The Effect of Carbodiimide Crosslinkers on Gelatin Hydrogel as a Potential Biomaterial for Gingival Tissue Regeneration

Connective tissue grafts for gingival recession treatment present significant challenges as they require an additional surgical site, leading to increased morbidity, extended operative times, and a more painful postoperative recovery for patients. Gelatin contains the arginine–glycine–aspartic acid (RGD) sequence, which supports cell adhesion and interactions. The development of gelatin hydrogels holds significant promise due to their biocompatibility, ease of customization, and structural resemblance to the extracellular matrix, making them a potential candidate for gingival regeneration. This study aimed to assess the physical and biological properties of crosslinked gelatin hydrogels using EDC/NHS with two crosslinker concentrations (GelCL12 and GelCL24) and compare these to non-crosslinked gelatin. Both groups underwent morphological, rheological, and chemical analysis. Biological assessments were conducted to evaluate human gingival fibroblast (HGF) proliferation, migration, and COL1 expression in response to the scaffolds. The crosslinked gelatin group exhibited greater interconnectivity and better physical characteristics without displaying cytotoxic effects on the cells. FTIR analysis revealed no significant chemical differences between the groups. Notably, the GelCL12 group significantly enhanced HGF migration and upregulated COL1 expression. Overall, GelCL12 met the required physical characteristics and biocompatibility, making it a promising scaffold for future gingival tissue regeneration applications

Reactive magnetron plasma modification of electrospun PLLA scaffolds with incorporated chloramphenicol for controlled drug release

Surface modification with the plasma of the direct current reactive magnetron sputtering has demonstrated its efficacy as a tool for enhancing the biocompatibility of polymeric electrospun scaffolds. Improvement of the surface wettability of materials with water, as well as the formation of active chemical bonds in the near-surface layers, are the main reasons for the described effect. These surface effects are also known to increase the release rate of drugs incorporated in fibers. Herein, we investigated the effect of plasma modification on the chloramphenicol release from electrospun poly (lactic acid) fibrous scaffolds. Scaffolds with high—50 wt./wt.%—drug content were obtained. It was shown that plasma modification leads to an increase in the drug release rate and drug diffusion coefficient, while not deteriorating surface morphology and mechanical properties of scaffolds. The materials’ antibacterial activity was observed to increase in the first day of the experiment, while remaining on the same level as the unmodified group during the next six days. The proposed technique for modifying the surface of scaffolds will be useful for obtaining drug delivery systems with controlled accelerated release, which can expand the possibilities of local applications of antibiotics and other drugs

Electrospinning and emerging healthcare and medicine possibilities

Electrospinning forms fibers from either an electrically charged polymer solution or polymer melt. Over the past decades, it has become a simple and versatile method for nanofiber production. Hence, it has been explored in many different applications. Commonly used electrospinning assembles fibers from polymer solutions in various solvents, known as solution electrospinning, while melt and near-field electrospinning techniques enhance the versatility of electrospinning. Adaption of additive manufacturing strategy to electrospinning permits precise fiber deposition and predefining pattern construction. This manuscript critically presents the potential of electrospun nanofibers in healthcare applications. Research community drew impetus from the similarity of electrospun nanofibers to the morphology and mechanical properties of fibrous extracellular matrices (ECM) of natural human tissues. Electrospun nanofibrous scaffolds act as ECM analogs for specific tissue cells, stem cells, and tumor cells to realize tissue regeneration, stem cell differentiation, and in vitro tumor model construction. The large surface-to-volume ratio of electrospun nanofibers offers a considerable number of bioactive agents binding sites, which makes it a promising candidate for a number of biomedical applications. The applications of electrospinning in regenerative medicine, tissue engineering, controlled drug delivery, biosensors, and cancer diagnosis are elaborated. Electrospun nanofiber incorporations in medical device coating, in vitro 3D cancer model, and filtration membrane are also discussed

3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research in Regenerative Medicine

Regenerative medicine has been coming into spotlight ever since the realisation that conventional treatments are not enough, and the need for specific therapies has emerged. This, however, has paved way for cell-free therapy using extracellular vesicles. A two-dimensional (2D) cell culture model is widely recognised as the “gold standard” for researching cellular communications ex vivo. Although the 2D culture technique is straightforward and easy to use, it cannot replicate the in vivo ECM interactions & microenvironment. On the contrary, 3D culture culturing technology has emerged which include structures such as spheroids and organoids. Organoids are small replicas of in vivo tissues and organs, which faithfully recreate their structures and functions. These could be used as models to derive stem cells based EVs for manufacturing purposes. The linkages between infection and cancer growth, as well as mutation and carcinogenesis, may be modelled using this bioengineered platform. All in all, 3D culturing derived EVs serves as a novel platform for diagnostics, drug discovery & delivery, and therapy

Biodegradable polylactides scaffolds with pharmacological activity by means of ultrasound micromolding technology

Ultrasound micromolding technology has been applied to get microporous polylactide scaffolds from the subsequent leaching of incorporated NaCl salts. A small amount of water-soluble polyethylene glycol (PEG) was required in order to improve the leaching process and get compact pieces with interconnected pores. Distribution of polymers in the processed specimens was quite homogeneous due to the small PEG content, although it was more concentrated in the regions close to the feeding channels due to its higher viscosity. Hydrophobic drugs like triclosan could be incorporated causing a minimum degradation during ultrasound processing and suffering an insignificant solubilization during the leaching step. Final scaffolds showed clear bactericide or bacteriostatic effects before and after 10 h of exposure. Cell proliferation of MDCK epithelial cells was higher for TCS loaded porous scaffolds (200%) than for unloaded samples (170%) and non-porous polylactide (PLA) specimens (100%, control). Micrographs showed the absence of non-inhibition areas in both the specimens and the container, confirming the biocompatibility of PLA specimensPeer ReviewedPostprint (published version

BIOENGINEERING APPROACHES FOR IMPROVED DIFFERENTIATION OF CULTURED RETINAL TISSUES FROM PLURIPOTENT STEM CELLS

Sight is the most powerful of all human senses. For the vast majority of people on Earth, the loss of that sense would be unimaginable. Without assistive technology, it would separate them from their ability to work, their ability to travel, and their ability to interact with their loved ones. And yet, this extraordinary process, carefully refined by billions of years of evolution, is threatened for millions of people all over the world from a wide array of diseases of the retina. Many of these diseases arise from malnutrition and infection and are being rapidly eradicated. However, many dozens more result from convoluted permutations of genetics, age, and diet that threaten blindness for millions more with little hope of treatment, even with the best of modern medicine. As our life expectancies extend and our population ages, these diseases will only become more prevalent. In humanity's ever-present pursuit of medicine and knowledge to improve our quality of life, cutting-edge treatments offer promise that one day soon, even these diseases may be eradicated. One key technology capable of treating these devastating illnesses, on the precipice of being translated to real-world clinical treatments, is pluripotent stem cell-derived therapies. Patient-specific pluripotent stem cells, meaning pluripotent stem cells sourced directly from the patient, have a wealth of applications ranging from drug identification to disease modeling to implantation and regeneration. This research has been developed and advanced remarkably in the approximately two decades since the early isolation of pluripotent stem cells. Naturally, this advancement has predominantly been focused on cell and molecular biology. However, this focus has left significant research questions to be answered from engineering perspectives across a wide latitude of sub-disciplines. This dissertation explores three independent avenues of engineering principles as they relate to improving 2D and 3D retinal tissues derived from pluripotent stem cells in materials, devices, and computation. The first aim explores how plant protein-based nanofibrous scaffolds can marry the advantages and minimize the disadvantages of synthetic and animal-derived scaffolds for the culture of 2D retinal pigment epithelium (RPE) constructs. The second aim describes the development and testing of a novel, perfusing rotating wall vessel (RWV) bioreactor to support culture of 3D retinal organoids. Finally, the third aim performs a meta-analysis of published RNA-Seq datasets to determine the precise mechanisms by which bioreactors support organoid growth and extrapolate how these conclusions can support future experiments.Bioengineerin

Non-contact strain determination of cell traction effects

Irreversible tissue damage leading to organ failure is a common health problem in today's world. Regenerating these damaged tissues with the help of scaffolds is the solution offered by tissue engineering. In cases where the extra-cellular matrix (ECM) is to be replaced by an artificial substrate (scaffold) or matrix, cellular traction forces (CTF) are exerted by the cells on the scaffold surface. An ideal scaffold should exhibit mechanical characteristics similar to those of the ECM it is intended to replace. In other words, the capacity of a scaffold to withstand deformation should be comparable to that of a natural ECM. And with knowledge of those forces and deformations the properties of the scaffolds may be inferred. Digital Image Correlation (DIC), a non-contact image analysis technique enables us to measure point to point deformation of the scaffold by comparing a sequence of images captured during the process of scaffold deformation. This review discusses the methodology involved and implementation of DIC to measure displacements and strain.Irreversible tissue damage leading to organ failure is a common health problem in today's world. Regenerating these damaged tissues with the help of scaffolds is the solution offered by tissue engineering. In cases where the extra-cellular matrix (ECM) is to be replaced by an artificial substrate (scaffold) or matrix, cellular traction forces (CTF) are exerted by the cells on the scaffold surface. An ideal scaffold should exhibit mechanical characteristics similar to those of the ECM it is intended to replace. In other words, the capacity of a scaffold to withstand deformation should be comparable to that of a natural ECM. And with knowledge of those forces and deformations the properties of the scaffolds may be inferred. Digital Image Correlation (DIC), a non-contact image analysis technique enables us to measure point to point deformation of the scaffold by comparing a sequence of images captured during the process of scaffold deformation. This review discusses the methodology involved and implementation of DIC to measure displacements and strain

Assessment of printability and mechanical performance of scaffolds produced by SLA with two thermosetting resins

LAUREA MAGISTRALELo sviluppo di scaffold non degradabili rappresenta una strategia promettente per la risoluzione di difetti ossei di dimensioni critiche e aree soggette a resezione tumorale, in cui la rigenerazione ossea risulta inadeguata e si rende necessario un supporto meccanico a lungo termine. In questo contesto, gli scaffold svolgono un ruolo fondamentale imitando la struttura dell’osso naturale e fornendo supporto strutturale per l’adesione, la proliferazione e la differenziazione cellulare. Uno scaffold ideale deve presentare elevata porosità e interconnessione, proprietà meccaniche adeguate e biocompatibilità. Grazie alla loro biocompatibilità, stabilità nel tempo e bassa percentuale di ritiro, le resine Dental Tray e Dental Yellow Clear si mostrano particolarmente promettenti per la realizzazione di scaffold non degradabili. La stereolitografia (SLA) è stata selezionata per la fabbricazione degli scaffold per la sua elevata risoluzione e precisione progettuale, permettendo così un controllo accurato della geometria. Sono state analizzate diverse configurazioni di scaffold, tra cui una griglia 0/90°, una griglia 0/90° con valori di offset differenti, e una griglia 0/45°, al fine di valutare l’influenza dell’architettura sulle prestazioni morfologiche e meccaniche. Le dimensioni dei pori interni sono state variate per determinare i limiti di stampabilità; l’aggiunta di un colorante ha migliorato la risoluzione e ha consentito l’identificazione di pori più piccoli. La caratterizzazione morfologica è stata condotta tramite microscopia confocale. La caratterizzazione delle travi ha mostrato un’elevata accuratezza dimensionale; in particolare, Dental Yellow Clear ha mostrato una maggiore fedeltà di stampa rispetto a Dental Tray. Gli scaffold stampati sono stati caratterizzati mediante misurazioni accurate delle dimensioni esterne, della dimensione dei pori, dello spessore e dell’altezza delle pareti, rivelando che la configurazione con offset intermedio, 90_P2_O2, rappresentava il miglior compromesso tra separazione strutturale e fedeltà di stampa. Le prove meccaniche di flessione a tre punti svolte su provini a trave hanno mostrato un modulo elastico di 941.07 ± 211.61 MPa per Dental Yellow Clear e 810.71 ± 59.98 MPa per Dental Tray. Gli scaffold sono stati sottoposti a prove meccaniche di compressione, mostrando un modulo elastico compreso tra 62.25 ± 19.38 MPa e 115.37 ± 12.21 MPa per Dental Yellow Clear, valori comparabili al limite inferiore della rigidità dell’osso trabecolare naturale (E = 100–2000 MPa). L’analisi agli elementi finiti, basata su un modello di plasticità reattiva, ha riprodotto accuratamente il comportamento delle travi con errori minimi (<21.10%), sebbene la rigidità sia stata generalmente sovrastimata (fino a oltre il 130%), ad eccezione della configurazione 45_P2_O0, per la quale si è registrato un errore limitato al 4.21%. Questi risultati confermano la fattibilità dell’utilizzo della tecnologia SLA e delle resine dentali per la fabbricazione di scaffold non degradabili caratterizzati da elevata fedeltà morfologica e adeguata resistenza meccanica, evidenziando al contempo l’importanza di affinare i modelli computazionali applicati a strutture porose complesse.The development of non-degradable scaffolds represents a promising strategy for addressing critical-size bone defects and post-tumour resection sites, where natural bone regeneration is insufficient and long-term mechanical support is essential. In this context, scaffolds play a fundamental role by mimicking the native bone, providing structural support for cellular attachment, proliferation, and differentiation. An ideal scaffold must exhibit high porosity and interconnectivity, suitable mechanical properties, and biocompatibility. Thanks to their biocompatibility, durability, stability and low shrinkage rate Dental Tray and Dental Yellow Clear resins show promise in non-degradable implants. SLA was selected for scaffold fabrication due to its high resolution and design precision, enabling fine control of geometry. Several scaffold configurations were explored, including a 0/90° grid, a 0/90° grid with different offset values, and a 0/45° grid, in order to assess the influence of architecture on morphological and mechanical performance. Internal pore sizes were varied to assess printability limits, with the addition of a dye improving resolution and enabling detection of the smallest pores. Morphological characterization was conducted using a confocal microscope. Beam characterization showed high dimensional accuracy, with Dental Yellow Clear outperforming Dental Tray in terms of shape fidelity. Printed scaffolds were characterised with accurate measurements of external dimensions, pore size, wall thickness, and wall height, revealing that an intermediate offset configuration, 90_P2_O2, offered the best compromise between inter-strut spacing and shape fidelity. Mechanical testing included three-point bending tests for beam specimens, which yielded an average elastic modulus of 941.07 ± 211.61 MPa for Dental Yellow Clear and 810.71 ± 59.98 MPa for Dental Tray. Scaffold specimens underwent compression tests, showing an elastic modulus ranging from 62.25 ± 19.38 MPa to 115.37 ± 12.21 MPa for Dental Yellow Clear, values that are comparable to the lower bound of native trabecular bone stiffness (E = 100–2000 MPa). FEA using a reactive plasticity material model accurately reproduced beam behaviour with minimal error (<21.10%), though stiffness predictions for scaffold structures were generally overestimated (up to >130%), except for the 45_P2_O0 configuration, where the error was only 4.21%. These results confirm the feasibility of using SLA and dental resins to fabricate non-degradable scaffolds with high morphological fidelity and adequate mechanical strength, while also underscoring the importance of refining computational models for complex porous designs

Characterisation of laser processed bio-compatible materials and the realisation of electro optical diffraction gratings

Laser processing methods using excimer lasers have become very attractive for processing materials and the fabrication of micro and nano optical components. Diffraction gratings are used in a wide range of applications and require different fabrication methods. These components can be fabricated from a variety of biocompatible polymers. In this work, an Argon Fluoride (ArF) excimer laser operating at a wavelength of 193 nm has been used to process chitosan and agarose substrates. These materials have been characterised for differing laser processing conditions. Diffraction gratings and component demonstrators have been realised using Laser Direct writing (LDW) and nanoimprinting lithography (NIL). Characterisation of the ArF 193 nm laser work involves ablation threshold, optical absorption measurements and quantification of structural and morphological changes. This results can be used to identify the ideal laser fluence to be used for the production of a diffraction grating and similar optical components fabricated from chitosan. An ablation threshold of chitosan at 193 nm wavelength has been measured as 85 mJcm⁻² and an optical absorption coefficient of 3×10³ cm⁻¹.A diffraction grating structure, measuring 12 μm, was generated in biocompatible materials films; chitosan and agarose, using a laser processing method. The results showed that the interaction between the laser and these materials can potentially open the pathway for a wide range of practical, real world applications such as optical and biomedical applications. Diffraction gratings with a feature size of 1 μm were successfully formed on the biocompatible material free standing films using a NIL technique. Microstructure cross grating patterning made of chitosan and agarose have been fabricated by ArF excimer laser processing using a mask projection ablation technique. Temperature rise calculations have been carried out by COMSOL™ Multi-Physics v5.3 using a Finite Element Method (FEM), to predict the temperature rise during laser ablation processing of chitosan and agarose. In addition, COMSOL™ Multi-physics v5.3 has been used to simulate the electric field in the vicinity of a diffraction grating that is illuminated with light from a HeNe laser emitting at a wavelength of 632.8 nm.The final experimental work investigated the possibility of realising 5CB liquid crystal doped chitosan diffraction gratings doped with Sudan Black B (SBB) dye to enhance the absorption properties at 632.8 nm. Diffraction gratings was fabricated using two intersecting beams from a HeNe laser. Polymer Dispersed Liquid Crystal (PDLC) chitosan doped with 5CB and SBB dye diffraction gratings were experimentally characterised