Iocono J A, Ehrlich H P, Keefer K A, Krummel T M
Department of Surgery, Milton S. Hershey Medical Center, Hershey, PA 17033-0850, USA.
J Pediatr Surg. 1998 Apr;33(4):564-7. doi: 10.1016/s0022-3468(98)90317-7.
BACKGROUND/PURPOSE: 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.
背景/目的：从妊娠两个不同时间点采集的受伤胎鼠肢体在器官培养中的愈合情况不同。妊娠第14天肢体的愈合方式为无瘢痕修复，而妊娠第18天（gd 18）肢体的愈合方式为瘢痕形成。透明质酸（HA）水平持续升高是无瘢痕修复的细胞外基质的一个主要差异。本研究的目的是证明向gd 18肢体的切口创面长期添加HA可诱导无瘢痕修复。
在妊娠第18天处死定时妊娠的CD-1小鼠（孕期20天），通过剖腹术取出胎儿。用1毫米显微手术刀在每个前肢上做一个贯通的刺伤伤口，并用一根10-0尼龙缝线缝合伤口。在前肢肩部水平处截肢，并置于器官培养中。每天更换1毫升不含血清的BGJb培养基。一半培养物在每次更换培养基时直接向伤口部位加入10微升HA（4毫克/毫升）。另一半培养物加入10微升磷酸盐缓冲盐水（PBS对照）。在第7天，取出肢体，固定于甲基卡诺氏液中，石蜡包埋，切成5微米连续切片。切片用苏木精和伊红（H&E）或天狼星红/固绿染色。由两名观察者以盲法观察切片。缝线确定伤口部位，对切片的瘢痕愈合情况进行分级。
对照肢体和HA处理的肢体均有最小程度的生长。大体上，对照肢体和处理过的肢体在培养7天时均愈合了切口创面。通过显微镜分析，治疗组和对照组的肢体均显示有活力。在有明显上皮凹陷和缝线的切片中，用HA处理的肢体在形态上没有明显瘢痕。对照伤口中胶原纤维束的方向呈平行排列，垂直于切口。用HA处理的肢体中胶原纤维束的方向呈篮状编织模式，与未受伤的真皮难以区分。向妊娠第18天胎鼠愈合的器官培养肢体植块直接反复添加HA可促进无瘢痕修复。
该结果表明，伤口微环境中HA的长期升高通过促进伤口部位沉积更类似真皮的结缔组织基质来影响愈合。维持HA的高水平在临床环境中可能有助于改善结缔组织的结构，从而减少瘢痕并发症。
