Dana Kubies

Microdomain structure in polylactide-block-poly(ethylene oxide) copolymer films

Structured surface is an important property of polymer biomaterials for tissue engineering, for its capacity to expose domains with different surface energy and functional groups. For this purpose, amphiphilic A-B-A block copolymers with polylactide (PLA) as A blocks and poly(ethylene oxide) (PEO 3, Mn = 3090; PEO6, Mn = 6110) as B block were synthesized by ring-opening polymerization of either L-lactide (L-LA) or DL-lactide (DL-LA), using poly(ethylene glycol)s as macroinitiators and tin(II) octanoate (Sn(Oct)2) as a catalyst. Differential scanning calorimetry (DSC) and electron microscopy were used to study the phase separation of the hydrophobic (PLA) and hydrophilic (PEO) segments in films made of the copolymers and their blends with high-molecular-weight PLA homopolymers. Hydrophilic (PEO) and hydrophobic (PLA) domains were formed at the polymer film surface due to the separation of phases. The phase separation was affected by the copolymer composition and the stereoregularity of PLA blocks in the copolymers.

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

Summary 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.

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.

Functionalized surfaces of polylactide modified by Langmuir-Blodgett films of amphiphilic block copolymers.

To modify the surface of poly(L-lactide) (PLA) supports, we have investigated the feasibility to deposit on the PLA surface Langmuir–Blodgett films of amphiphilic block copolymers based on poly(L-lactide). AB and ABA block copolymers were prepared with PLA as the A block and either poly(ethylene oxide), α-methoxy-ω-hydroxy poly(ethylene oxide), α-carboxy-ω-hydroxy poly(ethylene oxide) or poly(L-aspartic acid) as the B blocks. Films with phase-separated hydrophilic and hydrophobic blocks in a bilayer “brush” structure were prepared by compression of the copolymer Langmuir films on the water/air interface. The interfacial behavior of the monolayers and the effect of the copolymer composition on the phase separation was followed by measurements of the surface-pressure/area isotherms using a Langmuir trough and by contact angle measurement of deposited Langmuir–Blodgett (LB) films. The phase separation of the hydrophilic and PLA blocks is more effective in diblock AB copolymers compared with triblock ABA copolymers. The presence of ionic groups in the hydrophilic chains facilitates penetration of hydrophilic segments into the water subphase. Dynamic contact angle measurements were used to study the stability of the LB-films transferred on the PLA support and the changes in the surface properties upon incubation of surfaces in water.

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.

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.

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 scaffolds 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/cm2. 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/cm2) than on surfaces with a higher concentration of FGF-2 (120 ng/cm2).