Bone, 14, 35-40, (1993)

8756-3282/93 $6 .00 + .00Printed in the USA. All rights reserved .

Copyright ® 1993 Pergamon Press Ltd .

An Ultrastructural Study of the Mitotic Preosteoblasts in thePrimary Spongiosa of the Rat Mandibular CondyleS . SHIBATA, O . BABA, Y . SAKAMOTO, M. OHSAKO, Y . YAMASHITA and T . ICHIJO

Department ofOral Anatomy, School of Dentistry, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku. Tokyo 113, Japan

Address for correspondence and reprints : Shunichi Shibata, D.D .Sc ., Department of Oral Anatomy, School of Dentistry, Tokyo Medical andDental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan .

Abstract

In this study, we observed mitotic preosteoblasts that havethe structural features of osteoblasts in the primary spon-giosa of the rat mandibular condyle . The rough endoplasmicreticulum and the Golgi apparatus showed remarkable dis-organization during mitosis . The Golgi saccules were re-placed by groups of large vacuoles and small vesicles . Thecisterne of the rough endoplasmic reticulum also were vac-utilized . Since this disorganization occurred in conjunctionwith the formation of the mitotic spindle, it is probably re-lated to the change! of the microtubular cytoskeleton . Fur-ther, secretory granules were arrayed along the mitotic spin-dle microtubules at the metaphase, and concentrated aroundthe midbody at the telophase . These findings indicate aclose relationship exists between secretary granules andmicrotubules .

Key Words: Osteoblast-Mitosis-Golgi apparatus-Roughendoplasmic reticulum-Microtubules .

Introduction

There have been many morphological studies with regard tomitosis, and initially these studies mainly focused on the behav-ior of the chromosomes, the kinetochores, and the formation ofthe mitotic spindle, and a general consensus concerning theseaspects has been reached (Sanger & Sanger 1988) . Additionally,the behavior of the other cell organelles during mitosis have beenstudied, such as the endoplasmic reticulum, the Golgi apparatus,and the secretory granules in many cell types (Robbins & Go-natas 1964 ; Chang & Gibley 1968 ; Kimura & Onoe 1970 ; Maul& Brinkley 1970; Erlandson & de Harven 1971 ; Melmed et al .1973; Moskalewski et al . 1977; Zeligs & Wollman 1979;Paweletz & Finze 1981 ; Schroeter et al . 1985 ; Koch et al . 1987 ;Lucocq & Warren 1987 ; Lucocq et al . 1987, 1989 ; Moskalewski& Thyberg 1990) . In general, these organelles have been foundto be morphologically disorganized during mitosis, although thedegree of this disorganization varies from one cell type to an-other.

However, few uhrastructural studies with regard to the mi-totic osteogenic cells have appeared . To add to what little isknown, this study reports on the ultrastructure of the mitoticpreosteoblasts in the primary spongiosa of the rat mandibularcondyle, specifically focusing on the behavior seen in the roughendoplasmic reticulum and the Golgi apparatus .

Materials and Methods

Sprague-Dawley rats, three weeks old, were anaesthetized withether and sacrificed by CO, asphyxiation . The mandibularcondyles then were removed and immediately immersed in 5%glutaraldehyde-4% paraformaldehyde solution (0 .1M phosphatebuffer, pH 7 .4, room temperature) for three hours . For lightmicroscopic study, the tissues were decalcified in 10% EDTA at4° C for seven days, and embedded in Technovit 7100 (Kulzer),Sections were then cut in frontal plane at 1 Jim thickness andstained with 1 % toluidine blue . For electron microscopic study,undecalcified or decalcified tissues were post-fixed in 1% os-mium tetroxide (0 .1 M phosphate buffer, pH 7 .4, 4° C) for threehours, after which they were embedded in Epon 812 (TAAB) .Ultrathin sections were stained with uranyl acetate (U) andlead citrate (Pb), and examined by HITACHI HS-9 electronmicroscopy .

Additionally, other decalcified tissues were immersed in 2%phosphotungstic acid (PTA) for six hours, then post-fixed in I%osmium tetroxide for three hours. After being embedded in Epon812, ultrathin sections were examined without counterstaining .

Light microscopic observations

Many osteoblasts were observed in the primary spongiosa of therat mandibular condyle . Each cytoplasm was strongly stainedwith toluidine blue and the Golgi area was distinguishable as alight region (Fig . 1) . Also, mitotic cells having the structuralfeatures of the osteoblasts were sometimes observed . From theprophase to the prometaphase, many chromosomes were formed,but the cytoplasm was still strongly stained and the Golgi areawas distinguishable (Figs . 2 and 3) . At the metaphase, the chro-mosomes were lined up in the equatorial plate, and the Golgiarea became inconspicuous . However, the peripheral region ofthe cytoplasm showed denser stainings than did the central re-gion (Fig. 4) . At the anaphase, the chromosomes were separatedand moved to the opposite poles, but the staining pattern of thecytoplasm remained similar to that of the metaphase (Fig . 5) . Atthe telophase, the chromosomes grouped at the opposite polesand the division of the cytoplasm was nearly completed . Thestainings of the cytoplasm at this stage was uniform (Fig . 6) .

Electron microscopic observations

As has been previously described (Leblond & Weinstock 1976),the osteoblasts at the interphase had an extensive lamellar rough

Figs . 1-6 . Light microscopy, x 1500 . An osteoblast at the interphase (Fig . I) and mitotic preosteoblasts at the prophase (Fig . 2), prometaphase (Fig .3), metaphase (Fig . 4), anaphase (Fig. 5), and telophase (Fig . 6) . respectively .Fig. 7. An osteoblast at the interphase . The rough endoplasmic reticulum (RER) are arranged concentrically around the Golgi area (G) . Ovoidmitochondria (M) are scattered among the endoplasmic reticulum . U-Pb staining, x 7700, electron microscopy .Fig . S . The Golgi area in an osteoblast at the interphase . The Golgi apparatus consists of many small vesicles and several stacks of saccules with"spherical portions" (SP) and "cylindrical portions" (CP) . Secretory granules (SG) are also seen . U-Pb staining, x 20000, electron microscopy .Fig . 9 . A secretory granule (SG) observed in the peripheral part of the cytoplasm in an osteoblast . U-Pb staining, x 36000, electron microscopy .Fig . 10 . Collagen fibrils (CF) and secretory granules (SG) show relatively high affinity for phosphotungstic acid . PTA staining, x 36000, electronmicroscopy .Fig . 11 . A mitotic preosteobtast at the prophase . Chromatin (C) in nucleus has starts to condense . No significant changes arc observed in the roughendoplasmic reticulum (RER) and the Gulp area (G) . SP: "spherical portions - ' of the Golgi saccules . U-Pb staining, x 27000, electron microscopy .Fig . 12. A mitotic preosteoblast at the prometaphase . While the chromosomes (CH) are formed, the nuclear envelope (NE) starts to disorganize . Therough endoplasmic reticulum (RER) still maintains lamellar profile . U-Pb staining, x 17000, electron microscopy .

S. Shibata et al. : Mitotic preosteoblasts

endoplasmic reticulum that was concentrically arranged aroundthe Golgi area. Many ovoid mitochondria were scattered amongthe rough endoplasmic reticulum (Fig. 7). The Golgi area wasfound to have extensive Golgi apparatuses consisting of manysmall vesicles and several stacks of saccules with distended por-tions that Leblond and Weinstock have termed "spherical por-tions" and "cylindrical portions ." Cylindrical dense bodieswere also seen in both the Golgi area and the peripheral part ofthe cytoplasm (Figs . 8 and 9) . These dense bodies have beenwell studied . They had been labelled with 'H-proline (Weinstock1975), reacted with the antibody against Type I procollagen(Wright & Leblond 1981), and were found to have an affinity forphosphotungstic acid (Scherft & Heersche 1975 ; Ohya 1978) .Similar structures also have been observed in odontoblasts(Weinstock & Leblond 1974), in fibroblasts (Cho & Garant1981), and in chondrocytes (Marchi et al . 1991) . Because thesedense bodies contain at the very least collagen precursors, thesereferenced studies have regarded them as secretory granules .Therefore, since our investigations also confirmed their affinityfor phosphotungstic acid (Fig . 10), we too refer to them as beingsecretory granules in this paper .

At the prophase of the mitotic preosteoblast, chromatin beganto condense, but no significant changes were observed in thecytoplasm (Mig. 11).

At the prometaphase, many chromosomes were formed, andthe nuclear envelope started to disorganize . The rough endoplas-mic reticulum still remained lamellarly (Fig. 12), and the Golgiapparatus also maintained the structural features observed in theinterphase osteoblast (Figs. 13 and 14) .

At the metaphase, the chromosomes were Tined up in theequatorial plate (Fig. 15) . In the cell shown in Fig . 15, thechromosomes haf, just started to separate . In the central areaaround the chromosomes, the microtubules formed the mitoticspindle between the opposed sets of centrioles (either of themcould be observed in this section) . Most of the cell organelleswere located at the poles of the cytoplasm . In particular, therough endoplasmic reticulum was seen mainly in the peripheralpart of the cytoplasm, whereas the Golgi apparatus was seenbetween the mitotic spindle and the rough endoplasmic reticu-lum. Further, most of the cisternae of the rough endoplasmicreticulum showed vacuolar profiles, although a few narrowlamellar types were seen among them . The Golgi apparatus wasfound to consist mainly of large vacuoles and small vesicles, andno saccules were present, although a few cylindrical vacuoleswere observed (Figs . 16 and 17) .

The higher magnification revealed that the microtubules thatconstituted the mitotic spindle were attached to the chromosomesat the kinetochores (Fig. 18) . Further, the phosphotungstic acid-stained secretory granules were often seen near the chromosomes(Fig . 19). As seen in an uranyl acetate-lead citrate-stained sec-tion (Fig . 20), these secretory granules were located within themitotic spindle, and arrayed closely along the microtubules .

Figure 21 shows the telophase of the mitotic preosteoblast .The mitotic spindle no longer could be seen, and although thecytoplasmic division was nearly completed, both daughter cellswere still linked with the midbody . The chromosomes werefused and the nuclear envelopes were reformed . The cell or-ganelles had been equally distributed to the daughter cells, withthe rough endoplasmic reticulum seen located in the peripheralpart of the cytoplasm, and the Golgi apparatus located aroundthe nuclei . The cisterae of the rough endoplasmic reticulum andthe Golgi apparatus, however, still mainly showed vacuolar pro-files .

As can be seen in Fig . 22, the secretory granules were con-centrated around the midbody, which consisted of the bundles ofmicrotubules .

Discussion

In general, well differentiated cells exhibit low mitotic activity .As for osteogenic cells, mature osteoblasts rarely undergo mito-sis (Jee 1988) . Mitotic osteogenic cells found in the endosteumare termed osteoprogenitor cells or preosteoblasts, which arespindle-shaped with oval nuclei and inconspicuous cytoplasm(Jee 1988), and located between osteoblasts and capillaries(Scott & Glimchcr 1971) . In the primary spongiosa of the ratmandibular condyle, however, we have encountered mitotic cellshaving the structural features of osteoblasts, such as the exten-sive rough endoplasmic reticulum and the Golgi apparatus, andwe thus have termed these cells preosteoblasts, although they arewell differentiated .

Examples of other well differentiated, mitotic cells discussedin the literature are hepatocytes in the repairing liver (Kimura &Onod 1970), drug-stimulated thyroid cells (Zeligs & Wollman1979), and pancreatic acinar cells (Melmed et al . 1973) . A com-mon phenomenon is that the tissues of these cells require rapidprotein synthesis . This is also true for the primary spongiosa ofthe rat mandibular condyle, which is actively involved in boneformation and probably accounts for the presence of mitoticpreosteoblasts .

A series of common mitotic phenomena were also observedin these mitotic preosteoblasts, such as the formation of chro-mosomes, the duplication of centrioles, the disorganization ofthe nuclear membrane, the formation of the mitotic spindle, andthe subsequent, equal distribution of cell organelles to the daugh-ter cells . Further, the Golgi apparatus and the rough endoplasmicreticulum of these preosteoblasts showed remarkable disorgani-zation during mitosis . The Golgi saccules were replaced bygroups of large vacuoles and small vesicles during the latterstages of mitosis, and these changes occurred in conjunction withthe formation of the mitotic spindle .

In this regard, a similar disorganization was seen when os-teogenic cells of the rat were injected with antimicrotubular re-agents (Sherft & Heersche 1975 ; Ohya 1978) . Therefore, it maybe that this disorganization of the Golgi apparatus is closelylinked to the changes that occur in the distribution of the micro-tubular cytoskeleton . Moskalewski and Thyberg (1990) havealso presented a similar speculation with regard to the L929 cells .

The disorganization of the Golgi apparatus during mitosis hasbeen observed in many cell types . The duration of this disorga-nization among these cell types, which starts at the prometaphaseor metaphase and continues until the end of the telophase, ap-pears to be the same, although the degree of disorganizationvaries . In Hella cells (Robbins & Gonatas 1964; Erlandson & DeHarven 1971; Lucocq & Warren 1987 ; Lucocq et al. 1987,1989), PtK-l cells (Schroeter et al . 1985), thyroid cells (Zeligs& Wollman 1979), and hepatocytes (Kimura & Onod 1970), theGolgi stacks were mainly replaced by a cluster of small vesicles .On the other hand, in hepatoma cells (Chang & Gibley 1968),melanoma cells (Maul & Brinkley 1970), pancreatic acinar cells(Melmed 1973), chondrocytes (Moskalewski et al . 1977), andL929 cells (Moskalewski & Thyberg 1990), the Golgi stackswere changed but were still discretely perceived throughout themitosis. In this study, however, the Golgi stacks in the mitoticpreosteoblasts were replaced by groups of large vacuoles, Thistype of change has not been reported .

Further, it also was noted that cisternae of the rough endo-plasmic reticulum were also vacuolized, although the area of therough endoplasmic reticulum was maintained within the cyto-plasm. Similar results have been reported in thyroid cells (Zeligs& Wollman 1979), chondrocytes (Moskalewski et al . 1977),anterior pituitary gland cells (Noguchi et al . 1986), and hepato-cytes (Kimura & Onu6 1970), although other studies have indi-

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Fig. 13. Another mitotic preosteoblast at the prometaphase that cut through the Golgi area . No significant changes are observed in the roughendoplasmic reticulum (RER) and the Golgi area (G). CH: chromosomes. U-Pb staining, x 8100, electron microscopy .Fig . 14 . Higher magnification of the Golgi area in Fig . 13 . Secretory granules (SC), "spherical portions" (SP) and "cylindrical portions" (CP) ofthe Golgi saccules are observed . U-Pb staining, x 29000 .Fig. 15 . A mitotic preosteoblast at the metaphase . Chromosomes (CH) are lined up in the equatorial plate . The cistemae of the rough endoplasmicreticulum (RER) mostly show vacuolar profiles . G : Golgi area . * : Centriole . U-Pb staining, x 6000, electron microscopy .Fig . 16 . Higher magnification of Fig . 15 . Centrioles (*) and microtubules (MT) which constitute mitotic spindle are observed . Although theendoplasmic reticulum (RER) and the Golgi apparatus (G) maintain their areas, they mostly show vacuolar profiles . Secretory granules (SC) are alsoseen in the peripheral part of the cytoplasm . U-Pb staining, x 14000 .Fig . 17 . Higher magnification of the Golgi area in Fig . 15 . Golgi apparatus mostly consists of large vacuoles and small vesicles. A few cylindricalvacuoles (arrowhead) are also observed among them . U-Pb staining, x 42500.Fig . 18 . Higher magnification of the mitotic spindle in Fig . IS . The microtubules are radiating from the cenhiole (*) and attached to the chromosomes(CH) at the kinetochore (K) . U-Pb staining, x 34000 .

S. Shibata et al . : Mitotic preosteoblasts

Fig. 19 . Phosphotungs' :ic acid stained secretory granules (arrowhead) arc observed near the chromosomes (CH) in a mitotic preosteoblast at themetaphase . PTA staining, x 11000, electron microscopy .Fig. 20 . Secretory granules (SG) are arrayed closely along the microtubules (MT) which constitute the mitotic spindle . CH: chromosomes . U-Pbstaining, x 60000, electron microscopy .Fig. 21 . A mitotic preusteoblast at the telophase . The daughter cells are linked with midbody (MB) . The rough endosplasmic reticulum (RER) andthe Golgi apparatus (G) still mainly show vacuolar profiles . U-Pb staining, x 5700, electron microscopy .Fig. 22 . Higher magni ication of the midbody in Fig . 21 . Many secretory granules (arrowhead) are concentrated around the midbody (MB) whichconsists of the bundles of microtubutes, U-Pb staining, x 16000 .

cated that such vacuolization does not always occur (Melmed etal . 1973; Redman & ;Sreebny 1970 ; Pictet et al . 1972 ; Jimbow etal . 1975 ; Koch et al . 1987) .

The disorganization of the cell organelles has been consideredhelpful for capturing the space of the mitotic spindle and for theequal distribution to the daughter cells (Moskalewski & Thyberg1990). From this viewpoint, well differentiated cells, which gen-

39

erally contain many cell organelles, would require extensive dis-organization . Therefore, we feel that the variation seen in thedisorganization of many cell types may reflect their degree ofdifferentiation .

On the other hand, some researchers have suggested the pos-sibility that artifacts from the fixation process may be responsiblefor the morphological disorganization seen in the endoplasmic

40

reticulum (Koch et al . 1987 ; Hepler 1989) . Although we cannotdispute this possibility at this point, improved fixation methods,quick-freezing and freeze-substitution techniques (Hunziker etal . 1984; Akisaka et al . 1987) may soon clarify whether thisdisorganization results from some artifacts .

Microtubules have been found to be involved in the transportof the secretory granules to the cell membrane in some collagen-secreting cells (Ehrlich et al . 1974 ; Scherft & Heersche 1975 ;Ohya 1978 ; Cho & Garant 1981) . In this regard, we observedphosphotungstic acid stained dense bodies in the mitotic preo-steoblasts, and some at the metaphase were arrayed closely alongthe mitotic spindle microtubules . In the telophase, they wereconcentrated around the midbody, which consisted of bundles ofmicrotubules . As described in our results section, these densebodies are thought to be secretary granules . Thus, our observa-tions have indicated that a close relationship exists between thesecretory granules and the microtubules in osteogenic cells . Fur-ther, since exocytosis is inhibited during mitosis (Hesketh et al .1984), the secretory granules arrayed along the microtubulesseemed to accumulate within the cytoplasm .

As has been pointed out, secretary granules were found toaccumulate after an injection of antimicrotubular reagents(Scherft & Heersche 1975 ; Ohya 1978) . Also, Cho and Garant(1981) have demonstrated a definite association between colla-gen secretary granules and the microtubules in fibroblasts . Ourspeculations are thus supported by these experiments .

Acknowledgment: This work was supported by Grant-in-Aid for Scien-tific Research (No. 02404070) from the Ministry of Education, Scienceand Culture of Japan .

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Date received October 2, 1991Date revised.: July 14, 1992Date accepted : August 25, 1992