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Ultrastructural and biochemical characterization of extracellular matrix vesicles in healing alveolar bone sockets: Preliminary indications for the presence of contractile proteins
Metab. Bone Dis. & Rel. Res. 1, 185191 (1978)
Metabolic Bone Disease & Related Research @ by S.N.P.M.D. (Paris
1978)
Ultrastructural and Biochemical Characterization of Extracellular Matrix Vesicles in Healing Alveolar Bone Sockets: Preliminary Indications for the Presence of Contractile Proteins J. SELA’,
I.A. BAB”,
AND A. MUHLBAD”
Departments of Oral Pathology” and Oral Biology**, The Hebrew University-Hadassah ded by the Alpha Omega Fraternity, P.O. Box 1172, Jerusalem, Israel. Address for correspondence School of Dental Medicine,
and reprints: Professor J. Sela, P.O.B. 1172, Jerusalem, Israel.
Abstract Extracellular matrix vesicles were identified and isolated from healing bone sockets. The diameters of these trilaminar membrane-invested organelles was 0.05 to 0.45 pm. Ca’+-, Mg’+-, and K+ (EDTA)mediated ATPases; alkaline and acid phosphatase; and pyrophosphatase activities of isolated matrix vesicles were compared with the membrane and cellular fractions. The vesicle fraction showed high alkaline phosphatase activity, which correlated with PyrophosphaMg’+-, and Ca’+- mediated ATPases. tase activity was highest in the vesicle fraction as well. The presence of K+ (EDTAI-mediated ATPase in all the fractions indicated the occurrence of myosin. These findings were supported by electrophoretic patterns which revealed the presence of another contractile protein-actin. Key Words: Matrix Alkaline phosphatase
vesicles - Contractile
Healing socket proteins.
-
Introduction It is widely accepted that primary mineralization is associated with formation of extracellular matrix These trilaminar membrane-bound orgavesicles. nelles, 0.05-0.3 pm in diameter, were defined with transmission electron microscope (TEMI in several normal and pathologic mammalian tissues. It was surmised that the vesicles bud from the cell membrane into the forming extracellular organic matrix and serve as initial loci of calcification (Anderson, 19761. Isolated matrix vesicles show high activity of alkaline phosphatase and different ATPases. These enzymes are operative in the increase of inorganic phosphate concentration both within the vesicles and the milieu. In addition, pyrophosphatase and ATPase fulfil an important role in removal of the inhibitory effect of ATP and pyrophosphate (PPi) on calcification [Ali, 1977). ATP was also shown to be potent in stabilization of amorphous calcium phosphate, thus preventing the crystallization of the hy-
Division
of Oral
School of Dental Medicine,
Pathology,
Hebrew
Foun-
University-Hadassah
droxyapatite. The high levels of ATPase activity in the vesicle fraction are most probably operative in the onset of development’ of crystalline mineral (Betts et al., 1975; Hsu and Anderson, 1977). Recently the lipid spectrum of the vesicles was characterized. The lipids play a role in the increase of calcium concentration within the vesicle by virtue of a lipid-calcium interaction. The purpose of the present work was to study extracellular matrix vesicles in instances of primary bone formation in alveolar sockets following teeth extraction.
Materials
and
Methods
Two hundred rats of either sex of the Hebrew University (Sabra) strain, weighing 100-130 g each, were used. The animals were anesthetized and the first and second maxillary molar teeth on both sides were extracted. One week later, the alveolar bone sockets were curetted. The tissues were obtained for TEM, isolation of cells, membrane and matrix vesicle fractions. A similar procedure was performed on a dog, only in this instance all the premolars were extracted and the sockets were curetted twice, after one and two weeks. Five of the rats were separated after anesthesia and killed by heart perfusion with 4% glutaraldehyde in 0.01 M cacodylate buffer, . pH 7.2; the obtained tissues were processed for TEM. Preparation
of fractions
This was carried out essentially according to Majeska and Wuthier (1975). The tissues from the sockets were mechanically macerated into small pieces and collected in cold Gey’s solution. The material was repeatedly washed to remove blood, weighed and digested in 1% hyaluronidase (4 ml/g tissue) at 37% for one hour in a shaking water bath. The supernatant was decanded and the material was further digested for 120-180 min in 0.2% collagenase at 37%. The digest was centrifuged at 27,000 x g for one hour to remove aggregates possibly contaminant to the vesicle fractions, The released cells, membrane fragments and vesicles obtained by decantation of the collagenase digest were separated by differential centrifugation. Fraction I contained cells fosteoblasts. osteoclasts, blood cells and
J. Sela et al: Matrix
186
mineralized aqgreqates) in the pellet of the first centrifugation (600-g g-for i0 min.).. The supernatant of the first centrifugation was recentrifuged at 12,000 x g for 15 min and the pellet comprised Fraction II, which contained mainly membrane fraoments. The supernatant was centrifuged-at 127.000 x g for one hour. The pellet of this spin contained Fraction III. mainly comprising extracellular matrix vesicles. The centrifugal forces are presented as maximal values computed at the bottom of the tube. Samples of the pellets obtained from the fractions were processed for TEM. This was followed by resuspension of the pellets in IO mM Tris-acetate buffer, pH 7.0, for biochemical analysis. Transmission
electron
microscopy
Representative samples, including soft matrix and bone tissue were fixed in 4% olutaraldehyde solution in 0.1 M sodium cacodylate buffe;, pH 7.2: Post fixation was performed for one hour at 4% in 1% osmium tetraoxide in 0.1 M sodium cacodylate buffer, pH 7.2. After dehydratation in a graded series of alcohols and propylene oxide, the specimens were embedded in epon, 0.06 pm thin sections were cut, stained with 3% uranyl acetate for 5 min followed by lead citrate. and examined with a Philips 300 electron microscope. The isolated fractions were fixed with 2% glutaraldehyde and processed for TEM examination, as above. Enzymatic
assays
Mg’+--. Ca’+-and K+ (EDTAI-mediated ATPase activities were determined in the presence of 2 mM MgC%. IO mM imidazole, pH 7.0, or 5 mM CaC12. IO mM imidazole, pH 7.0, or 600 mM KCI, 6 mM EDTA and 20 mM Tris-HCI, pH 8.0, respectively. ATP concentration was 1.6 mM in all experiments, samples of fractions containing 40-200 pg protein were incubated in 1 ml reaction mixture at 37% for 30 min. The reaction was stopped by addition of trichloroacetic acid in 5% final concentration. Inorganic phosphate (P,) liberated during the reaction was determined by the method of Fiske and Subbarow (19251. Pyrophosphatase activity was assayed identically, using 1.6 mM Na-pyrophosphate as substrate in the presence of 1 mm MgCl* and 20 mM imidazole. pH 7.0. Alkaline phosphatase activity was determined in a test solution containing 100 mM glycine, pH 10.5, 1 mM MgCI?, 0.1 mM ZnCl?, 6mM Na-p-nitrophenylphosphate and IO-50 pg protein from the respective fractions. The spectral change at 405 nm due to the appearance of p-nitrophenyl was followed directly in a Unicam 800 spectrophotometer.
(1977). brilliant
vesicles
in healing
sockets
The gels were stained for proteins by Coomassie blue.
Results Ultrastructural
observations
Examination of bone and matrix obtained from the sockets one week post exodontia revealed typical primary bone formation. At the cellular level, the specimens showed a predominance of osteoblasts and osteoclasts. The extra-cellular matrix was composed of bundles of collagen fibrils, organized predominately in a longitudinal pattern. The calcifying front was in continuity with the collagenous bundles since it could be clearly demonstratdd that the calcifying crystals were patterned alongside the collagen fibrils (Fig. 1). The extra-cellular matrix vesicles were enveloped by a trilaminar membrane. Their diameters ranged from 0.05 to 0.45 pm. Most vesicles were found in the collagenous matrix (Fig. 2). Some vesicles were located close to the calcifying front and occasionally embedded in it (Figs 1 and 3). TEM examination of pellets from the different fractions was performed in order to verify the separation of the tissue elements. The matrix vesicle fraction contained large amounts of trilaminar membrane-bound vesicles, the diameters of which ranged between 0.05 and 0.45 pm. The vesicles often contained osmiophilic material which (Fig. 4). The memcould not yet be characterized brane fraction contained lamellar and saccular structures of different size and shape (Fig. 51, most probably resulting from disintegration of cells and organelles. The cellular fractions contained two major types, osteoblasts and osteoclasts. Blood cells and osmiophilic aggregates could occasionally be located. Enzymatic
activity
A general pattern of higher specific activity (sp act) was noted in the material obtained from the rat sockets than that from the dog. The data are summarized in Fig. 6. Pyrophosphatase
Acid phosphatase activity was measured by incubation of fraction samples (40-200 pg protein) in 1 ml reaction mixture containing 50 mM Na-acetate, pH 5.0, and 18 nM Na-p-nitrophenyl-phosphate at 37% for 15 min. The reaction was stopped by addition of 2 ml of 0.5 M Tris base in 5% trichloroacetlc acid and the optical density of 405 nm was recorded immediately (Aas = 18.5OOl. Enzymatic activity was expressed in all cases as n mole Pi liberated per mg protein per minute.
The sp act was highest in the vesicle fraction. Similar levels of activity were found in the vesicle fractions obtained from the dog sockets at the first and second curettages. The sp act of the membrane fraction was 33-76% of that of the vesicles, while in the cell fraction the activity was very low.
Protein
Myosin activity, characterized by K+ (EDTA) activated ATPase (Muhlrad et al., 19641, was found in all fractions. In fractions obtained from the dog sockets the activities were the same order of magnitude, while in the rat sockets the sp act was highest in the cell fraction.
determination
This was carried (19511. SDS-polyacrylamide
out by the
method
of Lowry
et
al
gel electrophoresis
50 pl of 8% SDS. ImM, EDTA, 4% P-mercaptoethanol and 20 mM Tris-HCI, pH 8.0, was added to 200 p.1 of susoension of the resoective fractions containina 0.52.0 ‘mg protein and the’mixture was incubated at ‘iOO% for 5 min. Cooling was followed by addition of 250 ~1 of 8 M urea. 50-f50 pl samples were applied to 10% polyacrylamide gels, which were prepared and electrophorised according to the method of Porzio and Pearson
ATPases
The sp act of Mg’+ mediated ATPase was similar in the membrane and vesicle fractions while a relatively lower sp act was found in the cellular fractions. Ca’+-dependent
ATPase
activity
was
sim’ilar
to
J. Sela
et
al:
Matrix
vesicles
in healing
sockets
Fig. 1. Matrix vesicles (MV) embedded in the calcifying front (CL) and the collagenous matrix (CO) of the healing socket (x 51,000, scale marker = 0.2 pm).
Fig. 2. Higher magnification marker = 0.2 pm).
of
matrix
vesicles
(MV).
Note
trimilar
membrane
(2a:
x
100,000;
2b:
x
80,000;
scale
188
J. Sela et al: Matrix
vesicles
in healing
sockets
Fig. 3. Matrix vesicles (MV) located close to the calcifying front which is constructed of hydroxyapatite crystals (HA1 fx 80,000, scale marker = 0.2 urn).
Fig. 4. Matrix vesicles from isolated fraction (x 60,000, scale marker = 0.2 pm). Note trilaminar membrane (inset x 132.000, scale marker = 0.1 ~1.
Fig. 6. Membrane fraction containing lamellar and saccular structure (x 17,000 scale marker = 1.0 vml.
J. Sela et al:
Matrix
vesicles
in healing
sockets
189
Mg 2--mediated ATPase activity in all instances. Higher Ca’+-dependent ATPase activity was observed in the vesicle and membrane fractions compared to the cellular fractions in all experiments. Alkaline
DOG 30t PYRO PHOSPHATASE
phosphatase
The sp act of alkaline phosphatase was highest in the vesicle fraction, while the membrane and cellular fractions manifested medium and low activities, The membrane fraction in the dog respectively. showed 50% sp act when compared to that found in the vesicles; the rat membrane fraction showed a relatively small sp act (33%) as compared to the vesicle fraction of the same source. The sp act of the cellular fraction was found to be 8%, 2% and 2% in the first and second dog socket and in the rat socket curettages, respectively, when compared to their vesicle fractions.
* > + c ”
u ” L
w
Mg++ ATPose
10 IL 300
l!h
20 K!d
150 100 50 0 F IXIL
CP++ ATPase
0
150 100 50 0i
VI
The sp act in the fractions obtained from the two curettages in the dog sockets was of similar magnitude. In rats, the vesicle fraction showed highest and the cellular fraction lowest sp act.
2* extr.
RAT SOCKET
20
‘“01
w
”
Acid phosphatase
K+ ATPase
extr.
I”
SOCKET
ALKALINE PHOSPHATASE
IZL
1200 a00 400 1 t%L
Total protein 6! The highest protein content was found in the cellular fraction in all experiments: it was 20-30-fold higher than in the vesicle fraction, while the amount of protein in the membrane fraction was about two-fold compared to the amount of the vesicle fraction
(Fig.
61.
SDS-polyacrylamide
gel
electrophoresis
The electrophoretic pattern of proteins obtained from the different fractions are presented in Fig. 7. In different fractions the presence of 200,000 and 43,000 daltons polypeptide chains were shown to correspond with the molecular weight of myosin heavy chain and actin, respectively.
ACID PHOSPHATASE
TOTAL PROTEIN fmgl
40 20 1 so0 LO ‘0”i
VESICLE FRACTION 0
ISI
ti
_I4
MEMBRANE FRACTION
CELL FRACTION
Fig. 6. Enzymatic activities and total protein of vesicle membrane and cellular fraction from alveolar bone sockets. (‘n moles of P, liberated per mg protein per minutes.)
Discussion Ample evidence has accumulated in recent years on the role of extra-cellular matrix vesicles in primary mineralization. The studies, by and large, were performed on cartilage. There are, however, sufficient data to evince that a similar process is operative in bone, dentine and diverse tissues. In the present study we characterized and identified the vesicles in bone repair by both ultrastructural and enzymatic methods. Matrix vesicles were found in membrane bone (Bernard and Pease, 1969), cortical bone of developing embryos (Ascenzi and Bonucci, 1971; Anderson, 1973; Anderson and Reynolds, 1973) and fracture callus (Schenk et al., 1970). The study of matrix vesicles during bone healing is of prime importance for the understanding of this basic biological process. The simplicity of the model used in this, and previous studies (Sela and Jaffe, 1977) i.e., new bone formation in sockets after extraction of teeth, makes it highly recommendable for further studies on matrix vesicles in bone. Moreover, it should be emphasized that, according to the best of our knowledge, the isolation of matrix vesicles from bone and characte-
rization of their enzymatic properties, is described for the first time in the present work. It is therefore of interest to compare the characteristics of bone matrix vesicles to those obtained from other sources. At the end of one-week post injury, large amounts of matrix vesicles were found in the tissue, and at that stage their morphology was established with no difficulties. The same was true for fractionation and collection of the matrix vesicles. TEM performed on the different fractions completed the picture, proving that they contained the different elements i.e., cells membranes and vesicles. Remarkably high specific activities of alkaline phosphatase and pyrophosphatase could be detected in the matrix vesicle fraction when compared to the cellular and membrane fractions, in spite of the fact that the sp act of alkaline phosphatase was highest in the vesicle fraction in all instances, it is of importance to mention that due to the relatively small amounts of the proteins in the vesicle fractions, the majority of the total activity of alkaline phosphatase was not found in this fraction. This finding is in accordance with the results of Felix and Fleisch
190
200 K
J. Sela et al: Matrix
vesicles
in healing
sockets
200 K
43 K 43 K
16K 16 K
Fig. 7a
Fig. 7‘b
Fig. 7. SDS polyacrylamide gel electrophoresis of vesicle, membrane and cellular fractions from alveolar bone sockets. (a = dog, first extraction; b = rats; V = vesicle; M = membrane; C = cell: PL = human platelets - controls -; 16 K, 43 K, 200 K molecular weight in daltons represent hemoglobin, actin and myosin heavy chain, respectively.) (1976) but not with the studies of Majeska and Wuthier (1975) and Ali et al. (1970). It should be noted that while our work was carried out on bone, the other studies were performed on cartilage. The enzymatic activities of the matrix vesicle and of the membrane fractions were similar to each other in almost all cases, thus supporting the assumption that the matrix vesicles originate in the plasma membrane. The specific activities of Ca2+ and Mg*+ mediated ATPases and alkaline phosphatase doubled from the first to the second curettage in matrix vesicles and membrane fractions obtained from dog sockets. Thus, there is a close correlation between the specific activities of Ca2+ and Mg*+ mediated ATPase and alkaline phosphatase in dog sockets. This corroborates the suggestion of Majeska and Wuthier (1975) that the same molecule is responsible for the activities of the above mentioned enzymes. No correlation was found, however, between the activities of these enzymes and pyrophosphatase. This result does not agree with the results of Majeska and Wuthier (1975) who studied cartilage. Similarly, no correlation was observed between the K-l- (EDTA) mediated ATPase and other ATPases. It is suggested that different enzyme molecules are res-
ponsible for the activities of K+ (EDTA) mediated ATPase and the rest of the ATPases studied. At present, no other enzyme is known to have ATPase activity in the presence of EDTA except myosin. It was surmised that this molecule is responsible for the K+ (EDTA) mediated ATPase activity in the matrix vesicles as well as in the different other fractions. This assumption is also supported by the results of the electrophoresis, where the presence of a polypeptide with molecular weight similar to that of the heavy chain myosin, was observed. A polypeptide with electrophoretic mobility similar to actin was also present in all fractions. Actin has been found in cultured bone cells (King and Holtrop, 1975). No previous studies indicated, however, the presence of actin in matrix vesicles. It is possible that contractile proteins serve either in maintaining the surface tension of the matrix vesicle membrane or, even more intriguing, they might account for a possible capacity of vesicle mobility It is of interest and/or their adhesion to the matrix. that there was a notable difference, with respect to the enzymatic activities, in the repair between the dog and the rat. As no morphologically detectable differences could be noted, we can not suggest any
J. Sela
et al:
Matrix
vesicles
in healing
sockets
191
possible explanation for the biochemical findings. It is of great importance to point out in this context that the levels of enzymatic specific activities recorded in human osteosarcoma (Muhlrad et al., 1978) were much higher than those found in the sockets. There is no information available at present to determine whether the differences found between the human osteosarcoma, and rat and dog socket healing are related to either differences in
species or to the nature of the mineralizing process. It is possible that a specific alkaline phosphatase isoenzyme with outstanding high sp act is operative in osteosarcoma process.
and
characteristic
of the
neoplastic
Acknowledgements: This project was supported by grants from the Ernst David Bergmann Foundation and the Joint Research Fund of the Hebrew University-Hadassah School of Dental Medicine founded by the Alpha Omega Fraternity.
Betts, G.L. and Lehninger, A.L.: Atomic structure of intracellular amorphous calcium phosphate deposits. Proc. Nat/. Acad. Sci. USA 72: 2088-2090. 1975.
Felix, R. and Fleisch, H.: Role of matrix vesicles cation. Fed. Proc. 35: 169-171, 1976.
in calcifi-
Fiske, C.H. and Subblarow, Y.: The colorometric determination of phosphorus. J. Biol. Chem. 66: 375-400, 1925.
Hsu, H.H.T. and Anderson, H.C.: A simple and define method to study calcification by isolated matrix vesicles (effect of ATP and vesicle phosphatase). Biochim. Biophys. Acta 500: 162-172, 1977. King, G.J. and Holtrop. M.E.: Actin-like filaments in bone cells of cultured mouse calvaria as demonstrated by binding to heavy meromyosin. J. Cell B/o/. 66: 445-451. 1975. Lowry, O.H., Rosebrough. J.J., Farr, A.L. and Randall, R.J.: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951.
References Ali, S.Y.: Matrix vesicles and apatite nodules cartilage. In Perspective in Inflammation. loughby, D.A., Giroud, J.P. and Velo, G.P.). Ltd., Lancaster UK, pp. 211-223, 1977.
Bernard, G.W. and Pease, DC.: An electron microscope study of initial intramembranous osteogenesis. Am. J. Anat. 125: 271-290. 1969.
in arthritic (Eds WilMTP Press
Ali. S.Y., Sajdera, S.W. and Anderson, H.C.: Isolation characterization of calcifying matrix vesicles from Proc. Nat/. Acad. Sci.. USA physeal cartilage. 1513-1520. 1970.
and epi67:
Anderson, H.C.: Calcium-accumulating vesicles in the intercellular matrix of bone. In Hard Tissue Growth, Repair and Remineralization. CIBA Foundation Symposium II (New Series), Elsevier, Excerpta Medica. Amsterdam, pp. 213-246, 1973. Anderson, H.C.: Matrix vesicles of cartilage and bone. In The Biochemistry and Physiology of Bone. (Ed. Bourne, G.H.). Academic Press, New York, vol. IV, pp. 135-157, 1976. Anderson, H.C. and Reynolds, J.J.: Pyrophosphate lation of calcium uptake into cultured embryonic Fine structure of matrix vesicles and their calcification. Dev. Biol. 34: 211-227.1973.
stimubones. role in
Ascenzi, A. and Bonucci, E.: Etudes comparees au microscope electronique des phases initiales de la calcification de 1’0s et du cartilage. In Phosphate et Metabolisme Phosphacalcique. (Ed. Hioco, A.J.). Sandoz, Paris, pp. 65-77, 1971.
Majeska, R.J. and Wuthier, R.E.: Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase. Biochim. Biophys. Acta 391: 51-60, 1975. Muhlrad, A., Fabian, F., and Biro, N.A.: of myosin ATPase by EDTA. Biochim. 186-192, 1964.
On the activation Biophys. Acta 89:
Muhlrad, A., Stein, C.. Bab.. I.A. and Sela, J.: Fine structure and enzymes of matrix vesicles in osteosarcoma. Metab. Bone Dis. Rel. Res. 1: 227-233, 1978. Porzio, M.A. and Pearson, A.M.: Improved resolution of myofibrillar protein with SDS-polyacrylamide gel electrophoresis. Biochim. Biophys. Acta 490: 27-34. 1977. Schenk, R.K., Miller, J., Zinkernagel, R. and Willenegger, H.: Ultrastructure of normal and abnormal bone repair. Calcif. Tissue Res. 4 (suppl.): 110-111, 1970. Sela, J. and Jaffe, A.: The role of bone remodelling in the healing of extraction socket in rats. Acta Anat. 97: 241-247. 1977. Wuthier, R.E.: Lipid composition of isolated epiphyseal cartilage cells membranes and matrix vesicles. Biochim. Biophys. Acta 409: 128-143, 1975.
RESUME Des vesicules matrlcielles extracellulalres ont et6 identlfides et isokes i partir d’os alveolaire en cows de clcatrisation.
Le diambtre de ces organelfes entourues d’une triple membrane variait de 0.05 B 0,45 u. Les activit&s enxymatiques suivantes des vesicules matrlcieiles isolues ont 6th compaties u celles des fractions celullaires et membranaires : ATpases_ Ca’f, M$+ et K+ (EDTAI d@endantes, les phosphatases acldes et alcalines, les pyvs. La fraction vesrculatre a montr6 une forte activite phosphatasique/alcallne u laquelle correspondent des actlvlt6s ATPases Mg’+ et Cal+ d6pendantea. L’actlvitd pyrophosphataslque &it elle aussl plus 6levCe dans les v6sicules. La pr6sence d’ao tivite ATPase K+ [EDTAI ddpe&ute au sein de toutes les fractions slgne la pr6aence da myoslns. Cats r&&tats ont et6 confirm& par I’Ctude Blectrophor&lque qui a r6v616 la prdsence d’une autre protelne acth&pre contractlle.
Metabolic Bone Disease & Related Research @ by S.N.P.M.D. (Paris
1978)
Ultrastructural and Biochemical Characterization of Extracellular Matrix Vesicles in Healing Alveolar Bone Sockets: Preliminary Indications for the Presence of Contractile Proteins J. SELA’,
I.A. BAB”,
AND A. MUHLBAD”
Departments of Oral Pathology” and Oral Biology**, The Hebrew University-Hadassah ded by the Alpha Omega Fraternity, P.O. Box 1172, Jerusalem, Israel. Address for correspondence School of Dental Medicine,
and reprints: Professor J. Sela, P.O.B. 1172, Jerusalem, Israel.
Abstract Extracellular matrix vesicles were identified and isolated from healing bone sockets. The diameters of these trilaminar membrane-invested organelles was 0.05 to 0.45 pm. Ca’+-, Mg’+-, and K+ (EDTA)mediated ATPases; alkaline and acid phosphatase; and pyrophosphatase activities of isolated matrix vesicles were compared with the membrane and cellular fractions. The vesicle fraction showed high alkaline phosphatase activity, which correlated with PyrophosphaMg’+-, and Ca’+- mediated ATPases. tase activity was highest in the vesicle fraction as well. The presence of K+ (EDTAI-mediated ATPase in all the fractions indicated the occurrence of myosin. These findings were supported by electrophoretic patterns which revealed the presence of another contractile protein-actin. Key Words: Matrix Alkaline phosphatase
vesicles - Contractile
Healing socket proteins.
-
Introduction It is widely accepted that primary mineralization is associated with formation of extracellular matrix These trilaminar membrane-bound orgavesicles. nelles, 0.05-0.3 pm in diameter, were defined with transmission electron microscope (TEMI in several normal and pathologic mammalian tissues. It was surmised that the vesicles bud from the cell membrane into the forming extracellular organic matrix and serve as initial loci of calcification (Anderson, 19761. Isolated matrix vesicles show high activity of alkaline phosphatase and different ATPases. These enzymes are operative in the increase of inorganic phosphate concentration both within the vesicles and the milieu. In addition, pyrophosphatase and ATPase fulfil an important role in removal of the inhibitory effect of ATP and pyrophosphate (PPi) on calcification [Ali, 1977). ATP was also shown to be potent in stabilization of amorphous calcium phosphate, thus preventing the crystallization of the hy-
Division
of Oral
School of Dental Medicine,
Pathology,
Hebrew
Foun-
University-Hadassah
droxyapatite. The high levels of ATPase activity in the vesicle fraction are most probably operative in the onset of development’ of crystalline mineral (Betts et al., 1975; Hsu and Anderson, 1977). Recently the lipid spectrum of the vesicles was characterized. The lipids play a role in the increase of calcium concentration within the vesicle by virtue of a lipid-calcium interaction. The purpose of the present work was to study extracellular matrix vesicles in instances of primary bone formation in alveolar sockets following teeth extraction.
Materials
and
Methods
Two hundred rats of either sex of the Hebrew University (Sabra) strain, weighing 100-130 g each, were used. The animals were anesthetized and the first and second maxillary molar teeth on both sides were extracted. One week later, the alveolar bone sockets were curetted. The tissues were obtained for TEM, isolation of cells, membrane and matrix vesicle fractions. A similar procedure was performed on a dog, only in this instance all the premolars were extracted and the sockets were curetted twice, after one and two weeks. Five of the rats were separated after anesthesia and killed by heart perfusion with 4% glutaraldehyde in 0.01 M cacodylate buffer, . pH 7.2; the obtained tissues were processed for TEM. Preparation
of fractions
This was carried out essentially according to Majeska and Wuthier (1975). The tissues from the sockets were mechanically macerated into small pieces and collected in cold Gey’s solution. The material was repeatedly washed to remove blood, weighed and digested in 1% hyaluronidase (4 ml/g tissue) at 37% for one hour in a shaking water bath. The supernatant was decanded and the material was further digested for 120-180 min in 0.2% collagenase at 37%. The digest was centrifuged at 27,000 x g for one hour to remove aggregates possibly contaminant to the vesicle fractions, The released cells, membrane fragments and vesicles obtained by decantation of the collagenase digest were separated by differential centrifugation. Fraction I contained cells fosteoblasts. osteoclasts, blood cells and
J. Sela et al: Matrix
186
mineralized aqgreqates) in the pellet of the first centrifugation (600-g g-for i0 min.).. The supernatant of the first centrifugation was recentrifuged at 12,000 x g for 15 min and the pellet comprised Fraction II, which contained mainly membrane fraoments. The supernatant was centrifuged-at 127.000 x g for one hour. The pellet of this spin contained Fraction III. mainly comprising extracellular matrix vesicles. The centrifugal forces are presented as maximal values computed at the bottom of the tube. Samples of the pellets obtained from the fractions were processed for TEM. This was followed by resuspension of the pellets in IO mM Tris-acetate buffer, pH 7.0, for biochemical analysis. Transmission
electron
microscopy
Representative samples, including soft matrix and bone tissue were fixed in 4% olutaraldehyde solution in 0.1 M sodium cacodylate buffe;, pH 7.2: Post fixation was performed for one hour at 4% in 1% osmium tetraoxide in 0.1 M sodium cacodylate buffer, pH 7.2. After dehydratation in a graded series of alcohols and propylene oxide, the specimens were embedded in epon, 0.06 pm thin sections were cut, stained with 3% uranyl acetate for 5 min followed by lead citrate. and examined with a Philips 300 electron microscope. The isolated fractions were fixed with 2% glutaraldehyde and processed for TEM examination, as above. Enzymatic
assays
Mg’+--. Ca’+-and K+ (EDTAI-mediated ATPase activities were determined in the presence of 2 mM MgC%. IO mM imidazole, pH 7.0, or 5 mM CaC12. IO mM imidazole, pH 7.0, or 600 mM KCI, 6 mM EDTA and 20 mM Tris-HCI, pH 8.0, respectively. ATP concentration was 1.6 mM in all experiments, samples of fractions containing 40-200 pg protein were incubated in 1 ml reaction mixture at 37% for 30 min. The reaction was stopped by addition of trichloroacetic acid in 5% final concentration. Inorganic phosphate (P,) liberated during the reaction was determined by the method of Fiske and Subbarow (19251. Pyrophosphatase activity was assayed identically, using 1.6 mM Na-pyrophosphate as substrate in the presence of 1 mm MgCl* and 20 mM imidazole. pH 7.0. Alkaline phosphatase activity was determined in a test solution containing 100 mM glycine, pH 10.5, 1 mM MgCI?, 0.1 mM ZnCl?, 6mM Na-p-nitrophenylphosphate and IO-50 pg protein from the respective fractions. The spectral change at 405 nm due to the appearance of p-nitrophenyl was followed directly in a Unicam 800 spectrophotometer.
(1977). brilliant
vesicles
in healing
sockets
The gels were stained for proteins by Coomassie blue.
Results Ultrastructural
observations
Examination of bone and matrix obtained from the sockets one week post exodontia revealed typical primary bone formation. At the cellular level, the specimens showed a predominance of osteoblasts and osteoclasts. The extra-cellular matrix was composed of bundles of collagen fibrils, organized predominately in a longitudinal pattern. The calcifying front was in continuity with the collagenous bundles since it could be clearly demonstratdd that the calcifying crystals were patterned alongside the collagen fibrils (Fig. 1). The extra-cellular matrix vesicles were enveloped by a trilaminar membrane. Their diameters ranged from 0.05 to 0.45 pm. Most vesicles were found in the collagenous matrix (Fig. 2). Some vesicles were located close to the calcifying front and occasionally embedded in it (Figs 1 and 3). TEM examination of pellets from the different fractions was performed in order to verify the separation of the tissue elements. The matrix vesicle fraction contained large amounts of trilaminar membrane-bound vesicles, the diameters of which ranged between 0.05 and 0.45 pm. The vesicles often contained osmiophilic material which (Fig. 4). The memcould not yet be characterized brane fraction contained lamellar and saccular structures of different size and shape (Fig. 51, most probably resulting from disintegration of cells and organelles. The cellular fractions contained two major types, osteoblasts and osteoclasts. Blood cells and osmiophilic aggregates could occasionally be located. Enzymatic
activity
A general pattern of higher specific activity (sp act) was noted in the material obtained from the rat sockets than that from the dog. The data are summarized in Fig. 6. Pyrophosphatase
Acid phosphatase activity was measured by incubation of fraction samples (40-200 pg protein) in 1 ml reaction mixture containing 50 mM Na-acetate, pH 5.0, and 18 nM Na-p-nitrophenyl-phosphate at 37% for 15 min. The reaction was stopped by addition of 2 ml of 0.5 M Tris base in 5% trichloroacetlc acid and the optical density of 405 nm was recorded immediately (Aas = 18.5OOl. Enzymatic activity was expressed in all cases as n mole Pi liberated per mg protein per minute.
The sp act was highest in the vesicle fraction. Similar levels of activity were found in the vesicle fractions obtained from the dog sockets at the first and second curettages. The sp act of the membrane fraction was 33-76% of that of the vesicles, while in the cell fraction the activity was very low.
Protein
Myosin activity, characterized by K+ (EDTA) activated ATPase (Muhlrad et al., 19641, was found in all fractions. In fractions obtained from the dog sockets the activities were the same order of magnitude, while in the rat sockets the sp act was highest in the cell fraction.
determination
This was carried (19511. SDS-polyacrylamide
out by the
method
of Lowry
et
al
gel electrophoresis
50 pl of 8% SDS. ImM, EDTA, 4% P-mercaptoethanol and 20 mM Tris-HCI, pH 8.0, was added to 200 p.1 of susoension of the resoective fractions containina 0.52.0 ‘mg protein and the’mixture was incubated at ‘iOO% for 5 min. Cooling was followed by addition of 250 ~1 of 8 M urea. 50-f50 pl samples were applied to 10% polyacrylamide gels, which were prepared and electrophorised according to the method of Porzio and Pearson
ATPases
The sp act of Mg’+ mediated ATPase was similar in the membrane and vesicle fractions while a relatively lower sp act was found in the cellular fractions. Ca’+-dependent
ATPase
activity
was
sim’ilar
to
J. Sela
et
al:
Matrix
vesicles
in healing
sockets
Fig. 1. Matrix vesicles (MV) embedded in the calcifying front (CL) and the collagenous matrix (CO) of the healing socket (x 51,000, scale marker = 0.2 pm).
Fig. 2. Higher magnification marker = 0.2 pm).
of
matrix
vesicles
(MV).
Note
trimilar
membrane
(2a:
x
100,000;
2b:
x
80,000;
scale
188
J. Sela et al: Matrix
vesicles
in healing
sockets
Fig. 3. Matrix vesicles (MV) located close to the calcifying front which is constructed of hydroxyapatite crystals (HA1 fx 80,000, scale marker = 0.2 urn).
Fig. 4. Matrix vesicles from isolated fraction (x 60,000, scale marker = 0.2 pm). Note trilaminar membrane (inset x 132.000, scale marker = 0.1 ~1.
Fig. 6. Membrane fraction containing lamellar and saccular structure (x 17,000 scale marker = 1.0 vml.
J. Sela et al:
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189
Mg 2--mediated ATPase activity in all instances. Higher Ca’+-dependent ATPase activity was observed in the vesicle and membrane fractions compared to the cellular fractions in all experiments. Alkaline
DOG 30t PYRO PHOSPHATASE
phosphatase
The sp act of alkaline phosphatase was highest in the vesicle fraction, while the membrane and cellular fractions manifested medium and low activities, The membrane fraction in the dog respectively. showed 50% sp act when compared to that found in the vesicles; the rat membrane fraction showed a relatively small sp act (33%) as compared to the vesicle fraction of the same source. The sp act of the cellular fraction was found to be 8%, 2% and 2% in the first and second dog socket and in the rat socket curettages, respectively, when compared to their vesicle fractions.
* > + c ”
u ” L
w
Mg++ ATPose
10 IL 300
l!h
20 K!d
150 100 50 0 F IXIL
CP++ ATPase
0
150 100 50 0i
VI
The sp act in the fractions obtained from the two curettages in the dog sockets was of similar magnitude. In rats, the vesicle fraction showed highest and the cellular fraction lowest sp act.
2* extr.
RAT SOCKET
20
‘“01
w
”
Acid phosphatase
K+ ATPase
extr.
I”
SOCKET
ALKALINE PHOSPHATASE
IZL
1200 a00 400 1 t%L
Total protein 6! The highest protein content was found in the cellular fraction in all experiments: it was 20-30-fold higher than in the vesicle fraction, while the amount of protein in the membrane fraction was about two-fold compared to the amount of the vesicle fraction
(Fig.
61.
SDS-polyacrylamide
gel
electrophoresis
The electrophoretic pattern of proteins obtained from the different fractions are presented in Fig. 7. In different fractions the presence of 200,000 and 43,000 daltons polypeptide chains were shown to correspond with the molecular weight of myosin heavy chain and actin, respectively.
ACID PHOSPHATASE
TOTAL PROTEIN fmgl
40 20 1 so0 LO ‘0”i
VESICLE FRACTION 0
ISI
ti
_I4
MEMBRANE FRACTION
CELL FRACTION
Fig. 6. Enzymatic activities and total protein of vesicle membrane and cellular fraction from alveolar bone sockets. (‘n moles of P, liberated per mg protein per minutes.)
Discussion Ample evidence has accumulated in recent years on the role of extra-cellular matrix vesicles in primary mineralization. The studies, by and large, were performed on cartilage. There are, however, sufficient data to evince that a similar process is operative in bone, dentine and diverse tissues. In the present study we characterized and identified the vesicles in bone repair by both ultrastructural and enzymatic methods. Matrix vesicles were found in membrane bone (Bernard and Pease, 1969), cortical bone of developing embryos (Ascenzi and Bonucci, 1971; Anderson, 1973; Anderson and Reynolds, 1973) and fracture callus (Schenk et al., 1970). The study of matrix vesicles during bone healing is of prime importance for the understanding of this basic biological process. The simplicity of the model used in this, and previous studies (Sela and Jaffe, 1977) i.e., new bone formation in sockets after extraction of teeth, makes it highly recommendable for further studies on matrix vesicles in bone. Moreover, it should be emphasized that, according to the best of our knowledge, the isolation of matrix vesicles from bone and characte-
rization of their enzymatic properties, is described for the first time in the present work. It is therefore of interest to compare the characteristics of bone matrix vesicles to those obtained from other sources. At the end of one-week post injury, large amounts of matrix vesicles were found in the tissue, and at that stage their morphology was established with no difficulties. The same was true for fractionation and collection of the matrix vesicles. TEM performed on the different fractions completed the picture, proving that they contained the different elements i.e., cells membranes and vesicles. Remarkably high specific activities of alkaline phosphatase and pyrophosphatase could be detected in the matrix vesicle fraction when compared to the cellular and membrane fractions, in spite of the fact that the sp act of alkaline phosphatase was highest in the vesicle fraction in all instances, it is of importance to mention that due to the relatively small amounts of the proteins in the vesicle fractions, the majority of the total activity of alkaline phosphatase was not found in this fraction. This finding is in accordance with the results of Felix and Fleisch
190
200 K
J. Sela et al: Matrix
vesicles
in healing
sockets
200 K
43 K 43 K
16K 16 K
Fig. 7a
Fig. 7‘b
Fig. 7. SDS polyacrylamide gel electrophoresis of vesicle, membrane and cellular fractions from alveolar bone sockets. (a = dog, first extraction; b = rats; V = vesicle; M = membrane; C = cell: PL = human platelets - controls -; 16 K, 43 K, 200 K molecular weight in daltons represent hemoglobin, actin and myosin heavy chain, respectively.) (1976) but not with the studies of Majeska and Wuthier (1975) and Ali et al. (1970). It should be noted that while our work was carried out on bone, the other studies were performed on cartilage. The enzymatic activities of the matrix vesicle and of the membrane fractions were similar to each other in almost all cases, thus supporting the assumption that the matrix vesicles originate in the plasma membrane. The specific activities of Ca2+ and Mg*+ mediated ATPases and alkaline phosphatase doubled from the first to the second curettage in matrix vesicles and membrane fractions obtained from dog sockets. Thus, there is a close correlation between the specific activities of Ca2+ and Mg*+ mediated ATPase and alkaline phosphatase in dog sockets. This corroborates the suggestion of Majeska and Wuthier (1975) that the same molecule is responsible for the activities of the above mentioned enzymes. No correlation was found, however, between the activities of these enzymes and pyrophosphatase. This result does not agree with the results of Majeska and Wuthier (1975) who studied cartilage. Similarly, no correlation was observed between the K-l- (EDTA) mediated ATPase and other ATPases. It is suggested that different enzyme molecules are res-
ponsible for the activities of K+ (EDTA) mediated ATPase and the rest of the ATPases studied. At present, no other enzyme is known to have ATPase activity in the presence of EDTA except myosin. It was surmised that this molecule is responsible for the K+ (EDTA) mediated ATPase activity in the matrix vesicles as well as in the different other fractions. This assumption is also supported by the results of the electrophoresis, where the presence of a polypeptide with molecular weight similar to that of the heavy chain myosin, was observed. A polypeptide with electrophoretic mobility similar to actin was also present in all fractions. Actin has been found in cultured bone cells (King and Holtrop, 1975). No previous studies indicated, however, the presence of actin in matrix vesicles. It is possible that contractile proteins serve either in maintaining the surface tension of the matrix vesicle membrane or, even more intriguing, they might account for a possible capacity of vesicle mobility It is of interest and/or their adhesion to the matrix. that there was a notable difference, with respect to the enzymatic activities, in the repair between the dog and the rat. As no morphologically detectable differences could be noted, we can not suggest any
J. Sela
et al:
Matrix
vesicles
in healing
sockets
191
possible explanation for the biochemical findings. It is of great importance to point out in this context that the levels of enzymatic specific activities recorded in human osteosarcoma (Muhlrad et al., 1978) were much higher than those found in the sockets. There is no information available at present to determine whether the differences found between the human osteosarcoma, and rat and dog socket healing are related to either differences in
species or to the nature of the mineralizing process. It is possible that a specific alkaline phosphatase isoenzyme with outstanding high sp act is operative in osteosarcoma process.
and
characteristic
of the
neoplastic
Acknowledgements: This project was supported by grants from the Ernst David Bergmann Foundation and the Joint Research Fund of the Hebrew University-Hadassah School of Dental Medicine founded by the Alpha Omega Fraternity.
Betts, G.L. and Lehninger, A.L.: Atomic structure of intracellular amorphous calcium phosphate deposits. Proc. Nat/. Acad. Sci. USA 72: 2088-2090. 1975.
Felix, R. and Fleisch, H.: Role of matrix vesicles cation. Fed. Proc. 35: 169-171, 1976.
in calcifi-
Fiske, C.H. and Subblarow, Y.: The colorometric determination of phosphorus. J. Biol. Chem. 66: 375-400, 1925.
Hsu, H.H.T. and Anderson, H.C.: A simple and define method to study calcification by isolated matrix vesicles (effect of ATP and vesicle phosphatase). Biochim. Biophys. Acta 500: 162-172, 1977. King, G.J. and Holtrop. M.E.: Actin-like filaments in bone cells of cultured mouse calvaria as demonstrated by binding to heavy meromyosin. J. Cell B/o/. 66: 445-451. 1975. Lowry, O.H., Rosebrough. J.J., Farr, A.L. and Randall, R.J.: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951.
References Ali, S.Y.: Matrix vesicles and apatite nodules cartilage. In Perspective in Inflammation. loughby, D.A., Giroud, J.P. and Velo, G.P.). Ltd., Lancaster UK, pp. 211-223, 1977.
Bernard, G.W. and Pease, DC.: An electron microscope study of initial intramembranous osteogenesis. Am. J. Anat. 125: 271-290. 1969.
in arthritic (Eds WilMTP Press
Ali. S.Y., Sajdera, S.W. and Anderson, H.C.: Isolation characterization of calcifying matrix vesicles from Proc. Nat/. Acad. Sci.. USA physeal cartilage. 1513-1520. 1970.
and epi67:
Anderson, H.C.: Calcium-accumulating vesicles in the intercellular matrix of bone. In Hard Tissue Growth, Repair and Remineralization. CIBA Foundation Symposium II (New Series), Elsevier, Excerpta Medica. Amsterdam, pp. 213-246, 1973. Anderson, H.C.: Matrix vesicles of cartilage and bone. In The Biochemistry and Physiology of Bone. (Ed. Bourne, G.H.). Academic Press, New York, vol. IV, pp. 135-157, 1976. Anderson, H.C. and Reynolds, J.J.: Pyrophosphate lation of calcium uptake into cultured embryonic Fine structure of matrix vesicles and their calcification. Dev. Biol. 34: 211-227.1973.
stimubones. role in
Ascenzi, A. and Bonucci, E.: Etudes comparees au microscope electronique des phases initiales de la calcification de 1’0s et du cartilage. In Phosphate et Metabolisme Phosphacalcique. (Ed. Hioco, A.J.). Sandoz, Paris, pp. 65-77, 1971.
Majeska, R.J. and Wuthier, R.E.: Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase. Biochim. Biophys. Acta 391: 51-60, 1975. Muhlrad, A., Fabian, F., and Biro, N.A.: of myosin ATPase by EDTA. Biochim. 186-192, 1964.
On the activation Biophys. Acta 89:
Muhlrad, A., Stein, C.. Bab.. I.A. and Sela, J.: Fine structure and enzymes of matrix vesicles in osteosarcoma. Metab. Bone Dis. Rel. Res. 1: 227-233, 1978. Porzio, M.A. and Pearson, A.M.: Improved resolution of myofibrillar protein with SDS-polyacrylamide gel electrophoresis. Biochim. Biophys. Acta 490: 27-34. 1977. Schenk, R.K., Miller, J., Zinkernagel, R. and Willenegger, H.: Ultrastructure of normal and abnormal bone repair. Calcif. Tissue Res. 4 (suppl.): 110-111, 1970. Sela, J. and Jaffe, A.: The role of bone remodelling in the healing of extraction socket in rats. Acta Anat. 97: 241-247. 1977. Wuthier, R.E.: Lipid composition of isolated epiphyseal cartilage cells membranes and matrix vesicles. Biochim. Biophys. Acta 409: 128-143, 1975.
RESUME Des vesicules matrlcielles extracellulalres ont et6 identlfides et isokes i partir d’os alveolaire en cows de clcatrisation.
Le diambtre de ces organelfes entourues d’une triple membrane variait de 0.05 B 0,45 u. Les activit&s enxymatiques suivantes des vesicules matrlcieiles isolues ont 6th compaties u celles des fractions celullaires et membranaires : ATpases_ Ca’f, M$+ et K+ (EDTAI d@endantes, les phosphatases acldes et alcalines, les pyvs. La fraction vesrculatre a montr6 une forte activite phosphatasique/alcallne u laquelle correspondent des actlvlt6s ATPases Mg’+ et Cal+ d6pendantea. L’actlvitd pyrophosphataslque &it elle aussl plus 6levCe dans les v6sicules. La pr6sence d’ao tivite ATPase K+ [EDTAI ddpe&ute au sein de toutes les fractions slgne la pr6aence da myoslns. Cats r&&tats ont et6 confirm& par I’Ctude Blectrophor&lque qui a r6v616 la prdsence d’une autre protelne acth&pre contractlle.