Hyaluronan induces scarless repair in mouse limb organ culture

Wounded fetal mouse limbs harvested from two distinct time points in gestation heal differently in organ culture. The healing of a gestational day 14 limb is by scarless repair, whereas gestational day 18 (gd 18) limbs heal by scarring. The persistence of elevated levels of hyaluronic acid (HA) is a major difference in the extracellular matrix of scarless repair. The purpose of this study was to demonstrate that chronic additions of HA to incisional wounds of gd 18 limbs induces scarless repair.

Time-dated pregnant CD-1 mice (term, 20 days) were killed on gestational day 18 and fetuses were harvested via laparotomy. A through and through stab wound was made in each forelimb with a 1-mm microscapel, and the wound was closed with a single 10-0 nylon suture. The forelimbs were amputated at the level of the shoulder and placed in organ culture. Daily medium changes with 1 mL of BGJb (devoid of serum) were made. Half the cultures received 10 microL of HA (4 mg/mL) directly to the wound site with each medium change. The other half of the cultures received 10 microL of phosphate-buffered saline (PBS-control). At day 7, the limbs were harvested, fixed in methyl Carnoys solution, paraffin embedded, and 5-microm serial sections cut. The sections were stained with H&E or Sirius red/fast green. The sections were viewed in a blinded fashion by two observers. Suture defined the wound site, and the sections were graded for healing by scarring.

Minimal limb growth occurred in both control and HA-treated limbs. Grossly, both control and treated limbs healed incisional wounds by 7 days in culture. Limbs from both treatment and control groups showed viability by microscopic analysis. The limbs treated with HA had no appreciable scar morphologically in sections in which epithelial dimpling and suture were evident. The orientation of the collagen fiber bundles in the control wounds were in parallel arrays perpendicular to the incision. The orientation of the collagen fiber bundles in the HA-treated limbs had a basket weave pattern that was indistinguishable from unwounded dermis. The direct repeated additions of HA to healing organ cultured limb explants of gestational day 18 fetal mice promoted scarless repair.

This result demonstrates that chronic elevation of HA in the microenvironment of a wound affects healing by promoting the deposition of a more dermal-like connective tissue matrix in the wound site. The maintenance of elevated levels of HA could have utility in the clinical setting to improve the organization of connective tissue, leading to the reduction of scar complications.