[Show abstract][Hide abstract] ABSTRACT: This study investigates the effect of insulin-like growth factor (IGF) -I on the development of anatomically shaped alginate menisci seeded with meniscal fibrochondrocytes. To accomplish this, bovine meniscal fibrochondrocytes were seeded into 2% w/v alginate, crosslinked with CaSO4, and injected into anatomical molds derived from μCT scans. The meniscal constructs were then cultured for up to 4 weeks with or without 100 ng/mL IGF-I supplemented in the media. Histological, immunohistological, biochemical and mechanical analyses were performed to characterize tissue development, accumulation and localization of ECM, and mechanical properties. After 4 weeks of culture, IGF-I treatment significantly improved mechanical and biochemical properties while maintaining DNA content, with a 26 fold increase in GAG content and 10 fold increase in collagen content compared to 0 week controls, and a 3 fold increase in the equilibrium modulus at 2 weeks compared to controls. IGF-I treated menisci had approximately 60% of the GAG content of native tissue and the compressive equilibrium modulus matched native properties by 2 weeks of culture. Further, IGF-I treated menisci developed a distinct surface layer similar to native tissue with elongated cells and collagen fibers aligned parallel to the surface, the presence of type I and II collagen, and accumulation of lubricin. This study demonstrates that IGF-I treatment can greatly increase the mechanical and biochemical properties of engineered tissues and aid in the development of a distinct surface zone similar to the superficial zone of native menisci.
Full-text · Article · Jan 2013 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: This study investigated the hypothesis that timing and duration of dynamic compression are integral to regulating extracellular matrix (ECM) assembly of tissue-engineered (TE) menisci. The goal of this study was to examine the effects of varying load and static culture duration on structure, composition, and mechanical properties of TE menisci. We accomplished this by varying the duration of dynamic loading over 4 weeks of culture, and by examining increasing periods of static culture after 2 weeks of dynamic loading. Bovine meniscal fibrochondrocytes were seeded into 2% w/v alginate, crosslinked with CaSO(4), injected into anatomical micro-computed tomography-based molds, and post-crosslinked with CaCl(2). Meniscal constructs were dynamically compressed three times a week via a custom bioreactor for a total of 2 h, with an hour of rest between loading cycles, for 1, 2, or 4 weeks. They were then placed in static culture. After 4 weeks of culture, increased load duration was found to be beneficial to matrix formation and mechanical properties, with superior mechanical and biochemical properties in samples loaded for 2 or 4 weeks. Further, the mechanical properties of these constructs were similar, suggesting that the additional 2 weeks of loading may not be necessary. Samples loaded for 2 weeks followed by a 4-week static culture period yielded the most mature matrix with significant improvements in collagen bundle formation, 2.8-fold increase in the glycosaminoglycan content, 2-fold increase in the collagen content, and 4.3-fold increase in the compressive equilibrium modulus. Overall, this study demonstrated the importance of timing and duration of loading. By switching to prolonged static culture after 2 weeks of loading, we decreased the amount of ECM lost to the media, while significantly increasing biochemical and mechanical properties of TE menisci.
Full-text · Article · Mar 2012 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: This study investigated the hypothesis that controlled media mixing will enhance tissue formation and increase mechanical properties of anatomically-shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were seeded in 2% w/v alginate, cross-linked with 0.02 g/mL CaSO(4), and injected into molds of menisci. Engineered menisci were incubated for up to 6 weeks. A mixing media bioreactor was designed to ensure proper mixing of culture medium while protecting constructs from the spinning impeller. Impeller speeds were calibrated to produce Reynolds number (Re) of 0.5, 2.9, 5.8, 10.2, and 21.8. Constructs were divided a tested in confined compression and in tension to determine the equilibrium and tensile moduli, respectively. Media stimulation resulted in a 2-5 fold increase in mechanical properties and a 2-3 fold increase in matrix accumulation in constructs over 6 weeks in culture. Benefits from mixing stimulation for collagen accumulation and compressive modulus appeared to peak near Re 2.9, and decreased with increased mixing intensity. This study suggests that fluid mixing can be optimized to enhance mechanical properties of anatomically-shaped engineered constructs.
[Show abstract][Hide abstract] ABSTRACT: This study investigated the hypothesis that dynamic compression loading enhances tissue formation and increases mechanical properties of anatomically shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were seeded in 2%w/v alginate, crosslinked with CaSO(4), injected into μCT based molds, and post crosslinked with CaCl(2). Samples were loaded via a custom bioreactor with loading platens specifically designed to load anatomically shaped constructs in unconfined compression. Based on the results of finite element simulations, constructs were loaded under sinusoidal displacement to yield physiological strain levels. Constructs were loaded 3 times a week for 1 h followed by 1 h of rest and loaded again for 1 h. Constructs were dynamically loaded for up to 6 weeks. After 2 weeks of culture, loaded samples had 2-3.2 fold increases in the extracellular matrix (ECM) content and 1.8-2.5 fold increases in the compressive modulus compared with static controls. After 6 weeks of loading, glycosaminoglycan (GAG) content and compressive modulus both decreased compared with 2 week cultures by 2.3-2.7 and 1.5-1.7 fold, respectively, whereas collagen content increased by 1.8-2.2 fold. Prolonged loading of engineered constructs could have altered alginate scaffold degradation rate and/or initiated a catabolic cellular response, indicated by significantly decreased ECM retention at 6 weeks compared with 2 weeks. However, the data indicates that dynamic loading had a strikingly positive effect on ECM accumulation and mechanical properties in short term culture.
No preview · Article · Sep 2010 · Journal of Biomechanics
[Show abstract][Hide abstract] ABSTRACT: Quantification of shape fidelity of complex geometries for tissue-engineered constructs has not been thoroughly investigated. The objective of this study was to quantitatively describe geometric fidelities of various approaches to the fabrication of anatomically shaped meniscal constructs. Ovine menisci (n = 4) were imaged using magnetic resonance imaging (MRI) and microcomputed tomography (microCT). Acrylonitrile butadiene styrene plastic molds were designed from each imaging modality and three-dimensional printed on a Stratasys FDM 3000. Silastic impression molds were fabricated directly from ovine menisci. These molds were used to generate shaped constructs using 2% alginate with 2% CaSO(4). Solid freeform fabrication was conducted on a custom open-architecture three-dimensional printing platform. Printed samples were made using 2% alginate with 0.75% CaSO(4). Hydrogel constructs were scanned via laser triangulation distance sensor. The point cloud images were analyzed to acquire computational measurements for key points of interest (e.g., height, width, and volume). Silastic molds were within + or - 10% error with respect to the native tissue for seven key measurements, microCT molds for six of seven, microCT prints for four of seven, MRI molds for five of seven, and MRI prints for four of seven. This work shows the ability to generate and quantify anatomically shaped meniscal constructs of high geometric fidelity and lends insight into the relative geometric fidelities of several tissue engineering techniques.
Full-text · Article · Sep 2009 · Tissue Engineering Part C Methods
[Show abstract][Hide abstract] ABSTRACT: Replication of anatomic shape is a significant challenge in developing implants for regenerative medicine. This has lead to significant interest in using medical imaging techniques such as magnetic resonance imaging and computed tomography to design tissue engineered constructs. Implementation of medical imaging and computer aided design in combination with technologies for rapid prototyping of living implants enables the generation of highly reproducible constructs with spatial resolution up to 25 microm. In this paper, we review the medical imaging modalities available and a paradigm for choosing a particular imaging technique. We also present fabrication techniques and methodologies for producing cellular engineered constructs. Finally, we comment on future challenges involved with image guided tissue engineering and efforts to generate engineered constructs ready for implantation.
Full-text · Article · Aug 2009 · Journal of Cellular and Molecular Medicine