Biomaterials (v.29, #1)

Heparin intercalation into reconstituted collagen I fibrils: Impact on growth kinetics and morphology by Dimitar Stamov; Milauscha Grimmer; Katrin Salchert; Tilo Pompe; Carsten Werner (1-14).
Collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape. A detailed structural analysis by scanning force and scanning electron microscopy revealed a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of glycosaminoglycan (GAG) concentrations. By varying molecular weight, degree of sulphation, charge, and concentration of different GAGs we are able to correlate the morphological data with kinetic turbidimetric measurements, and quantitation of fibril-bound GAG. The experiments imply a pronounced impact of the prenucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with tropocollagen. Heparin is assumed to stabilize the collagen microfibrils and to enhance their parallel accretion during cofibrillogenesis with preservation of the typical asymmetric collagen banding pattern. The heparin quantitation data show heparin to be intercalated as a linker molecule with one specific binding site inside the cofibrils. The reconstituted cofibrils with their unusual morphology and GAG intercalation—a phenomenon not reported in vivo—can be expected to exhibit interesting mechanical and biochemical behaviours as a biomaterial for extracellular matrix scaffolds.
Keywords: Collagen; Heparin; Glycosaminoglycan; Molecular weight; ECM; Charge;

A multifunctional and reversibly polymerizable carrier for efficient siRNA delivery by Xu-Li Wang; Thanh Nguyen; David Gillespie; Randy Jensen; Zheng-Rong Lu (15-22).
In this study a multifunctional carrier (MFC), 1,4,7-triazanonylimino-bis[N-(oleicyl-cysteinyl-histinyl)-1-aminoethyl)propionamide] (THCO), containing protonatable amines of different pK as, polymerizable cysteine residues and hydrophobic groups, was designed, synthesized and evaluated for efficient small interfering RNAs (siRNA) delivery. THCO showed pH-sensitive cellular membrane disruption at the endosomal–lysosomal pH to facilitate intracellular siRNA delivery. THCO formed stable and compact nanoparticles with siRNA through charge complexation, hydrophobic condensation and reversible polymerization. The THCO/siRNA nanoparticles were readily modified with PEG-Mal by reacting with remaining thiol groups at the surface. The siRNA delivery efficiency of THCO was comparable to that of Transfast™, much higher than that of N-(2,3-dioleoyloxy-1-propyl)trimethylammonium methyl sulphate (DOTAP) in serum-free medium. PEGylated THCO/siRNA nanoparticles resulted in higher transfection efficiency than those of Transfast™ and DOTAP in the presence of serum. This study demonstrated that the MFC-THCO is promising for efficient siRNA delivery.
Keywords: RNAi; THCO; Multifunctional siRNA carrier; Nanoparticles; Surface modification;

Chondrogenic differentiation of human mesenchymal stem cells on photoreactive polymer-modified surfaces by Likun Guo; Naoki Kawazoe; Yujiang Fan; Yoshihiro Ito; Junzo Tanaka; Tetsuya Tateishi; Xingdong Zhang; Guoping Chen (23-32).
Human mesenchymal stem cells (MSCs) were cultured on polystyrene surfaces modified with photoreactive azidophenyl-derivatives of three different chargeable polymers, poly(acrylic acid) (PAAc), polyallylamine (PAAm), and poly(ethylene glycol) (PEG). The MSCs adhered and spread both on a PAAm-modified surface and on PAAc-modified and polystyrene (control) surfaces. However, the cells adhered more easily to the PAAm-modified surface. The MSCs did not attach to the PEG-modified surface and aggregated to form pellets immediately after cell seeding. The cells proliferated on the PAAc-, PAAm-modified and control surfaces with culture time, formed a monolayer, and aggregated to form pellets. The cells in the pellets that formed on the PAAm- and PEG-modified surfaces after 2 weeks culture had a round morphology and the extracellular matrices were positively stained by safranin O and toluidine blue, while those that formed on the PAAc-modified and control surfaces had a spindle, fibroblast-like morphology and were not positively stained by safranin O and toluidine blue. The pellets that formed on the PAAm- and PEG-modified surfaces contained significantly higher levels of sulfated glycosaminoglycans than did those that formed on the PAAc-modified and control surfaces. Type II collagen and cartilage proteoglycan were immunohistologically detected in the pellets that formed on PAAm- and PEG-modified surfaces, but not those that formed on the PAAc-modified and control surfaces. The MSCs cultured on the PAAm- and PEG-modified surfaces expressed a high level of cartilaginous genes encoding type II collagen and aggrecan, while the MSCs cultured on the PAAc-modified and control surfaces did not express these genes. These results suggest that the PAAm-modified surface supported cell adhesion and proliferation and also promoted chondrogenic differentiation of the MSCs. The PAAc-modified and polystyrene surfaces supported cell adhesion and proliferation, but not chondrogenic differentiation. The PEG-modified surfaces did not support cell adhesion, but did promote chondrogenic differentiation. The adhesion, proliferation, and differentiation of the MSCs could be controlled by surface chemistry.
Keywords: Mesenchymal stem cells; Chondrogenic differentiation; Surface modification; Surface grafting; Surface property;

Anisotropic scaffolds of agarose hydrogels containing gradients of laminin-1 (LN-1) and nerve growth factor (NGF) molecules were used to promote sciatic nerve regeneration across a challenging 20 mm nerve gap in rats. Step and continuous gradient anisotropic scaffolds were fabricated and characterized, and regeneration was compared to that in isotropic scaffolds with uniform concentrations of LN-1 and NGF and sciatic nerve grafts harvested from syngenic rats. Polysulfone tubular guidance channels were used to present the agarose-based scaffolds to the nerve stumps. Four months after implantation, regenerating axons were observed in animals implanted with anisotropic scaffolds with gradients of both LN-1 and NGF molecules and nerve grafts, but not in animals with isotropic scaffold implants. Also, the scaffolds with gradients of either LN-1 or NGF, with the other component being uniformly distributed in the scaffold, did not elicit axonal regeneration. The total number of myelinated axons was similar for the anisotropic scaffold and the nerve graft conditions, with the anisotropic scaffolds having a higher density of axons than the nerve grafts. Axonal diameter distribution was similar for the anisotropic scaffolds and the nerve grafts. The nerve grafts and anisotropic scaffolds resulted in better functional outcome compared to isotropic scaffolds as measured by the relative gastrocnemius muscle weight (RGMW). Additionally the state of neuromuscular junctions as assessed by pre- and post-synaptic staining revealed that both the anisotropic scaffolds performed as well as nerve grafts.
Keywords: Agarose; Laminin; Nerve growth factor; Nerve regeneration; Protein gradients;

Characterisation of a soft elastomer poly(glycerol sebacate) designed to match the mechanical properties of myocardial tissue by Qi-Zhi Chen; Alexander Bismarck; Ulrich Hansen; Sarah Junaid; Michael Q. Tran; Siân E. Harding; Nadire N. Ali; Aldo R. Boccaccini (47-57).
The myocardial tissue lacks significant intrinsic regenerative capability to replace the lost cells. Therefore, the heart is a major target of research within the field of tissue engineering, which aims to replace infarcted myocardium and enhance cardiac function. The primary objective of this work was to develop a biocompatible, degradable and superelastic heart patch from poly(glycerol sebacate) (PGS). PGS was synthesised at 110, 120 and 130 °C by polycondensation of glycerol and sebacic acid with a mole ratio of 1:1. The investigation was focused on the mechanical and biodegrading behaviours of the developed PGS. PGS materials synthesised at 110, 120 and 130 °C have Young's moduli of 0.056, 0.22 and 1.2 MPa, respectively, which satisfy the mechanical requirements on the materials applied for the heart patch and 3D myocardial tissue engineering construction. Degradation assessment in phosphate buffered saline and Knockout™ DMEM culture medium has demonstrated that the PGS has a wide range of degradability, from being degradable in a couple of weeks to being nearly inert. The matching of physical characteristics to those of the heart, the ability to fine tune degradation rates in biologically relevant media and initial data showing biocompatibility indicate that this material has promise for cardiac tissue engineering applications.
Keywords: Myocardial tissue engineering; Heart patch; Poly(glycerol sebacate); Mechanical property; Degradation; Biocompatibility;

Genetic manipulation of human mesenchymal progenitors to promote chondrogenesis using “bead-in-bead” polysaccharide capsules by Jodie C. Babister; Rahul S. Tare; David W. Green; Stefanie Inglis; Stephen Mann; Richard O.C. Oreffo (58-65).
Articular cartilage defects arising from trauma or degenerative diseases fail to repair spontaneously. We have adopted a non-viral gene delivery and tissue engineering strategy, in which Sox-9 transfected human mesenchymal progenitors have been encapsulated within alginate/chitosan polysaccharide capsules to promote chondrogenesis. Human bone marrow stromal cells and articular chondrocytes were transfected with flag-tagged Sox-9 plasmid and after 7 days in static culture, large regions of cell-generated matrix containing cartilage proteoglycans were observed as confirmed by positive Alcian blue staining and Sox-9 immunohistochemistry. Further, after 28 days, in vitro and in vivo, samples encapsulated with Sox-9 transfected cells demonstrated large regions of cartilaginous matrix as confirmed by positive Alcian blue staining, Sox-9 and type-II collagen immunohistochemistry, absent in samples encapsulated with untransfected cells. Extracted protein from in vivo constructs was further assessed by western blot analysis and positive expression of Sox-9 and type-II collagen was observed in Sox-9 transfected constructs which was absent in untransfected cells. Regions of cartilage-like matrix were significantly increased in Sox-9 constructs in comparison with untransfected constructs, confirming Sox-9 gene delivery enhances chondrogenesis in targeted cell populations, outlining the potential to promote cartilaginous construct formation with therapeutic implications for regeneration of human articular cartilage tissue defects.
Keywords: Cartilage; Alginate; Polysaccharide; Gene transfer; Gene expression;

The effect of cross-linking of collagen matrices on their angiogenic capability by Chang Yao; Marta Markowicz; Norbert Pallua; Ernst Magnus Noah; Guy Steffens (66-74).
The poor vascularization rate of matrices following cell invasion is considered to be one of the main shortcomings of scaffolds used in tissue engineering. In the past decade much effort has been directed towards enhancing the angiogenic potential of biomaterials. A great many studies have appeared reporting about enhancement of vascularization by immobilizing angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-2 (FGF-2). We have also tried to achieve this goal by modifying collagen matrices by covalent incorporation of heparin into the matrices and loading them with VEGF. We and others have observed that loading angiogenic factors to heparinized materials markedly increases angiogenic capacity. In the present paper we also investigated the angiogenic properties of collagen matrices which were only cross-linked, i.e. in the absence of heparin. The angiogenic capacity of the modified matrices was evaluated using the chorioallantoic membrane assay. Differences in angiogenic potential were deduced from macroscopic and microscopic analyses of the chorioallantoic membrane, as well as from dry weight changes. Cross-linked only matrices and matrices both cross-linked and heparinized appeared to show a significantly larger angiogenic potential than unmodified matrices. As previously observed, loading VEGF to these matrices further stepped up angiogenic potential. Quite surprisingly, cross-linking had a substantial impact on angiogenic potential. In terms of magnitude, this effect was similar to the effect of loading VEGF to heparinized matrices. Both modification procedures resulted in an increase of average pore size within the collagen matrices, and this observation may explain the more rapid invasion of mouse fibroblasts into cross-linked and heparinized matrices. Form changes of the implants were also monitored during the in vivo contacts: cross-linked and heparinized matrices showed far better resistance against contraction, as compared to unmodified matrices.Results from the chorioallantoic membrane assay experiments were compared with data obtained from rat model experiments, which confirmed the results from the chorioallantoic membrane assay. This relatively simple assay was again shown to be extremely helpful in evaluating and predicting the angiogenic capabilities of biomaterials for use in tissue engineering and wound healing.
Keywords: Collagen; Angiogenesis; Cross-linking; Heparin; VEGF;

Growth, differentiation, transplantation and survival of human skeletal myofibers on biodegradable scaffolds by Lieven Thorrez; Janet Shansky; Lin Wang; Loren Fast; Thierry VandenDriessche; Marinee Chuah; David Mooney; Herman Vandenburgh (75-84).
Skeletal muscle transplantation strategies for muscle repair or gene therapy involve either the injection of proliferating myoblasts followed by fusion with host myofibers or implantation of ex vivo differentiated myofibers; however, both implant procedures are associated with significant cell loss. Biodegradable porous, gas-foamed poly-lactide-co-glycolide (PLG) scaffolds have desirable characteristics for cell transfer and were used to study attachment, growth, differentiation and survival of human myogenic cells. Primary human myoblasts suspended in clinical grade extracellular matrixes (ECMs) and adhered to PLG scaffolds differentiated in vitro into high-density tropomyosin positive myofibers. An immunodeficient non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse implant model was used to study the transfer and in vivo survival of differentiated human myofibers on these scaffolds. Scaffold rigidity allowed the myofibers to be maintained under tension in vitro and following subcutaneous transplantation in vivo. Following implantation, myofiber density on the PLG scaffolds decreased linearly by 78% over a 4-week period. ECM composed of either Tisseel® fibrin or Zyderm® collagen type I did not significantly affect in vivo cell viability over the 4-week period. Varying PLG scaffold microsphere content (10–100%) also had little effect on cell survival in vivo. In contrast, when the residual NK cell population in the immunodeficient NOD/SCID mouse model was depleted with anti-asialo GM1 (ASGM1) antiserum, in vivo cell survival significantly increased from 22% to 34% after 4 weeks. With further improvements in cell survival, PLG scaffolds may prove useful for the implantation of primary human myofibers in future clinical applications.
Keywords: Cell viability; Muscle; Biodegradation; Tension; Poly-lactide-co-glycolide; Cell differentiation;

Human mesenchymal stem cell differentiation to NP-like cells in chitosan–glycerophosphate hydrogels by Stephen M. Richardson; Nesta Hughes; John A. Hunt; Anthony J. Freemont; Judith A. Hoyland (85-93).
Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, interest in methods focused on biological repair has increased. Several tissue engineering approaches using different cell types and hydrogels/scaffolds have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering attention has focused on the use of mesenchymal stem cells (MSCs). Additionally, while rigid scaffolds have been demonstrated to allow MSC differentiation to the chondrocyte-like cells of the IVD, hydrogels are being increasingly studied as they allow minimally invasive implantation without extensive damage to the IVD. Here, we have studied the temperature-sensitive hydrogel chitosan–glycerophosphate (C/Gp), seeded with human MSCs and cultured for 4 weeks in standard medium. We have analysed the gene and protein expression profile of the MSCs and compared it to that of both nucleus pulposus (NP) cells and articular chondrocytes cultured in C/Gp. Gene expression analysis for chondrocytic-cell marker genes demonstrated differentiation of MSCs to a phenotype which showed similarities to both articular chondrocytes and NP cells. Conventional PCR demonstrated a lack of expression of osteogenic marker genes and the hypertrophic marker gene type X collagen. MSCs also secreted both proteoglycans and collagens in a ratio, which more closely resembled that of NP cells than articular chondrocytes. These results therefore suggest that MSC-seeded C/Gp gels could be used clinically for the regeneration of the degenerate human IVD.
Keywords: Mesenchymal stem cell; Nucleus pulposus; Chondrocyte-like; Chitosan glycerophosphate; Differentiation;

Multiwall carbon nanotube scaffolds for tissue engineering purposes by Ander Abarrategi; María C. Gutiérrez; Carolina Moreno-Vicente; María J. Hortigüela; Viviana Ramos; José L. López-Lacomba; María L. Ferrer; Francisco del Monte (94-102).
The use of scaffolds composed of a major fraction of multiwall carbon nanotubes (MWCNT, up to 89 wt.%) and a minor one of chitosan (CHI), and with a well-defined microchannel porous structure as biocompatible and biodegradable supports for culture growth is described. Cell adhesion, viability and proliferation onto the external surface of MWCNT/CHI scaffolds with C2C12 cell line (myoblastic mouse cell), which is a multipotent cell line able to differentiate towards different phenotypes under the action of some chemical or biological factors, has been evaluated in vitro and quantified by MTT assays. The evolution of the C2C12 cell line towards an osteoblastic lineage in presence of the recombinant human bone morphogenetic protein-2 (rhBMP-2) has also been studied both in vitro (e.g., following the appearance of alkaline phosphatase activity) and in vivo (e.g., by implantation of MWCNT/CHI scaffolds adsorbed with rhBMP-2 in muscle tissue and evaluation of the ectopic formation of bone tissue).
Keywords: Scaffolds; Freeze-drying; Bone tissue engineering; rhBMP-2;

Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid—An experimental study in rats by Henning Schliephake; Herbert A. Weich; Christian Dullin; Rudolf Gruber; Sarah Frahse (103-110).
The aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-dl-lactic acid discs were produced and loaded with 48 and 96 μg rhBMP-2 and inserted into non-healing defects of the mandible of 45 wistar rats. Fifteen rats received implants with 96 μg rhBMP-2 (Group 2), 48 μg rhBMP-2 (Group 1) and blank implants without BMP (Group 0) each on one side of the mandible. Unfilled defects of the same size on the contralateral sides of the mandibles served as empty controls. After 6, 13 and 26 weeks, implants of each group were retrieved from five animals each and submitted to flat panel detector computed tomography. Bone formation and thickness of augmentation was assessed by computer-assisted histomorphometry. In Group 2 significantly more bone was produced than in Group 1. Implants of Group 1 induced significantly more bone than the blank controls only after 6 weeks, whereas the difference was not significant after 13 and 26 weeks. Differences between Group 2 and Group 1 were clearly significant after 26 weeks. The thickness of bone tissue was maintained in Group 2 whereas it decreased in Group 1 and was negligible in Group 0. It is concluded that the PLA implants with 96 μg rhBMP-2 were able to bridge a non-healing defect in the rat mandible and maintained the thickness of an augmented volume. However, continuous supply of osteogenic signals appears to be required to compensate for adverse effects during polymer degradation.
Keywords: Bone morphogenetic proteins (BMP); Bone tissue engineering; Drug delivery; Polylactic acid; Recombinant protein;

The effect of conjugation to gold nanoparticles on the ability of low molecular weight chitosan to transfer DNA vaccine by Xianfeng Zhou; Xizhen Zhang; Xianghui Yu; Xiao Zha; Qiuan Fu; Bin Liu; Xueyun Wang; Yan Chen; Yue Chen; Yaming Shan; Yinghua Jin; Yongge Wu; Junqiu Liu; Wei Kong; Jiacong Shen (111-117).
Nonviral gene delivery systems based on conventional high molecular weight chitosans are efficient as DNA vaccine delivery system, but have poor physical properties such as aggregated shapes, low solubility at neutral pH, high viscosity at concentrations used for in vivo delivery and a slow onset of action. Furthermore, Chitosan oligomers shorter than 14 monomers units were recently found to form only weak complexes with DNA, resulting in physically unstable polyplexes in vitro and in vivo. Here, low molecular weight chitosans with an average molecular mass of 6 kDa (Chito6) have been covalently attached to gold nanoparticles (GNPs), and the potency of the resulting Chito6-GNPs conjugates as vectors for the delivery of plasmid DNA has been investigated in vitro and in vivo. After delivery by intramuscular immunization in BALB/c mice, the Chito6-GNPs conjugates induced an enhanced serum antibody response 10 times more potent than naked DNA vaccine. Additionally, in contrast to naked DNA, the Chito6-GNPs conjugates induced potent cytotoxic T lymphocyte responses at a low dose.
Keywords: Chitosan modified gold nanoparticle; DNA vaccination; Gene delivery; Immunogenicity; Nanocarriers;

The use of keratin biomaterials derived from human hair for the promotion of rapid regeneration of peripheral nerves by Paulina Sierpinski; Jeffrey Garrett; Jianjun Ma; Peter Apel; David Klorig; Thomas Smith; L. Andrew Koman; Anthony Atala; Mark Van Dyke (118-128).
The management of trauma-associated nerve defects is difficult because of the absence of autologous donor motor or sensory nerves. Pre-clinical development and clinical experience has shown that damaged nerves can be surgically repaired using a tubular conduit interposed across the defect. Acceptable patient outcomes are achieved so long as the gap distance does not exceed a few centimeters. Although research in animals has demonstrated that nerve repair can be facilitated across slightly larger gaps by introducing a biomaterial filler into the conduit lumen, these biomaterials are not typically “neuroinductive” (i.e. capable of acting directly on regenerative cells to enhance nerve tissue formation beyond clinical limits). Moreover, their use does not often result in functional recovery equivalent to nerve autograft, the clinical gold standard. Here we show that a biomaterial gel made from the proteins found in human hair can mediate a robust nerve regeneration response, in part through activation of Schwann cells. In vitro, keratins extracted from human hair enhance the activity of Schwann cells by a chemotactic mechanism, increase their attachment and proliferation, and up-regulate expression of important genes. Moreover, these characteristics translate to improved functional nerve recovery in an animal model. These results suggest that a biomaterial derived from human hair keratins is neuroinductive and can facilitate an outcome comparable to autograft in a nerve injury model.
Keywords: Nerve regeneration; Nerve guide; ECM; Schwann cell; Keratin;