静电纺丝纳米纤维支架结合脂肪干细胞构建组织工程尿道
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摘要
组织工程是一门融合了多种学科知识的综合性学科,是再生医学的重要组成部分。组织工程的研究方法很多,但其基本策略包括种子细胞的选择、支架材料的研制和细胞与支架的相互作用。
脂肪组织是一种新的成体干细胞来源。因为取材方便、侵入小、产量大等优点,脂肪干细胞已成为组织工程研究中一线种子细胞。许多研究表明,脂肪组织的生物学特性与解剖部位有关。脂肪干细胞来源于脂肪组织,其取材部位对细胞特性是否也存在这种相关性,目前尚无明确结论。
人工合成支架在组织工程研究中的地位越来越重要。高分子聚合物因其稳定而可控的特性应用广泛。聚乳酸质脆,降解度高,而聚己内酯质韧,降解度低,两者不同比例的复合物可提供各种性状的支架。我们利用静电纺丝技术制备了两者的共混物,这种混合物支架由纳米级纤维编织成三维结构,适合模拟细胞外基质。
在本研究中,我们比较了不同解剖部位来源的脂肪干细胞特性,结果显示兔腹股沟皮下部位是最佳取材部位。同时研究了聚乳酸/聚己内酯共混支架中两种成分的混合比例对其性能的影响,发现按1/1比例制备的支架表现出更好的性能。在上述研究的基础上,我们以脂肪干细胞为种子细胞,以1/1比例的静电纺丝聚乳酸/聚己内酯共混纤维为支架,通过细胞在支架上的平滑肌诱导,探索其构建组织工程化尿道的可行性。
第一部分不同解剖部位来源的脂肪干细胞生物学特性比较
目的研究并比较脂肪干细胞取材的解剖部位对脂肪干细胞生物学特性的影响,为脂肪干细胞作为组织工程种子细胞的选择策略提供理论基础。
方法5只同龄雌性新西兰大白兔从出生起在同等环境中饲养,4月龄时用于实验研究,平均体质量约2.4kg(2.37～2.42kg).经耳缘静脉麻醉后,每只兔在无菌术下于3个部位手术切取脂肪组织团块,包括腹股沟皮下、颈背部皮下和腹膜后肾周部位。所得脂肪组织经常规分离培养,按来源部位分为三个实验组培养。三组脂肪干细胞分别用以下实验方法比较生物学行为：(1)倒置相差显微镜下形态观察和流式细胞术检测细胞表面标志物CD105和CD166以鉴定细胞；(2)细胞计数法和MTT法检测细胞增殖能力；(3)标准曲线法和活死细胞染色检测细胞活力；(4)组织化学染色(碱性磷酸酶染色和油红染色)、Real-time PCR和Western blot检测成骨、成脂诱导分化能力。
结果(1)细胞鉴定：①三个部位的脂肪干细胞均呈典型间充质干细胞形态,肉眼观察未见明显差异；②流式检测三组细胞CD105、CD166标志物,其阳性率未见统计学差异。(2)增殖能力：①4天时皮下组织腹股沟和颈背部细胞数量均高于腹膜后肾周组,差异有显著性(均p<0.05)。两组皮下组织组之间无差异(p>0.05)。7天时三组细胞数量未检出统计学差异(p>0.05)。②单因素方差分析比较三组细胞MTT法测定的增殖曲线,腹股沟皮下和颈背部皮下组细胞都较腹膜后肾周组有统计学差异(均p<0.05),而两者之间无差异(p>0.05)。(3)细胞活力：①腹股沟组、颈背组、腹膜后组活细胞数量逐步递减,组间两两比较均有差异(均p<0.05)。②三组细胞经活死细胞染色计算的活细胞数量各组间比较未见统计学差异(均p>0.05)。(4)分化能力：①定量碱性磷酸酶染色示腹股沟皮下来源脂肪干细胞成骨分化能力优于另两组,差别均有显著性(均p<0.05)。定量油红染色示三组细胞成脂分化能力相似(均p>0.05)。②Real-time PCR检测成骨、成脂分化标志物均有表达,各组间比较显示皮下部位(腹股沟和颈背部)来源的细胞表达量明显高于腹膜后组(p<0.05),而皮下部位中腹股沟组优于颈背部组(p<0.05)。③Western blot检测到各标志物的蛋白表达,与Real-time PCR结果一致。
结论(1)脂肪干细胞是一种典型的间充质干细胞,其形态和表型均符合间充质干细胞特征；(2)来源部位对脂肪干细胞的增殖、活力和分化能力有影响,皮下来源的细胞在各方面优于腹膜后来源,同为皮下组织来源的腹股沟部位又优于颈背部位；(3)腹股沟皮下部位是脂肪干细胞现对较理想的取材部位；(4)脂肪干细胞可向骨细胞、脂肪细胞分化,可应用于组织工程研究；(5)脂肪是一种具有高度非均一性的组织,这种非均一性不仅存在于不同个体之间,也存在于同一个体不同部位之间。
第二部分静电纺丝聚乳酸/聚己内酯共混纤维支架与脂肪干细胞的生物相容性研究
目的研究利用静电纺丝技术制备的聚乳酸/聚己内酯共混纤维的生物相容性,探讨不同的重量混合比对该聚合物支架生物相容性的影响。
方法利用静电纺丝技术将聚乳酸(PLLA)和聚己内酯(PCL)的混合物(PLLA/PCL)制备成纳米纤维支架,两种聚合物共混时的重量比为3/1,2/1,1/1(PLLA/PCL)。对这三种不同比例的支架进行形态学、孔隙率、体外降解率的比较；将纳米纤维支架作为异物接种到大鼠背部皮下口袋中,以观察局部炎症反应,评价材料的组织相容性；以脂肪干细胞为种子细胞,接种到三种比例的支架上,检测细胞的增殖、粘附、活力和多向分化(成骨分化、成软骨分化、成脂分化)能力,评价材料的细胞相容性。
结果1.形态学观察：(1)纤维直径：3/1支架为1.25±0.27μm,2/1支架为870±45nm,1/1支架为452±24nm,1/1支架较3/1,2/1 NFS差异有非常显著性(均p<0.01)：(2)孔隙率：三种比例支架孔隙率分别为78.3±3.4%,78.7±5.1%和82.2±4.8%,三者间未检出统计学差异；(3)降解率：根据降解曲线显示,PLLA含量越大,支架体外降解率越高。2.组织相容性：三种支架移植均导致炎性细胞浸润,急性炎性反应过程约2周时间,4周后炎症反应消失。3.细胞相容性：脂肪干细胞可在支架上粘附、增殖并多向分化,脂肪干细胞与1/1支架组装的复合体有最好的生物学行为表现。
结论(1)PLLA和PCL的共混溶液可经静电纺丝技术制备具有纳米结构的纤维支架；(2)PLLA、PCL混合的重量比率对两者的静纺共混纤维在形态、孔隙率、降解率有影响,1/1比例的材料性能最优；(3)静纺PLLA/PCL支架生物相容性好,可作为组织工程候选支架材料。
第三部分尿道组织工程：脂肪干细胞在静电纺丝纳米纤维支架上的平滑肌分化
目的研究脂肪干细胞在静电纺丝聚乳酸/聚己内酯共混纳米纤维支架上的平滑肌分化,探索该支架材料作为尿道组织工程的可行性。
方法静电纺丝技术制备重量比为1：1的聚乳酸/聚己内酯共混纳米纤维支架。从兔腹股沟皮下分离脂肪干细胞,接种到支架上组装成为复合体。用平滑肌诱导培养基诱导支架上的脂肪干细胞,通过形态学观察、免疫荧光、细胞增殖检测、基因转录、蛋白印记和胶原收缩法比较脂肪干细胞在有无支架条件下诱导的平滑肌细胞有无差别。
结果诱导6周后,脂肪干细胞诱导的平滑肌细胞具有平滑肌细胞形态,能够表达α-actin和MHC两种平滑肌标志物,胞体中F-actin含量接近正常平滑肌细胞。细胞增殖显示,诱导3周时,支架上诱导的平滑肌细胞数量较无支架诱导的细胞无差别(p>0.05),而在6周时,两者有非常显著性差别(p<0.01)。基因转录和蛋白表达均显示诱导的脂肪干细胞能够表达平滑肌特异性基因或蛋白,且支架对脂肪干细胞向平滑肌分化无影响。收缩功能检测显示诱导的平滑肌细胞具有很好的收缩能力。
结论(1)ASCs可直接在静电纺丝PLLA/PCL纳米纤维支架上诱导分化为具有收缩功能的SMCs;(2)ASCs与NFS构建的复合体可用于尿道组织工程研究；(3)静电纺丝PLLA/PCL纳米纤维可作为平滑肌组织工程候选支架材料。
Tissue engineering, an important component of regenerative medicine, is a comprehensive discipline combining many kinds of sciences and technologies. The approaches of tissue engineering may very, but most involve the essential strategies including seed-cells selection, scaffolds design and interaction of cells and scaffolds.
Adipose tissue is a new source reservoir for adult stem cells. Because of convenient harvesting, minimal invasion and abundant quantity, adipose-derived stem cells (ASCs) have been regarded as forefront seed-cells in tissue engineering. Many studies have revealed a close relationship between anatomic site and cellular behavior. ASCs originate from adipose tissues. However, the impacts of anatomic site of harvesting on ASCs are obscure till now.
The synthetic scaffold plays an increasing role in tissue engineering field. The polymers are preferable for various purposes due to stable and controllable properties. Poly (L-lactic acid) (PLLA) is brittle with high degradation and poly (ε-caprolactone) (PCL) is flexile with low degradation. The composites of PLLA and PCL of different weight ratios offer the scaffolds of different properties. The blends of PLLA and PCL, which we prepared from electrospinning method, are three-dimensional in weaving fibers in nanometer scale, indicating their replacement for extracellular matrix.
In this study, we compared the biology of ASCs from three anatomic sites and discovered the subcutaneous inguinal region was the best site for cell harvesting. Moreover, we evaluated the effects of weight ratio of PLLA/PCL blend and confirmed the 1/1 synthesized scaffold had the highest performance. On the basis of the above data, we employed ASCs to differentiate towards smooth muscle cells on the electrospun PLLA/PCL blend nanofibers of 1/1 blending weight ratio to explore the feasibility of such construct for tissue engineered-urinary tract.
Objectives To study and compare the impacts of harvesting anatomic site on the biological behaviors of adipose-derived stem cells (ASCs), and to provide theoretical support for the strategy of seed-cell selection in tissue engineering.
Materials and Methods Five female New Zealand white rabbits born simultaneously were used in our study. They were raised in the identical environment until to 4 months age old and weighed averagely 2.4kg (ranging from 2.37 to 2.42kg). With the general anesthesia by auricular vein injection, the adipose tissues were resected under the asepsis condition from three different anatomic sites of each rabbit, including subcutaneous inguinal (SI), subcutaneous dorsocervical (SD) and retroperitoneal perinephric (RP) regions. The ASCs obtained from removed tissues were isolated and cultured conventionally and categorized into three experimental groups according to their region of origin. The ASCs of three groups were compared on the biological properties by the following methods:(1) ASCs were characterized by morphological observation under an inverted phase contrast microscope and the expression of surface markers, CD 105 and CD 166, by fluorescence-activated cell sorting (FACS). (2) Cell counting and MTT assay were employed to evaluate proliferation. (3) A known standard curve and Live/Dead cells staining were employed to evaluate viability. (4) Histochemistry staining (ALP staining and Oil red O staining), Real-time PCR and Western blot were employed to evaluate the multilineage differentiation towards to osteogenesis and adipogenesis.
Results (1) Characterization of ASCs:①ASCs from three anatomic sites demonstrated typical forms of the mesenchymal stem cells (MSCs). No obvious difference was found by macroscopic observation.②The positive percentages of CD 105 and CD 166 were assessed by FACS and analyzed to nonsignificant difference. (2) Proliferation assay:①On day 4, the average numbers of the ASCs in SI group and SD groups were significantly higher than those in RP group (both p<0.05), with no significant difference between SI and SD group (p>0.05). On day 7, no significant difference was detected among the ASCs from three anatomic sites (p>0.05).②The one way ANOVA was used to compare the growth curves of three groups generated from MTT assay data. The subcutaneous (SI and SD) ASCs had both statistical significance to RP ASCs (both p< 0.05), while nonsignificance was determined between SI and SD ASCs (p>0.05). (3) Viability assay:①The three groups had significant differences in the number of living cells, with a decline from SI to SD to RP ASCs (p<0.05 for these comparisons).②The living cells were counted in five random views of Live/Dead staining. The differences between any two or all of the sites were not statistically significant (all p>0.05). (4) Differentiation capacity:①At the end of osteogenetic culture, the SI ASCs had higher ALP activity than SD or RP cells (both p<0.05). At the end of adipogenic culture, there were no significant differences among the three groups (p>0.05).②All of the gene markers were detected by Real-time PCR during osteogenesis and adipogenesis. In the evaluation for each gene, the expression levels of the subcutaneous (SI and SD) groups were significant higher than that of RP group (both p<0.05). In the comparison between two subcutaneous groups, SI ASCs acted better than SD ASCs (p<0.05).③The results of protein expression levels determined by Western blot were in agreement with Real-time PCR.
Conclusions (1) ASCs are a typical type of MSCs, whose morphology and phenotype complied with the MSCs'characteristics; (2) The derived anatomic site has impacts on the proliferation, viability and differentiation capacity of ASCs. The subcutaneous ASCs are preferable than retroperitoneal ASCs, and the SI ASCs are better than SD ASCs. (3) The inguinal region may be a applicable resource reservoir for cell harvesting. (4) The ASCs are capable of multipotential to osteogenesis and adipogeneous, implying their application in tissue engineering. (5) The adipose tissue is highly heterogeneous, both among individuals and also within an individual.
PartⅡ. Biocompatibility of electrospun PLLA/PCL blend nanofibrous scaffold and adipose-derived stem cells
Objectives To study the biocompatibility of PLLA/PCL blend fibers prepared from electrospinning method and to discuss the effects of blending weight ration on it.
Materials and Methods PLLA and PCL blends with weight ratio of 3/1,2/1 and 1/1 were electrospun into nanofibrous scaffold (NFS). The morphology, porosity and degradation in vitro of such three NFS were compared. The histocompatibility was observed on the inflammatory responses of NFS implanted into subcutaneous pockets of rats. The cytocompatibility was evaluated by proliferation, attachment, viability and multipotential towards to osteogenesis, chondrogensis and adipogenesis.
Results 1.Mophology:(1) Average diameter (AD):AD3/1=1.25±0.27μm, AD2/1= 870±45nm, AD1/1=452±24nm. The fibers of 1/1 NFS were significantly thinner than those of 3/1 and 2/1 NFS (both p<0.01); (2) Porosity: The porosity rates of three NFSs were respectively 78.3±3.4%,78.7±5.1% and 82.2±4.8%, with no significant difference among them; (3) Degradation:According to the degradation curve, the rate of degradation was accelerated with the increasing content of PLLA.2. Histocompatibility:The inflammatory responses were all observed during the NFS transplants. The response course was as long as about 2 weeks and vanished at the end of 4 weeks.3. Cytocompatibility:The ASCs were able to attach, grow and differentiate into multilineages on NFSs. The NFS of 1/1 blending ratio was the most suitable scaffold to be fabricated with ASCs.
Conclusions (1) The blend composite of PLLA and PCL can be electrospun into nano-scale fibrous scaffold; (2) The weight ratio of PLLA and PCL blending has impacts on morphology, porosity and degradation of electrospun fibrous scaffold; (3) The electrospun PLLA/PCL blend nanofibrous scaffold has good biocompatibility and can be a candidate biomaterial for tissue engineering.
PartⅢ. Urethra tissue engineering:leiomyogenic differentiation of adipose-derived stem cells on electrospun nanofibrous scaffold
Objectives To study the leiomyogenic differentiation of adipose-derived stem cells(ASCs) on electrospinning PLLA/PCL blend nanafibrous scaffold and to explore the feasibility of the application of such novel material in urethra tissue engineering.
Materials and Methods ASCs obtained from subcutaneous adipose tissue in inguinal region were seeded on the electrospun PLLA/PCL blend nanofibrous scaffold (NFS), which was blended in 1:1 weight ratio, to fabricate ASCs-NFS constructs. The smooth muscle inductive medium was used for ASCs differentiating towards smooth muscle cells (SMCs). The difference between the ASCs-derived smooth muscle cells (AD-SMCs) with and without scaffolds were evaluated by morphological observation, immuno fluorescence, cell proliferation, gene and protein expression assay.
Results After 6 weeks, the AD-SMCs demonstrated the typical morphology of SMCs and expressed specific markers, a-actin and myosin heavy chain (MHC). The contents of F-actin of the AD-SMCs were similar with the SMCs. During the proliferation assay, there was no statistical significance between the AD-SMCs with and without the scaffolds (p> 0.05) for 3-week induction. However, great significant difference was detected when it went to the 6th week. Both of the gene transcription and protein expression showed the AD-SMCs were positive to SMCs specific genes or proteins. No effect of the existence of scaffold was found in the course of differentiation from ASCs to SMCs. The AD-SMCs posed good contraction ability by using collagen gel assay.
Conclusions (1) ASCs can differentiate into contractile SMCs on PLLA/PCL NFS prepared by electrospinning; (2) The constructs combined ASCs and NFS can be used for the researches of urethra reconstruction; (3) The electrospun PLLA/PCL blend nanofiber can be a candidate material for SMCs tissue engineering.
脂肪组织是一种新的成体干细胞来源。因为取材方便、侵入小、产量大等优点,脂肪干细胞已成为组织工程研究中一线种子细胞。许多研究表明,脂肪组织的生物学特性与解剖部位有关。脂肪干细胞来源于脂肪组织,其取材部位对细胞特性是否也存在这种相关性,目前尚无明确结论。
人工合成支架在组织工程研究中的地位越来越重要。高分子聚合物因其稳定而可控的特性应用广泛。聚乳酸质脆,降解度高,而聚己内酯质韧,降解度低,两者不同比例的复合物可提供各种性状的支架。我们利用静电纺丝技术制备了两者的共混物,这种混合物支架由纳米级纤维编织成三维结构,适合模拟细胞外基质。
在本研究中,我们比较了不同解剖部位来源的脂肪干细胞特性,结果显示兔腹股沟皮下部位是最佳取材部位。同时研究了聚乳酸/聚己内酯共混支架中两种成分的混合比例对其性能的影响,发现按1/1比例制备的支架表现出更好的性能。在上述研究的基础上,我们以脂肪干细胞为种子细胞,以1/1比例的静电纺丝聚乳酸/聚己内酯共混纤维为支架,通过细胞在支架上的平滑肌诱导,探索其构建组织工程化尿道的可行性。
第一部分不同解剖部位来源的脂肪干细胞生物学特性比较
目的研究并比较脂肪干细胞取材的解剖部位对脂肪干细胞生物学特性的影响,为脂肪干细胞作为组织工程种子细胞的选择策略提供理论基础。
方法5只同龄雌性新西兰大白兔从出生起在同等环境中饲养,4月龄时用于实验研究,平均体质量约2.4kg(2.37～2.42kg).经耳缘静脉麻醉后,每只兔在无菌术下于3个部位手术切取脂肪组织团块,包括腹股沟皮下、颈背部皮下和腹膜后肾周部位。所得脂肪组织经常规分离培养,按来源部位分为三个实验组培养。三组脂肪干细胞分别用以下实验方法比较生物学行为：(1)倒置相差显微镜下形态观察和流式细胞术检测细胞表面标志物CD105和CD166以鉴定细胞；(2)细胞计数法和MTT法检测细胞增殖能力；(3)标准曲线法和活死细胞染色检测细胞活力；(4)组织化学染色(碱性磷酸酶染色和油红染色)、Real-time PCR和Western blot检测成骨、成脂诱导分化能力。
结果(1)细胞鉴定：①三个部位的脂肪干细胞均呈典型间充质干细胞形态,肉眼观察未见明显差异；②流式检测三组细胞CD105、CD166标志物,其阳性率未见统计学差异。(2)增殖能力：①4天时皮下组织腹股沟和颈背部细胞数量均高于腹膜后肾周组,差异有显著性(均p<0.05)。两组皮下组织组之间无差异(p>0.05)。7天时三组细胞数量未检出统计学差异(p>0.05)。②单因素方差分析比较三组细胞MTT法测定的增殖曲线,腹股沟皮下和颈背部皮下组细胞都较腹膜后肾周组有统计学差异(均p<0.05),而两者之间无差异(p>0.05)。(3)细胞活力：①腹股沟组、颈背组、腹膜后组活细胞数量逐步递减,组间两两比较均有差异(均p<0.05)。②三组细胞经活死细胞染色计算的活细胞数量各组间比较未见统计学差异(均p>0.05)。(4)分化能力：①定量碱性磷酸酶染色示腹股沟皮下来源脂肪干细胞成骨分化能力优于另两组,差别均有显著性(均p<0.05)。定量油红染色示三组细胞成脂分化能力相似(均p>0.05)。②Real-time PCR检测成骨、成脂分化标志物均有表达,各组间比较显示皮下部位(腹股沟和颈背部)来源的细胞表达量明显高于腹膜后组(p<0.05),而皮下部位中腹股沟组优于颈背部组(p<0.05)。③Western blot检测到各标志物的蛋白表达,与Real-time PCR结果一致。
结论(1)脂肪干细胞是一种典型的间充质干细胞,其形态和表型均符合间充质干细胞特征；(2)来源部位对脂肪干细胞的增殖、活力和分化能力有影响,皮下来源的细胞在各方面优于腹膜后来源,同为皮下组织来源的腹股沟部位又优于颈背部位；(3)腹股沟皮下部位是脂肪干细胞现对较理想的取材部位；(4)脂肪干细胞可向骨细胞、脂肪细胞分化,可应用于组织工程研究；(5)脂肪是一种具有高度非均一性的组织,这种非均一性不仅存在于不同个体之间,也存在于同一个体不同部位之间。
第二部分静电纺丝聚乳酸/聚己内酯共混纤维支架与脂肪干细胞的生物相容性研究
目的研究利用静电纺丝技术制备的聚乳酸/聚己内酯共混纤维的生物相容性,探讨不同的重量混合比对该聚合物支架生物相容性的影响。
方法利用静电纺丝技术将聚乳酸(PLLA)和聚己内酯(PCL)的混合物(PLLA/PCL)制备成纳米纤维支架,两种聚合物共混时的重量比为3/1,2/1,1/1(PLLA/PCL)。对这三种不同比例的支架进行形态学、孔隙率、体外降解率的比较；将纳米纤维支架作为异物接种到大鼠背部皮下口袋中,以观察局部炎症反应,评价材料的组织相容性；以脂肪干细胞为种子细胞,接种到三种比例的支架上,检测细胞的增殖、粘附、活力和多向分化(成骨分化、成软骨分化、成脂分化)能力,评价材料的细胞相容性。
结果1.形态学观察：(1)纤维直径：3/1支架为1.25±0.27μm,2/1支架为870±45nm,1/1支架为452±24nm,1/1支架较3/1,2/1 NFS差异有非常显著性(均p<0.01)：(2)孔隙率：三种比例支架孔隙率分别为78.3±3.4%,78.7±5.1%和82.2±4.8%,三者间未检出统计学差异；(3)降解率：根据降解曲线显示,PLLA含量越大,支架体外降解率越高。2.组织相容性：三种支架移植均导致炎性细胞浸润,急性炎性反应过程约2周时间,4周后炎症反应消失。3.细胞相容性：脂肪干细胞可在支架上粘附、增殖并多向分化,脂肪干细胞与1/1支架组装的复合体有最好的生物学行为表现。
结论(1)PLLA和PCL的共混溶液可经静电纺丝技术制备具有纳米结构的纤维支架；(2)PLLA、PCL混合的重量比率对两者的静纺共混纤维在形态、孔隙率、降解率有影响,1/1比例的材料性能最优；(3)静纺PLLA/PCL支架生物相容性好,可作为组织工程候选支架材料。
第三部分尿道组织工程：脂肪干细胞在静电纺丝纳米纤维支架上的平滑肌分化
目的研究脂肪干细胞在静电纺丝聚乳酸/聚己内酯共混纳米纤维支架上的平滑肌分化,探索该支架材料作为尿道组织工程的可行性。
方法静电纺丝技术制备重量比为1：1的聚乳酸/聚己内酯共混纳米纤维支架。从兔腹股沟皮下分离脂肪干细胞,接种到支架上组装成为复合体。用平滑肌诱导培养基诱导支架上的脂肪干细胞,通过形态学观察、免疫荧光、细胞增殖检测、基因转录、蛋白印记和胶原收缩法比较脂肪干细胞在有无支架条件下诱导的平滑肌细胞有无差别。
结果诱导6周后,脂肪干细胞诱导的平滑肌细胞具有平滑肌细胞形态,能够表达α-actin和MHC两种平滑肌标志物,胞体中F-actin含量接近正常平滑肌细胞。细胞增殖显示,诱导3周时,支架上诱导的平滑肌细胞数量较无支架诱导的细胞无差别(p>0.05),而在6周时,两者有非常显著性差别(p<0.01)。基因转录和蛋白表达均显示诱导的脂肪干细胞能够表达平滑肌特异性基因或蛋白,且支架对脂肪干细胞向平滑肌分化无影响。收缩功能检测显示诱导的平滑肌细胞具有很好的收缩能力。
结论(1)ASCs可直接在静电纺丝PLLA/PCL纳米纤维支架上诱导分化为具有收缩功能的SMCs;(2)ASCs与NFS构建的复合体可用于尿道组织工程研究；(3)静电纺丝PLLA/PCL纳米纤维可作为平滑肌组织工程候选支架材料。
Tissue engineering, an important component of regenerative medicine, is a comprehensive discipline combining many kinds of sciences and technologies. The approaches of tissue engineering may very, but most involve the essential strategies including seed-cells selection, scaffolds design and interaction of cells and scaffolds.
Adipose tissue is a new source reservoir for adult stem cells. Because of convenient harvesting, minimal invasion and abundant quantity, adipose-derived stem cells (ASCs) have been regarded as forefront seed-cells in tissue engineering. Many studies have revealed a close relationship between anatomic site and cellular behavior. ASCs originate from adipose tissues. However, the impacts of anatomic site of harvesting on ASCs are obscure till now.
The synthetic scaffold plays an increasing role in tissue engineering field. The polymers are preferable for various purposes due to stable and controllable properties. Poly (L-lactic acid) (PLLA) is brittle with high degradation and poly (ε-caprolactone) (PCL) is flexile with low degradation. The composites of PLLA and PCL of different weight ratios offer the scaffolds of different properties. The blends of PLLA and PCL, which we prepared from electrospinning method, are three-dimensional in weaving fibers in nanometer scale, indicating their replacement for extracellular matrix.
In this study, we compared the biology of ASCs from three anatomic sites and discovered the subcutaneous inguinal region was the best site for cell harvesting. Moreover, we evaluated the effects of weight ratio of PLLA/PCL blend and confirmed the 1/1 synthesized scaffold had the highest performance. On the basis of the above data, we employed ASCs to differentiate towards smooth muscle cells on the electrospun PLLA/PCL blend nanofibers of 1/1 blending weight ratio to explore the feasibility of such construct for tissue engineered-urinary tract.
Objectives To study and compare the impacts of harvesting anatomic site on the biological behaviors of adipose-derived stem cells (ASCs), and to provide theoretical support for the strategy of seed-cell selection in tissue engineering.
Materials and Methods Five female New Zealand white rabbits born simultaneously were used in our study. They were raised in the identical environment until to 4 months age old and weighed averagely 2.4kg (ranging from 2.37 to 2.42kg). With the general anesthesia by auricular vein injection, the adipose tissues were resected under the asepsis condition from three different anatomic sites of each rabbit, including subcutaneous inguinal (SI), subcutaneous dorsocervical (SD) and retroperitoneal perinephric (RP) regions. The ASCs obtained from removed tissues were isolated and cultured conventionally and categorized into three experimental groups according to their region of origin. The ASCs of three groups were compared on the biological properties by the following methods:(1) ASCs were characterized by morphological observation under an inverted phase contrast microscope and the expression of surface markers, CD 105 and CD 166, by fluorescence-activated cell sorting (FACS). (2) Cell counting and MTT assay were employed to evaluate proliferation. (3) A known standard curve and Live/Dead cells staining were employed to evaluate viability. (4) Histochemistry staining (ALP staining and Oil red O staining), Real-time PCR and Western blot were employed to evaluate the multilineage differentiation towards to osteogenesis and adipogenesis.
Results (1) Characterization of ASCs:①ASCs from three anatomic sites demonstrated typical forms of the mesenchymal stem cells (MSCs). No obvious difference was found by macroscopic observation.②The positive percentages of CD 105 and CD 166 were assessed by FACS and analyzed to nonsignificant difference. (2) Proliferation assay:①On day 4, the average numbers of the ASCs in SI group and SD groups were significantly higher than those in RP group (both p<0.05), with no significant difference between SI and SD group (p>0.05). On day 7, no significant difference was detected among the ASCs from three anatomic sites (p>0.05).②The one way ANOVA was used to compare the growth curves of three groups generated from MTT assay data. The subcutaneous (SI and SD) ASCs had both statistical significance to RP ASCs (both p< 0.05), while nonsignificance was determined between SI and SD ASCs (p>0.05). (3) Viability assay:①The three groups had significant differences in the number of living cells, with a decline from SI to SD to RP ASCs (p<0.05 for these comparisons).②The living cells were counted in five random views of Live/Dead staining. The differences between any two or all of the sites were not statistically significant (all p>0.05). (4) Differentiation capacity:①At the end of osteogenetic culture, the SI ASCs had higher ALP activity than SD or RP cells (both p<0.05). At the end of adipogenic culture, there were no significant differences among the three groups (p>0.05).②All of the gene markers were detected by Real-time PCR during osteogenesis and adipogenesis. In the evaluation for each gene, the expression levels of the subcutaneous (SI and SD) groups were significant higher than that of RP group (both p<0.05). In the comparison between two subcutaneous groups, SI ASCs acted better than SD ASCs (p<0.05).③The results of protein expression levels determined by Western blot were in agreement with Real-time PCR.
Conclusions (1) ASCs are a typical type of MSCs, whose morphology and phenotype complied with the MSCs'characteristics; (2) The derived anatomic site has impacts on the proliferation, viability and differentiation capacity of ASCs. The subcutaneous ASCs are preferable than retroperitoneal ASCs, and the SI ASCs are better than SD ASCs. (3) The inguinal region may be a applicable resource reservoir for cell harvesting. (4) The ASCs are capable of multipotential to osteogenesis and adipogeneous, implying their application in tissue engineering. (5) The adipose tissue is highly heterogeneous, both among individuals and also within an individual.
PartⅡ. Biocompatibility of electrospun PLLA/PCL blend nanofibrous scaffold and adipose-derived stem cells
Objectives To study the biocompatibility of PLLA/PCL blend fibers prepared from electrospinning method and to discuss the effects of blending weight ration on it.
Materials and Methods PLLA and PCL blends with weight ratio of 3/1,2/1 and 1/1 were electrospun into nanofibrous scaffold (NFS). The morphology, porosity and degradation in vitro of such three NFS were compared. The histocompatibility was observed on the inflammatory responses of NFS implanted into subcutaneous pockets of rats. The cytocompatibility was evaluated by proliferation, attachment, viability and multipotential towards to osteogenesis, chondrogensis and adipogenesis.
Results 1.Mophology:(1) Average diameter (AD):AD3/1=1.25±0.27μm, AD2/1= 870±45nm, AD1/1=452±24nm. The fibers of 1/1 NFS were significantly thinner than those of 3/1 and 2/1 NFS (both p<0.01); (2) Porosity: The porosity rates of three NFSs were respectively 78.3±3.4%,78.7±5.1% and 82.2±4.8%, with no significant difference among them; (3) Degradation:According to the degradation curve, the rate of degradation was accelerated with the increasing content of PLLA.2. Histocompatibility:The inflammatory responses were all observed during the NFS transplants. The response course was as long as about 2 weeks and vanished at the end of 4 weeks.3. Cytocompatibility:The ASCs were able to attach, grow and differentiate into multilineages on NFSs. The NFS of 1/1 blending ratio was the most suitable scaffold to be fabricated with ASCs.
Conclusions (1) The blend composite of PLLA and PCL can be electrospun into nano-scale fibrous scaffold; (2) The weight ratio of PLLA and PCL blending has impacts on morphology, porosity and degradation of electrospun fibrous scaffold; (3) The electrospun PLLA/PCL blend nanofibrous scaffold has good biocompatibility and can be a candidate biomaterial for tissue engineering.
PartⅢ. Urethra tissue engineering:leiomyogenic differentiation of adipose-derived stem cells on electrospun nanofibrous scaffold
Objectives To study the leiomyogenic differentiation of adipose-derived stem cells(ASCs) on electrospinning PLLA/PCL blend nanafibrous scaffold and to explore the feasibility of the application of such novel material in urethra tissue engineering.
Materials and Methods ASCs obtained from subcutaneous adipose tissue in inguinal region were seeded on the electrospun PLLA/PCL blend nanofibrous scaffold (NFS), which was blended in 1:1 weight ratio, to fabricate ASCs-NFS constructs. The smooth muscle inductive medium was used for ASCs differentiating towards smooth muscle cells (SMCs). The difference between the ASCs-derived smooth muscle cells (AD-SMCs) with and without scaffolds were evaluated by morphological observation, immuno fluorescence, cell proliferation, gene and protein expression assay.
Results After 6 weeks, the AD-SMCs demonstrated the typical morphology of SMCs and expressed specific markers, a-actin and myosin heavy chain (MHC). The contents of F-actin of the AD-SMCs were similar with the SMCs. During the proliferation assay, there was no statistical significance between the AD-SMCs with and without the scaffolds (p> 0.05) for 3-week induction. However, great significant difference was detected when it went to the 6th week. Both of the gene transcription and protein expression showed the AD-SMCs were positive to SMCs specific genes or proteins. No effect of the existence of scaffold was found in the course of differentiation from ASCs to SMCs. The AD-SMCs posed good contraction ability by using collagen gel assay.
Conclusions (1) ASCs can differentiate into contractile SMCs on PLLA/PCL NFS prepared by electrospinning; (2) The constructs combined ASCs and NFS can be used for the researches of urethra reconstruction; (3) The electrospun PLLA/PCL blend nanofiber can be a candidate material for SMCs tissue engineering.
引文
1. Griffith LG, Naughton G. Tissue engineering--current challenges and expanding opportunities. Science.2002 Feb 8;295(5557):1009-14.
2. Atala A. Tissue engineering and regenerative medicine: concepts for clinical application. Rejuvenation Res.2004 Spring;7(1):15-31.
3. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res.2007 May 11;100(9):1249-60.
4. Atala A. Tissue engineering, stem cells and cloning: current concepts and changing trends. Expert Opin Biol Ther.2005 Jul;5(7):879-92.
5. Skalak R, Fox CF. Tissue Engineering: Proceedings of a workshop held at Granlibakken, Lake Tahoe, CA, New York, NY:Liss; 1988:26-9.
6. Langer R, Vacanti JP. Tissue engineering. Science.1993 May 14;260(5110):920-6.
7. Butler DL, Goldstein SA, Guilak F. Functional tissue engineering: the role of biomechanics. J Biomech Eng.2000 Dec; 122(6):570-5.
8. Nirmalanandhan VS, Sittampalam GS. Stem cells in drug discovery, tissue engineering, and regenerative medicine:emerging opportunities and challenges. J Biomol Screen. 2009 Aug;14(7):755-68.
9. Nakahara H, Goldberg VM, Caplan AI. Culture-expanded human periosteal-derived cells exhibit osteochondral potential in vivo. J Orthop Res.1991 Jul;9(4):465-76.
10. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science.1988 Jul 1;241(4861):58-62.
11. Stemple DL, Anderson DJ. Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell.1992 Dec 11;71(6):973-85.
12. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol.2000 Apr;109(1):235-42.
13. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng.2001 Apr;7(2):211-28.
14. Mo XM, Xu CY, Kotaki M, et al. Electrospun P(LLA-CL) nanofiber:a biomimetic extracellular matrix for smooth muscle cell and endothelial cell proliferation. Biomaterials.2004 May;25(10):1883-90.
15. Calandrelli L, Calarco A, Laurienzo P, et al. Compatibilized polymer blends based on PDLLA and PCL for application in bioartificial liver. Biomacromolecules.2008 Jun;9(6):1527-34.
16. Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, et al. Electrospun poly(epsilon-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials. 2008 Dec;29(34):4532-9.
17. Chen ZG, Wang PW, Wei B, et al. Electrospun collagen-chitosan nanofiber: a biomimetic extracellular matrix for endothelial cell and smooth muscle cell. Acta Biomater.2010 Feb;6(2):372-82.
18. Awad HA, Wickham MQ, Leddy HA, et al. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials.2004 Jul;25(16):3211-22.
19. Liu Q, Cen L, Yin S, et al. A comparative study of proliferation and osteogenic differentiation of adipose-derived stem cells on akermanite and beta-TCP ceramics. Biomaterials.2008 Dec;29(36):4792-9.
20. Haimi S, Suuriniemi N, Haaparanta AM, et al. Growth and osteogenic differentiation of adipose stem cells on PLA/bioactive glass and PLA/beta-TCP scaffolds. Tissue Eng Part A.2009 Jul; 15(7):1473-80.
21. Itoi Y, Takatori M, Hyakusoku H, et al. Comparison of readily available scaffolds for adipose tissue engineering using adipose-derived stem cells. J Plast Reconstr Aesthet Surg.2010 May;63(5):858-64.
22. Kasten P, Beyen I, Niemeyer P, et al. Porosity and pore size of beta-tricalcium phosphate scaffold can influence protein production and osteogenic differentiation of human mesenchymal stem cells:an in vitro and in vivo study. Acta Biomater.2008 Nov;4(6):1904-15.
23. Marino G, Rosso F, Cafiero G, et al. Beta-tricalcium phosphate 3D scaffold promote alone osteogenic differentiation of human adipose stem cells:in vitro study. J Mater Sci Mater Med.2010 Jan;21(1):353-63. Epub 2009 Aug 5.
24. Min BM, Lee G, Kim SH, et al. Electrospinning of silk fibroin nanofibers and its effect on the adhesion and spreading of normal human keratinocytes and fibroblasts in vitro. Biomaterials.2004 Mar-Apr;25(7-8):1289-97.
25. Mehlhorn AT, Zwingmann J, Finkenzeller G, et al. Chondrogenesis of adipose-derived adult stem cells in a poly-lactide-co-glycolide scaffold. Tissue Eng Part A.2009 May; 15(5):1159-67.
26. Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, et al. Electrospun poly(epsilon-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials.2008 Dec;29(34):4532-9.
27. Mo SM, Oh IJ. Release of adriamycin from poly(lactide-co-glycolide)-polyethylene glycol nanoparticles. J Nanosci Nanotechnol.2011 Feb;11(2):1795-8.
28. Peng J, Li X, Guo G, et al. Preparation and characterization of poly(vinyl alcohol) /poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone)/ nano-hydroxyapatite composite membranes for tissue engineering. J Nanosci Nanotechnol.2011 Mar;11(3):2354-60.
29. Itaka K, Ishii T, Hasegawa Y, et al. Biodegradable polyamino acid-based polycations as safe and effective gene carrier minimizing cumulative toxicity. Biomaterials.2010 May;31(13):3707-14.
30.金锡御,宋波.《临床尿动力学》.第一版.北京：人民卫生出版社,2002,p：10-11
31. Shokeir A, Osman Y, Gabr M, et al. Acellular matrix tube for canine urethral replacement: is it fact or fiction? J Urol.2004 Jan;171(1):453-6.
32. De Filippo RE, Yoo JJ, Atala A. Urethral replacement using cell seeded tubularized collagen matrices. J Urol.2002 Oct; 168(4 Pt 2):1789-92; discussion 1792-3.
33. Fu Q, Cao YL. Use of tissue engineering in treatment of the male genitourinary tract abnormalities. J Sex Med.2010 May;7(5):1741-6.
34. Barbagli G, Lazzeri M. Urethral reconstruction. Curr Opin Urol.2006 Nov;16(6):391-5.
35. Shokeir AA, Harraz AM, El-Din AB.Tissue engineering and stem cells:basic principles and applications in urology. Int J Urol.2010 Dec;17(12):964-73.
1. Vacanti J.Tissue engineering and regenerative medicine:from first principles to state of the art. J Pediatr Surg.2010 Feb;45(2):291-4.
2. Chapekar MS. Tissue engineering:challenges and opportunities. J Biomed Mater Res. 2000;53(6):617-20.
3. Meyer U, Meyer T, Handschel J, et al. Fundamentals of tissue engineering and Regenerative medicine. Springer.2009;26:224.
4. Khademhosseini A, Vacanti JP, Langer R. Progress in tissue engineering. Sci Am.2009 May;300(5):64-71.
5. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res.2007 May 11; 100(9):1249-60.
6. Nakahara H, Goldberg VM, Caplan AI. Culture-expanded human periosteal-derived cells exhibit osteochondral potential in vivo. J Orthop Res.1991 Jul;9(4):465-76.
7. Stemple DL, Anderson DJ. Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell.1992 Dec 11;71(6):973-85.
8. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol.2000 Apr; 109(1):235-42.
9. Asakura A, Komaki M, Rudnicki M. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation.2001 Oct;68(4-5):245-53.
10. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng.2001 Apr;7(2):211-28.
11. Cherubino M, Marra KG. Adipose-derived stem cells for soft tissue reconstruction. Regen Med.2009 Jan;4(1):109-17.
12. Mizuno H. Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review. J Nippon Med Sch.2009 Apr;76(2):56-66.
13. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell.2002 Dec;13(12):4279-95.
14. Hauner H, Wabitsch M, Pfeiffer EF. Differentiation of adipocyte precursor cells from obese and nonobese adult women and from different adipose tissue sites. Horm Metab Res Suppl.1988; 19:35-9.
15. Hauner H, Entenmann G. Regional variation of adipose differentiation in cultured stromal-vascular cells from the abdominal and femoral adipose tissue of obese women. Int J Obes.1991 Feb;15(2):121-6.
16. Shahparaki A, Grunder L, Sorisky A. Comparison of human abdominal subcutaneous versus omental preadipocyte differentiation in primary culture. Metabolism.2002 Sep;51(9):1211-5.
17. Van Harmelen V, Rohrig K, Hauner H. Comparison of proliferation and differentiation capacity of human adipocyte precursor cells from the omental and subcutaneous adipose tissue depot of obese subjects. Metabolism.2004 May;53(5):632-7.
18. Prunet-Marcassus B, Cousin B, Caton D, et al. From heterogeneity to plasticity in adipose tissues: site-specific differences. Exp Cell Res.2006 Apr 1;312(6):727-36.
19. Fraser JK, Wulur I, Alfonso Z, et al. Differences in stem and progenitor cell yield in different subcutaneous adipose tissue depots. Cytotherapy.2007;9(5):459-67.
20. Hausman DB, Park HJ, Hausman GJ. Isolation and culture of preadipocytes from rodent white adipose tissue. Methods Mol Biol.2008;456:201-19.
21. Jurgens WJ, Oedayrajsingh-Varma MJ, Helder MN, Zandiehdoulabi B et al. Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies. Cell Tissue Res.2008 Jun;332(3):415-26.
22. Toyoda M, Matsubara Y, Lin K, et al. Characterization and comparison of adipose tissue-derived cells from human subcutaneous and omental adipose tissues. Cell Biochem Funct.2009 Oct;27(7):440-7.
23. Schipper BM, Marra KG, Zhang W, et al. Regional anatomic and age effects on cell function of human adipose-derived stem cells. Ann Plast Surg.2008 May;60(5):538-44.
24. Strem BM, Hicok KC, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med.2005 Sep;54(3):132-41.
25. Gronthos S, Franklin DM, Leddy HA, et al. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol.2001 Oct;189(1):54-63.
26. de Girolamo L, Lopa S, Arrigoni E, et al. Human adipose-derived stem cells isolated from young and elderly women:their differentiation potential and scaffold interaction during in vitro osteoblastic differentiation. Cytotherapy.2009;11(6):793-803.
27. Masoud I, Shapiro F, Kent R, et al. A longitudinal study of the growth of the New Zealand white rabbit:cumulative and biweekly incremental growth rates for body length, body weight, femoral length, and tibial length. J Orthop Res.1986;4(2):221-31.
28. Kang HM, Kim J, Park S, Kim J et al. Insulin-secreting cells from human eyelid-derived stem cells alleviate type I diabetes in immunocompetent mice. Stem Cells.2009 Aug;27(8):1999-2008.
29. Arrigoni E, Lopa S, de Girolamo L, et al. Isolation, characterization and osteogenic differentiation of adipose-derived stem cells:from small to large animal models. Cell Tissue Res.2009 Dec;338(3):401-11.
1. Griffith LG, Naughton G. Tissue engineering--current challenges and expanding opportunities. Science.2002 Feb 8;295(5557):1009-14.
2. Atala A. Tissue engineering and regenerative medicine:concepts for clinical application. Rejuvenation Res.2004 Spring;7(1):15-31.
3. Atala A. Tissue engineering, stem cells and cloning: current concepts and changing trends. Expert Opin Biol Ther.2005 Jul;5(7):879-92.
4. Bhatia SK. Tissue engineering for clinical applications. Biotechnol J.2010 Dec;5(12):1309-23.
5. Yarlagadda PK, Chandrasekharan M, Shyan JY. Recent advances and current developments in tissue scaffolding. Biomed Mater Eng.2005; 15(3):159-77.
6. Mo XM, Xu CY, Kotaki M, et al. Electrospun P(LLA-CL) nanofiber: a biomimetic extracellular matrix for smooth muscle cell and endothelial cell proliferation. Biomaterials.2004 May;25(10):1883-90.
7. Chen ZG, Wang PW, Wei B, et al. Electrospun collagen-chitosan nanofiber: a biomimetic extracellular matrix for endothelial cell and smooth muscle cell. Acta Biomater.2010 Feb;6(2):372-82.
8. Calandrelli L, Calarco A, Laurienzo P, et al. Compatibilized polymer blends based on PDLLA and PCL for application in bioartificial liver. Biomacromolecules.2008 Jun;9(6):1527-34.
9. Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, et al. Electrospun poly(epsilon-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials.2008 Dec;29(34):4532-9.
10. Wu D, Zhang Y, Zhang M, et al. Selective localization of multiwalled carbon nanotubes in poly(epsilon-caprolactone)/polylactide blend. Biomacromolecules.2009 Feb 9;10(2):417-24.
11. Gunn J, Zhang M. Polyblend nanofibers for biomedical applications:perspectives and challenges. Trends Biotechnol.2010 Apr;28(4):189-97.
12. Jiang W, Kim BY, Rutka JT, et al. Nanoparticle-mediated cellular response is size-dependent. Nat Nanotechnol.2008 Mar;3(3):145-50.
13. McCullen SD, Stevens DR, Roberts WA, et al. Characterization of electrospun nanocomposite scaffolds and biocompatibility with adipose-derived human mesenchymal stem cells. Int J Nanomedicine.2007;2(2):253-63.
14. Li WJ, Tuli R, Okafor C, et al. A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials.2005 Feb;26(6):599-609.
15. Mehlhorn AT, Zwingmann J, Finkenzeller G, et al. Chondrogenesis of adipose-derived adult stem cells in a poly-lactide-co-glycolide scaffold. Tissue Eng Part A.2009 May; 15(5):1159-67.
16. Lee JH, Rhie JW, Oh DY, et al. Osteogenic differentiation of human adipose tissue-derived stromal cells (hASCs) in a porous three-dimensional scaffold. Biochem Biophys Res Commun.2008 Jun 6;370(3):456-60.
17. Baker SC, Rohman G, Southgate J, et al. The relationship between the mechanical properties and cell behaviour on PLGA and PCL scaffolds for bladder tissue engineering. Biomaterials.2009 Mar;30(7):1321-8.
18. Ajami-Henriquez D, Rodriguez M, Sabino M, et al. Evaluation of cell affinity on poly(L-lactide) and poly(epsilon-caprolactone) blends and on PLLA-b-PCL diblock copolymer surfaces. J Biomed Mater Res A.2008 Nov;87(2):405-17.
19. Kwon IK, Kidoaki S, Matsuda T. Electrospun nano-to microfiber fabrics made of biodegradable copolyesters:structural characteristics, mechanical properties and cell adhesion potential. Biomaterials.2005 Jun;26(18):3929-39.
20. Xu CY, Inai R, Kotaki M, et al. Aligned biodegradable nanofibrous structure: a potential scaffold for blood vessel engineering. Biomaterials.2004 Feb;25(5):877-86.
21. Xu C, Inai R, Kotaki M, et al. Electrospun nano fiber fabrication as synthetic extracellular matrix and its potential for vascular tissue engineering. Tissue Eng.2004 Jul-Aug;10(7-8):1160-8.
22. He W, Yong T, Teo WE, et al. Fabrication and endothelialization of collagen-blended biodegradable polymer nano fibers:potential vascular graft for blood vessel tissue engineering. Tissue Eng.2005 Sep-Oct;11(9-10):1574-88.
23. Sarazin P, Orts WJ, Favis BD. Binary and ternary blends of polylactide, polycaprolactone and thermoplastic starch. Polymer.2008 Jan;49(2):599-609.
24. Lopez-Rodriguez N, Lopez-Arraiza A, Meaurio E, et al. Crystallization, morphology, and mechanical behavior of polylactide/poly(ε-caprolactone) blends. Polym Eng Sci. 2006 Sep;46(9):1299-1308.
25. Sarazin P, Roy X, Favis BD. Controlled preparation and properties of porous poly(L-lactide) obtained from a co-continuous blend of two biodegradable polymers. Biomaterials.2004 Dec;25(28):5965-78.
26. Roy X, Sarazin P, Favis BD. Ultraporous Nanosheath Materials by Layer-by-Layer Deposition onto Co-continuous Polymer-Blend Templates. Adv Mater.2006 Apr;18(8):1015-9.
27. Wu DF, Zhang YS, Zhang M, et al. Phase behavior and its viscoelastic response of polylactide/poly(ε-caprolactone) blend. Eur Polym J.2008 Jul;44(7):2171-83.
28. Tsuji H, Ishizaka T. Blends of aliphatic polyesters. VI. Lipase-catalyzed hydrolysis and visualized phase structure of biodegradable blends from poly(epsilon-caprolactone) and poly(L-lactide). Int J Biol Macromol.2001 Aug 20;29(2):83-9.
29. Nakagami H, Morishita R, Maeda K, et al. Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy. J Atheroscler Thromb.2006 Apr;13(2):77-81.
30. Zhu Y, Liu T, Song K, et al. Adipose-derived stem cell:a better stem cell than BMSC. Cell Biochem Funct.2008 Aug;26(6):664-75.
31. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res.2007 May 11;100(9):1249-60.
32. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng.2001 Apr;7(2):211-28.
33. van Aalst JA, Reed CR, Han L, et al. Cellular incorporation into electrospun nanofibers: retained viability, proliferation, and function in fibroblasts. Ann Plast Surg.2008 May;60(5):577-83.
34. Heydarkhan-Hagvall S, Schenke-Layland K, Dhanasopon AP, et al. Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering. Biomaterials.2008 Jul;29(19):2907-14.
35. Schenke-Layland K, Rofail F, Heydarkhan S, et al. The use of three-dimensional nanostructures to instruct cells to produce extracellular matrix for regenerative medicine strategies. Biomaterials.2009 Sep;30(27):4665-75.
36. McCullen SD, Zhu Y, Bernacki SH, et al. Electrospun composite poly(L-lactic acid)/tricalcium phosphate scaffolds induce proliferation and osteogenic differentiation of human adipose-derived stem cells. Biomed Mater.2009 Jun;4(3):035002.
37. Shanti RM, Janjanin S, Li WJ, et al. In vitro adipose tissue engineering using an electrospun nanofibrous scaffold. Ann Plast Surg.2008 Nov;61(5):566-71.
38. McCullen SD, Stevens DR, Roberts WA, et al. Characterization of electrospun nanocomposite scaffolds and biocompatibility with adipose-derived human mesenchymal stem cells. Int J Nanomedicine.2007;2(2):253-63.
39. Noh HK, Lee SW, Kim JM, et al. Electrospinning of chitin nanofibers:degradation behavior and cellular response to normal human keratinocytes and fibroblasts. Biomaterials.2006 Jul;27(21):3934-44.
40. Liao GY, Chen L, Zeng XY, et al. Electrospun poly(L-lactide)/poly(ε-caprolactone) blend fibers and their cellular response to adipose-derived stem cells. J APPL POLYM SCI.2011 May;120(4):2154-65.
41. Huang ZM, Zhang YZ, Kotaki M, et al. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Compos Sci Technol.2003 Nov;63(15):2223-53.
42. You Y, Min BM, Lee SJ, et al. In vitro degradation behavior of electrospun polyglycolide, polylactide, and poly(lactideco-glycolide). J Appl Polym Sci 2005 Jan;95(2):193-200.
43. Chen DR, Bei JZ, Wang SG. Polycaprolactone microparticles and their biodegradation. Polym Degrad Stab 2000 Mar;67(3):455-9.
1.金锡御,宋波.《临床尿动力学》.第一版.北京：人民卫生出版社,2002,p：10-11
2. Mathur RK, Himanshu A, Sudarshan O. Technique of anterior urethra urethroplasty using tunica albuginea of corpora cavernosa. Int J Urol.2007 Mar;14(3):209-13.
3. Shokeir A, Osman Y, Gabr M, et al. Acellular matrix tube for canine urethral replacement: is it fact or fiction? J Urol.2004 Jan;171(1):453-6.
4. De Filippo RE, Yoo JJ, Atala A. Urethral replacement using cell seeded tubularized collagen matrices. J Urol.2002 Oct;168(4 Pt 2):1789-92; discussion 1792-3.
5. McAninch JW. Urethral reconstruction: a continuing challenge. J Urol.2005 Jan;173(1):7.
6. Shastri VP. In vivo engineering of tissues:Biological considerations, challenges, strategies, and future directions. Adv Mater.2009 Sep 4;21(32-33):3246-54.
7. Rodriguez LV, Alfonso Z, Zhang R, et al. Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells. Proc Natl Acad Sci U S A.2006 Aug 8; 103(32):12167-72.
8. Wang C, Yin S, Cen L, et al. Differentiation of adipose-derived stem cells into contractile smooth muscle cells induced by transforming growth factor-betal and bone morphogenetic protein-4. Tissue Eng Part A.2010 Apr; 16(4):1201-13.
9. Rohman G, Pettit JJ, Isaure F, et al. Influence of the physical properties of two-dimensional polyester substrates on the growth of normal human urothelial and urinary smooth muscle cells in vitro. Biomaterials.2007 May;28(14):2264-74.
10. Lin HK, Cowan R, Moore P, et al. Characterization of neuropathic bladder smooth muscle cells in culture. J Urol.2004 Mar; 171 (3):1348-52.
11. Lai JY, Yoon CY, Yoo JJ, et al. Phenotypic and functional characterization of in vivo tissue engineered smooth muscle from normal and pathological bladders. J Urol.2002 Oct;168(4 Pt 2):1853-7; discussion 1858.
12. Jeon ES, Moon HJ, Lee MJ, et al. Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smooth-muscle-like cells through a TGF-beta-dependent mechanism. J Cell Sci.2006 Dec 1;119(Pt 23):4994-5005.
13. Basu J, Genheimer C, Guthrie KI, et al. Expansion of the Human Adipose-derived Stromal Vascular Cell Fraction Yields a Population of Smooth Muscle-like Cells with Markedly Distinct Phenotypic and Functional Properties Relative to Mesenchymal Stem Cells. Tissue Eng Part C Methods.2011 Apr 2. [Epub ahead of print]
14. Marra KG, Brayfield CA, Rubin JP. Adipose stem cell differentiation into smooth muscle cells. Methods Mol Biol.2011;702:261-8.
15. de Villiers JA, Houreld N, Abrahamse H. Adipose derived stem cells and smooth muscle cells:implications for regenerative medicine. Stem Cell Rev.2009 Sep;5(3):256-65.
16. Lee WC, Maul TM, Vorp DA, et al. Effects of uniaxial cyclic strain on adipose-derived stem cell morphology, proliferation, and differentiation. Biomech Model Mechanobiol. 2007 Jul;6(4):265-73.
1. Bunnell BA, Flaat M, Gagliardi C, et al. Adipose-derived stem cells:isolation, expansion and differentiation. Methods.2008 Jun;45(2):115-20.
2. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res.2007 May 11;100(9):1249-60.
3. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell.2002 Dec;13(12):4279-95.
4. Utsunomiya T, Shimada M, Imura S, et al.Human adipose-derived stem cells:potential clinical applications in surgery. Surg Today.2011 Jan;41(1):18-23.
5. Wu H, Ren K, Zhao W, et al. Effect of electromagnetic fields on proliferation and differentiation of cultured mouse bone marrow mesenchymal stem cells. J Huazhong Univ Sci Technolog Med Sci.2005;25(2):185-7.
6. Wang FS, Yang KD, Chen RF, et al. Extracorporeal shock wave promotes growth and differentiation of bone-marrow stromal cells towards osteoprogenitors associated with induction of TGF-betal. J Bone Joint Surg Br.2002 Apr;84(3):457-61.
7. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng.2001 Apr;7(2):211-28.
8. Mizuno H, Zuk PA, Zhu M, et al. Myogenic differentiation by human processed lipoaspirate cells. Plast Reconstr Surg.2002 Jan;109(1):199-209; discussion 210-1.
9. Safford KM, Hicok KC, Safford SD, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun.2002 Jun 7;294(2):371-9.
10. Ashjian PH, Elbarbary AS, Edmonds B, et al. In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast Reconstr Surg.2003 May; 111(6):1922-31.
11. Rangappa S, Entwistle JW, Wechsler AS, et al. Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. J Thorac Cardiovasc Surg.2003 Jul;126(1):124-32.
12. Cao Y, Meng Y, Sun Z, et al. Potential of human adipose tissue derived adult stem cells differentiate into endothelial cells. Zhongguo Yi Xue Ke Xue Yuan Xue Bao.2005 Dec;27(6):678-82.
13. Romanov YA, Darevskaya AN, Merzlikina NV, et al. Mesenchymal stem cells from human bone marrow and adipose tissue:isolation, characterization, and differentiation potentialities. Bull Exp Biol Med.2005 Jul;140(1):138-43.
14. Prichard HL, Reichert WM, Klitzman B. Adult adipose-derived stem cell attachment to biomaterials. Biomaterials.2007 Feb;28(6):936-46.
15. Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet.1987 May;20(3):263-72.
16. Halvorsen YD, Franklin D, Bond AL, et al. Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells. Tissue Eng. 2001 Dec;7(6):729-41.
17. Halbleib M, Skurk T, de Luca C, et al. Tissue engineering of white adipose tissue using hyaluronic acid-based scaffolds. I:in vitro differentiation of human adipocyte precursor cells on scaffolds. Biomaterials.2003 Aug;24(18):3125-32.
18. Aust L, Devlin B, Foster SJ, et al. Yield of human adipose-derived adult stem cells from liposuction aspirates. Cytotherapy.2004;6(1):7-14.
19. Wall ME, Rachlin A, Otey CA, et al. Human adipose-derived adult stem cells upregulate palladin during osteogenesis and in response to cyclic tensile strain. Am J Physiol Cell Physiol.2007 Nov;293(5):C1532-8.
20. Yuksel E, Weinfeld AB, Cleek R, et al. De novo adipose tissue generation through long-term, local delivery of insulin and insulin-like growth factor-1 by PLGA/PEG microspheres in an in vivo rat model:a novel concept and capability. Plast Reconstr Surg.2000 Apr;105(5):1721-9.
21. Heydarkhan-Hagvall S, Schenke-Layland K, Dhanasopon AP, et al. Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering. Biomaterials.2008 Jul;29(19):2907-14.
22. Dudas JR, Losee JE, Penascino VM, et al. Leporine-derived adipose precursor cells exhibit in vitro osteogenic potential. J Craniofac Surg.2008 Mar;19(2):360-8.
23. Kisiday JD, Kopesky PW, Evans CH, et al. Evaluation of adult equine bone marrow-and adipose-derived progenitor cell chondrogenesis in hydrogel cultures. J Orthop Res. 2008 Mar;26(3):322-31.
24. Zhao LR, Duan WM, Reyes M, et al. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Exp Neurol.2002 Mar; 174(1):11-20.
25. Bacou F, el Andalousi RB, Daussin PA, et al. Transplantation of adipose tissue-derived stromal cells increases mass and functional capacity of damaged skeletal muscle. Cell Transplant.2004;13(2):103-11.
26. Jack GS, Almeida FG, Zhang R, et al. Processed lipoaspirate cells for tissue engineering of the lower urinary tract: implications for the treatment of stress urinary incontinence and bladder reconstruction. J Urol.2005 Nov;174(5):2041-5.
27. Martines E, McGhee K, Wilkinson C, et al. A parallel-plate flow chamber to study initial cell adhesion on a nanofeatured surface. IEEE Trans Nanobioscience.2004 Jun;3(2):90-5.
28. Liu J, Zou L, Wang J, et al. Hydrostatic pressure promotes WntlOb and Wnt4 expression dependent and independent on ERK signaling in early-osteoinduced MSCs. Biochem Biophys Res Commun.2009 Feb 6;379(2):505-9.
29. Miyanishi K, Trindade MC, Lindsey DP, et al. Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro. Tissue Eng.2006 Jun;12(6):1419-28.
30. Hamilton DW, Maul TM, Vorp DA.Characterization of the response of bone marrow-derived progenitor cells to cyclic strain:implications for vascular tissue-engineering applications. Tissue Eng.2004 Mar-Apr;10(3-4):361-9.
31. Hamilton DW, Maul TM, Vorp DA, et al. An analysis of the complete strain field within Flexercell membranes. J Biomech.2004 Dec;37(12):1923-28.
32.李德强,戴冠戎.生物反应器在组织工程中的应用进展[J].国际骨科学杂志.2008,29(1)：8-10.
33. Knippenberg M, Helder MN, Doulabi BZ, et al. Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation. Tissue Eng.2005 Nov-Dec; 11(11-12):1780-8.
34. Tjabringa GS, Vezeridis PS, Zandieh-Doulabi B,et al. Polyamines modulate nitric oxide production and cox-2 gene expression in response to mechanical loading in human adipose tissue-derived mesenchymal stem cells. Stem Cells.2006 Oct; 24 (10): 2262-9.
35. Lee WC, Maul TM, Vorp DA, et al. Effects of uniaxial cyclic strain on adipose-derived stem cell morphology, proliferation, and differentiation. Biomech Model Mechanobiol. 2007 Jul;6(4):265-73.
36. Fischer LJ, Mcllhenny S, Tulenko T, et al. Endothelial differentiation of adipose-derived stem cells:effects of endothelial cell growth supplement and shear force. J Surg Res.2009 Mar; 152(1):157-66.
37.康红军,卢世璧,张莉,等.人脂肪干细胞复合脱细胞软骨基质支架在生物反应器中构建组织工程软骨[J].中国组织工程研究与临床康复.2007,10(11)：1801-4.
38.梁慧,王庆胜,方海滨,等.脂肪干细胞体外快速扩增实验研究[J].中国误诊学杂志.2008,21(8)：5047—9.
39.戴景兴,杨林林,曲戎梅,等.差速贴壁法分离培养脂肪源间充质干细胞[J].中国临床解剖学杂志.2007,25(3)：313-6.
40.管利东,王韫芳,刘大庆,等.人脂肪来源干细胞在模拟微重力环境中的生长特性[J].自然科学进展.2007,17(1)：22-8.
41.冯振洲,蒋淳,王晓峰.微重力旋转培养系统对脂肪干细胞分化增殖的影响.中国临床医学[J].2007,14(6)：843-5.
42.周晓东,王常勇,毛天球,等.微载体悬浮培养体系对人脂肪基质干细胞增殖与分化的影响[J].中国临床康复.2005,9(22)：62-4.
43. Wang JH, Yang G, Li Z. Controlling cell responses to cyclic mechanical stretching. Ann Biomed Eng.2005 Mar;33(3):337-42.
44. Hughes-Fulford M. Signal transduction and mechanical stress. Sci STKE.2004 Aug 31;2004(249):RE12.
45. Banes AJ, Lee G, Graff R, et al. Mechanical forces and signaling in connective tissue cells:cellular mechanisms of detection, transduction, and responses to mechanical deformation. Current Opin Orthopedics.2001,12:389—96.
46. Stegemann JP, Nerem RM. Phenotype modulation in vascular tissue engineering using biochemical and mechanical stimulation. Ann Biomed Eng.2003 Apr;31(4):391-402.
47. Stolberg S, McCloskey KE. Can shear stress direct stem cell fate? Biotechnol Prog. 2009 Jan-Feb;25(1):10-9.
48.宋克东,刘天庆,崔占峰,等.生物反应器在组织工程中的应用进展.中国临床康复[J].2004,32(8)：7252-4.
49. Plett PA, Abonour R, Frankovitz SM, et al. Impact of modeled microgravity on migration, differentiation, and cell cycle control of primitive human hematopoietic progenitor cells. Exp Hematol.2004 Aug;32(8):773-81.
2. Atala A. Tissue engineering and regenerative medicine: concepts for clinical application. Rejuvenation Res.2004 Spring;7(1):15-31.
3. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res.2007 May 11;100(9):1249-60.
4. Atala A. Tissue engineering, stem cells and cloning: current concepts and changing trends. Expert Opin Biol Ther.2005 Jul;5(7):879-92.
5. Skalak R, Fox CF. Tissue Engineering: Proceedings of a workshop held at Granlibakken, Lake Tahoe, CA, New York, NY:Liss; 1988:26-9.
6. Langer R, Vacanti JP. Tissue engineering. Science.1993 May 14;260(5110):920-6.
7. Butler DL, Goldstein SA, Guilak F. Functional tissue engineering: the role of biomechanics. J Biomech Eng.2000 Dec; 122(6):570-5.
8. Nirmalanandhan VS, Sittampalam GS. Stem cells in drug discovery, tissue engineering, and regenerative medicine:emerging opportunities and challenges. J Biomol Screen. 2009 Aug;14(7):755-68.
9. Nakahara H, Goldberg VM, Caplan AI. Culture-expanded human periosteal-derived cells exhibit osteochondral potential in vivo. J Orthop Res.1991 Jul;9(4):465-76.
10. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science.1988 Jul 1;241(4861):58-62.
11. Stemple DL, Anderson DJ. Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell.1992 Dec 11;71(6):973-85.
12. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol.2000 Apr;109(1):235-42.
13. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng.2001 Apr;7(2):211-28.
14. Mo XM, Xu CY, Kotaki M, et al. Electrospun P(LLA-CL) nanofiber:a biomimetic extracellular matrix for smooth muscle cell and endothelial cell proliferation. Biomaterials.2004 May;25(10):1883-90.
15. Calandrelli L, Calarco A, Laurienzo P, et al. Compatibilized polymer blends based on PDLLA and PCL for application in bioartificial liver. Biomacromolecules.2008 Jun;9(6):1527-34.
16. Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, et al. Electrospun poly(epsilon-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials. 2008 Dec;29(34):4532-9.
17. Chen ZG, Wang PW, Wei B, et al. Electrospun collagen-chitosan nanofiber: a biomimetic extracellular matrix for endothelial cell and smooth muscle cell. Acta Biomater.2010 Feb;6(2):372-82.
18. Awad HA, Wickham MQ, Leddy HA, et al. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials.2004 Jul;25(16):3211-22.
19. Liu Q, Cen L, Yin S, et al. A comparative study of proliferation and osteogenic differentiation of adipose-derived stem cells on akermanite and beta-TCP ceramics. Biomaterials.2008 Dec;29(36):4792-9.
20. Haimi S, Suuriniemi N, Haaparanta AM, et al. Growth and osteogenic differentiation of adipose stem cells on PLA/bioactive glass and PLA/beta-TCP scaffolds. Tissue Eng Part A.2009 Jul; 15(7):1473-80.
21. Itoi Y, Takatori M, Hyakusoku H, et al. Comparison of readily available scaffolds for adipose tissue engineering using adipose-derived stem cells. J Plast Reconstr Aesthet Surg.2010 May;63(5):858-64.
22. Kasten P, Beyen I, Niemeyer P, et al. Porosity and pore size of beta-tricalcium phosphate scaffold can influence protein production and osteogenic differentiation of human mesenchymal stem cells:an in vitro and in vivo study. Acta Biomater.2008 Nov;4(6):1904-15.
23. Marino G, Rosso F, Cafiero G, et al. Beta-tricalcium phosphate 3D scaffold promote alone osteogenic differentiation of human adipose stem cells:in vitro study. J Mater Sci Mater Med.2010 Jan;21(1):353-63. Epub 2009 Aug 5.
24. Min BM, Lee G, Kim SH, et al. Electrospinning of silk fibroin nanofibers and its effect on the adhesion and spreading of normal human keratinocytes and fibroblasts in vitro. Biomaterials.2004 Mar-Apr;25(7-8):1289-97.
25. Mehlhorn AT, Zwingmann J, Finkenzeller G, et al. Chondrogenesis of adipose-derived adult stem cells in a poly-lactide-co-glycolide scaffold. Tissue Eng Part A.2009 May; 15(5):1159-67.
26. Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, et al. Electrospun poly(epsilon-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials.2008 Dec;29(34):4532-9.
27. Mo SM, Oh IJ. Release of adriamycin from poly(lactide-co-glycolide)-polyethylene glycol nanoparticles. J Nanosci Nanotechnol.2011 Feb;11(2):1795-8.
28. Peng J, Li X, Guo G, et al. Preparation and characterization of poly(vinyl alcohol) /poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone)/ nano-hydroxyapatite composite membranes for tissue engineering. J Nanosci Nanotechnol.2011 Mar;11(3):2354-60.
29. Itaka K, Ishii T, Hasegawa Y, et al. Biodegradable polyamino acid-based polycations as safe and effective gene carrier minimizing cumulative toxicity. Biomaterials.2010 May;31(13):3707-14.
30.金锡御,宋波.《临床尿动力学》.第一版.北京：人民卫生出版社,2002,p：10-11
31. Shokeir A, Osman Y, Gabr M, et al. Acellular matrix tube for canine urethral replacement: is it fact or fiction? J Urol.2004 Jan;171(1):453-6.
32. De Filippo RE, Yoo JJ, Atala A. Urethral replacement using cell seeded tubularized collagen matrices. J Urol.2002 Oct; 168(4 Pt 2):1789-92; discussion 1792-3.
33. Fu Q, Cao YL. Use of tissue engineering in treatment of the male genitourinary tract abnormalities. J Sex Med.2010 May;7(5):1741-6.
34. Barbagli G, Lazzeri M. Urethral reconstruction. Curr Opin Urol.2006 Nov;16(6):391-5.
35. Shokeir AA, Harraz AM, El-Din AB.Tissue engineering and stem cells:basic principles and applications in urology. Int J Urol.2010 Dec;17(12):964-73.
1. Vacanti J.Tissue engineering and regenerative medicine:from first principles to state of the art. J Pediatr Surg.2010 Feb;45(2):291-4.
2. Chapekar MS. Tissue engineering:challenges and opportunities. J Biomed Mater Res. 2000;53(6):617-20.
3. Meyer U, Meyer T, Handschel J, et al. Fundamentals of tissue engineering and Regenerative medicine. Springer.2009;26:224.
4. Khademhosseini A, Vacanti JP, Langer R. Progress in tissue engineering. Sci Am.2009 May;300(5):64-71.
5. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res.2007 May 11; 100(9):1249-60.
6. Nakahara H, Goldberg VM, Caplan AI. Culture-expanded human periosteal-derived cells exhibit osteochondral potential in vivo. J Orthop Res.1991 Jul;9(4):465-76.
7. Stemple DL, Anderson DJ. Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell.1992 Dec 11;71(6):973-85.
8. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol.2000 Apr; 109(1):235-42.
9. Asakura A, Komaki M, Rudnicki M. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation.2001 Oct;68(4-5):245-53.
10. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng.2001 Apr;7(2):211-28.
11. Cherubino M, Marra KG. Adipose-derived stem cells for soft tissue reconstruction. Regen Med.2009 Jan;4(1):109-17.
12. Mizuno H. Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review. J Nippon Med Sch.2009 Apr;76(2):56-66.
13. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell.2002 Dec;13(12):4279-95.
14. Hauner H, Wabitsch M, Pfeiffer EF. Differentiation of adipocyte precursor cells from obese and nonobese adult women and from different adipose tissue sites. Horm Metab Res Suppl.1988; 19:35-9.
15. Hauner H, Entenmann G. Regional variation of adipose differentiation in cultured stromal-vascular cells from the abdominal and femoral adipose tissue of obese women. Int J Obes.1991 Feb;15(2):121-6.
16. Shahparaki A, Grunder L, Sorisky A. Comparison of human abdominal subcutaneous versus omental preadipocyte differentiation in primary culture. Metabolism.2002 Sep;51(9):1211-5.
17. Van Harmelen V, Rohrig K, Hauner H. Comparison of proliferation and differentiation capacity of human adipocyte precursor cells from the omental and subcutaneous adipose tissue depot of obese subjects. Metabolism.2004 May;53(5):632-7.
18. Prunet-Marcassus B, Cousin B, Caton D, et al. From heterogeneity to plasticity in adipose tissues: site-specific differences. Exp Cell Res.2006 Apr 1;312(6):727-36.
19. Fraser JK, Wulur I, Alfonso Z, et al. Differences in stem and progenitor cell yield in different subcutaneous adipose tissue depots. Cytotherapy.2007;9(5):459-67.
20. Hausman DB, Park HJ, Hausman GJ. Isolation and culture of preadipocytes from rodent white adipose tissue. Methods Mol Biol.2008;456:201-19.
21. Jurgens WJ, Oedayrajsingh-Varma MJ, Helder MN, Zandiehdoulabi B et al. Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies. Cell Tissue Res.2008 Jun;332(3):415-26.
22. Toyoda M, Matsubara Y, Lin K, et al. Characterization and comparison of adipose tissue-derived cells from human subcutaneous and omental adipose tissues. Cell Biochem Funct.2009 Oct;27(7):440-7.
23. Schipper BM, Marra KG, Zhang W, et al. Regional anatomic and age effects on cell function of human adipose-derived stem cells. Ann Plast Surg.2008 May;60(5):538-44.
24. Strem BM, Hicok KC, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med.2005 Sep;54(3):132-41.
25. Gronthos S, Franklin DM, Leddy HA, et al. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol.2001 Oct;189(1):54-63.
26. de Girolamo L, Lopa S, Arrigoni E, et al. Human adipose-derived stem cells isolated from young and elderly women:their differentiation potential and scaffold interaction during in vitro osteoblastic differentiation. Cytotherapy.2009;11(6):793-803.
27. Masoud I, Shapiro F, Kent R, et al. A longitudinal study of the growth of the New Zealand white rabbit:cumulative and biweekly incremental growth rates for body length, body weight, femoral length, and tibial length. J Orthop Res.1986;4(2):221-31.
28. Kang HM, Kim J, Park S, Kim J et al. Insulin-secreting cells from human eyelid-derived stem cells alleviate type I diabetes in immunocompetent mice. Stem Cells.2009 Aug;27(8):1999-2008.
29. Arrigoni E, Lopa S, de Girolamo L, et al. Isolation, characterization and osteogenic differentiation of adipose-derived stem cells:from small to large animal models. Cell Tissue Res.2009 Dec;338(3):401-11.
1. Griffith LG, Naughton G. Tissue engineering--current challenges and expanding opportunities. Science.2002 Feb 8;295(5557):1009-14.
2. Atala A. Tissue engineering and regenerative medicine:concepts for clinical application. Rejuvenation Res.2004 Spring;7(1):15-31.
3. Atala A. Tissue engineering, stem cells and cloning: current concepts and changing trends. Expert Opin Biol Ther.2005 Jul;5(7):879-92.
4. Bhatia SK. Tissue engineering for clinical applications. Biotechnol J.2010 Dec;5(12):1309-23.
5. Yarlagadda PK, Chandrasekharan M, Shyan JY. Recent advances and current developments in tissue scaffolding. Biomed Mater Eng.2005; 15(3):159-77.
6. Mo XM, Xu CY, Kotaki M, et al. Electrospun P(LLA-CL) nanofiber: a biomimetic extracellular matrix for smooth muscle cell and endothelial cell proliferation. Biomaterials.2004 May;25(10):1883-90.
7. Chen ZG, Wang PW, Wei B, et al. Electrospun collagen-chitosan nanofiber: a biomimetic extracellular matrix for endothelial cell and smooth muscle cell. Acta Biomater.2010 Feb;6(2):372-82.
8. Calandrelli L, Calarco A, Laurienzo P, et al. Compatibilized polymer blends based on PDLLA and PCL for application in bioartificial liver. Biomacromolecules.2008 Jun;9(6):1527-34.
9. Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, et al. Electrospun poly(epsilon-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials.2008 Dec;29(34):4532-9.
10. Wu D, Zhang Y, Zhang M, et al. Selective localization of multiwalled carbon nanotubes in poly(epsilon-caprolactone)/polylactide blend. Biomacromolecules.2009 Feb 9;10(2):417-24.
11. Gunn J, Zhang M. Polyblend nanofibers for biomedical applications:perspectives and challenges. Trends Biotechnol.2010 Apr;28(4):189-97.
12. Jiang W, Kim BY, Rutka JT, et al. Nanoparticle-mediated cellular response is size-dependent. Nat Nanotechnol.2008 Mar;3(3):145-50.
13. McCullen SD, Stevens DR, Roberts WA, et al. Characterization of electrospun nanocomposite scaffolds and biocompatibility with adipose-derived human mesenchymal stem cells. Int J Nanomedicine.2007;2(2):253-63.
14. Li WJ, Tuli R, Okafor C, et al. A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials.2005 Feb;26(6):599-609.
15. Mehlhorn AT, Zwingmann J, Finkenzeller G, et al. Chondrogenesis of adipose-derived adult stem cells in a poly-lactide-co-glycolide scaffold. Tissue Eng Part A.2009 May; 15(5):1159-67.
16. Lee JH, Rhie JW, Oh DY, et al. Osteogenic differentiation of human adipose tissue-derived stromal cells (hASCs) in a porous three-dimensional scaffold. Biochem Biophys Res Commun.2008 Jun 6;370(3):456-60.
17. Baker SC, Rohman G, Southgate J, et al. The relationship between the mechanical properties and cell behaviour on PLGA and PCL scaffolds for bladder tissue engineering. Biomaterials.2009 Mar;30(7):1321-8.
18. Ajami-Henriquez D, Rodriguez M, Sabino M, et al. Evaluation of cell affinity on poly(L-lactide) and poly(epsilon-caprolactone) blends and on PLLA-b-PCL diblock copolymer surfaces. J Biomed Mater Res A.2008 Nov;87(2):405-17.
19. Kwon IK, Kidoaki S, Matsuda T. Electrospun nano-to microfiber fabrics made of biodegradable copolyesters:structural characteristics, mechanical properties and cell adhesion potential. Biomaterials.2005 Jun;26(18):3929-39.
20. Xu CY, Inai R, Kotaki M, et al. Aligned biodegradable nanofibrous structure: a potential scaffold for blood vessel engineering. Biomaterials.2004 Feb;25(5):877-86.
21. Xu C, Inai R, Kotaki M, et al. Electrospun nano fiber fabrication as synthetic extracellular matrix and its potential for vascular tissue engineering. Tissue Eng.2004 Jul-Aug;10(7-8):1160-8.
22. He W, Yong T, Teo WE, et al. Fabrication and endothelialization of collagen-blended biodegradable polymer nano fibers:potential vascular graft for blood vessel tissue engineering. Tissue Eng.2005 Sep-Oct;11(9-10):1574-88.
23. Sarazin P, Orts WJ, Favis BD. Binary and ternary blends of polylactide, polycaprolactone and thermoplastic starch. Polymer.2008 Jan;49(2):599-609.
24. Lopez-Rodriguez N, Lopez-Arraiza A, Meaurio E, et al. Crystallization, morphology, and mechanical behavior of polylactide/poly(ε-caprolactone) blends. Polym Eng Sci. 2006 Sep;46(9):1299-1308.
25. Sarazin P, Roy X, Favis BD. Controlled preparation and properties of porous poly(L-lactide) obtained from a co-continuous blend of two biodegradable polymers. Biomaterials.2004 Dec;25(28):5965-78.
26. Roy X, Sarazin P, Favis BD. Ultraporous Nanosheath Materials by Layer-by-Layer Deposition onto Co-continuous Polymer-Blend Templates. Adv Mater.2006 Apr;18(8):1015-9.
27. Wu DF, Zhang YS, Zhang M, et al. Phase behavior and its viscoelastic response of polylactide/poly(ε-caprolactone) blend. Eur Polym J.2008 Jul;44(7):2171-83.
28. Tsuji H, Ishizaka T. Blends of aliphatic polyesters. VI. Lipase-catalyzed hydrolysis and visualized phase structure of biodegradable blends from poly(epsilon-caprolactone) and poly(L-lactide). Int J Biol Macromol.2001 Aug 20;29(2):83-9.
29. Nakagami H, Morishita R, Maeda K, et al. Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy. J Atheroscler Thromb.2006 Apr;13(2):77-81.
30. Zhu Y, Liu T, Song K, et al. Adipose-derived stem cell:a better stem cell than BMSC. Cell Biochem Funct.2008 Aug;26(6):664-75.
31. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res.2007 May 11;100(9):1249-60.
32. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng.2001 Apr;7(2):211-28.
33. van Aalst JA, Reed CR, Han L, et al. Cellular incorporation into electrospun nanofibers: retained viability, proliferation, and function in fibroblasts. Ann Plast Surg.2008 May;60(5):577-83.
34. Heydarkhan-Hagvall S, Schenke-Layland K, Dhanasopon AP, et al. Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering. Biomaterials.2008 Jul;29(19):2907-14.
35. Schenke-Layland K, Rofail F, Heydarkhan S, et al. The use of three-dimensional nanostructures to instruct cells to produce extracellular matrix for regenerative medicine strategies. Biomaterials.2009 Sep;30(27):4665-75.
36. McCullen SD, Zhu Y, Bernacki SH, et al. Electrospun composite poly(L-lactic acid)/tricalcium phosphate scaffolds induce proliferation and osteogenic differentiation of human adipose-derived stem cells. Biomed Mater.2009 Jun;4(3):035002.
37. Shanti RM, Janjanin S, Li WJ, et al. In vitro adipose tissue engineering using an electrospun nanofibrous scaffold. Ann Plast Surg.2008 Nov;61(5):566-71.
38. McCullen SD, Stevens DR, Roberts WA, et al. Characterization of electrospun nanocomposite scaffolds and biocompatibility with adipose-derived human mesenchymal stem cells. Int J Nanomedicine.2007;2(2):253-63.
39. Noh HK, Lee SW, Kim JM, et al. Electrospinning of chitin nanofibers:degradation behavior and cellular response to normal human keratinocytes and fibroblasts. Biomaterials.2006 Jul;27(21):3934-44.
40. Liao GY, Chen L, Zeng XY, et al. Electrospun poly(L-lactide)/poly(ε-caprolactone) blend fibers and their cellular response to adipose-derived stem cells. J APPL POLYM SCI.2011 May;120(4):2154-65.
41. Huang ZM, Zhang YZ, Kotaki M, et al. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Compos Sci Technol.2003 Nov;63(15):2223-53.
42. You Y, Min BM, Lee SJ, et al. In vitro degradation behavior of electrospun polyglycolide, polylactide, and poly(lactideco-glycolide). J Appl Polym Sci 2005 Jan;95(2):193-200.
43. Chen DR, Bei JZ, Wang SG. Polycaprolactone microparticles and their biodegradation. Polym Degrad Stab 2000 Mar;67(3):455-9.
1.金锡御,宋波.《临床尿动力学》.第一版.北京：人民卫生出版社,2002,p：10-11
2. Mathur RK, Himanshu A, Sudarshan O. Technique of anterior urethra urethroplasty using tunica albuginea of corpora cavernosa. Int J Urol.2007 Mar;14(3):209-13.
3. Shokeir A, Osman Y, Gabr M, et al. Acellular matrix tube for canine urethral replacement: is it fact or fiction? J Urol.2004 Jan;171(1):453-6.
4. De Filippo RE, Yoo JJ, Atala A. Urethral replacement using cell seeded tubularized collagen matrices. J Urol.2002 Oct;168(4 Pt 2):1789-92; discussion 1792-3.
5. McAninch JW. Urethral reconstruction: a continuing challenge. J Urol.2005 Jan;173(1):7.
6. Shastri VP. In vivo engineering of tissues:Biological considerations, challenges, strategies, and future directions. Adv Mater.2009 Sep 4;21(32-33):3246-54.
7. Rodriguez LV, Alfonso Z, Zhang R, et al. Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells. Proc Natl Acad Sci U S A.2006 Aug 8; 103(32):12167-72.
8. Wang C, Yin S, Cen L, et al. Differentiation of adipose-derived stem cells into contractile smooth muscle cells induced by transforming growth factor-betal and bone morphogenetic protein-4. Tissue Eng Part A.2010 Apr; 16(4):1201-13.
9. Rohman G, Pettit JJ, Isaure F, et al. Influence of the physical properties of two-dimensional polyester substrates on the growth of normal human urothelial and urinary smooth muscle cells in vitro. Biomaterials.2007 May;28(14):2264-74.
10. Lin HK, Cowan R, Moore P, et al. Characterization of neuropathic bladder smooth muscle cells in culture. J Urol.2004 Mar; 171 (3):1348-52.
11. Lai JY, Yoon CY, Yoo JJ, et al. Phenotypic and functional characterization of in vivo tissue engineered smooth muscle from normal and pathological bladders. J Urol.2002 Oct;168(4 Pt 2):1853-7; discussion 1858.
12. Jeon ES, Moon HJ, Lee MJ, et al. Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smooth-muscle-like cells through a TGF-beta-dependent mechanism. J Cell Sci.2006 Dec 1;119(Pt 23):4994-5005.
13. Basu J, Genheimer C, Guthrie KI, et al. Expansion of the Human Adipose-derived Stromal Vascular Cell Fraction Yields a Population of Smooth Muscle-like Cells with Markedly Distinct Phenotypic and Functional Properties Relative to Mesenchymal Stem Cells. Tissue Eng Part C Methods.2011 Apr 2. [Epub ahead of print]
14. Marra KG, Brayfield CA, Rubin JP. Adipose stem cell differentiation into smooth muscle cells. Methods Mol Biol.2011;702:261-8.
15. de Villiers JA, Houreld N, Abrahamse H. Adipose derived stem cells and smooth muscle cells:implications for regenerative medicine. Stem Cell Rev.2009 Sep;5(3):256-65.
16. Lee WC, Maul TM, Vorp DA, et al. Effects of uniaxial cyclic strain on adipose-derived stem cell morphology, proliferation, and differentiation. Biomech Model Mechanobiol. 2007 Jul;6(4):265-73.
1. Bunnell BA, Flaat M, Gagliardi C, et al. Adipose-derived stem cells:isolation, expansion and differentiation. Methods.2008 Jun;45(2):115-20.
2. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res.2007 May 11;100(9):1249-60.
3. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell.2002 Dec;13(12):4279-95.
4. Utsunomiya T, Shimada M, Imura S, et al.Human adipose-derived stem cells:potential clinical applications in surgery. Surg Today.2011 Jan;41(1):18-23.
5. Wu H, Ren K, Zhao W, et al. Effect of electromagnetic fields on proliferation and differentiation of cultured mouse bone marrow mesenchymal stem cells. J Huazhong Univ Sci Technolog Med Sci.2005;25(2):185-7.
6. Wang FS, Yang KD, Chen RF, et al. Extracorporeal shock wave promotes growth and differentiation of bone-marrow stromal cells towards osteoprogenitors associated with induction of TGF-betal. J Bone Joint Surg Br.2002 Apr;84(3):457-61.
7. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng.2001 Apr;7(2):211-28.
8. Mizuno H, Zuk PA, Zhu M, et al. Myogenic differentiation by human processed lipoaspirate cells. Plast Reconstr Surg.2002 Jan;109(1):199-209; discussion 210-1.
9. Safford KM, Hicok KC, Safford SD, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun.2002 Jun 7;294(2):371-9.
10. Ashjian PH, Elbarbary AS, Edmonds B, et al. In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast Reconstr Surg.2003 May; 111(6):1922-31.
11. Rangappa S, Entwistle JW, Wechsler AS, et al. Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. J Thorac Cardiovasc Surg.2003 Jul;126(1):124-32.
12. Cao Y, Meng Y, Sun Z, et al. Potential of human adipose tissue derived adult stem cells differentiate into endothelial cells. Zhongguo Yi Xue Ke Xue Yuan Xue Bao.2005 Dec;27(6):678-82.
13. Romanov YA, Darevskaya AN, Merzlikina NV, et al. Mesenchymal stem cells from human bone marrow and adipose tissue:isolation, characterization, and differentiation potentialities. Bull Exp Biol Med.2005 Jul;140(1):138-43.
14. Prichard HL, Reichert WM, Klitzman B. Adult adipose-derived stem cell attachment to biomaterials. Biomaterials.2007 Feb;28(6):936-46.
15. Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet.1987 May;20(3):263-72.
16. Halvorsen YD, Franklin D, Bond AL, et al. Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells. Tissue Eng. 2001 Dec;7(6):729-41.
17. Halbleib M, Skurk T, de Luca C, et al. Tissue engineering of white adipose tissue using hyaluronic acid-based scaffolds. I:in vitro differentiation of human adipocyte precursor cells on scaffolds. Biomaterials.2003 Aug;24(18):3125-32.
18. Aust L, Devlin B, Foster SJ, et al. Yield of human adipose-derived adult stem cells from liposuction aspirates. Cytotherapy.2004;6(1):7-14.
19. Wall ME, Rachlin A, Otey CA, et al. Human adipose-derived adult stem cells upregulate palladin during osteogenesis and in response to cyclic tensile strain. Am J Physiol Cell Physiol.2007 Nov;293(5):C1532-8.
20. Yuksel E, Weinfeld AB, Cleek R, et al. De novo adipose tissue generation through long-term, local delivery of insulin and insulin-like growth factor-1 by PLGA/PEG microspheres in an in vivo rat model:a novel concept and capability. Plast Reconstr Surg.2000 Apr;105(5):1721-9.
21. Heydarkhan-Hagvall S, Schenke-Layland K, Dhanasopon AP, et al. Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering. Biomaterials.2008 Jul;29(19):2907-14.
22. Dudas JR, Losee JE, Penascino VM, et al. Leporine-derived adipose precursor cells exhibit in vitro osteogenic potential. J Craniofac Surg.2008 Mar;19(2):360-8.
23. Kisiday JD, Kopesky PW, Evans CH, et al. Evaluation of adult equine bone marrow-and adipose-derived progenitor cell chondrogenesis in hydrogel cultures. J Orthop Res. 2008 Mar;26(3):322-31.
24. Zhao LR, Duan WM, Reyes M, et al. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Exp Neurol.2002 Mar; 174(1):11-20.
25. Bacou F, el Andalousi RB, Daussin PA, et al. Transplantation of adipose tissue-derived stromal cells increases mass and functional capacity of damaged skeletal muscle. Cell Transplant.2004;13(2):103-11.
26. Jack GS, Almeida FG, Zhang R, et al. Processed lipoaspirate cells for tissue engineering of the lower urinary tract: implications for the treatment of stress urinary incontinence and bladder reconstruction. J Urol.2005 Nov;174(5):2041-5.
27. Martines E, McGhee K, Wilkinson C, et al. A parallel-plate flow chamber to study initial cell adhesion on a nanofeatured surface. IEEE Trans Nanobioscience.2004 Jun;3(2):90-5.
28. Liu J, Zou L, Wang J, et al. Hydrostatic pressure promotes WntlOb and Wnt4 expression dependent and independent on ERK signaling in early-osteoinduced MSCs. Biochem Biophys Res Commun.2009 Feb 6;379(2):505-9.
29. Miyanishi K, Trindade MC, Lindsey DP, et al. Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro. Tissue Eng.2006 Jun;12(6):1419-28.
30. Hamilton DW, Maul TM, Vorp DA.Characterization of the response of bone marrow-derived progenitor cells to cyclic strain:implications for vascular tissue-engineering applications. Tissue Eng.2004 Mar-Apr;10(3-4):361-9.
31. Hamilton DW, Maul TM, Vorp DA, et al. An analysis of the complete strain field within Flexercell membranes. J Biomech.2004 Dec;37(12):1923-28.
32.李德强,戴冠戎.生物反应器在组织工程中的应用进展[J].国际骨科学杂志.2008,29(1)：8-10.
33. Knippenberg M, Helder MN, Doulabi BZ, et al. Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation. Tissue Eng.2005 Nov-Dec; 11(11-12):1780-8.
34. Tjabringa GS, Vezeridis PS, Zandieh-Doulabi B,et al. Polyamines modulate nitric oxide production and cox-2 gene expression in response to mechanical loading in human adipose tissue-derived mesenchymal stem cells. Stem Cells.2006 Oct; 24 (10): 2262-9.
35. Lee WC, Maul TM, Vorp DA, et al. Effects of uniaxial cyclic strain on adipose-derived stem cell morphology, proliferation, and differentiation. Biomech Model Mechanobiol. 2007 Jul;6(4):265-73.
36. Fischer LJ, Mcllhenny S, Tulenko T, et al. Endothelial differentiation of adipose-derived stem cells:effects of endothelial cell growth supplement and shear force. J Surg Res.2009 Mar; 152(1):157-66.
37.康红军,卢世璧,张莉,等.人脂肪干细胞复合脱细胞软骨基质支架在生物反应器中构建组织工程软骨[J].中国组织工程研究与临床康复.2007,10(11)：1801-4.
38.梁慧,王庆胜,方海滨,等.脂肪干细胞体外快速扩增实验研究[J].中国误诊学杂志.2008,21(8)：5047—9.
39.戴景兴,杨林林,曲戎梅,等.差速贴壁法分离培养脂肪源间充质干细胞[J].中国临床解剖学杂志.2007,25(3)：313-6.
40.管利东,王韫芳,刘大庆,等.人脂肪来源干细胞在模拟微重力环境中的生长特性[J].自然科学进展.2007,17(1)：22-8.
41.冯振洲,蒋淳,王晓峰.微重力旋转培养系统对脂肪干细胞分化增殖的影响.中国临床医学[J].2007,14(6)：843-5.
42.周晓东,王常勇,毛天球,等.微载体悬浮培养体系对人脂肪基质干细胞增殖与分化的影响[J].中国临床康复.2005,9(22)：62-4.
43. Wang JH, Yang G, Li Z. Controlling cell responses to cyclic mechanical stretching. Ann Biomed Eng.2005 Mar;33(3):337-42.
44. Hughes-Fulford M. Signal transduction and mechanical stress. Sci STKE.2004 Aug 31;2004(249):RE12.
45. Banes AJ, Lee G, Graff R, et al. Mechanical forces and signaling in connective tissue cells:cellular mechanisms of detection, transduction, and responses to mechanical deformation. Current Opin Orthopedics.2001,12:389—96.
46. Stegemann JP, Nerem RM. Phenotype modulation in vascular tissue engineering using biochemical and mechanical stimulation. Ann Biomed Eng.2003 Apr;31(4):391-402.
47. Stolberg S, McCloskey KE. Can shear stress direct stem cell fate? Biotechnol Prog. 2009 Jan-Feb;25(1):10-9.
48.宋克东,刘天庆,崔占峰,等.生物反应器在组织工程中的应用进展.中国临床康复[J].2004,32(8)：7252-4.
49. Plett PA, Abonour R, Frankovitz SM, et al. Impact of modeled microgravity on migration, differentiation, and cell cycle control of primitive human hematopoietic progenitor cells. Exp Hematol.2004 Aug;32(8):773-81.