Exploring the application of poly(1,2-ethanediol citrate)/polylactide nonwovens in cell culturing.

Biomaterials containing citric acid as a building unit show potential for use as blood vessel and skin tissue substitutes. The success in commercializing implants containing a polymer matrix of poly(1,8-octanediol citrate) provides a rationale for exploring polycitrates based on other diols. Changing the aliphatic chain length of the diol allows functional design strategies to control the implant's mechanical properties, degradation profile and surface energy. In the present work, poly(1,2-ethanediol citrate) was synthesized and used as an additive to polylactide in the electrospinning process. It was established that the content of polycitrate greatly influences the nonwovens' properties: an equal mass ratio of polymers resulted in the best morphology. The obtained nonwovens were characterized by surface hydrophilicity, tensile strength, and thermal properties. L929 cell cultures were carried out on their surface. The materials were found to be non-cytotoxic and the degree of porosity was suitable for cell colonization. On the basis of the most important parameters for assessing the condition of cultured cells (cell density and viability, cell metabolic activity and lactate dehydrogenase activity), the potential of PLLA + PECit nonwovens for application in tissue engineering was established.

Biological Activity of Poly(1,3-propanediol citrate) Films and Nonwovens: Mechanical, Thermal, Antimicrobial, and Cytotoxicity Studies.

Polymers are of great interest for medical and cosmeceutical applications. The current trend is to combine materials of natural and synthetic origin in order to obtain products with appropriate mechanical strength and good biocompatibility, additionally biodegradable and bioresorbable. Citric acid, being an important metabolite, is an interesting substance for the synthesis of materials for biomedical applications. Due to the high functionality of the molecule, it is commonly used in biomaterials chemistry as a crosslinking agent. Among citric acid-based biopolyesters, poly(1,8-octanediol citrate) is the best known. It shows application potential in soft tissue engineering. This work focuses on a much less studied polyester, poly(1,3-propanediol citrate). Porous and non-porous materials based on the synthesized polyesters are prepared and characterized, including mechanical, thermal, and surface properties, morphology, and degradation. The main focus is on assessing the biocompatibility and antimicrobial properties of the materials.

Poly(glycerol itaconate) Crosslinking via the aza-Michael Reaction-A Preliminary Research.

In unsaturated glycerol polyesters, the C=C bond is present. It makes it possible to carry out post-polymerisation modification (PPM) reactions, such as aza-Michael addition. This reaction can conduct crosslinking under in-situ conditions for tissue engineering regeneration. Until now, no description of such use of aza-Michael addition has been described. This work aims to crosslink the synthesised poly(glycerol itaconate) (PGItc; P3), polyester from itaconic acid (AcItc), and glycerol (G). The PGItc syntheses were performed in three ways: without a catalyst, in the presence of p-toluenesulfonic acid (PTSA), and in the presence of zinc acetate (Zn(OAc)2). PGItc obtained with Zn(OAc)2 (150 °C, 4 h, G:AcItc = 2:1) was used to carry out the aza-Michael additions. Crosslinking reactions were conducted with each of the five aliphatic diamines: 1,2-ethylenediamine (1,2-EDA; A1), 1,4-butanediamine (1,4-BDA; A2), 1,6-hexanediamine (1,6-HDA; A3), 1,8-octanediamine (1,8-ODA; A4), and 1,10-decanediamine (1,10-DDA; A5). Four ratios of the proton amine group: C=C bond were investigated. The maximum temperature and crosslinking time were measured to select the best amine for the addition product's application. FTIR, 1H NMR, DSC, and TG analysis of the crosslinked products were also investigated.

A Study of the Properties of Scaffolds for Bone Regeneration Modified with Gel-like Coatings of Chitosan and Folic Acid.

The research has been conducted to obtain scaffolds for cancellous bone regeneration. Polylactide scaffolds were made by the phase inversion method with a freeze-extraction variant, including gelling polylactide in its non-solvent. Substitutes made of polylactide are hydrophobic, which limits cell adhesion. For this reason, the scaffolds were modified using chitosan and folic acid by forming gel-like coatings on the surface. The modification aimed to improve the material's surface properties and increase cell adhesion. Analyses of obtained scaffolds confirmed the effectiveness of performed changes. The presence of chitosan and folic acid was confirmed in the modified scaffolds, while all scaffolds retained high open porosity, which is essential for proper cell growth inside the scaffold and the free flow of nutrients. Hydrostatic weighing showed that the scaffolds have high mass absorbability, allowing them to be saturated with biological fluids. There were also cytotoxicity tests performed on 24 h extracts of the materials obtained, which indicated a lack of cytotoxic effect.

From Poly(glycerol itaconate) Gels to Novel Nonwoven Materials for Biomedical Applications.

Electrospinning is a process that has attracted significant interest in recent years. It provides the opportunity to produce nanofibers that mimic the extracellular matrix. As a result, it is possible to use the nonwovens as scaffolds characterized by high cellular adhesion. This work focused on the synthesis of poly(glycerol itaconate) (PGItc) and preparation of nonwovens based on PGItc gels and polylactide. PGItc gels were synthesized by a reaction between itaconic anhydride and glycerol. The use of a mixture of PGItc and PLA allowed us to obtain a material with different properties than with stand-alone polymers. In this study, we present the influence of the chosen ratios of polymers and the OH/COOH ratio in the synthesized PGItc on the properties of the obtained materials. The addition of PGItc results in hydrophilization of the nonwovens' surface without disrupting the high porosity of the fibrous structure. Spectral and thermal analyzes are presented, along with SEM imagining. The preliminary cytotoxicity research showed that nonwovens were non-cytotoxic materials. It also helped to pre-determine the potential application of PGItc + PLA nonwovens as subcutaneous tissue fillers or drug delivery systems.

Synthesis and Cytotoxicity Studies of Poly(1,4-butanediol citrate) Gels for Cell Culturing.

One of the main branches of regenerative medicine is biomaterials research, which is designed to develop and study materials for regenerative therapies, controlled drug delivery systems, wound dressings, etc. Research is continually being conducted to find biomaterials-especially polymers-with better biocompatibility, broader modification possibilities and better application properties. This study describes a potential biomaterial, poly(1,4-butanediol citrate). The gelation time of poly(1,4-butanediol citrate) was estimated. Based on this, the limiting reaction time and temperature were determined to avoid gelling of the reaction mixture. Experiments with different process conditions were carried out, and the products were characterised through NMR spectra analysis. Using statistical methods, the functions were defined, describing the dependence of the degree of esterification of the acid groups on the following process parameters: temperature and COOH/OH group ratio. Polymer films from the synthesised polyester were prepared and characterised. The main focus was assessing the initial biocompatibility of the materials.

Chitosan-Based High-Intensity Modification of the Biodegradable Substitutes for Cancellous Bone.

An innovative approach to treating bone defects is using synthetic bone substitutes made of biomaterials. The proposed method to obtain polylactide scaffolds using the phase inversion technique with a freeze extraction variant enables the production of substitutes with morphology similar to cancellous bone (pore size 100-400 µm, open porosity 94%). The high absorbability of the implants will enable their use as platelet-rich plasma (PRP) carriers in future medical devices. Surface modification by dipping enabled the deposition of the hydrophilic chitosan (CS) layer, maintaining good bone tissue properties and high absorbability (850% dry weight). Introducing CS increases surface roughness and causes local changes in surface free energy, promoting bone cell adhesion. Through this research, we have developed a new and original method of low-temperature modification of PLA substitutes with chitosan. This method uses non-toxic reagents that do not cause changes in the structure of the PLA matrix. The obtained bone substitutes are characterised by exceptionally high hydrophilicity and morphology similar to spongy bone. In vitro studies were performed to analyse the effect of morphology and chitosan on cellular viability. Substitutes with properties similar to those of cancellous bone and which promote bone cell growth were obtained.

Optimization of the Poly(glycerol citraconate) Synthesis Using the Box-Behnken Design.

This work aimed to obtain poly(glycerol citraconate) (PGCitrn) for biomedical applications, analyze the obtained polyester by spectroscopic methods, and optimize its preparation. Polycondensation reactions of glycerol and citraconic anhydride were carried out. It was provided that the results in the reaction are oligomers of poly(glycerol citraconate). Optimization studies were carried out based on the Box-Behnken design. The input variables in this plan were the ratio of functional groups, temperature, and time and occurrence in coded form: -1, 0, or 1. Three output variables were optimized: the degree of esterification, the percentage of Z-mers, and the degree of carboxyl group conversion; they were determined by titration and spectroscopic methods. The optimization criterion was to maximize the values of output variables. A mathematical model and an equation describing it were determined for each output variable. The models predicted the experimental results well. An experiment was conducted under determined optimal conditions. The experimental results were very close to the calculated values. Poly(glycerol citraconate) oligomers with an esterification degree of 55.2%, a Z-mer content of 79.0%, and a degree of rearrangement of carboxyl groups of 88.6% were obtained. The obtained PGCitrn can serve as a component of an injectable implant. The obtained material can be used to produce nonwoven fabrics (with the addition of PLLA, for example), which can be subjected to a cytotoxicity test which can then serve as a dressing material.

Stress-Induced Intensification of Deoxyshikonin Production in Rindera graeca Hairy Root Cultures with Ester-Based Scaffolds.

In vitro plant cell and tissue culture systems allow for controlling a wide range of culture environmental factors selectively influencing biomass growth and the yield of secondary metabolites. Among the most efficient methods, complex supplementation of the culture medium with elicitors, precursors, and other functional substances may significantly enhance valuable metabolite productivity through a stress induction mechanism. In the search for novel techniques in plant experimental biotechnology, the goal of the study was to evaluate stress-inducing properties of novel biodegradable ester-based scaffolds made of poly(glycerol sebacate) (PGS) and poly(lactic acid) (PLA) influencing on the growth and deoxyshikonin productivity of Rindera graeca hairy roots immobilized on the experimental constructs. Rindera graeca hairy roots were maintained under the dark condition for 28 days in three independent systems, i.e., (i) non-immobilized biomass (a reference system), (ii) biomass immobilized on PGS scaffolds, and (iii) biomass immobilized on PLA scaffolds. The stress-inducing properties of the applied polymerized esters selectively impacted R. graeca hairy roots. The PGS scaffolds caused the production of deoxyshikonin, which does not occur in other culture systems, and PLA promoted biomass proliferation by doubling its increase compared to the reference system.

Synthesis of Poly(glycerol butenedioate)-PGB-Unsaturated Polyester toward Biomedical Applications.

A new polyester poly(glycerol butenedioate) (PGB) was obtained in the bulk polycondensation of glycerin and maleic anhydride. Glycerol polyesters are new biomaterials commonly used in tissue engineering. PGB, containing the α,ß-unsaturated moiety, could be very interesting due to potential modifications such as additions or oxidation. Such modifications are not possible on the heretofore known glycerol polyesters due to their structure without α,ß-unsaturated moieties. In this work, the developed process was optimized by applying the design of experiments. The optimization criterium was the minimization of the E/Z isomer ratio. Applying the two-stage process, the E/Z isomer ratio was reduced from 5.5 to 0.5 compared to the one-stage process. The degree of branching was also reduced from 17 to 9%, as well as the degree of esterification from 0.89 to 0.72. The obtained structure can be used in modifying or cross-linking via Michael additions.

Optimisation of Glycerol and Itaconic Anhydride Polycondensation.

Glycerol polyesters have recently become objects of interest in tissue engineering. Barely known so far is poly(glycerol itaconate) (PGItc), a biocompatible, biodegradable polyester. Due to the presence of a C=C electron-acceptor moiety, it is possible to post-modify the product by Michael additions to change the properties of PGItc. Thus, using PGItc as one of the elements of cellular scaffold crosslinked in situ for bone tissue regeneration seems to be a very attractive yet unexplored solution. This work aims to optimize the synthesis of PGItc to obtain derivatives with a double bond in the side chain with the highest conversion rates. The experiments were performed with itaconic anhydride and glycerol using mathematical planning of experiments according to the Box-Behnken plan without solvent and catalyst. The input variables of the process were the ratio of the OH/COOH, temperature, and reaction time. The optimised output variables were: the degree of esterification (EDtitr), the degree of esterification calculated from the analysis of 1H NMR spectra (EDNMR), and the degree of itaconic anhydride conversion-calculation based on 13C NMR spectra (%X13CNMR). In each of statistical models, the significance of the changed synthesis parameters was determined. Optimal conditions are when OH/COOH ratio is equal to 1.5, temperature is 140 °C and time of reaction is 5 h. The higher OH/COOH ratio, temperature and longer the experiment time, the higher the value of the degree of esterification and the degree of anhydride conversion.

Into the Tissues: Extracellular Matrix and Its Artificial Substitutes: Cell Signalling Mechanisms.

The existence of orderly structures, such as tissues and organs is made possible by cell adhesion, i.e., the process by which cells attach to neighbouring cells and a supporting substance in the form of the extracellular matrix. The extracellular matrix is a three-dimensional structure composed of collagens, elastin, and various proteoglycans and glycoproteins. It is a storehouse for multiple signalling factors. Cells are informed of their correct connection to the matrix via receptors. Tissue disruption often prevents the natural reconstitution of the matrix. The use of appropriate implants is then required. This review is a compilation of crucial information on the structural and functional features of the extracellular matrix and the complex mechanisms of cell-cell connectivity. The possibilities of regenerating damaged tissues using an artificial matrix substitute are described, detailing the host response to the implant. An important issue is the surface properties of such an implant and the possibilities of their modification.

Rapid and Efficient Optimization of Poly(1,2-Ethanediol Citrate) Synthesis Based on Magic Squares' Various Methods.

New biomaterials among aliphatic polyesters are in demand due to their potential applications in tissue engineering. There is a challenge not only to design scaffolds to regenerate defects in load-bearing tissues but also to ensure a proper blood supply to the reconstructed tissues. Poly-(1,2-ethanediol citrate) is one of the novel citrate-based polymers that could have the desired properties for cell scaffold fabrication and for enhancing cell adhesion. Both citric acid and 1,2-ethanediol are used in medicine and are fully resorbable by cells. This work aimed to synthesize poly(1,2-ethanediol citrate) in a catalyzed reaction with water removed by the Dean-Stark apparatus. The polyester structure was characterized by FTIR and NMR spectroscopy, and the HMBC experiment was performed to support the theory of successful polymer synthesis. The molecular weight was determined for the products obtained at 140 °C. The process was described via non-linear mathematical models. The influence of temperature and catalyst content on the degree of esterification and the conversion of acid groups in citric acid is described. The optimal process parameters are determined at 140 °C and 3.6% of p-toluenesulfonic acid content. The presented results are the starting point for scaffold design and scaling-up the process.

Cytotoxicity Study of UV-Laser-Irradiated PLLA Surfaces Subjected to Bio-Ceramisation: A New Way towards Implant Surface Modification.

In this research we subjected samples of poly(L-lactide) (PLLA) extruded film to ultraviolet (193 nm ArF excimer laser) radiation below the ablation threshold. The modified film was immersed in Simulated Body Fluid (SBF) at 37 °C for 1 day or 7 days to obtain a layer of apatite ceramic (CaP) coating on the modified PLLA surface. The samples were characterized by means of optical profilometry, which indicated an increase in average roughness (Ra) from 25 nm for the unmodified PLLA to over 580 nm for irradiated PLLA incubated in SBF for 1 day. At the same time, the water contact angle decreased from 78° for neat PLLA to 35° for irradiated PLLA incubated in SBF, which suggests its higher hydrophilicity. The obtained materials were investigated by means of cell response fibroblasts (3T3) and macrophage-like cells (RAW 264.7). Properties of the obtained composites were compared to the unmodified PLLA film as well as to the UV-laser irradiated PLLA. The activation of the PLLA surface by laser irradiation led to a distinct increase in cytotoxicity, while the treatment with SBF and the deposition of apatite ceramic had only a limited preventive effect on this harmful impact and depended on the cell type. Fibroblasts were found to have good tolerance for the irradiated and ceramic-covered PLLA, but macrophages seem to interact with the substrate leading to the release of cytotoxic products.

Kinetics of neomycin release from polylactide spheres and its antimicrobial activity.

BACKGROUND: Neomycin is a natural aminoglycoside antibiotic produced by actinomycete Streptomyces fradiae. It exerts bacteriostatic and bactericidal activity against Gram-negative bacteria, certain Gram-positive bacteria and Mycobacterium tuberculosis. Neomycin inhibits the biosynthesis of bacterial proteins by impairing their life functions, leading to death of cells. OBJECTIVES: To examine the effect of molecular weight of polylactide (PLA), the applied stabilizer as well as mixing speed used in the encapsulation process on the size of obtained spheres. Examination of the kinetics of neomycin release from the obtained PLA spheres and determination of the antimicrobial activity of the neomycin-containing spheres against selected strains of bacteria, yeast and fungi have also been necessary. MATERIAL AND METHODS: Polylactide (Mn 3000-40,000 g/mol) was obtained in-house. Other materials used in the study were as follows: L-lactic acid (PLLA; Mn 66,500 g/mol and 86,000 g/mol), polyvinyl alcohol (PVA) as a stabilizer of emulsion (Mw 30,000 g/mol, 130,000 g/mol; degree of hydrolysis 88%) as well as dichloromethane, p.a. and dimethyl sulfoxide (DMSO), p.a. as solvents. Distilled water was obtained in-house. Neomycin sulfate was used for encapsulation; phosphate (pH 7.2) and acetate (pH 4.5) buffers were used for the examination of the active pharmaceutical ingredient (API) dissolution profile. Antimicrobial activity was tested using commercial cell lines and the following media: Mueller-Hinton agar (MHA), Mueller-Hinton broth (MHB), yeast extract peptone dextrose (YPD), and potato dextrose agar (PDA). RESULTS: Neomycin-containing PLA spheres were obtained using an emulsion method. The average molecular weight of PLA, the average molecular weight of PVA and mixing speed on the size of obtained spheres were investigated. Furthermore, the profile of API dissolution from the spheres and antimicrobial activity of neomycin-containing spheres against certain strains of bacteria, yeast and fungi were determined. CONCLUSIONS: We demonstrated that efficient encapsulation of neomycin requires spheres of a <200 mm diameter.

Poly(Glycerol Succinate) as an Eco-Friendly Component of PLLA and PLCL Fibres towards Medical Applications.

This study was conducted as a first step in obtaining eco-friendly fibres for medical applications using a synthesised oligomer poly(glycerol succinate) (PGSu) as an additive for synthetic poly(L-lactic acid) (PLLA) and poly (L-lactide-co-caprolactone) (PLCL). The effects of the oligomer on the structure formation, morphology, crystallisation behaviour, and mechanical properties of electrospun bicomponent fibres were investigated. Nonwovens were investigated by means of scanning electron microscopy (SEM), wide angle X-ray scattering (WAXS), differential scanning calorimetry (DSC), and mechanical testing. The molecular structure of PLLA fibres is influenced by the presence of PGSu mainly acting as an enhancer of molecular orientation. In the case of semicrystalline PLCL, chain mobility was enhanced by the presence of PGSu molecules, and the crystallinity of bicomponent fibres increased in relation to that of pure PLCL. The mechanical properties of bicomponent fibres were influenced by the level of PGSu present and the extent of crystal formation of the main component. An in vitro study conducted using L929 cells confirmed the biocompatible character of all bicomponent fibres.

Poly-L-lactide scaffolds with super pores obtained by freeze-extraction method.

A nonplanar polylactide scaffold to be used in tissue engineering was obtained by freeze-extraction method. Properties of the scaffold were modified by adding Eudragit® E100. The impact of the modification on morphology, porosity and pore size, mass absorbability, mechanical properties was determined. Scanning electron microscopy (SEM), hydrostatic weighing test, static compression test was used to this end. The chemical composition of the scaffold was defined based on infrared spectroscopy (FTIR) and energy-dispersive X-ray spectroscopy (EDX). Biocompatibility was confirmed by quantitative tests and microscopic observation. The obtained results show that the obtained scaffolds may be applied as a carrier of hydrophilic cellular growth factors for more efficient tissue regeneration.

Biomineralization of poly-l-lactide spongy bone scaffolds obtained by freeze-extraction method.

Implants in the form of polymer scaffolds are commonly used to regenerate bone tissue after traumas or tooth extractions. However, few implant formation methods enable building polymer scaffolds allowing to reconstruct larger bone losses without immune response. Spacious, porous poly-l-lactide implants with considerable volume were obtained using the phase inversion method with the freeze-extraction variant. The calcium phosphate (CaP) coating was deposited on implant surfaces with the biomimetic method to improve the implant's osteoconductivity. The substitues morphology was characterized-porosity, size and shape of pores; mechanical properties, mass absorbability of implants before and after mineralization. The characteristics were provided with scanning electron microscopy (SEM), static compression test and hydrostatic weighing, respectively. The presence of CaPs in the entire volume of the implant was confirmed with SEM and infrared spectroscopy with Fourier transform (FTIR). The biocompatibility of scaffolds was confirmed with in vitro quantitative test and microscopic observations. The obtained results show that the implants can be used in tissue engineering as a vehicle of platelet-rich plasma to regenerate critical spongy bone losses.

Poly(Glycerol Sebacate)-Poly(l-Lactide) Nonwovens. Towards Attractive Electrospun Material for Tissue Engineering.

Two types of poly(glycerol sebacate) (PGS) prepolymers were synthesized and electrospun with poly(l-lactic acid) (PLA), resulting in bicomponent nonwovens. The obtained materials were pre-heated in a vacuum, at different times, to crosslink PGS and investigate morphological and structural dependencies in that polymeric, electrospun system. As both PGS and PLA are sensitive to pre-heating (crosslinking) conditions, research concerns both components. More interest is focused on the properties of PGS, considering further research for mechanical properties and subsequent experiments with PGS synthesis. Electrospinning of PGS blended with PLA does not bring difficulties, but obtaining elastomeric properties of nonwovens is problematic. Even though PGS has many potential advantages over other polyesters when soft tissue engineering is considered, its full utilization via the electrospinning process is much harder in practice. Further investigations are ongoing, especially with the promising PGS prepolymer with a higher esterification degree and its variations.

Influence of liquid pore precursors on morphology and mechanical properties of 3D scaffolds obtained by dry inversion phase method.

Polyester 3D scaffolds were obtained by dry inversion phase method. The influence of a polymer and liquid pore precursor type on the 3D scaffolds morphology, porosity and mechanical properties was tested. Polymers and precursors forming a porous structure were identified. It was found that 3D scaffolds having the most preferable structure for cell cultures were obtained from polylactide with the addition of n-butanol as the liquid pore precursor. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1079-1087, 2019.