Al-Azhar Assiut Medical Journal

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 14  |  Issue : 4  |  Page : 158--168

The potential osteogenic effect of stem cells in mandibular distraction in goats


Eman B Elshal1, Sayed B Ahmed2, Alaa El Deen Jamal Ben Taleb3, Yasser N El Hadidi3, Marwa El Kassaby3, Khaled Abd El Meneim Abd El Kader3, Salah Abd El Fatah3,  
1 Department of Anatomy and Embryology, Faculty of Medicine for Girls, Al-Azhar University, Cairo, Egypt
2 Laboratory of Department of Laboratory Molecular Biology, Faculty of Science, Al-Azhar University, Cairo, Egypt
3 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Ain Shams University, Cairo, Egypt

Correspondence Address:
Eman B Elshal
Department of Anatomy and Embryology, Faculty of Medicine for Girls, Al-Azhar University, Cairo
Egypt

Abstract

Background Mandibular distraction osteogenesis is a powerful reconstructive tool for the repair of lower-jaw deformities. Mesenchymal stem cells (MSCs) have been initially identified in bone marrow as nonhematopoietic stem cells that may differentiate into different tissues. Objective The objective of this study was to construct an original experimental model for mandibular distraction osteogenesis in the lower jaw, followed by MSC injection that could produce a sufficient quantity and quality of intramembranous bone. Materials and methods Seventeen goats (Capra aegagrus hircus), each weighing about 10–15 kg, were divided into three groups: zero control (n=3), positive control (injured with spontaneous cure) (n=7), and treated (injured and treated by stem cells) groups (n=7). A monodirectional distractor was designed and fixed. Distraction was performed at a rate of 1 mm per day for 10 days to create a distracted gap of 10 mm. In the treated group, after 10 days of distraction, the prepared stem cells (three million cells) were applied in the distracted gap on two doses every 10 days. The treated and positive control group had 30 days of consolidation to allow healing and maturation of the distracted bone. After animal sacrification, histological and radiographic assessments were carried out. Results Cone beam computed tomography examined the radiographic bone density of the newly formed bone, and there was a statistically significant increase in the bone density in the treated group compared with the control group. There was a significant increase in trabecular bone thickness and decrease in osteoid bone percentage in the treated group as compared with the positive control, indicating more rapid bone maturation. Conclusion MSC injection into the distracted mandible induced osteogenesis in the lower jaw of the goats and improvement in bone regeneration.



How to cite this article:
Elshal EB, Ahmed SB, Jamal Ben Taleb AE, El Hadidi YN, El Kassaby M, Abd El Kader KA, El Fatah SA. The potential osteogenic effect of stem cells in mandibular distraction in goats.Al-Azhar Assiut Med J 2016;14:158-168


How to cite this URL:
Elshal EB, Ahmed SB, Jamal Ben Taleb AE, El Hadidi YN, El Kassaby M, Abd El Kader KA, El Fatah SA. The potential osteogenic effect of stem cells in mandibular distraction in goats. Al-Azhar Assiut Med J [serial online] 2016 [cited 2019 Oct 22 ];14:158-168
Available from: http://www.azmj.eg.net/text.asp?2016/14/4/158/208934


Full Text



 Introduction



Bone is a multifunctional organ that provides protective structure and mechanical support to the body. Repair and regeneration of bone are a series of biological events involving a number of cell types and signaling pathways in a limited time and space [1],[2]. Some of the congenital anomalies of the jaw require orthognathic surgery for reposition of the jaws. Distraction osteogenesis (DO) is a surgical technique that corrects jaw and craniofacial deformities when the bone in the jaw region requires lengthening. In addition, soft tissues surrounding the distracted bone started the process for regeneration. These procedures attracted the attention of a large number of authors to pursue a huge number of DO experimental studies to follow up the events associated with DO of craniofacial skeleton in rats [3]. These studies were carried out on many species including dog [4], pig [5], rabbit mandibles [6],[7], and sheep maxillae [8],[9]. Mesenchymal stem cells (MSCs) have been initially identified in bone marrow as nonhematopoietic stem cells that may differentiate into tissues of mesodermal origin, such as adipocytes, osteoblasts, chondrocytes, tenocytes, skeletal myocytes, and visceral stromal cells [10]. Safety of this procedure has been confirmed on 1873 patients by Hernigou et al. [11]. For the cultured MSCs, several reports found no evidence of deleterious changes or malignant transformation of the cultured MSCs used in two national metacentric immunohematology trials [12].

Several attempts were carried out to improve bone quality and quantity generated through DO. However, injection of stem cells improved the bone quality of distracted bone and improved bone density when assessed by cone beam computed tomography (CBCT) in a clinical trial on achondroplastic patients. Bone marrow stem cells were added to distraction gaps, which resulted in improved bone quality [13],[14].

Cancellous bone architecture is an important determinant of bone strength that depends on the bone density, as well as structure of the trabecular network [15]. The ability of bone substitutes to promote bone fusion is contingent upon the presence of osteo-inductive factors in the bone environment at the fusion site. Osteoblast progenitor cells are among these environmental osteo-inductive factors, and one of the most abundant and available sources of osteoblasts in the bone marrow [16]. The aim of this study was to shed more light on the effect of bone-marrow-derived stem cells on DO by assessment of bone density.

 Materials and methods



Animals

The study included 17 goats (Capra aegagrus hircus), each weighing 10–15 kg, with ages ranging between 6 and 12 months. They were housed in the animal house of Pancreas Transplant Unit in Ain Shams University provided with a controlled light (dark–light cycle 12 h) and temperature 25±2°C [14]. Animals were kept for 1 week for adaptation and observation before being subjected to this study. Animals were fed a soft diet of standard Lucerne and hay. Animals were divided into three groups: zero control (n=3), positive control (injured with spontaneous cure) (n=7), and treated (injured and treated by stem cells) groups (n=7). They were marked to identify animals of each group.

Surgical protocol

The submandibular area of the right side was shaved and scrubbed with povidone–iodine 10–12% and draped with sterile towels. Local anesthetic articaine 4% with 1 : 200 000 epinephrine field blocks was injected around the submandibular area as pre-emptive anesthesia and to provide homeostasis [14]. The incision was done by size 10 Bade Parker (Aspen Surgical, Michigan, USA) surgical blades on two layers at the submandibular region of the goat mandible, which was followed by dissection of superficial fascia, platysma, and periosteum until the bone was reached. Mental nerve dissection followed to preserve the nerve ([Figure 1]a). A vertical corticotomy from the superior border to the inferior border of the mandible ([Figure 1]b) was made in the mandibular body in the right side using fissure bur number one distal to first premolar by a straight low-speed hand piece associated with copious irrigation by saline followed by complete osteotomy of mandibular body using chisel and mallet [17]. A modified custom was made monodirectional. A stainless steel (Medical 316L) distractor was fixed in place at both osteotomy ends by two 2.0-mm miniscrews self-threading after drilling by the 2.0 system drill ([Figure 1]c). Trial activation and deactivation were made to assure function. An anterior skin puncture at the lip region was made to allow exit of distractor activation screw through the skin for activation during distraction procedure. Layered closure of the wound was done using size 0 black silk sutures for skin and 3-0 vicryl for the deep muscle layer and fascia. Deep tissues were closed first followed by the superficial layer. Activation screw of the distractor was kept extending outside the wound, allowing distraction following the surgical procedure [14].{Figure 1}

Distraction protocol

Distractors were kept in a state of latency for 5 days, allowing blood clot formation and hematoma followed by callus formation before active distraction. The active distraction of the goats followed latency at a rate of 1 mm per day for 10 days to create a distraction gap of 10 mm between both osteotomy ends by rotation of the distractor screw extruded from the skin of the goat mandible. During the time interval, between 10 and 20 days, MSCs were injected into the distraction gap and then followed by consolidation period [18].

Stem cell preparation

At the time of experiment termination, goats were anesthetized using 4% isoflurane in O2 and subsequently euthanized by CO2 asphyxiation followed by a bilateral thoracotomy to ensure death. Bone marrow aspiration was carried out from iliac crest using wide-pore gauge spinal needle to aspirate 10–20 ml under aseptic conditions on preservative-free heparin ([Figure 1]d). Bone marrow aspirate was washed with Dulbecco’s low-glucose modified Eagle’s medium (Hyclone, Logan, Utah, USA) containing 1% antibiotic–antimycotic (Gibco, Grand Island, New York, USA) and centrifuged through a density gradient (Ficoll-Plaque 1.077 g/ml; GE Biosciences, Piscataway, New Jersey, USA) for 30 min in a centrifugal field of 9000 m/s to remove lymphocyte and erythrocyte populations [19]. Mononuclear cells were resuspended in complete culture medium [minimum essential media (MEM); Gibco/BRL, Gaithersburg, Maryland, USA]; 2 mm of the goat’s own serum was selected for rapid growth of MSC (Atlanta Biologicals, Atlanta, Georgia, USA), as well as 100 U/ml of penicillin, 100 mg/ml streptomycin, and 2 mmol/l l-glutamine (Gibco/BRL). All cells were plated in 15 ml of medium and incubated in T25 flasks (Falcon Plastics, Los Angeles, California, USA) at 37°C, with 95% humidity and 5% CO2, in complete Dulbecco’s modified Eagle’s medium in concentrations of 500 000 cells/ml. After 24 h, nonadherent cells were discarded, and of the adherent cells the spindle-shaped cells were morphologically observed and evaluated on a regular basis using an inverted light microscope. The adherent cells were then washed with PBS and harvested by incubation for 4 min at 37°C in 4 ml of 0.25% trypsin/1-methyl ethylene diamine tetra acetic acid (EDTA). After incubation, the trypsin was inactivated by the addition of 5 ml of MEM. The cells were washed twice with PBS. Cell viability was determined using the trypan blue exclusion test. The cell population was characterized by typical fibroblast-like morphology, immunophenotyping (CD34 negative and CD44 positive), and the ability to differentiate. The cell numbers were counted with a hemocytometer. Flow cytometric enumeration of CD44+ cells was done using a FACS Caliber flow cytometer (Becton Dickinson, Franklin Lake, New Jersey, USA) with the use of phycoerythrin-conjugated anti-CD44 [19]. In this study, we designated the adherent cell population as ‘MSCs’ given the established osteogenic potential of these cells under proper culture conditions. In the treated group, after 10 days of distraction, the prepared stem cells (1.5 million cells) were applied using 18-G needles in the distracted gap every 10 days. The positive control and treated group had 30 days of reconsolidation following the active distraction to allow healing and maturation of the distracted bone.

Flow cytometric and phenotypic analysis of marrow-derived mesenchymal stem cells

Flow cytometry of a cell suspension carried out using samples up to 1×107 cells can measure multiple cellular constituents and activities [20] and enables correlation of these measurements with other cellular parameters, such as cell size, lineage, and viability [21]. Passage 3–5 cultured cells were used for the analysis of cell surface molecule pattern. The cells were washed with PBS and detached with trypsin/EDTA suspended in complete culture medium and analyzed according to Dvorakova et al. [22]. Controls and MSCs (3×105 per sample) were incubated with primary antibodies in 10× dilution on ice in the dark for 20 min. Cells were washed, resuspended in 50 ml of PBS, and vital staining with Hoechst 33258 (Molecular Probes, Carlsbad, California, USA) was carried out at room temperature for 10 min. Immunophenotyping analysis was performed against the following antigens: CD29, CD34, and CD90 [23].

Osteogenic differentiation

For the osteogenic differentiation studies on 2D petri dishes, hAFSCs or hMSCs were seeded at a density of 3000 cells/cm2 and were cultured in low-glucose DMEM medium with 10% FBS (Invitrogen, Carlsbad, CA), Pen/Strep and osteogenic supplements (50 ng/mL rhBMP-7 from Stryker Biotech (Hopkinton, MA) or 100 nM dexamethasone, 10 mM b-glycerophosphate, 50 mg/mL ascorbic acid-2-phosphate (Sigma-Aldrich, St. Louis, MO)). MSCs were incubated for 21 days and medium was changed every third day [24]. Undifferentiated mesenchymal stem cells have no extracellular calcium deposits, whereas differentiated osteoblasts feature vast extracellular calcium deposits in vivo and in vitro. Calcium deposits are, therefore, an indication of successful differentiation of MSC into osteoblasts and in vitro bone formation. Calcium deposits can specifically be stained bright orange-red using Alizarin Red S [25].

Mesenchymal stem cells transplantation

MSCs were transplanted into the distracted bone of lower jaw of the goat in the treated group, and after 10 days of distraction the prepared stem cells (three million cells) were applied using 18-G needles in the distracted gap on two doses every 10 days. The treated and control group had 30 days of reconsolidation following the active distraction to allow healing and maturation of the distracted bone. Therefore, assessment of bone quality was carried out after 30 days of transplantation [14].

Bone formation assessment

Assessment of bone quality by CBCT was applied with fixed exposure dose, using standardized two examiner inspection (inter examiner reliability) at fixed positions in axial, coronal, and sagittal cuts followed by tabulation and calculation [26],[27].

Histomorphometric evaluation

Sagittal sections from the mandible of the animal groups were dissected for histological study. The 10-mm distraction regenerate was osteomized and from normal bony mandible was fixed in 10% buffered formalin for 24 h. The specimens were decalcified in 10% EDTA for about 3 weeks before paraffin embedding. Sections, 5–7 μm thick, were cut and stained with hematoxylin and eosin (H&E) and Masson’s trichrome stain [28].

Trabeculae and cell numbers were estimated by H&E slide examination by light microscope and were tabulated [29].

Masson’s trichrome stain was performed to detect collagen fibers; the collagen fibers were stained blue, whereas the nuclei were black and the background was red. Soft and hard tissues changes, particularly bony maturation, were analyzed and related to the consolidation period. The results were tabulated and the average was calculated [6],[28]. The histomorphometric parameters of the stained sections were measured by quantitative evaluations of the parameters of the bone, as described by Balena et al. [30]. It was performed using Image Analyser (Leica Q Win and Q Go program; Leica GMBH, Weilburg, Germany) in the Histology Department, Faculty of Medicine, Ain Shams University measuring the following: (a) outer cortical bone thickness (μm): mean width of outer cortical bone measured in H&E slides; (b) trabecular bone thickness in μm: the mean width of trabeculae measured in H&E slides; (c) percentage area of osteoid tissue: the mean percentage of osteoid tissue measured in Masson’s trichrome slides.

Data management and analysis

The collected data were revised, coded, tabulated, and introduced to the computer using SPSS, 22.0 (IBM), USA for Windows for analysis according to the type of data obtained for each parameter. Unpaired Student’s t-test was used to compare the two groups. P less than 0.05 was considered statistically significant. Data were presented as mean±SD.

 Results



Bone formation assessment and radiographic evaluation

CBCT was adopted for bone density assessment. Obtained images were examined using Planmeca Romexis Viewer for examining bone density of newly formed bone and assessed and compared between treated and positive control groups regarding bone density, and it showed high statistical significance with the appearance of dense and high-quality newly formed bone in the lower jaw, as seen in [Figure 2]a and [Figure 2]b. t-Test revealed a high statistical significance between the treated and control groups (P<0.05). The sample size effect for Cohen’s d was 7.9, which is considered to be a large effective size to obtain acceptable statistical data ([Figure 2] and [Table 1]).{Figure 2}{Table 1}

Stem cell isolation and characterization

The initial culture of the isolated stem cells contained a crowded cell population with a majority of small spherical cells. However, the number of rounded-shape cells showed a gradual apparent decrease and the fibroblast-like cells showed an apparent increase over time and upon subculture, as seen in [Figure 3]. The Initial culture of the isolated stem cells contained a crowded cell population with a majority of small spherical cells (A&B). A gradual decrease in the number of rounded shape cells (C). The appearance of fibroblast-like mesenchymal stem cells (D). Osteogenic differentiation of mesenchymal stem cells into osteoblasts and acquiring of Alizarin red S stain (E&F). MSCs were identified by surface markers; cells were stained with the CD34, CD29, and CD90 antibodies and analyzed by flow cytometry. Bone marrow-derived MSCs were shown as the expression levels of CD34 negative (a), CD29 positive (b), and CD90 positive (c) bone-marrow-derived MSCs were presented as a histogram, as seen in [Figure 4].{Figure 3}{Figure 4}

Histological examination

Examination of H&E-stained sections from the zero control group showed the normal architecture of the bone ([Figure 5] and [Figure 6]). The outer cortical layer of the mandibular bone was covered from the outside by the periosteum, which was formed of an outer fibrous layer and an inner osteogenic layer. The trabeculae of the cancellous bone had a network of irregular bone lamellae containing dense collagenous fibers and blood vessels. The bony trabeculae were separated by interconnecting spaces containing bone marrow cavities filled with blood elements and blood vessels ([Figure 6]). The osteoblasts were cuboidal to ovoid basophilic cells that constituted a single layer surrounding the surface of the bone trabeculae. Osteocytes were located within the lacunae in the bone trabeculae. Osteoclasts were multinucleated giant cells located in shallow depressions called Howships lacunae lying in cavities in the trabecular surface ([Figure 6]). The Masson’s trichrome showed that most of the bone matrix was formed of collagen fibers, which had a regular arrangement mainly in the periosteum in the outer cortical layer and in the bone trabeculae ([Figure 7]).{Figure 5}{Figure 6}{Figure 7}

Examination of the positive control goat’s mandible showed that the outer cortical layer of the mandibular bone was covered by a thin layer of periosteum in comparison with the zero control and treated groups. The trabeculae of the inner cancellous bone lost their normal architecture and appeared few, thin, and interrupted. They were separated by widened bone marrow spaces. Some bone trabeculae showed irregular eroded surface and others were observed as the island of widely separated spicules. Moreover, some trabeculae appeared thin as compared with zero control and treated groups. It showed areas of low density with a decrease in the stained bone matrix ([Figure 8]). The osteoblast and osteocytes were few, ill-defined, and small in size. The osteoclast decreased in number and some of them were enlarged and contain a large number of nuclei ([Figure 9]). By Masson’s trichrome stain the healed woven bone (nonlamellar bone, reticulated bone) was characterized by an increase in the amount of collagen fibers of the matrix and was arranged irregularly in the form of interlacing networks in the cortical layer and in between bone lamellae ([Figure 10]).{Figure 8}{Figure 9}{Figure 10}

Examination of H&E stained sections from the treated group showed the nearly normal architecture of the bone. The outer cortical layer of the mandibular bone was covered by the periosteum, which was formed of an outer fibrous layer and an inner osteogenic layer ([Figure 11]). The cancellous bone was seen formed of branching and anastomosing trabeculae with bone forming marrow spaces or cavities filled with blood elements and blood vessels. The trabeculae were thicker than that of the control groups. The matrix of some trabeculae showed more basophilic staining ([Figure 12]). The osteoblasts were ovoid basophilic cells that constituted a single layer surrounding the surface of the bone trabeculae. Inside the trabeculae, osteocytes were located within their lacunae. Osteoclasts were multinucleated giant and occasionally seen situated adjacent to resorbed bone in their Howship’s lacunae. The space between the trabeculae was seen to be filled with bone marrow among the hematopoietic tissue ([Figure 12]). By Masson’s trichrome, the healed bone is characterized by a decrease in the amount of collagen fibers of the matrix, and thus the mineralized bone appeared red. The cancellous bone appeared thin with less cavity formation and a reduction in green-stained collagen in the trabeculae, whereas the outer cortex has a normal amount of collagen fibers ([Figure 13]).{Figure 11}{Figure 12}{Figure 13}

Histomorphometric evaluation

Outer cortical bone thickness and trabecular bone thickness

Measurement of the outer cortical bone and trabecular bone thickness of H&E-stained sections histomorphometrically showed an increase in the outer cortical bone and trabecular bone thickness in the treated group as compared with the control group ([Figure 8] and [Figure 11]). The independent-samples t-test revealed no statistical significance between the treated and positive control group in the outer cortical layer (P>0.05). The sample size effect Cohen’s d was 1.0098, which is considered to be a large effective size to get acceptable statistical data ([Table 2]). The independent-samples t-test revealed high statistical significance between the treated and positive control group in the trabecular bone thickness (P<0.05). The sample size effect Cohen’s d was 2.318, which is considered to be a large effective size to obtain acceptable statistical data ([Table 2]).{Table 2}

Area percentage of osteoid tissue

By using Masson’s trichrome-stained samples the histomorphometrical examination showed a decrease in the osteoid percentage in the treated group as compared with positive controls ([Figure 10] and [Figure 13]). The independent-samples t-test revealed a high statistical significance between the treated and positive control groups (P<0.05). The sample size effect Cohen’s d was 1.94, which is considered to be a large effective size to obtain statistical data ([Table 3]).{Table 3}

 Discussion



Cellular therapy using MSCs has been an extremely attractive cell model for research into the treatment of a variety of diseases over the past decades. In this study, MSCs were isolated and transplanted into the distracted bone of the lower jaw of the goat aiming to evaluate the role of stem cell in regeneration of the distracted bone through osteogenesis. After 30 days of MSC transplantation, CBCT was adopted for bone density assessment, which revealed formation of a new bone via the ability of MSCs to transdifferentiate into osteoblasts, and this bone was dense with high quality. In addition, bone density was statistically highly significant in the treated group as compared with the positive control group. This study attributed the osteogenesis of the distracted jaw to the ability of transplanted MSCs to transdifferentiate into osteoblasts with improved bone quality. Our results were supported by the studies of Hernigou et al. [11] and Tarte et al. [12] who used autologous MSCs for bone repair cell therapy. This is because of their capacity for self-renewal and their ability to differentiate into numerous different tissue types, such as bone, cartilage, and fat [31].

In this study, there was enhancement of bone consolidation in mandibular DO in the goat; this was confirmed by Alkaisi et al. [32], as he reported that transplantation of human dental pulp stem cells enhances bone consolidation in mandibular DO.

The results of this study were confirmed by Bayati et al. [33] as he said that MSCs exert their therapeutic effects through four axes, which are as follows: (a) the ability to reside in injured tissues when injected intravenously; (b) the ability to differentiate into various cell types; (c) the ability to secrete a wide variety of bioactive molecules that act in the repair of injured cells and inhibit inflammation; and (d) the lack of immunogenicity and an immunomodulatory capacity. This study was in agreement with several studies that investigated transplantation of MSCs into distraction sites and evaluated the mechanical strength of distraction gaps treated with MSCs injected on the first day of consolidation in a sheep model, choosing this animal model because of the similarity of the sheep mandible to the human jaw. The results of such studies revealed that Hemi-mandibles treated with MSCs had greater total and compact bone ratios in the regenerated zone [34],[35].

In this experiment, MSC transplantation showed that the bone marrow stem cells accelerated the bone healing and improved the quality of bone, as well as shortened the distraction or consolidation time. This was in agreement with Qi et al. [14] through his attempts to evaluate the effect of bone marrow stem cells injection as a medical line of treatment on distraction gaps made in 40 rats for 27 and 55 days, respectively. The newly formed bone was assisted by using radiographic density evaluation, histological examination, and histomorphometric analysis. The advantage of using the MSCs combined with DO carries a new hope of improving the bone quality and quantity of the distracted bone regenerate.

In addition, endogenous MSCs were attracted to sites of new bone formation, much like leukocytes hone in on sites of inflammation [36]. In this study, there was an increase of osteogenic cells and the trabecular thickness; these results were in agreement with those of Aykan et al. [37] who explained that the recruitment of MSCs by the stromal-cell-derived factor-1 or chemokine receptor-4 pathway was studied in a rat model of mandibular DO. The results suggested that stromal-cell-derived factor-1 facilitates migration of MSCs in vitro and in vivo, and can be inhibited by an antagonist of chemokine receptor-4. This growth factor pathway provides another potential target that can be manipulated to upregulate an ongoing endogenous process. MSCs cultured with or without osteogenic media may enable maintenance of osteogenic differentiation of MSCs transplanted into distracted zone or constructed scaffolds and/or osteogenically stimulated host cells without a need for an exogenous drug delivery [38],[39],[40],[41],[42].

Histomorphometric assessment is considered the reliable method in measurement of bone quantity to express the bone quality of newly formed bone in general [26] and distracted bone specifically [6]. Many studies supported the technique of use of histomorphometry combined with H&E stain in bone quantity assessment by measuring trabecular bone thickness and cortical bone thickness [43],[44],[45]. Masson’s trichrome stain was used to assess the percentage of osteiod in newly formed bone and is considered a reliable method in the assessment of new bone formation [46],[47]. There was a significant increase in trabecular bone thickness in the treated group compared with that of the positive control, with a high statistical significance, which may be attributed to the addition of MSCs and evidence for their effect in improving bone quality. Cohen’s d test showed that the sample size is large enough to give statistical data of high significance in agreement with [14]. Histomorphometric assessment of cortical bone thickness was done in previous studies conducted on DO to assess the regenerate quality [48]. There was an increase in the cortical bone thickness in the treated group as compared with the control, in agreement with Qi et al. [14]. However, there was no statistical significance, which may be attributed to the early sacrification of the experiment, because the cortical bone usually develops after two to three months during the phase of remodeling [49]. Histomorphometrical assessment of Masson’s trichrome sample showed a large decrease in the percentage of the osteoid and newly formed bone in the treated group compared with the positive control group, and this decrease was associated with an increase of the mature bone percentage. The previous results were in agreement with a study conducted by Song et al. [50] as he said the decrease in osteiod percentage and the increase in the mature bone formation indicated faster bone healing. There was a significant correlation between the increase in bone density in the treated group compared with the positive control group and the increase in trabecular bone thickness in the treated group compared with the positive control group, confirming the effect of MSC application. In addition, the decrease in the percentage of osteoid bone was directly proportional to the increase of mature bone percentage, which was more calcified and having more radiographic density, confirming the increased radiographic density of the formed bones.

Referring to the route of MSC transplantation, some studies revealed that MSCs infused in children with osteogenesis imperfecta not only engrafted without any side effect but also increased 3 months later the mean number of osteoblasts, the formation of new lamellar bone, and the total body mineral content. In addition, they eventually lowered the frequency of fractures and enhanced the body growth rate [51]. Other studies in animals showed that the best route of MSC transplantation to induce local repair or regeneration of bone, cartilage, or tendon is the in-situ injection or implant [52],[53]. Currently, an efficient approach to repair bone defects seems to be the local implantation of porous cell–matrix composites loaded with autologous bone marrow MSCs, previously collected from the patient and expanded in vitro under stringent culture conditions [54]. This study concluded that MSC transplantation locally into distracted lower jaw showed promising results in improving the bone quality of the DO animal model for bone regeneration.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Dimitriou R, Jones E, McGonagle D, Giannoudis PV. Bone regeneration: current concepts and future directions. BMC Med 2011; 9:66.
2Ramamoorthi M, Bakkar M, Jordan J, Simon D, Tran O. Osteogenic potential of dental mesenchymal stem cells in preclinical studies: a systematic review using modified ARRIVE and CONSORT guidelines. Stem Cells Int 2015; 2015:1–28.
3Bigi MM, Lewicki M, Ubios AM, Mandalunis PM. Experimental model of distraction osteogenesis in edentulous rats. Braz Oral Res 2011; 25:217–224.
4Snyder CC, Levine GA, Swanson HM, Browne EZ. Mandibular lengthening by gradual distraction. Plast Reconstr Surg 1973; 51:506–508.
5Troulis MJ, Glowacki J, Perrott DH, Kaban L. Effects of latency and rate on bone formation in a porcine mandibular distraction model. J Oral Maxillofac Surg 2000; 58:507–514.
6Al Ruhaimi KA. Comparison of different distraction rates in the mandible: an experimental investigation. Int J Oral Maxillofac Surg 2001; 30:220–227.
7Cheung LK, Zheng LW, Ma L. Effect of distraction rates on expression of bone morphogenetic proteins in rabbit mandibular distraction osteogenesis. J Craniomaxillofac Surg 2006; 34:263–269.
8Rachmiel A, Potparic Z, Jackson IT, Sugihara T, Clayman L, Topf JS. Midface advancement by gradual distraction. Br J Plast Surg 1993; 46:201–207.
9Lewinson D, Maor G, Rozen N, Rabinovich I, Stahl S, Rachmeil A. Expression of vascular antigens by bone cells in a membranous bone distraction system. Histochem Cell Biol 2001; 116:381–388.
10Barrena GE, Rosset P, Lozano D, Stanovici J, Gerbhard F. Bone fracture healing: cell therapy in delayed unions and nonunions. Bone 2015; 70:93–101.
11Hernigou P, Homma Y, Flouzat-Lachaniette CH, Poignard A, Chevallier N, Rouard H. Cancer risk is not increased in patients treated for orthopedic diseases with autologous bone marrow cell concentrate. J Bone Joint Surg Am 2013; 95:2215–2221.
12Tarte K, Gaillard J, Lataillade JJ, Fouillard L, Becker M, Mossafa H. Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation. Blood 2010; 115:1549–1553.
13Kitoh HT, Kitakoji T, Tsuchiya H, Mitsuyama H, Nakamura H, Katoh M, Ishiguro N. Transplantation of marrow-derived mesenchymal stem cells and platelet-rich plasma during distraction osteogenesis—a preliminary result of three cases. Bone 2004; 35:892–898.
14Qi M, Hu J, Zou S, Zhou H, Han L. Mandibular distraction osteogenesis enhanced by bone marrow mesenchymal stem cells in rats. J Craniomaxillofac Surg 2006; 34:283–289.
15Croucher PI, Garrahan NJ, Compston JE. Assessment of cancellous bone structure: comparison of strut analysis, trabecular bone pattern factor and marrow space star volume. J Bone Miner Res 1996; 11:955–961.
16Romih M, Delecrin J, Heymann D, Passuti N. The vertebral interbody grafting site’s low concentration in osteogenic progenitors can greatly benefit from addition of iliac crest bone marrow. Eur Spine J 2005; 14:645–648.
17Zou S, Hu J, Wang D, Li J, Tang Z. Changes in the temporomandibular joint after mandibular lengthening with different rates of distraction. Int J Adult Orthodon Orthognath Surg 2001; 16:221–225.
18Cai M, Shen G, Cheng AH, Lin Y, Yu D, Ye M. Navigation-assisted mandibular body distraction osteogenesis: a preliminary study in goats. J Oral Maxillofac Surg 2014; 72:168e1–168e7.
19Kishk N, Abokrysha NT, Gabr H. Possible induction of acute disseminated encephalomyelitis (ADEM)-like demyelinating illness by intrathecal mesenchymal stem cell injection. J Clin Neurosci 2013; 20:310–312.
20Kurtz JW, Wells WW. Automated fluorometric analysis of DNA, protein and enzyme activities: application of methods in cell culture. Anal Biochem 1979; 94:166–174.
21Al-Rubeai M, Welzenbach K, Lloyd DR, Emery AN. A rapid method for evaluation of cell number and viability by flow cytometry. Cytotechnology 1997; 24:161–168.
22Dvorakova J, Hruba A, Velebny V, Kubala L. Isolation and characterization of mesenchymal stem cell population entrapped in bone marrow collection sets. Cell Biol Int 2008; 32:1116–1125.
23Klingel S, Rothe G, Kellermann W, Valet G. Flow cytometric determination of cysteine and serine proteinase activities in living cells with rhodamine 110 substrates. Methods Cell Biol 1994; 41:449–459.
24Sun H, Feng K, Hu J, Soker S, Atala A, Peter X. Osteogenic differentiation of human amniotic fluid-derived stem cells induced by bone morphogenetic protein-7 and enhanced by nanofibrous scaffolds. Biomaterials, 2010; 31:1133–1139.
25Yamamoto N, Isobe M, Negishi A, Yoshimasu H, Shimokawa H. Effects of autologous serum on osteoblastic differentiation in human bone marrow cells. J Med Dent Sci 2003; 50:63–70.
26Loubele M, Guerrero ME, Jacobs R, Suetens P, van DS. A comparison of jaw dimensional and quality assessments of bone characteristics with cone-beam CT, spiral tomography, and multi-slice spiral CT. Int J Oral Maxillofac Implants 2007; 22:446–454.
27Rice JA. Mathematical statistics and data analysis. 3rd ed. Australia, United Kingdom, United States: Duxbury Advanced; 2010. 16–23.
28Bancroft JD, Stevens A. Theory and practice of histological techniques”. 4th ed. Edinburgh, London: Churchill Livingstone; 1996. 585–626.
29Wei H, Zili L, Yuanlu C, Biao Y, Cheng L, Xiaoxia W, Yang L. Effect of cariin on bone formation during distraction osteogenesis in the rabbit mandible. Int J Oral Maxillofac Surg 2011; 40:413–418.
30Balena R, Toolan BC, Shea M, Markatos A, Myers ER, Lee SC et al. The effects of 2-year treatment with the aminobisphosphonate alendronate on bone metabolism, bone histomorphometry, and bone strength in ovariectomized nonhuman primates. J Clin Invest 1993; 92:2577–2586.
31Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006; 8:315–317.
32Alkaisi A, Ismail AR, Mutum SS, Ahmad ZA, Masudi S, Abd Razak NH. Transplantation of human dental pulp stem cells: enhance bone consolidation in mandibular distraction osteogenesis. J Oral Maxillofac Surg 2013; 71:e1–13.
33Bayati V, itabar MH, Gazor R, Nejatbakh R, Bijannejad D. Expression of surface markers and myogenic potential of rat bone marrow- and adipose-derived stem cells: a comparative study. Anat Cell Biol 2013; 46:113–121.
34Kim IS, Cho TH, Lee ZH, Hwang SJ. Bone regeneration by transplantation of human mesenchymal stromal cells in a rabbit mandibular distraction osteogenesis model. Tissue Eng Part A 2013; 19:66–78.
35Ma D, Ren L, Yao H. Locally injection of cell sheet fragments enhances new bone formation in mandibular distraction osteogenesis: a rabbit model. J Orthop Res 2013 31:1082–1088.
36Cao J, Wang L, Lei DL, Liu YP, Cui FZ. Local injection of nerve growth factor via a hydrogel enhances bone formation during mandibular distraction osteogenesis, Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2012; 113:48–53.
37Aykan A, Ozturk S, Sahin I, Ural A, Oren NC, Isik S. Biomechanical analysis of the effect of mesenchymal stem cells on mandibular distraction osteogenesis. J Craniofac Surg 2013; 24:e169–e175.
38Thibault R, Baggett A, Mikos G, Kasper F. Osteogenic differentiation of mesenchymal stem cells on pregenerated extracellular matrix scaffolds in the absence of osteogenic cell culture. Tissue Eng Part A 2010; 16:431–440.
39Deshpande SS, Weiss DM, Donneys A. An isogenic model of murine mandibular distraction osteogenesis. J Cranifac Surg. 2013; 24:540–544.
40Sun Z, Tee BC, Kennedy KS. Scaffold-based delivery of autologous mesenchymal stem cells for mandibular distraction osteogenesis: preliminary studies in a porcine model. PLoS One 2013; 8:74672.
41Wu G, Hu C, He X. Effect of gene transfecting at different times on mandibular distraction osteogenesis. J Craniofac Surg 2013; 24:232–236.
42Earleya M, Butts SC. Update on mandibular distraction osteogenesis. Curr Opin Otolaryngol Head Neck Surg 2014; 22:276–283.
43Choi IH, Chung CY, Cho TJ, Yoo WJ. Angiogenesis and mineralization during distraction osteogenesis. J Korean Med Sci 2002; 17:435–447.
44Pampu AA, Ozkaynak O, Senel FC, Cankaya M. The effects of osteoformin on mineralisation and quality of newly formed bone during mandibular distraction osteogenesis in rabbits. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108:833–837.
45Cao J, Wang L, Du ZJ. Recruitmen to fexogenousmesenchymal stem cells in mandibular distraction osteogenesis by the stromal cell-derived factor-1/chemokinereceptor 4pathwayin rats. Br J Oral Maxillofac Surg 2013; 51:937–941.
46Hagiwara T, Bell WH. Effect of electrical stimulation on mandibular distraction osteogenesis. J Craniomaxillofac Surg 2000; 28:12–19.
47Liang L, Liu C, Bu R. Distraction osteogenesis for bony repair of cleft palate by using persistent elastic force: experimental study in dogs. Cleft Palate Craniofac J 2005; 42:231–238.
48Jacobsen KA, Al-Aql ZS, Wan C, Fitch JL. Bone formation during distraction osteogenesis is dependent on both VEGFR1 and VEGFR2 signaling. J Bone Miner Res 2008; 23:596–609.
49Samchukov ML, Cope JB, Cherkashin AM. Biological foundation. In: Samchukov ML, Cope JB, Cherkashin AM, editors Distraction osteogenesis interactive course on CD-ROM. Vol. 1. Dallas, Texas: Global MedNet Inc.; 1999. pp. 77–99.
50Song S, Yun Y, Kim H, Park K. Bone formation in a rat tibial defect model using carboxymethyl cellulose/bioc/bone morphogenic protein-2 hybrid materials. Biomed Res Int 2014; 2014:230152.
51Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WW, Gordon PL. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999; 5:262–264
52Kadiyala S, Young RG, Thiede MA, Bruder SP. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant 1997; 6:125–134.
53Richards M, Huibregtse BA, Caplan AI, Goulet JA, Goldstein SA. Marrow-derived progenitor cell injections enhance new bone formation during distraction. J Orthop Res 1999; 17:900–908.
54Krampera M, Pizzolo G, Aprili G, Franchini M. Mesenchymal stem cells for bone, cartilage, tendon and skeletal muscle repair. Bone 2006; 39:678–683