rhBMP-2/Co/PLGA复合生物膜对腭裂骨缺损修复的实验研究
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摘要
目的 要研究应用引导性骨再生（guided bone regeneration，GBR）技术能否在腭板缺损中形成良好的骨形成，为临床治疗腭裂提供更加科学有效的方法。腭部骨生成中细胞生成、分化、以及腭板骨架桥形成的时期和机理，胶原-聚羟基乙酸对腭裂骨缺损引导性再生的影响。
方法 1.实验所用胶原溶液为可溶性牛腱胶原，其主要成份为I型胶原。将牛腱剔下，浸入70%酒精溶液中，20min后在无菌条件下抽出牛腱并将其剪碎，置入0.5%的醋酸溶液中，在4℃条件下不时搅拌。48小时后离心（10000r/min）约1.5小时，去渣。上清液用100g/L NaCl溶液盐析，最后将沉淀的蛋白质溶于4℃ 1mmol/L的HCl溶液中备用。配制胶原蛋白浓度为84 g/L（Lowry法）。将直径15μm的聚羟基乙酸纤维无纺网与交联胶原相混合，注入24孔培养板中，混匀，4℃ 过夜，置于序列冻干机中，真空冷冻干燥48小时，即制成多孔状胶原包埋处理的PlGA支架。将其在无菌Tris-HCl缓冲液中漂洗7天，自然干燥，将基因重组人骨形成蛋白(rhBMP) 的醋酸溶液按每培养板孔4mg量滴入PLGA支架中即制成rhBMP–2/Co/PLGA复合生物膜60Co照射1至2小时灭菌备用。2. 8只成年实验犬分为二组：实验组4只，对照组4只。全身麻醉后切开腭部粘膜，去除高4cm，宽1.5cm的三角形骨板，切透鼻侧的粘骨膜，对照线直接以1号丝线间断结节缝合粘骨膜。实验组剪取预制完成的4×1.5大小的三角形PLGA膜，植入骨缺损区，粘骨膜1号丝线间断结节缝合。分别于术后4周、8周、12周、24周，成对处死实验组和对照组各一对实验犬。对上颌骨进行X线CT腭板冠状位扫描观察。切取长5cm宽3cm的腭部标本切片HE染色观察。
结果 X线CMR影像检查在术后对照组见腭部骨板后缘见三角型的透光区，裂隙边缘骨密度略有增强，裂隙明显地较切除的范围变小，骨缺
损尖端变成圆钝。实验组：8周骨缺损区的透光率略高于原有腭部的骨板的透光率，但明显低于对照组的缺损区，骨缺损区的骨密度自缺损周边向中间及均匀相同，后缘密度降低与8周相比骨缺损区密度增加与正常的腭部板的透光率仍有差距，但边界已开始模糊，至24周裂隙后缘中部向内前方透光率降低缺损区的密度均匀与12周相比明显增加，与周边的正常的腭板无明显的界线，腭后缘骨板连续完整。向后缘密度略有降低。CT 检查24周实验组从腭板水平前部到后缘全层腭板连续骨缺损区完全修复成形，骨架桥区肉齿至硬腭后缘与两侧正常腭板厚度均完全一致。
组织学所见实验组4周膜材料大部分降解，可见少量的纤维结缔组织，异物巨细胞少量的局灶性淋巴细胞浸润，新骨形成明显，膜周围有多处新生编织骨和较完整的板层骨下可见大量的成骨细胞。8周两侧的新生骨在中间呈指状交叉。残存少量局灶性淋巴C浸润区，该区新生骨表面形略低于中间区，为残存少量的膜。12周新骨进一步致密，骨细胞较少骨陷密不典型，呈典型的密质骨。24周新骨形成仍然活跃，骨板后份较前份略薄。
讨论 引导骨再生(GBR)是对引导组织再生（GTR）理论的丰富和完善。引导性骨再生的过程实际上是应用膜技术，为骨修复的“引导成骨”和“诱导成骨”造成良好环境完成骨再生的修复过程，即利用人工生物膜选择引导具有成骨能力的细胞占据骨缺损区，防止纤维性结缔组织向骨缺损内长入，改变骨创愈合环境，排除其它组织细胞干扰，引导骨再生，促进新骨形成，加速骨连接实验实验组术后4周，可见新生骨，腭板骨缺损内只有少量纤维性结缔组织长入；对照组存在大量致密结缔组织，新生骨不明显。实验组8周，新生骨组织充满整个骨缺损区，腭板裂隙骨缺损区内的新骨组织覆盖腭板骨裂隙缘。结果分析对照组新生骨量少，可能是骨修复过程中骨诱导因子的表达受到限制，骨缺损使其相对含量下降，加上骨诱导因子向周围扩散，绝对含量也随之下降，出现骨诱导障碍。而实验组出现大量新生骨，说明引导性骨再生成功的关键是放置屏障膜，由于
PLGA复合膜发挥引导组织再生作用，一方面阻止成纤维细胞等周围结缔组织向缺损区内生长，将其排斥在骨缺损区外，引导迁移速率较慢的成骨细胞向缺损区内迁移、增殖。另一方面在骨缺损局部形成了一个相对独立的骨再生环境，排除周围组织的干扰，防止骨诱导因子扩散，使骨缺损局部的骨诱导因子含量相对增加，从而诱导骨膜、骨内膜及骨髓中的骨原细胞成骨，修复骨缺损。与此同时rhBMP–2/Co/PLGA复合生物膜包绕覆盖缺损区，对骨组织再生还起到搭桥连接作用。本实验实验组术后8周X线CMR实验组显示各阶段在骨缺损区骨密度均匀，组织学观察新生骨在裂边缘和中缝都有产生，新生骨不断成熟并在其表面生成新骨。对照组的新生骨见于裂隙边缘，而中间缺损区为大量胶原纤维。结果分析胶原膜作为骨膜再生支架，使腭部粘骨膜参与成骨，对粘骨膜成骨的外骨痂起导向性生长作用，胶原膜在引导性骨再生中具有以下作用：①为骨再生组织提供空间；②阻止周围组织长入，排除外界干扰；③防止骨诱导因子扩散、丢失，使骨诱导因子收集、浓缩；④引导骨再生后经过一段时间隔膜降解不影响骨再生过程[145]。rhBMP–2/Co/PLGA复合生物膜是由基因重组人骨形成蛋白(rhBMP)、胶原、PLGA微粒混合制成，主要成份是胶原，实
Aim: To study whether the guided bone regeneration (GBR) technique can be used to achieve good bone formation in the defects in the palatal plate, providing a more scientific and effective method for clinical treatment of cleft palate, as well as cell generation and differentiation in palatal bone generation and the formation period and mechanism of the bony framework bridge of the palatal plate, and the effect of collagen-polyglycollic acid on guided regeneration in bony defects in cleft palate.
Methods: 1. The collagen solution used in the experiment was the soluble beef tendon collagen, with the main ingredient being type I collagen. The beef tendon was picked and immerged into 70% alcohol solution. 20 min later, under the aseptic condition, the beef tendon was taken out and cut into pieces with a pair of scissors. Then it was placed into 0.5% acetate solution and stirred at intervals at the temperature of 4℃. 48 h later, it was centrifuged (10000r/min) for 1.5 h and then residues were removed. The supernate fluid was salted out with 100g/L NaCl solution. Finally, the precipitated protein was dissolved in 1mmol/L HCl solution at 4℃. The concentration of the prepared collagen protein was 84 g/L (Lowry method). Cross linking: after the commercially available 25% glutaraldehyde was purified by air distillation, the measured final concentration was 11%. The glutaraldehyde solution was dripped into the purified concentrated collagen solution, making the final concentration of glutaraldehyde reach 0.06%. The nonwoven mesh of polyhydroxy-acetic acid fibre 15 μm in diameter was mixed with cross linked collagen and poured into a 24-hole culture plate, mixed to uniformity overnight at 4℃ and placed in the series freeze dryer for 48 h vacuum freeze drying to make the PLGA scaffold embedded in porous collagen, which was rinsed for 7 days in sterile Tris-HCl buffer solution and after natural drying, cross rhBMP 4mg in PLGA and make the rhBMP–2/Co/PLGA membrane sterilized by 60Co radiation for 1-2 h. 2. 8 adult experimental dogs were divided into two groups, with 4 dogs in the experimental group and 4 in the control group. After general anesthesia, the palatal mucosa was cut open to remove the triangular bone plate 4 cm high and 1.5 cm wide and the paranasal
mucoperiosteum was cut off. The mucoperiosteum was closed directly with #1 silk thread using interrupted nodal suture in the control group. In the experimental group, the readymade 4×1.5 cm triangular rhBMP-2/Co/ PLGA membrane was cut out and implanted in the bone defect area and the mucoperiosteum was closed with #1 silk thread using interrupted nodal suture. 4, 8, 12 and 24 weeks respectively after the surgical operation, a pair of experimental dogs was euthanized in pairs in the experimental group and control group each. The maxillary bone was observed by the palatal plate coronal position X-ray CT scan. A slice of the palatal specimen 5 cm long and 3 cm wide was cut out and observed after HE dyeing.
Results: X-ray CMR imaging after the surgical operation showed a triangular transparent area on the posterior border of the palatal bone plate, slightly increased bone density at the edge of the cleft, a significantly smaller cleft than the removal scope and a blunted tip of the bone defect in the control group. In the experimental group; the transmittance in the bone defect area was slightly higher than that of the original palatal bone plate after 8 weeks, but significantly lower than that in the bone defect area in the control group. The bone density in the bone defect area was uniform from the edge to the center of the defect, but the bone density on the posterior border decreased. The bone density in the defect area increased compared to that after 8 weeks, but there was still a gap in the transmittance compared to that of the normal palatal plate, and the border began to blur. After 24 weeks, the transmittance decreased from the middle posterior border to the interior front of the cleft and the density in the defect area was uniform and significantly
方法 1.实验所用胶原溶液为可溶性牛腱胶原，其主要成份为I型胶原。将牛腱剔下，浸入70%酒精溶液中，20min后在无菌条件下抽出牛腱并将其剪碎，置入0.5%的醋酸溶液中，在4℃条件下不时搅拌。48小时后离心（10000r/min）约1.5小时，去渣。上清液用100g/L NaCl溶液盐析，最后将沉淀的蛋白质溶于4℃ 1mmol/L的HCl溶液中备用。配制胶原蛋白浓度为84 g/L（Lowry法）。将直径15μm的聚羟基乙酸纤维无纺网与交联胶原相混合，注入24孔培养板中，混匀，4℃ 过夜，置于序列冻干机中，真空冷冻干燥48小时，即制成多孔状胶原包埋处理的PlGA支架。将其在无菌Tris-HCl缓冲液中漂洗7天，自然干燥，将基因重组人骨形成蛋白(rhBMP) 的醋酸溶液按每培养板孔4mg量滴入PLGA支架中即制成rhBMP–2/Co/PLGA复合生物膜60Co照射1至2小时灭菌备用。2. 8只成年实验犬分为二组：实验组4只，对照组4只。全身麻醉后切开腭部粘膜，去除高4cm，宽1.5cm的三角形骨板，切透鼻侧的粘骨膜，对照线直接以1号丝线间断结节缝合粘骨膜。实验组剪取预制完成的4×1.5大小的三角形PLGA膜，植入骨缺损区，粘骨膜1号丝线间断结节缝合。分别于术后4周、8周、12周、24周，成对处死实验组和对照组各一对实验犬。对上颌骨进行X线CT腭板冠状位扫描观察。切取长5cm宽3cm的腭部标本切片HE染色观察。
结果 X线CMR影像检查在术后对照组见腭部骨板后缘见三角型的透光区，裂隙边缘骨密度略有增强，裂隙明显地较切除的范围变小，骨缺
损尖端变成圆钝。实验组：8周骨缺损区的透光率略高于原有腭部的骨板的透光率，但明显低于对照组的缺损区，骨缺损区的骨密度自缺损周边向中间及均匀相同，后缘密度降低与8周相比骨缺损区密度增加与正常的腭部板的透光率仍有差距，但边界已开始模糊，至24周裂隙后缘中部向内前方透光率降低缺损区的密度均匀与12周相比明显增加，与周边的正常的腭板无明显的界线，腭后缘骨板连续完整。向后缘密度略有降低。CT 检查24周实验组从腭板水平前部到后缘全层腭板连续骨缺损区完全修复成形，骨架桥区肉齿至硬腭后缘与两侧正常腭板厚度均完全一致。
组织学所见实验组4周膜材料大部分降解，可见少量的纤维结缔组织，异物巨细胞少量的局灶性淋巴细胞浸润，新骨形成明显，膜周围有多处新生编织骨和较完整的板层骨下可见大量的成骨细胞。8周两侧的新生骨在中间呈指状交叉。残存少量局灶性淋巴C浸润区，该区新生骨表面形略低于中间区，为残存少量的膜。12周新骨进一步致密，骨细胞较少骨陷密不典型，呈典型的密质骨。24周新骨形成仍然活跃，骨板后份较前份略薄。
讨论 引导骨再生(GBR)是对引导组织再生（GTR）理论的丰富和完善。引导性骨再生的过程实际上是应用膜技术，为骨修复的“引导成骨”和“诱导成骨”造成良好环境完成骨再生的修复过程，即利用人工生物膜选择引导具有成骨能力的细胞占据骨缺损区，防止纤维性结缔组织向骨缺损内长入，改变骨创愈合环境，排除其它组织细胞干扰，引导骨再生，促进新骨形成，加速骨连接实验实验组术后4周，可见新生骨，腭板骨缺损内只有少量纤维性结缔组织长入；对照组存在大量致密结缔组织，新生骨不明显。实验组8周，新生骨组织充满整个骨缺损区，腭板裂隙骨缺损区内的新骨组织覆盖腭板骨裂隙缘。结果分析对照组新生骨量少，可能是骨修复过程中骨诱导因子的表达受到限制，骨缺损使其相对含量下降，加上骨诱导因子向周围扩散，绝对含量也随之下降，出现骨诱导障碍。而实验组出现大量新生骨，说明引导性骨再生成功的关键是放置屏障膜，由于
PLGA复合膜发挥引导组织再生作用，一方面阻止成纤维细胞等周围结缔组织向缺损区内生长，将其排斥在骨缺损区外，引导迁移速率较慢的成骨细胞向缺损区内迁移、增殖。另一方面在骨缺损局部形成了一个相对独立的骨再生环境，排除周围组织的干扰，防止骨诱导因子扩散，使骨缺损局部的骨诱导因子含量相对增加，从而诱导骨膜、骨内膜及骨髓中的骨原细胞成骨，修复骨缺损。与此同时rhBMP–2/Co/PLGA复合生物膜包绕覆盖缺损区，对骨组织再生还起到搭桥连接作用。本实验实验组术后8周X线CMR实验组显示各阶段在骨缺损区骨密度均匀，组织学观察新生骨在裂边缘和中缝都有产生，新生骨不断成熟并在其表面生成新骨。对照组的新生骨见于裂隙边缘，而中间缺损区为大量胶原纤维。结果分析胶原膜作为骨膜再生支架，使腭部粘骨膜参与成骨，对粘骨膜成骨的外骨痂起导向性生长作用，胶原膜在引导性骨再生中具有以下作用：①为骨再生组织提供空间；②阻止周围组织长入，排除外界干扰；③防止骨诱导因子扩散、丢失，使骨诱导因子收集、浓缩；④引导骨再生后经过一段时间隔膜降解不影响骨再生过程[145]。rhBMP–2/Co/PLGA复合生物膜是由基因重组人骨形成蛋白(rhBMP)、胶原、PLGA微粒混合制成，主要成份是胶原，实
Aim: To study whether the guided bone regeneration (GBR) technique can be used to achieve good bone formation in the defects in the palatal plate, providing a more scientific and effective method for clinical treatment of cleft palate, as well as cell generation and differentiation in palatal bone generation and the formation period and mechanism of the bony framework bridge of the palatal plate, and the effect of collagen-polyglycollic acid on guided regeneration in bony defects in cleft palate.
Methods: 1. The collagen solution used in the experiment was the soluble beef tendon collagen, with the main ingredient being type I collagen. The beef tendon was picked and immerged into 70% alcohol solution. 20 min later, under the aseptic condition, the beef tendon was taken out and cut into pieces with a pair of scissors. Then it was placed into 0.5% acetate solution and stirred at intervals at the temperature of 4℃. 48 h later, it was centrifuged (10000r/min) for 1.5 h and then residues were removed. The supernate fluid was salted out with 100g/L NaCl solution. Finally, the precipitated protein was dissolved in 1mmol/L HCl solution at 4℃. The concentration of the prepared collagen protein was 84 g/L (Lowry method). Cross linking: after the commercially available 25% glutaraldehyde was purified by air distillation, the measured final concentration was 11%. The glutaraldehyde solution was dripped into the purified concentrated collagen solution, making the final concentration of glutaraldehyde reach 0.06%. The nonwoven mesh of polyhydroxy-acetic acid fibre 15 μm in diameter was mixed with cross linked collagen and poured into a 24-hole culture plate, mixed to uniformity overnight at 4℃ and placed in the series freeze dryer for 48 h vacuum freeze drying to make the PLGA scaffold embedded in porous collagen, which was rinsed for 7 days in sterile Tris-HCl buffer solution and after natural drying, cross rhBMP 4mg in PLGA and make the rhBMP–2/Co/PLGA membrane sterilized by 60Co radiation for 1-2 h. 2. 8 adult experimental dogs were divided into two groups, with 4 dogs in the experimental group and 4 in the control group. After general anesthesia, the palatal mucosa was cut open to remove the triangular bone plate 4 cm high and 1.5 cm wide and the paranasal
mucoperiosteum was cut off. The mucoperiosteum was closed directly with #1 silk thread using interrupted nodal suture in the control group. In the experimental group, the readymade 4×1.5 cm triangular rhBMP-2/Co/ PLGA membrane was cut out and implanted in the bone defect area and the mucoperiosteum was closed with #1 silk thread using interrupted nodal suture. 4, 8, 12 and 24 weeks respectively after the surgical operation, a pair of experimental dogs was euthanized in pairs in the experimental group and control group each. The maxillary bone was observed by the palatal plate coronal position X-ray CT scan. A slice of the palatal specimen 5 cm long and 3 cm wide was cut out and observed after HE dyeing.
Results: X-ray CMR imaging after the surgical operation showed a triangular transparent area on the posterior border of the palatal bone plate, slightly increased bone density at the edge of the cleft, a significantly smaller cleft than the removal scope and a blunted tip of the bone defect in the control group. In the experimental group; the transmittance in the bone defect area was slightly higher than that of the original palatal bone plate after 8 weeks, but significantly lower than that in the bone defect area in the control group. The bone density in the bone defect area was uniform from the edge to the center of the defect, but the bone density on the posterior border decreased. The bone density in the defect area increased compared to that after 8 weeks, but there was still a gap in the transmittance compared to that of the normal palatal plate, and the border began to blur. After 24 weeks, the transmittance decreased from the middle posterior border to the interior front of the cleft and the density in the defect area was uniform and significantly
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In de Braekt MM，Maltha JC，Kuijpers-Jugtman AM．Cleft Palate Craniofac J，1995；32（4）：290-298
Vacanti CA,Vacanti JP. Bone and cartilage reconstruction with tissue engineering approaches.Otolargngo Clin North Am,1994,27:263～269
Dahlin C，linde A，Gottow J，et al．Healing of bone defects guided tissue regeneration [J]．Plast and Reconstr Surger,1988;(5):672-676
Becker J, Nekam FW,Schilephake H.Restoration of the lateral sinus wall using a collagen tape Imembrane for guided tissue regeneration [J].Int J Oral Maxillofac Surg,1992;21(4):243-246
Cook SD，Baffes GC，Wolfe MW，et al。J Bone Joint Surg．1994；76-A（6）：827-837
Stone CA。Br J Oral Maxillofac Surg．1997；50（4）：369-373
Fujimura K，Besslao K，Kusumoto K，et al．Int J Oral Maxillofac Surg。1997；26（Suppl）：88
林崇韬 崔新明 崔丽等 复合生物膜的制备及扫描电镜观察 电子显微学报 2003；22（6）：661
林崇韬 文胡实 孙宏晨等 乙酸溶解-酶消化法制备医用胶原膜 吉林大学学报（医学版）2004；30（1）：75-78
Buser D，Dura K，Belser U，et al。Localized ridge augmentation using guided bone regeneration．I Surgical procedure in the maxilla．Int J Periodontics Restorative Dent．1993；13（1）：29-45
Lundgren AK，Sennerby L，Lundgren D．Guided jaw-bone regeneration using an experimental rabbit model．Int J Pral Maxillofac Surg．1998；27（2）：135-140
Hammerle CHF，Schmid J，Lang NP，et al．Schmid J，Lang NP，et al．Temporal dynamics of healing in rabbit cranial difects using guided bone regeneration．J Oral Maxillofac Surg．1995；53（2）：167-174．
Nielsen FF，Karring T，Gogolewski S．Biodegradable guided for bone regeneration。Acta Orthop Scand．1992；63（1）：66-68
Breitbart S, Graride DA, Keler R.Tissue engineered bone repair of calvarial defects using cultured periosteal cells. Plast Reconstr Surg
1998;101(3):567
Isogaid N, Landis W, kim TH, et al.Formation of phalanges and small joint by tissue-engineering. J Bone Joint Surg, 1999; 81A:306
Zhang QQ，Ren L，Wang C，et al．Porous hydroxyapatite reinforced with collagen protein．Artif Cells Blood Substit Immobil Biotechnol，1996；24（6）：693-702
郭洪刚，顾云武等，胶原膜及同种异体骨引导兔桡骨再生的特点及机制. 中华创伤杂志，1999；15（1）：15～17
Basle, MF. Grizon,F. Pascaretti,C. Shape and orientation of osteoblast-like cells(Saos-2) are influenced by collagen fibers in xenogenic bone biomaterial. J Biomed Mater Res,1998;40(3):350
Shi,S. Kirk, M. Kahn, AJ. The role of type I collagen in the regulation of osteoblast phenotype. J Bone Miner Res , 1996;11(8):1139
Mizuno,M.Shindo, M. Kobayashi, D. Osteogenesis by bone marrow stromal cells maintained on type I collagen matrix gels in vivo. Bone, 1997;20(2);101
Urist MR, Mcleam FC. Recent advances in physiology of bone . J Bone Joint Surg (Am). 1963; 45:1305-1309
Urist MR, Dowell TA, Hay PH, et al. Inductive substrates for bone formation. J Clin Orthop, 1968; 59:59-74
Minabe M. A critical review of the biologic rationale for guided tissue regeneration. J Periodontot, 1991; 62(3) :171-174
Niederman R. Is "Guided tissue regeneration" really guided tissue regeneration. J Periodontol, 1995；65(10) :804-805.
Sandberg E, Dahlin C, Linde A. Bone Regeneration by the
osteopromotion technique using bioabsorbable membranes : An experimental study in rats. J Oral Maxillofac Surg. 1993,51:1106-1110.
傅豫川，黄洪章，李金荣腭裂胎鼠上颌畸形发育的实验研究，中华口腔医学杂志。1994；29（5）：288-290
Bardach J，Kelly KM..Does interference with mucoperiosteum and palatal bone affect maxillofacial surgery?Plast Reconstr Surgery。1991；88(4)：545-548
平云飞，严奉国，陈军。腭板缺失修复的动物模型实验研究 浙江大学学报（医学板）2001；30（1）：25-28
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Cottrell DA，Wolford LM。J Oral Maxillofac Surg 1998；56（6）：935-941
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Matzen M，Kostopoulos L，Karring T．Scand J Plast Reconstr Surg。1996；30（2）：161-167
Meinig RP，Buesing CM，Helm J，et al． J Orthop Trauma，1997；11（5）：551-558
In de Braekt MM，Maltha JC，Kuijpers-Jugtman AM．Cleft Palate Craniofac J，1995；32（4）：290-298
Vacanti CA,Vacanti JP. Bone and cartilage reconstruction with tissue engineering approaches.Otolargngo Clin North Am,1994,27:263～269
Dahlin C，linde A，Gottow J，et al．Healing of bone defects guided tissue regeneration [J]．Plast and Reconstr Surger,1988;(5):672-676
Becker J, Nekam FW,Schilephake H.Restoration of the lateral sinus wall using a collagen tape Imembrane for guided tissue regeneration [J].Int J Oral Maxillofac Surg,1992;21(4):243-246
Cook SD，Baffes GC，Wolfe MW，et al。J Bone Joint Surg．1994；76-A（6）：827-837
Stone CA。Br J Oral Maxillofac Surg．1997；50（4）：369-373
Fujimura K，Besslao K，Kusumoto K，et al．Int J Oral Maxillofac Surg。1997；26（Suppl）：88
林崇韬 崔新明 崔丽等 复合生物膜的制备及扫描电镜观察 电子显微学报 2003；22（6）：661
林崇韬 文胡实 孙宏晨等 乙酸溶解-酶消化法制备医用胶原膜 吉林大学学报（医学版）2004；30（1）：75-78
Buser D，Dura K，Belser U，et al。Localized ridge augmentation using guided bone regeneration．I Surgical procedure in the maxilla．Int J Periodontics Restorative Dent．1993；13（1）：29-45
Lundgren AK，Sennerby L，Lundgren D．Guided jaw-bone regeneration using an experimental rabbit model．Int J Pral Maxillofac Surg．1998；27（2）：135-140
Hammerle CHF，Schmid J，Lang NP，et al．Schmid J，Lang NP，et al．Temporal dynamics of healing in rabbit cranial difects using guided bone regeneration．J Oral Maxillofac Surg．1995；53（2）：167-174．
Nielsen FF，Karring T，Gogolewski S．Biodegradable guided for bone regeneration。Acta Orthop Scand．1992；63（1）：66-68
Breitbart S, Graride DA, Keler R.Tissue engineered bone repair of calvarial defects using cultured periosteal cells. Plast Reconstr Surg
1998;101(3):567
Isogaid N, Landis W, kim TH, et al.Formation of phalanges and small joint by tissue-engineering. J Bone Joint Surg, 1999; 81A:306
Zhang QQ，Ren L，Wang C，et al．Porous hydroxyapatite reinforced with collagen protein．Artif Cells Blood Substit Immobil Biotechnol，1996；24（6）：693-702
郭洪刚，顾云武等，胶原膜及同种异体骨引导兔桡骨再生的特点及机制. 中华创伤杂志，1999；15（1）：15～17
Basle, MF. Grizon,F. Pascaretti,C. Shape and orientation of osteoblast-like cells(Saos-2) are influenced by collagen fibers in xenogenic bone biomaterial. J Biomed Mater Res,1998;40(3):350
Shi,S. Kirk, M. Kahn, AJ. The role of type I collagen in the regulation of osteoblast phenotype. J Bone Miner Res , 1996;11(8):1139
Mizuno,M.Shindo, M. Kobayashi, D. Osteogenesis by bone marrow stromal cells maintained on type I collagen matrix gels in vivo. Bone, 1997;20(2);101
Urist MR, Mcleam FC. Recent advances in physiology of bone . J Bone Joint Surg (Am). 1963; 45:1305-1309
Urist MR, Dowell TA, Hay PH, et al. Inductive substrates for bone formation. J Clin Orthop, 1968; 59:59-74
Minabe M. A critical review of the biologic rationale for guided tissue regeneration. J Periodontot, 1991; 62(3) :171-174
Niederman R. Is "Guided tissue regeneration" really guided tissue regeneration. J Periodontol, 1995；65(10) :804-805.
Sandberg E, Dahlin C, Linde A. Bone Regeneration by the
osteopromotion technique using bioabsorbable membranes : An experimental study in rats. J Oral Maxillofac Surg. 1993,51:1106-1110.
傅豫川，黄洪章，李金荣腭裂胎鼠上颌畸形发育的实验研究，中华口腔医学杂志。1994；29（5）：288-290
Bardach J，Kelly KM..Does interference with mucoperiosteum and palatal bone affect maxillofacial surgery?Plast Reconstr Surgery。1991；88(4)：545-548
平云飞，严奉国，陈军。腭板缺失修复的动物模型实验研究 浙江大学学报（医学板）2001；30（1）：25-28