Accepted Manuscript Title: In vivo study of chitosan-natural nano hydroxyapatite scaffolds for bone tissue regeneration Author: Jong Seo Lee Sang Dae Baek Jayachandran Venkatesan Ira Bhatnagar Hee Kyung Chang Hui Taek Kim Se-Kwon Kim PII: DOI: Reference:
S0141-8130(14)00225-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.03.053 BIOMAC 4265
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
12-2-2014 19-3-2014 26-3-2014
Please cite this article as: http://dx.doi.org/10.1016/j.ijbiomac.2014.03.053 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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In vivo study of chitosan-natural nano hydroxyapatite scaffolds for bone tissue
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regeneration
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Jong Seo Lee a, Sang Dae Baek b, Jayachandran Venkatesanc, Ira Bhatnagard, Hee Kyung Change, Hui
Taek Kima and Se-Kwon Kim c
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a
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b
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c
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University, Busan, 608-737, Republic of Korea
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Department of Orthopaedic Surgery, Pusan National University Hospital, Busan 602-739, Republic of Korea
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Department of Medicine, Graduate School, Pusan National University, Busan, 602-739, Republic of Korea
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Department of Marine Bio Convergence Science and Marine Bioprocess Research Center, Pukyong National
Nanotheranostics Laboratory, Centre for Cellular and Molecular Biology, Hyderabad 500-007, India
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Department of Pathology, Medical College, Kosin University, Busan, 602-739, Republic of Korea
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Significant development has been achieved with bioceramics and biopolymer scaffolds in
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the construction of artificial bone. In the present study, we have developed and compared
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chitosan-micro hydroxyapatite (chitosan-mHA) and chitosan-nano hydroxyapatite (chitosan-
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nHA) scaffolds as bone graft substitutes. The biocompatibility and cell proliferation of the
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prepared scaffolds were checked with preosteoblast (MC3T3-E1) cells. Total Volume (TV),
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Bone Volume (BV), Bone Surface (BS), Trabecular Thickness (Tb.Th), Trabecular Number
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(Tb.N) and Trabecular Separation (Tb.Sp) were found to be higher in chitosan-nHA than
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chitosan-mHA scaffold. Hence, we suggest that chitosan-nHA scaffold could be a promising
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biomaterial for bone tissue engineering.
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Corresponding author; Department of Orthopaedic Surgery, Pusan National University Hospital, 1-10 Ami-dong, Seo-gu, Busan 602-739, Korea TEL:+82-51-240-7248 FAX:+82-51-247-8395 E-
[email protected] Tel: +82 51 629 7097; Fax: +82 51 628 8147. E-mail address:
[email protected];
[email protected] 1
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Keywords: Chitosan; Natural Hydroxyapatite; Marine Biomaterials.
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1.
Introduction The repair and replacement of injured or defect bone is a critical problem in orthopedic
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treatment worldwide. In recent years, significant development has been made in organ
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replacement, surgical reconstruction and the use of artificial prostheses to treat the loss or failure
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of an organ or tissue [1]. Autograft and allograft are considered as ideal procedures for bone
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grafting. However, both grafting procedures have own disadvantages such as insufficient donor
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site, secondary operation and transmissible diseases (HIV and diabetics). These drawbacks could
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be overwhelmed by synthetic graft by using metals, ceramics and polymers. Nano based
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synthetic graft biomaterials for tissue-engineering treatments are being explored for a better
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treatment of bone related ailments. Numerous techniques and biomaterials have been employed
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for the orthopedic treatment for the past several decades [2, 3].
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Chitosan is a natural biopolymer consisting of β-(1→4)-2-acetamido-d-glucose and β-
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(1→4)-2-amino-d-glucose unit linkage [4, 5] and a promising biomaterial for bone tissue
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engineering. In addition, chitosan can be easily modified into various forms like films, fibers,
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beads, sponges and more complex shapes for orthopedic treatment [6-8]. The drawbacks of
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chitosan scaffolds are flexibility and poor mechanical properties, which are inferior to those of
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normal bone, because of which, they are unable to support load bearing bone implants. Calcium
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phosphate mineral, hydroxyapatite [Ca10(PO4)6(OH)2] (HA) is considered to play a vital role in
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various fields including spinal fusion, craniomaxillofacial reconstruction, bone defects, fracture
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treatment, total joint replacement (bone augmentation) and revision surgery [9, 10]. Micro HA
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(mHA) and nano HA (nHA) can be made either synthetically [11, 12] or isolated from natural
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sources [13]. However, instead of natural HA, the composite of chitosan with synthetic HA is
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more widely used as a bone graft substitute [1]. The addition of calcium phosphate in the
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chitosan scaffold has been shown to increase cell adhesion, cell proliferation, mechanical
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strength, alkaline phosphatase activity, protein adsorption, Type I collagen production as well as
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expression of other osteogenic differentiation markers [1, 14-39]. The combination of chitosan-
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nHA with stem cells and growth factors has recently emerged as a new strategy for promoting
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bone regeneration [40, 41].
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Different kind of methods have been used to prepare the HA from natural bone such as
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thermal calcination, alkaline hydrolysis and subcritical water method [42-44]. Thermal
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calcination method usually produces mHA, whereas, alkaline hydrolysis method, produces
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cabonated nHA [42]. Literature suggests that carbonated HA has comparatively higher
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osteoconduction, bio resorption and biocompatibility with regard to bone formation [45, 46] than
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synthetic HA. Naturally derived mHA combined chitosan has already been investigated for bone
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formation [47, 48]. In the present study, we are intended to compare the effect of particle size of
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HA in bone formation. In the present study, mHA and nHA particles isolated from marine fish
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bone [42] have been used to prepare chitosan-mHA and Chitosan-nHA scaffolds with freeze-
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drying method to be used as bone graft substitutes. The prepared scaffolds were subjected to in
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vivo study with rabbit model and the Total Volume (TV), Bone Volume (BV), Bone Surface
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(BS), Trabecular Thickness (Tb.Th), Trabecular Number (Tb.N) and Trabecular Separation
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(Tb.Sp) were measured.
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Materials and methods
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Chitosan (310 KDa; Degree of deacetylation 90%) was purchased from Kitto Chemicals,
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South Korea. mHA and nHA were isolated from Thunnus obesus bone as per previous literature
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[42]. Osteoblast MC3T3-E1 cell line was obtained from American Type Culture Collection
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(Manassas, VA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from
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Gibco BRL, Life Technology. MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide)
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was purchased from Molecular Probes (Eugene, OR, USA). Other reagents used in this study
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were all of analytical grade.
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Preparative procedure of Chitosan-mHA and Chitosan-nHA scaffold
Chitosan-mHA and Chitosan-nHA were prepared according to previous literature with
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slight modification [49, 50]. Briefly, 2.5 g of chitosan was dissolved in 250 ml of 2% acetic acid
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solution. The solution was stirred overnight on a mechanical stirrer (RW 20.n Labortechik) and
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sonicated for 1 h to remove any air bubbles. 2.5 g each of tuna bone derived mHA and nHA were
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suspended separately in 50 ml of water and carefully transferred into the chitosan solution
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prepared as described before, with the help of a dropper. The solution was mechanically stirred
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for 24 h to disperse the HA particles in the polymer matrix in a homogeneous manner. The milky
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white resultant solution was then transferred to small petri dish (35 x 10 mm) with 5 to 6 g of
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solution/dish to freeze at -80 °C for 5 h and finally lyophilized in freeze dryer to form scaffolds.
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These scaffolds were immersed in 10% NaOH solution for 1 d and then washed with excess
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amount of water till the pH became neutral. All the neutralized scaffolds were then lyophilized
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again to make them ready for experimentation. Morphology of scaffolds were studied by
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Scanning Electron Microscopy (SEM HITACHI S-2400, Japan)
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2.2.
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Cytotoxicity Study
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MC3T3-E1 cells (Osteoblast cell line) were cultured in alpha-MEM medium
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supplemented with 5% fetal bovine serum, 2mM glutamine and 100 µg/ml penicillin-
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streptomycin and incubated at 37 °C, humidified atmosphere with 5% CO2. The in vitro effects
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(cytotoxicity and cell proliferation) of scaffolds on MC3T3-E1 were determined by measuring 4
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MTT dye. Human osteoblast cell with 90% confluence obtained from cell culture flask (75cm2,
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filter cap) were used to seed onto the scaffolds and investigate the cytocompatibility and cell
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proliferation of chitosan and their composite scaffolds. Earlier to cell seeding, the scaffolds were
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placed in 24-well culture plate with 400µl of cell culture media and incubated for 4 h at 37 °C in
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a humidified incubator with 5% CO2 and 95% air. 100 µl of 5 x103 cells were seeded drop wise
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onto the top of the scaffolds, allowing cells to distribute throughout the scaffolds. Consequently,
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the cell-seeded scaffolds were kept at 37 °C in a humidified incubator with 5% CO2 for 4 h in
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order to allow the cells to attach to the scaffolds. Fresh medium was replaced every 2 days till
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the cultivation period. Three replicates were used for each scaffold type experiment.
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In vitro cell proliferation assay
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Cell viability on the scaffolds was measured using MTT (3-(4, 5-dimethylthiazol-2-yl)-2,
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5-diphenyltetrazolium bromide) assay (Sigma, St. Louis, USA). Cleavage of the tetrazolium
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rings turns the pale yellow MTT into dark blue formazan, the concentration of which is directly
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proportional to the number of metabolically active cells. This reduction takes place only when
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mitochondrial reductase enzymes are active. The media on the scaffolds were removed on the
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respective days and incubated with fresh culture medium containing 400 µl of MTT (5 mg ml−1
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medium) at 37 °C for 4 h in darkness. Then the unreacted dye was removed and 400 µl of
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DMSO was added to dissolve the intracellular insoluble purple formazan product into a colored
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solution. The absorbance of this solution was quantified by spectrophotometer at 540 nm with
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aGENios® microplate reader (Tecan Austria GmbH, Austria). Cell viability and proliferation of
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cells was quantified as a percentage compared to that of control.
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2.4.
In vivo Study A total of 32 New Zealand White Rabbits (3.5-4.5Kg in weight) were used in this study.
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The rabbits were separated into Group A and Group B. In Group A, 8 rabbits were used for
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chitosan-mHA, whereas, in Group B, 6 rabbits were used for chitosan-nHA according to
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materials inserted into bone gap.
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The rabbit were anesthetized using a mixture of Ketamine (5 mg/kg), Xylazine (0.25
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mg/kg) and Acepromazine (0.75 mg/kg). The tibia of the rabbit was shaved and draped with 10
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% Betadine solution. In rabbit’s tibia, four 0.062-inch diameter K-wires were inserted from the
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lateral aspect pointing towards the center of the medullary cavity. The most proximal K-wire was
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aimed at the proximity of the tibial tuberosity, and the next K-wire was inserted at 1 cm interval
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below the first one whereas the third K-wire was inserted at 2 cm interval below the second one,
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the fourth 1cm below the third one in the same manner.
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External fixators (Dyna-Exter-(ST) Finger: Dynamic External Fixator for finger with
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distraction and compression, BK Meditech, Seoul, Korea) were applied on both sides of the tibia
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following K-wire insertion. A longitudinal skin incision between the second and third K-wires
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was made through the anterior tibial crest and the muscles as well as periosteums were carefully
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dissected. Osteotomy was then performed in the diaphysis with a 1-mm diameter dental drill, run
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at low speed. Intermittent sterilized normal saline was dropped to avoid bony necrosis at the
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osteotomy site. 1.5 cm of diaphyseal bone was excised between the second and third K-wire. In
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the bony defect area, chitosan-mHA and chitosan-nHA composites scaffolds were inserted
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carefully in both the experimental groups, respectively. Periosteum and skin were repaired with
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4-0 nylon suture threads. To prevent inflammation, Kanamycin (15 mg/kg, once a day) was
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injected intramuscularly for three consecutive days. Radiography (anteroposterior view of the
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tibia) was performed every 2 weeks to monitor the process of bone formation.
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Histologic examination Histologic examination was performed when the rabbits were sacrificed. All rabbits were
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killed by intravenous injection of pentobarbital (150 mg/kg) followed by potassium phosphate.
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In order to prevent fracture or deformity in the bone gap area while harvesting the sample, tibias
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were removed with the external fixator still in place, and preserved in a 4% neutral formalin
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solution. Histologic samples were taken from one end of the defect to the other end including
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areas where it joined the original bone. Bone tissues were fixed in phosphate buffer containing
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10% formalin and then cleaned by immersion in 10% nitric acid for 1 week. The specimens were
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embedded in paraffin, and 4μm sections were cut and stained with Hematoxylin-Eosin and
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Masson-Trichrome stains. Histologic observation was done with a light microscope at 40X and
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100X magnification.
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Micro-computed tomography (µCT) analysis
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Micro-computerized tomographic evaluation was performed for the newly formed bone in
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all rabbits and the trabecular patterns and its quality were measured. All specimens were scanned
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with a high-resolution micro-computed tomography (µCT) system (Skyscan 1173; SKYSCAN,
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Kontich, Belgium) at a resolution of 29.8 μm pixel. Source voltage was 130 kV, source current
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was 25 µA, with a 1.0 mm aluminium filter and 250 ms exposure time. The images were
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reconstructed using NRecon software. Region of interest (ROI) was defined as from proximal to
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distal interface between original bone and bony defect on both ends in longitudinal section, as
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defined as from medial tibial cortex to lateral tibial cortex except fibular portion in frontal
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section, as around outer cortical margins excluding soft tissue portion in axial section.
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Parameters determined in the metaphyseal trabecular bone included Total volume (TV), bone
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volume (BV), percent bone volume (BV/TV), bone surface density (BS/TV), trabecular number
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(Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp).
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Statistical analysis
All the data were expressed as means ± standard deviation of a minimum of three replicates for each scaffold in each experiment, measured by Graphpad Prism 5.
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Results and discussion
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3.1.
Gross Examination of the scaffolds
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Scanning electron microsgraph images of chitosan-mHA and chitosan-nHA have been
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shown in the Fig. 1 mHA and nHA have been isolated as per previous literature [42]. mHA
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scaffolds were obtained in pure white color, whereas, nHA were slight yellow in color. However,
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the both the prepared scaffolds are in pure white color.
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Cell Proliferation Assay
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Scaffold for bone tissue engineering requires highly porous structure to ensure that the
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biological environment is conductive to cell attachment, proliferation, tissue growth and
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adequate nutrient flow [51]. The cytotoxic and cell proliferative effects of respective scaffolds
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were investigated through MTT assay. The chitosan and its scaffolds were not found to be
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cytotoxic to MC3T3-E1 cell line. Fig. 2 depicts the MC3T3-E1 cell proliferation on chitosan and
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their mHA and nHA composite scaffolds as the function of time, measured by MTT assay which
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represents active mitochondrial activity of living cells. The cell viability of chitosan-nHA
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scaffolds was higher than that of chitosan-mHA scaffold; this might be due to the presence and
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proper distribution of HA in the scaffold. Nanophase ceramics have superior capacity to
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osteoblast adhesion then compared to micron-sized ceramics [52].
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3.3.
Radiological evaluation Chitosan-mHA and chitosan-nHA scaffolds were fixed in the defective area of rabbit
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tibia. Ten of these rabbits were alive more than 8 weeks after surgery, and the six (3 rabbits in
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each), which successfully completed all the process without wound infection and fractures were
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selected for this study. First, the defect regions were analyzed by radiography to evaluate
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healing, every two weeks after operation (Fig. 3). In-group A (chitosan-mHA), substantial
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detectable changes were not seen within 4 weeks on radiographic images. Marginal changes
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were observed at 6 weeks on an anteroposterior image and only a small amount of bone
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formation was observed at lateral aspect of the distal end of tibia. Clearly, no solid union was
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achieved in Group A by 8 weeks postoperatively. Most area of defect exclusive of bone
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formation showed even white density, which was assumed to originate from inserted material. In
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group B (chitosan-nHA), overall sequential change was similar to the one of group A. Up to 4
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weeks, no remarkable changes were visible on any radiographic images. On the images checked
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at 6 and 8 weeks, not only marginal changes but also bony consolidation were apparent as
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compared with group A. In most remaining part within defect, increase in white density was also
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scanty. In group A, all 3 were graded as “poor” and in group B, 2 were graded as “fair” but one
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was also estimated as “poor”.
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3.4.
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Micro-CT evaluation
Microcomputed tomography (µCT) is fully nondestructive and well suited for assessing truly three-dimensional (3D) microstructural bone properties.
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The longitudinal reconstructions of the rabbit tibia 8 weeks postoperatively are shown in
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Fig. 4. Group A shows cortical bone formation stemming from distal end to proximal end on
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lateral side and small bony particles within implanted material. Group B also shows cortical bone
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formation on lateral side but differs from group A in the sense that the formation was made on
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both proximal and distal ends. 3D reconstruction images make us to identify differences between
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group A and group B more clearly. Marginal cortical bone formation in group A looks like a
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narrow stalk and small bony particles are scattered irregularly in embedded material. In group B,
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the regenerated bone at both the proximal and distal ends were observed greater than group A
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around interfaces. Additionally, implanted materials were distributed more evenly within the
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remaining defect. Values for structural parameters measured in μCT are presented in Table 1.
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Comparing the two groups, BV/TV, Tb.Th, Tb.N are higher in Group B than in Group A and
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Tb.Sp is higher in Group A than in Group B.
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Histological evaluation
All microscopic examination showed that the bony defect was filled with amorphous
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implanted material and degenerated cells. In group A, there was no distinct evidence of
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ossification. Additionally, implanted material did not adhere to original bone, so free gap existed
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in nearly whole interface area. Some of the defects were partially filled with degenerated
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amorphous biomaterial. In group B, most defect area was also filled with implanted material
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mixed with degenerated cells. But some margins of defect showed evidence of focal ossification
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and implanted material adhered to original bone in a wider area (Fig. 5 (I & II). From this
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observation, nHA plays superior role in the bone ossification.
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Conclusion
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We have developed chitosan-mHA and chitosan-nHA composite scaffolds by freeze
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drying method to mimic the function of extracellular matrix of bone. Total volume, Bone
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volume, Bone surface, Trabecular thickness, Trabecular number, Trabecular separation in nHA
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containing chitosan polymer were found to be higher than chitosan-mHA scaffolds.
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conclude that chitosan-nHA is a suitable composite scaffold that will have great potential
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applications in the field of bone tissue engineering.
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Acknowledgement This research was supported by a grant from Marine Bioprocess Research Center of the
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Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries, Republic of
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Korea.
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313
Figure Captions
314
Fig. 1 Scanning electron microcopic images of (A and B) chitosan-mHA and (C and D) chitosan-
315
nHA. Fig. 2 Viability of MC3T3-E1 osteoblast like cell on chitosan and their mHA and nHA
317
composite scaffolds as the function of time, measured by MTT assay which represents
318
active mitochondrial activity of living cells. Cell density was significantly higher on the
319
composite scaffold after day 5 onwards, (n=3).
cr
ip t
316
Fig. 3 Micro-CT evaluation (A) Coronal longitudinal section (group A), (B) Coronal longitudinal
321
section (group B), (C and D) 3D reconstruction image (group A) and (E and F) 3D
322
reconstruction image (group B) for bone regeneration and remodeling in the bone defect.
324
Fig. 4 Serial radiographs of groups A (Chitosan-mHA) and group B (Chitosan - nHA) for bone
an
323
us
320
formation.
Fig 5. Fig. 5 (I) Microscopic findings of new bone at 8 weeks after transplantation of chitosan-
326
mHA (A & B) Microscopic examination showed the bony defect filled with biomaterial
327
() (H-E, 20X), no distinct ossification was seen, (C) Bony defect was filled with
328
biomaterial () and osteoblasts () (H-E 100X), (D) The defect was partially filled with
329
osteoid islands () and degenerated amorphous biomaterial mixed with osteoblasts ()
330
(H-E, 100X)
te
d
M
325
Fig. 5 (II) Microscopic findings of new bone at 8 weeks after transplantation of chitosan-nHA
332
(A & B) Microscopic examination showed that the bony defect was filled with
333
biomaterial mixed with osteoblasts () (A: H-E, 20X, B: 100X) (C) The margin of defect
334
showed the biomaterial and cells associated with ossification ( ) (H-E, 200X) (D) The
335
margin of defect shows bone trabeculae (⏎) and a few islands of ossification () (H-E,
336
100X).
337
Ac ce p
331
338
15
Page 15 of 23
nHA
ce
pt
ed
M an
mHA
u
Graphical Abstract
Chitosan-nHA
Ac
Chitosan-mHA
Page 16 of 23
Figure(s)
(A)
us
cr
ip t
(B)
(D)
d
M
an
(C)
Ac ce p
chitosan-nHA.
te
Fig. 1 Scanning electron microcopic images of (A and B) chitosan-mHA and (C and D)
1
Page 17 of 23
1st Day 3rd Day 5th Day 7th Day
0.6
ip t
0.4 0.2
cr
Absorbance @540nm
0.8
Ch
itos
a
m n-
HA Ch
io
tsan
nH
an
Scaffold Type
A
us
0.0
M
Fig. 2 Viability of MC3T3-E1 osteoblast like cell on chitosan and their mHA and nHA composite scaffolds as the function of time, measured by MTT assay, which
d
represents active mitochondrial activity of living cells. Cell density was
Ac ce p
te
significantly higher on the composite scaffold after day 5 onwards, (n=3).
2
Page 18 of 23
(B)
(C)
cr
ip t
(A)
M
an
us
(D)
(F)
Ac ce p
te
d
(E)
Fig. 3 Micro-CT evaluation (A) Coronal longitudinal section (group A), (B). Coronal longitudinal section (group B), (C and D) 3D reconstruction image (group A) and (E and F) 3D reconstruction image (group B) for bone regeneration and remodeling in the bone defect.
3
Page 19 of 23
PostOP (12.05.31)
12.06.21 (4w)
12.06.07 (2w)
12.07.5 (6w)
12.07.19 (8w)
Ac ce p
te
d
M
an
Group B Chitosan-nHA
us
PreOP (12.05.29)
cr
ip t
Group A Chitosan-mHA
PreOP (12.05.29)
PostOP (12.05.31)
12.6.14 (2w)
12.06.21 (4w)
12.07.06 (6w)
12.07.20 (8w)
Fig. 4 Serial radiographs of groups A (Chitosan-mHA) and group B (Chitosan - nHA) for bone formation.
4
Page 20 of 23
ip t cr us an M d
te
Fig. 5 (I) Microscopic findings of new bone at 8 weeks after transplantation of chitosan-
Ac ce p
mHA (A & B) Microscopic examination showed the bony defect filled with biomaterial () (H-E, 20X), no distinct ossification was seen, (C) Bony defect was filled with biomaterial () and osteoblasts () (H-E 100X), (D) The defect was partially filled with osteoid islands () and degenerated amorphous biomaterial mixed with osteoblasts () (H-E, 100X)
5
Page 21 of 23
ip t cr us an M d
te
Fig. 5 (II) Microscopic findings of new bone at 8 weeks after transplantation of chitosan-
Ac ce p
nHA (A & B) Microscopic examination showed that the bony defect was filled with biomaterial mixed with osteoblasts () (A: H-E, 20X, B: 100X) (C) The margin of defect showed the biomaterial and cells associated with ossification ( ) (H-E, 200X) (D) The margin of defect shows bone trabeculae (⏎) and a few islands of ossification () (H-E, 100X).
6
Page 22 of 23
Table 1. The average value of micro-CT parameters according to the group Group B
TV (mm3)
1179.449
1207.216
BV (mm3)
58.979
Percent bone volume, BV/TV (%)
5.001
BS (mm2) Bone surface density, BS/TV (mm2/ mm3)
an
0.944
Tb.Th (mm)
M
Tb.N (1/mm)
10.426
2160.444 1.790
0.458
0.709
0.109
0.147
1.228
0.757
d
Tb.Sp (mm)
125.864
us
1113.327
ip t
Group A
cr
Parameters
te
TV: Total volume, BV: Bone volume, BS: Bone surface, Tb.Th: Trabecular thickness,
Ac ce p
Tb.N: Trabecular number, Tb.Sp: Trabecular separation
8
Page 23 of 23