组织工程骨修复颅骨极限缺损种子细胞归宿的探讨

目的：检测纳米羟基磷灰石复合胶原／聚乳酸材料（nHAC／PLA,nano-hydroxyapatite／collagen／Polyacticacid）与骨髓基质细胞（BMSCs,bone marrow stroma cells）在体外复合构建的组织工程骨修复兔颅骨极限缺损的能力,并探讨种子细胞的归宿。方法：将圆盘状nHAC／PLA与BrdU标记的自体BMSCs复合后,植入新西兰大白兔颅骨直径1．5cm全层缺损中。设自体髂骨组、空白对照组及nHAC／PLA组,术后8w、16w通过X线片、HE染色、Masson’s三色法染色、荧光组织学和免疫组织化学染色,观察比较颅骨缺损的修复情况及种子细胞的归宿。结果：nHAC／PLA＋自体BMSCs组修复颅骨缺损的能力强,与自体髂骨组类似,nHAC／PLA组材料修复骨缺损的能力较弱,但强于空白对照组,组织工程骨的成骨细胞由植入的种子细胞演化而来。结论：nHAC／PLA复合自体BMSCs具有良好的修复骨缺损能力。     

bFGF基因修饰的骨髓基质细胞复合多孔矿化Bio-Oss胶原修复下颌骨缺损的实验研究

目的:观察经bFGF基因转染的兔骨髓基质细胞复合多孔矿化Bio-Oss骨胶原修复下颌骨缺损的效果.方法:标准成年新西兰白兔12 只随机分3 组,单纯Bio-Oss骨胶原组,BMSCs 凝胶 Bio-Oss骨胶原组,bFGF基因转染BMSCs 胶原 Bio-Oss骨胶原组于术后8 周取材,行肉眼、组织学观察骨缺损的修复情况.结果:单纯Bio-Oss骨胶原材料组:缺损区大量的未吸收的支架材料周围可见有类骨样组织;BMSCs 凝胶 Bio-Oss骨胶原材料组:缺损区大量的未吸收的支架材料周围可见有大量间充质细胞聚集、分化,有较幼稚的编织骨形成;bFGF基因转染BMSCs 凝胶 Bio-Oss骨胶原材料组:支架材料周围,大量新骨形成,新骨表面可见活跃的成骨细胞,部分区域有成熟骨组织形成,并有新生的毛细血管形成.结论:Bio-Oss骨胶原有较强的诱导成骨能力;Bio-Oss骨胶原为载体的自体骨髓基质细胞移植能有效修复骨缺损,是骨组织工程良好的支架材料;兔骨髓基质细胞体外表达人bFGF基因,并且经基因修饰的组织工程骨修复骨缺损效果最佳.     

骨髓基质细胞-Bio-oss修复颅骨缺损的实验研究

目的：分析和评价体外培养扩增的骨髓基质细胞（BMSCs）/Bio-oss修复大鼠颅骨极量骨缺损的效果.方法：取同基因大鼠的BMSCs,经体外分离培养扩增,与Bio-oss复合后充填颅骨极量骨缺损.在不损伤硬脑膜的情况下,磨除全层颅骨,造成动物标准缺损,用含或不含BMSCs的Bio-oss充填颅骨缺损.不充填物缺损作为空白对照.术后12周处死动物,取缺损区及周围骨质进行肉眼、X线片及组织学观察.结果：所有植入物无排除或感染.无论X线摄片及组织切片均表明,BMSCs/Bio-oss骨修复能力明显优于的空白组及平片用Bio-oss组.结论：BMSCs-Bio-oss复合物充填相对于单纯材料充填具有明显的骨缺损修复能力,是治疗各种骨缺损较有效的方法.     

应用牙周膜干细胞—牙囊干细胞复合细胞膜片同种异体移植修复比格犬牙周组织缺损的研究

目的 拟构建比格犬牙周缺损模型,观察牙周膜干细胞—牙囊干细胞复合细胞膜片修复牙周组织缺损的效果.方法 分离培养比格犬牙囊干细胞、牙周膜干细胞,制备牙囊干细胞细胞膜片、牙周膜干细胞细胞膜片以及以上2种细胞的复合细胞膜片,将膜片混合支架材料同种异体植入成年雄性健康比格犬牙周缺损模型,对照组只植入支架材料.分别于移植后1周、2周、4周、6周、8周、12周检测探诊深度、牙龈退缩、附着丧失情况.12周后观察分析牙周再生的组织形态学变化.结果 牙周临床检查及组织学形态学分析显示,3个膜片移植组牙周再生效果较对照组效果明显,其中复合细胞膜片移植组明显高于其余两个单一膜片移植组(P<0.05).结论 牙周膜干细胞—牙囊干细胞复合膜片同种异体移植更有利于修复比格犬牙周缺损.     

载辛伐他汀纳米羟基磷灰石支架材料对兔下颌骨缺损修复的影响

目的:采用骨髓间充质干细胞(BMSCs)复合辛伐他汀支架材料修复兔下颌骨局部缺损,观察辛伐他汀对BMSCs复合支架材料修复兔骨缺损的影响。方法:24只新西兰大白兔随机分为2组,每组12只,制备下颌骨缺损模型。A组为实验组,在缺损区植入复合BMSCs的载辛伐他汀纳米羟基磷灰石支架材料;B组为对照组,植入BMSCs复合羟基磷灰石材料。分别于术后2、4、8、12周处死各组动物,行影像学分析、HE染色、扫描电镜观察等检查。结果:影像学检查及组织学染色结果显示,A组骨缺损处愈合程度、成骨速度及骨质量明显优于B组。扫描电镜显示,A组复合材料与组织相容性好,材料吸收优于B组。统计各组各期牙CT分析数据的骨密度值,结果表明A组的骨密度值明显高于B组(P<0.05)。结论:实验表明经BMSCs复合的辛伐他汀支架材料具有明显的促进成骨能力,可加速骨缺损的修复效果,提高修复质量。     

纳米羟基磷灰石/聚酰胺66复合人工骨修复颅骨缺损的动物实验研究

目的研究纳米羟基磷灰石/聚酰胺66复合生物活性人工骨作为骨缺损修复材料的可行性。方法将人工骨材料的预制件植入人工形成的兔颅骨临界缺损区域内,通过大体标本观察、X线及锝(99mTc)亚甲基二磷酸盐核素骨显像检查评价该材料的骨缺损修复能力,并动态评价修复后受植区内的骨代谢变化。结果纳米羟基磷灰石/聚酰胺生物活性人工骨具有良好的生物相容性,对颅骨及其颅内代谢无影响,可在兔颅骨临界缺损区域内引导骨再生。结论纳米羟基磷灰石/聚酰胺生物活性人工骨材料具备骨缺损修复材料所需的良好生物学特性。     

Ⅰ型胶原凝胶/BMSCs复合体修复颅骨骨缺损的实验研究

目的　观察骨髓基质细胞 (BMSCs) /Ⅰ型胶原凝胶复合体修复颅骨骨缺损的情况。方法　 2 0只兔颅骨上分别制作一直径 1.5cm圆形全层骨缺损。其中 8只抽取骨髓行骨髓基质细胞培养 ,细胞与Ⅰ型胶原凝胶复合制成复合体 ,修复颅骨缺损为 1组 ;8只单纯以I型胶原凝胶修复为 2组 ;4只不修复 ,为空白对照 (3组 )。术后 8周处死全部兔 ,观察骨修复面积 ;组织学观察骨修复情况 ;测量修复骨灰重与湿重及比例。结果　骨缺损部被新生骨及纤维结缔组织所覆盖 ,1组骨修复面积多于 2组 ,2组多于 3组。组织学观察见缺损边缘有新骨形成 ,但 1组中间部分亦有成骨 ,新骨生成均明显多于 2、3组。修复骨灰重与湿重比显示 1组骨量及钙化高于 2组、3组。结论　Ⅰ型胶原凝胶可作为骨髓基质细胞的细胞外基质 ,复合骨髓基质细胞修复骨缺损。     

犬骨髓间充质干细胞复合磷酸钙骨水泥修复骨缺损的实验研究

目的:评价骨髓间充质干细胞复合磷酸钙骨水泥支架材料修复下颌骨缺损的效果。方法:分离培养犬骨髓间充质干细胞(BMSCs)。成骨诱导培养14 d后,分别采用茜素红染色与碱性磷酸酶染色,观察其诱导效果。将细胞与磷酸钙骨水泥(CPC)支架材料复合,用于动物实验。在4只Beagle犬的下颌骨每侧制作3处大小一定的骨缺损。随机将骨缺损分为3组进行处理:BMSCs-CPC组(移植复合种子细胞的支架材料)、CPC组(只移植支架材料)和空白组(不做任何处理)。分别于移植后第4、8周处死2只犬,行大体、X线、骨缺损修复区组织形态观察与计量分析。采用SPSS 13.0软件包对数据进行统计学分析。结果:术后各组均有不同程度的骨再生。BMSCs-CPC组、CPC组中骨组织再生情况优于空白组。BMSCs-CPC组支架材料降解程度与新骨形成情况优于CPC组。术后第4、8周,BMSCs-CPC、CPC组新生骨面积百分比均显著高于空白对照组(P<0.05),BMSCs-CPC组新生骨面积百分比显著高于CPC组(P<0.01);BMSCs-CPC组中剩余支架材料面积百分比显著低于CPC组(P<0.01)。结论:BMSCs种子细胞复合CPC支架材料是一种有效的、促进新骨再生的骨缺损修复方法,有利于颌骨高度及宽度的保存。     

人骨髓间充质干细胞/细胞外基质复合体用于骨缺损再生修复的研究

目的:评估人骨髓间充质干细胞/细胞外基质复合体(C-BMSC)的骨缺损再生修复能力。方法:体外构建C-BMSC,通过大体观察、HE染色、扫描电镜进行组织形态学检测;TUNEL染色法检测细胞凋亡水平;免疫荧光染色和PCR法检测成骨相关因子的表达水平;大鼠颅骨缺损模型评估C-BMSC体内骨缺损再生修复能力。结果:组织形态学显示5 d组C-BMSC的细胞外基质(ECM)最丰富,ECM将细胞完全包裹,ECM纤维粗壮,小分子物质附着;TUNEL染色结果提示3 d与5 d组细胞凋亡数量接近,而7 d时细胞凋亡显著增加(P<0.001);PCR结果表明C-BMSC对ALP、COL-1、Runx2和OCN表达均高于细胞膜片(P<0.05);免疫荧光结果显示C-BMSC中ECM对ALP、COL-1、BSP表达均呈阳性;大鼠颅骨移植实验结果表明C-BMSC组骨体积与软组织体积比显著高于空白组。结论:本研究证明C-BMSC具有良好的成骨分化能力,这为骨缺损再生修复提供了一种新的策略。     

天然煅烧骨修复材料（骼瑞）修复动物骨缺损的有效性研究

目的:观察煅烧牛骨(骼瑞)修复动物骨缺损的有效性。方法:分别建立犬牙槽骨缺损修复模型、SD大鼠和新西兰白兔颅骨极限缺损模型,对照组骨缺损不植入修复材料,实验组骨缺损中植入骼瑞,每个组取材6只动物。术后用HE染色、Micro CT、Masson三色染色等方法观察缺损修复效果。结果:犬牙槽骨缺损修复8周后牙槽骨缺损修复良好。兔的颅骨修复实验中,实验组术后6周,煅烧骨颗粒周围有大量新骨形成。术后12周,骨缺损区新骨部分连接成片,已改建成板层骨,煅烧骨颗粒大多被新生骨包围。术后24周,新生骨成熟度进一步提高。SD大鼠颅骨缺损修复实验,术后8周,实验组骨缺损区域愈合,且填充修复区域的骨密度几乎接近于正常骨的骨密度。3种动物实验中,实验组骨缺损修复均优于对照组。结论:骼瑞材料对于骨缺损修复具有显著的促进作用。     

Coculture of peripheral blood CD34+ cell and mesenchymal stem cell sheets increase the formation of bone in calvarial critical-size defects in rabbits

The reconstruction of large bony defects remains a clinical challenge, and angiogenesis and neovascularisation are being given more attention in bone tissue engineering. In this study we cocultured peripheral blood CD34+ cells (PB-CD34+ cells), an endothelial progenitor cell/haematopoietic stem cell-enriched population, with bone marrow-derived mesenchymal stem cells (MSC) to investigate their potential for bony regeneration. Cocultured cells showed better osteogenic differentiation than MSC alone in vitro. The cocultured cells and MSC sheets were also composited with hydroxyapatite and implanted in calvarial critical-size defects in rabbits. The rabbits were killed before microcomputed tomographic (MicroCT) and histological analysis. The results showed that cocultured cell composites had promoted bony regeneration more efficiently by 8 weeks after implantation. Our results indicate that the coculture of PB-CD34+ cells and MSC increases bony regeneration in calvarial critical-size defects in rabbits, and provide a new promising therapeutic strategy to aid skeletal healing.     

纳米羟基磷灰石复合胶原材料负载骨髓基质干细胞修复颅骨极限缺损的实验研究

目的:探讨纳米羟基磷灰石复合胶原材料(nHAC)负载骨髓基质干细胞作为组织工程骨在修复颅骨极限缺损中的作用。方法:20只新西兰大白兔随机分为复合细胞组、单纯材料组、空白对照组。复合细胞组取自体骨髓基质干细胞并扩增诱导培养后通过负压抽吸及细胞基质膜包裹将细胞与材料复合。材料植入颅骨缺损后8周取材,行大体、X线、组织学观察及灰量测量来研究它们的骨修复能力。结果:复合细胞组成骨能力最强,且材料边缘及内部均有较好的成骨,单纯材料组有部分成骨,空白对照组未修复骨缺损。结论:纳米羟基磷灰石复合胶原材料负载骨髓基质干细胞后是一种较为理想的骨替代材料。     

大鼠骨髓间充质干细胞与脂肪间充质干细胞生物学特性的比较

目的:本研究旨在比较同一个体来源的脂肪间充质干细胞(Adipose tissue-derived mesenchymal stem cells,ADSCs)和骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)的生长特点和诱导分化能力。方法:取7～10 d SD大鼠的脂肪和骨髓,体外分离、纯化脂肪和骨髓来源间充质干细胞,分别从细胞形态、生长动力学、表面标志、分化潜能等方面进行鉴定和比较。结果:脂肪间充质干细胞的增殖速度明显大于骨髓间充质干细胞(P<0.05),两种细胞所表达的表面蛋白标志物基本相似,但脂肪间充质干细胞表面蛋白标志CD106呈阴性,而骨髓间充质干细胞CD106呈阳性。在成软骨分化中脂肪间充质干细胞弱表达Ⅱ型胶原,而骨髓间充质干细胞不表达。第5、10、15、20代ADSCs及BMSCs经成骨诱导后茜素红染色均呈阳性且有"矿化"结节形成,碱性磷酸酶活性以第5代最强,随着细胞代次的递增,碱性磷酸酶活性呈递减趋势。结论:ADSCs较BMSCs更易于分离培养及体外扩增,诱导条件下成骨、成脂、成软骨能力较强,适合作为再生医学的种子细胞。     

Use of a mineralized collagen membrane to enhance repair of calvarial defects in rats

The aim of the present study was to evaluate mineralized collagen membranes for enhancement of bone regeneration in calvarial defects. Forty adult female Sprague-Dawley rats received calvarial full thickness defects with a diameter of 8 mm. In 20 animals, the defects were covered with a mineralized collagen membrane, and 20 animals served as controls. After 6, 13, 26 and 52 weeks, bone regeneration was evaluated using undecalcified thick-section histometry. There was no clear enhancement of bone regeneration during the first 26 weeks after the operation. Bone regeneration underneath the membrane produced consistently thicker bone, albeit without statistical significance. Accumulation of membrane material occurred in the center of the defects surrounded by multinuclear giant cells during early stages of healing. After complete resorption of the membrane, significantly increased bone formation was seen after 52 weeks in the defects that had received membrane coverage. It was concluded that mineralization in the present form did not increase mechanical strength of the membrane to prevent interference of the membrane with bone regeneration in the defect. The reason for the increase in bone formation after resorption of the membrane after 26 weeks remains to be clarified.     

骨髓间充质干细胞复合多孔纳米羟磷灰石/聚酰胺6整复大鼠颅骨缺损的实验研究

目的初步探讨纳米羟磷灰石/聚酰胺6（n-HA/PA6）多孔材料对体外培养的大鼠骨髓间充质干细胞（BMSCs）增殖及分化的影响,以及BMSCs作为种子细胞、多孔n-HA/PA6作为支架材料构建组织工程化骨修复大鼠颅骨极限骨缺损的可行性及整复效果。方法矿化诱导的第3代BMSCs与n-HA/PA6多孔材料复合培养,MTT检测细胞增殖,ALP染色检测骨向分化。将BMSCs与n-HA/PA6复合物植入大鼠颅骨8 mm骨缺损处,4、8、16周时,应用组织学、扫描电镜观察植入物与骨组织交界处的成骨修复情况,并与单纯n-HA/PA6植入组修复效果进行比较。结果与n-HA/PA6复合培养的BMSCs生长良好,细胞增殖未受影响,ALP染色阳性。BMSCs复合n-HA/PA6植入4周时,有较多新骨长入支架孔穴;8周时,材料和宿主骨融为一体,接近正常骨;16周时,材料和天然骨形成骨性结合。单纯n-HA/PA6组植入4周时,新骨形成较少;8周时,新骨明显增加,但骨钙化程度较低;16周时,2组无明显差异。结论多孔n-HA/PA6支架材料对种子细胞BMSCs的增殖和骨向分化无影响;BMSCs作为种子细胞、n-HA/PA6多孔复合体作为支架材料构建组织工程化骨能够加速界面骨愈合,有效修复颅骨缺损,具有潜在的骨组织工程应用前景。     

Osseous response to implanted natural bone mineral and synthetic hydroxylapatite ceramic in the repair of experimental skull bone defects.

The purpose of this study was to assess and compare the osseous responses to implanted particles of resorbable anorganic xenograft bone mineral and non-resorbable dense synthetic hydroxylapatite of two different granule sizes. Four trephine calvarial defects were produced in each of 13 adult rabbits. The experimental materials were subsequently implanted in three defects, leaving the fourth defect for control purposes. Six animals were killed 4 weeks after surgery and seven at 14 weeks. The tissue responses were assessed by contact radiography, light microscopy, and histometry. The biocompatibility of the implants was confirmed. All defects healed uneventfully, although the resorbable hydroxylapatite seemed to promote initial bone regeneration. The importance to orthognathic surgery of early and effective healing of bone gaps, as well as of the advantage of implant resorbability to bone remodeling, are discussed.     

Effect of platelet-derived growth factor-BB on bone formation in calvarial defects: an experimental study in rabbits

The effect of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) on bone healing was examined in calvarial defects in rabbits. Bicortical circular (critical size) defects were prepared in the calvarial bone of 16 rabbits. The defects were closed on the dural side and covered externally with expanded polytetrafluoroethylene membranes to prevent interference with osteogenesis within the defect by the surrounding tissue and to keep the growth factor in place. A single dose of methylcellulose gel (4.4%) with ( n = 8) or without rhPDGF-BB (50/jg/ml) ( n = 8) was applied to the defects, and the bone formation was evaluated after 8 weeks. Healing of detects in both groups was characterized by the presence of newly formed bone along the edges of the original defect and by a central area of fibrous connective tissue. The newly formed bone in the rhPDGF-BB treated defects had a trabecular structure; in contrast, a more compact structure was found in the control defects. In the rhPDGF-BB-treated defects, the bone ingrowth was 51.8 ± 7.1% compared to 30.5 ± 3.3% in the control defects. Furthermore, the amount of mineralized tissue was increased 112% in the rhPDGF-BB group. The amount of bone marrow was increased 75% in the rhPDGF-BB-treated defect. The porosity of cortical lamella in the newly formed bone was 84% higher in the rhPDGF-BB-trealed defects compared to the control. These results show that administration of a single dose of rhPDGF-BB stimulates bone formation in critical size calvarial defects.     

PRF复合自体骨髓间充质干细胞修复兔牙槽骨缺损的研究

目的:观察富血小板纤维蛋白(platelet-rich fibrin, PRF)复合自体第3代骨髓间充质干细胞(bone marrow stem cells, BMSCs)修复牙槽骨缺损的能力。方法:取健康雄性2月龄新西兰兔36只,随机分为4组:PRF+BMSCs组、PRF组、BMSCs组、空白对照组,均在全麻下微创拔除下颌左侧中切牙。4组实验动物分别在4、8、12周处死。取其下颌骨进行牙槽骨标本测量,X线观察,组织学观察。结果:牙槽骨标本测量比较:4组牙槽嵴宽度、高度丧失值均存在差异(P＜0.05);析因分析示PRF与BMSCs复合使用修复牙槽骨的效果优于单独使用PRF或BMSCs(P<0.05)。X线、组织学观察表明术后4、8、12周时拔牙窝骨吸收程度:PRF+BMSCs组＜PRF组＜BMSCs组＜空白对照组,PRF+BMSCs组成骨情况均优于PRF组、BMSCs组、空白对照组。结论:PRF复合自体BMSCs可以促进牙槽骨的再生与修复,具有潜在的临床应用价值。     

载辛伐他汀PLGA/CPC支架材料复合BMSCs修复大鼠颅骨缺损的实验研究

目的:探讨载辛伐他汀PLGA/CPC支架材料复合骨髓间充质干细胞(BMSCs)构建组织工程骨,修复大鼠颅骨临界尺寸骨缺损的可行性以及效果。方法:24只雄性大鼠随机均分为3组;在大鼠颅骨人字缝两侧各做一直径5 mm的骨缺损。每组16个骨缺损再随机分为:载辛伐他汀PLGA/CPC/BMSCs(Brdu标记的BMSCs)组(n=4);载辛伐他汀PLGA/CPC组(n=4);单纯PLGA/CPC组(n=4)以及空白对照组(n=4)。术后4、8、12周取标本分别进行大体观察,HE染色及免疫组织化学染色来评价骨再生情况。结果:术后4、8、12周组织学观察表明载辛伐他汀PLGA/CPC/BMSCs组的成骨质量和速度明显优于其他3组,免疫组化结果显示载辛伐他汀PLGA/CPC/BMSCs组骨钙素(OC)阳性表达IOD值明显高于其他3组。结论:载辛伐他汀PLGA/CPC支架材料复合BMSCs可以诱导大鼠颅骨临界尺寸骨缺损内的新骨形成,成骨质量优于其他3组,而且可以明显缩短骨愈合时间。     

Guided bone regeneration in pig calvarial bone defects using autologous mesenchymal stem/progenitor cells – A comparison of different tissue sources

Due to donor side morbidity and the absence of osteogenic properties in bone substitutes, there is a growing need for an alternative to traditional bone grafting within the scope of tissue engineering. This animal study was conducted to compare the in vivo osteogenic potential of adipose-derived (AD), periosteum-derived (PD) and bone marrow-derived (BM) mesenchymal stem/progenitor cells (MSC). Autologous mesenchymal stem/progenitor cells of named tissue origin were induced into osteogenic differentiation following in vitro cell expansion. Ex vivo cultivated cells were seeded on a collagen scaffold and subsequently added to freshly created monocortical calvarial bone defects in 21 domestic pigs. Pure collagen scaffold served as a control defect. The animals were sacrificed at specific time points and de novo bone formation was quantitatively analyzed by histomorphometry. Bone volume/total defect volume (BV/TV) and the mineralization rate of newly formed bone were compared among the groups. In the early stages of wound healing, up to 30 days, the test defects did not show better bone regeneration than those in the control defect, but the bone healing process in the test defects was accelerated in the later stage compared to those in the control defect. All the test defects showed complete osseous healing after 90 days compared to those in the control defect. During the observation period, no significant differences in BV/TV and mineralization of newly formed bone among the test defects were observed. Irrespective of the tissue sources of MSC, the speed and pattern of osseous healing after cell transplantations into monocortical bone defects were comparable. Our results indicate that the efficiency of autologous AD-MSC, PD-MSC and BM-MSC transplantation following ex vivo cell expansion is not significantly different for the guided regeneration of bone defects.