Abstract: BACKGROUND: Clinically, drugs such as povidone-iodine cream and fusidic acid cream are commonly used to control infection in infected skin wounds, but regulation of the wound microenvironment, such as inflammation, is neglected. Therefore, it is necessary to develop biomaterials that are effective in antibacterial and can modulate inflammation to promote skin healing.
OBJECTIVE: To investigate the antibacterial and anti-inflammatory properties of the vancomycin-porcine acellular extracellular matrix hydrogel, as well as its therapeutic effects on the healing of infected skin wounds.
METHODS: (1) Porcine skin acellular extracellular matrix hydrogels containing 0, 1, 2, and 4 mg/mL vancomycin were prepared and designated PAEH, VA-PAEH1, VA-PAEH2, and VA-PAEH4, respectively. The drug encapsulation efficiency and drug release properties of each hydrogel were tested in each group. Mouse embryonic fibroblasts (NIH-3T3) were co-cultured with the four groups of hydrogel extracts. The cytotoxicity of the hydrogels was assessed by live-dead staining. Mouse embryonic fibroblasts (NIH-3T3) were co-cultured with the four groups of hydrogel extracts. Cell viability was assessed by CCK-8 assay. The four groups of hydrogels were co-cultured with rat red blood cell suspensions to detect the hemolysis rate. Methicillin-resistant Staphylococcus aureus was co-cultured with the four groups of hydrogels to detect the antibacterial properties of the hydrogels. Based on the above experimental results, the VA-PAEH1 hydrogel with excellent performance was screened for subsequent experiments. The micromorphology, swelling properties, degradation properties and rheological properties of the VA-PAEH1 hydrogel were characterized. (2) PAEH hydrogel, VA-PAEH1 hydrogel, and chitosan hydrogel were co-cultured with methicillin-resistant Staphylococcus aureus (or Escherichia coli) respectively. The antibacterial properties of the hydrogels were evaluated by agar plate coating test, inhibition zone test, and scanning electron microscopy. Mouse mononuclear macrophage RAW264.7 cells were cultured in five groups. A blank group received no treatment. A control group received lipopolysaccharide to induce an inflammatory response. The remaining three groups received lipopolysaccharide for 24 hours followed by the addition of PAEH hydrogel, chitosan hydrogel, and VA-PAEH1 hydrogel, respectively. After an additional 24 hours of culture, immunofluorescence staining for CD86 (a marker of M1 macrophages) and CD206 (a marker of M2 macrophages) was performed. PAEH hydrogels, chitosan hydrogels, and VA-PAEH1 hydrogels were co-cultured with mouse embryonic fibroblasts NIH-3T3 cells in a non-contact manner. Cell proliferation was assessed by EdU staining. Cell migration was assessed by wound healing assay and Transwell assay. (3) A 12 mm diameter full-thickness skin defect was created on the back of 28 SD rats. Methicillin-resistant Staphylococcus aureus solution was then dripped onto the rats to simulate infection. The rats were randomly divided into four intervention groups. A control group (n=7) received normal saline injection, while the PAEH group (n=7), chitosan hydrogel group (n=7), and VA-PAEH1 group (n=7) received injections of the corresponding hydrogels. Wound healing was observed. Skulls were harvested 14 days postoperatively for hematoxylin-eosin and Masson staining, and 3 days postoperatively for myeloperoxidase and CD206 immunohistochemical staining.
RESULTS AND CONCLUSION: (1) The drug encapsulation efficiencies of VA-PAEH1, VA-PAEH2, and VA-PAEH4 hydrogels were 98.34%, 98.15%, and 97.68%, respectively. All three kinds of hydrogels exhibited good sustained drug release, but the cumulative drug release from the VA-PAEH2 and VA-PAEH4 hydrogel might pose a safety risk. CCK-8 assay and live/dead staining revealed that PAEH and VA-PAEH1 hydrogels exhibited no significant cytotoxicity and demonstrated good cytocompatibility. Hemolysis assay revealed that the hemolysis rates of PAEH and VA-PAEH1 hydrogels were within a safe range, demonstrating good hemocompatibility. VA-PAEH1, VA-PAEH2, and VA-PAEH4 all exhibited excellent antibacterial properties. VA-PAEH1 hydrogels were composed of interwoven fibers and exhibited excellent swelling, degradation, and rheological properties. (2) Agar plate coating experiments, inhibition zone assays, and scanning electron microscopy revealed that VA-PAEH1 hydrogels effectively inhibited the growth and reproduction of methicillin-resistant Staphylococcus aureus and Escherichia coli compared with PAEH and chitosan hydrogels. Compared with chitosan hydrogels, PAEH and VA-PAEH1 hydrogels promoted the transformation of macrophages from the M1 to M2 phenotype, demonstrating excellent anti-inflammatory properties. Compared with chitosan hydrogels, PAEH and VA-PAEH1 hydrogels promoted NIH-3T3 cell proliferation and migration. (3) Compared with the other three groups, the VA-PAEH1 group showed the fastest wound healing. Hematoxylin-eosin and Masson staining revealed that the VA-PAEH1 group had less inflammatory cell infiltration, denser collagen tissue, and the highest wound healing quality. Myeloperoxidase immunohistochemical staining showed that VA-PAEH1 hydrogel significantly reduced bacterial-induced inflammatory cell infiltration compared with PAEH and chitosan hydrogels. CD206 immunohistochemical staining showed that VA-PAEH1 hydrogel increased the proportion of M2 macrophages compared with PAEH and chitosan hydrogels, promoting wound healing. 

Key words: infected skin wound, acellular extracellular matrix, hydrogel, anti-infection, anti-inflammatory, tissue repair, bioengineered material