The reaction of osteoclasts when releasing osteocytes from osteocytic lacunae in the bone during bone modeling.

Osteocytes are released from the osteocytic lacunae when osteoclasts resorb the bone matrix during bone modeling and remodeling. It remains unknown how osteoclasts react when releasing osteocytes during bone modeling, and the fate of these released osteocytes is also unclear. Femoral mid-shafts of 2-day-old kittens were sectioned into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and examined by light microscopy (LM) and transmission electron microscopy (TEM). The sections showed many osteoclasts at the endosteum but there were no osteoblasts. There were many half-released, fully released, half-exposed, and fully exposed osteocytes on the bone surfaces. Many cell-like structures were seen in the cell bodies of osteoclasts by LM, and some semithin sections were re-sectioned into ultrathin sections for re-observation by TEM. By TEM, these were determinated to be mononuclear cells. The serial ultrathin sections showed that the mononuclear cells appeared to be engulfed in osteoclasts on one section but that the cell was connected with the bone surface of the osteocytic lacuna on another section. These results show that the mononuclear cells in the osteoclasts were osteocytes. The present study suggests that osteoclasts engulf some osteocytes but do not engulf others when releasing osteocytes during bone modeling.

Organization of cytoskeletal F-actin, G-actin, and gelsolin in the adhesion structures in cultured osteoclast.

Immunofluorescence using Gc protein (group-specific component or vitamin D binding protein [DBP]) as a marker of G-actin showed that nonfilamentous, monomeric G-actin is a component of the podosomes of osteoclasts cultured on glass plates or bone slices. Typical individual podosomes of the well-spread cells on glass plates were rosette in form. When viewed from the basolateral surface, the core portion of the dotlike podosomes was associated with packed F-actin filaments surrounded by G-actin organized in a ringlike structure. The podosomes, when viewed perpendicular to the substrate, showed a conical shape as a bundle of short F-actin core and a ring of G-actin. With cell spreading on glass plates, the clustering of the podosomes formed a continuous belt of tightly packed podosomes as an adhesion structure at the paramarginal area. In addition, these structures were seen on the ventral cell surface. Similar changes in cell shape were seen in the osteoclasts when they were plated on bone slices. With the loss of dotlike podosomes, a continuous band of F-actin was formed around the resorption lacunae. It became evident then that F- and G-actin dissociated from each other in the podosomes. The staining patterns of G-actin varied from a discrete dot to a diffuse one. Toward the nonresorption phase, the osteoclasts lost their continuous F-actin band but dotlike podosomes appeared in the leading and the trailing edges. In such a cell undergoing translational movements, G-actin was located diffusely in the cytoplasm behind the lamellipodia and along some segments of the leading edge. Cytochalasin B treatment caused cells to disorganize the actin cytoskeletal architecture, which indicated the disassembling of F-actin into G-actin in podosomes and disappearance of actin-ring of cultured osteoclasts. Staining with polyclonal actin antibody or monoclonal beta-actin was overlapped with the distribution pattern of G- and F-actin. Gelsolin was detected in the region of the adhesion area corresponding to the podosome. The observation that F-actin, G-actin, and gelsolin were detected in the osteoclastic adhesion structures suggests that the podosomes may represent sites where a rapid polymerization/depolymerization of actin occurs. These dynamic changes in cytoskeletal organization and reorganization of G-actin may reflect changes in the functional polarization of the osteoclast during the bone resorption cycle and suggest the important role of G-actin in the regulation of osteoclast adhesion.

Visualization of acidic compartments in cultured osteoclasts by use of an acidotrophic amine as a marker for low pH.

Using the acidotrophic amine 3-(2,4-dinitroanillino)-3'-amino-N-methyldipropylamine (DAMP) as a marker for low pH and immunofluorescence cytochemistry, we examined acidic compartments of osteoclasts cultured on cover glasses or bone slices, where they could resorb the bone surface, forming resorptive lacunae. DAMP-positive structures were seen as vesicular and tubular forms in the cytoplasm, indicating lysosomes and endosomes. Not only the osteoclastic cytoplasm but also the extracellular area around the ruffled border and resorptive lacunae were stained with DAMP, suggesting acidic regions. Immunofluorescence was localized predominantly on the substratum side of actively resorbing osteoclasts, whereas an evenly distributed staining pattern was seen in the nonactive cell. The most intensive reaction was seen at the advancing front of resorptive lacunae within the actively resorbing osteoclasts. The distribution pattern of DAMP seemed to be correlated with the osteoclastic activity, since osteoclasts exhibit alternating resorption and migration phases during the bone-remodeling cycle. In this culture system, the resorptive lacunae were left behind after the osteoclasts had completed resorption and migrated along the bone surface. These exposed resorptive lacunae were also stained with DAMP, which were presumably kept at an acidic pH. The effect of treatment with monensin, chloroquine, ammonium chloride, or nigericin was varied in terms of the immunoreactivity for DAMP, but not complete abolition of the staining was obtained. Weak bases such as chloroquine or ammonium chloride inhibited both intra- and extracellular immunoreactivity. Immunoreactivity for the vacuolar type of proton ATPase (V-ATPase) was demonstrable in the cytoplasm of the osteoclasts but was weakened by the addition of bafilomycin. Immunofluorescence of the resorptive lacunae was still retained even after the treatment with bafilomycin and acetazolamide. Besides, both bafilomycin and acetazolamide reversibly inhibited cellular acidity as judged by DAMP immunocytochemistry, which agrees with the fact that ostoeclastic acidification results from the action of vacuolar proton-pump ATPase coupled with carbonic anhydrase.

Ultrastructural modifications of the extracellular matrix upon calcification of growth plate cartilage as revealed by quick-freeze deep etching technique.

The ultrastructural changes in cartilage matrix that occur during calcification have been examined in chick epiphyseal growth plate cartilage prepared by quick-frozen, deep-etched, and rotary shadowed replicas. The extracellular cartilage matrix contains a reticular network closely associated with an extensive network of collagen. The components of the reticular network, including thick and thin filaments, are attached directly to the cell membrane, matrix vesicle membrane, and collagen fibrils. This network, which interconnects the matrix vesicles and collagen, fills the extracellular matrix. The dimensions of the reticular network seem to remain almost constant in size from the reserve and proliferative zones to the calcifying zone. The collagen fibrils seem to consist of subfibrillar structures that branch and anastomose. In optimally quick-frozen, deep-etched, prepared collagen, a cross-banding pattern was exposed. Globular structures stud the collagen fibrils, which gradually diminish in number from the reserve zone down to the calcifying zone. The matrix vesicles, when fractured, showed a granular appearance. In most cases, the fracture plane passed through the bilayer of the matrix vesicle membrane. The true surface of the matrix vesicle membrane, therefore, was exposed after deep etching. At the calcifying zone, crystal deposition had occurred in needle-like and/or plate-like form within the membrane-bound matrix vesicles. The reticular network was still intact in the vicinity of the calcified matrix, but in the intercrystalline space, neither the reticular structure nor the globular structure was detectable. Within the calcified matrix, both reticular and granular structures had disappeared from the interfibrillar space of the collagen fibrils.

Ultrastructure of quick-frozen and freeze-substituted chick osteoclasts.

For comparison with chemically fixed osteoclasts, we prepared chick osteoclasts by quick freezing followed by freeze-substitution. In spite of technical difficulties this demonstrated that osteoclasts can be satisfactorily frozen in situ by the metal contact method. Ultrastructural differences were revealed between conventional fixation and quick freezing. Compared with conventional fixation, the quick freezing method appeared to improve preservation: (1) a discrete trilaminar plasma membrane and other intracellular membranes showed a smooth profile without undulation or rupture; (2) cytoskeletal components appeared to be clearer, straighter, and more numerous; (3) the interior of the ruffled finger contained interconnected lattice structures whereas highly organised microfilaments were seen in the clear zone; (4) well developed tubulovesicular structures (TVSs) that branched or anastomosed with each other were revealed in the cytoplasm; (5) the contents of intracellular membrane systems including the nuclear envelope, endoplasmic reticulum, and Golgi complex were stained to a various extent; (6) vesicles and vacuoles were much smaller, round and well-defined with electron-dense contents; (7) crystalline structures were seen at the extracellular channels of the ruffled border, in the lumen of TVSs, and in vesicles; (8) in some instances mitochondrial granules were visible; (9) within the resorptive lacuna, osteoclasts adhered to the degraded bone matrix without any intervening empty space.

The cytoskeletal framework of chick osteoclasts in resin-less sections.

The cytoskeletal framework of chick osteoclasts was examined by the resin-less polyethylene glycol (PEG) method. Resin-less thin sections revealed changes in the 3-dimensional organisation of a microtrabecular lattice during different phases of osteoclast activity. This lattice, composed of interconnecting strands, occupied part of the osteoclast cytoplasm including the ruffled border and clear zone areas. In firmly attached, active osteoclasts, the clear zone exhibited a compact and orderly array with a meshwork appearance. This organelle-free area could be clearly distinguished from other cytoplasmic regions by the compactness of its lattice structure. Well developed membrane infoldings of the ruffled border next to the resorption lacunae were composed of interconnected strands, some of which were in direct contact with the ruffled border membrane. Migrating osteoclasts remote from the bone surface lacked these membrane modifications. Their peripheral cytoplasm exhibited a disorganised meshwork of strands without the clear zone. While migrating, osteoclasts which still adhered to the bone surface, appeared to form loci within the compact lattice structure referred to as podosomes. These results demonstrate the dynamic morphological changes in the organisation of the trabecular lattice that occur within attached and migrating osteoclasts.

Ultrastructure of quick-frozen secretory ameloblasts of the rat molar tooth.

Ameloblasts in molar tooth germs from 4 to 12-day-old neonatal rats were quick-frozen and then freeze-substituted in acetone-tannic acid and acetone-osmium tetroxide. Following quick freezing and freeze-substitution, the preservation of ameloblasts was principally the same as seen in conventionally-fixed ones. Several differences, however, became apparent between two methods. Concerning enamel formation, the quick-freezing procedure greatly improved the preservation including that of the extrusion of secretory granules into the extracellular space. The exocytosis is a type of merocrine secretion. The precursor stippled material was demonstrated at the front of Tomes' processes and in the intercellular space of the distal terminal junction. In the more advanced stage, the stippled material tended to decrease gradually in amount. Prior to the formation of the typical Tomes' process, large secretory granules fused directly with the distal plasma membrane, which plays a role in the formation of prismless enamel. After the typical Tomes' process had formed, secretory granules containing electron-dense material instead of the large secretory granules accumulated in the distal portion of the Tomes' process. These latter secretory granules had two different destinations; i.e. the granules discharged their contents into the infoldings of the distal part of Tomes' process and into the infoldings of the lateral cell membrane. These types of secretion seem to have a functional role in the rod or inter-rod enamel formation. The ultrastructure of the stippled material consisted of 3-dimensionally interconnected strands after quick freezing, which is different from the appearance of granules embedded in amorphous material after conventional fixation. The stippled material was seen in the intercrystal space among the newly formed crystals. Also, an extensive tubulo-vesicular structure was preserved in both the cell body and Tomes' process. The present data suggest that the membrane of this structure showed a smooth contour opening to the extracellular space, and also an intimate relationship with the secretory granules.

Ultrastructural and ultracytochemical characteristics of multinucleated cells after hydroxyapatite implantation into rat periodontal tissue.

Multinucleated cells (MNCs) that appeared after hydroxyapatite (HAP) implantation into experimentally-produced bone defects in rat periodontal tissues were investigated both ultrastructurally and ultracytochemically. At day 5 after implantation, MNCs first appeared along the HAP surface. They had no features of typical osteoclasts such as ruffled border and clear zone. By d 14, these cells acquired features similar to osteoclasts, including ruffled border and clear zone. With the appearance of ruffled borders in MNCs, new bone deposited around the implanted HAP. MNCs appeared to excavate both newly-formed bone and implanted HAP simultaneously. Ingested HAP particles were observed not only in MNCs but also in macrophages. MNCs contained both tartrate-resistant acid phosphatase (ACPase) and carbonic anhydrase (CAase). ACPase activity was detected along all the biosynthesizing pathways in MNCs. Extracellular ACPase activity around the ruffled border region was also demonstrable. CAase activity could be detected only in the cytosol, vesicles and mitochondrial cristae of the MNCs. These cytochemical characteristics were almost the same regardless of the time elapsed after implantation.

Heterogeneity of distribution pattern at the electron microscopic level of heparan sulfate in various basement membranes.

Using the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining technique and enzymatic digestion, we investigated the ultrastructural distribution pattern of heparan sulfate side chains of heparan sulfate proteoglycan (HSPG) in various basement membranes (nerve, capillary, oral epithelial, muscle, and dental basement membranes). Four different distribution patterns of stain deposits were identified as heparan sulfate on the basis of enzymatic degradation by heparitinase. In some basement membranes associated with tooth germs and oral epithelium, HID-TCH-SP stain deposits were regularly located at both sides of the lamina densa, but few were observed in the lamina densa itself. In nerve, muscle, and capillary basement membranes, the stain deposits were localized at the external side of the lamina densa adjacent to the underlying connective tissue, but were not found in the laminae lucida and densa. In the internal basal lamina of junctional epithelium of gingiva, the stain deposits were detected mainly in the lamina lucida region. Finally, in some dental and oral epithelial basement membranes, the stain deposits were randomly distributed throughout both laminae lucida and densa. Thus, the present study demonstrated distinct differences in heparan sulfate distribution pattern among various basement membranes, suggesting their architectural heterogeneity.

Membrane modifications in chick osteoclasts revealed by freeze-fracture replicas.

Remarkable differences among various membranes of bone cells became evident by examination of freeze-fracture replicas. In osteoclasts, three types of intramembranous particles (IMPs) were identified based on their size and shape: two sizes of isolated globular particles (8 and 12 nm in diameter) and rod-shaped, linear aggregates (8 x 30 nm in dimension). Furthermore, the density and distribution pattern of these IMPs enabled us to distinguish three different domains of membranes of osteoclasts including ruffled border, clear zone, and basolateral regions, as were also observed in thin sections. The highest density of IMPs was 3,500-4,000/microns2 in the ruffled border membrane, and these IMPs included linear aggregates among the usual globular particles. Linear aggregated particles were also observed in the membrane of cytoplasmic vesicles in the vicinity of the ruffled border region, but not in this membrane in other bone cells. In attached osteoclasts, the distribution patterns and densities of IMPs in each ruffled-finger and -plate were extremely variable, from closely to the loosely packed membrane particles. Focal aggregates of membrane particles were also frequently encountered. An important outcome of the present study was the finding that the presence of linear aggregated particles proved to be an additional criterion for distinguishing membrane domains in freeze-replicas of osteoclasts. The surface of the clear zone membrane was not smooth in profile, but revealed a number of eminences that were almost free of particles. Basolateral membranes exhibited a particle density of 2,400/microns2. Globular particles were homogeneously scattered in random fashion on their exposed fracture faces. In some cases, aggregates of IMPs on the basolateral membranes were encountered. In comparison with the ruffled fingers, microprojections from the basolateral surface showed a lesser density of IMPs and were devoid of rod-shaped or linear aggregated particles. Differences between osteoblasts and osteocytes were apparent in the density and the size of IMPs. The membranes of osteoblasts and osteocytes contained the same types of globular particles as seen in osteoclasts. Various sizes of gap junctions were located only on basolateral membranes of the osteoblasts. In contrast, no cellular junctions were observed between osteoclasts and any other type of cells.

Changes in ultrastructural distribution of dental basement membrane heparan sulfate during early mouse tooth development.

With the invagination of the deep surface of the tooth buds of mouse embryos (15 days of gestation), heparan sulfate in the region of the lamina lucida of the dental basement membrane began to disappear. Subsequently, as the outline of the future dentino-enamel junction, which elaborates the molar cusp pattern, was formed (16-17 days of gestation), heparan sulfate disappeared not only in the lamina lucida but also at the external side of the lamina densa. However, prior to or during predentin matrix deposition (18 days of gestation), heparan sulfate reappeared in the dental basement membrane.

Histochemical localization at the electron microscopic level of sulfated glycosaminoglycans in the rat gingiva.

Using the high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining technique, we investigated ultrastructural localization of sulfated glycosaminoglycans (GAGs) in the rat gingiva shortly after eruption, especially those associated with internal and external basal laminae. In the apical portion of the internal basal lamina, HID-TCH-SP stain deposits were distributed mainly in the region of the lamina lucida located between the lamina densa and the distal surface membrane of the junctional epithelium and inside the depression of the distal surface membrane adjacent to the basal lamina. Stain deposits were also detected on the surface membrane of the cytoplasmic protrusion. Interestingly, the density of HID-TCH-SP stain deposits in the internal basal lamina was highest in the apical portion of the junctional epithelium and decreased in the coronal direction, finally tending to disappear completely. On the other hand, in the external basal lamina the deposits were localized in the whole region of the basal lamina or at both sites of the lamina densa. HID-TCH-SP stain deposits were also detected external to the lamina densa in the basement membrane associated with capillaries and in the connective tissue where they were distributed in close relation to collagen fibrils. Testicular hyaluronidase digested most HID-TCH-SP stain deposits in the connective tissue, whereas those in the region of basement membranes resisted this enzymatic digestion.

Different tartrate sensitivity and pH optimum for two isoenzymes of acid phosphatase in osteoclasts. An electron-microscopic enzyme-cytochemical study.

By differentiation of substrate specificity, pH optimum range, and sensitivity to various inhibitors, 2 isoenzymes of acid phosphatase in bone cells have been studied at the electron-microscopic level. When p-nitrophenyl phosphate was used for the substrate, the demonstrable enzyme activity was affected by neither tartrate nor sodium fluoride. The reaction product, when incubated at pH 5-6, was detected in all sites along the pathway for the biosynthesis of acid phosphatase in the osteoclast, including the perinuclear space, cisternae of the endoplasmic reticulum, Golgi complex, various vesicles, and vacuoles. In the osteoclasts attached to bone, the enzymatic activity was demonstrated at the extracellular ruffled border and on the eroded bone surface. Reaction products became confined to lysosomes and extracellular ruffled border when incubated at pH 6-7. Unattached osteoclasts showed a similar intracytoplasmic localization of enzyme as the attached ones, except for the absence of the extracellular enzyme activity. The mononuclear, immature type of osteoclast also resembled the mature osteoclast in terms of enzymatic localization. Except for the osteoclasts, the acid p-nitrophenyl phosphatase activity was restricted to lysosomal vesicles in various bone cells, monocytes, and macrophages. Such activity was inhibited by adding 50 mM tartrate to the p-nitrophenyl phosphate medium. When beta-glycerophosphate or p-nitrocatechol sulfate was the substrate, most of the reaction product was localized intracellularly. Unlike the acid p-nitrophenyl phosphatase, the acid beta-glycerophosphatase or arylsulfatase activity in osteoclasts and other bone cells was inhibited completely by 10 mM tartrate or 10 mM sodium fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface modifications at the periosseous region of chick osteoclast as revealed by freeze-substitution.

Improved preservation of osteoclast fine structure can be achieved by quick freezing, freeze-substitution, or detergent extraction. With such techniques the ruffled border mainly contains a disorganized, interconnected meshwork of microfilaments (5-7 nm in diameter), whereas in the clear zone a few ordered arrays of intermediate-type filaments (10-12 nm in diameter) are detectable among the network of microfilaments. In well-frozen samples, well-preserved matrix may have occluded the cytoskeleton; detergent extraction permits visualization of the cytoskeletal components. In fresh-frozen cells an extracellular fuzzy coat overlays the ruffled border. At the site of attachment of the clear zone to the bone surface, extracellular cementing material is detected only after quick freezing. The superiority of quick freezing to preserve ultrastructure is shown in various cytoplasmic organelles. Most vesicles and vacuoles found close to the ruffled border seemed not to make contact with the extracellular matrix. Anhydrous procedures using quick freezing and freeze-substitution stabilize bone mineral in some vacuoles and in the channels of the ruffled border.

Ultrastructure of matrix vesicles in chick growth plate as revealed by quick freezing and freeze substitution.

The ultrastructure of extracellular membrane-bound matrix vesicles (MVs), their biogenesis, and the surrounding matrix in chick tibial growth plate were studied after quick freezing and freeze substitution (FS) in an organic solvent. There were several notable differences in the ultrastructural preservation of cartilage when FS was used as compared with conventional fixation. The ultrastructural appearance of MVs after FS was extremely variable. Within the MVs, intravesicular filaments, amorphous material, and membrane-associated undercoat structures were observed. Intravesicular filaments, similar in diameter to microfilaments seen in the cytoplasm, were attached to the inside of MV membranes. This observation indicates the similarity of MV membranes and the plasma membrane. In some MVs in the proliferative zone an electron-dense material was present along the inner side of the MV membrane. In the prehypertrophic zone, crystalline material often appeared within the electron-dense material, which may be a precursor form of hydroxyapatite. The earliest crystals observed were in MVs but not in the extracellular matrix. Regarding MV formation, in addition to budding from cell surfaces and to cellular disintegration, this study also indicates that a sequential process of extrusion of preformed cytoplasmic structures may occur. Also, small MVs measuring 25-40 nm seem to arise from the disruption of large MVs. This is a previously unreported observation on MV biogenesis. FS preserves proteoglycans in the cartilage matrix as a fine, filamentous network. Initial extracellular calcification was not associated with this network.

Ultracytochemical investigation of calcium-activated adenosine triphosphatase (Ca++-ATPase) in chick tibia.

The ultrastructural distribution of Ca++-ATPase in bone cells of growing chick tibia was investigated by a cytochemical method in order to gain insight into possible sites of calcium ion translocation. Both osteoclasts and osteoblasts showed a polar distribution of reaction product along the plasma membrane. In osteoclasts, enzymatic activity occurred along the portion of the plasma membrane facing the marrow but not along the ruffled border or clear zone. The reaction product in these cells was due solely to Ca++-ATPase action. In osteoblasts, the plasma membrane facing away from bone (apical and lateral membrane) was very intensely stained, whereas the basal membrane was unstained. The reaction product in these cells appeared to be the result of both Ca++-ATPase and Ca++,Mg++-ATPase. In osteocytes, no plasma membrane staining was detectable. Mitochondrial staining in all three types of cells was more sensitive to fixation than was the plasma membrane enzyme, suggesting that mitochondrial and plasma membrane Ca++-ATPases are chemically distinct, as biochemical studies have shown. In general, mitochondria in osteoclasts stained more intensely than those in osteoblasts or osteocytes. Mitochondrial and vesicular sites of activity may be related to intracellular calcium storage, whereas calcium ATPases of the plasma membrane are presumed to be involved in calcium efflux from the cells. Calcitonin treatment did not alter the enzymatic distribution or intensity in osteoclasts. The striking polar distribution of both osteoclast and osteoblast plasma-membrane activity suggests that directional calcium pumping by these cells may be of importance in bone-forming and bone-resorbing mechanisms.

Improved ultrastructural preservation of epiphyseal chondrocytes by the freeze-substitution method.

The ultrastructure of epiphyseal chondrocytes was studied following quick-freezing and freeze-substitution, and was compared to that of cells fixed with aqueous aldehydes. The former approach provided an improved ultrastructural preservation whereby every type of chondrocyte exhibited a smoother cell contour. The plasma membrane as well as intracytoplasmic membranes revealed a trilaminar substructure. The intracytoplasmic ground substance was composed of flocculent materials which were in direct contact with the inner leaflet of the plasma membrane. Within the extracellular matrix the proteoglycan network adhered to the outer leaflet of the plasma membrane. Whenever cellular shrinkage took place, the flocculent matrix within the cytoplasm and the proteoglycan network in the pericellular matrix disappeared. The contents of the RER, the Golgi apparatus, and the intracellular vesicles and vacuoles were well retained. In the proliferative zone, the Golgi saccules of young cells contained a thread-like structure showing a clear periodicity. The cytoplasmic vesicles and vacuoles showed marked variation in their electron density. Intramitochondrial granules were sensitive to aqueous treatments, as evidenced by the observation that they disappeared after either floating on water or staining with aqueous solution. In the calcifying zone, mitochondrial granules were noted within hypertrophic chondrocytes, a feature that was not observed following conventional processing. Cytoskeletal elements were well preserved in all types of cells. A dense microfilamentous network occupied the pericellular cytoplasm. Bundles of microfilaments were seen in the cellular peripheral processes. Microtubules were distributed throughout the cytoplasm, and the Golgi complex was intimately associated with the microtubule network; it appears that the secretory processes are involved with the microtubules.

Ultrastructural observations on chick bone processed by quick-freezing and freeze-substitution.

The ultrastructure of newly formed bone was examined with the use of quick-freezing followed by freeze-substitution. Osteoblasts and young osteocytes were characterized by a smooth cell contour, whereas old osteocytes were irregular in shape. The plasma and intracytoplasmic membranes were clearly identifiable as trilaminar substructures. With the method described herein the tissue is handled in the anhydrous state. Thus mitochondrial granules could be demonstrated in all samples, since their preservation is not affected by non-aqueous solutions. The matrices of intact mitochondria were densely stained with post-staining. The contents of the Golgi complex, rough-surfaced endoplasmic reticulum (RER), nuclear envelope, vesicles, and vacuoles were stained to various degrees. Lacunar spaces were always filled with flocculent and filamentous materials, and the plasma membrane was in direct contact with them. Membrane-bounded matrix vesicles were clearly visible within the osteoid extracellular matrix which was the initial site of mineral crystal deposition. In heavily mineralized bone matrix, the periodic pattern of collagen fibrils was retained, and the electron density of mineralized matrix in freeze-substituted and unstained sections which had been floated on ethylene glycol was greater than that encountered in sections processed in aqueous reagents.

The distribution of muscle primary afferents from the masseter nerve to the trigeminal sensory nuclei.

Transganglionic transport of horseradish peroxidase--wheat germ agglutinin conjugate was used to study the pattern of termination of somatic afferent fibers innervating the masseter muscle within the trigeminal sensory nuclear complex (TSNC) of the cat. The central processes of the masseteric nerve terminated in the caudal third of the pars interpolaris, and laminae I/V through the caudal two-thirds of caudalis and rostral parts of the C1 spinal cord segment. The functional significance of the masseteric afferent projections to the TSNC with a preferential pattern was discussed, particularly with respect to muscle pain.