Decalcified, Lyophilized Bone Allografts for Use in Human Periodontal Defects

MATERIALS

AND

METHODS

The three case histories presented here were of two male patients and one female undergoing periodontal treatment at New Y o r k University College of Dentistry. These patients had individual teeth demonstrating severe periodontal involvement, with a poor prognosis. Each patient was required to sign the informed consent form prepared by the Human Research Committee at New Y o r k University. A l l patients received thorough initial preparation consisting of home care instruction, scaling, occlusal equilibration, splinting (where indicated), soft tissue currettage, and root planing prior to the experimental procedure.

by BARRY M . LIBIN, B.A., D.D.S., M.S.D.* HOWARD L . WARD, B.A., D.D.S., M . A . | LOUIS FISHMAN, A.B., M.S., p h . D . J R E C E N T E M P H A S I S O N regeneration of periodontal osse­ ous defects has led to extensive evaluation of bone allograft transplantation techniques. Urist, using both animals and humans, showed consistently reproducible regenerative results in over 90% of cases following implantation of decalcified, lyophilized dentine and bone matrix. Register, Scopp, Kassouny, Pfau and Peskin have reviewed Urist's findings and presented two cases of successful bone induction in the human gingiva utilizing a substrate prepared from allogenic dentin.

Presurgical Records Presurgical records were obtained by an independent examiner. Pocket depths were measured with the Simring probe, utilizing either the cementoenamel junction or the gingival margin of a restoration as a fixed point from which measurements were taken. Positions and topogra­ phy of osseous levels were evaluated by clinical probing during the surgical procedure and again postoperatively by reentry procedures and by "sounding" procedures through the intact soft tissue attachment to the bony crest. Osseous levels were also evaluated by the roentgenographic technique described by Patur and Glickman. Further, clinical photographs were taken of all lesions pre and postoperatively as well as during the surgical procedure.

1

2

3

Narang, Reuben, Harris and Wells placed decalcified allogenic bone into surgical defects of canine mandibles. Histologic study of their specimens taken from two to eight weeks after implant placement showed that the graft underwent complete resorption. Bone formation appeared to take place more rapidly than with autolo­ gous grafts. In a second publication, Narang and Wells utilized decalcified allogenic bone to add height to edentulous ridges, and width to buccal plates in dogs. Their results indicated that "within the experimental period (18 weeks), grafts of decalcified allogenic bone matrix are not rejected, and that they induce the formation of new bone at the site of their placement." To date there has been no attempt to generate such an induction system within human periodontal osseous defects. The purpose of this investigation was to study bone induction following transplantation of the decalci­ fied lyophilized bone allograft into human periodontal osseous lesions. It is hoped that such an allograft can be 1) readily obtained; 2) easily stored; 3) easily shaped to fit the defect; 4) adaptable for short office procedures, and 5) accepted by the host's immunologic system.

5

1

Preparation of the Allograft (from Urist )

4

Bone samples from human sternums (cancellous bone) and humeri (cortical bone) obtained from accident victims were excised under aseptic conditions at autopsy. A l l bone samples were cut into 1.5 mm. lengths and decalcified, in 0.6 N H C L (1 gram of bone/100 ml. of solution) for four days at 2 ° C . The acid was removed by prolonged washing in cold distilled water. Bone was then placed into cold, 100% ethyl alcohol at - 1 8 ° C for one hour, and then put into individual plastic vials which had been sterilized by immersion for one hour in 100% ethyl alcohol. Subsequently the matrix within the vials was frozen in a mixture of dry ice and acetone ( - 5 0 ° C ) , and immediately dehydrated in vacuo using a lyophilizing apparatus and the vacuum immediately filled with sterile air. A i r sterilization was accomplished by connecting a millipore filter to the air inlet. Sterile caps were immedi­ ately placed tightly on the vials, taped, and labelled. The capsules were stored in the freezer at - 1 8 ° C , until one-half hour before their implantation.

* Department of Periodontics, New York University College of Dentistry, Brookdale Dental Center. Presently Assistant Clinical Professor, Department of Periodontics, School of Dental Medicine, State University of New York at Stony Brook, New York. f Professor and Chairman, Department of Preventive Dentistry and Community Health, Former Chairman, Department of Periodontics, New York University, College of Dentistry, Brookdale Dental Center. J Associate Professor, Department of Biochemistry, New York University College of Dentistry, Brookdale Dental Center.

Surgical Technique Minimal soft tissue was removed during the flap retraction in order to secure complete coverage of the allograft following the procedure. The site was subjected

51

J. Periodontol. January, 1975

52 Libin, Ward, Fishman to soft tissue currettage before retraction of the flap, or by removing the sulcular lining with a scissors after flap rretracion. A full thickness mucoperiosteal flap was utilized, the roots were thoroughly planed of all debris, and the chronic inflammatory tissue overlying the osse­ ous defects was completely removed. The osseous con­ tours were then charted from the cemento-enamel junc­ tion. Placement of the Cancellous Allograft When the operative site was completely prepared to receive the implant, the vial was removed from the freezer, warmed, and the core removed. The allograft was placed in sterile saline until sponge-like, and sec­ tioned, as necessary, to fill the defect site. Where ever possible the graft was wedged into place. If overfill was attempted, it was noted at this time. The flaps were replaced to allow complete coverage of the implant using 3-0 or 4-0 nylon suture, and covered with dryfoil and a periodontal dressing (Coe-Pak). The patient was placed on an antibiotic for five days following surgery. The antibiotic used most frequently was tetracycline one G m / d a y . Postoperative dressing and suture removal was carried out one week later, and a new dressing was placed for a second week. Placement of the Cortical

Allograft

The cortical allograft was prepared in the same manner as the cancellous graft. However, the cortical allograft did not become pliable when placed in sterile saline. Thus, it was necessary to section the allograft into pieces with a rongeur and place it to fill the defect. A l l other procedures utilizing the cortical allograft were identical to those described for the cancellous allograft.

SELECTED

REPORTS

Patient No. I. The patient was a 47 year old male with severe periodontal disease necessitating the extraction of the lower right canine. The canine displayed a 3+ mobility with complete loss of the lingual plate of bone to within two mm. of the apex (Fig. 1). The buccal plate was fairly well intact. The soft tissue lesion extended 15 mm. apically from the C E J on the lingual (Fig. 2A). The bony lesion measured 13 mm. from coronal crest apical­ ly, and seven mm. in width (Fig. 2B). Due to the complete abscence of lingual bone (almost horizontal bone loss) there was no bony undercut into which the graft could be placed. It was decided therefore, to attempt complete regeneration of the lingual plate by placing one piece of cancellous bone allograft matrix between the tooth and tissue (Fig. 2C). The soft tissue was replaced and interproximal sutures used to hold the graft in place. A t 17 weeks post surgically the patient was reevalu­ ated. Pocket depth at this time was two millimeters from the C E J (Fig. 2D) and a flap procedure revealed an entire lingual wall of bone extending to within three mm. of the C E J (Figs. 3 A ) . The material was slightly soft and

F I G U R E 1. Patient No. extension of lesion.

1. Preoperative

radiograph

showing

spongy when probed with an explorer tip. The middle portion of the apparent bone was removed and placed immediately in 10% formalin, in preparation for histo­ logical examination (Figs. 3 B - D ) . See Tables 1 and 2. Two year re-examination disclosed a two mm. soft tissue sulcus, and no tooth mobility. The x-ray, using a silver wire, revealed that the regenerated lingual plate had remained intact. The crest of bone measured five mm. from the C E J (Fig. 4). Histology The histological evidence presented at seventeen weeks indicates that the material was related to bone regenera­ tion. Although it has been reported that the graft will eventually resorb, it would appear that at this time in the allografts "life" it serves as a scaffold upon which the new bone is being built. Whether the new bone is being induced to differentiate from the connective tissues by the graft is not fully clear, but it is evident that adjacent to the new bone there is a vascular connective tissue bed with apparent associated osteoblast formation. Regard­ less of the interpretation, the evidence of new bone formation, in large quantities within the graft, indicates that osseous regeneration has occurred. 1

Patient No. 2. The patient was a 49 year old male with advanced periodontitis. The past medical history was

Volume 46 Number 1

Decalcified, Lyophilized

F I G U R E 2 A . Patient No. 1. Preoperative probing of lesion on lingual of the mandibular canine. Probe is completely within soft tissue defect. FIGURE apically, FIGURE FIGURE

2 B . Following flapping procedure. The extent of the osseous lesion is observed and in a mesio-distal direction. 2 C . Allograft matrix placed between soft tissue flap and tooth. 2 D . Seventeen weeks post surgically. Pocket depth two millimeters from CEJ.

F I G U R E 3 A . Patient No. I. Re-entry—seventeen weeks postsurgically. Lingual plate extend­ ing to within three mm. of CEJ. F I G U R E 3 B . Histologic section from specimen of new bone removed at seventeen weeks following graft implantation. The allograft (a) is still present. It can be distinguished by its empty lacunae. New osseous deposits (E) appear adjacent to the allograft. Large numbers of osteocytes are present in the newly formed bone. A cementing line appears present between the old and newly formed bone. The connective tissue lies adjacent to the new bone. WOx, H & E. F I G U R E 3 C . View of allograft ((A) new bone (E) and connective tissue association. lOOx, H &E. F I G U R E 3 D . High magnification of new bone and connective tissue interface. It would appear that bone matrix (F) is forming within the connective tissue stroma (T). 160x, H & E.

Allografts

53

J. Periodontol. January, 1975

54 Libin, Ward, Fishman TABLE 1

Soft Tissue Measurements

Patient No.

Type Implant

Tooth No.

Patient's Age (yrs.)

Time (wks.)

1

Cancellous

27

47

104

2

Cortical

22

49

64

3

Cortical

28

55

32

Depth to EA. from C E J Pre

Post 2

15 M 13

Change in Attachment Level

B 12 7

M 8

Pocket Depth Pre

13 M 5

B 4

2

15 B 8

M 13

4

3

Post

B 12

M 5

7

B 2 3

Key: E A — epithelial attachment; B—buccal; M—mesial.

TABLE 2

Osseous Measurements

Patient No.

Type Implant

Tooth No.

Age (yrs.)

Time (wks. graft in place)

Type of Defect

Cancellous

27

47

104

2

Cortical

22

49

64

2 wall

3

Cortical

28

55

32

1 wall

1

1 wall infrabony

Depth of Osseous Lesion from C E J (mm.) Pre

Re-entry

L 15

L 5

14 (Mesial) 7.5 (Distal)

Amt. of New Bone (mm.)

10

9 (Mesial)

5

3.5

4

Key: L—lingual.

uneventful. A cortical bone allograft grafting procedure was performed on the mandibular left canine which had an osseous defect extending almost to the apex on both the mesial and labial aspects of the tooth (Fig. 5A). The tooth exhibited a class III mobility. A summary of the pre and postoperative depths of the soft and hard tissue lesions appears in Tables 1 and 2. Sixteen months postoperatively the area was re-entered. Labial pocket depth now measured one millimeter (Fig. 5B). Tooth mobility was now minimal. The preoperative osseous level, 14 mm. apical to the C - E junction now measured nine mm. indicating there had been five mm. of new bone growth (Figs. 6 A - B ) . Patient No. 3. The patient, a 55 year old female, had an isolated 7.5 mm. combination one wall-no wall osseous lesion on the distal of the mandibular right first premolar. The patient was in good medical health. A n allograft of cortical bone was utilized to fill the defect. The area was re-entered eight months postoperatively. Examination revealed four mm. of bone regeneration within the combination defect (Figs. 7 A - B ) . DISCUSSION

The use of the decalcified, lyophilized bone allograft as presented here is based upon work by Urist. He suggests that the implanted prepared bone matrix is capable of producing new bone formation by emiting a bone inducing principle (B.I.P.). Extensive research has led him to the conclusion that this B . L P . largely involves the insoluble, three-dimen­ sional cross-linked collagenous structure of the bone 1,

F I G U R E 4. Patient No. 1. Two year postsurgically. Silver point to within 6 mm of CEJ.

6,

1 3

Volume 46 Number 1

Decalcified, Lyophilized

F I G U R E 5 A . Patient No. 2. Preoperative probing of lesion on labial of the mandibular canine. Probe is completely within soft tissue defect. F I G U R E 5B. Sixteen months postsurgically. The pocket has been eliminated. (Five mm. recession).

F I G U R E 6 A . Patient No. 2. Preoperative radiographic measurement. F I G U R E 6B. Sixteen months postsurgically.

F I G U R E 7 A . Patient No. 3. Preoperative radiograph. Note relationship of gold post and bone level. F I G U R E 7B. Eight months postoperatively. Bone level now appears at level of gold post.

Allografts

55

56

J. Periodontol. January, 1975

Libin, Ward, Fishman

matrix, which is able to emit a signal for mesenchymal cells to secrete an osteogenic substrate. In 1965, U r i s t first utilized this system in humans by treating various bone defects in 21 patients. Assessing the results by radiographic and histological methods he tabulated osseous regeneration in 90% of his cases. The observations made following the implantation of the allografts into the patients reported on here indi­ cated that: 1. The grafting material was easily obtained. Fresh human autopsy material from accident cases at city hospitals was readily available. For example, a section of sternum, five inches long, provided enough material for twelve large grafts. 2. Storage of the material posed no difficulty. Sam­ ples to be used immediately (within 48 hours) were kept at temperatures of - 5 ° C . Graft cores not to be utilized until a later date were kept in their individual vials and frozen at - 1 8 ° C . Defrosting took less than 30 minutes. Strates and Urist, have reported, however, that after two months there was some loss in the osteogenic inductance capacity of the material. 3. The material was easily shaped to fit defects. In this respect the cancellous material was ideal. Following removal from the vial, the defrosted graft is hard and not easily cut. When placed in sterile saline it swells and becomes spongelike in consistency. A t this point the material can be cut to proper size with a scissors or blade. After it is placed in a sanguinous field it becomes so pliable that it is easily shaped to fill any osseous defect. The cortical graft was more difficult to shape, in that it did not attain the same degree of flexibility as the cancellous graft. Sharp rongeurs were utilized to cut the cores into proper shape.

kind of tissues being regenerated and the nature of their attachment.

1

6

4. The technique is suitable for short office proce­ dures. In many of today's grafting procedures, a second operative site is necessary to obtain autogenous bone. The extension into a tuberosity to obtain marrow length­ ens the operative procedure and may add to postopera­ tive discomfort. The utilization of iliac crest transplants not only involves a second site, but removes that part of the procedure from the dental office, a situation not acceptable to many dental patients. In terms of ease and time, the stored allograft appears to be a more preferable type of grafting material. 5. The grafting material appeared to be accepted immunologically by the host. Urist's experiments since 1965 have reported no foreign body reactions, and there was no apparent clinical manifestation of rejection in these patients. 6. These case reports indicate it is possible to restore hard and soft tissue attachment in areas of severe periodontal destruction. The results indicate a very positive capability for this material to regenerate osseous tissue. This research is being expanded to include more patients, and with an additional objective of determining histologically the

SUMMARY

AND

CONCLUSIONS

Three reports of patients with severe periodontal defects treated with a decalcified, lyophilized bone allo­ graft, prepared as described by Urist, have been pre­ sented. Two patients received grafts of cancellous bone and one patient received a cortical bone graft. The patients were observed for up to two years following implantation. Clinical and histological data obtained from these patients makes possible the following conclu­ sions: A . The implantation of decalcified, lyophilized bone allografts of both the cortical and cancellous types resulted in new bone formation and a gain in attachment level. B. There has been no apparent evidence of rejection of the graft material for up to two years following implanta­ tion. ACKNOWLEDGEMENTS

We gratefully acknowledge the aid of D r . Edward Tonna with the photomicrographs and Drs. Enid Neidle and S. Sigmund Stahl for their counsel and editing. REFERENCES

1. Urist, M . R.: Bone Formation by Autoinduction Science, 150:893, 1965. 2. Register, A . A . , Scopp, I. W . , Kassouny, D . Y . , Pfau, F . P. and Peskin, D.: Human Bone Induction by Allogeneic Dentin Matrix. J . Periodontal., 43:459, 1972. 3. Narang, R., Reuben, M . P., Harris, M . H . , and Wells, H . : Improved Healing of Experimental Defect in the Canine Mandible by Grafts of Decalcified Allogenic Bone. Oral Surg., 30:151, 1970. 4. Narang, R., and Wells, H . : Stimulation of New Bone Formation on Intact Bones by Decalcified Allogenic Bone Matrix. Oral Surg., 32:668, 1971. 5. Patur, B., and Glickman, I.: Clinical and Roentgenographic Evaluation of the Post Treatment Healing of Infrabony Pockets. J . Periodontol., 33:164, 1962. 6. Strates, B. S., and Urist, M . R.: Origin of the Inductive Signal in Implants of N o r m a l and Iathyritic Bone Matrix. Clin. Orthop., 66:226, 1969. 7. Urist, M . R.: Personal Communication, 1970. 8. Pappas, A . M . , and Beisaw, N . E . : Bone Transplantation: Correlation of Physical and Histologic Aspects of Graft Incorporation. Clin. Orthop., 61:79, 1968. 9. H a m , A . W . : Histology, 6 ed., Philadelphia, J . B. Lippincott C o . , 1969. 10. Scopp, I. W . Morgan, F. H . , Dooner, J . J . , Fredrics, H . J . , and Heyman, R. A . : Bone (Boplant) Implants for Infrabony Oral Lesions. J . Periodontol., 4:169, 1966. 11. Schallhorn, R. G , Hiatt, W . H . , and Boyce, W . : Iliac Transplants in Periodontal Therapy. J . Periodontol., 41:566, 1970. 12. Ramfjord, S. P., and Costich, E . R.: Healing After Simple Gingivectomy. J . Periodontol., 34:401, 1963. 13. Urist, M . R., Jurist, J . M . , Dubuc, F . L . , and Strates, B. S.: Quantitation of New Bone Formation in Intramuscular Implants of Bone Matrix in Rabbits. Clin. Orthop., 55:279, 1967. 250 Patchogue-Yaphank R d . East Patchogue, New Y o r k 11772

Decalcified, lyophilized bone allografts for use in human periodontal defects.

Decalcified, Lyophilized Bone Allografts for Use in Human Periodontal Defects MATERIALS AND METHODS The three case histories presented here were o...
2MB Sizes 0 Downloads 0 Views