Degradation behaviour of porous poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) scaffolds in cell culture.

Exploring the degradation behaviour of biomaterials in a complex in vitro physiological environment can assist in predicting their performance in vivo, yet this aspect remains largely unexplored. In this study, the in vitro degradation over 12 weeks of porous poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) bone scaffolds in human osteoblast (hOB) culture was investigated. The objective was to evaluate how the presence of cells influenced both the degradation behaviour and mechanical stability of these scaffolds. The molecular weight (Mw) of the scaffolds decreased with increasing incubation time and the Mw reduction rate (6.2 ± 0.4 kg mol-1 week-1) was similar to that observed when incubated in phosphate buffered saline (PBS) solution, implying that the scaffolds underwent hydrolytic degradation in hOB culture. The mass of the scaffolds increased by 0.8 ± 0.2 % in the first 4 weeks, attributed to cells attachment and extracellular matrix (ECM) deposition including biomineralisation. During the first 8 weeks, the nominal compressive modulus, E⁎, of the scaffolds remained constant. However, it increased significantly from Week 8 to 12, with increments of 55 % and 42 % in normal and lateral directions, respectively, attributed to the reinforcement effect of cells, ECM and minerals attached on the surface of the scaffold. This study has highlighted, that while the use of PBS in degradation studies is suitable for evaluating Mw changes it cannot predict changes in mechanical properties to PHBV scaffolds in the presence of cells and culture media. Furthermore, the PHBV scaffolds had mechanical stability in cell culture for 12 weeks validating their suitability for tissue engineering applications.

Targeted Nanoparticles Based on Alendronate Polyethylene Glycol Conjugated Chitosan for the Delivery of siRNA and Curcumin for Bone Metastasized Breast Cancer Applications.

Bone metastasized breast cancer reduces the quality of life and median survival. Targeted delivery of small interfering RNA (siRNA) and chemotherapeutic drugs using nanoparticles (NPs) is a promising strategy to overcome current limitations in treating these metastatic breast cancers. This research develops alendronate conjugated polyethylene glycol functionalized chitosan (ALD-PEG-CHI) NP for the delivery of cell death siRNA (CD-siRNA) and curcumin (CUR) and explores its targeting ability and in vitro cell cytotoxicity. Polyethylene glycol functionalized CHI (mPEG-CHI) NPs serve as control. The size of CD-siRNA loaded NPs is below 100 nm while CUR loaded NPs is below 200 nm, with near neutral zeta potential for all NPs. The CUR encapsulation efficiency (EE) is 70% and 88% for targeted and control NPs, respectively, while complete encapsulation of CD-siRNA is achieved in both NP systems. The bone targeting ability of CY5-dsDNA loaded ALD-PEG-CHI NPs using hydroxyapatite discs is fivefold compared to control indicating ALD presentation at the targeting NP surface. Delivery of CD-siRNA loaded NPs and CUR loaded NPs show synergistic and additive growth inhibition effects against MCF-7 cells by mPEG-CHI and ALD-PEG-CHI NPs, respectively. Overall, these in vitro results illustrate the potential of the targeted NPs as an effective therapeutic system toward bone metastasized breast cancer.

Physicochemical and in vitro biological evaluation of an injectable self-healing quaternized chitosan/oxidized pectin hydrogel for potential use as a wound dressing material.

Injectable self-healing hydrogels are attractive materials for use as wound dressings. To prepare such hydrogels, the current study used quaternized chitosan (QCS) to improve the solubility and antibacterial activity and oxidized pectin (OPEC) to introduce aldehyde groups for Schiff's base reaction with the amine groups from QCS. Self-healing hydrogels were made by co-injection of polymer solutions at specific polymer concentrations and reagent ratios that optimized both Schiff's base reactions and ionic interactions. The optimal hydrogel displayed self-healing 30 min after cutting and continuous self-healing during continuous step strain analysis, rapid gelation (< 1 min), a storage modulus of 394 Pa, and hardness of 700 mN, and compressibility of 162 mN s. The adhesiveness of this hydrogel (133 Pa) was within a suitable range for application as a wound dressing. The extraction media from the hydrogel displayed no cytotoxicity to NCTC clone 929 cells and higher cell migration than the control. While the extraction media from the hydrogel was found not to have antibacterial properties, QCS was verified as having MIC50 of 0.04 mg/mL against both E. coli and S. aureus. Therefore, this injectable self-healing QCS/OPEC hydrogel has the potential use as a biocompatible hydrogel material for wound management.

Characterisation of products from EDC-mediated PEG substitution of chitosan allows optimisation of reaction conditions.

PEGylation is a common method use to modify the physiochemical properties and increase the solubility of chitosan (CHI). Knowledge of optimal reaction conditions for PEGylation of CHI underpins its ongoing use in nanomedicine. This study synthesised methoxy polyethyleneglycol grafted CHI (mPEG-CHI) using carbodiimide-mediated coupling. The effect of reagent concentrations and pH on the degree of substitution (DS) and the PEGylation yield (conversion of free PEG to conjugated PEG) was evaluated through detailed chemical characterisation. Within the parameter space investigated, optimised reaction conditions (NH2: COOH:NHS:EDC of 3.5:1:1:10, pH = 5) resulted in a DS of 24 % and a PEGylation yield of 84 %. An EDC-derived adduct formed at pH ≥ 5.5 and at a 15-fold excess of EDC relative to COOH. The adduct was evaluated to be a guanidine derivative formed by the reaction of the amine group of CHI directly with EDC. DS ≥ 12 % imparted water solubility to CHI at physiological pH and mPEG-CHI (0.2-1.0 mg/mL) was not cytotoxic against the breast cancer cell lines MCF-7 and MDA-MB-231, indicating its suitability for medical applications.

In vitro evaluation of porous poly(hydroxybutyrate-co-hydroxyvalerate)/akermanite composite scaffolds manufactured using selective laser sintering.

Incorporation of a bioactive mineral filler in a biodegradable polyester scaffold is a promising strategy for scaffold assisted bone tissue engineering (TE). The current study evaluates the in vitro behavior of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV)/Akermanite (AKM) composite scaffolds manufactured using selective laser sintering (SLS). Exposure of the mineral filler on the surface of the scaffold skeleton was evident from in vitro mineralization in PBS. PHBV scaffolds and solvent cast films served as control samples and all materials showed preferential adsorption of fibronectin compared to serum albumin as well as non-cytotoxic response in human osteoblasts (hOB) at 24 h. hOB culture for up to 21 days revealed that the metabolic activity in PHBV films and scaffolds was significantly higher than that of PHBV/AKM scaffolds within the first two weeks of incubation. Afterwards, the metabolic activity in PHBV/AKM scaffolds exceeded that of the control samples. Confocal imaging showed cell penetration into the porous scaffolds. Significantly higher ALP activity was observed in PHBV/AKM scaffolds at all time points in both basal and osteogenic media. Mineralization during cell culture was observed on all samples with PHBV/AKM scaffolds exhibiting distinctly different mineral morphology. This study has demonstrated that the bioactivity of PHBV SLS scaffolds can be enhanced by incorporating AKM, making this an attractive candidate for bone TE application.

Evaluation of the in vivo fate of ultrapure alginate in a BALB/c mouse model.

The linear anionic polysaccharide alginate (ALG) has been comprehensively studied for biomedical applications, yet thus far the in vivo fate of this polymer has not been explored in detail. The current study therefore evaluates the biodistribution of ultrapure ALG (M/G ratio ≥ 0.67 with a measured Mw of 530 kg/mol and polydispersity index; PDI of 1.49) over a 14-day period in BALB/c mice. The biodistribution pattern over 2-days after sample administration using PET imaging with 64Cu-labelled ALG showed liver and spleen uptake. This was confirmed by the 14-day biodistribution profile of cyanine 5-labelled ALG from in vivo and ex vivo fluorescence imaging. Using MacGreen mice confirmed the uptake of the ALG by macrophages in the spleen at the 2-day time point. This extended biodistribution study confirmed the clearance of only a portion of the administered ALG biopolymer, but also uptake by macrophage populations in the spleen over a 14-day period.

Morphology and Composition of Immunodiffusion Precipitin Complexes Evaluated via Microscopy and Proteomics.

New approaches to rapid, simple, in vitro diagnostic immunoassays that do not rely on centralized laboratory facilities are urgently needed for disease diagnosis and to inform treatment strategies. The recent and ongoing COVID-19 pandemic has emphasized that rapid diagnostics are needed to help guide government policies on quarantines, social distancing measures, and community lockdowns. A common approach to developing new immunoassays is to modify existing platforms (e.g., automated ELISA and lateral flow assays) for the new analyte, even though this does not address the drawbacks of existing platforms. An alternate approach is to search for robust assays that have been superseded but could in fact solve important challenges using modern technologies. Immunodiffusion is one such platform based on unique "precipitin ring" patterns formed in gels or paper following interactions between proteins and cognate antibodies in diffusion/reaction systems. Herein, we investigate the microstructure of these precipitin rings using a combination of fluorescence and electron microscopy and also perform a mass spectrometry investigation to determine the proteomic composition of the rings. We observed that the rings were composed of microparticles, which we termed "precipitin complexes", and that these complexes were composed of at least 19 key proteins, including immunoglobulins and complement factors along with a range of plasma proteins, possibly related to immune complexes and/or high-density lipoprotein particles. This information will be useful in developing new in vitro diagnostics using reaction/diffusion systems-techniques that require a single assay step and that only require calibrated length measurements for target protein quantification.

Chitosan Nanomedicine in Cancer Therapy: Targeted Delivery and Cellular Uptake.

Nanomedicine has gained much attention for the management and treatment of cancers due to the distinctive physicochemical properties of the drug-loaded particles. Chitosan's cationic nature is attractive for the development of such particles for drug delivery, transfection, and controlled release. The particle properties can be improved by modification of the polymer or the particle themselves. The physicochemical properties of chitosan particles are analyzed in 126 recent studies, which allows to highlight their impact on passive and active targeted drug delivery, cellular uptake, and tumor growth inhibition (TGI). From 2012 to 2019, out of 40 in vivo studies, only 4 studies are found reporting a reduction in tumor size by using chitosan particles while all other studies reported tumor growth inhibition relative to controls. A total of 23 studies are analyzed for cellular uptake including 12 studies reporting cellular uptake mechanisms. Understanding and exploiting the processes involved in targeted delivery, endocytosis, and exocytosis by controlling the physicochemical properties of chitosan particles are important for the development of safe and efficient nanomedicine. It is concluded based on the recent literature available on chitosan particles that combination therapies can play a pivotal role in transformation of chitosan nanomedicine from bench to bedside.

Evaluation of surface layer stability of surface-modified polyester biomaterials.

Surface modification of biomaterials is a strategy used to improve cellular and in vivo outcomes. However, most studies do not evaluate the lifetime of the introduced surface layer, which is an important aspect affecting how a biomaterial will interact with a cellular environment both in the short and in the long term. This study evaluated the surface layer stability in vitro in buffer solution of materials produced from poly(lactic-co-glycolic acid) (50:50) and polycaprolactone modified by hydrolysis and/or grafting of hydrophilic polymers using grafting from approaches. The data presented in this study highlight the shortcomings of using model substrates (e.g., spun-coated films) rather than disks, particles, and scaffolds. It also illustrates how similar surface modification strategies in some cases result in very different lifetimes of the surface layer, thus emphasizing the need for these studies as analogies cannot always be drawn.

Enhanced Oral Vaccine Efficacy of Polysaccharide-Coated Calcium Phosphate Nanoparticles.

Oral administration of vaccines has been limited due to low immune response compared to parenteral administration. Antigen degradation in the acidic gastrointestinal environment (GI), mucus barriers, and inefficient cellular uptake by immune cells are the major challenges for oral vaccine delivery. To solve these issues, the current study investigates calcium phosphate nanoparticles (CaP NPs) coated with polysaccharides as nanocarriers for oral protein antigen delivery. In this design, the CaP NP core had an optimized antigen encapsulation capacity of 90 mg (BSA-FITC)/g (CaP NPs). The polysaccharides chitosan and alginate were coated onto the CaP NPs to protect the antigens against acidic degradation in the GI environment and enhance the immune response in the small intestine. The antigen release profiles showed that alginate-chitosan-coated CaP NPs prevented antigen release in a simulated gastric fluid (pH 1.2), followed by sustained release in simulated intestinal (pH 6.8) and colonic (pH 7.4) fluids. Cellular uptake and macrophage stimulation data revealed that the chitosan coating enhanced antigen uptake by intestine epithelia cells (Caco-2) and macrophages and improved surface expression of costimulatory molecules on macrophages. In vivo test further demonstrated that oral administration of alginate-chitosan-coated CaP@OVA NPs significantly enhanced the mucosal IgA and serum IgG antibody responses as compared to naked OVA, indicating that the CaP-Chi-Alg nanoparticle can potentially be used as a promising oral vaccine delivery system.

Protein adsorption to poly(tetrafluoroethylene) membranes modified with grafted poly(acrylic acid) chains.

Protein adsorption to biomaterial surfaces is important for the function of such materials with anchorage-dependent cell adhesion requiring the presence of adsorbed proteins. The current study evaluated five solid surfaces with poly(acrylic acid) (PAA) grafted from the surface of a poly(tetrafluoroethylene) membrane with respect to the adsorption of serum albumin (SA), lactoferrin (Lf), and lysozyme (Lys) from a phosphate buffer and NaCl solution or water for specific combinations. With the use of x-ray photoelectron spectroscopy, the relative amounts and protein layer thickness were evaluated. SA adsorption was governed by ionic repulsive forces and hydrophobic interactions as evidenced from an increase in the protein adsorption at lower pH (6.5 compared to 7.4) and a correlation with surface coverage when water (pH 6.5) was used as the medium. The adsorption of Lf and Lys followed similar trends for all samples. In general, ionic attractive forces dominated and a strong correlation of increasing protein adsorption with the PAA chain length was evident. This study concluded that all surfaces appear suitable for use in biomaterial applications where tissue ingrowth is desired and that the enhanced protein adsorption in a medium with high ionic strength (e.g., biological fluid) correlates with the PAA chain length rather than the surface coverage.

Akermanite reinforced PHBV scaffolds manufactured using selective laser sintering.

Scaffold assisted tissue engineering presents a promising approach to repair diseased and fractured bone. For successful bone repair, scaffolds need to be made of biomaterials that degrade with time and promote osteogenesis. Compared to the commonly used ß-tricalcium phosphate scaffolds, Akermanite (AKM) scaffolds were found to degrade faster and promote more osteogenesis. The objective of this study is to synthesize AKM micro and nanoparticle reinforced poly(3-hydroxybutyrate-co-3-hydroxyvalerate; PHBV) composite scaffolds using selective laser sintering (SLS). The synthesized composite scaffolds had an interconnected porous microstructure (61-64% relative porosity), large specific surface areas (31.1-64.2 mm-1 ) and pore sizes ranging from 303 to 366 and 279 to 357 µm in the normal and lateral direction, respectively, which are suitable for bone tissue repair. The observed hydrophilic nature of the scaffolds and the swift water uptake was due to the introduction of numerous carboxylic acid groups on the scaffold surface after SLS, circumventing the need for postprocessing. For the composite scaffolds, large amounts of AKM particles were exposed on the skeleton surface, which is a requirement for cell attachment. In addition, the particles embedded inside the skeleton helped to significantly reinforce the scaffold structure. The compressive strength and modulus of the composite scaffolds were up to 7.4 and 103 MPa, respectively, which are 149 and 197% of that of the pure PHBV scaffolds. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2596-2610, 2019.

Challenges for the development of surface modified biodegradable polyester biomaterials: A chemistry perspective.

The design of current implants produced from biodegradable polyesters is based on strength and rate of degradation and tailored by the choice of polyester used. However, detailed knowledge about the degradation mechanism of surface modified materials with applications in biomaterials science and tissue engineering is currently lacking. This perspective aims to outline the need for a greater focus on analyzing the degradation of modified polyesters to ensure they can fulfil their intended function and that degradation products can effectively be cleared from the body. The status of the literature regarding surface modified polyesters is summarized to illustrate the main aspects investigated in recent studies and specifically the number of studies investigating the fate of the materials upon degradation.

In vitro mineralization of dual grafted polytetrafluoroethylene membranes.

The modification of biomaterials by radiation induced grafting is a promising method to improve their bioactivity. Successful introduction of carboxyl and amine functional groups on the surface of a polytetrafluoroethylene membrane was achieved by grafting of acrylic acid (AA) and 2-aminoethyl methacrylate hydrochloride (AEMA) using simultaneous gamma irradiation grafting. Chemical characterization by attenuated total reflectance Fourier transform infrared spectroscopy and x-ray photoelectron spectroscopy confirmed the presence of amine and carboxylate functionalities and indicated that all protonated amines formed ion pairs with carboxyl groups, but not all carboxyl are involved in ion pairing. It was found that the irradiation doses (2, 5, or 10 kGy) affected the grafting outcome only when sulfuric acid (0.5 or 0.9 M) was added as a polymerization enhancer. The use of the inorganic acid successfully enhanced the total graft yield (GY), but the changes in the graft extent (GE) were not conclusive. Dual functional films were produced by either a one- or a two-step process. Generally, higher GY and GE values were observed for the samples produced by the two-step grafting of AA and AEMA. The in vitro mineralization in 1.5× simulated body fluid (SBF) induced the formation of carbonated hydroxyapatite as verified by FITR. All samples showed an increase in weight after mineralization with significantly larger increases observed for the samples which had the 1.5× SBF changed every third day compared to every seventh. For the dual functional samples, it was found that the sample grafted by the one-step method shows a significantly higher increase in weight despite a much lower GY compared to the sample prepared by the two-step method and this was attributed to the different architecture of grafted chains.

Electrospinning and mechanical properties of P(TMC-co-LLA) elastomers.

P(TMC-co-LLA) elastomers have shown great potential for various biomaterial and tissue engineering applications. This study systematically investigated properties key to such applications. Three P(TMC-co-LLA) copolymers with 9 to 32 mol% TMC were synthesised and processed into 3D fibrous scaffolds using solution electrospinning. A range of fiber diameters (0.5-5.9 µm) and pore sizes (3.5-19.8 µm) were achieved simply by adjusting the voltage, collector distance and feed rate during electrospinning. The morphological features of the electrospun scaffolds were affected by the copolymer composition such that the average fiber diameters decreased in the order of P(TMC20-co-LLA80) > P(TMC32-co-LLA68) > P(TMC9-co-LLA91), which suggests inherent properties of the copolymers influence the electrospinning process. In addition, the specific parameter combinations applied during electrospinning did not affect the thermal properties of the scaffolds, however, it was confirmed that rapid solidification of the fibers occurred during electrospinning which lowered the inherent crystallinity and caused deviations of the thermodynamic state from equilibrium. Mechanical testing revealed that the Young's modulus and ultimate tensile strength were dependent on the morphology of the fibrous scaffolds, while in contrast, the ductility and toughness were strongly composition dependent with P(TMC20-co-LLA80) scaffolds displaying lower ductility and toughness than P(TMC32-co-LLA68) scaffolds.

Investigating the Effect of Substrate Materials on Wearable Immunoassay Performance.

Immunoassays are ubiquitous across research and clinical laboratories, yet little attention is paid to the effect of the substrate material on the assay performance characteristics. Given the emerging interest in wearable immunoassay formats, investigations into substrate materials that provide an optimal mix of mechanical and bioanalytical properties are paramount. In the course of our research in developing wearable immunoassays which can penetrate skin to selectively capture disease antigens from the underlying blood vessels, we recently identified significant differences in immunoassay performance between gold and polycarbonate surfaces, even with a consistent surface modification procedure. We observed significant differences in PEG density, antibody immobilization, and nonspecific adsorption between the two substrates. Despite a higher PEG density formed on gold-coated surfaces than on amine-functionalized polycarbonate, the latter revealed a higher immobilized capture antibody density and lower nonspecific adsorption, leading to improved signal-to-noise ratios and assay sensitivities. The major conclusion from this study is that in designing wearable bioassays or biosensors, the design and its effect on the antifouling polymer layer can significantly affect the assay performance in terms of analytical specificity and sensitivity.

In vitro mineralisation of grafted ePTFE membranes carrying carboxylate groups.

In vitro mineralisation in simulated body fluid (SBF) of synthetic polymers continues to be an important area of research as the outcomes cannot be predicted. This study evaluates a series of ePTFE membranes grafted with carboxylate-containing copolymers, specifically using acrylic acid and itaconic acid for grafting. The samples differ with regards to graft density, carboxylate density and polymer topology. The type and amount of mineral produced in 1.5 × SBF was dependent on the sample characteristics as evident from XPS, SEM/EDX, and FTIR spectroscopy. It was found that the graft density affects the mineral phases that form and that low graft density appear to cause co-precipitation of calcium carbonate and calcium phosphate. Linear and branched graft copolymer topology led to hydroxyapatite mineralisation whereas crosslinked graft copolymers resulted in formation of a mixture of calcium-phosphate phases. This study demonstrates that in vitro mineralisation outcomes for carboxylate-containing graft copolymers are complex. The findings of this study have implications for the design of bioactive coatings and are important for understanding the bone-biomaterial interface.

Dispersion of hydroxyapatite nanoparticles in solution and in polycaprolactone composite scaffolds.

The dispersion behaviour of hydroxyapatite nanoparticles (HAP) and surface-modified HAP was studied in 1,4-dioxane (DO), water and poly(ε-caprolactone) (PCL) solutions and the relationship between these and the dispersion in composite PCL scaffolds prepared by thermally induced phase separation (TIPS) was examined. Investigation of the change in particle sizes by dynamic light scattering, showed that the modification of HAP by adsorption or covalent attachment of heparin via a 3-aminopropyltriethoxysilane (APTES) layer improved the dispersion stability of the particles in water/DO mixtures, while no improvement was observed in DO. The distribution of the particles within the composite scaffolds was determined using a combination of transmission electron microscopy and a calcium quantification method which was used to determine distribution of the particles in the vertical direction. While the scaffolds fabricated in DO had particles embedded within the walls of the scaffold, the scaffolds fabricated in a DO/water mixed solvent showed the particles partitioned to the surface of the scaffold walls, which is likely because the particles acted as interface stabilisers and were not miscible with the PCL rich phase. Therefore, it can be concluded that the polymer-solvent system used, as well as the phase separation mechanism that occurs, significantly influences the distribution of the particles in the scaffolds and thus the particle behaviour in solution is not necessarily a good predictor for the ability to fabricate scaffolds with a high degree of particle dispersion and hence for overall materials performance. Bulk crystallinity and compressive modulus were examined and it was determined that no significant changes occurred compared with the pristine PCL, while the surface bioactivity of the scaffolds had improved significantly, indicating that the particles were present at the polymer-solution interface.

Exciting new developments at the 5th International Symposium on Surface and Interface of Biomaterials.

Materials intended for use as implantable or diagnostic devices are required not only to display the required functional bulk properties but also have surface properties that elicit a desired biological response, and do so with high selectivity. The area of surface functionalization approaches and bioactive coatings for biomaterials and biomedical devices has been the subject of much research over several decades; yet, many challenges still remain to be solved. The 5th International Symposium on Surface and Interface of Biomaterials (ISSIB) held in Sydney (Australia) in April 2015 was an ideal forum to discuss the most recent developments in biomaterial surface modification, characterization, and evaluation of biological responses. The conference covered a range of topics including antimicrobial coatings, analysis of biomaterial surfaces and interfaces, biomolecules and cells at surfaces and interfaces, nanoparticles, functional coatings, patterned biomaterials, nanofabrication, bioreactors, and biosensors. In this special conference issue, the authors include papers that detail some of the highlights from the meeting.