系统归巢的间充质干细胞在牙周组织修复再生过程中的作用研究
|
摘要
牙周组织的修复包含了一部分在牙周区域存在的前体细胞的参与,并且这些细胞具备自我更新、多向分化(可向成纤维细胞、成骨细胞和成牙骨质母细胞分化)的间充质干细胞的特性[1,2],但对于这些前体细胞的来源问题,截至目前一直没有明确的答案。有研究表明这些前体细胞主要集中于牙周韧带血管旁区域；并且牙槽骨骨内膜区域的前体细胞也具备从牙槽骨向牙周韧带迁移的潜能[3,4]。有意义的是,这些牙周韧带前体细胞的血管旁定位提示我们骨髓可能是这些前体细胞的来源[4]。
体外实验研究发现骨髓基质干细胞(bone marrow stem cells, BMSCs)与牙周成纤维细胞共同培养时,具有体内牙周成纤维细胞的特性,能够使骨桥素(osteopontin, OPN)和骨钙素(osteocalcin, OC)的表达显著增加,而骨涎蛋白(bone sialoprotein, BSP)的表达明显降低[5]。体内研究显示,白体BMSCs移植入犬牙周骨缺损区,BMSCs能在缺损区成活、增殖,并促使牙周组织再生[6,7]；将复合到明胶微载体颗粒中的GFP+(green fluorescent protein)的BMSCs局部应用于大鼠牙周缺损模型中,可观察到阳性细胞参与到牙周组织的各个部分(牙周膜、牙槽骨和牙骨质)的重建[8]。这些事实说明：BMSCs具有分化为牙周前体细胞并进一步促进牙周组织再生的能力。
有研究表明,当器官受损时,移植的骨髓基质细胞在损伤期间能迁移/归巢至受损部位,并修复该受损区[9],而经静脉移植的MSCs可以特异性的迁移到病变组织,在适合其生存的微环境下分化,最终改善器官的功能。已有的研究结果提示病变组织可能存在某些分子,在其形成的浓度梯度的诱导下实现了MSCs的趋化。而聚集到损伤部位的成体干细胞行使其修复功能的机制可能有：分化为适合的细胞类型；提供一定的细胞因子和其它类型的因子以增强内源性细胞的修复功能等[10]。
而Li shu等通过将荧光素酶和GFP双标的BMSCs方法经尾静脉输入小鼠体内,已证实系统骨髓来源的MSCs能够参与颌骨和颅骨的骨再生[11],这一结果更加坚定了我们的猜测：1、系统移植的骨髓基质细胞能够归巢至牙周缺损区；2在炎症刺激作用下,骨髓来源的MSC也可募集牙周受损区域周围的MSCs迁移到受损区域,并与之共同参与及调控牙周组织的修复与再生；3、牙周膜干细胞或牙周区域的成体干细胞部分来源于系统骨髓。故本研究中我们经过骨髓腔内注射移植增强型绿色荧光蛋白标记的骨髓来源的间充质干细胞(BM-MSCs),通过建立牙周急性炎症缺损模型来探讨系统骨髓源性细胞在牙周组织的创伤修复与再生过程中的作用,为下一步利用趋化因子趋化BM-MSCs和内源性的其它干细胞应用于原位组织工程的可行性提供一定的理论依据。材料和方法
第一部分：本实验采用Percoll密度梯度离心的方法,分离培养大鼠骨髓来源的间充质干细胞(rBM-MSCs)。通过有限稀释法来判断rBM-MSCs的克隆形成能力；通过诱导其三向分化来鉴定分离的细胞,在体外以0.μM地塞米松,10mMβ-甘油磷酸钠,50μM抗坏血酸磷酸盐诱导rBM-MSCs向成骨细胞分化,并以茜素红染色鉴定；以1μM地塞米松,200μM吲哚美辛,10μM胰岛素和0.5mM 3—异丁基—1—甲基黄嘌呤诱导rBM-MSCs向脂肪细胞分化,并用油红O(Oil Red O)染色鉴定；以50nM抗坏血酸,10ng/ml转录生长因子β1(transforming growth factor-β1,TGF-β1)和6.25μg/ml胰岛素诱导rBM-MSCs向软骨细胞分化,并以阿尔新兰染色鉴定。应用脂质体转染的方法,将第三代慢病毒包装系统,包括携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的慢病毒表达载体pBPLV、慢病毒包装质粒pLP1、pLP2和包膜质粒pLP/VSVG转入包装细胞293FT。进而用获得的慢病毒攻击靶细胞rBM-MSCs,实现EGFP基因的转入,并通过流式分选的方法对转染后的细胞进行分选,以获得稳定强表达EGFP基因的rBM-MSCs用于后续的实验。通过MTT实验检测转染前后细胞增殖的变化,以确定转染对细胞的影响。第二部分：为实现EGFP+ rBM-MSCs体内的顺利移植,我们对SD大鼠进行60Coγ,射线8.0Gy全身照射(total body irradiation, TBI)的预处理方案,分别通过骨髓腔内注射和尾静脉注射的方法将细胞输入动物体内。移植后3周,建立牙周组织炎症缺损模型,并在术后的1周、2周、4周、6周通过荧光显微镜直接观察确定EGFP+ rBM-MSCs在牙周缺损手术区域的分布情况,DAPI(4’,6-二乙酰基-2-苯基吲哚)染色,荧光显微镜下计数确定迁移的阳性细胞数,并通过H&E染色、Masson染色观察比较牙周组织修复在组间和时相之间的差异,通过免疫组化检测GFP、骨桥素和Ⅰ型胶原在牙周组织缺损区域内的表达,进一步验证我们上述的观察结果。
结果：
第一部分：经过Percoll密度梯度离心法我们获得了比较纯的、细胞的大小和形态接近一致的rBM-MSCs。克隆形成实验结果,CFU-F的形成率＞50%,证明rBM-MSCs具备较强的克隆形成能力。经骨向诱导4周,茜素红染色后可见致密红染的细胞外基质沉积和矿化结节的形成。经脂向诱导后,油红O(Oil Red O)染色可观察到阳性染色的脂肪滴形成。经软骨细胞分化诱导后,阿尔新兰染色(Alcian blue)结果观察到rBM-MSCs培养物内可见类软骨样嗜阿尔新兰染色的基质。说明分离培养的rBM-MSCs具备三向分化的潜能。采用第三代的慢病毒包装系统包装293FT细胞,效率达到95%以上,收集的病毒施行靶细胞的转染,荧光显微镜下可观察到大量GFP阳性细胞的存在。将转染后的细胞经过流式细胞分选术后,确定阳性比例为60%左右,并将其中40%强表达EGFP的rBM-MSCs分选收集,进行扩大培养。通过MTT检测确定,转染后EGFP+的rBM-MSCs的增殖与转染前相比无显著性差异,细胞形态和生长状态均良好,体外连续培养可稳定传代。
第二部分：为提高移植细胞的移植效率,我们采用60Coγ射线,8.0Gy总量(剂量率1.0Gy/min)进行全身照射(TBI)的预处理方案。照射后,经过骨髓腔内注射移植细胞的实验大鼠生存状态比尾静脉移植组的大鼠好,且持续到整个实验结束。通过DAPI染色,荧光显微镜直接观察显示EGFP+的rBM-MSCs在术后1周即可迁移到牙周缺损手术区域,到2周时,迁移的细胞数达到最高,并且在新形成的骨岛中可观察到绿色阳性细胞的存在,且新生血管周围分布着大量阳性细胞。在术后4周,新生牙周膜和牙骨质中也观察到阳性细胞的存在。通过计数统计,与非炎症缺损模型组相比,炎症缺损模型组在相同时间点,迁移的细胞数目高,且有统计学差异(P<0.05)。与尾静脉移植组相比,骨髓腔内移植组在术后1w和2w迁移的阳性细胞数较多,且有统计学差异(P＜0.05)。H&E染色和Masson染色显示,骨髓腔内移植炎症缺损模型组具有较强的新骨形成能力、牙周膜重建和新牙骨质形成的能力。骨桥素(OPN)免疫组化显示,缺损区内见大量阳性细胞,细胞外基质中也有较强骨桥素表达。新骨形成越活跃的地方,骨桥素阳性表达也越强。Ⅰ型胶原免疫组化染色也显示缺损区内部见Ⅰ型胶原染色呈棕色,主要分布在新骨形成区。而GFP免疫组化结果也证实上述棕染区为移植的EGFP+ rBM-MSCs来源的细胞。
结论：
1、rBM-MSCs可以在体外成功分离培养、扩增和纯化,并且具备干细胞的特性即较强的克隆形成能力和向成骨细胞、脂肪细胞和软骨细胞分化的潜能。慢病毒包装系统可成功转染rBM-MSCs且转染效率高,不影响干细胞增殖能力。标记后的EGFP+的rBM-MSCs可用于观察rBM-MSCs在体内的存活、迁徙及分化。表明慢病毒包装细胞是一种高效、安全的转染方式。
2、经8.0Gy全身照射(TBI)的预处理方案,尾静脉和骨髓腔内注射移植EGFP+的rBM-MSCs均可到达手术区域牙周组织的各个部分(新生牙周膜、牙槽骨和牙骨质),并且与尾静脉移植组相比,骨髓腔内注射移植是一种有效的干细胞移植法。荧光显微镜的观察结果和H&E染色、Masson染色和免疫组化相结合,证实急性炎症缺损能够迅速的趋化大量的EGFP+的rBM-MSCs迁移到缺损区,迁移到的部分阳性细胞可分化为成骨细胞,且绿色阳性细胞在新生牙骨质内的定位提示成牙骨质细胞部分来源于系统骨髓。同时新生血管周围分布大量阳性细胞,且阳性细胞同新生的牙周膜纤维伴行,提示牙周膜干细胞部分来源于骨髓来源的间充质干细胞,并提示迁移来的EGFP+ rBM-MSCs通过部分直接分化为功能细胞,并且通过旁分泌的方式分泌大量生物活性因子,促进血管形成,而新生区非阳性功能细胞的存在提示EGFP+ rBM-MSCs趋化受体大鼠牙周受损区域周围的干细胞或前体细胞迁移到受损区域,并通过促进这些细胞的分裂、增殖和分化来促进牙周组织的修复和再生。
Progenitor cells that may participate into the repair of the periodontal tissues have been reported to possess properties of mesenchymal stem cells such as self-renewal and multi-lineage differentiation ability(differentiate to fibroblasts, osteoblasts, and cementoblasts). However, the origin of this population is not well identified. In addition, the previous reports showed that these progenitor cells mainly located in the paravascular region of the periodontal tissues and were able to migrate from alveolar bone into the periodontal ligament. All of these suggest that bone marrow may be the origin of these local progenitor cells.
Co-cultured with periodontal fibroblasts in ex-vivo sdudy, bone marrow stem cells (BMSCs) exhibited the characteristics of periodontal fibroblasts, in which the expression of osteopontin and osteocalcin were dramatically improved and lower expression of bone sialoprotein was observed. Previously, in the beagle dog periodontal defect model, the autologous BM-MSCs were shown to be able to improve cementum, bone and periodontal ligament regeneration. And in another in vivo study, when GFP+ rat BM-MSCs expanded ex vivo on microcarrier gelatin beads were transplanted into a surgically created rat periodontal defect, the evidence of the GFP positive cells participating directly into the regeneration of bone, cementum and periodontal ligament was observed. The above reports indicate that BMSCs possess the capability to differentiate into the periodontal progenitor cells and enhance the regeneration of the periodontal tissues.
BMSCs are known to migrate or home preferentially to injured sites when transplanted in animal models of injury, which demonstrates that MSCs can migrate into the specific niche, adapt to the local microenvironment and improve the function of the tissue. Chemotaxis assays showed that cultured MSCs migrated to the injured sites due to the existence of growth factor, chemokine and extracellular matrix, and chemokines in a dose-dependent fashion drived the migration. The mechanisms involved in the restoration ability of somatic stem cells docking at sites of injured tissues include transdifferentiating to some available cell type, secreting cytokines and other molecules to enhance the function of the endogenous cell population.
The previous study demonstrated that luciferase and GFP double-labeled BMSCs systemically transplanted into irradiated mice could be detected in the bone wound site created in the mandibles and calvaria, which imply transplanted BMSCs can migrate to bone defect and participate in bone regeneration in orocraniofacial region. The above reports encourage our hypothesis that systemically transplanted BMSCs can home or dock at the sites of the periodontal defect, systemic bone marrow-derived MSCs can recruit the endogenous MSCs and exert the ability of regulation and synergetic effects on the repair and regeneration of the periodontal tissues, and periodontal ligament stem cells (PDLSCs) or cementoblasts partly derived from bone marrow.
Materials and methods:
Part I:Rat bone marrow-derived mesenchymal stem cells were isolated by density gradient centrifugation method with percoll. To assess colony-forming efficiency, CFU-F assay was performed by the limiting dilution method. To assess the tri-lineage differentiation potential, rBM-MSCs were induced to differentiate towards osteoblasts(cells cultured in osteogenic induction medium supplemented with a-MEM containing 10% FCS,0.1μM dexamethasone, 10mMβ-glycerophosphate,50μM ascorbate-2-phosphate and 1% antibiotic/antimycotic.), adipocytes(adipogenic induction medium supplemented with a-MEM containing 10% FCS,1μM dexamethasone,200μM indomethacin,10μM insulin,0.5mM isobutyl-methylxanthine and 1% antibiotic/antimycotic) and chondrocytes(chondrogenic induction medium supplemented with a-MEM containing 1% FCS,50nM ascorbate-2-phosphate,10 ng/ml TGF-β1 and 6.25μg/ml insulin and 1% antibiotic/antimycotic), respectively. Alizarin red staining, oil red O staining, and alcian blue staining were performed to detect the differentiation. To trace directly the distribution and differentiation of rBM-BMSCs in vivo, the lentiviral vector with enhanced green fluorescent protein (pBPLV-EGFP) was used to label rBM-BMSCs. The lentiviral system includes pBPLV, pLP1, pLP2 and pLP/VSVG. Lentiviral expression vector was firstly transfected into the packaging cells 293FT, then the viral supernatant were applied for transfection of rBM-BMSCs. To obtain the positive cells with high expression of EGFP, rBM-BMSCs were then sorted by FACS. Meanwhile, the difference of cell proliferation between transfected and untransfected cells was analyzed by using MTT assay.
Part II:To improve the efficiency of cell transplantation, we adopted the preparative regimen with 8.0 Gy (dose rate 1.0 Gy/min) total body irradiation on the SD rats. Then we applied the method of intra bone marrow (IBM) and intravenous (IV) transplantation 4h after irradiation, respectively. The periodontal inflammatory defect model was established 3 weeks after cell transplantation. The location of transplanted EGFP positive cells and the precise numbers of the migration and the difference of migrated positive cells among all of the groups and multi-time dots were determined by direct observation with fluorescence microscope 1w,2w,4w,6w after the surgery. H&E staining and Masson staining histomorphometric were performed to observe the ability to restore and repair periodontal tissues in experimental groups. Immunohistochemical staining for GFP, OPN and type I collagen were performed to verify the results of the above observation.
Results:
Part I:The pure rBM-MSC cells population with stable morphology was obtained by density gradient centrifugation. By classical CFU-F assay, we confirmed that rBM-MSCs were clonogenic with high colony-forming efficiency (CFU-F>50%). After induction under osteogenic medium for 4 weeks and alizarin red staining, deposition of densely stained extracellular matrix and mineralized nodules were observed. After induction under adipogenic medium for 2 weeks and oil red O staining, aggregation and deposition of lipid-rich vacuoles in the rBM-MSCs were detected. Correspondingly, after chondrogenic induction, the positive staining density was detected. The above results demonstrated that rBM-MSCs owned the potential of tri-lineage differentiation (osteoblasts, adipocytes, and chondrocytes). In addition, high purity of 60.02% EGFP+ rBM-MSCs was achieved using the lentiviral system, and 40% of purified cells by FACS with high level of GFP expression were collected for expansion and further study. MTT assay showed that transfected rBM-MSCs displayed well-spread in appearance and slightly lower proliferation compared with untransfected cells, but no statistically difference was observed (P>0.05).
Part II:After irradiation by 8.0Gy gamma ray with total body irradiation, all of the rats kept alive and displayed better mental status in the whole experiment in the IBM group compared with the IV group. After the periodontal surgery for 1 week, the EGFP+ rBM-MSCs were observed homing/docking into the injury sites by fluorescence microscope. Moreover, the numbers of EGFP positive cells in the defect reached the maximum at 2w after surgery. Meanwhile, some positive cells were observed locating in the newly formed bone islands, and majority of positive cells docked surrounded the neovessels. In addition, the positive cells were also existed in the newly formed periodontal ligament and cementum 4 weeks after surgery. Compared with the non-inflammatory defect model, larger numbers of homed positive cells were observed visualized by direct observation of cells using a fluorescence microscope in the inflammatory defect model. Compared with the IV group, the homed positive cells number was larger in the IBM group 1 week and 2 weeks after surgery. Histological analysis demonstrated that the ability of new bone formation, osseous maturation, periodontal ligament reconstruction and new cementum deposition were much more stronger in the IBM inflammatory defect model than that in the other groups. Immunohistochemical staining for OPN showed that cells and cell extracellular matrix in the injured sites were positive staining especially in the region of the newly formed bone, and the immunohistochemical staining for CoL I got the same results. Moreover, the expression of GFP was detected in the same area, suggesting that the above cells with the brown signal were derived from transplanted EGFP+ rBM-MSCs.
Conclusions:
1. rBM-MSCs can be easily isolated and expanded in vitro and possess classical stem cell properties with single colony forming ability and multipotent differentiation potential. The lentiviral system possesses a high efficiency of packaging cells which improve the transfection efficiency and enhance the procedure of our attempt.
2. Systemically transplanted EGFP+ rBM-MSCs can home or dock at the periodontal defect sites, and in addition, these positive cells maily locate in the new bone, new periodontal ligament and new cementum. Compared with intravenous transplantation model, the intra-bone marrow transplantation is an efficient method for cell transplantation. H&E staining, Masson staining and immunohistochemical staining combined with the visualization of fluorescence microscope demonstrate that acute inflammatory defect can recruit more EGFP+ rBM-MSCs which participate in the regeneration of the periodontal tissue and directly differentiate into the osteoblasts and osteocytes. Despite lack of cementum-specific markers, the morphology and position in correlation with the new cementum indicated that EGFP positive cells underwent the differentiation to cementoblasts. Some EGFP positive cells in the periodontal ligament region located in the paravascular site indicating that periodontal ligment stem cell partially come from bone marrow and transplanted cells not only participate directly in bone repair but promote new vessel formation by paracrine effects. However, not all fibroblast cells in the new bone, periodontal ligament and new cementum were positive for GFP. This suggests host endogenous progenitor cells may have been recruited by BM-MSCs through secretion of trophic factors.
体外实验研究发现骨髓基质干细胞(bone marrow stem cells, BMSCs)与牙周成纤维细胞共同培养时,具有体内牙周成纤维细胞的特性,能够使骨桥素(osteopontin, OPN)和骨钙素(osteocalcin, OC)的表达显著增加,而骨涎蛋白(bone sialoprotein, BSP)的表达明显降低[5]。体内研究显示,白体BMSCs移植入犬牙周骨缺损区,BMSCs能在缺损区成活、增殖,并促使牙周组织再生[6,7]；将复合到明胶微载体颗粒中的GFP+(green fluorescent protein)的BMSCs局部应用于大鼠牙周缺损模型中,可观察到阳性细胞参与到牙周组织的各个部分(牙周膜、牙槽骨和牙骨质)的重建[8]。这些事实说明：BMSCs具有分化为牙周前体细胞并进一步促进牙周组织再生的能力。
有研究表明,当器官受损时,移植的骨髓基质细胞在损伤期间能迁移/归巢至受损部位,并修复该受损区[9],而经静脉移植的MSCs可以特异性的迁移到病变组织,在适合其生存的微环境下分化,最终改善器官的功能。已有的研究结果提示病变组织可能存在某些分子,在其形成的浓度梯度的诱导下实现了MSCs的趋化。而聚集到损伤部位的成体干细胞行使其修复功能的机制可能有：分化为适合的细胞类型；提供一定的细胞因子和其它类型的因子以增强内源性细胞的修复功能等[10]。
而Li shu等通过将荧光素酶和GFP双标的BMSCs方法经尾静脉输入小鼠体内,已证实系统骨髓来源的MSCs能够参与颌骨和颅骨的骨再生[11],这一结果更加坚定了我们的猜测：1、系统移植的骨髓基质细胞能够归巢至牙周缺损区；2在炎症刺激作用下,骨髓来源的MSC也可募集牙周受损区域周围的MSCs迁移到受损区域,并与之共同参与及调控牙周组织的修复与再生；3、牙周膜干细胞或牙周区域的成体干细胞部分来源于系统骨髓。故本研究中我们经过骨髓腔内注射移植增强型绿色荧光蛋白标记的骨髓来源的间充质干细胞(BM-MSCs),通过建立牙周急性炎症缺损模型来探讨系统骨髓源性细胞在牙周组织的创伤修复与再生过程中的作用,为下一步利用趋化因子趋化BM-MSCs和内源性的其它干细胞应用于原位组织工程的可行性提供一定的理论依据。材料和方法
第一部分：本实验采用Percoll密度梯度离心的方法,分离培养大鼠骨髓来源的间充质干细胞(rBM-MSCs)。通过有限稀释法来判断rBM-MSCs的克隆形成能力；通过诱导其三向分化来鉴定分离的细胞,在体外以0.μM地塞米松,10mMβ-甘油磷酸钠,50μM抗坏血酸磷酸盐诱导rBM-MSCs向成骨细胞分化,并以茜素红染色鉴定；以1μM地塞米松,200μM吲哚美辛,10μM胰岛素和0.5mM 3—异丁基—1—甲基黄嘌呤诱导rBM-MSCs向脂肪细胞分化,并用油红O(Oil Red O)染色鉴定；以50nM抗坏血酸,10ng/ml转录生长因子β1(transforming growth factor-β1,TGF-β1)和6.25μg/ml胰岛素诱导rBM-MSCs向软骨细胞分化,并以阿尔新兰染色鉴定。应用脂质体转染的方法,将第三代慢病毒包装系统,包括携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的慢病毒表达载体pBPLV、慢病毒包装质粒pLP1、pLP2和包膜质粒pLP/VSVG转入包装细胞293FT。进而用获得的慢病毒攻击靶细胞rBM-MSCs,实现EGFP基因的转入,并通过流式分选的方法对转染后的细胞进行分选,以获得稳定强表达EGFP基因的rBM-MSCs用于后续的实验。通过MTT实验检测转染前后细胞增殖的变化,以确定转染对细胞的影响。第二部分：为实现EGFP+ rBM-MSCs体内的顺利移植,我们对SD大鼠进行60Coγ,射线8.0Gy全身照射(total body irradiation, TBI)的预处理方案,分别通过骨髓腔内注射和尾静脉注射的方法将细胞输入动物体内。移植后3周,建立牙周组织炎症缺损模型,并在术后的1周、2周、4周、6周通过荧光显微镜直接观察确定EGFP+ rBM-MSCs在牙周缺损手术区域的分布情况,DAPI(4’,6-二乙酰基-2-苯基吲哚)染色,荧光显微镜下计数确定迁移的阳性细胞数,并通过H&E染色、Masson染色观察比较牙周组织修复在组间和时相之间的差异,通过免疫组化检测GFP、骨桥素和Ⅰ型胶原在牙周组织缺损区域内的表达,进一步验证我们上述的观察结果。
结果：
第一部分：经过Percoll密度梯度离心法我们获得了比较纯的、细胞的大小和形态接近一致的rBM-MSCs。克隆形成实验结果,CFU-F的形成率＞50%,证明rBM-MSCs具备较强的克隆形成能力。经骨向诱导4周,茜素红染色后可见致密红染的细胞外基质沉积和矿化结节的形成。经脂向诱导后,油红O(Oil Red O)染色可观察到阳性染色的脂肪滴形成。经软骨细胞分化诱导后,阿尔新兰染色(Alcian blue)结果观察到rBM-MSCs培养物内可见类软骨样嗜阿尔新兰染色的基质。说明分离培养的rBM-MSCs具备三向分化的潜能。采用第三代的慢病毒包装系统包装293FT细胞,效率达到95%以上,收集的病毒施行靶细胞的转染,荧光显微镜下可观察到大量GFP阳性细胞的存在。将转染后的细胞经过流式细胞分选术后,确定阳性比例为60%左右,并将其中40%强表达EGFP的rBM-MSCs分选收集,进行扩大培养。通过MTT检测确定,转染后EGFP+的rBM-MSCs的增殖与转染前相比无显著性差异,细胞形态和生长状态均良好,体外连续培养可稳定传代。
第二部分：为提高移植细胞的移植效率,我们采用60Coγ射线,8.0Gy总量(剂量率1.0Gy/min)进行全身照射(TBI)的预处理方案。照射后,经过骨髓腔内注射移植细胞的实验大鼠生存状态比尾静脉移植组的大鼠好,且持续到整个实验结束。通过DAPI染色,荧光显微镜直接观察显示EGFP+的rBM-MSCs在术后1周即可迁移到牙周缺损手术区域,到2周时,迁移的细胞数达到最高,并且在新形成的骨岛中可观察到绿色阳性细胞的存在,且新生血管周围分布着大量阳性细胞。在术后4周,新生牙周膜和牙骨质中也观察到阳性细胞的存在。通过计数统计,与非炎症缺损模型组相比,炎症缺损模型组在相同时间点,迁移的细胞数目高,且有统计学差异(P<0.05)。与尾静脉移植组相比,骨髓腔内移植组在术后1w和2w迁移的阳性细胞数较多,且有统计学差异(P＜0.05)。H&E染色和Masson染色显示,骨髓腔内移植炎症缺损模型组具有较强的新骨形成能力、牙周膜重建和新牙骨质形成的能力。骨桥素(OPN)免疫组化显示,缺损区内见大量阳性细胞,细胞外基质中也有较强骨桥素表达。新骨形成越活跃的地方,骨桥素阳性表达也越强。Ⅰ型胶原免疫组化染色也显示缺损区内部见Ⅰ型胶原染色呈棕色,主要分布在新骨形成区。而GFP免疫组化结果也证实上述棕染区为移植的EGFP+ rBM-MSCs来源的细胞。
结论：
1、rBM-MSCs可以在体外成功分离培养、扩增和纯化,并且具备干细胞的特性即较强的克隆形成能力和向成骨细胞、脂肪细胞和软骨细胞分化的潜能。慢病毒包装系统可成功转染rBM-MSCs且转染效率高,不影响干细胞增殖能力。标记后的EGFP+的rBM-MSCs可用于观察rBM-MSCs在体内的存活、迁徙及分化。表明慢病毒包装细胞是一种高效、安全的转染方式。
2、经8.0Gy全身照射(TBI)的预处理方案,尾静脉和骨髓腔内注射移植EGFP+的rBM-MSCs均可到达手术区域牙周组织的各个部分(新生牙周膜、牙槽骨和牙骨质),并且与尾静脉移植组相比,骨髓腔内注射移植是一种有效的干细胞移植法。荧光显微镜的观察结果和H&E染色、Masson染色和免疫组化相结合,证实急性炎症缺损能够迅速的趋化大量的EGFP+的rBM-MSCs迁移到缺损区,迁移到的部分阳性细胞可分化为成骨细胞,且绿色阳性细胞在新生牙骨质内的定位提示成牙骨质细胞部分来源于系统骨髓。同时新生血管周围分布大量阳性细胞,且阳性细胞同新生的牙周膜纤维伴行,提示牙周膜干细胞部分来源于骨髓来源的间充质干细胞,并提示迁移来的EGFP+ rBM-MSCs通过部分直接分化为功能细胞,并且通过旁分泌的方式分泌大量生物活性因子,促进血管形成,而新生区非阳性功能细胞的存在提示EGFP+ rBM-MSCs趋化受体大鼠牙周受损区域周围的干细胞或前体细胞迁移到受损区域,并通过促进这些细胞的分裂、增殖和分化来促进牙周组织的修复和再生。
Progenitor cells that may participate into the repair of the periodontal tissues have been reported to possess properties of mesenchymal stem cells such as self-renewal and multi-lineage differentiation ability(differentiate to fibroblasts, osteoblasts, and cementoblasts). However, the origin of this population is not well identified. In addition, the previous reports showed that these progenitor cells mainly located in the paravascular region of the periodontal tissues and were able to migrate from alveolar bone into the periodontal ligament. All of these suggest that bone marrow may be the origin of these local progenitor cells.
Co-cultured with periodontal fibroblasts in ex-vivo sdudy, bone marrow stem cells (BMSCs) exhibited the characteristics of periodontal fibroblasts, in which the expression of osteopontin and osteocalcin were dramatically improved and lower expression of bone sialoprotein was observed. Previously, in the beagle dog periodontal defect model, the autologous BM-MSCs were shown to be able to improve cementum, bone and periodontal ligament regeneration. And in another in vivo study, when GFP+ rat BM-MSCs expanded ex vivo on microcarrier gelatin beads were transplanted into a surgically created rat periodontal defect, the evidence of the GFP positive cells participating directly into the regeneration of bone, cementum and periodontal ligament was observed. The above reports indicate that BMSCs possess the capability to differentiate into the periodontal progenitor cells and enhance the regeneration of the periodontal tissues.
BMSCs are known to migrate or home preferentially to injured sites when transplanted in animal models of injury, which demonstrates that MSCs can migrate into the specific niche, adapt to the local microenvironment and improve the function of the tissue. Chemotaxis assays showed that cultured MSCs migrated to the injured sites due to the existence of growth factor, chemokine and extracellular matrix, and chemokines in a dose-dependent fashion drived the migration. The mechanisms involved in the restoration ability of somatic stem cells docking at sites of injured tissues include transdifferentiating to some available cell type, secreting cytokines and other molecules to enhance the function of the endogenous cell population.
The previous study demonstrated that luciferase and GFP double-labeled BMSCs systemically transplanted into irradiated mice could be detected in the bone wound site created in the mandibles and calvaria, which imply transplanted BMSCs can migrate to bone defect and participate in bone regeneration in orocraniofacial region. The above reports encourage our hypothesis that systemically transplanted BMSCs can home or dock at the sites of the periodontal defect, systemic bone marrow-derived MSCs can recruit the endogenous MSCs and exert the ability of regulation and synergetic effects on the repair and regeneration of the periodontal tissues, and periodontal ligament stem cells (PDLSCs) or cementoblasts partly derived from bone marrow.
Materials and methods:
Part I:Rat bone marrow-derived mesenchymal stem cells were isolated by density gradient centrifugation method with percoll. To assess colony-forming efficiency, CFU-F assay was performed by the limiting dilution method. To assess the tri-lineage differentiation potential, rBM-MSCs were induced to differentiate towards osteoblasts(cells cultured in osteogenic induction medium supplemented with a-MEM containing 10% FCS,0.1μM dexamethasone, 10mMβ-glycerophosphate,50μM ascorbate-2-phosphate and 1% antibiotic/antimycotic.), adipocytes(adipogenic induction medium supplemented with a-MEM containing 10% FCS,1μM dexamethasone,200μM indomethacin,10μM insulin,0.5mM isobutyl-methylxanthine and 1% antibiotic/antimycotic) and chondrocytes(chondrogenic induction medium supplemented with a-MEM containing 1% FCS,50nM ascorbate-2-phosphate,10 ng/ml TGF-β1 and 6.25μg/ml insulin and 1% antibiotic/antimycotic), respectively. Alizarin red staining, oil red O staining, and alcian blue staining were performed to detect the differentiation. To trace directly the distribution and differentiation of rBM-BMSCs in vivo, the lentiviral vector with enhanced green fluorescent protein (pBPLV-EGFP) was used to label rBM-BMSCs. The lentiviral system includes pBPLV, pLP1, pLP2 and pLP/VSVG. Lentiviral expression vector was firstly transfected into the packaging cells 293FT, then the viral supernatant were applied for transfection of rBM-BMSCs. To obtain the positive cells with high expression of EGFP, rBM-BMSCs were then sorted by FACS. Meanwhile, the difference of cell proliferation between transfected and untransfected cells was analyzed by using MTT assay.
Part II:To improve the efficiency of cell transplantation, we adopted the preparative regimen with 8.0 Gy (dose rate 1.0 Gy/min) total body irradiation on the SD rats. Then we applied the method of intra bone marrow (IBM) and intravenous (IV) transplantation 4h after irradiation, respectively. The periodontal inflammatory defect model was established 3 weeks after cell transplantation. The location of transplanted EGFP positive cells and the precise numbers of the migration and the difference of migrated positive cells among all of the groups and multi-time dots were determined by direct observation with fluorescence microscope 1w,2w,4w,6w after the surgery. H&E staining and Masson staining histomorphometric were performed to observe the ability to restore and repair periodontal tissues in experimental groups. Immunohistochemical staining for GFP, OPN and type I collagen were performed to verify the results of the above observation.
Results:
Part I:The pure rBM-MSC cells population with stable morphology was obtained by density gradient centrifugation. By classical CFU-F assay, we confirmed that rBM-MSCs were clonogenic with high colony-forming efficiency (CFU-F>50%). After induction under osteogenic medium for 4 weeks and alizarin red staining, deposition of densely stained extracellular matrix and mineralized nodules were observed. After induction under adipogenic medium for 2 weeks and oil red O staining, aggregation and deposition of lipid-rich vacuoles in the rBM-MSCs were detected. Correspondingly, after chondrogenic induction, the positive staining density was detected. The above results demonstrated that rBM-MSCs owned the potential of tri-lineage differentiation (osteoblasts, adipocytes, and chondrocytes). In addition, high purity of 60.02% EGFP+ rBM-MSCs was achieved using the lentiviral system, and 40% of purified cells by FACS with high level of GFP expression were collected for expansion and further study. MTT assay showed that transfected rBM-MSCs displayed well-spread in appearance and slightly lower proliferation compared with untransfected cells, but no statistically difference was observed (P>0.05).
Part II:After irradiation by 8.0Gy gamma ray with total body irradiation, all of the rats kept alive and displayed better mental status in the whole experiment in the IBM group compared with the IV group. After the periodontal surgery for 1 week, the EGFP+ rBM-MSCs were observed homing/docking into the injury sites by fluorescence microscope. Moreover, the numbers of EGFP positive cells in the defect reached the maximum at 2w after surgery. Meanwhile, some positive cells were observed locating in the newly formed bone islands, and majority of positive cells docked surrounded the neovessels. In addition, the positive cells were also existed in the newly formed periodontal ligament and cementum 4 weeks after surgery. Compared with the non-inflammatory defect model, larger numbers of homed positive cells were observed visualized by direct observation of cells using a fluorescence microscope in the inflammatory defect model. Compared with the IV group, the homed positive cells number was larger in the IBM group 1 week and 2 weeks after surgery. Histological analysis demonstrated that the ability of new bone formation, osseous maturation, periodontal ligament reconstruction and new cementum deposition were much more stronger in the IBM inflammatory defect model than that in the other groups. Immunohistochemical staining for OPN showed that cells and cell extracellular matrix in the injured sites were positive staining especially in the region of the newly formed bone, and the immunohistochemical staining for CoL I got the same results. Moreover, the expression of GFP was detected in the same area, suggesting that the above cells with the brown signal were derived from transplanted EGFP+ rBM-MSCs.
Conclusions:
1. rBM-MSCs can be easily isolated and expanded in vitro and possess classical stem cell properties with single colony forming ability and multipotent differentiation potential. The lentiviral system possesses a high efficiency of packaging cells which improve the transfection efficiency and enhance the procedure of our attempt.
2. Systemically transplanted EGFP+ rBM-MSCs can home or dock at the periodontal defect sites, and in addition, these positive cells maily locate in the new bone, new periodontal ligament and new cementum. Compared with intravenous transplantation model, the intra-bone marrow transplantation is an efficient method for cell transplantation. H&E staining, Masson staining and immunohistochemical staining combined with the visualization of fluorescence microscope demonstrate that acute inflammatory defect can recruit more EGFP+ rBM-MSCs which participate in the regeneration of the periodontal tissue and directly differentiate into the osteoblasts and osteocytes. Despite lack of cementum-specific markers, the morphology and position in correlation with the new cementum indicated that EGFP positive cells underwent the differentiation to cementoblasts. Some EGFP positive cells in the periodontal ligament region located in the paravascular site indicating that periodontal ligment stem cell partially come from bone marrow and transplanted cells not only participate directly in bone repair but promote new vessel formation by paracrine effects. However, not all fibroblast cells in the new bone, periodontal ligament and new cementum were positive for GFP. This suggests host endogenous progenitor cells may have been recruited by BM-MSCs through secretion of trophic factors.
引文
1. Gronthos S, Mrozik K, Shi S, Bartold PM. Ovine periodontal ligament stem cells: isolation, characterization, and differentiation potential[J]. Calcif Tissue Int 2006;79(5):310-7.
2. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S. Investigation of multipotent postnatal stem cells from human periodontal ligament[J]. Lancet 2004;364(9429):149-55.
3. McCulloch CA. Progenitor cell populations in the periodontal ligament of mice[J]. Anat Rec 1985;211(3):258-262
4. McCulloch CA, Nemeth E, Lowenberg B, Melcher AH. Paravascular cells in endosteal spaces of alveolar bone contribute to periodontal ligament cell populations[J]. Anat Rec 1987;219(3):233-242
5. Kramer PR, Nares S, Kramer SF, Grogan D, Kaiser M. Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro[J]. J Dent Res 2004;83(1):27-34
6. Hasegawa N, Kawaguchi H, Hirachi A, Takeda K, Mizuno N, Nishimura M, Koike C, Tsuji K, Iba H, Kato Y, Kurihara H. Behavior of transplanted bone marrow-derived mesenchymal stem cells in periodontal defects[J].J Periodontol 2006;77(6):1003-7
7. Kawaquchi H, Hirachi A, Haseqawa N, Iwata T, Hamaguchi H, Shiba H, Takata T, Kato Y, Kurihara H. Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cell[J]. J Periodontol 2004; 75(9):1281-7
8. Yang Y, Rossi FM, Putnins EE. Periodontal regeneration using engineered bone marrow mesenchymal stromal cells[J]. Biomaterials 2010; 31(33):8574-82.
9. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone marrow as a potential source of hepatic oval cells[J]. Science 1999;284(5417):1168-70
10. Chen FM, Wu LA, Zhang M, Zhang R, Sun HH. Homing of endogenous stem/progenitor cells for in situ tissue regeneration:Promises, strategies, and translational perspectives [J]. Biomaterials 2011;32(12):3189-3209.
11. Li S, Tu Q, Zhang J, Stein G, Lian J, Yang PS, Chen J. Systemically transplanted bone marrow stromal cells contributing to bone tissue regeneration [J]. J Cell Physiol 2008;215(1):204-209
12. Kawaguchi H, Ogawa T, Kurihara H, Nanci A. Immunodetection of noncollagenous matrix proteins during periodontal tissue regeneration[J]. J Periodontal Res 2001;36(4): 205-213.
13. Bajada S, Mazakova I, Richardson JB, Ashammakhi N. Updates on stem cells and their applications in regenerative medicine[J]. J Tissue Eng Regen Med 2008;2:169-83.
14. Nakao K, Morita R, Saji Y, Ishida K, Tomita Y, Ogawa M, et al. The development of a bioengineered organ germ method[J]. Nat Methods 2007;4:227-30.
15. de Vries RB, Oerlemans A, Trommelmans L, Dierickx K, Gordijn B. Ethical aspects of tissue engineering:a review[J]. Tissue Eng Part B Rev 2008;14:367-75.
16. Daley GQ, Scadden DT. Prospects for stem cell-based therapy[J]. Cell 2008;132:544-8.
17. El Tamer MK, Reis RL. Progenitor and stem cells for bone and cartilage regeneration[J].J Tissue Eng Regen Med 2009;3:327-37.
18. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament[J]. Lancet 2004;364:149-55.
19. Chen FM, Jin Y. Periodontal tissue engineering and regeneration:current approaches and expanding opportunities [J]. Tissue Eng Part B Rev 2010;16:219-55.
20. Bible E, Chau DY, Alexander MR, Price J, Shakesheff KM, Modo M. Attachment of stem cells to scaffold particles for intra-cerebral transplantation[J]. Nat Protoc 2009;4:1440-53.
21. Yoshikawa T, Mitsuno H, Nonaka I, Sen Y, Kawanishi K, Inada Y, et al. Wound therapy by marrow mesenchymal cell transplantation[J]. Plast Reconstr Surg 2008;121:860-77.
22. Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM. Self-renewal and expansion of single transplanted muscle stem cells[J]. Nature 2008;456:502-6.
23. Quante M, Wang TC. Stem cells in gastroenterology and hepatology[J]. Nat Rev Gastroenterol Hepatol 2009;6:724-37.
24. Sieveking DP, Ng MK. Cell therapies for therapeutic angiogenesis:back to the bench[J]. Vasc Med 2009;14:153-66.
25. Teo AK, Vallier L. Emerging use of stem cells in regenerative medicine[J]. Bio chem J 2010;428:11-23.
26. Griffith LG, Naughton G. Tissue engineeringecurrent challenges and expanding opportunities[J]. Science 2002;295:1009e14.
27. Lutolf MP, Gilbert PM, Blau HM. Designing materials to direct stem-cell fate[J]. Nature 2009;462:433-41.
28. Mrozik KM, Zilm PS, Bagley CJ, Hack S, Hoffmann P, Gronthos S, Bartold PM. Proteomic characterization of mesenchymal stem cell-like populations derived from ovine periodontal ligament, dental pulp and bone marrow:analysis of differentially expressed proteins[J]. Stem Cells Dev 2010;19(10):1485-99
29. Trubiani O, Isgro A, Zini N, Antonucci I, Aiuti F, Di Primio R, Nanci A, Caputi S, Paganelli R. Functional interleukin-7/interleukin-7Ralpha, and SDF-1alpha/CXCR4 are expressed by human periodontal ligament derived mesenchymal stem cells[J]. J Cell Physiol 2008;214(3):706-13.
30. Chen FM, Zhang J, Zhang M, An Y, Chen F, Wu ZF. A review on endogenous regenerative technology in periodontal regenerative medicine[J]. Biomaterials 2010;31:7892-927.
31. Liu Y, Zheng Y, Ding G, Fang D, Zhang C, Bartold PM, Gronthos S, Shi S, Wang S. Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine [J]. Stem Cells 2008;26(4):1065-1073
32. Crisan M, Yap S, Casteilla L. Chen CW, Corselli M, Park TS, et al. A perivascular origin for mesenchymal stem cells in multiple human organs[J]. Cell Stem Cell 2008;3:301-13.
33. Krankel N, Spinetti G, Amadesi S, Madeddu P. Targeting stem cell niches and trafficking for cardiovascular therapy[J]. Pharmacol Ther 2011;129:62-81.
34. Muller R, Lengerke C. Patient-specific pluripotent stem cells:promises and challenges[J]. Nat Rev Endocrinol 2009;5:195-203.
35. Fodor WL. Tissue engineering and cell based therapies, from the bench to the clinic: the potential to replace, repair and regenerate[J]. Reprod Biol Endocrinol 2003;1:102.
36. Prockop DJ. Repair of tissues by adult stem/progenitor cells (MSCs):controversies, myths, and changing paradigms[J]. Mol Ther 2009;17:939-46.
37. Muschler GF, Raut VP, Patterson TE, Wenke JC, Hollinger JO. The design and use of animal models for translational research in bone tissue engineering and regenerative medicine[J]. Tissue Eng Part B Rev 2010;16:123-45.
38. Mao JJ, Stosich MS, Moioli EK, Lee CH, Fu SY, Bastian B, et al. Facial reconstruction by biosurgery:cell transplantation versus cell homing[J]. Tissue Eng Part B Rev 2010;16:257-62.
39. Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ. Stromal cell-derived factor-1 alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury[J]. Circulation 2004;110 (21):3300-3305.
40. Kushida T, Inaba M, Hisha H, Ichioka N, Esumi T, Ogawa R, Iida H, Ikehara S. Intra-bone marrow injection of allogeneic bone marrow cells:a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice[J]. Blood 2001;97(10):3292-9.
41. Ikehara S. A novel method of bone marrow transplantation (BMT) for intractable autoimmune diseases[J]. J Autoimmun 2008;30(3):108-115.
42 陈伟,陈建梅,徐皓.内源性骨髓间充质干细胞定向迁移的影响因素[J].实用医学杂志2007；23(20)：3292-3294.
43. Nyman S, Lindhe J, Karring T. Reattachment-new attachment. In:Lindhe J, ed. Textbook of clinical periodontology,2nd edn. Copenhagen [M]:Munksgaard,1989; 450-476.
44. Chirmule N, Xiao W, Truneh A, Schnell MA, Hughes JV, Zoltick P, Wilson JM. Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle[J]. J Virol 2000;74(5):2420-2425.
45. Zhang Q, Shi S, Liu Y, Uyanne J, Shi Y, Shi S, Le AD. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis[J]. J Immunol 2009;183(12):7787-7798.
46. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue:implications for cell-based therapies[J]. Tissue Eng 2001;7(2):211-228.
47. Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein(GFP)[J]. Gene 1996;173(1):33-38.
48. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression [J]. Science 1994;263(5148):802-805.
49. Wang F, Yu MJ, Yan XL, Wen Y, Zeng Q, Yue W, Yang P, Pei X. Gingiva-derived mesenchymal stem cells-mediated therapeutic approach for bone tissue regeneration[J]. Stem Cells Dev 2011,in press.
50.于美娇,杨丕山,葛少华.苯妥英钠对人牙周膜成纤维细胞生物学活性影响的实验研究[J].华西口腔医学杂志2008；26(2)：215-8.
51. Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy[J]. Proc Natl Acad Sci U S A 1994;91(10):4407-4411.
52. Palella TD, Hidaka Y, Silverman LJ, Levine M, Glorioso J, Kelley WN. Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector[J]. Gene 1989;80(1):137-44.
53. Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells[J]. Exp Hematol 2003; 31(10):890-6.
54. Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan EC. Suppression of allogeneic T-cell proliferation by human marrow stromal cells:implications in transplantation[J]. Transplantation 2003;75(3):389-397.
55. Devine SM, Cobbs C, Jennings M, Bartholomew A, Hoffman R. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates[J]. Blood 2003;101(8):2999-3001.
56. King GN, Hughes FJ. Bone morphogenetic protein-2 stimulates cell recruitment and cementogenesis during early wound healing[J]. J Clin Periodontol 2001;28(5):465-75.
57. Huang KK, Shen C, Chiang CY, Hsieh YD, Fu E. Effects of bone morphogenetic protein-6 on periodontal wound healing in a fenestration defect of rats[J]. J Periodontal Res 2005;40(1):1-10.
58. Talwar R, Di Silvio L, Hughes FJ, King GN. Effects of carrier release kinetics on bone morphogenetic protein-2-induced periodontal regeneration in vivo[J]. J Clin Periodontol 2001;28(4):340-7.
59. Wikesjo UM, Selvig KA, Zimmerman G, Nilveus R. Periodontal repair in dogs: healing in experimentally created chronic periodontal defects[J]. J Periodontol 1991; 62(4):258-63.
60. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases[J]. Lancet 2005;366(9499):1809-1820.
61. Chen T, Duncan MJ. Gingipain adhesion domains mediate Porphyromonas gingivalis adherence to epithelial cells[J]. Microb Pathog 2004;36(4):205-9.
62. Dierickx K, Pauwels M, Laine ML, Van Eldere J, Cassiman JJ, van Winkelhoff AJ, van Steenberghe D, Quirynen M. Adhesion of Porphyromonas gingivalis serotypes to pocket epithelium[J]. J Periodontol 2003;74(6):844-8.
63. He H, Liu R, Desta T, Leone C, Gerstenfeld LC, Graves DT. Diabetes causes decreased osteoclastogenesis, reduced bone formation, and enhanced apoptosis of osteoblastic cells in bacteria stimulated bone loss[J]. Endocrinology 2004;145(1):447-452.
64. Leone CW, Bokhadhoor H, Kuo D, Desta T, Yang J, Siqueira MF, Amar S, Graves DT. Immunization enhances inflammation and tissue destruction in response to Porphyromonas gingivalis[J]. Infect Immun 2006;74(4):2286-2292.
65. Graves DT, Fine D, Teng YT, Van Dyke TE, Hajishengallis G. The use of rodent models to investigate host-bacteria interactions related to periodontal diseases[J]. J Clin Periodontol 2008; 35(2):89-105.
66. Fibbe WE, Noort WA. Mesenchymal stem cells and hematopoietic stem cell transplantation [J]. Ann N Y Acad Sci 2003;996:235-244.
67.张建霞.苯妥英钠聚乳酸-羟基乙酸微球泊洛沙姆407凝胶对大鼠牙周缺损愈合影响的研究[D].济南：山东大学硕士研究生论文,2008.
68.程敏,马宁,张泽兵,孙宏晨,李成库.牙胚牙体组织切片的特殊染色方法及应用探讨[J].现代口腔医学杂志2007；21(4)：441-442.
69. Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow [J]. Blood 2001;98(8):2396-2402.
70. Noort WA, Kruisselbrink AB, in't Anker PS, Kruger M, van Bezooijen RL, de Paus RA, Heemskerk MH, Lowik CW, Falkenburg JH, Willemze R, Fibbe WE. Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34 (+) cells in NOD/SCID mice[J]. Exp Hematol 2002;30(8):870-878
71. Devine SM, Cobbs C, Jennings M, Bartholomew A, Hoffman R. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates[J]. Blood 2003;101(8):2999-3001.
72. Sodek J, Ganss B, McKee MD. Osteopontin[J]. Crit Rev Oral Biol Med 2000;11(3):279-303.
73. Morinobu M, Ishijima M, Rittling SR, Tsuji K, Yamamoto H, Nifuji A, Denhardt DT, Noda M. Osteopontin expression in osteoblasts and osteocytes during bone formation under mechanical stress in the calvarial suture in vivo[J]. J Bone Miner Res 2003;18(9):1706-15.
74. Block GJ, Ohkouchi S, Fung F, Frenkel J, Gregory C, Pochampally R, DiMattia G, Sullivan DE, Prockop DJ. Multipotent stromal cells are activated to reduce apoptosis in part by upregulation and secretion of stanniocalcin-1[J]. Stem Cells 2009;27(3):670-81.
75. Rehman J, Traktuev D, Li J, Merfeld-Clauss S, Temm-Grove CJ, Bovenkerk JE, Pell CL, Johnstone BH, Considine RV, March KL. Secretion of angiogenic and antiapoptotic factors by human adipose tromal cells[J].Circulation 2004;109(10):1292-8.
76. Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, Grisanti S, Gianni AM. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli[J]. Blood 2002;99(10):3838-43.
77. Krampera M, Glennie S, Dyson J, Scott D, Laylor R, Simpson E, Dazzi F. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide[J]. Blood 2003;101(9):3722-9.
78. Li L, Zhang S, Zhang Y, Yu B, Xu Y, Guan Z. Paracrine action mediate the antifibrotic effect of transplanted mesenchymal stem cells in a rat model of global heart failure[J]. Mol Biol Rep 2009;36(4):725-31.
79. Ohab JJ, Fleming S, Blesch A, Carmichael ST. A neurovascular niche for neurogenesis after stroke[J]. J Neurosci 2006;26(50):13007-16.
80. Seo J, Kim YO, Jo I. Differential expression of stromal cell-derived factor 1 in human brain microvascular endothelial cells and pericytes involves histone modifications[J]. Biochem Biophys Res Commun 2009;382(3):519-24.
81. Sundberg C, Kowanetz M, Brown LF, Detmar M, Dvorak HF. Stable expression of angiopoietin-1 and other markers by cultured pericytes:phenotypic similarities to a subpopulation of cells in maturing vessels during later stages of angiogenesis in vivo[J]. Lab Invest 2002;82(4):387-401.
82. Sorrell JM, Baber MA, Caplan AI. Influence of adult mesenchymal stem cells on in vitro vascular formation[J]. Tissue Eng Part A 2009; 15(7):1751-61.
83. Penn S, Mangi AA. Genetic enhancement of stem cell engraftment, survival, and efficacy[J]. Circ Res 2008;102:1471-82.
84. Fong EL, Chan CK, Goodman SB. Stem cell homing in musculoskeletal injury[J]. Biomaterials 2011;32:395-409.
85. Chute JP. Stem cell homing[J]. Curr Opin Hematol 2006; 13:399-406.
86. Lapidot T, Dar A, Kollet O. How do stem cells find their way home[J]? Blood 2005;106:1901-10.
87. Szekanecz Z, Kim J, Koch AE. Chemokines and chemokines receptors in rheumatoid arthritis[J]. Semin Immunol 2003;15(1):15-21.
2. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S. Investigation of multipotent postnatal stem cells from human periodontal ligament[J]. Lancet 2004;364(9429):149-55.
3. McCulloch CA. Progenitor cell populations in the periodontal ligament of mice[J]. Anat Rec 1985;211(3):258-262
4. McCulloch CA, Nemeth E, Lowenberg B, Melcher AH. Paravascular cells in endosteal spaces of alveolar bone contribute to periodontal ligament cell populations[J]. Anat Rec 1987;219(3):233-242
5. Kramer PR, Nares S, Kramer SF, Grogan D, Kaiser M. Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro[J]. J Dent Res 2004;83(1):27-34
6. Hasegawa N, Kawaguchi H, Hirachi A, Takeda K, Mizuno N, Nishimura M, Koike C, Tsuji K, Iba H, Kato Y, Kurihara H. Behavior of transplanted bone marrow-derived mesenchymal stem cells in periodontal defects[J].J Periodontol 2006;77(6):1003-7
7. Kawaquchi H, Hirachi A, Haseqawa N, Iwata T, Hamaguchi H, Shiba H, Takata T, Kato Y, Kurihara H. Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cell[J]. J Periodontol 2004; 75(9):1281-7
8. Yang Y, Rossi FM, Putnins EE. Periodontal regeneration using engineered bone marrow mesenchymal stromal cells[J]. Biomaterials 2010; 31(33):8574-82.
9. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone marrow as a potential source of hepatic oval cells[J]. Science 1999;284(5417):1168-70
10. Chen FM, Wu LA, Zhang M, Zhang R, Sun HH. Homing of endogenous stem/progenitor cells for in situ tissue regeneration:Promises, strategies, and translational perspectives [J]. Biomaterials 2011;32(12):3189-3209.
11. Li S, Tu Q, Zhang J, Stein G, Lian J, Yang PS, Chen J. Systemically transplanted bone marrow stromal cells contributing to bone tissue regeneration [J]. J Cell Physiol 2008;215(1):204-209
12. Kawaguchi H, Ogawa T, Kurihara H, Nanci A. Immunodetection of noncollagenous matrix proteins during periodontal tissue regeneration[J]. J Periodontal Res 2001;36(4): 205-213.
13. Bajada S, Mazakova I, Richardson JB, Ashammakhi N. Updates on stem cells and their applications in regenerative medicine[J]. J Tissue Eng Regen Med 2008;2:169-83.
14. Nakao K, Morita R, Saji Y, Ishida K, Tomita Y, Ogawa M, et al. The development of a bioengineered organ germ method[J]. Nat Methods 2007;4:227-30.
15. de Vries RB, Oerlemans A, Trommelmans L, Dierickx K, Gordijn B. Ethical aspects of tissue engineering:a review[J]. Tissue Eng Part B Rev 2008;14:367-75.
16. Daley GQ, Scadden DT. Prospects for stem cell-based therapy[J]. Cell 2008;132:544-8.
17. El Tamer MK, Reis RL. Progenitor and stem cells for bone and cartilage regeneration[J].J Tissue Eng Regen Med 2009;3:327-37.
18. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament[J]. Lancet 2004;364:149-55.
19. Chen FM, Jin Y. Periodontal tissue engineering and regeneration:current approaches and expanding opportunities [J]. Tissue Eng Part B Rev 2010;16:219-55.
20. Bible E, Chau DY, Alexander MR, Price J, Shakesheff KM, Modo M. Attachment of stem cells to scaffold particles for intra-cerebral transplantation[J]. Nat Protoc 2009;4:1440-53.
21. Yoshikawa T, Mitsuno H, Nonaka I, Sen Y, Kawanishi K, Inada Y, et al. Wound therapy by marrow mesenchymal cell transplantation[J]. Plast Reconstr Surg 2008;121:860-77.
22. Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM. Self-renewal and expansion of single transplanted muscle stem cells[J]. Nature 2008;456:502-6.
23. Quante M, Wang TC. Stem cells in gastroenterology and hepatology[J]. Nat Rev Gastroenterol Hepatol 2009;6:724-37.
24. Sieveking DP, Ng MK. Cell therapies for therapeutic angiogenesis:back to the bench[J]. Vasc Med 2009;14:153-66.
25. Teo AK, Vallier L. Emerging use of stem cells in regenerative medicine[J]. Bio chem J 2010;428:11-23.
26. Griffith LG, Naughton G. Tissue engineeringecurrent challenges and expanding opportunities[J]. Science 2002;295:1009e14.
27. Lutolf MP, Gilbert PM, Blau HM. Designing materials to direct stem-cell fate[J]. Nature 2009;462:433-41.
28. Mrozik KM, Zilm PS, Bagley CJ, Hack S, Hoffmann P, Gronthos S, Bartold PM. Proteomic characterization of mesenchymal stem cell-like populations derived from ovine periodontal ligament, dental pulp and bone marrow:analysis of differentially expressed proteins[J]. Stem Cells Dev 2010;19(10):1485-99
29. Trubiani O, Isgro A, Zini N, Antonucci I, Aiuti F, Di Primio R, Nanci A, Caputi S, Paganelli R. Functional interleukin-7/interleukin-7Ralpha, and SDF-1alpha/CXCR4 are expressed by human periodontal ligament derived mesenchymal stem cells[J]. J Cell Physiol 2008;214(3):706-13.
30. Chen FM, Zhang J, Zhang M, An Y, Chen F, Wu ZF. A review on endogenous regenerative technology in periodontal regenerative medicine[J]. Biomaterials 2010;31:7892-927.
31. Liu Y, Zheng Y, Ding G, Fang D, Zhang C, Bartold PM, Gronthos S, Shi S, Wang S. Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine [J]. Stem Cells 2008;26(4):1065-1073
32. Crisan M, Yap S, Casteilla L. Chen CW, Corselli M, Park TS, et al. A perivascular origin for mesenchymal stem cells in multiple human organs[J]. Cell Stem Cell 2008;3:301-13.
33. Krankel N, Spinetti G, Amadesi S, Madeddu P. Targeting stem cell niches and trafficking for cardiovascular therapy[J]. Pharmacol Ther 2011;129:62-81.
34. Muller R, Lengerke C. Patient-specific pluripotent stem cells:promises and challenges[J]. Nat Rev Endocrinol 2009;5:195-203.
35. Fodor WL. Tissue engineering and cell based therapies, from the bench to the clinic: the potential to replace, repair and regenerate[J]. Reprod Biol Endocrinol 2003;1:102.
36. Prockop DJ. Repair of tissues by adult stem/progenitor cells (MSCs):controversies, myths, and changing paradigms[J]. Mol Ther 2009;17:939-46.
37. Muschler GF, Raut VP, Patterson TE, Wenke JC, Hollinger JO. The design and use of animal models for translational research in bone tissue engineering and regenerative medicine[J]. Tissue Eng Part B Rev 2010;16:123-45.
38. Mao JJ, Stosich MS, Moioli EK, Lee CH, Fu SY, Bastian B, et al. Facial reconstruction by biosurgery:cell transplantation versus cell homing[J]. Tissue Eng Part B Rev 2010;16:257-62.
39. Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ. Stromal cell-derived factor-1 alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury[J]. Circulation 2004;110 (21):3300-3305.
40. Kushida T, Inaba M, Hisha H, Ichioka N, Esumi T, Ogawa R, Iida H, Ikehara S. Intra-bone marrow injection of allogeneic bone marrow cells:a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice[J]. Blood 2001;97(10):3292-9.
41. Ikehara S. A novel method of bone marrow transplantation (BMT) for intractable autoimmune diseases[J]. J Autoimmun 2008;30(3):108-115.
42 陈伟,陈建梅,徐皓.内源性骨髓间充质干细胞定向迁移的影响因素[J].实用医学杂志2007；23(20)：3292-3294.
43. Nyman S, Lindhe J, Karring T. Reattachment-new attachment. In:Lindhe J, ed. Textbook of clinical periodontology,2nd edn. Copenhagen [M]:Munksgaard,1989; 450-476.
44. Chirmule N, Xiao W, Truneh A, Schnell MA, Hughes JV, Zoltick P, Wilson JM. Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle[J]. J Virol 2000;74(5):2420-2425.
45. Zhang Q, Shi S, Liu Y, Uyanne J, Shi Y, Shi S, Le AD. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis[J]. J Immunol 2009;183(12):7787-7798.
46. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue:implications for cell-based therapies[J]. Tissue Eng 2001;7(2):211-228.
47. Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein(GFP)[J]. Gene 1996;173(1):33-38.
48. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression [J]. Science 1994;263(5148):802-805.
49. Wang F, Yu MJ, Yan XL, Wen Y, Zeng Q, Yue W, Yang P, Pei X. Gingiva-derived mesenchymal stem cells-mediated therapeutic approach for bone tissue regeneration[J]. Stem Cells Dev 2011,in press.
50.于美娇,杨丕山,葛少华.苯妥英钠对人牙周膜成纤维细胞生物学活性影响的实验研究[J].华西口腔医学杂志2008；26(2)：215-8.
51. Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy[J]. Proc Natl Acad Sci U S A 1994;91(10):4407-4411.
52. Palella TD, Hidaka Y, Silverman LJ, Levine M, Glorioso J, Kelley WN. Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector[J]. Gene 1989;80(1):137-44.
53. Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells[J]. Exp Hematol 2003; 31(10):890-6.
54. Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan EC. Suppression of allogeneic T-cell proliferation by human marrow stromal cells:implications in transplantation[J]. Transplantation 2003;75(3):389-397.
55. Devine SM, Cobbs C, Jennings M, Bartholomew A, Hoffman R. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates[J]. Blood 2003;101(8):2999-3001.
56. King GN, Hughes FJ. Bone morphogenetic protein-2 stimulates cell recruitment and cementogenesis during early wound healing[J]. J Clin Periodontol 2001;28(5):465-75.
57. Huang KK, Shen C, Chiang CY, Hsieh YD, Fu E. Effects of bone morphogenetic protein-6 on periodontal wound healing in a fenestration defect of rats[J]. J Periodontal Res 2005;40(1):1-10.
58. Talwar R, Di Silvio L, Hughes FJ, King GN. Effects of carrier release kinetics on bone morphogenetic protein-2-induced periodontal regeneration in vivo[J]. J Clin Periodontol 2001;28(4):340-7.
59. Wikesjo UM, Selvig KA, Zimmerman G, Nilveus R. Periodontal repair in dogs: healing in experimentally created chronic periodontal defects[J]. J Periodontol 1991; 62(4):258-63.
60. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases[J]. Lancet 2005;366(9499):1809-1820.
61. Chen T, Duncan MJ. Gingipain adhesion domains mediate Porphyromonas gingivalis adherence to epithelial cells[J]. Microb Pathog 2004;36(4):205-9.
62. Dierickx K, Pauwels M, Laine ML, Van Eldere J, Cassiman JJ, van Winkelhoff AJ, van Steenberghe D, Quirynen M. Adhesion of Porphyromonas gingivalis serotypes to pocket epithelium[J]. J Periodontol 2003;74(6):844-8.
63. He H, Liu R, Desta T, Leone C, Gerstenfeld LC, Graves DT. Diabetes causes decreased osteoclastogenesis, reduced bone formation, and enhanced apoptosis of osteoblastic cells in bacteria stimulated bone loss[J]. Endocrinology 2004;145(1):447-452.
64. Leone CW, Bokhadhoor H, Kuo D, Desta T, Yang J, Siqueira MF, Amar S, Graves DT. Immunization enhances inflammation and tissue destruction in response to Porphyromonas gingivalis[J]. Infect Immun 2006;74(4):2286-2292.
65. Graves DT, Fine D, Teng YT, Van Dyke TE, Hajishengallis G. The use of rodent models to investigate host-bacteria interactions related to periodontal diseases[J]. J Clin Periodontol 2008; 35(2):89-105.
66. Fibbe WE, Noort WA. Mesenchymal stem cells and hematopoietic stem cell transplantation [J]. Ann N Y Acad Sci 2003;996:235-244.
67.张建霞.苯妥英钠聚乳酸-羟基乙酸微球泊洛沙姆407凝胶对大鼠牙周缺损愈合影响的研究[D].济南：山东大学硕士研究生论文,2008.
68.程敏,马宁,张泽兵,孙宏晨,李成库.牙胚牙体组织切片的特殊染色方法及应用探讨[J].现代口腔医学杂志2007；21(4)：441-442.
69. Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow [J]. Blood 2001;98(8):2396-2402.
70. Noort WA, Kruisselbrink AB, in't Anker PS, Kruger M, van Bezooijen RL, de Paus RA, Heemskerk MH, Lowik CW, Falkenburg JH, Willemze R, Fibbe WE. Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34 (+) cells in NOD/SCID mice[J]. Exp Hematol 2002;30(8):870-878
71. Devine SM, Cobbs C, Jennings M, Bartholomew A, Hoffman R. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates[J]. Blood 2003;101(8):2999-3001.
72. Sodek J, Ganss B, McKee MD. Osteopontin[J]. Crit Rev Oral Biol Med 2000;11(3):279-303.
73. Morinobu M, Ishijima M, Rittling SR, Tsuji K, Yamamoto H, Nifuji A, Denhardt DT, Noda M. Osteopontin expression in osteoblasts and osteocytes during bone formation under mechanical stress in the calvarial suture in vivo[J]. J Bone Miner Res 2003;18(9):1706-15.
74. Block GJ, Ohkouchi S, Fung F, Frenkel J, Gregory C, Pochampally R, DiMattia G, Sullivan DE, Prockop DJ. Multipotent stromal cells are activated to reduce apoptosis in part by upregulation and secretion of stanniocalcin-1[J]. Stem Cells 2009;27(3):670-81.
75. Rehman J, Traktuev D, Li J, Merfeld-Clauss S, Temm-Grove CJ, Bovenkerk JE, Pell CL, Johnstone BH, Considine RV, March KL. Secretion of angiogenic and antiapoptotic factors by human adipose tromal cells[J].Circulation 2004;109(10):1292-8.
76. Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, Grisanti S, Gianni AM. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli[J]. Blood 2002;99(10):3838-43.
77. Krampera M, Glennie S, Dyson J, Scott D, Laylor R, Simpson E, Dazzi F. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide[J]. Blood 2003;101(9):3722-9.
78. Li L, Zhang S, Zhang Y, Yu B, Xu Y, Guan Z. Paracrine action mediate the antifibrotic effect of transplanted mesenchymal stem cells in a rat model of global heart failure[J]. Mol Biol Rep 2009;36(4):725-31.
79. Ohab JJ, Fleming S, Blesch A, Carmichael ST. A neurovascular niche for neurogenesis after stroke[J]. J Neurosci 2006;26(50):13007-16.
80. Seo J, Kim YO, Jo I. Differential expression of stromal cell-derived factor 1 in human brain microvascular endothelial cells and pericytes involves histone modifications[J]. Biochem Biophys Res Commun 2009;382(3):519-24.
81. Sundberg C, Kowanetz M, Brown LF, Detmar M, Dvorak HF. Stable expression of angiopoietin-1 and other markers by cultured pericytes:phenotypic similarities to a subpopulation of cells in maturing vessels during later stages of angiogenesis in vivo[J]. Lab Invest 2002;82(4):387-401.
82. Sorrell JM, Baber MA, Caplan AI. Influence of adult mesenchymal stem cells on in vitro vascular formation[J]. Tissue Eng Part A 2009; 15(7):1751-61.
83. Penn S, Mangi AA. Genetic enhancement of stem cell engraftment, survival, and efficacy[J]. Circ Res 2008;102:1471-82.
84. Fong EL, Chan CK, Goodman SB. Stem cell homing in musculoskeletal injury[J]. Biomaterials 2011;32:395-409.
85. Chute JP. Stem cell homing[J]. Curr Opin Hematol 2006; 13:399-406.
86. Lapidot T, Dar A, Kollet O. How do stem cells find their way home[J]? Blood 2005;106:1901-10.
87. Szekanecz Z, Kim J, Koch AE. Chemokines and chemokines receptors in rheumatoid arthritis[J]. Semin Immunol 2003;15(1):15-21.