Biomaterials (v.28, #25)

Biologic scaffolds composed of naturally occurring extracellular matrix (ECM) have received significant attention for their potential therapeutic applications. The full potential of the ability of ECM scaffolds to promote constructive remodeling will not be realized, however, until an understanding of the biology and the external influences that affect biology, are better achieved. The factors that appear important for the constructive remodeling of ECM biologic scaffolds are its ability to be rapidly and completely degraded with the generation of downstream bioactive molecules, the bioinductive properties of the functional molecules that compose native ECM material and the ability to engineer its mechanical properties at the time of implantation through an understanding of its collagen fiber microstructure.
Keywords: Extracellular matrix; Biologic scaffolds; SIS; Biomaterial;

Neural probe design for reduced tissue encapsulation in CNS by John P. Seymour; Daryl R. Kipke (3594-3607).
This study investigated relationships between a microscale neural probe's size and shape and its chronic reactive tissue response. Parylene-based probes were microfabricated with a thick shank (48 μm by 68 μm) and an integrated thin lateral platform (5 μm by 100 μm, either solid or one of three lattice sizes). Devices were implanted in rat cerebral cortex for 4 weeks before immunostaining for neurons, astrocytes, microglia, fibronectin, laminin, and neurofilament. While nonneuronal density (NND) generally increased and neuronal density decreased within 75 μm of a probe interface compared to unimplanted control regions, there were significant differential tissue responses within 25 μm of the platform's lateral edge compared to the shank. The NND in this region of the lateral edge was less than one-third of the corresponding region of the shank (129% and 425% increase, respectively). Moreover, neuronal density around the platform lateral edge was about one-third higher than at the shank (0.70 and 0.52 relative to control, respectively). Also, microglia reactivity and extracellular protein deposition was reduced at the lateral edge. There were no significant differences among platform designs. These results suggest that neural probe geometry is an important parameter for reducing chronic tissue encapsulation.
Keywords: Biosensor; Cell encapsulation; Foreign body response; Neural prosthesis;

The effect of 3-hydroxybutyrate and its derivatives on the growth of glial cells by Xiao-Qiang Xiao; Yan Zhao; Guo-Qiang Chen (3608-3616).
Sodium salts of d-3-hydroxybutyrate (D-3-HB), dl-3-hydroxybutyrate (DL-3-HB) and methyl (d)-3-hydroxybutyrate (M-3-HB), are derivatives of 3-hydroxybutyric acid (3-HB), a ketone body that is produced in vivo in animals including human. D-3-HB is the most common degradation product of microbial polyhydroxyalkanoates (PHA) that have been investigated for tissue engineering application. This study evaluated the in vitro effect of PHA degradation product 3-HB and its derivatives (collectively called 3-HB derivatives) on cell apoptosis and cytosolic Ca2+ concentration of mouse glial cells. Results showed that the percentage of cells undergoing apoptosis decreased significantly in the presence of 3-HB and its derivatives as evidenced by flow cytometry. The in vitro study on cytosolic Ca2+ concentration demonstrated that 3-HB derivatives dramatically elevated the cytosolic Ca2+ concentration. Both the extracellular and intracellular Ca2+ contributed as the sources of such Ca2+ concentration elevation. The effect of 3-HB derivatives on cytosolic Ca2+ concentration could be reduced by nitredipine, an l-type voltage-dependent calcium channel antagonist. In comparison, M-3-HB worked more efficiently than D-3-HB and DL-3-HB did as M-3-HB is more efficient in permeation into the cells. All results indicated that 3-HB derivatives had an inhibitory effect on cell apoptosis which is mediated by signaling pathways related to the elevation of cytosolic Ca2+ concentration. This positive effect helps explain the biocompatibility observed for PHA, it also points to the possibility of 3-HB derivatives regardless of chirality to become an effective neural protective agent.
Keywords: 3-HB; PHB; Apoptosis; Cytosolic Ca2+ concentration; Glial cell; Polyhydroxyalkanoates;

In vitro blood reactivity to hydroxylated and non-hydroxylated polymer surfaces by Claudia Sperling; Manfred F. Maitz; Sandra Talkenberger; Marie-Françoise Gouzy; Thomas Groth; Carsten Werner (3617-3625).
Complement activation on hydroxyl-group-bearing surfaces is regarded as the main reason for granulocyte activation in applications of blood-contacting medical devices such as extracorporeal blood purification. However, the factors inducing the cell adhesion so far remained ambiguous. For a dedicated research, whole blood was incubated with a set of structurally similar polymer coatings on glass with either hydroxy or ether functionalities. By co-incubation of an activating with a non-activating surface, the reaction of granulocytes activated by complement fragments on non-activating surfaces could be evaluated. As expected, hydroxyl-terminated polymer layers induced much higher levels of complement activation than those with ether functionalities. Leukocyte activation, as measured by the expression of CD11b, correlated closely with the presence of free complement fragment C5a. However, adhesion of leukocytes was rather associated with the adsorption of activated fragments of C3 than with the activation level of the cells. Moreover, it was found that adsorbed quantities of fibrin and fibrinogen had little influence on leukocyte adhesion. It is concluded that the activation of leukocytes is triggered by soluble complement factors such as C5a while their adhesion on hydroxy-bearing surfaces is mainly triggered by the presence of surface-bound complement fragment C3b.
Keywords: Blood compatibility; Complement; Immune response; Chemotaxis; Neutrophil; In vitro test;

Role of plasma fibronectin in the foreign body response to biomaterials by Benjamin G. Keselowsky; Amanda W. Bridges; Kellie L. Burns; Ciara C. Tate; Julia E. Babensee; Michelle C. LaPlaca; Andrés J. García (3626-3631).
Host responses to biomaterials control the biological performance of implanted medical devices. Upon implantation, synthetic materials adsorb biomolecules, which trigger an inflammatory cascade comprising coagulation, leukocyte recruitment/adhesion, and foreign body reaction. The foreign body reaction and ensuing fibrous encapsulation severely limit the in vivo performance of numerous biomedical devices. While it is well established that plasma fibrinogen and secreted cytokines modulate leukocyte recruitment and maturation into foreign body giant cells, mediators of chronic inflammation and fibrous encapsulation of implanted biomaterials remain poorly understood. Using plasma fibronectin (pFN) conditional knock-out mice, we demonstrate that pFN modulates the foreign body response to polyethylene terephthalate disks implanted subcutaneously. Fibrous collagenous capsules were two-fold thicker in mice depleted of pFN compared to controls. In contrast, deletion of pFN did not alter acute leukocyte recruitment to the biomaterial, indicating that pFN modulates chronic fibrotic responses. The number of foreign body giant cells associated with the implant was three times higher in the absence of pFN while macrophage numbers were not different, suggesting that pFN regulates the formation of biomaterial-associated foreign body giant cells. Interestingly, cellular FN (cFN) was present in the capsules of both normal and pFN-depleted mice, suggesting that cFN could not compensate for the loss of pFN. These results implicate pFN in the host response to implanted materials and identify a potential target for therapeutic intervention to enhance the biological performance of biomedical devices.
Keywords: Foreign body giant cells; Fibrosis; Inflammation; Integrin;

In the present study, we report advanced patterned biofunctionalization of thermoresponsive surfaces for achievement of spatiotemporally controlled cell adhesion, growth, and thermally induced detachment. These patterned biofunctional thermoresponsive surfaces were prepared using dual surface modification techniques: electron beam-induced surface patterning of carboxyl-functional thermoresponsive polymers with appropriate metal masks and following site-selective biofunctionalization with biomolecules, the cell adhesive peptide (RGDS) and/or the cell growth factor (insulin; INS). Patterned co-immobilization of RGDS–INS onto thermoresponsive surfaces dominated site-selective cell adhesion and growth along with patterned biofunctional domains in the serum-free culture. Spatiotemporal detachment of sparsely adherent and confluent cells from these patterned biofunctional thermoresponsive surfaces were both achieved only by reducing temperature. Furthermore, RGDS–INS-patterned thermoresponsive surfaces also successfully demonstrated the selective fabrication and recovery of either contiguous monolayer or mesh-like designed monolayer tissue constructs on the identical surfaces. Thus, patterned biofunctional designs would be utilized for the creation and harvest of biomimetic-designed vascular networks having sufficient biofunctional activities in facilitated cell sheet engineering and regenerative medicine.
Keywords: Patterned biofunctional thermoresponsive surfaces; RGDS; Insulin (INS); Spatiotemporal control; Designed monolayer tissues;

Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix by Marta B. Evangelista; Susan X. Hsiong; Rui Fernandes; Paula Sampaio; Hyun-Joon Kong; Cristina C. Barrias; Roberto Salema; Mário A. Barbosa; David J. Mooney; Pedro L. Granja (3644-3655).
There is a need for new therapeutic strategies to treat bone defects caused by trauma, disease or tissue loss. Injectable systems for cell transplantation have the advantage of allowing the use of minimally invasive surgical procedures, and thus for less discomfort to patients. In the present study, it is hypothesized that Arg–Gly–Asp (RGD)-coupled in a binary (low and high molecular weight) injectable alginate composition is able to influence bone cell differentiation in a three-dimensional (3D) structure. Viability, metabolic activity, cytoskeleton organization, ultrastructure and differentiation (alkaline phosphatase (ALP), von Kossa, alizarin red stainings and osteocalcin quantification) of immobilized cells were assessed. Cells within RGD-modified alginate microspheres were able to establish more interactions with the synthetic extracellular matrix as visualized by confocal laser scanning microscope and transmission electron microscopy imaging, and presented a much higher level of differentiation (more intense ALP and mineralization stainings and higher levels of osteocalcin secretion) when compared to cells immobilized within unmodified alginate microspheres. These findings demonstrate that peptides covalently coupled to alginate were efficient in influencing cell behavior within this 3D system, and may provide adequate preparation of osteoblasts for cell transplantation.
Keywords: Osteoblasts; Cell encapsulation; Alginate; RGD peptide; ECM (extracellular matrix); Bone regeneration;

Transient inter-cellular polymeric linker by Siew-Min Ong; Lijuan He; Nguyen Thi Thuy Linh; Yee-Han Tee; Talha Arooz; Guping Tang; Choon-Hong Tan; Hanry Yu (3656-3667).
Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell–cell and cell–matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell–cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell–cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell–cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.
Keywords: PEI-hydrazide; Cell–ECM interaction; Cell aggregates; Tissue engineering; Organ printing; Collagen assay;

Expression of cell adhesion receptors in human osteoblasts cultured on biofunctionalized poly-(ε-caprolactone) surfaces by Ilaria Amato; Gabriela Ciapetti; Stefania Pagani; Giovanni Marletta; Cristina Satriano; Nicola Baldini; Donatella Granchi (3668-3678).
This study was aimed to investigate whether the activation of poly-(ε-caprolactone) (PCL) surface by low-energy irradiation and/or the biofunctionalization by absorption of arginine–glycine–aspartic sequences (RGD), can modify the expression of integrins closely related to the osteoblast activity. For this purpose, we analysed the physicochemical changes induced by irradiation and RGD immobilization, the consequences on cell adhesion and spreading, and the effects on integrin expression. PCL irradiated with 5×1015He+/cm2 (10 keV energy) (irr-PCL) showed an altered surface layer with a partial loss of carboxyl species and the formation of carbonyl groups. Moreover, irr-PCL showed a small smoothening effect and a less polar character in comparison to the pristine ones. The RGD immobilization was observed only on irr-PCL (surface coverage: 7.0 pmol/cm2). Human osteoblasts (hOB) were cultured on untreated PCL (ut-PCL), ut-PCL+RGD, irr-PCL, and irr-PCL+RGD. After 24 h, ut-PCL hindered the cell adhesion, while a discrete layer of hOB with a good cytoskeleton organization was detected on irr-PCL and irr-PCL+RGD. Before seeding, the single hOB suspension expressed α1, α2, α3, α5, β1, and αVβ3; after 24 h, cells cultured on tissue-plastic expressed high levels of β1 and αVβ3, while α1 showed a low intensity and α2, α3, and α5 were negative. β1 and αVβ3 were selected to evaluate the interaction between cells and PCL samples. The β1 expression was higher in hOB cultured on irr-PCL than on the other samples. A significant increase in αVβ3 expression was observed only in irr-PCL+RGD, and confirmed by the gene expression analysis. In conclusion, ion irradiation and RGD adsorption on PCL surfaces modulate the expression of integrin involved in hOB growth and function, indicating the effectiveness of biomimetic surfaces in promoting cell adhesion. Ultimately, the study of integrin expression may suggest proper changes to the surface structure in order to improve the osteoconductivity of selected materials.
Keywords: Poly-(ε-caprolactone); Bioactivity; Osteoblast; Cell adhesion; Integrin;

The angiogenic potential of three-dimensional open porous synthetic matrix materials by M. Oates; R. Chen; M. Duncan; J.A. Hunt (3679-3686).
Angiogenesis is a complex multistage process involving multiple factors and numerous cells. The use of the Chorioallantoic Membrane (CAM) assay is well documented as a method to investigate angiogenesis. This technique is ideal for screening samples, but requires an objective analysis technique. The angiogenic response of vascular endothelial growth factor (VEGF) was used to confirm that computer-based image analysis was able to quantify angiogenesis. Image analysis was used on samples of increasing porosity of PLLA to determine the effect of pore size on angiogenesis. Another effect also noted was that of an inflammatory response co-incident with angiogenesis. The difference in pore size made a difference to both angiogenesis and inflammation. Real-time polymerase chain reaction (PCR) was used with primers for TNF-α to demonstrate and measure the presence of an inflammatory response.
Keywords: Angiogenesis; CAM; PLLA; Inflammation;

To date, there have been a number of cases where glucose sensors have performed well over long periods of implantation; however, it remains difficult to predict whether a given sensor will perform reliably, will exhibit gradual degradation of performance, or will fail outright soon after implantation. Typically, the literature emphasizes the sensor that performed well, while only briefly (if at all) mentioning the failed devices. This leaves open the question of whether current sensor designs are adequate for the hostile in vivo environment, and whether these sensors have been assessed by the proper regimen of testing protocols. This paper reviews the current in vitro and in vivo testing procedures used to evaluate the functionality and biocompatibility of implantable glucose sensors. An overview of the standards and regulatory bodies that govern biomaterials and end product device testing precedes a discussion of up-to-date invasive and non-invasive technologies for diabetes management. Analysis of current in vitro, in vivo, and then post explantation testing is presented. Given the underlying assumption that the success of the sensor in vitro foreshadows the long-term reliability of the sensor in the human body, the relative merits of these testing methods are evaluated with respect to how representative they are of human models.
Keywords: Biosensor; Foreign body response; In vitro test; In vivo test; wound healing;

Postoperative peritoneal adhesions can have serious, potentially lethal consequences. Pharmacotherapy and barrier devices can reduce adhesion formation to varying degrees, but their efficacy is limited by rapid clearance from the peritoneum and lack of biological activity, respectively. To overcome these limitations, we have delivered tissue-type plasminogen activator (tPA), which is deficient in the first 2–3 postoperative days, using a highly cross-linked in situ forming hyaluronan gel (HAXhx). We demonstrated this formulation's anti-adhesion activity in a rigorous animal model that involved recurrent adhesions. While non-treated or saline-treated animals developed widespread adhesions (frequency, both 100%; median adhesion area, 12.7 and 15.4 cm2, respectively), tPA delivered by HAXhx (tPA-HAXhx) was highly effective in preventing recurrent adhesions (frequency, 44%; median adhesion area, 0.1 cm2). HAXhx itself, tPA solution, and inactivated tPA in HAXhx did not provide comparable anti-adhesion activity. tPA-HAXhx is a system that is easy to use and potentially promising for adhesion prevention.
Keywords: Peritoneal adhesion; Hyaluronan hydrogel; Tissue-type plasminogen activator; Drug delivery; Repeated injury;

Elucidation of the physicomechanical and ab initio quantum energy transitions of a crosslinked PLGA scaffold by Sibongile R. Sibambo; Viness Pillay; Yahya E. Choonara; Riaz A. Khan; Joe L. Sweet (3714-3723).
This study elucidated the in vitro physicomechanical transitions of a crosslinked polylactic-co-glycolic acid (PLGA) scaffold, utilizing quantum mechanics to compute the ab initio energy requirements of a salted-out and subsequently crosslinked PLGA scaffold interacting with simulated physiological fluid, phosphate buffered saline (PBS) (pH 7.4, 37 °C) at a molecular level. Twenty-six salted-out PLGA scaffolds were formulated using a four factor, two centerpoint quadratic Face-Centered Central Composite Design (FCCD). PLGA molecular mass, PLGA concentration, water volume and salting-out reaction time were the dependant formulation variables. Subsequent to PLGA solubilization in dimethyl formamide (DMF), protonated water was added to induce salting-out of PLGA into a scaffolds that were immersed in PBS, oscillated at 100 rpm, and analyzed at pre-determined time intervals for their physicomechanical and ab initio quantum energy transitions. Results indicated that the matrix resilience (MR) decreased with longer incubation periods (MR=35–45%) at day 30. Scaffolds salted-out using higher PLGA concentrations exhibited minimal changes in MR and the matrix ability to absorb energy was found to closely correlate with the scaffold residence time in PBS. Spartan-based ab initio quantum energy predictions elucidated the potential scaffold stability from a molecular viewpoint and its suitability for use in rate-modulated drug delivery.
Keywords: Polylactic-co-glycolic acid; Crosslinking; Physicomechanical; Ab initio quantum mechanics; Drug delivery;

Paclitaxel and etanidazole are hypoxic radiosensitizers that exhibit cytotoxic action at different mechanisms. The poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel, etanidazole and paclitaxel+etanidazole were prepared by o/w and w/o/w emulsification-solvent evaporation method. The morphology of the nanoparticles was investigated by scanning electron microscope (SEM). The drug encapsulation efficiency (EE) and release profile in vitro were measured by high-performance liquid chromatography (HPLC). The cellular uptake of nanoparticles for the human breast carcinoma cells (MCF-7) and the human carcinoma cervicis cells (HeLa) was evaluated by transmission electronic microscopy and fluorescence microscopy. Cell viability was determined by the ability of single cell to form colonies in vitro. The prepared nanoparticles were spherical shape with size between 80 and 150 nm. The EE was higher for paclitaxel and lower for etanidazole. The drug release was controlled over time. The cellular uptake of nanoparticles was observed. Co-culture of the two tumor cell lines with drug-loaded nanoparticles demonstrated that released drug effectively sensitized hypoxic tumor cells to radiation. The radiosensitization of paclitaxel+etanidazole nanoparticles was more significant than that of single drug-loaded nanoparticles.
Keywords: Combined radiosensitizers; Paclitaxel; Etanidazole; Nanoparticle; Hypoxia; Human tumor cells;

Synthesis and characterization of chemically condensed oligoethylenimine containing beta-aminopropionamide linkages for siRNA delivery by Peter J. Tarcha; Jaroslav Pelisek; Thomas Merdan; Jan Waters; Kent Cheung; Katharina von Gersdorff; Carsten Culmsee; Ernst Wagner (3731-3740).
Polyethylenimines (PEI) are often inefficient in gene knockdown experiments with small interfering RNA (siRNA), presumably due to the strong complexing properties. A more efficient and potentially degradable oligoethylenimine-based carrier was synthesized by the condensation of 800 molecular weight PEI oligomers with hexanedioldiacrylate. Reaction conditions were chosen such that Michael reaction occurs followed by complete N-acylation of all residual ester bonds resulting in beta-aminopropionamide linkage sites and an average molecular weight of 30,000. Based on NMR analysis, these conditions produced 38% tertiary amides and 62% secondary amides, with about 2% residual carboxylate, presumably from hydrolysis. The ionizable equivalent weight of the carrier increased to 51, compared to a value of 43 for standard PEI. Sensible in vitro knockdown of the luciferase gene in stably transfected HUH7 cells, up to 80% in comparison to non-specific siRNA, demonstrated its suitability for siRNA delivery.
Keywords: siRNA delivery; Biodegradable nucleic acid carrier; Condensation of polyethylenimine;

Specific molecularly imprinted polymers (MIPs) for the drug reserpine (RES) using methacrylic acid (MAA) as the functional monomer were developed and characterised for the first time in this study. Evaluation of the various polymers by binding assays indicated that the optimum ratio of functional monomer to template was 4:1. Furthermore, the imprinting effect of the MIPs was assessed by the chromatographic method, which demonstrated that the MIPs had better chromatographic behavior and selectivity than those of the corresponding NIPs. A combination of BET, NMR, UV spectroscopy, and MISPE analyses for investigation of the imprinting and recognition properties revealed that strong specific interactions between the functional monomer and RES in the prepolymerization solutions and the aqueous solutions were probably responsible for RES recognition. The preparation of RES MIPs and elucidation of imprinting and recognition mechanisms may serve as useful references for other drug MIPs.
Keywords: Molecularly imprinted polymers; Solid-phase extraction; Reserpine; Drugs;