Experimental osteogenesis in periapical areas with decalcified allogeneic bone matrix

Experimental osteogenesisin periapical areas with decalcified allogeneic bone matrix :Vw~~~~g, B.D.S., M.Sc.D.,

Humcsh

D.L!f.D.,** BOSTON

D.Sc.D.,*

and Herbert

Wells,

Bostm, Uass.

USIVERSITY

SCHOOL

OF GRADUATE

DENTISTRY

Decalcified nllogeneic bone matrix grafts were implanted in fourteen teeth after pulpotomy, in twenty root canals after pulp extirpation, and in the periapical areas of forty-eight teeth after apicoectomy. These procedures were carried out in five dogs and three monkeys. Results of the present investigation reveal that DAB&i grafts stimulate osteogenesis and cementogenesis. Although present data are insufficient, it can be speculated that DABM grafts would result in physiologic seal at the apical foramen of apieoeet.omizecl teeth with new bone and cementum. Thus, this increased osteogenesis and cementogenesis will prolong the survival of nonvital teeth in human beings.

W

henever routine cndodontic treatment fails or when nonvital teeth are Sound to have wide apical foramina, an operator is called upon to perform pcriapical surgery (retrograde) for apical closure. Various materials, such as silver amalgam, gutta-percha, zinc oxyphosphate cement., and calcium hydroxide, have been tried in order to seal the apical foramen, but all these materials achieve is a mechanical seal. Thus, there are chances of reinfection into periapical areas which could result in a failure of the objectives of retrograde treatment. This will necessitate another retrograde procedure or ultimate extraction of the involved tooth. Thus, the development of any materials that could achieve a physiologic seal would be a definite advancement in the field of endodontic surgery. Decalcified allogeneic bone matrix (DABM) is a recent osteogenic material which has an excellent potential in new bone formation in skeletal and extraskeletal sitcs.1-13 We have induced new bone in bone defects,1* 4 in fracture sites,’ around plastic tooth implantq3 on intact bones,” and in periodontal bony This research was supported in part by Research Grants DE 02963, FR 65437~67, and DE 00265-01 from the National Institutes of Health. *Former Research Fellow, Boston University School of Graduate Dentistry; at present Resident in Oral Surgery at University of Illinois Hospital, Chicago, Ill. **Professor

136

of Pharmacology.

Experimental

Volume 35 Number 1

osteogenesis

137

defects6 in animals. On the basis of our previous results in four species,l-B the present experiments were designed to st.udy implants of DABM in monkey and dog teeth subjected to apicoectomy. The purpose of this experiment was to make an attempt to get a physiologic seal, thus decreasing the chances of periapical infection. This was achieved by proliferation of connective tissue and its differentiation into new bone and cementum. It can be concluded from this preliminary experiment that the osteogenesis and cementogenesis in periapieal areas could achieve physiologic seal of the apical foramen and thus may preserve nonvital teeth for a much longer time in human beings. MATERIALS

AND

METHODS

Five mongrel dogs of both sexes, each weighing 15 to 20 kilograms, were caged individually and fed Purina dog chow and water ad libitum. Three female monkeys weighing 4 to 6 kilograms each were also caged individually and fed standard primate diet and water ad libitum. Decalcification

of allogeneic

bone

One dog and one monkey unrelated to the above animals were killed with lethal doses of pentobarbital sodium. The long diaphyseal cortical bones were excised under aseptic conditions, cut into smaller pieces, and decalcified in 0.6N hydrochloric acid at 2O C. for 6 to 7 days. The further preparation of allogeneic bone was carried out as in previous experiments.le6 DABM grafts were stored in separate bottles for further implantation. Anesthesia

and

surgical

procedure

Dogs were anesthetized with intravenous pentobarbital sodium (32 mg. per kilogram). The monkeys were anesthetized with 0.5 ml. of Seranyl and 16 mg. of pentobarbital sodium. Lidocaine 2 per cent with 1 :lOO,OOOepinephrine was infiltrated for hemostasis in the areas where surgical procedures were to be performed. Four different procedures were performed in eight animals. In Group A, fourteen molars were subjected to removal of pulp from the pulp chamber (pulpotomy), and insertion of DABM in the pulp chamber, and these cavities were sealed with silver amalgam. In Group B, pulp extirpation was performed in twenty canines. These root canals were sealed with silver amalgam after insertion of DABM grafts in root canals. In group C, incisions were made along the gingival margins and mucoperiosteal flaps were retracted. The overlying bone around the apices of forty-eight anterior teeth were removed with a surgical bur. Extreme care was taken not to expose too much of the roots. The apical fourth of each root was excised with a fissure bur. The resected root portions were removed with curettes, and a small cavity was prepared in the apical portion of each tooth. DABM grafts were implanted into the periapical area of every tooth operated upon (Fig. l), and the flaps were approximated with 3-O chromic catgut sutures. Similarly, fifteen anterior teeth were subjected to apicoectomies and flaps were closed without DABM grafts (control). The animals were fed ad libitum and observed daily for signs of infection or rejection of these grafts after every surgical procedure. No special postopera-

Oral Surg. January, 1973

Pig. 1. Placement of decalcified xllogeneic (h’) containing remnnnt, pulp (I’). C, Crown.

I. DABM

Table

Group

All

bone

(DABM)

grafts in root canals and periapical Procedure

A

Pulpotomy DABM

and

R

Pulp extirpation and DABM

C

Apicoectomy DABM

D

Apicoectomy (control)

and

c~iyht :~nimals were killed

Five

dogs

in apicoectomized

(a)

root

areas Three monkeys

Total

6

8

14

8

12

20

36

12

48

8

7

15

12 weeks postoperatively.

tivc cart’ was necessary in these animals, and antibiotics were not used. All eight animals wcrc killed at 12 week interrals with lethal (loses of pentobarbital sodium (Table I). All operative sites were excised in toto. These specimens were stored in 10 per cent formalin for fixation, decalcified in 5 per cent nitric acid for 5 tlays, and washed for 24 hours in running water. After other appropriate histologic preparations, the spccimcns were serially sectioned at a thickness of 7 microns and stained with hematoxylin and eosin. RESULTS

All animals were examined, and no obvious signs of infection or of graft re,jection wcrc observed in the periapical areas of forty-eight teeth (Group C). Clinical observation disclosed that all grafts were accepted in periapical regions of the host tissues, and radiologic examination of the operated teeth revealed generalized radiopacity in the apical areas, simulating new bone. Radiographs of periapical areas were free of any pathologic radiolucencp. Most of the DABM grafts in Group A were lost because of loss of occlusal amalgam fillings and the remaincler did not stimulate any osteogenesis or dentinogenesis. Similarly, DABM grafts in root canals of Group B did not stimulate new bone formation but were nccrosed within the E-week interval. Microscopic

Volume 35 Number 1

Fig. 8. Microscopic appearance ce,mentum formation (open. ~TTOWS) (Hematoxylin at 12-week interval. re!spectively.)

Experimental

osteogenesis

139

of tooth showing new bone formation (ClOSea arrows) and around dentin (D) and in root canal with DABM q rafts and eosin stain. Magnifications: x3, x.50, x100, and x50,

140

Sarang

asd Wells

Oral Surg. January, 1973

E‘iy. 3. Microscopic appearance at 12.week interval demonstrating increased cem entogenes is (open arrows) and osteogenesis (closed arrows) around dentin (D) with DJ9BM graft s at periapical and areas of apicoectomized tooth. CC, Cellular Cementum. (Hematoxylin c%osin stain. Magnifications: x100, x2OQ, x200, and x435, respectively.)

stud y of DABM grafts implanted in pcriapical areas (Group C) revealed fol :mation of new bone and cementum in root canals (Figs. 2, 3, and 4). From prev .ious espe,rinlents,*-G it is revealed that DABM grafts, when implanted in tisr mes, stim ulate perivascular mesenchymal tissue and differentiate the latter into ostcc jhlasts and osteoclasts, thus resulting in resorption of DABM grafts and

Volume 35 Number

1

Experimental

osteogenesis 141

Fig. 4. DABM grafts in periapical areas at la-week interval. Microscopic appearance of new bone formation (closed avows) and cementurn formation (open arrows) in a root canal. Note the presenceof osteoeytes (0~) in new bone. (Hematoxylin and eosin stain. Magnifications:

x50, x100, x100, and x435, respectively.)

replacement by new bone within 8 weeks. The new bone formation was demonstrated by the presence of osteoid tissue and oseteoblasts and its union to the host bone. There was also evidence of increased cementum formation at the root resection areas. Increased cementogenesis was seen at the excised margins of roots and on the inner surface of dentin in the root canals. The new bone thus formed depicted viable osteocytes. The periapical areas were completely free of any remnant DABM grafts at the end of the 12-week interval. in Group D,

Oral Surg. January, 1973

Fi,c/. 5. Physiologic seal (F’S) can he achieved with use of DABM graft in apicoectomized (.4) root (R) after root canal keatment (RCT). C, Crown.

however, fifteen teeth subjected to apicoectomy without DABM grafts demonst.rated very little new bone. The .periapical areas in this group demonstrated inflammatory cells, necrotic tissue, empty bone defect, and occasional osteoid tissue. DISCUSSION

The results of our present experiment indicate that decalcified allogcneic bone matrix grafts arc not rejected within 12 weeks and that they are capable of new bone formation in pcriapical areas. In previous experiments, we have demonstrated that DABM implants were gradually removed by osteoclastic resorption and replaced by new bone and bone marrow.1-7 The new bone formation is much more rapid than the osteoclastic resorption of DABM grafts, and these grafts are not exfoliated from the tissues. These grafts, to our knowledge, do not cause any immunologic response. In the present experiment there is evidence of increased osteogenesis and cementogencsis in periapical areas with the use of DABM grafts. There were certain drawbacks in the present experiment, such as remnant pulp tissue and apicoectomy without root canal treatment. In spite of these drawbacks, DABM grafts were able to form new bone and stimulated cementogenesis, as they have a powerful osteogenic potential. These results could have been superior if proper root canal therapy were carried out before or at the time of the apicoectomy (Fig. 5). lmproper seal of the apical foramen generally results in failure of root canal therapy, quite often necessitating surgical endodontic therapy. The present root canal sealing materials cause a mechanical seal of the apical foramen. These materials sometimes shrink or are partly dissolved in tissues, thus cdreating a leakage which often results in periapical infection. On the other hand, a physiologic seal can be predicted with the use of DABM grafts at the apes. Another advantage of IIABX grafts is that these grafts are soft, can be shaped in any form, and thus can be used in any area without much difficulty. In our other experiments we arc implanting DABM grafts in rootcanal-trcatcd teeth and will report the results in a future publication.

Volume Number

Experimental

35 1

We are indebted to Mr. Arthur help in preparation of the histologic

Bloom, Chief specimens.

of the Oral Pathology

osteogenesis

Laboratory,

143

for his

REFERENCES

Improved Healing of Experimental Defects in the Canine R., and others: Mandible bv Grafts of Decalcified Allogeneic Bone, ORAL SURG. 30: 142. 1970. Grafts of -Decalcified’ Allogeneic Bone Matrix Promote the Narang, RI, and others: Healing of Fibular Fracture Gaps in Rats Clin. Orthop. 80: 174, 1971. Narang, R., and Wells, H.: Decalcified Allogeneic Bone Enhances the Successful Maintenance of Implanted Acrylic Teeth, IADR Abst. 259, March, 1971. Bone Matrix Implantation in Bone Defects, Narang, R., and Wells, H.: Decalcified Trans. Fourth Int. Conf. Oral Surg., Amsterdam, May, 1971. Narang, R., and Wells, H.: Stimulation of New Bone Formation on Intact Bones by Decalcified Allogeneic Bone Matrix, ORAL SURG. 32: 668, 1971. Narang, R., and Wells, H.: Bone Induction in Experimental Periodontal Bony Defects in Dogs With Decalcified Allogeneic Bone Matrix Grafts, ORAL SURG. 33: 306, 1972. Urist, M. R.: Bone Formation by Auto Induction, Science 150: 893, 1965. Urist, M. R., and others: The Bone Induction Principle, Clin. Orthop. 63: 243, 1967. Urist, M. R., and others: Inductive Substrates for Bone Formation, Clin. Orthop. 53: 59, 1968. Urist, M. R., and others: Osteogenic Competence, Clin. Orthop. 64: 194, 1969. Van de Putte, K. A., and Urist, M. R.: Experimental Mineralization of Collagen Sponge and Decalcified Bone, Clin. Orthop. 40: 48, 1965. Van de Putte, K. A., and Urist, M. R.: Osteogenesis in the Interior of Intramuscular Implants of Decalcified Bone Matrix, Clin. Orthop. 43: 257, 1965. Yeomans, J. D., and Urist, M. R.: Bone Induction by Decalcified Dentine Implanted Into Oral, Osseous and Muscle Tissue, Arch. Oral Biol. 12: 999, 1967.

1. Narang,

2. 3. 4. 5. 6. i: 9. 10. 11. 12. 13.

Reprint requests to : Dr. Ramesh Narang and Dr. Herbert Wells Boston University School of Graduate Dentistry 100 East Newton St. Boston, Mass. 02118