Cosmin Catalin Mustaciosu

Laser micro-patterning of biodegradable polymer blends for tissue engineering

We propose a multistep all laser, maskless, and solvent free synthesis of micro-patterned substrates of biodegradable polymer blends, with applicability for guided cell adhesion and localized hyaluronic acid (HA) immobilization. The polymer blends comprised polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), and polylactide-polyethylene glycol-polylactide (PPP) in 1:1:1 blending ratios. Polymer patterning was performed by laser processing in two steps. First, the polymers were patterned with periodic micro-channels by direct femtosecond laser ablation, which provided flexibility in design and spatial accuracy for the patterns. As a second step, the micro-patterned polymers were coated with thin layers of polymer blends using matrix assisted pulsed laser evaporation (MAPLE). The resulted sandwich substrates were composed of a bottom, micro-patterned layer and thin, top layer which conserved the patterns from the underlying layer and preserved the polymers chemical composition. Depending on the bottom/top layers, the substrates were denominated PU/PU:PLGA:PPP and PU:PLGA:PPP/PU:PLGA:PPP, respectively. The laser generated micro-patterns were used for selective attachment of oral keratinocyte stem cells and for HA immobilization. The highest cellular density was found on the PU:PLGA:PPP/PU:PLGA:PPP substrate, where the spongy-like micro-channels provided multiple anchoring points for the cells. For both substrates, the micro-channels enabled localized immobilization of HA. The effectiveness of HA immobilization was tested against cell adhesion and protein adsorption.

Electrically stimulated osteogenesis on Ti-PPy/PLGA constructs prepared by laser-assisted processes.

This work describes a versatile laser-based protocol for fabricating micro-patterned, electrically conductive titanium-polypyrrole/poly(lactic-co-glycolic)acid (Ti-PPy/PLGA) constructs for electrically stimulated (ES) osteogenesis. Ti supports were patterned using fs laser ablation in order to create high spatial resolution microstructures meant to provide mechanical resistance and physical cues for cell growth. Matrix Assisted Pulsed Laser Evaporation (MAPLE) was used to coat the patterned Ti supports with PPy/PLGA layers acting as biocompatible surfaces having chemical and electrical properties suitable for cell differentiation and mineralization. In vitro biological assays on osteoblast-like MG63 cells showed that the constructs maintained cell viability without cytotoxicity. At 24 h after cell seeding, electrical stimulation with currents of 200 μA was applied for 4 h. This treatment was shown to promote earlier onset of osteogenesis. More specifically, the alkaline phosphatase activity of the stimulated cultures reached the maximum before that of the non-stimulated ones, i.e. controls, indicating faster cell differentiation. Moreover, mineralization was found to occur at an earlier stage in the stimulated cultures, as compared to the controls, starting with Day 6 of cell culture. At later stages, calcium levels in the stimulated cultures were higher than those in control samples by about 70%, with Ca/P ratios similar to those of natural bone. In all, the laser-based protocol emerges as an efficient alternative to existing fabrication technologies.

3D Biomimetic Magnetic Structures for Static Magnetic Field Stimulation of Osteogenesis

We designed, fabricated and optimized 3D biomimetic magnetic structures that stimulate the osteogenesis in static magnetic fields. The structures were fabricated by direct laser writing via two-photon polymerization of IP-L780 photopolymer and were based on ellipsoidal, hexagonal units organized in a multilayered architecture. The magnetic activity of the structures was assured by coating with a thin layer of collagen-chitosan-hydroxyapatite-magnetic nanoparticles composite. In vitro experiments using MG-63 osteoblast-like cells for 3D structures with gradients of pore size helped us to find an optimum pore size between 20–40 µm. Starting from optimized 3D structures, we evaluated both qualitatively and quantitatively the effects of static magnetic fields of up to 250 mT on cell proliferation and differentiation, by ALP (alkaline phosphatase) production, Alizarin Red and osteocalcin secretion measurements. We demonstrated that the synergic effect of 3D structure optimization and static magnetic stimulation enhances the bone regeneration by a factor greater than 2 as compared with the same structure in the absence of a magnetic field.

Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium

Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human uterine myometrium and their gene expression regulation by beta-estradiol in pregnant conditions. Further exploration of the calcium signaling in TCs and its modulation by estrogen hormones will contribute to the understanding of labor and pregnancy mechanisms and to the development of effective strategies to reduce the risk of premature birth.

Smart Thermoresponsive Surfaces Based on pNIPAm Coatings and Laser Method for Biological Applications

Various applications within last decades such as bacterially resistant surfaces, soft robotics, drug delivery systems, sensors and tissue engineering are poised to feature the importance of the ability to control bio-interfacial interactions. An enhanced attention is dedicated to designing smart stimuli-responsive interfaces for DNA, drug delivery, protein and cell based applications. Within this context, the thermoresponsive materials, especially poly(N-isopropylacrylamide) (pNIPAm) have been intensively used in tissue engineering applications for a controlled detachment of proteins and cells with a minimum of invasive effect on protein and cell structural conformation. The properties of smart bio-interfaces can be controlled by its composition and polymer architecture. Therefore, appropriate methods for obtaining controlled coatings are necessary. Laser methods were successfully used in the last decades for obtaining controlled organic and inorganic coatings for various types of applications, from electronics to tissue engineering. Among these, Matrix-Assisted Pulsed Laser Evaporation (MAPLE) technique bring us a step forward to other laser methods by avoiding damage and photochemical decomposition of materials. In this chapter we describe materials and approaches used for design of smart bio-interfaces aimed at controlling protein and cells behavior in vitro, focusing MAPLE method for tuning coatings characteristics in relation with biological response.

Capsaicin is a key molecule in neuropathic pain induced by diabetes

Murine kodecytes bearing both blood group A antigen and biotin (A+biotin kodecytes) were created by incubating equal volumes of packed murine red cells with a solution containing ( ) FSL-biotin and (10 ) of FSL-A (or FSL-GB3 as negative control). These A+biotin kodecytes or GB3+biotin kodecytes were then transfused (2 into the circulation of laboratory mice with or without anti-A (stimulated by immunisation with salivary blood group A substance). Blood was sampled ( ) at specific time points post transfusion and, using the secondary reagent surviving kodecytes could be identified in blood films and fluorescence microscopy (figure 2). Additionally b