Evaluation of Osteoinductivity of Atorvastatin Combined with

β-TCP Versus β-TCP Alone in Filling Bone Defects after Large Mandibular
Radicular Jaw Cyst Enucleation
– A Randomized Clinical Trial

‫تقييم قدرة تحفيز تكوين الخاليا العظمية لألتورفاستاتين مع فوسفات بيتا ثالثي الكالسيوم مقابل فوسفات بيتا‬
‫ثالثي الكالسيوم وحده في معالجة العيوب العظمية بعد استئصال كيس الفك السفلي‬

Protocol
Submitted to the Faculty of Dentistry – Cairo University, in Partial Fulfillment of the
Requirements for Master Degree in Oral and Maxillofacial Surgery Department

By:
Yara Gamal Mohamed
B.D.S (2018)
Faculty of Oral and Dental Medicine,
Misr University for Science and Technology, Egypt

2023

Code:

Supervisors’ Signature Head of department’s Signature

1-
2-

Date
Protocol checklist
Reported
Item Reviewer’s
Section and topic Checked item on page
no. check
No.
1 Title 1
2 Protocol registration 1
I. Administrative
3 Protocol version 1
information
4 Funding 1
5 Roles and responsibilities 2

II. Introduction

Research question 3
Statement of the problem 3
A) Background and 6a Rationale for carrying out 4
Rationale the trial
Review of literature 4
6b Choice of comparators 9
Aim of the study 9
B) Objectives 7
Hypothesis 9
C) Trial design 8 Trial design 10

III. Methods

9 Study setting 11
10 Eligibility criteria 11
A) Participants, 11 Interventions 11
interventions & 12 Outcomes 14
outcomes 13 Participant timeline 15
14 Sample size 16
15 Recruitment 16
16 Allocation 16
16 a Random sequence 16
B) Assignment of generation
interventions (Randomization)
16 b Allocation concealment 16
mechanism
 16 c Implementation 16
17 Blinding (masking) 16
C) Data collection, 18 Data collection methods 16
management, and 19 Data management 17
analysis 20 Statistical methods 17
21 Data monitoring 17
D) Monitoring 22 Harms 17
23 Auditing 17

24 Research ethics approval 18

25 Protocol amendments 18
26 Informed Consent 18
IV. Ethics and 27 Confidentiality 18
dissemination 28 Declaration of interests 18
29 Access to data 18
30 Ancillary and post-trial care 18
31 Dissemination policy 18

V. Appendices 32 Informed consent 19

materials
33 Biological specimens 22

VI. References 23

Evidence based committee (Reviewers)

Name Signature Date

1.

2.

Research plan committee

Name Signature Date

1.
2.
 INDEX

Tables and Figures Index……………………………………………………………..…… I

List of abbreviations…………………………………………………………………….…....II
Administrative information……………………………….……………….……………...…..1
1. Title……………………………………………………………………….….…...…....1
2. Protocol registration…………………………………………….……………….…....1
3. Protocol version…………………………………………………...........................….1
4. Funding…………………………………………………………………………..…...1
5. Roles and responsibilities…………………………….…………………………….....2
Introduction……………………………………………………………………….………….3
A. Background and rationale……………………………………………………………3
6a. Research question………………………………………………………………...3
6a. Statement of the problem…………………………………………………………3
6a. Rationale for carrying out the trial……………………………………….………4
6a. Review of literature……………………………………………………………….4
6b. Choice of comparator………………………………………………………….…9
B. Objectives………………………………………………………………………….….5
7. Aim of the study…………………………………………………………………....9
7. Hypothesis………………………………………………………………………....9
C. Trial design…………………………………………………………..…………..…10
8. Trial design……………………………………………………………………...10
Methods………………………………………………………………………………………11
A. Participants, interventions and outcomes…………………………………………11
9. Study setting………………………………………………………………………11
10. Eligibility criteria…………………………………………………………..……11
11. Intervention………………………………………………………………………14
12. Outcomes…………………………………………………………………...……15
13. Participant timeline…………………………………………………………...…16
14. Sample size………………………………………………………………….……16
15. Recruitment………………………………………………………………..……16
B. Assignment of interventions………………………………………………………..16
16- Allocation …………………………………………………………….…………16
16a. Random sequence generation (Randomization)……………………………16
16b. Allocation concealment mechanism…………………………………..….…..16
16c. Implementation…………………………………………………………..……16
17. Blinging (masking)……………………………………………………………16
C. Data collection, management, and analysis…………………………………………….16
18. Data collection method………………………………………………………..…16
19. Data management………………………………………………………….…..…17
 20. Statistical methods……………………………………………………..………....17
21. Data Monitoring……………………………………………………….……..….17
22. Harms…………………………………………………………………………..…17
23. Auditing…………………………………………………………………..……….17
Ethics and dissemination……………………………………………………………………18
24. Research ethic approval…………………………………………………………….18
25. Protocol amendments………………………………………………………...……..18
26. Informed consent…………………………………………………………………….18
27. Confidentiality…………………………………………………………………...…..18
28. Declaration of interest…………………………………………………………..…...18
29. Access to data…………………………………………………………………………18
30. Ancillary and post-trial care………………………………………………………….18
31. Dissemination policy…………………………………………………………….........18
Appendices………………………………………………………………………………...…19
32. Informed consent materials…………………………………………………………..19
33. Biological specimens.....................................................................................................21
References……………………………………………………………………………………22
 Tables and Figures Index
Table Description
Table 1 Outcomes’ table
Table 2 Participants timeline
 LIST OF ABBREVIATIONS
Abbreviations Word(s)
BMP-2 Bone morphogenic protein-2
LDL-C Low density low molecular weight-
cholesterol
HMG-CoA 3-hydroxy-3methylglutaryl
coenzyme
NCT3T3E1 Cells Non-transformed osteoblastic cells
VEGF Vascular endothelial growth factor
COX-inhibitors Cyclo-oxygenase- inhibitors
SIM/GEL Simvastatin in methylcellulose gel
in a polylactic membrane
SIM Simvastatin
PLA Polylactic acid
ALP Alkaline phosphatase
COL-1 Type 1 collagen
BSP Bone sialoprotein
OCN Osteocalcin
CS Calcium sulfate
RANK-L Receptor activator of nuclear factor
kappa-Β ligand
α-TCP Alpha-Tricalcium phosphate
Β-TCP Beta-Tricalcium phosphate
HA Hydroxyapatite
IOAP Intra-oral periapical radiograph
 I. ADMINISTRATIVE INFORMATION:

1. Title:

Evaluation of the Osteoinductivity of Atorvastatin Combined with β-TCP Versus β-TCP

Alone in the Treatment of Residual Bone Defects after Large Mandibular Radicular
Jaw Cyst Enucleation – A Randomized Clinical Trial.

2. Protocol Registration:

This research will be registered on ClinicalTrials.gov.

3. Protocol Version:

-Date:
-Protocol number:

4. Funding:

This trial will be self- funded. Dental units and other consumables will be provided by
the department of Oral and Maxillofacial Surgery, Cairo university.

1
5. Roles and responsibilities:

2
 II. INTRODUCTION:
6a. Background and Rationale:
Research question:
Is atorvastatin an effective osteoinductive agent when combined with β-TCP versus β-TCP
alone for filling the residual bone defects after large mandibular radicular jaw cyst enucleation?
Statement of the problem:
Jaw cysts are a common disease of the maxillofacial region, and odontogenic cysts represent
the majority of cases1. The radicular cysts are the most common type of jaw cysts2. These cysts
arise from the epithelial rests of malassez in the periodontal ligament as a result of an
inflammatory process in the dental pulp region.3
They appear as defects in the epithelial lining in the mandible and maxilla which gradually
increase in volume without invading the bone or damaging the nerves4. However, their
enucleation results in a bony defect that especially in the case of larger cysts, may lead to
pathological fracture, loss of teeth, infection, and other symptoms4.
The treatment of choice for radicular cysts is based on the largest diameter, accordingly, some
researchers considered jaw cysts as three different types.5 For those larger than 4 cm,
decompression is required first before enucleation, which usually takes 6-18 months.6 For those
less than 1 cm, it is preferred to adopt endodontic treatment rather than surgery.7 Finally, for
cysts between 1 and 4 cm, enucleation is the most frequent treatment.8
However, decompression and enucleation are not always sufficient to achieve complete
regeneration and the healing process can be lengthy.1 Furthermore, without any bone-filling
material, other studies revealed that the spontaneous bone healing index can be from 25.85%
to 76% and from 43.46% to 81.03% after 6 and 12 months postoperative, respectively.9-11 These
results indicate that spontaneous bone healing is not efficient enough, especially for patients
with the extraction of relevant teeth who may process a subsequent surgery for implantation.12
Therefore, cavities are often filled with bone regeneration materials to reduce infection,
accelerate bone healing, prevent soft tissue collapse into the defect, and improve bone
strength.13 Some researchers suggest that when the size of the defect reaches 1-2 cm or 50%
of the circumference of the bone, filling materials are needed.14
Over time, various biomaterials were used to fill the bone defects; Autologous bone, xenograft,
allografts, and alloplasts.13 Autologous bone is considered the gold standard of bone because it
possesses osteoinductive, osteoconductive, and osteogenic properties.15-18 However, several
potential complications are involved with autogenous bone such as donor site morbidity,
limited availability for harvest, and increased blood loss. Thus, the demand for the use of
alloplastic materials is increasing.19
In order to develop more effective enhanced osteogenic properties, alloplastic materials are
combined with osteoinductive biological growth factors such as BMP-2, which are essential
regulators of osteogenic differentiation during bone repair.20 However, protein growth factors
are expensive, may degrade rapidly at the treatment site, and could potentially elicit an immune

3
response. If pharmacological compounds such as the lipid-lowering drugs, statins, can
upregulate the necessary autogenous growth factors to stimulate bone growth, this approach
may prove to be a cost-effective way to correct bone defects.20
The Rationale for Conducting the Research:
Studies have shown that the local application of lipid-lowering drugs, statins21, induces bone
growth by stimulating BMP-2.22 therefore, the addition of these pharmacological agents to
osteoconductive bone graft materials, which lack osteogenic properties, could be a promising
approach for bone regeneration.
However, there is still no consensus in the literature on the optimal therapeutic dose and mode
of application for statins.23 Furthermore, limited studies are available in the literature regarding
the use of statins as a biological modifier in filling bone defects after cyst enucleation.24
Atorvastatin was chosen to be the statin of choice in this study, as it is considered the second
most potent LDL-C lowering drug, which may hypothetically reflect a more potent
osteoinductivity.25-26
Therefore, the purpose of this randomized clinical trial is to validate and evaluate the
osteoinductivity of statins + β-TCP combination using the Gouda et al.,42 methodology used
in maxillary sinus lifting but instead in filling the residual defect of large odontogenic radicular
cysts (1.5 – 4 cm), which shows suppressive influence on bone formation, as well as a greater
risk for pathological fracture, infection, and insufficient bone healing.
Review of Literature:
Goldstein and Brown et al., [1990]21; reported that statins inhibit the function of 3-hydroxy-
3-methylglutrayl coenzyme A (HMG-COA) reductase to inhibit the conversion of 3-HMG-
CoA to mevalonate, which is a rate-limiting step in cholesterol synthesis.
Hamelin and Turgeon., [1998]25; Maron et al., [2000]26; These drugs, including simvastatin
and atorvastatin, therefore, are potent inhibitors of cholesterol biosynthesis that are widely
prescribed to lower cholesterol in hyperlipidemic patients at risk for cardiovascular disease. -
23.
Data from comparative trials confirm that rosuvastatin is the most effective statin for
lowering LDL-C, followed by atorvastatin, simvastatin, and pravastatin. The heavenly
highlighted study.
Statins are known to undergo efficient hepatic-first pass and are predominantly metabolized by
the cytochrome P450 (CYP450) family of enzymes. According to the literature, the dose at
which there is an increased risk for SAMSs is 80 mg/day. SAMSs are by far the most prevalent
and important adverse event. These can present as myalgia, myopathy, myositis with elevated
creatinine kinase, or at its most severe, rhabdomyolysis, with some people reporting additional
joint and abdominal pain. Natalie et al., [2023].27
Besides decreasing the production of cholesterol, statins possess several LDL-independent
pleiotropic actions. Mundy et al., [1999]28; were the first to report the possible effects of statins
on bone metabolism by testing the effects of more than 30,000 compounds on bone formation.
They found that adding statins to the neonatal murine calvarial bone in organ culture increased

4
new bone formation by two- to threefold. They also explained that the osteoblast-differentiation
effect found in vitro and in vivo in rats may be due to BMP-2-mediated action.
Moreover, they reported fewer osteoclasts on bone surfaces after statin treatment in vivo, which
suggests an anti-resorptive effect as well as an anabolic effect on bone. However, statins don’t
have the selective bone-targeting property of bisphosphonates in vivo. Furthermore, even
though oral administration of statins seemed to be effective in rats, the statins now available
are probably unsuitable as bone anabolic agents in humans, due to their low systemic
availability. Additionally, if the dose was adjusted for humans, the dose would be too high that
it would risk myalgia and other side effects; M.J.Rogers et al.,[2000]29
Further evidence of BMP-2 involvement in the osteoblast-differentiating effect of statins has
been provided by several groups; Sugiyama et al., [2000]30 found that adding lipophilic statins;
namely compactin (mevastatin), and simvastatin to HOS cells (in vitro), induced BMP-2 by
inhibiting HMG-CoA reductase. Whereas the hydrophilic pravastatin didn’t stimulate the
BMP-2 promoter.
Ohanka et al., [2001]31; reported that just like other lipophilic statins, pitavastatin increased
the BMP-2 mRNA expression in cultured human osteoblasts in addition to enhanced expression
of osteocalcin much more effectively than that of BMP-2, through the inhibition of Rho-kinase
activation.
Maeda et al., [2001]32; reported that statins promote osteoblast differentiation and
mineralization in MCT3T3-E1 cells derived from new bone mouse calvaria. Furthermore,
Maeda et al., [2003]33; were the first to report that statins stimulate gene expression for a bone
anabolic factor (VEGF) in osteoblasts other than BMP-2 in a time- and concentration-
dependent manner. They tested the effect of statins on VEGF mRNA expression in osteoblastic
cells, using MC3T3-E1 non-transformed cells which is a cell line derived from mouse calvaria.
They found a marked enhancement of VEGF mRNA gene expression with 12 hrs exposure to
hydrophobic statins including simvastatin, cerivastatin, and atorvastatin at the concentrations
of 10-7, 10-8, 10-6 M respectively. However, simvastatin at high concentrations (10-4 M) reduced
VEGF mRNA expression in MC3T3-E1 cells.
Stein et al., [2003]34; used an adult female rat bilateral mandible model to evaluate the topically
applied simvastatin (at different doses using Methycellulose gel in a PLA membrane as a
carrier) on the histologic bone growth and inflammation in vivo; the effects of anti-
inflammatory COX inhibitors, including selective COX-2 inhibitors Ns-398 and non-selective
COX inhibitor indomethacin on this topically applied simvastatin-induced bone growth and
inflammation. They concluded that locally applied SIM in methylcellulose gel/PLA membrane
can stimulate significant bone growth at an optimal dose of 0.5 mg where clinical inflammation
is reduced, while at 0.1 mg dose, minimal inflammation was demonstrated with little bone
growth; The COX pathway of inflammation appears to play a partial role in SIM-induced bone
growth in vivo, in that both NS-398 and especially indomethacin inhibited SIM/GEL form
causing significant bone growth; and that osteoblast activation and bone growth by SIM may
be mediated by fibroblasts and collagen turnover.

5
Maeda et al., [2004]35; investigated changes in marker expression corresponding to stages of
osteoblast differentiation in statin-treated MC3T3-E1 cells (in vitro). They found that statins
stimulate BMP-2 expression, promoting osteoblast differentiation at the culture's early and
middle stages. Furthermore, it is likely that VEGF stimulated by statins enhances the
differentiation and mineralization at the late stage of the culture. They concluded that statins
stimulated osteoblast differentiation and matrix mineralization in vitro through the sequentially
stimulated expression of different markers (BMP-2, ALP, COL-I, BSP, VEGF, and OCN) by
osteoblasts,, which suggests the possible clinical applicability of statins to the treatment of
osteoporosis. In vivo
Nyan et al., [2007]36; investigated the effects of locally administered simvastatin combined
with calcium sulfate as an osteoconductive carrier on the healing of a critical-sized bone defect
(8 mm) in the rats. 1 mg of simvastatin was incorporated into 60 mg CS, then, the powder was
mixed with distilled water in a ratio of 1:0.4, and the mix was poured in a template to form a
disc of 8mm diameter and 1 mm thickness. They concluded that the 1.0 mg simvastatin and
calcium sulfate graft combination appeared to promote bone formation in the critical-sized rat
calvarial defect at 8 weeks after a significant soft tissue inflammation over the defect area up
to 5 weeks, during which there was no significant bone formation. Furthermore, it was unclear
whether the previously released simvastatin or that released by a small amount of CS, which
possibly remained unreleased after 4-5 weeks was responsible for that bone formation.
Therefore, they suggested that a much slower resorption of the carrier with consequently the
gradual release of simvastatin is preferable for a faster bone formation in the defect without
inducing inflammation. Further studies should be performed using an appropriate carrier
scaffold that can release an optimal amount of simvastatin gradually through a slow
degradation and also provide an osteoconductive surface over which bone formation can take
place.
Ayukaway et al., [2009]37; reported that although there was a significant increase in new bone
areas under the influence of simvastatin local injection for 3 consecutive days in surgically
created bone defects in rats, that effect didn’t continue when the administration was terminated.
They collected and examined the bone on day 5 and found a significant increase in new bone
areas in comparison to the control group. Furthermore, using r-TPCR, both ALP and BMP-2
mRNA expression significantly increased in the simvastatin group. While the expression of
RANKL responsible for osteoclast differentiation was depressed in the same group. On day 10,
there was no significant difference between the test and control groups.
Nyan et al., [2009]38; tested the hypothesis that maximum bone regeneration with less
inflammation would be achieved by combining an optimal dose of simvastatin with α-TCP,
which is an osteoconductive biomaterial capable of releasing the drug gradually. A bilateral 5-
mm diameter calvarial defects were created in adult Wistar rats and filled with the preparation
of different doses of simvastatin dissolved in ethanol (0, 0.01, 0.1, 0.25, and 0.5 mg) combined
with α-TCP particles (per 14 mg) or left empty. The animals were sacrificed at 2, 4, and 8 weeks
and analyzed radiographically and histologically. They concluded that simvastatin when
combined with α-TCP particles, 0.1 mg simvastatin is the optimal dose for stimulation of the
maximum bone regeneration in rat calvarial defects without inducing inflammation and it could

6
be applied as an effective bone graft material. Furthermore, they suggested that more studies
are required to validate the effect of this optimal dose of simvastatin with α-TCP combination
in different clinical situations.
Mouhamed et al., [2009]39; were the only authors to report the radiological and
histomorphological evaluations of grafted residual bone defects resulting from the cystectomy
of 16 periapical lesions using tricalcium phosphate with simvastatin, which revealed significant
differences from the control tricalcium phosphate alone group. Simvastatin was added to the
tricalcium phosphate graft in a percentage of 5;100 grams.
Rojbani et al., [2011]40; followed Nyan et al., [2009] methodology but this time Evaluated the
osteoinductivity of 2 other alloplastic grafting materials in addition to the α-TCP; β-TCP and
HA combined with or without simvastatin. 72 Wistar rats were divided into 7 groups; α-TCP,
β-TCP, HA, α-TCP with SIM, β-TCP with SIM, HA with SIM, and the control (left empty).
Animals were sacrificed at 6 and 8 weeks after the surgery for histomorphometric analysis. The
quantitative results show the percentage of new bone formation among 6 + 8 weeks groups. In
the α-TCP + statin group. The percentage was higher compared with the other bone substitute
materials, while in the control group, the percentage was the lowest, furthermore, the
percentage was higher within the same material when sim was added in both α-TCP and β-TCP
groups, while no significance was found in the HA group, however, the percentage has a
tendency to increase when SIM was added. However, all simvastatin groups showed a reddish
inflammation in the skin area overlying the bone defects, the inflammation subsided to normal
skin within 10 days. The wounds showed complete healing after 2 weeks. This study confirmed
α-TCP, β-TCP, and HA are osteoconductive materials that can be used as space maintainers for
bone formation when applied to a bone defect, and demonstrated that combining these
materials with simvastatin stimulates bone regeneration and also affects the degradability of α-
TCP and β-TCP. Conclusively, α-TCP has the advantage of a higher rate of degradation
allowing more bone formation, and combining α-TCP with simvastatin enhances this property,
although the difference in new bone formation between α-TCP and β-TCP was statistically
insignificant.
Chauhan et al., [2015]41; assessed the efficacy of simvastatin in bone regeneration in the
extraction sockets of mandibular third molars. A total of 30 patients in each group; both female
and male aged between 18 and 40 years, with bilateral similarly impacted mandibular third
molars, with no active infection, and who had no systemic problems nor any abusive habits
were divided into two groups. The dose of simvastatin used was 10 mg mixed with 2 ml saline
solution and the gel foam was used as a carrier. The patients were followed up radiographically
and clinically (for pain and swelling assessment) on days 1, 2, 3, 30, 60, and 90. Bone
regeneration was evaluated with the help of standardized intraoral periapical radiographs. The
IOPA radiographs were taken and evaluated by the grey histogram on day 1, 1st month, and 3rd
month. The mean values of the grey level histogram were highly significant during the
immediate postoperative and first-month post-operative periods. Data suggested evidence of
early bone formation and maturation radiographically in the study group as compared to the
control group at 1st and 3rd month post-operatively. Moreover, facial swelling was numerically
greater on the study side as compared to the control side on the 2nd and 3rd postoperative days.

7
But it was clinically insignificant. The severity of pain was equal in both the study and control
groups and the results were not significant. The study
Gouda et al., [2017]42; Investigated the available bone quality and quantity after performing
sinus augmentation in a total of 6 patients; 4 sinuses in each group, using SIM/β-TCP
combination versus β-TCP alone. They reported a statistically significant difference in the
percentage of new bone formation between the test group (β-TCP+SIM) and the control group
(β-TCP+SIM); ( 26.25 +- 4.35 and 19.5 +- 2.38 respectively). Whereas, the percentage of bone
loss in the test group (12.53%) was higher than that of the control group (9.02%), Although it
was an insignificant difference. Moreover. They reported radiographic signs of sinusitis in the
CBCT one week post-operatively without any clinical signs of sinusitis, which was attributed
to the burst release of the simvastatin from the β-TCP on the first day as reported by Nyan et
al., [2009]38 and Rojbani et al., [2011]40. Finally, the histological examination of the new bone-
formed biopsies during the second stage surgery for implant placement revealed more mature
lamellar bone in the test group than in the control group.

Swati et al., [2019]23; suggested that simvastatin alone has a promising potential for alveolar
bone regeneration in the optimal dose of 0.5-10 mg depending on the route of administration.
Furthermore, The use of simvastatin with other bone grafts poses an additional advantage,
however, there are contradictory results in the literature; hence, more studies are needed to
reach a definitive consensus on the use of carriers with simvastatin, as the usage of the carrier
is determined by the site and type of surgical procedure. This systematic review and meta-
analysis investigated the impact of simvastatin intervention on the healing of bone, soft tissue,
and TMJ cartilage in dentistry. A total of 130 articles from the period of 2017-2005 were
screened, of which 38 articles were selected. and after evaluation of these articles, only 16
adequate studies; including 10 animal studies and 6 human studies with low risk of bias were
considered for review analysis. The meta-analysis was performed only with human studies.
whereas, animal studies were not considered for quantitative analysis due to the difference in
methodologies, protocols, defect types, animal models, and outcome variables. on the healing
of periodontal defects, There was evidence of a significant benefit of simvastatin for both CAL
gain (mean difference 1.50 mm, p < 0.001, 95% CI = 1.19, 1.82 mm) and PD reduction (mean
difference 2.01 mm, p < 0.001, 95% CI = 1.41, 2.61 mm). on the healing of bone defects (bone
defect fill); the changes in the width of the alveolar ridge radiographically, there was a
significant improvement of alveolar ridge width in the simvastatin group than its control group
(mean difference = 1.40 mm, p < 0.001, CI = 0.99, 1.81 mm). on the healing articular cartilage,
according to the animal studies investigating the outcome of SIM injection on experimental
TMA in rats, The SIM showed an anti-inflammatory property and that it could preserve normal
condylar growth of cartilaginous tissue at the dose of 0.1 mg (for the least inflammatory
reaction) or 0.5 mg (for increased bone formation). On the healing of soft tissue, a human
clinical study assessed palatal healing with SIM and chitosan gel (10 mg/ml) after free gingival
graft procedure. It was concluded that the SIM combination improved healing and reduced pain
following the Free gingival graft procedure.

Finally, the most recent systematic review Maria et al., [2022]21; suggested that more
randomized clinical trials are conducted with the objective of better elucidating the dosage and

8
the pharmacological association for certain procedures in dentistry. This systematic review
investigated the effectiveness of statins (rosuvastatin, simvastatin and atorvastatin) in alveolar
bone regeneration in dentistry by analyzing and evaluating 1,721 studies published in the last
10 years (2011-2021), of which only 17 clinical trials met the eligibility criteria adopted and
were included in the review. The majority of the studies (n = 14, 82%) were related to chronic
periodontitis where 1.2% statin gel form was used, followed by implantology (n = 2, 12%), and
lastly oral surgery (n = 1, 6%) Only 2 studies out of the 17 studies demonstrated statistical
insignificance when statins were administered alone; the first study in the area of implantology
where the use of simvastatin was ineffective in gaining bone tissue in the maxillary sinus
region, and the second study is in the area of periodontics, where the addition of 1.2% of
atorvastatin to PRF failed to increase the regenerative capacity of intraosseous defects in
chronic periodontitis.

6b. Explanation for choice of comparators

Synthetic grafting materials can provide osteo-conduction and osseointegration. The idea of
using these osteoconductive materials is to stabilize the blood clot in the defect avoiding
infection and to advance bone regeneration by enhancing the migration of osteoprogenitor cells
Horch et al., 200643. TCPs especially pure-phase β-TCP are regarded as being biocompatible,
osteoconductive, and resorbable Buser et al., 199844; Gassbeek et al., 200545; Xin et al.,
200546; Horch et al., 200647. Various studies utilized β-TCP to fill bone defects after cyst
enucleation ( Zerbo et al., 200148; Palti and Hoch., 200249; Bicsak et al., 200650; Horch et al.,
200651. In all these studies with radiographic and partly histological follow-up investigations,
the β-TCP was widely resorbed after 12 months.
Aim of the study:
To evaluate the effects of atorvastatin when combined with β-TCP versus β-TCP alone on
the quantity of bone formed and in hastening bone regeneration of the residual bone defects
after cyst enucleation.
Hypothesis
Null Hypothesis:

• There is no difference in the clinical and radiographical parameters between the use
of atorvastatin combined with β-TCP and β-TCP alone
• If the results are significant (i.e. there is a difference between the test group and the
control group) then the null hypothesis is rejected.

7. Trial Design:
1. Randomised clinical trial
2. Two arms trial;
3. Parallel group trial
4. Allocation ratio 1:1;
5. Superiority trial;

9
III. METHODS:

A) Participants, interventions & outcomes

8. Study setting:
It is a single-centred randomized clinical trial. All the patients will be selected from the
outpatient clinic of the oral and maxillofacial surgery department at Cairo University.
9. Eligibility Criteria:
Inclusion Criteria:
(1) Male and female patients
(2) Patients 18 to 40 years old
(3) Radicular cyst with a maximum diameter from 1.5 to 4.9 cm;
(4) Focal teeth were preserved with root canal treatment;
(5) No previous surgical treatment of the cyst site;
(6) No evidence of acute inflammation;
(7) In good physical status and oral health;
(8) Regular attendance at control visits
Exclusion Criteria:
(1) Patients < 17 years old
(2) Radicular cyst with a maximum diameter < 1.5 cm.
(3) Pregnancy or lactation
(4) Aggregate systemic pathologies such as diabetes, thyroid disorders, and bone
metabolism diseases, among others;
(5) Patients taking calcium, bisphosphonates, glucocorticoids, or other drugs that can
interfere with the metabolism of bone;
(6) Patients with uncontrolled periodontal conditions, endodontic conditions, and other oral
disorders;
(7) Heavy smokers (10 cigarettes/day or more)

10. Interventions:
i. Presurgical preparations:
A. Clinical Examination:
• A thorough medical and dental history will be taken from every patient
• A clinical examination will be carried out for every patient, to assess pain,
swelling, drainage, and the source of infection. An aspiration test will be
included to assess the presence or the absence of fluid and its nature, if present.
• All patients will be referred to the endodontic department to undergo an
endodontic treatment for the teeth involved with the lesion prior to the surgery.

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 B. Radiographic Examination:
1. Preoperative Radiograph:
• A routine pre-operative panoramic radiography will be made for every patient
as a preliminary step to assess the size and MD extension of the cystic lesion

• A pre-operative CBCT will be done for every patient, in order to assess the 3D
dimensions of the cystic lesion, to detect perforations, or any erosion in the
bone cortex, and to aid in the assessment of bone formation after surgery.

2. Preoperative Radiographic planning:

• The DICOM files of the test and control groups will be imported into 3D slicer
5.4.0 software, and a three-dimensional model of the cyst will be manually
labelled with a sphere brush and subsequently will be re-established.
• The volume of the cysts will then be calculated by a model segment. This is
important in;
o Determining the amount/volume of bone graft required for each defect
o Calculating and measuring the amount of new bone formed; how much
new bone has filled the defect by tracing the margin of the defect pre-
operatively and comparing it with the new margin of the newly formed
bone as well as comparing both the test and control groups.

ii. Surgical procedure:

A. Preparation of β-TCP-Atorvastatin complex (for the test group):

The β-Atorvastatin preparation will be done as described by Gouda et al.,

[2017]41 except for the step of dissolving the statin in ethanol, as atorvastatin,
unlike simvastatin, is already in its active form25. Atorvastatin pure powder form
supplied by (Atorvastatin Calcium analytical standard (≥ 98%), AMM0221020,
NAWAH scientific, Cairo, Egypt) and will be added to the β-TCP supplied by
(power bone, Bonegraft Granules (Crunch) 2-4 mm) in the ratio of 0.1 mg
(atorvastatin):14 mg (β -TCP) so that each gram of β-TCP would contain 7.21
mg of atorvastatin. Then, the mix will be soaked/hydrated with saline upon its
application to the defects.

B. Intraoperative procedure (for both groups):

All the surgical procedures will be carried out under local anesthesia
(Alexadricaine; articaine 1:100,000) using an inferior alveolar nerve block and
long buccal nerve block; if required. (1) Cyst enucleation for both groups;

11
 • A trapezoidal or triangular flap gingival sulcus incision will be used, and
an anterior vertical releasing incision will be provided for optimum
visualization
• A sharp dissection will be done superficially to the cyst wall in order to
separate the mucoperiosteal flap without compromising its integrity
• The cyst will be completely enucleated, then, placed immediately in a
biopsy jar containing 10% formalin, to be sent to the pathologist
• The cavity will be rinsed with saline, followed by scraping off the cavity
by a bone curette to completely remove any remnants of the cyst lining
• Any sharp bony margins will be smoothened with a bone file
• Thorough irrigation with povidone-iodine followed by isotonic saline
(2) Bone grafting; filling the bone defect:
• Control group; β bone graft material will be hydrated with saline (how
much), then will be condensed into the defect (the specific volume will
be determined by the size of the cavity), and the flap will be closed using
Vicryl 4-0 sutures.
• Test group; The β+atorvastatin combination will be hydrated with saline,
and then, the defect will be filled with that combination, followed by the
closure of the flap using Vicryl 4-0 sutures.
C. Post-operative instructions:
• Ice packs for the 1st 24 hrs
• Warm Fomentation for the 2nd 24 hrs
• Amoxicillin + clavulanic acid 1 g, every 12 hrs for one week
• Diclofenac Sodium 75 mg Intramuscular Injection, twice for 4 days
• Dexamethasone sodium phosphate 8 mg / 2 ml Intramuscular Injection, once
immediately post-operatively
• 2% Chlorhexidine gluconate mouth rinse, starting from the 2nd 24 hrs, twice
daily for one week
• The patients will all be advised to maintain good oral hygiene
D. Post-operative follow-up:
1) Clinical examination:
• All the patients will be called one week post-operatively for suture removal. The
site of surgery will be evaluated for any sign of inflammation, infection, wound
dehiscence, or loss of bone graft material.

12
 • Criteria for discontinuing or modifying intervention; If the test group showed
an intense inflammatory reaction, the atorvastatin dose was to be re-evaluated.
There is no protocol for the discontinuation of the procedure.
2) Radiographic examination:
• Post-operative CBCT radiographs will be taken for each patient at 3 months and
6 months postoperatively; to evaluate the shrinkage rate of the bone defect and
assess the difference between the test and control group in the amount of new
bone formation.
11. Outcomes:
• Primary outcome:
I. Shrinkage rate of the bone defect:
• This outcome will be measured radiographically;
• Radiographically using CBCT-based model segmentation and calculating the
volume of the model segment.
• The phased SR of cystic lesion will be calculated by (pre-operative cyst volume
– 3/6 months postoperative defect volume)/pre-operative cyst volume x
100%.
• Secondary outcome:
I. Inflammation over the surgical site:
• This outcome will be measured clinically;
• Redness
• Edema
• Drainage
•

Prioritization of Outcome Method of Measurement Unit of

outcome Measurement
• CBCT-based model • Cm3
segmentation and
Primary outcome Shrinkage rate of calculating the volume
the bone defect of the model segment

• Phased SR calculation • %

Binary
Secondary outcome Inflammation Visually method
Yes/no

12. Participant timeline:

13
 Enrolment Allocation Post-Allocation Close-out

Time point t-1* t0** t1*** t2 tx*****

Eligibility Screen X
Enrolment

Informed X
Consent

Allocation X

Β + Atorvastatin
combination bone
Intervention

grafting X

Β alone bone
grafting X

Base Line Data X

Outcomes
Assessment

Shrinkage rate of
bone defect X

Inflammation
X X

Complications
X X

Clinical Follow-up X X

*; Pre-operative time, ***; 1-week post-operative time,

**; Intra-operative time, ****; 4 months post-operative time.

13. Sample size:

14
 14. Recruitment:
Patients will be selected by the supervisors and the researcher from the outpatient clinic of the
OMFS department at the faculty of dentistry, Cairo University until the target population is
achieved.
B) Assignment of interventions:
15. Allocation:
1- Method of random sequence generation ( computerized random number generator)
2- Allocation ratio (1:1)
3- Type of randomization: simple

16a. Randomization:
The total sample size (n=) and the sample sizes in each group are pre-specified exactly
and are under the direct control of the investigator.

16b. Allocation concealment mechanism:

The generated sequence number will be written on the cards, one number per card, and
then these cards will be placed in an opaque sealed envelope. Then these envelopes will
be placed in a container so that each participant will pick one envelope blindly during
the pre-operative preparation.
16c. Implementation:
The principal investigator will generate the allocation sequence, enroll the participants,
and assign the participants to intervention

16. Masking/blinding:
Because the two interventions used in this trial are easily recognized by the investigator, this
study will be double-blinded; the participants and the statistician will be blinded.
C) Data collection, management, and analysis:
17. Data collection methods:
The author will be responsible for the outcome assessment. information on demographic
data, and past, concurrent medical history will be obtained by interviewing the patient.
During (T-1 pre-operative visit); Data regarding personal information, medical history, and
familial history will be obtained. At (T0 surgery day) operative procedure (including surgical
procedure- intraoperative complications- operation time) and post-operative complication
will be recorded in the specified section in prepared form. At (T1 one-week post-operative)
follow-up will be scheduled for stitch removal and any complications will be recorded as
well as a radiological follow-up. In the visit (Tx at 6 months post-operative) patient will be
recalled for routine follow-up to evaluate clinical outcomes.

15
 - For outcome evaluation and instruments used:
In the first week post-operative, panoramic radiograph will by requested to evaluate the
bone graft and surrounding bone to exclude any complications
Clinically, follow-up interval will be day 1, week 1, and 6 months post-operatively.
Plans to promote participant retention and complete follow-up: telephone number and
address of the patient included in the study will be recorded. All patients will be given a
phone call and a text message before the next appointment.
18. Data Management:
All data will be entered electronically. Patient files are to be stored in numerical order and
stored in a secure and accessible place. All data will be stored for 1 year after completion of
the study.
19. Statistical methods:
a. Outcome: A suitable statistical test will be used
b. Methods for any additional analysis: No additional subgroup analysis

20. Data Monitoring:

21. Harms:
- Intra-operative complications:
• Bleeding:
- Proper hemostatic measure
• Adhesion of the cystic lesion to the periosteum:
- Careful and proper dissection to preserve the periosteum.
- Post-operative complications:

• Infection, and wound dehiscence:

- Antibiotic
- Proper hygienic measures
• Loss of bone graft particles:
- Proper suturing technique and under no tension
22. Auditing:
Auditing of the study design will be done by the evidence-based committee, faculty of
dentistry- Cairo University.

16
 IV. Ethics and dissemination
23. Research ethics approval:
24. Protocol amendments:
25. Informed consent:
26. Confidentiality:
27. Declaration of interest:
28. Access to data:
29. Post-trial care:
30. Dissemination policy:

17
 V. APPENDICES:

1) Informed consent. 2) Diagnostic chart. 3) Registration on clinical trial;

31. Informed consent
Appendix 1: Informed consent

Appendix 2: Diagnostic chart

18
19
Appendix 3: Registration on clinical trial

32. Biological specimens:

Not applicable.

20
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