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Dynamics of the healing of skin wounds in the horse as compared with the rat
EXPERIMENTAL
AND
Dynamics
MOLECULAR
PATHOLOGY
30, 349-359
( 1979)
of the Healing of Skin Wounds as Compared with the Rat
in the
Horse
MILOS CHVAPIL, TAD PFISTER, SIMON ESCALADA, JANET LUDWIG, AND ERLE E. PEACOCK, JR.
Animal
Arizona Health Sciences Center, University Hospital, Nogales, Arizona, and Tulane Received
May
19, 1978;
and
in revised
of Arizona, Ukversity, form
Tucson, Arizona, New Orleans, Louisiana
November
27, 1978
The dynamics of skin wound healing were studied in three horses with either full thickness skin excision or with subcutaneously implanted polyvinyl alcohol sponges. Granulation tissue and reactive granuloma were harvested from three anatomical sites and were analyzed by morphological and biochemical methods at three time intervals. PVA sponges were also implanted in rats and studied by similar methods. No effect of wound or implant location on morphology or density of the sample tissue was found. In the horse, neutrophil infiltration was found in the tissue of wounds less than one day old. This tissue actively synthesized collagen and showed high activity of collagenase. The activity of proryl hydroxylase (PH), however, was low. At later time sampling period (3, 7, 14 days ), both granulation and granuloma tissue showed increasing PH activity, high activity of collagenase, and decreasing rate of collagen synthesis. Collagen content also increased with time. The reactive granuloma tissue found in rats showed less connective tissue reactivity than in the horse as seen by the dynamics of the morphological and biochemical changes of the tissue. We conclude that the healing in the horse is rather prompt and excessive and may tend toward abnormal repair reactions.
INTRODUCTION Although many attempts have been made to demonstrate qualitative differences in hypertrophic scar and normal scar, the preponderence of evidence presently supports the hypothesis that “overhealing” resulting in a hypertrophic scar or keloid is the result of quantitative abnormalities in connective tissue metabolism. Specifically, abnormal deposits of dense connective tissue in skin wounds seem most likely now to be the result of collagen synthesis and deposition exceeding normal collagen degradation by tissue collagenase acting at neutral pH. Of course, the same result also would occur if tissue collagenolytic activity was abnormally reduced in the presence of normal kinetics of net collagen synthesis and deposition. A major problem in testing the quantitative abnormality hypothesis has been lack of an appropriate laboratory model to study hypertrophic scar and keloid formation. Most fur bearing laboratory animals simply do not develop excessive surface scar tissue. About the only exception to this statement is the scar tissue which occasionally is produced in skin wounds of horses, particularly in the non-hair bearing area of the lower legs. 349 0014-4800/79/030349-11$02.00/0 All
Copyright 0 1979 rights of reproduction
by Academic Press, Inc. in any form reserved.
350
CHVAPIL
ET
AL.
It was indicated that the formation of exuberant granulation tissue often accompanies the healing ‘of skin wounds in the horse. In superficial wounds excessive granulation tissue interferes with epithelization dynamics or in full thickness wounds the proud flesh often delays the wound healing process (Meager and Adams, 1970). In fact, the horse is reported to be the only animal species, other than humans, which develops keloid. Although the topic of keloid in horses is a subject of controversy, the empirical fact remains that this species forms excessive amounts of granulation tissue. Still, not a study is available which documents and analyzes the process of wound healing in horses by established methods. The hypothesis that “overhealing” is related to quantitative rather than qualitative differences in connective tissue biology could be investigated further by comparative studies of connective tissue metabolism between the only known animal which forms hypertrophic scars and a common laboratory animal which does not form hypertrophic surface scars. Such a comparison is possible between wound healing in the horse and the rat. To test this hypothesis the following studies of wound healing biology have been performed in horses and SpragueDawley rats. MATERIALS
AND
METHODS
Full-thickness circular skin defects were created in two gelding horses 12 and 15 years old weighing 410 and 595 kg each. The wounds were 2 cm in diameter and were created by removing full thickness skin from the foreleg, knee, and cannon bone regions. Three separate wounds were made in each area and normal skin was excised from the shoulder of each horse to provide control data from normal skin. One of the wounds was excised after 18 hr, the second wound was excised after 3 days and the third wound in each area was excised after 12 ,days. In a third horse (g-year old mare weighing 386 kg) a rectangular 5 x 1 x 1 cm Ivalon sponge obtained from Unipoint Laboratories (N.C.) was implanted in a subcutaneous pocket in each of the three locations where wounds were placed in the other two horses. Sponges were inserted through a 1 cm incision and were placed in a subcutaneous pocket approximately 1 ‘cm away from the skin wound. A sponge was removed from each of the three areas after 19 hr, 7 days and 14 days. Sixty Sprague-Dawley male rats weighing approximately 200 g were operated upon. Two Ivalon sponges measuring 2.5 x 1 x 1 cm each were implanted subcutaneously in the dorsal region parallel to the spine. Prior to implantation sponges were sterilized in boiling distilled water. Sponges were removed at intervals ranging between 18 hr and 61 days as indicated in Table III. Biochemical
Methods
Total collagen was calculated in sponge tissue and wound tissue specimens by measuring hydroxyproline content. Sponges and tissue specimens were weighed and hydrolyzed in 6 N HCl, 105°C for 16 hr. The hydrolysate was decolorized with active charcoal and evaporated to dryness before being dissolved in distilled water. Hydroxyproline was measured in a standard Technicon Autoanalyzer technique. Rate of collagen synthesis and deposition was measured by a combination of the Uitto (1970) and Juva and Prockop (1966) techniques. Wound
SKIN
WOUNDS
IN
HORSES
AN11
RATS
351
tissue and sponge samples were incubated with carbon-14-proline under tissue culture conditions as described by Uitto. l’C-hydroxyproline activity was determined by the method of Juva and Prockop. The data are displayed as DPM of 14C-hydroxyproline/p mole total hydroxyproline. For the assay of prolyl hydroxylase the granuloma tissue and the wound granulation tissue specimens were homogenized with a Brinkman Polytron for 30 set in medium containing 0.23 Al sucrose, 0.014 A1 Tris-HCI buffer (pH 7.5), 50 pg/ml of phenylmethylsulfonylfluoride and 50 & dithiothreitol. The homogenate was centrifuged at 4” for 20 min at 15,000 g and the supernatant
Histology Strips of wound granulation tissue and 3 mm cross sections of sponge were fixed in 10%’ buffered formalin for histological studies. Tissues were stained by hemotoxyline and eosin and Masson’s trichrome stain.
352
CHVAPIL
ET
AL.
RESULTS Full thickness
excised skin wound
Morphology. Seventeen hours after full thickness skin excisions, a thin layer of tissue was collected from the edematous wound bed. This showed mostly blood clots infiltrated with polymorphonuclear leucocytes. The inflammatory infiltration was irregular, occurring in streaks and had the same appearance at all three sites. There is an indication that these forms of early granulocytic infiltration correspond with the topography of vessels in wound bed. Three days after excision, a soft, edematous and highly cellular granulation tissue completely filled the skin defect. The cells were granulocytes, mostly macrophages and many spindle-shaped cells, possibly fibroblasts. The granulation tissue was highly vascularized. At this time period distinct collagen fibrous deposits were seen in trichrome stain. Greater density of collagen layers was evident in the proximity of deep fascia. Although difficult to assess quantitatively, we found that denser collagenous stroma was present in the granulation tissue formed over the horse knee joint. TABLE Skin Parameter
Wound
I
Healing
Studied
in Horses Age of the wound
0
0.75
(days) 3
12
10,650 10,340 -
7,280 9,608 7,450
Collagen synthesis W-Hyp dpm/pmole Thigh Knee Foreleg Intact Collagen HYP
38,260 24,540 skin
5,900
content w/g
W.W.
Thigh Knee Foreleg Intact
0.42 0.59 1.89 skin
0.4’2 0.7’2 1.47
1.68 2.58 2.22
11.6
Prolyl hydroxylase cpm/mg protein Thigh Knee Foreleg Intact Collagenase y0 digested/48
690 400 214 skin
2,950 3,200 3,990
3,590 3,160 3,310
560
hr Thigh Knee Foreleg Intact
60.5 65.5 72.5 skin
95.0 73.0 69.0
91.5 70.0 90.2
2.3
Three similar, full thickness skin excision wounds were inflicted in a 12-year old gelding horse in three different locations. Samples of granulation tissue were collected after 0.75 days 3 and 12 days and analyzed for parameters indicating collagen metabolism. Each value is the average of two parallel determinations. Similar results obtained in two other horses.
SKIh’ WOUNDS
IN HORSES AND
RATS
FIG. 1. Polyvinyl alcohol sponge 19 hr after subcutaneous implantation in horse shows pronounced granulocytic infiltration. Left top shows part of the sponge. The matrix of the sponge filled with blood. Approximately 250X magnification.
days after excision, the granulation tissue was highly collagenous with Twelve several vesselspresent. Biochemistry. It was rather surprising to find that the wound tissue collected 17 hr after excising the skin was already very actively synthesizing collagen (Table I). In fact, the highest rate of coZZugenbio.sz@hesiswas found in tissue at this earliest sampling period. At the two later sampling periods the rate of collagen synthesis decreased at all three wound sites. Our previous experience indicates that the activity of prolyl hydroxylase reflects the functional state of fibroblasts and the potential for production of fully hydroxylated, collagen molecules. In this experiment, prolyl hydroxylase activity wCa.squite low 17 hr after skin excision. In fact, the activity of this enzyme in the wound tissue was the same as that in intact skin. It was, however, almost 10 times more active in granulation tissue 3 and 12 days old. The data on the activity of collagenase were unexpected. Only marginal activity was found in intact skin (2.3% ), while in wound granulation tissue, irrespective of sampling period or localization of the wound, the activity ranged within 60 to 90% (Table I). There is an indication of an increasing activity of collagenase with the age of the granulation tissue.
3.54
CHVAPIL
ET
AL.
Collagen concentration measured by its hydroxyproline content shows the presence of collagen in the tissue at the first sampling period. At 12 days the values were higher, indicating more collagen deposition in the older granulation tissue. Formation of reactive granuloma tissue was studied after subcutaneous implantation of Ivalon sponge in one horse and in rats. Morphology. In the horse, 19 hr after the implantation of the sponge blood is present and inflammatory cells have infiltrated outer layer of the sponge matrix. These cells are mainly granulocytes (Fig. 1). After the same time interval in rats, the sponge pores are clean, intact, without any cells or blood. At 7 days in the horse, regardless of site, a rather solid capsule had formed around the sponge implants. Inside the sponge granulation tissue with collagen structures and prevalent fibroblastic cell population was evident (Fig. 2). In rats after 7 days, the sponge is surrounded by a thin membranous capsule and collagenous structures have not invaded the matrix of the sponge. The granuloma tissue displays rich cellular infiltration with several macrophages and fusiformshape cells, possibly, fibroblasts. There are also many vessels ingrown into the sponge at this time.
FIG. 2. PoIyvinyl alcohol sponge, 7 days after subcutaneous implantation. The matrix is filled with loose connective tissue with many spindle shaped cells. At this stage a striking amount of collagenous structures occupies the space. Approximately 100X magnification.
SKIN
FIG. 3. At 14 days a solid mass
penetrates
the matrix.
WOUNDS
fibrotic capsule Approximately
IN
HORSES
surrounds
AND
the
3%
RATS
implanted
sponge.
Solid
collagen
100X magnification.
At 14 days the sponge in the horse subcutis was encapsulated with solid, thick, collagenous structures. The sponge matrix was filled with collagenous fibrosis and few dispersed cells (Fig. 3). In rat, the reactive granuloma showed some collagenous infiltration of the sponge. The nature of this ingrowth as seen from the cell types, collagen and vessel content indicated that the process was much slower than in the horse. The biochemical comparison of the grnnuloma tissue of the horse and rat at three time sampling periods is summarized in Table II. The results for the horse derived tissue are similar to those of the granulation tissue as reported above. The rate of collagen synthesb was also highest in the first sampling period. In later periods after the implantation the synthesis of collagen decreased and was almost 100 times less at 14 days than at 19 hr. Similar to the granulation tissue, the activity of prolyl hydroxylase in the granuloma tissue is very low at I9 hr after implantation of the sponge and is highest at 14 days. Collagenase activity was measured only at 7 and I4 days sampling periods. The activity was high at both periods ranging between 50 and 60% and no trends as to time or implant location could be seen. Finally it is worth mentioning, that the cell content as evidenced by DNA
CHVAPIL
356
ET
TABLE Comparison
AL.
II
of Wound Healing Dynamics in the Horse and Rat
of Implanted Subcutis Sampling
PVA
period
(18 to 19 hr)
Horseb Collagen
Sponges
Rat
content
(HYP mg/g wet wt) Collagen synthesis (I%-Hyp dpmlpmole Prolyl Hydroxylase (cpm/mg protein)
0.014 X 103)
No capsule”
formed
82.3
No capsulea
formed
13
No capsule”
formed
Sampling Collagen content Collagen synthesis Prolyl Hydroxylase
period
0.645 13.8 3070 Sampling
Collagen content Collagen synthesis Prolyl Hydroxylase
0.11 f 0.003 4.8 f 0.4 6400 period
15.1 0.83 7290
n Eighteen hours after implanting subcutaneously tissue formed for biochemical analysis. b Refers to sponges implanted in the foreleg area. results obtained in two other horses.
(14 days) 0.78 1.8 6300
the sponge Data
(7 days)
to rats
are the average
there
was no granuloma
of three
analyses.
Similar
analysis of the tissue samples was already high at the first day sampling period and showed an increasing trend with time of implantation. One day after the implantation of the Ivalon sponge to rats, not enough reactive tissue was formed to allow a biochemical study. At later sampling periods, collagen was detected and its content increased with time. The rate of collagen biosynthesis in rat granuloma tissue was highest at 10 days, at 7 and 14 days it was significantly lower. From Table II it is evident that in the horse, collagen is TABLE Biochemistry
Days after implant,ation
1 (4) 7 (8) 10 (12) 14 (13) 18 (14) 61 (7)
of Granuloma
DNA b&d
1.00 1.00 1.09 0.98 0.71
ND f f f f f
Tissue Induced of Ivalon Sponges Collagen content (HYP mg/g WWt)
0.07 0.09 0.15 0.08 0.04
III
ND 0.11 f 0.48 f 0.78 f 1.3 f 1.4 f
0.003 0.07 0.10 0.05 0.06
by Subcutaneous in Rats Collagen synthesis (I%-Hyp/dpm/ pmole X 103)
4.8 8.0 1.8 1.2 0.44
Number of rats in each group is given in parenthesis, 220 g body Data are presented as X f SE. ND refers to non-detectable.
ND f f f f f
Implantation
Prolyl hydroxylase (cpm/protein x 103) ND f f f f f
0.4 0.7 0.09 0.05 0.02
6.4 5.8 6.3 4.1 4.5
weight,
Sprague-Dawley
0.70 0.40 0.32 0.29 0.20 males.
SKIN WOUNDS
IN HORSES
AND
RATS
357
synthesized at higher rates, especially in earlier time periods. The rate of synthesis also declines faster. Furthermore, the total collagen content of sponge granuloma tissue from rats is less at all time intervals than that in horses. More details on the biochemistry of the granuloma tissue in rats are presented in Table III. The data show that the maximal rate of collagen synthesis is reached on day 10, while the activity of prolyl hydroxylase remains the same for the first 14 days and then declines. With time, collagen deposition within the granuloma tissue decreases. DISCUSSION The repair process that occurs in full thickness excised skin wounds and the reactive granuloma tissue formed around and within the framework of subcutaneously implanted PVA sponges are forms of fibroproliferative inflammation (Chvapil, 1967). By definition, this is an inflammatory growth of connective tissue characterized by collagen formation and deposition. The dynamics of the reactivity of the connective tissue components during the repair process can be classified into three phases: (a) mobilization and proliferation of cells, (b) synthesis and deposition of products of fibrogenic cells (col’lagen and glycosaminoglycans ), ( c) final organization or remodeling of the wound scar. It is important to stress that the sequence of individual biological-chemical reactions during the repair process is subject to definite biological principles, which govern the kinetics of the migration of various cell lines into the lesion, communication among cells, the local and systemic effect of their products, etc. Keeping this in mind, it was rather unexpected to find that in less than 1 day after the injury to horse skin or subcutis a tissue with high capacity to synthesize collagen appeared in defect. Such a tissue should contain fibroblasts, which normally appear in the repair tissue on the 3rd or 4th day after wounding (for review see Chvapil, 1967, Peacock and Van Winkle, 1976). The explanation for this early incidence of fibroblastic reaction may be due to the method of sampling of the tissue specimen which is misnamed granulation tissue. In fact, after excision of the skin the wound bed bleeds and swells. Increased water content in injured tissue in early inflammatory stages is a common phenomenon due to changes in microcirculation, the release of biologic amines, various activators, or mediators of inflammatison released by injured tissue cells, and by infiltrating inflammatory cells such as granulocytes and macrophages (James, 1969). Thus, the swollen hydrated tissues of the wound bed or the subcutaneous tissue in contact with Ivalon sponge and contraction of the edges of the excised skin were dissected and analyzed biochemically as “granulation tissue.” Granulation tissue, per definition is, a nezoly formed composite of cells, cell products and vessels. Thus, the high rate of collagen synthesis reflects rather prompt activation of the surface tissue edges of the wound than newly formed tissue. We believe, that a similar explanation holds for the data on high rate of collagen synthesis in wound tissue in l-day old excised skin wounds of rats, as reported by Digelman and Cohen (1974). Our morphological as well ‘as biochemical data indicate that the connective tissue structures are activated in the skin wound of a horse to a greater extent and at earlier periods than in the rat. This was also documented by macroscopic inspection and comparison of the granuloma tissue from both the horse and rats.
CHVAPIL
3%
ET AL.
An active infiltration of PVA sponge by granulocytes was already noticeable 17 hr after implantation. Sponge implanted into subcutis of rats was without any cellular inflammatory infiltrate within the first 24 hr. This may be explained by absence of bleeding during implanting the sponge in rats, while the horse bled profusely into the sponge. Three-day old granulation tissue in the horse contained high amounts of collagen, which was also seen in histological specimens. This was further evidence of a rather fast healing process in the horse, much faster than we have seen in comparable skin wounds in rats. As shown in Tables I and II, there exists a definite similarity in the general trend of the biochemical results in both granulation and granuloma tissue. Two parameters reflecting connective tissue reactivity, i.e., rate of collagen synthesis and the activity of prolyl hydroxylase seems to be in conflict in the first sampling period. The tissue analyzed shows synthesis of collagen at the highest rate, but the activity of prolyl hydroxylase in the sample is low. Although we have no experimental support to our view, we would assume that underhydroxylated collagen might be synthesized at this time. The disparity between the rate of collagen biosynthesis and the activity of prolyl hydroxylase in the analyzed tissue supports our view that the activity of prolyl hydroxylase is not indicative of the rate of collagen biosynthesis ( Chvapil et nl., 1976). The high activity of collagenase in the wound tissue collected 17 hr after full skin excision is of interest. The activity ‘of this enzyme in the wound bed was almost 30 times higher than in intact skin. We assume that such a high activity reflects infiltration and activation of inflammatory cells, since both activated polymorphonuclear leucocytes and macrophages contain collagenase (Oronsky et al., 1973, Robertson et al., 1972). ACKNOWLEDGMENT Financial
support
for
this
work
was provided
in part
by
NIH
Grant
GM
25159.
REFERENCES BURTON, K. (1956). Study of conditions and mechanism of diphenylamine reaction for calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. CHVAPIL, M. ( 1987). Physiology of Connective Tissue. London, Butterworth’s Scientific Publications, pp. 246-286. CHVAPIL, M. (1975). Pharmacology of Fibrosis: Definitions, Limits and Perspectives. Life Sciences 16 (9), 134551362. DIEGELMANN, R. F., ROTHKOPF, L. C., and COHEN, I. K. (1974). In: Wound Healing (Tom Gibson and J. C. van der Meulen, Eds.), pp. 55-58. Published by the Foundation International Cooperation in the Medical Sciences, Rotterdam. HUTTON, J. J., TAPPEL, A. L., and UDENFRIEND, S. ( 1966). A rapid assay for collagen proline hydroxylase. Anal. Biochem. 16, 384-394. JAMES, D. W., (1969). In; Repair and Regeneration: The Scientific Basis for Surgical Practice (J. Englebert Dunphy and H. Walton Van Winkle, Jr., Eds.), pp. 169-184. Pub. McGraw-Hill, Inc., New York. JUVA, K., and PROCKOP, D. J. (1966). Modified procedure for the assay of Ha or C14-labelled hydroxyproline. Anal. Biochem. 15, 77-83. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANIJAL.L, R. J. ( 1951). Protein measurement with the fohn phenol reagent. Z. Biol. Chem. 193, 265-275. MEAGER, D. M., and ADAPTS, 0. R. (1970). Skin transplantation in horses. Can. Vet. Jour. 11 (12), 239-248.
SKIN
WOUNDS
IN
HORSES
AND
RATS
359
ORONSKY, A. L., PEHPEH, R. J,, and SCHRO~ER, H. C. (1973). Phngocytic Release and Activation of Human Leukocyte Procollagenase. Nature 246, 417-419. PEACOCK, ERLE E., JR., and VAN WINKLE, W. ( 1976). Wound Repair, 2nd Ed., Pul). W. B. Saunders Company, Philadelphia. ROBERTSON, P. B., RYEL, R. B., TAYLOH, R. E., SHYU, K. W., and FULLhIm, H. M. (1972). Collagenase: localization in polymorphonuclear leukocyte granules in the rahhit. Science 177, BP6Ei. RYAN, J. N., and WOESSNEH, J. F., JR. ( 1971). M ammalian coKagenasc-Direct demonstration in homogenates of involuting rat uterus. Biochem. Biol>hys. Res. Commun. 44, 144. UITTO, J. (1970). A method for studying collagen biosynthesis in human skin biopsies in aitro. Biochim. Biophyn. Acta 201, 438-415.
AND
Dynamics
MOLECULAR
PATHOLOGY
30, 349-359
( 1979)
of the Healing of Skin Wounds as Compared with the Rat
in the
Horse
MILOS CHVAPIL, TAD PFISTER, SIMON ESCALADA, JANET LUDWIG, AND ERLE E. PEACOCK, JR.
Animal
Arizona Health Sciences Center, University Hospital, Nogales, Arizona, and Tulane Received
May
19, 1978;
and
in revised
of Arizona, Ukversity, form
Tucson, Arizona, New Orleans, Louisiana
November
27, 1978
The dynamics of skin wound healing were studied in three horses with either full thickness skin excision or with subcutaneously implanted polyvinyl alcohol sponges. Granulation tissue and reactive granuloma were harvested from three anatomical sites and were analyzed by morphological and biochemical methods at three time intervals. PVA sponges were also implanted in rats and studied by similar methods. No effect of wound or implant location on morphology or density of the sample tissue was found. In the horse, neutrophil infiltration was found in the tissue of wounds less than one day old. This tissue actively synthesized collagen and showed high activity of collagenase. The activity of proryl hydroxylase (PH), however, was low. At later time sampling period (3, 7, 14 days ), both granulation and granuloma tissue showed increasing PH activity, high activity of collagenase, and decreasing rate of collagen synthesis. Collagen content also increased with time. The reactive granuloma tissue found in rats showed less connective tissue reactivity than in the horse as seen by the dynamics of the morphological and biochemical changes of the tissue. We conclude that the healing in the horse is rather prompt and excessive and may tend toward abnormal repair reactions.
INTRODUCTION Although many attempts have been made to demonstrate qualitative differences in hypertrophic scar and normal scar, the preponderence of evidence presently supports the hypothesis that “overhealing” resulting in a hypertrophic scar or keloid is the result of quantitative abnormalities in connective tissue metabolism. Specifically, abnormal deposits of dense connective tissue in skin wounds seem most likely now to be the result of collagen synthesis and deposition exceeding normal collagen degradation by tissue collagenase acting at neutral pH. Of course, the same result also would occur if tissue collagenolytic activity was abnormally reduced in the presence of normal kinetics of net collagen synthesis and deposition. A major problem in testing the quantitative abnormality hypothesis has been lack of an appropriate laboratory model to study hypertrophic scar and keloid formation. Most fur bearing laboratory animals simply do not develop excessive surface scar tissue. About the only exception to this statement is the scar tissue which occasionally is produced in skin wounds of horses, particularly in the non-hair bearing area of the lower legs. 349 0014-4800/79/030349-11$02.00/0 All
Copyright 0 1979 rights of reproduction
by Academic Press, Inc. in any form reserved.
350
CHVAPIL
ET
AL.
It was indicated that the formation of exuberant granulation tissue often accompanies the healing ‘of skin wounds in the horse. In superficial wounds excessive granulation tissue interferes with epithelization dynamics or in full thickness wounds the proud flesh often delays the wound healing process (Meager and Adams, 1970). In fact, the horse is reported to be the only animal species, other than humans, which develops keloid. Although the topic of keloid in horses is a subject of controversy, the empirical fact remains that this species forms excessive amounts of granulation tissue. Still, not a study is available which documents and analyzes the process of wound healing in horses by established methods. The hypothesis that “overhealing” is related to quantitative rather than qualitative differences in connective tissue biology could be investigated further by comparative studies of connective tissue metabolism between the only known animal which forms hypertrophic scars and a common laboratory animal which does not form hypertrophic surface scars. Such a comparison is possible between wound healing in the horse and the rat. To test this hypothesis the following studies of wound healing biology have been performed in horses and SpragueDawley rats. MATERIALS
AND
METHODS
Full-thickness circular skin defects were created in two gelding horses 12 and 15 years old weighing 410 and 595 kg each. The wounds were 2 cm in diameter and were created by removing full thickness skin from the foreleg, knee, and cannon bone regions. Three separate wounds were made in each area and normal skin was excised from the shoulder of each horse to provide control data from normal skin. One of the wounds was excised after 18 hr, the second wound was excised after 3 days and the third wound in each area was excised after 12 ,days. In a third horse (g-year old mare weighing 386 kg) a rectangular 5 x 1 x 1 cm Ivalon sponge obtained from Unipoint Laboratories (N.C.) was implanted in a subcutaneous pocket in each of the three locations where wounds were placed in the other two horses. Sponges were inserted through a 1 cm incision and were placed in a subcutaneous pocket approximately 1 ‘cm away from the skin wound. A sponge was removed from each of the three areas after 19 hr, 7 days and 14 days. Sixty Sprague-Dawley male rats weighing approximately 200 g were operated upon. Two Ivalon sponges measuring 2.5 x 1 x 1 cm each were implanted subcutaneously in the dorsal region parallel to the spine. Prior to implantation sponges were sterilized in boiling distilled water. Sponges were removed at intervals ranging between 18 hr and 61 days as indicated in Table III. Biochemical
Methods
Total collagen was calculated in sponge tissue and wound tissue specimens by measuring hydroxyproline content. Sponges and tissue specimens were weighed and hydrolyzed in 6 N HCl, 105°C for 16 hr. The hydrolysate was decolorized with active charcoal and evaporated to dryness before being dissolved in distilled water. Hydroxyproline was measured in a standard Technicon Autoanalyzer technique. Rate of collagen synthesis and deposition was measured by a combination of the Uitto (1970) and Juva and Prockop (1966) techniques. Wound
SKIN
WOUNDS
IN
HORSES
AN11
RATS
351
tissue and sponge samples were incubated with carbon-14-proline under tissue culture conditions as described by Uitto. l’C-hydroxyproline activity was determined by the method of Juva and Prockop. The data are displayed as DPM of 14C-hydroxyproline/p mole total hydroxyproline. For the assay of prolyl hydroxylase the granuloma tissue and the wound granulation tissue specimens were homogenized with a Brinkman Polytron for 30 set in medium containing 0.23 Al sucrose, 0.014 A1 Tris-HCI buffer (pH 7.5), 50 pg/ml of phenylmethylsulfonylfluoride and 50 & dithiothreitol. The homogenate was centrifuged at 4” for 20 min at 15,000 g and the supernatant
Histology Strips of wound granulation tissue and 3 mm cross sections of sponge were fixed in 10%’ buffered formalin for histological studies. Tissues were stained by hemotoxyline and eosin and Masson’s trichrome stain.
352
CHVAPIL
ET
AL.
RESULTS Full thickness
excised skin wound
Morphology. Seventeen hours after full thickness skin excisions, a thin layer of tissue was collected from the edematous wound bed. This showed mostly blood clots infiltrated with polymorphonuclear leucocytes. The inflammatory infiltration was irregular, occurring in streaks and had the same appearance at all three sites. There is an indication that these forms of early granulocytic infiltration correspond with the topography of vessels in wound bed. Three days after excision, a soft, edematous and highly cellular granulation tissue completely filled the skin defect. The cells were granulocytes, mostly macrophages and many spindle-shaped cells, possibly fibroblasts. The granulation tissue was highly vascularized. At this time period distinct collagen fibrous deposits were seen in trichrome stain. Greater density of collagen layers was evident in the proximity of deep fascia. Although difficult to assess quantitatively, we found that denser collagenous stroma was present in the granulation tissue formed over the horse knee joint. TABLE Skin Parameter
Wound
I
Healing
Studied
in Horses Age of the wound
0
0.75
(days) 3
12
10,650 10,340 -
7,280 9,608 7,450
Collagen synthesis W-Hyp dpm/pmole Thigh Knee Foreleg Intact Collagen HYP
38,260 24,540 skin
5,900
content w/g
W.W.
Thigh Knee Foreleg Intact
0.42 0.59 1.89 skin
0.4’2 0.7’2 1.47
1.68 2.58 2.22
11.6
Prolyl hydroxylase cpm/mg protein Thigh Knee Foreleg Intact Collagenase y0 digested/48
690 400 214 skin
2,950 3,200 3,990
3,590 3,160 3,310
560
hr Thigh Knee Foreleg Intact
60.5 65.5 72.5 skin
95.0 73.0 69.0
91.5 70.0 90.2
2.3
Three similar, full thickness skin excision wounds were inflicted in a 12-year old gelding horse in three different locations. Samples of granulation tissue were collected after 0.75 days 3 and 12 days and analyzed for parameters indicating collagen metabolism. Each value is the average of two parallel determinations. Similar results obtained in two other horses.
SKIh’ WOUNDS
IN HORSES AND
RATS
FIG. 1. Polyvinyl alcohol sponge 19 hr after subcutaneous implantation in horse shows pronounced granulocytic infiltration. Left top shows part of the sponge. The matrix of the sponge filled with blood. Approximately 250X magnification.
days after excision, the granulation tissue was highly collagenous with Twelve several vesselspresent. Biochemistry. It was rather surprising to find that the wound tissue collected 17 hr after excising the skin was already very actively synthesizing collagen (Table I). In fact, the highest rate of coZZugenbio.sz@hesiswas found in tissue at this earliest sampling period. At the two later sampling periods the rate of collagen synthesis decreased at all three wound sites. Our previous experience indicates that the activity of prolyl hydroxylase reflects the functional state of fibroblasts and the potential for production of fully hydroxylated, collagen molecules. In this experiment, prolyl hydroxylase activity wCa.squite low 17 hr after skin excision. In fact, the activity of this enzyme in the wound tissue was the same as that in intact skin. It was, however, almost 10 times more active in granulation tissue 3 and 12 days old. The data on the activity of collagenase were unexpected. Only marginal activity was found in intact skin (2.3% ), while in wound granulation tissue, irrespective of sampling period or localization of the wound, the activity ranged within 60 to 90% (Table I). There is an indication of an increasing activity of collagenase with the age of the granulation tissue.
3.54
CHVAPIL
ET
AL.
Collagen concentration measured by its hydroxyproline content shows the presence of collagen in the tissue at the first sampling period. At 12 days the values were higher, indicating more collagen deposition in the older granulation tissue. Formation of reactive granuloma tissue was studied after subcutaneous implantation of Ivalon sponge in one horse and in rats. Morphology. In the horse, 19 hr after the implantation of the sponge blood is present and inflammatory cells have infiltrated outer layer of the sponge matrix. These cells are mainly granulocytes (Fig. 1). After the same time interval in rats, the sponge pores are clean, intact, without any cells or blood. At 7 days in the horse, regardless of site, a rather solid capsule had formed around the sponge implants. Inside the sponge granulation tissue with collagen structures and prevalent fibroblastic cell population was evident (Fig. 2). In rats after 7 days, the sponge is surrounded by a thin membranous capsule and collagenous structures have not invaded the matrix of the sponge. The granuloma tissue displays rich cellular infiltration with several macrophages and fusiformshape cells, possibly, fibroblasts. There are also many vessels ingrown into the sponge at this time.
FIG. 2. PoIyvinyl alcohol sponge, 7 days after subcutaneous implantation. The matrix is filled with loose connective tissue with many spindle shaped cells. At this stage a striking amount of collagenous structures occupies the space. Approximately 100X magnification.
SKIN
FIG. 3. At 14 days a solid mass
penetrates
the matrix.
WOUNDS
fibrotic capsule Approximately
IN
HORSES
surrounds
AND
the
3%
RATS
implanted
sponge.
Solid
collagen
100X magnification.
At 14 days the sponge in the horse subcutis was encapsulated with solid, thick, collagenous structures. The sponge matrix was filled with collagenous fibrosis and few dispersed cells (Fig. 3). In rat, the reactive granuloma showed some collagenous infiltration of the sponge. The nature of this ingrowth as seen from the cell types, collagen and vessel content indicated that the process was much slower than in the horse. The biochemical comparison of the grnnuloma tissue of the horse and rat at three time sampling periods is summarized in Table II. The results for the horse derived tissue are similar to those of the granulation tissue as reported above. The rate of collagen synthesb was also highest in the first sampling period. In later periods after the implantation the synthesis of collagen decreased and was almost 100 times less at 14 days than at 19 hr. Similar to the granulation tissue, the activity of prolyl hydroxylase in the granuloma tissue is very low at I9 hr after implantation of the sponge and is highest at 14 days. Collagenase activity was measured only at 7 and I4 days sampling periods. The activity was high at both periods ranging between 50 and 60% and no trends as to time or implant location could be seen. Finally it is worth mentioning, that the cell content as evidenced by DNA
CHVAPIL
356
ET
TABLE Comparison
AL.
II
of Wound Healing Dynamics in the Horse and Rat
of Implanted Subcutis Sampling
PVA
period
(18 to 19 hr)
Horseb Collagen
Sponges
Rat
content
(HYP mg/g wet wt) Collagen synthesis (I%-Hyp dpmlpmole Prolyl Hydroxylase (cpm/mg protein)
0.014 X 103)
No capsule”
formed
82.3
No capsulea
formed
13
No capsule”
formed
Sampling Collagen content Collagen synthesis Prolyl Hydroxylase
period
0.645 13.8 3070 Sampling
Collagen content Collagen synthesis Prolyl Hydroxylase
0.11 f 0.003 4.8 f 0.4 6400 period
15.1 0.83 7290
n Eighteen hours after implanting subcutaneously tissue formed for biochemical analysis. b Refers to sponges implanted in the foreleg area. results obtained in two other horses.
(14 days) 0.78 1.8 6300
the sponge Data
(7 days)
to rats
are the average
there
was no granuloma
of three
analyses.
Similar
analysis of the tissue samples was already high at the first day sampling period and showed an increasing trend with time of implantation. One day after the implantation of the Ivalon sponge to rats, not enough reactive tissue was formed to allow a biochemical study. At later sampling periods, collagen was detected and its content increased with time. The rate of collagen biosynthesis in rat granuloma tissue was highest at 10 days, at 7 and 14 days it was significantly lower. From Table II it is evident that in the horse, collagen is TABLE Biochemistry
Days after implant,ation
1 (4) 7 (8) 10 (12) 14 (13) 18 (14) 61 (7)
of Granuloma
DNA b&d
1.00 1.00 1.09 0.98 0.71
ND f f f f f
Tissue Induced of Ivalon Sponges Collagen content (HYP mg/g WWt)
0.07 0.09 0.15 0.08 0.04
III
ND 0.11 f 0.48 f 0.78 f 1.3 f 1.4 f
0.003 0.07 0.10 0.05 0.06
by Subcutaneous in Rats Collagen synthesis (I%-Hyp/dpm/ pmole X 103)
4.8 8.0 1.8 1.2 0.44
Number of rats in each group is given in parenthesis, 220 g body Data are presented as X f SE. ND refers to non-detectable.
ND f f f f f
Implantation
Prolyl hydroxylase (cpm/protein x 103) ND f f f f f
0.4 0.7 0.09 0.05 0.02
6.4 5.8 6.3 4.1 4.5
weight,
Sprague-Dawley
0.70 0.40 0.32 0.29 0.20 males.
SKIN WOUNDS
IN HORSES
AND
RATS
357
synthesized at higher rates, especially in earlier time periods. The rate of synthesis also declines faster. Furthermore, the total collagen content of sponge granuloma tissue from rats is less at all time intervals than that in horses. More details on the biochemistry of the granuloma tissue in rats are presented in Table III. The data show that the maximal rate of collagen synthesis is reached on day 10, while the activity of prolyl hydroxylase remains the same for the first 14 days and then declines. With time, collagen deposition within the granuloma tissue decreases. DISCUSSION The repair process that occurs in full thickness excised skin wounds and the reactive granuloma tissue formed around and within the framework of subcutaneously implanted PVA sponges are forms of fibroproliferative inflammation (Chvapil, 1967). By definition, this is an inflammatory growth of connective tissue characterized by collagen formation and deposition. The dynamics of the reactivity of the connective tissue components during the repair process can be classified into three phases: (a) mobilization and proliferation of cells, (b) synthesis and deposition of products of fibrogenic cells (col’lagen and glycosaminoglycans ), ( c) final organization or remodeling of the wound scar. It is important to stress that the sequence of individual biological-chemical reactions during the repair process is subject to definite biological principles, which govern the kinetics of the migration of various cell lines into the lesion, communication among cells, the local and systemic effect of their products, etc. Keeping this in mind, it was rather unexpected to find that in less than 1 day after the injury to horse skin or subcutis a tissue with high capacity to synthesize collagen appeared in defect. Such a tissue should contain fibroblasts, which normally appear in the repair tissue on the 3rd or 4th day after wounding (for review see Chvapil, 1967, Peacock and Van Winkle, 1976). The explanation for this early incidence of fibroblastic reaction may be due to the method of sampling of the tissue specimen which is misnamed granulation tissue. In fact, after excision of the skin the wound bed bleeds and swells. Increased water content in injured tissue in early inflammatory stages is a common phenomenon due to changes in microcirculation, the release of biologic amines, various activators, or mediators of inflammatison released by injured tissue cells, and by infiltrating inflammatory cells such as granulocytes and macrophages (James, 1969). Thus, the swollen hydrated tissues of the wound bed or the subcutaneous tissue in contact with Ivalon sponge and contraction of the edges of the excised skin were dissected and analyzed biochemically as “granulation tissue.” Granulation tissue, per definition is, a nezoly formed composite of cells, cell products and vessels. Thus, the high rate of collagen synthesis reflects rather prompt activation of the surface tissue edges of the wound than newly formed tissue. We believe, that a similar explanation holds for the data on high rate of collagen synthesis in wound tissue in l-day old excised skin wounds of rats, as reported by Digelman and Cohen (1974). Our morphological as well ‘as biochemical data indicate that the connective tissue structures are activated in the skin wound of a horse to a greater extent and at earlier periods than in the rat. This was also documented by macroscopic inspection and comparison of the granuloma tissue from both the horse and rats.
CHVAPIL
3%
ET AL.
An active infiltration of PVA sponge by granulocytes was already noticeable 17 hr after implantation. Sponge implanted into subcutis of rats was without any cellular inflammatory infiltrate within the first 24 hr. This may be explained by absence of bleeding during implanting the sponge in rats, while the horse bled profusely into the sponge. Three-day old granulation tissue in the horse contained high amounts of collagen, which was also seen in histological specimens. This was further evidence of a rather fast healing process in the horse, much faster than we have seen in comparable skin wounds in rats. As shown in Tables I and II, there exists a definite similarity in the general trend of the biochemical results in both granulation and granuloma tissue. Two parameters reflecting connective tissue reactivity, i.e., rate of collagen synthesis and the activity of prolyl hydroxylase seems to be in conflict in the first sampling period. The tissue analyzed shows synthesis of collagen at the highest rate, but the activity of prolyl hydroxylase in the sample is low. Although we have no experimental support to our view, we would assume that underhydroxylated collagen might be synthesized at this time. The disparity between the rate of collagen biosynthesis and the activity of prolyl hydroxylase in the analyzed tissue supports our view that the activity of prolyl hydroxylase is not indicative of the rate of collagen biosynthesis ( Chvapil et nl., 1976). The high activity of collagenase in the wound tissue collected 17 hr after full skin excision is of interest. The activity ‘of this enzyme in the wound bed was almost 30 times higher than in intact skin. We assume that such a high activity reflects infiltration and activation of inflammatory cells, since both activated polymorphonuclear leucocytes and macrophages contain collagenase (Oronsky et al., 1973, Robertson et al., 1972). ACKNOWLEDGMENT Financial
support
for
this
work
was provided
in part
by
NIH
Grant
GM
25159.
REFERENCES BURTON, K. (1956). Study of conditions and mechanism of diphenylamine reaction for calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. CHVAPIL, M. ( 1987). Physiology of Connective Tissue. London, Butterworth’s Scientific Publications, pp. 246-286. CHVAPIL, M. (1975). Pharmacology of Fibrosis: Definitions, Limits and Perspectives. Life Sciences 16 (9), 134551362. DIEGELMANN, R. F., ROTHKOPF, L. C., and COHEN, I. K. (1974). In: Wound Healing (Tom Gibson and J. C. van der Meulen, Eds.), pp. 55-58. Published by the Foundation International Cooperation in the Medical Sciences, Rotterdam. HUTTON, J. J., TAPPEL, A. L., and UDENFRIEND, S. ( 1966). A rapid assay for collagen proline hydroxylase. Anal. Biochem. 16, 384-394. JAMES, D. W., (1969). In; Repair and Regeneration: The Scientific Basis for Surgical Practice (J. Englebert Dunphy and H. Walton Van Winkle, Jr., Eds.), pp. 169-184. Pub. McGraw-Hill, Inc., New York. JUVA, K., and PROCKOP, D. J. (1966). Modified procedure for the assay of Ha or C14-labelled hydroxyproline. Anal. Biochem. 15, 77-83. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANIJAL.L, R. J. ( 1951). Protein measurement with the fohn phenol reagent. Z. Biol. Chem. 193, 265-275. MEAGER, D. M., and ADAPTS, 0. R. (1970). Skin transplantation in horses. Can. Vet. Jour. 11 (12), 239-248.
SKIN
WOUNDS
IN
HORSES
AND
RATS
359
ORONSKY, A. L., PEHPEH, R. J,, and SCHRO~ER, H. C. (1973). Phngocytic Release and Activation of Human Leukocyte Procollagenase. Nature 246, 417-419. PEACOCK, ERLE E., JR., and VAN WINKLE, W. ( 1976). Wound Repair, 2nd Ed., Pul). W. B. Saunders Company, Philadelphia. ROBERTSON, P. B., RYEL, R. B., TAYLOH, R. E., SHYU, K. W., and FULLhIm, H. M. (1972). Collagenase: localization in polymorphonuclear leukocyte granules in the rahhit. Science 177, BP6Ei. RYAN, J. N., and WOESSNEH, J. F., JR. ( 1971). M ammalian coKagenasc-Direct demonstration in homogenates of involuting rat uterus. Biochem. Biol>hys. Res. Commun. 44, 144. UITTO, J. (1970). A method for studying collagen biosynthesis in human skin biopsies in aitro. Biochim. Biophyn. Acta 201, 438-415.