Analysis of the Material Properties of Early ChondrogenicDifferentiated Adipose-Derived Stromal Cells (ASC) Using an invitro Three-dimensional Micromass Culture System

Yue Xu, M.D. Ph.D.1, Guive Balooch, Ph. D1, Michael Chiou, B.S.1, Elena Bekerman, B.A.1,Robert O. Ritchie, Ph.D.2, and Michael T. Longaker, M.D., MBA1,*

1Pediatric Surgical Research Program and Department of Surgery, Stanford University School of Medicine

2Department of Materials Science Engineering, University of California, Berkeley

AbstractCartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro havepoor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a greatpotential for application to tissue engineering due to their ability to differentiate into cartilage, bone,and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model systemof early chondrogenesis with ASC. The material properties of these micromasses showed a significantincrease in dynamic and static elastic modulus during the early chondrogenic differentiation process.These data suggest that the 3D micromass culture system represents an in vitro model of earlychondrogenesis with dynamic cell signaling interactions associated with the mechanical propertiesof chondrocyte differentiation.

Keywordschondrocyte; mesenchymal stem cells; high density culture; cartilage repair; mechanical property;tissue regeneration; nanoindentation

IntroductionChondrogenesis is a well-orchestrated, multicellular process driven by chondrogenicprogenitors that undergo mesenchymal condensation, proliferation, chondrocytedifferentiation and maturation [1,2]. The environment in which the chondro-progenitors resideduring this process is crucial in establishing chondrogenesis in vitro [3,4]. Studies using bonemarrow-derived stromal cells (BMSCs) and articular chondrocytes have achievedchondrogenesis in three-dimensional (3D) culture using either a micromass technique, pelletculture, agarose suspension culture, or by seeding cells into a matrix platform such as alginatebeads or a PLGA scaffold [5-10]. Most recently we have demonstrated that ambient oxygenis a critical micro-enviromental regulator during chondrogenic differentiation and influencesthe fate of chondrocyte [11].

*Corresponding author address: Michael T. Longaker, MD, MBA, FACS Pediatric Surgical Research Laboratory 257 Campus DriveStanford, CA 94305 Phone: 650-736-1707 Fax: 650-736-1705 Email: E-mail: Longaker@stanford.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

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Published in final edited form as:Biochem Biophys Res Commun. 2007 July 27; 359(2): 311–316. doi:10.1016/j.bbrc.2007.05.098.

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Adipose-derived stromal cells (ASC) are a promising cell source for tissue engineering becauseof their abundant supply and their capacity for differentiating to bone, cartilage, muscle andfat [12-16]. ASC are readily accessible, easily expandable and have low donor site morbidity;therefore, applying ASC as an alternative cell source for cartilage repair is an area of intenseinterest [17-19]. Most of our current research is focusing on studying the chondrogenic andosteogenic capacities of these cells [11,16,20-23]. Because of the low proliferation capabilityof explanted chondrocytes our group and others are investigating several in vitrochondrogenesis differentiation systems using ASC.

Recently, the nanomechanical properties (e.g. hardness and elastic modulus) have been usedto measure the material properties of calcified biological tissues [24,25]. Therefore,nanoindentation as a high-resolution probe has received a great deal of attention and provideda method for determining material properties on a highly reduced scale [25,26]. With thistechnique, direct physical property measurements of heterogeneous biological materials, suchas friction, adhesion and elasticity, can be determined with high spatial resolution. In addition,the time-dependent mechanical properties of these materials can be obtained in aqueousenvironments, providing a means for property determination that approximates the in situ tissueenvironment. In particular, cartilage behaves mechanically as a viscoelastic solid due to itscomposition and structure [27-29]. The mechanical properties of cartilage, such as elasticmodulus, depend on the rate of strains which are controlled by the amount of extracelluar matrix(ECM) formation, collagen deposition and degradation.

In this study, we utilized a 3D micromass culture system of ASC to create a structural micro-environment for these cells undergoing early chondrogenic differentiation. Subsequently, wehave observed the significant differentiation dynamics through the material properties of thedifferentiated micromasses. We conclude that this in vitro, 3D micromass system will be usefulfor elucidating the mechanism(s) underlying early chondrocyte differentiation and thatnanoindentation is a powerful tool to evaluate the material properties of cartilage/chondrocytesduring early chondrogenic differentiation.

Materials and MethodsChemicals and Media

Dulbecco’s modified Eagle’s medium (DMEM) media was purchased from Mediatech, Inc(Herndon, VA). Fetal bovine serum (FBS) was purchased from Omega Scientific, Inc (Tarzana,CA). Other chemicals were from Sigma-Aldrich (St. Louis, MO). ITS premix was from BDBiosciences (Bedford, MA), and TGF-β1 was from Research and Diagnosis, Inc. (Concord,MA)

Animals and AMC HarvestingAll experiments were performed in accordance with Stanford University Animal Care and UseCommittee guidelines. FVB mice were purchased from Charles River Laboratories Inc.(Wilmington, MA). The inguinal fat pads from three-week old FVB mice were carefullydissected and washed sequentially in a betadine and a phosphate buffered saline (FisherScientific, New Jersey, NJ) solution. The hip cartilage tissue (used for the positive control ofchondrocytes) from the same mice (N=4) was isolated and snap frozen samples were embeddedfor histology. The fat pads were finely minced and digested with 0.075% collagenase A (Sigma-Aldrich, St. Louis, MO)/Hanks’s Balanced Salt Solution (HBSS) with vigorous shaking in a37 °C water bath for 30 minutes. The collagenase A was neutralized with an equal volume ofgrowth media containing DMEM (Mediatech, Inc. Herndon,VA) and 10% FBS. The cellsuspension was filtered with a cell strainer (Falcon, 100um pore size) to remove larger,undigested fat tissue. After filtration, the cells were pelleted by centrifugation and resuspended

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in fresh growth media. In approximately two days, cells became confluent and were passagedby trypsin/EDTA.

In vitro Chondrogenic Experimental DesignThe micromass technique was modified from Ahrens et al. [4,11,20]. Briefly, passage two cellswere trypsinized and resuspended in growth media at a density of 100,000 cells/10μl. Ten-microliter droplets were seeded in culture dishes and allowed to form cell aggregates andsubstratum at 37°C for two and half hours. Chondrogenic media (containing DMEM, 1% FBS,1% penicillin/streptomycin, 37.5 μg/ml ascorbate-2-phophate, ITS premix, and 10ng/ml TGF-β1) was carefully added around the cell aggregates. The chondrogenic media was replenishedevery three days (Figure 1).

Micromass HarvestingFor the measurement of material properties, micromasses (N=4) were carefully harvested atday 3, day 6, day 9, day 12 and day 15. In order to maintain the properties of the differentiatedtissue, the micromasses were maintained in PBS at 4°C until they were loaded onto the platformof nanoindentation. Micromass slides were stained with hematoxylin and eosin (H&E) and foralcian blue to evaluate the chondrogenic differentiation (data not shown). After observing theconsistent chondrogenic differentiation results as reported our previous publications [11,20],we then proceeded with the nanoindentation experiments with these micromasses.

Material Properties of Differentiated MicromassesIn the present experiments, the conventional atomic force microscope (AFM) head wasreplaced by an electrostatic operated transducer that allowed for topographic imaging as wellas local indentation capability (TriboScope nanoindenter, Hysitron, Minneapolis, MN, USA)and mounted on a multimode AFM controlled by NanoScope IIIa electronics (Veeco, SantaBarbara, CA). In the indentation mode, the electrostatic force acting on the spring-suspendedcenter plate of the force—displacement transducer allows the detecting of the time dependenceforce behavior for prescribed displacement. The detailed description of the instruments hasbeen previously published [25,26]. All experiments were performed in a fluid cellencapsulating samples. A quartz sphere, glued to tungsten rode that is connected to the middleplate of the transducer (Figure 2A and 2B) acts as an indenter. The samples, sphere and portionof the tungsten rod were immersed in water during the experiments.

The typical force-displacement curve for cartilage sample is shown in Figure 3A using a 164μm sphere radius. The results were analyzed based on Kelvin model:

(1)

where F(t) , d(t), S, and t are applied force, displacement, stiffness, and time, respectively. Themoduli are calculated from Hertz formula for sphere with radius R:

(2)

where Poisson’s ratio, ν, is assumed 0.5.

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Statistical AnalysisAt each time point, micromass samples (N=4) were measured and mean and SD werecalculated. The statistical analysis was performed using Student’s t test. Material propertymodulus differences were measured using an ANOVA and post-hoc multiple comparison test.With all tests, *p≤0.01 was considered statistically significant.

ResultsMicromass formation, histology and staining for chondrogenesis

By 24 hours, the aggregates generally coalesced and became spherical (Figure 1A and 1B).The micromasses condensed as early chondrogenesis proceeded (Figure 1C). At laterchondrogenesis (day 12 and day 15), micromasses condensed significantly (data not shown).Beginning at day three, the ECM composition between the cells increased markedly and themicromass diameter decreased throughout differentiation, compared to the day 1 micromass(Figure 1C). We have previously reported the histology and ECM analysis of the condensedmicromasses [11, 20]. Hematoxylin and Eosin (H&E) staining indicated the structure of themicromasses during this chondrogenic process; alcian blue staining indicated progressivelyintense staining over time and suggested proteoglycan accumulation within the micromasses.In addition, staining for collagen II, a main ECM component of chondrogenesis which formsthe bulk of the ECM fiber network, was correlated with the alcian blue staining. Mouse fatpads and hips were analyzed as negative and positive controls respectively (data not shown).

Material properties of micromassesTo determine the differences in mechanical properties of native cartilage (as a positive control)and differentiated micromasses, we employed Atomic Force Microscopy-based (AFM)nanoindentation on micromasses at day 3, 6, 9, 12, and 15. By attaching a glass spherical bead(Figure 2B) to a diamond pyramid-shaped nanoindentation tip (Figure 2A), indentations weredone on the chondrogenic-differentiated micromasses enabling us to measure the materialproperties based on the load displacement curves of the micromasses (Figure 2C). The strainand stress relaxation were determined by applying Kelvin model (Figure 2C and 2D).

The dynamic and relaxed modulus of the micromasses significantly increased with time. Atlater time points (day 6, 9, 12 15), significant inductions of the relaxed modulus were observedcompared to the early differentiated micromasses suggesting greater changes occurred afterday 3 micromass differentiation (Figure 3C). Relaxed moduli values were 3 to 5 times lowerthan dynamic moduli, possibly due to the ability of water to flow through the organic structure.Therefore, the increase in material properties may reflect a greater degree of chondrogenicdifferentiation within the 3D structure. A significant increase in relaxed modulus was seen atdays 6-15 compared to only day 15 in dynamic modulus, possibly due to the highly viscoelasticnature of the micromasses. Indentations on adult mouse femur head (mature cartilage) revealeda modulus of 1.27 KPa, 10-fold larger than that of the micromasses at day 15 (data not shown).

DiscussionArticular cartilage in adults has limited capacity for self-repair following injury. Autologouschondrocyte transplantation is a cell-based orthobiologic technology used for the treatment ofcartilage defects [30]. Surgical therapeutic efforts to treat cartilage defects have focused ondelivering new, in vitro-cultured cells capable of chondrogenesis into the lesions [31].However, the poor proliferative potential of the chondrocytes in vitro has been a major obstaclefor stem cell research and the development of clinical therapies. Much of the current researchis focused on developing strategies to improve chondrocyte culture expansion and selection

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[20,31]. Therefore, using ASC, a readily available and abundant potential source ofmesenchymal stem cells is a promising approach for cartilage repair/regeneration.

The influence of mechanical stimuli within micro-environments has been extensivelydescribed as a large contributor to chondrogenic differentiation in vitro. Chondrocytes appearto maintain their phenotype in a 3D culture [5,32]. Consistent with previous reports, we haveachieved successful early chondrogenic differentiation in ASC by using the 3D micromassculture system [11,20]. Alcian blue and collagen II staining indicated the progression ofchondrogenesis in the 3D micromasses. In addition to the histology, we have assessed sulfatedglycosaminoglycan (sGAG) quantities to evaluate this micromass culture system [33]. Geneexpression data demonstrated that ASC underwent early chondrogenesis and obtained thecharacteristics of prechondrocytes: such as significantly higher levels of Sox-9, the criticaltranscription factor of chondrogenesis; elevated aggrecan and type II collagen, majorcomponents of chondrogenesis [11,20]. Collectively, these analyses provide details of spatial-temporal expression of molecules regulating chondrogenic differentiation in ASCmicromasses.

Building on our previous reported changes in histology and marker genes, in this study we alsoobserved significant changes in material properties of the differentiated micromasses. Lackingthe technological capabilities of high spatial resolution, analysis of the mechanical propertiesof differentiated mesenchymal stem cells is still in its infancy. However, in this study, by usinga spherical glass bead tip in AFM nanoindentation, we were able to increase the surface areawhile not compromising the high spatial resolution of the nanoindentation technique.Furthermore, the use of nanoindentation allowed us to assess the properties of thesemicromasses in nano-scale measurements. Therefore, a significant increase of mechanicalintegrity was observed over the time of differentiation. Our data indicated that the surroundingmicro-environmental factors combined with the 3D structure provide appropriate stimuli andcreate a niche for early ASC chondrogenesis.

ASC are a heterogeneous cell population consisting of cells in various stages of differentiationand levels of potency. Defining which cells within this population differentiate along specificlineages is a central goal of our research. Factors involved in the regulation of earlychondrogenesis will be further determined by utilizing the in vitro 3D chondrogenic system.Novel methods of priming and selecting for chondro-progenitors within the heterogeneouspopulation of ASC will contribute to translational therapies utilizing ASC for cartilage repair/replacement. In conclusion, we report that a 3D in vitro micromass system represents a dynamicmulti-cellular model of early chondrogenesis associated with the mechanical properties of earlychondrocyte differentiation.

AcknowledgementsThis work was supported by NIH grants R01 DE-14526 and R01 DE-13194 and the Oak Foundation to MTL. Wegreatly thank Dr. M. Balooch for providing us an excellent facility of AFM.

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Figure 1. Schematic process of experimental design for 3D micromasses creation1A) Schematic for cell harvest and high density seeding technique: mouse inguinal fat padswere harvested, digested, and pelleted. Cells were resuspended in culture media at a densityof 100,000 cells/10 μl; 10 μl droplets were placed in tissue culture dishes and allowed to dryfor two hours.1B) Schematic for micromass formation: chondrogenic media containing TGF-β1 at 10ng/mlwas added to the cell aggregates. Aggregates condensed and formed spheroids within 24 hoursof chondrogenic media addition.1C) Microscopic view of cell aggregates and condensation of 3D micromasses. Themicromasses are shown post-seeding from 12 hour to 96 hours of condensation and aggregationin chondrogenic culture (10x magnification).

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Figure 2. Nanoindentation of the differentiated micromasses2A) Schematic of indentation technique to measure material properties of micromasses.2B) A digital picture of the glass bead attached to the diamond tip.2C) Theoretical load displacement curves from micromasses. Displacement is shown in red,load is shown in black.2D) Experimental load vs. time curves indicating the error associated with the moduluscalculations.

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Figure 3. Changes of material properties during micromass differentiation3A) The theoretical load displacement curve used to determine the relaxed modulus of themicromasses. The force (red) and displacement (blue) are used to determine the stiffness.3B) The dynamic modulus, and 3C) the relaxed modulus of micromasses at days 3, 6, 9, 12,and 15. A significant increase in dynamic modulus (*p<.001) is seen at day 15 compare to day3. The relaxation moduli values showed significant differences at days 6, 9, 12, and 15compared to day 3 relaxed modulus (*p<.001).

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