Analisis Bioactivity Dari Scaffold Komposit Β-Tcp/Alginate Sebaagai Pelestarian Tulang Alveolar Pada Larutan Sbf Dan Domba Sebagai Hewan
BIOACTIVITY ASSESMENT OF β-TCP/ALGINATE
COMPOSITE FOR PRESERVATION ALVEOLAR RIDGE IN
SBF SOLUTION AND SHEEP AS ANIMAL MODEL
LIZA MARYETI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
STATEMENT OF THESIS
I, Liza Maryeti, hereby stated that this thesis entitled “Bioactivity
Assessment of β-TCP/alginate Composite for Preservation Alveolar Ridge in SBF
solution and Sheep as Animal Model” is true of my own work under the
supervisor advisory board and that it has not been submitted before in any form to
any university. The content of this thesis has been examined by the advising
advisory board and external examiner. Sources of information which is derived or
cited either from published or unpublished scientific paper from other writers have
mentioned in the script and listed in the References at the end part of this thesis.
I hereby assign the copyright of my thesis to Bogor Agricultural
University.
Bogor, Mei 2016
Liza Maryeti
G751130211
RINGKASAN
LIZA MARYETI. Analisis Bioactivity dari scaffold komposit β-TCP/alginate
sebaagai pelestarian tulang alveolar pada larutan SBF dan domba sebagai hewan
uji. Supervised by KIAGUS DAHLAN, GUNANTI dan YOSHIKI
MATSUMOTO.
Resopsi tulang alveolar adalah proses fisiologi yang umum terjadi setelah
kehilangan gigi. Pelestarian tulang alveolar setelah pencabutan gigi menjadi
perhatian yang penting dalam kedokteran gigi sebelum penempatan implant.
Penggunaan implant tulang sebagai pengisi pada soket alveolar setelah
pencabutan gigi sangat disarankan untuk mencegah resopsi tulang dan
membangun arsitektur tulang yang baik untuk penempatan implant. Dalam
penelitian ini kami menggunakan scaffold betha tricalcium fospat dikombinasikan
dengan alginate sebagai matriks untuk pelestraian dimensi alveolar ridge setelah
proses kehilangan gigi. Penelitian ini bertujuan untuk mengetahui laju degradasi
dari scaffold β–TCP/alginate dalam larutan SBF dengan variasi waktu yaitu
selama 0 sampai 90 hari perendaman. Utuk mengetahui sifat biokompatibiltas dari
scaffold digunakan domba sebagai hewan uji.
Laju degradasi scaffold dalam larutan SBF menunjukkan penurunan berat
sampel selama waktu perendaman. Hal ini mengindikasikan pertumbuhan apatite
yang mengacu pada component tulang karena presipitasi ion Ca dan PO dalam
larutan SBF. Selain itu, pelepasan calcium dan phosphate dari komposit juga di
ukur dalam penelitian ini, dimana terjadi penurunan ion calcium.
Proses penyembuhan tulang pada soket setelah pencabutan gigi juga
diamati pada hari ke 90 pasca operasi. Terlihat adanya pertumbuhan tulang baru
berupa woven bone pada kelompok control dan kelopmok yang di beri implan.
Data radiografi menunjukan hanya sedikit perubahan pada dimensi mesiodistal di
daerah edontulus pada kelompok yang di beri perlakuan. Data histologi dan
histomorphometry juga mengindikasikan presentasi kehadiran tulang baru yang
lebih besar pada socket yang di isi dengan scaffold β–TCP/alginate (78%)
dibandingkan dengan control (31%). Selain itu, kehadiran osteoid yang di deteksi
dengan pewarnaan azan lebih banyak pada kelompok yang di isi dengan scaffold
daripada yang dibiarkan kosong. Hal ini membuktikan sifat osteoinductivity dari
scaffold komposit β–TCP/alginate. Hasil penelitian ini menunjukkan bahwa
scaffold komposit β–TCP/alginate bisa digunakan sebagai pelestarian dimensi
alveolar ridge.
Kata Kunci : biodegradasi, scaffold β-TCP, Simulated Body Fluid, domba, ridge
preservation
SUMMARY
LIZA MARYETI. Bioactivity assessment of scaffold β-TCP/alginate composite
for preservation alveolar ridge in SBF solution sheeps as animal model.
Supervised by KIAGUS DAHLAN, GUNANTI and YOSHIKI MATSUMOTO.
Bone resorption is a physiological process after tooth extraction.
Preservation of alveolar bone following tooth extraction is among the important
goals in dental practices before dental implant placement. The use of bone
substitutes to fill the tooth socket is suggested to prevent bone resorption and
establish good bone architecture for implant placement. In this study we used
scaffold beta-tricalcium phosphate (ß-TCP) combine with alginate for
preservation alveolar ridge after extraction. This investigation purpose was to
study degradation of scaffold β–TCP/alginate in simulated body fluid (SBF)
solution and biocompatibility of scaffold in animal model.
Degradation rate with various immersing times for 0-90 days were
conducted. This result showed a decrease in weight of sample during different
time. This is indicates of growth the apatite who is a constituent component of
bone, that is occur because of the precipitation of Ca and PO4 in the SBF solution.
Furthermore, a continuous release of calcium and phosphate from the composite
was measured, whereas in SBF, decrease of the amount of the two ions in the
solution was observed accompanied with the formation of a CaP layer on the
surface.
The extraction socket healing process is considered complete (90d) when
the dental socket is filled by woven bone; it being the expression of mature bone
markers prevalent at this period. The x-ray radiograph of sheep’s incisor indicated
small change on mesio-distal of the edentulous area. Histological and
histomorphometric confirms the area of new bone formation higher percentage in
treatment than control. Histomorphometric analysis of the alveolar bone showed
that it contained 78% new bone formation in extraction socket, and 31% new bone
in socket left empty. In addition, the abundant of osteoid cell in socket filled with
β-TCP/Alginate was the proof of osteoinductivity of the composite. It was
obvious that the β-TCP/alginate scaffold composite could preservation alveolar
ridge dimension on sheep.
Keyword : biodegradation, scaffold β-TCP, SBF solution, sheeps, ridge
preservation
© Copyright of IPB, the year 2016
Copyright reserved by the law
Forbidden to quote part or all of these writings without including or
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not harm the interests of fair Bogor Agricultural University.
Prohibited announced and reproduce part or the whole paper in any form
without permission from Bogor Agricultural University.
BIOACTIVITY ASSESMENT OF β-TCP/ALGINATE
COMPOSITE FOR PRESERVATION ALVEOLAR RIDGE IN
SBF SOLUTION AND SHEEP AS ANIMAL MODEL
LIZA MARYETI
A Thesis submitted in partial fulfillment of the
requirement for Master Degree
in Biophysics Program
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
External examiner : Dr. Akhirudin Maddu, M.Si
Thesis title : Bioactivity Assesment of Scaffold β-TCP/aginate Composite for
Preservation Alveolar Ridge in SBF Solution and Sheep As Animal
Model
Name
: Liza Maryeti
ID
: G751130211
Approved by
The Commission of Supervisors
Dr Kiagus Dahlan
Supervisor
Dr drh Gunanti, MS
Co-Supervisor
Yoshiki Matsumoto, PhD
Co-Supervisor
Certified by:
Head of Biophysics
Graduate Program
Dean of the IPB Graduate School
Dr Mersi Kurniati, M.Si
Dr Ir Dahrul Syah, MSc Agr
Examination Date :
May 20, 2016
Graduation date :
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FOREWORD
First and foremost, I would humbly distinguish the Most Gracious Allah
SWT, all praises to Allah for the gifts and His blessing in completing this thesis
with title Bioactivity Assessment of Scaffold β-TCP/Alginate Composite for
Preservation Alveolar Ridge in SBF Solution and Sheeps as Animal Model. This
thesis submitted for the Degree Programs of Master Science in Master of Science
of Biophysics. I would like to sincerely deliver my greatest gratitude to my
advisor: Dr. Ir. Kiagus Dahlan, M.Si, Dr. drh. Gunanti, MS, Matsumoto Yoshiki,
Ph.D for their advice, expertness, encouragement, and support. I thank to Dr.
Akhirudin Maddu, M.Si as my examiner and representative of Biophysics
program for the suggestion, Dr. Toni Sumariada who give me inspiration, and Mr.
Rustami, M.Si for the useful discussion. I also thanks to my best friend Jayanti
Dwi Hamdila, Fitri Afriani, Marliani, and Ibu Eli Aisah Sugiarti, for our
togetherness about 3 years in Bogor, and thank a lot to my partner in crime
Wahyu Kumala Sari for always listening and supporting me. So many thankful to
Nur Aisyah Nuzulia and all friends in 2013’s Biophysics. To all of these people, I
owe its whole-hearted gratitude that impossible to describe.
My appreciations were also extended to Japan Student Services
Organization (JASSO) Scholarship for granting scholarship during the study and
experiment in Kagawa University and also for SUIJI-JDP (Six University
Initiative Japan-Indonesia Joint Degree Program) who allowed me to expand my
knowledge and experiences in Japan. This master thesis would not have been
possible unless the funding of Indonesian Government scholarship (Beasiswa
Freshgraduate). So many thankful to my all lab member (nao, kunikata, tagaki,
imade ect) animal science, Kagawa University Japan, for unforgettable moment
and precious togetherness and I also thank to all SUIJI-JDP student (UGM-IPBUNHAS).
I would like to take this moment to deeply express my thankful feeling to
my family Ibu, Ayah (alm), Uni, Abg, Niwit, Rezki and all my cousin and whole
families members who have been praying, loving and supporting as always. I give
all respect that impossible to describe, thank you so much. Finally, I hope this
thesis can give information about advanced biomaterial from natural source.
Nonetheless, I also welcome any critical feedback and advice from readers in
order to maintain it as successful project. I do hope this thesis could be useful.
Bogor, Mei 2016
Liza Maryeti
TABLE OF CONTENS
TABLE LIST
ii
FIGURE LIST
iii
APPENDICES LIST
iv
1 INTRODUCTION
Background
Objective
Benefit
1
1
2
3
2 MATERIALS AND METHODS
Place and Time Schedule
Materials and Equipments
Experimental Method
Preparation of SBF
Animal and teeth
Surgical and grafting Procedure
Radiographic observation
Histological and Histomorphometric analysis
3
3
3
4
4
4
4
4
4
3 RESULTS AND DISCUSSION
Characterization of β-TCP/alginate composite scaffold
Biodegradation Test in SBF solution
Biocompatibility Test on Sheep
Radiographic Evaluation
Histological and Histomorphometry Analysis
5
5
5
8
8
10
4 CONCLUSION AND SUGGESTION
Conclusion
Suggestion
27
27
27
REFERENCES
27
APPENDICES
31
BIOGRAPHY
32
TABLE LIST
1
2
3
Animal codes
Mass of β-TCP/Alginate scaffold after different degradation time in SBF
solution
Mesiodistal of the edentulous area
FIGURE LIST
1
2
3
4
5
6
7
8
9
10
11
12
13
SEM image of the β-TCP/alginate scaffold (a) scale bars=1 mm,
5
pore size 150–210 μm prepared in the β-TCP/alginate scaffold
scaffold. The appearance of scaffold β- TCP/alginate (b) scale bars
= 10 mm, height 1.6 cm and diameter 1cm
Wet and dry weights of the β-TCP/alginate scaffold upon
6
immersion in SBF over a period of 3 month
Weight loss versus degradation time for the materials studied
7
The weight loss curve of scaffold has also been included to
illustrate its degradation behavior
Concentration ion Ca2+ and PO4 in SBF solution
8
The radiograph of sheep ’s jaw pre-operation a (control) b (A3)
9
The radiograph of sheep (control) at (a) day+0 post operation (b)
9
day+7 post operation (c) day+30 post operation (d) day+60 post
operation (e) day+90 post operation
The radiograph of sheep (treatment) at (a) day+0 post operation
10
(b) day+7 post operation (c) day+30 post operation (d) day+60
post operation (e) day+90 post operation
Macroscopic results of sheeps mandible at Day+90 harvesting
10
(a) control (b) A1 (c) A2 (d) A3
Schema picture of alveolar ridge augmentation; untreated (a)
11
treated with β-TCP/Alginate
H&E staining of the extraction sockets following 3 month
12
treatments: (a) control group: new bone formation (*) mainly
composed of connective tissue (arrow); (b) filling with
β-TCP/alginate scaffold (A1): new bone formation (*)
almost completely filled by compact bone tissue,
arrows show connective tissue; (c) filling with β-TCP/alginate scaffold
(A2): mostly new bone tissue (d) filling with β-TCP/alginate scaffold
(A3); Specimens exhibited particles of involved by a thin calcified
tissue, and fissures were observed in the particles (arrows), while the
central portion showed connective cells (arrows). HE staining.
Magnification: 40x, Scale bar 100μm
Histomhorphometric showing the new one formation for control
13
and treatment (Using WinRoof Software)
Azan staining of extraction sockets following 3 month treatments:
13
(a) control (b)treatment. Magnification: 40×
14 MALDI-TOF MS imaging of alveolar bone, expressed localization of
optical image, upper : (a) control (b) treatment;
localization of collagen type 1 under (c) control (d) teatment
15 Histomhorphometric showing the area of collagen type 1 for
control and treatment (Using WinRoof Software)
APPENDIX LIST
1.
2.
3.
4.
5.
Flow chart of the research
SBF result of Scaffold at D+7 PO
SBF result of Scaffold at D+30 PO
SBF result of Scaffold at D+60 PO
Ion release of scaffod in SBF rsolution at differenet time
15
16
1
INTRODUCTION
Background
The most common dental diseases are periodontal disease, feline
odontoclastic resorptive lesions and feline chronic gingivostomatitis. These
diseases often cause the loss of teeth or require dental extraction. Alveolar bone
loss can occur after tooth extraction, as a result of advanced periodontal disease or
failed endodontic therapy. The resorption and remodeling of the alveolar ridge
after tooth removal is a natural healing phenomenon, which is physiologically
undesirable and possibly inevitable and can negatively impact implant placement
(Zeeshan et al. 2015). If the alveolar ridge is not preserved at the time of tooth
extraction or loss, alveolar ridge height and width may be lost, particularly in the
area of the facial plate. Several system reviews have reported losses between 3 &
6 mm horizontally and 2 mm vertically (Araujo et al. 2005). Reduction of bone in
the horizontal socket dimension of approximately 50% takes place over 1 year of
healing. The early resorption of buccal bundle bone, which takes place during the
first 8 weeks following extraction, proceeds with a marked reduction
predominantly in the horizontal dimension (Mahmoud-A et al. 2013). A reduction
in vertical ridge height of 0.8 mm over a 3 month period also predominates on the
buccal aspect. Adequate volumes of alveolar bone which are close to the original
dimensions of the alveolar process are necessary to provide favorable esthetics
and successful long-term outcomes for dental implants (Phunke et al. 2012).
Therefore, preservation of extraction socket dimensions has been attempted by
many investigators immediately following tooth extraction.
Conventional tissue replacements, such as autografts (i.e.,the patient’s own
bone, which requires multiple and potentially painful procedures), allografts
(i.e.,human bone, not from the patient), and xenografts (i.e.,animal bone) cannot
meet the quantity and performance needed by the patients. Advances in
biomaterials research and development of new and improved surgical techniques
and armamentarium have resulted in an ever increasing use of dental implants for
tooth replacement. A large number of 3-dimensional (3D) porous scaffolds have
been developed to overcome traditional limitations and have been applied to
repair bone defects. However, there are still many problems that need to be
resolved to meet clinical requirements (Zeeshaan et al. 2015). Bone is a complex
tissue mainly composed of nonstoichiometric hydroxyapatite [Ca10(PO4)6(OH)2]
and collagen (Turek et al. 1985). Approximately 30–35% of dry bone is of
organic materials, 95% of which is type I collagen. It has been widely used as a
skin substitute material. As the main inorganic component of bone, β-TCP has
been widely used in many orthopedic and dental implant materials because of its
bioactive, osteoconductive, and osteoinductive properties (Keilman et al. 2000).
Beta-tricalcium phosphate (β-TCP) is one of the representative calcium
phosphate-based alloplastic materials that exhibit biodegradation properties,
defined as the replacement of implanted material by newly formed organs. The
material has been widely applied in orthopedic and alveolar reconstruction
surgery. It biodegrades relatively slowly, which is generally recognized to be in
harmony with bone modeling (Kokovic et al. 2011). As a result, β-TCP is
2
accepted as a bioactive scaffold material for guided bone regeneration. In addition
to the requirements for chemical composition of the scaffold material, an
interconnected porous structure is necessary to allow cell attachment, proliferation,
and differentiation, and to provide pathways for biofluids (Hyong-ho et al. 2012).
Cell scaffolds provide the initial structural support and retain cells in the defective
area for cell growth, metabolism and matrix production, thus playing an important
role during the development of engineered tissues. Therefore, much attention is
focused on porous composites of β-TCP and biodegradable polymers, such as,
polylactic acid, gelatin, and chitosan and alginate (Lee et al. 2001).
Alginate is a biocompatible, hydrophilic, and biodegradable anionic
polymer under normal physiological conditions and is widely used as an instant
gel for bone tissue engineering (Hyon-ho et al. 2012). It has been studied as a
useful biomaterial in diverse tissue engineering applications because of its
hydrophilic surface promoting cell adhesion, proliferation and differentiation,
good biocompatibility and good host response, high biochemical significance in
hemostasis, angiogenesis and macrophage activation, biodegradability by
lysozyme and other enzymes, bactericidal/bacteriostatic activity, and capacity to
maintain a predefined shape after cross-linking (Bose et al. 2012). Therefore,
alginate appears to be a very promising candidate for building a bone engineering
scaffold as a natural 3D porous matrix.
Prior to the clinical use, biocompatibility and mechanical stability of new
materials should be test under both initial in vitro and in vivo conditions (Nuzulia
2014). In vitro testing is used primarily as a first stage test for obtaining a
bioactive scaffold for bone tissue engineering. Biomimetic mineralization is a
process by which organisms form minerals in a bioenvironmental acute toxicity
and cytocompatibility. Simulated body fluid (SBF) with ion concentrations nearly
equal to those of human blood plasma has been proposed by Kokubo with the
purpose of identifying a material with in vivo bone bioactivity instead of using
animal for the experiments. Recently, it has been used as a biomimetic
mineralization method to prepare biomaterials. While in vivo testing is used to
demonstrate the tissue response to materials (Arora et al. 2011).
Objectives
The objectives of this study was to evaluate the bioactivity, biocompatible
and biodegradable characteristics of β-TCP/Alginate composite material as a tooth
preservation in SBF solution and sheep as animal models. There have been no
reports on the use of β-TCP combined with alginate for post extraction socket
preservation.
Benefits
This research is expected to obtain composite scaffold has high mechanical
properties and advantages for the cell attachment. β-TCP used in this experiments
based on chicken eggshells as a calcium source and alginate due to its abundant
content and low price. Therefore, it has great potential application for the mass
production of high performance bone scaffolds.
3
2
MATERIALS AND METHOD
Place and Time Schedule
This research was conducted from July 2014 through March 2016 which
took place in Biophysics Laboratory-IPB, Faculty of Veterinary Medicine-IPB
and Faculty of Agriculture-Kagawa University Japan.
Material and Equipment’s
The β-TCP/Alginate scaffold composite was prepared by Fitri Afriani from
department of Biophysics-IPB with precipitation method. The ratio of β-TCP to
Alginate were 70/30. Materials and equipment were used for degradation rate
were NaCl, NaHCO2, KCl, K2HPO43H2O, MgCl2.6H2O, HCl 1M, CaCl2, Na2SO4,
(CH2OH)2CNH2, and AAS spectroscopy. While materials and equipment’s used
for biocompatibility testing were sheep as animal model, minor surgery set, dental
surgery tools, anesthetic material, and surgery room for aseptical insertion. Then,
materials used for histopathological examination were formalin 10%, nitric acid
5%, aquadest, aquabidest, alcohol, xylol, Hematoxilin-Eosin (HE), azocarmine G
or aniline blue, sodium citrate buffer, EDTA, alcohol.
Experimental Method
Preparation of SBF
Simulated body fluid (SBF) with ion concentration nearly equal to those of
human blood plasma has been proposed by Kokubo with the purpose of
identifying a material instead of using animal for the experiments (Kokubo et al.
2002). SBF was prepared in accordance with Kokubo’s method. The ion
concentration (mM) are as follows: 7,996 gr NaCl; 0,350 gr NaHCO2; 0,224 gr
KCl; 0,228 gr K2HPO43H2O; 0,305 gr MgCl2.6H2O; 40 mL HCl 1M; 0,278 gr
CaCl2; 0,071 gr Na2SO4; and 6,057 gr (CH2OH)2CNH2. Samples were placed in a
shaking waterbath at 37 °C for a maximum of three months. Calcium and
phosphate concentration were analyzed after 7, 30, 60 and 90 days. Calcium and
phosphate concentration were determined using AAS spectroscopy. For
degradation tests, the scaffold were accurately weighed before and after
immersion in SBF. The weight loss (WL) was calculated through Equation 1.
WL =
(1)
where W0 is the initial weight of the specimen and W is the weight of the
specimen dried after different degradation times (7, 30, 60, 90 days).
Animal and teeth
A total of 4 teeth from four healthy I year old sheep were involved in this
study (Table 1). The sheep were kept under appropriate farm conditions with food
4
and water ad libitum. All experiments were conducted along the institutional
guidelines for the care and use of laboratory animals. Prior to surgery, the animal
models were observed closely for a week in order to check their health status.
They were maintained under identical environment, management and standard
diet with ad libitum supply of drinking water.
Table 1 Animal code
Animal
Control
Animal 1
Animal 2
Animal 3
Code
Control
A1
A2
A3
Surgical and grafting procedure
Surgery was done on the lower incisor. Before surgery, sheeps were
anaesthetized by injection of ketamine 10% and xylazin 10%. Under aseptically
conditions, the lower incisor was extracted and immediately after tooth extraction,
the alveolar socket was filled with synthesized β-TCP/Alginate composites
(height 1.6 cm, diameter 1 cm) that were sterilized initially by exposure to
ultraviolet light. As control requirement, one sheep’s lower incisor were extracted
in the same manner and unfilled with any tooth filler and the other sheep’s fill
with β-TCP/Alginate. Each surgery was performed under same veterinary surgeon.
Then, the sheep were housed under a climate-controlled environment in stall of
animal used of FVM Bogor Agricultural University.
Radiographic observation
The alveolar sockets that filled with β-TCP/Alginate tooth filler and
unfilled with any tooth filler were monitored using a set of x-ray radiographic
apparatus in day D+ 0 pre operation, D+0 post operation (PO), D+7 PO and D+30
PO, D+60, and D+90 PO for observing the alveolar bone healing on sheep.
Histological and Histomorphometric analysis
The sheeps were sacrificed at 90 days after surgery. For observed new
bone formation, general histological was done using HE staining method. The
mandibular was thoroughly washed with water and fixed in 10% neutral formalin.
After fixation, the mandibular was dissected out, decalcified, and processed for
paraffin embedding. The decalcification process was carried out using 10% EDTA
solution and the specimens was dehydrated in ascending grades of ethyl alcohol,
cleared in xylene and embedded in paraffin wax. For observed osteoid cell in
socket after extraction, azan staining method were used in this study. Tissue
sections with a thickness 4-6 μm were cut using microtome and deparaffinized
using xylene, then treated with graded series of alcohol [100, 95, 80% and 70 %
ethanol] and rehydrated in PBS (pH 7.5). Fixed samples stained with azan method
using azocarmine G or aniline blue to evaluate the number of osteoid cell.
5
The histological examination was done for observing the degree of new
bone formation and the number of osteoid cell on alveolar ridge after extraction.
Sections were examined with (10x magnification) using Olympus CX20
microscope (Olympus, Japan) attached to a camera and computer. All the stained
sections were examined by image analyzer computer system using the Image
software (NIH, version v1.45e, Japan) capable of performing high speed digital
image processing for the purpose of tissue measurements. Image software was
calibrated and the images were opened on the computer screen for pre-analysis
adjustments. For histomorphometric analysis of the area percent of bone, the color
of bone trabecular was automatically selected, converted into grey then masked by
a red color which allowed automatic measurement by the computer system using
WinRoof Software version 7.4 (Japan).
3
RESULT AND DISCUSSION
Characterization of scaffold β-TCP/Alginate composite
Pore structure is an essential consideration in the development of scaffolds
for tissue engineering. Pores must be interconnected to allow for cell growth,
migration and nutrient flow. If pores are too small cell migration is limited,
resulting in the formation of a cellular capsule around the edges of the scaffold
(Murphy et al. 2009). Scaffolds with mean pore sizes ranging from 20 mm to 150
mm have been used in bone tissue engineering applications. Kalfas (2007)
reported, the pore sizes are good for bone growth ranged between 200-400 µm.
Pores allow for cell infiltration, tissue growth and facilitate the formation of new
cells. Therefore, samples were used in this study has pores in the range of 150-300
µm and porosity 67%.
Figure 1a demonstrates the microstructure of the porous β-TCP/Alginate
(pore size 150-300 µm). The appearance of β-TCP/alginate scaffold was a block
mass (Figure 1b).
(a)
(b)
Figure 1 SEM image of the β-TCP/alginate scaffold (a) scale bars=1 mm,
pore size 150–240 μm prepared in the β-TCP/Alginate scaffold
scaffold. The appearance of scaffold β- TCP/Alginate (b) scale
bars = 10 mm, height 1.6 cm and diameter 1cm.
6
Biodegradation Test in SBF solution
Simulated body fluid (SBF) is a solution with ion concentration and pH
value similar to that of human blood plasma. SBF is known to cause the
production of bioactive calcium phosphate precipitation similar to biological
mineralization. The purpose for the use of SBF was to simulate human
physiological condition. Human body fluid is supersaturated with respect to
biological apatite, which constitutes the mineral phase of calcified tissue such as
bone, dentine, and enamel in the body and also some pathological calcifications
(Alonso et al. 2012).
In this experiment mass change was used to determine apatite formation or
degradation of β-TCP/Alginate. The data collected was the mass change of the
post SBF. An initial mass was measured before SBF treatment and compared to
the final mass after treatment. Weight loss is a sign of occurring degradation
because ions are being released from the composite causing a slight decrease in
bulk sample weight; however, when these Ca-P ions react with other ions in the
SBF solution it is possible to form a calcium phosphate apatite layer on the
surface or inside the sample; when this happens the apatite formation has a greater
effect on the weight property of the ceramic than does the dissolution effect. As
expected, the samples that showed a decreased in mass (Fig. 2) also showed
apatite formation on the surface and inside the samples.
Table 2 Mass of β-TCP/Alginate scaffold after different degradation time in SBF
solution
Sampel
Mass
βTCP/Alginate
Data observation (gram)
D + 30
Day + 60
Day + 90
0.1805
0.1892
0.1887
0.1445
0.1386
0.0089
D+7
0.1831
0.1403
Initial
Final
initial
final
120
100
100
100
100
weight ratio (%)
100
80
77.87
75.13
73.3
60
40
20
20
0
D+7
Figure 2
D+30
Time/Days
D+60
D+90
Wet and dry weights of the β-TCP/alginate scaffold upon
immersion in SBF over a period of 3 month.
7
Figure 2 shows the initial and final setting time of scaffold βTCP/Alginate weight different time. Biodegradation processes of scaffold βTCP/Alginate was 80 % weight loss from the beginning. Degradation is a critical
parameter of biomaterials. In the case of bone graft substitutes, degradation rate of
the material should be comparable to the rate of new bone formation, in order for
the material to provide sufficient support while leaving space for tissue growth.
90
β-TCP/alginate
80
weight loss (%)
70
60
50
40
30
20
10
0
7
30
60
90
Time/days
Figure 3 Weight loss versus degradation time for the materials was studied.
The weight loss curve of scaffold has also been included to
illustrate its degradation behavior.
Yuliana (2015) reported that degradation of sample β-TCP alone the initial
and final setting times were lower than scaffold β-TCP/Alginate by 10% on times
experiments days 15, while in this study the scaffold β-TCP/Alginate degradation
by 20% over 7 days (Figure 3). This phenomena on that cells immersion into SBF
solution could remarkably slow down. The reason may be explained in this way,
early bone formation and mineralization deposited could cover the inner surface
which might have limited its exposure to surrounding body fluid. This might have
prevented the implanted grafts exposed to solution, slowed down of material
degradation. Another explanation might be associated with mechanical forces.
Navaro et al reported that β-TCP was hardly resorbed in a non-loading calvarias
model, while it could be easily degraded in the load-bearing area as other
researchers observed (Zhao et al. 2011; Shahabooi et al. 2015)
This results indicates that scaffold β-TCP/Alginate more rapidly degraded
because β-TCP has similar composition with bone and in addition to the pore,
adding alginate also affect the rate of degradation. On the other hand, release of
calcium and phosphate from β-TCP/Alginate scaffold was measured in SBF
solution over the time degradation of this study.
8
ion Ca and Po Dissolution (%)
60
50
40
30
ca
20
Po
10
0
0
D+7
D+30
D+60
D+90
days
Figure 4 Concentration ion Ca2+ and PO4 in SBF solution over a period
of 3 month. It could be seen, increase of ion calcium
concentration and decrease of ion phosphate concentration in
SBF solution during period time.
Figure 4 shows the release of calcium and phosphate from the βTCP/Alginate composite was measured in SBF solutions over the time of the
degradation study. It could be seen from the Figure 4, when ion calcium
concentration increase, the concentration of phosphate was decrease. It might be
caused by the release of ion calcium from the scaffold could absorbed the ion
phosphate from solution to scaffold. β-TCP/Alginate exhibited little ability to
induce calcium phosphate apatite formation on its surface; Xin has also reported
similar trends with β-TCP in SBF (Zhao et al. 2011). After 90 days some of the
pores were filled with apatite. The apatite formation on the inside of the sample
may have been due to the local release of calcium and phosphorous in the pores
which may be more favorable to promote apatite growth in the enclosed
environment rather than the free surface. Release of calcium from a Ca-P ceramic
is accompanied by other events, such as phosphate release, reprecipitation of a
biological apatite layer, possibly containing endogenous proteins and other factors,
and change of surface topography, all of which can affect bioactivity of the
material as well. We can conclude that degradation was actively occurring in the
β-TCP/Alginate compoiste over the 90 days SBF treatment.
Radiographic Evaluation
There were no clinical complications and the extraction wounds healed
uneventfully. Studies have shown long-term successful osseointegrated implant
with the satisfactory esthetic outcome can be achieved when applied with alveolar
ridge preservation after extraction. Whereas, studies conducted in the past by
various authors have also shown that the socket without graft has significant
resorption rate in both vertical as well as buccolingual dimensions. X-ray images
were taken under general anesthesia at 7, 30, 60, 90 days postoperation to follow
9
up the scaffold degradation as well as the new bone formation. Radiographic
evidence of scaffold resorption as well as new bone formation was highly variable
among the socket left empty and socket fill with β-TCP/Alginate.
(a)
(b)
Figure 5 The radiograph of dorsoventral (DV) position of sheep ’s jaw preOperation (Scale bar 15mm).
The radiograph of sheeps jaw pre operation was shows in Figure 5a as
control and figure 5b as treatment. This demonstrate the alveolar bone resorption
that could be seen from the narrower mesiodistal of edentulous are along the
observing time. As seen in Figure 6a, there was a larger distance of the alveolar
ridge after tooth extraction due to the unfilled socket. The radiopacity in control
could be visualized easily at 7 days after implantation (Fig. 6b), but it decreased
dramatically overtime due to scaffold degradation. The dimension of the alveolar
ridge had changed at D+7 PO (Figure 6b) and there is no significant change at
D+30 (Figure 6c), but clearly followed by significant change at D+60 (Figure 6c)
and D+90 PO (Figure 6e). Nevertheless, most of the β-TCP/Alginate implants had
been resorbed at 90 days post-operation with a lower radiopacity and minor
augmented alveolar ridge remained.
(a)
(b)
(c)
(d)
(e)
Figure 6 The radiograph of dorsoventral (DV) position of sheep’s jaw of control
(unfilled with sample). There was large mesio-distal and low opacity on
the extraction site ( ) at (a) day+0 post operation (b) day+7 post
operation that become narrower at (c) day+30 post operation (d)
day+60 post operation (e) and there was high alveolar bone resorption
shown by significant change on mesio-distal day+90 (e) post operation.
10
In socket that filled with β-TCP/Alginate showed the expected goal. It was
shown in Figure 7 that resubstituting process of β-TCP/Alginate and preservation
on the alveolar ridge. The implant was radiopaque due to the property of β-TCP
scaffold itself. However, the interface between the implant and the host bone
began to show radiopacity indicating new bone formation and remodeling at this
area. It was obvious from width of the edentulous area that has no significant
difference. The same alveolar ridge width was observed at D+7 PO with βTCP/Alginate resorption due to the decreasing of opacity on the alveolar socket
(Figure 7b). There was a little change on alveolar ridge at D+60 PO as shows in
Figure 7d. Certain degree of degradation was evident based on the appearance of
radiotranslucent area inside the graft. The interface between the graft and the host
bone remained radioopaque. At 90 days post-operation, radiopacity of elevated
alveolar began to increase and the remodeling was obvious by bone texture,
indicating newly formed trabecular bone, and at this time point augmented
alveolar ridge maintained the original height, and the interfaces between the grafts
and host bones were hard to distinguish.
(a)
(b)
(c)
(d)
(e)
Figure 7 The radiograph of sheep (treatment) at (a) day+0 post operation (b)
day+7 post operation (c) day+30 post operation (d) day+60 post
operation (e) day+90 post operation. The same width of mesio-distal
indicated there was no alveolar bone resorption.
The detailed mesiodistal of the edentulous area is shows in Table 3. It is
worth to note that β-TCP/Alginate as a tooth filler could stimulate the alveolar
bone healing process and maintain the desired alveolar ridge. This preservation
could be done before prostheses or implant placement.
Table 3 Width of alveolar ridge in socket fill with β-TCP/Alginate and control
Animal
Control
A1
A2
A3
D-0
5.7
5
5.8
5.5
Width of alveolar ridge (mm)
D+0 PO D+7 PO D+30 PO
5.7
5.4
4.8
5
5
5
5.8
5.7
5.7
5.5
5.4
5.4
D+60 PO
4.6
5
5.6
5.4
D+90 PO
4.5
4.9
5.2
5.2
11
To evaluate the effects of augmented alveolar bone, the width was measured.
As the shows in Table 3, the augmented width decreased significantly at 90 days
to only 5.7- 4.5 mm for control group. On the contrary, alveolar augmentation
width remained 5.0-4.9 mm (A1), 5.8- 5.2 mm (A2) and 5.5-5.2 mm (A3). This
width indicating the equivalent bone formation between the tissue engineered
construct and the autologous bone implants
Macroscopic result of D+90 harvesting day confirms the bone healing
process on postextraction socket shows in Figure 8. This funding was also
consistent with the results reported by Nuzulia (2014), which observed a reduction
in the early (30 days) phase of healing.
(a)
(b)
(c)
(d)
Figure 8 Macroscopic results of sheeps mandible at Day+90 harvesting.
Narrower mesio distal occurred on control (a) but the width of
mesio-distal remained same on the treatment filled with β-TCP
product (b) A1 (c) A2 (d) A3.
Biocompatibility test
Biocompatibility test was also done in sheep to observe the alveolar bone
healing process by resubstituting process of the corresponding β-TCP/Alginate.
Histological examination showed formation of woven bone within the extraction
socket. Areas of connective tissue were seen among bone trabeculae, indicating
that the regeneration of extraction socket occurred by intramembranous
ossification. The percentage of newly formed bone within the extraction socket
was measured by the histophotometry. The general findings showed evidence of
remodeling and new bone surrounding the graft fragments.
(a)
(b)
Figure 9 Schema picture of alveolar ridge augmentation; untreated (a);
treated with β-TCP/Alginate (b) (Alonso, 2012)
12
(a)
(b)
(
(c)
Figure 10
(d)
H&E staining of the extraction sockets following 3 month
treatments: low magnifications image of extraction site (a)
control group (b) treatment group (magnification: 40x, scale
bar 100μm). Representative high magnifications view (c)
control group; new bone formation (*) mainly composed of
connective tissue (arrow); (d) filling with β-TCP/alginate
scaffold; new bone formation (*) almost completely filled by
compact bone tissue. HE staining. Magnification: 100x, Scale
bar 100μm.
Figure 10 shows the area of woven bone in control group was lower than
treatment group. The general findings showed evidence of remodeling and new
bone surrounding the graft fragments. Three month after extraction of sheep lower
incisor, the extraction socket was filled with trabeculae of cancellous bone. The
result of histological examination of this study is in agreement with those obtained
in experiments in which the observation period was extended by several weeks.
Indovina Jr and Block evaluated the healing response with different bone
substitute materials in extraction sockets in dogs. Histological examination made
after an observation period of 8 weeks showed that the sockets left empty
contained woven bone.
The percentage of newly formed bone within the extraction socket was
measured by the histophotometry.
13
80
new bone formation (%)
70
60
50
40
30
20
10
0
control
treatment
Figure 11 Histomhorphometric showing the new bone formation for control
and treatment (Using WinRoof Software). The graph shows the
percentage of the area bone formation in treatment higher than
control.
For histomorphometric analysis of the area percent of bone, the color of woven
bone was automatically selected, converted into grey then masked by a red color
which allowed automatic measurement by the computer system. The
histomorphometric data of the present study showed that there was significant
difference among groups, i.e.,the percentage of new bone tissue in extraction
sockets filled with woven bone (78%) was different to that observed in sockets
left empty (31%), three month after extraction. In the β-TCP alone as Nuzulia
(2014) reported, however, the augmented alveolar ridge could have preserved
dimension of alveolar ridge overtime but there is still residual material on the graft,
indicating that β-TCP alone has served only as an osteoconductivity scaffold and
less biodegradable. This present study indicates that alginate has been shown to
induce osteoblastic, differentiation and proliferation of bone marrow
mesenchymal stem cells, could promote new bone formation and mineralization
inside the elevated space at much early stage.
Β-TCP/Alginate works through both osteoinduction and osteoconduction.
The material induces osteoblasts and chondroblasts differentiation from
mesenchymal cells. With its osteoinductive properties alginate leads to an increase
the number of available osteoblasts at the graft site (Wen sun et al. 2010). The
presence of β-TCP which supports osteoblasts adhesion (Bozidar et al. 2008),
combined with alginate at the defect site, can explain the higher percentage of
woven bone that was found in the treatment group.
This result confirm by the increase toluidine blue staining images in
Figure 12. We observed abundant newly formed osteoid in treatment group;
however, in the control group the osteoid exhibited closer proximity and contact
with the demineralized granule, as shown by the slightly lighter staining than the
osteoid. Thus, the new bone tissue in the site implanted with the composite
14
scaffold was actively generated, suggesting that degradation of β-TCP/Alginate
promoted osteoid production (Navaro et al. 2004).
(a)
(b)
Figure 12 Azan staining of extraction sockets following 3 month
treatments: (a) control (b) treatment. The increase toluidine
blue staining in treatment group shows the high of osteosid
cell. Magnification: 40x.
70
60
osteoid
number of cell
50
40
30
20
10
0
control
treatment
Figure 13 Histomhorphometric showing the total osteoid cell for control
and treatment (Using WinRoof Software). This graph shows the
number of osteosid cell in socket that fill with β-TCP/Alginate
higher than socket left empty.
Figure 13 shows histomorphomtric result of socket that filled with βTCP/Alginate higher (58%) than control (25%). These findings indicate that the
scaffolds β-TCP/Alginate exhibited superior osteoinduction compared with
control. Bone formation in grafts is thought to occur via three mechanisms of
bone deposition (1) osteogenesis (i.e.,signals the presence of osteoblasts, or boneforming cells, that directly deposit bone), (2) osteoinduction (i.e.,which is the
ability of the material to act as passive scaffolding that supports new bone
15
formation ingrowth of capillaries and bone), and (3) osteoconduction (i.e.,defined
as the presence of differentiating factors that facilitate the recruitment and
differentiation of mesenchymal stem cells and specifically induce them to form
osteoblasts which deposit the new bone), in which the graft acts as a scaffold for
deposition of new bone by adjacent living bone (Tuerky et al, 2000). The β-TCP
combine with alginate has good biocompatibility and osteoconductive capacity
(Charlene et al. 2014). Compared with other bone substitutes (e.g. PLGA
scaffolds), β-TCP is characterized by its precisely defined physical and chemocrystalline properties, high level of purity and uniformity of chemical composition,
so that its biological reactions can be predicted reliably (Shahabooi et al. 2015). It
can be fabricated into high porosity scaffolds with good interconnectivity, which
will ensure intercellular communication among osteogenic cells rested in lacunae
(Dubravka et al. 2002). The macro-porosity of the material will facilitate cells
adhesion and growth, and facilitate bone ingrowth and especially vascularization
(Shahabooi et al. 2015). These findings suggested that β-TCP/Alginate had served
as a good scaffold for osteoblasts to increase the bone area and mineralization
which has led the tissue-engineered complex to maintain the width of the
augmented alveolar ridge throughout the experiment.
Yuan J et al. implanted porous β-TCP to repair canine mandibular bone
segmental defects, and found that most of the material was degraded in loadbearing area 26 weeks post-operation. Lu J et al. has reported that 55% of β-TCP
could be degraded after 24 weeks of implantation in a rabbit model of femoral
condyle implantation (David t al. 2013). The degradation of β-TCP/Alginate in
vivo is believed to involve in two pathways: solution-mediated dissolution and a
cell-mediated resorption process (Middleton et al. 2000; David t al. 2013).
In this experiment, from the dramatically decreased width of augmented
alveolar ridge based on X-radiograph images, biodegradation on SBF solution and
general observations, we found that β-TCP/Alginate degraded rapidly.
Histological results demonstrating of β-TCP/Alginate are in accordance with
those of other studies which have shown that the material can be replaced
completely by new bone within a 3 months healing period. Results of this study
demonstrated higher percentage of new bone formation and high number of
osteoid cell. This is in agreement with the in vitro analysis of a previous study that
showed that the rate of degradation of the graft material was proportional to the
amount of new bone regeneration.
4
CONCLUSION AND SUGGESTION
Conclusion
In the present study, degradation of scaffold β-TCP/Alginate was analyzed
during a three month period in SBF solution and bioactivity also done in sheeps as
animal model. Degradation test in SBF solution showed that scaffold βTCP/alginate more rapidly degraded because in addition of the pore, adding
alginate also give effect to the rate of degradation. The degradation was actively
occurring in the β-TCP/Alginate composite over the 90 days SBF treatment.
16
Biocompatibility test of β-TCP/Alginate composite for preservation
alveolar ridge post extraction shows a promising challenge in dental practices.
The alveolar bone healing process is obviously observed on sheep showed by
mesio-distal of the edentulous area that remains same alveolar ridge width and the
presence of new bone formation proved by histological analysis.
Histomorphometry results also show that this scaffold is biocompatible, attested
by presence new bone and osteoid cell in treatment higher than the control. This
indicates that β-TCP with calcium sources from egg shells combine with alginate
potential as a tooth filler.
Suggestion
It is needed to analyze of collagen contain surrounding area implantation
and bone mineralized. It is important as a proof that scaffold were used in this
study was osteoinductive. In addition, histological section of alveolar bone should
be taken every period time to observe the existence of collagen and others protein.
17
REFERENCES
Afriani, Fitri. 2015. Perancah Berpori dan Betha Tricalsium Phosphate dari
limbah cangkang telur ayam dengan porogen alginate. [Thesis]. Bogor :
Faculty of Math and Natural Science : IPB.
Alonso L, Rasal G, Luis A. 2012. Development and characterization of αtricalcium phosphate/monocalcium aluminate composite bone cement. J.
Biomedical Science and Engineering. 5 :448-456.
Amler, M. H. 1980. The time sequence of tissue regeneration in human extraction
wounds. Oral Surgery, Oral Medicine and Oral Pathology. 27 : 309– 318.
Araoujo MG, Lindhe. 2005. Dimensional ridge alterations following tooth
extraction. An experimental study in the dog. J Clin Periodontol. 32 (20) :
212-218.
Arora N et al. 2011. Alveolar ridge preservation after dentalextraction and before
implant placement:A literature review. Rev Odonto Cienc 26 : 77-83.
Bozidar, Kovazdiz. 2008. Simple Preservation of a Maxillary Extraction Socket
Using Beta-tricalcium Phosphate with Type I Collagen: Preliminary
Clinical and Histomorphometric Observations. JDCA. 74 : 66 – 74.
Bose F, Hamel L., Lerouxel E., Baslé M.F, Chappard D. 2005.Bone loss and teeth.
Joint Bone Spine. 72: 215–221.
Charlène B Danoux, Davide Barbieri, Huipin Yuan, Joost D de Bruijn, Clemens A
van Blitterswijk. 2014. In vitro and in vivo bioactivity assessment of a
polylactic acid/hydroxyapatite composite for bone regeneration.
Biomatteral (12) : 31-40.
David L, Lian P. 2013. Soft and Hard Tissue Histologic Dimensions Around
Dental Implants in the Canine Restored with Smaller-Diameter Abutments:
A Paradigm Shift in Peri-implant Biology. The International Journal of
Oral & Maxillofacial Implants. 28 (2): 494-502.
Dubravka K, Asja C. 2002. Resorptive Changes of Maxillary and Mandibular
Bone Structures in Removable Denture Wearers. Acta Stomat Croat.
36(2) : 261-265.
Hoffmann, Oliver. 2008.Alveolar Bone Preservation in Extraction Sockets
Using Non-Resorbable dPTFE Membranes: A Retrospective NonRandomized Study .J. Periodontal. 79) : 8 – 20.
Horowiz R et al. 2011. A Review on Alveolar Ridge Preservation Following
Tooth Extraxtion. J Evid Base Dent Pract 31 (13): 149-160.
Hyeong-Ho J, Dong-Hyun, Tae W. 2012. In vivo evaluation of porous
hydroxyapatite/chitosan–alginate composite scaffolds for bone tissue
engineering. International Journal of Biological Macromolecules. 51(1) :
1079-1085.
Jayanti Y. 2015. Uji In vitro Hidroxyapatite dan β-
COMPOSITE FOR PRESERVATION ALVEOLAR RIDGE IN
SBF SOLUTION AND SHEEP AS ANIMAL MODEL
LIZA MARYETI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
STATEMENT OF THESIS
I, Liza Maryeti, hereby stated that this thesis entitled “Bioactivity
Assessment of β-TCP/alginate Composite for Preservation Alveolar Ridge in SBF
solution and Sheep as Animal Model” is true of my own work under the
supervisor advisory board and that it has not been submitted before in any form to
any university. The content of this thesis has been examined by the advising
advisory board and external examiner. Sources of information which is derived or
cited either from published or unpublished scientific paper from other writers have
mentioned in the script and listed in the References at the end part of this thesis.
I hereby assign the copyright of my thesis to Bogor Agricultural
University.
Bogor, Mei 2016
Liza Maryeti
G751130211
RINGKASAN
LIZA MARYETI. Analisis Bioactivity dari scaffold komposit β-TCP/alginate
sebaagai pelestarian tulang alveolar pada larutan SBF dan domba sebagai hewan
uji. Supervised by KIAGUS DAHLAN, GUNANTI dan YOSHIKI
MATSUMOTO.
Resopsi tulang alveolar adalah proses fisiologi yang umum terjadi setelah
kehilangan gigi. Pelestarian tulang alveolar setelah pencabutan gigi menjadi
perhatian yang penting dalam kedokteran gigi sebelum penempatan implant.
Penggunaan implant tulang sebagai pengisi pada soket alveolar setelah
pencabutan gigi sangat disarankan untuk mencegah resopsi tulang dan
membangun arsitektur tulang yang baik untuk penempatan implant. Dalam
penelitian ini kami menggunakan scaffold betha tricalcium fospat dikombinasikan
dengan alginate sebagai matriks untuk pelestraian dimensi alveolar ridge setelah
proses kehilangan gigi. Penelitian ini bertujuan untuk mengetahui laju degradasi
dari scaffold β–TCP/alginate dalam larutan SBF dengan variasi waktu yaitu
selama 0 sampai 90 hari perendaman. Utuk mengetahui sifat biokompatibiltas dari
scaffold digunakan domba sebagai hewan uji.
Laju degradasi scaffold dalam larutan SBF menunjukkan penurunan berat
sampel selama waktu perendaman. Hal ini mengindikasikan pertumbuhan apatite
yang mengacu pada component tulang karena presipitasi ion Ca dan PO dalam
larutan SBF. Selain itu, pelepasan calcium dan phosphate dari komposit juga di
ukur dalam penelitian ini, dimana terjadi penurunan ion calcium.
Proses penyembuhan tulang pada soket setelah pencabutan gigi juga
diamati pada hari ke 90 pasca operasi. Terlihat adanya pertumbuhan tulang baru
berupa woven bone pada kelompok control dan kelopmok yang di beri implan.
Data radiografi menunjukan hanya sedikit perubahan pada dimensi mesiodistal di
daerah edontulus pada kelompok yang di beri perlakuan. Data histologi dan
histomorphometry juga mengindikasikan presentasi kehadiran tulang baru yang
lebih besar pada socket yang di isi dengan scaffold β–TCP/alginate (78%)
dibandingkan dengan control (31%). Selain itu, kehadiran osteoid yang di deteksi
dengan pewarnaan azan lebih banyak pada kelompok yang di isi dengan scaffold
daripada yang dibiarkan kosong. Hal ini membuktikan sifat osteoinductivity dari
scaffold komposit β–TCP/alginate. Hasil penelitian ini menunjukkan bahwa
scaffold komposit β–TCP/alginate bisa digunakan sebagai pelestarian dimensi
alveolar ridge.
Kata Kunci : biodegradasi, scaffold β-TCP, Simulated Body Fluid, domba, ridge
preservation
SUMMARY
LIZA MARYETI. Bioactivity assessment of scaffold β-TCP/alginate composite
for preservation alveolar ridge in SBF solution sheeps as animal model.
Supervised by KIAGUS DAHLAN, GUNANTI and YOSHIKI MATSUMOTO.
Bone resorption is a physiological process after tooth extraction.
Preservation of alveolar bone following tooth extraction is among the important
goals in dental practices before dental implant placement. The use of bone
substitutes to fill the tooth socket is suggested to prevent bone resorption and
establish good bone architecture for implant placement. In this study we used
scaffold beta-tricalcium phosphate (ß-TCP) combine with alginate for
preservation alveolar ridge after extraction. This investigation purpose was to
study degradation of scaffold β–TCP/alginate in simulated body fluid (SBF)
solution and biocompatibility of scaffold in animal model.
Degradation rate with various immersing times for 0-90 days were
conducted. This result showed a decrease in weight of sample during different
time. This is indicates of growth the apatite who is a constituent component of
bone, that is occur because of the precipitation of Ca and PO4 in the SBF solution.
Furthermore, a continuous release of calcium and phosphate from the composite
was measured, whereas in SBF, decrease of the amount of the two ions in the
solution was observed accompanied with the formation of a CaP layer on the
surface.
The extraction socket healing process is considered complete (90d) when
the dental socket is filled by woven bone; it being the expression of mature bone
markers prevalent at this period. The x-ray radiograph of sheep’s incisor indicated
small change on mesio-distal of the edentulous area. Histological and
histomorphometric confirms the area of new bone formation higher percentage in
treatment than control. Histomorphometric analysis of the alveolar bone showed
that it contained 78% new bone formation in extraction socket, and 31% new bone
in socket left empty. In addition, the abundant of osteoid cell in socket filled with
β-TCP/Alginate was the proof of osteoinductivity of the composite. It was
obvious that the β-TCP/alginate scaffold composite could preservation alveolar
ridge dimension on sheep.
Keyword : biodegradation, scaffold β-TCP, SBF solution, sheeps, ridge
preservation
© Copyright of IPB, the year 2016
Copyright reserved by the law
Forbidden to quote part or all of these writings without including or
mentioning the source. Citing is only for educational purposes, research, writing
papers, drafting reports, writing criticism, or review an issue, and citing it does
not harm the interests of fair Bogor Agricultural University.
Prohibited announced and reproduce part or the whole paper in any form
without permission from Bogor Agricultural University.
BIOACTIVITY ASSESMENT OF β-TCP/ALGINATE
COMPOSITE FOR PRESERVATION ALVEOLAR RIDGE IN
SBF SOLUTION AND SHEEP AS ANIMAL MODEL
LIZA MARYETI
A Thesis submitted in partial fulfillment of the
requirement for Master Degree
in Biophysics Program
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
External examiner : Dr. Akhirudin Maddu, M.Si
Thesis title : Bioactivity Assesment of Scaffold β-TCP/aginate Composite for
Preservation Alveolar Ridge in SBF Solution and Sheep As Animal
Model
Name
: Liza Maryeti
ID
: G751130211
Approved by
The Commission of Supervisors
Dr Kiagus Dahlan
Supervisor
Dr drh Gunanti, MS
Co-Supervisor
Yoshiki Matsumoto, PhD
Co-Supervisor
Certified by:
Head of Biophysics
Graduate Program
Dean of the IPB Graduate School
Dr Mersi Kurniati, M.Si
Dr Ir Dahrul Syah, MSc Agr
Examination Date :
May 20, 2016
Graduation date :
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FOREWORD
First and foremost, I would humbly distinguish the Most Gracious Allah
SWT, all praises to Allah for the gifts and His blessing in completing this thesis
with title Bioactivity Assessment of Scaffold β-TCP/Alginate Composite for
Preservation Alveolar Ridge in SBF Solution and Sheeps as Animal Model. This
thesis submitted for the Degree Programs of Master Science in Master of Science
of Biophysics. I would like to sincerely deliver my greatest gratitude to my
advisor: Dr. Ir. Kiagus Dahlan, M.Si, Dr. drh. Gunanti, MS, Matsumoto Yoshiki,
Ph.D for their advice, expertness, encouragement, and support. I thank to Dr.
Akhirudin Maddu, M.Si as my examiner and representative of Biophysics
program for the suggestion, Dr. Toni Sumariada who give me inspiration, and Mr.
Rustami, M.Si for the useful discussion. I also thanks to my best friend Jayanti
Dwi Hamdila, Fitri Afriani, Marliani, and Ibu Eli Aisah Sugiarti, for our
togetherness about 3 years in Bogor, and thank a lot to my partner in crime
Wahyu Kumala Sari for always listening and supporting me. So many thankful to
Nur Aisyah Nuzulia and all friends in 2013’s Biophysics. To all of these people, I
owe its whole-hearted gratitude that impossible to describe.
My appreciations were also extended to Japan Student Services
Organization (JASSO) Scholarship for granting scholarship during the study and
experiment in Kagawa University and also for SUIJI-JDP (Six University
Initiative Japan-Indonesia Joint Degree Program) who allowed me to expand my
knowledge and experiences in Japan. This master thesis would not have been
possible unless the funding of Indonesian Government scholarship (Beasiswa
Freshgraduate). So many thankful to my all lab member (nao, kunikata, tagaki,
imade ect) animal science, Kagawa University Japan, for unforgettable moment
and precious togetherness and I also thank to all SUIJI-JDP student (UGM-IPBUNHAS).
I would like to take this moment to deeply express my thankful feeling to
my family Ibu, Ayah (alm), Uni, Abg, Niwit, Rezki and all my cousin and whole
families members who have been praying, loving and supporting as always. I give
all respect that impossible to describe, thank you so much. Finally, I hope this
thesis can give information about advanced biomaterial from natural source.
Nonetheless, I also welcome any critical feedback and advice from readers in
order to maintain it as successful project. I do hope this thesis could be useful.
Bogor, Mei 2016
Liza Maryeti
TABLE OF CONTENS
TABLE LIST
ii
FIGURE LIST
iii
APPENDICES LIST
iv
1 INTRODUCTION
Background
Objective
Benefit
1
1
2
3
2 MATERIALS AND METHODS
Place and Time Schedule
Materials and Equipments
Experimental Method
Preparation of SBF
Animal and teeth
Surgical and grafting Procedure
Radiographic observation
Histological and Histomorphometric analysis
3
3
3
4
4
4
4
4
4
3 RESULTS AND DISCUSSION
Characterization of β-TCP/alginate composite scaffold
Biodegradation Test in SBF solution
Biocompatibility Test on Sheep
Radiographic Evaluation
Histological and Histomorphometry Analysis
5
5
5
8
8
10
4 CONCLUSION AND SUGGESTION
Conclusion
Suggestion
27
27
27
REFERENCES
27
APPENDICES
31
BIOGRAPHY
32
TABLE LIST
1
2
3
Animal codes
Mass of β-TCP/Alginate scaffold after different degradation time in SBF
solution
Mesiodistal of the edentulous area
FIGURE LIST
1
2
3
4
5
6
7
8
9
10
11
12
13
SEM image of the β-TCP/alginate scaffold (a) scale bars=1 mm,
5
pore size 150–210 μm prepared in the β-TCP/alginate scaffold
scaffold. The appearance of scaffold β- TCP/alginate (b) scale bars
= 10 mm, height 1.6 cm and diameter 1cm
Wet and dry weights of the β-TCP/alginate scaffold upon
6
immersion in SBF over a period of 3 month
Weight loss versus degradation time for the materials studied
7
The weight loss curve of scaffold has also been included to
illustrate its degradation behavior
Concentration ion Ca2+ and PO4 in SBF solution
8
The radiograph of sheep ’s jaw pre-operation a (control) b (A3)
9
The radiograph of sheep (control) at (a) day+0 post operation (b)
9
day+7 post operation (c) day+30 post operation (d) day+60 post
operation (e) day+90 post operation
The radiograph of sheep (treatment) at (a) day+0 post operation
10
(b) day+7 post operation (c) day+30 post operation (d) day+60
post operation (e) day+90 post operation
Macroscopic results of sheeps mandible at Day+90 harvesting
10
(a) control (b) A1 (c) A2 (d) A3
Schema picture of alveolar ridge augmentation; untreated (a)
11
treated with β-TCP/Alginate
H&E staining of the extraction sockets following 3 month
12
treatments: (a) control group: new bone formation (*) mainly
composed of connective tissue (arrow); (b) filling with
β-TCP/alginate scaffold (A1): new bone formation (*)
almost completely filled by compact bone tissue,
arrows show connective tissue; (c) filling with β-TCP/alginate scaffold
(A2): mostly new bone tissue (d) filling with β-TCP/alginate scaffold
(A3); Specimens exhibited particles of involved by a thin calcified
tissue, and fissures were observed in the particles (arrows), while the
central portion showed connective cells (arrows). HE staining.
Magnification: 40x, Scale bar 100μm
Histomhorphometric showing the new one formation for control
13
and treatment (Using WinRoof Software)
Azan staining of extraction sockets following 3 month treatments:
13
(a) control (b)treatment. Magnification: 40×
14 MALDI-TOF MS imaging of alveolar bone, expressed localization of
optical image, upper : (a) control (b) treatment;
localization of collagen type 1 under (c) control (d) teatment
15 Histomhorphometric showing the area of collagen type 1 for
control and treatment (Using WinRoof Software)
APPENDIX LIST
1.
2.
3.
4.
5.
Flow chart of the research
SBF result of Scaffold at D+7 PO
SBF result of Scaffold at D+30 PO
SBF result of Scaffold at D+60 PO
Ion release of scaffod in SBF rsolution at differenet time
15
16
1
INTRODUCTION
Background
The most common dental diseases are periodontal disease, feline
odontoclastic resorptive lesions and feline chronic gingivostomatitis. These
diseases often cause the loss of teeth or require dental extraction. Alveolar bone
loss can occur after tooth extraction, as a result of advanced periodontal disease or
failed endodontic therapy. The resorption and remodeling of the alveolar ridge
after tooth removal is a natural healing phenomenon, which is physiologically
undesirable and possibly inevitable and can negatively impact implant placement
(Zeeshan et al. 2015). If the alveolar ridge is not preserved at the time of tooth
extraction or loss, alveolar ridge height and width may be lost, particularly in the
area of the facial plate. Several system reviews have reported losses between 3 &
6 mm horizontally and 2 mm vertically (Araujo et al. 2005). Reduction of bone in
the horizontal socket dimension of approximately 50% takes place over 1 year of
healing. The early resorption of buccal bundle bone, which takes place during the
first 8 weeks following extraction, proceeds with a marked reduction
predominantly in the horizontal dimension (Mahmoud-A et al. 2013). A reduction
in vertical ridge height of 0.8 mm over a 3 month period also predominates on the
buccal aspect. Adequate volumes of alveolar bone which are close to the original
dimensions of the alveolar process are necessary to provide favorable esthetics
and successful long-term outcomes for dental implants (Phunke et al. 2012).
Therefore, preservation of extraction socket dimensions has been attempted by
many investigators immediately following tooth extraction.
Conventional tissue replacements, such as autografts (i.e.,the patient’s own
bone, which requires multiple and potentially painful procedures), allografts
(i.e.,human bone, not from the patient), and xenografts (i.e.,animal bone) cannot
meet the quantity and performance needed by the patients. Advances in
biomaterials research and development of new and improved surgical techniques
and armamentarium have resulted in an ever increasing use of dental implants for
tooth replacement. A large number of 3-dimensional (3D) porous scaffolds have
been developed to overcome traditional limitations and have been applied to
repair bone defects. However, there are still many problems that need to be
resolved to meet clinical requirements (Zeeshaan et al. 2015). Bone is a complex
tissue mainly composed of nonstoichiometric hydroxyapatite [Ca10(PO4)6(OH)2]
and collagen (Turek et al. 1985). Approximately 30–35% of dry bone is of
organic materials, 95% of which is type I collagen. It has been widely used as a
skin substitute material. As the main inorganic component of bone, β-TCP has
been widely used in many orthopedic and dental implant materials because of its
bioactive, osteoconductive, and osteoinductive properties (Keilman et al. 2000).
Beta-tricalcium phosphate (β-TCP) is one of the representative calcium
phosphate-based alloplastic materials that exhibit biodegradation properties,
defined as the replacement of implanted material by newly formed organs. The
material has been widely applied in orthopedic and alveolar reconstruction
surgery. It biodegrades relatively slowly, which is generally recognized to be in
harmony with bone modeling (Kokovic et al. 2011). As a result, β-TCP is
2
accepted as a bioactive scaffold material for guided bone regeneration. In addition
to the requirements for chemical composition of the scaffold material, an
interconnected porous structure is necessary to allow cell attachment, proliferation,
and differentiation, and to provide pathways for biofluids (Hyong-ho et al. 2012).
Cell scaffolds provide the initial structural support and retain cells in the defective
area for cell growth, metabolism and matrix production, thus playing an important
role during the development of engineered tissues. Therefore, much attention is
focused on porous composites of β-TCP and biodegradable polymers, such as,
polylactic acid, gelatin, and chitosan and alginate (Lee et al. 2001).
Alginate is a biocompatible, hydrophilic, and biodegradable anionic
polymer under normal physiological conditions and is widely used as an instant
gel for bone tissue engineering (Hyon-ho et al. 2012). It has been studied as a
useful biomaterial in diverse tissue engineering applications because of its
hydrophilic surface promoting cell adhesion, proliferation and differentiation,
good biocompatibility and good host response, high biochemical significance in
hemostasis, angiogenesis and macrophage activation, biodegradability by
lysozyme and other enzymes, bactericidal/bacteriostatic activity, and capacity to
maintain a predefined shape after cross-linking (Bose et al. 2012). Therefore,
alginate appears to be a very promising candidate for building a bone engineering
scaffold as a natural 3D porous matrix.
Prior to the clinical use, biocompatibility and mechanical stability of new
materials should be test under both initial in vitro and in vivo conditions (Nuzulia
2014). In vitro testing is used primarily as a first stage test for obtaining a
bioactive scaffold for bone tissue engineering. Biomimetic mineralization is a
process by which organisms form minerals in a bioenvironmental acute toxicity
and cytocompatibility. Simulated body fluid (SBF) with ion concentrations nearly
equal to those of human blood plasma has been proposed by Kokubo with the
purpose of identifying a material with in vivo bone bioactivity instead of using
animal for the experiments. Recently, it has been used as a biomimetic
mineralization method to prepare biomaterials. While in vivo testing is used to
demonstrate the tissue response to materials (Arora et al. 2011).
Objectives
The objectives of this study was to evaluate the bioactivity, biocompatible
and biodegradable characteristics of β-TCP/Alginate composite material as a tooth
preservation in SBF solution and sheep as animal models. There have been no
reports on the use of β-TCP combined with alginate for post extraction socket
preservation.
Benefits
This research is expected to obtain composite scaffold has high mechanical
properties and advantages for the cell attachment. β-TCP used in this experiments
based on chicken eggshells as a calcium source and alginate due to its abundant
content and low price. Therefore, it has great potential application for the mass
production of high performance bone scaffolds.
3
2
MATERIALS AND METHOD
Place and Time Schedule
This research was conducted from July 2014 through March 2016 which
took place in Biophysics Laboratory-IPB, Faculty of Veterinary Medicine-IPB
and Faculty of Agriculture-Kagawa University Japan.
Material and Equipment’s
The β-TCP/Alginate scaffold composite was prepared by Fitri Afriani from
department of Biophysics-IPB with precipitation method. The ratio of β-TCP to
Alginate were 70/30. Materials and equipment were used for degradation rate
were NaCl, NaHCO2, KCl, K2HPO43H2O, MgCl2.6H2O, HCl 1M, CaCl2, Na2SO4,
(CH2OH)2CNH2, and AAS spectroscopy. While materials and equipment’s used
for biocompatibility testing were sheep as animal model, minor surgery set, dental
surgery tools, anesthetic material, and surgery room for aseptical insertion. Then,
materials used for histopathological examination were formalin 10%, nitric acid
5%, aquadest, aquabidest, alcohol, xylol, Hematoxilin-Eosin (HE), azocarmine G
or aniline blue, sodium citrate buffer, EDTA, alcohol.
Experimental Method
Preparation of SBF
Simulated body fluid (SBF) with ion concentration nearly equal to those of
human blood plasma has been proposed by Kokubo with the purpose of
identifying a material instead of using animal for the experiments (Kokubo et al.
2002). SBF was prepared in accordance with Kokubo’s method. The ion
concentration (mM) are as follows: 7,996 gr NaCl; 0,350 gr NaHCO2; 0,224 gr
KCl; 0,228 gr K2HPO43H2O; 0,305 gr MgCl2.6H2O; 40 mL HCl 1M; 0,278 gr
CaCl2; 0,071 gr Na2SO4; and 6,057 gr (CH2OH)2CNH2. Samples were placed in a
shaking waterbath at 37 °C for a maximum of three months. Calcium and
phosphate concentration were analyzed after 7, 30, 60 and 90 days. Calcium and
phosphate concentration were determined using AAS spectroscopy. For
degradation tests, the scaffold were accurately weighed before and after
immersion in SBF. The weight loss (WL) was calculated through Equation 1.
WL =
(1)
where W0 is the initial weight of the specimen and W is the weight of the
specimen dried after different degradation times (7, 30, 60, 90 days).
Animal and teeth
A total of 4 teeth from four healthy I year old sheep were involved in this
study (Table 1). The sheep were kept under appropriate farm conditions with food
4
and water ad libitum. All experiments were conducted along the institutional
guidelines for the care and use of laboratory animals. Prior to surgery, the animal
models were observed closely for a week in order to check their health status.
They were maintained under identical environment, management and standard
diet with ad libitum supply of drinking water.
Table 1 Animal code
Animal
Control
Animal 1
Animal 2
Animal 3
Code
Control
A1
A2
A3
Surgical and grafting procedure
Surgery was done on the lower incisor. Before surgery, sheeps were
anaesthetized by injection of ketamine 10% and xylazin 10%. Under aseptically
conditions, the lower incisor was extracted and immediately after tooth extraction,
the alveolar socket was filled with synthesized β-TCP/Alginate composites
(height 1.6 cm, diameter 1 cm) that were sterilized initially by exposure to
ultraviolet light. As control requirement, one sheep’s lower incisor were extracted
in the same manner and unfilled with any tooth filler and the other sheep’s fill
with β-TCP/Alginate. Each surgery was performed under same veterinary surgeon.
Then, the sheep were housed under a climate-controlled environment in stall of
animal used of FVM Bogor Agricultural University.
Radiographic observation
The alveolar sockets that filled with β-TCP/Alginate tooth filler and
unfilled with any tooth filler were monitored using a set of x-ray radiographic
apparatus in day D+ 0 pre operation, D+0 post operation (PO), D+7 PO and D+30
PO, D+60, and D+90 PO for observing the alveolar bone healing on sheep.
Histological and Histomorphometric analysis
The sheeps were sacrificed at 90 days after surgery. For observed new
bone formation, general histological was done using HE staining method. The
mandibular was thoroughly washed with water and fixed in 10% neutral formalin.
After fixation, the mandibular was dissected out, decalcified, and processed for
paraffin embedding. The decalcification process was carried out using 10% EDTA
solution and the specimens was dehydrated in ascending grades of ethyl alcohol,
cleared in xylene and embedded in paraffin wax. For observed osteoid cell in
socket after extraction, azan staining method were used in this study. Tissue
sections with a thickness 4-6 μm were cut using microtome and deparaffinized
using xylene, then treated with graded series of alcohol [100, 95, 80% and 70 %
ethanol] and rehydrated in PBS (pH 7.5). Fixed samples stained with azan method
using azocarmine G or aniline blue to evaluate the number of osteoid cell.
5
The histological examination was done for observing the degree of new
bone formation and the number of osteoid cell on alveolar ridge after extraction.
Sections were examined with (10x magnification) using Olympus CX20
microscope (Olympus, Japan) attached to a camera and computer. All the stained
sections were examined by image analyzer computer system using the Image
software (NIH, version v1.45e, Japan) capable of performing high speed digital
image processing for the purpose of tissue measurements. Image software was
calibrated and the images were opened on the computer screen for pre-analysis
adjustments. For histomorphometric analysis of the area percent of bone, the color
of bone trabecular was automatically selected, converted into grey then masked by
a red color which allowed automatic measurement by the computer system using
WinRoof Software version 7.4 (Japan).
3
RESULT AND DISCUSSION
Characterization of scaffold β-TCP/Alginate composite
Pore structure is an essential consideration in the development of scaffolds
for tissue engineering. Pores must be interconnected to allow for cell growth,
migration and nutrient flow. If pores are too small cell migration is limited,
resulting in the formation of a cellular capsule around the edges of the scaffold
(Murphy et al. 2009). Scaffolds with mean pore sizes ranging from 20 mm to 150
mm have been used in bone tissue engineering applications. Kalfas (2007)
reported, the pore sizes are good for bone growth ranged between 200-400 µm.
Pores allow for cell infiltration, tissue growth and facilitate the formation of new
cells. Therefore, samples were used in this study has pores in the range of 150-300
µm and porosity 67%.
Figure 1a demonstrates the microstructure of the porous β-TCP/Alginate
(pore size 150-300 µm). The appearance of β-TCP/alginate scaffold was a block
mass (Figure 1b).
(a)
(b)
Figure 1 SEM image of the β-TCP/alginate scaffold (a) scale bars=1 mm,
pore size 150–240 μm prepared in the β-TCP/Alginate scaffold
scaffold. The appearance of scaffold β- TCP/Alginate (b) scale
bars = 10 mm, height 1.6 cm and diameter 1cm.
6
Biodegradation Test in SBF solution
Simulated body fluid (SBF) is a solution with ion concentration and pH
value similar to that of human blood plasma. SBF is known to cause the
production of bioactive calcium phosphate precipitation similar to biological
mineralization. The purpose for the use of SBF was to simulate human
physiological condition. Human body fluid is supersaturated with respect to
biological apatite, which constitutes the mineral phase of calcified tissue such as
bone, dentine, and enamel in the body and also some pathological calcifications
(Alonso et al. 2012).
In this experiment mass change was used to determine apatite formation or
degradation of β-TCP/Alginate. The data collected was the mass change of the
post SBF. An initial mass was measured before SBF treatment and compared to
the final mass after treatment. Weight loss is a sign of occurring degradation
because ions are being released from the composite causing a slight decrease in
bulk sample weight; however, when these Ca-P ions react with other ions in the
SBF solution it is possible to form a calcium phosphate apatite layer on the
surface or inside the sample; when this happens the apatite formation has a greater
effect on the weight property of the ceramic than does the dissolution effect. As
expected, the samples that showed a decreased in mass (Fig. 2) also showed
apatite formation on the surface and inside the samples.
Table 2 Mass of β-TCP/Alginate scaffold after different degradation time in SBF
solution
Sampel
Mass
βTCP/Alginate
Data observation (gram)
D + 30
Day + 60
Day + 90
0.1805
0.1892
0.1887
0.1445
0.1386
0.0089
D+7
0.1831
0.1403
Initial
Final
initial
final
120
100
100
100
100
weight ratio (%)
100
80
77.87
75.13
73.3
60
40
20
20
0
D+7
Figure 2
D+30
Time/Days
D+60
D+90
Wet and dry weights of the β-TCP/alginate scaffold upon
immersion in SBF over a period of 3 month.
7
Figure 2 shows the initial and final setting time of scaffold βTCP/Alginate weight different time. Biodegradation processes of scaffold βTCP/Alginate was 80 % weight loss from the beginning. Degradation is a critical
parameter of biomaterials. In the case of bone graft substitutes, degradation rate of
the material should be comparable to the rate of new bone formation, in order for
the material to provide sufficient support while leaving space for tissue growth.
90
β-TCP/alginate
80
weight loss (%)
70
60
50
40
30
20
10
0
7
30
60
90
Time/days
Figure 3 Weight loss versus degradation time for the materials was studied.
The weight loss curve of scaffold has also been included to
illustrate its degradation behavior.
Yuliana (2015) reported that degradation of sample β-TCP alone the initial
and final setting times were lower than scaffold β-TCP/Alginate by 10% on times
experiments days 15, while in this study the scaffold β-TCP/Alginate degradation
by 20% over 7 days (Figure 3). This phenomena on that cells immersion into SBF
solution could remarkably slow down. The reason may be explained in this way,
early bone formation and mineralization deposited could cover the inner surface
which might have limited its exposure to surrounding body fluid. This might have
prevented the implanted grafts exposed to solution, slowed down of material
degradation. Another explanation might be associated with mechanical forces.
Navaro et al reported that β-TCP was hardly resorbed in a non-loading calvarias
model, while it could be easily degraded in the load-bearing area as other
researchers observed (Zhao et al. 2011; Shahabooi et al. 2015)
This results indicates that scaffold β-TCP/Alginate more rapidly degraded
because β-TCP has similar composition with bone and in addition to the pore,
adding alginate also affect the rate of degradation. On the other hand, release of
calcium and phosphate from β-TCP/Alginate scaffold was measured in SBF
solution over the time degradation of this study.
8
ion Ca and Po Dissolution (%)
60
50
40
30
ca
20
Po
10
0
0
D+7
D+30
D+60
D+90
days
Figure 4 Concentration ion Ca2+ and PO4 in SBF solution over a period
of 3 month. It could be seen, increase of ion calcium
concentration and decrease of ion phosphate concentration in
SBF solution during period time.
Figure 4 shows the release of calcium and phosphate from the βTCP/Alginate composite was measured in SBF solutions over the time of the
degradation study. It could be seen from the Figure 4, when ion calcium
concentration increase, the concentration of phosphate was decrease. It might be
caused by the release of ion calcium from the scaffold could absorbed the ion
phosphate from solution to scaffold. β-TCP/Alginate exhibited little ability to
induce calcium phosphate apatite formation on its surface; Xin has also reported
similar trends with β-TCP in SBF (Zhao et al. 2011). After 90 days some of the
pores were filled with apatite. The apatite formation on the inside of the sample
may have been due to the local release of calcium and phosphorous in the pores
which may be more favorable to promote apatite growth in the enclosed
environment rather than the free surface. Release of calcium from a Ca-P ceramic
is accompanied by other events, such as phosphate release, reprecipitation of a
biological apatite layer, possibly containing endogenous proteins and other factors,
and change of surface topography, all of which can affect bioactivity of the
material as well. We can conclude that degradation was actively occurring in the
β-TCP/Alginate compoiste over the 90 days SBF treatment.
Radiographic Evaluation
There were no clinical complications and the extraction wounds healed
uneventfully. Studies have shown long-term successful osseointegrated implant
with the satisfactory esthetic outcome can be achieved when applied with alveolar
ridge preservation after extraction. Whereas, studies conducted in the past by
various authors have also shown that the socket without graft has significant
resorption rate in both vertical as well as buccolingual dimensions. X-ray images
were taken under general anesthesia at 7, 30, 60, 90 days postoperation to follow
9
up the scaffold degradation as well as the new bone formation. Radiographic
evidence of scaffold resorption as well as new bone formation was highly variable
among the socket left empty and socket fill with β-TCP/Alginate.
(a)
(b)
Figure 5 The radiograph of dorsoventral (DV) position of sheep ’s jaw preOperation (Scale bar 15mm).
The radiograph of sheeps jaw pre operation was shows in Figure 5a as
control and figure 5b as treatment. This demonstrate the alveolar bone resorption
that could be seen from the narrower mesiodistal of edentulous are along the
observing time. As seen in Figure 6a, there was a larger distance of the alveolar
ridge after tooth extraction due to the unfilled socket. The radiopacity in control
could be visualized easily at 7 days after implantation (Fig. 6b), but it decreased
dramatically overtime due to scaffold degradation. The dimension of the alveolar
ridge had changed at D+7 PO (Figure 6b) and there is no significant change at
D+30 (Figure 6c), but clearly followed by significant change at D+60 (Figure 6c)
and D+90 PO (Figure 6e). Nevertheless, most of the β-TCP/Alginate implants had
been resorbed at 90 days post-operation with a lower radiopacity and minor
augmented alveolar ridge remained.
(a)
(b)
(c)
(d)
(e)
Figure 6 The radiograph of dorsoventral (DV) position of sheep’s jaw of control
(unfilled with sample). There was large mesio-distal and low opacity on
the extraction site ( ) at (a) day+0 post operation (b) day+7 post
operation that become narrower at (c) day+30 post operation (d)
day+60 post operation (e) and there was high alveolar bone resorption
shown by significant change on mesio-distal day+90 (e) post operation.
10
In socket that filled with β-TCP/Alginate showed the expected goal. It was
shown in Figure 7 that resubstituting process of β-TCP/Alginate and preservation
on the alveolar ridge. The implant was radiopaque due to the property of β-TCP
scaffold itself. However, the interface between the implant and the host bone
began to show radiopacity indicating new bone formation and remodeling at this
area. It was obvious from width of the edentulous area that has no significant
difference. The same alveolar ridge width was observed at D+7 PO with βTCP/Alginate resorption due to the decreasing of opacity on the alveolar socket
(Figure 7b). There was a little change on alveolar ridge at D+60 PO as shows in
Figure 7d. Certain degree of degradation was evident based on the appearance of
radiotranslucent area inside the graft. The interface between the graft and the host
bone remained radioopaque. At 90 days post-operation, radiopacity of elevated
alveolar began to increase and the remodeling was obvious by bone texture,
indicating newly formed trabecular bone, and at this time point augmented
alveolar ridge maintained the original height, and the interfaces between the grafts
and host bones were hard to distinguish.
(a)
(b)
(c)
(d)
(e)
Figure 7 The radiograph of sheep (treatment) at (a) day+0 post operation (b)
day+7 post operation (c) day+30 post operation (d) day+60 post
operation (e) day+90 post operation. The same width of mesio-distal
indicated there was no alveolar bone resorption.
The detailed mesiodistal of the edentulous area is shows in Table 3. It is
worth to note that β-TCP/Alginate as a tooth filler could stimulate the alveolar
bone healing process and maintain the desired alveolar ridge. This preservation
could be done before prostheses or implant placement.
Table 3 Width of alveolar ridge in socket fill with β-TCP/Alginate and control
Animal
Control
A1
A2
A3
D-0
5.7
5
5.8
5.5
Width of alveolar ridge (mm)
D+0 PO D+7 PO D+30 PO
5.7
5.4
4.8
5
5
5
5.8
5.7
5.7
5.5
5.4
5.4
D+60 PO
4.6
5
5.6
5.4
D+90 PO
4.5
4.9
5.2
5.2
11
To evaluate the effects of augmented alveolar bone, the width was measured.
As the shows in Table 3, the augmented width decreased significantly at 90 days
to only 5.7- 4.5 mm for control group. On the contrary, alveolar augmentation
width remained 5.0-4.9 mm (A1), 5.8- 5.2 mm (A2) and 5.5-5.2 mm (A3). This
width indicating the equivalent bone formation between the tissue engineered
construct and the autologous bone implants
Macroscopic result of D+90 harvesting day confirms the bone healing
process on postextraction socket shows in Figure 8. This funding was also
consistent with the results reported by Nuzulia (2014), which observed a reduction
in the early (30 days) phase of healing.
(a)
(b)
(c)
(d)
Figure 8 Macroscopic results of sheeps mandible at Day+90 harvesting.
Narrower mesio distal occurred on control (a) but the width of
mesio-distal remained same on the treatment filled with β-TCP
product (b) A1 (c) A2 (d) A3.
Biocompatibility test
Biocompatibility test was also done in sheep to observe the alveolar bone
healing process by resubstituting process of the corresponding β-TCP/Alginate.
Histological examination showed formation of woven bone within the extraction
socket. Areas of connective tissue were seen among bone trabeculae, indicating
that the regeneration of extraction socket occurred by intramembranous
ossification. The percentage of newly formed bone within the extraction socket
was measured by the histophotometry. The general findings showed evidence of
remodeling and new bone surrounding the graft fragments.
(a)
(b)
Figure 9 Schema picture of alveolar ridge augmentation; untreated (a);
treated with β-TCP/Alginate (b) (Alonso, 2012)
12
(a)
(b)
(
(c)
Figure 10
(d)
H&E staining of the extraction sockets following 3 month
treatments: low magnifications image of extraction site (a)
control group (b) treatment group (magnification: 40x, scale
bar 100μm). Representative high magnifications view (c)
control group; new bone formation (*) mainly composed of
connective tissue (arrow); (d) filling with β-TCP/alginate
scaffold; new bone formation (*) almost completely filled by
compact bone tissue. HE staining. Magnification: 100x, Scale
bar 100μm.
Figure 10 shows the area of woven bone in control group was lower than
treatment group. The general findings showed evidence of remodeling and new
bone surrounding the graft fragments. Three month after extraction of sheep lower
incisor, the extraction socket was filled with trabeculae of cancellous bone. The
result of histological examination of this study is in agreement with those obtained
in experiments in which the observation period was extended by several weeks.
Indovina Jr and Block evaluated the healing response with different bone
substitute materials in extraction sockets in dogs. Histological examination made
after an observation period of 8 weeks showed that the sockets left empty
contained woven bone.
The percentage of newly formed bone within the extraction socket was
measured by the histophotometry.
13
80
new bone formation (%)
70
60
50
40
30
20
10
0
control
treatment
Figure 11 Histomhorphometric showing the new bone formation for control
and treatment (Using WinRoof Software). The graph shows the
percentage of the area bone formation in treatment higher than
control.
For histomorphometric analysis of the area percent of bone, the color of woven
bone was automatically selected, converted into grey then masked by a red color
which allowed automatic measurement by the computer system. The
histomorphometric data of the present study showed that there was significant
difference among groups, i.e.,the percentage of new bone tissue in extraction
sockets filled with woven bone (78%) was different to that observed in sockets
left empty (31%), three month after extraction. In the β-TCP alone as Nuzulia
(2014) reported, however, the augmented alveolar ridge could have preserved
dimension of alveolar ridge overtime but there is still residual material on the graft,
indicating that β-TCP alone has served only as an osteoconductivity scaffold and
less biodegradable. This present study indicates that alginate has been shown to
induce osteoblastic, differentiation and proliferation of bone marrow
mesenchymal stem cells, could promote new bone formation and mineralization
inside the elevated space at much early stage.
Β-TCP/Alginate works through both osteoinduction and osteoconduction.
The material induces osteoblasts and chondroblasts differentiation from
mesenchymal cells. With its osteoinductive properties alginate leads to an increase
the number of available osteoblasts at the graft site (Wen sun et al. 2010). The
presence of β-TCP which supports osteoblasts adhesion (Bozidar et al. 2008),
combined with alginate at the defect site, can explain the higher percentage of
woven bone that was found in the treatment group.
This result confirm by the increase toluidine blue staining images in
Figure 12. We observed abundant newly formed osteoid in treatment group;
however, in the control group the osteoid exhibited closer proximity and contact
with the demineralized granule, as shown by the slightly lighter staining than the
osteoid. Thus, the new bone tissue in the site implanted with the composite
14
scaffold was actively generated, suggesting that degradation of β-TCP/Alginate
promoted osteoid production (Navaro et al. 2004).
(a)
(b)
Figure 12 Azan staining of extraction sockets following 3 month
treatments: (a) control (b) treatment. The increase toluidine
blue staining in treatment group shows the high of osteosid
cell. Magnification: 40x.
70
60
osteoid
number of cell
50
40
30
20
10
0
control
treatment
Figure 13 Histomhorphometric showing the total osteoid cell for control
and treatment (Using WinRoof Software). This graph shows the
number of osteosid cell in socket that fill with β-TCP/Alginate
higher than socket left empty.
Figure 13 shows histomorphomtric result of socket that filled with βTCP/Alginate higher (58%) than control (25%). These findings indicate that the
scaffolds β-TCP/Alginate exhibited superior osteoinduction compared with
control. Bone formation in grafts is thought to occur via three mechanisms of
bone deposition (1) osteogenesis (i.e.,signals the presence of osteoblasts, or boneforming cells, that directly deposit bone), (2) osteoinduction (i.e.,which is the
ability of the material to act as passive scaffolding that supports new bone
15
formation ingrowth of capillaries and bone), and (3) osteoconduction (i.e.,defined
as the presence of differentiating factors that facilitate the recruitment and
differentiation of mesenchymal stem cells and specifically induce them to form
osteoblasts which deposit the new bone), in which the graft acts as a scaffold for
deposition of new bone by adjacent living bone (Tuerky et al, 2000). The β-TCP
combine with alginate has good biocompatibility and osteoconductive capacity
(Charlene et al. 2014). Compared with other bone substitutes (e.g. PLGA
scaffolds), β-TCP is characterized by its precisely defined physical and chemocrystalline properties, high level of purity and uniformity of chemical composition,
so that its biological reactions can be predicted reliably (Shahabooi et al. 2015). It
can be fabricated into high porosity scaffolds with good interconnectivity, which
will ensure intercellular communication among osteogenic cells rested in lacunae
(Dubravka et al. 2002). The macro-porosity of the material will facilitate cells
adhesion and growth, and facilitate bone ingrowth and especially vascularization
(Shahabooi et al. 2015). These findings suggested that β-TCP/Alginate had served
as a good scaffold for osteoblasts to increase the bone area and mineralization
which has led the tissue-engineered complex to maintain the width of the
augmented alveolar ridge throughout the experiment.
Yuan J et al. implanted porous β-TCP to repair canine mandibular bone
segmental defects, and found that most of the material was degraded in loadbearing area 26 weeks post-operation. Lu J et al. has reported that 55% of β-TCP
could be degraded after 24 weeks of implantation in a rabbit model of femoral
condyle implantation (David t al. 2013). The degradation of β-TCP/Alginate in
vivo is believed to involve in two pathways: solution-mediated dissolution and a
cell-mediated resorption process (Middleton et al. 2000; David t al. 2013).
In this experiment, from the dramatically decreased width of augmented
alveolar ridge based on X-radiograph images, biodegradation on SBF solution and
general observations, we found that β-TCP/Alginate degraded rapidly.
Histological results demonstrating of β-TCP/Alginate are in accordance with
those of other studies which have shown that the material can be replaced
completely by new bone within a 3 months healing period. Results of this study
demonstrated higher percentage of new bone formation and high number of
osteoid cell. This is in agreement with the in vitro analysis of a previous study that
showed that the rate of degradation of the graft material was proportional to the
amount of new bone regeneration.
4
CONCLUSION AND SUGGESTION
Conclusion
In the present study, degradation of scaffold β-TCP/Alginate was analyzed
during a three month period in SBF solution and bioactivity also done in sheeps as
animal model. Degradation test in SBF solution showed that scaffold βTCP/alginate more rapidly degraded because in addition of the pore, adding
alginate also give effect to the rate of degradation. The degradation was actively
occurring in the β-TCP/Alginate composite over the 90 days SBF treatment.
16
Biocompatibility test of β-TCP/Alginate composite for preservation
alveolar ridge post extraction shows a promising challenge in dental practices.
The alveolar bone healing process is obviously observed on sheep showed by
mesio-distal of the edentulous area that remains same alveolar ridge width and the
presence of new bone formation proved by histological analysis.
Histomorphometry results also show that this scaffold is biocompatible, attested
by presence new bone and osteoid cell in treatment higher than the control. This
indicates that β-TCP with calcium sources from egg shells combine with alginate
potential as a tooth filler.
Suggestion
It is needed to analyze of collagen contain surrounding area implantation
and bone mineralized. It is important as a proof that scaffold were used in this
study was osteoinductive. In addition, histological section of alveolar bone should
be taken every period time to observe the existence of collagen and others protein.
17
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