Development and potential of a biomimetic chitosan/type Ⅱ collagen scaffold for cartilage tissue engineering

Background Damaged articular cartilage has very limited capacity for spontaneous healing. Tissue engineering provides a new hope for functional cartilage repair. Creation of an appropriate cell carrier is one of the critical steps for successful tissue engineering. With the supposition that a biomimetic construct might promise to generate better effects, we developed a novel composite scaffold and investigated its potential for cartilage tissue engineering.Methods Chitosan of 88% deacetylation was prepared via a modified base reaction procedure. A freeze-drying process was employed to fabricate a three-dimensional composite scaffold consisting of chitosan and type Ⅱcollagen. The scaffold was treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Ultrastructure and tensile strength of the matrix were carried out to assess its physico-chemical properties. After subcutaneous implantation in rabbits, its in vivo biocompatibility and degradability of the scaffold were determined. Its capacity to sustain chondrocyte growth and biosynthesis was evaluated through cell-scaffold co-culture in vitro. Results The fabricated composite matrix was porous and sponge-like with interconnected pores measuring from 100－250 μm in diameter. After cross-linking, the scaffold displayed enhanced tensile strength. Subcutaneous implantation results indicated the composite matrix was biocompatible and biodegradable. In intro cell-scaffold culture showed the scaffold sustained chondrocyte proliferation and differentiation, and maintained the spheric chondrocytic phenotype. As indicated by immunohistochemical staining, the chondrocytes synthesized type Ⅱ collagen. Conclusions Chitosan and type Ⅱ collagen can be well blended and developed into a porous 3-D biomimetic matrix. Results of physico-chemical and biological tests suggest the composite matrix satisfies the constraints specified for a tissue-engineered construct and may be used as a chondrocyte carrier for cartilage tissue engineering.     

COA: Bone Morphogenetic Protein 2 Promoted Transforming Growth Factor β3 Induced Chondrogenesis of Human Osteoarthritic Synovium-Derived Stem Cells

Background Synovium-derived stem cells (SDSCs) with greater chondrogenic potential are attracting more considerable attention as a cell source for cartilage regeneration. The aim of this study was to investigate the effect of bone morphogenetic protein-2 (BMP-2) on transforming growth factor-beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system. Methods Nucleated cells isolated from human osteoarthritic synovium were plated at an optimal cell density to allow the selective proliferation of SDSCs. The clonogenicity, stem cell marker expression and multi-differentiation potential were determined by CFU assay, flow cytometry assay and specific staining including alizarin red S staining, Oil red staining and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium without or with TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type II, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type II (COL2A1), aggrecan (ACAN), SOX9, link-protein (HAPLN1), collagen type X (COL10A1) and BMP receptor II (BMPR-II). Results Cells isolated under the optimized culturing density (104/60cm2) showed clonogenicity and multi-differentiation potential. These cells were positive (>99% positive) for CD44, CD90, CD105 and negative (<10% positive) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Metachromatic staining of the extracellular matrix with Safarnin O was positive and the expression of collagen type II was detected. The combination of TGF-β3 and BMP-2 produced cell pellets with larger diameter and weight, produced more sGAGs, expression higher levels of collagen type II and chondrogenic markers, except COL10A1, than medium with TGF-β3 alone. Conclusions SDSCs could be isolated from human osteoarthritic synovium. Supplementation of BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.     

In vitro chondrogenesis of the goat bone marrow mesenchymal stem cells directed by chondrocytes in monolayer and 3-dimetional indirect co-culture system

Background  Cartilage injury has a very poor capacity for intrinsic regeneration. The cell-based treatment strategy for the cartilage repair using differentiated bone marrow mesenchymal stem cells (BMSCs) is, however, a promising approach to the chondral repair. This study was aimed to explore the chondrogenic potential of the goat BMSCs in the Transwell co-culture system and the poly-laetide-co-glycolide (PLGA) scaffolds.

Methods  The BMSCs were isolated from the goat iliac crest while the chondrocytes were obtained from the goat’s last costal cartilage. In the Transwell co-culture system, the BMSCs co-cultured with chondrocytes were designed as group A, whereas the goat’s BMSCs induced with the chondrogenic medium were group B. Both groups A and B were the experimental groups, while group C that only contained BMSCs was the control group. In the PLGA scaffolds co-culture system, BMSCs were seeded into the PLGA scaffolds, which were suspended in the 24-well plate, and the control group was established by presence or absence of chondrocytes at the bottom of the 24-well plate. Toluidine blue staining, Alcian blue staining, collagen II immunofluoresence, collagen II immunochemical staining, collagen I, collagen II, COL2a Q-PCR and osteopontin Q-PCR were used to examine the chondrogenic conditions as well as the expressions of chondrogenic and osteogenic genes.

Results  Cells isolated from the aspirates of the goat bone marrow proliferated rapidly and gained characteristics of stem cells in Passage 4. However, the differentiations of chondrocytes were not apparent in Passage 3. The results from Toluidine blue staining, collagen II immunofluoresence and PCR showed the transformation of BMSCs to chondrocytes in the Transwell co-culture system and PLGA scaffolds. Although the cartilage gene expressions were upgraded in both chondrogenesis group and co-culture system, the osteopontin gene expression, which represents osteogenic level, was also up-regulated.

Conclusions  The Transwell co-culture system and the PLGA scaffolds co-culture system can promote the chondrogenic differentiation of the goat’s BMSCs, while up-regulated osteopontin gene expression in the Transwell co-culture system implies the osteogenic potential of BMSCs.

     

Expression of Transforming Growth Factor β1 in Mesenchymal Stem Cells: Potential Utility in Molecular Tissue Engineering for Osteochondral Repair

Summary: The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor (TGF)-β1 genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. The full-length rat TGF-β1 cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418,a synthetic neomycin analog. The transient and stable expression of TGF-β1 by MSCs was detected by using immunohistochemical staining. The lipofectamine-mediated gene therapy efficiently trans fected MSCs in vitro with the TGF-β1 gene causing a marked up-regulation in TGF-β1 expression as compared with the vector-transfected control groups, and the increased expression persisted for at least 4 weeks after selected with G418. It was suggested that bone marrow-derived MSCs were sus ceptible to in vitro lipofectamine mediated TGF-β1 gene transfer and that transgene expression persist-ed for at least 4 weeks. Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, I.e.molecular tissue engineering, are put forward for the first time. As a new branch of tissue engineer-ing, it represents both a new area and an important trend in research. Using this technique, we have a new powerful tool with which: (1) to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and (2) to affect a better repair of full-thickness artic ular cartilage defects that occur as a result of injury and osteoarthritis.     

Synthesis and characterization of UPPE-PLGA-rhBMP2 scaffolds for bone regeneration

A novel unsaturated polyphosphoester(UPPE) was devised in our previous research,which is a kind of promising scaffold for improving bone regeneration.However,the polymerization process of UPPE scaffolds was unfavorable,which may adversely affect the bioactivity of osteoinductive molecules added if necessary,such as recombinant human bone morphogenetic protein-2(rhBMP2).The purpose of this study was to build a kind of optimal scaffold named UPPE-PLGA-rhBMP2(UPB) and to investigate the bioactivity of rhBMP2 in this scaffold.Furthermore,the cytotoxicity and biocompatibility of UPB scaffold was assessed in vitro.A W1/O/W2 method was used to fabricate PLGA-rhBMP2 microspheres,and then the microspheres were added to UPPE for synthesizing UPB scaffold.The morphological characters of PLGA-rhBMP2 microspheres and UPB scaffolds were observed under the scanning electron microscopy and laser scanning confocal microscopy.The cumulative release of UPB scaffolds was detected by using ELISA.The cytotoxicity and biocompatibility of UPB scaffolds were evaluated through examining the adsorption and apoptosis of bone marrow stromal cells(bMSCs) seeded on the surface of UPB scaffolds.The bioactivity of rhBMP2 in UPB scaffolds was assessed through measuring the alkaline phosphates(ALP) activity in bMSCs seeded.The results showed that UPB scaffolds sequentially exhibited burst and sustained release of rhBMP2.The cytotoxicity was greatly reduced when the scaffolds were immersed in buffer solution for 2 h.bMSCs attached and grew on the surface of soaked UPB scaffolds,exerting well biocompatibility.The ALP activity of bMSCs seeded was significantly enhanced,indicating that the bioactivity of rhBMP2 remained and still took effect after the unfavorable polymerization process of scaffolds.It was concluded that UPB scaffolds have low cytotoxicity,good biocompatibility and preserve bioactivity of rhBMP2.UPB scaffolds are promising in improving bone regeneration.     

Bone morphogenetic protein 2 promotes transforming growth factor β3-induced chondrogenesis of human osteoarthritic synovium-derived stem cells

Background Synovium-derived stem cells （SDSCs） with higher chondrogenic potential are attracting considerable attention as a cell source for cartilage regeneration. We investigated the effect of bone morphogenetic protein 2 （BMP-2） on transforming growth factor beta3 （TGF-β3）-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system. Methods The clonogenicity, stem cell marker expression and multi-differentiation potential of isolated SDSCs were determined by colony forming unit assay, flow cytometry and specific staining including alizarin red S, Oil red O and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium with or without TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type Ⅱ, sulfated glycosaminoglycan （sGAG） synthesis and mRNA expression of collagen type Ⅱ, aggrecan, SOX9, link-protein, collagen type X and BMP receptor Ⅱ. Results Cells isolated under the optimized culturing density （104/60 cm2） showed clonogenicity and multi-differentiation potential. These cells were positive （〉99%） for CD44, CD90, CD105 and negative （〈10%） for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Safranin O staining of the extracellular matrix was positive and the expression of collagen type Ⅱ was detected. Cell pellets treated with TGF-β3 and BMP-2 were larger in diameter and weight, produced more sGAGs, and expressed higher levels of collagen type Ⅱ and other chondrogenic markers, except COL10A1, than medium with TGF-β3 alone. Conclusions SDSCs could be isolated from human osteoarthritic synovium. Supplementation with BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.     

EXPRESSION OF BONE MORPHOGENETIC PROTEIN-1 IN FETAL HUMAN CARTILAGE AND BONE DURING ENDOCHONDRAL OSSIFICATION

The expression ot mRNA for bone morphogenetie protein-l(BMP-1) was investigated in fetal human cartilage and bone during the developing endoehondral ossification by using in situ hybridization. BMP-1 mRNA was localized in the hypertrophic choadrocytes, the cells of perichondrium, osteoblasts and marrow cells. The results suggest that BMP-1 may involve in cartilage and bone formation, and in regulation of calcification of growth plate. As a comparative study, the expression of TGF-β1 protein was investigated in cartilage and bone using immunohistoehemical staining, correlated expression of TGF-β1 and BMP-1 was found. The results suggest that BMP-1 and TGF-β1 act synergistically to influence endochroodral ossification.     

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.     

Glucan HBP-A Increase Type Ⅱ Collagen Expression of Chondrocytes in Vitro and Tissue Engineered Cartilage in Vivo

Objective:Although chondroprotective activities have been documented for polysaccharides,the potential target of different polysaccharide may differ.The study was aimed to explore the effect of glucan HBP-A in chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs in vivo,especially on the expression of type Ⅱ collagen.Methods:Chondrocytes isolated from rabbit articular cartilage were cultured and verified by immunocytochemical staining of type Ⅱ collagen.Chondrocyte viability was assessed after being treated with HBP-A in different concentrations.Morphological status of chondrocytes-alginate hydrogel constructs in vitro was observed by scanning electron microscope(SEM).The constructs were treated with HBP-A and then injected to nude mice subcutaneously.Six weeks after transplantation,the specimens were observed through transmission electron microscopy(TEM).The mRNA expressions of disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTs-5),aggrecan and type Ⅱ collagen in both monolayer culture and constructs were determined by real time polymerase chain reaction(PCR).The expression of typeⅡ collagen and matrix metalloproteinases-3(MMP-3) in chondrocyte monolayer culture was also tested through Western blot and enzyme linked immunosorbent assay(ELISA),respectively.Results:MMP-3 secretion and ADAMTs-5 mRNA expression in vitro were inhibited by HBP-A at 0.3 mg/mL concentration.In morphological study,there were significant appearance of collagen in those constructs treated by HBP-A.Accordingly,in both chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs,the expression of type Ⅱcollagen was increased significantly in HBP-A group when compared with control group(P0.001).Conclusions:The study documented that the potential pharmacological target of glucan HBP-A in chondrocytes monolayer culture and tissue engineered cartilage in vivo may be concerned with the inhibition of catabolic enzymes MMP-3,ADAMTs-5,and increasing of type Ⅱ collagen expression.     

Induction of chondrogenesis of adipose-derived stem cells by novel recombinant TGF-β3 fusion protein

A new type of TGF-β3 fusion protein with targeted therapy function was constructed,and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells (ADSCs).The recombinant pIRESEGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFP.LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively,and the recombinant plasmid of pIRES-EGFPLAP-MMP-mTGF-β3 was constructed,which was transferred to ADSCs.The ADSCs were cultured and divided in three groups:experimental group (MMP group),negative control group (no MMP) and non-transfection group.The morphological changes were observed microscopically,and the expression of proteoglycan and type Ⅱ collagen (ColⅡ) was detected by using Alcian blue staining and immunohistochemistry staining at 7th,14th and 21st day after culture.The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and sequencing analysis.The mTGF-β3 fusion protein was successfully expressed after transfection,and in the presence of the MMP,active protein mTGF-β3 was generated,which significantly promoted differentiation of ADSCs into chondrocytes and the expression of cartilage matrix.The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes,which would open up prospects for target therapy of cartilage damage repair in future.     

负载TGF-β1微球的壳聚糖-丝素支架复合骨髓间充质干细胞修复兔关节软骨缺损的实验研究

目的研制负载转化生长因子β1（transforming growth factor—β1,TGF—B。）壳聚糖微球的壳聚糖-丝素支架复合骨髓间充质干细胞（mesenchymal stem cells,MSCs）体外构建组织工程软骨,观察将其移植修复兔膝关节软骨缺损的效果。方法通过密度梯度离心法及贴壁培养法分离纯化MSCs,以抗兔CD14、CD44等单抗采用流式细胞仪进行MSCs表面抗原鉴定,获得纯化的MSCs。采用乳化交联法获得包裹TGF—B,壳聚糖缓释微球,并将该微球负载在冷冻干燥获得的壳聚糖一丝素支架上。将培养的MSCs种植到支架上,体外构建组织工程软骨,移植到兔关节软骨缺损处。移植空白支架的为实验对照组,移植体外构建的组织工程软骨为实验组。主要观察指标,流式检测的MSCs细胞表面标记情况,微球的形态,支架与细胞共培养后电镜下情况,支架移植后的兔关节修复情况及组织学检查。结果流式检测MSCs第4代及第9代细胞的CD14及CD44表面抗原的表达情况发现两代MSCsCD14表达基本呈阴性,CD44表达呈阳性,符合间充质干细胞的特性。扫描电镜观察所制备的微球基本呈球形,表面光滑,直径约为1μm,大小较均一,分散度好。细胞在壳聚糖-丝素三维支架上生长状态良好,电镜观察,可见支架孔隙内有细胞黏附生长。移植治疗后发现实验组修复明显好于对照组,实验组术后3个月实验组已经有较硬的类软骨组织填充修复,切片显示有软骨细胞规则排列,甲苯胺蓝染色为阳性,而对照组仅为纤维组织修复,切片显示为纤维组织或纤维软骨组织修复,软骨细胞排列紊乱,甲苯胺蓝染色阴性或弱阳性。结论负载TGF—β1壳聚糖微球的壳聚糖-丝素支架复合骨髓间充质干细胞体外复合培养后移植治疗修复兔膝关节软骨缺损,具有较好的疗效。     

兔实验性骨关节病髁突软骨细胞表达软骨基质分子的变化及白细胞介素1β的影响

目的 :研究外源性炎症因子白细胞介素 1β(interleukin 1β,IL 1β)对颞下颌关节骨关节病与正常髁突软骨细胞代谢活动的影响 ,探讨其在颞下颌关节骨关节病进展过程中所发挥的作用。方法 :采用 2 0 μg·L-1重组白介素1β(recombinedhumaninterleukin 1β ,rhIL 1β)刺激体外贴壁培养条件下的成兔正常成熟髁突软骨细胞与兔实验性颞下颌关节骨关节病模型髁突软骨细胞 ,RT PCR方法检测、比较细胞对软骨基质成分Ⅱ型胶原、蛋白多糖聚糖体、胶原酶以及内源性生长因子胰岛素样生长因子 1(insulin likegrowthfactor 1,IGF 1)和转移生长因子 β1(trans forminggrowthfactor β1,TGF β1)的mRNA表达变化。结果 :在 2 0 μg·L-1rhIL 1β的刺激下 ,(1)正常成熟髁突软骨细胞Ⅱ型胶原和软骨蛋白多糖聚糖体的表达均明显降低 ,胶原酶表达水平变化不明显 ;(2 )在IL 1β的作用下 ,骨关节病软骨细胞对Ⅱ型胶原和胶原酶的表达水平降低 ,对软骨蛋白多糖聚糖体的表达水平无明显影响 ;(3)IL 1β对正常和骨关节病髁突软骨细胞内源性生长因子IGF 1和TGF β1的表达均无明显影响。 结论 :蛋白多糖聚糖体的合成 ,导致髁突软骨病变发生 ,而且能不断干扰骨关节病髁突软骨细胞代谢 ,导致关节软骨基质环境进一步恶化。     

Preparation of gastro-resistant pellets containing chitosan microspheres for improvement of oral didanosine bioavailability

The purpose of this work was to introduce a new concept of coated pellets containing chitosan microspheres loaded with didadosine for oral administration, aiming at reducing the frequency of administration and improving the bioavailability by a suitable release profile. Chitosan microspheres were produced under fluidized bed, followed by extrusion and spheronization to obtain pellets with a mean diameter of about 1 mm. The pellets were then coated with Kollidon^® VA64 and Kollicoat^® MAE100P in water dispersion to depict a sustained release profile. Conventional hard gelatine capsules were loaded with these pellets and tested in vitro for their release profile of didadosine. Dissolution testing confirmed that chitosan microsphere pellets provides appropriate sustained release up to 2 h behavior for didanosine.     

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.     

壳聚糖作为缓释重组人骨形态发生蛋白-2载体的实验研究

目的:将壳聚糖作为重组人骨形态发生蛋白2(rhBMP2)的载体,通过体外释放实验观测rhBMP2在壳聚糖中释放的动力学过程,探讨壳聚糖对rhBMP2的缓释作用。方法:采用乳液冷冻干燥法制作rhBMP2/壳聚糖支架,单纯壳聚糖支架为对照组。对材料进行扫描电镜观察;将支架材料在磷酸盐缓冲液(PBS)中持续浸泡,通过高效液相色谱分析仪检测不同时间点浸出液中rhBMP2的含量,进行累积释放量的动态观察,绘制rhBMP2释放曲线。结果:支架材料的形态学观察呈疏松多孔海绵状;rhBMP2从支架材料中释放的动力学过程第1天表现为“爆发性”释放(33.2%);第27天rhBMP2出现快速释放;第814天释放速度减慢;第1530天出现缓慢而持久的释放,至1个月释放量达81.3%。结论:多孔壳聚糖支架对rhBMP2有缓慢而持久的体外释放作用,可作为rhBMP2的缓释载体,为rhBMP2持续发挥骨诱导作用提供初步的实验基础。     

软骨细胞与复合水凝胶的生物相容性研究

目的构建光固化壳聚糖与自固化海藻酸钠复合水凝胶体系,研究其与软骨细胞的体外生物相容性,探索其作为软骨组织工程支架的可行性。方法以三维多孔光固化壳聚糖水凝胶作为细胞的空间载体,利用自固化海藻酸钠水凝胶将软骨细胞胶封入光固化壳聚糖水凝胶支架,构成复合水凝胶-细胞复合体。采用扫描电子显微镜观察光固化壳聚糖的结构形貌以及细胞在支架内部的分布与形态;CCK8法绘制细胞增殖曲线;DAPI细胞核染色荧光显微镜观察细胞核形态变化,判断细胞活性。结果纯光固化壳聚糖水凝胶材料呈孔隙较为均匀一致的多孔结构,平均孔径为300μm左右。复合水凝胶能够模拟天然软骨细胞外基质环境,使细胞保持圆形生长状态;促进成软骨细胞的增殖,具有良好的生物相容性。结论复合水凝胶能够为软骨细胞提供一个类似天然的生活环境,并能够促进其增殖、生长,有望作为软骨组织工程支架应用于软骨缺损修复的实验研究。     

转化生长因子β1诱导时间对骨髓基质细胞体外成软骨分化的影响

目的：探讨转化生长因子β1（TGF-β1）诱导时间对骨髓基质细胞（BMSC）体外成软骨分化,及构建组织工程化软骨的影响。方法：将体外培养扩增的第二代猪BMSC按5．0×10^7个／ml的密度接种于聚羟基乙酸（PGA）制成的圆柱形支架材料上,第5天开始应用含TGF-β1（10μg/L）、IGF-I（50μg/L）和地塞米松（40μg/L）的软骨诱导液培养。按TGF-β1诱导时间的不同分为A（诱导2周）、B（诱导4周）、C（诱导6周）、D（诱导8周）和E（诱导10周）5组。体外培养10周后取材行大体观察、组织学和组织化学检测、聚合蛋白多糖（GAG）定量分析和生物力学测定,同时利用Western印迹和免疫组织化学进行Ⅱ型胶原表达的分析。结果：随诱导时间延长,软骨陷窝由周边开始逐渐向内部增多,排列渐趋均匀,蛋白聚糖等软骨特异性基质成分积聚。免疫组化染色及Western印迹结果均显示Ⅱ型胶原含量随诱导时间延长逐渐递增。这种情况至诱导6周后逐渐稳定,诱导6周和诱导10周间差异无统计学意义。诱导6周以上组弹性模量和抗压强度均明显高于A、B两组。结论：利用BMSC作为种子细胞构建组织工程化软骨,诱导持续时间与形成的软骨质量相关,6周的诱导时间基本可以形成具有良好组织结构、化学组成和力学性质的软骨组织。     

壳聚糖胶原支架复合胰岛素样生长因子-1在牙周组织工程中的应用

目的:观察复合胰岛素样生长因子-1(IGF-1)的壳聚糖胶原支架对体外培养的人牙周韧带细胞(PDLC)的影响,以探讨复合IGF-1的壳聚糖胶原支架在人PDLC增殖中的作用。方法:将体外培养PDLC接种于复合IGF-1的壳聚糖胶原支架上,通过MTT法测定对细胞增殖的影响,同时通过半定量PCR检测细胞mRNA合成变化。结果:PDLC在两种不同支架上的增殖及mRNA合成情况有显著性差异(P<0.5)。结论:该复合支架可以作为IGF-1的载体;复合在支架上的IGF-1对PDLC的粘附与增殖及细胞外基质的合成均有较好地促进作用。     

壳聚糖微球/小牛松质骨支架复合缓释体系的制备及体外生物活性评价

目的：通过乳液交联法合成壳聚糖微球并负载骨形态发生蛋白（bone morphogenetic protein-2,BMP-2）,再将其复合于脱细胞基质的小牛松质骨支架,制备壳聚糖微球/小牛松质骨支架复合缓释体系。方法： 使用红外光谱仪、扫描电镜对合成的复合缓释系统进行结构表征和形貌观察,并对微球的包封率和载药量进行检测。用动态浸泡的方法来检测BMP-2的体外释放特征,通过体外检测复合体系的细胞毒性和对细胞增殖的影响。结果： 壳聚糖发生了交联并成功包载了BMP-2,微球平均直径为5.982 μm,表面光滑,且成功负载于小牛松质骨支架,形成了新型的微球/支架药物缓释体系。缓释数据表明,5 mg微球在体外释放21 d,BMP-2逐渐释放,第21天时浓度仍达到（239.1±20.0） mg/L。体外测试表明,该缓释体系有利于小鼠前成骨细胞MC3T3-E1的生长,促进向成骨方向分化。结论： 壳聚糖微球/小牛松质骨支架复合缓释体系实现了BMP 2的生物学功能及其在骨修复部位的持续、缓慢释放,为骨组织缺损的修复治疗及骨组织工程支架材料的选择提供了依据。     

鼻用氟尿嘧啶壳聚糖微球的制备及其特性研究

目的: 以壳聚糖为载体材料,氟尿嘧啶为模型药物, 制备鼻腔给药脑靶向微球.方法: 以液体石蜡为油相,span-80为乳化剂,采用乳化化学交联技术制备氟尿嘧啶鼻用微球.正交实验设计优化制备工艺,动态透析法检测微球的体外释放特性及其影响因素.用微球吸水能力表示微球的溶胀度.测定纤毛输送速率来评价微球的黏膜粘附力.结果:所得微球形态良好,粒径分布较为均匀,平均粒径(43±4) μm,载药量38.5%±1.0%,包封率79.0%±1.8%.体外释放符合Higuichi方程Q=0.103 5t1/2 0.028 4 (r=0.996 5).壳聚糖微球具有良好的生物粘附性,可显著降低纤毛输送速率(P<0.01),有效地延长微球在鼻腔的滞留时间.结论:所优化的制备工艺稳定,包封率较高,适于鼻黏膜用氟尿嘧啶壳聚糖微球的制备.壳聚糖是良好的鼻用制剂的载体材料,应用前景广阔.