Abstract
BACKGROUND:Regeneration of type Ⅱ furcation defects of periodontal tissues is still a great clinical challenge.
OBJECTIVE: To compare different densities of autologous bone marrow mesenchymal stem cells (auto-BMSCs) for repairing canine experimental class Ⅱ furcation defects of periodontal tissues.
DESIGN: A randomized controlled trial.
SETTING: Laboratory in Stomatological Hospital Affiliated to Fujian Medical University and Department of Animal Experiment in Fuzhou General Hospital.
MATERIALS: Experiments were performed at the Laboratory in Stomatological Hospital Affiliated to Fujian Medical University and Department of Animal Experiment in Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA from July 2005 to September 2006. Six 18-month Beagle dogs were provided by Department of Animal Experiment in Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA. Animal intervention met animal ethical standards. Bio-Gide collagen membrane and BME-10X collagen membrane were used in the study.
METHODS: Class Ⅱ furcation defects were induced surgically on the buccal side of canine mandibular second and third premolar (P2, P3) and first molar (M1). The ex vivo expanded auto-BMSCs from six 18-month Beagle dogs were seeded in BME-10X collagen membranes at cell density of 5×108 L-1，5×109 L-1，5×1010 L-1, and delivered into experimental class Ⅱ furcation defects, underneath a Bio-Gide membrane. Bio-Gide membrane alone was used as a control. The percentage of new cementum length and percentage of new alveolar bone area were measured on OLYPUS IX 71 inverted research microscope and OLYSIA BioAutoCell software in a computer.
MAIN OUTCOME MEASURES: Each specimen was stained with hematoxylin and eosin. The lengths of new cementum and the area of new alveolar bone were calculated.
RESULTS: The percentage of newly formed cementum length and the percentage of newly formed alveolar bone area were (51.5±5.6)% and (27.1±7.7)% in the control group,（84.8±8.9）% and（30.6±7.7）% in the 5×108 L-1 BMSCs group, (91.8±5.2)% and (68.3±11.4)% in the 5×109 L-1 BMSCs group and (88.8±7.2)% and (78.5±12.7)% in the 5×1010 L-1 BMSCs group. There were significant differences when comparing the BMSCs groups to the control group (P < 0.01), but there was no significant difference in each BMSCs group. There were significant differences in the percentage of newly formed alveolar bone when comparing the 5×109 L-1 and 5×1010 L-1 BMSCs groups to 5×108 L-1 BMSCs group and control group (P < 0.05), but there was no significantly difference between the first two groups, and neither was the later.
CONCLUSION: Periodontal regeneration can be induced by BMSCs transplantation. The mechanism of regeneration is associated with inoculated density.


INTRODUCTION

Periodontal disease can lead to periodontal tissue defects. It is difficult to achieve ideal periodontal regeneration by current regenerative therapies. Periodontal tissue engineering is one of hot re-searches on periodontal tissue regeneration. Bone marrow mesenchymal stem cells (BMSCs) can be easily obtained, expand in vitro and differentiate into various cells. Therefore, BMSCs served as an ideal seed cells have been used for periodontal tissue re-generation. It was reported that the osteogenesis abil-ity of BMSCs was related to the initial cells-seeding density [1]. In this study, periodontal regeneration capacity of BMSCs seeded in collagen membrane at different densities was evaluated after transplantation by using a Beagle canine model of experimentally induced class Ⅱ furcation defects.

MATERIALS AND METHODS

Materials
Experiments were performed at the laboratory Laboratory in Stomatological Hospital Affiliated to Fujian Medical University and Department of Animal Experiment in Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA from July 2005 to September 2006. Six 18-month old Beagle dogs were obtained from Department of Animal Experiment in Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA. Animal intervention met the animal ethical standards. DMEM culture medium (Gibico, America), trypsin (Gibico, America), newborn calf serum (Every Green Organism Engineering Mate-rials Company, China), OLYPUS IX 71 inverted research microscope (Olympus, Japan), BME-10X collagen membrane (Institute of Biomedical Engi-neering, Chinese Academy of Medical Sciences, China), Bio-Gide collagen membrane (Osteohealth, Switzerland), SUNCON medical biogel (Beijing Suncon Biogel Limited Company, China) were used in the study.

Methods
Creation of class Ⅱ furcation defects
In accordance with the model described by Yan [2],

all surgical procedures were performed under anesthesia, which was induced with ketamine (10 mg/kg) administered intramuscularly. Aneshesia was maintained intervenously by ketamine (1-2 mg/kg) and diazepam (0.1 mg/kg). The third, fourth premolars and the first molars (P3, P4 and M1) in each dog were selected for experimentation. Mucoperiosteal flaps were raised, and class Ⅱ furcation defects were cre-ated surgically on the buccal side of mandibular P3, P4 and M1. The "U" shape defect's height from the roof of the fur-cation and mesial-distal width were 5 mm and buccal-lingual depth was 3 mm. Furcation and root surface at the buccal side were exposed. Periodontal ligament was fully removed to denude root surface. The bone cavities were filled with gutta to prevent spontaneous regeneration of the lesions and the flaps were repositioned and sutured for 6 weeks.

Culture of auto-BMSCs
Based on the technique described by Yan [3], under intra-muscular induction of anesthesia with ketamine (10 mg/kg) and general aseptic conditioning, 5-7 mL bone mar-row aspirates of each dog were taken from the femoral bone proximal end with number 16 transfixion pin, placed in a 50 mL heparinized centrifuge tube with 30 mL DMEM. The mixture was centrifugated at 1 500 rpm for 10 minutes. After supernatant was removed, the mixture was centrifugated with additional 10 mL DMEM. Supernatant was removed before 10 mL culture medium was added, misce bene. The cells were seeded at 1×105/cm2 in culture dishes and incu-bated in a 100% saturated humidified atmosphere at 37 ℃ with 0.05 volume fraction of CO2. Five days after seeding, floating cells were removed and the medium was replaced with fresh medium. Thereafter, medium was changed every 3 days. When cultured dishes became near confluent, cells were harvested with 0.25% trypsin plus 0.02% EDTA and reseeded onto new plates at 1:4 proportion for continued passage. In this study, cell passage 1 was used for transplan-tation.

Treatment of furcation defects
Thirty-six teeth were divided at random into 5 groups: Bio-Gide membrane alone group, as a control group (n=7), 5×108 L-1 BMSCs group (n=7), 5×109 L-1BMSCs group (n=8), 5×1010 L-1BMSCs group (n=7), other 7 teeth for an-other study.
First passage of BMSCs were seeded at different densities in 4 mm×4 mm BME-10X collagen membrane rinced with medium, and cultured for 24 hours (Figure 1). A full thick-ness flap was raised after general anesthesia. The furcation lesions were submitted to curettage and the dental surfaces debrided and planned. Reference notches were placed at the base of the defect as a guideline for histological analysis. BME-10X membrane mixture was transplanted into the defects underneath a Bio-Gide membrane and the Bio-Gide membrane alone as a control. Membranes, fixed by biogel, were extended 2-3 mm over the bone tissue adjacent to the lesion and remaining at gingival margin level. Gentamicin 80,000 U/d was taken for 3 days postoperatively.

Preparation of tissue sample
All the dogs were sacrificed twelve weeks after transplantation. The teeth were cut in the mesial-distal plane, fixed with 10% formalin for three days, decalcificated with Krinstensen's, de-hydrated through graded ethanol, and embedded in paraffin. Sections (5 μm) were cut and stained with hematoxylin and eosin (HE) staining.

Histological and morphometric analysis
The percentage of new cementum length and percentage of new alveolar bone area were measured on OLYPUS IX 71 inverted research microscope and OLYSIA BioAutoCell software in a computer. The lengths of new cementum formed along the denuded root surface on each specimen were added, and the percentage of the lengths to the total root surface length from one notch to the next notch was calculated; the area of new bone on each specimen was cal-culated as a percentage of the area surround with reference notches at mesial and distal root surfaces facing the bone defect [4] (Figure 2).

Statistical analysis
All data were statistically analyzed using the variance analy-sis by the first author with SPSS 11.0 software.

RESULTS

Clinical observations
All rats tolerated the procedure well in the observational period. Membrane exposure was noted mostly two weeks after surgery in four teeth (12.1%, in the control group).
Histological analysis
Two teeth were evacuated during the sample were prepared because of improper treatment. Thirty teeth remained, in-cluding seven teeth in the control group, six teeth in the 5×108 L-1 BMSCs group, eight teeth in the 5×109 L-1 BMSCs group, and six teeth in the 5×1010 L-1 BMSCs group.
In the BMSCs-membrance groups (experimental groups), significant amount of new bone and new ce-mentoid tissue were observed above the notch in the furcation. Inclined and parallel fibers, as well as ne-ovascularization were found. Epithelial cell invasion, bone ankylosis, and root resorption were not observed. In the Bio-Gide membrane alone group (control group), epithelial cells invading into the top of the furcation were observed in four teeth. Less bone and cementum regeneration was observed. Bone ankylosis, and root resorption were not observed (Figures 3 and 4).

Histomorphology analysis
The proportion of new cementum length in the BMSCs-membrane groups was significantly higher than in the control group (P < 0.01), but there was no signifi-cantly difference in the three experimental groups. The percentage of new alveolar bone area in the 5× 109 L-1 and 5×1010 L-1 BMSCs groups was significantly higher than in the control and 5×108 L-1 BMSCs groups (P < 0.05), but there was no significantly difference between the first two groups, and neither was the later. (Table 1, Figure 5).

There were significant differences in the percentage of newly formed cementum when comparing BMSCs groups to the control group (P < 0.01), but there was no significant difference in each BMSCs group. There were significant differences in the percentage of newly formed alveolar bone when comparing the 5×109 L-1 and 5× 1010 L-1 BMSCs groups to 5×108 L-1 BMSCs group and control group (P < 0.05), but there was no significantly difference between the first two groups, and neither was the later.

DISCUSSION

BMSCs could be induced to differentiate into various cell types in different experimental conditions. For example, to promote chondrogenic differentiation, BMSCs were cultured in serum-free medium with transforming growth factor-β 3[5]; to promote osteogenic differentiation, BMSCs were cultured with dexamethasone, β-glycerophosphate, and ascorbate in the presence of mineralized fluid and fetal bo-vine serum[6].
Several cell types, including cementoblasts, osteoblasts, and periodontal ligament fibroblasts, were required for periodontal tissue regeneration. Recent studies demonstrated that transplanted BMSCs differentiated into various connec-tive tissue cells in vivo under the influence of host fac-tors[7-10], pointing to the local microenvironment as an im-portant factor regulating cell activity. The results of the cur-rent studies demonstrated that auto-transplantation of BMSCs can promote periodontal tissue regeneration[4, 11].
Initial cell-seeding density is an important factor for tissue regeneration. Sufficient tissue fluids and blood supply might be necessary for cells to survive after transplantation, and subsequently differentiate into various tissues. A high cell density might disturb the infiltration of nutrients into the deeper areas of transplanted cells. Meanwhile, a low density might influence the regenerative effect. It was reported that the osteogenic ability of BMSCs was in correlation with the initial cells-seeding density. In vivo, only the cell density over 5×108 L-1, BMSCs would form bone tissue [1]. Other types of cells and their biological function were re-ported [12-14]. Iwasa et al[12] examined the effects of cell den-sity（2×108 L-1, 2×109 L-1 and 2×1010 L-1） on the pro-liferation and chondroitin sulfate synthesis of chondrocytes embedded in collagen gel that was incubated for 4 weeks. They found that chondrocytes in the 2×108 L-1 BMSCs group gradually proliferated more than the other groups. In contrast, most chondrocytes in the 2×1010 L-1 BMSCs group showed low proliferation, even decreased in number, but increased capability to produce chondroitin 6-sulfate. Thus, they supposed that the difference in cell density influenced the interaction between chondrocytes and phenotype in col-lagen gel. Jensen et al[13] also found that the release of he-patic lipase from cultured rat hepatocytes was cell den-sity-dependent under certain conditions. In our study, the percentage of new alveolar bone area in the 5×109 L-1 and 5×1010 L-1 BMSCs groups were significantly higher than in the control group (P < 0.05), but there was no significant difference between the control groups and the 5×108 L-1 BMSCs group. The findings indicated that the osteogenous capability of BMSCs-membrane is related to the cell-seeding density. It is evident that the different tissue regeneration might require different cells' quantity.
In the present study, no significant differences were ob-served in bone or cementum formation between the high-density groups (5×109 L-1 and 5×1010 L-1 BMSCs groups), which was similar with others. Hiroyuki et al[4] mixed BMSCs with atelocollagen at concentrations of 2×109 L-1, 5×109 L-1, 1×1010 L-1, 2×1010 L-1 auto-transplanted into acute experimental Class Ⅲ defects. They found that significant periodontal tissue regeneration was observed in the BMSCs-atelocollagen compared to the control (atelocollagen alone) groups. However, no signifi-cant difference was observed among the cell groups. Ac-cordingly, it was indicated that tissue regeneration is not dependent on the concentration when the cells were reached to a certain concentration. Many scholars studed the adhe-sion efficiency of cells and scaffolds[15-18]. Chantal et al[15] seeded BMSCs (0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0×106/cm3) onto 5 mm×7 mm×7 mm scaffolds PLGA[poly(lactide-coglycolide)]. After 1 hour in culture, they found that a maximum of 25% of the initial cell seeding concentration adhered to the scaffolds in static culture con-ditions. In fact, a plateau of 1.5×106 cells/cm3 was reached regardless of the initial cell seeding concentration. Accord-ing to the findings, 109 L-1 might be the suitable concentra-tion for BMSCs transplantation into periodontal tissue de-fects.
The precise mechanisms of periodontal tissue regeneration with BMSCs remain to be elucidated. It is possible that the local microenvironment simulates transplanted BMSCs to exhibit their multilineage differentiation potential in vivo and undergo site-specific differentiation into the appropriate periodontal cells. They could also stimulate residual perio-dontal cells or periodontal precursor cell to differentiate into the periodontal cells. Thus, further studies are needed to understand the mechanism of periodontal tissue regeneration in molecular biology and histo-kinetics. The adhesion, pro-liferation and differentiation of BMSCs are various in dif-ferent scaffolds [19-22]. More investigation on enhancing the cells adhesion in scaffolds is needed, such as using adher-ence factor.

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自体骨髓间充质干细胞移植密度与
Beagle犬牙Ⅱ度根分叉病变组织
修复的关系**★○

詹 暶1，闫福华1，肖 毅○2
1福建医科大学附属口腔医院牙周科，福建省福州市 350002； 2 Institute of Health and Biomedical Innovation Queensland University of Technology, Australia
詹 暶★，女，1975年生，福建省三明市人，汉族，2005年福建医科大学毕业，硕士，主治医师，主要从事牙周组织工程方面的研究。
通讯作者：闫福华，主任医师，教授，博士生导师, 福建医科大学附属口腔医院牙周科，福建省福州市 350002
福建省教育厅科技项目基金（JA05267）*，福建医科大学第六批青年骨干教师基金（闽医大[2006]124号）*
摘要
背景：目前临床上II度根分叉病变区牙周组织的再生仍是一项难题。
目的：比较不同密度自体骨髓间充质干细胞修复II度牙根分叉病变区牙周组织缺损的能力。
设计：随机对照观察实验。
单位：福建医科大学附属口腔医院实验室和福州总医院动物实验科。
材料：实验于2005-07/2006-09在解放军南京军区福州总医院动物实验科和福建医科大学附属口腔医院实验室完成。6只1岁半雄性Beagle犬由解放军南京军区福州总医院动物实验科提供，实验过程中对动物处置符合动物伦理学标准。实验中选用Bio-Gide胶原膜和BME-10X胶原膜。
方法：在犬的下颌第2，3前磨牙和第1磨牙颊侧根分叉处制备慢性II度牙根分叉病变模型。从6只18月龄的Beagle犬抽取自体骨髓间充质干细胞。应用5×108 L-1，5×109 L-1，5×1010 L-1的骨髓间充质干细胞复合BME-10X胶原膜移植治疗牙根分叉病变，表面覆盖Bi0-Gide胶原膜，单纯Bi0-Gide胶原膜组为对照组。采用OLYPUS IX 71显微照相系统和OLYSIA BioAutoCell软件计算新生牙骨质长度的百分比和新生牙槽骨面积的百分比。
主要观察指标：苏木精-伊红染色观察并测量牙周组织再生(新生牙骨质长度和新生牙槽骨面积的百分比)情况。
结果： 对照组的新生牙骨质长度和新生牙槽骨面积的百分比分别为：（51.5±5.6）%和（27.1±7.7）%；5×108 L-1骨髓间充质干细胞组为：（84.8±8.9）%和（30.6±7.7）%； 5×109 L-1骨髓间充质干细胞组为：（91.8±5.2）%和（68.3±11.4）%；5×1010 L-1骨髓间充质干细胞组为：（88.8±7.2）%和（78.5±12.7）%。细胞组的新生牙骨质量与对照组相比，差异有显著性意义（P < 0.01），但各细胞组间相互比较差异无显著性意义；5×109 L-1组和5×1010 L-1组的新生牙槽骨量与 5×108 L-1组和对照组相比差异有显著性 (P < 0.05)，但前两组间和后两组间比较则差异无显著性意义。
结论：骨髓间充质干细胞移植能促进牙根分叉病变区的牙周组织再生，其作用与细胞的接种密度有关。
关键词：牙周组织再生；骨髓间充质干细胞；细胞密度
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)16-03193-05
詹暶，闫福华，肖毅.自体骨髓间充质干细胞移植密度与Beagle犬牙II度根分叉病变组织修复的关系[J].中国组织工程研究与临床康复，2008，12(16):3193-3197
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3193(ps).pdf]
(Edited by Zoran Ivanovicp/Qiu Y/Wang L)