Location: Cotton Chemistry and Utilization Research
Project Number: 6054-41430-009-005-A
Project Type: Cooperative Agreement
Start Date: May 1, 2023
End Date: Sep 1, 2023
Objective:
The objective of this effort is to develop and deploy a family of affordable, US sourced and manufactured cotton based wound treatments with protective and antimicrobial properties as well as advanced wound care products for burns and slowly healing wounds. To this end the first step is to develop “proof of principle” for the characterization and evidence demonstrating the feasibility and validity of wound repair and antimicrobial activity of the cotton-based scaffolds and combination products with acellular stem cell extracts shown to promote cell proliferation, and having potential to improve wound healing.
Approach:
General Experimental Guidelines:a) All studies will be performed with cryopreserved human adipose-derived stromal/stem cells (ASCs) and human dermal fibroblast cells (DFCs) pre-selected by USDA ARS. b) All studies will be performed with IRB reviewed and approved protocols with consenting participants undergoing routine orthopedic procedures where study participation has minimal impact on patient risk of adverse effects of any kind. c) All studies will be performed according to Standard Operating Procedures developed at the Cooperators.
ARS will provide nanocellulose hydrogels (NCHs) from varying source raw materials. Preparation of ASC:NCH formulations containing stem cell extract (ASC) gels as assembled by the Cooperator will be made at different ratios of ASC:NCH ranging from 5:1 to 2:1 by weight.
2. Physicochemical characteristics: The ASC/NCH formulations will be evaluated for stiffness, crosslinking time, protein content, and scaffold stability. In addition, the scaffolds will be characterized using scanning electron microscopy (SEM), swelling, and biodegradation studies by the Cooperator.
Biocompatibility studies: The combination formulations will be subject to in vitro studies that will demonstrate the biocompatibility of the individual and combination scaffolds with ASCs and DFCs. The in vitro studies will be performed by co-culturing the Cooperator's proprietary primary adipose (ASC) and skin (DFC) cells with the USDA cotton-derived scaffolds, both alone and in combination with the biological scaffolds prepared by the Cooperator. Assays will assess cell viability, cellular proliferation, and the differentiation potential of ASC:NCHs under culture conditions in the presence of standard growth media.
ARS will prepare encapsulation studies of ASC:NCH leads as gels and powders on formulations showing promise.
2. In vitro wound healing model: Wound healing transwell system with DFCs will be used to create a wound area that can be visualized over time for closure in presence of the scaffolds. (Serine proteases (HNE) and metalloproteinases can be tested for HNE, MMP2 and MMP9 via zymogram).
3. Antimicrobial studies: The prepared scaffolds will be tested in presence of Gram-positive and Gram-negative bacterial strains to demonstrate antibacterial activity. P. aeruginosa may delay the proliferative phase of wound repair by secreting proteins leading to delayed or impaired wound healing. Antimicrobial activity against S. aureus and P. aeruginosa will be tested in scaffolds, a one-third dilution of each scaffold, a negative control of media alone, and positive controls of antibiotics (ex. vancomycin for S. aureus). Each sample will be tested in duplicate. The bacterial growth in each well will be monitored over the course of 24 h using absorbance readings at 570 nm with microplate reader. Plate counts of total viable cells and flow cytometry (viability assay) will be used to assess the antimicrobial activity on Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacteria.
