Functional Tissue Engineering of Articular Cartilage Through Dynamic ...

Robert L. Mauck Cellular Engineering Laboratory, Department of Biomedical Engineering, Columbia University, New York, NY 10027

Michael A. Soltz Department of Mechanical Engineering, Columbia University, New York, NY 10027

Christopher C. B. Wang Cellular Engineering Laboratory, Department of Biomedical Engineering, Department of Mechanical Engineering, Columbia University, New York, NY 10027

Dennis D. Wong Pen-Hsiu Grace Chao Cellular Engineering Laboratory, Department of Biomedical Engineering, Columbia University, New York, NY 10027

Wilmot B. Valhmu Orthopædic Research Laboratory, Department of Orthopædic Surgery, Columbia University, New York, NY 10032

Clark T. Hung Cellular Engineering Laboratory, Department of Biomedical Engineering, Columbia University, New York, NY 10027

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 ⫻ (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 共 100⫾ 16 k Pa versus 15⫾ 8 k Pa, p⬍0.0001兲 . This represented a 21-fold increase over the equilibrium modulus of day 0 共 4.8⫾ 2.3 k Pa 兲 . Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 共 p⬍0.0001 and p ⫽ 0.002, respectively). 关S0148-0731共00兲00703-2兴

Gerard A. Ateshian Department of Mechanical Engineering, Columbia University, New York, NY 10027 e-mail: [email protected]

Introduction Articular cartilage serves as the load-bearing material of joints, with excellent lubrication and wear characteristics. Due to its avascular nature, cartilage exhibits a very limited capacity to regenerate and to repair. Moreover, it has been stated that the natural response of articular cartilage to injury is variable and, at best, unsatisfactory. The need for improved treatment options for patients with cartilage injuries has motivated tissue engineering studies aimed at the in vitro generation of cartilage replacement Contributed by the Bioengineering Division for publication in the JOURNAL OF BIOMECHANICAL ENGINEERING. Manuscript received by the Bioengineering Division November 3, 1999; revised manuscript received February 6, 2000. Associate Technical Editor: R. Vanderby, Jr.

252 Õ Vol. 122, JUNE 2000

tissues 共or implants兲 using chondrocyte-seeded scaffolds 关1–6兴. 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 and requires a considerable culture period to develop. It is believed that the ability to restore the tissue’s normal material properties will ameliorate the pain and suffering arising from osteoarthritis 共OA兲, a debilitating disease that results in the erosion of diarthrodial joint cartilage. Currently, OA affects 5 percent of the general population and 70 percent of the population over age 65 and costs nearly $8 billion annually in health care 关7兴. Given the lengthy period of time required for development of mechanical properties under conditions of static or free swelling

Copyright © 2000 by ASME

Transactions of the ASME

culture, many researchers have sought to design bioreactors to speed the production of cartilage in vitro. A prevailing thought is that ‘‘sophisticated culture systems, involving precise control of media, mixing, shear force, and hydrodynamic pressure, are often necessary to culture chondrocytes successfully’’ 关8兴. Malaviya and Nerem 关9,10兴 have adopted a parallel-plate flow chamber system to use fluid-induced shear stress as a modulator of chondrocyte activities for tissue engineering of cartilage. Perfusion systems, rotating wall vessels, and spinner flasks have also been adopted with some success 关2,11–16兴. Polyglycolic acid 共PGA兲 felts in a closed bioreactor system require 4–6 weeks for chondrocytes to synthesize a cartilage-like appearance 共as reviewed by Ehrenreich 关8兴兲. Bioresorbable scaffolds encapsulated by agarose were found to improve retention and accumulation of extracellular matrix proteins from chondrocytes in perfusion culture 关5兴. Interestingly, many of these bioreactors seem to be generic or nonspecific in that they foster tissue growth of many tissue types in culture 共e.g., 关17–20兴兲. Unlike the case of articular cartilage, mechanical loading may only play a minor role in the development and maintenance of other tissues. Therefore, one of our objectives has been to create a bioreactor that is tailored specifically for cartilage tissue growth. In our efforts to bioengineer cartilage, we have proceeded with the underlying premise that ‘‘cartilage with appropriate form and function for in vivo implantation can be created by selectively stimulating the growth and differentiated function of chondrocytes 共i.e., proteoglycan and collagen synthesis兲 through optimization of the in vitro culture environment’’ 关12兴. The mechanical environment is known to influence the phenotypic expression and activity of the chondrocytes within the matrix; immobilization is clearly detrimental to cartilage development and repair 共e.g., 关21,22兴兲. Under normal conditions, chondrocytes are able to balance their synthetic and catabolic activities to maintain the integrity of articular cartilage in vivo. In view of this fact, we desired to restore aspects of the physiological environment to articular chondrocytes in vitro with the rationale that a physiological environment is paramount to producing a tissue that is able to perform the loadbearing and lubrication function of natural cartilage. From the literature, only short-term 共typically one week or less兲 deformational loading studies of cell-seeded scaffolds have been performed. In these studies, parameters such as cell deformation, cell signaling pathways, and chondrocyte biosynthesis rates have been monitored 关23–26兴. In a variation of the short term loading study, Buschmann and co-workers 关27兴 assessed the response of cell-seeded agarose disks cultured statically with time to shortterm dynamic loading to assess biosynthetic activity of chondrocytes in a cell-elaborated matrix and reported that cell biosynthetic activity in cultured cell-seeded agarose disks resemble that for articular cartilage. No studies to our knowledge have investigated the efficacy of applied deformational loading in enhancing matrix elaboration in long-term cultures of chondrocyte-seeded scaffolds. In the present study, we hypothesized that the application of deformational loading at physiological levels would enhance chondrocyte matrix elaboration in cell-seeded hydrogel scaffolds, thereby resulting in a more mechanically competent tissue. To test our hypothesis, we performed three independent studies. Study 1 assessed the material properties of acellular agarose and alginate constructs, whereas Study 2 evaluated how each could support matrix elaboration and development of mechanical properties over time in free swelling cultures when seeded with chondrocytes. From the findings of these studies it was concluded that agarose was preferable to alginate for achieving the objectives of Study 3. In this last study, material and biochemical testing were performed on chondrocyte-seeded agarose disks that were subjected to physiological deformational loading over a one-month culture period. Agarose and alginate polysaccharide hydrogels were selected for study since they exhibit an open lattice structure when Journal of Biomechanical Engineering

polymerized, minimizing diffusional distances but maintaining the three-dimensional structure necessary for the fixation of cells 关4,5,26–35兴.

Methods A general outline of each study is presented first, followed by the specific methodology for the various aspects of the three studies. In Study 1, acellular hydrogel disks of various concentrations 共agarose: 1, 2, 3, and 5 percent; alginate: 2, 3, and 5 percent兲 were prepared and tested in confined compression stress-relaxation to determine their material properties. Four disks were used at each concentration and for each hydrogel. The confined compression aggregate modulus was acquired from the equilibrium response; furthermore, the resultant transient stress-relaxation curves were fitted with the linear isotropic biphasic theory of Mow et al. 关36兴 to determine whether this model could successfully describe agarose and alginate hydrogels. Statistical comparisons were performed between the aggregate modulus of agarose and alginate at concentrations of 2, 3, and 5 percent. In Study 2, 2 percent agarose and 2 percent alginate cell-laden disks and acellular controls were cultured under free swelling conditions for up to 56 days. Confined compression stress-relaxation testing and sulfated glycosaminoglycan 共S-GAG兲 compositional analyses were carried out on cell-laden and control disks at 0, 7, 14, 28, 42, and 56 days of culture. Three samples were used for each hydrogel at each time point. Statistical comparisons were performed to detect differences in the aggregate modulus and S-GAG content for agarose and alginate as a function of time. In Study 3, cell-seeded 2 percent agarose gels were either loaded dynamically in unconfined compression or maintained in free swelling static culture over a one-month period. On days 0, 3, 7, 14, 21, and 28, individual agarose disks were removed from culture to be tested in confined and unconfined compression stress-relaxation, as well as for S-GAG and hydroxyproline 共OH-Pro兲 content, with n⫽3 – 4 disks per group. Statistical comparisons were performed to detect differences in the aggregate modulus, peak stress response in confined compression stress-relaxation, unconfined compression modulus, and S-GAG and OH-Pro content, as a function of time, for free swelling versus dynamically loaded disks. All statistical analyses were performed using ANOVA and Fisher’s leastsignificance-difference test, with ␣ ⫽0.05. All three studies required hydrogel preparations, acellular for Study 1 and cell-laden for Studies 2 and 3. The preparation of these hydrogels and cell cultures is described below. The protocol for mechanical testing in all three studies is described in the next section, followed by a description of the testing protocol and custom-designed loading apparatus 共bioreactor兲 employed in Study 3. Biochemical composition measurements are described last. Cell Culture. Articular chondrocytes were harvested from the carpo-metacarpal joint of freshly slaughtered 3–5 month bovine calves obtained from a local abattoir. Articular cartilage specimens were diced into approximately 1 mm3 chunks, rinsed in Dulbecco’s Modified Essential Medium 共DMEM兲 supplemented with 10 percent fetal bovine serum 共FBS兲, amino acids 共0.5 ⫻minimal essential amino acids, 1⫻nonessential amino acids兲, buffering agents 共10 mM HEPES, 10 mM sodium bicarbonate, 10 mM TES, 10 mM BES兲, and antibiotics 共100 U/ml penicillin, 100 g/ml streptomycin兲. The cartilage chunks were digested with 50 mg of bovine testicular hyaluronidase type I-S 共Sigma Chemical Company, St. Louis, MO兲 in 100 ml of DMEM for 30 minutes at 37°C. After removal of the hyaluronidase solution, the cartilage specimens were digested at 37°C overnight with 50 mg of clostridial collagenase type II 共Sigma兲 in 100 ml of DMEM. The cell suspension was then sedimented in a benchtop clinical centrifuge at 4°C for 5 minutes. After rinsing the pellets by resuspension in 20 ml of DMEM and centrifugation, the cells were finally resuspended in 10 ml of DMEM. Cell concentration and viabiliJUNE 2000, Vol. 122 Õ 253

ties were monitored using a hemacytometer and trypan blue exclusion, respectively. Fully supplemented media, as described above, were used for all studies. Hydrogel Preparation. The two hydrogels used in this study were low melt agarose 共type VII兲 and medium viscosity alginate 共Sigma, St. Louis, MO兲. Alginate slabs were prepared using a modification of the method by Ragan et al. 关34兴. Briefly, a 1:1 mixture of 4, 6, or 10 percent alginate and either a cell suspension 共22⫻106 cells/ml兲 or PBS was cast between a sandwich of parallel glass plates and Whatman filter paper soaked in 100 mM CaCl2 , separated by 1.7 mm spacers. Agarose disks 共with and without cells兲 were made in a similar fashion, without the filter paper. The hydrogels were each allowed to set for 30 minutes at room temperature, after which individual 6.76 mm diameter disks were cored with a punch. These disks were cultured in 24 well plates 共Falcon兲 in a humidified incubator at 37°C and 5 percent CO2 . For long-term static culture, disks were cultured on top of 1 mm pore size nylon filters to promote nutrient diffusion from above and below, in fully supplemented DMEM and 50 g/ml ascorbic acid 共Sigma兲. Media 共with fresh ascorbate兲 were changed daily. Control disks for preliminary mechanical characterization studies were made up at various concentrations for both alginate and agarose 共Study 1兲. A final concentration of 2 percent 共wt/vol兲 of agarose 共Studies 2 and 3兲 and alginate 共Study 2兲 was used for studies involving encapsulated cells. Mechanical Testing. Confined compression stress-relaxation testing was performed on all the samples produced in these studies to evaluate the equilibrium confined compression aggregate modulus. Furthermore, in Study 3, unconfined compression stressrelaxation was also performed to evaluate the equilibrium unconfined compression 共Young’s兲 modulus. Confined compression tests were performed using a rigid-porous permeable sintered steel indenter, while unconfined compression tests employed rigid-impermeable glass loading platens. Before each set of mechanical tests, free swelling disk thickness was measured via a custom current sensing micrometer. All tests were performed on a custom-designed computer-controlled testing apparatus 共Fig. 1共a兲兲 consisting of a stepper micrometer displacement actuator 共Oriel Corp., Model 18515, Stratford, CT兲, a Linear Variable Differential Transformer 共LVDT, Schaevitz, Model HR100, Hampton, VA兲 for measuring specimen deformation, and a load cell 共Sensotec, Columbus, OH, range ⫾22 N, 0.01 percent precision兲 for measuring the reaction force. The confined compression chamber, Fig. 1共b兲, described previously 关37兴, employs a solid-state piezoresistive microchip pressure transducer 共NPC 1210-100G-3N; Lucas NovaSensor, Fremont, CA; range 0-690 kPa gage pressure; ⫾0.1 percent accuracy; linearity of 0.1 percent of fullscale output; pressure port 1 mm diameter; 1 mm deep; frequency response

Fig. 1 „a… Loading device used for material testing of hydrogels; „b… confined compression chamber equipped with a microchip pressure transducer „inset…

254 Õ Vol. 122, JUNE 2000

⬎1 kHz兲 for measuring the interstitial fluid pressure at the center of the bottom sample surface. Specimens were equilibrated under a tare load of 0.02 N prior to all mechanical tests. In Studies 1 and 3, the confined compression stress-relaxation test consisted of applying a ramp-and-hold surface displacement, with a ramp speed of 2 ␮m/s until reaching 10 percent strain; the test was completed after the reaction force relaxed to an equilibrium value. Both the total stress 共reaction force/area兲 and fluid pressure were acquired throughout the loading cycle. In Study 2, confined compression stress-relaxation was also performed on statically cultured disks with and without cells, however using four consecutive ramp-and-hold surface displacement profiles, each increasing the equilibrium strain by 5 percent. For this testing protocol, the equilibrium aggregate modulus was computed from the equilibrium stress at the 20 percent strain level. In Study 3, an additional unconfined compression stress-relaxation test was performed on the same samples, using a ramp-and-hold surface displacement with a ramp speed of 2 ␮m/s until reaching 10 percent strain, similar to the confined compression protocol. For all three studies, after equilibrium was reached on the final stress cycle, the disks were unloaded and allowed to recover for 1 hour at 4°C. Disks were frozen at ⫺30°C for subsequent biochemical analyses. Long-Term Dynamic Loading for Study 3. Before studies using chondrocyte-seeded disks were initiated, cyclical unconfined compression loading of 2 percent agarose hydrogels, 1.5 mm thick, at a frequency of 1 Hz and axial strain amplitudes of 10 and 20 percent were performed to determine whether lift-off of the loading platen would occur at these relatively large strain amplitudes, indicating incomplete recovery of the sample after each loading cycle. Each sample was first subjected to a tare strain of 3 percent; following tare equilibrium, a cyclical 共1 Hz兲 displacement with a saw-tooth profile was applied at the loading platen and the resulting compressive stress response and interstitial fluid pressure were measured for 50 cycles. From these preliminary tests, it was concluded that the strain amplitudes that can be used for cyclical loading at 1 Hz in Study 3 should remain at 10 percent or below. To carry out the physiological loading studies, cell-seeded agarose gels, 2 percent w/v in fully supplemented medium, were either loaded dynamically in unconfined compression 共using impermeable platens兲 or maintained in free swelling static culture over

Fig. 2 Schematic of the custom loading device capable of simultaneously deforming multiple chondrocyte-seeded agarose disks. A cam-follower system is used to impose the dynamic loading on unconfined samples. Overlap between the petri dish lid and the petri dish base, which houses the disks, maintains sterility of the samples in a 5 percent CO2 , humidified, 37°C incubator during periods of loading and rest. A custom agarose template prevents shifting of the cell-seeded disks during loading and transport.


a one-month period. Dynamic loading was applied with a custom loading device 共see below and Fig. 2兲, under displacement control, using a sinusoidal waveform with a physiological 10 percent peak-to-peak compressive strain amplitude at a frequency of 1 Hz. This loading regimen, which was deemed physiological and likely to encourage matrix development in the hydrogels, was carried out five days a week, with dynamic loading applied for three consecutive one-hour-on-one-hour-off cycles per day. To maintain up to 16 disks properly seated in the base of a Falcon 60 mm petri dish 共Fisher Scientific兲, a custom agarose template with 8-mm-dia wells was employed to prevent shifting of the agarose disks during dynamic unconfined compression loading and transport; a tare strain of ⬃2 percent was also initially applied to the disks. Two template-configured petri dishes were used to culture the loading and free swelling disks, respectively, for a total of 32 disks per group 共day 0兲. Fully supplemented culture medium 共5 ml兲 with 50 ␮g/ml ascorbic acid was changed every other day. Free swelling controls were handled in the same fashion, but without the application of tare or dynamic strain. Loading took place inside a 5 percent CO2 , humidified incubator at 37°C. Free swelling control disks were positioned adjacent to the loading device in the incubator during loading periods. Loading Device. To carry out this study, we designed a custom bioreactor 共Fig. 2兲 capable of applying a sinusoidal strain to cell-seeded hydrogels. This device consisted of an eccentric circular cam driving a spring-loaded translating cam-follower connected to a loading platen. The camshaft was connected via a flexible shaft to a motor 共Superior Electric, Model M062CS09, Bristol, CT兲 controlled by a signal generator 共BK Precision, Model 4017, Placentia, CA兲 used for adjusting the loading frequency. The cylindrical acrylic loading platen 共12.7 mm thick, 44.7 mm diameter兲 was covered with a 60 mm petri dish lid extending over the 60 mm petri dish base containing the agarose disks, to maintain sterility; sufficient clearance was provided to avoid interference of the lid with the base. The circular eccentric cam produced a simple harmonic 共sinusoidal兲 motion of the follower and loading platen. The circular cam eccentricity was adjusted so as to produce a follower rise of 165 ␮m, corresponding to ⬃10 percent of the disk thickness, as verified with a dial gage. Determination of Total Sulfated Glycosaminoglycan and Collagen Contents. For the determination of total sulfated glycosaminoglycan 共S-GAG兲 and collagen contents of each chondrocyte-seeded agarose construct, disks were digested with papain for 16 hours at 60°C in 0.1 M sodium acetate 共pH 5.6兲 containing 0.05 M EDTA and 0.01 M cysteine-HCl. S-GAG content of the digests was determined using the 1,9dimethylmethylene blue dye-binding assay 关38兴, as modified for microtiter plates and using absorbance at 540 nm and 595 nm for improved detectability 关39兴. Shark chondroitin sulfate 共5 to 50 ␮g/ml兲 was used as the standard. S-GAG for alginate samples were measured similarly using a pH modification that accounted for the anionic nature of the hydrogel 关40兴. Collagen content was determined from aliquots of the same papain digestion used for the S-GAG assay. Samples were hydrolyzed by heating with equal volume of 12 M HCl at 107°C for 18 hours and dried. The hydrolysates were assayed for hydroxyproline content using a colorimetric procedure 关41兴 adapted for use with a microtiter plate reader.

Results Study 1. The stress-relaxation response of a typical 2 percent agarose sample is presented in Fig. 3, together with the corresponding transient interstitial fluid pressure. For both agarose and alginate, it was found that the peak fluid pressure nearly matched the peak stress at the end of the ramp-displacement, indicating significant interstitial fluid load support, and that stress-relaxation equilibrium occurred when the fluid pressure had subsided. The Journal of Biomechanical Engineering

Fig. 3 Stress relaxation experiment for 2 percent agarose: „a… Experimentally measured total stress during a single 10 percent strain cycle, with a biphasic curve-fit superimposed „curve-fit hydraulic permeabilityÄ1.0Ã10À14 m4ÕN"s, curve-fit equilibrium aggregate modulusÄ1.22 kPa „ r 2 Ä0.881…, aggregate modulus from equilibrium responseÄ14.55 kPa. „b… Compares predicted fluid pressure, using curve-fitted parameters from „a…, and experimentally measured fluid pressure „ r 2 Ä0.368…. These results and previous direct measurements of permeability „2.2Ã10À12 m4ÕN"s †65‡… indicate that the linear isotropic biphasic theory does not properly model the agarose response.

Fig. 4 Equilibrium aggregate modulus for acellular alginate and agarose constructs of varying wÕv composition „Study 1…. * Indicates significant difference from alginate construct having the same wÕv composition, p Ä0.03 and p Ë0.0001 for 3 and 5 percent, respectively.

aggregate modulus of acellular agarose was found to increase significantly with concentration from 3 to 5 percent 共Fig. 4兲, while no significant concentration dependence was found for acellular alginate 共p⬎0.96兲. Agarose was found to have a greater aggregate modulus than alginate for concentrations of 3 percent (p⫽0.03) and 5 percent (p⬍0.0001) 共Fig. 4兲. When curve-fitting the linear isotropic biphasic theory to the stress-relaxation response, results demonstrated a uniformly poor fit for both agarose and alginate. A curve fit for 2 percent agarose is shown in Fig. 3. Study 2. The equilibrium aggregate modulus of chondrocyteseeded agarose disks was significantly greater than that of cellfree agarose disks for all time points tested 共p values of 0.003 to ⬍0.0001兲 except for day 0 共Fig. 5兲. The equilibrium modulus increased until day 28, and then exhibited a decreasing trend on days 42 and 56. The equilibrium modulus of cell-free cultures did not change significantly over time in culture. In contrast, chondrocyte-seeded alginate disks displayed no appreciable development of tissue stiffness over time in culture. Except for day 14 cultures (p⫽0.0002), the equilibrium modulus of the cell-seeded alginate disks was comparable to their respective cell-free controls. Compared to cell-free agarose control disks, chondrocyteJUNE 2000, Vol. 122 Õ 255

Fig. 7 Stress versus time response of 2 percent agarose hydrogel under cyclical „1 Hz…, sawtooth profile, axial compression at: „a… 20 percent, and „b… 10 percent compression. Lift-off of the loading platen, as indicated by the arrows, was observed at 20 percent compression. Fig. 5 Equilibrium aggregate modulus for unloaded „static… free-swelling, cell-seeded 2 percent agarose and alginate constructs over time in culture „Study 2…. * Indicates significant difference with respect to cell-free disks of the same hydrogel at the same time point „p values of 0.003 to Ë0.0001….

Fig. 6 Glycosaminoglycan content over time in culture for cell-seeded, 2 percent agarose and alginate free-swelling disks mechanically tested in Fig. 5 „Study 2…. * Indicates significant difference with respect to previous time point „p values of 0.01 to p Ë0.0001….

Fig. 8 Graph of the equilibrium aggregate modulus for dynamically loaded chondrocyte-seeded 2 percent agarose disks and their free-swelling controls over 4 weeks in culture „Study 3…. * Indicates significant difference from respective freeswelling control „ p Ë0.0001… and ** indicates significant difference between stiffness of day 21 and day 28 loaded samples „ p Ë0.0001….

seeded disks displayed significantly greater levels of S-GAG 共Fig. 6兲 at all time points examined except day 0 共p⬍0.0001 relative to day 0兲. A significant time-dependent increase in the accumulation of S-GAG was observed in chondrocyte-seeded agarose cultures up to day 28 共p values of 0.005 to ⬍0.0001 relative to previous time point兲. On days 42 and 56, this trend reversed with a significant decrease 共p values of 0.01 to p⬍0.0001 relative to previous time point兲. Chondrocyte-seeded alginate disks also demonstrated significantly greater S-GAG content than respective cell-free controls for all time points except day 0 共p values of 0.009 to ⬍0.0001 relative to day 0兲. Peak S-GAG content was observed for day 7 and day 14 cell-seeded alginate disks, followed by a significant decrease for days 28 and 42 共p⫽0.024 and p ⬍0.0001 relative to previous time point兲. Study 3. Cyclical unconfined compression loading of 2 percent agarose hydrogels indicated that lift-off of the loading platen occurs at a frequency of 1 Hz and an axial strain amplitude of 20 percent whereas no lift-off was observed at 10 percent 共Fig. 7兲. For the dynamic study, the confined compression tests demonstrated that dynamically loaded disks yielded a threefold increase in the equilibrium aggregate modulus over free swelling controls after 21 days of loading (p⬍0.0001) 共Fig. 8兲. After 28 days of loading, a sixfold increase in the modulus was observed over free swelling controls (p⬍0.0001). This represents a 21-fold increase over the equilibrium modulus of day 0. The day 28 loaded group was significantly stiffer than its day 21 counterpart (p⬍0.0001). 256 Õ Vol. 122, JUNE 2000

Fig. 9 Graph of the peak stress reached during 10 percent strain stress relaxation tests for the same dynamically loaded chondrocyte-seeded 2 percent agarose disks and their freeswelling controls analyzed in Fig. 8 „Study 3…. * Indicates significant difference from respective free-swelling control „p Ä0.007 and p Ë0.0001… and ** indicates significant difference between peak stress of day 21 and day 28 loaded samples „ p Ë0.0001….


Fig. 10 Equilibrium unconfined compression modulus for free-swelling and dynamically loaded 2 percent agaroseseeded disks over time in culture „Study 3…. * Indicates significant difference with respect to free swelling controls at the same time point „p Ä0.009 and p Ë0.0001….

Similarly, compared to the free swelling control, the respective peak stress increased fourfold in day 21 dynamically loaded samples (p⫽0.007) and 12-fold in day 28 loaded samples (p ⬍0.0001) 共Fig. 9兲. A fivefold increase in peak stress over free swelling controls observed for day 14 samples was not statistically significant (p⫽0.16). The unconfined equilibrium modulus for dynamically loaded chondrocyte-seeded agarose disks was significantly greater than that of free swelling disks for day 21 and day 28 samples 共p ⫽0.009 and p⬍0.0001 respectively兲, equivalent to a 2.5-fold increase over day 0 dynamically loaded disks 共Fig. 10兲. Similarly, the peak stress for days 21 and 28 were significantly increased from free swelling disks (p⬍ 0.0001兲, equivalent to a ⬃twofold increase over day 0 disks. The unconfined modulus and peak stress showed no statistical difference at day 21 and day 28 共p ⫽0.36 and p⫽0.1, respectively兲. As observed in Study 2, the S-GAG content of free swelling agarose disks seeded with chondrocytes increased with time in culture 共Fig. 11兲. Disks exhibited a time-dependent increase in S-GAG content from day 7 through day 21 共p⬍0.0001 relative to previous time point兲 for dynamically loaded disks and from day 3

Fig. 12 „a… Photo showing the gross appearance „from left to right… of a day 28 dynamically loaded chondrocyte-seeded disk, a day 28 chondrocyte-seeded free-swelling control disk, and a day 0 free swelling control disk. Notice that the cell-seeded disks are nearly transparent at day 0 and become opaque due to matrix elaboration by the chondrocytes over time in culture. The samples loaded dynamically exhibit the greatest opaqueness. Lower images show bright-field images at 10Ã magnification of the same cell-seeded agarose disks demonstrating the increased opaqueness of chondrocyte-seeded agarose disks with time in culture and applied loading. „b… Dynamically loaded. „c… Free swelling. „d… Cell-seeded, day 0.

through day 14 共p⬍0.0001 relative to previous time point兲 for the free swelling controls. Dynamic loading of chondrocyte-seeded agarose disks did not alter the disk S-GAG composition from that of respective free swelling controls through day 14 in culture. At day 21, a significant difference was noted, with the S-GAG composition of free swelling controls plateauing at the day 14 levels whereas the loaded disk composition further increased (p ⬍0.0001). Hydroxyproline content at day 21 averaged 54.8 ⫾5.3 ␮ g/disk (n⫽4) and 74.9⫾5.5 ␮ g/disk (n⫽4) for the free swelling and dynamically loaded specimens, respectively, demonstrating a significant difference (p⫽0.002). Day 28 data were not available for S-GAG and hydroxyproline analysis. Finally, as a more subjective measure, after 28 days in culture we observed that the dynamically loaded agarose disks were visibly more opaque than the free swelling controls, whereas free swelling control disks at day 0 appear transparent 共Fig. 12兲.

Discussion

Fig. 11 Graph of glycosaminoglycan „S-GAG… content of freeswelling and dynamically loaded agarose-seeded disks over time in culture „Study 3…. The free-swelling and loaded groups displayed similar S-GAG levels through day 14. At day 21, the loaded disks continued to accumulate S-GAG significantly, whereas the free-swelling controls plateaued at day 14 levels „ ** indicates significant difference from previous time point and free swelling control at same time point, p Ë0.0001…. Free swelling disks on all days tested had a significantly higher S-GAG content than free swelling disks at day 0 „ p values of 0.003 to Ë0.0001…. Loaded disk samples had significantly greater S-GAG content than that of loaded disks at day 0 for all time points „ p Ë0.0001… except day 3. * Indicates significant difference compared to previous time point „ p Ë0.0001….

Journal of Biomechanical Engineering

In the present study, we investigated a novel strategy to grow a more mechanically functional articular cartilage tissue from chondrocyte-seeded scaffolds in vitro. Study 1 was motivated by our desire to define a suitable scaffold material and assess its mechanical response and material properties in comparison to previously reported properties for articular cartilage. We considered two hydrogels that have been used extensively in cartilage biology and that have been adopted for tissue engineering applications. We demonstrated that the interstitial water of agarose and alginate will pressurize substantially relative to the total applied stress during confined compression stress relaxation, as has been observed previously in articular cartilage 关37兴 and that the viscoelastic response of these hydrogels is governed, at least in part, by this interstitial fluid pressurization and concomitant flow-dependent effects. However, we observed that even for relatively high concentrations 共up to 5 percent兲, neither agarose 共Fig. 3兲 nor alginate achieved aggregate moduli in the range of normal articular cartilage (⬃0.15– 0.80 MPa兲. The application of physiological cartilage strain levels 共e.g., on the order of 10 percent兲 to agarose JUNE 2000, Vol. 122 Õ 257

hydrogels yielded subphysiological peak stresses 共⬃8 kPa兲, producing interstitial fluid pressure several orders of magnitude lower than that achievable in articular cartilage 共e.g., 1–10 MPa兲. Therefore, for the purpose of functional tissue engineering, these hydrogels were found to be good candidates for monitoring tissue growth by measuring the equilibrium moduli and peak stress response. Importantly, we also observed that the linear isotropic biphasic theory 关36兴, which has been shown to predict the response of articular cartilage in confined compression successfully 关36,37,42兴, is not appropriate for modeling the response of agarose or alginate, even though the qualitative response of these hydrogels during stress-relaxation appeared similar to that of cartilage 共Fig. 3兲. This signified that neither the aggregate modulus nor the hydraulic permeability of the hydrogels could be reliably predicted from curve-fitting the transient stress-relaxation response; indeed, in the typical case represented in Fig. 3, the curve-fitted permeability parameter was found to be 1.0⫻10⫺14 m4/N•s, whereas our own recent direct measurements of permeability averaged 2.2⫻10⫺12 m4/N•s at 10 percent strain for 2 percent agarose gels 关37兴. Therefore, in the current study, the modulus is evaluated from the equilibrium stress–strain response. Following our findings in Study 1, and since the equilibrium aggregate modulus for the hydrogels at 2 percent were similar, we initiated Study 2 to characterize further the suitability of alginate and agarose as scaffold materials by monitoring the development of material properties and matrix elaboration in long-term free swelling cultures. Over the course of the 56 day experiment, we found that the peak equilibrium aggregate modulus achieved for cell-agarose constructs 共at day 28兲 was threefold greater than the peak equilibrium aggregate modulus measured for cell-alginate constructs 共at day 14兲. We also observed an initial increasing trend in S-GAG accumulation up to four weeks in culture for cell-agarose constructs, followed by a striking reverse trend. S-GAG release into the medium was assayed and found to be below 13 ␮g/day per disk on day 7, and below 10 ␮g/day by day 28. Our results are not entirely consistent with those of Buschmann and co-workers 关43兴 who observed that S-GAG accumulation in chondrocyte-seeded disks continued to increase through day 70 in culture. The basis for the disparate findings is unknown and may be related to differences in the agarose they used 共SeaPlaque兲 as well as their source of chondrocytes 共isolated from the femoropatellar groove of 1–2 week old calves兲 in addition to material testing methodology 共e.g., dynamic testing in unconfined compression and ⬃20 percent tare strain兲 relative to the current study. With respect to alginate, we observed modest increases in S-GAG accumulation that plateaued at day 7 and day 14. S-GAG accumulation then dropped successively for days 28 and 42. Contrary to our findings, a steady increase in matrix accumulation of S-GAG was reported by Ragan and co-workers 关34兴 for just over two weeks in culture. In another study however, Onobakhare and co-workers 关40兴 reported that the S-GAG content of chondrocyteseeded alginate plateaus at a peak value from day 14 through day 24 in culture, which is not necessarily inconsistent with our findings 共Fig. 5兲. Other investigators have demonstrated that alginate significantly softens in culture over time, due perhaps to leaching of calcium and replacement with sodium ions 关44,45兴. The anionic nature of the gel may also simulate the negative fixed charge density associated with the S-GAG in cartilage, thus depressing matrix synthetic activities. Considering these factors and our current findings, we concluded that alginate may not be a suitable scaffold for functional cartilage tissue engineering. For Study 3 we adopted agarose scaffolds to investigate whether dynamic loading could enhance the growth of a functional cartilage-like tissue, due to the superior development of tissue stiffness and matrix elaboration of agarose over alginate constructs reported for Study 2. Mechanical loading has been demonstrated in several in vivo 关22,46兴 and in vitro 关47–51兴 investigations to be important for the normal maintenance of articular cartilage. Our mechanical loading regimen was motivated by what we considered to be physiological compressive strain mag258 Õ Vol. 122, JUNE 2000

nitudes and cyclical frequencies. Under normal circumstances, the loading duration of diarthrodial joints is generally cyclical and/or intermittent 关52,53兴, even for seemingly static activities such as standing or sitting, which involve back and forth shifting of the body weight to relieve loading of the lower extremity joints. Similarly, upper extremity activities rarely involve sustained static loading for durations in excess of a few minutes, though sustained dynamic 共cyclical兲 loading may occur over a half-hour or more. Levels of in situ cartilage deformation have been measured in a number of studies, with one radiographic cadaver study of hip joints reporting a reduction of cartilage thickness by 20 percent or less in normal intact joints under physiological loading of five body weights 共BW兲关54兴. Ultrasound measurements of cartilage thickness in a cadaver hip experiment similarly demonstrated changes in cartilage thickness on the order of 10 percent or less, under 1.2 BW 关55兴 while in vivo magnetic resonance imaging 共MRI兲 measurements of cartilage volumetric changes in the knees of human volunteers, prior and subsequent to strenuous activities, has demonstrated a reduction of 6 percent in cartilage volume 关56兴. Our own theoretical contact analyses of biphasic cartilage layers under rolling or sliding motion have demonstrated that in a congruent joint, the cartilage layer thickness decreases by 6 percent under a contact load of one BW 关57兴. Thus, in situ cartilage deformation is generally of moderate magnitude 共⬃20 percent of the thickness or less兲 and for the current study we chose to subject the chondrocyte-agarose constructs to a 10 percent tissue deformational loading at 1 Hz. Interestingly, Lee and Bader 关24兴 examined 15 percent gross strain of chondrocyte-seeded agarose at 0.3, 1 and 3 Hz over a 48 hour period and found that static and lowfrequency strain inhibited S-GAG synthesis, whereas 1 Hz stimulated synthesis. From Study 1 it was observed that agarose did not experience permanent 共plastic兲 deformation for strains of 10 percent; this finding was supplemented with dynamic unconfined compression loading tests to confirm that the agarose disks did not experience a measurable loading platen lift-off under these conditions, further supporting the choice of agarose as a suitable scaffold material to test our hypothesis. Moreover, the biocompatibility and mechanical properties of agarose make it possible to apply load to chondrocyte-seeded agarose cultures immediately upon seeding of cells 共e.g., 关24兴兲. In contrast to agarose, three-day cell seeded PGA constructs, containing mostly cells, were found to be too fragile to be mechanically tested, whereas six-week constructs could support load, presumably due to their accumulation of proteoglycans and collagen and their assembly into a cartilaginous matrix 关58兴. Unconfined compressive loading was selected because it subjects the agarose disks to both compressive normal strains 共in the axial direction兲 and tensile normal strains 共in the radial and circumferential directions兲, which more closely reproduces the physiological environment of cartilage in situ than the exclusively compressive strain state of confined compression. The most significant result of this study is that dynamic loading of chondrocyte-seeded disks over 28 days showed a remarkable ⬃21-fold increase of the equilibrium aggregate modulus over day 0 loaded disks. In the absence of loading, cell-seeded free swelling disks exhibited a less dramatic threefold increase over day 0 disks. The latter is in close agreement with the results of Buschmann and co-workers 关43兴. Although the hydraulic permeability was not directly measured or estimated in this study, an indirect measure of its magnitude may be given by the peak stress response during confined compression stress-relaxation 共Fig. 9兲, which is also related to the dynamic confined compression modulus. The finding that peak stresses are much greater in dynamically loaded disks at days 21 and 28 suggests that their permeability is lower than that of free swelling disks. Expressed per volume of the disks, the S-GAG content of dynamically loaded samples on day 21 (768⫾71 ␮ g/disk) is equivalent to 12.9 mg/ml disk volume. This is nearly one-quarter of that reported for calf cartilage plugs 共57⫾11 mg/ml disk volume 关50兴兲 and is similar 共15.7 Transactions of the ASME

⫾1.4 mg/ml disk volume兲 to the S-GAG content observed in a previous study for day 36 chondrocyte-seeded free swelling agarose constructs 关43兴. The equilibrium unconfined compression modulus demonstrated similar trends to the equilibrium aggregate modulus, though not as impressive 共Fig. 10兲. An unexpected result is that the unconfined compression modulus was greater than the aggregate modulus for free swelling disks at all time points, and for dynamically loaded disks from day 0 to day 14. Since this condition would violate stability constraints, the likely explanation is that friction between the agarose disks and loading platens in unconfined compression is not negligible, as was assumed implicitly in our calculation of the unconfined compression modulus. Therefore, our findings on the unconfined compression modulus should be interpreted with caution. With this caveat, it remains of interest that the unconfined compression modulus of dynamically loaded samples averaged only 42 kPa at day 28 共Fig. 10兲, compared to the value of 100 kPa for the aggregate modulus 共Fig. 8兲. While this difference may simply be related to Poisson ratio effects, it is also possible that the dynamic loading conditions may have produced microfissures in the hydrogel, the effects of which are less apparent in the confined compression loading configuration. No such fissures were visible under 10⫻magnification 共Fig. 12兲, though for future studies, to avoid possible damage to the disks during the early weeks of culture, it may be preferable to apply smaller magnitude strains initially, which can be increased over time in culture.

Conclusion The major finding of this study is that applied physiological, deformational loading accelerated the growth of a cartilage-like tissue having the appearance of cartilage and material properties approaching ⬃1/4 of the natural tissue. To our knowledge, the ⬃21-fold enhancement of the equilibrium aggregate modulus with respect to day 0 disks, observed after only 28 days in culture, is unprecedented. In the absence of loading, chondrocyte-agarose constructs exhibited a ⬃3 – 4-fold increase over initial stiffness values after 28 days in culture 关43兴. For comparison, Freed and co-workers 关3兴 have reported the development of tissue having a modulus comparable to the present study for chondrocyte-seeded PGA scaffolds cultured in a rotating wall bioreactor for 3 months. In another study using PGA scaffolds, Ma and Langer 关59兴 reported a modulus level of ⬃1/5 of our values after 10 weeks in culture. It should be noted that the agarose scaffold employed in this study was chosen in part because of its well-characterized nature as attested by numerous basic science and tissue engineeringbased studies; we do not claim that it necessarily represents the scaffold material that will perform best as a cartilage implant in vivo. In this context, Rahfoth and co-workers 关4兴 investigated the use of allografting chondrocytes cultured in agarose gels to repair full-thickness articular cartilage defects. Control implants of pure agarose produced poor fibrous substitute tissue, insufficient healing and incomplete filling of the cartilage defects. Morphologically stable hyaline cartilage, with new subchondral bone formation at the level of the previous subchondral bone was noted for some cell-laden agarose constructs. Agarose has also been used to encapsulate cell-polymer integrates 共bioresorbable polymer fleeces兲 to improve retention and accumulation of extracellular matrix components synthesized by isolated human articular chondrocytes 关5兴. Moreover, in long-term cultures, chondrocytes have been found to synthesize a matrix in agarose having properties approaching one-quarter those of articular cartilage 共⬃1/4 the streaming potential, S-GAG content and modulus兲关43兴. The specific loading-induced stimuli to which the chondrocytes are responding remain to be elucidated. In addition to the tissue deformation, the unconfined nature of the bioreactor loading results in higher pressures in the central region and greater flow velocities in the periphery region of each disk 关60–62兴. Since Journal of Biomechanical Engineering

these radial pressure gradients are several orders of magnitude smaller than what has been observed to affect chondrocytes, fluid flow related phenomena 共e.g., streaming potentials, convective nutrient transport兲 may be contributing factors 关42,63,64兴. The favorable findings of this study have provided the rationale for studies presently underway to optimize bioreactor conditions and loading protocol further with the ultimate aim of achieving a functional articular cartilage substitute.

Acknowledgments This study was supported in part by funds from the National Institutes of Health 关R29 AR43628 共GAA兲, R01 AR46568 共CTH兲, and R01 AR43597 共WBV兲兴 and a graduate fellowship from the Whitaker Foundation 共RLM兲.

References 关1兴 Chu, C. R., Coutts, R. D., Yoshioka, M., Harwood, F. L., Monosov, A. Z., and Amiel, D., 1995, ‘‘Articular Cartilage Repair Using Allogenic Perichondrocyte-Seeded Biodegradable Porous Polylactic Acid 共PLA兲: A Tissue Engineering Study,’’ J. Biomed. Mater. Res., 29, No. 9, pp. 1147–1154. 关2兴 Dunkelman, N. S., Zimber, M. P., Lebaron, R. G., Pavelec, R., Kwan, M., and Purchio, A. F., 1995, ‘‘Cartilage Production by Rabbit Articular Chondrocytes on Polyglycolic Acid Scaffolds in a Closed Bioreactor System,’’ Biotechnol. Bioeng., 46, pp. 299–305. 关3兴 Freed, L. E., Langer, R., Martin, I., Pellis, N. R., and Vunjak-Novakovic, G., 1997, ‘‘Tissue Engineering of Cartilage in Space,’’ Proc. Natl. Acad. Sci., 94, pp. 13885–13890. 关4兴 Rahfoth, B., Weisser, J., Sternkopf, F., Aigner, T., Von Der Mark, K., and Brauer, R., 1998, ‘‘Transplantation of Allograft Chondrocytes in Agarose Gel Into Cartilage Defects in Rabbits,’’ Osteoarthritis Cartilage, 6, No. 1, pp. 50– 65. 关5兴 Sittinger, M., Bujia, J., Minuth, W. W., Hammer, C., and Burmester, G. R., 1994, ‘‘Engineering of Cartilage Tissue Using Bioresorbable Polymer Carriers in Perfusion Culture,’’ Biomaterials, 15, No. 6, pp. 451–456. 关6兴 Wakitani, S., Goto, T., Young, R. G., Mansour, J. M., Goldberg, V. M., and Caplan, A. I., 1998, ‘‘Repair of Large Full-Thickness Articular Cartilage Defects With Allograft Articular Chondrocytes Embedded in a Collagen Gel,’’ Tissue Eng., 4, No. 4, pp. 429–444. 关7兴 Kuettner, K. E., and Goldberg, V. M., 1995, Osteoarthritic Disorders, American Academy of Orthopaedic Surgeons, Rosemont, IL, Preface p:xix. 关8兴 Ehrenreich, M., 1999, ‘‘Articular Cartilage Repair: Tissue Engineering’s Killer Application?’’ Techvest, LLC Equity Research. 关9兴 Malaviya, P., Hunter, C., Seliktar, D., Schreiber, R., Symons, K., Ratcliffe, A., and Nerem, R., 1998, ‘‘Fluid-Induced Shear Stresses Promote Chondrocyte Phenotype Alteration,’’ Trans. Annu. Meet.—Orthop. Res. Soc., 23, No. 1, pp. 228. 关10兴 Malaviya, P., and Nerem, R. M., 1999, ‘‘Steady Shear Stress Stimulates Bovine Chondrocyte Proliferation in Monolayer Cultures,’’ Trans. Annu. Meet.— Orthop. Res. Soc., 24, p. 8. 关11兴 Freed, L. E., Vunjak-Novakovic, G., and Langer, R., 1993, ‘‘Cultivation of Cell-Polymer Cartilage Implants in Bioreactors,’’ J. Cell. Biochem., 51, pp. 257–264. 关12兴 Freed, L. E., Grande, D. A., Lingbin, Z., Emmanual, J., Marquis, J. C., and Langer, R., 1994, ‘‘Joint Resurfacing Using Allograft Chondrocytes, and Synthetic Biodegradable Polymer Scaffolds,’’ J. Biomed. Mater. Res., 28, pp. 891–899. 关13兴 Freed, L. E., Marquis, J. C., Vunjak-Novakovic, G., Emmanual, J., and Langer, R., 1994, ‘‘Composition of Cell-Polymer Cartilage Implants,’’ Biotechnol. Bioeng., 43, pp. 605–614. 关14兴 Carver, S. E., and Heath, C. A., 1999, ‘‘Influence of Intermittent Pressure, Fluid Flow, and Mixing on the Regenerative Properties of Articular Chondrocytes,’’ Biotechnol. Bioeng., 65, No. 3, pp. 274–281. 关15兴 Carver, S. E., and Heath, C. A., 1999, ‘‘Semi-Continuous Perfusion System for Delivering Intermittent Physiological Pressure to Regenerating Cartilage,’’ Tissue Eng., 5, No. 1, pp. 1–11. 关16兴 Davisson, T. H., Wu, F. J., Jain, D., Sah, R. L., and Ratcliffe, A. R., 1999, ‘‘Effect of Perfusion on the Growth of Tissue Engineered Cartilage,’’ Trans. Annu. Meet.—Orthop. Res. Soc., 45, p. 811. 关17兴 Goodwin, T. J., Jessup, J. M., and Wolf, D. A., 1992, ‘‘Morphologic Differentiation of Colon Carcinoma Cell Lines HT-29, and HT-29KM in RotatingWall Vessels,’’ In Vitro Cell Dev. Biol., 28A, pp. 47–60. 关18兴 Goodwin, T. J., Schroeder, W. F., Wolf, D. A., and Moyer, M. P., 1993, ‘‘Rotating-Wall Vessel Co-Culture of Small Intestine as a Prelude to Tissue Modeling: Aspects of Simulated Microgravity,’’ Proc. Soc. Exp. Biol. Med., 202, pp. 181–191. 关19兴 Duke, P. J., Daane, E. L., and Montufar-Solis, D., 1993, ‘‘Studies of Chondrogenesis in Rotating Systems,’’ J. Cell. Biochem., 51, pp. 274–282. 关20兴 Spaulding, G. F., Jessup, J. M., and Goodwin, T. J., 1993, ‘‘Advances in Cellular Construction,’’ J. Cell. Biochem., 51, pp. 249–251. 关21兴 Grumbles, R. M., Howell, D. S., Howard, G. A., 1995, ‘‘Cartilage Metalloproteases in Disuse Atrophy,’’ J. Rheumatol., 43 共Supplement兲, pp. 146–148.

JUNE 2000, Vol. 122 Õ 259

关22兴 Setton, L. A., Mow, V. C., and Howell, D. S., 1995, ‘‘Mechanical Behavior of Articular Cartilage in Shear Is Altered by Transection of the Anterior Cruciate Ligament,’’ J. Orthop. Res., 13, No. 4, pp. 473–482. 关23兴 Lee, D. A., and Bader, D. L., 1995, ‘‘The Development, and Characterization of an in Vitro System to Study Strain-Induced Cell Deformation in Isolated Chondrocytes,’’ In Vitro Cell Dev. Biol., 31, pp. 828–835. 关24兴 Lee, D. A., and Bader, D. L., 1997, ‘‘Compressive Strains at Physiological Frequencies Influence the Metabolism of Chondrocytes Seeded in Agarose,’’ J. Orthop. Res., 15, pp. 181–188. 关25兴 Lee, D. A., Frean, S. P., Lees, P., and Bader, D. L., 1998, ‘‘Dynamic Mechanical Compression Influences Nitric Oxide Production by Articular Chondrocytes Seeded in Agarose,’’ Biochem. Biophys. Res. Commun., 251, pp. 580– 585. 关26兴 Knight, M. M., Lee, D. A., and Bader, D. L., 1998, ‘‘The Influence of Elaborated Pericellular Matrix on the Deformation of Isolated Articular Chondrocytes Cultured in Agarose,’’ Biochim. Biophys. Acta, 1405, pp. 67–77. 关27兴 Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J., and Hunziker, E. B., 1995, ‘‘Mechanical Compression Modulates Matrix Biosynthesis in Chondrocyte/Agarose Cultures,’’ J. Cell. Sci., 108, pp. 1497–1508. 关28兴 Benya, P. D., and Shaffer, J. D., 1982, ‘‘Dedifferentiated Chondrocytes Reexpress the Differentiated Collagen Phenotype When Cultured in Agarose Gels,’’ Cell, 30, pp. 215–224. 关29兴 Atala, A., Cima, L. G., Kim, W., Paige, K. T., Vacanti, J. P., Retik, A. B., and Vacanti, C. A., 1993, ‘‘Injectable Alginate Seeded With Chondrocytes as a Potential Treatment for Vesicoureteral Reflux,’’ J. Urol., 150, pp. 745–747. 关30兴 Hauselmann, H. J., Fernandes, R. J., and Mok, S. S., 1994, ‘‘Phenotypic Stability of Bovine Articular Chondrocytes After Long-Term Culture in Alginate Beads,’’ J. Cell. Sci., 107, pp. 17–27. 关31兴 Hauselmann, H. J., Masuda, K., Hunziker, E. B., Neidhart, M., Mok, S. S., Michel, B. A., and Thonar, E. J., 1996, ‘‘Adult Human Chondrocytes Cultured in Alginate Form a Matrix Similar to Native Human Articular Cartilage,’’ Am. J. Physiol., 271 共No. 3, Pt 1兲, pp. C742–752. 关32兴 Paige, K. T., and Vacanti, C. A., 1995, ‘‘Engineering New Tissue, Formation of Neo-Cartilage,’’ Tissue Eng., 1, p. 97. 关33兴 Van Susante, J. L., Buma, P., Van Osch, G. J., Versleyen, D., Van Der Kraan, P. M., Van Der Berg, W. B., and Homminga, G. N., 1995, ‘‘Culture of Chondrocytes in Alginate, and Collagen Carrier Gels,’’ Acta Orthop. Scand., 66, No. 6, pp. 549–556. 关34兴 Ragan, P. M., Staples, A. K., Hung, H. K., Chin, V., Binette, F., and Grodzinsky, A. J., 1998, ‘‘Mechanical Compression Influences Chondrocyte Metabolism in a New Alginate Disk Culture System,’’ Trans. Annu. Meet.— Orthop. Res. Soc., 23, No. 2, p. 918. 关35兴 Rowley, J. A., Madlambayan, G., and Mooney, D. J., 1999, ‘‘Alginate Hydrogels as Synthetic Extracellular Matrix Materials,’’ Biomaterials, 20, pp. 45– 53. 关36兴 Mow, V. C., Kuei, S. C., Lai, W. M., and Armstrong, C. G., 1980, ‘‘Biphasic Creep, and Stress Relaxation of Articular Cartilage in Compression: Theory, and Experiments,’’ ASME J. Biomech. Eng., 102, pp. 73–84. 关37兴 Soltz, M. A., and Ateshian, G. A., 1998, ‘‘Experimental Verification, and Theoretical Prediction of Cartilage Interstitial Fluid Pressurization at an Impermeable Contact Interface in Confined Compression,’’ J. Biomech., 31, pp. 927–934. 关38兴 Farndale, R. W., Sayers, C. A., and Barrett, A. J., 1982, ‘‘A Direct Spectrophotometric Microassay for Sulfated Glycosaminoglycans in Cartilage Cultures,’’ Connect. Tissue Res., 9, pp. 247–248. 关39兴 Seibel, M. J., Macauley, W., Jelsma, R., Saed-Nejad, F., and Ratcliffe, A., 1992, ‘‘Antigenic Properties of Keratin Sulfate: Influence of Antigen Structure, Monoclonal Antibodies, and Antibody Valency,’’ Arch. Biochem. Biophys., 296, pp. 410–418. 关40兴 Onobakhare, B. O., Bader, D. L., and Lee, D. A., 1996, ‘‘Quantification of Sulfated Glycosaminoglycans in Chondrocyte/Alginate Cultures, by Use of 1,9-Dimethylmethylene Blue,’’ Anal. Biochem., 243, pp. 189–191. 关41兴 Stegeman, H., and Stalder, K., 1967, ‘‘Determination of Hydroxyproline,’’ Clin. Chim. Acta, 19, pp. 267–273. 关42兴 Frank, E. H., and Grodzinsky, A. J., 1987, ‘‘Cartilage Electromechanics —II. A Continuum Model of Cartilage Electrokinetics and Correlation With Experiments,’’ J. Biomech., 20, No. 6, pp. 629–639. 关43兴 Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J., Kimura, J. H., and Hunziker, E. B., 1992, ‘‘Chondrocytes in Agarose Culture Synthesize a Mechanically Functional Extracellular Matrix,’’ J. Orthop. Res., 10, pp. 745–758. 关44兴 Baer, A. E., Setton, L. A., Wang, J. Y., Nickisch, F., and Guilak, F., 1999,

260 Õ Vol. 122, JUNE 2000

关45兴

关46兴关47兴关48兴关49兴

关50兴关51兴关52兴关53兴关54兴关55兴关56兴

关57兴关58兴

关59兴关60兴关61兴关62兴

关63兴

关64兴

关65兴

‘‘Static Compression of Chondrocytes in Alginate Culture Does Not Alter Aggrecan Gene Expression as Measured by Competitive PCR,’’ Trans. Annu. Meet.—Orthop. Res. Soc., 45, p. 726. Leroux, M. A., Hernandez, C. H., Guilak, F., and Setton, L. A., 1999, ‘‘Characterization of the Mechanical Behavior of Alginate Gel for in Vitro Chondrocyte Culture Applications,’’ Trans. Annu. Meet.—Orthop. Res. Soc., 45, p. 657. Palmoski, M., Perricone, E., and Brandt, K. D., 1979, ‘‘Development and Reversal of a Proteoglycan Aggregation Defect in Normal Canine Knee Cartilage After Remobilization,’’ Arthritis Rheum. 22, pp. 508–517. Jones, I. L., Klamfeldt, A., and Sanstrom, T., 1982, ‘‘The Effect of Continuous Mechanical Pressure Upon the Turnover of Articular Cartilage Proteoglycans in Vitro,’’ Clin. Orthop., 165, pp. 283–289. Gray, M. L., Pizzanelli, A. M., Grodzinsky, A. J., and Lee, R. C., 1988, ‘‘Mechanical, and Physicochemical Determinants of the Chondrocyte Biosynthetic Response,’’ J. Orthop. Res., 6, pp. 777–792. Sah, R. L. Y., Doong, J.-Y. H., Grodzinsky, A. J., Plaas, A. H. K., and Sandy, J. D., 1991, ‘‘Effects of Compression on the Loss of Newly Synthesized Proteoglycans, and Proteins From Cartilage Explants,’’ Arch. Biochem. Biophys., 286, pp. 20–29. Sah, R. L. Y., Kim, Y. J., Doong, J.-Y. H., Grodzinsky, A. J., Plaas, A. H. K., and Sandy, J. D., 1989, ‘‘Biosynthetic Response of Cartilage Explants to Dynamic Compression,’’ J. Orthop. Res., 7, pp. 619–636. Guilak, F., Meyer, B. C., Ratcliffe, A., and Mow, V. C., 1994, ‘‘The Effects of Matrix Compression on Proteoglycan Metabolism in Articular Cartilage Explants,’’ Osteoarthritis Cartilage, 2, pp. 91–101. Paul, J. P., 1967, ‘‘Forces Transmitted by Joints in the Human Body,’’ Proc. Inst. Mech. Eng., 181 共No. 3J兲, p. 8. Dillman, C. J., 1975, ‘‘Kinematic Analyses of Running,’’ in: Exercise and Sport Sciences Review, Academic Press, New York, pp. 193–218. Armstrong, C. G., Bahrani, A. S., and Gardner, D. L., 1979, ‘‘In Vitro Measurement of Articular Cartilage Deformations in the Intact Human Hip Joint Under Load,’’ J. Bone Jt. Surg., 61A, No. 5, pp. 744–755. Macirowski, T., Tepic, S., and Mann, R. W., 1994, ‘‘Cartilage Stresses in the Human Hip Joint,’’ ASME J. Biomech. Eng., 116, pp. 10–18. Eckstein, F., Tieschky, M., and Faber, S., 1998, ‘‘In Vivo Quantification of Patellar Cartilage Volume, and Thickness Changes After Strenuous Dynamic Physical Activity—A Magnetic Resonance Imaging Study,’’ Trans. Annu. Meet.—Orthop. Res. Soc., 23, p. 486. Ateshian, G. A., and Wang, H., 1995, ‘‘A Theoretical Solution for the Frictionless Rolling Contact of Cylindrical Biphasic Articular Cartilage Layers,’’ J. Biomech., 28, pp. 1341–1355. Vunjak-Novakovic, G., Martin, I., Obradovic, B., Treppo, S., Grodzinsky, A. J., Langer, R., and Freed, L. E., 1999, ‘‘Bioreactor Cultivation Conditions Modulate the Composition, and Mechanical Properties of Tissue-Engineered Cartilage,’’ J. Orthop. Res., 17, pp. 130–138. Ma, P. X., and Langer, R., 1999, ‘‘Morphology, and Mechanical Function of Long-Term in Vitro Engineered Cartilage,’’ J. Biomed. Mater. Res., 44, No. 2, pp. 217–221. Armstrong, C., Lai, W., and Mow, V., 1984, ‘‘An Analysis of the Unconfined Compression of Articular Cartilage,’’ ASME J. Biomech. Eng., 106, pp. 165– 173. Kim, Y. J., Sah, R. L., Grodzinsky, A. J., Plaas, A. H., and Sandy, J. D., 1994, ‘‘Mechanical Regulation of Cartilage Biosynthetic Behavior: Physical Stimuli,’’ Arch. Biochem. Biophys., 311, No. 1, pp. 1–12. Mow, V. C., Bachrach, N. M., Setton, L. A., and Guilak, F., 1994, ‘‘Stress, Strain, Pressure, and Flow Fields in Articular Cartilage and Chondrocytes,’’ in: Cell Mechanics and Cellular Engineering, Springer-Verlag, New York, pp. 345–379. Garcia, A. M., Frank, E. H., Grimshaw, P. E., and Grodzinsky, A. J., 1996, ‘‘Contributions of Fluid Convection, and Electrical Migration to Transport in Cartilage: Relevance to Loading,’’ Arch. Biochem. Biophys., 333, No. 2, pp. 317–325. Buschmann, M. D., Kim, Y.-J., Wong, M., Frank, E., Hunziker, E. B., and Grodzinsky, A. J., 1999, ‘‘Stimulation of Aggrecan Synthesis in Cartilage Explants by Cyclic Loading Is Localized to Regions of High Interstitial Fluid Flow,’’ Arch. Biochem. Biophys., 366, No. 1, pp. 1–7. Soltz, M. A., Stankiewicz, A., Mauck, R. L., Hung, C. T., and Ateshian, G. A., 1999, ‘‘Direct Hydraulic Permeability Measurements of Agarose Hydrogels Used As Cell Scaffolds,’’ Advances in Bioengineering, ASME BED-Vol. 43, pp. 229–230.
