A new continuous cell line (GF‐1) was established and characterized. The GF‐1 cell line, derived from the fin tissue of a grouper, Epinephelus coioides (Hamilton), was maintained in L15 medium containing 5% foetal bovine serum (FBS) at 28 °C, and has been subcultured more than 160 times since 1995. The majority of GF‐1 cells are fibroblast‐like, together with some epithelioid cells. Spontaneous transformation of GF‐1 cells occurred during subculture 50 to subculture 80, and led to an increase of plating efficiency, less requirement of FBS and de novo susceptibility to grouper nervous necrosis virus (GNNV). Cytopathic effects (CPEs) could be observed in GF‐1 cells 3–5 days post‐infection with pancreatic necrosis virus (IPNV), hard clam reovirus (HCRV), eel herpes virus Formosa (EHVF) and GNNV. In addition, abundant GNNV particles were found in the cytoplasm of GNNV‐infected GF‐1 cells using electron microscopy and nucleic acids of GNNV virus were detected by polymerase chain reaction in the culture medium of GNNV‐infected cells after CPE appeared. The experimental results indicated that GF‐1 can effectively proliferate fish nodavirus and is a promising tool for studying fish nodavirus.

A lyophilization method was developed to locally release adenoviral vectors directly from biomaterials for in situ regenerative gene therapy. Adenovirus expressing a b-galactosidase reporter gene (AdLacZ) was mixed with different excipient formulations and lyophilized on hydroxyapatite (HA) disks followed by fibroblasts culturing and 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) staining, suggesting 1 M sucrose in phosphate-buffered saline had best viability. Adenovirus release studies showed that greater than 30% virus remained on the material surface up to 16 h. Lyophilized adenovirus could be precisely localized in defined patterns and the transduction efficiency was also improved. To determine if the lyophilization formulations could preserve viral bioactivity, the lyophilized AdLacZ was tested after being stored at varying temperatures. Bioactivity of adenovirus lyophilized on HA was maintained for greater than 6 months when stored at À801C. In vivo studies were performed using an adenovirus encoding BMP-2 (AdBMP-2). AdBMP-2 was lyophilized in gelatin sponges and placed into rat critical-size calvarial defects for 5 weeks. Micro-computed tomography (m-CT) analysis demonstrated that free-form delivery of AdBMP-2 had only modest effects on bone formation. In contrast, AdBMP-2 lyophilized in gelatin sponges led to more than 80% regeneration of critical-size calvarial defects. Gene Therapy (2007) 14, 891-901.

To functionalize biomaterials for bioconjugation, a chemical vapor deposition (CVD) polymerization technique was utilized to modify material surfaces. Poly [(4-amino-p-xylylene)-co-(p-xylylene)] (PPX-NH 2 ) was deposited on inert polycaprolactone (PCL) surfaces to provide a reactive amine layer on the substrate surfaces. The biocompatibility of PPX-NH 2 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and lactate dehydrogenase (LDH) assays. The results demonstrated that cells continuously proliferated on CVD treated PCL surfaces with high survival rates. Biotin was conjugated on modified PCL surfaces to immobilize avidin for binding of biotinylated adenovirus. Scanning electron microscopy (SEM) examination illustrated that adenoviruses were evenly bound on both 2-D films and 3-D scaffolds, suggesting CVD was capable of modifying various substrates with different geometries. Using a wax masking technique, the biotin conjugation was controlled to immobilize avidin on specific sites. Due to the virus binding specificity on CVD modified surfaces, cell transduction was restricted to the pattern of immobilized virus on biomaterials, by which transduced and nontransduced cells were controlled in different regions with a distinct interface. Because CVD demonstrated excellent controllability in different hierarchies, this surface modification should be able to tailor bioconjugation and expand the possibility of material application in different fields.

Background-Regenerative gene therapy using viral vectors enables transduced cells to express bioactive factors in vivo. Viral delivery with spatial control can enhance transduction efficiency and avoid systemic infection. Consequently, we tethered biotinylated adenovirus via interactions with avidin on chitosan surfaces to gain robust control for in situ transduction.

Biodegradable porous scaffolds have been investigated as an alternative approach to current metal, ceramic, and polymer bone graft substitutes for lost or damaged bone tissues. Although there have been many studies investigating the effects of scaffold architecture on bone formation, many of these scaffolds were fabricated using conventional methods, such as salt leaching and phase separation, and were constructed without designed architecture. To study the effects of both designed architecture and material on bone formation, we designed and fabricated three types of porous scaffold architecture from two biodegradable materials, poly (L-lactic acid) (PLLA) and 50:50Poly (lactic-co-glycolic acid) (PLGA) using image based design and indirect solid freeform fabrication techniques, seeded them with bone morphogenic protein-7 transduced human gingival fibroblasts and implanted them subcutaneously into mice for 4 and 8 weeks. Micro-computed tomography data confirmed that the fabricated porous scaffolds replicated the designed architectures. Histological analysis revealed that the 50:50PLGA scaffolds degraded and did not maintain their architecture after 4 weeks. The PLLA scaffolds maintained their architecture at both time points and showed improved bone ingrowth which followed the internal architecture of the scaffolds. Mechanical properties of both PLLA and 50:50PLGA scaffolds decreased, but PLLA scaffolds maintained greater mechanical properties than 50:50PLGA after implantation. The increase of mineralized tissue helped to support mechanical properties of bone tissue and scaffold constructs from 4 to 8 weeks. The results indicated the importance of choice of scaffold materials and computationally designed scaffolds to control tissue formation and mechanical properties for desired bone tissue regeneration.

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To explore the effect of electrical stimulation (ES) on osteogenesis, a polypyrrole (PPy)‐made electrical culture system was developed to provide a direct‐current electric field (DCEF). This DCEF device was applied to treat differentiated rat bone marrow stromal cells (rBMSCs) once in different stages of osteo‐differentation to investigate its temporal effects. The mineralization results showed that the DCEF treatment not only accelerated cell differentiation but also promoted the saturation levels, and the ES on day 8 was the group demonstrated the optimal result. The gene regulation analysis indicated that the DCEF treatment immediately increased the levels of genes related to osteo‐differentiation, especially Runx2. Because Runx2 is a crucial transcriptional factor of osteogenesis, the ES‐caused improvement of mineralization was likely contributed by the extension of its expression. Further, different ES modes were investigated of their efficacy on bone matrix deposition. Square waves with different parameters including frequency, offset, amplitude, and duty cycle were systematically examined. In contrast to constant voltage, square waves demonstrated periodical changes of current through substrate to significantly improve mineralization, and the efficiencies highly depended on both frequency and intensity. Through this comprehensive study, DCEF treating condition was optimized, which should be beneficial to its application on osteogenesis promotion. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1607–1619, 2019.

Critical-sized defects were created in rat calvariae previously treated with 12 Gy irradiation 2 weeks before surgery. Gelatin scaffolds containing lyophilized AdBMP-2, freely suspended AdBMP-2, or negative controls were transplanted in the defects for 5 weeks. Lyophilized AdBMP-2 treatment significantly improved both bone quality and quantity over free AdBMP-2 administration. The effects of radiotherapy on osteogenesis were also compared. Bone mineral density was reduced after radiotherapy. Histological analyses demonstrated that radiation damage led to less bone regeneration. Woven bone and immature marrow formed in the radiated defects indicated that preoperative radiotherapy retarded normal bone development and turnover. Finally, the gelatin scaffolds with lyophilized AdBMP-2 were stored at -80°C to determine adenovirus stability. Micro-CT quantification demonstrated that there were no significant differences between bone regeneration treated with lyophilized AdBMP-2 before and after 1-month storage, suggesting that virus-loaded scaffolds should be convenient for application as pre-made constructs for surgical applications.