Regulation of smoot muscle actin expression

Experimental Cell Research 278, 72–83 (2002)doi:10.1006/excr.2002.5561

Regulation of Smooth Muscle Actin Expression and Contractionin Adult Human Mesenchymal Stem Cells

B. Kinner,1 J. M. Zaleskas, and M. Spector2

Department of Orthopedic Surgery, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street,

Prior studies have demonstrated the expression ofa contractile actin isoform, �-smooth muscle actin,in bone marrow stromal cells. One objective of thecurrent study was to correlate contractility with�-smooth muscle actin expression in human bonemarrow stroma-derived mesenchymal stem cells. Asecond objective was to determine the effects oftransforming growth factor-�1, platelet derivedgrowth factor-BB, and a microfilament-modifyingagent on �-smooth muscle actin expression and�-smooth muscle actin-enabled contraction. Adulthuman bone marrow stromal cells were passaged inmonolayer and their inducibility to chondrocytic,osteoblastic, and adipogenic phenotypes was dem-onstrated. Western blot analysis was employed alongwith densitometry to quantify the �-smooth muscleactin content of the cells and their contractility wasevaluated by their contraction of a type I collagen–glycosaminoglycan sponge-like matrix into whichthey were seeded. Transforming growth factor-�1 (1ng/ml) significantly increased and platelet-derivedgrowth factor-BB (10 ng/ml) decreased �-smoothmuscle actin expression and the contractility of thecells. Cytochalasin D also blocked cell contraction.There was a notably high correlation of cell-mediated contraction normalized to the DNA con-tent of the matrices with �-smooth muscle actin con-tent of the cells by linear regression analysis (R2 �

0.88). These findings lay the groundwork for consid-ering the role of �-smooth muscle actin-enabled con-traction in mesenchymal stem cells and in their con-nective tissue cell progeny. © 2002 Elsevier Science (USA)

Key Words: smooth muscle actin; contraction; stemcells; TGF-�1; PDGF-BB.

1 Current address: Department of Trauma Surgery, Clinics of theUniversity of Regensburg, Regensburg, Germany.

2 To whom correspondence and reprint requests should be ad-dressed at Department of Orthopedic Surgery, MRB 106, Brighamand Women’s Hospital, 75 Francis Street, Boston, MA 02115. Fax:
(617) 732-6705. E-mail: [email protected].
720014-4827/02 $35.00© 2002 Elsevier Science (USA)All rights reserved.

INTRODUCTION

The biology of the hematopoietic stem cell has beenthe subject of intensive investigative work for decades,and the therapeutic value of hematopoietic stem cellimplantation has been demonstrated in the clinic formany years. Interestingly, during most of this periodthe mesenchymal stem cell (viz., the bone marrow stro-mal cell) has been of sole utility as a required supportcell in coculture with the hematopoietic stem cell toensure the survival and proliferation of the latter invitro [1, 2]. In recent years, however, the inducibility ofthe marrow stromal cell itself to differentiate into con-nective tissue cells—qualifying the cell as a mesenchy-mal stem cell [3, 4]—has drawn attention to its use forcell therapy and tissue engineering modalities for thetreatment of musculoskeletal problems [4, 5].

The finding that the adherent cell population derivedfrom whole marrow included mesenchymal stem cellsdates back more than 2 decades [2, 6]. Numerous sub-sequent studies have investigated culture methods [7]to separate and enrich subpopulations of adherent cellsfor differentiation along certain pathways. One of thehallmarks of the so-called pluripotent mesenchymalstem cell population in vitro is their well-spread mor-phology, with the display of prominent stress fibers(i.e., actin microfilament bundles) [3]. Prior in vitrostudies of mouse [8], lapine [9], canine [9], and human[10–13] marrow stroma-derived stem cells have dem-onstrated that they express the gene for a contractileactin isoform, �-smooth muscle actin (SMA), that canbe demonstrated to be colocalized on the stress fibers.Of related interest was that the adherent marrow stro-mal cells contained other proteins specific for smoothmuscle [12, 14] and displayed features of smooth mus-cle cells in their endocytotic behavior [10, 15] and syn-thesis of selected proteoglycans [16]. These findings ledto the conclusion that bone marrow stromal cells havea phenotypic similarity to a subset of vascular smoothmuscle cells [12]. Of importance is recent work that hasdemonstrated that the SMA-expressing stem cells can

Boston, Massachusetts 02115 and VA Boston Hea
are System, West Roxbury, Massachusetts 02132 lthc
actually contract a collagen–glycosaminoglycan analog

of extracellular matrix in vitro [9]. This investigation[9] provided a quantitative analysis of the contractilebehavior of mesenchymal stem cells that was noted inan earlier study [17].

An understanding of the regulators of SMA expres-sion in, and contraction of, marrow-derived stem cellsis important with respect to the adverse effects thatSMA-enabled contraction can have if the cells distort(a) scaffolds into which they are seeded for tissue en-gineering purposes or (b) newly synthesized matrix invivo in cases in which they are employed in cell ther-apy. It has been proposed that the positive effects ofSMA-enabled cell contraction relate to the generationof the higher forces required of cells to model newlyforming extracellular matrix and to impart architec-tural features such as crimp to the tissue [18].

That so little is known of the expression of this actinisoform in these cells and its effects on other cell func-tions such as biosynthesis also prompts investigationof these relationships through the stimulus and inhi-bition of SMA expression. This understanding may alsoshed light on SMA expression and contractile behaviorin the musculoskeletal connective tissue cell progeny ofthe mesenchymal stem cell in which SMA expressionand contraction have recently been discovered [18]:articular chondrocytes [19–21], meniscal cells [21, 22],ligament and tendon cells [23, 24], intervertebral disccells [25], and osteoblasts [26].

The specific objectives of the current study were toinvestigate the effects of transforming growth factor(TGF)-�1 and platelet-derived growth factor (PDGF)-BB on the SMA expression and contractility of adulthuman mesenchymal stem cells. These growth factorshave been demonstrated to up and down-regulateSMA, respectively, in other cell types in several previ-ous studies [27–29]. A fungal metabolite, cytochalasinD, known to disrupt the cytoskeleton was also investi-gated for its effects on SMA expression in and contrac-tion of these cells.

MATERIALS AND METHODS

Cell isolation and culture conditions. Samples of human bonemarrow were obtained from 10 (adult) patients undergoing total hiparthroplasty for osteoarthritis and one young healthy 10-year-oldfemale marrow transplant donor (Table 1). In the latter case themarrow was discarded material. Cell isolation followed principallythe protocol originally established by Friedenstein et al. [30] andrecently outlined in detail by others [3, 7].

In the arthroplasty patients, bone marrow removed from the re-sected femoral head was transferred to sterile tubes containing coldphosphate-buffered saline (PBS)/1 mM ethylenediamine tetraaceticacid (EDTA). The marrow and adherent bone trabeculae wereminced and vortexed to disperse the marrow cells. After beingstrained through a 70-�m nylon mesh filter, in order to removeremaining bone and debris, the cell suspension was centrifuged at1500 rpm for 10 min. Cells were resuspended, loaded onto a Ficoll-Histopaque gradient, and again centrifuged at 1500 rpm for 30 min.The cells at the interface (1.073 g/ml) were collected and resuspended

in fresh PBS followed by three washes in PBS. The cells werecounted and their viability was assessed using a trypan blue exclu-sion test. They were then plated into 25-cm2 flasks at a density of400,000 cells per cm2.

Medium was first changed after 48 h. With this most of the non-adherent hematopoietic cells were removed. Only the adherent cellpopulation was cultured subsequently. Cells from subconfluent cul-tures were detached and collected after approximately 10 days using0.05% trypsin and 0.52 mM EDTA for 5 min and then subcultured;cells were replated at 5000 cells/cm2. Cells were cultured in Dulbec-co’s modified Eagle’s medium/F12 containing 10% fetal bovine serum(FBS) from selected lots and 1% antibiotics/antimycotics. Mediumwas changed every other day. The adherent cell population grew assymmetric colonies. Cells were repeatedly subcultured just beforeconfluence to expand their number.

Growth kinetics were calculated using the formula

Population doublings per day � lnNN0

� t �1,

where t is the time period, N is the cell number at the end of the timeperiod, t, and N0 is the cell number at the beginning of the timeperiod.

The cells from the 11 individuals were grown separately. The cellswere expanded through monolayer for three passages and seeded aspassage 4 onto the collagen–glycosaminoglycan matrices (see below).The passage 3 cells were also used for the Western blot analyses. Forthe differentiation assays, cells from passages 2 through 5 wereemployed.

Treatment groups. Cells were grown both in the standard me-dium with 10% FBS and in a medium with a reduced FBS (2%)concentration (see table below). Dosages of the TGF-�1 andPDGF-BB were based on the levels used in several prior studiesreported in the literature [27–29]. The growth factors were used assupplements in the standard medium after it was found that therewas no significant difference in the SMA expression in the 10% FBSand reduced FBS (2%) cultures. This allowed for comparison of theresults with the prior work that had been performed using the 10%FBS medium. The dose of cytochalasin D was chosen based on theperformance in a proliferation assay and its effect on cell morphologyin a pilot study. A dose of cytochalasin D was chosen such that thecells displayed a complete collapse of the actin cytoskeleton, assessed

TABLE 1

Patient Information

Patient No. Age (years) Gender

1 43 F2 59 M3 67 M4 78 M5 66 M6 57 M7 54 M8 36 M9 47 F

10 41 F11 10a F

Mean 55.5

Note. All patients had the diagnosis of osteoarthritis, except forpatient 11, who was a healthy donor.

a Not included in the mean age.

73SMOOTH MUSCLE ACTIN IN ADULT STEM CELLS

by a rounded phenotype, but for which cell proliferation was notdecreased to more than 50%. This dose was found to be 10 �M.

The following were the treatment groups investigated in thisstudy:

Serum (control) 10% FBSReduced-serum medium (ITS) 2% FBS, supplemented with ITS

(insulin 5 mg/L, transferrin5 mg/L, selenium 5 �g/L

TGF-�1 1 ng/ml (in control serum),2 ng/ml (ITS)

PDGF-BB 10 ng/ml (in control serum)Cytochalasin D 10 �M (in control serum)

Cell proliferation assay. Cells were seeded at a density of 5000cells/cm2 in a 96-well plate and treated with serial dilutions ofcytochalasin D. After 5 and 10 days a colorimetric (MTT) kit(Chemicon International, Inc., Temecula, CA) was used to assessproliferation according to the manufacturer’s specifications (http://www.chemicon.com/catalog/mds/CT01.pdf). Briefly, each well wasincubated with 0.01 ml MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) solution for 4 h at 37°C. After 4 h, 0.1ml isopropanol with 0.04 N HCl was added to convert the phenol redof the medium to yellow and dissolve the formazan to give a homog-enous blue solution. Absorption was read on a multichannel platereader at 570-nm wavelength.

�-Smooth muscle actin Western blot analysis. For Western blotanalysis, cells were seeded onto 6-well plates. Treatment with thegrowth factors or other agents was started before confluence andlasted for 5 days (n � 4–8 patients). The samples were treated thesame way to avoid bias due to a difference in cell density. The celllayers were subsequently collected by trypsinization and washed inPBS. Proteins for SMA Western blot analysis were extracted and theWestern blot analysis was performed using a protocol previouslydescribed in detail [21]. Films were digitized and densitometric anal-ysis was performed using NIH Image Processing and Analysis soft-ware. Results are reported as the percentage of the densitometricreading of the positive control (protein extract of human aortasmooth muscle cells).

Type I collagen–glycosaminoglycan matrices. The type I colla-gen–glycosaminoglycan copolymer sponge-like matrices used in thisstudy were produced using a method previously described in detail[31]. Briefly, type I bovine tendon collagen (Integra LifeScience,Plainboro, NJ) was blended with chondroitin 6-sulfate (Chondroitinsulfate C, Sigma Chemical Co.). After being mixed, the coprecipitatewas degassed and lyophilized to prepare the highly porous scaffolds.Subsequently the matrices were cross-linked by dehydrothermaltreatment at 105°C and 1 mm Hg for 24 h. The resulting matriceshad interconnecting pores with a mean pore diameter of 85 �m aspreviously determined by quantitative light microscopy analysis ofmicrotomed sections [32]. Samples, 9 mm in diameter, were punchedfrom the approximately 3-mm thick sheets.

Contraction assay using cell-seeded collagen–glycosaminoglycanmatrices. The contractility of the cells was evaluated by measuringthe cell-mediated contraction of the collagen–glycosaminoglycan ma-trices as has previously been done [19, 21, 33]. The sponge-likematrices were transferred to agarose-coated 12-well plates andseeded with the cell suspension by pipetting cell aliquots of 25 �l oneach side of the matrix (2 � 106 cells per 9-mm-diameter sample).For this experiment cells from a different individual were used foreach treatment group. The cell-seeded matrices were treated withthe selected agent for the entire culture period using the mediumwith 10% FBS (n � 6). Other cell-seeded specimens were grown inthe reduced serum medium. Another group of seeded matrices usingcells from the 10-year-old subject was grown in the reduced serummedium supplemented with 2 ng/ml TGF-�1 (n � 6). Cells from the

same patient were used as the untreated cell-seeded control matrices(n � 6).

The diameters of the matrices were measured at the time that thecell-seeded specimens were placed in the agarose-coated wells andthen every other day using circular templates. The percentage con-traction was determined by dividing the diameter of each matrix atthe specific time period by its initial diameter. The dimensions ofnonseeded collagen–glycosaminoglycan matrix controls cultured un-der identical conditions were also recorded at these time periods.Cell-mediated contraction was computed by subtracting the meanvalue of the percentage contraction of the unseeded scaffold controlsat a particular time period from the percentage contraction of eachcell-seeded matrix sample at that time period as reported previously[19, 33]. The cell-mediated contraction of each matrix was normal-ized to the DNA content of the scaffolds, reflecting the cell numbercontributing to the contraction, by dividing by the value for cell-mediated contraction by the value of the DNA content.

For DNA analysis, the matrices were washed with PBS, lyophi-lized, and solubilized in 1 ml papain buffer (6 �g papain in 0.1 Msodium phosphate, 5 mM Na2EDTA, and 10 mM cysteine–HCl). TheDNA content of the constructs was measured using the Hoechst dyemethod. A 100-�l aliquot of papain digest was mixed with 2 ml ofDNA Dye solution (Hoechst 33258 dye solution in TEN: 10 mM Tris,1 mM Na2EDTA, and 1 M NaCl at pH 7.4) and assayed fluorometri-cally. Calf thymus DNA was used as the standard. The backgroundfluorescence of the matrix was accounted for by subtracting thevalues obtained for the unseeded matrices.

Immunohistochemistry for �-smooth muscle actin. The immuno-localization of SMA in monolayer culture and in microtomed sectionsof paraffin-embedded cell-seeded collagen–glycosaminoglycan matri-ces employed previously described protocols [21, 28]. In brief, afterfixation in formalin, the specimens were rinsed with distilled waterand dehydrated. For immunohistochemical staining of the paraffin-embedded samples, microtomed sections were deparaffinized in xy-lene and rehydrated through ethanol baths and PBS. The sectionswere digested with 0.1% trypsin or 0.1% proteinase XIV for 1 h. Afterbeing rinsed with PBS, the endogenous peroxidase was quenchedwith hydrogen peroxide, followed by blocking of nonspecific bindingwith 30% goat/horse serum. The monoclonal anti-SMA antibody(Product No. A-2547, Clone 1A4, Monoclonal Anti-� Smooth MuscleActin, Sigma Chemical Co.) was applied at a dilution of 1:400. ThisSMA antibody has been used in numerous previous studies [20, 23,34–36].

A section allocated as a negative control was contained on eachslide and was incubated with IgG2a diluted to the same proteinconcentration, instead of the primary antibody. A secondary anti-body composed of biotinylated anti-mouse IgG in PBS was appliedfor 30 min. The slides were subsequently incubated with extra-avidin-conjugated peroxidase (Sigma) for 30 min. For detection of theantibody the 3-amino-9-ethylcarbazole chromogen kit (Sigma) wasused, followed by counterstaining with hematoxylin for 20 min.

RESULTS

Cell Growth in Monolayer and in theCollagen–Glycosaminoglycan Matrices

After three passages, at the time that the cells wereseeded into the matrices, they underwent 9–11 popu-lation doublings. Growth kinetics varied among thepatients, ranging from 0.24 to 0.45 population dou-blings per day (0.4 � 0.05 doublings/day; mean �SEM). The MTT test demonstrated that cytochalasin Dcaused a dose-dependent inhibition of proliferation ofthe marrow stromal cells; the dose at which cell prolif-

74 KINNER, ZALESKAS, AND SPECTOR

eration decreased to 50% was 10 �M/ml (range 8.9–10.9 �M/ml) over a 10-day treatment period.

The distribution of the cells in the collagen–glyco-saminoglycan matrices and the changes in the porecharacteristics of the matrices as they underwent con-traction were similar to the histological findings previ-ously published using comparable matrices seededwith mesenchymal stem cells [9] and chondrocytes[21].

�-Smooth Muscle Actin Expression

Western blot analysis demonstrated the presence ofSMA in the mesenchymal stem cells from all of thepatients (Fig. 1a). The cytoplasmic protein extractsfrom the stem cells (passage 3) contained 112 � 8%(mean � standard error of the mean) of the SMA iso-lated from human arterial smooth muscle cells used ascontrols (Table 2). There was no significant effect ofFBS concentration in the medium (10% FBS versus 2%� ITS) on SMA expression (Table 2 and Fig. 1d; one-factor ANOVA with Bonferroni–Dunn posthoc test formultiple comparison, P � 0.05). TGF-�1 and PDGF-BB had significant effects on SMA expression by thestem cells obtained from the joint arthroplasty patientsand the 10-year-old healthy donor (Table 2 and Figs.1a, 1b, and 1d; one-factor ANOVA, P � 0.0001).TGF-�1 (n � 5 patients) increased by almost 40% theSMA content of the cells (Table 2 and Fig. 1d; Bonfer-roni–Dunn, P � 0.05) and PDGF-BB significantly de-creased the SMA content (by 33%; Bonferroni–Dunn,P � 0.001; Fig. 1d).

The effect of cytochalasin D on different cell typeshas not been previously studied to the same extent asfor the other agents. Therefore different doses andintervals of treatment were studied. Time but not dosehad an effect on SMA expression as shown by Westernblot analysis (Fig. 1c).

These Western blot results were confirmed by theimmunohistochemical findings. Stem cells grown in a10% FBS-containing medium showed a marked expres-sion of SMA incorporated into prominent stress fibersin �80% of the cells (Fig. 2a), as has been previouslyreported using SMA immunohistochemistry [8, 10].With time in culture these cells obtained the typicalflat morphology and a large size. The stress fibers couldbe easily seen, even without staining, in the interfer-ence contrast image as observed in prior investigations[8, 10]. The increase in SMA expression by TGF-�1 andthe decrease by PDGF-BB were also noticeable in theimmunohistochemistry of the monolayer preparations(Figs. 2b and 2c, respectively). TGF-�1 increased thenumber of cells incorporating SMA into their promi-nent stress fibers. PDGF-BB treatment, in contrast,led to a significant decrease of the number of cellsexpressing SMA at detectable levels immunohisto-

chemically as well as in the number of stress fibersincorporating SMA. This coincided with a significantchange in cell shape. The effect of the growth factorsseemed to be more obvious when using a low-serum-containing medium.

Disruption of the actin microfilaments, as achievedby 10 �M cytochalasin D, led to a quick collapse of thecell body, even before all attachment sites were re-leased, resulting in the altered morphology (Fig. 2d).This change of cell shape made the immunohistochem-ical identification of SMA difficult (Fig. 2d).

When cytochalasin D treatment was terminated af-ter 1 day, cells were able to recover (within 4 days)their typical morphology (Fig. 2e). SMA expression,however, continued to remain very low. Again no dosedependence could be seen. This underscored the dualeffect of disruption of stress fibers and decrease of SMAexpression.

Cell-Mediated Contraction of theCollagen–Glycosaminoglycan Matrices

All cell-seeded matrices contracted to a significantlygreater degree than unseeded controls. Contraction fol-lowed a typical time course with the most contractionoccurring during the first 3 days (Figs. 3a, 3b, and 3c).Of note were the complete absence of contraction by thecytochalasin D-treated cells (Fig. 3a) and a clear inhi-bition of contraction by PDGF-BB treatment (Fig. 3b).There was no evident effect of TGF-�1 on the decreasein the diameter of the matrices, prior to normalizing tocell number (Fig. 3c, two-factor ANOVA, P � 0.05).

Unseeded matrices also underwent a slight decreasein diameter as previously reported [19, 24]. Therefore,unseeded matrices were included as controls and thechange in diameter was subtracted from the totalamount of contraction. At 10 days the mean contribu-tion of the matrix/medium alone accounted for 10% oftotal contraction, with a range of 3 to 13% (one-factorANOVA, P � 0.0001, with several groups differingsignificantly from one another using Bonferroni–Dunnposthoc testing). As a result of these differences inchange of shape of the unseeded matrices alone, com-parison among the different experimental groups wasnot possible. However, for each experimental subgroupan untreated cell-seeded control was carried along,which allowed comparison of one specific treatmentwith its own cell-seeded control group.

Contraction was normalized to the number of cellsable to contribute to the reduction in diameter. Therewas no significant effect of FBS concentration (10%FBS versus low-serum formulation with ITS supple-ment) on matrix contraction (Student’s t test, P �0.12). All treatments, however, had a significant effecton cell-mediated contraction normalized to DNA (Table2 and Fig. 4). Because of the unremarkable, though


FIG. 1. Western blot results showing the bands appearing at the location of SMA (42 kDa). The FBS concentrations were 10% unlessotherwise noted. (a) Selected Western blot results showing the increase in SMA content of the cells from two patients induced by treatmentwith TGF-�1 and the decrease resulting from treatment with PDGF-BB. SMC, smooth muscle cell controls. (b) Films showing the Westernblot results after different treatments using cells isolated from a healthy 10-year-old subject. The medium comprising 2%FBS/ITS appearedto increase the SMA content of the cells. TGF-�1 increased the levels of SMA. (c) Western blot results showing the effects of increasingexposure to cytochalasin D and increasing the concentration of the agent (using cells from patient 9). (d) Graph showing the percentage


significant (Student’s t test, P � 0.05), difference inmatrix contraction between the TGF-�1-treated groupand its control (Fig. 4a), the experiment was repeatedwith 2 ng/ml TGF-�1 in a reduced-serum formulationof the medium using the cells from the 10-year-oldsubject. In this case an increase of 135% in matrixcontraction, compared to the untreated control group,was observed (Fig. 4b; Table 2).

Extraction of cytoplasmic protein from the cells inthe cell-seeded collagen–glycosaminoglycan scaffoldsdemonstrated that the cells had SMA levels compara-ble to those of cells grown in monolayer culture (Fig.1e). In addition, the cells in the matrices demonstratedthe same type of increase in SMA in response toTGF-�1 treatment as shown for cells in two-dimen-sional culture.

Linear regression analysis of the relationship be-tween the SMA content of the cells and the cell-medi-ated contraction normalized to DNA (Fig. 4c) yielded anotably high correlation with a coefficient of determi-nation, R2, of 0.88 (P � 0.01). The results for cytocha-lasin D were not included in the regression analysisbecause its prevention of contraction was to be ex-pected as a result of its complete disruption of the actincytoskeleton and not to any effect it may have had onSMA expression.

Histology and SMA Immunohistochemistry of Cell-Seeded Collagen–Glycosaminoglycan Matrices

Histological preparations demonstrated the distribu-tion of cells throughout the matrices with a general

preponderance of cells near the periphery. The de-crease in diameter of the matrices was reflected in thereduction of pore diameter evident in histological sec-tions. SMA-containing cells could be found throughoutthe matrices but generally appeared in greater per-centage near the surface.

DISCUSSION

This is the first report demonstrating SMA-enabledcontraction of human mesenchymal stem cells and theregulation of this behavior by selected growth factorsand a microfilament-modifying agent. The bone mar-row-derived stromal cells used in this study were qual-ified as mesenchymal stem cells on the basis of theirinducibility to chondrogenic, osteogenic, and adipo-genic phenotypes (data not presented) according towell-documented differentiation assays [3, 5, 37]. Thedemonstration of the SMA-enabled contractility of hu-man mesenchymal stem cells serves as a basis foraccepting this behavior as a phenotypic trait of connec-tive tissue cell progeny of stem cells, including chon-drocytes, fibrochondrocytes, osteoblasts, and musculo-skeletal fibroblasts [18].

A notable finding was the high correlation betweenthe SMA content of the stem cells and their contractil-ity as demonstrated by the cell-mediated contraction ofa preformed type I collagen–glycosaminoglycan analogof extracellular matrix, normalized to cell number. Al-most 90% of the variation of the cell-mediated contrac-tion could be explained by the SMA content of the cells.

TABLE 2

Summary of Findings

Treatment groupsAmount of SMA

(% SMC) naSignif.b

P values

Cell-mediatedcontraction(%/�g DNA) nc

%d

ChangeCMC/DNA

Signif.b

P values

Serum control 112 � 8 8 14.0 � 0.9 30ITS (2% FBS) 109 � 4 5 �0.05 13.2 � 0.2 6 �56 NS (0.1)Serum control

TGF-�1 (1ng/ml) 151 � 10 5 �0.001 18.3 � 0.5 6 �11 0.05PDGF-BB 70 � 4 4 �0.001 11.6 � 1.1 6 �50 0.012Cytochalasin D — — — 2.0 � 0.5 6 �88 0.0001

ITSTGF (2 ng/ml)e — — — 31.0 � 4 6 �135 0.01

a Number of patients.b Significance determined by Student’s t test using the respective controls for each treatment group.c Cells in each treatment group obtained from a different patient.d The percentage change was based on the respective untreated cell-seeded controls in serum.e Cells from the 10-year-old subject were used.

change in the SMA content of the cells relative to the respective serum control group. (e) Western blot showing the expression of SMA by cellsgrowing in monolayer and in the collagen–glycosaminoglycan matrix after 10 days (using cells from patient 3). 2D, two-dimensional culture;3D, three-dimensional culture.


This finding of the dependence of stem cell contractionon SMA content supports earlier work [38] concludingthat fibroblast contraction of collagen gels was depen-

dent on SMA expression. In that study electroinjectionof cells with an antibody to SMA reduced gel contrac-tion [38]. In more recent work [39], cells with increas-

FIG. 2. Light micrographs showing the SMA immunohistochemical preparations of the monolayer culture. (a) Cells grown in the controlmedium showing the well-spread polygonal morphology. SMA-labeled stress fibers can be seen in many of the cells. Scale bar, 20 �m. (b)TGF-�1-treated cells show the prominent SMA-containing stress fibers. Scale bar, 40 �m. (c) PDGF-BB-treated cells demonstrating anoticeably lower percentage of cells staining for SMA. Scale bar, 40 �m. (d) Cytochalasin D treatment (10 �M) resulted in a dramaticalteration of cell morphology with a loss of cytoplasmic volume. Cells processes appeared to remain attached to the substrate (see inset). Scalebar, 40 �m. (e) After the cytochalasin D (10 �M) was rinsed from the cells they recovered their normal morphology. Scale bar, 40 �m.


ing amounts of SMA were found to cause a greaterdegree of wrinkling of flexible silicone substrate onwhich they were grown.

One of the limitations of the model system used inthe current study is the determination of the number ofcells that are actually contributing to the dimensionalchange of the collagen–glycosaminoglycan scaffold.Several factors govern which cells are contributing tothe cell-mediated contraction of the matrix: cells thathave (a) SMA in the filamentous form and contain theother cytoplasmic proteins required for contraction and(b) integrin attachments to the collagen–glycosamin-oglycan scaffold or adhesion to other cells in the case ofconcerted contraction of a cell layer. Moreover, it islikely that the percentage of cells contributing to con-traction changes with time. In the current study weelected to normalize the cell-mediated contraction bythe DNA content of the matrices at the termination ofthe experiment, at 10 days. The supposition was that agreater percentage of the cells at that time period mayhave been contributing to the contracture than at the3-day period. Of the 2 million cells seeded into each ofthe matrices only a fraction attach to the scaffold andprobably contribute to the contraction measured after3 days. Many more (noncontracting) cells may havebeen loosely contained within the pores of the scaffold,eventually being lost from the collagen–glycosamin-oglycan sponge. We estimated that approximately200,000 cells were in the matrices (e.g., the TGF-�1-treated sample) at the 10-day termination of the exper-iment based on a nominal value of 10 pg DNA/cell. Atthe same time, prior work has suggested that contrac-tile connective tissue cells, like smooth muscle cells,can “maintain a state of long-term, steady contraction”(i.e., “tone”) [40]. The supposition, then, was that thecells responsible for the contraction recorded after 3days in culture and the additional dimensional changein the ensuing week would be captured in the DNAanalysis performed at the termination of the experi-ment, while spurious cells would have been lost by thattime. It is clear, however, that future work is needed toemploy methodology to determine which cells are ac-tually contributing to the dimensional change of thematrix.

The current study assayed cell contraction by eval-uating the dimensional change of a collagen–glyco-saminoglycan polymeric scaffold, serving as an analogof extracellular matrix, as has been done in severalprior studies [18]. For many years investigators havestudied the forces generated by cells by analyzing theirwrinkling of a silicone membrane [39, 41] and by thecell mediated contracture of collagen lattices (also re-ferred to as gels) in which they were cast [42, 43].While the wrinkling of the silicone membrane and di-mensional change of the gel have been correlated withSMA expression (as noted above), tractional as well as

FIG. 3. Graphs showing the change in the diameter of the cell-seeded collagen–glycosaminoglycan matrices as a percentage of theoriginal diameter of the scaffolds, prior to normalizing to cell num-ber. In each of the graphs, cells from the same patient served ascontrols. (a) The untreated mesenchymal stem cells contracted thematrices by 25% after the first day in culture. Treatment withcytochalasin D prevented contraction of the matrices. Mean � SEM.The error bars are contained entirely within the area of the symbolsat several time points. (b) PDGF-BB reduced the degree of contrac-tion when compared to its control. (c) TGF-�1 treatment resulted ina greater degree of contraction after the 10-day period of the exper-iment.


FIG. 4. (a) Graph showing the cell-mediated contraction normalized to cell number for the cell-seeded collagen–glycosaminoglycanscaffolds cultured in the control serum supplemented with 1 ng/ml TGF-�1 and the respective controls (mean � SEM). (b) Graph showingthe cell-mediated contraction normalized to cell number for the cell-seeded collagen–glycosaminoglycan scaffolds cultured in the reducedserum (ITS) supplemented with 2 ng/ml TGF-�1 and the respective controls (mean � SEM). (c) Linear regression analysis of thecell-mediated contraction of the collagen–glycosaminoglycan matrices, normalized to DNA content, as a function of the SMA content of thecells as assessed by densitometric analysis of Western blot films (n � 4–7). The coefficient of determination, R2, was 0.88 and the P valuefrom ANOVA was �0.01. The data for each of the groups were obtained using cells from different individuals (i.e., 4 patients). Mean � SEM.


SMA-enabled contractional forces may be contributingto these processes. The same may be said for the re-duction in diameter of the collagen–glycosaminoglycanmatrices in the current work. However, the preformedcollagen sponge-like scaffolds [32] used in this studydiffer from the collagen gels (lattices) cast with thecells in place [44–46] based on pore diameter (approx-imately 85 �m versus 1–10 �m) and the fiber thickness(10 �m versus from 50 to 500 nm). Moreover, it hasbeen proposed that the cell-mediated contracture ofcollagen–glycosaminoglycan matrices of the type usedin this investigation is more much likely due to thehigher forces produced by frank cell contraction thanmere cell motility because the scaffolds are stiffer thanthe collagen lattices [24, 33].

The effect of TGF-�1 in up-regulating the expressionof SMA in stem cells is consistent with prior findings ofsimilar effects of this growth factor on the SMA contentof cells from Dupuytren’s disease [47] and cells fromhuman gingiva [28], rotator cuff [27], and articularcartilage [21]. Furthermore, in a collagen gel contrac-tion assay, TGF-�1 has been shown to increase theforce of contraction generated by myofibroblasts ob-tained from Dupuytren’s tissue [48]. Also of impor-tance is a prior study [21] that directly measured theforce generated by chondrocytes in collagen–glycos-aminoglycan matrices comparable with those used inthe present work and demonstrated the increase inforce with treatment with the same dose of TGF-�1used in this investigation. These data indicate thatSMA expression and cell contractility need be added tothe list of TGF-�-stimulated cellular processes whichplay important roles in cellular physiology and pathol-ogy and wound healing, including mitogenesis [49],chemotaxis [50], and synthesis of collagen and fi-bronectin [51].

Of interest is the association of the characteristicformation of a spherical pellet in the cartilage differ-entiation assay with the presence of SMA-expressingand contracting cells [3, 5, 37]. In our own immunohis-tochemical evaluation (data not shown) we identifiedSMA-containing cells in the pellets containing type IIcollagen, with the SMA-expressing cells principally inthe outer region of the pellet. How does one resolve theissue that a critical ingredient of the chondrogenicmedium that induces stem cells to differentiate tochondrocytes is TGF-�1 at a dose of 10 ng/ml, whenTGF-�1 at a dose of 1 ng/ml has been shown to be apotent regulator of SMA expression and contraction? Itis likely that it is the SMA-enabled circumferentialcontraction of cells that causes the formation of thespherical structure. This raises questions about thelinks between cytoskeletal dynamics and the differen-tiation process. Is the contraction of the cells in thepellet a determinant of cartilage differentiation as aresult of (i) better containing newly synthesized matrix

molecules or (ii) a linked signaling effect on the biosyn-thetic activity of the cells? Further investigation will berequired to better understand the links between con-tractile actin expression and the differentiation pro-cess.

Another interesting finding in the present investiga-tion was the significant decrease in SMA and contrac-tility of stem cells caused by PDGF-BB treatment. Acomparable result was obtained previously in a studyemploying human gingival fibroblasts [28]. It may bethat the effect of PDGF in down-regulating SMA ex-pression is a homeostatic mechanism to counter theup-regulating effect of TGF-�. Alternatively, it may bethat the rapid cell proliferation stimulated by PDGFprecludes the expression of this particular actin iso-form. These findings warrant further study.

Cytochalasins, a family of metabolites excreted byvarious species of molds, paralyze many different kindsof vertebrate cell movement, inhibiting cell locomotion,phagocytosis, and cytokinesis. In the current investi-gation cytochalasin D blocked SMA-enabled contrac-tion of the collagen–glycosaminoglycan scaffolds by thestem cells. The stellate shape of cytochalasin D-treatedcells coincided with findings in the literature [52]. Thecytochalasins act by binding specifically to one end ofactin microfilaments, thereby preventing the additionof actin molecules to that end and by inducing depoly-merization of filamentous actin [53]. Cytochalasin Dhas been used in numerous studies to inhibit actinfilament polymerization [52, 54–56]. A prior studydemonstrated that treatment of gingival fibroblastswith cytochalasin D increased SMA expression [28],indicating that the effects of this agent on the contrac-tility of the stem cells were probably due principally toits inhibition of the polymerization of the actin micro-filaments.

The role of SMA-enabled contraction in stem cellswill require further investigation. Its role in fibroblastsin skin wound closure and Dupuytren’s contracture iswell documented [57]. It has been proposed that thehigher forces that can be generated by musculoskeletalconnective tissue cells as a result of expression of SMAmay facilitate the modeling of extracellular matrix toimpart tissue-specific architecture [18]. SMA-depen-dent contraction of the stromal cell in bone marrowmay enable the cell to tension the fine collagen networkthat serves as a framework for the cellular suspensioncomprising the marrow. The current findings of theeffects of selected growth factors on SMA expressionand stem cell contraction suggest that these processesmay be closely controlled during tissue developmentand regeneration. Such controls may also be importantfor tissue repair procedures using cell-based therapy ortissue engineering approaches.


This work was supported in part by the Robert Bosch Foundation(BK), the Brigham Orthopaedic Foundation, and the RehabilitationResearch and Development Service of the U.S. Department of Vet-erans Affairs, Veterans Health Administration.

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Received January 7, 2002Revised version received April 26, 2002Published online June 20, 2002


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Regulation of smoot muscle actin expression