A serum amyloid P-binding hydrogel speeds healing of partial thickness wounds in pigs

Abstract

During wound healing, some circulating monocytes enter the wound, differentiate into fibroblast-like cells called fibrocytes, and appear to then further differentiate into myofibroblasts, cells that play a key role in collagen deposition, cytokine release, and wound contraction. The differentiation of monocytes into fibrocytes is inhibited by the serum protein serum amyloid P (SAP). Depleting SAP at a wound site thus might speed wound healing. SAP binds to some types of agarose in the presence of Ca(2+). We found that human SAP binds to an agarose with a K(D) of 7 x 10(-8) M and a B(max) of 2.1 microg SAP/mg wet weight agarose. Mixing this agarose 1 : 5 w/v with 30 microg/mL human SAP (the average SAP concentration in normal serum) in a buffer containing 2 mM Ca(2+) reduced the free SAP concentration to approximately 0.02 microg/mL, well below the concentration that inhibits fibrocyte differentiation. Compared with a hydrogel dressing and a foam dressing, dressings containing this agarose and Ca(2+) significantly increased the speed of wound healing in partial thickness wounds in pigs. This suggests that agarose/Ca(2+) dressings may be beneficial for wound healing in humans.

Figure 1

Binding of human SAP to SP agarose. (A) Binding data were plotted and a one-site binding model (solid line) was fitted to the data. (B) The SAP concentration before and after adding a 1: 5 w/v ratio of agarose beads to the SAP solution. Values are mean ± SEM (n= 3). The absence of an error bar indicates that the error was smaller than the plot symbol. Arrow indicates an input concentration of 30 μg/mL SAP on the X axis (roughly the normal serum concentration), which the addition of agarose then decreased to ~0.02 μg/mL (Y axis). SAP, serum amyloid P.

Figure 2

Specificity of the high-affinity binding of human and porcine serum proteins to SP agarose. (A) Human and porcine sera were incubated with SP agarose, washed, and bound material was eluted and separated on an SDS-polyacrylamide gel which was then stained with Coomassie. Lanes are M, molecular mass markers (molecular masses in kDa are indicated at left); (1) 1 μg human SAP; (2) 0.3 μg human SAP; (3) 0.1 μg human SAP; (4) 0.03 μg human SAP; (5) 0.01 μg human SAP; (6) 0.003 μg human SAP; (7) 10 μL of the eluted material from human serum, (8) 10 μL of the eluted material from porcine serum. (B) A Western blot of the protein eluted from SP agarose was stained with anti-human SAP antibodies. H is the material from human serum; P is the material from porcine serum. The position of molecular mass markers (in kDa) is indicated at left. SAP, serum amyloid P.

Figure 3

Sections of wounds stained with hematoxylin and eosin. (A) A section of skin before wounding. (B) A section of an untreated wound at day 4. The *shows an area of crust. (C) An agarose in carbomer-treated wound at day 4. (D) An untreated wound at day 7. The arrow shows a region of epidermis under the crust. (E) An agarose in carbomer-treated wound at day 7. Scale bar is 0.5mm.

Figure 4

Detection of cytokeratin and collagen-I in day 10 wounds. (A–D) Cryosections were stained with anti-cytokeratin antibodies to show reepithelialization. (A) Normal skin, (B) day 10 untreated wound (the arrow shows a hair follicle), (C) agarose in carbomer-treated wound, and (D) IntraSite hydrogel-treated wound. (E–H) Sections were stained with anti-collagen-I antibodies to show dermal remodeling. (E) Normal skin, (F) day 10 untreated-wounds compared with wounds treated with (G) agarose in carbomer, or (H) IntraSite hydrogel. Scale bars are 0.2mm.