Decalcified, Lyophilized Bone
The three case histories presented here were of two
Allografts for Use in Human
Periodontal Defects
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
1
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
2 which measurements were taken. Positions and topogra­
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.
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.
4
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.
* 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.
51
MATERIALS AND METHODS
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.
Presurgical Records
Presurgical records were obtained by an independent
examiner. Pocket depths were measured with the Simr-
ing probe, utilizing either the cementoenamel junction or
the gingival margin of a restoration as a fixed point from
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 roent-
genographic technique described by Patur and
5
Glickman. Further, clinical photographs were taken of
all lesions pre and postoperatively as well as during the
surgical procedure.
Preparation of the Allograft (from Urist ) 1
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.
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
 J. Periodontol.
52 Libin, Ward, Fishman January, 1975

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. 1. Preoperative radiograph showing
extension of lesion.
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
1
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.
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 Allografts 53

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 2 B . Following flapping procedure. The extent of the osseous lesion is observed
apically, and in a mesio-distal direction.
FIGURE 2 C . Allograft matrix placed between soft tissue flap and tooth.
FIGURE 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.
 J. Periodontol.
54 Libin, Ward, Fishman January, 1975

TABLE 1

Soft Tissue Measurements

Patient's Depth to EA. from C E J Change in Pocket Depth

Patient Type Tooth Time
Age Attachment
No. Implant No. (wks.)
(yrs.) Pre Post Level Pre Post

1 Cancellous 27 47 104 15 2 13 15 2
2 Cortical 22 49 64 M B M B M B M B M B
13 12 8 4 5 8 13 12 5 2
3 Cortical 28 55 32 7 3 4 7 3

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

TABLE 2

Osseous Measurements

Time Depth of Osseous Lesion from C E J

Patient Type Tooth Age (wks. Type of (mm.) Amt. of New
No. Implant No. (yrs.) graft in Defect Bone (mm.)
place) Pre Re-entry

1 Cancellous 27 47 104 1 wall infra- L L 10

bony 15 5
2 Cortical 22 49 64 2 wall 14 (Mesial) 9 (Mesial) 5
3 Cortical 28 55 32 1 wall 7.5 (Distal) 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

F I G U R E 4. Patient No. 1. Two year postsurgically. Silver point
to within 6 mm of CEJ.
1, 6, 1 3
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
Volume 46
Number 1 Decalcified, Lyophilized Allografts 55

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.

56 Libin, Ward, Fishman
matrix, which is able to emit a signal for mesenchymal
cells to secrete an osteogenic substrate.
1
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.
6
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.
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
J. Periodontol.
January, 1975
kind of tissues being regenerated and the nature of their
attachment.
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 Roentgeno-
graphic 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