Characterization of a microbial transglutaminase cross-linked type II collagen scaffold

This study investigated the effect on the mechanical and physicochemical properties of type II collagen scaffolds after cross-linking with microbial transglutaminase (mTGase). It is intended to develop a collagen-based scaffold to be used for the treatment of degenerated intervertebral discs. By measuring the amount of epsilon-(gamma-glutamyl)lysine isodipeptide formed after cross-linking, it was determined that the optimal enzyme concentration was 0.005% (w/v). From the production of covalent bonds induced by mTGase cross-linking, the degradation resistance of type II collagen scaffolds can be enhanced. Rheological analysis revealed an almost sixfold increase in storage modulus (G') with 0.005% (w/v) mTGase cross-linked scaffolds (1.31 +/- 0.03 kPa) compared to controls (0.21 +/- 0.01 kPa). There was a significant reduction in the level of cell-mediated contraction of scaffolds with increased mTGase concentrations. Cell proliferation assays showed that mTGase crosslinked scaffolds exhibited similar cytocompatibility properties in comparison to non-cross-linked scaffolds. In summary, cross-linking type II collagen with mTGase imparted more desirable properties, making it more applicable for use as a scaffold in tissue engineering applications.     

N-telopeptide of type II collagen interacts with annexin V on human chondrocytes

Type II collagen binds to chondrocytes through integrins and annexin V. While the potential integrin binding sites have been identified, it is unclear which domains bind to annexin V. Proteolytic fragments of collagen are known to modulate cell signaling pathways resulting in degradation of articular cartilage; it is unknown whether annexin V binds to the fragments. The focus of our study was to determine the binding of type II collagen and its fragments to chondrocytes using flow cytometry and fluorescence microscopy. The N-telopeptide binds to annexin V, whereas the C-telopeptide and triple helical peptides do not. These data suggest that the binding of the N-telopeptide of type II collagen is through annexin V, whereas binding of the C-telopeptide and the triple helical peptide to the surface of chondrocytes are potentially facilitated through other collagen receptors, such as integrins or cell-associated matrix proteins.     

Impact of cAMP on the T-cell response to type II collagen

There is considerable interest in the possible use of cAMP-elevating agents in the treatment of autoimmune diseases such as rheumatoid arthritis. The objective of this study was to evaluate the impact of different cAMP-elevating agents on the T-cell response to type II collagen within the context of collagen-induced arthritis, a murine model of rheumatoid arthritis. Spleen cells or lymph node cells from type-II-collagen-immunized DBA/1 mice were cultured in the presence of type II collagen plus one of five different cAMP-elevating agents: rolipram, forskolin, prostaglandin E2, 8-bromo-cAMP, or cholera toxin. Levels of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and IL-5 were measured in culture supernatants by enzyme-linked immunosorbent assay. All of the cAMP-elevating agents tested were found to profoundly suppress IFN-gamma production in a dose-dependent manner. IL-4 and IL-5 production was slightly up-regulated at low concentrations of the cAMP-elevating agents and was modestly suppressed at the highest concentrations of cAMP-elevating agents. Experiments were then carried out to determine whether T cells were directly affected by cAMP-elevating agents or whether the immunomodulatory effects were mediated via antigen-presenting cells. Pulsing T cells alone for a brief period with cholera toxin produced an almost identical effect to pulsing antigen-presenting cells alone, i.e. down-regulation of proliferation, down-regulation of IFN-gamma production with little effect on IL-5 production. It was concluded that cAMP-elevating agents suppressed T helper type 1 responses to type II collagen to a greater extent than T helper type 2 responses. The cAMP-elevating agents could directly influence the activity of T cells but, in addition, influenced the ability of antigen-presenting cells to support T helper type 1 responses.     

Comparative phenotypic analysis of articular chondrocytes cultured within type I or type II collagen scaffolds

Among the existing repair strategies for cartilage injury, tissue engineering approach using biomaterials and chondrocytes offers hope for treatments. In this context, collagen-based biomaterials are good candidates as scaffolds for chondrocytes in cell transplantation procedures. These scaffolds are provided under different forms (gel or crosslinked sponge) made with either type I collagen or type I or type II atelocollagen molecules. The present study was undertaken to investigate how bovine articular chondrocytes sense and respond to differences in the structure and organization of these collagen scaffolds, over a 12-day culture period. When chondrocytes were seeded in the collagen scaffolds maintained in free-floating conditions, cells contracted gels to 40-60% and sponges to 15% of their original diameter. Real-time polymerase chain reaction analysis indicated that the chondrocyte phenotype, assessed notably by the ratio of COL2A1/COL1A2 mRNA and alpha10/alpha11 integrin subunit mRNA, was comparatively better sustained in type I collagen sponges when seeded at high cell density, also in type I atelocollagen gels. Besides, proteoglycan accumulation in the different scaffolds, as assessed by measuring the sulfated glycosaminoglycan content, was found be highest in type I collagen sponges seeded at high cell density. In addition, gene expression of matrix metalloproteinase-13 increased dramatically (up to 90-fold) in chondrocytes cultured in the different gels, whereas it remained stable in the sponges. Our data taken together reveal that type I collagen sponges seeded at high cell density represent a suitable material for tissue engineering of cartilage.     

表皮生长因子对传代培养的大鼠生长板软骨细胞增殖与胶原合成的影响

目的：探讨不同浓度的表皮生长因子（EGF）对传代培养的大鼠肋生长板软骨细胞（RGC）增殖和胶原合成的影响。 方法： 分离、培养RGC，分别用Western blot和阿尔新蓝（Alcine blue）染色检测各代RGC中Ⅱ型胶原和蛋白多糖的表达；用［³H］-TdR和［³H］-proline掺入分别检测1、10和100 μg/L EGF对第1、3和5代RGC增殖和胶原合成的影响。 结果： 原代培养的RGC表达Ⅱ型胶原和蛋白多糖，从第4代起Ⅱ型胶原和蛋白多糖的表达迅速降低，RGC发生了去分化。［³H］-TdR掺入表明，EGF促进第1代RGC的增殖（P<0.01），3个浓度的作用依次为：1 μg/L>10 μg/L>100 μg/L，差异显著（P<0.01）；3个浓度的EGF对第3代RGC保持了相近的促增殖作用（P<0.01）；对第5代RGC均无促增殖作用(P>0.05)。与促增殖作用不同，不同浓度EGF促不同传代的RGC的［³H］-proline掺入率比对照组均高20%左右（P<0.01）。 结论： EGF具有促进培养RGC增殖和胶原合成的作用，连续传代引起的去分化降低了RGC对EGF促增殖作用的反应，但对EGF的促胶原合成作用没有产生影响。     

Enhancing effects of tilorone on collagen arthritis and humoral immune response to type II collagen

The effect of tilorone, which is known to suppress adjuvant arthritis, on the induction of collagen arthritis in rats was investigated. Combined data of the present experiments show that all of the tilorone-treated rats except one in the lowest dosage group developed arthritis but that the incidence of arthritis in the tilorone-treated groups was not significantly different from that of the control group. The results also show that the two higher dosages (12.5 and 25 mg/kg/day) of tilorone caused a significant increase in the severity of collagen arthritis. Humoral immune response to type II collagen was significantly augmented in these two higher dosage groups; however, delayed-type hypersensitivity response to type II collagen was suppressed while tilorone was administered continuously. In addition, treatment with tilorone caused a significant increase in the concentration of anticollagen IgG extractable from the joint tissue. Anticollagen IgG subclass analysis revealed that the major subclass was IgG2a in both the serum and paw extract, with minor amounts of IgG2b, IgG2c, and IgG1. The response of all these subclasses was almost equally activated by tilorone treatment.     

The immune response of guinea-pigs to type II collagen: poor cross-reactivity with homologous type II collagen accounts for resistance to collagen-induced arthritis.

In order to determine the susceptibility of guinea-pigs to collagen-induced arthritis (CIA), Hartley and Strain 13 guinea-pigs were immunized with heterologous or homologous type II collagen. None of the animals developed CIA. Because immunity to type II collagen plays a critical role in CIA, we characterized the guinea-pig's immune response to determine the basis for this resistance. Guinea-pigs develop cellular and humoral reactivity to heterologous type II collagen similar to that of CIA-susceptible rats. The reactions distinguish type I from type II collagen but not among several heterologous type II collagens. The cell-mediated immune (CMI) response was specific for determinants on the primary amino acid structure of collagen, whether native or denatured collagen was used for immunization; however, the humoral response was specific for the form of the molecule used for immunization. Guinea-pigs differ from CIA-susceptible rats in that immunization with homologous or heterologous type II collagen fails to induce significant cross-reactive immunity with the homologous antigen. A transient arthritis could be induced in animals immunized with heterologous type II collagen by injecting them intra-articularly with heterologous but not with homologous type II collagen. Our results show that the disparity between immunity to type II collagen and the susceptibility to develop CIA in guinea-pigs is due to their poor cross-reactive immune response to autologous type II collagen.     

Humoral immune response to citrullinated collagen type II determinants in early rheumatoid arthritis

Collagen type II (CII) is a relevant joint-specific autoantigen in the pathogenesis of rheumatoid arthritis (RA). Whereas the reasons for the breakage of self tolerance to this major cartilage component are still enigmatic, T cell responses to glycosylated CII determinants in RA patients indicate that post-translational modifications play a role. Since the conversion of arginine into citrulline by peptidylarginine deiminases (PAD) in some non-joint-specific antigens such as filaggrin or fibrin has been shown to give rise to RA-specific humoral immune responses, we investigated whether PAD modification of cartilage-specific CII might affect its recognition by circulating autoantibodies in early RA. In vitro treatment with purified PAD led to arginine deimination of native CII or of synthetic CII peptides as evidenced by amino acid analysis. The citrullination resulted in modified recognition of the immunodominant CII epitope C1(III) (amino acid residues 359-369) by murine and human antibodies. In a cohort of early RA patients (n=286), IgG antibodies directed toward a synthetic citrullinated C1(III) peptide (citC1(III)-P) were detectable with a prevalence of 40.4%. The partial autoantibody cross-reactivity between citC1(III)-P and citrullinated peptides mimicking epitopes of the cytoskeletal autoantigen filaggrin suggests that autoimmunity to cartilage-specific modified self might be a critical intermediate bridging recognition of PAD-modified extra-articular autoantigens with the disruption of tolerance to native cartilage constituents.     

载穿心莲内酯胶原缓释支架促进体外炎症环境下软骨细胞表型维持的研究

本文主要研究载穿心莲内酯胶原缓释支架对炎症环境下软骨细胞维持表型的作用。通过物理共混结合真空冷冻干燥的方法制备载穿心莲内酯胶原缓释支架,采用环境扫描电子显微镜(ESEM)、真密度仪和紫外可见分光光度计表征载药支架的形貌、开孔率和药物释放情况。将经分离、扩增培养后的兔关节软骨细胞接种于载穿心莲内酯胶原缓释支架上,在常规培养液条件下培养3 d、白细胞介素1β(IL-1β)模拟的炎症环境下培养7 d,期间采用阿尔玛蓝(Alamar Blue)、荧光素二乙酸(FDA)染色、实时定量聚合酶链式反应(RT-qPCR)等方法研究所制备支架对软骨细胞的增殖和表型的影响。实验结果表明,真空冷冻干燥法制备的胶原支架孔连通性良好,平均孔径为(120.7±17.8)μm,开孔率可达96%,该载药支架可在两周内实现药物的持续释放。Alamar Blue实验结果表明,质量分数为2.22%的载穿心莲内酯胶原缓释支架可显著抑制软骨细胞增殖,而其它载药浓度的胶原缓释支架对炎症环境下软骨细胞的增殖无显著影响。FDA染色结果表明,质量分数为0.44%的载穿心莲内酯胶原缓释支架可降低IL-1β对软骨细胞形貌的改变,且该浓度载药支架可显著抑制基质金属蛋白酶-1(MMP-1)和基质金属蛋白酶-13(MMP-13)的转录,促进基质金属蛋白酶抑制剂-1(TIMP-1)、软骨细胞外基质相关基因II型胶原(COL II)和聚集蛋白聚糖(Aggrecan)的转录,提高COL II/I型胶原(COL I)的比值。基于以上研究结果,表明质量分数为0.44%的载穿心莲内酯胶原缓释支架可以抑制IL-1β模拟的炎症环境下软骨细胞内不利于维持表型的基因(如:MMP-1、MMP-13)的转录,促进利于维持表型的基因(如:COL II、Aggrecan、TIMP-1)的转录,从而增强炎症环境下的软骨细胞维持其表型的能力,有望用于关节炎软骨损伤修复。     

I型胶原-壳聚糖复合材料的生物相容性研究*

目的评价I型胶原-壳聚糖复合材料作为组织工程支架材料的生物相容性。方法体外培养兔骨髓间充质干细胞﹙BMSCs﹚,采用四甲基偶氮唑盐﹙MTT﹚比色法检测不同分组的BMSCs生长情况,酶标仪测吸光度值﹙OD﹚,计算细胞相对增值率﹙RGR%﹚;利用材料浸提液与兔新鲜血混合观察红细胞溶解情况,评价材料细胞生物相容性。结果空白组﹙A组﹚,50%浓度组﹙B组﹚和100%浓度组﹙C组﹚BMSCs均生长良好,并于1、3、5天逐渐增殖,阳性对照组﹙D组﹚细胞于第1天开始变圆、皱缩,3天和5天均皱缩死亡。A、B、C三组细胞在第1、3、5天各组吸光度值无显著差异﹙P>0.05﹚,但均与D组有明显统计学差异﹙P<0.05﹚。材料相对溶血率为4.1%。结论 I型胶原-壳聚糖复合材料无明显细胞毒性,有良好的生物相容性。     

Simultaneous anabolic and catabolic responses of human chondrocytes seeded in collagen hydrogels to long-term continuous dynamic compression

Cartilage repair strategies increasingly focus on the in vitro development of cartilaginous tissues that mimic the biological and mechanical properties of native articular cartilage. However, current approaches still face problems in the reproducible and standardized generation of cartilaginous tissues that are both biomechanically adequate for joint integration and biochemically rich in extracellular matrix constituents. In this regard, the present study investigated whether long-term continuous compressive loading would enhance the mechanical and biological properties of such tissues. Human chondrocytes were harvested from 8 knee joints (n=8) of patients having undergone total knee replacement and seeded into a collagen type I hydrogel at low density of 2×10(5)cells/ml gel. Cell-seeded hydrogels were cut to disks and subjected to mechanical stimulation for 28 days with 10% continuous cyclic compressive loading at a frequency of 0.3 Hz. Histological and histomorphometric evaluation revealed long-term mechanical stimulation to significantly increase collagen type II and proteoglycan staining homogenously throughout the samples as compared to unstimulated controls. Gene expression analyses revealed a significant increase in collagen type II, collagen type I and MMP-13 gene expression under stimulation conditions, while aggrecan gene expression was decreased and no significant changes were observed in the collagen type II/collagen type I mRNA ratio. Mechanical propertywise, the average value of elastic stiffness increased in the stimulated samples. In conclusion, long-term mechanical preconditioning of human chondrocytes seeded in collagen type I hydrogels considerably improves biological and biomechanical properties of the constructs, corroborating the clinical potential of mechanical stimulation in matrix-associated autologous chondrocyte transplantation (MACT) procedures.     

Subculture of chondrocytes on a collagen type I-coated substrate with suppressed cellular dedifferentiation

To evaluate the degree of cellular dedifferentiation, subculture of chondrocytes was conducted on a surface coated with collagen type I at a density of 1.05 mg/cm(2). In the primary culture, most of the cells were round in shape on the collagen (CL) substrate, whereas fibroblastic and partially extended cells were dominant on the polystyrene plastic (PS) substrate. Stereoscopic observation revealed that the round-shaped cells on the CL substrate were hemispherical with nebulous and punctuated F-actin filaments, whereas the fibroblastic cells on the PS substrate were flattened with fully developed stress fibers. This suggested that cell polarization was suppressed during culture on the former substrate. Although serial passages of chondrocytes through subcultures on the CL and PS substrates caused a decrease in the number of round-shaped cells, the morphological change was appreciably suppressed on the CL substrate, as compared with that on the PS substrate. It was found that only round-shaped cells formed collagen type II, which supports the view that cellular dedifferentiation can be suppressed to some extent on the CL substrate. Three-dimensional cultures in collagen gel were performed with cells isolated freshly and passaged on the CL or PS substrate. Cell density at 21 days in the culture of cells passaged on the CL substrate was comparable to that in the culture of freshly isolated cells, in spite of a significant reduction in cell density observed in the culture of cells passaged on the PS substrate. In addition, histological analysis revealed that the expression of glycosaminoglycans and collagen type II was of significance in the collagen gel with cells passaged on the CL substrate, and likewise in the gel with freshly isolated cells. This indicated that the CL substrate could offer a monolayer culture system for expanding chondrocyte cells.     

Specificity of an HLA-DRB1*0401-restricted T cell response to type II collagen

A panel of HLA-DRB1*0401-restricted CD4⁺ mouse T cell hybridomas specific for bovine type II collagen were generated from transgenic mice expressing the human HLA-DRA1*0101/-DRB1*0401 and CD4 molecules. The vast majority recognized a single peptide determinant corresponding to residues 261–273 (CII 261–273). This determinant was rapidly defined by the use of a predictive algorithm for peptide binding to DRB1*0401. CII 261–273 is conserved in bovine and human type II collagen and overlaps with an important I-A^q-restricted T cell determinant in mice with collagen-induced arthritis. This study demonstrates how HLA-DR and human CD4-transgenic mice can be used to identify a T cell epitope in a potential or candidate autoantigen.     

Immunogenetics of type II collagen autoimmunity and susceptibility to collagen arthritis.

The MHC restriction of the antibody response and development of arthritis after immunization with autologous or heterologous type II collagens in mice have been investigated. Mice from three different H-2q-carrying strains (DBA/1, NFR/N and B10.G) with different non-MHC genes, as well as B10-congenic strains carrying wild type H-2q-related or H-2r haplotypes, were susceptible for collagen arthritis. All strains tested developed an antibody response cross-reacting with autologous type II collagen after immunization with heterologous type II collagen; H-2q predisposes for a high response against chick, rat and bovine type II collagen, H-2r and H-2b for a high response against bovine type II collagen. Only mouse strains with H-2q, H-2r, H-2w3 or H-2w17 were responders to mouse type II collagen, and only these strains developed arthritis after immunization with heterologous or autologous type II collagens. These findings indicate that the ability to mount an immune response against autologous type II collagen is a prerequisite for the susceptibility to collagen arthritis. A cross-reactive autoimmune response after immunization with various heterologous type II collagen may enhance further the development of arthritis.     

骨桥蛋白干预体外培养人膝骨关节炎软骨细胞蛋白多糖和Ⅱ型胶原的表达

BACKGROUND: Osteopontin mRNA and protein expressions are highly correlated with the severity of osteoarthritis. OBJECTIVE: To investigate the effect of osteopontin on the gene expression of aggrecan and type II collagen in  the human knee osteoarthritic chondrocytes in vitro. METHODS: Chondrocytes were harvested from human osteoarthritic knees and cultured in vitro. The chondrocytes were cultured with 0 (blank control group), 0.1, 1 mg/L osteopontin, respectively, for 48 hours. Real-time PCR was employed to detect the mRNA expression of aggrecan and type II collagen. RESULTS AND CONCLUSION: After 0.1 and 1 mg/L osteopontin intervention, the mRNA expression of aggrecan and type II collagen in osteoarthritic chondrocytes was increased significantly (P < 0.05), and the mRNA expression of aggrecan and type II collagen was higher in the 1 mg/L osteopontin group than the 0.1 mg/L osteopontin group (P < 0.05). In addition, the mRNA expression of aggrecan and type II collagen was positively correlated with the concentration of osteopontin (r=0.751, P < 0.01; r=0.676, P < 0.01). These findings indicate that osteopontin up-regulates the mRNA expression of aggrecan and type II collagen in osteoarthritic chondrocytes of human knee in vitro in a dose-dependent manner.       

Centrifugation assay for measuring adhesion of serially passaged bovine chondrocytes to polystyrene surfaces

A major obstacle in chondrocyte-based therapy for cartilage repair is the limited availability of cells that maintain their original phenotype. Propagation of chondrocytes as monolayer cultures on polystyrene surfaces is used extensively for amplifying cell numbers. However, chondrocytes undergo a phenotypic shift when propagated in this manner and display characteristics of more adherent fibroblastic cells. Little information is available about the effect of this phenotypic shift on cellular adhesion properties. We evaluated changes in adhesion property as bovine chondrocytes were serially propagated up to five passages in monolayer culture using a centrifugation cell adhesion assay, which was based on counting of cells before and after being exposed to centrifugal dislodgement forces of 120 and 350 g. Chondrocytes proliferated well in a monolayer culture with doubling times of 2-3 days, but they appeared more fibroblastic and exhibited elongated cell morphology with continued passage. The centrifugation cell adhesion assay showed that chondrocytes became more adhesive with passage as the percentage of adherent cells after centrifugation increased and was not statistically different from the adhesion of the fibroblast cell line, L929, starting at passage 3. This increased adhesiveness correlated with a shift to a fibroblastic morphology and increased collagen I mRNA expression starting at passage 2. Our findings indicate that the centrifugation cell adhesion assay may serve as a reproducible tool to track alterations in chondrocyte phenotype during their extended propagation in culture.     

京尼平苷干预体外培养大鼠软骨细胞Ⅱ型胶原蛋白的合成

背景：近年来的研究表明,中药栀子可能具有活化软骨细胞的功能,而京尼平苷是栀子中重要成分之一。 目的：观察京尼平苷对体外培养大鼠膝关节软骨细胞Ⅱ型胶原蛋白合成的影响。 方法：体外分离培养大鼠膝关节软骨细胞,分别应用25,50及100 mmol/L京尼平苷干预,并设置正常对照组。RT-PCR和Western blot法观察京尼平苷对各组细胞Ⅱ型胶原mRNA和蛋白表达的影响。 结果与结论：RT-PCR和Western blot法检测结果显示,与正常对照组相比,25,50及100 mmol/L京尼平苷干预可以明显促进Ⅱ型胶原mRNA及蛋白的表达(P < 0.01)。结果证实,京尼平苷可以促进大鼠膝关节软骨细胞Ⅱ型胶原蛋白的合成。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Regulation of cellular and humoral immune responses to collagen type I or collagen type II.

We have investigated the characteristics of antigen-specific reductions in murine immune responses to rat collagen type I (R-CI), chick collagen type II (C-CII) or bovine collagen type II (B-CII). Intravenous pretreatment with the appropriate soluble collagen or collagen-coupled spleen cells led to the development of antigen-specific reduced immune responses, the former treatment being more effective than the latter. In the case of CII, pretreatment with R-CI or non-related antigens was ineffective. However, pretreatment with denatured bovine-CII, native bovine-CII or chick-CII led to immune hyporesponsiveness for either the homologous or heterologous CII molecule. A delayed development of the diminished immune responses was observed for the cell-mediated immune response (CMI), as measured by in vivo delayed-type hypersensitivity (DTH), in that no reduction was evident at Day 7 but a significantly decreased response was observed at Day 14. Collagen-specific IgG and IgM antibody responses were consistently reduced by the pretreatment and remained reduced during the study period. The antigen-specific hyporesponsive state was not sensitive to cyclophosphamide treatment and was not transferable with hyporesponsive spleen cells. Additionally, we have induced unresponsiveness to CII by treating mice with an antibody directed to T helper cells (GK1.5). This treatment led to profound reductions in CII CMI responses as well as CII antibody levels. However, this unresponsive state is not permanent and not transferable with spleen cells from treated mice. These two types of procedures, soluble B-CII i.v. or GK1.5 treatment, not only resulted in CII hyporesponsive states, but also produced delayed onset and decreased incidence of arthritis in the appropriate strains.     

Fluid flow increases type II collagen deposition and tensile mechanical properties in bioreactor-grown tissue-engineered cartilage

A novel parallel-plate bioreactor has been designed to apply a consistent level of fluid flow-induced shear stress to tissue-engineered articular cartilage in order to improve the matrix composition and mechanical properties and more nearly approximate to that of native tissue. Primary bovine articular chondrocytes were seeded into the bioreactor at high densities (1.7 x 10(6) cell/cm2) without a scaffold and cultured for two weeks under static, no-flow conditions. A mean fluid flow-induced shear stress of 1 dyne/cm2 was then applied continuously for 3 days. The application of flow produced constructs with significantly (p < 0.05) higher amounts of total collagen (via hydroxyproline) and specifically type II collagen (via ELISA) (25.3 +/- 2.5% and 22.1 +/- 4.7% of native tissue, respectively) compared to static controls (22.4 +/- 1.7% and 9.5 +/- 2.3%, respectively). Concurrently, the tensile Young's modulus and ultimate strength were significantly increased in flow samples (2.28 +/- 0.19 MPa and 0.81 +/- 0.07 MPa, respectively) compared to static controls (1.55 +/- 0.10 MPa and 0.62 +/- 0.05 MPa, respectively). This study suggests that flow-induced shear stresses and/or enhanced mass transport associated with the hydrodynamic environment of our novel bioreactor may be an effective functional tissue-engineering strategy for improving matrix composition and mechanical properties in vitro.