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Wound healing in the dog
Wound Healing in the Dog Radioisotope
Studies of Developing
Connective
Tissue and Fluid in an Artificial Dead Space JOHN A. SCHILLING,
M.D., Oklahoma
RElTY N. WHITE, MS., Oklahoma M. S. LOCKHART, B.S., Oklahoma H. M. SHURLEY, B.A., Oklahoma
Wound healing is a primary survival mechanism which has been the subject of a vast amount of research. Much of the published work in the field has been reviewed recently [l] where it was pointed out that a great deal of the newer information on cellular mechanisms has a direct bearing on the understanding of repair processes. Likewise, the use of wound models has revealed important knowledge about basic growth phenomena. This report concerns the latter, namely, the use of a wound model which is uniquely suited to studies with radioisotopes, and which has provided valuable information concerning the origin and utilization of specific metabolites during the healing of a wound. A stainless steel wire mesh cylinder implanted subcutaneously creates an artificial dead space within which extracellular fluid and wound connective tissue accumulate. In comparison to the animal’s serum, “wound fluid” has been found to contain a greater proportion of low molecular weight proteins like albumin and a proportionately higher concentration of carbohydrate-containing proteins like alpha-l glycoprotein [Z]. The wound granulation tissue of the implanted cylinder has been analyzed chemically [3] and histochemically [4] and was found to consist mainly of protein, the major type of which is collagen. There are smaller amounts of carbohydrates in the
From the Department of Surgery, homa Medical Center, Oklahoma This worw was supported by the search and Development Command,
330
The University of OklaCity, Oklahoma 73104. U. S. Army Medical ReContract NO. MD-2811.
City, Oklahoma
City, Oklahoma City, Oklahoma City, Oklahoma
ground substance which make up less than 4 per cent of the total dry weight of the tissue, but they have been found to undergo a high rate of metabolism, especially in the first and second weeks of tissue development. Radioisotope studies of carbohydrate-containing proteins of this granulation tissue and wound fluid were carried out in rats using carbohydrate precursors, (lJ4C)glucosamine [5,6], and (1-14C)galactosamine [7’], and using s5S in dogs [8]. When precursors were injected intraperitoneally, they were transported to and preferentially concentrated in the wound where rapid metabolic processes were taking place. Wound metabolites need not be blood-borne. When precursors were injected locally into implanted cylinders, they were used directly by cells in the wound area [5,6]. Potentially, this may have significance for wound treatment. The independence of the wound because of local syntheses was further demonstrated by local incorporation of radioactivity into carbohydrate-containing proteins which took place despite the absence of the liver, the biosynthetic site of serum glycoproteins [5,6]. When structured in tubular form in viva, cylinder-induced connective tissue has been tolerated for many months as aortic replacements in dogs [8,9].Electron microscopy studies of wound tissue from implanted cylinders in dogs have revealed specific data concerning the collagen molecule as well as information about the atypical and characteristic lamellar structure of the canine fibrocollagenous tube which forms within the implanted cylinder [1,10,11]. The present series of experiments were deThe
American
Journal
of Surgery
Wound
signed to study synchronously the metabolism of both collagen and ground Substance constituents of wound tissue and to relate this to changes which take place during the developmental aging of the tissue. The local and systemic influences were examined in relation-
ship to changes in fluid and wound tissue contents. The collagen-labelling precursor selected was
(UJ4C) proline.
(l-l%)
Glucosamine
was
used to study the complex polysaccharides and (1J4C> glycine was used for protein-labelling in general. Dogs were employed to allow study of wound tissue of varying age within the same animal. It was also possible to monitor the labelling pattern in blood components over a long period of time in the same animal. The disadvantages of the canine experimental model lie in the quantity of required radioisotope and the cost per animal. The latter was restrictive in the number of animaXs used per group.
Material and Methods Carefully selected young adult healthy mongrel male dogs with an average weight of 23 kg. were used. Twenty cylinders were implanted 14,LZl subcutaneously on the dorsum of each of nine animals as outlined in Table I. Cylinders of heavy No. 46 stainless steel wire mesh were employed to collect tissue for nine weeks or longer. Cylinders of a lighter No. 40 mesh were used for harvesting tissue younger ‘;han nine weeks. Three implantation schedules were followed. In each of the three dogs on the first schedule ten cylinders of the heavier mesh type were implanted
TABLE
Group” A
6
c
I
Schedule of Cylinder Implant and Radioisotope Injection in Dogs
Dog Number 1 2
Number of Weeks of Number Of Implantation of Cylindetst Implanted Cylinders Light Mesh Heavy Mesh 20
1
3
20 20
2 3
4 5 6 7 8 9
20 20 20 20 20 20
1 2 3 1 2 3
9
13.5 17.5 9 13.5 17.5 9 13.5 17.5
* Group A received (I-W)glucosamine. group B received (I-W)glycine, and group C received (U-‘4C)proline. f Ten cylinders of light No. 40 wire mesh and ten cylinders of heavy No. 46 wire mesh were implanted in each dog giving a total of twenty cylinders. Vol. 117. March U69
Healing
in the Dog
for 17.5 weeks prior to sacrifice to be followed subsequently with ten cylinders of the lighter mesh tYPe for three weeks prior to sacrifice. The three dogs on the second schedule also had implantation first with ten cylinders of the heavier mesh type and sukquently with ten cylinders of the lighter mesh type for periods of 33.5 weeks and two weeks, respectively, prior to sacrifice. The three dogs on the third scheduIe had cylinders implanted in a similar manner, but for periods of nine weeks and one week, respectively, prior to sacrifice. These nine dogs were divided into three groups for injection of the three different radioisotopes. The three groups were designated as A, B, and C. Group A dogs were given (I-%)gIucosamine, group B dogs were given (1-W) glycine, and group C dogs were given (U-W)proline. The three radioisotope groups were established by selecting one animal at random from each of the three aforementioned implantation schedules. By this method each radioisotope group of three dogs was made up of one dog with cylinders implanted for one week and nine weeks, a second dog with cylinders implanted for two weeks and 13.5 weeks, and a third dog with cylinders implanted for three weeks and 17.5 weeks. Thus, each radioisotope group of dogs contained fibrocollagenous tissue and wound fluid of one, two, three, nine, thirteen and a half, and seventeen and a half weeks of age within the implanted cylinders. The number of cylinders on each dog was sufficient to allow duphcate and triplicate anaIyses, thus com.pensating for the necessary limitation of the total number that could be used in each group. The age-related accumulation of fibrocollagenous tissue, its weight, total protein, and collagen content was established by implantation of similar cylinders, by similar technics in a separate control colony of twenty&eight dogs. Radioisotopes* were given intraperitoneally once daily for a period of four days prior to sacrific~. In the glucosamine and proline groups each dog received a total of 200 PC. (50 ~c./10 ml. of saline solution/day). In the glycine group each dog received a total of 400 PC. (100 FL/IO ml. of saline solution/day). Blood samples were collected at 0.5, three, six, twenty-four, seventy-two, and ninety-six hours after the first radioisotope injection in each dog. Injections were given in the morning after collection of blood and urine. At sacrifice dogs were anesthetized with NembutaP. Cylinders were excised, aspirated for wound fluid contents, rinsed in saline solution and tap water, and Iyophilized. Animals were then exssnguinated by carotid cannulation. Abdominal organs were removed, perfused with saline solution, and stored at -20°C. * Obtained from the New England Boston, Massachusetts.
Nuclear
CorPJratiM1, 331
chromatographed on paper in a butanol-pyridine0.1 N HCl solvent system and stained with an 0-aminodiphenyl preparation [IT]. At the same time, amino acids were chromatographed two dimensionally in butanol-formic acid-water and in propanol-water and were then stained with ninhydrin. Stained spots from chromatograms were analyzed for radioactivity by liquid scintillation counting.
Results
00 6
24
49
72
96
HOURSAFTERFIRST INJECTION
lime course of incorporation of various Fig. 1. precursors into nondialyzable portion of blood serum as a result of repeated injections of (I-lIC)glucosamine (A); (l-14C)gjycine (0 ); or (C-14C)proline (0 ). Radioactivity expressed as counts per minute x 10’ per ml. x total ml. blood volume. (Blood volume estimated at 4 per cent of body weight.). Each point represents mean from three dogs. Arrows indicate times of injections.
The following analytical determinations were made : protein-bound hexcse by a tryptophan method [IJ], hexosamine by a modification of the Boas method [14], protein by the Lowry method 1151, and hydroxyproline by the method of Neuman and Logan [16]. A Nuclear-Chicago gas-flow system was used to measure radioactivity of samples air-dried on stainless steel planchets. Radioactivities of sera and wound fluids were measured before and after dialysis against tap water. To remove non-protein bound radioactivity, connective tissue and liver were sequentially extracted with 7 per cent trichloroacetic acid, 95 per cent ethanol, and 96 per cent ethanol-ether of 1: 1 proportions at 45’~. Measured portions of the final suspension in water were placed on planchets for assay of radioactivity. Aliquots of serum and wound fluid were subjected to paper electrophoresis studies with Spinco Model R electrophoresis cells [z]. Proteins were stained with bromphenol blue and quantitated with a Spinco Analytrola. A Nuclear-Chicago liquid scintillation system was used to measure radioactivity of proteins on unstained paper sections in liquid scintillation solvent. Hydrolysates of sera and wound fluid proteins were applied to columns of Ag50-X12 resin. Neutral sugars, hexosamines, and amino acids were eluted in sequence 171. TO isolate radioactive substances, carbohydrates were 332
The protein-bound radioactivity measured in the blood of animals at intervals after radioisotope administration is shown in Figure 1. Reflecting either a smaller pool size or a faster turnover, the injection of (l-r4C)glucosamine resulted in a level of serum radioactivity three times higher than that obtained by the injection of either (l-l%) glycine or (UJ4C)proline. The radioactivity in urine excreted by dogs given (l-l’(Z) glucosamine was also greater than the radioactivity in urine excreted by dogs injected with (l-r4C)glycins or (UJ4C)proline. (Table II.) The greatest amount of administered radioactivity in the serum appeared six hours after the injection of all three radioisotopes. After hydrolysis of the proteins of sera and wound fluid, examination of component sugars by paper chromatography revealed very little conversion of glucosamine to other sugars since most of the Cl4 from (1J4C)glucosamine was located in glucosamine spots on chromatograms. Some 7 per cent of the radioactivity
TABLE II
Hours after First Injection 0.5 3 6 24
48 72 96
Recovery of Administered Radioactivity in Serum and Urine Percentage of Administered Radioactivity* (l-14C) (l-14C) ($!zrz Glycine Glucosamine Serum 3.6 24.3 31.9 23.5 20.3 17.9 12.0
Urine Serum Urine Serum
10.8 5.4 3.7 1.8
3.2 4.8 5.0 4.3 3.4 2.9 2.5
1.4 2.0 1.8 2.0
3.2 6.5 6.6 5.1 4.0 3.8 3.3
Urine
0.5 0.8 1.1 0.6
*Animals were given radioisotope injections at twentyfour hour intervals (see methods). Percentage of administered radioactivity was based on ratio of pc. found in total volume to cumulative total of pc. injected at time indicated. Serum volume was estimated as 4 per cent of body weight. Urine volume was that collected in each twenty-four hour period. The American
Journal of Surgery
Wound
TABLE
III
Healinginthe Dog
Specific Radioactivityof Proteins Separated by Paper Electrophoresis CountsperMinuteper Mg. Protein inElectrophoretic Components
Group* A
B
C
No.of Samplest
Globulins Albumin
alpha-l
alpha-2
beta-l
6 Sera 3 Fluids(new)
55 f 14$ 17zk 4
1895 f 419 3043 * 218
1563 f 218 1197 i 399
3 Fluids (old) 6Sera
12k 5 87 f 19
1571* 375 263 + 42
513 k 182 270~ 49
178 f 301*
3 Fluids(new) 3 Fluids(old) 6 Sera 3 Fluids (new) 3 Fluids (old)
112 f 14 37& 8 64 f 19 58 f 25 27zk 8
588~11 50 168& 36 203 i 65 476 z.t272 165 zt 90
225 f 64 73;t 1 168 * 40 176& 88 74* 33
197 f 61 74* 17 192 f 47 162 f 105 58z1z 34
*Group A dogs given(l-"C)glucosamine,groupB dogsgiven
713 =I=138 289 f 45 80 66
Vol. 117. March 1969
271 f 17 130 f. 26 67 f 37 270 f 72 205 zt 32 59 f 14 159 -+.40 132 rt 66 42 f 21
gamma 171 f 130 f 70 f 307 f 243 + 77 f 199 f 151 f 66 f
26 25 10 82 14 24 43 49 15
Total 4668 f 524 4807*400 2411 f 665 1496 f 317 1571* 298 489zt 66 983 f 246 1141 f 610 433 f 195
(I-"C)glycine,and group C dogs given(U-14C)proline.
t Fluid samples (new) were from cylinders implanted one, two, and three weeks. implanted approximately nine, thirteen, and eighteen weeks (see methods). t Values given are means + standard error. -
was found in galactosamine and 3 per cent in mannosamine. With the injection of (UJ*C)proline, essentially all of the radioactivity of sera and fluids was located in proline spots of chromatograms. Glycine was, however, converted to other amino acids. Approximately 40 per cent of the radioactivity was found in serine of sera and wound fluids after the injection of (lJ*C)glycine. The fate of injected radioactivity into specific proteins of sera and wound fluids is shown in Table III. The radioactivity of both serum and fluid protein fractions separated by paper electrophoresis in dogs given (l-l*C) glucosamine was detected in highest amounts in the alpha globulins. Lowest specific activity was located in the albumin fraction. Most of the globulins and albumin of serum are synthesized in the liver [IS], but albumin has a very low carbohydrate content. In dogs receiving Cl4 amino acids, the radioactivity of serum proteins was distributed rather nonspecifically in all globulin fractions; but again albumin had the lowest specific activity. In contrast to the serum, the fluid radioactivity in these dogs tended to be concentrated more in the alpha-l globulins, findings similar to those in the fluid of dogs injected with (lJ*C) glucosamine. The fluid found within the cylinders of the dogs was in contact with the inner surface of the newly synthesized connective tissue for varying lengths of time depending upon the period of cylinder implant. In all three radioisotope groups of dogs, the distribution of ra-
beta-2
Fluid samples (old) were from cylinders
dioactivity among the proteins of fluid separated by electrophoresis was not found to vary with the time-length of cylinder implant. It was always found in highest amounts in the alpha-l globulins. The total amount of radioactivity, however, did change with time. The total radioactivity of protein fractions was shown to be highest in fluid of the younger tissue which was quite similar to that in serum, but the fluid radioactivity subsequently decreased to about one half or less in cylinders implanted for nine weeks 01‘more. The total protein and hydroxyproline content of the induced connective tissue within cylinders implanted for varying times was examined. The results in Figure 2 show that the tissue has a characteristically age-related accumulation of collagen. There is a rapid linear increase in collagen in the younger tissue up to four weeks, after which the collagen synthesis slows down. In contrast to the collagen, the total protein content of the tissue was highest in the early stages of development. Measurement of Cl* uptake by this connective tissue at each developmental age, plotted in Figure 3, showed that tissue synthesized in the first three weeks contained a higher amount of protein-bound radioactivity than did the connective tissue produced in the later nine week period. The same was true in the fluid of the cylinders. So that while the radioactivity in the fluid may very well originate from the serum, it is under the influence of the local cellular metabolism. This is an important con333
Schilling et al
O-123
16 AGE
OF TISSUE
30
32
IN WEEKS
Age of tissue in weeks. The age-related Fig. 2. changes in total protein (0) and collagen ( l ) content of connective tissue synthesized de novo in cylinders implanted in dogs. Amount of collagen per unit of dry weight based on hydroxyproline content of tissue of each age. Total protein data and collagen data were based on values from a separate control colony of twenty-eight dogs.
cept which should be understood and appreciated by surgeons as they handle tissues during surgical procedures and as they care for wounds postoperatively. Comments Although the use of radioisotopes represents a valuable tool for metabolic studies, it seems not to have been utilized to its fullest potential in regard to wound healing. It was attempted in this study to inject labelled precursors and follow their systemic route to the wound, to determine the origin of wound metabolites, and, if possible, to bring into focus some metabolic relationship between the major constituent, collagen, and the ground substance of wound connective tissue. In an earlier investigation with rats [Z], it was shown that wound tissue may derive some of the hexosamine used for biosynthesis of ground substance components from plasma protein. High levels of radioactivity, found in small alpha globulin glycoproteins which are capable of diffusing into extracellular fluids, were thought to be of possible significance in this regard. Indeed, the main source of radio334
123
9
I4
18
AGE OF TISSUE IN WEEKS Fig. 3. Rate of incorporation of radioactivity in cylinder connective tissue with relation to age of tissue. Tissue was removed twenty-four hours after last of four (one per day) injections. Specific activity expressed as counts per minute x 1V per mg. hexosamine for dogs given (I-14C)glucosamine (A) and counts per minute x 10” per mg. hydroxyproline for dogs given (I-14C)glycine ( 0 ) or (U-14C)proline ( 0 ).
activity for the wound site which was encountered in the present study was serum. The radioactivity derived from each of the radioisotopes was conveyed to cylinders in serum protein form. The radioactivity from injected amino acids was distributed rather uniformly in the serum proteins. Only in the dogs injected with glucosamine was there a localization of radioactivity in a specific protein fraction, that is, the alpha-l globulins. In the fluid of the wound, however, specific activity in all three radioisotope groups was highest in the alpha-l globulins. The total amount of radioactivity in protein fractions of wound fluid decreased in older tissue ; but the location of highest specific activity in alpha-l globulin fractions of wound fluid did not change with the age of the tissue. How the cells in the wound locale were able to utilize these radioactive proteins and glycoproteins for their major activity-synthesis of collagen and ground substance-is not known. It is known, however, that levels of serum alpha globulins are altered as a result of injury [19]. The seromucoid fraction of serum, for The American
Journaj
of Surgery
Wound Healing in the Dog
example, is a mixture of glycoproteins and is known to migrate electrophoretically with the alpha globulins, some of which might readily diffuse through capillary walls and find way into the extracellular fluid. In an earlier study with dogs, wound fluid and sera were found to contain similar amounts of the seromucoid fraction [2]. Also, there is evidence that the seromucoid fraction is involved in serum protein changes resulting from injury [20,21]. It was concluded that the present study definitely showed the presence of small glycoproteins of high specific radioactivity in newly formed wound tissue of the dog. It aIso seemed likely that the serum alpha globulins were the main source of carbohydrate used by this wound tissue. Examination of the composition of the wound tissue shows an inverse relationship between the total protein content and the collagen content as the tissues develop after implantation of the cylinder. Although the total protein was at its very highest at one week, collagen content was at its very lowest. This high content of noncollagen protein in the initial development of the tissue is very likely due to serum or serum-like proteins because of the close association of serum and considerable quantities of fluid found within the wound area at this time. This illustrates the very early and dominant influence of serum on wound healing. The age-related increase in collagen is both unique and typical of wound tissue. In other tissues an increase in collagen with developmental age is mostly an embryonic phenomenon since these tissues after birth, except perhaps in the uterus, show either no change or a fall in collagen concentration with age [22]. But, throughout adult life the body, under both normal and many abnormal circumstances, never seems to Iose its embryonic ability to produce the collagen of wound connective tissue. With regard to radioisotope usage, wound tissue of all developmental ages in this study utilized both (lJ4C)glycine and W-14C)Proline, but the youngest tissue exhibited the highest specific activity. A higher metabolism was noted in the first three weeks of growth of the tissue with a rapid linear decrease in specific activity of collagen at this time. A definite change in rate of incorporation occurred around four weeks as the specific activity of the older tissue was definitely lower. These obVol.117,March 1969
servations on collagen metabolism during development in the same animal have some parallel to the differences noted by Neuberger, Perrone, and Slack [23] in their comparison of collagen metabolism of young and adult animals. CoIlagen fractions, made soluble by djfferent means and in different stages of maturity, have been found to vary in their rate of amino acid incorporation. But, on the whole, it has been said that collagen metabolism in most parts of the body is relatively inert in the adult. Recent evidence has shown that this is not true [I]. Studies of chronic labelling of wound tissue with radioisotopes by Klein and Weiss [.%I have led them to believe that the turnover of mature collagen is the result of reutilization of collagen subunits. Thus, the metabolic inertness of collagen is really only apparent. The classic isotope turnover study measures only the net change in incorporation so that the mature fibrous collagen appears to lose the incorporated radioisotope very slowly. Not only does this show that collagen is not really inert and is indeed dynamic in the adult, but this reutilization of collagen subunits shows the extremely important role of the cells and tissues in the locale of the wound. The incorporation of ( 1-14C) glucosamine by noncollagenous protein of the ground substance of the dogs also occurred very rapidly in the young connective tissue. A high Ievel of incorporation of glucosamine was previously observed in studies with rat wound tissue of two weeks’ developmental age [1,5,6]. The rate of synthesis of the noncollagenous proteins in the older tissue was also similar to that of collagen, being lower and more constant, but nevertheless remaining higher than that of collagen in the same age of tissue. The latter may be related to a higher turnover of mucopolysaccharides and possibIy glycoproteins than that of collagen. Part of the incorporation of glucosamine by young connective tissue of dogs was due no doubt to glycoproteins since they are known to predominate in the early wound [~,zx]. Another portion of the (1-14C)glucosamine was very likely incorporated into mueopolysaccharides of the ground substance since this was specifically demonstrated in an earlier study with rats [5,6]. The parallel incorporation of both the carbohydrate and amino acid precursors, and especially the change in rate which occurs simultaneously about the fourth week of tissue development, implies their in335
Schilling et al.
terrelation during the production of wound tissue and in the maturation of collagen. These changes in rates of synthesis during wound healing have been observed with radioisotope studies of nucleic acids [26,27]. The rate of formation of both RNA and DNA has been found to be more rapid in the early?tages of wound tissue regeneration. In past years and decades, there have been numerous expressions and generalities concerning the autonomy of the local wound and the priority of its metabolic demands. These studies would indicate that the local wound, particularly early in its time course, does have priority in its metabolic demand for glucosamine and to a lesser extent proline. Because of its widespread distribution and its interconversion into several other amino acids, the priority of the metabolic demand by the wound for glycine is difficult to assess. Obviously, these are only three of a very large number of substances that are required in growth, regeneration, and repair, and additional data are required for more definitive statements concerning priority of the metabolic demands of a wound for specific constituents.
growth period showed a higher level of incorporation than did collagen which was maturing in the nine to eighteen weeks’ growth period. Similar to the amino acid uptake by collagen, the carbohydrate-containing proteins of wound tissue exhibited a change from fast to slow in rate of incorporation of ( 1J4C) glucosamine. The significance of the change in rate of incorporation of radioisotopes by both the carbohydrates and amino acids which took place at about the fourth week in the wound tissue growth is not known. However, it provides additional evidence for the metabolic interrelation thought to occur between the ground substance and collagen during the maturation of the latter. References
2.
3.
Summary Radioisotopes were used to study the granulation-type connective tissue induced within dead spaces created by the subcutaneous implantation of stainless steel wire mesh cylinders on the backs of dogs. The incorporation of radioactivity by proteins and carbohydratecontaining proteins of the newly developing wound tissue and its associated wound fluid was compared by the use of the precursors (1J4C)glucosamine, (l-l%)glycine, and (U“C)proline. After intraperitoneal injection, the major portion of radioactivity was conveyed in protein-bound form via serum to the site of new wound tissue synthesis. With all three radioactive precursors, the highest specific activity in wound fluid proteins of the wound site was found electrophoretically in the alpha-l globulin fractions. (l-l%) Glucosamine invoked a higher specific activity in serum, wound fluid, and wound connective tissue than did either (1-14C)glycine or (U14C)proline. The rate of incorporation of amino acid radioisotopes by developing granulation tissue was related to the maturity of the collagen at each developmental age. Young collagen produced in the first three weeks’ 336
healing. Physiol. Rev., 48 ~374, 1968. WHITE, B. N., SHETLAR, M. R., SHURLEY, H. M., and SCHILLING,J. A. Wound healing: investigation of proteins, glycoproteins, and lipids of experimental wound fluid in the dog. Proc. Sot. Exper. Biol. & Med., 101:353,1969. SHETLAR, M. R., LACEFIELD,E. G., WHITE, B. N., and SHILLING,J. A. Wound healing: glycoproteins of wound tissue. I. Studies of hexosamine, hexose and uranic acid content. Proc. Sot. Exper. Biol. & Med., 100: 501, 1959. SCHILLING, J. A., JOEL, W., and SHURLEY, H. M. Wound healing: a comparative study
1. SCHILLING, J. A. Wound
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of the histochemical changes in granulation tissue contained in stainless steel wire mesh and polyvinyl sponge cylinders. SUVgery, 46 :702,1959. 5.
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WHITE, B. N. Incorporation of Glucosamine1-C” by Rat Connective Tissue. (M. S. Thesis). Oklahoma Ciy, 1963. University of Oklahoma Graduate School of Medicine. WHITE, B. N., SHETLAR, M. R., SHURLEY, H. M., and SCHILLING,J. A. Incorporation of (l-l%) glucosamine into mucopolysaccharides of rat connective tissue. Biochim. et biophgs. acta, 101:97,1965. WHITE, B. N., SHETLAR, M. R., SHURLEY, H. M., and SCHILLING,J. A. Incorporation of D- (l-l%) galactosamine into serum proteins and tissues of the rat. Biochim. et biophys. acta, 101:269,1966. SCHILLING,J. A., SHURLEY,H. M., JOEL, W., WHITE, B. N., and BRADFORD,R. H. Abdominal aortic grafts: use of in tivo strut?tured autologous and homologous fibrocollagenous tubes. Ann. SUT~., 169:819, 1964. The American
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Wound Healing in the Dog
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RICHTER,K. M., and WHITE, B. N. Fibrocollagenous tubes structured in vivo. Morphological and biological characteristics. Arch. Path., 71:548,1961. 10. RICHTER,K. M., SCHILLING,J. A., and SHURLEY, H. M. UItrastructure of experimentally induced scar issue. J. Appl. Ph2/sioE.,
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RICHTER,K. M., SCHILLING,J. A., and SHURLEY, H. M. A possible E.M. demonstration of part of the intramolecular character of the tropocollagcn molecule. In : Proceedings of the International Congress of Electron Microscopy (Sixth) _Tokyo, 1966, Maruzen. 12. SCHILLING, J. A. Technique of implanted steel mesh cylinders in studies of fibroplasia. In: Wound Healing, Proceedings of a Workshop p. 2’70. Edited by Levenson, S. M., Stein, J. M., and Grossblatt, N. Washington, D. C., 1966. National Academy of Sciences-National Research Council. 13. SHETLAR,M. R., FOSTER,J. V., and EVERETT, M. R. Determination of serum polysaccharides by tryptophane reaction. Proc. SOC. Exper.
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MILLER, L. L., BLY, C. G., WATSON, M. L., and BALE, W. F. The dominant role of the liver in plasma protein synthesis, J. Exper. Med., 94:431, 1951. PETERMANN,M. L. Alterations in plasma protein patterns in disease. In: The Plasma Proteins, vol. 2, p. 309. Edited by Putman, F. W. New York, 1960. Academic Press, Inc. NEUHAUS, 0. W. and LIU, A. Biochemical significance of serum glycoproteins. III. Hepatic production of al and a2 globulins responding to injury. PTOC. Sot. Exper.
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337
Studies of Developing
Connective
Tissue and Fluid in an Artificial Dead Space JOHN A. SCHILLING,
M.D., Oklahoma
RElTY N. WHITE, MS., Oklahoma M. S. LOCKHART, B.S., Oklahoma H. M. SHURLEY, B.A., Oklahoma
Wound healing is a primary survival mechanism which has been the subject of a vast amount of research. Much of the published work in the field has been reviewed recently [l] where it was pointed out that a great deal of the newer information on cellular mechanisms has a direct bearing on the understanding of repair processes. Likewise, the use of wound models has revealed important knowledge about basic growth phenomena. This report concerns the latter, namely, the use of a wound model which is uniquely suited to studies with radioisotopes, and which has provided valuable information concerning the origin and utilization of specific metabolites during the healing of a wound. A stainless steel wire mesh cylinder implanted subcutaneously creates an artificial dead space within which extracellular fluid and wound connective tissue accumulate. In comparison to the animal’s serum, “wound fluid” has been found to contain a greater proportion of low molecular weight proteins like albumin and a proportionately higher concentration of carbohydrate-containing proteins like alpha-l glycoprotein [Z]. The wound granulation tissue of the implanted cylinder has been analyzed chemically [3] and histochemically [4] and was found to consist mainly of protein, the major type of which is collagen. There are smaller amounts of carbohydrates in the
From the Department of Surgery, homa Medical Center, Oklahoma This worw was supported by the search and Development Command,
330
The University of OklaCity, Oklahoma 73104. U. S. Army Medical ReContract NO. MD-2811.
City, Oklahoma
City, Oklahoma City, Oklahoma City, Oklahoma
ground substance which make up less than 4 per cent of the total dry weight of the tissue, but they have been found to undergo a high rate of metabolism, especially in the first and second weeks of tissue development. Radioisotope studies of carbohydrate-containing proteins of this granulation tissue and wound fluid were carried out in rats using carbohydrate precursors, (lJ4C)glucosamine [5,6], and (1-14C)galactosamine [7’], and using s5S in dogs [8]. When precursors were injected intraperitoneally, they were transported to and preferentially concentrated in the wound where rapid metabolic processes were taking place. Wound metabolites need not be blood-borne. When precursors were injected locally into implanted cylinders, they were used directly by cells in the wound area [5,6]. Potentially, this may have significance for wound treatment. The independence of the wound because of local syntheses was further demonstrated by local incorporation of radioactivity into carbohydrate-containing proteins which took place despite the absence of the liver, the biosynthetic site of serum glycoproteins [5,6]. When structured in tubular form in viva, cylinder-induced connective tissue has been tolerated for many months as aortic replacements in dogs [8,9].Electron microscopy studies of wound tissue from implanted cylinders in dogs have revealed specific data concerning the collagen molecule as well as information about the atypical and characteristic lamellar structure of the canine fibrocollagenous tube which forms within the implanted cylinder [1,10,11]. The present series of experiments were deThe
American
Journal
of Surgery
Wound
signed to study synchronously the metabolism of both collagen and ground Substance constituents of wound tissue and to relate this to changes which take place during the developmental aging of the tissue. The local and systemic influences were examined in relation-
ship to changes in fluid and wound tissue contents. The collagen-labelling precursor selected was
(UJ4C) proline.
(l-l%)
Glucosamine
was
used to study the complex polysaccharides and (1J4C> glycine was used for protein-labelling in general. Dogs were employed to allow study of wound tissue of varying age within the same animal. It was also possible to monitor the labelling pattern in blood components over a long period of time in the same animal. The disadvantages of the canine experimental model lie in the quantity of required radioisotope and the cost per animal. The latter was restrictive in the number of animaXs used per group.
Material and Methods Carefully selected young adult healthy mongrel male dogs with an average weight of 23 kg. were used. Twenty cylinders were implanted 14,LZl subcutaneously on the dorsum of each of nine animals as outlined in Table I. Cylinders of heavy No. 46 stainless steel wire mesh were employed to collect tissue for nine weeks or longer. Cylinders of a lighter No. 40 mesh were used for harvesting tissue younger ‘;han nine weeks. Three implantation schedules were followed. In each of the three dogs on the first schedule ten cylinders of the heavier mesh type were implanted
TABLE
Group” A
6
c
I
Schedule of Cylinder Implant and Radioisotope Injection in Dogs
Dog Number 1 2
Number of Weeks of Number Of Implantation of Cylindetst Implanted Cylinders Light Mesh Heavy Mesh 20
1
3
20 20
2 3
4 5 6 7 8 9
20 20 20 20 20 20
1 2 3 1 2 3
9
13.5 17.5 9 13.5 17.5 9 13.5 17.5
* Group A received (I-W)glucosamine. group B received (I-W)glycine, and group C received (U-‘4C)proline. f Ten cylinders of light No. 40 wire mesh and ten cylinders of heavy No. 46 wire mesh were implanted in each dog giving a total of twenty cylinders. Vol. 117. March U69
Healing
in the Dog
for 17.5 weeks prior to sacrifice to be followed subsequently with ten cylinders of the lighter mesh tYPe for three weeks prior to sacrifice. The three dogs on the second schedule also had implantation first with ten cylinders of the heavier mesh type and sukquently with ten cylinders of the lighter mesh type for periods of 33.5 weeks and two weeks, respectively, prior to sacrifice. The three dogs on the third scheduIe had cylinders implanted in a similar manner, but for periods of nine weeks and one week, respectively, prior to sacrifice. These nine dogs were divided into three groups for injection of the three different radioisotopes. The three groups were designated as A, B, and C. Group A dogs were given (I-%)gIucosamine, group B dogs were given (1-W) glycine, and group C dogs were given (U-W)proline. The three radioisotope groups were established by selecting one animal at random from each of the three aforementioned implantation schedules. By this method each radioisotope group of three dogs was made up of one dog with cylinders implanted for one week and nine weeks, a second dog with cylinders implanted for two weeks and 13.5 weeks, and a third dog with cylinders implanted for three weeks and 17.5 weeks. Thus, each radioisotope group of dogs contained fibrocollagenous tissue and wound fluid of one, two, three, nine, thirteen and a half, and seventeen and a half weeks of age within the implanted cylinders. The number of cylinders on each dog was sufficient to allow duphcate and triplicate anaIyses, thus com.pensating for the necessary limitation of the total number that could be used in each group. The age-related accumulation of fibrocollagenous tissue, its weight, total protein, and collagen content was established by implantation of similar cylinders, by similar technics in a separate control colony of twenty&eight dogs. Radioisotopes* were given intraperitoneally once daily for a period of four days prior to sacrific~. In the glucosamine and proline groups each dog received a total of 200 PC. (50 ~c./10 ml. of saline solution/day). In the glycine group each dog received a total of 400 PC. (100 FL/IO ml. of saline solution/day). Blood samples were collected at 0.5, three, six, twenty-four, seventy-two, and ninety-six hours after the first radioisotope injection in each dog. Injections were given in the morning after collection of blood and urine. At sacrifice dogs were anesthetized with NembutaP. Cylinders were excised, aspirated for wound fluid contents, rinsed in saline solution and tap water, and Iyophilized. Animals were then exssnguinated by carotid cannulation. Abdominal organs were removed, perfused with saline solution, and stored at -20°C. * Obtained from the New England Boston, Massachusetts.
Nuclear
CorPJratiM1, 331
chromatographed on paper in a butanol-pyridine0.1 N HCl solvent system and stained with an 0-aminodiphenyl preparation [IT]. At the same time, amino acids were chromatographed two dimensionally in butanol-formic acid-water and in propanol-water and were then stained with ninhydrin. Stained spots from chromatograms were analyzed for radioactivity by liquid scintillation counting.
Results
00 6
24
49
72
96
HOURSAFTERFIRST INJECTION
lime course of incorporation of various Fig. 1. precursors into nondialyzable portion of blood serum as a result of repeated injections of (I-lIC)glucosamine (A); (l-14C)gjycine (0 ); or (C-14C)proline (0 ). Radioactivity expressed as counts per minute x 10’ per ml. x total ml. blood volume. (Blood volume estimated at 4 per cent of body weight.). Each point represents mean from three dogs. Arrows indicate times of injections.
The following analytical determinations were made : protein-bound hexcse by a tryptophan method [IJ], hexosamine by a modification of the Boas method [14], protein by the Lowry method 1151, and hydroxyproline by the method of Neuman and Logan [16]. A Nuclear-Chicago gas-flow system was used to measure radioactivity of samples air-dried on stainless steel planchets. Radioactivities of sera and wound fluids were measured before and after dialysis against tap water. To remove non-protein bound radioactivity, connective tissue and liver were sequentially extracted with 7 per cent trichloroacetic acid, 95 per cent ethanol, and 96 per cent ethanol-ether of 1: 1 proportions at 45’~. Measured portions of the final suspension in water were placed on planchets for assay of radioactivity. Aliquots of serum and wound fluid were subjected to paper electrophoresis studies with Spinco Model R electrophoresis cells [z]. Proteins were stained with bromphenol blue and quantitated with a Spinco Analytrola. A Nuclear-Chicago liquid scintillation system was used to measure radioactivity of proteins on unstained paper sections in liquid scintillation solvent. Hydrolysates of sera and wound fluid proteins were applied to columns of Ag50-X12 resin. Neutral sugars, hexosamines, and amino acids were eluted in sequence 171. TO isolate radioactive substances, carbohydrates were 332
The protein-bound radioactivity measured in the blood of animals at intervals after radioisotope administration is shown in Figure 1. Reflecting either a smaller pool size or a faster turnover, the injection of (l-r4C)glucosamine resulted in a level of serum radioactivity three times higher than that obtained by the injection of either (l-l%) glycine or (UJ4C)proline. The radioactivity in urine excreted by dogs given (l-l’(Z) glucosamine was also greater than the radioactivity in urine excreted by dogs injected with (l-r4C)glycins or (UJ4C)proline. (Table II.) The greatest amount of administered radioactivity in the serum appeared six hours after the injection of all three radioisotopes. After hydrolysis of the proteins of sera and wound fluid, examination of component sugars by paper chromatography revealed very little conversion of glucosamine to other sugars since most of the Cl4 from (1J4C)glucosamine was located in glucosamine spots on chromatograms. Some 7 per cent of the radioactivity
TABLE II
Hours after First Injection 0.5 3 6 24
48 72 96
Recovery of Administered Radioactivity in Serum and Urine Percentage of Administered Radioactivity* (l-14C) (l-14C) ($!zrz Glycine Glucosamine Serum 3.6 24.3 31.9 23.5 20.3 17.9 12.0
Urine Serum Urine Serum
10.8 5.4 3.7 1.8
3.2 4.8 5.0 4.3 3.4 2.9 2.5
1.4 2.0 1.8 2.0
3.2 6.5 6.6 5.1 4.0 3.8 3.3
Urine
0.5 0.8 1.1 0.6
*Animals were given radioisotope injections at twentyfour hour intervals (see methods). Percentage of administered radioactivity was based on ratio of pc. found in total volume to cumulative total of pc. injected at time indicated. Serum volume was estimated as 4 per cent of body weight. Urine volume was that collected in each twenty-four hour period. The American
Journal of Surgery
Wound
TABLE
III
Healinginthe Dog
Specific Radioactivityof Proteins Separated by Paper Electrophoresis CountsperMinuteper Mg. Protein inElectrophoretic Components
Group* A
B
C
No.of Samplest
Globulins Albumin
alpha-l
alpha-2
beta-l
6 Sera 3 Fluids(new)
55 f 14$ 17zk 4
1895 f 419 3043 * 218
1563 f 218 1197 i 399
3 Fluids (old) 6Sera
12k 5 87 f 19
1571* 375 263 + 42
513 k 182 270~ 49
178 f 301*
3 Fluids(new) 3 Fluids(old) 6 Sera 3 Fluids (new) 3 Fluids (old)
112 f 14 37& 8 64 f 19 58 f 25 27zk 8
588~11 50 168& 36 203 i 65 476 z.t272 165 zt 90
225 f 64 73;t 1 168 * 40 176& 88 74* 33
197 f 61 74* 17 192 f 47 162 f 105 58z1z 34
*Group A dogs given(l-"C)glucosamine,groupB dogsgiven
713 =I=138 289 f 45 80 66
Vol. 117. March 1969
271 f 17 130 f. 26 67 f 37 270 f 72 205 zt 32 59 f 14 159 -+.40 132 rt 66 42 f 21
gamma 171 f 130 f 70 f 307 f 243 + 77 f 199 f 151 f 66 f
26 25 10 82 14 24 43 49 15
Total 4668 f 524 4807*400 2411 f 665 1496 f 317 1571* 298 489zt 66 983 f 246 1141 f 610 433 f 195
(I-"C)glycine,and group C dogs given(U-14C)proline.
t Fluid samples (new) were from cylinders implanted one, two, and three weeks. implanted approximately nine, thirteen, and eighteen weeks (see methods). t Values given are means + standard error. -
was found in galactosamine and 3 per cent in mannosamine. With the injection of (UJ*C)proline, essentially all of the radioactivity of sera and fluids was located in proline spots of chromatograms. Glycine was, however, converted to other amino acids. Approximately 40 per cent of the radioactivity was found in serine of sera and wound fluids after the injection of (lJ*C)glycine. The fate of injected radioactivity into specific proteins of sera and wound fluids is shown in Table III. The radioactivity of both serum and fluid protein fractions separated by paper electrophoresis in dogs given (l-l*C) glucosamine was detected in highest amounts in the alpha globulins. Lowest specific activity was located in the albumin fraction. Most of the globulins and albumin of serum are synthesized in the liver [IS], but albumin has a very low carbohydrate content. In dogs receiving Cl4 amino acids, the radioactivity of serum proteins was distributed rather nonspecifically in all globulin fractions; but again albumin had the lowest specific activity. In contrast to the serum, the fluid radioactivity in these dogs tended to be concentrated more in the alpha-l globulins, findings similar to those in the fluid of dogs injected with (lJ*C) glucosamine. The fluid found within the cylinders of the dogs was in contact with the inner surface of the newly synthesized connective tissue for varying lengths of time depending upon the period of cylinder implant. In all three radioisotope groups of dogs, the distribution of ra-
beta-2
Fluid samples (old) were from cylinders
dioactivity among the proteins of fluid separated by electrophoresis was not found to vary with the time-length of cylinder implant. It was always found in highest amounts in the alpha-l globulins. The total amount of radioactivity, however, did change with time. The total radioactivity of protein fractions was shown to be highest in fluid of the younger tissue which was quite similar to that in serum, but the fluid radioactivity subsequently decreased to about one half or less in cylinders implanted for nine weeks 01‘more. The total protein and hydroxyproline content of the induced connective tissue within cylinders implanted for varying times was examined. The results in Figure 2 show that the tissue has a characteristically age-related accumulation of collagen. There is a rapid linear increase in collagen in the younger tissue up to four weeks, after which the collagen synthesis slows down. In contrast to the collagen, the total protein content of the tissue was highest in the early stages of development. Measurement of Cl* uptake by this connective tissue at each developmental age, plotted in Figure 3, showed that tissue synthesized in the first three weeks contained a higher amount of protein-bound radioactivity than did the connective tissue produced in the later nine week period. The same was true in the fluid of the cylinders. So that while the radioactivity in the fluid may very well originate from the serum, it is under the influence of the local cellular metabolism. This is an important con333
Schilling et al
O-123
16 AGE
OF TISSUE
30
32
IN WEEKS
Age of tissue in weeks. The age-related Fig. 2. changes in total protein (0) and collagen ( l ) content of connective tissue synthesized de novo in cylinders implanted in dogs. Amount of collagen per unit of dry weight based on hydroxyproline content of tissue of each age. Total protein data and collagen data were based on values from a separate control colony of twenty-eight dogs.
cept which should be understood and appreciated by surgeons as they handle tissues during surgical procedures and as they care for wounds postoperatively. Comments Although the use of radioisotopes represents a valuable tool for metabolic studies, it seems not to have been utilized to its fullest potential in regard to wound healing. It was attempted in this study to inject labelled precursors and follow their systemic route to the wound, to determine the origin of wound metabolites, and, if possible, to bring into focus some metabolic relationship between the major constituent, collagen, and the ground substance of wound connective tissue. In an earlier investigation with rats [Z], it was shown that wound tissue may derive some of the hexosamine used for biosynthesis of ground substance components from plasma protein. High levels of radioactivity, found in small alpha globulin glycoproteins which are capable of diffusing into extracellular fluids, were thought to be of possible significance in this regard. Indeed, the main source of radio334
123
9
I4
18
AGE OF TISSUE IN WEEKS Fig. 3. Rate of incorporation of radioactivity in cylinder connective tissue with relation to age of tissue. Tissue was removed twenty-four hours after last of four (one per day) injections. Specific activity expressed as counts per minute x 1V per mg. hexosamine for dogs given (I-14C)glucosamine (A) and counts per minute x 10” per mg. hydroxyproline for dogs given (I-14C)glycine ( 0 ) or (U-14C)proline ( 0 ).
activity for the wound site which was encountered in the present study was serum. The radioactivity derived from each of the radioisotopes was conveyed to cylinders in serum protein form. The radioactivity from injected amino acids was distributed rather uniformly in the serum proteins. Only in the dogs injected with glucosamine was there a localization of radioactivity in a specific protein fraction, that is, the alpha-l globulins. In the fluid of the wound, however, specific activity in all three radioisotope groups was highest in the alpha-l globulins. The total amount of radioactivity in protein fractions of wound fluid decreased in older tissue ; but the location of highest specific activity in alpha-l globulin fractions of wound fluid did not change with the age of the tissue. How the cells in the wound locale were able to utilize these radioactive proteins and glycoproteins for their major activity-synthesis of collagen and ground substance-is not known. It is known, however, that levels of serum alpha globulins are altered as a result of injury [19]. The seromucoid fraction of serum, for The American
Journaj
of Surgery
Wound Healing in the Dog
example, is a mixture of glycoproteins and is known to migrate electrophoretically with the alpha globulins, some of which might readily diffuse through capillary walls and find way into the extracellular fluid. In an earlier study with dogs, wound fluid and sera were found to contain similar amounts of the seromucoid fraction [2]. Also, there is evidence that the seromucoid fraction is involved in serum protein changes resulting from injury [20,21]. It was concluded that the present study definitely showed the presence of small glycoproteins of high specific radioactivity in newly formed wound tissue of the dog. It aIso seemed likely that the serum alpha globulins were the main source of carbohydrate used by this wound tissue. Examination of the composition of the wound tissue shows an inverse relationship between the total protein content and the collagen content as the tissues develop after implantation of the cylinder. Although the total protein was at its very highest at one week, collagen content was at its very lowest. This high content of noncollagen protein in the initial development of the tissue is very likely due to serum or serum-like proteins because of the close association of serum and considerable quantities of fluid found within the wound area at this time. This illustrates the very early and dominant influence of serum on wound healing. The age-related increase in collagen is both unique and typical of wound tissue. In other tissues an increase in collagen with developmental age is mostly an embryonic phenomenon since these tissues after birth, except perhaps in the uterus, show either no change or a fall in collagen concentration with age [22]. But, throughout adult life the body, under both normal and many abnormal circumstances, never seems to Iose its embryonic ability to produce the collagen of wound connective tissue. With regard to radioisotope usage, wound tissue of all developmental ages in this study utilized both (lJ4C)glycine and W-14C)Proline, but the youngest tissue exhibited the highest specific activity. A higher metabolism was noted in the first three weeks of growth of the tissue with a rapid linear decrease in specific activity of collagen at this time. A definite change in rate of incorporation occurred around four weeks as the specific activity of the older tissue was definitely lower. These obVol.117,March 1969
servations on collagen metabolism during development in the same animal have some parallel to the differences noted by Neuberger, Perrone, and Slack [23] in their comparison of collagen metabolism of young and adult animals. CoIlagen fractions, made soluble by djfferent means and in different stages of maturity, have been found to vary in their rate of amino acid incorporation. But, on the whole, it has been said that collagen metabolism in most parts of the body is relatively inert in the adult. Recent evidence has shown that this is not true [I]. Studies of chronic labelling of wound tissue with radioisotopes by Klein and Weiss [.%I have led them to believe that the turnover of mature collagen is the result of reutilization of collagen subunits. Thus, the metabolic inertness of collagen is really only apparent. The classic isotope turnover study measures only the net change in incorporation so that the mature fibrous collagen appears to lose the incorporated radioisotope very slowly. Not only does this show that collagen is not really inert and is indeed dynamic in the adult, but this reutilization of collagen subunits shows the extremely important role of the cells and tissues in the locale of the wound. The incorporation of ( 1-14C) glucosamine by noncollagenous protein of the ground substance of the dogs also occurred very rapidly in the young connective tissue. A high Ievel of incorporation of glucosamine was previously observed in studies with rat wound tissue of two weeks’ developmental age [1,5,6]. The rate of synthesis of the noncollagenous proteins in the older tissue was also similar to that of collagen, being lower and more constant, but nevertheless remaining higher than that of collagen in the same age of tissue. The latter may be related to a higher turnover of mucopolysaccharides and possibIy glycoproteins than that of collagen. Part of the incorporation of glucosamine by young connective tissue of dogs was due no doubt to glycoproteins since they are known to predominate in the early wound [~,zx]. Another portion of the (1-14C)glucosamine was very likely incorporated into mueopolysaccharides of the ground substance since this was specifically demonstrated in an earlier study with rats [5,6]. The parallel incorporation of both the carbohydrate and amino acid precursors, and especially the change in rate which occurs simultaneously about the fourth week of tissue development, implies their in335
Schilling et al.
terrelation during the production of wound tissue and in the maturation of collagen. These changes in rates of synthesis during wound healing have been observed with radioisotope studies of nucleic acids [26,27]. The rate of formation of both RNA and DNA has been found to be more rapid in the early?tages of wound tissue regeneration. In past years and decades, there have been numerous expressions and generalities concerning the autonomy of the local wound and the priority of its metabolic demands. These studies would indicate that the local wound, particularly early in its time course, does have priority in its metabolic demand for glucosamine and to a lesser extent proline. Because of its widespread distribution and its interconversion into several other amino acids, the priority of the metabolic demand by the wound for glycine is difficult to assess. Obviously, these are only three of a very large number of substances that are required in growth, regeneration, and repair, and additional data are required for more definitive statements concerning priority of the metabolic demands of a wound for specific constituents.
growth period showed a higher level of incorporation than did collagen which was maturing in the nine to eighteen weeks’ growth period. Similar to the amino acid uptake by collagen, the carbohydrate-containing proteins of wound tissue exhibited a change from fast to slow in rate of incorporation of ( 1J4C) glucosamine. The significance of the change in rate of incorporation of radioisotopes by both the carbohydrates and amino acids which took place at about the fourth week in the wound tissue growth is not known. However, it provides additional evidence for the metabolic interrelation thought to occur between the ground substance and collagen during the maturation of the latter. References
2.
3.
Summary Radioisotopes were used to study the granulation-type connective tissue induced within dead spaces created by the subcutaneous implantation of stainless steel wire mesh cylinders on the backs of dogs. The incorporation of radioactivity by proteins and carbohydratecontaining proteins of the newly developing wound tissue and its associated wound fluid was compared by the use of the precursors (1J4C)glucosamine, (l-l%)glycine, and (U“C)proline. After intraperitoneal injection, the major portion of radioactivity was conveyed in protein-bound form via serum to the site of new wound tissue synthesis. With all three radioactive precursors, the highest specific activity in wound fluid proteins of the wound site was found electrophoretically in the alpha-l globulin fractions. (l-l%) Glucosamine invoked a higher specific activity in serum, wound fluid, and wound connective tissue than did either (1-14C)glycine or (U14C)proline. The rate of incorporation of amino acid radioisotopes by developing granulation tissue was related to the maturity of the collagen at each developmental age. Young collagen produced in the first three weeks’ 336
healing. Physiol. Rev., 48 ~374, 1968. WHITE, B. N., SHETLAR, M. R., SHURLEY, H. M., and SCHILLING,J. A. Wound healing: investigation of proteins, glycoproteins, and lipids of experimental wound fluid in the dog. Proc. Sot. Exper. Biol. & Med., 101:353,1969. SHETLAR, M. R., LACEFIELD,E. G., WHITE, B. N., and SHILLING,J. A. Wound healing: glycoproteins of wound tissue. I. Studies of hexosamine, hexose and uranic acid content. Proc. Sot. Exper. Biol. & Med., 100: 501, 1959. SCHILLING, J. A., JOEL, W., and SHURLEY, H. M. Wound healing: a comparative study
1. SCHILLING, J. A. Wound
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of the histochemical changes in granulation tissue contained in stainless steel wire mesh and polyvinyl sponge cylinders. SUVgery, 46 :702,1959. 5.
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WHITE, B. N. Incorporation of Glucosamine1-C” by Rat Connective Tissue. (M. S. Thesis). Oklahoma Ciy, 1963. University of Oklahoma Graduate School of Medicine. WHITE, B. N., SHETLAR, M. R., SHURLEY, H. M., and SCHILLING,J. A. Incorporation of (l-l%) glucosamine into mucopolysaccharides of rat connective tissue. Biochim. et biophgs. acta, 101:97,1965. WHITE, B. N., SHETLAR, M. R., SHURLEY, H. M., and SCHILLING,J. A. Incorporation of D- (l-l%) galactosamine into serum proteins and tissues of the rat. Biochim. et biophys. acta, 101:269,1966. SCHILLING,J. A., SHURLEY,H. M., JOEL, W., WHITE, B. N., and BRADFORD,R. H. Abdominal aortic grafts: use of in tivo strut?tured autologous and homologous fibrocollagenous tubes. Ann. SUT~., 169:819, 1964. The American
Journal
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Wound Healing in the Dog
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RICHTER,K. M., and WHITE, B. N. Fibrocollagenous tubes structured in vivo. Morphological and biological characteristics. Arch. Path., 71:548,1961. 10. RICHTER,K. M., SCHILLING,J. A., and SHURLEY, H. M. UItrastructure of experimentally induced scar issue. J. Appl. Ph2/sioE.,
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RICHTER,K. M., SCHILLING,J. A., and SHURLEY, H. M. A possible E.M. demonstration of part of the intramolecular character of the tropocollagcn molecule. In : Proceedings of the International Congress of Electron Microscopy (Sixth) _Tokyo, 1966, Maruzen. 12. SCHILLING, J. A. Technique of implanted steel mesh cylinders in studies of fibroplasia. In: Wound Healing, Proceedings of a Workshop p. 2’70. Edited by Levenson, S. M., Stein, J. M., and Grossblatt, N. Washington, D. C., 1966. National Academy of Sciences-National Research Council. 13. SHETLAR,M. R., FOSTER,J. V., and EVERETT, M. R. Determination of serum polysaccharides by tryptophane reaction. Proc. SOC. Exper.
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WILLIAMSON, M. B. and GUSCHLBAUER, W. Metabolism of nucleic acids during regeneration of wound tissue. II. The rate of formation of RNA. Arch. Biochem., 100: 246, 1968. WILLIAMSON, M. B. and GUSCHLBAUER, W. Metabolism of nucleic acids during regeneration of wound tissue. II. The rate of formation of RNA. Arch. Biochem., 100: 251,1963.
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LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. Protein measurement with the folin phenol reagent. J. Bi01. Chem., 193:265,1951. 16. NEUMAN, R. E. and LOGAN,M. A. The determination of hydroxyproiine. J. Biol. Chem., 184 :299, 1950. 17. GORDON,H. T., THORNBURG,W., and WERNUM, L. N. Rapid paper chromatography of carbohydrates and related compounds.
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CHANDLER,A. M. and NEUHAUS,0. W. Synthesis of serum glycoproteins in response to injury. Am. J. Physiol., 206:169, 1964. 22. HARKNESS,R. D. Biological functions of collagen. Biol. Rev., 36:399, 1961. 23. NEUBERGER, A., PERRONE,J. C., and SLACK, H. G. B. The relative metabolic inertia of tendon collagen in the rat. Biochem. J., 49: 199, 1951. 24. KLEIN, L. and WEISS, P. H. Induced connective tissue metabolism in vivo: reutilization of pre-existing collagen. Proc. Nat.
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MILLER, L. L., BLY, C. G., WATSON, M. L., and BALE, W. F. The dominant role of the liver in plasma protein synthesis, J. Exper. Med., 94:431, 1951. PETERMANN,M. L. Alterations in plasma protein patterns in disease. In: The Plasma Proteins, vol. 2, p. 309. Edited by Putman, F. W. New York, 1960. Academic Press, Inc. NEUHAUS, 0. W. and LIU, A. Biochemical significance of serum glycoproteins. III. Hepatic production of al and a2 globulins responding to injury. PTOC. Sot. Exper.
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