The aim of this study was to demonstrate the use of magnetic resonance imaging to evaluate the function of tissue engineered cartilage constructs. Function was evaluated in an articular cartilage defect model in terms of displacement fields computed throughout the tissue construct. Primary results in this study were (1) deformation fields varied nonuniformly depending on spatial position, (2) strains were highest in the hydrogel constructs compared to surrounding cartilage, and (3) [GAG] was lowest in the hydrogel constructs compared to the surrounding cartilage. This work represents the first application of the DENSE-FSE technique to engineered cartilage and in combination with techniques for biochemical assessment such as dGEMRIC.
The ability to measure in situ
tissue quality is essential for assessing the success of cartilage repair techniques. Current techniques for measuring tissue quality include optical coherence tomography (Han et al. 2003
) and photoacoustic (Ishihara et al. 2005
) techniques. Optical coherence tomography allows for the identification of subtle changes in tissue structure, including surface fibrillations comparable to histological analysis at low magnification. Photoacoustic measurements provide data regarding bulk viscoelastic relaxation times. In comparison, while the spatial resolution of images from the MRI techniques discussed herein does not approach the optical scale, the present technique provides spatially-resolved characterization of mechanical deformation with minimal error. The DENSE-FSE technique used herein has been demonstrated to exhibit minimal error, with zero bias and a displacement and strain precision of 8.8 μ
m and 0.17%, respectively. Thus, the technique is appropriate for characterizing tissue-level deformations noninvasively in engineered tissue.
There has been limited use of MRI to investigate mechanical function in the emerging fields of tissue engineering and regenerative medicine. Routinely, standard mechanical testing techniques have assessed bulk properties of engineered cartilage (e.g. (Hofmann et al. 2006
)). Additionally, bulk properties have been shown to correlate with MRI parameters such as T1
, and the diffusion coefficient (Miyata et al. 2007
). Only recently have MRI techniques been developed to assess and characterize tissue-level deformation patterns throughout the volume of the material under the application of physiologically-relevant mechanical loading (Neu and Walton 2008
Precise timing of MRI actions was required. T1
measures were taken prior to mechanical loading (pre-Gd(DTPA)2−
) and following the fluid exchange (post-Gd(DTPA)2−
) to ensure that tissue morphology was similar for the two sets of images. Differences in morphology (due to mechanical loading and tissue creep) would have precluded registration of T1
measures and calculation of [GAG]. Also, the integration of cyclic loading and MRI pulse sequence actions was required for images free of motion artifacts (Neu and Hull 2003
Strain fields in the hydrogel constructs were typically larger compared to those in the surrounding cartilage. The mismatch was attributed to at least three factors. First, there was a lack of GAGs in the repair tissue compared to the surrounding cartilage. Proteoglycans are known to contribute to the load support of cartilage through interactions with water in the highly hydrated tissue (Maroudas 1976
). The lack of GAGs in the repair tissue may have not provided sufficient load support and resulted in increased deformations. Second, there were observed differences and variability in the morphology of unloaded tissue that corresponded to strain fields (). Differences in material properties (e.g. creep under cyclic loading or swelling behavior resulting from altered biochemical content) may have contributed to surface geometry irregularities. Thus, there is a need for appropriate matching of material properties for a successful repair. In the case of articular cartilage, the study of allograft or autograft tissues may be of interest considering the variation of tissue even within the same joint of an individual (Neu et al. 2007
). Further studies with larger sample sizes are required to address these concerns. Third, the lack of integration in the model system resulted in alterations of the strain field data that depended on load (). Thus, the study of tissue integration is critical in future cartilage repair experiments.
The concentration of glycosaminoglycans in the hydrogel constructs was not restored to levels of the surrounding cartilage for the treatments investigated. The use of BMP-7 resulted in [GAG] that was lower compared to the use of 10% FBS-supplemented media. This finding may be attributed to the milieu of molecules present in serum that may include BMPs in addition to other morphogens and growth factors (Reddi 1998
). Importantly, the heterogeneous biochemical composition of degenerated and repaired articular cartilage (Gray et al. 2007
) has been explored and monitored using dGEMRIC both in vitro
(e.g. (Allen et al. 1999
)) and in vivo
(e.g. (Trattnig et al. 2007
)). Further, while the measurement of [GAG] using dGEMRIC has been directly validated versus the dimethylmethylene blue (DMMB) biochemical assay in human cartilage (Bashir et al. 1999
), the use of the polar agarose hydrogel could have interfered with Gd-DTPA2−
actions and resulted in false positives (i.e. elevated relative [GAG]) in this tissue engineering application. Thus, for the application of dGEMRIC to measure [GAG] restoration in engineered cartilage, a careful consideration of the scaffold material is required in addition to the optimum type, concentration, and incubation period for media supplements. The general tissue engineering strategy for restoration of GAG concentrations will likely involve the novel combinations of cells, acellular biomaterials, drugs, gene products, or genes that may be designed, specified, fabricated, and delivered either simultaneously or sequentially as therapeutic agents (Boyce 2002
Integration strategies are required to aide in distributing mechanical force from the repair to the surrounding tissue. The defect model used herein did not allow for tissue integration seen especially in the mismatch in strain fields (). A model of successful repair is therefore required that is based on increasing incubation times or altering other parameters (e.g. use of growth factors or enzymatic pretreatments (Quinn and Hunziker 2002
)) to allow for integration between the tissue surfaces. The application of the techniques described herein to an animal model (Chan et al. 2007
) of a tissue repair would address the mechanical function of integrated tissues.
This study demonstrated the ability of MRI to noninvasively evaluate mechanical function in a defect repair model in tissue explants. In particular, strain fields were evaluated to a high precision. The technique in its current state is appropriate to analyze novel biomimmetic scaffolding materials alone or in combination with stem cells and inductive signals (e.g. morphogens, growth factors, or mechanical forces), and may readily be applied for study of natural or synthetic scaffolding-based constructs for cartilage repair. Regenerative success, defined in terms of deformation fields, and the integration of native tissue with allograft/autograft or other repair tissues, may be studied post-removal from the living organism. Further development of the techniques for use in (e.g. the whole joints of) animals or humans will require the appropriate development of MRI pulse sequences and methods for in vivo application of mechanical forces to normal, diseased, and regenerated tissue of interest.