Biomaterials (v.29, #22)

Universal cell labelling with anionic magnetic nanoparticles by Claire Wilhelm; Florence Gazeau (3161-3174).
Magnetic labelling of living cells creates opportunities for numerous biomedical applications, from individual cell manipulation to MRI tracking. Here we describe a non-specific labelling method based on anionic magnetic nanoparticles (AMNPs). These particles first adsorb electrostatically to the outer membrane before being internalized within endosomes. We compared the labelling mechanism, uptake efficiency and biocompatibility with 14 different cell types, including adult cells, progenitor cells, immune cells and tumour cells. A single model was found to describe cell/nanoparticle interactions and to predict uptake efficiency by all the cell types. The potential impact of the AMNP label on cell functions, in vitro and in vivo, is discussed according to cellular specificities. We also show that the same label provides sufficient magnetization for MRI detection and distal manipulation.
Keywords: Iron oxide nanoparticles; Magnetism; MRI; Biocompatibility; Cell therapy; Cell manipulation;

Thermoresponsive nanocomposite hydrogels with cell-releasing behavior by Yaping Hou; Andrew R. Matthews; Ashley M. Smitherman; Allen S. Bulick; Mariah S. Hahn; Huijie Hou; Arum Han; Melissa A. Grunlan (3175-3184).
Poly(N-isopropylacrylamide) (PNIPAAm) hydrogels become more hydrophobic when they reversibly switch from a water-swollen to a deswollen state above the volume phase transition temperature (VPTT, ∼33 °C) which has been used to modulate cell adhesion. In the current work, we prepared novel thermoresponsive nanocomposite hydrogels comprised of a PNIPAAm hydrogel matrix and polysiloxane colloidal nanoparticles (∼220 nm average diameter) via in situ photopolymerization of aqueous solutions of NIPAAm monomer, N,N′-methylenebisacrylamide (BIS, crosslinker), photoinitiator and polysiloxane nanoparticles (0.5–2.0 wt% based on solution weight) at ∼7 °C. The VPTT of the nanocomposite hydrogels is not altered versus the pure PNIPAAm hydrogel. Dynamic mechanical analysis and tensile tests revealed that higher nanoparticle content generally produced improved hydrogel mechanical properties. Surfaces of nanocomposite hydrogels became increasingly more hydrophobic at all temperatures between 10 and 40 °C as the amount of hydrophobic polysiloxane nanoparticles was increased. When cooled from 37 to 25 °C, mouse smooth muscle precursor cells (10T1/2) were effectively detached from nanocomposite hydrogel surfaces. The utility of photopatterning to create surface micropillars comprised of nanocomposite hydrogels was demonstrated.
Keywords: Hydrogel; Thermally responsive material; Siloxane; Nanoparticles; Cell adhesion; Photopolymerization;

Thermo-responsive multiblock poly(ester urethane)s comprising poly(ε-caprolactone) (PCL), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) segments were synthesized. The copolymers were characterized by GPC, NMR, FTIR, XRD, DSC and TGA. Water-swelling analysis carried out at different temperatures revealed that the bulk hydrophilicity of the copolymers could be controlled either by adjusting the composition of the copolymer or by changing the temperature of the environment. These thermo-responsive copolymer films formed highly swollen hydrogel-like materials when soaked in cold water and shrank when soaked in warm water. The changes are reversible. The mechanical properties of the copolymer films were assessed by tensile strength measurement. These copolymers were ductile when compared to PCL homopolymers. Young's modulus and the stress at break increased with increasing PCL content, whereas the strain at break increased with increasing PEG content. The results of the cytotoxicity tests based on the ISO 10993-5 protocol demonstrated that the copolymers were non-cytotoxic and could be potentially used in biomedical applications.
Keywords: Biodegradable; Stimuli-responsive; Poly(ester urethane); Poly(ε-caprolactone); Poly(ethylene glycol); Poly(propylene glycol);

Simultaneous in vivo comparison of bone substitutes in a guided bone regeneration model by Dieter Busenlechner; Stefan Tangl; Birgit Mair; Georg Fugger; Reinhard Gruber; Heinz Redl; Georg Watzek (3195-3200).
A direct, simultaneous comparison of bone substitutes is hampered by the limited number of samples that can be tested simultaneously. The goal of this study was to establish a preclinical model for guided bone regeneration that offers testing of different bone substitutes in a one-wall defect situation. We show here that up to eight titanium hemispheres can be placed on the calvaria of minipigs. To establish our model, titanium hemispheres were filled with and without Bio-Oss®, a deproteinized bovine bone mineral, Ostim®, an aqueous paste of synthetic nanoparticular hydroxyapatite, and Osteoinductal®, an oily calcium hydroxide suspension, before being positioned on the calvaria. After 6 and 12 weeks, titanium hemispheres were subjected to histological and histomorphometric analysis. We show here that bone filled approximately one-tenth of the area below the hemispheres which were left empty, indicating a critical size model for guided bone regeneration. In accordance with the documented osteoconductive properties of Bio-Oss® and Ostim®, titanium hemispheres were almost completely filled with bone. Moreover, the expected degradation profile of Bio-Oss® and Ostim® could be confirmed by histologic and histomorphometric analysis. Under the same conditions, Osteoinductal® failed to exert osteoconductive properties, rather a progressive resorption of the host bone was observed. These results demonstrate that the preclinical model presented here is suitable to simultaneously compare bone substitutes with different material properties. Our model based on the titanium hemispheres allows evaluation of graft consolidation under standardized conditions thereby avoiding intra-individual variations.
Keywords: In vivo test; Bone graft; Bone ingrowth; Bone regeneration; Osteoconduction; Degradation;

Given the inadequacies of existing repair strategies for cartilage injuries, tissue engineering approach using biomaterials and stem cells offers new hope for better treatments. Recently, we have fabricated injectable collagen–human mesenchymal stem cell (hMSC) microspheres using microencapsulation. Apart from providing a protective matrix for cell delivery, the collagen microspheres may also act as a bio-mimetic matrix facilitating the functional remodeling of hMSCs. In this study, whether the encapsulated hMSCs can be pre-differentiated into chondrogenic phenotype prior to implantation has been investigated. The effects of cell seeding density and collagen concentration on the chondrogenic differentiation potential of hMSCs have been studied. An in vivo implantation study has also been conducted. Fabrication of cartilage-like tissue micro-masses was demonstrated by positive immunohistochemical staining for cartilage-specific extracellular matrix components including type II collagen and aggrecan. The meshwork of collagen fibers was remodeled into a highly ordered microstructure, characterized by thick and parallel bundles, upon differentiation. Higher cell seeding density and higher collagen concentration favored the chondrogenic differentiation of hMSCs, yielding increased matrix production and mechanical strength of the micro-masses. These micro-masses were also demonstrated to integrate well with the host tissue in NOD/SCID mice.
Keywords: Chondrogenic differentiation; Mesenchymal stem cell; Collagen; Microspheres; Cartilage; Tissue engineering;

Transient elastic support for vein grafts using a constricting microfibrillar polymer wrap by Mohammed S. El-Kurdi; Yi Hong; John J. Stankus; Lorenzo Soletti; William R. Wagner; David A. Vorp (3213-3220).
Arterial vein grafts (AVGs) often fail due to intimal hyperplasia, thrombosis, or accelerated atherosclerosis. Various approaches have been proposed to address AVG failure, including delivery of temporary mechanical support, many of which could be facilitated by perivascular placement of a biodegradable polymer wrap. The purpose of this work was to demonstrate that a polymer wrap can be applied to vein segments without compromising viability/function, and to demonstrate one potential application, i.e., gradually imposing the mid-wall circumferential wall stress (CWS) in wrapped veins exposed to arterial levels of pressure.Poly(ester urethane)urea, collagen, and elastin were combined in solution, and then electrospun onto freshly-excised porcine internal jugular vein segments. Tissue viability was assessed via Live/Dead™ staining for necrosis, and vasomotor challenge with epinephrine and sodium nitroprusside for functionality. Wrapped vein segments were also perfused for 24 h within an ex vivo vascular perfusion system under arterial conditions (pressure = 120/80 mmHg; flow = 100 mL/min), and CWS was calculated every hour.Our results showed that the electrospinning process had no deleterious effects on tissue viability, and that the mid-wall CWS vs. time profile could be dictated through the composition and degradation of the electrospun wrap. This may have important clinical applications by enabling the engineering of an improved AVG.
Keywords: Electrospinning; Vein graft; Intimal hyperplasia; Vein wrap;

The application of human bone marrow stromal cells and poly(dl-lactic acid) as a biological bone graft extender in impaction bone grafting by Benjamin J.R.F. Bolland; Janos M. Kanczler; Patrick J. Ginty; Steve M. Howdle; Kevin M. Shakesheff; Douglas G. Dunlop; Richard O.C. Oreffo (3221-3227).
Concerns over disease transmission, high costs and limited supply have led to interest in synthetic grafts in the field of impaction bone grafting (IBG). Poly(dl-lactic acid) (PLA) grafts are attractive alternatives due to their biocompatibility, established safety and versatile manufacturing process. This study examined the potential of PLA scaffolds augmented with human bone marrow stromal cells (HBMSCs) in IBG. In vitro and in vivo studies were performed on impacted morsellised PLA seeded with HBMSC and compared to PLA alone. In vitro samples were incubated under osteogenic conditions and in vivo samples were implanted subcutaneously into severely compromised immunodeficient mice, for 4 weeks. Biochemical, histological, mechanical and 3D micro-computed tomography analyses were performed. HBMSC viability, biochemical activity and histological evidence of osteogenic cellular differentiation, post-impaction were observed in vitro and in vivo in PLA/HBMSC samples compared to impacted PLA alone. In vitro PLA/HBMSC samples demonstrated evidence of mechanical enhancement over PLA alone. In vivo studies showed a significant increase in new bone and blood vessel formation in the PLA/HBMSC constructs compared to PLA alone. With alternatives to allograft being sought, these studies have demonstrated PLA/HBMSC living composites, to be a potential prospect as a biological bone graft extender for future use in the field of IBG.
Keywords: Impaction bone grafting; Bone graft extenders; Poly(dl-lactic acid); Tissue engineering; Neovascularisation;

Tissue-to-cellular level deformation coupling in cell micro-integrated elastomeric scaffolds by John A. Stella; Jun Liao; Yi Hong; W. David Merryman; William R. Wagner; Michael S. Sacks (3228-3236).
In engineered tissues we are challenged to reproduce extracellular matrix and cellular deformation coupling that occurs within native tissues, which is a meso-micro scale phenomenon that profoundly affects tissue growth and remodeling. With our ability to electrospin polymer fiber scaffolds while simultaneously electrospraying viable cells, we are provided with a unique platform to investigate cellular deformations within a three dimensional elastomeric fibrous scaffold. Scaffold specimens micro-integrated with vascular smooth muscle cells were subjected to controlled biaxial stretch with 3D cellular deformations and local fiber microarchitecture simultaneously quantified. We demonstrated that the local fiber geometry followed an affine behavior, so that it could be predicted by macro-scaffold deformations. However, local cellular deformations depended non-linearly on changes in fiber microarchitecture and ceased at large strains where the scaffold fibers completely straightened. Thus, local scaffold microstructural changes induced by macro-level applied strain dominated cellular deformations, so that monotonic increases in scaffold strain do not necessitate similar levels of cellular deformation. This result has fundamental implications when attempting to elucidate the events of de-novo tissue development and remodeling in engineered tissues, which are thought to depend substantially on cellular deformations.
Keywords: Cellular deformations; Tissue engineering; Elastomeric scaffolds; Mechanobiology;

A gel-free 3D microfluidic cell culture system by Siew-Min Ong; Chi Zhang; Yi-Chin Toh; So Hyun Kim; Hsien Loong Foo; Choon Hong Tan; Danny van Noort; Sungsu Park; Hanry Yu (3237-3244).
3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.
Keywords: 3D in vitro cell culture; Cell aggregates; Microfluidics; Perfusion culture; Transient inter-cellular polymeric linker; Hydrogel;

Retention of in vitro and in vivo BMP-2 bioactivities in sustained delivery vehicles for bone tissue engineering by Diederik H.R. Kempen; Lichun Lu; Teresa E. Hefferan; Laura B. Creemers; Avudaiappan Maran; Kelly L. Classic; Wouter J.A. Dhert; Michael J. Yaszemski (3245-3252).
In this study, we investigated the in vitro and in vivo biological activities of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated into (1) a gelatin hydrogel, (2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a gelatin hydrogel, (3) microspheres embedded in a poly(propylene fumarate) (PPF) scaffold and (4) microspheres embedded in a PPF scaffold surrounded by a gelatin hydrogel. A fraction of the incorporated BMP-2 was radiolabeled with 125I to determine its in vitro and in vivo release profiles. The release and bioactivity of BMP-2 were tested weekly over a period of 12 weeks in preosteoblast W20-17 cell line culture and in a rat subcutaneous implantation model. Outcome parameters for in vitro and in vivo bioactivities of the released BMP-2 were alkaline phosphatase (AP) induction and bone formation, respectively. The four implant types showed different in vitro release profiles over the 12-week period, which changed significantly upon implantation. The AP induction by BMP-2 released from gelatin implants showed a loss in bioactivity after 6 weeks in culture, while the BMP-2 released from the other implants continued to show bioactivity over the full 12-week period. Micro-CT and histological analysis of the delivery vehicles after 6 weeks of implantation showed significantly more bone in the microsphere/PPF scaffold composites (Implant 3, p  < 0.02). After 12 weeks, the amount of newly formed bone in the microsphere/PPF scaffolds remained significantly higher than that in the gelatin and microsphere/gelatin hydrogels (p  < 0.001), however, there was no statistical difference compared to the microsphere/PPF/gelatin composite. Overall, the results from this study show that BMP-2 could be incorporated into various bone tissue engineering composites for sustained release over a prolonged period of time with retention of bioactivity.
Keywords: Bioactivity; Bone morphogenetic protein; Controlled drug release; Ectopic bone formation; Gelatin; Poly(lactic-co-glycolic acid) microspheres;

The use of RANKL-coated brushite cement to stimulate bone remodelling by Damien Le Nihouannen; S. Adam Hacking; Uwe Gbureck; Svetlana V. Komarova; Jake E. Barralet (3253-3259).
Calcium phosphate cements were first proposed as synthetic bone substitutes over two decades ago, however, they are characterised by slow chemical or cellular resorption and a slow osteointegration. In contrast, bone autograft has been shown to stimulate osteoclastogenesis and angiogenesis resulting in active bone remodelling and rapid graft incorporation. Therefore, we aimed to develop a biomaterial able to release a key stimulator of the bone remodelling process, cytokine RANKL. Cylinders of brushite cement, hydroxyapatite cement and sodium alginate were loaded with RANKL either by incorporation into the cement or by coating the material with soluble RANKL. To test the biological activity of these formulations, we assessed their effectiveness in inducing osteoclast formation from RAW 264.7 monocytic cell line. Only brushite and hydroxyapatite cements coated with RANKL allowed for retaining sufficient biological activity to induce osteoclast formation. Most efficient was coating 40 mg cylinder of brushite cement with 800 ng RANKL. We have found that RANKL-coated brushite cement exhibits osteoclastogenic activity for at least 1 month at 37 °C. Thus, we developed a formulation of brushite cement with RANKL – a synthetic bone graft that is similar to autografts in its ability to actively induce osteoclastogenesis.
Keywords: Calcium phosphate cement; Brushite; RANKL; Osteoclast; Controlled drug release;

To mimic the high affinity of heparin-binding proteins to heparin/heparan sulfate, the uronic acids in non-sulfated alginate were sulfated, and hydrogels of mixed alginate/alginate-sulfate were fabricated. Surface plasmon resonance analysis probed the interactions of 13 proteins with alginate-sulfate. Of these, the 10 heparin-binding proteins revealed strong binding to alginate-sulfate and heparin, but not to alginate. The equilibrium binding constants to alginate-sulfate were comparable or one order of magnitude higher than those obtained between the proteins and heparin. Only the fibroblast growth factors (FGFs) revealed higher affinity for heparin than to alginate-sulfate. Sulfation of hyaluronan, as well, resulted in strong binding of basic FGF to hyaluronan-sulfate, but not to hyaluronan. Mixed hydrogels of alginate/alginate-sulfate sustained the release of basic FGF, with the release rate being dependent on the percentage of bFGF bound to the hydrogels. In vivo, the delivery of bFGF bound to alginate/alginate-sulfate scaffolds induced the formation of twice the number of blood vessels compared to when bFGF was delivered adsorbed to the matrix and 51% of the vessels were matured, as judged by pericyte coverage of the vessels. Our results thus describe the engineering of alginate hydrogels for the spatially presentation and controlled delivery of heparin-binding proteins.
Keywords: Affinity binding; Biomimetics; Angiogenesis; bFGF; Hyaluronic acid; Sulfation of polysaccharides;

Biodegradable arginine-based poly(ester-amide)s as non-viral gene delivery reagents by Dai Yamanouchi; Jun Wu; Andrew N. Lazar; K. Craig Kent; Chih-Chang Chu; Bo Liu (3269-3277).
A novel family of synthetic biodegradable poly(ester-amide)s (Arg-PEAs) was evaluated for their biosafety and capability to transfect rat vascular smooth muscle cells, a major cell type participating in vascular diseases. Arg-PEAs showed high binding capacity toward plasmid DNA, and the binding activity was inversely correlated to the number of methylene groups in the diol segment of Arg-PEAs. All Arg-PEAs transfected smooth muscle cells with an efficiency that was comparable to the commercial transfection reagent Superfect®. However, unlike Superfect®, Arg-PEAs, over a wide range of dosages, had minimal adverse effects on cell morphology, viability or apoptosis. Using rhodamine-labeled plasmid DNA, we demonstrated that Arg-PEAs were able to deliver DNA into nearly 100% of cells under optimal polymer-to-DNA weight ratios, and that such a high level of delivery was achieved through an active endocytosis mechanism. A large portion of DNA delivered, however, was trapped in acidic endocytotic compartments, and subsequently was not expressed. These results suggest that with further modification to enhance their endosome escape, Arg-PEAs can be attractive candidates for non-viral gene carriers owning to their high cellular uptake nature and reliable cellular biocompatibility.
Keywords: Gene transfer; Nanoparticle; Smooth muscle cell; MTT assay; Image analysis; Fluorescence;

An electrochemical fabrication process for the assembly of anisotropically oriented collagen bundles by Xingguo Cheng; Umut A. Gurkan; Christopher J. Dehen; Michael P. Tate; Hugh W. Hillhouse; Garth J. Simpson; Ozan Akkus (3278-3288).
Controlled assembly of collagen molecules in vitro remains a major challenge for fabricating the next generation of engineered tissues. Here we present a novel electrochemical alignment technique to control the assembly of type-I collagen molecules into highly oriented and densely packed elongated bundles at the macroscale. The process involves application of electric currents to collagen solutions which in turn generate a pH gradient. Through an isoelectric focusing process, the molecules migrate and congregate within a plane. It was possible to fabricate collagen bundles with 50–400 μm diameter and several inches length via this process. The current study assessed the orientational order, and the presence of fibrillar assembly in such electrochemically oriented constructs by polarized optical microscopy, small angle X-ray scattering, second harmonic generation, and electron microscopy. The mechanical strength of the aligned crosslinked collagen bundles was 30-fold greater than its randomly oriented-crosslinked counterpart. Aligned crosslinked collagen bundles had about half the strength of the native tendon. Tendon-derived fibroblast cells were able to migrate and populate multiple macroscopic bundles at a rate of 0.5 mm/day. The anisotropic order within biocompatible collagenous constructs was conferred upon the nuclear morphology of cells as well. These results indicate that the electrochemically oriented collagen scaffolds carry baseline characteristics to be considered for tendon/ligament repair.
Keywords: Assembly; Collagen; Electrochemistry; Biomimetic materials; Connective tissues;