成人骨髓源成骨细胞体内异位成骨的实验研究

目的　观察成人骨髓源成骨细胞与珊瑚羟基磷灰石 (CHA)复合构建的组织工程化骨组织的体内异位成骨能力 ,探讨适宜的组织工程化骨组织的构建方式。　方法　抽取健康成人骨髓 ,采用全骨髓法培养 ,使成人骨髓基质干细胞 (hBMSCs)定向诱导分化为成骨细胞 ,然后种植于CHA上 ,复合培养 5d后植入裸鼠股部肌袋内 ,以未种植细胞的CHA作为对照。术后 4,8,12周取材作一般观察、X线摄片、组织学检查和源于成人的碱性磷酸酶 (ALP)及骨钙素 (OCN)的RT PCR检测。　结果　原代和传代培养的细胞具有活跃的增殖能力 ,成骨细胞与CHA复合生长良好。实验组术后 4、8、12周均有新骨形成 ,随着时间延长 ,新骨生成量增多 ;对照组则均无新骨形成。术后 4周实验组RT PCR检测源于人的ALP及OCN表达均为阳性 ,对照组阴性。　结论　成人骨髓源成骨细胞与CHA复合培养后构建的组织工程化骨组织 ,具有良好的异位成骨能力 ,可望应用于临床修复骨缺损。     

陶瓷样异种骨复合骨髓异位成骨的实验研究

目的了解陶瓷样异种骨（ＣＸＢ）与骨髓（ＢＭ）复合移植的成骨作用。方法将ＣＸＢ与ＢＭ复合及单纯ＣＸＢ植入兔的骶棘肌内,术后２、４、８、１２、１６及２４周取出植入材料作组织学及组织化学检查,观察植入材料的成骨作用。结果ＣＸＢ与ＢＭ复合植入后２周开始有软骨和新骨生成,以后软骨逐渐钙化成骨,８周时出现髓腔,并随植入时间的延长,新骨生成增多,成骨更为典型。而单纯ＣＸＢ植入后无新骨或软骨形成。结论ＣＸＢ与ＢＭ复合移植通过骨传导、骨诱导及提供成骨细胞或成软骨细胞而成骨,是一种理想的骨移植替代材料,可望在临床上用于骨缺损的修复。     

人骨髓基质细胞接种珊瑚构建组织工程骨

目的：观察体外培养的人骨髓基质细胞与珊瑚复合物植入体内后的成骨能力。方法：穿刺抽吸入髂骨区骨髓基质细胞，体外培养扩增、诱导，将其与珊瑚复合后植入裸鼠体内，以单纯珊瑚作为对照。术后4、8周取材，通过大体、组织学、扫描电镜观察植入物体内成骨情况。结果：术后4周，复合物中有少量新骨形成；术后8周，复合物中出现大量成熟骨组织。而对照组无骨组织形成。结论：人骨髓基质细胞复合珊瑚在无免疫动物体内具有成骨能力，穿刺抽吸的人骨髓基质细胞可作为骨组织工程的种子细胞。     

自体脂肪干细胞复合珊瑚修复犬颅骨标准缺损的初步研究

目的 应用自体脂肪干细胞(adipose-derived stem cells,ADSCs)复合珊瑚构建组织工程化骨,修复犬颅骨标准缺损.方法 体外扩增培养、成骨诱导Beagle犬ADSCs,将第2代细胞接种在珊瑚支架上共同培养.制造实验犬双侧颅骨全层标准缺损(20 mm×20 mm),一侧以细胞材料复合物修复作为实验组(n=7),另一侧以单纯珊瑚材料修复作为对照组(n=7).术后24周分别通过影像学、大体形态观察、生物力学检测、组织学方法检测颅骨缺损的修复效果.结果 成骨诱导的犬ADSCs体外呈现成骨特性,在珊瑚支架上生长良好.3D-CT重建显示术后12周实验组有新生骨痂形成,对照组材料大部分降解;24周时实验组为骨性愈合,对照组为骨不连.24周时实验组缺损修复百分比为(84.19±6.45)%,显著高于对照组的(25.04 ±18.82)%(P<0.01).大体观察见实验组由新生骨痂修复缺损,对照组缺损边缘可见少量骨痂形成,主要为软组织充填;24周生物力学检测修复组织能耐受的最大压力载荷,实验组为(73.45±17.26)N,为犬顶骨最大压力负荷(104.27±22.71)N的70%,两者比较差异有统计学意义(P<0.01),对照组为软组织无法完成上述检测.HE染色见实验组有较多成熟骨呈骨性愈合,对照组为纤维性愈合.结论 自体成骨诱导的ADSCs复合珊瑚形成的组织工程化骨可修复犬颅骨标准缺损.     

构建组织工程骨修复兔颅骨极限缺损的实验研究

目的观察以胶原缓释重组人骨形成蛋白2(recom b inan t hum an bone m orphogenetic prote in 2,rhBM P-2)复合骨髓间充质干细胞(m arrow m esenchym a l stem ce lls,M SC s)及珊瑚构建的组织工程骨修复兔颅骨极限缺损的能力。方法新西兰大白兔40只,制备颅骨极限缺损,按植入的修复物不同随机分为5组,每组8只。Ⅰ组:自体髂骨,为阳性对照组;Ⅱ组:珊瑚,为阴性对照组;Ⅲ组:rhBM P-2+珊瑚;Ⅳ组:胶原+rhBM P-2+珊瑚;Ⅴ组:M SC s+胶原+rhBM P-2+珊瑚。将其分别植入兔颅骨极限缺损处,术后8、16周行大体观察、X线片、HE染色及M asson三色染色法观察比较骨缺损修复的情况。结果术后Ⅴ组材料与Ⅰ组修复颅骨极限缺损的效果相近,缺损区大体标本可见骨样组织充填,硬度与周边骨质相近,并与周边骨质形成明显骨融合;X线阻射程度高,16周时达80.45%±2.52%;组织学观察为板层状结构的新骨组织,空白孔隙区较少。Ⅳ组修复效果次之,Ⅲ组材料成骨能力较弱,Ⅱ组大部为半透明的纤维薄膜,缺损区界限清晰。结论胶原是rhBM P-2适宜的缓释载体,胶原及M SC s对促进复合支架材料修复骨缺损有重要意义。以M SC s+胶原+rhBM P-2+珊瑚构建的组织工程骨可成为一种良好的骨缺损修复材料。     

以藻酸钙为载体的可注射组织工程骨行隆鼻术的实验研究

目的：研究可注射组织工程骨做为隆鼻术植入材料的可行性。方法：从兔髂骨中获取骨髓基质成骨细胞，将骨髓基质成骨细胞与25g/L藻酸钠溶胶混合形成藻酸钠/骨髓基质成骨细胞复合物，取其2mL与0.17g硫酸钙粉末混合均匀，注射于新西兰兔鼻背部骨膜下，观察成骨情况。结果：藻酸钙/骨髓基质成骨细胞复合物植入兔鼻背部皮下4周后有类软骨样组织形成，8周时有骨小梁、骨髓腔等骨组织结构。结论：以藻酸钙为载体的可注射性组织工程骨可用于隆鼻术植入材料。     

组织工程方法修复羊腭裂骨缺损的初步研究

目的对羊骨髓基质干细胞（Bone marrow stromal cells，BMSCs）复合珊瑚修复腭裂骨缺损的可行性进行初步探讨。方法体外培养扩增、成骨诱导羊BMSCs。将第3代细胞复合珊瑚修复羊完全性腭裂骨缺损，以单纯珊瑚植入缺损作为对照组。术后16周头颅CT扫描、大体观察、评价骨缺损的修复效果。结果三维CT显示，实验组可见珊瑚被新生骨替代，对照组可见珊瑚明显降解，裂隙仍然存在。大体观察显示实验组骨缺损基本愈合；对照组中珊瑚明显降解，裂隙仍然存在。结论初步证明羊BMSCs成骨诱导后与珊瑚复合能修复羊腭裂骨缺损。     

自体骨髓基质干细胞复合珊瑚修复犬下颌骨节段缺损的初步研究

目的应用自体骨髓基质干细胞(bonemarrowstromalcells,BMSCs)复合珊瑚构建组织工程化骨,修复犬下颌骨节段性缺损。方法体外扩增培养、成骨诱导犬BMSCs。将第二代细胞复合珊瑚后修复犬自体右侧3cm的下颌骨节段缺损(n=6);以单纯珊瑚植入缺损处为对照(n=6),术后12、32周分别通过影像学,大体形态观察,组织学和生物力学的方法检测骨缺损的修复效果。结果成骨诱导的BMSCs在珊瑚支架上生长良好。X线片显示12周时实验组骨痂较多,对照组材料明显吸收;32周时CT、X线片和大体观察显示术后实验组骨愈合良好,对照组为骨不连;骨密度检测示实验组显著高于对照组(P<0.05);组织学示实验组有较多成熟骨呈骨性愈合,对照组为纤维性愈合;生物力学测试实验组与正常下颌骨力学强度差异无统计学意义(P>0.05)。结论自体成骨诱导BMSCs复合珊瑚形成的组织工程化骨可修复犬下颌骨节段缺损。     

Bone tissue engineering in a critical size defect compared to ectopic implantations in the goat.

Since the application of the autologous bone graft, the need for an alternative has been recognized. Tissue engineering (TE) of bone by combining bone marrow stromal cells (BMSCs) with a porous scaffold, is considered a promising technique. In this study we investigated the potential of tissue engineered bone to heal a critical sized defect in the goat. Orthotopic bone formation was compared to ectopic bone formation in comparable constructs. TE constructs were prepared from goat BMSCs and porous biphasic calcium phosphate ceramic scaffolds. These constructs and scaffolds without cells were implanted paired in critical sized iliac wing defects. Comparable samples were implanted intramuscularly. After 9 (n=7) and 12 (n=8) weeks implantation, the samples were analyzed histomorphometrically. After 9-weeks implantation in the iliac wing defect, significantly more bone apposition was found in the TE condition. After 12 weeks, the defects were almost completely filled with bone, but no significant advantage of TE was determined anymore. This contrasted with the intramuscular samples where TE implants showed significantly more bone at both time points. In conclusion, bone TE is feasible in critical sized defects. However, when appropriate osteoconductive/inductive materials are applied the effect of cell seeding may be temporary.     

Qualitative and quantitative analysis of orthotopic bone regeneration by marrow

A rat model of a femoral segmental defect was used to specifically test the hypothesis that autogenous marrow has the osteogenic capability to heal a bone defect. The variables analyzed included the ratio of the marrow volume to the defect, implantation of live or dead marrow, and remodeling of established nonunions by implantation of live marrow. The uniqueness of this model allows biomechanical evaluation of the new bone formed by the implant. When live marrow was implanted, woven bone formed at 3 weeks, progressing to early lamellar bone at 6 weeks, with subsequent remodeling for as long as 12 weeks in a volumetric fashion (p < 0.05). Bone marrow, when placed in a fresh femoral defect and given in sufficient amounts, produced a rate of union comparable with that of autologous bone grafts. Mature lamellar bone formed by marrow was evaluated biomechanically: the results were statistically comparable with those of cancellous bone grafts at 12 weeks. Significant bone formation occurred when marrow was percutaneously injected in femoral nonunions, although union and remodeling did not take place in this rat model. Implantation of dead marrow resulted in rare cellular infiltration and minimal bone formation in a manner comparable with that of autogenous cancellous bone grafts. These results indicate that bone marrow can lead to structurally functional bone regeneration in an orthotopic location.     

骨髓基质成骨细胞-松质骨基质复合人工骨修复颅骨缺损的实验研究

目的 观察松质骨基质－骨髓基质成骨细胞复合工人骨在骨缺损区的成骨作用，探索该组织工程化人工骨修复颅骨缺损的可行性。方法 成年新西兰兔骨髓细胞体外培养、诱导后，接种于藻酸盐－松质骨基质中，形成松质骨基质－藻酸盐－骨髓基质成骨细胞复合人工骨，修复自体颅骨缺损。分别植入松质骨基质和骨髓基质成骨细胞作为对照。植入4周和8周后行X线摄片和组织学检查，观察骨形成情况。结果 复合人工成骨量优于单纯植入松质骨基持或骨髓基质成骨细胞组，并明显优于空白对照。结论 松质骨基质－骨髓基质成骨细胞复合人工骨髓修复颅骨缺损效果良好，可以作为临床大型骨缺损修复的途径之一。     

Autologous osteoblasts enhance osseointegration of porous titanium implants.

The goal of this study was to assess the osseointegration of porous titanium implants by means of coating with autologous osteoblasts. Titanium implants (8 x 5 x 4 mm) having drill channels with diameters of 400, 500, and 600 microm were coated with autologous osteoblasts obtained from spongiosa chips. The implants were inserted into the distal femora of 17 adult Chinchilla Bastard rabbits (group I). Uncoated implants were inserted as controls in the contralateral femur (group II). The animals were sacrificed after 5, 11, and 42 days. Intravital fluorochrome labeling and microradiography were used for the assessment of bone ingrowth into the titanium channels. In both groups, no bone tissue was formed in the channels up to day 5. On day 11, group I exhibited significantly more (p<0.05) bone tissue (19.8+/-14.0% vs. 5.8+/-9.1%) with greater bone-implant contact (13.3+/-15.1% vs. 5.7+/-5.3%, p<0.05) at the channel mouths than group II. Bone tissue was formed mainly between day 15 and 30 in group I, in group II between day 25 and 40. Six weeks after implantation, bone tissue filled on an average 68.8+/-15.1% of the mouths of the drill channels in implants in group I, the filling for group II was 49.8+/-18.1% (p<0.05). The average bone-implant contact at the channel mouths after six weeks was 56.5+/-13.5% in group I, 40.2+/-21.9% in group II (p<0.05). 600-microm channels showed at this time point the best osseous integration (p<0.05). Coating with autologous osteoblasts accelerates and enhances the osseointegration of titanium implants and could be a successful biotechnology for future clinical applications.     

种植体联合骨组织工程技术修复犬下颌骨节段缺损的实验研究

目的 观察应用种植体联合骨组织工程技术,修复犬下颌骨节段缺损的效果。方法 体外扩增培养、成骨诱导犬BMSCs。将第2代细胞复合珊瑚后修复犬自体右侧下颌骨3 cm的节段缺损,术后32周植入种植体(实验组n=3);同时,以邻近正常骨植入种植体作为对照(n=3)。植入4周、12周、26周后,分别通过影像学、大体形态观察、组织学和生物力学等方法,检测骨缺损的修复效果。结果 植入后26周,X线片和CT均显示种植体与实验组及对照组骨质为良好骨性愈合,实验组种植体周围新生骨密度较高。Micro-CT显示,实验组骨密度和对照组间无显著性差别(P>0.05)。大体观察见种植体与组织工程骨和正常骨均形成紧密连接。组织学显示,实验组与对照组均有较多成熟骨结构。生物力学测试结果表明,实验组与正常下颌骨力学强度无显著性差异(P>0.05)。结论 自体成骨诱导BMSCs复合珊瑚形成的组织工程化骨,可较好地修复犬下颌骨节段缺损,植入种植体后可进一步促进骨成熟。     

预构骨肌皮瓣修复下颌骨和皮肤复合缺损的实验研究

目的 探讨BMP-2与胶原复合材料在骨骼肌中异位成骨预构骨肌皮瓣,修复自体下颌骨和皮肤复合缺损的可行性. 方法 4～6周龄新西兰白兔24只,体重2.0～2.5 kg,雌雄不拘,随机分成3组,即实验组、对照组和空白组(n=8).将BMP-2与胶原复合,植入兔背阔肌,预构异位新生骨组织,2、4、6周后行X线片、ALP、Von Kossa、CD31免疫组织化学血管标记及组织学观察.6周后,实验组动物于同侧下颌骨体部制备2 cm × 3 cm皮肤缺损,并形成直径8 mm洞穿性骨缺损;以同侧胸背动脉体表投影为皮瓣纵轴,设计含预制新骨组织的背阔肌肌皮瓣,带蒂移位修复下颌骨及皮肤复合缺损.对照组和空白组实验动物下颌骨体部均造成直径8 mm的洞穿性骨缺损,对照组将复合组织瓣中的骨性部分游离移植修复缺损;空白组缺损不予修复.术后6周,各组取材行四环素荧光染色、X线摄片、组织学观察以及新骨计量等,观察骨和皮肤复合缺损修复效果. 结果 BMP-2与胶原复合材料在兔背阔肌中4～6周成骨,胶原材料于植入后3～5周降解,成骨过程以软骨成骨为主,新骨形态为编织骨,可见明显的微血管分布.修复自体下颌骨缺损6周后,实验组皮肤及骨缺损均愈合良好,对照组缺损修复区基本骨化,空白组尚残留大块骨缺损实验组、对照组及空白组新骨计量分别为(1.594 ±0.674)、(0.801 ±0.036)和(0.079±0.010)mm2,组间两两比较,差异均有统计学意义(P＜0.05). 结论 预构的骨肌皮瓣带血管蒂移位修复兔自体下颌骨和皮肤复合缺损具有可行性和明显优势,有望成为一种血管化骨移植的供区预构形式.     

Enhanced healing of goat femur‐defect using BMP7 gene‐modified BMSCs and load‐bearing tissue‐engineered bone

Segmental defect regeneration is still a clinical challenge. In this study, we investigated the feasibility of bone marrow stromal cells (BMSCs) infected with adenoviral vector containing the bone morphogenetic protein 7 gene (AdBMP7) and load‐bearing to enhance bone regeneration in a critically sized femoral defect in the goat model. The defects were implanted with AdBMP7‐infected BMSCs/coral (BMP7 group) or noninfected BMSCs/coral (control group), respectively, stabilized with an internal fixation rod and interlocking nails. Bridging of the segmental defects was evaluated by radiographs monthly, and confirmed by biomechanical tests. Much callus was found in the BMP7 group, and nails were taken off after 3 months of implantation, indicating that regenerated bone in the defect can be remodeled by load‐bearing, whereas after 6 months in control group. After load‐bearing, it is about 5 months; the mechanical property of newly formed bone in the BMP7 group was restored, but 8 months in control group. Our data suggested that the BMP7 gene‐modified BMSCs and load‐bearing can promote bone regeneration in segmental defects. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:412–418, 2010     

以自体诱导和无诱导的骨髓基质干细胞复合珊瑚修复犬下颌骨节段缺损的比较研究

目的分别采用诱导和无诱导的自体骨髓基质干细胞（Bone marrow stromal cells，BMSCs）复合珊瑚构建组织工程化骨，修复犬下颌骨节段性缺损，比较修复效果。方法体外扩增、成骨诱导或无诱导培养犬BMSCs，分别将第2代细胞复合珊瑚后修复犬自体右侧3cm的下颌骨节段缺损（诱导组n=6，无诱导组n=6）。术后32周，分别通过Micro-CT、大体形态观察和组织学方法检测骨缺损的修复效果。结果32周时，Micro-CT检测示诱导组骨容积率和密度均显著高于对照组（P〈0.05）；大体观察示诱导组骨愈合良好，无诱导组中的3条犬为骨不连；组织学检测诱导组有较多成熟骨形成，缺损部分均呈骨性愈合。无诱导组中的3只犬有新骨形成，但形态不完整，另3只犬的缺损部分呈纤维性愈合。结论成骨诱导的自体BMSCs复合珊瑚形成的组织工程化骨修复犬下颌骨节段缺损效果优于无诱导组。     

Mandibular reconstruction using intraoral trifocal bone transport: report of a case.

On the mandibular reconstruction after tumor resection, it is easy to achieve esthetic and functional results when mandibular defect is relative small, however, it is difficult to reconstruct adequately larger defected mandible. Recently, with progress of devices, distraction osteogenesis that is the method of tissue regeneration is used as mandibular reconstruction. A 19-year-old male patient presented complaining of right lower jaw swelling. Biopsy suspected a multiple-cystic ameloblastoma in mandible. Under the general anesthesia, a mandibulectomy was performed from the right side ramus to the left side incisor. A mandibular reconstruction plate was attached to the proximal and distal bone segments. 2 types of intraoral distraction devices were placed inside the plate. These devices had 25 mm and 60 mm distraction length. After 9 days of latency, trifocal bone transport was started by 0.5 mm 2 times activation per day. After consolidation for 23 weeks, reconstruction plate and distraction devices were removed. 2.5 m x 2.0 cm iliac bone and cancellous bone were placed in the docking site with platelet rich plasma. The mandibular defect (85 mm) was reconstructed adequately using intraoral distraction osteogenesis trifocal bone transport technique. Symmetric facial balance was achieved. Now there is no recurrence and dental implants were placed on new bone.     

Repair of mandible defect with tissue engineering bone in rabbits

BACKGROUND: The aim of the present study was to investigate the effect of tissue engineering bone composed of bone marrow-derived osteoblasts and demineralized bone in repairing mandible defect. METHODS: Bone marrow-derived osteoblasts of 20 rabbits were cultured and seeded into scaffold of allogeneic demineralized bone to construct tissue engineering bone graft in vitro, which was used to repair the 10 x 5-mm bone defect made in the same rabbit mandible edge. Implant of demineralized bone alone was as the control. Rabbits were killed according to the schedule: five after 2 weeks, five after 4 weeks, five after 8 weeks, five after 12 weeks, and the implants were harvested for gross, radiographic, and histological observation. RESULTS: New bone formation at the margin region of defect and osteogenesis at the centre were observed in the implant of tissue engineering bone, and the bone formation pattern included osteogenesis, osteoconduction, and osteoinduction. In the implant of demineralized bone alone, the major bone formation pattern was 'creeping substitute'. CONCLUSIONS: The tissue engineering bone graft constructed by autogenous bone marrow-derived osteoblasts and allogeneic demineralized bone was better than demineralized bone alone in bone formation capability, which might be an ideal graft for bone defect repair.     

The study of the feasibility of segmental bone defect repair with tissue- engineered bone membrane: a qualitative observation

The objective of the study was to investigate the feasibility of intramembranous osteogenesis from tissue-engineered bone membrane in vivo. Bone marrow mesenchymal stem cells (MSCs) of rabbits were harvested, expanded and some of them were induced into osteoblasts. Porcine small intestinal submucosa (SIS) was converted by a series of physical and chemical procedures into a scaffold. MSCs and induced osteoblasts were seeded separately onto the scaffold, thus fabricating two kinds of tissue-engineered bone membrane. A total of 12 New Zealand rabbits were subjected to a surgical operation; a 15 mm bone segment, including the periosteum, was resected from the radius on both sides of each rabbit to create critical bone defects. The two kinds of tissue-engineered bone membrane and SIS (as control) were implanted randomly into the site of bone defect. The animals had radiographs and were killed after 4 weeks. The specimens were harvested and histological examination performed for evidence of osteogenesis. Bone tissue had formed in defects treated by the two kinds of tissue-engineered bone membrane at 4 weeks. This was supported by the X-ray and histological examination, which confirmed the segmental gap bridged by bone. There was no attempt to bridge in the bone defect treated by SIS. Tissue-engineered bone membrane, constructed by seeding allogeneic cells on an xenogeneic and bio-derived scaffold, can repair critical bone defects successfully.     

In vivo osteogenic durability of cultured bone in porous ceramics: a novel method for autogenous bone graft substitution

BACKGROUND: Bone marrow cells differentiate into bone-forming osteoblasts when cultured in medium supplemented with 15% fetal bovine serum, ascorbic acid, beta-glycerophosphate, and dexamethasone. METHODS: To investigate in vivo osteoblastic activity and bone matrix formation by cultured bone marrow cells, Fischer rat marrow cells were cultured for 2 weeks in porous hydroxyapatite (HA) and then subcutaneously implanted into 7-week-old male syngeneic rats. The implants were harvested after 8 and 52 weeks for biochemical and histological analyses. RESULTS: At both times, formation of lamellar bone accompanied by regeneration of marrow were seen in many of the HA pores. When a fluorochrome (calcein) was administered at 50 weeks after implantation, it was detected in the pores of implants harvested at 52 weeks. Osteoclastic resorption followed by new bone formation was seen in some pores at 52 weeks, indicating that bone remodeling was continuing. The alkaline phosphatase activity of implants harvested at 52 weeks was comparable to that at 8 weeks, whereas the osteocalcin content of the implants harvested at 52 weeks was about twice that at 8 weeks. CONCLUSION: These results demonstrated that there was persistent in vivo osteogenic and hematopoietic activity in the prefabricated bone/HA constructs, and indicated that normal bone tissue was regenerated after grafting of the constructs, which were brittle before implantation. Tissue engineering using HA and cultured marrow cells culture may provide an alternative method of bone transplantation for patients with skeletal disorders, although further in vivo and in vitro experiments are needed.