascular tissue engineering applications.

Effect of enzymatic degradation on the release kinetics of model drug from Pluronic F127/poly(lactic acid) nano-particles

Poly(lactic acid) (PLA) was successfully grafted to both ends of Pluronic F127 block copolymer (PEO-PPO-PEO) to obtain amphiphilic PLA-F127-PLA block copolymers. The effect of enzymatic degradation on the release behaviors of hydrophobic model drug 9-(methylaminomethyl)anthracene (MAMA) from PLA-F127-PLA nano-particles with vesicular structure was studied by UV-Vis spectroscopy. It was observed that the release rate of MAMA from PLA-F127-PLA nano-particles with the enzymatic degradation varied with temperature due to the activity of the enzyme with temperature. However, the enzyme concentration has negligible effect on the release rates of MAMA.

Effects of enzymatic degradation on the frictional response of articular cartilage in stress relaxation

It was recently shown experimentally that the friction coefficient of articular cartilage correlates with the interstitial fluid pressurization, supporting the hypothesis that interstitial water pressurization plays a fundamental role in the frictional response by supporting most of the load during the early time response. A recent study showed that enzymatic treatment with chondroitinase ABC causes a decrease in the maximum fluid load support of bovine articular cartilage in unconfined compression. The hypothesis of this study is that treatment with chondroitinase ABC will increase the friction coefficient of articular cartilage in stress relaxation. Articular cartilage samples (n=34) harvested from the femoral condyles of five bovine knee joints (1–3 months old) were tested in unconfined compression with simultaneous continuous sliding (±1.5mm at 1mm/s) under stress relaxation. Results showed a significantly higher minimum friction coefficient in specimens treated with 0.1μ/ml of chondroitinase ABC for 24h (μmin=0.082±0.024) compared to control specimens (μmin=0.047±0.014). Treated samples also exhibited higher equilibrium friction coefficient (μeq=0.232±0.049) than control samples (μeq=0.184±0.036), which suggest that the frictional response is greatly influenced by the degree of tissue degradation. The fluid load support was predicted from theory, and the maximum value (as a percentage of the total applied load) was lower in treated specimens (77±12%) than in control specimens (85±6%). Based on earlier findings, the increase in the ratio μmin/μeq may be attributed to the decrease in fluid load support.

T1rho-relaxation in articular cartilage: effects of enzymatic degradation.

Spin-lattice relaxation in the rotating frame (T1rho) dispersion spectroscopy and imaging were used to study normal and enzymatically degraded bovine articular cartilage. Normal specimens demonstrate significant T1rho "dispersion" (approximately 60 to approximately 130 ms) in the 100 Hz to 9 kHz frequency range. Proteoglycan-degraded specimens have 33% greater T1rho values than collagen-degraded or normal samples. T1rho-weighted images reveal structure not found in conventional T1- or T2-weighted images. Our results suggest that T1rho measurements are selectively sensitive to proteoglycan content. The potential of this method in distinguishing the early degenerative changes in cartilage associated with osteoarthritis is discussed.

Effects of Enzymatic Degradation on the Subunit Composition and Biologic Properties of Human Factor VIII

Human factor VIII was purified from cryoprecipitate and subjected to enzymatic degradation with plasmin, trypsin, chymotrypsin, and thrombin. The reactions were monitored by sodium dodecyl sulfate (SDS)-Polyacrylamide gel electrophoresis and by measurement of four biologic properties, namely, procoagulant activity, ristocetin cofactor activity, precipitation by heterologous antibody, and neutralization of a human factor VIII inhibitor. The electrophoretic patterns of reduced digests showed a reproducible sequence of degradation of the 215,000 molecular weight subunit by plasmin, trypsin, and chymotrypsin, each with well-defined intermediate and enzyme-resistant chain remnants. There were striking similarities in the sizes of several intermediate chain remnants produced by these three enzymes, suggesting the presence of regions on the factor VIII molecule that are especially susceptible to enzymatic degradation, but each enzyme also produced distinctive proteolytic derivatives. The most obvious differences included (1) a single-chained 40,000 molecular weight remnant cleaved by plasmin and trypsin which was either absent or degraded in chymotryptic digests, (2) large intermediate chain remnants of 176,000 and 157,000 molecular weight which were produced only by trypsin, and (3) differences in size and content of nonreduced fragments in the final digests. Thrombin caused no detectable change in electrophoretic patterns. The immunoprecipitation reaction was sensitive to minor degrees of proteolytic cleavage, showing a marked increase in rocket size even when the SDS-treated subunit chains showed virtually no change in electrophoretic mobility. Procoagulant and inhibitor neutralizing activities were rapidly destroyed by trypsin and plasmin, but this destruction did not correlate with specific changes in electrophoretic patterns. Both thrombin and chymotrypsin caused an increase in procoagulant activity prior to destruction of activity; changes in this property were therefore unrelated to changes in the protein visualized by gel electrophoresis. Ristocetin cofactor activity was considerably more resistant to plasmin than to trypsin or chymotrypsin, and roughly paralleled the persistence of highly aggregated forms in the digests.

Effects of enzymatic degradation on the subunit composition and biologic properties of human factor VIII

Effects of enzymatic degradation on the subunit composition and biologic properties of human factor VIII

Spectral characteristics of metachromatically stained cartilage: Effects of enzymatic degradation and dehydration

When a microspectrophotometer was used to study cartilage, stained metachromatically with Azure A and Safranin O, the following 3 points were made: 1. the absorption maximum of stained cartilage matrix is identical to that reported for solutions of these dyes containing chondroitin sulfate; 2. partial enzymatic degradation of the cartilage matrix has no effect on the position of the absorption peak (although the intensity is greatly diminished); 3. ethanolic dehydration shifts the absorption peak somewhat toward the orthochromatic position, and decreases the intensity of the stain.

Absorption microscopy of enzymatically treated cell-free chloroplasts.

Monochromatic light microscopy at 435 mmicro shows in Euglena gracilis, the distribution of chlorophyll and the general orientation and geometry of chloroplasts in vivo. In addition it discloses, in swelling chloroplasts, a lamellar pigmented structure. Changes in this structure are observed in extruded swollen chloroplasts treated with lipolytic or proteolytic enzymes. Lipolytic enzymes produce an increase in the number of visible lamellae while proteolytic enzymes disrupt the lamellar array. Correlation of chloroplast swelling behavior and the effects of enzymatic degradation with current electron microscope observations support the following: (1) the pigment lamellae observed in vivo consist of component laminae; (2) the lamellae are separated by sites of swelling; and (3) the integrity of the lamellar structure is primarily dependent upon the intact state of the protein.