Activation of latent transforming growth factor .beta. by matrix vesicles

A latent growth factor such as transforming growth factor beta (TGF.beta.) is converted to active form by matrix vesicles or an extract from matrix vesicles. The matrix vesicles may be stimulated with a Regulator of Enhancing Factor (REF) such as 1,25-dihydroxy vitamin D (1,25-(OH).sub.2 D.sub.3) or steroid hormones which may be intercalated into the vesicle membrane. The latent growth factor may be activated in culturing cells such as chondrocytes that have been pretreated with 24,25-(OH).sub.2 D.sub.3 to activate cell differentiation, or in healing of bone or cartilage defects, and activation can be carried out in vivo or in vitro. Biodegradable polymeric implants may be prepared containing latent growth factor, REF, matrix vesicle or matrix vesicle extract.

FIELD OF THE INVENTION 
This invention lies in the field of compositions and methods for effecting 
wound healing, specifically, the activation of latent growth factor 
through matrix vesicles by stimulation with Regulator of Enhancing Factor 
(REF). 
BACKGROUND OF THE INVENTION 
Endochondral bone formation consists of a developmental cascade of cellular 
differentiation that culminates in extracellular matrix mineralization. 
The process is required for normal growth and development of long bones 
and for certain kinds of bone repair. During the chondrogenic phase of the 
process, chondrocytes are responsible for the synthesis, maintenance and 
maturation of a calcifiable extracellular matrix that is composed mainly 
of proteoglycan and collagen. (Boskey, A. L. (1991), "Current concepts of 
the physiology and biochemistry of calcification," Clin. Orthop. 
157:225-257; Howell, D. S. and Dean, D. D. (1992), "Biology, chemistry and 
biochemistry of the mammalian growth plate," In: Disorders of Bone and 
Mineral Metabolism, Coe, F. L. and Favus, M. J. (eds), Raven Press Ltd., 
New York 313-353.) 
The complex regulation of chondrocyte differentiation by growth factors 
such as TGF.beta. and other hormones has been shown by numerous 
investigators. (Crabb, I. D., et al. (1990), "Synergistic effect of 
transforming growth factor-.beta. and fibroblast growth factor on DNA 
synthesis in chick growth plate chondrocytes," J. Bone Min. Res. 
5:1105-1112; Kinoshita, A., et al. (1992), "Demonstration of receptors for 
epidermal growth factor on cultured rabbit chondrocytes and regulation of 
their expression by various growth and differentiation factors," Biochem. 
Biophys. Res. Comm. 183:14-20; Suzuki, F. (1992), "Effects of various 
growth factors on a chondrocyte differentiation model," Adv. Exper. Med. 
and Biol. 324:101-106; Thorp, B. H., et al. (1992), "Transforming growth 
factor-.beta.1, -.beta.2, and -.beta.3 in cartilage and bone cells during 
endochondral ossification in the chick," Development 114:907-911). 
Vitamin D.sub.3 is known to be an essential regulator of this complex 
process, and both 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 are 
involved. (Raisz, L. G. and Kream, B. E. (1983), "Regulation of bone 
formation," (first of two parts), N. Engl. J. Med. 309:29-35; Raisz, L. G. 
and Kream, B. E. (1983), "Regulation of bone formation," (second of two 
parts), N. Engl. J. Med. 309:83-89; Canterbury, J. M., et al. (1980), 
"Metabolic Consequences of oral administration of 24,25 
hydroxycholecalciferol to uremic dogs," J. Clin. Invest. 65:571-580; 
Liberherr, M. et al. (1979), "Interaction of 24,25-dihydroxyvitamin 
D.sub.3 and parathyroid hormone on bone enzymes in vitro," Calcif. Tissue 
Int. 27:47-53; Ornoy, A., et al. (1978), "24,25-Dihydroxyvitamin D.sub.3 
is a metabolite of vitamin D essential for bone formation," Nature 
276:517-520; and Norman, A. W. (1980), "1,25-Dihydroxyvitamin D.sub.3 and 
24,25-dihydroxyvitamin D.sub.3 : key components of the vitamin D endocrine 
system. Contr. Nephrol. 18:1-11; Grigoriadis, A. E., et al. (1989), 
"Effects of dexamethasone and vitamin D.sub.3 on cartilage differentiation 
in a clonal chondrogenic cell population," Endocrinology 125:2103-2110; 
Schwartz, Z., et al. (1992), "Direct effects of transforming growth factor 
.beta. on chondrocytes are modulated by vitamin D metabolites in a cell 
maturation specific manner," Endocrinology 132:1544-1552; Schwartz, Z. et 
al., "Differential Regulation of prostaglandin E2 synthesis and 
phospholipase A.sub.2 activity by 1,25-(OH).sub.2 D.sub.3 in three 
osteoblast-like cell lines (MC-373-E1), ROS 17/2.8 and MG-63", Bone (1992) 
13:51-58.) 
Matrix vesicles, and the phospholipids present in them, are involved in 
initial formation of calcium hydroxyapatite crystals via the interaction 
of calcium and phosphate ions with phosphatidylserine to form 
phospholipid:Ca:Pi complexes (CPLX). CPLX is present in tissues which are 
undergoing initial mineral deposition but are absent from nonmineralizing 
tissues. Evidence suggests that CPLX resides in the interior of matrix 
vesicles where the earliest mineral crystals are formed in association 
with the vesicle membrane. More recently, it has been determined that 
specific membrane proteins, called proteolipids, participate in CPLX 
formation and hydroxyapatite deposition, in part by structuring 
phosphatidylserine in an appropriate conformation. Phosphatidylserine 
involvement in the initiation of mineralization has been extensively 
investigated because of its extremely high binding affinity for Ca.sub.2+. 
In addition to structuring a specific phospholipid environment, 
proteolipids may also act as ionophores, promoting export of protons and 
import of calcium and phosphate, both requirements of biologic 
calcification (Boyan, B. D. et al., "Role of lipids in calcification of 
cartilage," Anat. Rec. (June 1989) 224(2):211-219). 
There is a known correlation between in vivo bone formation and in vitro 
production of normal matrix vesicles (Boyan, B. D. et al., "Epithelial 
cell lines that induce bone formation in vivo produce alkaline 
phosphatase-enriched matrix vesicles in culture," Clin. Orthop. (April 
1992) 266-276). 
Many cells produce growth factors in latent form and store them in their 
extracellular matrix, or they may store them in an inactive form via 
specific binding proteins. These growth factors may be activated at a 
later time and act on the original cell as autocrine factors, or a 
neighboring cell as paracrine factors, or they may be released into the 
circulation and have a systemic effect as endocrine agents. One function 
of the extracellular matrix vesicles is to transport enzymes for matrix 
modification (Boskey, A. L. et al., "Studies of matrix-vesicle-induced 
mineralization in a gelatin gel," Bone Miner. 17:257-262). Matrix vesicles 
are selectively enriched in enzymes that degrade proteoglycans (Dean, D. 
D. et al., "Matrix vesicles contain metaloproteinases that degrade 
proteoglycans," Bone Miner. (1992) 17:172-176). 
Transforming growth factor beta (TGF.beta.) is an important regulator of 
cartilage development and chondrocyte differentiation (Seyedin, S. M., et 
al., J. Biol. Chem (1987) 262:1946-1949; Seyedin, S. M., et al., Proc. 
Natl. Acad. Sci. USA (1985) 82:2267-2271; Seyedin, S. M., et al., J. Biol. 
Chem. (1986) 261:5693-5695). It is synthesized by chondrocytes and appears 
to act in an autocrine manner (Gelb, D. E., et al., Endocrinology (1990) 
127:1941-1947; Schwartz, Z., et al., "Direct effects of transforming 
growth factor-beta on chondrocytes are modulated by vitamin D metabolites 
in a cell maturation-specific manner," Endocrinology (1993) 132:1544-1552; 
Rosier, R. N., et al., Connect. Tissue Res. (1989) 20:295-301). TGF.beta. 
production varies with stage of chondrocyte differentiation. 
TGF.beta. is produced by many cell types in a latent form which may be 
released into the circulation, as during platelet lysis (Wakefield, L. M., 
et al., J. Biol. Chem. (1988) 263:7646-7654; Miyazono, K., et al., J. 
Biol. Chem. (1988) 263:6407-6415) or targeted for storage in the 
extracellular matrix (Dallas, S. L., et al., J. Biol. Chem. (1994) 
269:6815-6822). Latent TGF.beta. exists in a number of macromolecular 
forms. Recombinant human TGF.beta..sub.1 is a homodimer of 100 kD which 
contains a latency-associated peptide non-covalently bound to the mature 
TGF.beta. molecule (Gentry, L. E., et al. (1987), Mol. Cell. Biol. 
7:3418-3427). Latent TGF.beta. synthesized by fibroblasts consists of a 
similar or identical 100 kD homodimer covalently bound through a cysteine 
residue to a 190 kD TGF.beta. binding protein (Kanzaki, T., et al. (1990), 
Cell 61:1051-1061; Tsujmi, T., et al. (1990), Proc. Natl. Acad. Sci. 
U.S.A. 87:8835-8839). Platelets produce a latent TGF.beta. that contains a 
truncated form of the 190 kD binding protein (Wakefield, et al. (1988), J. 
Biol. Chem. 263:7646-7654). Bone cells produce large amounts of the 100 kD 
complex (Bonewald, L. et al. (1991), Mol. Endocrinol. 5:741-751) in 
addition to the fibroblast form of latent TGF.beta. (Dallas, S. L., et al. 
(1994), J. Biol. Chem. 269:6815-6822). 
Storage of latent TGF.beta. and the mechanism, as well as timing, of 
activation of latent TGF.beta. appears to be specific for each cell and 
tissue type. A variety of factors may stimulate cells to activate latent 
TGF.beta.. For example, macrophages treated with .gamma.-interferon 
activate latent TGF.beta. (Twardzik, D. R., et al., Ann. N.Y. Acad. Sci. 
(1990) 593:276-284), as will osteoclasts treated with retinol (Oreffo, 
R.O.C., et al., Biochem. Biophys. Res. Comm. (1989) 153:817-823). 
Local production of acid may be one mechanism by which latent TGF.beta. is 
activated. For example, it is believed that latent TGF.beta. in milk is 
activated by stomach acid and that the active form is transported through 
the gut (Saito, S., et al., Clin. Exp. Immunol. (1993) 94:220-224). While 
acid pH can activate latent TGF.beta., it is clear that proteases play an 
important role in most systems. Endothelial cells activate latent 
TGF.beta. through the plasmin system (Sato, Y. and Rifkin, D. B., J. Cell 
Biol. (1989) 109:309-315). Arian osteoclasts appear to use multiple 
proteases in addition to acid pH (Oursler, M. J., J. Bone Min. Res. (1994) 
9:443-452). In growth plate cartilage and unmineralized osteoid in bone, 
where local generation of acid has not been reported, participation of 
proteases is an attractive option. 
Recent studies have shown that proteinases, including neutral and acid 
metalloproteinases and plasminogen activator, and various peptidases are 
present at high levels in matrix vesicles (Hirschman, A., et al., Calcif. 
Tissue Int. (1983) 35:791-797; Einhorn, T. A., et al., J. Orthop. Res. 
(1989) 7:792-805; Dean, D. D., et al., "Matrix vesicles are enriched in 
metalloproteinases that degrade proteoglycans," Calcif. Tissue Int. (1992) 
50:342-349). These extracellular organelles are membrane bounded, produced 
by chondrocytes and osteoblasts in vivo (Anderson, H. C., J. Cell Biol. 
(1969) 41:59-72; Schwartz, Z., et al., Bone (1989) 10:53-60) and in vitro 
(Boyan, B. D., et al., "Differential expression of phenotype by resting 
zone and growth region costochondral chondrocytes in vitro," Bone (1988) 
9:185-194; Boyan, B. D., et al., J. Biol. Chem. (1989) 264:11879-11886; 
Ecarot-Charrier, B., et al., Bone (1988) 9:147-154), are found in the 
extracellular matrix, and are associated with modification of the 
extracellular matrix prior to calcification. 
Matrix vesicles have a distinctive phospholipid composition and enzyme 
activity. Their characteristics are cell-maturation dependent. Regulation 
of matrix vesicle structure and function occurs at the genomic and 
non-genomic levels. By following alkaline phosphatase gene transcription, 
protein concentration, and enzyme specific activity, it has been shown 
that steroid hormones and growth factors exhibit a regulatory influence 
over gene transcription, protein synthesis, and matrix vesicle activity. 
Matrix vesicles respond to peptide hormones such as testosterone 
(Schwartz, Z., et al. "Gender-specific, maturation-dependent effects of 
testosterone on chondrocytes in culture," Endocrinology (1994) 
134:1640-1647); estrogen (Nasatzky, E., et al., "Sex-dependent effects of 
17-beta-estradiol on chondrocyte differentiation in culture," J. Cell 
Physiol. (1993) 154:359-367); growth factors such as TGF.beta. (Bonewald, 
L. F., et al., "Stimulation of plasma membrane and matrix vesicle enzyme 
activity by transforming growth factor-beta in osteosarcoma cell 
cultures," J. Cell Physiol (1990) 145:200-206); other matrix proteins, 
like alpha 2-HS-glycoprotein (Yang, F. et al., "Alpha 2-HS-glycoprotein: 
expression in chondrocytes and augmentation of alkaline phosphatase and 
phospholipase A2 activity," Bone (1991) 12:7-15); and autocoid mediators 
like prostaglandins as well. Calcifying cells can modulate events in the 
matrix via direct autocrine/paracrine stimulation or inhibition of the 
matrix vesicles. 1,25-dihydroxy vitamin D.sub.3 (1,25-(OH).sub.2 D.sub.3) 
and 24,25-dihydroxy vitamin D.sub.3 (24,25-(OH).sub.2 D.sub.3) regulate 
matrix vesicle phospholipase A.sub.2 activity, fatty acid turnover, 
arachidonic acid release, PGE2 production, and membrane fluidity, which 
can act on the matrix vesicle to alter enzyme activity (Boyan, B. D., et 
al., "Cell maturation-specific autocrine/paracrine regulation of matrix 
vesicles," Bone Miner. (May 1992) 17(2):263-268). 
Matrix vesicle structure and function, as well as extracellular matrix 
synthesis by osteoblasts and chondrocytes, are regulated by TGF.beta. as 
well as vitamin D metabolites (Schwartz, Z., et al., Endocrinology (1993) 
132:1544-1552; Miyazono, K., et al., J. Biol. Chem. (1988) 263:6407-6415; 
Bonewald, L., et al., J. Cell Physiol. (1990) 145:200-206; Boyan, B. D., 
et al., "In vitro studies on the regulation of endochondral ossification 
by vitamin D," Crit. Rev. Oral Biol. Med. (1992) 3(1/2):15-30; Schwartz, 
Z., et al., Endocrinology (1988) 123:2878-2884; Boyan, B. D. et al., 
"Matrix vesicles as a marker of endochondral ossification," Connect. 
Tissue Res. (1990) 24:67-75; Bonewald, L. F. et al., "Stimulation of 
matrix vesicle enzyme activity in osteoblast-like cells by 1,25-(OH).sub.2 
D.sub.3 and transforming growth factor beta (TGF beta)," Bone Miner. 
(1992) 17:139-144); Swain, L. D. et al., "Regulation of matrix vesicle 
phospholipid metabolism is cell maturation-dependent," Bone Miner. (1992) 
17:192-196). Moreover, it appears that these two regulators interact in a 
specific manner during cell differentiation. The details of this 
interaction have been partially elucidated by using chondrocytes derived 
from costochondral cartilage. Resting zone and growth zone chondrocytes 
constitutively produce 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 
D.sub.3, and TGF.beta. regulates this production (Schwartz, Z., et al., 
Endocrinology (1993) 132:1544-1552). Vitamin D metabolites alter membrane 
fluidity (Swain, L. D., et al., "Nongenomic regulation of chondrocyte 
membrane fluidity by 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 
is dependent on cell maturation," Bone (1993) 14:609-617) and enzyme 
activity (Schwartz, Z. and Boyan, B., Endocrinology (1988) 122:2191-2198) 
of isolated matrix vesicles in vitro. Nongenomic effects of 
1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 have been reported 
and include alterations in arachidonic acid turnover (Schwartz, Z., et 
al., "Regulation of arachidonic acid turnover by 1,25-(OH).sub.2 D.sub.3 
and 24,25-(OH).sub.2 D.sub.3 in growth zone and resting zone chondrocyte 
cultures," Biochim. Biophys. Acta (1990) 102:278-286; Swain, L., et al., 
Biochim. Biophys. Acta (1992) 1136:45-51; Boyan, B. et al., Connect. 
Tissue Res. (1989) 22:3-16), calcium ion flux (Langston, G. G., et al., 
Calcif. Tissue Int. (1990) 17:230-236; Schwartz, Z. et al., "Inhibition of 
1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 -dependent 
stimulation of alkaline phosphatase activity by A23187 suggests a role for 
calcium in the mechanism of vitamin D regulation of chondrocyte cultures," 
J. Bone Min. Res. (1991) 6:709-718), and protein kinase C activity 
(Sylvia, V. L., et al., "Maturation-dependent regulation of protein kinase 
C activity by vitamin D.sub.3 metabolites in chondrocyte cultures," J. 
Cell Physiol. (1993) 157:271-278). 
TGF.beta. and vitamin D have been shown to synergize with respect to 
alkaline phosphatase induction in bone cell lines (Bonewald, L. F., et 
al., Mol. Endocrinol. (1991) 5:741-751; Bonewald, L. F., et al., "Effects 
of combining transforming growth factor beta and 1,25-dihydroxyvitamin 
D.sub.3 on differentiation of a human osteosarcoma (MG-63)," J. Biol. 
Chem. (1992) 267:8943-8949), primary human bone cells (Wegedahl, J. E., et 
al., Metabolism (1992) 41:42-48), and rat resting zone chondrocytes 
(Schwartz, Z., et al., Endocrinology (1993) 132:1544-1552). Both TGF.beta. 
and vitamin D regulate chondrocyte differentiation. Exogenous TGF.beta. 
stimulates DNA synthesis and matrix formation in chick growth plate 
chondrocytes (Rosier, R. N., et al., Calcif. Tissue Res. (1988) 
20:295-301; Crabb, I. D., et al., J. Bone Min. Res. (1990) 5:1105-1112; 
O'Keefe, R., et al., J. Bone Min. Res. (1988) 3:S67). In rat growth plate 
chondrocytes, rhTGF.beta.1 regulates alkaline phosphatase, phospholipase 
A.sub.2 (Schwartz, Z., et al., Endocrinology (1993) 132:1544-1552), as 
well as vitamin D metabolite production (Schwartz, Z., et al., 
Endocrinology (1992) 130:2495-2504). Cellular response to TGF.beta. 
depends on the state of endochondral maturation, with resting zone cells 
exhibiting a differential response compared to that observed in growth 
zone cell cultures. Similarly, vitamin D metabolites also regulate the 
expression of alkaline phosphatase (Schwartz, Z. and Boyan, B., 
Endocrinology (1988) 122:2191-2198), phospholipase A.sub.2, and protein 
kinase C (Sylvia, V. L., et al., "Maturation-dependent regulation of 
protein kinase C activity by vitamin D.sub.3 metabolites in chondrocyte 
cultures," J. Cell Physiol. (1993) 157:271-278) in chondrocytes in a cell 
maturation-specific manner. 
Active metalloproteinases are present in matrix vesicles (Hirschman, A., et 
al., Calcif. Tissue Int. (1983) 35:791-797; Einhorn, T. A., et al., J. 
Orthop. Res. (1989) 7:792-805; Dean, D. D., et al., Calcif. Tissue Int. 
(1992) 50:342-349). In growth plate, the immunohistochemical distribution 
of TGF.beta.1 (Jingushi, S., et al., Calcium Regulation and Bone 
Metabolism, Cohn, D. V., Glorieux, F. H., and Martin, T. J. (eds.), 
Elsevier Science Publishers (Biomedical Division) New York, (1990) Vol. 
10,298-303) coincides with the localization of matrix vesicles in the 
territorial matrix of the cells (Anderson, H. C., J. Cell Biol. (1969) 
41:59-72). Active acid and neutral metalloproteinases, as well as 
plasminogen activator, are present in matrix vesicles and require physical 
destruction of the matrix vesicle membrane for their release (Dean, D. D., 
et al., Calcif. Tissue Int. (1992) 50:342-349). 
Other enzymes present in matrix vesicles are sensitive to regulation by 
TGF.beta. and vitamin D metabolites (Schwartz, Z., et al., Endocrinology 
(1993) 132:1544-1552; Schwartz, Z., et al., Endocrinology (1988) 
123:2878-2884; Sylvia, V. L., et al., J. Cell Physiol. (1993) 157:271-278; 
Boyan, B. D., et al., Endocrinology (1988) 122:2851-2860). In both 
instances the effects are cell maturation-dependent and vitamin D 
metabolite-specific. 1,25-(OH).sub.2 D.sub.3 stimulates matrix vesicle 
phospholipase A.sub.2 (Schwartz, Z. and Boyan, B., Endocrinology (1988) 
122:2191-2198), increasing the production of lyso derivatives, resulting 
in loss of membrane integrity (Ginsburg, L. et al., Inflammation (1992) 
16:519-538). In contrast, 24,25-(OH).sub.2 D.sub.3 inhibits matrix vesicle 
phospholipase A.sub.2 (Schwartz, Z. and Boyan, B., Endocrinology (1988) 
122:2191-2198), potentially resulting in a more stable membrane and 
retention of metalloproteinases within the matrix vesicle. 
Matrix vesicle membrane fluidity (Swain, L. D., et al., Bone (1993) 
14:609-617) and enzyme activity (Schwartz, Z., et al., Endocrinology 
(1988) 123:2878-2884) can be directly and specifically regulated by 
1,25-(OH).sub.2 D.sub.3 in the absence of the cell and its molecular and 
protein synthetic machinery. 
Matrix vesicles have been associated with wound healing (Schmitz, J. et 
al., Acta Anatomica (1990) 138:185-192; Einhorn, T. A. et al., J. Orthop. 
Res. (1989) 7:792-805; Brighton, C. T. and Hunt, R. M., Clin. Orth. Rel. 
Res. (1974) 100:406-416), however the role of matrix vesicles in wound 
healing has not previously been known. Endochondral wound healing is 
stimulated by application of electrical energy possibly through 
stimulation of matrix vesicle production by cells. C. T. Brighton and R. 
M. Hunt noted that stimulation of non-union tissue with electromagnetic 
fields causes an increase in the number of matrix vesicles as well as in 
the formation of crystals and calcification of the matrix. This was 
followed by healing of the nonunion defect with calcified cartilage and 
bone. 
Cartilage and bone wound healing are also aided through placing implants 
made of bioerodible polymers into the defects. Such bioerodible polymers 
are described, e.g. in U.S. patent application Ser. No. 08/123,812 filed 
Sep. 20, 1993, and corresponding PCT publication WO/9315694, published 
Aug. 19, 1993, and U.S. Pat. No. 08/196,970 filed Feb. 15, 1994, all of 
which are incorporated herein by reference. Such implants may contain 
growth factors and other agents for promotion of wound healing. 
Bone-bonding implants such as KG Cera, Mina 13, and titanium support an 
increase in matrix vesicle concentration compared with nonbone-bonding 
implants (Schwartz, Z. et al., "Effect of glass ceramic and titanium 
implants on primary calcification during rat fibial bone healing," Calcif. 
Tissue Int. (1991) 49:359-364) and also lead to increased alkaline 
phosphatase and phospholipase A.sub.2 (Schwartz, Z. et al., "In vivo 
regulation of matrix vesicle concentration and enzyme activity during 
primary bone formation," Bone Miner. (1992) 17:134-138; Schwartz, Z. et 
al., "Modulation of matrix vesicle enzyme activity and phosphatidylserine 
content by ceramic implant materials during endosteal bone healing," 
Calcif. Tissue (1992) 51:429-437). Hydroxyapatite implants behave like 
bone-bonding implants in that there is a stimulation of matrix vesicle 
enzymes, increased phosphatidylserine content and increased numbers of 
matrix vesicles (Schwartz, Z. et al., "Effects of hydroxyapatite implants 
on primary mineralization during rat fibial healing: biochemical and 
morphometric analysis," J. Biomed. Mater. Res. 27:1029-1038). 
Biodegradable polymeric scaffold systems seeded with cells are useful for 
culture of specific types of cells in vitro. U.S. Pat. No. 4,963,489 to 
Naughton et al. issued Oct. 16, 1990 for "Three-Dimensional Cell and 
Tissue Culture System," incorporated herein by reference, discloses the 
use of a polymeric matrix for culture of cells such as skin, liver, 
pancreas, bone marrow, osteoblasts and chondrocytes, etc. in vitro. The 
seeded matrix may be transplanted in vivo. Related U.S. Pat. No. 5,032,508 
to Naughton et al. for "Three-Dimensional Cell and Tissue Culture System," 
also incorporated herein by reference, contains a similar disclosure. A 
further related U.S. Pat. No. 5,160,490 to Naughton et al. issued Nov. 3, 
1992 for "Three-Dimensional Cell and Tissue Culture Apparatus," 
incorporated herein by reference, discloses that hip prostheses coated 
with three-dimensional cultures of cartilage may be implanted into 
patients. This patent also discloses that proteins can be "added to" the 
matrix or coated on. 
SUMMARY OF THE INVENTION 
This invention provides compositions and methods useful in wound healing. 
One such composition comprises matrix vesicles and/or matrix vesicle 
extract and Regulator of Enhancing Factor (REF). Another such composition 
also comprises latent growth factor in addition to the matrix vesicles and 
REF. A further composition comprises latent growth factor in combination 
with matrix vesicles and/or matrix vesicle extract. Further compositions 
of this invention comprise latent growth factors in combination with REF 
which are applied to a medium comprising matrix vesicles. 
Matrix vesicles are membrane-bounded bodies secreted by cells involved in 
matrix formation, such as bone, cartilage and tendon cells. The matrix 
vesicles contain enzymes, hormones, and other factors which aid in matrix 
formation and which stimulate the cells in an autocrine manner. As 
secreted by the cells, they do not contain genetic material. Matrix 
vesicles may be isolated from a mammalian source, preferably from the 
patient (which may be a human or other mammal) to be treated with the 
matrix vesicles. A source of the same species, preferably a source known 
to be histocompatible with the patient, may also be used. Procedures for 
isolating matrix vesicles are described herein. Matrix vesicles may also 
be synthesized as described herein. Matrix vesicle extract may be used in 
place of matrix vesicles and may be prepared from isolated matrix vesicles 
as described herein. 
The compositions of this invention enhance wound healing when targeted to a 
wound site, preferably a cartilage or bone wound site. The compositions 
may be administered by means known to the art such as injection in a 
suitable pharmaceutical carrier, encapsulation in microspheres, e.g. for 
timed release, or incorporation into a biodegradable implant such as those 
described in U.S. patent application Ser. No. 08/123,812 filed Sep. 20, 
1993, and corresponding PCT publication WO/9315694, published Aug. 19, 
1993, and U.S. Pat. No. 08/196,970 filed Feb. 15, 1994, all of which are 
incorporated herein by reference. Preferably the compositions are 
incorporated into a timed release implant providing for release of the 
composition at the appropriate time during the wound healing. The implant 
may be a continuous release implant or may provide for release of the 
composition at specific times during wound healing for appropriate 
activation of the matrix. For example, Schmitz et al. (Acta Anatomica 
(1990) 138:185-192) have shown that critical size defects in the cranium 
of rats fail to heal. The failure of bone to form is evident 17 days post 
surgery, indicating that intervention must occur before this time. As 
known to the art, an initial burst of active growth factor is required for 
optimal healing in some instances. In other cases, continuous steady 
release is preferred. Implants of this invention incorporating latent 
growth factor may be designed to provide an initial burst of released 
latent growth factor or a continuous steady release of latent growth 
factor. Extraneous REF and matrix vesicles or extraneous matrix vesicle 
extract may be added to the wound site at the desired times, e.g. 
initially within 24 hours to provide an initial burst of active growth 
factor and/or when cell differentiation is desired, such as after about 
three days, and at about three-day intervals up to about 17 days. 
The composition may also be used in vitro to stimulate growth and 
differentiation of cells, preferably osteoblasts or chondrocytes, in cell 
culture. It may also be incorporated onto scaffolding material for cell 
growth for later implantation into the host such as that described in U.S. 
Pat. Nos. 4,963,489, 5,032,508, and 5,160,490 to Naughton et al., also 
incorporated herein by reference. 
REF is a substance which acts upon matrix vesicles to cause release of 
activating factors for growth factors, such as TGF.beta. activating 
factor. As discussed above, TGF.beta. is produced in latent form by many 
cell types and consists of a homodimer of 100 kD bound to an additional 
protein which may be a binding protein. TGF.beta. activating factor is a 
protease contained within matrix vesicles which releases the 100 kD active 
form of TGF.beta.. 
As is known to the art, other growth factors are also produced in naturally 
latent form or may be synthesized in latent form. Such growth factors 
include insulin-like growth factors, fibroblast growth factors, bone 
morphogenetic proteins, and platelet-derived growth factors. 
Matrix vesicles contain growth factor activating factors which, upon 
release from matrix vesicles stimulated with REF, activate such latent 
growth factors. 
Some REFs are produced in vitro by the cells which produce matrix vesicles, 
and, as described in this invention, may also be added to a medium 
containing matrix vesicles either in vivo or in vitro to enhance release 
of factors which convert growth factors from latent to active form. As the 
matrix vesicles in their natural state contain no genetic machinery, the 
REFs act in a non-genomic manner, e.g., by altering membrane fluidity. 
Examples of REFs include vitamin D metabolites such as 
1,25-dihydroxy-vitamin D (1,25-(OH).sub.2 D.sub.3). When 1,25-(OH).sub.2 
D.sub.3 is used to stimulate activation of growth factor in vitro, it is 
preferably applied to cartilage cells in the growth zone stage of 
maturation or to differentiated osteoclasts. 
When it is desired to stimulate activation of growth factor in resting zone 
stage cells, 24,25 dihydroxy-vitamin D (24,25-(OH).sub.2 D.sub.3) or 
active TGF.beta. may be used in combination with 1,25-(OH).sub.2 D.sub.3 
to stimulate production of matrix vesicles. The 24,25-(OH).sub.2 D.sub.3 
stimulates new matrix vesicle production by the cells through a genomic 
mechanism. When 24,25-(OH).sub.2 D.sub.3 is used directly on matrix 
vesicles, it may regulate the rate at which they are activated by 
inhibiting breakdown of the matrix vesicle membrane. 
Steroid hormones are another class of REF, including estrogen, e.g. 
17-beta-estradiol, testosterone, and dexamethasone. Thyroid hormone 
(T.sub.3) is also considered to be a REF. 
Prostaglandins and other lipophilic mediators of membrane action such as 
leukotrienes and platelet activating factor comprise a useful class of 
REFs. 
The components of the compositions of this invention are present in 
pharmaceutically effective amounts, which means amounts effective to 
convert latent growth factor to active growth factor in measurable 
quantities, such that measurable effects on wound healing and tissue 
growth and/or differentiation occur. A pharmaceutically effective amount 
of REF is an amount sufficient to stimulate release by matrix vesicles of 
TGF.beta. activating factor in an amount sufficient to convert latent 
TGF.beta. to active TGF.beta.. Such amounts of REF may vary from minimal 
amounts necessary to produce a measurable amount of active TGF.beta., 
assayed directly or by means of enhancement of wound healing, to a maximal 
amount equalling or exceeding the amount necessary to convert all latent 
TGF.beta. to active form. 
In a preferred embodiment involving the use of 1,25-(OH).sub.2 D.sub.3, a 
10.sup.-8 to 10.sup.-12 M solution is used, preferably a 10.sup.-8 to 
10.sup.-9 M solution. Such a solution incubated with a suspension of 
matrix vesicles containing 1.6 mg protein per ml will activate a 
measurable amount of latent TGF.beta., i.e. use of 8 .mu.l of a 10.sup.-7 
M solution of 1,25-(OH).sub.2 D.sub.3 in 80 .mu.l of the matrix vesicle 
suspension, resulting in a final concentration of 10.sup.-8 M 
1,25-(OH).sub.2 D.sub.3, will activate 0.6 ng/ml of latent TGF.beta.. When 
latent growth factors other than TGF.beta. are present, analogous molar 
ratios of matrix vesicles and REFs are used. Similarly analogous molar 
ratios of REFs other than 1,25-(OH).sub.2 D.sub.3 may be used. The REF may 
be injected locally into a healing wound, incorporated into an implant, or 
delivered by other means known to the art. 
In healing bone and cartilage defects, matrix vesicles are present in a 
concentration of about 2-10 .mu.g per .mu.m.sup.2 area of matrix. In a 
growing culture of bone or cartilage cells, matrix vesicles are naturally 
present at a concentration of about 5-50 .mu.g matrix vesicle protein per 
150 cm.sup.2 of confluent monolayer cells. When REF is added to a healing 
wound or culture, it is preferably added in an amount sufficient to 
stimulate matrix vesicles present to release TGF.beta. activating factor, 
e.g., in an amount of about 100 pico M/cm.sup.3. 
When extraneous matrix vesicles are added to a defect in vivo or to a 
culture, they are added in amount which will produce measurable 
enhancement of wound healing or activation of latent TGF.beta.. A useful 
amount of matrix vesicles is between about 10 ng/cm.sup.3 and about 2.0 
mg/cm.sup.3, preferably between about 1.8 mg/cm.sup.3 and about 1.4 
mg/cm.sup.3 and more preferably between about 1.5 and about 1.7 
mg/cm.sup.3. Matrix vesicle extract may be used instead of or in addition 
to whole matrix vesicles. A useful amount of matrix vesicle extract is 
between about 200 .mu.g protein/cm.sup.3 and about 800 .mu.g 
protein/cm.sup.3, preferably between about 400 and about 600 and more 
preferably between about 450 and about 550. 
REF may be added in addition to matrix vesicles, preferably at a ratio to 
the added matrix vesicles as set forth above, i.e. about 1:10 by volume of 
REF solution to matrix vesicle suspension, said REF solution having a 
concentration between about 10.sup.-8 and about 10.sup.-12 M and said 
matrix vesicle suspension having about 1.6 mg protein/ml. 
The inventors have discovered that resting zone chondrocytes are activated 
by the vitamin D metabolite 24,25-(OH).sub.2 D.sub.3. The methods of this 
invention include pretreatment of cultures and/or healing wounds to 
activate cell differentiation prior to treatment with 1,25-(OH).sub.2 
D.sub.3 or other REFs and/or matrix vesicles or matrix vesicle extract. 
Preferably such pre-treatment occurs about 36 to 72 hours prior to 
treatment with 1,25-(OH).sub.2 D.sub.3 or other REFs. Preferably the 
pre-treatment includes serum such as fetal bovine serum (FBS). 
Some agents inhibit matrix vesicles. For example, 24,25-(OH).sub.2 D.sub.3 
inhibits some matrix vesicle enzymes which may be important for the 
release of growth factor activating factor. 
This invention also provides matrix vesicles which have been treated so as 
to incorporate REF into their membranes. Preferably, such matrix vesicles 
are incubated with the desired REF, preferably 1,25-(OH).sub.2 D.sub.3, so 
that the REF is intercalated into the matrix vesicle membrane as described 
herein. The REF does not act immediately to break down the membrane, but 
rather the membrane breaks down over time to allow delivery of growth 
factor activating factors into the cellular matrix at a controlled rate. 
Varying the phospholipid composition of the matrix vesicles allows control 
of the release of the REF. 
Latent growth factor capable of being converted to active form by matrix 
vesicle secretions containing growth factor activating factor may be added 
to a healing wound or culture. Wound healing and cell growth and/or 
differentiation are stimulated by activation of the latent growth factor 
by means of added REF and/or matrix vesicles, or matrix vesicle extract. 
The latent growth factor should be added in an amount sufficient to 
provide measurable enhancement of wound healing or culture growth and/or 
differentiation. Preferably, it is added in an amount between about 1 and 
about 2000 ng per cc of wound or culture volume, more preferably in an 
amount between about 10 and about 1000 ng and most preferably in an amount 
between about 50 and about 500 ng. 
The administration of latent growth factors to healing wounds and to cell 
cultures for stimulation of cell growth and differentiation is especially 
useful when it is desired to control the timing of activation of the 
growth factor, for example so as to favor proliferation versus 
differentiation at appropriate times. By activating the growth factor 
responsible for regulating each event, cells can be modulated in a manner 
that is more physiological than present technology permits. 
Matrix vesicles and/or REF may also be added in combination with the latent 
growth factor, in amounts as set forth above. 
This invention also provides biodegradable polymeric implants or 
scaffolding materials (referred to generically herein as implant 
materials) comprising latent growth factors, REF, matrix vesicles or 
matrix vesicle extract in pharmaceutically effective amounts. 
Pharmaceutically effective amounts of latent growth factor are amounts 
sufficient to stimulate cell proliferation and/or differentiation upon 
activation during use. A preferred implant of this invention comprises 
between about 0.1 .mu.g and about 2,500 .mu.g latent growth factor per cc 
of polymeric material, or between about 10 pmoles and about 1000 pmoles 
REF per cc of polymeric material, or between about 10 ng and about 1000 ng 
of matrix vesicle protein per cc of polymeric material, or between about 5 
ng and about 500 ng of matrix vesicle extract per cc of polymeric 
material. Any combination of latent growth factor, REF, matrix vesicles 
and matrix vesicle extract may be incorporated into such implant material, 
and the remaining components necessary for activation of latent growth 
factor may be added to the culture or wound site separately. 
It is preferred that the polymeric implant material be designed for 
controlled release of the active components. Such polymeric implant 
materials are known to the art and are described hereinabove. In one 
embodiment, the polymeric implant is designed to continuously release 
active ingredients over its entire degradation period, as described in 
U.S. patent application Ser. No. 08/196,970 incorporated herein by 
reference. 
The polymeric implant material may also comprise cells compatible with the 
host for which it is intended, for example as described in the 
above-referenced Naughton et al. patents. 
This invention also provides a method for stimulating activation of a 
latent growth factor in a cellular matrix, which matrix comprises matrix 
vesicles, comprising contacting said matrix vesicles with a 
pharmaceutically effective amount of REF. If desired, additional latent 
growth factor may be added to the cellular matrix along with a sufficient 
amount of REF to activate it. Additional matrix vesicles or matrix vesicle 
extract may also be added. 
This invention further provides a method of converting a growth factor or 
other cytokine from latent to active form comprising adding to a medium 
containing said growth factor in latent form matrix vesicles and/or matrix 
vesicle extract in an amount sufficient to activate said growth factor. 
REF may also be added to the medium along with matrix vesicles in an 
amount sufficient to convert said growth factor from latent to active 
form. 
The method may be performed in vitro or in vivo. When the method is 
performed in vitro, it may be performed by adding matrix vesicles or 
matrix vesicle extract to a medium containing latent growth factor, or to 
a cell culture comprising latent growth factor. Cultures of cartilage, 
bone and tendon cells may be treated with the addition of matrix vesicles 
or matrix vesicle extract, as may defects in cartilage, bone and tendon 
tissue. When matrix vesicles are added to a medium containing latent 
growth factor which does not contain cells, it will be necessary to 
stimulate release of growth factor activating factor from the matrix 
vesicles by adding an effective amount of REF. When matrix vesicle extract 
is used, REF may not be required. 
In methods involving activation of latent growth factor in cellular 
matrices in vivo or in vitro to which latent growth factor has been added, 
matrix vesicles may be provided by stimulating the cells with electricity, 
ultrasound or physical stress sufficient to increase production of matrix 
vesicles by the cells. REF in an amount sufficient to activate said latent 
growth factor may also be provided to the cellular matrices. 
Healing of a wound, preferably a bone or cartilage defect, may be enhanced 
by a method of this invention comprising locally administering to said 
defect a composition comprising matrix vesicles or matrix vesicle extract 
in an amount sufficient to activate latent growth factor present in said 
defect. REF may also be added in an amount sufficient to stimulate said 
matrix vesicles to produce TGF.beta. activating factor. If desired, 
additional latent growth factor may also be added to the defect. 
Healing of such wounds may also be enhanced by treating the defect with 
electrical energy in an amount sufficient to stimulate production of 
matrix vesicles or with ultrasound, physical stress or other means known 
to the art in an amount sufficient to stimulate production of matrix 
vesicles. 
These healing methods may involve implanting into the wound or defect a 
biodegradable polymeric implant comprising an amount of latent growth 
factor sufficient to stimulate cell proliferation and/or differentiation 
upon activation during use.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As shown in FIG. 1, a number of factors affect cellular production of 
matrix vesicles and REFs, and matrix vesicles themselves can be stimulated 
by REFs to release growth factor activating factors. New protein is 
synthesized within the cells, proteins are incorporated into the membranes 
and matrix vesicles are released. 
Specifically, FIG. 1a depicts a cell 10, such as a cartilage, bone, or 
tendon cell, preferably a chondrocyte or osteoblast, containing a nucleus 
12, rough endoplasmic reticulum 14, and golgi bodies 16. 
The cell 10 may be stimulated by endogenous or added growth factors 
including TGF.beta. 18 which regulate the cell, REFs such as 
1,25-(OH).sub.2 D.sub.3, and 24,25-(OH).sub.2 D.sub.3 and steroid hormones 
such as estrogen and testosterone. Electrical energy 22, ultrasound 24 or 
physical stress may be applied to cell 10 to stimulate production of 
matrix vesicles 28 and promote wound healing. Peptide hormones 26, 
endogenous or added, also regulate the cell 10. 
The stimulated cell 10 produces matrix vesicles 28 and latent growth factor 
30 as well as vitamin D metabolites 32 such as 1,25-(OH).sub.2 D.sub.3 and 
24,25-(OH).sub.2 D.sub.3. 
The vitamin D metabolites 32, specifically 1,25-(OH).sub.2 D.sub.3, act on 
the matrix vesicle 28 shown enlarged in FIG. 1b surrounded by collagen 35 
and proteoglycans 36. Other REFs 34 which may be produced by the cell or 
matrix vesicles or added to the system, and which can include added 
1,25-(OH).sub.2 D.sub.3, act on the matrix vesicle. The REFs 34 and 
vitamin D metabolites 32 produced by cell 10 act on matrix vesicle 28 to 
cause release of growth factor activating factors 42 (indicated by black 
arrow) to convert latent growth factors 30 to active growth factors 40. 
This invention is based on the discovery that latent growth factor can be 
converted to active form by matrix vesicle extract or through the medium 
of matrix vesicles stimulated with REF. 
These components can be added to healing wounds by direct injection or by 
means of implants or cell-seeded scaffolds cultured in vitro. This 
combination can also be used to stimulate cell growth and differentiation 
in cell cultures. 
This discovery is specifically described in detail in the following 
examples using TGF.beta. as the growth factor, 1,25-(OH).sub.2 D.sub.3 as 
the REF, and isolated matrix vesicles from chondrocyte cultures incubated 
with 1,25-(OH).sub.2 D.sub.3. 
As will be appreciated by those skilled in the art, substitutions of 
additional growth factors, REFs and matrix vesicle materials as described 
herein and as known to the art may be made as equivalents to the preferred 
embodiments described in detail herein. 
The following examples provide detailed enablement for the compositions and 
methods of this invention. 
EXAMPLES 
Example 1 
Activation of Latent TGF.beta. by 1,25-(OH).sub.2 D.sub.3 
The aim of this study was to examine the production of TGF.beta. by vitamin 
D metabolites and TGF.beta.. The model has the advantage of allowing 
comparison of chondrocytes at two different stages of cell maturation. In 
addition, by using matrix vesicles isolated from these cultures, we can 
determine what role non-genomic regulation plays in TGF.beta. activation 
in the extracellular matrix. The results demonstrate that extracellular 
matrix vesicles derived from growth zone chondrocytes have the capacity to 
activate latent TGF.beta.; that production and activation of TGF.beta. by 
these chondrocytes is regulated by 1,25-(OH).sub.2 D.sub.3 ; and that the 
effect of 1,25-(OH).sub.2 D.sub.3 is cell maturation-dependent occurring 
through a non-genomic mechanism. 
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 
antibiotics (penicillin, streptomycin, fungizone), trypsin, and other 
tissue culture reagents were from GIBCO Laboratories (Grand Island, N.Y.). 
Collagenase (Type II) was purchased from Worthington (Freehold, N.J.). 
24R,25-(OH).sub.2 D.sub.3 and 1.alpha.,25-(OH).sub.2 D.sub.3 were kind 
gifts of Dr. Milan Uskokovic (Hoffman LaRoche, Nutley, N.J.). Recombinant 
simian latent TGF.beta..sub.1 and TGF.beta..sub.2 were prepared as 
described below. Recombinant human TGF.beta..sub.1 and specific antibodies 
to TGF.beta. or TGF.beta..sub.2 were purchased from R & D Systems 
(Minneapolis, Minn.). Spin-X filters were purchased from Costar 
(Cambridge, Mass.); reagents for Northern analysis, including Nitroplus 
2000 filters, were purchased from Micron Separation, Inc. (Westborough, 
Mass.); guanidine thiocyanate was purchased from Fluka, Inc. (Ronkonkoma, 
N.Y.). The human TGF.beta..sub.1 cDNA (1.1 kb) used to prepare radioactive 
probes was a gift from Genentech, Inc. The glyceraldehyde-3-phosphate 
dehydrogenase (GAPDH) cDNA probe was isolated from a pHcGAP clone obtained 
from the American Type Culture Collection (Bethesda, Md.). 
The culture system used in this study has been described in detail 
previously (Boyan, B. D., et al. (1988) Bone 9:185-194). Briefly, rib 
cages were removed from 125 g Sprague-Dawley rats by sharp dissection and 
placed in DMEM until microdissection could be performed. The resting zone 
and adjacent growth zone cartilage were separated, and care was taken to 
dissect out intervening tissue so that cross contamination of cell zones 
would be decreased. Perichondrium and calcified cartilage were discarded 
to limit contamination by fibroblasts, osteoblasts, and osteoclasts. 
When the dissection was complete, cartilage from each zone was sliced, 
placed in DMEM containing 10% FBS and incubated overnight in a 5% CO.sub.2 
atmosphere at 37.degree. C. The DMEM was then replaced by two 20-minute 
washes of Hank's balanced salt solution (HBSS), followed by sequential 
incubations in 1% trypsin for one hour and 0.02% collagenase for three 
hours. After enzymatic digestion of the extracellular matrix was complete, 
cells were separated from tissue debris by filtration through 40-mesh 
nylon and collected from the filtrate by centrifugation at 500.times. g 
for ten minutes, resuspended in DMEM, counted and plated at an initial 
density of 10,000 cells/cm.sup.2 for resting zone cells or 25,000 
cells/cm.sup.2 for growth zone cells. 
Cells were incubated in DMEM containing 10% FBS, 1% 
penicillin-streptomycin-fungizone, and 50 .mu.g/ml vitamin C in an 
atmosphere of 5% CO.sub.2 at 37.degree. C. and 100% humidity for 24 hours. 
The culture medium was replaced at that time and then at 72-hour intervals 
until the cells reached confluence. At confluence, cells were subcultured 
to T75 flasks at the same plating densities as before and allowed to 
return to confluence. Cells were only subcultured a maximum of three times 
to ensure retention of phenotype. Fourth passage cells were used for all 
experiments. Previous studies have shown that these cells retain their 
chondrocytic phenotype and differential responsiveness to 1,25-(OH).sub.2 
D.sub.3 and 24,25-(OH).sub.2 D.sub.3. 
Vitamin D metabolite stock solutions were prepared by using ethanol as the 
solvent. Before addition to the cultures, each hormone stock solution was 
diluted at least 1:5000 (v/v) to minimize any toxic effects of ethanol. 
For the experiments, final concentrations were 10.sup.-8 M or 10.sup.-9 M 
1,25-(OH).sub.2 D.sub.3 or 10.sup.-7 M or 10.sup.-8 M 24,25-(OH).sub.2 
D.sub.3. Each experiment included control cultures that contained ethanol 
at the highest concentration used in the vitamin D metabolite-treated 
groups. 
Fourth passage cells were cultured in 24-well culture dishes as described 
above. At confluence, the medium was replaced with DMEM containing 10% 
FBS, antibiotics, ascorbic acid, and appropriate concentrations of vitamin 
D metabolites. Medium was also added to 24-well plates without cells to 
measure the amount of active and latent TGF.beta. derived from 10% FBS. At 
harvest, media were analyzed for their content of both active and latent 
TGF.beta.. The cell layers were trypsinized (1% trypsin), the cells 
counted, and the amount of TGF.beta. per ml or 10.sup.5 cells calculated. 
TGF.beta. activity was assayed by stimulation of alkaline phosphatase 
specific activity in cultures of ROS 17/2.8 cells. This microassay was 
performed as described previously (Schwartz, Z., et al., Endocrinology 
(1993) 132:1544-1552; Bonewald, L. F., et al., Mol. Endocrinol. (1991) 
5:741-751; Oreffo, R.O.C., et al., Biochem. Biophys. Res. Comm. (1989) 
153:817-823). The CCL64 mink lung epithelial cell assay was also performed 
as described by Danielpour et al. (Danielpour, D. et al., J. Cell. 
Physiol. (1989) 138:79-86) and is based on the ability of TGF.beta. to 
inhibit [.sup.3 H]-thymidine incorporation by these cells. A TGF.beta. 
standard curve (0.02 to 5 ng/ml) was performed in each assay. Specificity 
for TGF.beta.1 or TGF.beta.2 was confirmed by neutralization of activity 
with specific antibodies to TGF.beta.1 or TGF.beta.2. 
To determine the amount of latent TGF.beta., conditioned media were 
acid-activated by addition of 4 .mu.l 4N HCl to 100 .mu.l of the medium 
and incubation for 15-20 minutes at 20.degree. C. The reaction was then 
neutralized by addition of 4 .mu.l 4N NaOH, aseptically filtered using 
Spin-X filters, and TGF.beta. activity in the filtrate measured as 
described above. The amount of latent TGF.beta. was determined by 
subtracting the amount of activity in the pre-acidified samples from the 
total activity following acid activation. 
Total cellular RNA was isolated from fourth passage, confluent cultures of 
growth zone and resting zone chondrocytes by lysing cells in guanidinium 
thiocyanate, followed by phenol:chloroform extraction (Chomczynski, P. and 
Sacchi, N., Anal. Biochem. (1987) 162:156-159). Poly(A.sup.+) RNA was 
obtained by fractionating total RNA using oligo(dT) cellulose 
chromatography. Northern blot analysis was performed as described by 
Fourney et al. (Fourney, R. M. et al., Bethesda Res. Lab, Inc. Focus 
(1988) 10:5-7), with a modified procedure for formaldehyde agarose gel 
electrophoretic separation of RNA (Lehrach, H. et al., Biochemistry (1977) 
16:4743-4751; Davies, L. G. et al., in: Basic Methods in Molecular 
Biology, Elsevier, New York, N.Y. (1977) 143-149). Preparations of RNA 
loaded onto 1% agarose gels were electrophoresed at 4 volts/cm gel length 
for six hours and transferred to Nitroplus 2000 filters in 10.times. SSC 
at room temperature. The filter was baked at 80.degree. C. under vacuum 
and prehybridized in 50% formamide, 5.times. SSPE, 5.times. Denhardt's 
buffer, and 250 .mu.g/ml denatured E. coli DNA at 37.degree. C. for three 
to five hours. The filter was then hybridized at 37.degree. C. overnight 
in the same solution containing 10% dextran sulfate and .sup.32 P-labeled 
probe. The cDNA probes were labeled with .sup.32 P as described by 
Feinberg and Vogelstein (Feinberg, A. P. and Vogelstein, B. Anal. Biochem. 
(1983) 132:6-13). After hybridization, filters were washed and 
autoradiographed (Fourney, R. M. et al., Bethesda Res. Lab, Inc. Focus 
(1988) 10:5-7). The RNA blots were analyzed by a Beta Scope 603 Blot 
Analyzer (Betagen, Waltham, Mass.). Relative intensities of the 
hybridization signals were calculated with the aid of a GS 370 program 
(Hoefer Scientific Instruments, San Francisco, Calif.). The relative 
amount of mRNA loaded in each lane of the agarose gel was determined by 
the amount of GADPH mRNA detected in each lane. Filters were stripped in 
50% formamide containing 10 mM Tris, 1 mM ethylenediamine tetraacetic acid 
(EDTA), and 0.1% sodium dodecyl sulfate (SDS) at pH 7.5 for two to four 
hours at 65.degree. C. before hybridization with a second probe. 
Matrix vesicles were prepared from chondrocyte cultures as described 
previously (Boyan, B. D., et al., Bone (1988) 9:185-194). At harvest, the 
conditioned media were decanted, and the cells were released by 
trypsinization (1% in HBSS). The reaction was stopped with DMEM containing 
10% FBS, and the cells were collected by centrifugation at 500.times. g 
for 10 minutes, resuspended in saline, washed twice, and counted. The 
supernatant from the trypsin digest was centrifuged for 20 minutes at 
13,000.times. g to pellet a mitochondria/membrane fraction, and the 
resulting supernatant was centrifuged for one hour at 100,000.times. g to 
pellet matrix vesicles. Matrix vesicles were resuspended in 1 ml 0.9% 
NaCl. Detergents such as Triton X-100 were not used to solubilize the 
membranes, since they inhibit phospholipase A.sub.2, an enzyme which is 
sensitive to vitamin D metabolites. All samples used in subsequent assays 
represent the combination of three cultures (i.e., three T75 flasks). The 
protein content of each fraction was determined (Lowry, O. H. et al., J. 
Biol. Chem. (1951) 193:265-275). 
Alkaline phosphatase [orthophosphoric monoester phosphohydrolase alkaline 
(EC 3.1.3.1)] was measured as a function of the release of 
para-nitrophenol from para-nitrophenylphosphate at pH 10.2 (Bretaudiere, 
J. P. and Spillman, T., In: Methods of Enzymatic Analysis, Bergmeyer, H. 
U. (ed.), Verlag Chemica, Weinheim, Germany (1984) Vol. 4, 75-93). These 
techniques resulted in matrix vesicle preparations that were enriched in 
alkaline phosphatase-specific activity that was two to ten times greater 
than that of the plasma membrane. Previous studies have shown that there 
is a differential distribution of other plasma membrane marker enzymes in 
matrix vesicles and that contamination of other organelles in either 
membrane preparation is minimal. 
Preparation of Recombinant Latent TGF.beta.. The source of latent TGF.beta. 
for these experiments was NH.sub.4 SO.sub.4 precipitated protein derived 
from Chinese Hamster ovary (CHO) cells transfected with the gene coding 
for either simian TGF.beta.1 (Gentry, L. E. et al., Mol. Cell. Biol. 
(1987) 7:3418-3427) or TGF.beta.2 (Madisen, L. et al., Growth Factors 
(1990) 3:129-138). The recombinant TGF.beta.1 preparation was &gt;90% latent 
and contained approximately 60-100 ng/ml of latent TGF.beta.. The 
recombinant TGF.beta.2 preparation was also &gt;90% latent and contained 
approximately 150-200 ng/ml of latent TGF.beta.. 
Activation of Latent TGF.beta. by Chondrocytes. These experiments were 
performed as described by Oreffo et al. (Oreffo, R.O.C., et al., Biochem. 
Biophys. Res. Comm. (1989) 153:817-823) except that chondrocytes were 
incubated with recombinant simian latent TGF.beta.-instead of latent 
TGF.beta. purified from bone. Resting zone and growth zone chondrocytes 
were cultured to confluence, the media removed, and DMEM containing 1% 
FBS, ascorbic acid, antibiotics, and recombinant latent TGF.beta.1 (0.6 
ng/ml).+-.1,25-(OH).sub.2 D.sub.3 (10.sup.-7 M) or 24,25-(OH).sub.2 
D.sub.3 (10.sup.-6 M) was added. Cultures were incubated for 24 hours at 
37.degree. C. in an atmosphere of 5% CO.sub.2. Antibody specific for 
TGF.beta.1 was used to prove specificity. The conditioned media were then 
tested for their content of active and latent TGF.beta. in the ROS 17/2.8 
alkaline phosphatase microassay. 
Activation of Latent TGF.beta. by Isolated Matrix Vesicles. Matrix vesicles 
were diluted to 1.6 mg protein/ml in PBS. All incubations were performed 
in 96-well microtiter plates in a total volume of 200 .mu.l. First, 80 
.mu.l of the matrix vesicle suspension were added, followed by 8 .mu.l of 
either 10.sup.-7 M 1,25-(OH).sub.2 D.sub.3 or 10.sup.-6 M 24,25-(OH).sub.2 
D.sub.3 in DMEM containing 2% FBS, resulting in a 10.sup.-8 M or 10.sup.-7 
M concentration, respectively. The plate was incubated for three hours at 
room temperature. After the matrix vesicles had been pre-incubated with 
vitamin D, recombinant simian latent TGF.beta.1 or TGF.beta.2 was added 
and the incubation continued for an additional 24 hours at room 
temperature. TGF.beta. activity was then measured using the ROS 17/2.8 
microassay. To ensure that changes in alkaline phosphatase specific 
activity were entirely due to active TGF.beta., pan-neutralizing antibody 
for all TGF.beta. isoforms was added at 40 .mu.g/ml (sufficient to block 2 
ng/ml TGF.beta.) and incubated for 30 minutes before addition of the 
samples to the ROS 17/2.8 cells. 
The data are from representative experiments and are expressed as mean .+-. 
standard error of the mean. For any particular experiment, each data point 
represents six individual cultures. For studies using matrix vesicles 
(n=3), each "n" represents the matrix vesicles isolated from two to three 
T-75 flasks. Data were analyzed by analysis of variance with statistical 
significance between treatment and control being assessed by Bonferroni's 
modification of the t-test. To verify the consistency of the observations, 
experiments were repeated two or more times. Treatment/control ratios were 
derived from five or more independent experiments and were compared using 
the Wilcoxon2. 
Virtually all TGF.beta. present in the conditioned media produced by either 
growth zone or resting zone chondrocytes was in latent form. In the 
present study, growth zone chondrocytes produced 12.90.+-.0.7 ng latent 
TGF.beta./ml or 8.2.+-.1.7 pg/10.sup.5 cells. In contrast, resting zone 
chondrocytes produced 9.7.+-.0.6 ng latent TGF.beta./ml or 4.8.+-.0.5 
pg/10.sup.5 cells. Attempts to measure active TGF.beta. (i.e., activity 
prior to acidification of the conditioned medium) in these cultures were 
unsuccessful, even though the assay could detect active TGF.beta. at 
concentrations of 0.1 ng/ml or more. 
Anti-TGF.beta.1 antibody inhibited the majority of the TGF.beta. activity 
in DMEM+10% FBS, as well as conditioned media, indicating that TGF.beta.1 
was the predominant isoform produced by the chondrocytes. Anti-TGF.beta.2 
antibody inhibited approximately 25% of the activity present in growth 
zone chondrocyte conditioned media, indicating that these cells also 
produced the TGF.beta.2 isoform. In contrast, resting zone chondrocytes 
only produced TGF.beta.1. 
The production of latent TGF.beta. by growth zone or resting zone 
chondrocytes was unaffected by addition of rhTGF.beta.1 to the culture 
medium. Pan-neutralizing anti-TGF.beta. antibody blocked the activity of 
acid-activated culture media. In addition, no active TGF.beta. was 
detected when exogenous active TGF.beta. was added to the cells for 24 
hours, growth factor-containing medium removed, and the conditioned media 
examined 24 or 48 hours later. Similarly, TGF.beta.1 mRNA levels were 
unaffected by addition of rhTGF.beta.1 to cultures of either cell type. 
Treatment of growth zone chondrocytes for 24 hours with 1,25-(OH).sub.2 
D.sub.3 significantly reduced the amount of latent TGF.beta. found in the 
conditioned media in a dose-dependent manner. This was true whether 
pre-confluent or confluent cultures were used. However, the effect of 
serum concentration in the medium was dependent on the confluency of the 
cells. 24,25-(OH).sub.2 D.sub.3 had no effect on the production of latent 
TGF.beta. by these cells (FIG. 2). Resting zone chondrocytes behaved in a 
similar manner, but to a lesser degree. As before, no active TGF.beta. 
could be detected in these cultures. 1,25-(OH).sub.2 D.sub.3 did not alter 
the level of mRNA for TGF.beta.1 in chondrocytes, as determined by 
Northern analysis using total RNA. 
When exogenous latent TGF.beta.2 or TGF.beta.1 was added to either resting 
zone or growth zone chondrocyte cultures in the presence of 
1,25-(OH).sub.2 D.sub.3 or 24,25-(OH).sub.2 D.sub.3, no active TGF.beta. 
was detected in the conditioned media. This indicates that no cellular 
activation of latent TGF.beta. occurred with 1,25-(OH).sub.2 D.sub.3 or 
24,25-(OH).sub.2 D.sub.3 treatment. 
When matrix vesicles were isolated from cultures of growth zone or resting 
zone chondrocytes and assayed for their content of active TGF.beta., no 
activity was found. Further, when exogenous latent TGF.beta.1 or 
TGF.beta.2 was added to these membrane fractions, no activation of latent 
growth factor occurred. Pretreatment of isolated matrix vesicles with 
1,25-(OH).sub.2 D.sub.3 or 24,25-(OH).sub.2 D.sub.3, followed by 
incubation with latent TGF.beta.1, resulted in a detectable increase in 
active TGF.beta. in all samples; however, highly significant increases 
(4.4-fold) were only seen in matrix vesicles isolated from growth zone 
chondrocytes. The 1,25-(OH).sub.2 D.sub.3 -dependent increase in active 
TGF.beta. was inhibited greater than 50% by pan-neutralizing TGF.beta. 
antibody. Depending on the experiment, matrix vesicles isolated from 
growth zone chondrocyte cultures activated 25-70% of the total latent 
TGF.beta.. Although total activation varied among experiments, a 
significant increase was always found after treatment with 1,25-(OH).sub.2 
D.sub.3. Treatment/control ratios derived from five experiments showed a 
five-fold increase in TGF.beta. activation over control levels (FIG. 3). 
Activation of latent TGF.beta.2 was also regulated by 1,25-(OH).sub.2 
D.sub.3 in a manner comparable to that seen for latent TGF.beta.1. 
This study emphasizes the complex interactions that can occur between 
vitamin D metabolites and TGF.beta.. Costochondral chondrocytes, like 
epiphyseal chondrocytes, secrete primarily latent TGF.beta. of the .beta.1 
isoform. Unlike many other cell types, however, exogenously added 
TGF.beta. had no effect on TGF.beta.1 mRNA levels or on release of latent 
TGF.beta. into the conditioned media. 1,25-(OH).sub.2 D.sub.3 reduced the 
amount of latent TGF.beta. produced by chondrocytes, whereas, 
24,25-(OH).sub.2 D.sub.3 had no effect. Furthermore, 1,25-(OH).sub.2 
D.sub.3 had a direct effect on isolated matrix vesicles, inducing them to 
activate latent TGF.beta.. 
Both TGF.beta. and vitamin D alone have significant effects on expression 
of the chondrocyte or osteoblast phenotype, but in conjunction, the 
effects can be dramatic. TGF.beta. and vitamin D have been shown to 
synergize with respect to alkaline phosphatase induction in bone cell 
lines, primary human bone cells, and rat resting zone chondrocytes. 
TGF.beta. may act as a "coupling" factor in bone remodeling, and vitamin D 
has been shown to be essential for proper endochondral ossification. The 
present data suggest that vitamin D can stimulate activation of latent 
TGF.beta., thereby increasing the chance that both factors will be present 
simultaneously. 
Both TGF.beta. and vitamin D regulate chondrocyte differentiation. 
Exogenous TGF.beta. stimulates DNA synthesis and matrix formation in chick 
growth plate chondrocytes. In rat growth plate chondrocytes, rhTGF.beta.1 
regulates alkaline phosphatase, phospholipase A.sub.2, and protein kinase 
C activities, as well as vitamin D metabolite production. Cellular 
response to TGF.beta. depends on the state of endochondral maturation, 
with resting zone cells exhibiting a differential response compared to 
that observed in growth zone cell cultures. Similarly, vitamin D 
metabolites also regulate the expression of alkaline phosphatase, 
phospholipase A.sub.2, and protein kinase C in chondrocytes in a cell 
maturation-specific manner. These studies demonstrated that production of 
1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 is sensitive to 
TGF.beta., and the actions of TGF.beta. and the vitamin D metabolites are 
interdependent. The present study demonstrates that latent TGF.beta. 
production and activation are sensitive to 1,25-(OH).sub.2 D.sub.3 and 
suggests a potential feedback mechanism. 
Regulation of TGF.beta. production and activation in cartilage has not been 
previously described. In many cell types, synthesis of TGF.beta. is 
sensitive to exogenous TGF.beta., suggesting an autocrine loop. Unlike 
these cell types, however, the costochondral chondrocytes do not appear to 
respond to exogenous TGF.beta.1 by increasing their levels of TGF.beta.1 
mRNA or of either latent or active TGF.beta. in their conditioned media. 
This reproducibly occurs under the culture conditions used in this study. 
Using comparable culture conditions, the effect of TGF.beta. on 
chondrocyte alkaline phosphatase is ten times greater than that seen in 
osteoblast cell lines. 
The failure of exogenous TGF.beta. to autoregulate TGF.beta. production by 
the chondrocytes may be an adaptive result of the high levels of this 
growth factor stored in cartilage. Although exogenous TGF.beta. may not 
have an autocrine effect on the production of latent TGF.beta. and its 
release into the culture media, it is likely in cartilage that such an 
autocrine loop is maintained by growth factor stored in the matrix in 
latent form and activated locally. The results of this study support this 
hypothesis. 
We have previously shown that exogenous TGF.beta.1 regulates production of 
vitamin D metabolites by chondrocytes in a cell maturation-specific and 
time-dependent manner. The present study demonstrated an effect of 
1,25-(OH).sub.2 D.sub.3 on TGF.beta., resulting in a marked decrease in 
the amount of latent factor in the media. The effects of vitamin D were 
both metabolite-specific and cell maturation-dependent. There was a 
1,25-(OH).sub.2 D.sub.3 -dependent decrease in latent TGF.beta. in both 
chondrocyte populations, although the effect was greater in growth zone 
cell cultures. The role of 1,25-(OH).sub.2 D.sub.3 in this process appears 
to be specific, since 24,25-(OH).sub.2 D.sub.3 did not elicit a comparable 
response. The preincubation period was long enough for the chondrocytes to 
convert 24,25-(OH).sub.2 D.sub.3 to 24,25-(OH).sub.2 D.sub.3 (Schwartz, 
Z., et al., Endocrinology (1992) 130:2495-2504), further supporting the 
specificity of the 1,25-(OH).sub.2 D.sub.3 effect. 
The data support the activation of existing latent TGF.beta.1 and 
TGF.beta.2 by matrix vesicles via direct interaction of 1,25-(OH).sub.2 
D.sub.3 with the organelle. Active metalloproteinases present in matrix 
vesicles may be prime candidates for accomplishing this process. In growth 
plate, the immunohistochemical distribution of TGF.beta.1 coincides with 
the localization of matrix vesicles in the territorial matrix of the 
cells, providing support for potential activation of latent TGF.beta. in 
the matrix by matrix vesicle proteases. 
The results of this study are consistent with the hypothesis that 
1,25-(OH).sub.2 D.sub.3, secreted by the chondrocyte, regulates matrix 
vesicle via direct, nongenomic mechanisms. Matrix vesicle membrane 
fluidity and enzyme activity can be directly and specifically regulated by 
1,25-(OH).sub.2 D.sub.3 in the absence of the cell and its molecular and 
protein synthetic machinery. In the present study, direct incubation of 
isolated matrix vesicles with 1,25-(OH).sub.2 D.sub.3 resulted in 
activation of latent TGF.beta.1, as well as latent TGF.beta.2. Matrix 
vesicles produced by osteoblast-like cells also contain matrix processing 
enzymes which indicates that a similar mechanism of TGF.beta. activation 
plays a role in bone, as well as cartilage. 
In summary, these studies show that TGF.beta. and vitamin D metabolites 
have complex and interactive roles in chondrogenesis. The effects of these 
factors vary, depending on the stage of differentiation of the 
chondrocyte. TGF.beta. is produced in a latent form by these cells. 
Whereas autocrine effects have been observed with respect to chondrocyte 
phenotype expression, none were observed in the present study with respect 
to TGF.beta. messenger RNA levels or protein production in active or 
latent forms. In contrast, 1,25-(OH).sub.2 D.sub.3 reduces the level of 
latent TGF.beta. produced by these cells by an unknown mechanism, as 
messenger RNA was not affected, and activation of the latent form did not 
appear to be occurring, at least with respect to the conditioned media. 
Matrix vesicles are excellent targets for the nongenomic effects of 
vitamin D, as these are located in the matrix at a distance from the cell 
and adjacent to the mineralization front. In vitro, matrix vesicles are 
inert with respect to activation of latent TGF.beta. unless exposed to 
1,25-(OH).sub.2 D.sub.3, which triggers the activation process. 
Example b 2 
Effects of 1,25-(OH).sub.2 D.sub.3 in calcium ion flux and Protein Kinase C 
activity 
It is well accepted that 1,25-(OH).sub.2 D.sub.3 alters Ca ion flux in 
osteoblasts. However, little is known concerning the role of this vitamin 
D metabolite in chondrocytes, particularly with respect to its nongenomic 
action. Even less information is available concerning the effects of 
24,25-(OH).sub.2 D.sub.3 . To examine this, we characterized the uptake 
and release of .sup.45 Ca by resting zone and growth zone chondrocytes in 
the presence of 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3. At 1 
minute, 24,25-(OH).sub.2 D.sub.3 inhibited .sup.45 Ca efflux from resting 
zone cells and 1,25-(OH).sub.2 D.sub.3 stimulated .sup.45 Ca efflux from 
growth zone cells. 
Changes in arachidonic acid metabolism and Ca ion flux suggested that 
membrane signal transduction pathways might also be affected by vitamin D. 
To determine if this was the case, we assayed cultures for protein kinase 
C activity in the presence of inhibitors of gene transcription and 
translation. The results of the studies demonstrate that 1,25-(OH).sub.2 
D.sub.3 stimulated protein kinase C activity in growth zone chondrocytes 
but had no effect on resting zone cells. In contrast, 24,25-(OH).sub.2 
D.sub.3 stimulated enzyme activity in resting zone cells but had no effect 
on growth zone cells. Moreover, the time course of response was different. 
Stimulation was more rapid in the growth zone cells (9 to 90 minutes), but 
the effect of 24,25-(OH).sub.2 D.sub.3 on the resting zone cells was 
delayed but sustained over a longer time (90 to 360 minutes). The 
inhibitor studies demonstrated clearly that the 1,25-(OH).sub.2 D.sub.3 
-dependent effect was non-genomic, requiring no new gene transcription or 
translation, whereas both processes were required for the 24,25-(OH).sub.2 
D.sub.3 -dependent effect. 
These studies showed that at the cellular level, the action of 
24,25-(OH).sub.2 D.sub.3 involved genomic mechanism while the action of 
1,25-(OH).sub.2 D.sub.3, at least at short time periods, did not. There 
remained the question of whether this enzyme activity was also found in 
matrix vesicles and, if so, if it could be regulated directly by the 
hormones. Our results show that protein kinase C-.zeta. is preferentially 
localized in matrix vesicles produced by both cell types. Anti PKC.alpha. 
antibody inhibits PKC activity in plasma membranes and anti PKC .zeta. 
antibody inhibits PKC activity in matrix vesicles. Both metabolites 
regulate matrix vesicle PKC .zeta. in a nongenomic manner. When matrix 
vesicles from growth zone cell cultures are incubated directly with 
1,25-(OH).sub.2 D.sub.3 enzyme activity is inhibited. Similarly, when 
matrix vesicles isolated from resting zone cell cultures are incubated 
with 24,25-(OH).sub.2 D.sub.3 PKC .zeta. activity is decreased. 
Both 1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 can exert their 
effects on chondrocytes by nongenomic mechanisms. The actions include 
changes in membrane fluidity, phospholipid metabolism, Ca ion flux, and 
protein kinase C activity. Matrix vesicles are regulated independently of 
the cell. While their composition may be under genomic control, it is 
likely that once in the extracellular matrix they are regulated by direct 
action of vitamin D metabolites secreted by the chondrocyte. 
Example 3 
Activation of resting zone chondrocytes by 24,25-(OH).sub.2 D.sub.3. 
Studies suggest that 24,25-(OH).sub.2 D.sub.3 has an important role in the 
early stages of chondrocyte differentiation, whereas 1,25-(OH).sub.2 
D.sub.3 has an important role in the later stages of chondrocyte 
differentiation. Based on previous in vivo and in vitro observations, a 
hypothesis can be made that 24,25-(OH).sub.2 D.sub.3 induces resting zone 
chondrocytes to progress down the endochondral pathway and acquire a 
growth zone-like phenotype. 
To test this hypothesis, we assessed whether resting zone cells acquired 
responsiveness to 1,25-(OH).sub.2 D.sub.3 following exposure to 
24,25-(OH).sub.2 D.sub.3. The ability of 24,25-(OH).sub.2 D.sub.3 
-stimulated resting zone chondrocytes to respond to 1,25-(OH).sub.2 
D.sub.3 was compared to that of authentic growth zone chondrocytes with 
respect to DNA synthesis, alkaline phosphatase activity, RNA synthesis, 
collagen and noncollagen protein synthesis, and proteoglycan production. 
To assess whether cells already in the endochondral lineage (i.e., from 
the resting zone to calcified cartilage) differ from hyaline chondrocytes 
in their response to 24,25-(OH).sub.2 D.sub.3, we also examined cells from 
the xiphoid process. 
Fourth passage resting zone or xiphoid chondrocytes were grown to 
confluence. At confluence, the media were replaced with media containing 
10.sup.-7 M 24,25-(OH).sub.2 D.sub.3 or vehicle alone for 24, 36, 48, 72 
or 120 hours. For those cells pretreated for 120 hours, fresh media 
containing the appropriate concentration of 24,25-(OH).sub.2 D.sub.3 was 
added at 72 hours. At the end of the pretreatment period, the media were 
replaced again with medium containing 1,25-(OH).sub.2 D.sub.3 at a 
concentration of 10.sup.-10 to 10.sup.-8 M or vehicle alone and grown for 
an additional 24 hours. At that time, the cells were harvested and assayed 
as described below. To determine if the effect of pretreatment with 
24,25-(OH).sub.2 D.sub.3 was metabolite-specific and not due to a general 
steroid hormone effect, resting zone cells were pretreated with 10.sup.-8 
M 1,25-(OH).sub.2 D.sub.3 for 24, 36, 48, 72, or 120 hours, followed by 
treatment with 10.sup.-10 to 10.sup.-8 M 1,25-(OH).sub.2 D.sub.3 and then 
assayed for alkaline phosphatase specific activity. 
DNA synthesis by nonquiescent resting zone cells was estimated by measuring 
[.sup.3 H]-thymidine incorporation into trichloroacetic acid (TCA) 
insoluble precipitates (Langston, G. G. et al., "Effect of 1,25-(OH).sub.2 
D.sub.3 and 24,25-(OH).sub.2 D.sub.3 on calcium influxes in costochondral 
chondrocyte cultures", Calcif. Tissue Int. (1990) 47:230-236). 
Chondrocytes were grown to confluence in 6 mm diameter microwells and 
[.sup.3 H]-thymidine (50 .mu.l) added two hours prior to harvest so that 
the final concentration in the medium was 2 .mu.Ci/ml. At harvest, the 
cell layers were washed twice with cold phosphate-buffered saline, twice 
with 5% TCA, and then treated with saturated TCA for 30 minutes. 
TCA-precipitable material was dissolved in 0.2 ml 1% sodium dodecyl 
sulfate (SDS), and the radioactivity measured by scintillation 
spectroscopy. 
Resting zone and xiphoid cells were cultured in 24-well culture dishes 
(Corning, N.Y.). At harvest, the media were decanted and the cell layers 
washed twice with phosphate-buffered saline (PBS) before removal with a 
cell scraper. Enzyme assays were performed using lysates of the cell 
layers (Schwartz, Z. et al., "Localization of Vitamin D.sub.3 responsive 
alkaline phosphatase in cultured chondrocytes," J. Biol. Chem (1988) 
263:6023-6026; Hale, L. V. et al., "Effect of vitamin D metabolites on the 
expression of alkaline phosphatase activity by epiphyseal hypertrophic 
chondrocytes in primary cell culture", J. Bone Min. Res. (1986) 
1:489-495). After centrifugation, the cell layer pellet was washed two 
times with PBS and resuspended by vortexing in 500 .mu.l deionized water 
containing 25 .mu.l of 1% Triton X-100. Alkaline phosphatase 
[orthophosphoric monoester phospho-hydrolase alkaline (EC 3.1.3.1)] 
specific activity was measured as a function of para-nitrophenol release 
from para-nitrophenylphosphate at pH 10.2, as previously described 
(Bretaudier, J. P. and Spillman, T., "Alkaline phosphatases", In: 
Bergmeyer HU (ed) Methods Enzymatic Anal. Verlag Chemie, Weinheim (1984) 
4:75-81). 
RNA synthesis was estimated by measuring [.sup.3 H]-uridine incorporation 
into TCA-insoluble cell precipitates. Resting zone cells were grown to 
confluence in 6 mm diameter microwells and [.sup.3 H]-uridine (50 .mu.l) 
added two hours before harvest so that the final concentration in the 
medium was 14 .mu.CI/ml. From this point, the protocol described above for 
quantitating [.sup.3 H]-thymidine incorporation was followed exactly. 
Incorporation of labeled proline into collagenase-digestible protein (CDP) 
and collagenase-nondigestible protein (NCP) was used to estimate matrix 
protein synthesis by resting zone cells (Raisz, L. G. et al., "Comparison 
of the effects of a potent synthetic analog of bovine parathyroid hormone 
with native bPTH-(1-84) and synthetic bPTH-(1-34) on bon resorption and 
collagen synthesis," Calcif. Tissue Int. (1979) 29:215-218). Percent 
collagen synthesis was calculated after multiplying the labeled proline in 
NCP by 5.4 to correct for its relative abundance in collagen (Beresford, 
J. N. et al., "1,25-Dihydroxyvitamin D.sub.3 and human bone-derived cells 
in vitro: Effects on alkaline phosphatase, type I collagen and 
proliferation", Endocrinology (1986) 119:1776-1785). 
Twenty-four hours before harvesting, 5 .mu.Ci of L-[G.sup.3 H]-proline (New 
England Nuclear, Boston, Mass.) in 1.0 ml medium was added. At harvest, 
the media were decanted and the cell layer collected in two 0.2 ml 
portions of 0.2N NaOH. Proteins present in the cell layer were first 
precipitated with 0.1 ml 100% TCA containing 10% tannic acid. The 
resultant precipitate was washed three times with 10% TCA-1% tannic acid 
and then twice with ice-cold acetone. The final pellet was dissolved in 
500 .mu.l 0.05N NaOH. 
The amount of radio-labeled proline incorporated into CDP and NCP was 
determined according to the method of Peterkofsky and Diegelmann 
(Peterkofsky, B., and Diegelmann, R., "Use of a mixture of proteinase-free 
collagenases for the specific assay of radioactive collagen in the 
presence of other proteins," Biochemistry (1971) 10:988-994). Data were 
expressed as dpm and were calculated with respect to protein content. 
Highly purified clostridial collagenase, 158 U/mg protein, was obtained 
from Calbiochem (San Diego, Calif.). This batch of enzyme was found to be 
very low in nonspecific proteolytic activity. Less than 5% of the total 
incorporated radioactivity was released from [.sup.3 H]-tryptophan-labeled 
chondrocytes. The protein content of each fraction was determined by a 
miniaturization of the method of Lowry et al. (Lowry, O. H. et al., 
"Protein measurement with the folin phenol reagent," J. Biol. Chem. (1951) 
193:265-275). For most experiments, CDP and NCP were only measured in the 
cell layer, not the media, because more than 80% of the total CDP was 
incorporated into the cell layer. This assay did not take into account any 
degradation that may have occurred. 
Proteoglycan synthesis was assessed by measuring [.sup.35 S]-sulfate 
incorporation according to the method of Regis et al. (Regis, J. O. et 
al., "Effects of transforming growth factor .beta. on matrix synthesis by 
chick growth plate chondrocytes," Endocrinology (1988) 122:2953-2961). In 
prior studies, we have found that the amount of radiolabeled proteoglycan 
released by growth zone and resting zone chondrocytes into the medium was 
less than 15% of the total radiolabeled proteoglycan (media and cell 
layer) synthesized (Nasatzky, E., et al., "Sex dependent effects of 
17.beta. estradiol on chondrocyte differentiation in culture," J. Cell. 
Phys. (1993) 156:359-367). Because of this, we only examined the effects 
of hormone treatment on .sup.35 SO.sub.4 incorporation in the cell layer. 
This assay does not measure any degradation that may occur during the 
culture. 
For assay, fourth passage resting zone chondrocytes were grown to 
confluence in 24-well culture plates (Corning, Corning, N.Y.) with media 
containing 10% FBS, antibiotics, and 50 .mu.g/ml ascorbic acid. 
Twenty-four hours prior to harvest, fresh media containing vehicle alone 
or vitamin D was added to the cells. Four hours prior to harvest, 
50.mu.DMEM containing 18 .mu.Ci/ml .sup.35 SO.sub.4 and 0.814 mM carrier 
sulfate was added to each culture. At harvest, the conditioned media were 
removed and the cell layers (cells and matrix) collected in two 0.25 ml 
portions of 0.25M NaOH. The protein content was determined by the method 
of Lowry et al., (Lowry, O. H. et al., "Protein measurement with the folin 
phenol reagent," J. Biol. Chem. (1951) 193:265-275). The total volume was 
adjusted to 0.75 ml by the addition of 0.15M NaCl and the sample dialyzed 
in a 12,000-14,000 molecular weight cut off membrane against buffer 
containing 0.15M NaCl, 20 mM Na.sub.2 SO.sub.4, and 20mM Na.sub.2 
HPO.sub.4, pH 7.4, at 4.degree. C. The dialysis solution was changed until 
the radioactivity in the dialysate reached background. The amount of 
.sup.35 SO.sub.4 incorporated was determined by liquid scintillation 
spectrometry and calculated as DPM/mg protein in the cell layer. This 
protocol was also used for assessing the change in phenotype induced by 
24,25-(OH).sub.2 D.sub.3 -pretreatment of resting zone chondrocytes. 
The data generated were from one experiment that was repeated three or more 
times with comparable results. For any given experiment, each data point 
represents the mean .+-. SEM for six individual cultures. 
Treatment/control ratios were derived from five or more independent 
experiments, with controls having a ratio of 1.0. 
The data were analyzed by analysis of variance, and statistical 
significance determined by comparing each data point to the control 
(containing ethanol vehicle) using Bonferroni's modification of the 
t-test. Treatment/control ratios were compared using the Wilcoxon matched 
pair rank sum test. P&lt;0.05 was considered significant. 
Addition of 10.sup.-8 to 10.sup.-10 M 1,25-(OH).sub.2 D.sub.3 to resting 
zone cells pretreated with 10.sup.-7 M 24,25-(OH).sub.2 D.sub.3 rods for 
24 or 48 hours caused a dose-dependent inhibition in [.sup.3 H]-thymidine 
incorporation. The inhibitory effect was also observed in chondrocytes 
pretreated for up to 120 hours with 24,25-(OH).sub.2 D.sub.3. Resting zone 
cells pretreated with vehicle alone and challenged with 1,25-(OH).sub.2 
D.sub.3 incorporated [.sup.3 H]-thymidine at levels comparable to cells 
that were pretreated with 24,25-(OH).sub.2 D.sub.3 followed by treatment 
with 10.sup.-8 M 1,25-(OH).sub.2 D.sub.3. 
Addition of 1,25-(OH).sub.2 D.sub.3 had no effect on alkaline phosphatase 
specific activity of resting zone chondrocytes pretreated with 10.sup.-7 M 
24,25-(OH).sub.2 D.sub.3 in for 24 hours. Enzyme activity in these 
cultures was comparable to that of cells incubated with 10.sup.-8 M 
1,25-(OH).sub.2 D.sub.3 with no 24,25-(OH).sub.2 D.sub.3 pretreatment. 
However, when resting zone cells were pretreated with 24,25-(OH).sub.2 
D.sub.3 for 48 hours, there was a dose-dependent increase in alkaline 
phosphatase specific activity which was significant at concentrations of 
10.sup.-9 M and 10.sup.-8 M 1,25-(OH).sub.2 D.sub.3. 
The effect of 24,25-(OH).sub.2 D.sub.3 pretreatment was observed by 36 
hours. Pretreatment with 24,25-(OH).sub.2 D.sub.3 enhanced the stimulation 
of alkaline phosphatase specific activity by 1,25-(OH).sub.2 D.sub.3 in a 
time-dependent manner. Maximum effects were observed in cultures incubated 
for 72 hours with 24,25-(OH).sub.2 D.sub.3, and the effect was maintained 
in cells pre-cultured for 120 hours. In contrast, resting zone cells 
pretreated with vehicle alone failed to exhibit 1,25-(OH).sub.2 D.sub.3 
-dependent increases in enzyme activity. Alkaline phosphatase activity in 
these cultures was comparable to that seen in cultures pretreated with 
24,25-(OH).sub.2 D.sub.3, but challenged with vehicle alone. Pretreatment 
of resting zone chondrocytes with 1,25-(OH).sub.2 D.sub.3 had no effect on 
the responsiveness of cells to 1,25-(OH).sub.2 D.sub.3. 
Xiphoid cells responded to pretreatment with 24.25-(OH).sub.2 D.sub.3 in a 
manner distinct from the resting zone cells. Cultures preincubated with 
vehicle alone and challenged with vehicle exhibited comparable enzyme 
activity, regardless of the length of pretreatment. In cultures pretreated 
with vehicle alone or with 24,25-(OH).sub.2 D.sub.3 for 24 hours, 
1,25-(OH).sub.2 D.sub.3 inhibited alkaline phosphatase specific activity 
in a dose-dependent manner. The effect of 1,25-(OH).sub.2 D.sub.3 was not 
seen in xiphoid cells preincubated with 24,25-(OH).sub.2 D.sub.3 for 36, 
48 or 72 hours. 
[.sup.3 H]-Uridine incorporation was unaffected by any of the treatment 
regimens used. 
Following a 24-hour pretreatment with 24,25-(OH).sub.2 D.sub.3, resting 
zone chondrocytes exhibited a dose-dependent decrease in synthesis of 
collagenase-digestible protein when exposed to 1,25-(OH).sub.2 D.sub.3. At 
the highest concentration of 1,25-(OH).sub.2 D.sub.3, CDP synthesis was 
comparable to that seen in chondrocytes pretreated with vehicle alone. NCP 
synthesis was unaffected by any of the treatment protocols. The percent 
collagen production calculated from the CDP/NCP ratio also demonstrated a 
dose-dependent inhibition when the pretreated chondrocytes were exposed to 
1,25-(OH).sub.2 D.sub.3. These observations were consistent among 
experiments. 
Following a 48-hour exposure to 24,25-(OH).sub.2 D.sub.3, 1,25-(OH).sub.2 
D.sub.3 stimulated CDP synthesis, with a maximum increase at 10.sup.-9 M. 
A corresponding effect was observed in percent collagen production. When 
resting zone chondrocytes were pretreated with vehicle alone and then 
challenged with 1,25-(OH).sub.2 D.sub.3, CDP synthesis and percent 
collagen production were decreased in comparison to cultures pretreated 
with 24,25-(OH).sub.2 D.sub.3 and challenged with vehicle only. These 
observations were consistent among experiments. As found in the 24 hour 
pre-treatment group above, NCP synthesis was unaffected by 48 hours of 
pretreatment as well. 
The effect of 24,25-(OH).sub.2 D.sub.3 pretreatment on CDP production was 
time-dependent. In cultures pre-incubated with 24,25-(OH).sub.2 D.sub.3 
but challenged with vehicle alone, CDP production was unchanged, 
regardless of the length of pretreatment. When 24,25-(OH).sub.2 D.sub.3 
pretreated cells were subsequently incubated with 1,25-(OH).sub.2 D.sub.3, 
CDP production was decreased in cultures exposed for 24 hours, but by 36 
hours of exposure, there was a marked increase in CDP synthesis. The 
effect of pretreatment was maximal at 48 hours and was sustained in 
cultures pretreated for 120 hours. In contrast, in cultures preincubated 
with vehicle alone and challenged with 1,25-(OH).sub.2 D.sub.3, CDP 
production remained decreased, regardless of the length of pre-incubation. 
NCP was unaffected under all treatment protocols. Consequently, the 
effects of treatment on percent collagen production mirrored those on CDP 
production. 
The effect of vitamin D metabolites on sulfate incorporation by growth zone 
and resting zone chondrocytes has not been reported, so before examining 
the effect of pretreatment with 24,25-(OH).sub.2 D.sub.3, we characterized 
the baseline effects of both vitamin D metabolites on the two cells. 
1,25-(OH).sub.2 D.sub.3 stimulated .sup.35 SO.sub.4 incorporation by growth 
zone chondrocytes. The effect was significant at 10.sup.-9 M to 10.sup.-8 
M. No effect was observed when 1,25-(OH).sub.2 D.sub.3 was added to 
resting zone cells. 24,25-(OH).sub.2 D.sub.3 had no effect on .sup.35 
SO.sub.4 incorporation by growth zone cells. In resting zone cells 
incubated with 24,25-(OH).sub.2 D.sub.3, there was a dose-dependent 
increase in proteoglycan production at 10.sup.-9 M to 10.sup.-8 M, with a 
peak at 10.sup.-8 M. 
The addition of 1,25-(OH).sub.2 D.sub.3 to resting zone cells pretreated 
for 24 hours with 24,25-(OH).sub.2 D.sub.3 produced no effect on sulfate 
incorporation. A similar level of .sup.35 SO.sub.4 incorporation was found 
in cultures pre-incubated with vehicle and challenged with 1,25-OH).sub.2 
D.sub.3. However, if resting zone cells were pretreated with 
24,25-(OH).sub.2 D.sub.3 for 48 hours, and then incubated with 
1,25-(OH).sub.2 D.sub.3, a dose-dependent increase in SO.sub.4 
incorporation was observed. 1,25-(OH).sub.2 D.sub.3 -dependent increases 
in .sup.35 SO.sub.4 incorporation were seen only in cultures pretreated 
for a minimum of 48 hours. 
The results of the present study provide evidence that fourth passage 
chondrocytes derived from the resting zone of rat costochondral cartilage 
exhibit a distinct phenotype compared with cells derived from the growth 
zone. Incorporation of [.sup.35 S]-sulfate by these cultures, presumably 
into proteoglycan, was dependent on both the state of cell maturation and 
vitamin D metabolite used. 1,25-(OH).sub.2 D.sub.3 affected cells derived 
from the growth zone, whereas 24,25-(OH).sub.2 D.sub.3 affected cells 
derived from the resting zone. 
Resting zone chondrocytes appear to be specific target cells for 
24,25-(OH).sub.2 D.sub.3. While previous studies have shown that cell 
metabolism is affected by 24,25-(OH).sub.2 D.sub.3, this is the first 
study to provide a definitive demonstration that this hormone induces 
differentiation. Resting zone chondrocytes pre-treated with 
24,25-(OH).sub.2 D.sub.3 not only acquired responsiveness to 
1,25-(OH).sub.2 D.sub.3, a growth zone chondrocyte trait, but exhibited a 
phenotype consistent with authentic growth zone cells. 
The ability of 24,25-(OH).sub.2 D.sub.3 to induce this effect was not due 
to a nonspecific phenomenon during pre-incubation of the cells. Neither 
pre-incubation with vehicle alone for up to 120 hours, nor pre-incubation 
with 1,25-(OH).sub.2 D.sub.3 induced differentiation of these cells. In 
fact, the response of the cells pretreated with vehicle alone or with 
1,25-(OH).sub.2 D.sub.3 to challenge with 1,25-(OH).sub.2 D.sub.3 was 
entirely consistent with their being resting zone chondrocytes. Alkaline 
phosphatase specific activity and sulfate incorporation were unchanged, 
but collagen production was inhibited. These experiments also confirmed 
our previous observation that exposure to the ethanol vehicle alone had no 
measurable effect on these cells. 
Resting zone chondrocytes required a minimum of 36-48 hours exposure to 
24,25-(OH).sub.2 D.sub.3 before responsiveness to 1,25-(OH).sub.2 D.sub.3 
was detectable. For example, there was no difference in [.sup.3 H]-proline 
incorporation into collagenase-digestible protein in chondrocytes treated 
with 1,25-(OH).sub.2 D.sub.3 for 24 hours, whether or not they were 
pretreated with 24,25-(OH).sub.2 D.sub.3. In both instances, CDP 
production was decreased by treatment with 1,25-(OH).sub.2 D.sub.3. In 
contrast, after 48 hours of pretreatment with 24,25-(OH).sub.2 D.sub.3, 
CDP production was significantly higher than in the non-24,25-(OH).sub.2 
D.sub.3 pretreated cells and was further stimulated by 1,25-(OH).sub.2 
D.sub.3. 
The data suggest that 24,25-(OH).sub.2 D.sub.3 initiates a differentiation 
cascade. This hypothesis is supported by the observation that maximal 
response to 1,25-(OH).sub.2 D.sub.3 is not achieved until the resting zone 
cells have been pretreated with 24,25-(OH).sub.2 D.sub.3 for 72 hours. 
Further, this was the case for all parameters examined. 
Although 24,25-(OH).sub.2 D.sub.3 has the ability to induce differentiation 
of resting zone cells in vitro, it probably promotes its effect in concert 
with other local factors and hormones. When fetal mouse bones are exposed 
to 24,25-(OH).sub.2 D.sub.3, the effects of the hormone on growth and 
development are observed only in serum-containing media (Schwartz, Z. et 
al., "A direct effect of 24,25-(OH).sub.2 D.sub.3 and 1,25-(OH).sub.2 
D.sub.3 on the modeling of fetal mice long bones in vitro," J. Bone Min. 
Res. (1989) 4:157-163). Effects of another steroid hormone, 
17.beta.-estradiol, on the chondrocytes were also dependent on the 
presence of FBS in the medium (Nasatzky, E. et al., "Sex dependent effects 
of 17.beta. estradiol on chondrocyte differentiation in culture," J. Cell. 
Phys. (1993) 156:359-367). The requirement for serum may be due in part to 
the presence of binding proteins needed for proper presentation of the 
hormone to the cell. In addition, growth factors in the serum may play a 
role. For example, as discussed above TGF.beta. has a synergistic effect 
with 24,25-(OH).sub.2 D.sub.3 on resting zone chondrocytes; the complex 
regulation of chondrocyte differentiation by other factors and hormones 
has been shown by numerous investigators. 
The regulation of chondrocyte differentiation by 24,25-(OH).sub.2 D.sub.3 
involves at least two major steps. As shown by this study, it causes the 
less mature resting zone chondrocyte to advance in the endochondral 
differentiation cascade and develop a growth zone chondrocyte phenotype. 
24,25-(OH).sub.2 D.sub.3 also regulates production of vitamin D 
metabolites by the chondrocytes (Schwartz, Z. et al., "Production of 
1,25-(OH).sub.2 D.sub.3 and 24,25-(OH).sub.2 D.sub.3 by growth zone and 
resting zone chondrocytes is dependent on cell maturation and is regulated 
by hormones and growth factor," Endocrinology (1992) 130:2495-2504). While 
it down-regulates production of 24,25-(OH).sub.2 D.sub.3 by resting zone 
cells, it up-regulates production of 1,25-(OH).sub.2 D.sub.3 by growth 
zone cells. Thus, as the resting zone cells acquire a growth zone 
phenotype, production of 1,25-(OH).sub.2 D.sub.3 may be stimulated, 
regulating the next stage of differentiation in an autocrine manner. 
This study also indicates that cells isolated from xiphoid cartilage are 
distinct from resting zone chondrocytes in their phenotype, although both 
cell types are derived from proteoglycan-rich cartilaginous tissues. 
Whereas alkaline phosphatase activity in resting zone cell cultures is 
unaffected by 1,25-(OH).sub.2 D.sub.3, it is inhibited in xiphoid cell 
cultures. Only after a minimum of 36 hours exposure to 24,25-(OH).sub.2 
D.sub.3 do these cells become nonresponsive to 1,25-(OH).sub.2 D.sub.3, 
suggesting that they may have acquired a different phenotype, perhaps a 
resting zone chondrocyte phenotype. This is consistent with the hypothesis 
that there is a chondrocyte lineage continuum from the noncalcifying 
hyaline xiphoid cartilage cell through the calcifying chondrocyte, with 
the time spent in the resting zone maturation state being dependent on 
anatomic site and physiology of the animal. 
While 24,25-(OH).sub.2 D.sub.3 appears to promote xiphoid differentiation, 
1,25-(OH).sub.2 D.sub.3 appears to inhibit this process. In contrast, 
chondrocytes derived from embryonic chick sternum, also a hyaline type of 
cartilage, can become hypertrophic in vitro following 12 days of exposure 
to 1,25-(OH).sub.2 D.sub.3 (Schwartz, Z. et al., "Regulation of 
prostaglandin E.sub.2 synthesis by vitamin D metabolites in growth zone 
and resting zone chondrocyte cultures is dependent on cell maturation," 
Bone (1992) 13:395-401). It is likely that the differences in the two 
model systems account for some of the apparent inconsistency in the 
observations. The length of treatment, species and age of the animal 
model, and selection criteria of cells for culture all varied. Even with 
these differences in experimental design, both models support the concept 
of a chondrogenic differentiation cascade. 
The results of our study provide further evidence of the importance of 
24,25-(OH).sub.2 D.sub.3 in chondrocyte differentiation and confirm 
previous observations, and those of other laboratories, that 
24,25-(OH).sub.2 D.sub.3 can regulate cartilage cell proliferation and 
matrix production and growth plate maturation. This study demonstrates for 
the first time that 24,25-(OH).sub.2 D.sub.3 specifically targets resting 
zone cells, inducing their differentiation along the endochondral 
developmental pathway. Moreover, it shows for the first time that xiphoid 
cartilage cells are regulated by 24,25-(OH).sub.2 D.sub.3 in a manner 
distinct from resting zone cells. The role of 24,25-(OH).sub.2 D.sub.3 in 
resting zone cell differentiation appears to be specific to this 
metabolite, since pretreatment with 1,25-(OH).sub.2 D.sub.3 was not 
effective. 
Example 4 
Isolation of matrix vesicles. 
Matrix vesicles, extracellular organelles that are membrane bound and have 
diameters of approximately 200-450 Angstroms, are isolated from calcifying 
tissues and have a characteristic alkaline phosphatase specific activity 
that is greater than 2-fold the activity found in the plasma membranes of 
the cells which formed the matrix vesicles. Matrix vesicles also tend to 
be high in phosphatidylserine content. 
Matrix vesicles are prepared from cell cultures as follows. At harvest, the 
conditioned media are decanted and the cells are released by 
trypsinization (1% in Hank's balanced salt solution). The reaction is 
stopped with Dulbecco's modified Eagle's medium containing 10% fetal 
bovine serum. The cells are collected by centrifugation at 500.times. g 
for 10 minutes. The supernatant from the trypsin digest is centrifuged for 
20 minutes at 13,000.times.g to pellet a mitochondria/membrane fraction, 
and the resulting supernatant is centrifuged for one hour at 
100,000.times. g to pellet matrix vesicles. Matrix vesicles are 
resuspended in 1 ml 0.9% NaCl and stored frozen at -20.degree. to 
-70.degree. C. until used. 
Example 5 
Matrix Vesicle Extract. 
Matrix vesicle extract is made using the following protocol. Equal volumes 
of the matrix vesicle suspension (1 mg protein/ml in 0.9% NaCl) are mixed 
with 0.1M Tris buffer, pH 7.5, containing 4M guanidine HCl, 0.02M 
CaCl.sub.2 and 0.4% Triton X-100. The membrane suspension is briefly mixed 
for 20-30 seconds with a ground glass homogenizer (Duall #20, Kontes Co., 
Vineland, N.J.) and then stirred for 2 hours at 4.degree. C. The extract 
is then centrifuged at 106,000.times. g for 1 hour and the supernatants 
dialyzed into metalloproteinase or plasminogen activator assay buffer. 
Matrix vesicles prepared in this manner exhibit neutral metalloproteinase 
activity (specifically stromelysin), acid metalloproteinase activity, and 
plasminogen activator. Gelatinase activity may also be present. 
Example 6 
Method of making matrix vesicles having intercalated REF. 
Matrix vesicles isolated as described in Example 4 are incubated with 
1,25-(OH).sub.2 D.sub.3 as described in Example 1 for activation of latent 
TGF.beta. by isolated matrix vesicles to allow intercalation of the 
1,25-(OH).sub.2 D.sub.3 into the matrix vesicle membranes. The matrix 
vesicles are then assayed for the presence of 1,25-(OH).sub.2 D.sub.3 by 
means known to the art and a significant amount is found to have been 
taken up. The treated matrix vesicles are then tested for their ability to 
activate latent growth factors, TGF.beta., insulin-like growth factor, 
bone morphogenic protein, platelet-derived growth factor, and fibroblast 
growth factor, i.e, by the method of Example 1. Significant activation is 
demonstrated in all cases. 
The procedure is repeated incubating the additional REFs estrogen and 
testosterone with the matrix vesicles. Significant growth factor 
activation is shown. 
Example 7 
Conversion of latent growth factor to active growth factor. 
The procedure of Example 1 for activation of latent TGF.beta. by isolated 
matrix vesicles is followed, successively using latent insulin-like growth 
factor, latent fibroblast growth factor, latent bone morphogenic protein, 
and latent platelet-derived growth factor in place of latent TGF.beta., 
and assaying for growth factor activity by bioassays known to the art. 
Significant conversion of latent to active growth factor is demonstrated in 
each instance. 
The procedure is repeated deleting the step of incubating the matrix 
vesicles with 1,25-(OH).sub.2 D.sub.3, and significant conversion of each 
latent growth factor to active form is found. 
The procedure is repeated, substituting in turn the REFs estrogen and 
testosterone for the 1,25-(OH).sub.2 D.sub.3, and significant conversion 
of latent growth factor to active form is seen for all growth factors. 
The procedure is repeated omitting the step of incubating the matrix 
vesicles with 1,25-(OH).sub.2 D.sub.3 and instead adding the REF to the 
latent growth factor and incubating with the matrix vesicles as described. 
This procedure is repeated with estrogen, testosterone and prostaglandin 
E.sub.2. Significant conversion of latent growth factor to active form is 
seen for all growth factors. 
Example 8 
Implants for enhancing activation of latent growth factor. 
Two-phase biodegradable implants are designed and constructed using 50:50 
poly(DL-lactide-co-glycolide)(PLG) with inherent viscosity of 0.71 dl/gm 
(weight average molecular weight 65 kD). The implant consists of a "bone" 
phase that abuts against the underlying bone for anchoring and a 
"cartilage" phase which interfaces with the adjacent layer of articular 
cartilage. The polymer is solubilized in acetone and precipitated with 
ethanol. The gummy "bone" composite is placed under 10 m Tort vacuum for 
six hours and then packed into a Teflon mold under 10 m Torr and 
24.degree. C. for 24 hours. The implants are then partially removed and 
allowed to remain under the same conditions for 24 hours. New polymer is 
then solubilized in acetone and combined with the appropriate amount of 
TGF.beta.. Latent recombinant human TGF.beta..sub.1 (approximately 4 g) is 
solubilized in 0.2 ml sterile water, stirred overnight and added to the 
soft polymer. The appropriate volume of solution to give a total of 500 ng 
of latent TGF.beta. is used in the "cartilage" phase only of each implant. 
The two-phase implants are placed in the mold under 10 m Torr and 
4.degree. C. for 24 hours, partially removed, and placed in a lyophilizer 
under the same conditions for another 24 hours. At the end of the curing 
period, the implants are completely removed from the mold and stored in 
the lyophilizer until required for implantation into the host. The curing 
techniques used for the two phases render the implant porous and the 
"cartilage" phase softer than the "bone" phase. The two phases are 
mechanically tested using an automated indenter and modeled using the 
linear biphasic theory (Mow, V. C. et al., J. Biomech. Eng. (1980) 
102:73-84). 
At the same time the TGF.beta..sub.1 is added, 1 ml of a solution of 
1,25-(OH).sub.2 D.sub.3 as described in Example 1, a sufficient amount to 
activate said growth factor, is added to the implant. 
Cylindrical, 4 mm.times.6 mm, full-thickness defects are created with a 
low-speed drill, under saline irrigation, in the central posterior medial 
condyle of each right knee joint, through a posteromedial approach. 
Defects are filled with implants containing 500 ng of latent TGF.beta., 
implants without latent growth factor, implants with active 
rhTGF.beta..sub.1 or are left empty as controls. The animals are allowed 
free cage activity for either four or eight weeks, prior to sacrifice. A 
total of 96 New Zealand male white rabbits are used. The quality of 
healing is examined at four weeks (48 rabbits) and at eight weeks (48 
rabbits) using gross morphology, biomechanics, and histomorphometry. 
Statistically the results are compared with analysis of variance and 
multiple comparisons tests. 
The repair osteochondral defect and adjacent site are biomechanically 
tested using an automated indenter under conditions of biphasic creep 
indentation. The three intrinsic material properties of repair and 
adjacent cartilage are obtained using a numerical algorithm (Athanasiou et 
al., Trans. Orth. Res. Soc. (1992) 17(1):172) based on biphasic finite 
element methods (Spilker et al., J. of Biomech. Eng. (1990) 112:138) and 
nonlinear optimization techniques. The adjacent site is tested 3 mm 
anterior to the defect. After biomechanical testing, each osteochondral 
specimen is sectioned, stained with Alcian blue, and digitized to obtain 
the geometric parameters needed in the finite element modeling. The Cray 
supercomputer is used for these analyses. Histologically, each 
osteochondral specimen is decalcified and stained with hematoxylin and 
eosin. Sections are analyzed with an image analysis system to measure the 
percent of trabecular bony repair in each defect. 
The group having the implant with latent TGF.beta. shows significant 
healing after eight weeks, similar to that with active TGF.beta., compared 
to the group having the implant without TGF.beta. and the unimplanted 
control group. 
This procedure is repeated using the additional latent growth factors, 
insulin-like growth factor, platelet-derived growth factor, and fibroblast 
growth factor, with similar results. 
The foregoing procedures are repeated successively using estrogen, 
testosterone, dexamethasone, prostaglandin E.sub.2, thyroid, leukotrienes 
and platelet activating factors instead of 1,25-(OH).sub.2 D.sub.3 with 
comparable results. 
The foregoing procedures are repeated without incorporating REF into the 
implant, but injecting 1 ml of a 10.sup.-12 M solution into the wound site 
at intervals of 24 hours during the eight-week period. Significant healing 
is shown compared to controls with and without implants. 
The procedures are repeated incorporating 1 ml per cc of polymer of a 
suspension of matrix vesicles into the implant with and without latent 
growth factors and REFs. Matrix vesicles having REF intercalated into the 
cell membrane as described in Example 6 are also incorporated into the 
polymer, with and without latent growth factor. In the implants without 
latent growth factors and/or REFs, the missing component(s) are injected 
into the wound site. 1 ml of a 1% solution of latent growth factor is 
used. The results indicate significant wound healing compared to controls. 
The procedures are repeated incorporating 1 ml of a matrix vesicle extract 
as described in Example 5 with and without latent growth factor. When 
latent growth factor is not incorporated into the polymer, it is 
periodically injected into the wound site as described above. Significant 
healing compared to controls is observed. 
Example 9 
Cell seeded scaffolding. 
Polymeric materials incorporating the full range of combinations of latent 
growth factors, REFs, matrix vesicles with and without intercalated REFs, 
and matrix vesicle extracts described above are prepared as described in 
Example 8, except that rather than forming cylindrical implants with such 
polymers, three-dimensional scaffolds as described in U.S. Pat. No. 
5,160,490, incorporated herein by reference, are prepared. The scaffolds 
are seeded with osteoblasts, chondrocytes or tendon cells, and cultured as 
described in said patent. In each instance where the necessary REF, latent 
growth factor, or matrix vesicle material required for activation of 
latent growth factor is not incorporated into the polymer, it is added to 
the culture medium. Significantly, enhanced growth and differentiation of 
cells is shown. 
This invention has been described with reference to preferred embodiments; 
however, it will be apparent to those skilled in the art that additional 
equivalent procedures and compositions may be substituted in the practice 
of this invention for those disclosed herein within the scope and spirit 
of applicants contribution to the art. The appended claims are to be 
interpreted to include all such modifications and equivalents.