Fabrication of a Novel pH-Sensitive PLLA/PVA/Hyaluronic acid/Tragacanth 3D Printing Wound Healing Scaffold with Antagonistic Bacteria as the Antibacterial Agent

3D Bio-printing technique has emerged as an accurate manufacturing technique for the production of bio-engineered structures. 3D Bio-printing uses a computer-aided transfer process for the simultaneous deposition of living cells and biomaterials (bio-ink) in predefined spatial locations to construct bio-engineered structures in regenerative medicine, wound healing scaffold and basic cell biology studies.[1] The main advantages of 3D bio-printing that distinguish it from the traditional methods of freeze-drying and electrospinning are the possibility of accurately controlling the porosity of the scaffolds and high speed of printing. In this study, we use the 3D-printing technology as a new method to produce poly-l-lactic acid (PLLA) scaffolds. PLLA is a polyester which is biodegradable in alkali pH [2] (pH of granulation and epithelialization of wound healing process) and carries the nutrients that are needed for tissue regeneration and on the other hand, provides mechanical strength in order to maintain the scaffold structure. In the next phase we use polyvinyl alcohol (PVA) as a hydrogel that resists to degradation in alkali pH but degrades in acidic pH, therefore in the early inflammation stages of the skin wound, it can be gradually degraded and release an intended antibacterial agent simultaneously. In addition, 90% of PVA hydrogel is consist of water hence it can provide moisture requirement of the wound region and decrease the scar after healing. [3] Recent studies have shown great interest in using natural polymers incorporation with synthetic polymers to improve the efficiency of the final scaffold. Hyaluronic acid (HA) and tragacanth are among the natural polymers that are suitable for scaffold fabrication. Hyaluronic acid (hyaluronan) is a natural non-antigenic polymer. Current studies demonstrate that HA has the ability to induce angiogenesis and enhance the healing of chronic wounds. [4,5] Gum tragacanth is a viscose and water-soluble mixture of polysaccharides obtained from sap which is drained from the root of the Astragalus plant and dried. It absorbs water to become a gel, which can be stirred into a paste and has been used as a traditional treatment for skin burns. [6] These natural polymers mimic the extracellular matrix (ECM) and also provide the main components for ECM regeneration such as D-glucuronic acid and N-acetyl-glucosamine. Therefore, in order to improve the beneficial application of the PLLA scaffolds, we coated the PLLA pores with HA and tragacanth. The microbial microflora can adversely affect the wound healing process. Specific bacteria, such as Staphylococcus aureus, have been correlated with infections and complications in the wound healing process. More specifically, Anaerococcus, Corynebacterium, Porphyromonas and, Streptococcus are some of the most common bacterial genera which have been found to be abundant in the microbiota of chronic wounds. [7] It is possible to biologically control these pathogens by employing antagonistic and probiotic bacteria which can cause disorders in their function and effectively suppress them. Lactic acid bacteria is an antagonist for the Staphylococcus aureus which is the main bacterial population of the wound region. Also, lactic acid bacteria alongside bifidobacteria are the most studied probiotics. Probiotics are microorganisms that have been used to improve the healing of intestinal ulcers and infected cutaneous wounds. [8[Probiotics have many advantages that make them a good choice against the wound region’s pathogens. Some of the most important features of probiotics are (a) inactivation of toxins and metabolites of pathogenic bacteria, (b) production of antimicrobial substances that inhibit the growth of pathogenic microorganisms, like bacteriocins which represent small secretory peptides that show wide range of antimicrobial activity both in-vitro and in-vivo, (c) Stimulation of host immune response, and (d) Probiotics can also induce re-epithelialization through induction of chemokines. [8] The antibacterial activity of probiotic strains towards skin pathogens has been shown in many in-vitro studies with human fibroblast and keratinocytes. Recently two types of bacteria have been investigated in some in-vitro studies: (a) L.rhamnosus GG which promote cell migration, and allowing the cells to close the artificial wound rapidly, and (b) L.reuteri which increase the rate of cell division, helping to reproduce the pool of cells destroyed by the creation of the wound. We have used the extraction of antagonistic and probiotic bacteria which contain all the bacteriocins secretory peptides in the PVA. Therefore, during the inflammation phase of the wound healing, the PVA will be degraded and the antibacterial activity of the scaffold will be fulfilled. In this work, we hypothesized that the PLLA/PVA scaffold coated with HA/ tragacanth would have an improved tissue regeneration ability in the wound region and also it would show an effective antibacterial activity due to the presence of antagonist/probiotics bacteria in the PVA.

Keywords:

Wound Healing , 3D Bio-printing , Smart Scaffold , PLLA , PVA , Antagonistic Bacteria