SUMMARY. The chick embryo chorioallantoic membrane (CAM) was used as an in vivo wound healing model. A full excision of a 1 mm2 CAM area was filled by ...
Path. Res. Pract. 192, 1068-1076 (1996)
The Chick Embryo Chorioallantoic Membrane as an in vivo Wound Healing Model D. Ribatti\ A. Vacca 2 , G. Ranieri 2 , S. Sorino1 and L. Roncalj1 11nstitute of Human Anatomy, Histology and Embryology, 2Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy
SUMMARY The chick embryo chorioallantoic membrane (CAM) was used as an in vivo wound healing model. A full excision of a 1 mm 2 CAM area was filled by a granulation tissue after 96-120 h, which eventually formed a scar in 75% of the cases. In the remaining 25%, a solution of continuity was left which, however, was smaller in size than the One observed immediately after the excision. Under the microscope, the CAM area involved in the repair process showed: i. hyperplasia of the chorionic epithelium; ii. about three times as many microvessels and fibroblasts in the mesenchyme as in the normal adjacent control regions; iii. an inflammatory infiltrate mostly consisting of macrophages; and iv. a strong positivity for fibronectin in the extracellular matrix. The validity of this experimental model appears to be confirmed by the fact that we were able to reproduce all the critical events controlling the wound healing process, such as re-epithelization, angiogenesis, formation of an inflammatory infiltrate and deposition of one of the main constituents of the extracellular matrix, such as fibronectin.
Introduction Wound healing (WH) is characterized by the formation of a granulation tissue consisting of inflammatory cells, newly-formed blood vessels and fibroblasts embedded in a loose collagenous extracellular matrix. Re-epithelization, angiogenesis and matrix deposition are critical events controlling this process 8• Angiogenesis is confined to the wound site 1 and plays a pivotal role for successful WHo Revascularization is required to furnish the new tissue and metabolities, and to dispose of the waste products of metabolism. Angiogenesis occurs as a higher regulated process which is rapidly stimulated after injury and ceases when WH is complete l2 • 0344-0338/96/0192-1068$3.50/0
Extracellular matrix proteins are known to be important in many phases of WH. Among these proteins, fibronectin, a glycoprotein secreted by proliferating endothelial cells ,21, forms the provisional extracellular matrix of wounds 14,19 and promotes migration of endothelial cells over the wound 27• A frequently employed method to stimulate WH in vivo is the subcutaneous implantation of sponges 9 • In this work, we examined the modes of WH development in an experimentally induced lesion in the chick embryo chorioallantoic membrane (CAM). Specifically, we examined WH with respect to the amount of the associated vasoproliferative processes, the characteristics of the inflammatory infiltrate and of the extracellular matrix adjacent to the site of the lesion. Our © 1996 by Gustav Fischer Verlag, Stuttgart
The CAM as an in vivo Wound Healing Model· 1069 Table 1. Quantitation of microvessels in the CAM's intermediate mesenchyme
Under the wound Distant from the wound (Control)
Microvessel density (%)a
No. of intersection points b
11.2 3.3
54.5±5.2 C 16.2±1.6
a For specification see text, b Mean ± SD, C Statistically significant difference p < 0.001 vs number of intersection points distant from the wound. Table 2. Quantitation of fibroblasts in the CAM's intermediate mesenchyme
Under the wound Distant from the wound (Control)
Fibroblast density(%)a
No. of intersection points b
16.5 5.2
80.4±12.5 C 25.2±6.3
a For specification see text, b Mean+SD, C Statistically significant difference p < 0.001 vs number of intersection points distant from the wound.
findings suggest that CAM can be regarded as an in vivo WH model. Results
Macroscopic Examination The CAM region surrounding the continuity solution showed swift vasoproliferative processes as early as 24 h after excision (HH stage 36) (Fig.1a). In 75% of the cases, the solution of continuity was filled by a granulation tissue within 48 to 72 h (HH stages 37-3 8). The scar developed upon this tissue during the next 24-48 h (HH stages 39-40) (Fig.1b). In the remaining 25% of the specimens, a solution of continuity smaller than the initial one was still present. Microscopic Examination Where the macroscopic examination showed that the solution of continuity had been filled by granulation tissue, the histologic examination confirmed that CAM continuity had been fully restored. Conversely, when a macroscopic analysis revealed that the continuity solution had only been filled by the scar tissue, an optical empty space between the two juxtaposed CAM stumps (Fig.2a) could be observed under the microscope. The chorionic epithelium of the stumps, normally consisting of 1 or 2 cell layers, was here formed by 2 to 6 layers (Fig.2 a-c). Within 96120 h following the lesion, microvessels and fibroblasts density in the underlying mesenchyme was about three times as high as in the adjacent normal control regions (Fig. 2 b, c Tables 1, 2}. The mesenchyme also showed the presence of an inflammatory infiltrate, mostly distributed in a perivascular position (Fig. 2 d} and consisting prevailingly of macrophages that reacted positively to naphthol-AS-D-chloroace-
tate esterase activity. Under normal conditions these cells are located almost exclusively inside the vessels (Fig.3 a); instead, where the lesion had been performed experimentally, their location was mostly interstitial (Fig. 3 b). Lastly, the extracellular matrix reacted with fibronectin in the mesenchyme beneath the lesion. Fibronectin deposition started 24 h after the lesion had been produced and reached its maximum extension after 24-48 h (Fig.4). Discussion In this work, we have followed the time-course of the repair process of an experimentally induced wound in the chick embryo CAM. In 75% of the cases, a 1 mm 2 resection area forms a scar some 96 -120 h later. The scar itself consists of a granulation tissue formed by newly-formed vessels, fibroblasts and inflammatory infiltrate essentially composed of macrophages. The process of WH depends upon a variety of interactions between cells and the extracellular matrix components, and wound repair is in part mediated by multiple growth factors released by inflammatory cells and from their storage sites of the extracellular matrix. In the CAM mesenchyme beneath the lesion we demonstrated that the number of microvessels was three times higher than in the normal adjacent regions which were considered as the control. WH angiogenesis has been extensively studied 1,22 and is characterized by disruption of the integrity of endothelial cell monolayers in pre-existing microvessels, basement membrane lysis, endothelial migration and proliferation, lumen and capillary loop formation. Angiogenesis is an absolute requirement for the development of the granulation tissue during WH. For example, under the category of wound repair the healing of peptic ulcers and of myocardial infarction is dependent upon angiogenesis 11. Healing is accelerated by the use of exogenous angio-
1070 . D. Ribatti et al.
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a
b. Fig. 1 a - b a: 1O-day old CAM (HH stage 36), 24 h after a full thickness excision of a small CAM's area (asterisk). The margins of the lesion show an intense vasoproliferative response - b: 14-day old CAM (HH stage 40), 120 h after the excision. The solution of continuity has been filled by a scar tissue (arrows). Original magnifications: a, x30; b, x20.
The CAM as an in vivo Wound Healing Model· 1071
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Fig. 2 a-d. Light micrographs showing in (a) an optically empty space (arrow) between the two juxtaposed CAM stumps of an experimental case in which continuity solution had been partially filled by the scar tissue. Note in (b, c) the two stumps photographed at higher magnification showing the hyperplasia of the chorionic epithelium (C) with the increase of vessels and fibroblasts in the underlying mesenchyme (M), and in (d) inflammatory cells located in perivascular position. Original magnifications: a, x 100; b, d, x400; c, x250.
1072 . D. Ribatti et al.
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The CAM as an in vivo Wound Healing Model· 1073
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