Biomaterials (v.29, #4)

Prosthetic heart valves: Catering for the few by Peter Zilla; Johan Brink; Paul Human; Deon Bezuidenhout (385-406).
Prosthetic heart valves epitomize both the triumphant advance of cardiac surgery in its early days and its stagnation into a retrospective, exclusive first world discipline of late. Fifty-two years after the first diseased heart valve was replaced in a patient, prostheses largely represent the concepts of the 1960s with many of their design-inherent complications. While the sophisticated medical systems of the developed world may be able to cope with sub-optimal replacements, these valves are poorly suited to the developing world (where the overwhelming majority of potential valve recipients reside), due to differences in age profiles and socio-economic circumstances. Therefore, it is the latter group which suffered most from the sluggish pace of developments. While it previously took less than 7 years for mechanical heart valves to develop from the first commercially available ball-in-cage valve to the tilting pyrolytic-carbon disc valve, and another 10 years to arrive at the all-carbon bi-leaflet design, only small incremental improvements have been achieved since 1977. Similarly, bioprosthetic valves saw their last major break-through development in the late 1960s when formalin fixation was replaced by glutaraldehyde cross linking. Since then, poorly understood so-called ‘anti-calcification’ treatments were added and the homograft concept rediscovered under the catch-phrase ‘stentless’. Still, tissue valves continue to degenerate fast in younger patients, making them unsuitable for developing countries. Yet, catheter-delivered prostheses almost exclusively use bioprosthetic tissue, thereby reducing one of the most promising developments for patients of the developing world into a fringe product for the few first world recipients. With tissue-engineered valves aiming at the narrow niche of congenital malformations and synthetic flexible leaflet valves being in their fifth decade of low-key development, heart valve prostheses seem to be destined to remain an unsatisfying and exclusive first world solution for a long time to come.
Keywords: Heart valve; Bioprosthesis; Calcification; Immune response; Degradation; Thrombogenicity;

Complement activation on surfaces carrying amino groups by Mitsuaki Toda; Takayuki Kitazawa; Isao Hirata; Yoshiaki Hirano; Hiroo Iwata (407-417).
The complement system is strongly activated by surfaces carrying nucleophilic groups, such as hydroxyl (OH) groups, and triggered by deposition of complement protein fragment, C3b. Surfaces carrying amino groups, the other representative nucleophilic group, are expected to be potential activators of the complement system through the alternative pathway. Few studies thus far have examined the potential of artificial materials carrying amino groups in activating the complement system. In this study, we employed a self-assembled monolayer (SAM) of 11-amino-1-undecanethiol (NH2-SAM) and a polyethyleneimine (PEI)-coated surface as model surfaces to study interactions between amino groups and serum complement pathway. SAMs of 11-mercaptoundecanol (OH-SAM) and 1-dodecanethiol (CH3-SAM) were used as control surfaces, respectively. Although much protein was adsorbed from serum solutions on the two types of amino surfaces, amounts of C3b deposition were much less than those observed on OH-SAM. Amounts of C3a released on the amino surfaces were same levels as that of CH3-SAM, but significantly smaller than that on OH-SAM. These facts suggest that the nucleophilic amino groups on NH2-SAM and PEI-coated surfaces do not directly activate the alternative pathway, but the protein adsorbed layers formed on amino surfaces activate it, but to an extent much smaller than that on OH-SAM. In addition, we found no deposition of C1q molecules on the amino surfaces, suggesting that these surfaces fail to activate the classical pathway. However, more careful studies are needed to conclude it, because it is known that C1q is only transiently detected at typical classical activation interfaces.
Keywords: Complement; Protein adsorption; Blood compatibility; Surface analysis; Surface plasmon resonance;

A microfabricated scaffold for retinal progenitor cell grafting by William L. Neeley; Stephen Redenti; Henry Klassen; Sarah Tao; Tejal Desai; Michael J. Young; Robert Langer (418-426).
Diseases that cause photoreceptor cell degeneration afflict millions of people, yet no restorative treatment exists for these blinding disorders. Replacement of photoreceptors using retinal progenitor cells (RPCs) represents a promising therapy for the treatment of retinal degeneration. Previous studies have demonstrated the ability of polymer scaffolds to increase significantly both the survival and differentiation of RPCs. We report the microfabrication of a poly(glycerol-sebacate) scaffold with superior mechanical properties for the delivery of RPCs to the subretinal space. Using a replica molding technique, a porous poly(glycerol-sebacate) scaffold with a thickness of 45 μm was fabricated. Evaluation of the mechanical properties of this scaffold showed that the Young's modulus is about 5-fold lower and the maximum elongation at failure is about 10-fold higher than the previously reported RPC scaffolds. RPCs strongly adhered to the poly(glycerol-sebacate) scaffold, and endogenous fluorescence nearly doubled over a 2-day period before leveling off after 3 days. Immunohistochemistry revealed that cells grown on the scaffold for 7 days expressed a mixture of immature and mature markers, suggesting a tendency towards differentiation. We conclude that microfabricated poly(glycerol-sebacate) exhibits a number of novel properties for use as a scaffold for RPC delivery.
Keywords: Progenitor cell; Scaffold; Retina; Biocompatibility; Elastomer;

Bioluminescence imaging of calvarial bone repair using bone marrow and adipose tissue-derived mesenchymal stem cells by Irene R. Dégano; Marta Vilalta; Juli R. Bagó; Annette M. Matthies; Jeffrey A. Hubbell; Helen Dimitriou; Paolo Bianco; Nuria Rubio; Jerónimo Blanco (427-437).
A combined strategy using bioluminescence imaging, bone densitometry and histology was used to analyze the bone regeneration capacity of human bone marrow (hBMSC) and adipose tissue (hAMSC) mesenchymal stem cells, seeded in an osteoconductive arginine–glycine–aspartate (RGD) crosslinked hydrogel scaffold, implanted in a mouse calvarial bone defect. We show that firefly luciferase labeled stem cells can be monitored in vivo through a prolonged 90 days period, during which hBMSCs survive better than hAMSCs and that the density of scaffold bearing defects increased significantly more than that of defects without scaffolds.
Keywords: In vivo imaging; Cell differentiation; Scaffold; Luciferase; Enhanced green fluorescent protein (eGFP);

An injectable cross-linked scaffold for nucleus pulposus regeneration by Damien O. Halloran; Sibylle Grad; Martin Stoddart; Peter Dockery; Mauro Alini; Abhay S. Pandit (438-447).
Incorporation of scaffolds has long been recognized as a critical element in most tissue engineering strategies. However with regard to intervertebral disc tissue engineering, the use of a scaffold containing the principal extracellular matrix components of native disc tissue (i.e. collagen type II, aggrecan and hyaluronan) has not been investigated. In this study the behavior of bovine nucleus pulposus cells that were seeded within non-cross-linked and enzymatically cross-linked, atelocollagen type II based scaffolds containing varying concentrations of aggrecan and hyaluronan was investigated. Cross-linking atelocollagen type II based scaffolds did not cause any negative effects on cell viability or cell proliferation over the 7-day culture period. The cross-linked scaffolds retained the highest proteoglycan synthesis rate and the lowest elution of sulfated glycosaminoglycan into the surrounding medium. From confined compression testing and volume reduction measurements, it was seen that the cross-linked scaffolds provided a more stable structure for the cells compared to the non-cross-linked scaffolds. The results of this study indicate that the enzymatically cross-linked, composite collagen–hyaluronan scaffold shows the most potential for developing an injectable cell-seeded scaffold for nucleus pulposus treatment in degenerated intervertebral discs.
Keywords: Intervertebral disc; Scaffold; Extracellular matrix (ECM); Cell signalling;

Medical applications of poly(styrene-block-isobutylene-block-styrene) (“SIBS”) by Leonard Pinchuk; Gregory J. Wilson; James J. Barry; Richard T. Schoephoerster; Jean-Marie Parel; Joseph P. Kennedy (448-460).
Poly(Styrene-block-IsoButylene-block-Styrene) (“SIBS”) is a biostable thermoplastic elastomer with physical properties that overlap silicone rubber and polyurethane. Initial data collected with SIBS stent-grafts and coatings on metallic stents demonstrate hemocompatibility, biocompatibility and long-term stability in contact with metal. SIBS has been used successfully as the carrier for a drug-eluting coronary stent; specifically Boston Scientific's TAXUS® stent, and its uses are being investigated for ophthalmic implants to treat glaucoma, synthetic heart valves to possibly replace tissue valves and other applications. At present, researchers developing medical devices utilizing SIBS have found the following: (1) SIBS does not substantially activate platelets in the vascular system; (2) polymorphonuclear leukocytes in large numbers are not commonly observed around SIBS implants in the vascular system or in subcutaneous implants or in the eye; (3) myofibroblasts, scarring and encapsulation are not clinically significant with SIBS implanted in the eye; (4) embrittlement has not been observed in any implant location; (5) calcification within the polymer has not been observed; and (6) degradation has not been observed in any living system to date. Some deficiencies of SIBS that need to be addressed include creep deformation in certain load-bearing applications and certain sterilization requirements. The reason for the excellent biocompatibility of SIBS may be due to the inertness of SIBS and lack of cleavable moieties that could be chemotactic towards phagocytes.
Keywords: Biostable; Poly(styrene-b-isobutylene-b-styrene); SIBS; Glaucoma; Polyurethane;

Roles of bone scintigraphy and resonance frequency analysis in evaluating osseointegration of endosseous implant by Yi Zhou; Tao Jiang; Mingbo Qian; Xiangyuan Zhang; Jiawei Wang; Bin Shi; Haibin Xia; Xiangrong Cheng; Yining Wang (461-474).
The purpose of this study is to analyze the roles of two non-invasive techniques, bone scintigraphy and resonance frequency analysis (RFA), in the osseointegration assessment. Sixty implants with sandblasted/acid-etched (SA) or machined (MA) surface were placed into the distal femur condyles of 30 rabbits. At 1, 2, 4, 6, 8, and 12 weeks postsurgery, they were subjected to bone scintigraphy, digital radiographic examination, histological and histomorphometric analysis. RFA was performed on each implant both at the time of implant placement and animal sacrifice. The results showed that variation of Tc-99m-MDP uptake (bone scintigraphy value) coincided with that of new bone formation activity and accumulation of osteoblasts. Bone scintigraphy was more sensitive to the change of peri-implant bone than the digital radiographic examination. But it did not correlate with histomorphometric data and failed to detect the difference between SA and MA implants. It was found that RFA value increased with the bone-to-implant contact during the healing phase and correlated with the histomorphometric data. Moreover, RFA distinguished between the SA and MA implants. It is concluded, therefore, that bone scintigraphy may be a dynamic method on peri-implant bone healing, while RFA may be a reliable biomechanical technique that can monitor the osseointegration at macro level. We propose that the combination of these non-destructive techniques may facilitate the identification of the nature of osseointegration.
Keywords: Osseointegration; Implant; Bone scintigraphy; Resonance frequency analysis; In vivo test;

This paper continued our earlier work on the poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles (PLGA/MMT NPs), which were further decorated by human epidermal growth factor receptor-2 (HER2) antibody Trastuzumab for targeted breast cancer chemotherapy with paclitaxel as a model anticancer drug. Such a NP system is multifunctional, which formulates anticancer drugs with no harmful adjuvant, reduces the side effects of the formulated anticancer drug, promotes synergistic therapeutic effects, and achieves targeted delivery of the therapy. The paclitaxel-loaded PLGA/MMT NPs were prepared by a modified solvent extraction/evaporation technique, which were then decorated with Trastuzumab. The effects of the surface decoration on particle size and size distribution, surface morphology, drug encapsulation efficiency, as well as the drug release kinetics, were investigated. The NP formulation exhibited a biphasic drug release with a moderate initial burst followed by a sustained release profile. The surface decoration speeded the drug release. Surface chemistry analysis was conducted by X-ray photoelectron spectroscopy (XPS), which confirmed the presence of Trastuzumab on the NP surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the stability of the antibody in the NP preparation process. Internalization of the coumarin-6-loaded PLGA/MMT NPs with or without the antibody decoration by both of Caco-2 colon adeno carcinoma cells and SK-BR-3 breast cancer cells was visualized by confocal laser scanning microscopy and quantitatively analyzed, which shows that the antibody decoration achieved significantly higher cellular uptake of the NPs. The results of in vitro cytotoxicity experiment on SK-BR-3 cells further proved the targeting effects of the antibody decoration. Judged by IC50 after 24 h culture, the therapeutic effects of the drug formulated in the NPs with surface decoration could be 12.74 times higher than that of the bare NPs and 13.11 times higher than Taxol®.
Keywords: Biodegradable polymers; Cancer nanotechnology; Medical clay; Nanomedicine; Herceptin; HER2;

Iron oxide nanoparticles as a drug delivery vehicle for MRI monitored magnetic targeting of brain tumors by Beata Chertok; Bradford A. Moffat; Allan E. David; Faquan Yu; Christian Bergemann; Brian D. Ross; Victor C. Yang (487-496).
This study explored the possibility of utilizing iron oxide nanoparticles as a drug delivery vehicle for minimally invasive, MRI-monitored magnetic targeting of brain tumors. In vitro determined hydrodynamic diameter of ∼100 nm, saturation magnetization of 94 emu/g Fe and T 2 relaxivity of 43 s−1  mm 1 of the nanoparticles suggested their applicability for this purpose. In vivo effect of magnetic targeting on the extent and selectivity of nanoparticle accumulation in tumors of rats harboring orthotopic 9L-gliosarcomas was quantified with MRI. Animals were intravenously injected with nanoparticles (12 mg Fe/kg) under a magnetic field density of 0 T (control) or 0.4 T (experimental) applied for 30 min. MR images were acquired prior to administration of nanoparticles and immediately after magnetic targeting at 1 h intervals for 4 h. Image analysis revealed that magnetic targeting induced a 5-fold increase in the total glioma exposure to magnetic nanoparticles over non-targeted tumors (p=0.005) and a 3.6-fold enhancement in the target selectivity index of nanoparticle accumulation in glioma over the normal brain (p=0.025). In conclusion, accumulation of iron oxide nanoparticles in gliosarcomas can be significantly enhanced by magnetic targeting and successfully quantified by MR imaging. Hence, these nanoparticles appear to be a promising vehicle for glioma-targeted drug delivery.
Keywords: Drug delivery; Nanoparticle; MRI; Magnetism; Image analysis;

Biotinylated thermoresponsive micelle self-assembled from double-hydrophilic block copolymer for drug delivery and tumor target by Cheng Cheng; Hua Wei; Bao-Xian Shi; Han Cheng; Cao Li; Zhong-Wei Gu; Si-Xue Cheng; Xian-Zheng Zhang; Ren-Xi Zhuo (497-505).
A multifunctional micellar drug carrier formed by the thermosensitive and biotinylated double-hydrophilic block copolymer (DHBC), biotin-poly(ethylene glycol)-block-poly(N-isopropylacrylamide-co-N-hydroxymethylacrylamide) (biotin-PEG-b-P(NIPAAm-co-HMAAm)), was designed and prepared. The P(NIPAAm-co-HMAAm) block with an molar feed ratio of NIPAAm and HMAAm (10:1) was identified to exhibit the reversible phase transition at the lower critical solution temperature (LCST) of 36.7 °C. Cytotoxicity study indicated that the biotin-PEG-b-P(NIPAAm-co-HMAAm) copolymer did not exhibit obvious cytotoxicity. The block copolymer was capable of self-assembling into micelle in water. Transmission electron microscopy showed that the self-assembled micelles were regularly spherical in shape. The anticancer drug methotrexate (MTX) was loaded in the micelles and the in vitro release behaviors of MTX at different temperatures were investigated. The association of biotin molecule with the copolymer was confirmed by a unique capillary electrophoresis immunoassay (CEIA) method based on enhanced chemiluminescence (CL) detection. The fluorescence spectroscopy analysis as well as confocal microscopy studies confirmed the DHBC drug carriers could specifically and efficiently bind to cancer cells with pretreatment of biotin-transferrin, suggesting that the multifunctionalized DHBC micelle may be a useful drug carrier for tumor targeting.
Keywords: DHBC; Biotinylated thermoresponsive micelle; CEIA-CL; Controlled release; Tumor target;

Delivery of siRNA from lyophilized polymeric surfaces by Morten Ø. Andersen; Kenneth A. Howard; Søren R. Paludan; Flemming Besenbacher; Jørgen Kjems (506-512).
Standard in vitro gene silencing protocols are performed using aqueous formulations of transfection reagents and small interfering RNAs (siRNA) reconstituted immediately prior to use. In this study, we describe a method for producing gene silencing-active lyophilized cationic polymer (chitosan) or lipid (TransIT-TKO) siRNA formulations. We demonstrate specific and efficient knockdown of enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells transfected in plates pre-coated with both TransIT-TKO/siRNA (∼85%) and a chitosan/siRNA formulation containing sucrose as lyoprotectant (∼70%). This method removes the necessity for both siRNA reconstitution immediately prior to use and addition onto cells. Furthermore, silencing activity of the chitosan/siRNA formulation was shown over the period studied (∼2 months) when stored at room temperature. Higher cell viability was observed using the chitosan system compared to the lipid formulation. Silencing of the proinflammatory cytokine tumour necrosis factor (TNF-α) was also demonstrated in the RAW macrophage cell line using the lyophilized chitosan/siRNA system suggesting that the coating can improve the biocompatibility of medical implants. This work describes an efficient gene silencing methodology using freeze-dried formulations with potential applications as a high throughput screening tool for gene function, biocompatible medical implant components and longer shelf-life therapeutics.
Keywords: SiRNA; Chitosan; Lyophilization; RNA interference; Drug delivery; Macrophages.;