Biomaterials (v.28, #21)

The effect of strontium incorporation into CaSiO3 ceramics on their physical and biological properties by Chengtie Wu; Yogambha Ramaswamy; Danielle Kwik; Hala Zreiqat (3171-3181).
CaSiO3 ceramics have been regarded as a potential bioactive material for bone regeneration. Strontium (Sr) as a trace element in human body has been found to have beneficial effects on bone formation. The aim of this study was to incorporate Sr into CaSiO3 bioactive ceramics and to investigate their effect(s) on phase transition, sintering property, apatite-formation ability, ionic dissolution, and human bone-derived cells (HBDC) proliferation. Sr containing CaSiO3 (Sr-CaSiO3) ceramics at various concentrations (0–10% Sr) were prepared. The incorporation of Sr into CaSiO3 promoted the phase transition from β to α-CaSiO3 and enhanced ceramic densification but did not alter the mechanism and ability of apatite formation in SBF. The ionic dissolution rate of the Sr-CaSiO3 decreased compared to the CaSiO3. The addition of Sr decreased pH value in SBF. The effect of Sr-CaSiO3 extracts, carried out according to the International Standard Organization, on HBDC proliferation was evaluated. At high extract concentration (100 and 200 mg/mL), CaSiO3 was found to stimulate HBDC proliferation, however, the incorporation of Sr into CaSiO3 stimulated HBDC proliferation even at low extract concentration (ranging from 12.5, 25 to 50 mg/mL). Our results indicate that Sr-CaSiO3 ceramics improved the physical and biological properties of the pure CaSiO3 ceramics.
Keywords: Sr-CaSiO3; Phase transition; Apatite-formation ability; Dissolution; Proliferation;

On imparting radiopacity to a poly(urethane urea) by Nirmala R. James; A. Jayakrishnan (3182-3187).
A poly(urethane urea) (PUU) synthesized from 2,4-toluene diisocyanate (TDI) and polyethylene glycol (PEG) with ethylenediamine (ED) as the chain extender was rendered radiopaque by attaching 3,4,5-triiodobenzoic acid (TIB) onto the polymer backbone. The radiopaque polyurethane obtained was characterized by infra red (IR) spectroscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and X-radiography. By optimizing the reaction conditions, it was possible to carry out the modification without adversely affecting the properties of the starting polymer significantly. IR spectral evidence suggested that the hydrogen bonded structure of PUU remained undisrupted even after modification. However, the product exhibited altered thermal characteristics when compared to the parent polymer. Degradation characteristics as observed from the TGA remained unchanged, while one of the glass transitions got shifted to a lower temperature. The observed changes in thermal characteristics were explained on the basis of possible inter-phase mixing and the changes in the close packing of the polymer chains by the introduction of bulky iodine atoms.
Keywords: Poplyurethane; Polymerization; Radiopacity; 3,4,5-triiodobenzoic acid; Polymer modification;

Influence of engineered titania nanotubular surfaces on bone cells by Ketul C. Popat; Lara Leoni; Craig A. Grimes; Tejal A. Desai (3188-3197).
A goal of current orthopedic biomaterials research is to design implants that induce controlled, guided, and rapid healing. In addition to acceleration of normal wound healing phenomena, these implants should result in the formation of a characteristic interfacial layer with adequate biomechanical properties. To achieve these goals, however, a better understanding of events at the bone–material interface is needed, as well as the development of new materials and approaches that promote osseointegration. Using anodization, titania interfaces can be fabricated with controlled nanoarchitecture. This study demonstrates the ability of these surfaces to promote osteoblast differentiation and matrix production, and enhance short- and long-term osseointegration in vitro. Titania nanotubular surfaces were fabricated using an anodization technique. Marrow stromal cells (MSCs) were isolated from male Lewis rats and seeded on these surfaces along with control surfaces. The interaction of cells with these surfaces was investigated in terms of their ability to adhere, proliferate and differentiate on them. The experiments were repeated three times with cells from different cultures. All the results were analyzed using analysis of variance (ANOVA). Statistical significance was considered at p<0.05. Furthermore, in vivo biocompatibility was assessed by implanting surfaces subcutaneously in male Lewis rat and performing histological analysis after 4 weeks. Our results indicate that the nanotubular titania surfaces provide a favorable template for the growth and maintainence of bone cells. The cells cultured on nanotubular surfaces showed higher adhesion, proliferation, ALP activity and bone matrix deposition compared to those grown on flat titanium surfaces. In vivo biocompatibility results suggest that nanotubular titania does not cause chronic inflammation or fibrosis. The fabrication routes of titania nano-architectures are flexible and cost-effective, enabling realization of desired platform topologies on existing non-planar orthopedic implants.
Keywords: Nanotopography; Bone; Alkaline phosphatase; Titanium oxide;

The effect of three-dimensional demineralized bone matrix on in vitro cumulus-free oocyte maturation by Suofeng Ma; Hang Lin; Yiliang Miao; Xinyong Liu; Bin Wang; Jianwu Dai (3198-3207).
The physiological role of cumulus cell surrounding oocytes is particularly important for normal cytoplasmic maturation of oocytes. Collagen-based demineralized bone matrix (DBM) is a valuable biomaterial for the three-dimensional (3-D) cell culture. The present study was designed to determine whether in vitro maturation (IVM) of cumulus-free oocytes in mice could be improved by using the 3-D DBM co-culture system. The results indicated that the denuded oocytes cultured in 3-D DBM co-culture system with cumulus cells showed close similarity of cortical granules (CGs) distribution pattern, had more normal maturation-promoting factor (MPF) level and zona pellucida (ZP) hardening level to the in vivo matured oocytes, and the best preimplantation development after being activated by in vitro fertilization (IVF) or parthenogenetic activation. Thus, 3-D DBM collagen scaffold could serve as a tool for fundamental in vitro studies of cells or tissues under the environment that closely assembles the in vivo conditions.
Keywords: Co-culture; Demineralized bone matrix; In vitro maturation; Oocyte;

Cell patterning chip for controlling the stem cell microenvironment by Adam Rosenthal; Alice Macdonald; Joel Voldman (3208-3216).
Cell–cell signaling is an important component of the stem cell microenvironment, affecting both differentiation and self-renewal. However, traditional cell-culture techniques do not provide precise control over cell–cell interactions, while existing cell-patterning technologies are limited when used with proliferating or motile cells. To address these limitations, we created the Bio Flip Chip (BFC), a microfabricated polymer chip containing thousands of microwells, each sized to trap down to a single stem cell. We have demonstrated the functionality of the BFC by patterning a 50×50 grid of murine embryonic stem cells (mESCs), with patterning efficiencies >75%, onto a variety of substrates—a cell-culture dish patterned with gelatin, a 3-D substrate, and even another layer of cells. We also used the BFC to pattern small groups of cells, with and without cell–cell contact, allowing incremental and independent control of contact-mediated signaling. We present quantitative evidence that cell–cell contact plays an important role in depressing mESC colony formation, and show that E-cadherin is involved in this negative regulatory pathway. Thus, by allowing exquisite control of the cellular microenvironment, we provide a technology that enables new applications in tissue engineering and regenerative medicine.
Keywords: Cell patterning; Embryonic stem cells; Cell signaling; Microwells;

Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering by Hansoo Park; Johnna S. Temenoff; Yasuhiko Tabata; Arnold I. Caplan; Antonios G. Mikos (3217-3227).
We investigated the development of an injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) with encapsulated rabbit marrow mesenchymal stem cells (MSCs) and gelatin microparticles (MPs) loaded with transforming growth factor-β1 (TGF-β1) for cartilage tissue engineering applications. Rabbit MSCs and TGF-β1-loaded MPs were mixed with OPF, a poly(ethylene glycol)-diacrylate crosslinker and the radical initiators ammonium persulfate and N,N,N′,N′-tetramethylethylenediamine, and then crosslinked at 37 °C for 8 min to form hydrogel composites. Three studies were conducted over 14 days in order to examine the effects of: (1) the composite formulation, (2) the MSC seeding density, and (3) the TGF-β1 concentration on the chondrogenic differentiation of encapsulated rabbit MSCs. Bioassay results showed no significant difference in DNA amount between groups, however, groups with MPs had a significant increase in glycosaminoglycan content per DNA starting at day 7 as compared to controls at day 0. Chondrocyte-specific gene expression of type II collagen and aggrecan were only evident in groups containing TGF-β1-loaded MPs and varied with TGF-β1 concentration in a dose-dependent manner. Specifically, type II collagen gene expression exhibited a 161±49-fold increase and aggrecan gene expression a 221±151-fold increase after 14 days with the highest dose of TGF-β1 (16 ng/ml). These results indicate that encapsulated rabbit MSCs remained viable over the culture period and differentiated into chondrocyte-like cells, thus suggesting the potential of OPF composite hydrogels as part of a novel strategy for localized delivery of stem cells and bioactive molecules.
Keywords: Cartilage tissue engineering; Marrow mesenchymal stem cells; Gelatin microparticles; Injectable hydrogels; Tgf-β1;

Biomolecular surface coating to enhance orthopaedic tissue healing and integration by Catherine D. Reyes; Timothy A. Petrie; Kellie L. Burns; Zvi Schwartz; Andrés J. García (3228-3235).
Implant osseointegration is a prerequisite for clinical success in orthopaedic and dental applications, many of which are restricted by loosening. Biomaterial surface modification approaches, including calcium-phosphate ceramic coatings and macro/microporosity, have had limited success in promoting integration. To improve osseointegration, titanium surfaces were coated with the glycine–phenylalanine–hydroxyproline–glycine–glutamate–arginine (GFOGER) collagen-mimetic peptide, selectively promoting α 2 β 1 integrin binding, a crucial event for osteoblastic differentiation. Titanium surfaces presenting GFOGER triggered osteoblastic differentiation and mineral deposition in bone marrow stromal cells, leading to enhanced osteoblastic function compared to unmodified titanium. Furthermore, this integrin-targeted coating significantly improved in vivo peri-implant bone regeneration and osseointegration, as characterized by bone–implant contact and mechanical fixation, compared to untreated titanium in a rat cortical bone–implant model. GFOGER-modified implants also significantly enhanced osseointegration compared to surfaces modified with full-length type I collagen, highlighting the importance of presenting specific biofunctional domains within the native ligand. In addition, this biomimetic implant coating is generated using a simple, single-step procedure that readily translates to a clinical environment with minimal processing and cytotoxicity concerns. Therefore, this study establishes a biologically active and clinically relevant implant-coating strategy that enhances bone repair and orthopaedic implant integration.
Keywords: Biomimetic material; Cell adhesion; Collagen; Osseointegration; Integrin;

A novel trans-lymphatic drug delivery system: Implantable gelatin sponge impregnated with PLGA–paclitaxel microspheres by Jiang Liu; Dale Meisner; Elizabeth Kwong; Xiao Y. Wu; Michael R. Johnston (3236-3244).
A translymphatic drug delivery system which incorporates poly-lactide-co-glycolide–paclitaxel (PLGA–PTX) or PLGA–rhodamine microspheres into gelatin sponge matrix is described. The system combines the sustained release properties of PLGA–PTX with the structural advantages of gelatin matrix that can be implanted directly to the lymphatic site for both therapeutic and prophylactic purposes. The PLGA microspheres were prepared using spray drying technique. The particles were in the size range of 1–8 μm, suitable for intraperitoneal and intrapleural lymphatic targeting delivery. Scanning electron microscopy revealed the homogeneous distribution of PLGA microspheres in the porous sponge network. The release of PTX was mainly controlled by the degradation of the PLGA. Crosslinking gelatin using carbodiimide reduced the biodegradation of the sponge and thereby delayed the release of the PLGA in vitro. In vivo lymphatic delivery was assessed in both healthy rats and rats bearing orthotopic lung cancer. Intraperitoneal and intrapleural implantation of the sponge impregnated with PLGA microspheres resulted in spontaneous absorption of the particles in the lymphatic system. It is concluded that the system provides great potential for targeted delivery of therapeutic agent to the lymphatic system especially for the control of lymphatic metastasis in cancer.
Keywords: Lymphatic drug delivery; Gelatin sponge; PLGA-paclitaxel microspheres; Controlled release; Cancer;

A series of novel cationic star polymers were synthesized by conjugating multiple oligoethylenimine (OEI) arms onto an α-cyclodextrin (α-CD) core as nonviral gene delivery vectors. The molecular structures of the α-CD-OEI star polymers, which contained linear or branched OEI arms with different chain lengths ranging from 1 to 14 ethylenimine units, were characterized by using size exclusion chromatography, 13C and 1H NMR, and elemental analysis. The α-CD-OEI star polymers were studied in terms of their DNA binding capability, formation of nanoparticles with plasmid DNA (pDNA), cytotoxicity, and gene transfection in cultured cells. All the α-CD-OEI star polymers could inhibit the migration of pDNA on agarose gel through formation of complexes with pDNA, and the complexes formed nanoparticles with sizes ranging from 100 to 200 nm at N/P ratios of 8 or higher. The star polymers displayed much lower in vitro cytotoxicity than that of branched polyethylenimine (PEI) of molecular weight 25K. The α-CD-OEI star polymers showed excellent gene transfection efficiency in HEK293 and Cos7 cells. Generally, the transfection efficiency increased with an increase in the OEI arm length. The star polymers with longer and branched OEI arms showed higher transfection efficiency. The best one of the star polymers for gene delivery showed excellent in vitro transfection efficiency that was comparable to or even higher than that of branched PEI (25K). The novel α-CD-OEI star polymers with OEI arms of different chain lengths and chain architectures can be promising new nonviral gene delivery vectors with low cytotoxicity and high gene transfection efficiency for future gene therapy applications.
Keywords: Cyclodextrin; Star polymer; Polyethylenimine; Gene transfer;

Development of an antigen-presenting cell-targeted DNA vaccine against melanoma by mannosylated liposomes by Yan Lu; Shigeru Kawakami; Fumiyoshi Yamashita; Mitsuru Hashida (3255-3262).
As part of our research involving the targeted delivery of plasmid DNA (pDNA) to antigen-presenting cells (APCs), we developed mannosylated cationic liposomes: N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/cholesten-5-yloxy-N-(4-((1-imino-2-d-thiomannosyl-ethyl)amino)butyl)formamide (Man-C4-Chol)/Chol (Man liposomes). In this study, we used melanoma-associated antigen expressing pDNA; pUb-M and Man liposomes to create a novel APC-targeted DNA vaccine against melanoma and examined its potency by measuring the Ub-M mRNA expression in splenic dendritic cells and macrophages, the cytotoxic T lymphocyte (CTL) activity against melanoma B16BL6 cells and the melanoma B16BL6-specific anti-tumor effect after intraperitoneal (i.p.) administration. We verified that Man lipoplex induces significantly higher pUb-M gene transfection into dendritic cells and macrophages than unmodified lipoplex and naked DNA and it also strongly induces CTL activity against melanoma, inhibits its growth and prolongs the survival after tumor challenge compared with unmodified liposomes and the standard method (naked pDNA, intramuscular (i.m.)). These results demonstrate that Man liposomes are a potent APCs-targeted vector that induce strong immunopotency of DNA vaccine against melanoma.
Keywords: Mannosylated cationic liposome; DNA vaccine; Melanoma; Non-viral vector; Gene therapy;