Relationships between tooth eruption, occlusion and alveolar bone resorption: histochemical and cytological studies of bone remodeling on rat incisor alveolar bone facing the enamel after root resection

The labial side of rat incisor alveolar bone facing the enamel is continuously resorbed as the result of compressive force produced by the occlusion and eruption of incisors. In order to clarify the relationship between this mechanical compressive force and the bone cells involved in bone remodeling, we examined morphological changes occurring in the alveolar bone once the compressive force was eliminated by the removal of the proliferative odontogenic base (root resection according to Berkovitz and Thomas, 1969). After root resection, the incisor migrated halfway along the socket. On the crestal part where incisor still existed, active osteoclasts were prominent on the bone surface, and flattened mononuclear cells lay close upon active osteoclasts. Sinusoidal blood vessels or capillaries were observed at short distances from the bone surface. On the basal part where socket was vacant, osteoblasts lined up on the newly formed bone, and the osteogenic cell layer lay on the osteoblasts. Between the two parts, which correspond to the reversal phase proposed by Baron (1977), osteoblastic cells with developed cell organelles increased in number and the distance between blood vessels and bone surface increased. Osteoclasts reduced their activities, and osteoblastic cells often wedged themselves between the osteoclasts and bone surface. These findings indicate that the elimination of compressive force mediated by incisors leads to the activation of osteoblastic cells and inactivation of osteoclasts, which results in a conversion from bone resorption to bone formation. Thus, osteoblastic cells may play an important role in controlling osteoclastic activity in conversion from bone resorption to bone formation, partly by a direct effect and partly by controlling the access of blood vessels to the bone surface.     

Some osteocytes released from their lacunae are embedded again in the bone and not engulfed by osteoclasts during bone remodeling

It is generally accepted that osteocytes derive from osteoblasts that have secreted the bone around themselves. Osteocytes are cells embedded in the lacunae in the bone, and they are characteristically in contact with other cells by many slender cytoplasmic processes in canaliculi. During bone remodeling, many osteocytes in the bone are released from their lacunae by osteoclasts; however it remains unclear what happens to these released osteocytes. The cortical bone of the rat mandibular body was used in this study. Mandibles were fixed, decalcified, and then embedded in Epon 812. Specimens were sectioned in the frontal direction into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and then examined by light or transmission electron microscopy. Cells that fitted in the osteocytic lacunae with canaliculi extending to the bone were identified as osteocytes in this study. Among many osteocytes released by osteoclasts in cutting cones, there were osteocytes half-released from their lacunae. These cells fitted in their lacunae with canaliculi extending to the bone and showed developed cell organelles in the cytoplasm. In closing cones, many osteocytes were situated in the bone away from cement lines; however, there were half-embedded osteocytes in the bone formed on cement lines. These cells fitted in their lacunae with canaliculi extending to the bone formed below cement lines and showed developed cell organelles in the cytoplasm. These results show that half-embedded osteocytes in closing cones derive from half-released osteocytes in cutting cones. Osteocytes encircled by osteoclasts were sometimes observed on one section, but serial sections showed that these osteocytes fitted in their remaining lacunae in the bone on other sections. This shows that not all osteocytes released from their lacunae are engulfed by osteoclasts. Consequently, the present results suggests that some osteocytes released from their lacunae are embedded again in the bone and not engulfed by osteoclasts during bone remodeling.     

Morphological relationships between osteoclasts and bone resorption surfaces on mouse parietal bones

Parietal bones from mice 1-20 weeks of age were histochemically stained for detection of acid-phosphatase activity and then observed by the light microscope to evaluate the distribution and shape of osteoclasts on the inner surface of their bones. After microscopic examination, the same bones were macerated by NaOCl to both remove organic materials and expose the mineralized surface. The inner surface was then examined by scanning electron microscopy and the observations were compared with the light micrographs of the areas where osteoclasts were located. The bone resorption areas were identified as well-demarcated rough areas, and corresponded to the areas where osteoclasts were distributed. In young mice, osteoclasts observed in the bone resorption areas, which were composed of accumulations of irregular concavities, were mainly polygonal or round in shape. In adult mice, elongated osteoclasts with longer or shorter cytoplasmic processes were predominant; the bone concavities were also elongated and gathered in a flame-like pattern. The findings suggest that osteoclasts change shape according to their resorptive activities and that the activities differ between growing bones and those where growth has ceased, probably in relation to the modeling and remodeling of the bone.     

Two types of bone resorption lacunae in the mouse parietal bones as revealed by scanning electron microscopy and histochemistry

To understand the bone resorption process on the basis of the morphology of bone resorption lacunae, the inner surface of parietal bones in juvenile mice was exposed with a treatment of ultrasonic waves or NaOCl treatment and examined by scanning electron microscopy (SEM). The bone resorption lacunae were divided into two types (I and II) according to differences in morphological features of their walls; the wall of type I lacunae was covered with loose collagen fibrils, while that of type II lacunae was smooth with almost no fibrillar structures. Collagen fibrils in type I lacunae treated with ultrasonic waves differed in appearance from those treated with NaOCl; the collagen fibrils were thin and displayed a smooth surface in type I lacunae treated with ultrasonic waves, while they were thick and showed a rough surface in those treated with NaOCl-probably because superficial uncalcified collagen fibrils were digested with the chemical. The results indicated that type I lacunae occupied 77% of all of the bone resorption lacunae treated with ultrasonic waves, but 51% of those treated with NaOCl. This finding led to the idea that type I lacunae can be subdivided into two: lacunae (Ia), covered with partially calcified fibrils as well as superficial uncalcified fibrils; and lacunae (Ib), covered only with uncalcified fibrils. The presence of uncalcified fibrils in the bone resorption lacunae was further confirmed by backscattered electron (BSE) imaging of SEM. Histochemistry for acid phosphatase or immuno-histochemistry for cathepsin B or carbonic anhydrase in combination with SEM revealed that type I lacunae were located under osteoclasts but type II lacunae were not. These findings indicate that type I lacunae are in the process of bone resorption by osteoclasts, while type II lacunae are in the final stage of bone resorption and free from osteoclasts. Bone resorption may thus proceed in the order of Ia, Ib, and II.     

成人牙根唇舌侧牙槽骨的厚度

背景：牙根在牙槽骨的位置及周围骨板厚度影响着口腔治疗,治疗过程中如果对牙齿控制不当可造成医源性并发症。以往对颌骨的研究主要针对解剖学、骨厚度或骨密度,对于牙根在牙槽骨内的空间位置及其与周围骨骼的关系,研究关注较少。 目的：建立颌骨的数字化计算机三维模型,测量牙根的唇舌侧牙槽骨厚度。 方法：选择牙列完整无明显骨骼吸收的年轻成人70例,采用牙科专用锥形束CT机进行颌面部扫描,将扫描中采集的容积信息传入计算机工作站,以及冠状位或矢状位多平面重建,获得高质量的重建图像,原始数据以DICOM格式导入计算机,并输出到整合的3D设计软件Invivo5软件进行测量。 结果与结论：重建的颌骨数字化模型可从多平面进行观察及测量,实验测得70 例患者各个牙根唇舌侧牙槽骨厚度的均值：上下前牙舌侧牙槽骨厚度大于唇侧(P < 0.05);除上前磨牙的牙颈部唇侧牙槽骨较厚外, 其他前磨牙舌侧牙槽骨厚度大于唇侧(P < 0.05);上磨牙和下颌第一磨牙唇舌侧牙槽骨厚度接近,下第二磨牙唇侧牙槽骨厚度大于舌侧(P < 0.01)。结果证实,成人不同牙位的唇舌侧牙槽骨厚度差异较大。     

Disaggregated osteoclasts increase in resorption activity in response to roughness of bone surface.

The roughness of the bone matrix surface affects osteoblastic differentiation. However, the effect of the roughness of the matrix surface on osteoclastic bone resorption remains to be studied. We examined the latter effect using disaggregated osteoclasts from neonatal rats. The resorption pit number and the total pit area on the rough surface were not different from those on smooth surfaces after 1 day, but they were 2 or more times higher after 3 days. The number of osteoclasts was not different on bone slices with either smooth or rough surfaces at 3 days. The alkaline phosphatase (ALP)-positive osteoblasts were relatively rare in both types of slices at first, then the number and the diameter of the enzyme-positive cells and the clusters preferentially increased on the rough bone slices. When hydroxyurea was added to the culture in order to suppress the proliferation and the subsequent differentiation of osteoblastic cells on rough surfaces, the increase in resorption on the rough surfaces was effaced; however, this agent had little affect on resorption of the smooth surfaces. The addition of ALP-positive cells to disaggregated osteoclasts increased bone resorption on the smooth surface. The results suggest that osteoblast development and subsequently bone resorption by osteoclasts is enhanced by the roughness of matrix surfaces.     

Surface and cross-sectional comparisons of bone growth remodeling

Broad, regional surface and cross-sectional assessments of growth remodeling in the macaque and human lower limb bones were compared using thin sections, stereomicroscopy and scanning election microscopy. In general, areas identified by cortical tissue type in thin sections as depository showed smooth surfaces under the SEM. Areas identified as resorptive in cross section always showed resorption lacunae with the SEM. Actively forming bone surfaces as described by previous SEM studies were not encountered even though rapidly forming cortical tissues were encountered with the light microscope. These results are consistent both for fresh and exhumed bone.     

Identification of cell types responsible for bone resorption in rheumatoid arthritis and juvenile rheumatoid arthritis.

Focal resorption of bone at the bone-pannus interface is common in rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA) and can result in significant morbidity. However, the specific cellular and hormonal mechanisms involved in this process are not well established. We examined tissue sections from areas of bone erosion in patients with RA and JRA. Multinucleated cells (MNCs) were present in resorption lacunae in areas of calcified cartilage and in subchondral bone immediately adjacent to calcified cartilage, as previously described. mRNA for the calcitonin receptor (CTR) was localized to these MNCs in bone resorption lacunae, a finding that definitively identifies these cells as osteoclasts. These MNCs were also positive for tartrate-resistant acid phosphatase (TRAP) mRNA and TRAP enzymatic activity. Occasional mononuclear cells on the bone surface were also CTR positive. Mononuclear cells and MNCs not on bone surfaces were CTR negative. The restriction of CTR-positive cells to the surface of mineralized tissues suggests that bone and/or calcified cartilage provide signals that are critical for the differentiation of hematopoietic osteoclast precursors to fully differentiated osteoclasts. Some MNCs and mononuclear cells off bone and within invading tissues were TRAP positive. These cells likely represent the precursors of the CTR-TRAP-positive cells on bone. Parathyroid hormone receptor mRNA was present in cells with the phenotypic appearance of osteoblasts, in close proximity to MNCs, and in occasional cells within pannus tissue, but not in the MNCs in bone resorption lacunae. These findings demonstrate that osteoclasts within the rheumatoid lesion do not express parathyroid hormone receptor. In conclusion, the resorbing cells in RA exhibit a definitive osteoclastic phenotype, suggesting that pharmacological agents that inhibit osteoclast recruitment or activity are rational targets for blocking focal bone erosion in patients with RA and JRA.     

Ultrastructure of alveolar bone during tooth eruption in the dog

Previous work from our laboratories has established that eruption of the permanent mandibular premolars in dogs is dependent upon the presence of the dental follicle and that it involves resorption of alveolar bone and the roots of the deciduous predecessor above and formation of alveolar bone below the developing crown. This study illustrates the topography of the bone surfaces of the crypt by scanning electron microscopy and the ultrastructure of the cells on alveolar bone surfaces during tooth eruption. Above the developing crown where the eruption pathway forms, the bone surface is a pitted sheet, the characteristic topographic feature of bone resorption; between the crown and the mandibular canal, the bone surface has numerous interconnecting trabeculae. Transmission electron microscopy of bone cells lining the eruption pathway area of the crypt showed numerous osteoclasts with adjacent mononuclear cells. Both cell types contained specific, membrane-bound cytoplasmic vesicles shown by the work of others to be characteristic features of osteoclasts and their precursors. Basal trabeculated bone in the crypt was covered by plump osteoblasts. These data show that the metabolic events in alveolar bone associated with tooth eruption have the appropriate cellular and bony surface correlates and that the suspected control of alveolar bone resorption by the dental follicle may be mediated by its recruiting and directing to adjacent bone surfaces the mononuclear precursors of osteoclasts.     

In vitro bone resorption by isolated multinucleated giant cells from giant cell tumour of bone: Light and electron microscopic study

Summary The behaviour of multinucleated giant cells (GCs), obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized bone slices, was studied using light and electron microscopy. Monitoring the GCs on bone slices by phase-contrast microscopy revealed that they had removed calcified bone matrix resulting in excavation of lacunae, with subsequent lateral extension and perforation of the bone slices. Electron microscopy demonstrated for the first time that the GCs responsible for exavating lacunae had two specific membrane modifications, ruffled border and clear zone, and showed basically similar cytoplasmic fine structures to those of osteoclasts. Fluorescence images of the GCs on glass and on bone after rhodamine-conjugated phalloidin staining revealed that most of the GCs had an intensely fluorescent peripheral band composed of a number of F-actin dots called podosomes. Some GCs showed unusual arrangements of podosomes suggesting abortive attempts at GC formation. We have demonstrated that the band structure of the GCs cultured on bone is intimately involved in bone resorption. Two stromal cell types could be recognized. The predominant type, which seemed to be the only neoplastic element because of its proliferative capability, showed quite different fine structural and cytoskeletal features from the GCs. The other type, which was much less frequent and seemed not to proliferate, had morphological similarities to the GCs, and seemed to be their precursor. Importantly GCs cultured on bone and the osteoclasts share common structures for adhesion to and resorption of bone, strongly supporting the view that the GCs of the giant cell tumour of bone are potentially active bone resorbers and can be regarded as osteoclasts.     

Growth and the Modeling/Remodeling of the Alveolar Bone of the Rat Incisor

The modeling and remodeling of the rat incisor alveolar bone was followed as the animals grew. The weight of the hemimandible, the length of the socket, and the width of the lower incisor were measured. Osteoclasts and resorption areas were identified by tartrate‐resistant acid phosphatase staining. Fluorochrome markers were used to detect and measure osteogenic activities. In the socket related to the periodontal ligament, osteoclasts appeared in scattered sites as well as isolated sites of osteogenic activity, apparently without any variation related to the age of the animals. At the socket facing the dental follicle of young rats, the inner surface was lined with osteoclasts. The number of osteoclasts decreased steadily as the rats grew. In 1‐year‐old rats, in addition to a few scattered osteoclasts, the internal aspect of the labial wall showed some sites lined with osteoblasts and cement lines indicative of prior bone formation. In young rats, there was a continuous osteogenic activity at the external surface of this wall. The thickness of the labial wall of the socket remained apparently constant; therefore, bone resorption must have occurred at the internal side of the wall. Such osteogenic activity was not observed in old rats. The main forces acting on rat incisors, biting and eruption, are continuous through the life of the animals. Thus, these results indicate that the modeling of the alveolar bone related to the dental follicle, in young rats, can only be associated with another force, specifically, the growth of the incisor. Anat Rec, 291:827‐834, 2008. © 2008 Wiley‐Liss, Inc.     

First histological observations on the incorporation of a novel calcium phosphate bone substitute material in human cancellous bone

Calcium phosphates are frequently used as bone substitute materials because of their similarity to the mineral phase of bone, absence of antigenicity, and excellent osteoconductivity. However, in most currently available mineral substitutes, resorption occurs slowly if at all. In contrast, calcium phosphate cements have shown rapid resorption and remodeling in animal studies. In two prospective studies, a novel amorphous calcium phosphate cement (Biobon) was implanted in human patients for the first time. After 2-12 months, ten biopsies were obtained from nine individuals during secondary surgical interventions, for example, for implant removal. In all specimens, partial replacement of the material by new bone was observed, while residues of the cement were still visible. Undecalcified sections revealed extensive bone formation in immediate contact to the cement without fibrous interface. Polynucleated cells and superficial lacunae were indicative of resorptive activity, but inflammatory tissue response was absent. The new bone displayed regular trabecular and osteonal patterns. The histologic findings are in accordance with the excellent biocompatibility observed in the clinical follow-up. Though still incomplete, the resorbability of this cement appears superior to sintered calcium phosphates in these biopsy specimens. Presumably this is due to its amorphous crystalline structure. Biobon merits further studies as a promising substance for bone defect reconstruction in non-stress-bearing areas.     

叉头框蛋白O1在正畸力介导的牙槽骨改建中的表达改变

目的观察大鼠正畸牙移动牙槽骨的改建及叉头框蛋白O1(forkhead box O1,FOXO1)的表达改变,初步探讨FOXO1在正畸力介导的牙槽骨改建中的作用。方法构建大鼠牙移动模型,左侧上颌第1磨牙在50 g力作用下近中移动。分别于牙移动1、3、7 d处死大鼠。通过HE染色及免疫组织化学法观察牙移动不同时间点第1磨牙根分叉区牙槽骨的改建及FOXO1的表达水平。结果正畸力作用后,大鼠上颌第1磨牙不断近中移动。牙移动组根分叉区牙槽骨内破骨细胞较对照组明显增加,牙移动第3 d时破骨活性最强。加载正畸力后,根分叉区牙槽骨内活跃的成骨细胞逐渐增加,成骨活性增强;根分叉区牙槽骨内FOXO1主要表达于成骨细胞内,且随着成骨细胞的增加,FOXO1的表达逐渐增强。结论正畸力介导了根分叉区牙槽骨的改建,FOXO1的表达改变可能与正畸牙移动骨改建相关。     

The possible role of the fibroblast in granuloma-induced bone resorption in the rat.

Granulomas were produced over the calvaria of rats by injection of a mixture of turpentine and peanut oil. The granulomas induced localised areas of bone resorption and these resorptive lesions were examined at the fine structural level. A close association was found between osteoclasts and fibroblasts at resorbing surfaces. Furthermore, fibroblasts but not osteoclasts were observed to engulf and phagocytose bone collagen fibrils. It is suggested that the fibroblast either alone or in collaboration with the osteoclast is involved in the destruction of bone collagen during chronic inflammatory disorder of bone.     

Quantitative determination of alveolar bone density using digital image analysis of microradiographs

Horizontal 100 microns ground sections of 20 alveolar bone specimens from adult human mandibles obtained from autopsies were prepared for microradiography. Quantitative analysis of bone density of the alveolar cortex was performed using a semiautomatic digital image analysis system (KONTRON). The results demonstrated that bone density was higher in the lingual than in the labial alveolar cortex (p less than 0.05). In addition, the coronal portion of alveolar cortical bone was significantly more porous than the medical and apical ones (p less than 0.05). These variances were primarily due to increased canal size rather than to an increased number of canals. No significant age dependent changes in bone density could be determined.     

Transmission and scanning electron microscope studies of calcified cartilage resorption

The authors' previous report (Savostin-Asling and Asling, 1973) demonstrated that Meckel's cartilage is a favorable site for study of calcified cartilage resorption. In the present study the ultrastructural features at this resorption front have been examined by transmission and scanning electron microscopes (19-day rat fetus). Multinucleated giant cells (chondroclasts) dominated the erosion front. The many features which they showed in common with osteoclasts included abundant mitochondria, vacuolation, lysosomes, sparsity of roughsurfaced endoplasmic reticulum, and deep infoldings at loci of contact with calcified matrix. Crumbling of matrix (with mineral crystals penetrating between these foldings) and fragmentation of collagen fibrils were also seen. The propensity of chondroclasts for spanning several opened lacunae provided special opportunity to demonstrate cell surface modifications in presence or absence of matrix contact. Ameboid processes extending into lacunae were seen by both transmission and scanning procedures; they were sometimes tipped with a veil of filamentous processes as small as 0.3 μm in diameter. Most hypertrophic chondrocytes, when released from lacunae, appeared to be disintegrating. However, in accord with previous evidence of their possible merger with chondroclasts (in light microscopic studies) there was also evidence for breakdown of cell walls between a chondroclast and a chondrocyte in intimate contact, with possibility of cytoplasmic continuity.     

Acute effects of ovariectomy on wound healing of alveolar bone after maxillary molar extraction in aged rats

Acute effects of ovariectomy on the bone wound healing processes after maxillary molar extraction in aged rats were examined by means of quantitative scanning electron microscopy (SEM), backscattered electron image (BSE) analysis and energy-dispersive X-ray (EDX) microanalysis. Six-month-old female rats underwent either sham operation or bilateral ovariectomy, and 7 days postoperatively, the maxillary first molars were extracted. On post-extraction days 7, 30 and 60, the dissected maxillary bone surfaces were examined by SEM to reveal the bone formative and resorptive areas around the extracted alveolar sockets. In addition, the resin-embedded maxillae were micromilled in the transverse direction through the extracted alveolar sockets, and the newly-formed bone mass on the buccal bone surfaces and within the extracted sockets was examined by BSE analysis. Compared with sham-operated controls, the extent of newly-formed bone mass on the buccal bone surfaces in OVX rats was significantly decreased, due to increased bone resorption. On the other hand, new bone formation within the extracted sockets was similar in the experimental groups. In EDX microanalysis of these newly-formed bone matrices, both Ca and P weight % and Ca/P molar ratio were similar in the experimental groups. Our results suggest that 1) acute estrogen deficiency induced by ovariectomy stimulates sustained bone resorption, but has less effect on bone formation, and 2) bone wound healing after maxillary molar extraction within extracted alveolar sockets is not significantly delayed by ovariectomy, but bony support by newly-formed bone mass on the maxillary bone surfaces at the buccal side of the extracted sockets is significantly decreased, due to increased bone resorption.     

Transmission and scanning electron microscope studies of calcified cartilage resorption.

The authors' previous report (Savostin-Asling and Asling, '73) demonstrated that Meckel's carilage is a favorable site for study of calcified cartilage resorption. In the present study the ultrastructural features at this resorption front have been examined by transmission and scanning electron microscopes (19-day rat retus). Multinucleated giant cells chondroclasts) dominated the erosion front. The many features which they showed in common with osteoclasts included abundant mitochondria, vacuolation, lysonsomes, sparsity of rough-sufaced endoplasmic reticulum, and deep infoldings at loci of contact with calcified matrix. Crumbling of matrix (with mineral crystals penetrating between these foldings) and fragmentation of collagen fibrils were also seen. The propensity of chondroclasts for spanning several opened lacunae provided special opportunity to demonstrate cell surface modifications in presence or absence of matrix contact. Amebiod processes extending into lacunae were seen by both transmission and scanning procedures; they were sometimes tipped with a veil of filamentous processes as small as 0.3 mum in diameter. Most hypertrophic chondrocytes. when released from lacunae, appeared to be disintegrating. However, in accord with previous evidence of their possible merger with chondroclasts (in light microscopic studies) there was also evidence for breakdown of cell walls between a chondroclast and a chondrocyte in intimate contact, with possibility of cytoplasmic continuity.     

Effect of mineral content of human bone on in vitro resorption

Summary Osteoclasts, mechanically isolated from chick long bones, were grown in vitro on slices of human rib and femur. Evidence of their activity was assessed by secondary electron and backscattered electron (BSE) imaging in the SEM. BSE imaging was also used to study the relative degree of mineralisation of the bone matrix in which resorption had taken place. All bone phases were resorbed, from osteoid through to densely mineralised interstitial bone and reversal (cement) lines. Resorbing osteoclasts crossed reversal lines between osteons of different mineral density and moved both from higher to lower and lower to higher density phases. Where single loci spanned reversal lines, and thus breached bone of two different mineral densities, depth of demineralisation was inversely related to mineral density. The presence of an annular zone around some resorption loci, which may be caused by demineralisation beneath the osteoclast clear zone, was confirmed. Also, BSE imaging of polished substrata showed that significantly more osteoclastic activity had occurred at their surfaces than was apparent from the amount of cavitation present.