Biomaterials (v.27, #15)

Calendar (I).

How useful is SBF in predicting in vivo bone bioactivity? by Tadashi Kokubo; Hiroaki Takadama (2907-2915).
The bone-bonding ability of a material is often evaluated by examining the ability of apatite to form on its surface in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma. However, the validity of this method for evaluating bone-bonding ability has not been assessed systematically. Here, the history of SBF, correlation of the ability of apatite to form on various materials in SBF with their in vivo bone bioactivities, and some examples of the development of novel bioactive materials based on apatite formation in SBF are reviewed. It was concluded that examination of apatite formation on a material in SBF is useful for predicting the in vivo bone bioactivity of a material, and the number of animals used in and the duration of animal experiments can be reduced remarkably by using this method.
Keywords: Bioactivity; Bone; Hydroxyapatite; In vitro test; Osteoconduction; SBF;

Synthesis and characterization of single-phase silicon-substituted α-tricalcium phosphate by Joel W. Reid; Loughlin Tuck; Michael Sayer; Karen Fargo; Jason A. Hendry (2916-2925).
Silicon-substituted calcium phosphate (CaP) powders with a Ca/(P+Si) ratio of 1.50 have been prepared by a wet chemical method, with silicon contents up to 2.16 weight percent (wt%). Sintering for 2 h at 1250 °C yields single-phase silicon-substituted alpha tricalcium phosphate (Si-α-TCP) for compositions between 0.59 and 1.14 wt% silicon. The sintered powders have been characterized with X-ray fluorescence (XRF) spectrometry, X-ray diffraction (XRD), attenuated total reflection infrared spectroscopy (ATR-IR) and transmission electron microscopy (TEM). Compositions with less than 0.59 wt% silicon result in mixtures of Si-α-TCP, β-TCP, and calcium hydroxyapatite (HA), while compositions with more than 1.14 wt% silicon result in mixtures of Si-α-TCP and HA. The lattice parameters of single-phase Si-α-TCP prepared with 0.87 wt% silicon are a = 1 2.8 7 4 ( 1 ) Å , b = 2 7.3 7 2 ( 2 ) Å , c = 1 5.2 2 5 ( 1 ) Å , and β = 1 2 6.3 8 ( 1 ) ° .
Keywords: Tricalcium phosphate; Hydroxyapatite; α-TCP; Silicon; Bioceramics; Rietveld refinement;

The effect of the amelogenin fraction of enamel matrix proteins on fibroblast-mediated collagen matrix reorganization by Rachel E. Grayson; Y. Yamakoshi; Edward J. Wood; Magnus S. Ågren (2926-2933).
Enamel matrix proteins (EMP), extracted from developing porcine teeth, promote not only periodontal regeneration but also cutaneous wound healing presumably via the amelogenin fraction. Because it is unclear whether the effect of EMP can be ascribed to amelogenins, we compared EMP with recombinant amelogenin in the relaxed dermal equivalent (DE) in vitro model for early wound contraction. EMP and recombinant porcine amelogenin (rP172) at 1 mg/ml were incorporated into DEs composed of human dermal fibroblasts and a type I collagen matrix. The area reduction, as a measure of contraction, as well as fibroblast numbers and TGF-β1 levels, were quantified over 7 days in culture in the presence of 10% foetal bovine serum. Both EMP and recombinant amelogenin increased contraction ( p < 0.0 0 5 ) and fibroblast numbers ( p < 0.0 0 5 ) compared with controls (acetic acid vehicle and 1 mg/ml porcine serum albumin) and the positive control TGF-β1 added at 10 ng/ml. Increased contraction with EMP and recombinant amelogenin was most pronounced after the first day of incubation and was associated with elevated ( p < 0.0 0 5 ) TGF-β1 levels in conditioned medium. In conclusion, the amelogenin component of EMP augmented fibroblast-driven collagen matrix remodelling, at least partially, by increasing the endogenous production of TGF-β1. These effects of EMP/amelogenin may be beneficial for cutaneous wound healing.
Keywords: Adhesion molecule; Amelogenin; Apoptosis; Extracellular matrix (ECM); MTT assay; TGF-β1;

The modulation of gene expression in osteoblasts by thrombin coated on biphasic calcium phosphate ceramic by Gilles Bluteau; Paul Pilet; Xavier Bourges; Melitta Bilban; Reiner Spaethe; Guy Daculsi; Jérôme Guicheux (2934-2943).
For many years, fibrin sealants were associated with bone substitutes to promote bone healing. However, the osteoblastic response to fibrin sealant components remains poorly documented. In this study, MC3T3-E1 osteoblastic cells were cultured on biphasic calcium phosphate ceramic (MBCP®) coated with Tissucol® components (thrombin and fibrinogen). Analysis of osteoblastic differentiation markers by RT-PCR revealed that MBCP® coated with Tissucol® stimulated mRNA levels for osteocalcin and alkaline phosphatase (ALP). Of all the components of Tissucol®, thrombin has been reported to affect osteoblastic behavior. Our results demonstrated that low thrombin concentrations (0.5–5 U/ml) stimulated mRNA levels for ALP, whereas high thrombin concentrations (50–100 U/ml) decreased mRNA levels for ALP and PTH/PTHrP receptor and also increased mRNA level for the osteoclastogenesis inhibitor OPG. As thrombin stimulated angiogenesis, we then wondered whether thrombin could influence the expression of angiogenic factors. Low thrombin concentrations were shown to up-regulate mRNA levels for VEGF-B and VEGF-R1, suggesting an autocrine/paracrine role for VEGF-B. Higher thrombin concentrations also up-regulated mRNA for VEGF-A and neuropilin-1. In conclusion, the association of MBCP® with thrombin and fibrinogen appears to be a convenient scaffold for bone cell differentiation. Thrombin could also acts at the cellular level by increasing the angiogenic potential of osteoblasts as well as their responsiveness to thrombin and VEGF.
Keywords: Fibrin glue; Osteoblast; Thrombin; Differentiation; VEGF; Biphasic calcium phosphate;

In this paper, comprehensive characteristics including cell attachment, cell proliferation status, cell cycle progression and phenotypic changes of smooth muscle cells from rabbit aorta (RaSMCs) were studied on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 0–20% HHx (mol%) in comparison with tissue culture plates (TCPs). Results demonstrated that RaSMCs adhered better on PHBHHx containing 12% HHx (12%HHx) although they proliferated better on 20%HHx-containing PHBHHx films (20%HHx). This was explained by the difference in cell cycle progression observed using flow cytometry, as it was found that only 20%HHx-containing polymer could maintain the normal cell cycle evolution as TCPs did after 3 d incubation. The highest expression level and typical spindle-like distribution of α-actin on 20%HHx-containing polymer were characterized as the contractile-like phenotype, suggesting that RaSMCs tended to differentiate rather than proliferate compared to the cells grown on 12%HHx polymer. Results obtained above suggested that 20%HHx was suitable for RaSMCs proliferation, leading to its change to contractile phenotype. This study extends the potential applications of PHBHHx in SMCs-related graft scaffold fabrication for tissue engineering.
Keywords: PHB; PHBHHx; Smooth muscle cells; α-actin; Crystallization; Cell cycle;

Collagen scaffolds derived from a marine source and their biocompatibility by Eun Song; So Yeon Kim; Taehoon Chun; Hyun-Jung Byun; Young Moo Lee (2951-2961).
The primary sources of industrial collagens are calf skin and bone. However, these carry a high risk of bovine spongiform encephalopathy or transmissible spongiform encephalopathy. In this study, a novel form of acid-soluble collagen was extracted from jellyfish in an effort to obtain an alternative and safer collagen. Porous scaffolds composed of jellyfish collagen were prepared by freeze-drying and cross-linking with 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide to be used in tissue engineering applications. Enzymatic degradation kinetics of jellyfish collagen scaffolds were controlled by EDC/NHS-cross-linking density. Results from an MTT assay indicated that jellyfish collagen exhibited higher cell viability than other naturally derived biomaterials, including bovine collagen, gelatin, hyaluronic acid, and glucan. Jellyfish collagen scaffolds also had a highly porous and interconnected pore structure, which is useful for an high-density cell seeding, an efficient nutrient and an oxygen supply to the cells cultured in the three-dimensional matrices. To determine whether jellyfish collagen evokes any specific inflammatory response compared to that induced by bovine collagen or gelatin, we measured the levels of pro-inflammatory cytokines and antibody secretions and monitored the population changes of immune cells after in vivo implantation. Jellyfish collagen was found to induce an immune response at least comparable to those caused by bovine collagen and gelatin.
Keywords: Collagen; Jellyfish; Scaffold; Tissue engineering; Immune response;

Peptide-surface modification of poly(caprolactone) with laminin-derived sequences for adipose-derived stem cell applications by Lizzie Y. Santiago; Richard W. Nowak; J. Peter Rubin; Kacey G. Marra (2962-2969).
Human adipose tissue has been recognized as a source of adult stem cells for tissue engineering applications such as bone, cartilage, and soft tissue repair. For the success of these tissue-engineering approaches, a cell delivery vehicle such as a hydrogel or scaffold is required to position the stem cells at the site of need. Surface modification techniques have been instrumental in the development of scaffolds that promote cell-surface interactions. In this study, poly(caprolactone) (PCL), surfaces were modified in order to promote the attachment and proliferation of adipose-derived stem cells (ASCs). RGD, YIGSR, and IKVAV peptide sequences derived from the extracellular matrix protein laminin were each covalently attached to an aminated polymer surface using carbodiimide chemistry. The surface was characterized using scanning electron microscopy (SEM), goniometry and X-ray photoelectron spectroscopy (XPS). The attachment and proliferation of ASCs was assessed on the different peptide-treated surfaces. XPS analysis confirmed the presence of the peptide sequences on the surface of the polymer as indicated by the increase in the nitrogen/carbon ratio on the surface of the polymer. Among all peptide sequences tested, IKVAV-treated surfaces had a significantly greater number of ASCs bound 2 and 3 days after cell seeding. SEM confirmed differences in the morphology of the cells attached to the three peptide-treated surfaces. These results indicate that IKVAV is a suitable peptide sequence for use in surface modification techniques aimed at improving the attachment of ASCs to a tissue-engineered scaffold.
Keywords: Adipose tissue engineering; Poly(caprolactone); Laminin; Stem cell; Surface modification;

Expansion of chondroprogenitor cells on macroporous microcarriers as an alternative to conventional monolayer systems by Juan M. Melero-Martin; Mary-Ann Dowling; Mark Smith; Mohamed Al-Rubeai (2970-2979).
Routine tissue culture methodologies can hardly cope with the scale of cell production required for the manufacture of engineered cartilage tissue products. In vitro cell expansion has become an essential step in the process of tissue engineering of articular cartilage and the optimization of expansion protocols is a fundamental issue that needs to be addressed. The expansion suitability of chondroprogenitor cells isolated from the superficial zone of articular cartilages was evaluated in both conventional monolayer and macroporous microcarrier in spinner flask cultures. Although monolayer systems promoted rapid in vitro expansion of undifferentiated cells, they present limited scalability. Alternatively, the use of CultiSpher-G microcarriers resulted in cell densities of 5.5×105  cell/ml, representing a 17-fold expansion in batch cultures. In addition, chondroprogenitor cells were capable of undergoing bead-to-bead migration, which allowed subcultivation to be performed without a harvesting step, thus improving the scalability of the expansion process. By employing macroporous microcarrier cultures it will be possible to obtain large number of chondroprogenitor cells for tissue engineering applications. Not only its satisfactory expansion potential, but more importantly the cost and operational advantages over traditional monolayer culture make this system a feasible alternative method for the extensive expansion of chondroprogenitor cells.
Keywords: Expansion; Monolayer; Chondroprogenitor; Cartilage; Macroporous microcarriers; Tissue engineering;

Osteoprogenitor response to semi-ordered and random nanotopographies by Matthew John Dalby; David McCloy; Mary Robertson; Hossein Agheli; Duncan Sutherland; Stanley Affrossman; Richard O.C. Oreffo (2980-2987).
In bone tissue engineering, it is desirable to use materials to control the differentiation of mesenchymal stem cell populations in order to gain direct bone apposition to implant materials. It has been known for a number of years that microtopography can alter cell adhesion, proliferation and gene expression. More recently, the literature reveals that nanotopography is also of importance. Here, the reaction of primary human osteoprogenitor cell populations to nanotopographies down to 10 nm in size is considered. The topographies were originally produced by colloidal lithography and polymer demixing on silicon and then embossed (through an intermediate nickel shim) into polymethylmethacrylate. The biological testing considered cell morphology (image analysis of cell spreading and scanning electron microscopy), cell cytoskleton and adhesion formation (fluorescent staining of actin, tubulin, vimentin and vinculin) and then subsequent cell growth and differentiation (fluorescent staining of osteocalcin and osteopontin). The results demostrated that the nanotopographies stimulated the osteoprogenitor cell differentiation towards an osteoblastic phenotype.
Keywords: Nanomaterials; Nanobiotechnology; Nanotopography; Osteoprogenitor cells; Stem cells; Differentiation;

In vivo enhancement of sensory perception recovery in a tissue-engineered skin enriched with laminin by René Caissie; Marie Gingras; Marie-France Champigny; François Berthod (2988-2993).
The use of autologous reconstructed skin appears to be a promising treatment for the permanent coverage of deep and extensive burns. However, the capability of reconstructed skin transplanted on wounds to promote recovery of sensory perception is a major concern. Our aim was to assess the effect of laminin on cutaneous nerve regeneration. We prepared collagen-chitosan sponges enriched with 0, 1, 10 or 50 μg of laminin/sponge to produce tissue-engineered reconstructed skins by culture of human fibroblasts and keratinocytes, then grafted on the back of athymic mice for 120 days. Immunohistochemical studies demonstrated that there were 7 times more neurofilament 150 kD-positive nerve fibers migrating in the graft in the samples enriched with 10 μg laminin/sponge, compared to reconstructed skin without laminin, 120 days after graft. A significant improvement in the current perception threshold of the Aβ and Aδ nerve fibers was measured using a Neurometer® in all grafts enriched with laminin. In addition, the type C nerve fibers reached an identical current perception threshold than mouse skin, in all reconstructed skins enriched or not with laminin. We conclude that the use of a tissue-engineered autologous skin graft enriched with laminin has the potential to efficiently optimize cutaneous sensory nerve regeneration in vivo.
Keywords: Burn; Collagen scaffold; Nerve regeneration; Wound healing;

Tissue engineering of vascular elastin matrices disrupted by mechanical injury, disease, or congenitally absent, is among other factors, limited by the lack of suitable cell scaffolds to up-regulate and guide innately poor elastin synthesis by adult vascular smooth muscle cells (SMCs). Evidence suggests that scaffolds based on hyaluronan (HA), a glycosaminoglycan, may be useful to elicit elastogenic cell responses, although these effects appear to be dictated by HA fragment size and/or dose. This study investigates the efficacy of a simple, frequently adopted exogenous HA supplementation model to test this hypothesis. Rat aortic SMCs were cultured with HA (2×106  Da (HMW)⩾MW⩽2.2×104  Da) supplemented at doses between 0.2 and 200 μg/ml. Cell layers were biochemically assayed for DNA, elastin and collagen content. Fragmented, but not high molecular weight (HMW) HA, stimulated cell proliferation in inverse correlation fragment size while the opposite effect was observed for synthesis of soluble and matrix elastin; almost no dose effects were observed within any group. SDS-Page/Western Blot and a desmosine assay semi-quantitatively confirmed the observed biochemical trends for tropoelastin and matrix elastin, respectively. Quantitative differences in elastin deposition were mirrored in TEM micrographs. Elastin was mostly deposited in the form of amorphous clumps but fibers were increasingly present in cell layers cultured with HMW HA. HA and its fragments did not disrupt normal fibrillin-mediated mechanisms of elastin matrix deposition. While the current outcomes confirm that the effects of HA on elastin synthesis are fragment size-specific, this study shows that an exogenous supplementation model does not necessarily simulate cellular matrix synthesis responses to HA-based biomaterial scaffolds.
Keywords: Elastin; Regeneration; Vascular; Extracellular matrix; Glycosaminoglycan; Hyaluronan;

Maskless liquid-crystal-display projection photolithography for improved design flexibility of cellular micropatterns by Kazuyoshi Itoga; Jun Kobayashi; Masayuki Yamato; Akihiko Kikuchi; Teruo Okano (3005-3009).
We previously developed an all-in-one photopolymerization device by modifying a commercially available liquid crystal display projector (LCDP) for the preparation of micropatterned surfaces and microfluidic channels without the need for expensive photomasks. In the present study, we demonstrate a simple and reliable method for rapid prototyping of cell micropatterning with high resolution using the modified LCDP device. Fabrication of complicated and flexible patterns was achieved using this device with positive-type photoresist in a two-step process. First, micropatterns on the silanized coverslips were fabricated from positive photoresist. Second, acrylamide monomer solution containing polymerization initiator was dropped onto the micropatterned positive photoresist and copolymerized on the silanized coverslips in situ by thermally initiated radical polymerization. After the reaction, the remnant micropatterned photoresist is easily dissolved, resulting in a polyacrylamide-silane micropattern on the coverslip. The resultant polyacrylamide layer is highly hydrophilic and repels both protein adsorption and cell adhesion. Cells seeded on the micropatterned surfaces therefore attach and spread only on unpolymerized silanized glass surfaces, conforming to the pattern design. This technique is therefore useful for inexpensive, rapid prototyping of surface micropatterns using polymer materials.
Keywords: Liquid crystal display projector; Maskless; Cell micropatterning; Photolithography; Photoresist; Polyacrylamide;

Over-expression of p53/BAK in aseptic loosening after total hip replacement by Stefan Landgraeber; Martin Toetsch; Christian Wedemeyer; Guido Saxler; Michael Tsokos; Fabian von Knoch; Markus Neuhäuser; Franz Löer; Marius von Knoch (3010-3020).
Particle-induced osteolysis is a major cause of aseptic loosening after total joint replacement. The possible induction of apoptosis has not been addressed in great detail. Thus far, it has been shown that ceramic and polyethylene particles can induce apoptosis of macrophages in vitro. The purpose of this study was to test the hypothesis that wears debris generated from total hip arthroplasty could induce cellular damage and apoptosis in vivo. We therefore determined by immunohistochemical methods if increased expression of p53, an important transcription factor, and BAK and Bcl-2, two important regulators of apoptosis, can be found in interface membranes and capsules of hips with aseptically loose implants.Strongly positive immunohistochemical staining for p53 and BAK was found in peri-implant tissues from patients with aseptic hip implant loosening. Differentiation of various cell types showed that macrophages stained positive for p53 in all capsule and interface specimens. p53 was frequently detected in giant cells. Positive staining of BAK in macrophages and giant cells was seen in all specimens. Some positive reactions were observed in fibroblasts, only two of 19 cases stained for p53 and three cases for BAK within synovial cells. Positive macrophages and giant cells were localized around polyethylene particles. While T-lymphocytes showed a regular BAK-staining, the other leukocytes were negative. Statistical analyses showed significant positive correlations ( p < 0.0 0 1 ) between the presence of polyethylene and metal debris and the expression of BAK and p53. Polyethylene particles were surrounded by more positive macrophages and giant cells than were metal particles, indicating that polyethylene debris may be a stronger inductor of cell cycle arrest and apoptosis than metal debris.In this study apoptosis of macrophages, giant cells and T-lymphocytes in capsules and interface membranes of patients with aseptic hip implant loosening has been demonstrated in vivo. It is possible that the apoptotic cascade could evolve as a novel therapeutic target to prevent particle-induced osteolysis.
Keywords: Apoptosis; Hip replacement prosthesis; Immunochemistry; Macrophage; Polyethylene;

Towards developing surface eroding poly(α-hydroxy acids) by Xiao-Jun Xu; Jay C. Sy; V. Prasad Shastri (3021-3030).
We have prepared a library of biodegradable polyesters derived from poly(α-hydroxy acids) (PHAs) that appear to primarily exhibit surface erosion behavior. This was achieved by increasing the hydrophobicity of the polymers in two distinct steps, namely: macromer formation and a coupling step. In the first step, macromerdiols (MDs) with varying lipophilicities were prepared by polymerization of l-lactide or mixture of l-lactide and glycolide (3/1 by mole) to various lengths ( n = 1 0 , 20, 30, and 40) using alkanediols of increasing C-chain length (C6, C8, and C12) as initiators in the presence of Tin(II) catalyst. In the second step, the macromer diols were linked together with diacid dichlorides of varying C-chain lengths (C6, C8, C10, and C12) to yield polyesters ranging in molecular weight (M w) from 20 to 130 KDa and polydispersity of 1.5–6. These polyesters exhibited different thermal behavior from pure PHAs that can be tuned by changing the initiator core, the lactide/glycolide chain length, and diacid dichloride type. In addition, all these polymers showed solubility in tetrahydrofuran unlike poly(l-lactic acid) (PLLA) and poly(lactide-co-glycolide) (PLGA). In contrast to PLLA and PLGA, the degradation behavior of these novel polyesters exhibited linear profiles consistent with a surface erosion behavior. Release studies using Congo red as a model drug from microspheres prepared from these polyesters showed linear release profiles with correlation constants of least-square fits approaching a value of unity. Degradable polyesters with tunable thermal and degradation behavior may find applications in drug delivery and tissue engineering, where control over these parameters is critical to ensure predictable outcomes.
Keywords: Poly(α-hydroxy acids); Surface erosion; Drug delivery; Microparticles;

The release of the anti-inflammatory agent dexamethasone (DEX) from nanoparticles of poly(lactic-co-glycolic acid) (PLGA) embedded in alginate hydrogel (HG) matrices was investigated. DEX-loaded PLGA nanoparticles were prepared using a solvent evaporation technique and were characterized for size, drug loading, and in-vitro release. The crosslinking density of the HG was studied and correlated with drug release kinetics. The amount of DEX loaded in the nanoparticles was estimated as ∼13 wt%. The typical particle size ranged from 400 to 600 nm. The in-vitro release of DEX from NPs entrapped in the HG showed that 90% of the drug was released over 2 weeks. The impedance of the NP-loaded HG coatings on microfabricated neural probes was measured and found to be similar to the unmodified and uncoated probes. The in-vivo impedance of chronically implanted electrodes loaded with DEX was maintained at its initial level, while that of the control electrode increased by 3 times after about 2 weeks after implantation until it stabilized at approximately 3 MΩ. This improvement in performance is presumably due to the reduced amount of glial inflammation in the immediate vicinity of the DEX-modified neural probe.
Keywords: Dexamethasone; Controlled delivery; Neural prosthetics; Hydrogel; PLGA nanoparticles;