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Rapid maxillary expansion in alveolar cleft repaired with a tissue-engineered bone in a canine model Jialiang Huang, Bo Tian, Fengting Chu, Chenjie Yang, Jun Zhao, Xinquan Jiang, Yufen Qian

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S1751-6161(15)00126-5 http://dx.doi.org/10.1016/j.jmbbm.2015.03.029 JMBBM1444

To appear in: Journal of the Mechanical Behavior of Biomedical Materials

Received date:8 December 2014 Revised date: 17 March 2015 Accepted date: 24 March 2015 Cite this article as: Jialiang Huang, Bo Tian, Fengting Chu, Chenjie Yang, Jun Zhao, Xinquan Jiang, Yufen Qian, Rapid maxillary expansion in alveolar cleft repaired with a tissue-engineered bone in a canine model, Journal of the Mechanical Behavior of Biomedical Materials, http://dx.doi.org/10.1016/ j.jmbbm.2015.03.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Rapid maxillary expansion in alveolar cleft repaired with a tissue-engineered bone in a canine model Jialiang Huang1,2,#, Bo Tian3,#, Fengting Chu1,2, Chenjie Yang1, Jun Zhao1,2, Xinquan Jiang2,4,*, Yufen Qian1,* 1

Department of Orthodontics, Ninth People’s Hospital Affiliated to Shanghai Jiao

Tong University, School of Medicine, Shanghai, People’s Republic of China. 2

Oral Bioengineering Lab/Oral Tissue Engeneering Lab, Shanghai Research Institute

of Stomatology, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai, People’s Republic of China. 3

Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics,

Ninth People's Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, People’s Republic of China. 4

Department of Prosthodontics, Ninth People’s Hospital Affiliated to Shanghai Jiao

Tong University, School of Medicine, Shanghai, People’s Republic of China. * Corresponding authors: 1. Yufen Qian, email: [email protected] (YFQ) 2. Xinquan Jiang, email: [email protected] (XQJ)

#

The authors contributed equally to this work.

1

Abbreviations TEB

tissue-engineered bone

RME

rapid maxillary expansion

BMSCs

bone marrow stromal cells

ȕ-TCP

ȕ-tricalcium phosphate

OM

osteogenic media

GM

growth media

A590

absorbance at 590 nm

PCR

polymerase chain reaction

Runx2

runt-related transcription factor 2

OPN

osteopontin

BSP

bone sialoprotein

OCN

osteocalcin

GADPH

Glyceraldehyde-3-phosphate dehydrogenase

TRITC-phalloidin tetraethyl rhodamine isothiocyanat-phalloidin, red DAPI

4'-6-diamidino-2-phenylindole, blue

HE

hematoxylin–eosin

SD

standard deviation

ALP

alkaline phosphatase

TE

Tetracycline, yellow

AL

alizarin red S, red

CA

calcein-AM, green

2

Abstract This study aims to investigate the effects of orthodontic expansion on graft area of a tissue-engineered bone (TEB) BMSCs/ȕ-TCP, and to find an alternative strategy for the therapy of alveolar cleft. A unilateral alveolar cleft canine model was established and then treated with BMSCs/ȕ-TCP under rapid maxillary expansion (RME). Sequential fluorescent labeling, radiography and helical computed tomography were used to evaluate new bone formation and mineralization in the graft area. Hematoxylin-eosin staining and Van Gieson’s picro fuchsin staining were performed for histological and histomorphometric observation. ALP activity, mineralization and the expression of osteogenic differentiation related genes of BMSCs that grew on the ȕ-TCP scaffold were promoted by their cultivation in osteogenic medium. Based on fact, TEB was constructed. After 8 weeks of treatment with BMSCs/ȕ-TCP followed by RME, new bone formation and mineralization of the dogs were markedly accelerated, and bone resorption was significantly reduced, compared with the untreated dogs, or those only treated with autogenous iliac bone. The treatment with both TEB and RME evidently made the bone trabecula more abundant and the area of bone formation larger. What’s more, there were no significant differences between BMSCs/ȕ-TCP group and the group treated with autogenous bone and RME. This study further revealed that TEB was not only a feasible clinical approach for patients with alveolar cleft, but also a potential substituent of autogenous bone, and its combination with RME might be an alternative strategy for the therapy of alveolar cleft. 3

Keywords: rapid maxillary expansion (RME); alveolar cleft; cleft lip and palate; tissue engineering; bone marrow stromal cells (BMSCs); ȕ-tricalcium phosphate (ȕ-TCP)

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1. Introduction Cleft lip and/or palate is the third common congenital malformation, and it occurs frequently around the world, even worse, the morbidity is still climbing (Cooper et al., 2000; DeVolder et al., 2013). Among these cleft lip and/or palate patients, 75% are coupled with alveolar cleft, an osseous defect of the premaxillary alveolar (Waite and Waite, 1996). Secondary alveolar bone grafting with particulate cancellous bone and marrow from the iliac crest bone is, at present, considered as one of the most acceptable procedures to fill the bony gap, stabilize the premaxilla and overall dental arch, to provide bony support for the teeth adjacent to the cleft site, to prevent the residual oro-nasal fistula and segmental collapse, to reestablish dentoalveolar bony contour, and to support the lip and nose (Harrison, 2014; Lee et al., 2009; Yang et al., 2012). Although autogenous bone graft is a gold standard for alveolar cleft repair, this treatment is influenced by the location and shape of bone source, and the restricted bone mass (Gimbel et al., 2007). Additionally, this approach also brings new traumas to the site where autogenous bone is taken (Moreau et al., 2007; Paganelli et al., 2006; Schultze-Mosgau et al., 2003). Therefore autogenous bone grafting is still not perfect in a way. The burgeoning tissue engineering is, at present, widely thought to be the best potential technique to repair alveolar cleft instead of autogenous bone grafting, because it is much more achievable to obtain both naturally derived and synthetic biomaterials without any inflicted traumas than autogenous bone (Gimbel et al., 2007; Hibi et al., 2006; Pradel et al., 2008; Zhang et al., 2011). Beta-tricalcium phosphate 5

(ȕ-TCP), as a type of resorbable biomaterial, could be used for bone regeneration of canine artificial alveolar clefts, which has already been authenticated in various studies (Tokugawa et al., 2012). Bone resorption is the most key issue that alveolar cleft patients faced with after bone grafting (Feichtinger et al., 2007; Schultze-Mosgau et al., 2003). Our research team previously found in series of animal experiments that, mechanical stress stimulation provided by orthodontic tooth movement appliances could reduce bone resorption in bone graft area after autogenous bone grafting, which was in favor of the secular stability of bone graft area (Zhang et al., 2011). However, the effect of TEB combined with RME on the repair of alveolar cleft and whether TEB resembles autogenous bone in repairing alveolar cleft are still unclear. The present study aims to investigate the effect of rapid orthodontic expansion on graft area of TEB BMSCs/ȕ-TCP in an alveolar cleft canine model, and find an alternative feasible approach to the therapy of alveolar cleft.

2. Materials and methods 2.1 Cell isolation, induction and differentiation BMSCs (bone marrow stromal cells) were obtained from autogenous bone marrow. In brief, autogenous bone marrow aspirate was obtained from iliac crests of the beagle dogs mentioned below under anesthesia using a heparinized biopsy needle. Then nucleated cells were successively collected and incubated after the centrifugation. Subsequently, nonadherent cells were removed, and the remaining cells were detached 6

with 0.25% trypsin/ethylenediaminetetraacetic acid and passaged. The cells after the second passage were chosen for the following study. Osteogenic differentiation of BMSCs was performed via cultivating the cells in osteogenic media (OM). Meanwhile, BMSCs cultivated in growth media (GM), namely Dulbecco's modified eagle media, served as a control.

2.2 Confirmation of mature osteoblasts by ALP activity, mineralization and osteogenic genes To confirm the maturation of osteoblasts, the following experiments were performed, and all the experiments were performed in triplicate. a. After cultivation for 0, 6 and 9 d, respectively, ALP staining was performed. In order to evaluate osteogenic differentiation of BMSCs, ALP activity was quantitatively assessed using Bradford’s method with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). b. Mineralization of the BMSCs after 0, 15 and 21 d of cultivation was quantified by the absorbance at 590 nm (A590) after alizarin red staining. c. Quantitative real-time PCR (polymerase chain reaction) was used to analyze the expressions of osteogenic genes in osteoblasts. Total RNA extracted from the cells cultured in GM or OM at day 1, 6 and 9, was used as templates to synthesize the complementary DNA. Markers of mature osteoblasts were detected here, including runt-related transcription factor 2 (Runx2), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) 7

gene served as a normalizer. Primer sequences for the real-time PCR were provided in supplementary material Table S1.

2.3 Cell seeding and osteogenic differentiation of BMSCs on ȕ-TCP scaffolds Beta-TCP (Shanghai Bio-Lu Biomaterials Co. Ltd., Shanghai, China) with a diameter of 1.5 ~ 2.5 mm was used as scaffolds after sterilization. The cells cultured in GM or OM were suspended and dropped onto the ȕ-TCP scaffolds using pipetting technique. After 4 h of incubation in 24-well plates, the BMSCs/ȕ-TCP constructs were then cultured in GM or OM. A scanning electron microscope (SEM, Philips SEM XL-30, The Netherlands) was used to display the microcosmic surface morphology of the scaffolds. On day 4 post cell seeding, cytoskeletal filamentous actin was observed using confocal laser scanning microscopy (CLSM, Leica TCS SP2; Leica Microsystems, Heidelberg, Germany) to investigate cytoskeleton organization and spreading of BMSCs on the scaffolds. Briefly, BMSCs were seeded onto the ȕ-TCP granules (3 × 104 cells/sample) in a 12-well plate (n = 3). After cultivation for 4 d, the samples were gently washed with PBS and incubated in 4% paraformaldehyde for 15 min, then they were immersed in 0.1% Triton X for 15 min. Actin filaments and nuclei were visualized by staining with TRITC-phalloidin (tetraethyl rhodamine isothiocyanat-phalloidin, red) and DAPI (4'-6-diamidino-2-phenylindole, blue), respectively. To analyze the osteogenic differentiation of BMSCs on ȕ-TCP scaffolds, ALP 8

activity assay, quantitative alizarin red mineralization analysis, and quantitative real-time PCR analysis of the 4 aforesaid osteogenic markers, were performed as previously described. TEB BMSCs/ȕ-TCP, which was constructed with osteogenically induced BMSCs and ȕ-TCP scaffolds, was used as a graft to the surgically created alveolar cleft. In brief, the construct was directly put into the defect area and surrounded by the soft tissue. Then, TEB was sewed and sealed into the defect area (supplementary materials, Figure. S1).

2.4 Experimental animals and grouping In various animals, dogs have enough oral cavity volume to place and retain the expansion screws that can make effect observation easier than in smaller animals’ oral cavity, and their alveolar can be easily excised to form a full-thickness alveolar bone defect. So, they are ideal for the establishment of alveolar cleft animal model. Fourteen 24-week-old male beagles (purchased from Experimental Animal Centre of Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai, China), weighing 9.5 ± 0.5 kg, were selected for this study. Alveolar of each dog was unilaterally (right) cleaved by surgery after tooth extraction (detailed in section 2.5), and the dogs were randomly divided into four groups: A. Blank control (n = 2), without bone graft and RME, to observe if the alveolar cleft could heal spontaneously during the experiment period; B. Autogenous group, autogenous bone (harvested from the top of the anterior iliac crest after anesthesia) 9

graft without RME (n = 4); C. Positive control group, treated with autogenous bone and RME (n = 4); D. Experimental group, treated with TEB BMSCs/ȕ-TCP constructs graft and RME (n = 4). The animal study was permitted by the Animal Care and Experiment Committee of Ninth People’s Hospital affiliated to School of Medicine, Shanghai Jiao Tong University (approval NO. HKDL[2013]51).

2.5 Alveolar cleft surgery The surgery was performed according to the previous literature (Zhang et al., 2011). Shortly, the maxillary second and third incisors of all anaesthetic dogs were unilaterally (right) extracted, following the intramuscular injection of ketamine (10 mg/kg). Using a physiologic saline-cooled round carbide bur, the alveolar cleft (defect width: ~15 mm) canine model was established through the resection of all bone substance from axiodistal of central incisor to axiomesial of canine. The graft from each group was transplanted to fill in the alveolar cleft made by surgery. The wound was closed in layers, and all of the alveolar cleft dogs were injected with penicillin for 1 week and fed with soft diet during the research. After the surgery and during the maxillary expansion, all efforts were made to minimize animal suffering, such as injecting with analgesic tramadol. Every 2 days, the animals were assessed for health and welfare, in which, behavioral signs and clinical symptoms were monitored. If the dogs had polished furs and behaved actively, instead of dull fur, piloerection and reduced activities, they would be considered in good health with a good welfare. The animals were euthanatized before harvesting the premaxillas for undecalcified bone 10

sections and paraffin sections.

2.6 Rapid maxillary expansion Ultra-anhydrite models were cast with a silicone impression material (Die stone IX, Heraeus Kulzer Dental Ltd, Germany) (Figure. S2 A). For all experimental animals, arch expansion devices were individually redesigned and recast based on the memory expansion screws (Forestadent, Pforzheim, Germany) according to their canines on both sides. The recast expansion device which can supply and maintain a constant force was applied to the maxillary of each experimental dog in groups C and D (Figure. S2 B). Specifically, the maxillary soft tissue was pulled open to make maxillary dentition and palate tissue fully exposed, and the appliance was fixed in lateral position. Four weeks after surgery, thrust started in groups C and D, and it was augmented 4 weeks later (8 weeks after surgery). According to clinical experience, the appliance expanded 2 mm each time, and finally expanded 4 mm in total (Yang et al., 2012). The expansion lasted for 8 weeks at a constant force of about 1000 g (Wichelhaus et al., 2004b).

2.7 Vertical height determination of the bone graft using radiography and helical computed tomography Maxillary occlusal films were taken immediately after surgery, 4, 8, and 12 weeks after surgery, respectively, using a dental X-ray machine (Trophy Radiology, Marne-la-ValleÂe, France). 11

Based on the maxillary occlusal films, vertical heights of the alveolar bone was measured immediately and 12 weeks after surgery, respectively. The alteration ratio of the bone graft vertical heights was evaluated with the formula given below: Alteration ratio = (R1 × L0 / L1) / R0

(I)

R0: The height of the alveolar crest immediately after surgery; R1: The height of the alveolar crest 12 weeks after surgery; L0: The root length of the maxillary central incisor immediately after surgery; L1: The root length of the maxillary central incisor 12 weeks after surgery. The ratio of L0 to L1 was used to eliminate the influence caused by different scalings of the maxillary occlusal films. For a better understanding of the formula (I), our previous study (Zhang et al., 2011) and the schematic diagram (Fig. 1) are suggested to refer to.

Figure. 1 To confirm the alteration of vertical height of the bone graft during recovery after surgery, helical computed tomography (CT) with a slice thickness of 0.625 mm (GE LightSpeed Qxi/Plus/16 Helical CT, USA) was performed under general anesthesia, immediately, 4 w, 8 w, and 12 w after surgery, respectively. The CT films were processed with the interactive medical image control system Mimics 10.01 (Materialise, Leuven, Belgium). The vertical height was measured using the method shown in supplementary materials (Figure. S3).

12

2.8 Observation of new bone formation and mineralization through sequential fluorescent labeling. The dogs were intraperitoneally injected with tetracycline (TE) hydrochloride (Sigma) at a dose of 25 mg/kg immediately after surgery, and then alizarin red S (AL; Sigma) at a dose of 30 mg/kg 4 weeks after surgery. Finally, the dogs were intraperitoneally injected with 20 mg/kg calcein (CA; Sigma) 3 days before euthanasia. The results were analyzed according to the methods described by Wang et al. (Wang et al., 2009) and Zhao et al. (Zhao et al., 2013). In order to quantitatively evaluate the bone formation and mineralization in the grafted alveolar, the fluorescent staining at the same locations, namely the top, middle, and bottom of the central graft area between the maxillary central incisor and the canine, was respectively scanned. The images of different fluorescent-labeled undecalcified bone sections were merged. The mineral apposition rate was calculated with the vertical distances between TE and AL, and between AL and CA fluorescent labels, using an image analysis system (Image-Pro PlusTM, Media Cybernetics, Silver Springs, MD, USA). Four mean values were individually calculated for four merged images in each group, and finally they were used to evaluate the average mineral apposition rate for each group.

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2.9 Histological and histomorphometrical observation after hematoxylin-eosin staining and Van Gieson’s picro fuchsin staining The premaxillas of all 14 dogs were harvested after euthanasia 12 weeks after surgery, and then fixed in a 4% paraformaldehyde buffer (pH = 7.4) for 4 d. The sample was bisected into two blocks at the intermediate line. One half was decalcified, embedded with paraffin, then sliced into 4-mm-thick sections, and finally stained with hematoxylin–eosin (HE). The other half was dehydrated in increasing concentrations (from 75% to 100%) of ethyl alcohol, and embedded in polymethylmethacrylate (Sigma, USA) for 14 d before polymerization. A diamond-coated internal-hole saw microtome (SP1600; Leica Microsystems, Wetzlar, Germany) was used to cut the specimens into 120-ȝm-thick sections, which were then ground and polished to a final thickness of about 50 ȝm for the observation of fluorescent labeling mentioned above. The undecalcified sections were subsequently stained with Van Gieson’s picro fuchsin for histological observation. General histological analysis was performed using the image analysis system (Image-Pro Plus). From the serial sections of each sample, representative HE slides were randomly selected for the analysis of the area of newly formed bone. The fluorescent labeling of the undecalcified sections was analyzed under a confocal laser scanning microscope (Leica TCS Sp2 AOBS; Leica, Heidelberg, Germany). Excitation/emission wavelengths were respectively set at 405/580 nm for TE 14

(Tetracycline, yellow), 543/617 nm for AL (alizarin red S, red), and 488/517 nm for CA (calcein-AM, green). Three sections were randomly selected from the serial mesio-distal sections for each sample, and analyzed with the image analysis system (Image-Pro Plus). The percentage of newly formed bone and the ȕ-TCP scaffolds residues in graft areas were calculated under a 20- or 40-fold magnification.

2.10 Statistical analysis All data were processed with SPSS v.10.1 software (SPSS Inc., Chicago, Illinois, USA) and expressed as mean ± SD (standard deviation). Differences between two groups were statistically analyzed by Student’s t-tests, and the comparison of more than two groups was performed using analysis of variance (ANOVA) with Student-Newman-Keuls (SNK) post hoc test. Differences were considered significant if p < 0.05.

3. Results 3.1 The surgically created canine alveolar cleft model The canine alveolar cleft (defect width: 15 mm) model was successfully established by resecting all bone substance between axiodistal of maxillary central incisor and axiomesial of maxillary canine (Figure. 2 B, C and D). The alveolar defects unilaterally went deep into the lateral wall of palatal bone, extended to the nasopalatine foramen, and reached up toward the nasal floor. 15

3.2 The constructed TEB BMSCs-ȕ-TCP 3.2.1 BMSCs cultured in OM or GM After cultivation for 9 d, BMSCs cultured in OM (Figure. 3 B3) showed obviously stronger coloration than the cells grew in GM (Figure. 3 A3), indicating stronger ALP activity in the cells in OM. After 15 d of cultivation, a significant crimson started to show in the cells in OM (Figure. 3 D2), compared with those in GM (Figure. 3 C2). What’s more, a more significant difference in color was observed after 21 d of cultivation (Figure. 3 C3, D3). Consistently, ALP activity of BMSCs in OM was stronger than that in GM according to the quantitative ALP activity assay (Figure. 3 E), and the absorbance values at 590 nm (A590) of BMSCs in OM were also significantly higher than those in GM, based on the quantitative mineral deposition assay (Figure. 3 F). These results suggested that BMSCs produced extensive sheets of calcified deposits and differentiated into mature osteoblasts more easily in OM. Consistent with the above results, at transcriptional level, real-time PCR analysis showed that mRNA levels of all osteogenic differentiation-related genes in the cells in OM were significantly (p < 0.05) upregulated after 6 d of cultivation, and the upregulation became very significant (p < 0.01) after 9 d of cultivation, compared with those in GM (Figure. 3 G ~ J). 3.2.2 BMSCs on ȕ-TCP scaffolds cultured in OM or GM As shown in the SEM image, the ȕ-TCP that we used owns a high interconnected porosity (volume porosity is about 70%; diameter = 450 ± 50 ȝm) (Figure. 4 A) as before (Zhang et al., 2011). The morphological characteristics of cytoskeletal 16

filamentous actin (Figure. 4 B ~ D), obtained by confocal laser scanning microscopy, demonstrated that BMSCs spread and grew very well on ȕ-TCP scaffolds in vitro 24 h after cell seeding. Like the growth of BMSCs cultivated in OM or GM without ȕ-TCP scaffolds, the growth of BMSCs in the presence of ȕ-TCP scaffolds was not influenced by the scaffolds, which can contrarily provide a favorable environment for cells. However, the media significantly affected BMSCs. In this experiment, stronger ALP activity (Figure. 4 E), higher A590 value (Figure. 4 F), and 4 more highly expressed osteogenic genes (Figure. 4 G ~ J) of BMSCs cultivated in OM suggested that ȕ-TCP was suitable for cell attachment and differentiation and therefore eligible in the following studies.

3.3 The alteration of the bone graft height Occlusal radiography demonstrated that both groups C and D possessed significantly higher ratios of the residual alveolar (bone graft) height (Figure. 5 a and A), compared with group B, which was subsequently confirmed by the following helical computed tomography. Eight or twelve weeks after surgery (expansion had been lasted for 4 and 8 weeks, respectively), vertical height of the graft area in groups C and D showed significantly greater value than that in group B (Figure. 5 b and B). What’s more, the graft area in group D was vertically higher than that in group C without statistical significance. In this experiment, TEB BMSCs/ȕ-TCP showed favorable performance against bone resorption, therefore it may be used as a potential substitute of autogenous bone. 17

3.4 Fluorescent labeling and histomorphometrical analysis To determine the rate of bone formation, sequential fluorescent labeling with TE (yellow), AL (red), and CA (green) were performed. All dogs were respectively intraperitoneally administered with TE (immediately), AL (4 weeks after surgery), and CA (12 weeks after surgery), which represented the mineralization level in different periods. So, the bone between TE and AL lines is that formed in 0 ~ 4 w after surgery (without expansion), and the bone between AL and CA lines is that formed in 4 ~ 12 w after surgery (with expansion). In this experiment, we determined vertical distances between different fluorescent labels reflecting the quantity of newly formed bone to show the rate of bone formation. As shown in Figure. 6, neither TE nor AL labeling was observed in group A (Figure. 6 A1, A2), and the vertical distance between TE and AL in group C or D was not significantly longer than that in group B (Figure. 6 E). But, the vertical distance between AL and CA in groups C and D were found significantly greater than that in group B (Figure. 6 F). Taken together, these results indicated that the mineralization of TEB BMSCs/ȕ-TCP and autogenous bone in the presence of RME were significantly (p < 0.05) more rapid than those treated without RME. Further, BMSCs/ȕ-TCP bone showed higher bone formation rate (compared with group B) with a non-significant difference between it and autogenous bone, which was similar with the detection results of residual bone graft height shown in Figure. 5.

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3.5 Histologcal and histomorphological analysis of bone regeneration In group A, only a small amount of newly formed bone and abundant connective tissue were observed (Figure. 7 A1 and A2). And in group B, the newly formed bone was relatively poor, though it could be found (Figure. 7 B1 and B2). While in groups C and D, significantly more active bone reconstruction, more obvious newly formed bone and more abundant thick bone trabecula in bone graft area could be easily and clearly observed (Figure. 7 C1, C2, D1 and D2), instead of poor new bone and abundant connective tissue. These results demonstrated that RME evidently accelerated the formation of new bone. Consistent with the above results of HE staining, groups C and D showed more newly formed bone and abundant bone trabecula than groups A and B (Figure. 8 A1/A2 ~ D1/D2). This result once again proved the fact that RME can accelerate new bone formation. Besides, the area of new bone formation in the grafted region was calculated based on the result of Van Gieson’s picro fuchsin staining. As expected, both groups C and D presented significantly larger area of bone formation than groups A and B (Figure. 8 E). But between groups C and D, no significant difference in the area of newly formed bone was found.

4. Discussion The present study focuses on the effect of rapid orthodontic expansion on graft area of TEB BMSCs/ȕ-TCP, and to find an alternative approach to the therapy of alveolar cleft. 19

ȕ-TCP scaffolds own both osteoinduction capacity and mechanical strength that can resist external pressure to a certain extent (Tokugawa et al., 2012). BMSCs are known as pluripotent cells, which can differentiate into osteoblasts, chondrocytes, etc. (Tokugawa et al., 2012). The promising TEB BMSCs/ȕ-TCP based on autogenous cells could eliminate the issues like donor scarcity, supply limitation, and pathogen transfer and immune rejection (Liu and Ma, 2004). In our study, based on the results of ALP activity assay, alizarin red staining, TRITC-phalloidin staining and osteogenic differentiation gene level determination (Figure. 3, 4), we likewise found that BMSCs grew, spread and differentiated very well on ȕ-TCP, when cultivated in OM. So, this construct was qualified to be used in the next experiments, and might be qualified to be applied in clinical trials as well. Numbers of researchers have proved that mechanical stresses could be transmitted to bone graft area through sutura and parodontium, can induce osteoblast differentiation, and can reduce bone resorption via various ways (Ikegame et al., 2001; Jiang et al., 2006; Kido et al., 2009; Marzban and Nanda, 1999). In terms of tissue repair, mechanical stimulation can actually promote it through not only the secretion of reparative cytokines, such as TGF-ȕ, IGF-1 and IL-6 (Knoll et al., 2005; Raab Cullen et al., 1994), but also chemokine release that guides the chemotaxis and recruitment of osteoblast in graft area, which accelerates the proliferation and differentiation of osteogenic cells, results in increased deposition and mineralization of osteoid matrix, and finally causes increased formation of new bone. As a biologically functional stimulation, expansion after bone grafting has been clinically 20

proved not to compromise the final result of the bone grafting but to promote tissue regeneration as reported (da Silva Filho et al., 2009; de Oliveira Cavassan et al., 2004; Hou et al., 2007). But, we took one more step to think it probably served as a promoter during the recovery of alveolar cleft, according to various reports (Knoll et al., 2005; Mitsui et al., 2005; Yang et al., 2005). Through the recast expansion screws we used here, mechanical stress was well delivered to the grafted bone (Wichelhaus et al., 2004a), which successfully laid a solid foundation for this study. In this study, TEB together with RME was used to treat alveolar cleft. Simultaneously, the group treated with both iliac bone and RME served as a positive control. Reports showed that functions of TEB, including direct osteogenesis, paracrine and vascularization, were approximated to those of autogenous bone, and TEB were therefore better than those of pure biomaterials (Granero

Moltó et al., 2011; Zhang et al., 2011). Furthermore, the effect of TEB was

also equivalent to that of autogenous bone in the presence of RME, and was stronger than the effect of sole autogenous bone treatment (Knoll et al., 2005). In our present study, the same phenomenon was observed. Specifically, after the rapid expansion for 8 weeks,

! occlusal film and helical CT showed significantly greater heights of

TEB and autogenous bone, compared with those treated without expansion (Figure.

" fluorescent labeling suggested that the rates of formation of new bone and bone mineralization were higher for TEB and autogenous bone (Figure. 6); # 5);

histological and histomorphological observation found significantly more newly formed bone and significantly more abundant bone trabecula in the group treated with 21

TEB or the autogenous bone

(Figure. 7, 8). But what’s most important of all is the

fact that there was no significant difference between TEB and the autogenous bone, and in some specific experiments, the former showed better performances to some extent. Based on the above results from different angles, we could speculate that this treatment might have a long-term stability, although 12 weeks was not long enough. In accordance with the literatures (Jiang et al., 2006; Kido et al., 2009; Knoll et al., 2005), several following aspects might be involved in the mechanism of RME’s working on TEB. Mechanical stresses facilitated the differentiation and osteogenesis of seed cells, enhanced paracrine actions of BMSCs to promote bone-formation, induced host osteoblasts to be chemotactic and to aggregate in bone graft area, and/or improved vascularization of bone graft. If this treatment is intended to be applied in clinic, some significant obstacles that we are facing may stand in its way. A second general anesthetic is frequently required to harvest the bone marrow. The second general anesthesia will burden the patients to various degrees. However, scientists’ unceasing explorations in translational medicine are presently expected to solve this issue. Tan et al. (Tan et al., 2014) have developed a method that has potential to facilitate the development of large-scale human induced pluripotent stem cell (hiPSC) banking worldwide using human finger-prick blood. These hiPSCs have shown significant performance which is similar with that of human embryonic stem cells. So, the novel strategies like this will make tissue engineering easier and bring no trauma to the patient. 22

Repairing alveolar defects to support canine eruption and orthodontic tooth movement is one of the important steps in the serial therapy of cleft lip and palate (Bajaj et al., 2003; Batra et al., 2004). The study by Zhang et al. (Zhang et al., 2011) reported that TEB from the combination of ȕ-TCP and BMSCs is a feasible clinical approach for patients with alveolar cleft and the subsequent orthodontic tooth movement, and it would be a new method to repair alveolar cleft as an alternative of autogenous bone. The repaired bone not only allowed the immigration of adjacent teeth and provided favorable support, but also physiologically functioned like the normal alveolar bone. Alveolar bone grafting is carried out mostly in children, who are capable of growth and development and are often in the phase of canine eruption. Studies demonstrated that canine eruption functionally stimulated the bone graft and resulted in the reduced bone resorption, and enhanced the regeneration and assimilation of bone graft (da Silva Filho et al., 2000; Dempf et al., 2002). The use of the teeth that immigrated into graft area can also bring about favorable functional stimulations and reduce bone resorption. Researchers found that the force provided by endosseous implants can also reduce the resorption of bone graft (Isono et al., 2002). Therefore, an effective and persistent functional stimulus after bone grafting will contribute to the decrease of bone resorption and sustaining the long term stability and normal function. Meantime, it lays a solid foundation for the orthognathic surgery that is possibly going to be performed in patients with cleft lip and palate in future. In short, this treatment probably has a long-term stability, but it is still need to be further 23

confirmed in the future. The basic thinking of tissue engineering is to replant the autologous cells cultivated in vitro into bodies. However, the ordinary two-dimension cell culture can’t work efficiently. Therefore, a special 3D cell culture method is in great need. This method will well simulate the surroundings of the cells grow in vivo, thus resulting in a higher efficiency of in-vitro cell differentiation. Nowadays, this technic has made a dent in tissue engineering to some extents. For instance, a rotating wall vessel bioreactor designed by John Space Center and Ray Schwarz can simulate microgravity for 3D cell culture and help signal transduction between cells through affecting gene expression directly, or promoting cell autocrine/paracrine indirectly (Su et al., 2013). Summarily, TEB BMSCs/ȕ-TCP might be a potential substituent of autogenous bone for alveolar cleft patients, and RME had a promoting effect on tissue repair in the graft area. The combination of TEB and RME may serve as an alternative strategy for the therapy of alveolar cleft in the upcoming future. However, the exact mechanism of RME’s promoting effect on TEB, the long-term stability of the new bone formed by TEB under RME, and routine clinical application of this technique are still in great need of investigation in the next moment.

5. Conclusion Beta-TCP possesses favorable biocompatibility that makes it a type of excellent scaffold biomaterial in tissue engineering. Complexus constructed with BMSCs and 24

ȕ-TCP can be used as a fungible mimic of autogenous bone in repairing bone defects. RME after bone grafting probably provides functional stimulation for bone graft area, which is in favor of reducing bone resorption and accelerates new bone formation. As a result, RME will probably benefit the long-term stability of grafted bone. When applied to repairing alveolar cleft, the treatment with TEB and RME has the same ability with the strategy of the autogenous bone treatment assisted with RME, indicating that TEB is a favorable exogenous graft, and can be alternatively chosen to repair bone defects rather than just autogenous bone.

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Acknowledgments This study was financially supported by National Natural Science Foundation of China (Grant No.: 81170989).

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Figure legends: Figure. 1 Schematic diagram of the height measurement of the repaired alveolar from the occlusal films: taking the front point of piriform aperture as a center, a tangent circle was drawn through the bottom point of alveolar bone in graft area.

Figure. 2 Surgically created alveolar cleft model in dogs. A. Scheme designed to show the size and shape of the alveolar cleft that would be created by surgery. B. Photograph of the surgically created alveolar cleft after surgery. C. Occlusal view of the alveolar cleft was taken immediately after operation by X-ray. D. Reconstructed three-dimensional tomogram by computer tomography immediately after surgery operation.

Figure. 3 Osteogenic differentiation of BMSCs cultured in the growth media (GM) or osteogenic media (OM). A/B. ALP staining after cultivation in GM for 0 d (A1), 6 d (A2) and 9 d (A3), or in OM for 0 d (B1), 6 d (B2) and 9 d (B3), respectively. C/D. Mineralized calcium nodules stained with alizarin red (AL) in GM for 0 d (C1), 15 d (C2) and 21 d (C3), or in OM for 0 d (D1), 15 d (D2) and 21 d (D3), respectively. Magnification fold: 40×. E. ALP activity of BMSCs measured using quantitative ALP assay. F. Quantitative mineral deposition of BMSCs. G ~ J. Real-time PCR analysis of osteogenic differentiation-related genes in BMSCs: Runx2 (Runt-related transcription factor 2) (G), OPN (osteopontin) (H), BSP (bone sialoprotein) (I) and OCN (osteocalcin) (J); *p < 0.05 indicated significant differences; 32

**p < 0.01 indicated very significant differences.

Figure. 4 Spreading and osteogenic differentiation of BMSCs on ȕ-TCP scaffolds. A. SEM images (100×) of ȕ-TCP. B ~ D. Cytoskeletal morphology of BMSCs after 24 h of growth on ȕ-TCP: nuclei were stained blue by DAPI (B) and actin filaments were stained red by TRITC phalloidin (C); figure D was merged with figures B and C. E. The ALP activity of BMSCs which was cultivated in GM or OM on ȕ-TCP was quantitatively measured. F. Quantitative mineral deposition in BMSCs. G ~ J. Expressions of osteogenic differentiation related genes at transcript level of BMSCs in GM or OM were analyzed by real-time PCR: Runx2 (G); OPN (H); BSP (I); OCN (J) (*p < 0.05 and **p < 0.01 respectively indicated significant and very significant differences between GM and OM group).

Figure. 5 Height of the bone graft. a. Sequential occlusal radiographs taken immediately (A1, B1, C1, D1), 4 (A2, B2, C2, D2), 8 (A3, B3, C3, D3), and 12 (A4, B4, C4, D4) weeks after surgery, respectively. b. Images of helical computed tomography performed immediately (A1, B1, C1, D1), 4 (A2, B2, C2, D2), 8 (A3, B3, C3, D3), and 12 (A4, B4, C4, D4) weeks after surgery. A. Based on sequential occlusal radiography, the percentage of the remaining bone graft height of three different groups (group B, C and D) 12 weeks after surgery compared with the height immediately after surgery. B. Vertical height of the grafted region measured by helical computed tomography immediately, 4, 8, and 12 weeks after durgery. 33

Figure. 6 Sequential fluorescent labeling of TE (tetracycline), AL (alizarin red S) and CA (calcein-AM) intraperitoneally administered immediately, 4, and 12 weeks after surgery, respectively. A4 ~ D4. Merged images of the former three fluorographs of undecalcified sections in the same group (50×), which were respectively taken at excitation/emission wavelengths of 405/580 nm (TE, yellow), 543/617nm (AL, red), and 488/517 nm (CA, green). A5 ~ D5. Merged images of the three fluorochromes and plain confocal laser microscope (CLSM) images of the same group (50×). E. Vertical distance between TE and AL. F. Vertical distance between AL and CA. *p < 0.05 represented significant differences between groups.

Figure. 7 Histological and histomorphological analysis of alveolar bone repair 12 weeks post-surgery using HE staining. A1/A2. Blank control (group A), without treatment. B1/B2. Group B, treated with autogenous bone graft. C1/C2. Group C, treated with autogenous bone graft and RME. D1/D2. Group D, treated with BMSCs/ȕ-TCP tissue-engineered bone graft and RME.

Figure. 8 Histological and histomorphological analysis of alveolar bone repair via Van Gieson’s picro fuchsin staining and the area of bone formation in the grafted region. A1/A2. Blank control (group A), without treatment. B1/B2. Group B, treated with autogenous bone graft. C1/C2. Group C, treated with autogenous bone graft and RME. D1/D2. Group D, treated with BMSCs/ȕ-TCP tissue-engineered bone graft and 34

RME.

35

Figure 1

L0, L1

R0, R1

Figure. 1

Figure 2

Figure. 2

Figure 3

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OM

GM

OM

0d

0d

6d

15 d

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Figure. 3

Figure 4

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B

C

E

D (merged)

F

G **

**

** **

** *

I

H

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** **

**

** *

Figure. 4

Figure 5

Group A

Group B

Group C

Group D

a Immediately

4 weeks

8 weeks (Expansion for 4 weeks)

12 weeks (Expansion for 8 weeks)

b Immediately

4 weeks

8 weeks (Expansion for 4 weeks)

12 weeks (Expansion for 8 weeks)

A

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*

* *

Figure. 5

* *

Figure 6

TE, immediately

AL, week 4

CA, week 12

Merged

Group A

Group B

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Figure. 6

Figure 7

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Figure. 7

Figure 8

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C. Autogenous + Expansion

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D. BMSCs/β-TCP + Expansion *

Figure. 8

*