血管化组织工程骨修复牙槽嵴裂的实验研究
|
摘要
研究背景
牙槽嵴裂是一种常见的先天性疾病，伴发于唇腭裂，严重影响患者的面容及功能。一般在唇裂和腭裂治疗后一段时间(9-11岁)进行修复，在这段时间内前颌骨生长发育的变化临床上尚无定论。常规的治疗方法是取自体松质骨(髂骨或下颌骨)植入缺损部位，以恢复上颌牙槽骨连续性，同时使尖牙从植骨部位萌出，并矫正鼻翼基底部畸形。植骨后有相当多的病例存在不同程度的植入骨吸收以及取骨区疼痛，跛行等并发症。近十余年发展起来的组织工程骨技术在修复躯干与四肢的长骨部分缺损上取得了进展，但工程骨的血管化目前仍然是一个悬而未决的问题，在牙槽嵴裂修复中的应用也较少。
因此，有必要对唇裂和腭裂修复后前颌骨的生长发育，组织工程骨及血管化的组织工程骨修复牙槽嵴裂后的形态改变等进行研究，为其临床应用提供理论依据。
第一部分双侧牙槽嵴裂动物模型的建立及对上颌骨生长发育的影响
目的：建立双侧牙槽嵴裂大动物模型，研究其对上颌骨发育的影响。
方法：选用8只12W龄的同窝实验犬，随机分成正常对照组和实验动物模型组，对实验组的动物采用外科手术的方法建立裂隙程度一致的双侧上颌牙槽嵴裂，术后12W处死动物，通过实体标本、三维CT对两组动物行头颅测量。
结果：实验模型组牙槽嵴裂裂隙为(10.81±0.41)mm，其上颌骨长度、前部宽度和前部高度较正常对照组明显缩小(P＜0.05)。
结论：牙槽嵴裂对上颌骨生长发育具有一定的影响。
第二部分组织工程骨修复牙槽嵴裂的实验研究
目的：探讨骨髓来源的种子细胞复合胶原蛋白海绵构建组织工程骨修复牙槽嵴裂的可行性。
方法：16条实验犬被分成4组。于双侧上颌第三切牙处去除15mm牙槽骨形成牙槽嵴裂动物模型；经股骨骨髓穿刺，分离骨髓基质细胞，培养、传代扩增诱导后，与胶原蛋白海绵混合培养72h，植入骨缺损处。饲养12W后处死动物，通过三维CT及组织学检查评价骨缺损修复的效果。
结果：实验组牙槽骨断端间形成完整的骨连接，切片可见髓腔通畅，新形成的牙槽嵴宽度与松质骨植入对照组相似(P＞0.05)，但高度不足(P＜0.05)。
结论：组织工程骨修复牙槽嵴裂具有一定的可行性，有望成为修复牙槽嵴裂的治疗方法。
第三部分血管化组织工程骨修复牙槽嵴裂的实验研究
目的：探讨应用经血管内皮细胞生长因子(VEGF)转染的骨髓基质干细胞(BMSC)复合胶原蛋白海绵构建的血管化组织工程骨修复牙槽嵴裂的可行性。
方法：经股骨骨髓穿刺获取犬BMSC经分离、培养和鉴定后，转染pAdMV-VEGF165质粒，流式细胞仪、RT-PCR和免疫组化检测；转染细胞与蛋白胶原海绵混合培养72h形成复合体；将复合体植于犬牙槽嵴裂模型的骨缺损处，12W后处死动物，通过大体形态及X线、三维CT重建、组织学检查等方法评价修复牙槽嵴裂的效果。
结果：转染组骨缺损完全愈合，骨髓腔通畅，组织工程骨高度及宽度与自体松质骨植入组无明显差异(P＞0.05)。
结论：由经VEGF转染的BMSC复合胶原蛋白海绵构建的组织工程骨可以较好地修复牙槽嵴裂，在修复后的形态、结构等方面均与自体松质骨修复相似，是一种牙槽嵴裂修复的良好方法。
第四部分内皮前体细胞在组织工程骨修复牙槽嵴裂中的应用
目的：探讨血管内皮前体细胞(EPC)联合骨髓基质干细胞(BMSC)复合胶原蛋白海绵构建的血管化组织工程骨修复牙槽嵴裂的可行性。
方法：20只实验犬被分成模型组、单纯材料组、BMSC组、实验组、松质骨植入组；经股骨穿刺获得犬骨髓分离BMSC、EPC，差异消化法纯化EPC，经培养和鉴定后，与蛋白胶原海绵混合培养72h形成复合体；将复合体植于犬牙槽嵴裂模型的骨缺损处，12W后处死动物，通过大体形态及X线、三维CT重建、组织学检查等方法评价修复牙槽嵴裂的效果。
结果：实验组骨缺损完全愈合，骨髓腔通畅，骨密度、高度及宽度与自体松质骨植入组无明显差异(P＞0.05)；单位面积血管数较对照组明显增加(P＜0.05)。
结论：EPC可以促进组织工程骨血管化及骨化，与BMSC联合形成组织工程骨可以较好地修复牙槽嵴裂，有望成为修复牙槽嵴裂的治疗方法。
Background
Alveolus cleft is a congenital disease accompanied with cleft lip andpalate, which seriously affects the facial complexion and function. Thedefect will be repaired after the restoration of cleft lip and palate (whenpatient are 9-11 years old). There isn't any clinic verdict of the growth ofthe maxilla during the stage. As a general operative mode, autogolouscancellous (from the ilium or the mandible) is implanted into thedefective part to restore the continuity of the maxilla, to help thegeneration of canine, and to rectify the malformation of the nose. Manycomplications such as pain in the bone taking and bone reception area, etc,are reported during or after the operation. Engineered bone have beenused to restore the bone defect in the limbs during the past 10 years, butthe problem of vascularization remains unsolved, and it is rarely used inthe restoration of alveolus cleft.
Therefore, it is necessary to study the growth of pre-maxilla after therestoration of cleft lip and palate, the use of engineered bone, and thechange of form after the use of vascular engineered bone to restorealveolus cleft, and to provide theoretical base for clinical application.
Part 1 Influence of surgical induced bilateral cleft alveolus onmaxillary growth in dogs
Objective To establish an animal model of bilateral alveolus cleftand study the influence of cleft on maxillary growth.
Methods Eight dogs (12 weeks old ) were divided into unoperatedcontrol groups (n=4)and the model group (n=4) .The model dogs wereoperated onto establish a bilateral alveolus cleft. All the animals werekilled and the craniofacial morphology on clean skull was analyzed bythe direct detection and CT.
Results The length ,the foreside width and the foreside height ofthe maxilla in the model group were shrunken than those of control group(P＜0.05)
Conclusion The alveolus cleft plays an important role in themaxillary aberration.
Part 2 An Investigation of Restoration of Alveolus Cleft withEngineered Bone
Objective To investigate the feasibility of the restoration ofalveolus cleft with engineered bone constructed by sponge collagenprotein combined bone mesenchymal stem cells (BMSC).
Methods Sixteen dogs were divided into 4 groupes, the thirdincisor and alveolus bone with peri0steum in bilateral maxilla wereremoved to form alveolus cleft model. The BMSCs were isolated from dog bone marrow. After being cultured and induced, the BMSCs wereseeded in sponge collagen protein and cultured for 72 hours. Thecomposites of BMSCs and collagen were implanted into the defect ofalveolus cleft. After being fed for 12 weeks, those dogs were killed.Three-dimensional CT and histological examination were used to observethe progress of bone formation.
Results The defects healed 12 weeks after BMSCs-collagencomposites were implanted, the width of engineered bone resembledpositive control(implant with autologous cancellous), but the height isless than positive control(P＜0.05).
Conclusion The engineered bone can restore the defect ofalveolus cleft, it may be used in the clinical treatment of the restoration ofalveolus cleft.
Part 3 An Investigation of Restoration of Cleft Alveolus withVascularised Engineered Bone
Objective To investigate the feasibility of the restoration of cleftalveolus with vascularised engineered bone constructed by collagencombined with vascular endothelial growth factor (VEGF) genetransfected bone mesenchymal stem cells.
Methods The BMSCs were isolated by gradient densitycentrifugation with Percoll solution from dog bone marrow. After beingcultured and VEGF165 gene transfected, Verified by FCM, RT-PCR and IHC, BMSCs were seeded in collagen for 72 hours. The compositesof BMSCs and collagen were implanted into the defect of cleft alveolusof dog. After being fed for 12 weeks, those dogs were sacrificed. X-ray,three-dimensional CT and histological examination were used to observethe bone formation.
Results 52% of BMSCs were transfected with VEGF165plasmid successfully. The defects of cleft alveolus healed at 12 weeksafter being implant collagen and VEGF 165 plasmid transfected BMSCcomposites and the forms of engineered bone resembled those implantedby autologous cancellous bone.
Conclusion The vascularised engineered bone can repair thedefect of alveolus cleft effectively. It may be used in the clinic.
Part 4 Application of Endothelial Progenitor Cell in Restoration ofCleft Alveolus with Engineered Bone
Objective To investigate the feasibility of the restoration of cleftalveolus bridge with vascularised engineered bone which wereconstructed by collagen combined with endothelial progenitor cells(EPC)and bone mesenchymal stem cells (BMSC).
Methods Twenty dogs were divided into 5 groups.The BMSC andEPC were isolated from dog bone marrow. EPC were purified bytime-limited digestive method. After being cultured and identified, BMSC and EPC were seeded in collagen for 72 hours. The composites wereimplanted into the defect of cleft alveolus model of dogs. After being fedfor 12 weeks, 20 dogs were sacrificed. X-ray, three-dimensional CT andhistological examination were used to observe the bone formation.
Results The alveolus defects healed 12 weeks after beingimplanted composites and the forms of engineered bone resembled thoseimplanted by autologous cancellous bone. The engineered bone had morevessels than control groups in section.
Conclusion EPC may promote the vascularization and ossificationof the engineered bone. Combined with BMSC, the vascular engineeredbone can repair the defect of alveolus cleft effectively, and it may be usedin clinical practice.
牙槽嵴裂是一种常见的先天性疾病，伴发于唇腭裂，严重影响患者的面容及功能。一般在唇裂和腭裂治疗后一段时间(9-11岁)进行修复，在这段时间内前颌骨生长发育的变化临床上尚无定论。常规的治疗方法是取自体松质骨(髂骨或下颌骨)植入缺损部位，以恢复上颌牙槽骨连续性，同时使尖牙从植骨部位萌出，并矫正鼻翼基底部畸形。植骨后有相当多的病例存在不同程度的植入骨吸收以及取骨区疼痛，跛行等并发症。近十余年发展起来的组织工程骨技术在修复躯干与四肢的长骨部分缺损上取得了进展，但工程骨的血管化目前仍然是一个悬而未决的问题，在牙槽嵴裂修复中的应用也较少。
因此，有必要对唇裂和腭裂修复后前颌骨的生长发育，组织工程骨及血管化的组织工程骨修复牙槽嵴裂后的形态改变等进行研究，为其临床应用提供理论依据。
第一部分双侧牙槽嵴裂动物模型的建立及对上颌骨生长发育的影响
目的：建立双侧牙槽嵴裂大动物模型，研究其对上颌骨发育的影响。
方法：选用8只12W龄的同窝实验犬，随机分成正常对照组和实验动物模型组，对实验组的动物采用外科手术的方法建立裂隙程度一致的双侧上颌牙槽嵴裂，术后12W处死动物，通过实体标本、三维CT对两组动物行头颅测量。
结果：实验模型组牙槽嵴裂裂隙为(10.81±0.41)mm，其上颌骨长度、前部宽度和前部高度较正常对照组明显缩小(P＜0.05)。
结论：牙槽嵴裂对上颌骨生长发育具有一定的影响。
第二部分组织工程骨修复牙槽嵴裂的实验研究
目的：探讨骨髓来源的种子细胞复合胶原蛋白海绵构建组织工程骨修复牙槽嵴裂的可行性。
方法：16条实验犬被分成4组。于双侧上颌第三切牙处去除15mm牙槽骨形成牙槽嵴裂动物模型；经股骨骨髓穿刺，分离骨髓基质细胞，培养、传代扩增诱导后，与胶原蛋白海绵混合培养72h，植入骨缺损处。饲养12W后处死动物，通过三维CT及组织学检查评价骨缺损修复的效果。
结果：实验组牙槽骨断端间形成完整的骨连接，切片可见髓腔通畅，新形成的牙槽嵴宽度与松质骨植入对照组相似(P＞0.05)，但高度不足(P＜0.05)。
结论：组织工程骨修复牙槽嵴裂具有一定的可行性，有望成为修复牙槽嵴裂的治疗方法。
第三部分血管化组织工程骨修复牙槽嵴裂的实验研究
目的：探讨应用经血管内皮细胞生长因子(VEGF)转染的骨髓基质干细胞(BMSC)复合胶原蛋白海绵构建的血管化组织工程骨修复牙槽嵴裂的可行性。
方法：经股骨骨髓穿刺获取犬BMSC经分离、培养和鉴定后，转染pAdMV-VEGF165质粒，流式细胞仪、RT-PCR和免疫组化检测；转染细胞与蛋白胶原海绵混合培养72h形成复合体；将复合体植于犬牙槽嵴裂模型的骨缺损处，12W后处死动物，通过大体形态及X线、三维CT重建、组织学检查等方法评价修复牙槽嵴裂的效果。
结果：转染组骨缺损完全愈合，骨髓腔通畅，组织工程骨高度及宽度与自体松质骨植入组无明显差异(P＞0.05)。
结论：由经VEGF转染的BMSC复合胶原蛋白海绵构建的组织工程骨可以较好地修复牙槽嵴裂，在修复后的形态、结构等方面均与自体松质骨修复相似，是一种牙槽嵴裂修复的良好方法。
第四部分内皮前体细胞在组织工程骨修复牙槽嵴裂中的应用
目的：探讨血管内皮前体细胞(EPC)联合骨髓基质干细胞(BMSC)复合胶原蛋白海绵构建的血管化组织工程骨修复牙槽嵴裂的可行性。
方法：20只实验犬被分成模型组、单纯材料组、BMSC组、实验组、松质骨植入组；经股骨穿刺获得犬骨髓分离BMSC、EPC，差异消化法纯化EPC，经培养和鉴定后，与蛋白胶原海绵混合培养72h形成复合体；将复合体植于犬牙槽嵴裂模型的骨缺损处，12W后处死动物，通过大体形态及X线、三维CT重建、组织学检查等方法评价修复牙槽嵴裂的效果。
结果：实验组骨缺损完全愈合，骨髓腔通畅，骨密度、高度及宽度与自体松质骨植入组无明显差异(P＞0.05)；单位面积血管数较对照组明显增加(P＜0.05)。
结论：EPC可以促进组织工程骨血管化及骨化，与BMSC联合形成组织工程骨可以较好地修复牙槽嵴裂，有望成为修复牙槽嵴裂的治疗方法。
Background
Alveolus cleft is a congenital disease accompanied with cleft lip andpalate, which seriously affects the facial complexion and function. Thedefect will be repaired after the restoration of cleft lip and palate (whenpatient are 9-11 years old). There isn't any clinic verdict of the growth ofthe maxilla during the stage. As a general operative mode, autogolouscancellous (from the ilium or the mandible) is implanted into thedefective part to restore the continuity of the maxilla, to help thegeneration of canine, and to rectify the malformation of the nose. Manycomplications such as pain in the bone taking and bone reception area, etc,are reported during or after the operation. Engineered bone have beenused to restore the bone defect in the limbs during the past 10 years, butthe problem of vascularization remains unsolved, and it is rarely used inthe restoration of alveolus cleft.
Therefore, it is necessary to study the growth of pre-maxilla after therestoration of cleft lip and palate, the use of engineered bone, and thechange of form after the use of vascular engineered bone to restorealveolus cleft, and to provide theoretical base for clinical application.
Part 1 Influence of surgical induced bilateral cleft alveolus onmaxillary growth in dogs
Objective To establish an animal model of bilateral alveolus cleftand study the influence of cleft on maxillary growth.
Methods Eight dogs (12 weeks old ) were divided into unoperatedcontrol groups (n=4)and the model group (n=4) .The model dogs wereoperated onto establish a bilateral alveolus cleft. All the animals werekilled and the craniofacial morphology on clean skull was analyzed bythe direct detection and CT.
Results The length ,the foreside width and the foreside height ofthe maxilla in the model group were shrunken than those of control group(P＜0.05)
Conclusion The alveolus cleft plays an important role in themaxillary aberration.
Part 2 An Investigation of Restoration of Alveolus Cleft withEngineered Bone
Objective To investigate the feasibility of the restoration ofalveolus cleft with engineered bone constructed by sponge collagenprotein combined bone mesenchymal stem cells (BMSC).
Methods Sixteen dogs were divided into 4 groupes, the thirdincisor and alveolus bone with peri0steum in bilateral maxilla wereremoved to form alveolus cleft model. The BMSCs were isolated from dog bone marrow. After being cultured and induced, the BMSCs wereseeded in sponge collagen protein and cultured for 72 hours. Thecomposites of BMSCs and collagen were implanted into the defect ofalveolus cleft. After being fed for 12 weeks, those dogs were killed.Three-dimensional CT and histological examination were used to observethe progress of bone formation.
Results The defects healed 12 weeks after BMSCs-collagencomposites were implanted, the width of engineered bone resembledpositive control(implant with autologous cancellous), but the height isless than positive control(P＜0.05).
Conclusion The engineered bone can restore the defect ofalveolus cleft, it may be used in the clinical treatment of the restoration ofalveolus cleft.
Part 3 An Investigation of Restoration of Cleft Alveolus withVascularised Engineered Bone
Objective To investigate the feasibility of the restoration of cleftalveolus with vascularised engineered bone constructed by collagencombined with vascular endothelial growth factor (VEGF) genetransfected bone mesenchymal stem cells.
Methods The BMSCs were isolated by gradient densitycentrifugation with Percoll solution from dog bone marrow. After beingcultured and VEGF165 gene transfected, Verified by FCM, RT-PCR and IHC, BMSCs were seeded in collagen for 72 hours. The compositesof BMSCs and collagen were implanted into the defect of cleft alveolusof dog. After being fed for 12 weeks, those dogs were sacrificed. X-ray,three-dimensional CT and histological examination were used to observethe bone formation.
Results 52% of BMSCs were transfected with VEGF165plasmid successfully. The defects of cleft alveolus healed at 12 weeksafter being implant collagen and VEGF 165 plasmid transfected BMSCcomposites and the forms of engineered bone resembled those implantedby autologous cancellous bone.
Conclusion The vascularised engineered bone can repair thedefect of alveolus cleft effectively. It may be used in the clinic.
Part 4 Application of Endothelial Progenitor Cell in Restoration ofCleft Alveolus with Engineered Bone
Objective To investigate the feasibility of the restoration of cleftalveolus bridge with vascularised engineered bone which wereconstructed by collagen combined with endothelial progenitor cells(EPC)and bone mesenchymal stem cells (BMSC).
Methods Twenty dogs were divided into 5 groups.The BMSC andEPC were isolated from dog bone marrow. EPC were purified bytime-limited digestive method. After being cultured and identified, BMSC and EPC were seeded in collagen for 72 hours. The composites wereimplanted into the defect of cleft alveolus model of dogs. After being fedfor 12 weeks, 20 dogs were sacrificed. X-ray, three-dimensional CT andhistological examination were used to observe the bone formation.
Results The alveolus defects healed 12 weeks after beingimplanted composites and the forms of engineered bone resembled thoseimplanted by autologous cancellous bone. The engineered bone had morevessels than control groups in section.
Conclusion EPC may promote the vascularization and ossificationof the engineered bone. Combined with BMSC, the vascular engineeredbone can repair the defect of alveolus cleft effectively, and it may be usedin clinical practice.
引文
[1] Hermann NV, Darvann TA, Jensen BL, et al. Early craniofacial morphology and growth in children with bilateral complete cleft lip and palate. Cleft Palate Craniofac J. 2004, 41:424-438.
[2] Dodson TB, Schmidt B, Longaker MT, et al. Fetal cleft lip repair in rabbits: postnatal facial growth after repair. J Oral Maxillofac Surg. 1991, 49:603-611.
[3] Bardach J, Kelly KM, Salyer KE.Relationship between the sequence of lip and palate repair and maxillary growth: an experimental study in beagles. Plast Reconstr Surg. 1994;93:269-278.
[4] Bardach J, Kelly KM, Salyer KE. A comparative study of facial growth following lip and palate repair performed in sequence and simultaneously: an experimental study in beagles. Plast Reconstr Surg. 1993; 91:1008-1016.
[5] Swennen GR, Grimaldi H, Berten JL, et al. Reliability and validity of a modified lateral cephalometric analysis for evaluation of craniofacial morphology and growth in patients with clefts. J Craniofac Surg. 2004, 15:399-412; discussion 413-4.
[6] Young DL, Schneider RA, Hu D, et al. Genetic and teratogenic approaches to craniofacial development. Crit Rev Oral Biol Med. 2000, 11:304-317.
[7] Longaker MT, Dodson TB, Kaban LB. A rabbit model for fetal cleft lip repair. J Oral Maxillofac Surg. 1990, 48:714-9.
[8] Nagata M, Amin NM, Kannari Y, et al. Isolated maxillary bending in CLFR strain mice observation of craniofacial deformity and inheritance pattern. Cleft Palate Craniofac J. 1997;34:101-105.
[9] 石冰，龙洁，王晴等，唇裂伴牙槽嵴裂动物模型的建立及对上颌骨生长发育影响的观察。口腔颌面外科杂志2000，10：138-140。
[10] Da Silva FOG, Valladares NJ, Capelloza FL, et al. Influence of lip repair on craniofacial morphology of patients with complete bilateral cleft lip and palate. Cleft Palate Craniofac J. 2003, 40:144-53.
[11] Sasaki A, Takeshita S, Publico AS, et al. Finite element growth analysis for the craniofacial skeleton in patients with cleft lip and palate. Med Eng Phys. 2004, 26:109-18.
[12] Bardach J, Kelly KM, Salyer KE. The effects of lip repair with and without soft-tissue undermining and delayed palate repair on maxillary growth: an experimental study in beagles. Plast Reconstr Surg. 1994 Aug;94:343-51.
[1] Guy W, Johan VC, Emil A, et al. Retrospective image based surgery assessment for uni lateral Alveolus cleft patients.J Radiology 2001 9:1-10.
[2] Murthy AS, Lehman JA. Evaluation of Alveolus Bone Grafting: A Survey of ACPA Teams. Cleft Palate Craniofac J. 2005 42:99-101.
[3] Schultze-Mosgau S, Nkenke E, Schlegel AK, et al. Analysis of bone resorption after secondary Alveolus cleft bone grafts before and after canine eruption in connection with orthodontic gap closure or Drosthodontic treatment. J Oral Maxillofac Surg. 2003;61:1245-1248.
[4] 王永刚，裴国献，张洪涛等，兔股骨干缺损模型的制备及在组织工程骨实验中的应用，中华创伤骨科杂志．2005 7：971-974．
[5] Lew D, Farrell B, Bardach J, et al. Repair of craniofacial defects with hydroxyapatite cement. J Oral Maxillofac Surg. 1997;55:1441-1449; discussion 1449-51.
[6] Salyer KE, Bardach J, Squier CA, et al.A comparative study of the effects of biodegradable and titanium plating systems on cranial growth and structure: experimental study in beagles. Plast Reconstr Surg. 1994;93(4):?05-13.
[7] Deliloglu-Gurhan I, ruglu I, Vatansever HS, et al The effect of osteogenic medium on the adhesion of rat bone marrow stromal cell to the hydroxyapatite. Saudi Med J. 2006;27:305-311.
[8] Montjovent MO, Burri N, Mark S, et al. Fetal bone cells for tissue engineering. Bone. 2004 35:1323-1333.
[9] El-Amin SF, Kofron MD, Attawia MA, et al. Molecular regulation of osteoblasts for tissue engineered bone repair. Clin Orthop. 2004 427:220-225.
[10] Cancedda R, Mastrogiacomo M, Bianchi G, et al. Bone marrow stromal cells and their use in regenerating bone. Novartis Found Symp. 2003;249:133-43.
[11] 盛辉，王洪复，高建军，等。不同浓度地塞米松诱导大鼠间充质干细胞向成骨细胞的分化。复旦学报(医学版)2003 30：164-7
[12] Moreira PL, An YH, Santos AR Jr, et al. In vitro analysis of anionic collagen scaffolds for bone repair. J Biomed Mater Res. 2004;71B: 229-237.
[13] Oshima Y, Watanabe N, Matsuda K, et al. Fate of transplanted bone-marrow-derived mesenchymal cells during osteochondral repair using transgenic rats to simulate autologous transplantation. Osteoarthritis Cartilage. 2004 12:811-817.
[14] Gaggl A, Schultes G, Karcher H. Aesthetic and functional outcome of surgical and orthodontic correction of bilateral clefts of lip, palate, and alveolus. Cleft Palate Craniofac J. 1999;36:407-412.
[1] 顾祖超 李起鸿 赵玉峰，等，自体骨髓基质细胞复合支架材料修复兔尺骨干节段骨缺损的实验研究 中华创伤骨科杂志 2004，6：651～656。
[2] Henrik E, Kristian G, Knud Set al. Effects of locally applied vascular endothelial growth factor (VEGF) and VEGF-inhibitor to the rabbit tibia during distraction osteogenesis. Journal of Orthopaedic Research 2003;21;335～340。
[3] Masashi N, Anthony A, Paolo DC, et al. Principals of neovascularization for tissue engineering. Molecular Aspects of Medicine, 2002;23:463～483。
[4] Al-Salihi KA, Samsudin AR. Bone marrow mesenchymal stem cells differentiation and proliferation on the surface of coral implant. Med J Malaysia. 2004; 59 Suppl B:45-46.
[5] Guy W, Johan VC, Emil A, et al. Retrospective image based surgery assessment for uni lateral alveolar cleft patients. J Radiology 2001; 9:1～10.
[6] Murthy AS, Lehman JA. Evaluation of Alveolar Bone Grafting: A Survey of ACPA Teams. Cleft Palate Craniofac J. 2005 Jan 42:99～101.
[7] 李永东 血管内皮生长因子的研究进展 中国误诊学杂志2002；2：200～203
[8] John S, Min B, Leode G, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. PNAS 2002;99:9656～9661
[9] Kassem M, Kristiansen M, Abdallah BM. Mesenchymal stem cells: cell biology and potential use in therapy. Basic Clin Pharmacol Toxicol. 2004;95:209～214。
[10] Kim HJ, Jang SY, Park JI, et al Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease. Exp Mol Med. 2004;36:336-344.
[11] Martine ML, Marcel K, Chris VD, et al Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endo 2000;141: 1667～1674.
[12] Moreira PL, An YH, Santos AR Jr, et al. In vitro analysis of anionic collagen scaffolds for bone repair. J Biomed Mater Res. 2004; 71B:229～237.
[13] 陈滨，裴国献，王珂，等，大动物体内促组织工程骨成骨及血管化手段的研究 中国医学科学院学报 2003 25：26～33。
[1] Fang TD, Salim A, Xia W, et al. Angiogenesis is required for successful bone induction during distraction osteogenesis. J Bone Miner Res. 2005;20:1114-24.
[2] Masashi N, Anthony A, Paolo DC, et al. Principals of neovascularization for tissue engineering. Molecular Aspects of Medicine, 2002 23:463～483。
[3] Kawamoto A, Asahara T, Losordo DW. Transplantation of endothelial progenitor cells for therapeutic neovascularization. Cardiovasc Radiat Med. 2002 3:221-225.
[4] Armoneva DA, Vukmirovic R, Davis ME, et al. Endothelial ceils promote cardiac myocyte survival and spatial reorganization: implications for cardiac regeneration. Circulation. 2004, 110:962-968.
[5] Stahl A, Wu X, Wenger A, Endothelial progenitor cell sprouting in spheroid cultures is resistant to inhibition by osteoblasts: A model for bone replacement grafts. FEBS Lett. 2005;579:5338-5342
[6] 汪保和，王绮如．人骨髓内皮细胞的分离、纯化和培养．中华血液学杂志，1998．19：327-328．
[7] David AI, Laura EM, Hiromi T, et al. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. BLOOD, 2004, 104: 2752-2760.
[8] Moreira PL, An YH, Santos AR Jr, et al. In vitro analysis of anionic collagen scaffolds for bone repair. J Biomed Mater Res. 2004:71B:229～37.
[9] 曲哲，孙宏晨，郭英等 血管内皮细胞对骨髓间充质干细胞成骨能力影响的体外研究。口腔医学研究2003；19：480-482．
[10] Zisch AH. Tissue engineering of angiogenesis with autologous endothelial progenitor cells. Curr Opin Biotechnol. 2004, 15: 424-429.
[11] Kassem M, Kristiansen M, Abdallah BM. Mesenchymal stem cells: cell biology and potential use in therapy. Basic Clin Pharmacol Toxicol. 2004 95: 209-214.
[1] Willems Guy, Van Cleynenbreugel Johan, Alberga Emile, et al Retrospective image based surgery assessment for unilateral alveolar cleft patients. Journal of Radiology 2001, 25: 1～10。
[2] 裴国献 金 丹 骨组织工程学种子细胞研究进展 中华创伤骨科杂志2003，5：55～57。
[3] Montjovent MO, Burri N, Mark S,et al. Fetal bone cells for tissue engineering. Bone. 2004, 35:1323～1333.
[4] Al-Salihi KA, Samsudin AR. Bone marrow mesenchymal stem cells differentiation and proliferation on the surface of coral implant.Med J Malaysia. 2004, 59 Suppl B:45～46.
[5] Chen F, Chen S, Tao K, et al. Marrow-derived osteoblasts seeded into porous natural coral to prefabricate a vascularised bone graft in the shape of a human mandibular ramus: experimental study in rabbits. Br J Oral Maxillofac Surg. 2004, 42:532～537.
[6] Uji M. Histological study on experimental tooth movement in alveolar bone defects implanted with decalcified bone powder. Kokubyo Gakkai Zasshi. 1996, 63:208～221.
[7] Kawamoto T, iotohashi N, Kitamura A et al, A histological study on experimental tooth movement into bone induced by recombinant human bone morphogenetic protein-2 in beagle dogs. Cleft Palate Craniofac J. 2002, 39:439～448.
[8] Mayer M, Hollinger J, Ron E, et al Maxillary alveolar cleft repair in dogs using recombinant human bone morphogenetic protein-2 and a polymer carrier. Plast Reconstr Surg. 1996, 98:247～259.
[9] 何创龙，王远亮，杨立华等，骨组织工程天然衍生细胞外基质材料．中国生物工程杂志2003，23：11～17
[10] Kuyl MH, Thierens H, Dermaut Let al A multidisciplinary approach to the healing of cranial and residual maxillary cleft defects by means of allogenous demineralized osseous implants andpolylactic acid casts in dogs. Cleft Palate Craniofac J. 1999, 36:207～216.
[11] Kawamoto T, Motohashi N, Kitamura Aet al, Experimental tooth movement into bone induced by recombinant human bone morphogenetic protein-2. Cleft Palate Craniofac J. 2003, 40:538～543.
[12] Shimakura Y, Yamzaki Y, Uchinuma E. Experimental study on bone formation potential of cryopreserved human bone marrow mesenchymal cell/hydroxyapatite complex in the presence of recombinant human bone morphogenetic protein-2. J Craniofac Surg. 2003, 14:108～116.
[13] Linton JL, Sohn BW, Yook JI et al Effects of calcium phosphate ceramic bone graft materials on permanent teeth eruption in beagles. Cleft Palate Craniofac J. 2002, 39:197～207.
[14] Cottrell DA, Wolford LM, Long-term evaluation of the use of coralline hydroxyapatite in orthognathic surgery. J Oral Maxillofac Surg. 1998, 56(8):935-41; discussion 941～942.
[15] Moreira PL, An YH, Santos AR Jr, et al. In vitro analysis of anionic collagen scaffolds for bone repair. J Biomed Mater Res. 2004, 71B:229～237.
[16] Masashi N, Anthony A, Paolo DC, et al. Principals of neovascularization for tissue engineering. Molecular Aspects of Medicine, 2002, 23:463～483。
[17] 李永东 血管内皮生长因子的研究进展．中国误诊学杂志2002，2：200～203
[18] Martine ML, Marcel K, Chris VD, et al Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endo 2000, 141:: 1667～1674.
[19] John Street, Min Bao, Leode Guzman, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. PNAS 2002, 99: 9656～9661
[20] Armoneva DA, Vukmirovic R, Davis ME, et al. Endothelial cells promote cardiac myocyte survival and spatial reorganization: implications for cardiac regeneration. Circulation. 2004, 110:962～8.
[21] Fang TD, Salim A, Xia W, et al. Angiogenesis is required for successful bone induction during distraction osteogenesis. J Bone Miner Res. 2005, 20:1114～1124.
[22] 陈滨，裴国献，王珂等 动物体内促组织工程骨成骨及血管化手段的研究中国医学科学院学报 2003，25：26～32。
[2] Dodson TB, Schmidt B, Longaker MT, et al. Fetal cleft lip repair in rabbits: postnatal facial growth after repair. J Oral Maxillofac Surg. 1991, 49:603-611.
[3] Bardach J, Kelly KM, Salyer KE.Relationship between the sequence of lip and palate repair and maxillary growth: an experimental study in beagles. Plast Reconstr Surg. 1994;93:269-278.
[4] Bardach J, Kelly KM, Salyer KE. A comparative study of facial growth following lip and palate repair performed in sequence and simultaneously: an experimental study in beagles. Plast Reconstr Surg. 1993; 91:1008-1016.
[5] Swennen GR, Grimaldi H, Berten JL, et al. Reliability and validity of a modified lateral cephalometric analysis for evaluation of craniofacial morphology and growth in patients with clefts. J Craniofac Surg. 2004, 15:399-412; discussion 413-4.
[6] Young DL, Schneider RA, Hu D, et al. Genetic and teratogenic approaches to craniofacial development. Crit Rev Oral Biol Med. 2000, 11:304-317.
[7] Longaker MT, Dodson TB, Kaban LB. A rabbit model for fetal cleft lip repair. J Oral Maxillofac Surg. 1990, 48:714-9.
[8] Nagata M, Amin NM, Kannari Y, et al. Isolated maxillary bending in CLFR strain mice observation of craniofacial deformity and inheritance pattern. Cleft Palate Craniofac J. 1997;34:101-105.
[9] 石冰，龙洁，王晴等，唇裂伴牙槽嵴裂动物模型的建立及对上颌骨生长发育影响的观察。口腔颌面外科杂志2000，10：138-140。
[10] Da Silva FOG, Valladares NJ, Capelloza FL, et al. Influence of lip repair on craniofacial morphology of patients with complete bilateral cleft lip and palate. Cleft Palate Craniofac J. 2003, 40:144-53.
[11] Sasaki A, Takeshita S, Publico AS, et al. Finite element growth analysis for the craniofacial skeleton in patients with cleft lip and palate. Med Eng Phys. 2004, 26:109-18.
[12] Bardach J, Kelly KM, Salyer KE. The effects of lip repair with and without soft-tissue undermining and delayed palate repair on maxillary growth: an experimental study in beagles. Plast Reconstr Surg. 1994 Aug;94:343-51.
[1] Guy W, Johan VC, Emil A, et al. Retrospective image based surgery assessment for uni lateral Alveolus cleft patients.J Radiology 2001 9:1-10.
[2] Murthy AS, Lehman JA. Evaluation of Alveolus Bone Grafting: A Survey of ACPA Teams. Cleft Palate Craniofac J. 2005 42:99-101.
[3] Schultze-Mosgau S, Nkenke E, Schlegel AK, et al. Analysis of bone resorption after secondary Alveolus cleft bone grafts before and after canine eruption in connection with orthodontic gap closure or Drosthodontic treatment. J Oral Maxillofac Surg. 2003;61:1245-1248.
[4] 王永刚，裴国献，张洪涛等，兔股骨干缺损模型的制备及在组织工程骨实验中的应用，中华创伤骨科杂志．2005 7：971-974．
[5] Lew D, Farrell B, Bardach J, et al. Repair of craniofacial defects with hydroxyapatite cement. J Oral Maxillofac Surg. 1997;55:1441-1449; discussion 1449-51.
[6] Salyer KE, Bardach J, Squier CA, et al.A comparative study of the effects of biodegradable and titanium plating systems on cranial growth and structure: experimental study in beagles. Plast Reconstr Surg. 1994;93(4):?05-13.
[7] Deliloglu-Gurhan I, ruglu I, Vatansever HS, et al The effect of osteogenic medium on the adhesion of rat bone marrow stromal cell to the hydroxyapatite. Saudi Med J. 2006;27:305-311.
[8] Montjovent MO, Burri N, Mark S, et al. Fetal bone cells for tissue engineering. Bone. 2004 35:1323-1333.
[9] El-Amin SF, Kofron MD, Attawia MA, et al. Molecular regulation of osteoblasts for tissue engineered bone repair. Clin Orthop. 2004 427:220-225.
[10] Cancedda R, Mastrogiacomo M, Bianchi G, et al. Bone marrow stromal cells and their use in regenerating bone. Novartis Found Symp. 2003;249:133-43.
[11] 盛辉，王洪复，高建军，等。不同浓度地塞米松诱导大鼠间充质干细胞向成骨细胞的分化。复旦学报(医学版)2003 30：164-7
[12] Moreira PL, An YH, Santos AR Jr, et al. In vitro analysis of anionic collagen scaffolds for bone repair. J Biomed Mater Res. 2004;71B: 229-237.
[13] Oshima Y, Watanabe N, Matsuda K, et al. Fate of transplanted bone-marrow-derived mesenchymal cells during osteochondral repair using transgenic rats to simulate autologous transplantation. Osteoarthritis Cartilage. 2004 12:811-817.
[14] Gaggl A, Schultes G, Karcher H. Aesthetic and functional outcome of surgical and orthodontic correction of bilateral clefts of lip, palate, and alveolus. Cleft Palate Craniofac J. 1999;36:407-412.
[1] 顾祖超 李起鸿 赵玉峰，等，自体骨髓基质细胞复合支架材料修复兔尺骨干节段骨缺损的实验研究 中华创伤骨科杂志 2004，6：651～656。
[2] Henrik E, Kristian G, Knud Set al. Effects of locally applied vascular endothelial growth factor (VEGF) and VEGF-inhibitor to the rabbit tibia during distraction osteogenesis. Journal of Orthopaedic Research 2003;21;335～340。
[3] Masashi N, Anthony A, Paolo DC, et al. Principals of neovascularization for tissue engineering. Molecular Aspects of Medicine, 2002;23:463～483。
[4] Al-Salihi KA, Samsudin AR. Bone marrow mesenchymal stem cells differentiation and proliferation on the surface of coral implant. Med J Malaysia. 2004; 59 Suppl B:45-46.
[5] Guy W, Johan VC, Emil A, et al. Retrospective image based surgery assessment for uni lateral alveolar cleft patients. J Radiology 2001; 9:1～10.
[6] Murthy AS, Lehman JA. Evaluation of Alveolar Bone Grafting: A Survey of ACPA Teams. Cleft Palate Craniofac J. 2005 Jan 42:99～101.
[7] 李永东 血管内皮生长因子的研究进展 中国误诊学杂志2002；2：200～203
[8] John S, Min B, Leode G, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. PNAS 2002;99:9656～9661
[9] Kassem M, Kristiansen M, Abdallah BM. Mesenchymal stem cells: cell biology and potential use in therapy. Basic Clin Pharmacol Toxicol. 2004;95:209～214。
[10] Kim HJ, Jang SY, Park JI, et al Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease. Exp Mol Med. 2004;36:336-344.
[11] Martine ML, Marcel K, Chris VD, et al Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endo 2000;141: 1667～1674.
[12] Moreira PL, An YH, Santos AR Jr, et al. In vitro analysis of anionic collagen scaffolds for bone repair. J Biomed Mater Res. 2004; 71B:229～237.
[13] 陈滨，裴国献，王珂，等，大动物体内促组织工程骨成骨及血管化手段的研究 中国医学科学院学报 2003 25：26～33。
[1] Fang TD, Salim A, Xia W, et al. Angiogenesis is required for successful bone induction during distraction osteogenesis. J Bone Miner Res. 2005;20:1114-24.
[2] Masashi N, Anthony A, Paolo DC, et al. Principals of neovascularization for tissue engineering. Molecular Aspects of Medicine, 2002 23:463～483。
[3] Kawamoto A, Asahara T, Losordo DW. Transplantation of endothelial progenitor cells for therapeutic neovascularization. Cardiovasc Radiat Med. 2002 3:221-225.
[4] Armoneva DA, Vukmirovic R, Davis ME, et al. Endothelial ceils promote cardiac myocyte survival and spatial reorganization: implications for cardiac regeneration. Circulation. 2004, 110:962-968.
[5] Stahl A, Wu X, Wenger A, Endothelial progenitor cell sprouting in spheroid cultures is resistant to inhibition by osteoblasts: A model for bone replacement grafts. FEBS Lett. 2005;579:5338-5342
[6] 汪保和，王绮如．人骨髓内皮细胞的分离、纯化和培养．中华血液学杂志，1998．19：327-328．
[7] David AI, Laura EM, Hiromi T, et al. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. BLOOD, 2004, 104: 2752-2760.
[8] Moreira PL, An YH, Santos AR Jr, et al. In vitro analysis of anionic collagen scaffolds for bone repair. J Biomed Mater Res. 2004:71B:229～37.
[9] 曲哲，孙宏晨，郭英等 血管内皮细胞对骨髓间充质干细胞成骨能力影响的体外研究。口腔医学研究2003；19：480-482．
[10] Zisch AH. Tissue engineering of angiogenesis with autologous endothelial progenitor cells. Curr Opin Biotechnol. 2004, 15: 424-429.
[11] Kassem M, Kristiansen M, Abdallah BM. Mesenchymal stem cells: cell biology and potential use in therapy. Basic Clin Pharmacol Toxicol. 2004 95: 209-214.
[1] Willems Guy, Van Cleynenbreugel Johan, Alberga Emile, et al Retrospective image based surgery assessment for unilateral alveolar cleft patients. Journal of Radiology 2001, 25: 1～10。
[2] 裴国献 金 丹 骨组织工程学种子细胞研究进展 中华创伤骨科杂志2003，5：55～57。
[3] Montjovent MO, Burri N, Mark S,et al. Fetal bone cells for tissue engineering. Bone. 2004, 35:1323～1333.
[4] Al-Salihi KA, Samsudin AR. Bone marrow mesenchymal stem cells differentiation and proliferation on the surface of coral implant.Med J Malaysia. 2004, 59 Suppl B:45～46.
[5] Chen F, Chen S, Tao K, et al. Marrow-derived osteoblasts seeded into porous natural coral to prefabricate a vascularised bone graft in the shape of a human mandibular ramus: experimental study in rabbits. Br J Oral Maxillofac Surg. 2004, 42:532～537.
[6] Uji M. Histological study on experimental tooth movement in alveolar bone defects implanted with decalcified bone powder. Kokubyo Gakkai Zasshi. 1996, 63:208～221.
[7] Kawamoto T, iotohashi N, Kitamura A et al, A histological study on experimental tooth movement into bone induced by recombinant human bone morphogenetic protein-2 in beagle dogs. Cleft Palate Craniofac J. 2002, 39:439～448.
[8] Mayer M, Hollinger J, Ron E, et al Maxillary alveolar cleft repair in dogs using recombinant human bone morphogenetic protein-2 and a polymer carrier. Plast Reconstr Surg. 1996, 98:247～259.
[9] 何创龙，王远亮，杨立华等，骨组织工程天然衍生细胞外基质材料．中国生物工程杂志2003，23：11～17
[10] Kuyl MH, Thierens H, Dermaut Let al A multidisciplinary approach to the healing of cranial and residual maxillary cleft defects by means of allogenous demineralized osseous implants andpolylactic acid casts in dogs. Cleft Palate Craniofac J. 1999, 36:207～216.
[11] Kawamoto T, Motohashi N, Kitamura Aet al, Experimental tooth movement into bone induced by recombinant human bone morphogenetic protein-2. Cleft Palate Craniofac J. 2003, 40:538～543.
[12] Shimakura Y, Yamzaki Y, Uchinuma E. Experimental study on bone formation potential of cryopreserved human bone marrow mesenchymal cell/hydroxyapatite complex in the presence of recombinant human bone morphogenetic protein-2. J Craniofac Surg. 2003, 14:108～116.
[13] Linton JL, Sohn BW, Yook JI et al Effects of calcium phosphate ceramic bone graft materials on permanent teeth eruption in beagles. Cleft Palate Craniofac J. 2002, 39:197～207.
[14] Cottrell DA, Wolford LM, Long-term evaluation of the use of coralline hydroxyapatite in orthognathic surgery. J Oral Maxillofac Surg. 1998, 56(8):935-41; discussion 941～942.
[15] Moreira PL, An YH, Santos AR Jr, et al. In vitro analysis of anionic collagen scaffolds for bone repair. J Biomed Mater Res. 2004, 71B:229～237.
[16] Masashi N, Anthony A, Paolo DC, et al. Principals of neovascularization for tissue engineering. Molecular Aspects of Medicine, 2002, 23:463～483。
[17] 李永东 血管内皮生长因子的研究进展．中国误诊学杂志2002，2：200～203
[18] Martine ML, Marcel K, Chris VD, et al Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endo 2000, 141:: 1667～1674.
[19] John Street, Min Bao, Leode Guzman, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. PNAS 2002, 99: 9656～9661
[20] Armoneva DA, Vukmirovic R, Davis ME, et al. Endothelial cells promote cardiac myocyte survival and spatial reorganization: implications for cardiac regeneration. Circulation. 2004, 110:962～8.
[21] Fang TD, Salim A, Xia W, et al. Angiogenesis is required for successful bone induction during distraction osteogenesis. J Bone Miner Res. 2005, 20:1114～1124.
[22] 陈滨，裴国献，王珂等 动物体内促组织工程骨成骨及血管化手段的研究中国医学科学院学报 2003，25：26～32。