Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

Passage in monolayer influences the response of chondrocytes to dynamic compression

This study tests the hypothesis that expansion by passage in monolayer influences the response of isolated articular chondrocytes to dynamic compression. Chondrocytes, isolated from bovine articular cartilage, were seeded in monolayer and passaged 4 times (P1-4). For assessment of chondrocytic and fibroblastic phenotype, freshly isolated and passaged cells were seeded on glass coverslips or in 2% alginate beads and cultured for 7 days in DMEM + 10% FCS. Samples were assayed for DNA and GAG content and stained for collagen types I and II. In separate experiments, freshly isolated or passaged chondrocytes were seeded at 10 x 10(6) cells.ml(-1) in 4% cylindrical agarose constructs and subjected to 15% dynamic compressive strain at 1 Hz for 24 hours. [(3)H]-thymidine incorporation, SO(4) incorporation and nitrite release were analysed. Immediately following isolation (P0), chondrocytes seeded in alginate expressed high levels of type II collagen, but did not stain for type I collagen. Following repeat passage the cells expressed enhanced levels of type I collagen, with an associated reduction in type II collagen staining. These data indicate a modulation to a fibroblastic phenotype during monolayer expansion which was not rapidly reversed by culture in a 3D hydrogel. Dynamic compression down-regulated SO(4) incorporation at P0, but did not affect [(3)H]-thymidine incorporation. By contrast the incorporation of both SO(4) and [(3)H]-thymidine was enhanced by dynamic compression at both P1 and to a lesser extent P2. SO(4) and [(3)H]-thymidine incorporation were inhibited at P3 and P4. Nitrite release was down-regulated by dynamic compression at all passages. These data demonstrate a clear modulation in the response of bovine articular chondrocytes to dynamic compression following passage in monolayer.     

Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells

The objective of this study was to investigate the influence of dynamic compressive loading on chondrogenesis of mesenchymal stem cells (MSCs) in the presence of TGF-β3. Isolated porcine MSCs were suspended in 2% agarose and subjected to intermittent dynamic compression (10% strain) for a period of 42 days in a dynamic compression bioreactor. After 42 days in culture, the free-swelling specimens exhibited more intense alcian blue staining for proteoglycans, while immunohistochemical analysis revealed increased collagen type II immunoreactivity. Glycosaminoglycan (GAG) content increased with time for both free-swelling and dynamically loaded constructs, and by day 42 it was significantly higher in both the core (2.5 ± 0.21%w/w vs. 0.94 ± 0.03%w/w) and annulus (1.09 ± 0.09%w/w vs. 0.59 ± 0.08%w/w) of free-swelling constructs compared to dynamically loaded constructs. This result suggests that further optimization is required in controlling the biomechanical and/or the biochemical environment if such stimuli are to have beneficial effects in generating functional cartilaginous tissue.     

Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.     

Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds

Dynamic mechanical loading has been reported to affect chondrocyte biosynthesis in both cartilage explant and chondrocyte-seeded constructs. In this study, the effects of dynamic compression on chondrocyte-seeded peptide hydrogels were analyzed for extracellular matrix synthesis and retention over long-term culture. Initial studies were conducted with chondrocyte-seeded agarose hydrogels to explore the effects of various non-continuous loading protocols on chondrocyte biosynthesis. An optimized alternate day loading protocol was identified that increased proteoglycan (PG) synthesis over control cultures maintained in free-swelling conditions. When applied to chondrocyte-seeded peptide hydrogels, alternate day loading stimulated PG synthesis up to two-fold higher than that in free-swelling cultures. While dynamic compression also increased PG loss to the medium throughout the 39-day time course, total PG accumulation in the scaffold was significantly higher than in controls after 16 and 39 days of loading, resulting in an increase in the equilibrium and dynamic compressive stiffness of the constructs. Viable cell densities of dynamically compressed cultures differed from free-swelling controls by less than 20%, demonstrating that changes in PG synthesis were due to an increase in the average biosynthesis per viable cell. Protein synthesis was not greatly affected by loading, demonstrating that dynamic compression differentially regulated the synthesis of PGs. Taken together, these results demonstrate the potential of dynamic compression for stimulating PG synthesis and accumulation for applications to in vitro culture of tissue engineered constructs prior to implantation.     

Design and application of an oscillatory compression device for cell constructs

Mechanical compression has been shown to impact cell activity; however a need for a single device to perform a broader range of parametric studies exists. We have developed an oscillatory displacement controlled device to uniaxially strain cell constructs under both static and dynamic compression and used this device to investigate gene expression in cell constructs. The device has a wide stroke (0.25-4 mm) and frequency range (0.1-3 Hz) and several loading waveforms are possible. Alginate cellular constructs with embedded equine chondrocytes were tested and viability was maintained for the 24 h test period. Off-line mechanical testing is described and a modulus value of 18.2 +/- 1.3 kPa found for alginate disks which indicates the level of stress achieved with this deformation profile. Static (15% strain) and dynamic (15% strain, 1 Hz, triangle waveform) testing of chondrocyte constructs was performed and static compression showed significantly higher collagen II expression than dynamic using quantitative RT-PCR. In contrast, differences in matrix metalloproteinase-3 (MMP-3) expression were statistically insignificant. These studies indicate the utility of our device for studying cell activity in response to compression and suggest further studies regarding how the load and strain spectrum impact chondrocyte activity.     

Dynamic compressive loading of image-guided tissue engineered meniscal constructs

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.     

Role of cell-associated matrix in the development of free-swelling and dynamically loaded chondrocyte-seeded agarose gels

Chondrocytes embedded in agarose and subjected to dynamic deformational loading produce a functional matrix with time in culture, but there is usually a delay in the development of significant differences compared to free swelling. In this study, we hypothesized that the initial presence of a cell-associated matrix would expedite construct development in response to dynamic deformational loading. Seeded samples with enzymatically isolated chondrocytes and chondrons (the chondrocyte and its pericellular matrix) and examined the effects of seeding density and dynamic loading on the development of tissue properties. At 60 million/ml, dynamic loading significantly augmented the development of material properties in chondrocyte- and chondron-seeded constructs. Biochemical content and histological analysis indicated that the deposition of GAG, link protein and collagens are affected by the pre-existing cell-associated matrix of the chondron-seeded samples. The pericellular matrix associated with the chondrons did not expedite the development of material properties in response to deformational loading, disproving our hypothesis. The relative concentration and distribution of matrix proteins may play a major role in the disparate responses observed for the chondrocyte- and chondron-seeded cultures. In further support of these findings, culturing chondrocytes in agarose for two weeks prior to the application of deformational loading also did not exhibit expedited construct development.     

Rapid regulation of collagen but not metalloproteinase 1, 3, 13, 14 and tissue inhibitor of metalloproteinase 1, 2, 3 expression in response to mechanical loading of cartilage explants in vitro

This study analyzes the molecular response of articular chondrocytes to short-term mechanical loading with a special focus on gene expression of molecules relevant for matrix turnover. Porcine cartilage explants were exposed to static and dynamic unconfined compression and viability of chondrocytes was assessed to define physiologic loading conditions. Cell death in the superficial layer correlated with mechanical loading and occurred at peak stresses >or=6 MPa and a cartilage compression above 45%. Chondrocytes in native cartilage matrix responded to dynamic loading by rapid and highly specific suppression of collagen expression. mRNA levels dropped 11-fold (collagen 2; 6 MPa, P=0.009) or 14-fold (collagen 1; 3 and 6 MPa, P=0.009) while levels of aggrecan, tenascin-c, matrix metalloproteinases (MMP1, 3, 13, 14), and their inhibitors (TIMP1-3) did not change significantly. Thus, dynamic mechanical loading rapidly shifted the balance between collagen and aggrecan/tenascin/MMP/TIMP expression. A better knowledge of the chondrocyte response to mechanical stress may improve our understanding of mechanically induced osteoarthrits.     

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints

The effect of dynamic mechanical shear and compression on the synthesis of human tissue‐engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA–alginate scaffolds were precultured in shaking T‐flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak‐to‐peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T‐flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3‐ and 10‐fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4‐fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue‐engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II. Biotechnol. Bioeng. 2012; 109:1060–1073. © 2011 Wiley Periodicals, Inc.     

Applied osmotic loading for promoting development of engineered cartilage

This study investigated the potential use of static osmotic loading as a cartilage tissue engineering strategy for growing clinically relevant grafts from either synovium-derived stem cells (SDSCs) or chondrocytes. Bovine SDSCs and chondrocytes were individually encapsulated in 2% w/v agarose and divided into chondrogenic media of osmolarities 300 (hypotonic), 330 (isotonic), and 400 (hypertonic, physiologic) mOsM for up to 7 weeks. The application of hypertonic media to constructs comprised of SDSCs or chondrocytes led to increased mechanical properties as compared to hypotonic (300 mOsM) or isotonic (330 mOsM) media (p<0.05). Constant exposure of SDSC-seeded constructs to 400 mOsM media from day 0 to day 49 yielded a Young's modulus of 513±89 kPa and GAG content of 7.39±0.52%ww on day 49, well within the range of values of native, immature bovine cartilage. Primary chondrocyte-seeded constructs achieved almost as high a Young's modulus, reaching 487±187 kPa and 6.77±0.54%ww (GAG) for the 400 mOsM condition (day 42). These findings suggest hypertonic loading as a straightforward strategy for 3D cultivation with significant benefits for cartilage tissue engineering strategies. In an effort to understand potential mechanisms responsible for the observed response, cell volume measurements in response to varying osmotic conditions were evaluated in relation to the Boyle–van't Hoff (BVH) law. Results confirmed that chondrocytes behave as perfect osmometers; however SDSCs deviated from the BVH relation.     

Integrin-mediated mechanotransduction processes in TGFbeta-stimulated monolayer-expanded chondrocytes

Previous studies have demonstrated that passage in monolayer detrimentally affects the response of articular chondrocytes to the application of dynamic compression. Transforming growth factor beta (TGFbeta) is known to regulate metabolic processes in articular cartilage and can enhance the re-expression of a chondrocytic phenotype following monolayer expansion. The current study tests the hypothesis that TGFbeta also modulates the response of monolayer-expanded human chondrocytes to the application of dynamic compression, via an integrin-mediated mechanotransduction process. The data presented demonstrate that TGFbeta3 enhanced 35SO4 and [3H]thymidine incorporation and inhibited nitrite release after 48 h of culture when compared to unsupplemented constructs. Dynamic compression also enhanced 35SO4 and [3H]thymidine incorporation and inhibited nitrite release in the presence of TGFbeta3. By contrast, dynamic compression did not alter these parameters in the absence of the growth factor. The addition of the peptide, GRGDSP, which acts as a competitive ligand for the alpha5beta1 integrin, reversed the compression-induced stimulation of 35SO4 incorporation, [3H]thymidine incorporation, and suppression of nitrite release. No effect was observed when the control peptide, GRADSP, was used. The current data clearly demonstrate that the dynamic compression-induced changes observed in cell metabolism for human monolayer-expanded chondrocytes were dependent on the presence of TGFbeta3 and are integrin-mediated.     

Low oxygen tension is a more potent promoter of chondrogenic differentiation than dynamic compression

During fracture healing and microfracture treatment of cartilage defects mesenchymal stem cells (MSCs) infiltrate the wound site, proliferate extensively and differentiate along a cartilaginous or an osteogenic lineage in response to local environmental cues. MSCs may be able to directly sense their mechanical environment or alternatively, the mechanical environment could act indirectly to regulate MSC differentiation by inhibiting angiogenesis and diminishing the supply of oxygen and other regulatory factors. Dynamic compression has been shown to regulate chondrogenesis of MSCs. In addition, previous studies have shown that a low oxygen environment promotes in vitro chondrogenesis of MSCs. The hypothesis of this study is that a low oxygen environment is a more potent promoter of chondrogenic differentiation of MSCs embedded in agarose hydrogels compared to dynamic compression. In MSC-seeded constructs supplemented with TGF-β3, GAG and collagen accumulation was higher in low oxygen conditions compared to normoxia. For normoxic and low oxygen culture GAG accumulation within the agarose hydrogel was inhomogeneous, with low levels of GAG measured in the annulus of constructs maintained in normoxic conditions. Dynamic compression did not significantly increase GAG or collagen accumulation in normoxia. However under low oxygen conditions, dynamic compression reduced GAG accumulation compared to free-swelling controls, but remained higher than comparable constructs maintained in normoxic conditions. This study demonstrates that continuous exposure to low oxygen tension is a more potent pro-chondrogenic stimulus than 1 h/day of dynamic compression for porcine MSCs embedded in agarose hydrogels.     

Cultivation of cell-polymer tissue constructs in simulated microgravity

Tissue-engineered cartilage was cultivated under conditions of simulated microgravity using rotating bioreactors. Rotation randomized the effects of gravity on inoculated cells (chondrocytes) and permitted their attachment to three-dimensional (3D) synthetic, biodegradable polymer scaffolds that were freely suspended within the vessel. After 1 week of cultivation, the cells regenerated a cartilaginous extracellular matrix (ECM) consisting of glycosaminoglycan (GAG) and collagen types I and II. Tissue constructs grown in simulated microgravity had higher GAG contents and thinner outer capsules than control constructs grown in turbulent spinner flasks. Two fluid dynamic regimes of simulated microgravity were identified, depending on the vessel rotation speed: (i) a settling regime in which the constructs were maintained in a state of continuous free-fall close to a stationary point within the vessel and (ii) an orbiting regime in which the constructs orbited around the vessel spin axis. In the settling regime, the numerically calculated relative fluid-construct velocity was comparable to the experimentally measured construct settling velocity (2-3 cm/s). A simple mathematical model was used in conjunction with measured construct physical properties to determine the hydrodynamic drag force and to estimate the hydrodynamic stress at the construct surface (1.5 dyn/cm(2)). Rotating bioreactors thus provide a powerful research tool for cultivating tissue-engineered cartilage and studying 3D tissue morphogenesis under well-defined fluid dynamic conditions. (c) 1995 John Wiley & Sons, Inc.     

The influence of mechanical loading on isolated chondrocytes seeded in agarose constructs

Articular cartilage is subjected to dynamic compressive loading during normal activity which influences chondrocyte metabolism through various mechanotransduction pathways. A well characterised and reproducible model system, involving chondrocytes embedded in agarose gel, has been used to investigate the effects of mechanical compression on chondrocytes, isolated from full depth cartilage or separately from the superficial and deep zone tissue. The role of nitric oxide as a mediator of mechanical-induced effects has also been studied. Chondrocytes were isolated, separately, from full depth, superficial and deep zone cartilage and seeded in 3% agarose constructs. Dynamic compressive strain was applied to the constructs using a range of frequencies (0.3, 1 and 3 Hz). Glycosaminoglycan synthesis, cell proliferation and nitrite production were assessed. In further experiments, constructs were compressed in the presence of 1 mM L-NAME or 10 microM dexamethasone. Glycosaminoglycan synthesis by full depth chondrocytes was affected by compressive strain in a frequency dependent manner. Dynamic strain at all frequencies induced an increase in [3H]-thymidine incorporation. Glycosaminoglycan synthesis by deep zone cells was affected by the strain regimes in a similar fashion to full depth cells, while superficial cells exhibited a similar proliferative response to full depth cells. Dynamic compression inhibited nitrite production, the effect being reversed by L-NAME. Compression induced stimulation of [3H]-TdR incorporation was reversed by L-NAME. These studies demonstrate that glycosaminoglycan synthesis and proliferation are influenced by the dynamic strain regimes in a distinct manner. Indeed the data suggest that these processes occur in different chondrocyte sub-populations. It may be speculated that nitric oxide acts as a mediator of mechanotransduction processes affecting proliferation primarily in the superficial cell sub-population.     

Restoration by parathyroid hormone and dibutyryl cyclic AMP of expression of the differentiated phenotype of chondrocytes inhibited by a tumor promoter, 12-0-tetradecanoylphorbol-13-acetate

12-0-Tetradecanoylphorbol-13-acetate (TPA) inhibited expression of the differentiated phenotype of chondrocytes in rabbit costal chondrocytes in culture. TPA transformed typical polygonal chondrocytes into multilayered, fibroblastic cells and also inhibited the rate of [35S]sulfate incorporation into glycosaminoglycan (GAG), a differentiated phenotype of chondrocytes. These changes were apparent within 24 h and reached a plateau at 48 h after the addition of TPA. Phorbol didecanoate and phorbol dibenzoate also inhibited sulfation of GAG, even though the effect was weaker than that of TPA. Phorbol diacetate and 4-0-methyl TPA did not inhibit sulfation of GAG. Addition of parathyroid hormone (PTH) or dibutyryl cyclic AMP simultaneously with TPA overcame the inhibition caused by TPA. PTH and dibutyryl cyclic AMP also reversed the inhibition and stimulated expression of the differentiated phenotype of chondrocytes even in de-differentiated cells which had been pretreated for 3 days with TPA. These findings suggest that cyclic AMP plays an important role in the restoration of the differentiated phenotype of chondrocytes in TPA-treated chondrocytes, and that the TPA-treated cells retain some of the differentiated phenotype of the original cells, such as responsiveness to PTH.     

In situ chondrocyte viscoelasticity

It has been proposed, based on theoretical considerations, that the strain rate-dependent viscoelastic response of cartilage reduces local tissue and cell deformations during cyclic compressions. However, experimental studies have not addressed the in situ viscoelastic response of chondrocytes under static and dynamic loading conditions. In particular, results obtained from experimental studies using isolated chondrocytes embedded in gel constructs cannot be used to predict the intrinsic viscoelastic responses of chondrocytes in situ or in vivo. Therefore, the purpose of this study was to investigate the viscoelastic response of chondrocytes in their native environment under static and cyclic mechanical compression using a novel in situ experimental approach. Cartilage matrix and chondrocyte recovery in situ following mechanical compressions was highly viscoelastic. The observed in situ behavior was consistent with a previous study on in vivo chondrocyte mechanics which showed that it took 5-7min for chondrocytes to recover shape and volume following virtually instantaneous cell deformations during muscular loading of the knee in live mice. We conclude from these results that the viscoelastic properties of cartilage minimize chondrocyte deformations during cyclic dynamic loading as occurs, for example, in the lower limb joints during locomotion, thereby allowing the cells to reach mechanical and metabolic homeostasis even under highly dynamic loading conditions.