利用软骨细胞外基质来源的多孔支架与骨髓基质干细胞体外长期培养组织工程化软骨的试验研究

研究背景 关节软骨损伤临床常见,但损伤后自我修复能力极差,目前的修复方法均有其局限性。在先前的研究中,我们研制了关节软骨细胞外基质来源的多孔支架（Cartilage ECM-derived porous scaffold, CEDPS）并观察了并在裸鼠体内异位构建软骨。但在进一步可能的临床应用之前,应进一步评估其体外培养组织工程软骨的特点及可行性。 方法 本研究利用关节软骨细胞外基质来源的支架及骨髓基质干细胞体外长时间培养构建软骨组织。粉碎人关节软骨,脱细胞处理后差速离心法收集细胞外基质悬液,采用冷冻干燥技术制备三维多孔支架。扫描电镜及Micro-CT观察其微观结构,并进行细胞毒性试验,组织学观察,生化成分定量检测其胶原、氨基葡聚糖（GAG）、DNA含量,生物力学方法测量其干性及覆水状态下压缩弹性模量;骨髓基质干细胞经含TGF-β1, bFGF的条件培养基成软骨诱导后鉴定,种植到支架上,荧光显微镜及扫描电镜观察细胞黏附情况,Dead/Live免疫荧光染色观察支架内部细胞活性,体外培养1,3周后观察大体形态和组织学形态变化,同时行II型胶原免疫组织化学分析。 结果 制备的CEDPS支架无细胞碎片残留,软骨细胞外基质特异性染色阳性,具有相互贯通的三维孔隙结构;支架生化成分定量检测：总胶原含量为708.2?44.7μg/mg,GAG含量为254.7?25.9μg/mg;支架纵向压缩弹性模量E = 1.226?0.288MPa,覆水后压缩弹性模量E = 0.052?0.007MPa。体外培养的BMSCs-CEDPS复合体形成了类软骨样组织,Dead/Live染色表明支架内部均为活细胞,电镜检查结果表明细胞广泛均匀的分布在支架内部,呈圆形或椭圆形,在支架上增殖显著,细胞基质分泌明显。组织学结果表明蕃红花“O”、Ⅱ型胶原免疫组化染色阳性,表明随着培养时间的增加,细胞在支架中增殖显著,细胞外有大量基质分泌。 结论 CEDPS支架在生化组成和结构上与软骨细胞外基质成分类似,去细胞彻底,具有良好的生物力学特性,是一种较为理想的软骨组织工程支架载体;成软骨诱导的BMSCs与CEDPS支架在体外可初步构建类软骨样组织。     

In vitro cartilage production using an extracellular matrix-derived scaffold and bone marrow-derived mesenchymal stem cells

Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy （SEM）,micro-computed tomography （micro-CT）,histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was （708.2±44.7）μg/mg,with GAG （254.7±25.9） μg/mg.Mechanical testing showed the compression moduli （E） were （1.226±0.288） and （0.052±0.007） MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells （BMSCs） were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained pink,smooth and translucent cartilage-like tissue after 3 weeks of culture.We observed evenly distributed cartilage ECM proteoglycans and collagen type Ⅱ around seeded BMSCs on the surface and inside the pores throughout the scaffold.Conclusion This study stuggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.     

Repair of Rabbit Segmental Defects with the Hybrid RP Scaffolds

Objective The objective of this experiment is to observe the effects of hybrid RP scaffolds on repairing segmental bony defects. Methods BMSCs were seeded into the hybrid and original RP scaffolds in order to repairing 15 mm segmental defect in the radius of rabbits. Radiograph, Micro CT and histology were used to evaluate the scaffolds degrading rate and new bone formation. Results The results suggested that the apatite/collagen sponge composite coating could improve the RP scaffolds degrading rate and new bone forming in vivo. Bony union was observed in the hybrid scaffolds group after 1 year post-operation. Conclusion The PLGA/β-TCP scaffolds With apatite/collagen sponge composite coating is promising as a candidate 3D substrate for bone tissue engineering.     

In vitro and In vivo Evaluation of the Developed PLGA/HAp/Zein Scaffolds for Bone-Cartilage Interface Regeneration

Objective To investigate the effect of electronspun PLGA/HAp/Zein scaffolds on the repair of cartilage defects.
Methods The PLGA/HAp/Zein composite scaffolds were fabricated by electrospinning method. The physiochemical properties and biocompatibility of the scaffolds were separately characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), and fourier transform infrared spectroscopy (FTIR), human umbilical cord mesenchymal stem cells (hUC-MSCs) culture and animal experiments.
Results The prepared PLGA/HAp/Zein scaffolds showed fibrous structure with homogenous distribution. hUC-MSCs could attach to and grow well on PLGA/HAp/Zein scaffolds, and there was no significant difference between cell proliferation on scaffolds and that without scaffolds (P>0.05). The PLGA/HAp/Zein scaffolds possessed excellent ability to promote in vivo cartilage formation. Moreover, there was a large amount of immature chondrocytes and matrix with cartilage lacuna on PLGA/HAp/Zein scaffolds.
Conclusion The data suggest that the PLGA/HAp/Zein scaffolds possess good biocompatibility, which are anticipated to be potentially applied in cartilage tissue engineering and reconstruction.     

Study of vascular smooth muscle cell calcification induced by hyperphosphate and intervented by phosphonoformic acid

Objective:To evaluate the effects of different concentrations of phosphate on calcium deposition and osteocalcin level in cultured bovine aortic smooth muscle cell,investigate the mechanism of hyperphosphatemia to evoke calcification of vascular smooth muscle cell and observe the effects of phosphonoformic acid(PFA)in different concentrations on vascular calcification.Methods:The bovine aortic smooth muscle cells(BASMC)were cultured.Calcium deposition and the expression of osteocalcin of BASMC in different concentrations of phosphate(1.5mmol/Land2.0mmol/L)and PFA were determined by o-cresolphthalein complexone and radioimmunity methods,respectively.Osteocalcin mRNA expressions were determined by RT-PCR.Results:After six or nine days of BASMC cultured,the calcium deposition in Pi 2.0 mmol/L group was more than that in Pi 1.5 mmol/L group[(77.187±11.692)μg/(mg·protein)vs(25.768±1.750)μg/(mg·protein),P＜0.01 and(125.399±16.677)μg/(mg·protein)vs(29.046±2.635)μg/(mg·protein),P＜0.01 respectively].The calcium deposition was dependent on time and dosage of phosphate treatment.After 72 h culture the osteocalcin in Pi2.0 mmol/L group was more than that in Pi1.5 mmol/L group[in supernatant,(1.503±10-2±2.601×10-3)ng/(μg·protein)vs(2.981×10-3±8.382×10-4)ng/(μg·protein),P＜0.001],the same was found in osteocalcin mRNA expression[OC/GAPDH,(1.906±0.132)vs(0.748±0.037),P＜0.001].Compared to Pi 1.5mmol/L group,bovine smooth muscle cells(BSMC)cultured in media containing Pi 2.0 mmol/L phosphate levels increased calcium deposition[On day 6,(77.187±11.692)μg/(mg·protein)vs (25.768±1.750)μg/(mg·protein),P＜0.001].Elevated phosphate treamaent of BSMCs also enhanced the expression of the osteoblastic differentiation marker osteocalcin[On day 3,Pi 2.0 mmol/L group vs Pi 1.5mmol/L group,(1.503×10-2±2.601×10-3)ng/(μg·protein)vs(2.981×10-3±8.382×10-4)ng/(ug·protein),P＜0.001].PFA decreased ciacium deposition and osteocalcin expression statistically[Pi 2.0 mmol/L PFA1.0 mmol/L group vs Pi 2.0 mmol/L group,ciacium deposition,(37.729±5.899)μg/(mg·protein)vs(77.187±11.692)μg/(mg·protein),P＜0.001];Osteocalcin in supernatant,(4.529±10-3±1.250×10-3)ng/(μg·protein)vs(1.503×10-2±2.601×10-3)ng/(μg·protein),P＜0.001;osteocalcin mRNA expression,OC/GAPDH,(0.642±0.092)vs(1.89±0.165),P＜0.01].Conclusion:Hyperphosphate may directly promote calcium deposition and the osteocalcin expression of BASMCs.It may be a new explanation for the phenomenon of vascular calcification in hyperphosphatemic conditions.Hyperphosphatemia is an independent factor to stimulate vascular calcification.PFA can inhibit calcium deposition and osteocalcin expression induced by elevated phosphate.PFA may be a new medicine to treat vascular calcification induced by elevated phosphate.     

Dilated intercellular spaces in gastroesophageal reflux disease patients and the changes of intercellular spaces after omeprazole treatment

Background Gastroesophageal reflux disease （GERD） is a common disorder. Dilation of intercellular spaces of esophageal epithelium has been revealed at transmission electron microscopy both in the rabbit acid-perfused esophagus and in esophageal biopsies from GERD patients. This study aimed to observe the changes of the intercellular spaces of squamous epithelium of lower esophagus in patients with GERD and the changes of intercellular spaces of patients with erosive esophagitis （EE） before and after omeprazole treatment.
Methods Outpatients having GERD symptoms for more than 3 months and volunteers were collected. All of them underwent gastroendoscopy and 24-hour ambulatory pH monitoring. Biopsies were taken from the lower esophagus （2 cm above Z-line） for electron microscope examination. Five healthy volunteers, six non-erosive reflux disease （NERD） patients, and five EE patients were enrolled. Intercellular spaces of GERD patients and controls were calculated. Then we selected 20 patients with EE diagnosed by gastroendoscopy. All of them were treated with omeprazole （Losec, 20 mg bid） for 4 weeks then underwent gastroendoscopy again. Biopsies were taken from 2 cm above Z-line for electron microscope examination. All the patients completed the questionnaire about reflux symptoms before and after treatment.
Results Intercellular spaces of esophageal epithelial cell in volunteers, NERD patients and EE patients were （0.37±0.07）μm, （1.31±0.08）μm, and （1.33±0.14）μm, respectively, with significant differences between the control group and the NERD group （P=0.000）. In the 20 EE patients, the mean intercellular space before treatment was （1.14±0.15） μm After treatment the intercellular space was （0.51±0.18）μm, a significant difference compared with pre-treatment measurements （P=0.000）. Conclusions Dilated intercellular spaces （DIS） were seen in both NERD and EE cases. The dilated intercellular spaces of esophaaeal epithelium in EE patients could be recove     

Effect of Yiqi Bushen Granule (益气补肾颗粒) on the Peripheral Natural Killer Cell and γ δ T-cell in the Patients with Minimal Residual Leukemia

Objective: To analyze the changes in peripheral natural killer T-cells (NKT) and gammadelta T-cells (γδT-cell) in patients with minimal residual leukemia (MRL) before and after being treated with Yiqi Bushen Granule (益气补肾颗粒, YBG) in order to determine their significance in prognosis of the disease. Methods: Before and after treatment, the changes in 36 patients (16 males and 20 females) receiving long-term (more than 3 months) YBG therapy were analyzed using multi-parameter flow cytometry, with 34 healthy persons (19 males and 15 females) acting as controls. Results: The absolute value and percentage of NKT cells and γδT-cells were all significantly raised after treatment, for NKT cells, 0.52%±0.39% to 0.83%±0.66% and 7.25±7.77 cell/μL to 12.86±11.99 cell/μL, for γδT-cells, 6.08%±3.03% to 7.24%±2.78% and 83.97±48.09 cell/μL to 110.53±54.12 cell/μL, respectively (P0.05 or P0.01). Conclusion: YBG could regulate the immune function and elevate the amount of NKT cells and γδT-cells, thus to kill or suppress the residual leukemic cell in the body, which might be one of the mechanisms of YBG in prolonging the disease-free survival in MRL patients.     

Effect of electroacupuncture preconditioning on serum S100β and NSE in patients undergoing craniocerebral tumor resection

Objective：To investigate the effect of electroacupuncture preconditioning on the serum level of S100 calcium-binding protein beta（S100β）and neuron-specific enolase（NSE）in patients undergoing craniocerebral tumor operation.Methods：A total of 32 patients,who would go through craniocerebral tumor resection under general anesthesia,were randomly assigned to two groups,16 in each group.Patients in the electroacupuncture（EA）group received electroacupuncture on Fengfu acupoint（Du16）and Fengchi acupoint （GB20）for 30 min,2 h before operation.The stimulus is 1-4 mA with a density wave frequency of 2/15 Hz. Patients in the control group received no pretreatment.Anesthesia was maintained with remifentanil at the dose of 4-8 mg/kg per hour,pumped intravenous drip of vecuronium at 1.0-2.0μg/kg each hour,and discontinuous intravenous dripped with vecuronium bromide at 0.5-1 mg.The serum levels of S100βand NSE were measured with ELISA before operation,before skin incision,after tumor removal,at the end of operation,and at 24 h after operation.Results：The serum level of S100βand NSE did not change before skin incision.The serum level of NSE increased significantly and the level of S100βincreased insignificantly after the tumor resection. The serum levels of S100βand NSE in the EA group and the control group were 1.16±0.28μg/L vs 1.47±0.33μg/L,24.7±13.3μg/L vs 31.4±14.1μg/L at the end of the operation,respectively.Twenty-four h after operation,the correspondence indices were 1.18±0.31μg/L vs 1.55±0.26μg/L,and 25.5±12.4μg/L vs 32.4±11.7μg/L.The two indices at these two time points were significantly increased than those before operation, respectively（P〈0.05）.At the end of the operation and 24 h post-operation,the serum levels of S100βand NSE in the EA group were significantly lower than those in the control group（P〈0.05）.Conclusion：Electroacupuncture Fengchi and Fengfu for 30 min before craniocerbral tumor operation could decrease the serum level of S100βand NSE,thus may have potential protective effect on brain damage,which needs to befurther studied.     

Effect of perindopril and metoprolol on left ventricular hypertrophy and performance in essential hypertension.

The effects of perindopril and metoprolol on left ventricular hypertrophy (LVH) and function were studied in 47 essential hypertensive patients with LVH. Previous antihypertensive drugs were discontinued for at least 2 weeks, after which patients were randomly divided into 2 groups. 25 subjects were treated with perindopril 4 to 8 mg once daily in the morning (Group A) and 22 subjects with metoprolol 25 to 62.5 mg twice daily (Group B). The subjects were evaluated before and after 4 and 8 weeks of treatment by use of echocardiography. Before treatment LV mass indexes (LVMI) of two groups were respectively 143.2 ± 21.3 g / m2 and 140.6 ± 23.7 g / m2 (P>0.05). In Group A, reduction of LVMI occurred after 4 weeks of treatment, and more pronounced after 8 weeks (from 143.2 ± 21.3 g / m2 to 126.6 ± 15.3 g / m2, P< 0.001), whereas reduction of LVMI occurred only after 8 weeks in Group B (from 140.6 ± 23.7 g / m2 to 133.4 ± 13.2 g / m2, P< 0.001). In addition, there was a significant (P<0.05) difference i     

松质骨骨基质明胶复合软骨细胞构建组织工程软骨

目的 观察软骨细胞在同种异体松质骨骨基质明胶(BMG)上的生长、增殖和分化,探讨用松质骨BMG与软骨细胞体外构建组织工程软骨的效果.方法 分离1月龄兔关节软骨细胞,扩增后种植在松质骨BMG上体外构建组织工程软骨.于1、2、4、6周取材进行组织学、Ⅱ型胶原免疫组织化学及透射电镜观察.结果 软骨细胞在BMG上生长良好,分泌蛋白聚糖和胶原.随培养时间延长,细胞增殖,松质骨BMG空隙内细胞数量增多,软骨陷窝形成,基质分泌增强.培养6周时软骨细胞在BMG表面及孔隙内形成软骨样组织,免疫组织化学染色显示细胞周围基质富含Ⅱ型胶原,分布均匀:透射电镜显示软骨细胞超微结构正常,细胞周围有大量基质产生.结论 同种异体松质骨BMG可以促进软骨细胞的生长增殖,维持软骨细胞的分化表型;在体外成功构建了组织工程软骨,它是一种较好的软骨组织工程支架材料.

Abstract：

Objective To evaluate the use of cancellous bone matrix gelatin (BMG) combined with chondrocytes in constructing tissue-engineered cartilage by observing the growth, proliferation and differentiation of chondrocytes on allogeneic cancellous BMG. Methods The articular chondrocytes isolated from a 1-month-old rabbit were multiplied to a monolayer and seeded onto cancellous BMG to construct tissue-engineered cartilage in vitro during a period of 6 weeks. Samples were taken from the construct after 1, 2,4, and 6 weeks of culture and evaluated by histology, immunohistochemistry and transmission electron microscopy (TEM). Results The chondrocytes excreted matrix proteoglycan and collagen on cancellous BMG. With the prolongation of the culture time, the cells proliferated in the construct and the cells in the lacunae increased. Numerous chondrocytes were present the central region of the cancellous BMG and surrounded by extracellular matrix. By 6 weeks of culture, the BMG was covered with 15-20 layers of chondrocytes and cartilaginous tissue occurred in the pores throughout the cancellous BMG. Immunohistochemical staining showed rich and evenly distributed type Ⅱ collagen around the chondrocytes, and TEM revealed an ultrastructure of the chondrocyte similar to that of native chondroctyes, with abundant extracellular matrix produced around the cells. Conclusion Tissue-engineered cartilage can be constructed in vitro using allogeneic cancellous BMG combined with chondrocytes. Allogeneic cancellous BMG serves as a good scaffold material for tissue-engineered cartilage to promote the growth and proliferation of the seeded chondrocytes and allows maintenance of the differentiation phenotype of the cells.     

Evaluation of an extracellular matrix-derived acellular biphasic scaffold/cell construct in the repair of a large articular high-load-bearing osteochondral defect in a canine model

Background  Osteochondral lesion repair is a challenging area of orthopedic surgery. Here we aimed to develop an extracellular matrix-derived, integrated, biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically-induced bone marrow-derived mesenchymal stem cells (BMSCs) in the repair of a large, high-load-bearing, osteochondral defect in a canine model.

Methods  The biphasic scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and characterized by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). Osteochondral constructs were fabricated in vitro using chondrogenically-induced BMSCs and a biphasic scaffold, then assessed by SEM for cell attachment. Osteochondral defects (4.2 mm (diameter) × 6 mm (depth)) were created in canine femoral condyles and treated with a construct of the biphasic scaffold/chondrogenically-induced BMSCs or with a cell-free scaffold (control group). The repaired defects were evaluated for gross morphology and by histological, biochemical, biomechanical and micro-CT analyses at 3 and 6 months post-implantation.

Results  The osteochondral defects of the experimental group showed better repair than those of the control group. Statistical analysis demonstrated that the macroscopic and histologic grading scores of the experimental group were always higher than those of the control group, and that the scores for the experimental group at 6 months were significantly higher than those at 3 months. The cartilage stiffness in the experimental group (6 months) was (6.95±0.79) N/mm, 70.77% of normal cartilage; osteochondral bone stiffness in the experimental group was (158.16±24.30) N/mm, 74.95% of normal tissue; glycosaminoglycan content of tissue-engineered neocartilage was (218±21.6) μg/mg (dry weight), 84.82% of native cartilage. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at 3 and 6 months, with no significant difference between the experimental and control groups.

Conclusion  The extracellular matrix-derived, integrated, biphasic scaffold shows potential for the repair of large, high-load-bearing osteochondral defects.

     

COA:细胞外基质来源的双层支架的研制及其修复犬膝关节负重区骨软骨缺损的实验研究

背景与目的 骨软骨损伤常见且修复困难,本研究探讨采用软骨细胞外基质（CECM)与脱细胞骨基质（ACBM)为材料制作新型组织工程骨软骨双层支架的可行性,并检测其联合骨髓基质干细胞（bone marrow mesenchymal stem cells,BMSCs）修复犬膝关节负重区骨软骨联合缺损的疗效。 方法 ⑴新型组织工程骨软骨双层支架的骨部分以犬松质骨制备的ACBM为原料,软骨部分以人CECM为材料：将天然软骨粉碎成微丝并脱细胞处理后配成30 g/L的乳状悬液,将ACBM浸于盛CECM悬液的模具中,采用冷冻冻干法制备CECM/ACBM双层支架并交联。进行组织学、扫描电镜、Micro-CT观察,测定支架孔隙率,采用MTT法分析支架浸提液毒性。⑵ 将成软骨诱导的BMSCs种植到双相支架上体外构建组织工程骨软骨复合体,并以此复合体修复犬膝关节股骨髁负重区骨软骨缺损,分为细胞-双相支架组(实验组)和单纯支架组（对照组）,分别在3和6个月时取材,根据大体、组织学、生物力学、生物化学、Micro-CT等检测结果进行半定量或定量评估。 结果 支架评估：组织学显示双层支架去细胞彻底,无细胞碎片残留;CECM部分番红O及Ⅱ型胶原免疫荧光染色呈阳性。扫描电镜及Micro-CT观察显示支架内孔洞相互贯通呈海绵状,CECM部分孔径(155±34）μm,孔隙率为91.3%±2%;ACBM部分具有天然骨的孔径和空隙率,骨软骨部分结合良好。MTT法显示,不同浓度支架浸提液与对照培养液吸光度值比较,差异无统计学意义（P>0.05）。在修复犬膝关节骨软骨缺损的实验中,结果显示两组以纤维软骨或透明软骨对缺损有不同的修复,大体以及组织学评分表明实验组明显优于对照组,生物力学测试表明6月实验组修复软骨的刚度为正常膝关节软骨的70.1%,与生物化学分析结果一致;软骨下骨的刚度达到正常膝关节的74.96%。Micro-CT结果表明实验组与对照组软骨下骨重建不具备明显差异。结论 CECM /ACBM骨软骨双层支架保留了骨、软骨的细胞外基质成分,具备良好的孔径和孔隙率,骨-软骨两层间结合良好,无毒,生物相容性良好,可作为支架载体用于组织工程骨软骨复合体的构建。骨软骨双相支架复合成软骨诱导的BMSCs能成功修复犬膝关节负重区的骨软骨缺损,     

胶原凝胶包埋软骨细胞接种骨胶原基质支架体外构建组织工程软骨

目的探讨复合应用胶原凝胶和骨胶原基质（BCM）支架作为软骨细胞载体体外构建组织工程软骨的有效性和可行性.方法将胶原凝胶包埋的软骨细胞接种BCM支架并在体外培养,应用倒置相差显微镜和扫描电镜观察软骨细胞的粘附、生长和增殖情况,培养14d,行HE、TB（甲苯胺蓝）染色观察软骨组织形成情况.结果软骨细胞在支架上粘附、生长和增殖良好,体外培养14d能形成较成熟的软骨组织.结论胶原凝胶复合BCM支架可作为软骨细胞的载体体外构建组织工程软骨.     

Ⅱ型胶原糖胺聚糖复合支架对 hUCM SCs成软骨诱导的实验研究

目的：探讨以人脐带间充质干细胞（hUCMSCs）作为软骨组织工程种子细胞,Ⅱ型胶原复合糖胺聚糖支架材料作为细胞载体及其细胞‐支架复合体成软骨的可行性。方法制备Ⅱ型胶原复合糖胺聚糖多孔支架,电镜观察测定支架材料的孔径、孔隙率和亲水性,对支架材料进行相应的组织学分析。培养鉴定 P3代的hUCMSCs ,将hUCMSCs混悬液接种到Ⅱ型胶原复合糖胺聚糖支架上,不加诱导剂进行培养,3周后取出样品行甲苯胺蓝、番红精O染色,Ⅱ型胶原免疫组织化学染色及扫描电镜观察。结果第3代hUCMSCs高度表达CD29、CD105等间充质细胞标志,几乎不表达CD34等内皮细胞、造血细胞标志。Ⅱ型胶原复合糖胺聚糖支架材料呈白色多孔泡沫状,孔隙率为（91．8±2．17）％,孔径110～230μm ,分布均匀,相互贯通。吸水膨胀率为（213．71±1．31）％,亲水性良好。甲苯胺蓝、番红精O及Ⅱ型胶原免疫组织化学染色呈阳性。细胞‐支架复合体在培养过程中可观察到有软骨样组织形成并逐步增多,甲苯胺蓝、番红精O及Ⅱ型胶原免疫组织化学染色均呈阳性,电镜观察显示细胞在支架上增殖活跃,与材料黏附紧密,可见软骨样细胞及其周围大量交错连接的胶原纤维。结论 hUCMSCs与Ⅱ型胶原复合糖胺聚糖支架材料复合,可在体外不经诱导而初步构建组织工程软骨,为软骨损伤的修复奠定了一定的实验基础。     

应用明胶甲基丙烯酰胺低温3D打印组织工程软骨

目的：探讨应用低温3D打印制作的明胶甲基丙烯酰胺（gelatin methacrylamide,GelMA）支架能否构建高质量组织工程软骨。方法：经预冷处理后,利用低温沉积3D打印技术制备低浓度GelMA支架。大体观察、扫描电镜观察支架宏观、微观结构,并对水凝胶进行流变学和生物力学检测,以DMEM培养液培养大鼠骨髓间充质干细胞作为对照,细胞增殖（CCK?8法）和细胞活性（Live/Dead染色法）检测支架生物相容性,组织学染色定性分析支架成分。裸鼠皮下包埋支架,苏木素?伊红（HE）染色、阿利新蓝染色、番红染色评估软骨细胞外基质结构。结果：大体观察和扫描电镜观察示5％GelMA支架孔径均一,结构规整。与纯GelMA相比,添加细胞的支架力学强度稍强。骨髓间充质干细胞能在支架上黏附并增殖。体内体外组织学染色均表明构建的组织工程软骨在大小、色泽、氨基葡糖聚糖分泌等方面均满足组织工程软骨要求。结论：5％GelMA生物墨水可以在低温条件进行3D打印并成功构建组织工程软骨。     

含天然钙化层的骨软骨材料修复猪膝关节软骨缺损

目的 构建含和不含天然钙化层的修复材料,比较两者在猪膝关节软骨缺损修复中的效果,探讨钙化层在骨软骨组织工程中的重要性.方法 选取普通实验猪膝关节骨,软骨通过Ⅱ型胶原水凝胶冻干技术和天然骨软骨脱细胞技术构建含有钙化层和无钙化层的骨软骨支架,以贵州小香猪自体骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)为种子细胞,构建移植修复材料.30只小香猪按随机数字表法分为空白对照组、无钙化层组、含钙化层组(n=10).双膝关节滑车骨软骨缺损造模,直径8 mm,深至软骨下骨,植入对应的修复材料.分别于12、24周取材,采用大体、体式显微镜观察,以及MRI检测缺损填充情况,HE染色、番红0-固绿染色,以及Ⅰ、Ⅱ型胶原蛋白免疫组化染色观察新生修复物的组织类别,O'Driscoll组织学评分评价修复效果.结果 各组术后24周修复效果较12周均有改善.大体和体式显微镜观察,24周空白对照组新生组织部分填充缺损,为纤维组织,软骨表面凹陷深大;无钙化层组移植物填充缺损,软骨表面凹陷;含钙化层组缺损填充较好,表面微凹陷,三层结构可见,与宿主组织整合佳.组织学观察提示空白对照组为纤维组织填充修复;无钙化层组为骨和纤维软骨修复,三层结构不明显;含钙化层组钙化层结构清晰,移植物与宿主整合,软骨表面微凹陷,透明软骨修复.O'Driscoll组织学评分,12、24周空白对照组分别为(3.80±0.83)、(5.50 ±0.52)分,无钙化层组分别为(10.30±0.63)、(14.20±0.68)分,含钙化层组分别为(15.10±0.58)、(18.80±0.87)分,各组评分差异均有统计学意义(P＜0.01),含钙化层组评分最高.结论 含有天然钙化层的骨软骨支架,在猪骨软骨缺损修复中取得了很好的修复效果,明显优于无钙化层支架和空白对照,是今后软骨组织工程支架设计的重要参考.     

壳聚糖-明胶多孔复合支架构建自体组织工程化软骨组织的实验研究

目的探讨壳聚糖一明胶多孔复合支架作为软骨组织工程支架的可行性。方法消化分离猪耳廓软骨细胞,接种于自制壳聚糖一明胶多孔复合支架上培养1周,将细胞一生物支架复合物种植于猪自体腹外侧壁皮下,第10、16周取材,分别从大体、组织学、Ⅱ型胶原免疫组化和生物化学对再生软骨组织进行评价。结果HE染色见10周时有软骨组织形成,所形成的软骨组织见软骨细胞均匀分布,包埋在软骨陷窝内,还可见未降解的支架材料。16周时软骨组织完成成熟,软骨细胞包埋在软骨陷窝内,支架材料完全降解,结构与正常软骨组织相似。Masson’s trichrome染色见所形成的软骨组织间质中都有被染成绿色的胶原分布。Vehoeff染色见16周时形成的软骨间质内含大量被染成蓝黑色弹性纤维。免疫组化证实形成的软骨组织有Ⅱ型胶原分布,生物化学证实所形成的软骨组织的蛋白聚糖含量与正常猪耳软骨的含量接近。结论利用自制壳聚糖一明胶多孔复合支架上可在具有免疫功能的自体动物内形成软骨组织,但形成的软骨不充分,壳聚糖一明胶多孔复合支架有以作为软骨组织工程支架的应用前景。     

软骨细胞与骨髓间充质干细胞隔离共培养构建组织工程化软骨

目的探讨隔离共培养条件下,软骨细胞诱导骨髓间充质干细胞（BMSCs）向软骨细胞分化并形成软骨组织的能力。方法分别提取并培养兔BMSCs和耳软骨细胞,以5．0×10^7／ml的终浓度接种于聚羟基乙酸／聚乳酸（PGA／PLA）支架上并置于Transwell室内。实验组Transwell下方为贴壁软骨细胞,对照组Transwell下方为DMEM培养液（含10％胎牛血清）。两组BMSCs-支架复合物体外培养8周后植入裸鼠皮下,继续培养6周后取材。通过大体观察、逆转录-聚合酶链反应（RT-PCR）检测、组织学及免疫组织化学等相关检测对新生组织进行评价。结果体外培养期间,实验组和对照组BMSCs在支架上黏附良好并能分泌细胞外基质,RT-PCR检测示实验组Ⅱ型胶原和蛋白聚糖有较强的表达,而对照组两者的表达较低或检测不到。裸鼠皮下培养6周,实验组BMSCs-支架复合物能基本保持原形,组织学染色及免疫组织染色显示新生组织有明显的软骨陷窝形成并表达软骨特异性细胞外基质。对照组BMSCs-支架复合物逐渐皱缩变形,未见形成软骨样组织。结论采用Transwell隔离共培养的方法,软骨细胞能够诱导BMSCs向软骨细胞分化。     

PVA/ι-CA软骨组织工程支架对ATDC-5细胞生物学行为和组织相容性的影响

目的：探讨PVA/ι-CA软骨组织工程支架对ATDC-5细胞生物学行为的影响,评价其用于构建组织工程软骨的可行性。方法：采用物理共混技术和反复冷冻-解冻方法,将聚乙烯醇（PVA）和卡拉胶按照一定比例制作成复合支架材料PVA/ι-CA并测定其孔径和孔隙率。将ATDC-5细胞接种于支架材料,观察细胞的黏附生长情况;免疫组织化学法和免疫荧光法检测ATDC-5细胞中Ⅱ型胶原的表达情况;甲苯胺蓝检测ATDC-5细胞形态表现;扫描电镜（SEM）下观察细胞生长及细胞外基质（ECM）分泌情况。将ATDC-5细胞分为阴性对照组（加入空白培养液）和实验组（加入含材料的培养液）,MTT法检测支架材料上2组ATDC-5细胞的增殖率。将支架材料植入SD大鼠皮下,评价该支架材料的组织相容性和血管化能力。结果：PVA/ι-CA软骨组织工程支架平均孔隙率为（86.88±3.88）%,平均孔径为20~40 μm。HE染色,ATDC-5细胞在支架材料上贴附生长良好,呈多角形,形态饱满;免疫组织化学和免疫荧光染色,ATDC-5细胞分泌Ⅱ型胶原蛋白;甲苯胺蓝染色,ATDC-5细胞在支架材料上保持软骨细胞的特性,随着培养时间的延长,支架材料上阳性细胞数量明显增加;ATDC-5细胞与支架材料共培养7 d时,SEM下可见少量细胞呈多角形;培养14 d时细胞数量增多,形态饱满、相互融合,伸入材料间形成锚状结构,牢固地黏附于材料表面;培养21~28 d时材料上附着的细胞分泌大量的ECM包裹材料;ATDC-5细胞在材料上于7~14 d增殖较快,21~28 d增殖较慢,实验组增殖率与对照组比较差异无统计学意义（P>0.05）。皮下包埋早期有轻微的炎症反应,随着时间延长而消退;后期有血管化发生,材料降解吸收比较缓慢。结论：PVA/ι-CA支架材料有望成为的软骨组织工程支架材料。     

黄芪-壳聚糖/聚乳酸多孔支架对犬骨髓基质细胞生物学行为的影响

目的:观察黄芪-壳聚糖/聚乳酸、壳聚糖/聚乳酸多孔支架复合材料对犬骨髓基质细胞(BMSCs)的黏附、生长及分化的影响,寻找较合适的牙周骨组织工程复合物支架材料。方法:将诱导分化的犬BMSCs分别与黄芪-壳聚糖/聚乳酸、壳聚糖/聚乳酸支架体外培养。第5天取材,测定2种材料的吸附率,用扫描电镜观察超微结构,以免疫组织化学检测I型胶原,放射免疫法检测骨钙素的分泌情况。结果:BMSCs与2组材料均能形成良好贴附,黄芪-壳聚糖/聚乳酸复合材料上贴附的细胞吸附率、Ⅰ型胶原及骨钙素的分泌量高于壳聚糖/聚乳酸组。结论:黄芪-壳聚糖/聚乳酸能促进BMSCs生长、分化及基质分泌,是一应用前景良好的组织工程骨构建复合材料。