Antimicrobial activities of silver used as a polymerization catalyst for a wound-healing matrix

Abstract

Wound healing is a complex and orchestrated process that re-establishes the barrier and other functions of the skin. While wound healing proceeds apace in healthy individual, bacterial overgrowth and infection disrupts this process with significant morbidity and mortality. As such, any artificial matrix to promote wound healing must also control infecting microbes. We had earlier developed a two-part space-conforming gel backbone based on polyethyleneglycol (PEG) or lactose, which used ionic silver as the catalyst for gelation. As silver is widely used as an in vitro antimicrobial, use of silver as a catalyst for gelation provided the opportunity to assess its function as an anti-microbial agent in the gels. We found that these gels show bacteriostatic and bactericidal activity for a range of Gram-negative and Gram-positive organisms, including aerobic as well as anaerobic bacteria. This activity lasted for days, as silver leached out of the formed gels over a day in the manner of second-order decay. Importantly the gels did not limit either cell growth or viability, though cell migration was affected. Adding collagen I fragments to the gels corrected this effect on cell migration. We also found that the PEG gel did not interfere with hemostasis. These observations provide the basis for use of the gel backbones for incorporation of anesthetic agents and factors that promote wound repair. In conclusion, silver ions can serve dual functions of catalyzing gelation and providing anti-microbial properties to a biocompatible polymer.

Fig. 1

Silver leaches out of the gels over several days as determined by atomic absorption spectroscopy. (a) Upper graph shows cumulative silver concentration over a period of 10 days in broth in which the gels were immersed (one to three gels were tested). (b) Lower graph shows silver concentrations in the broth for tubes that were seeded between one and three gels and switched to fresh broth daily for 4 days.

Fig. 2

(a) UV spectrum of DOPA with maximal absorbance in aqueous solution at 280 nm. (b) The standard curve of DOPA was obtained by the determination of OD at 280 nm and the correlation coefficient of the standard curve was 0.9958. (c) Degradation of various DOPA-containing polymer gels in PBS. The polymer gels were incubated in PBS for 3 weeks and the degradation was tested by the concentration of DOPA released from the polymer gel into the solution.( PEG SG-solid gel with PEG, LDI, DOPA, Ag and peroxydiphosphate; LAC LG- liquid gel with lactose, LDI, DOPA, Ag and peroxydiphosphate).

Fig. 3

(a) Swelling property of the gels as a function of time at 37 °C. (b) Change in diameter of the gels as a function of time at 37 °C. (PEG SGPEG, LDI, DOPA, Ag, peroxydiphosphate; LAC SG-lactose, LDI, DOPA, Ag, peroxydiphosphate).

Fig. 4

Effect of gels on human dermal fibroblasts: (a) cell viability and (b) cell proliferation. Cells were exposed to gels (PEG SG-solid gel with PEG, LDI, DOPA, Ag, and peroxydiphosphate; PEG LG-liquid gel with PEG, LDI, and DOPA; LAC SG-solid gel with lactose, LDI, DOPA, Ag, and peroxydiphosphate; LAC LG-liquid gel with lactose, LDI, and DOPA) after 48 h in serum-depleted media. Cell viability and proliferation were assessed at 24, 48 and 72 h. Values are expressed as mean ±SEM (n = 3). NS = not significant, *p<0.05, **p<0.001 compared to diluent alone (No tx-No treatment).

Fig. 5

Effect of gels on human keratinocytes: (a) cell viability and (b) cell proliferation. Cells were exposed to gels with composition as described in previous figure legend after 48 h in serum-depleted media. Cell viability and proliferation were assessed at 24, 48 and 72 h. Values are expressed as mean ±SEM (n = 3). NS = not significant, * = p<0.05 compared to diluent alone (No tx).

Fig. 6

Effect of gels on (a) human dermal fibroblast and (b) keratinocyte cell motility. Gels composition was as described in previous figure legends. Cells were exposed to gels after 48 h in serum depleted media and motility assessed by an in vitro wound healing assay. Values are expressed as mean ±SEM (n = 3), *p<0.05 compared to diluent alone (No tx).

Fig. 7

Effects of individual components of gel on (a) human dermal fibroblast and (b) keratinocyte cell motility. The cells were exposed to gels after 48 h treatment in serum depleted media and motility assessed by an in vitro wound healing assay. Values are expressed as mean ±SEM (n = 3), NS = not significant, *p<0.05, **p<0.01 compared to diluent alone (No tx).

Fig. 8

Effect of gels with collagen on (a) human dermal fibroblast and (b) keratinocyte cell motility. Cells were exposed to gels. PEG SG-solid gel having PEG, LDI, DOPA, Ag, and peroxydiphosphate; PEG LG-liquid gel having PEG, LDI, and DOPA; both with collagen after 48 h treatment in serum depleted media. Cell motility was assessed by in vitro wound healing assay. Values are expressed as mean±SEM (n = 3).

Fig. 9

Effect of gels on (a) clotting time (b) prothrombin time and (c) partial thromboplastin time as assessed by the tube method. Gel composition was as described previously. Values are expressed as mean±SEM (n = 3), *p<0.05,**p<0.01. Bars above the graphs indicate the groups compared.