Scanning Microscopy Volume 4 Number 4 Article 7 9-19-1990 Characteristics of Granulation Tissue which Promote Hypertrophic Scarring C. Ward Kischer The University of Arizona Jana Pindur The University of Arizona Peggy Krasovitch The University of Arizona Eric Kischer The University of Arizona Follow this and additional works at: https://digitalcommons.usu.edu/microscopy Part of the Biology Commons Recommended Citation Kischer, C. Ward; Pindur, Jana; Krasovitch, Peggy; and Kischer, Eric (1990) "Characteristics of Granulation Tissue which Promote Hypertrophic Scarring," Scanning Microscopy: Vol. 4 : No. 4 , Article 7. Available at: https://digitalcommons.usu.edu/microscopy/vol4/iss4/7 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. Scanning Microscopy, Vol. 4, No. 4, 1990 (Pages 877-888) 0891-7035/90$3.00+.00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA CHARACTERISTICS OF GRANULATION TISSUE WHICH PROMOTE HYPERTROPHIC SCARRING C. Ward Kischer*, Jana Pindur, Peggy Krasovitch and Eric Kischer Department of Anatomy, The University of Arizona, College of Medicine Tucson, Arizona 85724 (Received for publication June 22, 1990, and in revised form September 19, 1990) Abstract Introduction Hyper trophic scars and keloids are Hypertrophic scars are peculiar to humans characterized by nodules of collagen that (Kischer et al., 1982a) and occur as sequelae originate in granulation tissue arising from to deep injury of the body surface. A related full thickness or deep 2° injuries to the lesion is the keloid, which, unlike the skin. Fifty-six granulation tissues of varying hypertrophic scar, tends to outgrow the ages post-injury were examined morphologically boundaries of the original injury (Peacock for evidences of how the nodules and, thus, the et al., 1970). Both types of lesions may be scar form. New microvessels grow in ascension prompted by the surgeon's knife, a thermal burn towards the free surface in a milieu of or equivalent-type injury. They may undergo inf lammatory cells and fibroblasts. Collagen self-limiting resolution (not so prevalent in deposition increases with time from the base of the case of the keloid). The time for the the wound to the free surface and begins to self-limiting process to occur may range from a concentrate between lateral branching of the few months to ma ny years and cannot be new microvessels. Computer derived serial predicted. These fibrotic lesions are hard, reconstructions of hypertrophic scar nodules e levated, erythemic and contracting. indicate they are of varying shape and size Every hypertrophic scar and keloid contains probably due to fusion of adjacent structural units of collagen called nodules, microvascular collagen masses between lateral which are never present in fully mature scars branches. This is accomplished by the gradual (Kischer and Brody, 1981). Therefore, these but persistent degeneration of microvascular anatomical units actually define the scar and endothel.i.a and pericyte s. Fifty-six pieces of keloid. How do these nodules develop? From granulation tissue taken from 5 cases of where and from what do they arise? varying age post-injury were implanted into An earlier study strongly suggested the nude mice. Several proceeded to develop scar origin of nodules to be in granulation tissue and some of those developed nodules. The (Linares et al., 1973), but did not specify how latter developed only when the zero-time they might be formed. The nodules have long implant contained lateral microvascular axes parallel to one another and, branches. Hypertrophic scars and keloids are a predominantly, the free surface of the skin. product of granulation tissue elements, the They are rarely aligned perpendicular to the most important of which are primed active free surface. They also are oriented parallel fibroblasts and excessive microvascular to the lines of flexion across joints. Nodules regeneration, including lateral branching, are composed of unidirectionally aligned which subsequently degenerates, in part, collagen (parallel to the long axis of the promoting nodule formation and remodeling. nodule), which shows virtually no undulation. Interstitial space is not observed by electron microscopy within the nodule (Kischer, 1974). The fibroblasts are similarly oriented and the nodules are avascular. However, the nodules KEY WORDS: granulation, hypertrophic scar, are circumscribed with a network of keloid, light microscopy, electron microscopy, microvessels (Kischer and Brody, 1981). tissue culture, implantation, nude mice, Is it a .foregone conclusion that a deep fibronectin, platelet derived growth factor. inJury will always result in a hypertrophic scar (in some cases a keloid)? If nodules *Address for Correspondence: form, the answer is yes. C. Ward Kischer, Ph.D., Department of Anatomy We need to know how the nodule is formed, The University of Arizona College of Medicine and why. This knowledge might provide the Tucson, Arizona 85724 means by which development of the nodule could Phone No. (602) 626-6090 be prevented, which might then obviate the 877 C.W. Kischer, et al . development of the hypertrophic scar. and coated with 300A0 of gold in a Polaron Therefore, the present study was designed Sputter Coater, Model #5100, using argon gas. to investigate the following: 1) the organiza­ Examination of the tissues was conducted in an tion of granulation tissues over time after ETEC Autoscan using 20 kV. injury with particular attention to micro­ vascular distribution, collagen deposition and Implants cellular orientation; 2) to determine if there might be some developmental relationship Pieces of full thickness granulation between the above (#1) information and the tissues were implanted into subcutaneous nodules; 3) to determine if pieces of pockets over the scapular areas of nude mice. granulation tiss ue implanted into the nude Five cases of granulation tissues, three, mouse will proceed to develop nodules and/or 1 month or younger, and two, 4 months scar. post-injury, provided 56 i mplants into 22 mice. The implants were carried from 6 to 160 days. Materials and Methods All procedures from acquisition of the tissue to implantation were carried out under sterile conditions. Fifty-six different human granulation Nude mice (nu/nu), which are also athymic tissues, each from full thickness wounds, vary­ (Krueger and Briggaman, 1982) and do not reject ing in age post-injury from less than 1 month foreign tissue implants, were obtained from to 4 years, obtained by biopsy or surgical Harlan Sprague Dawley, Inc., Indianapolis, IN, scrapings prior to grafting, have been analyzed and from Charles River, Inc., Charles River, by light and electron microscopy. In terms of MA. All mice used were males and were kept in post-injury age, 13 were 1 month or less, 8 were presterilized microisolator units with filter 2 months, 4 were 3 months, and 6 were 4 months bonnets on top and lined up in front of laminar post-injury. Twenty-two were 5 months or older flow units. All handling of these mice was and 3 were of unknown age. done in a laminar flow hood under sterile Pieces of these tissues were processed for conditions. The mice were fed with sterilized all subsequent microscopic analyses by initial Purina Mouse Chow and given sterilized water to fixing in Karnovsky's fixative (Karnovsky, drink. They were housed one mouse to a unit. 1965). Granulation tissues from full thickness wounds were obtained under sterile conditions, Light Microscopy (LM) kept in sterile containers over ice, and transported to the animal laboratory for For light microscopic analysis, the tissues surgical implantation into the mice. The mice were dehydrated in graded ethanols then through were anesthetized with sodium pentobarbital xylene and embedded in paraffin. Subsequent using approximately 60-70 mg/kg of body tissue sections of 10 pm thickness were stained weight. Before the tissue pieces were by Hematoxylin and Eosin or by Masson' s tri­ implanted, smaller representative pieces were chrome method. General morphology and the taken for microscopic study. Those pieces for distribution and location of collagen for each microscopic studies were taken in full sample of tissue was recorded. Transverse thickness, placed in Karnovsky's fixative, and sections were mostly studied, although a few later processed for light and electron tissues were examined that were cut parallel to microscopy. the free surface. The implantation procedure has been previously described (Kischer et al. 1989a). Electron Microscopy (EM) The suprascapular areas on the back were incised 1 cm in length and the incision Transmission EM (TEM): Smaller pieces of slightly widened with tips of fine scissors to the same tissues were processed from the initial form a subcutaneous pouch. The tissue piece, Karnovsky's fixative through dehydration in standardized at approximately 5 x 8 x 5 mm in graded ethanols, to absolute alcohol to size, were inserted into this pouch; two pieces propylene oxide, and embedded in Epox 812 (Ladd were implanted in each mouse, one on each side, Research Co.). Thin sections were cut by although some mice received 3 or 4 implants. diamond knives and stained with lead citrate In these cases the pouch was widened to and examined in a Philips 300 electron accommodate 2 pieces but which