骨形态发生蛋白的提取,纯化及诱导成骨的实验报告

目的 提取，纯化牛骨形态发生蛋白（ＢＭＰ）并验证其异位诱导成骨的活性。方法采用生物化学方法提取并纯化出ＢＭＰ，植入家兔肌肉和豚鼠颅骨进行动物实验研究，结果植入ＢＭＰ组１４ｄ即有成纤维细胞转化为软骨细胞；第２１，２８天形成软骨和骨组织，成年豚鼠颅骨缺损修复过程中，植入ＢＭＰ后第２８天即有骨痂生成，８４ｄ时颅骨缺损修复完整，结论ＢＭＰ有明显的异位诱导骨形成作用。     

牛骨形态发生蛋白异位诱导小鼠成骨的超微结构观察

目的：牛骨形态发生蛋白（ｂＢＭＰ）植入小鼠肌袋后可诱导血管周围的间充质细胞分化形成软骨和骨组织．但ｂＢＭＰ诱导异位成骨过程的超微结构研究报道甚少．本研究的目的是利用透射电子显微镜（ＴＥＭ）观察ｂＢＭＰ植入区间充质细胞向生骨细胞分化及成骨的超微结构变化．方法：将牛骨中提取的ｂＢＭＰ５ｍｇ植入小鼠股部肌间隙内，术后５，９，１４和２１ｄ取材，制备光镜及电镜标本，镜下观察组织超微结构的变化．结果：ｂＢＭＰ植入后５ｄ，植入区内发现大量肥大的间充质细胞及纤维母细胞样细胞聚集；术后９ｄ，成熟的软骨细胞出现并可见丰富的细胞外软骨基质；术后１４ｄ，软骨细胞变性，基质钙化明显，骨母细胞功能活跃；术后２１ｄ，骨母细胞深陷于钙化基质中形成骨细胞，骨髓腔出现，原始骨髓形成．结论：ｂＢＭＰ能诱导具有分化潜能的间充质细胞进行分化，产生一个完整的骨发育过程．     

重组人骨形态发生蛋白诱导异位成骨的病理学观察

目的：本文报告了重组人骨形态发生蛋白－２（ｒｈＢＭＰ－２）在小鼠肌肉内诱导异位成骨的生物学作用，方法：将大肠杆功表达的ｒｈＢＭＰ－２成熟肽植入小鼠股部肌肉。结果：植入ｒｈＢＭＰ－２后ｄ１就有间充质细胞向植入区内聚集；ｄ５出现典型的软骨细胞；ｄ７钙化开始；ｄ１０形成雏形的骨髓腔，ｄ１４骨髓腔内出现幼稚的造血细胞；２１ｄ－３０ｄ形成大量网状骨小梁、骨单位、在骨小梁间隙出现骨髓细胞。结论：大肠杆菌中表达     

珊瑚/胶原/重组人骨形成蛋白-2复合人工骨的异位骨诱导(英文)

目的：评价珊瑚／胶原／重组人骨形成蛋白－２ （ｒｈＢＭＰ－２）复合人工骨的骨诱导活性。　方法：将珊瑚／胶原／ｒｈＢＭＰ－２ 植入大鼠背部肌肉内,以珊瑚／胶原或珊瑚／ｒｈＢＭＰ－２ 植入作对照。术后１ 周和４周取材,通过组织学和计算机图像分析评价其异位诱导成骨情况。　结果：珊瑚／胶原／ｒｈＢＭＰ－２ 植入后１ 周,在局部诱导软骨形成;４ 周,形成含骨髓的板层骨;诱导成骨的量有明显的ｒｈＢＭＰ－２ 剂量依赖性（Ｐ＜ ０．００１）。珊瑚／胶原或珊瑚／ｒｈＢＭＰ－２ 植入区无骨或软骨形成。　结论：珊瑚／胶原／ｒｈＢＭＰ－２ 复合人工骨具有良好的异位骨诱导活性,是一种较理想的骨移植替代材料     

异体脱钙骨基质骨粒骨水泥骨形态发生蛋白复合材料的成骨诱导活性

目的：探讨异体脱钙骨基质（ＤＢＭ）骨粒、骨水泥（ＢＣ）及重组人骨形态发生蛋白－２（ｒｈＢＭＰ－２）复合骨制损修复材料成骨诱导活性。方法：小鼠股部股袋内植入复合材料,不同时间分别取材ＨＥ染色观察复合骨制损修复材料的异位诱导成骨活笥;检测不同复合质量比的复合材料的聚合温度。结果：第７日时材料外周即形成间充质组织包膜,并沿材料不规则裂隙向植入材料内部长入间充质组织。第１４日间充质组织继续大量长入且逐渐深     

骨不连与骨形成蛋白

研究骨不连发生的原理及骨不连与骨诱导因子的关系是抗击有不连发生机理的重要课题。方法：将兔桡骨中段告成ｃｍ的缺损，Ａ组术后即放入ＢＭＰ，Ｂ组术后４周放入ＢＭＰ，Ｃ组为在Ａ组术后８周放入ＢＭＰ。结果：发现ＢＭＰ植入８周时形成骨小梁。Ｂ组和Ｃ组，骨缺损修复仍然取得满意的效果。     

牛骨形态发生蛋白异位诱导小鼠成央骨的超微结构观察

牛骨形态发生蛋白植入小鼠肌袋后可诱导血管周围的间充质细胞分化形成软骨和骨组织。但ｂＢＭＰ诱导异位成骨过程的超微结构研究报道甚少。本研究的目的是利用透射电子显微镜观察ｂＢＭＰ植入区间充质细胞向生骨细胞分化及成骨的超微结构变化。     

猪骨松质复合材料修复颅骨大面积缺损的实验研究

本文报道了猪骨形态发生蛋白与猪骨松质复合材料修复颅骨大面积缺损的实验结果。以家兔颅顶骨的两侧制成２个１０ｍｍ圆形骨缺损作为颅骨大面积缺损的动物模型，分别植入猪骨形态发生蛋白（ｐＢＭＰ）与猪骨松质复合材料、猪骨松质和羟基磷灰石（ＨＡ）颗粒。术后２、４、８周进行Ｘ线摄片和组织学检查，结果表明ｐＢＭＰ与骨松质复合材料无排斥反应，有良好的骨诱导作用，术后８周已将骨缺损修复，而不含ＢＭＰ的骨松质和羟基磷灰石颗粒未见有骨组织生成。说明猪骨形态发生蛋白与猪骨松质复合材料可以作为小儿颅骨大面积缺损的修复材料。     

骨形态形成蛋白和纤维蛋白粘合剂复合物修复骨缺损的放射性核素骨显像研究

在家兔桡骨中段造成１５ｍｍ骨缺损，分别植入骨形态形成蛋白和纤维蛋白粘合剂及二者复合物，并设空白对照。术后第二、四、八周分别行Ｘ线及核素骨显像检查，发现复合物和骨形态形成蛋白组缺损均骨性愈合，纤维蛋白粘合剂和空白组则形成骨不连，两周时含Ｆ５组缺损区血流量大于其它组。定量分析表明，复合物组缺损区新骨量大于骨形态形成蛋白组（Ｐ＜０．０５），说明ＦＳ与ＢＭＰ复合后可增强ＢＭＰ修复骨缺损的能力，骨显像检查可定量观察修复过程中缺损区血流量及成骨活动。     

家兔骨生长诱导修复腭裂缺损的实验研究

目的 研究评价牛骨形成蛋白（ｂＢＭＰ）－白蛋白－成纤维细胞生长因子（ｂＦＧＦ）复合材料修复腭裂骨缺损的可行性。方法 建立家兔腭骨缺损模型，将固化的ｂＦＧＦ复合材料植入骨缺损区，通过其骨诱导作用，诱导新骨生成，修复骨缺损。经Ｘ线、组织学观察和钙含量测定。结果 ｂＢＭＰ－白蛋白－ｂＦＧＦ有很强的骨诱导作用，可以诱导骨形成、实验组与对照组Ｘ线骨修复分数评估分别为１２分，０分，组织学检查实验组骨修复完成，     

Gene expression analysis of ectopic bone formation induced by electroporatic gene transfer of BMP4

Implantation of bone morphogenetic protein (BMP) using a carrier or by BMP gene transfer into rodent muscle can induce bone formation. Implanted BMP becomes bioactive immediately after implantation. In BMP gene transfer, there is a time-lag between the secretion of gene products and bone formation. We analyzed the gene expression of chondrogenic and osteogenic specific markers in the process of ectopic bone formation by using semi-quantitative RT-PCR. A plasmid vector containing mouse BMP4 gene (pCAGGS-BMP4) was transferred into the gastrocnemius muscles of mice using electroporation. Histological examination revealed the process of endochondral bone formation in the pCAGGS-BMP4 transferred muscles. As chondrogenic markers, aggrecan gene maximal expression was detected on day 7 and decreased by day 14, and for collagen X the gene maximal expression was on day 10. As osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP) and osteopontin (OPN) gene expressions were clearly detected from day 10 and then increased by day 14. In conclusion, the present study proved that ectopic bone formation by BMP4 gene transfer into the muscle induced endochondral ossification that corresponded well with that to that by implantation of demineralized bone matrix.     

重组人BMP-2骨诱导中神经生长因子的表达

目的通过观察重组人骨形态发生蛋白-2(rhBMP-2)异位诱导成骨过程中神经生长因子(NGF)的表达,探讨NGF在BMP骨诱导中的作用。方法采用ICR小鼠48只,随机分为实验组和对照组,行右侧股后部肌袋异位成骨模型,实验组植入含0．125mg rhBMP-2胶原海绵复合物,对照组仅植入相同体积胶原海绵,分别于术后3、7、14、21d取材,进行放射学、组织学及免疫组化检测。结果放射学和组织学检测均证实,rhBMP-2胶原海绵复合物具有良好的诱导成骨能力;术后第7d,在软骨大量形成期,NGF阳性染色达到高峰．在成纤维细胞、软骨前体细胞、软骨细胞、肥大软骨细胞和成骨细胞中均有阳性表达;术后14、2ld,阳性细胞数量和染色强度有所回落。对照组未见成骨现象。结论首次证实在外源性BMP诱导成骨过程中有明显的NGF表达,提示NGF可能通过直接或间接的方式参与并协同BMP的诱导成骨过程。     

Gene Expression Analysis of Ectopic Bone Formation Induced by Electroporatic Gene Transfer of BMP4

Implantation of bone morphogenetic protein (BMP) using a carrier or by BMP gene transfer into rodent muscle can induce bone formation. Implanted BMP becomes bioactive immediately after implantation. In BMP gene transfer, there is a time-lag between the secretion of gene products and bone formation. We analyzed the gene expression of chondrogenic and osteogenic specific markers in the process of ectopic bone formation by using semi-quantitative RT-PCR.

A plasmid vector containing mouse BMP4 gene (pCAGGS-BMP4) was transferred into the gastrocnemius muscles of mice using electroporation. Histological examination revealed the process of endochondral bone formation in the pCAGGS-BMP4 transferred muscles. As chondrogenic markers, aggrecan gene maximal expression was detected on day 7 and decreased by day 14, and for collagen X the gene maximal expression was on day 10. As osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP) and osteopontin (OPN) gene expressions were clearly detected from day 10 and then increased by day 14. In conclusion, the present study proved that ectopic bone formation by BMP4 gene transfer into the muscle induced endochondral ossification that corresponded well with that to that by implantation of demineralized bone matrix.     

Experimental Research on Ectopic Osteogenesis of BMP2-derived Peptide P24 Combined with PLGA Copolymers

To experimentally evaluate the ectopic osteogenetic capacity of synthesized BMP2-derived peptide P24 combined with poly lactic-co-glycolic acid (PLGA), Wistar rats were di- vided into two groups: group A, in which BMP2-derived peptide P24/PLGA complex was implanted, and group B which received simple PLGA implant. The complex was respectively implanted into the back muscles of rats. Samples were taken the 1st, 4th, 8th, and the 12th week after the implantation. Their bone formation was detected by X-ray examination, and tissue response was histologically ob- served. Western blotting was used for the detection of the expression of collagen Ⅰ (Col-Ⅰ) and osteopontin (OPN). There was acute inflammation in the tissue around both types of implants at early stage. The cartilage was found around implant areas 4 weeks after the implantation of BMP2-derived peptide p24/PLGA complex, 8 weeks after the implantation, osteoblasts were found, and 12 weeks after the implantation, typical trabecular bone structure was observed. In group B, after 12 weeks, no osteoblasts were found. It is concluded that PLGA is an ideal scaffold material for bone tissue engi- neering. BMP2-derived peptide can start endochondral ossification and is more effective in inducing ectopic osteogenesis.     

异种脱钙骨基质诱导异位骨髓形成的实验研究

目的：开展诱导异位骨髓形成的研究,探讨骨髓形成及其调控机理方法：采用异种（人）脱钙骨基质粉,植入Ｓｐｒａｑｕｅ－Ｄａｗｌｅｙ大鼠腹壁两侧皮下,建立异种脱钙骨基质粉诱导异位骨髓形成的动物实验模型。结果：在诱导第７天可见间充质细胞包绕并迁入骨基质性植入物的间隙,分化成原始软骨细胞、形成软骨组织。诱导第１３天,新生软骨组织进一步增加,成骨细胞,破骨细胞和血管位于其中,呈现软骨化骨现象,类骨样基质产生,成     

负载BMP的新型组织工程骨的构建及骨缺损修复实验

OBJECTIVE: To construct a new tissue-engineered bone with poly (D, L-lactide-co-glycolide) (PLGA), bone morphogenetic protein (BMP) and bone marrow-derived stem cells (BMSCs) and observe its effect in repairing segmental bone defects. METHODS: A 15-mm bone defect in the right radius was induced in New Zealand white rabbits, and the models were randomized into three groups to receive implantation of the tissue-engineered bone grafts constructed with PLGA carrying 5 mg BMP and about 1 x 10(6) BMSCs (experimental group), grafts of PLGA with about 1 x 10(6) BMSCs (control group), or grafts of exclusive PLGA (blank control group), respectively. The osteogenesis in the bone defect after the implantation on was evaluated X-ray films, and the histological changes of the tissues sampled from the bone defect 4, 8, and 12 weeks after operation were observed and new bone formation was measured by image analysis. RESULTS: The bone defect was completely repaired in the experimental group 12 weeks after the implantation, showing the best results among the 3 groups. The bone defects in the blank control group was filled with only fibrous and connective tissues at 12 weeks. CONCLUSION: This tissue-engineered bone constructed with PLGA, BMP and BMSCs possesses good ability in repairing segmental bone defect.     

骨保护素在兔下颌牵张后新骨组织中的表达

目的 研究骨保护素（osteoprotegerin,OPG）在不同时期下颌骨牵张成骨过程中表达水平的变化,探讨其可能发挥的作用.方法在30只成年大耳白兔的一侧下颌骨前部行骨切开术,用牵引器延长一侧下颌骨4mm,分别于牵张完成后的第1,3,7,14,28天处死一组动物,取牵引区新生骨痂行组织学及OPG免疫组化染色.结果下颌牵引延长后牵引间隙均有新骨形成,免疫组化染色OPG主要定位于成骨细胞的胞浆中.其中以牵引结束的第1,7天的表达最强,以后逐渐下降,第28天仅有微弱表达.结论OPG可能在牵引成骨过程中特别是调控组织细胞应力信号传递的早期发挥重要作用.     

负载BMP的新型组织工程骨的构建及骨缺损修复实验

目的 探讨以聚乳乙醇酸(poly-(D,L-lactide-co-glycolide),PLGA)、骨形态发生蛋白(BMP)、骨髓基质干细胞(BM-SCs)构建成新型组织工程骨并观察其在动物体内的成骨能力。方法 制作新西兰大白兔右桡骨中段15mm骨缺损实验模型,随机分为实验组、对照组和空白组,实验组植入同时负载5mgBMP及1×10⁶个已向成骨细胞诱导的BMSCs的PLGA、对照组植入负载1×10⁶个已向成骨细胞诱导的BMSCs的PLGA、空白组仅植入PLGA。术后对动物进行大体观察、摄X线片观察各组不同时相骨缺损修复情况、比较不同时相的骨缺损区X线阻射密度、并于术后第4、8、12周取出骨缺损区标本进行大体观察和组织学切片观察,图像分析骨小梁的生成数量。结果 实验组在12周内骨缺损完全修复,且同时期内新生骨的数量和质量显著优于对照组,空白材料组骨缺损主要由纤维结缔组织填充。结论 利用含BMP的PLGA支架与BMSCs复合构建的新型组织工程骨具有良好的骨缺损修复能力。     

PRF 对下颌骨牵引成骨区骨保护素表达的影响

目的 探讨富血小板纤维蛋白（PRF）对牵引成骨（DO）区骨保护素（OPG）表达的影响。方法 将25 只大耳白兔随机分5 组，分别行双侧下颌骨皮质骨切开术，一侧下颌骨牵引间隙放置PRF 膜，作为实验组，对侧作为对照组，分别于牵引稳定期1、3、7、14 和28 d 各处死一组动物，将下颌骨DO 区骨块制成脱钙石蜡切片，进行苏木精- 伊红染色及OPG 免疫组织化学染色，细胞图像分析仪测量牵引间隙处骨痂OPG 表达情况。结果 下颌骨牵引处形成新生骨，免疫组织化学OPG 染色主要表达在成骨细胞的胞浆中。实验组与对照组在稳定期1、3、7、14 和28 d 的OPG 阳性表达细胞数比较，差异有统计学意义（P <0.05）。结论 PRF 能促进兔下颌骨DO 区新骨的生成，OPG 可能在DO 过程的早期调控组织细胞应力信号传递，发挥成骨作用。