Hong In Shin

Surface immobilization of MEPE peptide onto HA/β-TCP ceramic particles enhances bone regeneration and remodeling†

Calcium phosphate ceramics have been widely used as scaffolds for bone regeneration. Here, to improve the osteogenic potential of hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) and to apply the bioactive peptide in situ, matrix extracellular phosphoglycoprotein (MEPE) peptide, which has been shown to stimulate osteoblast differentiation, was covalently and directionally immobilized on HA/β-TCP particles. The free-hydroxyl groups on the surface of the HA/β-TCP particles were sequentially conjugated with APTES, PEG-(SS)(2), and the synthetic MEPE peptide. Using FTIR and XPS, immobilization of the MEPE peptide on the HA/β-TCP was confirmed. Implantation of the MEPE peptide-immobilized HA/β-TCP into calvarial defect and subsequent analyses using a micro CT and histology showed significant bone regeneration and increased bone area (9.89-fold) as compared to that of unmodified HA/β-TCP. Moreover, tartrate-resistant acid phosphatase-positive osteoclasts were observed in regenerated bone by the MEPE peptide-immobilized HA/β-TCP, indicating that the bones newly formed by the MEPE peptide-immobilized HA/β-TCP are actively remodeled by osteoclasts. Therefore, our data demonstrate that MEPE peptide immobilization onto the HA/β-TCP surface stimulates bone regeneration associated with physiological bone remodeling.

Cytotoxicity and Antimicrobial Effect of Ag Doped Hydroxyapatite

In the synthesis of antimicrobial hydroxyapatite doped with silver, the amount of silver is the critical element affecting its properties. And also depending on the synthesis route, not only the amount of silver but also the release behavior was found to be altered. The cytotoxicity, antimicrobial effects and stability of effects were observed with Ag doped hydroxyapatite fabricated through various routes. The results suggested that the optimal antimicrobial property with none or mild cytotoxicity may be produced by modulating not only the doped silver amount but also their shape and synthesis route.

Effect of Ag-Doped Hydroxyapatite as a Bone Filler for Inflamed Bone Defects

Silver (Ag)-doped hydroxyapatite (HAp) agglomerates containing 0.15%, 1.5% and 4.3% mole % of silver among total cations, respectively, were evaluated in vitro and in vivo to explore their potential application as a bone filler with antibacterial properties. The 0.15% Ag-doped HAp was mildly cytotoxic, whereas the 1.5% and 4.3% Ag-doped HAps were moderately cytotoxic in the standard agar overlay cytotoxicity assay. The in vivo test was carried out by implanting Ag-HAps in artificial bone defects at the periapical area of both mandibular 1 molar of rats and no remarkable cytotoxicity was found unlike what was observed in the in vitro data. All of the implanted Ag-doped HAp particles, regardless of their Ag contents, allowed appropriated cellular proliferation and favorable bone repair without remarkable inflammatory reaction through 3 week healing periods, in spite of the mild delay in organization of fibrin and inflammatory reaction with the 4.3% Ag-doped HAp at the early healing phase. They supported well new bone formation with osteointegrative and osteoconductive properties. The results suggest that HAps doped with Ag up to 4.3 % of total cations can be applied for repair of infection-associated bony defects.

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.

Evaluation of 5% Na2O-Incorporated Calcium Metaphosphate as a Scaffold for Tissue-Engineered Bone Regeneration

As a part of the effort to develop a suitable scaffold for tissue-engineered bone regeneration, we modified calcium metaphosphate (CMP) ceramic with Na20 and evaluated its efficiency as a scaffold. We incorporate 5% Na20 into pure CMP and prepare for an average pore size of 250 or 450 µm average pore sizes. The incorporation of 5% Na2O caused reduced compressive strength and there was no change in biodegradability. The in vitro cellular attachment and proliferation rate, however, were slightly improved. The 5% Na2O-incorporated macroporous CMP ceramic-cell constructs treated with Emdogain induced ectopic bone formation more effectively than those without Emdogain treatment. These results suggest that the incorporation of 5% Na2O into pure CMP is not effective for improving the physical characteristics of pure CMP but it is positive for improving the cellular reaction and osteogenic effect with the addition of Emdogain.

Successful Osteoinduction by Cell-Macroporous Biphasic HA-TCP Ceramic Matrix

To improve the potential of osteogenic repair, we developed macroporous biphasic hydroxyapatite-tricalcium phosphate (HA-TCP) ceramic and evaluated its efficiency as a scaffold for tissue engineered bone regeneration, which allows for appropriate cellular attachment and proliferation with osteogenic differentiation by evaluating ectopic bone formation ability after the implantation of cell-matrix construct in the skid mice subcutaneous pouches for 3 weeks. The macroporous biphasic HA-TCP ceramic matrix, with an average porosity of 86% and 200 µm mean pore size, provided favorable conditions for the attachment of cultured bone marrow derived osteoblastic cells along its inner surfaces in a filed up pattern and the active proliferation of them. The implanted cell-matrix constructs in the subcutaneous pouches induced favorable ectopic bone formation without any remarkable inflammatory reactions. These findings suggest that the biphasic HA-TCP ceramic matrix with macroporous structure has excellent biocompatibility, and that it allows for favorable cellular attachment with the acceleration of cellular proliferation and osteogenic differentiation support as well. Thus, with the controlled biodegradability, the biphasic HA-TCP ceramic may be a promising scaffold for tissue engineered bone regeneration technology.

Human Cartilage Tissue Engineering with Pluronic and Cultured Chondrocyte Sheet

Despite many outstanding research works on cartilage tissue engineering, actual clinical application is not quite successful because of the absorption and progressive distortion of tissue engineered cartilage. We have developed a new method of cartilage tissue engineering comprising chondrocyte mixed Pluronic F-127 and cultured chondrocyte cell sheet which entirely cover the cell-Pluronic complex. We believe the addition of cultured chondrocyte cell sheet enhances the efficacy of chondrogenesis in vivo. Human ear cartilage piece was enzymatically dissociated and chondrocyte suspension was acquired. Chondrocytes were cultured and expanded as the routine manner. Cultured chondrocytes were plated in high-density monolayer and cultured with Chondrogenic media in 5% CO2 incubator. After 3 weeks of culture, chondrocyte cell sheet was formed and complete single sheet of chondrocyte could be harvested by gentle manipulation of culture plate with a cell scraper. Chondrocyte-Pluronic complex was established by mixing 1x 106 cells with 0.5 of Pluronic F- 127. Chondrocyte-Pluronic complex was completely covered with a sheet of cultured chondrocyte. The completed tissue engineered constructs were implanted into the subcutaneous tissue pocket of nude mice on the back. Tissue engineered constructs without cultured cell sheet were used as control. Samples were harvested at 8 weeks postoperatively and they were subjected to histological analysis and assayed for glycosaminoglycan (GAG), and type II collagen. Grossly, the size of cartilage specimen of cultured chondrocyte cell sheet covered group was larger than that of the control. On histologic examination, the specimen of cultured chondrocyte cell sheet covered group showed lacunae-containing cells embedded in a basophilic matrix. The chondrocyte cell sheet covered group specimen resembled mature or immature cartilage. The result of measurement of GAG and type II collagen of cartilage specimen of cultured chondrocyte sheet covered group was higher than that of the control. In conclusion, the new method of cartilage tissue engineering using chondrocyte cell sheet seems to be an effective method providing higher cartilage tissue gain and reliable success rate for cartilage tissue engineering.

In Vivo Evaluation of Macroporous Calcium Metaphosphate Ceramic as a Bone Substitute

Calcium metaphosphate(CMP) ceramic in the form of a macroporous block was evaluated as a bone substitute in vivo. Macroporous calcium metaphosphate ceramic blocks with a mean pore size of 250 μm were implanted into either subcutanous pouches or artificial tibial bone defects in rats, and biodegradibility, biocompatibility and osteoconductivity were analyzed, respcectively. The macroporous CMP ceramic blocks implanted into subcutanous pouches permitted ingrowth of vascularized connective tissue without an inflammatory response or a foreign body reaction for 3 weeks after implantation. In addition, there was no remarkable weight change during 3 weeks implanted in subcutanous pouches. The macroporous CMP ceramic blocks implanted into bony defects revealed a favorable connection and direct fusion of newly formed bone from local osseous margin with their framework without insertion of fibrous connective tisssue and did not evoke an inflammatory reaction. The excellent biocompatibility,osteoconductivity, biomechanical strength, and ease of handling fullfill the requirement as a promising bone substitute.

Osteogenic Repair by Bovine Bone Ash Derived Porous HA Ceramic Formed by Foaming Method

To develop a suitable scaffold optimizing bone regeneration, we developed bovine bone ash derived fully connected porous HA ceramic scaffolds adopting a foaming method. They revealed excellent biocompatibility. The attached cells on the scaffolds proliferated in multi-layers with osteoblastic differentiation. The bone defects grafted with bovine bone ash derived fully interconnected porous HA ceramics having average 500 μm sized spherical pores and average 150 μm sized interconnecting interpores with average 80% porosity were favorably healed without any pathologic changes within 3 weeks. New bone ingrowth with excellent osteoconduction through the spherical pores along the inner surface was noted from 1 week after implantation. Each spherical pore was filled with hematopoietic marrow and newly formed bone which with time was well integrated with the porous HA ceramic scaffold with time. These findings suggest that the bovine bone ash-derived fully interconnected porous HA ceramic formed by foaming method can be a promising bone substitute and a scaffold for bone tissue engineering.

Bone Repair Efficiency by Various Round Granular Bone Substitutes

To improve the efficiency of osteogenic repair, we compared 3 types of round granular bone substitutes composed of hydroxyapatite (HA) in a single opened large pore covered by one or more thin shell structure, biphasic HA and tricalcium phosphate (TCP) in a compact granules with small uniform interconnected internal pores, and bioglass(BG) in a compact granules with hierarchical interconnected pores its bone repair efficiency by evaluation of cellular toxicity, cellular attachment and proliferation rate, and osteogenic supportive effect. They were nontoxic and revealed no noxious effect on cellular proliferation and osteoblastic differentiation. The cultured cells were most effectively proliferated on HA granular bone substitute surface. However, the bony repair of calvarial defects was most effective by BCP granular bone substitutes. The implanted BCP and HA granular bone substitutes showed excellent osteoconductive bone growth and favorable bone regeneration within 3 weeks compared to BG granular bone substitutes. All type granular bone substitutes were well incorporated into newly formed bone without foreign body reaction. Except for HA granular bone substitute, some implanted BG and BCP granular bone substitutes were partially resobed by TRAP positive multinucletated cells. These findings suggest that round granular biphasic calcium phosphate bone substitute structured with fully interconnected uniform sized internal pore might be a more promising bone substitute for small-sized none load-bearing bone defects.