Background SARS-CoV-2-neutralizing antibodies (nABs) showed great promise in the early phases of the COVID-19 pandemic. The emergence of resistant strains, however, quickly rendered the majority of clinically approved nABs ineffective. This underscored the imperative to develop nAB cocktails targeting non -overlapping epitopes. Methods Undertaking a nAB discovery program, we employed a classical workflow, while integrating artificial intelligence (AI) -based prediction to select non -competing nABs very early in the pipeline. We identified and in vivo validated (in female Syrian hamsters) two highly potent nABs. Findings Despite the promising results, in depth cryo-EM structural analysis demonstrated that the AI -based prediction employed with the intention to ensure non -overlapping epitopes was inaccurate. The two nABs in fact bound to the same receptor -binding epitope in a remarkably similar manner. Interpretation Our findings indicate that, even in the Alphafold era, AI -based predictions of paratope-epitope interactions are rough and experimental validation of epitopes remains an essential cornerstone of a successful nAB lead selection. Copyright (c) 2023 Published by Elsevier B.V. This is an open access article under the CC BY -NC -ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Manufacturing of a tissue-engineered product (TEP) is an essential part of the road from “lab to patient,” with its own set of challenges which are different from those covered in other chapters of this book. In manufacturing, TEP quality is the main consideration, and this is also reflected in the regulatory framework facilitating the commercial manufacturing of these products, ensuring their safety and efficacy for patients. This regulatory framework specifies most of the technical demands that the manufacturing process needs to meet, which are often underdeveloped when the TEP enters preclinical phases. Thus, a certain amount of process development is still needed, and existing strategies allow to do this efficiently and effectively. Besides quality, there are other important considerations such as process scalability and manufacturing cost. From these perspectives, a lot of attention is being devoted in recent years to next-generation processes which are automated, utilize single-use equipment, and rely on mathematical process models for efficient process development and effective process monitoring and control.

The hydrophobic nature and the regular scaffold architecture of bioplotted poly(ε-caprolactone) (PCL) scaffolds present some hurdles for homogeneous tissue formation and differentiation. The current hypothesis is that a synergistic effect of applied surface modification and scaffold design enhances colonization and osteogenic differentiation. First, PCL scaffolds with a 0/90° lay-down pattern (0/90) were plotted and subjected to an oxygen plasma (O2) or multistep surface modification, including post-argon 2-amino-ethylmethacrylate grafting (AEMA), followed by immobilization of gelatin type B (gelB) and physisorption of fibronectin (gelB Fn). Secondly, scaffolds of different designs were plotted (0/90° shift (0/90 S), 0/45° and 0/90° with narrow pores (0/90 NP)) and subjected to the double protein coating. Preosteoblasts were cultured on the scaffolds and the seeding efficiency, colonization and differentiation were studied. The data revealed that a biomimetic surface modification improved colonization (gelB Fn>gelB>AEMA>O2). Compact scaffold architectures (0/90 NP, 0/45, 0/90 S>0/90) positively influenced the seeding efficiency and differentiation. Interestingly, the applied surface modification had a greater impact on colonization than the scaffold design. In conclusion, the combination of a double protein coating with a compact design enhances tissue formation in the plotted PCL scaffolds.

In the present study a structural characterization and in vitro cell-biological evaluation was performed on polycaprolactone (PCL) scaffolds that were produced by the additive manufacturing technique selective laser sintering (SLS), followed by a plasma-based surface modification technique, either non-thermal oxygen plasma or double protein coating, to functionalize the PCL scaffold surfaces. In the first part of this study pore morphology by means of 2D optical microscopy, surface chemistry by means of hydrophilicity measurement and X-ray photoelectron spectroscopy, strut surface roughness by means of 3D micro-computed tomography (CT) imaging and scaffold mechanical properties by means of compression testing were evaluated before and after the surface modifications. The results showed that both surface modifications increased the PCL scaffold hydrophilicity without altering the morphological and mechanical properties. In the second part of this study the in vitro cell proliferation and differentiation of human osteoprogenitor cells, over 14 days of culture in osteogenic and growth medium were investigated. The O2 plasma modification gave rise to a significant lower in vitro cell proliferation compared to the untreated and double protein coated scaffolds. Furthermore the double protein coating increased in vitro cell metabolic activity and cell differentiation compared to the untreated and O2 plasma PCL scaffolds when OM was used.

Scaffold architecture and composition are crucial parameters determining the initial cell spatial distribution and consequently bone tissue formation. Three-dimensional poly-ε-caprolactone (PCL) scaffolds with a 0/90° lay-down pattern were plotted and subjected to (1) an oxygen plasma (PCL O) or (2) a postargon plasma modification with gelatin and fibronectin (PCL Fn). These scaffolds with an open pore structure were compared with more compact scaffolds fabricated by conventional processing techniques: oxidized polylactic acid (LA O) and collagen (COL) scaffolds. Human adipose tissue-derived stem cell/scaffold interactions were studied. The study revealed that the biomimetic surface modification of plotted scaffolds did not increase the seeding efficiency. The proliferation and colonization was superior for PCL Fn in comparison with PCL O. The plotted PCL Fn was completely colonized throughout the scaffold, whereas conventional scaffolds only at the edge. Protein-based scaffolds (PCL Fn and COL) enhanced the differentiation, although plotted scaffolds showed a delay in their differentiation compared with compact scaffolds. In conclusion, protein modification of plotted PCL scaffolds enhances uniform tissue formation, but shows a delayed differentiation in comparison with compact scaffolds. The present study demonstrates that biomimetic PCL scaffolds could serve as a guiding template to obtain a uniform bone tissue formation in vivo.

This study covers the quantification of the covalent attachment of gelatin type B (GelB) and the subsequent adsorption of Fibronectin (Fn) on poly-ε-caprolactone (PCL) surfaces, functionalised with 2-aminoethyl methacrylate (AEMA) by means of post-plasma UV-irradiation grafting. As typical surface characterisation tools do not allow quantification of deposited amounts of GelB or Fn, radiolabeled analogues were used for direct measurement of the amount of immobilized material. Bolton-Hunter GelB (BHG) and Fn were radioiodinated with 131I and 125I respectively and S-Hynic GelB (SHG) was labeled with 99mTc. Immobilisation of 131I-BHG or 99mTc-SHG on both PCL and PCL-AEMA scaffolds was performed in analogy with earlier work. SPECT images on scaffolds coated with 99mTc-SHG conjugates were acquired on a U-SPECT II camera. There was a clear difference in the amount of deposited 131I-BHG between blanco and AEMA-grafted PCL on 2D samples. No significant differences in immobilization behaviour were observed between 99mTc-SHG and 131I-BHG. Subsequent immobilisation of Fn was successful and depended on the amounts of deposited GelB. SPECT imaging on cylindrical 3D scaffolds confirmed these findings and showed that the amount of immobilized 99mTc-SHG was depth dependant. The architecture of the scaffolds strongly influences the distribution of GelB within these structures. Furthermore, there is a clear difference in the homogeneity of the protein coating when different GelB immobilization protocols were applied. This study shows that radiolabeled compounds are a rapid and accurate tool in the quantitative and qualitative evaluation of the biofunctionalisation of AEMA grafted PCL scaffolds.

Electron paramagnetic resonance (EPR) measurements were performed on poly-epsilon-caprolactone (PCL) films at different stages of the postplasma-grafting process. PCL films prepared by solvent casting (SC) or (ESP) yield very similar EPR spectra after Ar-plasma treatment and subsequent exposure to air, but the EPR signal is much stronger in the PCL-ESP films. The free radicals appear to be mainly, and possibly exclusively, oxygen centered. The radicals generated by UV irradiation in PCL-ESP films were studied in situ with EPR, using a UV-LED (lambda = (285 +/- 5) nm). Their EPR spectrum is distinctly different from the plasma-induced signal, indicative of carbon-centered radicals, and appears to be independent of the plasma pretreatment. UV-induced homolytic splitting of (hydro) peroxide bonds was not observed. Both the plasma- and UV-induced radicals decay at room temperature (RT), even in an inert atmosphere. This study demonstrates the potential of electrospun films and UV-LEDs for the study of plasma- and UV-generated free radicals with EPR in polyesters, and raises questions with respect to the validity of some generally accepted molecular mechanisms underpinning the postplasma grafting technique for polyesters. (C) 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 50: 2142-2149, 2012

A dielectric barrier discharge (DBD) discharge is used to modify the surface properties of 3-D porous polycaprolactone (PCL) scaffolds. After plasma treatment, the penetration of blue ink into the samples was used to determine the effectiveness of the plasma treatment inside the structures. It was found that the ink could penetrate deeper into the scaffolds after plasma treatment.

Polycaprolactone (PCL) is a biodegradable and biocompatible polymer which has acquired much attention the last few years for its potential use in biomedical applications, like tissue engineering and bone repair. Due to its hydrophobic nature, cell adhesion, proliferation and differentiation are however far less than optimal. To overcome this drawback, the surface properties of PCL need to be improved. In this work, a medium pressure dielectric barrier discharge (DBD) operating in different atmospheres (dry air, argon and helium) is employed to alter the surface properties of PCL. Chemical changes on the plasma-treated surfaces are examined using contact angle measurements and XPS analysis. Results show that discharge gas can have a significant influence on the chemical composition of the PCL surfaces: air and argon plasmas introduce oxygen-containing groups, while helium plasmas incorporate both oxygen and nitrogen-containing functionalities. This latter observation can however be explained by the fact that the helium discharge operates in the glow mode.

The presence of polyamine groups on the surface of dielectric resins potentially improves the adhesion with electrochemically deposited metals. In this article, first cyanuric chloride is covalently bound to the surface hydroxyl groups of the epoxy resin. The remaining reactive sites on the coupled cyanuric chloride molecule are then used to anchor polyamines. New data on the triazine coupling is presented. The surface reactions are monitored and characterized by means of ATR-IR, SEM-EDS, XPS and ToF-S-SIMS.

In the last decade, substantial research in the field of post-plasma grafting surface modification has focussed on the introduction of carboxylic acids on surfaces by grafting acrylic acid (AAc). In the present work, we report on an alternative approach for biomaterial surface functionalisation. Thin poly-ε-caprolactone (PCL) films were subjected to a dielectric barrier discharge Ar-plasma followed by the grafting of 2-aminoethyl methacrylate (AEMA) under UV-irradiation. X-ray photoelectron spectroscopy (XPS) confirmed the presence of nitrogen. The ninhydrin assay demonstrated, both quantitatively and qualitatively, the presence of free amines on the surface. Confocal fluorescence microscopy (CFM), atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to visualise the grafted surfaces, indicating the presence of pAEMA. Static contact angle (SCA) measurements indicated a permanent increase in hydrophilicity. Furthermore, the AEMA grafted surfaces were applied for comparing the physisorption and covalent immobilisation of gelatin. CFM demonstrated that only the covalent immobilisation lead to a complete coverage of the surface. Those gelatin-coated surfaces obtained were further coated using fibronectin. Osteosarcoma cells demonstrated better cell-adhesion and cell-viability on the modified surfaces, compared to the pure PCL films.

The introduction of amine groups on the surface of dielectric resins improves the adhesion with electrochemically deposited metals. In this work, etched epoxy resin surfaces are modified with aliphatic amines via a two step wet chemical reaction approach. First, cyanuric chloride is introduced on the surface. Next, the remaining reactive sites of cyanuric chloride are used to couple an aliphatic polyamine. Both reaction steps are optimized by variation of reaction parameters such as concentration, chemicals, temperature and time. A detailed surface characterization after each reaction step is provided using following techniques: ATR-IR, SEM–EDS, XPS and AFM.

In modern technology, there is a constant need to solve very complex problems and to fine-tune existing solutions. This is definitely the case in modern medicine with emerging fields such as regenerative medicine and tissue engineering. The problems, which are studied in these fields, set very high demands on the applied materials. In most cases, it is impossible to find a single material that meets all demands such as biocompatibility, mechanical strength, biodegradability (if required), and promotion of cell-adhesion, proliferation, and differentiation. A common strategy to circumvent this problem is the application of composite materials, which combine the properties of the different constituents. Another possible strategy is to selectively modify the surface of a material using different modification techniques. In the past decade, the use of nonthermal plasmas for selective surface modification has been a rapidly growing research field. This will be the highlight of this review. In a first part of this paper, a general introduction in the field of surface engineering will be given. Thereafter, we will focus on plasma-based strategies for surface modification. The purpose of the present review is twofold. First, we wish to provide a tutorial-type review that allows a fast introduction for researchers into the field. Second, we aim to give a comprehensive overview of recent work on surface modification of polymeric biomaterials, with a focus on plasma-based strategies. Some recent trends will be exemplified. On the basis of this literature study, we will conclude with some future trends for research.

In the current review, we aim to give an overview of different rapid prototyping (RP) methods which have been applied so far for the production of polymeric scaffolds for tissue engineering applications. After a general introduction on tissue engineering, scaffolds and biomaterials, both the well established RP techniques and the novel more recent developments in the field will be discussed. Comparison between RP and the classical techniques for producing scaffolds, each with their specific advantages and drawbacks will enable the reader to gain insight in the power of RP as an elegant scaffold production technique. Finally, an overview of some commonly used techniques for characterising the materials developed will be given.