The aim of the present study was to investigate an isolation procedure to culture mesenchymal stem cells derived from bone marrow and evaluate their potential in periodontal regeneration. Potential stem cells from bone marrow, aspirated from the iliac crest of nine mongrel canines 1 to 2 years of age, were cultivated. After the examination of surface epitopes of the isolated cells, the total RNA from osteogenic, adipogenic, and chondrogenic cell cultures were analyzed by reverse transcription polymerase chain reaction (RT-PCR) to confirm stem cell gene expressions. 2 × 107 mL of the stem cells were loaded on 0.2 mL of anorganic bovine bone mineral (ABBM) granules. In each animal, bilateral acute/chronic intrabony periodontal defects were created surgically and by placement of ligatures around the cervical aspect of the teeth. At week 5, after flap debridement, the bilateral defects were randomly assigned to 2 treatment groups: the control group received ABBM, and the test group received BMSCs-loaded ABBM. Eight weeks after transplantation, regenerative parameters were analyzed histologically and histometrically. The RNA expressions confirmed the cultivation of mesenchymal stem cell. More new cementum and periodontal ligament (PDL) were measured in the test group (cementum: 3.33 ± 0.94 vs 2.03 ± 1.30, P = 0.027; PDL: 2.69 ± 0.73 vs 1.53 ± 1.21, P = 0.026). New bone formation was similar in both groups (2.70 ± 0.86 vs 1.99 ± 1.31; P = 0.193). Mesenchymal stem cells derived from bone marrow should be considered a promising technique for use in patients with periodontal attachment loss and merits further investigations.
Introduction
Periodontium is a complex tissue comprised of two hard (bone and cementum) and two soft tissues (periodontal ligament and gingiva) with a limited capacity of regeneration. The three key elements required for tissue regeneration include cells, scaffolds, and signaling molecules. Conventional regenerative therapies are generally based on the application of scaffolds or signaling molecules. Previous systematic reviews have shown that these approaches can partially regenerate periodontal tissues.1–5 The clinical results of such procedures are variable,6,7 and histological studies evidenced that the application of graft materials often leads to healing with long junctional epithelium rather than periodontal regeneration.8,9
The current research trends are toward developing cell-based techniques for periodontal regeneration. Recently, investigators consider mesenchymal stem cells (MSCs) as seeding cells. Periodontal ligament (PDL) progenitor/stem cells are the most commonly studied cell population. The results of previous studies demonstrated that PDL-derived stem cells have a good capacity to generate dental-associated structures.7,10–20 The culture of PDL cells and differentiation control of these cells is difficult.21 One important limiting factor is the extraction of at least one healthy tooth.7,22 We have reported that dental pulp derived stem cells could be effective in regeneration of attachment apparatus in a canine model.23
Recent studies have suggested bone marrow as a source of mesenchymal stem cells (BMSCs) to regenerate lost periodontal tissue.22,24–26 Kawaguchi et al reported that the application of BMSCs in the furcation defects has the potential to regenerate new cementum and alveolar bone in a beagle dog model.24
The aim of the present study was to investigate an isolation procedure to culture mesenchymal stem cells derived from bone marrow and evaluate their potential in periodontal regeneration.
Materials and Methods
Nine male mongrel dogs, ages 1–2 years and weighing 14–22 kg, constituted our study sample. The experimental animal study was approved by the Ethical Committee for animal experiments of Tehran University of Medical Sciences (No: 88-02-70-8722).
Animals were determined to be healthy through clinical examination and hematologic analysis. Dogs were housed in individual cages with free access to water and commercially balanced dry food (Friskies, Purina, Marne La Valle, France). The animals were allowed to acclimate to their diet and housing condition for two weeks before starting the experimental procedures. During this period, dogs were vaccinated against contagious diseases and medicated against parasitic infections.
Isolation and cultivation of bone marrow MSCs
Under general anesthesia with acepromazine (0.05 mg/kg), diazepam (0.5 mg/kg), and ketamine (8 mg/kg), 1 mL bone marrow was taken from the iliac crest of each animal. Canine BMSCs were isolated and characterized using the method previously reported by Eslaminejad et al.27 Briefly, bone marrow aspirates were seeded with culture medium (Dulbecco's modified Eagle's medium, DMEM; Gibco BRL, Life Technologies, Grand Island, NY) and supplemented with 10% fetal bovine serum (Hyclone, South Logan, Utah), 100 U/mL penicillin (Invitrogen Corp, Carlsbad, Calif), and 100 U/mL streptomycin (Invitrogen Corp). To separate MSCs, 2 × 108 nucleated cells in 5 mL of media were layered over 20 mL of Lymphodex (Inno-Train, Sweden) and then were centrifuged at 400g for 20 minutes. Cells collected at the media-Lymphodex interface were plated in 15 mL low-glucose DMEM and incubated in a humidified 5% CO2 atmosphere and 37°C. After seven days, the non-adherent cells were removed and the medium was changed twice weekly. Cells were dissociated with trypsin plus EDTA (Invitrogen) and at third passage were used for transplantation.
Flow cytometric analysis
This analysis was employed to examine the nature of the isolated cells in terms of surface epitopes including anti-macrophage marker, endothelial marker (CD146), embryonic stem cell marker (SSEA-4), and MSC markers (CD44 and CD90). About 1.5 × 105 marrow-derived third-passage cells were suspended in 5 mL tubes containing 100 μl PBS and 5 μl FITC-conjugated antibodies (Becton Dickenson, Franklin Lakes, NJ). The tubes were incubated in a dark room at 4°C for 30 minutes and centrifuged at 1200 rpm for 4 minutes. The pellet was dispersed in 300–500 μl washing buffer and analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif) equipped with a 488 nm argon laser. IgG1 and IgG2 were used as isotype controls. Data was analyzed using WinMDI software.
Differentiation potential
Osteogenesis
Osteogenic medium consisting of DMEM supplemented with 50 μg/mL ascorbic 2-phosphate (Sigma), 10 nM dexametazone (Sigma), and 10 mM β-glycerole phosphate (Sigma) was added to confluent third passage culture. At 21 days, Alizarin red staining as well as RT-PCR analysis, was used to detect differentiation.
Adipogenesis
Confluent cultures were exposed to adipogenic medium composed of DMEM supplemented with 100 nM dexametasone (Sigma) and 50 μg/mL indomethacin (Sigma) for a period of 3 weeks. At the end of the induction period, the cultures were stained with Oil Red O for detection of lipid droplets. In addition, RT-PCR analysis was used to examine adipogenic differentiation.
Chondrogenesis
BMSCs were suspended in 5 mL DMEM medium and centrifuged in 15 mL polypropylene tubes (Corning Incorporated Life Sciences, Acton, Mass) at 1200 rpm for 5 minutes. The chondrogenic differentiation medium was added to the cell pellet. Chondrogenic medium consisted of DMEM supplemented with 3 ng transforming growth factor beta, 50 mg/mL transferrin-selenium-insulin, 10 ng bone morphogenetic protein-6, 50 mg bovine serum albumin, and 1% FBS (Gibco BRL, Gaithersburg, Md). The culture was maintained for 21 days. Five-micrometer thick sections were stained with toluidine blue O, and RT-PCR was performed for expression of cartilage specific genes.
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression
The total RNA was isolated from osteogenic, adipogenic, and chondrogenic cultures using RNX-Plus solution (CinnaGen Inc, Tehran, Iran). The standard reverse-transcription reaction was performed using Oligo (dT)18 as a primer and RevertAid H Minus First Strand cDNA Synthesis Kit according to the manufacturer's instructions. Reaction mixtures for PCR included 2.5 μl cDNA, 1X PCR buffer (AMS), 200 μM dNTPs, 1 unit/25 μl reaction of Thermus aquaticus (Taq) DNA polymerase (Fermentas, Hunover, Md), and 0.5 μM of various specific primer sets. Each PCR was carried out in triplicate and under linear conditions. The products were analyzed by electrophoresis on 2% agarose gel and stained with ethidium bromide.
Preparation of cell-based construct
Before transplantation, 2 ×107 cells were loaded on 0.2 mL of anorganic bovine bone mineral (ABBM) granules (Bio-Oss, Geistlich Biomaterials, Wolhusen, Switzerland) and stored in 500 mL DMEM. The constructs were then incubated at 37°C for 2 hours prior to transplantation. To calculate the loading efficiency, the construct was removed, and the remaining cells were collected and counted using a hemocytometer. To ensure that BMSCs has been seeded within the scaffold, the loaded ABBM were fixed in 10% formaldehyde in PBS buffer, decalcified in 10% EDTA, and processed for light microscopic observation.
Surgical procedures
Each animal was premedicated with an intramuscular injection of 2 mg/kg xylazine-HCl. The general anesthesia was induced by an intravenous injection of ketamin-HCl (8 mg/kg) and diazepam (0.5 mg/kg), and maintained by the same drugs. The general anesthesia was monitored under the supervision of a veterinary surgeon (MMD).
Eight weeks after the extraction of mandibular first premolars, 3-wall intrabony defects were prepared at the mesial surfaces of the second premolars using burs and sterile saline coolant. The bony defects were 4 mm wide buccolingually and mesiodistally, and 4 mm deep apico-coronally.28 Following root planing, using a round bur, 2 reference notches were made on the root surfaces: one at the level of alveolar crest and one at the base of each defect. A 3-0 silk ligature was placed around the cervical aspect of the second premolars to cause plaque-induced periodontal attachment loss. Periodontal flaps were sutured at presurgical position. The animals received intramuscular analgesic medication (Tramadol, 2 mg/kg), twice daily for 3 days, and sutures were removed after 2 weeks. During this period, the animals were fed a soft diet to allow plaque accumulation.
Transplantation of BMSCs into experimental periodontal defects
Four weeks after surgery, bleeding on probing and attachment loss evidenced periodontitis. At this time, ligatures were removed and plaque and calculus eradicated. One week later, regenerative surgeries were performed.10 Under general anesthesia, full thickness flap was raised, and debridement, scaling, and root planing were carried out. The bilateral defects were randomly assigned to 2 treatment groups: the control group received ABBM, and the test group received 2 × 107 BMSCs-loaded ABBM. Tension free flaps were sutured with interrupted 3-0 nylon sutures. All animals received antibiotic (Cefazoline 22 mg/kg) and analgesic (Tramadol), intramuscularly 2 times daily for 3 days. Chlorhexidine solution (0.2%) was applied topically every day for 2 weeks. Moreover, all animals were fed a soft diet for 2 weeks to reduce potential mechanical trauma to target areas and sutures were removed after this period.
Histologic and histomorphometric analyses
Eight weeks after transplantation, all animals were sacrificed by an overdose of sodium thiopental. The experimental sites were dissected, fixed in 10% buffered formalin, demineralized in nitric acid (15%), dehydrated in graded alcohol, and embedded in paraffin. Serial sections (5μ) were cut mesiodistally, and three representative sections with the largest root surface area were selected for evaluation. All slides were stained with Hematoxylin-Eosin and analyzed descriptively and histometrically under a light microscope (BX51, Olympus Co, Tokyo, Japan) equipped with a digital camera (DP25, Olympus) and analysis software (DP2-BSW, Olympus). The Epithelial migration and newly formed cementum, PDL, and bone were assessed descriptively. Histometric analysis was based on the parameters described by Akizuki et al:19
Defect height: distance between the apical extension of apical notch and coronal extension of coronal notch.
New cementum length: length of newly formed cementum on the denuded root surface.
New bone length: length of newly formed bone along the denuded root surface.
New PDL length: length of newly formed periodontal ligament, which was oriented perpendicular to the root surface.
The percentages of newly formed cementum, PDL, and bone to the total denuded root surface (form one notch to the next notch) were calculated.
All microscopic evaluations were carried out by two examiners, and any disagreement resolved by consensus (SH, EM, and MA).
Statistical analysis
The experimental defect was used in the statistical analyses as the unit of measurement. For each parameter, the mean and the standard deviation were calculated. The student's t test was used to compare the mean values in both groups. The differences were considered statistically significant when P < .05.
Results
Cell culture
Multiple cell colonies were detected in primary cultures. At day 2, the adhering fibroblasts to culture surfaces were quiescent; however, they reached confluence at day 10 and maintained their fibroblastic morphology during each passage (Figures 1a and b).
(a) Marrow-derived cell culture. At primary culture, some cells tended to adhere on the culture surface. (b) The cells multiplied and the culture reached confluency at 10 days (Bar = 100 μm). (c) Flow cytometric analysis of the isolated cells from canine marrow. Majority of the cells tended to express mesenchymal markers including CD44 and CD90. Non-mesenchymal markers including CD146, SSEA-4, and anti-macrophage antigen were expressed in a small percentage of the cells.
(a) Marrow-derived cell culture. At primary culture, some cells tended to adhere on the culture surface. (b) The cells multiplied and the culture reached confluency at 10 days (Bar = 100 μm). (c) Flow cytometric analysis of the isolated cells from canine marrow. Majority of the cells tended to express mesenchymal markers including CD44 and CD90. Non-mesenchymal markers including CD146, SSEA-4, and anti-macrophage antigen were expressed in a small percentage of the cells.
Flow cytometry
CD90 and CD44 were expressed by the majority of cells, unlike CD146, SSEA-4, and anti-macrophage surface antigens, which were expressed in a very low percentage of cells (Figure 1c).
Tissue differentiation
Increasing nodule-like structures, stained heavily with alizarin red, were observed after the exposure of BMSCs to osteogenic-inducing media with a strong expression of osteopontin, ColIA1, and ColIA2, detected by the RT-PCR analysis.
Increasing numbers of Oil Red O positive lipid-laden droplets were observed after 5 days of culture with an adipogenic inducing media. Adipocyte gene expressions, such as LPL (lipoprotein lipase) and PPAR-gamma (peroxisome proliferators activated receptor-gamma) were confirmed by RT-PCR analysis (Figure 2).
Figure 2. Differentiation potential of isolated cells from canine marrow. (a) The osteogenic culture stained by alizarin red. (b) The adipogenic culture stained by Oil Red (Bar = 100 μm). Figure 3. Photomicrograph of a mesiodistal histologic section of one of the specimens in BMSC group including both reference notches. Thick new cementum (arrow) and new bone (asterisk) have been formed on the entire root surface between the notches. The PDL is well organized with collagen fibers oriented perpendicularly between the bone and cementum (double-chevron). Single chevron shows a blood vessel in the PDL (hematoxylin and eosin stain, original magnification ×40). Figure 4. Photomicrograph of a mesiodistal histologic section of one of the specimens in the control group, representing the apical notch. Arrow shows collagen fibers parallel to the root surface, and asterisk depicts new bone formation in the apical notch (hematoxylin and eosin stain, original magnification ×40; scale bar = 200 μm).
Figure 2. Differentiation potential of isolated cells from canine marrow. (a) The osteogenic culture stained by alizarin red. (b) The adipogenic culture stained by Oil Red (Bar = 100 μm). Figure 3. Photomicrograph of a mesiodistal histologic section of one of the specimens in BMSC group including both reference notches. Thick new cementum (arrow) and new bone (asterisk) have been formed on the entire root surface between the notches. The PDL is well organized with collagen fibers oriented perpendicularly between the bone and cementum (double-chevron). Single chevron shows a blood vessel in the PDL (hematoxylin and eosin stain, original magnification ×40). Figure 4. Photomicrograph of a mesiodistal histologic section of one of the specimens in the control group, representing the apical notch. Arrow shows collagen fibers parallel to the root surface, and asterisk depicts new bone formation in the apical notch (hematoxylin and eosin stain, original magnification ×40; scale bar = 200 μm).
After exposure to chondrogenic differentiation media, BMSCs expressed metachromasia with toluidine blue stain. BMSCs potential for chondrogenesis was further confirmed by the expression of cartilage specific genes including collagen II, decorin, and aggrecan.
Scaffolds
About 60% of the loaded cells were successfully trapped within the scaffold pore systems.
Histologic Evaluations
The apical migration of epithelium was observed in one sample in the test group and three samples in the control group. Bone ankylosis and root resorption were not observed in any sample. Formation of new cementum was observed in all samples of the test group and 8 samples of the control group. In the test group, new PDL was formed in all samples, of which 4 samples displayed well-organized perpendicular-oriented fibers (Figure 3). In 8 samples of the control group, PDL was formed, of which 2 samples displayed well-organized fibers (Figure 4). In the experimental group, bone formation was observed in all specimens. In the control group, bone regeneration occurred in 8 samples.
Histomorphometric Analyses
The Table provides mean and standard deviation values of defect height, new cementum, new PDL and new bone.
New cementum filled 80% of the defect height in the test group and 48% in the control group (Figure 5). The dimension of newly formed cementum was significantly higher in the test group (P < .02) (Table). New PDL filled 64% of the defect height in the test group and 36% in the control group (Figure 5). The dimension of the newly formed PDL was more significant in the test group (P < .02) (Table). The mean percentages of new bone formation in the test and control groups were 65% and 48%, respectively (Figure 5) with no statistically significant difference (P < .193) (Table).
Bars show the percentage distribution of the values of the different parameters in test and control groups. PDL indicates periodontal ligament. *P < .05.
Bars show the percentage distribution of the values of the different parameters in test and control groups. PDL indicates periodontal ligament. *P < .05.
Discussion
In the present study, mesenchymal stem cells were isolated from bone marrow, and their periodontal regenerative potential was investigated. After the examination of surface epitopes of the isolated cells, the total RNA from osteogenic, adipogenic, and chondrogenic cell cultures were analyzed by RT-PCR to confirm stem cell gene expressions. The outcome met the criteria of the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cell Therapy.29
The histological findings of the present investigation showed that the insertion of BMSCs into periodontal defects could promote formation of cementum and PDL. However, BMSCs did not accelerate bone formation. In a previous investigation with a similar design, using dental pulp stem cell, comparable results were obtained.23
The results of this study are consistent with the findings of previous animal studies that demonstrated enhancement of periodontal repair by transplantation of BMSCs in class III furcation defects.24,25 Kawaguchi et al24 reported that the mean percentage of new cementum length in the BMSCs group was 94%, significantly greater than that of the control group (70%). Corresponding numbers in the present study were 80% and 48%, respectively. Lower values obtained in the current project may be attributed to the differences in the study designs (defect types, osteoconductive materials, method of measurements, and duration of the studies). In contrast, an animal study on 4 dogs using BMSC sheets combined with β-tricalcium phosphate (β-TCP) and collagen in one-wall periodontal defect did not increase the thickness of newly formed cementum.22
The study results showed mean percentages of PDL formation in the test and the control groups to be 64% and 36%, respectively (P < .05). This finding is in agreement with the results reported by Tsumanuma et al.22 Compared to the control group, this study showed that the application of BMSCs sheet of woven polyglycolic acid on the denuded root surface of one-wall intrabony defects, filled with a mixture of β-TCP and collagen, resulted in denser and well-organized PDL.22
The height of the alveolar bone was higher in the test group compared to the control group. The difference was not statistically significant and can be explained by the experimental model. Three-wall periodontal defects have a self-contained morphology and provide stable environments for wound healing.
Tsumanuma et al22 reported no statistically significant difference between BMSCs-loaded and cell-free groups regarding the height of new bone when applied on the root surface of one-wall defects. The intrabony defect in the Tsumanuma et al study22 was a surgically created acute model vs the acute/chronic model of the present study,30 which may mimic the natural attachment loss more closely.31
Kawaguchi et al24 demonstrated that the percentage of new bone area in the class III furcation defect increased significantly when BMSCs were added to atelocollagen (63% vs 54%, P < .05).
The outcome of the present study is in accordance with previous studies using cells from other sources.10,19 A similar study design, using PDL-derived and cementum-derived cells in 3-wall defects, suggested that cell therapy promotes regeneration of periodontal tissues through the formation of cementum and PDL. In contrast, no difference in new bone formation was observed in cell-loaded and cell-free groups in Nunez et al study.10
In a study on peri-implant defects, BMSC-loaded hydroxyapatite/β-TCP showed promising results regarding new bone formation when compared with hydroxyapatite/β-TCP alone.11 Previous investigators have used BMSCs to regenerate periodontal tissues around a titanium dental implant placed in a fresh extraction socket in a goat experimental animal model. These investigators have demonstrated periodontal-like tissue and newly formed bone around the titanium fixture upon implantation of BMSCs.32 Khojasteh et al showed that application of combination of BMSC/particulate allograft/fibrin glue was useful for vertical bone augmentation around simultaneously inserted implants in rabbit tibia.33
Within the limitations of this study, it can be concluded that BMSCs should be considered a promising technique for use in patients with periodontal attachment loss and merits further investigations.
Abbreviations
