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Chondrogenic stimulation of embryonic chick limb mesenchymal cells by factors in bovine and human dentine extracts
0003-9969/90
oral Bid. Vol. 35, No. I, pp. 49-54, 1990 Printed in Great Britain. All rights reserved
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CHONDROGENIC STIMULATION OF EMBRYONIC CHICK LIMB MESENCHYMAL CELLS BY FACTORS IN BOVINE AND HUMAN DENTINE EXTRACTS T. RABINOWITZ,’ G. T. SYFTESTAD’and A. I. CAPLAN** Departments of ‘Pediatric Dentistry and *Biology, Case Western Reserve University, Cleveland, OH 44106, U.S.A. (Accepted 6 Jury 1989) Summary-Exl.racts of bovine or human dentine when incorporated into the medium bathing embryonic chick-limb mesenchymal cells caused the stimulation of chondrogenesis. Such extracts were prepared from demineralized bovine or human dentine by suspension in a 4 M guanidinium hydrochloride (GuHCl) solution. After stepwise dialysis down to distilled water to remove the GuHCI, the protein extracts were separated chromatographically on DEAE resin. The crude and separated extracts from both dentines produced morphological changes in the cultured mesenchymal cells similar to those reported for bovine and human adult bone extracts, as well as metachromatic staining with toluidine blue indicating that they stimulated chondrogenesis. Measurements of [35S]-S0, incorporation and DNA content in the cultures also indicated that the extracts stimulated chondrogenesis but not cell proliferation. The similarity between silver-stained patterns on sodium dodecyl sulphate polyacrylamide gel electrophoresis of corresponding fractions of the bovine and human dentine suggests that they may contain similar or identical proteins. Key words: dentine, cartilage, chondrogenesis.
chick-limb mesenchymal cell cultures provided an in vitro assay for chondrocyte stimulation (Syftestad and Caplan, 1984; Syftestad et al., 1984; Syftestad, Lucas and Caplan, 1985).
IhTRODUflION The non-collagenous proteins of dentine are acidic and may be grouped into classes: acidic glycoproteins, y -carboxy,glutamate-rich proteins, phos-
phoproteins, proteoglycans and serum proteins. It has been suggested that these molecules may play a part in the various steps of dental mineralization by virtue of their physico-chemical properties (Linde, 1984). In addition to the non-collagenous proteins, the extracellular matrix of dentine also contains factors that induce c:artilage or bone when tested at ectopic sites in vivo (Yoemans and Urist, 1967). This demineralized rabbit dentine, implanted into muscle pouches of allogeneic recipients, induced new bone, which was interpreted as indicating that the dentine matrix had released a bone morphogenetic protein that initiated the difierentiation of cartilage and bone in a manner identical to that observed with mineralfree adult bone matrix (Uris& 1965). Later, Conover and Urist (1979) demonstrated that in viuo bone induction could occur outside of a double-walled diffusion chamber containing demineralized dentine. This finding suggested that the active agent(s) were diffusible at body temperature in body fluids and could therefore be removed from dentine by protein solvents. We have tested this hypothesis using extracts of both bovine and human dentine; preliminary work had shown that a crude extract from human dentine would stimulate chondrogenesis. Embryonic
MATERIALS AND METHODS Tissue processing
Mandibles were removed from freshly slaughtered cows that were less than 2 yr old, and stored at 0°C in sealed dry plastic bags at a local slaughterhouse for no longer than 5 days before processing. Human teeth were collected for us by local oral surgeons, and were stored in sealed dry containers at 0°C. The bovine mandibles and attached teeth were cut into 0.5 cm thick slices with a band saw and stirred in a 0.6 M HCl solution at 4°C for 10-14 days, until they could be cut easily with a razor blade. Human teeth were demineralized in a similar fashion and were processed separately from the bovine teeth. After this, the teeth were scraped clean of any remaining soft tissue or demineralized alveolar bone and cut in half through their long axes to remove the pulps. Teeth that were badly decayed or judged unsuitable for these experiments were discarded. The remaining teeth were cut into small pieces with razor blades and were stirred in distilled water for 24 h to remove all traces of HCl. The demineralized pieces were then suspended in a solution of chloroformmethanol (1: 1, v/v) and stirred for 24 h to remove lipid. The dentine residue was allowed to air-dry for 1 day to constant weight.
*Address all correspondence to: A. I. Caplan, Department of Biology, Case Western Reserve University, Cleveland, OH 44106, U.S.A. 49
50
T.
RABINOWITZ et
Extraction and fractionation
The demineralized dentine matrix was placed in a solution of 4 M guanidinium hydrochloride (GuHCl) containing 50 mM Tris (hydroxymethyl) amino methane (Tris) plus 5 mM each of iodoacetic acid, phenylmethyl sulphonyl fluoride, and sodium azide as protease inhibitors. The pH of the solution was adjusted to 7.0. The volume in ml of GuHCl solution used was equal to 4 times the weight in grammes of the dentine matrix to be processed. Extraction proceeded for 72 h with constant stirring at 4°C. The turbid GuHCl solution was then passed through cheesecloth and centrifuged at 16,000 g for 20 min. The supernatant was decanted, the volume measured and transferred to tubular dialysis membranes (molecular weight cut-off: 12-14 kdalton). The membranes were suspended in a volume of distilled water equal to 7 times the volume of the fluid contained within the dialysis tubing; this was done to give a GuHCl concentration of 0.5 M at the end of 72 h of dialysis. After this step, a precipitate usually formed; therefore, the solutions were centrifuged as described above and the precipitate discarded. The supernatant was transferred to fresh dialysis membranes and dialysed against distilled water at 4°C for 5-7 days. The water was changed every 12-24 h. If a precipitate formed, the solution was centrifuged and the precipitate discarded. The remaining supernatant comprised the cold water-soluble GuHCl extract of bovine or human dentine. This was frozen in flasks in a mixture of dry ice and ethanol and lyophilized. A portion of this crude extract was dissolved in a small volume of a 6 M urea solution containing 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]lpropane-sulphonate (CHAPS), 0.5 mM Tris and the protease inhibitors described above; the pH of the solution was adjusted to 8.0. This solution was introduced into a 50 ml cylindrical column (2.0 cm dia) containing DEAE bound to Sepharose, followed by 250 ml of the urea/CHAPS solution; this 250ml comprised the unbound fraction. The next fraction was eluted by 250ml of the urea/CHAPS solution containing 0.6 M NaCl. All fractions were dialysed against distilled water at 4°C for 5-7 days, frozen and lyophilized. Thus, three fractions of bovine or human dentine extract were prepared-rude, unbound and 0.6 M NaCl-eluted fractions. Electrophoresis
Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) was performed on all extracts tested using the Tri-glycine buffer system of Laemmli (1970). A 5% acrylamide upper gel was used for stacking the samples, which were then separated by a l&22% acrylamide gradient in the running gel. After electrophoresis, the gels were stained with silver (Merril, Switzer and Van Keuren, 1979). In vitro assay and quantitation
All lyophilized fractions were dissolved in l-2ml of distilled water and were analysed for protein content (Bradford, 1976). Aliquots of the dissolved extracts were added to Eagle’s minimal essential medium supplemented with 3% fetal calf serum, 5% chick embryo extract and 7% horse serum (Syftestad
al.
and Caplan, 1984) to give final protein concentrations of 0 (control) 1, 5, IO, 20, 50, 100 and 25Opg protein/ml of medium. These extracts were applied to embryonic stage 24 (Hamburger and Hamilton, 1951) chick limb/mesenchymal cell cultures, plated at a density of 0.5 x lo6 cells/well on 24-well sterile plastic culture plates (16 mm dia) previously coated with a 1% gelatin solution. These cells had been dissected from the embryo limbs, dissociated with trypsin, and passed through a 20pm filter to give a single cell suspension (Caplan, 1972). They were allowed to incubate for 2 days after plating during which time they were given control medium. Nutrient medium supplemented with dentine extract was applied to the cell cultures in 250 ~1 at daily intervals beginning 2 days after plating (day 2). The plates were incubated at 37°C in an atmosphere of 5% CO1 and 95% humidity for B-10 days and viewed daily with phasecontrast optics. At the end of the incubation period the cultures were rinsed twice with Tyrode’s balanced salt solution, fixed with 70% ethanol for 20min, rinsed in decreasing concentrations of ethanol, rinsed with water, stained with 0.1% toluidine blue for 3.5 min (pH 6.0), destained with 70% ethanol until the destaining fluid was clear and allowed to air-dry. For some cell cultures exposed to the 0.6 M NaCleluted fraction of bovine dentine, radioactive sulphate incorporation into macromolecules was quantitated as a measure of cartilage proteoglycan biosynthesis after pulsing for 3 h with 5 PCi of [35S]-S0, (carrier-free sodium salt) per well, after which the cell layers were rinsed with cold Tyrode’s solution, precipitated with cold 10% trichloroacetic acid, solubilized with NCS (Amersham Corp., Arlington Heights, Ill., U.S.A.), and subjected to scintillation counting. In duplicate cultures, the DNA content (as a measure of cell number) was estimated by the diphenylamine method of Patterson (1979). Quantitative measurements on cells exposed to the human dentine extracts were not done because only enough extract was available to observe living and stained cultures. RESULTS
Composition of extracts
A diagram of silver-stained PAGE patterns obtained for the bovine and human dentine extracts is shown in Text Fig. 1. The crude dentine extracts from both types of teeth produced a heavy smear on the gel indicating that many proteins with a variety of molecular weights were present in these fractions (not depicted in Text Fig. 1). The unbound fraction of bovine dentine showed enrichment at approx. 45 and 31 kdalton; the fraction eluted with 0.6 M NaCl produced an identical pattern except for an additional heavy band at 55 kdalton. The unbound fraction of human dentine produced a gel pattern the same as the one produced by the corresponding fraction from the bovine dentine described above. The fraction eluted by 0.6 M NaCl contained a heavy band at approx. 55 kdalton and a lighter band at 45 kdalton; no 31 kdalton band was seen in this fraction. These major bands were present on a background of a light smear indicating the presence of a complex array of various proteins.
51
Chondrogenic stimulation by dentine
92.5 -
66.2 45.0 -
I-
-
-
31.0-
‘-
-
-
-
21.514.4 A
B
C
D
E
Fig. I. Diagram of silver-stained PAGE pattern of fractions of bovine and human dentine. Column A: molecular weight markers ( x 1000 dalton); column B: unbound fraction from bovine dentine with banding at 45 and 31 kdalton; column C: 0.6 M NaCI-eluted fraction of bovine dentine with bands at 55, 45 and 31 kdalton; column D: unbound fraction from human dentine with bands at 45 and 31 kdalton; column E: 0.6 M NaCl-eluted fraction of human dentine with bands at 55 and 45 kdalton.
Mesenchymal
cell response
When observed wi1.h phase-contrast optics, all cell cultures that had a positive response to the dentine extracts had a distinct change in morphology as early as 2 days after the beginning of the exposure, i.e. the fourth day after plating. By this time the stimulated cells were plumper and rounder than those in the unstimulated controls. The volume of the extracellular matrix and, therefore, the spacing between the cells, was also increased in stimulated cultures (Plate Figs 2 and 3). By the fifth or sixth day after plating, small nests of cells were seen in control wells. These cells (early chondrocytes) formed cartilage nodules that usually appeared as discrete round isiands several cell layers thiclc (Caplan, 1970; Osdoby and Caplan, 1979). Cells that did not respond to the dentine extracts formed chondrocyte islands identical to those in control wells (Plate Fig. 4). By the seventh or eighth day after plating, we observed a perichondrium-like structure surrounding the cartilage nodules in the unstimulared plates, and this signalled the end of further nodule formation or growth. About 5-10% of the cells in the well could be identified as chondrocytes (Plate Fig. 5). Cells that showed evidence of chondrogenic stimulation did not form chondrocyte islands. Instead, there was an obvious increase in the volume of the extracellular matrix surrounding a greater proportion of the cells (Plate Fig. 6). The stimulated cells formed large aggregates of chondrocytes that covered a much greater area than that of the cartilage nodules in control or unstimulated wells. No surrounding perichondrial structure was seen in the stimulated wells. By the ninth or tenth day after plating, control cultures had shown no increase in the number or size of cartilage nodules, while maximally stimulated wells were covered by a layer of chondrocytes (Plate Figs 7-9). When the cell cultures were stained with toluidine blue, those that had shown a more pronounced response to the extracts also retained more stain; a typical staining pattern is shown in Plate Fig. 8. In these cultures, we tested the bovine dentine
extract eked from the DEAE column by the urea/CHAPS solution containing 0.6 M NaCl; stimulation occurred at protein concentrations as low as 5 lg/ml and was maximal at 20 pg/ml. Occasionally, a particular extract was toxic to the cell layer; this was usually apparent by the second or third day of exposure. By this time, the cells were pyknotic, and by the fifth or sixth day of exposure, the cell layer had usually become detached from the well and was non-viable. Toxicity usually occurred at protein concentrations greater than 20 or 50pg/ml, whereas lower doses had stimulated chondrogenesis. Human dentine extracts also stimulated chondrogenesis in the cell cultures. Plate Fig. 9 shows the staining pattern of cultures exposed to different concentrations of the unbound fraction of the extract. A maximal response was observed at protein concentrations of 50 pgg/ml. For both the bovine and human dentine extracts, all three fractions (crude, unbound and 0.6 M NaCl-eluted) stimulated chondrogenesis in a dose-dependent fashion. The crude extract fraction, however, generally produced toxic effects at lower protein concentrations than the other two fractions. Estimations of [?S]-SO, incorporation into macromolecules and DNA quantitation were variable but clear trends were observed. Because only a limited amount of extract or partially purified fractions was available, only material from day 8 or 10 of culture was assayed. In these cases, a single 3 h exposure to [3’S]-S0., , followed by trichloroacetic acid precipitation of macromolecules, was accomplished; likewise, DNA measurements were made at these time points. The experiments were repeated several times with different cell preparations using the same batch of bovine dentine extract. The data were analysed by Student’s t-test. In some experiments, there was almost a five-fold increase in labelled sulphate incorporation compared to controls (p < O.Ol), while other experiments resulted in only a two-fold increase; this occurred under conditions where stimulation of chondrogenesis had been clearly observed morphologically. For DNA measurements, we found no significant increase in [3H]-thymidine incorporation into the experimental cells compared to controls (p > 0.1) indicating no increase in cell number and thus no stimulation of mitosis; higher concentrations of protein produced toxic effects in the [3H]-thymidine experiment shown (Table 1). DISCUSSION
We have made use of the novel in vitro model developed by Syftestad and Caplan (1984) to demonstrate the chondrogenic stimulatory effects of watersoluble extracts of bovine and human dentine. The responses to these extracts were identical to those reported for similarly prepared extracts from adult chicken and beef bone (Syftestad and Caplan, 1984; Syftestad et al., 1985), which were said to contain chondrogenic stimulating activity (Syftestad et al., 1984). Usually, under control conditions at the plating densities used in these experiments, a small fraction of cells will differentiate into chondrocytes, but when the cells are exposed to extracts containing chrondrogenic stimulating activity, changes occur in the cell layer. A greater number of undifferentiated
52
T. RABINOWITZet al.
Plate 1
Chondrogenic stimulation by dentine
53
Table 1. Metabolism: DNA and proteoglycan synthesis. Effects of the 0.6 M NaCI-eluted fraction of bovine dentine extract* [‘q-so, [‘HI-thymidine (p) pg Protein/ml counts/min counts/min (PI 0 1151*43 48,913 k 5270 5 1201 If: 97 (>O.l) 54,188 & 5180 (>O.l) 10 2069 + 392 (cO.01) 49,395 * 13,300 (>O.l) 20 5314 + 54 (
similar results. cells are stimulated ultimately to become chondrocytes with a concomitant increase in the amount of cartilaginous extracellular matrix. Mitogenic effects are sometimes observed, but are always less pronounced than the chondrogenic stimulatory ones. These stimulatory effects can be demonstrated by fixing the cell cultures and staining them with toluidine blue, which reacts orthochromatically (giving a blue colour) with rnany cellular constituents, but
metachromatically (giving a purplish colour) with acidic or sulphated glycosaminoglycans, a major extracellular matrix component of cartilage. The cell cultures shown increased [35S]-S0, incorporation, indicating an increase in proteoglycan synthesis, a major component of cartilage. Variability in the amount of sulphate incorporation and DNA content in cell cultures indicates the technical limitations of attempts to relate microscopic observations to the synthetic expression of chondrocytes. Because the effects of dentine extracts on the chick limb-bud
system so closely parallel those for chondrogenic stimulating activity in bone extracts (Syftestad and Caplan, 1984) we conclude that dentine extracts contain chondrogenic stimulatory components, but we cannot state whether these are identical to those of bone. Seyedin et al. (1985) have purified and characterized two cartilage-inducing factors from demineralized bovine bone, with molecular weights of approx. 26 kdalton as shown by their PAGE patterns. These factors, called cartilage-inducing factor A and B induce rat muscle cells to synthesize cartilage-specific proteoglycan in vitro. A subsequent examination of cartilage-inducing factor A (Seyedin et al., 1986) has shown that the sequence of its N-terminal 30 amino acids is identical to that of human transforming growth factor /?, and that both factors have similar activities in vitro, leading to the conclusion that they are closely related or identical molecules. We do not believe that the chondro-stimulatory fractions we
Plate 1 Note: Figs 227 were photographed
Fig. 2. Stage-24
embryonic
using phase-contrast optics (at x 100) and printed at similar magnifications, i.e. x 335
chick limb-bud
mesenchyme cells 4 days after plating. control medium.
These cells received only
Fig. 3. Stage-24 embryonic chick limb-bud mesenchyme cells that had been exposed to a stimulatory extract of bovine dentine (20 pg/ml protein concentration) for 2 days. The cells are rounder and plumper than those in Fig. 2. Intercellular spacing (extracellular matrix) is also increased, seen here as a refractile halo (arrows) about individual cells. Fig. 4. A cartilage
nodule
Fig. 5. A perichondrium-like
in a control
structure
well composed of several layers of chondrocytes nest (arrows). (arrows)
between
individual
cartilage
nodules
forming
a circular
in a control
well.
Fig. 6. Stimulated cells in culture: a thicker region of chondrocytes is present in the middle of the figure, and two thinr.er areas of chondrocytes (arrows) surround it. In wells containing cells showing a positive response to the extracts, this was a typical pattern of chondrocyte presentation. Fig. 7. An agpregate entire field contains
of chondrocytes in a well exposed to a stimulatory extract of bovine dentine. The chondrocytes and is typical of the morphological response of cells exposed to stimulatory extracts of human or bovine dentine.
Fig. 8. Toluidine blue-stained cell cultures prepared on day 8. Bovine dentine extract (0.6 M NaCl-eluted fraction) was applied at concentrations of 0 (control; column A), 5 (column B), 10 (column C), and 20 (column D) pg protein/ml per day. A dose-dependent increase in stain incorporation is apparent. Each black dot (arrow) is a nest of chondrocytes. x 1.1 Fig. 9. Toluidine blue-stained cell cultures prepared on day 8. Human dentine extract (unbound fraction) was applied at concentrations of 0 (control; column A), 5 (column B), 10 (column C), 20 (column D) and 50 (column E) pg protein/ml per day. Each black dot (arrow) is a nest of chondrocytes. x0.94
T. RAMNOU mz et al.
54
have prepared from bovine and human dentine contain transforming growth factor /I because these fractions produced different PAGE patterns than those reported either for cartilage-inducing factor A or transforming growth factor )!I(Seyedin et al., 1985, 1986) with no banding at 26 kdalton. The similarity in the silver-stained gel patterns between corresponding fractions from the bovine and human dentine suggests that similar or identical proteins may be responsible for the chondro-stimulatory effects we observed. The in vitro limb-bud mesenchyme assay has several advantages over the in vivo assays described earlier (Yoemans and Urist, 1967): much less material is required for testing (mg of material in vivo versus pg in vitro), the cultures can be examined every day or more often if necessary; the system responds much more quickly; and dose-dependent responses are more easily quantified. A potential disadvantage lies in the fact that this model contains a fraction of cells that normally differentiate into chondrocytes; thus, it is difficult to discriminate between inductive and enhancement effects. It is of interest to speculate why chondro-stimulatory substances comprise a fraction of the noncollagenous proteins of demineralized bovine and human dentine because cartilage is not found in dentine, although Cummings et al. (198 1) have shown that dental mesenchyme can be directed to differentiate into cartilage under appropriate conditions in vitro. These substances may simply have an affinity for the dentine matrix, or they may indeed be products of odontoblasts. Thus far, we have evidence for the presence of small amounts of chondrogenic stimulatory substances in several soft tissues and body fluids (Syftestad et al., 1987), but the greatest concentrations of these (mg/g dry tissue) have been found in bone and dentine. Whether they play a role in chondro- or osteogenesis or in bone repair of the periodontal tissues requires further investigation. Work continues in our laboratory aimed at the purification and precise identification of these interesting components.
Caplan A. I. (1972) The effect of the nicotinamide sensitive teratogen 3-acetylpyridine on chick limb mesodermal cells in culture: biochemical parameters. J. exp. Zool. 180, 351-362. Conover M. A. and Urist M. R. (1979) Transmembrane bone morphogenesis by implants of dentine matrix. J. dent. Res. 58, 1911. Cummings E. G., Bringas P. Jr, Grodin M. S. and Slavkin H. C. (1981) Epithelial-directed mesenchyme differentiation in vitro. Model of murine odontoblast differentiation mediated by quail epithelia. Differentiation 20, 1-9. Hamburger V. and Hamilton H. L. (1951) A series of normal stages in the development of the chick embryo.
thank Dr D. Lennon for providing the stage-24 limb mesenchymal cell cultures and Mr J. Holecek and Mr S. P. Bruder for technical assistance. This work was supported by grants from the National Institute of Health (U.S.A.).
Syftestad G. T., Lucas P. A. and Caplan A. I. (1985) The in vitro chondrogenic response of limb-bud mesenchyme to a water-soluble fraction prepared from demineralized bone matrix. Differentiation 29, 230-237. Syftestad G. T., Lucas P. A., Ohgushi H. and Caplan A. I. (1987) Chondrogenesis as an in vitro response to bioactive factors extracted from adult bone and non-skeletal tissues. In: Development and Diseases of Cartilage and Bone Matrix (Edited by Sen A. and Thornhill T.) pp. 187-199. Alan R. Liss, New York. Urist M. R. (1965) Bone formation by auto-induction. Science 150, 893-899. Yoemans J. D. and Urist M. R. (1967) Bone induction by decalcified dentine implanted into oral osseous and muscle tissues. Archs oral Biol. 12, 999-1008.
J. Morph. 88, 49-92.
Laemmli U. R. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T,. Nature 227, 680-685.
Linde A. (1984) Non-collagenous proteins and proteoglycans in dentinogenesis. In: Dentin and Dentinogenesis (Edited by Linde A.) Vol. 2, pp. 55-92. CRC Press, Florida. Merril C. R., Switzer R. C. and Van Keuren M. L. (1979) Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain. Proc. natn. Acad. Sci. U.S.A. 79, 43354339.
Osdoby P. and Caplan A. I. (1979) Osteogenesis in cultures of limb mesenchymal cells. Deul Biol. 73, 84-102. Patterson M. K. J. (1979) Measurement of growth and viability of cells in culture. Meth. Enzym. 58, 141-151.
Seyedin S. M., Thomas T. C., Thompson A. Y., Rosen D. M. and Piez K. A. (1985) Purification and characterization of two cartilage-inducing factors from bovine demineralized bone. Proc. natn. Acad. Sci. U.S.A. 82, 2267-227
1.
Seyedin S. M., Thompson A. Y., Bentz H., Rosen D. M., McPherson J. M., Conti A., Siegel N. R., Gallupi G. R. and Piez K. A. (1986) Cartilage-inducing factor-A. Apparent identity to transforming growth factor-j. J. biol. Chem. 261, 5693-5695.
Syftestad G. T. and Caplan A. I. (1984) A fraction from extracts of demineralized adult bone stimulates the conversion of mesenchymal cells into chondrocytes. Deal Biol. 104, 348-356. Syftestad G. T., Triffit J. T., Urist M. R. and Caplan A. I. (1984) An osteo-inductive bone matrix extract stimulates the in vitro conversion of mesenchyme into chondrocytes. Calc. Tissue Int. 36, 625-627.
Acknowledgements-We
REFERENCES
Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254.
Caplan A. I. (1970) Effects of the nicotinamide sensitive teratogen 3-acetylpyridine on chick limb bud cells in culture. Expl CeN Res. 62, 341-348.
oral Bid. Vol. 35, No. I, pp. 49-54, 1990 Printed in Great Britain. All rights reserved
Archs
%3.00
+ 0.00
Copyright 0 1990Pergamon Press plc
CHONDROGENIC STIMULATION OF EMBRYONIC CHICK LIMB MESENCHYMAL CELLS BY FACTORS IN BOVINE AND HUMAN DENTINE EXTRACTS T. RABINOWITZ,’ G. T. SYFTESTAD’and A. I. CAPLAN** Departments of ‘Pediatric Dentistry and *Biology, Case Western Reserve University, Cleveland, OH 44106, U.S.A. (Accepted 6 Jury 1989) Summary-Exl.racts of bovine or human dentine when incorporated into the medium bathing embryonic chick-limb mesenchymal cells caused the stimulation of chondrogenesis. Such extracts were prepared from demineralized bovine or human dentine by suspension in a 4 M guanidinium hydrochloride (GuHCl) solution. After stepwise dialysis down to distilled water to remove the GuHCI, the protein extracts were separated chromatographically on DEAE resin. The crude and separated extracts from both dentines produced morphological changes in the cultured mesenchymal cells similar to those reported for bovine and human adult bone extracts, as well as metachromatic staining with toluidine blue indicating that they stimulated chondrogenesis. Measurements of [35S]-S0, incorporation and DNA content in the cultures also indicated that the extracts stimulated chondrogenesis but not cell proliferation. The similarity between silver-stained patterns on sodium dodecyl sulphate polyacrylamide gel electrophoresis of corresponding fractions of the bovine and human dentine suggests that they may contain similar or identical proteins. Key words: dentine, cartilage, chondrogenesis.
chick-limb mesenchymal cell cultures provided an in vitro assay for chondrocyte stimulation (Syftestad and Caplan, 1984; Syftestad et al., 1984; Syftestad, Lucas and Caplan, 1985).
IhTRODUflION The non-collagenous proteins of dentine are acidic and may be grouped into classes: acidic glycoproteins, y -carboxy,glutamate-rich proteins, phos-
phoproteins, proteoglycans and serum proteins. It has been suggested that these molecules may play a part in the various steps of dental mineralization by virtue of their physico-chemical properties (Linde, 1984). In addition to the non-collagenous proteins, the extracellular matrix of dentine also contains factors that induce c:artilage or bone when tested at ectopic sites in vivo (Yoemans and Urist, 1967). This demineralized rabbit dentine, implanted into muscle pouches of allogeneic recipients, induced new bone, which was interpreted as indicating that the dentine matrix had released a bone morphogenetic protein that initiated the difierentiation of cartilage and bone in a manner identical to that observed with mineralfree adult bone matrix (Uris& 1965). Later, Conover and Urist (1979) demonstrated that in viuo bone induction could occur outside of a double-walled diffusion chamber containing demineralized dentine. This finding suggested that the active agent(s) were diffusible at body temperature in body fluids and could therefore be removed from dentine by protein solvents. We have tested this hypothesis using extracts of both bovine and human dentine; preliminary work had shown that a crude extract from human dentine would stimulate chondrogenesis. Embryonic
MATERIALS AND METHODS Tissue processing
Mandibles were removed from freshly slaughtered cows that were less than 2 yr old, and stored at 0°C in sealed dry plastic bags at a local slaughterhouse for no longer than 5 days before processing. Human teeth were collected for us by local oral surgeons, and were stored in sealed dry containers at 0°C. The bovine mandibles and attached teeth were cut into 0.5 cm thick slices with a band saw and stirred in a 0.6 M HCl solution at 4°C for 10-14 days, until they could be cut easily with a razor blade. Human teeth were demineralized in a similar fashion and were processed separately from the bovine teeth. After this, the teeth were scraped clean of any remaining soft tissue or demineralized alveolar bone and cut in half through their long axes to remove the pulps. Teeth that were badly decayed or judged unsuitable for these experiments were discarded. The remaining teeth were cut into small pieces with razor blades and were stirred in distilled water for 24 h to remove all traces of HCl. The demineralized pieces were then suspended in a solution of chloroformmethanol (1: 1, v/v) and stirred for 24 h to remove lipid. The dentine residue was allowed to air-dry for 1 day to constant weight.
*Address all correspondence to: A. I. Caplan, Department of Biology, Case Western Reserve University, Cleveland, OH 44106, U.S.A. 49
50
T.
RABINOWITZ et
Extraction and fractionation
The demineralized dentine matrix was placed in a solution of 4 M guanidinium hydrochloride (GuHCl) containing 50 mM Tris (hydroxymethyl) amino methane (Tris) plus 5 mM each of iodoacetic acid, phenylmethyl sulphonyl fluoride, and sodium azide as protease inhibitors. The pH of the solution was adjusted to 7.0. The volume in ml of GuHCl solution used was equal to 4 times the weight in grammes of the dentine matrix to be processed. Extraction proceeded for 72 h with constant stirring at 4°C. The turbid GuHCl solution was then passed through cheesecloth and centrifuged at 16,000 g for 20 min. The supernatant was decanted, the volume measured and transferred to tubular dialysis membranes (molecular weight cut-off: 12-14 kdalton). The membranes were suspended in a volume of distilled water equal to 7 times the volume of the fluid contained within the dialysis tubing; this was done to give a GuHCl concentration of 0.5 M at the end of 72 h of dialysis. After this step, a precipitate usually formed; therefore, the solutions were centrifuged as described above and the precipitate discarded. The supernatant was transferred to fresh dialysis membranes and dialysed against distilled water at 4°C for 5-7 days. The water was changed every 12-24 h. If a precipitate formed, the solution was centrifuged and the precipitate discarded. The remaining supernatant comprised the cold water-soluble GuHCl extract of bovine or human dentine. This was frozen in flasks in a mixture of dry ice and ethanol and lyophilized. A portion of this crude extract was dissolved in a small volume of a 6 M urea solution containing 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]lpropane-sulphonate (CHAPS), 0.5 mM Tris and the protease inhibitors described above; the pH of the solution was adjusted to 8.0. This solution was introduced into a 50 ml cylindrical column (2.0 cm dia) containing DEAE bound to Sepharose, followed by 250 ml of the urea/CHAPS solution; this 250ml comprised the unbound fraction. The next fraction was eluted by 250ml of the urea/CHAPS solution containing 0.6 M NaCl. All fractions were dialysed against distilled water at 4°C for 5-7 days, frozen and lyophilized. Thus, three fractions of bovine or human dentine extract were prepared-rude, unbound and 0.6 M NaCl-eluted fractions. Electrophoresis
Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) was performed on all extracts tested using the Tri-glycine buffer system of Laemmli (1970). A 5% acrylamide upper gel was used for stacking the samples, which were then separated by a l&22% acrylamide gradient in the running gel. After electrophoresis, the gels were stained with silver (Merril, Switzer and Van Keuren, 1979). In vitro assay and quantitation
All lyophilized fractions were dissolved in l-2ml of distilled water and were analysed for protein content (Bradford, 1976). Aliquots of the dissolved extracts were added to Eagle’s minimal essential medium supplemented with 3% fetal calf serum, 5% chick embryo extract and 7% horse serum (Syftestad
al.
and Caplan, 1984) to give final protein concentrations of 0 (control) 1, 5, IO, 20, 50, 100 and 25Opg protein/ml of medium. These extracts were applied to embryonic stage 24 (Hamburger and Hamilton, 1951) chick limb/mesenchymal cell cultures, plated at a density of 0.5 x lo6 cells/well on 24-well sterile plastic culture plates (16 mm dia) previously coated with a 1% gelatin solution. These cells had been dissected from the embryo limbs, dissociated with trypsin, and passed through a 20pm filter to give a single cell suspension (Caplan, 1972). They were allowed to incubate for 2 days after plating during which time they were given control medium. Nutrient medium supplemented with dentine extract was applied to the cell cultures in 250 ~1 at daily intervals beginning 2 days after plating (day 2). The plates were incubated at 37°C in an atmosphere of 5% CO1 and 95% humidity for B-10 days and viewed daily with phasecontrast optics. At the end of the incubation period the cultures were rinsed twice with Tyrode’s balanced salt solution, fixed with 70% ethanol for 20min, rinsed in decreasing concentrations of ethanol, rinsed with water, stained with 0.1% toluidine blue for 3.5 min (pH 6.0), destained with 70% ethanol until the destaining fluid was clear and allowed to air-dry. For some cell cultures exposed to the 0.6 M NaCleluted fraction of bovine dentine, radioactive sulphate incorporation into macromolecules was quantitated as a measure of cartilage proteoglycan biosynthesis after pulsing for 3 h with 5 PCi of [35S]-S0, (carrier-free sodium salt) per well, after which the cell layers were rinsed with cold Tyrode’s solution, precipitated with cold 10% trichloroacetic acid, solubilized with NCS (Amersham Corp., Arlington Heights, Ill., U.S.A.), and subjected to scintillation counting. In duplicate cultures, the DNA content (as a measure of cell number) was estimated by the diphenylamine method of Patterson (1979). Quantitative measurements on cells exposed to the human dentine extracts were not done because only enough extract was available to observe living and stained cultures. RESULTS
Composition of extracts
A diagram of silver-stained PAGE patterns obtained for the bovine and human dentine extracts is shown in Text Fig. 1. The crude dentine extracts from both types of teeth produced a heavy smear on the gel indicating that many proteins with a variety of molecular weights were present in these fractions (not depicted in Text Fig. 1). The unbound fraction of bovine dentine showed enrichment at approx. 45 and 31 kdalton; the fraction eluted with 0.6 M NaCl produced an identical pattern except for an additional heavy band at 55 kdalton. The unbound fraction of human dentine produced a gel pattern the same as the one produced by the corresponding fraction from the bovine dentine described above. The fraction eluted by 0.6 M NaCl contained a heavy band at approx. 55 kdalton and a lighter band at 45 kdalton; no 31 kdalton band was seen in this fraction. These major bands were present on a background of a light smear indicating the presence of a complex array of various proteins.
51
Chondrogenic stimulation by dentine
92.5 -
66.2 45.0 -
I-
-
-
31.0-
‘-
-
-
-
21.514.4 A
B
C
D
E
Fig. I. Diagram of silver-stained PAGE pattern of fractions of bovine and human dentine. Column A: molecular weight markers ( x 1000 dalton); column B: unbound fraction from bovine dentine with banding at 45 and 31 kdalton; column C: 0.6 M NaCI-eluted fraction of bovine dentine with bands at 55, 45 and 31 kdalton; column D: unbound fraction from human dentine with bands at 45 and 31 kdalton; column E: 0.6 M NaCl-eluted fraction of human dentine with bands at 55 and 45 kdalton.
Mesenchymal
cell response
When observed wi1.h phase-contrast optics, all cell cultures that had a positive response to the dentine extracts had a distinct change in morphology as early as 2 days after the beginning of the exposure, i.e. the fourth day after plating. By this time the stimulated cells were plumper and rounder than those in the unstimulated controls. The volume of the extracellular matrix and, therefore, the spacing between the cells, was also increased in stimulated cultures (Plate Figs 2 and 3). By the fifth or sixth day after plating, small nests of cells were seen in control wells. These cells (early chondrocytes) formed cartilage nodules that usually appeared as discrete round isiands several cell layers thiclc (Caplan, 1970; Osdoby and Caplan, 1979). Cells that did not respond to the dentine extracts formed chondrocyte islands identical to those in control wells (Plate Fig. 4). By the seventh or eighth day after plating, we observed a perichondrium-like structure surrounding the cartilage nodules in the unstimulared plates, and this signalled the end of further nodule formation or growth. About 5-10% of the cells in the well could be identified as chondrocytes (Plate Fig. 5). Cells that showed evidence of chondrogenic stimulation did not form chondrocyte islands. Instead, there was an obvious increase in the volume of the extracellular matrix surrounding a greater proportion of the cells (Plate Fig. 6). The stimulated cells formed large aggregates of chondrocytes that covered a much greater area than that of the cartilage nodules in control or unstimulated wells. No surrounding perichondrial structure was seen in the stimulated wells. By the ninth or tenth day after plating, control cultures had shown no increase in the number or size of cartilage nodules, while maximally stimulated wells were covered by a layer of chondrocytes (Plate Figs 7-9). When the cell cultures were stained with toluidine blue, those that had shown a more pronounced response to the extracts also retained more stain; a typical staining pattern is shown in Plate Fig. 8. In these cultures, we tested the bovine dentine
extract eked from the DEAE column by the urea/CHAPS solution containing 0.6 M NaCl; stimulation occurred at protein concentrations as low as 5 lg/ml and was maximal at 20 pg/ml. Occasionally, a particular extract was toxic to the cell layer; this was usually apparent by the second or third day of exposure. By this time, the cells were pyknotic, and by the fifth or sixth day of exposure, the cell layer had usually become detached from the well and was non-viable. Toxicity usually occurred at protein concentrations greater than 20 or 50pg/ml, whereas lower doses had stimulated chondrogenesis. Human dentine extracts also stimulated chondrogenesis in the cell cultures. Plate Fig. 9 shows the staining pattern of cultures exposed to different concentrations of the unbound fraction of the extract. A maximal response was observed at protein concentrations of 50 pgg/ml. For both the bovine and human dentine extracts, all three fractions (crude, unbound and 0.6 M NaCl-eluted) stimulated chondrogenesis in a dose-dependent fashion. The crude extract fraction, however, generally produced toxic effects at lower protein concentrations than the other two fractions. Estimations of [?S]-SO, incorporation into macromolecules and DNA quantitation were variable but clear trends were observed. Because only a limited amount of extract or partially purified fractions was available, only material from day 8 or 10 of culture was assayed. In these cases, a single 3 h exposure to [3’S]-S0., , followed by trichloroacetic acid precipitation of macromolecules, was accomplished; likewise, DNA measurements were made at these time points. The experiments were repeated several times with different cell preparations using the same batch of bovine dentine extract. The data were analysed by Student’s t-test. In some experiments, there was almost a five-fold increase in labelled sulphate incorporation compared to controls (p < O.Ol), while other experiments resulted in only a two-fold increase; this occurred under conditions where stimulation of chondrogenesis had been clearly observed morphologically. For DNA measurements, we found no significant increase in [3H]-thymidine incorporation into the experimental cells compared to controls (p > 0.1) indicating no increase in cell number and thus no stimulation of mitosis; higher concentrations of protein produced toxic effects in the [3H]-thymidine experiment shown (Table 1). DISCUSSION
We have made use of the novel in vitro model developed by Syftestad and Caplan (1984) to demonstrate the chondrogenic stimulatory effects of watersoluble extracts of bovine and human dentine. The responses to these extracts were identical to those reported for similarly prepared extracts from adult chicken and beef bone (Syftestad and Caplan, 1984; Syftestad et al., 1985), which were said to contain chondrogenic stimulating activity (Syftestad et al., 1984). Usually, under control conditions at the plating densities used in these experiments, a small fraction of cells will differentiate into chondrocytes, but when the cells are exposed to extracts containing chrondrogenic stimulating activity, changes occur in the cell layer. A greater number of undifferentiated
52
T. RABINOWITZet al.
Plate 1
Chondrogenic stimulation by dentine
53
Table 1. Metabolism: DNA and proteoglycan synthesis. Effects of the 0.6 M NaCI-eluted fraction of bovine dentine extract* [‘q-so, [‘HI-thymidine (p) pg Protein/ml counts/min counts/min (PI 0 1151*43 48,913 k 5270 5 1201 If: 97 (>O.l) 54,188 & 5180 (>O.l) 10 2069 + 392 (cO.01) 49,395 * 13,300 (>O.l) 20 5314 + 54 (
similar results. cells are stimulated ultimately to become chondrocytes with a concomitant increase in the amount of cartilaginous extracellular matrix. Mitogenic effects are sometimes observed, but are always less pronounced than the chondrogenic stimulatory ones. These stimulatory effects can be demonstrated by fixing the cell cultures and staining them with toluidine blue, which reacts orthochromatically (giving a blue colour) with rnany cellular constituents, but
metachromatically (giving a purplish colour) with acidic or sulphated glycosaminoglycans, a major extracellular matrix component of cartilage. The cell cultures shown increased [35S]-S0, incorporation, indicating an increase in proteoglycan synthesis, a major component of cartilage. Variability in the amount of sulphate incorporation and DNA content in cell cultures indicates the technical limitations of attempts to relate microscopic observations to the synthetic expression of chondrocytes. Because the effects of dentine extracts on the chick limb-bud
system so closely parallel those for chondrogenic stimulating activity in bone extracts (Syftestad and Caplan, 1984) we conclude that dentine extracts contain chondrogenic stimulatory components, but we cannot state whether these are identical to those of bone. Seyedin et al. (1985) have purified and characterized two cartilage-inducing factors from demineralized bovine bone, with molecular weights of approx. 26 kdalton as shown by their PAGE patterns. These factors, called cartilage-inducing factor A and B induce rat muscle cells to synthesize cartilage-specific proteoglycan in vitro. A subsequent examination of cartilage-inducing factor A (Seyedin et al., 1986) has shown that the sequence of its N-terminal 30 amino acids is identical to that of human transforming growth factor /?, and that both factors have similar activities in vitro, leading to the conclusion that they are closely related or identical molecules. We do not believe that the chondro-stimulatory fractions we
Plate 1 Note: Figs 227 were photographed
Fig. 2. Stage-24
embryonic
using phase-contrast optics (at x 100) and printed at similar magnifications, i.e. x 335
chick limb-bud
mesenchyme cells 4 days after plating. control medium.
These cells received only
Fig. 3. Stage-24 embryonic chick limb-bud mesenchyme cells that had been exposed to a stimulatory extract of bovine dentine (20 pg/ml protein concentration) for 2 days. The cells are rounder and plumper than those in Fig. 2. Intercellular spacing (extracellular matrix) is also increased, seen here as a refractile halo (arrows) about individual cells. Fig. 4. A cartilage
nodule
Fig. 5. A perichondrium-like
in a control
structure
well composed of several layers of chondrocytes nest (arrows). (arrows)
between
individual
cartilage
nodules
forming
a circular
in a control
well.
Fig. 6. Stimulated cells in culture: a thicker region of chondrocytes is present in the middle of the figure, and two thinr.er areas of chondrocytes (arrows) surround it. In wells containing cells showing a positive response to the extracts, this was a typical pattern of chondrocyte presentation. Fig. 7. An agpregate entire field contains
of chondrocytes in a well exposed to a stimulatory extract of bovine dentine. The chondrocytes and is typical of the morphological response of cells exposed to stimulatory extracts of human or bovine dentine.
Fig. 8. Toluidine blue-stained cell cultures prepared on day 8. Bovine dentine extract (0.6 M NaCl-eluted fraction) was applied at concentrations of 0 (control; column A), 5 (column B), 10 (column C), and 20 (column D) pg protein/ml per day. A dose-dependent increase in stain incorporation is apparent. Each black dot (arrow) is a nest of chondrocytes. x 1.1 Fig. 9. Toluidine blue-stained cell cultures prepared on day 8. Human dentine extract (unbound fraction) was applied at concentrations of 0 (control; column A), 5 (column B), 10 (column C), 20 (column D) and 50 (column E) pg protein/ml per day. Each black dot (arrow) is a nest of chondrocytes. x0.94
T. RAMNOU mz et al.
54
have prepared from bovine and human dentine contain transforming growth factor /I because these fractions produced different PAGE patterns than those reported either for cartilage-inducing factor A or transforming growth factor )!I(Seyedin et al., 1985, 1986) with no banding at 26 kdalton. The similarity in the silver-stained gel patterns between corresponding fractions from the bovine and human dentine suggests that similar or identical proteins may be responsible for the chondro-stimulatory effects we observed. The in vitro limb-bud mesenchyme assay has several advantages over the in vivo assays described earlier (Yoemans and Urist, 1967): much less material is required for testing (mg of material in vivo versus pg in vitro), the cultures can be examined every day or more often if necessary; the system responds much more quickly; and dose-dependent responses are more easily quantified. A potential disadvantage lies in the fact that this model contains a fraction of cells that normally differentiate into chondrocytes; thus, it is difficult to discriminate between inductive and enhancement effects. It is of interest to speculate why chondro-stimulatory substances comprise a fraction of the noncollagenous proteins of demineralized bovine and human dentine because cartilage is not found in dentine, although Cummings et al. (198 1) have shown that dental mesenchyme can be directed to differentiate into cartilage under appropriate conditions in vitro. These substances may simply have an affinity for the dentine matrix, or they may indeed be products of odontoblasts. Thus far, we have evidence for the presence of small amounts of chondrogenic stimulatory substances in several soft tissues and body fluids (Syftestad et al., 1987), but the greatest concentrations of these (mg/g dry tissue) have been found in bone and dentine. Whether they play a role in chondro- or osteogenesis or in bone repair of the periodontal tissues requires further investigation. Work continues in our laboratory aimed at the purification and precise identification of these interesting components.
Caplan A. I. (1972) The effect of the nicotinamide sensitive teratogen 3-acetylpyridine on chick limb mesodermal cells in culture: biochemical parameters. J. exp. Zool. 180, 351-362. Conover M. A. and Urist M. R. (1979) Transmembrane bone morphogenesis by implants of dentine matrix. J. dent. Res. 58, 1911. Cummings E. G., Bringas P. Jr, Grodin M. S. and Slavkin H. C. (1981) Epithelial-directed mesenchyme differentiation in vitro. Model of murine odontoblast differentiation mediated by quail epithelia. Differentiation 20, 1-9. Hamburger V. and Hamilton H. L. (1951) A series of normal stages in the development of the chick embryo.
thank Dr D. Lennon for providing the stage-24 limb mesenchymal cell cultures and Mr J. Holecek and Mr S. P. Bruder for technical assistance. This work was supported by grants from the National Institute of Health (U.S.A.).
Syftestad G. T., Lucas P. A. and Caplan A. I. (1985) The in vitro chondrogenic response of limb-bud mesenchyme to a water-soluble fraction prepared from demineralized bone matrix. Differentiation 29, 230-237. Syftestad G. T., Lucas P. A., Ohgushi H. and Caplan A. I. (1987) Chondrogenesis as an in vitro response to bioactive factors extracted from adult bone and non-skeletal tissues. In: Development and Diseases of Cartilage and Bone Matrix (Edited by Sen A. and Thornhill T.) pp. 187-199. Alan R. Liss, New York. Urist M. R. (1965) Bone formation by auto-induction. Science 150, 893-899. Yoemans J. D. and Urist M. R. (1967) Bone induction by decalcified dentine implanted into oral osseous and muscle tissues. Archs oral Biol. 12, 999-1008.
J. Morph. 88, 49-92.
Laemmli U. R. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T,. Nature 227, 680-685.
Linde A. (1984) Non-collagenous proteins and proteoglycans in dentinogenesis. In: Dentin and Dentinogenesis (Edited by Linde A.) Vol. 2, pp. 55-92. CRC Press, Florida. Merril C. R., Switzer R. C. and Van Keuren M. L. (1979) Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain. Proc. natn. Acad. Sci. U.S.A. 79, 43354339.
Osdoby P. and Caplan A. I. (1979) Osteogenesis in cultures of limb mesenchymal cells. Deul Biol. 73, 84-102. Patterson M. K. J. (1979) Measurement of growth and viability of cells in culture. Meth. Enzym. 58, 141-151.
Seyedin S. M., Thomas T. C., Thompson A. Y., Rosen D. M. and Piez K. A. (1985) Purification and characterization of two cartilage-inducing factors from bovine demineralized bone. Proc. natn. Acad. Sci. U.S.A. 82, 2267-227
1.
Seyedin S. M., Thompson A. Y., Bentz H., Rosen D. M., McPherson J. M., Conti A., Siegel N. R., Gallupi G. R. and Piez K. A. (1986) Cartilage-inducing factor-A. Apparent identity to transforming growth factor-j. J. biol. Chem. 261, 5693-5695.
Syftestad G. T. and Caplan A. I. (1984) A fraction from extracts of demineralized adult bone stimulates the conversion of mesenchymal cells into chondrocytes. Deal Biol. 104, 348-356. Syftestad G. T., Triffit J. T., Urist M. R. and Caplan A. I. (1984) An osteo-inductive bone matrix extract stimulates the in vitro conversion of mesenchyme into chondrocytes. Calc. Tissue Int. 36, 625-627.
Acknowledgements-We
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Caplan A. I. (1970) Effects of the nicotinamide sensitive teratogen 3-acetylpyridine on chick limb bud cells in culture. Expl CeN Res. 62, 341-348.