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Biomaterials (v.29, #14)

Editorial board (pp. ifc).

Mechanically strong double network photocrosslinked hydrogels from N, N-dimethylacrylamide and glycidyl methacrylated hyaluronan by Lihui Weng; Andrew Gouldstone; Yuhong Wu; Weiliam Chen (pp. 2153-2163).
Hyaluronan (HA) is a natural polysaccharide abundant in biological tissues and it can be modified to prepare biomaterials. In this work, HA modified with glycidyl methacrylate was photocrosslinked to form the first network (PHA), and then a series of highly porous PHA/ N, N-dimethylacrylamide (DAAm) hydrogels (PHA/DAAm) with high mechanical strength were obtained by incorporating a second network of photocrosslinked DAAm into PHA network. Due to the synergistic effect produced by double network (DN) structure, despite containing 90% of water, the resulting PHA/DAAm hydrogel showed a compressive modulus and a fracture stress over 0.5MPa and 5.2MPa, respectively. Compared to the photocrosslinked hyaluronan single network hydrogel, which is generally very brittle and fractures easily, the PHA/DAAm hydrogels are ductile. Mouse dermal fibroblast was used as a model cell line to validate in vitro non-cytotoxicity of the PHA/DAAm hydrogels. Cells deposited extracellular matrix on the surface of these hydrogels and this was confirmed by positive staining of Type I collagen by Sirius Red. The PHA/DAAm hydrogels were also resistant to biodegradation and largely retained their excellent mechanical properties even after 2 months of co-culturing with fibroblasts.

Keywords: Hyaluronan; N; ,; N; -dimethylacrylamide; Hydrogels; Photocrosslinkable; Double network

Biodegradable thermogelling poly(ester urethane)s consisting of poly(lactic acid) – Thermodynamics of micellization and hydrolytic degradation by Xian Jun Loh; Yun Xuan Tan; Ziyun Li; Lin Shin Teo; Suat Hong Goh; Jun Li (pp. 2164-2172).
Multiblock poly(ether ester urethane)s comprising of poly(lactic acid) (PLA), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) segments were synthesized, and their aqueous solutions exhibited thermogelling behavior at critical gelation concentrations (CGC) ranging from 7 to 9wt%. The chemical structures and molecular characteristics of the copolymers were studied by GPC,1H NMR,13C NMR and FTIR. The thermal stability of the poly(PEG/PPG/PLA urethane)s was studied by thermogravimetry analysis (TGA), and the PLA contents were calculated based on the thermal degradation profile. The results were in good agreement with those obtained from the1H NMR measurements. The critical micellization concentration (CMC) of these water-soluble poly(ether ester urethane)s was determined at different temperatures using a dye solubilization method. The thermodynamic parameters for micelle formation were calculated, indicating that the process is largely entropy-driven. Interestingly, it appears that there exists a requirement for the system to possess a minimum gain in entropy before the thermogelling effect can be observed. Dilute copolymer solutions showed a lower critical solution temperature (LCST) behavior similar to pNIPAM dissolved in aqueous solutions. The thermogels hydrolytically degraded to polymer fragments corresponding to the constituent segment blocks within 3 months.

Keywords: Thermogelling polymer; Poly(lactic acid); Poly(ethylene glycol); Poly(propylene glycol)

The effect of water-soluble chitosan on macrophage activation and the attenuation of mite allergen-induced airway inflammation by Chin-Lung Chen; Yu-Ming Wang; Chia-Fang Liu; Jiu-Yao Wang (pp. 2173-2182).
Chitin and chitosan have versatile anti-tumor, anti-fungal, and antimicrobial biological properties. Oral intakes and intranasal administration of chitin attenuated allergen-induced airway inflammation in sensitized mice, which may be due to its Th1 adjuvant properties. However, their mechanism of action is not entirely clear. In this report, we demonstrate that water-soluble chitosan (WSC) has specific immunomodulatory effects on dust mite allergen Dermatophagoides farinae (Der f)-stimulated, monocyte-derived macrophages (MDM). These effects include polarizing the cytokine balance towards Th1 cytokines, decreasing the production of the inflammatory cytokines IL-6 and TNF-α, down-regulating CD44 and TLR4 receptor expression, and inhibiting T cell proliferation. Scanning electron microscope (SEM) examination found that WSC reduced the rate of pseudopodia formation in Der f-stimulated MDM from allergic asthma patients. The effect of WSC on allergen-stimulated MDM may be mediated via inhibition of PKCζ phosphorylation and NF-κB pathway activation. In a murine model of asthma, we found that intranasal application of WSC attenuates Der f-induced lung inflammation by reducing infiltration of inflammatory cells, epithelial damage, and goblet cell hyperplasia and markedly decreasing production of Arg I, iNOs, and thymic stromal lymphopoietin (TSLP) in the bronchial epithelium. Therefore, we believe that WSC may provide a new therapeutic modality for allergic asthma.

Keywords: Macrophage; Chitosan; AllergyAbbreviations; Arg I; arginase I; Der f; Dermatophagoides farinae; Der p; Dermatophagoides pteronyssinus; iNOs; inducible nitric oxide synthase; HDM; house dust mite; MDM; monocyte-derived macrophages; PBMCs; peripheral blood mononuclear cells; PKCζ; protein kinase Cζ; SEM; scanning electronic microscopy; TSLP; thymic stromal lymphopoietin

The dependence of in vivo stable ectopic chondrogenesis by human mesenchymal stem cells on chondrogenic differentiation in vitro by Kai Liu; Guang Dong Zhou; Wei Liu; Wen Jie Zhang; Lei Cui; Xia Liu; Tian Yi Liu; Yilin Cao (pp. 2183-2192).
In vivo niche plays an important role in determining the fate of implanted mesenchymal stem cells (MSCs) by directing committed differentiation. An inappropriate in vivo niche can also alter desired ultimate fate of exogenous MSCs even they are in vitro induced to express a specific phenotype before in vivo implantation. Studies have shown that in vitro chondrogenically differentiated MSCs are apt to lose their phenotype and fail to form stable cartilage in subcutaneous environment. We hypothesized that failure of maintaining the phenotype of induced MSCs in subcutaneous environment is due to the insufficient chondrogenic differentiation in vitro and fully differentiated MSCs can retain their chondrocyte-like phenotype and form stable ectopic cartilage. To test this hypothesis, extended in vitro chondrogenic induction and cartilage formation were carried out before implantation. Human bone marrow stem cells (hBMSCs) were seeded onto polylactic acid coated polyglycolic acid scaffolds. The cell-scaffold constructs were chondrogenically induced from 4 to 12 weeks for in vitro chondrogenesis, and then implanted subcutaneously into nude mice for 12 or 24 weeks. The engineered cartilages were evaluated by gross view, glycosaminoglycan content measurement, and histological staining before and after implantation. Histological examination showed typical cartilage structure formation after 8 weeks of induction in vitro. However, part of the constructs became ossified after implantation when in vitro induction lasted 8 weeks or less time. In contrast, those induced for 12 weeks in vitro could retain their cartilage structure after in vivo implantation. These results indicate that a fully differentiated stage achieved by extended chondrogenic induction in vitro is necessary for hBMSCs to form stable ectopic chondrogenesis in vivo.

Keywords: Human mesenchymal stem cells; Chondrogenic induction; Ectopic chondrogenesis; Subcutaneous environment

Effect of hydrogel porosity on marrow stromal cell phenotypic expression by Mahrokh Dadsetan; Theresa E. Hefferan; Jan P. Szatkowski; Prasanna K. Mishra; Slobodan I. Macura; Lichun Lu; Michael J. Yaszemski (pp. 2193-2202).
This study describes investigation of porous photocrosslinked oligo[(polyethylene glycol) fumarate] (OPF) hydrogels as potential matrix for osteoblastic differentiation of marrow stromal cells (MSCs). The porosity and interconnectivity of porous hydrogels were assessed using magnetic resonance microscopy (MRM) as a noninvasive investigative tool that could image the water construct inside the hydrogels at a high-spatial resolution. MSCs were cultured onto the porous hydrogels and cell number was assessed using PicoGreen DNA assay. Our results showed 10% of cells initially attached to the surface of scaffolds. However, cells did not show significant proliferation over a time period of 14 days. MSCs cultured on porous hydrogels had increased alkaline phosphatase activity as well as deposition of calcium, suggesting successful differentiation and maturation to the osteoblastic phenotype. Moreover, continued expression of type I collagen and osteonectin over 14 days confirmed osteoblastic differentiation of MSCs. MRM was also applied to monitor osteogenesis of MSCs on porous hydrogels. MRM images showed porous scaffolds became consolidated with osteogenic progression of cell differentiation. These findings indicate that porous OPF scaffolds enhanced MSC differentiation leading to development of bone-like mineralized tissue.

Keywords: Hydrogel; Oligo[(polyethylene glycol) fumarate] (OPF); Marrow stromal cells; Magnetic resonance microscopy; Osteogenesis

The influence of proepicardial cells on the osteogenic potential of marrow stromal cells in a three-dimensional tubular scaffold by Mani T. Valarmathi; Michael J. Yost; Richard L. Goodwin; Jay D. Potts (pp. 2203-2216).
It is well established that the process of neovascularization or neoangiogenesis is coupled to the development and maturation of bone. Bone marrow stromal cells (BMSCs) or mesenchymal stem cells (MSCs) comprise a heterogeneous population of cells that can be differentiated in vitro into both mesenchymal and non-mesenchymal cell lineages. When both rat BMSCs and quail proepicardia (PEs) were seeded onto a three-dimensional (3-D) tubular scaffold engineered from aligned collagen type I strands and co-cultured in osteogenic media, the maturation and co-differentiation into osteoblastic and vascular cell lineages were observed. In addition, these cells produced abundant mineralized extracellular matrix materials and vessel-like structures. BMSCs were seeded at a density of 2×106cells/15mm tube and cultured in basal media for 3 days. Subsequently, on day 3, PEs were seeded onto the same tubes and the co-culture was continued for another 3, 6 or 9 days either in basal or in osteogenic media. Differentiated cells were subjected to immunohistochemical, cytochemical and biochemical analyses. Phenotypic induction was analyzed at mRNA level by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Immunolocalization of key osteogenic and vasculogenic lineage specific markers were examined using confocal scanning laser microscopy. In osteogenic tube cultures, both early and late osteogenic markers were observed and were reminiscent of in vivo expression pattern. Alkaline phosphatase activity and calcium content significantly increased over the observed period of time in osteogenic medium. Abundant interlacing fascicles of QCPN, QH1, isolectin and α-smooth muscle actin (α-SMA) positive cells were observed in these tube cultures. These cells formed extensive arborizations of nascent capillary-like structures and were seen amidst the developing osteoblasts in osteogenic cultures. The 3-D culture system not only generated de novo vessel-like structures but also augmented the maturation and differentiation of BMSCs into osteoblasts. Thus, this novel co-culture system provides a useful in vitro model to investigate the functional role and effects of neovascularization in the proliferation, differentiation and maturation of BMSC derived osteoblasts.

Keywords: Bone marrow stromal cells; Mesenchymal stem cells; Proepicardial cells; Osteogenesis; Vasculogenesis; Bone tissue engineering

In vitro evaluation of electrospun silk fibroin scaffolds for vascular cell growth by Xiaohui Zhang; Cassandra B. Baughman; David L. Kaplan (pp. 2217-2227).
Human aortic endothelial (HAEC) and human coronary artery smooth muscle cell (HCASMC) responses on electrospun silk fibroin scaffolds were studied to evaluate potential for vascular tissue engineering. Cell proliferation studies supported the utility of this biomaterial matrix by both HAECs and HCASMCs. Alignment and elongation of HCASMCs on random non-woven nanofibrous silk scaffolds was observed within 5 days after seeding based on SEM and confocal microscopy. Short cord-like structures formed from HAECs on the scaffolds by day 4, and a complex interconnecting network of capillary tubes with identifiable lumens was demonstrated by day 7. The preservation of cell phenotype on the silk fibroin scaffolds was confirmed by the presence of cell-specific markers, including CD146, VE-cadherin, PECAM-1 and vWF for HAECs, and SM-MHC2 and SM-actin for HCASMCs at both protein and transcription levels using immunocytochemistry and real-time RT-PCR, respectively. Formation of ECM was also demonstrated for the HCASMCs, based on the quantification of collagen type I expression at protein and transcription levels. The results indicate a favorable interaction between vascular cells and electrospun silk fibroin scaffolds. When these results are factored into the useful mechanical properties and slow degradability of this protein biomaterial matrix, potential utility in tissue-engineered blood vessels can be envisioned.

Keywords: Silk; Electrospinning; Endothelial cells; Smooth muscle cells; Vascular

Using living radical polymerization to enable facile incorporation of materials in microfluidic cell culture devices by Helen M. Simms; Christopher M. Bowman; Kristi S. Anseth (pp. 2228-2236).
High throughput screening tools are expediting cell culture studies with applications in drug discovery and tissue engineering. This contribution demonstrates a method to incorporate 3D cell culture sites into microfluidic devices and enables the fabrication of high throughput screening tools with uniquely addressable culture environments. Contact lithographic photopolymerization (CLiPP) was used to fabricate microfluidic devices with two types of 3D culture sites: macroporous rigid polymer cell scaffolds and poly(ethylene glycol) (PEG) encapsulated cell matrices. Cells were cultured on-device with both types of culture sites, demonstrating material cytocompatibility. Multilayer microfluidic devices were fabricated with channels passing the top and bottom sides of a series of rigid porous polymer scaffolds. Cells were seeded and cultured on device, demonstrating the ability to deliver cells and culture cells on multiple scaffolds along the length of a single channel. Flow control through these rigid porous polymer scaffolds was demonstrated. Finally, devices were modified by grafting of PEG methacrylate from surfaces to prevent non-specific protein adsorption and ultimately cell adhesion to channel surfaces. The living radical component of this CLiPP device fabrication platform enables facile incorporation of 3D culture sites into microfluidic cell culture devices, which can be utilized for high throughput screening of cell-material interactions.

Keywords: Three-dimensional; Scaffold; Hydrogel; Photopolymerization; Photolithography; Tissue engineering

Regeneration of abdominal wall musculofascial defects by a human acellular collagen matrix by Denis Dufrane; Michel Mourad; Mathieu van Steenberghe; Rose-Marie Goebbels; Pierre Gianello (pp. 2237-2248).
This work studied the reconstruction of an abdominal wall defect by a human acellular collagen matrix.The abdominal wall defect was cured in 40 rats by implanting (i) polypropylene (Pro), (ii) polyester (Mers) meshes, and (iii) human acellular collagen matrix with two orientations: fibres in parallel (fascia lata longitudinal [FLL]) or perpendicular (fascia lata transversal [FLT]) to native rats' abdominal walls. Hernia recurrence, adhesions, and histology (for inflammation and remodelling) were assessed at 4 and 8 weeks after implantation. Two large abdominal eventrations were cured by a human acellular matrix in human patients.A higher hernia recurrence rate was observed for rats transplanted with FLL than with FLT/Pro/Mers at 4 and 8 weeks after implantation. A lower adhesion rate was achieved for FLL/FLT than for Pro/Mers meshes ( p<0.05). A decrease in immunologic cell infiltrations in FLL/FLT was observed between day 30 and day 60 ( p<0.05). Collagen, elastin, and muscular tissues were found only in FLL/FLT matrix; a weaker muscular cell infiltration for FLL occurred at 8 weeks. Human abdominal eventrations were totally cured by using FLT as confirmed by computed tomography scanning at 12 and 16 months after implantation. In conclusion, human acellular collagen matrix, placed in an FLT position, can induce an abdominal wall reconstitution without adhesions and hernia recurrence.

Keywords: Abdomen; Joint replacement; Collagen structure; Connective tissue; In vivo; test; Histomorphometry

Effect of β-tricalcium phosphate particles with varying porosity on osteogenesis after sinus floor augmentation in humans by Christine Knabe; Christian Koch; Alexander Rack; Michael Stiller (pp. 2249-2258).
This study examines the effect of two β-tricalcium phosphate (TCP) particulate bone grafting materials with varying porosity on bone formation and on osteogenic marker expression 6 months after sinus floor augmentation. Unilateral sinus grafting was performed in 20 patients using a combination (4:1 ratio) of β-TCP particles with 35% porosity (TCP-C) or 65% porosity (TCP-CM) and autogenous bone chips. At implant placement cylindrical biopsies were sampled and processed for immunohistochemical analysis of resin embedded sections. Sections were stained for collagen type I (Col I), alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP). Furthermore, the area fraction of newly formed bone as well as the particle area fraction were determined histomorphometrically first, apically close to the Schneiderian membrane and second, in the center of the cylindrical biopsies. In the TCP-CM patient group a larger amount of bone formation and particle degradation was observed in the apical area and thus at the largest distance from the crestal bone compared to the TCP-C group. Good bone bonding behaviour was observed with both materials. This was accompanied by expression of ALP, Col I, BSP and OC in the newly formed bone and osteogenic mesenchym in contact with the degrading particles. Both TCP materials supported bone formation in the augmented sinus floor. Six months after implantation of both types of β-TCP particles, bone formation and matrix mineralization was still actively progressing in the tissue surrounding the particles. Consequently, a greater porosity appears to be advantageous for enhancing bone formation and particle degradation.

Keywords: Bone substitute materials; Tricalcium phosphate ceramics; Porosity; In vivo; Osteoblast differentiation; Hard tissue histology

Extended delivery of ophthalmic drugs by silicone hydrogel contact lenses by Jinah Kim; Anthony Conway; Anuj Chauhan (pp. 2259-2269).
We developed extended wear silicone hydrogel soft contact lenses that deliver ophthalmic drugs for an extended period of time ranging from weeks to months. Silicone hydrogels comprising of N, N-dimethylacrylamide, 3-methacryloxypropyltris(trimethylsiloxy)silane, bis-alpha,omega-(methacryloxypropyl) polydimethylsiloxane, 1-vinyl-2-pyrrolidone, and ethylene glycol dimethacrylate were prepared with varying ratios of monomers and transport of three different ophthalmic drugs, timolol, dexamethasone, and dexamethasone 21-acetate was explored. All the silicone hydrogels of 0.1mm thickness exhibit diffusion limited transport and extended release varying 20 days up to more than three months depending on the compositions of hydrophobic and hydrophilic components of silicone hydrogels. Also, there are multiple time scales in transport of at least certain molecules, which is perhaps due to the complex microstructure of these gels. The mechanical and physical properties of lenses such as ion permeability, equilibrium water content, transparency, and surface contact angles of some of the gels are suitable for contact lens application.

Keywords: Silicone hydrogels; Extended wear contact lens; Extended release; Timolol; Dexamethasone; Ion permeability

HER-2-mediated endocytosis of magnetic nanospheres and the implications in cell targeting and particle magnetization by Shy Chyi Wuang; Koon Gee Neoh; En-Tang Kang; Daniel W. Pack; Deborah E. Leckband (pp. 2270-2279).
Polypyrrole–Fe3O4 nanospheres were synthesized via an emulsion polymerization method with hyaluronic acid as the surfactant. Hyaluronic acid offers the advantages of biocompatibility, cell adhesive property and the availability of functional groups for attachment of other molecules. The nanospheres were further functionalized with herceptin, and the efficacy of uptake of the functionalized nanospheres by human breast cancer cells was evaluated. It is envisioned that the combination of hyaluronic acid with its cell adhesive property and herceptin would result in high efficacy of internalization of the nanospheres by the cancer cells via a HER-2-mediated endocytosis. Our results showed that this is indeed the case and that the high concentration of herceptin-functionalized magnetic nanospheres in the cancer cells offers great potential in cancer cell targeting and treatment. In addition, the magnetic property of these nanospheres was also critically investigated and the magnetization was found to be affected by the particles' environment. The combination of these cell-targeting magnetic carriers with chemotherapeutic agents will be highly advantageous for the preferential killing of cancer cells in hyperthermia treatment.

Keywords: Polypyrrole–Fe; 3; O; 4; nanospheres; Herceptin; HER-2-mediated endocytosis; Cell targeting; Magnetization

Enhancement of PDGF-BB mitogenic activity on human dermal fibroblasts by biospecific dextran derivatives by Anne-Sophie Vercoutter-Edouart; Guy Dubreucq; Barbara Vanhoecke; Christelle Rigaut; François Renaux; Latifa Dahri-Correia; Jérôme Lemoine; Marc Bracke; Jean-Claude Michalski; José Correia (pp. 2280-2292).
Dextran derivatives are biosynthetic polyanionic polymers which exert some of the heparin properties such as regulating the activity of several heparin-binding growth factors. Based on a reproducible synthetic procedure, we have synthesized a new generation of dextran derivatives named dextran methylcarboxylate benzylamide sulfate (DMCBSu). Here we investigated the ability of a library of well-characterized DMCBSu to interact with platelet-derived growth factor-BB (PDGF-BB), which has essential roles during wound healing. Using gel mobility shift assay, our results indicate that benzylamide and sulfate groups act synergically to bind to PDGF-BB. Furthermore we show that depending on their chemical composition, functionalized dextrans are able to potentiate the mitogenic activity of PDGF-BB on human dermal fibroblasts. This enhancing effect is accompanied with changes in PDGF-BB-induced signaling events, as determined by the use of specific inhibitors and by western blot. Our results suggest that the use of such biopolymers combined with a local administration of the growth factor could increase the efficiency of the biomolecule activity in future therapeutic strategies.

Keywords: Dextran derivative; Platelet-derived growth factor; Human dermal fibroblast; Wound healing

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