Dynamic Deformational Loading Research Articles

Tissue-engineered articular cartilage exhibits tension–compression nonlinearity reminiscent of the native cartilage

The tensile modulus of articular cartilage is much larger than its compressive modulus. This tension–compression nonlinearity enhances interstitial fluid pressurization and decreases the frictional coefficient. The current set of studies examines the tensile and compressive properties of cylindrical chondrocyte-seeded agarose constructs over different developmental stages through a novel method that combines osmotic loading, video microscopy, and uniaxial unconfined compression testing. This method was previously used to examine tension–compression nonlinearity in native cartilage. Engineered cartilage, cultured under free-swelling (FS) or dynamically loaded (DL) conditions, was tested in unconfined compression in hypertonic and hypotonic salt solutions. The apparent equilibrium modulus decreased with increasing salt concentration, indicating that increasing the bath solution osmolarity shielded the fixed charges within the tissue, shifting the measured moduli along the tension–compression curve and revealing the intrinsic properties of the tissue. With this method, we were able to measure the tensile (401±83kPa for FS and 678±473kPa for DL) and compressive (161±33kPa for FS and 348±203kPa for DL) moduli of the same engineered cartilage specimens. These moduli are comparable to values obtained from traditional methods, validating this technique for measuring the tensile and compressive properties of hydrogel-based constructs. This study shows that engineered cartilage exhibits tension–compression nonlinearity reminiscent of the native tissue, and that dynamic deformational loading can yield significantly higher tensile properties.

A Trabecular Bone Explant Model of Osteocyte–Osteoblast Co-Culture for Bone Mechanobiology

The osteocyte network is recognized as the major mechanical sensor in the bone remodeling process, and osteocyte-osteoblast communication acts as an important mediator in the coordination of bone formation and turnover. In this study, we developed a novel 3D trabecular bone explant co-culture model that allows live osteocytes situated in their native extracellular matrix environment to be interconnected with seeded osteoblasts on the bone surface. Using a low-level medium perfusion system, the viability of in situ osteocytes in bone explants was maintained for up to 4 weeks, and functional gap junction intercellular communication (GJIC) was successfully established between osteocytes and seeded primary osteoblasts. Using this novel co-culture model, the effects of dynamic deformational loading, GJIC, and prostaglandin E(2) (PGE(2)) release on functional bone adaptation were further investigated. The results showed that dynamical deformational loading can significantly increase the PGE(2) release by bone cells, bone formation, and the apparent elastic modulus of bone explants. However, the inhibition of gap junctions or the PGE(2) pathway dramatically attenuated the effects of mechanical loading. This 3D trabecular bone explant co-culture model has great potential to fill in the critical gap in knowledge regarding the role of osteocytes as a mechano-sensor and how osteocytes transmit signals to regulate osteoblasts function and skeletal integrity as reflected in its mechanical properties.

Duty Cycle of Deformational Loading Influences the Growth of Engineered Articular Cartilage

This study examines how variations in the duty cycle (the duration of applied loading) of deformational loading can influence the mechanical properties of tissue engineered cartilage constructs over one month in bioreactor culture. Dynamic loading was carried out with three different duty cycles: 1 h on/1 h off for a total of 3 h loading/day, 3 h continuous loading, or 6 h of continuous loading per day, with all loading performed 5 days/week. All loaded groups showed significant increases in Young's modulus after one month (vs. free swelling controls), but only loading for a continuous 3 and 6 h showed significant increases in dynamic modulus by this time point. Histological analysis showed that dynamic loading can increase cartilage oligomeric matrix protein (COMP) and collagen types II and IX, as well as prevent the formation of a fibrous capsule around the construct. Type II and IX collagen deposition increased with increased with duration of applied loading. These results point to the efficacy of dynamic deformational loading in the mechanical preconditioning of engineered articular cartilage constructs. Furthermore, these results highlight the ability to dictate mechanical properties with variations in mechanical input parameters, and the possible importance of other cartilage matrix molecules, such as COMP, in establishing the functional material properties of engineered constructs.

Physiologic deformational loading does not counteract the catabolic effects of interleukin-1 in long-term culture of chondrocyte-seeded agarose constructs

An interplay of mechanical and chemical factors is integral to cartilage maintenance and/or degeneration. Interleukin-1 (IL-1) is a pro-inflammatory cytokine that is present at elevated concentrations in osteoarthritic joints and initiates the rapid degradation of cartilage when cultured in vitro. Several short-term studies have suggested that applied dynamic deformational loading may have a protective effect against the catabolic actions of IL-1. In the current study, we examine whether the long-term (42 days) application of dynamic deformational loading on chondrocyte-seeded agarose constructs can mitigate these catabolic effects. Three studies were carried out using two IL-1 isoforms (IL-1 α and IL-1 β) in chemically defined medium supplemented with a broad range of cytokine concentrations and durations. Physiologic loading was unable to counteract the long-term catabolic effects of IL-1 under any of the conditions tested, and in some cases led to further degeneration over unloaded controls.

The beneficial effect of delayed compressive loading on tissue-engineered cartilage constructs cultured with TGF-β3

To determine whether the functional properties of tissue-engineered constructs cultured in a chemically-defined medium supplemented briefly with TGF-beta3 can be enhanced with the application of dynamic deformational loading. Primary immature bovine cells (2-3 months old) were encapsulated in agarose hydrogel (2%, 30 x 10(6)cells/ml) and cultured in chemically-defined medium supplemented for the first 2 weeks with transforming growth factor beta 3 (TGF-beta3) (10 microg/ml). Physiologic deformational loading (1 Hz, 3 h/day, 10% unconfined deformation initially and tapering to 2% peak-to-peak deformation by day 42) was applied either concurrent with or after the period of TGF-beta3 supplementation. Mechanical and biochemical properties were evaluated up to day 56. Dynamic deformational loading applied concurrently with TGF-beta3 supplementation yielded significantly lower (-90%) overall mechanical properties when compared to free-swelling controls. In contrast, the same loading protocol applied after the discontinuation of the growth factor resulted in significantly increased (+10%) overall mechanical properties relative to free-swelling controls. Equilibrium modulus values reach 1306+/-79 kPa and glycosaminoglycan levels reach 8.7+/-1.6% w.w. during this 8-week period and are similar to host cartilage properties (994+/-280 kPa, 6.3+/-0.9% w.w.). An optimal strategy for the functional tissue engineering of articular cartilage, particularly to accelerate construct development, may incorporate sequential application of different growth factors and applied deformational loading.

Spatial and temporal development of chondrocyte-seeded agarose constructs in free-swelling and dynamically loaded cultures

Dynamic deformational loading has been shown to significantly increase the development of material properties of chondrocyte-seeded agarose hydrogels, however little is known about the spatial development of the material properties within these constructs. In this study, a technique that combines video microscopy and optimized digital image correlation, was applied to assess the spatial development of material properties in tissue-engineered cartilage constructs cultured in free-swelling and dynamically-loaded conditions (3 h/day, 5 days/week, and maintained in free-swelling conditions when not being loaded) over a 6-week period. Although homogeneous at day 0, both free-swelling and dynamically loaded samples progressively developed stiffer outer edges and a softer central region. The distribution of GAGs and collagens were shown to mimic this profile. These results indicate that although dynamic loading augments the development of bulk properties in these samples, possibly by overcoming some of the diffusion limitation and nutrient transport issues, the overall profile of construct properties in the axial direction remains qualitatively the same as in free-swelling culture conditions. Poisson's ratio of these constructs increased over time in culture with increased fixed charged density contributed by the GAGs, but this increase was significantly less in dynamically loaded samples by day 42. Polarized light microscopy of Picrosirius Red labeled samples, at an angle perpendicular to the direction of loading, suggests that these differences in Poisson's ratio may be due to improved organization of collagen network in the dynamically loaded samples.

The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading

Objective: Functional tissue engineering (FTE) of articular cartilage involves the use of physiologically relevant mechanical signals to encourage the growth of engineered constructs. The goal of this study was to determine the utility of deformational loading in enhancing the mechanical properties of chondrocyte-seeded agarose hydrogels, and to investigate the role of initial cell seeding density and nutrient supply in this process. Design: Chondrocyte-seeded agarose hydrogels were cultured in free-swelling conditions or with intermittent deformational loading (10% deformation, 1 Hz, 1 h on/1 h off, 3 h per day, five days per week) over a two-month culture period. Disks were seeded at lower (10 million cells/ml) and higher (60 million cells/ml) seeding densities in the context of a greater medium supply than previous studies (decreasing the number of cells/ml feed medium/day) and with an increasing concentration of fetal bovine serum (10 or 20% FBS). Results: Under these more optimal nutrient conditions, at higher seeding densities and high serum concentration (20% FBS), dynamically loaded constructs show >2-fold increases in material properties relative to free-swelling controls. After two months of culture, dynamically loaded constructs achieved a Young’s modulus of ∼185 kPa and a dynamic modulus (at 1 Hz) of ∼1.6 MPa, with a frequency dependent response similar to that of the native tissue. These values represent ∼3/4 and ∼1/4 the values measured for the native tissue, respectively. While significant differences were found in mechanical properties, staining and bulk measurements of both proteoglycan and collagen content of higher seeding density constructs revealed no significant differences between free-swelling and loading groups. This finding indicates that deformational loading may act to increase material properties via differences in the structural organization, the production of small linker ECM molecules, or by modulating the size of macromolecular proteoglycan aggregates. Conclusions: Taken together, these results point to the utility of dynamic deformational loading in the mechanical preconditioning of engineered articular cartilage constructs and the necessity for increasing feed media volume and serum supplementation with increasing cell seeding densities.

Synergistic action of growth factors and dynamic loading for articular cartilage tissue engineering.

It has previously been demonstrated that dynamic deformational loading of chondrocyte-seeded agarose hydrogels over the course of 1 month can increase construct mechanical and biochemical properties relative to free-swelling controls. The present study examines the manner in which two mediators of matrix biosynthesis, the growth factors TGF-beta1 and IGF-I, interact with applied dynamic deformational loading. Under free-swelling conditions in control medium (C), the [proteoglycan content][collagen content][equilibrium aggregate modulus] of cell-laden (10 x 10(6) cells/mL) 2% agarose constructs reached a peak of [0.54% wet weight (ww)][0.16% ww][13.4 kPa]c, whereas the addition of TGF-beta1 or IGF-I to the control medium led to significantly higher peaks of [1.18% ww][0.97% ww][23.6 kPa](C-TGF) and [1.00% ww][0.63% ww][19.3 kPa](C-IGF), respectively, by day 28 or 35 (p<0.01). Under dynamic loading in control medium (L), the measured parameters were [1.10% ww][0.52% ww][24.5 kPa]L, and with the addition of TGF-beta1 or IGF-I to the control medium these further increased to [1.49% ww][1.07% ww][50.5 kPa](L-TGF) and [1.48% ww][0.81% ww][46.2 kPa](L-IGF), respectively (p<0.05). Immunohistochemical staining revealed that type II collagen accumulated primarily in the pericellular area under free-swelling conditions, but spanned the entire tissue in dynamically loaded constructs. Applied in concert, dynamic deformational loading and TGF-beta1 or IGF-I increased the aggregate modulus of engineered constructs by 277 or 245%, respectively, an increase greater than the sum of either stimulus applied alone. These results support the hypothesis that the combination of chemical and mechanical promoters of matrix biosynthesis can optimize the growth of tissue-engineered cartilage constructs.

Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels.

Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue. In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls. A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 x (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks. Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading (100 +/- 16 kPa versus 15 +/- 8 kPa, p < 0.0001). This represented a 21-fold increase over the equilibrium modulus of day 0 (4.8 +/- 2.3 kPa). Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 (p < 0.0001 and p = 0.002, respectively).