Quality Of Tissue-engineered Cartilage Research Articles

Construction of Tissue-engineered Cartilage In Vivo from Microtia Chondrocytes After Transfection with Human VEGF165 Genes Mediated by a Recombinant Adeno-Associated Viral Vector.

To evaluate the transfection efficiency of cultured chondrocytes from individuals with microtia (microtia chondrocytes) with the recombinant adeno-associated virus vector rAAV2-hVEGF165-IRES-EGFP and hVEGF165 in vitro. To test whether VEGF165 gene-modified microtia chondrocytes can enhance the survival and quality of tissue-engineered cartilage. The recombinant plasmid rAAV2-hVEGF165-IRES-EGFP was inserted into rAAV2 virus vectors to construct rAAV2-hVEGF165-IRES-EGFP using the AATMaxTM system. The second-passage microtia chondrocytes were divided into 3 groups in vitro: the Ctr group (without transfection), Exp1 group (transfected with rAAV2-IRES-EGFP), and Exp2 group (transfected with rAAV2-hVEGF165-IRES-EGFP). At 24 h, 48 h, 72 h and 7 d after transfection, cell viability was measured by MTT staining. Transfection efficiency was determined by the rate of fluorescence-positive cells. The mRNA expression of hVEG165 was detected by RT-PCR (reverse transcription PCR) and agarose gel electrophoresis, and the VEGF165 protein levels in the supernatant fluids were measured by ELISAs. The second passage microtia chondrocytes with (Exp) and without (Ctr) transfection of VEGF165 genes were mixed with 0.5 ml 30% Pluronic F-127 at 4°C and then injected subcutaneously into the opposing side of the back of nude mice. Eight weeks after injection, the cartilage-like tissues of nude mice were harvested for morphological and histologic examination. Chondrocyte viability increased in a time-dependent manner but did not differ among the 3 groups at the same time point. The mRNA and protein levels of VEGF increased in a time-dependent manner in the 3 groups. The mRNA and protein levels of VEGF165 were much higher in the Exp 2 group than in the Ctr and Exp 1 groups at the same time point, but the levels were not significantly different between the Exp 1 and Exp 2 groups. Both the Ctr group and the Exp1 group formed mature cartilage with mature lacunar structures, metachromatic matrices, collagen, and elastic fibers, and the structure of neonatal cartilage was not significantly different between the 2 groups. However, the wet weight of the neonatal cartilage was much larger in the Exp group (127.4±12.4 mg) than in the Ctr group (58.5±12.2 mg, p < 0.05). VEGF protein staining also showed a higher level in the Exp group. The HVEGF165 gene was transfected efficiently into microtia chondrocytes using the recombinant adeno-associated virus vector rAAV2-hVEGF165-IRES-EGFP. After transfection, the mRNA and protein levels of hVEGF165 increased in a time-dependent manner. VEGF165 gene-modified microtia chondrocytes showed enhanced survival in vivo but did not improve the texture of tissue-engineered cartilage. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description ofthese Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints

The effect of dynamic mechanical shear and compression on the synthesis of human tissue-engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA-alginate scaffolds were precultured in shaking T-flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak-to-peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T-flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3- and 10-fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4-fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue-engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II.

The evaluation of chrongenic potential for construction of tissue-engineered cartilage by combination of oscillationg seeding and ADSCs encapsulated with gels compound

Objective To analyze the osteogenic potential of tissue-engineered bone in vitro and in vivo, and to evaluate an effective way in constructing tissue-engineered bone. Methods Tissue-engineered cartilage was constructed in the following ways: ADSCs were encapsulated with gels compound and oscillating seeding ( group A ), ADSCs were encapsulated with gels compound and static culture ( group B), ADSCs were seeded with oscillating seeding (group C), and ADSCs were seeded with static seeding ( group D). There were 48 pieces of constructs for each group, and 6 piece of them in each group were examined at different periods. SEM and CLM were used to observe cellular pattern, survival rate, cell activity, and collagen type Ⅱ level and contents of DNA and GAG were detected at the 1st, 4th, 8th,12th, 16th, 20th, 24th, and 28th day. Results Under observation of SEM and CLM, new tissue showed more remarkable cell proliferation with abundant ECM in group A. As compared with other groups, the survival rate in group A was significantly higher ( 86. 39 ± 5.05, P < 0.05 ) and level of cell activity,contents of collagen type Ⅱ , DNA and GAG were significantly increased (0. 57 ± 0. 12, 71.30 ± 2.51,73.21 ± 1.38, 81.25 ± 1.29, P < 0. 05). Conclusion Gels encapsulation ADSCs combined with oscillating seeding promotes the cell augmentation and improves the quality of tissue-engineered cartilage. Key words: Adipose derived stromal cells; Gels; Culture; Tissue-engineered cartilage

The application of atelocollagen gel in combination with porous scaffolds for cartilage tissue engineering and its suitable conditions

For improving the quality of tissue-engineered cartilage, we examined the in vivo usefulness of porous bodies as scaffolds combined with an atelocollagen hydrogel, and investigated the suitable conditions for atelocollagen and seeding cells within the engineered tissues. We made tissue-engineered constructs using a collagen sponge (CS) or porous poly(L-lactide) (PLLA) with human chondrocytes and 1% hydrogel, the concentration of which maximized the accumulation of cartilage matrices. The CS was soft with a Young's modulus of less than 1 MPa, whereas the porous PLLA was very rigid with a Young's modulus of 10 MPa. Although the constructs with the CS shrank to 50% in size after a 2-month subcutaneous transplantation in nude mice, the PLLA constructs maintained their original sizes. Both of the porous scaffolds contained some cartilage regeneration in the presence of the chondrocytes and hydrogel, but the PLLA counterpart significantly accumulated abundant matrices in vivo. Regarding the conditions of the chondrocytes, the cartilage regeneration was improved in inverse proportion to the passage numbers among passages 3-8, and was linear with the cell densities (10(6) to 10(8) cells/mL). Thus, the rigid porous scaffold can maintain the size of the tissue-engineered cartilage and realize fair cartilage regeneration in vivo when combined with 1% atelocollagen and some conditioned chondrocytes.

Effect of Seeding and Bioreactor Culture Conditions on the Development of Human Tissue-Engineered Cartilage

Human cartilage was produced using fetal chondrocytes seeded into polyglycolic acid (PGA) mesh scaffolds and cultured in recirculation bioreactors. The effect of scaffold thickness, seeding cell density, and bioreactor operating conditions on the quality of the engineered cartilage was investigated. Thin (2.15-mm-thick) PGA scaffolds lost their structural integrity during bioreactor culture and the resulting constructs were small and misshapen compared with tissues generated using 4.75-mm-thick scaffolds. Increasing the seeding cell number from 1.2 x 10(7) to 2.2 x 10(7 )per 4.75-mm-thick scaffold resulted in a doubling of the construct wet weight, a 4.4-fold increase in glycosaminoglycan (GAG) concentration, and a 2.9-fold increase in total collagen concentration in the tissues. Levels of GAG and total collagen were also improved significantly when 100 mL or 50% v/v of the culture medium was replaced periodically during operation of the bioreactors compared with 50, 25, or 5 mL. The proportion of GAG lost from the tissues into the medium was reduced by increasing the seeding cell number and replaced medium volume. This work demonstrates that the quality of tissue-engineered cartilage can be manipulated substantially depending on the cell seeding and bioreactor culture conditions employed.

Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds

Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage.

Effect of a degraded core on the mechanical behaviour of tissueengineered cartilage constructs: A poro-elastic finite element analysis

The structure and functionality of tissue-engineered cartilage is determined by the tissue culture conditions and mechanical conditioning during growth. The quality of tissue-engineered cartilage can be evaluated using tests such as the confined compression test. Tissue-engineered cartilage constructs usually consist of an outer layer of cartilage and an inner core of either undeveloped cartilage or degrading scaffold material. A biphasic poro-elastic finite element model was used to demonstrate how such a core influences the reaction force-time curve obtained from a confined compression test. The finite element model predicted that higher volumes of degraded scaffold in the inner core would reduce the aggregate modulus calculated from the confined compression test and raised the estimate of tissue permeability. The predicted aggregate modulus reduced from 0.135 MPa, for a homogenous construct, to 0.068 MPa, for a construct that was only 70% cartilaginous. It was found that biphasic poro-elastic finite modelling should be used in preference to a one-dimensional model that assumed homogeneity in estimating the properties of tissue-engineered cartilage.