Journal of Orthopedic Research 16:181-189 Thc Journal olBone and Joint Surgery, Inc Q 1998 Orthopaedic Research Society

In V i m Differentiation of Chick Embryo Bone Marrow Stromal Cells into Cartilaginous and Bone-like Tissues

"tIvan Martin, "Robert F. Padera, "Gordana Vunjak-Novakovic, and *Lisa E. Freed

*Division of Health Science5 und Technology, Massarhusetts Instrliile of TechnologL; Crinzbridge, iWassachusettg U.S A, arid ?National Cancer Resew h InJtitiirr/Advrmcedn~e~ Biotechnology Center, Genoa, Italy

Summary: Bone marrow stromal cells. progenitor cells involved in repair of bone and cartilage. can poten- tially provide a sourcc for autologous skeletal tissue engineering. We investigated which factors were required to induce in vitro differentiation of avian bone marrow stromal cells into three-dimensional cartilaginous and bone-like tissues. Bone marrow stromal cells from embryonic chicks were expanded i n monolayers, seeded onto biodegradable polyglycolic acid scaffolds, and cultured for 4 weeks in orbitally mixed Petri dishes. Cell-polymer constructs developed an organized extracellular matrix containing glycosaminoglycans and col- lagen, whereas control bone marrow stromal cell pellet cultures were smaller and consisted predominantly of fibrous tissue. Bone marrow stromal cells expanded with fibroblast growth factor-2 and seeded onto polymer rcaffolds formed highly homogeneous three-dimensional tissues that contained cartilage-specific molecular markers and had biochemical compositions comparable with avian epiphyseal cartilage. When cell-polymer constructs were cultured in the presence of beta-glycerophosphale and dcxamethasone. the extracellular matrix mineralized and bone-specific proteins were expressed. Our work shows that cell expansion in the presence of fibroblast growth factor-2 and cultivation on a three-dimensional polymer scaffold allows differ- entiation of chick bone marrow stromal cells into three-dimensional cartilaginous tissues. In the in vitro system studied, the same population could be selectively induced to regenerate either cartilaginous or bone- like tissue.

Bone marrow stroma contains a heterogeneous population of precursor cells that, depending on the local microenvironment, can differentiate into several mesenchymal lineages: osteoblasts, chondrocytes. ad- ipocytes, or myocytes (2,4,10,18,40). The potential use of this population (here called bone marrow stromal cells) has recently been explored for bone and carti- lage repair (9,21,29,38,49). The advantages of use of progenitor bone marrow stromal cells rather than dif- ferentiated cells (e.g., chondrocytes or osteoblasts) are that (a) a lower initial number of cells is needed, because human bone marrow stromal cells proliferate quickly in monolayers and can be serially passaged without adverse effects (7): (b) the number of human bone marrow stromal cells per total nucleated cells in marrow decreases as a function of age, but their mi- totic potential and biosynthetic activity are compara- ble in newborns and the elderly (26); (c) small (5-10 ml) marrow aspirates are easily harvested using local anesthesia; and (d) composite tissues consisting of car-

Received September 15,1997; accepted January 6,1998. Address correspondence and reprint requests to L. E. Freed

at Massachusetts Institute of 'l'echnology, E25-342,77 Massachu- setts Avenue, Camhridge, MA 02139, U.S.A. E-mail: Ifreed@ init.edu

tilagc and bone could be engineered starting from only one cell type, allowing the repair of cartilage defects involving both the articular surface and the underlying bone (49).

Several studies have shown that bone marrow stro- ma1 cells obtained Crom different species, when im- planted in vivo, are able to reconstitute bone and, under certain conditions, cartilage (2,18,23,25,34,35). Mineralized bone-like tissue was also obtained in vitro when mammalian bone marrow stromal cells were cultured in the presence of dexamcthasone, ascor- bic acid, and beta-glycerophosphate (28.33-3539). Al- though avian ( 5 ) and mammalian (9.42) bone marrow stromal cells were previously reported to express a chondrocytic phenotype in culture: to thc best of our knowledge, they have not yet been successfully used to engineer large three-dimensional cartilaginous tis- sue in vitro.

In the present work, we investigated which factors were required to achieve a full and selective in vitro differentiation of chick embryonic bonc marrow stro- ma1 cells into either cartilaginous or bone-like tissue. Our hypothesis was that a three-dimensional scaffold could provide the cells with an appropriate microen- vironment for tissue organization and that the pres- ence ol spccific biochemical factors could regulate cell

182 I. MARTIN E?' AL.

Phase 11: Cell seeding in spinner flask (3 days)

FIG. 1. Model system. Phase I: expansion of bone marrow stromal cells in monolayers. Phasc I1 seeding of bone marrow stromal cells onto fibrous polyglycolic acid scaffolds in spinner flasks. Phase 111: cultivation of three-dimensional cell-polymer constructs in mixed Petri dishes. In some groups, fibroblast growth factor-2 was addcd to the culture medium during phases I and 11. In other groups, dexamethasone and bcta-glycerophosphate were added to the culture medium during phase ITI.

Phase 111: Construct culture in mixed Petri dish (25 days)

Phase I: Cell expansion in monolayer (= 2 weeks)

expansion in monolayers and their subsequent differ- entiation in three-dimensional cultures.

MATERIALS AND METHODS Materials

Fibroblast growth factor-2 (FGF-2) was from R and D Systems (Minneapolis. MN. LJ.S.A.). Dexamethasone (water soluble), beta- glycerophosphate, thiazolyl blue (MTT). p-nitrophenyl phosphate, and alkaline bufCer solution were from Sigma (St. Louis, MO, U.S.A.). The alkaline phosphatase LSAB 2 kit was from DAKO (Carpinteria, CA, U.S.A.). Anti-collagen type-I1 antibody (II- I16B3) was from the Developmental Studies Hyhridoma Bank (Baltimore, MD, U.S.A.). Anti-collagen type-X antibody was a kind gift from T. Linsenmayer, Tufts Univcrsity (Boston, MA, U.S.A.). Anti-Ch21 antibody was kindly provided by R. Cancedda. Advanced Biotechnology Center (Genos, Italy). Anti-bone sialo- protein and osteopontin antibodies wcrc kind gifts from L. Ger- stenfeld, Children's Hospital (Boston, MA, U.S.A.). All other culture materials and chemical reagents were from previously specified sources (1 7).

Cultivation of Chick Bone Marrow Stromd Cells Phase I: Cell Isolation and Expansion Bone marrow cultures were established with use of cells

isolated from 16-day-old embryonic chicks (n = 12 per primary culture) as previously dcscribed (5). Tibiae and femora were asep- tically harvested, and the adhercnt soft tissue and cartilaginous ends of the bones were removed. Marrow contents were flushed from the bones with Dulbecco's modificd Eaglc medium. with use of a 10 nil syringe fitted with a 25-gaugc needle (1 ml of Dulbecco's modified Eagle medium per bone). Single-cell suspensions were made by repeatedly passing the marrow through the needle. Cell suspensions were then centrifuged (10 minutes at 1,000 rpm) and resuspended in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, 100 Uiml penicillin. and 100 pg/ml streptomycin (PIS). White blood cells were counted with use o f a hemocytomctcr, plated in 100 mm Petri dishes at 2 X lo6 cells per dish in 10 ml of medium, and cultured in a humidified 37'C/.5% C 0 2 incubator (Fig. 1, phase I). Each marrow sample was cultured cither with or without 1 ng/ml FGF-2. Bone marrow stromal cells were selected on the basis of their ability to adhere to thc Petri dish; nonadhcrent hematopoi- etic cells were removed with culture medium during refeeding (25). The medium was changed after 3 days, at which point colonies o f bone marrow stromal cells were evident, and twice per week

thereafter. When the Petri dishes became nearly confluent (ap- proximately 10 days after the primary culture was established), the cells were detached with 0.25% trypsinil mM EDTA and replated in 100 mm dishes at 3 X 10j cells per dish. After 1 more week, when the dishes again became confluent, first-passage cells were trypsinized and either seeded onto polymer scaffolds or cultured as pellets as will be described.

Phase II: Cell Seeding onto Polymer Scaffolds Polyglycolic acid scaffolds were made as previously dcscribed

(16). In brief, polyglycolic acid was extruded into 13 pm diamcter fibers. processed to form a 96% porous mesh, and die-punchcd into discs (5 mm diameter X 2 mm thick). Prior to ccll seeding. scaffolds were prewetted in culture medium, threaded onto needles (two scaffolds per needle; two needles per ilask). positioned with 3 mm segments of silicone tubing, and fixed to a stopper in the mouth of a spinner flask (1967-0025; Bellco, Vineland, N.J, U.S.A.) (48). The flasks were filled with 30 ml of medium, placed in a humidified 37"C/S% C 0 2 incubator with the side-arm caps loosencd to permit gas cxchange, and magnetically stirred at 7.5 rpm. After 8-12 hours, the flasks were inoculated with bone marrow stromal cells using 1,3. or 5 X lo6 cells per scaffold (Fig. 1, phase 11). FGF-2 (1 ngiml) was addcd to the tlasks containing cells previously cxpanded with FGF-2.

Phase III: Three-Dimensional Construct Cultivation After 3 days, when cell attachment to the scaffolds was coni-

plctc (as inferred from the absence of cells in the culture medium [48]), cell-polymer constructs were transferred to 3.5 mm diameter dishes coated with a thin layer of 1% agarose and were placed on an orbital shakcr (75 rpm) for further cultivation (Fig. 1, phase 111). Unless otherwise stated, each construct was cultured in 5 ml of Uulbccco's modified Eagle medium conlaining 10% fctal bovine serum, 0.1 mM nonessential amino acids, 50 pgIml ascorbic acid, 0.4 mM proline, and PIS. In this phase, FGF-2 was not added to the culture medium. In some groups, the medium was further supplemented with 7 mM beta-glyccrophosphate and 10" M dexa- methasone to induce mineralization and osteogenic differenlia- tion (28,33,34,39). The medium was completely replaced twice per week.

Control pellet culturcs of bone marrow stromal cells were es- tablished as follows. A suspension of 5 X 10h first-passage cells in 5 ml of medium was placcd in a 15 nil Falcon tube, centrifuged (1,000 rpm lor 10 minutes), and cultured under conditions (e.g.. medium compositions and exchangc rates) identical to those used for constructs cultured in mixed dishes.

J Orlhop Rn, Vol 16, No 2, 1998

IN VITRO DIFFEKENl IATION OF STROMAL CEI*I,S 183

FIG. 2. Histological appearance of samples cultured for 4 weeks. Chick bone marrow strotnal cells (5 X loh) were seeded in pellets (A and B) or onto polyglycolic acid scaffolds (C-J). after expansion without (A-D) or with (E-J) fibroblast growth factor-2. In oiic group (I and J), the medium used during three-dimensional cultivation also included dexamethasone and beta-glycerophosphate. Cross sections were stained with safranin 0 (A-F) or alizarin red (G-J). Scale bar = 1 mm for A, C, E, G, and 1 and 100 pm for B, D, F, H, and J . Arrows indicate undegraded polyglycolic acid fibers.

Assays Cell Proliferution mi l Alkaline Phosphutuse Activity in Monoluyers After expansion of bone marrow stromal cells in monolayers

with or without FGF-2, first-passagc cells were plated in 10 nim wells (7 X lo3 ccllsiwcll). Cell number was evaluated at timed intervals in triplicate wells by MTT staining (11). In brief. the culturc medium in each well was replaced with 0.5 ml of rnediurri without fetal bovine serum and phenol red, including 25 ul of 5 mg/ml MTT. After 3 hours of incubation at 37”C/5% C 0 2 , the medium was replaced with 1 ml of 0.04 N HC1 in absolute iso- propanol. The amount of convertcd dye was measured spectropho- tometrically at 570 nm with background subtraction at 670 nm. Conversion to cell numbcr was based on a standard curve derived by assaying known numbers of plated cells. The doubling time of the cell population during the exponential growth phase was cal- culated as the slope of’1versus logz (N/No), where No was the initial

cell number and N was the number of cells after time T of expo- nential growth (8).

Alkaline phosphatase activity was assessed from the rate of conversio~~ of y-nitrophenyl phosphate to p-nitrophenol ( 6 ) . First- passage hone marrow stromal cells were plated in triplicatc 10 mm wells (2.5 X lo4 cellsiwell) and allowed to adhere for 20 hours. The cells were thcn washcd with phosphate buffered saline and incu- bated at 37°C for 10 minutes in 50 p1 of 0.01% sodium dodecyl sulphate and for 15 more minutes after the addition of 250 p1 of p-nitrophenyl phosphate and 250 pl of alkaline buffer. The coil- tents of each well were then transferred into a vial containing 5 ml of 0.05 N NaOH, and the absorbance was read at 410 nm. Alkaline phosphatasc activity was normalized by cell number, nieasured with use of the MTT assay, and for each experiment alkaline phos- phatase activity of cells expanded with FGF-2 was expressed as a percentage of the activity measured in parallel cultures expanded without FGF-2.

J Orthop Rcs, L’ol 16, No. 2, 1998

184 I. MARTIN ET A L .

A

B

C

40

30

20

10

0

DNA (%ww)

GAG (%ww) COL (%ww)

FIG. 3. Size and composition of cell pellets (white bars) and cell- polyglycolic acid constructs based on bone marrow stromal cells expanded without (gray bars) or with (black bars) fibroblast growth Cactor-2 after 4 weeks of culture. A: Wet wcight (ww) and dry to wet weight ratio (dwiwwj. B: DNA (YO ww). C Glycos- aminoglycan (GAG) (YO ww) and total collagen (COL) (5% ww). Data represent the average 2 SD of four independent measure- ments. Dashed lines indicate the composition of adult chick cpiph- yseal cartilage (43.45).

Histology and Immidnohistochenlistry o f the Constructs Samples for histological analysis were cross sectioned, exten-

sively rinsed in phosphate buffercd saline, fixed in 4% neutral buffered lormalin for 24 hours, dehydrated, cmbedded in paraffin, and scctioned (5 pm thick). The sections were staincd with hema- toxylin and cosin for general eva1uation.w-ith safranin 0 for sulfatcd glycosaminoglycan, and with alizarin red for niineralizcd CXtrdcellU- '

lar matrix (41). Quantification of mineralized matrix was performed by computer-assisted analysis of digitized imagcs of the sections

stained with alizarin red as follows. Black and white images were acquired by a solid-stare charge-coupled dcvice camera (Hitachi, Tokyo, Japan) mounted on an inverted microscope (Diaphot; Ni- kon. Tokyo, Japan). digtized by a LG-3 frame grabber (Scion Cor- poration. Frederick, MD, U.S.A.) into a Power Macintosh 7100, and analyzed with the NIH-Image public domain program (version 1.60; National Institutes of Health. Bethesda, MD, lJ.S.A.). Thc amount of mineralized area was calculated as the percentage of the total cross-sectional conslrucl area stained red. with a minimum intensity that could be distinguished by our system from the background level of the nonminerali,xxl controls. Beyond this threshold, the intensity level of the stain was not taken into account (35.3')).

The sections were inimunohistochemically assessed with anti- bodies against chick type-I1 (32) and type-X (44) collagens, Ch21 (a specific marker for chick hypertrophic chondrocytes 1121). bone sialoprotcin (the antibody was against the intact molecule and purificd according to a previously described method [20]), and osteopontin (1Y) as follows. The sections were deparaffinized, pre- treated for 30 minutes at 37°C with teslicular hyaluronidase (1 mg/ml). rinsed with phosphate buffered saline. and incubated for 15 minutes at 25°C in normal goat scrum (diluted 1:10 in phos- phatc buffercd salinc). Tlic scctions were thcn incubated with the appropriale primary antibody for 1 hour at 25°C: and stained with use of an alkaline phosphatase kit.

Biochemical Analyses o,f the Conslriicts Samples for biochemical analyses were fro7en, lyophili7ed, and

papain-digested as previously described (IS). DNA content was measured spectrofluorometrically with Hoechst dye; purificd calf thymus DNA was uscd as a standard (30). The amount of sulfated glycosaminoglycan was dctcrmined spectrophotornetrically with use of dimcthylmethylcnc blue dye and bovine chondroitin sulfate as a standard (13). Total collagen content was determined spec- trophotometrically from the hydroxyproline content after acid hydrolysis and reaction with p-dimelhylaminobenzaldehyde and chloramine-T (50) with use of a hydroxyproline to collagen ratio of 0.1 (27).

Osteocalcin Assay of Medium Samples A radioiinmunoassay using an anti-chicken osteocalcin anli-

body was performed on aliquots of culturc medium as described previously (24). Osteocalcin release was calculatcd as the average amount released per construct per day over the 4-week cultivation period.

Statistical Analysis Statistical significance (p < 0.02) was asscsscd by analysis of

variance (ANOVA) ( a = 0.05) in conjunction with Tukcy's studcnt- ized range test.

RESULTS Cell Expansion

The addition o f FGF-2 to the culture medium dur- ing phase I increased the proliferation rate of embry- onal chick bone marrow stromal cells, such that the doubling time during exponential growth decreased from 33.3 2 0.5 to 26.9 2 0.3 hours (n = 3 data sets). The alkaline phosphatase activity of bone marrow stromal cells expanded without FGF-2 averaged 3.8 -t 2.7 IJ/105 cells (n = 9 measurements from three inde- pendent studies). Alkaline phosphatase activity in cul- tures of bone marrow stromal cells expanded with FGF-2 was 32.8 -+ 8.0% that measurcd in cultures cxpanded without FGF-2.

J Orthop Kes V d 16, No 2, I998

IN VITRO DIFFEKC'N7'1ATIO:V OF STROMAL CELLS 185

FIG. 4. I m m u n ( ~ h i s t ~ c h ~ m i ~ t r ~ of constructs seedrd with 5 X 10" chick bone marrow stronial cells and cultured for 4 weeks without (A-D) and w-ith (E-H) the addition of beta-glycerophosphatc and dcxamcthasone. Scctionq wcrc stained with antibodies against type-IT (A) and type-X collagen (B and E), Ch21 (C). bone sialoprotein (F), and osleopontin ( C ) . In control sections, the primary antibody was replaced with nonspecific inousehbbit inmunoglobulins (D) or with diluted goat serum (H). Scale bar - SO pni. Arrows indicate undcgradcd polymer fibers.

Cartilaginous Tissue Formation Cultivation of 5 X 106 bone marrow stromal cells in

pellets for 4 weeks resulted in constructs with two distinct regions: a lower region containing glycos- aminoglycans, as assessed by safranin 0 stain: and an upper region consisting of undifferentiated fibrous tis- sue (Fig. 2A and B). When the same number of bone mai-row stromal cells was cultivated on polyglycolic acid scaffolds for 4 weeks, the constructs were larger and consisted of a single tissue phasc containing gly- cosaminoglycans (Fig. 2C and D). In the same group, a significant percentage oC the total cross-sectional

area (9.75 ? 0.92%) was mineralized in the form of focal areas at the construct surface. as assessed with use of alizarin red stain (data not shown). Cell- polymer constructs based on bone marrow stromal cclls expanded in thc presencc ol FGF-2 maintained the dimensions of the original scaffold (Fig. 2E). con- tained a homogeneous extracellular matrix rich in glycosaminoglycans (Fig. 2E and F), and displayed a negligible percentage of mineralized area (0.55 2 0.45%) (Fig. 2G and H).

After 4 weeks of cultivation. undegraded polygly- colic acid represented less than 5% of the wet weight

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I86 1. MARTIN ET A L .

TABLE 1. Size und coniposition of #-week constructsn

Constructs

Medium supplement (during two-dimcnsional expansion)

No. of cells seeded (per polyglycolic acid scaffold, x l U h )

Medium supplement (during three-dimensional cultivation)

Wet weight (mg)

Dry to wet weight ratio (%)

Glycosaniinoglycan (YO wet weight)

Collagen (YO wet weight)

DNA (YO wet weight)

Polyglycolic acid (% wet weighty

Mineralized area (YO total)

Osteocalcin release (ngiday)"

FGF-2

1 ~

36.1 t 2.7

20.0 t 0 7

5.05 ? 0.26"

4.49 2 0.05b

0.62 2 0.02

3.89 % 0.29

1.6 -+ 1.3

NA

FGF-2

3 -

40.7 -t 3.3

20.5 i 0.7

1.96 5 0.13*

4.67 i 0.31h

0.61 -t 0.05

3.46 5 0.29

0.7 2 0.8

NA

FGF-2

5 -

43.1 f 2.0

19.8 ? 1.3

4.69 t 0.22h

4.52 -t 0.24h

0.59 ? 0.01

3.26 2 0.14

0.6 It 0.5

10.7 +- 5.6

FGF-2

5

bGP. dex

43.8 2 10.5

24.5 +- 5.3

2.90 +- 1.39

1.85 5 0.3s

0.42 2 0.13

3.30 t- 0.79

35.6 2 5.@ 101.6 f 36.5"

-

5

bGP. dex

31.7 t 11.2

27.6 ? 4.6

2.64 2 0.81

2.66 t 0.82

0.50 t 0.09

3.72 ? 1.68

33.9 I l S h

NA

FGF-2 = Cibroblast growth factor-2, bGP = beta-glycerophosphate, dex = dexamethasone. and NA = not assessed. "Data represent average 2 SD of three or four independent measuremcnts.

'Calculated on the basis of initial polymer mass of 2.8 nig (dry weight) and previously determined polyglycolic acid degradation

dBackground values of 2.1 t (J.2 @day were mcasured in control culture medium.

Statistically significant differences.

kinetics (16).

of the construct (Table 1). Cell-polyglycolic acid con- structs had higher wet weights. lower DNA contents, and higher glycosaminoglycan and collagcn contcnts than did cell pellet cultures (Fig. 3). The presence of FGF-2 during expansion of bone marrow stromal cells lurther increased wet weight and glycosaminoglycan content of the construct but did not aifect DNA or collagen content (Fig. 3). No differences in construct biochemistry ('Table 1) or histology (data not shown) were observcd between polyglycolic acid scaffolds seeded with 1, 3, or 5 million hone marrow ~tromal cells expanded in the presence of FGF-2. Moreover, 4-week conslructs based on FGF-2 expanded cells and polyglycolic acid scaffolds (Fig. 3 and Table 1) had dry to wet weight ratios and DNA, glycosaminoglycan, and collagen fractions (% wet weight) comparable with those previously reported for epiphyseal carti- lagc of the adult chick (43.45).

The expression of cartilage-specific molecular markers was demonstrated immunohistochemically as lollows. Type-I1 collagen was abundant and homo- geneously distributed in the extracellular matrix (Fig. 4A). whereas type-X collagen was mostly localizd around cells (Fig. 4B). Ch21 was also clearly expressed (Fig. 4C). Control sections in which the primary anti- body was replaced with nonspecific immunoglobulins or serum showed that endogenous alkaline phospha- tase activity was negligible and confirmed the speci- ficity of the immunostaining (Fig. 4D and H).

Bone-like Tissue Formation

The addition of beta-glycerophosphate and dexa- methasone to the culture medium during three-

dimensional cultivations resulted in significantly dif- ferent construct composition; under these conditions, tissue charactcristics shifted from cartilaginous to bone-like, glycosaminoglycan and collagen fractions decreased (Table l), and the matrix mineralized at the construct periphery. The mineralized area increased with cultivation time (Fig. 5) , reaching a thickness of 0.3-0.5 mm at 4 weeks: this corresponded to more than

40 1 T

- FGF-2

+FGF-2

1

2 weeks 4 weeks

Cultivation time FIG. 5. Mineralized area (expressed as YO of lolal cross-sectional area) of polyglycolic acid scaffolds seeded with 5 X lo6 chick bone marrow stromal cells and cultured in medium supplemented with beta-glycerophosphate and dexamethasone. Cells were expanded without or with fibroblast growth factor-2 (FGF-2) (gray and black bars. respectively).

IN VITRO DIFFERENTlATION OF STROMAL CELLS IN7

one-third of the cross-sectional area of the total con- struct (Fig. 21). The presence of FGF-2 during expan- sion of bone marrow stromal cells did not result in any apparent difference in construct histology (data not shown), mineralized area (Fig. S), or biochemical com- position (Table 1).

In constructs cultured in the presence of beta- glycerophosphate and dexamethasone, osteocalcin re- lease into the medium was significantly higher than in controls (Table l), type-X collagen was expressed only at the construct center (Fig. 4E), and bone sialoprotein and osteopontin were expressed only at the construct periphery (Fig. 4F and G). These patterns were consis- tent with those obtained with use of alizarin red stain (Fig. 21).

DISCUSSION This work represents, to the best of our knowledge,

the first demonstration that embryonal chick bone marrow stromal cells can be differentiated in vztro on three-dimensional polymer scaffolds to form cartilag- inous tissues of significant size (5 mm diameter X 2 mm thick discs). Two factors were identified as essen- tial in engineering homogeneous tissue constructs with an extracellular matrix composition cornparablc with natural avian cartilage: expansion of bone mar- row strornal cells in monolayers in the presence of FGF-2, and three-dimensional cultivation on a poly- mer scafold. When these criteria were met, the char- acteristics ol' the constructs did not depend on thc initial number of cells secdcd; this suggests that in an appropriate in v i m environment, chick bone marrow stromal cells can self-regulate their growth to obtain physiological tissue cellularities and compositions.

Previous studies showed that chick bone marrow stromal cells include chondrocytic colonies ( 5 ) and that mammalian bonc marrow stromal cells can be induced to express a chondrogenic phenotype in Pc- tri dishes if cultured at initial cell densities greater than 1 Oh cells/cm2 (9.42). Recent preliminary works suggested that chondrogenic differcntiation of bone marrow stromal cells can be achieved in a three- dimensional environment, such as in small pellet cul- tures (1-2 X 10' cells/pellet) (3 ) , and in fibrin gels (14). In the present work, we studied chondrogenic differ- entiation of chick hone marrow stromal cells cultured on three-dimensional biodegradable polymer scaf- folds and demonstrated that it was superior to that observed in large pellet cultures (1-5 X lo6 cells/pel- let), as ascertained by histological and biochemical evaluation. The three-dimensional structure of the scaffold appeared to prevent bone marrow stromal cells from becoming too densely packed but allowed cell-to-cell contacts, which have been suggested to ac- tivate differentiation of progenitor mesenchymal cells into chondrocytes (47). Our results suggest that the

three-dimensional polymer scaffold provided a per- missive environment for cell seeding, extracellular matrix production, and cartilaginous tissue formation.

FGF-2 is a potent modulator of the growth and differentiation of marrow stromal cells (22,33,35,39). Increased cell proliferation and decreased alkaline phosphatase activity suggest that chick bone marrow stromal cells expanded in the presence of FGF-2 are maintained in a more stem cell-like state, which is consistent with previous reports (33,35). This hypoth- esis might explain why bone marrow stromal cells that were expanded with FGF-2 were able to regenerate cartilaginous tissue with biochemical compositions closer to natural cartilage, as compared with bone marrow stromal cells expanded without FGF-2 and still retained their osteogenic potential. An alternative explanation of this result is that chick bone marrow stromal cells. similar to chick periosteum-derived mes- enchymal cells (37), include subpopulations of osteo- progenitor and chondroprogenitor cells: osteogenic cells would proliferate relatively faster in the absence of FGF-2, whereas FGF-2 would allow expansion of both subpopulations. As a third possibility. one might speculate that cell division. enhanced by FGF-2, is an important preliminary event for differentiation of bonc marrow stromal cells, as previously proposed for other mesenchymal precursor cell systems (33,51).

Calcification of the extracellular matrix was negli- gible in the carti1aginou.l constructs seeded with bone marrow stromal cells expanded with FGF-2. In con- trast, the addition of beta-glycerophosphate and dcx- amethasone to the culture medium, in concentrations previously shown to stimulate ostcogenic differentia- tion of bone marrow stromal cells (28,33,34,39), in- duced expression of specific molecular bone markcrs and extensive mineralization at the construct periph- ery, where expression of type-X collagen could not be detected. This demonstrated that bone marrow stro- ma1 cells expanded with FGF-2 could subsequently differentiate into either cartilaginous or bone-like tis- sue, depending on the biochemical mediators added to the culture medium during three-dimensional culti- vation. The finding that mineralization occurred only at the construct periphery is consistent with previous studies of chick mesenchymal precursors from perios- teum (36) and might be attributed to environmental factors as follows. Relatively high rates of nutrient and gas transfer at the interface of the medium and the construct would promote mineralization at the con- struct surfaces and result in increasingly limited mass transfer into the inner tissue phase.

Our culture system displays characteristics that re- semble in vzvo chondrocyte differentiation in the epi- physeal plates of long bones; therefore, it offers an in vitro three-dimensional model to study endochondral ossification starting from precursor cells. Further stud-

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188 I. MARTIN ET A / .

ies are needcd to determine whether each bone mar- 1mv stromal cell in the construct is able LO differentiate into either a chondrocyte or an osteoblast, or if there are different cell subpopulations. each responsible for a specific differentiation pattern in response to cnvi- ronmental conditions.

One of the requircmcnts lor most tissue-enginecring applications is the availability of a cell population that can be quickly expanded and then used to produce a tissue equivalent with clinically useful dimensions (31). In this respect, the exlension of the present work to mammalian bone marrow stromal cells would permit the engineering of autologous cartilaginous and bone- like tissues starting from small bone marrow aspirates. To achieve this goal, in vitro cultivation conditions might need to be optimized by varying scaffold struc- ture and degradation time (16), using bioreactors to enhance gas and nutrient transport (17), and intro- ducing mechanical or electromagnetic stimulation, or both, during cultivation (1,46). Different molecular cues: such as exogenous growth factors and hormones, might also be necessary to modulate cell commitment (3.14). Studies are underway in our laboratory to in- vestigate these variables with the goal of improving skeletal tissue engineering starting from bone marrow stromal cells.

Acknowledgment: We thank K. Skazina and P. Hauschka. Chil- dren's Hospital, Boston. MA. for essing osteocalcin in medium. We are indebted to K. Canccdda and P. Hauschka for helpful discussions. Funding of this hark was provided by the National Aeronautics and Space Administration (Grant NAG9-836).

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