The influence of parathyroid hormone-related protein (PTHrP) on tooth-germ development and osteoclastogenesis in alveolar bone of PTHrP-knock out and wild-type mice in vitro

In a previous study, it was shown that tooth germs of neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are penetrated or compressed by the surrounding alveolar bone, suggesting an important role for PTHrP in the formation and activation of osteoclasts around growing tooth germs. In order to elucidate the role of PTHrP during the development of the tooth germ and related structures, mandibular explants containing cap stage tooth germs of embryonic day 14, homozygous mice were here cultured with or without surrounding alveolar bone. There was no difference in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells around the first molars of homozygous and wild-type mice. After 10 days of culture, osteoclastic cells were rarely present in explants from homozygous mice and penetration of alveolar bone into the dental papilla was observed. The decline in osteoclast number was partly restored by the addition of PTHrP to the culture. Tooth germs of both wild-type and homozygous mice cultured without alveolar bone developed well, with no apparent structural abnormality; dentine formation was evident after 10 days. These data suggest that PTHrP is not required for the development of the tooth germ proper but is indispensable in promoting the osteoclast formation required to accommodate that development.     

Comparative effects of parathyroid hormone on osteoblasts and cementoblasts

Although bone, dentin and dental cementum are mesenchymal mineralized tissues composed mainly of collagen and hydroxy apatite, they differ markedly in their suceptibility to resorption. Bone undergoes physiological resorption to which the dental tissues appear to be resistant. Recently, the cells covering bone surfaces have been attributed a regulatory role in osteoclastic bone resorption by exposing the bone surface following stimulation with hormones and inflammatory mediators, thereby allowing osteoclasts to colonize the bone surface. In the present study, it was shown that reparative cementum-forming cells covering an experimental cavity in the root surface of replanted monkey incisors were unaffected by parathyroid hormone, a potent mediator of bone resorption. In parallel experiments, endocranial osteoblasts exposed bone surface by widening their inercellular spaces. It was concluded that the layer of cells covering the root surface forms a protective barrier against resorption which serves to preserve the integrity of the dental root.     

Immunohistochemical localization of parathyroid hormone-related protein in developing mouse Meckel''s cartilage and mandible

In order to clarify the role of parathyroid hormone-related protein (PTHrP) during Meckel's cartilage and mandibular development, an immunohistochemical study of PTHrP and its receptor, PTH/PTHrP receptor, was designed to examine their localization in the anterior region of Meckel's cartilage including the rostrum, which is known to contribute to the development of the mandible. Meckel's cartilage was first observed on day 13 of gestation and PTHrP was faintly localized in the chondrocytes. On day 16 of gestation, at the stage of elongation and initiation of endochondral ossificastitial in Meckel's cartilage, PTHrP was localized in the chondrocytes located in the area showing interstitial growth and in and around the nuclei of hypertrophic chondrocytes undergoing endochondral ossification. At day 18 of gestation, endochondral ossification was spread over the entire area proximal to the molar region in Meckel's cartilage, except in the mesial fusion site formed by immature chondrocytes. PTHrP was localized in the osteoblasts adjacent to the calcified matrix, but had disappeared from the chondrocytes forming Meckel's cartilage. The localization of PTH/PTHrP receptor was similar to that of PTHrP. These results show that localization of PTHrP is spatially and temporally related to the growth of Meckel's cartilage.     

前伸大鼠下颌后髁突局部甲状旁腺激素相关蛋白含量的季节性变化规律

目的 :探讨不同季节给生长发育期大鼠戴用功能矫治器后 ,其髁突局部的甲状旁腺激素相关蛋白 (PTHrP)含量的季节性变化规律。方法 :选用SD大鼠 ,采用免疫放射法计测大鼠髁突局部甲状旁腺激素相关蛋白含量的变化 ,应用宏观、微观分析法进行统计分析。结果 :生长发育期的大鼠在自然状态下 ,其髁突局部甲状旁腺激素相关蛋白含量存在着季节性变化规律。戴用功能矫治器后 ,其髁突局部甲状旁腺激素相关蛋白含量的季节性变化规律仍存在 ,冬末春初含量最高 ,夏季最低。结论 :季节变化对功能矫形治疗有影响。     

牛成牙骨质细胞在体内形成牙骨质样组织的初步研究

目的：培养成牙骨质细胞（cementoblasts,CBs)，观察以CBs自身作为组织工程支架在裸鼠内形成牙骨质的可行性。方法：在矿化培养液中培养新生牛的成牙骨质细胞，使其形成多层CBs作为载体（CBs-made carrier)与CBs混合，植入裸鼠皮下。以CBs与胶原膜载体（collagen carrier) 复合体作为对照。7周后取材并做HE、von Kossa、茜素红及牙骨质附着蛋白单克隆抗体免疫组化染色。结果：CBs在体内可形成牙骨质样矿化组织，中心矿化程度较高，四周有尚未钙化的类牙骨质及CBs包绕，并有牙骨质细胞埋入矿化组织。以CBs自身作为载体所形成的牙骨质较以胶原膜作为载体所形成的牙骨质样组织多。结论：本组分离培养的CBs在体内可形成牙骨质样矿化组织；以多层细胞自身作为载体是一种较理想的生物支架材料。     

功能矫治前伸大鼠下颌髁突中甲状旁腺相关蛋白(PTHrP)基因表达的季节性变化规律

目的探讨生长发育期大鼠下颌髁突软骨中甲状旁腺相关蛋白mRNA的表达特征及其季节性变化规律。方法采用SD大鼠，应用原位杂交技术检测大鼠下颌髁突软骨中甲状旁腺相关蛋白的mRNA表达，图像分析半定量研究甲状旁腺相关蛋白mRNA的季节性变化规律，应用宏观、微观分析法进行统计分析。结果髁突软骨细胞中有甲状旁腺相关蛋白mRNA的表达，并有季节性变化规律；戴用功能矫治器后其表达信号增强。一月中旬到二月初增强的最明显。结论功能矫形治疗受季节变化影响，模拟功能矫形治疗的生长发育期大鼠在二月份左右戴用矫治器矫治效果更明显。     

Distribution of parathyroid hormone-related protein (PTHrP) and type I parathyroid hormone (PTH) PTHrP receptor in developing mouse mandibular condylar cartilage.

The mandibular condylar cartilage undergoes endochondral bone formation and is an important growth site in the mandible. Parathyroid hormone-related protein (PTHrP) has received attention as a physiological regulator attenuating chondrocytic differentiation and preventing apoptotic cell death. In order to examine the localization of PTHrP and its receptor during fetal development of the condylar cartilage, an immunohistochemical study of PTHrP and the type I PTH/PTHrP receptor was carried out. At day 15 of gestation, the condylar cartilage was evident and some chondrocytes showed positive staining for PTHrP. At day 16, the cartilage was increasing in length and width, and PTHrP was localized in the flattened and hypertrophic cell layers. After day 17, when endochondral bone formation had already started, PTHrP was mainly observed in the flattened cell layer and in a few layers of the hypertrophic chondrocytes. The localization of the type I PTH/PTHrP receptor was similar to that of PTHrP on days 15 and 16, and was broadly distributed at day 18. Apoptotic chondrocytes were scarcely observed on days 15 and 16, and only a few cells were present in the erosion front at day 18. This temporal and spatial localization of PTHrP and the type I PTH/PTHrP receptor suggests that PTHrP is a possible autocrine/ paracrine factor regulating condylar chondrocytic differentiation during development.     

Chronological gene expression of parathyroid hormone-related protein (PTHrP) in the stellate reticulum of the rat: implications for tooth eruption

OBJECTIVE: Tooth eruption is a localized event that requires the expression of certain molecules at precise times to regulate bone resorption and bone formation. Parathyroid hormone-related protein (PTHrP) may be one of those molecules. Although PTHrP is produced in the stellate reticulum (SR) of the tooth and exerts its effect on the adjacent dental follicle, its expression pattern in the SR is unknown. Thus, it was the objectives of this study to determine the chronology of expression of PTHrP, and then to determine its effect on vascular endothelial growth factor (VEGF) expression for osteoclastogenesis and on bone morphogenetic protein-2 (BMP-2) for bone growth. DESIGN: Laser capture microdissection and RT-PCR were used to determine the chronological expression of PTHrP in vivo. In vitro, dental follicle cells were incubated with PTHrP and RT-PCR was conducted to determine its effect on VEGF and BMP-2 gene expression. RESULTS: PTHrP was maximally expressed at day 7 postnatally in the SR with the level of expression still high at day 9. In vitro, PTHrP upregulated VEGF120 and VEGF164 expression after 4h of incubation with a maximum effect at 6h. PTHrP upregulated BMP-2 gene expression with a maximal effect at 2h. CONCLUSIONS: Because the secondary burst of osteoclastogenesis needed for eruption occurs around day 10, it is possible that PTHrP is stimulating this osteoclastogenesis by upregulating VEGF. Concurrently, the upregulation of BMP-2 by PTHrP may stimulate bone growth at the base of the bony crypt to promote eruption.     

Immunohistochemical localization of parathyroid hormone-related protein (PTHrP) and serum PTHrP in normocalcemic patients with oral squamous cell carcinoma

Cancer cells produce parathyroid hormone-related protein (PTHrP) in the early phase of malignancy development, before hypercalcemia occurs. The relationship between PTHrP and the clinicopathologic features of oral squamous cell carcinoma is poorly understood. We studied 60 patients (43 men, 17 women; mean age, 64.8 ± 11.2 years) with primary oral squamous cell carcinoma, from whom pretreatment biopsy specimens were obtained. We examined the relationship among immunohistochemical PTHrP expression, serum PTHrP levels, clinical characteristics of the tumor, and histopathologic aspects of the tumor. The mean calcium concentration for the 60 patients was 9.1 ± 0.4 mg/dl. No patients had laboratory evidence of hypercalcemia before treatment. Six patients had serum levels of C-terminal (C)-PTHrP higher than the normal level of 55.3 pmol/l. There were no significant differences in serum C-PTHrP levels according to TNM stages. Abundant positive immunoreactivity for anti-PTHrP (1-34) antibody was recognized diffusely in the whole cytoplasm of many tumor cells. Anti-PTHrP (38-64) antibody staining tended to localize as small granules in the cytoplasm, especially close to the nuclear periphery. There was no correlation between the serum C-PTHrP concentration and the intensity of either immunostain. The intensity of PTHrP was proportionally related to the degree of differentiation or extent of keratinization (P < 0.05) and the histologic malignancy grade of the tumor (P < 0.05), when using antibody against PTHrP (1-34), but not when using antibody against PTHrP (38-64). Serum C-PTHrP levels did not correlate with the intensity of cellular PTHrP expression and characteristics of the tumor at the initial patient visit. The fragment that includes PTHrP (1-34) may be involved in the differentiation of oral squamous cell carcinoma. The differences between immunoreactivities may have been due to differing tissue malignancies and the use of different antibodies. The results suggest the need for caution when interpreting immunoreactivities of PTHrP in malignancies.     

Changes in parathyroid hormone-related protein and 3-dimensional trabecular bone structure of the mandibular condyle following mandibular distraction osteogenesis in growing rats.

PURPOSE: Distraction osteogenesis (DO) is commonly performed for mandibular reconstruction during the growth period. We tested the hypothesis that parathyroid hormone-related protein (PTHrP) in mandibular condylar cartilage and underlying trabecular bone in growing individuals undergo changes in response to distraction forces. MATERIALS AND METHODS: Forty-eight 6-week-old male Sprague-Dawley rats were used. Animals underwent unilateral mandibular distraction using a distractor that we devised, and unoperated animals were evaluated as controls. DO procedure was performed: 3 days' latency period, 0.4 mm/day rate, total 4.0 mm. Changes in cartilage morphology, PTHrP activity, and 3-dimensional trabecular bone structure changes measured by micro-computed tomography were examined at 0, 2, 4, and 6 weeks of consolidation. RESULTS: A marked irregularity was noted in the superior portion of the distracted side's condylar cartilage that resolved after distraction ceased. PTHrP was more strongly expressed in the hypertrophic layer of condylar cartilage on the distracted side than in controls, up to 6 weeks after the end of distraction. Subchondral trabecular bone volume, percent bone volume, and trabecular number in the superior and posterior regions of the condyle decreased significantly by 2 weeks after distraction. These parameters returned to normal in the posterior condyle, but not in the superior part of the condyle by 6 weeks following distraction. CONCLUSION: These results suggest that unilateral mandibular distraction in growing rats causes temporary morphologic alterations of trabecular bone structure on the distracted side accompanied by increased production of PTHrP in the mandibular condyle.     

CSF-1 regulation of osteoclastogenesis for tooth eruption

The dental follicle regulates the alveolar bone resorption needed for tooth eruption. In the rat first mandibular molar, a decrease in the expression of osteoprotegerin (OPG) in the dental follicle at day 3 enables the osteoclastogenesis needed for eruption to occur. Because colony-stimulating factor-1 (CSF-1) is maximally expressed in the dental follicle at day 3, it was hypothesized that CSF-1 down-regulates OPG gene expression in the dental follicle in vivo. To test this, we compared the expression of OPG in osteopetrotic toothless (tl/tl) rats deficient in CSF-1 with expression in their normal littermates for given ages. OPG gene expression was found to be higher in the dental follicle of the tl/tl mutants than in normals. Transfecting short interfering RNA specific for CSF-1 mRNA into dental follicle cells resulted in an up-regulation of OPG expression. Thus, these studies support our hypothesis that the down-regulation of OPG needed for tooth eruption is mediated by CSF-1.     

Possible role of cementoblasts in the resorbant organ of human deciduous teeth during root resorption

Human deciduous teeth undergoing physiologic root resorption were extracted and fixed with a mixture of formaldehyde and glutaraldehyde and processed for scanning (SEM) and transmission (TEM) electron microscopy, and for acid (ACPase) and alkaline phosphatase (ALPase) cytochemistry. The resorbant organ, rich in odontoclasts, cementoblasts, fibroblasts, and macrophages, formed prominent resorption lacunae in root dentin. SEM observations of resorption lacunae treated with trypsin solution showed islands of newly-formed cementum matrix in part of the resorbing dentin surfaces. Such cementum consisted of bundles of densely-arranged collagen fibrils and, in part, contained forming cementocytic lacunae and canaliculi. Active cementoblasts adjacent to odontoclasts on resorbing dentin surfaces showed cuboidal outlines and were characterized by the presence of numerous cisterns of rough endoplasmic reticulum, well-developed Golgi complexes, secretion granules, and many mitochondria. They sometimes formed a thin layer of cementoid and/or cementum matrix upon the resorbing dentin surface. These cementoblasts had ACPase-positive lysosomes in the cell bodies and exhibited intense ALPase activity along the plasma membranes of whole cell surfaces. These results suggest that, during root resorption, 1) active cementoblasts are present adjacent to active odontoclasts and 2) these cementoblasts are involved in remodeling the resorbing dentin surfaces.     

Parathyroid hormone-related protein (PTHrP) expression in ameloblastoma

OBJECTIVE: Parathyroid hormone-related protein (PTHrP) production has been demonstrated in a variety of tumor subtypes. Local production of PTHrP by metastatic tumor cells in bone has been linked to bone destruction and tumor growth. Ameloblastoma (AB) is a relatively common odontogenic epithelial neoplasm that manifests local infiltrative intraosseous growth. AB recapitulates the developing enamel epithelium, in which PTHrP recently has been demonstrated. Yet PTHrP expression in a series of ABs has not been studied to date. The purpose of this investigation is to assess the expression of PTHrP in ameloblastoma. STUDY DESIGN: Formalin-fixed, paraffin-embedded tissue sections of ameloblastoma (n = 30; 24 conventional, 4 unicystic, and 2 arising in dentigerous cyst) were immunostained with anti-PTHrP antibody using a multistep streptavidin-peroxidase technique. Semiquantitative scoring of immunoreactivity was assessed as mild, moderate, and intense. RESULTS: All cases (100%) demonstrated positive immunoreactivity, with mild reaction in 3 conventional ABs, 1 unicystic and 1 AB arising in dentigerous cyst, and with moderate reaction in 12 conventional ABs, 3 unicystic and 1 AB arising in dentigerous cyst. Intense immunoreactivity was seen in 9 cases of conventional AB. This difference in immunostaining was not statistically significant (Sigma2 = 4.41, df = 4, P = .358). CONCLUSION: The results of this investigation suggest that PTHrP may play a significant role in local bone resorption, offering at least partial explanation for the tumor's infiltrative growth and destructive behavior. The uniformity of PTHrP expression by AB, as detailed in this study, may harbor significant therapeutic implications, particularly through PTHrP-blocking treatment modalities.     

实验性单侧前牙反牙合修复体对大鼠髁突软骨中甲状旁腺激素相关蛋白和PTH/PTHrP受体-1表达的影响

目的 探讨实验性单侧前牙反牙合修复体对大鼠髁突软骨中甲状旁腺激素相关蛋白（PTHrP）及PTH/PTHrP 受体-1（PTH1R）表达的影响。方法 在6周龄SD大鼠左侧上、下切牙粘接金属不良修复体,使其呈反牙合关系,2、 4、8周后取颞下颌关节（TMJ）,苏木精-伊红（HE）染色观察组织形态学变化,免疫组织化学染色及实时定量聚合酶链反应检测PTHrP、PTH1R的表达情况。结果 8周时,实验组髁突软骨出现明显退行性变,PTHrP阳性细胞较对照组显著增多（P<0.01）, PTH1R阳性细胞明显减少（P<0.01）;实验组髁突软骨PTHrP mRNA的表达显著高于对照组（P<0.01）,而PTH1R mRNA表达明显降低（P<0.01）。结论 髁突软骨PTHrP的高表达因PTH1R的低表达不能有效发 挥作用,可能是髁突软骨骨关节病样变的重要分子机制之一。