Annals of Biomedical Engineering ( 2015) DOI: 10.1007/s10439-015-1347-y

Bone Tissue Engineering by Using Calcium Phosphate Glass Scaffolds and the Avidin–Biotin Binding System MIN-CHUL KIM,1 MIN-HO HONG,2 BYUNG-HYUN LEE,1 HEON-JIN CHOI,2 YEONG-MU KO,3 and YONG-KEUN LEE3 1

Department of Applied Life Science, Yonsei University College of Dentistry, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea; 2Department of Materials Science and Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea; and 3Research Center for Oral Disease Regulation of the Aged, Chosun University School of Dentistry, 309 Pilmundaero, Dong-gu, Gwangju 501-759, Korea (Received 10 March 2015; accepted 25 May 2015) Associate Editor Jane Grande-Allen oversaw the review of this article.

Abstract—Highly porous and interconnected scaffolds were fabricated using calcium phosphate glass (CPG) for bone tissue engineering. An avidin–biotin binding system was used to improve osteoblast-like cell adhesion to the scaffold. The scaffolds had open macro- and micro-scale pores, and continuous struts without cracks or defects. Scaffolds prepared using a mixture (amorphous and crystalline CPG) were stronger than amorphous group and crystalline group. Cell adhesion assays showed that more cells adhered, with increasing cell seeding efficiency to the avidin-adsorbed scaffolds, and that cell attachment to the highly porous scaffolds significantly differed between avidin-adsorbed scaffolds and other scaffolds. Proliferation was also significantly higher for avidin-adsorbed scaffolds. Osteoblastic differentiation of MG-63 cells was observed at 3 days, and MG-63 cells in direct contact with avidin-adsorbed scaffolds were positive for type I collagen, osteopontin, and alkaline phosphatase gene expression. Osteocalcin expression was observed in the avidin-adsorbed scaffolds at 7 days, indicating that cell differentiation in avidin-adsorbed scaffolds occurred faster than the other scaffolds. Thus, these CPG scaffolds have excellent biological properties suitable for use in bone tissue engineering. Keywords—Bone tissue engineering, Calcium phosphate glass, Scaffold, Avidin–biotin binding system.

Address correspondence to Yong-Keun Lee, Research Center for Oral Disease Regulation of the Aged, Chosun University School of Dentistry, 309 Pilmun-daero, Dong-gu, Gwangju 501-759, Korea. Electronic mail: [email protected] Min-Chul Kim and Min-Ho Hong have contributed equally to this work.

INTRODUCTION Because of the limitations of autografts and allografts, with respect to limited donor tissue sites, tissue rejection, and disease transfer, the use of synthetic bone graft substitute materials has increased.4,11 Tissue engineering has been defined as an interdisciplinary field that applies biotechnology principles towards the development of biological substitutes.23 Thus, the engineering of bone for tissue regeneration, as opposed to using a simple bone replacement, presents an alternative approach to repair bony defects by encouraging new bone ingrowth at the damaged site. There are numerous aspects involved in tissue engineering, which are attributed to the significant undertaking of this work.29 Several groups are investigating the optimal chemical and physical properties of new materials and how they interact with cells to produce neo-tissue constructs. These materials can be biodegradable or permanent, and may be categorized as natural, synthetic, or hybrid materials. Biomaterials must be congruous with tissues or with cells in vitro and in vivo, and the biomaterial interface with cells and the defect site must be clearly understood so that optimal interactions are attained. Identification of the most favorable biomaterial design characteristics presents a major challenge for the field, as the molecular chemical level must be considered. Biomaterial design should take into account the specific replacement therapy being considered, and recently, biomaterials incorporating biological signaling molecules have been designed.29 Scaffolds for bone tissue engineering require basic characteristics, such as adequate mechanical and

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biological properties for tissue regeneration. First, the scaffold must have high porosity and adequate pore size with a high surface area. Second, biodegradability is generally required with a proper degradation rate. Third, the scaffold must have the required mechanical strength to maintain the pre-designed tissue structure. Forth, the scaffold should not be cytotoxic. Fifth, the scaffold should enhance cell adhesion, structural support for cell function of proliferation and differentiation, and scaffold architect proliferation, and differentiation.8,26 Scaffolds with sufficient mechanical strength provide the sure has been shown to ultimately define the shape of regenerated bone.7 In addition, the scaffold should have an ideal tissue replacement function through sequential regeneration cycles.12 Therefore, when osteoblasts are seeded into a scaffold, the scaffold should be controlled the degradation and resorption rate for the neo-tissue structure growth.23 In hard tissue engineering, a scaffold is used as a temporary carrier for seeded osteoblastic cells. Scaffold structure has been shown to control osteoblast behavior.18,25 In general, there are two different approaches to tissue engineering. In the first strategy, cells are seeded into the scaffold to enhance the secretion of extracellular matrix (ECM) and the regeneration of tissue in vitro, before implantation. The second strategy consists of the development of porous and interconnective materials with the capacity to cells and guide hard tissue regeneration in vivo. In this respect polymers and ceramics have been proposed for the research and development of these scaffolds. Bioglasses consisting of calcium phosphate (CaP) represent an interesting form of biodegradable material for hard tissue engineering constructs.27 Since there is some flexibility in the composition of glass-based materials, they offer a significant advantage in that their chemical/physical properties and microstructures after crystallization are controllable, thereby resulting in improved biomaterial performance. Calcium phosphate glass (CPG)-based materials have great potential for use as bone grafts because their chemical composition closely resembles that of hard tissues.19 Cell attachment is a critical consideration to biotechnological applications.23 Adhesion to biological factors absorbed onto the surface of scaffold is especially important to host-implant interactions in the field of tissue engineering. Immediately immersing in physiological fluids, many biological factors adsorb onto the surface of scaffold and control the subsequent inflammatory response.2 Consequently, the modified surface has abilities that either prevent or enhance the adhesion of specific cells represents an immensely fruitful field of biomaterial research. Cell attachment to the ECM is principally mediated by integrins.15 Integrin-mediated adhesion is related to a complex

cascade of biochemical and biomechanical events regulating through several mechanisms.9,10,15 Avidin and biotin are broadly used in the field of biotechnology because of their highly specific and stable complex. The avidin–biotin binding system (ABBS) has been demonstrated to convert non-adhesive Ehrlich ascites carcinoma cells to anchoragedependent cells.6 Recently, research groups reported using ABBS to attach cells to the surfaces of nonporous two-dimensional (2D) and three-dimensional (3D) scaffolds.1,5 These studies demonstrated that ABBS increased cell attachment and migration. Other research groups also reported using ABBS for cell attachment to the surface of 2D materials made of several biodegradable polymers.28 This binding system may allow easier cell handling for tissue engineering purposes, because it does not severely inhibit cell proliferation and differentiation.22 However, there have been no reports of using ABBS to seed osteoblasts onto the surface of highly porous and interconnective 3D CaP scaffolds. The effect of ABBS reactions on the efficiency of the initial attachment, proliferation, and differentiation of a human osteoblast-like cell line, MG-63, to flat dense disks and highly porous 3D scaffolds was described in this paper. For this study, we fabricated CPG scaffolds in the form of sponges, consisting of amorphous and crystallized CPG to enhance the scaffold mechanical properties in our previous study.20 In this study, an ABBS was applied to the scaffold to improve adhesion between osteoblast-like cells and the fabricated scaffold. The objective of this study was to examine the scaffolds for bone tissue engineering, thus, we determined the biocompatibility of the scaffolds using ABBS.

MATERIALS AND METHODS Fabrication of Calcium Phosphate Glass Scaffold CPG (CaO–CaF2–P2O5–Na2O) was prepared with a Ca/P ratio of 0.55 using raw materials, consisting of CaCO3, CaF2, H3PO4, and Na2CO3.13 Mixed batches were dried and melted in a platinum crucible at 1200 C. After melting, the solution was poured onto a copper plate at room temperature (RT) to prepare the amorphous structure. The crystallized CPG was prepared by crystallizing as-quenched glasses at 600 C. Amorphous and crystallized glasses were crushed and sieved to produce particles less than 45 lm in size. Two types of polyurethane (PU) sponges, one with 45-ppi (pores per inch) and another with 60-ppi (Regicell, Jehil Urethane Co., Asan-si, Korea), were used in this study.14 CaP slurry was prepared by

Bone Tissue Engineering by Using CPG Scaffolds and the ABBS

dispersing the prepared amorphous and crystallized CPG, or various contents of mixed powders, into distilled water with organic additives. Polyvinyl alcohol (PVA, Duksan Pure Chemical Co., Ansan-si, Korea), polyethylene glycol (PEG, Duksan Pure Chemical Co., Ansan-si, Korea), and dimethylformamide (DMF, Aldrich, Milwaukee, WI) were used as the binder, dispersant, and drying chemical control additive, respectively. First, PVA was hydrolyzed at 5 wt%. PEG was then added at 5 wt%, which was followed by the addition of DMF at 10 wt%. Preparation of the CaP slurry was completed by dispersing the prepared powders into the organic solution at ratio 1.5 of powder/liquid (P/L) (g/mL). After the PU was coated with CaP slurry, it was then dried and heat-treated in a Kanthal furnace. The conditions for heat-treatment of the PU and the CPG were determined based on differential thermal analysis (DTA, TG/DTA92, Setaram, France).21 First, the temperature was increased to 600 C at 3 C/min to burn out the PU entirely, and the temperature was held constant for 60 min to volatize the organic additives. The remaining CPG was then sintered for 2 h at 800 C. In Vitro Biocompatibility A human osteoblast-like cell line, MG-63, was purchased from Korean cell line bank (Seoul, Korea). The culture medium consisted of Dulbecco’s Modification of Eagle’s Medium (DMEM; WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY), and 100 units/mL penicillin and 100 lg/mL streptomycin (Pen/Strep; Gibco, Grand Island, NY), and cells were cultured in a 37 C, 5% CO2 humidified atmosphere incubator. Cells were biotinylated with a reagent, EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL), according to the manufacturer’s instructions. The MG-63 cells were suspended in a dish, and were treated with the biotinylation reagent. Dense disks of the A25C75, A75C25, and hydroxyapatite (HAp) were prepared by uniaxial pressing (Table 1). A quantity of 0.5 g of the powder was placed in a metal mold (d = 12 mm) and pressed under 90 MPa of pressure. The A25C75 (or A75C25) and HAp disks were then sintered at either 800 or 1200 C, at a heating rate of 3 C/min. The surface of the disks was modified using two different coating methods: simple adsorption of avidin or avidin–biotin binding. The simple adsorption of avidin was performed using 1 mg/mL of avidin in phosphate-buffered saline (PBS) solution, which was adsorbed onto the disks. For the avidin–biotin binding method, after the disks were cleaned and dried, they were placed in a vacuum oven

with a 1-mL aliquot of aminopropyl-trimethoxysilane (Aldrich, Milwaukee, WI) at 140 C. After 60 min, the vacuum was released, and the disks were allowed to cool. After cooling, the disks were coated with SulfoNHS-LC-biotin (Pierce, Rockford, IL). A biotin solution (500 lg/mL) was prepared in PBS solution and 200 lL of the solution was placed on top of the silanized disks to cover one surface of each slide. The biotinylated disks were rinsed with deionized (DI) water and allowed to dry. The biotinylated disks were then coated with avidin (Sigma, St. Louis, MO). The disks were placed in a Petri dish, and a 1 mg/mL solution of avidin in PBS was placed on the surface of the biotinylated disks, ensuring that each side coated was the same side previously coated with the biotin reagent. To attach cells to the dense disks, biotinylated or untreated MG-63 cells, suspended in 200 lL of fresh medium containing 10% FBS, were seeded into avidin-, avidin–biotin, or non-adsorbed disks placed in a 24well culture plate (5 9 104 cells/sample). After 10 min, 60 min, and 24 h, unattached cells were removed by washing out with PBS solution, and attached cells were observed with a scanning electron microscope (SEM; S4200, Hitachi, Tokyo, Japan). The control and experimental groups for the cell adhesion test are shown in Table 2. MG-63 cell adhesion to the disks was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cell culture medium was removed, and 1 mL of MTT solution was added to each well. Subsequently, the MTT solution was discarded, the precipitated formazan was dissolved in dimethyl sulfoxide (DMSO; 500 lL/well), and 200 lL of DMSO from each well was transferred to a 96-well culture plate. The optical density (OD) at 570 nm was measured using an ELISA reader (Molecular Devices, Sunnyvale, CA). After each cell culture time point, the morphological characteristics of the attached cells on the disks were observed with SEM. Based on the results of the cell adhesion test on the dense disks, scaffolds fabricated with A25C75 were selected for the 3D cell test. The codes assigned to scaffolds for the cell test are shown in Table 2. First, the optimal cell culture conditions for the scaffold were determined using two types of scaffolds (45- and 60ppi) and measuring the degree of cell attachment observed by varying the culturing conditions, including the cell seeding efficiency and the incubation period. MG-63 cells were seeded on the top of the avidin- or non-absorbed scaffolds with 200 lL of osteogenic culture medium containing cells at different concentrations (1 9 104, 5 9 104, and 1 9 105 cells/scaffold), and then the cell/scaffold constructs were placed in the 24-well culture plate. The medium consisted of

KIM et al. TABLE 1. Codes assigned to scaffolds prepared by mixing various amounts of amorphous and crystallized CPG.

TABLE 3. RT-PCR oligonucleotides. Gene

Code A75C25 A25C75

Amorphous CPG content (wt%)

Crystallized CPG content (wt%)

75 25

25 75

GAPDH Collagen type I Osteopontin ALP

TABLE 2. Codes assigned to groups for testing cells on dense disks and porous scaffolds. Code of groups

Surface modification

Cell test on dense disks Control Well culture D-non Non-adsorbed D-A Avidin-adsorbed D-B Non-adsorbed D-AB Avidin-adsorbed D-BAB Biotinylated + avidin-adsorbed Cell test on porous scaffolds S-non Non-adsorbed S-AB Avidin-adsorbed

Osteocalcin

Primer sequence F R F R F R F R F R

5¢-TGACATCAAGAAGGTGGTGA-3¢ 5¢-TCCACCACCCTGTTGCTGTA-3¢ 5¢-ATGTCTAGGGTCTAGACATGTTCA-3¢ 5¢-CCTTGCCGTTGTCGCAGACG-3¢ 5¢-GACCACATGGACCGACGATG-3¢ 5¢-TGGAACTTGCTTGACTATCGA-3¢ 5¢-ACGTGGCTAAGAATGTCATC-3¢ 5¢-CTGGTAGGCGATGTCCTTA-3¢ 5¢-ATGAGAGCCCTCACACTCCTC-3¢ 5¢-GCCGTAGAAGCGCCGATAGGC-3¢

Cell biotinylation

Untreated Untreated Untreated Biotinylated Biotinylated Biotinylated Untreated Biotinylated

DMEM supplemented with 10 mM b-glycerophosphate, 50 lg/mL ascorbic acid, 0.1 mM dexamethasone, and 10% FBS. The viability of cells attached to the scaffolds was determined using a water-soluble tetrazolium salt (WST) assay. After culturing at each condition, the scaffolds were removed and washed twice with PBS solution. The WST-1 reagent (Premix WST-1; TaKaRa, Tokyo, Japan) was added and incubated. A 200-lL aliquot of the added WST-1 reagent from each well was then transferred to a 96-well culture plate, and the OD at 450 nm was measured using an ELISA reader. Based on the cell attachment results, the cell concentration and attachment period for subsequent cellular assays were set at 1 9 105 cells/ scaffold and 10 min, respectively. For the proliferation tests, cells were first allowed to attach to scaffolds in undisturbed medium, and then, the scaffolds were placed into another plate and cultured for 1, 3, and 5 days. At each cell culture time point, the proliferation was measured with the WST assay, and cell morphology was observed with SEM. The differentiation level of the MG-63 cells was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR was performed using osteoblast-specific gene primers designed from the sequence of each cDNA, including type I collagen (COL I), osteopontin (OP), alkaline phosphatase (ALP), and osteocalcin (OC). After 3, 7, 14, 21, and 28 days, the RNA was extracted from the scaffolds using TRIzol

reagent. The TRIzol reagent from each well was transferred to a 1.5-mL tube. Chloroform was added, and the RNA was centrifuged at 12,000 rpm. Subsequently, the upper aqueous phase containing the RNA was transferred to another tube and was mixed with isopropanol to obtain an RNA pellet. After mixing, a RNA pellet was obtained by centrifuging at 12,000 rpm, and the medium was carefully discarded. The RT-PCR reaction was performed according to the manufacturer’s instructions (Maxime RT-PCR PreMix; iNtRON, Seongnam-si, K1orea), where 1 lL of template RNA and 1.5 lL of each specific primer (15 pmol/lL of forward and reverse primer) were added into the Maxime RT-PCR PreMix tubes. Table 3 shows the oligonucleotides used for PCR primers. Autoclaved DI water was added to obtain a total volume of 20 lL, and RT-PCR reactions for the samples were performed using the following conditions for the PCR machine. The RT reaction was heated at 45 C for 30 min, and RTase was inactivated by heating at 94 C for 5 min. The PCR reaction was continuously followed by 32 denaturation cycles at 94 C for 30 s, annealing at 48 C (COL I, ALP), 52 C (OP), and 56 C (OC) each for 60 s, extension at 72 C for 60 s, and final extension at 72 C for 5 min. Each PCR product was analyzed by separation on a 1.2% agarose gel by electrophoresis and stained with ethidium bromide (EthB) for visualization using the Bio-Rad ChemiDoc (Bio-Rad, Hercules, CA) with UV rays.

RESULTS The final step for scaffold fabrication was the heat treatment, with conditions defined based on the DTA results. At 430 C in the TG trace, the PU was almost removed (Fig. 1), and the DTA trace of the CPG powder exhibited a clear Tg (590 C), Tc (616 C), Tm1 (787 C), and Tm2 (960 C) peaks.

Bone Tissue Engineering by Using CPG Scaffolds and the ABBS

FIGURE 1. DTA data for the CPG powder (left) and TG data for the polymeric sponge (right).

FIGURE 2. MG-63 cell adhesion on dense disks after 10 min, 60 min, and 24 h.

Cell Adhesion on Dense Avidin–Biotin Binding System Disks The efficiency of ABBS-mediated cell adhesion was determined by MTT assay. Figure 2 shows the OD values for MG-63 cells adhered to the dense disks. For all groups, the OD values of adhered cells increased with the cell culture time. After 10 min, more biotinylated cells were attached to the avidinadsorbed surfaces of D-AB and D-BAB than to the other disks. The OD for the D-A samples was similar to the control group, but that of the D-non and D-B groups were less than the control group. After 60 min and 24 h, the OD of adhered cells for the D-AB and D-BAB groups was still higher than the others, and after 24 h, the adhered cells were similar to the control group, D-A, D-AB, and D-BAB. For prolonged adhesion times, the difference in adhered cells decreased among the groups. The cell culture time had no effect on OD values for D-AB and D-BAB, and OD values of D-non and D-B were also comparable. For the D-AB and D-BAB samples, the more cells adhered to A25C75 and A75C25 than to HAp for all cell culture times.

Figure 3 shows the morphology of MG-63 cells adhered to the A25C75 disks, after 10 min, 60 min, and 24 h. D-AB resulted in more cells attaching than D-non within 10 min. After 10 min, some cells adhered to D-AB had already spread, whereas cells adhered to D-non remained rounded in shape. After 60 min, cells in D-non had spread, and after 24 h, cell spreading for both D-non and D-AB was comparable.

Cell Adhesion and Proliferation on Scaffolds ABBS was applied to the highly porous 3D scaffolds to aid in uniform and rapid MG-63 cell adhesion, where the scaffolds were biotinylated and adsorbed with avidin, respectively. Figure 4a shows the degree of cell adhesion on A25C75 scaffolds with 45- and 60-ppi under different cell culture conditions. The cell concentrations used were 1 9 104, 5 9 104, and 1 9 105 cells/scaffold. Greater cell adhesion, with increasing cell seeding efficiency, was observed for SAB, whereas the level of cell adhesion for S-non had no effect. Cell adhesion was not affected by pore sizes of 45- and 60-ppi for S-non, but for S-AB, the degree

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FIGURE 3. SEM images of MG-63 cells adhered to A25C75 disks after 10 min, 60 min, and 24 h.

of cell adhesion in 60-ppi was higher than that in 45ppi, except for the cell density of 1 9 104 cells/scaffold. For the proliferation and differentiation tests, the cell density and adhesion time were fixed at 1 9 105 cells/scaffold for 10 min, respectively. Figure 5a shows SEM images of the cells adhered to the scaffolds. The cell viability on the scaffolds after 1, 3, and 5 days was quantified using a WST assay, as shown in Fig. 4b. The cells on the scaffolds continued to proliferate over increasing cell culture time. The OD values for the S-AB group were significantly higher than those of the S-non group at each time point, and the proliferation for 60-ppi samples was much higher than that for the 45-ppi samples. Figure 5b shows SEM images of cells seeded and proliferated on scaffolds after 1 and 5 days.

Osteogenic Differentiation on Scaffolds Gene expression for MG-63 cells proliferated and differentiated on the scaffolds was analyzed using RTPCR and the EthB-stained agarose gel electrophoresis, as shown in Fig. 6. Bone-specific genes, including COL I, ALP, OC, and OP were evaluated, and the data for S-non samples were compared with those for S-AB samples. GAPDH was expressed at a similar level for all samples at each cell culture time point, except at 3 days. At 3 days, COL I in the S-AB samples appeared to be expressed at higher levels than the Snon group, and OP and thin ALP expression were observed only in the S-AB group. After 7 days, OP, ALP, and OC were expressed in both the S-non and SAB groups, with similar levels of COL I, OP, and ALP

Bone Tissue Engineering by Using CPG Scaffolds and the ABBS

FIGURE 4. (a) Adhesion of MG-63 cells to the scaffolds after 10 min. (b) Proliferation of MG-63 cells on the scaffolds after 1, 3, and 5 days. Cell seeding density was 1 3 105 cells/scaffold for proliferation.

FIGURE 5. (a) SEM images of MG-63 cells adhered to the S-AB after 10 min. (b) SEM images of MG-63 cells adhered to the S-AB after 1 day and 5 days.

expression. However, OC expression for the S-non group was higher than that in the S-AB group. After 14 days, COL I, OP, ALP, and OC was expressed in all groups, and ALP expression decreased in comparison to levels expressed at 7 days. At 21 and 28 days, all genes were expressed for both the S-non and S-AB groups, and ALP was again higher than that that at 14 days. OC expression was shown to increase as cell culture time increased. Figure 7 shows SEM images of MG-63 cells proliferated and differentiated in the SAB scaffold. After 14 days, the cultured cells were perfectly spread over the scaffold strut, and calcification of the cell surface could was confirmed by observing the morphology at higher magnification. The level of calcification at 28 days was greater than that at 14 days.

DISCUSSION In this study, highly porous scaffolds were successfully fabricated into CPG. The scaffolds exhibited sufficient mechanical strength, in spite of their high porosity, appropriate pore size, and highly interconnected pore structure in our previous study.20 In this study, these scaffolds were biocompatible and were able to support MG-63 cell adhesion, proliferation, and differentiation in vitro. Initial adhesion of MG-63 cells to the scaffolds was enhanced by using ABBS, and cells attached by ABBS functioned as osteoblasts, as indicated by cell spreading, growth, and calcification. Before applying the treatment to the scaffolds, cell adhesion to dense disks was evaluated using ABBS. Biotinylated cells attached to avidin-adsorbed disk

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FIGURE 6. RT-PCR analysis of the bone-associated genes expressed by MG-63 cells on the scaffold after 3, 7, 14, 21, and 28 days.

surfaces (D-AB and D-BAB) within 10 min (Figs. 2 and 3). In contrast, similar to the control group, 10 min was insufficient for untreated cell attachment to avidin-adsorbed disk surfaces (D-A). After 24 h, most cells were stably attached to the surfaces, were spread out, and proliferated, suggesting that the ABBS

significantly improved initial amounts of attached cells to the disks. Observation of cell behavior over time showed the process of cell spreading, growth, and proliferation by MG-63 cells by ABBS. After 10 min, biotinylated and untreated cells attached to the surface of the non-adsorbed disks, but did not spread after 10 min, whereas after 10 min the ABBS-treated cells spread on the avidin-adsorbed surface. Therefore, cells on an avidinadsorbed surface by ABBS were finally able to spread for proliferation. The avidin-adsorbed surface (D-A) showed lower cell adhesion than D-AB, but adhesion for the D-A surface was greater than D-non, as shown in Fig. 2. Pre-adsorption of avidin protein could modify the properties of the material surfaces for cell attachment. The untreated cells could also attach and spread on the protein-adsorbed surface for a sufficient period. The level of initial cell adhesion increased with increasing cell density for the two scaffolds with 45and 60-ppi in ABBS. And we performed the WST assay, which is a series of other water soluble dyes for MTT assays and does not need a solubilization step. To analyze the cell adhesion on the scaffolds, we need more simple protocol than the MTT assay, because the scaffolds have more complex structure than the disk type. For the S-AB group, the degree of cell adhesion for the 60-ppi was higher than that for the 45-ppi, except for the 1 9 104 cell density. The scaffolds with 60-ppi had smaller pores, and thus, the binding affinity was higher. That is, for the static cell culture seeding system used in this study, the distance between the biotinylated cell and the surface adsorbed avidin of the scaffold was closer as cell density increased. As shown in Fig. 5a, the cells inoculated into the scaffold homogenously adhered to the inside of the scaffold. These experiments were performed using common static seeding conditions as opposed to dynamic methods (i.e., dynamic perfusion, centrifugal, agitation, or rotating seeding). Based on these findings, it is suggested that transient contact may be sufficient to attach cells stably on surfaces by ABBS, as demonstrated by effective attachment in the advanced static cell culture. The S-AB group exhibited great initial cell adhesion and a significantly higher degree of proliferation (Fig. 5b). The cells in scaffolds were observed to attach, spread, and proliferate, suggesting positive cellular response to the scaffold. In addition, there was a common problem that is rapid tissue formation on the outside of the scaffold. This is most likely due to the effects of limited diffusion, which is thought attributed to limited cell migration and nutrient supply with waste exchange. Therefore, an optimal scaffold design would improve cell and nutrient transfer inside of the

Bone Tissue Engineering by Using CPG Scaffolds and the ABBS

FIGURE 7. SEM images of MG-63 cells adhered to the S-AB after 7, 14, and 28 days.

scaffold.16,17 In the present study, the highly porous and interconnective structured scaffolds facilitated cell infiltration into the scaffolds structure. Based on these results, we determined that the S-AB did not have a negative effect on the adhesion and proliferation of MG-63 cells, and that they are biocompatible and noncytotoxic. In our in vitro test, the osteogenic differentiation of MG-63 cells was determined by evaluating the expression of bone-specific genes, COL I, ALP, OP, and OC, using RT-PCR. ALP activity has been used as a biomarker for determining the osteoblast phenotype and is considered an important factor in determining osteogenic differentiation and mineralization. Thus, ALP expression is an initial stage indicator of osteogenic differentiation. During bone differentiation, markers of the osteoblast phenotype appear, such as the expression of ALP and the accumulation of hard tissue ECM proteins, of which COL I is the most general. Osteoblasts are defined by their site on bone and their ability to mineralize matrix, and in addition, osteoblasts are characterized by their ability to synthesize COL I, osteonectin, OP, bone sialoprotein (BSP), and OC, and their high ALP activity. And responsiveness to hormones and growth factors are also characteristic of osteoblasts populations. It has been shown that changes in the levels of expression of these osteoblast-associated

molecules properties associated with osteoblasts may be characteristic of cells at different stages of development and maturation. For example, some in vitro time course model systems of osteoblast differentiation showed that COL I expression is initially relatively high, and then expression decreases; ALP increases in initial stage, but decreases as mineralization progresses; OP appears including BSP and OC; BSP is first detected in osteogenic differentiated; and OC appears in the lately stage, i.e. mineralization. Based on observations of osteoblast proliferation over time, in connection with the onset of detectable nodule formation and changes in mRNA levels, the sequence of osteoblast development has been defined and is as comprised of proliferation, matrix maturation, and mineralization.3,24 As shown by the results of our present study, after 3 days of osteogenic culture, MG-63 cells in direct contact with S-AB exhibited positive COL I, OP, and ALP expression; OC in the S-AB group was expressed at 7 days. Thus, these findings demonstrated that the scaffold material had no negative influence on MG-63 osteogenic differentiation.

CONCLUSION The goal of the present study was to develop a highly porous and interconnected scaffold for bone

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tissue engineering. Bone tissue engineering scaffolds require basic attributes, such as proper mechanical and biological properties for tissue regeneration. In addition, scaffolds should have high porosity with adequate pore size, sufficient mechanical properties, biodegradability, biocompatibility, and positively interact with cells to support cell adhesion, growth, migration, and differentiation. The MG-63 cells may then be expanded in culture and seeded on an ideal scaffold with all of properties previously stated. We fabricated highly porous, interconnected scaffolds using CPG in the previous study.20 After confirming the results of their mechanical strength and structure properties, the biocompatibility of scaffolds using ABBS was evaluated and was proven the improvement of adhesion between osteoblast-like cells and the fabricated scaffold in this study. Based on this work, we determined that the CPG scaffold has the proper biological properties suitable for use in bone tissue engineering applications.

ACKNOWLEDGEMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government MSIP (No. 2008-0062283). This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2014M3A7B4051594). This work was supported (in part) by the Yonsei University Yonsei-SNU Collaborative Research Fund of 2014.

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Bone Tissue Engineering by Using Calcium Phosphate Glass Scaffolds and the Avidin-Biotin Binding System.

Highly porous and interconnected scaffolds were fabricated using calcium phosphate glass (CPG) for bone tissue engineering. An avidin-biotin binding s...
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