Abstract



BACKGROUND: The peripheral blood stem cell is a multi-differentiation precursor cell, and it can differentiate into osteoblasts. Tissue engineered bone, which is regarded as a vector of cell transplantation, has good compatibility with receptor tissue and seed cells. Transforming growth factor-β (TGF-β) is an important regulatory factor for repairing bone injury. Additionally, TGF-β can induce peripheral blood stem cells to differentiate and proliferate into osteoblasts.
OBJECTIVE: To study TGF-β expression in repairing alveolar bone during synergetic transplantation of peripheral blood stem cells and tissue engineered bone.
DESIGN: Observational study.
SETTING: Stomatology Hospital of Xi’an Jiaotong University.
MATERIALS: This study was performed at the Stomatology Hospital of Xi’an Jiaotong University from 2003 to 2006. Experimental animals were provided by the Animal Experimental Center, Medical College of Xi’an Jiaotong University (original Xi’an Medical University). All animals were intramuscularly induced with ketamine, intramuscularly anesthetized with sumianxin, and then sacrificed for surgery. The experiment was approved by the local ethics committee.
METHODS: Peripheral blood stem cells were extracted from dog and prepared as a cell suspension. Iliac bone was obtained from healthy pig to prepare decalcifying-deproteinic tissue engineered bone. The tissue engineered bone was then dipped into peripheral blood stem cell suspension. Ten healthy hybrid dogs were randomly divided into an experimental group and a control group, with 5 dogs in each group. An incision was made from left to right along the canine teeth of the lower mandible, along the lip, lateral to the gingival sulcus, to the alveolar crest, and then along the bilateral vestibular groove to form a trapezoid segment. Subsequently, the segment was turned downward to expose the bone lamella lateral to the lip. In addition, a bone defect region of 2 cm × 2 cm × 1 cm was drilled between the lateral incisor of lower mandible using a turbine drill. Peripheral blood stem cell-tissue engineered bone was implanted in the experimental group but tissue engineered bone only was implanted in the control group. At 2, 3, 4, 8 and 12 weeks after surgery, during the differentiation and proliferation of peripheral blood stem cell into osteoblasts, TGF-β expression was measured using immunohistochemistry.
MAIN OUTCOME MEASURES: ① Morphological changes of peripheral blood stem cells differentiating into osteoblasts and structural function of organoid were observed under optical microscopy and by transmission electron microscopy. ② TGF-β expression was measured using immunohistochemistry during the differentiation and proliferation of peripheral blood stem cells into osteoblasts.
RESULTS: Two weeks after peripheral blood stem cell-tissue engineered bone transplantation in the experimental group, TGF-β expression was mildly positive at the fringe of the bone defect. Four to eight weeks after the transplantation, high numbers of osteoblasts, fibroblasts and collagenous fibers were found at the center of the bone defect region, and TGF-β expression was strongly positive. The bone defect was completely repaired after 12 weeks. In the control group, 8-12 weeks after tissue engineered bone transplantation, TGF-β expression was mildly positive only at the fringe of the bone defect.
CONCLUSION: During dog alveolar bone defect repair, TGF-β can induce peripheral blood stem cells, in combination with tissue engineered bone, to differentiate and to proliferate into osteoblasts.

INTRODUCTION

Periodontal tissue bone defect is one of important manifestations of alveolysis. Repairing bone defect is a complex physiological process. Osteoblasts are the key cells to repair bone defect. Autologous osteoblasts are hard to be used in clinical treatment. Autologous peripheral blood stem cell is a multi-differentiation precursor cell, and it can differentiate into osteoblasts induced by transforming growth factor-β (TGF-β); therefore, peripheral blood stem cell is an important cell during bone growth. TGF-βhas significantly regulatory effects on bone formation and reconstruction[1]. This study was designed to examine TGF-βexpression during bone formation, to investigate the effects of TGF-βon the differentiation of autologous stem cells into osteoblasts, and to provide clinical evidences to repair alveolar bone defect by synergetic transplantation between peripheral blood stem cell and tissue engineered bone.

MATERIALS AND METHODS

Materials
This study was performed at the Stomatology Hospital of Xi’an Jiaotong University from 2003 to 2006. Experimental animals were provided by the Animal Experimental Center, Medical College of Xi’an Jiaotong University (original Xi’an Medical University). All animals were intramuscularly induced with ketamine, intramuscularly anesthetized with sumianxin, and then sacrificed for surgery. The experiment was approved by the local ethics committee.

Methods
Extraction of peripheral blood stem cell from dogs
Granulocyte colony-stimulating factor (batch number: 2000-3-21; ChuGAI Pharmaceutical Co., Ltd., Japan) was intravenously injected into dog’s ear once a day, three days before surgery. On the surgical day, 10 mL peripheral blood was extracted from femoral vein and added with the same volume of lymphocyte separation liquid (batch number: 1996-5-21; Shanghai Huajing Biological High-technological Co., Ltd.). Subsequently, the mixture was centrifuged at 2 000 r/min for 20 minutes. At that time, there were four blood layers in the tube. Monocyte on the second layer was drawn off using pipette and poured into another aseptic-dry tube, while the same volume of saline was added. The mixture was centrifuged again at the same speed for 5 minutes. Additionally, monocyte on the bottom of tube was drawn off and poured into saline containing 1.06/mL heparin to make cell suspension.

Preparation of tissue engineered bone
Iliac bone in the size of 2 cm × 2 cm × 1 cm was obtained from healthy piggy to prepare decalcification-deproteinization-derosination tissue engineered bone based on establishment of tissue engineered bone by Pelton et al[2]. The tissue engineered bone was dipped into above-mentioned cell suspension for preparation.

Experimental procedures
Ten healthy hybrid dogs were randomly divided into two groups: experimental and control groups, with 5 dogs in each group. After routine sterilization, dogs were intramuscularly injected with 10 mg/kg ketamine, and intramuscularly anesthetized with 0.1 mL/kg sumianxin. Scalpel started from left to right canine teeth of lower mandible, went along lip-lateral gingival sulcus to alveolar crest, and turned to bilateral vestibular groove to form a trapezoid segment. Subsequently, the segment was turned downwardly to expose lip-lateral bone lamella. In addition, a bone defect region in the size of 2 cm × 2 cm × 1 cm was drilled between lateral incisors of lower mandible using turbine drill. Peripheral blood stem cell-tissue engineered bone was implanted in the experimental group but only tissue engineered bone was implanted in the control group. Incision was sutured after implantation. At 2, 3, 4, 8 and 12 weeks after surgery, one dog in the experimental group was sacrificed and one dog in the control group. Bone defect mass containing left and right canine teeth of lower mandible in the size of 2 cm × 2 cm × 1 cm was cut for histological processing.

Histological examination
The obtained samples were fixed with 10% formaldehyde, decalcified, embedded in paraffin, cut into sections; stained with HE staining, and observed under optic microscope.

Observation under transmission electron microscope
The above-mentioned fresh samples were fixed with 2% glutaraldehyde, washed with PBS, fixed with 1% osmic acid, dehydrated with a series of acetone, embedded in epoxide resin, cut into ultrathin section, and stained with 2% acetic acid oil and lead citrate. Activities of osteoblasts and fibroblasts, and formation of collagenous fiber were observed under transmission electron microscope.

Immunohistochemical examination
The above-mentioned samples were stained with ABC method. PBS was used as negative control. Anti-TGF-β monoclonal antibody was provided by Beijing Jingmei Biological Co., Ltd.; ABC immune kit by Vector-lab Company, USA. The stained samples were cut into sections, gradually dehydrated with alcohol, cleared with dimethyl benzene, and sealed with gum. A dark brown fluorescence was found at the positive site under fluorescent microscope.

RESULTS

Two weeks after peripheral blood stem cell-tissue engineered bone transplantation in the experimental group, a lot of peripheral blood stem cells and fibroblasts were found in tissue engineered bone stent under optic microscope; meanwhile, there were also a lot of blood capillaries (Figure 1). On the other hand, under transmission electron microscope, tissue engineered bone in the bone defect region was complete; while, peripheral blood stem cells in the tissue engineered bone stent deformed from ellipse to multi-angles. The nucleoli were enlarged and mitochondria were developed. Four to eight weeks after the transplantation, peripheral blood stem cells gradually differentiated and proliferated into osteoblasts. Cytoplasm was projected, formed round or shuttle-shaped tentacles, connected to the proximal osteoblasts, and expanded into collagenous fibers. There were numerous petty ribose granules having increasing electron density on the surface of expanded rough endoplasmic reticulum. By this time, tissue engineered bone was gradually absorbed. During 8-12 weeks, pieces of bone trabeculas were formed, and it replaced tissue engineered bone. In the experimental group, immunohistochemistry indicated that TGF-β expression was negative in the bone defect region; in the fourth week, a few of osteoblasts showed mildly positive TGF-β expression near the bone defect region; in 8-12 weeks, the whole bone defect region, and all osteoblasts, collagenous fibers and fibroblasts presented a strongly positive TGF-β expression (Figure 2). In the control group, 8-12 weeks after tissue engineered bone transplantation, TGF-β expression was mildly positive in only bone defect fringe (Figures 3 and 4). There were no inflammatory infiltrations in both groups.

DISCUSSION

Alveolar bone tissue is hard to repair by itself due to alveolysis defect. Peripheral blood stem cell is a multi-differentiation precursor cell, and it can differentiate into osteoblasts[3]. While, tissue engineered bone which is regarded as a vector of cell transplantation has a good compatibility to receptor tissue and seed cells. Transforming growth factor-β (TGF-β) can induce the differentiation and proliferation of peripheral blood stem cell into osteoblasts. Some researches demonstrate that TGF-β is an important regulatory factor to repair bone defect[4]. Exogenous TGF-β can remarkably promote bone trauma healing and bone tissue repairing and induce new bone formation in vivo.
In this study, two weeks after peripheral blood stem cell-tissue engineered bone transplantation in the experimental group, TGF-β expression was mildly positive in the bone defect fringe; under electron microscope, peripheral blood stem cells deformed from ellipse to multi-angles. The nucleoli were enlarged and cytoplasm was projected. This suggested that TGF-β was produced in the bone defect fringe and its activity was taken into effects. During 8-12 weeks, new bone tissue completely repaired the bone defect, and bone trabecula was formed. The whole bone defect region, and all osteoblasts, collagenous fibers and fibroblasts presented a strongly positive TGF-β expression in the experimental group. However, in the control group, TGF-β expression was mildly positive in only bone defect fringe. This suggested that TGF-β took part in bone reconstruction and played a key role in inducing the differentiation and proliferation of peripheral blood stem cells into osteoblasts.
Researches indicate that stem cell is a multi-differentiation precursor cell, and it has a strong differentiation into osteoblasts. Therefore, it becomes a hot seed cell for repairing bone defect now[5]. Stem cells are derived from embryonic tissues, bone marrow, other organs, and peripheral blood[6]. The collection is limited to embryo and other organs and harmful to bone marrow, but peripheral blood stem cells are rich in resources. The results in this study indicated that peripheral blood stem cells differentiated and proliferated into a lot of osteoblasts based on TGF-β induction 4-8 weeks after implantation. Moreover, a plenty of fibroblasts and collagenous fibers formed into new bone tissues to repair the bone defect. By 12 week, pieces of bone trabeculas were formed. This suggested that bone repairing was generally finished. However, there was no bone trabecula in the control group. Granulation tissue filled in the bone defect region, and TGF-β expression was mildly positive in the bone defect fringe.
It is important for tissue engineered bone, as a vector of seed cell transplantation, to have a good compatibility to seed cells and receptor bone bed[7-21]. Tissue engineered bone becomes a soft, stretchable, and cancellated biomaterial following deproteinization and decalcification. Results in this study demonstrated that tissue engineered bone was gradually absorbed and replaced by new bone tissue in the experimental group 4 weeks after transplantation. None of inflammatory reactions were found. Tissue engineered bone is a biomaterial and can be absorbed by organism. Iliac bone collected from piggy was used to make tissue engineered bone following deproteinization and decalcification. It was safe and cheap, while there were no immunological rejections.

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外周血干细胞与组织工程骨复合移植
过程中转化生长因子β在修复犬牙
周骨缺损的表达*

赵淑贤,王　敏,张建国,董 凯,张 兰,赵东方
西安交通大学口腔医学院口腔内科教研室,陕西省西安市 710004
赵淑贤，女，1950年生，陕西省西安市人，汉族，1977年四川医学院毕业，副教授，副主任医师，主要从事牙周病的病因及临床治疗学研究。
陕西省科技攻关基金资助项目(2004-K16-G16)*
摘要
背景：外周血干细胞是一类具有多分化潜能的前体细胞,有分化为成骨的潜能,组织工程骨作为细胞移植的载体,与受体组织和种子细胞有良好的相容性。转化生长因子β是骨缺损修复的重要调节因子，有诱导外周血干细胞分化增殖为成骨细胞的作用。
目的：观察外周血干细胞与组织工程骨复合移植修复牙周骨缺损中转化生长因子β的表达。
设计：以细胞为对象的观察实验。
单位：西安交通大学口腔医院。
材料：实验于2003/2006年在西安交通大学口腔医院完成。实验动物由西安交通大学医学院（原西安医科大学）动物试验中心提供。实验动物均采用氯胺酮肌肉诱导后,肌内注射速眠新麻醉后进行手术或处死取材，对动物处置符合动物伦理学标准。
方法：抽取犬外周血干细胞，制成细胞悬液备用。取健康仔猪髂骨制作脱钙脱蛋白生物组织工程骨,浸入犬外周血干细胞细胞悬液中备用。将10 只健康杂种犬分成实验组和对照组,每组5 只。自犬下颌左右侧尖牙之间沿唇侧牙龈沟处达牙槽嵴,再转向双侧前庭沟切开,形成一个梯形瓣，向下翻瓣暴露唇侧骨板，在下颌侧切牙之间用涡轮钻制备 2 cm×2 cm×1 cm 的骨缺损区,实验组植入外周血干细胞-组织工程骨，对照组不移植外周血干细胞,仅移植组织工程骨。术后2，3，4，8，12周采用免疫组织化学方法观察外周血干细胞分化增殖成为成骨细胞过程中转化生长因子β的表达。
主要观察指标：①光镜和透射电镜观察外周血干细胞转化成为成骨细胞的形态学变化和细胞器的结构功能。②免疫组织化学方法测量外周血干细胞在转化成为成骨细胞的过程中转化生长因子β的表达。
结果：实验组在外周血干细胞-组织工程骨移植后2 周,即见骨缺损区边缘转化生长因子β呈弱阳性表达,4~8 周骨缺损区中心区域可见大量的成骨细胞、成纤维细胞和胶原纤维呈现强阳性表达,12 周骨缺损区已完全修复。对照组组织工程骨移植后8~12 周,仅在骨缺损边缘区域呈转化生长因子β弱阳性表达。
结论：当用外周血干细胞和组织工程修复犬牙周骨缺损时，转化生长因子β能诱导外周血干细胞分化和增殖为成骨细胞。
关键词：外周血干细胞; 免疫组织化学; 转化生长因子β;犬
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)08-1573-04
赵淑贤,王敏,张建国,董凯,张兰,赵东方.外周血干细胞与组织工程骨复合移植修复犬牙周骨缺损过程中转化生长因子β的表达[J].中国组织工程研究与临床康复，2008，12(8):1573-1576
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1573(ps).pdf]
(Edited by Asok Mukhopadhyay/Ji H/Wang L)