Jiwon Lim

Secretome analysis of human BMSCs and identification of SMOC1 as an important ECM protein in osteoblast differentiation.

Extracellular matrix proteins have been implicated in the regulation of osteoblast differentiation of bone marrow derived mesenchymal stem cells (BMSCs) through paracrine or autocrine mechanisms. In the current study, we analyzed the secretory protein profiles of BMSCs grown in osteogenic medium (OSM) and identified SPARC-related modular calcium-binding protein 1 (SMOC1), a member of the SPARC family, as a regulator of osteoblast differentiation of BMSCs. BMSCs with high and low osteogenic potential were grouped and stimulated with OSM, after which conditioned medium was collected and analyzed by LC-MS/MS. We identified 410 proteins, 64 of which were selectively secreted by high osteogenic potential BMSCs. Of these 64 secreted proteins, we selected extracellular matrix proteins for validation in BMSCs undergoing osteoblast differentiation and found that SMOC1 is highly expressed and secreted in BMSCs stimulated with OSM. To examine the role of SMOC1 in osteoblast differentiation, we analyzed the effect of SMOC1 knockdown and overexpression using shRNAs and wild-type cDNA, respectively. Knockdown of SMOC1 significantly inhibited mineralization and the expression of osteoblast differentiation markers, while overexpression of SMOC1 substantially increased the expression of osteoblast differentiation-related genes. Thus, validation of secretome profiling data identified SMOC1 as a putative regulator of osteoblast differentiation of BMSCs.

Enhanced ex vivo expansion of human adipose tissue-derived mesenchymal stromal cells by fibroblast growth factor-2 and dexamethasone.

In the present study, we investigated the ex vivo expansion of human adipose tissue-derived mesenchymal stromal cells (ATSCs) to identify factors that promoted efficient expansion while preserving stem cell potential. We examined several growth factors and steroids, and found that the combination of a low concentration of fibroblast growth factor-2 (FGF-2) (1 ng/mL) and dexamethasone (DEX) or betamethasone (BET) enhanced the proliferation of ATSCs by approximately 30-60% as compared to control. Enhanced proliferation under these conditions was confirmed using ATSCs isolated from three independent donors. ATSCs that were expanded in the presence of FGF-2 and DEX for 5 days were capable of differentiating into either osteoblastic or adipogenic cells, and the cells were positive for the mesenchymal stem cell markers such as CD29, CD44, CD90, CD105, and CD146, suggesting that the stem cell potential of the ATSCs was preserved. Analysis of signaling pathway revealed that tyrosine phosphorylation of Src kinase was dramatically increased in response to FGF-2 and DEX, suggesting the involvement of Src-dependent pathways in the stimulatory mechanism of proliferation of ATSCs by FGF-2 and DEX. Moreover, Src family kinase inhibitors (SU6656 and Src kinase inhibitor I) substantially reduced the FGF-2 and DEX-induced proliferation of ATSCs. SU6656 also inhibited the osteogenic and adipogenic differentiation of ATSCs. The results of the current study demonstrate that FGF-2 in combination with DEX stimulates the proliferation and osteoblastic and adipogenic differentiation of ATSCs through a Src-dependent mechanism, and that FGF-2 and DEX promote the efficient ex vivo expansion of ATSCs.

A simultaneous process of 3D magnesium phosphate scaffold fabrication and bioactive substance loading for hard tissue regeneration

A novel room temperature process was developed to produce a 3D porous magnesium phosphate (MgP) scaffold with high drug load/release efficiency for use in hard tissue regeneration through a combination of a paste extruding deposition (PED) system and cement chemistry. MgP scaffolds were prepared using a two-step process. The first step was fabrication of the 3D porous scaffold green body to control both the morphology and pore structure using a PED system without hardening. The second step was cementation, which was carried out by immersing the scaffold green body in the binder solution for hardening instead of the typical sintering process in ceramic scaffold fabrication. Separation of the manufacturing process and cement reaction was important to secure enough time to fabricate a 3D scaffold with various sizes and architectures under homogeneous extruding conditions. Because the whole process is carried out at room temperature, the bioactive molecules, which are easily denatured by heat, may apply to scaffolds during the process. Lysozyme was selected as a model bioactive substance to demonstrate the efficiency of this process; this was directly mixed into MgP powder to introduce homogeneous distribution in the scaffold. The extruding paste for the PED system was prepared using the MgP-lysozyme blended powder as starting materials. That is, both 3D scaffold fabrication and functionalization of the scaffold with bioactive substances could be carried out simultaneously. This process significantly enhanced both drug loading efficiency and release performance compared to the typical sintering process, where the drug is generally loaded by adsorption after heat treatment. The MgP scaffold developed in this study satisfied the required conditions for scaffolding in hard tissue regeneration in an ideal manner, including 3 dimensionally well-interconnected pore structures, favorable mechanical properties, biodegradability, good cell affinity and in vitro biocompatibility; thus, it has excellent potential for application in the field of biomaterials.

Effect of the biodegradation rate controlled by pore structures in magnesium phosphate ceramic scaffolds on bone tissue regeneration in vivo.

UNLABELLED Similar to calcium phosphates, magnesium phosphate (MgP) ceramics have been shown to be biocompatible and support favorable conditions for bone cells. Micropores below 25μm (MgP25), between 25 and 53μm (MgP53), or no micropores (MgP0) were introduced into MgP scaffolds using different sizes of an NaCl template. The porosities of MgP25 and MgP53 were found to be higher than that of MgP0 because of their micro-sized pores. Both in vitro and in vivo analysis showed that MgP scaffolds with high porosity promoted rapid biodegradation. Implantation of the MgP0, MgP25, and MgP53 scaffolds into rabbit calvarial defects (with 4- and 6-mm diameters) was assessed at two times points (4 and 8weeks), followed by analysis of bone regeneration. The micro-CT and histologic analyses of the 4-mm defect showed that the MgP25 and MgP53 scaffolds were degraded completely at 4weeks with simultaneous bone and marrow-like structure regeneration. For the 6-mm defect, a similar pattern of regeneration was observed. These results indicate that the rate of degradation is associated with bone regeneration. The MgP25 and MgP53 scaffold-implanted bone showed a better lamellar structure and enhanced calcification compared to the MgP0 scaffold because of their porosity and degradation rate. Tartrate-resistant acid phosphatase (TRAP) staining indicated that the newly formed bone was undergoing maturation and remodeling. Overall, these data suggest that the pore architecture of MgP ceramic scaffolds greatly influence bone formation and remodeling activities and thus should be considered in the design of new scaffolds for long-term bone tissue regeneration. STATEMENT OF SIGNIFICANCE The pore structural conditions of scaffold, including porosity, pore size, pore morphology, and pore interconnectivity affect cell ingrowth, mechanical properties and biodegradabilities, which are key components of scaffold in bone tissue regeneration. In this study, we designed hierarchical pore structure of the magnesium phosphate (MgP) scaffold by combination of the 3D printing process, self-setting reaction and salt-leaching technique, and first studied the effect of pore structures of bioceramic scaffolds on bone tissue regeneration through both in vitro and in vivo studies (rabbit calvarial model). The MgP scaffolds with higher porosity promoted more rapid biodegradation and enhanced new bone formation and remodeling activities at the same time.

Gene expression profiling of oral epithelium during tooth development.

OBJECTIVE Tooth development is regulated by the complex interplay of various regulatory molecules. To identify new regulatory genes released from the dental epithelium, gene expression profiling of dental epithelium was analysed. DESIGN ICR mouse dental epithelia were isolated from the initiation (E10.5) and bell (E16.5) stages, and microarray analysis was performed using Affymetrix GeneChip(®). Microarray data were validated using reverse transcriptase polymerase chain reaction (RT-PCR), and gene ontology and signalling network were analysed. RESULTS Detection signals more than 300 and changes more than two folds were considered as positive signals and were further analysed. Expressions of 193 genes in the E10.5 epithelium and 582 genes in the E16.5 epithelium were significantly increased. Validation of the selected genes using RT-PCR showed a well correlation with microarray data. Subsequent signalling network analysis revealed that at E10.5 and 16.5, nine genes such as histones, signalling molecules and transcription factors were closely related with neighbouring molecules. Moreover, gene ontology analysis showed that seven growth factors/receptors or secreted proteins were highly expressed at E10.5, including the platelet-derived growth factor, C polypeptide (Pdgfc), insulin-like growth factor binding protein 2 (Igfbp2) and Igfbp5. At E16.5, nine growth factors/receptors or secreted proteins, including Igfbp3, Igfbp10/Cyr61 and heparin-binding EGF-like growth factor (Hbegf) were highly expressed. CONCLUSIONS These data suggest that the regulatory genes newly identified in this study may play significant roles in tooth development.

Comparison of bone regeneration rate in flat and long bone defects: Calvarial and tibial bone

An ideal scaffold for bone tissue regeneration should be dissolved at the same rate of host bone growth into the defect. Therefore, to produce such a scaffold, it is necessary to obtain a standard healing rate of bone defects. In this study, we compared healing rate of bone defects in calvarial and long bones, which have differential developmental and regenerative mechanisms. In the calvaria and tibia, 3 mm defects were made, and healing was analyzed using microcomputed tomography (microCT) and histology up to six weeks. MicroCT analysis showed that in calvarial defects, an unhealed gap remained until six weeks, whereas tibial defects had healed after three weeks. H&E and Trichrome staining consistently showed that calvarial defects were not completely healed by six weeks, however, a tibial defect started to heal from three weeks. Results of histomorphometric analysis showed that 60% of calvarial defects had healed at six weeks after surgery, whereas 80% of tibial defects showed regeneration at three weeks. Cartilage formation was detected only in tibial defects, suggesting endochondral regeneration in long bone, but not in flat bone. Collectively, these results demonstrate that healing of a long bone defect is faster than that of flat bone by approximately two folds. Therefore, our data suggest that dissolution of scaffold should be optimized based on the type of bone defect.

Fully Interconnected Globular Porous Biphasic Calcium Phosphate Ceramic Scaffold Facilitates Osteogenic Repair

An appropriate scaffold, which provides structural support for transplanted cells and acts as a vehicle for the delivery of biologically active molecules, is critical for tissue engineering. We developed a fully interconnected globular porous biphasic calcium phosphate ceramic scaffold by adopting a foaming method, and evaluated its efficiency as a bone substitute and a scaffold for bone tissue engineering by in vitro and in vivo biocompatible analysis and its osteogenic healing capacity in rat tibial bone defects. They have spherical pores averaging 400um in diameter and interconnecting interpores averaging 70um in diameter with average 85% porosity. They elicited no cytotoxicity and noxious effect on cellular proliferation and osteoblastic differentiation during the cell-scaffold construct formation. Also the bone defects grafted with fully interconnected globular porous biphasic calcium phosphate ceramic blocks revealed excellent bone healing within 3 weeks. These findings suggest that the fully interconnected porous biphasic calcium phosphate scaffold formed by the foaming method can be a promising bone substitute and a scaffold for bone tissue engineering.

Effect of Fibroblast Growth Factor-2 and Retinoic Acid on Lineage Commitment of Bone Marrow Mesenchymal Stem Cells

In this study, we examined the effect of a combination of fibroblast growth factor-2 (FGF-2) and retinoic acid (RA) on osteoblast and adipocyte lineage commitment and differentiation of human bone marrow mesenchymal stem cells (BMSCs). Pretreatment of human BMSCs with FGF-2 or RA for 5 days followed by osteoblast differentiation induction showed high calcium deposition compared to control. A combination of FGF-2 and RA further induced calcium deposition compared to FGF-2 or RA alone. The enhanced mineral deposition was accompanied with the increased expression of osteoblast differentiation markers, alkaline phosphatase and osteocalcin. On the other hand, FGF-2 pretreatment followed by adipocyte differentiation induction also showed increased formation of lipid droplets in human BMSCs, whereas RA pretreatment suppressed formation of lipid droplets. However, a combination of FGF-2 and RA increased formation of lipid droplets and expression of adipocyte marker genes, including adiponectin, ADIPOQ, FABP4, peroxisome proliferator-activated receptor γ (PPARγ), and C/EBPα. During pretreatment of BMSCs with FGF-2, RA or in combination, the cells expressed similar levels of MSC surface markers such as CD29, CD44, CD90, and CD105, indicating that they maintain stem cell potential. To determine how RA cooperates with FGF-2 in osteoblast and adipocyte lineage commitment, the expression of RA receptors and intracellular lipid-binding proteins was examined. A combination of FGF-2 and RA strongly induced the expression of RA receptor α, β, γ, PPAR β/δ, CRABP-II, and FABP5. Collectively, these results demonstrate that combined pretreatment of human BMSCs with FGF-2 and RA enhances the commitment into osteoblast and adipocyte lineages through modulation of the expression of RA-related genes.