/. Periodontal Rex. 11: 368-373, 1976.

Histological evaluation of the osteogenic capacity of sclera R. S. TDKNBULL, E. FREEMAN, AND A. H. MELX;HER

Faculty of Dentistry and MRC Group in Periodontal Physiology, University of Toronto, Canada Sclera was obtained from the eyes of New Zealand white rabbits and immediately stored at —79 °C. Later, after thawing, a piece 4 mm square was implanted into each of the anterior chamber of 14 eyes and under the renal capsule of 28 kidneys of rabbits of the same strain., Allografts of skin, muscle or tendon were implanted subcapsularly in the contralateral kidneys as controls. Two animals were sacrificed postoperatively each week up to 4 weeks, 6 animals at 6 weeks, 4 each at S and 9 weeks and 6 at 12 weeks. Serial paraffin sections of the implants and host tissues were examined by light microscopy. Osteogenesis occurred in 7 of 12 implants of sclera implanted beneath the renal capsule at 6 and 12 weeks, but was not seen prior to 6 weeks or at 8 or 9 weeks. Osteogenesis did not occur in sclera implanted into the anterior chamber of the eye or in control implants. (Received for publication January 30, 1976, accepted April 27, 1976)

Introduction

Klingsberg (1972a, b, 1974) has used implaots of selera to treat defects in periodontium resulting from periodontal disease. The allografts were apparently well tolerated by the periodontal tissues and were reported to aid in "tissue augmentation". The observations were made on patients treated clinically aod were not derived from carefully controlled studies. Because of this fact, and on account of the paucity of laboratory studies on the fate of implanted sclera, little is known about the response of host tissues to scleral allografts. The present study was undertaken to determine whether sclera can ioduce osteogenesis. Materials and Methods

Male adult New Zealand white rabbits were used in the study. After sacrifice the eye-

balls were enucleated, and the sclera cut into strips and rinsed in antibiotic-antimycotic solution. The sclera was then sealed in plastic packages aod stored at —79°C. Later, after thawing, a piece 4 mm square was utilized as an implant. Uoder intravenovis sodium pentobarbital anaesthesia, single implaots were placed into the anterior chamber of each of 14 eyes and under the renal capsule of each of 28 kidneys of 28 rabbits of the same strain. Eyes An incision was made at the corneal-scieral line angle to permit access into the anterior chamber. A piece of sclera was then inserted into the anterior chamber and ophthalmic ointment placed over the surface of the operative site without suturing (Fig. 1). Two animals were sacrificed postoperatively at weekly intervals up to 4 weeks and 6, 8 and 12 weeks.

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Fig. 1, Scleral graft in the anterior chamber of the eye of the rabbit. Fig.

2. ScEeral graft mseried between the capsule and the renal parenchyma ol the rabbit.

Fig. 3. Scierai graft (S) in the anterior chamber of the eye twelve weeks postoperativeiy. The gTaft is avascular and no new bone is evident. C.,, corr^ea. Haematoxylin and eosin. x 180.

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Kidneys The kidoey was exposed by a lumbar approach. A small incision was made into the capsule, care being takeo to avoid cutting the parenchyma of the kidney. A blunt instrument was then inserted between the capsule and the renal paretichyma to make a space, into which a piece of sclera was placed (Fig. 2). The tension of the capsule retained the implant securely. Two animals were sacrificed postoperatively at weekly intervals tip to 4 weeks, 6 animals at 6 weeks, 4 each at 8 and 9 weeks and 6 at 12 weeks.

c) Sclera implanted under the renal capsule The scleral implaots did not exhibit osteogenesis prior to 6 weeks nor at 8 or 9' weeks (Fig. 4). However, deposits of bone with marrow aod blood vessels were seen in 3 implants at 6 weeks and 4 implants at 12 weeks postoperatively (Fig. 5 & 6). The bone was always found within the sclera and was always associated with proliferatitig host blood vessels. A summary of the observations for the scleral grafts and controls is shown in Figure 7.

Controls Siio, muscle and tendon were obtained from rabbits aod stored in the same manner as the sclera. A 4 mm piece of one of these samples was implanted subcapsularly in the contralateral kidney of each rabbit as a control.

Discussion

The anterior chamber of the eye (Urist & McLean 1952) and the reoal capsule of the kidney (Buyse 1933) have been used to evaluate the osteogenic capacity of various implant materials. By placing implants into a non-bony connective tissue site it can be determined if the implant material can Histology induce host stem cells to differentiate into AH implants and host tissues were fixed in hone cells. The finding that sclera was able 10 % neutral btiffered formalin and pre- to produce new bone under the renal cappared by routine histological methods for sule in 25 % of all the animals, aod 7 of the preparation of paraffin sections. Serial 20 animals between 6 and 12 weeks postsections were cot, stained with haematoxylin operatively, suggests that sclera may have and eosin and examined by light micro- the capacity to induce osteogenesis. The scopy. finding that formation of new bone was invariably absent from sclera implanted into the anterior chamber of the eye, and Resuits that these grafts did not appear to be vasa) Sclera placed into anterior chamber of cularized, suggests that ingrowth of new the eye blood vessels may constitute an important There was no evidence of osteogenesis in step in the induction process. This belief relation to the sclera that was implanted is strengthened by the observation that in the anterior chamber of the 14 eyes blood vessels were always seen in sub{Fig. 3). capstolar sclera! grafts in which osteogenesis had occurred,, but could not be identified b) Control implants placed under the renal using routine histology io those grafts from capsule which osteogenesis appeared to be absent. No new hone was found in relation to the No vascularizatioo of control implaots was allografts of skin, mtjscle or teodon in the evident. These observations are consistent 28 implants used as controls. with those of Scott (1967) who found that

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Fig. 4. Scleral graft (S) under the renal capsule eight weeks postoperaiiuely. Blood vessels cannot be found within the graft and no new bone is present. The margin of the implant and the renal capsuie can be seen (arrow). K., kidney. Haematoxylin and eosin. X 180. Fig. S, Bone and marrow within the sclera. twelve weeks po'stoperatively. K., kidney, Undemineralized section. Haematoxylin and eosin. x 180. Fig. 6. Blood vessels (arrows) associated witli bone formation within the scEera[ graft six weeks postoperativeiy. K., kidney, B., bone. Undemineraiized section, iiaematoxylin and eosiri. x 450.

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osteogenic cells in fetal rat tibia are associated with capillaries. Since the ittiplanted sclera stitnulated formation of new bone in a non-bony connective tissue site, this itivestigation suggests that sclera tnay, under some circumstances, be ahle to induce the differentiation of osteobiasts from host cells. It is not possible to explain why this occurred in sotne itnplaots and not others. The identity of the cells that are induced letnains to be elucidated, and the mechanistn by which induction occurs is unknown. In this regard, it is of interest that Urist et al. (1967) have provided some evidence for a bone-inducing principle, which they refer to as "bone morphogenetic protein" (BMP). BMP is believed to be an acid-insoluble macrotnolecular complex that occurs in the highly crosslinked collagen matrix of bone and detitin (Urist & Strates 1971). Morris (1967) maintains that the rabbit in an unsuitable experimental animal for studies on the induction of bone, since this atiimal exhibits a propensity to form bone as a non-specific reaction to injtiry. It

could therefore be argued that the new bone that was formed in the scleral implants of - 7 animals was a non-specific response. However, since no new bone was found to be formed in relation to any of the control allografts, our results suggest that the implanted sclera may have induced osteogenesis. These observations appear to be interesting in relation to the current use of sclera in attempts to regenerate periodontium lost in periodontal disease. However, it is perhaps important to emphasize the fact that treatment of periodontal defects should aim at restoration of the periodootium as a whole and not just alveolar bone (Melcher 1976). Acknowledgements

The authors would like to acknowledge Mr. D. Wagner and Mrs. N. Yaroshevsky for their exceUent technical assistance. References Buyse, A. 1933. The transplantation of em-

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bryonic mammalian primordia. Abstract Amer. Ass. of Anat. 59th Session. Anat. Rec. 55.- 10: Klingsberg, J. 1972a. Scleral allografts in the repair of periodontal osseous detects. N. Y. State Dent. 3. 38: 418-420. Klingsberg, J. 1972b. Preserved sclera in periodontal surgery. /. Periodontol. 43: 634639. Klingsberg, J. 1974. Periodontal scleral grafts and combined grafts of sclera and bone: two-year appraisal. /. Periodontol. 45: 252272. Melcher, A. H. 1976. On the repair potential periodontal surgery. J. Periodontol. 43: 634press. Morris, M. L. 1967. The implantation of human dentin and cementum into the subcutaneous tissues of the rat. Periodontics. 5: 113-122. Scott, B. L. 1967. Thymidine-»H electron microscope radioautography of osteogenic

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cells in the fetal rat. /. Cell Biol. 35: 115125. Urist, M. R. & McClean, F. C. 1952. Osteogenetic potency and new-bone formation by induction in transplants to the anterior chamber of the eye. /. Bone Joint Surg. (Am.) Vol. 34: 443-476. Urjst, M. R., Siivcrman, B. F., Biiring, K.,, Dubuc, F. L. & Rosenberg, J. M. 1967. The bone induction principle. Clin. Orthop. 53: 243-283. Urist, M. R. & Strates, B. S. 1971. Bone morphogenetic protein. /. Dent. Res. 50: 1392-1406.

Address: Faculty of Dentistry 124 Edward Street Toronto, Ontario M5G 1G6 Canada

Histological evaluation of the osteogenic capacity of sclera.

/. Periodontal Rex. 11: 368-373, 1976. Histological evaluation of the osteogenic capacity of sclera R. S. TDKNBULL, E. FREEMAN, AND A. H. MELX;HER F...
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