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Regeneration of Hair Follicles and Sebaceous Glands from the Epithelium of Scars in the Rabbit* CHARLESBREEDIS (Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pa.) It has been generally accepted that, when all the boiling, during which time the skin was painted with iodine and hair follicles and sebaceous glands in a region of then washed with ether. A disc of skin about 25 mm. in diame ter was cut with a scissors from within the circle of ink and con adult skin are destroyed, or the entire region is centric with it. The disc included the total thickness of skin as extirpated, the new epithelium growing in from the well as the underlying panniculus carnosus. side will not regenerate these structures (14). The wire ring was now pushed through the wound into the On the basis of this concept, attempts were subcutaneous tissue and was so placed that its upward pro jecting prongs coincided with the marks on the India ink made to obtain a purely squamous sheet of epi circle. The skin was punctured at each mark with a No. 11 thelium in the rabbit for the purpose of inoculating Bard-Parker blade, thus allowing the prongs to project through it with the Shope papilloma virus. A region of skin the skin. The appearance of the anchor ring in relation to a was surgically removed from the back, and the healed wound is shown in Figure 3. The wound was protected by a glass-covered chamber wound was allowed to heal under conditions that fastened to the ring, as shown in Figure 2. The chamber con largely prevented contraction and drying. Con sisted of a heavy stainless steel ring with three projecting lugs trary to expectation, functioning hair follicles and (Fig. 1 [B]) and an ordinary chemical watch glass (Fig. 1 [A]). sebaceous glands appeared in the scars, sometimes The under surface of the ring had a shallow groove into which in great profusion. the seven prongs of the underlying wire ring were fitted. The heavy ring, placed on top of the prongs, was firmly In the experiments here reported, wounds in attached to each with 28-gauge stainless steel wire. The glass various stages of healing were studied in an at cover was then fastened in place, as shown in Figure 2, with tempt to determine the origin of the new hair 25-gauge stainless steel wire. follicles and glands. The chamber was left in place, except for temporary re The "hair follicle" is here considered a purely moval of the glass cover when observations were made or dressings applied, until the end of the experiment. Then the epithelial derivative, as defined by Cowdry (3) and animals were sacrificed with Nembutal. For microscopic by Ham (6). examination sections were cut perpendicular to the skin sur face after fixation in Bouin's solution. Whole mounts of the MATERIALS AND METHODS epithelium were prepared by the acetic acid method described Rabbits of mixed breeds weighing 2,800-3,500 gm. were by Cowdry (4). Only the epithelium of the scar separated used. They were fed commercial rabbit pellets, supplemented readily by this method. Mature hair follicles in the surround in some experiments with greens. All operations described ing skin were too deep to be pulled out. The best results below were performed under Nembutal anesthesia and as were obtained by soaking in ice-cold 1 per cent acetic acid for aseptically as possible. 2-3 days, fixing in Bouin's solution for 1 day at room tempera To obtain a uniform and large sheet of scar epithelium it ture, and then lifting off the scar epithelium. was thought desirable to hinder as much as possible the con Preparations of the dermal papillae were made after re traction of the wound during healing. For this purpose a wire moving the epithelium. Using a razor blade, a thin slice of ring was devised that could be anchored in the skin surrounding the dermal surface was shaved off and stained with hema- the wound. The ring consisted of a 22-gauge surgical stainless toxy lin. steel wire bent into the form shown in Figure 1 (C). The ring was fastened in the skin as follows: The back of the EXPERIMENTAL RESULTS rabbit was shaved with an electric clipper and was depilated Dressing of the wound and rate of healing.—In with a mixture of equal parts of barium sulfide and corn- starch made to a thin paste with water. The ring was placed on the first five rabbits the wound was kept moist the middle region of the back, 2 or 8 cm. from the midline. Its with vaseline gauze dressings. With this treatment outline was traced with India ink, thus forming a circle on the the wound did not develop a base that moved with skin 33 mm. in diameter. The positions of the seven prongs the skin, but remained open to the subcutaneous were marked on the circle. The ring was then sterilized by tissue until granulation from the side closed the *This investigation was supported by a research grant defect. The closure required 18-30 days and com (C-1116) from the National Cancer Institute of the National plete epithelialization several additional days. Institutes of Health, Public Health Service. Three of the wounds became infected. Received for publication April 21, 1954. Leaving the wound alone resulted in more rapid 575 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1954 American Association for Cancer Research. 576 Cancer Research healing and only an occasional infection. This pro lium, there were other possible explanations. The cedure was adopted for the remaining 33 animals. trauma of making the wound and of affixing the The chamber allowed slow evaporation to occur, chamber may have loosened viable fragments of h. and the wound healed by scab formation and hair follicles, which could either be scattered over granulation. the wound or be carried into it by the migrating The sequence of events was as follows : As dry surface epithelium. Some parts of a follicle can ing proceeded, the upper layer of the loose sub regenerate the whole (6). cutaneous tissue became attached to the edges of To determine the distribution of the new fol the wound and moved with the skin. In 7-10 days licles and to study their structure in three dimen this membrane became thick and opaque. In 10-14 sions, whole mounts of the wound epithelium were days a brown scab covered the entire wound. The made as described above. granulation tissue growing in from the side reached Mounts prepared at various stages of healing the center in 15-22 days. Epithelialization was revealed the following sequence of events : Before usually complete several days thereafter, and the the epithelium had completely covered the wound scab separated. it had numerous bands or bars of thickening that Beginning at about the time of separation of the consisted of five or more layers of cuboidal cells. scab or several days before this occurred, vaseline Most of them were radially arranged, but many gauze dressings were applied every 3-6 days. formed a reticular pattern that connected with the When the new epithelium was not protected in radial bars. This arrangement is illustrated in Fig this way, and especially when the glass cover was ure 7 (27 days). The bars correspond to the thick left off the chamber, there was excessive drying enings of epithelium shown in cross-section in Fig and contraction of the wound with little or no ure 4. No organized structure could be seen in regeneration of hair follicles and sebaceous glands. them. Some of the bars could be traced back to Organization of the scar epithelium into hair fol hair follicles at the edge of the wound. licles and sebaceous glands.—Microscopic observa As epithelialization proceeded, most of the con tions were made on sections through the wounds necting bars disappeared while the radial ones per from 23 rabbits and on whole epidermal mounts sisted. Then numerous budlike projections ap from fifteen additional rabbits. peared on the bars, first at the periphery of the None of the sections showed evidence that hair wound and later near the center. This budding is follicles or sebaceous glands had been left within illustrated in Figures 8 and 9 (31 days). Each bud the wound area at the time of operation. All epi was an immature hair follicle that in cross section thelium either was confined to the surface of the would appear about as shown in Figure 5. granulation tissue or appeared to be projecting Sometimes the radial bars could be seen through from this surface epithelium into the granulation the dissecting microscope in the living animal. tissue. Radially arranged blood vessels were also some Sections of wounds not yet completely epithe- times seen under the wound epithelium. lialized showed scattered diffuse thickenings of the As the hair follicles matured and began to pro epithelium. These are illustrated in Figure 4, duce hair, the radial bars gradually disappeared, which represents a wound of 23 days. The thick leaving radially arranged rows of follicles. These ened epithelium consisted of five or more layers of rows are seen at the periphery of the scar in Figure cuboidal cells and showed no evidence of organiza 8 (31 days) and near its center in Figure 10 (33 tion. At about 30 days, when epithelialization was days). Still later, owing perhaps to local contrac usually complete, the thickened regions developed tions of the healing wounds, the linear arrange downward projections into the granulation tissue.

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