Biomaterials (v.29, #6)

The use of elastin-like polypeptide–polyelectrolyte complexes to control hepatocyte morphology and function in vitro by Amol V. Janorkar; Padmavathy Rajagopalan; Martin L. Yarmush; Zaki Megeed (625-632).
Both tissue engineering and biological science will benefit from improved methods to control the morphology, differentiated state, and function of primary cells. In this paper, we show that surface modification of tissue culture polystyrene (TCPS) with chemically derivatized elastin-like polypeptides (ELPs) enables control over the in vitro morphology and liver-specific function of primary rat hepatocytes. The ELP (VPGVG)40 was produced in Escherichia coli and conjugated with polyacrylic acid (PAA) and polyethyleneimine (PEI) using carbodiimide activation chemistry. These conjugates were characterized by transmission Fourier transform infrared (FTIR) spectroscopy, mass spectroscopy, and the ninhydrin assay. We demonstrated that the ELP–polyelectrolyte conjugates profoundly influenced the morphology, aggregation, and differentiated function of primary rat hepatocytes, where hepatocytes plated on the ELP–PAA and ELP–PEI surfaces formed spread and spheroidal morphologies with corresponding low and high liver-specific function, respectively. These materials may have utility as substrata for in vitro studies of hepatocyte biology and tissue engineering applications.
Keywords: Elastin; Hepatocyte; Polyelectrolyte; Liver; Tissue engineering;

The effect of hyaluronic acid on silk fibroin conformation by Marcos Garcia-Fuentes; Elisabeth Giger; Lorenz Meinel; Hans P. Merkle (633-642).
The molecular conformation of silk fibroin drastically changes the physical properties of this biomaterial. Herein, we investigated the capacity of hyaluronic acid to modify the conformational transition of silk fibroin into its crystalline β-sheet form. For this aim, matrices composed of these two polymers were prepared and studied. Instrumental analysis confirmed the presence of two intermixed phases: one of pure hyaluronic acid, and another consisting of a molecular dispersion of silk fibroin and hyaluronic acid. Studies performed with silk fibroin/hyaluronic acid matrices indicated that hyaluronic acid induces molecular transition of silk fibroin into a β-sheet structure when incubated in water, and that it synergistically enhances β-sheet formation together with methanol treatment. The enhancement of β-sheet content observed for silk fibroin/hyaluronic acid matrices correlated with improved mechanical properties: blended matrices had higher compressive moduli and higher breaking strengths than pure silk fibroin matrices. These new properties, together with the capacity of silk fibroin/hyaluronic acid to form partially insoluble matrices without any treatment with organic solvents, make this blend composition an interesting material for biomedical applications.
Keywords: Silk; Hyaluronic acid; Crystallization; Secondary structure; Mechanical properties;

Biphasic scaffold for annulus fibrosus tissue regeneration by Yuqing Wan; Gang Feng; Francis H. Shen; Cato T. Laurencin; Xudong Li (643-652).
Intervertebral disc (IVD) degeneration is the major cause of lower back pain, while the currently available treatments are symptomatic rather than curative. Tissue engineering is a powerful therapeutic strategy that can restore the normal biomechanical motion of the human spine. The ability of a biphasic elastic scaffold to structurally and elastically simulate the annulus fibrosus (AF) tissue of the IVD was explored. The outer phase of the scaffold was a ring-shaped demineralized bone matrix gelatin (BMG) extracted from cortical bone, which mimicks the type I collagen structure and ligamentous properties of outer AF. The inner phase of the scaffold was a bio-biomaterial poly(polycaprolactone triol malate) (PPCLM) orientated in concentric sheets and seeded with chondrocytes to recapitulate the inner layer of the AF, which is rich in type II collagen and proteoglycan. The mechanical properties and degradation of PPCLM could be adjusted by controlling the post-polymerization time of the pre-polymer. PPCLM also demonstrated good biocompatibility in a foreign body response in vivo assay. Incorporation of BMG into the scaffold enhanced the compressive strength compared with PPCLM alone. In addition, the tensile stress of the BMG/PPCLM scaffold was 50-fold greater than that of PPCLM alone, and close to that of normal rabbit AF. Finally, the biphasic scaffold supported the growth of rabbit chondrocytes, as confirmed by Safranin-O and type II collagen immunostaining. The excellent mechanical properties and biocompatibility of the BMG/PPCLM scaffold make it a promising candidate for AF repair.
Keywords: Polycaprolactone; Biphasic scaffold; Tissue engineering; Intervertebral disc; Annulus fibrosus;

The effect of the alignment of electrospun fibrous scaffolds on Schwann cell maturation by Sing Yian Chew; Ruifa Mi; Ahmet Hoke; Kam W. Leong (653-661).
Peripheral nerve regeneration can be enhanced by the stimulation of formation of bands of Büngner prior to implantation. Aligned electrospun poly(ε-caprolactone) (PCL) fibers were fabricated to test their potential to provide contact guidance to human Schwann cells. After 7 days of culture, cell cytoskeleton and nuclei were observed to align and elongate along the fiber axes, emulating the structure of bands of Büngner. Microarray analysis revealed a general down-regulation in expression of neurotrophin and neurotrophic receptors in aligned cells as compared to cells seeded on two-dimensional PCL film. Real-time-PCR analyses confirmed the up-regulation of early myelination marker, myelin-associated glycoprotein (MAG), and the down-regulation of NCAM-1, a marker of immature Schwann cells. Similar gene expression changes were also observed on cells cultured on randomly oriented PCL electrospun fibers. However, up-regulation of the myelin-specific gene, P0, was observed only on aligned electrospun fibers, suggesting the propensity of aligned fibers in promoting Schwann cell maturation.
Keywords: Electrospinning; Peripheral nerve regeneration; Contact guidance; Neural tissue engineering; Schwann cell;

Cell seeding on knitted scaffolds often require a gel system, which was found to be practically unsuitable for anterior cruciate ligament (ACL) reconstruction as the cell–gel composite often gets dislodged from the scaffold in the in vivo dynamic situations. In order to solve this problem, we fabricated this combined silk scaffold with weblike microporous silk sponges formed in the openings of a knitted silk scaffold and subsequently combined with adult human bone marrow-derived mesenchymal stem cells (hMSCs) for in vitro ligament tissue engineering. Human MSCs adhered and grew well on the combined silk scaffolds. Moreover, in comparison with the knitted silk scaffolds seeded with hMSCs in fibroin gel the cellular function was more actively exhibited on the combined silk scaffolds, as evident by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ligament-related gene markers (e.g., type I, III collagen and tenascin-C), immunohistochemical and western blot evaluations of ligament-related extracellular matrix (ECM) components. While the knitted structure holds the microporous silk sponges together and provides the structural strength of the combined silk scaffold, the microporous structure of the silk sponges mimic the ECM which consequently promotes cell proliferation, function, and differentiation. This feature overcomes the limitation of knitted scaffold for ligament tissue engineering application.
Keywords: Ligament tissue engineering; Combined silk scaffold; Mesenchymal stem cell;

Bone response and mechanical strength of rabbit femoral defects filled with injectable CaP cements containing TGF-β1 loaded gelatin microparticles by Dennis P. Link; Juliette van den Dolder; Jeroen J. van den Beucken; Joop G. Wolke; Antonios G. Mikos; John A. Jansen (675-682).
This study focused at the potential of transforming growth factor β1 (TGF-β1) loaded gelatin microparticles to enhance the bone response and mechanical strength of rabbit femoral defects filled with injectable calcium phosphate (CaP)/gelatin microparticle composites. Therefore, TGF-β1 loaded composites and non-loaded controls were injected in circular defects as created in the femoral condyles of rabbits and were left in place for 4, 8 and 12 weeks. The specimens were evaluated mechanically (push-out test), and morphologically (scanning electron microscopy (SEM), histology, and histomorphometry). The results showed a gradual increase in mechanical strength with increasing implantation periods. Histological and histomorphometrical evaluation showed similar results for both composite formulations regarding histological aspect, new bone formation and bone/implant contact. However, TGF-β1 loading of the composites demonstrated a significant effect on composite degradation after twelve weeks of implantation. The results of this study showed that CaP/gelatin composites show excellent osteogenic properties and a rapid increase in mechanical strength. The addition of TGF-β1 significantly enhances the bone remodeling process.
Keywords: Injectable CaP cement; Gelatin microparticles; TGF-β1; Mechanical properties; Bone ingrowth;

The use of biodegradable scaffold as an alternative to silicone implant arthroplasty for small joint reconstruction: An experimental study in minipigs by Eero Waris; Nureddin Ashammakhi; Mauri Lehtimäki; Riitta-Mari Tulamo; Minna Kellomäki; Pertti Törmälä; Yrjö T. Konttinen (683-691).
Biodegradable poly-l/d-lactide (P(l/d)LA) 96/4 joint scaffold arthroplasty is a recently clinically introduced concept in the reconstruction of small joints, however its histology and function in vivo is unknown. In this experimental study on 11 minipigs the fifth metacarpophalangeal joints were reconstructed using a P(l/d)LA 96/4 joint scaffold or Swanson silicone implant. They were evaluated until 3 years. The P(l/d)LA 96/4 joint scaffold formed a porous interposition spacer, which maintained the arthroplasty space and induced fibrous tissue in-growth in situ. No differences were found in the range of motion or arthroplasty space width between the study groups. The P(l/d)LA 96/4 joint scaffold was initially filled with vascular, loose connective tissue. Along with degradation of the scaffold, the in-grown connective tissue matured and condensed turning into dense fibrous connective tissue. After 3 years, the P(l/d)LA 96/4 joint scaffold had almost completely degraded and been replaced by dense fibrous connective tissue. These findings indicate that P(l/d)LA 96/4 joint scaffold arthroplasty leads to the formation of a functional fibrous joint. The avoidance of permanent foreign material makes the biodegradable joint scaffold an attractive alternative for small joint arthroplasty.
Keywords: Arthroplasty; Bioprosthesis; Fibrous tissue; Polylactic acid; Scaffold; Tissue engineering;

Ordered mesoporous silica (MCM-41) particles with different morphologies were synthesized through a simple hydrothermal process. Then these silica particles were functionalized with luminescent YVO4:Eu3+ layers via the Pechini sol–gel process. The obtained YVO4:Eu3+and MCM-41 composites, which maintained the mesoporous structure of MCM-41 and the red luminescence property of YVO4:Eu3+, were investigated as drug delivery systems using ibuprofen (IBU) as model drug. The physicochemical properties of the samples were characterized by X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), N2 adsorption, and photoluminescence (PL) spectra, respectively. It is found that the specific surface area and pore volume, which are directly correlated with the drug-loading amount and drug release rate, decrease in sequence after the deposition of YVO4:Eu3+ layer and the further incorporation of IBU. Additionally, the drug release test indicated that the IBU release rate could be controlled by regulating the morphology of the materials. It is worth noting that the IBU-incorporated samples still exhibit red luminescence under UV irradiation. Furthermore, the emission intensities of Eu3+ can be tailored as a function of the released amount of IBU, thus making the drug release be easily tracked and monitored.
Keywords: Sol–gel techniques; Porosity; In vitro test; Drug delivery; Simulated body fluids (SBF); Silica;

The use of deoxycholic acid to enhance the oral bioavailability of biodegradable nanoparticles by Robert M. Samstein; Karlo Perica; Fanor Balderrama; Michael Look; Tarek M. Fahmy (703-708).
Oral delivery of nanoparticles encapsulating drugs and proteins remains a challenging route for administration due to the many barriers in the gastrointestinal tract that limit bioavailability. We hypothesized that bile salts could be used to improve the bioavailability of poly(lactide-co-glycolide) (PLGA) nanoparticles by protecting them during their transport through the gastrointestinal tract and enhancing their absorption by the intestinal epithelia. A deoxycholic acid emulsion is shown to protect PLGA nanoparticles from degradation in acidic conditions and enhance their permeability across a Caco-2 cell monolayer, an in vitro model of human epithelium. Oral administration of loaded PLGA nanoparticles to mice, using a deoxycholic acid emulsion, produced sustained levels of the encapsulant in the blood over 24–48 h with a relative bioavailability of 1.81. Encapsulant concentration was highest in the liver, demonstrating a novel means for targeted delivery to the liver by the oral route.
Keywords: Nanoparticles; Oral delivery; Deoxycholic acid; PLGA;

Development of lipid particles targeted via sugar–lipid conjugates as novel nuclear gene delivery system by Tomoya Masuda; Hidetaka Akita; Takashi Nishio; Kenichi Niikura; Kentaro Kogure; Kuniharu Ijiro; Hideyoshi Harashima (709-723).
Efficient nuclear gene delivery is essential for successful gene therapy. This study developed a novel system that mimics the mechanism of nuclear entry of adenovirus (Ad) by means of a Multifunctional Envelope-type Nano Device (MEND). In this system, plasmid DNA (pDNA) was condensed with polycation, followed by encapsulation in a lipid membrane. To target MEND to the nuclear pore complex (NPC), sugar served as a NPC-mediated nuclear targeting device was modified on the surface of the lipid envelope. This was accomplished via synthesis of a sugar–cholesterol conjugate. After binding of the MEND to the NPC, the pDNA core was transferred into the nucleus in conjunction with a breakdown of the lipid envelope. Sugar-modified MEND showed higher transfection efficiency compared with unmodified MEND, in non-dividing and dividing cells. Confocal microscopy confirmed that nuclear transfer of pDNA was improved by sugar modification of MEND. Furthermore, destabilization of the lipid envelope significantly enhanced transfection activity: therefore, nuclear-delivery efficiency was closely related to lipid envelope stability. Moreover, quantitative evaluation of cellular uptake and nuclear transfer processes by real-time PCR confirmed that the surface sugars affected nuclear transfer, but not cellular uptake. In summary, a novel system for the nuclear delivery of pDNA was successfully developed by using a sugar-modified MEND and by optimizing the lipid envelope stability.
Keywords: Non-viral vector; Gene delivery; Nuclear delivery; Sugar; Envelope-type nano device;

Determination of nanoparticle vehicle unpackaging by MR imaging of a T 2 magnetic relaxation switch by In-Kyu Park; Chee-Ping Ng; Jinnan Wang; Baocheng Chu; Chun Yuan; Shanrong Zhang; Suzie H. Pun (724-732).
Non-invasive imaging of gene and drug delivery is an important tool in understanding the biodistribution and pharmacokinetics of vectors after in vivo administration. In this work, we demonstrate the utility of a multifunctional delivery vector comprised of polyethylenimine conjugated to ultrasmall, superparamagnetic iron oxide (USPIO). The conjugate (USPIO-PEI) is capable of complexing plasmid DNA into nanoparticles (SPIO-polyplex) with diameters ∼100 nm and protecting the DNA from nuclease degradation. SPIO-polyplexes transfect cells with high efficiency and low toxicity. In addition, the T 2 relaxation time of water enhanced by USPIO is shown to be a function of the packaging state of the vector. Thus, this material integrates capabilities of gene delivery with magnetic resonance (MR) contrast and also provides an MR-based read-out for vector unpackaging.
Keywords: Gene transfer; Gene therapy; Magnetic resonance imaging (MRI); Nanoparticle;

Biodegradable polycation and plasmid DNA multilayer film for prolonged gene delivery to mouse osteoblasts by Zhen-Zhen Lu; Juan Wu; Tian-Meng Sun; Jing Ji; Li-Feng Yan; Jun Wang (733-741).
Sustained release of functional plasmid DNA from the surfaces of materials which support cell adhesion for tissue formation could have a significant impact on gene therapy and tissue engineering. We report here layer-by-layer assembled multilayer film from a degradable cationic poly(2-aminoethyl propylene phosphate) and plasmid DNA encoding for enhanced green fluorescent protein (EGFP) for mouse osteoblast cell adhesion and prolonged gene delivery. Multilayer film growth was monitored by UV spectrophotometry and intensity of absorbance at 260 nm related to incorporated DNA increased in an exponential manner with increase the number of deposited polymer and plasmid layers. It degraded upon incubation in phosphate-buffered saline (PBS) at 37 °C and sustained the release of bioactive plasmid DNA up to 2 months. The multilayer film facilitated initial mouse osteoblast cell adhesion onto the surface and enhanced cellular alkaline phosphatase activity and calcium accumulation. It sustained delivering transcriptional active DNA to mouse osteoblast cells cultured on the film, and directly prolonged gene expression in the presence of serum without any exogenous transfection agent. This biodegradable multilayer assembly is promising for the local and sustained delivery of plasmid DNA and such a layer-by-layer system suggests an alternative method for plasmid DNA incorporation which may be useful for surface modification of implanted materials or scaffold for gene therapy and tissue regeneration.
Keywords: Gene delivery; Layer-by-layer assembly; Biodegradable polyphosphoester; Osteoblast cells; Tissue engineering;

The use of biodegradable polymeric nanoparticles in combination with a low-pressure gene gun for transdermal DNA delivery by Po-Wei Lee; Shu-Fen Peng; Chun-Jen Su; Fwu-Long Mi; Hsin-Lung Chen; Ming-Cheng Wei; Hao-Jan Lin; Hsing-Wen Sung (742-751).
Gold particles have been used as a carrier for transdermal gene delivery, which may cause adverse side effects when accumulated. In this study, biodegradable nanoparticles, composed of chitosan (CS) and poly-γ-glutamic acid (γ-PGA), were prepared by an ionic-gelation method for transdermal DNA delivery (CS/γ-PGA/DNA) using a low-pressure gene gun. The conventional CS/DNA without the incorporation of γ-PGA was used as a control. Small-angle X-ray scattering (SAXS) was used to examine the internal structures of test nanoparticles, while identification of their constituents was conducted by Fourier transformed infrared (FT-IR) spectroscopy. The CS/γ-PGA/DNA were spherical in shape with a relatively homogeneous size distribution. In contrast, CS/DNA had a heterogeneous size distribution with a donut, rod or pretzel shape. Both test nanoparticles were able to effectively retain the encapsulated DNA and protect it from nuclease degradation. As compared with CS/DNA, CS/γ-PGA/DNA improved their penetration depth into the mouse skin and enhanced gene expression. These observations may be attributed to the fact that CS/γ-PGA/DNA were more compact in their internal structures and had a greater density than their CS/DNA counterparts, thus having a larger momentum to penetrate into the skin barrier. The results revealed that CS/γ-PGA/DNA may substitute gold particles as a DNA carrier for transdermal gene delivery.
Keywords: Transdermal gene delivery; Gene carrier; Degradable nanoparticle; Gene gun;

A microwell array system for stem cell culture by Hannes-Christian Moeller; Matthew K. Mian; Shamit Shrivastava; Bong Geun Chung; Ali Khademhosseini (752-763).
Directed embryonic stem (ES) cell differentiation is a potentially powerful approach for generating a renewable source of cells for regenerative medicine. Typical in vitro ES cell differentiation protocols involve the formation of ES cell aggregate intermediates called embryoid bodies (EBs). Recently, we demonstrated the use of poly(ethylene glycol) (PEG) microwells as templates for directing the formation of these aggregates, offering control over parameters such as size, shape, and homogeneity. Despite these promising results, the previously developed technology was limited as it was difficult to reproducibly obtain cultures of homogeneous EBs with high efficiency and retrievability. In this study, we improve the platform by optimizing a number of features: material composition of the microwells, cell seeding procedures, and aggregate retrieval methods. Adopting these modifications, we demonstrate an improved degree of homogeneity of the resulting aggregate populations and establish a robust protocol for eliciting high EB formation efficiencies. The optimized microwell array system is a potentially versatile tool for ES cell differentiation studies and high-throughput stem cell experimentation.
Keywords: Microwells; Poly(ethylene glycol) (PEG); Embryoid bodies (EBs);

Pseudo-hyperelastic model of tendon hysteresis from adaptive recruitment of collagen type I fibrils by Pasquale Ciarletta; Paolo Dario; Silvestro Micera (764-770).
Understanding the functional relationship between the viscoelasticity and the morphology of soft collagenous tissues is fundamental for many applications in bioengineering science. This work presents a pseudo-hyperelastic constitutive theory aiming at describing the time-dependant hysteretic response of tendons subjected to uniaxial tensile loads. A macroscopic tendon is modeled as a composite homogeneous tissue with the anisotropic reinforcement of collagen type I fibrils. The tissue microstructure is considered as an adaptive network of fibrillar units connected in temporary junctions. The processes of breakage and reformation of active fibrils are thermally activated, and are occurring at random times. An internal softening variable and a dissipation energy function account for the adaptive arrangement of the fibrillar network in the pseudo-hyperelastic model. Cyclic uniaxial tensile tests have been performed in vitro on porcine flexor digital tendons. The theoretical predictions fit accurately the experimental stress–strain data both for the loading and the unloading processes. The hysteresis behavior reflects the improvement in the efficiency and performance of the motion of the muscle-tendon unit at high strain rates. The results of the model demonstrate the microstructural importance of proteoglycans in determining the functional viscoelastic adaptability of the macroscopic tendon.
Keywords: Pseudo-hyperelasticity; Tendon replacements; Proteoglycans; Collagen cross-linking; Hysteresis; Tendon biomechanics;