Biomaterials (v.29, #21)

Trends in biomaterials research: An analysis of the scientific programme of the World Biomaterials Congress 2008 by Sander C.G. Leeuwenburgh; John A. Jansen; Jos Malda; Wouter A. Dhert; Jeroen Rouwkema; Clemens A. van Blitterswijk; C. James Kirkpatrick; David F. Williams (3047-3052).

Cell based bone tissue engineering in jaw defects by Gert J. Meijer; Joost D. de Bruijn; Ron Koole; Clemens A. van Blitterswijk (3053-3061).
In 6 patients the potency of bone tissue engineering to reconstruct jaw defects was tested. After a bone marrow aspirate was taken, stem cells were cultured, expanded and grown for 7 days on a bone substitute in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. At the end of the procedure, this viable bone substitute was not only re-implanted in the patient, but also simultaneously subcutaneously implanted in mice to prove its osteogenic potency. In all patients, a viable bone substitute was successfully constructed, which was proven by bone formation after subcutaneous implantation in mice (ectopic bone formation). However, the same construct was reluctant to form bone in patients with intra-oral osseous defects (orthotopic bone formation). Although biopsies, taken 4 months after reconstructing the intra-oral bone defect, showed bone formation in 3 patients, only in 1 patient bone formation was induced by the tissue-engineered construct. Although bone tissue engineering has proven its value in animal studies, extra effort is needed to make it a predictable method for reconstruction jaw defects in humans. To judge its benefit, it is important to differentiate between bone formation induced by cells from the border of the osseous defect (osteoconduction) in relation to bone matrix produced by the implanted cells (osteogenesis).
Keywords: Bone tissue engineering; Cell culture; Osteogenesis; Stem cell;

Volumetric interpretation of protein adsorption: Kinetic consequences of a slowly-concentrating interphase by Naris Barnthip; Hyeran Noh; Evan Leibner; Erwin A. Vogler (3062-3074).
Time-dependent energetics of blood-protein adsorption are interpreted in terms of a slowly-concentrating three-dimensional interphase volume initially formed by rapid diffusion of protein molecules into an interfacial region spontaneously formed by bringing a protein solution into contact with a physical surface. This modification of standard adsorption theory is motivated by the experimental observation that interfacial tensions of protein-containing solutions decrease slowly over the first hour to a steady-state value while, over this same period, the total adsorbed protein mass is constant (for lysozyme, 15 kDa; α-amylase, 51 KDa; albumin, 66 kDa; prothrombin, 72 kDa; IgG, 160 kDa; fibrinogen, 341 kDa studied in this work). These seemingly divergent observations are rationalized by the fact that interfacial energetics (tensions) are explicit functions of solute chemical potential (concentration), not adsorbed mass. Hence, rates of interfacial tension change parallel a slow interphase-concentration effect whereas solution depletion detects a constant interphase composition within the timeframe of experiment. A straightforward mathematical model approximating the perceived physical situation leads to an analytic formulation that is used to compute time-varying interphase volume and protein concentration from experimentally-measured interfacial tensions. Derivation from the fundamental thermodynamic adsorption equation verifies that protein adsorption from dilute solution is controlled by a partition coefficient at equilibrium, as is observed experimentally at steady state. Implications of the alternative interpretation of adsorption kinetics on biomaterials and biocompatibility are discussed.
Keywords: Protein adsorption; Kinetics; Interfacial energetics; Interphase; Surface; Radiometry;

The effect of RGD peptides on osseointegration of hydroxyapatite biomaterials by Kristin M. Hennessy; Will C. Clem; Matthew C. Phipps; Amber A. Sawyer; Faheem M. Shaikh; Susan L. Bellis (3075-3083).
Given that hydroxyapatite (HA) biomaterials are highly efficient at adsorbing proadhesive proteins, we questioned whether functionalizing HA with RGD peptides would have any benefit. In this study, we implanted uncoated or RGD-coated HA disks into rat tibiae for 30 min to allow endogenous protein adsorption, and then evaluated mesenchymal stem cell (MSC) interactions with the retrieved disks. These experiments revealed that RGD, when presented in combination with adsorbed tibial proteins (including fibronectin, vitronectin and fibrinogen), has a markedly detrimental effect on MSC adhesion and survival. Moreover, analyses of HA disks implanted for 5 days showed that RGD significantly inhibits total bone formation as well as the amount of new bone directly contacting the implant perimeter. Thus, RGD, which is widely believed to promote cell/biomaterial interactions, has a negative effect on HA implant performance. Collectively these results suggest that, for biomaterials that are highly interactive with the tissue microenvironment, the ultimate effects of RGD will depend upon how signaling from this peptide integrates with endogenous processes such as protein adsorption.
Keywords: Bioadsorption; Bone tissue engineering; Cell adhesion; Hydroxyapatite; Osseointegration; RGD peptide;

The formation of platelet–neutrophil microaggregates and successive activation of neutrophils are closely related to hemodialysis-associated complications. The microaggregate is mediated primarily by the interaction between P-selectin (CD62P) expressed on activated platelets and P-selectin glycoprotein ligand-1 (PSGL-1, CD162) expressed on neutrophils. We previously reported that the clustered distribution of PSGL-1 on the cell membranes of chemokine-treated neutrophils caused upregulation of the microaggregate formation. In this study, we found that neutrophils treated with human plasma that had been incubated with hemodialysis membranes greatly enhanced the microaggregate formation. The membrane-treated plasma also induced PSGL-1 to form a cap-like cluster on the neutrophil surface. Analysis of several hemodialysis membranes with different materials indicated that the inducibility for the cap-like cluster formation of PSGL-1 parallels their ability to activate the complement system. Both the enhancement of microaggregate formation and the redistribution of PSGL-1 induced by the hemodialysis membrane-treated plasma were almost completely abrogated in the presence of a specific antagonist for the complement component C5a receptor, W-54011. These results strongly suggest that the generation of anaphylatoxin C5a through complement activation induced by hemodialysis membranes is responsible for the clustered redistribution of PSGL-1 in neutrophils leading to the increase in the platelet–neutrophil microaggregate formation. The present study indicates the importance of synergistic exacerbation of complement activation and platelet–neutrophil microaggregate formation in developing hemodialysis-associated complications.
Keywords: Anaphylatoxin; Cell adhesion; Complement; Hemodialysis; Platelet–neutrophil microaggregate; P-selectin glycoprotein ligand-1;

Development of biodegradable electrospun scaffolds for dermal replacement by Keith A. Blackwood; Rob McKean; Irene Canton; Christine O. Freeman; Kirsty L. Franklin; Daryl Cole; Ian Brook; Paula Farthing; Stephen Rimmer; John W. Haycock; Anthony J. Ryan; Sheila MacNeil (3091-3104).
Our objective is to develop a synthetic biodegradable replacement dermal substitute for tissue engineering of skin and oral mucosa. Our in vivo criteria were that candidate scaffolds should allow surrounding cells to migrate fully into the scaffolds, enabling vasculogenesis and remodelling without invoking a chronic inflammatory response. We examined a total of six experimental electrospun polymer scaffolds: (1) poly-l-lactide (PLLA); (2) PLLA + 10% oligolactide; (3) PLLA + rhodamine and (4–6) three poly(d,l)-lactide-co-glycolide (PLGA) random multiblock copolymers, with decreasing lactide/glycolide mole fractions (85:15, 75:25 and 50:50). These were evaluated for degradation in vitro up to 108 days and in vivo in adult male Wistar rats from 4 weeks to 12 months. In vivo, all scaffolds permitted good cellular penetration, with no adverse inflammatory response outside the scaffold margin and with no capsule formation around the periphery. The breakdown rate for each scaffold in vitro versus in vivo was similar, and an increase in the ratio of polyglycolide to polylactide correlated with an increase in breakdown rate, as expected. Scaffolds of PLLA were stable in vivo even after 12 months whereas scaffolds fabricated from PLGA 85:15 and 75:25 revealed a 50% loss of mass after 4 and 3 months, respectively. In vitro PLGA 85:15 and 75:25 scaffolds were able to support keratinocyte, fibroblast and endothelial cell growth and extracellular matrix production, with evidence of new collagen production after 7 days. In conclusion, the data supports the development of PLGA 85:15 and 75:25 electrospun polymer scaffolds as potential degradable biomaterials for dermal replacement.
Keywords: Polymers; Biocompatibility; Tissue engineering; Electrospinning;

Development of specific collagen scaffolds to support the osteogenic and chondrogenic differentiation of human bone marrow stromal cells by Jonathan I. Dawson; Denys A. Wahl; Stuart A. Lanham; Janos M. Kanczler; Jan T. Czernuszka; Richard O.C. Oreffo (3105-3116).
Type I Collagen matrices of defined porosity, incorporating carbonate substituted hydroxyapatite (HA) crystals, were assessed for their ability to support osteo- and chondrogenic differentiation of human bone marrow stromal cells (HBMSCs). Collagen–HA composite scaffolds supported the osteogenic differentiation of HBMSCs both in vitro and in vivo as demonstrated by histological and micro-CT analyses indicating the extensive penetration of alkaline phosphatase expressing cells and new matrix synthesis with localised areas immunologically positive for osteocalcin. In vivo, extensive new osteoid formation of implant origin was observed in the areas of vasculature. Chondrogenic matrix synthesis was evidenced in the peripheral regions of pure collagen systems by an abundance of Sox9 expressing chondrocytes embedded within a proteoglycan and collagen II rich ECM. The introduction of microchannels to the scaffold architecture was seen to enhance chondrogenesis. Tissue specific gene expression and corresponding matrix synthesis indicate that collagen matrices support the growth and differentiation of HBMSCs and suggest the potential of this platform for understanding the ECM cues necessary for osteogenesis and chondrogenesis.
Keywords: Bone tissue engineering; Cartilage tissue engineering; Collagen; Scaffold; Biomimetic material;

The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps by Young-tae Kim; Valerie K. Haftel; Satish Kumar; Ravi V. Bellamkonda (3117-3127).
Peripheral nerve regeneration across long nerve gaps is clinically challenging. Autografts, the standard of therapy, are limited by availability and other complications. Here, using rigorous anatomical and functional measures, we report that aligned polymer fiber-based constructs present topographical cues that facilitate the regeneration of peripheral nerves across long nerve gaps. Significantly, aligned but not randomly oriented fibers elicit regeneration, establishing that topographical cues can influence endogenous nerve repair mechanisms in the absence of exogenous growth promoting proteins. Axons regenerated across a 17 mm nerve gap, reinnervated muscles, and reformed neuromuscular junctions. Electrophysiological and behavioral analyses revealed that aligned but not randomly oriented constructs facilitated both sensory and motor nerve regeneration, significantly improved functional outcomes. Additionally, a quantitative comparison of DRG outgrowth in vitro and nerve regeneration in vivo on aligned and randomly oriented fiber films clearly demonstrated the significant role of sub-micron scale topographical cues in stimulating endogenous nerve repair mechanisms.
Keywords: Topographical guidance; Nerve regeneration; Cell migration;

Preparation and cell affinity of microtubular orientation-structured PLGA(70/30) blood vessel scaffold by Xixue Hu; Hong Shen; Fei Yang; Jianzhong Bei; Shenguo Wang (3128-3136).
In this study, a kind of microtubular orientation-structured blood vessel mimicking natural structure was fabricated with poly(lactide-co-glycolide)(70/30) (PLGA(70/30)) solutions in 1,4-dioxane by an improved thermal-induced phase separation (TIPS) technique. The effect of main factors of the TIPS technique, such as environmental temperature, temperature gradient and concentration of the polymer solution on the structure and morphology of formed vessel scaffold was investigated. It was observed that the outer-wall of the scaffold became thick obviously and the microtubules neighboring the outer-wall became disordered with environmental temperature increasing. The diameter of microtubules of vessel scaffolds reduced with temperature gradient increasing or concentration of the polymer solution increasing. By controlling parameters of the TIPS, the scaffolds with various morphologies could be manufactured, which had different diameters of microtubules. On the other hand, inner-diameter and outer-diameter of the vessel scaffolds could be controlled by adjusting size of the polyethylene mould. Cell affinity of the scaffolds was tested in vitro by using A10 cell as model cells. Results showed that the cells grew well in the vessel scaffolds which were modified by ammonia plasma treatment and then anchored with collagen. The cells could array along the direction of the microtubules.
Keywords: Blood vessel scaffolds; PLGA(70/30); Microtubular structure; Thermal-induced phase separation (TIPS) technique; Surface modification; Cell affinity;

The objective of this work was to control the orientation of bioactive molecules immobilized on a biodegradable substrate to improve their accessibility for binding to cell surface receptors and, therefore, to increase bioactivity. The osteotropic peptide, parathyroid hormone (1–34) (PTH(1–34)), was used to demonstrate the approach. To this end, the intrinsic N-terminal serine residue was oxidized to create an aldehyde group that specifically bound to hydrazide-derivatized poly(lactide-co-glycolide) under neutral conditions to form a hydrazone bond. Use of dihydrazide spacers significantly increased the amount of peptide immobilized compared to simple adsorption or direct, random attachment. In probing accessibility of immobilized PTH(1–34), attachment using longer dihydrazide spacers enhanced binding of an antibody against an epitope in the N-terminal region of the peptide. The longest spacer also increased binding of a C-terminal antibody. Furthermore, substrates with peptide tethered via spacers stimulated intracellular synthesis of cAMP, with activity increasing with dihydrazide length. PTH(1–34) immobilized using the longest spacer was significantly more effective than both random binding and adsorption. Site-directed binding of bioactive peptides to surfaces presents biomolecules for binding with cells so as to enhance interaction with receptors, and therefore the approach may be useful for obtaining preferred localized tissue responses.
Keywords: Cell activation; Osteoblast; Peptide; Surface grafting; Surface modification;

The use of therapeutic gene eNOS delivered via a fibrin scaffold enhances wound healing in a compromised wound model by Ailish M. Breen; Peter Dockery; Timothy O'Brien; Abhay S. Pandit (3143-3151).
Diabetic healing is marked by a reduced nitric oxide (NO) production at the wound site. This study aimed to investigate whether a fibrin scaffold would enhance the delivery of adenovirus encoding endothelial nitric oxide synthase (eNOS), one of the enzymes responsible for NO production, resulting in more NO production, and enhanced healing. An alloxan rabbit ear ulcer model was used to investigate healing, in response to the following treatments: fibrin containing AdeNOS, AdeNOS alone, fibrin alone and no treatment. Immunohistochemistry to detect eNOS expression and histological evaluation of healing were assessed at 7 and 14 days. eNOS expression was significantly greater in the fibrin containing AdeNOS group at 14 days compared to all other groups. Furthermore, this group showed a significantly faster rate of epithelialisation than all other groups. The volume of inflammatory cells was highest in the fibrin containing AdeNOS group at 7 days, which dropped significantly by 14 days. Likewise, the surface area and length of vessels reduced significantly in the fibrin containing AdeNOS group between 7 and 14 days indicating tissue remodelling, but remained stable in all other groups. Regression analysis showed that the epithelialisation rate was significantly affected by change in eNOS expression, inflammation, and surface area and length of vessels over time in the fibrin containing AdeNOS group. It was concluded that fibrin delivery of AdeNOS resulted in enhanced eNOS expression, inflammatory response, and a faster rate of re-epithelialisation.
Keywords: Fibrin; Scaffold; Wound healing; Gene transfer; Nitric oxide;

The synthetic peptide RAD16-II has shown promise in tissue engineering and drug delivery. It has been studied as a vehicle for cell delivery and controlled release of IGF-1 to repair infarcted cardiac tissue, and as a scaffold to promote capillary formation for an in vitro model of angiogenesis. The structure of RAD16-II is hierarchical, with monomers forming long β-sheets that pair together to form filaments; filaments form bundles approximately 30–60 nm in diameter; branching networks of filament bundles form macroscopic gels. We investigate the mechanics of shearing between the two β-sheets constituting one filament, and between cohered filaments of RAD16-II. This shear loading is found in filament bundle bending or in tensile loading of fibers composed of partial-length filaments. Molecular dynamics simulations show that time to failure is a stochastic function of applied shear stress, and that for a given loading time behavior is elastic for sufficiently small shear loads. We propose a coarse-grained model based on Langevin dynamics that matches molecular dynamics results and facilities extending simulations in space and time. The model treats a filament as an elastic string of particles, each having potential energy that is a periodic function of its position relative to the neighboring filament. With insight from these simulations, we discuss strategies for strengthening RAD16-II and similar materials.
Keywords: Peptide; Self-assembly; Scaffold; Hydrogel; Modeling;