Biomaterials (v.30, #21)
The antibacterial activity of Magainin I immobilized onto mixed thiols Self-Assembled Monolayers
by Vincent Humblot; Jean-Fabrice Yala; Pascal Thebault; Kada Boukerma; Arnaud Héquet; Jean-Marc Berjeaud; Claire-Marie Pradier (pp. 3503-3512).
An antibacterial peptide, Magainin I, was covalently bound to a mixed 11-mercaptoundecanoïc acid (MUA) and 6-mercaptohexanol (C6OH) (ratio 1:3) Self-Assembled Monolayer (SAM) on gold surfaces. Each step of the surface functionalization was characterized by Polarization Modulation Reflection Absorption InfraRed Spectroscopy (PM-RAIRS) and X-ray Photoelectron Spectroscopy (XPS). The antibacterial activity of the anchored Magainin was tested against three Gram-positive bacteria ( Listeria ivanovii, Enterococcus faecalis and Staphylococcus aureus), and the results revealed that the adsorbed Magainin I reduced by more than 50% the adhesion of bacteria at the surface, together with the killing of the bacteria that nonetheless adhered to the surface. No release of the peptide was observed upon contact with the bacterial suspension; the activity has proven to be persistent overtime, up to six months after the first use.
Keywords: Self-assembled monolayers; XPS; PM-RAIRS; Bacteria; Magainin; Antibacterial surfaces
The use of phospholipase A2 to prepare acellular porcine corneal stroma as a tissue engineering scaffold
by Zheng Wu; Yang Zhou; Naiyang Li; Minghai Huang; Haoyun Duan; Jian Ge; Peng Xiang; Zhichong Wang (pp. 3513-3522).
This study was to develop a method using phospholipase A2 (PLA2) to prepare acellular porcine corneal stroma (APCS) for tissue engineering. The APCS was prepared from native porcine cornea (NPC) that was treated with 200U/ml PLA2 and 0.5% sodium deoxycholate (SD). The removal of DNA content, representing decellularization efficiency, reached to 91%, while all hydroxyproline and 80% of glycosaminoglycan were retained in the APCS when compared with NPC. The residual PLA2 and SD were 0.35±0.04U/mg dry weight and 4.3±0.8ng/mg dry weight respectively. The extracts of APCS had no inhibitory effects on proliferation of corneal epithelial and endothelial cells as well as keratocytes. There was no sign of infiltration of neutrophilic leukocytes or leukomonocytes at 2 weeks after subcutaneous implantation of APCS. The prepared APCS displayed similar light transmittance to NPC. There were no significant differences in the areal modulus and curvature variation between APCS and NPC. Rabbit lamellar keratoplasty showed that the grafts of APCS were epithelialized completely in 8±2 days, and their transparency was restored in 84±11 days when the light transmittance of APCS-transplanted corneas displayed no significant difference compared with native corneas. Corneal neovascularization, corneal deformation, and graft degradation were not observed within 12 months.
Keywords: Corneal tissue engineering; Scaffold; Phospholipase A; 2; Sodium deoxycholate; Decellularization
Cell immobilization in gelatin–hydroxyphenylpropionic acid hydrogel fibers
by Min Hu; Motoichi Kurisawa; Rensheng Deng; Choon-Meng Teo; Annegret Schumacher; Ya-Xuan Thong; Lishan Wang; Karl M. Schumacher; Jackie Y. Ying (pp. 3523-3531).
Gelatin–hydroxyphenylpropionic acid (Gtn–HPA) hydrogels are highly porous and biodegradable materials. Herein we report a fiber spinning method that can produce cell-seeded solid and hollow hydrogel fibers by enzymatically cross-linking Gtn–HPA in solutions flowing within a capillary tube. The cell-immobilized hydrogel fibers, with feature sizes down to 20μm, are formed as a result of continuous cross-linking of cell-mixed hydrogel precursors in a multiphase laminar flow. This fiber formation process is mild enough to retain the cell viability. The continuous fiber formation, simultaneous cell encapsulation, as well as versatile combination of fiber structures provided by this approach make it a promising and effective technique for the preparation of cell-seeded hydrogel scaffolds and carriers for tissue engineering.
Keywords: Cell encapsulation; Cross-linking; Gelatin; Hydrogel; Scaffold
Osteoblastic differentiation of human bone marrow stromal cells in self-assembled BMP-2 receptor-binding peptide-amphiphiles
by Jue-Yeon Lee; Jung-Eun Choo; Young-Suk Choi; Jin-Sook Suh; Seung-Jin Lee; Chong-Pyoung Chung; Yoon-Jeong Park (pp. 3532-3541).
Self-assembled nanostructures consisting of BMP receptor-binding peptides, termed osteopromotive domains (OPDs), and hydrophobic alkyl chains were fabricated with the aim of developing three-dimensional scaffolding materials for osteoblastic differentiation. OPD peptide was identified from BMP-2 and had an affinity for BMP receptors thereby inducing differentiation of human bone marrow stromal cells into osteoblastic cells. The peptide-hydrophobic alkyl chain amphiphiles were designed to mimic nanofibrous extracellular structures and to add osteogenic ligands to enhance osteoblastic cell function. The OPD peptide-amphiphiles (OPDAs) that end with the alkylation of the N-terminus of the OPD peptide were synthesized by standard solid phase chemistry. The self-assembly was triggered by mixing OPDA solution with calcium ions. Observation using scanning electron microscopy (SEM) revealed the formation of nanofibrous structures with extremely high aspect ratios and high surface areas. The FT-IR and circular dichroism (CD) spectrophotometry demonstrated that self-assembled nanofibers have a β-sheet structure. The activation of Smad, an osteoblastic differentiation marker, was obtained in the cell culture gel of self-assembled OPDA; therefore, the intracellular signal transduction for osteogenesis was performed like an OPD peptide. Cell survival was supported in the OPDA gel for 10 days, and osteoblastic differentiation of human bone marrow stromal cells (hBMSCs) was evident as demonstrated by calcein staining and ALP activity measurement. These results revealed that self-assembled OPDA maintained osteogenic activity by the surface-exposed OPD peptide. Taken together, the self-assembled OPDA nanofibrous gel can be utilized as a cell culture scaffold in bone regeneration.
Keywords: Osteopromotive domain (OPD); BMP-2; Peptide-amphiphiles; Nanofibers; Scaffold; Osteoblastic differentiation
Differentiation of fetal osteoblasts and formation of mineralized bone nodules by 45S5 Bioglass® conditioned medium in the absence of osteogenic supplements
by Olga Tsigkou; Julian R. Jones; Julia M. Polak; Molly M. Stevens (pp. 3542-3550).
Bioactive glasses bond strongly to bone in vivo and their ionic dissolution products have previously been shown to have stimulatory properties on adult and fetal osteoblasts and to induce the differentiation of embryonic stem cells towards the osteoblastic lineage in vitro. In the present study, the effect of 45S5 Bioglass® conditioned medium with two different Si concentrations (15 μg/ml (BGCM/15) and 20 μg/ml (BGCM/20)) on human fetal osteoblast growth, differentiation and extracellular matrix production and mineralization was investigated. In the first instance, primary fetal osteoblasts were examined for the osteoblast phenotypic markers alkaline phosphatase (ALP), collagen type I (Col I) and OB Cadherin (Cadherin 11) (OB Cad) as well as for the mesenchymal stem cell markers CD105 and CD166. At passage 0 more than 50% of the population was positive for Col I and ALP, but at passage 2, the proportion of cells expressing ALP increased. In addition at passage 0 more than 50% of the fetal osteoblasts expressed the mesenchymal stem cell surface markers CD105 and CD166. Treatment with BGCM/15 and BGCM/20 in the absence of osteogenic supplements increased the gene expression of the bone extracellular matrix proteins alkaline phosphatase, osteonectin and bone sialoprotein as determined by quantitative real time reverse transcriptase-polymerase chain reaction (rt RT-PCR) analysis. Extracellular matrix production was also enhanced in the absence of osteogenic supplements by the 45S5 Bioglass® conditioned medium as demonstrated by ALP enzymatic activity, osteocalcin and Col I protein synthesis. Furthermore, BGCM/15 and BGCM/20 significantly enhanced the formation of mineralized nodules, based on alizarin red histochemical staining, without necessitating the addition of β-glycerophosphate,l-ascorbate-2-phosphate or dexamethasone (commonly used osteogenic supplements).
Keywords: Bioactive glass; Bone tissue engineering; Osteoblasts
Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering
by Pinar Yilgor; Kadriye Tuzlakoglu; Rui L. Reis; Nesrin Hasirci; Vasif Hasirci (pp. 3551-3559).
The aim of this study was to develop a 3-D construct carrying an inherent sequential growth factor delivery system. Poly(lactic acid- co-glycolic acid) (PLGA) nanocapsules loaded with bone morphogenetic protein BMP-2 and poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) nanocapsules loaded with BMP-7 made the early release of BMP-2 and longer term release of BMP-7 possible. 3-D fiber mesh scaffolds were prepared from chitosan and from chitosan–PEO by wet spinning. Chitosan of 4% concentration in 2% acetic acid (CHI4–HAc2) and chitosan (4%) and PEO (2%) in 5% acetic acid (CHI4–PEO2–HAc5) yielded scaffolds with smooth and rough fiber surfaces, respectively. These scaffolds were seeded with rat bone marrow mesenchymal stem cells (MSCs). When there were no nanoparticles the initial differentiation rate was higher on (CHI4–HAc2) scaffolds but by three weeks both the scaffolds had similar alkaline phosphatase (ALP) levels. The cell numbers were also comparable by the end of the third week. Incorporation of nanoparticles into the scaffolds was achieved by two different methods: incorporation within the scaffold fibers (NP–IN) and on the fibers (NP–ON). It was shown that incorporation on the CHI4–HAc2 fibers (NP–ON) prevented the burst release observed with the free nanoparticles, but this did not influence the total amount released in 25 days. However NP–IN for the same fibers revealed a much slower rate of release; ca. 70% released at the end of incubation period. The effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from the CHI4–HAc2 scaffolds was studied in vitro using samples prepared with both incorporation methods. The effect of delivered agents was higher with the NP–ON samples. Delivery of BMP-2 alone suppressed cell proliferation while providing higher ALP activity compared to BMP-7. Simultaneous delivery was not particularly effective on cell numbers and ALP activity. The sequential delivery of BMP-2 and BMP-7, on the other hand, led to the highest ALP activity per cell (while suppressing proliferation) indicating the synergistic effect of using both growth factors holds promise for the production of tissue engineered bone.
Keywords: Bone tissue engineering; Sequential delivery; BMP; PLGA; PHBV; Chitosan
The promotion of the vascularization of decalcified bone matrix in vivo by rabbit bone marrow mononuclear cell-derived endothelial cells
by Hongbo Tan; Bin Yang; Xiaojun Duan; Fuyou Wang; Ying Zhang; Xuhong Jin; Gang Dai; Liu Yang (pp. 3560-3566).
The neovascularization of bone grafting represents an important challenge in bone regeneration. Prevascularization of tissue-engineered bone using endothelial cells (ECs) in vitro sheds light on accelerating the vascularization of bone replacements. In the present study, decalcified bone matrix (DBM) was prevascularized by seeding fibrin gels with ECs that are derived from rabbit bone marrow mononuclear cells (BMMNCs). The compound was then transplanted autologously into bone defects of rabbits to observe the vascularization in vivo. At 2, 4 and 8 weeks after grafting, the microvessel density of new bone tissues was significantly higher in the experimental group than in the control group ( P<0.05), which suggests that prevascularization of BMMNC-derived cells may be suitable for improving vascularization in tissue-engineered bone.
Keywords: Bone marrow; Endothelial cell; Bone regeneration; ECM (extracellular matrix)
Node of Ranvier formation on motoneurons in vitro
by John W. Rumsey; Mainak Das; Maria Stancescu; Marga Bott; Cristina Fernandez-Valle; James J. Hickman (pp. 3567-3572).
One of the most significant interactions between Schwann cells and neurons is myelin sheath formation. Myelination is a vertebrate adaptation that enables rapid conduction of action potentials without a commensurate increase in axon diameter. In vitro neuronal systems provide a unique modality to study both factors influencing myelination and diseases associated with myelination. Currently, no in vitro system for motoneuron myelination by Schwann cells has been demonstrated. This work details the myelination of motoneuron axons by Schwann cells, with complete Node of Ranvier formation, in a defined in vitro culture system. This defined system utilizes a novel serum-free medium in combination with the non-biological substrate, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA). The myelinated segments and nodal proteins were visualized and quantified using confocal microscopy. This defined system provides a highly controlled, reproducible model for studying Schwann cell interactions with motoneurons as well as the myelination process and its effect on neuronal plasticity. Furthermore, an in vitro system that would allow studies of motoneuron myelination would be beneficial for understanding peripheral demyelinating neuropathies such as diabetes induced peripheral neuropathy and could lead to a better understanding of CNS demyelinating diseases like multiple sclerosis, as well as neuromuscular junction maturation and maintenance.
Keywords: Schwann cells; Nerve tissue engineering; Silane; Biomimetic material; Growth factors; Co-culture
Electrochemical desorption of self-assembled monolayers for engineering cellular tissues
by Rina Inaba; Ali Khademhosseini; Hiroaki Suzuki; Junji Fukuda (pp. 3573-3579).
Adherent cells, cell sheets, and spheroids were harvested noninvasively from a culture surface by means of electrochemical desorption of a self-assembled monolayer (SAM) of alkanethiol. The SAM surface was made adhesive by the covalent bonding of Arg-Gly-Asp (RGD)-peptides to the alkanethiol molecules. The application of a negative electrical potential caused the reductive desorption of the SAM, resulting in the detachment of the cells. Using this approach greater than 90% of adherent cells detached within 5min. Furthermore, this approach was used to obtain two-dimensional (2D) cell sheets. The detached cell sheets consisted of viable cells, which could be easily attached to other cell sheets in succession to form a multilayered cell sheet. Moreover, spheroids of hepatocytes of a uniform diameter were formed in an array of cylindrical cavities at a density of 280 spheroids/cm2 and were harvested by applying a negative electrical potential. This cell manipulation technology could potentially be a useful tool for the fabrication and assembly of building blocks such as cell sheets and spheroids for regenerative medicine and tissue engineering applications.
Keywords: Cell sheet; Spheroid; Fibroblast; Hepatocyte; Self-assembled monolayer; Electrochemistry
The growth of a vascular network inside a collagen–citric acid derivative hydrogel in rats
by Toshio Takayama; Tetsushi Taguchi; Hiroyuki Koyama; Matomo Sakari; Wataru Kamimura; Tsuyoshi Takato; Tetsuro Miyata; Hirokazu Nagawa (pp. 3580-3587).
Three-dimensional regenerative tissue with a certain bulk cannot survive without sufficient blood perfusion in vivo, so construction of a vascular system in regenerative tissue is a key technology in tissue engineering. In order to construct such a vascular system, we attempted to create a scaffold material that induces neovascular growth from the recipient bed into the material. This material, an ion complex gel matrix (IC gel) consisting of collagen and a citric acid derivative, enabled it to associate with basic fibroblast growth factor (bFGF). The IC gel was implanted in the subfascial space of the rat rectus muscle and excised 5 days later. Cross-sections of the excised samples were stained for von Willebrand factor, and then neovascular development into the gel was observed and also quantified by image analysis. These data showed that the IC gel markedly induced growth of vascular-rich tissue into the inside of the gel by day 5, which surpassed that after implantation of Matrigel® or gelated collagen. Further, combination with bFGF significantly enhanced the vascularization ability of IC gel. These findings suggest that IC gel functioned as a scaffold material for neovascular ingrowth and a reservoir of bFGF.
Keywords: Neovascularization; In vivo test; Collagen structure; Biodegradation; Scaffold; Fibroblast growth factor
Dextran conjugated dendritic nanoconstructs as potential vectors for anti-cancer agent
by Abhinav Agarwal; Umesh Gupta; Abhay Asthana; Narendra K. Jain (pp. 3588-3596).
The purpose of the present investigation was to evaluate the potential of surface engineered polypropylene imine (PPI) dendrimers as nanoscale drug delivery units for site-specific delivery of a model anti-cancer agent, doxorubicin·hydrochloride (DOX). Dextran conjugated PPI dendrimers were synthesized, characterized and further loaded with DOX. The developed formulation was characterized by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and transmission electron microscopic (TEM) studies. Dendrimer formulation was evaluated for in vitro drug release and haemolytic studies under various pH conditions. Cell uptake and cytotoxicity studies were performed on A549 cell lines using MTT cell proliferation assay. In vivo studies were conducted for evaluation of various pharmacokinetic parameters and tissue distribution pattern. In vitro, formulation displayed initial rapid release of the drug followed by rather slow release. Further, the dextran conjugated dendrimer formulation was found to be least haemolytic but more cytotoxic as compared to free drug. Cell uptake studies depicted that the formulation was preferably taken up by the tumor cells when compared to free drug. The conjugation of oxidized polyaldehyde dextran imparts macromolecular nature to the dendritic carrier, consequently the formulation was found to selectively enter highly porous mass of tumor cells at the same time precluding normal tissues. Thus it was concluded that the drug loaded dendrimer formulation would selectively localize in the tumor mass, increasing the therapeutic margin of safety while reducing the side effects associated with anti-cancer agents.
Keywords: Dextran; Dendrimers; Cancer; Doxorubicin; Targeted drug delivery
Block-copolymer micelles as carriers of cell signaling modulators for the inhibition of JNK in human islets of Langerhans
by Radoslav Savić; Tony Azzam; Adi Eisenberg; Hinyu Nedev; Lawrence Rosenberg; Dusica Maysinger (pp. 3597-3604).
Here we investigate the potential of PCL-b-PEO micelles in preventing the cell death of isolated human islets of Langerhans. PCL-b-PEO micelles were loaded with c-Jun NH2-terminal kinases inhibitor SP600125 to rescue the isolated islets. Mechanistic studies of the uptake were conducted in PC12 cells. Incorporation of SP600125 afforded 8.2 fold greater solubility of SP600125 in micelle suspension. To investigate the effectiveness of micelle-incorporated SP600125 in preventing the islet cell death, we challenged the islets with TNF-alpha, IL-1, and IFN gamma. Micelle-incorporated SP600125 did not lose its inhibitory activity during incorporation into micelles, and it protected the islets against cytokine-induced loss of viability to the same extent as control SP600125. Moreover, the concentration of micelle-incorporated SP600125 used was 13-fold lower, demonstrating the greater efficacy of micelle delivered SP600125. Micelles maintained their cytoplasmic distribution without detectable nuclear localization in islets. The inhibition of JNK was confirmed by western blots. This study suggests that micelle-based intracellular delivery of potent, poorly water soluble, cell-death-pathway inhibitors may represent a valuable addition to established delivery of cytocidal block-copolymer micelle-incorporated bioactives.
Keywords: Micelle; Islet; Drug delivery; Diabetes; Polycaprolactone; Polyethyleneoxide
A chitosan–dipotassium orthophosphate hydrogel for the delivery of Doxorubicin in the treatment of osteosarcoma
by Hang T. Ta; Crispin R. Dass; Ian Larson; Peter F.M. Choong; Dave E. Dunstan (pp. 3605-3613).
The current management of primary osteosarcoma (OS) and its secondary metastasis is limited by the lack of an efficient drug delivery system. Here we report an in situ gelling chitosan/dipotassium orthophosphate hydrogel system designed to directly deliver the frontline chemotherapeutic agent (Doxorubicin) in a sustained time period to tumor sites. A significant reduction of both primary and secondary OS in a clinically relevant orthotopic model was measured when doxorubicin was administered with the hydrogel. This hydrogel delivery system also reduced cardiac and dermal toxicity of Doxorubicin in mice. The results obtained from this study demonstrate the potential application of a biodegradable hydrogel technology as an anti-cancer drug delivery system for successful chemotherapy.
Keywords: Chitosan; Orthophosphate; Drug delivery; Hydrogel; Osteosarcoma; Cancer
Dual chemotherapy and photodynamic therapy in an HT-29 human colon cancer xenograft model using SN-38-loaded chlorin-core star block copolymer micelles
by Cheng-Liang Peng; Ping-Shan Lai; Feng-Huei Lin; Steven Yueh-Hsiu Wu; Ming-Jium Shieh (pp. 3614-3625).
Chlorin-core star-shaped block copolymer (CSBC) may self-assemble to form micelles, which act as nanosized photosensitizing agents for photodynamic therapy (PDT) and further encapsulate hydrophobic drugs. This functionalized micellar delivery system is a potential dual carrier for the synergistic combination of photodynamic therapy and chemotherapy for the treatment of cancer. In this study, SN-38 encapsulated CSBC micelles were successfully prepared using a lyophilization–hydration method. Our results show that the prolonged plasma residence time of SN-38/CSBC micelles as compared with free CPT-11 permit increased tumor accumulation and consequently, improved antitumor activity. The combined effects of SN-38/CSBC micelles with PDT were evaluated in an HT-29 human colon cancer xenograft model. Interesting, SN-38/CSBC-mediated PDT synergistically inhibited tumor growth, resulting in up to 60% complete regression of well-established tumors after 3 treatments. These treatments also decreased the microvessel density (MVD) and cell proliferation within the subcutaneous tumors. Therefore, this SN-38/CBSC delivery system has the potential to offer dual therapies for the synergistic combination of PDT and chemotherapy for the treatment of cancer.
Keywords: Star-shaped copolymer; Micelle; Photodynamic therapy; SN-38; Chemotherapy; Colon cancer
Surface mediated in situ differentiation of mesenchymal stem cells on gene-functionalized titanium films fabricated by layer-by-layer technique
by Yan Hu; Kaiyong Cai; Zhong Luo; Rui Zhang; Li Yang; Linhong Deng; Klaus D. Jandt (pp. 3626-3635).
In this work, multilayered and gene-functionalized titanium films composed of chitosan (Chi) and plasmid DNA (pEGFP–hBMP2, pGB) were employed to investigate the surface mediated in situ differentiation of mesenchymal stem cells (MSCs). The Chi/pGB multilayered structures were fabricated by layer-by-layer (LbL) assembly technique and degraded to release plasmid DNA complexes depending on bilayer numbers over 7 days. Therefore, the differentiation behaviors of MSCs cultured onto Chi/pGB multilayered titanium films surface were investigated. Chi/pGB LbL-modified titanium films show significant higher (p<0.01) transfection efficiency than those of other groups transfected by lipofectamine 2000 regarding the expression of green fluorescent protein (GFP). Reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that MSCs adhered onto Chi/pGB LbL-modified titanium films could still express hBMP2 mRNA over 7 days culture. Compared with control groups, MSCs cultured onto Chi/pGB LbL-modified titanium films display significantly higher (p<0.01 or p<0.05) production levels of alkaline phosphatase (ALP) and osteocalcin over 7 days and 14 days culture, respectively. These results demonstrate that Chi/pGB LbL-modified titanium films are beneficial for sustained in situ inducing osteoprogenitor cells to differentiate into mature osteoblasts over long time. The approach presented here has potential applications in the development of gene-stimulating biomaterials and implant technology.
Keywords: Titanium; Layer-by-layer; Mesenchymal stem cells; Differentiation; Gene transfection