Periodonlology 2000, Viol.
19, 1999, 74-86
Pririted in Denmark All rights reserved
Bone and bone substitutes
HISHAMF. NASR,MARYELIZABETH
AICHELMANN-REIDY
& RAYMOND A. YUKNA
The ultimate goal in periodontal therapy is creation
of an environment that is conducive to maintaining
the patient’s dentition in health, comfort, and func-
tion. The shift in therapeutic concepts from resec-
tion to regeneration has significantly impacted the
practice of periodontology in the last quarter of this
century. The objective of this text is to provide an
overview of bone and bone substitutes intended for
periodontal reconstructive therapy and their biologi-
cal potential.
The role of bone replacement
grafts
Interest in bone replacement grafts has emerged
from the desire to “fill” an intrabony or furcation de-
fect rather than radically resect surrounding intact
bone tissue. Albeit true or false, the assumption that
their application would potentially manipulate the
biological response into a regenerative rather than a
predominantly reparative pattern has rendered the
Fig. 1. Samples of commercially available bone replace-
ment grafts (the samples do not represent any particular
preference by the authors)
74
C o p y r i g h t 0 M u n k s g a a r d 1999
PERIODONTOLOGY 2000
I S S N 0906-6713
use of bone replacement grafts an attractive choice
in certain periodontal defect configurations.
Increasing demand and interest in market share
stimulated tissue banks and manufacturers to claim
superiority of one product over another (Fig. 1).Due
to the variable physical and chemical nature among
bone replacement grafts (Table 11, the goal of repro-
duction or reconstitution of lost periodontal struc-
ture (alveolar bone, periodontal ligament and ce-
mentum) has been met with varying success or with
failure. Historically, autogenous and allogeneic bone
have been used with some success (11, 23, 44, 57).
Several other bone replacement grafts have been de-
veloped for use in periodontal therapy to support
bone formation and defect fill. These latter ma-
terials, synthetically derived (alloplastic or synthetic)
or processed from skeletal structures of other species
(xenogeneic), are biocompatible and non-organic.
Their purpose is to substitute for autogenous bone.
Oftentimes sufficient autogenous bone is unavail-
able, or harvesting would require additional surgical
sites, increasing patient morbidity and clinician
chair time. Allogeneic bone obtained from a different
person and commonly processed by tissue banks
provides an alternative to autogenous bone, but the
fear of disease transmission persists despite evi-
dence to the contrary (15, 62). This concern has
driven the market to produce biocompatible alterna-
tives to allograft bone. Bone substitutes have filled
this niche in periodontal practice, providing defect
fillers of variable biological activity. Plaster of Paris
(81) and Boplant (bovine derived) (80) represent
some of the early non-human bone replacement
grafts. Both are associated with inherent difficulties
and minimal clinical success. The focus began to
shift to calcium phosphate ceramics in the 1970s
(69). With the exploration of bioceramics; biocom-
patible, generally inert materials became available.
Clinical success has been reported in terms of defect
bone fill, but with little evidence of periodontal re-
generation.

Table 1. Bone replacement grafts
Human bone
Autogenous grafts (autografts)
Extraoral
Intraoral
Allogeneic grafts (allografts)
Fresh frozen bone
Freeze-dried bone all
Demineralized freeze
Bone substitutes
Xenogeneic grafts (xenografts)
Bovine-derived hydroxyapatite
Coralline calcium carbonate
Alloplastic grafts (alloplasts)
Polymers
Bioceramics
Tricalcium phosphate
Hydroxyapatite
dense, nonporous, nonresorbable
porous, nonresorbable (xenograft)
resorbable hydroxyapatite derived at
low temperature
Bioactive glasses
Rationale and objectives of
bone replacement grafts
Periodontal therapy is directed not only at inflam-
mation control but also at pocket reduction and cor-
rection of associated bone defects. Moderate to se-
vere periodontal defects are often not amenable to
osseous resection without further compromising the
support of the involved and adjacent teeth. When
applicable, regeneration of the lost bone and peri-
odontal attachment improves the support of the
tooth and hopefully its long-term prognosis. Bone
replacement grafts have been used to achieve this
end. Bone replacement grafts have been shown to
produce greater clinical bone defect fill than flap de-
bridement alone (11, 23, 44, 57). Histological reports
have confirmed their ability to support new attach-
ment in the apical portion of periodontal defects (18,
23, 68). Complete restitution is not likely; however,
with bone replacement grafts some regeneration or
new attachment is more likely to occur (12).
The ideal bone replacement graft should be able
to trigger osteogenesis, cementogenesis and forma-
tion of a functional periodontal ligament. Osteogen-
esis, the formation of mineralized bone by trans-
planted osteoblasts, is only achieved with autogen-
ous grafts (76). Cellular elements or progenitor cells
of the autogenous graft have to be present for this to
occur. Other types of bone replacement grafts do not
provide any cellular elements. The best case scenario
for these bone replacement grafts would be osteoin-
Bone and bone substitutes
duction, which is the stimulation of phenotypic con-
version of progenitor cells within the healing wound
to those that can form osseous tissue. Currently, de-
mineralized freeze-dried human bone and bone
morphogenetic proteins may fulfill this role (95, 96).
Most bone replacement grafts are osteoconductive,
relatively inert filling materials, and integrate with
new bone. Osteoconductive materials provide a scaf-
fold to allow bone ingrowth and deposition and may
support significant improvement in clinical probing
depth and attachment levels. Histologically, these
bone replacement grafts have produced limited re-
generation.
History of bone replacement grafts
Historically, autogenous bone grafts were the first
bone replacement grafts to be reported for peri-
odontal applications. Autogenous bone from extra-
oral and intraoral sources was the focus of initial
attention, being an accessible and desirable graft.
Allogeneic freeze-dried bone was introduced to peri-
odontics in the early 1970s, while demineralized
allogeneic freeze-dried bone gained wider appli-
cation in the late 1980s with an even increasing mar-
ket share in the 1990s. The introduction of xeno-
geneic and alloplastic bone replacement grafts for
periodontal use occurred during the same time.
Regenerative therapy with bone replacement
grafts did not gain acceptance as predictable therapy
until the 1980s. Controversy still remains as to
whether new attachment (regeneration) at a coronal
level is achieved or whether primarily a long junc-
tional epithelium (repair) occurs as found with flap
debridement surgery alone. Currently, the ability of
bone replacement grafts to bond to bone and to be-
come slowly replaced with host bone is being exam-
ined. Hence, resorbability and the ability of a graft
material to enhance osteoconduction are the im-
petus behind the development of bone replacement
grafts available today.
Autogenous grafts (autografts)
The use of extraoral autogenous bone grafts has
been scarcely reported in the periodontal literature.
Iliac cancellous bone and marrow were of initial in-
terest due to a high potential for true osteogenesis
(20, 87). Despite a relatively low frequency of root
resorption associated with the use of fresh iliac grafts
(45), this observation has limited their clinical use.
75
 Since periodontal regenerative procedures gener-
ally are conducted in an outpatient environment, in-
traoral autogenous bone grafts are utilized widely
and have been shown to produce favorable defect
fill (31). Common donor sources are the maxillary
tuberosity and other edentulous alveolar areas, in-
cluding healing extraction sockets (33),and osseous
coagulum harvested from osteoplasty procedures at
the surgical site. Other donor sites for cortical and/
or cancellous bone include mental and mandibular
retromolar areas. While autogenous bone is still the
standard, the relatively limited amount of con-
veniently available autogenous bone and the harvest
time involved in obtaining these grafts have led clini-
cians to utilize other bone replacement grafts. Often-
times whatever amount of autogenous bone that can
be obtained is used in combination with other bone
replacement grafts (75).
AUogeneic grafts (allografts)
Frozen iliac cancellous bone and marrow
The possibility of disease transfer, antigenicity and
the need for extensive cross-matching has precluded
the use of frozen iliac allografts in modern peri-
odontics.
Mineralized freeze-dried bone allografts
The evidence that freeze-drying markedly reduces
the health risks associated with fresh frozen bone
(27),as well as the favorable results obtained in field
trials with freeze-dried bone allografts (581, have led
to the extensive use of freeze-dried bone allografts
in the treatment of periodontal osseous defects. Cor-
tical bone allografts preserved by freeze-drying do
not appear to elicit an immune response in nonhu-
man primates (94). Freeze-drying may partially dis-
tort the three-dimensional presentation of human
leukocyte antigens on freeze-dried bone allografts,
affecting immune recognition (28, 71). The American
Academy of Periodontology recommends use of cor-
tical rather than cancellous bone allografts since
cancellous bone is more antigenic and there is more
bone matrix and consequently more inductive com-
ponents in cortical bone (2). Moreover, experimental
studies have shown that addition of autogenous
bone to freeze-dried bone allografts enhances its
osteogenic potential (61). Freeze-dried bone allograft
is regarded as osteoconductive (2, 36).
76
Demineralized freeze-dried bone allografts
Demineralization of allografts was performed be-
cause the bone mineral blocked the effect of the fac-
tors stimulating bone growth sequestered in bone
matrix including bone morphogenetic proteins (97).
Bone morphogenetic proteins are a group of acidic
polypeptides belonging to the transforming growth
factor-p gene super-family. They stimulate bone for-
mation through osteoinduction by inducing pleuri-
potential stem cells to differentiate into osteoblasts
(38, 98). Experimental animal studies have shown
that demineralized freeze-dried bone allograft has
osteogenic potential (59, 60). Further experimental
studies have indicated that the addition of autogen-
ous bone to demineralized freeze-dried bone allo-
graft does not dramatically enhance its osteogenic
potential (61). The bioactivity of demineralized
freeze-dried bone allograft appears to be age de-
pendent. Bone from younger animals has been
shown to have an increased osteogenic potential
compared with bone from older animals (22, 48, 91).
A major concern with allografts in general is the
potential for disease transfer, particularly viral trans-
mission, and even more particularly HIV. Tissue
banks have adopted rigorous exclusionary tech-
niques, testing for HIV antigen, and HIV antibody
and lymph node biopsy in order to reduce this po-
tential risk. Additionally, mere freezing of bone allo-
grafts reduces the risk of disease transfer to 1 in 8
million (15). Treatment of cadaveric bone spiked
with viral particles and cortical bone procured from
a donor who had died of AIDS with a viricidal agent
and demineralization in HC1 has been found to inac-
tivate HIV in both cases (62). The probability of HIV
transfer following appropriate demineralized freeze-
dried bone allograft preparation has been calculated
to be 1 in 2.8 billion (74).To date there is no reported
case of disease transmission from freeze-dried bone
allografts used for dental purposes in over 1 million
cases over 25 years of use.
A rare disease entity, Creutzfeldt-Jacobs disease,
transmitted via preons (smallest proteinaceous in-
fectious particles) has also never been reported from
a bone-derived material. However, due to difficult
exclusionary technicalities, Creutzfeldt-Jacobs dis-
ease may remain a remote potential risk.
The availability of bone morphogenetic proteins
in demineralized freeze-dried bone allograft has
raised the issue of whether use of demineralized
freeze-dried bone allograft over other bone replace-
ment grafts is advantageous in periodontal therapy
(8) and of additional value when combined with

guided tissue regeneration (34). Schwartz et al. have
found that while demineralized freeze-dried bone
allograft samples from some tissue banks showed os-
teoinductive potential in mice, samples from other
tissue banks did not (84).At this time, demineralized
freeze-dried bone allograft remains the only bone re-
placement graft proven to result in periodontal re-
generation in a controlled human histological study
(11) and is recognized in the consensus report by
the 1996 World Workshop in Periodontics to fulfill
all criteria considered for promotion of periodontal
regeneration (3).
Xenogeneic grafts (xenografts)
Xenografts are grafts shared between different spe-
cies. Currently, there are two available sources of
xenografts used as bone replacement grafts in peri-
odontics; bovine bone and natural coral. Both
sources, through different processing techniques,
provide products which are biocompatible and
structurally similar to human bone. Xenografts are
osteoconductive, readily available and risk free of
disease transmission. The latter point has been
questioned with the discovery of bovine spongiform
encephalopathy, particularly in Great Britain. Ma-
terials marketed in the United States certify that they
originate in the United States as bovine-derived
bone material from animals approved by the US De-
partment of Agriculture. Since United States cattle
have remained unaffected, the risk of disease trans-
mission should be nonexistent with materials pro-
duced in the United States.
Bovine derived bone replacement grafts
Commercially available bovine bone is processed to
yield natural bone mineral minus the organic com-
ponent. A purported advantage of these products is
that they provide structural components similar to
that of human bone, with improved osteoconductive
capability compared with synthetically derived ma-
terials. Anorganic bovine bone is the hydroxylapatite
“skeleton” that retains the macroporous and micro-
porous structure of cortical and cancellous bone (47)
remaining after chemical or low-heat extraction of
the organic component. Historically, bovine xeno-
grafts have failed due to rejection (561, probably be-
cause earlier materials used chemical detergent ex-
traction, which left residual protein and therefore
produced adverse reactions and clinically unaccept-
able results (25).
Bone and bone substitutes
Currently available bovine-derived hydroxyapatite
is deproteinated, retaining its natural microporous
structure, which supports cell-mediated resorption
(47). This becomes important if the product is to be
replaced with new bone. Two products are currently
available, Osteograf/N (CeraMed Dental, LLC, Lake-
wood, CO) and Bio-OsP (Osteohealth Co., Shirley,
NY).Both have been reported to have good tissue
acceptance with natural osteotrophic properties (17,
19). Histologically, no fibrous tissue or space be-
tween the hydroxyapatite and newly formed bone is
found (17).
In summary, bovine-derived hydroxypatite bone
replacement grafts increase the available surface
area that can act as an osteoconductive scaffold due
to their porosity and have a mineral content com-
parable to that of human bone, allowing them to in-
tegrate with host bone. They have been used with
success for the treatment of intrabony defects and
ridge augmentation (14).
Coralline calcium carbonate
Biocoral (Inoteb, Saint Gonnery, France) is calcium
carbonate obtained from a natural coral, genus Por-
ites, and is composed primarily of aragonite (>98%
CaCO,). Its pore size of 100 to 200 pm is similar to
the porosity of spongy bone (37). Its porosity, at
greater than 45%, provides a large surface area for
resorption and replacement by bone (104). Unlike
porous hydroxyapatite, derived from the same coral
by heat conversion and made non-resorbable, cal-
cium carbonate is resorbable. It does not require a
surface transformation into a carbonate phase as do
other bone replacement grafts to initiate bone for-
mation (37); hence, it should more rapidly initiate
bone formation. Biocoral has a high osteoconductive
potential because no fibrous encapsulation has been
reported (70). Coralline calcium carbonate produces
comparable results to other bone replacement grafts
with significant gain in clinical attachment, reduc-
tion of probing depth and defect fill (35, 51, 64).
Alloplastic (synthetic) grafts
(alloplasts)
The 1996 World Workshop in Periodontics has con-
cluded that “synthetic graft materials function pri-
marily as defect fillers. If regeneration is the desired
treatment outcome, other materials are rec-
ommended” (3).
77
Nasr et al.
Polymers: HTR polymer
HTR"M Synthetic Bone (Bioplant, Norwalk, CT) is a
biocompatible microporous composite of poly-
methylmethacrylate, polyhydroxylethylmethacrylate
and calcium hydroxide. Favorable clinical results
have been achieved with HTRTM (the acronym
stands for hard tissue replacement) in the treatment
of intrabony and furcation defects (102, 105). How-
ever, improved clinical results with this bone re-
placement graft have not always been achieved (82).
Histologically, new bone growth has been found de-
posited on HTRTMparticles (29, 89, 106). Its hydro-
philicity enhances clotting, and its negative particle
surface charge allows adherence to bone. It appears
to serve as a scaffold for bone formation when in
close contact with alveolar bone. Clinical defect fill
and resolution can be achieved supporting its use as
a biocompatible alloplastic bone substitute (15, 67).
Bioceramics
Bioceramic alloplasts are comprised primarily of cal-
cium phosphate, with the proportion of calcium and
phosphate similar to bone. The two most widely
used forms are tricalcium phosphate and hydroxy-
apatite.
Tricalcium phosphate
Tricalcium phosphate is a porous form of calcium
phosphate, the most commonly used form of which
is p-tricalcium phosphate. It serves as a biological
filler which is partially resorbable and allows bone
replacement. Conversion of the graft is pivotal to
periodontal regeneration; first, serving as a scaffold
for bone formation, and then permitting replace-
ment with bone (39, 83).
Tricalcium phosphate as a bone substitute has
gained clinical acceptance, but the results are not al-
ways predictable. In direct comparison with allogen-
eic cancellous grafts, the allogeneic grafts appear to
outperform tricalcium phosphate (90). The tricalci-
um phosphate particles generally become encapsu-
lated by fibrous connective tissue and do not stimu-
late bone growth (4, 6). However, some bone depo-
sition has been reported with tricalcium phosphate
grafts (4, 6, 13, 88, 90).
Hydroxyapatite
Hydroxyapatite, Calo (PO4I6 (OH12, is the primary
mineral component of bone. Synthetic hydroxyapa-
78
tites have been marketed in a variety of forms, pri-
marily as a porous nonresorbable, a dense or solid
nonresorbable, and a resorbable (non-ceramic, po-
rous) form. Processing of the basic calcium phos-
phate mixture dictates which of the listed properties
it will possess. Hydroxyapatite resorbability is deter-
mined by the temperature at which it is processed.
Resorbability is desirable if the graft is eventually to
be replaced by the host bone.
When prepared at high temperature (sintered), hy-
droxyapatite is nonresorbable, nonporous, dense,
and has a larger crystal size (53). Dense hydroxyapa-
tite grafts are osteophillic, osteoconductive and act
primarily as inert biocompatible fillers. They have
produced clinical defect fill greater than flap de-
bridement alone in the treatment of intrabony de-
fects (55, 72). Histologically, new attachment is not
achieved (30). They yield similar defect fill as other
bone replacement grafts and the clinical improve-
ment is more stable than with debridement alone.
Yukna et al. demonstrated over a five-year period
that open flap debridement was not stable and re-
gressed three to five times faster than sites having
received hydroxyapatite (108).
Porous hydroxyapatite (Interpore 200, Irvine, CA)
is obtained by the hydrothermal conversion of the
calcium carbonate exoskeleton of the natural coral
genus Porites into the calcium phosphate hydroxy-
apatite. It has a pore size of 190 to 200 pm, which
allows bone ingrowth (63, 100) into the pores and
ultimately within the lesion itself (50). Clinical defect
fill, probing depth reduction, and attachment gain
have been reported (49). As with dense hydroxyapa-
tite, any regeneration appears limited to the apical
aspect of the defect. Kenney et al. provided histologi-
cal evidence suggesting that porous hydroxyapatite
supports bone formation, but since no evidence of a
new connective tissue attachment or cementum was
noted, it should be considered a biocompatible fill-
ing material (50).
Another form of synthetic hydroxyapatite is a re-
sorbable, particulate material processed at a low
temperature (OsteoGen, Impladent, Holliswood, NY;
OsteoGraf LD, CeraMed Dental, LLC, Lakewood,
CO). This resorbable form is a non-sintered (non-
ceramic) precipitate with particles measuring 300 to
400 pm. It has been proposed that non-sintered hy-
droxyapatite resorbs acting as a mineral reservoir in-
ducing bone formation via osteoconductive mech-
anisms (73, 99). Its reported advantage is the slow
resorption rate, allowing it to act as a mineral reser-
voir at the same time acting as a scaffold for bone
replacement (73, 99).

Combinations of the two primary forms of cal-
cium phosphate have been studied to take advan-
tage of the rapid resorption of p-tricalcium phos-
phate and the inert scaffold of dense hydroxyapatite.
In a histological study, Hashimoto-Uoshima et al. re-
ported that biphasic calcium phosphate supported
active bone replacement from surrounding bone
which may have been triggered by macrophages
(39). However, further studies are needed before
clinical acceptance.
Generally, hydroxyapatite is not osteoinductive,
but osteoconductive. A few studies demonstrate os-
teoinductive activity associated with porous hy-
droxyapatite (43, 101). Takata et al. provided evi-
dence that cementum could be deposited on porous
hydroxyapatite in uitro (92). However, this report
does not claim cementum induction, because the
study included periodontal ligament cells which may
inherently differentiate into cementoblasts.
In summary, there are three available forms of hy-
droxyapatite, a solid particulate, nonresorbable
form; a porous nonresorbable form derived from the
exoskeleton of coral; and a resorbable non-ceramic
hydroxyapatite. Hydroxyapatite is not osteogenic nor
osteoinductive, but rather is osteophillic and osteo-
conductive. It acts as a trellis for the ingrowth and
subsequent deposition of new bone.
Bioactive glasses
There are two forms of bioactive glass currently
available. PerioGlas@(Block Drug Co., Jersey City, NJ)
and BiogranTM(Orthovita, Malvern, PA). Bioactive
glasses are composed of CaO, Na,O, SiO,, P205 and
bond to bone through the development of a surface
layer of carbonated hydroxyapatite (40, 41). When
exposed to tissue fluids, bioactive glasses are covered
by a double layer composed of silica gel and a cal-
cium-phosphorous rich (apatite) layer. The calcium
phosphate-rich layer promotes adsorption and con-
centration of proteins utilized by osteoblasts to form
a mineralized extracellular matrix (24). It has been
theorized that these bioactive properties guide and
promote osteogenesis (50, 51), allowing rapid forma-
tion of bone (42).
PerioGlaP has a particle size ranging from 90 to
710 pm, which facilitates manageability and packing
into osseous defects. In surgically created defects in
nonhuman primates, 68% defect repair was achieved
as new attachment (26). Compared to tricalcium
phosphate, hydroxyapatite and unimplanted con-
trols, Fetner et al. showed that PerioGlasa produced
significantly greater bone and cementum repair (26).
Bone and bone substitutes
Observations of the material suggest good manage-
ability, hemostatic properties, and the possibility
that PerioGlas@not only is osteoconductive, but may
also act as a barrier retarding epithelial downgrowth.
BiogranTMhas a narrower range of particle sizes
of the purportedly critical 300 to 355 pm size range
which has been reported to be advantageous for
guiding osteogenesis (78). Formation of hollow cal-
cium phosphate growth chambers occurs with this
particle size because phagocytosing cells can pen-
etrate the outer silica gel layer by means of small
cracks in the calcium-phosphorous layer and par-
tially resorb the gel. This resorption leads to the for-
mation of protective pouches where osteoprogenitor
cells can adhere, differentiate and proliferate. Ac-
cording to the manufacturer, larger particles do not
resorb in the same manner which slows the healing
process theoretically because bone healing must
progress from the bony walls of the defect (79); and
smaller particles cause a transient inflammatory re-
sponse, which retards the stimulation of osteopro-
genitor cells.
It is argued that the more uniform sized BiogranT"
would have a clinical advantage over the PerioGlas@
preparation, which has multiple particle sizes. Clin-
ically, no comparison has been made between the
products, and no human periodontal studies are
available. Schepers et al. have demonstrated that Bi-
ogranTMcan be used successfully in the treatment of
human oral osseous defects (79).
Morphological and biological
implications
The morphology of bone replacement grafts has
been postulated to contribute to their osteoconduc-
tive capacity mainly due to the influence of particle
size and shape on the resorption and replacement
phenomenon, as well as the influence of interpar-
ticulate space on infiltration of vascular cellular ele-
ments and bone formation. It would seem rational
to suggest that particles too large in size will resorb
at a slower rate and offer an overall reduced surface
area, while particles too small in size may induce
inflammation, be readily resorbed or phagocytosed
and result in an interparticulate space of a reduced
dimension that would not be conducive to cellular
migration and ingrowth. Trabeculae of bone vary in
size from 20 to over 100 pm. When a trabecula
reaches about 100 pm, it carries its own blood ves-
sels, much in the same way an osteon does via the
79
Nasr et al.
Fig. 2. Scanning electron micrograph of a human peri-
odontal ligament fibroblast cultured on freeze-dried bone
allografts for four hours. Cells are well spread and display
numerous processes ( ~ 7 5 0 )Micrograph
. courtesy of R. L.
Moses.
Haversian canal. Compact bone has Haversian sys-
tems or osteons of between 50 to 250 pm. Thus, to
support trabecular bone ingrowth, the pores would
need to be at least 40 to 100 pm, and to support
osteonal bone ingrowth, pores of at least 100 pm
would appear necessary (52). Particle sizes of about
380 pm in diameter would yield this minimal dimen-
sion (46).To date, little or no histological evidence is
available to support this claim.
In a small clinical study, Fucini et al. found that
there was no difference in defect fill between demin-
eralized freeze-dried bone allograft particles of 250
to 500 pm size compared to those of 850 to 1000 pm
(32). In uitro analysis of the interparticulate space
among bone replacement grafts condensed under a
uniform standard force showed that autogenous
bone harvested by low- and high-speed rotary in-
struments, freeze-dried bone allograft (250-7 10 pm),
Bio-Oss@ (cancellous and cortical), Osteograf LD,
PerioGlasa and Osteogen yielded a 40 to 100 pm in-
terparticulate space. Autogenous bone harvested by
back action chisels, demineralized freeze-dried bone
allograft (350-500 pm), Osteograf N (300 and 700
pm), Biocoral, BiogranTM,Interpore 200 and Calcitite
(40-60 pm) hydroxyapatite granules yielded an inter-
particulate space that was equal to or greater than
100 pm (54). Thus, evaluated bone replacement
grafts seem to yield an interparticulate space large
enough for osteoid cells to migrate and for bone to
form. Hypothetically, particles yielding under 100
pm space dimensions may possess less mineraliza-
tion potential (52); however, recent animal studies
appear to challenge this assumption (9).
80
Human periodontal ligament fibroblasts were
grown on a variety of bone replacement grafts in-
cluding demineralized freeze-dried bone allograft,
freeze-dried bone allograft, coral, silicon-glass, poly-
mers, and various hydroxyapatites in order to ana-
lyze differences in cell binding and spreading as a
function of the bone replacement graft substrata.
Scanning electron microscopy observations demon-
strated that adherence dynamics varied among bone
replacement grafts, with cell spreading occurring
most rapidly on materials derived directly from bone
(Fig. 2). Cell spreading was slower on non-osseous
hydroxylapatites and other synthetic surfaces, al-
though there was considerable variability within
classes of these materials (65). Evaluation of the time
course of cell spreading indicated that periodontal
ligament fibroblasts cultured on bovine-derived
natural bone mineral with a synthetic cell-binding
peptide (P-15) attached to its surface spread as
rapidly as those seeded onto bone of human origin.
These results suggested that the addition of P-15 to
bone replacement graft surfaces may enhance bind-
ing of periodontal ligament fibroblasts during the
early stages of wound repair (66).
Current studies at Louisiana State University are
utilizing fluorescently labeled human periodontal
ligament fibroblasts in order to quantify cell binding
to different bone replacement grafts, as well as using
fluorescent techniques to assess periodontal liga-
ment fibroblasts for the presence of bone specific
cell products such as alkaline phosphatase and os-
teonectin.
Technical implications
The success rates with clinical application of bone
replacement grafts could be enhanced by appropri-
ate selection of a defect configuration that is amen-
able to grafting and by following a thorough surgical
technique. While bone replacement grafts have been
used in a combined approach with guided tissue re-
generation barriers, both to benefit from the biologi-
cal effects of each material and maintain a blood clot
space beneath membranes, bone replacement grafts
used exclusively in osseous defects may result in dis-
appointing outcomes if not appropriately nego-
tiated. The bone graft technique has been previously
outlined to include many of the basic periodontal
surgery procedures and recommendations that
should lead to success a majority of the time (103).
This section is intended to address some of the criti-
cal procedures that may enhance clinical outcomes.
 Bone and bone substitutes

Preparation of graft material
It is suggested that bone replacement grafts be wet-
ted with the patient’s own blood from the surgical
site, rather than saturated with sterile water or sa-
line, which may hinder vascular infiltration of the
saturated particles.
level of the defect walls. There is little suggestion that
overfilling with these materials results in supracres-
tal bone formation. Overfilling may actually be
counterproductive in that it may preclude proper
flap closure, thereby retarding healing and possibly
resulting in loss of the graft material.

Promotion of a bleeding surface

This is generally already accomplished by proper de-
fect debridement (Fig. 3, 4). However, if the defect
walls are relatively dry, and/or glistening, healing
may be enhanced by intramarrow penetration to en-
courage bleeding and allow the ingress of reparative
cells, vessels and other tissues. Such penetration can
be accomplished with a small round bur or hand in-
struments.

Presuturing
Fig. 3. Intrabony defect on mandibular right first molar
Loose placement of sutures, left untied, prior to the
filling of the defect reduces the possibility of dis-
placing the bone replacement graft during the sutur-
ing process. It also simplifies the last steps of the
procedure, in that once defect fill has been com-
pleted, the already placed sutures need only to be
tied to complete the surgical procedure. Vertical
mattress sutures that pass over the incision would
offer an alternative to presuturing that would not
displace the bone replacement graft.

Adequate condensation of graft material

The commercial availability of allogeneic and allo-
plastic materials eliminates the problem of not
having enough graft material. The graft material Fig*4* Lingud view Of defect
should be placed in small increments. Useful in this
regard are sterile plastic or Teflon-lined amalgam
carriers to place the material and sterile amalgam
squeeze-cloths to use over the suction tip to dry the
defect without removing any of the bone replace-
ment graft material. Small increments of material are
placed in the defect, gently packed into the angles
and base of the defect with small pluggers or cu-
rettes and dried with a squeeze-cloth covered suc-
tion tip. The process is repeated until the defect is
filled (Fig. 5).

Fill to a realistic level

Except in unusual circumstances, defects should be
filled with the bone replacement grafts only to the Fig. 5. Bone replacement graft in place

81
Nasr et al.
Achievement of tissue coverage
With adequate flap design, primary closure with re-
placed flaps and contact of the interproximal papil-
lae can usually be obtained. If tissue coverage of the
bone replacement graft is not satisfactory, additional
releasing incisions and/or flap reflection may be
necessary. Another possible treatment is the use of
an autogenous free gingival graft, freeze-dried skin
or dura mater allograft, or collagen barrier to cover
the bone graft site. Modification of incision designs
has been suggested to reduce exfoliation potential
and aid in primary coverage (93).
Placement of a periodontal dressing
The use of a firm, protective periodontal dressing for
ten days following bone replacement graft surgery is
suggested. An antibiotic ointment under the dress-
ing to help seal the area may be useful. It has be-
come popular not to use dressing for many peri-
odontal surgical procedures, but prudence would
seem to suggest that the possible impingement of
foreign materials into the graft site, flap displace-
ment and loss of the bone replacement graft ma-
terial that would jeopardize the success of treatment
make the use of protective dressings preferable.
Administration of antibiotics
A growing body of evidence lends support to empiri-
cal clinical use of antibiotics in conjunction with the
use of bone replacement grafts. In our judgment, te-
tracyclines represent a group of antibiotics of choice
for immediate post-surgery plaque suppression due
to their broad spectrum of activity, attraction to
healing wound sites, and concentration in gingival
crevicular fluid. They may be administered in thera-
peutic doses for the first 20 days post-surgery or un-
til the patient can practice proper plaque control in
the area.
Clinical comparison and
comparative histology of bone
replacement grafts
Clinical performance of bone replacement grafts for
the treatment of periodontal osseous defects has
usually been reported relative to surgical debride-
ment (11, 26, 35, 42, 64, 108). A mean defect fill of
60-70% can be anticipated with similar clinical effec-
82
tiveness among bone replacement grafts (70). Surgi-
cal debridement alone yields between 10-30% defect
fill (11, 55, 57, 72). Human derived graft materials,
autogenous or allogeneic, when placed in intrabony
defects yield similar clinical defect fill as do the other
types of bone replacement grafts (7, 10). Overall,
probing depth reduction and attachment level gain
are similar for all bone replacement grafts.
Differences between graft materials become ap-
parent when considering histological observations.
Hydroxyapatite has been one of the most criticized
due to product exfoliation, problematic manage-
ability and limited ability to promote new attach-
ment. And yet, clinical success in the form of re-
duced probing depth, bony defect fill, and gain in
clinical attachment are repeated findings (49, 50, 55,
72, 108).Histologically, dense hydroxyapatite has not
produced new attachment. Healing is generally by
formation of a long junctional epithelium and hy-
droxyapatite particles are generally found encapsu-
lated in fibrous connective tissue and may be associ-
ated with some osseous regeneration, but new bone
formation is not a predictable result.
Human histology of bone replacement grafts is
limited. Most comparative histology of bone replace-
ment grafts is found in animal models. In a single
human case report by Froum that compared HTRTM
polymer with freeze-dried bone allograft in a patient
that had not been directly monitored for two years,
neither graft material produced new attachment.
HTRTM particles were surrounded by connective
tissue capsules and lamellar bone with evidence of
new bone formation, but no evidence of cemento-
genesis (29). In a controlled human histological
study, Bowers et al. reported formation of a new
attachment apparatus averaging 1.2 mm from the
calculus notch in sites with implanted demineralized
freeze-dried bone allograft, while limited regenera-
tion was found in non-grafted sites (11).
Other comparative studies involving coralline cal-
cium carbonate, bioactive glass, p-tricalcium phos-
phate and hydroxyapatite have been conducted in
animal models. In a sheep tibia1 model, coralline cal-
cium carbonate showed better integration and more
haversian systems than tricalcium phosphate and
both had no interposed fibrous tissue ( 3 5 ) . A com-
parison of bioactive glass, dense hydroxyapatite and
tricalcium phosphate in monkeys showed more
favorable results for PerioGlasa with respect to new
attachment (26). With limited human histological
studies available, it is difficult to make generalities
regarding any bone replacement grafts except for de-
mineralized freeze-dried bone allograft (11).

The clinical significance of the histological differ-
ences among bone replacement grafts may be more
dependent on the clinician’s objective. At dental im-
plant site preparation, bone density may have
greater importance, but not the same significance as
for the treatment of periodontal intrabony defects.
Likewise, new attachment would have greater im-
portance for the treatment of periodontal defects.
Clinical bone fill with graft material encapsulated
within fibrous connective tissue have produced clin-
ically acceptable results. The future of newer bone
replacement grafts will be based on both the clinical
and histological results obtained.
Future research and new trends
Anorganic bovine mineral, natural and converted
corals and other materials may have potential as car-
riers for recombinant bone morphogenetic protein-
2 (Genetics Institute, Inc., Andover, MA) (85) or other
biological modulators (21, 107), which may increase
their usefulness. With source limitations for
autogenous bone and concerns regarding allogeneic
bone, the role of bone substitutes will likely increase.
Development and manufacture of effective, safe, and
user-friendly bone replacement grafts will increase
their use in oral and maxillofacial and periodontal
reconstructive therapy. Already bone replacement
grafts are finding increased use as adjuncts to guided
tissue regeneration barriers in an attempt to improve
results with a combined technique.
Summary
Bone replacement grafts will play a continuing role
in periodontal and other regenerative therapy. Sev-
eral choices are available to the clinician including
autogenous, allogeneic, xenogeneic and a variety of
alloplastic materials. Except for fresh autogenous
bone, bone replacement graft(s) do not provide the
cellular elements necessary for osteogenesis nor can
they reliably be considered truly osteoinductive, but
instead are mostly osteoconductive, providing a
scaffold for bone deposition. Currently, significant
decrease in clinical probing depth and gain of clin-
ical attachment have been reported following use of
bone replacement grafts when compared to flap de-
bridement surgery alone for periodontal osseous de-
fects. Reported differences among bone replacement
grafts (autogenous, allogeneic, xenogeneic, and allo-
plastic) occur with respect to histological outcomes.
Bone and bone substitutes
Overall, probing depth reduction, attachment level
gain and degree of defect fill are similar for all bone
replacement grafts.
Acknowledgment
We thank Connie Holland Gandy for assistance in
preparing this manuscript.
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