evaluation of the effects of biphasic calcium phosphate ... · this project would not have been...

Evaluation of the effects of biphasic calcium

phosphate and growth factors on bone

regeneration in one-wall defects around implant:

an experimental study in mongrel dogs

Young Woo SON, DDS, MS

The Graduate School

Yonsei University

Department of Prosthodontics





(Directed by Prof. Hong Seok Moon, DDS, MS, Ph.D)

A Doctoral Dissertation

Submitted to the Department of Prosthodontics

and the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy of Dental Science

Young Woo SON

JUNE 2014

Dedication

This thesis is dedicated to:

My lovely wife, who supported me each step of my life with great love,

My beloved kids: Sung Won, and Jun Won, who are the meaning and purpose of my life,

My dear mother, who made me be who I am,

My respected mother in law, who supported and encouraged me to believe in myself,

Without their love, unconditional support and encouragement, this thesis could not

have been possible.

Acknowledgments

I would like to gratefully acknowledge the enthusiastic supervision of my mentor

Dr. Hong Seok Moon during this work. Without his common-sense, knowledge

and perceptiveness, I would never have finished. Also, I would like to thank my

advisor, Dr. Young Bum Park. My warmest and deepest gratitude is expressed for

his scientific guidance and for his continuous support and encouragement.

This project would not have been possible without the support of Dr. Jae Hoon

Lee, Dr. Sung Tae Kim and Dr. Jee Hwan Kim respectively. I would like to

express my appreciation for their advice during my research.

I would like to express my deep appreciation to Dr. Han Sung Jung who has

supported me all the way since the beginning of my studies.

I would like to thank Dr. Min Young Kim, Ms. Chae Eun Lee, Dr. Hyun Min Choi,

Mr. Sang Hyun Park and Ms. Min Jung Lee for their unlimited help.

TABLE OF CONTENTS

LIST OF FIGURES ·························································································· iii

LIST OF TABLES····························································································· v

ABSTRACT ································································································· vi

. INTRODUCTION ························································································ 2

. MATERIALS AND METHODS ······································································· 5

Part I: In vitro study ····················································································· 5

Materials and Methods ············································································ 5

Part II: Animal study ······················································································ 8

A. Animal and materials ·········································································· 8

B. Methods ·························································································· 9

1. Surgical procedures and implant placement with bone graft ··························· 9

2. Animal sacrifice ············································································ 12

3. Histomorphometric analysis ······························································ 13

4. Micro-computed tomographic evaluation ··············································· 17

5.Statistical Analysis ·········································································· 18

. RESULTS ································································································ 19

Part I: In vitro study ···················································································· 19

Part II: Animal study ···················································································· 24

A. Micro-computed tomographic measurement ············································· 24

B. Histomorphometric analysis ································································ 25

1. Contact area ·················································································· 25

1.1 Bone to implant contact ratio (BIC) ···················································· 25

1.2 Inter-thread bone volume (BV) ························································· 27

2. Distance area ················································································· 29

2.1 Unfilled area ·············································································· 29

2.2 New bone formation area ································································ 30

2.3 Remained graft materials area ·························································· 32

. DISCUSSION ··························································································· 34

. CONCLUSION ·························································································· 39

. REFERENCES ·························································································· 40

ABSTRACT(KOREAN) ··················································································· 43

LIST OF FIGURES

Figure 1. Picture showing experimental groups ········································ 6

Figure 2. Analyzing of a wound healing assay ········································· 7

Figure 3. Clinical photographs ··························································· 10

Figure 4. Schematic diagram depicting the implantation at the surgically created

one wall bony defects ························································· 11

Figure 5. A. Clinical photographs

B. Radiographic image ························································ 11

Figure 6. Diagram for experimental design time table ······························· 13

Figure 7.Calculation of bone-to- implant contact ···································· 14

Figure 8. Measurement of bone volume (inter-thread BV) ·························· 15

Figure 9. Region of interest (ROI) ······················································ 16

Figure 10. Unfilled area in the defect area ············································· 16

Figure 11. Region of interest (ROI) ···················································· 17

Figure 12. Wound-healing assay of control group ···································· 20

Figure 13. Wound-healing assay of Osteon II group ·································· 20

Figure 14. Wound-healing assay of Osteon II + BMP (0.01 mg/mL) group ······· 21

Figure 15. Wound-healing assay of Osteon II + BMP (0.1 mg/mL) group ········ 21

Figure 16. Wound-healing assay of Osteon II + FGF (0.01 mg/mL) group ········ 22

Figure 17. Wound-healing assay of Osteon II + FGF (0.1 mg/mL) group ········· 22

Figure 18. Comparison of experimental groups for percentage of wound closure at

different time points ························································· 23

Figure 19. Comparison of bone volume (%) between 4 weeks and 8 weeks ······ 25

Figure 20. Comparison of normalized BIC between 4 weeks and 8 weeks ········ 27

Figure 21. Comparison of normalized inter-thread bone volume between 4 weeks

and 8 weeks ·································································· 28

Figure 22. Comparison of % of unfilled area 4 weeks vs. 8 weeks ················· 30

Figure 23. Comparison of % of new bone area 4 weeks vs. 8 weeks ··············· 31

Figure 24. Comparison of % of remained graft materials between 4 weeks vs. 8

weeks ·········································································· 33

LIST OF TABLES

Table 1. Table showing experimental groups classified by graft materials ········· 7

Table 2. Table showing experimental groups classified by graft materials ········ 12

Table 3. Percentage of wound closure at different time points ······················ 19

Table 4. Means and standard deviations of bone volume value in each groups ··· 24

Table 5. Comparisons of means and standard deviations of bone to implant contact

ratio (BIC) value between experimental groups at 4 weeks and 8 weeks ···

····················································································· 26

Table 6. Comparisons of means and standard deviations of inter-thread bone

volume value between experimental groups at 4 weeks and 8 weeks ···· 28

Table 7. Comparisons of means and standard deviations of unfilled area value

between experimental groups at 4 weeks and 8 weeks ····················· 29

Table 8. Comparisons of means and standard deviations of new bone formation

area value between experimental groups at 4 weeks and 8 weeks ······ 31

Table 9. Comparisons of means and standard deviations of remained graft

materials area value between experimental groups at 4 weeks and 8

weeks ············································································· 32

ABSTRACT





Young Woo SON, DDS, MS

The Graduate School

Yonsei University


(Directed by Prof. Hong Seok Moon, DDS, MS, Ph.D)

Objectives. The aim of this study was to evaluate the effect of Biphasic Calcium

Phosphate(Osteon II), Bone Morphogenic Protein-2 (BMP-2) and Fibroblast

Growth Factor-2 (FGF-2) when simultaneously placed with SLA surface implant

on surgically created one wall critical bone defect

Materials & Methods. In vitro wound-healing assay was used to evaluate the

effect of BMP-2, FGF-2 and Osteon II, which were treated to the scrape-wounded

cultures. The cell migration toward the scratched area was evaluated at 0, 12, 24

and 48 h. For animal study, one-wall critical bone defects (5x5x8 mm) were

created in 6 male mongrel dogs. A total of 8 implants were placed for each

mongrel dog, having 4 implants at both sides of mandible. At each side, one

control (No graft) and three type grafts (Osteon II, OsteonII+BMP-2,

OsteonII+FGF-2) were done within defect area. Total 48 titanium implants were

placed. Bone to implant contact (BIC), the inter-thread bone volume, the length of

bone loss, and defect area volume (unfilled area, new bone area, remained bone

graft area) were investigated using micro-computed tomographic evaluation and

histomorphometric analysis at 4 and 8 weeks.

Results. In vitro study, Osteon II did not greatly influence the migration of

MC3T3-E1 cells, but BMP inhibited the migration of MC3T3-E1 cells regardless

of BMP concentration and FGF facilitated the migration of MC3T3-E1 cells

regardless of FGF concentration. In animal study, the bone volume value of

control (no graft) group in micro CT evaluation was significantly lower than other

grafted groups (Osteon II, Osteon II+BMP, Osteon II + FGF) at both 4 weeks and

8 weeks. The new bone formation volume in Osteon II + BMP groups was

significantly better than that of other experimental groups. Osteon II + BMP graft

could enhance the BIC in the critical defect site around the implant both at 4

weeks and 8 weeks. However, Osteon II + FGF graft could enhance the BIC only

at 4 weeks as compared with Osteon II group. 90% of grafts materials had

disappeared in the critical defects at 4 weeks and there was no significant

difference in remained bone graft percentage between 4 weeks and 8 weeks

groups.

Conclusions. Bone graft materials may have a role to play in critical bone defect

reconstruction and BMP may enhance new bone formation. Osteon II acted as a

good scaffold for new bone formation during whole healing period and BMP

played a critical role of enhancing BIC around implant during whole

oseeointegration period, while FGF had contributed to enhance BIC at initial

period, but the effect of FGF graft faded over time.

Keywords: biphasic calcium phosphate, bone morphogenic protein-2, fibroblast

growth factor-2, wound healing assay, critical bone defect, BIC, bone

graft, new bone formation, dental implant





Young Woo SON

The Graduate School

Yonsei University


I. INTRODUCION

The use of dental implants is becoming a standard method of oral rehabilitation.

However, in the case of insufficient bone volume, a procedure of ridge

augmentation is needed additionally. Therefore, during the past several decades,

tissue engineering has been considered as one of the important technologies in

regenerative dentistry. Even though several trials have been used to reconstruct

bone defects, each method has some advantages and disadvantages.

Autogenous bone grafts continue to be the gold standard because autogenous

bone grafts have shown a remarkable success rate because of their own osteogenic

potentiality. However, there are major impediments such as the limited availability

of donor site.1

Allogeneic bone is commonly used for alternative to the autogenous bone, but

these materials offer the potential risk of disease transmission and potential

foreign body reaction, and also xenogenic bone grafts have a possibility of leading

to cross species antigenic reaction.

Alloplastic bone substitutes such as hydroxyapatite (HAP) and tricalcium

phosphate (TCP) have been investigated extensively because their composition of

minerals is similar to natural bone tissue structure.2 Alloplastic materials have

several advantages. No additional surgery is needed for gathering bone and

supplies of the material are unlimited and there are no concerns of disease

transmission.

Hyroxyapatite is the major mineral component in human bone, and synthetic

apatites have been considered as a common osteoconductive substituted material

for bone defects. Synthetic calcium phosphate ceramics such as tricalcium

phosphate and hydroxyapatite are commonly used in the form of blocks, cements,

pastes, powders, or granules. Synthetic calcium phosphate ceramics, with their

excellent biocompatibility, are common alternatives to autogenous bone,

xenograft, or allograft materials.2-7

Biphasic calcium phosphate (BCP) is one of alloplastic bone substitutes and

these compositions are mixture of hydroxyapatite (HA) and �-tricalcium

phosphate (TCP). Also BCP has a similar structure with human bone.3,8 As the

ratio of �-TCP becomes higher, the resorption rate becomes higher accordingly.

However, as the ratio of HA becomes higher, the resorption rate becomes lower

accordingly. It was proved that the resorption rate is regulated by their proper ratio.

The extent of bone formation and the degree of resorption of the ceramic particles

were significantly higher in the mixture composed of 25% HAP-75% TCP.9

Surface modifications have been attempted to improve osteoinductive effect on

BCP. Many studies reported that bone morphogenetic protein (BMP) and basic

fibroblast growth factor (bFGF) can induce new bone formation in vitro and in

vivo.10-12Since BMP-2 has an osteoinductive activity, some studies have been

performed for examining the induction of bone formation combining rhBMP-2

with various carriers.13-15 It is believed that the rh-BMP is a growth factor which

is useful for increasing new bone formation.9,16

Fibroblast growth factor (FGF) was originally extracts of the pituitary gland and

brain, and has been known to stimulate the proliferation of fibroblasts. Some

studies have reported that bFGF can enhance osteogenic differentiation of bone

marrow stromal cells (BMSCs).17-20 The application of exogenous bFGF to a

large bone defect during the early healing stage accelerates new bone formation.21

Previous studies reported that growth factors such as bFGF and BMP accelerated

biological activities in vivo when combined with various carrier

scaffolds.22,23Previous studies showed that if the BMP-2 or FGF was combined

with calcium phosphate ceramics, CPC acted as an appropriate scaffold material

and both BMP-2 and bFGF enhanced bone formation.13,24,25However, the potential

applicability of CPC and BMP-2 and/or FGF for enhancing oseeointegration was

limited. Therefore, we wanted to verify the ability of bone regeneration in a

critical bone defect around the implant combining with bone graft using graft

materials such as biphasic calcium phosphate and growth factors. Also we wanted

to verify whether the graft materials enhanced osseointegration while the critical

bone defect healed.

The aims of this study were to evaluate the effects of biphasic calcium phosphate

and growth factors (bone morphogenetic protein, fibroblast growth factor) on

bone regeneration in one-wall critical defects when implant fixture was placed

simultaneously.

. MATERIALS AND METHODS

Part I: In vitro study

Materials and Methods

MC3T3-E1 cells were purchased from ATCC (Manassas, VA) and grown in

ascorbic acid-free �-MEM ( WelGene, Daegu, Korea) containing 10% FBS, 100

units/ml penicillin, and 100 �g/ml streptomycin. The cells were seeded on 24-well

plates at a density of 2 × 105 cells/well in culture medium. Then, the cells were

incubated at 37 °C in a humidified, 5 % CO2 atmosphere. At 24 h after seeding, a

wound was created using 10 �L micropipette tip. The scrape-wounded cultures

were treated with Osteon II, Osteon II + BMP-2 and Osteon II + FGF-2.

Experimental groups were divided as one control (No graft) and three type grafts

(Osteon II, Osteon II+BMP-2, Osteon II+FGF-2). The alloplastic material Osteon

II (Genoss, Suwon, Korea), Bone Morphogenetic Protein-2 (BMP-2) and

Fibroblast Growth Factor- 2(FGF-2) (GENOSS, Suwon, Korea) were used.

Osteon II volume was 0.007mg and the each volume of BMP-2 and FGF-2 was

10ul. The specific concentrations of GFs (0.1 mg/mL and 0.01 mg/mL of FGF-2;

0.1 mg/mL and 0.01 mg/mL of BMP-2) were used. Sample sizes were as followed:

4 of Control group, 3 of Osteon II+BMP-2(0.1 mg/mL of BMP-2), 3 of Osteon

II+BMP-2(0.01 mg/mL of BMP-2 ), 3 of Osteon II+FGF-2(0.1 mg/mL of FGF-2 ),

3 of Osteon II+FGF-2(0.01 mg/mL of FGF-2 ) and 4 of Osteon II group (Fig-

1)(Table 1). The migration of cells towards the wound was visualized by

microscope and magnification, and images were captured at 0, 12, 24 and 48 h

time points in the same position using a Nikon Eclipse TE2000-5 microscope.

Images at time zero (0 h) were captured to record the initial area of the wounds,

and the cell migration toward the scratched area was evaluated at 12, 24 and 48 h.

The area of wound was quantified by Java’s Image J software. Five regions of

interest (ROI) per well per wound edge were chosen for imaging over period. The

migration of cells toward the wounds was evaluated as mean value of arbitrary

five lines width, which is the width from one side of wound margin to the

opposing wound margin (Fig- 2). The percentage of wound closure, which is that

the each mean value of 12, 24 and 48h was divided by the value of 0 h, was

calculated and compared.

Figure 1. Picture showing experimental groups.

Number of samples Experimental periods

Control 4 0,12,24,48 h

Osteon II 4 0,12,24,48 h

Osteon II+BMP(0.01 mg/mL) 3 0,12,24,48 h

Osteon II+BMP(0.1 mg/mL) 3 0,12,24,48 h

Osteon II+FGF(0.01 mg/mL) 3 0,12,24,48 h

Osteon II+FGF(0.1 mg/mL) 3 0,12,24,48 h

Figure 2. Analyzing of a wound healing assay:

Arbitrary five lines width, which is the width from one side of wound

margin to the opposing wound margin, were measured.

Table 1. Table showing experimental groups classified by graft materials.

Part II: Animal study

A. Animal and materials

Six mongrel dogs of 18-24 months old, weighting approximately 30 kg each

were used in this study. After complete blood count (CBC; Hemavet TM 950,

CDC Technologies, Irvine, CA, USA) and serum test had been carried out to

check oral health status a period of one week was allowed for an environmental

adaptation. Prestudy preparation including scaling and plaque control was also

carried out to obtain the optimum gingival health. The animals had free access to

water and were fed with canned dog food diet. Animal selection and management,

surgical protocol and procedures for this study were reviewed and approved by

the Institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul,

Korea.

SLA (Sand-blasted, Large-grit, Acid-etched) surfaced internal type implants

(Dentium, Seoul, Korea) with 3.8 mm in diameter and 8 mm in length were used

in this study.

The alloplastic material Osteon II (Genoss, Suwon, Korea), developed in Korea,

has an HA surface coated with -TCP; it consists of 30% HA and 70% -TCP.

The pore size of Osteon II is 250 m. The volumetric porosity of Osteon is

approximately 70% and 0.5-1 mm particle size was used.

Bone Morphogenetic Protein-2 (0.01 mg/mL of BMP-2) and Fibroblast Growth

Factor-2 (0.01 mg/mL of FGF-2) (GENOSS, Suwon, Korea) were used.

B. Methods

1. Surgical procedures and implant placement with bone graft

All surgical procedures were performed under general anesthesia. Pre-operative

antibiotics, 22 mg/kg of cefazolin sodium (Cefazolin®, Chongkundang pharm. Co.,

Seoul, Korea) were injected intravenously. 0.1mg/kg of atropine sulfate(Atropine®,

Dai Han Pharm, Co., Seoul, Korea) was also injected as a premedication 15

minutes prior to anesthesia. The animals were anesthetized with intravenous

administered mixture of 5 mg/kg of ketamine (Ketalar® Yuhan Co., Seoul, Korea)

and 0.5 mg/kg of Xylazine HCI(Rumpun , Bayer Korea, Seoul, Korea), followed

by inhalation anesthesia with 100% oxygen and 2% isoflurane. Electrocardiogram,

oxygen saturation and body temperature were consistently monitored by

component monitor system (Omicare M1205A, Hewlett Packerd, Palo Alto, CA,

USA). The operative sites were then further anesthetized with 2% lidocane HCI

with epinephrine 1:80000 by infiltration.

8 weeks after extraction of P1, P2, P3, P4, M1 on mandible, implants were

placed. Healing period of 4 weeks and 8 weeks were allowed after implantation

and bone grafts.

For implant placement, a crestal incision was made to preserve keratinized tissue,

and mucoperiosteal flaps were carefully reflected on the buccal and lingual

aspects and “cuboid shape” one-wall bony defects (5x5x8 mm) were prepared

bilaterally (four on each side) using a high-speed fissure bur and a bone chisel on

mandible. Total of eight defects were made on the left and the right sides of the

mandibles of each of the six mongrels, for a total of 48 defects. Osteotomy was

performed under copious saline irrigation and 4 implants with 3.8 mm in diameter

and 8 mm in length were placed. The center of implant was placed on the edge of

cuboid. All implants were placed in parallel. A total of 8 implants were placed

for each mongrel dog, having 4 implants at both sides of mandible (Fig-3). At

each side, one control (No graft) and three type grafts (Osteon II, Osteon II+BMP-

2, Osteon II+FGF-2) were done within defect area. Total 48 titanium implants

were placed (Fig-4,5)(Table 2).

After implant placement, the mucoperiosteal flaps were sutured using a

resorbable suture material (Monosyn 4/0 Glyconate monofilament absorbable, B-

Braun, Aesculap, PA, USA). Post surgery care included intravenous injection of

antibiotics(Ampicillin, Jong- geun Dang Pharmaceutical Company, Seoul, Korea:

500 mg/day IV) and analgegic(Ketopro®, Unibiotech Co., Seoul, Korea 3 mg/kg)

for 3 days as well as daily topical application of a 0.2% chlorhexidine

solution(Chlorhexamed, Bukwang Pharmaceutical Compaceutical Company,

Seoul, Korea) for infection control. Sutures were removed after 2 weeks.

Figure 3. Clinical photographs showing “cuboid shape” one-wall bony defects and all implants

were placed in parallel.

Figure 4. Schematic diagram depicting the implantation at the

surgically created one wall bony defects.

Figure 5.

A. Clinical photograph showing one control (no graft)

and three type grafts (OsteonII, OsteonII+rh-BMP2,

OsteonII+rh-FGF2) were filled in defect areas.

B. Radiographic image of bone graft materials filled in

defect areas.

Left Right

Dog No. Defect 1 Defect 2 Defect 3 Defect 4 Defect 1 Defect 2 Defect 3 Defect 4

1 Osteon II Osteon II + BMP Control Osteon II +

FGF Osteon II Osteon II + BMP Control Osteon II +

FGF

2 Osteon II Control Osteon II + BMP

Osteon II + FGF Osteon II Control Osteon II +

BMP Osteon II +

FGF

3 Osteon II + FGF

Osteon II + BMP Osteon II Control Osteon II +

FGF Osteon II +

BMP Osteon II Control

4 Osteon II + BMP Osteon II Osteon II +

FGF Control Osteon II + BMP Osteon II Osteon II +

FGF Control

5 Control Osteon II + BMP


BMP Osteon II +

FGF Osteon II

6 Osteon II Control Osteon II + BMP


BMP Osteon II +

FGF

2. Animal sacrifice

The animals were sacrificed 4 weeks (3 dogs) and 8 weeks (3 dogs) after

implantation with overdose of ketamin HCI(Ketalar®Yuhan Co., Seoul, Korea)

intravenously (Fig-6). Mandibular block sections including implants were

preserved and fixed in 10% neutral buffered formalin for 2 weeks. The x-ray(Digi

X® , Digident Co., Seoul, Korea) was taken for the collected mandibular block

section to check the axis of the implant. Mandibular block sections were also used

for histomorphometric analysis.

Table 2. Table showing experimental groups classified by graft materials.

3. Histomorphometric analysis

The specimens were dehydrated through graded alcohols of 70%, 80%, 90%,

95%, 100% at 2 hour intervals for 1 week. The specimens were embedded in

Technovit 7200(Heraeus KULZER, Dormagen, Germany) and alcohols (1:3, 1:1,

3:1 ratio) and sectioned in the buccolingual plane using a diamond saw (Exakt 300,

Kulzer, Norderstedt, Germany). From each implant site, the central section was

reduced to a final thickness of about 35 by micro grinding and polishing with

a cutting-grinding device (EXAKT 400 CS, EXAKT Apparatebau, Norderstedt,

Germany) along the longitudinal axis of the dental implant fixture, and a section

was made in the sagittal plane at the center of the implant site, with a thickness of

4 �m so that it would include the surrounding mandible. Grinding was done, and

the specimen was attached to an acrylic slide and stained with hematoxylin and

eosin (H&E). The binding between the bone and the implant was observed using

light microscopy.

The region of interest (ROI) was defined in the grafted bone area from the

native bottom and axial side bone, up to the top of implant fixture. The stained

specimens were scanned and captured using optical microscopy (Leica DM-LB,

Leica Microsystems, Wetzlar, Germany) at x 12.5 and x 50 magnification

accordingly and bone-to-implant contact ratio (BIC) and bone volume (BV) was

measured using imaging analysis system (Image-Pro Plus, 4.5 Media Cybernetics

Figure 6. Diagram for experimental design time table.

Inc., Silver Springs, MD, USA). The bone to implant contract ratio around

macrothread as well as total surface area in ROI from the top of implant fixture

were calculated on both experimental group and internal control group (native

bone side of implant). The investigated length of internal control group was same

with that of experimental side. (Fig-7). Also bone volume (inter-thread BV)

around macrothread in experimental group and internal control group was

measured at ROI from the top of implant fixture (Fig-8). The value of internal

control group was used for normalization of the value of experimental group.

Normalized value means that each value of experimental group was divided by

each value of internal control group. The degree of new bone formation,

remaining bone graft materials and unfilled area (amount of bone loss) were

calculated in ROI. (Fig-9,10).

Figure 7.Calculation of bone-to- implant contact. Total length of consecutive macrothreads in ROI (A), bone-to- implant contact (B)

Figure 8. Measurement of bone volume

(inter-thread BV) in ROI.

Figure 9. Green line shows region of interest (ROI), which consists of new bone,

remained graft materials and unfilled area.

Figure 10. Green line shows unfilled area in the defect area.

4. Micro-computed tomographic evaluation.

The mandibles (including specimen-graft sites) were also evaluated using

micro-computed tomography (micro-CT; Skyscan 10763, Aartselaar Micro-CT,

Belgium) on the entire specimen at a medium resolution of 18 �m, with a 0.5 mm

brass filter, at 100 kV and 100 �A. The region of interest (ROI) for defect area

was examined in a rectangular column area, 1.6 mm (Bucco-lingual) and 5 mm

(from the center of implant fixture) in width and 5 mm in length (from the top of

the implant fixture) and ROI of internal control (native bone side) was examined

in a rectangular column area, 1.6 mm (Bucco-lingual) and 3 mm (from the center

of implant fixture) in width and 5 mm in length (from the top of the implant

fixture) (Fig-11).

Figure 11. The region of interest (ROI) was examined in a rectangular column area.

A: ROI of defect area (1.6x5x5 mm) B: ROI of native bone side (1.6x3x5 mm)

5. Statistical Analysis

Statistical analysis was performed using the SAS V 8.1(SAS Institute, Cary,

NC USA). Student’s t-test and Fisher’s exact test were used to compare the two

groups for the tooth defect experiment. The Kruskal–Wallis test was used for

nonparametric analysis and ANOVA for parametric analysis to compare the four

groups for the assessment of improvement in the quality of bone surrounding the

dental implant. P values <0.05 were considered significant.

. RESULTS

Part I: In vitro study

Phase contrast images of MC3T3-E1 cells at different time points taken (Fig-12-

17). In control group, 52% wound closure was visible after 12 hours and 79%

wound closure was visible after 24 hours. Complete closure occurred after 48 hours.

In Osteon II group, 41% wound closure was visible after 12 hours and 64% wound

closure was visible after 24 hours. However, complete closure did not occur after 48

hours and only 85% wound closure is visible after 48 hours. In Osteon II +

BMP(0.01 mg/mL) group, 43% wound closure was visible after 12 hours and 73%

wound closure was visible after 24 hours. However, complete closure did not occur

after 48 hours and only 76% wound closure is visible after 48 hours. In Osteon II +

BMP(0.1 mg/mL) group, 35% wound closure was visible after 12 hours and 62%

wound closure was visible after 24 hours. However, complete closure did not occur

after 48 hours and only 43% wound closure is visible after 48 hours. In Osteon II +

FGF(0.01 mg/mL) group, 84% wound closure was visible after 12 hours. Complete

closure occurred after 24 hours and 48 hours. In Osteon II + FGF(0.1 mg/mL)

group, 75% wound closure was visible after 12 hours. Complete closure occurred

after 24 hours and 48 hours (Table3)(Fig-18).

0h 12h 24h 48h Control 0% 52% 79% 100% Osteon II 0% 41% 64% 85% Osteon II+BMP(0.01 mg/mL) 0% 43% 73% 76% Osteon II+BMP(0.1 mg/mL) 0% 35% 62% 43% Osteon II+FGF(0.01 mg/mL) 0% 84% 100% 100% Osteon II+FGF(0.1 mg/mL) 0% 75% 100% 100%

Table 3. Percentage of wound closure at different time points.

Figure 12. Wound-healing assay of control group: Cells were scraped and measured after

0,12,24,48h. The “wounded” distances were examined at the indicated time points.

Figure 13. Wound-healing assay of Osteon II group.

Figure 14. Wound-healing assay of Osteon II + BMP (0.01 mg/mL) group.

Figure 15. Wound-healing assay of Osteon II + BMP (0.1 mg/mL) group.

Figure 17. Wound-healing assay of Osteon II + FGF (0.1 mg/mL) group.

Figure 16. Wound-healing assay of Osteon II + FGF (0.01 mg/mL) group.

Figure 18. Comparison of experimental groups for percentage of wound closure at different

time points.

Part II: Animal study

A. Micro-computed tomographic measurement

Reconstruction by micro-CT showed mineralized bone and graft material in the

specimens. Graft material was seen throughout the entire length of the specimens.

The volume of newly formed mineralized bone with graft materials was highest in

the Osteon II at 8 weeks, similar to Osteon II + BMP and Osteon II + FGF groups

at 8 weeks (Table 4)(Fig -19).

The bone volume of 8 weeks specimens increased comparing to that of 4 weeks

specimens.

Groups Mean (SD)of 4 weeks experimental group(%)

Mean (SD) of 8 weeks experimental group(%)

Control group 18(3) 29(6)

Osteon II 28(5) 38(7)

Osteon II + BMP 22(7) 37(4)

Osteon II + FGF 26(5) 35(7)

Table 4. Means and standard deviations of bone volume value in each groups.

B. Histomorphometric analysis

Two parts were divided as contact area and distance area.

The parameters analyzed were as follows:

1) Contact area: BIC (Bone to Implant Contact ratio), Bone volume (inter-thread)

2) Distance area: % of unfilled area, % of new bone area, and % of remained graft

material

1. Contact area

1.1 Bone to implant contact ratio (BIC)

Mean values and errors were recorded for each BIC measurement, and simple

0

20

40

60

80

100

4weeks 8weeks

%�of�b

one�volume�

Comparison�of�bone�volume�(%)�between�4�weeks�and�8�weeks

CONTROL

OSTEON�II

OSTEON�II+�BMP

OSTEON�II+�FGF

Figure 19. Comparison of bone volume (%) between 4 weeks and 8 weeks.

comparison showed that increased BIC value was found in 8 weeks groups

compared to corresponding 4 weeks groups.

Osteon II + BMP experimental group at 8 weeks showed the greatest BIC value

in all experimental groups. However, the BIC value of Osteon II + BMP is lower

than mean BIC value of internal control groups.

The BIC value of the internal control group significantly differed from that of the

experimental groups at both 4 weeks and 8weeks groups (P <0 .05).

At 4 weeks, the BIC of the control group significantly differed from that of the

other experimental groups (P <0 .05).

At 8 weeks, the BIC of the control group significantly differed from that of

Osteon II, and Osteon II + BMP groups (P <0 .05). However, the BIC value of the

Osteon II + FGF groups did not significantly differ from that of the control groups

(P >0 .05)(Table 5)(Fig- 20).

Groups Mean (SD)of 4 weeks

experimental group(%)

Mean (SD)of 4 weeks internal control

group(%)

Mean (SD)of 8 weeks experimental group(%)


group(%)

Control group 11(10) 43(18) 29(9) 66(16)

Osteon II 22(12) 53(13) 40(16) 60(26)

Osteon II+ BMP 29(14) 53(13) 46(8) 74(10)

Osteon II +FGF 21(7) 43(13) 25(13) 66(18)

Table 5. Comparisons of means and standard deviations of bone to implant contact ratio (BIC)

value between experimental groups at 4 weeks and 8 weeks.

1.2 Inter-thread bone volume (BV)

Each inter-thread bone volume area measurement was done for experimental

groups and internal control groups in 4 weeks groups and 8 weeks groups. Simple

comparison with mean values showed that the mean of BV (%) around implant

was greatest in OsteonII + BMP experimental group sacrificed at 8 weeks. The

bone volume in OsteonII + BMP groups was significantly better than that in other

experimental groups, although no significant difference was observed between the

control, OsteonII and Osteon II + FGF groups(P >0 .05)(Table 6)(Fig- 21).

0.00

0.20

0.40

0.60

0.80

1.00

1.20

4�weeks 8�weeks

norm

alize

d�BIC�ratio

Comparison�of�normalized�BIC�ratio

CONTROL

OSTEON�II

OSTEON�II+�BMP

OSTEON�II+�FGF

Figure 20. Comparison of normalized BIC between 4 weeks and 8 weeks.

Normalized value means that each value of experimental group was divided by each value of

internal control group.

Groups Mean (SD)of 4 weeks

experimental group(%)


group(%)

Mean (SD)of 8 weeks experimental group(%)


group(%)

Control group 17(8) 25(13) 29(17) 34(14)

Osteon II 23(9) 23(5) 34(8) 30(7)

Osteon II+ BMP 28(12) 28(11) 46(9) 41(12)

Osteon II +FGF 26(7) 18(4) 29(11) 42(20)

0.00

0.50

1.00

1.50

2.00

2.50

3.00

4�weeks 8�weeks

norm

alize

d��bon

e�volume�ratio

Comparison�of�normalized�inter�thread�bone�volume�ratio

CONTROL

OSTEON�II

OSTEON�II+�BMP

OSTEON�II+�FGF

Table 6. Comparisons of means and standard deviations of inter-thread bone volume value

between experimental groups at 4 weeks and 8 weeks.

Figure 21. Comparison of normalized inter-thread bone volume between 4 weeks and 8 weeks.

Normalized value means that each value of experimental group was divided by each value of

internal control group.

2. Distance area

2.1 Unfilled area

Differences between the 4 weeks and 8 weeks were seen (Table 7) (Fig-22). The

8 weeks group had more bone graft loss (increasing percentage of unfilled area)

comparing 4 weeks group.

At 4 weeks, the percentage of unfilled area in the control group significantly

differed from that in the other experimental groups (P <0 .05) and the percentage

of unfilled area in the Osteon II group was lowest among all the groups.

At 8 weeks, the percentage of unfilled area in the control group significantly

differed from that in the other experimental groups (P <0 .05) and the percentage

of unfilled area in the Osteon II + BMP group was lowest among all the groups.

Especially, there was a noticeable increase in percentage of unfilled area in the

Osteon II group from the 4 weeks to 8 weeks time period.




Osteon II 8(5) 22(10)

Osteon II + BMP 17(10) 16(9)

Osteon II + FGF 11(2) 21(11)

Table 7. Comparisons of means and standard deviations of unfilled area value between

experimental groups at 4 weeks and 8 weeks.

2.2 New bone formation area

When the 4 weeks and 8 weeks specimens were compared, there was increasing

tendency of new bone formation.

In both groups (at the 4 weeks and 8 weeks) , the new bone volume in Osteon II

+ BMP groups was significantly better than that in other experimental groups,

although no significant difference was observed between the control, Osteon II

and Osteon II + FGF groups(P >0 .05)(Table 8)(Fig-23).

0

20

40

60

80

100

4�weeks(%) 8�weeks(%)

Unfilled

�area(%)�

Unfilled�area(%)�of�4�weeks�vs.�8�weeks

CONTROL

OSTEON�II

OSTEON�II+�BMP

OSTEON�II+�FGF

Figure 22. Comparison of % of unfilled area 4 weeks vs. 8 weeks.




Osteon II 25(4) 39(14)

Osteon II + BMP 31(11) 45(5)

Osteon II + FGF 24(6) 36(14)

0

20

40

60

80

100

4�weeks 8�weeks

New

�bon

e�area(%

)�

New�bone�area(%)�of�4�weeks�vs.�8�weeks

CONTROL

OSTEON�II

OSTEON�II+�BMP

OSTEON�II+�FGF

Figure 23. Comparison of % of new bone area 4 weeks vs. 8 weeks.

Table 8. Comparisons of means and standard deviations of new bone formation area value


2.3 Remained graft materials area

When the 4 weeks and 8 weeks specimens were compared, the percentage of

remained graft materials at 4 weeks did not significantly differ from that of 8

weeks (P >0 .05)(Table 9)(Fig-24). By 8 weeks there was little change comparing

4 weeks.



Control group 0 0

Osteon II 9(3) 6(2)

Osteon II + BMP 7(6) 5(4)

Osteon II + FGF 7(3) 10(4)

Table 9. Comparisons of means and standard deviations of remained graft materials area value


0

20

40

60

80

100

4�weeks 8�weeks

Remaine

d�graft�a

rea(%)�

Remained�graft�area(%)�of�4�weeks�vs.�8�weeks

OSTEON�II

OSTEON�II+�BMP

OSTEON�II+�FGF

Figure 24. Comparison of % of remained graft materials between 4 weeks vs. 8 weeks.

IV. DISCUSSION

A critical size bone defect cannot heal spontaneously without any kind of a graft

material during the time period allowed for healing.26 Studies of not employing a

critical size defect model showed little or no difference in bone healing between

test and control sites.27 Therefore, the model for evaluating the regenerative

potential of graft bone materials is important.26 This study used a 5 mm x 5 mm x

8 mm defect, which represented one wall critical size defect. As a result, none of

the control defects in this study showed complete bone healing after 4 weeks and

8 weeks.

Efficient new bone formation on defect area is essential for successful oral

implant placement. The reason of using biphasic calcium phosphate for bone graft

on defect area in implant surgery is based on the fact that this material consists of

a sintered mixture of HA and �-TCP. The �-TCP will dissolve over time while the

HA will maintain the structure as a scaffold for new bone formation.28 It has been

proven that topical application of recombinant BMP stimulates long bone reunion

in large bone defects. 29For enhancing the bioactivity of calcium phosphate

compounds in bone regeneration, rhBMP-2 has been used together with HAP/TCP

ceramics to induce new bone formation more efficiently. BMP has been

considered as having strong capability of bone regeneration in vivo30,31, and

applying FGF to a bone defect during the early healing period can accelerate bone

formation.21

These days, for promoting new bone formation, growth factors critical to new

bone formation like bone morphogenetic protein (BMP) and fibroblast growth

factor (FGF) are often combined with synthetic grafts. Considering the previous

studies with BMP-2 and bFGF, we assumed that it was appropriate to investigate

the regeneration of critical size bone defects in mongrels.

Even though extensive trials were done in effectiveness of alloplastic bone

materials, there was a big gap between basic research and clinical application.

Especially, the effectiveness of using alloplastic bone materials for bone

regeneration of critical size bone defect area in implant dentistry is lacking

because many clinicians prefer doing the staged approach procedure for implant

placement instead of performing the combined graft implant procedure. That is

originated from the concerns that the combined graft implant procedure might be

graft failure leading to implant failure and insufficient osseointegration.

The overall aim of the present investigation was to study the effects of biphasic

calcium phosphate and growth factors (bone morphogenetic protein, fibroblast

growth factor) on bone regeneration in one-wall defects around implant. Therefore,

we studied two experiments in vitro study and animal study.

To evaluate the physiological activity of FGF-2 and BMP-2, we used MC3T3-E1

cell which is a cloned mouse osteoblast-like cell. Present in vitro study showed

that in control group, complete closure occurred after 48 hours. When treated with

Osteon II, the migration of MC3T3-E1 cells in the Osteon II group was lower than

in the control group over all time periods. However, there were no significant

differences between the control and Osteon II groups. When treated with Osteon

II+ BMP, the migration of MC3T3-E1 cells appears to be inhibited at both low

BMP concentration (0.01 mg/mL) and higher BMP concentrations (0.1 mg/mL).

In contrast, when treated with Osteon II+ FGF, at both low FGF concentration

(0.01 mg/mL) and higher FGF concentrations (0.1 mg/mL) the migration of

MC3T3-E1 cells clearly faster than that of control group. These data showed that

Osteon II did not greatly influence the migration of MC3T3-E1 cells, but BMP

inhibited the migration of MC3T3-E1 cells regardless of BMP concentration and

FGF facilitated the migration of MC3T3-E1 cells regardless of FGF concentration.

This finding may help explain previous studies that FGF has a strong proliferative

effect on various cells, which is a process for early bone formation and

remodeling.32,33 As described by previous study, bone formation is divided into

two phases, proliferation and mineralization.34 This finding may help explain

previous studies that FGF-2 plays a critical role of osteoblast growth in early bone

repair while BMP-2 is instrumental in stimulating mineralization35 and that a

delayed administration of BMP-2 to a fracture resulted in better repair of critical

size defects.36

In micro computed tomographic evaluation, we found that the bone volume of

defect area increased at 8 weeks as compared with the specimen at 4 weeks and

the bone volume value of control (no graft) group was significantly lower than

other grafted groups (Osteon II, Osteon II+BMP, Osteon II + FGF) at both 4

weeks and 8 weeks. These data suggested that bone graft materials might have a

role to play in critical bone defect reconstruction.

In histomorphometric analysis, two parts were divided as contact area and

distance area and analyzed. In all groups, the values of BIC at 8 weeks were

higher than those at 4 weeks. At 4 weeks, the BIC value of Osteon II + BMP

group was highest comparing other groups. However, the BIC value of the Osteon

II + FGF groups did not significantly differ from that of the Osteon II + BMP

group (P >0 .05). At 8 weeks, the BIC value of Osteon II group was highest

comparing other groups. However, the BIC value of the Osteon II group did not

significantly differ from that of the Osteon II + BMP group. Also the BIC value of

the Osteon II + FGF group was lowest among the graft groups. From the results,

we found that Osteon II + BMP graft contributed to increase the BIC value at both

4 weeks and 8 weeks. However, Osteon II + FGF graft contributed to increase the

BIC value only at 4 weeks as compared with Osteon II graft. The reasons for this

difference are not clear. One explanation for the difference between the BIC value

of Osteon II + FGF group at 4 weeks and at 8 weeks might be that FGF had

influenced positively during initial period and the efficiency of FGF faded over

time. This finding may be explained by our in vitro study and other study that

FGF-2 plays a critical role of osteoblast growth in early bone repair while BMP-2

is instrumental in stimulating mineralization.35 It means that Osteon II acted as a

good scaffold for new bone formation during whole healing period and BMP

contributed to enhance BIC around implant during whole oseeointegration period.

However, FGF contributed to enhance BIC at initial period but the effect of FGF

graft faded over time.

In all groups, the values of inter-thread bone volume (BV) at 8 weeks were

higher than those at 4 weeks. The BV values of Osteon II + BMP group at 4

weeks and Osteon II + FGF group at 8 weeks were highest. However, there was

no statistically significant difference between the four groups at both 4 weeks and

8 weeks respectively (P >0.05).These data suggested that the Osteon II, BMP, and

FGF combination did not contribute to the enhanced inter-thread bone volume in

bone defects around implants.

The value of unfilled area can be considered as amount of graft bone loss in the

defect area.

In all groups, the values of unfilled area at 8 weeks were higher than those at 4

weeks. The value of control (no graft) group was highest among all groups. And

the difference was significant. Previous study showed that a BMSCs/CPC/BMP-

2/bFGF composite is capable of repairing critical bone defects around implants.37

In present study, we found that Osteon II, BMP, and FGF combination contributed

to maintain bone volume in critical bone defects.

Comparing the percent of new bone formation area at 4 weeks and 8 weeks , the

new bone volume in Osteon II + BMP groups was significantly better than that in

other experimental groups, although no significant difference was observed

between the control, Osteon II and Osteon II + FGF groups. These data showed

that BMP contributed to enhance new bone formation in critical defect area. The

reason that control group did not differ from Osteon II and Osteon II + FGF

groups may be explained that natural healing had occurred in a significant portion

of the defect area because the sacrifices of mongrel dogs were done at 4 weeks

and 8 weeks after doing bone graft. Therefore, if time passed more and it still

continued to occur bone healing in progress later, the results could be changed.

Almost 90% of grafts materials had disappeared in the defects filled with Osteon

II, Osteon II + BMP, and Osteon II + FGF at 4 weeks. By comparison, we

observed that there was no significant difference in remained bone graft

percentage between 4 weeks and 8 weeks groups. Therefore, we found that

Osteon II graft had begun to resorb noticeably in the initial stages and had been

maintained since then.

V. CONCLUSION

Within the limitations of this study, the following conclusions can be proposed:

1) In vitro study, we found that Osteon II did not greatly influence the migration

of MC3T3-E1 cells, but BMP inhibited the migration of MC3T3-E1 cells

regardless of BMP concentration and FGF facilitated the migration of MC3T3-

E1 cells regardless of FGF concentration.

2) The bone volume value of control (no graft) group in micro CT evaluation was

significantly lower than other grafted groups (Osteon II, Osteon II+BMP,

Osteon II + FGF) at both 4 weeks and 8 weeks.

3) In critical defect area, the new bone formation volume in Osteon II + BMP

groups was significantly better than that of other experimental groups.

4) This study showed that Osteon II + BMP graft could enhance the BIC in the

critical defect site around the implant both at 4 weeks and 8 weeks. However,

Osteon II + FGF graft could enhance the BIC only at 4 weeks as compared

with Osteon II group.

Further studies should establish the suitability of combined BMP and FGF for

treating critical bone defects.

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evaluation of the effects of biphasic calcium phosphate ... · this project would not have been...

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