骨形态发生蛋白7局部基因治疗和组织工程化骨对大鼠脊柱融合的影响

目的 评价腺病毒介导的人骨形态发生蛋白7 (Ad-hBMP-7)基因转染的骨髓间充质干细胞(BMSCs)和纳米羟基磷灰石/胶原复合材料(NHAC)复合后对脊柱融合的影响. 方法 抽取大鼠骨髓获得BMSCs,应用Ad-hBMP-7转染大鼠BMSCs,将NHAC材料和密度为2×105/ml的Ad-BMP-7转染大鼠的BMSCs细胞孵育2h后备用;Wistar大鼠56只,随机分成4组,建立脊柱后外侧融合模型,分别植入自体髂骨(A组)、NHAC材料(B组)、NHAC材料和BMSCs(C组)以及Ad-hBMP-7-BMSCs与NHAC材料混合物(D组).每组行RT-PCR 及Western blotting检测,8周和12周后分别行X线摄片,并对标本进行手触检测硬度、生物力学检测和组织学观察. 结果 D组10只大鼠的脊柱L4/5节段全部融合;A组12周0例融合;B组12周0例融合;C组12周3例融合.D组同A、B、C组结果 相比均有显著性差异( P <0.001). 结论 BMP-7局部基因治疗和组织工程化骨能促进脊柱融合.     

表面矿化修饰煅烧骨/BMP2活性多肽复合MC3T3-E1细胞构建组织工程骨

探讨表面矿化修饰煅烧骨/BMP₂活性多肽复合材料、表面矿化修饰煅烧骨和单纯煅烧骨复合MC3T3-E1细胞构建的组织工程骨在体内的成骨能力。实验分3组,A组：表面矿化修饰煅烧骨/BMP₂活性多肽复合材料,B组：表面矿化修饰煅烧骨,C组：单纯煅烧骨。诱导培养MC3T3-E1细胞,将其分别接种于3组材料,倒置相差显微镜和环境扫描电镜分别观察细胞生长情况。将18只新西兰大白兔随机分成3组,每只大白兔作两侧骶棘肌肌袋模型,然后将3组材料分别植入大白兔骶棘肌内,分别于术后第4、8周各组处死大白兔3只,行组织学观察及新生骨面积测定。通过观察发现MC3T3-E1细胞在3组材料表面生长良好,表面矿化修饰煅烧骨/BMP₂活性多肽复合材料能促进MC3T3-E1细胞在其表面粘附与增殖并能较好地保持细胞的形态。术后第4、8周组织学观察A组材料新生骨明显多于B组,B组多于C组。术后4周和8周新生骨面积测定值A、B、C组分别为（19 712.524±3 782.126）μm²、（28 227.617±2 455.375）μm²,（11 648.507±1 047.221）μm²、（14 592.892±899.532）μm²,（7 986.655±903.487）μm²,（11 254.822±669.508）μm²。表面矿化修饰煅烧骨/BMP₂活性多肽复合材料是一种较理想的骨组织工程复合材料,有望应用于临床。     

不同融合方式对多节段腰椎椎管狭窄合并腰椎间盘突出症患者应激反应的影响#br#

目的 探讨多节段椎管减压配合不同融合方式对多节段腰椎椎管狭窄（LSS）合并腰椎间盘突出症（LDH）患者植骨融合情况及应激反应的影响。 方法 选取2017年2月～2019年2月我院多节段LSS合并LDH患者85例,按照治疗方案不同分为A组（n=43）、B组（n=42）。A组采取多节段椎管减压配合横突间植骨融合内固定术,B组采取多节段椎管减压配合椎间植骨融合术。比较两组手术有关指标情况、术前、术后1 d、3 d血清应激反应有关指标［血管紧张素Ⅱ（AngⅡ）、肾素（R）、超敏C反应蛋白（hs-CRP）、白细胞介素-6（IL-6）］水平、并发症、疗效、植骨融合情况、术前、术后1周、12个月、24个月腰腿痛视觉模拟评分量表（VAS）评分、日本骨科学会（JOA）评分、功能障碍指数量表（ODI）评分。 结果 A组手术时间、住院时间短于B组,术中出血量与住院费用低于B组（P<0.05）;两组术后1 d、3 d血清AngⅡ、R、hs-CRP、IL-6水平均较本组术前提高（P<0.05）,A组术后1 d、3 d上述血清指标水平均低于B组（P<0.05）;两组并发症发生率相比,差异无统计学意义（P>0.05）;两组手术优良率、植骨融合率相比,差异无统计学意义（P>0.05）;两组术后1周、术后3个月、12个月、24个月腰腿痛VAS评分、ODI评分均较本组术前降低,JOA评分较本组术前提高（P<0.05）,但组间对比,差异无统计学意义（P>0.05）。 结论 多节段椎管减压配合横突间植骨融合内固定术或椎间植骨融合术均是治疗多节段LSS合并LDH值得肯定的术式,临床疗效满意,而横突间植骨融合内固定术具有创伤小、出血少、恢复快、住院费用低的优势。     

一种兔腰椎间融合模型的建立及效果评价

目的　利用兔腰椎骨性结构测量数据建立一种稳定的腰椎椎体间植骨融合模型,为组织工程学研究提供标准化的模型参考。 方法　选用健康新西兰大白兔（2~2.5 kg）44只,随机分为A、B、C、D四组, A组（n=10）用于解剖进行腰椎椎体测量,B组（n=12）行L4/5椎体间椎骨融合术加内固定;C组（n=12只）行L4/5椎体间植骨融合术未进行内固定,D组（n=10）行单纯显露加横突破坏。B、C、D 3组造模后4周行X线检查,术后12周取标本进行大体观察、Micro-CT、生物力学及组织学切片检查。 结果　B组仅出现1例因固定螺钉进入椎管导致脊髓损伤造成双下肢瘫痪。手触法检测B组融合率为100%（12/12）,C组融合率为75%(9/12),D组标本未见椎间盘损伤及间隙破坏。影像学B组植入骨块位置明显优于C组,融合评分明显高于C组（P<0.05）。组织学提示B、C两组椎体间融合部位均可见新生软骨,C组中可见植骨块脱出后形成软组织空腔。生物力学显示B组最大载荷显著高于C组（P<0.05）。 结论　在兔腰椎解剖基础上进行兔腰椎间植骨融合模型制作是安全、可靠的。内固定装置可以为椎间植骨块提供稳定的融合空间,防止植骨块脱离融合位置,有助于提高该模型的融合效率。     

表面修饰羟基磷灰石修复骨缺损骨形态发生蛋白-2的表达

目的了解精氨酸-甘氨酸-天冬氨酸多肽表面修饰的羟基磷灰石（hydroxyapatite,HA）修复节段性骨缺损局部骨形态发生蛋白-2（bone morphogenefic protein-2，BMP-）的表达。方法以骨髓基质干细胞（marrow stromal cels，MSCs）复合Arg-Gly-Asp（RGD）多肽表面修饰的HA或单纯材料培养制备组织工程骨，选择60只新西兰白兔。制作15mm长的桡骨节段性骨缺损模型，根据植入不同的材料分为A、B、C、D组。A组：骨缺损区植入MSCs复合RGD多肽表面修饰的HA培养制备的组织工程骨；B组：骨缺损区植入MSCs复合HA培养制备的组织工程骨；C组：骨缺损区植入RGD多肽表面修饰的HA；D组：骨缺损区植入HA。术后4周取材，行修复区局部BMP-2免疫组化分析。结果术后4周各组骨缺损区均有新骨生成，修复区局部BMP-2表达水平依次为：A〉B〉C〉D（P〈0．05）。结论RGD多肽表面修饰对以HA为支架材料组织工程骨的修复作用有明显优化作用。     

骨形态发生蛋白2基因与重组蛋白缓释方法修复骨缺损的比较研究

为比较骨形态发生蛋白2(BMP-2)基因与重组蛋白缓释方法修复节段性骨缺损的效果,于兔双侧桡骨中段造成1.5cm骨缺损,采用四种方法修复:A组植入转基因骨髓基质干细胞(MSCs)与PLA/PCL(聚乳酸/聚己内酯)支架的复合物;B组植入单纯MSCs与含重组BMP-2的PLA/PCL缓释载体的复合物;C组植入单纯MSCs与PLA/PCL复合物;D组植入单纯PLA/PCL。术后4、8、12周行X线、组织学、生物力学和骨密度等检测。结果表明,A组体内植入4周后,成骨细胞和间质细胞呈BMP-2强阳性表达;其成骨速度及成骨质量均明显优于B组,12周时骨缺损完全修复。C组成骨能力较弱,而D组则无新骨形成,残留骨缺损。BMP-2基因治疗是修复节段性骨缺损的好方法。     

不同骨移植材料修复骨缺损的初步实验研究

目的对比观察不同骨移植材料对兔桡骨节段性骨缺损的修复效果。方法48只新西兰大白兔采用桡骨15mm节段性骨缺损模型,随机分为4组,每组12只,A组植入深冻异体骨复合自体骨髓,B组植入深冻异体骨,C组植入自体骨,D组植入新鲜同种异体骨。术后不同阶段分别行组织学、透射电镜及X线检测。结果四组骨缺损均有成骨现象发生,骨生成、骨连接情况A、C组优于B组,B组优于D组;A、C组细胞增生活跃、核呈分裂相、胞质丰富、核膜光整、细胞器丰富,同比均优于B、D组;骨缺损愈合时间A、C组为8-10周,B组为12周,D组骨缺损在术后12周仍未愈合。结论提示兔深冻异体骨复合自体骨髓具有良好的组织相容性及成骨作用,其取代自体骨植骨修复骨缺损具有可能性。     

自体颗粒植骨与髂骨块植骨治疗单节段胸腰椎结核的疗效对比

目的　回顾性观察自体颗粒植骨和髂骨块植骨在后路单节段胸腰椎结核手术中的临床疗效差异。 方法　2012年8月至2016年6月行后路病灶清除、椎体间植骨融合内固定术治疗的58例单节段胸腰椎结核患者,按植骨方式不同分为两组,颗粒组31例,髂骨组27例。比较两组手术时间、术中出血量,手术前后的VAS、ODI评分、神经功能恢复,在影像学资料上记录两组节段后凸Cobb角和椎间高度的改善及末次随访时的丢失、植骨融合时间。 结果　术后平均随访26(15~38)月,颗粒组手术时间(194.2±34.9) min、术中出血量(282.2±130.0) ml、植骨融合时间(5.8±1.0)月均优于髂骨组（240.0±61.5 min、540.7±276.6 ml、8.3±3.6月）（P<0.05）。两组术后、末次随访时节段后凸Cobb角和椎间高度均较术前明显改善（P<0.05）,末次随访时颗粒组节段后凸Cobb角和椎间高度丢失稍大于髂骨块组,但两组间无明显统计学差异（P>0.05）。 结论　和髂骨植骨相比,颗粒骨植骨方便易行,术中出血少,植骨融合快,应用于单节段胸腰椎结核手术中,是一种安全、有效的植骨方式。     

后路长、短节段固定结合有限减压治疗退变性腰椎侧凸的比较研究

目的　比较两种后路减压、矫形术式治疗退变性腰椎侧凸症的临床和影像学结果。 方法 回顾两种后路减压、内固定术式治疗46例退变性腰椎侧凸的临床和影像资料,A组：选择性责任节段有限减压、短节段固定（2.7±0.5）26例;B组：责任节段减压、长节段固定（5.8±1.7）20例。应用JOA、VAS评价腰腿痛和活动功能,用Cobb法测量腰椎侧凸角、前凸角;比较两组临床和影像学结果,手术时间、出血量和手术并发症。 结果 平均随访2.8年（2～6.5年）,手术时间和出血量B组显著大于A组（Pt<0.001, Pb<0.001）;术前侧凸角分别为(22±4.6)°(A组)和(28±7.3)°(B组),术后随访末期分别为(11±3.8)°(A组)和(12±5.9)°(B组),B组改善率显著优于A组(P<0.001);术前腰椎前凸角分别为(19±4.6)°(A组)和(18±7.3)°　(B组),术后随访末期分别为(29±3.8)°(A组)和(32±5.9)°(B组),两组矫正率无显著差别（P=0.17）。术前、随访末期JOA评分为14±3.6、23±4.7(A组)和13±3.8、21±5.9（B组）,分别较术前显著改善（PA<0.001, 　PB<0.001）,改善率两组无显著差别(P=0.24);术前、随访末期VAS评分为8±0.16、3±0.07(A组)和8±1.8、4±　2.7（B组）,分别较术前显著改善（PA<0.001, PB<0.001）,两组改善率无显著差别(P=0.22);并发症发生率B组显著大于A组（P<0.001）。 结论　长节段固定侧凸矫正率高于短节段固定,但两组矢状面矫正和临床症状改善率相似,长节段固定手术并发症多、术后腰椎活动功能差,应该谨慎选择。     

脊髓型颈椎病动物模型的初步建立

目的　建立脊髓型颈椎病动物模型。方法　 36只短毛豚鼠按处理方法不同随机分成 3组 ,A组 :在C4～C5或C5～C6 椎间盘后缘植入骨形态发生蛋白 (BMP)与聚乙烯吡咯烷酮 (PVP)复合制剂 ;B组 :在椎间盘后缘植入PVP ,C组系假手术组。分别于手术后 4、8、1 2周以斜板试验观察实验动物的运动功能 ,并按计划处死动物 ,对实验节段颈椎进行组织学观察。结果　A组术后 8周起出现运动功能减退 ;B组和C组动物术后各期运动功能无改变。组织学观察 ,A组术后 4周起出现椎体后缘骨质增生 ,占据椎管 ,并出现不同程度的脊髓受压变形和脱髓鞘、神经元脱失等改变 ;B、C组术后各期椎管形态、脊髓等无改变。结论　植入颈椎间盘后缘的BMP可诱导椎间盘及韧带组织骨化、椎体后缘骨质增生 ,并对颈脊髓造成恒定的慢性压迫 ,为开展脊髓型颈椎病的实验研究提供了重要的动物模型。     

Evaluation of autologous bone marrow mesenchymal stem cell-calcium phosphate ceramic composite for lumbar fusion in rhesus monkey interbody fusion model

Autologous bone marrow mesenchymal stem cell (BMSC)-calcium phosphate ceramic composites were constructed in vitro and implanted as a bone graft substitute for lumbar anterior interbody fusion in rhesus monkeys to determine the osteogenic capacity of the composites. Nine adult rhesus monkeys underwent lumbar L3-L4 and L5-L6 diskectomy and interbody fusion via an anterior retroperitoneal approach. Two fusion sites in each animal were randomly assigned to two of three treatments: autogenous tricortical iliac crest bone graft (autograft group), cell-free ceramic graft (ceramic group), or BMSC-ceramic composite graft (BMSC group). Autologous BMSCs were expanded in culture and stimulated with osteogenic supplement. The spinal fusion segments were evaluated by radiography, biomechanical testing, histologic analysis, and histomorphometric analysis 3 months postsurgery. The BMSC group achieved lumbar interbody fusion superior to that of the ceramic group, both biomechanically and histologically. The BMSC group and the autograft group showed equivalent biomechanical stiffness. Ceramic residues were significantly greater in the ceramic group versus the BMSC group. The results indicate that BMSC-ceramic composites can enhance bone regeneration and achieve osseous spinal fusion 3 months after implantation in the rhesus monkey interbody fusion model.     

Autogenous bone marrow stromal cell sheets-loaded mPCL/TCP scaffolds induced osteogenesis in a porcine model of spinal interbody fusion

This study was designed to investigate whether a tissue-engineered construct composed of autogenous cell sheets and a polycaprolactone-based bioresorbable scaffold would enhance bone regeneration and spinal interbody fusion in a large animal model. Porcine-derived autogenous bone marrow stromal cells (BMSCs) cultured into multilayered cell sheets were induced into osteogenic differentiation with dexamethasone, l-ascorbic acid, and β-glycerol phosphate. These cell sheets were assembled with bioresorbable scaffolds made from medical-grade poly(epsilon-caprolactone) incorporating 20% β-tricalcium phosphate (mPCL/TCP) as tissue-engineered BMSC constructs. L2/3, L4/5 discectomies and decortication of the vertebral end plates were performed on 16 SPF Yorkshire pigs through an anterolateral approach. The tissue-engineered BMSC constructs were transplanted into the prepared intervertebral disc spaces of half of the pigs (n?=?8), whereas cell-free mPCL/TCP served as controls in the remaining pigs. New bone formation and spinal fusion were evaluated at 3 and 6 months using microcomputed tomography, histology, fluorochrome bone labeling, and biomechanical testing. New bone formation was evident as early as 3 months in the BMSC group. At 6 months, bony fusion was observed in >60% (5/8) of segments in the BMSC group. None of the control animals with cell-free scaffold showed fusion at both time points. Biomechanical evaluation further revealed a significantly increased segmental stability in the BMSC group compared with the cell-free group at 6 months postimplantation (p?相似文献   

A prospective consecutive study of instrumented posterolateral lumbar fusion using synthetic hydroxyapatite (Bongros-HA) as a bone graft extender

A prospective, single institution, clinical case-matched, radiographic study was undertaken. Thirty-two patients underwent posterior lumbar interbody fusion with cages containing laminectomized bone chips and posterolateral lumbar fusion with pedicle screws. Autogenous bone graft (3 mL) plus 3 mL of hydroxyapatite was placed in one side of a posterolateral gutter, and 6 mL of autogenous iliac bone graft was placed on the other side. Bony union, volumes of fusion mass, and bone absorption rates were postoperatively evaluated using simple radiographs and 3D-CT scans. Average postoperative Lenke scores at 3 and 6 months in the hydroxyapatite group were statistically higher than in the autograft group, but at 12 months no difference was found between the hydroxyapatite and autograft groups in terms of fusion rate. Complete fusion rates by 3D-CT were 86.7% in the hydroxyapatite group and 88.9% in the autograft group, which are not significantly different. Volumes of fusion mass and bone absorption rates at 12 months were 2.35 mL in the hydroxyapatite group and 1.31 mL in the autograft group. The mean fusion mass volume was greater in the hydroxyapatite group than in the autograft group. Lumbar posterolateral fusion using a mixture of hydroxyapatite artificial bone and autogenous bone graft showed good bony union similar to that shown with autogenous bone only. This study suggests that hydroxyapatite bone chips could be used usefully as a bone-graft extender in short-segment posterolateral spinal fusion.     

Evaluation of an injectable silk fibroin enhanced calcium phosphate cement loaded with human recombinant bone morphogenetic protein-2 in ovine lumbar interbody fusion

The objective of this study was to investigate the efficacy of an injectable calcium phosphate cement/silk fibroin/human recombinant bone morphogenetic protein-2 composite (CPC/SF/rhBMP-2) in an ovine interbody fusion model. Twenty-four mature sheep underwent anterior lumbar interbody fusion at the levels of L1/2, L3/4, and L5/6 with random implantation of CPC/SF, CPC/rhBMP-2, CPC/SF/rhBMP-2, or autogenous iliac bone. After the sheep were sacrificed, the fusion segments were evaluated by manual palpation, CT scan, undestructive biomechanical testing, undecalcified histology, and histomorphology. The fusion rates of CPC/SF/rhBMP-2 were 55.56% and 77.78% at 6 and 12 months, respectively. The fusion was superior to all the biomaterial grafts in stiffness, and reached the same stiffness as the autograft at 12 months. The new bone formation was less than autograft at 6 months, but similar with that at 12 months. However, the ceramic residue volume of CPC/SF/rhBMP-2 was significantly decreased compared with CPC/SF and CPC/rhBMP-2 at both times. The results indicated that CPC/SF/rhBMP-2 composite had excellent osteoconduction and osteoinduction, and balanced degradation and osteogenesis.     

Biological performance of a polycaprolactone-based scaffold used as fusion cage device in a large animal model of spinal reconstructive surgery

A bioactive and bioresorbable scaffold fabricated from medical grade poly (epsilon-caprolactone) and incorporating 20% beta-tricalcium phosphate (mPCL–TCP) was recently developed for bone regeneration at load bearing sites. In the present study, we aimed to evaluate bone ingrowth into mPCL–TCP in a large animal model of lumbar interbody fusion. Six pigs underwent a 2-level (L3/4; L5/6) anterior lumbar interbody fusion (ALIF) implanted with mPCL–TCP + 0.6 mg rhBMP-2 as treatment group while four other pigs implanted with autogenous bone graft served as control. Computed tomographic scanning and histology revealed complete defect bridging in all (100%) specimen from the treatment group as early as 3 months. Histological evidence of continuing bone remodeling and maturation was observed at 6 months. In the control group, only partial bridging was observed at 3 months and only 50% of segments in this group showed complete defect bridging at 6 months. Furthermore, 25% of segments in the control group showed evidence of graft fracture, resorption and pseudoarthrosis. In contrast, no evidence of graft fractures, pseudoarthrosis or foreign body reaction was observed in the treatment group. These results reveal that mPCL–TCP scaffolds could act as bone graft substitutes by providing a suitable environment for bone regeneration in a dynamic load bearing setting such as in a porcine model of interbody spine fusion.     

Dual hydroxyapatite composite with porous and solid parts: experimental study using canine lumbar interbody fusion model

Hydroxyapatite (HA) has been evaluated for use in a variety of applications in bone reconstruction surgery because of its high affinity with host bone and biocompatibility. However, because of the difficulty in combining porosity (for bone ingrowth) and strength in HA, it is generally considered inappropriate to use HA for high-load applications such as spinal interbody fusion. In the present study, we constructed a HA implant for spinal interbody fusion, composed of a dual HA composite (DHC) that combines two HA materials with different porosities: HA with 75% porosity, for bone ingrowth; and HA with 0% porosity, for load bearing. We used a canine lumbar interbody fusion model to evaluate bone conduction of the implant and its efficacy for bony fusion. Six months after the operation, DHC exhibited almost the same efficacy for bony fusion as iliac bone grafts. Moreover, pores of the porous part of the DHC were completely filled with newly formed bone and bone marrow cells. The present findings indicate that DHC is suitable for use as an implant material for spinal interbody fusion as a substitute for iliac bone grafts, which could eliminate the disadvantages associated with autograft harvesting.     

Different mechanisms of spinal fusion using equine bone protein extract,rhBMP-2 and autograft during the process of anterior lumbar interbody fusion

To elucidate the molecular mechanisms of spinal fusion with different graft materials during an anterior lumbar interbody fusion, we examined the gene-expression profiles after implantation of equine bone protein extract, rhBMP-2 and autograft using microarray technology and data analysis, including hierarchical clustering, self-organizing maps (SOM), KEGG pathway and Biological process GO analyses in a porcine model. The results suggest that equine bone protein extract exhibited a more similar expression pattern with autograft than that of rhBMP-2. rhBMP-2 recruits progenitor cells, proliferation and differentiation possibly by inducing various factors including PGHS-2, IFGBP-2, VEGF and chemokines and then leads to preferable membranous ossification and bone remodeling. Conversely, equine bone protein extract results in endochondral ossification via upregulation of cartilage-related genes. Ossification by inducing direct osteoblastic differentiation and obviating the cartilaginous intermediate phases may increase spinal fusion rate.     

聚氨基酸/羟基磷灰石椎间融合器植入山羊颈椎

背景：聚氨基酸/纳米羟基磷灰石复合材料已被研究证实具有良好的生物安全性。 目的：观察聚氨基酸/羟基磷灰石椎间融合器对山羊颈椎的融合效果。 方法：取成年雄性山羊,切除C3、4椎间盘,清除软骨终板,随机分成实验组与对照组,分别植入聚氨基酸/羟基磷灰石颈椎间融合器与自体髂骨。术后2,4,8,12周行X射线检查;术后4,12周行CT扫描和组织学检查。 结果与结论：术后2周,实验组平均椎间高度高于对照组(P < 0.05),术后4~12周,两组平均椎间高度差异无显著性意义。术后4周,实验组融合度评分差于对照组(P < 0.05),术后12周两组融合度评分差异无显著性意义。术后4,12周,两组均可观察到从成骨细胞集聚、胶原增生到新骨形成的过程。表明聚氨基酸/羟基磷灰石椎间融合器具有良好的初始稳定性和融合效果,是一种理想的椎间融合装置。     

颈椎间不同植入物拔出载荷的比较

目的比较颈前路减压后分别植入钛合金螺纹融合支架、异体骨螺纹融合支架和自体髂骨的拔出载荷。方法以人尸体颈椎为标本,颈前路环锯减压后,分别植入钛合金螺纹融合支架、异体骨螺纹融合支架和自体髂骨,测试以上植入物的最大拔出载荷。结果减压后植入钛合金螺纹融合支架、异体骨螺纹融合支架、自体骼骨的最大拔出载荷分别为(114.7±23.3)、(101.5±15.7)、(58.7±8.3)N。结论Cloward术减压后,钛合金螺纹融合支架、异体骨螺纹融合支架拔出载荷均大于植入自体髂骨。