Hideaki Nagaoka

Tissue Engineering Strategies for Immature Teeth with Apical Periodontitis

INTRODUCTION Regenerative endodontic treatment on immature teeth with apical periodontitis is promising but still not well-established. The purpose of this study was to explore novel strategies to engineer a vital support structure within a root canal space by a combination of induced blood clot, exposure of dentin matrix, and a cross-linked collagen scaffold. METHODS Apical periodontitis was induced in 6 dogs with immature teeth (n = 64). After disinfection, the following groups were randomly assigned: blood clot (BC) alone, BC with a cross-linked collagen scaffold (CCS), BC with exposure of dentin matrix by ethylenediaminetetraacetic acid (EDTA), and BC with CCS and EDTA. Positive (infected only) and negative controls (untreated) were also included. The dogs were followed up for 3.5 months and killed. Periradicular healing and root wall thickening were radiographically analyzed and statistically evaluated. The jaws were then fixed, demineralized, and subjected to histologic analyses. Newly formed mineralized tissues were histomorphometrically analyzed, quantified, and statistically evaluated. RESULTS Radiographically there was significant difference in periradicular healing and root wall thickening (P < .05). Histomorphometric analysis showed significantly more mineralized tissue formation in the groups containing the scaffold (P < .05). Exposure of the dentin matrix by EDTA appeared to increase the adherence of the newly formed mineralized tissue to the root walls. CONCLUSIONS The use of cross-linked collagen scaffold and exposure of dentin matrix combined with blood clot might provide an efficient approach to generate a vital support structure for the treatment of immature teeth with apical periodontitis.

Immunohistological Characterization of Newly Formed Tissues after Regenerative Procedure in Immature Dog Teeth

INTRODUCTION In a previous report, we showed that 2 types of mineralized tissues were formed in the canal spaces of dogs after tissue engineering treatments of immature teeth with apical periodontitis: (1) dentin- associated mineralized tissue (DAMT) and (2) bony islands (BIs). The objective of this study was to characterize these mineralized tissues. METHODS The maturation and organization of collagen matrices in DAMT, BIs, and the interface between DAMT and the dentin wall were characterized using a histochemical method with picrosirius red staining under polarized light microscopy. In addition, the distribution of 2 noncollagenous proteins (ie, dentin sialoprotein and bone sialoprotein) in these tissues was investigated by immunohistochemical methods with specific antibodies. RESULTS The results showed that DAMT is distinct from dentin, bone, or BIs. Although it resembled cementum to an extent showing similar immunoreactivity to the noncollagenous proteins, the organization and maturation of collagen matrix was significantly different from cementum. BIs resembled a bone matrix in terms of morphology, collagen organization, and immunoreactivity. CONCLUSIONS The results indicate that DAMT and BIs formed in the canal space are distinct from each other, one exhibiting a unique mineralized tissue and the other a bone-like tissue.

Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance.

Background: Type I collagen is the most abundant organic component in bone, providing form and stability. Results: Lysyl hydroxylase 3-mediated glucosylation occurs at specific sites in collagen, including cross-linking sites, and suppression of this modification results in defective collagen and mineralization. Conclusion: The data indicate the critical importance of this modification in bone physiology. Significance: Alterations of this collagen modification may cause bone defects. Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.

1,25(OH)2D3 regulates collagen quality in an osteoblastic cell culture system

The active form of vitamin D, 1,25(OH)(2)D(3), has a broad range of effects on bone, however, its role in the quality of bone matrix is not well understood. In this study, using an osteoblastic cell (MC3T3-E1) culture system, the effects of 1,25(OH)(2)D(3) on collagen cross-linking and related enzymes, i.e., lysyl hydroxylases (LH1-3) and lysyl oxidases (LOX, LOXL1-4), were examined and compared to controls where cells were treated with cholecalciferol or ethanol. When compared to the controls, gene expressions of LH1, LH2b and LOXL2 were significantly upregulated by 1,25(OH)(2)D(3) up to 72h of culture. In addition, hydroxylysine (Hyl), Hyl aldehyde (Hyl(ald)), Hyl(ald)-derived cross-links and a total number of cross-links of collagen were significantly higher and the cross-link maturation was accelerated in the 1,25(OH)(2)D(3) treated group. These results demonstrate that 1,25(OH)(2)D(3) directly regulates collagen cross-linking in this culture system likely by upregulating gene expression of specific LH and LOX enzymes.

Lysyl Hydroxylase 3 Glucosylates Galactosylhydroxylysine Residues in Type I Collagen in Osteoblast Culture

Lysyl hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technology in a mouse osteoblastic cell line, MC3T3-E1; generated single cell-derived clones stably suppressing LH3 (short hairpin (Sh) clones); and characterized the phenotype. Plod3 expression and the LH3 protein levels in the Sh clones were significantly suppressed when compared with the controls, MC3T3-E1, and the clone transfected with an empty vector. In comparison with controls, type I collagen synthesized by Sh clones (Sh collagen) showed a significant decrease in the extent of glucosylgalactosylhydroxylysine with a concomitant increase of galactosylhydroxylysine, whereas the total number of hydroxylysine residues was essentially unchanged. In an in vitro fibrillogenesis assay, Sh collagen showed accelerated fibrillogenesis compared with the controls. In addition, when recombinant LH3-V5/His protein was generated in 293 cells and subjected to GGT/GT activity assay, it showed GGT but not GT activity against denatured type I collagen. The results from this study clearly indicate that the major function of LH3 in osteoblasts is to glucosylate galactosylhydroxylysine residues in type I collagen and that an impairment of this LH3 function significantly affects type I collagen fibrillogenesis.

Loss of fibulin-4 disrupts collagen synthesis and maturation: implications for pathology resulting from EFEMP2 mutations

Homozygous recessive mutations in either EFEMP2 (encoding fibulin-4) or FBLN5 (encoding fibulin-5), critical genes for elastogenesis, lead to autosomal recessive cutis laxa types 1B and 1A, respectively. Previously, fibulin-4 was shown to bind lysyl oxidase (LOX), an elastin/collagen cross-linking enzyme, in vitro. Consistently, reported defects in humans with EFEMP2 mutations are more severe and broad in range than those due to FBLN5 mutations and encompass both elastin-rich and collagen-rich tissues. However, the underlying disease mechanism in EFEMP2 mutations has not been fully addressed. Here, we show that fibulin-4 is important for the integrity of aortic collagen in addition to elastin. Smooth muscle-specific Efemp2 loss in mouse (termed SMKO) resulted in altered fibrillar collagen localization with larger, poorly organized fibrils. LOX activity was decreased in Efemp2-null cells, and collagen cross-linking was diminished in SMKO aortas; however, elastin cross-linking was unaffected and the level of mature LOX was maintained to that of wild-type aortas. Proteomic screening identified multiple proteins involved in procollagen processing and maturation as potential fibulin-4-binding partners. We showed that fibulin-4 binds procollagen C-endopeptidase enhancer 1 (Pcolce), which enhances proteolytic cleavage of the procollagen C-terminal propeptide during procollagen processing. Interestingly, however, procollagen cleavage was not affected by the presence or absence of fibulin-4 in vitro. Thus, our data indicate that fibulin-4 serves as a potential scaffolding protein during collagen maturation in the extracellular space. Analysis of collagen in other tissues affected by fibulin-4 loss should further increase our understanding of underlying pathologic mechanisms in patients with EFEMP2 mutations.

Alfacalcidol enhances collagen quality in ovariectomized rat bones

The aim of this study was to investigate the effects of alfacalcidol (1α(OH)D3: ALF) on bone collagen employing an ovariectomized rat model. Thirty‐five 16‐week‐old female Sprague‐Dawley rats were divided into five groups: SHAM (sham‐operated + vehicle), OVX (ovariectomy + vehicle), and three ALF‐treated groups, that is, ovariectomy + 0.022 µg/kg/day ALF, ovariectomy + 0.067 µg/kg/day ALF, and ovariectomy + 0.2 µg/kg/day ALF. After 12 weeks of treatment, tibiae were subjected to histological, biochemical and immunohistochemical analyses. Collagen matrices in OVX bone appeared as immature and poorly organized; however, with the ALF treatment, it was improved in a dose‐dependent manner. Contents of collagen and pyridinoline cross‐link were decreased in OVX compared with SHAM, but they increased to the level comparable to SHAM in ALF‐treated groups. The total aldehyde, that is, a sum of free and those involved cross‐links, in the highest dose of ALF was significantly higher than the rest of the groups (p < 0.05). In addition, the expression of lysyl oxidase was increased in the all ALF‐treated groups compared with OVX (p < 0.05). In conclusion, ALF increases not only the amount of collagen but also enhances the maturation of collagen in ovariectomy‐induced osteoporotic bones, which likely contributes to the improvement of bone quality.

Characterization of Genipin-Modified Dentin Collagen

Application of biomodification techniques to dentin can improve its biochemical and biomechanical properties. Several collagen cross-linking agents have been reported to strengthen the mechanical properties of dentin. However, the characteristics of collagen that has undergone agent-induced biomodification are not well understood. The objective of this study was to analyze the effects of a natural cross-linking agent, genipin (GE), on dentin discoloration, collagen stability, and changes in amino acid composition and lysyl oxidase mediated natural collagen cross-links. Dentin collagen obtained from extracted bovine teeth was treated with three different concentrations of GE (0.01%, 0.1%, and 0.5%) for several treatment times (0–24 h). Changes in biochemical properties of NaB3H4-reduced collagen were characterized by amino acid and cross-link analyses. The treatment of dentin collagen with GE resulted in a concentration- and time-dependent pigmentation and stability against bacterial collagenase. The lysyl oxidase-mediated trivalent mature cross-link, pyridinoline, showed no difference among all groups while the major divalent immature cross-link, dehydro-dihydroxylysinonorleucine/its ketoamine in collagen treated with 0.5% GE for 24 h, significantly decreased compared to control (P < 0.05). The newly formed GE-induced cross-links most likely involve lysine and hydroxylysine residues of collagen in a concentration-dependent manner. Some of these cross-links appear to be reducible and stabilized with NaB3H4.

Identification of the effector domain of biglycan that facilitates BMP-2 osteogenic function

We have reported that recombinant biglycan (BGN) core protein accelerates bone formation in vivo by enhancing bone morphogenetic protein (BMP)-2 function. The purpose of the present study was to identify the specific domain (“effector”) within the BGN core protein that facilitates BMP-2 osteogenic function. Thus, we generated various recombinant and synthetic peptides corresponding to several domains of BGN, and tested their effects on BMP-2 functions in vitro. The results demonstrated that the leucine-rich repeats 2–3 domain (LRR2-3) of BGN significantly enhanced the BMP-2 induced Smad1/5/9 phosphorylation, osteogenic gene expression, and alkaline phosphatase activity in myogenic C2C12 cells. Furthermore, addition of LRR2-3 to osteoblastic MC3T3-E1 cells accelerated in vitro mineralization without compromising the quality of the mineral and matrix. These data indicate that LRR2-3 is, at least in part, responsible for BGN’s ability to enhance BMP-2 osteogenic function, and it could be useful for bone tissue regeneration.