Chapter 20 Book Biomedical Technology and Devices Handbook

20

Shear Stress and

Chondrocytes

CONTENTS

20.1 Introduction

Significance • Shear Stress

20.2 Chondrocytes and Cell Adhesion

Micropipette Aspiration • Cytodetachment • Microplates • Fluid-Induced Shear

20.3 Mechanical Property Measurement of Single Cells

Micropipette Aspiration • Single-Cell Cantilever Techniques • Atomic Force Microscopy • Laser Tracking Microrheology • Magnetic Beads

20.4 Effect of Shear Stress on Chondrocytes

Metabolism and Proliferation • Bioreactors • Mechanotransduction

20.5 Conclusions

Acknowledgments

References

20.1 Introduction

20.1.1 Significance

Articular cartilage failure is currently a cause of much morbidity and patient suffering, and few solutionscurrently exist for any but the smallest defects. Though articular cartilage is relatively thin, avascular,and aneural,

1

replacement through tissue engineering still remains elusive. However, the field of tissueengineering has entered an exponential phase of growth and discovery, in particular in terms of eluci-dating the effects of the mechanical and biochemical environment on chondrocytes, as well as the synergyof the two.

2

Osteoarthritis (OA), while affecting millions, is not fully understood. OA affects over a fifth of Amer-

icans over age 45, almost half of Americans over age 65, and affects women more often than men.

3

Predisposing factors include previous injury, fracture, ligament tear, and many other causes of misalignedor abnormal force transduction across a joint.

4

Also, heavily trained horses showed increased cartilagefibrillation and decreased stiffness compared to lightly trained horses.

5

Thus, mechanical forces are criticalin cartilage pathology. Patients who have had their joints immobilized, resulting in much lower mechan-ical loading, eventually experienced cartilage thinning and alteration in composition in addition to boneloss.

6-8

These studies illustrate the critical importance of mechanical loading to normal cartilage main-tenance as well as pathology.

C. Corey Scott

Rice University

Kyriacos A. Athanasiou

Rice University

1140_C20.fm Page 1 Tuesday, July 15, 2003 12:34 PM

© 2004 by CRC Press LLC

Applying different forces to cartilage or chondrocytes has been shown to result in diverse genetic andmetabolic effects. Compressive forces and hydrostatic pressure on cartilage and chondrocytes are knownto be beneficial in certain amounts at certain frequencies and can be synergistic with particular growthfactors, although some specifics are still unknown.

9-12

Shear stress, another form of mechanical loadingon chondrocytes and cartilage, is not as fully understood.

20.1.2 Shear Stress

Shear stress has been shown to have diverse effects on chondrocytes through mechanotransductivemechanisms and cell adhesion studies. The difficulty of applying a known and uniform shear stress tocells adds complexity to the issue of shear and chondrocytes.

One method of exploring the effect of shear stress on chondrocytes involves cell adhesion or the forceof detaching a cell from a substrate, particularly if detached by fluid flow or a force parallel to the substratesurface.

13-17

Also, cell adhesion is vital for cells and tissues to function, particularly for connective tissues.

18

Many techniques have been used to measure the detachment force or strength of cells on different surfaces,either individually, or in monolayer culture. In addition to knowing the adhesive strength of cells onvarious materials in different media, these experiments have led to some understanding of the surfacecharacteristics and cytoskeletal behavior of chondrocytes with respect to resisting shear stress. The sametechniques used for cell adhesion can be employed to apply a known amount of shear to chondrocytes,gauge the genetic and phenotypic response, and gather the mechanical properties. Cell adhesion tech-niques are used to understand chondrocyte metabolism and mechanotransduction, but the materialproperties must be known for the cell’s mechanical environment to be characterized.

To obtain the shear mechanical environment experienced by the cell, the material properties of thechondrocyte must be identified.

19

Many models of the techniques used to study cell adhesion, such asmicropipette adhesion

20,21

and microplates,

22

allow the material properties of the chondrocytes to becalculated. Other techniques, such as cytoindentation,

23

magnetic microbeads,

24-26

or atomic forcemicroscopy

27,28

are also used to attain the mechanical properties of the whole cell or a locality of the cell.The mechanotransduction of chondrocytes in response to shear stress has mostly been studied

through fluid-induced shear. These studies have shown an array of effects in response to shear, someregenerative and some degenerative. Several investigations explore the changes in morphology andgene expression of chondrocytes under shear by applying a uniform average shear or simple shear tocartilage explants, while others explore cartilage tissue constructs in bioreactors. Many bioreactorstudies compare static versus perfused culture

29-35

or attempt to tissue engineer cartilage constructs ina low shear or simulated microgravity environment.

36-39

Static versus perfused versus rotating-wallbioreactor comparisons allow conjecture as to the relative shear levels experienced by the cells andtheir phenotypic and genotypic response.

Consequently, biomechanical shear forces are important and integral in OA and tissue engineering ofcartilage. While compressive, cyclical forces of a certain level are beneficial to chondrocytes, the effectsof shear stress are largely unknown. One particularly difficult task is separating out the effects of shearstress from mass transit effects, while another intricacy entails knowledge of the forces transduced to thecell from the extracellular or pericellular matrix. Therefore, shear stress plays a vital and as yet not fullyrecognized function for chondrocytes.

20.2 Chondrocytes and Cell Adhesion

Cell adhesion, which is important to tissue formation, growth, and maturation, involves straightforwardattempts to understand how well chondrocytes adhere to a material, which can indicate the level ofbiocompatibility or the receptors present in the membrane. Generally, a method is devised to remove cellsfrom a substrate and measure the force or pressure of removal. The substrate may be coated with anextracellular matrix or basement membrane protein, such as fibronectin or laminin. Many studies wereperformed demonstrating how certain cells adhered better to certain substrates. The methods utilized



included micropipette aspiration,

14,40-42

cytodetachment,

13,17

fluid-induced shear and cone viscometry,

15,43-

45

and could include microplates.

46

The techniques used for cell detachment are not only important foradhesion studies, but they can also be modified to examine the effect of shear on genetic response.

20.2.1 Micropipette Aspiration

Micropipette aspiration uses a glass pipette with a micron scale tip combined with controlled negativepressure to pluck cells off of a surface (Figure 20.1a).

14,40-42,47

The pressure is increased in small steps untilthe cell is detached from the substrate. Moussy et al.

40

investigated the adhesion strength of endothelialcells on four different surfaces in either phosphate buffer solution (PBS) or culture medium, finding thatthe force of adhesion on both surfaces increased with time. In PBS, the force of detachment increasedwith surface tension, while in culture medium the opposite occurred.

Other experiments utilized chondrocytes or connective tissue cells. Lee and associates

14

performedexperiments that measured the pressure necessary to pull chondrocytes off of the surface of cut cartilagethat was treated with different amounts of chondroitinase ABC. Chondrocytes were seeded onto differentzones of the transversely cut cartilage. Adhesion pressure increased with the duration of seeding four tosix times and with chondroitinase treatment, though to a lesser extent. Sung and associates,

41,42,47

in threedifferent studies, used micropipette aspiration to explore differences in adhesion of fibroblasts from twoligaments of the knee, the anterior cruciate ligament (ACL) and the posterior cruciate ligament (PCL).These studies are analogous to using chondrocytes from different zones, as the fibroblasts are twopopulations of the same cell type. The ACL fibroblasts adhered better to laminin,

41

while the MCLfibroblasts adhered better to fibronectin.

47

These results are thought to be due to the different receptortypes present in different densities on the fibroblasts.

The micropipette studies demonstrate how cell adhesion studies extract detailed information aboutcell behavior and allow inferences as to cell membrane composition and organization. Using micropi-pette aspiration for cell adhesion studies does not measure shear, as the pipette pulls the cell perpen-dicular to the surface. The complex mechanical environment complicates attempts to compare thistechnique to others.

20.2.2 Cytodetachment

Cytodetachment is a method that measures the adhesion force by applying shear through a thin cantileverprobe under microscopy.

13

A similar method also allows measuring cell adhesion.

49

The force is measured

FIGURE 20.1

Techniques for cell adhesion experiments. (a) Micropipette aspiration — the pressure is recorded atthe time the cell detaches; (b) cytodetachment — the deflection of the probe is recorded and calculated into theapplied force at the time of detachment; (c) microplate adhesion rupture — the deflection of the flexible plate canbe recorded and calculated into the applied force at the time of detachment.

Micropipette

Cell

Cell

Probe

Cell

Thick Plate(Rigid)

Thin Plate(Flexible)

Cell

a b c

∆P

∆P



through the deflection of the probe by a fiber optic sensor

48

or a photodiode (Figure 20.1b).

13

Athanasiouand associates

13

found that chondrocytes adhered to fibronectin-coated glass better than plain glass andbovine serum albumin-coated glass. This study also examined the cytoskeletal organization of chondro-cytes on the different substrata, finding that the cytoskeleton was more organized on fibronectin thanglass or bovine serum albumin. Hoben and associates

17

used cytodetachment and found fixed cells hadmore adhesiveness than living cells and that seeding time increased the adhesiveness. Using the cytode-tacher, a study by Huang et al

.

49

showed adhesion forces increased and cell height decreased with seedingtime. Yamamoto et al

.

48, 50

used fibroblasts and calculated the shear strength and adhesive energy by usingthe force to detach the cell and the projected area of the cell. The adhesion shear strength and detachmentsurface energy found necessary to detach the fibroblasts were 1.5 kPa and 29 pJ on collagen-coatedpolystyrene and 1 kPa and 16 pJ on fibronectin polystyrene, while these values were approximately 420to 670 kPa and 7 to 11 pJ for both glass and uncoated polystyrene. These studies were able to find theshearing force of detachment, which allows inference as to the surface composition and binding sitedensity. These methods also lend themselves to mechanical modeling. Further, cytodetachment could beused to explore the mechanical properties of single chondrocytes and combined with reverse transcriptasepolymerase chain reaction (RT-PCR) to investigate the genetic and phylogenic response to shear stress.

20.2.3 Microplates

Microplates are glass beams of micron-scale thickness pulled from rectangular glass that are used to applyforces to single cells.

22,51

The microplates are coated with fibronectin or laminin to allow the cells toadhere more firmly and specifically. One of the plates is rigid, while the other plate is flexible and ofknown elastic moduli (Figure 20.1c). A cell is attached to the rigid plate, and then to the flexible plate.For testing cell adhesion, the rigid plate applies a force pulling the cell away from the flexible plate. Theflexible plate’s deflection allows the calculation of the force, from 1 to 1000 nN, exerted upon the cellusing beam theory since its modulus and thickness is known.

51

Thoumine and Ott

22

tested chick embryonic cardiac fibroblasts in tension and compression, definingthree regimens of mechanical behavior delineated by time scale. During the initial seconds of deformation,the cells behaved elastically, while on the scale of several minutes, the cells behaved viscoelastically. After10 min, the cells were found to have contractile forces dependent upon the actin cytoskeleton. Thebehavior of the cell was modeled using a three-element standard linear solid model. The cells’ two elasticmoduli were found to be from 0.6 to 1.0 kPa with an apparent viscosity of 10

4

Pa-s.

20.2.4 Fluid-Induced Shear

Fluid-induced shear was one of the first methods used to study cellular adhesion. Most fluid-inducedshear devices were created to test endothelial cells, but have also been used for connective tissue cells,including chondrocytes. Several types of devices have been created for inducing shear stress with fluidflow, including parallel plate flow chambers,

15

coaxial parallel plate rotation,

52

cone and plate viscome-ters,

43

and jet impingement (Figure 20.2).

53

These methods attempt to create a constant shear force acrossa monolayer culture of cells. To test adhesion, the percent of cells that detach can be noted, a standardnumber being the shear stress at which half of the cells detach.

15

Some studies indirectly study celladhesion through bioreactors. For instance, Vunjak-Novakovic and colleagues

54

found that seeding thickscaffolds uniformly requires fluid-flow in mixed flasks versus static culture. The seeding kinetics weremodeled with fluid flow, though the role of the resulting shear stress in chondrocyte adhesion to thescaffold is unclear.

Bussolari et al.

43

described a cone and plate viscometer that applies a constant shear stress over asurface, including modeling the flow, which must be maintained at a very low Reynolds number (Figure20.2a). Blackman and colleagues

44

described an advanced design of a cone and plate viscometer thatincludes a microstepper motor to allow close control of the rotation of the cone and the ensuing shearstress. The modeling of this device includes a nonsteady-state startup three-dimensional flow solution,



as well as the steady-state solution. Wendl

52

described a model of a parallel coaxial disk device thatsimulates shear loading, solving for oscillatory flow conditions in addition to steady state (Figure 20.2b).The models of these devices have increased in complexity over time, demonstrating the difficulty ofachieving a steady shear stress over groups of cells. Schnittler et al.

45

developed a cone plate viscometerfor specifically measuring shear detachment force and showed endothelial cell adhesion increased withlaminin and that stress fiber formation occurred under fluid-induced shear.

Schinagl and associates

15

used a parallel plate shear flow chamber, seeding chondrocytes onto a cartilagesurface for between 5 and 40 min, inverting the chamber to count the percentage of cells still adherent,and then applying between 6 and 90 Pa of shear stress through fluid flow (Figure 20.2c). The fraction ofdetached cells was then measured. At 9 min of seeding time, half of the cells were detached due to theinversion and gravity. At 40 min of seeding time, half of the cells were detached by 26 Pa of shear stress.The study also found that increasing seeding time increased adhesion.

Bundy and colleagues

53

used jet impingement to measure adhesion forces of fibroblasts and the bacteria

Staphylococcus aureus

(Figure 20.2d). This technique uses a submerged jet of fluid to apply shear to asurface. The jet of fluid causes peak shear around its diameter and a gradient of less shear farther awayfrom the stream. The jet detaches cells within a certain area on a plate, the diameter of which is fit witha computer. By modeling the shear stress as a function of radius and noting the greatest average diameterat which cells are detached, the approximate detachment force is calculated. This study found fibroblaststend to detach due to shear, while the bacteria detached more due to pressure. Further, fibroblasts adheredto titanium and tissue culture plastic approximately the same, likely due to adsorbed proteins. Theadhesion strength of bacteria was not as dependent on seeding time as fibroblasts, possibly due tofibroblasts’ more complex cytoskeleton organization.

FIGURE 20.2

Techniques to apply fluid-induced shear stress over a population of cells. (a) Cone and plate viscom-eter; (b) coaxial plate viscometer; (c) parallel plate flow chamber; (d) jet impingement and lesion size.

a b

c d

FluidFlow

Plate with Cells

Cells Cells

Nozzle

Lesion

d



All of the fluid-induced shear studies of cell adhesion test groups of cells. The fluid-induced shearstress is an overall calculated average. While these studies are important to understanding how cellsbehave under shear, applying a uniform mechanical environment to each cell is not possible, due to smalldifferences in adhesion, extracellular matrix composition, and cell geometry. The genetic response of thecells gauged by these studies is also of the whole population of cells and does not account for possibledifferences between cells of the same type, such as fibroblasts from different tendons or chondrocytesfrom different zones.

20.3 Mechanical Property Measurement of Single Cells

Cell mechanical properties must be obtained to understand the stress environment of the cell. Cellsexhibit a wide array of mechanical properties. For example, while neutrophils tend to behave as a fluid,

55

the chondrocyte exhibits viscoelastic behavior.

56

Koay and colleagues

57

demonstrated viscoelastic chon-drocyte behavior in response to a step load using cytoindentation. The material properties of the chon-drocyte were obtained using a standard linear solid model. As the chondrocyte and its cytoskeleton arecomplex, different models are used depending on the type of loading and properties desired. Thus,different cells and different loading regimes require different models. Many of the techniques used forcell adhesion strength are also used for calculating mechanical properties. Once cell mechanical propertiesare known, the stress and strain profile of a cell undergoing a specific deformation can be interpretedand compared to other mechanical, genetic, and biochemical environmental factors.

20.3.1 Micropipette Aspiration

Micropipette aspiration to obtain mechanical, viscoelastic properties of chondrocytes has used a homo-geneous elastic solid model

56,58

or a standard linear solid model,

20,59-61

which assumes an axisymmetrichalf-space and incompressibility (Figure 20.3a). The differences between using this technique for detach-ment and using it for measuring cell properties include a pipette tip diameter that is smaller than thecell and recording time displacement curves for the displacement of the cell membrane in the center ofthe pipette, L. Using an elastic solid model, the chondrocyte was found to behave as a viscoelastic solid;the Young’s modulus of both osteoarthritic and nonosteoarthritic chondrocytes was calculated to be 0.67and 0.65 kPa, respectively, and not significantly different.

56

The volume change after aspiration was greaterin osteoarthritic chondrocytes. Trickey and colleagues

20

employed a more complex standard linear solidmodel with three parameters to characterize normal and osteoarthritic chondrocytes, finding thatosteoarthritic chondrocytes displayed an increased instantaneous and equilibrium modulus, as well as ahigher apparent viscosity. This model employs a Kelvin body to model the chondrocytes’ viscoelasticityphenomenologically (Figure 20.4a).

20

Another study found decreasing osmolarity led to decreased moduliand apparent viscosity concurrently with dissociation of actin, though no change to hyperosmotic stress.

59

A study of the pericellular matrix with a chondrocyte found that the pericellular matrix was twice as stiffas the chondrocyte.

62

These models demonstrate viscoelastic solid behavior by the chondrocyte andalterations in mechanical properties that were due to loading, osmotic environment, and disease states.Further, the surrounding pericellular matrix was found to be stiffer than the cell and thought to be lessstiff than the extracellular matrix. These results have implications for the transduction of force from thetissue to the cell and may be used in future modeling.

20.3.2 Single-Cell Cantilever Techniques

Another method of obtaining the material properties of a cell involves applying a force to the cell via acantilever beam. Cytoindentation uses a thin probe to compress the cell (Figure 20.3b).

23

Modeling thecell with two continuum mechanics models, including a mixed-boundary value, linear elastic solid withBousinesq-Papkovitch potential functions and a linear biphasic model, the compressive modulus of acell line was found. This apparatus allows a prescribed displacement and force profile on a single cell.



Another indentation technique used a glass probe to indent the surface of mouse embryonic carcinomacells, looking at differences between cells with and without vinculin.

63

Vinculin-deficient cells were lessresistant to indentation, indicating that vinculin is an important part of the cytoskeleton. Similar canti-lever techniques, such as microplates,

22

have also been used to obtain cell mechanical properties, whileother cantilever techniques could be modeled to obtain properties. Cytodetachment, discussed earlier,could be modeled to further understand how a single cell behaves under shear.

13

20.3.3 Atomic Force Microscopy

Atomic force microscopy has been used to investigate the differences in mechanical properties of variouscells, including endothelial, cardiac and skeletal muscle,

64

liver endothelial cells and fibroblasts,

27

aorticendothelial cells,

28

osteoblasts,

65

and a fibroblast cell line (Figure 20.3c).

66

The Hertz model is often usedand assumes a pointed or spherical tip indentation probe and an elastic, homogenous material. Thedeflection of the probe is measured, and the applied force can be calculated using beam theory. Thedeformations are infinitesimal. Sato and colleagues

28

looked at the changes in local cell properties inendothelial cells exposed to fluid-induced shear stress, similar to studies of chondrocyte metabolism.Comparison of cardiac and skeletal muscle viscoelastic properties is similar to ascertaining the differencesbetween zonal chondrocytes or between fibroblasts from two similar ligaments.

64

20.3.4 Laser Tracking Microrheology

The procedure of using laser tracking microrheology involves implanting small spheres into the cyto-skeleton and recording their micromotion.

67

Yamada and colleagues tracked polystyrene particles in

FIGURE 20.3

Techniques to obtain mechanical properties. (a) Micropipette aspiration; (b) cytoindentation; (c)atomic force microscopy; (d) magnetic bead microrheometry.



polymerized actin and COS-7 cell cytoplasm without applying any forces. The Brownian motion of theparticles is indicative of the local mechanical properties. The technique demonstrated the viscoelasticnature of cytoplasm and measured local differences in the cytoplasm.

20.3.5 Magnetic Beads

One method that can test local membrane mechanical properties is called magnetic bead microrhe-ometry.

26,68

A 4.5-

m

m magnetic bead is coated with a molecule that will bind a specific receptor anda magnetic field is applied to the bead, which applies a force to the membrane (Figure 20.3d). Thosebeads transmitting force onto the cells are tracked versus time, allowing force response curves to begenerated. The viscoelastic behavior is modeled as a dashpot in series with a Kelvin body (Figure 20.4).The data are curve fit and an effective modulus, relaxation time, and viscosity are calculated. A similarstudy utilized the same size beads and applied tension on part of the cell membrane, but did notinclude modeling.

24

Force-displacement curves were compared between different cells with and withoutvinculin.

20.4 Effect of Shear Stress on Chondrocytes

While compressive or hydrostatic forces have been studied and shown to be beneficial at specific fre-quencies and levels, the effect of shear stress on chondrocytes remains controversial. While shear hasbeen shown to increase proliferation,

69

glycosaminoglycan (GAG) size and amount,

70

proteoglycan size,and protein amount,

71

other effects of shear appear injurious to chondrocytes. Fluid-induced shear hasbeen shown to increase pro-inflammatory mediators such as interleukin-6,

72

nitric oxide, and prostag-landin E2.

73

Protective molecules such as tissue inhibitor of metalloproteinase (TIMP-1)

70

and interleukin-4

74

are also induced by shear, possibly indictating activation of protective mechanisms.

70,72,73,75

The effects

FIGURE 20.4

Phenomenological models of viscoelastic behavior. (a) Kelvin body with a spring in parallel with aseries of a dashpot and a spring; (b) Kelvin body in series with a dashpot.

a

b

µ

K1

K2

K1

K2

Bead

µ1

µ2



of shear on metabolism and proliferation are summarized in Table 20.1. Further adding to confusion,many studies compare static to perfused to rotating wall bioreactors. Rotating wall constructs attain thebest results, yet the level of shear stress in these reactors is an average over a population and not alwayscharacterized.29-32,35 Furthermore, mass transport effects cannot be separated from the mechanical effectsof the shear using fluid-induced shear in a viscometer or bioreactor.

20.4.1 Metabolism and Proliferation

The metabolism of chondrocytes in response to shear appears increased and reflective of an injuriousstate. Smith and colleagues70 found 1.6 Pa of fluid-induced shear in a cone viscometer caused bovinechondrocytes to align along the axis of flow and doubled GAG synthesis. Proteoglycan molecule size wasincreased, while prostaglandin E2 (PGE2) increased by a factor of nine. Other pro-inflammatory medi-ators were not increased, including matrix metalloproteinase-3 (MMP-3), collagenase, and stromolyesin,but an inhibitor of MMP-3, tissue inhibitor of metalloproteinase 1 (TIMP-1), was increased by a factorof 10. Archambault et al.76 found that rabbit fibroblasts produce MMP-1, MMP-3, and cyclooxygenase-2 under fluid-induced shear. Das and colleagues73 found that GAG and nitric oxide synthesis was increasedin response to fluid-induced shear. Nitric oxide increased in proportion to the magnitude and durationof the shear stress. Mohtai et al.72 explored the expression of the pro-inflammatory cytokine interleukin6 (Il-6) in chondrocytes. Osteoarthritic chondrocytes produce Il-6, while normal chondrocytes do not.When fluid-induced shear was applied at 1.6 Pa for 48 h, Il-6 mRNA and protein increased approximatelytenfold. Signals for mRNA from interferon-1a and 1b, tumor necrosis factor-a, and transforming growthfactor-b were not affected by shear. Il-6 mRNA was increased within 1 h of applied shear, indicating apossible role in mechanotransduction. Under 1.64 Pa of fluid-induced shear stress, chondrocytes aug-mented the production of the nitric oxide synthase gene and increased nitric oxide delivery by the cellsup to 3.5-fold at 24 h.77 Collagen type II mRNA and aggrecan mRNA were both inhibited by shear stress,but inhibiting nitric oxide production increased both mRNA levels. Based on these studies and others,Smith and colleagues75 put forth a model where shear stress causes chondrocytes to convert to an injuriousstate characterized by certain metabolic products, such as Il-6, PGE2, and TIMP-1.

Other studies confirmed the increase in GAG synthesis and showed other beneficial effects of shear.Gooch et al.32 explored static versus mixed flasks and rotating bioreactors with and without IGF-I. The

TABLE 20.1 Effect of Shear Stress on Metabolism and Proliferation of Chondrocytes

Authors Parameter Change from Baseline Shear Stress Time

Smith et al., 1995 Cell orientation, peak angle 35–45o and 125–135o 1.6 Pa 48 h Glycosaminoglycans Increased twofold 1.6 Pa 24, 48 h Glycosaminoglycan size Increased 1.6 Pa 48 h Proteoglycan size Increased 1.6 Pa 48 h TIMP-1 Increased ninefold 1.6 Pa 48 h Prostaglandin E2 Increased tenfold 1.6 Pa 48 hMohtai et al., 1996 Interleukin-6, protein Increased 11-fold 1.6 Pa 48 h Interleukin-6, mRNA Increased 10- to 15-fold 1.6 Pa 48 h Interleukin-1a Not detected 1.6 Pa 48 h Interleukin-1b Not detected 1.6 Pa 48 h Tumor necrosis factor-a Not detected 1.6 Pa 48 h Transforming growth

factor-b

Not detected 1.6 Pa 48 hDas et al., 1997 Nitric oxide (NO) Increased twofold 0.41 Pa 24 h Increased sixfold 0.82 Pa 24 h Increased fivefold 1.6 Pa 4 h Increased 18-fold 1.6 Pa 24 hMalaviya et al., 1999 Proliferation Increased 44% 3.5 Pa 4 daysJin et al., 2001 Protein Increased 50% 1–3% strain 24 h Proteoglycans Increased 25% 1–3% strain 24 h



mixing in bioreactors causes fluid-induced shear; the rotating bioreactor likely has less shear stress thanthe mixed flasks. The mixed flask constructs proliferated more than the static, though not significantlydifferent from the rotating bioreactor. IGF-I caused an increase in cell count in all conditions and anincreased amount of GAGs in the rotating bioreactor. Interestingly, a decrease in GAG content was seenin the mixed flask versus static when IGF-I was not present. Collagen was increased in the mixed flasksand rotating bioreactors, while GAG was increased the most in the rotating bioreactors with added IGF-I. This study found a nonlinear correlation between GAG content and equilibrium compressive modulus.Although these results were rigorously obtained, comparison of these results with other studies onchondrocyte metabolism is confounded due to the uncharacterized nature of the flow and stress fieldswithin each bioreactor. Millward-Sadler et al.78 studied the effect of Il-4 on chondrocytes, which induceshyperpolarization of the membrane under hydrostatic pressure. Antibodies to Il-4 blocked the mechan-ical-induced hyperpolarization, and Il-4 knock-out mice did not exhibit hyperpolarization. These resultsindicate a role in mechanotransduction for Il-4, but do not indicate a mechanism. In another study,normal, but not osteoarthritic, chondrocytes under intermittent hydrostatic pressure decreased theamount of MMP-3 and increased the amount of aggrecan.74 These changes in metabolism for normalchondrocytes were found to be coupled to mechanotransduction through Il-4. Malaviya and Nerem69

found an increase in proliferation of chondrocytes under shear of 3.5 Pa (35 dynes/cm2) after 4 days. Jinet al.71 applied 1 to 3% simple shear strain at frequencies of 0.01 to 1.0 Hz to cartilage explant disks,illustrating an increase of protein and proteoglycans of approximately 50 and 25%, respectively. The onlysignificant difference was between the static culture and shear; no significant differences were foundbased on the level of shear stress or frequency. These increases only occurred with fetal bovine serum,not in serum-free media. Jin and colleagues71 concluded that the stimulatory effect of shear is mostlydue to deformation, not fluid flow.

These studies demonstrate that shear stress, fluid-induced or not, has a dramatic effect on the metab-olism and gene expression of chondrocytes. Somewhat contradictory data are also present in these studies;for example, one found MMP-3 mRNA levels change with intermittent hydrostatic pressure and anotherfound no MMP-3.70,74 The experiments involving fluid flow pose the problem of delineating the effectsof mass transport and mechanical shear. Though mechanically induced shear stress did not exhibit adose-dependent effect, the range of shear stresses was small, and the force transmission to the cells inmatrix is not wholly known. So, a dose-dependence of cartilage gene expression and shear stress ispossible. The pro-inflammatory and cytoprotective cytokines produced by chondrocytes in response toshear stress can be interpreted as a response to damage.75 The increase in ECM production likewise maybe a consequence of attempts at repair. However, the differential effects of fluid-induced shear, mechanicalshear, and mass transport remain unidentified.

20.4.2 Bioreactors

A major thrust of tissue engineering involves developing cartilage constructs in bioreactors. Cartilagedevelops better in bioreactors that have some perfusion or flow vs. static conditions, and fluid flowinduces shear stress. These observations can indirectly aid in understanding the effect of shear stress onchondrocytes, though the exact levels of shear are unknown for each cell and mass transport effects areagain inseparable. Two major classes of bioreactor studies involve comparing static to perfused to rotatingwall bioreactors29-35 and simulated microgravity bioreactors.36-39

20.4.2.1 Static vs. Mixed vs. Rotating Bioreactors

Comparative studies between static culture, mixed flasks, and rotating bioreactors have shown thatrotating bioreactors tend to produce the best constructs, followed by mixed flasks. Pazzano et al.29

compared static to perfused bioreactors, finding that the perfusion of 1 mm/sec of flow increased DNAcontent, GAGs, and hydroxyproline by 118, 184, and 155%, respectively. Dunkelman et al.80 built abioreactor with a continuous flow rate of 50 mm/min (0.83 mm/sec), achieving 15 and 25% dry weightsof collagen and GAG, respectively. Freed and colleagues33 demonstrated that doubling time decreases



approximately 60% with mixing vs. static, which was attributed to increased mass transport. Freed andcolleagues34 attempted to recapitulate native cartilage in a rotating wall bioreactor using bovine chon-drocytes on PGA for 40 days, finding comparable cellularity, 68% GAG, and 33% collagen of nativecartilage. Gooch et al.,32 also using PGA and bovine chondrocytes, compared static culture to mixed petridishes and flasks and a rotating wall bioreactor with and without IGF-I. In all cases, IGF-I increased wetweight. IGF-I also increased GAG content 1.7-fold in mixed flask and 2.9-fold in the rotating bioreactorand increased collagen 1.6-fold in the rotating bioreactor, while decreasing collagen in static culture.GAG content was found to be correlated to the equilibrium modulus. Mizuno et al.30 used bovinechondrocytes on collagen sponges with and without perfusion. In contrast to other studies, the staticcultures were found to have 3.5- and 2.4-fold increases in aggrecan and collagen type II compared toperfused cultures, respectively. Martin et al.31 compared the mechanical properties of PGA-bovine chon-drocyte constructs cultured in static vs. mixed flask vs. rotating bioreactors. Grossly, the rotating biore-actor construct was the most like native cartilage with 75% of the GAG, 39% of the collagen, and 20%of the equilibrium modulus. The study also included up to 7 months of culture in a rotating bioreactorand achieved the equilibrium modulus and GAG content of native cartilage, though not the stiffness orcollagen content. While these studies have some conflicting results, rotating bioreactors seem to producethe best constructs. However, the particular combination of increasing mass transport and addingmechanical stimulation that will optimize cartilage tissue engineering is not fully known.

One other study that may shed light upon the issue of shear’s effect on chondrocytes combines differentbioreactors for the same culture. Carver and Heath35 used five experimental groups with differentcombinations of spinner flasks (s), intermittent compression/perfusion system (p), and a control withoutthe intermittent compression (c). The fluid flow in the spinner flasks was turbulent, while the rotatingbioreactor likely had less fluid-induced shear, though neither was modeled. The five groups were notedby 1s5c, 1s5p, 2s4p, 4s2p, and 6s. The 2s4p experimental group outperformed the others in collagen andGAG content, though the 4s2p group was next best. This study, though the mechanics and mass transportare uncharacterized, put the chondrocytes in a higher-shear environment initially and switched to a lowershear environment, producing better constructs.

20.4.2.2 Bioreactors and Microgravity

Mathematical models of bioreactors attempt to characterize the mechanical and mass transport envi-ronment due to fluid flow, though this characterization is not always included in bioreactor studies.Much of the mathematical modeling of bioreactors explores simulated microgravity. The rotating wallbioreactor was designed to minimize shear stress and still provide mass transport. While severalbioreactors have been designed to simulate microgravity for cells or aggregates of cells, some mechan-ical stresses are present.

The simplest model of the rotating wall bioreactor involves a force balance on a PGA construct.37 Theforces balanced were the gravitational, hydrodynamic, and centrifugal, resulting in a hydrodynamic forcedue to drag of 0.15 Pa (1.5 dyne/cm2). This study assumed two-dimensional flow and that the constructsdid not interrupt the fluid flow. The model also assumes a consistent construct size. Another early modelof the rotating wall bioreactor by Tsao and colleagues81 assumed steady, Couette flow (flow between twoparallel plates), modeled with Navier-Stokes equations. This model showed a shear stress component inunit gravity up to approximately 0.1 Pa (1 dyne/cm2), especially as the particle size increased. In themodel, space-based operation of the bioreactor substantially decreased the shear stress component. Begleyand Kleis36 modeled the rotating wall bioreactor only assuming axisymmetric flow and validated themodel with laser velocimetry. Their model also found large differences between space-based operationand simulated microgravity and used the inner and outer rotation regimes normal for space (innerrotation slower) or earth-based (inner and outer rotation equal) operation. Mean shear was found to beincreased two- to threefold in earth-based operation. Though the shear levels were still low, the masstransport effects cannot be separated from the hydrodyamically imposed shear effects.

A new design for a bioreactor, the hydrodynamic focusing bioreactor designed for NASA, wasmodeled with results showing even less shear stress on constructs than the rotating wall bioreactor.39



This model assumed that mean shear stress is the same in space- and earth-based applications, whichwas shown not to be the case for the rotating wall bioreactor. Using FLUENT, software to model fluid-mechanics, the model of the dome-shaped bioreactor was calculated to have a maximum shear of0.001 Pa (0.01 dyne/cm2).

The bioreactor studies show promising results for tissue culture of cartilage, but the mass transportand mechanical environment, even if well characterized, are only averages over a population of cells. Thegenetic and phenotypic response of cell subpopulations, for example, different zones of chondrocytes,would be masked in these experiments. In both the bioreactor and metabolism studies, the reaction ofa single chondrocyte to a particular level of shear is unknown and inseparable from the different levelsof mass transport.

20.4.3 Mechanotransduction

The surface and cytoskeleton of the chondrocyte interact with each other and their environment totransduce mechanical signals into genetic expression and biochemical states. Schmidt et al.82 found thatstressing b1 or a2 integrins on osteoblast membranes caused an increase in tyrosine phosphorylation,as well as increased phosphorylation of mitogen-activated kinases. The stresses were induced magneticallyand nonspecifically. Das et al.73 established that nitric oxide increases in response to shear 18-fold in2 hours. By utilizing inhibitors of cellular cascade pathways, the investigators discovered that G-proteins,nitric oxide, and phospholipase C are involved in the transduction of GAGs. Calcium and potassiumchannel blockers did not inhibit GAG synthesis.

While studies revealed that ion channel blockers did not inhibit GAG synthesis, several studies doimplicate ion transients as a method of mechanotransduction.59,83,84 Guilak and colleagues59 foundchanges in the viscoelastic moduli with osmotic stress. Hypo-osmotic stress generated decreased instan-taneous and equilibrium moduli, as well as apparent viscosity. Hypo-osmotic stress also caused actincytoskeleton dissociation. Erickson et al.83 illustrated that hyper-osmotic stress increased calcium in thecell and decreased cell volume.

Mobasheri et al.84 put forth a mechanoreceptor model involving integrins, ion channels, extracellularmatrix (ECM), and cytoskeleton based on the co-localization of the ion channels and integrins. Thesemechanoreceptor complexes are thought to respond to deformation, changes in ion concentration, andpossibly streaming potentials. The specific configuration or operation of these complexes is still unknown.Much of the chondrocyte mechanotransduction apparatus remains to be deciphered.

20.5 Conclusions

The relationship between shear and chondrocytes is not fully elucidated, though some aspects aremore explicitly grasped than others. Cell adhesion studies result in information about chondrocytemechanotransduction. The same techniques, accompanied by appropriate continuum mechanics mod-els, furnish viscoelastic cell mechanical properties. Knowledge of these properties permits acquisitionof the amount of shear stress the cell experiences when loaded in a certain way. Understanding themechanical properties and the mechanical environment of the chondrocytes allows detailed study ofhow shear affects chondroctyes.

The metabolism, gene expression, proliferation, and phenotype of the chondrocyte in response toshear loading has been demonstrated to have various results. While pro-inflammatory cytokinesincrease in response to fluid-induced shear stress, proliferation, and extracellular matrix products havealso been found to increase.69-72,74,75,77 From the perspective of tissue engineering, increasing prolifer-ation and extracellular matrix is beneficial, possibly decreasing the currently lengthy culture times forchondrocytes. However, the increase in pro-inflammatory cytokines due to shear stress is likely unsuit-able for chondrocyte culture. Bioreactor studies have also had mixed results, demonstrating that staticculture is not as suitable as perfusion or rotating wall bioreactor cultures, but the studies are unableto differentiate mechanical and mass transport effects on the culture. Simulated microgravity bioreactor



studies have had success in improving tissue constructs, but have not reached native cartilage levelsand cannot separate out effects of mass transport and mechanical environment. Further, earth- andspace-based operation of the same bioreactor results in different mechanical and flow environments,which also depend on the particle or construct size. While shear stress has both regenerative anddegenerative effects on chondrocytes, the amount of stress and the effects of mass transport on thecells to achieve these effects remain unknown.

Future work in the form of a single cell approach19 to shear stress and chondrocytes is needed to beable to fully understand effects of shear in regenerative and degenerative processes. The single cellapproach involves applying a known shear stress to a single chondrocyte with known cell materialproperties and measuring its genetic and phenotypic response. Using reverse transcriptase polymerasechain reaction (RT-PCR), the mRNA levels of degenerative and regenerative genes can be attained inchondrocytes that experience different shear stress regimes. By understanding what levels of shear induceparticular cytokines and at what level, inhibitors of injurious cytokines at specific concentrations can beused to counteract negative effects. Knowledge of the intensity and duration of shear stress that inducesproliferation and extracellular matrix permits shorter culture time and improved constructs. Combiningcurrent studies with a single cell approach will result in more comprehensive strategies for tissue engi-neering cartilage.

Acknowledgments

We gratefully acknowledge the support of The Whitaker Foundation and The Arthritis Foundation.

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Chapter 20 Book Biomedical Technology and Devices Handbook