Synthesis of Type I Collagen in Healing Wounds in Humans
KARI HAUKIPURO, M.D.,* JUKKA MELKKO, CAND. MED.,tt LEILA RISTELI, M.D., PH.D.,tt MATTI 1. KAIRALUOMA, M.D., PH.D.,* and JUHA RISTELI, M.D., PH.D.t§
To quantify wound healing in surgical patients, samples of wound fluid were collected through a silicone rubber tube for 7 postoperative days and their concentrations of the carboxyterminal propeptide of type I procollagen (PICP) and the aminoterminal propeptide of type III procollagen (PIIINP) were measured with specific radioimmunoassays. The mean concentration of PICP in wound fluid on day 1 was 207 ± 92 (SD),ug/L, and on day 2 908 ± 469 gg/L (p < 0.001, signed rank test). On day 7, the mean concentration reached was 380 times higher than that of day 1 (79,330 ± 54,151 gg/L). Only one peak of PICP antigenicity, corresponding to the intact propeptide as set free during synthesis of type I procollagen, was detected on Sephacryl S300 gel filtration analysis of wound fluid samples. The mean concentration of PIIINP was 70 ± 61 gg/L on day 1, 86 ± 88 gg/L on day 2, and 180 ± 129 gg/L on day 3 (p < 0.001 when compared with day 1). Finally on day 7, a 250-fold concentration (17,812 ± 9839 jig/L), compared with day 1, was reached. Methods described in the present paper allow separate and repetitive quantification of the synthesis of both type I and type III procollagen during human wound healing.
From the Departments of Surgery,* Medical Biochemistry,t and Clinical Chemistry,§ and the Collagen Research Unit,f University of Oulu, Oulu, Finland
peptides is necessary for proper fiber formation, where as not all the aminoterminal propeptides, especially those of type III procollagen, are cleaved off. Such type III pN collagen molecules are found on the surface of the fibers, where they probably prevent further growth of the fibril.6 Previously we showed7 that type III procollagen synthesis can be measured directly in a healing human wound in situ applying a specific radioimmunoassay for the aminoterminal propeptide of type III procollagen (PIIINP). The PIIINP antigen in wound fluid was derived mainly from new formation of type III collagen. Type I collagen is the most abundant protein component of the granulation tissue.8 Because of its complete
T n HE STRENGTH OF a healing wound ultimately is based on collagen.' Proliferation of fibroblasts and synthesis of new collagen predominate in the proliferative phase of the healing process. Both type I and type III collagens are found in the granulation tissue, the
removal and stoichiometric release during the formation of fibers, the carboxyterminal propeptide of type I procollagen (PICP) in wound fluid is theoretically a good indicator ofthe synthesis of this collagen type. We recently developed a radioimmunoassay for human PICP.9 In the present study, the PICP concentration was measured in human wound fluid for up to 7 days after wounding. The size distribution of the antigen also was studied, as was the possible sequential appearance of collagen types I and III.
proportion of the latter being higher than in the surrounding dermis.2 A sequential appearance of type I and III collagens has been suggested on the basis of immunohistochemical studies.3'4 The fiber-forming collagens (types I, II, III, and V) are synthesized as procollagens, which contain additional propeptide extensions at both ends.5 These are cleaved off en bloc when collagen molecules are deposited in the tissue. The complete removal of the carboxyterminal pro-
Materials and Methods
Patients Twenty consecutive patients, 4 men and 16 women, were studied (mean age, 65 years; range, 44 to 82 years). They were electively operated on at Oulu University Central Hospital for colorectal cancer. Standard operative methods (resection of the colon or rectum in 14 patients,
This study was supported in part by the Paulo Foundation and the Medical Research Council of the Academy of Finland. Address reprint requests to Kari Haukipuro, M.D., Department of Surgery, University of Oulu, SF-90220 Oulu, Finland. Accepted for publication February 19, 1990.
75
76
HAUKIPURO AND OTHERS
abdominoperineal excision of the rectum in 5, and exploratory laparotomy in 1) were used. For wound infection prophylaxis, single doses of doxycycline (400 mg) and metronidazole (1 g) were given intravenously at the induction of anesthesia. The tumor staging'0 was as follows: stage A, 5 patients; stage B, 5 patients; stage C, 6 patients; and stage D, 4 patients. Ten of the patients had a previous operation through the same incision 4 to 20 years earlier. Two of the patients had diabetes, one being treated with glibenclamid and the other with a sugar-free diet only. None of the patients used steroid medication. The predefined exclusion criteria included a recent (less than 1 year) operation through the same incision and the patient's unwillingness to participate in the study. The study was approved of by the Ethical Committee of the Medical Faculty of the University of Oulu and was carried out according to the provisions of the Declaration of Helsinki. Collection of Wound Fluid
For collecting wound fluid, two 15-cm Silastic® silicone rubber tubings (Dow Corning [Midland, MI] medical grade tubing no. 602-235, external diameter 2 mm) were left subcutaneously in the wound at the end of the operation, as previously described.7 Samples of wound fluid were then drawn daily for 7 days by puncturing the tubing. The volume of the fluid (maximum amount drawn, 1 mL) was measured and the appearance determined visually as bloody, semiserous, or serous. All the samples were stored at -20 C until analyzed. Before analysis all the wound fluid samples were diluted 1:10 with phosphatebuffered saline, pH 7.2, containing 0.04% of Tween 20 (ICI Americas, Wilmington, DE). Further dilutions, when necessary, also were made in this buffer. The tubes of one patient were detached before collection of the fourth day sample and her data were excluded. Some samples were inadvertently not taken or had other technical problems (e.g., too high a dilution of two samples). Thus the total number of wound fluid samples examined was 18, 18, 16, 15, 16, 15, and 18 for days 1 to 7, respectively. No wound healing complications were seen. A blood sample also was taken from all the patients before operation to determine serum PICP and PIIINP concentrations. Radioimmunoassay for Analyzing Wound Fluid PICP A recently developed radioimmunoassay9 was used for analyzing the concentration of the carboxyterminal PICP. The assay was established by isolating type I procollagen from the medium of primary cultures of human skin fibroblasts and by digesting it with highly purified bacterial collagenase to liberate PICP. The released PICP finally was purified with lectin affinity, gel filtration, and anion exchange chromatographies. The polyclonal antiserum
Ann. Surg. * January 1991
against the propeptide was raised in rabbits. The radioimmunoassay procedure was briefly as follows: 0.1 mL of an antiserum dilution capable of binding about 50% of the labeled antigen was incubated for 2 hours at +37 C with 0.1 mL of unlabeled standard antigen or unknown sample before adding 0.1 mL of 1251-labeled PICP. After a further incubation of hour at +37 C, the free and bound antigens were separated by overnight precipitation at +4 C with a goat antiserum (second antibody) against rabbit IgG. The precipitate was collected by centrifugation for 30 minutes at +4 C, dissolved in mL of phosphatebuffered saline, and recentrifuged. The radioactivity in the precipitate then was counted using an LKB-Wallac Multigamma gamma counter (Turku, Finland). Dilutions of 1:40 to 1:160 could be used for the first- to third-day samples, but up to 1:10,000 (most often 1:4000) dilutions of the seventh-day samples were required for reaching the measuring range of the assay. All the samples of each patient were determined in the same run. The intra-assay coefficient of variation was 5%. Radioimmunoassay for Analyzing Wound Fluid PIIINP
The concentration of the aminoterminal propeptide of was analyzed with a sequential modification of a radioimmunoassay" based on the human antigen (Farmos Diagnostica, Oulunsalo, Finland). The antiserum and the assay conditions have been selected so that the authentic propeptide and another, somewhat larger, PIIINP related antigen are detected, but the assay is not sensitive to the smaller degradation products of the propeptide. With this method the standards and samples of biologic fluids give parallel inhibition curves. We previously demonstrated that this assay reflects the synthesis of type III procollagen in human wounds.7"2 The actual laboratory procedure was comparable to that of the PICP assay described above. The volumes used were 0.1 mL for antiserum, 0.1 mL for standard, and 0.2 mL for samples. Dilutions of 1:20 to 1:100 were suitable for the first- to third-day samples, but up to 1:8000 (usually 1:4000) dilutions of the seventh-day samples were necessary. The intra-assay coefficient of variation was 4%.
type III procollagen (PIIINP)
Gel Filtration Analysis of Wound Fluid PICP
Gel filtration of 100-,uL samples of the first-day and fourth-day wound fluid and a 50-,uL sample of the seventhday wound fluid was carried out on a column (1.5 X 110 cm) of Sephacryl S-300 (Pharmacia, Uppsala, Sweden) equilibrated in 0.2 mol/L (molar) (NH4)HCO3, pH 7.9. Seventy fractions of 2.5 mL were collected from each sample. Aliquots (400, 100, and 25 ,uL) of each fraction of the first-, fourth-, and seventh-day wound fluid, respectively, then were analyzed with the PICP radioimmunoassay using the conditions described above.
COLLAGEN TYPES IN WOUND FLUID
Vol. 213 - No. I
Clinical Variables The clinical variables recorded included the type and the interval from any previous operation and specifically the operations through the same incision, the staging of the tumor, operative blood loss, duration of the operation, postoperative complications, the hematocrit value, and the hemoglobin and serum albumin concentrations.
Statistical Analysis The statistical analyses were done with Statgraphics® Software (STSC, Inc., Rockville, MD). The results of wound fluid PICP and PIIINP concentrations are given as means with standard deviations (SD) or with standard errors as indicated in the text and the figures. Wilcoxon signed rank test for paired observations was used when comparing later concentrations of PICP and PIIINP with those of day 1, p < 0.05, evaluated from standard tables for small samples, was considered as statistically significant. The Spearman rank correlation coefficient was calculated when clinical and sampling variables were compared to the concentrations of PICP and PIIINP. It also was used for comparisons between the PICP and PIIINP concentrations on the different postoperative days. Results PICP and PIIINP in Wound Fluid The mean concentration of PICP in wound fluid on day 1 was 207 ± 92 (SD) ,g/L. Already on day 2 it was 908 ± 469 ,ug/L (t = 0, z = 3.8, p < 0.001), and 3246 100 80 r-'
I
60 D- 40 _
u0L
24 1
20 F -J
_-- 16
%j 12 0-
z _8
CL
4
1
2
3
4
5
6
7
DAYS AFTER SURGERY FIG. 2. Effect of time on the concentration of the aminoterminal propeptide of type III procollagen (PIIINP) in wound fluid of patients operated on for colorectal cancer (mean, SE).
± 2858 ,ug/L on day 3 (Fig. 1). On day 7 the mean concentration had reached a level 380 times that of day 1 (79,330 ± 54,151 ,ug/L). The individual differences were great, e.g., at day 7 the range was from 19,750 to 198,813 jug/L. The mean PIIINP concentrations (Fig. 2) changed more slowly, no increase being found between the first 2 days (70 ± 61 u g/L on day 1 and 86 ± 88 ,ug/L on day 2; t = 64, z = 1.2, p = 0.2), but on day 3 the concentration was increased to 180 ± 129 ,ug/L (t = 0, z = 3.48, p
KARI HAUKIPURO, M.D.,* JUKKA MELKKO, CAND. MED.,tt LEILA RISTELI, M.D., PH.D.,tt MATTI 1. KAIRALUOMA, M.D., PH.D.,* and JUHA RISTELI, M.D., PH.D.t§
To quantify wound healing in surgical patients, samples of wound fluid were collected through a silicone rubber tube for 7 postoperative days and their concentrations of the carboxyterminal propeptide of type I procollagen (PICP) and the aminoterminal propeptide of type III procollagen (PIIINP) were measured with specific radioimmunoassays. The mean concentration of PICP in wound fluid on day 1 was 207 ± 92 (SD),ug/L, and on day 2 908 ± 469 gg/L (p < 0.001, signed rank test). On day 7, the mean concentration reached was 380 times higher than that of day 1 (79,330 ± 54,151 gg/L). Only one peak of PICP antigenicity, corresponding to the intact propeptide as set free during synthesis of type I procollagen, was detected on Sephacryl S300 gel filtration analysis of wound fluid samples. The mean concentration of PIIINP was 70 ± 61 gg/L on day 1, 86 ± 88 gg/L on day 2, and 180 ± 129 gg/L on day 3 (p < 0.001 when compared with day 1). Finally on day 7, a 250-fold concentration (17,812 ± 9839 jig/L), compared with day 1, was reached. Methods described in the present paper allow separate and repetitive quantification of the synthesis of both type I and type III procollagen during human wound healing.
From the Departments of Surgery,* Medical Biochemistry,t and Clinical Chemistry,§ and the Collagen Research Unit,f University of Oulu, Oulu, Finland
peptides is necessary for proper fiber formation, where as not all the aminoterminal propeptides, especially those of type III procollagen, are cleaved off. Such type III pN collagen molecules are found on the surface of the fibers, where they probably prevent further growth of the fibril.6 Previously we showed7 that type III procollagen synthesis can be measured directly in a healing human wound in situ applying a specific radioimmunoassay for the aminoterminal propeptide of type III procollagen (PIIINP). The PIIINP antigen in wound fluid was derived mainly from new formation of type III collagen. Type I collagen is the most abundant protein component of the granulation tissue.8 Because of its complete
T n HE STRENGTH OF a healing wound ultimately is based on collagen.' Proliferation of fibroblasts and synthesis of new collagen predominate in the proliferative phase of the healing process. Both type I and type III collagens are found in the granulation tissue, the
removal and stoichiometric release during the formation of fibers, the carboxyterminal propeptide of type I procollagen (PICP) in wound fluid is theoretically a good indicator ofthe synthesis of this collagen type. We recently developed a radioimmunoassay for human PICP.9 In the present study, the PICP concentration was measured in human wound fluid for up to 7 days after wounding. The size distribution of the antigen also was studied, as was the possible sequential appearance of collagen types I and III.
proportion of the latter being higher than in the surrounding dermis.2 A sequential appearance of type I and III collagens has been suggested on the basis of immunohistochemical studies.3'4 The fiber-forming collagens (types I, II, III, and V) are synthesized as procollagens, which contain additional propeptide extensions at both ends.5 These are cleaved off en bloc when collagen molecules are deposited in the tissue. The complete removal of the carboxyterminal pro-
Materials and Methods
Patients Twenty consecutive patients, 4 men and 16 women, were studied (mean age, 65 years; range, 44 to 82 years). They were electively operated on at Oulu University Central Hospital for colorectal cancer. Standard operative methods (resection of the colon or rectum in 14 patients,
This study was supported in part by the Paulo Foundation and the Medical Research Council of the Academy of Finland. Address reprint requests to Kari Haukipuro, M.D., Department of Surgery, University of Oulu, SF-90220 Oulu, Finland. Accepted for publication February 19, 1990.
75
76
HAUKIPURO AND OTHERS
abdominoperineal excision of the rectum in 5, and exploratory laparotomy in 1) were used. For wound infection prophylaxis, single doses of doxycycline (400 mg) and metronidazole (1 g) were given intravenously at the induction of anesthesia. The tumor staging'0 was as follows: stage A, 5 patients; stage B, 5 patients; stage C, 6 patients; and stage D, 4 patients. Ten of the patients had a previous operation through the same incision 4 to 20 years earlier. Two of the patients had diabetes, one being treated with glibenclamid and the other with a sugar-free diet only. None of the patients used steroid medication. The predefined exclusion criteria included a recent (less than 1 year) operation through the same incision and the patient's unwillingness to participate in the study. The study was approved of by the Ethical Committee of the Medical Faculty of the University of Oulu and was carried out according to the provisions of the Declaration of Helsinki. Collection of Wound Fluid
For collecting wound fluid, two 15-cm Silastic® silicone rubber tubings (Dow Corning [Midland, MI] medical grade tubing no. 602-235, external diameter 2 mm) were left subcutaneously in the wound at the end of the operation, as previously described.7 Samples of wound fluid were then drawn daily for 7 days by puncturing the tubing. The volume of the fluid (maximum amount drawn, 1 mL) was measured and the appearance determined visually as bloody, semiserous, or serous. All the samples were stored at -20 C until analyzed. Before analysis all the wound fluid samples were diluted 1:10 with phosphatebuffered saline, pH 7.2, containing 0.04% of Tween 20 (ICI Americas, Wilmington, DE). Further dilutions, when necessary, also were made in this buffer. The tubes of one patient were detached before collection of the fourth day sample and her data were excluded. Some samples were inadvertently not taken or had other technical problems (e.g., too high a dilution of two samples). Thus the total number of wound fluid samples examined was 18, 18, 16, 15, 16, 15, and 18 for days 1 to 7, respectively. No wound healing complications were seen. A blood sample also was taken from all the patients before operation to determine serum PICP and PIIINP concentrations. Radioimmunoassay for Analyzing Wound Fluid PICP A recently developed radioimmunoassay9 was used for analyzing the concentration of the carboxyterminal PICP. The assay was established by isolating type I procollagen from the medium of primary cultures of human skin fibroblasts and by digesting it with highly purified bacterial collagenase to liberate PICP. The released PICP finally was purified with lectin affinity, gel filtration, and anion exchange chromatographies. The polyclonal antiserum
Ann. Surg. * January 1991
against the propeptide was raised in rabbits. The radioimmunoassay procedure was briefly as follows: 0.1 mL of an antiserum dilution capable of binding about 50% of the labeled antigen was incubated for 2 hours at +37 C with 0.1 mL of unlabeled standard antigen or unknown sample before adding 0.1 mL of 1251-labeled PICP. After a further incubation of hour at +37 C, the free and bound antigens were separated by overnight precipitation at +4 C with a goat antiserum (second antibody) against rabbit IgG. The precipitate was collected by centrifugation for 30 minutes at +4 C, dissolved in mL of phosphatebuffered saline, and recentrifuged. The radioactivity in the precipitate then was counted using an LKB-Wallac Multigamma gamma counter (Turku, Finland). Dilutions of 1:40 to 1:160 could be used for the first- to third-day samples, but up to 1:10,000 (most often 1:4000) dilutions of the seventh-day samples were required for reaching the measuring range of the assay. All the samples of each patient were determined in the same run. The intra-assay coefficient of variation was 5%. Radioimmunoassay for Analyzing Wound Fluid PIIINP
The concentration of the aminoterminal propeptide of was analyzed with a sequential modification of a radioimmunoassay" based on the human antigen (Farmos Diagnostica, Oulunsalo, Finland). The antiserum and the assay conditions have been selected so that the authentic propeptide and another, somewhat larger, PIIINP related antigen are detected, but the assay is not sensitive to the smaller degradation products of the propeptide. With this method the standards and samples of biologic fluids give parallel inhibition curves. We previously demonstrated that this assay reflects the synthesis of type III procollagen in human wounds.7"2 The actual laboratory procedure was comparable to that of the PICP assay described above. The volumes used were 0.1 mL for antiserum, 0.1 mL for standard, and 0.2 mL for samples. Dilutions of 1:20 to 1:100 were suitable for the first- to third-day samples, but up to 1:8000 (usually 1:4000) dilutions of the seventh-day samples were necessary. The intra-assay coefficient of variation was 4%.
type III procollagen (PIIINP)
Gel Filtration Analysis of Wound Fluid PICP
Gel filtration of 100-,uL samples of the first-day and fourth-day wound fluid and a 50-,uL sample of the seventhday wound fluid was carried out on a column (1.5 X 110 cm) of Sephacryl S-300 (Pharmacia, Uppsala, Sweden) equilibrated in 0.2 mol/L (molar) (NH4)HCO3, pH 7.9. Seventy fractions of 2.5 mL were collected from each sample. Aliquots (400, 100, and 25 ,uL) of each fraction of the first-, fourth-, and seventh-day wound fluid, respectively, then were analyzed with the PICP radioimmunoassay using the conditions described above.
COLLAGEN TYPES IN WOUND FLUID
Vol. 213 - No. I
Clinical Variables The clinical variables recorded included the type and the interval from any previous operation and specifically the operations through the same incision, the staging of the tumor, operative blood loss, duration of the operation, postoperative complications, the hematocrit value, and the hemoglobin and serum albumin concentrations.
Statistical Analysis The statistical analyses were done with Statgraphics® Software (STSC, Inc., Rockville, MD). The results of wound fluid PICP and PIIINP concentrations are given as means with standard deviations (SD) or with standard errors as indicated in the text and the figures. Wilcoxon signed rank test for paired observations was used when comparing later concentrations of PICP and PIIINP with those of day 1, p < 0.05, evaluated from standard tables for small samples, was considered as statistically significant. The Spearman rank correlation coefficient was calculated when clinical and sampling variables were compared to the concentrations of PICP and PIIINP. It also was used for comparisons between the PICP and PIIINP concentrations on the different postoperative days. Results PICP and PIIINP in Wound Fluid The mean concentration of PICP in wound fluid on day 1 was 207 ± 92 (SD) ,g/L. Already on day 2 it was 908 ± 469 ,ug/L (t = 0, z = 3.8, p < 0.001), and 3246 100 80 r-'
I
60 D- 40 _
u0L
24 1
20 F -J
_-- 16
%j 12 0-
z _8
CL
4
1
2
3
4
5
6
7
DAYS AFTER SURGERY FIG. 2. Effect of time on the concentration of the aminoterminal propeptide of type III procollagen (PIIINP) in wound fluid of patients operated on for colorectal cancer (mean, SE).
± 2858 ,ug/L on day 3 (Fig. 1). On day 7 the mean concentration had reached a level 380 times that of day 1 (79,330 ± 54,151 ,ug/L). The individual differences were great, e.g., at day 7 the range was from 19,750 to 198,813 jug/L. The mean PIIINP concentrations (Fig. 2) changed more slowly, no increase being found between the first 2 days (70 ± 61 u g/L on day 1 and 86 ± 88 ,ug/L on day 2; t = 64, z = 1.2, p = 0.2), but on day 3 the concentration was increased to 180 ± 129 ,ug/L (t = 0, z = 3.48, p
