The Influence of Early Postoperative Intraperitoneal Chemotherapy onHuman Wound Healing

WILHELM GRAF, M.D., PH.D.,*,l MIKAEL IvARssoN, PHARM.D., t BENGT GERDIN, M.D., PH.D.,*KRISTOFFER HELLSING, M.D., PH.D.,:j: LARS PAHLMAN, M.D., PH.D.,* ANO BENGT GLIMELIUS, M.D., PH.D.§

Departments o{ .Surgery, :l:Clinical Chemistry, and §Oncology, Akademiska sjukhuset, and the tDepartment o{ Medical and PhysiologicalChemistry, Biomedical Centre, Universityo{ Uppsala, 751 85 Uppsala, Sweden

Submitted far publicatian March 1, 1993

Cell ingrowth, hydroxyproline accumulation, andmRN A expression of collagen 1 were measured in two

polytetraftuoroethylene grafts implanted subcutane-ously at the time of colorectal cancer surgery to evalu-ate tbe inftuence of early postoperative cbemotherapyon buman wound healing. Eleven patients treated with

intraperitoneal 5-ftuorouracil and intravenous folinicacid Days 1-6 after operation were compared with 15patients wbo underwent surgcry alone. At 1 wcek, cbc-

motherapy-treated paticnts had accumulatcd less by-droxyproline (mean 0.35 :t 0.33 p.gfcm) comparcd withuntreated patients (mean 0.73 :t 0.37 p.gfcm, P < 0.05).By 2 wecks, the hydroxyprolinc content bad increasedsixfold in thc chemotherapy group (P < 0.01) andthrcefold in thc nonchcmothcrapy group (P < 0.01) andthere was no difTcrencc bctwccn the groups. Ccll andconnectivc tissuc ingrowth and total RNA content didnot difTcr betwecn the groups at any point in time, butat 1 week thc mRN A cxprcssion of collagen 1 washigbcr in the chemothcrapy group (P < 0.05). Theseresults indicate that collagen accumulation in humansubjects is reduced during a short course of postopera-tive cbemotherapy and normalizes after the end oftreatment. (!;) 1994 Acodcmic Prcss. loco

toneal administration appears to impair postoperativecollagen synthesis [7]. Although numerous animal stud-ies have shown that 5-FU inhibits wo1.Jnd healing, it isnot known if there is an analogous effect in human sub-jects treated with clases commonly used in adjuvant ther-apy. Such knowledge would be valuable for the applica-tion of results from animal studies to humans and hencefor assessing the risks connected with early postopera-tive chemotherapy.

This study was undertaken to analyze the efrects ofearly postoperative intraperitoneal 5-FU infusion onhuman wound healing through comparing treated pa-tients with those submitted to surgery alone with respectto cell migration and collagen accumulation in a stan-dardized subcutaneous wound.

PATIENTS ANO METHOOS

Patients

Surgical morbidity and tolerance to adjuvant intraper-itoneal chemotherapy were studied in a randomizedmulticenter phase 1 trial involving patients not olderthan 75 years who were scheduled for a colorectal cancerresection with curative intent between February 1990and April 1992. Fifty patients were randomized to re-ceive intraperitoneal 5-FU and intravenous folinic acid,and 51 to receive placebo, in a double-blind manner. Fo-linic acid was given with the aim of improving the anti-tumor activity of 5-FU [8]. The active treatment con-sisted of 500 mg/m2/day 5-FU (Cyanamid Nordiska AB,Stockholm, Sweden) as an intraperitoneal infusiongiven over a 30-min period and 60 mg/m2/day folinicacid (Leucovorin, Cyanamid Nordiska AB) as an intrave-nous infusion lasting 5-10 min and commencing 1 hrafter the start ofthe intraperitoneal infusion. The 5-FUwas dissolved in 500 mI of 0.9% NaCI and the Leuco-vorin in 100 mI of 0.9% NaCI. Patients in the placeboarm received only the vehicle according to the sameschedule. The treatment started on the day after surgery

INTRODUCTION

Adjuvant intraportal 5-fluorouracil (5-FU) infusionhas been associated with an improved survival after cur-ative surgery for colorectal cancer [1]. Intraperitonealadministration has been suggested as an alternative tointraportal infusion to expose algo peritoneal and localtumor remnants to the cytotoxic drug [2]. However, sev-eral experimental studies have shown that 5-FU reducesthe strength of surgical wounds [3-+5], most likely due toreduced collagen formation [5,6]. Particularly, intraperi-

ITo whom correspondence and reprint requests should be addressed.

3940022-4804/94 $5.00Copyright @ 1994 by Academic Press, lnc.AII rights of reproduction in any form reserved.

GRAF ET AL.: CHEMOTHERAPY AND WOUND HEALING 395

The grafts were cut into 7 -cm lengths and sterilized in anautoclave. During anaesthesia for colorectal cancel sur-gery, the extensor part of the right upper arm wascleaned with chlorohexidine and wiped dry. A small stabwound was made with a surgical blade approximatelyhalfway between the elbow and shoulder. A 2 X 50-mmcrista biopsy needle (Cameco AB, Tiiby, Sweden) wasinserted subcutaneously, with the tip emerging at a dis-tance large enough to allow the graft to be placed instraight position. The tlachar was removed and a guidewire with the graft threaded on it was inserted throughthe needle, and while the graft was held with a forceps,first the guide wire and then the needle were withdrawn.Two 7 -cm grafts were placed parallel to each other ap-proximately 3-cm aparto One centimeter of the graftprotruded outside the ski n and was fixed with a suture.The grafts were covered with a sterile dressing. Thismethod was originally described by Goodson and Hunt[9]. One graft was extracted after 1 wliek and the otherafter 2 weeks. After extraction, the grafts were divided ina standardized fashion; the innermost 8 mm was takenfor histology, the next 20 mm for RNA extraction, andthe next 30 mm for determination of hydroxyproline; thelast part, the 1 cm outside the skin and a further 2 mm,was discarded. The pieces were immediately measured tothe nearest millimeter, weighed, and thereafter frozen inliquid N2 and stored at -70°C until analysed.

TABLEl

Patient Characteristics

Chemotherapy Nonchemotherapy(n = 11) (n = 15)

5:666 (52-75)

11:462 (41-75)

155O

2742

22

2131

2118

3

Men:womenMean age (range)Dukes' stage

ABCD.

SurgeryRight hemicolectomyTransverse resectionLeft hemicolectomySigmoid resectionSub total colectomyAnterior resectionAbdominoperineal resection

.Metastatic disease.

and continued for 6 consecutive days with a break onSundays.

Twenty-two patients treated within the trial at ourdepartment were included in the present study. Also in-cluded in the study were four patients eligible preopera-tively for the randomized trial, but in whom exclusioncriteria were found at surgery (in two cases metastaticdisease and in two suspected Dukes',A lesions), giving atotal of 11 patients treated with chemotherapy postoper-atively and 15 undergoing surgery alone (1'able 1).

The average (:tSD) preoperative weight loss was 2.9 :t4.3 in the chemotherapy group and 2.5 :t 2.9 in the non-chemotherapy group. The corresponding values for bodymass index (body weightflength2) and S-albumin were24.8 :t 5.0 and 23.9 :t 3.7 and 39 :t 4.9 and 41 :t 3.4,respectively. There was no indication of a difference inimmunocompetence between the groups as reflected bypreoperative hematological values (data not shown). Nei-ther did variables reflecting the surgical trauma (i.e.,operating time, preoperative bleeding, and number ofunits of blood transfused) differ between the groups(data not presented). During the conduct of the analy-ses, the investigators had no knowledge about the ran-doro allocation of the patients in the trial but knewwhich patients were outside the trial. The studywas ap-proved by the regional ethics committee and informedconsent was obtained from all patients.

Hydroxyproline As.s-ay

The hydroxyproline content was measured as de-scribed by Stegemann and Stalder [10]. Briefiy, the graftwas cut into 2-mm pieces and hydrolyzed in 1 mI HCI 6mole/liter at 110°C for 21 hr. The hydrolysate was eva-porized to dryness and dissolved in 500 11.1 deionizedwater. Buffer was added to a volume of 2 mI. The samplewas then incubated with 1 mI chloramine T at room tem-perature for 20 mino One milliliter of aldehyde/perchlo-ric acid solution was then added, and the admixture wasshaken thoroughly and immersed in a 60°C water bathfor 15 mino After cooling, the absorbance was read in aspectrophotometer at 550 nm. To assess the variation inthe hydroxyproline content along the graft, determina-tions based on two different parts of the 30-mm graftsegment were performed in 35/52 measurements. Therewas a close correlation between the two determinations(r = 0.93, P < 0.0001) (Fig. 1). The total amount of hy-droxyproline per totallength of graft (i.e., the weightedmean) was used in the presentation of results. Thecompositions of the various solutions are detailed else-where [10].

Technique

Polytetratluoroethylene (PTFE) grafts, 1 mm in diam-eter and with a 90-}J.m pore size, were purchased fromGore, Swedish AB (Molndal, Sweden). The weightflength ratio was close to constant (0.46 ::!: 0.03 mgfmm).

RNA Analyses

Total RNA was extracted by the acid-guanidinium-phenol-chloroform method [11] and quantitated by spec-trophotometry at 260 nm, and the results were expressed

396 JOURNAL OF SURGICAL RESEARCH: VOL. 57, NO. 3, SEPTEMBER 1994

5.0 tometry of autoradiographs from the hybridized dot blotfilters, a pro a1(I)/oligo(dT) index was created for eachsample and these figures were compared for relative val-ues ofpro a1(I) mRNA levels. Absolute levels ofmRNAexpression of collagen 1 were calculated only in the 14patients evaluated with the oligo(dT) method, but rela-tive changes were calculated for all patients.

x

xx~

'Eti

DI

~11

.: 2.5~

x/

H istological Examinations

After fixation in 10% formaldehyde overnight, thegrafts were dehydrated in ethanol and embedded in theplastic medium JB4 (Bio-Rad, Richmond, CA), and 2- to3-ILm thick sections were cut 10ngitudinaUy. A hematox-ylin-eosin-stained coded section was inspected for ceUand connective tissue ingrowth (Figs. 2 and 3). A quanti-fication of the degree of invasion of tissue into the poreswas performed after identification of the fibroblast-con-taining organized granulation tissue and for each poredescribed as the location ofthe edge ofllthis compartment(arrowhead in Fig. 3). This was estimated to be O, 25, 50,75, or 100% of the totallength of the pore. The mean of40 randomly selected transversaUy sectioned pores wasgiven for each patient and each time.

~

x xXxx

x x

¿-0.0 J .I .I ., .I .I .I .I .I .I .I

0.0 2.5 ~.o

-1HydroHyproline (~g C" )

FIG. l. Determination of hydroxyproline content (Jlg/cm) in twoadjacent parts of the polytetrafluoroethylene graft.

as /lg/cm. Serial dilutions of formaldehyde-denaturedRNA were dot blotted anta nylon membranes (Amer-sham, UK) with the aid of a vacuum chamber device(Bio-Rad, Richmond, CA), fixed on a bed of 0.05 mole/liter NaOH, and washed briefty in standard saline phos-phate EOTA (0.30 mole/liter NaCl, 20 mmole/liter,NaH;¿PO4' 2 mmole/liter EOTA, pH 7.4). The mem-branes were then subjected to hybridization with a 372-bp cONA probe corresponding to a conserved regían ofthe collagen al(I) chain (a gift from Dr. E. Vuorio, Uni-versity of Turku, Finland). The probe was labeled with32p by the random priming method (Amersham, UK).Autoradiographs were evaluated by laser densitometry.As an internal reference for the mRNA measurements,two different methods were used. Initially, the glyceral-dehyde-3-phosphate dehydrogenase (GAPOH) methodwas employed in which the membranes were reprobedwith a 700-bp cDNA probe corresponding to theGAPDH enzyme ofthe glycolysis, labeled exactly in thesame way. This probe served as a positive control and asa reference for comparison ofthe different RNA samplesin the densitometry assay, as it is considered to be aconstitutively expressed gene producto In the later partofthe study (n = 14), the oligo(dT) method was used inwhich mRNA was semiquantitated as described by Har-ley [12]. Briefty, RNA was dot blotted anta a nitrocellu-lose filter and subsequently hybridized with a 32p end-Ia-beled 0Iigo(dT)18 probe which detects total mRNA bybinding to its poly(A) tail. The oligo(dT) 18 was removedby stringent washing and the filter was finally exposedto the collagen pro al(I) cDNA probe. After laser densi-

Stati,-;tical Method.'i

Results are presented as means ::t standard deviation.Statistical comparisons between the groups were roadewith the Mann-Whitney U test and temporal changeswithin the groups with the Wilcoxon signed rank test.All presented P values are two-tailed.

RESULTS

Postoperatiue Course

Anastomotic dehiscence was diagnosed in one patientin each group, one of whom required a second laparot-omy. Furthermore, two patients treated with 5-FU expe-rienced surgical complications; one had severe localizedabdominal pain and rebound tenderness, which resolvedspontaneously, a condition judged as chemical peritoni-tis [13], and the other had a perineal infection after anabdominoperineal resection. A leukopenia was observedin one patient in the chemotherapy group (white bloodcell count 1.1 X 109jliter on the third day of treatment).In all other patients the postoperative course was un-eventful. Among the 11 patients treated with chemother-apy, 8 received the intended six treatments, whereas itwas terminated on Days 2, 3, and 4 in the remainingthree patients.

Graft Weights and Hydroxyproline

No signs of infection appeared around the grafts andremoval was always easy. At 1 week, the graft weight was1.43 :t 0.22 mg/mm in the chemotherapy group and 1.52

GRAF ET AL.: CHEMOTHERAPY AND WOUND HEALING 397

¡.'IG.2. Longitudinnl Rcction 01' III)olytctrnfluorocthylcnc I{rnl't removed IIl'tcr ] wcck, Rhowinl{ nn nvcrllgc ofO% ccllnnd conncctivc tissue

ingrowtll (62..5X mngnilicntion).

:!: 0.28 mg/mm in the nonchemotherapy group (P >0.10). By two weeks the corresponding figures were 1.65:!: 0.34 and 1.69 :!: 0.23 mg/mm, respectively (P > 0.10).The difference ayer time was significant in both groups(P < 0.05). By 1 week, the hydroxyproline content in thechemotherapy group was lower than that in the nonche-motherapy group (P < 0.05). This was algo true whenhydroxyproline accumulation was expressed as concen-tration (P < 0.05, Table 2). Compared with 1 week, amarked increment in hydroxyproline accumulation hadoccurred at 2 weeks in both groups (P < 0.01), when itwas approximately equal in the two groups (Table 2).

of collagen 1 was 0.70 :t 0.40 in the chemotherapy group(n = 7) and 0.28:t 0.19 in the nonchemotherapy group (n= 7) (P < 0.03). By 2 weeks the corresponding figureswere 0.88 :t 0.64 and 0.38 ::!: 0.27, respectively (P = 0.10).The relative change in mRNA expression oí collagen 1was 223 ::!: 213% in the chemotherapy group and 191 :t165% in the nonchemotherapy group (P> 0.10).

Histology

There was no difference in the average percentage oíingrowth between the groups at either measurementtime (P > 0.10) with values oí 5.5 :t 7.8 and 5.8:t 8.8% at1 week and 17.1 :t 12.6 and 19.8:t 13.1 % at 2 weeks in thechemotherapy and nonchemotherapy groups, respec-tively. The increase between 1 and 2 weeks was statisti-cally significant in both groups (chemotherapygroup P< 0.05; nonchemotherapy group P < 0.01).

RNA Analyses

The total RNA content at 1 week was 5.64 :t 2.02Jl,gfcm in the chemotherapy group and 6.17 :t 4.26 Jl,gfcmin the nonchemotherapy group (P > 0.10). At 2 weeksthe corresponding values were 7.53 :t 3.13 and 10.7 :t5.24 Jl,gfcm, respectively (P > 0.10). The difference inRNA content between 1 and 2 weeks was significant inthe nonchemotherapy group (P < 0.01), but not in thechemotherapy group. By 1 week, the mRNA expression

DISCUSSION

A large number of studies have explored the effects ofchemotherapy on wound healing in experimental ani-

398 JOURNAL OF SURGICAL RESEARCH: VOL. 57, NO. 3, SEPTEMBER 1994

FIG. 3. Longitudinal section oí a polytctranuoroethylcnc grnft removed aítcr 2 wcck!l, showing IIn IIvcrllgc oí 25% ingrowth. Thc nrrowindicntcs thc cdgc oí thc organized fibroblll!lt- IInd conncctivc ti!l!luc-contllining compllrtrncnt (G2.I>X mllgniliclltion).

mals, but this is, to the best of our knowledge, the firststudy of this issue in human subjects. A standardizedsubcutaneous wound was employed since it is unethical

TABLE2

Hydroxyproline Content (¡¡.g/cm) and Concentration(¡¡.g/mg) at 1 and 2 Weeks

Chemotherapy Nonchemotherapy(n = 11) (n = 15)

0.35 :t 0.332.25 :t 1.12**

0.73 :f: 0.37.2.38 :f: 1.24..

0.025:!: 0.0200.142 :!: 0.065**1.90 :!: 1.010.118:!: 0.064

0.051 :0.151 :1.65 :0.101 :

to biopsy a newly constructed anastomosis in humans.The subcutaneous wound and the anastomosis wereboth exposed to 5-FU in the systemic circulation, but thelatter site might algo have been exposed to local 5-FUpenetration [14]. As far as the anastomosis is concerned,this model is therefore likely to reflect a "minimum"level of influence. In this study, hydroxyproline was de-termined as a marker of collagen [15]. In contrast totissue biopsies, hydroxyproline in the PTFE graft repre-sents only "de novo" collagen and is therefore poten-tially better suited as a marker of collagen synthesis.Histology and total RNA content might be viewed asindicative of the amount of cells in the graft. Finally,mRNA expression of collagen I shows the cellular signalfor collagen formation. The variables in this study thusreflect basic events in wound healing, and a defect in anyof them might theoretically lead to serious complica-tions.

The present numerical values of hydroxyproline con-tent were in clase proximity to those observed in a pre-vious study, where the hydroxyproline accumulationafter 1 week was 0.45 ILg/cm in control subjects and 0.25ILg/cm in "debilitated" patients [9]. Trauma [16], a briefpreoperative illness [17], chronic uremia [18], and poor

Hydroxyproline content1 week2 weeks

Hydroxyproline concentration1 week2 weeks

~ Content~ Concentration

Note. The in crease in hydroxyproline content (delta content, JJg/cm) and concentration (delta concentration, JJg/mg) is alBo shown.

.Difference between groups, P = 0.02 (Mann-Whitney U test)...Difference between 1 and 2 weeks, P < 0.01 (Wilcoxon signed

rank test).

t

0,022-t

0.074--t

1.23

1: 0.083

GRAF ET AL.: CHEMOTHERAPY AND WOUND HEALING 399

ACKNOWLEDGMENTS

This study was supported by grants from the Swedish Cancer Soci-ety, Project Nos. 1921-B90-07XA and 2729-B91-02XAC, The Swed-'ish Medical Research Council, Project No. 09102, and The Lions Fundfor Cancer Research at Akademiska sjukhuset.

REFERENCES

oxygenation [19] have been associated with depressedcollagen accumulation in humans, whereas protein defi-ciency negatively influenced collagen deposition in ex-perimental animals [20].

Hydroxyproline accumulation in the chemotherapygroup was reduced to 50% of the control level after 1week. Because of the larger proportion of women in thechemotherapy group, separate analyses were made ac-cording to gender. It was found that the men and thewomen in the chemotherapy group showed a similar re-duction in the hydroxyproline level compared with thecorresponding sex in the nonchemotherapy group (datanot shown), suggesting that the sex imbalance could notexplain the difference. Separate analyses were algo per-formed with exclusion of patients with surgical compli-cations, with essentially similar results (data notshown). The preoperative registration of other parame-ters that might affect wound healing (tumor stage, nu-tritional status, hematological values, extent of surgicaltrauma) did not reveal any majar differences betweenthe groups. The present findings thus indicate a de-pressed collagen accumulation during the chemotherapycourse. Another observation in this study was the sub-stantial increase in hydroxyproline amount during thesecond week which took place irrespective of chemother-apy treatment, showing that the negative eff'ect duringchemotherapy ceased after the end ofthe course. A simi-lar rige have previously been observed in experimentalanimals [9, 16] and hydroxyproline levels were also ele-vated from Day 5 through Day 7 in human subjects [9].The present study demonstrates that if the time of graftextraction is extended to 2 weeks, there is a continuingsteep rige in hydroxyproline deposition.

rrhe mRNA expression of collagen 1 was higher in thechemotherapy group, suggesting that treatment with 5-FU results in a post-transcriptional inhibition of colla-gen synthesis. l'he mRNA elevation was still present at2 weeks although the difference was not statistically sig-nificant at this time. There was no clear difference ingraft weight, histological ingrowth, or total RNA con-tent, suggesting that treatment did not affect cell migra-tion to the same extent as collagen turnover.

This study suggests that collagen accumulation is re-duced during a short course ofpostoperative intraperito-neal 5-FU. Since col1.agen is the main determinant ofstrength in the anastomosis, the present findings indi-cate that anastomotic complications might result fromintraperitoneal 5-FU therapy shortly after surgery, notonly in experimental animals, but algo in human sub-jects. The minimum requirements for uneventful heal-ing is however not known, and there has been no greatincrease in surgical complications reported after adju-vant intraportal 5-FU infusion [21-25]. Nevertheless,the present results call for considerable caution whentreating patients with cytotoxic drugs shortly after gas-trointestinal surgery.

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