Induced oxidative stress management in wounds through phenolic acids engineered fibrous protein: An in vitro assessment using polymorphonuclear (PMN) cells.

The present study explores the preparation, characterization and the role of phenolic acid tethered fibrous protein in the management of induced oxidative stress studied under in vitro conditions. In brief, the biomaterial is prepared by engineering the fibrous protein with dihydroxy and trihydroxy phenolic acid moieties and subjected to characterization to ensure the tethering. The resultant biomaterial studied for its efficacy as a free radical scavenger using polymorphonuclear (PMN) cells with induced oxidative stress and also as an agent for cell migration using fibroblasts cells. Results revealed that induced oxidative stress in PMN cells after exposure to UVB radiation managed well with the prepared biomaterial by reducing the levels of superoxide anion, oxygen and hydroxyl radicals. Further, the protein and the phenolic acid interaction supports the cell migration as evidenced from the scratch assay. In conclusion, though phenolic acids are well known for their antimicrobial and antioxidant potential, indenting these acids directly to the wounds is not sensible, but tethering to protein explored the scavenging activity as expected. The present study infers that phenolic acid engineered protein has a significant role in managing the imbalance in the redox state prevailing in wounds and supports the healing at appreciable level.

pH and redox sensitive albumin hydrogel: A self-derived biomaterial.

Serum albumin can be transformed to a stimuli (pH and redox) responsive hydrogel using the reduction process followed by oxidative refolding. The preparation of albumin hydrogel involves a range of concentrations (75, 150, 300, 450, 600 and 750 µM) and pH (2.0-10.0) values and the gelation begins at a concentration of 150 µM and 4.5-8.0 pH value. The hydrogel shows maximum swelling at alkali pH (pH > 9.0). The increase in albumin concentration increases hydrogel stability, rheological property, compressive strength, proteolytic resistance and rate of in vivo biodegradation. Based on the observed physical and biological properties of albumin hydrogel, 450 µM was determined to be an optimum concentration for further experiments. In addition, the hemo- and cytocompatibility analyses revealed the biocompatibility nature of albumin hydrogel. The experiments on in vitro drug (Tetracycline) delivery were carried out under non reducing and reducing conditions that resulted in the sustained and fast release of the drug, respectively. The methodology used in the preparation of albumin hydrogel may lead to the development of autogenic tissue constructs. In addition, the methodology can have various applications in tissue engineering and drug delivery.

In vitro and in vivo assessments of a 3-(3,4-dihydroxyphenyl)-2-propenoic acid bioconjugated gelatin-based injectable hydrogel for biomedical applications.

Imparting functional properties on a biomaterial for high end applications is always a challenging task. In the present study, an attempt was made to construct an injectable hydrogel through bioconjugation of dihydroxy phenolic acids to a gelatin backbone. Bioconjugating caffeic acid with gelatin followed by oxidation with mild oxidation agents provided a hydrogel with all the requisite properties (biocompatibility, controlled biodegradability, and antioxidant, antimicrobial and wound healing ability). Bioconjugation was performed using EDC/NHS and the resultant gel named as caffeic acid bioconjugated gel (CBG gel). The physicochemical, rheological, swelling, in vitro (biocompatibility, biodegradability, antimicrobial properties, antioxidant properties and drug release properties) and in vivo (biocompatibility, biodegradability and wound healing properties) studies on the CBG gel were carried out using standard protocols. The bioconjugation was confirmed by 1H NMR and UV-Vis analysis. Rheological analysis of the CBG gel revealed that the storage modulus was greater than the loss modulus at all the frequencies and suggested the elastic nature of the gel. About 50% weight gain within 12 hours during swelling studies and 50% weight loss within 12 hours during evaporation suggested the suitability of the CBG gel as a drug carrier. The drug release studies implied that there was an initial burst and later the release was sustained. The CBG gel promotes cell migration and demonstrates radical scavenging behavior. When subcutaneously injected into the animal, as in situ CBG gel, the gel was highly biocompatible and did not cause any necrosis. The crosstalk with adjacent tissue cells was smooth and the gel completely degraded within 24 days. The wound healing efficacy on full-thickness wounds suggested that the CBG gel accelerated healing and imparted high strength on the healed skin at an appreciable level. With all these additional functional properties, the CBG gel could be useful for biomedical applications.

Rejoining of cut wounds by engineered gelatin-keratin glue.

BACKGROUND: Rejoining of cut tissue ends of a critical site challenges clinicians. The toxicity, antigenicity, low adhesive strength, flexibility, swelling and cost of the currently employed glue demands an alternative. Engineered gelatin-keratin glue (EGK-glue) described in the present study was found to be suitable for wet tissue approximation. METHODS: EGK-glue was prepared by engineering gelatin with caffeic acid using EDC and conjugating with keratin by periodate oxidation. UV-visible, (1)H NMR and circular dichroism analyses followed by experiments on gelation time, rheology, gel adhesive strength (in vitro), wet tissue approximation (in vivo), H&E staining of tissue sections at scheduled time intervals and tensile strength of the healed skin were carried out to assess the effectiveness of the EGK-glue in comparison with fibrin glue and cyanoacrylate. RESULTS: Results of UV-visible, NMR and CD analyses confirmed the functionalization and secondary structural changes. Increasing concentration of keratin reduces the gelation time (<15s). Lap-shear test demonstrates the maximum adhesive strength of 16.6±1.2kPa. Results of hemocompatibility and cytocompatibility studies suggested the suitability of the glue for clinical applications. Tissue approximation property assessed using the incision wound model (Wistar strain) in comparison with cyanoacrylate and fibrin glue suggested, that EGK-glue explicitly accelerates the rejoining of tissue with a 1.86 fold increase in skin tensile strength after healing. CONCLUSIONS: Imparting quinone moiety to gelatin-keratin conjugates through caffeic acid and a weaker oxidizing agent provides an adhesive glue with appreciable strength, and hemocompatible, cytocompatible and biodegradable properties, which, rejoin the cut tissue ends effectively. GENERAL SIGNIFICANCE: EGK-glue obtained in the present study finds wide biomedical/clinical applications.

Bonding interactions and stability assessment of biopolymer material prepared using type III collagen of avian intestine and anionic polysaccharides.

The present study demonstrate bonding interactions between anionic polysaccharides, alginic acid (AA) and type III collagen extracted from avian intestine used for the preparation of thermally stable and biodegradable biopolymer material. Further the study describes, optimum conditions (pH, temperature and NaCl concentration) required for the formation of fibrils in type III collagen, assessment on degree of cross-linking, nature of bonding patterns, biocompatibility and biodegradability of the cross-linked biomaterial. Results revealed, the resultant biopolymer material exhibit high thermal stability with 5-6 fold increase in tensile strength compared to the plain AA and collagen materials. The degree of cross-linking was calculated as 75%. No cytotoxicity was observed for the cross-linked biopolymer material when tested with skin fibroblast cells and the material was biodegradable when treated with enzyme collagenase. With reference to bonding pattern analysis we found, AA cross-linked with type III collagen via (i) formation of covalent amide linkage between -COOH group of AA and ε-NH2 group of type-III collagen as well as (ii) intermolecular multiple hydrogen bonding between alginic acid -OH group with various amino acid functional group of type-III collagen. Comparisons were made with other cross-linking agents also. For better understanding of bonding pattern, bioinformatics analysis was carried out and discussed in detail. The results of the study emphasize, AA acts as a suitable natural cross-linker for the preparation of wound dressing biopolymer material using collagen. The tensile strength and the thermal stability further added value to the resultant biopolymer.

Preparation and characterization of a thermostable and biodegradable biopolymers using natural cross-linker.

The present study describes preparation and characterization of a thermally stable and biodegradable biopolymer using collagen and a natural polymer, alginic acid (AA). Required concentration of alginic acid and collagen was optimized and the resulting biopolymer was characterized for, degree of cross-linking, mechanical strength, thermal stability, biocompatibility (toxicity) and biodegradability. Results reveal, the degree of cross-linking of alginic acid (at 1.5% concentration) with collagen was calculated as 75%, whereas it was 83% with standard cross-linking agent, glutaraldehyde (at 1.5% concentration). The AA cross-linked biopolymer was stable up to 245°C and Exhibits 5-6-fold increase in mechanical (tensile) strength compared to plain collagen (native) materials. However, glutaraldehyde cross-linked material exhibits comparatively less thermal stability and brittle in nature (low tensile strength). With regard to cell toxicity, no cytotoxicity was observed for AA cross-linked material when tested with mesenchymal cells and found degradable when treated with collagenase enzyme. The nature of bonding pattern and the reason for thermal stability of AA cross-linked collagen biopolymer was discussed in detail with the help of bioinformatics. A supplementary file on efficacy of AACC as a wound dressing material is demonstrated in detail with animal model studies.