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Comparison of bone regeneration with the use of mineralized and demineralized freeze-dried bone allografts: a histological and histochemical study in man
Biomaterials 17 (1996) 1127-1131 0 1996 Elscvier Science Limited Printed in Great Britain. All rights reserved 014%9612/96/$15.00
ELSEVIER
Comparison of bone regeneration with the use of mineralized and demineralized freeze-dried bone allografts: a histological and histochemical study in man A. Piattelli, A. Scarano, M. Corigliano and M. Piattelli Dental School, University of Chiefi, Mineralized
(FDBA)
substitutes rodents,
to determine
poor clinical light
and demineralized
for autologous
microscopical
results
in the mineralization
from
the host bone were
formed
bone.
These
Keywords:
analysis
Our histological
farthest
bone,
in tissues
and histochemical
involved
osteoinduction
freeze-dried
in oral surgery.
results
was observed
Bone
regeneration,
osteoblasts,
osteoclasts
lined
probably with
(DFDBA)
than bone.
Other
of bone regeneration that in DFDBA while
by osteoblasts,
phosphatase,
study
processes,
have
secreting
1996 Elsevier acid phosphatase,
as in
reported
was a comparative in man, with the use
only the particles
osteoconductive 0
been proposed
has been shown,
investigators
in FDBA even
actively
to a more
have
process
The aim of the present
showed
FDBA or DFDBA.
alkaline
bone allografts
other
processes, point
h/y
The demineralization
in man, with the use of DFDBA.
of FDBA and DFDBA. bone were
bone
osteoinduction
results,
63, 66100 Chieti,
Via F. Sciucchi
the particles osteoid
near the host that were
matrix
effect
of FDBA.
Science
Limited
and newly No
demineralized
freeze-dried
Received 21 June 1995; accepted 5 September 1995
demonstrated the capability of demineralized bone powder to induce bone formation in tissues other than bone7-‘. In periodontology, DFDBA has been shown to be highly osteoinductive when compared to blood coagulum, bone blend and freeze-dried bone”. Some investigators, however, have reported poor clinical results with the use of DFDBAl’-13. Moreover, the real usefulness of these two substitutes in human jawbone defect restoration and the precise mechanism for the bone formation induced by them have not yet been elucidated. The aim of the present study was a light microscopical and histochemical analysis of bone regeneration, under expanded polytetrafluoroethylene membranes, in the presence of FDBA and DFDBA.
The use of implants in athophic bone can be a problem in oral implantology, and the bone should be of the correct height and thickness to receive the implant. Many regenerative techniques and materials have been such as resorbable and non-resorbable studied, materials’“. Membrane or filling membranes regenerative techniques have been demonstrated to be effective in filling peri-implant defects, but bone be further techniques need to augmentation investigated. Some authors suggest the use of filling materials under the membrane for creating the space needed to allow bone formation. The materials used are different in composition and source, but one of the most important factors is the possibility of their being resorbed and substituted by newly formed bone. tricalcium phosphate (TCP) or Resorbable hydroxyapatite (HA) can be used5, but autologous bone, when available, is probably the material of choice. Mineralized (FDBA) and demineralized freezedried bone allografts (DFDBA) could be good substitutes for autologous bone. FDBA has been demonstrated by some investigators to be a successful osseous grafting material”, and other researchers have
MATERIALS AND METHODS Eight patients, who gave their informed consent, participated in this study. Gore-Tex membranes (W.L. Gore and Associates, Flagstaff, AZ, USA) were used to cover post-extraction Branemark titanium implants (Nobelpharma, Sweden). In four patients commercially available freeze-dried demineralized bone (Life-Net Services, Norfolk, VA, USA) was put around the
Correspondence to Professor A. Piattelli. 1127
Biomaterials
1996,
Vol. 17 No. 11
Bone
1128
implants, while in the other four patients mineralized freeze-dried bone (Miami Tissue Bank, Florida, USA) was used. After 6 months, during the re-entry and bone abutment connection procedure, a small specimen was retrieved from around the implants. The specimens were washed in saline solution and immediately fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.15~ cacodylate buffer at 4°C and pH 7.4, to be processed for histology. The specimens were processed to obtain thin ground sections with the Precise 1 Automated System (Precise, Pescara, Italy). The specimens were put in graded alcohol rinses and embedded in a glycol methacrylate resin (Technovit 7200 VLC, Kulzer, Wehrheim, Germany). After polymerization the specimens were sectioned with a high-precision diamond disc at about 150pm and ground down to about ~O,LL~.The slides were stained with basic fuchsin and toluidine blue. von Kossa staining was also done to visualize the calcified structures. For the enzyme histological staining of alkaline phosphatase (ALP) and acid phosphatase (ACP), the procedure has already been described14.
regeneration
with FDBA and DFDBA:
A. Piattelli
et al.
Figure 2 It is possible to observe newly formed bone (B), with wide osteocytic lacunae, very close to the FDBA particles (F). Alkaline phosphatase-positive osteoblasts are present on the external surface of the FDBA (arrowheads) and of the newly formed bone (arrowheads). Basic fuchsintoluidine blue. Original magnification x200.
RESULTS FDBA At low magnification it was possible to see that particles that were in close contact with pre-existing bone were completely surrounded by newly formed bone (Figure 2). However, a thin layer of newly formed bone was found around the particles that were at a distance from pre-existing bone. At the interface between pre-existing and newly formed bone it was possible to observe in all specimens the absence of a gap. Newly formed bone presented wide osteocytic lacunae (Figure 2). In all particles of FDBA it was possible to observe the presence of cells inside the osteocytic lacunae. No inflammatory infiltrate was present. In some areas it was possible to see osteoblasts secreting osteoid matrix directly on the external surface
Figure 1 Some of the FDBA particles (F) appear embedded in newly formed bone. The FDBA has a lower staining affinity for basic fuchsin. Other particles seem to be connected, only in a small portion of the perimeter, by the newly formed bone (arrowhead). Basic fuchsin-toluidine blue. Original magnification x50. Biomaterials
1996. Vol. 17 No. 11
Figure 3 At higher magnification it is possible to observe mineralized bone (MB), osteoid matrix (0) and osteoblasts positive for alkaline phosphatase (ALP) (small arrowheads). Some osteoblasts negative for ALP are also present (big arrowheads). The blue halo is probably due to the diffusion of the enzyme, caused by cell swelling due to the fixation with acetic acid. Basic fuchsin-toluidine blue. Original magnification x1200.
of the FDBA particles. With von Kossa it was possible to observe that the newly formed bone was highly mineralized. With the histochemical staining for ALP it was possible to observe in some areas the presence of a few positive osteoblasts, located directly on the particles’ surface (Figures z and 3). Osteoblasts negative for ALP could be observed nearby. All the specimens were negative for ACP. In some specimens it was possible to observe the presence of osteons with Haversian canals, with capillaries and connective tissue cells at the centre (Figure 4). On the outer surface of a few particles, which were at a distance from pre-existing bone, it was possible to observe the presence of osteoclasts which were in the process of actively resorbing bone. DFDBA DFDBA particles were still almost intact in the central zone of the microscopic field. At the edge of
Bone
regeneration
with
FDBA and DFDBA:
1129
A. Piattelli et al.
Figure 4 Newly formed bone (B) is found between two FDBA particles (F). Inside the FDBA osteocytic lacunae are filled by the osteocytes. In one of the particles it is possible to see an Haversian canal (H) filled by cells. On one side of the canal it is possible to observe a small quantity of osteoid material with the very close presence of osteoblast-like cells. At the centre of the canal it is possible to observe a capillary (c) lined by endothelial cells. Some osteocytes are very close to the outer border of the Haversian canal. Basic fuchsin-toluidine blue. Original magnification x200.
the newly formed bone, DFDBA particles seemed to be engaged in the process of bone forming activity (Figure 5). However, the particles located at a distance from the newly formed bone showed no signs of mineralization or osteogenesis. At higher magnification, examination of this zone showed that small, flat and elongated macrophages were resorbing the demineralized matrix at a slow rate, because almost all the particles were still present. Many fibroblasts, connective tissues and capillaries surrounded the remaining DFDBA particles. Still unmineralized particles were encased in the matrix of the newly formed bone produced by active osteoblasts (Figure 6). The borderline between newly formed bone and demineralized matrix showed signs of ongoing mineralization. Basic fuchsin-positive rounded nuclei seemed to come from the newly formed bone into the DFDBA particles. In other regions these nuclei seemed to fuse together, making the particles similar, in staining properties, to normal bone. In some instances DFDBA particles seemed to be completely encased in newly formed bone that surrounded the whole particle. In this area the bone trabeculae showed a central area with unmineralized matrix showing not yet fused nodules of mineralization with empty lacunae (Figure 7). The newly formed bone trabeculae showed different aspects: some of these had the aspect of mature osteonic lamellar bone, while others showed the aspect of dystrophic mineralized tissue without a lamellar aspect and with numerous large osteocytic lacunae, with the aspect of interglobular dentin (Figure 8). The histochemical staining for mineralized tissue showed that the rounded nodules colonizing the DFDBA particles were mineralized (Figure 9). In other words the DFDBA particles at a distance from the bone were not positive for basic fuchsin or von Kossa staining, while the particles
Figure 5 Basic
DFDBA particles (D) near newly formed bone. fuchsin-toluidine blue. Original magnification x200.
Figure 6
Still demineralized particle (D) in close with newly formed bone. Basic fuchsin-toluidine Original magnification x 100.
Figure 7 nodules Original
Unmineralized particle of mineralization. Basic magnification x100.
that were adjacent to newly three aspects simultaneously: (1) (2) (3)
contact blue.
(D) with not yet fuchsin-toluidine
formed bone
fused blue.
showed
aspects of demineralized particles; areas positive for basic fuchsin; areas positive for von Kossa.
In other areas it was possible to observe a complete positivity for von Kossa. In some fields it was possible to see some cells positive for ACP. Biomaterials
1996,
Vol.
17 No. 11
1130
Figure 8
Bone
Newly formed
near a not yet fuchsin-toluidine
bone with wide osteocytic lacunae remineralized DFDBA particle (D). Basic blue. Original magnification x100.
(b) Figure 9 The nodules inside the DFDBA particles are highly mineralized. Silver nitrate-basic fuchsin-toluidine blue. Original magnification x100.
In a previous study from our laboratory we compared bone augmentation under Gore-Tex membrane with and without DFDBA used as a space maker, and the qualitative results showed more bone formation in the cases treated with DFDBA particlesi5. Some researchers have suggested placing autologous bone chips or DFDBA under the membranes16-18. There are, however, studies that question the utility of DFDBA11-13. The aim of the present study was to elucidate the mechanism by which FDBA and DFDBA particles induced bone formation. Urist and that demineralization by Dowell1g demonstrated hydrochloric acid exposed the bone-inductive proteins located in bone matrix, thus enhancing the osteogenic potential. Many studies have been performed in animals7,* demonstrating the ability of demineralized bone powder in inducing bone formation in non-osseous sites such as muscle. Recently, Katagiri et ~1.” demonstrated that bone morphogenetic protein-2 converted the differentiation pathway of myoblasts into that of osteoblasts. The DFDBA particles were involved in the process of mineralization, but only near the newly forming Biomaterials
1996, Vol. 17 No. 11
with
FDBA and DFDBA:
A. Piattelli
et al.
bone, while no signs of mineralization were visible in the particles far away from the host bone, and these particles were not positive for basic fuchsin or for von Kossa. These observations may indicate that the matrix of DFDBA particles first undergoes biochemical changes involving diffusion of acidic proteins from the neighbouring bone or directly from the osteoblasts. It is possible that there is a maximum distance of diffusion of these proteins inside the particles and this fact could explain the incomplete mineralization in the centre of the particles. The second step could be the mineralization of the modified matrix by heterogeneous nucleation, with the appearance of calcification nodules which tend to grow and fuse together. The formation of acellular mineral deposition, inside demineralized matrix implanted in muscle of rats, was shown by an ultrastructural study’l. This mineralization was not mediated by cells or matrix vesicles, and was probably induced by the intrinsic nucleation of the DFDBA. This mineralization properties stimulated the resorption by osteoclasts and the deposition by osteoblasts of new bone. Our study showed a similar pattern of mineralization inside the DFDBA matrix. It is possible that DFDBA may serve as a scaffold for bone formation in an osteoconductive fashion. The mechanisms for bone formation induced by DFDBA could be: (a)
DISCUSSION
regeneration
the demineralized particles undergo a biochemical change that brings about the remineralization of the particles; the demineralized particles are colonized by mononuclear osteoclasts from the neighbouring bone, that are able to attract osteoblasts on their surface, thus allowing successive bone layering”.
In the FDBA particles we found, on the contrary, that many particles were completely surrounded by newly formed bone. Even the particles that were farthest from the host bone were lined by osteoblasts actively secreting osteoid and newly formed bone. In conclusion, according to our histological results, the main differences between these two allografts seem to be as follows. (1)
(2)
(3)
(4)
In FDBA the resorption processes are very scarce, and it has not been possible to find cells positive for ACP, while, on the contrary, in the DFDBA particles the resorption processes were present and cells positive for ACP were found. In FDBA even the particles farthest from the host bone were lined by or embedded in newly formed bone, while the DFDBA particles located far from the host bone tended to be surrounded by a scarcely cellular connective tissue, composed mainly of collagen fibres. In FDBA all the osteocytic lacunae were filled by osteocytes and in some areas Haversian systems with a capillary at the centre were found, while in the DFDBA the osteocytic lacunae tended, for the most part, to remain empty. FDBA and DFDBA did not show any osteoinductive effects.
Bone
regeneration
with
FDBA and DFDBA:
A. Piaffelli
et al.
ACKNOWLEDGEMENTS
1131
11
Becker W, Lynch SE, Lekholm U et al. A comparison of e-PTFE membranes alone or in combination with platelet-derived growth factors and insulin-like growth factor-I or demineralized freeze-dried bone in promoting bone formation around immediate extraction sockets implants. J Periodonfol 1992; 63:
12
Becker W, Becker BE, Caffese R. A comparison of demineralized freeze-dried bone and autologous bone to induce bone formation in human extraction sockets.
This work was partially supported by the Ministry of University, Research, Science and Technology (MURST) and by the National Research Council (CNR).
929-940.
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Becker W, Becker BE, Handelsman M, Ochsenbein C, Albrektsson T. Guided tissue regeneration for implants placed into extraction sockets: a study in dogs. / Periodantol
2
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Implants
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Biomaferials
47: 50-56.
Pettis GY, Kaban LB, Glowacki J. Tissue response to composite ceramic hydroxyapatite/demineralized bone implants. J Oral Maxillofac Surg 1990; 46: 1068-1074. Katz RW, Hollinger JO, Reddi AH. The functional equivalence of demineralized bone and tooth matrices in ectopic bone induction. J Biomed Mater Res 1994; Blumenthal N, Steinberg J. The use of collagen membrane barriers in conjunction with combined demineralized bone-collagen gel implants in human infrabony defects. JPeriodonfol 1990; 61: 319-327.
16: 1333-1338.
Simion M, Dahlin C, Trisi P, Piattelli A. Qualitative and quantitative comparative study on different filling materials used in bone tissue regeneration: a controlled clinical study. Inf JPeriodanf Rest Dent 1994; 14: 199-
16
Buser D, Bragger U, Lang NP, Nyman S. Regeneration and enlargement of jaw bone using guided tissue regeneration. Chin Oral ImpI Res 1990; 1:22-32. Nevins M, Mellonig JT. Enhancement of the damaged edentulous ridge to receive dental implants: a combination of allografts and the Gore-Tex membrane.
215.
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Inf JPeriodonf
Rest Dent 1992;
12: 91-111.
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Mellonig JT, Triplett J. Guided tissue regeneration and endosseous dental implants. Inf J Periodonf Rest Dent
19
Urist MR, Dowel1 TA. Inductive substratum for osteogenesis in pellets of particulate bone matrix. Clin
20
Katagiri T, Yamaguchi A, Komaki M et al. Bone morphogenetic protein-2 converts the differentiation pathway of CXX? myoblasts into the osteoblast lineage. J Cell Biol1994; 127: 1755-1766. Yamashita K, Takagi T. Ultrastructural observation of calcification preceding new bone formation induced by demineralized bone matrix gelatin. Acfa Anaf 1992;
1993;
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Drury GI, Yukna RA. Histologic evaluation of combining tetracycline and allogeneic freeze-dried bone on bone regeneration in experimental defects in baboons. JPeriodonfoll991; 62: 652-658. El Deeb M, Hosny M, Sharawy M. Osteogenesis in composite grafts of allogenic demineralized bone powder and porous hydroxylapatite. J Oral Maxillofac Surg 1989;
8
1991;
Gotfredsen K, Warrer K, Hjorting-Hansen E, Karring T. Effect of membranes and porous hydroxyapatite on healing in bone defects around titanium dental implants. An experimental study in monkeys. Clin Oral Imp1 Res 1991;
6
143-154.
Zablotsky M, Meffert RM, Caudill R, Evans G. Histological and clinical comparisons of guided tissue regeneration on dehisced hydroxylapatite-coated and titanium endosseous implant surfaces: a pilot study. Inf J Oral Maxillofac
5
Piattelli A, Scarano A, Piattelli M. Detection of alkaline and acid phosphatases around titanium implants: a light microscopical histochemical and study.
Wachtel HC, Langford A, Bernimoulin JP, Reichart P. Guided bone regeneration next to osseointegrated implants in humans. Inf J Oral Maxillofac Implants 1991;
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62: 703-709.
Rest Dent 1990;
1994;
Becker W, Schenk R, Higuchi K, Lekholm LJ, Becker BE. Variations in bone regeneration adjacent to implants augmented with barrier membranes alone or with demineralized freeze-dried bone or autologous grafts: a study in dogs. Inf J Oral Maxillofac Implants 1995; 10:
Becker W, Becker BE. Guided tissue regeneration for implants placed into extraction sockets and for implant dehiscences: surgical techniques and case reports. Int 1 Periodont
3
1991;
JPeriodonfol 13
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Galvin RJ, Cullison JW, Avioli LV, Osdoby PA. Influence of osteoclasts and osteoclast-like cells on osteoblasts alkaline phosphatase activity and collagen synthesis. /Bone Miner Res 1994; 9: 1167-1178.
Biomaterials 1996, Vol. 17 No. 11
ELSEVIER
Comparison of bone regeneration with the use of mineralized and demineralized freeze-dried bone allografts: a histological and histochemical study in man A. Piattelli, A. Scarano, M. Corigliano and M. Piattelli Dental School, University of Chiefi, Mineralized
(FDBA)
substitutes rodents,
to determine
poor clinical light
and demineralized
for autologous
microscopical
results
in the mineralization
from
the host bone were
formed
bone.
These
Keywords:
analysis
Our histological
farthest
bone,
in tissues
and histochemical
involved
osteoinduction
freeze-dried
in oral surgery.
results
was observed
Bone
regeneration,
osteoblasts,
osteoclasts
lined
probably with
(DFDBA)
than bone.
Other
of bone regeneration that in DFDBA while
by osteoblasts,
phosphatase,
study
processes,
have
secreting
1996 Elsevier acid phosphatase,
as in
reported
was a comparative in man, with the use
only the particles
osteoconductive 0
been proposed
has been shown,
investigators
in FDBA even
actively
to a more
have
process
The aim of the present
showed
FDBA or DFDBA.
alkaline
bone allografts
other
processes, point
h/y
The demineralization
in man, with the use of DFDBA.
of FDBA and DFDBA. bone were
bone
osteoinduction
results,
63, 66100 Chieti,
Via F. Sciucchi
the particles osteoid
near the host that were
matrix
effect
of FDBA.
Science
Limited
and newly No
demineralized
freeze-dried
Received 21 June 1995; accepted 5 September 1995
demonstrated the capability of demineralized bone powder to induce bone formation in tissues other than bone7-‘. In periodontology, DFDBA has been shown to be highly osteoinductive when compared to blood coagulum, bone blend and freeze-dried bone”. Some investigators, however, have reported poor clinical results with the use of DFDBAl’-13. Moreover, the real usefulness of these two substitutes in human jawbone defect restoration and the precise mechanism for the bone formation induced by them have not yet been elucidated. The aim of the present study was a light microscopical and histochemical analysis of bone regeneration, under expanded polytetrafluoroethylene membranes, in the presence of FDBA and DFDBA.
The use of implants in athophic bone can be a problem in oral implantology, and the bone should be of the correct height and thickness to receive the implant. Many regenerative techniques and materials have been such as resorbable and non-resorbable studied, materials’“. Membrane or filling membranes regenerative techniques have been demonstrated to be effective in filling peri-implant defects, but bone be further techniques need to augmentation investigated. Some authors suggest the use of filling materials under the membrane for creating the space needed to allow bone formation. The materials used are different in composition and source, but one of the most important factors is the possibility of their being resorbed and substituted by newly formed bone. tricalcium phosphate (TCP) or Resorbable hydroxyapatite (HA) can be used5, but autologous bone, when available, is probably the material of choice. Mineralized (FDBA) and demineralized freezedried bone allografts (DFDBA) could be good substitutes for autologous bone. FDBA has been demonstrated by some investigators to be a successful osseous grafting material”, and other researchers have
MATERIALS AND METHODS Eight patients, who gave their informed consent, participated in this study. Gore-Tex membranes (W.L. Gore and Associates, Flagstaff, AZ, USA) were used to cover post-extraction Branemark titanium implants (Nobelpharma, Sweden). In four patients commercially available freeze-dried demineralized bone (Life-Net Services, Norfolk, VA, USA) was put around the
Correspondence to Professor A. Piattelli. 1127
Biomaterials
1996,
Vol. 17 No. 11
Bone
1128
implants, while in the other four patients mineralized freeze-dried bone (Miami Tissue Bank, Florida, USA) was used. After 6 months, during the re-entry and bone abutment connection procedure, a small specimen was retrieved from around the implants. The specimens were washed in saline solution and immediately fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.15~ cacodylate buffer at 4°C and pH 7.4, to be processed for histology. The specimens were processed to obtain thin ground sections with the Precise 1 Automated System (Precise, Pescara, Italy). The specimens were put in graded alcohol rinses and embedded in a glycol methacrylate resin (Technovit 7200 VLC, Kulzer, Wehrheim, Germany). After polymerization the specimens were sectioned with a high-precision diamond disc at about 150pm and ground down to about ~O,LL~.The slides were stained with basic fuchsin and toluidine blue. von Kossa staining was also done to visualize the calcified structures. For the enzyme histological staining of alkaline phosphatase (ALP) and acid phosphatase (ACP), the procedure has already been described14.
regeneration
with FDBA and DFDBA:
A. Piattelli
et al.
Figure 2 It is possible to observe newly formed bone (B), with wide osteocytic lacunae, very close to the FDBA particles (F). Alkaline phosphatase-positive osteoblasts are present on the external surface of the FDBA (arrowheads) and of the newly formed bone (arrowheads). Basic fuchsintoluidine blue. Original magnification x200.
RESULTS FDBA At low magnification it was possible to see that particles that were in close contact with pre-existing bone were completely surrounded by newly formed bone (Figure 2). However, a thin layer of newly formed bone was found around the particles that were at a distance from pre-existing bone. At the interface between pre-existing and newly formed bone it was possible to observe in all specimens the absence of a gap. Newly formed bone presented wide osteocytic lacunae (Figure 2). In all particles of FDBA it was possible to observe the presence of cells inside the osteocytic lacunae. No inflammatory infiltrate was present. In some areas it was possible to see osteoblasts secreting osteoid matrix directly on the external surface
Figure 1 Some of the FDBA particles (F) appear embedded in newly formed bone. The FDBA has a lower staining affinity for basic fuchsin. Other particles seem to be connected, only in a small portion of the perimeter, by the newly formed bone (arrowhead). Basic fuchsin-toluidine blue. Original magnification x50. Biomaterials
1996. Vol. 17 No. 11
Figure 3 At higher magnification it is possible to observe mineralized bone (MB), osteoid matrix (0) and osteoblasts positive for alkaline phosphatase (ALP) (small arrowheads). Some osteoblasts negative for ALP are also present (big arrowheads). The blue halo is probably due to the diffusion of the enzyme, caused by cell swelling due to the fixation with acetic acid. Basic fuchsin-toluidine blue. Original magnification x1200.
of the FDBA particles. With von Kossa it was possible to observe that the newly formed bone was highly mineralized. With the histochemical staining for ALP it was possible to observe in some areas the presence of a few positive osteoblasts, located directly on the particles’ surface (Figures z and 3). Osteoblasts negative for ALP could be observed nearby. All the specimens were negative for ACP. In some specimens it was possible to observe the presence of osteons with Haversian canals, with capillaries and connective tissue cells at the centre (Figure 4). On the outer surface of a few particles, which were at a distance from pre-existing bone, it was possible to observe the presence of osteoclasts which were in the process of actively resorbing bone. DFDBA DFDBA particles were still almost intact in the central zone of the microscopic field. At the edge of
Bone
regeneration
with
FDBA and DFDBA:
1129
A. Piattelli et al.
Figure 4 Newly formed bone (B) is found between two FDBA particles (F). Inside the FDBA osteocytic lacunae are filled by the osteocytes. In one of the particles it is possible to see an Haversian canal (H) filled by cells. On one side of the canal it is possible to observe a small quantity of osteoid material with the very close presence of osteoblast-like cells. At the centre of the canal it is possible to observe a capillary (c) lined by endothelial cells. Some osteocytes are very close to the outer border of the Haversian canal. Basic fuchsin-toluidine blue. Original magnification x200.
the newly formed bone, DFDBA particles seemed to be engaged in the process of bone forming activity (Figure 5). However, the particles located at a distance from the newly formed bone showed no signs of mineralization or osteogenesis. At higher magnification, examination of this zone showed that small, flat and elongated macrophages were resorbing the demineralized matrix at a slow rate, because almost all the particles were still present. Many fibroblasts, connective tissues and capillaries surrounded the remaining DFDBA particles. Still unmineralized particles were encased in the matrix of the newly formed bone produced by active osteoblasts (Figure 6). The borderline between newly formed bone and demineralized matrix showed signs of ongoing mineralization. Basic fuchsin-positive rounded nuclei seemed to come from the newly formed bone into the DFDBA particles. In other regions these nuclei seemed to fuse together, making the particles similar, in staining properties, to normal bone. In some instances DFDBA particles seemed to be completely encased in newly formed bone that surrounded the whole particle. In this area the bone trabeculae showed a central area with unmineralized matrix showing not yet fused nodules of mineralization with empty lacunae (Figure 7). The newly formed bone trabeculae showed different aspects: some of these had the aspect of mature osteonic lamellar bone, while others showed the aspect of dystrophic mineralized tissue without a lamellar aspect and with numerous large osteocytic lacunae, with the aspect of interglobular dentin (Figure 8). The histochemical staining for mineralized tissue showed that the rounded nodules colonizing the DFDBA particles were mineralized (Figure 9). In other words the DFDBA particles at a distance from the bone were not positive for basic fuchsin or von Kossa staining, while the particles
Figure 5 Basic
DFDBA particles (D) near newly formed bone. fuchsin-toluidine blue. Original magnification x200.
Figure 6
Still demineralized particle (D) in close with newly formed bone. Basic fuchsin-toluidine Original magnification x 100.
Figure 7 nodules Original
Unmineralized particle of mineralization. Basic magnification x100.
that were adjacent to newly three aspects simultaneously: (1) (2) (3)
contact blue.
(D) with not yet fuchsin-toluidine
formed bone
fused blue.
showed
aspects of demineralized particles; areas positive for basic fuchsin; areas positive for von Kossa.
In other areas it was possible to observe a complete positivity for von Kossa. In some fields it was possible to see some cells positive for ACP. Biomaterials
1996,
Vol.
17 No. 11
1130
Figure 8
Bone
Newly formed
near a not yet fuchsin-toluidine
bone with wide osteocytic lacunae remineralized DFDBA particle (D). Basic blue. Original magnification x100.
(b) Figure 9 The nodules inside the DFDBA particles are highly mineralized. Silver nitrate-basic fuchsin-toluidine blue. Original magnification x100.
In a previous study from our laboratory we compared bone augmentation under Gore-Tex membrane with and without DFDBA used as a space maker, and the qualitative results showed more bone formation in the cases treated with DFDBA particlesi5. Some researchers have suggested placing autologous bone chips or DFDBA under the membranes16-18. There are, however, studies that question the utility of DFDBA11-13. The aim of the present study was to elucidate the mechanism by which FDBA and DFDBA particles induced bone formation. Urist and that demineralization by Dowell1g demonstrated hydrochloric acid exposed the bone-inductive proteins located in bone matrix, thus enhancing the osteogenic potential. Many studies have been performed in animals7,* demonstrating the ability of demineralized bone powder in inducing bone formation in non-osseous sites such as muscle. Recently, Katagiri et ~1.” demonstrated that bone morphogenetic protein-2 converted the differentiation pathway of myoblasts into that of osteoblasts. The DFDBA particles were involved in the process of mineralization, but only near the newly forming Biomaterials
1996, Vol. 17 No. 11
with
FDBA and DFDBA:
A. Piattelli
et al.
bone, while no signs of mineralization were visible in the particles far away from the host bone, and these particles were not positive for basic fuchsin or for von Kossa. These observations may indicate that the matrix of DFDBA particles first undergoes biochemical changes involving diffusion of acidic proteins from the neighbouring bone or directly from the osteoblasts. It is possible that there is a maximum distance of diffusion of these proteins inside the particles and this fact could explain the incomplete mineralization in the centre of the particles. The second step could be the mineralization of the modified matrix by heterogeneous nucleation, with the appearance of calcification nodules which tend to grow and fuse together. The formation of acellular mineral deposition, inside demineralized matrix implanted in muscle of rats, was shown by an ultrastructural study’l. This mineralization was not mediated by cells or matrix vesicles, and was probably induced by the intrinsic nucleation of the DFDBA. This mineralization properties stimulated the resorption by osteoclasts and the deposition by osteoblasts of new bone. Our study showed a similar pattern of mineralization inside the DFDBA matrix. It is possible that DFDBA may serve as a scaffold for bone formation in an osteoconductive fashion. The mechanisms for bone formation induced by DFDBA could be: (a)
DISCUSSION
regeneration
the demineralized particles undergo a biochemical change that brings about the remineralization of the particles; the demineralized particles are colonized by mononuclear osteoclasts from the neighbouring bone, that are able to attract osteoblasts on their surface, thus allowing successive bone layering”.
In the FDBA particles we found, on the contrary, that many particles were completely surrounded by newly formed bone. Even the particles that were farthest from the host bone were lined by osteoblasts actively secreting osteoid and newly formed bone. In conclusion, according to our histological results, the main differences between these two allografts seem to be as follows. (1)
(2)
(3)
(4)
In FDBA the resorption processes are very scarce, and it has not been possible to find cells positive for ACP, while, on the contrary, in the DFDBA particles the resorption processes were present and cells positive for ACP were found. In FDBA even the particles farthest from the host bone were lined by or embedded in newly formed bone, while the DFDBA particles located far from the host bone tended to be surrounded by a scarcely cellular connective tissue, composed mainly of collagen fibres. In FDBA all the osteocytic lacunae were filled by osteocytes and in some areas Haversian systems with a capillary at the centre were found, while in the DFDBA the osteocytic lacunae tended, for the most part, to remain empty. FDBA and DFDBA did not show any osteoinductive effects.
Bone
regeneration
with
FDBA and DFDBA:
A. Piaffelli
et al.
ACKNOWLEDGEMENTS
1131
11
Becker W, Lynch SE, Lekholm U et al. A comparison of e-PTFE membranes alone or in combination with platelet-derived growth factors and insulin-like growth factor-I or demineralized freeze-dried bone in promoting bone formation around immediate extraction sockets implants. J Periodonfol 1992; 63:
12
Becker W, Becker BE, Caffese R. A comparison of demineralized freeze-dried bone and autologous bone to induce bone formation in human extraction sockets.
This work was partially supported by the Ministry of University, Research, Science and Technology (MURST) and by the National Research Council (CNR).
929-940.
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Biomaterials 1996, Vol. 17 No. 11