TISSUE ENGINEERINGVolume 1, Number 3, 1995Mary Ann Liebert, Inc.

TGF-/3 Promotes the Growth of Bovine Chondrocytes inMonolayer Culture and the Formation of Cartilage Tissue

on Three-Dimensional Scaffolds

MICHAEL P. ZIMBER, B.S.,1 BETTY TONG, M.S.,2 NOUSHIN DUNKELMAN, B.S.,1

REBECCA PAVELEC, B.S.,1 DAN GRANDE, Ph.D.,3 LIGOU NEW, Ph.D.,1

and A.F. PURCHIO, Ph.D.1

ABSTRACT

We have investigated the ability of transforming growth factor p (TGF-/5) to promote thegrowth and differentiation of chondrocytes in monolayer and on three-dimensional scaffolds.Treatment of chondrocytes with TGF-/J and ascorbate individually stimulated the prolifer-ation of bovine articular chondrocytes about 2-fold when cells were grown in monolayer cul-ture: the combination of TGF-/J and ascorbate resulted in a 3-fold increase in cell numberover a 72-h period. Peak stimulation with TGF-jS occurred at about 1.0 ng/ml: bFGF wasslightly inhibitory in these assays. TGF-/3 led to an increase in glycosaminoglycan synthesisas detected by Western blotting using anti-chondroitin sulfate antibodies. No significantchange in collagen type II mRNA or protein was observed. When cells were grown on three-dimensional scaffolds composed of polyglyocolic acid, TGF-/J treatment led to an increasein the size of the cartilage-like constructs produced. This was accompanied by increases incollagen and glycosaminoglycan deposition; immunohistochemical staining showed that thepredominant collagen was type II. These results indicate that TGF-/3 is capable of increas-ing the proliferation rate of chondrocytes in monolayer as well as increasing cartilage pro-duction on three-dimensional scaffolds and may find utility in the in vitro engineering of car-tilage tissue.

INTRODUCTION

TRANSFORMING GROWTH FACTOR j8 (TGF-/3) refers to a family of homodimeric proteins that regulates thegrowth, differentiation, and function of multiple cell types.1"3 Three isotypes are found in mammalian

cells: TGF-/31,4"7 TGF-j32,8"13 and TGF-j33.14~16 The pleiotropic effects of these proteins are mediated bybinding to specific cell surface receptors that are serine/threonine kinases.17"22

TGF-/3 has been shown to play a role in soft tissue repair including bone growth and collagen synthesis.TGF-/3 stimulates bone formation23"28 and accelerated the healing of incisional wounds.29 TGF-/3 induces

Advanced Tissue Sciences, 10933 North Torrey Pines Road, La Jolla, California 92037-1005.2Georgia Institute of Technology, The George Woodruff School of Mechanical Engineering, Atlanta, Georgia 30332-

0405.3North Shore University Hospital, 350 Community Drive, Manhassett, New York 11030.

289

ZIMBER ET AL.

rat muscle cells to produce cartilage-specific macromolecules13'30 and enhances sulfated-proteoglycan coreprotein synthesis in rabbit chondrocytes.31 TGF-/3 increased collagen type II production in rabbit articularchondrocytes32 and type I and type III collagen synthesis in human dermal fibroblasts.33 Kulyk et al.34

showed that TGF-/3 led to an increase in type II collagen mRNA in embryonic chick limb bud chondro-cytes. In contrast, Horton et al.35 reported that TGF-/3 and fibroblast growth factor acted synergistically toinhibit the synthesis of collagen type II by chicken sternal chondrocytes. Rosen et al.36 also reported de-creased type II collagen production after treatment of rat chondrocytes with TGF-/3.

Since adult cartilage has a limited ability for self-repair and due to the large number of orthopedic surg-eries performed to treat damaged cartilage, several groups have attempted to grow cartilage grafts in vitrofor in vivo use. Cima et al.37 seeded polyglycolic acid (PGA) mesh with bovine chondrocytes and implantedthem subcutaneously into nude mice. After 7 weeks, mice were sacrificed and cartilaginous formation wasshown to have taken place on the implants. Vacanti et al.38 also succeeded in growing cartilage in nudemice and more recently produced cartilage in the shape of a human ear.39 Nixon et al.40 demonstrated car-tilage formation on porous collagen matrices by equine articular chondrocytes. Vacanti et al.41 producedcartilage implants on PGA scaffolds in tissue culture dishes under a variety of seeding and growth condi-tions.

In this report we examine the effect of TGF-/3 on the growth of bovine articular chondrocytes in mono-layer and on three-dimensional scaffolds; we also investigate the effect of this growth factor on collagenand GAG synthesis by these cells.

MATERIALS AND METHODS

Cell Culture and Tissue Growth

Bovine articular chondrocytes (BACs) were obtained from articular ankle joints of healthy mature (2-3years old) cows (obtained from a local slaughterhouse) by collagenase digestion and were propagated inDMEM containing 10% fetal bovine serum, 2 mM L-glutamine, nonessential amino acids, 50 /Jig/ml pro-line, 1 mM sodium pyruvate, and 25 jug/ml gentamicin (complete medium). Cells were seeded at 106 cellsper T-150 flask and passaged by trypsinization approximately every 7 days. For proliferation studies, cellswere seeded into T-25 flasks at 200,000 cells per flask in complete medium: the next day they were treatedwith TGF-/3 (20 ng/ml), ascorbate (50 )u,g/ml), or both. Seventy-two hours later, cells were trypsinized andcounted. For the dose-response studies with TGF-/3, the concentrations used ranged from 0.1 to 20 ng/ml.Chondrocytes are designated in the following manner; cells isolated from cow number one are designatedas BAC 1; BAC 2 refers to cells isolated from cow number two, etc. The designation PI refers to pas-sage 1.

PGA mesh [45 mg/cm3; non-heat plated (N.H.P.); 2 mm thick; 1 cm diam.] was seeded with 3 X 106

cells in 6-well dishes, in complete media: 24 h later, fresh medium containing ascorbate (50 jug/ml) withor without TGF-/3 (20 ng/ml) was added. Medium was changed every 3 days. Recombinant TGF-/31 (rTGF-/31) was purified as described42 and used at the concentration given in the figure legends. bFGF was pur-chased from PeproTech, Inc. (Rocky Hill, NJ).

Immunoblotting

Confluent monolayers of bovine articular chondrocytes were scraped in phosphate-buffered saline (PBS)containing 1% Tween 20. Lysis buffer43 was added and cell lysates were fractionated by sodium dodecylsulfate-polyacrylamide gel electrophersis (SDS-PAGE) and analyzed by immunoblotting as described.44

Antibodies to collagen type I and II were from Southern Biochemicals Inc. (Birmingham, AL) and anti-chondroitin sulfate antibody was from Sigma (St. Louis, MO).

Histology and Immunohistochemistry

After 3 weeks of growth, tissues were washed in PBS and photographed. They were fixed in 10% bufferedformalin, paraffin embedded, and stained with hematoxylin and eosin (H&E), trichrome, and Safranin O.

290

TGF-j3 PROMOTES CHONDROCYTE GROWTH

Immunohistochemical staining was also performed on paraffin embedded tissue using biotin-streptavidinamplified system (Biogenex, Inc.). Sections were incubated with chondroitinase, ABC enzyme (SeikagukuCorp.) to unmask antigenic sites. They were then incubated with 3% hydrogen peroxide to block endoge-nous peroxidase activity, rinsed with Tris-saline buffer, and incubated with biotin block serum (Dako, Inc.)to reduce nonspecific background. Blocking serum was tapped off and primary antibodies to collagen typeI and collagen type II were added. Sections were then incubated with biotinylated anti-IgG, and washedwith Tris-saline buffer. They were then incubated with horseradish peroxidase-conjugated streptavidin andwashed with Tris-saline. Sections were then incubated with 3,3-diaminobenzidine substrate and counter-stained with hematoxylin.

Quantitation of Collagen and GAG

At 3 weeks, constructs were frozen lyophylized and stored at -70°C until analysis. The constructs weredigested with papain (1 mg/ml) in 100 mM phosphate buffer (pH 6.5) containing 5 mM cysteine and 5 mMEDTA at 65°C overnight. Quantitation of collagen and GAG were determined as described.4546

Northern Blot Analysis

RNA was isolated as described,47 fractionated on agarose-formaldehyde gels,48 and probed with 32P-la-beled type II collagen cDNA as described.49

4e+6

3e+6 "

# Cells/T25Flask 2e+6 "

1e+6

3.00e+6 -

U Cells /T25Flask 2.00e+6

Oe"fO Control Ascorbate TGFIi TGFR+Ascorbate

24 48 72

Hours After Addition B5e+6

# Ce l l s /T25Flask

4e+6 -

3e+6

2e+6 "

1e+6

0e+0

3e+6

2e+6# Cells/T25

Flask

1e+6

0e+00 0.1 0.5 1 10 20

[TGF-fi, ng/ml]

Control AA bFGF AA+bFGF

Sample D

FIG. 1. TGF-/3 and ascorbate increase the proliferation of bovine articular chondrocytes. (A) Bovine articular chon-drocytes (BAC 10, P3) were treated with TGF-/3 (20 ng/ml), ascorbate (50 /xg/ml), or both as described in Materialsand Methods. Seventy-two hours later, cells were trypsinized and counted. Cell counts shown represent average val-ues of triplicate flasks. Error bars represent standard deviation. (B) Bovine articular chondrocytes (BAC 6, P2) wereseeded and treated with TGF-/3 (20 ng/ml), ascorbate (50 jug/ml), or both as described in Materials and Methods. Cellswere trypsinized and counted at the indicated times. All cell counts represent average values of triplicate flasks. (C)Bovine articular chondrocytes (BAC 10, P3) were grown for 72 h in media containing 50 /ig/ml ascorbate and the in-dicated amounts of TGF-/3. They were then trypsinized and counted. Cell counts represent averages of triplicate flasks.(D) Bovine articular chondrocytes (BAC 10, P3) were treated with bFGF (10 ng/ml), ascorbate (50 ju,g/ml), or both asdescribed in Materials and Methods. Seventy-two hours later, cells were trypsinized and counted. Cell counts shownrepresent average values of triplicate flasks.

291

ZIMBER ET AL.

RESULTS

TGF-(3 Increases the Proliferation of Bovine Chondrocytes in Monolayer

The effects of TGF-/3 on chondrocytes were first examined on cells growing in monolayer. Bovine chon-drocytes were seeded and treated with ascorbate, TGF-/3, or both as described in Materials and Methods.Figure 1A shows that TGF-/3 stimulated the growth of bovine chondrocytes: this effect was noted usingchondrocytes isolated from different cows and at several passages up to passage four (Fig. 1 and data notshown). Note that the addition of ascorbate also had a stimulatory effect, and this was additive with TGF-j8. While the absolute fold increase varied about 30% from animal to animal (compare Fig. 1A with Fig.IB and Fig. ID), the stimulatory effect of TGF-/3 and ascorbate on these cells was always a consistent ob-servation. Figure IB shows a time course for the proliferative effect of TGF-/3 and Figure 1C shows adose-response curve for TGF-/3 in the presence of ascorbate: the stimulation of cell growth can be seen aslow as 0.1 ng/ml. Photomicrographs of these cells 48 h after treatment (Fig. 2) did not show any grosschange in morphology other than that the TGF-/3-treated cells appeared slightly rounder and smaller. FigureID shows that bFGF had a slight inhibitory effect on the growth of these cells when assayed in a similarmanner.

Effects ofTGF-(3 on Collagen and GAG Synthesis

Our previous results indicated that TGF-/3 enhanced sulfated proteoglycan core synthesis in rabbit chon-drocytes.31 To further extend these studies, bovine chondrocytes were treated with TGF-/3 and cell ly sateswere prepared as described in Materials and Methods. They were then fractionated by SDS-PAGE and an-

FIG. 2. Photomicrographs of bovine chondrocytes grown with TGF-j8 and ascorbate. Bovine articular chondrocytesgrown for 48 h in complete media containing no additives (A), 50 ju.g/ml ascorbate (B), 20 ng/ml TGF-/3 (C), 50 /ig/mlascorbate + 20 ng/ml TGF-/3 (D).

292

TGF-/3 PROMOTES CHONDROCYTE GROWTH

Anti-CS NRS

207 -

112 -

80 -

45 -

36 -

1 2 3 4 5 6 7 8

200 —

135 -

81 -

32 -

28S -

18S -

B1 4 C

FIG. 3. Detection of collagen type II and GAGs in bovine articular chondrocyte lysates. (A) Bovine articular chon-drocytes were grown in complete media containing no additives (lanes 1 and 5), ascorbate (50 /xg/ml, lanes 2 and 6),TGF-/3 (20 ng/m, lanes 3 and 7), or TGF-/3 + ascorbate (lanes 4 and 8). Cell lysates were prepared, fractionated bySDS-PAGE and analyzed by immunoblotting using anti-chondroitin sulfate antibody (anti-CS; lanes 1-4) or normalrabbit serum (NRS; lanes 5-8). (B) Bovine articular chondrocyte lysates were fractionated as in A and immunoblottedwith antibody against collagen type II (lanes 1-4) or normal rabbit serum (lanes 5-8). Lanes 1 and 5, no additives;lanes 2 and 6, plus ascorbate; lanes 3 and 7, plus TGF-/3; lanes 4 and 8, plus TGF-/3 and ascorbate. (C) RNA was pre-pared from chondrocytes that were untreated (lane 1), or treated with ascorbate (50 /xg/ml, lane 2), TGF-/3 (20 ng/ml,lane 3), or TGF-j3 plus ascorbate (lane 4) and analyzed by Northern blotting using anti-collagen type II probe as de-scribed in Materials and Methods.

alyzed by Western blotting using anti-chondroitin sulfate and anti-collagen type II antibodies. Figure 3

shows that TGF-/3 treatment led to a substantial increase in GAG synthesis (Fig. 3A) and no detectable ef-

fect on collagen type II (Fig. 3B). Northern blot analysis showed that TGF-/3 had no effect on collagen type

II mRNA levels (Fig. 3C). Immunoblotting with anti-type I collagen antibody was negative (Fig. 7 and data

not shown).

Growth of Cartilage Constructs on Three-Dimensional Scaffolds

The effects of TGF-/3 were next examined on cells grown on three-dimensional scaffolds. Cells wereseeded onto PGA discs and grown as described in Materials and Methods. Figure 4 shows the gross pic-

293

ZIMBER ET AL.

B

FIG. 4. Cartilage-like tissue produced by bovine chondrocytes grown on PGA scaffolds. Bovine chondrocytes seededonto PGA scaffolds and grown for 3 weeks as described in Materials and Methods with (A) or without (B) TGF-/3 (20ng/ml).

tures of the cartilage constructs. They were smooth and glistening; note that the constructs grown in thepresence of TGF-/3 were almost three times larger (Fig. 4 and Table 1). H&E staining of the cartilage-liketissues showed that after 3 weeks of growth, cells were mostly concentrated on the outside edge while thecenters were less dense and contained more undegraded PGA fibers (Fig. 5). Note the increase cellularityof the TGF-/3-treated constructs. Staining with trichrome and Safranin O shows deposition of collagen andGAG, respectively (Fig. 6).

We also examined the cartilage tissue for collagen deposition by immunostaining. Constructs stained pos-itively for type II collagen (Fig. 7E and F) but not for collagen type I (Fig. 7C and D), consistent with typeII collagen being the predominant collagen type in cartilage. Note that in all of the photomicrographs, con-structs grown in the presence of TGF-/3 appear thicker. Collagen and GAG were also examined quantita-

TABLE 1. EFFECTS OF TGF-/S AND ASCORBIC ACID ON CARTILAGE CONSTRUCTS3

Sample TGF-pDry weight

(mg)

Totalcollagen Total GAG

content (/xg)

123456

4.54.23.6

11.711.011.2

31.529.325.281.876.978.3

71.071.669.3

171.6180.5189.4

aSamples shown in Figure 4 were processed for collagen and GAG analysis as de-scribed in Materials and Methods. Note the 2- to 3-fold increases in dry weight, collagen,and GAG values for the contructs grown in the presence of TGF-/3.

294

TGF-/3 PROMOTES CHONDROCYTE GROWTH

A B

• ' • • • • • .

-

.7

• > : •

D

L r N - . :

;

. . • ' • • ' ' • • • • • •

FIG. 5. Hematoxylin and eosin staining of in vitro cartilage tissue. The samples shown in Figure 4 were sectionedand stained with hematoxylin and eosin. Samples shown in A and B were grown without TGF-/3 and samples shownin C and D were grown with TGF-/3.

tively. Table 1 shows that TGF-/3 treatment resulted in an approximately 2.5-fold increase in the dry weightof the constructs as well as in collagen and GAG.

DISCUSSION

The effects of TGF-/3 and ascorbate on collagen production in fibroblasts grown in monolayer have beenwell documented. TGF-/3 has been shown to stimulate the incorporation of collagen and fibronectin intothe extracellular matrix.50 TGF-/3 and ascorbate act synergistically to enhance the production of type I andtype III collagen in human dermal fibroblasts.5152 Ascorbate treatment resulted in an increase in prolifer-ation of dermal fibroblasts isolated from newborn and elderly sources.53 Phillips et al.33 also showed thatTGF-/3 and ascorbate both led to an increase in collagen type I and type III biosynthesis in dermal fibro-blasts grown in monolayer while Geesin et al.54 made similar observations for cells grown in collagen gels.

The reported effects of TGF-/3 on chondrocytes grown in monolayer have been mixed. Horton et al.35

reported that TGF-/3 inhibits the synthesis of collagen type II mRNA in chick chondrocytes and Rosen etal.36 demonstrated that treatment of rat chondrocytes with TGF-/3 led to a decrease in type II collagen pro-tein. However, both Kuyle et al.34 and Galera et al.32 reported that TGF-/3 increased the levels of type IIcollagen mRNA in chick limb buds and rabbit chondrocytes, respectively.

In experiments presented here, we first examined the effects of TGF-/3 and ascorbate on the proliferation ofbovine chondrocytes in monolayer. Both TGF-/3 and ascorbate resulted in an increase in proliferation of thesecells and the increases were additive (Fig. 1A and B). bFGF had an inhibitory effect on cell proliferation whetheror not cells were grown in the presence of ascorbate (Fig. 1C). Western blot analysis with anti-chondroitin sul-fate antibodies showed that TGF-/3 led to a marked increase in the synthesis of GAGs (Fig. 3) in agreement

295

ZIMBER ET AL.

B

D

.'

FIG. 6. Trichrome and Safrannin O staining of cartilage constructs. Samples shown in Figure 4 were sectioned andstained with trichrome (A and C) or Safrannin O (B and D). Samples shown in A and B were grown without TGF-/3and samples shown in C and D were grown with TGF-/3.

with previously reported experiments.31 Our results on collagen type II biosynthesis showed that neither TGF-/3 nor ascorbate had a detectable effect on collagen synthesis either at the mRNA or protein level.

The proliferative effect of ascorbate on bovine chondrocytes is similar to its effect on dermal fibroblasts53;however, unlike the case with chondrocytes, ascorbate also increases the rate of collagen type I and colla-gen type III in these fibroblasts. One biological role of ascorbate is to function as a cofactor in the hy-droxylation of proline and lysine residues: its effect on proliferation and gene transcription has been sug-gested to be due to its ability to stimulate lipid peroxidation which in turn can affect various signal transducingsystems.54 TGF-/3 has been shown to stimulate ascorbate transport activity in osteoblastic cells55; it will beinteresting to determine if the same holds true for chondrocytes, since this may contribute to the prolifera-tive effect of TGF-/3 in these cells.

The effect of TGF-/3 on the production of cartilage-like tissue by bovine chondrocytes grown on PGAscaffolds was significant; constructs grown in the presence of the growth factor weighed 2.5 times morethan controls and contained similar increases in GAGs and collagen type II. These differences are likelydue in part to increases in cell numbers in the TGF-/3 treated constructs since the percents dry weight ofcollagen and GAGs are similar for both. Our results are the first to show that TGF-/3 enhances the forma-tion of cartilage tissue by chondrocytes grown in this fashion. As several groups are engaged in producingcartilage tissue in vitro for use in joint resurfacing and reconstructive surgery, the addition of TGF-/3 shouldbe useful for enhancing the growth of these tissues for clinical use.

ACKNOWLEDGMENT

We thank Patrice Wick for excellent manuscript preparation.

296

TGF-j6 PROMOTES CHONDROCYTE GROWTH

A B

D

. • - • .

/ ' . . . ' ' - • •

FIG. 7. Immunohistochemical staining of cartilage constructs. Samples shown in Figure 4 were stained with normalrabbit serum (A and B), anticollagen type I (C and D), or anticollagen type II (E and F). Samples shown in A, C, andE were grown without TGF-/3 while samples shown in B, D, and F were grown with TGF-/3.

REFERENCES

1. Barnard, J.A., Lyons, R.M., and Moses, H.L. The cell biology of transforming growth factor /3. Biochim. Biophys.Acta 1032, 79, 1990.

2. Massague, J. The transforming growth factor-/? family. Annu. Rev. Cell. Biol. 6, 697, 1990.3. Roberts, A.B., and Sporn, M.B. The transforming growth factor-/3s. In Sporn, M.B., and Roberts, A.B., eds. Peptide

Growth Factors and Their Receptors. Heidelberg: Springer-Verlag, 1990, pp. 419^72.4. Derynck, R., Jarret, J.A., Chen, E.Y., Eaton, D.H., Bell, J.R., Assoian, R.K., Roberts, A.B., Sporn, M.S., and

297

ZIMBER ET AL.

Goeddel, D.V. Human transforming growth factor-beta complementary DNA sequence and expression in normaland transformed cells. Nature (London) 316, 701, 1985.

5. Moses, H.L., Branum, E.B., Proper, J.A., and Robinson, R.A. (1981) Transforming growth factor production bychemically transformed cells. Cancer Res. 41, 2842, 1981.

6. Roberts, A.B., Anzano, M.A., Lamb, L.C., Smith, J.M., and Sporn, M.B. New class of transforming growth fac-tors potentiated by epidermal growth factor. Proc. Natl. Acad. Sci. U.S.A. 78, 5339, 1981.

7. Sharpies, K., Plowman, G.D., Rose, T.M., Twardzik, D.R., and Purchio, A.P. Cloning and sequence analysis ofsimian transforming growth factor-/3 cDNA. DNA 6, 239, 1987.

8. Demartin, R., Haendler, B., Hoeffer-Warbinek, R., Gaugitsch, H., Wrann, M., Schlusener, H., Seifert, J.M., Bodmer,S., Fontana, A., and Hofer, E. Complementary DNA for human glioblastoma-derived T cell suppressor factor, anovel member of the transforming growth factor-j8 gene family. EMBO J. 6, 3673, 1987.

9. Hanks, S.K., Armour, R., Baldwin, J.H., Maldonado, F., Spiess, J., and Holley, R.W. Amino acid sequence of theBSC-1 cell growth inhibitor (Polyergin) deduced from the nucleotide sequence of the cDNA. Proc. Natl. Sci. U.S.A.85, 79, 1988.

10. Ikeda, T., Lioubin, M.N., and Marquardt, H. Human transforming growth factor type j82: Production by a prosta-tic and adenocarcinoma cell line, purification, and initial characterization. Biochemistry 26, 2406, 1987.

11. Madisen, L., Webb, N.R., Rose, T.M., Marquardt, H., Ikeda, T., Twardzik, D., Seyedin, S., and Purchio, A.F.Transforming growth factor-/32: cDNA cloning and sequence analysis. DNA 7, 1, 1988.

12. Marquardt, H., Lioubin, M.N., and Ikeda, T. Complete amino acid sequence of human transforming growth factorbeta 2. J. Biol. Chem. 262, 12127, 1987.

13. Seyedin, S.M., Segarini, P.R., Rosen, D.M., Thompason, A.Y., Bentz, H., and Graycar, J. Cartilage inducing fac-tor-B is a unique protein structurally and functionally related to transforming growth factor-beta. J. Biol. Chem.262, 1946, 1987.

14. Derynck, R., Lindquist, P.B., Lee, A., Wen, D., Tamm, J., Graycar, J.L., Mason, H.L., Miller, D.A., Coffey, R.J.,Moses, H.L., and Chen, E.L. A new type of transforming growth factor-/}, TGF-/33. EMBO J. 7, 3737, 1988.

15. Jakowlew, S.B., Dillard, P.J., Kondaiah, P., Sporn, M.B., and Roberts, A.B. Complementary deoxyribonucleic acidcloning of a novel transforming growth factor /34 from chicken chondrocytes. Mol. Endocrinol. 2, 747, 1988.

16. ten Dijke, P., Iwata, K.K., Pieler, C , and Foulkes, J.G. Identification of a new member of the transforming growthfactor-j3 gene family. Proc. Natl. Acad. Sci. U.S.A. 85, 4715, 1988.

17. Attisano, L., Carcamo, J., Ventura, F., Weis, F.M.B., Massague, J., and Wrana, J.L. Identification of human ac-tivin and TGF-/3 type I receptors that form heteromeric kinase complexes with type II receptors. Cell 75, 671, 1993.

18. Ebner, R., Chen, R.-H., Shum, L., Lawler, S., Zioncheck, T.F., Lee, A., Lopez, A.R., and Derynck, R. Cloning ofa type I TGF-/3 receptor and its effect on TGF-/3 binding to the type II receptor. Science 260, 1344, 1993.

19. Franzen, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., Heldin, C.-H., and Miyazono, K. Cloning of aTGF-/3 type I receptor that forms a heteromeric complex with the TGF-/3 type II receptor. Cell 75, 681, 1993.

20. Lin, H.Y., Wang, X.F., Ng-Eaton, E., Weinberg, R.A., and Lodish, H.F. Expression cloning of the TGF-j3 type IIreceptor, a functional transmembrane serine/threonine kinase. Cell 68, 775, 1992.

21. ten Dijke, P., Yamashita, H., Ichijo, H., Franzen, P., Laiho, M., Miyazono, K., and Heldin, C.-H. Characterizationof type I receptors for transforming growth factor-/? and activin. Science 264, 101, 1994,

22. Wang, X.-F., Lin, H.Y., Ng-Eaton, E., Downward, J., Lodish, H.R., and Weinberg, R.A. Expression cloning andcharacterization of the TGF-/3 type III receptor. Cell 67, 797, 1991.

23. Noda, M., and Camilliere, J.J. In vivo stimulation of bone formation by transforming growth factor-j8. Endocrinology124,2991, 1989.

24. Joyce, M.E., Roberts, A.B., Sporn, M.B., and Bolander, M.E. Transforming growth factor-/? and the initiation ofchondrogenesis and osteogenesis in the rat femur. J. Cell Biol. 110, 2195, 1990.

25. Mackie, E.J., and Trechsel, U. Stimulation of bone formation in vivo by transforming growth factor-j8: Remodelingof woven bone and lack of inhibition by indomethacin. Bone 11, 295, 1990.

26. Marcelli, C , Yates, A.J., and Mundy, G.R. In vivo effects of human recombinant transforming growth factor j8 onbone turnover in normal mice. J. Bone Min. Res. 5, 1087, 1990.

27. Beck, L.S., Ammann, A.J., Aufdemorte, T.B., Deguzman, L., Xu, Y., Lee, W.P., McFatridge, L.A., and Chen, T.L.In vivo induction of bone by recombinant human transforming growth factor /31. J. Bone Min. Res. 6, 961, 1991.

28. Kibblewhite, D.J., Bruce, A.G., Strong, D.M., Ott, S.M., Purchio, A.F., and Larrabbee, W.F., Jr. Transforminggrowth factor-/3 accelerates osteoinduction in a craniofacial onlay model. Growth Factors 9, 185, 1993.

29. Mustoe, T.A., Pierce, G.F., Thomason, A., Gramates, P., Sporn, M.B., and Deuel, T.F. Accelerated healing of in-cisional wounds in rats induced by transforming growth factor-/3. Science 237, 1333, 1987.

30. Seyedin, S.M., Thompason, A.Y., Bentz, H., Rosen, D.M., McPherson, J.M., Contin, A., Siegel, N.R., Galluppi,

298

TGF-/3 PROMOTES CHONDROCYTE GROWTH

G.R., and Pietz, K.A. Cartilage-inducting factor-A: Apparent identity of TGF-j3. J Biol. Chem. 261, 5693,1986.

31. Malemud, C.J., Killeen, W., Hering, T.M., and Purchio, A.F. Enhanced sulfated-proteoglycan core protein syn-thesis by incubation of rabbit chondrocytes with recombinant transforming growth factor-j81. J. Cell Physiol. 149,152, 1991.

32. Galera, P., Vivien, D., Pronost, S., Bonaventure, J., R6dini, F., Loyau, G., and Pujol, J-P. Transforming growthfactor-/31 (TGF-/31) up-regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC)involves increased mRNA levels without affecting mRNA stability and procollagen processing. J. Cell Physiol.153, 596, 1992.

33. Phillips, C.L., Combs, S.B., and Pinnell, S.R. Effects of ascorbate acid on proliferation and collagen synthesis inrelation to the donor age of human dermal fibroblasts. Soc. Inv. Dermatol. 103-2, 228, 1994.

34. Kulyk, W.M., Rodgers, B.J., Greer, K., and Kosher, R.A. Promotion of embryonic chick limb cartilage differenti-ation by transforming growth factor-/3. Dev. Biol. 135, 424, 1989.

35. Horton, W.E., Higginbotham, J.D., and Chandrasekhar, S. Transforming growth factor-beta and fibroblast growthfactor act synergistically to inhibit collagen II synthesis through a mechanism involving regulatory DNA sequences.J. Cell. Physiol. 141, 8, 1989.

36. Rosen, D.M., Stempien, S.A., Thompson, A.Y., and Seyedin, S.M. Transforming growth factor-beta modulates theexpression of osteoblast and chondroblast phenotypes in vitro. J. Cell. Physiol. 134, 337, 1988.

37. Cima, L.G, Vacanti, J.P., Vacanti, C , Ingber, D., Mooney, D., and Langer, R. Tissue engineered by cell trans-plantation using degradable polymer substrates. J. Biomed. Eng. 113, 143, 1991.

38. Vacanti, C.A., Kim, J., Upton, M.P., Vacanti, D., Mooney, D., Schloo, B., and Vacanti, J.P. Tissue-engineeredgrowth of bone and cartilage. Trans. Proc. 25, 1019, 1993.

39. Vacanti, C.A., Cima, L.G, Ratkowski, D., Upton, J., and Vacanti, J.P. Tissue Engineered growth of new cartilagein the shape of a human ear using synthetic polymers seeded with chondrocytes. Mat. Res. Soc. Symp. Proc. 252,367, 1992.

40. Nixon, A.J., Sams, A.E., Lust, G, Grande, D.A., and Mohammed, H.O. Temporal matrix synthesis and histologicfeatures of a chondrocyte-laden porous collagen cartilage analogue. Am. J. Vet. Res. 54, 349, 1993.

41. Vacanti, C.A., WooSeob, K., Schloo, B., Upton, J., and Vacanti, J.P. Joint resurfacing with cartilage grown in situfrom cell-polymer structures. Am. J. Sport Med. 22, 485, 1994.

42. Gentry, L.E., Lioubin, M., Purchio, A.F., and Marquardt, H. Molecular events in the processing of combinant typeI pre-pro-transforming growth factor beta to the mature polypeptide. Mol. Cell. Biol. 8, 4162, 1988.

43. Laemmli, U.K. Cleavage of structural proteins during the assemble of the head of bacteriophage Tr. Nature (London)227, 680, 1970.

44. Burnette, W.N. Western blotting: Electrophoretic transfer of proteins from sodium dodecyl sulphate polyacrylamidegels to unmodified nitrocellulose and radiographic detection with antibody and radioionated protein. Anal. Biochem.112, 195, 1981.

45. Farndale, R.W., Buttle, D.J., and Barrett, A.J. Improved quantitation and discrimination of sulphated gly-cosaminoglycans by the use of dimethylmethylene blue. Biochim. Biophys. Acta 883, 73, 1986.

46. Woessner, J.F. The determination of hydroxyproline in tissue and protein samples containing small proportions ofthis imino acid. Arch. Biochem. Biophys. 93, 440, 1961.

47. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chlo-roform extraction. Anal. Biochem. 162, 6, 1987.

48. Lehrach, H., Diamond, D., Wozney, J.M., and Boedtker, H. RNA molecular weight determination by gel elec-trophoresis under denaturing conditions: A critical re-examination. Biochemistry 16, 4743, 1977.

49 Madisen, L., Webb, N.R., Rose, T.M., Marquardt, H., Ikeda, T., Twardzik, D., Seyedin, S., and Purchio, A.F.Transforming growth factor-/32: cDNA cloning and sequence analysis. DNA 7, 1, 1986.

50. Ignotz, R.A., and Massague, J. Transforming growth factor-/3 stimulates the expression of fibronectin and colla-gen and their incorporation into the extracellular matrix. J. Biol. Chem. 261, 4337, 1986.

51. Appling, W.D., O'Brien, W.R., Johnston, D.A., and Duvic, M. Synergistic enhancement of type I and II collagenproduction in cultured fibroblasts by transforming growth factor-j8 and ascorbate. FEBS Lett. 250-2, 541, 1989.

52. Geesin, J.C., Brown, L.J., Gordon, J.S., Berg, R.A. Regulation of collagen synthesis in human dermal fibroblastsin contracted collagen gels by ascorbic acid, growth factors, and inhibitors of lipid peroxidation. Exp. Cell Res.

206, 283, 1993.53. Phillips, C.L., Tajima, S., and Pinnell, S.R. Ascorbate acid and transforming growth factor-/31 increase collagen

biosynthesis via different mechanisms: Coordinate regulation of Proal(I) and Proal(III) collagens. Arch. Biochem.Biophys. 295, 397, 1992.

299

ZIMBER ET AL.

54. Geesin, J.C., Hendricks, L.J., Gordon, J.S., and Berg, R.A. Modulation of collagen synthesis by growth factors:The role of ascorbate-stimulated lipid peroxidation. Arch. Biochem. Biophys. 289, 6, 1991.

55. Dixon, S.J., and Wilson, J.X. Transforming growth factor-/3 stimulates ascorbate transport activity in osteoblasticcells. Endocrinology 130, 484, 1992.

Address reprint requests to:A.F. Purchio

Advanced Tissue Sciences

10933 North Torrey Pines Road

La Jolla, California 92037-1005

300