In vitro differentiation of chick embryo bone marrow stromal cells into cartilaginous and bone-like tissues

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Bone marrow stromal cells, progenitor cells involved in repair of bone and cartilage, can potentially provide a source for autologous skeletal tissue engineering. We investigated which factors were required to induce in vitro differentiation of avian
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  Journal of Orthopedic Research 16:181-189 Thc Journal olBone and Joint Surgery, Inc Q 1998 Orthopaedic Research Society In Vim 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 zyxwvutsrq f Health Science5 und Technology, Massarhusetts Instrliile of TechnologL; Crinzbridge, Wassachusettg U.S z   arid ?National Cancer Resew h InJtitiirr/Advrmcedn~e~ iotechnology Center, Genoa, Italy zy 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 in 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@ 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 zyx n 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 zyxwvu 1: Cell seeding in spinner flask (3 days) zyxwv IG. 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 : Construct culture in mixed Petri dish (25 days) Phase I: Cell expansion in monolayer zyxwvutsrq = 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 of a hemocytomctcr, plated in zyxwvu 00 mm Petri dishes at 2 X lo6 cells per dish in 10 ml of medium, and cultured in a humidified 37'C/.5% C02 ncubator (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 of 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% C02 ncubator with the side-arm caps loosencd to permit gas cxchange, and magnetically stirred at 75 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 irst-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 zyxwvutsrq   998  IN VITRO DIFFEKENlIATION OF STROMAL CEI*I,S 183 FIG. 2. Histological appearance of samples cultured for 4 weeks. Chick bone marrow strotnal cells (5 zyx   loh) were seeded in pellets A and B) or onto polyglycolic acid scaffolds zyxwvuts 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 and 100 pm for B, D, F, H, and J. Arrows indicate undegraded polyglycolic acid fibers. Assays Cell Proliferution mil 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% C02, he 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 zyxw   was the number of cells after time T of expo- nential growth (8). Alkaline phosphatase activity was assessed from the rate of conversio~~ f y-nitrophenyl phosphate to p-nitrophenol zy 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 zyx 6, No. 2 998  184 I. MARTIN ET AL. A B C zyxwvutsrqpo 40 zyxwvutsrq 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 zyxwvut YO ww). C Glycos- aminoglycan (GAG) YO ww) and total collagen (COL) zyxwvu 5 ww). Data represent the average zyxwvuts   SD of four independent measure- ments. Dashed lines indicate the composition of adult chick cpiph- yseal cartilage (43.45). Histology and Immidnohistochenlistry of he 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 or sulfatcd glycosaminoglycan, and with alizarin red for niineralizcd CXtrdcellU- zyxwvut   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 of 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 0.5 to 26.9 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 zyxwvutsrqponm es Vd 16, No zyxwvutsr   998  IN VITRO DIFFEKC'N7'1ATIO:V OF STROMAL CELLS 185 FIG. zyxwvutsrqp   Immun(~hist~ch~mi~tr~ f constructs seedrd with 5 zyxwvut   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 zyxwvut C). n control sections, the primary antibody was replaced with nonspecific inousehbbit inmunoglobulins D) or with diluted goat serum H). Scale bar zy O 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 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 srcinal 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 z   0.45%) (Fig. 2G and H). After 4 weeks of cultivation. undegraded polygly- colic acid represented less than 5% of the wet weight I Orthop Re,, Vul. 16. No 2, 1998
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