Polydopamine Interfacial Coating for Stable Tumor-on-a-Chip Models: Application for Pancreatic Ductal Adenocarcinoma.

Addressing current challenges in solid tumor research requires advanced in vitro three-dimensional (3D) cellular models that replicate the inherently 3D architecture and microenvironment of tumor tissue, including the extracellular matrix (ECM). However, tumor cells exert mechanical forces that can disrupt the physical integrity of the matrix in long-term 3D culture. Therefore, it is necessary to find the optimal balance between cellular forces and the preservation of matrix integrity. This work proposes using polydopamine (PDA) coating for 3D microfluidic cultures of pancreatic cancer cells to overcome matrix adhesion challenges to sustain representative tumor 3D cultures. Using PDA's distinctive adhesion and biocompatibility, our model uses type I collagen hydrogels seeded with different pancreatic cancer cell lines, prompting distinct levels of matrix deformation and contraction. Optimizing the PDA coating enhances the adhesion and stability of collagen hydrogels within microfluidic devices, achieving a balance between the disruptive forces of tumor cells on matrix integrity and the maintenance of long-term 3D cultures. The findings reveal how this tension appears to be a critical determinant in spheroid morphology and growth dynamics. Stable and prolonged 3D culture platforms are crucial for understanding solid tumor cell behavior, dynamics, and responses within a controlled microenvironment. This advancement ultimately offers a powerful tool for drug screening, personalized medicine, and wider cancer therapeutics strategies.

Collagen-Laponite Nanoclay Hydrogels for Tumor Spheroid Growth.

The extracellular matrix (ECM) plays an important regulatory role in the development and progression of tumoral tissue. Its functions and properties are crucial in determining tumor cell behavior such as invasion, migration, and malignancy development. Our study explores the role of collagen type I in cancer development and spread using engineered tumor models like multicellular spheroids grown in collagen-based hydrogels to simulate early tumor formation. We employ microfluidic techniques to test the hypothesis that (i) adding Laponite nanoclay to collagen hydrogels modifies mechanical and rheological properties and (ii) changing the stiffness of the collagen microenvironment affects tumor spheroid growth. Our findings support our theories and suggest the use of ECM components and engineered tumor models in cancer research, offering a biocompatible and biomimetic method to tailor the mechanical properties of conventional collagen hydrogels.

Engineering the migration and attachment behaviour of primary dermal fibroblasts.

The availability of primary cells present in pathological conditions is often very limited due to stringent ethical regulation and patient consent. One such condition is chronic wounds, where dermal fibroblasts show a deficient migration. In vitro models with cellular tools that mimic the in vivo scenario would be advantageous to test new therapies for these challenging wounds. Since the availability of primary dermal fibroblasts present in chronic wounds is restricted and their "shelf-life" limited due to the increased senescence, our aim was to engineer human dermal fibroblasts with impaired migration using synthetic Arg-Gly-Asp (RGD) peptides. We studied fibroblast behaviour on three different two dimensional (2D) surfaces, representative of the dermal extracellular matrix and the materials used in the development of dermal scaffolds, in addition to commercially available, collagen-based 3D dermal scaffolds, demonstrating that the concentration of synthetic RGD peptides necessary to impair migration of dermal fibroblasts should be tailored to the particular surface/material and cell population used. The described technology could be translated to other cell types including established cell lines. A wide range of synthetic peptides exists, which differ in the amino acid sequence, thus increasing the possibilities of this technology.

Apoptotic primary normal human dermal fibroblasts for in vitro models of fibrosis.

Recent studies show that apoptosis affects surrounding tissue, playing a role in diseases such as fibrosis, a significant global disease burden. Elucidating the mechanisms by which the different apoptotic cells present during fibrotic wound healing affect their environment would enable development of new therapies. We describe here a simple, rapid, and cost-effective method for inducing apoptosis of primary normal human dermal fibroblasts without affecting the overall cell viability of the population. Such population could be used for in vitro models of fibrotic wound healing in co-culture with other cells involved in this process to study events such as apoptosis-induced proliferation.

Collagen gels and the 'Bornstein legacy': from a substrate for tissue culture to cell culture systems and biomaterials for tissue regeneration.

As collagen is the main structural component of connective tissues and skin, much effort was made in the past and still today to use it in cell culture applications. Moreover, collagen biomaterials are widely used in tissue regeneration, including the treatment of burns and chronic wounds. The great implications of the research carried out by Bornstein, Ehrmann and Gey on collagen preparations in the 1950s for cell culture and more recently tissue engineering and regeneration are described in this commentary. Specifically, it is explored why the 1958 paper on 'Reconstituted Rat-Tail Collagen Used as Substrate for Tissue Cultures on Coverslips in Maximow Slides and Roller Tubes' by M. B. Bornstein has made an invaluable contribution to the field.

Stem cell engineered bone with calcium-phosphate coated porous titanium scaffold or silicon hydroxyapatite granules for revision total joint arthroplasty.

Aseptic loosening in total joint replacements (TJRs) is mainly caused by osteolysis which leads to a reduction of the bone stock necessary for implant fixation in revision TJRs. Our aim was to develop bone tissue-engineered constructs based on scaffolds of clinical relevance in revision TJRs to reconstitute the bone stock at revision operations by using a perfusion bioreactor system (PBRS). The hypothesis was that a PBRS will enhance mesenchymal stem cells (MSCs) proliferation and osteogenic differentiation and will provide an even distribution of MSCs throughout the scaffolds when compared to static cultures. A PBRS was designed and implemented. Scaffolds, silicon substituted hydroxyapatite granules and calcium-phosphate coated porous TiAl6V4 cylinders, were seeded with MSCs and cultured either in static conditions or in the PBRS at 0.75 mL/min. Statistically significant increased cell proliferation and alkaline phosphatase activity was found in samples cultured in the PBRS. Histology revealed a more even cell distribution in the perfused constructs. SEM showed that cells arranged in sheets. Long cytoplasmic processes attached the cells to the scaffolds. We conclude that a novel tissue engineering approach to address the issue of poor bone stock at revision operations is feasible by using a PBRS.

Ex Ovo Chorioallantoic Membrane Assay as a Model of Bone Formation by Biomaterials.

Biomaterials play an increasingly critical role in bone tissue engineering. However, achieving effective clinical translation requires a careful choice of biomimetic materials and thorough assessment of their efficacy and safety. Existing in vitro and in vivo models have drawbacks including time and cost constraints, invasive procedures, and discordance between animal models and clinical outcomes. Therefore, there is a demand for an alternative model. We hypothesized that the chick embryo chorioallantoic membrane can serve as a bioreactor to evaluate the initial sign of bone formation on scaffolds. In parallel, we investigated the osteogenic potential of a previously fabricated fibrin-alginate-calcium phosphate biomaterial (FACaP). Blood vessels were observed to infiltrate the scaffolds with early signs of bone formation, confirmed via RUNX-2 and alpha smooth muscle actin markers. The scaffolds' chemical composition was evaluated by Fourier-transform infrared spectroscopy, and ion chromatography was used to assess calcium ion release. Finally, the topography was examined by atomic force microscopy. In conclusion, this system offers simple refinement for in vivo models in bone tissue engineering and highlights the great potential of FACaP as an angiogenic and osteogenic biomaterial for non-load-bearing applications.

3D Porous Binary Composites of Collagen, Elastin, and Fibrin Proteins Orchestrate Adipose Tissue Regeneration.

The objective for this study is to advance the development of a specialized biomaterial that can effectively facilitate the regeneration of adipose tissue. In prior studies, the assessment of collagen (Col), elastin (Ela), and fibrin (Fib) unary scaffolds has been conducted. However, it is important to note that native adipose tissue is comprised of a diverse array of extracellular matrix (ECM) constituents. To mimic this behavior, binary compositions of collagen, elastin, and fibrin are fabricated in a 1:1 ratio, resulting in the formation of Col/Ela, Col/Fib, and Ela/Fib composites through a customized fabrication procedure. The physical properties of these scaffolds are comprehensively analyzed using a range of material characterization techniques. Additionally, the biological properties of the scaffolds are investigated by examining the survival, proliferation, and phenotype of adipose-derived stem cells. Subsequently, the aforementioned binary scaffolds are implanted into a rodent model for 28 days. the explants are analysed through X-ray microtomography, histology, and immunohistochemistry. The findings of the study demonstrate that the utilization of binary combinations of Col/Ela, Col/Fib, and Ela/Fib has a discernible impact on the physical and biological characteristics of the scaffolds. Nevertheless, Ela/Fib exhibits characteristics that make it a suitable candidate for adipogenesis due to its notable upregulation of caveolin-1 expression in both acellular and cellular cohorts. The combination of two natural polymers in this cell-material interaction has significantly enhanced the comprehension of adipogenesis.

Physico-chemical characterization of the tumour microenvironment of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy that accounts for more than 90% of pancreatic cancer diagnoses. Our research is focused on the physico-chemical properties of the tumour microenvironment (TME), including its tumoural extracellular matrix (tECM), as they may have an important impact on the success of cancer therapies. PDAC xenografts and their decellularized tECM offer a great material source for research in terms of biomimicry with the original human tumour. Our aim was to evaluate and quantify the physico-chemical properties of the PDAC TME. Both cellularized (native TME) and decellularized (tECM) patient-derived PDAC xenografts were analyzed. A factorial design of experiments identified an optimal combination of factors for effective xenograft decellularization. Our results provide a complete advance in our understanding of the PDAC TME and its corresponding stroma, showing that it presents an interconnected porous architecture with very low permeability and small pores due to the contractility of the cellular components. This fact provides a potential therapeutic strategy based on the therapeutic agent size.

Comparison of mesenchymal stem cell proliferation and differentiation between biomimetic and electrochemical coatings on different topographic surfaces.

The hypothesis for this study was that there is no difference in mesenchymal stem cells (MSCs) proliferation and osteogenic differentiation between calcium-phosphate (CaP) coatings with different crystal size deposited on different topographic surfaces of metal discs. Polished (P) and sand-blasted (SB) tantalum and TiAl6V4 discs were CaP coated by three methods-biomimetic (BioM), electrochemical at 20 mA/cm(2) and at 6.5 mA/cm(2)-and cultured with MSCs. At days 4, 7 and 14, cell proliferation-alamarBlue(®) activity and DNA quantification-and differentiation down the osteogenic lineage-ALP activity normalised per amount of DNA and SEM (morphology)-were analysed. Results showed that MSCs proliferated more when cultured on the nano-sized BioM coatings compared to uncoated and electrochemically coated discs. MSCs also proliferated more on P surfaces than on SB and or electrochemical coatings. All the coatings induced osteogenic differentiation, which was greater on electrochemical coatings and SB discs.

The rise of mechanical metamaterials: Auxetic constructs for skin wound healing.

Auxetic materials are known for their unique ability to expand/contract in multiple directions when stretched/compressed. In other words, they exhibit a negative Poisson's ratio, which is usually positive for most of materials. This behavior appears in some biological tissues such as human skin, where it promotes wound healing by providing an enhanced mechanical support and facilitating cell migration. Skin tissue engineering has been a growing research topic in recent years, largely thanks to the rapid development of 3D printing techniques and technologies. The combination of computational studies with rapid manufacturing and tailored designs presents a huge potential for the future of personalized medicine. Overall, this review article provides a comprehensive overview of the current state of research on auxetic constructs for skin healing applications, highlighting the potential of auxetics as a promising treatment option for skin wounds. The article also identifies gaps in the current knowledge and suggests areas for future research. In particular, we discuss the designs, materials, manufacturing techniques, and also the computational and experimental studies on this topic.

A bone-on-a-chip collagen hydrogel-based model using pre-differentiated adipose-derived stem cells for personalized bone tissue engineering.

Mesenchymal stem cells have contributed to the continuous progress of tissue engineering and regenerative medicine. Adipose-derived stem cells (ADSC) possess many advantages compared to other origins including easy tissue harvesting, self-renewal potential, and fast population doubling time. As multipotent cells, they can differentiate into osteoblastic cell linages. In vitro bone models are needed to carry out an initial safety assessment in the study of novel bone regeneration therapies. We hypothesized that 3D bone-on-a-chip models containing ADSC could closely recreate the physiological bone microenvironment and promote differentiation. They represent an intermedium step between traditional 2D-in vitro and in vivo experiments facilitating the screening of therapeutic molecules while saving resources. Herein, we have differentiated ADSC for 7 and 14 days and used them to fabricate in vitro bone models by embedding the pre-differentiated cells in a 3D collagen matrix placed in a microfluidic chip. Osteogenic markers such as alkaline phosphatase activity, calcium mineralization, changes on cell morphology, and expression of specific proteins (bone sialoprotein 2, dentin matrix acidic phosphoprotein-1, and osteocalcin) were evaluated to determine cell differentiation potential and evolution. This is the first miniaturized 3D-in vitro bone model created from pre-differentiated ADSC embedded in a hydrogel collagen matrix which could be used for personalized bone tissue engineering.

Hydrocolloids of Egg White and Gelatin as a Platform for Hydrogel-Based Tissue Engineering.

Innovative materials are needed to produce scaffolds for various tissue engineering and regenerative medicine (TERM) applications, including tissue models. Materials derived from natural sources that offer low production costs, easy availability, and high bioactivity are highly preferred. Chicken egg white (EW) is an overlooked protein-based material. Whilst its combination with the biopolymer gelatin has been investigated in the food technology industry, mixed hydrocolloids of EW and gelatin have not been reported in TERM. This paper investigates these hydrocolloids as a suitable platform for hydrogel-based tissue engineering, including 2D coating films, miniaturized 3D hydrogels in microfluidic devices, and 3D hydrogel scaffolds. Rheological assessment of the hydrocolloid solutions suggested that temperature and EW concentration can be used to fine-tune the viscosity of the ensuing gels. Fabricated thin 2D hydrocolloid films presented globular nano-topography and in vitro cell work showed that the mixed hydrocolloids had increased cell growth compared with EW films. Results showed that hydrocolloids of EW and gelatin can be used for creating a 3D hydrogel environment for cell studies inside microfluidic devices. Finally, 3D hydrogel scaffolds were fabricated by sequential temperature-dependent gelation followed by chemical cross-linking of the polymeric network of the hydrogel for added mechanical strength and stability. These 3D hydrogel scaffolds displayed pores, lamellae, globular nano-topography, tunable mechanical properties, high affinity for water, and cell proliferation and penetration properties. In conclusion, the large range of properties and characteristics of these materials provide a strong potential for a large variety of TERM applications, including cancer models, organoid growth, compatibility with bioprinting, or implantable devices.

Decellularization of tumours: A new frontier in tissue engineering.

Cancer is one of the leading causes of death worldwide. The tumour extracellular matrix (ECM) has unique features in terms of composition and mechanical properties, resulting in a structurally and chemically different ECM to that of native, healthy tissues. This paper reviews to date the efforts into decellularization of tumours, which in the authors' view represents a new frontier in the ever evolving field of tumour tissue engineering. An overview of the ECM and its importance in cancer is given, ending with examples of research using decellularized tumours, which has already indicated potential therapeutic targets, unravelled malignancy mechanisms or response to chemotherapy agents. The review highlights that more research is needed in this area, which can answer important questions related to tumour formation and progression to ultimately identify new and effective therapeutic targets. Within the near-future of personalized medicine, this research can create patient-specific tumour models and therapeutic regimes.

In Vitro Hydrolytic Degradation of Polyester-Based Scaffolds under Static and Dynamic Conditions in a Customized Perfusion Bioreactor.

Creating biofunctional artificial scaffolds could potentially meet the demand of patients suffering from bone defects without having to rely on donors or autologous transplantation. Three-dimensional (3D) printing has emerged as a promising tool to fabricate, by computer design, biodegradable polymeric scaffolds with high precision and accuracy, using patient-specific anatomical data. Achieving controlled degradation profiles of 3D printed polymeric scaffolds is an essential feature to consider to match them with the tissue regeneration rate. Thus, achieving a thorough characterization of the biomaterial degradation kinetics in physiological conditions is needed. Here, 50:50 blends made of poly(ε-caprolactone)-Poly(D,L-lactic-co-glycolic acid (PCL-PLGA) were used to fabricate cylindrical scaffolds by 3D printing (⌀ 7 × 2 mm). Their hydrolytic degradation under static and dynamic conditions was characterized and quantified. For this purpose, we designed and in-house fabricated a customized bioreactor. Several techniques were used to characterize the degradation of the parent polymers: X-ray Photoelectron Spectroscopy (XPS), Gel Permeation Chromatography (GPC), Scanning Electron Microscopy (SEM), evaluation of the mechanical properties, weigh loss measurements as well as the monitoring of the degradation media pH. Our results showed that flow perfusion is critical in the degradation process of PCL-PLGA based scaffolds implying an accelerated hydrolysis compared to the ones studied under static conditions, and up to 4 weeks are needed to observe significant degradation in polyester scaffolds of this size and chemical composition. Our degradation study and characterization methodology are relevant for an accurate design and to tailor the physicochemical properties of polyester-based scaffolds for bone tissue engineering.

Inclusion of calcium phosphate does not further improve in vitro and in vivo osteogenesis in a novel, highly biocompatible, mechanically stable and 3D printable polymer.

At a time of unpredictable challenges for health, one trend is certain: there is an exceedingly high demand for functional implants, particularly bone grafts. This has encouraged the emergence of bone tissue engineering substitutes as an alternative method to conventional bone grafts. However, the current approaches in the field face several limitations that have prevented the ultimate translation into clinical settings. As a result, many attempts have been made to fabricate synthetic bone implants that can offer suitable biological and mechanical properties.Light curable methacrylate-based polymers have ideal properties for bone repair. These materials are also suitable for 3D printing which can be applicable for restoration of both function and aesthetics. The main objective of this research was to investigate the role of calcium phosphate (CaP) incorporation in a mechanically stable, biologically functional and 3D printable polymer for the reconstruction of complex craniofacial defects. The experimental work initially involved the synthesis of (((((((((((3R,3aR,6S,6aR)- hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(ethane-2,1- 48 diyl))bis(oxy))bis(carbonyl))bis(azanediyl))bis(3,3,5-trimethylcyclohexane-5,1- 49 diyl))bis(azanediyl))bis(carbonyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) referred to as CSMA and fabrication of composite discs via a Digital Light Printing (DLP) method. The flow behaviour of the polymer as a function of CaP addition, surface remineralisation potential, in vitro cell culture, using MC3T3 and Adipose-Derived Mesenchymal Stem Cells (ADSCs) and ex ovo angiogenic response was assessed. Finally, in vivo studies were carried out to investigate neo-bone formation at 4- and 8-weeks post-implantation. Quantitative micro-CT and histological evaluation did not show a higher rate of bone formation in CaP filled CSMA composites compared to CSMA itself. Therefore, such polymeric systems hold promising features by allowing more flexibility in designing a 3D printed scaffold targeted at the reconstruction of maxillofacial defects.

Chronic Leg Ulcers: Are Tissue Engineering and Biomaterials Science the Solution?

Chronic leg ulcers (CLUs) are full thickness wounds that usually occur between the ankle and knee, fail to heal after 3 months of standard treatment, or are not entirely healed at 12 months. CLUs present a considerable burden on patients, subjecting them to severe pain and distress, while healthcare systems suffer immense costs and loss of resources. The poor healing outcome of the standard treatment of CLUs generates an urgent clinical need to find effective solutions for these wounds. Tissue Engineering and Biomaterials Science offer exciting prospects for the treatment of CLUs, using a broad range of skin substitutes or scaffolds, and dressings. In this review, we summarize and discuss the various types of scaffolds used clinically in the treatment of CLUs. Their structure and therapeutic effects are described, and for each scaffold type representative examples are discussed, supported by clinical trials. Silver dressings are also reviewed due to their reported benefits in the healing of leg ulcers, as well as recent studies on new dermal scaffolds, reporting on clinical results where available. We conclude by arguing there is a further need for tissue-engineered products specifically designed and bioengineered to treat these wounds and we propose a series of properties that a biomaterial for CLUs should possess, with the intention of focusing efforts on finding an effective treatment.

Role of hydrogen peroxide in intra-operative wound preparation based on an in vitro fibrin clot degradation model.

Three per cent hydrogen peroxide (H2O2) is widely used to irrigate acute and chronic wounds in the surgical setting and clinical experience tells us that it is more effective at removing dried-on blood than normal saline alone. We hypothesise that this is due to the effect of H2O2 on fibrin clot architecture via fibrinolysis. We investigate the mechanisms and discuss the clinical implications using an in vitro model. Coagulation assays with normal saline (NaCl), 1% and 3% concentrations of H2O2 were performed to determine the effect on fibrin clot formation. These effects were confirmed by spectrophotometry. The effects of 1%, 3% and 10% H2O2 on the macroscopic and microscopic features of fibrin clots were assessed at set time intervals and compared to a NaCl control. Quantitative analysis of fibrin networks was undertaken to determine the fibre length, diameter, branch point density and pore size. Fibrin clots immersed in 1%, 3% and 10% H2O2 demonstrated volume losses of 0.09-0.25mm3/min, whereas those immersed in the normal saline gained in volume by 0.02±0.13 mm3/min. Quantitative analysis showed that H2O2 affects the structure of the fibrin clot in a concentration-dependent manner, with the increase in fibre length, diameter and consequently pore sizes. Our results support our hypothesis that the efficacy of H2O2 in cleaning blood from wounds is enhanced by its effects on fibrin clot architecture in a concentration- and time-dependent manner. The observed changes in fibre size and branch point density suggest that H2O2 is acting on the quaternary structure of the fibrin clot, most likely via its effect on cross-linking of the fibrin monomers and may therefore be of benefit for the removal of other fibrin-dependent structures such as wound slough.

Cell morphology as a design parameter in the bioengineering of cell-biomaterial surface interactions.

Control of cell-surface interaction is necessary for biomaterial applications such as cell sheets, intelligent cell culture surfaces, or functional coatings. In this paper, we propose the emergent property of cell morphology as a design parameter in the bioengineering of cell-biomaterial surface interactions. Cell morphology measured through various parameters can indicate ideal candidates for these various applications thus reducing the time taken for the screening and development process. The hypothesis of this study is that there is an optimal cell morphology range for enhanced cell proliferation and migration on the surface of biomaterials. To test the hypothesis, primary porcine dermal fibroblasts (PDF, 3 biological replicates) were cultured on ten different surfaces comprising components of the natural extracellular matrix of tissues. Results suggested an optimal morphology with a cell aspect ratio (CAR) between 0.2 and 0.4 for both increased cell proliferation and migration. If the CAR was below 0.2 (very elongated cell), cell proliferation was increased whilst migration was reduced. A CAR of 0.4+ (rounded cell) favoured cell migration over proliferation. The screening process, when it comes to biomaterials is a long, repetitive, arduous but necessary event. This study highlights the beneficial use of testing the cell morphology on prospective prototypes, eliminating those that do not support an optimal cell shape. We believe that the research presented in this paper is important as we can help address this screening inefficiency through the use of the emergent property of cell morphology. Future work involves automating CAR quantification for high throughput screening of prototypes.

Poly-ε-Caprolactone/Fibrin-Alginate Scaffold: A New Pro-Angiogenic Composite Biomaterial for the Treatment of Bone Defects.

We hypothesized that a composite of 3D porous melt-electrowritten poly-ɛ-caprolactone (PCL) coated throughout with a porous and slowly biodegradable fibrin/alginate (FA) matrix would accelerate bone repair due to its angiogenic potential. Scanning electron microscopy showed that the open pore structure of the FA matrix was maintained in the PCL/FA composites. Fourier transform infrared spectroscopy and differential scanning calorimetry showed complete coverage of the PCL fibres by FA, and the PCL/FA crystallinity was decreased compared with PCL. In vitro cell work with osteoprogenitor cells showed that they preferentially bound to the FA component and proliferated on all scaffolds over 28 days. A chorioallantoic membrane assay showed more blood vessel infiltration into FA and PCL/FA compared with PCL, and a significantly higher number of bifurcation points for PCL/FA compared with both FA and PCL. Implantation into a rat cranial defect model followed by microcomputed tomography, histology, and immunohistochemistry after 4- and 12-weeks post operation showed fast early bone formation at week 4, with significantly higher bone formation for FA and PCL/FA compared with PCL. However, this phenomenon was not extrapolated to week 12. Therefore, for long-term bone regeneration, tuning of FA degradation to ensure syncing with new bone formation is likely necessary.