Bone regeneration using a synthetic matrix containing enamel matrix derivate

Bone regeneration using a syntheticmatrix containing enamelmatrix derivate

David SchneiderFranz E. WeberChristoph H. F. HammerleAndreas FeloutzisRonald E. Jung

Authors’ affiliations:David Schneider, Christoph H. F. Hammerle,Ronald E. Jung, Center for Dental and OralMedicine and Cranio-Maxillofacial Surgery, Clinicfor Fixed and Removable Prosthodontics and DentalMaterial Science, University of Zurich, Zurich,SwitzerlandFranz E. Weber, Department of Cranio-Maxillofacial Surgery, Section of OralBiotechnology & Bioengineering, UniversityHospital Zurich, Zurich, SwitzerlandAndreas Feloutzis, Private Practice, Maroussi,Greece

Corresponding author:Dr Ronald E. JungCenter for Dental and Oral Medicine and Cranio-Maxillofacial SurgeryClinic for Fixed and Removable Prosthodontics andDental Material ScienceUniversity of ZurichPlattenstrasse 11CH-8032 ZurichSwitzerlandTel.: þ 41 44 634 32 51Fax: þ 41 44 634 43 05e-mail: [email protected]

Key words: animal study, bioactive factor, bone regeneration, EMD, enamel matrix protein,

graft material, polyethylene glycols, RGD

Abstract

Purpose: The aim of the present study was to test whether the delivery of enamel matrix

derivate (EMD) via synthetic polyethylene glycol (PEG)-based hydrogels with and without

RGD sequences enhances bone formation in vivo.

Material and methods: In each of 10 rabbits, four titanium cylinders were placed on the

external cortical bones of their calvaria. The following four treatment modalities were

randomly allocated: One of the four cylinders was left empty (control), the other three were

filled with a combination of PEG matrix with hydroxyapatite/tricalciumphosphate (HA/TCP)

granules and EMD in a concentration of 100mg/ml (test 1) or 500mg/ml (test 2) or 500mg/ml

and RGD peptide (test 3). After 8 weeks, the animals were sacrificed and ground sections

were obtained for histological analysis. For statistical analysis, the Kruskal–Wallis test was

applied (Po0.05).

Results: The histomorphometric analysis revealed a statistically larger area fraction of

newly formed bone in the EMD 500/RGD group (54.8 � 14.5%) compared with the control

group (28.7 � 10.3%) and the EMD 500 group (31.2 � 14.1%) and non-significantly higher

area fraction compared with the EMD 100 group (38.2 � 10.4%). The percentage of

mineralized bone showed no statistically significant differences among the four groups.

The mean percentage of mineralized bone was 13.6 � 3.3% in the control group,

14.2 � 5.8% in the EMD 100 group, 11.69 � 5.9% in the EMD 500 group and 15.66 � 5.2%

in the EMD 500/RGD group. No statistically significant difference regarding the bone-to-

graft contact between the EMD 100 group (23 � 15.7%), the EMD 500 group

(22.2 � 14.6%) and the EMD 500/RGD group (21.6 � 8.8%) was observed.

Conclusions: The combination of a PEG matrix containing EMD with HA/TCP granules had

no effect on the formation of mineralized bone tissue in rabbit calvaria. The addition of

RGD peptide to the PEG/EMD 500 combination increased the area fraction of newly formed

bone compared with the other treatment groups. Further studies are indicated to study a

possible synergistic effect of EMD and RGD.

Tooth extraction is followed by a remodel-

ing processes of the alveolar ridge resulting

in partial loss of the buccal bone contour

(Araujo & Lindhe 2005). A large variety of

methods and materials for the regeneration

of lost alveolar bone have been described

(Hammerle & Karring 1998). Although

autologous bone is still considered to be

the ‘‘gold standard,’’ the limited availabil-

ity and the donor site morbidity have led to

a shift toward the use of xenogenic or

allogenic materials (Nkenke et al. 2001,

2002). Some of these materials are well

documented and have proven their efficacy

Date:Accepted 14 May 2010

To cite this article:Schneider D, Weber FE, Hammerle CHF, Feloutzis A,Jung RE. Bone regeneration using a synthetic matrixcontaining enamel matrix derivateClin. Oral Impl. Res 22, 2011; 214–222.doi: 10.1111/j.1600-0501.2010.01985.x

214 c� 2011 John Wiley & Sons A/S

mailto:[email protected]

and safety over a long period, but they are

associated with long healing periods be-

cause they lack osseoinductive properties

(Iezzi et al. 2007; Traini et al. 2007).

Grafting materials originating from animal

sources can cause difficulties in terms of

patient acceptance, immune response and a

possible transmission of infectious agents

that can never be completely excluded. As

a fully synthetic material, hydroxyapatite/

tricalciumphosphate (HA/TCP) overcomes

these shortcomings and has shown effec-

tiveness as a bone substitute for guided

bone regeneration (GBR) in several studies

(Froum et al. 2001; Schwarz et al. 2007;

Zafiropoulos et al. 2007; Artzi et al. 2008;

Cordaro et al. 2008)

The induction of bone formation re-

mains a challenge in tissue regeneration.

Numerous preclinical and clinical studies

have shown a positive effect of growth

factors and differentiation factors in hard

and soft tissue regeneration (Lynch et al.

1991; Howell et al. 1997; Cochran et al.

1999; Jung et al. 2003, 2005, 2008). The

regenerative potential also depends on the

method of application and release kinetics

of the bioactive substances from their car-

rier (Sigurdsson et al. 1996; Hunt et al.

2001).

Enamel matrix derivate (EMD) is an

extract from porcine tooth buds. It is com-

posed of several proteins, up to 90% of

which are amelogenins. The residual 10%

are proline-rich non-amelogenins. Enamel

matrix proteins are secreted by ameloblasts

during tooth formation and regulate en-

amel mineralization (Simmer & Fincham

1995). They are also secreted by epithelial

cells of the Hertwig’s root sheath during

root formation and affect the formation of

periodontal tissues, primarily acellular ce-

mentum (Hammarstrom 1997). Therefore,

EMD has been mainly used in periodontal

regenerative treatment, especially for furca-

tion and intrabony defects (Froum et al. 2001;

Tonetti et al. 2002; Donos et al. 2003; Jepsen

et al. 2004; Meyle et al. 2004; Sanz et al.

2004; Hoffmann et al. 2006).

Very little data are available regarding the

effect of EMD for periimplant bone regen-

eration (Casati et al. 2002). As matrix

proteins, EMD shows very low solubility

at physiological pH, but is soluble under

acidic conditions. Commercially available

EMD (Emdogains

, Institut Straumann AG,

Basel, Switzerland) is dissolved in a slightly

acidic propylene glycol alginate (PGA) gel.

One problem in applying Emdogains

for

periimplant bone regeneration is that its

PGA carrier gel is designed to collapse and

release the EMD at the tooth root under

physiological conditions and thus does not

provide space-keeping properties or the

retention of the protein in a larger volume.

The use of an optimized matrix as a deliv-

ery system for EMD could overcome this

problem. The combination of a bone sub-

stitute (e.g. HA/TCP) potentially provides

additional stability of the compound, help-

ing to keep the substances at the site of

bone regeneration.

Polyethylene glycol (PEG)-based hydro-

gels have been shown to be effective delivery

systems for bioactive substances because

these matrices allow optimal cell ingrowth

as well as retention and release of bioactive

proteins such as recombinant human bone

morphogenetic protein-2 (rhBMP-2) and

parathyroid hormone (PTH) (Lutolf et al.

2003; Jung et al. 2007a, 2007b, 2008). The

ingrowth of cells into these matrices can be

facilitated by the presence of RGD-contain-

ing peptides, an amino acid sequence com-

posed of arginine, glycine and aspartate.

RGD sequences are mainly found in extra-

cellular matrix proteins (e.g. fibronectin)

and mediate cell adhesion, cell migration

and signal transduction via binding to integ-

rins, a subgroup of cell surface receptors

(Akiyama 1996). The possible application

of EMD with an optimized carrier material

with and without RGD has not been in-

vestigated yet.

Hence, the aim of the present study was

to test if the delivery of EMD in different

concentrations via synthetic PEG-based

hydrogels with and without RGD se-

quences enhances bone formation in vivo.

Materials and methods

The present animal investigation was eval-

uated and approved by the responsible An-

imal Research Ethics Committee at the

University of Zurich, Switzerland.

Synthetic matrix and bioactive peptides

The synthetic matrix used as the carrier in

the present study was a PEG-based hydro-

gel (Institut Straumann AG, Basel, Swit-

zerland). This gel was formed by a reaction

of a 4-arm PEG with acrylate endgroups

with a linear PEG with thiol endgroups in

an aqueous buffer system (triethanola-

mine/HCl) (Elbert et al. 2001). The PEG

termini connected through a highly self-

selective addition reaction, forming an

elastic gel network. Immediately before

application, both PEG solutions were ster-

ile filtered and mixed with 0.15 g of HA/

TCP granulate in a distribution of 60% :

40% and a particle size of 400–700mm

(Straumann BoneCeramic, Institut Strau-

mann AG).

For the activated gels, stock solutions of

a lyophilized EMD and a nine amino acid

cys-RGD peptide (Bachem, Bubendorf,

Switzerland) in dilute (0.1%) acetic acid

were added first to the PEG-acrylate solu-

tion, resulting in the formation of covalent

bonds between the cystein residues and the

PEG-acrylate upon gelation, which was

started by adding the triethanolamine buf-

fer solution. The final concentrations for

the peptides were 350 mg/ml gel for cys-

RGD and 100 or 500mg/ml gel for EMD.

The surgical procedure, the histological

preparation, the histomorphometric and

statistic evaluation were performed accord-

ing to a previous animal study (Jung et al.

2007b).

Surgical procedure

The surgical procedure was performed in

10 adult New Zealand white rabbits. Dur-

ing a standardized surgical procedure, four

evenly distributed circular slits, 6 mm in

diameter and 1 mm in depth, were prepared

with a trephine bur bilaterally in the par-

ietal and frontal bones. Without removal

but after perforation of the external cortical

plate inside the created circles with a round

bur, specially designed 7 � 7 mm large

cylinders made of c.p. titanium with a

machined surface on their inside were

screwed into the slits.

One of the four cylinders served as a

control and was left empty. Another cylin-

der contained the EMD in a concentration

of 100mg/ml gel (test 1), and a third cylin-

der contained EMD in a concentration of

500 mg/ml gel (test 2). The fourth cylinder

contained EMD in a concentration of

500 mg/ml gel in combination with cys-

RGD (test 3). The position of the cylinders

in each animal was randomly assigned by

rotating the position of the treatment mod-

alities in a clockwise direction. After appli-

cation of the test substances into the

Schneider et al �Bone regeneration using a synthetic matrix and EMD

c� 2011 John Wiley & Sons A/S 215 | Clin. Oral Impl. Res. 22, 2011 / 214–222

cylinders, they were closed with a titanium

lid and the skin flap was adapted and

sutured for primary healing (Figs 1–3).

Histologic preparation

After 8 weeks, the rabbits were sedated

with barbiturates and sacrificed by an over-

dose of ketamin. The specimens were sec-

tioned in the frontal plane through the

middle of the cylinders. 200-mm-thick sec-

tions were obtained, ground and polished to

a uniform thickness of 60–80mm (Donath &

Breuner 1982). The specimens were surface-

stained with toluidine blue (Schenk et al.

1984). One central section was selected

to perform histomorphometric measure-

ments.

Histomorphometry

The mineralized bone tissue content was

assessed by applying standard morphome-

trical techniques (Weibel 1980; Gundersen

et al. 1988). Measurements were carried

out using a light microscope at a magnifi-

cation of � 160, using an optically super-

imposed eyepiece test grid composed of 100

points and 10 cycloid lines (Schenk &

Olah 1980). The graft to bone contact

was calculated by the number of intersec-

tions between graft particles and the

outlines of either mineralized bone or non-

mineralized tissue.

In order to respect not only the amount

of newly formed bone but also its extension

and distribution within the cylinders, a

quantitative evaluation of the area of bone

regeneration within the cylinders was per-

formed using a pixel count of histological

digital images in an image analysis program

(Adobes

Photoshop 7.0.1) (area of bone

regeneration [%]¼ pixel number of the

bone area � 100/total pixel number of the

cylinder) (Jung et al. 2008).

Statistical analysis

Mean values and standard deviations were

calculated based on point measurements or

cycloid measurements. The primary unit

for statistical analysis was the cylinder. All

values are displayed including the median.

Significant differences were identified by a

Kruskal–Wallis test and confirmed be-

tween groups by a Mann–Whitney U-

test. Statistical significance was set at

Po0.05. The statistical analysis was per-

formed using a statistical software package

(SPSS 16 for Windows).

Fig. 1. Bone preparation: four circular slits (6 mm in diameter, 1 mm in depth) and five cortical perforation

holes.

Fig. 2. Titanium cylinders screwed into the slits.

Fig. 3. Closed cylinders after the augmentation procedure.


216 | Clin. Oral Impl. Res. 22, 2011 / 214–222 c� 2011 John Wiley & Sons A/S

Results

During the experiment, all animals showed

an uneventful healing in the area of sur-

gery. No reductions in body weights were

noted, and no postoperative infections were

observed. Upon specimen retrieval, 33 cy-

linders were found to be stable and in the

same position as at placement. However,

seven out of 40 cylinders were loose and

therefore excluded from further analysis

(two in the control group, two in test 1,

two in test 2 and one in the test 3 group).

Descriptive histology

The qualitative evaluation of the histologi-

cal specimens showed variable amounts

and patterns of bone formation in the four

treatment groups. The empty cylinders

(control group) presented a structure with

few trabeculae and large marrow spaces.

Adjacent to the inner walls of these cylin-

ders, the bone trabeculae were oriented in a

parallel manner. The bone formation was

limited to the lower third of the cylinders

(Fig. 4).

In cylinders containing the PEG matrix

with EMD in a concentration of 100mg/g

gel (test 1 group), the amount of newly

formed bone varied considerably. Similar to

the control group, the trabeculae neighbor-

ing the inner walls were aligned parallel to

the titanium surface. The extension of the

newly formed bone varied between the

lower to the upper third of the cylinders

(Fig. 5).

A similar situation was found in the

cylinders filled with PEG matrix with

EMD in a concentration of 500mg/g gel

(test 2 group). A large variety of new bone

was observed (Fig. 6).

The highest amount of newly formed

bone was observed in the group containing

EMD 500 mg/g combined with RGD (test 3

group). In most cases, the new bone

reached the upper third of the cylinders

and was evenly distributed among the

lower two thirds, embedding the HA/

TCP granules (Figs 7 and 8). All features

of lamellar bone structure (osteons, Haver-

sian canals, osteoblasts and -clasts, etc.)

could be observed.

Histomorphometry

The quantitative histomorphometric ana-

lysis revealed that the content of miner-

alized bone tissue was very similar in all

the groups (Fig. 9). Therefore, no signifi-

cant difference was detectable between the

groups (Table 1). The difference between

the means was highest between EMD

500 (11.7� 5.6%) and EMD 500/RGD

(15.7� 5.2%). On the other hand, the

quantitative evaluation of the area of bone

regeneration revealed a statistically signifi-

cant larger bone area in the EMD 500/RGD

(test 3) group compared with the control

Fig. 4. Histological section through an initially empty cylinder (control group) (original magnification � 10).

Fig. 5. Histological section through a cylinder containing polyethylene glycolþenamel matrix derivate 100

(test 1) (original magnification � 10).



group (Fig. 10; Table 2). Among the groups

containing EMD, the most extended area of

bone formation was found in the EMD

500/RGD (test 3) group (54.8� 14.5%)

compared with the other EMD groups

(test 1: 38.2� 10.4%; test 2: 31.2�

14.1%). The area of bone formation was

significantly increased in the EMD 500-

containing groups, when RGD peptides

were added.

Regarding the bone-to-graft contact, the

EMD 100 group, the EMD 500 group and

the EMD 500/RGD group showed no statis-

tically significant difference (23� 15.7%,

22.2� 14.6% and 21.6� 8.8%, respec-

tively) (Fig. 11; Table 3).

Discussion

The results of the present study indicate

that the combination of a PEG matrix

containing enamel matrix protein (EMD)

with HA/TCP granules has a limited effect

on the formation of mineralized bone tissue

in the rabbit calvaria. However, the area

fraction of bone formation was signifi-

cantly increased by the addition of RGD

peptide to the PEG/EMD500 group with-

out affecting the graft to bone contact.

The topical application of enamel matrix

protein is well documented in periodontal

therapy and is effective in the regeneration

of periodontal tissues including bone. Ani-

mal and clinical studies have shown the

effectiveness of EMD in the treatment of

infrabony (Heijl et al. 1997; Froum et al.

2001; Tonetti et al. 2002; Cochran et al.

2003; Silvestri et al. 2003; Sanz et al. 2004;

Sculean et al. 2004) and furcation type

defects (Donos et al. 2003; Jepsen et al.

2004; Meyle et al. 2004; Hoffmann et al.

2006), providing superior clinical results

than open flap surgery alone and similar

results as by applying GTR techniques.

Although it stimulates osteoblasts in

vitro (Jiang et al. 2006), EMD has not

been tested clinically for the regeneration

of non-periodontal bone defects. In an ani-

mal study, EMD was combined with de-

proteinized bovine bone mineral (DBBM)

in one group and with a PGA carrier in

another and inserted into muscular tissue,

without contact to any bone tissue (Donos

et al. 2006). After 2 and 4 months, the

DBBM particles were encapsulated, regard-

less of the presence of PGA and no bone

formation was observed indicating that

neither EMD alone nor in combination

with PGA is able to induce bone formation.

A similar study in mice has shown that

EMD is not osteoinductive but it can be

osteoconductive in certain concentrations

in combination with demineralized freeze-

dried bone allograft (DFDBA) (Boyan et al.

2000). A comparative study in rabbits

with a similar design as the present study

showed that EMD had little effect on bone

formation in combination with b-TCP

Fig. 6. Histological section through a cylinder containing polyethylene glycolþenamel matrix derivate 500

(test 2) (original magnification � 10).

Fig. 7. Histological section through a cylinder containing polyethylene glycolþenamel matrix derivate

500þRGD (test 3) (original magnification � 10).



compared with b-TCP alone (Murai et al.

2005). Using EMD alone or in combination

with DBBM for bone regeneration at the

lateral rat ramus, no effect on the forma-

tion of new bone under dome-shaped PTFE

capsules was observed compared with the

use of the capsule alone in another inves-

tigation (Donos et al. 2005). These results

are in agreement with the present study

revealing no effect of EMD in combination

with HA/TCP on bone regeneration, even

in higher dosages.

In a recent animal model, EMD has been

compared with rhBMP-2 and DFDBA for

the regeneration of critical-size calvaria

bone defects. It was shown that, without

the use of a bone substitute, unlike rhBMP-

2, both DFDBA and EMD did not lead to a

regeneration of the bone defect (Intini et al.

2008).

EMD has also been investigated for peri-

implant bone regeneration. An in vitro

study demonstrated a concentration-de-

pendent positive effect of EMD on human

osteoblast-like cells on a titanium surface

(Schwarz et al. 2004). In an animal study

with artificial dehiscence-type defects, it

was observed that the application of EMD

resulted in a similar amount of bone for-

mation as GBR with a resorbable mem-

brane (Casati et al. 2002). However, only

the combination of EMD and the mem-

brane showed a significantly higher area of

bone formation compared with the un-

treated control group in that study, suggest-

ing that EMD has the potential to

stimulate bone formation even in the

absence of periodontal ligament cells. The

addition of EMD into drill holes before

implant placement did not have a benefi-

cial effect on the osseointegration in

another animal study (Franke Stenport &

Johansson 2003). According to the observa-

tion of the growth pattern in the present

study, a layer of newly formed bone

was found on the inner wall of the titanium

cylinders in all groups. A difference in

the amount or the pattern of growth along

the titanium surface was not found among

the groups. The titanium surface appears

to be a guiding structure for the

cells involved in bone formation indepen-

dently of the presence or absence of EMD

or RGD.

The addition of RGD to the PEG–EMD

500 combination has led to a significant

increase in the area of bone formation

within the titanium cylinders. Several ex-

perimental studies have shown an in-

creased bone formation on RGD-coated

implant surfaces compared with uncoated

implants (Schaffner et al. 1999; Elmen-

gaard et al. 2005; Schliephake et al.

2005). Because RGD sequences play an

important role in cell adhesion, cell migra-

tion and signal transduction (Pierschbacher

& Ruoslahti 1984; Akiyama 1996), it can

be assumed that these properties are bene-

ficial for the formation of new bone in

connection with the bone substitute HA/

TCP. However, although RGD has been

suggested to be a mediator for osteoblast

adhesion to HA (Okamoto et al. 1998), the

bone-to-HA/TCP contact appears not to

have been influenced by RGD in the pre-

sent study. The fact that the bone-to-graft

contact is not influenced by the presence of

the RGD peptides might indicate their

absence from the HA/TCP surface, most

likely due to the fact that the cys-RGD

peptides were allowed to covalently bind to

the PEG hydrogel by their addition to the

Fig. 8. Histological section through a cylinder containing polyethylene glycolþenamel matrix derivate

500þRGD (test 3), detail (original magnification � 160).

Fig. 9. The percentage of mineralized bone generated within the cylinders in each group is displayed. The lines to

the right of the values indicate the median of the group. No significant differences between the groups were observed.

Table 1. Percentage of mineralized bone

Condition Number ofsamples

Mean(%)

SD

Empty 8 13.6 3.3EMD 100 8 14.2 5.8EMD 500 8 11.7 5.9EMD 500/RGD 9 15.7 5.2

EMD, enamel matrix derivate.



PEG acrylate solution before their first

contact with the HA/TCP particles.

According to a recent in vitro study

(Lutolf et al. 2003), the ingrowth of osteo-

blasts into the PEG matrix is facilitated by

the presence of RGD. This might explain

the finding that the addition of RGD to

500 mg/ml EMD significantly increased the

area of newly formed bone. It can be

speculated that the presence of RGD

within the PEG gel facilitates the ingrowth

of osteoblasts and leads to a broader dis-

tribution of the bone tissue within the

cylinders. Thus, also in this animal model,

the presence of RGD peptides in the matrix

is beneficial for bone formation.

The same study design and the same

carrier (PEG) were used to investigate the

effect on bone formation of rhBMP-2 (Jung

et al. 2008) and PTH (Jung et al. 2007a).

Therefore, a comparison of these sub-

stances and those used in the present study

can be performed. Compared with PTH

(53.5� 22.7%), the mean area of newly

formed bone was similar in the present

study in the EMD 500/RGD group

(54.8� 14.5%). The percentage of miner-

alized bone, however (15.7� 5.2%), was

lower than observed in the investigations

with BMP (30.2� 7.6%) and PTH

(19.6� 6%).

Based on the comparison of these three

studies, it appears that the effect on bone

regeneration was similar between EMD

and PTH, whereas rhBMP-2 revealed al-

most twice as much newly formed bone

compared with either EMD or PTH. All

three bioactive factors reveal a favorable

ingrowth and distribution of newly formed

bone within the entire titanium cylinder.

This might indicate a good matrix structure

given by the PEG hydrogel.

Conclusion

It can be concluded that the combination of

a PEG matrix containing enamel matrix

protein (EMD) with HA/TCP granules has

no effect on the percentage of mineralized

bone tissue in the rabbit calvaria compared

with spontaneous healing, independent of

the EMD concentration. However, the ad-

dition of RGD peptide to the PEG/EMD

500 group revealed an increased area

fraction of new bone compared with the

other treatment groups. Further studies are

Fig. 10. Area fraction of newly formed bone within the cylinder. The lines to the right of the values indicate the

median of the group. Application of the Kruskal–Wallis test revealed a significant difference between the four

groups (P¼ 0.004). Significant differences between groups determined by the Mann–Whitney U-test are

indicated nnPo0.05.

Table 2. Area fraction of newly formed bone

Condition Number of samples Mean (%) SD

Empty 8 28.7 10.3EMD 100 8 38.2 10.4EMD 500 8 31.2 14.1EMD 500/RGD 9 54.8 14.5


Fig. 11. Surface fraction of bone-to-graft contact. The lines to the right of the values indicate the median of the

group. No significant differences between the groups were observed.

Table 3. Surface fraction of bone to graft contact

Condition Number of samples Mean (%) SD

EMD 100 8 23 15.7EMD 500 8 22.2 14.6EMD 500/RGD 9 21.6 8.8




indicated to investigate a possible synergis-

tic effect of EMD and RGD.

Acknowledgements: The authors

express their gratitude to Aart

Molenberg (PhD, Institut Straumann

AG) and Michel Dard (PhD, Institut

Straumann AG) for providing expertise

and support. The animal care by F.

Nicholls (University Hospital, Zurich,

Switzerland) and the technical

assistance by J. Fierz and A.

Tchouboukov are also highly

acknowledged.

References

Akiyama, S.K. (1996) Integrins in cell adhesion and

signaling. Human Cell 9: 181–186.

Araujo, M.G. & Lindhe, J. (2005) Dimensional ridge

alterations following tooth extraction. An experi-

mental study in the dog. Journal of Clinical

Periodontology 32: 212–218.

Artzi, Z., Weinreb, M., Carmeli, G., Lev-Dor, R.,

Dard, M. & Nemcovsky, C.E. (2008) Histomor-

phometric assessment of bone formation in sinus

augmentation utilizing a combination of autoge-

nous and hydroxyapatite/biphasic tricalcium

phosphate graft materials: at 6 and 9 months in

humans. Clinical Oral Implants Research 19:

686–692.

Boyan, B.D., Weesner, T.C., Lohmann, C.H., An-

dreacchio, D., Carnes, D.L., Dean, D.D., Co-

chran, D.L. & Schwartz, Z. (2000) Porcine fetal

enamel matrix derivative enhances bone forma-

tion induced by demineralized freeze dried bone

allograft in vivo. Journal of Periodontology 71:

1278–1286.

Casati, M.Z., Sallum, E.A., Nociti, F.H. Jr., Caf-

fesse, R.G. & Sallum, A.W. (2002) Enamel matrix

derivative and bone healing after guided bone

regeneration in dehiscence-type defects around

implants. A histomorphometric study in dogs.

Journal of Periodontology 73: 789–796.

Cochran, D.L., King, G.N., Schoolfield, J., Velas-

quez-Plata, D., Mellonig, J.T. & Jones, A. (2003)

The effect of enamel matrix proteins on perio-

dontal regeneration as determined by histological

analyses. Journal of Periodontology 74: 1043–

1055.

Cochran, D.L., Schenk, R., Buser, D., Wozney, J.M.

& Jones, A.A. (1999) Recombinant human bone

morphogenetic protein-2 stimulation of bone for-

mation around endosseous dental implants. Jour-

nal of Periodontology 70: 139–150.

Cordaro, L., Bosshardt, D.D., Palattella, P., Rao, W.,

Serino, G. & Chiapasco, M. (2008) Maxillary

sinus grafting with bio-oss or straumann bone

ceramic: histomorphometric results from a rando-

mized controlled multicenter clinical trial. Clin-

ical Oral Implants Research 19: 796–803.

Donath, K. & Breuner, G. (1982) A method for the

study of undecalcified bones and teeth with at-

tached soft tissues. The Sage-Schliff (sawing and

grinding) technique. Journal of Oral Pathology

11: 318–326.

Donos, N., Bosshardt, D., Lang, N., Graziani, F.,

Tonetti, M., Karring, T. & Kostopoulos, L. (2005)

Bone formation by enamel matrix proteins and

xenografts: an experimental study in the rat ra-

mus. Clinical Oral Implants Research 16: 140–

146.

Donos, N., Glavind, L., Karring, T. & Sculean, A.

(2003) Clinical evaluation of an enamel matrix

derivative in the treatment of mandibular degree II

furcation involvement: a 36-month case series.

International Journal of Periodontics and Re-

storative Dentistry 23: 507–512.

Donos, N., Kostopoulos, L., Tonetti, M., Karring, T.

& Lang, N.P. (2006) The effect of enamel matrix

proteins and deproteinized bovine bone mineral on

heterotopic bone formation. Clinical Oral Im-

plants Research 17: 434–438.

Elbert, D.L., Pratt, A.B., Lutolf, M.P., Halstenberg,

S. & Hubbell, J.A. (2001) Protein delivery from

materials formed by self-selective conjugate addi-

tion reactions. Journal of Controlled Release 76:

11–25.

Elmengaard, B., Bechtold, J.E. & Soballe, K. (2005)

In vivo study of the effect of RGD treatment on

bone ongrowth on press-fit titanium alloy im-

plants. Biomaterials 26: 3521–3526.

Franke Stenport, V. & Johansson, C.B. (2003) En-

amel matrix derivative and titanium implants.

Journal of Clinical Periodontology 30: 359–363.

Froum, S.J., Weinberg, M.A., Rosenberg, E. & Tar-

now, D. (2001) A comparative study utilizing

open flap debridement with and without enamel

matrix derivative in the treatment of periodontal

intrabony defects: a 12-month re-entry study.


Gundersen, H.J., Bendtsen, T.F., Korbo, L., Mar-

cussen, N., Moller, A., Nielsen, K., Nyengaard,

J.R., Pakkenberg, B., Sorensen, F.B. & Vesterby,

A. (1988) Some new, simple and efficient stereo-

logical methods and their use in pathological

research and diagnosis. APMIS 96: 379–394.

Hammarstrom, L. (1997) Enamel matrix, cemen-

tum development and regeneration. Journal of

Clinical Periodontology 24: 658–668.

Hammerle, C.H. & Karring, T. (1998) Guided bone

regeneration at oral implant sites. Periodontology

2000 17: 151–175.

Heijl, L., Heden, G., Svardstrom, G. & Ostgren, A.

(1997) Enamel matrix derivative (EMDOGAIN)

in the treatment of intrabony periodontal defects.


Hoffmann, T., Richter, S., Meyle, J., Gonzales, J.R.,

Heinz, B., Arjomand, M., Sculean, A., Reich, E.,

Jepsen, K., Jepsen, S. & Boedeker, R.H. (2006) A

randomized clinical multicentre trial comparing

enamel matrix derivative and membrane treat-

ment of buccal class II furcation involvement in

mandibular molars. Part III: patient factors and

treatment outcome. Journal of Clinical Perio-

dontology 33: 575–583.

Howell, T.H., Fiorellini, J., Jones, A., Alder, M.,

Nummikoski, P., Lazaro, M., Lilly, L. & Co-

chran, D. (1997) A feasibility study evaluating

rhBMP-2/absorbable collagen sponge device for

local alveolar ridge preservation or augmentation.

International Journal of Periodontics and Re-

storative Dentistry 17: 124–139.

Hunt, D.R., Jovanovic, S.A., Wikesjo, U.M., Woz-

ney, J.M. & Bernard, G.W. (2001) Hyaluronan

supports recombinant human bone morphoge-

netic protein-2 induced bone reconstruction of

advanced alveolar ridge defects in dogs. A pilot

study. Journal of Periodontology 72: 651–658.

Iezzi, G., Degidi, M., Scarano, A., Petrone, G. &

Piattelli, A. (2007) Anorganic bone matrix re-

trieved 14 years after a sinus augmentation pro-

cedure: a histologic and histomorphometric

evaluation. Journal of Periodontology 78: 2057–

2061.

Intini, G., Andreana, S., Buhite, R.J. & Bobek, L.A.

(2008) A comparative analysis of bone formation

induced by human demineralized freeze-dried

bone and enamel matrix derivative in rat calvaria

critical-size bone defects. Journal of Perio-

dontology 79: 1217–1224.

Jepsen, S., Heinz, B., Jepsen, K., Arjomand, M.,

Hoffmann, T., Richter, S., Reich, E., Sculean,

A., Gonzales, J.R., Bodeker, R.H. & Meyle, J.

(2004) A randomized clinical trial comparing

enamel matrix derivative and membrane treat-

ment of buccal class II furcation involvement in

mandibular molars. Part I: study design and re-

sults for primary outcomes. Journal of Perio-

dontology 75: 1150–1160.

Jiang, J., Goodarzi, G., He, J., Li, H., Safavi, K.E.,

Spangberg, L.S. & Zhu, Q. (2006) Emdogain-gel

stimulates proliferation of odontoblasts and osteo-

blasts. Oral Surgery, Oral Medicine, Oral Pathol-

ogy, Oral Radiology, and Endodontology 102:

698–702.

Jung, R.E., Cochran, D.L., Domken, O., Seibl, R.,

Jones, A.A., Buser, D. & Hammerle, C.H. (2007a)

The effect of matrix bound parathyroid hormone

on bone regeneration. Clinical Oral Implants

Research 18: 319–325.

Jung, R.E., Glauser, R., Scharer, P., Hammerle,

C.H., Sailer, H.F. & Weber, F.E. (2003) Effect of

rhBMP-2 on guided bone regeneration in humans.

Clinical Oral Implants Research 14: 556–568.

Jung, R.E., Hammerle, C.H., Kokovic, V. & Weber,

F.E. (2007b) Bone regeneration using a synthetic

matrix containing a parathyroid hormone peptide

combined with a grafting material. The Interna-

tional Journal of Oral & Maxillofacial Implants

22: 258–266.

Jung, R.E., Schmoekel, H.G., Zwahlen, R., Koko-

vic, V., Hammerle, C.H. & Weber, F.E. (2005)

Platelet-rich plasma and fibrin as delivery systems

for recombinant human bone morphogenetic

protein-2. Clinical Oral Implants Research 16:

676–682.

Jung, R.E., Weber, F.E., Thoma, D.S., Ehrbar, M.,

Cochran, D.L. & Hammerle, C.H. (2008) Bone

morphogenetic protein-2 enhances bone forma-

tion when delivered by a synthetic matrix con-



taining hydroxyapatite/tricalciumphosphate.


Lutolf, M.P., Weber, F.E., Schmoekel, H.G.,

Schense, J.C., Kohler, T., Muller, R. & Hubbell,

J.A. (2003) Repair of bone defects using synthetic

mimetics of collagenous extracellular matrices.

Nature Biotechnology 21: 513–518.

Lynch, S.E., Buser, D., Hernandez, R.A., Weber,

H.P., Stich, H., Fox, C.H. & Williams, R.C.

(1991) Effects of the platelet-derived growth fac-

tor/insulin-like growth factor-I combination on

bone regeneration around titanium dental im-

plants. Results of a pilot study in beagle dogs.


Meyle, J., Gonzales, J.R., Bodeker, R.H., Hoffmann,

T., Richter, S., Heinz, B., Arjomand, M., Reich,

E., Sculean, A., Jepsen, K. & Jepsen, S. (2004) A

randomized clinical trial comparing enamel ma-

trix derivative and membrane treatment of buccal

class II furcation involvement in mandibular mo-

lars. Part II: secondary outcomes. Journal of


Murai, M., Sato, S., Koshi, R., Yokoyama, K., Ikeda,

K., Narukawa, M., Takayama, T., Yoshinuma, N.

& Ito, K. (2005) Effects of the enamel matrix

derivative and beta-tricalcium phosphate on bone

augmentation within a titanium cap in rabbit

calvarium. Journal of Oral Science 47: 209–217.

Nkenke, E., Radespiel-Troger, M., Wiltfang, J.,

Schultze-Mosgau, S., Winkler, G. & Neukam,

F.W. (2002) Morbidity of harvesting of retromolar

bone grafts: a prospective study. Clinical Oral

Implants Research 13: 514–521.

Nkenke, E., Schultze-Mosgau, S., Radespiel-Troger,

M., Kloss, F. & Neukam, F.W. (2001) Morbidity

of harvesting of chin grafts: a prospective study.


Okamoto, K., Matsuura, T., Hosokawa, R. & Aka-

gawa, Y. (1998) RGD peptides regulate the spe-

cific adhesion scheme of osteoblasts to

hydroxyapatite but not to titanium. Journal of

Dental Research 77: 481–487.

Pierschbacher, M.D. & Ruoslahti, E. (1984) Cell

attachment activity of fibronectin can be dupli-

cated by small synthetic fragments of the mole-

cule. Nature 309: 30–33.

Sanz, M., Tonetti, M.S., Zabalegui, I., Sicilia, A.,

Blanco, J., Rebelo, H., Rasperini, G., Merli, M.,

Cortellini, P. & Suvan, J.E. (2004) Treatment of

intrabony defects with enamel matrix proteins or

barrier membranes: results from a multicenter

practice-based clinical trial. Journal of Perio-

dontology 75: 726–733.

Schaffner, P., Meyer, J., Dard, M., Wenz, R., Nies,

B., Verrier, S., Kessler, H. & Kantlehner, M.

(1999) Induced tissue integration of bone implants

by coating with bone selective RGD-peptides in

vitro and in vivo studies. Journal of materials

science. Materials in Medicine 10: 837–839.

Schenk, R. & Olah, A.J. (1980) Histomorphometrie.

In: Kuhlencordt, F. & Bartelheimer, H., eds.

Handbuch Der Inneren Medizin, 437–494. Ber-

lin: Springer.

Schenk, R.K., Olah, A.J. & Herrmann, W. (1984)

Preparation of calcified tissues for light micro-

scopy. In: Dickson, G.R., ed. Methods of Calci-

fied Tissue Preparation, 1–56. Amsterdam:

Elsevier.

Schliephake, H., Scharnweber, D., Dard, M., Sew-

ing, A., Aref, A. & Roessler, S. (2005) Functiona-

lization of dental implant surfaces using adhesion

molecules. Journal of Biomedical Materials Re-

search. Part B, Applied Biomaterials 73: 88–96.

Schwarz, F., Herten, M., Ferrari, D., Wieland, M.,

Schmitz, L., Engelhardt, E. & Becker, J. (2007)

Guided bone regeneration at dehiscence-type

defects using biphasic hydroxyapatiteþbeta

tricalcium phosphate (bone ceramic) or a

collagen-coated natural bone mineral (biooss

collagen): an immunohistochemical study in

dogs. The International Journal of Oral and

Maxillofacial Surgery 36: 1198–1206.

Schwarz, F., Rothamel, D., Herten, M., Sculean, A.,

Scherbaum, W. & Becker, J. (2004) Effect of

enamel matrix protein derivative on the attach-

ment, proliferation, and viability of human

SaOs(2) osteoblasts on titanium implants. Clin-

ical Oral Investigations 8: 165–171.

Sculean, A., Donos, N., Schwarz, F., Becker, J.,

Brecx, M. & Arweiler, N.B. (2004) Five-year

results following treatment of intrabony defects

with enamel matrix proteins and guided tissue

regeneration. Journal of Clinical Periodontology

31: 545–549.

Sigurdsson, T.J., Nygaard, L., Tatakis, D.N., Fu, E.,

Turek, T.J., Jin, L., Wozney, J.M. & Wikesjo,

U.M. (1996) Periodontal repair in dogs: evaluation

of rhBMP-2 carriers. International Journal

of Periodontics and Restorative Dentistry 16:

524–537.

Silvestri, M., Sartori, S., Rasperini, G., Ricci, G.,

Rota, C. & Cattaneo, V. (2003) Comparison of

infrabony defects treated with enamel matrix

derivative versus guided tissue regeneration with

a nonresorbable membrane. Journal of Clinical


Simmer, J.P. & Fincham, A.G. (1995) Molecular

mechanisms of dental enamel formation. Critical

Reviews in Oral Biology and Medicine 6:

84–108.

Tonetti, M.S., Lang, N.P., Cortellini, P., Suvan,

J.E., Adriaens, P., Dubravec, D., Fonzar, A., Four-

mousis, I., Mayfield, L., Rossi, R., Silvestri, M.,

Tiedemann, C., Topoll, H., Vangsted, T. & Wall-

kamm, B. (2002) Enamel matrix proteins in the

regenerative therapy of deep intrabony defects.


Traini, T., Valentini, P., Iezzi, G. & Piattelli, A.

(2007) A histologic and histomorphometric eva-

luation of anorganic bovine bone retrieved 9 years

after a sinus augmentation procedure. Journal of


Weibel, E.R. (1980) Stereological Methods: Practical

Methods for Biological Morphometry. New York:

Academic Press.

Zafiropoulos, G.G., Hoffmann, O., Kasaj, A., Will-

ershausen, B., Weiss, O. & Van Dyke, T.E. (2007)

Treatment of intrabony defects using guided tissue

regeneration and autogenous spongiosa alone or

combined with hydroxyapatite/beta-tricalcium

phosphate bone substitute or bovine-derived xe-

nograft. Journal of Periodontology 78: 2216–2225.



Bone regeneration using a synthetic matrix containing enamel matrix derivate

Documents

Transcript of Bone regeneration using a synthetic matrix containing enamel matrix derivate