FACTORS AFFECTING CHINESE HAMSTER OVARY CELL PROLIFERATION AND VIABILITY

Advantageous cultivation procedures for the Chinese hamster ovary (CHO) cells are necessary for the productive commercial production of biopharmaceuticals. A main challenge that needs to be addressed during the process development is the differences in each cell line requirements concerning the nutrients and feed strategies in order to achieve the desired growth characteristics. Therefore, within the current research, a naïve high cell density serum free suspension adapted CHO cell line was tested with glucose and glutamine rich feeds in fed-batch Erlenmeyer shake flask cultures. Glucose consumption rate was adjusted to develop the optimal feed strategies. Obtained results indicated that high glucose and l-glutamine feeding did not improve maximum viable cell density compared to the control samples. During the exponential phase, cell proliferation and viability of all feeds showed no statistically significant difference. Instead, the fed-batch processes tested led to statistically significant differences in viable cell density and cell viability during the decline phase, compared to control (batch) culture. The difference between glucose and glutamine feeding was indistinguishable, most probably due to the concentration imbalance with the rest of the nutrients in feed. The overall study presented a method to slow down the decrease in CHO cell proliferation and viability during the decline phase, instead of increasing the maximum cell density at the plateau phase.

Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction

The inhibition of gamma-butyrobetaine (GBB) hydroxylase, a key enzyme in the biosynthesis of carnitine, contributes to lay ground for the cardioprotective mechanism of action of mildronate. By inhibiting the biosynthesis of carnitine, mildronate is supposed to induce the accumulation of GBB, a substrate of GBB hydroxylase. This study describes the changes in content of carnitine and GBB in rat plasma and heart tissues during long-term (28 days) treatment of mildronate [i.p. (intraperitoneal) 100 mg/kg/daily]. Obtained data show that in concert with a decrease in carnitine concentration, the administration of mildronate caused a significant increase in GBB concentration. We detected about a 5-fold increase in GBB contents in the plasma and brain and a 7-fold increase in the heart. In addition, we tested the cardioprotective effect of mildronate in isolated rat heart infarction model after 3, 7, and 14 days of administration. We found a statistically significant decrease in necrotic area of infarcted rat hearts after 14 days of treatment with mildronate. The cardioprotective effect of mildronate correlated with an increase in GBB contents. In conclusion, our study, for the first time, provides experimental evidence that the long-term administration of mildronate not only decreases free carnitine concentration, but also causes a significant increase in GBB concentration, which correlates with the cardioprotection of mildronate.publishersversionPeer reviewe

Fusion and classification algorithm of octacalcium phosphate production based on XRD and FTIR data

The present manuscript tested an automated analysis sequence to provide a decision support system to track the OCP synthesis from -TCP over time. Initially, the XRD and FTIR signals from a hundredfold scaled-up hydrolysis of OCP from -TCP were fused and modeled by the curve fitting based on the significantly established maxima from the literature and nine features extracted from the fitted shapes. Afterward, the analysis sequence enclosed the machine learning techniques for feature ranking, spatial filtering, and dimensionality reduction to support the automatic recognition of the synthesis stages. The proposed analysis pipeline for OCP identification might be the foundation for a decision support system explicitly targeting OCP synthesis. Future projects will exploit the suggested methodology for pinpointing the OCP production over time (including the intermediary phases present in the OCP formation) and for evaluating whether biological variables might be merged with biomaterial properties to build a unified model of tissue response to the implant

Incorporation of Barium Ions into Biomaterials: Dangerous Liaison or Potential Revolution?

In the present manuscript, a brief overview on barium, its possible utilization, and the aftermath of its behavior in organisms has been presented. As a bivalent cation, barium has the potential to be used in a myriad of biochemical reactions. A number of studies have exhibited both the unwanted outcome barium displayed and the advantages of barium laden compounds, tested in in vitro and in vivo settings. The plethora of prospective manipulations covered the area of hydrogels and calcium phosphates, with an end goal of examining barium’s future in the tissue engineering. However, majority of data revert to the research conducted in the 20th century, without investigating the mechanisms of action using current state-of-the-art technology. Having this in mind, set of questions that are needed for possible future research arose. Can barium be used as a substitute for other biologically relevant divalent cations? Will the incorporation of barium ions hamper the execution of the essential processes in the organism? Most importantly, can the benefits outweigh the harm

Doxorubicin loaded octacalcium phosphate particles as controlled release drug delivery systems: Physico-chemical characterization, in vitro drug release and evaluation of cell death pathway

: Mastering new and efficient ways to obtain successful drug delivery systems (DDS) with controlled release became a paramount quest in the scientific community. Increase of malignant bone tumors and the necessity to optimize an approach of localized drug delivery require research to be even more intensified. Octacalcium phosphate (OCP), with a number of advantages over current counterparts is extensively used in bone engineering. The aim of the present research was to synthesize bioactive and biocompatible doxorubicin (DOX) containing OCP particles. DOX-OCP was successfully obtained in situ in an exhaustive range of added drug (1-20 wt%, theoretical loading). Based on XRD, above 10 wt% of DOX, OCP formation was inhibited and the obtained product was low crystalline α-TCP. In-vitro drug release was performed in pH 7.4 and 6.0. In both pH environments DOX had a continuous release over six weeks. However, the initial drug burst for pH 7.4, in the first 24 h, ranged from 15.9 ± 1.3 % to 33.5 ± 12 % and for pH 6.0 23.7 ± 1.5 % to 36.2 ± 12 %.The DOX-OCP exhibited an inhibitory effect on viability of osteosarcoma cell lines MG63, U2OS and HOS. In contrast, MC3T3-E1 cells (IC50 > 0.062 μM) displayed increased viability and proliferation from 3rd to 7th day. Testing of the DDS on ferroptotic markers (CHAC1, ACSL4 and PTGS2) showed that OCP-DOX does not induce ferroptotic cell death. Moreover, the evaluation of protein levels of cleaved PARP, by western blotting analysis, corroborated that apoptosis is the main pathway of programmed cell death in osteosarcoma cells induced by DOX-OCP

Octacalcium Phosphate-Laden Hydrogels on 3D-Printed Titanium Biomaterials Improve Corrosion Resistance in Simulated Biological Media

| openaire: EC/H2020/860462/EU//PREMUROSA Funding Information: Financial support from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie ITN “Premurosa” (GA 860462) is gratefully acknowledged. The authors also acknowledge the access to the infrastructure and expertise of the BBCE—the Baltic Biomaterials Centre of Excellence (European Union’s Horizon 2020 research and innovation program under the grant agreement No. 857287). Publisher Copyright: © 2023 by the authors.The inflammatory-associated corrosion of metallic dental and orthopedic implants causes significant complications, which may result in the implant’s failure. The corrosion resistance can be improved with coatings and surface treatments, but at the same time, it might affect the ability of metallic implants to undergo proper osteointegration. In this work, alginate hydrogels with and without octacalcium phosphate (OCP) were made on 3D-printed (patterned) titanium alloys (Ti Group 2 and Ti-Al-V Group 23) to enhance their anticorrosion properties in simulated normal, inflammatory, and severe inflammatory conditions in vitro. Alginate (Alg) and OCP-laden alginate (Alg/OCP) hydrogels were manufactured on the surface of 3D-printed Ti substrates and were characterized with wettability analysis, XRD, and FTIR. The electrochemical characterization of the samples was carried out with open circuit potential, potentiodynamic polarization, and electrochemical impedance spectroscopy (EIS). It was observed that the hydrophilicity of Alg/OCP coatings was higher than that of pure Alg and that OCP phase crystallinity was increased when samples were subjected to simulated biological media. The corrosion resistance of uncoated and coated samples was lower in inflammatory and severe inflammatory environments vs. normal media, but the hydrogel coatings on 3D-printed Ti layers moved the corrosion potential towards more nobler values, reducing the corrosion current density in all simulated solutions. These measurements revealed that OCP particles in the Alg hydrogel matrix noticeably increased the electrical charge transfer resistance at the substrate and coating interface more than with Alg hydrogel alone.Peer reviewe

Apatite/Chitosan Composites Formed by Cold Sintering for Drug Delivery and Bone Tissue Engineering Applications

In the biomedical field, nanocrystalline hydroxyapatite is still one of the most attractive candidates as a bone substitute material due to its analogies with native bone mineral features regarding chemical composition, bioactivity and osteoconductivity. Ion substitution and low crystallinity are also fundamental characteristics of bone apatite, making it metastable, bioresorbable and reactive. In the present work, biomimetic apatite and apatite/chitosan composites were produced by dissolution–precipitation synthesis, using mussel shells as a calcium biogenic source. With an eye on possible bone reconstruction and drug delivery applications, apatite/chitosan composites were loaded with strontium ranelate, an antiosteoporotic drug. Due to the metastability and temperature sensitivity of the produced composites, sintering could be carried out by conventional methods, and therefore, cold sintering was selected for the densification of the materials. The composites were consolidated up to ~90% relative density by applying a uniaxial pressure up to 1.5 GPa at room temperature for 10 min. Both the synthesised powders and cold-sintered samples were characterised from a physical and chemical point of view to demonstrate the effective production of biomimetic apatite/chitosan composites from mussel shells and exclude possible structural changes after sintering. Preliminary in vitro tests were also performed, which revealed a sustained release of strontium ranelate for about 19 days and no cytotoxicity towards human osteoblastic-like cells (MG63) exposed up to 72 h to the drug-containing composite extract

Challenging applicability of ISO 10993-5 for calcium phosphate biomaterials evaluation: Towards more accurate in vitro cytotoxicity assessment

: Research on biomaterials typically starts with cytocompatibility evaluation, using the ISO 10993-5 standard as a reference that relies on extract tests to determine whether the material is safe (cell metabolic activity should exceed 70 %). However, the generalized approach within the standard may not accurately reflect the material's behavior in direct contact with cells, raising concerns about its effectiveness. Calcium phosphates (CaPs) are a group of materials that, despite being highly biocompatible and promoting bone formation, still exhibit inconsistencies in basic cytotoxicity evaluations. Hence, in order to test the cytocompatibility dependence on different experimental setups and material-cell interactions, we used amorphous calcium phosphate, α-tricalcium phosphate, hydroxyapatite, and octacalcium phosphate (0.1 mg/mL to 5 mg/mL) with core cell lines of bone microenvironment: mesenchymal stem cells, osteoblast-like and endothelial cells. All materials have been characterized for their physicochemical properties before and after cellular contact and once in vitro assays were finalized, groups identified as 'cytotoxic' were further analyzed using a modified Annexin V apoptosis assay to accurately determine cell death. The obtained results showed that indirect contact following ISO standards had no sensitivity of tested cells to the materials, but direct contact tests at physiological concentrations revealed decreased metabolic activity and viability. In summary, our findings offer valuable guidelines for handling biomaterials, especially in powder form, to better evaluate their biological properties and avoid false negatives commonly associated with the traditional standard approach

Eggshell-derived amorphous calcium phosphate: Synthesis, characterization and bio-functions as bone graft materials in novel 3D osteoblastic spheroids model

A multitude of autogenous/allogeneic and semi-synthetic bone graft materials have been developed to reconstruct the defective bone tissue but with high bio-cost and potential environmental pollution. With high calcium content and several trace elements, chicken eggshells are no longer considered as wastes but attractive sources of high-value-added biomaterials. This study used chicken eggshells and synthetic hydroxyapatite (HAp) to synthesize amorphous calcium phosphate (ACP) bone graft materials, namely Control and Eggshell. The physiochemical characteristics, biosafety, and immunocompatibility of synthetic ACP particles were inspected. Their osteogenic activity was further investigated in a novel osteoblastic spheroids model. Eggshell ACP particles exhibited ideal cytocompatibility compared to the control ACP and were more resistant to re-crystallization. In osteoblastic spheroids, Eggshell ACP mediated typical osteogenic mRNA profiles of MC-3T3-E1 cells, accompanied by the increased formation of mineralized nodules and boosted synthesis of ECM proteins represented by OPN and collagen I. This study establishes a promising technique to synthesize stable, safe, and osteoinductive ACP graft particles from eggshell waste. Furthermore, the osteoblastic spheroids constructed in the present study provide a more practical model for biomaterial research, which reflect the three-dimensional interaction between host bone tissue and graft materials more realistically