Evaluation of implant osseointegration with different regeneration techniques in the treatment of...

Evaluation of implant osseointegration withdifferent regeneration techniques in thetreatment of bone defects around implants:an experimental study in a rabbit model

Isabel GuerraFernando Morais BrancoMario VasconcelosAmerico AfonsoHelena FigueiralRaquel Zita

Authors’ affiliations:Isabel Guerra, Raquel Zita, Private Practice,Oporto, PortugalFernando Morais Branco, Helena Figueiral,Department of Prosthodontics, School of DentalMedicine, University of Oporto, PortugalMario Vasconcelos, Department of Materials, School ofDental Medicine, University of Oporto, PortugalAmerico Afonso, Department of Histology, School ofDental Medicine, University of Oporto, Portugal

Corresponding author:Dr Isabel GuerraSchool of Dental MedicineUniversity of OportoRua Dr. Manuel Pereira da Silva4200-393 PortoPortugalTel.: þ 35 1918 162129Fax: þ 35 1227 162383e-mail: [email protected]

Key words: bone regeneration, collagen membrane, deproteinized bovine bone, guided tissue

regeneration, osseointegration, peri-implant defects, platelet rich in growth factors

Abstract

Aim: The aim of this study was to evaluate the osseointegration of implants placed in areas with

artificially created bone defects, using three bone regeneration techniques.

Material and methods: The experimental model was the rabbit femur (16), where bone defects were

created and implants were placed. The peri-implant bone defects were filled with a deproteinized

bovine bone mineral, NuOsst (N), NuOsst combined with plasma rich in growth factors (PRGF)

(Nþ PRGF), NuOsst covered by an RCM6 membrane (NþM), or remained unfilled (control group [C]).

After 4 and 8 weeks, the animals were euthanized and bone tissue blocks with the implants and the

surrounding bone tissue were removed and processed according to a histological protocol for hard

tissues on non-decalcified ground sections. The samples were studied by light and electron scanning

microscopy, histometric analysis was performed to assess the percentage of bone in direct contact with

the implant surface and a statistical analysis of the results was performed.

Results: In the samples analyzed 4 weeks after implantation, the percentage of bone tissue in direct

contact with the implant surface for the four groups were 57.66 � 24.39% (N), 58.62 � 20.37%

(Nþ PRGF), 70.82 � 20.34 % (NþM) and 33.07 � 5.49% (C). In the samples with 8 weeks of

implantation time, the percentage of bone in direct contact was 63.35 � 27.69% (N), 58.42 � 24.77%

(Nþ PRGF), 78.02 � 15.13% (NþM) and 40.28 � 27.32% (C). In terms of the percentage of bone

contact, groups N and NþM presented statistically significant differences from group C in the 4-week

trial test (Po0.05; ANOVA). For the 8-week results, only group NþM showed statistically significant

differences when compared with group C (Po0.05; ANOVA).

Conclusion: In conclusion, the NuOsst granules/RCM6 membrane combination presented a

percentage of bone contact with the implant surface statistically greater than in the other groups.

Oral rehabilitation with implants has allowed for

therapeutic solutions that are unquestionably

more advantageous for the patient, as it pertains

to function, esthetics and comfort.

Osseointegration was defined as the direct

contact between bone and implant detected by

light microscopy (Albrektsson et al. 1981). Clini-

cally, osseointegration may be defined as the rigid

fixation of an implant to the surrounding bone,

maintained during functional load (Cooper 1998).

However, the osseointegration is a concept that is

based on a histologic definition as well as on

clinical and radiographic definitions. Therefore,

the classification of an implant as osseointegrated

only by the elements obtained through clinical

and radiographic examinations is incorrect (Al-

brektsson et al. 1986).

Implant success could be defined, in the first

place, according to its osseointegration. There are

numerous published articles that confirm im-

plant success in both the medium and long-

term time frames (Albrektsson et al. 1986; Adell

et al. 1990). Insufficient amounts of bone in the

implant beds decrease the success rates (Lekholm

et al. 1986). Hence, considerable efforts have

been made to develop techniques and materials

that increase the host bone volume, thus increas-

ing the bone-to-implant contact. The most com-

monly used techniques in clinical practice that

promote bone regeneration around exposed im-

plant threads involve the four processes that

promote new bone formation: osteogenesis that

can be achieved with the use of autogenous hu-

man bone (Lang et al. 2003); osteoinduction,

creating cell differentiation by means of specific

growth factors (GFs) (Misch & Dietsh 1993);

osteoconduction, where a grafting material serves

as a scaffold for new bone formation (Jensen et al.

1996); and guided bone regeneration (GBR),

which allows space maintenance through the

Date:Accepted 29 May 2010

To cite this article:Guerra I, Branco FM, Vasconcelos M, Afonso A, Figueiral H,Zita R. Evaluation of implant osseointegration with differentregeneration techniques in the treatment of bone defectsaround implants: an experimental study in a rabbit model.Clin. Oral Impl. Res. 22, 2011; 314–322.doi: 10.1111/j.1600-0501.2010.02002.x

314 c� 2010 John Wiley & Sons A/S

mailto:[email protected]

use of barrier membranes, into which osteoblas-

tic cells can migrate and promote bone growth

(Buser et al. 1993; Dahlin 1994).

There are several bone-grafting materials that

can be used in bone regeneration procedures.

These include autogenous human bone, demi-

neralized freeze-dried human bone allograft, xe-

nographic bone substitutes such as deproteinized

bovine bone mineral (DBBM) and synthetic bone

substitutes such as hydroxyapatite and calcium-

phosphate compounds. Among these materials,

DBBM, alone or in association with membranes,

has been used successfully in several clinical

situations: periodontal defects (Camelo et al.

1998), post-extraction defects (Artzi et al.

2001), dehiscence defects around implants (Ber-

glundh & Lindhe 1997; Hammerle et al. 1998;

Mayfield et al. 2001) and sinus augmentation

(Valentini et al. 1998).

Among the techniques described, there is a

consensus among researchers that GBR is the

most predictable technique, presenting the best

results in bone regeneration within cavities peri-

implant defect (Lynch et al. 1991; Hammerle

et al. 1998; Hurzeler et al. 1998; Hockers et al.

1999; Zitmann et al. 2001; Oh et al. 2003).

Lately, considerable emphasis has been placed

on resorbable membranes, because they present a

great advantage over non-resorbable membranes,

given that the former does not need a second

surgical intervention for its removal. Several

investigations have demonstrated the success of

bone regeneration utilizing the resorbable mem-

branes alone (Hurzeler et al. 1998; Wang & Car-

roll 2001; Mardas et al. 2003) or associated with

grafting materials (Buser et al. 1996; Proussaefs

& Lozada 2003; Donos et al. 2004).

In the last few years, there has been intensive

research in the biomaterials area, namely in the

area of GFs. The efficacy of GFs, contained in

plasma rich in growth factors (PRGF) and in

platelet-rich plasma (PRP), in tissue regeneration

has been documented in numerous studies (Marx

et al. 1998; Anitua 1999; Fennis et al. 2002;

Furst et al. 2003). Marx (2001) was the first

author to describe the success of local application

of PRP to achieve bone regeneration in oral and

maxillofacial surgery. Although the regeneration

potential of PRP and PRGF has been highlighted

by several authors (Marx et al. 1998; Anitua

1999; Kassoulis et al. 2000), to date, the exact

role of PRP on bone processes is still unclear

(Schmitz & Hollinger 2001).

The aim of the present investigation was to

evaluate the osseointegration of implants placed

in bone defect areas. The filler material of the

peri-implant dehiscence was a DBBM alone,

together with PRGF, or in association with a

collagen membrane.

Material and methods

Surgical procedures

Sixteen adult rabbits, with a median weight of

3.5 � 0.6 kg, were used in this study. The ani-

mals were divided into four groups, with four

rabbits in each group, according to four treatment

modalities. The protocol was approved by the

ethical committee of the Portuguese Veterinary

Association.

All surgical procedures were performed under

general anesthesia, achieved by pre-anesthetic

sedation with 2% xylazine (Rompuns

2%, Bayer,

Kiel, Germany, 5 mg/kg, IM), and general an-

esthesia with 1% ketamine (Clorketams

1000,

Vetoquinol, Lure, France, 25 mg/kg, IM). These

procedures were followed by intubation and

maintenance with 2–3% isoflurane (Forenes

,

Abbot Lab., Kaveenborough, UK) and oxygen

(Gasin, Oporto, Portugal) for the duration of the

surgery.

The posterior legs of the animals were shaved

and disinfected with an iodide dermic solution

(Betadines

, Asta Medica, Lisbon, Portugal).

The surgical procedure was initiated by a 4 cm

incision on the top of the crest of the femur, and

mucoperiosteal flaps were gently raised. Irregula-

rities on the exposed bone surface were removed,

and the osseous cortical bone was removed in a

5 mm circular area in order to obtain similar bone

conditions to a D3/D4 bone density.

Preparation of the implant bed was carried out

according to the Aces

system protocol (ACE

Surgical Supply Co., Brockton, MA, USA). Be-

fore implant placement, standardized bone de-

fects were surgically prepared, involving three

walls of the implant bed (anterior, medial and

lateral), and measuring approximately 8 mm in

both length and width, to simulate a critical size

defect. A round burr was used under slow rota-

tion and with copious sterile saline irrigation.

Although individual differences in the ridge di-

mensions did not allow perfect standardization of

the defects, special effort was made to keep the

defect dimensions relatively constant.

Thirty-two ACEs

Surgical, commercially pure

titanium resorbable blast media (RBM) implants,

with a diameter of 3.3 mm and a length of

13 mm, were inserted. The implants were placed

so that four threads were exposed in the anterior,

medial and lateral walls of the implant bed. Only

the posterior wall was maintained intact, and in

that wall, the implant was fully covered with

bone (Fig. 1).

Both femurs of all animals underwent the

described surgical procedures, with the place-

ment of two implants in each animal.

In the right and left femur of each rabbit, the

defects were treated with one of the following

four treatment modalities: (1) grafting with

DBBM (NuOsst, ACE Surgical Supply Co.,

‘‘N’’) – animals 1, 2, 3 and 4; (2) grafting with

DBBM in association with PRGF (NuOsst, ACE

Surgical Supply Co., ‘‘NþPRGF’’) – animals 5,

6, 7 and 8; (3) grafting with DBBM in association

with a collagen membrane (NuOsst, RCM6

membrane, ACE Surgical Supply Co., ‘‘NþM’’)

– animals 9, 10, 11 and 12; and (4) no treatment

(control ,‘‘C’’) – animals 13, 14, 15 and 16 (Figs 1

and 2). The grafts used overbuilt the area of the

defects. The rabbits were randomly chosen for

each group.

All the implants were submerged with tension-

free mucoperiosteal flaps and suturing was car-

ried out in layers. The deep tecidular layer was

sutured using the horizontal mattress technique

Fig. 1. Group C (control). Alveolar crest exhibiting the implant after its placement.

Guerra et al �Evaluation of implant osseointegration with different regeneration techniques

c� 2010 John Wiley & Sons A/S 315 | Clin. Oral Impl. Res. 22, 2011 / 314–322

with a resorbable 3/0 Vicryls

suture (Johnson &

Johnson, New Brunswick, NJ, USA) and the

superficial layer was sutured with 3/0 silk suture

(B. Braun SSC AG, Sempach, Switzerland).

At the end of each surgical procedure, the

animals were radiographed using a 18 � 24 cm

standard film, which allowed the simultaneous

imaging of both femurs.

In the post-operative period, the rabbits were

given an analgesic (50% dipirone-Vetalgins

, In-

tervet, Munchen, Germany, 50 mg/kg, IM) as

well as antibiotic therapy with 2.5% eurofloxa-

cine (Baytril, KVP Pharma, Kiel, Germany,

5 mg/kg, SC). This medication was given once

a day for a period of 5 days.

At 4 and 8 weeks after implantation, the

animals were sacrificed with an overdose of

intravenous 5% sodium tiopental (Tiopental

0.5 g Brauns

, B. Braun Medical SA, Barcelona,

Spain, 30 mg/kg), to obtain biopsies representing

4 and 8 weeks of healing after implant installa-

tion. Animals 1, 2, 5, 6, 9, 10, 13 and 14 were

sacrificed at 4 weeks. Animals 3, 4, 7, 8, 11, 12,

15 and 16 were sacrificed at 8 weeks. Block

sections containing the implants and the over-

lying bone were harvested with extreme care to

preserve the area where the grafting materials

were implanted.

PRGF preparation

Blood was obtained several minutes before the

administration of anesthesia. Ten milliliters of

blood were drawn from each rabbit using 5 ml

tubes, which contained 10% trisodium citrate

solution as an anticoagulant. The tubes were

centrifuged at 391.3 g force for 8 min in accor-

dance with the protocol elaborated by Anitua

1999. The blood was thus separated into its three

basic components: red blood cells, which ap-

peared at the bottom of the tube; PRGF, which

appeared in the middle of the tube; and plasma

poor in growth factors (PPGF), which appeared at

the top of the tube. One milliliter of the PPGF

from each 5 ml tube was discarded. The remain-

ing plasma was collected and transferred to ep-

pendorf tubes, and 50ml of 10% calcium chloride

were added to each tube containing 1 ml of

PRGF. This mixture was then immediately

added to DBBM (NuOsst). After 15–20 min, a

PRGF gel formed. The time delay between the

PRGF gel formation and the filling of the defect

was standardized to 5–10 min.

Histological preparation

Before initiating histological preparation, the

block sections containing the test and control

sites were reduced in size, in order to decrease

fixation, dehydratation, impregnation and inclu-

sion times.

The first step in histological preparation was

fixation. The samples were fixed in 4% neutral

formaldehyde with a pH of 7.4, for a 24-h period.

The specimens were dehydrated in an ascending

series of alcohol rinses, 70%, 80%, 90% and

100% ethylic alcohol, respectively, for a period of

96 h in each alcohol concentration. The blocks

were impregnated with a 75% methyl–metha-

crylate solution for a period of 72 h. The samples

were finally placed in a solution contain-

ing 800 ml/l of methyl–methacrylate (BDH,

Merck Chemicals Ltd, Nottingham, UK),

200 ml/l of Plastoid (Plastoid N, Rohm Pharma,

San Diego, CA, USA) and 1 g/l of Perkadox

(Akzo Chemicals BV, Amersfoort, the Nether-

lands). To remove the air bubbles that usually

occur in the solution before polymerization, the

open bottles were placed in vacuum equipment for

10–15 min, then hermetically sealed and placed

in GFL water bath equipment (Gesellschaft fur

Labortechnik GmbH, Burgwedel, Germany) at

371C temperature for 48 h. After polymerization,

the specimens were sectioned along their long-

itudinal axis with a slow-speed diamond disc

(Acuttom, Struers, Denmark) into approximately

150–200-mm-thick sections. These sections were

processed in accordance with the requirements

for light and scanning electron microscopy.

The sections to be observed with the light

microscope (Leica DMLB, Heerbrugg, Switzer-

land) were ground and polished to a final thick-

ness of 40 � 10mm (P1200, 3M 314, AZoM,

Stoke-on-Trent, UK), and surface stained with

two colorizing techniques: (1) hematoxylin

(Harris Hematoxylin Acidified, Shandon Sci.

Ltd, Eppelheim, Germany) and eosin (Eosin Y,

Shandon Sci. Ltd, Eppelheim, Germany); (2)

Solocromo Cianine R (Polysciences Inc., War-

rington, UK).

The 150–200-mm-thick sections to be observed

with electron scanning microscopy (JEOL JSM-

35C, Tokyo, Japan) underwent careful silica disc

polishing, with decreasing granulometries of

#1000 and #1200. The samples were then coated

with a thin gold film through cathodic deposition

in Ion sputter equipment (Jeol Fine Coat, Ion

Sputter JFC 1100).

Histometry

The determination of the percentage of bone

contact between the implant surface and the

newly formed bone was carried out through

sample photographs with the utilization of a

curvimeter (Jansen et al. 1993). Five sections

were randomly selected, surface stained with

hematoxylin and eosin and Solocromo Cianine

R in each of the four treatment groups and for

each time period. The � 100 magnification in

light microscopy was used for histometric analy-

sis. In each section, two threads were isolated and

photographed, using an Olympus BH-2 photo-

graphy system (Olympus, Tokyo, Japan). This

corresponded to the first thread closest to implant

shoulder and the thread in the middle of the

surgically prepared bone defect. Linear measure-

ments were carried out directly with a curvi-

meter, first from the total perimeter of the two

selected threads in each implant, and then from

the perimeter of direct bone contact between

newly formed bone and implant surface of those

two threads.

Bone contact percentage was calculated in

accordance with the following formula:

% bone contact ¼bone contact perimeter of two threads

total perimeter of two threads

� 100

Statistics

The data were analyzed with Statistical Pack-

age for Social Sciences version 18.0 (PASW

Fig. 2. The exposed threads of the implants undergoing regenerative procedures. (a) Group N (NuOsst); (b) Group NþPRGF (NuOsstþPRGF); (c) Group NþM (NuOsstþRCM6

membrane).


316 | Clin. Oral Impl. Res. 22, 2011 / 314–322 c� 2010 John Wiley & Sons A/S

Statisticss

18, IBM, Armonk, NY, USA) using

the most appropriate techniques for the variables

involved. Given the nature of the variables in-

volved and the experimental design carried out,

the statistical analysis was the application of

techniques of ANOVA (one-way ANOVA) for

each time period separately. All the assumptions

associated with ANOVA were evaluated pre-

viously (normality of the dependent variable

was determined using the Kolmogorov–Smirnov

test and homogeneity of variance was determined

using Levene’s test).

The decision rule used is to detect statistically

significant evidence for probability values (value

of the evidence test, P) o0.05.

Results

All rabbits recovered well from the anesthesia

and the surgical interventions. They all behaved

normally throughout the remainder of the study.

At sacrifice, no clinical signs of inflammation or

adverse tissue reactions could be seen. All im-

plants were still in situ at sacrifice.

On histological examination, apparent osseoin-

tegration of all experimental groups and the con-

trol group were observed. No space was observed

between the implant surface and the newly

formed bone. No tissue specimens were lost or

damaged during the processing procedures and all

were available for analysis.

Qualitative histology

Under light microscopy, the two staining proce-

dures allowed the distinction between pre-exist-

ing and newly formed bone. In hematoxylin and

eosin staining, the new bone was stained in light

stained pink, while in Solocrome Cianine R,

the new bone assumed a red color. In all groups,

the new bone primarily consisted of intensely

stained woven bone with a high number of

osteoblasts and blood vessels. The regenerated

bone close to the pre-existing bone, however,

exhibited a more lamellar structure. A high

number of osteocytes and numerous Haversion

systems were observed in the newly formed bone

compared with the pre-existing bone. In addition,

the vascularization of the regenerated bone was

more pronounced.

The scanning electron microscopy allowed a

more detailed analysis of the interfaces between

the newly formed bone and the implant surface,

and between the grafting material and the newly

formed bone. The observations with this type of

microscopy allowed for the viewing of different

mineralization stages of the bone matrix, which

presented three different stains: dark gray for the

newly formed bone; lightly stained gray for the

mature bone tissue; and an even lighter gray stain

for the NuOsst granules.

Figs 3 and 4 show the histological evaluation of

all grafting materials at 4 and 8 weeks.

4 Weeks

Group 1(N). There was great NuOsst granule

dispersion. All histological samples presented

Fig. 3. Histologic evaluation of all grafted materials at 4 weeks. Group N (NuOsst): (a) and (b); Group NþPRGF: (c) and (d);

Group NþM: (e) and (f); Group C: (g) and (h). Hematoxylin and eosin, magnification � 25 – (a), (c), (e) and (g). Electron

scanning microscopy, magnification � 54 – (b), (d) and (f); magnification � 30 – (h).



reduced immature bone tissue formations. The

bone fill around the implant threads were very

restricted, with some cases of uncovered threads.

The newly formed bone between the NuOsst

granules was also very deficient. Every sample

showed that the growth of bone tissue was more

pronounced near the implant surface, surround-

ing the existing NuOsst granules in most of their

perimeter.

Group 2 (NþPRGF). A large number of the

NuOsst granules were identified near each

other, and near the implant surface. New bone

tissue deposition was in evidence along the im-

plant threads and surrounding the granules that

were near the surface. However, the bone contact

with the implant surface was still deficient

around some of the threads.

Group 3 (NþM). There were a high number of

NuOsst granules near the implant surface and

near each other. The samples presented signs of

high osteoconductivity with the NuOsst granules

almost totally surrounded by the newly formed

bone and with depth contact in the interface. Bone

deposition was verified, not only in the NuOsst

granules nearest the implant surface, surrounding

these in almost the total length of their perimeter,

but also of the furthest granules. Bone fill in all

implant threads also occurred. Some samples

showed traces of the RCM6 membrane delimiting

the NuOsst granules placed in the defected bone

area.

Group 4 (C). The control group presented the

worst bone regeneration, evidenced by absence of

bone growth surrounding some implant threads.

Immature bone deposition was observed in the

threads nearest to the base of the bone defect. In

the exposed thread areas small amounts of fi-

brous connective tissue were found between the

bone and the implant. In the bone defect area

further from the implant there were several

empty interstitial spaces without bone fill.

8 Weeks

Group 1(N). The contact between the newly

formed bone and the implant surface had in-

creased. A greater maturation of the bone tissues

deposited in the interface with the implant was

also observed. The NuOsst granules nearest to

the implant surface were surrounded by bone

tissue in most of their perimeter. The bone growth

in the defected area furthest from the implant was

still very deficient, without any bone tissue sur-

rounding the NuOsst granules. It was also possi-

ble to observe interstitial spaces of large

dimensions.

Group 2 (NþPRGF). This group presented a

large number of NuOsst granules near each other

and near the implant surface. The quantity of new

bone tissue rose along the experimental time and

showed a more developed maturation status, pre-

senting a large number of Haversion systems.

New bone depositions in the implant threads

rose along the experimental trial. However, the

contact between the bone tissue and the implant

surface was still deficient in some of the threads.

Fig. 4. Histologic evaluation of all grafted materials at 8 weeks. Group N (NuOsst): (a) and (b); Group NþPRGF: (c) and (d);

Group NþM: (e) and (f); Group C: (g) and (h). Hematoxylin and eosin, magnification � 25 – (a) and (e). Solocromo Cianine

R, magnification � 25 – (c) and (g). Electron scanning microscopy, magnification � 54 – (b), (d) and (f); magnification

� 30 – (h).



The interstitial spaces between the NuOsst gran-

ules nearest the implant surface were almost

entirely fulfilled by new bone tissue. The

NuOsst granules furthest from the implant still

showed reduced bone tissue between them, pre-

senting several unfulfilled interstitial spaces.

Group 3 (NþM). This group showed the

presence of a high quantity of NuOsst granules

near the implant surface and near each other. The

percentage of newly formed bone increased dur-

ing trial time, resulting in homogeneous filling of

all implant threads, without exception, and with

depth contact with the interface. There was also

an increase of bone tissue surrounding the

NuOsst granules nearest the implant surface,

covering almost the total perimeter by newly

formed bone. The samples showed a more devel-

oped maturation status. The matured bone tissue

presented reduced quantities of osteoid tissue, a

smaller number of osteoblasts as well as dimin-

ished number and caliber of blood vessels. None

of the samples revealed traces of the RCM6

membrane.

Group 4 (C). The growth of bone tissue pre-

sented by this group remained deficient, showing

absence of bone tissue in the first thread of the

implant. New bone deposition only occurred near

the implant surface, with no bone formation in

areas far from the implant. A layer of unminer-

alized tissue was identified in the defected area

near the implant. Trial time presented a matura-

tion of the newly formed bone tissue. Detailed

observation of the interface showed, in the ma-

jority of the analyzed sites, lack of direct contact

between the implant surface and bone tissue.

Histometric analysis

The mean percentages of new bone formation in

groups 1, 2, 3 and 4 are shown in Table 1.

The percentage of bone tissue in direct contact

with the implant surface, 4 weeks after implanta-

tion, was 57.66� 24.39% for the N group;

58.62� 20.37% for the NþPRGF group;

70.82� 20.34% for the NþM group; and

33.07� 5.49% for the C group. In the sections

with 8 weeks of implantation time, the percentage

of bone in direct contact was 63.35� 27.69% for

the N group; 58.42� 24.77% for the NþPRFC

group; 78.02� 15.13% for the NþM group; and

40.28� 27.32% for the C group.

From the Kolmogorov–Smirnov test for nor-

mality of the dependent variable (the amount of

bone-to-implant contact) at 4 and 8 weeks, it was

found that this could be considered normal

(Z¼0.957, P¼0.319 and Z¼0.774, P¼0.587,

respectively).

To evaluate the homogeneity of variance, Le-

vene’s test was carried out at 4 and 8 weeks, and

homogeneity of variance was observed among

groups [F(3,16)¼2.935, P¼0.065 and F(3,16)¼1.662, P¼0.215, respectively].

Given these results, the conditions of applic-

ability of the ANOVA for comparison between

groups are satisfied.

Based on ANOVA results, with regard to direct

implant–bone contact, the NþM group pre-

sented statistically significant differences when

compared with the C group, in the 4-week trial

test (Bonferroni multiple comparisons test pre-

sents Po0.05). At 8 weeks, any group presented

statistically significant differences when com-

pared with the C group (F (3, 16)¼2.058,

P40.05; ANOVA).

In summary, the following findings were de-

termined:

1. The highest rate of bone growth occurred in

the NþM group.

2. The bone growth rate was very similar in the

N group and the NþPRGF group.

3. The lowest rate of bone growth was present

in the C group.

Discussion

The threaded portion of all ACE dental implants

in the present study was osseointegrated. The

results demonstrated that surgically created bone

defects healed with bone fill and osseointegration

to the implant surface when grafted with

NuOsst, whether or not the defect was grafted

with PRGF or covered with an RCM6 membrane.

Bone defects around dental implants are ob-

served frequently when implants are placed in

areas with inadequate bone, i.e., dehiscence de-

fects, fenestration defects, residual intraosseous

defects and defects associated with extraction

sockets. In all of these situations, bone regenera-

tion of the defect would improve the long-term

prognosis for the implant (Mayfield et al. 2001).

In the attempt to stimulate osteogenesis and

reconstruct bone defects in cases of various types

of injury, diseases and congenital malformation

in dentistry, various types of bone grafts or

biomaterials have been used to treat the defects

(Lekovic et al. 2002).

DBBM is one of the bone graft materials more

commonly used in bone regeneration procedures,

because it presents a chemical composition and

structure similar to human bone tissue (Yildirim

et al. 2000). Numerous studies have shown that

inorganic bovine bone exhibits osteoconductive

properties, providing a physical matrix where

osteogenic cells can migrate and settle, creating

a new bone tissue (Wetzel et al. 1995; Hammerle

et al. 1997). The results of the present study

demonstrated that the DBBM (NuOsst) exhibits

the same osteoconductive properties and that it

can successfully be used for GBR procedures in

dehiscence defects.

It has also been reported that DBBM is well

integrated by the host bone (Hammerle et al.

1998). In the present investigation, these earlier

findings have not only been confirmed in princi-

ple, but it has also been demonstrated that a very

high surface fraction of the grafting particles was

actually in direct contact with bone.

In some of the specimens of the present study,

contact between particles of NuOsst and the

implant surface was observed, indicating that the

use of grafting materials may hinder the migra-

tion of bone-forming cells onto the surface of the

implants. These findings are in contrast with the

observations of some investigators that there is

no contact between particles of the graft materials

and the implant surface (Berglundh & Lindhe

1997; Hammerle et al. 1998; Hockers et al.

1999).

Conflicting results have been reported regard-

ing the long-term performance of DBBM. While

some investigators have reported lack of break-

down (Dies et al. 1996; Hammerle et al. 1997),

others have described signs of resorption (Klinge

et al. 1992; Wetzel et al. 1995), or even docu-

mented a decrease in area density of the graft over

time (Berglundh & Lindhe 1997). In the present

study, resorptive activity of the NuOsst particles

by osteoclasts has not been demonstrated.

The results of this study showed that a resorb-

able collagen membrane provided an effective

barrier, aiding in the regeneration of bone into

peri-implant defects. Clearly, better results were

obtained in the group with a membrane (NþM)

as compared with the three groups without

membrane placement (N, NþPRGF and con-

trol). Apparently, the use of an RCM6 membrane

had a more pronounced influence on the degree of

bone regeneration than the placement of the bone

Table 1. Percentage of direct bone-to-implant contact

Mean � SD

4 weeks 8 weeks

NuOsst 57.66 � 24.39 63.35 � 27.69NuOsst/PRGF 58.62 � 20.37 58.42 � 24.77NuOsst/RCM6 membrane 70.82 � 20.34 78.02 � 15.13Control group 33.07 � 5.49 40.28 � 27.32

PRGF, plasma rich in growth factors.



substitutes. This is in agreement with previously

published experiments, where the use of bone

grafts or substitute materials alone yielded poor

results (Pinholt et al. 1992; Jensen et al. 1995),

while better results were obtained when grafting

materials were combined with membranes (Jen-

sen et al. 1995). Hence, it seems that the collagen

membrane, with its bilayer structure used in this

experiment, improved soft tissue healing and

subsequent integrity. This effect may have been

caused by cellular adhesion to the membrane

surface, stabilization of the blood clot and inte-

gration of the membrane by the proliferating

connective tissues.

PRGF gel is one of the latest techniques for

promoting osteogenisis, thereby strengthening

the effects of bone-graft substitutes (Marx et al.

1998). PRGF is obtained by sequestering and

concentrating platelets, using gradient density

centrifugation (Anitua 1999). Platelets contain

angiogenic, mitogenic and vascular GFs in their

granules, which stimulate bone growth (Maloney

et al. 1998). Therefore, it is a reasonable hypoth-

esis that increasing the concentration of platelets

in a bone defect may lead to improved and faster

healing. Many researchers have described the

successful use of PRGF gel in oral and maxillo-

facial surgery, in conjuction with the following:

ablative surgery of the maxillofacial region, man-

dibular reconstruction, sinus lift bone augmenta-

tion, root apex removal, periapical cyst removal,

tooth extraction, alveolar ridge augmentation,

surgical repair of alveolar clefts and oro-antral/

oro-nasal fistulae, and in adjunctive procedures

related to the placement of osseointegrated im-

plants (Anitua 1999).

However, little evidence exists for the ability of

these GFs to improve bone healing when added to

osteoconductive materials (Kim et al. 2001,

2002). Nevertheless, some authors maintain

that the use of PRGF with allogenic or xenogenic

bone facilitates new bone formation when auto-

genous bone grafting is difficult or not indicated

for use (Kim et al. 2002). Kim et al. 2001 were

the first authors to introduce the rabbit as an

experimental animal, while utilizing the techni-

que of concentrating platelets. In that study, they

evaluated the effect of PRP on bone formation

when associated with inorganic bovine bone

(BioOsss

, Geistlich Pharma AG, Wolhusen,

Switzerland), in cranial defects in 20 New Zeal-

and rabbits. The defects were filled with

BioOsss

þPRP in the experimental group and

with BioOsss

alone in the control group. The

animals were sacrified at 4 and 8 weeks. The

bone formation was evaluated in periapical RX

and TC scan. These investigators described

greater bone density in the grafts with

BioOsss

þPRP, and concluded that the PRP

technique in association with bovine bone

mineral enhances bone formation.

Marx et al. 1998 also reported that adding PRP

to grafts resulted in a radiographic maturation

rate of 1.62–2.16 times that of grafts without

PRP. In that study on histomorphometric analy-

sis, there was also greater bone density in grafts to

which PRP had been added (74 � 11%) than in

grafts without PRP (55.1 � 8%).

The present study does not provide any quan-

titative analysis of the amount of regenerated

bone in all portions of the defect area. The

histomorphometric analysis remains to be done

on the regenerated bone. Future studies should

clarify these issues.

Nevertheless, from the histologic findings in

the present investigation, it can be concluded that

for bone formation, the combination of the

NuOsst granules with PRGF (NþPRGF) was

advantageous when compared with the NuOsst

group (N). In the histologic analysis, a greater

newly formed bone was observed in the

NþPRGF group, especially for the NuOsst

granules that were close to the implant surface.

One may theorize that the results could be

explained by the agglutinant effect of PRGF,

which allowed the granules to be placed both

closer to each other and in greater numbers,

compared with the N group, thus promoting

greater bone formation. These observations are

in agreement with the studies referred to pre-

viously, but are in contrast with other investiga-

tions. Wiltfang et al. 2004 compared the

regenerative potential of autogenous bone, trical-

cium-phosphate granules (CeraSorbt, Curasan

AG, Kleinostheim, Germany), bovine spongious

blocks (BioOsss

) and a bovine bone inducing

collagenous sponge (Collosst, Vetcell, Burford,

UK) with or without PRP, in critical-sized defects

in mini-pigs. The animals were sacrificed after 2,

4 and 12 weeks. The specimens were evaluated

microradiographically and immunohistologi-

cally. These authors reported that PRP did not

confer additional benefit when xenogenic bone

substitutes were applied.

The results about the effect of PRGF on bone

reossification are still controversial. Further ex-

perimental studies with a standardized critical-

sized defect, designed to evaluate this effect, are

necessary.

In the present study, it is noteworthy that the

combination of NuOsst with PRGF did not

increase the percentage of bone contact at the

implant interface, when compared with the

NuOsst group alone. In fact, the fraction of

bone-to-implant contact was very similar in the

two groups. The specific reason for this similarity

remains unknown, but one possible hypothesis is

that in the NþPRGF group, the space between

the granules is occupied by the PRGF, while in

the N group, the granules that are close to the

implant surface occupy a more condensed space,

promoting bone deposition in that area, namely

between the threads of the implant. These results

are in accordance with the study of Froum et al.

2002. They tested the efficacy of PRP in three

bilateral sinus graft cases with grafts of inorganic

bovine bone (BioOsss

), with or without PRP.

Histomorphometric analysis indicated that the

addition of PRP to the grafts did not make a

significant difference in interfacial bone contact

on the test implants, with percentages of bone-to-

implant contact of 37.6% and 38.8% in the

BioOsss

þPRP group and 33.8% in the BioOsss

group.

Furst et al. 2003, working with mini-pigs,

studied the efficacy of BioOsss

and the associa-

tion of BioOsss

with PRP, as graft materials in

sinus graft surgery with simultaneous implant

placement. The animals were sacrificed after 3, 6

and 12 weeks. At 6 weeks, the histomorpho-

metric analysis demonstrated lower percentages

of bone-to-implant contact in the group with PRP

(15.9%) compared with the group without PRP

(22.5%). At 12 weeks, the BioOsss

þPRP group

(34%) equaled the BioOsss

(34.8%) group. These

authors concluded that BioOsss

in association

with PRP was not more effective than the utili-

zation of BioOsss

alone.

The amount of bone-to-implant contact was

clearly higher in the group treated with a mem-

brane (NþM) compared with the other three

groups. The group with the RCM6 membrane

was the only one to present significant differ-

ences in direct bone-to-implant contact in

the previously created defect area at 4 weeks.

The NþM group presented a high rate of bone-

to-implant contact in the two implantation

times: 70.82 � 20.34% at 4 weeks and

78.02 � 15.13% at 8 weeks, respectively. The

immobilization of the NuOsst granules, created

by the RCM6 membrane, may be responsible for

these high percentages.

These results are in agreement with another

study that has demonstrated high bone-to-implant

contact fractions, as a result of GBR use in the

treatment of dehiscence defects (Hammerle et al.

1998). Four treatment modalities were evaluated

in that study: the application of ePTFE membranes

alone, ePTFE membranes in conjunction with

BioOsss

, BioOsss

alone and control sites receiving

neither membrane nor bone grafts. The groups

using membranes or membranes and BioOsss

revealed direct bone-to-implant contact fractions

of 55% and 65% respectively.

Other investigators, however, reported mini-

mal bone-to-implant contact following bone re-

generation (Wachtel et al. 1991; Simion 1994;



Becker et al. 1995). Only minimal contact was

found in an animal experimental study, where

dehiscence defects were augmented with ePTFE

membranes alone, ePTFE membranes and auto-

genic bone or ePTFE membranes and deminer-

alized freeze-dried bone allografts (Becker et al.

1995). A human pilot study similarly reported

excellent bone regeneration in dehiscence defects,

but only minimal new bone-to-implant contact

(Palmer et al. 1994). Hockers et al. 1999 also

reported low percentages of direct bone-to-

implant contact in the area intended for

bone regeneration: 20.3% for the BioGuides

(Geistlich Pharma AG, Wolhusen, Switzerland)

þBioOsss

group and 17.3% for the group with

BioGuides

alone. These results are in contrast

with the findings of the present study (70.82%

and 78.02%, respectively, at 4 and 8 weeks, for

the NþM group). The reasons for this difference

are obscure and need to be clarified in future

experiments. One possible reason for the low

percentages in the last study mentioned could

be related to the implant surface. In that experi-

ment, the implants had a machined surface (Im-

plant Innovation Inc., Biomet 3i, Warsaw, IN,

USA), while the present investigation made use

of the ACEs

implants with an RBM surface.

Studies by Piattelli et al. (1998, 1999) document

that the RBM surface leads to a higher contact

percentage with bone during osseointegration

than machined surfaces. Therefore, it is reason-

able to assume that the surface texture of the

titanium plays an important role when bone

grows into contact with an implant during a

GBR procedure.

In the present study, non-grafted defects (con-

trol group) demonstrated bone regeneration in

their lower portions only. The coronal portions

of the partially regenerated control defects con-

tained connective tissue. In addition, in all the

groups, there was a progressive increase in the

new bone volume over time.

In summary, despite the limitations of this

study, the results of the present experiments

demonstrate that the association of a bilayered

bioresorbable collagen membrane with DBBM

enhanced bone regeneration, with a large portion

of the regenerated bone in contact with the

implant surface. This study showed that the

implants placed in the host bone osseointegrated,

and a large amount of bone contact was achie-

ved with the implant in all the experimental

groups. However, the use of PRGF did not

facilitate bone-to-implant contact. In addition,

the DBBM graft was well integrated into the

regenerating bone, despite one part of DBBM

being surrounded by connective tissue. Results

of the present study indicate a positive effect of

the addition of an RCM6 membrane to NuOsst

in the treatment of bone defects around dental

implants.

Acknowledgements: The

investigators wish to express their special

thanks to Ana Mota for expert processing of

the histological specimens, Prof. Ramiro

Mascarenhas, Dr Andre Gomes, Dr Joao Nobre

and all the team of the National Zootechnic

Station of Santarem for their esteemed support

regarding the experimental surgery, and Prof.

A. Gonshor for valuable advice in the revision

of this paper. The present experiment was

funded partly by ACE Surgical Supply Co.,

which supplied the implants, the biomaterial

and the membranes. Pierre Fabre Dermo

Cosmetique also contributed to the graphic

impression of this work.

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