Biomaterials (v.28, #24)

Enhanced osseointegration by the chemotactic activity of plasma fibronectin for cellular fibronectin positive cells by Ryo Jimbo; Takashi Sawase; Yasuaki Shibata; Kazunari Hirata; Yoshitaka Hishikawa; Yasuhiro Tanaka; Kazuhisa Bessho; Tohru Ikeda; Mitsuru Atsuta (3469-3477).
Plasma fibronectin (pFN) is known to regulate cell growth, differentiation or survival of osteoblasts in vitro. It is also speculated to be important for the early phase of osseointegration, however, its actual in vivo behavior is unknown. The objective of this study is to clarify the role of pFN during osseointegration. We developed a titanium ion-plated acrylic implant (Ti-acryl) for thin sectioning without removal of the implant. Either Ti-acryl or pFN-coated Ti-acryl (FN-Ti-acryl) was implanted in the mouse femur. Samples were taken on days 1–7 and on day 14 after the operation, and were decalcified and paraffin embedded. The bone healing process and immunofluorescence localization of pFN and cellular fibronectin (cFN), a marker for fibroblastic cells were examined. Simultaneously, the effect of pFN on chemotaxis, proliferation and differentiation of bone marrow stromal cells (BMSCs) was analyzed in vitro. The in vivo results showed that faster direct bone formation was seen for the FN-Ti-acryl group compared to the Ti-acryl group. The in vitro results showed that pFN significantly promoted BMSCs chemotaxis, however, had no effect on proliferation or differentiation. The results indicate that pFN regulated chemotaxis of osteogenic cells and coating the implant with pFN enhanced earlier osseointegration.
Keywords: Implant; Fibronectin; Osseointegration; Histomorphometry; Chemotaxis;

The use of physical hydrogels of chitosan for skin regeneration following third-degree burns by Nadège Boucard; Christophe Viton; Diane Agay; Eliane Mari; Thierry Roger; Yves Chancerelle; Alain Domard (3478-3488).
Skin repair is an important field of the tissue engineering, especially in the case of extended third-degree burns, where the current treatments are still insufficient in promoting satisfying skin regeneration. Bio-inspired bi-layered physical hydrogels only constituted of chitosan and water were processed and applied to the treatment of full-thickness burn injuries. The aim of the study was at assessing whether this material was totally accepted by the host organism and allowed in vivo skin reconstruction of limited area third-degree burns. A first layer constituted of a rigid protective gel ensured good mechanical properties and gas exchanges. A second soft and flexible layer allowed the material to follow the geometry of the wound and ensured a good superficial contact. To compare, highly viscous solutions of chitosan were also considered. Veterinary experiments were performed on pig's skins and biopsies at days 9, 17, 22, 100 and 293, were analysed by histology and immuno-histochemistry. Only one chitosan material was used for each time. All the results showed that chitosan materials were well tolerated and promoted a good tissue regeneration. They induced inflammatory cells migration and angiogenetic activity favouring a high vascularisation of the neo-tissue. At day 22, type I and IV collagens were synthesised under the granulation tissue and the formation of the dermal–epidermal junction was observed. After 100 days, the new tissue was quite similar to a native skin, especially by its aesthetic aspect and its great flexibility.
Keywords: Chitosan; Physical hydrogel; Skin regeneration; Third-degree burns;

In this paper the mechanical properties of micropatterned polyester films prepared to serve as tissue engineering scaffolds of cornea were examined. Films were prepared by solvent casting of blends of poly(l-lactide-co-d,l-lactide) and poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid), on a micropatterned silicon template. They were seeded with keratocytes or retinal pigment epithelial cells and subjected to tensile testing to assess the contribution of cells and the deposited extra-cellular matrix (ECM) to the mechanical properties of the scaffold. In all the tests, the films used were wet and the cells were not fixed. Cell-free scaffolds showed a gradual deterioration in strength upon incubation in the cell culture medium at 37 °C; the deterioration rate was highest in the first week and decreased significantly over the second and third weeks. The ultimate strength of the cell-free scaffolds decreased from 0.99 to 0.42 N/mm after 21 days of incubation. Cell seeded scaffolds showed a more complicated mechanical strength profile. Their response was found to depend both on the extent of surface coverage and on the cell type. The results were examined after dividing the data into two groups of lower and higher stiffness. For keratocyte seeded scaffolds, the strength of the high stiffness groups continued to increase as the incubation period increased while the lower stiffness groups did not show a distinct change. For the keratocyte seeded scaffolds, tensile strength increased from 0.65 N/mm on Day 7 to 0.73 N/mm on Day 21. On the other hand, the scaffolds seeded with retinal pigment epithelial cells showed a gradual deterioration over time in both the higher and lower stiffness groups. For epithelial cell seeded scaffolds this was 0.98 N/mm on Day 7 and decreased to 0.77 N/mm on Day 21 still an improvement over the unseeded scaffolds. This most probably was a result of a lower rate of ECM secretion in comparison to keratocytes and the newly secreted ECM could not compensate for the influence of scaffold degradation on the mechanical properties. It could, therefore, be concluded that cell seeding plays a positive role in strengthening a tissue engineered construct, and cell type has a significant influence on the extent of this improvement.
Keywords: Cornea; Mechanical properties; Tissue engineering; Polyester;

Solid lipid templating of macroporous tissue engineering scaffolds by Michael Hacker; Michael Ringhofer; Bernhard Appel; Markus Neubauer; Thomas Vogel; Simon Young; Antonios G. Mikos; Torsten Blunk; Achim Göpferich; Michaela B. Schulz (3497-3507).
Macroporous biodegradable cell carriers (scaffolds) provide the three-dimensional matrix for tissue formation in vitro. In this study, we present the fabrication of macroporous scaffolds with high inter-pore connectivity from different biodegradable polymers using the recently developed solid lipid templating technique. Starting from a polymer solution and solid lipid microparticles, a dispersion is prepared and subsequently transferred into molds, which are finally submerged in warm hexane to precipitate the polymer and extract the porogens. The study shows how to control pore structure, pore size and porosity of the scaffold using this technique. The process parameters dispersion viscosity, porogen size and type of polymer are considered. Limits of viscosity are examined by macroscopic and microstructure evaluation of the scaffolds prepared at different viscosities. An approach to rationalize these data by oscillation rheometry is shown. Pore size can be controlled by porogen particle size and adaptation of the viscosity of the polymer solution. Porosity can be modified by changing the ratio of porogen to polymer. The suitability of the resulting scaffolds was shown using an established cartilage cell culture model.
Keywords: Scaffold; Lipid; Polylactic acid; Polyethylene oxide; Cartilage tissue engineering;

Creation of myocardial tubes using cardiomyocyte sheets and an in vitro cell sheet-wrapping device by Hirotsugu Kubo; Tatsuya Shimizu; Masayuki Yamato; Tetsuo Fujimoto; Teruo Okano (3508-3516).
Regenerative medicine involving injection of isolated cells and transplantation of tissue-engineered myocardial patches, has received significant attention as an alternative method to repair damaged heart muscle. In the present study, as the next generation of myocardial tissue engineering we demonstrate the in vitro fabrication of pulsatile myocardial tubes using cell sheet engineering technologies. Three neonatal rat cardiomyocyte sheets, which were harvested from temperature-responsive culture dishes, were wrapped around fibrin tubes using a novel cell sheet-wrapping device. The tubular constructs demonstrated spontaneous, synchronized pulsation within 3 h after cell sheet wrapping. Contractile force measurements showed that the contractile force increased in accordance with both increasing rest length (Starling mechanism) and increasing extracellular Ca2+ concentration. Furthermore, the tissue-engineered myocardial tubes presented measurable inner pressure changes evoked by tube contraction (0.11±0.01 mmHg, max 0.15 mmHg, n=5). Histological analyses revealed both well-differentiated sarcomeres and diffuse gap junctions within the myocardial tissues that resembled native cardiac muscle. These data indicate that tissue-engineered myocardial tubes have native heart-like structure and function. These new myocardial tissue constructs should be useful for future applications in physiological studies and pharmacological screening, and present a possible core technology for the creation of engineered tissues capable of independent cardiac assistance.
Keywords: Tissue engineering; Myocardial tube; Cell sheet; Wrapping device; Inner pressure;

Oriented immobilization of epidermal growth factor onto culture substrates for the selective expansion of neural stem cells by Tadashi Nakaji-Hirabayashi; Koichi Kato; Yusuke Arima; Hiroo Iwata (3517-3529).
To develop a culture substrate that allows efficient expansion of neural stem cells (NSCs), epidermal growth factor (EGF) was immobilized onto the Ni(II)-chelated surface of a glass-based substrate through coordination of Ni(II) to the histidine tag that was fused to the C-terminal of EGF using recombinant technology. For the preparation of the nickel-chelated surface, a thin gold layer was deposited to the glass surface, and then the self-assembled monolayer of alkanethiol terminated with trivalent carboxylic acids was formed on gold and chelated with Ni(II) ions. In the preparation of a monolayer, triethylene glycol-terminated alkanethiol was mixed with carboxylic acid-terminated alkanethiol at various compositions in order to reduce the non-specific adsorption of EGF. The surface analysis of the monolayers was performed by X-ray photoelectron spectroscopy, infrared reflection–absorption spectroscopy, and contact angle measurements. Surface plasmon resonance analyses and protein assays were performed for characterizing EGF-immobilized surfaces. The proliferation and differentiation of rat fetal NSCs were examined on the EGF-chelated substrates to assess quantitatively the effects of alkanethiol composition on the efficiency of stem cell amplification. It was shown that the amplification efficiency was dependent on the alkanethiol composition. This result could be attributed to the difference in the surface density of chelated EGF. Under the optimal condition, 98% of proliferated cells expressed NSC marker. In addition, these cells could be subcultured for further expansion, while retained their multipotency. We concluded that the substrate developed here provides the efficient method for the highly selective expansion of NSCs.
Keywords: Neural stem cell; Growth factor; Surface modification; Self assembly; Surface analysis;

Alkaline phosphatase-induced mineral deposition to anchor collagen fibrils to a solid surface by Agnes D. Berendsen; Theo H. Smit; Kees A. Hoeben; X. Frank Walboomers; Antonius L.J.J. Bronckers; Vincent Everts (3530-3536).
Reconstruction of tendon and ligament tissues requires proper attachment of the tissue-engineered construct to surrounding tissues. A problem of reconstructing collagen-rich tissues is that an in vitro engineered collagenous network containing fibroblasts will contract and detach from a solid surface. In vivo anchorage of soft connective tissues to mineralized tissues like bones and teeth is accomplished by embedding collagen fibrils into mineralized layers. Mineralization is partially the result of local activity of the enzyme alkaline phosphatase (ALP). In this study, we tested whether ALP-induced mineral deposition at the interface between a collagen gel and a polystyrene or polyetheretherketone (PEEK) surface could prevent gel detachment from the surface. Coating of culture wells with intestinal ALP prevented detachment of gels harbored with human periodontal ligament (PDL) fibroblasts in the presence of its substrate β-glycerophosphate. Mineral deposition was observed predominantly at the interface of collagen gel and well surface. The contractile properties of fibroblasts were not influenced by either ALP, β-glycerophosphate or both. The presence of ALP on a solid surface and providing its substrate to allow mineral deposition can prevent detachment of collagen matrices. Our findings provide a tool to induce attachment of fibrillar collagen to a solid surface; an approach that seems useful for reconstruction of load-bearing tissues and attachment of ligaments to implants.
Keywords: Alkaline phosphatase; Collagen; Calcium phosphate; Surface treatment; Fibroblast;

A biomimetic peptide fluorosurfactant polymer for endothelialization of ePTFE with limited platelet adhesion by Coby C. Larsen; Faina Kligman; Chad Tang; Kandice Kottke-Marchant; Roger E. Marchant (3537-3548).
Endothelialization of expanded polytetrafluoroethylene (ePTFE) has the potential to improve long-term patency for small-diameter vascular grafts. Successful endothelialization requires ePTFE surface modification to permit cell attachment to this otherwise non-adhesive substrate. We report here on a peptide fluorosurfactant polymer (FSP) biomimetic construct that promotes endothelial cell (EC)-selective attachment, growth, shear stability, and function on ePTFE. The peptide FSP consists of a flexible poly(vinyl amine) backbone with EC-selective peptide ligands for specific cell adhesion and pendant fluorocarbon branches for stable anchorage to underlying ePTFE. The EC-selective peptide (primary sequence: Cys–Arg–Arg–Glu–Thr–Ala–Trp–Ala–Cys, CRRETAWAC) has demonstrated high binding affinity for the α 5 β 1 integrin found on ECs. Here, we demonstrate low affinity of CRRETAWAC for platelets and platelet integrins, thus providing it with EC-selectivity. This EC-selectivity could potentially facilitate rapid in vivo endothelialization and healing without thrombosis for small-diameter ePTFE vascular grafts.
Keywords: Endothelial cells; ePTFE vascular grafts; Surface modification; Platelet adhesion;

Anabolic effects of bisphosphonates on peri-implant bone stock by Fabian von Knoch; Christina Eckhardt; Claude I. Alabre; Erich Schneider; Harry E. Rubash; Arun S. Shanbhag (3549-3559).
The long-term durability of total joint replacements is critically dependent on adequate peri-implant bone stock, which can be compromised by wear debris-mediated osteolysis. This study investigated the effects of bisphosphonates on enhancing peri-implant bone in the presence of clinically relevant ultra-high molecular weight polyethylene (UHMWPE) wear debris. Fiber-mesh coated titanium-alloy plugs were implanted bilaterally in the femoral condyles of 36 New Zealand white rabbits. Implants in the left femora were covered with submicron UHMWPE particles during surgery. Rabbits were administered either no drug, subcutaneous alendronate weekly (1.0 mg/kg/week) or a single dose of intravenous zoledronate (0.015 mg/kg). A total of 6/12 rabbits in each group were sacrificed at 6 weeks and the remainder at 12 weeks postoperatively. Peri-implant bone stock was analyzed radiographically and histomorphometrically. Radiographically, both bisphosphonates significantly increased periprosthetic cortical thickness at 6 weeks (p<0.0001; alendronate: +18%; zoledronate: +11%) and at 12 weeks (p=0.001; alendronate: +17%; zoledronate:+19%). Histomorphometrically, alendronate and zoledronate raised peri-implant bone volume (BV/TV) up to 2-fold after 6 weeks without added wear debris and more than 3-fold when wear debris was present. Furthermore a 6-week bisphosphonate treatment increased osteoid thickness in the absence of wear debris (alendronate: +132%, p=0.007; zoledronate: +67%, p=0.51) and in the presence of wear debris (alendronate: +134%, p=0.023; zoledronate: +138%, p=0.016). In summary, alendronate and zoledronate treatment increased periprosthetic bone stock in a rabbit femoral model, particularly in the presence of UHMWPE wear debris. These new findings suggest that bisphosphonates may more than compensate for the well-documented negative effects of wear debris on peri-implant bone stock. The combined antiresorptive and osteoanabolic effects of bisphosphonates on periprosthetic bone stock may have an important role for critically improving the biological fixation and ultimate durability of total joint arthroplasty.
Keywords: Bisphosphonates; Alendronate; Zoledronate; Total joint replacements; Wear debris; UHMWPE;

Practical recombinant hybrid mussel bioadhesive fp-151 by Dong Soo Hwang; Youngsoo Gim; Hyo Jin Yoo; Hyung Joon Cha (3560-3568).
Mussel adhesive proteins (MAPs) have received increased attention as potential environmentally friendly adhesives under aqueous conditions and in medicine. However, attempts to produce functional recombinant MAPs (mainly foot protein type 1, fp-1) by several expression systems have failed. Even though we previously reported a functional expression of recombinant foot protein type 5 (fp-5) with significant adhesive ability in Escherichia coli, its practical use was limited by several problems such as low production yield, low purification yield, and high levels of post-purification insolubility. Here, to overcome these limitations, we designed and constructed the novel type of hybrid mussel bioadhesive fp-151, a fusion protein comprising six fp-1 decapeptide repeats at each fp-5 terminus. Using micro- and bulk-scale characterization and mammalian cell-adhesion analyses, we demonstrate that fp-151 has the potential to be a practical bioadhesive with strong adhesive ability, a simple purification process (∼1 g-purified protein per 1 l-pilot-scale fed-batch bioreactor culture), proper manipulation properties (∼330 g/l solubility), and high biocompatibility.
Keywords: Mussel adhesive protein; Hybrid fp-151; Bioadhesive; Fusion protein; Escherichia coli; Cell adhesive;

The mathematical formulation of a generalized Hooke's law for blood vessels by Wei Zhang; Chong Wang; Ghassan S. Kassab (3569-3578).
It is well known that the stress–strain relationship of blood vessels is highly nonlinear. To linearize the relationship, the Hencky strain tensor is generalized to a logarithmic–exponential (log–exp) strain tensor to absorb the nonlinearity. A quadratic nominal strain potential is proposed to derive the second Piola–Kirchhoff stresses by differentiating the potential with respect to the log–exp strains. The resulting constitutive equation is a generalized Hooke's law. Ten material constants are needed for the three-dimensional orthotropic model. The nondimensional constant used in the log–exp strain definition is interpreted as a nonlinearity parameter. The other nine constants are the elastic moduli with respect to the log–exp strains. In this paper, the proposed linear stress–strain relation is shown to represent the pseudoelastic Fung model very well.
Keywords: Hooke's law; Constitutive relation; Strain measure; Nonlinearity;

A rate-insensitive linear viscoelastic model for soft tissues by Wei Zhang; Henry Y. Chen; Ghassan S. Kassab (3579-3586).
It is well known that many biological soft tissues behave as viscoelastic materials with hysteresis curves being nearly independent of strain rate when loading frequency is varied over a large range. In this work, the rate-insensitive feature of biological materials is taken into account by a generalized Maxwell model. To minimize the number of model parameters, it is assumed that the characteristic frequencies of Maxwell elements form a geometric series. As a result, the model is characterized by five material constants: μ 0, τ, m, ρ and β, where μ 0 is the relaxed elastic modulus, τ the characteristic relaxation time, m the number of Maxwell elements, ρ the gap between characteristic frequencies, and β=μ 1/μ 0 with μ 1 being the elastic modulus of the Maxwell body that has relaxation time τ. The physical basis of the model is motivated by the microstructural architecture of typical soft tissues. The novel model shows excellent fit of relaxation data on the canine aorta and captures the salient features of vascular viscoelasticity with significantly fewer model parameters.
Keywords: Viscoelasticity; Hysteresis; Relaxation; Microstructure;