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Dynamic changes in bone cells and extracellular matrix vesicles during healing of alveolar bones in rats : An ultrastructural and biochemical study
Metab.
Bone
Ok.
8, Rel. Res. 2, 347-356
(1981)
Matabok Bono Dboaso a Rdatod Ruemth 0 by S.N.P.M.D. (Park
s in Bene Cells and Extmettwtar HeatCng of Alveolar Bgne in Rats rat and Bteekenttcat Study.
W81)
:
A. MUHLRAD, I.A. BAB * and J. SELA * Departments Israel.
of Oral Biology
and Oral Pathology
**,
The Hebrew
University-Hadassah
for correspondence and reprints : Prof. J. Sela, Division of Oral Pathology, of Dental Medicine, P.0.B.1172, Jerusalem, Israel.
Address
Abstract The changes in ultrastructure, quantities and enxymatic activity of cells and extracellular matrix veslcles were studied during alveolar bone healing after tooth extraction in rats. Biochemical studies revealed a marked decrease hi protein content of the Isolated fractions of vesicles Immediately after the surgical procedure. Transmisston electron microscopy confirmed that an extensive tissue dIsruption occurred at that stage. These changes were accompanied by decreased activities of cellular and vesicular alkaline phosphatase and of different ATPases. The recovery of enzymatic actlvlty on the 9th day was expressed in cells and vesicles. The different stages of healing were chrmcteristic of primary calciftcation comprising active osteoblasts, matrix vesicles and calcifying nodules. It is suggested that the amounts and enzymatic activities of extracellular matrix vesicles may serve as a marker system to evaluate the different stages of primary mlneralizatlon. Key Words : Mineralization-Matrix Vesicles - Ajveolar Bone Healing - Alkaline Phosphatase - Rats.
Introduction Extracellular matrix vesicles have recently been described isolated and characterized by both morphological and biochemical methods in normal and healing alveolar bones of young rats (Sela et al., 1978, Bab et al., 1979, Sela and Bab, 1979b). It has been established that the vesicles are operative in the process of primary mineralization in cartilage (Anderson, 1969, Bonucci, 1970), developing bone (Bernard and Peaze, 1969) and osteosarcoma (Lee et al., 1975, Muhlrad et al., 1978, Sela and Bab, 1979a). In addition, matrix vesicles have been observed in the jaw bones of adult rats (Sela et al., 1981). It is widely accepted that the vesicles produced by the forming cell serve as initial loci for calcification by accumulation of calcium and phosphate from the milieu (Anderson, 1976, Ali, 1977, Dereszewski and Howell, 1978). Binding of calcium by the negatively charged
School
of Dental
Medicine,
Jerusalem,
Hebrew University-Hadassah
School
membrane linked phospholipids and liberation of orthophosphate by vesicular enzymes i.e. pyrophosphatase, alkaline phosphatase and ATPases is followed by formation of hydroxyapatite crystals (Anderson, 1973). Alkaline phosphatase is considered as a marker enzyme for these matrix vesicles (Ali et al., 1970, Majeska and Wuthier, 1975). Studies of matrix vesicles have centered on their characterization in different tissues in normal and pathological conditions (Anderson, 1978). However, no attempt has been made to follow sequential changes in the occurrence and activity of these vesicles during the different stages of bone mineralization. The purpose of the present work was to study cellular and vesicular changes in alveolar bone healing after the extraction of molar teeth in rats.
Materials and Methods 385 rats of either sex of the Hebrew University (Sabra) strain weighing 100-120 g each were used. The animals were separated into 5 groups. The groups were divided into subgroups of 15 rats each for biochemical studies ; 5 additional rats of each group were used for TEM (transmission electron microscope) studies. The maxillary first and second molar teeth were extracted in 4 groups. The animals were sacrificed and the maxillary processes including the socket tissue (Figure 1) were removed according to the following time table : Immediately, 3, 9 and 22 days after the extraction of the teeth ; the number of subgroups (each comprising 15 rats) sacrificed at each time period was 6, 4, 5 and 5, respectively. Another group comprising 4 subgroups served as control ; the maxillary processes of these rats were removed and the teeth were extracted from the dissected bone. Preparation
of fractions.
The separated maxillary processes were minced into small pieces and washed with cold Gey’s solution. Fractionation was performed according to a method described previously (Majeska and Wuthier, 1975). Digestion with crude collagenase was followed by centrifugations that yielded cellular, membranous and vesicular fractions. Samples of the fractions were processed for TEM.
A. Muhfrad
348
et al.
in matrix
: Changes
Enzymatic
vesicles
during
healing
of rat jaw
assays.
Enzymatic assays were performed on the various fractions suspended in a hypotonic solution of 5 mM Tris-acetate buffer pH 7.4 as described previously (Bab et al., 1979). Mgz+, Gas+ and K+ (EDTA) mediated ATPases activities were determined in the presence of the chloride salts of these cations, the first two in the presence of imidazole buffer, pH 7.0 and third in the presence of Tris-HCI, pH 8.0. Pi liberated during the reaction was determined according to a method described by Fiske and Subbarow (1925). The assay of pyrophosphatase activity was performed using sodium pyrophosphate as substrate in the presence of MgClz and imidazole pH 7.0. A/ka/ine phosphatase activity was determined at pH 10.5 in the presence of the chloride salts of Mg and Zn using p-nitrophenyl phosphate as substrate. Acid phosphatase activity was measured at pH 5.0 using a similar substrate as for alkaline phosphatase. Enzymatic activity was expressed in all cases as n mole Pi liberated per mg protein per minute (specific activity) and n mole Pi per g bone. Protein determination was performed according to a method described by Lowry and associates (1951). Statistical
Analysis.
Differences between different fractions and between time groups were analysed using the Mann-Whitney U-test. Differences were considered significant at pGO.05. Transmission
Fig. 1. Light micrograph of healing maxillary alveolar process in rat. Note young bone (YB) replacing the organized blood clot and outside socket. Tissue within the frame represents samples se,parated for isolation of matrix vesicles. Hematoxylin and eosin (Bar = 283 pm). x 53. Changes In pCoteln
,A
/ t--f
electron
microscopy
(TEM).
Anesthetized rats of the 4 experimental groups were killed by intracardiac perfusion of 4V0 glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. In the control group the sacrifice was followed by extraction of the maxillary first and second molar teeth. Representative samples including soft matrix and alveolar bone tissue were further treated with the same fixative, in the cold. Post fixation was performed for 1 h at 4°C in 1 o/o osmium tetroxide in the same buffer. After dehydration in a graded series of the alcohols and propylene oxide, the specimens were embedded in epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 400 electron microscope. Pellets of the isolated fractions were fixed in 2Vo glutaraldehyde and processed for TEM examination as above. Dlstrl
content
butionof protch amongrractlonr
b
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a_---
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9
3 Days
after
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Fig. 2. Changes in protein content (a) and distribution (b) in cell, membrane and vesicle fractions from alveolar bone of norMean -C SEM [standard error of mean) ; mal rats. (N.E. = non extracted) and at different time periods after tooth extraction. Groups of normal rats and animals examined after 0,3,9 and 22 days, respectively.
A. Muhlrad
et al.
: Changes in matrix vesicles during healing of rat jaw
Results
on the 9th day. This was followed by an increase to normal levels at the end of the experiment. The vesicular and membranous protein was low on the 9th day and increased on the 22nd day. The distribution of relative amounts of protein in all fractions reached normal levels on the 9th and 22nd days.
Protein content (Figure 2a) and distribution (Figure 2b) A statistically significant decrease (24 O/O) was observed in the concentration of total protein in the vesicle fraction immediately after extractions of teeth. This decrease was also expressed in the relative amounts of protein distribution among fractions (vesicle proteins decreased from 17.7% to 11.7 O/o). No such decrease could be detected in the membrane or cell fractions. Moreover, a relative increase was found in the amount of cellular protein at that stage. Concentrations of protein in the cellular and vesicular fractions, recorded 3 days after the operation, were similar to the values found immediately after the extractions. However, a significant decrease in protein concentration was observed in the membrane fraction. The relative amount of cellular protein was highest on the third day. This was accompanied by a slight, statistically significant increase in the relative amount of vesicular protein. The membrane fraction showed a decrease in the relative amount of protein. A marked decrease in protein concentration in the cellular fraction was observed A,ka,,nc
349
phosphatase
Enzymatic activities There was a significant decrease in the specific activity (Sp act) of alkaline phosphatgse (Flgure 3a) and activity on a per bone basis (Figufe 3b) following extraction of teeth in all fractions. A similar low level was observed in the sp act of alkaline phosphatase in the vesicle fraction on the third day. The sp act of the enzyme increased to a peak in the vesicle and membrane fractions on the ninth day. At the end of the experiment the sp act of the enzyme decreased to levels similar to those found immediately after the extraction in all fractions. Sp act of alkaline phosphatase in the cellular fraction showed maximal values on the 3rd and 9th days (Figure 3a). Alkaline phosphatase levels on a per bone basis were highest in the vesicle and membrane fractions prior to extraction of teeth. A decrease observed immediately after exodontia was followed by an increase in the vesicle and cell fractions on the 3rd day. The increase continued in the vesicles on the 9th and 22nd days. The cellular fraction manifested with a decrease on the 9th and 22nd days. The membrane fraction showed constant low levels (Figure 3b). The ratio of relative activity of alkaline phos,phatase distribution among the fractions in normal rats was 49.7:36.3:14.9 (49.7 O/o, 36.3 O/o and 14.0%) for the vesicles membranes and cells respectively. Immediately after exodontia, 3, 9 and 22 days after extractions the ratios were, 37.2:43.2:19.6 ; 36.8:36.0: 27.2 ; 48.5:37.9:13.6 and 51.3:34.4:14.3 respectively (Figure 3~). ,j,r,r,t,u,,on
_.&_________&
of olkalme
phospha'w
oc”““y
C=ll -----------__*________*
0 N.E.
0
3 Days
Alkaline
z
9 after
C
22 extraction
phowhataw
)+&zz--J
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cell ______----------
N.E.
0
3 Days
9 after
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---4
Fig. 3. Specific activity of alkaline phosphatase (n mole Pi liberated per mg protein per min.) (a) activity per unit weight (b) and distribution of activity among fractions (c) in healing alveo1a.r bone in rats. Values recorded in cell, membrane and vesicle fractions before tooth extraction (N.E.) and during the process (Mean * SEM ; number of groups as in Fig. 1).
350
A. Muhlrad
sp act of pyrophosphatase showed similar levels in the membrane and vesicle fractions throughout the experiment (Figure 4a). No significant differences could be observed between fractions obtained from tissues of normal animals and those examined immediately after exodontia and after 3 and 9 days. However, a marked decrease was observed on the 22nd day. Pyrophosphatase sp act in the cellular fraction remained at normal levels immediately after tooth extraction and on the 3rd day. A decrease was observed on the 9th day, followed by a further decrease on the 22nd day. tevels of pyrophosphatase activity calculated on a per bone basis decreased immediately after tooth extraction in the vesicular and membranous fractions (Figure 4b). The memThe
et al.
: Changes in matrix vesicles during healing of rat jaw
brane fraction showed similar low levels of activity throughout the experiment. Pyrophosphatase activity in the vesicle fraction increased slightly on the 3rd day and remained at this level on the 9th and 22nd days. The levels of activity in the cellular fraction immediately after the extraction and on the 3rd day were similar to the values found in normal rats. Values observed on the 9th and 22nd days showed a continuous decrease. Ca W- and Mg2+ activated ATPases showed similar patterns of activities and therefore only the pattern of Ca2* mediated ATPase activity is presented (FIgure 5). A significant decrease in the specific activity of this enzyme (Figure 5a) was observed immediately after the extractions in all fractions. In the vesi-
T
pYrOphosphatose
1
NE
9 after
3
3
Lws
22 extmction
.
Pyrophosphatasa
NE.0
3 Days
1 9 after
1 22 cxtmction
Fig. 4. Specific activity of pyrophosphatase (n mole Pi liberated per mg protein per min.) (a) and activity per unit weight (b) in healing alveolar bone in rats. Values recorded in cell, membrane and vesicle fractions before tooth extraction (N.E.) and during the process (Mean * SEM ; number of groups as in Fig. 1). Fig. 5. Specific activity of Can+ -ATPase (n mole Pi liberated per mg protein per min.) (a) and activity per unit weight (b) in healing alveolar bone in rats. Values recorded in cell, membrane and vesicle fractions before tooth extraction (N.E.) and during the process (Mean -C SEM ; number of groups as in Fig 1).
ccl**
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0 N.E.
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9
3 oays
after
22 extractIon
A. Muhlrad et al.
: Changes in matrix vesicles during healing of rat jaw
cular and cellular fractions the activity remained low on the 3rd and 9th days and increased to preextraction levels at the end of the experiment. In the membrane fractions the activity of Ca2+ mediated ATPase increased to a level maintained on the 3rd and 9th days. A further increase to normal values was observed on the 22nd day. Enzymatic activities assessed on a per bone basis showed a different pattern (Flgure 5b). The activity of Ca2+ mediated ATPase in the cellular fraction remained constant immediately after extraction and on the 3rd and 22nd days. A marked decrease was observed on the 9th day. Vesicular and membranous fractions showed a decrease on operation and a continuous slow increase towards the end of the experiment. Nevertheless, in both fractions restoration of normal levels of activity was not reached. K(EDTA)
mediated
351
The activity of K+ (EDTA) mediated ATPase decreased after tooth extraction in the vesicle and membrane fractions (Figure 6). The levels remained low on the 3rd and 9th days and increased to normal at the end of the experiment. In the cellular fraction K+ (EDTA) mediated ATPase activity was normal on operation and on the 9th and 22nd days. A marked decrease was observed however on the 3rd day. The enzymatic activity calculated on a per bone basis (Figure 6b) for K+ (EDTA) mediated ATPase showed a pattern similar to that described for the sp act (Figure 6a) of this enzyme. Acid phosphatase activity is summarized in Figure 7. The activity of this enzyme was lowest after the extraction. An increase* in activity was observed on the 3rd day and this activity remained constant throughout the experiment in all fractions.
ATPoSe
1
N.E. 0
K(EDTA)
mediated
ATPaSe
22
9
3
Fig. 6. Specific activity of K+ (EDTA) mediated ATPase (n mole Pi liberated per mg protein per min.) (a) and activity per unit weight (b) in healing alveolar bone in rats. Values recorded in cell, membrane and vesicle fraction before tooth.extraction (N.E.) and during the process (Mean f SEM ; number of groups as in Fig. 1). Fig. 7. Specific activity of acid phosphatase (n mole Pi liberated per mg protein per min.) (a) and activity per unit weight (b) in healing alveolar bone in rats. Values recorded in cell, membrane and vesicle fractions before tooth extraction (N.E.) and during the process (Mean * SEM ; number of groups as in Fig. 1).
Acid
N.E. 0
3
phosphataSS
Days
9 after
Acid
a
cxtraCtion
22
phoSphataSS
4
Q Days
2?2 after
eXttUCtiOn
A. Muhlrad et al. . Changes in matrix vesicles during healing of rat jaw
352
Alkollne
N.E. 0
phasphatase
I
3
Days
after
to add phosphatase
8
with different stages of onset of calcification. This was characterized by active osteoblasts with rich RER. The cells were embedded in a collagen-rich matrix with the fibers organized in a general longitudinal pattern (Figure 9). A thorough examination of the matrix in a large number of TEM fields revealed an abundance of extracellular matrix vesicles and calcifying nodules in all phases of healing (Figures 9, 11). The vesicles were located among the collagen fibers. Their diameters ranged between 0.05 and 0.24 Wm. Different stages of calcification with crystal formation, both within the vesicles and in the matrix were demonstrated (Figures 9, 10, 11).
ratio
212
extractlon
Fig. 8. Ratio of the activity of alkaline and acid phosphatase in healing alveolar bone in rats. Values calculated, for cell, membrane and vesicle fractions before tooth extraction (N.E.) and during the process (Mean f SEM : number of groups as in Fig. 1).
The ratio of alkaline to acid phosphatase activity showed the highest values in the vesicular and lowest values in the cellular fractions. The ratios for the vesicular fraction were lowest on the 3rd day and highest on the 9th day after teeth extractions. Ratio values were more or less constant in the membrane and cell fractions during the experiment (Figure 8).
Fig. 10. Higher magnification of matrix vesicles (MV) from Fig. 9 with a trilaminar membrane and onset of calcification. Note penetration of vesicular membrane by apatite cristal (arrow) (Bar = 0,ll urn). x 138,950.
TEM Observations TEM examination of the tissues obtained from the alveolar bone sockets of rats killed 3, 9 and 22 days
post exodontia
revealed formation
of primary
bone
Fig. 9. Part of osteoblast with RER. Note matrix vesicles (MV) in collagenous matrix (Bar = 0.25 pm). x 59,550.
Fig. 11. Collagenous matrix with calcifying nodules (arrows) and matrix vesicles (MV) (Bar = 0.24 pm). x 62,500.
A.
Muhlrad et al. : Changes in matrix vesicles during healing of rat jaw
This was accompanied by rupture of the vesicular membrane (Figure 10). Calcifying nodules 0.07 to 0.35 iLm in diameter were observed (Figure 11). The control specimens showed morphological characteristics which were essentially similar to those of the experimental rats. Tissue specimens from animals killed immediately after tooth extraction revealed disruption of cells, matrix and of calcified elements. TEM examination of pellets from the different fractions was performed in order to verify the separation of tissue ‘elements. The matrix vesicle fractions contained large amounts of trilaminar membrane invested organelles ; the diameter of these ranged between 0.05 and 0.34 pm (Figure 12). Fibrilar material was scattered among the vesicular structures. Some vesicles in both tissue and isolated fractions contained osmiophilic material. The membrane fractions contained lamellar and saccular structures of different size and shape which were most probably the result of disintegration of cells and organelles (Figure 13). The cellular fractions contained forming ceils as well as blood cells and electron dense aggregates. Large cellular organelles, i.e. broken nuclei, mitochondria and ribosomes could occasionally be located (Figure 14). No significant difference could be noted between fractions obtained from the different groups.
353
Discussion Alveolar bone formation after extraction of molar teeth in rats was used as a biological model for the investigation of primary mineralization. The different stages of healing were described from both light (Astrand and Carlsson, 1969, Huebsch et al., 1952) and electron microscopic aspects (Sela and Bab, 1979b, Sela and Jaffe, 1977). The advantages of this model over the healing of fractures in long bones are two fold. First, there is no need for splinting and second, no cartilage is involved in the formation of new bone. Isolated cells, vesicles and membranes were examined for their ultrastructure and enzymatic properties (Sela et al., 1978, Bab et al., 1979). The present study of the dynamic changes in cells and matrix vesicles during bone healing revealed a general pattern of decrease in protein content and enzymatic activities in the vesicle fraction immediately after exodontia. However, the marked decrease in cellular protein appeared on the 9th day. The biochemical findings coincide with TEM observations at these stages. It is suggested that the vesicles are the organelles most vulnerable to injury. The restoration of cellular and vesicular proteins to normal levels on the 22nd day of healing is attributed to cellular proliferation and production of vesicles. We assume
Fig. 12. Matrix vesic:les from isolated fraction on extra ction of teeth = ‘0.27 pm ; x 55,( db; ‘,“,Y, on the 3rd (b) (Bar zz 0.40 Nrn ; x 37,500). 9th (Bar = 0.5 vrn ; x 30,( NO) = and 22nd (d) (Bar 0.40 wrn ; x 37,500) d ays after exodontia.
A. Muhlrad et al. : Changes in matrix vesicles
354
Fig. 13. Membrane
fraction
(Bar
=
0.86 pm) x 17,550.
that the low values of sp act of alkaline phosphatase found on the 3rd day in the vesicle fraction when compared to the high levels recorded a week later indicate that vesicles in the early stage of repair are less active than those found on the 9th day. Study of the ratio alkaline to acid phosphatase which is considered an indicator of the functional integrity of matrix vesicles (Ali et al., 1970) renders further support to this observation. Lowest values of this ratio were recorded on the 3rd day after injury. This was followed by an increase to a peak higher than normal on the 9th day. A comparison between the activity of alkaline phosphatase in the vesicular and cellular fractions reveals that the increase in levels of activity occurs on the third day in the cellular fraction. However, in the vesicular fraction this increase was observed on the 9th day. These findings might indicate that either vesicles with alkaline phosphatase are formed in the cell up to the third day and released between the 3rd and the 9th days, or that alkaline phosphatase per se is formed in the cells and released into the matrix. The latter assumption is supported by observations of Lewinson and associates * who showed that alkaline phosphatase
* Lewinson D., Toister Z., Silberman M. : Alkaline phosphatase evolvment in the maturation of condylar cartilage in the mouse. Personal communication.
Fig. 14. Cell fraction. Note forming RER (Bar = 1.54 km) x 9,750.
during healing
of rat jaw
cell (OB) with abundant
released from the cells to the matrix is accumulated in extracellular vesicles. The specific activity of K+ (ETDA) mediated ATPase was highest in the cellular.fraction. This contrasted with the activity of other enzymes which showed highest activities in the vesicular and lowest in the cellular fractions. It is widely accepted that the myosin ATPase is the only ATPase active in the presence of EDTA (Muhlrad et al., 1964, Pollard and Weihig, 1974) and this contractile protein has been demonstrated previously in our fractions in both normal and healing bone and in osteosarcoma (Muhlrad et al., 1978, Sela et al., 1978). Since myosin is generally associated with the movement of cells and organelles, the enhanced activity of the K+ (E6TA) activated ATPase in the cellular fraction on the 9th day might indicate an increase of cellular motility at that stage. A further study should be performed to elucidate whether this activity is resident in the osteoblast, osteoclast or both. It has been claimed that the same molecule is responsible for ATPase and alkaline phosphatase activity (Majeska and Wuthier, 1975, Kahn et al., 1978). Our findings in a previous study (Bab et al., 1979) suggested that different enzymes or molecular sites are responsible for the activity of these two enzymes. The differences in patterns of activity of alkaline phosphatase and Ca*+ and Mg*+ ATPase
A. Muhlrad et al. : Changes in matrix vesicles during healing of rat jaw
in the present study previous suggestion.
lend further
support
to our
It was proposed that ATPase is indirectly responsible for the formation of hydroxyapatite crystals (Betts et al., 1975, Hsu and Anderson, 1977). The marked increase of Ca2+ and Mg2+ ATPase activity on the 22nd day is probably indicative of the progress of the process of calcification as characterized by abundance of hydroxyapatite crystals in our transmission electron micrographs. The differences between acid phosphatase activity in cartilage fractions and in our model could be related either to the lower purity of bone fractions than cartilagenous ones or to higher acid phosphatase activity in bone vesicles per se. The difficulty in obtaining isolated bone vesicles as compared to obtaining such preparations from cartilage growth plate is related to the small quantitites of osteoid in the healing bone. However, the profiles of alkaline and pyrophosphatase activities and ultrastructural observations revealed considerable amounts of matrix vesicles in the fractions. Thus cell damage during vesicle isolation is probably not a major factor. In conclusion extraction trauma is manifested by a marked decrease in protein content and enzymatic activities of all fractions. Recovery is expressed by cellular proliferation represented by an increase in protein content and enzymatic activity of the cells. This is most probably followed by production of vesicles as evidenced by the increase of enzymatic activity and protein content in the corresponding fractions. There is a distinct correlation between the restoration of vesicular enzymatic activity and the TEM observations of the occurrence of vesicles in the matrix and formation of calcifying nodules.
Acknowledgemenfs : This project was supported by grant from the P. Sapir Foundation. The authors wish to thank Mrs. M. Feinstein and Mr. E. Hattab for their devoted assistance.
355
Anderson H.C. : Matrix vesicles of cartilage and bone. In *The Biochemistry and Physiology of Bone * edited by G.H. Bourne. Academic Press (New York). Vol. IV, D. 135-157.
Anderson H.C. : Introduction matrix vesicle calcification. 1 : 83-87, 1978.
to the second conference on Metab. Bone Dis. and Rel. Res.
Astrand P. and Carlsson G.E. : Changes in the alveolar process after extractions in the white rat. Acta Odontol. Stand. 27 : 113-127, 1969. Bab I.A.. Muhlrad A. and Sela J. : Ultrastructural and biochemical properties of extracellular matrix vesicles in normal alveolar bone of rats. Cc/l Tiss. Res. 202 : 1-7, 1979. Bernard G.W. and Peaze D.C. : An electron microscope study of initial intramembranous osteogenesis. Am. J. Anat. 125 : 271-290, 1969. Betts F., Blumenthal N.C., Posner A.S., Becker G.L. and Lehninger A.L. : Atomic structure of intracellular amorphus calcium phosphate deposites, Proc. Nefl. Acad. Sci. U.S. 72 : 2068-2090. 1975. Bonucci E. : Fine structure and histochemistry of calcifying globules in epiphyseal cartilage. Zelleforsoh and Mkr. Anat. 103 : 192-217, 1970. Dereszewski G. and Howell D.S. : The role of matrix vesicles in calcification. Trends in Biochem. Sci. 31 : 115-153, 1978. Fiske C.H. and Subbarow Y. : The calorimetric determination of phosphorus. J. No/. Chem. 66 : 375-400, 1925. Hsu H.H.T. and Anderson H.C. : A simple and defined method to study calcification by isolated matrix vesicles (effect of ATP and vesicle phosphatase). Biochem. Biophys. Acfa. 500 : 162-172. 1977. Huebsch RF., Coleman RD., Frandsen A.M. and Becks H. : The healing process following molar extraction. I. Normal male rats. Oral Surg. 5 : 864676, 1951. Kahn S.E., Jafri A.M. and Arsenis C. : Purification of Alkaline phosphatase from extracellular vesicles of fracture callus cartilage. C&if Tiss. Res. 25 : 85-92, 1978. Lee W.R., Laurie J. and Townsend A.L. radiation induced osteogenic sarcoma. 1428. 1975.
Lowry O.H., Rosenbrough J.J.. Far A.L. and Randall R.J. : Protein measurement with the folin phenol reagent. J. Biof. Chem. 193 : 265-275. 1952.
References Ali
Ali
S.Y. : Matrix vesicles and apatite nodules in arthritic cartilage. fn * Perspectives in Inflammation l edited by D.A. Willoughby, J.P. Giroud and G.P. Veto. MTP Press Ltd (Lancaster U.K.). p. 211-233, t977. S.Y., Sajdera SW. and Anderson H.C. : Isolation and characterization of calcifying matrix vesicles from eplphyseal cartilage. Proc. Nat/. Acad. Sci. U.S. 67 : 1513-1520, 1970.
Anderson H.C. : Vesicles associated with calcification matrix of epiphyseal cartilage. J. Cell. B/o/. 41 1969.
: Fine structure of Cancer. 76 : 1414-
in the
Majeska R.J. and Wuthier R.E. : Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphate and ATPase activities with alkaline phosphatase. Biochem. Biophys. Acta. 391 : 51-60, 1975. Muhlrad A., Fabian myosin ATPase 186-188, 1964.
I. and Biros N.A. : On the activation of by EDTA. Biochem. Biophys. Acta. 89 :
: 69-72,
Anderson H.C. : Hard tissue growth and remtneralization. Fdn Simp. No. 11 New series, p. 224, 1973.
Ciba
Muhlrad A., Stein H.. Bab I.A. and Sela J. : Fine structure and enzymes of matrix vesicles in osteosarcoma : Posstble occurrence of contractile proteins. Meteb. Bone Dfs. and Rel. Res. 1 : 227-233, 1978.
356
A. Muhlrad
Pollard T.D. and Weihig R.R. : Actin movement. CRC Crit. Rev. Biochem.
and Myosin and cell 2 : l-68. 1974.
Sela J. and Bab LA. : The relationship between extracellular matrix vesicles and calcospherites In primary mineralization of neoplastic bone tissue (TEM and SEM studies on osteosarcoma). Virchows Arch. A, Path. Anat. and Histo/. 383 : 1-9, 1979a. Sela J. and Bab LA. : Correlative transmission and scanning electron microscopy of the initial mineralization of healing alveolar bone in rats. Acfa. Anat. 105 : 401-408, 1979b.
..-_..._
et al.
: Changes
in matrix vesicles
during healing
of rat jaw
Sela J., Bab LA. and Muhlrad A. : Ultrastructural and biochemical characterization of extracellular matrix vesicles in healing alveolar bone socket : Preliminary indications for the presence of contractile proteins. Metab. Bone D/s. and Rel. Res. 1 : 188-191, 1978. Sela J. and Jaffe A. : The role of bone remodelllng In the healing of extraction sockets in rats. Acta Anat. Q7 : 241247, 1977. Received Revised Accepted
: February
4, 1980.
: April 24, 1980. : May 27, 1980.
-~ -
Les changements ultrastructuraux et enzymatiques des cellules et des vCsicules matriciellee a,u cows de la cicatrisation de 1’0s alvt5oiaire apks extraction des dents ont Ctk itudk chez le rat. Les htudes biochimiques ant montrk une diminution marquhe du contenu protkique des fractions de v&icules isddes immbdiatement ap&e chirurgie. La microscopic Clectronique par transmission a confirm6 le fait qu’une importante destruction tissulaire est pr&+ente a ce stade. Lea changements sont accompagnks par une diminution des activitk alcatine phosphatase et de diihrentes ATPasee cellulaires et v&siculaires. Cette activith enrymatique dapparait au ge jo.ur. Les difftkents stadee de la cioatrisation sent caract4riatiques de la calcir fication primaire avec des ost4oblastes actifs, des visicules metricielles et des nodules de calcification. H est suggM que Ies quantitbs et les activitis enxymatiques des visicules matricielles puisaent servir comme marqueur permettant d%valuer les diffirents stades de la minkalisation primaire.
Bone
Ok.
8, Rel. Res. 2, 347-356
(1981)
Matabok Bono Dboaso a Rdatod Ruemth 0 by S.N.P.M.D. (Park
s in Bene Cells and Extmettwtar HeatCng of Alveolar Bgne in Rats rat and Bteekenttcat Study.
W81)
:
A. MUHLRAD, I.A. BAB * and J. SELA * Departments Israel.
of Oral Biology
and Oral Pathology
**,
The Hebrew
University-Hadassah
for correspondence and reprints : Prof. J. Sela, Division of Oral Pathology, of Dental Medicine, P.0.B.1172, Jerusalem, Israel.
Address
Abstract The changes in ultrastructure, quantities and enxymatic activity of cells and extracellular matrix veslcles were studied during alveolar bone healing after tooth extraction in rats. Biochemical studies revealed a marked decrease hi protein content of the Isolated fractions of vesicles Immediately after the surgical procedure. Transmisston electron microscopy confirmed that an extensive tissue dIsruption occurred at that stage. These changes were accompanied by decreased activities of cellular and vesicular alkaline phosphatase and of different ATPases. The recovery of enzymatic actlvlty on the 9th day was expressed in cells and vesicles. The different stages of healing were chrmcteristic of primary calciftcation comprising active osteoblasts, matrix vesicles and calcifying nodules. It is suggested that the amounts and enzymatic activities of extracellular matrix vesicles may serve as a marker system to evaluate the different stages of primary mlneralizatlon. Key Words : Mineralization-Matrix Vesicles - Ajveolar Bone Healing - Alkaline Phosphatase - Rats.
Introduction Extracellular matrix vesicles have recently been described isolated and characterized by both morphological and biochemical methods in normal and healing alveolar bones of young rats (Sela et al., 1978, Bab et al., 1979, Sela and Bab, 1979b). It has been established that the vesicles are operative in the process of primary mineralization in cartilage (Anderson, 1969, Bonucci, 1970), developing bone (Bernard and Peaze, 1969) and osteosarcoma (Lee et al., 1975, Muhlrad et al., 1978, Sela and Bab, 1979a). In addition, matrix vesicles have been observed in the jaw bones of adult rats (Sela et al., 1981). It is widely accepted that the vesicles produced by the forming cell serve as initial loci for calcification by accumulation of calcium and phosphate from the milieu (Anderson, 1976, Ali, 1977, Dereszewski and Howell, 1978). Binding of calcium by the negatively charged
School
of Dental
Medicine,
Jerusalem,
Hebrew University-Hadassah
School
membrane linked phospholipids and liberation of orthophosphate by vesicular enzymes i.e. pyrophosphatase, alkaline phosphatase and ATPases is followed by formation of hydroxyapatite crystals (Anderson, 1973). Alkaline phosphatase is considered as a marker enzyme for these matrix vesicles (Ali et al., 1970, Majeska and Wuthier, 1975). Studies of matrix vesicles have centered on their characterization in different tissues in normal and pathological conditions (Anderson, 1978). However, no attempt has been made to follow sequential changes in the occurrence and activity of these vesicles during the different stages of bone mineralization. The purpose of the present work was to study cellular and vesicular changes in alveolar bone healing after the extraction of molar teeth in rats.
Materials and Methods 385 rats of either sex of the Hebrew University (Sabra) strain weighing 100-120 g each were used. The animals were separated into 5 groups. The groups were divided into subgroups of 15 rats each for biochemical studies ; 5 additional rats of each group were used for TEM (transmission electron microscope) studies. The maxillary first and second molar teeth were extracted in 4 groups. The animals were sacrificed and the maxillary processes including the socket tissue (Figure 1) were removed according to the following time table : Immediately, 3, 9 and 22 days after the extraction of the teeth ; the number of subgroups (each comprising 15 rats) sacrificed at each time period was 6, 4, 5 and 5, respectively. Another group comprising 4 subgroups served as control ; the maxillary processes of these rats were removed and the teeth were extracted from the dissected bone. Preparation
of fractions.
The separated maxillary processes were minced into small pieces and washed with cold Gey’s solution. Fractionation was performed according to a method described previously (Majeska and Wuthier, 1975). Digestion with crude collagenase was followed by centrifugations that yielded cellular, membranous and vesicular fractions. Samples of the fractions were processed for TEM.
A. Muhfrad
348
et al.
in matrix
: Changes
Enzymatic
vesicles
during
healing
of rat jaw
assays.
Enzymatic assays were performed on the various fractions suspended in a hypotonic solution of 5 mM Tris-acetate buffer pH 7.4 as described previously (Bab et al., 1979). Mgz+, Gas+ and K+ (EDTA) mediated ATPases activities were determined in the presence of the chloride salts of these cations, the first two in the presence of imidazole buffer, pH 7.0 and third in the presence of Tris-HCI, pH 8.0. Pi liberated during the reaction was determined according to a method described by Fiske and Subbarow (1925). The assay of pyrophosphatase activity was performed using sodium pyrophosphate as substrate in the presence of MgClz and imidazole pH 7.0. A/ka/ine phosphatase activity was determined at pH 10.5 in the presence of the chloride salts of Mg and Zn using p-nitrophenyl phosphate as substrate. Acid phosphatase activity was measured at pH 5.0 using a similar substrate as for alkaline phosphatase. Enzymatic activity was expressed in all cases as n mole Pi liberated per mg protein per minute (specific activity) and n mole Pi per g bone. Protein determination was performed according to a method described by Lowry and associates (1951). Statistical
Analysis.
Differences between different fractions and between time groups were analysed using the Mann-Whitney U-test. Differences were considered significant at pGO.05. Transmission
Fig. 1. Light micrograph of healing maxillary alveolar process in rat. Note young bone (YB) replacing the organized blood clot and outside socket. Tissue within the frame represents samples se,parated for isolation of matrix vesicles. Hematoxylin and eosin (Bar = 283 pm). x 53. Changes In pCoteln
,A
/ t--f
electron
microscopy
(TEM).
Anesthetized rats of the 4 experimental groups were killed by intracardiac perfusion of 4V0 glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. In the control group the sacrifice was followed by extraction of the maxillary first and second molar teeth. Representative samples including soft matrix and alveolar bone tissue were further treated with the same fixative, in the cold. Post fixation was performed for 1 h at 4°C in 1 o/o osmium tetroxide in the same buffer. After dehydration in a graded series of the alcohols and propylene oxide, the specimens were embedded in epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 400 electron microscope. Pellets of the isolated fractions were fixed in 2Vo glutaraldehyde and processed for TEM examination as above. Dlstrl
content
butionof protch amongrractlonr
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9
3 Days
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Fig. 2. Changes in protein content (a) and distribution (b) in cell, membrane and vesicle fractions from alveolar bone of norMean -C SEM [standard error of mean) ; mal rats. (N.E. = non extracted) and at different time periods after tooth extraction. Groups of normal rats and animals examined after 0,3,9 and 22 days, respectively.
A. Muhlrad
et al.
: Changes in matrix vesicles during healing of rat jaw
Results
on the 9th day. This was followed by an increase to normal levels at the end of the experiment. The vesicular and membranous protein was low on the 9th day and increased on the 22nd day. The distribution of relative amounts of protein in all fractions reached normal levels on the 9th and 22nd days.
Protein content (Figure 2a) and distribution (Figure 2b) A statistically significant decrease (24 O/O) was observed in the concentration of total protein in the vesicle fraction immediately after extractions of teeth. This decrease was also expressed in the relative amounts of protein distribution among fractions (vesicle proteins decreased from 17.7% to 11.7 O/o). No such decrease could be detected in the membrane or cell fractions. Moreover, a relative increase was found in the amount of cellular protein at that stage. Concentrations of protein in the cellular and vesicular fractions, recorded 3 days after the operation, were similar to the values found immediately after the extractions. However, a significant decrease in protein concentration was observed in the membrane fraction. The relative amount of cellular protein was highest on the third day. This was accompanied by a slight, statistically significant increase in the relative amount of vesicular protein. The membrane fraction showed a decrease in the relative amount of protein. A marked decrease in protein concentration in the cellular fraction was observed A,ka,,nc
349
phosphatase
Enzymatic activities There was a significant decrease in the specific activity (Sp act) of alkaline phosphatgse (Flgure 3a) and activity on a per bone basis (Figufe 3b) following extraction of teeth in all fractions. A similar low level was observed in the sp act of alkaline phosphatase in the vesicle fraction on the third day. The sp act of the enzyme increased to a peak in the vesicle and membrane fractions on the ninth day. At the end of the experiment the sp act of the enzyme decreased to levels similar to those found immediately after the extraction in all fractions. Sp act of alkaline phosphatase in the cellular fraction showed maximal values on the 3rd and 9th days (Figure 3a). Alkaline phosphatase levels on a per bone basis were highest in the vesicle and membrane fractions prior to extraction of teeth. A decrease observed immediately after exodontia was followed by an increase in the vesicle and cell fractions on the 3rd day. The increase continued in the vesicles on the 9th and 22nd days. The cellular fraction manifested with a decrease on the 9th and 22nd days. The membrane fraction showed constant low levels (Figure 3b). The ratio of relative activity of alkaline phos,phatase distribution among the fractions in normal rats was 49.7:36.3:14.9 (49.7 O/o, 36.3 O/o and 14.0%) for the vesicles membranes and cells respectively. Immediately after exodontia, 3, 9 and 22 days after extractions the ratios were, 37.2:43.2:19.6 ; 36.8:36.0: 27.2 ; 48.5:37.9:13.6 and 51.3:34.4:14.3 respectively (Figure 3~). ,j,r,r,t,u,,on
_.&_________&
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---4
Fig. 3. Specific activity of alkaline phosphatase (n mole Pi liberated per mg protein per min.) (a) activity per unit weight (b) and distribution of activity among fractions (c) in healing alveo1a.r bone in rats. Values recorded in cell, membrane and vesicle fractions before tooth extraction (N.E.) and during the process (Mean * SEM ; number of groups as in Fig. 1).
350
A. Muhlrad
sp act of pyrophosphatase showed similar levels in the membrane and vesicle fractions throughout the experiment (Figure 4a). No significant differences could be observed between fractions obtained from tissues of normal animals and those examined immediately after exodontia and after 3 and 9 days. However, a marked decrease was observed on the 22nd day. Pyrophosphatase sp act in the cellular fraction remained at normal levels immediately after tooth extraction and on the 3rd day. A decrease was observed on the 9th day, followed by a further decrease on the 22nd day. tevels of pyrophosphatase activity calculated on a per bone basis decreased immediately after tooth extraction in the vesicular and membranous fractions (Figure 4b). The memThe
et al.
: Changes in matrix vesicles during healing of rat jaw
brane fraction showed similar low levels of activity throughout the experiment. Pyrophosphatase activity in the vesicle fraction increased slightly on the 3rd day and remained at this level on the 9th and 22nd days. The levels of activity in the cellular fraction immediately after the extraction and on the 3rd day were similar to the values found in normal rats. Values observed on the 9th and 22nd days showed a continuous decrease. Ca W- and Mg2+ activated ATPases showed similar patterns of activities and therefore only the pattern of Ca2* mediated ATPase activity is presented (FIgure 5). A significant decrease in the specific activity of this enzyme (Figure 5a) was observed immediately after the extractions in all fractions. In the vesi-
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1
NE
9 after
3
3
Lws
22 extmction
.
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3 Days
1 9 after
1 22 cxtmction
Fig. 4. Specific activity of pyrophosphatase (n mole Pi liberated per mg protein per min.) (a) and activity per unit weight (b) in healing alveolar bone in rats. Values recorded in cell, membrane and vesicle fractions before tooth extraction (N.E.) and during the process (Mean * SEM ; number of groups as in Fig. 1). Fig. 5. Specific activity of Can+ -ATPase (n mole Pi liberated per mg protein per min.) (a) and activity per unit weight (b) in healing alveolar bone in rats. Values recorded in cell, membrane and vesicle fractions before tooth extraction (N.E.) and during the process (Mean -C SEM ; number of groups as in Fig 1).
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A. Muhlrad et al.
: Changes in matrix vesicles during healing of rat jaw
cular and cellular fractions the activity remained low on the 3rd and 9th days and increased to preextraction levels at the end of the experiment. In the membrane fractions the activity of Ca2+ mediated ATPase increased to a level maintained on the 3rd and 9th days. A further increase to normal values was observed on the 22nd day. Enzymatic activities assessed on a per bone basis showed a different pattern (Flgure 5b). The activity of Ca2+ mediated ATPase in the cellular fraction remained constant immediately after extraction and on the 3rd and 22nd days. A marked decrease was observed on the 9th day. Vesicular and membranous fractions showed a decrease on operation and a continuous slow increase towards the end of the experiment. Nevertheless, in both fractions restoration of normal levels of activity was not reached. K(EDTA)
mediated
351
The activity of K+ (EDTA) mediated ATPase decreased after tooth extraction in the vesicle and membrane fractions (Figure 6). The levels remained low on the 3rd and 9th days and increased to normal at the end of the experiment. In the cellular fraction K+ (EDTA) mediated ATPase activity was normal on operation and on the 9th and 22nd days. A marked decrease was observed however on the 3rd day. The enzymatic activity calculated on a per bone basis (Figure 6b) for K+ (EDTA) mediated ATPase showed a pattern similar to that described for the sp act (Figure 6a) of this enzyme. Acid phosphatase activity is summarized in Figure 7. The activity of this enzyme was lowest after the extraction. An increase* in activity was observed on the 3rd day and this activity remained constant throughout the experiment in all fractions.
ATPoSe
1
N.E. 0
K(EDTA)
mediated
ATPaSe
22
9
3
Fig. 6. Specific activity of K+ (EDTA) mediated ATPase (n mole Pi liberated per mg protein per min.) (a) and activity per unit weight (b) in healing alveolar bone in rats. Values recorded in cell, membrane and vesicle fraction before tooth.extraction (N.E.) and during the process (Mean f SEM ; number of groups as in Fig. 1). Fig. 7. Specific activity of acid phosphatase (n mole Pi liberated per mg protein per min.) (a) and activity per unit weight (b) in healing alveolar bone in rats. Values recorded in cell, membrane and vesicle fractions before tooth extraction (N.E.) and during the process (Mean * SEM ; number of groups as in Fig. 1).
Acid
N.E. 0
3
phosphataSS
Days
9 after
Acid
a
cxtraCtion
22
phoSphataSS
4
Q Days
2?2 after
eXttUCtiOn
A. Muhlrad et al. . Changes in matrix vesicles during healing of rat jaw
352
Alkollne
N.E. 0
phasphatase
I
3
Days
after
to add phosphatase
8
with different stages of onset of calcification. This was characterized by active osteoblasts with rich RER. The cells were embedded in a collagen-rich matrix with the fibers organized in a general longitudinal pattern (Figure 9). A thorough examination of the matrix in a large number of TEM fields revealed an abundance of extracellular matrix vesicles and calcifying nodules in all phases of healing (Figures 9, 11). The vesicles were located among the collagen fibers. Their diameters ranged between 0.05 and 0.24 Wm. Different stages of calcification with crystal formation, both within the vesicles and in the matrix were demonstrated (Figures 9, 10, 11).
ratio
212
extractlon
Fig. 8. Ratio of the activity of alkaline and acid phosphatase in healing alveolar bone in rats. Values calculated, for cell, membrane and vesicle fractions before tooth extraction (N.E.) and during the process (Mean f SEM : number of groups as in Fig. 1).
The ratio of alkaline to acid phosphatase activity showed the highest values in the vesicular and lowest values in the cellular fractions. The ratios for the vesicular fraction were lowest on the 3rd day and highest on the 9th day after teeth extractions. Ratio values were more or less constant in the membrane and cell fractions during the experiment (Figure 8).
Fig. 10. Higher magnification of matrix vesicles (MV) from Fig. 9 with a trilaminar membrane and onset of calcification. Note penetration of vesicular membrane by apatite cristal (arrow) (Bar = 0,ll urn). x 138,950.
TEM Observations TEM examination of the tissues obtained from the alveolar bone sockets of rats killed 3, 9 and 22 days
post exodontia
revealed formation
of primary
bone
Fig. 9. Part of osteoblast with RER. Note matrix vesicles (MV) in collagenous matrix (Bar = 0.25 pm). x 59,550.
Fig. 11. Collagenous matrix with calcifying nodules (arrows) and matrix vesicles (MV) (Bar = 0.24 pm). x 62,500.
A.
Muhlrad et al. : Changes in matrix vesicles during healing of rat jaw
This was accompanied by rupture of the vesicular membrane (Figure 10). Calcifying nodules 0.07 to 0.35 iLm in diameter were observed (Figure 11). The control specimens showed morphological characteristics which were essentially similar to those of the experimental rats. Tissue specimens from animals killed immediately after tooth extraction revealed disruption of cells, matrix and of calcified elements. TEM examination of pellets from the different fractions was performed in order to verify the separation of tissue ‘elements. The matrix vesicle fractions contained large amounts of trilaminar membrane invested organelles ; the diameter of these ranged between 0.05 and 0.34 pm (Figure 12). Fibrilar material was scattered among the vesicular structures. Some vesicles in both tissue and isolated fractions contained osmiophilic material. The membrane fractions contained lamellar and saccular structures of different size and shape which were most probably the result of disintegration of cells and organelles (Figure 13). The cellular fractions contained forming ceils as well as blood cells and electron dense aggregates. Large cellular organelles, i.e. broken nuclei, mitochondria and ribosomes could occasionally be located (Figure 14). No significant difference could be noted between fractions obtained from the different groups.
353
Discussion Alveolar bone formation after extraction of molar teeth in rats was used as a biological model for the investigation of primary mineralization. The different stages of healing were described from both light (Astrand and Carlsson, 1969, Huebsch et al., 1952) and electron microscopic aspects (Sela and Bab, 1979b, Sela and Jaffe, 1977). The advantages of this model over the healing of fractures in long bones are two fold. First, there is no need for splinting and second, no cartilage is involved in the formation of new bone. Isolated cells, vesicles and membranes were examined for their ultrastructure and enzymatic properties (Sela et al., 1978, Bab et al., 1979). The present study of the dynamic changes in cells and matrix vesicles during bone healing revealed a general pattern of decrease in protein content and enzymatic activities in the vesicle fraction immediately after exodontia. However, the marked decrease in cellular protein appeared on the 9th day. The biochemical findings coincide with TEM observations at these stages. It is suggested that the vesicles are the organelles most vulnerable to injury. The restoration of cellular and vesicular proteins to normal levels on the 22nd day of healing is attributed to cellular proliferation and production of vesicles. We assume
Fig. 12. Matrix vesic:les from isolated fraction on extra ction of teeth = ‘0.27 pm ; x 55,( db; ‘,“,Y, on the 3rd (b) (Bar zz 0.40 Nrn ; x 37,500). 9th (Bar = 0.5 vrn ; x 30,( NO) = and 22nd (d) (Bar 0.40 wrn ; x 37,500) d ays after exodontia.
A. Muhlrad et al. : Changes in matrix vesicles
354
Fig. 13. Membrane
fraction
(Bar
=
0.86 pm) x 17,550.
that the low values of sp act of alkaline phosphatase found on the 3rd day in the vesicle fraction when compared to the high levels recorded a week later indicate that vesicles in the early stage of repair are less active than those found on the 9th day. Study of the ratio alkaline to acid phosphatase which is considered an indicator of the functional integrity of matrix vesicles (Ali et al., 1970) renders further support to this observation. Lowest values of this ratio were recorded on the 3rd day after injury. This was followed by an increase to a peak higher than normal on the 9th day. A comparison between the activity of alkaline phosphatase in the vesicular and cellular fractions reveals that the increase in levels of activity occurs on the third day in the cellular fraction. However, in the vesicular fraction this increase was observed on the 9th day. These findings might indicate that either vesicles with alkaline phosphatase are formed in the cell up to the third day and released between the 3rd and the 9th days, or that alkaline phosphatase per se is formed in the cells and released into the matrix. The latter assumption is supported by observations of Lewinson and associates * who showed that alkaline phosphatase
* Lewinson D., Toister Z., Silberman M. : Alkaline phosphatase evolvment in the maturation of condylar cartilage in the mouse. Personal communication.
Fig. 14. Cell fraction. Note forming RER (Bar = 1.54 km) x 9,750.
during healing
of rat jaw
cell (OB) with abundant
released from the cells to the matrix is accumulated in extracellular vesicles. The specific activity of K+ (ETDA) mediated ATPase was highest in the cellular.fraction. This contrasted with the activity of other enzymes which showed highest activities in the vesicular and lowest in the cellular fractions. It is widely accepted that the myosin ATPase is the only ATPase active in the presence of EDTA (Muhlrad et al., 1964, Pollard and Weihig, 1974) and this contractile protein has been demonstrated previously in our fractions in both normal and healing bone and in osteosarcoma (Muhlrad et al., 1978, Sela et al., 1978). Since myosin is generally associated with the movement of cells and organelles, the enhanced activity of the K+ (E6TA) activated ATPase in the cellular fraction on the 9th day might indicate an increase of cellular motility at that stage. A further study should be performed to elucidate whether this activity is resident in the osteoblast, osteoclast or both. It has been claimed that the same molecule is responsible for ATPase and alkaline phosphatase activity (Majeska and Wuthier, 1975, Kahn et al., 1978). Our findings in a previous study (Bab et al., 1979) suggested that different enzymes or molecular sites are responsible for the activity of these two enzymes. The differences in patterns of activity of alkaline phosphatase and Ca*+ and Mg*+ ATPase
A. Muhlrad et al. : Changes in matrix vesicles during healing of rat jaw
in the present study previous suggestion.
lend further
support
to our
It was proposed that ATPase is indirectly responsible for the formation of hydroxyapatite crystals (Betts et al., 1975, Hsu and Anderson, 1977). The marked increase of Ca2+ and Mg2+ ATPase activity on the 22nd day is probably indicative of the progress of the process of calcification as characterized by abundance of hydroxyapatite crystals in our transmission electron micrographs. The differences between acid phosphatase activity in cartilage fractions and in our model could be related either to the lower purity of bone fractions than cartilagenous ones or to higher acid phosphatase activity in bone vesicles per se. The difficulty in obtaining isolated bone vesicles as compared to obtaining such preparations from cartilage growth plate is related to the small quantitites of osteoid in the healing bone. However, the profiles of alkaline and pyrophosphatase activities and ultrastructural observations revealed considerable amounts of matrix vesicles in the fractions. Thus cell damage during vesicle isolation is probably not a major factor. In conclusion extraction trauma is manifested by a marked decrease in protein content and enzymatic activities of all fractions. Recovery is expressed by cellular proliferation represented by an increase in protein content and enzymatic activity of the cells. This is most probably followed by production of vesicles as evidenced by the increase of enzymatic activity and protein content in the corresponding fractions. There is a distinct correlation between the restoration of vesicular enzymatic activity and the TEM observations of the occurrence of vesicles in the matrix and formation of calcifying nodules.
Acknowledgemenfs : This project was supported by grant from the P. Sapir Foundation. The authors wish to thank Mrs. M. Feinstein and Mr. E. Hattab for their devoted assistance.
355
Anderson H.C. : Matrix vesicles of cartilage and bone. In *The Biochemistry and Physiology of Bone * edited by G.H. Bourne. Academic Press (New York). Vol. IV, D. 135-157.
Anderson H.C. : Introduction matrix vesicle calcification. 1 : 83-87, 1978.
to the second conference on Metab. Bone Dis. and Rel. Res.
Astrand P. and Carlsson G.E. : Changes in the alveolar process after extractions in the white rat. Acta Odontol. Stand. 27 : 113-127, 1969. Bab I.A.. Muhlrad A. and Sela J. : Ultrastructural and biochemical properties of extracellular matrix vesicles in normal alveolar bone of rats. Cc/l Tiss. Res. 202 : 1-7, 1979. Bernard G.W. and Peaze D.C. : An electron microscope study of initial intramembranous osteogenesis. Am. J. Anat. 125 : 271-290, 1969. Betts F., Blumenthal N.C., Posner A.S., Becker G.L. and Lehninger A.L. : Atomic structure of intracellular amorphus calcium phosphate deposites, Proc. Nefl. Acad. Sci. U.S. 72 : 2068-2090. 1975. Bonucci E. : Fine structure and histochemistry of calcifying globules in epiphyseal cartilage. Zelleforsoh and Mkr. Anat. 103 : 192-217, 1970. Dereszewski G. and Howell D.S. : The role of matrix vesicles in calcification. Trends in Biochem. Sci. 31 : 115-153, 1978. Fiske C.H. and Subbarow Y. : The calorimetric determination of phosphorus. J. No/. Chem. 66 : 375-400, 1925. Hsu H.H.T. and Anderson H.C. : A simple and defined method to study calcification by isolated matrix vesicles (effect of ATP and vesicle phosphatase). Biochem. Biophys. Acfa. 500 : 162-172. 1977. Huebsch RF., Coleman RD., Frandsen A.M. and Becks H. : The healing process following molar extraction. I. Normal male rats. Oral Surg. 5 : 864676, 1951. Kahn S.E., Jafri A.M. and Arsenis C. : Purification of Alkaline phosphatase from extracellular vesicles of fracture callus cartilage. C&if Tiss. Res. 25 : 85-92, 1978. Lee W.R., Laurie J. and Townsend A.L. radiation induced osteogenic sarcoma. 1428. 1975.
Lowry O.H., Rosenbrough J.J.. Far A.L. and Randall R.J. : Protein measurement with the folin phenol reagent. J. Biof. Chem. 193 : 265-275. 1952.
References Ali
Ali
S.Y. : Matrix vesicles and apatite nodules in arthritic cartilage. fn * Perspectives in Inflammation l edited by D.A. Willoughby, J.P. Giroud and G.P. Veto. MTP Press Ltd (Lancaster U.K.). p. 211-233, t977. S.Y., Sajdera SW. and Anderson H.C. : Isolation and characterization of calcifying matrix vesicles from eplphyseal cartilage. Proc. Nat/. Acad. Sci. U.S. 67 : 1513-1520, 1970.
Anderson H.C. : Vesicles associated with calcification matrix of epiphyseal cartilage. J. Cell. B/o/. 41 1969.
: Fine structure of Cancer. 76 : 1414-
in the
Majeska R.J. and Wuthier R.E. : Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphate and ATPase activities with alkaline phosphatase. Biochem. Biophys. Acta. 391 : 51-60, 1975. Muhlrad A., Fabian myosin ATPase 186-188, 1964.
I. and Biros N.A. : On the activation of by EDTA. Biochem. Biophys. Acta. 89 :
: 69-72,
Anderson H.C. : Hard tissue growth and remtneralization. Fdn Simp. No. 11 New series, p. 224, 1973.
Ciba
Muhlrad A., Stein H.. Bab I.A. and Sela J. : Fine structure and enzymes of matrix vesicles in osteosarcoma : Posstble occurrence of contractile proteins. Meteb. Bone Dfs. and Rel. Res. 1 : 227-233, 1978.
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Pollard T.D. and Weihig R.R. : Actin movement. CRC Crit. Rev. Biochem.
and Myosin and cell 2 : l-68. 1974.
Sela J. and Bab LA. : The relationship between extracellular matrix vesicles and calcospherites In primary mineralization of neoplastic bone tissue (TEM and SEM studies on osteosarcoma). Virchows Arch. A, Path. Anat. and Histo/. 383 : 1-9, 1979a. Sela J. and Bab LA. : Correlative transmission and scanning electron microscopy of the initial mineralization of healing alveolar bone in rats. Acfa. Anat. 105 : 401-408, 1979b.
..-_..._
et al.
: Changes
in matrix vesicles
during healing
of rat jaw
Sela J., Bab LA. and Muhlrad A. : Ultrastructural and biochemical characterization of extracellular matrix vesicles in healing alveolar bone socket : Preliminary indications for the presence of contractile proteins. Metab. Bone D/s. and Rel. Res. 1 : 188-191, 1978. Sela J. and Jaffe A. : The role of bone remodelllng In the healing of extraction sockets in rats. Acta Anat. Q7 : 241247, 1977. Received Revised Accepted
: February
4, 1980.
: April 24, 1980. : May 27, 1980.
-~ -
Les changements ultrastructuraux et enzymatiques des cellules et des vCsicules matriciellee a,u cows de la cicatrisation de 1’0s alvt5oiaire apks extraction des dents ont Ctk itudk chez le rat. Les htudes biochimiques ant montrk une diminution marquhe du contenu protkique des fractions de v&icules isddes immbdiatement ap&e chirurgie. La microscopic Clectronique par transmission a confirm6 le fait qu’une importante destruction tissulaire est pr&+ente a ce stade. Lea changements sont accompagnks par une diminution des activitk alcatine phosphatase et de diihrentes ATPasee cellulaires et v&siculaires. Cette activith enrymatique dapparait au ge jo.ur. Les difftkents stadee de la cioatrisation sent caract4riatiques de la calcir fication primaire avec des ost4oblastes actifs, des visicules metricielles et des nodules de calcification. H est suggM que Ies quantitbs et les activitis enxymatiques des visicules matricielles puisaent servir comme marqueur permettant d%valuer les diffirents stades de la minkalisation primaire.