Biomaterials (v.28, #20)

Si–C linked oligo(ethylene glycol) layers in silicon-based photonic crystals: Optimization for implantable optical materials by Kristopher A. Kilian; Till Böcking; Katharina Gaus; Michael Gal; J. Justin Gooding (3055-3062).
Porous silicon has shown potential for various applications in biology and medicine, which require that the material (1) remain stable for the length of the intended application and (2) resist non-specific adsorption of proteins. Here we explore the efficacy of short oligo(ethylene glycol) moieties incorporated into organic layers via two separate strategies in achieving these aims. In the first strategy the porous silicon structure was modified in a single step via hydrosilylation of α-oligo(ethylene glycol)-ω-alkenes containing three or six ethylene glycol units. The second strategy employs two steps: (1) hydrosilylation of succinimidyl-10-undecenoate and (2) coupling of an amino hexa(ethylene glycol) species. The porous silicon photonic crystals modified by the two-step strategy displayed greater stability relative to the single step procedure when exposed to conditions of physiological temperature and pH. Both strategies produced layers that resist non-specific adsorption of proteins as determined with fluorescently labelled bovine serum albumin. The antifouling behaviour and greater stability to physiological conditions provided by this chemistry enhances the suitability of porous silicon for biomaterials applications.
Keywords: Porous silicon; Photonic crystals; Self-assembled monolayers; Protein resistance; Oligo(ethylene glycol);

3-hydroxybutyrate (3HB), one of the degradation products of microbial biopolyesters polyhydroxyalkanoates (PHA), is a high energy metabolic substrate in animals. This study evaluated the effects of 3HB on growth of osteoblasts in vitro and on anti-osteoporosis in vivo. Alkaline phosphatase (ALP) assay, Van Kossa assay and Alizarin S red staining were used to study in vitro differentiation of murine osteoblast MC3T3-E1 cells. The intensity of in vitro cell differentiation measured in ALP was in direct proportion to the concentration of 3HB when it was lower than 0.01 g/L. Calcium deposition, a strong indication of cell differentiation, also showed an obvious increase with increasing 3HB concentration from 0–0.1 g/L, evidenced by Alizarin red S staining and Van Kossa assay. RT-PCR also showed significantly higher expression of osteocalcin (OCN) mRNA in MC3T3-E1 cells after 3HB administration. In vivo study using female Wistar rats (3 months old, n=80) allocated into normal, sham-operated or ovariectomized (OVX) group that led to decreasing bone mineral density (BMD), bone histomorphometry and biomechanics compared with normal and sham groups, had demonstrated that 3HB increased serum ALP activity and calcium deposition, decreased serum OCN, prevented BMD reduction resulting from OVX. All these led to enhanced femur maximal load and bone deformation resistance, as well as improved trabecular bone volume (TBV%). In conclusion, 3HB monomer containing PHA can be effective bone growth stimulating implant materials.
Keywords: 3-hydroxybutyrate; PHA; Osteoblast; Osteoporosis; Polyhydroxyalkanoate;

Self-assembled monolayers (SAMs) of alkanethiols, which can provide flat and chemically well-defined surfaces, were employed as model surfaces to understand cellular interaction with artificial materials. SAMs presenting a wide range of wettabilities were prepared by mixing two kinds of alkanethiols carrying terminal methyl (CH3), hydroxyl (OH), carboxylic acid (COOH), or amino (NH2) groups. Adhesion behavior of human umbilical vein endothelial cells (HUVECs) and HeLa cells on these mixed SAMs were examined. The number of adhered HUVECs reached a maximum on CH3/OH mixed SAMs with a water contact angle of 40°, while cell adhesion increased with decreasing water contact angle up to 60–70° and then leveled off on CH3/COOH and CH3/NH2 mixed SAMs. Numbers of adhered HeLa cells showed a maximum on CH3/OH and CH3/COOH mixed SAMs with a water contact angle of 50°. These facts suggest that cell adhesion is mainly determined by surface wettability, but is also affected by the surface functional group, its surface density, and the kinds of cells. The effect of exchange of adsorbed proteins on cell adhesion was also examined. HUVECs were cultured on the mixed SAMs preadsorbed with albumin. Cell adhesion was effectively prohibited on hydrophobic SAMs pretreated with albumin. Albumin strongly adsorbed and resisted replacement by cell adhesive proteins on hydrophobic SAMs. On the other hand, cells adhered to albumin-adsorbed hydrophilic SAMs. Displacement of preadsorbed albumin with cell adhesive proteins effectively occurs on these hydrophilic SAMs. This effect contributes to induce SAMs with moderate wettability to give suitable surfaces for cell adhesion.
Keywords: Cell adhesion; Protein adsorption; Wettability; Thiol; Self-assembly;

Assembled alginate/chitosan nanotubes for biological application by Yang Yang; Qiang He; Li Duan; Yue Cui; Junbai Li (3083-3090).
Biodegradable nanotubes were fabricated through the layer-by-layer (LbL) assembly technique of alternate adsorption of alginate (ALG) and chitosan (CHI) onto the inner pores of polycarbonate template with the subsequent removal of the template. The assembled materials present good film formation ability. The thickness of nanotubes wall can be controlled by changing the assembled layers. The assembled tubular structure was verified by the confocal laser scanning microscope (CLSM) using fluorescent-labeled ALG as well as the measurements of scan electron microscope (SEM) and transmission electron microscopy (TEM). Atomic force microscopy (AFM) images confirm the biodegradable feature of the assembled nanotubes as they are immersed in the pancreatin. Confocal microscopy images show that the assembled ALG/CHI nanotubes can be internalized into the cancer cell readily. The cell viability experiment proves the low cytotoxicity of ALG/CHI nanotubes. The final assembled nanotubes have presented good biodegradability and low cytotoxicity.
Keywords: Template synthesis; Nanotubes; Biodegradability; Layer-by-layer assembly; Chitosan; Alginate;

Osteochondral repair using the combination of fibroblast growth factor and amorphous calcium phosphate/poly(l-lactic acid) hybrid materials by Xin Huang; Disheng Yang; Weiqi Yan; Zhongli Shi; Jie Feng; Yanbo Gao; Wenjian Weng; Shigui Yan (3091-3100).
A novel amorphous calcium phosphate (ACP)/poly(l-lactic acid) (PLLA) material, which can experience morphological variations in the microstructure is supposed to be a suitable candidate as scaffold for cartilage tissue-engineering. The purpose of this study was to evaluate the efficacy of this scaffold combined with basic fibroblast growth factor (bFGF) to repair articular cartilage defects in a rabbit model. Forty-two osteochondral defects created in the femoral condyles were (a) left untreated, (b) treated by PLLA combined with bFGF, or (c) ACP/PLLA loaded with bFGF. The treatment of PLLA incorporated with bFGF improved defect filling compared with that left untreated, while the regenerated tissue was mainly fibrocartilage and showed little bone formation with only a small amount of collagen type II (Col II) and no aggrecan gene message measured. When implanted with ACP/PLLA and bFGF, most of the defects were filled with a well-established layer of cartilage tissue with abundance of cartilaginous extracellular matrix accumulation observed. Positive immunohistochemical staining of Col II was observed. High levels of Col II and aggrecan message were also detected by RT-PCR. These results indicate the feasibility of using the combination of ACP/PLLA with bFGF for cartilage repair.
Keywords: Cartilage tissue engineering; Amorphous calcium phosphate; Polylactic acid; Basic fibroblast growth factor; Gene expression;

The performance of poly-ε-caprolactone scaffolds in a rabbit femur model with and without autologous stromal cells and BMP4 by L. Savarino; N. Baldini; M. Greco; O. Capitani; S. Pinna; S. Valentini; B. Lombardo; M.T. Esposito; L. Pastore; L. Ambrosio; S. Battista; F. Causa; S. Zeppetelli; V. Guarino; P.A. Netti (3101-3109).
The ability of a cellular construct to guide and promote tissue repair strongly relies on three components, namely, cell, scaffold and growth factors. We aimed to investigate the osteopromotive properties of cellular constructs composed of poly-ε-caprolactone (PCL) and rabbit bone marrow stromal cells (BMSCs), or BMSCs engineered to express bone morphogenetic protein 4 (BMP4). Highly porous biodegradable PCL scaffolds were obtained via phase inversion/salt leaching technique. BMSCs and transfected BMSCs were seeded within the scaffolds by using an alternate flow perfusion system and implanted into non-critical size defects in New Zealand rabbit femurs. In vivo biocompatibility, osteogenic and angiogenic effects induced by the presence of scaffolds were assessed by histology and histomorphometry of the femurs, retrieved 4 and 8 weeks after surgery. PCL without cells showed scarce bone formation at the scaffold–bone interface (29% bone/implant contact and 62% fibrous tissue/implant contact) and scarce PCL resorption (16%). Conversely, PCL seeded with autologous BMSCs stimulated new tissue formation into the macropores of the implant (20%) and neo-tissue vascularization. Finally, the BMP4-expressing BMSCs strongly favoured osteoinductivity of cellular constructs, as demonstrated by a more extensive bone/scaffold contact.
Keywords: Poly-ε-caprolactone; Bone marrow stromal cells; BMP4 infection; Bone tissue engineering; In vivo test;

Bone marrow mesenchymal stromal cells (BM-MSC) are attractive candidates for connective tissue regeneration. Currently, their use is limited by poor overall cell survival and high apoptosis rates upon transplantation in vivo. We hypothesized that disruption of cell–extracellular matrix contact either during cell expansion or immediately prior to cell transplantation may impair cell viability and facilitate apoptosis. We therefore investigated whether BM-MSC can be expanded on microcarrier beads in spin culture and directly transplanted. This novel approach removes the need for the repeated trypsinizations that are usually required for expansion and transplantation. CultiSpher-S gelatin microcarrier beads supported Fisher and transgenic green fluorescent protein (GFP)+ Sprague Dawley rat BM-MSC expansion. Bead-expanded BM-MSC could still be differentiated along the chondrogenic, osteogenic and adipogenic lineages. In the short term, direct subcutaneous transplantation of cells expanded on microcarriers was associated with significantly less apoptosis than trypsinized control cells. In the long term, BM-MSC expanded on microcarrier beads induced de novo trabecular bone formation in vivo. This novel approach present several advantages over current expansion–transplantation protocols for mesenchymal tissue regeneration.
Keywords: Mesenchymal stromal cell; Gelatin microcarrier; Bone healing; Apoptosis; Transplantation; Cell culture;

High lubricious surface of cobalt–chromium–molybdenum alloy prepared by grafting poly(2-methacryloyloxyethyl phosphorylcholine) by Masayuki Kyomoto; Yasuhiko Iwasaki; Toru Moro; Tomohiro Konno; Fumiaki Miyaji; Hiroshi Kawaguchi; Yoshio Takatori; Kozo Nakamura; Kazuhiko Ishihara (3121-3130).
Osteolysis caused by wear particles from polyethylene in artificial hip joints is of great concern. Various bearing couple combinations, bearing material improvements, and surface modifications have been attempted to reduce such wear particles. With the aim of reducing the wear and developing a novel artificial hip-joint system, we created a highly lubricious metal-bearing material: A 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer was grafted onto the surface of the cobalt–chromium–molybdenum (Co–Cr–Mo) alloy. For ensuring the long-term retention of poly(MPC) on the Co–Cr–Mo alloy, we used a 4-methacryloxyethyl trimellitate anhydride (4-META) intermediate layer and photo-induced graft polymerization technique to create a strong bonding between the Co–Cr–Mo substrate and the poly(MPC) chain via the 4-META layer. The Co–Cr–Mo alloy was pretreated with nitric acid and O2 plasma to facilitate efficient interaction between the 4-META carboxyl group and the surface hydroxyl group on the Cr oxide passive layer of the Co–Cr–Mo alloy. After MPC grafting, the MPC unit peaks were clearly observed in the Fourier-transform infrared spectroscopy with attenuated total reflection (FT-IR/ATR) and X-ray photoelectron spectroscopy (XPS) spectra of the Co–Cr–Mo surface. Tribological studies with a pin-on-plate machine revealed that surface MPC grafting markedly lowered the friction coefficient. We concluded that the grafted poly(MPC) layer successfully provided high lubricity to the Co–Cr–Mo surface.
Keywords: Joint replacement; Cobalt alloy; Phosphorylcholine; Metal surface treatment;

Towards improved artificial lungs through biocatalysis by Joel L. Kaar; Heung-Il Oh; Alan J. Russell; William J. Federspiel (3131-3139).
Inefficient CO2 removal due to limited diffusion represents a significant barrier in the development of artificial lungs and respiratory assist devices, which use hollow fiber membranes (HFMs) as the blood–gas interface and can require large blood-contacting membrane area. To offset the underlying diffusional challenge, “bioactive” HFMs that facilitate CO2 diffusion were prepared via covalent immobilization of carbonic anhydrase (CA), an enzyme which catalyzes the conversion of bicarbonate in blood to CO2, onto the surface of plasma-modified conventional HFMs. This study examines the impact of enzyme attachment on the diffusional properties and the rate of CO2 removal of the bioactive membranes. Plasma deposition of surface reactive hydroxyls, to which CA could be attached, did not change gas permeance of the HFMs or generate membrane defects, as determined by scanning electron microscopy, when low plasma discharge power and short exposure times were employed. Cyanogen bromide activation of the surface hydroxyls and subsequent modification with CA resulted in near monolayer enzyme coverage (88%) on the membrane. The effect of increased plasma discharge power and exposure time on enzyme loading was negligible while gas permeance studies showed enzyme attachment did not impede CO2 or O2 diffusion. Furthermore, when employed in a model respiratory assist device, the bioactive membranes improved CO2 removal rates by as much as 75% from physiological bicarbonate solutions with no enzyme leaching. These results demonstrate the potential of bioactive HFMs with immobilized CA to enhance CO2 exchange in respiratory devices.
Keywords: Artificial respiratory device; Hollow fiber membrane; Carbon dioxide; Gas exchange; Carbonic anhydrase; Enzyme immobilization;

In the present study, effects of alterations in the chemical structure of polyester-co-polyether (PEPE) dendrimers on the encapsulation and release of methotrexate (MTX) was investigated. A series of PEPE dendrimers of different architecture were synthesized. Biocompatibility of the resulting dendrimers was evaluated in vitro by assessing their cytotoxicity on RAW 264.7 cells using lactate dehydrogenase (LDH) assay. Dendrimers caused no cell death even at the concentration of 250 μg/mL, suggesting that they are acceptable for pharmaceutical applications. They also showed good capacity to encapsulate MTX, with loading as high as 24.5% w/w. Increase in the number of branches and the size of internal voids were shown to enhance the encapsulation. On the other hand, absence of aromatic rings as branching units drastically reduced the loading capacity. Physical entrapment, weak hydrogen bonding and hydrophobic interactions were established to be the mechanisms of encapsulation. Release of MTX was biphasic, which included a burst release in 6 h followed by a slower release over a period of 50 or 168 h. Increase in the number of branches profoundly decreased this initial burst release; in contrast, absence of aromatic rings in the dendritic structure resulted in a very rapid release.
Keywords: Dendrimer; Methotrexate; Release; Encapsulation; Architecture;

Physical and biological properties of collagen-phospholipid polymer hybrid gels by Kwangwoo Nam; Tsuyoshi Kimura; Akio Kishida (3153-3162).
We successfully developed a novel method for immobilizing poly(2-methacryloyloxyethyl phosphorylcholine) [Poly(MPC)] polymer onto collagen using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as cross-linkers. In order to obtain the highest possible molar ratio of immobilized MPC moieties on the collagen gel, a collagen-phospholipid polymer hybrid gel was prepared by repeating the cross-linking process up to three times to create a dense network of collagen and PMA. Network formation by repeating the immobilization process was successful, resulting in decreased free amine group content and a low swelling ratio. The hybrid gel displayed very high stability against degradation by collagenase and possessed high hydrophilicity. Fibrinogen adsorption and cell adhesion were reduced and demonstrated less cell proliferation as compared to that by uncross-linked collagen gel. The collagen-phospholipid polymer hybrid gel did not exhibit toxicity, and the cell morphology remained intact (round); this implies that the interaction between the cell and the collagen-phospholipid polymer hybrid gel is safe and mild.
Keywords: Collagen; Phospholipid polymer; Immobilization; Protein adsorption; Cell adhesion;

We have developed collagenase-sensitive hydrogels by incorporating a collagenase-sensitive fluorogenic substrate (CS-FS) within the backbone of a polyethylene glycol (PEG) copolymer to visualize collagenase activity during three-dimensional cell migration. CS-FS was synthesized by conjugating Bodipy dyes to a peptide with collagenase-sensitive sequence, Leu-Gly-Pro-Ala (LGPA), and the products were grafted into the collagenase-sensitive PEG hydrogels. CS-FS both in solution and hydrogels had an increase in the fluorescence intensity after proteolytic degradation by collagenase, but not by non-targeted proteases nor in the absence of an enzyme. Fibroblasts inside the hydrogels conjugated with CS-FS spread and extended lamellipodia in three dimensions over several days, and their pericellular collagenase-mediated proteolysis of the hydrogel was visualized via confocal microscopy. A matrix metalloproteinase inhibitor, served as a negative control, significantly reduced the degradation rate of CS-FS by collagenase and prevented cell migration and cell-mediated collagenase activity inside these hydrogels. In summary, we have fabricated collagenase-sensitive hydrogels incorporated with CS-FS and successfully visualized the collagenase activity during three-dimensional cell migration.
Keywords: Hydrogel; Polyethylene oxide; Fluorescence; Peptide;