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«Pergamon Press.Printed inGt.Britain. Archs or01 Bid. Vol. 13, pp. 271-288, 1968. THE EFFECT OF FORMOCRESOL ON HAMSTER CONNECTIVE TISSUE CELLS, A ...»

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Pergamon

Press.Printed inGt.Britain.

Archs or01 Bid. Vol. 13, pp. 271-288, 1968.

THE EFFECT OF FORMOCRESOL ON HAMSTER

CONNECTIVE TISSUE CELLS, A HISTOLOGIC AND

QUANTITATIVE RADIOAUTOGRAPHIC STUDY WITH

PROLINE-H3

L. H. STRAFFON S. S. HAN

and

Departments of Oral Biology and Pedodontics, School of Dentistry and

Department of Anatomy, Medical School, The University of Michigan, Ann Arbor, Michigan 48104, U.S.A.

Summary-The reaction to formocresol of connective tissue cells has been examined in sponge implants and femur wounds, by means of quantitative radioautography following proline-H3 injection. For twenty-four hamsters, a sponge implant on the dorsum of the neck and a surgical opening in the right femur were prepared. Formocresol was diluted to l/50 of a normal concentration for the sponge implant study and the normal concentration was tested on the femur. The time intervals from application of the drug to sacrifice were 5 hr, 1 day, 3 and 10 days, and 1 month. Proline-H3 was injected 4 or 24 hr prior to sacrifice.

Three animals were used for the scintillation counting of blood level of tritium from 2.5 min to 6 days. The tissues were fixed in Bouin’s solution, embedded and sectioned in a routine manner. The sections were coated with Kodak NTB3 emulsion and exposed for suitable lengths of time. Formocresol used in l/50 concentration caused degeneration of cells in the immediate vicinity of the sponge implant as judged by histology and radioautographic grain counts. However, in all animals treated with formocresol a definite reduction in the number of infiltrating intlammatory cells was observed. By the tenth day both the experimental and control sponges showed a comparable recovery of the connective tissue ingrowth. This was also true for the repair in the femur wound area. It was concluded that formocresol does not interfere with prolonged recovery of connective tissue and might suppress initial inflammatory response, significantly.

INTRODUCTION

THE SOLUTION of formocresol (FC) used today, was introduced by BUCKLEY (1904) as an empirical means of pulp treatment. Prior to this and since then, different combinations of formaldehyde and cresol had been used along with arsenic to “mummify” the pulp, i.e. to fix and produce a rubbery mass of connective tissue (BONNECKER and PRINZ, 1899; BONSACK,1930; COOLIDGE,1932; FOSTER, 1936; HESS, 1920; LUTZ, 1923; MUELLER,1920; ORBAN, 1933, 1934). SWEET (1936) formulated a treatment regimen for the use of FC on exposed primary t

–  –  –

GROSSMAN, 1944; PECK, 1898; RUBBO, REICH and DIXSON, 1958; SCHILDERand AMSTERDAM, 1959), muscle (STANDISH, 1964), and bacteria (BARTELS,1941; KITCHIN and HORTON,1931; NEELY,1963a, b; PEAR,1942).

Despite these attempts, the basic effects of FC on the metabolism of connective tissue cells are not clearly understood and no unified views exist on this point. The purpose of this article is to evaluate the appearance of connective tissue cells and their capacity to incorporate radioactive proline by means of quantitative radioautography, as observed in the polyvinyl alcohol sponge implants in the presence or absence of FC. A limited observation on the histologic response of the bone will also be recorded.

MATERIALS AND METHODS

Experimental regimen A total of twenty-seven young adult golden Syrian hamsters (Mesocricetus auratus), weighing 100-130 g, were used throughout this investigation. All animals were maintained on a stock Purina chow and tap water ad libitum.

A sponge implant on the dorsum of the neck and a surgical opening in the right

femur were prepared in twenty-four hamsters. Formocresol (Buckley’s Formocresol :

Formaldehyde 19 per cent and cresol35 per cent in a vehicle of glycerine 15 per cent and water. Crosby Laboratories, Burbank, California) was diluted to a l/50 of a normal concentration for the sponge implant study. The wound placed in the femur received a normal concentration of formocresol. Physiologic saline was used as a control.

The intervals from the application of the drug to sacrifice were 5 hr, 1 day, 3 and 10 days, and one month. The injection time of radioactive proline with respect to that of sacrifice was 4 and 24 hr. Twelve animals were used for experiments and 12 for controls. Three animals were used for the scintillation counting of the blood level of H3 as a function of time.

Surgical procedure The hamsters were anaesthetized by an intraperitoneal injection of: nembutal, U.S.P., 1.0 ml; distilled water, 4.5 ml. For every 10 g of body weight, 0.05 ml of the above anaesthetic was injected. To maintain the anaesthesia, half of the initial dose was repeated.

The dorsal skin just posterior to the neck was shaved. A midline incision was made through the epidermis for about 3 mm, and a previously sterilized polyvinyl alcohol sponge measuring 5 mm in diameter was implanted. Metal clips were used as sutures. Throughout the procedure care was taken to maintain general principles of sterile surgical techniques.

Concurrently with the implantation of sponge, the right hind leg was shaved and an incision made into the skin for 20 mm. Blunt longitudinal dissection of the muscles was performed along the lateral intermuscular septum to expose the lower part of the femur, and approximately 3 mm of periosteum was removed. With a dental handpiece at 10,000 rev/min, a round carbide bur, No. l/2, was used to bore





FORMOCRESOL, CONN!XTlVE TISSUE AND PROLINE-HS RADIOAUTOGRAPHY

a hole into the femur. A sterile cotton pellet moistened with saline was used to wipe the area clean of debris. A medium-size paper point was dipped in the drug and applied into the open wound for 5 min. After the paper point was removed, the incision was sutured with metal clips, Injection of radioisotope and scintillation counting Tritiated L-Proline-3, 4-H3 (specific activity 5-O c/mM), obtained from New England Nuclear Corp., was the radioactive precursor administered. This will be referred to as proline-H3. The isotope was used in the amount of 5&g body weight.

In order to determine the blood level of available radioactive precursors, three

hamsters were injected with the same relative amounts of proline-H3 and bled at:

2 - 5, 5, 10,20, 30,40, 50 and 60 min, 6, 12, l&24, 30, 36,42 and 48 hr, and 4 and 6 days after injection (ANDERSON, 1965).

Blood samples, approximately 0.008 ml/animal, were collected in heparinized capillary tubes measuring I.15 mm x 75 mm in inner diameter and length, respectively. Blood was drawn from the choriod plexus of the eye by applying simultaneously a gentle pressure coupled with a rotary motion. The capillary tube of blood was immediately inverted into the counting vial for drainage. Then 4 drops of 30 per cent peroxide were added to cover the blood sample, which was allowed to settle for 30 min. Hyamine X-10 was added in the amount of 0 *5 ml and the vial was placed in a water bath for 30 min at 50-60°C. The vial was removed, allowed to cool and then 10 ml of the scintillation fluid was added and shaken to eliminate bubbles. Samples were stored in a refrigerator and counted a week later (FRENKEL al., 1962; GORDON et and WOLFE, 1960; NASJLETI,1965, personal communication).

Scintillation counting was carried out in a Packard Tri-Carb Liquid Scintillation Spectrometer, model 3000. The mean background count was 24.4 counts/min. The counting efficiency of the prepared system was 10 per cent.

Histologic and radioautographic preparations The tissues were fixed in Bouin’s fixative for 48 hr, washed overnight in running water and femurs were decalcified in the usual manner using Versene. They were then vacuum embedded in paraplast. Serial sections of the sponge implant at 6~ and longitudinal serial sections of femur at 10~ were made on a rotary microtome. The sections were mounted on slides which were pretreated with a subbing solution, composed of O-5 g of gelatin and 0.05 g of chromium potassium sulphate in 100ml of distilled water.

For radioautography, selected slides were freed from paraplast, hydrated and dipped into Kodak NTB-3 nuclear track emulsion in a routine manner (SMITH and HAN, 1967). Slides were placed on end and allowed to dry for $ hr. The slides were then arranged in an air-tight slide box containing small packs of Drierite, shielded with lead-sheets and kept in a refrigerator. Following 4-8 weeks of exposure, the slides were removed, developed in Dolmi solution and stained with haematoxylin and eosin.

STRAFFON AND HAN

L. H. S. S.

Slides not coated with the emulsion were stained either with haematoxylin and eosin, Masson’s trichrome or PAS-azure II.

RESULTS AND OBSERVATIONS

Scintillation counting Figure 27 records the average values of scintillation counts of the blood level of tritium obtained from three separate animals at various times following the injection of proline-H3 intraperitoneally. The vertical bars indicate the range in which the three counts fall. The level of tritium in the circulating blood rises rather rapidly during the early post-injection period, reaching a peak which diminishes gradually throughout the first week. It might be pointed out that there was about 15 per cent or more reduction in the scintillation count for 6-24 hr, a factor which should be considered in the evaluation of the grain counts.

Histological observations After 5 hr of implantation, the sponge from the control hamster showed a large number of polymorphonuclear leucocytes (PMN) along with some regressive changes of the surrounding connective tissue (Figs. 1 and 3). Degenerative changes were characterized by the disorganization of connective tissue fibres in the immediate vicinity of the implant. PMN were more numerous in the perivascular region, and many of them reached the surface of the sponge. On the other hand, the sponge treated with FC showed much less PMN infiltration as compared to the control (Figs. l-4). However, signs of the connective tissue degeneration were somewhat greater.

The sponge from the l-day control animal demonstrated again a larger number of PMN than in the experimental hamster (Figs. 5-8). Many of the PMN were degranulated and could be identified only on the basis of the nuclear morphology. Occasional mononuclear cells appeared at this time, some of which appeared to be hypertrophic and actively phagocytizing the nuclear debris of PMN (arrow, Fig. 7). The fibroblasts located along the periphery of the control sponge demonstrated a cytoplasm which was highly basophilic and contained a large nucleus with prominent nucleoli. In the experimental animals these changes did not appear to take place as fast as in the control (Fig. 8). Although there was a migration of mononuclear cells, they were few in number and connective tissue fibres remained as broken patches of homogeneous eosinophilic material. Few active fibroblasts were observed.

By the end of 3 days a greater number of active fibroblasts and newly developing connective tissue fibres were observed along the surface of the control sponge (Fig. 9).

The connective tissue covering the sponge from the experimental animal also contained numerous active fibroblasts infiltrating the area (Fig. 10). At the outer border of the sponge, typical giant cells were seen along with phagocytic macrophages in the control animal, while no giant cells were observed in the FC-treated sponge.

On the tenth day the control sponge showed few PMN and the ingrowth of connective tissue was well advanced (Figs. 11 and 13). Similar changes had occurred in the FORMOCRESOL, CONNECTIVE TISSUE AND PR3LINE-H3 RADIOAUTO03RAPHY 275 experimental animal by this time. However, the ingrowth of the tissue into the substance of the matrix might have been somewhat less than in the control (Figs. 11 and 12). Within the sponge matrix of the control there was finely fibrous material, while in the experimental such network of fibrous material was even finer. Along the trabecular surface of the sponge in the experimental animal was found a fair number of large giant cells with basophilic cytoplasm (Fig. 14). After 31 days, the capsule of the connective tissue in both experimental and control animals was well organized and the intra-trabecular space of both sponges was filled with advancing connective tissue elements along with giant cells and a number of macrophages.

Histologically, events occurring in the femur more or less paralleled the general changes which were characteristic of the connective tissue of the sponge implants.

Thus, the wound region of the femur treated with FC had a smaller number of PMN infiltrating the haemorrhagic mass than the control. On the third day the marrow space adjacent to the haematoma of the wound region showed many macrophages containing haemosiderin and the infiltration of capillaries and fibroblast-like cells in both groups, although the number of fibroblasts was greater in the control. Of significance was the observation that both the control and experimental animals presented a similar histological picture 10 days after surgery (Figs. 15 and 16). The degenerating tissues were replaced by active fibroblasts and connective tissue fibres and new bone formation was clearly evident in both groups. In the control the amount of newly formed bone was slightly greater than in the FC-treated femur. No difference between the two could be distinguished 3 1 days after the injury.

With the PAS-azure II stain, there was a greater number of mast cells in the immediate vicinity of the experimental sponge implant at 5 and 24 hr than in the control. At 24 hr, the mast cells next to the sponge in the experimental animal were degranulated and fewer in number than in the control. By the third day, the mast cells in the experimental animal were still in greater number than the control, but at 10 days and 1 month, the number was about equal.



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