Jeffrey H. Haynes

Hyaluronic Acid is a Major Component of the Matrix of Fetal Rabbit Skin and Wounds: Implications for Healing by Regeneration

The extracellular matrix (ECM) of fetal rabbit wounds contains an abundance of the glycosaminoglycan hyaluronic acid (HA) but is devoid of excessive collagen. Thus, fetal wounds heal without scarring, such that tissue repair grossly resembles regeneration. To obtain further insight into the process of fetal wound healing, the ECM of normal fetal rabbit skin was analyzed, thus providing a comparative endpoint for the ECM of healing fetal wounds. Similarities between the matrices would support the theory of healing by regeneration. The glycosaminoglycan (GAG) component of fetal rabbit skin from 24- and 29-day gestational age fetuses was extracted and then quantitated using an alcian blue binding assay. The extracted GAG was characterized by cellulose acetate electrophoresis and HA was identified by its selective digestion by Streptomyces hyaluronidase. The mean GAG content, measured as ng GAG per mg dry weight skin, was 260 +/- 200 for the 24-day group (n = 28) and 280 +/- 220 for the 29-day group (n = 26). The only GAG identified at both times of gestation was HA. This study has demonstrated that HA is the predominant GAG present in fetal rabbit skin and its quantity is stable during the period studied late in gestation. A major component of the ECM of both wounded and normal fetal skin is HA, indicating a close compositional similarity. These observations provide biochemical support for the hypothesis that the reparative process of injured tissue in the fetal rabbit proceeds in an attempt to reconstitute normality, i.e. regeneration.

Platelet-Derived Growth Factor Induces Fetal Wound Fibrosis

The fetal response to cutaneous injury differs markedly from that of the adult, proceeding with only minimal inflammation, minimal fibroblast proliferation, and only essential collagen deposition. Although the sequence of events in adult wound healing is well defined and thought to be controlled in part by potent polypeptide cytokines, relatively sparse information exists regarding growth factor involvement in fetal wound repair. Thus, the authors sought to examine the effect of platelet-derived growth factor (PDGF), a putative adult wound healing regulator, on the cellular and extracellular matrix events at a fetal wound site. SILASTIC wound implants containing 0, 1.0, 5.0, or 10.0 ng of human PDGF were placed subcutaneously on the backs of 24-day-gestation fetal rabbits (full term, 31 days) and then harvested after either 1, 3, or 5 days in utero. The specimens underwent standard histological processing and were evaluated in a blinded fashion. Compared with controls, PDGF-treated implants had a marked increase in acute inflammation, fibroblast recruitment, and collagen and hyaluronic acid deposition; these differences appeared to be largely time- and PDGF dose-dependent. Thus, the fetal system is responsive to an adult wound healing mediator, and these data suggest that fetal repair proceeds in the absence of PDGF.

Myofibroblast induction with transforming growth factor-β1 and -β3 in cutaneous fetal excisional wounds☆☆☆

Abstract Background/Purpose: In a noncontractile fetal rabbit model, the authors recently have shown the induction of excisional wound contraction with sustained-release cellulose implants formulated with transforming growth factor (TGF)-β. The purpose of this study was to test the hypothesis that the excisional wound contraction in this model is associated with the induction of myofibroblasts in the surrounding dermis, demonstrated by the presence of α-smooth muscle actin. Methods: Cellulose discs were formulated with either 1.0 μg of TGF-β 1 (n = 6); 1.0 μg of TGF-β 3 (n = 9); 10 μg of TGF-β 3 (n = 6); or their carrier protein, bovine serum albumin (BSA; n=9), for sustained-release over 5 days. Each disc was implanted into a subcutaneous pocket on the back of a fetal New Zealand White rabbit in utero on day 24 of gestation (term, 31 days). A full-thickness, 3-mm excisional wound (7.4 mm 2 ) was then made next to the implanted cellulose disc. All fetuses were harvested at 3 days. The amount of α-smooth muscle (SM) actin in the dermis around the implants and wounds was determined using immunohistochemical techniques. Results: Excisional wounds exposed to 1.0 μg of TGF-β 1 (5.6 ± 2.0 mm 2 ), 1.0 μg of TGF-β 3 (6.9 ± 1.0 mm 2 ), and 10 μg of TGF-β 3 (2.7 ± 1.0 mm 2 ) were significantly smaller when compared with the BSA control group (12.8 ± 1.1 mm 2 ; P 1 (1.8 ± 0.5) and 10 μg TGF-β 3 (2.8 ± 0.2) groups in comparison with the scant staining in the BSA control group (0.5 ± 0.2; P Conclusions: TGF-β 1 and -β 3 induce α-SM actin and contraction of cutaneous excisional wounds in a fetal noncontractile model. This model of inducible cutaneous excisional wound contraction may be useful in further determining the role of the myofibroblast in wound contraction and the physiology underlying this poorly understood aspect of wound healing. J Pediatr Surg 35:183-188. Copyright

TGF-β1 alters the healing of cutaneous fetal excisional wounds

Abstract Background/Purpose: In a number of species, fetal wound healing differs from the adult in the absence of inflammation, fibrosis, scar formation, and excisional wound contraction. The lack of inflammation also may explain the relative absence of any cytokine levels at the wound site, such as transforming growth factor (TGF)-β, and therefore the unique characteristics of fetal wound healing. The authors hypothesized that exogenous TGF-β1 would induce contraction, inflammation, fibrosis, and scar formation in cutaneous excisional wounds in the fetal rabbit. Methods: Cellulose discs (3 mm in diameter) were formulated with either 1.0 μg TGF-β1 (n = 6) or bovine serum albumin (BSA; n=7), as a control, for sustained-release over 3 days. Each disc was implanted into the subcutaneous tissue on the backs of fetal New Zealand White Rabbits in utero on day 24 of gestation (term, 31 days). A full-thickness, 3-mm excisional wound (7.4 mm 2 ) was then made next to the implanted cellulose disc. All wounds were harvested 3 days later. Results: At harvest, the excisional wounds in the TGF-β1 group had contracted (5.6 ± 2.0 mm 2 ), whereas those in the control group had expanded (13.5 ± 1.2 mm 2 , P P Conclusions: These results demonstrate that the cytokine TGF-β1 can induce fetal excisional wounds to contract, stimulate fibrosis, and increase procllagen type 1α1 gene expression. These findings further suggest that the absence of TGF-β1 at the wound site may be responsible in part for the lack of a postnatal healing response.

Operative techniques in the fetal rabbit.

The development of fetal surgical techniques has made the antenatal correction of congenital defects possible. These techniques have evolved from trials with animal models, permitting increasingly sophisticated operations with low morbidity and mortality. Experimental models range from large animals offering longer gestations but with single pregnancies and high cost, to smaller animals offering multiple pregnancies at reduced cost but with shorter gestations. This paper describes operative techniques in the fetal rabbit and its advantages as a fetal surgical model. Experience with the pregnant rabbit has shown it to be a suitable surgical model for several reasons. Pregnancies are multiple, increasing cost effectiveness and permitting operation on up to eight fetuses per litter without fetal loss. Techniques that promote fetal survival include local housing of does several days prior to operation and preoperative sedation. Spontaneous mask ventilation provides ease of anesthetic administration and titration. Overall surgery is well tolerated with a low incidence of intraoperative complications. Rabbit models have been used in the study of transamniotic fetal feeding, abdominal wall defects, and wound healing. These techniques have resulted in postoperative fetal viability approaching 90%, with negligible maternal mortality in over 4000 fetal operations, thereby making the rabbit a manageable cost-effective model of fetal surgery.

In vitro and in vivo analysis of the inability of fetal rabbit wounds to contract

Fetal rabbit wounds that are sutured show excellent repair without obvious scarring. In contrast, an unsutured wound in a rabbit fetus does not close, and it appears that the process of wound contraction does not occur. Experiments were carried out to illustrate the mechanisms responsible for the noncontraction of open fetal rabbit wounds. Results showed that the lack of wound contraction was not an artifact caused by rapid fetal growth. With regard to the ability of cultured fetal fibroblasts to show cytoplasmic muscle‐induced cell contraction, we found that, in cultured fetal fibroblasts, cell contraction was induced by adenosine triphosphate. Contractile abilities of fetal‐derived fibroblasts were equivalent to those of adult‐derived fibroblasts. The fetal fibroblasts also demonstrated the generation of superior contractile activity when examined in a fibroblast‐populated collagen lattice model. Finally, the ability of amniotic fluid to alter wound contraction was addressed by means of the fibroblast‐populated collagen lattice in vitro model. Increasing concentrations of amniotic fluid inhibited fetal fibroblast lattice contraction. Therefore, rabbit amniotic fluid contains an inhibitor that may be partially responsible for the noncontraction of fetal rabbit wounds in utero.

Hirschprung's disease and imperforate anus in Pallister-Hall syndrome: A new association

Hirschprungs disease and imperforate anus are described concurrently in a newborn with Pallister-Hall syndrome as well as the difficulties in making this diagnosis. Awareness of this new association should prompt the exclusion of Hirschprungs disease before repair of imperforate anus in infants with Pallister-Hall syndrome. The known genetic parallels between these conditions is discussed briefly in terms of etiology.

Management of persistent pediatric tracheostomal fistulas.

Decannulation of a tracheostomy generally results in spontaneous closure. Occasionally, epithelialization results in persistence of the fistula, which may be initially treated by local curettage or cautery. Failure of these methods constitutes an indication for surgical closure. Dissection of the entire tracheocutaneous tract permits fistula closure in juxtaposition to but outside the trachea and prevents any iatrogenic airway narrowing. Twelve patients have been so managed over the last 10 years, and there have been no immediate or long-term complications.

Ultrastructural analysis of fetal rabbit wounds

Fetal wound healing proceeds rapidly with minimal inflammation and fibroplasia and little or no scar formation. These observations have led to the hypothesis that fetal wound healing more closely resembles regeneration rather than adult wound repair. To test this hypothesis, this study used ultrastructural analysis of fetal and adult fibroblasts and collagen to gain greater insight into differences in the healing processes. Full‐thickness, primarily closed linear incisions were created dorsally on 24‐day gestational age fetal rabbits (n = 9). The fetuses were killed 5 days later, and the wounds were excised and evaluated with transmission electron microscopy. Similarly, uninjured fetal skin of the same gestational age was obtained and analyzed. Adult rabbit dermal wounds were analyzed after 8 days of healing. Resting adult dermal fibroblasts had features of quiescent, inactive cells, whereas adult wound fibroblasts were highly active and filled with secretory vesicles. In contrast, both fetal normal dermal and wound fibroblasts appeared highly active and contained numerous secretory vesicles. In the adult wound, collagen fibril diameter was only 45% of the diameter of normal dermal collagen. However, fetal wound collagen fibrils were basically the same as normal dermal collagen, having a diameter that was 82% of the size of dermal collagen. These observations suggest that fetal wound fibroblasts do not require activation from an inactivated state and that fetal wound collagen deposition undergoes more rapid organization and maturation. These findings have significance in extending our understanding of the rapidity and functional superiority of fetal wound healing compared with adult wound healing.

Fetal Skin Organ Culture

The fetal response to cutaneous injury has been investigated in a variety of models; most have studied the differences between fetal and adult healing mechanisms in vivo and in cell culture. Further disclosure of the cellular and biochemical events requires a model that can be manipulated to study single factors influencing fetal tissue repair without the complex interactions that occur in vivo, but in a system that more closely approximates normal skin than cell culture models. This paper presents a method for the organ culture of fetal skin and its advantages as a model to help elucidate fetal healing mechanisms. Skin sections (1 x 1 cm) excised from the backs of fetuses of New Zealand white rabbits on day 27 gestation (term = 31 days) were placed eccentrically in 65-mm culture dishes and fed daily with 2.5 mL of DMEM containing 10% fetal calf serum, antibiotics, and 10 mM ascorbic acid. A separate group, treated similarly, received 4-mm punch wounds to assess the in vitro response to wounding. The specimens were incubated at 37 degrees C in humidified room air on a rocker platform to provide alternate exposure of the skin to air and medium. Gross observation at 3 weeks showed cells extending into the central wound, indicating that viable cells were proliferating and/or migrating from the tissue. Skin was examined histologically and was viable over the 3-week period studied. Organ culture, by maintaining tissue in the natural extracellular matrix, allows cell-to-cell contact and communication to be maintained while allowing controlled environmental manipulation.(ABSTRACT TRUNCATED AT 250 WORDS)