Bone morphogenetic proteins (BMPs), which have been shown to be heparin-binding proteins, induce osteoblast differentiation in mesenchymal cells. In the present study, we examined the effects of heparin on the BMP activities in C2C12 myoblasts. Heparin dose dependently enhanced the osteoblast differentiation induced by not only homodimers of BMP-2 or BMP-4 but also heterodimers of BMP-2/6 or BMP-2/7. However, the osteoblast differentiation induced by the constitutively active BMPR-IA, a functional BMP type I receptor, was not affected by heparin. Heparan sulfate and dextran sulfate also enhanced the BMP-2 activity, although the chemically desulfated heparin-derivatives have lost this stimulatory capacity. Heparin dose-dependently suppressed the accumulation of BMP-2 from the culture media into the cell layer or BMPR-IA, and retained a large amount of BMP-2 in the culture media. The biological activity of BMP-2, which was evaluated using a BMP-responsive reporter gene expression, was prolonged in the presence of heparin. Taken together, these results suggest that sulfated polysaccharides enhance the biological activity of both homodimers and heterodimers of BMPs by continuously serving the ligands to their signaling receptors expressed on cell membranes. Bone morphogenetic proteins (BMPs)1 were originally identified as unique in demineralized bone matrix that could induce ectopic bone formation when implanted into muscular tissues (1, 2). More than 15 members of BMPs have been identified, and they are members of the TGF-␤ superfamily (3-7). They are classified into several BMP subfamilies based on their homology within the mature domains; the BMP-2 and BMP-4 subfamily, the BMP-5, BMP-6, BMP-7 (also called OP-1) and BMP-8 subfamily, the GDF-5, GDF-6 (BMP-13), and GDF-7 (BMP-12) subfamily, and the BMP-3 and GDF-10 (BMP-3b) subfamily (3-7). Most BMPs are first synthesized in large inactive preproproteins. They form homodimers or heterodimers via a disulfide bond in the mature domain, and then are secreted as active dimers after proteolytic processing (3-7). Several recombinant proteins of BMP dimers were reported to be active in an ectopic bone formation assay. Some heterodimers were shown to be more potent than each homodimer (8 -11). Several lines of evidence suggest that BMPs are key molecules for normal skeletal development in vertebrates (3, 5-7, 12). We previously reported that BMP-2 inhibits myogenic differentiation of C2C12 myoblasts, and converts their differentiation pathway into that of osteoblast lineage cells (13). Both the ectopic bone-inducing activity and the osteoblast differentiation-inducing activity appear to be unique for BMPs among members of the TGF-␤ superfamily.Signaling of BMPs is initiated by binding to the specific transmembrane receptors, type I and type II serine/threonine kinase receptors (6, 12, 14 -16). The type I receptors are activated by the ligand bound-type II receptors, and then phosphorylate Smad proteins as substrates in the cytoplasm. The phosphorylated Smad forms a complex ...

Previous studies have provided the biological basis for the therapeutic use of enamel matrix derivative (EMD) at sites of periodontal regeneration. A purpose of this study is to determine effects of EMD on cell growth, osteoblastic differentiation and insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 (TGF-beta 1) production in human periodontal ligament cells (HPLC). We also examined participation of endogenous IGF-I and TGF-beta 1 with EMD-stimulated cell growth in these cells. HPLCs used in this study were treated with EMD alone or in combination with antihuman IGF-I antibody (anti-hIGF-I) or anti-hTGF-beta 1, recombinant human bone morphogenetic protein-2 (rhBMP-2), 1,25-dihydroxyvitamin D3[1,25(OH)2D3], rhTGF-beta 1 or rhIGF-I. After each treatment, cell growth, the production of IGF-I and TGF-beta 1 and the expression of osteoblastic phenotypes were evaluated. EMD stimulated cell growth in dose-dependent and time-dependent manners. EMD was also stimulated to express IGF-I and TGF-beta 1 at protein and mRNA levels. The EMD-stimulated cell growth was partially suppressed by cotreatment with anti-hIGF-I or anti-hTGF-beta 1, and cell growth was also stimulated by treatment with rhIGF-I or rhTGF-beta 1. rhBMP-2 stimulated alkaline phosphatase (ALPase) activity and ALPase mRNA expression, and 1,25(OH)2D3 stimulated ALPase and osteocalcin mRNA expression. However, EMD showed no effect on the osteoblastic phenotypes expression. These results demonstrated that EMD has no appreciable effect on osteoblastic differentiation, however it stimulates cell growth and IGF-I and TGF-beta 1 production in HPLC, and that these endogenous growth factors partially relate to the EMD-stimulated cell growth in HPLC.

DPLC retain the capability to differentiate into an osteoblast lineage and may act in the regeneration of periodontal ligament with new cementum formation, whereas these cells may have a limited influence on alveolar bone formation during the course of periodontal regeneration.

We examined the possibility that periodontal ligament (PDL) cells can differentiate into osteoblasts and/or cementoblasts in freshly isolated PDL tissues and in cultured cells derived from PDL. PDL tissues were obtained from the incisor teeth of bovine lower jaws; gingival connective tissues of the same animals were used as controls. Freshly isolated PDL tissues and cultured PDL cells showed an intense alkaline phosphatase (ALPase) activity both histochemically and biochemically. The production of 3',5'-cyclic adenosine monophosphate (cAMP) was greatly increased in response to human parathyroid hormone [PTH(1-34)], in both freshly isolated PDL tissues and cultured PDL cells. In contrast, neither ALPase activity nor PTH-dependent cAMP production was detected in gingival connective tissues and cultured gingival fibroblasts. Furthermore, cultured PDL cells synthesized a protein immunologically cross-reactive with bovine bone gla protein (BGP), a highly reliable marker of osteoblastic cells. When 10(-8) M 1a, 25-dihydroxyvitamin D3 [1a,25(OH)2D3] was added to the PDL cell cultures, the synthesis of the BGP-like protein was increased 2- to 3-fold. The maximal level of the synthesis was obtained 72 h after the addition of 1a,25(OH)2D3. Gingival fibroblasts cultured with or without 1a,25(OH)2D3 did not produce any appreciable amounts of the BGP-like protein. These results indicate that the PDL cells have phenotypes typical of osteoblasts, indicating that they may differentiate into osteoblasts and/or cementoblasts.

We evaluated the clinical usefulness of an erbium:YAG laser for soft tissue surgery, and scaling. Thirty-one patients with soft tissue lesions (13 males and 18 females from 24 to 71 years old), and 60 patients with calculus deposits (21 males and 39 females from 19 to 72 years old) were treated with the laser. The clinical parameters evaluated were pain, redness, swelling of the gingiva, and the subjective patient comfort parameters including uneasiness with the sound and vibration associated with laser treatment. Additionally, hemorrhage and wound healing during and after the surgery, the roughness of the root surface after scaling, and the effectiveness of scaling using the laser were examined. Laser surgery caused less hemorrhage and resulted in better wound healing when compared with conventional methods. It was easy to remove calculi from the root surface with the laser in 95% of the cases. Although the scaled site showed some irregularity, it was not clinically significant in 98% of the cases. Only a few patients complained about the unpleasant sound and vibration. There were no complications or side effects during this clinical trial. Thus, this study suggests that an Er:YAG laser is useful for soft tissue surgery and scaling.

Human periodontal ligament cells activated with inflammatory factors such as IL-1beta and PGE2 may directly stimulate osteoclastogenesis through RANKL, which is stimulated to express by these factors.

Periodontal ligament cells may play an important role in the successful regeneration of the periodontium. We investigated the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2), one of the most potent growth factors that stimulates osteoblast differentiation and bone formation, on cell growth and osteoblastic differentiation in human periodontal ligament cells (HPLC) isolated from four adult patients. rhBMP-2 induced no significant changes in cell growth in any of the HPLCs. rhBMP-2 at concentrations over 50 ng/mL significantly stimulated alkaline phosphatase (ALPase) activity and parathyroid hormone (PTH)-dependent 3', 5'-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in the HPLCs. rhBMP-2 (500 ng/mL) also slightly enhanced the level of PTH/PTH-related peptide receptor mRNA expression in these cells. While interleukin-1 beta enhanced ALPase activity stimulated with rhBMP-2, tumor necrosis factor-alpha inhibited the rhBMP-2-stimulated activity. Interleukin-6 induced no significant changes in ALPase activity stimulated with rhBMP-2. Although HPLCs, whether treated with rhBMP-2 or not, could not produce measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced osteocalcin mRNA expression and protein synthesis in these cells. rhBMP-2 inhibited 1,25(OH)2D3-induced osteocalcin synthesis in HPLCs at both the mRNA and protein levels. These results suggest that rhBMP-2 provides an anabolic effect on periodontal regeneration by stimulation of osteoblastic differentiation in human periodontal ligament cells, and that this stimulatory effect is differentially modulated by inflammatory cytokines during the course of periodontal regeneration.

Mesenchymal stem cells (MSC), a distinct type of adult stem cell, are easy to isolate, culture, and manipulate in ex vivo culture. These cells have great plasticity and potential for therapeutic application, but their properties are poorly understood because of their low frequency and the lack of knowledge on cell surface markers and their location of origin. The present study was designed to address the undefined lineage relationship of hematopoietic and mesenchymal stem cells. Genetically marked, highly purified hematopoietic stem cells (HSCs) were transplanted into wild-type animals and, after bone marrow repopulation, the progeny were rigorously investigated for differentiation potential into mesenchymal tissues by analyzing in vitro differentiation into mesenchymal tissues. None/very little of the hematopoietic cells contributed to colony-forming units fibroblast activity and mesenchymal cell differentiation; however, unfractionated bone marrow cells resulted in extensive replacement of not only hematopoietic cells but also mesenchymal cells, including MSCs. As a result, we concluded that purified HSCs have no significant potency to differentiate into mesenchymal lineage. The data strongly suggest that hematopoietic cells and mesenchymal lineage cells are derived from individual lineage-specific stem cells. In addition, we succeeded in visualizing mesenchymal lineage cells using in vivo microimaging and immunohistochemistry. Flow cytometric analysis revealed CD140b (PDGFR␤) could be a specific marker for mesenchymal lineage cells. The results may reinforce the urgent need for a more comprehensive view of the mesenchymal stem cell identity and characteristics.