Extracellular Matrix and Bone Morphogenetic Proteins in Cartilage and Bone Development and Repair

EXTRACELLULAR MATRIX A N D BONE MORPHOGENETIC PROTEINS IN CARTILAGE A N D BONE DEVELOPMENT A N D REPAIR

Slobodan Vu kicevic, Vishwas M. Para1kar, and A.H. Reddi

I. INTRODUCTION

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208

11. CARTILAGE AND BONE DIFFERENTIATION . . . . . . . . . . . . . . . . 208

A. Demineralized Bone Matrix-Induced Osteogenesis . . . . . . . . . . . . B. Osteogenin and Bone Morphogenetic Proteins . . . . . . . . . . . . . . C. In Htro Activities of Cartilage and Bone Differentiation Factors . . . . . D. In Vivo Role of Cartilage and Bone Differentiation Factors . . . . . . . 111. INTERACTION OF GROWTH FACTORS WITH EXTRACELLULAR MATRIX . . . . . . . . . . . . . . . . . . . . . . . . . IV. DIFFERENTIATIONOF OSTEOBLAST-LIKECELLS ON RECONSTITUTED BASEMENT MEMBRANE: ANGIOGENESIS AND OSTEOGENESIS . . . . . . . . . . . . . . . . . .

Advances in Molecular and Cell Biology Volume 6, pages 207-224 Copyright 6 1993 by JAI Press Inc. All rights of repduction in any form reserved. ISBN: 1-55938-515-4

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SLOBODAN VUKICEVIC, VISHWAS M. PARALKAR, and A.H. REDDl

A. Osteoblastic Cells Recognize Basement Membrane Components B. Growth Factors in Basement Membrane Regulate Osteoblastic

. . . . . 220

221 Network Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. CHALLENGES FOR THE FUTURE . . . . . . . . . . . . . . . . . . . . . . 221 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,222

1. INTRODUCTION The origin and evolution of multicellular eukaryotes is accompanied with the appearance of the extracellular matrix (ECM). In certain skeletal tissues, such as bone and cartilage, the ECM is prominent and accounts for much of the mass and volume. The biosynthesis and supramolecular assembly of ECM in bone and cartilage is intimately associated with the development and morphogenesis of these tissues. The ECM provides the framework for the tissues of the skeletal system and, in addition, plays an important physiological role in sequestration of growth and differentiation factors involved in repair, regeneration, and remodeling. The development and differentiation of bone occurs by one of two processes. First, there is a direct development of bone from mesenchyme as in the bones of the skull and facial skeleton. This process is referred to as intramembranous bone formation. Second, a transient cartilage model precedes differentiation of bone and occurs in a majority of bones. This process is known as endochondral ossification. It is noteworthy that these two methods of ossification signify only the environment of bone formation, and there appears to be no distinct differences in the bone formed. Angiogenesis and vascular invasion are prerequisites for both intramembranous and endochondral ossification. However, the underlying mechanistic interactions between vascular invasion and osteogenesis are not clear. The aim of this chapter is to describe recent progress in the interface of extracellular matrix components and growth and differentiation factors with special reference to bone and cartilage development and repair.

II. CARTILAGE AND BONE DIFFERENTIATION The bone has a remarkable potential for repair and regeneration. Regeneration processes, as fracture repair, recapitulate events that occur in the course of embryonic bone development. The experimental means of inducing new bone formation at non-bony sites have been known for years. In early 1930, Huggins (1931a,b) discovered that surgical transplantation of living urinary bladder epithelium into abdominal muscles in dogs resulted in new cartilage and bone formation. The phenomenon was termed “epithelial osteogenesis.” Urist (1965) and Reddi and Huggins (1972) have shown that implantation of demineralized bone matrix into intramuscular or subcutaneous sites induced a sequence of cellular events leading to the formation of cartilage, bone, and bone marrow. The events were termed

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“matrix-induced endochondral bone formation.” Inductive properties of demineralized bone matrix implantation at heterotopic sites are also reminiscent of embryonic bone development. It, is believed that the putative molecules responsible for epithelial and matrix-induced osteogenesis may have common structural properties, although it has not yet been demonstrated. A. Demineralized Bone Matrix-Induced Osteogenesis

The cascade of matrix-induced endochondral bone formation consists of chemotaxis, attachment of mesenchymal stem cells to the matrix, proliferation of progenitor cells, and differentiation of cartilage. This is followed by cartilage resorption, bone formation, and finally culminating in the formation of an ossicle filled with hematopoietic marrow (Reddi, 1981). On day 1, fibronectin is deposited on the matrix facilitating cell attachment. On days 2 and 3, proliferation of mesenchymal cells takes place. This cell-matrix interaction results in the differentiation of chondroblasts at day 5, with appearance of cartilaginous matrix as shown by an increased 35S(sulfate) incorporation into proteoglycans, and the appearance of hypertrophic chondrocytes at day 7. Angiogenesis with cartilage resorption starts on day 9, and new bone formation in association with 4SCaincorporation into new bone mineral occurs at day 11. Extensive bone remodeling with hematopoietic marrow formation, as evidenced by %e incorporation, is maximal on day 21. This developmental program is a single cycle of cartilage and bone formation, and recapitulates the embryonic development of limbs. It could be a useful model for fracture healing. Moreover, this system enables molecular studies at the different stages of endochondral bone formation, and a better understanding of the role of endogenous and exogenous growth and differentiation factors involved.

B. Osteogenin and Bone Morphogenetic Proteins Identification of osteogenic proteins in mammalian bone matrix responsible for this cascade of cartilage and bone differentiation has been a difficult task due to the low abundance of osteogenin and related bone morphogenetic proteins (BMPs) tightly bound to the bone extracellular matrix. Progress in recent years in the study of BMPs has been aided by four important technical developments, namely: (1) the development of a functional bioassay in a subcutaneous site in the rat to monitor the specific biology of osteogenic activity (Sampath and Reddi, 1981, 1983); (2) the development of a specific purification procedure involving heparin affinity chromatography (Sampath et al., 1987); (3) the use of electroendosmotic elution techniques after preparative sodium dodecyl sulfate gel electrophoresis to achieve final purification homogeneity (Luyten et al., 1989);and (4) the use ofrecombinant DNA methodologies for the cloning and expression of several members of the BMP family (Wozney et al., 1988; Celeste et al., 1990; Ozkaynak et al., 1990; Hammonds et al., 1991).

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SLOBODAN VUKICEVIC, VISHWAS M. PARALKAR, and A.H. REDDI

A breakthrough in the isolation of osteogenic activity has been the dissociative extraction of demineralized bone matrix by 4-M guanidinium hydrochloride, or 8-M urea (Sampath and Reddi, 1981). Neither the soluble extract nor the residual bone powder alone had detectable activity when implanted subcutaneously in the rat. However, if the extract was reconstituted with the inactive residue, the biological activity of bone and cartilage formation was recovered. This reconstitution assay has permitted the further purification of osteogenic components. Additional experiments revealed that irrespective of the species, the lower molecular weight fractions of a4-M guanidinium hydrochloride extract following molecular sieve chromatography induced new cartilage and bone formation in rats (Sampath and Reddi, 1983). This discovery set the stage for the large scale purification of bone- and cartilageinducing proteins . Agroup of related bone morphogenetic factors has been discovered that include osteogenin, bone morphogenetic protein-2A (BMP-2A), BMP-2B (now BMP-4). BMP-3, BMPs 5-7, and osteogenic protein4 (OP-1) (Wozney et al., 1988; Luyten et al., 1989; Celeste et a]., 1990; Ozkaynak et al., 1990; Wang et al., 1990). Osteogenin was purified to homogeneity from bovine bone matrix, and the sequences of several tryptic peptides were determined (Luyten et al., 1989). The sequences were similar to portions of the amino acid sequence deduced from the cDNA clone of bone morphogenetic protein-3 (BMP-3) (Wozney et al., 1988). Osteogenin shows 49 and 48% sequence identity in its carboxyl-terminal quarter to BMP-2A and BMP-2B, respectively (Wozney et al., 1988). BMP-7 was found to be identical to OP-l (Wang et a]., 1990). Based on structural and amino acid sequence homologies, these osteogenic proteins are grouped among members of the transforming growth factor beta (TGF-P) superfamily, all of which share a striking homology in the C-terminal domain. As in the case with TGF-P, members of TGF-p superfamily are synthesized as a large precursor, four times greater than the mature form of the molecule. The mature proteins are processed from the precursor at the C-terminal as disulfide-linked homodimers containing a region that includes the highly conserved seven cystein residues (Massague, 1990). Osteogenin and related BMPs initiate cartilage and bone formation in vivo. BMP-3 is most abundant in the highly purified preparations, but other members, especially BMP-2 and BMP-7, are also present in the bioactive highly purified bone-derived fractions. BMPs 2-5 and BMP-7 have been reported to induce cartilage and bone as single recombinant proteins in conjunction with residue (Wozney et al., 1988; Ozkaynak et al., 1990; Wang et al., 1990; Hammonds et al., 1991). The availability of all the recombinant proteins will allow researchers to address the question of particular functions of different members in endochondral bone formation more accurately. The studies of the mechanism of action of the bone morphogenetic proteins will add considerable new information on the molecular signals controlling cartilage and bone formation, as well as cartilage and bone regeneration.

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C. In Vitro Activities of Cartilage and Bone Differentiation Factors Recently, experimental studies have been performed to define the role o f bone morphogenetic proteins i n development, interaction with extracellular matrix, cellular binding and localization, as well as in v i m and in vivo activities. There i s accumulating evidence that the TGF-P superfamily members are important regulators of many morphogenetic events duringearly vertebrateembryogenesis. Using iodinated osteogenin i n sections of developing rat embryo, maximal binding o f the protein was observed i n mesodermaltissues such as cartilage, bone, perichondrium, andperiosteum (Vukicevic et al., 1990a) (Figure 1). Applying in situ hybridization techniques, investigators have localized BMP-2, BMP-4, and Vgr- 1 (BMP-6)

Figure 1. Autoradiographic images of osteogenin (BMP-3) binding to longitudinal sectionsofan 18-day embryo. Magnification, 9x. (A) Section stained with hematoxylin and eosin. Vascular invasion into the cartilaginous centers of the long bones is seen. Arrows and arrowheads correspond to the osteogenin binding sites of section €3. (6) Total binding after incubation of an adjacent section with 2.5-ng/ml '251-osteogenin. Note the intensive binding to areas of endochondral ossification: ribs (arrow), femur (double arrow), tibia (small arrowheads), radius and ulna (double small arrowheads), humerus (thick arrow), as well as areas on intramembranous ossification; calvarial bones (big arrowheads). L stands for liver. (C)Nonspecific binding by incubation of an adjacent section with 2.5-ng/ml '251-osteogeninand 260-ng/mI unlabeled osteogenin. Reprinted with permission: Vukicevic et al., Dev. Biol. 140:209, 1990.

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SLOBODAN VUKICEVIC, VISHWAS M. PARALKAR, and A.H. REDDI

transcripts in several specialized organ systems during murine embryonic development. They include: the early development of the limb; myogenic layer of the atrioventricular cushions of the developing heart; developing hair and whisker follicles; tooth buds and palate; developing central nervous system; and craniofacial tissues (Lyons et al., 1989, 1990). Recent OP-1 (BMP-7) localization data during human development suggested that OP-1 is present during critical morphogenetic stages in early human embryonic development, particularly in tissues of mesodermal origin, as well as in basement membranes separating epithelium from the underlying stroma (Vukicevic et al., in preparation). These data, together with the specific affinity of osteogenin and BMP-2B for the type IV collagen of the basement membrane matrix (Paralkar et al., 1990; see later), underscores the potential critical role of the extracellular matrix in sequestering differentiation factors, and in regulating their bioavailability. Moreover, it also sheds light on the possible role of basement membrane components of blood vessel walls (as laminin and type IV collagen) in both bone formation and regeneration (Vukicevic et al., 1990~;Ripamonti and Reddi, 1992). In v i m studies allowed us to explore the effects of osteogenin and related bone morphogenetic proteins on both cartilage and bone cells. Osteogenin stimulated alkaline phosphatase activity and collagen synthesis in rat periosteal cells and calvarial osteoblasts (Vukicevic et a]., 1989). There was also an increase in the formation of alkaline phosphatase-positive colonies in rat bone marrow stromal cell cultures (Vukicevic et al., 1989). The promotion of the osteogenic phenotype was confirmed in experiments with MC3T3-El osteoblastic cells; osteogenin inhibited growth and stimulated alkaline phosphatase activity within 72 hours (Vukicevic et al., 1990b), while usually about 12 days are needed for their spontaneous differentiation in v i m (Kodama et al., 1981). In the same cells, high-affinity receptors for BMP-2B have been identified and partially characterized (Paralkar et al., 1991) (Figure 2, Table 1). Several studies using either BMP-2, 3, or 4 have confirmed and extended these original data on BMPspromotingtheosteoblasticphenotype(Chenetal., 1991a; Hiraki eta]., 1991; Takuwaet al., 1991; Yamaguchiet al., 1991).Aprofoundstimulationofproteoglycan synthesis in fetal rat chondroblasts and rabbit articular chondrocytes (Vukicevic et al., 1989), together with increased cartilage matrix synthesis in chick limb bud cell cultures (Carrington et al., 1991; Chen et al., 1991b), demonstrated the role of osteogenin in the promotion of the chondrogenic phenotype. Osteogenin and BMP-2B (BMP-4) were equipotent in the maintenanceofproteoglycan metabolism in articular cartilage explant cultures of young, adolescent and adult tissues by increasing proteoglycan synthesis and decreasing proteoglycan catabolism (Luyten et a]., 1992). Analysis of the size of the newly synthesized proteoglycans, the glycosaminoglycan chain size, and the glycosaminoglycan type of explants treated with osteogenin or BMP-4 indicated that they were very comparable to each other. Thus, both osteogenin and BMP-4 alone are capable of stimulating and maintaining the chondrocyte phenotype in virro (Luyten et al., 1992). Taken together, these in

Bone Proteins, Development, and Repair

21 3

6

5

4

T %-o x

z

3

0 2

1

0

2

BMP 28 ng/rnl

Figure 2. Binding of radiolabeled BMP 2 6 to MC3T3-El cells. Increasing concentrations of radiolabeled BMP-26 were incubated with MC3T3-El cells at room temperature for 1 h r and the cell-associated radioactivity was determined. Points represent specifically bound radioactivity determined by subtracting nonspecific binding (1 040% of total binding) from total radioactivity bound. (Inset) Scatchard analysis of the binding data. 6,bound; F, free. Reprinted with permission: Paralkar et al., Proc. Natl. Acad. Sci. USA 88:3397,1991. v i m data suggest that osteogenin and related bone morphogenetic proteins do not only play a role in the initiation of cartilage and bone formation, but clearly promote the expression and maintenance of the chondrogenic and osteogenic phenotype.

D. In Vivo Role of Cartilage and Bone Differentiation Factors The availability of bone morphogenetic proteins made it possible to extend the knowledge of their in vivo morphogenetic potential in rodents to primates, as a prerequisite for the exploration of potential therapeutic applications for the regeneration of bone in man. The healing potential of osteogenin on calvarial defects in a series of adult primates of genus Pupio (baboon) has been studied. Acritical size

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SLOBODAN VUKICEVIC, VISHWAS M. PARALKAR, and A.H. REDDI Table 1. Competition With Growth Factors For the Binding of lodinated BMP-26 to MC3T3-El and NIH 3T3 Cells CPM Boutid Growth Factors "'1-BMP 2 8 BMP 2B PDGF FGF IGF-I EGF TGF 01

MC3T3-El

NIH 3T3

9,600 * 800 4.000 t 230 11.720t 1000 8,210 2 217 8,930 2 110 9,806 * 600 9,260 t 460

10,480 * 680 2,300 * 265 10,050 * 202 9,510 2 230 9,670 * 215 9,070 * 660 9,570 + 330

Nore: Each factor was in a 100-fold excess of the iodinated BMP-2B. The values are mean i standard deviation of four observations. Reprinted with permission: Paralhr et al. Proc. Natl. Acad. Sci. U.S.A. 88. 3397. 1991.

defect (CSD)-dependent nonunion of the baboon calvaria model-that is, a defect that does not repair spontaneously with bone, requiring a graft of viable bone, or alternative substitutes to heal-has been established (Ripamonti et al., 1991). In 48 adult male baboons, calvarial defects were implanted with a graft of autogenous bone harvested from the iliac crest, and with different osteoconductiveandosteoinductive substrata. Before calvarial implantation, osteogenin, isolated from both baboon and bovine bone matrix (and with biological activity in rats), was tested for biological activity in the rectus abdominis of an additional 16 baboons (Ripamonti and Reddi, 1992). The extraskeletal implantation permitted the unequivocal histological investigation of bone formation by induction, avoidingpossible ambiguities of the orthotopic site. Reconstitution of calvarial defects with baboon osteogenin induced huge amounts of new bone as early as 30 days. At 3 months, bone formation was extensive, culminating in complete regeneration of the craniotomy defects (Ripamonti et al., 1992). Osteogenin was recently tested for its regenerating effects in human periodontal intrabonydefects (Bowers et al., 1991).Combined with demineralized freeze-dried bone allografts, osteogenin significantly enhanced regeneration of a new attachment apparatus and component tissues in a submerged environments (Bowers et al., 1991). Using transformation of rat muscle flaps, it was possible to generate in vivo, autogenous, well-perfused bones in the shapes of femoral heads and mandibles (Khouri et al., 1991). Significant progress has been made in the characterization of the cartilage and bone differentiating proteins. A family of unique proteins has been described, and

Bone Proteins, Development, and Repair

21 5

there is ample evidence that they are directly responsible for de mvo cartilage and bone formation in vivo. Extensive research is underway to develop appropriate and optimal delivery systems based on extracellular matrix components. It is likely that bone morphogenetic proteins will play a crucial role in bone regeneration and repair.

111. INTERACTION OF GROWTH FACTORS WITH EXTRACELLULAR MATRIX Renewed attention has recently been focused on extracellular matrix macromolecules with the discovery of the interactions of these macromolecules with growth and differentiation factors. The interaction of heparan sulfate proteoglycans with fibroblast growth factors (FGFs) has been known for some time (for review see Burgess and Maciag, 1989). Recently, it has been demonstrated that basic fibroblast growth factor (bFGF) binds to heparan sulfate prior to the binding to its receptor (Yayon et al., 1991). Many other growth and differentiation factors are known to bind heparin and these include granulocyte-macrophage colony-stimulating factor, neurite-promoting factor, pleitropin, and platelet factor 4 (Roberts et al., 1988; Li et at., 1990; Merenmies and Rauvala, 1990; Ruoslahti and Yamaguchi, 1991). Transforming growth factor beta (TGF-P) has been shown to interact with the protein core of proteoglycan, named betaglycan, which is present on the cell surface of many cell types (Andres et al., 1989). TGF-p has also been shown to bind to extracellular matrix proteoglycan, decorin (Yamaguchi et al., 1990). During studies on cartilage and bone development, we have shown that osteogeninBMP-3, isolated from bovine bone, binds to collagen type IV which is a major component of the basement membrane (Paralkar et al., 1990). Using radioactive osteogenin, we examined the affinity of osteogenin for various components of extracellular matrix. Osteogenin binds with highest affinity to collagen type IV, but also binds to collagen types I and IX, and to heparin (Table 2). Osteogenin binds to both chains of collagen type IV, and this binding is pH- and time-dependent. The binding is also reversible with an apparent Kd of 4 x lo-'' M. Since osteogenin is also a member of TGF-P supergene family, we also tested the ability of TGF-P to bind extracellular matrix macromolecules. TGF-P binds to collagen type IV with high affinity (Paralkar et al., 1991; Tables 3 and 4).As both osteogenin and TGF-P bind collagen type IV, we tested some of the other growth factors for their ability to bind collagen type IV. As can be seen from Table 3, basic FGF and plateletderived growth factor (PDGF) also bind collagen type IV. The ability of aFGF to bind collagen type IV has also been shown (Thompson et al., 1988). Thus, FGFs, TGF-f3, and osteogenin all bind to proteoglycans as well as to collagen type IV of the extracellular matrix.

Table 2. Binding of Osteogenin (Bone Morphogenetic Protein31 to Various Extracellular Matrix Macromolecules Immobilized on Nitrocellulose Macmmolecules

c p d p g Mucmrnoleculea

910 * 70

Heparin Collagen type 1 Collagen type II Collagen type 111 Collagen type IV Collagen type V Collagen type IX Collagen type X Laminin Fi bronec t i n Chondroitin sulfate Keratin sulfate Dermatan sulfate Heparan sulfate Hyaluronic acid

990 * 55 N D ~ ND

4540 * 100

ND 770 * 20 ND ND ND ND ND ND ND ND

Notes: %ach macromolecule (2 pg/spot) was spotted i n triplicate and the experiment was repeated three times. Each point represents mean i S.D.of triplicate experiments with nine observations. bND, not detectable. Reprinted with permission: Palalkar et al., J. Biol. Chem. 265, 17281, 1990

Table 3. Specificity of Binding of Various Growth Factors to Type IV Collagen 10 Type I V Collageri m mole of Grow111Fuaor/Mole of Collugeri

Growrli fucror biridirig

Growth Factors Transforming growth factor PI b Fibroblast growth factor Platelet-derived growth factor Epidermal growth factor Insulin-like growth factor-1

I 0.07 0.09 ND* ND

Notes: *ND = not detectable. Values shown are the mean of triplicate determinations. The various radioicdinated growth factors were incubated with type IV collagen in the solid state on nitrocellulose paper as described i n

Materials and Methods The specific activities were :TGF (80pcilpg); bFGF (52 pCilpg): Platele~-&nved growth factor (70 pCi/pg): Epidermal growth factor (174 pCi/pg); and Insulin-like growth factor- I (188 vCihg). Reprinted with permission: Parelkaret al.. Dev. Biol. 143. 303. 1991.

216

Bone Proteins, Development, and Repair

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Table 4. Binding of '251-TGF-fi1to type IV collagen in solution. Effect of TGF-fJ2, fibronectin and laminin Addition

CPM

Experiment I

1 ng '251-TGF-pt I ng l Z ? - ~ ~ ~+-200 p I ng TGF-p~

6515 i 1526 29+4

Experiment 2

Collagen alone Collagen + fibronectin Collagen + laminin

26160i2760 30560-c510

32890 i 1260

Notes: Experinlent 1. Collagen type IV (0.5 pg) was incubated with 1 ng radiolabeled TGF-PI (specific activity 1OOpCilyg)inthe presenceorabsence ofTGF-fJ2(200 ng) in a final volume of SO0 pl of PBS plus 0.05% Tween 20 (TPBS) for 12 hr at 4°C. Qpe IV collagen precipitates under these conditions. After centrifugation at 12OOOxg for 15 mintheprecipitatedcollagen pelletwas washed 2 times with TPBS. The radioactivity incorporated into the pellet was determined in a gamma counter. Each point represents mean t S.D. oftriplicateobservations. Experinietu 2. 1251-TGF-~~ (I00 yCi/pg) was incubated with 5 yg of type IV collagen in the presence ci absence of fibronectin and laminin (5 yg each) and the amount of radioactivity bound to collagen was determined by a g a m m a counter. Each point represents mean t S.D. of triplicate observations. Reprinted with permission: Paralkaret al., Dev. Biol. 143. 303, 1991.

These observations could have far reaching implications for the regulatory role of growth and differentiation factors in the solid state. It is plausible that the extracellular matrix might protect these factors from proteolytic degradation, or alter the activity of these factors by their interactions. The latter was seen to be the case in our studies (Paralkar et a]., 1991).In this work, MC3T3-EI cells were grown in 24 well-tissue culture plates precoated with laminin, collagen I, and collagen type IV, or on plastic without any precoating. TGF beta inhibits the proliferation of these cells grown on plastic. When TGF beta type 1 was added to these cells grown on various matrix macromolecules, a transient inhibition of cell proliferation (40 to 50%) for up to 18 hours was seen in cells grown on all substrata. These cells were seen to recover from the inhibitory effects of TGF-p after 36 hours. However, in the case of cells grown on collagen type IV, the inhibitory effects on proliferation were sustained for up to 54 hours (Figure 3). Thus, in vitro collagen type IV seems to be able to modulate the activity of TGF beta 1. Similarly, heparin and heparan sulfate have been shown to be able to alter the activity of FGF's in vitro (Damon et al., 1986). Heparin has also been shown to stimulate the EGF receptor mediated phosphorylation of tyrosine and threonine residues (Gupta et a]., 1991). Also

21 8

s n

.

SLOBODAN VUKICEVIC, VISHWAS M. PARALKAR, and A.H. REDDI

60

-

40

-

20

-

Plastic Laminin

E

8

Y

E

.-0

s g O = F O

c ) -

E:

5 b Q)

.-c

-20

-

-40

-

-0 .-

E

f

I-

F

0

u

Control Collagen I

0-

-60

!

10

Collagen IV

I

I

I

I

1

20

30

40

50

60

Time (hrs) Figure 3. Sustained inhibition of [3H]thymidine incorporation into acid-precitable DNA in osteoblasticcells by TGF-PI in the presenceoftype IVcollagen. The inhibition was transient (only at 18 hr) when cultured on plastic, collagen type I, and laminin. Osteoblastic cells (MC3T3-El) were plated at 10,000 cells per well in 24-well plates precoated with the indicated matrix macromolecules (50 &well), and half of the medium was exchanged daily. Cells were incubated in the presence or absence of TCF-f51 (50 pdml) added at time 0. [3H]Thymidine (5 pCi/ml) was added at the indicated time points in medium containing 0.5% fetal bovine serum for the last 4 hr of culture. Incorporation of [3H]thymidineinto DNA was determined as described. The results are presented as a percentage of control for each matrix substratum at 18, 36, and 54 hr. A control curve for each experimental substratum (presence of collagen type IV, laminin, collagen type I, and plastic) was determined in the absence of TCF-P. Each point is the mean 4 D of eight observations. Reprinted with permission: Paralkar et al., Dev. Biol. 143:303, 1991.

extracellular matrix-associated macromolecules act synergistically with ciliary neurotrophic factor to induce type two astrocyte development (Lillien et al., 1990). The interactions of growth factors with extracellular matrix macromolecules could result in tight physiological regulation. These interactions could localize a potent bioactive molecule to the appropriate site. Indeed, the Wtit genes present in vertebrates code for a protein that acts over short distances because the protein is bound tightly to the extracellular matrix (Tabin, 1991). It is also possible that, depending upon the developmental stage of the embryo, the growth factors could be localized by the extracellular matrix (thus providing a storage of active mole-

Bone Proteins, Development, and Repair

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cules at the appropriate site), or they may be in the soluble form, thus being able to act at sites distant from the site of synthesis. With the discovery that differentiation inhibiting activity (DIA) is synthesized both as a diffusible protein as well as one that is matrix bound, this hypothesis seems more plausible (Rathjen et al., 1990). The diffusible and matrix bound forms of DIA seem to be transcribed from different promoters, thus raising the possibility that the two proteins could be differentially regulated depending upon the need for DIA at a local or distant site during embryonic development. It is also possible that soluble factors can have differential activity on the cells, depending on the composition of the extracellular matrix surrounding the cells. Atypical example of the in vivo role of extracellular matrix is endochondral bone induction by demineralized bone matrix (Urist, 1965; Reddi and Huggins, 1972; Luyten et al., 1989). Bone induction is due to the combined action of osteogeninBMPs and the collagenous matrix (Reddi, 1981). It is known that vascular invasion is a prerequisite for new bone formation (Foidart and Reddi, 1980; Reddi and Kuettner, 1981). It is therefore possible that basement membrane, especially collagen type IV and heparan sulfate proteoglycan around the endothelial cells of the invading capillaries, may function as an affinity matrix to present soluble growthanddifferentiation factors in the solid state, and to responding mesenchymal cells and osteoprogenitors to initiate osteogenesis. We also propose that collagen type IV functions as a physiological delivery system by sequestering factors involved in embryonic induction, morphogenesis, and epithelial-mesenchymal interactions. These interactions may help explain the general role of basement membrane in embryonic development. The interaction of bone-inductive proteins with collagen type IV also helps to link demineralized bone matrix-induced bone formation to epithelium-induced bone formation.

IV. DIFFERENTIATION OF OSTEOBLAST-LIKE CELLS ON RECONSTITUTED BASEMENT MEMBRANE: ANGIOGENESIS AND OSTEOGENESIS Vascular invasion is a prerequisite for bone formation (Trueta, 1963; Foidart and Reddi, 1980). Since developing osteoprogenitorcells and osteoblasts are in contact with the basement membrane of the invading vascular system, it is possible that the endothelial cell matrix might function during critical phases of capillary invasion in endochondral bone formation (Rhodin, 1974),growth (Scott and Pease, 1956; Kimmel and Jee, 1980), remodelling (Jaworski, 1976), and fracture repair (Simmons, 1980). In the matrix-dependent bone induction model, angiogenesis is correlated with chondrolysis and concomitant osteogenesis. Basement membrane components including laminin, collagen IV, and factor VIII antigen are localized around invading capillaries prior to and during osteogenesis (Foidart and Reddi, 1980). These observations,together with data on TGF-P and osteogenin binding to

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type IV collagen (Paralkar et al., 1990, 1991), prompted us to examine whether basement membrane extracellular matrix components might influence bone cell differentiation in vitro. A. Osteoblastic Cel Is Recognize Basement Membrane Components

Primary calvarial cells and an osteoblastic cell line (MC3T3-El) recognized components of basement membrane and underwent profound morphological changes when cultured on the surface of basement membrane. Colonies and canalicular cell processes that required both collagen IV and laminin were observed (Vukicevic et al., 1990~).Antibodies to laminin and to a laminin receptor, as well as certain biologically active laminin-derived synthetic peptides, blocked the cellular differentiation on the basement membrane (Matrigel). Direct interaction of bone cells with the laminin-derived peptides was demonstrated in cell binding assays (Table 5). Type IV collagen also appeared to be an active component in the Matrigel, since its removal resulted in loss of bone cell organization (Vukicevic et a]., 1990~).In addition, ascorbic acid, which promotes collagen synthesis, together with P-glycerophosphate, increased calcified bone nodule formation on Matrigel. These data demonstrate that osteoblastic cells interact with basement membrane components, and suggest that such a matrix may modulate the phenotype of bone cells. Moreover, basement membrane plays a significant role in vitro in formation of a network of cytoplasmic processes resembling the osteocyte network in bone. Table 5. Attachment of Osteoblastic Cells to Laminin-Derived Peptides Subst rnre

Laminin CDPGYIGSR Ac-YIGER-* FALRGDNP FALRDGNP* CSRARKQAASIKVAVSADR CRKQAAS1K VAVA CIKVAVA KKSAVQARI VAS *

Residire (cliuIn)

Number of Wells

12 925-933 ( B I )

16

1120-1127 (A)

10 12 8

2091-2108 (A) 2094 = 2105 (A) 2099-2105 (A)

16

8 16

8

Cells per Field MC3T3-El Cells

32 = 4 35 = 5 0 23 2 4 0 33 2 3

29 3 28 + 4 _+

0

Notes: Peptideswere synthesizedtocomespond tomouse laminin Aand BI ChaincDNAsequences. The NH2termin;ll cysteineinrhepeptidesisnotpresentithe [amininchainsbut wasincludedinthesynrhesis tofacilitatecoupling. and an mi& group was present in all peptides at the carboxyl terminus. The peptides were applied to nitrocellulose at 1 nip/ml. b n i i n i n (100 pglml) served as a positive control. Values given a~ nieans t S.E.M. Asterisks indicate peptides a1 the carboxyl terminus. The peptides were applied to nitrocellulose at I mg/ml. Laminin(100Cl%ml)servedasapositive control. Values givenare means t S.E.M. Asterisks indicate peptides with scrambled sequence usedas control. Reprinted with permission: Vukicevic et al.. Cell 63,437. 1990.

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Osteocytes do not directly contact basement membrane in vivo. The osteocyte is a developmental stage of the osteoprogenitor osteoblast lineage, and thus may retain a “memory” of the initial contact of osteoblast with laminin. Most osteons demonstrate a central blood vessel with the basement membrane in contact with the first circle of concentric osteoblasts. This initial contact may set into motion a ripple-like cascade of cell differentiation.

B. Growth Factors in Basement Membrane Regulate Osteoblastic Network Formation

Additional studies have demonstrated the presence of TGF-P, epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-I), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) in the basement membrane, Matrigel (Vukicevic et al., 1992). These factors significantly contributed to the behavior of osteoblastic cells (MC3T3-EI) when cultured on Matrigel. Exogenous TGF-P blocked the migration and cellular network formation. Conversely, addition of TGF-P neutralizing antibodies to Matrigel stimulated the cellular network formation. bFGF, EGF, and PDGF all promoted cellular migration and organization on Matrigel. Addition of bFGF to MC3T3-EI cells grown on Matrigel overcame the inhibitory effect of TGF-P. TGF-P cannot be removed from Matrigel by repeated 20%ammonium sulfate precipitations. Some TGF-P also remained bound to type IV collagen purified from reconstituted basement membrane, suggesting caution in the interpretation of experiments on cellular activity related to Matrigel, collagen type IV, and possibly other extracellular matrix components (Vukicevic et al., 1992). These data confirmed the above mentioned results of Paralkar et al. (1991)of TGF-P binding to type IV collagen of the basement membrane, suggesting that TGF-P may be a basement membrane component in vivo. All of these investigations provide a conceptual framework to explain the physiological role of blood vessels in osteogenesis,and moreover, provide evidence for the role of basement membrane macromolecules in the phenotypic modulation of osteoblastic cells.

V. CHALLENGES FOR THE FUTURE What is the precise cellular and moleculy basis of the interaction of endothelial elements and the osteoblastic lineage? Is there a causal role for basement membrane components and osteoprogenitor differentiation? What are the molecular domains in laminin which signal the osteoblastic lineage? What are the signal transducing mechanisms? These are exciting challenges for the future and provide opportunities for molecular insights into morphogenesis of bone. A better understanding of these molecular interactions will pave the path to solving baffling clinical problems, such as avascular necrosis of the femoral head, nonunion of fractures, and optimal biological fixation of the implant interface in the prostheses.

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