壳聚糖-β-磷酸三钙作为可注射组织工程骨支架材料的可行性研究

目的 探讨以壳聚糖-β-磷酸三钙(β-TCP)作为骨髓基质干细胞(BMSCs)注射型骨组织工程支架材料的可行性.方法 将体外培养并扩增的中国青山羊BMSCs与可注射的壳聚糖-β-TCP支架复合培养(材料组),以单纯接种培养的BMSCs为对照组,倒置显微镜观察细胞生长情况,MTT法检测材料对细胞增殖的影响.将BMSCs与壳聚糖-β-TCP混匀成BMSCs/壳聚糖-β-TCP复合物,扫描电镜观察BMSCs在材料表面的生长情况.结果 倒置显微镜下见材料组细胞生长良好,与对照组无明显差异,MTT法显示材料组细胞增殖与对照组无显著性差异(P＞0.05),扫描电镜下见材料表面细胞增殖良好.结论 BMSCs/壳聚糖-β-TCP复合物表面细胞增殖良好,该生物材料具有良好的细胞相容性,可作为可注射型骨组织支架材料.     

多孔β-磷酸三钙修复骨缺损的实验研究

目的 制备主要成分为β-磷酸三钙(β-TCP)的多孔颗粒并探讨其修复骨缺损和作为骨组织T程支架材料的可行性.方法 健康牛松质骨经脱细胞、脱脂处理后在高温下经2次煅烧制备主要成分为β-TCP的脱有机质骨多孔颗粒,在兔股骨髁制作包容性骨缺损模型并采用多孔β-TCP修复骨缺损.术后4、8、12周取材并采用X线检查、钼靶照相、荧光标记和组织学染色方法观察骨修复效果.结果 主要成分为β-TCP脱有机质骨多孔颗粒保持天然松质骨的孔隙结构,植入骨缺损后外形保持完整.术后4周材料与周边组织紧密结合,新生骨组织长入β-TCP孔隙内,原有骨缺损部位界限清楚,植入材料无明显吸收变小,中心部位无软组织或骨组织长入.植入8周β-TCP周边组织进一步向内部生长.β-TCP中心部位新生成的骨组织较少.植入12周β-TCP被周边新生骨组织严密包裹,且与新生骨组织界限模糊,植人材料部分吸收变小,新生骨小梁增多增厚并长入植入材料内部,但新骨形成量及形成速度降低.组织学检查显示β-TCP降解缓慢.结论 多孔β-TCP修复包容性骨缺损具有优良的成骨性能,可作为一种骨组织工程多孔支架材料,但需要改善其降解速度.     

17β-雌二醇对大鼠骨髓基质干细胞成骨分化初期细胞骨架的影响

目的研究17β-雌二醇（17β-estriol,17β-E2）促进体外培养的大鼠骨髓幕质干细胞（bone mesenchymal stem cells,BMSCs）骨向分化过程中细胞骨架的变化。方法采用全骨髓培养法体外培养大鼠BMSCs,分为阴性对照组、阳性对照组（成骨细胞诱导液）和雌激素处理组（成骨细胞诱导液＋10^-6。mol／L。17β-E2）,使用碱性磷酸酶（alkaline phosphatase,AI,P）试剂盒检测BMSCs的ALP活性,激光扫描共聚焦显微镜观察各组BMSCs成骨分化初期的细胞骨架。结果BMSCs细胞ALP活性随时间的增加而增强,雌激素处理组表达ALP活性明显高于阴性对照组和阳性对照组（P〈0．01）;细胞骨架荧光强度定量分析显示,雌激素处理组（26．56％±9．87％）较阳性对照组（18．31％±5．76％）和阴性对照组（8．12％土4．42％）明显增强（P〈0．05）。结论17β—E2可能通过早期调节细胞骨架微丝聚合改变细胞形态,从而促进骨髓基质干细胞向成骨分化。     

壳聚糖/聚磷酸钙复合支架的生物相容性研究

目的:评价比较不同配比壳聚糖/聚磷酸钙(Chitosan/Calcium polyphosphate,CS/CPP)复合支架的细胞相容性和组织相容性,探讨其应用于组织工程半月板支架材料的可行性。方法:采用扫描电镜检测半月板细胞在支架材料上的生长情况。采用MTT法检测不同配比CS/CPP支架材料浸提液对第3代半月板细胞的毒性,通过肌袋内植入试验评价支架材料的体内组织相容性。结果:扫描电镜下可见:不同比例CS/CPP复合支架材料呈三维多孔的结构。复合第3代半月板细胞体外培养7 d,细胞在支架材料上均匀分布,紧贴支架铺展伸长,生长良好。MTT法显示不同浓度支架浸提液与对照DF培养液吸光度值比较,差异无统计学意义(P>0.05),支架无细胞毒性。肌袋内植入试验评价:肌袋内植入实验显示动物术后生存良好,各植入部位均未见组织坏死、积液及化脓感染。术后1周植入体周围有较多中性粒细胞浸润;术后2~8周植入体周围中性粒细胞浸润逐渐减少,出现淋巴细胞浸润,淋巴细胞多于中性粒细胞,纤维囊壁厚薄不均;3个月后植入体周围未见炎症细胞浸润,主要为胶原纤维和成纤维细胞。随植入时间的延长,炎症细胞浸润逐渐减少,浸润范围逐渐缩小,包膜先逐渐变厚再变薄,植入材料内部逐渐有组织长入,并且逐渐降解。结论:CS/CPP具有良好的细胞相容性和组织相容性,有望成为半月板组织工程的支架材料。     

巨噬细胞对掺锶聚磷酸钙降解行为影响的体外研究

目的 通过设计巨噬细胞在体外对SCPP的降解实验,为骨组织工程支架材料掺锶聚磷酸钙(SCPP)体内降解规律的研究提供基础和参考.方法 制备SCPP支架材料,将该材料与巨噬细胞RAW264.7培养6周以研究细胞介导的降解.用扫描电镜(SEM)观察细胞及材料形貌;复合电极测量培养液pH值变化和用等离子体发射光谱检测培养液中的Ca2+、磷(Pi)、Sr2+浓度.结果 SEM结果表明,培养细胞后材料孔径与粒径均减小,表面粗糙度增大,降解显著;细胞组培养液pH值在6.87～7.17之间,明显低于对照组;细胞组培养液中Ca2+、Pi、Sr2+释放量明显高于对照组.细胞介导的降解速率约为对照组的5倍,且Sr2+浓度在正常生理范围内.结论 在SCPP降解过程中,巨噬细胞加速了SCPP的降解,并在降解过程中起到主导作用.     

工程化骨构建策略及其进展

骨缺损的修复一直是热门课题,1995年Crane等[1]系统提出了骨组织工程的概念、研究方法、研究现状及发展前景,引起了广大学者的关注.目前的骨组织工程研究主要包括种子细胞、生物活性因子和支架材料三方面.骨髓间充质干细胞(BMSCs)由于具有多向分化的潜能,成为目前较为理想的骨种子细胞;应用于骨组织工程的生物活性因子主要有骨形态发生蛋白(BMP)、碱性成纤维细胞生长因子(BFGF)、血小板衍化生长因子(PDGF)及血管内皮细胞生长因子(VEGF)等;目前支架材料的新构建策略不断取得突破,使得组织工程骨部分进入临床应用阶段.本文就工程化骨研究中的构建策略转变作一综述.     

以纤维蛋白胶为载体的注射型骨修复材料对兔骨髓基质细胞增殖和分化的影响

目的 观察以纤维蛋白胶(FS)为载体的注射型骨修复材料对体外培养的兔骨髓基质细胞(MSCs)增殖和分化的影响.方法 取3天龄新西兰兔MSCs进行培养,实验分为5组,A组(实验组)培养液中加入含1μg/ml重组人骨形态发生蛋白2(rhBMP-2)和1μg/ml重组人碱性成纤维细胞生长因子(bFGF)的纤维蛋白胶,B组(对照1组)培养液中加入单纯的纤维蛋白胶,C组(对照2组)培养液中加入含lμg/ml bFGF的纤维蛋白胶,D组(对照3组)培养液中加入含1μg/ml rhBMP-2的纤维蛋白胶,E组为单纯对照组(培养液中不加入任何材料).采用细胞培养、组织化学及电镜观察等方法对各组细胞增殖情况、贴壁率、碱性磷酸酶(ALP)活性、Ⅰ型胶原表达、超微结构变化等进行研究.结果 各组材料的促增殖作用和对细胞贴壁率的影响由强到弱均依次为C组、A组、B组、D组、E组,差异有显著性(P＜0.05).各组细胞ALP活性及Ⅰ型胶原表达水平由强到弱均依次为A组、D组、B组、C组、E组,差异有显著性(P＜0.05).扫描电镜观察发现,以纤维蛋白胶为载体的注射型骨修复材料表面粗糙,有微孔存在,各组细胞与其融合生长.透射电镜观察发现:A组的绝大部分细胞呈成骨细胞表型,细胞增殖旺盛,分化好,胞质内有丰富的粗面内质网和线粒体,并明显扩张,内有蛋白样物质,细胞外基质丰富,细胞周围可见大量的胶原纤维;而各对照组或是细胞增殖活性高,但向成骨细胞表型分化差,或是向成骨细胞表型分化好,但细胞增殖活性较差,细胞外基质及胶原纤维少.结论 以纤维蛋白胶为载体的注射型骨修复材料(FS bFGF rhBMP-2)可显著促进MSCs的增殖及其向成骨细胞的分化.     

137 Cs γ射线对成骨细胞增殖、分化和矿化的影响

目的 探讨137Cs γ射线对成骨细胞形态、增殖、分化、矿化及细胞因子的影响及分子机制.方法 将成骨细胞分为对照组(0 Gy)及0.5、1.0、2.0和5.0 Gy组,分别接受137Cs γ射线照射,倒置相差显微镜观察各组细胞形态,MTT法测定细胞增殖,PNPP法检测碱性磷酸酶(ALP)活性,茜素红染色法观察矿化功能,RT-PCR半定量检测ALP、骨钙素(OC)、Ⅰ型胶原(collagen Ⅰ)、护骨素(OPG)和NF-kB受体活化因子配体(RANKL)等基因mRNA表达量.结果 1.0 Gy以上照射组抑制成骨细胞增殖(t=6.197～18.677,P＜0.05);2.0 Gy以上照射组致细胞数量减少,折光性减低,细胞间突起连接减少,并可抑制细胞ALP活性和矿化能力(t=2.790 ～ 21.374,P＜0.05).ALP和OC基因mRNA表达量在0.5 Gy以上剂量照射即明显下调(t=3.563～16.508,P＜0.05),OPG、OPG/RANKL在5.0 Gy剂量时表达明显下调(t=12.942、4.954,P＜0.05),Ⅰ型胶原和RANKL基因mRNA的表达未见明显改变.结论 137CsΥ射线照射致成骨细胞形态改变,增殖、分化和矿化能力下降,ALP、OC、OPG等相关基因表达下调,OPG/RANKL通路可能是大剂量电离辐射时骨损伤的主要作用途径之一.     

骨髓基质细胞的体外培养与临床应用

目的　验证骨髓基质细胞 (bonemarrowstromalcells,BMSCs)向成骨细胞转化的能力及其临床应用价值。方法　取健康成年大白兔股骨大转子红骨髓 ,在DMEM标准培养液中原代培养骨髓基质细胞 ,传代细胞分别用DMEM标准培养液和加入地塞米松、β 甘油磷酸钠、L 抗坏血酸的条件培养液培养 ,倒置显微镜观察不同培养条件下细胞的形态学特征 ,进行酶学测定 ,计数传代细胞中成骨样细胞转化率 ,免疫组化法检测Ι型胶原表达。同样方法培养人BMSCs,与同种异体骨基质明胶充分混合 ,应用于临床修复创伤所致的骨缺损、骨不连或骨折延迟愈合。结果　体外培养条件下 ,原代细胞呈长梭形 ,具有成纤维细胞的生长特性 ,条件培养的传代BMSCs变为三角形或锥形 ,呈多层生长 ,无接触抑制现象 ,具有与成骨细胞相似的形态和生长特点。ALP染色阳性率可达 85 %以上 ,Ι型胶原免疫组化染色呈强阳性 ,具有与成骨细胞类似的功能性表现。临床应用 4 2例病人 ,随访 8～ 2 6个月 ,取得了满意疗效。结论　BMSCs能够向成骨细胞分化和增殖 ,是骨组织工程学种子细胞的理想来源 ,并有广阔的临床应用前景     

三维多孔聚DL-乳酸/羟基磷灰石复合材料体外降解特征的研究

观察不同比例羟基磷灰石 (HA)复合聚DL 乳酸 (PDLLA)制备的泡沫材料的体外降解特性 ,将PDLLA、PDLLA/HA(5wt%)、PDLLA/HA(2 0wt%)及PDLLA/HA(35wt%)四种材料置入蒸馏水中降解 ,比较各组pH值、重量、分子量及抗压强度的变化。结果发现 ,复合材料PDLLA/HA(2 0wt%)降解各时期的各指标与其他组比较差异有显著性意义 (P <0 0 5 )。纯PDLLA的pH值呈逐渐下降的趋势 ,而PDLLA/HA(2 0wt%)在 6周后趋向于平稳 ,12周时pH值与原环境相近。说明HA的加入影响了PDLLA的体外降解性能 ,不同比例的复合材料性能不同 ,PDLLA/HA(2 0wt%)具有降解过程相对延迟、力学性能稳定和能缓冲酸性代谢产物的特点。     

Stress MR imaging for evaluation of popliteal artery entrapment

The popliteal artery entrapment (PAE) syndrome has been recognized as a cause of arterial occlusion in young people. It is the result of an anomaly of the relationship between the popliteal artery and the gastrocnemius muscle. Eight young healthy volunteers (16 legs) and six patients (10 legs) with suspected PAE underwent magnetic resonance (MR) imaging. Gradient-echo images were obtained in axial planes with the leg at rest and during active plantar flexion against resistance. Imaging at rest allowed identification of PAE signs in only one leg, which had an anomalous medial course of the popliteal artery. In the other cases, only the stress technique was able to show signal loss in the popliteal artery due to muscular compression (two legs) or the presence of accessory muscle slip around the vessel (two legs), as confirmed at surgery. MR imaging is therefore a useful technique for the diagnosis of PAE because of its capability of combining information obtainable with other modalities.     

MR imaging of muscle and tender points in fibromyalgia

Fibromyalgia is a syndrome manifested by chronic, diffuse muscu-loskeletal aching and soreness, palpable muscle tender points, and other symptoms. Standardized clinical diagnostic criteria have recently been developed. Skeletal muscle has been postulated as the end organ in this disease. Biochemical, histologic, electromyographic, and conventional radiographic studies have demonstrated no definitive abnormality. This study sought to establish whether magnetic resonance (MR) imaging could demonstrate any abnormality in these patients. Eighteen patients were entered in the study, 14 of whom were able to complete their examinations. T1 -weighted, T2-weighted, gradient-echo, and STIR (short-tau inversion-recovery) sequences were performed in all patients, with selected patients examined with T1weighted, gadopentetate dimeglu-mine-enhanced sequences. The trapezius and suboccipital regions were imaged in patients who, clinically, had active fibro-myalgia. No abnormalities could be detected. The authors conclude that the conventional MR imaging used in this study was unable to depict any primary skeletal muscle abnormality in fibromyalgia.     

Controversies in cervical spine imaging in trauma patients

No area of emergency radiology has generated as much discussion in recent years as the subject of cervical spine imaging for trauma patients. This review will be in three parts. The first will examine the indications for cervical imaging and will focus on those factors that make patients at high risk or low risk for cervical injury. The second part will discuss the merits of radiography and computed tomography as the main screening diagnostic examination. In addition to the roles of each modality in the evaluation process, such factors as efficacy of diagnosis, time (duration) of study, and cost will be discussed. Finally, the third part will explore the methods currently employed to clear the cervical spine in comatose patients.Presented at the Annual Meeting of the American Society of Emergency Radiology, Las Vegas, Nevada, 22–25 October, 2003     

Uterine contractions: Possible diagnostic pitfall at MR imaging

A total of 206 nongravid patients with various gynecologic problems underwent pelvic magnetic resonance (MR) examinations that included both sagittal T2-weighted and contrast agent–enhanced T1-weighted images. MR images were retrospectively reviewed to identify changes in endometrial configuration on serial images obtained during the same MR examination. In 20 MR examinations (all in women of reproductive age), endometrial distortion due to myometrial bulging was noted on T2-weighted or contrast-enhanced T1-weighted images. It was absent on other MR images obtained at different times. Myometrial bulging exhibited low signal intensity in 18 examinations. The finding resembled adenomyosis or leiomyoma on T2-weighted or contrast-enhanced T1-weighted images. These results evidence the presence of transient myometrial bulging and transient low-intensity myometrium in the nongravid uterus. This phenomenon is thought to represent uterine contraction. Clinicians should be aware of the potential presence of transient low-signal-intensity myometrial bulging that could present diagnostic problems in the normal uterus.     

Brodie Abscess: MR imaging appearance in 10 patients

The magnetic resonance (MR) imaging features of Brodie abscess have not yet been fully evaluated. Ten patients with Brodie abscess, eight of long bone and two of vertebra, were studied with MR imaging. Long bone abscess had a characteristic “target” appearance with four layers: (a) a center with low signal intensity on T1-weighted images and high signal intensity on T2-weighted and STIR (short-inversion-time inversion recovery) images, (b) an inner ring isointense to muscle on T1-weighted images and with high signal intensity on T2-weighted and STIR images, (c) an outer ring hypoin-tense on all images, and (d) a peripheral halo hypointense on T1-weighted images. In six of eight cases, a soft-tissue mass was found. The two vertebral abscesses had a less specific appearance, with low signal intensity on T1-weighted images and high signal intensity on T2-weighted and STIR images. Only the peripheral halo was clearly identified in both cases.     

Right-side aortic arch with aneurysm of aberrant left subclavian artery: MR imaging appearance.

Reports of aneurysms of the subclavian artery in both normal and anomalous aortic arches have been rare. The authors describe a patient with a right-side aortic arch and an aneurysm of the aberrant left subclavian artery, which, to the authors' knowledge, is a previously unreported association. At presentation, the aneurysm appeared as a calcified left superior mediastinal mass. Magnetic resonance imaging enabled preoperative diagnosis and guided surgical planning.     

MR angiography of congenital heart disease: Value of segmented two-dimensional inflow technique and maximum-intensity-projection display

Magnetic resonance (MR) angiography of the cardiovascular system was evaluated in 41 patients with congenital heart disease by using a two-dimensional (2D) inflow technique based on a magnetization-prepared gradient-echo pulse sequence with segmented k-space data acquisition and electrocardiographic gating at 0.5 T. Inversion and saturation prepulses were used to suppress stationary tissue and enhance intravascular signal. Presaturation slabs were applied where certain vascular structures had to be suppressed. Sequence parameters were optimized by evaluating signal intensity and contrast characteristics for various flip angles and inversion and saturation delay times. The heart and intrathoracic vasculature were encompassed with 40–50 overlapping sections. Both 2D angiograms and maximum-intensity-projection images were evaluated. Combining data sets acquired in the sagittal and transverse orientations provided the most satisfactory information about the pulmonary arteries. The highest signal-to-noise ratios were obtained with a flip angle of 65° and short prepulse delay times. Two-dimensional MR angiography can provide useful diagnostic information but requires a thorough understanding of in-plane and hemodynamically induced signal intensity changes.     

Adrenal tissue characterization with 0.5-T MR imaging: Value of T2*-weighted images

The authors investigated the value of magnetic resonance (MR) imaging at 0.5 T for distinguishing adrenal adenomas from adrenal metastases. The series included 23 adrenal adenomas (18 nonhyperfunctioning, five hyperfunctioning) and 23 adrenal metastases from various organs. Adrenal tumor–liver signal intensity ratios on T1-, T2-, and T2*-weighted images were calculated for adrenal tissue characterization. Adrenal adenomas were more precisely distinguished from adrenal metastases on T2*-weighted images (21 of 23, 91%) than on T2-weighted images (15 of 23, 65%). T1-weighted images were not useful for this distinction. In conclusion, T2*-weighted images were better than routine T2-weighted images for distinguishing adrenal adenomas from adrenal metastases. It can be postulated that the total signal intensity of adrenal adenomas, which contain some fat components, decreased on T2*-weighted images because of an out-of-phase effect.     

Dynamic contrast-enhanced MR imaging assessment of vascularized free fibular grafts

Magnetic resonance (MR) imaging may be a noninvasive method for assessing perfusion of vascularized bone grafts placed for treatment of avascular necrosis. One proximal femur of seven beagles was devascularized, with insertion of a vascularized fibular graft. MR imaging at 1 week (seven dogs) and 6 weeks (five dogs) after surgery included pre- and postcontrast spin-echo sequences, unenhanced twodimensional time-of-flight (TOF) vascular imaging, and dynamic gradient-echo imaging during infusion of gadolinium. Relative signal intensity values of selected regions obtained from the dynamic gradientecho images were plotted as percent enhancement versus time. In the operated hip, MR imaging did not show enhancement in six of seven femoral heads and greater trochanters at 1 week after surgery, with similar results after 6 weeks. MR imaging of fibular grafts 6 weeks after surgery showed an initial rapid increase in enhancement and a subsequent slower increase in five of five dogs, although no enhancement was seen in six of seven dogs at 1 week. These findings contrasted with a rapid initial increase in enhancement followed by slow decline in non-operated hips. Two-dimensional TOP imaging did not show the vascular pedicle of the graft in any dog. Findings of radionuclide bone scanning performed 1 week after surgery were consistent with devascularization of the operated femur and fibular graft. However, tetracycline distribution and histologic findings confirmed the viability of five of five grafts within the devascularized femurs 6 weeks after surgery. Thus, dynamic contrast-enhanced MR imaging at 6 weeks after surgery is valuable for assessing vascular bone graft perfusion, while similar imaging at 1 week may suggest otherwise.