Analysis of radial variations in material properties and matrix composition of chondrocyte-seeded agarose hydrogel constructs

SummaryObjectiveTo examine the radial variations in engineered cartilage that may result due to radial fluid flow during dynamic compressive loading. This was done by evaluating the annuli and the central cores of the constructs separately.MethodChondrocyte-seeded agarose hydrogels were grown in free-swelling and dynamic, unconfined loading cultures for 42 days. After mechanical testing, constructs were allowed to recover for 1–2h, the central 3mm cores removed, and the cores and annuli were retested separately. Histological and/or biochemical analyses for DNA, glycosaminoglycan (GAG), collagen, type I collagen, type II collagen, and elastin were performed. Multiple regression analysis was used to determine the correlation between the biochemical and material properties of the constructs.ResultsThe cores and annuli of chondrocyte-seeded constructs did not exhibit significant differences in material properties and GAG content. Annuli possessed greater DNA and collagen content over time in culture than cores. Dynamic loading enhanced the material properties and GAG content of cores, annuli, and whole constructs relative to free-swelling controls, but it did not alter the radial variations compared to free-swelling culture.ConclusionSurprisingly, the benefits of dynamic loading on tissue properties extended through the entire construct and did not result in radial variations as measured via the coring technique in this study. Nutrient transport limitations and the formation of a fibrous capsule on the periphery may explain the differences in DNA and collagen between cores and annuli. No differences in GAG distribution may be due to sufficient chemical signals and building blocks for GAG synthesis throughout the constructs

Influence of decreasing nutrient path length on the development of engineered cartilage

SummaryObjectiveChondrocyte-seeded agarose constructs of 4mm diameter (2.34mm thickness) develop spatially inhomogeneous material properties with stiffer outer edges and a softer central core suggesting nutrient diffusion limitations to the central construct region [Guilak F, Sah RL, Setton LA. Physical regulation of cartilage metabolism. In: Mow VC, Hayes WC, Eds. Basic Orthopaedic Biomechanics, Philadelphia 1997;179–207.]. The effects of reducing construct thickness and creating channels running through the depth of the thick constructs were examined.MethodsIn Study 1, the properties of engineered cartilage of 0.78mm (thin) or 2.34mm (thick) thickness were compared. In Study 2, a single nutrient channel (1mm diameter) was created in the middle of each thick construct. In Study 3, the effects of channels on larger 10mm diameter, thick constructs were examined.ResultsThin constructs developed superior mechanical and biochemical properties than thick constructs. The channeled constructs developed significantly higher mechanical properties vs control channel-free constructs while exhibiting similar glycosaminoglycan (GAG) and collagen content. Collagen staining suggested that channels resulted in a more uniform fibrillar network. Improvements in constructs of 10mm diameter were similarly observed.ConclusionsThis study demonstrated that more homogeneous tissue-engineered cartilage constructs with improved mechanical properties can be achieved by reducing their thickness or incorporating macroscopic nutrient channels. Our data further suggests that these macroscopic channels remain open long enough to promote this enhanced tissue development while exhibiting the potential to refill with cell elaborated matrix with additional culture time. Together with reports that <3mm defects in cartilage heal in vivo and that irregular holes are associated with clinically used osteochondral graft procedures, we anticipate that a strategy of incorporating macroscopic channels may aid the development of clinically relevant engineered cartilage with functional properties

Mechanical response of bovine articular cartilage under dynamic unconfined compression loading at physiological stress levels

AbstractObjective: The objective of this study was to characterize the dynamic modulus and compressive strain magnitudes of bovine articular cartilage at physiological compressive stress levels and loading frequencies.Design: Twelve distal femoral cartilage plugs (3mm in diameter) were loaded in a custom apparatus under load control, with a load amplitude up to 40N and loading frequencies of 0.1, 1, 10 and 40Hz, resulting in peak Cauchy stress amplitudes of 4.8MPa (engineering stress 5.7MPa).Results: The equilibrium Young's modulus under a tare load of 0.4N was 0.49±0.10MPa. In the limit of zero applied stress, the incremental dynamic modulus derived from the slope of the stress–strain curve increased from 14.6±6.9MPa at 0.1Hz to 28.7±7.8MPa at 40Hz. At 4MPa of applied stress, the corresponding increase was from 48.2±13.5MPa at 0.1Hz to 64.8±13.0MPa at 40Hz. Peak compressive strain amplitudes varied from 15.8±3.4% at 0.1Hz to 8.7±1.8% at 40Hz. The phase angle decreased from 28.8°±6.7° at 0.1Hz to−0.5°±3.8° at 40Hz.Discussion: These results are representative of the functional properties of articular cartilage under physiological load magnitudes and frequencies. The viscoelasticity and nonlinearity of the tissue helps to maintain the compressive strains below 20% under the physiological compressive stresses achieved in this study. These findings have implications for our understanding of cartilage metabolism and chondrocyte viability under various loading regimes. They also help establish guidelines for cartilage functional tissue engineering studies

Effects Of Fixed Charges On The Stress Relaxation Behavior Of Hydrated Soft Tissues In A Confined Compression Problem

The 0!D con_ned!compression stressrelaxation behavior of a charged\ hydrated!soft tissue was analyzed using the continuum mixture theory developed for cartilage &quot;Lai et al[\ 0880#[ A pair of coupled nonlinear partial di}erential equations governing the displacement component u s of the solid matrix and the cation concentration c were derived[ The initial!boundary value problem\ corresponding to a rampdisplacement stressrelaxation experiment was solved using a _nite!di}erence method to obtain the complete spatial and temporal distributions of stress\ strain\ interstitial water pressure &quot;including osmotic pressure#\ ion concentrations\ di}usion rates and water velocity within the tissue[ Using data available in the literature\ it was found that ] &quot;0# the equilibrium aggregate modulus of the tissue &quot;as commonly used in the biphasic theory# consists of two com! ponents ] the Donnan osmotic component and the intrinsic matrix component\ and that these two components are of similar magnitude[ &quot;1# For the rate of compression of 09) in 199 s\ during the compression stage\ the ~uid pressure at the impermeable boundary supports nearly all the load\ while near the free!draining boundary\ both the matrix sti}ness and the ~uid pressure support a substantial amount of the load[ &quot;2# Equivalent aggregate modulus and equivalent di}usive coe.cient used in the biphasic theory can be found\ which predict essentially the same stress relaxation behavior[ These equivalent parameters for the biphasic model embody the FCD e}ect of the triphasic medium[ The internal ~uid pressure predicted by the two models are however di}erent because of osmotic e}ects[ &quot;3# Peak stress at the end of the compression stage is higher for a tissue with higher FCD[We have obtained the strain\ str..

Development of multibody model for diathrodial joints using accurate 3-D cartilage and bone surfaces

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