Biomaterials (v.27, #24)

Calendar (I).

Injectable and macroporous calcium phosphate cement scaffold by Hockin H.K. Xu; Michael D. Weir; Elena F. Burguera; Alexis M. Fraser (4279-4287).
Calcium phosphate cement (CPC) can be molded and self-hardens in vivo to form resorbable hydroxyapatite with excellent osteoconductivity. The objective of this study was to develop an injectable, macroporous and strong CPC, and to investigate the effects of porogen and absorbable fibers. Water-soluble mannitol was used as porogen and mixed with CPC at mass fractions from 0% to 50%. CPC with 0–40% mannitol was fully extruded under a syringe force of 10  n. The paste with 50% mannitol required a 100-N force which extruded only 66% of the paste. At fiber volume fraction of 0–5%, the paste was completely extruded. However, at 6% and 7.5% fibers, some fibers were left in the syringe after the paste was extruded. The injectable CPC scaffold had a flexural strength (mean±sd; n = 5 ) of (3.2±1.0) MPa, which approached the reported strengths for sintered porous hydroxyapatite implants and cancellous bone. In summary, the injectability of a ceramic scaffold, a macroporous CPC, was studies for the first time. Processing parameters were tailored to achieve high injectability, macroporosity, and strength. The injectable and strong CPC scaffold may be useful in surgical sites that are not freely accessible by open surgery or when using minimally invasive techniques.
Keywords: Calcium phosphate cement; Injectability; Macroporous scaffold; Strength; Bone repair;

Shape memory properties of poly(d,l-lactide)/hydroxyapatite composites by Xiaotong Zheng; Shaobing Zhou; Xiaohong Li; Jie Weng (4288-4295).
Poly(d,l-lactide) (PDLLA) and Hydroxyapatite (HA) are compounded, which possess biodegradation, biocompatibility and shape memory properties. In the paper, we prepared serial imposing shape memory composites with different shapes, composite ratios and sample thicknesses. Scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) were carried out to examine surface morphology, glass transition temperature (T g), dynamic mechanical properties, and shape memory effect of PDLLA/HA composites, respectively. Moreover, some interesting shape memory behaviors were investigated. The results show that the better disperse morphology of HA grains using the experiment methods, and PDLLA/HA composites at a definite range of compound ratio have much better shape memory effect than pure PDLLA polymer. It indicates that HA particles can improve shape memory effect and PDLLA/HA composites are potential for biomedical applications.
Keywords: Shape memory polymer; Poly(d,l-lactide); Hydroxyapatite; Composite; Biodegradation;

Stem cell attachment to layer-by-layer assembled TiO2 nanoparticle thin films by Dinesh S. Kommireddy; Shashikanth M. Sriram; Yuri M. Lvov; David K. Mills (4296-4303).
Surface topography is one of the most important factors influencing the attachment and spreading of cells. In the present study, layer-by-layer assembled titanium dioxide (TiO2) nanoparticle thin films were chosen for attachment, proliferation and spreading studies on mouse mesenchymal stem cells (MSC). Increasing surface roughness was observed with increasing number of layer-by-layer assembled TiO2 thin films. Four layer TiO2 thin film showed higher number of attached cells than a one layer thin film and control surfaces. MSCs experienced no cytotoxic effects after culture on the TiO2 coated substrates as observed from the cytotoxicity tests. Cell spreading, visualized with scanning electron microscopy, showed a faster rate of spreading on a rougher surface. Cells on a four-layer substrate, at 12 h showed complete spreading, where as most of the cells on a control surface and a one-layer surface, at 24 h, retained a rounded morphology. In conclusion, TiO2 nanoparticle thin films were successfully assembled in alternation with polyelectrolytes and in-vitro studies with MSC showed an increase in the attachment and faster spreading of cells on rougher surfaces.
Keywords: Layer-by-layer assembly; Stem cells; Nanoparticles; Surface roughness; Surface modification; Titanium dioxide;

Antimicrobial activities of silver used as a polymerization catalyst for a wound-healing matrix by Ranjith Babu; Jianying Zhang; Eric J. Beckman; Mohammed Virji; William A. Pasculle; Alan Wells (4304-4314).
Wound healing is a complex and orchestrated process that re-establishes the barrier and other functions of the skin. While wound healing proceeds apace in healthy individual, bacterial overgrowth and infection disrupts this process with significant morbidity and mortality. As such, any artificial matrix to promote wound healing must also control infecting microbes. We had earlier developed a two-part space-conforming gel backbone based on polyethyleneglycol (PEG) or lactose, which used ionic silver as the catalyst for gelation. As silver is widely used as an in vitro antimicrobial, use of silver as a catalyst for gelation provided the opportunity to assess its function as an anti-microbial agent in the gels. We found that these gels show bacteriostatic and bactericidal activity for a range of Gram-negative and Gram-positive organisms, including aerobic as well as anaerobic bacteria. This activity lasted for days, as silver leached out of the formed gels over a day in the manner of second-order decay. Importantly the gels did not limit either cell growth or viability, though cell migration was affected. Adding collagen I fragments to the gels corrected this effect on cell migration. We also found that the PEG gel did not interfere with hemostasis. These observations provide the basis for use of the gel backbones for incorporation of anesthetic agents and factors that promote wound repair. In conclusion, silver ions can serve dual functions of catalyzing gelation and providing anti-microbial properties to a biocompatible polymer.
Keywords: Wound healing; Infections; Bacteria; Hemostasis;

Hemocompatibility evaluation of poly(glycerol-sebacate) in vitro for vascular tissue engineering by Delara Motlagh; Jian Yang; Karen Y. Lui; Antonio R. Webb; Guillermo A. Ameer (4315-4324).
Poly(glycerol-sebacate) (PGS) is an elastomeric biodegradable polyester that could potentially be used to engineer blood vessels in vivo. However, its blood-material interactions are unknown. The objectives of this study were to: (a) fabricate PGS-based biphasic tubular scaffolds and (b) assess the blood compatibility of PGS in vitro in order to get some insight into its potential use in vivo. PGS was incorporated into biphasic scaffolds by dip-coating glass rods with PGS pre-polymer. The thrombogenicity (platelet adhesion and aggregation) and inflammatory potential (IL-1β and TNFα expression) of PGS were evaluated using fresh human blood and a human monocyte cell line (THP-1). The activation of the clotting system was assessed via measurement of tissue factor expression on THP-1 cells, plasma recalcification times, and whole blood clotting times. Glass, tissue culture plastic (TCP), poly(l-lactide-co-glycolide) (PLGA), and expanded polytetrafluorethylene (ePTFE) were used as reference materials. Biphasic scaffolds with PGS as the blood-contacting surface were successfully fabricated. Relative to glass (100%), platelet attachment on ePTFE, PLGA and PGS was 61%, 100%, and 28%, respectively. PGS elicited a significantly lower release of IL-1β and TNFα from THP-1 cells than ePTFE and PLGA. Similarly, relative to all reference materials, tissue factor expression by THP-1 cells was decreased when exposed to PGS. Plasma recalcification and whole blood clotting profiles of PGS were comparable to or better than those of the reference polymers tested.
Keywords: Hemocompatibility; Blood compatibility; Coagulation; Thrombosis; Vascular grafts; Tissue engineering;

Autoactivation of blood factor XII at hydrophilic and hydrophobic surfaces by Rui Zhuo; Christopher A. Siedlecki; Erwin A. Vogler (4325-4332).
Contact activation of blood factor XII (FXII, Hageman factor) in neat-buffer solution is shown not to be specific for anionic hydrophilic procoagulants as proposed by the accepted biochemistry of surface activation. Rather, FXII activation in the presence of plasma proteins leads to an apparent specificity for hydrophilic surfaces that is actually due to a relative diminution of the FXII→FXIIa reaction at hydrophobic surfaces. FXII activation in neat-buffer solution was effectively instantaneous upon contact with either hydrophilic (fully water-wettable clean glass) or hydrophobic (poorly water-wettable silanized glass) procoagulant particles, with greater FXIIa yield obtained by activation with hydrophobic procoagulants. In sharp contrast, both activation rate and yield was found to be significantly attenuated at hydrophobic surfaces in the presence of plasma proteins. Putative FXIIa produced by surface activation with both hydrophilic and hydrophobic procoagulants was shown to hydrolyze blood factor XI (FXI) to the activated form FXIa ( FXI ⟶ FXIIa FXIa ) that causes FXI-deficient plasma to rapidly coagulate.
Keywords: Blood coagulation; FXII; Hageman factor; Contact activation; Autoactivation;

A critical element of any stem cell differentiation protocol is the ability to compare its effects relative to an undifferentiated population of the same cells. In an attempt to standardize pre-differentiation conditions of adult derived mesenchymal stem cells prior to neural induction experiments, we asked what is the simplest chemical surface that supports the growth and maintenance of these cells in a pre-differentiation state. Adult bone marrow-derived rat mesenchymal stem cells (BMSCs) were expanded in vitro on Permanox Lab-Tek® tissue culture treated plastic (TCP), poly-d-lysine (PDL) coated glass, PDL-laminin-1 coated glass, and untreated glass. TCP provided the best surface for maintaining morphologies generally considered to be undifferentiated, while PDL coated glass and uncoated glass provided the least suitable surfaces. Expansion of BMSCs on PDL-laminin-1 coated glass resulted in expression of nestin, a marker associated with neuronal and other progenitor cells, and therefore may confound experimental results if used as a pre-differentiation surface.
Keywords: Stem cells; Cell culture; Surface treatment; Neural cells;

Tissue-engineered muscle is a viable option for tissue repair, though presently technologies are not developed enough to produce tissue in vitro identical to that in vivo. One important step in generating accurate engineered muscle is to mimic natural muscle architecture. Skeletal muscle is composed of fibrils whose organization defines functionality. In musculoskeletal myogenesis, aligning myoblasts in preparation for myotube formation is a crucial step. The ability to efficiently organize myoblasts to form aligned myotubes in vitro would greatly benefit efforts in muscle tissue engineering. This paper reports alignment of prefused and differentiated skeletal muscle cells in vitro by use of continuous micropatterned wavy silicone surfaces, with features sized 3, 6 and 12 μm in periodicity. Wave features with 6 μm periodicity produced the most healthy, aligned myoblasts. Alignment was found to be a function of plating density. Further growth on these substrates with aligned myoblasts promoted fusion, yielding healthy aligned myotubes. This method will be useful for applications in which differentiated myogenic cells need to be aligned unidirectionally as in the development of engineered muscle.
Keywords: Muscle; Micropatterning; Silicone elastomer; Cell adhesion; Laminin; Surface topography;

Strontium-containing hydroxyapatite bioactive bone cement in revision hip arthroplasty by G.X. Ni; K.Y. Chiu; W.W. Lu; Y. Wang; Y.G. Zhang; L.B. Hao; Z.Y. Li; W.M. Lam; S.B. Lu; K.D.K. Luk (4348-4355).
Clinical outcome of cemented implants to revision total hip replacement (THR) is not as satisfactory as primary THR, due to the loss of bone stock and normal trabecular pattern. This study evaluated a bioactive bone cement, strontium-containing hydroxyapatite (Sr-HA) bone cement, in a goat revision hip hemi-arthroplasty model, and compared outcomes with polymethylmethacrylate (PMMA) bone cement. Nine months after operation, significantly higher bonding strength was found in the Sr-HA group (3.36±1.84 MPa) than in the PMMA bone cement group (1.23±0.73 MPa). After detached from the femoral component, the surface of PMMA bone cement mantle was shown relatively smooth, whereas the surface of the Sr-HA bioactive bone cement mantle was uneven, by SEM observation. EDX analysis detected little calcium and no phosphorus on the surface of PMMA bone cement mantle, while high content of calcium (14.03%) and phosphorus (10.37%) was found on the surface of the Sr-HA bone cement mantle. Even higher content of calcium (17.37%) and phosphorus (10.84%) were detected in the concave area. Intimate contact between Sr-HA bioactive bone cement and bone was demonstrated by histological and SEM observation. New bone bonded to the surface of Sr-HA cement and grew along its surface. However, fibrous tissue was observed between PMMA bone cement and bone. The results showed good bioactivity of Sr-HA bioactive bone cement in this revision hip replacement model using goats. This in vivo study also suggested that Sr-HA bioactive bone cement was superior to PMMA bone cement in terms of bone-bonding strength. Use of bioactive bone cement may be a possible solution overcoming problems associated with the use of PMMA bone cement in revision hip replacement.
Keywords: Strontium-containing hydroxyapatite bone cement; Revision hip arthroplasty; Goat; Bone-bonding strength;

Parameters influencing the stealthiness of colloidal drug delivery systems by Arnaud Vonarbourg; Catherine Passirani; Patrick Saulnier; Jean-Pierre Benoit (4356-4373).
Over the last few decades, colloidal drug delivery systems (CDDS) such as nano-structures have been developed in order to improve the efficiency and the specificity of drug action. Their small size permits them to be injected intravenously in order to reach target tissues. However, it is known that they can be rapidly removed from blood circulation by the immune system. CDDS are removed via the complement system and via the cells of the mononuclear phagocyte system (MPS), after their recognition by opsonins and/or receptors present at the cell surface. This recognition is dependent on the physicochemical characteristics of the CDDS. In this study, we will focus on parameters influencing the interactions of opsonins and the macrophage plasma membrane with the surface of CDDS, whereby parameters of the polymer coating become necessary to provide good protection.
Keywords: Complement; Liposome; Macrophage; Nanoparticle; Polyethylene oxide; Protein adsorption;

A defined system to allow skeletal muscle differentiation and subsequent integration with silicon microstructures by Mainak Das; Cassie A. Gregory; Peter Molnar; Lisa M. Riedel; Kerry Wilson; James J. Hickman (4374-4380).
This work documents the development of an in vitro cell culture model consisting of a novel serum-free medium and a non-biological growth substrate, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA), to enable functional myotube integration with cantilevers fabricated using MEMS technology. This newly developed, defined in vitro model was used to study the differentiation of fetal rat skeletal muscle and it promoted the formation of myotubes from the dissociated rat fetal muscle cells. The myotubes were characterized by morphological analysis, immunocytochemistry and electrophysiology. Further, it was demonstrated that when the dissociated muscle cells were plated on fabricated microcantilevers, the muscle cells aligned along the major axis of the cantilever and formed robust myotubes. This novel system could not only find applications in skeletal muscle differentiation and biocompatibility studies but also in bioartificial muscle engineering, hybrid actuation system development, biorobotics and for a better understanding of myopathies and neuromuscular disorders.
Keywords: Hybrid devices; Defined system; Serum-free; Myotubes; MEMS;

Protein-imprinted polymer with immobilized assistant recognition polymer chains by Min-Jie Guo; Zhuo Zhao; Yun-Ge Fan; Chun-Hong Wang; Lin-Qi Shi; Jian-Jun Xia; Yi Long; Huai-Feng Mi (4381-4387).
Here we introduce a new method for preparing a protein-imprinted polymer with immobilized assistant recognition polymer chains as an additional element of monomer to create effective recognition sites. In this work the bovine serum albumin was used as template and the template protein was selectively assembled with immobilized assistant recognition polymer chains from their library, numerous limited length polymer chains with randomly distributed recognition sites and immobilizing sites. These assemblies of protein and immobilized assistant recognition polymer chains would be adsorbed by the macro porous adsorbent spheres and immobilized by cross-linking polymerization. After removing the template, binding sites that were complementary to the target protein in size, shape and position of recognition groups were exposed, and their confirmation was preserved by the cross-linked structure. The synthesized imprinted polymer was used to adsorb BSA from protein mixtures, and showed a high selectivity.
Keywords: Protein-imprinted polymer; Assistant recognition polymer chains (ARPCs) as functional prepolymer; Selectively assembling template and ARPCs;

Elastic modulus and stress–strain response of human enamel by nano-indentation by Li Hong He; Naoki Fujisawa; Michael V. Swain (4388-4398).
Nano-indentation with a sharp (Berkovich) and two spherical indenters with nominal tip radii of 5 and 20 μm was used to determine the elastic modulus and stress–strain response of human enamel. Indentation tests were made over a wide range of peak loads from 1 to 450 mN in two orthogonal directions, i.e., parallel and perpendicular to enamel prisms. The elastic modulus and hardness (mean contact pressure) versus depth of penetration were determined for the three indenters. From the spherical indentation data, stress–strain curves (H−tan  θ curve) of enamel were determined in the two orthogonal directions and were found to be different. The elastic modulus showed load dependence for both orientations of the enamel rod structure that depended on the indenter. However, these differences could be normalized upon considering the contact diameter. The indented sample was imaged with an SEM to investigate the near surface damage. In conclusion, prism-sheath structure played an important role in determining the mechanical properties as well as the localized fracture of enamel.
Keywords: Nano-indentation; Elastic modulus; Stress–strain curve; Enamel;