Collagenase Activity Research Articles

Positive Effect of Cannabis sativa L. Herb Extracts on Skin Cells and Assessment of Cannabinoid-Based Hydrogels Properties.

The skin is an organ that is constantly exposed to many external factors that can affect its structure and function. Due to the presence of different cannabinoid receptors on many types of skin cells, cannabinoids can interact directly with them. Therefore, as part of this work, the impact of two types of Cannabis sativa L. herb extracts on keratinocytes and fibroblasts was assessed. The content of biologically active compounds such as phenols, flavonoids, chlorophylls and cannabinoids was evaluated. The antioxidant capacity of prepared extracts using the DPPH radical, H2DCFDA probe and measurement of superoxide dismutase activity was also assessed. The cytotoxicity of hemp extracts was determined using the Alamar Blue, Neutral Red and LDH assays. The ability of the extracts to inhibit the activity of matrix metalloproteinases, collagenase and elastase, was assessed. Preparations of model hydrogels were also prepared and their effect on transepidermal water loss and skin hydration was measured. The obtained results indicate that hemp extracts can be a valuable source of biologically active substances that reduce oxidative stress, inhibit skin aging processes and positively affect the viability of skin cells. The analysis also showed that hydrogels based on cannabis extracts have a positive effect on skin hydration.

In Vitro Investigation of Thiol-Functionalized Cellulose Nanofibrils as a Chronic Wound Environment Modulator.

There is currently a huge need for new, improved therapeutic approaches for the treatment of chronic wounds. One promising strategy is to develop wound dressings capable of modulating the chronic wound environment (e.g., by controlling the high levels of reactive oxygen species (ROS) and proteases). Here, we selected the thiol-containing amino acid cysteine to endow wood-derived cellulose nanofibrils (CNF) with bioactivity toward the modulation of ROS levels and protease activity. Cysteine was covalently incorporated into CNF and the functionalized material, herein referred as cys-CNF, was characterized in terms of chemical structure, degree of substitution, radical scavenging capacity, and inhibition of protease activity. The stability of the thiol groups was evaluated over time, and an in vitro cytotoxicity study with human dermal fibroblasts was performed to evaluate the safety profile of cys-CNF. Results showed that cys-CNF was able to efficiently control the activity of the metalloprotease collagenase and to inhibit the free radical DPPH (1,1-Diphenyl-2-picrylhydrazyl radical), activities that were correlated with the presence of free thiol groups on the nanofibers. The stability study showed that the reactivity of the thiol groups challenged the bioactivity over time. Nevertheless, preparing the material as an aerogel and storing it in an inert atmosphere were shown to be valid approaches to increase the stability of the thiol groups in cys-CNF. No signs of toxicity were observed on the dermal fibroblasts when exposed to cys-CNF (concentration range 0.1–0.5 mg/mL). The present work highlights cys-CNF as a promising novel material for the development of bioactive wound dressings for the treatment of chronic wounds.

In Vitro Anti-Collagenase Activity and Total Phenolic Content Of Five Selected Herbs: A Review

Etlingera elatior, Persicaria odorata, Centella asiatica, Senna alata and Phyllanthus emblica were reviewed for their in vitro anti-collagenase activity and its total phenolic contents. Plant herbs play a vital role to promote the production of collagen which is the component of the skin. Different solvents during plant extraction and various plant parts were used to determine anti-collagenase properties and total phenolic content. Generally, anti-collagenase assay and Folin-Ciocalteu method were used to determine the anti-collagenase activity and total phenolic content, respectively. The extracts of these herbs have the potential of inhibiting collagenase activity and they possess total phenolic content. The total phenolic was found to be important elements in collagenase inhibition activity other than flavonoid. The selected herbs are popular in Malaysia and this review can be a useful reference or information for future application in pharmaceutical, food and cosmetics fields.
 Keywords: Anti-collagenase activity, total phenolic contents; plant Extraction; flavonoids

Isolation of Murine Primary Aortic Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) have been cultured for decades to study the role of these cells in cardiovascular disorders. The most common source of VSMCs is the rat aorta. Here we show the adaptation of this method to isolate and culture mouse aortic VSMCs. The advantage of this method is that there are many more transgenic mouse lines available compared to rats. The protocol consists of the isolation of the aorta, the liberation of vascular cells by the action of collagenase, culturing of VSCMs, and analyzing filamentous actin and alpha smooth muscle actin by fluorescence microscopy. VSCMs can be further used to study mechanisms underlying cardiovascular diseases. Graphic abstract: Figure 1.Working steps.

Expression of Matrix Metalloproteinases-8 and Myeloperoxidase in Pulp Tissue after Pulpotomy with Calcium Silicate Cements

Objective: To evaluate the pulp tissue of rat molars after pulpotomies with mineral trioxide aggregate (MTA), BiodentineTM (BDT) and calcium hydroxide (CH) mixed with sterile saline solution (24 hours, 72 hours, 7 days and 15 days), through correlating MPO activity with active neutrophils and MMP8 activity with tissue remodeling. Material and Methods: Thirty-eight Wistar rats were randomly distributed into groups (control, I (MTA gray), II (BDT), and III (CH)) and subdivided according to the study period of 24 hours, 72 hours, 7 days or 15 days after pulpotomy. MMP8 activity was assessed through fluorescence technique, and MPO activity was determined using the MPO assay. Results: A gradual decrease of MPO and MM8 activity occurred in the group MTA over the experimental periods (p<0.05). Groups BDT and CH exhibited an increase in the activity at 7 and 15 days (p<0.05). Conclusion: MTA demonstrated a decrease in the values of MPO e MMP8. BDT and CH showed high neutrophilic and collagenase activity over the experimental periods.

A Face Serum Containing Palmitoyl Tripeptide-38, Hydrolyzed Hyaluronic Acid, Bakuchiol and a Polyherbal and Vitamin Blend Improves Skin Quality

A face serum composed of a combination of biologically active compounds was evaluated for safety and efficacy in vitro, in a repeat insult patch test and in a human clinical efficacy trial. The serum inhibited tyrosinase activity modestly, decreased collagenase activity and exhibited notable free radical scavenging activity in vitro. It is gentle to the skin, as the serum did not irritate the skin or produce symptoms of allergic contact dermatitis in the 55 healthy adults that participated in the repeat insult patch test. In the efficacy trial, daily application of the face serum for 30 days significantly increased skin hydration, with all 35 volunteers experiencing improvement. Substantial improvements in skin elasticity, roughness (fine lines and wrinkles), and brightness also occurred during the trial. Dermatological examination also revealed a trend for reduced comedone count with use of the serum. Self-assessment responses revealed that all volunteers experienced improvements in multiple skin quality parameters and that participant perceptions are consistent with the results of the instrumental analyses. These findings indicated that the measured improvements in skin quality are not only statistically significant but are also clinically relevant as they were great enough for users of the face serum to feel and recognize.

Antioxidant and Antiaging Assays of Ageratum conyzoides (L.) Ethanolic Extract

The declining ability of tissues to repair or replace themselves and maintain their structure and normal function is called aging. Aging is caused by free radical that can induce the activation of elastase and collagenase. Ageratum conyzoides (L.) or known as Babadotan is a medicinal plant that has been reported to be a good source of antioxidants that also inhibit aging process. The purpose of this research is to evaluate the antioxidant and antiaging properties of Babadotan Ethanolic Extract (BEE). Franswoth method was used to determine the phytochemical assay of BEE. Measurement of 2,2-diphenyl 1-picrylhydrazyl (DPPH) scavenger was used for antioxidant assay, and inhibitory of elastase and collagenase activities was used for antiaging assay. The analysis of phytochemical compounds exhibited the presence of flavonoids, alkaloids, tannins, polyphenols, steroids and triterpenoids, monoterpenoids and terpenoids, and the absence of saponin. BEE has lower activity on DPPH scavenging activity (IC50 = 80.7 μg/mL) than quercetin (IC50 = 3.25 μg/mL). BEE exhibited lower elastase and collagenase inhibitory activity (IC50 = 45.35 ± 2.2 μg/mL and 55.07 ± 1.1 μg/mL, respectively) compared to quercetin (IC50 = 11.64 ± 0.67 μg/mL and 19.91 ± 0.46 μg/mL). Overall, BEE possesses antioxidant and antiaging activities, although the activities are lower than quercetin.

괭생이모자반으로부터 미백과 주름개선 생리활성물질 생산을 위한 초음파 추출공정 개발

Statistically-based optimization using response surface methodology was performed to derive optimal extraction conditions for the extraction of bioactive compounds with antioxidant activity, skin whitening, and anti-wrinkle effects from Sargassum horneri, marine waste, using the ultrasound-assisted extraction (UAE). The main UAE process variables, extraction time (5.3~18.7 min, X₁), extraction temperature (22.4~79.6°C, X₂), and ethanol concentration (0~99.5% v/v, X₃), were selected through preliminary experiments and optimized using central composite design at five levels. Total phenolic content (TPC), total flavonoid content (TPC), DPPH radical scavenging activity (RSA), tyrosinase activity inhibition (TAI), and collagenase activity inhibition (CAI) were analyzed and second-order polynomial models were generated for predicting the responses. The optimum UAE conditions for enhancing antioxidants, skin whitening, and anti-wrinkle effects were extraction time of 11.7 min, extraction temperature of 54.8°C, and ethanol concentration of 73.0%, under which RSA, TAI, and CAI were 29.1%, 78.5%, and 83.6%, respectively. In this study, we proposed an extraction process for the production of cosmetic materials that have a combination of antioxidant, whitening, and wrinkle effects from S.horneri, which are marine waste.

Chemical composition of Prunus padus L. flower extract and its anti-inflammatory activities in primary bone marrow-derived macrophages

Ethnopharmacological relevancePrunus padus L. has been traditionally used in European ethnomedicine as a treatment for internal and external purposes and is mainly used to reduce inflammation, pain and fever. The activities of P. padus flower extracts are not well characterized, and additional experimental studies at the molecular level are needed to confirm the ethnobotanical findings. Aim of the studyTo assess the potential of P. padus flower extract (PPFE) as a source of bioactive compounds through the characterization of its chemical composition and antioxidant, anti-collagenase, and anti-inflammatory activities. Materials and methodsThe ethanolic extract (1:10 w/v in ethanol solution) from P. padus flowers was subjected to phytochemical analysis and evaluation of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Anti-collagenase activity was determined using a spectrophotometric method in vitro. The effect of PPFE on inflammation was evaluated by measuring specific markers using flow cytometry and assessing pro-inflammatory cytokine (IL-6) release by bone marrow-derived macrophages (BMDMs) ex vivo. ResultsThe major components of the ethanolic extract of P. padus flowers were quercetin diglycosides, chlorogenic acid and N′,N″-dicaffeoyl,N‴-coumaroyl spermidine. The total phenolic content of PPFE was 85.19 mg GAE/g extract, and the EC50 value in the DPPH assay was 0.55 mg/ml. PPFE exhibited the ability to inhibit collagenase activity in a dose-dependent manner. Preincubation of BMDMs with PPFE reduced the population of M1 (pro-inflammatory) and increased the population of M2 (anti-inflammatory) macrophages. Furthermore, PPFE decreased pro-inflammatory cytokine IL-6 release from BMDMs. ConclusionsPPFE is a rich source of bioactive compounds and possesses considerable anti-inflammatory properties, supporting its use in ethnomedicine for the reduction of inflammatory processes.

DETERMINATION OF ANTIOXIDANT, ANTI-AGING AND CYTOTOXICITY ACTIVITY OF THE ESSENTIAL OILS FROM CINNAMOMUM ZEYLANICUM

Antioxidant activity, anti-aging effects and cytotoxicity activity of cinnamon essential oils from Cinnamomum zeylanicum were investigated in this study. The antioxidant activities of the cinnamon essential oil at the concentrations of 125, 250, 500, and 1000 µg/mL were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2ʹ-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS). The inhibitory activities against collagenase, elastase and tyrosinase were evaluated for anti-aging effects. The antioxidant activity determined by DPPH and ABTS assays varied from 4.91 - 28.74% and 4.96 - 50.17%, respectively. In addition, cinnamon essential oil at all concentrations tested (100, 200, 500, and 1000 µg/mL) inhibited tyrosinase activity by 61.68 - 93.12 %, collagenase activity by 2.83 - 30.28 % and elastase activity by 4.37 - 33.92 %. The cytotoxicity activity determined by the diphenyltetrazolium (MTT) assay revealed that the cinnamon essential oil at the concentration less than 100 µg/mL did not exhibit cytotoxicity activity on human fibroblast cells while the percentage of cell viability decreased when exposed to this oil at the concentration higher than 150 µg/mL. These results demonstrated that the cinnamon essential oil has antioxidant, tyrosinase inhibitory, collagenase inhibitory, and elastase inhibitory activities. In addition, cinnamon essential oil at each effective concentration did not show any toxicity when tested on normal human fibroblast cell. Therefore, this essential oil could be a potential candidate for cosmetic and pharmaceutical products.

GENERATION OF BIOACTIVE PEPTIDES IN MEAT RAW MATERIALS EXPOSED TO LYSATES OF BACTERIAL STARTER CULTURES

Nowadays, preparations based on bacterial lysates are mainly applied in medicine. In food industry, bacterial lysates are still not widely used, in particular for manufacturing meat functional foodstuff. Though their potential for functional foodstuff production is predictable, the efficiency and specificity of action which depend on the characteristics of the strain and the method of cell disintegration require study. A set of peptidases identified in starter cultures, in particular endo-peptidases, aminopeptidases, dipeptidases, tripeptidases, and proline-specific peptidases stimulate interest in the lysates of these microorganisms for food biotechnology. In this work, we have shown that lysates of <em>Pediococcus pentosaceus</em> 28, <em>Staphylococcus carnosus </em>108, <em>Lactobacillus curvatus </em>1, <em>P</em>. <em>acidilactici</em> 38, <em>L</em>. <em>sakei</em> 103, <em>L</em>. <em>sakei</em> 105, <em>L</em>. <em>curvatus</em> 2, <em>L</em>. <em>acidophilus</em> AT-41 that we obtained by physical destruction of bacterial cells have the widest spectrum of enzymes and biologically active substances. Our goal was to determine the biochemical composition and enzymatic activity of the lysates of starting bacterial cultures and their role in the formation of biologically active peptides in raw meat. The bacterial suspensions were exposed either to lysozyme treatment followed by separation of the extract from the cell debris by centrifugation, or to ultrasonic treatment to compare two methods of cell destruction. The physical method was proved to be the most effective. For biochemical characterization, the proteolytic, lipolytic and collagenase activities of the lysates, and the concentration of organic acids, proteins, and free amino acids were measured. Enzymatic activities of the lysates were determined using API®ZYM tests. The <em>Lactobacillus curvatus</em> 2, <em>Lactobacillus acidophilus</em> AT-41, <em>Pediococcus acidilactici</em> 38 and <em>Staphylococcus carnosus</em> 108 lysates showed the widest range of intracellular enzymes, including leucine and valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and b-galactosidase. The proteolytic activity was the highest in <em>Staphylococcus carnosus</em> 108 (115.94 proteolytic capability PC units per mg protein), <em>Lactobacillus acidophilus </em>AT-41 (66.7 PC units per mg protein), <em>Lactobacillus curvatus </em>1 (91.03 PC units per mg protein), and <em>Lactobacillus curvatus</em> 2 (72.20 PC units per mg protein) as compared to other strains. The level of malic, lactic and succinic acids in the lysates varied in the range of 0.002-0.02, 0.02-0.06, and 0.2-0.9 mg/100 g, respectively. The highest enrichment in free amino acids with 13 AA detected out of 17 AA studied was characteristic of <em>P. acidilactici</em> 38 lysate while only 7 AA were detected in the <em>L. sakei</em> 105 lysate. A comparison of 2D electrophoregrams of fermented raw meat showed both general effects on reducing total proteins and the lysate-specific effects toward various proteins, e.g. formation of protein conjugates and cleavage of target proteins, in particular actin skeletal muscle. Therefore, lysates of the studied starter cultures can serve as a source of various enzymes for practical use in the food industry, for example to improve the functional, technological and biocorrective characteristics of meat products.

ОБРАЗОВАНИЕ БИОЛОГИЧЕСКИ АКТИВНЫХ ПЕПТИДОВ В МЯСНОМ СЫРЬЕ ПОД ВЛИЯНИЕМ ЛИЗАТОВ СТАРТОВЫХ БАКТЕРИАЛЬНЫХ КУЛЬТУР

Nowadays, preparations based on bacterial lysates are mainly applied in medicine. In food industry, bacterial lysates are still not widely used, in particular for manufacturing meat functional foodstuff. Though their potential for functional foodstuff production is predictable, the efficiency and specificity of action which depend on the characteristics of the strain and the method of cell disintegration require study. A set of peptidases identified in starter cultures, in particular endo-peptidases, aminopeptidases, dipeptidases, tripeptidases, and proline-specific peptidases stimulate interest in the lysates of these microorganisms for food biotechnology. In this work, we have shown that lysates of <em>Pediococcus pentosaceus</em> 28, <em>Staphylococcus carnosus </em>108, <em>Lactobacillus curvatus </em>1, <em>P</em>. <em>acidilactici</em> 38, <em>L</em>. <em>sakei</em> 103, <em>L</em>. <em>sakei</em> 105, <em>L</em>. <em>curvatus</em> 2, <em>L</em>. <em>acidophilus</em> AT-41 that we obtained by physical destruction of bacterial cells have the widest spectrum of enzymes and biologically active substances. Our goal was to determine the biochemical composition and enzymatic activity of the lysates of starting bacterial cultures and their role in the formation of biologically active peptides in raw meat. The bacterial suspensions were exposed either to lysozyme treatment followed by separation of the extract from the cell debris by centrifugation, or to ultrasonic treatment to compare two methods of cell destruction. The physical method was proved to be the most effective. For biochemical characterization, the proteolytic, lipolytic and collagenase activities of the lysates, and the concentration of organic acids, proteins, and free amino acids were measured. Enzymatic activities of the lysates were determined using API®ZYM tests. The <em>Lactobacillus curvatus</em> 2, <em>Lactobacillus acidophilus</em> AT-41, <em>Pediococcus acidilactici</em> 38 and <em>Staphylococcus carnosus</em> 108 lysates showed the widest range of intracellular enzymes, including leucine and valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and b-galactosidase. The proteolytic activity was the highest in <em>Staphylococcus carnosus</em> 108 (115.94 proteolytic capability PC units per mg protein), <em>Lactobacillus acidophilus </em>AT-41 (66.7 PC units per mg protein), <em>Lactobacillus curvatus </em>1 (91.03 PC units per mg protein), and <em>Lactobacillus curvatus</em> 2 (72.20 PC units per mg protein) as compared to other strains. The level of malic, lactic and succinic acids in the lysates varied in the range of 0.002-0.02, 0.02-0.06, and 0.2-0.9 mg/100 g, respectively. The highest enrichment in free amino acids with 13 AA detected out of 17 AA studied was characteristic of <em>P. acidilactici</em> 38 lysate while only 7 AA were detected in the <em>L. sakei</em> 105 lysate. A comparison of 2D electrophoregrams of fermented raw meat showed both general effects on reducing total proteins and the lysate-specific effects toward various proteins, e.g. formation of protein conjugates and cleavage of target proteins, in particular actin skeletal muscle. Therefore, lysates of the studied starter cultures can serve as a source of various enzymes for practical use in the food industry, for example to improve the functional, technological and biocorrective characteristics of meat products.

Cannabis-derived cannabidiol and nanoselenium improve gut barrier function and affect bacterial enzyme activity in chickens subjected to C. perfringens challenge

Revealing the multifocal mechanisms affecting cross-talk between Clostridium perfringens pathogenesis and the host response is an urgent need in the poultry industry. Herein, the activity of Cannabis sativa-derived cannabidiol (CBD) and selenium nanoparticles (Nano-Se) in modulating the host response to Clostridium perfringens challenge was investigated in broiler chickens subjected to a mild infection model. The infected chickens exhibited no clinical manifestations, confirming the potential hazard of pathogen transmission to the food chain in the commercial sector. However, both CBD and Nano-Se affected the responses of chickens to C. perfringens challenge. The beneficial actions of both agents were manifested in the upregulated expression of genes determining gut barrier function. Both CBD and Nano-Se promoted shifts in gut bacterial enzyme activity to increased energy uptake in challenged chickens and upregulated potential collagenase activity. There was no opposite effect of CBD and Nano-Se in mediating the host response to challenge, whereas an additive effect was evidenced on the upregulation of gene determining gut integrity. Collectively, these findings indicate that understanding the action mechanisms of CBD and Nano-Se is of great interest for developing a preventive strategy for C. perfringens infection in broilers.

A controlled release bupivacaine-alginate construct: Effect on chondrocyte hypertrophy conversion

ObjectiveOsteoarthritis is a degenerative disease of the joint, affecting over 30 million people in the US1. A key characteristic of OA is chondrocyte hypertrophy, characterized by chondrocyte changes to a more rounded and osteoblastic phenotype, characterized by increased IL-6 and IL-8 secretion2. While there are no cures for OA, treatments focus on mitigating pain and inflammation, the two main symptoms of OA. However, the analgesics, NSAIDS and corticosteroids commonly used, do not target regeneration and have negative side effects. Local anesthetics (LA) can be used as a pain management alternative but are usually short lasting and therefore, not suited for chronic conditions such as OA. Our engineered sustained release local anesthetic construct successfully delivers bupivacaine for an extended period of time3-5. This study is designed to evaluate the effect of the LA system on chondrocytes in an inflammatory OA-like environment. DesignChondrocytes were cultured with bolus, liposomal, or construct LA and either untreated or treated with TNF-α and IL-1α for 24 hrs, 48 hrs, or 96 hrs. Chondrocyte viability, interleukin-8 (IL-8), interleukin-6 (IL-6), collagenase activity and proteoglycan deposition were assessed. ResultsIn the presence of the engineered construct, the chondrocytes retained viability and regenerative function. Moreover, the construct allowed for higher initial doses to be used, which promoted more regeneration and decreased inflammation without compromising cellular viability. ConclusionsThe construct promotes a less hypertrophic chondrocyte environment while promoting a more anti-inflammatory environment. These two factors are consistent with a less OA progressive environment when using the engineered construct, compared to bolus LA.

Effect of Fermentation Time on Antioxidant and Anti-Ageing Properties of Green Coffee Kombucha Ferments.

Kombucha, also known as the Manchurian mushroom, is a symbiotic culture of bacteria and yeast, the so-called SCOBY. This paper presents a comprehensive evaluation of the ferments obtained from green coffee beans after different fermentation times with kombucha. Results for the ferments were compared to the green coffee extract that was not fermented. In this study, the antioxidant potential of obtained ferments was analyzed by assessing the scavenging of external and intracellular free radicals and the assessment of superoxide dismutase activity. Cytotoxicity of ferments on keratinocyte and fibroblast cell lines was assessed as well as anti-aging properties by determining their ability to inhibit the activity of collagenase and elastase enzymes. In addition, the composition of the obtained ferments and the extract was determined, as well as their influence on skin hydration and transepidermal water loss (TEWL) after application of samples on the skin. It has been shown that the fermentation time has a positive effect on the content of bioactive compounds and antioxidant properties. The highest values were recorded for the tested samples after 28 days of fermentation. After 14 days of the fermentation process, it was observed that the analyzed ferments were characterized by low cytotoxicity to keratinocytes and fibroblasts. On the other hand, the short fermentation time of 7 days had a negative effect on the properties of the analyzed ferments. The obtained results indicate that both green coffee extracts and ferments can be an innovative ingredient of cosmetic products.

Chitosan oligosaccharide-g-linalool polymer as inhibitor of hyaluronidase and collagenase activity

In this study, chitosan oligosaccharide (COS) was modified by grafting Linalool (Lin) on its backbone to improve its anti-inflammatory activity. By changing the molar ratios of COS to Lin, three different degrees of substitution COS-g-Lin1-3 were prepared. The degrees of substitution of derivatives were 0.65, 0.80 and 1.14 respectively. The structure of COS-g-Lin1-3 were characterized by UV–vis, FT-IR, 1H NMR and elemental analysis in order to show the COS-g-Lin1-3 successfully synthesized. Besides, the thermal stability, solubility, pH stability as well as crystallinity were also investigated. The results revealed that the derivatives exhibited higher thermal stability and more remarkable anti-inflammatory property against hyaluronidase and collagenase than that of COS. The good biocompatibility made this novel material a promising and effective compound for anti-inflammatory applications.

Engineered PLGA-PVP/VA based formulations to produce electro-drawn fast\xa0biodegradable microneedles for labile biomolecule delivery

Biodegradable polymer microneedles (MNs) are recognized as non-toxic, safe and stable systems for advanced drug delivery and cutaneous treatments, allowing a direct intradermal delivery and in some cases a controlled release. Most of the microneedles found in the literature are fabricated by micromolding, which is a multistep thus typically costly process. Due to industrial needs, mold-free methods represent a very intriguing approach in microneedle fabrication. Electro-drawing (ED) has been recently proposed as an alternative fast, mild temperature and one-step strategy to the mold-based techniques for the fabrication of poly(lactic-co-glycolic acid) (PLGA) biodegradable MNs. In this work, taking advantage of the flexibility of the ED technology, we engineered microneedle inner microstructure by acting on the water-in-oil (W/O) precursor emulsion formulation to tune drug release profile. Particularly, to promote a faster release of the active pharmaceutical ingredient, we substituted part of PLGA with poly(1-vinylpyrrolidone-co-vinyl acetate) (PVP/VA), as compared to the PLGA alone in the matrix material. Moreover, we introduced lecithin and maltose as emulsion stabilizers. Microneedle inner structural analysis as well as collagenase entrapment efficiency, release and activity of different emulsion formulations were compared to reach an interconnected porosity MN structure, aimed at providing an efficient protein release profile. Furthermore, MN mechanical properties were examined as well as its ability to pierce the stratum corneum on a pig skin model, while the drug diffusion from the MN body was monitored in an in vitro collagen-based dermal model at selected time points.

P.776 Targeting of an F-BAR domain protein to the synaptic periactive zone ensures uniform size of synaptic vesicles

Osteoarthritis is a degenerative disease of the joint, affecting over 30 million people in the US1. A key characteristic of OA is chondrocyte hypertrophy, characterized by chondrocyte changes to a more rounded and osteoblastic phenotype, characterized by increased IL-6 and IL-8 secretion2. While there are no cures for OA, treatments focus on mitigating pain and inflammation, the two main symptoms of OA. However, the analgesics, NSAIDS and corticosteroids commonly used, do not target regeneration and have negative side effects. Local anesthetics (LA) can be used as a pain management alternative but are usually short lasting and therefore, not suited for chronic conditions such as OA. Our engineered sustained release local anesthetic construct successfully delivers bupivacaine for an extended period of time3-5. This study is designed to evaluate the effect of the LA system on chondrocytes in an inflammatory OA-like environment.Chondrocytes were cultured with bolus, liposomal, or construct LA and either untreated or treated with TNF-α and IL-1α for 24 hrs, 48 hrs, or 96 hrs. Chondrocyte viability, interleukin-8 (IL-8), interleukin-6 (IL-6), collagenase activity and proteoglycan deposition were assessed.In the presence of the engineered construct, the chondrocytes retained viability and regenerative function. Moreover, the construct allowed for higher initial doses to be used, which promoted more regeneration and decreased inflammation without compromising cellular viability.The construct promotes a less hypertrophic chondrocyte environment while promoting a more anti-inflammatory environment. These two factors are consistent with a less OA progressive environment when using the engineered construct, compared to bolus LA.

P.774 Olfactory reference syndrome as a prodrome of Alzheimer disease

Osteoarthritis is a degenerative disease of the joint, affecting over 30 million people in the US1. A key characteristic of OA is chondrocyte hypertrophy, characterized by chondrocyte changes to a more rounded and osteoblastic phenotype, characterized by increased IL-6 and IL-8 secretion2. While there are no cures for OA, treatments focus on mitigating pain and inflammation, the two main symptoms of OA. However, the analgesics, NSAIDS and corticosteroids commonly used, do not target regeneration and have negative side effects. Local anesthetics (LA) can be used as a pain management alternative but are usually short lasting and therefore, not suited for chronic conditions such as OA. Our engineered sustained release local anesthetic construct successfully delivers bupivacaine for an extended period of time3-5. This study is designed to evaluate the effect of the LA system on chondrocytes in an inflammatory OA-like environment.Chondrocytes were cultured with bolus, liposomal, or construct LA and either untreated or treated with TNF-α and IL-1α for 24 hrs, 48 hrs, or 96 hrs. Chondrocyte viability, interleukin-8 (IL-8), interleukin-6 (IL-6), collagenase activity and proteoglycan deposition were assessed.In the presence of the engineered construct, the chondrocytes retained viability and regenerative function. Moreover, the construct allowed for higher initial doses to be used, which promoted more regeneration and decreased inflammation without compromising cellular viability.The construct promotes a less hypertrophic chondrocyte environment while promoting a more anti-inflammatory environment. These two factors are consistent with a less OA progressive environment when using the engineered construct, compared to bolus LA.

Characterization of collagenase found in the nonpathogenic bacterium Lysinibacillus sphaericus VN3.

High collagenolytic activity has been detected in pathogenic bacteria. Collagenase plays an essential role in the invasion step in animals and humans. In this study, we characterized collagenase found in the nonpathogenic bacterium Lysinibacillus sphaericus VN3, which was isolated from soil in Vietnam. The collagenase activity of the purified enzyme was strongly inhibited by Cu2+, but it was significantly increased by Zn2+. The purified enzyme with a molecular mass of approximately 110 kDa exhibited collagenolytic, gelatinolytic, and caseinolytic activity. The kinetic studies showed that this enzyme had greater hydrolyzing activity toward collagen and gelatin compared with casein. Based on the ratio V max/K m, collagen is likely to be the best substrate among three proteins. We found that this collagenase could digest small pieces of bovine skin and tendon into a collagen solution. Interestingly, at pH 6.0-8.0, the soluble collagen could form a collagen membrane, which is useful as a wound-healing biomaterial.