Chronic wounds remain a large problem in the field of medicine and are often associated with risk of infection and amputation. Recently, a commercially available human cryopreserved viable amniotic membrane (hCVAM) has been shown to effectively promote wound closure and reduce wound-related infections. A sprevious study indicates that hCVAM can inhibit the growth of bacteria associated with chronic wounds. In the present study, we investigated the mechanism of hCVAM antimicrobial activity. Our data demonstrate that antimicrobial activities against common pathogens in chronic wounds such as P.aeruginosa, S.aureus and Methicillin-resistant S.aureus (MRSA) are mediated via the secretion of soluble factors by viable cells in hCVAM and that these factors are proteins in nature. Further, we show that genes for antimicrobial peptides (AMPs) including human beta-defensins (HBDs) are expressed by hCVAM and that expression levels positively correlate with antimicrobial activity of hCVAM. At the protein level, our data indicate that HBD2 and HBD3 are secreted by hCVAM and directly contribute to its activity against P. aeruginosa. These data provide evidence that soluble factors including AMPs are hCVAM antimicrobial agents and are consistent with a role for AMPs in mediating antimicrobial properties of the membrane.
Background Human amniotic fluid (AF) contains numerous nutrients, trophic factors and defense proteins that provide a nurturing and protective environment for fetal development. Based on reports that AF has antibacterial, anti-inflammatory and regenerative properties, we designed a novel method to process AF for use in clinical care. Methods Six randomly selected lots of processed AF (pAF) were examined to determine whether they retained their antibacterial activity against a panel of wound-associated pathogens E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa , and E. aerogenes (ESKAPE). To identify proteins in pAF that might be responsible for its antibacterial activity, three different lots of pAF were analyzed with quantitative cytokine arrays that consisted of 400 unique human proteins. One protein identified by microarrays, lactoferrin, and a second prominent antibacterial protein that was not identified by microarrays, lysozyme, were examined by depletion experiments to determine their contribution to the antibacterial activity of pAF. Results All six lots of pAF exhibited antibacterial activity against ESKAPE microorganisms, especially against the pathogens predominately found in chronic wounds (i.e. S. aureus and P. aeruginosa ). Thirty-one of the peptides on the microarray were annotated as having antibacterial activity and 26 of these were detected in pAF. Cystatin C and lactoferrin were among the most highly expressed antibacterial proteins in pAF. Cystatin C and lactoferrin were confirmed by ELISA to be present in pAF along with lysozyme. Immunoprecipitation of lactoferrin and lysozyme reduced, but did not abolish the antibacterial activities of pAF. Conclusion Our data demonstrate that pAF maintains antibacterial activity via the preservation of antibacterial proteins against a broad spectrum of wound-associated pathogens.
Biofilm, a community of bacteria, is tolerant to antimicrobial agents and ubiquitous in chronic wounds. In a chronic DFU (Diabetic Foot Ulcers) clinical trial, the use of a human cryopreserved viable amniotic membrane (CVAM) resulted in a high rate of wound closure and reduction of wound-related infections. Our previous study demonstrated that CVAM possesses intrinsic antimicrobial activity against a spectrum of wound-associated bacteria under planktonic culture conditions. In this study, we evaluated the effect of CVAM and cryopreserved viable umbilical tissue (CVUT) on biofilm formation of S. aureus and P. aeruginosa, the two most prominent pathogens associated with chronic wounds. Firstly, we showed that, like CVAM, CVUT released antibacterial activity against multiple bacterial pathogens and the devitalization of CVUT reduced its antibacterial activity. The biofilm formation was then measured using a high throughput method and an ex vivo porcine dermal tissue model. We demonstrate that the formation of biofilm was significantly reduced in the presence of CVAM- or CVUT-derived conditioned media compared to control assay medium. The formation of P. aeruginosa biofilm on CVAM-conditioned medium saturated porcine dermal tissues was reduced 97% compared with the biofilm formation on the control medium saturated dermal tissues. The formation of S. auerus biofilm on CVUT-conditioned medium saturated dermal tissues was reduced 72% compared with the biofilm formation on the control tissues. This study is the first to show that human cryopreserved viable placental tissues release factors that inhibit biofilm formation. Our results provide an explanation for the in vivo observation of their ability to support wound healing.
Tissue regeneration often requires recruitment of different cell types and rebuilding of two or more tissue layers to restore function. Here, we describe the creation of a novel multilayered scaffold with distinct fiber organizations—aligned to unaligned and dense to porous—to template common architectures found in adjacent tissue layers. Electrospun scaffolds were fabricated using a biodegradable, tyrosine‐derived terpolymer, yielding densely‐packed, aligned fibers that transition into randomly‐oriented fibers of increasing diameter and porosity. We demonstrate that differently‐oriented scaffold fibers direct cell and extracellular matrix (ECM) organization, and that scaffold fibers and ECM protein networks are maintained after decellularization. Smooth muscle and connective tissue layers are frequently adjacent in vivo; we show that within a single scaffold, the architecture supports alignment of contractile smooth muscle cells and deposition by fibroblasts of a meshwork of ECM fibrils. We rolled a flat scaffold into a tubular construct and, after culture, showed cell viability, orientation, and tissue‐specific protein expression in the tube were similar to the flat‐sheet scaffold. This scaffold design not only has translational potential for reparation of flat and tubular tissue layers but can also be customized for alternative applications by introducing two or more cell types in different combinations.
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