CRANIOMAXILLOFACIAL DEFORMITIES/COSMETIC SURGERY
Transplantation of Human Dental Pulp Stem Cells: Enhance Bone Consolidation in Mandibular Distraction Osteogenesis Amera Alkaisi, PhD,* Abd Rashid Ismail, MSc,y Samarendra S. Mutum, PhD,z Zainal A. Rifin Ahmad, PhD,x Sam’an Masudi, MSc,k and Noor Hayati Abd Razak, MClin.Dent(OMFS){ Purpose: The main aim of the present study was to evaluate the capacity of stem cells from human exfoliated deciduous teeth (SHED) to enhance mandibular distraction osteogenesis (DO) in rabbits. Materials and Methods:
A randomized controlled trial was conducted. Eighteen skeletally immature New Zealand white rabbits were divided into 2 groups, with 9 in the control group and 9 in the SHED group. The SHED were isolated, expanded, and characterized. Six million cells were transplanted into the distracted area during the osteotomy period. After a 4-day latency period, a total of 6 mm was distracted for 6 days. The newly formed bone was analyzed radiologically, histologically, and histomorphometrically at 2, 4, and 6 weeks postoperatively. Nonparametric analysis of variance (Kruskal-Wallis test) was used for data analysis, and P < .05 was considered statistically significant.
Results: The cell lineage was positive for the 2 mesenchymal stem cell markers tested (CD105 and CD166). More mature bone in the SHED transplanted group was observed radiographically and histologically. Histomorphologically, the percentage of newly formed bone after 2, 4, and 6 weeks was 18.41% and 41.53%, 31.68% and 59.78%, and 52.34% and 65.24% in the control and SHED groups, respectively. The difference between the groups was statistically significant (P = .012). The bone union and stage of bone maturity scores were significantly different between the control and SHED groups (P = .006 and P = .011, respectively). Conclusions:
Our findings suggest that SHED can serve as an additional cell resource for DO enhancement in rabbits and might be a promising model for the reconstruction of large mandibular defects in human oral maxillofacial surgery. Ó 2013 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 71:1758.e1-1758.e13, 2013
Distraction osteogenesis (DO) is a surgical technique used to lengthen bone by creating a corticotomy, placing a rigid distractor across the cut bone and gradually activating the device.1 This technique was first
developed and defined by Codivilla2 and first reported by Ilizarov3-6 as a technique for limb lengthening. McCarthy et al7 reported the first clinical case of mandibular distraction using an extraoral device, and
*Lectural, PhD Student, Universiti Sains Malaysia; Lectural in University of Anbar, College of Dentistry; Oral and Maxillofacial
{Associated Professor, Universiti Sains Malaysia School of Dental Sciences, Head of Oral and Maxillofacial Surgery Department, Kubang
Department, Alramady, Iraq.
Kerian, Kelantan, Malaysia.
yProfessor, Community Dentistry, Faculty of Dentistry, Dean, Melaka Manipal Medical College, Bukit Baru, Melaka, Malaysia.
Address correspondence and reprint requests to Mrs Alkaisi: Universiti Sains Malaysia, Lectural in University of Anbar, College
zAssociated Professor, Universiti Sains Malaysia School of
of Dentistry, Oral and Maxillofacial Department, Alramady, Iraq;
Medical Sciences, Department of Pathology,16150, Kubang
e-mail:
[email protected]
Kerian, Kelantan, Malaysia.
Received December 19 2012
xHead, University Science Malaysia School of Materials and Mineral Resources Engineering, Department of Ceramics, Nibong
Accepted May 16 2013
Tebal, Penang, Malaysia.
0278-2391/13/00522-3$36.00/0
kAssociated Professor, Universiti Sains Malaysia School of Dental
Ó 2013 American Association of Oral and Maxillofacial Surgeons
http://dx.doi.org/10.1016/j.joms.2013.05.016
Sciences, Conservative Department, Kubang Kerian, Kelantan, Malaysia.
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after their successful clinical results, this concept was investigated further. A number of clinical and experimental studies have involved various anatomic regions of the mandible,8-11 and this technique has been widely used for severe craniomaxillofacial deformities.12,13 DO is becoming the treatment of choice for the surgical correction of congenital and acquired craniofacial skeletal anomalies. Unlike fracture healing, DO is an active process of bone regeneration under controlled mechanical stimulation, and it has been regarded as a type of endogenous tissue engineering.14,15 However, the duration of the bone consolidation phase is quite long, approximately 1 to 2 months for every 1 cm of lengthening,16 and patients are at risk of many complications, including pain, refracture, pin loosening, infection, and, occasionally, delayed union of the distraction regenerate. Clinically, both patients and surgeons would like to shorten the treatment time of DO, but this would require a reduction in the consolidation period or an increase in the distraction rate. Various approaches have been tested to accelerate the osteogenesis, such as electrical stimulation, low-level laser therapy, mechanical compression, low-intensity pulsed ultrasound stimulation, and the injection of growth factors, among others.17-21 Recently, bone regeneration with mesenchymal stem cells (MSCs) has attracted considerable interest. The first cellbased approaches for bone tissue engineering used unfractionated fresh autologous bone marrow.22-24 Friedenstein25 identified mesenchymal stromal cells with pluripotent abilities in human bone marrow. Since then, many researchers have successfully documented the ability of bone marrow mesenchymal stromal cells to differentiate down the osteogenic lineage and to regenerate bone.26,27 Few studies have investigated whether injecting cells with osteogenic potential into the distraction gap will enhance its consolidation.28,29 All these results suggested that the transplantation of cultured osteogenic cells will have a positive effect on promoting bone formation and shortening the consolidation period. Recently, Miura et al30 demonstrated that cells with multipotential abilities residing in the pulp of human exfoliated deciduous teeth (stem cells from human exfoliated deciduous teeth [SHED]) can be isolated from disposable resources and easily expanded in vitro. This discovery has important implications for future research and potential therapeutic applications. SHED exhibit a higher proliferation rate and a greater number of population doublings compared with adult bone marrow and dental pulp stem cells.30 Moreover, it has been found that human dental pulp stem cells (hDPSCs) injected into rats were not rejected by the rats and exhibited immunosuppressive activity that could have potential clinical applications in allogeneic
in vivo stem cell transplantation, in particular, for calcified tissue reconstruction.31 Because SHED originate from the cranial neural crest, which contributes to the development of the craniofacial skeleton, the use of these cells might be a better choice for bone regeneration in craniofacial construction.32,33 However, little is known about the interactions of this cell population in vivo, particularly about their ability to regenerate bone, and no study has examined its transplantation in DO. The present study addressed 1 of the many aspects in cell-based therapy: the potential of adult tissue-harvested SHED for use in future in vivo applications to enhance bone regeneration, in general, and, specifically, to be used to enhance the consolidation period in mandibular DO. The newly formed bone in the distraction gap was evaluated through radiographic analysis, which has been assessed in DO in support of histologic and histomorphometric data. To the best of our knowledge, ours is the first study to compare the osteopromoting activities of SHED with regard to their osteogenic potential in DO in nonimmunosuppressed rabbits.
Materials and Methods EXPERIMENTAL PROCEDURES
Isolation, Expansion, and Characterization of SHED Normal exfoliated human deciduous incisors and canine teeth were collected from 4 children aged 7 to 8 years old at the dental clinic, Universiti Sains Malaysia School of Dental Sciences. The human ethics committee of Universiti Sains Malaysia approved the experimental protocol (no. USMKK/PPP/JEPeM [211.3.(7)]), all the patients’ parents provided consent. Preparation of Primary Culture Each tooth was immediately washed using normal saline and placed into a 20-mL container of transporting medium containing Hank’s balanced salt solution (Gibco, Invitrogen, Carlsbad, CA) supplemented with 1% penicillin-streptomycin (Gibco, Invitrogen). The tooth was partially cut around the cementoenamel junction using a hard tissue cutter and soaked in 70% ethanol. It was then washed several times with Dulbecco’s phosphate-buffered saline (DPBS) (Gibco, Invitrogen). The teeth were maintained in DPBS and then transferred to a class II biosafety cabinet for the pulp extirpation procedure. Each tooth was completely cut into 2 pieces, and the pulp tissue was gently removed using a sterile endodontic barbed broach. It was then minced and centrifuged at 1000 rpm for 5 minutes in a 15-mL centrifuge tube containing DPBS. The pulp tissue was digested by adding 5 mL collagenase type II (5 mg/mL) and then incubated overnight in a 5% carbon dioxide incubator. A single
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cell suspension was obtained by passing the cells through a 70-mm cell strainer, and the filtered cells were centrifuged, the supernatant was removed, the procedure was repeated twice, and 1 mL growth medium was added and resuspended. The cell suspension was added to a T25 cm2 tissue culture flask containing 5 mL growth medium and then incubated in a 5% carbon dioxide incubator at 37 C for 24 hours and labeled as passage 0. After 24 hours, the medium was changed. The cells were observed daily to check the culture conditions. Fresh medium was replaced every 2 days until the cells reach 80% confluence, and then the cells were passaged and transferred into a T75 culture flask and labeled as passage 1. Cell Morphology The cellular morphology was observed daily in the 75-cm2 culture flask under 5 magnification with an inverted light microscope. Characterization of SHED Using Flow Cytometry At passage 3, the prepared fibroblast-like cells were characterized to confirm that they were MSCs using flow cytometry analysis against specific MSC markers, including CD105 (endoglin) monoclonal mouse antihuman (R&D Systems, Minneapolis, MN), and CD166 (activated leukocyte cell adhesion molecule) monoclonal mouse antihuman (BD Pharmingen, San Jose, CA) antibodies. Procedure The pulp cell suspensions were harvested and counted. The cell pellets were resuspended at a concentration of 1 105 cells/mL in tissue culture tubes (BD Bioscience). Approximately 20 mL of phycoerythrin conjugated antibodies were added as follows: anti–CD166-PE and anti–CD105-PE, with incubation for 30 minutes at room temperature in the dark. The suspension was washed with 2 mL DPBS twice and centrifuged at 1,000 rpm for 5 minutes; the supernatant was then discarded. The cell pellet was suspended with 200 mL DPBS, and the tube was covered with aluminum foil. Flow cytometry was performed using a BD FACSCanto II (BD Biosciences). Data were analyzed using BD FACSDIva software (BD Biosciences). Surgical Procedure A total of 22 skeletally immature New Zealand white rabbits, aged 3 to 5 months, with a mean weight of 2.7 0.31 kg, were used in the present study. The animal ethics committee of the Universiti Sains Malaysia [no. USM/Animal Ethics Approval/2010/(58)(226)] approved the experimental protocol. The rabbits were divided into 2 groups: group A, 9 rabbits as the control group; and group B, 9 rabbits as the experimental group. The rabbits were anesthetized preoperatively with an intramuscular administration
STEM CELL TRANSPLANTATION
FIGURE 1. Internal distractor. Alkaisi et al. Stem Cell Transplantation. J Oral Maxillofac Surg 2013.
of ketamine (35 mg/kg) and xylazine (5 mg/kg). Local administration of 2% lidocaine at the surgical site was performed. A longitudinal incision was made on the inferior border of the right mandible with the rabbit’s head hyperextended. An incomplete osteotomy cut was placed between the first premolar and the mental foramen using a water-cooled drill and a 1.0-mm carbide fissure bur. Great care was taken to preserve the inferior alveolar neurovascular bundle. Two small incisions penetrating all tissue layers were made in the submandibular area for the entrance of the distractor limbs to the lateral cortex. A SUPERscrew palatal expander (OrthoCare UK, Saltaire, West Yorkshire, UK), a 12 + 12-mmlong stainless steel device with full distraction capacity of 12 mm after modification (Fig 1), was fixed to either side of the osteotomy cut. The osteotomy was then completed with a chisel and mallet. Hemostasis was ensured, irrigation was performed, and the distractor was activated for several turns to confirm that the osteotomy was completed. The wound site was cleaned of any bone debris, and the periosteum and muscle were reapproximated. The wound was then closed in layers using 4-0 Vicryl suture, cleaned with povidone-iodine antiseptic, and neomycin ointment (antibiotic) was applied. DO Protocol The schedule for the procedures performed in the present study is shown in Figure 2. After a 4-day latency period, distraction began at a rate of 1 mm/day and continued for 6 days, resulting in a final lengthening of 6 mm. After completion of distraction, the rabbits were monitored for 2, 4, and 6 weeks postoperatively for consolidation. Preparation of SHED and Transplantation Protocol Approximately 4 to 6 T75 tissue culture flasks that were 80% to 100% confluent were passaged, and the
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FIGURE 2. Protocol method. Alkaisi et al. Stem Cell Transplantation. J Oral Maxillofac Surg 2013.
cells were counted and adjusted to 6 106. In group A, no transplantation was deposited in the osteotomy site, and in group B, 6 106 SHED were transplanted. Postoperative Care The rabbits were monitored for 2, 4, and 6 weeks postoperatively. Neomycin antibiotic cream was applied daily to the incision site and the entrance of the distractor for the first week. All the rabbits were given an intramuscular injection of enrofloxacin (Baytril) 10 mg/kg/day (Bayer, Shawnee Mission, KS) once daily for 7 days. Tramadol HCl 2 mg/kg was given once daily for pain relief for 7 days. ASSESSMENTS
Clinical Evaluation During the entire experimental period, the rabbits were observed for their diet, general condition, and behavior. They were examined for inflammation at the operation area and around the distractor, the degree of resistance when distracting, the changing aspects of occlusion, and the fixation condition of the distractors. After 2, 4, and 6 postoperative weeks, the bone segment of the sacrificed rabbits was measured and examined for mobility at the distracted site. Radiographic Examination (Soft Radiograph) After the postoperative periods of 2, 4, and 6 weeks, for each group, the rabbits were sacrificed, and the right bone segments of the mandible were separated. For plain radiography, each specimen was placed on an occlusal film with the lingual side touching the film, and a radiograph was taken with an Orthoralix 9200 x-ray machine (Gendex, Milan, Italy) using a standard of 66 KVp, 8 mA, and 12 seconds. A fixed distance of approximately 20 cm was used between the x-ray source and the object to obtain minimal distortion. The significance of callus formation, the formation of bone cortex, and
the bone marrow cavity were compared between the 2 groups. Histologic Examination The right hemimandible was dissected free of the surrounding tissue, immersed in 4% formalin for 24 hours, decalcified in a 10% solution of nitric acid for 4 days, automatically processed in a machine for dehydration, and embedded in paraffin wax. Sections 5 mm in thickness were cut longitudinally with a microtome and stained with hematoxylin and eosin for light microscopy examination. New bone formation, blood vessels, cartilage, and fibrous tissue were assessed. Histomorphometric Examination The samples were analyzed histomorphometrically using a Zeiss image analysis system (MMI 0684, Carl Zeiss, Oberkochen, Germany) to quantify the bone fill present in the defects, fibrous tissue, and cartilage. The grade of osseous regeneration was analyzed using an established numeric scoring system first described by Heilple et al34 and modified by Linde and Hedner35 for the assessment of bone healing. Serial sections were scored for 2 independent bone-forming indexes: the stage of bone union (range, 0 to 5) and the grade of bone maturity (range, 0 to 5). The images of the histologic sections were scanned by the system and saved on a computer. Using a computerized image analysis system, the regions of interest in the distracted area were outlined and calculated in square micrometers. The new bone trabeculae, cartilage, and remaining fibrous tissue were outlined and measured, and their percentages were calculated. STATISTICAL ANALYSIS
The histomorphometric data representing the percentage of bone and grade of osseous regeneration
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FIGURE 3. Cell morphology of primary culture (passage 0, 1, and 7) viewed under light microscope (magnification 10). Alkaisi et al. Stem Cell Transplantation. J Oral Maxillofac Surg 2013.
within the regions of interest were analyzed using nonparametric analysis of variance (Kruskal-Wallis test). P values
