Histochemical examination on the peri-implant bone with early occlusal loading after the immediate placement into extraction sockets.

Early and immediate loading of dental implants has become a routine procedure in dental practices throughout the world, but the histological feature of peri-implant bone has not been fully understood. Therefore, we aimed to elucidate the histological response of peri-implant bone bearing the early occlusal loading using rat models. Four-week-old male Wistar rats were subjected to extraction of their maxillary left first molars and had titanium implants inserted immediately into the post-extraction sockets. In experimental groups at 1 week after placement, implants were loaded for 1 or 2 weeks by adding adhesive resin on the top of the screws. In control groups, no adhesive resin was added to the implants. After 1 or 2 weeks with loading, rats were fixed with an aldehyde solution for histochemical assessment. Newly-formed bone adhered broadly to the implant surface in both the control and experimental groups. The experimental group loaded for 2 weeks showed thicker trabeculae between the implant threads compared to those in the control group. Osteopontin- and osteocalcin-positive cement lines, which are histological hallmarks of bone remodeling, were narrow and smooth in the experimental groups, while featuring a complex meshwork with thick scalloped lines in the control groups. The index of sclerostin-positive osteocytes located close to implants loaded for 2 weeks was significantly lower than in controls, suggesting that osteoblast activity was preserved. Summarizing, our experimental model suggested that early implant loading increases trabecular thickness in the peri-implant bone tissue in a process that involves the regulation of bone remodeling.

Molecular defect of tissue-nonspecific alkaline phosphatase bearing a substitution at position 426 associated with hypophosphatasia.

Mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNSALP) cause hypophosphatasia (HPP), a genetic disorder characterized by deficiency of serum ALP and hypomineralization of bone and teeth. Three missense mutations for glycine 426 (by standard nomenclature) of TNSALP have been reported: cysteine (p.G426C), serine (p.G426S), and aspartate (p.G426D). We expressed TNSALP mutants carrying each missense mutation in mammalian cells. All three TNSALP mutants appeared on the cell surface like the wild-type (WT) TNSALP, although the cells expressing each TNSALP mutant exhibited markedly reduced ALP activity. TNSALP (WT) was mainly present as a 140 kDa catalytically active dimeric form, whereas ~80 kDa monomers were the predominant molecular species in the cells expressing TNSALP (p.G426D) or TNSALP (p.G426S), suggesting that aspartate or serine at position 426 may hamper the subunit assembly essential for the enzymatic function of TNSALP. Alternatively, the subunits of TNSALP (p.G426C) were found to be aberrantly cross-linked by disulfide bonds, giving rise to a 200 kDa form lacking ALP activity. Taken together, our results reveal that the amino acid substitutions at position 426 of TNSALP differentially affect the structure and function of TNSALP, leading to understanding of the molecular and cellular basis of HPP.

Effects of drug discontinuation after short-term daily alendronate administration on osteoblasts and osteocytes in mice.

In order to determine whether osteoclastic bone resorption is restarted after withdrawn of bisphosphonates, we conducted histological examinations on murine osteoclasts, osteoblasts and osteocytes after discontinuation of a daily regimen of alendronate (ALN) with a dosage of 1 mg/kg/day for 10 days. After drug discontinuation, metaphyseal trabecular number and bone volume remained unaltered for the first 4 days. Osteoclast number did not increase, while the number of apoptotic osteoclasts was elevated. On the other hand, tissue non-specific alkaline phosphatase-immunoreactive area was markedly reduced after ALN discontinuation. In addition, osteocytes showed an atrophic profile with empty lacunar areas during and after ALN treatment. Interestingly, as early as 36 h after a single ALN injection, osteocytes show signs of atrophy despite the presence of active osteoblasts. Structured illumination microscopy system showed shortening of osteocytic cytoplasmic processes after drug cessation, suggesting a possible morphological and functional disconnection between osteocytes and osteoblasts. Taken together, it appears that osteoclastic bone resorption is not resumed after ALN discontinuation; also, osteoblasts and osteocytes hardly seem to recover once they are inactivated and atrophied by ALN. In summary, it seems that one must pay more attention to the responses of osteoblasts and osteocytes, rather focusing on the resuming of osteoclastic bone resorption after the ALN discontinuation.

Trans-Golgi protein p230/golgin-245 is involved in phagophore formation.

p230/golgin-245 is a trans-Golgi coiled-coil protein that is known to participate in regulatory transport from the trans-Golgi network (TGN) to the cell surface. We investigated the role of p230 and its interacting protein, microtubule actin crosslinking protein 1 (MACF1), in amino acid starvation-induced membrane transport. p230 or MACF1 knock-down (KD) cells failed to increase the autophagic flow rate and the number of microtubule-associated protein 1 light chain 3 (LC3)-positive puncta under starvation conditions. Loss of p230 or MACF1 impaired mAtg9 recruitment to peripheral phagophores from the TGN, which was observed in the early step of autophagosome formation. Overexpression of the p230-binding domain of MACF1 resulted in the inhibition of mAtg9 trafficking in starvation conditions as in p230-KD or MACF1-KD cells. These results indicate that p230 and MACF1 cooperatively play an important role in the formation of phagophore through starvation-induced transport of mAtg9-containing membranes from the TGN. In addition, p230 itself was detected in autophagosomes/autolysosome with p62 or LC3 during autophagosome biogenesis. Thus, p230 is an important molecule in phagophore formation, although it remains unclear whether p230 has any role in late steps of autophagy.

Molecular phenotype of tissue-nonspecific alkaline phosphatase with a proline (108) to leucine substitution associated with dominant odontohypophosphatasia.

Hypophosphatasia (HPP) is a genetic disease characterized by defective calcification of hard tissues such as bone and teeth accompanying deficiency of serum alkaline phosphatase (ALP) activity. Its development results from various mutations in the ALPL gene encoding tissue-nonspecific ALP (TNSALP). HPP is known to be transmitted in an autosomal recessive or autosomal dominant manner. A point mutation (c.323C>T) in the ALPL gene leading to a proline to leucine substitution at position 108 of TNSALP was first reported in a patient diagnosed with odonto-HPP (M Herasse et al., J Med Genet 2003;40:605-609), although the effects of this mutation on the TNSALP molecule have not been elucidated. To understand the molecular basis of this dominantly transmitted HPP, we first characterized TNSALP (P108L) by expressing it in COS-1 cells transiently. In contrast to wild-type TNSALP (WT), TNSALP (P108L) showed virtually no ALP activity. When coexpressed with TNSALP (WT), TNSALP (P108L) significantly inhibited the enzyme activity of TNSALP (WT), confirming that this mutant TNSALP exerts a dominant negative effect on TNSALP (WT). Using immunofluorescence and digestion with phosphatidylinositol-specific phospholipase C, we demonstrated that TNSALP (P108L) was anchored to the cell surface via glycosylphosphatidylinositol-like TNSALP (WT) in a Tet-On CHO cell expression system. Consistent with this, TNSALP (P108L) acquired endo-ß-N-acetylglucosaminidase H resistance and sialic acids, as evidenced by glycosidase treatments. Importantly, TNSALP (WT) largely formed a functional dimeric structure, while TNSALP (P108L) was found to be present as a monomer in the cell. This indicates that the molecular structure of TNSALP is affected by a missense mutation at position 108, which is in contact with the active site, such that it no longer assembles into the functional dimeric form. Collectively, these results may explain why TNSALP (P108L) loses its ALP activity, even though it is able to gain access to the cell surface.

TNFα triggers release of extracellular vesicles containing TNFR1 and TRADD, which can modulate TNFα responses of the parental cells.

Tumor necrosis factor-α (TNFα)-induced reactions are effective to maintain homeostasis; however, excessive responses play progressive roles in the pathogenesis of various chronic inflammatory diseases. We demonstrate that TNFα triggered the release of its receptor TNFR1 as a content of extracellular vesicles (EVs) from the human bronchial epithelial cell, BEAS-2b. The TNFR1 cytoplasmic domain binding partner, TNFR-associated death domain (TRADD), was released by TNFα treatment along with TNFR1. TNFα-triggered release of EVs was decreased in the presence of amitriptyline, an inhibitor of acid sphingomyelinase (A-SMase), or of GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), indicating that EVs containing TNFR1 and TRADD are released through A-SMase and N-SMase dependent manners. From sucrose density gradient analysis, each sphingomyelinase is involved in the generation of distinct populations of EVs. Inhibition of A-SMase or N-SMase resulted in significantly increased responses to TNFα in parental cells. Given that TRADD serves as a platform for the assembly of subsequent signaling molecules, the TNFα triggered release of TNFR1 and TRADD might be an effective strategy for down regulation of the TNFα responses of parental cells.

Inhibitory effect of bisphosphonate on osteoclast function contributes to improved skeletal pain in ovariectomized mice.

The aim of this study was to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effect of bisphosphonate (BP) on pain in an ovariectomized (OVX) mouse model. We evaluated skeletal pain in OVX mice through an examination of pain-like behavior as well as immunohistochemical findings. In addition, we assessed the effects of alendronate (ALN), a potent osteoclast inhibitor, on those parameters. The OVX mice showed a decrease in the pain threshold value, and an increase in the number of c-Fos immunoreactive neurons in laminae I-II of the dorsal horn of the spinal cord. Alendronate caused an increase in the pain threshold value and inhibited c-Fos expression. The serum level of tartrate-resistant acid phosphatase 5b, a marker of osteoclast activity, was significantly negatively correlated with the pain threshold value. Furthermore, we found that an antagonist of the transient receptor potential channel vanilloid subfamily member 1, which is an acid-sensing nociceptor, improved pain-like behavior in OVX mice. These results indicated that the inhibitory effect of BP on osteoclast function might contribute to an improvement in skeletal pain in osteoporosis patients.

Hertwig's epithelial root sheath cell behavior during initial acellular cementogenesis in rat molars.

This study was designed to examine developing acellular cementum in rat molars by immunohistochemistry, to elucidate (1) how Hertwig's epithelial root sheath disintegrates and (2) whether epithelial sheath cells transform into cementoblasts through epithelial-mesenchymal transition (EMT). Initial acellular cementogenesis was divided into three developmental stages, which can be seen in three different portions of the root: portion 1, where the epithelial sheath is intact; portion 2, where the epithelial sheath becomes fragmented; and portion 3, where acellular cementogenesis begins. Antibodies against three kinds of matrix proteinases, which degrade epithelial sheath-maintaining factors, including basement membrane and desmosomes, were used to investigate proteolytic activity of the epithelial sheath. Tissue non-specific alkaline phosphatase (TNALP) and keratin were used to investigate EMT. Epithelial sheath cells showed immunoreactivity for all three enzymes at fragmentation, which suggests that epithelial sheath disintegration is enzymatically mediated. Dental follicle cells and cementoblasts showed intense immunoreactivity for TNALP, and from portion 1 through to 3, the reaction extended from the alveolar bone-related zone to the root-related zone. Cells possessing keratin/TNALP double immunoreactivity were virtually absent. Keratin-positive epithelial sheath cells showed negligible immunoreactivity for TNALP, and epithelial cells did not appear to migrate to the dental follicle. Together, these findings suggest that a transition phenotype between epithelial cells and cementoblasts does not exist in the developing dental follicle and hence that epithelial sheath cells do not undergo EMT during initial acellular cementogenesis. In brief, this study supports the notion that cementoblasts derive from the dental follicle.

[Microscopic aspects on biomineralization in bone].

In bone, biomineralization induced by osteoblasts is known to be initiated by small extracellular vesicles referred to as "matrix vesicles". Matrix vesicles possess many enzymes and transporters, which synthesize and incorporate Ca²âº and PO4⁻ into the vesicles. Calcification initiates when crystalline calcium phosphates are nucleated inside these matrix vesicles, and calcium phosphates, i.e., hydroxyapatite crystals, grow and eventually break through the membrane to get out of the matrix vesicles. Exposed calcium phosphates featuring "ribbon-like" appearance assemble radially, forming spherical mineralized structure, referred to as "mineralized nodule" or "calcifying globule". This process is called "matrix vesicle mineralization". Thereafter, the mineralized nodules make contacts with surrounding collagen fibrils, extending mineralization along with their longitudinal axis from the contact points of collagen fibrils - collagen mineralization. Matrix vesicle mineralization and subsequent collagen mineralization are classified as primary mineralization associated with osteoblastic bone formation. After primary mineralization, secondary mineralization takes place, gradually increasing mineral density of bone matrix. This review will introduce the microscopic findings on matrix vesicle mineralization and subsequent collagen mineralization.

[Tissue-nonspecific alkaline phosphatase and hypophosphatasia].

There are four isozymes for human alkaline phosphatase (ALP) : tissue-nonspecific ALP (TNSALP), intestinal ALP, placental ALP and germ cell ALP. We present a brief history of TNSALP and review progress in research on it and a rare inborn error of metabolism called hypophosphatasia (HPP), which is caused by various loss-of-function mutations in the ALPL gene encoding TNSALP. HPP is characterized by decreased levels of serum ALP activity and defect in mineralization of bone and teeth, thus establishing the direct link between TNSALP and biomineralization. In addition to its 3D structure, studies on TNSALP mutants expressed in mammalian cells have revealed how each mutation affects the structure and function of TNSALP at the molecular level, which contributes to our understanding of the molecular basis of HPP.

Disulfide bonds are critical for tissue-nonspecific alkaline phosphatase function revealed by analysis of mutant proteins bearing a C(201)-Y or C(489)-S substitution associated with severe hypophosphatasia.

Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171-172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66kDa immature form was predominant with a marginal amount of a 80kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80kDa mature form, though most of the 66kDa form was found to be endo-ß-N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme.

An asparagine at position 417 of tissue-nonspecific alkaline phosphatase is essential for its structure and function as revealed by analysis of the N417S mutation associated with severe hypophosphatasia.

Various loss-of function mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene cause a rare genetic disorder called hypophosphatasia (HPP), which is characterized by defective mineralization in the bones and teeth and a deficiency in serum alkaline phosphatase. A point mutation (c.1250A>G), which leads to replacement of an asparagine at position 417 of TNSALP with serine [TNSALP (N417S)], has been reported in a patient diagnosed with perinatal HPP (Sergi C. et al. Am, J. Med. Genet. 103, 235-240, 2001). In order to characterize the molecular properties of TNSALP (N417S), we expressed and analyzed TNSALP (N417S) both in COS-1 cells (transient expression) and CHO K1 Tet-On cells (inducible cell system). In contrast to wild-type TNSALP [TNSALP (W)], cells expressing TNSALP (N417S) lacked its alkaline phosphatase activity. However, this mutant underwent N-linked oligosaccharide processing and appeared on the cell surface similar to TNSALP (W). Importantly, this mutant failed to assemble into a dimer structure, which is needed for the catalytic function of TNSALP, as evidenced by newly developed SDS-PAGE as well as sucrose-density-gradient centrifugation. Substitution of the asparagine at position 417 with structurally related amino acids such as an aspartate and a glutamine also abolished the dimerization of TNSALP without perturbing its cell surface localization. Taken together, the asparagine at position 417 is crucial for the assembly and function of TNSALP, which may explain the severity of the N417S mutation.

Identification of a soluble isoform of human IL-17RA generated by alternative splicing.

IL-17RA, a member of the interleukin (IL)-17 receptor family, is a single membrane-spanning protein that ubiquitously expressed on the cell surface. IL-17RA transduces IL-17A, IL-17F, and IL-17A/F heterodimer-mediated signals by forming a complex with IL-17RC, and also signals the IL-17E (also known as IL-25) response in combination with IL-17RB (also known as IL-25R). Previously, soluble isoforms of human IL-17RC and IL-17RB have been reported, but the existence of a soluble isoform of human IL-17RA has remained unclear. Here, we report the identification of a soluble isoform of human IL-17RA at the mRNA and protein levels. Reverse transcribed PCR experiments showed that the IL-17RA variant is generated by spliced out of exon 11 encoding the transmembrane region in a variety of human tissues. The soluble IL-17RA isoform was detected in the culture media of human cell lines by Western blotting. The existence of the soluble IL-17RA isoform sheds new light on the regulation of IL-17RA mediated responses.

Interaction of Golgin-84 with the COG complex mediates the intra-Golgi retrograde transport.

The coiled-coil Golgi membrane protein golgin-84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin-84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin-84 as the tether for COPI vesicles of intra-Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin-84 knockdown (KD) cells. The depletion of golgin-84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra-Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and cis-Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex-dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin-84. Surprisingly, the interaction between golgin-84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin-84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin-84 with COG plays an important role in the tethering process of intra-Golgi retrograde vesicle traffic.

Tissue nonspecific alkaline phosphatase is activated via a two-step mechanism by zinc transport complexes in the early secretory pathway.

A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.

Molecular characterization of tissue-nonspecific alkaline phosphatase with an Ala to Thr substitution at position 116 associated with dominantly inherited hypophosphatasia.

Mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia, an inborn error of bone and teeth metabolism associated with reduced levels of serum alkaline phosphatase activity. A missense mutation (c.346G>A) of TNSALP gene, which converts Ala to Thr at position 116 (according to standardized nomenclature), was reported in dominantly transmitted hypophosphatasia patients (A.S. Lia-Baldini et al. Hum Genet. 109 (2001) 99-108). To investigate molecular phenotype of TNSALP (A116T), we expressed it in the COS-1 cells or Tet-On CHO K1 cells. TNSALP (A116T) displayed not only negligible alkaline phosphatase activity, but also a weak dominant negative effect when co-expressed with the wild-type enzyme. In contrast to TNSALP (W, wild-type), which was present mostly as a non-covalently assembled homodimeric form, TNSALP (A116T) was found to exist as a monomer and heterogeneously associated aggregates covalently linked via disulfide bonds. Interestingly, both the monomer and aggregate forms of TNSALP (A116T) gained access to the cell surface and were anchored to the cell membrane via glycosylphosphatidylinositol (GPI). Co-expression of secretory forms of TNSALP (W) and TNSALP (A116T), which are engineered to replace the C-terminal GPI anchor with a tag sequence (his-tag or flag-tag), resulted in the release of heteromeric complexes consisting of TNSALP (W)-his and TNSALP (A116T)-flag. Taken together, these findings strongly suggest that TNSALP (A116T) fails to fold properly and forms disulfide-bonded aggregates, though it is indeed capable of interacting with the wild-type and reaching the cell surface, therefore explaining its dominant transmission.

Immunolocalization of sclerostin synthesized by osteocytes in relation to bone remodeling in the interradicular septa of ovariectomized rats.

This study aimed at elucidating whether estrogen deficiency would affect the synthesis of an osteocyte-derived factor, sclerostin, in the mesial region of alveolar bone. Eight 9-week-old Wistar female rats were ovariectomized (OVX) and eight other rats were Sham-operated (Sham). After 4 weeks, the interradicular septa of mandibular first molar were embedded in paraffin and then histochemically examined. Sclerostin-positive osteocytes were located in the superficial layer of the mesial region of Sham bones, whereas the OVX mesial region showed less sclerostin-reactive osteocytes. There was no significant difference in the distribution of estrogen receptor α and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling -positive cells in the groups studied. The Sham mesial region featured many osteoclasts, and OVX specimens showed numerous osteoclasts in association with intense immunolabeling of the receptor activator of the nuclear factor kB ligand. Contrary to the observations in Sham specimens, a complex meshwork of cement lines was seen in the OVX mesial region, accompanied by an irregularly distributed osteocytic lacunar-canalicular system. In conclusion, estrogen deficiency appears to inhibit osteocyte-derived sclerostin synthesis in the mesial region of the interradicular septum, in a process that seems to be mediated by accelerated bone remodeling rather than by direct effects on osteocytes.

Ultrastructural observation on cells meeting the histological criteria for preosteoblasts--a study in the mouse tibial metaphysis.

Preosteoblasts are currently defined as the precursors of mature osteoblasts. These cells are morphologically diverse and may represent a continuum during osteoblast differentiation. We have attempted to categorize the different preosteoblastic phenotypes in vivo by examining bone cells expressing the runt-related transcription factor 2, alkaline phosphatase and BrdU incorporation - histological traits of a preosteoblast - under transmission electron microscopy (TEM). TEM observations demonstrated, at least, in part two preosteoblastic subtypes: (i) a cell rich in cisterns of rough endoplasmic reticulum (rER) with vesicles and vacuoles and (ii) a subtype featuring extended cytoplasmic processes that connect with distant cells, with a small amount of scattered cisterns of rER and with many vesicles and vacuoles. ER-rich cells, whose cellular machinery is similar to that of an osteoblast, were often seen adjacent to mature osteoblasts, and therefore, may be ready for terminal differentiation. In contrast, ER-poor and vesicle-rich cells extended their cytoplasmic processes to mature osteoblasts and, frequently, to bone-resorbing osteoclasts. The abundant vesicles and vacuoles identified in this cell type indicate that this cell is involved in vesicular transport rather than matrix synthesis activity. In summary, our study verified the morphological diversity and the ultrastructural properties of osteoblastic cells in vivo.

FGFR3 down-regulates PTH/PTHrP receptor gene expression by mediating JAK/STAT signaling in chondrocytic cell line.

The signaling axis comprising the parathyroid hormone (PTH)-related peptide (PTHrP), the PTH/PTHrP receptor and the fibroblast growth factor receptor 3 (FGFR3) plays a central role in chondrocyte proliferation. The Indian hedgehog (IHH) gene is normally expressed in early hypertrophic chondrocytes, and its negative feedback loop was shown to regulate PTH/PTHrP receptor signaling. In this study, we examined the regulation of PTH/PTHrP receptor gene expression in a FGFR3-transfected chondrocytic cell line, CFK2. Expression of IHH could not be verified on these cells, with consequent absence of hypertrophic differentiation. Also, expression of the PTH/PTHrP receptor (75% reduction of total mRNA) and the PTHrP (50% reduction) genes was reduced in CFK2 cells transfected with FGFR3 cDNA. Interestingly, we verified significant reduction in cell growth and increased apoptosis in the transfected cells. STAT1 was detected in the nuclei of the CFK2 cells transfected with FGFR3 cDNA, indicating predominance of the JAK/STAT signaling pathway. The reduction in PTH/PTHrP receptor gene in CFK2 cells overexpressing FGFR3 was partially blocked by treatment with an inhibitor of JAK3 (WHI-P131), but not with an inhibitor of MAPK (SB203580) or JAK2 (AG490). Altogether, these findings suggest that FGFR3 down-regulates PTH/PTHrP receptor gene expression via the JAK/STAT signaling in chondrocytic cells.

Influence of heat stress to matrix on bone formation.

OBJECTIVES: It is important to know the etiology of implant failure. It has been reported that heat stress during drilling was one of the causes for failure and the threshold was 47 degrees C. However, clinically, we encounter cases in which overheating does not seem to affect osseointegration eventually. The purpose of this study was to assess histologically the spatio-temporal effect of heat stress on bone formation after overheating the bone matrix. MATERIAL AND METHODS: Rat calvarial bone was heated to 37 degrees C, 43 degrees C, 45 degrees C and 48 degrees C for 15 min by a temperature stimulator. Paraffin sections were prepared 1, 3 and 5 weeks after heating and investigated histologically under light microscopy. Hematoxylin and eosin staining, alkaline phosphatase (ALP), osteopontin (OPN), heat shock protein 27 (Hsp27) and heat shock protein 70 (Hsp70) immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry were carried out. The area of dead osteocytes was calculated and statistically analyzed. Apoptotic osteocytes were detected by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) method. RESULTS: Along with the temperature increase, the area of dead osteocytes increased and regeneration of the periosteal membrane was delayed. Hsps- and TUNEL-positive cells were only seen in the 48 degrees C group. Spatio-temporal changes of TRAP- and ALP-positive cell numbers were observed, while OPN expression was mostly absent. Even after 48 degrees C stimulation, bone formation on the calvarial surface was observed after 5 weeks. CONCLUSIONS: Although there was a temperature-dependent delay in bone formation after heat stress, the 48 degrees C heat stress did not obstruct bone formation eventually. This delay was probably caused by slow periosteal membrane regeneration.