(LR-037) Evaluation Of Collagen Wound Matrix-Micronized: Biochemical, Physical, Functional, and In Vitro Properties as a Wound Management Matrix

Justin Avery, PhD – Scientist II, Organogenesis; Jumana Alhamdi, PhD – Scientist II, Organogenesis; Pooja Nargunde, MS – Research Associate II, Organogenesis; Isabella Goh, BS – R&D Intern, Organogenesis; Katie Mowry, PhD – VP R&D, Organogenesis

Introduction: The extracellular matrix (ECM) is a 3D network comprised of structural proteins, with collagen being the most abundant. Fibroblasts and their interaction with ECM proteins are known to be a crucial process in the wound-healing cascade. Matrix metalloproteinases (MMP) are both responsible for ECM degradation and play an important role in remodeling and maintenance; however, often MMP levels are increased and dysregulated within chronic wounds. Collagen Wound Matrix-Micronized (CWM-MZ*) is an acellular, ECM-based biomaterial with a large surface area that increases contact with wounds. In this study, we characterized the biochemical and structural properties of CWM-MZ, inhibition of MMPs and proteases, and fibroblast attachment, proliferation, and migration using in vitro models.

Methods: Scanning electron microscopy (SEM) was used to show individual collagen fibrils and their respective D-period bands. Total collagen was quantified via hydroxyproline and Sircol Assays. Cell migration (scratch wound assay), adhesion, and proliferation were evaluated using primary fibroblasts cultured on surfaces coated with CWM-MZ, purified calf-skin collagen, or negative control (untreated). Total protease, collagenase, and gelatinase proteolytic activity were evaluated using gelatin or casein fluorescein-labeled substrates. MMP inhibition was evaluated using a fluorogenic substrate kit.

Results: SEM image analysis showed D-periods of 66.43 ± 6.40 nm in CWM-MZ, consistent with that of native collagen. CWM-MZ total collagen content was approximately 80.5% per mg of protein. We found that fibroblasts adhered and migrated at a higher capacity when cultured on the native collagen present in CWM-MZ compared to purified collagen. Fibroblasts proliferated more robustly on the CWM-MZ-coated surface as evidenced by the reduced doubling time (11 hours) compared to purified collagen and untreated surface (14 and 17.3 hours, respectively). We found that 10mg of CWM-MZ reduced total protease activity by 52% and collagenase/gelatinase activity by 82%. Additionally, CWM-MZ inhibited the activity of matrix metalloproteinases (MMPs)-1, -2, -8, and -9.

Discussion: CWM-MZ is comprised of collagen that retains the complex architecture and composition characteristic of native tissue. CWM-MZ significantly inhibits MMPs and proteases, which are often dysregulated in the chronic wound environment.