不同年龄人鼻中隔软骨细胞生物学特性观察

目的观察年龄因素对体外培养的人鼻中隔软骨细胞生物学特性的影响。方法体外培养不同年龄组人鼻中隔软骨细胞,观察其生物学形态、细胞增殖周期、甲苯胺蓝异染和Ⅱ型胶原的免疫组化染色,并测定35S-Na2SO4掺入量以观察对软骨细胞蛋白多糖合成量的影响,在细胞特性和增殖特征方面进行比较。结果各组间软骨细胞的形态、传代特征及传代数、组织学检测无明显差异,细胞倍增时间随年龄增大而下降,软骨细胞蛋白多糖合成量随体外时间延长而上升,不依赖年龄。结论人鼻中隔软骨细胞生物学特性总体上不依赖年龄,其作为软骨组织工程的潜在种子细胞来源具有广泛的年龄段。     

转化生长因子-β1和胰岛素对人鼻中隔软骨细胞增殖和分化影响的研究

目的 了解转化生长因子-β1(transforming growth factor-betal，TGF—β1)和胰岛素对人鼻中隔软骨细胞增殖和分化的影响。方法 体外培养人鼻中隔软骨细胞，采用四甲基偶氮唑蓝(MTT)代谢和^35S-Na2SO4掺入的检测比较不同浓度TGF-β1和胰岛素对人鼻中隔软骨细胞的增殖以及软骨基质蛋白多糖合成的影响，观察TGF-β1和胰岛素对软骨细胞表型的影响。结果 在15％血清的培养条件下，TGF-β1和胰岛素均能显著促进软骨细胞增殖，且在各自一定的浓度范围内呈量效关系，联合作用效果累加；TGF—β1、TGF—β1和胰岛素联合作用促使软骨基质蛋白多糖的合成量明显提高；TGF-β1使传代软骨细胞去分化提前。结论 一定浓度的TGF—β1、胰岛素和TGF-β1 胰岛素对体外培养的人鼻中隔软骨细胞具有显著的促增殖作用。     

转化生长因子-β1和胰岛素对人鼻中隔软骨细胞增殖和分化影响的研究

目的 了解转化生长因子-β1(transforminggrowth factor-beta 1,TGF-β1)和胰岛素对人鼻中隔软骨细胞增殖和分化的影响。方法 体外培养人鼻中隔软骨细胞,采用四甲基偶氮唑蓝(MTT)代谢和35S-Na2SO4掺入的检测比较不同浓度TGF-β1和胰岛素对人鼻中隔软骨细胞的增殖以及软骨基质蛋白多糖合成的影响,观察TGF-β1和胰岛素对软骨细胞表型的影响。结果在15％血清的培养条件下,TGF-β1和胰岛素均能显著促进软骨细胞增殖,且在各自一定的浓度范围内呈量效关系,联合作用效果累加;TGF-β1、TGF-β1和胰岛素联合作用促使软骨基质蛋白多糖的合成量明显提高;TGF-β1使传代软骨细胞去分化提前。结论一定浓度的TGF-β1、胰岛素和TGF-β1 胰岛素对体外培养的人鼻中隔软骨细胞具有显著的促增殖作用。     

人软骨细胞的培养及冷冻保存

由于新鲜软骨来源有限,有必要建立异体移植软骨库。冷冻保存法能使软骨细胞长期存活并维持其正常的代谢和生物学特性。鼻中隔软骨取自重建手术中或尸体材料,共15例,年龄5～80岁。经消化酶消化、孵育、过滤。从标本中的软骨中分离软骨细胞,进行细胞的计数,用台盼蓝染料测定细胞活性。以不同培养基孵育分离出的软骨细胞,倒置显微镜观察不同孵育期软骨细胞增殖情况。上述细胞经冷冻     

鼻中隔软骨细胞组织工程法构建预定形态软骨

目的探讨利用人鼻中隔软骨细胞组织工程方法构建预定形态软骨的可能性。方法将人鼻中隔软骨细胞播种在聚乙醇酸（polyglycolicacid,PGA）无纺网支架材料上,制成片状和管状结构,埋入裸鼠体内,经4、6、8周后取材作大体及组织学观察。结果大体观察见裸鼠体内形成了预定的片状和管状软骨。组织学观察6周时软骨细胞基本成熟,Masson三色染色显示胶原形成,番红花-“O”染色证实其基质中存在糖氨多糖。对照组于6周时PGA纤维基本消失。结论人鼻中隔软骨细胞与PGA无纺网复合可在裸鼠体内形成预定形态软骨。     

琼脂胰岛素对体外培养人鼻中隔软骨细胞生长增殖的影响

观察琼脂包埋培养皿后鼻中隔软骨细胞体外单层培养的生长状况 ,将胰岛素作用于鼻中隔软骨细胞 ,了解其对软骨细胞生长的影响 ,为软骨组织工程提供理论参考。一、材料和方法1.软骨细胞的培养 :无菌条件下取10例鼻中隔偏曲手术患者鼻中隔软骨 ,ＰＢＳ液 (含青霉素 2 0 0Ｕ/ｍｌ,链霉素 2 0 0μｇ/ｍｌ)漂洗 3次 ,剪成 1ｍｍ× 1ｍｍ× 1ｍｍ大小软骨碎片 ,ＰＢＳ液漂洗 2次 ,加入 2倍体积的Ⅱ型胶原酶 (3ｍｇ/ｍｌ,ｓｉｇｍａ公司 ,美国 ) ,37℃恒温震荡消化 ,过滤 ,离心 ,ＰＢＳ液漂洗 2次 ,离心 ,弃上清 ,16 40培养基 (ｓｉｇｍａ ,美国。含…     

培养状态下成人鼻中隔软骨细胞生物学特性的研究

目的：研究体外２的正常成人鼻中隔软骨细胞的生物学特性。方法：体外培养成我鼻中隔软骨细胞，观察原代及传代培养的形态学变化；测定细胞数量的变化及其生长曲线，观察细胞的增殖；借助特殊染色、免疫组织化学的方法了解糖胺聚糖、碱性磷酸酶、Ⅱ型胶原的合成情况，测定冷冻复苏后的细胞存活。结果：初始接种细胞为圆形，细胞贴壁后４ｄ细胞逐渐变为放射延异状；从第５代开始绝大部分细胞转变成纤维样细胞。２代细胞体外培养至第５     

鼻科学

20010713促进体外培养人鼻中隔软骨细胞增殖方法的研究/黄金中…//耳鼻咽喉一头颈外科一2000,7(5)一29尘一297 目的:研究促进人鼻中隔软骨细胞在体外增殖的方法,为少、鼻中隔软骨细胞在软骨组织工程的应用提供方法和理论依据。方法:研究三种培养基(DMEM、F一12、1640)、胰岛素、琼脂和肌昔对正常成人鼻中隔软骨细胞体外培养的影响。结果:164。培养基和新生牛血清适合进行人鼻中隔软骨细胞培养,胰岛素可保持软骨细胞生长,琼脂可促进软骨细胞表型稳定;在1640培养基条件下,肌昔对软骨细胞的生长无明显的促进作用。图l表2参10(原提要)20010714…     

耳廓软骨天然生物支架材料的制备及初步研究

目的探讨制备兔耳廓天然软骨细胞外基质材料的新方法,制备适用于组织工程应用的天然生物支架材料.方法①采用多步骤去污剂-酶处理技术抽提兔耳廓软骨细胞制备天然软骨细胞外基质(Nature Extracellular Matrix,NECM),对NECM行HE染色、澳新蓝染色、维多利亚蓝染色、VanGieson染色并进行组织学及扫描电镜观察;②以灰家兔NECM为支架材料和新西兰大白兔耳廓软骨细胞为种子细胞进行体外培养,倒置显微镜下观察其亲水性和对细胞的吸附力,并通过四氮唑盐比色法(MTT Assay)观察NECM对软骨细胞的增殖作用.结果通过HE染色、蛋白多糖、胶原及弹力纤维特染、扫描电镜观察分析证实经过多步骤去污剂-酶法抽提软骨细胞制备的天然生物支架材料里,不含细胞及细胞器成分,仅含有不溶解的胶原、弹性蛋白和蛋白多糖,亦即NECM;体外培养实验发现,兔耳廓软骨细胞易进入脱细胞异体软骨基质结构裸露的空穴,且生长旺盛,组织学检查证实兔耳廓软骨NECM上裸露的骨陷窝已部分被软骨细胞占据.MTT Assay检测证实NECM对软骨细胞增殖有促进作用.结论本实验制备的NECM脱细胞彻底、细胞外基质成分保留较完整,并具有促进细胞粘附、增殖和分化生长的作用,是一种较为理想的天然生物支架材料.     

鼻中隔软骨细胞细胞组织工程法构建预定形态软骨

目的 探讨利用人算中隔软骨细胞组织工程方法构建预定形态软骨的可能性。方法 将人鼻中隔软骨细胞播种在聚乙醇酸（polyglycoli acid,PGA)无纺网支架材料上，制成片状和管状结构，埋入裸裸鼠体内，经4、6、8周后取材作大体及组织学观察。结果 大体观察见裸 本内形成了预定的片状和管状软骨。组织学观察：6周时软骨细胞基本成熟，Masson三色染色显示胶原形成，番红花－“O”染色证实其基质中存在糖多糖。对照组于6周时PGA纤维基本消失。结论 人鼻中软骨细胞与PGA无纺网复合可在裸鼠体内形成预定形态软骨。     

Stored human septal chondrocyte viability analyzed by confocal microscopy

OBJECTIVES: To analyze the effects of prolonged storage time, at warm and cold temperatures, on the viability of human nasal septal chondrocytes and to understand the implications for tissue engineering of septal cartilage. DESIGN: Basic science. SUBJECTS: Septal cartilage was obtained from 10 patients and placed in bacteriostatic isotonic sodium chloride solution. Four specimens were kept at 23 degrees C, and 4 were kept at 4 degrees C. The viability of the chondrocytes within the cartilage was assessed using confocal laser scanning microscopy every 5 days. The 2 other specimens were assessed for viability on the day of harvest. RESULTS: Viability on the day of harvest was 96%, implying minimal cell death from surgical trauma. After 1 week, cell survival in all specimens was essentially unchanged from the day of harvest. At 23 degrees C, the majority (54%) of cells were alive after 20 days. At 4 degrees C, 70% of cells survived 1 month and 38% were alive at 2 months. Qualitatively, chondrocytes died in a topographically uniform distribution in warm specimens, whereas cold specimens displayed a more irregular pattern of cell death. CONCLUSION: Septal chondrocytes remain viable for prolonged periods when stored in simple bacteriostatic isotonic sodium chloride solution, and such survival is enhanced by cold storage.     

Regenerative and proliferative activities of chondrocyte based on the degree of perichondrial injury in rabbit auricular cartilage

Although the regeneration process for injured cartilage requires an intact perichondrium, few studies have addressed the importance of the intact perichondrial layer in the regeneration of damaged cartilage. In this study, we evaluated the role of the perichondrium on regenerative activities in injured cartilage according to different degrees of perichondrial injury. Auricular cartilage harvested from six New Zealand white rabbits was irradiated with a 1,460-nm diode laser at two different power settings (0.3 or 0.5 W). Irradiated cartilage was reimplanted into a subperichondrial pocket under three different conditions: non-injured perichondrium (NPI), unilaterally injured perichondrium (UPI), or bilaterally injured perichondrium (BPI). Rabbits were sacrificed at 1, 2, and 4 weeks after reimplantation and the auricular cartilage was reharvested. A histopathological study using hematoxylin and eosin staining, a live/dead viability assay, and immunohistochemical staining for proliferating cell nuclear antigen were performed to evaluate structural changes and regenerative and proliferative activities of the injured chondrocytes. A modified array and restored boundary of chondrocytes were observed in the NPI and UPI groups. Regeneration of chondrocytes was prominent in the NPI and UPI groups, but was not observed in the BPI group. Proliferative activity of chondrocytes was observed only when the perichondrium was preserved in the NPI and UPI groups. In contrast, proliferative activity was not observed until 4 weeks in the BPI group. The degree of perichondrial injury affected proliferation and regeneration in injured elastic cartilage. In the case of unilateral perichondrial injury, the surgeon should be careful to avoid damaging the other side of the perichondrium, because at least a unilateral perichondrial layer is needed for the regeneration of elastic cartilage.     

Tissue-engineered tracheal reconstruction using chondrocyte seeded on a porcine cartilage-derived substance scaffold

Objectives

Tracheal reconstruction with tissue-engineering technique has come into the limelight in the realm of head and neck surgery. We intended to evaluate the plausibility of allogenic chondrocytes cultured with porcine cartilage-derived substance (PCS) scaffold for partial tracheal defect reconstruction.

Methods

Powder made from crushed and decellularized porcine articular cartilage was formed as 5 mm × 12 mm (height × diameter) scaffold. Chondrocytes from rabbit articular cartilage were expanded and cultured with PCS scaffold. After 7 weeks culture, the scaffolds were implanted on a 5 mm × 10 mm artificial tracheal defect in six rabbits. Two, four and eight weeks postoperatively, the sites were evaluated endoscopically, radiologically, histologically and functionally.

Results

None of the six rabbits showed any sign of respiratory distress. Endoscopic examination did not show any collapse or blockage of the reconstructed trachea and the defects were completely covered with regenerated respiratory epithelium. Computed tomography showed good luminal contour of trachea. Postoperative histologic data showed that the implanted chondrocyte successfully formed neo-cartilage with minimal inflammatory response and granulation tissue. Ciliary beat frequency of regenerated epithelium was similar to those of normal adjacent mucosa.

Conclusions

The shape and function of reconstructed trachea using allogenic chondrocytes cultured with PCS scaffold was restored successfully without any graft rejection.     

同种异体软骨细胞修复耳廓缺损的动物实验研究

目的探讨兔同种异体软骨细胞和自行制备生物材料高孔隙率聚乳酸(poly—DL-lactide,PDLLA)支架复合物修复兔耳廓软骨缺损的可行性。方法 18只大白兔分为软骨细胞/PDLLA复合物移植组、单纯PDLLA对照组和空白对照组,将体外培养的兔同种异体软骨细胞和自行制备的PDLLA支架形成复合物,修复兔耳廓软骨缺损。分别于植入后6周、12周、18周取材,HE和甲苯胺蓝染色了解兔耳廓缺损修复情况。结果术后18周,软骨细胞/PDLLA复合物移植组软骨缺损区为软骨组织修复,新生软骨与正常软骨问连接好,单纯PDLLA对照组和空白对照组为纤维软骨或纤维组织修复。结论同种异体软骨细胞/自制PDLLA复合物移植可较好地修复兔耳廓软骨缺损。     

Tissue engineered cartilage: utilization of autologous serum and serum-free media for chondrocyte culture

BACKGROUND: Standard culture medium contains bovine serum. If standard culture methodology is used for future human tissue-engineering, unknown risks of infection from bovine disease or immune reaction to foreign proteins theoretically might occur. In this study we wished to evaluate the potential of chondrocyte expansion using autologous and serum free media. METHODS: Autologous auricular cartilage was harvested in a swine model. An initial concentration of 100x10(3) cells per group were expanded in three groups. Group A, F-12 with 10% fetal calf serum; Group B, F-12 supplemented with 10% autologous serum; Group C, F-12 supplemented with growth factors. Cell numbers were counted at days 3, 6, 9 and 12. RESULTS: The cells in all the three groups exhibited normal chondrocyte morphology. At early time points there was a statistically significant difference in the number of cells between Group A and the two other groups (p<0.05). By day 12, both Groups A and C demonstrated greater cell number as compared to Group B (p<0.05). CONCLUSION: The results suggest that both autologous serum as well as serum free media might be substituted for the expansion of the number of chondrocytes, thus avoiding the potential need for a bovine serum supplement.     

Human septal chondrocyte redifferentiation in alginate,polyglycolic acid scaffold,and monolayer culture

OBJECTIVES/HYPOTHESIS: Tissue engineering laboratories are attempting to create neocartilage that could serve as an implant material for structural support during reconstructive surgery. One approach to forming such tissue is to proliferate chondrocytes in monolayer culture and then seed the expanded cell population onto biodegradable scaffolds. However, chondrocytes are known to dedifferentiate after this type of monolayer growth and, as a result, decrease their production of cartilaginous extracellular matrix components such as sulfated glycosaminoglycans. The resultant tissue lacks the biomechanical properties characteristic of cartilage. The objective of the study was to determine whether different culture systems could induce monolayer-expanded human septal chondrocytes to redifferentiate and form extracellular matrix. STUDY DESIGN: Laboratory research. METHODS: Chondrocytes were isolated from human nasal septal cartilage of five donor patients (age, 35.8 +/- 9.3 y). Cell populations were seeded at low density (30,000 cells/cm2) into monolayer culture and expanded for 4 to 6 days. Following trypsin release, chondrocytes were placed into three different systems for neocartilage formation: alginate beads, polyglycolic acid scaffolds, and monolayer. After 7 and 14 days of growth, neocartilage was analyzed using histological and quantitative biochemical assessment of cellularity (Hoechst 33258 assay) and sulfated glycosaminoglycan content (dimethyl methylene blue assay). RESULTS: Histologically, alginate beads contained spherical chondrocytes surrounded by dense extracellular matrix, an appearance similar to that of native cartilage. In contrast, polyglycolic acid scaffolds and monolayer cultures contained elongated cells with scant staining for matrix sulfated glycosaminoglycans, which are features that are characteristic of dedifferentiated chondrocytes. Biochemical analysis demonstrated a lower level of cell proliferation (P <.001) in scaffolds (+52% over baseline) and alginate (+96% over baseline) than in monolayer (+366% over baseline), as well as a higher content of sulfated glycosaminoglycans per cell (P <.001), after 14 days of growth in alginate culture than in either polyglycolic acid scaffolds (19-fold difference) or monolayer (98-fold difference). CONCLUSIONS: Of the systems compared, monolayer-expanded human septal chondrocytes demonstrated the greatest accumulation of sulfated glycosaminoglycans per cell when grown in alginate beads. Future research on cartilage tissue engineering may use alginate culture for reverting dedifferentiated cells back to the chondrocytic phenotype.     

Ciliated cell observation by SEM on the surface of human incudo-malleolar-joint articular cartilage: are they a new chondrocyte phenotype?

Background: Scanning electron microscopy (SEM) study of the human incus bone is scanty whilst, to our knowledge, no information regarding human incudo-malleolar joint articular-cartilage morphology has previously been provided.

Aims/objectives: Our aim was to shed some light on this morphological issue and to propose some theoretical perspectives on its functional role.

Material and methods: The human incudo-malleolar joint was documented with field emission SEM on samples recovered during ear surgery procedures after patients’ informed consent.

Results: Normal articular cartilage chondrocytes, flattened cells with prominent nucleus and short microvilli were observed. Interestingly, cells provided with long cilia were identified. Type A cilia are arranged in a pyramidal formation with extra-long cilia stemming from the cluster, projecting upwards in an antenna-like formation ending with a dilated structure that as a whole, resembles the stereocilia with kinocilium. Types B, C and D cilia resemble those of the genital and respiratory tracts.

Conclusions and significance: It is therefore possible to hypothesize that the observed ciliated cells may be a new chondrocyte phenotype with sensory function. Motile cilia confer the ability to distinguish variations in synovial fluid chemical composition and, in addition, they perhaps may also play some role in the mechanism of sound transmission.     

转化生长因子β1和同种异体软骨细胞及高孔隙率聚乳酸复合物修复兔耳廓软骨缺损

目的　观察加入转化生长因子 β1(transforminggrowthfactor beta 1,TGF β1)的同种异体软骨细胞 /高孔隙率聚乳酸 (poly DL lactide,PDLLA)支架复合物修复兔耳廓软骨缺损的效果。 方法18只大白兔随机分为同种异体软骨细胞 /PDLLA复合物加TGF β1组 (复合物组 )、同种异体软骨细胞 /PDLLA复合物组 (复合物对照组 )和不用任何修复材料的空白对照组 ,每组 6只。分别于 4、12、18周取材 ,行HE和甲苯胺蓝染色 ,观察各组兔耳廓软骨缺损修复情况。结果　术后 18周 ,TGF β1复合物组修复软骨缺损的效果无论从大体标本观察或是病理组织学检查都比复合物对照组的修复效果好 ,空白对照组为纤维组织修复。结论　同种异体软骨细胞 /PDLLA复合物移植可形成组织工程化软骨修复兔耳廓软骨缺损 ,TGF β1可提高同种异体软骨细胞 /PDLLA复合物移植修复兔耳廓软骨缺损的质量。     

转化生长因子β1和同种异体软骨细胞及高孔隙率聚乳酸复合物修复兔耳廓软骨缺损

目的 观察加入转化生长因子-β1(transforming growth factor-beta1，TGF-β1)的同种异体软骨细胞／高孔隙率聚乳酸(poly-DL-lactide，PDLLA)支架复合物修复兔耳廓软骨缺损的效果。方法 18只大白兔随机分为同种异体软骨细胞／PDLLA复合物加TGF-β1组(复合物组)、同种异体软骨细胞／PDLLA复合物组(复合物对照组)和不用任何修复材料的空白对照组，每组6只。分别于4、12、18周取材，行HE和甲苯胺蓝染色，观察各组兔耳廓软骨缺损修复情况。结果 术后18周，TGF-β1复合物组修复软骨缺损的效果无论从大体标本观察或是病理组织学检查都比复合物对照组的修复效果好，空白对照组为纤维组织修复。结论 同种异体软骨细胞／PDLLA复合物移植可形成组织工程化软骨修复兔耳廓软骨缺损，TGF-β1可提高同种异体软骨细胞／PDLLA复合物移植修复兔耳廓软骨缺损的质量。