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Healing of incisional wounds in stomach and duodenum
Healing of Incisional Wounds in Stomach and Duodenum Collagen Distribution and Relation to Mechanical Strength
Finn Gottrup, MD, Aarhus, Denmark
Wound healing, as estimated by improvement in the mechanical properties of the scar, is more rapid in the gastrointestinal tract, such as stomach and duodenum [I] and colon [24], than in most other organs, such as skin [5] and tendon [6]. The tissue formed to repair the defect caused by wounding is granulation or scar tissue, in which newly laid down collagen fibers constitute a fibrous “skeleton” giving the tissue mechanical stability. It has been shown for healing skin wounds that there is a “biochemically active zone” which extends about 5 to 6 mm from the incision line into the old tissue [7]. However, no data are available on how far this zone extends from the incision line in stomach and duodenal wounds. Neither has the development and regression of this zone with time been investigated. As collagen is, from a functional point of view, the most important component of repair tissues, the aim of this study was to determine the changes in collagen concentration around a healing incisional wound 5 to 40 days postoperatively. These results were related to the mechanical properties reported previously
PI. Material
and Methods
Forty male Wistar rats weighing 302 f 34 g (mean f standard deviation) at the beginning of the experiment were randomly divided into five groups. Each group was operated on in the gastric wall and the duodenum and the animals were killed 5,7,10,20 and 40 days after wounding. Eight unoperated rats served as controls. Wounding: A midline abdominal wall incision was made, the stomach was mobilized and viscera were covered with saline solution-moistened towels. A 30 mm long incision was made in the anterior gastric wall, parallel to the greater curvature. Half of the incision was placed in the nonglandular part of the stomach (the rumen) and half of it in the glandular oxyntic part (the corpus) (Figure 1). The glandular pyloric part (the antrum) was not wounded. The wounds were immediately resutured with Prolene@ (polypropylene) 6-O suture using single suture technique. From the Department of Connective Tissue Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C. Denmark. Requests for reprints should be addressed to Finn Gottrup, MD, Department of Connective Tissue Biology, Institute of Anatomy, University of Aarhus. DK-8000 Aarhus C. Denmark.
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In duodenum a longitudinal 15 mm antimesenteric incision was made, starting 25 mm distal to pylorus and resutured as described before (Figure 1). To optimize the suturing technique the procedure was performed under a Zeiss operation microscope. The abdominal wall wound was closed in two layers; peritoneum, muscles and fasciae were resutured with 4-O catgut and skin was closed with 4-O Mersilenem suture. Further details of the anatomy, preoperative preparation and anesthesia, postoperative treatment, and killing are described in an earlier study [I 1. Sampling: The stomach was cut along the greater and lesser curvatures, and the incision line was straightened out. Then standardized strip specimens were cut parallel to the incision line with a multibladed cutting instrument containing parallel razor blades 1 mm apart. This procedure yields about 12 strips of rumen and corpus, numbered 1 to 6 on each side. The specimens with the same number from the two sides of the incision line were pooled because of the small amount of hydroxyproline present. Duodenum was opened longitudinally along along its mesenteric border and cut as just described. In addition one specimen was cut out from 40 mm distal to the wound in duodenum. Collagen determinations: After lyophilization dry defatted weight was determinated and collagen content estimated by measuring hydroxyproline according to the method of Grant [S] for rumen and corpus, and for the smaller duodenal specimen after the method of Woessner [9] (slightly modified: the reagents described by Grant were used). Collagen content was calculated from the hydroxyproline [IO], and collagen concentration was expressed in percent of dry defatted weight. In plotting the results, the concentration of collagen in the wounds was expressed as the difference between the concentration in the wounds and the concentration in the unwounded tissue. Statistical methods: Wilcoxon’s two-sample test (nonpaired) was used in all statistical analyses of the results. Mean values and standard error of the means were calculated. Results
Complications and weight changes were similar to those in the previous study [I]. Collagen distribution of unwounded stomach and duodenum are given in Figure 2. Rumen had twice the collagen concentration of corpus and duo-
The American Journal of Surgery
Healing of Incisional Wounds
denum, which did not differ from each other. Collagen concentration was constantly distributed laterally on each side, although there was for rumen a slight decrease 5 to 6 mm lateral to the area going to be wounded. The collagen concentration was significantly lower in the distal specimen of duodenum than in the area where the wound was placed (p
Collagen distribution in animals with incisional wounds in stomach and duodenum: Five days postoperatively (Figure 3): The collagen concentration in rumen had decreased significantly in the incision line and, 1 mm apart by 52 percent. Six to seven mm lateral to the incision line no significant difference between the wounded tissue and the intact tissue was found. A decrease in collagen concentration was also found in duodenum (50 percent within 1 mm of the incision line). There was a decrease of collagen concentration in the distal specimen in wounded animals compared with intact animals (26 percent). No significant decrease (7 percent lower mean value) in collagen concentration was found in specimens from the incision line or 1 mm apart in corpus, and no differences between wounded and unwounded tissue were found lateral to this area. Seven days postoperatively (Figure 3): In all types of tissue a marked increase of collagen concentration in the incision line had begun. The zone of biochemical activity was of the same lateral extent as found for 5 days. Ten days postoperatively (Figure 3): The same pattern with increase in collagen concentration was found after 10 days in all types of tissue. The increase was especially pronounced near the incision line (1 to 2 mm). The collagen concentration in corpus wounds was now significantly higher than in intact tissue near the incision line. The biochemically active zone was unchanged in size and in duodenum a “distant effect” was still recognized both 5 mm lateral to the incision line and in the distal specimen. Twenty days postoperatively (Figure 4): The pattern was the same as after 10 days: increasing
F/gore 1. Schematk anatomy of the rat sknnach and duodenum. C = corpus or glandular oxyntk part of fhe skmach; 0 = dwdenom; R = rumen or the non@andular part of the stomach. ( Ths lnclslon lines In stomach and duodenum are shown.) ( luodmed from [ I]).
collagen concentration in the biochemically active wound area and an unchanged biochemically active zone. Forty days postoperatively (Figure 4): The increase in collagen concentration continued between 20 and 40 days for all types of tissue, but the increase was marked only in corpus and duodenum. In rumen the collagen concentration had reached the intact level. In corpus and duodenum the collagen concentration was significantly increased 2 to 3 mm out on each side of the incision line. The increase at the incision line was 90 percent for corpus and 59 percent for duodenum. No differences from intact tissue were found either 5 or 40 mm from the incision line in duodenum. The collagen concentration in the distal specimen of duodenum did not differ significantly from that in intact tissue. Relation between collagen concentration and mechanical strength: In Figures 5 to 7, the relation between wound collagen concentration (incision line and 1 mm laterally) and two mechanical parameters,
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Flgure 2. Dlstrlbutlon of collagen concentratkn ( perced of dry defatfed welght [DOW]) in unwounded t&sues (mean f standard error of the mean).C= corpos;D= duodenum;DD= dktal duodenal specbnen; R = rumen.
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breaking strength (F,,,) and breaking energy (area under the curve), are shown. The mechanical parameters are from the previous study on rats from the same stock [I]. The development of collagen concentration was very similar to the development of breaking strength and breaking energy for all types of tissue. Only for corpus was there a slightly slower increase in breaking energy from day 7 to day 20, compared with the increase in collagen concentration.
Comments The submucous layer of the stomach and the bowel has a fibrous meshwork consisting mainly of collagen and elastin fibers, which besides giving these structures their mechanical strength in general is also re-
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Figure 3. Distribution of collagen concentration inwoundadtksuesve~~~specimendletanceIn mm from In&don Ilne 5.7 and 10 days after operat/on (mean f standard error of the mean) compared wifh fhaf fn lnfacf ffssues (mean f standatde#mrofthemeanehaded). c= oorpu9 D=~;DD=dieta~dvodenelepe&mr& DDW = dry defatled weight; R = rumen.
sponsible for the strength to hold sutures after surgery. Variation in the collagen content of the gastrointestinal wall in the postoperative period may thus be of great importance for healing wounds in stomach and intestine. In the present investigation collagen content was estimated by measurement of hydroxyproline as this amino acid is found almost exclusively in collagen and constitutes about 14 percent of the weight of collagen [JO]. Collagen was expressed as concentration in dry defatted tissue (percent of weight). In their study on wound healing Irvin and Hunt [II] advocated that collagen should be calculated as content in standardized biopsies and not as concentration in tissue, since the concentration is influenced by the amount of noncollagenous material present
The American Journal of Surgery
Healing of Incisional Wounds
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Ftgure 4. Dtstrlbutkm of collagen concetitratfon tn wounded ttssues versus qecbnen dtstance In mm from the Inch&n /km 20 and 40 days after qeratkm(meanfstanda~erfvrofthemean), compared wtth that of tntact tlssues (mean f standafdsworofthemeansfladsd).c=corpuq D = duodenum. DD = distal duodenum spectmen. DDW = dry defatted wefght; R = rumen.
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Ftgore 5. Changes In collagen concentration, breaking strength ( F,,,= ) and breaktng energy (ansa)dwwnds(meanfstandarftenwotthe mean) wtth heal@ time In rumen, compared wtththatoftntactttssue(meanfstandatdenvr of the mean shaded). DDW = dry defatted weight.
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Figure 6. Changes In collagen concentration, breaking strength (F,,,.= ) and breaking energy (an9a)ofwounds(meanfstandantenurofthe mean) wtth healing time In corpus. compared wNhthatoftntacttfssue(meanfsfanda~emx of the mean shaded). DDW = dry defatted wetght.
Volume 141, February 1991
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in the tissues. Standardization of biopsies from soft tissue with respect to exact surface is very difficult, especially in the early stages of healing with pronounced edema. To reduce the effect of changes in the amount of noncollagenous material on collagen concentration measurement, water and fat were eliminated by defatting and lyophilizing the specimens. Further, all suture material was removed before the analysis of collagen. Collagen concentration varied in different parts of intact stomach, and duodenum had a decreased collagen concentration in a distal direction. This necessitated a carefully standardized technique in order to ensure that the wound was in the same position throughout the series of sampling. Rumen (the nonglandular part) and corpus (the glandular oxyntic part) of the stomach differed in collagen concentration (32 and 14 percent), while the glandular oxyntic part of the stomach and the duodenum had the same concentration (14 and 15 percent). Also the changes in collagen concentration after wounding were different; the nonglandular part showed a pronounced decrease in collagen concentration after 5 days, while no change was found in the glandular oxyntic part. Both tissues thereafter showed an increase in collagen concentration and after 40 days that of the nonglandular part reached the intact level while that of the glandular oxyntic part overshot it by 90 percent. Duodenum also showed a decrease after 5 days, but no changes from the intact level were found after 7 days, and after 40 days the concentration was 59 percent higher than this level. The specimen of distal duodenum showed a decrease after 5 days but no change after 40 days, and this perhaps reflected a distant effect of the wound healing process. These findings agree with previous studies on wound healing in the colon [3,12], where the decrease in collagen concentration was most pronounced after 3 to 5 days while after 10to 14days the control level was reached. Jiborn et al [12] investigated the development of collagen concentration in colon up to 28 days after wounding and found that collagen concentration increased throughout this period.
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Flgure 7. Changes in collagen concentratkm, breaktng strength ( F,,,,= ) and breaking energy (ama)dwounds(meanfstand.miemudthe mean) wtthheatktgttmetnduo&nuqcoqwed wtththatdhtactthwe(meanfstamwdenur of the mean shaded). DDW = dry defatted wetght.
The present study also reveals that the lateral extent of the biochemically active zone around an incision in stomach and duodenum remain essentially unchanged for 40 days after operation, even though the biochemical changes within that zone are profound. The alteration in collagen concentration was limited to a 5 to 6 mm wide zone on each side of the incision, but the most pronounced changes were observed in a more narrow zone on each side of the incision. These findings are in accordance with the biochemical investigations of healing wounds in abdominal muscle fasciae in guinea pigs [7], while no such data are available for the gastrointestinal tract. In this study a small distant effect on collagen metabolism was found in duodenum which is consistent with changes in wound healing in colon and skin [3,12,13]. The development of collagen concentration and mechanical strength showed parallel increases after the fifth postoperative day and throughout the period of study, which illustrates that collagen plays the main role for the mechanical properties of wounds from these tissues. This is in agreement with studies on abdominal wall wounds in rats [14] and guinea pigs [l/j]. Sandberg and Zederfeldt [16] showed a related rate of gain in tensile strength and hydroxyproline concentration and total amount in granulation tissue of healing skin wounds from rats and rabbits during the first 13 days. Holm-Pedersen and Zederfeldt [l7] found the same relations for granulation tissue in “wounds” between subcutaneously implanted twin cellulose sponges which had been sutured to each other. Jiborn [4] showed a correlation between collagen concentration and breaking strength in colon, but found no correlation with bursting strength. Changes in collagen concentration result from synthesis and breakdown of collagen, The early decrease in collagen concentration must primarily be due to the increased breakdown, although in the early stage of healing plasma proteins and fibrin formation also increase the dry defatted weight, resulting in a somewhat diluted collagen concentration. The dif-
The American Journal of Surgery
Healing of Incisional Wounds
ferences between the various tissues investigated in this study could possibly be explained by differences in the concentration and organization of collagen in them. Tissues with high collagen concentration require after wounding extensive collagen synthesis and remodelling before a scar can reach the high strength and collagen concentration of unwounded tissue (such as skin and tendon), while scars in tissues with lower collagen concentration gain greater strength than that of intact tissue even before reorganization of collagen has taken place [18]. This is explained by the facts that collagen is the strongest structural protein in soft tissues and that the collagen content of scars in collagen-poor tissues rapidly exceeds that of intact tissue. Further details of wound healing in stomach and duodenum can be obtained by relating the absolute values of the changes in collagen content to the rate of collagen synthesis at various times. Such studies are in progress. It is evident from this study that collagen is the primary factor in the mechanical properties in healing wounds in stomach and duodenum. The width of the zone of biochemical activity remains constant throughout the healing period and measures approximately 5 to 7 mm on each side of the incisional line, with the highest activity close to the incision line. A distant effect of collagen metabolism in duodenum was found. Summary The present study was performed to determine the changes in and distribution of collagen concentration around a healing incision in rat stomach and duodenum. These concentrations were related to the mechanical properties presented previously. Wounds were made in the nonglandular (rumen) and the glandular oxyntic parts (corpus) of the stomach and in duodenum. Specimens were cut parallel to the incision line and hydroxyproline contents and dry defatted weight were measured. Wounds were investigated 5 to 40 days after operation. Of the intact tissues the nonglandular part of the stomach had twice the collagen concentration of the glandular oxyntic part and duodenum, which did not differ from each other. The healing wounds in the glandular oxyntic part of the stomach and duodenum showed the most rapid increase in collagen concentration in the incision line and 40 days postoperatively both had collagen concentrations significantly greater than those of intact tissues. Wounds in the nonglandular part of the stomach only reached the level of intact tissue after 40 days. The dimensions of the biochemically active zones around incisions in stomach and duodenum remain essentially unchanged for 40 days after operation. A relation be-
Volume 141, February 1981
tween the development of collagen concentration and mechanical strength was shown. These findings indicate that wound healing in stomach and duodenum is rapid, that collagen is the primary factor in the mechanical properties and that the highest activity is limited to a zone close to the incision line. The width of the biochemical zone remains constant. Acknowledgment: The author thanks Professor Viidik for his advice and review of the manuscript.
A.
References 1. Gottrup F. Healing of incisional wounds in stomach and duodenum. A biomechanical study. Am J Surg 1980;140:296. 2. Herrmann JB, Woodward S, Pulanski EJ. Healing of colonic anastomoses in the rat. Surg Gynecol Obstet 1964: 119: 269. 3. Cronin K, Jackson DS, Dunphy JE. Changing bursting strength and collagen content of the healing colon. Surg Gynecol Obstet 1966; 126:747. 4. Jiborn H. Healing of left colon anastomoses. An experimental study in the rat. Thesis. Malrrtb, Sweden: 1976. 5. Forrester JC, Zederfeldt BH, Hayes TL, Hunt T. Mechanical, biochemical and architectural features of repair. In: Dunphy JE, Van Winkle W, eds. Repair and regeneration. New York: McGraw-Hill, 1969:71. 6. Hirsch G. Tensile properties during tendon healing. A comparative study of intact and sutured rabbit peroneus brevis tendons. Acta Drthop Stand Suppl 1974; 153: 1. 7. Adamson RJ, Musco F, Enquist JF. The chemical dimensions of healing incision. Surg Gynecol Obstet 1966;123:515. 6. Grant RA. Application of the autoanalyzer to connective tissue analysis. J Clin Pathol 1964;17:665. 9. Woessner JF. Determination of hydroxyproline in tissue and protein samples containing small properties of the amino acid. Arch Biochem Biophys 1961;93:440. 10. Eastoe JE. Composition of collagen and allied proteins. In: Ramachandran GN. ed. Treatise in collagen. Vol I. London: Academic Press, 1967: 1. 11. Irvin TT, Hunt TK. Reappraisal of the healing process of anastomosis of the colon. Surg Gynecol Obstet 1974;136:741. 12. Jiborn K, Ahonen J, Zederfeldt B. Healing of experimental coIonic anastomosis. The effect cf suture technic on collagen concentration in the colonic wall. Am J Surg 1976135: 333. 13. Viidik A, Holm-Pedersen P. Rundgren A. Some observations on the distant collagen response to wound healing. Stand J Plast Reconstr Surg 1972;6:114. 14. Dunphy JE. Udupa KN. Chemical and histochemical sequences in the normal healing of wounds. N Engl J Med 1955;253: 647. 15. Adamson RJ, Musco F, Enquist JF. The relationship of collagen content to wound strength in normal and scorbutic animals. Surg Gynecol Obstet 1964; 119:323. 16. Sandberg N, Zederfeldt B. The tensile strength of healing wounds and collagen formation in rats and rabbits. Acta Chir Stand 1963;126:187. 17. Holm-Pedersen P, Zederfeldt B. Granulation tissue formation in subcutaneously implanted cellulose sponges in young and old rats. Stand J Plast Reconstr Sura 1971:5:13. 18. Adamson RJ, Kahan SA. The rate of healing of incised wounds of different tissues in rabbits. Surg Gynecol Obstet 1970; 130:837.
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Finn Gottrup, MD, Aarhus, Denmark
Wound healing, as estimated by improvement in the mechanical properties of the scar, is more rapid in the gastrointestinal tract, such as stomach and duodenum [I] and colon [24], than in most other organs, such as skin [5] and tendon [6]. The tissue formed to repair the defect caused by wounding is granulation or scar tissue, in which newly laid down collagen fibers constitute a fibrous “skeleton” giving the tissue mechanical stability. It has been shown for healing skin wounds that there is a “biochemically active zone” which extends about 5 to 6 mm from the incision line into the old tissue [7]. However, no data are available on how far this zone extends from the incision line in stomach and duodenal wounds. Neither has the development and regression of this zone with time been investigated. As collagen is, from a functional point of view, the most important component of repair tissues, the aim of this study was to determine the changes in collagen concentration around a healing incisional wound 5 to 40 days postoperatively. These results were related to the mechanical properties reported previously
PI. Material
and Methods
Forty male Wistar rats weighing 302 f 34 g (mean f standard deviation) at the beginning of the experiment were randomly divided into five groups. Each group was operated on in the gastric wall and the duodenum and the animals were killed 5,7,10,20 and 40 days after wounding. Eight unoperated rats served as controls. Wounding: A midline abdominal wall incision was made, the stomach was mobilized and viscera were covered with saline solution-moistened towels. A 30 mm long incision was made in the anterior gastric wall, parallel to the greater curvature. Half of the incision was placed in the nonglandular part of the stomach (the rumen) and half of it in the glandular oxyntic part (the corpus) (Figure 1). The glandular pyloric part (the antrum) was not wounded. The wounds were immediately resutured with Prolene@ (polypropylene) 6-O suture using single suture technique. From the Department of Connective Tissue Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C. Denmark. Requests for reprints should be addressed to Finn Gottrup, MD, Department of Connective Tissue Biology, Institute of Anatomy, University of Aarhus. DK-8000 Aarhus C. Denmark.
222
In duodenum a longitudinal 15 mm antimesenteric incision was made, starting 25 mm distal to pylorus and resutured as described before (Figure 1). To optimize the suturing technique the procedure was performed under a Zeiss operation microscope. The abdominal wall wound was closed in two layers; peritoneum, muscles and fasciae were resutured with 4-O catgut and skin was closed with 4-O Mersilenem suture. Further details of the anatomy, preoperative preparation and anesthesia, postoperative treatment, and killing are described in an earlier study [I 1. Sampling: The stomach was cut along the greater and lesser curvatures, and the incision line was straightened out. Then standardized strip specimens were cut parallel to the incision line with a multibladed cutting instrument containing parallel razor blades 1 mm apart. This procedure yields about 12 strips of rumen and corpus, numbered 1 to 6 on each side. The specimens with the same number from the two sides of the incision line were pooled because of the small amount of hydroxyproline present. Duodenum was opened longitudinally along along its mesenteric border and cut as just described. In addition one specimen was cut out from 40 mm distal to the wound in duodenum. Collagen determinations: After lyophilization dry defatted weight was determinated and collagen content estimated by measuring hydroxyproline according to the method of Grant [S] for rumen and corpus, and for the smaller duodenal specimen after the method of Woessner [9] (slightly modified: the reagents described by Grant were used). Collagen content was calculated from the hydroxyproline [IO], and collagen concentration was expressed in percent of dry defatted weight. In plotting the results, the concentration of collagen in the wounds was expressed as the difference between the concentration in the wounds and the concentration in the unwounded tissue. Statistical methods: Wilcoxon’s two-sample test (nonpaired) was used in all statistical analyses of the results. Mean values and standard error of the means were calculated. Results
Complications and weight changes were similar to those in the previous study [I]. Collagen distribution of unwounded stomach and duodenum are given in Figure 2. Rumen had twice the collagen concentration of corpus and duo-
The American Journal of Surgery
Healing of Incisional Wounds
denum, which did not differ from each other. Collagen concentration was constantly distributed laterally on each side, although there was for rumen a slight decrease 5 to 6 mm lateral to the area going to be wounded. The collagen concentration was significantly lower in the distal specimen of duodenum than in the area where the wound was placed (p
Collagen distribution in animals with incisional wounds in stomach and duodenum: Five days postoperatively (Figure 3): The collagen concentration in rumen had decreased significantly in the incision line and, 1 mm apart by 52 percent. Six to seven mm lateral to the incision line no significant difference between the wounded tissue and the intact tissue was found. A decrease in collagen concentration was also found in duodenum (50 percent within 1 mm of the incision line). There was a decrease of collagen concentration in the distal specimen in wounded animals compared with intact animals (26 percent). No significant decrease (7 percent lower mean value) in collagen concentration was found in specimens from the incision line or 1 mm apart in corpus, and no differences between wounded and unwounded tissue were found lateral to this area. Seven days postoperatively (Figure 3): In all types of tissue a marked increase of collagen concentration in the incision line had begun. The zone of biochemical activity was of the same lateral extent as found for 5 days. Ten days postoperatively (Figure 3): The same pattern with increase in collagen concentration was found after 10 days in all types of tissue. The increase was especially pronounced near the incision line (1 to 2 mm). The collagen concentration in corpus wounds was now significantly higher than in intact tissue near the incision line. The biochemically active zone was unchanged in size and in duodenum a “distant effect” was still recognized both 5 mm lateral to the incision line and in the distal specimen. Twenty days postoperatively (Figure 4): The pattern was the same as after 10 days: increasing
F/gore 1. Schematk anatomy of the rat sknnach and duodenum. C = corpus or glandular oxyntk part of fhe skmach; 0 = dwdenom; R = rumen or the non@andular part of the stomach. ( Ths lnclslon lines In stomach and duodenum are shown.) ( luodmed from [ I]).
collagen concentration in the biochemically active wound area and an unchanged biochemically active zone. Forty days postoperatively (Figure 4): The increase in collagen concentration continued between 20 and 40 days for all types of tissue, but the increase was marked only in corpus and duodenum. In rumen the collagen concentration had reached the intact level. In corpus and duodenum the collagen concentration was significantly increased 2 to 3 mm out on each side of the incision line. The increase at the incision line was 90 percent for corpus and 59 percent for duodenum. No differences from intact tissue were found either 5 or 40 mm from the incision line in duodenum. The collagen concentration in the distal specimen of duodenum did not differ significantly from that in intact tissue. Relation between collagen concentration and mechanical strength: In Figures 5 to 7, the relation between wound collagen concentration (incision line and 1 mm laterally) and two mechanical parameters,
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Flgure 2. Dlstrlbutlon of collagen concentratkn ( perced of dry defatfed welght [DOW]) in unwounded t&sues (mean f standard error of the mean).C= corpos;D= duodenum;DD= dktal duodenal specbnen; R = rumen.
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breaking strength (F,,,) and breaking energy (area under the curve), are shown. The mechanical parameters are from the previous study on rats from the same stock [I]. The development of collagen concentration was very similar to the development of breaking strength and breaking energy for all types of tissue. Only for corpus was there a slightly slower increase in breaking energy from day 7 to day 20, compared with the increase in collagen concentration.
Comments The submucous layer of the stomach and the bowel has a fibrous meshwork consisting mainly of collagen and elastin fibers, which besides giving these structures their mechanical strength in general is also re-
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Figure 3. Distribution of collagen concentration inwoundadtksuesve~~~specimendletanceIn mm from In&don Ilne 5.7 and 10 days after operat/on (mean f standard error of the mean) compared wifh fhaf fn lnfacf ffssues (mean f standatde#mrofthemeanehaded). c= oorpu9 D=~;DD=dieta~dvodenelepe&mr& DDW = dry defatled weight; R = rumen.
sponsible for the strength to hold sutures after surgery. Variation in the collagen content of the gastrointestinal wall in the postoperative period may thus be of great importance for healing wounds in stomach and intestine. In the present investigation collagen content was estimated by measurement of hydroxyproline as this amino acid is found almost exclusively in collagen and constitutes about 14 percent of the weight of collagen [JO]. Collagen was expressed as concentration in dry defatted tissue (percent of weight). In their study on wound healing Irvin and Hunt [II] advocated that collagen should be calculated as content in standardized biopsies and not as concentration in tissue, since the concentration is influenced by the amount of noncollagenous material present
The American Journal of Surgery
Healing of Incisional Wounds
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Ftgure 4. Dtstrlbutkm of collagen concetitratfon tn wounded ttssues versus qecbnen dtstance In mm from the Inch&n /km 20 and 40 days after qeratkm(meanfstanda~erfvrofthemean), compared wtth that of tntact tlssues (mean f standafdsworofthemeansfladsd).c=corpuq D = duodenum. DD = distal duodenum spectmen. DDW = dry defatted wefght; R = rumen.
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Ftgore 5. Changes In collagen concentration, breaking strength ( F,,,= ) and breaktng energy (ansa)dwwnds(meanfstandarftenwotthe mean) wtth heal@ time In rumen, compared wtththatoftntactttssue(meanfstandatdenvr of the mean shaded). DDW = dry defatted weight.
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Figure 6. Changes In collagen concentration, breaking strength (F,,,.= ) and breaking energy (an9a)ofwounds(meanfstandantenurofthe mean) wtth healing time In corpus. compared wNhthatoftntacttfssue(meanfsfanda~emx of the mean shaded). DDW = dry defatted wetght.
Volume 141, February 1991
10
,I
I 10
2b
I 40
I., 10
I 20
8 40
II,
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Gottrup
CollageniDDW %
s
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in the tissues. Standardization of biopsies from soft tissue with respect to exact surface is very difficult, especially in the early stages of healing with pronounced edema. To reduce the effect of changes in the amount of noncollagenous material on collagen concentration measurement, water and fat were eliminated by defatting and lyophilizing the specimens. Further, all suture material was removed before the analysis of collagen. Collagen concentration varied in different parts of intact stomach, and duodenum had a decreased collagen concentration in a distal direction. This necessitated a carefully standardized technique in order to ensure that the wound was in the same position throughout the series of sampling. Rumen (the nonglandular part) and corpus (the glandular oxyntic part) of the stomach differed in collagen concentration (32 and 14 percent), while the glandular oxyntic part of the stomach and the duodenum had the same concentration (14 and 15 percent). Also the changes in collagen concentration after wounding were different; the nonglandular part showed a pronounced decrease in collagen concentration after 5 days, while no change was found in the glandular oxyntic part. Both tissues thereafter showed an increase in collagen concentration and after 40 days that of the nonglandular part reached the intact level while that of the glandular oxyntic part overshot it by 90 percent. Duodenum also showed a decrease after 5 days, but no changes from the intact level were found after 7 days, and after 40 days the concentration was 59 percent higher than this level. The specimen of distal duodenum showed a decrease after 5 days but no change after 40 days, and this perhaps reflected a distant effect of the wound healing process. These findings agree with previous studies on wound healing in the colon [3,12], where the decrease in collagen concentration was most pronounced after 3 to 5 days while after 10to 14days the control level was reached. Jiborn et al [12] investigated the development of collagen concentration in colon up to 28 days after wounding and found that collagen concentration increased throughout this period.
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Flgure 7. Changes in collagen concentratkm, breaktng strength ( F,,,,= ) and breaking energy (ama)dwounds(meanfstand.miemudthe mean) wtthheatktgttmetnduo&nuqcoqwed wtththatdhtactthwe(meanfstamwdenur of the mean shaded). DDW = dry defatted wetght.
The present study also reveals that the lateral extent of the biochemically active zone around an incision in stomach and duodenum remain essentially unchanged for 40 days after operation, even though the biochemical changes within that zone are profound. The alteration in collagen concentration was limited to a 5 to 6 mm wide zone on each side of the incision, but the most pronounced changes were observed in a more narrow zone on each side of the incision. These findings are in accordance with the biochemical investigations of healing wounds in abdominal muscle fasciae in guinea pigs [7], while no such data are available for the gastrointestinal tract. In this study a small distant effect on collagen metabolism was found in duodenum which is consistent with changes in wound healing in colon and skin [3,12,13]. The development of collagen concentration and mechanical strength showed parallel increases after the fifth postoperative day and throughout the period of study, which illustrates that collagen plays the main role for the mechanical properties of wounds from these tissues. This is in agreement with studies on abdominal wall wounds in rats [14] and guinea pigs [l/j]. Sandberg and Zederfeldt [16] showed a related rate of gain in tensile strength and hydroxyproline concentration and total amount in granulation tissue of healing skin wounds from rats and rabbits during the first 13 days. Holm-Pedersen and Zederfeldt [l7] found the same relations for granulation tissue in “wounds” between subcutaneously implanted twin cellulose sponges which had been sutured to each other. Jiborn [4] showed a correlation between collagen concentration and breaking strength in colon, but found no correlation with bursting strength. Changes in collagen concentration result from synthesis and breakdown of collagen, The early decrease in collagen concentration must primarily be due to the increased breakdown, although in the early stage of healing plasma proteins and fibrin formation also increase the dry defatted weight, resulting in a somewhat diluted collagen concentration. The dif-
The American Journal of Surgery
Healing of Incisional Wounds
ferences between the various tissues investigated in this study could possibly be explained by differences in the concentration and organization of collagen in them. Tissues with high collagen concentration require after wounding extensive collagen synthesis and remodelling before a scar can reach the high strength and collagen concentration of unwounded tissue (such as skin and tendon), while scars in tissues with lower collagen concentration gain greater strength than that of intact tissue even before reorganization of collagen has taken place [18]. This is explained by the facts that collagen is the strongest structural protein in soft tissues and that the collagen content of scars in collagen-poor tissues rapidly exceeds that of intact tissue. Further details of wound healing in stomach and duodenum can be obtained by relating the absolute values of the changes in collagen content to the rate of collagen synthesis at various times. Such studies are in progress. It is evident from this study that collagen is the primary factor in the mechanical properties in healing wounds in stomach and duodenum. The width of the zone of biochemical activity remains constant throughout the healing period and measures approximately 5 to 7 mm on each side of the incisional line, with the highest activity close to the incision line. A distant effect of collagen metabolism in duodenum was found. Summary The present study was performed to determine the changes in and distribution of collagen concentration around a healing incision in rat stomach and duodenum. These concentrations were related to the mechanical properties presented previously. Wounds were made in the nonglandular (rumen) and the glandular oxyntic parts (corpus) of the stomach and in duodenum. Specimens were cut parallel to the incision line and hydroxyproline contents and dry defatted weight were measured. Wounds were investigated 5 to 40 days after operation. Of the intact tissues the nonglandular part of the stomach had twice the collagen concentration of the glandular oxyntic part and duodenum, which did not differ from each other. The healing wounds in the glandular oxyntic part of the stomach and duodenum showed the most rapid increase in collagen concentration in the incision line and 40 days postoperatively both had collagen concentrations significantly greater than those of intact tissues. Wounds in the nonglandular part of the stomach only reached the level of intact tissue after 40 days. The dimensions of the biochemically active zones around incisions in stomach and duodenum remain essentially unchanged for 40 days after operation. A relation be-
Volume 141, February 1981
tween the development of collagen concentration and mechanical strength was shown. These findings indicate that wound healing in stomach and duodenum is rapid, that collagen is the primary factor in the mechanical properties and that the highest activity is limited to a zone close to the incision line. The width of the biochemical zone remains constant. Acknowledgment: The author thanks Professor Viidik for his advice and review of the manuscript.
A.
References 1. Gottrup F. Healing of incisional wounds in stomach and duodenum. A biomechanical study. Am J Surg 1980;140:296. 2. Herrmann JB, Woodward S, Pulanski EJ. Healing of colonic anastomoses in the rat. Surg Gynecol Obstet 1964: 119: 269. 3. Cronin K, Jackson DS, Dunphy JE. Changing bursting strength and collagen content of the healing colon. Surg Gynecol Obstet 1966; 126:747. 4. Jiborn H. Healing of left colon anastomoses. An experimental study in the rat. Thesis. Malrrtb, Sweden: 1976. 5. Forrester JC, Zederfeldt BH, Hayes TL, Hunt T. Mechanical, biochemical and architectural features of repair. In: Dunphy JE, Van Winkle W, eds. Repair and regeneration. New York: McGraw-Hill, 1969:71. 6. Hirsch G. Tensile properties during tendon healing. A comparative study of intact and sutured rabbit peroneus brevis tendons. Acta Drthop Stand Suppl 1974; 153: 1. 7. Adamson RJ, Musco F, Enquist JF. The chemical dimensions of healing incision. Surg Gynecol Obstet 1966;123:515. 6. Grant RA. Application of the autoanalyzer to connective tissue analysis. J Clin Pathol 1964;17:665. 9. Woessner JF. Determination of hydroxyproline in tissue and protein samples containing small properties of the amino acid. Arch Biochem Biophys 1961;93:440. 10. Eastoe JE. Composition of collagen and allied proteins. In: Ramachandran GN. ed. Treatise in collagen. Vol I. London: Academic Press, 1967: 1. 11. Irvin TT, Hunt TK. Reappraisal of the healing process of anastomosis of the colon. Surg Gynecol Obstet 1974;136:741. 12. Jiborn K, Ahonen J, Zederfeldt B. Healing of experimental coIonic anastomosis. The effect cf suture technic on collagen concentration in the colonic wall. Am J Surg 1976135: 333. 13. Viidik A, Holm-Pedersen P. Rundgren A. Some observations on the distant collagen response to wound healing. Stand J Plast Reconstr Surg 1972;6:114. 14. Dunphy JE. Udupa KN. Chemical and histochemical sequences in the normal healing of wounds. N Engl J Med 1955;253: 647. 15. Adamson RJ, Musco F, Enquist JF. The relationship of collagen content to wound strength in normal and scorbutic animals. Surg Gynecol Obstet 1964; 119:323. 16. Sandberg N, Zederfeldt B. The tensile strength of healing wounds and collagen formation in rats and rabbits. Acta Chir Stand 1963;126:187. 17. Holm-Pedersen P, Zederfeldt B. Granulation tissue formation in subcutaneously implanted cellulose sponges in young and old rats. Stand J Plast Reconstr Sura 1971:5:13. 18. Adamson RJ, Kahan SA. The rate of healing of incised wounds of different tissues in rabbits. Surg Gynecol Obstet 1970; 130:837.
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