British Journal of Oral Surgery (1973), Io, 334-338

T HE P R O D U C T I O N OF A B O N E R E S O R B I N G F A C T O R BY DENTAL CYSTS I N VITRO

MALCOLM HARRIS, M.B., B.S., F.D.S., R.C.S. King's College Hospital, Denmark Hill, London

and

PAUL GOLDHABER, D.D.S. Harvard School of Dental Medicine, Boston, Mass., u.S.A.

THE enlargement of dental cysts within the jaws has been attributed to the gradient in osmolality between cyst fluid and plasma creating an expansile hydrostatic force (Toller, 1967, I97O).

One of the aspects of cyst growth that has not been explored is the production by the lesion of a bone resorbing agent. Gaillard (I955) first demonstrated the capacity of parathyroid tissue to produce bone resorption in vitro, and since then Goldhaber et al. (r96o, I964, I97 I) have achieved similar effects with fragments of mouse fibrosarcoma, bronchial carcinoma and gingiva. Myers et al. (I968) have produced the release of radiocalcium from prelabelled rat embryo bones with extracts of cervical, bronchial, penile and urethral carcinomas in organ culture.

The dental cyst by virtue of its intra-osseous growth seemed to be an appropri- ate tissue to study for a bone resorbing factor (BRF).

METHOD AND MATERIALS

Dental cysts were immersed in Geys I balanced salt solution containing IOO U/ml penicillin and 8 I~G/ml streptomycin immediately after enucleation, and transported in ice. All experiments were set up within two hours of the operation. The tissue was divided into portions for histology, direct tissue culture, freeze-- thawing, and elastase incubation.

I-2 mm cubes of the untreated cyst were washed in Geys solution and placed adjacent to the posterior border of a mouse calvarium on a coverslip. The cal- varium consisted of the frontal bone and both parietal bones aseptically dissected from five-day-old Swiss Albino mice of the Webster strain. Both the cyst tissue and bone were covered with a thin fibrin clot made up of two drops of heparinised chicken plasma to one drop of chicken embryo extract. Control calvaria were clotted without a cyst tissue fragment.

Each coverslip was then transferred to the well of a Leighton tube and 2 ml of medium added. This consisted of 7 ° per cent heated horse serum, 3 ° per cent Geys balanced salt solution, Ioo U/ml penicillin, 8 /~G/ml streptomycin, and heparin Io U/ml. The heparin enhances the activity of bone resorbing agents in tissue culture (Goldhaber, I965).

A portion of the tissue was devitalised by rapidly freezing in Geys solution surrounded by a mixture of dry ice and alcohol and then thawing in water at 6o°C,

1 Difco Laboratories, Detroit, Michigan.

334

PRODUCTION OF BONE RESORBING FACTOR BY DENTAL CYSTS I N V I T R O 3 3 5

three times. This tissue was also cut into small fragments, washed and cultured as above.

Similar fragments were incubated at 37°C in a solution of o'I ml elastase ~ in 2"5 ml. antibiotic-Geys solution for half an hour. The epithelium was then care- fully separated from the capsule using a dissecting microscope. These separated fragments were washed and cultured and in the same way as the whole tissue. Muscle incubated both in elastase and Geys was used as a control tissue.

The tubes were gassed with a mixture of 50 per cent oxygen and 50 per cent nitrogen, sealed with silicone stoppers and placed horizontally in a rotating drum at 37°C.

All calvaria were scored for resorption and their media changed and gassed every two days. The scoring is based on the surface area resorbed, one point being given for every sixth part of the frontal bone and one for each quarter of the parietals. Thus, a score of 14 represents resorption of the whole calvarium. As each group consisted of four calvaria, the results are expressed as a mean.

TABLE I

Calvaria cultured for seven days with vital cyst fragments all showed significant resorption compared with their controls apart from 2335 which proved to be an

enlarged dental follicle

Control Calvaria Cyst No. Cyst--Clinical Details ~ Calvaria plus cyst

23oo Dentigerous with parakeratinised 0.8 9"0

2304

2314a 2314c 2316 2332 2334

2335

2339

2 2 4 9

8/M. 3o yrs. epithelium /7M. 33 yrs. keratinising /8 M. 65 yrs. 3/3 F. 23 yrs.

Infected periapical. Non-

Non-keratinising Kerato-cyst ~

/~-8 F. 26 yrs. Infected ameloblastoma i/I M. ao yrs. Periapical non-keratinising i~/I M. 35 yrs. infected periapical. Non- keratinising 8//M. 18 yrs. Enlarged follicle--remr~nts of reduced enamel epithelium Nasopalatine. M. 55 yrs. I n f l a m e d ~ haemorrhagic s" ~! ,~=, 8/M. 55Yrs. Keratocyst. Parakeratinisedwith much old haemorr,hage ,

3 ' 0

3 " 0

3.0 2"0 2. 5 2. 5

4"5

3"5

2"75

9'O

7"o I 2 ' O IO'O

I I ' O

13 "5

6"25

10'5

9"o

Note: Total resorption of the calvarium would be a score of 14. Each of these results is the mean of four calvaria.

, , , ~ , i 2 ,. ~ . - ~ ~ ,

The experiments were terminated after seven d@s, when the calvaria were fixed ) ) , , : , . . ' r . ~ ,, ~ . o . ' ~ , r ~.

m Io per cent neutral f6rmalm. They we~e~Jater stained whole by yon Kossa s method or decalcified in the disodium salt of E ~ T A and sectioned at 6ff and stained

;.! ( ~'~ i [ 2- '

with haematoxylin and eosin. . " . '2 ' ~ '~ ' : : 7

Worthington Biochemical Corporation Freehold, N.J. < s"

336 BRITISH JOURNAL OF ORAL SURGERY

RESULTS

With one exception, all the explants produced marked bone resorption (Table I). The difference between the control and the cyst explanted calvaria was readily seen at 48 hours and by the end of seven days, many of the latter were totallyresorbed (Fig. I). Complete resorption of the control calvaria was never seen.

FIG. I

A, Control calvaria. B, Almost complete resorption by cyst B.R.F. C, Absence of resorption when incubated with non-vital cyst.

TABLE II

Non-vital cyst fails to produce or release B.R.F.

Freeze-thawed Tissue

Control Calvaria Cyst No. Calvaria plus cyst

2314a 2314c 2332 2334 2335 2249

2"5 2. 5 3"5 2. 5 3"5 2"75

2"0

3"5 2"25 2"0 3"0 3.0

There was no detectable difference in activity between simple and keratini- sing cysts, nor between those cysts that showed evidence of infection as compared with the non-infected. Two heavily infected cysts were omitted from the series because offungal contamination of the cultures after four days' incubation. How- ever, up to that time they also produced marked bone resorption.

Specimen 2335 which produced minimal bone resorption proved to be an enlarged follicle lined by remnants of reduced enamel epithelium.

PRODUCTION OF BONE RESORBING FACTOR BY DENTAL CYSTS IN VITRO 337

Case 2 3 I6 was of considerable interest in that it proved to be an ameloblastoma. Freezing and thawing the tissue three times prior to culture completely des-

troyed its capacity to resorb bone (Table II) indicating the need for vital tissue to synthesise the active agent.

The splitting of cyst epithelium from its capsule in order to culture them separately was an intriguing exercise. However, it was limited by the absence of epithelium on some of the incubated fragments or by difficulty in separation. Only those cases where clean atraumatic separation was achieved are included in Table III. These results are inconclusive and suggest that the epithelium only

TABLE III

Elastase Separated Cyst Components

Cyst No.

2332 2334 2339 2349

Control

2"5 2. 5 3"5 2"75

Whole Cyst

I I I3"5 xo'5

9"0

Epithelium Capsule

4"5 4"5 4"5 5"5 4. 0 i2-O ~r 4"25 8"25"

* Only these two capsules produced significant resorption.

produces slightly more resorption than the controls but significantly less than the whole cyst, whereas the capsular tissue could be highly active. Controls consisting of muscle preincubated both in Geys alone or elastase produced no resorption.

DISCUSSION

Bone resorption in vivo has been attributed to either the ill-defined activity of a vascular connective tissue network adjacent to the resorbing bone surface (Sognnaes, i963; Trueta, I963) or to the action of a bone resorbing agent working through the osteoclast (Gaillard, I955). These two concepts are by no means mutuallyexclusive (Goldhaber, r97~ ). The marked vascularity of cyst capsule is particularly obvious in preparations showing fibrinolytic cytokinase activity which appears to be associ- ated with the capillary walls (Harris, I97o).

Folkman (r97I) has shown that turnout vascularity and growth is regulated by a factor released by the neoplastic epithelium which he calls tumour angiogenesis factor (T.A.F.). As the name suggests, this factor can stimulate the formation of a capillary network. It is interesting to speculate that the same interdependence exists between cyst epithelium and its vascular capsule both for growth and bone resorption. The latter being due to the formation of a bone resorbing factor by vascular or perivascular cells.

The resorption stimulated by small fragments of vital cyst tissue in this investigation could only have been achieved through the synthesis and release of a potent bone resorbing agent, acting independently of any osmotic forces.

It is not difficult to conclude from these results that intra-osseous cyst expan- sion is facilitated by local enzyme or hormone induced bone resorption. The same

338 B R I T I S a JOURNAL OF ORAL SURGERY

process would also appear to be true for the ameloblastoma. Work being carried out at the present t ime suggests that the active principal is in fact a prostaglandin (Harris et al., I973).

S U M M A R Y

Vital explains of nine dental cysts and one ameloblastoma were maintained in tissue culture with mouse calvaria for seven days. Eight produced marked bone resorption which was not seen when devitalised tissue was similarly cultured.

I t is proposed that dental cyst and turnout growth within bone is dependent on the synthesis and release of a potent bone resorbing factor.

ACKNOWLEDGEMENTS

We are grateful for the assistance of Aurora Garcia, Joan Jennings, Elaine Paznick, Lorraine Stevens and Frederica Ward. The work was carried out at the Harvard School of Dental Medicine and was supported in part by grant DE 02849 from the United States Public Health Service. We are also indebted to the Oral Surgeons of the New England area who provided the cysts.

REFERENCES

FOLKMANN, ~. (1971). New England Journal of Medicine, 285, No. 2I, 1182. GAILLARD, P. J. (I955). Exp. Cell Res, 3, suppl. I54. GOLDHABER, P. (196o). Proc. Am. Assoc. Cancer Res., 3, n3 . GOLDHABER, P. (~965). Science, x47, 407. GOLDHABER, P. (I97I). Journal of Dental Research, Supplement to No. 2, 5o, 278. GOLDHABER, P., ROTH, S. I. & CIRULIS, G. (I964). Cancer Res, 24, No. 2, 254. GROSS, J. ~: LAPIERE, C. M. (I962). Proc. Natl. Acad. Sci. U.S., 48, lOI4. HARRIS, M. (I97O). In Transactions of third International Conference on Oral Surgery, New

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A. & WEBER, D. (1968). Calc. Tiss. Res., 2, 63 Suppl. SOGNNAES, R. F. (I963). In Mechanisms of Hard Tissue Destruction, ed. R. F. Sognnaes.

Am. Ass. Adv. Sci. Washington DC. TOLLER, P. A. (I967). Annals Royal College of Surgeons England, 4o, 306-336. TOLLER, P. A. (I97O). British Dental Journal, I29, 275. TRUETA, J., (I963). Journal of Bone and Jt. Surgery, 45B, 402.