Gingival mesenchymal stem cells as an alternative source to bone marrow mesenchymal stem cells in regeneration of bone defects: In vivo study

Gingival mesenchymal stem cells as an alternative source to bone marrow mesenchymal stem cells in regeneration of bone defects: In vivo study

Journal Pre-proof Gingival Mesenchymal Stem Cells as an Alternative Source to Bone Marrow Mesenchymal Stem Cells in Regeneration of Bone Defects: in v...

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Journal Pre-proof Gingival Mesenchymal Stem Cells as an Alternative Source to Bone Marrow Mesenchymal Stem Cells in Regeneration of Bone Defects: in vivo study Gamilah Al-Qadhi, Malak Soliman, Iman Abou-Shady, Laila Rashed

PII:

S0040-8166(19)30374-X

DOI:

https://doi.org/10.1016/j.tice.2019.101325

Reference:

YTICE 101325

To appear in:

Tissue and Cell

Received Date:

4 September 2019

Revised Date:

16 December 2019

Accepted Date:

16 December 2019

Please cite this article as: Gamilah A-Qadhi, Malak S, Iman A-Shady, Laila R, Gingival Mesenchymal Stem Cells as an Alternative Source to Bone Marrow Mesenchymal Stem Cells in Regeneration of Bone Defects: in vivo study, Tissue and Cell (2019), doi: https://doi.org/10.1016/j.tice.2019.101325

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier.

Gingival Mesenchymal Stem Cells as an Alternative Source to Bone Marrow Mesenchymal Stem Cells in Regeneration of Bone Defects: in vivo study

Gamilah Al-Qadhia,*, Malak SolimanError! Bookmark not defined., Iman AbouShadyError! Bookmark not defined., Laila Rashedb

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Biochemistry and Molecular Biology Unit, Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Cairo University, Kasr El Aini, Cairo, Egypt. 1

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Oral Biology Department, Faculty of Dentistry, Cairo University, Mathaf-El-Manial Street,11553, Cairo, Egypt. b

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Biochemistry and Molecular Biology Unit, Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Cairo University, Kasr El Aini, Cairo, Egypt.

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Graphical Abstract

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* Corresponding author: Gamilah Abdulhak Al-Qadhi, PhD student, Address: Oral Biology Department, Faculty of Dentistry, Cairo University, Mathaf-El-Manial Street, Cairo, 11553, Egypt Email: [email protected] Phone number: 00201019140459

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Critical sized bone defects represent a significant issue in clinical and research fields.



Stem cell-based therapy has emerged as a promising tool to regenerate

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bony defects.



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Highlights:

The most widely used mesenchymal stem cell source is the bone marrow. However; recently, gingiva represents a potential easily accessible and effective source

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Combination of stem cells and nano scaffolds provides favorable results in bone regeneration.

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Abstract: Healing of critical sized bone defects represents a challenging issue in clinical and research fields. Current therapeutic techniques, such as bone grafts or bone grafts substitutes, still have limitations and drawbacks. Therefore, stem cell-based therapy provides a prospective approach to enhance bone regeneration. The

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present study aimed to assess the regenerative capacity of Gingival mesenchymal stem cells (GMSCs) as well as Bone marrow mesenchymal stem cells (BMSCs)

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loaded on NanoBone scaffold, in comparison to the unloaded one, in surgically

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created bone defects in rabbits' tibiae. To achieve this aim, critical sized bone defects, of 6-mm diameter each, were unilaterally created in tibiae of adult New

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Zeeland male white rabbits (n=27). The rabbits were then divided randomly into three groups (9 each) and received the following: Group I: Unloaded NanoBone

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Scaffold, Group II: GMSCs Loaded on NanoBone Scaffold, and Group III: BMSCs Loaded on NanoBone Scaffold. Three rabbits from each group were then

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sacrificed at each time point (2, 4 and 6 weeks postoperatively), tibiae were dissected out to evaluate bone healing in the created bony defects; both histologically and histomorphometrically. The findings of this study indicate that both GMSCs and BMSCs exhibited fibroblast morphology and expressed

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phenotypic MSCs markers. Histologically, local application of GMSCs and BMSCs loaded on NanoBone scaffold showed enhanced the pattern of bone regeneration as compared to the unloaded scaffold. Histomorphometrically, there was astatistically insignificant difference in the new bone area % between the bony

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defects treated with GMSCs and BMSCs. Thus, GMSCs can be considered as a comparable alternative source to BMSCs in bone regeneration.

Key words: Mesenchymal stem cells; Bone marrow; Gingiva; NanoBone scaffold;

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Critical sized Bone defects; Bone regeneration.

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1. Introduction

Large or critical-sized bone defects represent a relevant clinical issue

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in orthopedic and oral surgery practice (Nather et al., 2010). Bone defects

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may result from traumatic injury, bone infection, congenital malformation, tumor, pathological fracture, and as an outcome of surgical procedures (Bostrom and Mikos, 1997).

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Different approaches are available to enhance bone regeneration

process, such as bone grafts; autologous grafts (gold standard), allografts and xenografts, as well as bone graft substitutes which are based on growth

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factors, osteoconductive ceramic, polymer and osteoprogenitor cells (Laurencin et al., 2006; Dimitriou et al., 2011a). However, several complications were associated with these approaches such as risk of immune rejection and infection (Lord et al., 1988), reduction of cell number and osteoinductive properties of material (Keating and McQueen, 2001),

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harvest-site morbidity and limited available donor bone (Dimitriou et al., 2011b; Kumar and Narayan, 2014). Alternatively, stem cell-based therapy, especially bone marrow mesenchymal stem cells (BMSCs), has been recognized as a hopeful solution to overcome these limitations in bone regeneration. Numerous preclinical studies have been established to assess the ability of BMSCs to

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enhance the regeneration of critical-sized long bone defects. The finding

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revealed that BMSCs loaded on scaffold was more efficient in generating new bone than the scaffold alone (Giannoni et al., 2008; Xing et al., 2014;

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Li et al., 2014).

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Recently; gingiva-derived mesenchymal stem cells (GMSCs) were introduced as a new easily accessible source, capable of differentiation,

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self-renewal and immunomodulation as well as their anti-inflammatory properties (Zhang et al., 2009). Furthermore, when GMSCs and BMSCs

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were loaded on HA/TCP scaffold and implanted subcutaneously into immunocompromised mice, a highly mineralized tissue was observed in both groups 10 weeks post operatively (Tomar et al., 2010). In contrary to the previous study, Wang et al., 2011 declared that no

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or minimal amount of new bone was formed after 8 weeks in mandibular and calvarial defects receiving GMSCs seeded on type I collagen gel. Using small animal model (mice) and non-critical sized defect size,

human GMSCs were transplanted systematically via tail vein into mandibular bony defect. The difference in new bone formation between the

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GMSCs and control groups was statistically significant (p<0.05), indicating the potential effect of GMSCs on bone regeneration (Xu et al., 2014). Although GMSCs showed reduction in osteogenic differentiation capacity, transplanted

GMSCs

encapsulated

in

a

RGD-modified

alginate

microspheres were able to repair critical sized calvarial bone defects in immunecompromised mice (Moshaverinia et al., 2014).

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Regarding stem cells carrier, combination of stem cells with Nano

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scaffolds gives considerable results in bone regeneration; as it promotes bone formation via acting as a biological osteoconductive surface for cells

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and/or growth factors (Götz et al., 2008), enhances stem cell adhesion,

2014; Xia et al., 2014).

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spreading, proliferation and osteogenic differentiation (Wittenburg et al.,

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The utilization of in vivo studies in cell-based research is fundamental to turn the basic research into an applied one (Berner et al., 2011).

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Collectively, the animal model, surgical site, type and size of the bony defect, design of the scaffold, type of stem cells and duration of the experiment were taken into consideration while designing the current study. Rabbits are the most widely used animals in bone-related

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researches and are the best models for stem cells applications regarding their life span, number and type of stem cells that can be extracted from a single animal than those of rodents (Harding et al., 2013). Moreover, rabbit’s bone is composed of primary longitudinal bone tissue with areas of dense Haversian systems in which primary and

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secondary osteons, containing short vascular canals, are present (Martiniaková et al., 2003). This histological pattern somewhat resembles the histology of human bone. On the other hand; the absence of osteons was a typical feature of cortical bone microstructure in the rat (Martiniaková et al., 2005). In comparison to other strains, New Zealand white strains are less aggressive in behavior (Thomas et al., 2012). The previous facts led to

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the use of New Zealand white rabbits as a model in the current study.

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Regarding the surgical site, tibia is away from the chewing site and is protected against bacterial infection and trauma. Additional protection can

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be also achieved through double layers suturing of the soft tissue and skin,

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which accelerates bone turnover (Gholami et al., 2010). The unilateral defect model was appropriate in case of major

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procedures since the bilateral defect model may cause ethically unacceptable disability to the experimental animal. The 6 mm diameter was

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considered as a critical sized defect in tibia of rabbits in different studies (Calvo-Guirado et al., 2012; Delgado-Ruiz et al., 2014). The three evaluation periods (2, 4 & 6 weeks post-operatively) were selected in order to assess early events of the regenerative process.

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Based upon the former background, the efficacy of applying GMSCs

in regeneration of critical sized bone defects in vivo studies still needs more work and investigation. Therefore, the current study designed to assess the regenerative capacity of GMSCs as well as BMSCs loaded on NanoBone

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scaffold, in comparison to the unloaded scaffold in surgically created, critical-sized tibial bone defect in a rabbit model. This capacity was assessed through the following outcomes; formation of new bone (primary outcome) and amount of new collagen fibers (secondary outcome). Measurement of these outcomes was based

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on the histological and histomorphometrical analysis.

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1. Materials and Methods 2.1. Ethical Statement The experiment was performed in the animal house, Experimental Surgery and Biological Center, Faculty of Medicine, Cairo University, Egypt. It was approved by the Research Ethics Committee of the Faculty

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of Dentistry, Cairo University (Approval Number: 151220) that comply with the ARRIVE guidelines (ARRIVE Guideline Checklist).

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Preparation of stem cells was implemented at the Biochemistry and Microbiology unit (B.M.B.U), Faculty of Medicine, Cairo University.

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2.2. Animal Model

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Healthy (Specific Pathogen Free) adult male rabbits of New Zealand white strain (n=27), mean weight = 2.7±0.567 kgs with an average age of

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6 months, were purchased from and housed at the animal house, Experimental Surgery and Biological Center, Faculty of Medicine, Cairo

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University, under the supervision of a veterinary team. Additionally, five rabbits were used for the purpose of BMSCs and GMSCs isolation. Animals were maintained under monitoring and constant conditions

(25± 2 C○ room temperature; 12/12 light/dark cycle and 50±5% relative

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humidity). They were given standard dried pelleted diet (IBEX® International Company for Feed Production, Egypt), fresh grass hay, vegetables and allowed free access to water ad libitum from nipple drinker. Each rabbit was kept in a single stainless steel - open sided wire mesh cage allowing rabbits to communicate with each other and provide

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good ventilation. The dimension of cage was (40 cm height x 48 cm width x 63 cm depth) and the floor was covered with a comfortable dimple plastic slat. The try underneath of cage was filled with sawdust to remove any unpleasant odor. Each large rack consisted of nine removable cages. All rabbits were acclimated to the research room for one week prior to beginning the surgical procedure and provided with environmental

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enrichment objects like; grass hay and hard chew toy (wooden sticks) (local

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toy store). 2.3. Sample Size Calculation

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The sample size was calculated in accordance with the Evidence

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Based Dentistry Committee (EBD) at the Faculty of Dentistry, Cairo University. Based on a previous study (Ai et al., 2012), the difference in

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bone formation between the studied experimental groups was about 4%, with an average variability of 1.4%. A total sample size of 27 rabbits (9 in

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each of the three groups) has power 80% and 5% significance. The sample size was calculated by power and sample size (PS).

2.4. Study Design

The experiment was conducted on March 2017. All rabbits were held

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under the similar housing (single housing cage) and feeding conditions. Each animal was matched with a specific number (from 1- 27) by the technician at the animal house. Twenty seven rabbits were divided randomly using computer random allocation program (Random Allocation Software Version 1.0) (Saghaei, 2004) into three groups (n=9 in each

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group) as follows: group I:Unloaded NanoBone Scaffold, group II: GMSCs loaded on NanoBone Scaffold and group III: BMSCs loaded on NanoBone Scaffold. The three main groups were further subdivided into three subgroups (n=3 in each group) according to the termination time points (2, 4 and 6 weeks postoperatively) (Table 1).

2.5.1 Aspiration, Isolation and Cultivation of BMSCs

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2.5. Preparation of Stem Cells and Scaffold

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The bone marrow was harvested from New Zealand white rabbits (n=5), with an average age of 6 months and weight of 2.5-3.0 kgs, under

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general anesthesia using intramuscular injection of a mixture of

Xylazine

Hydrochloride®

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Ketamine Hydrochloride® 10% (35 mg/kg) (Ketamar, Amoun Co.) and 2%

(5

mg/kg)

(Xyla-Ject®,

Phoenix

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Pharmaceutical Inc.) (Lipman et al., 1997). The detailed process was performed according to previous studies (Alhadlaq and Mao, 2004; Tan

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et al., 2013) (Supplementary Material). 2.5.2 Collection, Isolation and Cultivation of GMSCs: Stem cells were prepared following Zhang and his colleagues protocol

(Zhang et al., 2009) (Supplementary Material).

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2.5.3 Morphologic and Phenotypic Characterization of MSCs According to Mesenchymal and Tissue Stem Cell Committee of the

International Society for Cellular Therapy (ISCT), MSCs must be characterized by their adhesiveness, fibroblast-like shape and identified by expressing MSCs surface markers CD29, CD73, CD90 and CD105

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and lack the expression of CD45, CD34, CD14 or CD11b, CD79 alpha or CD19 and Human leukocyte antigen-antigen D related (HLADR) surface molecules. Moreover, MSCs must differentiate into adipocytes, chondroblasts and osteoblasts (Dominici et al., 2006). Flow cytometric analysis was used in the current study to examine the phenotype of cells.

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Fluorescence absorbance cell sorting (FACS) analysis was performed

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using the standard protocol. At the third passage, the media was removed from the flasks and both the BMSCs and GMSCs layers were

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washed twice with PBS then detached from the flask with 0.25% trypsin-

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EDTA for 5 min at 37°C. Cells were recovered by centrifugation at 2000 rpm for 6 min and 0.5-1x107 cells/mL were incubated at 4°C for 30 min

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in flow cytometry buffer containing 1 µg of Phycoerythrin (PE) or Fluorescent isothiocyanate (FITC)-conjugated monoclonal antibodies CD29-FITC, CD73-PE, CD90-PE,

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against the following markers:

CD105-PE, CD34-FITC and CD45-PE (Beckman coulter, USA). FITC and PE labeled rabbit non-specific isotype controls and Immunoglobulin G were used as negative controls to determine the background

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fluorescence emission. Flow cytometric cell analysis was performed using CYTOMICS FC

500 Flow Cytometer (Beckman coulter, Elite XL, USA) and data were analyzed using CXP Software version 2.2. At least 10000 events were

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captured by the system. The percentage of cells that were positive for a marker was reported (Majumdar et al., 1998; Tan et al., 2013). 2.5.4 MSCs tracking with PKH26 Following the characterization of cells, GMSCs and BMSCs were labeled with PKH26 (Sigma-Aldrich, Co. LLC, Saint Louis, MO). According to the manufacturer’s instructions, cells were washed once in

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medium without serum, centrifuged for 5 min, pelleted and suspended.

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Then, cell suspension was rapidly added to equal volumes of fluorescent dye (red) solution. Postoperatively, sectioned bone tissues were

Cells-NanoBone scaffold construct:

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2.6.

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examined with a fluorescence microscope to trace the cells

NanoBone® comes in a sterile vial. It was obtained from Artoss,

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Rostock Company, Germany via the International Company for Dental Supplies, Cairo, Egypt. According to the manufacturer, NanoBone® is a

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granular material consisting of nano-crystalline particles of hydroxyapatite (HA) (average size 3 nm x 50 nm) embedded in an amorphous silica gel (ASG) matrix. Un-sintered particles were achieved through a sol–geltechnique at temperatures below 700 °C. Open connection between HA

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particles and the silicon dioxide (SiO2) molecules is responsible for the appearance of interconnecting pores (10-20 nm) and promoting the protein adsorption. Different methods have been used to combine scaffolds with stem cells; either MSCs are placed within the scaffold and then cultured or

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cultured MSCs are placed within the scaffold during the operation (Salimi et al., 2010). In the current study, we applied the second method based on the available equipment. NanoBone granules were placed in a 48-well flat bottom plate (200 mg of NanoBone per well) and rehydrated with 2 mL of modified Eagle's medium (MEM) (GIBCO/BRL) for 2 hours before cells’ seeding. Then,

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scaffold incubation medium was removed and the resuspended cultured

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MSCs (with a density of 1x106 per well) were gently pipetted on to the scaffold. Afterward, plates were placed in a humidified incubator (5% CO2

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at 37 °C) for 24 h for cell attachment.” The homogenous MSCs- seeded

spatula.

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2.7. Surgical Procedures

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scaffolds were placed directly into the bony defects using small sterile

The surgical procedure was conducted in the light phase (morning)

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in the operating room at the animal house, Experimental Surgery and Biological Center, Faculty of Medicine, Cairo University by the experienced surgeon and under aseptic condition as described by Cacchioli and his colleagues (Cacchioli et al., 2006). Rabbits of the three experimental

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groups were anesthetized as previously mentioned. The anterior and medial portions of the tibial bones were shaved by a safety blade razor and cleaned using iodopovidone and alcohol (Betadine ® EL-Nile Co. for Pharmaceutical and Chemical Industries, Cairo, Egypt). Linear skin

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incision (3-4cm length) was made over the medial surface of the tibia using NO.22 blade. Care was taken to split the muscular layer by blunt dissection and to keep the periosteum intact apart from the longitudinal excision. Using trephine dental bur (6.0/5.0 mm diameter) (Doma Dent Company for Dental Supplies, Cairo, Egypt) at low speed turbine powered dental hand piece

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(25000 rpm) (STRONG 90 Micromotor; Shenzhen Novo Export Limited,

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Guangdong, China) with constant copious sterile saline irrigation, a circular unicortical 6 mm diameter full-thickness bone defect was created in the

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proximal diaphysis of the medial surface of the left or right tibia of each

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rabbit. The defects were filled according to their group with material of interest using metal sterile spatula (PRIMADENT SS®, Pakistan).

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Then, soft tissues were repositioned and sutured with absorbable suture material (2/0 catgut) (Ethicon® Edinburgh- UK) and the skin was

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sutured by 3/0 silk suture (Assut Medical Sarl- Switzerland). Tincture Iodine 2% was then applied to the skin (Fig. 1).

2.8. Postoperative management To prevent the infection and eliminate the pain, each rabbit was given

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topical antibiotic spray; Bivatracin (Egyptian Company for Advanced Pharmaceuticals (ECAP), Egypt), intramuscular injection of analgesic 75mg/3ml Cataflam® (Novartis Pharma, Egypt) and Cephotaxime® antibiotic (15-20mg/kg) (Cephotax, Egyptian International Pharmaceutical Co., Egypt) once daily for 3 days and all animals were kept on standard

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diet with free access to water. Rabbits were then euthanized at each time interval: 2, 4 and 6 weeks after surgery by an intravenous overdose of sodium pentobarbital 100mg/kg. According to the American veterinary medical association (AVMA) guidelines for the euthanasia of animals, an intravenous injection of sodium pentobarbital is the kindest and most compassionate method of

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euthanizing animals (Leary et al., 2001). Tibial specimens were explanted,

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dissected free from any soft tissues, fixed, processed and the bony defects

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were evaluated through histological and histomorphometric analysis.

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2.9. Examinations Following the standard technique (Bancroft, 2008), tibial specimens were fixed in 10% calcium formol solution for 48 hours, then washed and decalcified in 10% EDTA for 4-5 weeks. After decalcification, the specimens were dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin wax. Paraffin blocks were sectioned

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serially at 5µm thickness and stained for histological analysis with H & E

Egypt distributer; GeneTech company).

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and Masson Trichrome (MT) (H&E: ab245880, MT: ab150686; Abcam,

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The cross sectioned specimens were obtained as much possible as

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we could from the center of the defect gap. The specimens were examined under the light microscope linked to a digital camera (Leica DM300

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Microsystems, Inc., Switzerland) and subjected to histomorphometric analysis to measure the newly formed bone area % and mature collagen

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fibers % respectively.

Primary outcome: new bone formation. Secondary outcome: lamellar bone formation (maturation of collagen

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fibers).

Histomorphometric Analysis: The area % of the newly formed

bone (H&E) and matured collagen fibers (MT) were measured blindly by one investigator at the Oral Biology Department, Faculty of Dentistry, Cairo University. For each sample, three non-overlapping regions (one from the middle and one from each side of the defect) per slide were observed and

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photographed using a microscope linked to a digital camera (Leica DM300 Microsystems, Inc., Switzerland). This measurement was carried out using the objective lens of magnification x 100. Subsequently, area % of new bone and matured collagen fibers (Region of Interest, ROI) were created, analyzed and calculated separately using Image J 1.52C software program (National institute of health, USA). The standard measuring frame of 700

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mm width and 500 mm height was specified for each region.

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Image J program was calibrated automatically to produce actual micrometer units from the measurement units (pixels). Using the color

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detector, the newly formed bone trabeculae and matured collagen areas

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were automatically colored and masked by a blue binary color measurable by the computer. The results were transferred automatically to statistical

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worksheets. 2.10.Statistical Analysis

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Statistical analysis was performed using a commercially available software program (SPSS 19; SPSS, Chicago, IL, USA). As data was parametric, significance of the difference between different groups at each time point (data shown) and difference within the same group at different

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times (data not shown) was evaluated using one way analysis of variance (ANOVA) test, followed by Tukey’s post hoc test when ANOVA yielded a significant difference. The level of significance was set at P < 0.05. Data was presented as mean, standard deviation and 95% confidence interval for mean.

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3. Results: 3.1. Condition of Animals After surgery, all rabbits recovered and survived during the experimental period without any complications except two rabbits, where one of them suffered from fracture while other rabbit showed signs of sever inflammatory reaction.

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3.2. Morphologic Characterization of MSCs

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The morphology of MSCs was observed under phase-contrast inverted microscope. Both GMSCs and BMSCs were expanded

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successfully, adhered to culture flasks showing a heterogeneous

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population at first passage (3 days after primary culture) (Fig. 2 a,b) and exhibited fibroblast-like morphology forming a monolayer on

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confluence at the 3rd passage (after 14 days of culture) (Fig. 2c, d) 3.3. Phenotypic Characterization of MSCs

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The Flow cytometry of cell surface markers showed that GMSCs were stained positively for MSCs surface markers CD29 (+ve) (94.6%), CD73 (+ve) (85.3%), CD90 (+ve) (95.2%) and CD105 (+ve) (90.4%) and negatively for hematopoietic cell surface markers CD34 (-ve)

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(1.2%). And CD45 (+ve) (1.03%). Equivalently, BMSCs were stained positively for MSCs surface

markers CD29 (+ve) (93.4%), CD73 (+ve) (94.7%), CD90 (+ve) (89.4%) and CD105 (+ve) (92.8%) and negatively for hematopoietic cell surface markers CD34 (-ve) (1.6%) and CD45 (-ve) (1.02 %) (Fig. 3).

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The filled areas reflects MSCs stained with monoclonal antibodies directed against the indicated antigens and open areas represents isotype control antibodies (Ruster et al., 2017). The expression levels of surface markers were presented as histogram. Therefore, these cells were considered as multipotent mesenchymal stem cells. 3.4. MSCs tracking with PKH26

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Labeled GMSCs and BMSCs were observed at the site of bone

homed successfully (Fig. 4).

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3.5. Qualitative Histological Assessment

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defect at different time points of the experiment; confirming that cells

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3.5.1.Two weeks postoperatively: Thin irregular newly formed woven bone trabeculae appeared to originate partially from the inner

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surface in group I (Fig. 5a) & emerged laterally near the defect’s edge in group III (Fig. 5c), while defects treated with GMSCs loaded

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on NanoBone scaffold (group II) (Fig. 5b) showed thicker newly formed bone trabeculae emerging mainly from the lateral sides and to lesser extent from the bottom side spanning the defect gap but

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not completely bridging it. The histological evaluation (H&E) of bony defects of three groups showed remnants of the scaffold material entrapped within granulation tissue (Fig. 5d, e, f). These woven bone trabeculae enclosed multiple wide fibrocellular marrow cavities and were characterized by the absence of lamellar structure, numerous randomly distributed large sized osteocytes

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and continuous rim of osteoblasts (Fig. 5g, h, i). Additionally, numerous dilated, endothelial lined blood vessels, congested with red blood cells (RBCs) were found particularly in defects loaded with stem cells (Fig. 5h, i). The sections stained by Masson’s Trichrome exhibited noticeable

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amount of new collagen fibers in the newly formed bone trabeculae which attained a blue color implying an immature woven bone (Fig.

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5j, k, l). No evidence of red stain could be seen within the newly formed bone indicating the absence of progressive maturation of

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stain in group II (Fig. 5k).

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collagen fibers except for a very narrow area that attained a red

3.5.2. Four weeks postoperatively: Examination of the grafted bony

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defects showed further healing among the defects regardless of the experimental groups. The bony defect of group I showed a core of

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partially degraded residual scaffold material in the center of the defect encapsulated by granulation tissue (Fig. 6a, d). Furthermore, a granulation tissue was still present in the central part of the defect

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of group II (Fig. 6b, e) while remnants of the scaffold intermingled with granulation tissue filling the defect gap in group III (Fig. 6c, f). Regarding the newly formed bone, limited but thick newly formed bone trabeculae at the border of the defect gap was evident in groups I&II. These bone trabeculae enclosed multiple narrow marrow cavities and tend to be arranged in more organized

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lamellated bone (Fig. 6g, h), while relatively thin interconnecting woven bone trabeculae, with scattered osteoblastic cells along the their peripheries and containing numerous irregularly arranged entrapped osteocytes, were observed in group III (Fig. 6i). The Masson’s trichrome stained sections showed different degrees of maturation with more mature newly formed lamellated bone or

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Haversian systems, as indicated by the red color, intermingled with

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other areas of woven bone, as indicated by the blue color (Fig. 6j, k, l).

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3.5.3. Six weeks postoperatively: H&E stained sections of group I

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showed the defect hole filled with a dense core of granulation tissue intermixed with large remnants of the scaffold material. Regarding

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new bone formation, it was restricted to lateral walls of the defect (Fig. 7a, d). The defect hole of Group II revealed central areas of

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granulation tissue and newly formed bone trabeculae originating from the defect side (Fig. 7b, e). Group III presented with almost complete bridging of the defect from both the periosteal and

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endosteal sides (Fig. 7c, f). The new bone formation in group I appeared in the form of thick newly formed bone trabeculae with irregularly arranged osteocytes and small fibrocellular marrow cavities. Some osteocytes were arranged in a circular pattern around the marrow cavity forming few Haversian systems (Fig. 7g).

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In the most peripheral part of the bony defect in group II, a band of lamellar bone with few Haversian systems was detected. Moving toward the center, thin interconnected newly formed woven bone trabeculae was identified. Osteoblastic cells line the periphery of woven bone trabeculae as well as marrow cavities (Fig. 7h). There were areas of woven bone trabeculae with irregularly arranged

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osteocytes alternating with some areas of more organized

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Haversian conformation. Two layers of the periosteum; outer fibrous and inner osteogenic layers were located over the defect surface

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(Fig. 7i). On examining the Masson’s Trichrome stained sections of

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the bony defect in group I, areas of woven bone, as indicated by the blue color intermingled with very narrow areas of lamellated bone,

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as represented by the red color (Fig. 7j). The bony defect of groups II&III showed prevalence of the red color of lamellated bone (Fig.

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7k, l).

Detailed panoramic views and photomicrographs can be found in this link.

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https://data.mendeley.com/datasets/dwns5m3xxh/draft?a=f61c893 0-2875-410e-824e-9552ba8301a6 doi:10.17632/dwns5m3xxh.1

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3.1.Quantitative

Histological

Assessments

(Histomorphometric

Analysis): For H&E stained sections, the newly formed bone area % was compared between different groups within the same time point. At 2 and 4 weeks post-operatively, the highest mean value was recorded in group II,

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followed by group III, whereas the lowest mean value was recorded in

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group I. ANOVA test revealed a statistically significant difference between group I and both group II and group III where (p=0.009). Tukey’s post hoc

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test revealed no significant difference between groups II and III. At 6 weeks

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post-operatively, the highest mean value was recorded in group III, followed by group II, whereas the lowest mean value was recorded in group

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I. ANOVA test revealed a statistically significant difference between group I and both group II and group III where (p<0.0001). Tukey’s post hoc test

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revealed no significant difference between groups II and III (Table 2 & Fig. 8).

For Masson’s trichrome stained sections, the mature collagen fibers

area %, as represented by a red color, was estimated. At 2 and 4 weeks

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post-operatively, the highest mean value was recorded in group II, followed by group III, whereas the lowest mean value was recorded in group I. ANOVA test revealed a statistically significant difference between groups where (p<0.0001) and (p=0.003) at 2 and 4 weeks respectively. Tukey’s post hoc test revealed a statistically significant difference between each 2

24

groups. However; at 6 weeks post-operatively, the highest mean value was recorded in group III, followed by group II, whereas the lowest mean value was recorded in group I. ANOVA test revealed a statistically significant difference between groups (p<0.0001). Tukey’s post hoc test revealed no statistically significant difference between groups II and III (Table 3 & Fig.

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4.

Discussion: Various approaches are available to treat critical sized bone defects; however; their limitations and drawbacks have encouraged researchers toward regenerative medicine. Regenerative medicine depends on two principles: biocompatible scaffolds and appropriate stem cells. In the current study, combination of MSCs and NanoBone scaffold was applied. Nano-scaffold has

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a great impact on bone regeneration since nanoparticles are in the same size

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range as the constituents of bone (Andrades et al., 2013). Among the proposed nano-scaffolds for bone regeneration is NanoBone®. Gerber et al., (2006) clarified

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that bone proteins (like osteopontin (OPN) and BMP-2) were found in the new

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matrix following 5 weeks of NanoBone implantation in mandibular defects created in mini pigs. Additionally, alkaline phosphatase activity was located in osteoblasts

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and newly formed bone on the NanoBone® surface. Later, Abshagen et al., 2008 confirmed that NanoBone granules induced

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stronger angiogenic response in comparison to NanoBone plates and cancellous bone. Besides, Liu et al., (2011) compared BioOss® as a natural scaffold and NanoBone® as a synthetic one in terms of their biocompatibility and their ability to promote the proliferation of human osteoblasts. Both materials had low cytotoxicity

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and good biocompatibility but the proliferation test and the number of osteoblasts on the material surface were superior for NanoBone®. Wittenburg et al., (2014) recommended NanoBone® for use as a scaffold in bone regeneration that requires MSCs. BMSCs and ADMSCs were seeded and cultured separately on two types of hydroxyapatite (HA) bone substitutes: 26

BONITmatrix® and NanoBone®. BMSCs-derived osteoblast-like cells spread on the surface of NanoBone® while ADMSCs preferred to grow on BONITmatrix®. This preference was verified by the higher expression of runt-related transcription factor 2 (Runx2) in MSCs cultured on NanoBone®. Compared with BMSCs, GMSCs provide easier isolation method, faster

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healing of the donor site, faster proliferation without any growth factors, and stable phenotype; which all make this source attractive for researchers (Tomar

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et al., 2010). Therefore, BMSCs were chosen in this study as they represent

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the gold standard source for MSCs in bone regeneration as compared to GMSCs which may represent a promising alternative source.

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Likewise, Allogeneic MSCs was chosen in the present study. Injection of

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allogeneic MSCs prevented the irreversible bone and cartilage damage in arthritis mouse model (Augello et al., 2007), enhanced the regeneration of critical side bone defect in canine model (Arinzeh et al., 2003). On the contrary, allogeneic

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MSCs fail to form the new bone matrix in humanized mice model and the autologous source was more effective than allogeneic one; yet both autogenic and allogeneic MSCs proliferated successfully and expressed earlier angiogenic

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markers which were important for the bone regeneration (Rapp et al., 2018). This conflicting results have encouraged the researcher to establish more works pointing out this issue. The morphologic and phenotypic characterization of both GMSCs and BMSCs in the present work were in agreement with the criteria identified by the Committee of ISCT (Dominici et al., 2006). For labeling of MSCs, we used the 27

red fluorescent dye (PKH26) as it was stable during the integration into the cell membrane (Parish 1999). Interestingly, PKH26 was reported to be effective and efficient in labeling MSCs (Pratheesh et al., 2017). In comparison to the other cell tracer like CM-DiI (Cell Tracker TMCM-DiI) and CSFE (Vy-brant® CFDA-SE cell tracer kit), PKH26 has lower toxicity and appeared to be the most suitable reagent for tissue engineering certainly adipose tissue (Hemmrich et

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al., 2006).

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A Study by (Li et al, 2013) revealed that PKH26 has no toxic or inhibitory

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effects on the cells; however, when the cell debris from PKH26 labeled cells were cultured with the unlabeled cells, the unlabeled cells were react with dye

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and emit fluorescence. This issue should be taken into consideration in the

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future work.

Histologically, the defect gap at two weeks postoperatively was almost filled with granulation tissue intermingled with small remnants of scaffold

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material in all experimental groups, with less scaffold residuals in groups II and III. This may be related to the increased resorption of the scaffold material when MSCs were added. To some extent, such findings were confirmed by Tour and

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his colleagues (Tour et al., 2014). They contended that loading of BMSCs onto HA scaffold improved the new bone formation via modulation of foreign body reaction.

Degradation of NanoBone granules was concomitant with the deposition of bone-specific proteins, bone cells and hence, new bone matrix throughout the physiological stages of bone healing. The transformation of 28

NanoBone from graft status into the lamellar bone was reported at the early stage of bone remodeling (Götz et al., 2008). Two weeks after implantation of NanoBone into the adipose tissue of rat’s neck, the silica gel (component of NanoBone) was degraded faster especially in their peripheral region and was replaced by the carbohydrate host components (Xu et al., 2009).

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Further, in critical sized bone defects of rabbit’s tibia, both adipose derived-mesenchymal stem cells (ADMSCs) and unseeded scaffold groups

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showed bone formation initially in the peripheral part of the scaffolds close to

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the bone edge, However; the difference between peripheral and central parts of the constructs was less evident in the defects treated with stem cells-loaded

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scaffolds; this may be due to the endothelial differentiation ability of ADMSCs which would accelerate angiogenesis process (De Girolamo et al., 2011).

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The newly formed woven bone trabeculae of all studied groups emerged from different sites and contained numerous randomly distributed

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entrapped large sized osteocytes with wide lacunae, multiple wide fibrocellular marrow spaces lined with a continuous rim of osteoblastic cells. The finding of osteoblasts indicated a regulated process of bone formation. Additionally,

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large osteocytes “young osteocytes” represent the early stage of osteoblast to osteocyte differentiation (Baud, 1968). The density of osteocyte lacunae in woven bone from the center of the fracture site, subjected to rapid bone formation, was more than twice as large as that in the lamellar cortical bone (Hernandez et al., 2004).

29

The histological results at four weeks postoperatively showed progression in the healing process towards the center of the defect in an attempt to bridge the defect, despite being still partial. The thick newly formed bone at the lateral walls of the defect appeared to be of lamellated bone in groups I and II while group III showed thin interconnecting woven bone trabeculae arising primarily from the defect side walls. Similar observation was

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reported when the mandibular critical sized defects were implanted with

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BMSCs loaded on tricalcium phosphate scaffold (TCP) or scaffold alone. Large amounts of woven bone were observed after 4 weeks with osteoblasts

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actively covering the bone surfaces and enclosing fibrovascular marrow

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cavities of varying sizes (Saad et al., 2015).

At six weeks post-operatively, the defect gap was noticeably

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decreased in size with islands of new bone tissue occupying the defect area. These bone islands growth resulted in an extensive bridging of the defect in

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groups II and III from both the periosteal (outer) and endosteal (inner) sides. This outcome might be related to the osteogenic potential of BMSCs and GMSCs. A higher rate of bone formation was previously demonstrated in MSCs-platelet derived growth factor loaded nano-hydroxyapatite scaffold

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(Behnia et al., 2013) and strong osteocalcin expression was observed in GMSCs and BMSCs loaded biografts but not in the unloaded ones (Tomar et al., 2010). It is well-known that collagen is an important component of the ECM of bone. Any alteration in its distribution can be indicative for different 30

physiologic or mechanical changes in the bone tissue (Viguet-Carrin et al., 2006). In the current investigation, Masson’s Trichrome results were coinciding with the obtained H&E histological results where the defects loaded with GMSCs or BMSCs showed considerable amounts of matured collagen fibers, as indicated by the red reaction, reflecting accelerated bone remodeling and effective maturation of new bone tissue following MSCs

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therapy. These results are in accordance with previous studies which

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reported that BMSCs can significantly increase new bone formation which appeared to be mature with a dark red staining (Kim et al., 2007; Liu et al.,

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2014).

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The obtained results in the current study confirmed the involvement of MSCs in all phases of bone healing either directly or indirectly. Directly, via

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differentiation into bone-forming cells such as osteoblasts and osteocytes and indirectly, via secretion of trophic factors. Granero-moltó et al., (2009)

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demonstrated that MSCs attenuated the inflammatory response via increased anti-inflammatory cytokines and decreased proinflammatory cytokines which leads to limit tissue injury, prevent the development of fibrosis and

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fundamentally, to promote regeneration of damaged tissue. Generally, a combination of stem cells and nanoscaffold provides

considerable results in comparison to the scaffold alone. This may be due to the impact of surface nanotopography on cellular interactions and behavior, in addition to the osteogenic differentiation capability of MSCs. NanoBone material acts as a guide for new bone formation and it has a negative surface 31

charge that is rapidly covered with proteins and growth factors (Wittenburg et al., 2014). Interestingly, nanoscale features promote MSCs responses such as: cell attachment, spreading, proliferation and angioneic expression (Xia et al., 2014), osteogenic differentiation (Dalby et al., 2007; Xia et al., 2014)

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increased focal adhesion formation and enhanced cytoskeletal expression as well as organization (Dalby et al., 2006) and maintained MSCs

coincidence

with

the

current

histological

results,

the

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In

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phenotypes and multipotency (McMurray, 2011).

histomorphometric analysis of new bone area % revealed a statistically

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significant decrease in group I compared to both group II and group III at different time points. On the other hand, the obtained results revealed no

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significant difference in the mean area % of new bone between groups II

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and III throughout all experimental periods. Similarly, (Xu et al., 2014) assessed the newly formed bone area %

within mandibular defects of rats receiving either GMSCs or culture medium only, and they found a statistically significant difference between the two groups;

which

indicated

a

significant

effect

of

GMSCs

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studied

transplantation on bone regeneration. In addition, there was a significant difference in the new bone area % between BMSCs loaded on β-TCP group and the unloaded one at 2 weeks postoperatively in critical sized mandibular defects (Saad et al., 2015).

32

On the contrary, there was not a statistically significant difference in % of new bone formation between the unloaded biphasic calcium phosphate (BCP) scaffold and the BMSCs loaded BCP groups at 4 weeks following implantation of critical sized mandibular defects (Brennan et al., 2014). A popular explanation of these contradictory results may be related

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to the different animal model, size of the defect and type of scaffold used. With regards to the area % of mature collagen fibers in the newly

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formed bone, the histomorphometric results showed a statistically

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significant decrease in the mean area % of mature collagen fibers in group I compared to both group II and group III at different time points. On the

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other hand, a significant increase in the mature collagen fibers area % was noticed in group II more than in group III at 2 and 4 weeks postoperatively,

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while there was a non-significant difference between the two groups at 6 weeks postoperatively.

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Many factors could be responsible for the highest value of collagen

fiber maturation in GMSCs loaded group at 2 and 4 weeks postoperatively. Among these, the primary cultures of GMSCs were uniformly homogenous,

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whereas BMSCs became homogenous after 2–3 passages. Also, GMSCs proliferated faster than BMSCs and generated a larger number of MSCs in a very short duration (Tomar et al., 2010). Conclusively, GMSCs represent an abundant, easily accessible alternative source that obtained via non-invasive technique for stem cellbased therapies. In the current study, stem cell-loaded groups displayed 33

more new bone formation in comparison to the unloaded NanoBone group regardless of the MSCs source. Findings of the current study could add further support to the ability of GMSCs to regenerate bone defects in a way that may be greatly similar to that of BMSCs. The positive impact of GMSCs’ preclinical application on the

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regeneration of bone, tendon, skin and periodontal ligaments opens the door to a promising role of these cells in regenerative medicine.

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Nevertheless, many biological considerations and technical issues should

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be improved before translating the results from the preclinical side to the

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clinical side.

34

5.

Conclusion:

Local application of GMSCs and BMSCs loaded on NanoBone scaffold showed enhanced pattern of bone regeneration in critical sized bone defects compared to the unloaded one, which highlighted the importance of GMSCs, as a promising alternative to BMSCs, as well as the value of combination strategy (cells & scaffold) in regenerative medicine. Conflict of interest statement:

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6.

7.

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None Funding source:

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This research did not receive any specific grant from funding agencies in

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the

public, commercial, or not-for-profit sectors.

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Author’s contribution:

Gamilah Al-Qadhi: Conceptualization, Literature search, Methodology (in vivo

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procedures), Collection and presentation of data, Formal analysis, Writing (original draft). Malak Soliman: Conceptualization, Supervision, Result analysis and interpretation. Iman Abou-Shady: Conceptualization, Supervision, Result analysis and interpretation,

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Writing (review and editing).

Laila Rashed: Conceptualization, Supervision, Methodology (in vitro procedures).

8.

Acknowledgment: 35

Authors would like to thank Dr. Sara El Moshy for her assistance in

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capturing histological images.

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Fig. 1: Photographs showing the surgical procedures: 1) shaved, disinfected and

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incised medial aspect of tibia, 2) 0.6 mm trephine bur used to make a standardized defect size, 3) the created bone defect, 4) bony defect, 5) bony defect filled with

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material of interest and 6) skin sutures with (3/0) silk suture.

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Fig. 2: Photomicrographs showing the morphology of (a) GMSCs and (b) BMSCs at first passage (3 days after primary culture), (c) GMSCs and (d) BMSCs at third

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passage (after 14 days of culture). (Orig. Mag. x100) (Scale bar=100 µm)

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Fig. 3: Flow cytometric analysis of cell surface markers showing that GMSCs and BMSCs were positive for MSC markers CD29, CD73, CD90 and CD105, while

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negative for hematopoietic markers CD34 and CD45.

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Fig. 4: Photomicrographs showing detection of PKH26-labeled transplanted mesenchymal stem cells. (a,b) two weeks postoperatively, (c,d) four weeks postoperatively and (e,f) six

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weeks postoperatively. (Scale bar=100 µm)

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Fig. 5: Panoramic view of bony defects at two weeks postoperatively treated by (a) unloaded NanoBone scaffold, (b) GMSCs loaded on NanoBone scaffold and (c) BMSCs loaded on NanoBone scaffold showing old preexisting bone (OB) and areas of new bone (NB)/ (d,e,f) photomicrographs of dotted circle areas showing residuals of scaffold material (S) and granulation tissue (GT) (H&E X40)(Scale bar=500µm)/ (g,h,i) higher magnification of dotted

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square areas represent the newly formed bone area trabeculae (black asterisks), randomly

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distributed entrapped osteocytes (yellow arrows), osteoblastic cells (black arrows) and fibrocellular marrow cavities (MC) filled with blood vessels (BV) (H&E X100) (scale bar=

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200µm)/ (j,k,l) Masson’s Trichrome stained sections of experimental groups showing small areas of lamellated bone as represented by the red color (red asterisk) and collagen fibers

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of the newly formed woven bone as indicated by the blue color (yellow asterisks). (MTx100)

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(Scale bar=200um)

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Fig. 6: Panoramic view of bony defects at four weeks postoperatively treated by (a) unloaded NanoBone scaffold, (b) GMSCs loaded on NanoBone scaffold and (c) BMSCs loaded on NanoBone scaffold showing old preexisting bone (OB) and areas of new bone (NB)/ (d,e,f) photomicrographs of dotted circle areas showing residuals of scaffold material (S) and

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granulation tissue (GT) (H&E X40) (scale bar=500µm)/ (g,h,i) Higher magnification of dotted squared area represent the newly formed bone area which appeared to be lamellated bone

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(black asterisks), entrapped osteocytes (yellow arrows), osteoblast lining (black arrows), marrow cavities (MC) and rich vascular front growing from the bone marrow (blue arrows)

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(H&E X100) (scale bar=200µm)/ (j,k,l) Masson’s Trichrome stained sections of experimental

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groups showing more areas of lamellated bone as represented by the red color (red asterisk) and few areas reflecting woven bone as indicated by the blue color (yellow asterisks).

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(MTx100) (Scale bar=200µm)

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Fig. 7: Panoramic view of bony defects at six weeks postoperatively treated by (a) unloaded NanoBone scaffold, (b) GMSCs loaded on NanoBone scaffold and (c) BMSCs loaded on NanoBone scaffold showing old bone (OB) and areas of new bone (NB)/ (d,e,f) photomicrographs of dotted circle areas showing residual of scaffold material (S) and granulation tissue (GT) (H&E X40) (scale bar=500µm)/ (g,h,i) higher magnification

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of dotted squared area represent areas of newly formed bone trabeculae (black

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asterisks) enclosing marrow cavities (MC), entrapped osteocytes (yellow arrows), osteoblast lining (black arrows), haversian system (dotted circle), band of lamellar bone

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(green asterisk), periosteum (Pt) (H&E X100) (scale bar=200µm)/ (j,k,l) Masson’s Trichrome stained sections of experimental groups showing more areas of lamellated

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bone as represented by the red color; particularly in groups II&III (red asterisks) and few

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(Scale bar=200µm)

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areas reflecting woven bone as indicated by the blue color (yellow asterisks). (MTx100)

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Fig. 8: Column chart showing mean area percentage of newly formed bone at 2 weeks post operatively (a), 4 weeks post operatively (b) and 6 weeks post operatively (c). (Group I: Unloaded NanoBone Scaffold, Group II: GMSCs loaded

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on NanoBone Scaffold and Group III: BMSCs loaded on NanoBone Scaffold). Significance level p<0.05, * significant, ns non- significant difference.

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Fig. 9: Column chart showing mean area percentage of mature (calcified) collagen fibers in different groups at 2 weeks post operatively (a), 4 weeks post operatively (b) and 6 weeks post operatively (c). (Group I: Unloaded NanoBone Scaffold, Group II: GMSCs loaded on NanoBone Scaffold and Group III: BMSCs loaded on

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NanoBone Scaffold. Significance level p<0.05, * significant, ns non- significant difference.

48

Table 1: Study design (animal groups, animal number, materials used, dose, route of administration, termination time points and number of analyzed slides).

9

NanoBone® (Artoss, Rostock Company, Germany; The International Company for Dental Supplies, Cairo, Egypt)

GMSCs +NanoBone

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BMSCs + NanoBone

200 mg of NanoBone + 2 mL of modified Eagle's medium (MEM) (GIBCO/BRL).

200 mg of NanoBone + 2 mL of MEM containing 1x106 GMSCs

200 mg of NanoBone + 2 mL of MEM containing 1x106 BMSCs

Local

Local

Local

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Route of administration

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Dose for each rabbit

Group III: BMSCs Loaded on NanoBone Scaffold

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Material

Group II: GMSCs Loaded on NanoBone Scaffold

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Number of rabbits

Group I: Unloaded NanoBone Scaffold (free-cells)

2-4-6 weeks (3 rabbits at each time point).

2-4-6 weeks (3 rabbits at each time point).

2-4-6 weeks (3 rabbits at each time point).

Number of analyzed slides

Three representative slides (9 zones)/each group

Three representative slides (9 zones)/each group

Three representative slides (9 zones)/each group

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Termination time points

49

Table 2: Comparison of the mean area percentage of newly formed bone in different groups at different time points. (Group I: Unloaded NanoBone Scaffold, Group II: GMSCs loaded on NanoBone Scaffold and Group III: BMSCs loaded on NanoBone Scaffold).

Mean

Std. Dev

Std. Error

Group I

32.27b

3.29

1.90

24.10

40.45

29.34 35.83

Group II

47.25a

3.10

1.79

39.55

54.94

44.62 50.67

Group III

43.14a

5.11

2.95

30.44

55.85

37.78 47.97

40.11b

4.82

56.46 a

4.93

Group III

53.68a

2.42

P Group I Group II Group III

52.07

36.14 45.46

44.21

68.71

51.16 60.92

1.40

47.67

59.68

50.91 55.38 12.91 0.007*

4.32

2.49

25.11

46.57

31.14 39.63

63.99a

6.27

3.62

48.43

79.56

58.96 71.01

67.87a

3.97

2.29

58.00

77.74

63.45 71.15

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F P

11.75 0.009*

35.84b

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6 weeks postoperatively

28.15

Max

2.85

re

F

2.78

-p

4 weeks Group I Postoperatively Group II

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F P

Min

of

2 weeks postoperatively

Experimental Groups

95% Confidence Interval for Mean Lower Upper Bound Bound

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Significance level p<0.05, * significant, Tukey’s post hoc test: mean values sharing the same superscript letter are not significantly different, group I: Unloaded NanoBone Scaffold, group II: GMSCs loaded on NanoBone Scaffold and group III: BMSCs loaded on NanoBone Scaffold.

50

37.33 <0.0001*

Table 3: Comparison of the mean area percentage of mature collagen fibers in different groups at different time points. (Group I: Unloaded NanoBone Scaffold, Group II: GMSCs loaded on NanoBone Scaffold and Group III: BMSCs loaded on NanoBone Scaffold).

24.79b 51.51a 55.93a

3.21 1.12 1.15

1.85 0.65 0.66

Min 1.45 9.54 6.61

Max 1.81 12.26 8.55

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0.60 0.54 2.26

33.90 45.68 32.49

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1.05 0.93 3.91

Std. Error 0.10 0.80 0.57

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36.50c 48.01a 42.20b

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F P

Mean 1.65c 11.04a 7.46b

Std. Dev 0.18 1.38 0.99

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Experimental Groups 2 weeks Group I postoperatively Group II Group III F P 4 weeks Group I postoperatively Group II Group III F P 6 weeks Group I postoperatively Group II Group III

95% Confidence Interval for Mean Lower Upper Bound Bound 1.20 2.09 7.61 14.48 5.01 9.92

39.11 50.33 51.91

69.29 <0.0001* 35.30 37.23 46.93 48.60 38.86 46.50 17.26 0.003*

16.82 48.72 53.08

32.77 54.29 58.78

21.50 50.70 55.22

27.91 52.78 57.25 198.31 <0.0001*

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Significance level p<0.05, * significant, Tukey’s post hoc test: mean values sharing the same superscript letter are not significantly different, group I: Unloaded NanoBone Scaffold, group II: GMSCs loaded on NanoBone Scaffold and group III: BMSCs loaded on NanoBone Scaffold.

51

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biocompatibility and vascularization of the synthetic bone grafting substitute NanoBone®. Journal of Biomedical Materials Research Part A: 91(2), 557-566. doi: 10.1002/jbm.a.32237. 2. Ai, J., Ebrahimi, S., Khoshzaban, a, Jafarzadeh Kashi, S., Mehrabani, D., 2012. Tissue engineering using human mineralized bone xenograft and bone marrow mesenchymal stem cells allograft in healing of tibial fracture of experimental rabbit model. Iran. Red Crescent Med. J. 14(2), 96–103. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372046/ 3. Alhadlaq, A., Mao, J., 2004. Mesenchymal stem cells: isolation and therapeutics. Stem Cells Dev. 13, 436–448. https://doi.org/10.1089/scd.2004.13.436 4. Andrades, A., Narváez-Ledesma, L., Cerón-Torres, L., Cruz-Amaya, P., LópezGuillén, D., Laura Mesa-Almagro, M., Moreno-Moreno, A., 2013. Bone Engineering: A Matter of Cells, Growth Factors and Biomaterials. in Regenerative medicine and tissue engineering. InTech, 25, 616-618. https://doi.org/10.5772/56389 5. Arinzeh, T., Peter, S., Archambault, M., Van Den Bos, C., Gordon, S., Kraus, K., Smith, A. and Kadiyala, S., 2003. Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect. JBJS, 85(10), 1927-1935. doi: 10.2106/00004623-200310000-00010. 6. Augello, A., Tasso, R., Negrini, M., Cancedda, R. and Pennesi, G. 2007. Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen‐induced arthritis. Arthritis & Rheumatism, 56: 1175-1186. doi:10.1002/art.22511. 7. Bancroft, J., 2008. The Hematoxyline and Eeosin. In M, Gamble & J, Bancroft (Eds.). Bancroft's theory and practice of histological technique (6th ed., pp.121134). Edinburgh: Churchill Livingstone/Elsevier. 8. Baud C., 1968. Submicroscopic structure and functional aspects of the osteocyte. Clinical Orthopaedics and Related Research, 56, 227-236. 9. Behnia, H., Khojasteh, A., Kiani, T., Khoshzaban, A., Mashhadi Abbas, F., Bashtar, M., Dashti, G., 2013. Bone regeneration with a combination of nanocrystalline hydroxyapatite silica gel, platelet-rich growth factor, and mesenchymal stem cells: A histologic study in rabbit calvaria. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 115, e7–e15. https://doi.org/10.1016/j.oooo.2011.09.034 10. Berner, A., Reichert, C., Müller, B., Zellner, J., Pfeifer, C., Dienstknecht, T., Nerlich, M., Sommerville, S., 2011. Treatment of long bone defects and nonunions : from research to clinical practice. Cell &Tissue Research, 347(3), 501-519. https://doi.org/10.1007/s00441-011-1184-8 11. Bostrom, R., Mikos, G., 1997. Synthetic Biodegradable Polymer Scaffolds. In A, Atala & J, Mooney (Eds.). Tissue Engineering of Bone (pp. 215-234). Birkhäuser Basel:Springer. 12. Brennan, Á., Renaud, A., Amiaud, J., Rojewski, T., Schrezenmeier, H., Heymann, D., Trichet, V., Layrolle, P., 2014. Pre-clinical studies of bone regeneration with 52

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