Biology of Fetal Wound Healing: Collagen Biosynthesis During Dermal Repair ByFrazier
W. Frantz, Robert F. Diegelmann,
Bruce A. Mast, and I. Kelman Cohen
Richmond, Virginia l The rapid restoration of tissue integrity and breaking strength in healing fetal wounds is mainly a function of fetal wound collagen. In this study, the fetal and adult tissue responses to injury were characterized in terms of changes in collagen biosynthesis. Linear wounds and unwounded skin were incubated with radioactive proline, and collagen synthesis was measured as isotope incorporation into collagenasesensitive protein. Likewise, noncollagen protein synthesis was represented by isotope incorporation into collagenaseresistant protein. Adult wounds demonstrated a preferential stimulation of collagen as compared with noncollagen protein synthesis after wounding. In contrast, both collagen and noncollagen protein synthesis were significantly elevated in the fetus during the first 5 days postwounding. Despite marked increases in fetal wound collagen synthesis above both unwounded fetal skin and adult wound levels, fetal wounds exhibited no evidence of excessive collagen deposition or scar formation after wounding. These findings suggest that the fetal response to tissue injury is a function of the distinctive qualities of fetal fibroblasts associated with the extracellular wound matrix and may involve rapid collagen turnover and degradation. Copyright o 1992 by W.B. Saunders Company INDEX WORDS:
Fetal wound healing; collagen biosynthesis.
F
ETAL WOUND healing research has undergone tremendous growth over the last decade. First, in utero intervention to correct life-threatening congenital anomalies has mandated a thorough understanding of the mechanisms of fetal healing. Second, through an understanding of the biological mechanisms of “scarless” fetal repair, it is hoped that pathological conditions of adult healing may be prevented or more effectively treated. While the histological and biochemical aspects of this unique repair process have been well documented in several animal models,i-7 the mechanisms of fetal repair are only beginning to be elucidated. From histological evidence, fetal repair more closely resembles regeneration as opposed to classic adult healing by scar deposition. As in adult healing, the study of collagen biosynthesis is critical to understanding fetal tissue repair. The central role of collagen as the major structural protein of connective tissue is evidenced by the rapid restoration of tissue integrity and breaking strength reported in healing fetal wounds.8 In addition to its functional role in tissue repair, fetal collagen, alone or through interactions with other elements of the
JournalofPediatric Surgery, Vol27, No 8 (August), 1992: pp 945-949
extracellular matrix, is likely responsible for the lack of scarring in fetal wounds. In this study, collagen synthesis rates in healing fetal incisional wounds were quantitated. In addition, fetal and adult responses to tissue injury, as measured by alterations in collagen and total protein synthesis, were compared. MATERIALS
AND METHODS
Time-dated pregnant and 5-kg nonpregnant adult New Zealand white rabbits were obtained from Myrtle’s Rabbitry (Thompson Station, TN) and housed locally for 3 to 6 days prior to wounding to allow for acclimation to the new environment. All rabbits were fed ad lib rabbit chow and had free access to water.
Wounding Procedure Fetal wounding studies were performed at 24 days’ gestation (term = 31 days) using previously described fetal surgical techniques.9 Briefly, pregnant does were anesthetized with spontaneous halothaneioxygen ventilation and underwent midline celiotomy. Sequential hysterotomies were performed, through which each fetus (n = 6 per doe) received a single l- to l&m fullthickness dorsal midline incisional wound. After wound closure with interrupted sutures of 6-O silk, amniotic fluid volume was reconstituted with warmed plasmalyte solution (Travenol Laboratories Inc, Deerfield, lL), and hysterotomies were closed with full-thickness purse-string sutures of 4-O silk. Fetal wounds were excised at sacrifice with l-mm borders of normal surrounding tissue at 1,2,3.4,5, and 6 days postwounding. Unwounded skin served as controls. Fetal survival postwounding was 89% for 34 fetuses. Maternal survival was 100%. Adult wounding studies were accomplished using general halothaneloxygen anesthesia. Each animal (n = 6) received 5 dorsal paravertebral full-thickness incisional wounds, 3 to 4 cm in length, which were closed with interrupted sutures of 2-ODermalon (Davis and Geck, Danbury. CT). Adult wounds were excised at sacrifice with l-mm borders of normal surrounding tissue at 2, 3, 5, 7, 10, and 14 days postwounding. Survival was 100%.
From The Wound Healing Center, Division of Plastic Surgery Department of Surgery, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA. Supported in part by National Institutes of Health grant GM 20298. Second Place Winner in the Jens G. Rosenkranz Resident Competition. Presented at the 43rd Annual Meeting of the Surgical Section of the American Academy of Pediatrics, New Orleans, Louisiana, October 26-27, 1991. Address reprint requests to Frazier W. Frantz, MD, The Wound Healing Center, Box I1 7, MCVStation. Richmond. VA 23298-0117. Copyright Q I992 bv W.B. Saunders Company 0022-3468/9212708-0002$03.00/O
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At killing, sections from all adult and fetal wounds were fixed in 10% formalin, stained with hematoxylin and eosin, and examined by light microscopy to confirm histological progression of healing. Confirmation of full-thickness injury through the panniculus carnosis muscle at the time of original wounding was also achieved at this time.
Collagen Synthesis Assay Radioactive proline (L-[5-3H]-proline, 7.59 Ci/mmol) was obtained from Amersham Corporation (Arlington Heights, IL). Chromatographically purified bacterial collagenase was purchased from Worthington Biochemical Corporation (Freehold, NJ) and further purified on a Sephadex G-200 column to remove noncollagen-degrading proteinase activity.‘O Using previously described techniques11,12 as outlined in Fig 1, excised wounds or unwounded skin from fetal and adult animals were immediately placed in 20-mL flasks containing 3 mL of Hanks’ balanced salt solution supplemented with 0.1 mmol/L of ascorbate. The tissues were minced and pulse-labeled with 60 uCi (20~ Ci/mL) of [3H]-proline during a 2.5-hour incubation at 37°C in a shaking water bath. After homogenization, unincorporated [3H]-proline was separated from proteins by precipitation with cold trichloracetic acid (TCA). Quantities of 8 to 12 mg of each dried protein sample were redissolved in NaOH, and a portion of the solubilized substrate was used to quantitate total protein by the Lowry protein assay.i3 Equal
portions of the solution were then neutralized and incubated with or without purified bacterial collagenase. The proteins were reprecipitated with a mixture of TCA and tannic acid. In the samples that had collagenase added to them, the peptides derived from collagen remained in the TCA/tannic acid-soluble fraction, whereas noncollagen proteins were in the reprecipitated fraction. The minus enzyme control was used to correct for any free [3H]-proline that had been bound to proteins and was released by dissolution in NaOH. The radioactivity of the noncollagen peptide, collagen peptide, and blank vials solubilized in 10 mL of Tritonliquifluor was measured in a model 1500 Packard Tri-Carb liquid scintillation spectrometer (Packard Instrument Co, Downer’s Grove, IL) and expressed in disintegrations per minute (dpm). The amount of radioactivity solubilized by collagenase was a measurement of collagen synthesized, while the radioactivity in the precipitate correlated to noncollagen protein synthesized.
Calculations These data were used to calculate the following parameters of collagen synthesis: absolute collagen synthesis (ACS), absolute noncollagen protein synthesis (ANCPS), and relative collagen synthesis (RCS). ACS =
ANCPS =
6
dpm collagen mg total protein
dpm noncollagen protein mg total protein
RCS (%) =
e
Skin/Wounds Excised
dpm collagen x 100 (dpm noncollagen protein x 5.4) + dpm collagen ACS and ANCPS values represent quantitative measures of protein synthesized per milligram of total protein, whereas RCS values represent the ratio of collagen synthesized in relation to total protein synthesized. Of note, RCS calculations were corrected to reflect the relative imino acid content of collagen as compared with noncollagen protein (collagen is 5.4 times enriched in imino acids in relation to other proteins).‘” A fundamental assumption of this assay is that the same cellular pool of precursor amino acids is equally used for both collagen and noncollagen protein synthesis and that major differences in cell pool size between tissues do not exist. Collagen synthesis data shown represent the mean 2 standard error. Statistical comparisons of these data were performed using Student’s t test analysis. All procedures conducted were approved by the Medical College of Virginia Institutional Animal Care and Use Committee.
Incubation 3H-proline
I
Lab&d Tissue
Homogenization TCA Centrifugation
Collagen Psptides
Precipitate
Supematant
Precipitate
(+)
(4
(-1
Nonccllagen Peptides
Blank
Fig 1. Schematic drawing of steps involved in collagen synthesis assay. Radioactivity of samples treated with (+) and without (-) bacterial collagenase was measured using liquid scintillation counting.
RESULTS
Histological findings in adult14J5 and fetal wound&* were as previously described. In adult wounds, acute inflammation was predominant at 2 and 3 days postwounding, while fibroblast migration and collagen deposition prevailed from 5 days on. Fetal wounds demonstrated minimal acute inflammation, mild fibroblast infiltration, and no excessive collagen deposition or scar. Reepithelialization was complete by 48 hours, and dermal healing was histologically complete by 72 hours.
COLLAGEN
BIOSYNTHESIS
IN FETAL WOUND HEALING
Adult wounds showed significant increases in ACS at 5,7, and 10 days postwounding and in ANCPS at 2 and 3 days postwounding with subsequent rapid return to baseline levels (Fig 2). In contrast, fetal wounds demonstrated significant elevations in both ACS and ANCPS above baseline starting at 1 day and continuing until 5 days postwounding (Fig 3). Absolute synthesis rates for both collagen and noncollagen protein were lo-fold higher in fetal skin and wounds in comparison to their adult counterparts. Analysis of collagen synthesis relative to total protein synthesis showed significantly greater rates in adult incisional wounds when compared with unwounded skin values at 5, 7, 10, and 14 days postwounding, peaking at a near threefold difference (Fig 4). In marked contrast, RCS rates in fetal wounds remained essentially unchanged from unwounded levels throughout the study period. DISCUSSION
It is clear from histological and biochemical data that the fetal response to tissue injury is quite different from that of the adult.1-7 Of the many factors that contribute to the unique nature of fetal repair, it appears that fetal collagen metabolism has a central role in effecting rapid restoration of structure and function in fetal skin wounds. In this study, collagen biosynthesis analysis was applied to further characterize this role. Collagen synthesis rates were quantitated in unwounded skin during development and in incisional fetal wounds during healing. These data were then compared with similar parameters measured during healing of adult wounds to assess differences in tissue responses to injury as measured by changes in collagen biosynthesis. Adult collagen synthesis data generated in this study are consistent with previous applications of this assay in the rat model. l1 It is likely that protein production by inflammatory cells present in the adult wound accounts for the increases in noncollagen protein synthesis during the first 2 to 3 days after
947
Fig 3. Protein synthesis in thefetus. ACS and ANCPS of fetal linear wounds (0) and unwounded skin (0). lP c .05.
wounding. The delayed increase in collagen synthesis after wounding can be attributed to several factors. First, adult fibroblasts are quite sparse and exist in a quiescent state in normal, unwounded dermis. These cells require an activation stimulus (either humoral or chemical) and time to proliferate and migrate into the wound space before collagen synthesis can begin. Typically, these steps can take up to 2 to 3 days. Furthermore, a significant cell density is required to optimize or maximize collagen synthesis, a phenomenon reproduced in monolayer culture where fibroblasts in the log phase of growth produce reduced amounts of collagen. l4 It is only after confluence has been attained that increased amounts of collagen are produced. From the earliest timepoint observed, fetal wounds demonstrated rapid and significant increases in both collagen and noncollagen protein synthesis (Fig 3). This rapid timecourse of increased collagen synthesis is likely attributable to several fundamental differences between fetal and adult fibroblasts. First, the fetal fibroblast normally exists in a relatively active state and may require no specific stimulation to rapidly increase collagen synthesis. Evidence of this highly active state of protein synthesis in normal, developing fetal fibroblasts is manifested as extensive networks of rough endoplasmic reticulum and has been observed using transmission electronmicros6-
*
r T
6
w
5
Fig 2. Protein synthesis in the adult. ACS and ANCPS of adult linear wounds (0) and unwounded skin (0). lP c .05.
Fig 4. RCS of adult versus fetal linear wounds skin (0). lP < .05.
(0) and
unwounded
FRANTZ ET AL
949
copy.16 Second, relative to adult fibroblasts, fetal fibroblasts exist in a more “fluid” extracellular matrix rich in hyaluronic acid, an environment that facilitates proliferation and migration of these cells to the wound site.” Third, fetal fibroblasts, unlike adult fibroblasts, do not demonstrate contact-inhibition in vitro16 and, thus, may assemble in wounds in a more densely populated manner. We have observed rabbit fetal fibroblasts maintained under standard tissue culture conditions stacked 5 to 7 cells deep on electronmicroscopy sections. We hypothesize that this effect may be due to coats of pericellular hyaluronate that block cell-to-cell membrane interactions responsible for contact inhibition.i8 Finally, fetal fibroblasts are capable of simultaneously proliferating and synthesizing collagen in vitro (Mast B: personal communication) unlike adult fibroblasts that maximally produce collagen during quiescence.19 Increased noncollagen protein synthesis in fetal wounds is likely a manifestation of the rapid cellular proliferation induced by tissue injury in the fetal environment. In addition, in light of the marked increases in collagen synthesis and the documented absence of excessive collagen deposition and scar formation in fetal wounds, it is interesting to postulate that a portion of the increased protein synthesis
in these wounds includes collagenase and other enzymes that may modulate early remodeling, crosslinking, and orderly deposition of fetal collagen. From this study, it appears that the fetal response to tissue injury involves not only increased collagen synthesis but also increased noncollagen protein synthesis. The proportional increases in collagen and noncollagen protein synthesis after wounding in the fetus provide biochemical support for fetal repair through a “regeneration-like” process. In sharp contrast to the adult “repair” process, whereby tissue defects are filled with large deposits of collagen, fetal healing mimics regeneration. In fetal wounds, tissue structure and function are restored by rapid proliferation of cells to replace cellular elements lost to injury, and essential components of the extracellular matrix are restored through increased collagen synthesis. Attention must now be focused on characterizing the mechanisms that control these processes at the molecular level.
ACKNOWLEDGMENT The authors gratefully acknowledge the sponsorship of Dr Arnold Salzberg, Chairman, Division of Pediatric Surgery, Medical College of Virginia.
REFERENCES 1. Longaker MT, Adzick NS: The biology of fetal wound healing: A review. Plast Reconstr Surg 87:788-798, 1991 2. Krummel TM, Nelson JM, Diegelmann RF, et al: Fetal response to injury in the rabbit. J Pediatr Surg 22:640-644,1987 3. Hallock GG, Rice DC, Merkel JR, et al: Analysis of collagen content in the fetal wound. Ann Plastic Surg 21:310-315, 1988 4. Adzick NS, Harrison MR, Glick PL, et al: Comparison of fetal, newborn, and adult wound healing by histologic, enzymehistochemical, and hydroxyproline determinations. J Pediatr Surg 20:315-319, 1985 5. Burd DAR, Longaker MT, Adzick NS, et al: Foetal wound healing in a large animal model: The deposition of collagen is confirmed. Br J Plast Surg 43:571-577,199O 6. Merkel JR, DiPaolo BR, Hallock GG, et al: Type I and type III collagen content of healing wounds in fetal and adult rats. Proc Sot Exp Biol Med 187:493-497,1988 7. Longaker MT, Whitby DJ, Adzick NS, et al: Studies in fetal wound healing: VI. Second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation. J Pediatr Surg 25:63-69,199O 8. Frantz FW, Garrison FR, Mast BA, et al: Rapid restoration of breaking strength in healing fetal rabbit wounds. Surg Forum 42:661-663,199l 9. Adzick NS, Harrison MR: Surgical techniques in the fetal rabbit, in Nathanielsz P (ed): Animal Models in Fetal Medicine. Amsterdam, The Netherlands, North Holland, 1980, pp 68-99 10. Peterkofsky B, Diegelmann R: Use of a mixture of proteinasefree collagenase for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry 10:988-994,197l
11. Diegelmann RF, Rothkopf LC, Cohen IK: Measurement of collagen biosynthesis during wound healing. J Surg Res 19:239-243, 1975 12. Peterkofsky B, Chojkier M, Bateman J: Determination of collagen synthesis in tissue and cell culture systems, in Furthmayr H (ed): Immunochemistry of the Extracellular Matrix, vol II. Boca Raton, FL, CRC Press, 1982, pp 19-47 13. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275,195l 14. Madden JW, Arem AJ: Wound healing: Biologic and clinical features, in Sabiston DC (ed): Textbook of Surgery: The Biological Basis of Modern Surgical Practice. Philadelphia, PA, Saunders, 1986, pp 193-213 15. Hunt TK, Van Winkle W: Normal repair, in Hunt TK, Dunphy JE (eds): Fundamentals of Wound Management. New York, NY, Appleton-Century-Crofts, 1979, pp 2-67 16. Mast BA, Harris TH, Frantz FW, et al: Ultrastructural comparison of fetal wounds and skin: Mammalian healing by regeneration. (in preparation) 17. Depalma RL, Krummel TM, Durham LA, et al: Characterization and quantitation of wound matrix in the fetal rabbit. Matrix 9:224-231,1989 18. Underhill CB, Toole BP: Transformation-dependent loss of the hyaluronate-containing coats of cultured cells. J Cell Physiol 110:123-128,1982 19. Graham MF, Diegelmann RF, Cohen IK: An in vitro model of fibroplasia: Simultaneous quantification of fibroblast proliferation, migration, and collagen synthesis. Proc Sot Exp Bio Med 176:302-308,1984
COLLAGEN
BIOSYNTHESIS
IN FETAL WOUND HEALING
949
Discussion Michael Harrison (San Francisco, CA): Dr Frantz and the Richmond group have continued to search for the magic ingredient that allows the fetus to heal without scar formation. They have concentrated, logically, on collagen and its synthesis. The first reports, however, were that the fetal wound was deficient in collagen. And now we hear that in fact the synthesis of collagen has increased. So the first question is, what happened in the intervening years? Second, there’s no question that collagen is a key molecule, but our group feels it is the organization of the collagen and not its absence that distinguishes the fetal wound. You have very nicely demonstrated that synthesis of collagen has increased, and yet the total amount has not increased. It would seem obvious then that reorganization and remodeling by metalloproteins like collagenase, must be the key. So the second question is, have you looked at collagenase in this mode, or, better yet, inhibitors of metalloproteinase (TIMPS) which fine-tune collagen deposition. And, finally, I’m sure you are very aware of the problems with the fetal rabbit model. After developing it, we abandoned it because it’s a small animal model with a short gestation, and the wounds gape like they do in almost no other animal. So the final question is, are you looking at this in a larger animal model? Frazier Frantz (response): As I have pointed out, the role of collagen metabolism in fetal tissue repair appears obvious when you consider the rapid restoration of tissue integrity and breaking strength evidence in fetal wounds. In this study, we chose to use actual healing linear wounds to quantitate and further characterize collagen biosynthesis during fetal repair. As you have mentioned, in earlier reports from our lab, Dr Tom Krummel reported the absence of collagen (by HPLC) in polyvinyl alcohol (PVA) sponge implants placed in fetal rabbits. This is in sharp contrast to the marked fibroplasia seen in PVA implants placed in adult rabbits and serves as one of several examples of the unique responses of the fetus to wounding. However, it is important to note that the PVA implant model, like other wound chamber models, is dependent on a foreign-body response
which, in the fetus, appears to be minimal. This model has proved an invaluable tool for measuring the effects of adult growth factors and other manipulations on fetal wound healing because of its low background with regard to collagen deposition. We are very interested in the potential roles of collagenase and early collagen turnover in fetal wounds. The fact that you have increased collagen synthesis but no excessive collagen deposition or scar formation in fetal wounds invokes the role of collagenase or other metalloproteases in modulating early breakdown, turnover, cross-linking, and orderly deposition of fetal collagen. We have begun our investigation of this phenomenon at the molecular level looking for expression of fetal collagenase message. We have used polymerase chain reaction (PCR) technology to analyze RNA from 2%day fetal skin and wounds. cDNAs were synthesized from equal amounts of skin and wound RNAs and hybridized with oligonucleotide primers specific for collagenase message. While we found no expression of collagenase message in 2%day fetal skin, there were easily detectable levels of this message in 2%day wound tissue. Right now, we are investigating the complete time course of collagenase expression after fetal wounding. Additional studies we plan to undertake to characterize the role of collagenase in fetal repair are, first, analyzing collagenolytic activity on the protein level, and, second, applying collagenase inhibitors. Theoretically, if collagenase is so important in these wounds and you inhibit it, will we see further increases in collagen deposition in fetal wounds? Since coming into the lab last July, we really felt that there was a lot of work to be done in analyzing the mechanisms of fetal repair at the molecular level. Accordingly, we have continued to use the fetal rabbit model because it is easy to work with, has multiple gestations, is relatively inexpensive, and has provided for easily reproducible data. At this point, we are interested to see if our findings will be reproduced in a larger animal model and have, consequently, begun wounding experiments in fetal pigs.
W. Frantz, Robert F. Diegelmann,
Bruce A. Mast, and I. Kelman Cohen
Richmond, Virginia l The rapid restoration of tissue integrity and breaking strength in healing fetal wounds is mainly a function of fetal wound collagen. In this study, the fetal and adult tissue responses to injury were characterized in terms of changes in collagen biosynthesis. Linear wounds and unwounded skin were incubated with radioactive proline, and collagen synthesis was measured as isotope incorporation into collagenasesensitive protein. Likewise, noncollagen protein synthesis was represented by isotope incorporation into collagenaseresistant protein. Adult wounds demonstrated a preferential stimulation of collagen as compared with noncollagen protein synthesis after wounding. In contrast, both collagen and noncollagen protein synthesis were significantly elevated in the fetus during the first 5 days postwounding. Despite marked increases in fetal wound collagen synthesis above both unwounded fetal skin and adult wound levels, fetal wounds exhibited no evidence of excessive collagen deposition or scar formation after wounding. These findings suggest that the fetal response to tissue injury is a function of the distinctive qualities of fetal fibroblasts associated with the extracellular wound matrix and may involve rapid collagen turnover and degradation. Copyright o 1992 by W.B. Saunders Company INDEX WORDS:
Fetal wound healing; collagen biosynthesis.
F
ETAL WOUND healing research has undergone tremendous growth over the last decade. First, in utero intervention to correct life-threatening congenital anomalies has mandated a thorough understanding of the mechanisms of fetal healing. Second, through an understanding of the biological mechanisms of “scarless” fetal repair, it is hoped that pathological conditions of adult healing may be prevented or more effectively treated. While the histological and biochemical aspects of this unique repair process have been well documented in several animal models,i-7 the mechanisms of fetal repair are only beginning to be elucidated. From histological evidence, fetal repair more closely resembles regeneration as opposed to classic adult healing by scar deposition. As in adult healing, the study of collagen biosynthesis is critical to understanding fetal tissue repair. The central role of collagen as the major structural protein of connective tissue is evidenced by the rapid restoration of tissue integrity and breaking strength reported in healing fetal wounds.8 In addition to its functional role in tissue repair, fetal collagen, alone or through interactions with other elements of the
JournalofPediatric Surgery, Vol27, No 8 (August), 1992: pp 945-949
extracellular matrix, is likely responsible for the lack of scarring in fetal wounds. In this study, collagen synthesis rates in healing fetal incisional wounds were quantitated. In addition, fetal and adult responses to tissue injury, as measured by alterations in collagen and total protein synthesis, were compared. MATERIALS
AND METHODS
Time-dated pregnant and 5-kg nonpregnant adult New Zealand white rabbits were obtained from Myrtle’s Rabbitry (Thompson Station, TN) and housed locally for 3 to 6 days prior to wounding to allow for acclimation to the new environment. All rabbits were fed ad lib rabbit chow and had free access to water.
Wounding Procedure Fetal wounding studies were performed at 24 days’ gestation (term = 31 days) using previously described fetal surgical techniques.9 Briefly, pregnant does were anesthetized with spontaneous halothaneioxygen ventilation and underwent midline celiotomy. Sequential hysterotomies were performed, through which each fetus (n = 6 per doe) received a single l- to l&m fullthickness dorsal midline incisional wound. After wound closure with interrupted sutures of 6-O silk, amniotic fluid volume was reconstituted with warmed plasmalyte solution (Travenol Laboratories Inc, Deerfield, lL), and hysterotomies were closed with full-thickness purse-string sutures of 4-O silk. Fetal wounds were excised at sacrifice with l-mm borders of normal surrounding tissue at 1,2,3.4,5, and 6 days postwounding. Unwounded skin served as controls. Fetal survival postwounding was 89% for 34 fetuses. Maternal survival was 100%. Adult wounding studies were accomplished using general halothaneloxygen anesthesia. Each animal (n = 6) received 5 dorsal paravertebral full-thickness incisional wounds, 3 to 4 cm in length, which were closed with interrupted sutures of 2-ODermalon (Davis and Geck, Danbury. CT). Adult wounds were excised at sacrifice with l-mm borders of normal surrounding tissue at 2, 3, 5, 7, 10, and 14 days postwounding. Survival was 100%.
From The Wound Healing Center, Division of Plastic Surgery Department of Surgery, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA. Supported in part by National Institutes of Health grant GM 20298. Second Place Winner in the Jens G. Rosenkranz Resident Competition. Presented at the 43rd Annual Meeting of the Surgical Section of the American Academy of Pediatrics, New Orleans, Louisiana, October 26-27, 1991. Address reprint requests to Frazier W. Frantz, MD, The Wound Healing Center, Box I1 7, MCVStation. Richmond. VA 23298-0117. Copyright Q I992 bv W.B. Saunders Company 0022-3468/9212708-0002$03.00/O
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At killing, sections from all adult and fetal wounds were fixed in 10% formalin, stained with hematoxylin and eosin, and examined by light microscopy to confirm histological progression of healing. Confirmation of full-thickness injury through the panniculus carnosis muscle at the time of original wounding was also achieved at this time.
Collagen Synthesis Assay Radioactive proline (L-[5-3H]-proline, 7.59 Ci/mmol) was obtained from Amersham Corporation (Arlington Heights, IL). Chromatographically purified bacterial collagenase was purchased from Worthington Biochemical Corporation (Freehold, NJ) and further purified on a Sephadex G-200 column to remove noncollagen-degrading proteinase activity.‘O Using previously described techniques11,12 as outlined in Fig 1, excised wounds or unwounded skin from fetal and adult animals were immediately placed in 20-mL flasks containing 3 mL of Hanks’ balanced salt solution supplemented with 0.1 mmol/L of ascorbate. The tissues were minced and pulse-labeled with 60 uCi (20~ Ci/mL) of [3H]-proline during a 2.5-hour incubation at 37°C in a shaking water bath. After homogenization, unincorporated [3H]-proline was separated from proteins by precipitation with cold trichloracetic acid (TCA). Quantities of 8 to 12 mg of each dried protein sample were redissolved in NaOH, and a portion of the solubilized substrate was used to quantitate total protein by the Lowry protein assay.i3 Equal
portions of the solution were then neutralized and incubated with or without purified bacterial collagenase. The proteins were reprecipitated with a mixture of TCA and tannic acid. In the samples that had collagenase added to them, the peptides derived from collagen remained in the TCA/tannic acid-soluble fraction, whereas noncollagen proteins were in the reprecipitated fraction. The minus enzyme control was used to correct for any free [3H]-proline that had been bound to proteins and was released by dissolution in NaOH. The radioactivity of the noncollagen peptide, collagen peptide, and blank vials solubilized in 10 mL of Tritonliquifluor was measured in a model 1500 Packard Tri-Carb liquid scintillation spectrometer (Packard Instrument Co, Downer’s Grove, IL) and expressed in disintegrations per minute (dpm). The amount of radioactivity solubilized by collagenase was a measurement of collagen synthesized, while the radioactivity in the precipitate correlated to noncollagen protein synthesized.
Calculations These data were used to calculate the following parameters of collagen synthesis: absolute collagen synthesis (ACS), absolute noncollagen protein synthesis (ANCPS), and relative collagen synthesis (RCS). ACS =
ANCPS =
6
dpm collagen mg total protein
dpm noncollagen protein mg total protein
RCS (%) =
e
Skin/Wounds Excised
dpm collagen x 100 (dpm noncollagen protein x 5.4) + dpm collagen ACS and ANCPS values represent quantitative measures of protein synthesized per milligram of total protein, whereas RCS values represent the ratio of collagen synthesized in relation to total protein synthesized. Of note, RCS calculations were corrected to reflect the relative imino acid content of collagen as compared with noncollagen protein (collagen is 5.4 times enriched in imino acids in relation to other proteins).‘” A fundamental assumption of this assay is that the same cellular pool of precursor amino acids is equally used for both collagen and noncollagen protein synthesis and that major differences in cell pool size between tissues do not exist. Collagen synthesis data shown represent the mean 2 standard error. Statistical comparisons of these data were performed using Student’s t test analysis. All procedures conducted were approved by the Medical College of Virginia Institutional Animal Care and Use Committee.
Incubation 3H-proline
I
Lab&d Tissue
Homogenization TCA Centrifugation
Collagen Psptides
Precipitate
Supematant
Precipitate
(+)
(4
(-1
Nonccllagen Peptides
Blank
Fig 1. Schematic drawing of steps involved in collagen synthesis assay. Radioactivity of samples treated with (+) and without (-) bacterial collagenase was measured using liquid scintillation counting.
RESULTS
Histological findings in adult14J5 and fetal wound&* were as previously described. In adult wounds, acute inflammation was predominant at 2 and 3 days postwounding, while fibroblast migration and collagen deposition prevailed from 5 days on. Fetal wounds demonstrated minimal acute inflammation, mild fibroblast infiltration, and no excessive collagen deposition or scar. Reepithelialization was complete by 48 hours, and dermal healing was histologically complete by 72 hours.
COLLAGEN
BIOSYNTHESIS
IN FETAL WOUND HEALING
Adult wounds showed significant increases in ACS at 5,7, and 10 days postwounding and in ANCPS at 2 and 3 days postwounding with subsequent rapid return to baseline levels (Fig 2). In contrast, fetal wounds demonstrated significant elevations in both ACS and ANCPS above baseline starting at 1 day and continuing until 5 days postwounding (Fig 3). Absolute synthesis rates for both collagen and noncollagen protein were lo-fold higher in fetal skin and wounds in comparison to their adult counterparts. Analysis of collagen synthesis relative to total protein synthesis showed significantly greater rates in adult incisional wounds when compared with unwounded skin values at 5, 7, 10, and 14 days postwounding, peaking at a near threefold difference (Fig 4). In marked contrast, RCS rates in fetal wounds remained essentially unchanged from unwounded levels throughout the study period. DISCUSSION
It is clear from histological and biochemical data that the fetal response to tissue injury is quite different from that of the adult.1-7 Of the many factors that contribute to the unique nature of fetal repair, it appears that fetal collagen metabolism has a central role in effecting rapid restoration of structure and function in fetal skin wounds. In this study, collagen biosynthesis analysis was applied to further characterize this role. Collagen synthesis rates were quantitated in unwounded skin during development and in incisional fetal wounds during healing. These data were then compared with similar parameters measured during healing of adult wounds to assess differences in tissue responses to injury as measured by changes in collagen biosynthesis. Adult collagen synthesis data generated in this study are consistent with previous applications of this assay in the rat model. l1 It is likely that protein production by inflammatory cells present in the adult wound accounts for the increases in noncollagen protein synthesis during the first 2 to 3 days after
947
Fig 3. Protein synthesis in thefetus. ACS and ANCPS of fetal linear wounds (0) and unwounded skin (0). lP c .05.
wounding. The delayed increase in collagen synthesis after wounding can be attributed to several factors. First, adult fibroblasts are quite sparse and exist in a quiescent state in normal, unwounded dermis. These cells require an activation stimulus (either humoral or chemical) and time to proliferate and migrate into the wound space before collagen synthesis can begin. Typically, these steps can take up to 2 to 3 days. Furthermore, a significant cell density is required to optimize or maximize collagen synthesis, a phenomenon reproduced in monolayer culture where fibroblasts in the log phase of growth produce reduced amounts of collagen. l4 It is only after confluence has been attained that increased amounts of collagen are produced. From the earliest timepoint observed, fetal wounds demonstrated rapid and significant increases in both collagen and noncollagen protein synthesis (Fig 3). This rapid timecourse of increased collagen synthesis is likely attributable to several fundamental differences between fetal and adult fibroblasts. First, the fetal fibroblast normally exists in a relatively active state and may require no specific stimulation to rapidly increase collagen synthesis. Evidence of this highly active state of protein synthesis in normal, developing fetal fibroblasts is manifested as extensive networks of rough endoplasmic reticulum and has been observed using transmission electronmicros6-
*
r T
6
w
5
Fig 2. Protein synthesis in the adult. ACS and ANCPS of adult linear wounds (0) and unwounded skin (0). lP c .05.
Fig 4. RCS of adult versus fetal linear wounds skin (0). lP < .05.
(0) and
unwounded
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copy.16 Second, relative to adult fibroblasts, fetal fibroblasts exist in a more “fluid” extracellular matrix rich in hyaluronic acid, an environment that facilitates proliferation and migration of these cells to the wound site.” Third, fetal fibroblasts, unlike adult fibroblasts, do not demonstrate contact-inhibition in vitro16 and, thus, may assemble in wounds in a more densely populated manner. We have observed rabbit fetal fibroblasts maintained under standard tissue culture conditions stacked 5 to 7 cells deep on electronmicroscopy sections. We hypothesize that this effect may be due to coats of pericellular hyaluronate that block cell-to-cell membrane interactions responsible for contact inhibition.i8 Finally, fetal fibroblasts are capable of simultaneously proliferating and synthesizing collagen in vitro (Mast B: personal communication) unlike adult fibroblasts that maximally produce collagen during quiescence.19 Increased noncollagen protein synthesis in fetal wounds is likely a manifestation of the rapid cellular proliferation induced by tissue injury in the fetal environment. In addition, in light of the marked increases in collagen synthesis and the documented absence of excessive collagen deposition and scar formation in fetal wounds, it is interesting to postulate that a portion of the increased protein synthesis
in these wounds includes collagenase and other enzymes that may modulate early remodeling, crosslinking, and orderly deposition of fetal collagen. From this study, it appears that the fetal response to tissue injury involves not only increased collagen synthesis but also increased noncollagen protein synthesis. The proportional increases in collagen and noncollagen protein synthesis after wounding in the fetus provide biochemical support for fetal repair through a “regeneration-like” process. In sharp contrast to the adult “repair” process, whereby tissue defects are filled with large deposits of collagen, fetal healing mimics regeneration. In fetal wounds, tissue structure and function are restored by rapid proliferation of cells to replace cellular elements lost to injury, and essential components of the extracellular matrix are restored through increased collagen synthesis. Attention must now be focused on characterizing the mechanisms that control these processes at the molecular level.
ACKNOWLEDGMENT The authors gratefully acknowledge the sponsorship of Dr Arnold Salzberg, Chairman, Division of Pediatric Surgery, Medical College of Virginia.
REFERENCES 1. Longaker MT, Adzick NS: The biology of fetal wound healing: A review. Plast Reconstr Surg 87:788-798, 1991 2. Krummel TM, Nelson JM, Diegelmann RF, et al: Fetal response to injury in the rabbit. J Pediatr Surg 22:640-644,1987 3. Hallock GG, Rice DC, Merkel JR, et al: Analysis of collagen content in the fetal wound. Ann Plastic Surg 21:310-315, 1988 4. Adzick NS, Harrison MR, Glick PL, et al: Comparison of fetal, newborn, and adult wound healing by histologic, enzymehistochemical, and hydroxyproline determinations. J Pediatr Surg 20:315-319, 1985 5. Burd DAR, Longaker MT, Adzick NS, et al: Foetal wound healing in a large animal model: The deposition of collagen is confirmed. Br J Plast Surg 43:571-577,199O 6. Merkel JR, DiPaolo BR, Hallock GG, et al: Type I and type III collagen content of healing wounds in fetal and adult rats. Proc Sot Exp Biol Med 187:493-497,1988 7. Longaker MT, Whitby DJ, Adzick NS, et al: Studies in fetal wound healing: VI. Second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation. J Pediatr Surg 25:63-69,199O 8. Frantz FW, Garrison FR, Mast BA, et al: Rapid restoration of breaking strength in healing fetal rabbit wounds. Surg Forum 42:661-663,199l 9. Adzick NS, Harrison MR: Surgical techniques in the fetal rabbit, in Nathanielsz P (ed): Animal Models in Fetal Medicine. Amsterdam, The Netherlands, North Holland, 1980, pp 68-99 10. Peterkofsky B, Diegelmann R: Use of a mixture of proteinasefree collagenase for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry 10:988-994,197l
11. Diegelmann RF, Rothkopf LC, Cohen IK: Measurement of collagen biosynthesis during wound healing. J Surg Res 19:239-243, 1975 12. Peterkofsky B, Chojkier M, Bateman J: Determination of collagen synthesis in tissue and cell culture systems, in Furthmayr H (ed): Immunochemistry of the Extracellular Matrix, vol II. Boca Raton, FL, CRC Press, 1982, pp 19-47 13. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275,195l 14. Madden JW, Arem AJ: Wound healing: Biologic and clinical features, in Sabiston DC (ed): Textbook of Surgery: The Biological Basis of Modern Surgical Practice. Philadelphia, PA, Saunders, 1986, pp 193-213 15. Hunt TK, Van Winkle W: Normal repair, in Hunt TK, Dunphy JE (eds): Fundamentals of Wound Management. New York, NY, Appleton-Century-Crofts, 1979, pp 2-67 16. Mast BA, Harris TH, Frantz FW, et al: Ultrastructural comparison of fetal wounds and skin: Mammalian healing by regeneration. (in preparation) 17. Depalma RL, Krummel TM, Durham LA, et al: Characterization and quantitation of wound matrix in the fetal rabbit. Matrix 9:224-231,1989 18. Underhill CB, Toole BP: Transformation-dependent loss of the hyaluronate-containing coats of cultured cells. J Cell Physiol 110:123-128,1982 19. Graham MF, Diegelmann RF, Cohen IK: An in vitro model of fibroplasia: Simultaneous quantification of fibroblast proliferation, migration, and collagen synthesis. Proc Sot Exp Bio Med 176:302-308,1984
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Discussion Michael Harrison (San Francisco, CA): Dr Frantz and the Richmond group have continued to search for the magic ingredient that allows the fetus to heal without scar formation. They have concentrated, logically, on collagen and its synthesis. The first reports, however, were that the fetal wound was deficient in collagen. And now we hear that in fact the synthesis of collagen has increased. So the first question is, what happened in the intervening years? Second, there’s no question that collagen is a key molecule, but our group feels it is the organization of the collagen and not its absence that distinguishes the fetal wound. You have very nicely demonstrated that synthesis of collagen has increased, and yet the total amount has not increased. It would seem obvious then that reorganization and remodeling by metalloproteins like collagenase, must be the key. So the second question is, have you looked at collagenase in this mode, or, better yet, inhibitors of metalloproteinase (TIMPS) which fine-tune collagen deposition. And, finally, I’m sure you are very aware of the problems with the fetal rabbit model. After developing it, we abandoned it because it’s a small animal model with a short gestation, and the wounds gape like they do in almost no other animal. So the final question is, are you looking at this in a larger animal model? Frazier Frantz (response): As I have pointed out, the role of collagen metabolism in fetal tissue repair appears obvious when you consider the rapid restoration of tissue integrity and breaking strength evidence in fetal wounds. In this study, we chose to use actual healing linear wounds to quantitate and further characterize collagen biosynthesis during fetal repair. As you have mentioned, in earlier reports from our lab, Dr Tom Krummel reported the absence of collagen (by HPLC) in polyvinyl alcohol (PVA) sponge implants placed in fetal rabbits. This is in sharp contrast to the marked fibroplasia seen in PVA implants placed in adult rabbits and serves as one of several examples of the unique responses of the fetus to wounding. However, it is important to note that the PVA implant model, like other wound chamber models, is dependent on a foreign-body response
which, in the fetus, appears to be minimal. This model has proved an invaluable tool for measuring the effects of adult growth factors and other manipulations on fetal wound healing because of its low background with regard to collagen deposition. We are very interested in the potential roles of collagenase and early collagen turnover in fetal wounds. The fact that you have increased collagen synthesis but no excessive collagen deposition or scar formation in fetal wounds invokes the role of collagenase or other metalloproteases in modulating early breakdown, turnover, cross-linking, and orderly deposition of fetal collagen. We have begun our investigation of this phenomenon at the molecular level looking for expression of fetal collagenase message. We have used polymerase chain reaction (PCR) technology to analyze RNA from 2%day fetal skin and wounds. cDNAs were synthesized from equal amounts of skin and wound RNAs and hybridized with oligonucleotide primers specific for collagenase message. While we found no expression of collagenase message in 2%day fetal skin, there were easily detectable levels of this message in 2%day wound tissue. Right now, we are investigating the complete time course of collagenase expression after fetal wounding. Additional studies we plan to undertake to characterize the role of collagenase in fetal repair are, first, analyzing collagenolytic activity on the protein level, and, second, applying collagenase inhibitors. Theoretically, if collagenase is so important in these wounds and you inhibit it, will we see further increases in collagen deposition in fetal wounds? Since coming into the lab last July, we really felt that there was a lot of work to be done in analyzing the mechanisms of fetal repair at the molecular level. Accordingly, we have continued to use the fetal rabbit model because it is easy to work with, has multiple gestations, is relatively inexpensive, and has provided for easily reproducible data. At this point, we are interested to see if our findings will be reproduced in a larger animal model and have, consequently, begun wounding experiments in fetal pigs.
