Biomaterials (v.28, #29)

A new approach to the rationale discovery of polymeric biomaterials by Joachim Kohn; William J. Welsh; Doyle Knight (4171-4177).
This paper attempts to illustrate both the need for new approaches to biomaterials discovery as well as the significant promise inherent in the use of combinatorial and computational design strategies. The key observation of this Leading Opinion Paper is that the biomaterials community has been slow to embrace advanced biomaterials discovery tools such as combinatorial methods, high-throughput experimentation, and computational modeling in spite of the significant promise shown by these discovery tools in materials science, medicinal chemistry and the pharmaceutical industry. It seems that the complexity of living cells and their interactions with biomaterials has been a conceptual as well as a practical barrier to the use of advanced discovery tools in biomaterials science. However, with the continued increase in computer power, the goal of predicting the biological response of cells in contact with biomaterials surfaces is within reach. Once combinatorial synthesis, high-throughput experimentation, and computational modeling are integrated into the biomaterials discovery process, a significant acceleration is possible in the pace of development of improved medical implants, tissue regeneration scaffolds, and gene/drug delivery systems.
Keywords: Biomaterials design; Computational modeling; Combinatorial synthesis; High-throughput experimentation;

Molecular imprinting is an inexpensive method for the rapid fabrication of organic polymeric and inorganic network-structured materials that selectively bind a template molecule—in other words, materials that function as artificial antibodies. Imprints against small-molecule templates have been generated for decades, but attempts to prepare imprints against proteins have, until recently, been far less successful. The field has progressed rapidly, however, and a number of molecular imprints selective for protein ligands have now been reported. Given the enormous potential of replacing the antibodies used in a host of immunoassays with robust and inexpensive receptors, efforts in this area continue to intensify. This review begins with a brief analysis of two naturally occurring protein–ligand complexes, each of which illustrates the specific interactions essential for precise molecular recognition. Key developments—all appearing in 2006 and 2007—in the molecular imprinting of proteins, including many impressive advances, are then discussed.
Keywords: Protein recognition; Polymerization; Sol–gel; Molecular imprint; Sensor;

Inhibition of bacterial adhesion and biofilm formation on zwitterionic surfaces by Gang Cheng; Zheng Zhang; Shengfu Chen; James D. Bryers; Shaoyi Jiang (4192-4199).
In this work, we report a study of long-chain zwitterionic poly(sulfobetaine methacrylate) (pSBMA) surfaces grafted via atom transfer radical polymerization (ATRP) for their resistance to bacterial adhesion and biofilm formation. Previously, we demonstrated that p(SBMA) is highly resistant to nonspecific protein adsorption. Poly(oligo(ethylene glycol) methyl ether methacrylate) (pOEGMA) grafted surfaces were also studied for comparison. Furthermore, we quantify how surface grafting methods will affect the long-term biological performance of the surface coatings. Thus, self-assembled monolayers (SAMs) of alkanethiols with shorter-chain oligo(ethylene glycol) (OEG) and mixed SO3 /N+(CH3)3 terminated groups were prepared on gold surfaces. The short-term adhesion (3 h) and the long-term accumulation (24 or 48 h) of two bacterial species (Gram-positive Staphylococcus epidermidis and Gram-negative Pseudomonas aeruginosa) on these surfaces were studied using a laminar flow chamber. Methyl-terminated (CH3) SAM on gold and a bare glass were chosen as references. p(SBMA) reduced short-term adhesion of S. epidermidis and P. aeruginosa relative to glass by 92% and 96%, respectively. For long-term biofilm formation, qualitative images showed that p(SBMA) dramatically reduced biofilm formation of S. epidermidis and P. aeruginosa as compared to glass.
Keywords: Zwitterionic; ATRP; Bacteria; Biofilm; Nonfouling;

Use of telechelic cis-1,4-polyisoprene cationomers in the synthesis of antibacterial ionic polyurethanes and copolyurethanes bearing ammonium groups by Nasreddine Kébir; Irène Campistron; Albert Laguerre; Jean-François Pilard; Claude Bunel; Thierry Jouenne (4200-4208).
New crosslinked ionic polyurethanes and copolyurethanes were yielded by reaction of telechelic cis-1,4-oligoisoprenes, bearing a variable number of ammonium and hydroxy groups, with isocyanurate of isophorone diisocyanate (I-IPDI). Aiming for a comparative study, polyurethane elastomers based on non-ionic telechelic oligomers were also synthesized. Thermo-mechanical behavior and crosslinking density of these three families of materials were investigated by DMTA and swelling test, respectively. Surface properties were examined by static contact angle measurements and AFM imaging. The bactericidal activity of the polymers was investigated by enumerating living Pseudomonas aeruginosa on material surfaces and on water suspensions. The number of attached living bacteria was found to depend on the chemical structure of the material and on the contact time between the microorganisms and the surface. An exclusive bactericidal activity was obtained with the ionic copolyurethane family. Materials with weak crosslinking density were found to release bactericidal moieties. The abilities of the polymers to prevent bacterial growth were examined through zone of inhibition experiments against P. aeruginosa, which shown a bacteriostatical effect for each synthesized material. These experiments were not sufficiently sensitive to detect the leaching of bactericidal moieties from the materials with weak crosslinking density. When the zone of inhibition experiments was performed on more sensitive bacteria, namely Staphylococcus epidermidis, the leaching of bactericidal moieties as well as bacteriostatic effect was detected. This work demonstrates the potentiality for making functional biomaterials from natural rubber, a renewable resource.
Keywords: Biocide; Polyurethanes; Polyisoprene; Thermo-mechanical properties; Surface properties; Antibacterial properties;

Biological performance in goats of a porous titanium alloy–biphasic calcium phosphate composite by JiaPing Li; Pamela Habibovic; Huipin Yuan; Mirella van den Doel; Clayton E. Wilson; Joost R. de Wijn; Clemens A. van Blitterswijk; Klass de Groot (4209-4218).
In this study, porous 3D fiber deposition titanium (3DFT) and 3DFT combined with porous biphasic calcium phosphate ceramic (3DFT+BCP) implants, both bare and 1 week cultured with autologous bone marrow stromal cells (BMSCs), were implanted intramuscularly and orthotopically in 10 goats. To assess the dynamics of bone formation over time, fluorochrome markers were administered at 3, 6 and 9 weeks and the animals were sacrificed at 12 weeks after implantation. New bone in the implants was investigated by histology and histomorphometry of non-decalcified sections. Intramuscularly, no bone formation was found in any of the 3DFT implants, while a very limited amount of bone was observed in 2 BMSC 3DFT implants. 3DFT+BCP and BMSC 3DFT+BCP implants showed ectopic bone formation, in 8 and 10 animals, respectively. The amount of formed bone was significantly higher in BMSC 3DFT+BCP as compared to 3DFT+BCP implants. Implantation on transverse processes resulted in significantly more bone formation in composite structure as compared to titanium alloy alone, both with and without cells. Unlike intramuscularly, the presence of BMSC did not have a significant effect on the amount of new bone either in metallic or in composite structure. Although the 3DFT is inferior to BCP for bone growth, the reinforcement of the brittle BCP with a 3DFT cage did not negatively influence osteogenesis, osteoinduction and osteoconduction as previously shown for the BCP alone. The positive effect of BMSCs was observed ectopically, while it was not significant orthotopically.
Keywords: Porous Ti6Al4V; Biphasic calcium phosphate (BCP); BMSC; Osteoconduction; Osteogenesis;

Surface characteristics greatly influence attachment and growth of cells on biomaterials. Although polylactone-type biodegradable polymers have been widely used as scaffold materials for tissue engineering, lack of cell recognition sites, poor hydrophilicity and low surface energy lead to a bad cell affinity of the polymers, which limit the usage of polymers as scaffolds in tissue engineering. In the present study, surface of poly (l-lactide-co-glycolide) (PLGA) was modified by a method of combining oxygen plasma treatment with anchorage of cationized gelatin. Modification effect of the method was compared with other methods of oxygen plasma treatment, cationized gelatin or gelatin coating and combining oxygen plasma treatment with anchorage of gelatin. The change of surface property was compared by contact angles, surface energy, X-ray photoelectron spectra (XPS), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM) measurement. The optimum oxygen pretreatment time determined by surface energy was 10 min when the power was 50 W and the oxygen pressure was 20 Pa. Analysis of the stability of gelatin and cationized gelatin anchored on PLGA by XPS, ATR-FTIR, contact angles and surface energy measurement indicated the cationized gelatin was more stable than gelatin. The result using mouse NIH 3T3 fibroblasts as model cells to evaluate cell affinity in vitro showed the cationized gelatin-anchored PLGA (OCG-PLGA) was more favorable for cell attachment and growth than oxygen plasma treated PLGA (O-PLGA) and gelatin-anchored PLGA (OG-PLGA). Moreover cell affinity of OCG-PLGA could match that of collagen-anchored PLGA (AC-PLGA). So the surface modification method combining oxygen plasma treatment with anchorage of cationized gelatin provides a universally effective way to enhance cell affinity of polylactone-type biodegradable polymers.
Keywords: PLGA; Surface modification; Oxygen plasma treatment; Cationized gelatin anchorage; Cell affinity; Tissue engineering;

Response of monocytes exposed to phagocytosable particles and discs of comparable surface roughness by Dong-Hwan Kim; Matt T. Novak; Jamie Wilkins; Minkyu Kim; Anita Sawyer; William M. Reichert (4231-4239).
This in vitro study characterized the temporal cytokine expression profile from human monocytes exposed to phagocytosable Ti particles (0.78±0.12 μm) and to Ti discs of comparable surface roughness. Human THP-1 monocytes were cultured in six well tissue culture polystyrene (TCPS) plates. Each well was either bare, contained Ti particles (the particles were clearly engulfed by the monocytes), or contained a Ti disc. Half of the wells were treated with 1 μg/mL lipopolysaccharide (LPS), while the other half were left unstimulated. Unstimulated and LPS-stimulated cells in bare wells were the negative and positive controls, respectively. Supernatant was sampled from each well at 1, 6, 24, 48, and 72 h and assayed for the expression of nine different cytokines using a Luminex system. Three cytokines (IL-1β, GM-CSF and IL-13) gave little to no response under all conditions, while six cytokines (TNF-α, IL-6, MIP-1α, MCP-1, VEGF, and IL-1ra) were clearly detectable. Expression levels generally increased with culture time, particle concentration, and LPS stimulation. Most significantly, it was found that cells treated by Ti discs produced in many instances a higher cytokine expression than did particles.
Keywords: Ti; Cytokines; Monocytes; Endotoxin; Inflammation and wound healing;

A tissue engineering approach to bone repair in large animal models and in clinical practice by Ranieri Cancedda; Paolo Giannoni; Maddalena Mastrogiacomo (4240-4250).
The repair of large segmental bone defects due to trauma, inflammation and tumor surgery remains a major clinical problem. Animal models were developed to test bone repair by tissue engineering approaches, mimicking real clinical situations. Studies differed with regard to animals (dog, sheep, goat), treated bone (femur, tibia, mandible), chemistry and structure of the scaffolds. Still, an advantage in the bone formation and in the healing of the segmental defect was always observed when scaffolds were seeded with bone marrow derived stromal cells (BMSCs). In the year 1998 was performed the first implantation of a porous ceramic construct in a bone segmental defect of a patient; it was the first construct seeded with cultured autologous osteogenic cells. Since then, only few other similar cases were treated by the same approach. However, in other fields, such as oral and maxillofacial surgery, injectable cells/platelet-rich plasma composites have been used as grafting materials for maxillary sinus floor augmentation and/or onlay plasty. More recently, the reconstruction of a human mandible was also reported by means of a bone–muscle-flap in vivo prefabrication technique, where the patient served as his own bioreactor. Indeed continuous implementations test and provide new means of defects treatment and cure. However, based on results so far obtained in animal models and pilot clinical studies, one can affirm that the bone tissue engineering approaches, although successful in most cases, need further validation before a wide application in clinics. In particular, the supply of oxygen and nutrients to the cells in the inner part of the implanted scaffolds remains a major concern, requiring additional investigations.
Keywords: Bone graft; Scaffold; Stem cell; Transplantation;

Identification and characterization of bioactive factors in bladder submucosa matrix by So Young Chun; Grace Jeong Lim; Tae Gyun Kwon; Eun Kyoung Kwak; Bup Wan Kim; Anthony Atala; James J. Yoo (4251-4256).
In spite of long term clinical use of decellularized bladder submucosa matrix (BSM), little is known about the active factors within this material. In this paper, we analyzed the biological factors from the decellularized BSM using ELISA, Western blotting, and immunohistochemistry for the purpose of effective utilization of this material in the field of regenerative medicine. At least 10 growth factors, including VEGF, BMP4, PDGF-BB, KGF, TGFβ1, IGF, bFGF, EGF and TGFα were found to be preserved in the decellularized BSM . The existence of collagen (type 1, 2, 3, 4), laminin and elastin within the matrix was also demonstrated. The soluble BSM extracts showed a conspicuous effect on cell proliferation when added as a supplement in vitro. These findings demonstrate that growth factors and extracellular matrix in the BSM maintain valuable biological activity even after the decellularization and extraction processes, thus supporting the wide applicability of BSM in tissue regeneration. The identification and characterization of growth factors and extracellular matrix in the BSM is a prerequisite for understanding tissue regeneration using this scaffold.
Keywords: Growth factors; Bladder submucosa matrix (BSM); Extracellular matrix; Bioactive factors; Decellularized scaffold;

Assembly of collagen-binding peptide with collagen as a bioactive scaffold for osteogenesis in vitro and in vivo by Jue-Yeon Lee; Jung-Eun Choo; Young-Suk Choi; Jun-Bum Park; Do-Sik Min; Seung-Jin Lee; Hyung Keun Rhyu; In-Ho Jo; Chong-Pyoung Chung; Yoon-Jeong Park (4257-4267).
Bioactive scaffolds inducing cell adhesion, differentiation have been premise for optimal formation of target tissue. Collagen has been employed as a tissue regenerative scaffold especially for bone regeneration and has been chemically surface-modified to present bioactivity. Herein, we show that peptide, denoted as collagen-binding motif (CBM, GLRSKSKKFRRPDIQYPDATDEDITSHM) identified from osteopontin (OPN) protein, was able to specifically bind collagen without chemical conjugation, while presenting apatite forming capability in vitro and in vivo. Collagen surface alone was not able to induce noticeable apatite nucleation however, mineralization was evident when assembled with CBM peptide, implying that the collagen–CBM assembly played a pivotal role in biomineralization. In vivo result further demonstrated that the CBM peptide in complex with material was able to induce bone formation by helping mineralization in the bone defect. Taken together, the CBM peptide herein and its assembly with collagen can be applied as an inducer of biomineralization as well as a bioactive scaffold for bone regeneration.
Keywords: Bioactive scaffold; Collagen-binding motif peptide; Assembly; Osteopontin; Biomineralization; Bone regeneration;

Orthogonal scaffold of magnetically aligned collagen lamellae for corneal stroma reconstruction by Jim Torbet; Marilyne Malbouyres; Nicolas Builles; Virginie Justin; Muriel Roulet; Odile Damour; Åke Oldberg; Florence Ruggiero; David J.S. Hulmes (4268-4276).
The creation of 3D scaffolds that mimic the structure of physiological tissue required for normal cell function is a major bioengineering challenge. For corneal stroma reconstruction this necessitates the creation of a stroma-like scaffold consisting of a stack of orthogonally disposed sheets of aligned collagen fibrils. This study demonstrates that such a scaffold can be built up using magnetic alignment. By allowing neutralized acid-soluble type I collagen to gel in a horizontal magnetic field (7 T) and by combining a series of gelation–rotation–gelation cycles, a scaffold of orthogonal lamellae composed of aligned collagen fibrils has been formed. Although initially dilute, the gels can be concentrated without noticeable loss in orientation. The gels are translucent but their transparency can be greatly improved by the addition of proteoglycans to the gel-forming solution. Keratocytes align by contact guidance along the direction of collagen fibrils and respect the orthogonal design of the collagen template as they penetrate into the bulk of the 3D matrix. The scaffold is a significant step towards the creation of a corneal substitute with properties resembling those of native corneal stroma.
Keywords: Corneal stroma reconstruction; Collagen; Proteoglycans; Keratocytes; Stroma-like scaffold; Magnetic alignment;

In contractile tissues such as myocardium, functional properties are directly related to the cellular orientation and elongation. Thus, tissue engineering of functional cardiac patches critically depends on our understanding of the interaction between multiple guidance cues such as topographical, adhesive or electrical. The main objective of this study was to determine the interactive effects of contact guidance and electrical field stimulation on elongation and orientation of fibroblasts and cardiomyocytes, major cell populations of the myocardium. Polyvinyl surfaces were abraded using lapping paper with grain size 1–80 μm, resulting in V-shaped abrasions with the average abrasion peak-to-peak width in the range from 3 to 13 μm, and the average depth in the range from 140 to 700 nm (AFM). The surfaces with abrasions 13 μm wide and 700 nm deep, exhibited the strongest effect on neonatal rat cardiomyocyte elongation and orientation as well as statistically significant effect on orientation of fibroblasts, thus they were utilized for electrical field stimulation. Electrical field stimulation was performed using a regime of relevance for heart tissue in vivo as well as for cardiac tissue engineering. Stimulation (square pulses, 1 ms duration, 1 Hz, 2.3 or 4.6 V/cm) was initiated 24 h after cell seeding and maintained for additional 72 h. The cover slips were positioned between the carbon rod electrodes such that the abrasions were either parallel or perpendicular to the field lines. Non-abraded surfaces were utilized as controls. Field stimulation did not affect cell viability. The presence of a well-developed contractile apparatus in neonatal rat cardiomyocytes (staining for cardiac Troponin I and actin filaments) was identified in the groups cultivated on abraded surfaces in the presence of field stimulation. Overall we observed that (i) fibroblast and cardiomyocyte elongation on non-abraded surfaces was significantly enhanced by electrical field stimulation, (ii) electrical field stimulation promoted orientation of fibroblasts in the direction perpendicular to the field lines when the abrasions were also placed perpendicular to the field lines and (iii) topographical cues were a significantly stronger determinant of cardiomyocyte orientation than the electrical field stimulation. The orientation and elongation response of cardiomyocytes was completely abolished by inhibition of actin polymerization (Cytochalasin D) and only partially by inhibition of phosphatidyl-inositol 3 kinase (PI3K) pathway (LY294002).
Keywords: Cardiomyocyte; Fibroblast; Surface topography; Electrical stimulation; Actin; Cardiac tissue engineering;

Dynamic sealing of lung air leaks by the transplantation of tissue engineered cell sheets by Masato Kanzaki; Masayuki Yamato; Joseph Yang; Hidekazu Sekine; Chinatsu Kohno; Ryo Takagi; Hideyuki Hatakeyama; Tamami Isaka; Teruo Okano; Takamasa Onuki (4294-4302).
Current methods including the use of various biological and synthetic sealants are ineffective in the closure of intraoperative air leaks that often occur during cardiothoracic surgeries, resulting in a decreased quality of life for patients. We present the development of a novel lung air leak sealant using tissue engineered cell sheets. In contrast to previous materials such as fibrin glue, these bioengineered cell sheets immediately and permanently seal air leaks in a dynamic fashion that allows for the extensive tissue contraction and expansion involved in respiration, without any postoperative recurrences. Additionally, we demonstrate that mesothelial cells migrate to cover the transplanted cells sheets, thereby confirming excellent biocompatibility and integration with the host tissues. Finally, we present the use of skin fibroblasts as an effective and readily available autologous cell source that can be easily applied. This study shows for the first time, the development of an immediate and permanent lung air leak sealant, suitable for future clinical applications.
Keywords: Air leak; Cell sheet; Autologous cell; Temperature-responsive culture dish;

Effect of human corneal keratocytes and retinal pigment epithelial cells on the mechanical properties of micropatterned collagen films by Nihal E. Vrana; Ahmed Elsheikh; Nicolas Builles; Odile Damour; Vasif Hasirci (4303-4310).
Collagen-based micropatterned films were seeded with human corneal keratocyte and epithelial cells to study their mechanical properties as tissue engineering substrates. The patterns were in the form of parallel channels with slanted walls. Influence of cell presence, type and growth on the mechanical properties of the films was investigated. Unseeded films showed gradual strength reduction from an initial value of 0.046 N/mm, possibly due to degradation, down to 0.032±0.012 N/mm in 2 weeks. Keratocyte growth was found to significantly improve the mechanical behavior of the films upon 1 week of incubation (0.067±0.017 N/mm) and the improvement continued gradually over the next 2 weeks. Films seeded with D407 retinal pigment epithelial cells, on the other hand, experienced a decrease (0.023±0.011 N/mm), followed by a slight increase in mechanical properties in the 21-day period. A steady increase in the number of keratocytes along the channels, cytoskeleton alignment and extracellular matrix (ECM) secretion restricted to the channels was observed. Increase in strength observed with keratocytes and, to a lesser extent, with the epithelial cells can be attributed to directional ECM synthesis and the orientation of the cells and their cytoskeleton which contribute to the strength in the direction of the channels. This study showed that cell, especially keratocyte, presence compensates for the degradation of collagen films and improve the overall mechanical properties of the engineered tissue.
Keywords: Cornea; Collagen; Mechanical properties; Tissue engineering; Micropatterning; Keratocytes;

Cholesterol transport from liposomal delivery vehicles by Azadeh Kheirolomoom; Katherine W. Ferrara (4311-4320).
Rapid internalization of drugs from delivery vehicles via non-endocytotic pathways is an important goal. The transport of imaging probes attached to cholesterol and introduced via a liposomal formulation is considered here, in order to evaluate the intracellular distribution and kinetics of small molecular cargo that might be attached to cholesterol or phospholipids. The internalization efficiencies of two fluorescent cholesterol analogues, one carrying a fluorophore on the head of the cholesterol molecule 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate (BODIPY)-cholesteryl ester (CE) (BODIPY-CE) and the other on the tail (25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl]amino]-27-norcholesterol (NBD-cholesterol)), were compared with those of other phospholipid molecules (NBD-phosphatidylcholine (PC) and NBD-phosphatidylethanolamine (PE)) using a liposomal formulation (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 85.5  m%; 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2k), 9.5  m%; fluorescent analogue, 5  m%). The rate and transfer efficiency were NBD-cholesterol>BODIPY-CE>NBD-PC>NBD-PE. NBD-cholesterol, delivered by liposomes with an average diameter of 100 nm, localized in the perinuclear region and lipid storage droplets, with transfer observed in as little as 5 min. NBD-cholesterol transport was approximately constant with time, suggesting a unidirectional mode of entry. In the absence of PEG within the liposome, the transfer rate decreased. Filipin, a caveolae-blocking agent, caused 70% inhibition of cholesterol internalization in treated cells, suggesting that cholesterol internalization follows a caveolae-mediated pathway.
Keywords: Cholesterol internalization; Endocytosis pathway; Caveolae; Liposome; Drug delivery;

A naturally derived, cytocompatible, and architecturally optimized scaffold for tendon and ligament regeneration by Patrick W. Whitlock; Thomas L. Smith; Gary G. Poehling; Jeffrey S. Shilt; Mark Van Dyke (4321-4329).
Tissue-engineered tendon scaffolds have the potential to significantly improve the treatment of tendon and ligament injuries, especially those associated with tumors, trauma, and congenital deficiencies where autograft or allograft tissue might not be available in sufficient quantity for reconstruction. In this study, a tendon scaffold was produced that: (1) has decreased/absent cellular material histologically, as well as significantly decreased DNA content in comparison with the material it is derived from—fresh-frozen flexor digitorum profundus tendon; (2) is cytocompatible in vitro; (3) has been modified to produce increased pore size and porosity; (4) retains 76–78% of the tensile properties of the material it is derived from; (5) is readily infiltrated by fibroblast-like, mononuclear host cells; and (6) does not exhibit a host-cell-mediated foreign-body immune response after implantation in vivo.
Keywords: Ligament; Porosity; Scaffold; Tendon;