Biomaterials (v.27, #25)

Calendar (I).

Porogen-based solid freeform fabrication of polycaprolactone–calcium phosphate scaffolds for tissue engineering by Mark J. Mondrinos; Robert Dembzynski; Lin Lu; Venkata K.C. Byrapogu; David M. Wootton; Peter I. Lelkes; Jack Zhou (4399-4408).
Drop on demand printing (DDP) is a solid freeform fabrication (SFF) technique capable of generating microscale physical features required for tissue engineering scaffolds. Here, we report results toward the development of a reproducible manufacturing process for tissue engineering scaffolds based on injectable porogens fabricated by DDP. Thermoplastic porogens were designed using Pro/Engineer and fabricated with a commercially available DDP machine. Scaffolds composed of either pure polycaprolactone (PCL) or homogeneous composites of PCL and calcium phosphate (CaP, 10% or 20% w/w) were subsequently fabricated by injection molding of molten polymer-ceramic composites, followed by porogen dissolution with ethanol. Scaffold pore sizes, as small as 200 μm, were attainable using the indirect (porogen-based) method. Scaffold structure and porosity were analyzed by scanning electron microscopy (SEM) and microcomputed tomography, respectively. We characterized the compressive strength of 90:10 and 80:20 PCL–CaP composite materials (19.5±1.4 and 24.8±1.3 Mpa, respectively) according to ASTM standards, as well as pure PCL scaffolds (2.77±0.26 MPa) fabricated using our process. Human embryonic palatal mesenchymal (HEPM) cells attached and proliferated on all scaffolds, as evidenced by fluorescent nuclear staining with Hoechst 33258 and the Alamar Blue™ assay, with increased proliferation observed on 80:20 PCL–CaP scaffolds. SEM revealed multilayer assembly of HEPM cells on 80:20 PCL–CaP composite, but not pure PCL, scaffolds. In summary, we have developed an SFF-based injection molding process for the fabrication of PCL and PCL–CaP scaffolds that display in vitro cytocompatibility and suitable mechanical properties for hard tissue repair.
Keywords: Calcium phosphate; Polycaprolactone; Composite; Scaffold; Mechanical properties; Cell proliferation;

Poly(ε-caprolactone) (PCL) film was modified using specified amounts of poly(d,l-lactide-co-glycolide) (PLGA) to provide a means to control polymer degradation. The aim of the study was to determine the effects of doping PCL with PLGA on the materials degradation, morphology and cell adhesion, to determine the significant variables within the process that could provide further control of cell adhesion. PLGA-doped PCL films were aged in osteogenic medium at 37 °C for up to 28 days. The aged samples were analysed in terms of weight loss or weight gain, molecule deposition and surface morphology. Molecule deposition was determined using Fourier transform infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) and morphology was determined using scanning electron microscopy and interferometric microscopy. The loss of the PLGA doping during degradation enhanced the formation of nano-porous structures in the remaining PCL domains, which attracted the deposition of substances from the osteogenic medium, which favoured the attachment and growth of human osteoblasts. The growth of osteoblasts was influenced by the controlled release of acidic products through polymer blending. Two pairs of FTIR-ATR absorption bands at 1090 and 1110 cm−1, and at 1180 and 1190 cm−1 were found to correlate to both PLGA and PCL, respectively. Changing the level of PLGA doping in PCL provided an approach to control the acidic products which can direct the growth of osteoblast cells.
Keywords: PLGA-doped PCL films; Ageing; Cell culture medium; Nano-porous structures; Osteogenic surface smoothening;

Osteoinduction with highly purified β-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation by Naoki Kondo; Akira Ogose; Kunihiko Tokunaga; Hajime Umezu; Katsumitsu Arai; Naoko Kudo; Makiko Hoshino; Hikaru Inoue; Hiroyuki Irie; Koichi Kuroda; Hisashi Mera; Naoto Endo (4419-4427).
The aim of the study was to examine the chronological histology of osteoinduction of highly purified β-tricalcium phosphate (β-TCP) implanted in dog dorsal muscles. Specimens were harvested on days 14, 28, 42, 56, 112 and 168 after implantation, and were analyzed by hematoxylin and eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry, in situ hybridization, and silver impregnation. After day 28, abundant TRAP- and cathepsin K-positive multinucleated cells adhered to β-TCP, suggesting that these cells are osteoclasts that can resorb β-TCP. On day 56, new bone was formed and α1 chain of type I procollagen mRNA-positive osteoblasts lined the newly formed bone. Silver impregnation showed abundant collagen fibrils within the β-TCP micropores. These results suggest that micropores function as a storage space for extracellular matrix components, including collagen. Newly formed bone never degenerated in the late stage, suggesting that β-TCP has good biocompatibility and this material retains the conditions appropriate for osteointegration and bioresorption. In conclusion, β-TCP has osteoinductivity after implantation in dog dorsal muscles without use of bone marrow cells or osteoinductive cytokines. The appearance of a large number of active osteoclasts precedes new bone formation.
Keywords: Bioresorption; Calcium phosphate; Microstructure; Osteoblast; Osteoclast;

Nanocomposites of hydroxyapatite with aspartic acid and glutamic acid and their interaction with osteoblast-like cells by Elisa Boanini; Paola Torricelli; Massimo Gazzano; Roberto Giardino; Adriana Bigi (4428-4433).
The direct synthesis of hydroxyapatite (HA)–aspartic acid (ASP) and HA–glutamic acid (GLU) nanocrystals was carried out in presence of different amounts of the amino acids in solution. ASP and GLU incorporation into HA crystals reduces the coherent length of the perfect crystalline domains along the long dimension (0 0 2) and, even more, along the cross section (3 1 0) of the apatite crystals, suggesting a specific interaction of the amino acids with the HA structure. FTIR analysis indicates that the carboxylic groups of the acidic amino acids interact with the calcium ions of HA. The relative amount of ASP incorporation into HA nanocrystals is greater than that of GLU, suggesting a greater affinity of ASP for HA. Osteoblast-like, MG63, cells cultured on the composite nanocrystals display good proliferation and increased values of ALP activity, collagen type I, TGF-betaI and osteocalcin production, indicating that the presence of the acidic amino acids enhances osteoblast activation and extra-cellular matrix mineralization processes.
Keywords: Hydroxyapatite; Amino acid; Biomimetic material; Cell culture;

Cartilage tissue engineering with silk scaffolds and human articular chondrocytes by Yongzhong Wang; Dominick J. Blasioli; Hyeon-Joo Kim; Hyun Suk Kim; David L. Kaplan (4434-4442).
Adult cartilage tissue has poor capability of self-repair, especially in case of severe cartilage damage due to trauma or age-related degeneration. Autologous cell-based tissue engineering using three-dimensional (3-D) porous scaffolds has provided an option for the repair of full thickness defects in adult cartilage tissue. Mesenchymal stem cells (MSCs) and chondrocytes are the two major cell sources for cartilage tissue engineering. Silk fibroin as a naturally occurring degradable fibrous protein with unique mechanical properties, excellent biocompatibility and processability has demonstrated strong potential for skeletal tissue engineering [Wong Po Foo C, Kaplan DL. Genetic engineering of fibrous proteins: spider dragline silk and collagen. Adv Drug Deliv Rev 2002; 54: 1131–43; Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J, et al. Silk-based biomaterials. Biomaterials 2003; 24: 401–16; Altman GH, Horan RL, Lu HH, Moreau J, Martin I, Richmond JC, et al. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 2002; 23: 4131–41; Jin HJ, Kaplan DL. Mechanism of silk processing in insects and spiders. Nature 2003; 424: 1057–61; Jin HJ, Fridrikh SV, Rutledge GC, Kaplan DL. Electrospinning Bombyx mori silk with poly(ethylene oxide). Biomacromolecules 2002; 3: 1233–9]. The present study combined adult human chondrocytes (hCHs) with aqueous-derived porous silk fibroin scaffolds for in vitro cartilage tissue engineering. The results were compared with a previous study using the same scaffolds but using MSCs to generate the cartilage tissue outcomes. Culture-expanded hCHs attached to, proliferated and redifferentiated in the scaffolds in a serum-free, chemically defined medium containing TGF-β1, based on cell morphology, levels of cartilage-related gene transcripts, and the presence of a cartilage-specific ECM. Cell density was critical for the redifferentiation of culture-expanded hCHs in the 3-D aqueous-derived silk fibroin scaffolds. The level of cartilage-related transcripts (AGC, Col-II, Sox 9 and Col-II/Col-I ratio) and the deposition of cartilage-specific ECM were significantly upregulated in constructs initiated with higher seeding density. The hCH-based constructs were significantly different than those formed from MSC-based constructs with respect to cell morphology, zonal structure and initial seeding density needed to successfully generate engineered cartilage-like tissue. These results suggest fundamental differences between stem cell-based (MSC) and primary cell-based (hCH) tissue engineering, as well as the importance of suitable scaffold features, in the optimization of cartilage-related outcomes in vitro. The present work diversifies cell sources in combination with silk fibroin-based tissue engineering applications. Together with our previous studies, the present results show great promise for engineered 3-D silk fibroin scaffolds in autologous cell-based skeletal tissue engineering.
Keywords: Chondrocyte; Mesenchymal stem cell; Cartilage tissue engineering; Silk fibroin scaffold; Silk;

Dense fibrillar collagen matrices: A model to study myofibroblast behaviour during wound healing by Christophe Helary; Ludmila Ovtracht; Bernard Coulomb; Gaston Godeau; Marie Madeleine Giraud-Guille (4443-4452).
Fibroblastic cells play an important part in wound healing. Human dermal fibroblasts seeded onto three-dimensional fibrillar collagen matrices migrate into the collagen network and differentiate into myofibroblasts. In order to evaluate the use of collagen matrices as model systems for studying myofibroblast phenotype during wound healing, myofibroblast behaviour migrating into dense or loose matrices was compared. The effect of collagen concentration on cell morphology, remodelling, proliferation and apoptosis of human myofibroblasts was evaluated. Myofibroblasts within dense collagen matrices (40 mg/ml) were spindle shaped, similar to cells observed during tissue repair. In contrast, cells within loose matrices (5 mg/ml) were more rounded. Matrix hydrolysis activities (MT1-MMP and MMP2) did not differ between the two collagen concentrations. The myofibroblast proliferation rate was measured after 24 h bromodeoxyuridine incorporation (BrdU). Cells in dense collagen matrices proliferated at a higher rate than cells in loose matrices at each culture time point tested. For example, 40% of cells in dense matrices were replicating compared to 10% of cells in loose matrices after 28 days in culture. Apoptotic cells were only detected in dense matrices from day 21 onwards when cells had already migrated into the collagen network. Taken together, these results show that a high collagen concentration has a stimulatory effect on myofibroblast proliferation and apoptosis, two important events in wound healing. Thus, dense matrices can be used to create controlled conditions to study myofibroblast phenotype.
Keywords: Myofibroblast; Collagen matrices; Migration; Metalloproteinases; Proliferation; Apoptosis;

The use of biodegradable microcarriers as initial supports for tissue engineering has been demonstrated to be advantageous for maintaining a differentiated cell phenotype; the high surface area also allows rapid cell expansion. Poly l-lactide (PLLA) is a significant member of a group of polymers regarded as bioresorbable and has been widely used for manufacturing 3D scaffolds for tissue engineering. In this study, the hypothesis that PLLA microspheres could be surface modified using RGD peptide sequences to improve the cell adhesion and function of those cells in contact with PLLA was tested. Using this type of approach it may be possible to generate larger structures that contain a high cell number relative to the amount of polymer, whilst remaining free from mass transport limitations. PLLA microspheres were prepared using an oil-in-water solvent-evaporation technique and then an RGD-motif was incorporated onto the microspheres surface by conjugation to improve cell attachment and function. Both PLLA and GRGDSPK modified PLLA microspheres were used as cell microcarriers for chondrocytes cultured in a flow intermittency bioreactor. At the same time, the degradation of the microspheres has been studied after 7, 14, 21, 28, 35, 49 and 56 days. The molecular weight of the PLLA microspheres was determined by Gel Permeation Chromatography. The morphology was assessed by scanning electron microscopy, and the thermal properties determined by Differential Scanning Calorimetry. It was demonstrated that the RGD modified and pure PLLA microspheres degraded gradually at a steady rate over the experimental period, which would provide a controlled degradation profile, both could serve as cell microcarriers because of their thermal and mechanical stabilities. The microspheres with RGD surface modification enhanced cell adhesion and increased the cell numbers in the microspheres aggregates.
Keywords: Poly(l-lactide); Microspheres; Cartilage; Biodegradation;

In this study, the behavior of neural stem cells from embryonic rat cerebral cortex were compared on the chitosan and poly(vinylidene fluoride) (PVDF) substrates at single-cell and neurosphere level. It was found that chitosan and PVDF substrates inhibited the proliferation and differentiation of single neural stem cells. It seemed that single-cell cultures on both substrates show cells remained dormant. However, neurospheres could exhibit different or similar behavior on these two substrates, which is dependent on the presence or absence of serum. More cells migrated outside from the neurospheres and longer processes extended from differentiated cells on chitosan than on PVDF when neurospheres were cultured in the serum-free medium. On the contrary, when serum was added to the culture system, chitosan and PVDF could induce the neurosphere-forming cells into an extensive cellular substratum of protoplasmic cells upon which process-bearing cells spread. In addition, based on the immunocytochemical analysis, the percentages of differentiated cell phenotypes of neurospheres cultured on chitosan and PVDF substrates became similar in the presence of serum. Therefore, it is reasonable to suggest that biomaterials may stimulate or inhibit the proliferation and differentiation of neural stem cells according to the complex environmental conditions. The information presented here should be useful for the development of biomaterials to regulate the preservation, proliferation, and differentiation of neural stem cells.
Keywords: Chitosan; Poly(vinylidene fluoride) (PVDF); Neural stem cells;

Reactivation of inactivated endogenous proteolytic activities in phosphoric acid-etched dentine by etch-and-rinse adhesives by Annalisa Mazzoni; David H. Pashley; Yoshihiro Nishitani; Lorenzo Breschi; Ferdinando Mannello; Leo Tjäderhane; Manuel Toledano; Edna L. Pashley; Franklin R. Tay (4470-4476).
Auto-degradation of collagen matrices occurs in resin-infiltrated dentine by the slow action of host-derived matrix metalloproteinases. As phosphoric acid-etching inactivates these endogenous enzymes, it is puzzling how hybrid layers created by simplified etch-and-rinse adhesives can degrade in vivo. This study tested the null hypothesis that there are no differences in the relative proteolytic activities of mineralised dentine, acid-etched dentine, and etch-and-rinse adhesivetreated acid-etched dentine. Powdered dentine prepared from extracted human teeth was treated with 17% EDTA, 10% phosphoric acid, or with five simplified etch-and-rinse adhesives that were applied to 10% phosphoric acid-etched dentine. The gelatinolytic activity of the dentine powder was assayed using fluorescein-labelled gelatine. TEM examination of the air-dried, treated dentine powder was performed to confirm the presence of remnant mineralised dentine after acid-etching. 17% EDTA significantly reduced the relative proteolytic activity (73.2%) of the untreated mineralised dentine powder (control), while 10% phosphoric acid-etched dentine exhibited the highest reduction (98.1%). Treating the acid-etched dentine powder with any of the five simplified etch-and-rinse adhesives resulted in the reactivation of the proteolytic activity, with a significant negative linear correlation ( P < 0.05 ) between the increases in fluorescence and the corresponding pH values of the adhesives. It is concluded that simplified etch-and-rinse adhesives can reactivate endogenous enzymatic activities in dentine that are previously inactivated by phosphoric acid-etching. The amount of enzyme reactivated may even exceed the original quantity present in untreated mineralised dentine. This provides an explanation for the degradation of hybrid layers after acid-etched dentine matrices are infiltrated with these adhesives.
Keywords: Fluorometric enzymatic assay; Acid-etched dentine; Etch-and-rinse adhesives; Proteolytic activity; Fluorescence; Collagen degradation;

PEGylated albumin-heme as an oxygen-carrying plasma expander: Exchange transfusion into acute anemia rat model by Yubin Huang; Teruyuki Komatsu; Hisashi Yamamoto; Hirohisa Horinouchi; Koichi Kobayashi; Eishun Tsuchida (4477-4483).
Poly(ethylene glycol) (PEG) conjugated recombinant human serum albumin (HSA) incorporating the synthetic iron-porphyrin (FeP) [PEGylated albumin-heme, PEG(HSA–FeP)] is a unique albumin-based oxygen carrier as a red blood cell (RBC) substitute. The physiological responses to an exchange transfusion with PEG(HSA–FeP) into an acute anemia rat model were investigated. After a 65% isovolemic hemodilution with HSA, a 30% volume of the circulatory blood was withdrawn, affording a hemorrhaged state. The circulation parameters, blood parameters, renal cortical oxygen partial pressure [PtO2(R)], and muscle tissue oxygen partial pressure [PtO2(M)] were continuously monitored. The intravenous infusion of PEG(HSA–FeP) restored the reduced levels of the mean arterial pressure, heart rate, respiration rate, mixed venous PO2, and arterial PCO2. The increased arterial PO2 and pH also returned to their basal values. These effects were almost to the same extent as those observed after the administration of the RBC suspension. The relatively low recovery in PtO2(R) and PtO2(M) might be due to the Langmuir-type oxygen binding profile of PEG(HSA–FeP) (Hill coefficient: 1.0). All the animals survived during the experiments. In contrast, those injected with HSA died within 41 min. The PEG(HSA–FeP) solution is an oxygen-carrying plasma expander which can be used as a resuscitative fluid for hemorrhagic shock.
Keywords: Albumin; Biomimetic material; Blood; In vivo test; Oxygenation; Poly(ethylene glycol);

The effect of various forms of sterilisation on a novel thermoplastic polyurethane elastomer synthesised using poly(hexamethylene oxide) (PHMO) and poly(dimethylsiloxane) (PDMS) macrodiols has been studied. The five sterilisation methods investigated were ethylene oxide (EtO) (single and multiple cycles), gas plasma, steam, vapour phase liquid chemical and γ-irradiation (single and multiple cycles). Following sterilisation, scanning electron microscopy (SEM) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) were used to assess changes in the surface chemical structure and morphology, and gel permeation chromatography (GPC) and tensile testing were used to examine changes in bulk characteristics. Biostability was assessed using subcutaneous implantation of strained samples in sheep for 6 weeks. The results showed that the properties of the commercially available control material, Pellethane® 2363-80A, were significantly affected by exposure to γ-irradiation, steam and multiple cycles of EtO with aging and implantation compounding the effect. Exposure to a gas plasma sterilisation process resulted in significant degradation in both polyurethanes. A vapour phase liquid chemical sterilisation process caused minimal adverse effects. Sterilisation of the PDMS-based polyurethane using EtO, γ-irradiation and autoclaving resulted in no significant changes in properties. The material's biostability was also unaffected by exposure to each of these sterilisation processes followed by short-term implantation suggesting that this material is a potential candidate for use in a wide range of implantable medical devices sterilised using commercially available processes. Further biostability studies should be performed to assess the longer-term in vivo biostability of the PDMS-based material sterilised using autoclaving and γ-irradiation.
Keywords: Polyurethanes; Poly (dimethylsiloxane); Degradation; Biostability; Environmental stress cracking (ESC); Sterlisation;

Formulations of dextran methacrylate (dex-MA) and concanavalin A methacrylamide (con A-MA) were photopolymerised to produce covalently cross-linked glucose-responsive materials for the basis of a closed-loop insulin delivery device. The viscoelastic properties of these polymerised materials were tested rheologically in the non-destructive oscillatory mode within the linear viscoelastic range at glucose concentrations between 0% and 5% w/w. The degree of acrylic substitution was varied for the dex-MA and con A-MA, and as the formulation glucose concentration was raised, a graded decrease in storage modulus, loss modulus and complex viscosity when compared at 1 Hz was observed for each cross-linked material. Increasing the degree of substitution (DS) of the derivatised dextran produced viscosity profiles at higher values throughout the glucose concentration range. A comparison with non-polymerised mixtures shows similar rheological properties but at much lower values across the chosen glucose concentration range. High-pressure liquid chromatography analyses and in vitro diffusion experiments showed that there were optimum degrees of derivatisation to minimise dex-MA and con A-MA component leach from the material. The in vitro diffusion experiments also showed that differential delivery of insulin in response to glucose was possible with candidate polymerised glucose-responsive formulations, thus highlighting the potential of such a novel glucose-sensitive material to be used as part of implantable closed-loop insulin delivery device.
Keywords: Acrylics; Dextran; Diabetes; Drug delivery; Polymerisation; Viscoelasticity;

Crystal structure characterization of nautilus shell at different length scales by Rodrigo Rafael Velázquez-Castillo; Jose Reyes-Gasga; Domingo I. García-Gutierrez; Miguel Jose-Yacaman (4508-4517).
In this work, we studied the shell structure of the Nautilus pompilius Linnaeus by using analytical techniques of scanning (SEM) and transmission electron microscopy (TEM) and X-ray diffraction. The main objective of this study is the structural characterization of Nautilus shell at different length levels, from micron to nano-scale. The results were also used to try to determine the shell structure mechanism of formation. The information obtained in this work will place our particular knowledge a closer step to understand how self-assembly works in nature, and will increase the opportunities of using this information in the future synthesis of new advanced materials.
Keywords: Nautilus shell; SEM; TEM; HRTEM; X-ray diffraction;