Layer-by-layer assembly of poly-l-lysine/hyaluronic acid protein reservoirs on poly(glycerol sebacate) surfaces.

The modification of biomaterial surfaces has become increasingly relevant in the context of ongoing advancements in tissue engineering applications and the development of tissue-mimicking polymer materials. In this study, we investigated the layer-by-layer (LbL) deposition of polyelectrolyte multilayer protein reservoirs consisting of poly-l-lysine (PLL) and hyaluronic acid (HA) on the hydrophobic surface of poly(glycerol sebacate) (PGS) elastomer. Using the methods of isothermal titration calorimetry and surface plasmon resonance, we systematically investigated the interactions between the polyelectrolytes and evaluated the deposition process in real time, providing insight into the phenomena associated with film assembly. PLL/HA LbL films deposited on PGS showed an exceptional ability to incorporate bone morphogenetic protein-2 (BMP-2) compared to other growth factors tested, thus highlighting the potential of PLL/HA LbL films for osteoregenerative applications. The concentration of HA solution used for film assembly did not affect the thickness and topography of the (PLL/HA)10 films, but had a notable impact on the hydrophilicity of the PGS surface and the BMP-2 release kinetics. The release kinetics were successfully described using the Weibull model and hyperbolic tangent function, underscoring the potential of these less frequently used models to compare the protein release from LbL protein reservoirs.

Quaternized Chitosan/Heparin Polyelectrolyte Multilayer Films for Protein Delivery.

Layer-by-layer (LbL) polyelectrolyte coatings are intensively studied as reservoirs of bioactive proteins for modulating interactions between biomaterial surfaces and cells. Mild conditions for the incorporation of growth factors into delivery systems are required to maintain protein bioactivity. Here, we present LbL films composed of water-soluble N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC), heparin (Hep), and tannic acid (TA) fabricated under physiological conditions with the ability to release heparin-binding proteins. Surface plasmon resonance analysis showed that the films formed on an anchoring HTCC/TA bilayer, with TA serving as a physical crosslinker, were more stable during their assembly, leading to increased film thickness and increased protein release. X-ray reflectivity measurements confirmed intermixing of the deposited layers. Protein release also increased when the proteins were present as an integral part of the Hep layers rather than as individual protein layers. The 4-week release pattern depended on the protein type; VEGF, CXCL12, and TGF-ß1 exhibited a typical high initial release, whereas FGF-2 was sustainably released over 4 weeks. Notably, the films were nontoxic, and the released proteins retained their bioactivity, as demonstrated by the intensive chemotaxis of T-lymphocytes in response to the released CXCL12. Therefore, the proposed LbL films are promising biomaterial coating candidates for stimulating cellular responses.

Complexation of CXCL12, FGF-2 and VEGF with Heparin Modulates the Protein Release from Alginate Microbeads.

Long-term delivery of growth factors and immunomodulatory agents is highly required to support the integrity of tissue in engineering constructs, e.g., formation of vasculature, and to minimize immune response in a recipient. However, for proteins with a net positive charge at the physiological pH, controlled delivery from negatively charged alginate (Alg) platforms is challenging due to electrostatic interactions that can hamper the protein release. In order to regulate such interactions between proteins and the Alg matrix, we propose to complex proteins of interest in this study - CXCL12, FGF-2, VEGF - with polyanionic heparin prior to their encapsulation into Alg microbeads of high content of α-L-guluronic acid units (high-G). This strategy effectively reduced protein interactions with Alg (as shown by model ITC and SPR experiments) and, depending on the protein type, afforded control over the protein release for at least one month. The released proteins retained their in vitro bioactivity: CXCL12 stimulated the migration of Jurkat cells, and FGF-2 and VEGF induced proliferation and maturation of HUVECs. The presence of heparin also intensified protein biological efficiency. The proposed approach for encapsulation of proteins with a positive net charge into high-G Alg hydrogels is promising for controlled long-term protein delivery under in vivo conditions.

pH-responsive and antibacterial properties of self-assembled multilayer films based on chitosan and tannic acid.

Polyelectrolyte layer-by-layer (LbL) films that disintegrate under physiological conditions are intensively studied as coatings to enable the release of bioactive components. Herein, we report on the interactions and pH-stability of LbL films composed of chitosan (CH) or N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (CMCH) and tannic acid (TA), employed to guarantee the film disintegration. The self-assembly of TA with CH and CMCH at pH 5 and with CMCH at pH 7.4 were proven by turbidimetric, surface plasmon resonance and UV-Vis analyses. The LbL films exhibited pH-dependent properties; CMCH/TA films prepared at pH 7.4 showed exponential growth as well as a higher layer thickness and surface roughness, whereas films prepared at pH 5 grew linearly and were smoother. The film stability varied with the pH used for film assembly; CH/TA films assembled at pH 5 were unstable at pH 8.5, whereas CMCH/TA films assembled at pH 7.4 disintegrated at pH 4. All films exhibited a similar disassembly at pH 7.4. The coatings reduced the adhesion of E. coli and S. aureus by approximately 80%. CMCH-terminated CMCH/TA films were more resistant to bacterial adhesion, whereas CH-terminated CH/TA films demonstrated stronger killing activity. The prepared pH-triggered decomposable LbL films could be used as degradable coatings that allow the release of therapeutics for biomedical applications and also prevent bacterial adhesion.

Correction: Bioengineering a pre-vascularized pouch for subsequent islet transplantation using VEGF-loaded polylactide capsules.

Correction for 'Bioengineering a pre-vascularized pouch for subsequent islet transplantation using VEGF-loaded polylactide capsules' by Naresh Kasoju et al., Biomater. Sci., 2020, DOI: 10.1039/c9bm01280j.

Bioengineering a pre-vascularized pouch for subsequent islet transplantation using VEGF-loaded polylactide capsules.

The effectiveness of cell transplantation can be improved by optimization of the transplantation site. For some types of cells that form highly oxygen-demanding tissue, e.g., pancreatic islets, a successful engraftment depends on immediate and sufficient blood supply. This critical point can be avoided when cells are transplanted into a bioengineered pre-vascularized cavity which can be formed using a polymer scaffold. In our study, we tested surface-modified poly(lactide-co-caprolactone) (PLCL) capsular scaffolds containing the pro-angiogenic factor VEGF. After each modification step (i.e., amination and heparinization), the surface properties and morphology of scaffolds were characterized by ATR-FTIR and XPS spectroscopy, and by SEM and AFM. All modifications preserved the gross capsule morphology and maintained the open pore structure. Optimized aminolysis conditions decreased the Mw of PLCL only up to 10% while generating a sufficient number of NH2 groups required for the covalent immobilization of heparin. The heparin layer served as a VEGF reservoir with an in vitro VEGF release for at least four weeks. In vivo studies revealed that to obtain highly vascularized PLCL capsules (a) the optimal VEGF dose for the capsule was 50 µg and (b) the implantation time was four weeks when implanted into the greater omentum of Lewis rats; dense fibrous tissue accompanied by vessels completely infiltrated the scaffold and created sparse granulation tissue within the internal cavity of the capsule. The prepared pre-vascularized pouch enabled the islet graft survival and functioning for at least 50 days after islet transplantation. The proposed construct can be used to create a reliable pre-vascularized pouch for cell transplantation.

Effect of Blood Component Coatings of Enosseal Implants on Proliferation and Synthetic Activity of Human Osteoblasts and Cytokine Production of Peripheral Blood Mononuclear Cells.

The study monitored in vitro early response of connective tissue cells and immunocompetent cells to enosseal implant materials coated by different blood components (serum, activated plasma, and plasma/platelets) to evaluate human osteoblast proliferation and synthetic activity and inflammatory response presented as a cytokine profile of peripheral blood mononuclear cells (PBMCs) under conditions imitating the situation upon implantation. The cells were cultivated on coated Ti-plasma-sprayed (Ti-PS), Ti-etched (Ti-Etch), Ti-hydroxyapatite (Ti-HA), and ZrO2 surfaces. The plasma/platelets coating supported osteoblast proliferation only on osteoconductive Ti-HA and Ti-Etch whereas activated plasma enhanced proliferation on all surfaces. Differentiation (BAP) and IL-8 production remained unchanged or decreased irrespective of the coating and surface; only the serum and plasma/platelets-coated ZrO2 exhibited higher BAP and IL-8 expression. RANKL production increased on serum and activated plasma coatings. PBMCs produced especially cytokines playing role in inflammatory phase of wound healing, that is, IL-6, GRO-α, GRO, ENA-78, IL-8, GM-CSF, EGF, and MCP-1. Cytokine profiles were comparable for all tested surfaces; only ENA-78, IL-8, GM-CSF, and MCP-1 expression depended on materials and coatings. The activated plasma coating led to uniformed surfaces and represented a favorable treatment especially for bioinert Ti-PS and ZrO2 whereas all coatings had no distinctive effect on bioactive Ti-HA and Ti-Etch.

Silk fibroin gelation via non-solvent induced phase separation.

Tissue engineering benefits from novel materials with precisely tunable physical, chemical and mechanical properties over a broad range. Here we report a practical approach to prepare Bombyx mori silk fibroin hydrogels using the principle of non-solvent induced phase separation (NIPS). A combination of reconstituted silk fibroin (RSF) and methanol (non-solvent), with a final concentration of 2.5% w/v and 12.5% v/v respectively, maintained at 22 °C temperature turned into a hydrogel within 10 hours. Freeze-drying of this gel gave a foam with a porosity of 88%, a water uptake capacity of 89% and a swelling index of 8.6. The gelation kinetics and the loss tangent of the gels were investigated by rheometry. The changes in the morphology of the porous foams were visualized by SEM. The changes in RSF chemical composition and the relative fraction of its secondary structural elements were analyzed by ATR-FTIR along with Fourier self-deconvolution. And, the changes in the glass transition temperature, specific heat capacity and the relative fraction of crystallinity of RSF were determined by TM-DSC. Data suggested that RSF-water-methanol behaved as a polymer-solvent-non-solvent ternary phase system, wherein the demixing of the water-methanol phases altered the thermodynamic equilibrium of RSF-water phases and resulted in the desolvation and eventual separation of the RSF phase. Systematic analysis revealed that both gelation time and the properties of hydrogels and porous foams could be controlled by the ratios of RSF and non-solvent concentration as well as by the type of non-solvent and incubation temperature. Due to the unique properties we envisage that the herein prepared NIPS induced RSF hydrogels and porous foams can possibly be used for the encapsulation of cells and/or for the controlled release of both hydrophilic and hydrophobic drugs.

Polymer scaffolds with no skin-effect for tissue engineering applications fabricated by thermally induced phase separation.

Thermally induced phase separation (TIPS) based methods are widely used for the fabrication of porous scaffolds for tissue engineering and related applications. However, formation of a less-/non-porous layer at the scaffold's outer surface at the air-liquid interface, often known as the skin-effect, restricts the cell infiltration inside the scaffold and therefore limits its efficacy. To this end, we demonstrate a TIPS-based process involving the exposure of the just quenched poly(lactide-co-caprolactone):dioxane phases to the pure dioxane for a short time while still being under the quenching strength, herein after termed as the second quenching (2Q). Scanning electron microscopy, mercury intrusion porosimetry and contact angle analysis revealed a direct correlation between the time of 2Q and the gradual disappearance of the skin, followed by the widening of the outer pores and the formation of the fibrous filaments over the surface, with no effect on the internal pore architecture and the overall porosity of scaffolds. The experiments at various quenching temperatures and polymer concentrations revealed the versatility of 2Q in removing the skin. In addition, the in vitro cell culture studies with the human primary fibroblasts showed that the scaffolds prepared by the TIPS based 2Q process, with the optimal exposure time, resulted in a higher cell seeding and viability in contrast to the scaffolds prepared by the regular TIPS. Thus, TIPS including the 2Q step is a facile, versatile and innovative approach to fabricate the polymer scaffolds with a skin-free and fully open porous surface morphology for achieving a better cell response in tissue engineering and related applications.

Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies.

In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm(2). The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm(2)) than on surfaces with a higher concentration of FGF-2 (120 ng/cm(2)).

Self-assembled anchor layers/polysaccharide coatings on titanium surfaces: a study of functionalization and stability.

Composite materials based on a titanium support and a thin, alginate hydrogel could be used in bone tissue engineering as a scaffold material that provides biologically active molecules. The main objective of this contribution is to characterize the activation and the functionalization of titanium surfaces by the covalent immobilization of anchoring layers of self-assembled bisphosphonate neridronate monolayers and polymer films of 3-aminopropyltriethoxysilane and biomimetic poly(dopamine). These were further used to bind a bio-functional alginate coating. The success of the titanium surface activation, anchoring layer formation and alginate immobilization, as well as the stability upon immersion under physiological-like conditions, are demonstrated by different surface sensitive techniques such as spectroscopic ellipsometry, infrared reflection-absorption spectroscopy and X-ray photoelectron spectroscopy. The changes in morphology and the established continuity of the layers are examined by scanning electron microscopy, surface profilometry and atomic force microscopy. The changes in hydrophilicity after each modification step are further examined by contact angle goniometry.

Dip TIPS as a facile and versatile method for fabrication of polymer foams with controlled shape, size and pore architecture for bioengineering applications.

The porous polymer foams act as a template for neotissuegenesis in tissue engineering, and, as a reservoir for cell transplants such as pancreatic islets while simultaneously providing a functional interface with the host body. The fabrication of foams with the controlled shape, size and pore structure is of prime importance in various bioengineering applications. To this end, here we demonstrate a thermally induced phase separation (TIPS) based facile process for the fabrication of polymer foams with a controlled architecture. The setup comprises of a metallic template bar (T), a metallic conducting block (C) and a non-metallic reservoir tube (R), connected in sequence T-C-R. The process hereinafter termed as Dip TIPS, involves the dipping of the T-bar into a polymer solution, followed by filling of the R-tube with a freezing mixture to induce the phase separation of a polymer solution in the immediate vicinity of T-bar; Subsequent free-drying or freeze-extraction steps produced the polymer foams. An easy exchange of the T-bar of a spherical or rectangular shape allowed the fabrication of tubular, open- capsular and flat-sheet shaped foams. A mere change in the quenching time produced the foams with a thickness ranging from hundreds of microns to several millimeters. And, the pore size was conveniently controlled by varying either the polymer concentration or the quenching temperature. Subsequent in vivo studies in brown Norway rats for 4-weeks demonstrated the guided cell infiltration and homogenous cell distribution through the polymer matrix, without any fibrous capsule and necrotic core. In conclusion, the results show the "Dip TIPS" as a facile and adaptable process for the fabrication of anisotropic channeled porous polymer foams of various shapes and sizes for potential applications in tissue engineering, cell transplantation and other related fields.

Polylactide nanofibers with hydroxyapatite as growth substrates for osteoblast-like cells.

Various types of nanofibers are increasingly used in tissue engineering, mainly for their ability to mimic the architecture of tissue at the nanoscale. We evaluated the adhesion, growth, viability, and differentiation of human osteoblast-like MG 63 cells on polylactide (PLA) nanofibers prepared by needle-less electrospinning and loaded with 5 or 15 wt % of hydroxyapatite (HA) nanoparticles. On day 7 after seeding, the cell number was the highest on samples with 15 wt % of HA. This result was confirmed by the XTT test, especially after dynamic cultivation, when the number of metabolically active cells on these samples was even higher than on control polystyrene. Staining with a live/dead kit showed that the viability of cells on all nanofibrous scaffolds was very high and comparable to that on control polystyrene dishes. An enzyme-linked immunosorbent assay revealed that the concentration of osteocalcin was also higher in cells on samples with 15 wt % of HA. There was no immune activation of cells (measured by production of TNF-alpha), associated with the incorporation of HA. Moreover, the addition of HA suppressed the creep behavior of the scaffolds in their dry state. Thus, nanofibrous PLA scaffolds have potential for bone tissue engineering, particularly those with 15 wt % of HA.

Adhesion and growth of vascular smooth muscle cells in cultures on bioactive RGD peptide-carrying polylactides.

The surface of poly(L-lactide) (PLLA) films deposited on glass coverslips was modified with poly(DL-lactide) (PDLLA), or 1:4 mixtures of PDLLA and PDLLA-b-PEO block copolymers, in which either none, 5% or 20% of the copolymer molecules carried a synthetic extracellular matrix-derived ligand for integrin adhesion receptors, the GRGDSG oligopeptide, attached to the end of the PEO chain. The materials, perspective for vascular tissue engineering, were seeded with rat aortic smooth muscle cells (11,000 cells/cm(2)) and the adhesion, spreading, DNA synthesis and proliferation of these cells was followed on inert and bioactive surfaces. In 24-h-old cultures in serum-supplemented media, the number of cells adhering to the PDLLA-b-PEO copolymer was almost eight times lower than that on the control PDLLA surface. On the surfaces containing 5% and 20% GRGDSG-PEO-b-PDLLA copolymer, the number of cells increased 6- and 3-fold respectively, compared to the PDLLA-b-PEO copolymer alone. On PDLLA-b-PEO copolymer alone, the cells were typically round and non-spread, whereas on GRGDSG-modified surfaces the cell spreading areas approached those found on PDLLA, reaching values of 991 microm(2) and 611 microm(2) for 5% and 20% GRGDSG respectively, compared to 958 microm(2) for PDLLA. The cells on GRGDSG-grafted copolymers were able to form vinculin-containing focal adhesion plaques, to synthesize DNA and even proliferate in a serum-free medium, which indicates specific binding to the GRGDSG sequences through their adhesion receptors.

Adhesion and growth of rat aortic smooth muscle cells on lactide-based polymers.

Biodegradable materials based on polymers of hydroxy acids are studied for application in artificial vascular substitutes. Polymers with functional surfaces are being developed, carrying specific recognition structures to affect selectively the adhesion and proliferation of endothelial cells (EC) and vascular smooth muscle cells (VSMC). This preliminary study focuses on evaluation of adhesion and growth of VSMC on surfaces of polylactide polymers and those modified by amphiphilic polylactide/poly(ethylene oxide) copolymers. Poly(L-lactic acid), PLLA, and poly(DL-lactic acid), PDLLA, and a block copolymer of lactide with a carboxylated poly(ethylene oxide) segment, PLLA-b-PEO-COOH, were synthesized by controlled polymerization of L and D,L-lactide, respectively, and using delta-hydroxy-Z-carboxymethyl-PEO as a macroinitiator for the copolymer. Films of polymers were deposited on glass coverslips by a spin-coating method. Uncoated glass coverslips and Falcon dishes were used as control substrates. VSMC were obtained from the thoracic aorta of young adult male Wistar rats by explantation method and seeded in Dulbecco-Modified Eagle MEM with 10% foetal bovine serum. The number of adhering cells, their shape, size of cell-material contact area and cell population doubling time were evaluated from day 1 to 7 after seeding. It was found that both PLLA and especially PDLLA relatively well supported adhesion and growth of VSMC. However, on carboxylated surfaces of the PLLA-b-PEO-COOH copolymer, a lower number of initially adhering cells (by 37% than on Falcon dishes, pdelta0.05), smaller cell spreading area (by 45% and 37% than on glass and Falcon dishes, respectively, pdelta0.01) and longer doubling time (by 49% and 31% than on glass and Falcon dishes, pdelta0.001). Thus, surfaces coated by a PLA/PEO-COOH copolymer can be used as minimum background surface to reveal the effect of other more specific adhesion structures.