Soo-Boo Han

Tissue-engineered growth of bone by marrow cell transplantation using porous calcium metaphosphate matrices

In this study we investigated not only osteoblastic cell proliferation and differentiation on the surface of calcium metaphosphate (CMP) matrices in vitro but also bone formation by ectopic implantation of these cell-matrix constructs in athymic mice in vivo. Interconnected porous CMP matrices with pores 200 microm in size were prepared to use as scaffolds for rat-marrow stromal-cell attachment. Cell-matrix constructs were cultured in vitro, and cell proliferation and ALPase activities were monitored for 56 days. In addition to their being cultured in vitro, cell-matrix constructs were implanted into subcutaneous sites of athymic mice. In vitro these porous CMP matrices supported the proliferation of osteoblastic cells as well as their differentiation, as indicated by high ALPase activity. In vivo the transplanted marrow cells gave rise to bone tissues in the pores of the CMP matrices. A small amount of woven bone formation was detected first at 4 weeks; osteogenesis progressed vigorously with time, and thick lamellar bones that had been remodeled were observed at 12 weeks. These findings demonstrate the potential for using a porous CMP matrix as a biodegradable scaffold ex vivo along with attached marrow-derived mesenchymal cells for transplantation into a site for bone regeneration in vivo.

N-acetylcysteine prevents LPS-induced pro-inflammatory cytokines and MMP2 production in gingival fibroblasts.

Periodontitis is an inflammatory process that ultimately results in tooth loss. Although the primary etiologic agent for periodontitis is bacteria, the majority of periodontal tissue destruction is thought to be caused by an inappropriate host response. Reactive oxygen species (ROS) have been known to be involved in periodontal tissue destruction. We treated human gingival fibroblasts with lipopolysaccharide (LPS) obtained fromE. coli and the periodontopathogensActinobacillus actinomycetemcomitans andPorphyromonas gingivalis, and examined their inflammatory responses in the presence and absence of the antioxidant N-acetylcysteine (NAC). LPS enhanced ROS production, as well as, expression of pro-inflammatory cytokines such as interleukin-1β, interleukin-6, interleukin-8 and tumor necrosis factor-α, and the production and activation of MMP2. NAC suppressed all LPS-induced inflammatory responses examined, suggesting that LPS-induced ROS may play a major regulatory role in these responses in gingival fibroblasts. In addition, NAC prevented LPS-induced activation of p38 MAPK and JNK but not phosphorylation and subsequent degradation of IkB. These results indicate that NAC exerts anti-inflammatory effects in LPS-stimulated gingival fibroblasts, functioning at least in part via down-regulation of JNK and p38 MAPK activation. Furthermore, this work suggests that antioxidants may be useful in adjunctive therapies that complement conventional periodontal treatments.

The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells

Platelet-rich plasma (PRP) is an emerging therapeutic application because PRP contains various growth factors that have beneficial effects on tissue regeneration and engineering. Mesenchymal stem cells and PRP derived from peripheral blood have been well studied. In this study, we investigated the effects of PRP derived from human umbilical cord blood (UCB-PRP) on proliferation, alkaline phosphatase (ALP) activity, and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs), dental pulp stem cells (DPSCs), and periodontal ligament stem cells (PDLSCs). Three types of dental stem cells were primarily isolated and characterized by flow cytometric analysis. Dental stem cells were exposed to various concentrations of UCB-PRP, which resulted in the proliferation of dental stem cells. Treatment with 2% UCB-PRP resulted in the highest level of proliferation. The ALP activity of DPSCs and PDLSCs increased following treatment with UCB-PRP in a dose-dependent manner up to a concentration of 2%. ALP activity decreased with higher concentration of UCB-PRP. The effects of UCB-PRP on calcium deposition were similar to those on proliferation and ALP activity. Treatment with 2% UCB-PRP resulted in the highest calcium depositions in DPSCs and PDLSCs; however, treatment with 1% UCB-PRP resulted in the highest calcium deposition in SHEDs. The concentrations of platelet-derived growth factor-AB and transforming growth factor-β1 in UCB-PRP were investigated and found to be comparable to the amounts in peripheral blood. Overall, UCB-PRP had beneficial effects on the proliferation and osteogenic differentiation of dental stem cells. Determination of the optimal concentration of UCB-PRP requires further investigation for clinical applications.

Design and biological activity of synthetic oligopeptides with Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) motifs for human osteoblast-like cell (MG-63) adhesion

A synthetic oligopeptide, composed of Arg-Gly-Asp (RGD) and its synergistic Pro-His-Ser-Arg-Asn (PHSRN) motifs and a six glycines (G6) linker, promoted human osteoblast-like cell (MG-63) adhesion, spreading and mitogen-activated protein kinase (MAPK) activity in a similar manner to a positive fibronectin control. This synthetic oligopeptide may therefore be a useful osteo-inductive material.

Photoemission, soft x-ray absorption, and magnetic circular dichroism spectroscopy study of Fe1−xCuxCr2S4 (0.1≤x≤0.5) spinel sulfides

The electronic and magnetic structures of Fe1−xCuxCr2S4 (0.1≤x≤0.5) spinel sulfides have been investigated systematically by performing photoemission spectroscopy (PES), soft x-ray absorption spectroscopy (XAS), and soft x-ray magnetic circular dichroism (XMCD) measurements using synchrotron radiation. Cr and Cu ions are found to be nearly trivalent (Cr3+) and monovalent (Cu+), respectively, and their valence states do not change with x. The Fe 2p XAS spectra of Fe1−xCuxCr2S4 are very similar to that of Fe metal, indicating that the Fe 3d electrons are strongly hybridized to other valence electrons. The Fe and Cr 2p XMCD spectra show that the magnetic moments of Cr ions and Fe ions are aligned antiparallel to each other and that both the Cr and Fe magnetic moments increase with increasing x. The valence-band PES study reveals that the Cr3+ () 3d states are located at ~1.5 eV below EF. The occupied Fe 3d states consist of the broad states, the states at ~4 eV below EF, and the states very close to EF. The filled Cu 3d10 states lie at ~2.5 eV below EF. This study suggests that the hybridized Fe and S 3p states near EF play an important role in determining the transport properties of Fe1−xCuxCr2S4 for x≤0.5.

Osteogenic effects of bone‐morphogenetic‐protein‐2 plasmid gene transfer

The aim of the present study was to test the osteogenic effects of BMP‐2 (bone morphogenetic protein‐2) gene transfer in BMSCs (bone‐marrow stromal cells) and rabbit calvarial bone defects. The pBMP‐2‐cDNA3.1 plasmid was constructed by subcloning hBMP‐2 (human BMP‐2) cDNA into the plasmid pcDNA3.1. BMSCs were transfected with a pBMP‐2‐cDNA3.1–Lipofect‐amine™ complex. Transfected cells were observed for localization of the BMP‐2 coding plasmid. Also, the level of BMP‐2 in the culture medium of transfected cells was measured. The culture medium was collected and we tested whether this medium could induce non‐transfected BMSCs to express ALP (alkaline phosphatase) and osteocalcin. The pBMP‐2‐cDNA3.1 complexes were incorporated into the collagen scaffold and the plasmid‐loaded collagen scaffolds were then grafted into rabbit calvarial defects. After 2 weeks, granulation tissue at the grafted site was obtained and mRNA of BMP‐2 was examined via RT (reverse transcriptase)–PCR. After 4 and 8 weeks, the animals were killed and the calvarial tissue was excised. After specimen preparation, optical microscopical examination was performed to evaluate bone formation. The results show that transfected cells were able to incorporate the BMP‐2 gene into their nuclei. Also, the level of expressed and secreted BMP‐2 was significantly higher in transfected cells than in untransfected cells (P<0.01). The retrieved culture medium could induce the expression of ALP and osteocalcin in non‐transfected BMSCs. hBMP‐2 mRNA was detected at the granulation tissue of experimental animals, but not in control animals after 2 weeks. At both 4 and 8 weeks, experimental groups showed significantly more newly formed bone area than the control group (P<0.01). Therefore pBMP‐2‐cDNA3.1 gene delivery could induce BMSCs into osteoblastic phenotype cells and enhance bone regeneration in rabbit calvarial bone defects.