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The role of rhFGF-2 soaked polymer membrane

for enhancement guided bone regeneration

Sang-hoon Lee

Department of Dentistry

The Graduate School, Yonsei University

[UCI]I804:11046-000000514267[UCI]I804:11046-000000514267


for enhancement guided bone regeneration

Directed by professor Young-Bum Park

The Doctoral Dissertation

Submitted to the Department of Dentistry

and The Graduate School of Yonsei University

in partial fulfillment of the requirements

for the degree of Ph.D. in dental science

Sang-hoon Lee

December 2017

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF TABLES

ABSTRACT ······································································· 1

I. INTRODUCTION ····························································· 3

II. MATERIALS AND METHODS ·········································· 8

1. In vitro release kinetics of FGF ············································· 8

2. Animals and materials ························································ 8

A. Experimental animals ····················································· 8

B. Materials ···································································· 9

3. Methods ······································································ 11

A. Control / Experimental group ·········································· 11

B. Surgical procedures ····················································· 13

C. Sacrifice ··································································· 14

4. Histological preparation ···················································· 15

5. Histological and histomorphometric Analysis··························· 15

6. Immunohistochemical / TRAP stain analysis ··························· 16

7. Statistical analysis··························································· 17

III. RESULTS ··································································· 18

1. In vitro release kinetics of BMP2 and FGF2 ···························· 18

2. Histological findings ························································ 19

A. Analysis of tissue in the 2 week group ······························· 19

B. Analysis of tissue in the 4 week group ································ 21

C. Analysis of tissue in the 12 week group ······························ 23

3. Histomorphometric analysis ··············································· 25

A. New bone ································································· 25

B. Remaining bone graft materials ······································· 28

C. Histologic componants ·················································· 29

4. Immunohistochemical / TRAP stain findings ··························· 32

A. Osteocalcin ······························································· 32

B. Osteoclast ································································· 40

5. Russel-Movat Pentachrome stain findings ······························· 45

A. Analysis of tissue in the 2 week group ······························· 45

B. Analysis of tissue in the 4 week group ································ 46

C. Analysis of tissue in the 12 week group ······························ 47

IV. DISCUSSION ······························································ 49

V. CONCLUSION ······························································ 60

REFERENCES ································································· 61

ABSTRACT (KOREAN ) ···················································· 68

LIST OF FIGURES

Figure 1. Micro-structure of polymer collagen membrane ···· 10

Figure 2. Schematic diagram of surgery and sample labeling · 12

Figure 3. Photograph of surgery site ······························ 14

Figure 4. Cumulative concentration of FGF2 measured for 7

days ························································ 18

Figure 5. Histologic specimens undergone 2 weeks of healing

time ························································ 19


time ························································ 21


time ························································ 23

Figure 8. Percentage area of New bone in Histomorphometric

data ························································ 25

Figure 9. Percentage area of remaining bone in

Histomorphometric data ································ 28

Figure 10. Cumulative graph of histologic components at

Control group ············································ 30

Figure 11. Cumulative graph of histologic components at FGF

1mg/ml group ············································ 30


0.5mg/ml group ·········································· 31


0.1mg/ml group ·········································· 31

Figure 14. Immnnohistochemical localization of osteocalcin · 32

Figure 15. Division into 8 parts of region of interest(ROI) to

evaluate the expression of osteocalcin exactly ······ 33

Figure 16. Immunohistochemical specimens of expression of

osteocalcin undergone 2 weeks of healing time ····· 35


osteocalcin undergone 4 weeks of healing time ····· 37


osteocalcin undergone 12 weeks of healing time···· 39

Figure 19. TRAP staining specimens of expression of osteoclast

undergone 2 weeks of healing time ··················· 42


undergone 4 weeks of healing time ··················· 43


undergone 12 weeks of healing time ·················· 44


time (Russell-Movat Pentachrome Stain) ············ 45

Figure 23. Histologic specimens undergone 4weeks of healing




LIST OF TABLES

Table 1. Control / Experimental groups ·························· 11

Table 2. Percentage area of New bone in Histomorphometric

data ························································· 25

Table 3. Percentage area of remaining bone in

Histomorphometric data ································· 28

Table 4. Expression of osteocalcin ································ 33

Table 5. Expression of osteoclast ································· 40

1

ABSTRACT


for enhancement of guided bone regeneration

Sang-hoon Lee



(Directed by Professor Young-Bum Park)

Purpose : The purposes of this study are to confirm the role of FGF-2 in bone

regeneration by adding various concentrations of FGF-2 to the collagen

membrane and applying it to the BCP bone graft site for guided bone

regeneration, to explore the potential of collagen membrane as FGF-2 carrier,

and to determine the optimum FGF concentration for enhancement of bone

regeneration.

Methods : 4 bone defects of 8mm in diameter was created in 18 New Zealand

rabbits calvaria. After BCP bone graft, graft material was covered with collagen

membranes adding various concentration of FGF-2, and then sutured. The

concentration of FGF-2 was set at 1.0mg/ml, 0.5mg/ml, 0.1mg/ml, and same

amount of saline was used in the control group. To confirm the bone regeneration

2

over time, six New Zealand rabbits were sacrificed each at 2,4, and 12 weeks,

and the amounts of new bone and residual bone graft material were analyzed by

histologic and histomorphometric analysis. Qualitative analyses are also

conducted through immunohistochemistry, TRAP and Russell-Movat

pentachrome stain.

Results and Conclusion : As the healing period increased, the formation of new

bone increased and the amount of residual graft material decreased in all groups.

Immunohistochemistry, TRAP and pentachrome staining showed that the

addition of FGF-2 promoted bone regeneration in all experimental groups. But, it

was not confirmed whether FGF addition enhanced bone formation. It was also

confirmed that polymer collagen membrane can be used as a useful carrier of

FGF-2 when enhanced early stage of new bone formation is required.

Keyword : FGF-2, Collagen membrane, Guided Bone Regeneration, TRAP stain,

Immunohistochemistry, Pentachrome stain

3


for enhancement of guided bone regeneration

Sang-hoon Lee



(Directed by Professor Young-Bum Park)

I. INTRODUCTION

Guided bone regeneration(GBR) was introduced to the field of dentistry in

order to restore the bone defect around the implant,1 and it shows relatively good

results.2,3 However, the polymer membrane used in clinic, which is an important

factor in GBR, only plays a passive role in bone regeneration, because the

membrane only acts as a barrier, it does not directly affect bone formation.4

Therefore, the effect depends on the inherent healing power in the host, so the

4

larger the bone defect, the harder it becomes to heal. In addition, it takes more

than six months to obtain adequate clinical results, and the degree of ossification

is often questionable.5 As a consequence, various attempts have been made to

enhance bone regeneration ability.

Much of the study on maximizing bone regeneration had been performed on

growth factors, and in fact, growth factors play an important role in the

development of teeth and surrounding tissues. Among them, Bone morphogenic

protein (BMP) and Fibroblast growth factor (FGF) play an important role in bone

development and fracture healing process that occur naturally in the body and

their temporal and spatial manifestations are known to regulate the extent of limb

growth and bone formation.6 FGF is known to induce proliferation, chemotaxis,

and angiogenesis of undifferentiated ectodermal cells and it is also known to act

as an important regulator of periodontal ligament regeneration and bone

formation in the body.7-10 Also, in the absence of FGF-2 gene, both bone quality

and quantity decreased.11 Several studies using FGF-2 showed increased bone

regeneration and new bone formation.4,12-15 These studies show that FGF-2

plays an important role in bone regeneration in the body. FGF is the protein first

found in bovine pituitary gland in 1974, which strongly induces proliferation of

fibroblast.16 The action of FGF-2 increases laminin mRNA expression, which is

important for angiogenesis, and increases synthesis of Osteopontin, Hyaluronan.

It also increases early osteogenic marker, Cbfa-1.17-20 Conversely, the synthesis

5

of type Ⅰ collagen and the synthesis of osteocalcin, a late osteogenic marker, are

reduced. Alkaline phosphatase activity associated with osteogenic differentiation

levels also decreases.17,18,21 This phenomenon is interesting because FGF-2

increases the proliferation of immature mesenchymal cells, but also reduces the

differentiation and matrix synthesis of osteoblastic cells. In other words, although

not directly involved in osteogenesis, proliferation of immature mesenchymal

cells is sufficiently achieved at the early stage of osteogenesis, resulting in an

advantageous environment for continuous bone formation.

There have been two clinical trials on humans. In the case of periodontal disease

with 2 or 3 wall bony defects, it was reported that the bone regeneration effect

was significantly increased at a concentration of 0.3% FGF -2 when the various

concentration of FGF-2 was applied topically. Root resorption, ankylosis, and

epithelial migration were not found and there were no systemic side effects.22,23

However, although there have been reports of successful bone regeneration with

only FGF-2 alone without bone graft, in the case of large bone defects, only the

application of FGF-2 alone has a limitation on the bone regeneration.

In order to maximize bone regeneration effect of FGF-2, carriers that can slowly

and efficiently release FGF-2 are also important. In the past, viscous gel type

carriers were used for simplicity of application in periodontal surgery. This

method involves direct application to the periodontal tissue, in which gelatin is

disintegrated, so FGF is released in consistent manner. Although it has been

6

simple and successful in several experiments, this approach has the problem that

FGF-2 can easily migrate to other sites and disperse. Recently, various methods

have been introduced. It was reported that collagen minipellets contained FGF-2

inside and 80% of the total FGF was gradually released within 7 days.24 A

method of folding and wrapping FGF-2 containing collagen sponge was also

introduced.25 Santana et al.4 introduced polylactide carriers. A 0.8-mm-thick

polylactide membrane containing 5μg of FGF-2 released high concentrations of

FGF-2 over a period of one week.

As the whole process of bone formation is affected by the temporal flow of

various factors and FGF-2 acts in time-dependent manner,18,26 it can be

anticipated that proper amount of FGF-2 should be administered at an

appropriate time as well as an appropriate carriers to improve bone formation

ability. Throughout our previous study (data not shown), we have identified that

a consistent concentration of FGF-2 release (more than 100ng/ml of FGF-2) was

maintained for 7 days when collagen membrane with a size of 10×20 ㎟ soaked

in 50µL of FGF-2(1mg/ml) was used as a carrier. Compared with the above

mentioned study24,25,4 ,the release amount of our previous study was sufficient, so

in this study, we set 3 different concentration of FGF-2(1.0mg/ml, 0.5mg/ml,

0.1mg/ml). Therefore, based on our previous study, the purposes of this study are

to confirm the role of FGF-2 in bone regeneration by adding various

concentrations of FGF-2 to the collagen membrane and applying it to the BCP

7

bone graft site, and to determine the optimum FGF-2 concentration for bone

regeneration.

8

II. MATERIALS AND METHODS

1. In vitro release kinetics of FGF-2

In order to evaluate the release kinetics of FGF2, a collagen membrane with a

size of 10×20 ㎟ soaked in 50µL of FGF-2(1mg/ml) was prepared and using

enzyme-linked immunosorbent assay(ELISA) kit(Abcam, Cambridge, UK), the

release was measured according to the manufacturer’s instructions. The sample

was inserted into a 100µL PBS solution maintaining 37°C, 100µL PBS solution

was collected at 1,3,5,7,12-th hour and 1,3,5,7-th day, and replaced with a new

100µL PBS solution. The concentration released from the collected PBS solution

was measured using ELISA reader(Molecular devices. Workingham, UK) at 450

nm wavelength.

2. Animals and materials

A. Experimental animals

18 New Zealand white rabbits weighing 3-3.5 kg aged 16~20 weeks were

prepared. The animals were fed with a standard laboratory diet in a purpose-

designed room for experimental animals. All animal care and treatment protocols

9

followed the routines approved by the Animal Care and Use Committee, Yonsei

Medical Center, Seoul, Korea.

B. Materials

Osteon II (Genoss, Suwon, Korea) was used for biphasic calcium phosphate

(BCP). The ratio of Hydroxyapatite(HA)and β-Tricalcium phosphate(β-TCP) is

30:70, and has porous structure. Pore size is 250 μm, porosity is 70%, and each

particle size is 0.5 ~ 1.0mm. The appropriate volume for a bone defect size of 8

mm in the rabbit calvaria was 70 mg and the same volume was used for each

bone defect.

rhFGF-2 (E.coli-derived, GENOSS, Suwon, Korea) was used for FGF-2.

1.0mg/ml , 0.5mg/ml, and 0.1mg/ml concentration FGF-2 solutions were

prepared through dilution with saline. A collagen membrane with a size of

10×20 ㎟ soaked in 50µL of FGF-2 dilutions of each concentration. The

contents of rh-FGF2 in 50µL of FGF-2 dilutions was each 50µg, 25µg, 5µg

respective to concentration to yield 3 different concentration of FGF-2(1.0mg/ml,

0.5mg/ml, 0.1mg/ml). In the control group, 50 μL of normal saline was used.

For polymer membrane, highly pure type I collagen (Genoss, Seoul, Korea)

derived from bovine tendon was used. It has a thickness of 300μm and lasts more

than 6 months in the human body, providing sufficient time for clot stabilization.

The polymer collagen membrane has a dense multilayer structure with no pores

10

on both the upper and lower surfaces. Tensile strength of polymer collagen

membrane is 37.3MPa at dry and 9.7MPa at wet. (Fig.1) (the properties of the

membrane provided by the manufacturer)

Fig. 1 Micro-structure of polymer collagen membrane

11

3. Methods

A. Control / Experimental groups

Four concentrations of rhFGF-2 groups were set. (0.0mg/ml FGF-2

group<normal Saline, control>, 1.0mg/ml FGF-2 group, 0.5mg/ml FGF-2 group,

0.1mg/ml FGF-2 group) (Table 1)

Table. 1 Control / Experimental group

Control group Osteon Ⅱ+ collagen membrane

Experimental Group1 Osteon Ⅱ+ FGF-2 1.0mg/ml collagen membrane



In each of the 18 individuals, 4 bone defects were created in the calvaria. In

order to reduce the individual specificity, four concentration groups may be

arranged in each of the 4 bone defects of each individual. However, because it is

difficult to secure 4 collagen membranes of each concentration in each bone

defect in the surgical process, only two concentration groups were set for each of

12

the individual in each of the 2 left and right bone defects. (Fig.2) To minimize

the specificity of each individual, the groups were evenly divided.

The healing period was set at 2 weeks, 4 weeks, and 12 weeks. There are six

animals in each period.

Fig. 2 Schematic diagram of surgery and sample labeling

13

B. Surgical procedures

The New Zealand white rabbits were injected subcutaneously with Zolazepam

(Zoletil, virback Korea Co., Seoul, Korea) 1.5mg/kg and injected intramuscularly

with Xylazine HCl(Rompun, Bayer Korea Co., Seoul, Korea) 5mg/Kg for

general anesthesia. After 10 minutes of general anesthetic injection, the skull skin

was sterilized with Povidone-Iodine and local anesthesia was performed with 2%

lidocaine (lidocaine HCl, Huons, Seongnam, Korea) containing 1:80,000

epinephrine. The skin and periosteum were incised from the anterior part of the

frontal bone through the parietal bone to the anterior part of the occipital bone for

about 2.0-2.5cm and the periosteum was elevated.

On the exposed calvarial surface, 4 bone defects were created, 2 on each side

and 2 front and back, as shown in Fig.1. Trephine bur (MR.Curette Tech,

Seongnam, Korea) with an outer diameter of 8 mm was used to form a circular

bone defect with a depth of 1 ~ 2 mm to the inferior cortical bone so that the

underlying dura was not damaged. The bone fragments of the formed defect were

gently elevated using a curette. It was confirmed that the dura mater was not

damaged and there was no bleeding. The distance between each defect was at

least 3 mm.

BCP, FGF-2 diluents by concentration, 10×20 ㎟ collagen membrane, and

normal saline was prepared in advance, and collagen membrane soaked in 50µL

FGF-2 dilutions of each concentration for 30 minutes so that FGF-2 will be

14

absorbed into the collagen membrane. The control group was soaked in 50μL of

saline..

70 mg of BCP was implanted in each bone defect and the collagen membrane

soaked with FGF-2 was covered and fixed with micropin. The periosteum was

first sutured with an absorbable suture(4-0 Vicryl, Ethicon, Somerville, NJ, USA),

and the skin was closed. (Fig.3)

Fig. 3 Photograph of surgery site

C. Sacrifice

After 2, 4, 12 weeks of healing periods, each 6 rabbits were sacrificed. After

induction of deep anesthesia, skin was dissected and periosteum was elevated.

Using a handpiece, the marginal bone was removed and a specimen was taken at

a sufficient distance from the vicinity of the healed bone defect. The front side of

the specimens were marked to identify the direction.

15

4. Histological preparation

After washing the specimens fixed in 10% formalin for 6 weeks,

demineralization process was performed for 18 days in 2.5% NaOCl and 17%

EDTA solution. Dehydration and paraffin infiltration process were performed

using ethanol and xylene. After embedding, it was sectioned into 5μm

thicknesses. At this time, the specimen was cut along the sagittal plane passing

through the center of the defect. Hematoxylin-Eosin(HE) stain and Russell-

Movat pentachrome stain(American MasterTech) were conducted.

5. Histological and histomorphometric Analysis

The specimens were imaged at 12.5 times and 50 times magnification using an

optical microscope(Leica DM 2500, Leica Microsystems, wetzlar, Germany),

and histomorphometric measurements were performed with H-E stain tissue

slides. New bone area and residual bone graft material area were measured using

Image-pro plus program (Media cybernetics, Silver Spring, Maryland, USA).

The area of the new bone and the area of the residual bone graft material for the

total area were calculated as a percentage.

16

Russell-Movat pentachrome stain tissue slides were used to observe bone

development, and the degree of differentiation of new bone was analyzed. (Bone :

dark yellow & green, New osteoid : red)

New bone area (%) : The areas of new bone in the defect

(Newly formed bone area / total defect area x 100)

Residual particle area (%) : The areas of residual bone graft material

(Residual bone graft material area / total defect area x 100)

6. Immunohistochemical / TRAP stain analysis

Immunohistochemistry was performed to detect the expression of osteocalcin

using mouse anti-osteocalcin antibody (1:100, abcam, ab13420, Cambridge).

Immunohistochemical analysis of osteocalcin was performed using the avidin–

biotin DAB system. Endogenous peroxidase activity was blocked by incubation

with 1% hydrogen peroxide in PBS for 30 min. The sections were blocked with

PBS containing 5% BSA(bovine serum albumin) at room temperature for 1 h and

reacted with the primary antibody. Sections were washed with PBS, and reacted

with the secondary antibody biotinylated-conjugate (1:200, Vectastain ABC kit,

Vector laboratories) at room temperature for 1 h. The bound antibodies were

visualized using the avidin-biotin complex reagent, followed by 3, 3-

17

diaminobenzidine (Vector Laboratories, Burlinggsme, CA, USA). The sections

were washed, dehydrated, and mounted using mount solution (Fisher Scientific,

NJ, USA).

To detect expression of osteoclast, Tartrate-resistance acid phosphatase(TRAP)

stain was performed with the manufacture’s protocol.

7. Statistical analysis

Statistical analysis and validation were performed using histomorphometric data.

Since the number of samples was small and two specimens were excluded due to

errors in specimen production, a non-parametric test was performed.

Kruskal Wallis test was performed to compare between FGF-2 concentration

groups at 2, 4, and 12 weeks and to compare statistically significant differences

according to healing period within each FGF-2 concentration group. The

significance level was set at 0.05. Mann-Whitney U test was used for comparison

of each group. Statistical calculations were made using IBM SPSS Statistics 23

(IBM Corp., Armink, NY, USA).

18

III. Results

Although 18 individuals and a total of 72 specimens were produced in the study,

two specimens (1R1, 17L1) had damages during the specimen production

process and were excluded from statistics.

1. In vitro release kinetics of FGF-2

The concentration released by time was represented as a graph.

Within the first 24 hours, more than 50% of the total amount were released, and

after 1 day (24h), a relatively consistent concentration of release was maintained

until day 7 (168h). It was confirmed that a release amount of 100 ng/ml or more

per 48 hours was retained for 7 days.

Fig. 4 Cumulative concentration of FGF2 measured for 7 days

19

2. Histological findings (H-E Stain)

A. Analysis of tissue in the 2 week group

Fig. 5 Histologic specimens undergone 2 weeks of healing time (H&E Stain.

Upper raw original magnification X12.5, Bottom raw original magnification

20

X50). Yellow circles, blue stars and green triangles indicate new bone, fibrous

connective tissues and residual bone graft material.

The above tissue is a histological slide according to the concentration of FGF-2

in the 2-week group. The formation of new bone begins to be observed in the 2-

week group. Most of the new bone is in the form of woven bone, that begins to

be formed from the existing bone on the right and left border. The new bone

formed from the lower dura mater and the superior periosteum is almost invisible.

Overall, the amount of residual bone graft material is large, and the new bone is

formed surrounding the graft material. The boundaries between existing bone and

new bone in both sides are very clearly identified. As aspect of initial healing,

inflammatory cells are largely observed and most of the bone defects are filled

with loose fibrous connective tissue.

21

B. Analysis of tissue in the 4 week group





22


in the 4-week group. Compared to the 2-week group, new bone begins to form at

the center of the bone defect, and some of them are formed from the lower dura

mater and the upper periosteum. Histologically, it is more mature and bone

marrow is observed but it is still immature bone. Overall, the space of the

residual bone graft material becomes smaller and a new bone is formed

therebetween. The boundaries between the existing bone and the new bone in

both sides are more obscure. Inflammatory cells are still observed around the

fibrous connective tissue

23

C. Analysis of tissue in the 12 week group





24


in the 12-week group. At the center of the bone defect, an independent new bone

is found, and the amount of bone produced from lower dura mater and superior

periosteum is also increasing. Overall, it shows signs of more resorption of bone

graft material. The maturation of the new bone is improved, so mature lamellar

bone is seen, and bone marrow is well observed within the new bone. The

boundaries between the existing bone and the new bone on both sides are hardly

distinguishable, and inflammatory cells are rarely found.

25

3. Histomorphometric analysis

A. New Bone

Table. 2 Percentage area of New bone in Histomorphometric data

New Bone area (%)

2week

(mean±SD) (n)

4week

(mean±SD) (n)

12week

(mean±SD) (n)

Control 6.49 ± 5.15 (6) 11.95 ± 4.89 (6) 20.62 ± 10.14 (6)

FGF 1.0 6.22 ± 4.04 (5) 17.80 ± 4.21 (6) 27.19 ± 14.08 (6)

FGF 0.5 5.46 ± 3.17 (6) 14.11 ± 6.00 (5) 27.27 ± 3.68 (6)

FGF 0.1 5.02 ± 3.20 (6) 11.25 ± 6.32 (6) 23.88 ± 8.98 (6)

Fig. 8 Percentage area of New bone in Histomorphometric data

0

10

20

30

40

50

Control FGF 1.0 FGF 0.5 FGF 0.1

2week 4week 12week

26

First, when comparing the degree of formation of new bone according to the

healing period in each group, the amount of new bone increased with the

increasing healing period of 2, 4, and 12 weeks. (Kruskal Wallis test p <0.05)

There was no statistically significant difference in the control group between 2

weeks and 4 weeks, 4 weeks and 12 weeks, and 4 weeks and 12 weeks in FGF 1

mg/ml group, and in all other groups there were statistically significant

differences in new bone formation according to healing period. (Mann Whitney

test p <0.05)

Comparing new bone formation by groups in identical healing periods of 2

weeks, 4 weeks, and 12 weeks, there was no difference in new bone formation at

2 weeks between groups, but at 4 weeks, FGF 1mg/ml group showed the highest

level of new bone formation, and the FGF 0.5mg/ml group also showed higher

values than the FGF 0.1mg/ml group and the control group. At 12 weeks, all

experimental groups with FGF added showed a higher value of new bone than

the control group, among them, FGF 1mg/ml group and 0.5mg/ml group showed

higher values than FGF 0.1mg/ml group. As shown in Fig.8, it shows a tendency

of bone formation, At 4 weeks, except for the FGF 0.1mg/ml group, the higher

FGF concentration resulted in higher new bone formation, and at 12 weeks,

optimal bone formation was observed above 0.5mg/ml concentration and bone

formation was also increased in the concentration of 0.1mg/ml. However, the

non-parametric statistical test showed no statistically significant difference

27

between all groups in the same healing period, and these results need to be

discussed.

28

B. Remaining bone graft material

Table. 3 Percentage area of remaining bone in Histomorphometric data

Remaining bone graft material area (%)

2week

(mean±SD) (n)

4week

(mean±SD) (n)

12week

(mean±SD) (n)

Control 27.24 ± 3.26 (6) 19.80 ± 5.52 (6) 16.22 ± 3.03 (6)

FGF 1.0 28.07 ± 3.94 (5) 20.75 ± 2.22 (6) 16.30 ± 1.54 (6)

FGF 0.5 26.90 ± 5.64 (6) 18.55 ± 1.43 (5) 14.11 ± 1.51 (6)

FGF 0.1 24.27 ± 5.13 (6) 21.19 ± 2.25 (6) 14.60 ± 2.08 (6)

Fig. 9 Percentage area of remaining bone in Histomorphometric data

0

5

10

15

20

25

30

35

Control FGF 1.0 FGF 0.5 FGF 0.1

2week 4week 12week

29

First, when comparing the amount of residual bone graft material according to

the healing period in each group, as the healing period increased to 2, 4, and 12

weeks, the amount of residual bone graft material decreased. (Kruskal Wallis test

p <0.05) There was no statistically significant difference between the groups in

the control group for 4 and 12 weeks, in the FGF 0.5mg/ml group for 2 and 4

weeks, in the FGF 0.1mg/ml group for 2 and 4 weeks, and there was a

statistically significant difference in the amount of residual bone graft material

over the healing period in all other groups. (Mann Whitney test p <0.05)

There was overall a similar value in the comparison of residual bone graft

material in identical healing periods of 2, 4, and 12 weeks by groups and there

were no statistically significant differences. In other words, the addition of FGF

showed no significant effect on the resorption of residual bone graft, and

regardless of the addition of FGF, the bone graft material was resorbed at a

constant rate.

C. Histological components

To examine the ratio of new bone and residual bone graft materials according to

the healing period by group at a glance, it was represented as 100% cumulative

graph. Bone graft materials were resorbed at a consistent rate and the area of

fibrous connective tissue decreased with the degree of new bone formation in

each group.

30

(1) Control group

Fig. 10 Cumulative graph of histologic components at Control group

(2) FGF 1mg/ml group

Fig. 11 Cumulative graph of histologic components at FGF 1mg/ml group

0%

20%

40%

60%

80%

100%

2w 4w 12w

6.48 11.94 20.62

27.23 19.8 16.22

66.29 68.26 63.16 Fibrous connective

tissue

Residual bone graft

material

New Bone(%)

0%

20%

40%

60%

80%

100%

2w 4w 12w

6.22 17.79

27.18 28.07

20.74 16.29


tissue

Residual bone graft

material

New Bone(%)

31

(3) FGF 0.5mg/ml group

Fig. 12 Cumulative graph of histologic components at FGF 0.5mg/ml group

(4) FGF 0.1mg/ml group

Fig. 13 Cumulative graph of histologic components at FGF 0.1mg/ml group

0%

20%

40%

60%

80%

100%

2w 4w 12w

5.46 14.1

27.27 26.89

20.32

14.1

67.65 65.58 58.63

Fibrous connective

tissue

Residual bone graft

material

New Bone(%)

0%

20%

40%

60%

80%

100%

2w 4w 12w

5.01 11.25 23.87

24.27 21.18

14.6


tissue

Residual bone graft

material

New Bone(%)

32

4. Immunohistochemical / TRAP stain findings

A. Osteocalcin

As shown in Fig.14, it was expressed along the lining of osteoblast cells

covering the area of the bone graft material and when the bone was generated, it

was also strongly expressed in the bone.

Fig. 14 Immunohistochemical localization of osteocalcin

Helfrich27 suggested 4 step scoring method involving (-), (+), (++), (+++) as

an immunohistochemical analysis method. In this study, this was modified and as

it can be seen in fig.15, the osteocalcin expression was measured more precisely

by dividing the measurement range of each specimen into 8 regions and scoring

each region.

33

Fig. 15 Division into 8 parts of region of interest(ROI) to evaluate the

expression of osteocalcin exactly

It is measured (-): 0 point if there is almost no osteocalcin expression at each

site, (+++): 3 points when osteocalcin was expressed around almost of all bone

graft material, (++): 2 points if more than 50% were expressed, and (+): 1 point if

less than 50% was expressed, and with the combination of 8 sites, average was

calculated.

Table. 4 Expression of osteocalcin

Expression of osteocalcin

2week 4week 12week

Control 0.875 1.00 2.25

FGF 1.0 1.75 2.25 1.25

FGF 0.5 2.125 2.00 1.25

FGF 0.1 1.625 2.125 1.375

34

Table 4 shows that the experimental groups added with FGF started to express

osteocalcin strongly at 2 weeks and it showed the highest expression at 4 weeks.

However, control group without FGF had a weak expression at 2,4 weeks and it

showed the highest expression at 12 weeks. This is clearly evident in group-by-

group comparisons.

In the control group, osteocalcin was expressed only in the boundary area where

bone formation is vigorously progressing, but at the center of the bone defect it

was rarely expressed at 2 and 4 weeks. However, in the experimental group,

osteocalcin was expressed around the bone graft material at the center of the

bone defect at 2,4 weeks. In contrast, osteocalcin is expressed at the center of the

bone defect in the control group at 12 weeks and in the experimental group, the

expression of osteocalcin was weakened at 12 weeks. (Fig. 16, 17, 18)

In summary, osteoblastic activity was increased in the experimental group with

FGF over 2-4 weeks compared to the control group.

36

Fig. 16 Immunohistochemical specimens of expression of osteocalcin

undergone 2 weeks of healing time. Significantly higher staining level were

identified in FGF experimental groups.

38

Fig. 17 Immunohistochemical specimens of expression of osteocalcin undergone

4 weeks of healing time. Significantly higher staining level were identified in

FGF experimental groups

40

Fig. 18 Immunohistochemical specimens of expression of osteocalcin

undergone 12 weeks of healing time. Significantly higher staining level were

identified in control groups. The staining level of experimental groups was

decreased.

B. Osteoclast

Table. 5 Expression of osteoclast

Expression of osteoclast

2week 4week 12week

Control 0 0 118

FGF 1.0 112 152 45

FGF 0.5 61 132 85

FGF 0.1 105 92 51

Expression of osteoclast is purple in the TRAP stain, the number of osteoclasts

was counted and then the expression level of osteoclast was compared.

Looking at Table 5, there is slight variation but the expression of osteoclast was

higher in the experimental group with FGF than in the control group at 2 weeks

and 4 weeks. In control group, osteoclast was not expressed numerically at 2

41

weeks and 4 weeks. Even considering that TRAP staining does not detect all

osteoclasts, it could be seen that the expression of osteoclast was very limited.

(Fig. 19, 20)

Conversely, in 12-week specimens, osteoclast expression was higher in the

control group than in the experimental group, and expression of osteoclast was

significantly decreased in the experimental group compared to the 2 and 4 weeks.

(Fig. 21)

In summary, in the experimental group added with FGF, osteoclastic activity

was increased over 2-4 weeks compared to the control group.

42

Fig. 19 TRAP staining specimens of expression of osteoclast undergone 2

weeks of healing time. Significantly higher staining level were identified in

experimental groups. No staining was observed in the control group.

43


weeks of healing time. Significantly higher staining level were identified in

experimental groups. No staining was observed in the control group.

44


weeks of healing time. Slightly higher staining level were identified in control

groups. The staining level of experimental groups was decreased.

45

5. Russell-Movat Pentachrome stain findings

A. Analysis of tissue in the 2 week group

Fig. 22 Histologic specimens undergone 2 weeks of healing time (Russell-

Movat Pentachrome Stain) The new osteoid around new bone of the control

group was stained red.

Histomorphometrically, there was no significant difference in the degree of new

bone formation in each group at 2 weeks. However, looking at the pentachrome

stain findings, the experimental group with FGF showed higher histological

46

maturity of bone than the control group. In the control group, findings of red

dyeing which seems to be surrounding the new bone, this is the osteoid stained

before mineralization.

B. Analysis of tissue in the 4 week group


Movat Pentachrome Stain) The new osteoid around new bone of the control

group was stained red.

47

The 4 week findings were also similar to the 2 week findings. The experimental

group with FGF showed some osteoid but control group showed much more

osteoid. Thus the histological maturity of the control group was the lowest.

C. Analysis of tissue in the 12 week group


Movat Pentachrome Stain) The bone marrow of FGF 1.0mg/ml and 0.5mg/ml

groups were well developed compared to the another groups.

48

On the 12th weeks, bone formation progressed to the center of the bone defect.

There was no significant difference in the degree of osteoid expression at the new

bone formation site in the center of the bone defect. However, it could be

confirmed that, in the 1.0mg/ml group and the 0.5mg/ml group, the bone marrow

were well developed compared with the other groups.

Summarizing the pentachrome staining analysis, the bone maturity of the

experimental group added with FGF was higher than that of the control group in

the 2 and 4 week specimen.

49

IV. DISCUSSION

There are two mechanisms of bone formation : endochondral bone formation

and intramembranous bone formation. In Endochondral bone formation process,

ossification occurs in existing cartilage and in intramembranous bone formation

process, mesenchymal cells differentiate to osteoblasts that form bone.28

Calvaria is developed by intramembranous bone formation as in most of the

bones of the maxillofacial region, and it consists of two cortical plates with

regions of intervening cancellous bone, which is anatomically similar to the

mandible.29 In addition, non-load bearing bone with limited blood supply and

limited bone marrow space is physiologically similar to the mandible.30 Due to

the similarity in the formation of the maxillofacial region and the anatomical

physiological similarity of the mandible, calvaria is suitable as a research model

for bone regeneration in dental research.31,32

Lundgren et al.33 created a defect of 4.5mm diameter in the rabbit calvaria and

confirmed the degree of osteogenesis using an occlusive barrier. There was no

artificial bone regeneration process, such as GBR, but three months later, the

bone height was more than twice the original height. Thus, the contained defect

in the calvaria is good for bone formation and therefore, in order to accurately

50

determine the effect of FGF in this experiment, it was important to select the size

of bone defect.

Critical size defect (CSD) refers to the size of the defect in an animal that will

not be fully healed for life.34 According to Frame et al.30, CSD of a rabbit is a

circular bone defect with a diameter of 16 mm, Dodde et al.35 stated that it was

15 mm. Kramer et al.36 confirmed that when 8mm bone defect was formed in the

rabbit calvaria, bone bridge formation usually took 8-16 weeks, and at 16 weeks,

there were cases where bridges were not formed in the healing process. Also,

even after 24 weeks, the original thickness of the calvaria was not recovered.

Sohn et al.37 proposed two 11mm defect size to the left and right of sagittal

suturing and stated that four defects of size 8mm could be used to reduce

differences among individuals in the early phage healing studies. Dodde et al.35

reported that at least 12 weeks of observation was required in the CSD model,

also in the study by Sohn et al.37, even after 12 weeks of healing, defects of 8 mm

diameter were not fully healed. According to Misch38, the cycle of the bone

remodeling of the rabbit was 6 weeks, and that of human was 17 weeks,

approximately 3 times than rabbit. In addition, it was stated that, to achieve bone

maturation through the second mineralization stage, rabbits require 18 weeks and

humans require 54 weeks. Considering the above considerations, four 8mm defects

were created in this study and to observe both the early phase and the late phase in

bone healing process, healing periods of 2, 4, and 12 weeks were set.

51

In this study, the amount of new bone increased as the healing period

progressed in 2, 4, and 12 weeks in each group. However, there was no

statistically significant difference in the formation of new bone between groups

in the same healing period. Comparing the measured values, at 2 weeks, there

was no difference between 4 groups but at 4 weeks, FGF 1 mg/ml group showed

a new bone formation level about 6% points higher than control group, and FGF

0.5 mg/ml group also showed about 2% points higher level. At 12 weeks, the

FGF-2 1 mg/ml group and the FGF-2 0.5 mg/ml group showed levels about 7%

points higher than the control group, and FGF 0.1 mg/ml group showed more

than 3% points higher than control group. It is clear that it is not a small

difference in quantitative analysis. However, the non-parametric statistical test

showed no statistically significant difference between all groups in the same

healing period. These results seem to be due in part to the lack of experimental

populations and the differences in specificity between individuals. In fact, the

standard deviation was large due to large differences in new bone formation in

each individual and therefore it was a reason for the lack of statistically

significant difference. Another reason for these results may be due to the setting

of healing period.

Sohn et al.37 reported that at 8 weeks, independent bone tissue including bone

marrow began to appear at the center of the bone defect and lamellar bone

appeared at 12 weeks. In the cranial defect model, new bone formation starts

52

from the bone at the border of the bone defect, and new bone formation in the

center begins from the dura mater in the lower part and the periosteum in the

upper part. The periosteum supplies blood vessels to the cortical bone,29,34 and

dura mater has osteogenic potential and is known to contribute to the ossification

of the cranial defect.39 In this study, new bone formation was observed at the

center of the defect in the 4 week specimen, and in the 12 week specimen, a large

amount of new bone was formed in the center. Therefore, the dura mater in the

lower part should not be damaged in operation and periosteum should be restored

to its original position to maximize bone formation. Conversely, in order to

accurately verify the bone formation capacity of FGF-2, a method of deliberate

removing the dura mater and the periosteum can be considered. It is more

important in the CSD determining studies. In fact, there were a number of studies

that removed the lower dura mater and periosteum37,40. Hur et al. 40 showed that

the size of CSD varies with presence of periosteum. Though it is difficult that the

dura mater and the periosteum are accurately removed around the bone defect, it

is worth the attempt.

The amount of residual bone graft material tended to decrease as the healing

period was increased to 2, 4, and 12 weeks. The amount of residual bone graft

material per group during each healing period was comparable overall and there

was no statistically significant difference. The addition of FGF-2 does not have a

significant effect on the resorption of residual bone graft material and is resorbed

53

at a constant rate. The measured data show that the standard deviation is stable,

unlike in new bone formation.

Since bone formation by osteoblasts is associated with bone resorption by

osteoclasts in the bone remodeling process,41,42 the role of osteoclast is important

in osteogenesis process. After bone graft, osteoclast activity was observed on the

surface of bone graft material,43 the apatite contained in the bone graft material

and the apatite of the existing bone are biochemically bonded through the

dissolution / precipitation reaction.44 The resorption of bone graft material is

influenced by the solubility of bone graft material itself and the degree of

osteoclast activation. However, greater solubility does not mean better resorption

by osteoclasts. If the solubility of the graft material is too high, the concentration

of released calcium is high, thus limiting the activation of osteoclasts. According

to Yamada et al.45, although the solubility of β-TCP is higher, resorption of graft

material by osteoclasts was more active in the BCP where β-TCP : HA had a

ratio of 25:75.Also, BCP resorption pattern and histologic morphology of

osteoclasts in these ratios were similar to those in natural bone and dentin.

However, since the BCP of 25:75ratio in terms of mechanical strength is weak,

there is also a view that a 40:60 ratio BCP is more useful.46 The biphasic calcium

phosphate (BCP) used in the study has β-TCP and HA ratio of 70:30, and it was

verified in the previous studies that the volume stability is not compromised

compared to the BCP having a higher HA ratio. 47-49

54

In this study, besides quantitative measurement of bone formation, qualitative

measurement method was conducted. First, immunohistochemical staining was

used to confirm the degree of expression of osteocalcin, a potent marker of bone

formation, second, osteoclast expression, which plays an important role in the

bone regeneration process, was also confirmed through TRAP stain. In addition,

pentachrome staining was performed to analyze the bone formation stage

histologically.

Osteocalcin is a non-collagenous protein found in bone and dentin, which is

secreted by the osteoblast, and is known as a potent marker of the osteogenesis

process.50 It was expressed along the lining of osteoblast cells covering the area

of the bone graft material and when the bone was generated, it was also strongly

expressed in the bone.

As a result of osteocalcin expression analysis, osteoblastic activity was higher in

experimental group with FGF-2 compared to control group at 2 and 4 weeks.

Conversely, at 12 weeks, the control group had the highest osteoblastic activity.

As a result of osteoclast expression analysis, the experimental group also showed

higher osteoclastic activity at 2 weeks and 4 weeks. Conversely, at 12 weeks, the

control group had the highest osteoclastic activity. FGF-2 promoted both

osteoblastic and osteoclastic activities. In Bone regeneration, osteoclasts plays a

very important role41,42, these results can be considered that the addition of FGF-2

promoted bone regeneration process.

55

Pentachrome staining was devised by Movat in 1955 to compare the various

components of connective tissue, particularly cardiovascular tissue.51 It was then

modified by Ressell in 1972 to increase consistency and reliability.52 According

to Rentsh53, it was stated that pentachrome staining is an optimal staining method

to confirm both intramembranous bone formation and endochondral bone

formation, where bone is stained dark yellow, new osteoid stained red, and

cartilage is stained green. The osteoid secreted from the osteoblast at the initial

stage of bone formation appears as red, and as the mineralization progresses, it

becomes dark yellow, so the histological differentiation of the bone formation

process can be confirmed. The cartilage appearing in endochondral bone

formation process appears green. In other textbook54, the colors of pentachrome

staining were explained a little differently, the bone is green, the collagen is

yellow, and the osteoid is red. Our results showed that the bone is green and

yellow. The results of pentachrome stain showed that the experimental group

with FGF-2 had more histologic differentiation at 2 and 4 weeks than the control

group. In control group, red that means osteoid was more apparent at 2 and 4

weeks. On the other hand, in experimental groups, most of bones were yellow

and green, which means mature bone. At 12 weeks, bone formation progressed to

the center of the bone defect, there was no difference in histological maturity.

Summarizing the above immunohistochemical analysis and TRAP /

pentachrome staining analysis, it can be concluded that the addition of FGF-2

56

promoted osteogenesis process. However, there was no difference in bone

formation between the groups in histomorphometric results. As mentioned above,

this is thought to be due to the rabbit sacrifice timing in study design. The

addition of FGF-2 promoted the bone regeneration at 2-4 weeks, but the time of

12 weeks could be enough time for bone regeneration in control group, so at 12

weeks, there was no difference between experimental and control group. We

wanted to observe both early and late stage of bone regeneration, and thought 4

weeks’ time was enough to observe the difference of bone formation level, so we

set 2,4,12 weeks of healing time. But judging from the results of the study, 4

weeks’ time was not enough, and 12 weeks’ time was too long. If the study was

conducted with the addition of 6 and 8 week sacrifice points, the difference in

bone regeneration due to the addition of FGF-2 may have been observed as

differences in the amount of new bone formation. Nagayasu-Tanaka et al.

reported that FGF-2promoted and enhanced alveolar bone, cementum, and PDL

regeneration.55 In this study, although bone generation was promoted, it could

not be concluded that it was enhanced. If the study will be conducted with the

addition of 6 and 8 week sacrifice points, it will be possible to obtain a clear

conclusion.

The concentrations of FGF-2 used in this study were 1.0 mg/ml, 0.5 mg/ml

and 0.1 mg/ml, and doses used were 50 μg, 25 μg, and 5 μg, respectively.

Defect size was 8mm in diameter and 1 ~ 2mm in depth and 50 ~ 100mm³ in

57

terms of volume. The important factor in the growth factor experiment is the

total dose used rather than the concentration used. However, there will

certainly be a appropriate concentration that is gradually released and exhibits

the optimal effect, thus the study needs to mention both the concentration of

growth factors and the total usage. Looking at the studies up until now, there

were many cases where there was no specific mentioning of concentration of

growth factors and the total amount used. Also, when comparing different

studies, the defect size and size of experimental animals must be considered

together. Even so, there is ambiguity about whether the comparison of the

ratio of growth factor usage to defect size is sufficient to compare studies or

what additional criterion is needed for comparison between different species.

Oortigiesen et al .14 compared previous studies with use ratio for defect sizes

regardless of species. Considering that, we reviewed previous study according

to animal species used in each study and compared the defect size with the

amount used.

In the study using rats, Oortigiesen et al 14 used 2.5 µg of FGF for alveolar

defect of 6.8 mm³ size(0.37 µg/mm³), and Santana et al.4 used 5 µg of FGF in

a 1.6 mm diameter circular cavarial defect. Assuming calvarium thickness of

0.5∼1mm, volume is 1.0∼2.0mm³ and when this is converted into

concentration per unit volume, it is 2.5∼5.0µg/mm³.

58

In the study using Beagle dogs, Yosei et al.56 used 200 µg of FGF for

alveolar defect of 125 mm³ size(1.6µg/ mm³), Murakami et al.57 used 50 μg of

FGF for alveolar bone defect of 36 mm³(1.38 μg / mm³), Nakahara et al.25 used

100μg for alveolar bone defect of 48mm³ (≒ 2μg / mm³). There have been

many other studies, but there has been no accurate mention of defect size, or

defect sizes have not been uniform, thus the amount of FGF-2 used per unit

area could not be calculated accurately. In general, the larger the size and

weight of the animals used in the experiment, the larger the amount of FGF-2

used per unit volume. In this study, 0.5 ~ 1.0 μg / mm³ of FGF-2 was used

based on 1.0 mg/ml concentration. Although there were no data of using

rabbits as experimental animals, doses used in the study were similar to those

of the previous studies using other species. Future studies should accurately

address species of experimental animal, defect size, concentration and usage

amount of growth factors to make it easy to compare study results and when

these data are accumulated, it will be possible to infer the optimal

concentration and capacity of each growth factor.

To date, different types of bio-degradable polymer membranes are being widely

used in GTR and GBR in the field of dentistry, as a mean of barrier to isolate the

epithelium from other tissue to aid bone regeneration. Within the limitation of the

present study, polymer collagen membrane, used in dental clinic on the daily

basis, displayed promising results as a carrier of FGF-2 in order to enhance early

59

stage of new bone formation. Also, this simple, easy soaking-application without

further manufacturing process may contribute to increase clinical usability.

Nevertheless, further study regarding balancing physiochemical, mechanical and

biological properties of polymer membrane according to clinical demand may be

required.

60

V. CONCLUSION

As the healing period increased, regardless of different concentration

of FGF-2 used, the formation of new bone increased and the amount

of residual graft material decreased in all experimental FGF-2 groups..

FGF-2 promoted bone regeneration in all experimental groups.

Collagen membrane can be used as a useful carrier of FGF-2 in order

to enhance early stage of new bone formation.

61

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ABSTRACT(in Korean)

골유도 재생술의 증진을 위한

rhFGF-2 함유 Polymer membrane의 역할에 대한 연구

이 상 훈

연세대학교 대학원 치의학과

(지도교수 박영범)

Purpose : 이번 연구의 목적은 다양한 농도의 FGF-2 를 collagen

membrane 에 첨가하여 골유도재생술을 위한 biphasic calcium phosphate(BCP)

골 이식 부위에 국소 적용함으로써 골재생에 있어 FGF 의 역할을

확인하고, collagen membrane 의 carrier 로써의 가능성 및 골 재생에 최적의

FGF 농도를 결정하는 데 있다.

Methods : 18 마리의 New Zealand rabbit 두개골에 지름 8mm 의 골결손부를

형성하였다. BCP 골이식 후 농도를 달리한 FGF-2 를 흡수한 collagen

membrane 으로 덮고 봉합하였다. FGF-2 의 농도는 1.0mg/ml, 0.5mg/ml,

0.1mg/ml 로 설정하였고, control group 은 같은 양의 normal saline 을

69

사용하였다. 시간에 따른 골재생 양상을 확인하기 위하여 2, 4, 12 주 각각

6 마리의 New Zealand rabbit 을 희생한 후 조직계측학 분석을 통해 신생골,

잔여 골 이식재의 양을 분석하였다. 또한 면역조직화학법과 TRAP 및

Russell-Movat pentachrome stain 을 통해 정성적으로 조직을 분석하였다.

Results and Conclusions : 2, 4, 12 주로 치유시간이 길어질수록 모든

group 에서 신생골 형성이 증가하였고, 잔여 골 이식재의 양은 감소하였다.

면역조직화학 분석, TRAP stain 및 pentachrome 염색 분석 결과 FGF-2

첨가가 골재생을 촉진함을 확인할 수 있었다. 또한 collagen membrane 이

FGF-2 carrier 로 충분히 사용 가능하다고 확인하였다.

Keyword : FGF-2, Collagen membrane, BCP, Bone Regeneration, TRAP stain,

Immunohistochemistry, Pentachrome stain

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