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Cyclic distension of fibrin-based tissue constructs: evidence of adaptation during growth of engineered connective tissue.

ABSTRACT: Tissue engineering provides a means to create functional living tissue replacements. Here, we examine the effects of 3 weeks of cyclic distension (CD) on fibrin-based tubular tissue constructs seeded with porcine valve interstitial cells. CD with circumferential strain amplitude ranging from 2.5% to 20% was applied to evaluate the effects of CD on fibrin remodeling into tissue. We hypothesized that during long-term CD cells adapt to cyclic strain of constant strain amplitude (constant CD), diminishing tissue growth. We thus also subjected constructs to CD with strain amplitude that was incremented from 5% to 15% over the 3 weeks of CD [incremental CD (ICD)]. For constant CD, improvement occurred in construct mechanical properties and composition, peaking at 15% strain: ultimate tensile strength (UTS) and tensile modulus increased 47% and 45%, respectively, over statically incubated controls (to 1.1 and 4.7 MPa, respectively); collagen density increased 29% compared with controls (to 27 mg/ml). ICD further improved outcomes. UTS increased 98% and modulus increased 62% compared with the largest values with constant CD, and collagen density increased 34%. Only in the case of ICD was the ratio of collagen content to cell number greater (70%) than controls, consistent with increased collagen deposition per cell. Studies with human dermal fibroblasts showed similar improvements, generalizing the findings, and revealed a 255% increase in extracellular signal-regulated kinase signaling for ICD vs. constant CD. These results suggest cell adaptation may limit conventional strategies of stretching with constant strain amplitude and that new approaches might optimize bioreactor operation.

SUBMITTER: Syedain ZH

PROVIDER: S-EPMC2373356 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Cyclic distension of fibrin-based tissue constructs: evidence of adaptation during growth of engineered connective tissue.

Syedain Zeeshan H ZH Weinberg Justin S JS Tranquillo Robert T RT

Proceedings of the National Academy of Sciences of the United States of America 20080424 18

Tissue engineering provides a means to create functional living tissue replacements. Here, we examine the effects of 3 weeks of cyclic distension (CD) on fibrin-based tubular tissue constructs seeded with porcine valve interstitial cells. CD with circumferential strain amplitude ranging from 2.5% to 20% was applied to evaluate the effects of CD on fibrin remodeling into tissue. We hypothesized that during long-term CD cells adapt to cyclic strain of constant strain amplitude (constant CD), dimin ...[more]

PMID: 18436647

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Project description:Different tissue-engineering approaches have been developed to induce and promote cardiac regeneration; however, the impact of the immune system and its responses to the various scaffold components of the engineered grafts remains unclear. Fibrin-based engineered heart tissue (EHT) was generated from neonatal Lewis (Lew) rat heart cells and transplanted onto the left ventricular surface of three different rat strains: syngeneic Lew, allogeneic Brown Norway, and immunodeficient Rowett Nude rats. Interferon spot frequency assay results showed similar degrees of systemic immune activation in the syngeneic and allogeneic groups, whereas no systemic immune response was detectable in the immunodeficient group (p < .001 vs. syngeneic and allogeneic). Histological analysis revealed much higher local infiltration of CD3- and CD68-positive cells in syngeneic and allogeneic rats than in immunodeficient animals. Enzyme-linked immunospot and immunofluorescence experiments revealed matrix-directed TH1-based rejection in syngeneic recipients without collateral impairment of heart cell survival. Bioluminescence imaging was used for in vivo longitudinal monitoring of transplanted luciferase-positive EHT constructs. Survival was documented in syngeneic and immunodeficient recipients for a period of up to 110 days after transplant, whereas in the allogeneic setting, graft survival was limited to only 14 ± 1 days. EHT strategies using autologous cells are promising approaches for cardiac repair applications. Although fibrin-based scaffold components elicited an immune response in our studies, syngeneic cells carried in the EHT were relatively unaffected.An initial insight into immunological consequences after transplantation of engineered heart tissue was gained through this study. Most important, this study was able to demonstrate cell survival despite rejection of matrix components. Generation of syngeneic human engineered heart tissue, possibly using human induced pluripotent stem cell technology with subsequent directed rejection of matrix components, may be a potential future approach to replace diseased myocardium.

Project description:Cyclic stretching and growth factors like TGF-? have been used to enhance extracellular matrix (ECM) production by cells in engineered tissue to achieve requisite mechanical properties. In this study, the effects of TGF-?1 were evaluated during long-term cyclic stretching of fibrin-based tubular constructs seeded with neonatal human dermal fibroblasts. Samples were evaluated at 2, 5, and 7 weeks for tensile mechanical properties and ECM deposition. At 2 weeks, +TGF-?1 samples had 101% higher collagen concentration but no difference in ultimate tensile strength (UTS) or modulus compared to -TGF-?1 samples. However, at weeks 5 and 7, -TGF-?1 samples had higher UTS/modulus and collagen concentration, but lower elastin concentration compared to +TGF-?1 samples. The collagen was better organized in -TGF-?1 samples based on picrosirius red staining. Western blot analysis at weeks 5 and 7 showed increased phosphorylation of ERK in -TGF-?1 samples, which correlated with higher collagen deposition. The TGF-?1 effects were further evaluated by western blot for ?SMA and SMAD2/3 expression, which were 16-fold and 10-fold higher in +TGF-?1 samples, respectively. The role of TGF-?1 activated p38 in inhibiting phosphorylation of ERK was evaluated by treating samples with SB203580, an inhibitor of p38 activation. SB203580-treated cells showed increased phosphorylation of ERK after 1 hour of stretching and increased collagen production after 1 week of stretching, demonstrating an inhibitory role of activated p38 via TGF-?1 signaling during cyclic stretching. One advantage of TGF-?1 treatment was the 4-fold higher elastin deposition in samples at 7 weeks. Further cyclic stretching experiments were thus conducted with constructs cultured for 5 weeks without TGF-?1 to obtain improved tensile properties followed by TGF-?1 supplementation for 2 weeks to obtain increased elastin content, which correlated with a reduction in loss of pre-stress during preconditioning for tensile testing, indicating functional elastin. This study shows that a sequential stimulus approach - cyclic stretching with delayed TGF-?1 supplementation - can be used to engineer tissue with desirable tensile and elastic properties.

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Cyclic distension of fibrin-based tissue constructs: evidence of adaptation during growth of engineered connective tissue.

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Cyclic distension of fibrin-based tissue constructs: evidence of adaptation during growth of engineered connective tissue.

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