Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo ³H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3–5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage. Received: 30 November 1999 / Accepted: 21 March 2000     

自制NECM修复兔耳廓软骨缺损的试验研究

目的本研究的目的在于评价自制同种异体天然细胞外基质(Nature Extracellular Matrix,NECM)在修复兔耳廓软骨缺损中的价值,从而在体外研究的基础上进一步证实我们采用的去污剂-酶四步法制作的NECM在组织工程学中的可行性.方法选用10只新西兰大白兔,于其双耳廓制造1.0×.0cm大小的软骨缺损后,分别作以下研究:①灰兔NECM修复兔耳廓缺损.②灰兔NECM复合软骨膜修复兔耳廓缺损;③不植任何材料的空白对照.术后4、6、12、24周取材并行组织学检查.结果NECM植入后4、6周有轻重不一的炎症反应,第12周炎症反应消失,NECM周边无包裹现象.第12周时NECM复合软骨膜组有软骨膜细胞长入NECM,此时的软骨膜细胞多处于增殖期,24周时软骨膜细胞继续增殖分化细胞逐渐长入NECM深部,且部分软骨膜细胞发育成熟.单纯NECM修复组,第6周出现纤维结缔组织长入NECM,12、24周纤维结缔组织逐渐长入NECM深部,并逐渐取代NECM.结论①NECM可诱导软骨膜细胞增殖分化生长,修复软骨缺损:②NECM具有较好的组织亲和性和相容性;③软骨膜细胞可以作为NECM再生软骨的种子细胞的来源;④软骨膜可以阻止纤维结缔组织侵入修复区NECM.     

多克隆CNTF抗体对肉毒毒素引起肌麻痹超微结构的影响

目的研究多克隆睫状神经营养因子(c iliary neurotroph ic factor,CNTF)抗体对肉毒毒素(botu linum toxin,BTX)引起的肌麻痹的作用。方法10只新西兰兔,分别在双侧眼外肌肌腹和面部皮下注射A型肉毒毒素(botu linum toxin A,BTXA),注射后第4天在右侧眼外肌和面部皮下注射多克隆CNTF抗体,左侧相同部位注射等量生理盐水作为对照,在注射后14 d,行电镜观察双侧眼外肌和面肌的肌细胞、运动终板及神经纤维的改变。结果注射BTXA后14 d眼外肌和面肌的肌纤维均明显萎缩,线粒体肿胀,肌纤维结构无明显破坏;神经髓鞘板层松散,可见髓样小体,运动终板处可见清亮的兴奋性神经递质小泡增多密集。右侧注射CNTF组可见肌细胞核内移,胞质内线粒体肿胀聚集,肌细胞呈空泡样改变,局部肌丝断裂溶解,变性的肌细胞坏死崩解成细胞碎片;运动终板处也可见大量清亮小泡聚集,神经髓鞘增多。结论注射多克隆CNTF抗体后,与单纯注射BTXA者比较,局部肌细胞结构出现不可逆性破坏,延长了肌肉麻痹恢复时间,提示多克隆CNTF抗体可能延长BTX的肌麻痹作用。     

维拉帕米干预血管钙化作用的研究

目的观察维拉帕米在血管钙化时的作用及机制。方法将SD大鼠随机分为钙化组、维拉帕米组和对照组。5周后测定主动脉ALP、OC、ET水平及钙含量,Von Kossa染色行组织学观察。结果钙化组主动脉ALP、OC、ET水平及钙含量明显增高(P<0.01),主动脉组织中膜弹力纤维间可见大量钙沉积;经维拉帕米治疗后上述指标显著降低(P<0.05),钙沉积减少。结论维拉帕米可能通过抑制内皮素的生成减轻血管钙化的程度。     

小儿腔隙性脑梗死的病因与CT分析

目的探讨小儿腔隙性脑梗死的病因与CT表现。方法对43例腔隙性脑梗死的临床资料进行分析。结果(1)病因:头轻微外伤21例,上呼吸道感染8例,水痘2例,线粒体脑肌病(MELAS),烟雾病1例,系统性红斑狼疮1例,原因不明9例。(2)临床表现:偏瘫41例,失语5例,癫疒间10例,突发性耳聋2例。(3)CT:43例患儿发现51处病灶,其中15例患儿发现一侧或双侧基底节区点状或片状钙化,其附近存在类圆形点或片状低密度灶。(4)预后:40例患儿在12周内症状消失。结论小儿与成人引起腔隙性脑梗死病因不同,预后好。基底节钙化与外伤后急性偏瘫的发生有一定关系。     

藻酸包埋异体关节软骨细胞修复兔膝关节软骨缺损

目的 探讨异体关节软骨细胞作为软骨种子细胞修复关节软骨缺损的可行性。方法 无菌取成年新西兰大白兔四肢关节软骨，酶消化法分离细胞，条件培养基体外培养扩增，藻酸钙凝胶包埋培养1周，植入兔膝关节股骨髁间软骨缺损，对照组缺损旷置。术后3、6个月分别取材，观察缺损修复情况；切片特染和免疫组化观察修复组织病理，并Wakitnni评分评估修复质量。结果 7／8缺损为成熟透明软骨组织修复，1／8为纤维软骨修复；Wakitani平均评1．75分；空白对照组评7．65分。实验组3个月组标本未见淋巴细胞或白细胞浸润，6个月组标本未见明显退变；所有标本均未见藻酸残留。结论 用藻酸包埋异体关节软骨细胞修复兔膝关节软骨缺损的方法是可行的，异体关节软骨细胞在适当的处理后可成为关节软骨组织工程种子细胞。     

Could severity of mitral annular calcification predict other left sided structural or functional abnormalities?

ObjectiveThe prognostic value of MAC severity is important not only because it shares a common risk factors with vascular atherosclerotic changes but also through increasing the cardiovascular morbidity and mortality. This study was conducted in order to evaluate association between severity of MAC and left sided echocardiographic abnormalities mainly left sided valvular abnormalities as well as left atrial systolic and diastolic functional abnormalities.MethodsWe prospectively obtained 12-leads electrocardiograms and transthoracic echocardiograms (TTE) on patients scheduled for non-emergent echocardiographic assessment at tertiary care hospital. MAC was graded as mild, moderate and severe. LA linear dimensions, LA filling and emptying volumes and left atrium ejection fraction were done specifically in addition to commonly measured TTE parameters.ResultsFrom the 80 patients considered for the study, 47 patients had mild MAC, 29 patients had moderate MAC and 4 patients had severe MAC. Valvular affection through echo-doppler assessment that included mainly mitral stenosis, mitral incompetence, aortic sclerosis as well as aortic incompetence showed a highly significant statistical difference (p-values 0.00, 0.00, 0.00 and 0.00 respectively). There was a highly significant statistical difference between patients with different degrees of MAC and LA linear dimension (p-value 0.00), all LA filling and emptying volumes as well as LA ejection fractions with the exception of LA passive emptying volume and LA passive ejection fraction was insignificant.ConclusionsThere is a direct proportionate relationship between severity of MAC and associated left sided valvular affection, increase LA linear dimension as well as lower overall LA function.     

A New Method for in Vitro Calcification Using Acrylamide Gel and Bovine Serum

To investigate the mechanism of biological calcification in vitro a model system consisting of an acrylamide gel block (1×3×3 mm) and fetal bovine serum was developed. Mineral deposition was induced in gel blocks which were immersed in 300 μl of fetal bovine serum at 37°C for 7 days in a CO₂ incubator. X-ray diffraction indicated that the mineral was hydroxyapatite with low crystallinity. Effects of the concentration of acrylamide gel, the partial pressure of CO₂ and matrix proteins within the gel on the mineral formation were investigated. In the gel concentration range of 10–60%, the largest amount of crystal grew in 40% acrylamide gel, where the serum protein did not penetrate. With an increase in the partial pressure of CO₂ the Ca content in the gel block increased, reached the highest level at about 3.5% CO₂ and then began to decrease. In 40% gel and at 5% CO₂ the mineral formation was enhanced by phosvitin, phosphophoryn, demineralized dentin powder and alkaline phosphatase. Mineral deposition occurred around the collagen fibers immobilized in 40% acrylamide gel. These results indicate that 1) a putatively serum-derived inhibitor of calcification with high-molecular weight was prevented from penetrating into the 40% acrylamide gels, 2) immobilized polyanionic proteins and alkaline phosphatase were able to increase mineral deposition and 3) the partial pressure of CO₂ greatly influenced the mineral deposition. It was concluded that this gel system is useful to investigate the mechanism of biological calcification in vitro.