Pathophysiology 13 (2006) 81–84

Subcutaneous low molecular weight heparin administrationpromotes wound healing in rats

Ewa Kus a,∗, Andrzej Bienkiewicz b

a Department of High Risk Pregnancy, Medical University of Lodz, Wilenska 37 Str. 94-029, Lodz, Polandb Department of Gynecologic Oncology, Chair of Oncology, Medical University of Lodz, Poland

Received 27 May 2004; received in revised form 26 January 2006; accepted 29 January 2006

Abstract

The influence of subcutaneous low molecular weight heparin (LMWH) compared with physiologic saline on the healing of abdominalwounds with and without prolene mesh was studied in rats. The collagen concentration was determined in the wound tissue and in prolenemesh 14, 21, 28 and 42 days after the skin incision in controls and in two groups of rats treated either with LMWH for 7 (I group) or 14(II group) days after skin incision and prolene insertion. LMWH administration resulted in a significant increase of collagen content both inw©

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ound and in prolene mesh in total dose-dependent manner.2006 Elsevier Ireland Ltd. All rights reserved.

eywords: Wound healing; Collagen; Prolene mesh; Low molecular heparin

. Introduction

It is known that wound healing depends on tissue revascu-arisation. The wound healing is, however, a complex chain ofiochemical and other events which contribute to the repair.he mechanisms of all these processes are not yet well eluci-ated. The influence of e.g. various agents on wound repaireed thus to be investigated [1,2]. One of the crucial eventsn wound healing is the formation of collagen fibers [3].eparin decreases the stricture formation after esophagealurns as reported by Bingol-Kologlu [4].Heparin reducesdhesions. Heparin also promotes in dose-dependent way thebroblast invasion into collagen implants. Those implantsre more vascular than the control wounds, and they pro-ote the wound healing [5]. On the other hand standard

eparin interferes the blood coagulation, and therefore it issed e.g. in surgery to prevent thrombotic complications inlderly people as well as in gynecological oncology and afteralve replacement. If, instead of standard heparin, the ratseceive low molecular weight heparin (LMWH) the bleeding

tendency is diminished [6]. The vast majority of experimentsconcerning the effects of low molecular weight heparin onhealing deal with the repair of bones, scull defects, tympanicmembrane, bowels, tendon degeneration, burns and otherproblems [7–16]. Beneficial influence of LMWH adminis-tration on gastric ulcer healing, not affecting the coagula-tion activity has been described by Li et al. [11]. Saba etal. [17] have demonstrated that platelet-derived growth fac-tor (PDGF) enhances wound closure by re-epithelialization,and it prevents of wound contraction in similar way asheparin.

The aim of the present study was to evaluate the influenceof long term anticoagulant treatment with LMWH on thehealing of the skin wounds with and without inserted prolenemesh by measuring the collagen contents.

2. Materials and methods

Three-month-old female Wistar rats (n = 60) were used.They received commercial pellets prepared by Bacutil

∗ Corresponding author. +48 42 686 83 80; fax: +48 42 686 04 71.E-mail address: ewakadr@interia.pl (E. Kus).

(Poland) and water ad libitum. Commercial LMWH(enoxaparinum natricum, Clexane, Bellon, Rhone-Poulenc

928-4680/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.

oi:10.1016/j.pathophys.2006.01.001

82 E. Kus, A. Bienkiewicz / Pathophysiology 13 (2006) 81–84

Roer, France) was used as an anticoagulant. The formal,written approval of Ethics Committee for the Experimentson Animals, Medical University, Lodz was obtainedbefore the experiments started. The 30 animals weredivided into three groups. Thirty additional rats served ascontrols.

Under general intraperitoneal anaesthesia (0.05 mg/kgbody weight, Thiopental, Biochemie GmbH, Vienna, Aus-tria) the skin of the rats was shaved in the dorso-lateralregion, and a 4 cm longitudinal full thickness skin incisionwas made. The prolene mesh (0.25 cm2 PMM1, Ethicon,Johnson-Johnson Intl.) was inserted subcutaneously. Sub-sequently, the wound was closed by 4 single stitches ofEthibond 2.0 sutures. The control rats were given physiologicsaline solution subcutaneously every 24 h for 7 (control groupI) or 14 (control group II) days, respectively. The experimen-tal group I received subcutaneously LMWH (1 mg/kg bodyweight) once a day for 7 days and the experimental group IIfor 14 days.

Rats were killed by an intraperitoneal thiopental adminis-tration 14, 21, 28 and 42 days after the wound incision. Skinfrom the wound area and the prolene mesh were removedand cleaned from the subcutaneous tissue for the analysis.The water content of the tissue was defined as the differencebetween the wet and dry weight (dried to the constant weightat 90 ◦C).

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to constant weight at 60 C. Collagen was then hydrolysed,and the collagen determination was carried out as describedabove.

For the soluble collagen assessment, tissue homogenateswere mixed with 0.45 M NaCl (pH 7.0). Crystalline penicillin(10000 IU/sample) was added to inhibit the bacterial growth,and the samples were incubated at 4 ◦C for 48 h. After thatthey were centrifuged and the supernatant was collected anddried for the collagen analysis as described above.

For the soluble collagen assessment in the prolene meshsamples (after the removal of the subcutaneous tissue) 0.45 MNaCl (pH 7.0) was added and the samples were incubated asdescribed above.

In the statistical analysis ANOVA and Kruskal-Wallis nonparametric tests were used.

3. Results and discussion

The LMWH administration increased the collagen con-tent in skin wounds. The differences were significant bothafter 7 and 14 day treatments. The results in group II werestatistically different from those obtained in group I only at3 and 6 weeks (Table 1A; Fig. 1A).

The LMWH administration also significantly increasedthe collagen contents in the prolene mesh placed in the woundaiicshge

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For the total collagen assessment the tissue samples10 mg) were extracted with ether and acetone (1:3), vacuumried and then hydrolysed with 6N HCl, and finally theolution was evaporated to dryness. Then distilled waternd 5N NaOH were added, until pH 7.0 was achieved,p to 10 ml. The hydroxyproline was assessed usingoessner’s method [18]. In short, the hydroxyprolineas oxidized to pyrrole by chloramine T and then let

o react with p-dimethylaminobenzaldehyde. The opticalensity was measured at 560 nm using a Unicam SP00 B spectrophotometer. Collagen concentration wasalculated by using a coefficient 8.2 (12.2% of collagens hydroxyproline). The collagen content in prolene meshamples was analyzed after the removal of subcutaneousissue as follows: the samples were weighted, vacuum dried

able 1ollagen concentration (�g/mg dry tissue)

eeks Control Group I

2 405 ± 26 519 ± 203 479 ± 38 566 ± 374 550 ± 81 609 ± 276 651 ± 109 736 ± 43

2 47.0 ± 6.8 71.2 ± 8.3 64.9 ± 9.0 87.1 ± 104 88.4 ± 7.9 105.8 ± 8.6 110.3 ± 7.6 136.8 ± 7.

: in the skin wound tissue and B: in the prolene mesh under the skin woul) and 14 (Group II) days compared to saline treatment. Results are mean ±

rea. In experimental group I, where the anticoagulant admin-stration had lasted for 7 days, the differences were observedn the wound till the 6th week of healing. Thereafter theollagen contents did not differ from that observed in thealine treated controls. In the group of animals, where LMWHad been administered for 14 days, the differences in colla-en concentration were statistically significant till the end ofxperiment (Table 1B; Fig. 1B).

The results showed thus a positive effect of low molecu-ar heparin administration on wound healing seen as in thencreased collagen contents. Li et al. [11] have shown thathe promotion of low molecular heparin on the healing ofastric ulcer was independent from the anticoagulant effect.he present results are in line with their studies indicatingetter fibroblast invasion into collagen implants by heparin

Group II p, I vs. C, II vs. I

532 ± 30 0.002, <0.001, 0.430632 ± 21 0.003, 0.001, 0.014670 ± 14 0.015, 0.091, 0.106,895 ± 113 0.025, 0.152, 0.026

102.1 ± 9.2 0.003, 0.001, <0.001127.5 ± 8.9 0.003, 0.002, <0.001138.5 ± 17.0 0.002, 0.029, 0.001272.7 ± 66.5 0.001, 0.188, <0.001

after low molecular weight heparin administration (1 mg/kg) for 7 (Group

E. Kus, A. Bienkiewicz / Pathophysiology 13 (2006) 81–84 83

Fig. 1. Collagen concentration (�g/mg dry tissue). Panel A: in the skinwound tissue and panel B: in the prolene mesh placed under the skin woundafter subcutaneous low molecular weight heparin administration (1 mg/kg)for 7 or 14 days compared to saline injections to the rats. Results aremean ± S.D. Collagen concentration (�g/mg dry tissue). Panel A: in thewound and panel B: in the prolene mesh after low molecular weight heparin(clexane) administration to the rats. Results are mean ± S.D.

[6]. The collagen formation in the prolene mesh seemed tobe stimulated as well.

The influence of heparin administration on wound healingin rats has been investigated by Cen et al. [9]. The results oftheir studies on experimentally burned rats, were similar ifthey received subcutaneously heparin as this caused a signifi-cant shortening of healing time. Our results on LMWH are inline with this observation. Similar effect has been observedby Kweon et al. [19] who used heparin/chitosan complexadministered topically. After15 days of treatment, the dor-sal full thickness skin excision was nearly completely healedcompared to the respective controls.

Neoangiogenesis seems to play a crucial role in woundrepair. The in vitro experiments reported by Zamora et al.[20] have shown a positive influence of heparin on capillarytube formation of endothelial cells.

Interesting data concerning the promotion of wound heal-ing have also been published also by Victor-Vega et al. [21].In their experiments adenosine A2A receptor antagonist top-ically applied accelerated significantly the wound closure.This effect was even more remarkable when compared to therecombinant human platelet derived growth factor admin-istered locally. The positive influence of platelet-derived

growth factor on wound repair even in ischemic conditionshas been demonstrated by Uhl et al. [22].

In conclusion, the subcutaneous administration of lowmolecular weight heparin increased collagen levels both dur-ing the repair process in the skin wounds as well as insubcutaneous prolene mesh.

Acknowledgement

The study was supported by the internal grant of MedicalUniversity of Lodz, Poland, No. 502-11-028

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