Specific sizes of hyaluronan oligosaccharides stimulate fibroblast migration and excisional wound repair

Abstract

The extracellular matrix polysaccharide hyaluronan (HA) plays a key role in both fibrotic and regenerative tissue repair. Accumulation of high molecular weight HA is typical of regenerative repair, which is associated with minimal inflammation and fibrosis, while fragmentation of HA is typical of postnatal wounds, which heal in the presence of inflammation and transient fibrosis. It is generally considered that HA oligosaccharides and fragments of a wide size range support these processes of adult, fibrotic wound repair yet the consequences of sized HA fragments/oligosaccharides to each repair stage is not well characterized. Here, we compared the effects of native HA, HA oligosaccharide mixtures and individual sizes (4-10 mer oligosaccharides, 5 and, 40 kDa) of HA oligosaccharides and fragments, on fibroblast migration in scratch wound assays and on excisional skin wound repair in vivo. We confirm that 4-10 mer mixtures significantly stimulated scratch wound repair and further report that only the 6 and 8 mer oligosaccharides in this mixture are responsible for this effect. The HA 6 mer promoted wound closure, accumulation of wound M1 and M2 macrophages and the M2 cytokine TGFβ1, but did not increase myofibroblast differentiation. The effect of 6 mer HA on wound closure required both RHAMM and CD44 expression. In contrast, The 40 kDa HA fragment inhibited wound closure, increased the number of wound macrophages but had no effect on TGFβ1 accumulation or subsequent fibrosis. These results show that specific sizes of HA polymer have unique effects on postnatal wound repair. The ability of 6 mer HA to promote wound closure and inflammation resolution without increased myofibroblast differentiation suggests that this HA oligosaccharide could be useful for treatment of delayed or inefficient wound repair where minimal fibrosis is advantageous.

Figure 1. 6 and 8mer HA fragments stimulate but HMW HA inhibits rat dermal fibroblast migration in modified scratch wound assays.

Migration of rat dermal fibroblasts was analyzed using Ibidi culture inserts as described in Material and Methods. A: Mixture of HA oligomeres significantly stimulate migration of rat dermal fibroblasts. Cells were treated with DMEM 1% FBS containing a HA oligomere mixture consisting of equal parts of 4mer, 6mer, 8mer and 10mer HA at a final concentration of 10 µg/ml. Cell migration was quantified by measuring the area of cells that migrated into the empty space after removal of the culture insert. Graph represents Mean±SE of N = 6 culture wells B: HMW HA inhibits migration of rat dermal fibroblasts. Cells were treated with DMEM 1% FBS containing 10 µg/ml 500 kDa HA. Cell migration was quantified by measuring the area of cells that migrated into the empty space after removal of the culture insert. Graph represents Mean±SE of N = 6 culture wells C: 6 and 8mer significantly stimulate migration of rat dermal fibroblasts. Cells were treated with DMEM 1% FBS containing either 4mer, 6mer, 8mer, 10mer, 5 kDa, 40 kDa HA at 1 µg/ml or PBS over night. Cell migration was quantified by measuring the area of cells that migrated into the empty space after removal of the culture insert. Graph represents Mean±SE of N = 4 culture wells.

Figure 2. 6mer HA fragments accelerate repair of full thickness excisional wounds.

Full thickness excisional wounds were treated with a mixture of Collagen I and either 6mer, 8mer or 10mer 40(1–50 µg/ml) or PBS as described in Material and Methods. A: Wound edges were traced and remaining wound area quantified by image analysis. Graph shows remaining wound area relative to original wound size on day 7 after wounding. Mean±SE of N = 6 wounds. B: Wound closure is completed 2 weeks after wounding. Representative images of 2 weeks old wounds that were stained with Masson’s Trichrome are shown.

Figure 3. 6mer HA significantly increases TGFβ1 accumulation but has no effect on collagen accumulation during wound repair.

A, B: Cross sections of 7 days old wounds were stained with TGFβ1 specific antibodies as described in Materials and Methods. Positive stained area was quantified by image analysis using ImageJ. Graph shows Mean±SE of N = 18 (6 sections, three areas/section). C: Cross sections of 7 days old wounds were stained with Masson’s Trichrome as described in Materials and Methods. Blue staining (Collagen) was quantified by image analysis using ImageJ. Graph shows Mean±SE of N = 18 (6 sections, three areas/section).

Figure 4. 6mer and 40 kDa HA moderately increase macrophage infiltration during wound repair.

A: Cross sections of 7 days old wounds were stained with INOS specific antibodies as described in Materials and Methods. Positive cells were counted/area granulation tissue. Graph shows Mean±SE of N = 18 (6 sections, three areas/section). B: Cross sections of 7 days old wounds were stained with ARG1 specific antibodies as described in Materials and Methods. Positive cells were counted/area granulation tissue. Graph shows Mean±SE of N = 18 (6 sections, three areas/section).

Figure 5. 6mer HA and 40 kDa HA has no significant effect on Smooth muscle actin or Tenascin C expression during wound repair.

A: Cross sections of 7 days old wounds were stained with Smooth muscle actin specific antibodies as described in Materials and Methods. Positive stained area was quantified by image analysis using ImageJ. Graph shows Mean±SE of N = 18 (6 sections, three areas/section). B: Cross sections of 7 days old wounds were stained with Tenascin C specific antibodies as described in Materials and Methods. Positive stained area was quantified by image analysis using ImageJ. Graph shows Mean±SE of N = 18 (6 sections, three areas/section).

Figure 6. RHAMM and CD44 are both required for stimulation of wound repair by 6mer HA.

Wild type (wt), CD44−/− and RHAMM−/− (Rh−/−) were wounded as described in Material and Methods. Wounds were treated with Collagen I +/−6mer HA as described in Material and Methods. Wound edges were traced and remaining wound areas quantified by image analysis. Graph shows remaining wound area relative to original wound size on day 5 after wounding. Mean±SE of N = 6 wounds.