Durability assessment of hydrogel mountings for contrast-enhanced micro-CT.

Micro-computed tomography (micro-CT) provides valuable data for studying soft tissue, though it is often affected by sample movement during scans and low contrast in X-ray absorption. This can result in lower image quality and geometric inaccuracies, collectively known as 'artefacts'. To mitigate these issues, samples can be embedded in hydrogels and enriched with heavy metals for contrast enhancement. However, the long-term durability of these enhancements remains largely unexplored. In this study, we examine the effects of two contrast enhancement agents - iodine and phosphotungstic acid (PTA) - and two hydrogels - agarose and Poloxamer 407 - over a 14-day period. We used Drosophila melanogaster as a test model for our investigation. Our findings reveal that PTA and agarose are highly durable, while iodine and poloxamer hydrogel exhibits higher leakage rates. These observations lay the foundation for estimating contrast stabilities in contrast-enhanced micro-CT with hydrogel embedding and serve to inform future research in this field.

IVM Advances for Early Antral Follicle-Enclosed Oocytes Coupling Reproductive Tissue Engineering to Inductive Influences of Human Chorionic Gonadotropin and Ovarian Surface Epithelium Coculture.

In vitro maturation (IVM) is not a routine assisted reproductive technology (ART) for oocytes collected from early antral (EA) follicles, a large source of potentially available gametes. Despite substantial improvements in IVM in the past decade, the outcomes remain low for EA-derived oocytes due to their reduced developmental competences. To optimize IVM for ovine EA-derived oocytes, a three-dimensional (3D) scaffold-mediated follicle-enclosed oocytes (FEO) system was compared with a validated cumulus-oocyte complex (COC) protocol. Gonadotropin stimulation (eCG and/or hCG) and/or somatic cell coculture (ovarian vs. extraovarian-cell source) were supplied to both systems. The maturation rate and parthenogenetic activation were significantly improved by combining hCG stimulation with ovarian surface epithelium (OSE) cells coculture exclusively on the FEO system. Based on the data, the paracrine factors released specifically from OSE enhanced the hCG-triggering of oocyte maturation mechanisms by acting through the mural compartment (positive effect on FEO and not on COC) by stimulating the EGFR signaling. Overall, the FEO system performed on a developed reproductive scaffold proved feasible and reliable in promoting a synergic cytoplasmatic and nuclear maturation, offering a novel cultural strategy to widen the availability of mature gametes for ART.

3D-printed ß-TCP/S53P4 bioactive glass scaffolds coated with tea tree oil: Coating optimization, in vitro bioactivity and antibacterial properties.

Bone infection treatment is a significant challenge for the orthopedic field. 3D printing is a promising technology to produce scaffolds with customized architecture, able to stimulate and support bone growth. ß-TCP and S53P4 bioactive glass (BG) are well-known biomaterials for scaffold manufacturing. However, a multifunctional scaffold, able to inhibit microbial proliferation at the defect site, is of increasing interest to avoid infection recurrence. Tea tree oil (TTO) has aroused interest as an antimicrobial agent to minimize the use of antibiotics. Therefore, combining the regenerative potential of a bioceramic with TTO's antimicrobial properties could result in a scaffold capable of stimulating tissue growth and treating infections. In this context, this study aimed to produce and characterize 3D-printed ß-TCP/S53P4 BG scaffolds coated with TTO. Scaffolds morphological and chemical characterizations were carried out through XDR, SEM, and FTIR analysis. ß-TCP/S53P4 BG scaffolds showed a compressive strength of ~2 MPa and 53 ± 2% of porosity. The scaffolds were coated by two different procedures, using an ethanol/TTO (EtOH/TTO) and a gelatin/TTO (Gel/TTO) solution with 5, 10, and 15% (v/v) TTO. The addition of TTO decreased MG-63 cell viability for both coating groups, but the Gel/TTO group showed higher cell viability. The antibacterial activity of the coated scaffolds was evaluated against S. aureus and higher inhibition of colony formation was found for Gel/TTO group. Therefore, the coating with Gel/TTO was effective in terms of antibacterial activity and cell viability. Such Gel/TTO coated ß-TCP/S53P4 BG scaffolds are proposed for antibacterial bone tissue engineering.

Zn-Loaded and Calcium Phosphate-Coated Degradable Silica Nanoparticles Can Effectively Promote Osteogenesis in Human Mesenchymal Stem Cells.

Nanoparticles such as mesoporous bioactive glasses (MBGs) and mesoporous silica nanoparticles (MSN) are promising for use in bone regeneration applications due to their inherent bioactivity. Doping silica nanoparticles with bioinorganic ions could further enhance their biological performance. For example, zinc (Zn) is often used as an additive because it plays an important role in bone formation and development. Local delivery and dose control are important aspects of its therapeutic application. In this work, we investigated how Zn incorporation in MSN and MBG nanoparticles impacts their ability to promote human mesenchymal stem cell (hMSC) osteogenesis and mineralization in vitro. Zn ions were incorporated in three different ways; within the matrix, on the surface or in the mesopores. The nanoparticles were further coated with a calcium phosphate (CaP) layer to allow pH-responsive delivery of the ions. We demonstrate that the Zn incorporation amount and ion release profile affect the nanoparticle's ability to stimulate osteogenesis in hMSCs. Specifically, we show that the nanoparticles that contain rapid Zn release profiles and a degradable silica matrix were most effective in inducing hMSC differentiation. Moreover, cells cultured in the presence of nanoparticle-containing media resulted in the highest induction of alkaline phosphate (ALP) activity, followed by culturing hMSC on nanoparticles immobilized on the surface as films. Exposure to nanoparticle-conditioned media did not increase ALP activity in hMSCs. In summary, Zn incorporation mode and nanoparticle application play an important role in determining the bioactivity of ion-doped silica nanoparticles.

When Electrospun Fiber Support Matters: In Vitro Ovine Long-Term Folliculogenesis on Poly (Epsilon Caprolactone) (PCL)-Patterned Fibers.

Current assisted reproduction technologies (ART) are insufficient to cover the slice of the population needing to restore fertility, as well as to amplify the reproductive performance of domestic animals or endangered species. The design of dedicated reproductive scaffolds has opened the possibility to better recapitulate the reproductive 3D ovarian environment, thus potentially innovating in vitro folliculogenesis (ivF) techniques. To this aim, the present research has been designed to compare ovine preantral follicles in vitro culture on poly(epsilon-caprolactone) (PCL)-based electrospun scaffolds designed with different topology (Random vs. Patterned fibers) with a previously validated system. The ivF performances were assessed after 14 days under 3D-oil, Two-Step (7 days in 3D-oil and on scaffold), or One-Step PCL protocols (14 days on PCL-scaffold) by assessing morphological and functional outcomes. The results show that Two- and One-Step PCL ivF protocols, when performed on patterned scaffolds, were both able to support follicle growth, antrum formation, and the upregulation of follicle marker genes leading to a greater oocyte meiotic competence than in the 3D-oil system. In conclusion, the One-Step approach could be proposed as a practical and valid strategy to support a synergic follicle-oocyte in vitro development, providing an innovative tool to enhance the availability of matured gametes on an individual basis for ART purposes.

The Effect of Electrophoretic Deposition Parameters on the Microstructure and Adhesion of Zein Coatings to Titanium Substrates.

Zein coatings were obtained by electrophoretic deposition (EPD) on commercially pure titanium substrates in an as-received state and after various chemical treatments. The properties of the zein solution, zeta potential and conductivity, at varying pH values were investigated. It was found that the zein content and the ratio of water to ethanol of the solution used for EPD, as well as the process voltage value and time, significantly influence the morphology of coatings. The deposits obtained from the solution containing 150 g/L and 200 g/L of zein and 10 vol % of water and 90 vol % of ethanol, about 4-5 µm thick, were dense and homogeneous. The effect of chemical treatment of the Ti substrate surface prior to EPD on coating adhesion to the substrate was determined. The coatings showed the highest adhesion to the as-received and anodized substrates due to the presence of a thick TiO2 layer on their surfaces and the presence of specific surface features. Coated titanium substrates showed slightly lower electrochemical corrosion resistance than the uncoated one in Ringer's solution. The coatings showed a well-developed surface topography compared to the as-received substrate, and they demonstrated hydrophilic nature. The present results provide new insights for the further development of zein-based composite coatings for biomedical engineering applications.

Evaluation of mechanical properties, in vitro corrosion resistance and biocompatibility of Gum Metal in the context of implant applications.

In recent decades, several novel Ti alloys have been developed in order to produce improved alternatives to the conventional alloys used in the biomedical industry such as commercially pure titanium or dual phase (alpha and beta) Ti alloys. Gum Metal with the non-toxic composition Ti-36Nb-2Ta-3Zr-0.3O (wt. %) is a relatively new alloy which belongs to the group of metastable beta Ti alloys. In this work, Gum Metal has been assessed in terms of its mechanical properties, corrosion resistance and cell culture response. The performance of Gum Metal was contrasted with that of Ti-6Al-4V ELI (extra-low interstitial) which is commonly used as a material for implants. The advantageous mechanical characteristics of Gum Metal, e.g. a relatively low Young's modulus (below 70 GPa), high strength (over 1000 MPa) and a large range of reversible deformation, that are important in the context of potential implant applications, were confirmed. Moreover, the results of short- and long-term electrochemical characterization of Gum Metal showed high corrosion resistance in Ringer's solution with varied pH. The corrosion resistance of Gum Metal was best in a weak acid environment. Potentiodynamic polarization studies revealed that Gum Metal is significantly less susceptible to pitting corrosion compared to Ti-6Al-4V ELI. The oxide layer on the Gum Metal surface was stable up to 8.5 V. Prior to cell culture, the surface conditions of the samples, such as nanohardness, roughness and chemical composition, were analyzed. Evaluation of the in vitro biocompatibility of the alloys was performed by cell attachment and spreading analysis after incubation for 48 h. Increased in vitro MC3T3-E1 osteoblast viability and proliferation on the Gum Metal samples was observed. Gum Metal presented excellent properties making it a suitable candidate for biomedical applications.

Interaction of MOPS buffer with glass-ceramic scaffold: Effect of (PO4 )3- ions in SBF on kinetics and morphology of formatted hydroxyapatite.

The international standard ISO 23317:2014 for the in vitro testing of inorganic biomaterials in simulated body fluid (SBF) uses TRIS buffer to maintain neutral pH. In our previous papers, we investigated the interaction of a glass-ceramic scaffold with TRIS and HEPES buffers. Both of them speeded up glass-ceramic dissolution and hydroxyapatite (HAp) precipitation, thereby demonstrating their unsuitability for the in vitro testing of highly reactive biomaterials. In this article, we tested MOPS buffer (3-[N-morpholino] propanesulfonic acid), another amino acid from the group of "Goods buffers". A highly reactive glass-ceramic scaffold (derived from Bioglass®) was exposed to SBF under static-dynamic conditions for 13/15 days. The kinetics and morphology of the newly precipitated HAp were studied using two different concentrations of (PO4 )3- ions in SBF. The pH value and the SiIV , Ca2+ , and (PO4 )3- concentrations in the SBF leachate samples were measured every day (AAS, spectrophotometry). The glass-ceramic scaffold was monitored by SEM/EDS, XRD, WD-XRF, and BET before and after 1, 3, 7, 11, and 13/15 days of exposure. As in the case of TRIS and HEPES, the preferential dissolution of the glass-ceramic crystalline phase (Combeite) was observed, but less intensively. The lower concentration of (PO4 )3- ions slowed down the kinetics of HAp precipitation, thereby causing the disintegration of the scaffold structure. This phenomenon shows that the HAp phase was predominately generated by the presence of (PO4 )3- ions in the SBF, not in the glass-ceramic material. Irrespective of this, MOPS buffer is not suitable for the maintenance of pH in SBF.

Fabrication of Gelatin Methacrylate (GelMA) Scaffolds with Nano- and Micro-Topographical and Morphological Features.

The design of biomimetic biomaterials for cell culture has become a great tool to study and understand cell behavior, tissue degradation, and lesion. Topographical and morphological features play an important role in modulating cell behavior. In this study, a dual methodology was evaluated to generate novel gelatin methacrylate (GelMA)-based scaffolds with nano and micro topographical and morphological features. First, electrospinning parameters and crosslinking processes were optimized to obtain electrospun nanofibrous scaffolds. GelMA mats were characterized by SEM, FTIR, DSC, TGA, contact angle, and water uptake. Various nanofibrous GelMA mats with defect-free fibers and stability in aqueous media were obtained. Then, micropatterned molds produced by photolithography were used as collectors in the electrospinning process. Thus, biocompatible GelMA nanofibrous scaffolds with micro-patterns that mimic extracellular matrix were obtained successfully by combining two micro/nanofabrication techniques, electrospinning, and micromolding. Taking into account the cell viability results, the methodology used in this study could be considered a valuable tool to develop patterned GelMA based nanofibrous scaffolds for cell culture and tissue engineering.

Interaction of HEPES buffer with glass-ceramic scaffold: Can HEPES replace TRIS in SBF?

An international standard (ISO: 23317:2014) exists for the in vitro testing of inorganic biomaterials in simulated body fluid (SBF). This standard uses TRIS buffer to maintain neutral pH in SBF, but in our previous paper, we showed that the interaction of a tested glass-ceramic material with TRIS can produce false-positive results. In this study, we evaluated whether the HEPES buffer, which also belongs to the group of Good´s buffers, would be more suitable for SBF. We compared its suitability in two media: SBF with HEPES and demineralized water with HEPES. The tested scaffold (45S5 bioactive glass-based) was exposed to the media under a static-dynamic arrangement (solutions were replaced on a daily basis) for 15 days. Leachate samples were collected daily for the analysis of Ca2+ ions and Si (AAS), (PO4 )3- ions (UV-VIS), and to measure pH. The glass-ceramic scaffold was analyzed by SEM/EDS, XRD, and WD-XRF before and after 0.3, 1, 3, 7, 11, and 15 days of exposure. Our results confirmed the rapid selective dissolution of the glass-ceramic crystalline phase (Combeite) containing Ca2+ ions due to the presence of HEPES, hydroxyapatite supersaturation being reached within 24 h in both solutions. These new results suggest that, like TRIS, HEPES buffer is not suitable for the in vitro testing of highly reactive inorganic biomaterials (glass, glass-ceramics). The ISO standard for such tests requires revision, but HEPES is not a viable alternative to TRIS buffer. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 143-152, 2018.

Investigating the Vascularization of Tissue-Engineered Bone Constructs Using Dental Pulp Cells and 45S5 Bioglass® Scaffolds.

Identification of a suitable cell source combined with an appropriate 3D scaffold is an essential prerequisite for successful engineering of skeletal tissues. Both osteogenesis and angiogenesis are key processes for bone regeneration. This study investigated the vascularization potential of a novel combination of human dental pulp stromal cells (HDPSCs) with 45S5 Bioglass® scaffolds for tissue-engineered mineral constructs in vivo and in vitro. 45S5 Bioglass scaffolds were produced by the foam replication technique with the standard composition of 45 wt% SiO2, 24.5 wt% Na2O, 24.5 wt% CaO, and 6 wt% P2O5. HDPSCs were cultured in monolayers and on porous 45S5 Bioglass scaffolds under angiogenic and osteogenic conditions for 2-4 weeks. HDPSCs expressed endothelial gene markers (CD34, CD31/PECAM1, and VEGFR2) under both conditions in the monolayer. A combination of HDPSCs with 45S5 Bioglass enhanced the expression of these gene markers. Positive immunostaining for CD31/PECAM1 and VEGFR2 and negative staining for CD34 supported the gene expression data, while histology revealed evidence of endothelial cell-like morphology within the constructs. More organized tubular structures, resembling microvessels, were seen in the constructs after 8 weeks of implantation in vivo. In conclusion, this study suggests that the combination of HDPSCs with 45S5 Bioglass scaffolds offers a promising strategy for regenerating vascularized bone grafts.

Is non-buffered DMEM solution a suitable medium for in vitro bioactivity tests?

Several laboratories had tested bioactivity of the materials in commercially available solution DMEM (Dulbecco's Modified Eagle's Medium) that is normally used for cultivation of cell cultures. The objective of this work was to find out whether it is possible to replace TRIS-buffered SBF currently used for bioactivity tests with the non-buffered DMEM solution. To understand the role of the organic part of the DMEM solution in the process of crystallization, we have prepared non-buffered solution simulating only its inorganic part (identified as I-solution). It was found that under static-dynamic test conditions calcite (CaCO3) and the amorphous phase of calcium phosphate (ACP) formed on the surface of the glass-ceramic (45S5 bioactive glass based) scaffold exposed to both solutions. Additionally, halite (NaCl) formed at the beginning of exposure to DMEM. Hydroxyapatite phase was not detected on the surface in either non-buffered solution. Organic components contained in the DMEM solution failed to prevent formation of crystalline phases. The present results indicate that it is not recommendable to use DMEM for bioactivity tests of glass-ceramic materials due to its low concentration of Ca2+ ions, high concentration of HCO3 - ions and the necessity to maintain sterile environment during the test.

TRIS buffer in simulated body fluid distorts the assessment of glass-ceramic scaffold bioactivity.

The paper deals with the characterisation of the bioactive phenomena of glass-ceramic scaffold derived from Bioglass® (containing 77 wt.% of crystalline phases Na(2)O·2CaO·3SiO(2) and CaO·SiO(2) and 23 wt.% of residual glass phase) using simulated body fluid (SBF) buffered with tris-(hydroxymethyl) aminomethane (TRIS). A significant effect of the TRIS buffer on glass-ceramic scaffold dissolution in SBF was detected. To better understand the influence of the buffer, the glass-ceramic scaffold was exposed to a series of in vitro tests using different media as follows: (i) a fresh liquid flow of SBF containing tris (hydroxymethyl) aminomethane; (ii) SBF solution without TRIS buffer; (iii) TRIS buffer alone; and (iv) demineralised water. The in vitro tests were provided under static and dynamic arrangements. SBF buffered with TRIS dissolved both the crystalline and residual glass phases of the scaffold and a crystalline form of hydroxyapatite (HAp) developed on the scaffold surface. In contrast, when TRIS buffer was not present in the solutions only the residual glassy phase dissolved and an amorphous calcium phosphate (Ca-P) phase formed on the scaffold surface. It was confirmed that the TRIS buffer primarily dissolved the crystalline phase of the glass-ceramic, doubled the dissolving rate of the scaffold and moreover supported the formation of crystalline HAp. This significant effect of the buffer TRIS on bioactive glass-ceramic scaffold degradation in SBF has not been demonstrated previously and should be considered when analysing the results of SBF immersion bioactivity tests of such systems.

Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering.

Biodegradable polymers and bioactive ceramics are being combined in a variety of composite materials for tissue engineering scaffolds. Materials and fabrication routes for three-dimensional (3D) scaffolds with interconnected high porosities suitable for bone tissue engineering are reviewed. Different polymer and ceramic compositions applied and their impact on biodegradability and bioactivity of the scaffolds are discussed, including in vitro and in vivo assessments. The mechanical properties of today's available porous scaffolds are analyzed in detail, revealing insufficient elastic stiffness and compressive strength compared to human bone. Further challenges in scaffold fabrication for tissue engineering such as biomolecules incorporation, surface functionalization and 3D scaffold characterization are discussed, giving possible solution strategies. Stem cell incorporation into scaffolds as a future trend is addressed shortly, highlighting the immense potential for creating next-generation synthetic/living composite biomaterials that feature high adaptiveness to the biological environment.