Biomaterials (v.30, #14)

Intracellular chromosome breaks on silicon surface by Jiang Jiang; Kaifu Huo; Shaopeng Chen; Yunchang Xin; Yongjian Xu; Zhengwei Wu; Zengliang Yu; Paul K. Chu (2661-2665).
The genotoxicity of silicon (Si) is investigated by soaking crystalline Si in a complete culture medium for 60 days and conducting micronuclei tests (MNTs) utilizing hamster ovary (CHO) cells and its Ku80 deficient CHO mutant (xrs5) cells (DNA double-strand breaks repair deficiency). The intracellular concentrations of reactive oxygen/nitrogen species (ROS/RNS) on Si are determined to elucidate the relationship between ROS/RNS and Si-induced genotoxicity by using CHO cells. The cells are treated with ROS scavenger (dimethyl sulfoxide) and MNT are performed. The results indicate that the intracellular concentration of ROS and nitrogen oxide (NO) on Si is higher than those on the control group by about 38% and 12%. ROS/RNS include superoxide (O2 •−) anion, NO, and peroxynitrite (ONOO) which can injure chromosomes and induce high cellular DNA double-strand breaks (DSBs).
Keywords: Silicon; Reactive oxygen/nitrogen species; Micronuclei; DNA double-strand breaks; Biocompatibility;

Expansion and delivery of human fibroblasts on micronized acellular dermal matrix for skin regeneration by Xiaojun Zhang; Zhihong Deng; Hailun Wang; Zhenhua Yang; Weihua Guo; Yuan Li; Dandan Ma; Chunyan Yu; Yongjie Zhang; Yan Jin (2666-2674).
In order to obtain an abundant supply of autologous dermal fibroblasts for the manufacture of engineered autologous dermal substitutes, we fabricated the micronized acellular dermal matrix (MADM) microcarriers and expanded human fibroblasts on them. This novel approach eliminated the need for the repeated trypsinizations that may disrupt cell–extracellular matrix interactions and impair cell viability. This cell expansion protocol simultaneously formed an engineered particulate dermal substitute (EPDS) avoiding cell reseeded on the scaffolds process. We further tested its feasibility and effectiveness in athymic murine subcutaneous injection and full-thickness cutaneous wound model. Our results showed that MADM microcarriers retained the ultrastructure of the acellular dermal matrix, had good biocompatibility, and supported human fibroblast expansion either as a direct culture substrate or through culturing cells in conditioned medium prepared from them. In the animal study, EPDS formed a thick layer of tissue below the subcutaneous muscle tissue at 3 weeks following EPDS injection into subcutaneous tissue. In full-thickness cutaneous wound, the degree of wound healing with EPDS implantation was better than that without EPDS although healing rates were not significantly different between wounds implanted with or without EPDS. This demonstrates the potential utility of MADM not only as a cell culture substrate to expand fibroblasts but also as a cell transplantation vehicle for skin regeneration, with several advantages over current expansion–transplantation protocols for skin regeneration. In addition, EPDS may be used for cosmetic or reconstructive soft tissue augmentation in a minimally invasive fashion.
Keywords: Micronized acellular dermal matrix; Expansion; Transplantation; Fibroblasts; Engineered particulate dermal substitute;

Calcium phosphate cement (CPC) can be molded or injected to form a scaffold in situ, has excellent osteoconductivity, and can be resorbed and replaced by new bone. However, its low strength limits CPC to non-stress-bearing repairs. Chitosan could be used to reinforce CPC, but mesenchymal stem cell (MSC) interactions with CPC-chitosan scaffold have not been examined. The objective of this study was to investigate MSC proliferation and osteogenic differentiation on high-strength CPC-chitosan scaffold. MSCs were harvested from rat bone marrow. At CPC powder/liquid (P/L) mass ratio of 2, flexural strength (mean ± sd; n  = 5) was (10.0 ± 1.1) MPa for CPC-chitosan, higher than (3.7 ± 0.6) MPa for CPC (p  < 0.05). At P/L of 3, strength was (15.7 ± 1.7) MPa for CPC-chitosan, higher than (10.2 ± 1.8) MPa for CPC (p  < 0.05). Percentage of live MSCs attaching to scaffolds increased from 85% at 1 day to 99% at 14 days. There were (180 ± 37) cells/mm2 on scaffold at 1 day; cells proliferated to (1808 ± 317) cells/mm2 at 14 days. SEM showed MSCs with healthy spreading and anchored on nano-apatite crystals via cytoplasmic processes. Alkaline phosphatase activity (ALP) was (557 ± 171) (pNPP mM/min)/(μg DNA) for MSCs on CPC-chitosan, higher than (159 ± 47) on CPC (p  < 0.05). Both were higher than (35 ± 32) of baseline ALP for undifferentiated MSCs on tissue-culture plastic (p  < 0.05). In summary, CPC-chitosan scaffold had higher strength than CPC. MSC proliferation on CPC-chitosan matched that of the FDA-approved CPC control. MSCs on the scaffolds differentiated down the osteogenic lineage and expressed high levels of bone marker ALP. Hence, the stronger CPC-chitosan scaffold may be useful for stem cell-based bone regeneration in moderate load-bearing maxillofacial and orthopedic applications.
Keywords: Bone marrow mesenchymal stem cells; Calcium phosphate bone cement; Chitosan; Cell proliferation; Osteogenic differentiation; Bone tissue engineering;

Repair of articular cartilage defect in non-weight bearing areas using adipose derived stem cells loaded polyglycolic acid mesh by Lei Cui; Yaohao Wu; Lian Cen; Heng Zhou; Shuo Yin; Guangpeng Liu; Wei Liu; Yilin Cao (2683-2693).
The current study was designed to observe chondrogenic differentiation of adipose derived stem cells (ASCs) on fibrous polyglycolic acid (PGA) scaffold stabilized with polylactic acid (PLA), and to further explore the feasibility of using the resulting cell/scaffold constructs to repair full thickness articular cartilage defects in non-weight bearing area in porcine model within a follow-up of 6 months. Autologous ASCs isolated from subcutaneous fat were expanded and seeded on the scaffold to fabricate ASCs/PGA constructs. Chondrogenic differentiation of ASCs in the constructs under chondrogenic induction was monitored with time by measuring the expression of collagen type II (COL II) and glycosaminoglycan (GAG). The constructs after being in vitro induced for 2 weeks were implanted to repair full thickness articular cartilage defects (8 mm in diameter, deep to subchondral bone) in femur trochlea (the experimental group), while scaffold alone was implanted to serve as the control. Histologically, the generated neo-cartilage integrated well with its surrounding normal cartilage and subchondral bone in the defects of experimental group at 3 months post-implantation, whereas only fibrous tissue was filled in the defects of control group. Immunohistochemical and toluidine blue staining confirmed the similar distribution of COL II and GAG in the regenerated cartilage as the normal one. A vivid remolding process with post-operation time was also witnessed in the neo-cartilage as its compressive moduli increased significantly from 50.55% of the normal cartilage at 3 months to 88.05% at 6 months. The successful repair thus substantiates the potentiality of using chondrogenic induced ASCs and PGA/PLA scaffold for cartilage regeneration.
Keywords: Articular cartilage engineering; Adipose derived stem cells; Chondrogenic differentiation; Polyglycolic acid mesh;

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering by Zhi-Yong Zhang; Swee Hin Teoh; Woon-Shin Chong; Toon-Tien Foo; Yhee-Cheng Chng; Mahesh Choolani; Jerry Chan (2694-2704).
The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation in vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity (2×, p  < 0.01), and maintained high cellular viability in the core, which was 2000 μm from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression (1.5×p  < 0.01), calcium deposition (5.5×, p  < 0.001) and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice (3.2×, p  < 0.001) compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications.
Keywords: Bioreactor; Fetal mesenchymal stem cell; Bone tissue engineering; Polycaprolactone; NOD/SCID mice;

3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells by Wenjie Wang; Keiji Itaka; Shinsuke Ohba; Nobuhiro Nishiyama; Ung-il Chung; Yuichi Yamasaki; Kazunori Kataoka (2705-2715).
Multipotent mesenchymal stem cells (MSCs) are one of the most powerful tools in regeneration medicine. Their low differentiation efficiency, however, limits further application of MSCs in clinical therapy. Here we report that a much higher multipotent differentiation efficiency of MSCs to adult cells can be achieved using a 3D spheroid culture method based on photolithography and micropatterning techniques. MSC spheroid of precise dimension and uniform quality cultured on the microdomain substrates was prepared first, and then was induced into adipocytes and osteoblasts. Both gene expression results from RT-PCR and morphology observation results revealed that the 3D spheroid culture method could greatly improve differentiation efficiency. Gene expression profiles obtained from gene microarray analysis confirmed the high differentiation efficiency and revealed that MSCs induced in 3D spheroid culture system regulated gene expression not only by increasing the expression levels of genes related to adipogenesis and osteogenesis, but also by down-regulating the gene maintaining MSCs' self-renewal phenotypes. We conclude that our 3D spheroid culture system contributes to an optimization for efficient differentiation of MSCs, offers insight into the mechanism of efficient differentiation of engineered 3D culture system, and has promise for wide applications in regeneration medicine and drug discovery fields.
Keywords: Mesenchymal stem cell; Micropatterning; Three dimension cell culture; Spheroid; Differentiation efficiency;

Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model by Takanori Iwata; Masayuki Yamato; Hiroaki Tsuchioka; Ryo Takagi; Shigeki Mukobata; Kaoru Washio; Teruo Okano; Isao Ishikawa (2716-2723).
Periodontal regeneration has been challenged with chemical reagents and/or biological approaches, however, there is still no sufficient technique that can regenerate complete periodontium, including alveolar bone, cementum, and well-oriented collagen fibers. The purpose of this study was to examine multi-layered sheets of periodontal ligament (PDL)-derived cells for periodontal regeneration. Canine PDL cells were isolated enzymatically and expanded in vitro. The cell population contained cells capable of making single cell-derived colonies at an approximately 20% frequency. Expression of mRNA of periodontal marker genes, S100 calcium binding protein A4 and periostin, was observed. Alkaline phosphatase activity and gene expression of both osteoblastic/cementoblastic and periodontal markers were upregulated by osteoinductive medium. Then, three-layered PDL cell sheets supported with woven polyglycolic acid were transplanted to dental root surfaces having three-wall periodontal defects in an autologous manner, and bone defects were filled with porous β-tricalcium phosphate. Cell sheet transplantation regenerated both new bone and cementum connecting with well-oriented collagen fibers, while only limited bone regeneration was observed in control group where cell sheet transplantation was eliminated. These results suggest that PDL cells have multiple differentiation properties to regenerate periodontal tissues comprising hard and soft tissues. PDL cell sheet transplantation should prove useful for periodontal regeneration in clinical settings.
Keywords: Temperature-responsive culture surfaces; Periodontal ligament cells; β-tricalcium phosphate;

Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties by Oju Jeon; Kamal H. Bouhadir; Joseph M. Mansour; Eben Alsberg (2724-2734).
Photocrosslinked and biodegradable alginate hydrogels were engineered for biomedical applications. Photocrosslinkable alginate macromers were prepared by reacting sodium alginate and 2-aminoethyl methacrylate in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and N-hydroxysuccinimide. Methacrylated alginates were photocrosslinked using ultraviolet light with 0.05% photoinitiator. The swelling behavior, elastic moduli, and degradation rates of photocrosslinked alginate hydrogels were quantified and could be controlled by varying the degree of alginate methacrylation. The methacrylated alginate macromer and photocrosslinked alginate hydrogels exhibited low cytotoxicity when cultured with primary bovine chondrocytes. In addition, chondrocytes encapsulated in these hydrogels remained viable and metabolically active as demonstrated by Live/Dead cell staining and MTS assay. These photocrosslinked alginate hydrogels, with tailorable mechanical properties and degradation rates, may find great utility as therapeutic materials in regenerative medicine and bioactive factor delivery.
Keywords: Alginate; Biodegradable; Biomaterial; Hydrogel; Photopolymerization; Tissue engineering;

The engineering of patient-specific, anatomically shaped, digits by Peng Wang; Jiang Hu; Peter X. Ma (2735-2740).
It is now recognized that geometric structures of scaffolds at several size levels have profound influences on cell adhesion, viability, proliferation and differentiation. This study aims to develop an integrated process to fabricate scaffolds with controllable geometric structures at nano-, micro- and macro-scales. A phase-separation method is used to prepare interconnected poly(l-lactide) (PLLA) nanofibrous (NF) scaffolds. The pore size of the NF scaffold at the scale of several hundred micrometers is controlled by the size of porogen, paraffin spheres. At millimeter scale and above, the overall shape of the scaffold is defined by a wax mold produced using a three-dimensional printer. The printer utilizes a stereo lithographic file generated from computed tomographic files retrieved from the National Library of Medicine's Visual Human Project. NF PLLA scaffolds with a human digit shape are successfully prepared using this process. Osteoblast cell line MC3T3-E1 cells are then seeded and cultured in the prepared scaffolds. Cell proliferation, differentiation and biomineralization are characterized to demonstrate the suitability of the scaffolds for the digit bone tissue engineering application.
Keywords: Bone tissue engineering; Nanofibrous scaffold; 3D printing; Anatomical shape; Digit;

In vitro generation of an osteochondral construct using injectable hydrogel composites encapsulating rabbit marrow mesenchymal stem cells by Xuan Guo; Hansoo Park; Guangpeng Liu; Wei Liu; Yilin Cao; Yasuhiko Tabata; F. Kurtis Kasper; Antonios G. Mikos (2741-2752).
Injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) were utilized to fabricate a bilayered osteochondral construct consisting of a chondrogenic layer and an osteogenic layer, and to investigate the differentiation of rabbit marrow mesenchymal stem cells (MSCs) encapsulated in both layers in vitro. The results showed that MSCs in the chondrogenic layer were able to undergo chondrogenic differentiation, especially in the presence of TGF-β1-loaded MPs. In the osteogenic layer, cells maintained their osteoblastic phenotype. Although calcium deposition in the osteogenic layer was limited, cells in the osteogenic layer significantly enhanced chondrogenic differentiation of MSCs in the chondrogenic layer. The greatest effect was observed when MSCs were encapsulated with TGF-β1-loaded MPs and cultured with osteogenic cells in the bilayered constructs. Overall, this study demonstrates the fabrication of bilayered hydrogel composites that mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers.
Keywords: Mesenchymal stem cell; Cartilage tissue engineering; Hydrogel; Cell encapsulation; Drug delivery; Growth factors;

The effect of nanofibrous galactosylated chitosan scaffolds on the formation of rat primary hepatocyte aggregates and the maintenance of liver function by Zhang-Qi Feng; Xuehui Chu; Ning-Ping Huang; Tao Wang; Yichun Wang; Xiaolei Shi; Yitao Ding; Zhong-Ze Gu (2753-2763).
Liver tissue engineering requires a perfect extracellular matrix (ECM) for primary hepatocytes culture to maintain high level of liver-specific functions and desirable mechanical stability. The aim of this study was to develop a novel natural nanofibrous scaffold with surface-galactose ligands to enhance the bioactivity and mechanical stability of primary hepatocytes in culture. The nanofibrous scaffold was fabricated by electrospinning a natural material, galactosylated chitosan (GC), into nanofibers with an average diameter of ∼160 nm. The GC nanofibrous scaffolds displayed slow degradation and suitable mechanical properties as an ECM for hepatocytes according to the evaluation of disintegration and Young's modulus testing. The results of morphology characterization, double-staining fluorescence assay and function detection showed that hepatocytes cultured on GC nanofibrous scaffold formed stably immobilized 3D flat aggregates and exhibited superior cell bioactivity with higher levels of liver-specific function maintenance in terms of albumin secretion, urea synthesis and cytochrome P-450 enzyme than 3D spheroid aggregates formed on GC films. These spheroid aggregates could be detached easily during culture period from the flat GC films. We suggest such GC-based nanofibrous scaffolds could be useful for various applications such as bioartificial liver-assist devices and tissue engineering for liver regeneration as primary hepatocytes culture substrates.
Keywords: Chitosan; Scaffolds; Nanotopography; Hepatocyte; Cell culture;

In this study, to improve the attachment, growth and adhesion of endothelial cells (ECs) and thus accelerate the re-endothelialization of stents, a synthesized mussel adhesive polypeptide mimics containing dihydroxyphenylalanine and l-lysine (MAPDL) was immobilized onto 316L stainless steel (316LSS) with polyethylene glycol (PEG) molecule as spacer arm by using cold plasma-induced grafting technique. To immobilize MAPDL effectively, ethylene vinyl acetate (EVA) was first coated onto 316LSS. Different molecular weights of PEG and grafting times were tested to obtain the optimal cell bioactivity. XPS and water contact angles measurement indicated the successful immobilization of MAPDL. In vitro cell culture results showed that compared with the control of 316LSS, the attachment, adhesion and growth of cells on the MAPDL-coated EVA surface, in particular with PEG as spacer arm, were significantly enhanced, and a confluent endothelial cells layer was formed after a 2-day culture. A platelet adhesion experiment revealed that the platelet adhesion was also reduced on the MAPDL-coated EVA surface. The in vitro inflammatory assessment showed that the MAPDL coating inhibited the TNF-α and IL-1β release from monocyte cells, indicative of good anti-inflammation property. Therefore, it is concluded that the MAPDL coating developed here appeared to be a promising strategy for rapid re-endothelialization of intravascular stent devices.
Keywords: Stent; Restenosis; Endothelial cell; Polyethylene glycol; Cold plasma-induced grafting technique;

Immobilization of oligonucleotides on titanium based materials by partial incorporation in anodic oxide layers by René Beutner; Jan Michael; Anne Förster; Bernd Schwenzer; Dieter Scharnweber (2774-2781).
This paper describes the immobilization of bioactive molecules on titanium based surfaces through a combination of nano-mechanical fixation of nucleic acid anchor strands (ASs) by partial and regioselective incorporation within an anodic oxide layer and their hybridization with complementary strands (CSs) intended to be conjugated to bioactive molecules. We focus on the interaction between the substrate surface and the anchor strands, the integrity of ASs and their hybridization ability. The observed dependence of adsorption on pH suggests that initial interaction of terminally phosphorylated ASs with the substrate surface is mediated by electrostatic interaction. Using ASs labelled with 32P at different termini, it could be shown that strand breaks occur, which are attributed (i) to the formation of reactive oxygen species during anodic polarization, (ii) the photocatalytic activity of the titanium oxide and (iii) drying effects. Damage to AS could be considerably reduced if the electrolyte contained 5 mol l−1 ethanol, light was excluded during the experimental procedure, and the number of drying and rewetting steps was minimized. A total surface density of AS of 4.5 pmol cm−2 was reached and could be hybridized to CS with an efficiency of up to 100%. A non-complementary strand (NS) bound with less than 0.5% of the amount of CS under similar conditions. Therefore, non-specific binding of CS is considered as negligible.
Keywords: Implant; Titanium; Electrochemistry; Oligonucleotide; Immobilization; Surface hybridization;

Anti-biofilm efficacy of nitric oxide-releasing silica nanoparticles by Evan M. Hetrick; Jae Ho Shin; Heather S. Paul; Mark H. Schoenfisch (2782-2789).
The ability of nitric oxide (NO)-releasing silica nanoparticles to kill biofilm-based microbial cells is reported. Biofilms of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans were formed in vitro and exposed to NO-releasing silica nanoparticles. Replicative viability experiments revealed that ≥99% of cells from each type of biofilm were killed via NO release, with the greatest efficacy (≥99.999% killing) against gram-negative P. aeruginosa and E. coli biofilms. Cytotoxicity testing demonstrated that the highest dose of NO-releasing silica nanoparticles inhibited fibroblast proliferation to a lesser extent than clinical concentrations of currently administered antiseptics (e.g., chlorhexidine) with proven wound-healing benefits. This study demonstrates the promise of employing nanoparticles for delivering an antimicrobial agent to microbial biofilms.
Keywords: Nitric oxide; Nanoparticle; Biofilm; Antimicrobial; Cytotoxicity;

The uptake and intracellular fate of PLGA nanoparticles in epithelial cells by Malgorzata S. Cartiera; Katherine M. Johnson; Vanathy Rajendran; Michael J. Caplan; W. Mark Saltzman (2790-2798).
Biodegradable polymer nanoparticles (NPs) are a promising approach for intracellular delivery of drugs, proteins, and nucleic acids, but little is known about their intracellular fate, particularly in epithelial cells, which represent a major target. Rhodamine-loaded PLGA (polylactic-co-glycolic acid) NPs were used to explore particle uptake and intracellular fate in three different epithelial cell lines modeling the respiratory airway (HBE), gut (Caco-2), and renal proximal tubule (OK). To track intracellular fate, immunofluorescence techniques and confocal microscopy were used to demonstrate colocalization of NPs with specific organelles: early endosomes, late endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus. Confocal analysis demonstrated that NPs are capable of entering cells of all three types of epithelium. NPs appear to colocalize with the early endosomes at short times after exposure (∼2 h), but are also found in other compartments within the cytoplasm, notably Golgi and, possibly, ER, as time progressed over the period of 4–24 h. The rate and extent of uptake differed among these cell lines: at a fixed particle/cell ratio, cellular uptake was most abundant in OK cells and least abundant in Caco-2 cells. We present a model for the intracellular fate of particles that is consistent with our experimental data.
Keywords: Nanoparticle; Epithelial cell; Intracellular; Organelle; Confocal microscopy; PLGA;

Triggered release of insulin from glucose-sensitive enzyme multilayer shells by Wei Qi; Xuehai Yan; Jinbo Fei; Anhe Wang; Yue Cui; Junbai Li (2799-2806).
A glucose-sensitive multilayer shell, which was fabricated by the layer-by-layer (LbL) assembly method, can be used as a carrier for the encapsulation and controlled release of insulin. In the present report, glucose oxidase (GOD) and catalase (CAT) were assembled on insulin particles alternately via glutaraldehyde (GA) cross-linking. The resulting core–shell system has been proven to be glucose-sensitive. When the external glucose was introduced, the release ratio of insulin from the protein multilayer can be increased observably. This is likely attributed to the catalysis interaction of CAT/GOD shells to glucose, which leads to the production of H+ and thus drops the pH of the microenvironment. Under the acidic conditions, on the one hand, a part of C=N bond formed from Schiff base reaction can be broken and thus increasing the permeability of the capsule wall. On the other hand, the solubility of insulin can also be increased. The above factors may be the key control to increase the release of insulin from the multilayer. Therefore, such CAT/GOD multilayer may have a great potential as a glucose-sensitive release carrier for insulin, and may open the way for the further application of LbL capsules in the drug delivery and controlled release, etc.
Keywords: Glucose-sensitive; Release of insulin; Microcapsules; Glucose oxidase; Catalase;

On the spontaneous encapsulation of proteins in carbon nanotubes by Yu Kang; Ying-Chun Liu; Qi Wang; Jia-Wei Shen; Tao Wu; Wen-Jun Guan (2807-2815).
Biomolecules–carbon nanotube (CNT) interactions are of great importance in CNT-based drug delivery systems and biomedical devices. In this study, a spontaneous encapsulation of a globular protein into the CNT was observed through molecular dynamics simulation. The free energy of the system was found to be decreased after the encapsulation, which is the most fundamental reason for this spontaneous process. The system enthalpy decrease was found to make a dominant contribution to the free-energy change, and the system entropy increase also contributes to the spontaneous process. During the insertion, the protein makes a stepwise conformational change to maximize its affinity to the CNT walls as well as the protein–CNT interactions, mainly resulting in the deformation of the β-sheets in the protein. As a whole, the CNT was considered to attract protein molecules nonspecifically although the groups with high hydrophobicity and/or aromatic rings show great affinity.
Keywords: Drug delivery; Protein–CNT interaction; Spontaneous encapsulation; Free energy; Conformational change;

Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration by Diederik H.R. Kempen; Lichun Lu; Andras Heijink; Theresa E. Hefferan; Laura B. Creemers; Avudaiappan Maran; Michael J. Yaszemski; Wouter J.A. Dhert (2816-2825).
Bone regeneration is a coordinated cascade of events regulated by several cytokines and growth factors. Angiogenic growth factors are predominantly expressed during the early phases for re-establishment of the vascularity, whereas osteogenic growth factors are continuously expressed during bone formation and remodeling. Since vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are key regulators of angiogenesis and osteogenesis during bone regeneration, the aim of this study was to investigate if their sequential release could enhance BMP-2-induced bone formation. A composite consisting of poly(lactic-co-glycolic acid) microspheres loaded with BMP-2 embedded in a poly(propylene) scaffold surrounded by a gelatin hydrogel loaded with VEGF was used for the sequential release of the growth factors. Empty composites or composites loaded with VEGF and/or BMP-2 were implanted ectopically and orthotopically in Sprague–Dawley rats (n  = 9). Following implantation, the local release profiles were determined by measuring the activity of 125I-labeled growth factors using scintillation probes. After 8 weeks blood vessel and bone formation were analyzed using microangiography, μCT and histology. The scaffolds exhibited a large initial burst release of VEGF within the first 3 days and a sustained release of BMP-2 over the full 56-day implantation period. Although VEGF did not induce bone formation, it did increase the formation of the supportive vascular network (p  = 0.03) in ectopic implants. In combination with local sustained BMP-2 release, VEGF significantly enhanced ectopic bone formation compared to BMP-2 alone (p  = 0.008). In the orthotopic defects, no effect of VEGF on vascularisation was found, nor was bone formation higher by the combination of growth factors, compared to BMP-2 alone. This study demonstrates that a sequential angiogenic and osteogenic growth factor release may be beneficial for the enhancement of bone regeneration.
Keywords: Vascular endothelial growth factor; Bone morphogenetic protein-2; Vessel formation; Bone regeneration; Local release;

Silk fibroin/polyacrylamide semi-interpenetrating network hydrogels for controlled drug release by Biman B. Mandal; Sonia Kapoor; Subhas C. Kundu (2826-2836).
The present study describes a semi-interpenetrating network hydrogel fabricated using silk fibroin/polyacrylamide for controlled drug delivery applications. Hydrogels were synthesized using varied ratios of silk fibroin/acrylamide mixtures crosslinked by N,N′-Methylenebisacrylamide. Fourier-Transform Infrared analysis was performed suggesting β sheet transition of silk fibroin with hydrogels. Scanning electron microscopy revealed microporous surface with maximum pore size of 50 ± 11 μm. Rheological properties along with swellability, degradation, sol fraction estimation, equilibrium water content and swelling kinetics were evaluated. Compressive strength of 241.9 ± 5.5 kPa was observed suggesting mechanically stronger gels. MTT assay showed biocompatibility and absence of deleterious effects of hydrogel on cell viability and functionality. In vitro release studies using two model compounds i.e. trypan blue dye and FITC-inulin reveal their sustained release from the fabricated hydrogel constructs.
Keywords: Silk fibroin; Controlled release; Drug delivery; Hydrogel; Semi-interpenetrating networks;

The use of genetically modified islets is a potential strategy for overcoming pitfalls that currently plague islet transplantation. This study employed functional polymeric vectors specifically designed to transfect insulin-secreting cells and results were compared to various non-viral vectors. The evaluation included transfection efficiency, experimental condition effects, gross morphological observation, cytotoxicity, apoptosis, gene distribution in treated islets, insulin secretion function and time-dependent gene expression pattern. Observations from this study suggest that 1) the experimental conditions for islet transfection should be optimized, 2) the cytotoxicity of sulfonylurea containing vectors differs between the RINm5F cell line and primary pancreatic islets, 3) the non-viral vectors were primarily located in the peripheral region of an islet where the initial cell toxicity/apoptosis was apparent, 4) the genetic modification of pancreatic islets with genes for secretory proteins is more feasible than for residing proteins, and 5) the gene construct selection may prolong the gene expression period and oscillating pattern as demonstrated in this study. This study provides some fundamental background information that will aid in further designing polymeric gene vectors for the optimal manipulation of pancreatic islets prior to transplantation.
Keywords: Islet transfection; Non-viral gene delivery; Polymeric gene delivery; Receptor targeting gene carrier; Endosomolytic materials; Oscillating gene expression;

Ternary complexes of pDNA, polyethylenimine, and γ-polyglutamic acid for gene delivery systems by Tomoaki Kurosaki; Takashi Kitahara; Shintaro Fumoto; Koyo Nishida; Junzo Nakamura; Takuro Niidome; Yukinobu Kodama; Hiroo Nakagawa; Hideto To; Hitoshi Sasaki (2846-2853).
We discovered a vector coated by γ-polyglutamic acid (γ-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic–polycytidylic acid, α-polyaspartic acid, α-polyglutamic acid, and γ-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely high transgene efficiency although it agglutinated with erythrocytes and had extremely high cytotoxicity. Those polyanions changed the positive ζ-potential of pDNA/PEI complex to negative although they did not affect the size. They had no agglutination activities and lower cytotoxicities but most of the ternary complexes did not show any uptake and gene expression; however, the pDNA/PEI/γ-PGA complex showed high uptake and gene expression. Most of the pDNA/PEI/γ-PGA complexes were located in the cytoplasm without dissociation and a few complexes were observed in the nuclei. Hypothermia and the addition of γ-PGA significantly inhibited the uptake of pDNA/PEI/γ-PGA by the cells, although l-glutamic acid had no effect. These results strongly indicate that the pDNA/PEI/γ-PGA complex was taken up by γ-PGA-specific receptor-mediated energy-dependent process. Thus, the pDNA/PEI/γ-PGA complex is useful as a gene delivery system with high transfection efficiency and low toxicity.
Keywords: γ-Polyglutamic acid; Gene transfer; Self assembly; Nano particle; Surface modification; DNA;