BASIC BIOLOGICAL SCIENCES
Pre-apatitic Mineral Deposition in Bacterionema matruchotii BARBARA D. BOYAN-SALYERS, JAMES J. VOGEL, and JOHN ENNEVER
The University of Texas Health Science Center, Dental Science Institute at Houston, Houston, Texas 77025, USA A prelimznary examination of calcification of Bacterionema matruchotii was undertaken to provide a base-line for future kinetic and mechanistic studies. Specific ion ratios were correlated with crystallography and ultrastructure. Observations include: three consistent X-ray diffraction patterns; no resolution of cellular EMP by electron microscopy; increasing Ca /Mg, Ca /P during calcification. J Dent Res 57(2): 291-295, February 1978.
uniform mid-logarithmic growth inocula (250 x 107 colony-forming units) were incubated at 37 C in 250 ml volumes of a chemically defined culture medium supporting calcification (calcifying medium).' Noncalcifying cells grown in chemically defined maintenance medium2 served as controls. Calcifying and maintenance media differed only in calcium concentration. Cells were harvested at appropriate intervals. X-RAY DIFFRACTION.
-
At 24-hour in-
tervals for four days and twice daily for the The purpose of this investigation is to pro- next six days cells were harvested (35,000 vide a preliminary examination of biologic g, 5 minutes), washed three times with deapatite formation by the microorganism, ionized water, and stored at - 20 C. For Bacterionema matruchotii. The organism, each time interval, cells were ashed an actinomycete of the human oral biota, (throughout these experiments all ashing forms intracellular mineral, crystallog- was done in a muffle furnace at 700 C) and raphically indistinguishable from that of the residue was analyzed by X-ray diffracvertebrate bones and teeth, when grown 7 tion using the Debye-Scherer powder to 10 days in a chemically defined culture camera film technique (4 hours, nickel filmedium. ' The relative simplicity of micro- tered Cu K oc radiation, 30kV, 2OmA). bial calcification and the high degree of The films were either compared to known experimental control that can be exercised standards or their d-spaces determined. afford conditions unobtainable with verte- Ashed dentin was the apatite standard; brate tissues for studying the dynamics of ashed amorphous calcium phosphate was biologically mediated apatite formation. the whitlockite standard. By correlating specific ion ratios during SPECIFIC ION RATIOS. - Cells were harcalcification with crystallographic and ul- vested at 2, 4, 6, and 8 days, washed three trastructural data, a baseline for future times with deionized water and air-dried mechanistic and kinetic studies has been (50 C). Approximately 100 mg of dried established. cells were ashed and the residue diffracted. Another 100 mg were ashed, dissolved in Materials and Methods concentrated HCl and the volume adjusted to 10 ml with deionized water. Calcium Throughout these experiments cultures and magnesium were determined by of B matruchotii were prepared as follows: atomic absorption spectrophotometry.3 InReceived for publication March 3, 1977. organic phosphate was determined colorAccepted for publicationJuly 14, 1977. imetrically.4 This Investigation was supported by USPHS Grants DE00669, DE-04212, and DE-02232 from the National Institute of Dental Research.
ULTRASTRUCTURE. - Nonashed cells, harvested concurrently with those exam-
291 Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 17, 2015 For personal use only. No other uses without permission.
292
BO YAN- SA L YERS, VOGEL & ENNE VER
j Dent
Res
February
1978
FIG 1. - X-ray diffraction patterns observed during calcification of B matruchotii (a) Early mineral phase (EMP); (b) whitlockite; (c) biologic apatite.
ined by X-ray diffraction, were fixed in cacodylate buffered glutaraldehyde (2%), postfixed in OS04 (1%), dehydrated and embedded in a low viscosity epoxy resin.5 Ultrathin sections were examined unstained with an Hitachi 11-E electron microscope.
Ash from 6-day-cultures grown in calcifying medium was embedded without prior fixation and ultrathin sections examined unstained. Ultrathin sections of unashed cells were floated on 0.1 N HCl for one minute and re-examined. Cells grown in maintenance medium were examined for comparison.
Results
A reproducible crystallographic record of B matruchotii calcification was established in that three X-ray patterns were consistently observed. Ash from cultures grown 2 to 6 days in calcifying medium produced an X-ray pattern (Fig la), designated EMP (early mineral phase), which does not conform to known standards. Calculated d-spaces are presented in Table 1. The occurrence of the EMP pattern is independent of inoculum size and age. Be-
tween 6 and 10 days the crystal type changes to whitlockite (Fig Ib) and finally, biologic apatite (Fig Ic). Noncalcifying cells produced X-ray diffraction patterns of general salt mixtures. Ash from 6-day cultures of calcifying cells appeared ultrastructurally amorphous (Fig 2). Attempts to resolve similar material within the cells were unsuccessful. Treatment with dilute acid did not alter the appearance of the cells. Moreover, no differences were observed when the ultrastructure of the calcifying cells was compared to that of cells grown in maintenance medium (Fig 3). Ultrastructure of calcified cells (containing apatite) was identical to that already reported. 1 Calcium, magnesium, and phosphate concentrations determined for calcifying and noncalcifying B matruchotii are shown in Table 2 as Ca/Pi, Ca/Mg, and Ca + Mg/Pi molar ratios. Ca concentration was slightly higher in calcifying cells as early as day two. However, significant differences between calcifying and noncalcifying cells were not observed at this time. Elevated ratios observed throughout the incubation period in calcifying cells are a function of increasing Ca content. Cell Ca was approx-
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PRE-APA TITIC MINERAL DEPOSITION
Vol. 57 No. 2
293
imately equal to Mg in calcifying cells until day eight, when a marked Ca increase was noted. In contrast, the lower Ca concentration of the noncalcifying cells remained relatively constant. Discussion
There has been considerable speculation concerning the mineral phase sequence of biologic apatite formation in vertebrate calcifying tissues. Much of the research thus far has employed in vitro experimental conditions using highly supersaturated calcium phosphate solutions.6 Amor phous calcium phosphate (ACP) forms first and transforms autocatalytically to apatite. A role for ACP as the apatite precursor state has been postulated. 7 Hydrated CaHPO4 has also been proposed as a precursor of biologic apatite.8 An initial rapid formation of amorphous CaHPO4 followed by the slow formation of crystalline apatite from the precipitate has been postulated. Recently, evidence was obtained for the presence in viable bone of a calcium phosphate phase of high solubility, probably CaHPO4 * 2H20.9 Brushite has been identified by X-ray diffraction in newly mineralized chick embryo bone"' and in developing bone and callus. 1 1 A mechanism for membrane localized acidic phospholipid calcification has been proposed.'2"13 The initial stages of apatite formation involve Ca++ binding by acidic phospholipids (complexed as proteolipid) followed by addition of HPO4= and Ca" ions to form a (CaHOP4)2 intermediate. Such a role for proteolipid would be consistent with nucleation data reported for the organism.2 With additional Ca"t the dimers condense to form Cag(P04)6 clusters. Hydration of the clusters allows conversion to apatite. Whether the three X-ray diffraction patterns observed in this study represent a mineral phase sequence of calcification or unrelated events was not considered. The EMP and whitlockite patterns are artifacts reflecting cellular ion ratios as well as temperatures of pyrolysis. 14 Unlike precipitates from synthetic solutions, analysis of the X-ray patterns is further complicated by intimate organic-inorganic associations. For example, the occurrence of a whitlock-
FiG 2. - Electron micrograph of ash from 6-day cultures of calcifying B matruchotio. Orig mag 96,000 x unstained.
ite pattern indicates that there is some structure or organizing of Ca and Pi in a ratio of 1.5's despite the medium ratio of 1 and a cellular ratio of 0.6. Thorough washing of the cells prior to pyrolysis eliminates the contribution of ions from the medium to either the X-ray patterns or ion ratios. The phenomena analyzed are, therefore, biologically-mediated stable organizations of mineral ions. The early mineral phase is of particular interest in that it is consistently observed in cells grown in calcifying medium despite steadily increasing Ca/Mg and Ca/P ratios. Moreover, it appears by day two when no real differences in Ca/P, or Ca + Mg/Pi ratios were detected. These data, TABLE 1 EMP X RAY DIFFRACTION MAXIMA D-Space
Intensity
I)-Space
Intensity
4.68 4.32 3.38 3.15 2.98 2.82 2.71 2.62 2.52 2.35 2.25
8 8 8 7 9 10
2.17 2.03
8 3 2
2 2
1.93 1.83 1.77 1.72 1.68 1.66
Pre-apatitic Mineral Deposition in Bacterionema matruchotii BARBARA D. BOYAN-SALYERS, JAMES J. VOGEL, and JOHN ENNEVER
The University of Texas Health Science Center, Dental Science Institute at Houston, Houston, Texas 77025, USA A prelimznary examination of calcification of Bacterionema matruchotii was undertaken to provide a base-line for future kinetic and mechanistic studies. Specific ion ratios were correlated with crystallography and ultrastructure. Observations include: three consistent X-ray diffraction patterns; no resolution of cellular EMP by electron microscopy; increasing Ca /Mg, Ca /P during calcification. J Dent Res 57(2): 291-295, February 1978.
uniform mid-logarithmic growth inocula (250 x 107 colony-forming units) were incubated at 37 C in 250 ml volumes of a chemically defined culture medium supporting calcification (calcifying medium).' Noncalcifying cells grown in chemically defined maintenance medium2 served as controls. Calcifying and maintenance media differed only in calcium concentration. Cells were harvested at appropriate intervals. X-RAY DIFFRACTION.
-
At 24-hour in-
tervals for four days and twice daily for the The purpose of this investigation is to pro- next six days cells were harvested (35,000 vide a preliminary examination of biologic g, 5 minutes), washed three times with deapatite formation by the microorganism, ionized water, and stored at - 20 C. For Bacterionema matruchotii. The organism, each time interval, cells were ashed an actinomycete of the human oral biota, (throughout these experiments all ashing forms intracellular mineral, crystallog- was done in a muffle furnace at 700 C) and raphically indistinguishable from that of the residue was analyzed by X-ray diffracvertebrate bones and teeth, when grown 7 tion using the Debye-Scherer powder to 10 days in a chemically defined culture camera film technique (4 hours, nickel filmedium. ' The relative simplicity of micro- tered Cu K oc radiation, 30kV, 2OmA). bial calcification and the high degree of The films were either compared to known experimental control that can be exercised standards or their d-spaces determined. afford conditions unobtainable with verte- Ashed dentin was the apatite standard; brate tissues for studying the dynamics of ashed amorphous calcium phosphate was biologically mediated apatite formation. the whitlockite standard. By correlating specific ion ratios during SPECIFIC ION RATIOS. - Cells were harcalcification with crystallographic and ul- vested at 2, 4, 6, and 8 days, washed three trastructural data, a baseline for future times with deionized water and air-dried mechanistic and kinetic studies has been (50 C). Approximately 100 mg of dried established. cells were ashed and the residue diffracted. Another 100 mg were ashed, dissolved in Materials and Methods concentrated HCl and the volume adjusted to 10 ml with deionized water. Calcium Throughout these experiments cultures and magnesium were determined by of B matruchotii were prepared as follows: atomic absorption spectrophotometry.3 InReceived for publication March 3, 1977. organic phosphate was determined colorAccepted for publicationJuly 14, 1977. imetrically.4 This Investigation was supported by USPHS Grants DE00669, DE-04212, and DE-02232 from the National Institute of Dental Research.
ULTRASTRUCTURE. - Nonashed cells, harvested concurrently with those exam-
291 Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 17, 2015 For personal use only. No other uses without permission.
292
BO YAN- SA L YERS, VOGEL & ENNE VER
j Dent
Res
February
1978
FIG 1. - X-ray diffraction patterns observed during calcification of B matruchotii (a) Early mineral phase (EMP); (b) whitlockite; (c) biologic apatite.
ined by X-ray diffraction, were fixed in cacodylate buffered glutaraldehyde (2%), postfixed in OS04 (1%), dehydrated and embedded in a low viscosity epoxy resin.5 Ultrathin sections were examined unstained with an Hitachi 11-E electron microscope.
Ash from 6-day-cultures grown in calcifying medium was embedded without prior fixation and ultrathin sections examined unstained. Ultrathin sections of unashed cells were floated on 0.1 N HCl for one minute and re-examined. Cells grown in maintenance medium were examined for comparison.
Results
A reproducible crystallographic record of B matruchotii calcification was established in that three X-ray patterns were consistently observed. Ash from cultures grown 2 to 6 days in calcifying medium produced an X-ray pattern (Fig la), designated EMP (early mineral phase), which does not conform to known standards. Calculated d-spaces are presented in Table 1. The occurrence of the EMP pattern is independent of inoculum size and age. Be-
tween 6 and 10 days the crystal type changes to whitlockite (Fig Ib) and finally, biologic apatite (Fig Ic). Noncalcifying cells produced X-ray diffraction patterns of general salt mixtures. Ash from 6-day cultures of calcifying cells appeared ultrastructurally amorphous (Fig 2). Attempts to resolve similar material within the cells were unsuccessful. Treatment with dilute acid did not alter the appearance of the cells. Moreover, no differences were observed when the ultrastructure of the calcifying cells was compared to that of cells grown in maintenance medium (Fig 3). Ultrastructure of calcified cells (containing apatite) was identical to that already reported. 1 Calcium, magnesium, and phosphate concentrations determined for calcifying and noncalcifying B matruchotii are shown in Table 2 as Ca/Pi, Ca/Mg, and Ca + Mg/Pi molar ratios. Ca concentration was slightly higher in calcifying cells as early as day two. However, significant differences between calcifying and noncalcifying cells were not observed at this time. Elevated ratios observed throughout the incubation period in calcifying cells are a function of increasing Ca content. Cell Ca was approx-
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 17, 2015 For personal use only. No other uses without permission.
PRE-APA TITIC MINERAL DEPOSITION
Vol. 57 No. 2
293
imately equal to Mg in calcifying cells until day eight, when a marked Ca increase was noted. In contrast, the lower Ca concentration of the noncalcifying cells remained relatively constant. Discussion
There has been considerable speculation concerning the mineral phase sequence of biologic apatite formation in vertebrate calcifying tissues. Much of the research thus far has employed in vitro experimental conditions using highly supersaturated calcium phosphate solutions.6 Amor phous calcium phosphate (ACP) forms first and transforms autocatalytically to apatite. A role for ACP as the apatite precursor state has been postulated. 7 Hydrated CaHPO4 has also been proposed as a precursor of biologic apatite.8 An initial rapid formation of amorphous CaHPO4 followed by the slow formation of crystalline apatite from the precipitate has been postulated. Recently, evidence was obtained for the presence in viable bone of a calcium phosphate phase of high solubility, probably CaHPO4 * 2H20.9 Brushite has been identified by X-ray diffraction in newly mineralized chick embryo bone"' and in developing bone and callus. 1 1 A mechanism for membrane localized acidic phospholipid calcification has been proposed.'2"13 The initial stages of apatite formation involve Ca++ binding by acidic phospholipids (complexed as proteolipid) followed by addition of HPO4= and Ca" ions to form a (CaHOP4)2 intermediate. Such a role for proteolipid would be consistent with nucleation data reported for the organism.2 With additional Ca"t the dimers condense to form Cag(P04)6 clusters. Hydration of the clusters allows conversion to apatite. Whether the three X-ray diffraction patterns observed in this study represent a mineral phase sequence of calcification or unrelated events was not considered. The EMP and whitlockite patterns are artifacts reflecting cellular ion ratios as well as temperatures of pyrolysis. 14 Unlike precipitates from synthetic solutions, analysis of the X-ray patterns is further complicated by intimate organic-inorganic associations. For example, the occurrence of a whitlock-
FiG 2. - Electron micrograph of ash from 6-day cultures of calcifying B matruchotio. Orig mag 96,000 x unstained.
ite pattern indicates that there is some structure or organizing of Ca and Pi in a ratio of 1.5's despite the medium ratio of 1 and a cellular ratio of 0.6. Thorough washing of the cells prior to pyrolysis eliminates the contribution of ions from the medium to either the X-ray patterns or ion ratios. The phenomena analyzed are, therefore, biologically-mediated stable organizations of mineral ions. The early mineral phase is of particular interest in that it is consistently observed in cells grown in calcifying medium despite steadily increasing Ca/Mg and Ca/P ratios. Moreover, it appears by day two when no real differences in Ca/P, or Ca + Mg/Pi ratios were detected. These data, TABLE 1 EMP X RAY DIFFRACTION MAXIMA D-Space
Intensity
I)-Space
Intensity
4.68 4.32 3.38 3.15 2.98 2.82 2.71 2.62 2.52 2.35 2.25
8 8 8 7 9 10
2.17 2.03
8 3 2
2 2
1.93 1.83 1.77 1.72 1.68 1.66
