Effectiveness of tissue engineered chitosan-gelatin composite scaffold loaded with human platelet gel in regeneration of critical sized radial bone defect in rat

Accepted Manuscript Effectiveness of tissue engineered chitosan-gelatin composite scaffold loaded with human platelet gel in regeneration of critical sized radial bone defect in rat

Ahmad Oryan, Soodeh Alidadi, Amin Bigham-Sadegh, Ali Moshiri, Amir Kamali PII: DOI: Reference:

S0168-3659(16)31095-1 doi: 10.1016/j.jconrel.2017.03.040 COREL 8725

To appear in:

Journal of Controlled Release

Received date: Accepted date:

27 October 2016 21 March 2017

Please cite this article as: Ahmad Oryan, Soodeh Alidadi, Amin Bigham-Sadegh, Ali Moshiri, Amir Kamali , Effectiveness of tissue engineered chitosan-gelatin composite scaffold loaded with human platelet gel in regeneration of critical sized radial bone defect in rat. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Corel(2017), doi: 10.1016/j.jconrel.2017.03.040

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ACCEPTED MANUSCRIPT Effectiveness of tissue engineered chitosan-gelatin composite scaffold loaded with human platelet gel in regeneration of critical sized radial bone defect in rat Ahmad Oryan1* , Soodeh Alidadi1 , Amin Bigham-Sadegh2 , Ali Moshiri3 and Amir Kamali1 Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

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Department of Surgery and Radiology, School of Veterinary Medicine, Shahrekord University,

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Razi Drug Research Center, Iran University of Medical Sciences, Tehran, Iran

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Shahrekord, Iran

*Correspondence to: Ahmad Oryan, DVM, PhD, Professor of Comparative Pathology at

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Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.

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E-mail: [email protected]; Tel: +98-7132286950; Fax: +98-7132286940.

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Running title: Effectiveness of chitosan-gelatin composite loaded with platelet gel in bone

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healing

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ACCEPTED MANUSCRIPT Abstract Although many strategies have been utilized to accelerate bone regeneration, an appropriate treatment strategy to regenerate a new bone with optimum morphology and mechanical properties has not been invented as yet. This study investigated the healing potential of a

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composite scaffold consisting of chitosan (CS), gelatin (Gel) and platelet gel (PG), named CSGel-PG, on a bilateral critical sized radial bone defect in rat. Eighty radial bone defects were

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bilaterally created in 40 Sprague-Dawley rats and were randomly divided into eight groups

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including untreated, autograft, CS, Gel, CS-PG, Gel-PG, CS-Gel, and CS-Gel-PG treated defects. The bone defects were evaluated clinically and radiologically during the study and their

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bone samples were assessed by gross and histopathology, histomorphometry, CT-scan, scanning

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electron microscopy, and biomechanical testing after 8 weeks of bone injury. The autograft and CS-Gel-PG groups showed significantly higher new bone formation, density of osseous and

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cartilaginous tissues, bone volume, and mechanical performance than the defect, CS and Gel-PG

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groups (P˂0.05). In addition, bone volume, density of osseous and cartilaginous tissues, and numbers of osteons in the CS-Gel-PG group were significantly superior to the CS-PG, CS-Gel

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and Gel groups (P˂0.05). Increased mRNA levels of alkaline phosphatase, runt-related

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transcription factor 2, osteocalcin, collagen type 1 and CD31, vascular endothelial growth factor as osteogenic and angiogenic differentiation markers were found with the CS-Gel-PG scaffold by quantitative real-time PCR in vitro after 30 days of culturing on bone marrow-derived mesenchymal stem cells. In conclusion, the healing potential of CS-Gel scaffold embedded with PG was comparable to autografting and therefore, it can be offered as an appropriate scaffold in bone tissue engineering and regenerative applications. Keywords: chitosan, gelatin, platelet gel, bone tissue engineering, bone regeneration, radius 2

ACCEPTED MANUSCRIPT Chemical compounds used in this study Chitosan (PubChem CID: 71853); Methyl methacrylate (PubChem CID: 6658); Glutaraldehyde (PubChem CID: 3485); Acetic acid (PubChem CID: 176); Glycine (PubChem CID: 750); Nitric acid (PubChem CID: 944); Hydrochloric acid (PubChem CID: 313); Ethanol (PubChem CID:

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702); Formaldehyde (PubChem CID: 712); Formic acid (PubChem CID: 284); Calcium chloride

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(PubChem CID: 5284359)

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ACCEPTED MANUSCRIPT 1. Introduction Tissue-engineered constructs are considered as promising alternatives to bone grafts in regeneration of large bone defects [1-4]. Recently, natural polymers such as gelatin (Gel) and chitosan (CS) have been increasingly proposed as biological materials in designing scaffolds to

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be applied in bone tissue engineering [3,5-7]. They have several considerable advantages such as high biodegradability, biocompatibility, non-antigenicity, and non-toxicity [5,6,8,9]. Chitosan

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has a hydrophilic surface that promotes cell adhesion, proliferation and differentiation [2,5].

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Since neither CS nor Gel is sufficient to induce bone formation [10,11], incorporation of growth factors within the Gel and/or CS based scaffolds may improve the osteoconductive and

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osteoinductive properties and enhance new bone formation [1,12,13]. It is expected that the

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biological effects of several growth factors such as those present in platelet-rich plasma (PRP), due to their drawbacks, are greater in enhancement of tissue regeneration than a single growth

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factor such as bone morphogenetic proteins (BMPs) [12,14-16]. Platelets contain angiogenic,

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mitogenic and osteogenic growth factors in their α-granules [2,12,15,17]. Several studies have demonstrated the effectiveness of PRP in combination with biomaterials in promotion of bone [17-20].

Nonetheless,

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regeneration

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questionable

whether

combination

with other

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biomaterials allows the PRP to enhance its biological activity. Overall, composite scaffolds may help bone regeneration in a better way because of the beneficial properties of several biomaterials applied in the scaffold [3,20]. Biodegradable polymers such as CS and Gel have been widely used substrates for cells to attach and grow [5,21]. Moreover, they are able to interact ionically with growth factors having opposite charges [1,18,22]. Therefore, growth factors immobilized in these scaffolds could be released with degradation of the scaffold [18].

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ACCEPTED MANUSCRIPT Given the above explanations, application of a combination of CS and Gel combined with growth factors appears to provide promising results in bone engineering and regenerative medicine. Therefore, for the first time, this study investigated the role of

CS-Gel composite

scaffold incorporated with human xenogeneic platelet gel (PG) in bone healing and regeneration

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of experimentally induced radial bone defects in rats. Although CS and Gel have extensively

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been used in bone regeneration, the pure effects of CS and Gel, and their combination as CS-Gel,

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or with human PG as CS-PG and Gel-PG have not been investigated in the previous

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regenerative potential of the CS-Gel-PG scaffold.

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experimental studies. Thus, we used these five options as our control groups to evaluate the

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2. Materials and Methods

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2.1. Preparation of Scaffolds

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2.1.1. Gelatin Scaffold

Acidic Gel from the bovine skin (type B, ~225 g Bloom, isoelectric point ~5; Sigma-Aldrich,

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Germany) was added into acetone with an equal proportion to precipitate high molecular weight

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particles. A 10% Gel solution (100 mg/ml) was prepared by adding the residual Gel into deionized water at 45ºC. To crosslink the Gel particles, the solution was then mixed with a 0.25% (v/v) glutaraldehyde (GA, Acros Organics

TM

) solution in phosphate buffer solution (PBS)

at pH=7.4 by a homogenizer (300 rpm for 15 min). The resultant gel was maintained at -20 ºC for 24 h and then freeze-dried (Christ freeze dryer, ALPHA 2-4 LD plus, Germany) at -80 ºC and a pressure of 1 mBar for 48 h.

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ACCEPTED MANUSCRIPT 2.1.2. Chitosan Scaffold A 2% aqueous solution (w/v) of CS (medium molecular weight, 75-85% de-acetylation degree, Sigma-Aldrich, St. Louis, Germany) was made by dissolving CS into 1% (v/v) acetic acid by stirring at 500 rpm for 5 h to get a perfectly transparent solution. The 0.25% GA solution (v/v)

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was then added into the CS solution and homogenized to crosslink the solution. The resultant

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hydrogel was kept at 4 o C for 24 h to allow the gel to be polymerized, then maintained at -20ºC

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for 24 h and finally freeze-dried at -80 ºC for 48 h to become porous and ensure complete drying.

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2.1.3. Chitosan-Gelatin Composite Scaffold

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To prepare the CS-Gel composite scaffold, the 2% CS solution was dissolved in 1% acetic acid (v/v). The Gel (2% w/v) was then added to the CS (2% w/v) solution at 20 wt% and stirred for

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12 h at 37 ºC to form CS-Gel solution. A 0.25% GA solution was added to the solution as a

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cross-linker agent. The resultant cross-linked gel was frozen at -20 ºC for 24 h and then freeze-

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dried at -80 ºC for 48 h.

2.1.4. Post-fabrication Processing of the Scaffolds

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The active sites or non-reactive sites of GA in the Gel, CS and CS-Gel scaffolds were then

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deactivated by immersing the scaffolds in the 0.55 milimolar glycine solution for 2 h. All the scaffolds were then washed with distilled water at room temperature at triplicate. The scaffolds were then freeze dried again to remove the solvents without altering the scaffold structure and architecture. The scaffolds were then freeze-dried at -80 ºC for 48 h and sterilized under irradiation at a dose of 15 kGy and kept in sterile packs until surgical application. 2.1.5. Platelets 6

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Co γ-

ACCEPTED MANUSCRIPT Human platelets were provided from the Shiraz Blood Bank Center. The platelet solution was then immediately freeze-dried at -80 ºC for 48 h and transformed into platelet powder. The powder was then sterilized by

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Co γ-irradiation at a dose of 15 kGy (λ = 254 nm) for 10 min.

Number of platelets in the whole blood and PG was 259.4 ± 41.6 × 103 /µl and 1174.3 ± 261.3 ×

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103 /µl (4.5-fold increase), respectively. Health and activity of the platelets were checked and

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2.1.6. Preparation of the Scaffolds Loaded by Platelet Gel

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confirmed by the Blood Bank Center.

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To prepare the CS, Gel and CS-Gel scaffolds incorporated with PG, the fully dried CS, Gel and CS-Gel scaffolds were suspended in platelet solution (prepared by dissolving the platelet powder

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into sterile PBS) to absorb the platelets. After absorption of the plateletby the scaffolds, the

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platelet-scaffold composites were suspended in platelet activator solution (5000 U bovine

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thrombin + five ml of 10% CaCl2 ) [23]. As a result, PG was formed inside the scaffolds and the PG embedded CS (CS-PG), Gel (Gel-PG) and CS-Gel-PG scaffolds were fabricated. The

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scaffolds were then stored at -20 ºC for 24 h, freeze-dried at -80 ºC and pressure of 1 mBar for

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48 h, and placed in sterile packs until further use.

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2.2. In vivo biodegradation analysis of scaffolds through subcutaneous implantation The analysis was performed on five mature Sprague-Dawley rats. After anesthesia of the animals, the fabricated cylindrical scaffolds (CS, Gel and CS-Gel) with dimensions of 10 mm in length and 5 mm in diameter were implanted subcutaneously into the lower back of the rats. The incision sites were then closed in a routine fashion. 2.3. Quantitative real-time RT-PCR

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ACCEPTED MANUSCRIPT Total mRNA was extracted from 3D cultured bone marrow mesenchymal stem cells (BMSCs cellular scaffolds: CS, Gel, CS-PG, Gel-PG, CS-Gel, and CS-Gel-PG), using RNeasy Micro Kit (Qiagen-74004). The cDNA was synthesized from total RNA, using RevertAidTM first strand cDNA Synthesis Kit (Fermentas,

k1632) according to

the manufacturer’s instructions.

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Osteocalcin (OCN), collagen type 1 (Col1), alkaline phosphatase (ALP), and runt-related

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transcription factor 2 (Runx2) mRNA levels as osteogenic markers and CD31, vascular

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endothelial growth factor receptor 2 (VEGFr2) and angiogenic differentiation markers, were measured by real-time RT-PCR (Applied Bio-systems life technologies; ABi step one plus real-

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time PCR system). The 20-μL Q-RT PCR reaction contained 25 ng cDNA from each sample

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mixed with 10 μL SYBR® Green Master Mix (Applied biosystems life technologies, Inc, REF 4367659), 10 pmol of each forward and reverse primer and 6 μL RNase/DNase-free water. The

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Q-RT PCR Thermal conditions were: Pre-heating stage at 95 ˚C for 10 min followed by cycling

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stage: 95 ˚C for 15 sec and at 60 ˚C for 60 sec, 40 cycles. The gene expression levels of target genes: OCN, Col1, CD31, VEGFR2, ALP and Runx2 were determined based on the threshold

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PCR cycle-values (Ct) following the instructions of Applied Bio-systems. The relative

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quantification was performed, using comparative CT method (also known as the 2 -ΔΔCt method), where the amount of target genes normalized to an endogenous control (B2M) and relative to

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calibrator group (negative control group). All reactions were performed in duplicates and all samples were collected in three biological replicates. The specific primers designed for target genes are listed in Table 1.

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ACCEPTED MANUSCRIPT 2.4. Animals and Surgical Procedures This experimental study was approved by the local Ethics Committee of “Regulations for using animals in scientific procedures” in School of Veterinary Medicine, Shiraz University, Iran. The animals received human care in compliance with the Guide for Care and Use of Laboratory

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Animals published by the National Institutes of Health (NIH publication No. 85-23, revised 1985). Forty adult 8-week-old, male Sprague-Dawley rats, weighing 250 ± 25 g used in this

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study had full access to standard food and water ad libitum. They underwent general anesthesia

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via intramuscular injection of Ketamine hydrochloride 10% (50 mg/kg), Xylazine 2% (2 mg/kg) and Acepromazine maleate (1 mg/kg) (all purchased from Alfasan Co., Woerden, Netherland).

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Under aseptic condition, 2-cm incisions were made over both forelimbs of each animal and the

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radial bones were exposed. Bilateral 5 -mm osteotomies were then made in the diaphysis of the

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radii of both sides using electrical bone saw (Strong Co., Seoul, South Korea). The bone defects were randomly divided into eight equal groups (n=10defects/group). The

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defects in group 1 (untreated or defect group) were left untreated and empty. In group 2

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(autograft group), they were implanted with cortico-medullary autografts, which were provided from the harvested bone segments of the contralateral side in the same animal. In groups 3-8, the

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defects were implanted with CS, Gel, CS-PG, Gel-PG, CS-Gel and CS-Gel-PG scaffolds having the size and shape similar to the radial bone defects in this study (dimensions=2 × 2 × 5 mm3 ). Finally, the soft tissues and skin were sutured in a routine fashion. Post-operative analgesia and antibiotic therapy were provided by IM administration of flunixin meglumine (Razak Co., Tehran, Iran; 2.5 mg/kg) and enrofloxacin (Enrofan 5%, Erfan, Tehran, Iran), respectively for a 5-day period. Because of the fibro-osseous union between the ulna and radius and supportive role of the intact ulna, fixation of radial bones was not performed [18]. 9

ACCEPTED MANUSCRIPT 2.5. Clinical Examination The animals were evaluated for their clinical behavior, weight bearing and physical activity after surgery. In addition, edema, hyperemia and swelling of the defect areas were blindly assessed by

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two veterinary surgeons.

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2.6. Euthanasia

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Eight weeks after the operation, the rats were euthanized and the radius-ulna complexes were harvested [26]. Firstly, the rats were anesthetized and they were then euthanized by intra-cardiac

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injection of 1 mg/kg Gallamine triethiodide (Specia, Paris, France).

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2.7. Macroscopic Evaluation

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After euthanasia, the radial bone defects were evaluated for macroscopic signs of bone healing and blindly scored as follows: no union or instability at the defect site (0 score), incomplete

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union with the presence of soft tissue (+1) or cartilage (+2) within the defect, and complete union

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and presence of bridging bone (+3 score) [24,27].

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2.8. Diagnostic imaging evaluation

At the 2nd, 5th and 8th weeks after bone injury, lateral radiographs were provided from the radial

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bone defects of the anesthetized animals. To evaluate the degree of bone healing in the injured bones, each radiograph was blindly scored by two orthopedic surgeons [24,25]. In addition, the bone specimens were scanned using an Inveon TM unit (Siemens Healthcare, Inc., PA, USA) at sections with 0.06 mm thickness. The images of the newly formed bones and their gross profiles were reconstructed three-dimensionally via Inveon Research Workplace

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ACCEPTED MANUSCRIPT software (Siemens Healthcare Inc., PA, USA). Bone volume (%) of the specimens was then calculated from the images. 2.9. Histological and Ultrastructural Evaluations

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The bone samples (n=5/group) were fixed in neutral 10% buffered formalin and decalcified with

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15 % formic acid. They were then dehydrated in graded ethanol, embedded in paraffin, 5-µm

number

of

inflammatory

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thickness sections were provided and stained with hematoxylin and eosin. Cell counts including cells,

fibroblasts+fibrocytes,

chondroblasts+chondrocytes,

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osteoblasts+osteocytes, and osteoclasts were performed by digital morphometry, using Adobe Photoshop CC (extended version, Adobe Corp; CA). Furthermore, other constituents such as

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osteons, blood vessels and the density (%) of fibrous connective tissue (FCT), cartilaginous (CT)

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and osseous (OT) tissues were calculated and analyzed. A magnification of × 400 was employed

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for counting the cells.

For SEM analysis, the bone samples were fixed in cold 2.5% GA, dehydrated in graded series of

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ethanol and coated by gold. Using an SEM (S360, Cambridge, London, UK), high-qualified

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images were created. Quality and degree of repair of all the bone defects were examined and some tissue constituents such as collagen fibers and fibrils, degree of the matrix calcification,

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and presence of the hydroxyapatite (HA) crystals were evaluated. 2.10. Biomechanical Evaluation The radius-ulna complexes (n=5/group) were maintained in PBS-soaked gauzes at -20 ºC to perform the biomechanical testing as previously described [25,27]. Three-point bending test was performed, using a universal tensile testing machine (Instron, London, UK). The bone samples were placed horizontally on two supporting bars at a distance of 16 mm. The third bar was then 11

ACCEPTED MANUSCRIPT lowered to the middle of the diaphysis at a rate of 5 mm/min until fracturing. The biomechanical properties including maximum load, stiffness, strain, and stress were calculated from the loaddeformation and stress-strain curves and expressed as Mean ± standard deviation (SD).

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2.11. Statistical Analysis

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The quantitative data were expressed as mean ± SD and analyzed by one-way ANOVA with

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subsequent Tukey’s post-hoc tests. The scored values were statistically analyzed, using KruskalWallis H test, non-parametric ANOVA, and if they were significant by Mann-Whitney U test. A

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P<0.05 was considered to be statistically significant. Statistical analyses were performed using

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SPSS software, version 16.0 (SPSS, Inc, Chicago, USA).

3.1. In vivo biodegradation of scaffolds

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3. Results

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To evaluate in vivo biodegradation characteristics of different scaffolds the geometry of each

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scaffold was monitored on the 7th and 30th post-operative days. After 7 days, the length and diameter (Fig 1a and b, respectively) of the CS-Gel-PG (Fig. 2A), Gel-PG (Fig. 2B), and CS-PG

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(Fig. 2C) scaffolds were decreased, while the differences were not significant. Thirty days after

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implantation, the length and diameter of the scaffolds (Fig. 1a and b, respectively) were significantly decreased in comparison to the first week post-operation (P<0.05) (Fig 2. A', B', and C'). In addition, reduction in the dimension of the CS-Gel-PG scaffold (Fig. 2B') was significantly greater than the CS-PG scaffold (Fig. 2A') (P=0.039). During

the

first

week

after

implantation

of

different

scaffolds,

some

degenerating

polymorphonuclear leukocytes such as neutrophils or reactive lymphocytes were observed in the 12

ACCEPTED MANUSCRIPT exudates (Fig. 2a; thin arrow). Moreover, some parts of scaffold were replaced with soft connective tissue (Fig. 2a and b; thick arrows). Thirty days after surgery, the inflammatory response was significantly decreased. The porous structure of scaffolds was fills with the host tissues (Fig. 2c and d; thick arrows) and the process of angiogenesis was initiated (Fig. 2, A' and

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d; thin arrows). Overall, no obvious adverse chronic inflammatory responses was found in the H

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& E staining of different scaffolds after 30 days of implantation and the histopathological

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evaluation showed normal wound healing process in response to the fabricated scaffolds.

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3.2. Quantitative Real-time PCR

The BMSCs were seeded on the scaffolds for 30 days, and real-time PCR was performed to

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analyze the osteogenic gene expression. The exposure of BMSCs to the CS-Gel-PG scaffold

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resulted in an increase of VEGFr2 (Fig. 3a) and ALP (Fig. 3e) mRNA expression compared with

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the CS and Gel-PG scaffolds (P<0.05). Similarly, the ALP mRNA was also expressed higher in the Gel, CS-PG and CS-Gel scaffolds than those in the CS and Gel-PG ones (P<0.05).

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Regarding CD31 (Fig. 3b) and OCN (Fig. 3d), BMSCs cultured in the CS-Gel-PG followed by

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Gel scaffolds were expressed higher than those in other scaffolds (P<0.05). The Runx2 mRNA expression (Fig. 3c) decreased after exposure of all the scaffolds, while they showed no obvious

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difference (P˃0.05). For Col1 (Fig. 3f), it was expressed higher in the CS-Gel-PG followed by the Gel and CS-PG ones in comparison to other scaffolds (P<0.05). Together, these gene expression results demonstrated that the CS-Gel-PG scaffold enhanced the expression of osteogenic-related genes. 3.3. Clinical Evaluations

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ACCEPTED MANUSCRIPT None of the animals died during the course of the experiment and they had normal appetite, physical activity and weight bearing and weight gain At the first two weeks after bone injury, the CS and Gel-PG treated groups showed severe inflammatory signs including hyperemia, edema and swelling at the defect site which gradually diminished to normal status after 4-5 weeks. The

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animals treated with Gel, CS-PG and CS-Gel had moderate inflammatory signs, but the signs

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subsided to the normal level after 3-4 weeks. Mild inflammatory signs were seen in the autograft

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and CS-Gel-PG treated animals, while the sings were disappeared at the 2nd or 3rd weeks after operation. The edges of defect areas in the untreated group were palpable under digital palpation.

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A tissue with soft consistency was palpated in the defect areas treated with CS and Gel-PG,

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while the Gel, CS-PG, and CS-Gel treated defects were filled with tissues having semi-firm consistency. In contrast, the defect areas in the autograft and CS-Gel-PG groups were palpated as

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hard tissues with uniform firm consistency and both bone edges were not palpable.

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3.4. Gross Pathology

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In addition to the CS scaffold that was almost present in the defect area, eight weeks after

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surgery, the autograft, CS-PG, Gel-PG and CS-Gel scaffolds were not completely degraded and few remnants of the scaffolds existed after eight weeks (Fig. 4). The untreated defects were filled

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with a soft tissue similar to subcutaneous fascia so that no bone union was observed after eight weeks. In contrast, the defects treated with autograft and CS-Gel-PG were filled with a bonnylike hard tissue, either cartilage, bone or their mixture, and bone union seemed to be achieved over 75%. In the CS and Gel-PG groups, the defect sites were mostly replaced with soft tissue and bone union was seen in none of the samples. New tissues with variable consistency with different nature from fibrous- to cartilaginous-like tissues filled the defect sites in the Gel, CS-

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ACCEPTED MANUSCRIPT PG, and CS-Gel groups and the healing occurred similarly in these groups at the macroscopic

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level.

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ACCEPTED MANUSCRIPT Macroscopic scores related to each group on the basis of the type of newly formed tissue that filled the defect sites after eight weeks are available in Table 2. The autograft group achieved the highest macroscopic scores among other groups (P<0.05) excluding the CS-Gel-PG group

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this stage, the defects treated with the Gel, CS-PG and CS-Gel scaffolds had higher scores

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compared with the untreated and CS treated defects (P<0.05). In addition, the macroscopic

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scores in the CS-Gel-PG group were significantly greater than the untreated, CS, and Gel-PG

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groups (P=0.009, 0.015 and 0.018, respectively).

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3.5. Results of diagnostic imaging

Table 2 and Fig. 4 present the results achieved by the radiographic examination after two, five

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and eight weeks of bone injury. The radiographic scores in Table 2 reveal that bone formation

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and union of the autograft group was significantly superior to other groups at the 2 nd and 5th

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weeks (P˂0.05). In addition, such significant superiority to other groups with the exception of the CS-Gel-PG group existed in the 8th week (P˂0.05). At the 5th week, the CS-Gel-PG group

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had more significant bone union compared to the defect group (P=0.016). There were significant differences between radiographic scores of the Gel, CS-PG and CS-Gel groups with the defect

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group at the 8th week (P=0.039, 0.048 and 0.045, respectively). Additionally, the CS-Gel-PG

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group had more bone formation and union than the untreated, CS and Gel-PG groups (P=0.011, 0.017 and 0.032, respectively). After eight weeks, the defect sites in the autograft and CS-GelPG groups were radiopaque, whereas those in the CS and Gel-PG groups were radiolucent. , Over 75% and 50-75% of the defect areas in the autograft and CS-Gel-PG groups, respectively, were filled with newly regenerated bone after 8 weeks. Bone formation and union were less than 25% in the untreated, CS and Gel-PG groups, while the CS-Gel, CS-PG and Gel groups showed 25-50% bone formation. Moreover, none of the groups achieved complete union and remodeling. 17

ACCEPTED MANUSCRIPT The results achieved from the CT-scan evaluation have been reported as percentage of bone volume for all the radial bone defects after eight weeks of bone injury and these results are presented in Figs. 4 and 5. The bone volume, as an index of bone formation, was significantly higher in all the treated groups when compared with the untreated group, eight weeks after

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surgical operation (P˂0.05). Bone formation in the autograft and the CS-Gel-PG treated groups

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was significantly higher than other groups (P˂0.05). In addition, the bone volume of the

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autograft treated defects was significantly superior to the defects treated with the CS-Gel-PG composite scaffolds (P=0.025). There was no significant difference in the percentage of bone

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volume in the Gel, CS-PG and CS-Gel groups.

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3.6. Histopathologic and Histomorphometric Findings

Based on Fig. 6, minimum or no healing was observed in the untreated defects after eight weeks.

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A soft connective tissue consisting of loose areolar or fibrous connective tissue with randomly

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oriented low density collagen fibers and blood vessels filled the defect site of the untreated group. The implanted autograft was partially degraded after eight weeks so that the degraded

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parts were replaced with woven bone in the bone edges. The newly formed woven bones were

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connected with each other by a mixed tissue consisting of hyaline cartilage and dense connective tissue. The most proportion of CS scaffold remained intact and the residual particles were

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encapsulated by fibrous connective tissue infiltrated by numerous inflammatory cells . A mixed fibro-cartilaginous tissue was seen in other parts of the defect site in this group. In addition, there were few small islands of the newly formed bony matrix near the bone edges. A nonhomogenous matrix composed of fibrocartilage, hyaline cartilage and woven bone was observed in the defects treated with the Gel scaffold. The Gel scaffold was completely degraded and no remnants of this scaffold were seen. The newly formed bone was seen near the bone edges and 18

ACCEPTED MANUSCRIPT the cartilage and fibrous connective tissues were the dominant tissues in the middle part of the defect areas. Small particles of the CS-PG, CS-Gel and Gel-PG scaffolds remained in the defect areas after eight weeks.

These scaffolds’ remnants were surrounded by mononuclear

inflammatory cells and dense connective tissue. The defects in the CS-PG and CS-Gel groups

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were replaced with a mixture of fibrocartilage tissue, hyaline cartilage and woven bone from

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both the radial bone edges toward the central parts. In general, histopathologic features of the

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defects treated with CS-PG and CS-Gel were close to those treated with the Gel scaffold. However, the inflammatory reaction was more severe in the CS-PG and CS-Gel compared with

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the Gel group (P=0.034 and 0.025, respectively). The Gel-PG scaffold was mostly replaced with

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a non-homogenous fibrocartilaginous tissue and few newly formed bonny islands. Small remnants of scaffold particles were still present in the defect area and triggered a mild

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inflammatory reaction.

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The scaffold in the CS-Gel-PG treated defects was completely degraded and the newly

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regenerated bone was dominant in both bone edges and periosteum of the ulnar cortex with no remarkable inflammatory reaction. In this group, some ossified foci were present within the

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cartilaginous zone, reflecting the continuity of bone formation (Fig. 6).

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ACCEPTED MANUSCRIPT According to the histomorphometric findings in Table 3, all the treated groups had significantly higher number of chondroblasts + chondrocytes and fewer fibroblasts + fibrocytes when compared to the untreated defects (P<0.05). The CS group had significantly higher inflammatory cells compared with other treated and untreated groups (P<0.05). The CS-Gel-PG treated defects

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had significantly higher number of chondroblasts + chondrocytes, osteoblasts + osteocytes, and

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osteons and had lower number of fibroblasts + fibrocytes than other groups with the exception of

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the autograft group (P<0.05). Similar to the autograft, the number of osteoclasts and osteons in the CS-Gel-PG group was significantly superior to the defect, CS and Gel-PG groups (P<0.05).

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In addition, the number of osteons in the CS-Gel-PG group was significantly higher than the Gel,

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CS-PG and CS-Gel groups (P=0.032, P=0.025 and P=0.017, respectively).

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Moreover, the density of FCT, CT and OT as percentage are available in Table 3. Based on these data, the autograft and CS-Gel-PG treated defects had the highest OT and CT density and the

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least FCT density compared with other untreated and treated groups (P˂0.05). However, the

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density of OT and CT of these two groups was not significantly different (P>0.05).

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3.7. Scanning Electron Microscopy

Only a loose areolar connective tissue consisting of haphazardly oriented low density collagen

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fibrils without HA crystals was seen under SEM in the untreated group (Fig. 7).Many HA crystals reflecting high degree of matrix calcification were visible in all parts of the defect site in the autograft group. A dense fibrocartilaginous tissue filled the defect area, in the CS and Gel-PG groups, whereas the Gel, CS-PG and CS-Gel groups had low calcified cartilaginous matrix with few HA crystals. However, there was fibrous connective tissue structure in some parts of the defect areas in these groups. In the CS-Gel-PG group, a hard callus and cartilaginous matrix with 21

ACCEPTED MANUSCRIPT large amounts of HA crystals and variable degrees of calcification were observed at different parts of the defect areas. 3.8. Biomechanical Findings

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The biomechanical data achieved from the three-point bending test are available in Fig. 8. The

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autograft group demonstrated significantly higher maximum load (N) compared with other

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groups (P˂0.05) excluding the CS-Gel-PG group. The defects in the CS-Gel-PG group could tolerate significantly higher load when compared to those in the untreated and Gel-PG groups

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(P=0.011 and P=0.047, respectively). Additionally, the Gel, CS-PG, and CS-Gel groups showed greater maximum load compared with the defect group (P=0.025, P=0.034 and P=0.039,

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respectively).

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There were significant differences among the autograft group with other groups excluding the

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CS-Gel-PG group in terms of strain (%) and maximum stress (N/mm2 ) (P˂0.05). The untreated defects had the highest strain compared with those of the autograft, CS-Gel-PG and Gel groups

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(P˂0.05). In addition, the CS and Gel-PG groups had significantly higher strain in comparison to

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the CS-Gel-PG group (P=0.049 and P=0.041, respectively). The maximum stress of the CS-GelPG, Gel, CS-PG and CS-Gel groups was greater than that of the untreated group (P=0.011,

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P=0.025, P=0.034 and P=0.039, respectively). Furthermore, the defects in the CS-Gel-PG group indicated higher stress than those in the Gel-PG group (P = 0.034). The autograft group had the greatest stiffness among all the groups (P˂0.05). The CS-Gel-PG treated defects were associated with significantly greater stiffness than the Gel-PG, CS and CS-PG groups (P˂0.05). In addition, the stiffness of all treated groups was significantly superior to that of the untreated group

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ACCEPTED MANUSCRIPT (P˂0.05). The Gel, CS-PG, and CS-Gel groups had higher stiffness than the Gel-PG group (P=0.032, 0.035, and 0.043, respectively).

4. Discussion

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The focus of this study was to understand the effect of incorporating PG within the CS-Gel composite scaffold on healing of bilateral critical-sized radial bone defects in rats after eight

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weeks. This study indicated that the self-healing capacity of the body was not able to regenerate

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such a critical sized bone defect and treatment with autologous bone graft was found to be the best method. The untreated bone defects and those defects treated with CS and surprisingly Gel-

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PG failed to be regenerated and the bone healing delayed in these groups. Other groups were

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associated with different promotion in new bone formation and union compared with the defect and CS groups. Although the Gel alone enhanced bone regeneration compared with the untreated

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defects, incorporation of PG to the Gel hydrogel did not promote bone healing response. It seems

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that the rate of bone regeneration and scaffold biodegradation was lower than the rate of growth factors release; therefore the remaining scaffold in the defect area physically impaired bone

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regeneration [1,3,14]. Indeed, Gel seems to be an impediment to the efficacy of growth factors in

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PG and could not function as a suitable carrier for controlled and prolonged release of the growth factors. Ishida et al. [28] promoted regeneration of meniscal defects by Gel hydrogels incorporated autologous PRP after 12 weeks, while the Gel-PG scaffold did not enhance bone healing after eight weeks in the present study. Contrary to our findings, Hokugo et al. [18] showed the effectiveness of Gel hydrogel incorporated with allogeneic PRP on enhancement of bone healing of ulnar defects in rabbits after 4 weeks. However, the PRP source (allogeneic vs.

23

ACCEPTED MANUSCRIPT human xenogeneic), method of PRP formation and platelet activation (contact with Gel molecules), type of the defect and the animal model in these two studies were different with ours. Chitosan had low biocompatibility and biodegradability as the scaffold remnants were severely surrounded by inflammatory cells, after 8 weeks. However, addition of Gel, PG or Gel-PG to the

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CS scaffolds improved degradation rate, and biomechanical properties, enhanced cartilage and bone formation, and reduced the inflammatory reaction. In line with us, Oktay et al. [29] showed

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that a CS sponge incorporated with autologous PRP enhanced regeneration in the rabbits’ cranial

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defects after 8 weeks, while the CS sponge alone resulted in remarkable fibrous connective tissue formation. In addition, consistent with our findings regarding efficacy of CS-PG scaffold

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compared with CS alone, Bi et al. [30] showed an injectable composite tri-calcium phosphate

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/CS incorporated with PRP resulted in proper bone formation, promising mechanical strength, biocompatibility and osteoinductive properties in healing of tibial bone defects in goats after 16

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weeks.

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Gelatin rapidly dissolves in an aqueous environment at body temperature and exhibits

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uncontrolled and fast release of growth factors [9], thus we combined negatively charged type B Gel with cationic CS biopolymer [9,31]. Therefore, such oppositely charged molecules could

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readily bind and form intermolecular interactions. Similarly, Huang et al. [18] showed composite CS-Gel scaffolds cross-linked by GA significantly affected the biological characteristics and cell attachment and enhanced the biomechanical properties and degradation rate of the CS biomaterial. Huang et al. [18] and Kim et al. [9] also suggested that formation of such poly-ionic complexes can provide a good medium for controlled release of proteins. Furthermore, it seems that when the growth factors with positive charge interact with negatively charged biomaterials,

24

ACCEPTED MANUSCRIPT the resultant poly-ionic complexation allows the proteins to be absorbed into the scaffold while maintaining their bioactivity [9,18], therefore we fabricated and tested CS-Gel-PG scaffold. The CS-Gel-PG scaffold was biocompatible and biodegradable so that it was completely degraded

with no

remarkable inflammatory reaction. In addition, it showed promising

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osteoinductive and osteoconductive potentials and the CS-Gel scaffold acted as a suitable carrier for PG and resulted in a significantly more newly regenerated mature bone compared with other

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groups. In several studies, the CS-Gel composite scaffolds and also other biomaterials have

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successfully been used to deliver growth factors to enhance bone repair [10,11,32,33]. Kim et al. [9] developed a CS gel/Gel microsphere (MSs) dual delivery system for sequential release of

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BMP-2 and insulin-like growth factor (IGF)-I in vitro to enhance osteoblast differentiation. They

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showed that encapsulation of the Gel MSs into the CS positively affected the release of IGF-I and BMP-2 over a week by generating intermolecular interactions. Similar to our study, Lu et al.

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[32] loaded a CS-Gel sponge cross-linked by tannins (CSGT) with autogenous PRP for healing

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of skin wounds. They showed that the wounds covered with CSGT and PRP healed more quickly than those covered with CS-Gel, CS or Gel. Moreover, addition of PRP to the CSGT accelerated healing more effectively. They showed cross-linking improved the stability and

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wound

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mechanical properties of the sponge. The osteogenic differentiation process of BMSCs is usually divided into three discrete stages including commitment to osteogenic lineage, synthesis of bone matrix, and matrix mineralization [34]. Previous studies have shown that Runx2 is an essential transcriptional activator for osteogenic lineage commitment and a critical regulator of differentiation of chondrocytes and osteoblasts [35,36]. The Runx2 gene expression decreased in all experimental groups when compared to the 2D culture of BMSCs at day 30. This result might result from its gene 25

ACCEPTED MANUSCRIPT expression reduction over time [35]. Some studies have found that ALP plays important role in osteoid formation and bone mineralization and it is a marker of osteoblast differentiation [35,36]. The highest expression of ALP found with the CS-Gel-PG scaffold was consistent with its high mineralization progression and bone formation in the defect sites in vivo. On the other hand,

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OCN and Col1 are essential bone proteins produced by osteoblasts [36,37]. The OCN mRNA

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expression is used as a late marker of osteogenic differentiation [38] which was significantly

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increased with the CS-Gel-PG scaffolds. Similarly, increased expression of Col1 as the main organic component of bone ECM, with the CS-Gel-PG scaffold was confirmed by promoted new

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bone formation in this group. One of the most important and essential growth factors involved in

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bone healing process is VEGF which is present in alpha granules of platelets and results in angiogenesis induction especially at early phase of regeneration [39]. Similar to CD31, increased

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mRNA expression of the angiogenic VEGF gene showed that the CS-Gel-PG scaffold had

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controlled and prolonged release of growth factors until day 30 and it resulted in an optimum bone healing quality. Overall, our results were associated with increased levels of ALP, Runx2,

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VEGF, OCN, Col1, and CD31 measured in vitro by real time-PCR and they indicated that the

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other scaffolds.

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CS-Gel-PG scaffold stimulated osteoblast differentiation in a suitable manner compared with

5. Conclusions The present study suggests that the CS-Gel scaffold incorporated with PG enhances bone regeneration and mRNA expression of ALP, OCN, Col1, CD31, Runx2 genes and VEGF. Therefore, due to the role of growth factors in osteoblast differentiation and bone formation 26

ACCEPTED MANUSCRIPT along with high in vivo degradability of CS-Gel-PG, such an implant can be introduced as a promising option to substitute autologous bone grafting. In can be concluded that the CS-Gel scaffold rather than CS or Gel alone is a promising scaffold for PG loading; therefore, the composite CS-Gel-PG scaffold can assist bone regeneration.

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Acknowledgment

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The authors would like to thank the authorities of the Veterinary School, Shiraz University for their support and cooperation.

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Conflict of Interest: The authors declare that they have no conflict of interest.

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ACCEPTED MANUSCRIPT Figure legends Fig. 1. Length (a) and diameter (b) of different scaffolds after 7 and 30 days of subcutaneous implantation. Fig. 2. In vivo degradation of CS, Gel, and CS/Gel scaffolds after 7 and 30 days of subcutaneous

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implantation. Skin layers (a and c; star), scaffold (arrowheads), host tissue (a, b, c and d; tick

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arrows), angiogenesis (A' and d; thin arrows).

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Fig. 3. Effects of the scaffolds on mRNA expression of VEGFr2 (a), CD31 (b), Runx2 (c), OCN (d), ALP (e), and Col1 (f) on day 30. It was shown that the CS-Gel-PG scaffold could promote

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expression of all the genes were expressed higher compared with the control group and other

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scaffolds except with Runx2. CS: Chitosan, Gel: Gelatin; PG: Platelet gel; Runx2: Runt-related transcription factor 2; OCN: Osteocalcin; ALP: Alkaline phosphatase; Col: Collagen type 1;

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VEGFr2: Vascular endothelial growth factor receptor 2.

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Fig. 4. Macroscopic and diagnostic imaging of the radial bone defects after eight weeks of injury. The untreated defects were filled with soft tissue or still remained empty. Hard cartilage

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or bone tissues filled the defects in the autograft and CS-Gel-PG groups. The defects in other

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groups were filled with variable soft to hard tissues. The autograft group showed higher radio-opacity after two and five weeks than other groups

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excluding the CS-Gel-PG group at the 8th week. At this stage, the CS-Gel-PG group showed higher radiologic scores than the untreated, CS and Gel-PG groups (P=0.009, 0.018 and 0.025, respectively). After eight weeks, the autograft followed by the CS-Gel-PG group had the highest percentage of bone volume (P<0.05). Other treated groups had greater bone volume than the defect group (P˂0.05). 35

ACCEPTED MANUSCRIPT Fig. 5. Bone volumes of the healed radial bone defects at the 8th week after injury. The untreated defects had the least bone volume (%) compared with the treated groups (P˂0.05), while it was the highest in the autograft and CS-Gel-PG groups (P˂0.05). Fig. 6. Longitudinal histopathological sections of the radial bone defects in rats after eight weeks

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of bone injury. There are still some remnants of the CS, CS-PG, Gel-PG scaffolds, while CSGel-PG was completely degraded and replaced mostly with woven bone and fibrocartilage

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tissue. Moreover, the implanted autograft was still visible at the defect site. There was minimal

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healing and regeneration in the untreated (defect) group. CS: chitosan; Gel: gelatin; PG: platelet gel; LACT: loose areolar connective tissue; FCT: fibrous connective tissue; RBE: radial bone

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edge; BV: blood vessel; BM: bone marrow; DCT: dense connective tissue; CZ: cartilaginous

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zone, CCT: calcified cartilaginous tissue; HC: hyaline cartilage; WB: woven bone; R: remnants

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of scaffold; IC: inflammatory cells; UB: ulnar bone; O: osteon. Stained with H&E. Fig. 7. Colored scanning ultra-micrographs of the radial bone defects after eight weeks of injury.

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Collagen fibrils filled the defects in the untreated group (a), while those in the autograft group

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were filled with a calcified bone matrix composed of large number of HA crystals (b). Although the defects in the CS (c) and Gel-PG (e) groups were replaced with a fibrocartilage matrix, those

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in the Gel (d), CS-PG (f), and CS-Gel (g) groups were filled with cartilaginous tissue and few HA crystals. The defects in the CS-Gel-PG group were filled with a highly calcified cartilaginous and bone matrix with HA crystals (h). CF: collagen fibrils; CT: connective tissue; HCM: highly calcified matrix; HC: hydroxyapatite crystals; FCT: fibro-cartilaginous tissue; LCM: low calcified matrix.

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ACCEPTED MANUSCRIPT Fig. 8. Biomechanical properties of the healed radial bone defects after eight weeks of injury. These properties included maximum load (a), strain (b), stress (c) and stiffness (d) for all the

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ACCEPTED MANUSCRIPT Table1. Primer sequences used in quantitative real time-PCR Primer sequence

S ize (bp)

Gene bank code

Runx2

F:GGACGAGGCAAGAGTTTCAC R: GAGGCGGTCAGAGAACAAAC

165

NM _053470.2

Annealing Temperature (˚C) 60

OCN

F: GAGGGCAGTAAGGTGGTGAA R: GTCCGCTAGCTCGTCACAAT F:TACACTTATTTATGAACCAGCCCT R: TCTGCACACCCAACATTAACA F: GCACAACATCAAGGACATCG R: TCAGTGCGGTTCCAGACATA F:GAATATGTATCACCAGACGCAG R: AGCAAAGTTTCCTCCAAGAC F: CCCAGAAATGTACCAAACCA R: ACTTCCTCTTCCTCCATACAG

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NM _013414.1

60

105

NM _031591.1

60

195

NM _013059.1

60

186

NM _053304.1

225

NM _001106088.2

ALP Col1 VEGFr2

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CD31

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Gene

60 60

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Runx2: Runt-related transcription factor 2; OCN: Osteocalcin; ALP: Alkaline phosphatase; Col: Collage n type 1; VEGFr2: Vascular endothelial growth factor receptor 2.

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ACCEPTED MANUSCRIPT Table 2. Radiographical data related to the healed radial defects on 2nd , 5th and 8th weeks and macroscopic scores after 8 weeks of bone injury

T ype evaluation Radiology, W2 Radiology, W5 Radiology, W8 Macroscopic union *

Defect (1) Median (min - max) 0 (0-0) 1 (0-1) 1 (0-2) 1 (0-1)

Autograft (2) Median (min - max) 2 (1-3) b 4 (3-5) c 5 (4-7) e 3 (2-3) j

CS (3) Median (min - max) 0 (0-1) 1 (0-1) 2 (1-2) 1 (0-2)

Gel (4) Median (min - max) 0 (0-1) 2 (0-2) 3 (1-4) f 2 (1-3) k

CS-PG (5) Median (min - max) 0 (0-1) 1 (0-2) 2 (1-4) g 1(1-3) l

Gel-PG (6) Median (min - max) 0 (0-1) 1 (1-2) 1 (1-3) 1 (1-2)

CS-Gel (7) Median (min - max) 0 (0-1) 1 (1-2) 3 (1-3) h 2 (1-2) m

CS-Gel-PG (8) Median (min - max) 1 (0-1) 2 (1-3) d 3 (2-5) i 2 (2-3) n

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CS: Chitosan; Gel: Gelatin; PG: Platelet gel Complete union (+ 3 score), presence of cartilage (+ 2 score), presence of soft connective tissue (+ 1 score), nonunion (0 score)

Kruskal-Wallis non-parametric ANOVA; b and c P˂0.05 (2 vs. 1, 3, 4, 5, 6, 7, and 8); d P=0.016 and 0.32 (8 vs. 1 and 3); e P˂0.05 (2 vs. 1, 3, 4, 5, 6 and 8); f P=0.039 (4 vs. 1); g P=0.048 (5 vs. 1); h P=0.045 (7 vs. 1); and i P˂0.05 (8 vs. 1, 3, 6); j P˂0.05 (2 vs. 1, 3, 6 and 7); k P=0.016 (4 vs. 1); l P=0.034 (5 vs. 1); m P=0.032 (7 vs. 1); n P=0.009, 0.018 and 0.020) by M annWhitney U test

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Pa 0.006 0.002 0.002 0.003

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Table 3. Histomorphometric findings of healed bone defects after 8 weeks of injury Def ect (1) Mean ± SD

Value (number)

193.60 ± 15.52

Chondroblast + chondrocyte

5.40 ± 1.14 i

b

53.00 ± 6.82

CS (3) Mean ± SD c

G el (4) Mean ± SD

114.40 ± 12.30

131.40 ± 12.12 j n

d

28.20 ± 7.32 k

91.80 ± 5.07

CS-PG (5) Mean ± SD e

64.80 ± 8.61

96.40 ± 7.13

G el-PG (6) Mean ± SD f

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p

g

101.00 ± 8.46

h

CS-G el-PG (8) Mean ± SD

Pa

78.80 ± 8.26

0.000

87.80 ± 6.50 m

62.00 ± 7.28 q

0.000

0.00

72.00 ± 11.07

3.40 ± 1.52

36.80 ± 6.65

11.60 ± 2.7

31.20 ± 4.97

Osteoclast (n)

0.00

1.60 ± 0.55 s

0.00

1.00 ± 0.71

1.00 ± 0.71

0.00

0.60 ± 0.55

1.20 ± 0.84 t

0.001

Inflammatory cells

8.60 ± 2.41 u

19.80 ± 4.15

58.80 ± 9.34 v

16.20 ± 3.70

32.00 ± 5.10 w

23.60 ± 4.93 x

39.60 ± 5.46 y

13.40 ± 3.85

0.000

Blood vessels

16.40 ± 2.41 z

4.60 ± 2.41 ab

9.60 ± 2.41

10.00 ± 1.58

8.40 ± 2.97

11.20 ± 3.27

7.80 ± 2.39

7.20 ± 1.92

0.001

Osteon

0.00

6.40 ± 2.07 ac

0.00

2.60 ± 0.55 ad

2.10 ± 1.14 ae

0.00

1.80 ± 0.55 af

4.40 ± 1.14 ag

0.000

Density of FCT (%)

97.31 ± 0.37

20.48 ± 1.31

71.33 ± 4.43

42.30 ± 3.40

46.54 ± 2.84

66.27 ± 3.77

49.86 ± 3.50

31.97 ± 2.44

0.000

Density of CT (%)

2.69 ± 0.37

51.00 ± 4.36

23.36 ± 5.23

37.28 ± 3.67

35.08 ± 3.52

27.38 ± 2.94

33.75 ± 2.10

44.01 ± 1.64

0.000

Density of OT (%)

0.00

28.52 ± 3.67

5.31 ± 0.97

20.42 ± 2.97

18.37 ± 3.74

6.35 ± 0.95

16.38 ± 2.89

24.02 ± 2.55

0.000

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57.40 ± 6.51

r

Osteoblast + osteocyte

CR

33.00 ± 6.04

120.60 ± 9.34 49.80 ± 5.54 l

63.20 ± 8.62

CS-G el (7) Mean ± SD

IP

Fibroblast + fibrocyte

Autograf t (2) Mean ± SD

CS: Chitosan; Gel: Gelatin; PG: Platelet gel; SD: Standard deviation; FCT: Fibrous connective tissue; CT : Cartilaginous tissue; OT :

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One-way ANOVA followed by Tukey post-hoc test; b and i P˂0.01 (1 vs. 2, 3, 4, 5, 6, 7, and 8); c and j P˂0.05 (2 vs. 3, 4, 5, 6, 7, and 8); d and g P˂0.05 (3 and 6 vs. 4, 5, 7 and 8); e P=0.032 (4 vs. 8); f P=0.018 (5 vs. 8); h P=0.012 (7 vs. 8); i P˂0.05 (1 vs. 2, 3, 4, 5, 6, 7, and 8); j P˂0.05 (2 vs. 3, 4, 5, 6, 7, and 8); k P˂0.05 (3 vs. 4, 5, 6, 7 and 8); l P˂0.01 (6 vs. 4, 5, 7, and 8); m P˂0.01 (8 vs. 4, 5 and 7); n P˂0.01 (2 vs. 1, 3, 5, 6, and 7); o P˂0.05 (4 vs. 1, 3 and 6); p P˂0.05 (5 vs. 1, 3 and 6); q P˂0.05 (7 vs. 1, 3 and 6); r P˂0.01 (8 vs. 1, 3, 4, 5, 6, and 7); s P=0.000 (2 vs. 1, 3 and 6); t P=0.000 (8 vs. 1, 3 and 6); u P˂0.05 (1 vs. 2, 3, 4, 5, 6, and 7); v P=0.009 (3 vs. 2, 4, 5, 6, 7, and 8); w P˂0.01 (5 vs. 2, 4 and 8); x P˂0.01 (6 vs. 5, 7 and 8); y P=0.018 and 0.009 (7 vs. 4 and 8); z P˂0.05 (1 vs. 2, 3, 4, 5, 6, 7 and 8); ab P˂0.05 (2 vs. 3, 4, 5, 6, and 7); ac P˂0.05 (2 vs. 1, 3, 5, 6, and 7); ad P=0.000 (4 vs. 1, 3 and 6); ae P=0.000 (5 vs. 1, 3 and 6); af P=0.000 (7 vs. 1, 3 and 6); ag P˂0.05 (8 vs. 1, 3, 4, 5, 6, and 7)

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