Impaired wound healing in mice deficient in a matricellular protein SPARC (osteonectin, BM-40)

Abstract

Background: SPARC is a matricellular protein involved in cell-matrix interactions. From expression patterns at the wound site and in vitro studies, SPARC has been implicated in the control of wound healing. Here we examined the function of SPARC in cutaneous wound healing using SPARC-null mice and dermal fibroblasts derived from them.

Results: In large (25 mm) wounds, SPARC-null mice showed a significant delay in healing as compared to wild-type mice (31 days versus 24 days). Granulation tissue formation and extracellular matrix protein production were delayed in small 6 mm SPARC-null wounds initially but were resolved by day 6. In in vitro wound-healing assays, while wild-type primary dermal fibroblasts showed essentially complete wound closure at 11 hours, wound closure of SPARC-null cells was incomplete even at 31 hours. Addition of purified SPARC restored the normal time course of wound closure. Treatment of SPARC-null cells with mitomycin C to analyze cell migration without cell proliferation showed that wound repair remained incomplete after 31 hours. Cell proliferation as measured by 3H-thymidine incorporation and collagen gel contraction by SPARC-null cells were not compromised.

Conclusions: A significant delay in healing large excisional wounds and setback in granulation tissue formation and extracellular matrix protein production in small wounds establish that SPARC is required for granulation tissue formation during normal repair of skin wounds in mice. A defect in wound closure in vitro indicates that SPARC regulates cell migration. We conclude that SPARC plays a role in wound repair by promoting fibroblast migration and thus granulation tissue formation.

Figure 1

Healing of 25 mm oblong skin wounds (A) and histology of 6 mm circular skin wounds (B) in wild-type (+/+) and SPARC-null (-/-) mice. In (A) upper panel: an example of the progress of repair processes at the indicated times (days) is shown. S: scab; W: open wound field. In (A) lower panel: percent of wild-type and SPARC-null mice (6 in each group) with healed wounds, defined as loss of the wound scabs and complete covering of the wounds with epidermis, is plotted versus days after the excision. Panel (B) illustrates histologic appearance of 6-mm wounds in a wild-type (+/+) mouse and an SPARC-null (-/-) mouse 4 days after surgery. Granulation (G) tissue formation is more extensive in the wild-type mouse than in the SPARC-null mouse. Arrows indicate reepithelialized wound edge. Reepithelialization appears to be as efficient in mutant mice as in control mice. S: scab; P: panniculus carnosus. Hematoxylin and eosin staining. Scale bar, 210 μm.

Figure 2

Expression of fibronectin, α1(I) collagen and cytokeratin 14 (ck-14) transcripts (left panel) and levels of mRNA expression relative to that of GAPDH set at 100 (right panel) in wounds of wild-type (+/+) and SPARC-null (-/-) mice. Five 6 mm diameter wounds were made on the back of each mouse. Biopsies were collected from 3 wild-type and 3 SPARC-null mice 2, 4, and 6 days after the surgery. Skin from the backs of 2 unwounded wild-type and mutant mice (0 day) was collected and used as controls.

Figure 3

Phase-contrast photomicrographs showing the process of wound repair by wild-type (upper panel) and SPARC-null (lower panel) fibroblasts cultured in the absence of SPARC (A) and in the presence of SPARC (B). In (A), wild-type (upper panel) and SPARC-null (lower panel) fibroblasts were grown to confluence in DMEM containing 10% FBS and then wounded (example shown at 0 hour). Wound closure was monitored at various time points. Wound healing is delayed in SPARC deficient fibroblasts. In (B), wounded monolayers of wild-type (upper panel) and SPARC-null (lower panel) fibroblasts were cultured in DMEM containing 10% FBS and SPARC protein purified from human platelets. Exogenous SPARC restores wound healing ability of SPARC-null cells to the level of wild-type cells. Scale bar, 62.5 μm.

Figure 4

Wound repair by wild-type (A) and SPARC-null fibroblasts (B) cultured in the absence (upper panel) or presence (lower panel) of mitomycin C. Wounded monolayers of wild-type (A) and SPARC-null fibroblasts (B) were cultured in the absence (upper panel) or presence (lower panel) of mitomycin C and monitored for wound closure. Wound repair is not affected by the presence of mitomycin C, an inhibitor of cell mitosis. Scale bar, 62.5 μm.

Figure 5

Incorporation of ³H-thymidine by wild-type (+/+) and SPARC-null (-/-) dermal fibroblasts. Cells were plated and cultured overnight in DMEM containing 10% FBS. Cells were then washed with DMEM without FBS and incubated with ³H-thymidine in DMEM containing 10% FBS. Data were derived from two independent experiments each performed in triplicate. Bars show mean ± SD.

Figure 6

Contraction of collagen gels by wild-type (+/+) and SPARC-null (-/-) fibroblasts. Cells were incorporated in collagen gels and incubated in DMEM plus 2% FBS. Data represent the average of three independent experiments, each performed in triplicate.