Autogenous bone tissue has regeneration potential; however, this capacity may not be sufficient in larger bone defects. The aim of this study is to histologically evaluate anorganic bovine bone grafts (GenOx Inorg) with or without platelet-rich plasma (PRP). Two bone lesions were created in calvaria of 12 rabbits. The 24 surgical lesions were separated into 3 groups: coagulous, anorganic, and anorganic with PRP. At the 4-week time point, the animals were euthanized and the grafted area removed, fixed in formalin 10% with phosphate buffered saline, 0.1 M, and embedded in paraffin. The histologic parameters analyzed were new bone filling the defect area, presence of giant cells and particles of the graft, and new bone formation associated with the particles. In the coagulous group, defects were filled with fibrous tissue that attached the periosteum and little bone neoformation in the periphery. In anorganic groups with or without PRP, little new bone formation in the periphery of the defect was observed; however, in the center of some defects there was new bone. Moderate presence of giant cells and little new bone formation was associated with the innumerous graft particles. Histologic results revealed no statistically significant differences among the defects new bone fill between the studied groups (P  =  .64). There was no significant difference in the number of giant cells (P  =  .60), graft particles (P  =  .46), and new bone formation around graft particles (P  =  .26), whether PRP was added or not. Anorganic bone, isolated or mixed with PRP, was biocompatible and osteoconductive, while maintaining bone volume.

Introduction

Autogenous bone is currently used for treating bone defects.13 Advantages of autogenous bone are due to its biologic properties, determined by the terms osteoconduction, osteoinduction, and osteogenesis, and the lack of the possibility of disease transmission or host rejection.2,4 Nevertheless, donor site morbidity is a factor that limits the autograft procedure,2 and additional site-specific complications of harvesting must be taken into consideration, particularly if a second surgery is needed.5 

Researchers continuously strive to improve on current bone grafting techniques and provide faster and denser bone regeneration. In recent years, several therapeutic alternatives have been developed, such as synthetic bone substitutes,68 local growth factors, and composites that behave as repair promoters and carriers of bone induction factors.4 Anorganic biomaterials are important alternatives to autogenous grafts due to their biocompatibility9,10 and osteoconduction properties.11 These anorganic biomaterials can also enhance a high content of calcium and phosphorus essential for bone formation.12,13 

Bovine bone has been investigated and has shown favorable results.14,15 In vivo studies have demonstrated an increase in osteoblast activity and bone formation when mineralized,1618 and demineralized2 bone matrices are used in conjunction with growth factors.19 In vitro studies also demonstrated the benefits of anorganic bone matrices.20 Anorganic biomaterials require long periods to be resorbed, determining continuous flow of giant cells next to the material surface. The resorption process seems to be related to the amount of newly formed bone.14 

The use of platelet-rich plasma (PRP) can increase the benefits of bone grafts.21,22 Platelets release growth factors and cytokines that contribute to bone regeneration and vascular proliferation, essential to bone graft healing.23,24 Additional advantages include their adhesive nature due to the fibrin, which acts as a tissue glue25,26 and their capacity to accelerate deposition of new bone along the graft material.26 Some authors suggest that the addition of PRP to osteoconductive grafting materials can potentiate osteoinduction27,28; however, others did not observe any increase in the bone healing when using PRP.23,2931 The current study histologically evaluated bone repair in calvarial defects filled with bovine anorganic bone matrix, with or without PRP.

Material and Methods

Material

The tested material was bovine anorganic bone (BAB), thermally deproteinized at 100°C (GenOx Inorg, Baumer SA, Mogi Mirim, São Paulo, Brazil).

Animal surgical procedure

Twelve New Zealand white rabbits weighing between 2.5 and 3.5 kg were included in this randomized, blind study. The guidelines of the Brazilian College of Animal Experimentation were used in all animal protocols. Each rabbit was anesthetized with ketamine (25 mg/kg), xylazine (10 mg/kg), Acepran (0.2 mg/kg), midazolam (0.2 mg/kg), and local anesthesia with 0.9 mL of mepivacaine with epinephrine. A single prophylactic dose of antibiotic therapy with cephalosporin (30 mg/kg) was administered intravenously. After trichotomy and antisepsis with aqueous solution of povidone-iodine, a 2-cm incision was made along the anteroposterior surface of the bony cranium and a full-thickness flap was raised. Two defects of 8 mm diameter were created in parietal calvarial bone with a trephine burr under continuous irrigation with saline solution. Care was taken to make a full-thickness defect without damaging the underlying dura. The 24 defects were separated in 3 groups and randomly filled with coagulant in the control group, BAB alone, and BAB mixed with PRP. The wound was closed using nylon 4.0 suture, which was removed 7 days after surgery. All animals received the antibiotic Flotril (enrofloxacina, 2.5%, 2.5 mL/kg) subcutaneously during 5 days after surgery.

PRP preparation

Four and a half milliliters of autologous blood was drawn from each rabbit from the auricular vein to prepare the PRP several minutes before administration of anesthesia. The 4.5 mL of autologous blood was combined with anticoagulant, 0.5 mL of 3.8% sodium citrate, to prevent coagulation. The blood was centrifuged (206-BL-Fanem; Datamed, São Paulo, Brazil) according to the Sonnleitner modified method29,32 at 1000 rpm (160G) for 20 minutes to separate the plasma containing the platelets from the red cells. The supernatant and 2 mm below the dividing line between the phases was pipetted and transferred to a tube without anticoagulant. An additional centrifugation for 15 minutes at 1600 rpm (400G) was done to separate the platelets. The precipitate formed in the tube by this second centrifugation was the PRP. For each 0.5 mL of PRP, 25 µL of 10% calcium chloride was used as an activator.

Sample evaluation

The animals received a normal diet consisting of granular food and water ad libitum. At the 4-week survival period they were anesthetized with Pentothal sodium 2.5% and euthanized with an overdose of potassium chloride, 19.1%. The calvarial defects and surrounding tissue were removed and immediately fixed in 10% phosphate buffered formaldehyde solution during hour 48. Subsequently, the tissue blocks were decalcified in EDTA, 4.13% for 4 weeks, dehydrated with graded alcohols, and embedded in paraffin. The histologic specimens were prepared in the usual fashion, and the semi-serial sections of 5-µm thickness were stained with hematoxylin-eosin and Mallory trichrome. Histologic analysis of the bone defect area, performed under light microscope at ×10 and ×40 magnification in 3 sections for each animal revealed the defects new bone fill, presence of giant cells/graft particles, and new bone formation associated with the graft particles. The scores in Table 1 were employed for evaluation. The results obtained were submitted to normality test, Kruskal-Wallis, and Mann-Whitney U tests. Differences were considered statistically significant at P ≤ .05.

Table 1

Established criteria for evaluation

Established criteria for evaluation
Established criteria for evaluation

Results

During the experiment all animals remained in good health and did not experience complications. The histologic analysis of the defect area exhibited normal healing process. No inflammatory signs or adverse tissue was observed regardless of the evaluated groups.

In the coagulous group, the defects presented an extensive fibrous connective tissue and little bone ingrowth from the periphery of the defects (Figure 1a). The presence of giant cells was not observed. In the BAB and BAB/PRP groups, we observed little new bone formation, mainly from the periphery of the defects (Figure 1b,c). A moderate amount of multinucleated giant cells among an abundant quantity of graft particles (Figure 2a) and little new bone formation associated with these particles (Figure 2b) could be noted.

Figure 1.

Micrography of the defect (delimited by dotted). (a) Coagulous group. (b) Anorganic group. (c) Anorganic with PRP group. Hematoxylin-eosin, ×4.

Figure 1.

Micrography of the defect (delimited by dotted). (a) Coagulous group. (b) Anorganic group. (c) Anorganic with PRP group. Hematoxylin-eosin, ×4.

Figure 2.

(a) Micrography evidencing graft particles (GP), surrounded by multinucleated giant cells (GC). Hematoxylin-eosin, ×4. (b) Micrography evidencing bone neoformation (BN) associated with graft particles (GP). Hematoxylin-eosin, ×10.

Figure 2.

(a) Micrography evidencing graft particles (GP), surrounded by multinucleated giant cells (GC). Hematoxylin-eosin, ×4. (b) Micrography evidencing bone neoformation (BN) associated with graft particles (GP). Hematoxylin-eosin, ×10.

Histologic results revealed no statistically significant differences in defect bone filling between all studied groups (P  =  .64). There was no significant difference in the number of giant cells (P  =  .60), graft particles (P  =  .46), and new bone formation around graft particles (P  =  .26) between the grafted materials, whether PRP was added or not (Table 2).

Table 2

Mean values and standard deviation of histologic scoring of treated defects

Mean values and standard deviation of histologic scoring of treated defects
Mean values and standard deviation of histologic scoring of treated defects

Discussion

Rabbits, used in the current study, are useful animal models for preparation of platelet concentrates and bone repair.29 Generally, platelets of human and other mammalians have a similar ultrastructure and constituents. Physiologic and metabolic similarities are also seen between the bone tissues of both species.33,34 Additional advantages of using a rabbit include easy manipulation and sufficient blood volume for preparation of platelet concentrates.27 

In this study, care was taken to avoid damaging the underlying dura and the periosteum. Periosteum provides blood supply for bone and osteoprogenitor cells for bone regeneration, and its preservation is found to favorably influence graft revascularization and integrity.35 

Bovine anorganic bone is comparable to the mineralized matrix of human bone9,10; therefore, this material has been used as an alternative to autogenous bone grafts. Previous studies using anorganic bovine bone in bone defects demonstrate significant long-term bone formation, attributing osteoconductive properties to this biomaterial.14,15,17,18 In our study, the bone defects exhibited new bone formation in both experimental groups, varying between the central and peripheral areas, suggesting the influence of local tissue and the calvarial bone in providing osteogenic cells,36 and also the biomaterial osteoconductive capacity, which clearly maintained the original calvarial bone volume. In both experimental groups, there was bone ingrowth in contact with the surface of the biomaterial's particles. These particles seem to act like a scaffold, supporting the formation of new bone.37 Osteoconductive potential of bovine anorganic bone was confirmed, and the material porosity contributed to an increase in the surface area, favoring the recruitment of a large number of cells surrounding the graft particles.37 

Bovine anorganic bone resulted in a foreign body reaction with moderate presence of giant cells. However, the presence of these cells occurs in an attempt by the organism to resorb the material. These cells act as phagocytic macrophages, cleaning the graft surface and preparing it for deposition of newly formed bone. The presence of giant cells around the biomaterial particles and bone ingrowth along with graft material after a 4-week period suggest that anorganic bone may be slowly resorbed.38 The resorption rate appears to be directly related to the amount of new bone formation.14 

Marx39,40 demonstrated promising results when treating mandibular defects using a combination of autogenous bone graft with growth factors contained in PRP. It is reasonable to think that increasing the concentration of platelets in a bone defect may improve bone formation. The association of the biomaterial to the PRP allows the use of biomaterial's osteoconductive potential14,15 in conjunction with the osteoinductive properties of the PRP.27 In vivo studies claimed the successful use of PRP associated with artificial bone substitutes for sinus floor augmentation41 and treatment of intraosseous periodontal defects.28 Nevertheless, corroborating with our findings, other studies have failed to provide evidence of the positive effect of PRP combined with various artificial bone materials on bone regeneration. 23,42,43 

Platelets are known to be effective during the early stage of bone graft healing44,45 because the life span of a platelet in a wound and the period of direct influence of its growth factors are less than 5 days.46 Therefore, a pronounced effect of PRP supposedly occurs, especially during the early stages of bone regeneration.44,45 This suggests that its effectiveness cannot be seen in long-term evaluations47,48 of 4-week survival like in our study, but probably would be more significant if the analysis was made during the first weeks of bone healing.

Conclusion

Bovine anorganic bone, isolated or mixed with PRP, was biocompatible and osteoconductive, maintaining bone volume at the 4-week postoperative time point.

Abbreviations

     
  • BAB

    bovine anorganic bone

  •  
  • PRP

    platelet-rich plasma

References

References
1.
Foitzik
C
,
Staus
H
.
Le Fort I osteotomy in atrophied maxilla and bone regeneration with pure-phase beta-tricalcium phosphate and PRP
.
Implant Dent
.
2003
;
12
:
132
139
.
2.
Lye
KW
,
Deatherage
JR
,
Waite
PD
.
The use of demineralized bone matrix for grafting during Le Fort I and chin osteotomies: techniques and complications
.
J Oral Maxillofac Surg
.
2008
;
66
:
1580
1585
.
3.
Maus
U
,
Andereya
S
,
Gravius
S
,
et al.
How to store autologous bone graft perioperatively: an in vitro study
.
Arch Orthop Trauma Surg
.
2008
;
128
:
1007
1011
.
4.
Lohmann
H
,
Grass
G
,
Rangger
C
,
Mathiak
G
.
Economic impact of cancellous bone grafting in trauma surgery
.
Arch Orthop Trauma Surg
.
2007
;
127
:
345
348
.
5.
Younger
EM
.
Chapman
MW
.
Morbidity at bone graft donor sites
.
J Orthop Trauma
.
1989
;
3
:
192
195
.
6.
Barone
A
,
Crespi
R
,
Aldini
NN
,
Fini
M
,
Giardino
R
,
Covani
U
.
Maxillary sinus augmentation: histologic and histomorphometric analysis
.
Int J Oral Maxillofac Implants
.
2005
;
20
:
519
525
.
7.
Butz
SJ
,
Huys
LW
.
Long-term success of sinus augmentation using a synthetic alloplast: a 20 patients, 7 years clinical report
.
Implant Dent
.
2005
;
14
:
36
42
.
8.
Schizas
C
,
Triantafyllopoulos
D
,
Kosmopoulos
V
,
Tzinieris
N
,
Stafylas
K
.
Posterolateral lumbar spine fusion using a novel demineralized bone matrix: a controlled case pilot study
.
Arch Orthop Trauma Surg
.
2008
;
128
:
621
625
.
9.
Baptista
AD
,
Sornilha
A
,
Tormes
TAM
,
et al.
Estudo histológico dos enxertos ósseos homólogos humanos
.
Acta Ortop Bras
.
2003
;
11
:
220
224
.
10.
Grageda
E
.
Platelet-rich plasma and bone graft materials: a review and a standardized research protocol
.
Implant Dent
.
2004
;
13
;
301
309
.
11.
Silva
FM
,
Cortez
AL
,
Moreira
RW
,
Mazzonetto
R
.
Complications of intraoral donor site for bone grafting prior to implant placement
.
Implant Dent
.
2006
;
15
:
420
426
.
12.
Damien
CJ
,
Parsons
JR
,
Prewett
AB
,
Huismans
F
,
Shors
EC
,
Holmes
RE
.
Effect of demineralized bone matrix on bone growth within a porous material: a histologic and histometric study
.
J Biomater Appl
.
1995
;
9
:
275
288
.
13.
Sciadini
MF
,
Dawson
JM
,
Johnson
KD
,
Evaluation of bovine-derived bone protein with a natural coral carrier as a bone-graft substitute in a canine segmental defect model
.
J Orthop Res
.
1997
;
15
:
844
857
.
14.
Norton
MR
,
Odell
EW
,
Thompson
ID
,
Cook
RJ
.
Efficacy of bovine bone mineral for alveolar augmentation: a human histologic study
.
Clin Oral Implants Res
.
2003
;
14
:
775
783
.
15.
Sartori
S
,
Silvestri
M
,
Forni
F
,
Icaro Cornaglia
A
,
Tesei
P
,
Cattaneo
V
.
Ten-year follow-up in a maxillary sinus augmentation using anorganic bovine bone (Bio-Oss). A case report with histomorphometric evaluation
.
Clin Oral Implants Res
.
2003
;
14
:
369
372
.
16.
Sculean
A
,
Chiantella
GC
,
Windisch
P
,
Arweiler
NB
,
Brecx
M
,
Gera
I
.
Healing of intra-bony defects following treatment with a composite bovine-derived xenograft (Bio-Oss Collagen) in combination with a collagen membrane (Bio-Gide PERIO)
.
J Clin Periodontol
.
2005
;
32
:
720
724
.
17.
Zambuzzi
WF
,
Oliveira
RC
,
Alanis
D
,
et al.
Microscopic analysis of porous microgranular bovine anorganic bone implanted in rat subcutaneous tissue
.
J Appl Oral Sci
.
2005
:
13
:
382
386
.
18.
Zambuzzi
WF
,
Oliveira
RC
,
Pereira
FL
,
Cestari
TM
,
Taga
R
,
Granjeiro
JM
.
Rat subcutaneous tissue response to macrogranular porous anorganic bovine bone graft
.
Braz Dent J
.
2006
:
17
:
274
278
.
19.
Mott
DA
,
Mailhot
J
,
Cuenin
MF
,
Sharawy
M
,
Borke
J
.
Enhancement of osteoblast proliferation in vitro by selective enrichment of demineralized freeze-dried bone allograft with specific growth factors
.
J Oral Implantol
.
2002
;
28
:
57
66
.
20.
Herten
M
,
Rothamel
D
,
Schwarz
F
,
Friesen
K
,
Koegler
G
,
Becker
J
.
Surface- and nonsurface-dependent in vitro effects of bone substitutes on cell viability
.
Clin Oral Investig
.
2009
;
13
:
149
155
.
21.
Kassolis
JD
,
Rosen
PS
,
Reynolds
MA
.
Alveolar ridge and sinus augmentation utilizing platelet-rich plasma in combination with freeze-dried bone allograft: case series
.
J Periodontol
.
2000
;
71
:
1654
1661
.
22.
Wiltfang
J
,
Kloss
FR
,
Kessler
P
,
et al.
Effects of platelet-rich plasma on bone healing in combination with autogenous bone and bone substitutes in critical-size defects. An animal experiment
.
Clin Oral Implants Res
.
2004
;
15
:
187
193
.
23.
Aghaloo
TL
,
Moy
PK
,
Freymiller
EG
.
Evaluation of platelet-rich plasma in combination with freeze-dried bone in the rabbit cranium. A pilot study
.
Clin Oral Implants Res
.
2005
;
16
:
250
257
.
24.
Kim
E
,
Park
E
,
Choung
P
.
Platelet concentrates and its effect on bone formation in calvarial defects: an experimental study in rabbits
.
J Prosthet Dent
.
2001
;
86
:
428
433
.
25.
Landesberg
R
,
Burke
A
,
Pinsky
D
,
et al.
Activation of platelet-rich plasma using thrombin receptor agonist peptide
.
J Oral Maxillofac Surg
.
2005
;
63
:
529
535
.
26.
Lysiak-Drwal
K
,
Dominiak
M
,
Solski
L
,
et al.
Early histological evaluation of bone defect healing with and without guided bone regeneration techniques: experimental animal studies
.
Postepy Hig Med Dosw (online)
.
2008
;
62
:
282
288
.
27.
Kim
SG
,
Kim
WK
,
Park
JC
,
Kim
HJ
.
A comparative study of osseointegration of Avana implants in a demineralized freeze-dried bone alone or with platelet-rich plasma
.
J Oral Maxillofac Surg
.
2002
;
60
:
1018
1025
.
28.
Pradeep
AR
,
Shetty
SK
,
Garg
G
,
Pai
S
.
Clinical effectiveness of autologous platelet-rich plasma and peptide-enhanced bone graft in the treatment of intrabony defects
.
J Periodontol
.
2009
;
80
:
62
71
.
29.
Hatakeyama
M
,
Beletti
ME
,
Zanetta-Barbosa
D
,
Dechichi
P
.
Radiographic and histomorphometric analysis of bone healing using autogenous graft associated with platelet-rich plasma obtained by 2 different methods
.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod
.
2008
;
105
:
e13
e18
.
30.
Harris
D
,
Farrel
B
,
Block
MS
.
Zygomatic arch defects grafted with mineralized bone with PRP or PPP in dogs
.
J Oral Maxillofac Surg
.
2003
;
61
(
8 suppl 01
):
42
.
31.
Plachokova
AS
,
van den Dolder
J
,
Stoelinga
PJ
,
Jansen
JA
.
The bone regenerative effect of platelet-rich plasma in combination with an osteoconductive material in rat cranial defects
.
Clin Oral Implants Res
.
2006
;
17
:
305
311
.
32.
Santana
SI
,
Marques
LAP
,
Silva
CJ
,
Marquez
IM
,
Zanetta- Barbosa
D
.
Processo de reparo de cavidade óssea cirúrgica preenchida ou não com plasma rico em plaquetas: estudo radiográficoem calvária de coelhos
.
RBP: Rev Bras Implantodont Protese Implant
.
2006
;
13
:
51
60
.
33.
Harkness
JE
,
Wagner
JE
.
Biologia e Clínica de Coelhos e Roedores
.
São Paulo, Brazil
:
Roca;
1993
.
34.
Nunamaker
DM
.
Experimental model of fracture repair
.
Clin Orthop Relat Res
.
1998
;(
355 suppl
):
S56
S65
.
35.
Manson
PN
.
Facial bone healing and bone grafts. A review of clinical physiology
.
Clin Plast Surg
.
1994
;
21
:
331
348
.
36.
Gosain
AK
,
Santoro
TD
,
Song
LS
,
Capel
CC
,
Sudhakar
PV
,
Matloub
HS
.
Osteogenesis in calvarial defects: contribution of the dura, the pericranium, and the surrounding bone in adult versus infant animals
.
Plast Reconstr Surg
.
2003
;
112
:
515
527
.
37.
Xu
H
,
Shimizu
Y
,
Asai
S
,
Ooya
K
.
Experimental sinus grafting with the use of deproteinized bone particles of different sizes
.
Clin Oral Implants Res
.
2003
;
14
:
548
555
.
38.
Jensen
SS
,
Broggini
N
,
Hjørting-Hansen
E
,
Schenk
R
,
Buser
D
.
Bone healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs
.
Clin Oral Implants Res
.
2006
;
17
:
237
243
.
39.
Marx
RE
,
Carlson
ER
,
Eichstaedt
RM
,
Schimmele
SR
,
Strauss
JE
,
Georgeff
KR
.
Platelet-rich plasma
.
Oral Surg Oral Med Oral Pathol
.
1998
;
85
:
638
646
.
40.
Marx
RE
.
Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation
.
J Oral Maxillofac Surg
.
2000
;
58
:
300
301, discussion
.
41.
Okuda
K
,
Tai
H
,
Tanabe
K
,
et al.
Platelet-rich plasma combined with a porous hydroxyapatite graft for the treatment of intrabony periodontal defects in humans: a comparative controlled clinical study
.
J Periodontol
.
2005
;
76
:
890
898
.
42.
Aghaloo
TL
,
Moy
PK
,
Freymiller
EG
.
Investigation of platelet-rich plasma in rabbit cranial defects: a pilot study
.
J Oral Maxillofac Surg
.
2002
;
60
:
1176
1181
.
43.
Tsay
RC
,
Vo
J
,
Burke
A
,
Eisig
SB
,
Lu
HH
,
Landesberg
R
.
Differential growth factor retention by platelet-rich plasma composites
.
J Oral Maxillofac Surg
.
2005
;
63
:
521
528
.
44.
Gerard
MP
,
Wotman
KL
,
Komáromy
AM
.
Infections of the head and ocular structures in the horse
.
Vet Clin North Am Equine Pract
.
2006
;
22
:
591
631, x–xi
.
45.
Kanno
T
,
Takahashi
T
,
Tsujisawa
T
,
Ariyoshi
W
,
Nishihara
T
.
Platelet-rich plasma enhances human osteoblast-like cell proliferation and differentiation
.
J Oral Maxillofac Surg
.
2005
;
63
:
362
369
.
46.
Choi
BH
,
Im
CJ
,
Huh
JY
,
Suh
JJ
,
Lee
SH
.
Effect of platelet-rich plasma on bone regeneration in autogenous bone graft
.
Int J Oral Maxillofac Surg
.
2004
;
33
:
56
59
.
47.
Klongnoi
B
,
Rupprecht
S
,
Kessler
P
,
et al.
Lack of beneficial effects of platelet-rich plasma on sinus augmentation using a fluorohydroxyapatite or autogenous bone: an explorative study
.
J Clin Periodontol
.
2006
;
33
:
500
509
.
48.
Lee
C
,
Nishihara
K
,
Okawachi
T
,
Iwashita
Y
,
Majima
HJ
,
Nakamura
N
.
A quantitative radiological assessment of outcomes of autogenous bone graft combined with platelet-rich plasma in the alveolar cleft
.
Int J Oral Maxillofac Surg
.
2009
;
38
:
117
125
.