bio-oss® blocks combined with bmp-2 and vegf for the regeneration of bony defects and vertical...

Christian SchmittRainer LutzHendrik DoeringMichael LellJozsef RatkyKarl Andreas Schlegel

Bio-Oss® blocks combined with BMP-2 and VEGF for the regeneration ofbony defects and vertical augmentation

Authors’ affiliations:Christian Schmitt, Rainer Lutz, Hendrik Doering,Karl Andreas Schlegel, Department of Oral andMaxillofacial Surgery, University of Erlangen-Nuremberg, Erlangen, GermanyMichael Lell, Department of Radiology, Universityof Erlangen-Nuremberg, Erlangen, GermanyJozsef Ratky, Research Institute for AnimalBreeding and Nutrition, Herceghalom, Hungary

Corresponding author:Christian SchmittDepartment of Oral and Maxillofacial SurgeryUniversity of Erlangen-NurembergErlangen, GermanyTel.: +49 9131 8534191Fax: +49 9131 8534106e-mail: [email protected]

Key words: animal experiments, biomaterials, bone implant interactions, bone regeneration,

bone substitutes, growth factors, guided tissue regeneration

Abstract

Objectives: The aim of this study was to evaluate the bone formation rate and osseointegration of

Bio-Oss® blocks combined with rhBMP-2 and rhVEGF in bony defects and after vertical

augmentation.

Material and methods: Bio-Oss® blocks plus rhBMP-2 (BMP), Bio-Oss® blocks plus rhVEGF (VEGF),

or Bio-Oss® blocks plus rhBMP-2 and rhVEGF (BMPVEGF) were inserted in “critical size defects”

(CSD) in the calvariae of adult pigs. Control defects were filled with collagen carrier (Lyostypt®)

plus growth factors and untreated Bio-Oss® blocks (CO). In a second group, Bio-Oss® blocks plus

growth factors and untreated Bio-Oss® blocks were used for vertical augmentation of the calvariae.

In the first group, the investigation time was 30 days, in the second group it was 30 and 60 days.

The bone samples were investigated histomorphometrically, and the newly formed bone (BV/TV)

was judged by microradiographic investigation.

Results: In the CSD model, the newly formed bone in the region of interest was not significantly

different within the groups. In the second setting, the inserted bone blocks exhibited sufficient

volume stability with increasing bone formation up to 9.33% ± 3.92% for BMP, 10.42% ± 1.81%

for BMP/VEGF, 11.01% ± 4.78% for VEGF, and 10.02% ± 5.43% for the control group after 60 days.

Conclusion: In the chosen setting and time frame, de novo bone formation did not increase with

the additional use of growth factors.

The functional and esthetic rehabilitation of

atrophic edentulous jaws or jaw segments by

implant-fixed dentures often requires primary

surgical augmentation of the hard tissues

(Rocchietta et al. 2008). The aim of such pro-

cedures is to create sufficient vertical and

horizontal bone volume. Autogenous bone

block grafting is the accepted standard of care

(Hausamen & Neukam 1992; Neukam et al.

1994; Blokhuis & Arts 2011). These tissue

transplants have limited availability and

must be obtained in an accompanying proce-

dure, which involves risks such as infection,

bleeding, pain, swelling, and damage to

nerves and blood vessels (Ahlmann et al.

2002; Nkenke et al. 2004; Raghoebar et al.

2007; Weibull et al. 2009; Schaaf et al. 2010;

Blokhuis & Arts 2011). Alternatively, a vari-

ety of bone substitute materials are currently

available (Schlegel et al. 2003b, 2004; Thor-

warth et al. 2006; Zhou et al. 2011). The dis-

advantage is that these materials are not

suitable for vertical or horizontal augmenta-

tion due to lacking volume stability, and

their use is limited to space providing defects

(Schmitz & Hollinger 1986; Hollinger & Kle-

inschmidt 1990; Schlegel et al. 2006a; Por

et al. 2007). These disadvantages may be pre-

vented by implanting autogenous bone block

grafts or bone graft substitutes, such as Bio-

Oss® blocks (Chris Arts et al. 2006). To cir-

cumvent the osteo-inductive properties of

autogenous bone transplants for the regenera-

tion of “critical size defects” (CSD) with

bone substitute material, bioactive optimiza-

tion is a possible option (Stockmann et al.

2011).

Angiogenesis, osteogenesis, and bone

remodeling are closely associated processes

that share common mediators during repair

and bone development (Oringer 2002). Animal

experiments have shown that bone morpho-

genic proteins (BMPs) support bone repair

(Arosarena & Collins 2005). BMPs form a

unique group of proteins within the transform-

ing growth factor beta (TGF-beta) superfamily

Date:Accepted 18 September 2011

To cite this article:Schmitt C, Lutz R, Doering H, Lell M, Ratky J, Schlegel KA.Bio-Oss® blocks combined with BMP-2 and VEGF for theregeneration of bony defects and vertical augmentation.Clin. Oral Impl. Res. 00, 2011, 1–11doi: 10.1111/j.1600-0501.2011.02351.x

© 2011 John Wiley & Sons A/S 1

and play a crucial role in the regulation of bone

induction, maintenance, and repair (Kempen

et al. 2009; Bragdon et al. 2011). Experimental

studies confirmed that the administration of

recombinant BMP-2 proteins induces ortho-

topic and ectopic bone formation (Shore et al.

1995; Govender et al. 2002).

Blood vessels are an important component

of bone formation and maintenance, and bone

tissue differentiation is related to the local

presence of blood vessels (Amizuka et al.

2002; Amir et al. 2006). Vascular endothelial

growth factor (VEGF) has been shown to be a

key regulator of angiogenesis (Tille et al.

2003; Kempen et al. 2009). The activation of

angiogenesis by VEGF leads to enhanced bone

regeneration (Kleinheinz et al. 2005). The

combined delivery of these two factors pro-

motes new bone and vessel formation at ecto-

pic sites, leading to enhanced bone formation

(Shore et al. 1995; Govender et al. 2002).

Bone substitute materials enhancing bone

regeneration in the craniofacial complex and/

or other parts of skeleton have already been

tested in a variety of preclinical animal models

(Schlegel et al. 2004; Thorwarth et al. 2006).

New treatment strategies have to be estab-

lished in animal models ahead of clinical appli-

cation. The integration of bone substitutes

with different growth and differentiation

factors into routine clinical therapeutic proto-

cols requires extensive preclinical studies in

appropriate animal models. The morphological

and physiological characteristics of bone heal-

ing in pigs allow comparability with humans

(Schlegel et al. 2003a,b, 2004, 2006a,b).

It was hypothesized that the combined

administration of VEGF, an endothelial cell-

specific mitogen, and BMP-2, which stimu-

lates the osteogenic differentiation of mesen-

chymal cells, induces bone formation and

plays a crucial role in enhanced bone remod-

eling in a defect model.

The aim of the present study was to evalu-

ate the rate of bone formation and osseointe-

gration of scaffolds carrying bone

morphogenetic proteins and VEGF in combi-

nation with Bio-Oss® block materials into

freshly created bony defects. Successful

osseointegration of Bio-Oss® blocks in verti-

cal augmentation would provide an easily

available approach and valuable alternative to

autogenous bone grafts.

Material and methods

Study animal and test groups

The domestic pig is recognized as a valuable

model in biomedical research due to its ana-

tomical, physiological, and metabolic similar-

ities with humans (Schlegel et al. 2006a,b;

Wang et al. 2007). We selected the pig as our

study model because its bone regeneration

rate (1.2–1.5 lm/day) is comparable to that of

humans (1.0–1.5 lm/day) (Ahlmann et al.

2002; Schlegel et al. 2003a,b, 2004). The pig

is widely used as an established model of

bone regeneration (Schlegel et al. 2006a).

Bone remodeling, angiogenesis, and wound

healing in particular have been investigated

in the pig model (Schlegel et al. 2006a). The

chosen experimental setting was established

as shown in several previous studies (Schle-

gel et al. 2003a,b, 2004, 2006a,b).

Eighteen female adult domestic pigs (Breed-

ing Company Renner, Franconia) aged

18 months were included in the study, 6 in

the first phase and 12 in the second phase

(approval no. 54-2532.1-13/11).

Materials

The bone substitute material (Bio-Oss®

block; Geistlich Biomaterials GmbH, Baden-

Baden, Germany) is a natural, non-antigenic,

porous bone mineral matrix produced by

removing all organic components from

bovine bone. Due to its natural structure,

Bio-Oss® is physically and chemically compa-

rable to the mineralized matrix of human

bone (Tapety et al. 2004). The anorganic bone

matrix of Bio-Oss® has macroscopic and

microscopic structures with an interconnect-

ing pore system that serves as a physical scaf-

fold for the immigration of osteogenic cells

(Tapety et al. 2004). Bio-Oss® is a non-resorb-

able bone substitute (Schlegel 1996; Schlegel

& Donath 1998). In this study, convention-

ally available block forms 2 9 1 9 1 cm in

size were used.

Control defects were filled with a collagen

carrier (Lyostypt®; B. Braun AG, Melsungen,

Germany). Collagens have a hemostatic func-

tion that allows early wound stabilization

(Kruger 1992; Baumann et al. 2009), and their

chemotactic properties of attracting fibro-

blasts and semipermeability, which facili-

tates nutrient transfer, are advantageous over

other materials (Schwarz et al. 2006).

We used the fibrin glue Tissucol® (Duo s

1 ml immune; Baxter Germany GmbH, Un-

terschleißheim, Germany), a biological two-

component glue: one pre-filled syringe of

1 ml adhesive protein solution (human

plasma protein fraction, fibrinogen, coagula-

tion factor XIII, plasma fibronectin, and apro-

tinin) and one pre-filled syringe of 1 ml

thrombin solution (thrombin, calcium chlo-

ride). The onset of wound healing after

wound closure is characterized by the

ingrowth of fibroblasts into the wound area.

The next phase of wound healing is the deg-

radation of the fibrin by proteolysis and

phagocytosis.

Growth factors recombinant human bone

morphogenetic protein-2 (rhBMP-2) and

recombinant human vascular endothelial

growth factor (rhVEGF165) were used (R&D

Systems, Minneapolis, MN, USA).

Implantation of the constructs

All surgical procedures were performed under

general anesthesia. After intramuscular seda-

tion with azaperone (1 mg/kg body weight)

and midazolam (1 mg/kg body weight) and

intravenous ketamine (10 mg/kg body

weight; Ketanest S, Parke-Davis, Berlin, Ger-

many) and midazolam (1 mg/kg body weight;

Dormicum, Hoffmann-La Roche, Grenzach-

Whylen, Germany), the animals were anes-

thetized with isoflurane after oral intubation

(Isofluran, Curamed, Curamed Pharma, Kar-

lsruhe, Germany). Atropine (0.05 mg/kg body

weight; Atropinsulfat Braun, Braun Melsun-

gen, Melsungen, Germany) was administered

intravenously to avoid salivation and to stim-

ulate cardiac action.

The rhBMP-2 and rhVEGF165 were incor-

porated into the defects at a concentration of

8 lg/ml; 8 lg of rhBMP-2 and/or rhVEGF was

diluted in 1 ml of injectable distilled water

to yield a concentration of 8 lg/ml.

Growth factors were applied using Tissu-

col®. The growth factors were added to the

unfrozen fibrin glue components. The syringe

(Dubloject; Baxter Germany GmbH) was pre-

pared with the two components and the

whole portion (3 ml) fixed into the preformed

blocks. In the group with rhBMP-2 and

rhVEGF combined, the adsorbates were incor-

porated together into the blocks at the same

concentration, 8 lg/ml for each growth fac-

tor.

In the first phase, the materials were

inserted in CSD on the porcine calvariae

according to an established protocol (Schlegel

et al. 2006a). Access to the os frontale was

achieved using a coronal sagittal approach in

the forehead region. Identical bony defects

were created with a trephine burr (diameter

10 mm, depth 10 mm; Roland Schmid, Furth,

Germany) according to the established CSD

model described previously (Wiltfang et al.

2004; Schlegel et al. 2006a). The defects were

positioned at least 1 cm apart to avoid biolog-

ical interactions. Nine bony defects in three

rows were created. A total of three test

groups and four control groups were used for

each of the six animals. The seven constructs

were inserted in seven of the nine critical

size defects under randomized conditions in

2 | Clin. Oral Impl. Res. 0, 2011 / 1–11 © 2011 John Wiley & Sons A/S

Schmitt et al �Bio-Oss® blocks in combination with growth factors

each pigs calvaria. The remaining two defects

were filled with the materials were we imple-

mented the growth factors (1–6) (Fig. 1).

Testgroups:

1. Bio-Oss® block + rhBMP-2 (BMP),

2. Bio-Oss® block + rhVEGF (VEGF),

3. Bio-Oss® block + rhBMP-2 and rhVEGF

(BMPVEGF)

The control defects were filled with colla-

gen carriers (Lyostypt®) with the growth fac-

tors and Bio-Oss® block material without

growth factors (CO):

4. Lyostypt® + rhBMP-2

5. Lyostypt® + rhVEGF

6. Lyostypt® + rhBMP-2 + rhVEGF

7. Bio-Oss® (CO)

After implantation of the constructs, the

periosteum and skin over the defects were

sutured in two layers with resorbable mate-

rial (Vicryl 3.0 or Vicryl 1.0; Ethicon GmbH

& Co KG, Norderstedt, Germany).

Twelve experimental animals were

included in the second phase (vertical aug-

mentation). The Bio-Oss® blocks combined

with growth factors were fixed on the calva-

riae to simulate vertical augmentation. The

access to the pigs calvariae was prepared in

the same way as previously described. After

preparation of the cortical bone, the planned

fixation area of the blocks was pre-formed

using a bur (Hager & Meisinger GmbH,

Neuss, Germany) so that the block was

2 mm below the outer surface of the cortical

bone in a stable position. The blocks were

then fixed using osteosynthetic material

(2.0 9 14 mm screws; KLS Martin group

GmbH & Co. KG, Tuttlingen, Germany).

In both experimental groups (30 and

60 days) the test constructs were fixed on

five pigs calvariae under randomized condi-

tions (Fig. 1). The remaining pig was used for

fixation of the control group.

The following specific material combina-

tions were used:

1. Bio-Oss® block + rhBMP-2 (BMP),

2. Bio-Oss® block + rhVEGF (VEGF),

3. Bio-Oss® block + rhBMP-2 and rhVEGF

(BMPVEGF).

An untreated Bio-Oss® block was used as a

negative control (CO).

The growth factors were fixed to the blocks

in the same way and in the same concentra-

tions as in step one. After the augmentation

procedure, a bilateral periosteal incision was

made and the periosteum and skin over the

defects sutured in multiple layers to get a

secure wound closure and to avoid wound

healing complications.

For post-operative pain control, each ani-

mal received subcutaneous buprenorphine

(Temgesic®; 0.1 mg/kg body weight) every

12 h for 3 days. A peri-operative antibiotic

was administered 1 h before the operation

and 3 days postoperatively (streptomycin,

0.5 g s.i.d., Gruenenthal, Stolberg, Germany).

Preparation of the specimens

After an observation period of 30 days in

phase one and 30 and 60 days in phase 2 (ver-

tical augmentation), the animals were sacri-

ficed. Azaperone (1 mg/kg) and midazolam

(1 mg/kg) were injected intramuscularly as

sedatives and 20% pentobarbitone solution

(Luminal injectable solution 20%, Desitin

drugs GmbH, Hamburg, Germany) into an

ear vein until cardiac arrest occurred.

The resected skulls were scanned in three

dimensions using computed tomography

(Somatom Sensation 64; Siemens AG,

Munich, Germany) (Fig. 2). The skull was

separated using a precision saw (Exakt Appa-

ratebau GmbH, Norderstedt, Germany),

dehydrated in a series of alcohol solution,

and embedded in methacrylate-based resin

(Technovit® 9100 Neu, Haereus Kulzer, Ger-

many.

The embedded bone samples were prepared

for histomorphological evaluation according

to the cutting and grinding technique (Do-

nath & Breuner 1982; Donath 1985; Schlegel

et al. 2003a,b, 2004, Thorwarth et al. 2006;

Stockmann et al. 2011).

1. Bio-Oss® block + rhBMP-2 (BMP)

2. Bio-Oss® block + rhVEGF (VEGF)

3. Bio-Oss® block + rhBMP-2 and rhVEGF (BMPVEGF)

4. Lyostypt® + rhBMP-2

5. Lyostypt® + rhVEGF

6. Lyostypt® + rhBMP-2 + rhVEGF

7. Bio-Oss® (CO)

BMP VEGF

BMPVEGF

Fig. 1. Left Image showing the distribution of the control and test groups in phase one (CSD). Right image showing

the distribution of the block material test groups (vertical augmentation). BMP (Bio-Oss® block + rhBMP-2), VEGF

(Bio-Oss® block + rhVEGF), BMPVEGF (Bio-Oss® block + rhBMP-2 and rhVEGF).

Fig. 2. Computed tomography images identifying

defects (scarification after 60 days). De novo bone for-

mation can already be seen around the inserted bone

substitute material blocks.

© 2011 John Wiley & Sons A/S 3 | Clin. Oral Impl. Res. 0, 2011 / 1–11


Microradiography

The embedded bone samples were cut into

different segments. The bone samples in

group one (CSD) were cut in the median seg-

ment; the samples in step two (vertical

augmentation) were cut in the right and left

lateral and median segment (Fig. 3) and

ground into thin sections (180 lm) using a

precision saw and special grinding machine

(Exakt Apparatebau GmbH). Microradio-

graphs were taken using the Faxitron® X-ray

unit (exposure time: 3 minutes, tube voltage:

13.5 kV, intensity: 2.5 mA) on commercially

available dental X-ray films (ZF; Agfa, Koln,

Germany). The X-rays were digitized at a

high resolution of 1200 dpi and 12-bit gray

scales, and saved as a tagged image file (tif

format) (Fig. 4).

Light microscopy

Light microscopy allows qualitative analysis

of the tissue reactions and regeneration char-

acteristics within the defects. The slices were

grounded to 30-lm thickness, high gloss pol-

ished, and stained with toluidine blue-o for

histological examination. This specific stain-

ing causes mineralized laminated tissue to

stain as uncolored to pale blue cells; cell cores,

osteoid fringes, cement lines, collagen fibers,

and soft tissue colors a different blue; and car-

tilage matrix and early wound healing areas

stain metachromatic red-violet and calcified

matrix dark-blue. Samples were then digitized

using an optical microscope (Axioskop; Zeiss,

Jena, Germany) with integrated video camera.

Histomorphometric evaluation

Microradiographs were evaluated using image

processing software (Bioquant Osteo®, ver-

sion 7.10.10; BIOQUANT Image Analysis

Corporation, Nashville, TN, USA) as a basis

for the histomorphometric assessment. The

software distinguishes between different tis-

sue fractions via their individual color spec-

tra, marks it in a specific color, and assigns a

metric variable that allows the calculation of

different bone indices. As an evaluation

parameter, the area of the newly formed bone

(bone volume, BV) and residual bone substi-

tute material (bone substitute material vol-

ume, BSMV) was calculated in proportion to

the complete defect (tissue volume, TV).

Evaluation parameters in region of interest

1 (CSD) were:

1. Residual bone substitute material volume

(BSMV/TV)

2. Newly formed bone volume (BV/TV)

Evaluation parameters in region of interest

2 (vertical augmentation) were:

1. Residual bone substitute material volume

(BSMV/TV)

2. Newly formed bone volume (BV/TV)

Statistical analysis

The results were transferred into an Excel

2007 (Microsoft Corp., Redmond, WA, USA)

spreadsheet. Statistical analyses were per-

formed using the statistical package SPSS for

Windows version 18.0 (SPSS Inc., Chicago,

IL, USA). Mean values, the median and stan-

dard deviations were calculated for each sam-

ple in each animal. To evaluate the

differences between groups for each observa-

tion interval, a paired t-test was used, and an

unpaired t-test was used for the same group

between intervals. P-value 0.05 was consid-

ered significant.

Results

In the first phase, all the animals survived,

and 11 of the 12 animals survived in the sec-

ond phase to planned sacrifice at 60 days.

One animal died immediately after surgery

due to cardiac arrest.

Microradiography

Evaluation of the first experimental phase in

all test groups (BMP, VEGF, BMPVEGF)

showed comparable results regarding residual

bone substitution material and newly formed

bone in the region of interest. Measurements

of the newly formed bone (BV/TV) revealed

13.02% ± 5.66% for BMP, 14.49% ± 5.55%

for BMPVEGF, 18.69% ± 7.36% for VEGF,

15.49% ± 12.40% for Lyostypt® BMP,

13.50% ± 3.50% for Lyostypt® BMP/VEGF,

16.78% ± 12.28% for Lystoypt® VEGF, and

17.09% ± 5.96% for CO. A direct comparison

with CO indicated no increasing regeneration

of the CSD within the growth factor groups.

The results were not significant for any pair

combination in either evaluation parameter

(BV/TV, BSMV/TV). The use of Lyostypt® as

a scaffold resulted in comparable values

regarding new bone formation (BV/TV). In all

cases, bony regeneration from lateral and

basal was observed (Fig. 4). The measure-

ments are shown in Table 1.

Due to the comparability of the results,

study group selection was maintained.

The lack of dimensional stability in the

Lyostypt® scaffold made it impossible to

incorporate this control group in the second

part of the experiment (vertical augmenta-

tion). The experimental setup consisted of

the three test groups (BMP, VEGF, and

BMPVEGF) and one control group (CO). A

constant increase of new bone formation

(BV/TV) was demonstrated for all growth fac-

tor constructs and the control defect over the

observation period. Basal bony regeneration

and periosteal-induced regeneration were

observed on the lateral surfaces of the

inserted blocks. The periosteal bone forma-

tion nearly completely encircled the inserted

block after 60 days of observation (Fig. 5).

Compared to part one (CSD), less newly

Fig. 3. Exemplary presentation of the cut procedure. The block was cut in the right and left lateral and median seg-

ment. The bottom images show the corresponding microradiographic pictures.



VEGFBMP

COBMPVEGF

Lyostypt + BMP Lyostypt + VEGF

Lyostypt + BMP + VEGF

Fig. 4. Microradiographic results of the first phase (CSD).



formed bone was found in the region of inter-

est at 30 days (4.88% ± 1.09% for BMP,

6.22% ± 2.17% for BMPVEGF, 5.75% ±

0.92% for VEGF, and 3.92% ± 1.14% for the

control group).

After 60 days, bone formation had

increased to 9.33% ± 3.92% for BMP, 10.42%

± 1.81% for BMPVEGF, 11.01% ± 4.78% for

VEGF, and 10.02% ± 5.43% for the control

group (Table 2). The values of the newly

formed bone in the region of interest were

not significant between individuals. A signifi-

cant result was found when comparing

the newly formed bone values on the two

sacrifice dates (Fig. 6). The BSMV/TV in the

region of interest was comparable during the

whole period and not significant for any pair

combinations.

Light microscopy

At 30 days in the CSD group, we observed de

novo bone formation of woven bone from the

edges of the defects to the center. The

inserted bone substitute material exhibited

good tissue integrity. We found direct contact

between the material and the superimposed

newly formed bone (Fig. 7). The different test

groups with the inserted blocks had visually

identical results.

In the control group, the collagen scaffold

plus different growth factors, new bone for-

mation started from the local lateral and

basal bone. Moderate periosteal regeneration

was observed on the top of the defect and

sometimes bridged the defect.

Bone regeneration in the vertical augmen-

tation procedure started from the basal into

the block. Lateral bone formation did not

occur due to the missing contact with local

bone.

Bone formation increased after 60 days. In

contrast to the CSD, regeneration of the verti-

cally augmented blocks seemed to proceed

more slowly. We also observed periosteal-

induced bone formation on the lateral sur-

faces, which enclosed the blocks after 60 days

of healing, but there was no bone formation

starting from the lateral bone into the block.

Discussion

This study evaluated the value of bovine

bone substitute blocks alone or combined

with BMP-2 and VEGF for the regeneration of

bony defects, and particularly vertical aug-

mentation. For the esthetic and functional

reconstruction of atrophic jaws or jaw seg-

ments, the regeneration of bony defects in

oral and maxillofacial surgery represents a

widespread method for creating sufficient

bone prior to implant placement (Cordaro

et al. 2002; Dori et al. 2008; Nissan et al.

2009; Nkenke & Stelzle 2009; Urban et al.

2009; Acocella et al. 2010; Blokhuis & Arts

2011). Here, long-term stability of the graft

and the dental implants is desired. Various

transplants have been examined for their

potency in regenerating a CSD (Hollinger &

Kleinschmidt 1990; Schlegel et al. 2003b,

2004, 2006a; Thorwarth et al. 2006). Never-

theless, autologous transplantation is often

used as a standard of care, and its use seems

reasonable for larger and non-space-providing

defects. The higher regenerative potential of

autologous transplants may be explained by

the transfer of mesenchymal stem cells, vital

osteoblasts, and their precursors (Blokhuis &

Arts 2011). These cells are responsible for the

high regenerative potential of autologous

transplants (Cypher & Grossman 1996; Blo-

khuis & Arts 2011). On the other hand, the

disadvantages of autologous bone block trans-

plants have to be considered. The steady

resorption of autologous bone after augmen-

tation is a factor in long-term stability,

which is not desirable and often a compro-

mise (Cordaro et al. 2002; Donos et al. 2005;

Aghaloo & Moy 2007; Tonetti & Hammerle

2008). In addition, harvesting is associated

with risks and the patient experiences addi-

tional stress (Ahlmann et al. 2002; Nkenke

et al. 2004; Raghoebar et al. 2007; Weibull

et al. 2009; Schaaf et al. 2010). The detach-

ment of autologous transplants is still desired

in to minimize the risks and burden of the

patient and generate a comparable bioactive

material with long-term stability.

The bone substitute material we used is

comparable to the extensively researched

bovine bone substitute material Bio-Oss®,

which has shown good bone regeneration

potential in various indications (Thorwarth

et al. 2006). The structure of the evaluated

substitute consists of a wide interconnecting

pore system that serves as a physical scaffold

for the immigration of osteogenic cells (Tap-

ety et al. 2004). The production of the block

form allows extension of the range of applica-

tion.

The idea of this study was to add addi-

tional bioactivity to the natural bovine bone

block using bone growth stimulating

(rhBMP-2) and angiogenesis stimulating fac-

tors (rhVEGF). The optimal concentration of

injected growth factors to enhance bone for-

mation remains to be determined. A multi-

tude of scientific studies have achieved

controversial results regarding the optimal

concentration of BMP-2 (Sigurdsson et al.

1997, 2001; Tatakis et al. 2002; Kato

et al. 2006; Jung et al. 2008; Ramazano-

glu et al. 2011). One study reported that bone

formation is not affected by different concen-

trations of rhBMP-2 (Tatakis et al. 2002).

Other studies have demonstrated that bone

formation increases with a higher concentra-

tion of growth factor (Kato et al. 2006; Jung

et al. 2008). Due to different experimental

approaches, making comparisons and defin-

ing an optimal concentration is difficult. In

addition, the bony regeneration and the influ-

ence of growth factors depend on the species,

the study subjects, the delivery modes of the

carriers, and conditions for the bony regener-

ation(Liu et al. 2007). A relatively low con-

centration of BMP was used in the present

study compared to that used in other studies

(Sigurdsson et al. 1997, 2001; Kato et al.

2006; Jung et al. 2008). De novo bone forma-

tion can be induced with a relatively low

concentration of BMP-2 in sites with suffi-

cient blood supply (Marden et al. 1994). The

concentration of rhVEGF used in the present

study was based on the results of Kleinheinz

et al.; they reported that the activation of

angiogenesis using rhVEGF leads to more

intensive angiogenesis and bone regeneration

(Kleinheinz et al. 2005). Despite the com-

bined use of rhBMP-2 and rhVEGF in this

study, we found no benefit regarding bone

formation in the CSD or vertical augmenta-

tion. The addition of BMP and VEGF had no

promotional effect at any phase of the study

(CSD or vertical augmentation). This obser-

vation is in accordance with a previous study

that demonstrated no beneficial effect of

BMP-2 on vertical bone augmentation in one

Table 1. Values for the first phase (CSD)

BV/TV% BSMV/TV%

Mean Median SD Mean Median SD

Bio-Oss® block (CO) 17.09 17.35 5.96 22.46 22.45 3.65Bio-Oss® block+BMP (BMP) 13.02 14.45 5.66 26.91 27.79 4.40Bio-Oss® block+BMP+VEGF (BMPVEGF) 14.49 14.03 5.55 23.08 22.80 4.60Bio-Oss® block+VEGF (VEGF) 18.69 16.49 7.36 22.07 21.33 5.40Lyostypt®+BMP 15.49 17.16 12.40 .00 .00 .00Lyostypt®+BMP+VEGF 13.50 13.50 3.50 .00 .00 .00Lyostypt®+VEGF 16.78 11.96 12.28 .00 .00 .00



syad06PMBsyad03PMB

syad06FGEVsyad03FGEV

syad06FGEVPMBsyad03FGEVPMB

syad06OCsyad03OC

Fig. 5. Microradiographic results of the second phase (vertical augmentation).



wall defect onlay grafting combined with de-

proteinized bovine bone blocks (Kim et al.

2010). The comparison of bone regeneration

by using higher and different concentrations

of growth factors (BMP-2 and VEGF) could be

another approach for future studies.

Compared to other studies using Bio-Oss®

granulate in the same defect configuration

(Thorwarth et al. 2006), we observed less

new bone formation in the region of interest

in the CSD of the control group (Bio-Oss®

block).

The reason for decreased bone regeneration

in the CSD should be discussed. Regarding

the project settings, the method differs at

two points. In the present study, we used a

block material that has less contact with the

local bone than the inserted bone substitute

granules. In addition, the fibrin glue could

have an effect on bony consolidation.

Fibrin glue degrades through phagocytic and

enzymatic pathways in roughly 4–10 days

(Auger et al. 1993; Xu et al. 1996, Hallman

et al. 2001a) and acts as an ideal carrier and

delivery system for cultured cells, growth fac-

tors, and other drugs (Boyce et al. 1994; Horch

et al. 1998; Pandit et al. 2000). Fibrin also pro-

motes the migration of fibroblasts and endo-

thelial cells (Pandit et al. 1998). Thus, fibrin

can be considered an optimal medium for the

delivery of growth factors to the wound bed

and bone substitute. In addition, fibrin

releases growth factors at a steady rate until

total degradation. Other studies have exam-

ined the augmentation procedure in the max-

illary sinus using autologous bone combined

with fibrin glue (Lee et al. 2007) or a bovine

bone substitute in combination with autolo-

gous bone and fibrin glue (Hallman et al.

2001a,b). No negative effect of using fibrin

glue was found in regard to the consolidation

of defects. The insertion of fibrin glue can be

hypothesized to have no negative property

with respect to bony healing.

Furthermore, the results of our study

clearly show that the contact area of the

inserted material is crucial for the progres-

sion of bone formation and regeneration. Bio-

logical healing depends on the direct contact

of the transplant with the local bone and

mechanical immobility is needed (Hausamen

& Neukam 1992). In summary, bone regener-

ation was faster for the CSD filled with bone

substitute material then after vertical aug-

mentation. This difference may be due to the

configuration of the defect. Felice et al.

(2009, 2010a,b) achieved promising results

using bovine bone blocks for the augmenta-

tion of atrophic edentulous jaw segments

with an inlay technique. Thus, the surface of

P = 0.001*

P = 0.02*

Mea

n B

V/TV

%

Group

Fig. 6. Percentage of the newly formed bone (BV/TV) in the region of interest during the postoperative course (30

and 60 days). *Significant when comparing the groups with each other.

Table 2. Values for the second phase (vertical augmentation)

BV/TV% BSMV/TV%

Mean Median SD Mean Median SD

BMP 30 days 4.88 4.77 1.09 31.56 29.09 4.80BMP+VEGF 30 days 6.22 6.38 2.17 26.74 27.47 2.47VEGF 30 days 5.75 5.53 .92 33.23 31.98 5.60Control 30 days 3.92 3.85 1.14 32.34 33.73 6.88BMP 60 days 9.33 9.52 3.92 33.15 34.85 6.95BMP+VEGF 60 days 10.42 9.37 1.81 36.02 36.08 2.22VEGF 60 days 11.01 9.62 4.78 30.17 26.70 8.43Control 60 days 10.02 8.93 5.43 33.38 33.25 0.45

CSD, BMP group

CSD, BMP group 2, 5x, new bone formation in contact with the substitute material

CSD, BMP group 5x

Fig. 7. Light micrographs showing de novo bone forma-

tion in the region of interest (CSD) 30 days after aug-

mentation. The arrow shows the osteoconductive

property of the inserted material in close contact with

the substitute.



the local bone adjacent to the bone substitute

material seems to be crucial for regeneration.

This type of implementation makes transfor-

mation of a single-wall defect, such as super-

position of bone grafts, into a multi-walled

one. Bone regeneration is stimulated from

the ground and the opposite surface, and

promises better blood circulation and de novo

bone formation.

The increase in newly formed bone in the

region of interest from the 30th to the 60th

day showed progressive bone regeneration.

Periosteal regeneration was also seen around

the blocks, but we observed no bone forma-

tion starting from the newly formed bone on

the lateral surface into the block. In all cases

the bone formation started from the local

bone toward the center. Therefore, de novo

bone formation is limited to the endosteal

and periosteal vascularized areas. Defect con-

solidation may occur at a later observation

time, which can be investigated by a new

study with a later sacrifice time point.

In conclusion, the inserted and augmented

blocks showed sufficient form and primary

stability and can be used for augmentation

procedures. In this study the application of

growth factors (BMP-2 and VEGF) had no pro-

motional effect on de novo bone formation.

Due to the small sample number in this

study it should, however, be noted that there

is a low evidence regarding the functionality

of the implemented constructs. The use of

autologous bone blocks seems to remain the

gold standard of care in the regeneration of

non-space providing defects due to its osteo-

inductive properties, and is a safe procedure

(Hausamen & Neukam 1992; Neukam et al.

1994; Blokhuis & Arts 2011).

Acknowledgements: The study was

supported by grants of the Osteology

Foundation (09-107).

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