hyaluronate metabolism undergoes and ontogenic transition during fetal development: implications for...

Hyaluronate Metabolism Undergoes an Ontogenic Transition During Fetal Development: Implications for Scar-Free Wound Healing

By James M. Estes, N. Scott Adzick, Michael R. Harrison, Michael T. Longaker, and Robert Stern San Francisco, California

�9 Wound healing in the fetus occurs by a different process from that in the adult. Instead of healing with scar formation, fetal cutaneous wounds heal by regeneration that results in complete restoration of normal skin architecture. The mecha- nisms responsible for this remarkable phenomenon involve factors in the fetal environment and properties intrinsic to fetal cells. Hyaluronic acid (HA) is a major component of the fetal extracellular matrix (ECM) and is believed to play an important role in this process. In this study, HA and HA- stimulating activity (HASA) in fetal and adult wound fluid were examined using sensitive, newly developed assays. In an ovine model, higher levels of HA and HASA were observed in fetal as compared with adult wound fluid. This difference was most prominent in wound fluid from fetal lambs at 75 and 100 days gestation (term = 145 days); these samples contained persistently elevated HA and HASA levels for up to 2 weeks after wounding (HA peak levels 145 ~g/mL and 110 iLg/mL, respectively). In contrast, wound fluid from 120-day- gestation fetuses had significantly lower levels (P < .001) that were transient and similar to that in the adult (HA peak levels 70 i~g/mL and 10 i~g/mL, respectively). These observa- tions confirm an ontogenic transition in wound HA metabo- lism from a fetal to an adult-like phenotype. Levels of HASA as a function of time after wounding correlated with levels of HA, suggesting a role for HASA in controlling HA deposition during tissue repair. Two patterns of HASA and HA synthesis were noted. Wound fluid from early and midgestation fetal lambs exhibited markedly elevated and sustained HASA and HA levels, and this pattern correlates temporally with the scarless wound healing response noted prior to 120 days gestation. In contrast, wound fluid from 120-day-gestation lambs and adult samples exhibited a small and transient increase in HASA and HA; this response correlated with the adult wound healing pattern that produced a scar. Thus, HA and HASA are both phenotypic markers for scar-free wound healing in the fetus, suggesting a mechanistic role for HA and other factors that regulate its deposition during the tissue repair process. These observations have important implica- tions for devising new therapeutic agents to prevent scar formation. Copyright �9 1993 by W.B. Saunders Company

INDEX WORDS: Hyaluronic acid; fetal wound healing.

critical in several experimental models of embryogen- esis as the rapid cell movement, proliferation, and tissue generation that take place during this develop- mental phase occur in an HA-rich environment. 10

Elevated levels of HA occur in wound fluid ob- tained from midgestation fetal lambs, relative to that found in adults. 9 We have previously described a HA stimulating activity (HASA) present in the fetal circulation and in fetal wound fluid at 100 days gestation that is absent in the adult. 11,12 This factor may account for the elevated HA levels found in fetal wound fluid.

A transition in the mode of wound healing occurs in fetal lambs during the mid-third trimester. Just prior to 120 days gestation, fetal tissue repair trans- forms to a more adult-like pattern of scar formation, characterized by the deposition of disorganized colla- gen bundles. 13 In the present experiments, decreasing levels of HA and HASA in wounds correlated with the transition from a fetal to an adult-like wound healing pattern.

MATERIALS AND METHODS

Surgical Procedure The technique of fetal lamb surgery has been described previous-

ly. ~z Briefly, 18 time-dated pregnant ewes (Torrel Farms, Ukiah, CA) underwent midline laparotomy at 75, 100, and 120 days gestation (6 animals at each gestational age). Fetuses were exposed by hysterotomy of the gravid horn of the bicornuate uterus. Three to 4 Hunt-Schilling stainless steel no. 40-gauge wire mesh cylinders of appropriate size were placed in subcutaneous pockets in the inguinal and posterolateral thoracic areas in each fetal and maternal sheep. 14,15 The incisions were closed with 4-0 silk in adults and a running 6-0 Prolene suture (Ethicon, Inc, Somerville, NJ) in fetuses to assure a water-tight seal. Amniotic fluid and urine were obtained at the time of wound cylinder placement; fetal urine was sampled by percutaneous bladder aspiration using a no. 27-gauge needle. The harvest of wound fluid was performed by needle

T HE VERTEBRATE fetus has a unique ability to heal cutaneous wounds without scar forma-

tion. 1-6 Experience with fetal surgery at the Fetal Treatment Center of the University of California, San Francisco, confirms these observations in humans. 7 Understanding the mechanisms by which this remark- able process occurs may lead to rational therapy for the untoward consequences of postnatal healing, such as excessive fibrosis, scarring, and contracture. Hyaluronic acid (HA) is an important component of the matrix in fetal wounds. 8,9 Deposition of HA is

From the Departments of Surgery and Pathology, University of California, San Francisco, CA.

Presented at the 1992 Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, San Francisco, California, October 9-11, 1992. Tie for second place in the Resident Competion.

Supported in part by NIH grants HD-25505 and CA-44768; an NIH National Research Service Award HD-07462 (J.M.E. ); and an Ameri- can College of Surgeons Fellowship (N.S.A.).

Address reprint requests to Robert Stern, MD, Box 0506, Department of Pathology, University of California, San Francisco, CA 94143.

Copyright �9 1993 by W.B. Saunders Company 0022-3468/93/2810-0002503. 00/0

Journal of Pediatric Surgery, Vo128, No 10 (October), 1993: pp 1227-1231 1227

1228 ESTES ET AL

aspiration at 3, 7, and 14 days after implantation (two animals per time point). Wound fluid was discarded if any contamination with blood or amniotic fluid was suspected. Wound fluid samples were placed in EDTA-coated blood collection tubes (Becton Dickinson, Rutherford, NJ), centrifuged for 30 minutes, and the supernatant collected. Amniotic fluid, urine, and serum were collected in siliconized tubes without additive (Becton Dickinson). Serum samples were centrifuged 30 minutes and the supernatant col- lected. All samples were frozen immediately and stored at -70~ until assayed.

Hyaluronic Acid Assay

HA levels were determined using a sensitive, enzyme-linked immunosorbant-like assay that utilizes a high-affinity HA binding protein (HABP) derived from bovine nasal cartilage. 16,17 The bound HA-HABP complex was linked to peroxidase, which pro- duced a colorimetric reaction. Absorbance at 492 nm was deter- mined and HA values calculated by first plotting A492 against log [HA] for the standards, which routinely produced a straight line. The HA concentrations of unknowns, which required dilution in order to fall within the range of the standard curve, were calculated by least squares linear interpolation (Excel 3.0; Microsoft Corp, Redmond, WA). Samples were run in triplicate and the results expressed as the mean and standard deviation of at least 5 wound fluid samples. Statistical significance was determined using a two-tailed Student's t test.

HA SA Assay HS-27 human foreskin fibroblasts (Cell Culture Facility, Univer-

sity of California San Francisco) were seeded at a density of 2 x 105 in 2-cm-diameter 6-well plates (CoStar, Cambridge, MA) in 3 mL of DME-H16 media (Cell Culture Facility, UCSF) containing 10% heat-inactivated bovine calf serum (CS), 1% penicillin/streptomy- cin, and 1% glutamine (standard media) and incubated at 37~ The cells were allowed to grow for 2 weeks until confluence was reached, with a media change every 3 days. Calf serum rather than fetal calf serum (FCS) was used to avoid introducing HASA activity present in fetal serumJ 1 On day one of the experiment, the media was removed and 1.8 mL of media replaced together with 0.20 mL of sample, yielding a total volume of 2.0 mL per well. Two negative controls containing 2 mL of standard media only were run per assay. The cells were incubated 24 hours and then the conditioned media was removed and frozen at -70~ until assayed. The cell layer was removed by rinsing and then incubating 60 s with 0.4 mL 0.25% trypsin and 0.02% EDTA in 0.8% phosphate- buffered saline (Cell Culture Facility, UCSF). The dispersed cells were removed and the well rinsed with 0.4 mL media. Cell counts were then performed using a Coulter particle counter (Coulter Industries, Hialeah, FL). The HA determinations were performed on the samples and controls. HASA values were calculated by the following formula:

HASA = [(nmsarnpte - - HAcontrol) - - HAn,ia]/cellcount

where HAsamr and HAcomrot are the measured levels of HA in the sample and control wells, respectively, and HAn,id is the amount of HA present in the added wound fluid. Cellcount represents the ratio of total cells in the sample to cells in the control wells. Samples were run in triplicate and expressed as the mean and standard deviation. Statistical significance was determined using a two-tailed Student's t test.

RESULTS

Hyaluronic Acid Levels

A determination of HA concentrations was per- formed on fetal serum, urine, and amniotic fluid at 75, 100, and 120 days gestation (data not shown). Peak HA levels occurred in amniotic fluid and fetal serum at 100 days gestation (3.6 p~g/mL and 1.2 p~g/mL, respectively). Fetal urine HA levels were essentially unchanged throughout the sampling pe- riod (0.8 p~g/mL). The data obtained for HA levels in amniotic fluid are consistent with previously reported measurements in sheepJ 8

Wound fluid was aspirated from the subcutaneous cylinders placed in fetal and adult sheep at either 3, 7, or 14 days following implantation. The HA levels from fetal and adult wound fluid are shown in Fig 1. Levels were highest in wound fluid from 75 days gestation, with a peak occurring 7 days after wound- ing. Compared to 75-day samples, 100-day-gestation wound fluid had a similar profile, with slightly lower HA levels present at each time point (P < .001 at 7 days postwounding and not significant [NS] at 3 and 14 days). However, major differences were seen in the 120-day and adult wound fluid samples. These HA levels were significantly less than those of the 75- and 100-day specimens (P < .001 at all points). In addi- tion, at 14 days postwounding, HA levels in adult and 120-day fluid decreased to near zero, while samples from earlier gestational ages remained at high levels

200

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Fig 1. Levels of HA in fetal and adult wound fluid (in I ,g/mLI. Fluid obtained from Hunt.Schilling wound chambers in fetal and adult sheep was assayed for HA at 3, 7, and 14 days postwounding. Samples were taken and averaged from 3 to 4 wound cylinders in each of 2 animals and are expressed as the mean and standard deviation.

TRANSITION IN HA METABOLISM IN DEVELOPMENT 1229

(ie, > 100 ~g/mL). Thus after 7 days postwounding early fetal wound fluid (ie, 100 days gestation and less) had sustained, high levels of HA. In contrast, 120-day and adult samples demonstrated an HA peak with barely detectable levels thereafter. Significantly, HA concentrations in wound fluid were 1 to 2 orders of magnitude greater than that detected in any of the urine, serum, or amniotic fluid samples examined.

In order to validate this assay, it was necessary to establish whether endogenous HABPs affected the HA assay by making wound fluid HA less available for the biotinylated HABP probe. Digestion of wound fluid samples with papain (5.0 mg/mL) at 70~ and pH 5.0 for 18 hours did not significantly alter the results (data not shown). Therefore, we concluded that endogenous HA binding proteins do not inter- fere with this HABP-dependent assay for HA.

HASA in Wound Fluid

Levels of HASA were determined by measuring de novo synthesis of HA induced by the wound fluid samples i n cultured human dermal fibroblasts (Fig 2). Elevated levels were observed in 75- and 100-day gestation wound fluid samples. In contrast, barely detectable levels ( < 12 ~g/mL of HA) were observed with 120-day and adult wound fluid samples, in a profile similar to that observed with HA. The 75- and

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Fig 2. Levels of HASA in fetal and adult wound fluid at 3, 7, and 14 days after wound cylinder implantation, HASA was measured in fetal and adult wound fluid and expressed as the concentration of de novo synthesized HA by cultured fibroblasts, corrected for cell count. Samples were taken and averaged from 3 to 4 wound cylinders in each of 2 animals and are expressed as the mean and standard deviation.

100-day samples had rapid increases in HASA after wounding and maintained such levels even after 14 days postwounding (P < .001 for 7 and 14 days compared with adult and 120 days gestation).

Cell counts of the cultured HS27 indicator fibro- blasts were performed to permit normalization of HA synthesis on a per cell basis. No significant mitogenic effect was observed from any of the wound fluid samples, compared to untreated controls (data not shown).

DISCUSSION

HA plays a crucial role in the organization and structure of the extracellular matrix (ECM). It facili- tates cellular proliferation and motility, and promotes the maintenance of the undifferentiated state during early stages of embryonic organogenesis) ~ DePalma et al demonstrated that HA was prominent in the ECM of fetal rabbit excisional skin wounds. 8 Our laboratory group subsequently confirmed these obser- vations in large animal studies and hypothesized that the prolonged presence of HA in fetal wounds pro- moted the scar-free healing and regeneration ob- served during fetal wound healing. 9 In that study, greater levels of HA in 100-day-gestation fetal lamb wound fluid were present compared to adult samples. Histological studies subsequently documented a phe- notypic transition point in ovine fetal tissue repair. At 120 days gestation fetal lamb wounds were shown to heal with disorganized scar formation, characteristic of adult tissue repair} 3 Based on the importance of HA in embryonic morphogenesis, we postulated that elevated levels of fetal wound HA would correlate with scar-free tissue repair, and that HA content would fall to adult-like levels with the onset of scarring. The present studies were undertaken to test this hypothesis.

The predicted differences were indeed noted. HA levels were persistently elevated from 7 to 14 days postwounding in the 75- and 100-day samples. By 120 days, or midway through the last trimester, fetal wound fluid became more "adult-like" and only a transient increase in HA content was noted. Further- more, HA levels were substantially lower in urine, amniotic fluid, and serum than in wound fluid, estab- lishing that elevated HA levels are endogenous to wounded fetal tissue.

We have previously measured HA in adult and 100-day-gestation wound fluid. 9 In the present series of experiments we did not detect the early HA peak in adult wound fluid that was previously reported, yet the relative relationship between adult and fetal levels was preserved. Conceivably, the early peak of

1230 ESTES ET AL

H A occurred prior to the first sampling at 3 days postwounding.

HASA has been detected in fetal serum, amniotic fluid, fetal urine, and in a number of other fetal tissues. This activity stimulates H A synthesis in a consistent and dose-dependent manner using an in vitro cell culture assay. 11,19"21 The activity in fetal serum is associated with a glycoprotein that has a peak serum level at 120 days gestation in fetal calves u and 60 days gestation in fetal lambs. 19 This activity is the putative mechanism by which HA levels are elevated in fetal wounds. The activity of the HASA in wound fluid described in the present study resembles that in FCS 11 and in CS, 22 and the factor from FCS has been partially purified. 11 Here, we investigated HASA in fetal and adult wound fluid and found a correlation with HA levels such that levels of HASA parallel those of HA, and HASA is virtually absent in adult wound fluid. The HASA in wound fluid may be identical with that which occurs in the fetal circula- tion, but further isolation and purification of this factor are required to establish this fact.

HA can modulate the tissue repair process through several mechanisms. HA may influence the wound environment by binding growth factors and thereby modulate their activity in a manner similar to heparan sulfate. 23 Furthermore, cells expressing HA receptors have the capacity to organize their immediate pericel- lular matrix via HA and HA receptor interactions. 24 HABPs, or "hyaladherins" may also play a role in the regulation of fetal and adult wound healing. 24

Mammalian HABPs are important to the biologic activity of HA in wound healing. Burd et al demon- strated that HA purified from human skin increased epidermal cell proliferation in explant cultures, com- pared with commercially available sources of HA. This activity was attributed to associated proteins that copurified with HA. 25 Interestingly, the mammalian- derived HA also inhibited fibroblast proliferation in vitro. These results suggest that HABP's confer on H A much of its biologic wound healing activity.

A transition of the fetal wound healing response has also been described in rats. Ihara et al demon- strated differences in tissue repair at 16 and 18 days gestation. 26 At 16 days, 2-mm punch wounds in the paniculus carnosus of fetal rats healed by cellular migration and completely filled the defect with nor- mal appearing skin by 7 days. In contrast, wounds made at 18 days became covered with a thin layer of fibrin and immature epidermis and did not close.

A fetal-to-adult transformation in fibroblast func- tion has been characterized by Schor et al. They observed that fetal fibroblasts have greater migratory activity on collagen gels and are less inhibited by high cell density than their adult counterparts. 27 Further- more, they documented this transition from the fetal to adult-like phenotype when fetal fibroblasts were serially passaged in culture. This transition may parallel the phenotypic change of fetal to adult-type wound healing we have observed in vivo. Schor et al subsequently identified an autocrine factor secreted by fetal fibroblasts that appears to be responsible for the enhanced migratory properties of fetal cells. 28 This agent, termed migration stimulating factor, also stimulates the synthesis of HA by fibroblasts. 29 They showed that fetal fibroblasts in culture continue to produce large amounts of H A at confluence, in contrast to adult fibroblasts which down-regulate HA synthesis as cell density increases. 3~ These observa- tions confirm our findings and implicate H A as one biochemical basis for the transition of fetal healing by regeneration to the adult pattern of scarring.

From the present experiments we conclude that high levels of HASA and H A correlate with the occurrence of scar-free fetal wound healing. These factors may influence the complex interactions of cells and ECM that distinguish fetal from adult tissue repair.

A C K N O W L E D G M E N T

The skilled technical assistance of Jackson Hall is gratefully acknowledged.

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fetal, newborn, and adult wound healing by histologic, enzyme- histochemical, and hydroxyproline determinations. J Pediatr Surg 20:315-319, 1985

4. Rowsell AR: The intra-uterine healing of fetal muscle wounds: Experimental study in the rat. Br J Plast Surg 37:635-642, 1986

5. Krummel TM, Nelson JM, Diegelmann RF, et al: Fetal response to injury in the rabbit. J Pediatr Surg 22:640-644, 1987

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7. Harrison MR, Adzick NS: The fetus as a patient: Surgical implications. Ann Surg 213:279-290, 1991

8. De Palma RL, Krummel TM, Durham LA IIl: Characteriza- tion and quantitation of wound matrix in the fetal rabbit. Matrix 9:224-231, 1989

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11. Decker M, Chiu ES, Dollbaum C, et al: Hyaluronic acid stimulating activity in the sera from the bovine fetus and from breast cancer patients. Cancer Res 49:3499-3505, 1989

TRANSITION IN HA METABOLISM IN DEVELOPMENT 1231

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Discuss ion

T, Krummel (Hershey, PA): W h a t Dr Estes and his mentors have taught us is that fetal w o u n d healing is charac ter ized not only by high levels o f hyaluronic acid but also by high levels of hyaluronic acid stimulat- ing activity. The two are at least temporal ly related.

Hyaluronic acid is also associated with morphogen - esis; that is to say, with developmenta l biologic phenomenon . It is similarly associated with regenera- tion. I f you amputa te a Newt limb the extracellular matrix that accumula tes is also hyaluronic acid. A n d finally, hyaluronic acid and H A S A have also been descr ibed in a n u m b e r o f mal ignant condit ions, which makes for an interest ing sort o f po tpour r i o f H A activity. This gives rise to several questions.

Can you character ize H A S A ? Can you block it or can you enhance it and thus satisfy Koch ' s postulates as relates to its cause and effect in fetal wounds?

Could you suggest a unified hypothesis o f what this means in terms of H A S A and the fact that it's found in these different situations o f fetal wound repair, embryogenesis , regenerat ion, and mal ignancy?

J. Estes (response): To address your first question, H A S A is a 150-kd glycoprotein present in high levels

in fetal serum. Peaks o f H A S A activity have been identified just pr ior to midgesta t ion in fetal lambs and calves. Efforts are present ly underway to purify this activity and raise ant ibodies for immunohis to logic and biologic activity analysis.

In your second question, you men t ioned the very interesting parallel be tween w o u n d healing and malig- nancy. A simplistic view of this p h e n o m e n o n is tha t wound healing is a control led proliferative response to a defined insult; whereas in mal ignancy the re- sponse is unres t ra ined and the insult less well de- fined. N u m e r o u s factors and genes have been impli- cated in the development o f unrestrained cellular proliferation that results in the malignant transformation.

H A S A may be one o f those factors. Inc reased levels o f H A S A have been identified in the se rum of w o m e n with advanced breas t cancer. Fu r the rmore , Bryan Toole ' s early work implicated hyaluronic acid as an impor tan t med ia to r o f cellular de t achmen t and prol iferat ion dur ing embryogenesis . So presumably H A S A promotes an env i ronment where cellular pro- l iferation and invasion can occur in bo th mal ignancy and wound healing.