Biomaterials (v.27, #34)

Calendar (I).

Mesoporous carbide-derived carbon with porosity tuned for efficient adsorption of cytokines by Gleb Yushin; Elizabeth N. Hoffman; Michel W. Barsoum; Yury Gogotsi; Carol A. Howell; Susan R. Sandeman; Gary J. Phillips; Andrew W. Lloyd; Sergey V. Mikhalovsky (5755-5762).
Porous carbons can be used for the purification of various bio-fluids, including the cleansing blood of inflammatory mediators in conditions such as sepsis or auto-immune diseases. Here we show that the control of pore size in carbons is a key factor to achieving efficient removal of cytokines. In particular, the surface area accessible by the protein governs the rate and effectiveness of the adsorption process. We demonstrate that novel mesoporous carbon materials synthesized from ternary MAX-phase carbides can be optimized for efficient adsorption of large inflammatory proteins. The synthesized carbons, having tunable pore size with a large volume of slit-shaped mesopores, outperformed all other materials or methods in terms of efficiency of TNF-α removal and the results are comparable only with highly specific antibody–antigen interactions.
Keywords: Carbon; Protein adsorption; Adsorption; Cytokine;

The correlation between difference in foreign body reaction between implant locations and cytokine and MMP expression by Daniel T. Luttikhuizen; Machteld J. van Amerongen; Pieter C. de Feijter; Arjen H. Petersen; Martin C. Harmsen; Marja J.A. van Luyn (5763-5770).
The foreign body reaction (FBR) differs between subcutaneously and supra-epicardially implanted materials. We hypothesize that this is a result of differences in cytokine, chemokine and matrix metalloproteinase (MMP) dynamics. Therefore we applied collagen disks subcutaneously and on the epicardium in mice and analyzed the FBR from day 1 to 21. Both the influx of leukocytes and implant degradation were higher in supra-epicardially implanted collagen than in subcutaneously implanted material. This correlated with a higher gene expression of pro-inflammatory cytokines such as IL-1 and IL-6, and a lower expression of the anti-inflammatory cytokine IL-10. Furthermore, the higher supra-epicardial expression of PMN attractants CXCL1/KC and CXCL2/MIP2 correlated with a higher and prolonged PMN influx. The gene expression levels of collagen degrading MMPs, i.e. MMP8, MMP13 and MMP14 were similar in subcutaneous and supra-epicardial disks. However, the activity of these enzymes was markedly higher supra-epicardially. In addition, the MMP9 expression was higher supra-epicardially, suggesting a role for this enzyme in the degradation process. In conclusion, a strong pro-inflammatory milieu is generated after supra-epicardial implantation that enables prolonged PMN presence and activation. This, together with the high supra-epicardial MMP9 level, could explain the observed difference in Col-I degradation between locations.
Keywords: Matrix metalloproteinase; Cytokine; Gene expression; Inflammation; Rat; Collagen;

Stability, sterility, coagulation, and immunologic studies of salmon coagulation proteins with potential use for mammalian wound healing and cell engineering by Ivo Laidmäe; Margaret E. McCormick; Julia L. Herod; Jennifer J. Pastore; Tiit Salum; Evelyn S. Sawyer; Paul A. Janmey; Raivo Uibo (5771-5779).
Fibrin sealants made by polymerization of fibrinogen activated by the protease thrombin have many applications in hemostasis and wound healing. In treatments of acute injury or surgical wounds, concentrated fibrin preparations mimic the initial matrix that normally prevents bleeding and acts as a scaffold for cells that initiate tissue repair. However risks of infectious disease, immunogenic reaction, and the high cost of purified human or other mammalian blood proteins limit widespread use of these materials. Purified coagulation proteins from Atlantic salmon represent a potentially safer, equally effective, and less costly alternative in part because of the low ambient temperature of these farmed animals and the absence of endogenous agents known to be infectious in mammalian hosts. This study reports rheologic measurements of lyophilized salmon fibrinogen and thrombin that demonstrate stability to prolonged storage and gamma irradiation sufficient to reduce viral loads by over five orders of magnitude. Coagulation and immunologic studies in rats and rabbits treated intraperitoneally with salmon fibrin show no deleterious effects on coagulation profiles and no cross reactivity with host fibrinogen or thrombin. The results support the potential of salmon fibrin as an alternative to mammalian proteins in clinical applications.
Keywords: Fibrin sealant; Thrombin; Salmon; Fibrinogen; Wound healing; Coagulation;

The solution-depletion method of measuring protein adsorption is implemented using SDS gel electrophoresis as a separation and quantification tool. Experimental method is demonstrated using lysozyme (15 kDa), α-amylase (51 kDa), human serum albumin (66 kDa), prothrombin (72 kDa), immunoglobulin G (160 kDa), and fibrinogen (341 kDa) adsorption from aqueous-buffer solution to hydrophobic octyl-sepharose and silanized-glass particles. Interpretive mass-balance equations are derived from a model premised on the idea that protein reversibly partitions from bulk solution into a three-dimensional (3D) interphase volume separating the physical-adsorbent surface from bulk solution. Theory both anticipated and accommodated adsorption of all proteins to the two test surfaces, suggesting that the underlying model is descriptive of the essential physical chemistry of protein adsorption. Application of mass balance equations to experimental data quantify partition coefficients P, interphase volumes V I, and the number of hypothetical layers M occupied by protein adsorbed within V I. Partition coefficients quantify protein-adsorption avidity through the equilibrium ratio of interphase and bulk-solution-phase w/v (mg/mL) concentrations W I and W B, respectively, such that P ≡ W I / W B . Proteins are found to be weak biosurfactants with 45 < P < 520 and commensurately low apparent free-energy-of-adsorption - 6 RT < ( Δ G ads phobic 0 = - RT ln P ) < - 4 RT . These measurements corroborate independent estimates obtained from interfacial energetics of adsorption (tensiometry) and are in agreement with thermochemical measurements for related proteins by hydrophobic-interaction chromatography. Proteins with molecular weight MW < 100 kDa occupy a single layer at surface saturation whereas the larger proteins IgG and fibrinogen required two layers.
Keywords: Protein adsorption; Hydrophobic solid–liquid interface; Globular blood proteins; Octyl sepharose;

To elucidate a possible link between the cytotoxicity and Ca2+ mobilization by (meth)acrylates, we investigated the cell survival of and change in [Ca2+] i in human salivary gland (HSG) cells (salivary gland carcinoma cell line) and human gingival fibroblasts (HGF) cells treated separately with 9 (meth)acrylate monomers used in dentistry. The cell survival was determined by the MTT method, and the [Ca2+] i changes after the stimulation with the (meth)acrylate monomers were measured in floating indo-1/AM-loaded cells in Ca2+-free medium. For both HSG and HGF cells, the cytotoxicity of the monomers was approximately proportional to their hydrophobicity (log  P). No increase in [Ca2+] i was found with hydrophilic monomers, even with 10 mm stimulation. [Ca2+] i elevation by hydrophobic monomers occurred in a dose- and hydrophobic-dependent manner. The [Ca2+] i change in HSG cells appeared as twin peaks, i.e., an initial sharp peak followed by a delayed broad one; whereas with the HGF cells only a single broad peak was seen, possibly dependent on their membrane quality. Pretreatment with n-butanol or methylmethacrylate enhanced the butylmethacrylate-induced [Ca2+] i elevation, suggesting the [Ca2+] i elevation by (meth)acrylate may be related to monomer hydrophobicity and cell type. The causal link between the cytotoxicity and [Ca2+] i mobilization of monomers is discussed.
Keywords: Methacrylate; Resin monomer; Ca2+; Cytotoxicity; Hydrophobicity;

The solution-depletion method of measuring human serum albumin (HSA) adsorption to surface-modified glass-particle adsorbents with incrementally increasing hydrophilicity is implemented using SDS gel electrophoresis as a separation and quantification tool. It is shown that adsorbent capacity for albumin measured in interfacial–concentration units (mg/mL) decreases monotonically with increasing surface energy (water wettability) to detection limits near an adsorbent-particle water adhesion tension τ 0 = 30 dyne / cm (nominal water contact angle θ = 6 5 ∘ ) and that albumin does not adsorb to (concentrate within the surface region of) more hydrophilic adsorbents. These adsorbed-mass measurements corroborate predictions based on interfacial energetics and are consistent with AFM measurement of protein–surface adhesion. Interpretive mass-balance equations are derived from a model premised on the idea that protein reversibly partitions from bulk solution into a three-dimensional (3D) interphase volume separating the physical adsorbent surface from bulk solution. Theory is shown to both anticipate and accommodate experimental results for all test adsorbents, suggesting that the underlying model is descriptive of the essential physical chemistry of albumin adsorption to surfaces spanning the full range of observable water wetting. In particular, application of theory to experimental data shows that the free-energy cost of dehydrating the surface region by protein displacement upon adsorption increases with increasing adsorbent hydrophilicity in a manner that controls ultimate capacity for protein. It is concluded that a simple, three-component free-energy rule adequately describes protein adsorption from aqueous solution, at least for materials bearing varying surface concentrations of anionic (not cationic) functional groups.This work yields detailed insights into the physical chemistry of protein adsorption by elucidating relationships among adsorbent surface energy, capacity to adsorb the protein human serum albumin, and the energy required to displace vicinal water from the interface.
Keywords: Protein adsorption; Surface energy; Albumin;

Real-time analysis of cell–surface adhesive interactions using thickness shear mode resonator by Soonjin Hong; Ertan Ergezen; Ryszard Lec; Kenneth A. Barbee (5813-5820).
The cell adhesion process and the molecular interactions that determine its kinetics were investigated using a thickness shear mode (TSM) sensor. The goal of this study was to correlate sensor readings with the progression of cell adhesion. In particular, the specific effects of receptor-mediated adhesion, the glycocalyx, and surface charge on initial cell–surface attachment and steady-state adhesion of endothelial cells were investigated. We found a strong correlation between resistance changes (ΔR) and the development of cell adhesion strength by comparing the sensor readings with independently assessed cell adhesion. The result showed that integrin binding determines the kinetics of initial cell attachment while heparan sulfate proteoglycan (HSPG) modulates steady-state adhesion strength. Coating the sensor surface with the positively charged poly-d-lysine (PDL) enhanced the initial interaction with substratum. These data confirm our current understanding of the contribution of these three phenomena to the adhesion process. The real-time monitoring capability of this technique with high temporal resolution provides more detailed information on the kinetics of the different stages of the adhesion process. This technique has the potential to facilitate the evaluation of biomaterials and surface treatments used for implants and tissue-engineering scaffolds for their bioactive effects on the cell adhesion process.
Keywords: Cell adhesion; Biosensor; Endothelial cell; Surface treatment; Integrin; RGD peptide;

EDC cross-linking improves skin substitute strength and stability by Heather M. Powell; Steven T. Boyce (5821-5827).
Collagen-based scaffolds are extensively utilized as an analog for the extracellular matrix in cultured skin substitutes (CSS). To improve the mechanical properties and degradation rates of collagen scaffolds, chemical cross-linking is commonly employed. In this study, freeze-dried collagen-GAG sponges were crosslinked with increasing concentrations of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC; 0, 1, 5, 10, 50 mm). Cross-linking with EDC at concentrations >1 mm was shown to greatly decrease degradation by collagenase up to 21 days. Ultimate tensile strength (UTS) of acellular collagen sponges scaled positively with EDC concentration up to 10 mm. At 50 mm EDC, the UTS decreased dramatically likely due to the brittle nature of the highly crosslinked material. Co-culture of human fibroblasts (HF) and keratinocytes (HK) on these substrates reveals an apparent cytotoxicty of the EDC at high concentrations with reduced cell viability and poor cellular organization in CSS fabricated with scaffolds crosslinked with 10 or 50 mm EDC. From the data gathered in this study, intermediate concentrations of EDC, specifically 5 mm, increase collagen sponge stability and strength while providing an environment in which HF and HK can attach, proliferate and organize in a manner conducive to dermal and epidermal regeneration.
Keywords: Skin; Tissue engineering; Mechanical properties; Collagen; Wound healing;

The microencapsulation of islets of Langerhans (islets) has been studied as a safe and simple technique for islet transplantation without the need for immuno-suppressive therapy. However, thinner membranes are desired, because the increased total volume of the implant led to limited transplantation sites. Here, we propose a novel method for microencapsulation by polyion complex membrane formation on islets. Amino group-terminated poly(ethylene glycol)-conjugated phospholipids (PEG-lipids, M w: 5000) spontaneously formed a thin layer on cells existing in the outer layer of islets when they were added to islet suspension. This layer-by-layer membrane could be further formed on the PEG-lipid layer through polyion complex formation between amino groups at the end of PEG chains, sodium alginate and poly(l-lysine). Islets could be microencapsulated by this method without volume increase. Encapsulation of the islet surface with PEG-lipids and polyion complex membranes did not impair the insulin release function in response to glucose stimulation. Our method is promising to encapsulate islets without affecting cell viability or increasing volume.
Keywords: Bioartificial pancreas; Islet; Poly(ethylene glycol)-lipid (PEG-lipid); Polyion complex; Microencapsulation;

Enhanced angiogenesis through controlled release of basic fibroblast growth factor from peptide amphiphile for tissue regeneration by Hossein Hosseinkhani; Mohsen Hosseinkhani; Ali Khademhosseini; Hisatoshi Kobayashi; Yasuhiko Tabata (5836-5844).
In the present study, we hypothesized that a novel approach to promote vascularization would be to create injectable three-dimensional (3-D) scaffolds with encapsulated growth factor that enhance the sustained release of growth factor and induce the angiogenesis. We demonstrate that a 3-D scaffold can be formed by mixing of peptide-amphiphile (PA) aqueous solution with basic fibroblast growth factor (bFGF) suspension. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. A 3-D network of nanofibers was formed by mixing bFGF suspensions with dilute aqueous solutions of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. In vitro and in vivo release profile of bFGF from 3-D network of nanofibers was investigated while angiogenesis induced by the released bFGF was assessed. When aqueous solution of PA was subcutaneously injected together with bFGF suspension into the back of mice, a transparent 3-D hydrogel was formed at the injected site and induced significant angiogenesis around the injected site, in marked contrast to bFGF injection alone or PA injection alone. The combination of bFGF-induced angiogenesis is a promising procedure to improve tissue regeneration.
Keywords: Scaffold; Peptide amphiphile; Nanofibers; Self-assembly; Angiogenesis;

A citric acid-based hydroxyapatite composite for orthopedic implants by Hongjin Qiu; Jian Yang; Pradeep Kodali; Jason Koh; Guillermo A. Ameer (5845-5854).
We describe a novel approach to process bioceramic microparticles and poly(diol citrates) into bioceramic–elastomer composites for potential use in orthopedic surgery. The composite consists of the biodegradable elastomer poly(1,8-octanediol-citrate) (POC) and the bioceramic hydroxyapatite (HA). The objective of this work was to characterize POC–HA composites and assess the feasibility of fabricating tissue fixation devices using machining and molding techniques. The mechanical properties of POC–HA composites with HA (40, 50, 60, 65 wt.%) were within the range of values reported for tissue fixation devices (for POC–HA 65 wt.%, S b=41.4±3.1, E b=501.7±40.3, S c=74.6±9.0, E c=448.8±27.0, S t=9.7±2.3, E t=334.8±73.5, S s=27.7±2.4, T s=27.3±4.9, all values in MPa). At 20 weeks, the weight loss of POC–HA composites ranged between 8 and 12 wt.%, with 65 wt.% HA composites degrading the slowest. Exposure of POC–HA to simulated body fluid resulted in extensive mineralization in the form of calcium phosphate with Ca/P of 1.5–1.7 similar to bone. POC–HA supported osteoblast adhesion in vitro and histology results from POC–HA samples that were implanted in rabbit knees for 6 weeks suggest that the composite is biocompatible. Synthesis of POC–HA is easy and inexpensive, does not involve harsh solvents or initiators, and the mechanical properties of POC–HA with 65 wt.% HA are suitable for the fabrication of potentially osteoconductive bone screws.
Keywords: Composites; Mechanical properties; Biodegradation; Osteoblast; Hydroxyapatite;

In vivo comparison of various polymeric and low molecular mass inhibitors of intestinal P-glycoprotein by Florian Föger; Herbert Hoyer; Krum Kafedjiiski; Michael Thaurer; Andreas Bernkop-Schnürch (5855-5860).
Several polymers have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a direct in vivo comparison of delivery systems based on Pluronic P85, Myrj 52 and chitosan-4-thiobutylamidine (Ch-TBA) in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. Furthermore, the postulated low molecular mass P-gp inhibitors 6-mercaptopurine and reduced glutathione (GSH) were evaluated in vitro and in vivo. In vitro, the permeation enhancing effect of 6-mercaptopurine, GSH, Pluronic P85, Myrj 52, and the combination of Ch-TBA with GSH was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type diffusion chambers. In comparison to buffer only, Rho-123 transport in presence of 100 μm 6-mercaptopurine, 0.5% (w/v) GSH, 0.5% (w/v) Pluronic P85, 0.5% (w/v) Myrj 52 and the combination of 0.5% (w/v) Ch-TBA/ 0.5% (w/v) GSH, was 2.1, 1.6, 1.9, 1.8, 3.0-fold improved, respectively. In vivo in rat, enteric-coated tablets based on Pluronic P85, Myrj 52 or Ch-TBA/GSH increased the area under the plasma concentration time curve (AUC0–12) of Rho-123 1.6-fold, 2.4-fold, 4.3-fold, respectively, in comparison to control only. Contrariwise, the low molecular mass excipients 6-mercaptopurine and GSH showed no significant effect in vivo at all.This in vivo study showed that polymeric P-gp inhibitors and especially the delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.
Keywords: P-glycoprotein; Myrj; Pluronic; Thiomers; P-gp inhibition; Oral drug delivery;

Graded delamination behavior of human stratum corneum by Kenneth S. Wu; Morgan M. Stefik; K.P. Ananthapadmanabhan; Reinhold H. Dauskardt (5861-5870).
An in vitro adhesion test method has been adapted to quantify the through-thickness intercellular delamination energy of isolated human stratum corneum (SC). Both untreated and delipidized tissues were tested. Measured delamination energies were found to increase from ∼3 J/m2 near the surface to ∼15 J/m2 for the inner layers of the tissue. For delipidized SC, the location of the initial debond was located closer to the center of the tissue. Delamination energy values were elevated compared to untreated specimens, increasing from ∼7 J/m2 near the surface to ∼18 J/m2 for the inner layers of the SC. Further tests were run to measure delamination energies of SC as a function of hydration (15–100% relative humidity (RH)) at ∼25 °C and as a function of temperature (10–90 °C) at several hydrations (15, 45, 100% RH). Delamination energies were observed to decrease with increasing hydration and increasing temperature with the most significant changes occurring for 100% RH conditioned SC. Additional SC was treated with pH-buffered solutions (pH 4.2, 6.7, 9.9) and selected surfactant solutions (1%, 10% wt/wt sodium dodecyl sulfate (SDS)) for comparison to untreated controls. While statistically significant differences were observed, the SC was found to be resistant to large changes in delamination energy with pH and 1% wt/wt SDS treatments with values in the range 4.2–5.1 J/m2 compared to control values of 4.4 J/m2. More substantially elevated values were observed for SC treated with a 10% wt/wt SDS solution (6.6 J/m2) and a chloroform–methanol extraction (11.2 J/m2).
Keywords: Mechanical properties; Fracture toughness; Epithelial cell; Stratum corneum; Tissue treatment;