SUMMARY. Collagen structures of the keloids were studied by polarized light microscopy. The purpose of the investigation was to determine the histophysical characteristics of the collagenous fibrils before and after clinical treatment of keloids with Kenacort. The method of imbibition analysis was used. The total anisotropy of keloid collagen after the cure was measured and compared with the anisotropy of the collagen before treatment. An assessment of the rate of collagen rehydration was made by using total birefringence rates. It was found that the rate of collagen rehydration was faster in the group of treated keloids than in untreated lesions. Data for the histophysical state of the collagen were established. It is suggested that the variation of collagen rehydration could be explained by the alterations in the mucopolysaccharides of the ground substance. Two hypotheses for the explanation are put forward.

Formation of keloids over a period of many years is among the most common healing problems in patients who survive thermal injury. This type of bum complication, besides its significance as a cosmetic deformation, may be a cause of different functional disorders in the organism. This fact has drawn the interest of researchers to the unexplained moments of the mechanism of keloid formation and to the more or less successful efforts for their cure. The special literature offers detailed information regarding the histiotypic organization of keloids and hypertrophic scars and their specificity, after examination by scanning and transmission electron microscopy (4, 1, 2, 3). However, data on the morphological changes of the structural elements of the keloids, as treated in various manners, are scant. There are no reports on the physicochemical characteristics of these keloids.
The purpose of the present investigation is to determine this characteristic of keloid collagen before and after clinical treatment with Kenacort. This involves a determination of the histophysical changes in the collagen of the keloids, after the cure, as compared to the state of keloid collagenous fibre before attempts at treatment. Polarizing microscopy and the method of imbibition analysis were used to achieve our aim.

Steroid treatment on keloids in patient~volunteers was performed at the Pirogov Emergency Institute, Section of Bums and Plastic Surgery, Sofia, Bulgaria. Subjects from either sex in the age range of 12 to 50 yr were subjected to treatment with Kenacort. The keloids were treated with an average dose of 20 mg/ml triamcinolone acetate, applied sublesionally. All details of clinical treatment will be published in another report.
Materials from untreated keloids and from keloids treated with Kenacort were fixed in 10% neutral paraformaldehyde and prepared for observation according to the recommendations of Scheuner and Hutschenreiter for polarizing microscopy (6). Incubation of the slides was performed in dilutions of 1:1, 3:1 and 1:3 at time intervals of 30, 60 and 100 min and 5, 50, 100, 150 and 150 It. Total anisotropy (resulting retardation) was measured on the materials from the two groups, using the rotatable Ehringhaustype compensator with k 1/16 and k 1/32. The investigation was completed with a NU 2 Carl Zeisslena microscope. Five untreated (control) keloids and seven Kenacort-treated keloids were studied.
In polarizing microscopy intrinsic bireftingence is due to the effect of different chemical groups in an ordered configuration on the velocity of light. Form bireftingence occurs in the rod-like or plate-like bodies immersed in a medium having a different refractive index. Whenever the two forms of birefringence are present together in an object, the resulting retardation is the algebraic sum of both:

R = I + F (8).

It should be noted that total anisotropy of the collagenous fibre is also a function of the cross diameter of the fibre which in contemporary studies is measured by interference microscopy. For this reason the results of our investigations for the measuring of birefringence have only a relative value. All tissues were obtained from surgical excisions. Every keloid was cut into 5 small pieces for examination. Sixteen microscope slides were prepared for the investigation with each hydrophilic medium. The anisotropy rates given in Table I are an average of the anisotropy rates of 10 collagenous fibres per slide. Total anisotropy was calculated in nanometers by Wiener's formula (6) and presented with the meaning of X for every group and every interval of time. Data were elaborated statistically with the finding of t and R

The data for the total anisotropy of keloid collagen before and after clinical treatment, depending on the time intervals and the type of incubation medium, are shown in Table I.

Fig. I Curves of the rate of collagen rehydration in the incubation medium glycerol in untreated and treated keloids

.On the basis of these, the curves for the velocity of collagen rehydration in different immersion media are built. An assessment of the velocity is made by means of the rates of total birefringence in nanometers. It is clear that the velocity of collagen rehydration is faster in the group of treated keloids than in the group of untreated lesions in most of the investigative time intervals investigated. The rate velocity is highest in the group of treated keloids after 50 h (Figs. 2, 3, 4 and 5) or at exactly the fiftieth hour (Fig. 1). The curves show a diminution in the anisotropy rates after this peak without any further increase. The rates of bireftingence during incubation with water show a new increase after 150 h (Fig. 2). In the group of untreated keloids the curves demonstrate the variability of the rate of collagen rehydration. There are some considerable rises and reductions in the rates of anisotropy which cause changes of inconstant type (Figs. 1, 2, 3, 4 and 5). The distinctions between the rates of the two groups are statistically reliable (Table 1).

Fig. 2 Curves of the rate of collagen rehydration in the incubation medium water in untreated and treated keloids Fig. 3 Curves of the rate of collagen rehydration in the incubation media glycerol and water diluted 1: 1 in untreated and treated keloids.

Fig. 5 Curves of the rate of collagen rehydration in the incubation media glycerol and water diluted 3:1 in untreated and treated keloids. Fig. 4 Curves of the rate of collagen rehydration in the incubation media glycerol and water diluted 1:3 in untreated and treated keloids.

Data regarding the histophysical state of keloid collagen before and after clinical treatment are considered. The possibilities of polarizing microscopy were used to gather indirect information on the molecular organization, which defines the interrelations between the structural elements of collagen and the other components of the connective tissue. Total anisotropy of the collagenous fibres was used as one of the indices for the estimation of keloidal collagen. The imbibition curves gave information only regarding the velocity of collagen rehydration in different immersion media, as estimated by the variable rates of bireffingence. On the basis of these results we tried to discover whether the keloidal collagenous fibrils are rehydratable and whether there are any differences in their rehydration rates before and after clinical treatment.
The strong equilibristic correlations between the biological structures and the hydrophilic media are apparent (7). Every change in homeostasis is connected to structural changes in the biological object. Keloids, a pathological process of the connective tissue, transform the original structure of collagen. A corresponding change in the protein/medium interaction is expected. All this assumes an alteration in the capability of the collagenous fibrils for hydration - their main indicator in the normal and pathological state. Data for total anisotropy showed faster rehydration of the collagen in the group of treated keloids. The results are statistically reliable.
The extracellular matrix is a very complicated network of collagenous and elastic fibrils, inserted in a viscous ground substance, formed by proteoglycans and glycoproteins (5). The adaptors for watermucopolysaccharides are attached to the original collagen structure. The velocity of collagen rehydration may depend to a certain degree on the mucopolysaccharides of the ground substance. The variation in collagen rehydration might be explained by the qualitative and quantitative alterations in the mucopolysaccharides of the treated keloids. The interpretation of this problem is the purpose of our other investigation.
In conclusion, we offer two hypotheses for the changes in the clinical treated keloids: the marked variation in the rehydration of keloid collagen after clinical cure with Kenacort clearly shows the presence of a new type of collagen formed by the fibroblasts which in its physicochemical characteristics might be similar to the collagen of a normal scar; the other explanation involves the possibility of alterations of abnormal collagen, external to the cells, in which the histophysical parameters might achieve the parameters of normal wound collagen.

RESUME. Les auteurs ont étudié les structures collagènes moyennant la microscopie polarisante. Le but de l'investigation était de déterminer les charactéristiques histophysiques des fibrilles collagènes avant et après le traitement clinique des chéloïdes avec Kenacort. En utilisant la méthode de l'analyse de l'imbibition, ils ont mesuré l'anisotropie totale du collagène chéloïde après le traitement et ils l'ont confrontée avec l'anisotropie du collagène avant le traitement. En outre ils ont calculé la vélocité de la réhydratation collagène moyennant les vélocités de la biréfringence. Ils ont trouvé que la vélocité de la réhydratation collagène était majeure dans le groupe des chéloïdes traités par rapport aux lésions non traitées. Les données pour l'état histophysique du collagène ont été établies. Les auteurs proposent que la variation de la réhydratation collagène pourrait être causée par les altérations dans les mucopolysaccharides de la substance interstitielle, et ils avancent deux hypothèses pour justifier cette possibilité.

1. Kischer C.W.: Fibroblasts during hypertrophic scar formation and resolution. 31 st Ann. Proc. Electron. Microsc. Soc. Amer., New Orleans, La. C.J. Arceneaux (ed.), 1973. Invest. Dermatol., 59: 323, 1972.
2. Liotta L.A., Rao C.N., Barsky S.A.: Tumor invasion and the extracellular matrix. Lab. Investig., 49: 636, 1983.
3. Kischer C.W.: Fibroblasts of the hypertrophic scar, mature scar and normal skin. A study by scanning and transmission electron microscopy. Texas Rep. Biol. Med., 32, 3 & 4: 699, 1974.
4. Scheuner G., Hutschenreiter J.: Polarisations Mikroskopie in der Histophysik. Thieme, Leipzig, 1972.
5. Wojtowiez P.J.: Lyotropic liquid crystals and biological membranes: the crucial role of water. R.C.A. Review, 35, Princeton.
6. Kischer C.W.: Predictability of resolution of hypertrophic scars by scanning electron microscopy. J. Trauma, 15: 205, 1975.
7. Wolman M., Kasten F.H.: Polarized light microscopy in the study of the molecular structure of collagen and reticuline. Histochemistry, 85: 41, 1986.
8. Linares H.A., Kischer C.W., Dobrkovsky M. et al.: The histiotypic organization of the hypertrophic scar in human. J.