Masaru Kaku

Mechano-regulation of collagen biosynthesis in periodontal ligament

Periodontal ligament (PDL) plays critical roles in the development and maintenance of periodontium such as tooth eruption and dissipation of masticatory force. The mechanical properties of PDL are mainly derived from fibrillar type I collagen, the most abundant extracellular component. The biosynthesis of type I collagen is a long, complex process including a number of intra- and extracellular post-translational modifications. The final modification step is the formation of covalent intra- and intermolecular cross-links that provide collagen fibrils with stability and connectivity. It is now clear that collagen post-translational modifications are regulated by groups of specific enzymes and associated molecules in a tissue-specific manner; and these modifications appear to change in response to mechanical force. This review focuses on the effect of mechanical loading on collagen biosynthesis and fibrillogenesis in PDL with emphasis on the post-translational modifications of collagens, which is an important molecular aspect to understand in the field of prosthetic dentistry.

Cell-based bone regeneration for alveolar ridge augmentation – Cell source, endogenous cell recruitment and immunomodulatory function

Alveolar ridge plays a pivotal role in supporting dental prosthesis particularly in edentulous and semi-dentulous patients. However the alveolar ridge undergoes atrophic change after tooth loss. The vertical and horizontal volume of the alveolar ridge restricts the design of dental prosthesis; thus, maintaining sufficient alveolar ridge volume is vital for successful oral rehabilitation. Recent progress in regenerative approaches has conferred marked benefits in prosthetic dentistry, enabling regeneration of the atrophic alveolar ridge. In order to achieve successful alveolar ridge augmentation, sufficient numbers of osteogenic cells are necessary; therefore, autologous osteoprogenitor cells are isolated, expanded in vitro, and transplanted to the specific anatomical site where the bone is required. Recent studies have gradually elucidated that transplanted osteoprogenitor cells are not only a source of bone forming osteoblasts, they appear to play multiple roles, such as recruitment of endogenous osteoprogenitor cells and immunomodulatory function, at the forefront of bone regeneration. This review focuses on the current consensus of cell-based bone augmentation therapies with emphasis on cell sources, transplanted cell survival, endogenous stem cell recruitment and immunomodulatory function of transplanted osteoprogenitor cells. Furthermore, if we were able to control the mobilization of endogenous osteoprogenitor cells, large-scale surgery may no longer be necessary. Such treatment strategy may open a new era of safer and more effective alveolar ridge augmentation treatment options.

Canonical and noncanonical intraflagellar transport regulates craniofacial skeletal development

Significance Intraflagellar transport (IFT) plays a critical role in assembling primary cilia that mediate growth factor signaling. The disruption or dysfunction of IFT components can generate multiple diseases, including skeletal dysplasia. However, the mechanism by which IFT regulates skeletogenesis remains elusive. In the present study, we show that neural crest-specific deletion of the gene that encodes intraflagellar transport 20 (IFT20) in mice compromises ciliogenesis and the intracellular transport of collagen, leading to osteopenia in the face. Our findings highlight a unique function of IFT beyond its role in cilium assembly during craniofacial development, suggesting that IFT20 is indispensable for the regulation of not only ciliogenesis but also the intracellular trafficking of collagen in the unique multipotent stem cell population of cranial neural crests. The primary cilium is a cellular organelle that coordinates signaling pathways critical for cell proliferation, differentiation, survival, and homeostasis. Intraflagellar transport (IFT) plays a pivotal role in assembling primary cilia. Disruption and/or dysfunction of IFT components can cause multiple diseases, including skeletal dysplasia. However, the mechanism by which IFT regulates skeletogenesis remains elusive. Here, we show that a neural crest-specific deletion of intraflagellar transport 20 (Ift20) in mice compromises ciliogenesis and intracellular transport of collagen, which leads to osteopenia in the facial region. Whereas platelet-derived growth factor receptor alpha (PDGFRα) was present on the surface of primary cilia in wild-type osteoblasts, disruption of Ift20 down-regulated PDGFRα production, which caused suppression of PDGF-Akt signaling, resulting in decreased osteogenic proliferation and increased cell death. Although osteogenic differentiation in cranial neural crest (CNC)-derived cells occurred normally in Ift20-mutant cells, the process of mineralization was severely attenuated due to delayed secretion of type I collagen. In control osteoblasts, procollagen was easily transported from the endoplasmic reticulum (ER) to the Golgi apparatus. By contrast, despite having similar levels of collagen type 1 alpha 1 (Col1a1) expression, Ift20 mutants did not secrete procollagen because of dysfunctional ER-to-Golgi trafficking. These data suggest that in the multipotent stem cells of CNCs, IFT20 is indispensable for regulating not only ciliogenesis but also collagen intracellular trafficking. Our study introduces a unique perspective on the canonical and noncanonical functions of IFT20 in craniofacial skeletal development.

Distribution and relative activity of matrix metalloproteinase-2 in human coronal dentin

The presence of matrix metalloproteinase‐2 (MMP‐2) in dentin has been reported, but its distribution and activity level in mature human coronal dentin are not well understood. The purpose of this study was to determine the MMP‐2 distribution and relative activity in demineralized dentin. Crowns of twenty eight human molars were sectioned into inner (ID), middle (MD), and outer dentin (OD) regions and demineralized. MMP‐2 was extracted with 0.33 mol·L−1 EDTA/2 mol·L−1 guanidine‐HCl, pH 7.4, and MMP‐2 concentration was estimated with enzyme‐linked immunoabsorbant assay (ELISA). Further characterization was accomplished by Western blotting analysis and gelatin zymography. The mean concentrations of MMP‐2 per mg dentin protein in the dentin regions were significantly different (P=0.043): 0.9 ng (ID), 0.4 ng (MD), and 2.2 ng (OD), respectively. The pattern of MMP‐2 concentration was OD>ID>MD. Western blotting analysis detected ∼66 and ∼72 kDa immunopositive proteins corresponding to pro‐ and mature MMP‐2, respectively, in the ID and MD, and a ∼66 kDa protein in the OD. Gelatinolytic activity consistent with MMP‐2 was detected in all regions. Interestingly, the pattern of levels of Western blot immunodetection and gelatinolytic activity was MD>ID>OD. The concentration of MMP‐2 in human coronal dentin was highest in the region of dentin that contains the dentinoenamel junction and least in the middle region of dentin. However, levels of Western blot immunodetection and gelatinolytic activity did not correlate with the estimated regional concentrations of MMP‐2, potentially indicating region specific protein interactions.

FAM20A binds to and regulates FAM20C localization

Mutations in the Family with sequence similarity (FAM) 20 gene family are associated with mineralized tissue phenotypes in humans. Among these genes, FAM20A mutations are associated with Amelogenesis Imperfecta (AI) with gingival hyperplasia and nephrocalcinosis, while FAM20C mutations cause Raine syndrome, exhibiting bone and craniofacial/dental abnormalities. Although it has been demonstrated that Raine syndrome associated-FAM20C mutants prevented FAM20C kinase activity and secretion, overexpression of the catalytically inactive D478A FAM20C mutant was detected in both cell extracts and the media. This suggests that FAM20C secretion doesn’t require its kinase activity, and that another molecule(s) may control the secretion. In this study, we found that extracellular FAM20C localization was increased when wild-type (WT), but not AI-forms of FAM20A was co-transfected. On the other hand, extracellular FAM20C was absent in the conditioned media of mouse embryonic fibroblasts (MEFs) derived from Fam20a knock-out (KO) mouse, while it was detected in the media from WT MEFs. We also showed that cells with the conditioned media of Fam20a WT MEFs mineralized, but those with the conditioned media of KO MEFs failed to mineralize in vitro. Our data thus demonstrate that FAM20A controls FAM20C localization that may assist in the extracellular function of FAM20C in mineralized tissues.

Mechanical Loading Stimulates Expression of Collagen Cross‐Linking Associated Enzymes in Periodontal Ligament

Type I collagen, a major extracellular component of the periodontal ligament (PDL), is post‐translationally modified by a series of specific enzymes. Among the collagen‐modifying enzymes, lysyl oxidase (LOX) is essential to initiate collagen cross‐linking and lysyl hydroxylases (LHs) to regulate the cross‐linking pathways that are important for tissue specific mechanical properties. The purpose of this study was to investigate the effects of mechanical loading on the expression of collagen‐modifying enzymes and subsequent tissue changes in PDL. Primary human PDL cells were subjected to mechanical loading in a 3D collagen gel, and gene expression and collagen component were analyzed. Wistar rats were subjected to excessive occlusal loading with or without intra‐peritoneal injection of a LOX inhibitor, β‐aminopropionitrile (BAPN). Upon mechanical loading, gene expression of LH2 and LOX was significantly elevated, while that of COL1A2 was not affected on hPDL‐derived cells. The mechanical loading also elevated formation of collagen α‐chain dimers in 3D culture. The numbers of LH2 and LOX positive cells in PDL were significantly increased in an excessive occlusal loading model. Notably, an increase of LH2‐positive cells was observed only at the bone‐side of PDL. Intensity of picrosirius red staining was increased by excessive occlusal loading, but significantly diminished by BAPN treatment. These results demonstrated that mechanical loading induced collagen maturation in PDL by up‐regulating collagen‐modifying enzymes and subsequent collagen cross‐linking which are important for PDL tissue maintenance. J. Cell. Physiol. 231: 926–933, 2016.

Osteoblastic differentiation and mineralization ability of periosteum-derived cells compared with bone marrow and calvaria-derived cells.

PURPOSE Clinically, bone marrow stromal cells (BMCs) are the most common source of osteoprogenitor cells. Its harvest process, however, is invasive to patients. Previous reports have shown the potential advantages of using periosteum-derived cells (PDCs) as a source of cell-based transplant therapy. The objective of our study was to characterize the osteoblastic differentiation and mineralization ability of PDCs versus BMCs and osteoblasts (OBs). MATERIALS AND METHODS BMCs, OBs, and PDCs were isolated from 4-week-old male Wistar rats. To characterize the differentiation ability of the cells, MTS assay, alkaline phosphatase (ALP) activity staining, picrosirius red staining, and alizarin red staining were performed. Immunohistochemistry was performed on paraffin sections of calvarial periosteum to determine the presence of mesenchymal stem cells. RESULTS PDCs showed the greatest proliferation rate compared with BMCs and OBs. Matured collagenous matrix formation was observed in PDCs and BMCs. ALP-positive cells and in vitro mineralization were evident in all cell types analyzed; however, that of PDCs was not comparable to that of the OBs and BMCs. Immunohistochemistry revealed the presence of STRO-1-and CD105-positive cells in the cambium layer of the periosteum. CONCLUSIONS PDCs have remarkable proliferative ability, but contain only a small population of osteogenic cells compared with BMCs and OBs. Although cell activity can be affected by various factors, such as age, culture condition, additives, and so forth, PDCs are likely not the source of OBs, although they might provide matrices that indirectly aid in bone formation.

Recruitment of bone marrow-derived cells to the periodontal ligament via the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 axis

BACKGROUND/OBJECTIVES The periodontal ligament (PDL) is a non-mineralized connective tissue that exists between the alveolar bone and root surface cementum and plays important roles in tooth function. The PDL harbors a remarkable reserve of multipotent stem cells, which maintain various types of cells. However, the sources of these stem cells, other than their developmental origin, are not well understood. MATERIAL AND METHODS To elucidate the recruitment of bone marrow (BM)-derived stem cells in the PDL, green fluorescent protein (GFP)-expressing BM-derived cells were transplanted into the femoral BM of immunodeficient rats, and the distribution and expression of stem cell markers in the PDL were analyzed in vivo. To evaluate the functional significance of BM-derived cells to the PDL, tooth replantation was performed and the expression of stromal cell-derived factor (SDF)-1, a critical chemotactic signal for mesenchymal stem cell recruitment, was analyzed. To confirm the SDF-1-dependency of BM-derived cell migration to the PDL, PDL-conditioned medium (CM) was prepared, and BM-derived cell migration was analyzed using a transwell culture system. RESULTS Four weeks after cell transplantation, GFP-positive cells were detected in the PDL, and some of them were also positive for stem cell markers (i.e., CD29, SSEA4, and αSMA). Seven days after tooth replantation, the number of GFP- and SDF-1-positive cells significantly increased in PDL. Concurrently, the concentration of SDF-1 and the number of colony-forming units of fibroblasts in peripheral blood were increased. BM-derived cell migration increased in PDL-CM and was inhibited by an inhibitor of C-X-C chemokine receptor type 4 (CXCR4), an SDF-1 receptor. CONCLUSION These results indicate that stem cells and their progeny in PDL are not only derived from their developmental origin but are also supplied from the BM via the blood as the need arises. Moreover, this BM-derived cell recruitment appears to be regulated, at least partially, by the SDF-1/CXCR4 axis.

Functional Diversity of Ciliary Proteins in Bone Development and Disease

Purpose of ReviewThe primary cilium is a non-motile microtubule-based organelle that senses a diverse range of extracellular signals. While recent studies highlight the importance of ciliary-dependent developmental signals, including Hedgehog, Wnt, and platelet-derived growth factor, it is not well understood whether and how bone morphogenetic protein (BMP) signaling, a key regulator of skeletogenesis, is involved in cilia-related bone developmental aspects and in the etiology of skeletal disorders.Recent FindingsIncreasing evidence suggests that osteoblast- or osteocyte-specific deletion of ciliary proteins leads to diverse skeletal malformations, reinforcing the idea that primary cilia are indispensable for regulating bone development and maintenance. Furthermore, it became evident that ciliary proteins not only contribute to ciliogenesis but also orchestrate cellular trafficking.SummaryThis review summarizes the current understanding of ciliary proteins in bone development and discusses the potential role of BMP signaling in primary cilia, enabling us to unravel the potential pathogenesis of skeletal ciliopathies.

Immunohistochemical localization of tenascin-C in rat periodontal ligament with reference to alveolar bone remodeling

We investigated the immunohistochemical localization of tenascin-C in 8-week-old rat periodontal ligaments. Tenascin-C immunoreactivity was detected in zones along with cementum and alveolar bone, and more intensely on the resorption surface of alveolar bone than on the formation surface. On the resorbing surface, tenascin-C immunoreactivity was detected in Howship’s lacunae without osteoclasts, and in the interfibrous space of the periodontal ligaments, indicating that this molecule works as an adhesion molecule between bone and fibers of periodontal ligaments. Upon experimental tooth movement by inserting elastic bands (Waldo method), the physiological resorption surface of alveolar bone under compressive force showed enhanced bone resorption and enhanced tenascin-C immunoreactivity. However, on the physiological bone formation surface under compressive force, bone resorption was seen only occasionally, and no enhanced tenascin-C immunoreactivity was noted. In an experiment involving excessive occlusal loading to rat molars, transient bone resorption occurred within interradicular septa, but no enhanced tenascin-C immunoreactivity was seen in the periodontal ligaments. These results indicate that tenascin-C works effectively on the bone resorbing surface of physiological alveolar bone remodeling sites, rather than on the non-physiological transient bone resorbing surface. Fibronectin immunoreactivity was distributed evenly in the periodontal ligaments under experimental conditions. Co-localization of tenascin-C and fibronectin immunoreactivity was observed in many regions, but mutually exclusive expression patterns were also seen in some regions, indicating that fibronectin might not be directly involved in alveolar bone remodeling, but may play a role via interaction with tenascin-C.