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Calcium phosphate solid phase induction by dioleoylphosphatidate liposomes
Calcium Phosphate Solid Phase Induction by Dioleoylphosphatidate Liposomes JAMES J. VOGEL Dental Science Institute, University of Texas, Dental Branch, P.O. Box 20068, Houston, Texas 77225 Received June 25, 1985; accepted June 25, 1985
The effect of phospholipid liposomes on solid phase induction from metastable calcium phosphate solutions was evaluated. Dioleoylphosphatidate liposomes catalyzed formation of apatite in the presence of carbonate and octacalcium phosphate when carbonate was absent. Dioleoylphosphatidylglycerol liposomes did not induce solid phase formation. The phosphatidate molecules had to be packed into an extended lamellar phase in order to function as a catalyst. The study indicates that the dioleoylphosphatidate bilayer presents an electrostatic grid in which Ca 2+ binding and subsequent Ca. PO4 ion pairing provide nidi for crystal formation and growth. Acidic phospholipid microdomains in situ could provide similar catalyticsites. © 1986AcademicPress,Inc.
Certain acidic phospholipids have been found to be closely associated with the mineralization processes (calcification) of bones and teeth. Although a precise function remains to be defined, there is considerable evidence for a pivotal role of these lipids during initiation of a solid mineral phase (see recent review, (1)). Briefly, the acidic phospholipids, phosphatidylserine, and the phosphoinositides, are extracted totally only after demineralization of calcified matrices (2, 3) indicating that they are tightly bound to the mineral phase. While such data could mean that the phospholipids are merely adsorbed onto the preformed mineral phase, in vivo labeling studies with 32p-orthophosphate by Eisenberg et al. (4) demonstrated that incorporation of 32p label into both phosphatidylserine and phosphatidylinositol coincided with mineral uptake. Boskey and Posner (5) found that an initial interaction between phospholipids and mineral is reflected by the presence of defined calcium-phospholipid-orthophosphate (CaPL-Pi) complexes which could be extracted into organic solvents. The complexes are pres-
ent in a variety of calcifying matrices but absent from nonmineralizing tissues (6). Both extracted and synthetic Ca-PL-Pi complexes induced apatite formation during incubation in a metastable calcium phosphate solution (7). Parallel with the above studies, we found that virtually all calcified tissues contain proteolipids, nonpolar protein-phospholipid complexes which induce apatite formation in vitro. A calcifiable proteolipid was first isolated and characterized from the oral microorganism, Bacterionema matruchotii (8, 9). All normal and pathologic calcified tissues examined thus far contain a similar proteolipid (10-14). The integrity of the proteolipid complex is essential for calcification (15) and unlike CaPL-Pi complexes, total extraction of proteolipid requires complete decalcification (16). While a complete decalcification procedure results in the dissociation of Ca-PL-Pi complexes, Boyan and Boskey (17) recently established a correlation between the two; Ca-PLPi complexes form during the initial interactions of Ca 2÷ and HPO 2- ions with acidic 152
0021-9797/86 $3.00 Copyright© 1986by AcademicPress,Inc. All rightsof reproductionin any formreserved.
JournalofColloidand InterfaceScience, VoL 111,No. 1, May 1986
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Chlorpromazine HC1 was dissolved in 0.05 N succinate buffer, pH 6.0, at a concentration of 1 mg/ml and a dioleoylphosphatidate liposome suspension was also prepared in the same buffer. A chlorpromazine-liposome complex was prepared by mixing the two solutions at a 1:1 ratio and allowing to stand at 25°C for 1 h. In practice the complexes were prepared in the same centrifuge tubes used for subsequent calcification assays. Calcification assays. Two metastable calcium phosphate solutions (MCPS) were used: (A)--70.0 mMNaC1, 22.0 mMNaHCO3, 5.0 m M KC1, 1.94 m M Na2HPO4, and 2.0 m M CaC12 (from a standardized stock solution). The solution pH was adjusted to 6.0 with CO2 gassing prior to addition of Ca2÷ and final volume adjustment. MCPS (B)--71.0 mM NaC1, 5.0 m M KC1, 1.64 m M Na2HPO4, 2.36 m M CaC12 (from stock solution), and 20.0 mM EXPERIMENTAL PROCEDURES TES buffer, pH 7.2. MCPS (B) was prepared Materials. Sodium L-o~-dioleoylphosphati- and stored as two components with Ca> and date, ammonium L-~-dioleoylphosphatidyl- HPO 2- separate, then rapidly mixed in equal glycerol, L-polylysine.HBr (25 kDa), and volumes prior to use. All solutions were Milchlorpromazine HC1 were purchased from lipore-filtered (0.45 um) and stored at 4°C with Sigma Chemical Company. N-tris-(hydroxy- 200 mg ofthymol crystals/liter added to avoid methyl)methyl-2-aminoethanesulfonic acid microbial contamination. All assays were done (TES) and dimethyl-l-naphthalenesulfonyl- in duplicate in 50-ml screw-top polycarbonate phosphatidylethanolamine (DANSYL-PE) centrifuge tubes (Oak Ridge type). The soluwere purchased from Calbiochem-Behring. tions were prewarmed to 37°C and for MCPS All other chemicals were reagent grade ob- (A) pH was adjusted to 7.15 ___0.01 with N2 gassing. This bicarbonate buffer required sealtained from local suppliers. Liposome preparation. The salts of the two ing the tubes with paraffin lined caps to prephospholipids were each suspended in 0.12 N vent CO2 loss. After each tube was filled, the NaC1, 0.02 N TES buffer solution, pH 7.2, at liposome preparations were immediately ina concentration of 1 mg/ml and dispersed into jected into the solution with a 0.5-ml autoan opalescent suspension by sonication at matic pipet. Incubations were at 37°C without 55,000 cps for 10 min at 25°C in a Branson shaking. Upon termination the tubes were 120-W bath-type sonicator. L-Polylysine was centrifuged at 10,000g for 10 min at 25°C, dissolved in the NaCI-TES buffer solution at two 1-ml aliquots of the supernatant were a concentration of I mg/ml and complexes taken for Ca and Pi analyses and the remainder were prepared by mixing 1.5 ml of the phos- was aspirated off. The precipitates were washed phatidate liposome suspension with 0.5 ml of and recentrifuged 2>
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Calcium and inorganicphosphate analyses. The 1-ml aliquot for calcium determination was diluted to a 0- to 4-ug/ml range in 0.1 N HC1 containing 0.25% LaC13 to suppress phosphate interference (21). Calcium concentrations were determined with a U n i c a m SP1900 A.A. spectrophotometer. Inorganic phosphate (Pi) concentrations were determ i n e d using the spectrophotometric procedure described by H e i n o n e n and Lahti (22). Uptake was calculated from # M initial m i n u s # M at termination. X-Ray diffractionanalyses. T h e dried samples were finely powdered in an agate m o r t a r and packed into 0.5-ram lead-free glass capillaries. The capillary was m o u n t e d in a 114m m Debye-Scherer powder c a m e r a and run on a Philips X-ray generator with exposure to Ni-filtered C u radiation at 30 kV, 20 m A for 4 h. Identification was m a d e by c o m p a r i n g films with those o f k n o w n standards and calculation o f d spaces. Electron microscopy. The glutaraldehyde solution was r e m o v e d by centrifugation and the samples were then fixed in 1% OsO4 in cacodylate buffer for 1-2 h. The samples were dehydrated through a graded series o f ethanol and e m b e d d e d in a low viscosity epoxy resin
(23). Sections were cut at 60-nm thickness with a glass knife, m o u n t e d on copper grids, and examined in a Hitachi 11-E electron microscope without post-staining. Fluorescent probe analysis. Dansylphosphatidylethanolamine was incorporated into the dioleoylphosphatidate liposomes at a m o lar ratio o f 1:50 to function as an internal fluorescent probe (24). Small samples of the liposomes (0.2 rag) were suspended in 10 ml o f 0.12 NNaC1, 0.02 M T E S buffer, p H 7.2, alone or with either 2 m M Ca 2+ or 2 m M Ca z+ and 2 m M HPOZ4-. The suspensions were incubated at 37°C for 30 m i n then scanned from 400 to 600 n m in a Varian 330 spectrofluorometer with excitation at 340 nm. Both excitation and emission slitwidths were set at 10 nm. The spectral scans were recorded on a H e w l e t t - P a c k a r d X - Y recorder and plotted as uncorrected relative fluorescence intensity vs wavelength. RESULTS Ion uptake data and X-ray diffraction analyses from incubation o f liposomes in MCPSA are summarized in Table I. Dioleoylphosphatidate ( D O P A ) liposomes at a concentra-
TABLE I Results from Incubation of Phospholipid Liposomes in Bicarbonate-Buffered Metastable Calcium Phosphate Solution (MCPS-A)~ Total uptake (umole) Liposome
Incubation period (h)
Ca2+
PO]-
Molar Ca/Pi
DOPAc (17 uM) DOPAc (17 #M) DOPAc (17 ~M) DOPAc (17 uM) DOPA~(17 uM) DOPGa ( 16 izM)
2 4 8 18 24 24
27.8 40.0 49.8 51.0 52.0 4.5
20.6 27.6 29.6 30.4 31.0 0
1.35 1.45 1.68 1.68 1.68 --
XRDb
Weak apatite Apatite Apatite Apatite Apatite --
Incubations were in duplicate at 37°C. Uptake values are the average of duplicate samples, which were within 5% of each other. b XRD = X-ray diffractionanalysis. Apatite was confirmed by comparison with film of powdered bone and calculation of"d" spacings. Most prominent lines were 344, 281,271, and 172 pro. c DOPA = dioleoylphosphatidate. a DOPG = dioleoylphosphatidylglycerol. Journal of Colloid and InterfaceScience, Vol. 111, No. 1, May 1986
DIOLEOYLPHOSPHATIDATE LIPOSOME CALCIFICATION tion o f 17 u M initiated calcification as evidenced by a rapid uptake o f both Ca 2+ and HPO24- from solution and an X-ray diffraction-detectable apatite pattern within 2 h. After about 2 h o f incubation the samples became a colloidal suspension which then precipitated between 3 and 4 h. Molar ratios o f the mineral phases did not attain the theoretical value o f 1.67 for apatite until after 8 h o f incubation. The dioleoylphosphatidylglycerol ( D O P G ) liposomes did not calcify even after 24 h o f incubation and ion uptake reflected only Ca 2+ binding. Both M C P S solutions were stable when incubated alone at 37°C for at least 14 days. The p o l y l y s i n e - D O P A liposome complex was used to c o m p a r e solid phase induction from solutions with and without added carbonate. The results from a 48-h incubation period are given in Table II. Here, ion uptake was less than with D O P A liposomes alone because o f the modulating effect o f polylysine. The solid phase formed in the presence o f carbonate was apatite, while octacalcium phosphate was the only phase formed in absence o f carbonate, as shown by both the molar Ca/ P ratio and X-ray diffraction. Electron microTABLE II Comparison Between Incubation of Polylysine-Dioleoylphosphatidate Liposome Complex (1:3) in Metastable Calcium Phosphate Solutions (MCPS) with and without Added Carbonate"
155
I
b 11111
I
I
IIJI
FIG, 1. Electron micrographs of thin sections from dioleoylphosphatidate liposomes after incubation in MCPS(A) for 4 h. (a) Low magnification (40,000X), of unstained section showing apatite as small electron dense needles encrusting outer surfaces of liposomes. (b) Higher magnification (120,000X) where crystals can be seen aligned essentially parallel to bilayer surfaces (arrows). Virtually no crystalsare seen within the liposomes (I). Average dimensions of the crystals are 2-3 nm thick and 2050 nm long. Bar length equals 100 nm.
Total uplake (/~mole)
MCPS (A) + CO3zMCPS (B) - CO32-
Ca2~
PO~-
Molar Ca/Pi
40.7 34.3
25.5 24.8
1.59 1.38
XREP
Apatite OCW
a Incubations were in duplicate at 37°C for 48 h. Uptake values are the average of duplicate samples, which were within 5% of each other. b XRD = X-ray diffraction analysis. Identification as described in Table I legend. OCP = octacalcium phosphate. A comparison was made with a known standard kindly provided by Dr. W. E. Brown, National Bureau of Standards.
graphs showing ultrastructural features o f the calcified liposomes are given in Figs. 1 and 2. The apatite crystals appear as electron-dense needles radiating from the outer surface o f the liposomes (Fig. 1a). At a higher magnification some crystals can be seen aligned parallel to the bilayer surfaces (Fig. lb). The crystals averaged 2 - 3 n m in diameter and 2 0 - 5 0 n m in length. Virtually no calcification could be seen inside the liposomes. The octacalcium phosphate crystals appeared as m u c h larger plates extending well b e y o n d the bilayer surfaces Journal of Colloid and Interface Science, Vol. 111, No. 1, May 1986
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binding occurred at a molar Ca/DOPA ratio of 1.8. Fluorescent spectral scans of dansylphosphatidylethanolamine (DANSYL-PE) incorporated into DOPA liposomes are presented in Fig. 3. Incubation in buffer containing 2 mM Ca~÷ for 30 min resulted in marked enhancement of the probe fluorescence and in addition the wavelength maximum was blueshifted almost 40 nm. With both 2 m M Ca 2+ and 2 mM HPOZ4- in the buffer the increase in fluorescence intensity was about 50% lower. The scans remained essentially the same during l-h incubation, but afterward began to decrease because of aggregation of the liposomes that resulted as the mineral phase formed. DISCUSSION
FIG. 2. Electron micrographs of thin sections from dioleoylphosphatidate liposomes after incubation in MCPS(B) for 20 h. (a) Low magnification (40,000×) of unstained section showing octacalcium phosphate as platelike crystals surrounding the liposomes. (b) Higher magnification (120,000×) showing that crystals adjacent to bilayers (arrow) are similar in size to apatite. Bar length equals I00 nm.
(Fig. 2a). The platy features are more apparent at a higher magnification (Fig. 2b). The results from stabilization of DOPA liposomes as either a lamellar or hexagonal II phase and the effect on subsequent calcification are summarized in Table III. The complexes were incubated in MCPS (A) for at least 24 h in order to evaluate whether dissociation and secondary phase conversion could occur during incubation. Liposomes stabilized in the lamellar phase with polylysine (25) induced apatite formation whereas liposomes stabilized in the hexagonal II phase with chlorpromazine (26) did not calcify and even though Caz+ Journal of Colloid and Interface Science,
Vol. 11l, No. 1, May 1986
Both ion uptake data and X-ray diffraction analyses showed the dioleoylphosphatidate (DOPA) liposomes to be very efficient catalysts for crystalline phase induction from metastable calcium phosphate solutions. Detection of apatite within 2 h of incubation was much earlier than the 24-168 h required previously for either individual phospholipids (7), isolated proteolipid, or synthetic protein-acidic phospholipid complexes (20). These variations in the rate of nucleation are due in part to differences in solution composition and phospholipid concentration used in the cited references. The finding that dioleoylphosphatidylglycerol liposomes did not induce a solid phase even after 24 h of incubation indicates that either the monoesterphosphate group is required or that the glycerol moiety provides sufficient steric hindrance. The results also show that solid phase induction is not due merely to a physical effect of injecting the liposomes on solution metastability but to a more specific interaction. The solid phase which formed was either apatite or octacalcium phosphate (OCP) depending upon the presence of carbonate in the metastable solution. OCP is very unstable in the presence of CO~ 2 ions and rapidly hydro-
157
DIOLEOYLPHOSPHATIDATE LIPOSOME CALCIFICATION TABLE III Effect o f Phospholipid Phase on Apatite Induction by Dioleoylphosphatidatea
Polylysine-DOPA (1:3) Chlorpromazine-DOPA (1:1)
Phase
Ca2+uptake (/*mole)
XRDb
Lamellar Hexagonal 11
27,8 2,5
Apatite NEG
a Phases were induced by preparing complexesas described under ExperimentalProcedures. Incubationwas in MCPS(A) at 37°Cfor 24 h. b XRD = X-raydiffractionanalysis.
lyzes to apatite (27). Brown et aL (28) have proposed that because of a lower interfacial energy requirement, OCP is the first phase to form which then hydrolyzes to apatite. It is interesting that in the present study OCP was stable even after 48 h of incubation when carbonate was absent. More detailed analyses during the 0- to 2-h incubation period might provide valuable information concerning the OCP-apatite interrelationships occurring during nucleation. While the morphologic
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FIG. 3. Fluorescence spectral scans of dansylphosphatidylethanolamine probe incorporated into dioleoylphosphatidate liposomes (1:50). Scan A with addition of 2 mA/ Ca 2+, scan B with addition of 2 m M Ca 2+ and 2 m M HPO~-, and scan C (control) without either Ca 2+ or HPO4~-. Note spectral blue shift with added Ca 2+ (arrow). Excitation wavelength was 340 nm.
features of the crystals as seen by electron microscopy are distinctly different overall, the initial crystals observed along the liposome bilayers are remarkably similar for both phases. The results obtained in this study indicate that the bilayer surface provides the catalytic site for solid phase induction. This conclusion is reinforced by the observation that conversion of the bilayer into the hexagonal II phase with chlorpromazine effectively blocks nucleation. In the hexagonal II phase the phospholipid molecules are packed into long cylinders with the polar heads directed inward. Although there was sufficient aqueous space within the cylinders for Ca2+ binding to occur, it was not large enough to allow formation of a solid calcium phosphate phase. On the other hand, stabilization of the bilayer with polylysine allowed calcification to occur but the rate was altered by the blocking of some of the acidic phospholipid groups via binding with the basic polypeptide. The DOPA liposome bilayer surface can be visualized as an extended two-dimensional electrostatic grid. According to Hauser et aL (29) Ca2÷ binding occurs by 2point electrostatic attachment between adjacent phospholipid molecules forming a Stern layer. Such binding would allow further ion pairing between the Ca2+ ions and inorganic phosphate. The close proximity of these ion pairs could then generate (Ca. POa)n chains or columns which would provide nidi for crystal formation and growth. Charge neutralization due to Ca 2÷ binding causes the bilayer to become more rigid and dehydrated as indicated Journal of Colloid and Interface Science, Vol. 111,No. 1, May 1986
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by the increased fluorescence and spectral shift of the D A N S Y L - P E probe molecules. Such dehydration during Ca 2+ binding has been well d o c u m e n t e d (30) and would decrease the energy barrier that water molecules present to the incorporation of ions into a crystalline phase (31). Initial C a . PO4 ion pairing would also be enhanced because of the effect that a decrease in the dielectric constant of the m e m b r a n e e n v i r o n m e n t has u p o n the electrostatic contribution to the total free energy change (32). T h e decrease in fluorescence intensity o f the D A N S Y L - P E probe supports f o r m a t i o n of a C a . PO4 mineral phase on the bilayer surface. Addition of Ca 2+ and PO4 ions above Ca/+ initially b o u n d would result in partial rehydration. Since Ca 2+ in solution was 70-fold in excess of the a m o u n t b o u n d by liposomes, it is unlikely that the 50% decrease in fluorescence intensity was due to a net decrease in solution calcium ion activity as a result o f complexing with phosphate. Although not shown, spectral scans for 1 and 2 m M Ca concentration were essentially the same. T h e net result is that the AG* o f the bilayer surface is less than AG* in the bulk aqueous phase. That calcification is a surface catalyzed process is also evidenced by the ultrastructural features showing the mineral phase associated pred o m i n a n t l y with the outer surfaces, while virtually no mineral is found within liposomes. It should be mentioned that the ultrastructural features are complicated somewhat by calcium phosphate-induced fusion (33) which results in c l u m p i n g o f the liposomes. A m o r e definitive study using unilameUar liposomes is currently in progress. T h e dioleoylphosphatidate liposomes described here are an in vitro model. Because of their extremely low concentrations in biological m e m b r a n e s , phosphatidic acids are not likely to be involved in situ calcification. However, acidic phospholipids (such as phosphatidylserine and the phosphoinositides) associated with calcifiable proteolipids or present in matrix vesicle m e m b r a n e s , (34) could provide similar catalytic d o m a i n s in vivo. T h e Journal of Colloid and Interface Science, Vol. 111, No. 1, May 1986
D O P A liposome m o d e l should be useful in elucidating the m e c h a n i s m s involved in lipidinduced calcification, and because of its defined structure, it should be especially valuable in infrared and nuclear magnetic resonance spectrophotometric evaluations. ACKNOWLEDGMENTS The research was supported by USPH Grant DE-04439 from the National Institute for Dental Research. The author wishes to thank Mr. M. M. Campbell for preparation of the electron micrographs, Ms. M. S. Curry for technical assistance, and Ms. C. A. Williams for manuscript typing. REFERENCES 1. Wuthier, R. E., in "The Role of Calcium in Biological Systems" (L. J. Anghileri and A. M. Tuffet-Anghileri, Eds.), Vol. I, Chap. 3. CRC Press, Boca Raton, Fla., 1982. 2. Irving, J. T., and Wuthier, R. E., Clin. Orthop. Relat. Res. 56, 237 (1968). 3. Shapiro, I. M., Calcif TissueRes. 5, 21 (1970). 4. Eisenberg, E., Wuthier, R. E., Frank, R. B., and Irving, J. T., Calcif Tissue Res. 6, 32 (1970). 5. Boskey, A. L., and Posner, A. S., Calcif Tissue Res. 19, 273 (1976). 6. Boskey, A. L., Goldberg, M. R., and Posner, A. S., Proc. Soc. Exp. Biol. Med. 157, 590 (1978). 7. Boskey, A. L., and Posner, A. S., Calcif Tissue Res. 23, 251 (1977). 8. Ennever, J., Vogel, J. J., Rider, L. J., and Boyan-Salyers, B. D., Proc. Soc. Exp. Biol. Med. 152, 147 (1976). 9. Ennever, J., Riggan, L. J., Vogel, J. J., and BoyanSalyers, B. D., J. Dent. Res. 57, 637 (1978). 10. Ennever, J., Vogel, J. J., Riggan, L. J., and Paoloski, S. B., J. Dent. Res. 56, 140 (1977). 11. Ennever, J., Boyan-Salyers, B. D., and Riggan, L. J., J. Dent. Res. 56, 967 (1977). 12. Boyan-Salyers,B., Vogel,J. J., Riggan, L. J., Summers, F. M., and Howell, R. E., Metab. Bone Dis. Relat. Res. 1, 143 (1978). 13. Ennever, J., Vogel, J. J., and Riggan, L. J., Atherosclerosis 35, 209 (1980). 14. Ennever, J., and Riggan, L. J., Atherosclerosis 47, 27 (1983). 15. Ennever, J., and Vogel, J. J., J. Dent. Res. 59, 1175 (1980). 16. Ennever, J., Riggan, L. J., and Vogel, J. J., Cytobios 39, 151 (1984). 17. Boyan, B., and Boskey, A. L., Calcif. Tissue Int. 36, 214 (1984).
DIOLEOYLPHOSPHAT1DATE LIPOSOME CALCIFICATION 18. Vogel, J. J., Campbell, M. M., and Ennever, J., Proc. Soc. Exp. Biol. Med. 143, 677 (1973). 19. Vogel, J. J., and Boyan-Salyers, B. D., Clin. Orthop. Relat. Res. 118, 230 (1976). 20. Vogel, J. J., Boyan-Salyers, B. D., and Campbell, M. M., Metab. Bone Dis. Relat. Res. 1, 149 (1978). 21. Willis, J. B., Anal. Chem. 33, 556 (1961). 22. Heinonen, J. K., and Lahti, R. J., Anal. Biochem. 113, 313 (1981). 23. Spurt, A. R., J. Ultrastruct. Res. 26, 31 (1969). 24. Shechter, E., Gulik-Krzywicki, T., Azerad, R., and Gros, C., Biochim. Biophys. Acta 241,431 (1971). 25. DeKruijoff, B., and Cullis, P. R., Biochim. Biophys. Acta 601, 235 (1980). 26, Verkleij, A. J., DeMargd, R., Leunissen-Bijvelt, J., and DeKruijf, B., Biochim. Biophys. Acta 684, 255 (1982). 27. Chickerur, N. S., Tung, M. S., and Brown, W. E., Calcif TissueInt. 32, 55 (1980). 28. Brown, W. E., Mathew, M., and Chow, L. C., in "Adsorption on and Surface Chemistry of Hydroxy-
29. 30.
31.
32.
33.
34.
159
apatite" (D. N. Misra, Ed.), Chap. 2. Plenum, New York, 1984. Hauser, H., Chapman, D., and Dawson, R. M. C., Biochim. Biophys. Acta 183, 320 (1969). Dawson, R. M. C., and Hauser, H., in "Calcium and Cellular Function" (A. W. Cuthbert, Ed.), Chap. 3. MacMillan, London, 1970. Nielsen, A. E., and Christoffersen, J., in "Biological Mineralization and Demineralization" (G. H. Nancollas, Ed.), Chap. 3. Springer-Verlag, Berlin/ New York, 1982. Clarke, I. D., and Wayne, R. P., in "Comprehensive Chemical Kinetics" (C. H. Banford and C. F. H. Tipper, Eds.), Vol. II, Chap. 4. Elsevier, Amsterdam, 1969. Fraley, R., Wilschut, J., Duzgunes, N., Smith, C., and Papahadjopoulos, D., Biochemistry 19, 6021 (1980). Wuthier, R. E., Biochim. Biophys. Acta 409, 128 (1975).
Journal of Collotd and Interface Science, Vol. 111, No. 1, May 1986
The effect of phospholipid liposomes on solid phase induction from metastable calcium phosphate solutions was evaluated. Dioleoylphosphatidate liposomes catalyzed formation of apatite in the presence of carbonate and octacalcium phosphate when carbonate was absent. Dioleoylphosphatidylglycerol liposomes did not induce solid phase formation. The phosphatidate molecules had to be packed into an extended lamellar phase in order to function as a catalyst. The study indicates that the dioleoylphosphatidate bilayer presents an electrostatic grid in which Ca 2+ binding and subsequent Ca. PO4 ion pairing provide nidi for crystal formation and growth. Acidic phospholipid microdomains in situ could provide similar catalyticsites. © 1986AcademicPress,Inc.
Certain acidic phospholipids have been found to be closely associated with the mineralization processes (calcification) of bones and teeth. Although a precise function remains to be defined, there is considerable evidence for a pivotal role of these lipids during initiation of a solid mineral phase (see recent review, (1)). Briefly, the acidic phospholipids, phosphatidylserine, and the phosphoinositides, are extracted totally only after demineralization of calcified matrices (2, 3) indicating that they are tightly bound to the mineral phase. While such data could mean that the phospholipids are merely adsorbed onto the preformed mineral phase, in vivo labeling studies with 32p-orthophosphate by Eisenberg et al. (4) demonstrated that incorporation of 32p label into both phosphatidylserine and phosphatidylinositol coincided with mineral uptake. Boskey and Posner (5) found that an initial interaction between phospholipids and mineral is reflected by the presence of defined calcium-phospholipid-orthophosphate (CaPL-Pi) complexes which could be extracted into organic solvents. The complexes are pres-
ent in a variety of calcifying matrices but absent from nonmineralizing tissues (6). Both extracted and synthetic Ca-PL-Pi complexes induced apatite formation during incubation in a metastable calcium phosphate solution (7). Parallel with the above studies, we found that virtually all calcified tissues contain proteolipids, nonpolar protein-phospholipid complexes which induce apatite formation in vitro. A calcifiable proteolipid was first isolated and characterized from the oral microorganism, Bacterionema matruchotii (8, 9). All normal and pathologic calcified tissues examined thus far contain a similar proteolipid (10-14). The integrity of the proteolipid complex is essential for calcification (15) and unlike CaPL-Pi complexes, total extraction of proteolipid requires complete decalcification (16). While a complete decalcification procedure results in the dissociation of Ca-PL-Pi complexes, Boyan and Boskey (17) recently established a correlation between the two; Ca-PLPi complexes form during the initial interactions of Ca 2÷ and HPO 2- ions with acidic 152
0021-9797/86 $3.00 Copyright© 1986by AcademicPress,Inc. All rightsof reproductionin any formreserved.
JournalofColloidand InterfaceScience, VoL 111,No. 1, May 1986
DIOLEOYLPHOSPHAT1DATE
LIPOSOME
CALCIFICATION
153
Chlorpromazine HC1 was dissolved in 0.05 N succinate buffer, pH 6.0, at a concentration of 1 mg/ml and a dioleoylphosphatidate liposome suspension was also prepared in the same buffer. A chlorpromazine-liposome complex was prepared by mixing the two solutions at a 1:1 ratio and allowing to stand at 25°C for 1 h. In practice the complexes were prepared in the same centrifuge tubes used for subsequent calcification assays. Calcification assays. Two metastable calcium phosphate solutions (MCPS) were used: (A)--70.0 mMNaC1, 22.0 mMNaHCO3, 5.0 m M KC1, 1.94 m M Na2HPO4, and 2.0 m M CaC12 (from a standardized stock solution). The solution pH was adjusted to 6.0 with CO2 gassing prior to addition of Ca2÷ and final volume adjustment. MCPS (B)--71.0 mM NaC1, 5.0 m M KC1, 1.64 m M Na2HPO4, 2.36 m M CaC12 (from stock solution), and 20.0 mM EXPERIMENTAL PROCEDURES TES buffer, pH 7.2. MCPS (B) was prepared Materials. Sodium L-o~-dioleoylphosphati- and stored as two components with Ca> and date, ammonium L-~-dioleoylphosphatidyl- HPO 2- separate, then rapidly mixed in equal glycerol, L-polylysine.HBr (25 kDa), and volumes prior to use. All solutions were Milchlorpromazine HC1 were purchased from lipore-filtered (0.45 um) and stored at 4°C with Sigma Chemical Company. N-tris-(hydroxy- 200 mg ofthymol crystals/liter added to avoid methyl)methyl-2-aminoethanesulfonic acid microbial contamination. All assays were done (TES) and dimethyl-l-naphthalenesulfonyl- in duplicate in 50-ml screw-top polycarbonate phosphatidylethanolamine (DANSYL-PE) centrifuge tubes (Oak Ridge type). The soluwere purchased from Calbiochem-Behring. tions were prewarmed to 37°C and for MCPS All other chemicals were reagent grade ob- (A) pH was adjusted to 7.15 ___0.01 with N2 gassing. This bicarbonate buffer required sealtained from local suppliers. Liposome preparation. The salts of the two ing the tubes with paraffin lined caps to prephospholipids were each suspended in 0.12 N vent CO2 loss. After each tube was filled, the NaC1, 0.02 N TES buffer solution, pH 7.2, at liposome preparations were immediately ina concentration of 1 mg/ml and dispersed into jected into the solution with a 0.5-ml autoan opalescent suspension by sonication at matic pipet. Incubations were at 37°C without 55,000 cps for 10 min at 25°C in a Branson shaking. Upon termination the tubes were 120-W bath-type sonicator. L-Polylysine was centrifuged at 10,000g for 10 min at 25°C, dissolved in the NaCI-TES buffer solution at two 1-ml aliquots of the supernatant were a concentration of I mg/ml and complexes taken for Ca and Pi analyses and the remainder were prepared by mixing 1.5 ml of the phos- was aspirated off. The precipitates were washed phatidate liposome suspension with 0.5 ml of and recentrifuged 2>
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JAMES J. VOGEL
Calcium and inorganicphosphate analyses. The 1-ml aliquot for calcium determination was diluted to a 0- to 4-ug/ml range in 0.1 N HC1 containing 0.25% LaC13 to suppress phosphate interference (21). Calcium concentrations were determined with a U n i c a m SP1900 A.A. spectrophotometer. Inorganic phosphate (Pi) concentrations were determ i n e d using the spectrophotometric procedure described by H e i n o n e n and Lahti (22). Uptake was calculated from # M initial m i n u s # M at termination. X-Ray diffractionanalyses. T h e dried samples were finely powdered in an agate m o r t a r and packed into 0.5-ram lead-free glass capillaries. The capillary was m o u n t e d in a 114m m Debye-Scherer powder c a m e r a and run on a Philips X-ray generator with exposure to Ni-filtered C u radiation at 30 kV, 20 m A for 4 h. Identification was m a d e by c o m p a r i n g films with those o f k n o w n standards and calculation o f d spaces. Electron microscopy. The glutaraldehyde solution was r e m o v e d by centrifugation and the samples were then fixed in 1% OsO4 in cacodylate buffer for 1-2 h. The samples were dehydrated through a graded series o f ethanol and e m b e d d e d in a low viscosity epoxy resin
(23). Sections were cut at 60-nm thickness with a glass knife, m o u n t e d on copper grids, and examined in a Hitachi 11-E electron microscope without post-staining. Fluorescent probe analysis. Dansylphosphatidylethanolamine was incorporated into the dioleoylphosphatidate liposomes at a m o lar ratio o f 1:50 to function as an internal fluorescent probe (24). Small samples of the liposomes (0.2 rag) were suspended in 10 ml o f 0.12 NNaC1, 0.02 M T E S buffer, p H 7.2, alone or with either 2 m M Ca 2+ or 2 m M Ca z+ and 2 m M HPOZ4-. The suspensions were incubated at 37°C for 30 m i n then scanned from 400 to 600 n m in a Varian 330 spectrofluorometer with excitation at 340 nm. Both excitation and emission slitwidths were set at 10 nm. The spectral scans were recorded on a H e w l e t t - P a c k a r d X - Y recorder and plotted as uncorrected relative fluorescence intensity vs wavelength. RESULTS Ion uptake data and X-ray diffraction analyses from incubation o f liposomes in MCPSA are summarized in Table I. Dioleoylphosphatidate ( D O P A ) liposomes at a concentra-
TABLE I Results from Incubation of Phospholipid Liposomes in Bicarbonate-Buffered Metastable Calcium Phosphate Solution (MCPS-A)~ Total uptake (umole) Liposome
Incubation period (h)
Ca2+
PO]-
Molar Ca/Pi
DOPAc (17 uM) DOPAc (17 #M) DOPAc (17 ~M) DOPAc (17 uM) DOPA~(17 uM) DOPGa ( 16 izM)
2 4 8 18 24 24
27.8 40.0 49.8 51.0 52.0 4.5
20.6 27.6 29.6 30.4 31.0 0
1.35 1.45 1.68 1.68 1.68 --
XRDb
Weak apatite Apatite Apatite Apatite Apatite --
Incubations were in duplicate at 37°C. Uptake values are the average of duplicate samples, which were within 5% of each other. b XRD = X-ray diffractionanalysis. Apatite was confirmed by comparison with film of powdered bone and calculation of"d" spacings. Most prominent lines were 344, 281,271, and 172 pro. c DOPA = dioleoylphosphatidate. a DOPG = dioleoylphosphatidylglycerol. Journal of Colloid and InterfaceScience, Vol. 111, No. 1, May 1986
DIOLEOYLPHOSPHATIDATE LIPOSOME CALCIFICATION tion o f 17 u M initiated calcification as evidenced by a rapid uptake o f both Ca 2+ and HPO24- from solution and an X-ray diffraction-detectable apatite pattern within 2 h. After about 2 h o f incubation the samples became a colloidal suspension which then precipitated between 3 and 4 h. Molar ratios o f the mineral phases did not attain the theoretical value o f 1.67 for apatite until after 8 h o f incubation. The dioleoylphosphatidylglycerol ( D O P G ) liposomes did not calcify even after 24 h o f incubation and ion uptake reflected only Ca 2+ binding. Both M C P S solutions were stable when incubated alone at 37°C for at least 14 days. The p o l y l y s i n e - D O P A liposome complex was used to c o m p a r e solid phase induction from solutions with and without added carbonate. The results from a 48-h incubation period are given in Table II. Here, ion uptake was less than with D O P A liposomes alone because o f the modulating effect o f polylysine. The solid phase formed in the presence o f carbonate was apatite, while octacalcium phosphate was the only phase formed in absence o f carbonate, as shown by both the molar Ca/ P ratio and X-ray diffraction. Electron microTABLE II Comparison Between Incubation of Polylysine-Dioleoylphosphatidate Liposome Complex (1:3) in Metastable Calcium Phosphate Solutions (MCPS) with and without Added Carbonate"
155
I
b 11111
I
I
IIJI
FIG, 1. Electron micrographs of thin sections from dioleoylphosphatidate liposomes after incubation in MCPS(A) for 4 h. (a) Low magnification (40,000X), of unstained section showing apatite as small electron dense needles encrusting outer surfaces of liposomes. (b) Higher magnification (120,000X) where crystals can be seen aligned essentially parallel to bilayer surfaces (arrows). Virtually no crystalsare seen within the liposomes (I). Average dimensions of the crystals are 2-3 nm thick and 2050 nm long. Bar length equals 100 nm.
Total uplake (/~mole)
MCPS (A) + CO3zMCPS (B) - CO32-
Ca2~
PO~-
Molar Ca/Pi
40.7 34.3
25.5 24.8
1.59 1.38
XREP
Apatite OCW
a Incubations were in duplicate at 37°C for 48 h. Uptake values are the average of duplicate samples, which were within 5% of each other. b XRD = X-ray diffraction analysis. Identification as described in Table I legend. OCP = octacalcium phosphate. A comparison was made with a known standard kindly provided by Dr. W. E. Brown, National Bureau of Standards.
graphs showing ultrastructural features o f the calcified liposomes are given in Figs. 1 and 2. The apatite crystals appear as electron-dense needles radiating from the outer surface o f the liposomes (Fig. 1a). At a higher magnification some crystals can be seen aligned parallel to the bilayer surfaces (Fig. lb). The crystals averaged 2 - 3 n m in diameter and 2 0 - 5 0 n m in length. Virtually no calcification could be seen inside the liposomes. The octacalcium phosphate crystals appeared as m u c h larger plates extending well b e y o n d the bilayer surfaces Journal of Colloid and Interface Science, Vol. 111, No. 1, May 1986
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JAMES J. VOGEL
binding occurred at a molar Ca/DOPA ratio of 1.8. Fluorescent spectral scans of dansylphosphatidylethanolamine (DANSYL-PE) incorporated into DOPA liposomes are presented in Fig. 3. Incubation in buffer containing 2 mM Ca~÷ for 30 min resulted in marked enhancement of the probe fluorescence and in addition the wavelength maximum was blueshifted almost 40 nm. With both 2 m M Ca 2+ and 2 mM HPOZ4- in the buffer the increase in fluorescence intensity was about 50% lower. The scans remained essentially the same during l-h incubation, but afterward began to decrease because of aggregation of the liposomes that resulted as the mineral phase formed. DISCUSSION
FIG. 2. Electron micrographs of thin sections from dioleoylphosphatidate liposomes after incubation in MCPS(B) for 20 h. (a) Low magnification (40,000×) of unstained section showing octacalcium phosphate as platelike crystals surrounding the liposomes. (b) Higher magnification (120,000×) showing that crystals adjacent to bilayers (arrow) are similar in size to apatite. Bar length equals I00 nm.
(Fig. 2a). The platy features are more apparent at a higher magnification (Fig. 2b). The results from stabilization of DOPA liposomes as either a lamellar or hexagonal II phase and the effect on subsequent calcification are summarized in Table III. The complexes were incubated in MCPS (A) for at least 24 h in order to evaluate whether dissociation and secondary phase conversion could occur during incubation. Liposomes stabilized in the lamellar phase with polylysine (25) induced apatite formation whereas liposomes stabilized in the hexagonal II phase with chlorpromazine (26) did not calcify and even though Caz+ Journal of Colloid and Interface Science,
Vol. 11l, No. 1, May 1986
Both ion uptake data and X-ray diffraction analyses showed the dioleoylphosphatidate (DOPA) liposomes to be very efficient catalysts for crystalline phase induction from metastable calcium phosphate solutions. Detection of apatite within 2 h of incubation was much earlier than the 24-168 h required previously for either individual phospholipids (7), isolated proteolipid, or synthetic protein-acidic phospholipid complexes (20). These variations in the rate of nucleation are due in part to differences in solution composition and phospholipid concentration used in the cited references. The finding that dioleoylphosphatidylglycerol liposomes did not induce a solid phase even after 24 h of incubation indicates that either the monoesterphosphate group is required or that the glycerol moiety provides sufficient steric hindrance. The results also show that solid phase induction is not due merely to a physical effect of injecting the liposomes on solution metastability but to a more specific interaction. The solid phase which formed was either apatite or octacalcium phosphate (OCP) depending upon the presence of carbonate in the metastable solution. OCP is very unstable in the presence of CO~ 2 ions and rapidly hydro-
157
DIOLEOYLPHOSPHATIDATE LIPOSOME CALCIFICATION TABLE III Effect o f Phospholipid Phase on Apatite Induction by Dioleoylphosphatidatea
Polylysine-DOPA (1:3) Chlorpromazine-DOPA (1:1)
Phase
Ca2+uptake (/*mole)
XRDb
Lamellar Hexagonal 11
27,8 2,5
Apatite NEG
a Phases were induced by preparing complexesas described under ExperimentalProcedures. Incubationwas in MCPS(A) at 37°Cfor 24 h. b XRD = X-raydiffractionanalysis.
lyzes to apatite (27). Brown et aL (28) have proposed that because of a lower interfacial energy requirement, OCP is the first phase to form which then hydrolyzes to apatite. It is interesting that in the present study OCP was stable even after 48 h of incubation when carbonate was absent. More detailed analyses during the 0- to 2-h incubation period might provide valuable information concerning the OCP-apatite interrelationships occurring during nucleation. While the morphologic
100" 90
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80'
70. 60.
i ~ ~ t kA
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B
4030-
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2010. 0
i
400
i
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'
i
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i
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FIG. 3. Fluorescence spectral scans of dansylphosphatidylethanolamine probe incorporated into dioleoylphosphatidate liposomes (1:50). Scan A with addition of 2 mA/ Ca 2+, scan B with addition of 2 m M Ca 2+ and 2 m M HPO~-, and scan C (control) without either Ca 2+ or HPO4~-. Note spectral blue shift with added Ca 2+ (arrow). Excitation wavelength was 340 nm.
features of the crystals as seen by electron microscopy are distinctly different overall, the initial crystals observed along the liposome bilayers are remarkably similar for both phases. The results obtained in this study indicate that the bilayer surface provides the catalytic site for solid phase induction. This conclusion is reinforced by the observation that conversion of the bilayer into the hexagonal II phase with chlorpromazine effectively blocks nucleation. In the hexagonal II phase the phospholipid molecules are packed into long cylinders with the polar heads directed inward. Although there was sufficient aqueous space within the cylinders for Ca2+ binding to occur, it was not large enough to allow formation of a solid calcium phosphate phase. On the other hand, stabilization of the bilayer with polylysine allowed calcification to occur but the rate was altered by the blocking of some of the acidic phospholipid groups via binding with the basic polypeptide. The DOPA liposome bilayer surface can be visualized as an extended two-dimensional electrostatic grid. According to Hauser et aL (29) Ca2÷ binding occurs by 2point electrostatic attachment between adjacent phospholipid molecules forming a Stern layer. Such binding would allow further ion pairing between the Ca2+ ions and inorganic phosphate. The close proximity of these ion pairs could then generate (Ca. POa)n chains or columns which would provide nidi for crystal formation and growth. Charge neutralization due to Ca 2÷ binding causes the bilayer to become more rigid and dehydrated as indicated Journal of Colloid and Interface Science, Vol. 111,No. 1, May 1986
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JAMES J. VOGEL
by the increased fluorescence and spectral shift of the D A N S Y L - P E probe molecules. Such dehydration during Ca 2+ binding has been well d o c u m e n t e d (30) and would decrease the energy barrier that water molecules present to the incorporation of ions into a crystalline phase (31). Initial C a . PO4 ion pairing would also be enhanced because of the effect that a decrease in the dielectric constant of the m e m b r a n e e n v i r o n m e n t has u p o n the electrostatic contribution to the total free energy change (32). T h e decrease in fluorescence intensity o f the D A N S Y L - P E probe supports f o r m a t i o n of a C a . PO4 mineral phase on the bilayer surface. Addition of Ca 2+ and PO4 ions above Ca/+ initially b o u n d would result in partial rehydration. Since Ca 2+ in solution was 70-fold in excess of the a m o u n t b o u n d by liposomes, it is unlikely that the 50% decrease in fluorescence intensity was due to a net decrease in solution calcium ion activity as a result o f complexing with phosphate. Although not shown, spectral scans for 1 and 2 m M Ca concentration were essentially the same. T h e net result is that the AG* o f the bilayer surface is less than AG* in the bulk aqueous phase. That calcification is a surface catalyzed process is also evidenced by the ultrastructural features showing the mineral phase associated pred o m i n a n t l y with the outer surfaces, while virtually no mineral is found within liposomes. It should be mentioned that the ultrastructural features are complicated somewhat by calcium phosphate-induced fusion (33) which results in c l u m p i n g o f the liposomes. A m o r e definitive study using unilameUar liposomes is currently in progress. T h e dioleoylphosphatidate liposomes described here are an in vitro model. Because of their extremely low concentrations in biological m e m b r a n e s , phosphatidic acids are not likely to be involved in situ calcification. However, acidic phospholipids (such as phosphatidylserine and the phosphoinositides) associated with calcifiable proteolipids or present in matrix vesicle m e m b r a n e s , (34) could provide similar catalytic d o m a i n s in vivo. T h e Journal of Colloid and Interface Science, Vol. 111, No. 1, May 1986
D O P A liposome m o d e l should be useful in elucidating the m e c h a n i s m s involved in lipidinduced calcification, and because of its defined structure, it should be especially valuable in infrared and nuclear magnetic resonance spectrophotometric evaluations. ACKNOWLEDGMENTS The research was supported by USPH Grant DE-04439 from the National Institute for Dental Research. The author wishes to thank Mr. M. M. Campbell for preparation of the electron micrographs, Ms. M. S. Curry for technical assistance, and Ms. C. A. Williams for manuscript typing. REFERENCES 1. Wuthier, R. E., in "The Role of Calcium in Biological Systems" (L. J. Anghileri and A. M. Tuffet-Anghileri, Eds.), Vol. I, Chap. 3. CRC Press, Boca Raton, Fla., 1982. 2. Irving, J. T., and Wuthier, R. E., Clin. Orthop. Relat. Res. 56, 237 (1968). 3. Shapiro, I. M., Calcif TissueRes. 5, 21 (1970). 4. Eisenberg, E., Wuthier, R. E., Frank, R. B., and Irving, J. T., Calcif Tissue Res. 6, 32 (1970). 5. Boskey, A. L., and Posner, A. S., Calcif Tissue Res. 19, 273 (1976). 6. Boskey, A. L., Goldberg, M. R., and Posner, A. S., Proc. Soc. Exp. Biol. Med. 157, 590 (1978). 7. Boskey, A. L., and Posner, A. S., Calcif Tissue Res. 23, 251 (1977). 8. Ennever, J., Vogel, J. J., Rider, L. J., and Boyan-Salyers, B. D., Proc. Soc. Exp. Biol. Med. 152, 147 (1976). 9. Ennever, J., Riggan, L. J., Vogel, J. J., and BoyanSalyers, B. D., J. Dent. Res. 57, 637 (1978). 10. Ennever, J., Vogel, J. J., Riggan, L. J., and Paoloski, S. B., J. Dent. Res. 56, 140 (1977). 11. Ennever, J., Boyan-Salyers, B. D., and Riggan, L. J., J. Dent. Res. 56, 967 (1977). 12. Boyan-Salyers,B., Vogel,J. J., Riggan, L. J., Summers, F. M., and Howell, R. E., Metab. Bone Dis. Relat. Res. 1, 143 (1978). 13. Ennever, J., Vogel, J. J., and Riggan, L. J., Atherosclerosis 35, 209 (1980). 14. Ennever, J., and Riggan, L. J., Atherosclerosis 47, 27 (1983). 15. Ennever, J., and Vogel, J. J., J. Dent. Res. 59, 1175 (1980). 16. Ennever, J., Riggan, L. J., and Vogel, J. J., Cytobios 39, 151 (1984). 17. Boyan, B., and Boskey, A. L., Calcif. Tissue Int. 36, 214 (1984).
DIOLEOYLPHOSPHAT1DATE LIPOSOME CALCIFICATION 18. Vogel, J. J., Campbell, M. M., and Ennever, J., Proc. Soc. Exp. Biol. Med. 143, 677 (1973). 19. Vogel, J. J., and Boyan-Salyers, B. D., Clin. Orthop. Relat. Res. 118, 230 (1976). 20. Vogel, J. J., Boyan-Salyers, B. D., and Campbell, M. M., Metab. Bone Dis. Relat. Res. 1, 149 (1978). 21. Willis, J. B., Anal. Chem. 33, 556 (1961). 22. Heinonen, J. K., and Lahti, R. J., Anal. Biochem. 113, 313 (1981). 23. Spurt, A. R., J. Ultrastruct. Res. 26, 31 (1969). 24. Shechter, E., Gulik-Krzywicki, T., Azerad, R., and Gros, C., Biochim. Biophys. Acta 241,431 (1971). 25. DeKruijoff, B., and Cullis, P. R., Biochim. Biophys. Acta 601, 235 (1980). 26, Verkleij, A. J., DeMargd, R., Leunissen-Bijvelt, J., and DeKruijf, B., Biochim. Biophys. Acta 684, 255 (1982). 27. Chickerur, N. S., Tung, M. S., and Brown, W. E., Calcif TissueInt. 32, 55 (1980). 28. Brown, W. E., Mathew, M., and Chow, L. C., in "Adsorption on and Surface Chemistry of Hydroxy-
29. 30.
31.
32.
33.
34.
159
apatite" (D. N. Misra, Ed.), Chap. 2. Plenum, New York, 1984. Hauser, H., Chapman, D., and Dawson, R. M. C., Biochim. Biophys. Acta 183, 320 (1969). Dawson, R. M. C., and Hauser, H., in "Calcium and Cellular Function" (A. W. Cuthbert, Ed.), Chap. 3. MacMillan, London, 1970. Nielsen, A. E., and Christoffersen, J., in "Biological Mineralization and Demineralization" (G. H. Nancollas, Ed.), Chap. 3. Springer-Verlag, Berlin/ New York, 1982. Clarke, I. D., and Wayne, R. P., in "Comprehensive Chemical Kinetics" (C. H. Banford and C. F. H. Tipper, Eds.), Vol. II, Chap. 4. Elsevier, Amsterdam, 1969. Fraley, R., Wilschut, J., Duzgunes, N., Smith, C., and Papahadjopoulos, D., Biochemistry 19, 6021 (1980). Wuthier, R. E., Biochim. Biophys. Acta 409, 128 (1975).
Journal of Collotd and Interface Science, Vol. 111, No. 1, May 1986