骨髓间充质干细胞及其复合物对肌腱早期愈合影响的实验研究

[目的]探讨骨髓间充质干细胞(BMSCs)与纤维蛋白胶复合体对兔肌腱早期愈合的影响,为临床应用提供依据.[方法]家兔骨髓间充质干细胞离体培养、纯化、扩增后与纤维蛋白胶混合.36只家兔分为实验组和对照组,兔跟腱横断制作肌腱断裂模型.实验组应用自体BMSCs复合纤维蛋白胶移植于肌腱损伤区,对照组仅以纤维蛋白胶置于肌腱损伤区.分别于术后1周,3周,6周及12周对移植部位进行大体标本观察,细胞示踪、组织学检查、免疫组织化学检查、形态测定分析及生物力学测定.[结果]实验组相比对照组肌腱大体观察粘连差,肌腱活动性好.3周后纤维蛋白载体即降解,细胞示踪结果显示标记的骨髓间充质干细胞至少6周内仍可保持活性并存在于肌腱组织中,但之后逐渐扩散.3周时,实验组与对照组相比胶原纤维排列更为有序,且胞核形态结构更规则,但在6周及12周时,实验组与对照组胞核参数测定无统计学意义.3周时实验组比对照组具有更强的的生物力学特性,但之后则差别不明显.[结论]肌腱损伤后即给以腱内BMSCs复合纤维蛋白胶治疗可促进肌腱愈合早期组织形态学及生物力学修复.     

骨髓间充质干细胞促进骨-肌腱结合部位早期愈合的动物生物力学实验研究

[目的]通过生物力学研究,观察骨髓间充质干细胞对骨-肌腱结合部愈合的影响.[方法]采用骨髓穿刺、全骨髓培养法获取兔骨髓间充质干细胞.24只18周龄新西兰大白兔随机分为2组,实验组将骨髓间充质干细胞与Pluronic F-127载体材料结合后,植入兔髌骨部分切除模型,对照组只进行手术,不植入细胞.在术后6、12、18周处死动物取标本(n=4)进行生物力学检测评估骨-肌腱结合部位的愈合恢复情况.[结果]生物力学结果显示相同时间点实验组拉断负荷及极限拉应力均大于对照组,差异有统计学意义(P<0.05),提示实验组恢复较对照组迅速.[结论]相同时间点实验组拉断负荷、极限拉应力均大于对照组.实验组的力学特性明显高于对照组.骨髓间充质干细胞可以促进骨-肌腱结合部细胞早期愈合,提高其力学特性.     

骨髓间充质干细胞促进骨-肌腱结合部愈合的初步观察

[目的]观察骨髓间充质干细胞对骨-肌腱结合部愈合的影响.[方法]采用骨髓穿刺、全骨髓培养法获取兔骨髓间充质干细胞.36只18周龄新西兰大白兔随机分为2组,实验组将骨髓间充质干细胞与Pluronic F-127载体材料结合后,植入兔髌骨部分切除模型,对照组只进行手术,不植入细胞.在术后6、12、18周进行X线片、大体和组织学观察评估.[结果]X线片显示术后6、12、18周实验组骨-肌腱结合部新生骨面积显著大于对照组（P＜0.01）.大体观察可见实验组骨-腱结合部愈合较早.HE染色显示实验组术后6周有大量的纤维母细胞和类软骨细胞增生,并可见新骨形成;12周,髌腱与松质骨接触面软骨细胞移行带形成;18周,纤维软骨移行带排列更有序.Safranin 0染色显示实验组术后6周,基质染色较深,部分骨-腱结合处可见相对浓染区;12周,基质染色范围明显减少,沿类软骨细胞走行分布;18周,类软骨细胞集中的区域染色较12周有所减退.组织学检查显示实验组术后6、12、18周骨-腱结合部组织愈合明显,较对照组迅速.[结论]骨髓间充质干细胞可以促进骨-肌腱结合部细胞增生,促进其早期愈合.     

新型MRI磁标记技术对兔骨髓基质干细胞自体皮下移植进行活体示踪的研究

目的 利用超顺磁性氧化铁(SPIO)粒子标记骨髓基质干细胞(BMSCs),探讨用0.2T MRI活体示踪自体皮下移植磁化标记BMSCs的分布和迁移的可行性. 方法 从兔骨髓中分离培养BMSCs,实验组经体外采用SPIO和BrdU双重标记后,与壳聚糖复合植入兔自体大腿皮下.设立自体皮下移植未标记BMSCs组和皮下单纯注射SPIO组为对照组,每组6只.于术后1、5 d及2、4、8周连续行MRI梯度回波T2加权(GRE T2* WI)扫描序列对磁标记细胞成像示踪,并行组织学检查. 结果 磁标记细胞普鲁士蓝染色和电镜检查显示细胞胞浆内含致密铁颗粒.磁标记后自体皮下移植的兔BMSCs在GRE T2* WI序列成像时产生特征性的低信号改变至少维持8周,并观察到从移植部位发出低信号线进入宿主组织.组织学检查显示宿主组织中发现普鲁士蓝阳性细胞和BrdU阳性细胞.结论 体外SPIO能有效地标记BMSCs,利用0.2T MRI活体示踪体自体皮下移植的磁标记兔BMSCs分布和迁移是可行的.     

骨形态发生蛋白2基因修饰自体骨髓间充质干细胞移植促进兔胫骨牵张成骨的实验研究

[目的]观察人骨形态发生蛋白2基因修饰的自体骨髓间充质干细胞移植对兔胫骨牵张成骨新骨形成的促进作用。[方法]48只新西兰白兔随机摸球法分为3组。建立牵张成骨动物模型,在固定期第2 d,实验组于牵张间隙注射人骨形态发生蛋白2基因修饰的自体骨髓间充质干细胞;对照组注射等量自体骨髓间充质干细胞;空白组不注射任何物质。[结果]在固定期2周及6周实验组牵张区在大体观察、HE染色、X线观察方面成骨质量均好于对照组和空白组。12周后取牵张成骨区标本作骨组织骨密度和生物力学测定,结果显示实验组新生骨质量较高,骨愈合情况要优于对照组和空白组。[结论]骨形态发生蛋白2基因修饰的自体骨髓间充质干细胞移植能有效提高兔胫骨牵张成骨新骨形成质量。     

磷酸钙水泥对自体腘绳肌腱重建前交叉韧带后腱-骨愈合影响的生物力学研究

目的探讨骨形态发生蛋白结合型磷酸钙水泥(rhBMP-2-CPC)对自体腘绳肌腱重建前交叉韧带(ACL)后腱-骨愈合的影响。方法取54只成年新西兰大白兔,建立48只双膝关节ACL完全断裂的自身对照模型,24只用rhBMP-2-CPC充填移植腱-骨隧道界面(实验组),24只未用rhBMP-2-CPC(对照组),6只为生物力学试验的正常组。术后第2、4、8和12周取材进行生物力学检测。结果2、4、8和12周实验组肌腱移植物较对照组抗拉伸的最大载荷明显要大,差异有统计学意义(P<0.05)。结论实验组肌腱移植物的生物力学特性优于对照组;BMP-CPC能促进肌腱移植物在骨隧道内的早期腱-骨愈合。     

间充质干细胞体内成骨的实验研究

[目的]探讨间充质干细胞在体内的成骨能力。[方法]从羊髂嵴处抽取10～15ml骨髓组织，用Pereoll分离液按梯度离心法分离出间充质干细胞，培养、增殖后种植在多孔β-磷酸三钙上，构建组织工程化骨，植入8只羊的左后肢跖骨缺损区（长21mm）作为实验组；单纯植入多孔β-磷酸三钙陶瓷材料的8只羊作为对照组；分别在术后6、12、24周处死动物行放射学、组织学和生物力学检测。[结果]放射学和组织学检测，术后6周实验组即可见有新骨生成，对照组则无明显新骨生成；骨缺损部位新生骨样组织、编织骨和板状骨出现的时间实验组也都较对照组早，并且不经软骨介导即能直接成骨，而对照组从两端以“爬行替代”方式成骨。术后24周，放射学和生物力学检测显示实验组骨缺损几乎完全修复，对照组只有部分愈合。[结论]间充质干细胞不仅在体外具有良好的增殖能力，回植人体内后仍然具有良好的成骨能力，不需经过软骨过程就能直接形成新骨，显示了间充质干细胞作为骨组织工程种子细胞良好的应用前景。     

转血管内皮细胞生长因子基因干细胞结合珊瑚羟基磷灰石修复兔骨缺损的实验研究

目的探讨珊瑚羟基磷灰石（CHA）结合转血管内皮细胞生长因子（VEGF）基因的骨髓间充质干细胞构建的组织工程骨修复兔骨缺损的效果。方法 以CHA作为转VEGF基因骨髓间充质干细胞（BMSCs）的可吸附载体,体外构建组织工程骨,将其植入兔尺骨中段10mm骨缺损处,以CHA/BMSCs、自体骨移植组和空白组作为对照,在术后2、4、8、12周进行大体解剖、X线摄片、病理组织切片、生物力学测试,观察各组骨愈合程度及力学强度。结果 术后8周病理组织切片及X线摄片显示实验组骨缺损基本修复,效果优于对照组。生物力学测试显示术后12周组织工程骨组组抗扭转强度优于自体骨移植组。结论 CHA结合转VEGF基因BMSCs构建组织工程骨可用于骨缺损修复,优于自体骨移植组。     

SPIO标记骨髓间充质干细胞和软骨细胞共培养的研究

[目的]探讨SPIO标记骨髓间充质干细胞和软骨细胞共培养的可行性,为临床修复关节软骨损伤寻找新途径.[方法] 分离、扩增新西兰大白兔骨髓间充质干细胞(BMSCs)和软骨细胞,根据SPIO标记(50μg/ml)和不同培养环境(共培养、单独培养)分4组,每天在倒置显微镜观察共培养后BMSCs的形态变化,共培养14 d后,免疫组化检测Ⅱ型胶原表达情况,阿利新蓝法检测蛋白多糖(GAG)表达水平.[结果] 共培养7 d后标记的部分BMSCs变圆,14 d时BMSCs形态高度分化与成熟软骨细胞相似,其蛋白多糖和Ⅱ型胶原的基因转录和蛋白表达均增高,明显优于对照组,差异具有显著性意义(P<0.01).[结论]1.软骨细胞微环境能有效诱导BMSCs向软骨细胞分化;2.SPIO可以安全、有效地标记BMSCs.     

大鼠骨髓间充质干细胞复合n-HA/PLA支架异位成骨的实验研究

[目的]研究大鼠骨髓间充质干细胞(BMSCs)复合纳米羟基磷灰石/聚乳酸(n-HA/PLA)支架材料体内异位成骨情况.[方法]取SD大鼠股骨胫骨骨髓,进行贴壁培养BMSCs,将第3代BMSCs接种到n-HA/PLA支架材料上,加入成骨诱导液,3d后置人大鼠体内,设为实验组;将单纯支架材料置入组为对照组.4、8、12周时取材进行大体和组织学观察.[结果]8、12周时实验组可见类骨质成分形成,并且支架材料维持置入时形状,对照组无类骨质形成.[结论]BMSCs复合n-HA/PLA构建组织工程骨有很好的异位成骨能力,且能在体内维持良好塑型.     

跖骨感染骨外露的显微外科治疗

[目的]回顾总结跖骨骨感染骨外露的显微外科治疗方法。[方法]自1995年～2005年采用显微外科技术治疗214例跖骨骨外露骨感染患者。[结果]全部病例获得随访1～10年，平均随访3年，14例游离植皮术后皮肤成活良好，199例术后皮瓣全部成活，1例腓肠神经营养血管皮瓣移位修复术出现远端部分皮肤坏死，后经换药处理后，伤口自然愈合。皮瓣移植术后质地良好，无溃疡复发。患足均可负重走路。[结论]应用显微外科技术治疗跖骨骨感染骨外露可获得较好的疗效。     

Assessment of bone density and bone loss

Osteoporosis International - Dual-energy X-ray absorptiometry has become the standard for the evaluation of osteoporosis. It is useful both for identifying those people who are going to be at risk...     

Spinal fusions: bone and bone substitutes

Vertebral arthrodesis is one of the most commonly performed, yet incompletely understood, procedures in spinal surgery. Despite major progress in internal fixation techniques, the high rate of non-unions indicates that physiologic, biologic and molecular events that are crucial to this process are not well known. This article will analyze the general biology of bone regeneration, and particularly discuss the properties and use of various bone graft materials and graft substitutes. Received: 9 August 2000/Accepted: 11 August 2000     

Trabecular bone remodeling and bone balance in hyperthyroidism

In vivo tetracycline double-labeled iliac crest bone biopsies from 15 hyperthyroid patients were used for the reconstruction of curves describing the variation of resorption depth and formation thickness with time. The curves emerging were compared to curves reconstructed from 13 age- and sex-matched normal individuals (mean age 44 years). The median function period for resorptive cells in hyperthyroid patients (16 days) was about one-third the resorptive period in normals (51 days). No significant difference between the osteoclast-, mononuclear-, or preosteoblast-like cell resorption depths could be demonstrated between the two groups. Consequently, the median resorption rate in hyperthyroid patients (3.8 μm/day) was more than 3 times higher than the value in the control group (1.1 μm/day). Median Sigma_f, was shorter in the hyperthyroid group (109 days) than in the control group (151 days, P < 0.05), as was the median initial mineralization lag time (5 and 16 days, respectively, P < 0.01). No significant difference between the measured mean completed wall thickness (mcwT) values in the hyperthyroid groups and the control group could be demonstrated (58.1 and 60.5 μm respectively). Median initial mineralization rate in the hyperthyroid group (1.2 μm³/μm² per day) was not significantly higher than the value calculated in the control group (0.9 μm³/ μm² per day), but median initial matrix appositional rate in hyperthyroids (4.8 μm³/μm² per day) was 3 times higher than the value calculated for normals (1.6 μm³/μm² per day) (P < 0.01). Direct measurements of mean completed wall thickness in the hyperthyroid group gave results (58.1 μm) that were not in accordance with the mean completed wall thickness calculated from the growth curve (52.1 μm, P < 0.02). In normals no such discrepancy could be demonstrated. Using the mcwT value estimated from the growth curve, the bone formation period was calculated to 90 days for hyperthyroid patients. This maximal estimate for mcwT was also significantly lower than the mean resorption depth measured in the hyperthyroid group (61.7 μm, P < 0.05), which means that a net negative balance per remodeling cycle existed in the hyperthyroid group. Bone balance was preserved in the control group.     

骨胶原与骨质疏松骨密度及骨生物力学的相关性研究

目的探讨骨胶原含量在绝经后骨质疏松症的发生、发展及在骨质疏松性骨折中的作用。方法取7个月龄未交配雌性SD大鼠60只，随机分为四组，A组：对照组（sham组）；B组：切除卵巢组；C组：切除卵巢＋雌激素治疗组；D组：切除卵巢＋降钙素治疗组。除A组外，其他三组通过切除双侧卵巢法12周后制成骨质疏松模型，24周后分别行k的力学特性、右侧股骨三点弯曲试验、羟脯氨酸含量、k骨密度（BMD）测定，Masson三色染色法显示骨胶原形态。结果A、C、D组与B组在k羟脯氨酸含量、BMD、k压缩力学参数值、右侧股骨生物力学参数值、骨胶原染色含量及形态方面差异均有统计学意义（P〈0．05），而A、C、D组之间差异无统计学意义（P〉0．05）。统计学分析显示羟脯氨酸含量与BMD及骨生物力学参数值呈直线相关性。结论骨质疏松的发生与骨胶原含量下降有关。骨胶原含量的下降与BMD降低及骨生物力学改变呈相关性。应用雌激素和降钙素治疗去势后骨质疏松大鼠，不仅可以提高其BMD含量和骨生物力学性能，而且还可以提高骨胶原的含量。     

Repair of long bone defects with demineralized bone matrix and autogenous bone composite

Background:

Repair of diaphyseal bone defects is a challenging problem for orthopedic surgeons. In large bone defects the quantity of harvested autogenous bone may not be sufficient to fill the gap and then the use of synthetic or allogenic grafts along with autogenous bone becomes mandatory to achieve compact filling. Finding the optimal graft mixture for treatment of large diaphyseal defects is an important goal in contemporary orthopedics and this was the main focus of this study. The aim of this study is to investigate the efficacy of demineralized bone matrix (DBM) and autogenous cancellous bone (ACB) graft composite in a rabbit bilateral ulna segmental defect model.

Materials and Methods:

Twenty-seven adult female rabbits were divided into five groups. A two-centimeter piece of long bone on the midshaft of the ulna was osteotomized and removed from the rabbits’ forearms. In group 1 (n=7) the defects were treated with ACB, in group 2 (n=7) with DBM, and in group 3 (n=7) with ACB and DBM in the ratio of 1:1. Groups 4 and 5, with three rabbits in each group, were the negative and positive controls, respectively. Twelve weeks after implantation the rabbits were sacrificed and union was evaluated with radiograph (Faxitron), dual-energy x-ray absorptiometry (DEXA), and histological methods (decalcified sectioning).

Results:

Union rates and the volume of new bone in the different groups were as follows: group 1 - 92.8% union and 78.6% new bone; group 2 - 72.2% union and 63.6% new bone; and group 3 - 100% union and 100% new bone. DEXA results (bone mineral density [BMD]) were as follows: group 1 - 0.164 g/cm², group 2 - 0.138 g/cm², and group 3 - 0.194 g/cm².

Conclusions:

DBM serves as a graft extender or enhancer for autogenous graft and decreases the need of autogenous bone graft in the treatment of bone defects. In this study, the DBM and ACB composite facilitated the healing process. The union rate was better with the combination than with the use of any one of these grafts alone.