Introducing Dynamicity: Engineering Stress Relaxation Into Hydrogels Via Thiol-Ene Modified Alginate for Mechanobiological in vitro Modeling of the Cornea.

Developing biomaterials for corneal repair and regeneration is crucial for maintaining clear vision. The cornea, a specialized tissue, relies on corneal keratocytes, that respond to their mechanical environment. Altering stiffness affects keratocyte behavior, but static stiffness alone cannot capture the dynamic properties of in vivo tissue. This study proposes that the cornea exhibits time-dependent mechanical properties, similar to other tissues, and aims to replicate these properties in potential therapeutic matrices. First, the cornea's stress relaxation properties are investigated using nanoindentation, revealing 15% relaxation within 10 seconds. Hydrogel dynamicity is then modulated using a specially formulated alginate-PEG and alginate-norbornene mixture. The tuning of the hydrogel's dynamicity is achieved through a photoinitiated norbornene-norbornene dimerization reaction, resulting in relaxation times ranging from 30 seconds to 10 minutes. Human primary corneal keratocytes are cultured on these hydrogels, demonstrating reduced αSMA (alpha smooth muscle actin) expression and increased filopodia formation on slower relaxing hydrogels, resembling their native phenotype. This in vitro model can enable the optimization of stress relaxation for various cell types, including corneal keratocytes, to control tissue formation. Combining stress relaxation optimization with stiffness assessment provides a more accurate tool for studying cell behavior and reduces mechanical mismatch with native tissues in implanted constructs.

Flexible, Suturable, and Leak-free Scaffolds for Vascular Tissue Engineering Using Melt Spinning.

Coronary artery disease affects millions worldwide. Bypass surgery remains the gold standard; however, autologous tissue is not always available. Hence, the need for an off-the-shelf graft to treat these patients remains extremely high. Using melt spinning, we describe here the fabrication of tubular scaffolds composed of microfibers aligned in the circumferential orientation mimicking the organized extracellular matrix in the tunica media of arteries. By variation of the translational extruder speed, the angle between fibers ranged from 0 to ∼30°. Scaffolds with the highest angle showed the best performance in a three-point bending test. These constructs could be bent up to 160% strain without kinking or breakage. Furthermore, when liquid was passed through the scaffolds, no leakage was observed. Suturing of native arteries was successful. Mesenchymal stromal cells were seeded on the scaffolds and differentiated into vascular smooth muscle-like cells (vSMCs) by reduction of serum and addition of transforming growth factor beta 1 and ascorbic acid. The scaffolds with a higher angle between fibers showed increased expression of vSMC markers alpha smooth muscle actin, calponin, and smooth muscle protein 22-alpha, whereas a decrease in collagen 1 expression was observed, indicating a positive contractile phenotype. Endothelial cells were seeded on the repopulated scaffolds and formed a tightly packed monolayer on the luminal side. Our study shows a one-step fabrication for ECM-mimicking scaffolds with good handleability, leak-free property, and suturability; the excellent biocompatibility allowed the growth of a bilayered construct. Future work will explore the possibility of using these scaffolds as vascular conduits in in vivo settings.

Development of a biomimetic arch-like 3D bioprinted construct for cartilage regeneration using gelatin methacryloyl and silk fibroin-gelatin bioinks.

In recent years, engineering biomimetic cellular microenvironments have been a top priority for regenerative medicine. Collagen II, which is arranged in arches, forms the predominant fiber network in articular cartilage. Due to the shortage of suitable microfabrication techniques capable of producing 3D fibrous structures,in vitroreplication of the arch-like cartilaginous tissue constitutes one of the major challenges. Hence, in the present study, we report a 3D bioprinting approach for fabricating arch-like constructs using two types of bioinks, gelatin methacryloyl (GelMa) and silk fibroin-gelatin (SF-G). The bioprinted SF-G constructs displayed increased proliferation of the encapsulated human bone marrow-derived mesenchymal stem cells compared to the GelMA constructs. Biochemical assays, gene, and protein expression exhibited the superior role of SF-G in forming the fibrous collagen network and chondrogenesis. Protein-protein interaction study using Metascape evaluated the function of the proteins involved. Further GeneMANIA and STRING analysis using Col 2A1, SOX 9, ACAN, and the genes upregulated on day 21 in RT-PCR, i.e.ß-catenin, TGFßR1, Col 1A1 in SF-G and PRG4, Col 10A1, MMP 13 in GelMA validated ourin vitroresults. These findings emphasized the role of SF-G in regulating the Wnt/ß-catenin and TGF-ßsignaling pathways. Hence, the 3D bioprinted arch-like constructs possess a substantial potential for cartilage regeneration.

Fabrication of a mimetic vascular graft using melt spinning with tailorable fiber parameters.

Smooth muscle cells play a pivotal role in maintaining blood pressure and remodeling of the extracellular matrix. These cells have a characteristic spindle shape and are aligned in the radial direction to aid in the constriction of any artery. Tissue engineered grafts have the potential to recreate this alignment and offer a viable alternative to non-resorbable or autologous grafts. Specifically, with melt spinning small diameter fibers can be created that can align circumferentially on the scaffolds. In this study, a set of simplified equations were formulated to predict the final fiber parameters. Smooth muscle cell alignment was monitored on the fabricated scaffolds. Finally, a co-culture of smooth muscle cells in direct contact with endothelial cells was performed to assess the influence of the smooth muscle cell alignment on the morphology of the endothelial cells. The results show that the equations were able to accurately predict the fiber diameter, distance and angle. Primary vascular smooth muscle cells aligned according to the fiber direction mimicking the native orientation. The co-culture with endothelial cells showed that the aligned smooth muscle cells did not have an influence on the morphology of the endothelial cells. In conclusion, we formulated a series of equations that can predict the fiber parameters during melt spinning. Furthermore, the method described here can create a vascular graft with smooth muscle cells aligned circumferentially that morphologically mimics the native orientation.

Tuning Hydrogels by Mixing Dynamic Cross-Linkers: Enabling Cell-Instructive Hydrogels and Advanced Bioinks.

Rational design of hydrogels that balance processability and extracellular matrix (ECM) biomimicry remains a challenge for tissue engineering and biofabrication. Hydrogels suitable for biofabrication techniques, yet tuneable to match the mechanical (static and dynamic) properties of native tissues remain elusive. Dynamic covalent hydrogels possessing shear-thinning/self-healing (processability) and time-dependent cross-links (mechanical properties) provide a potential solution, yet can be difficult to rationally control. Here, the straightforward modular mixing of dynamic cross-links with different timescales (hydrazone and oxime) is explored using rheology, self-healing tests, extrusion printing, and culture of primary human dermal fibroblasts. Maintaining a constant polymer content and cross-linker concentration, the stiffness and stress relaxation can be tuned across two orders of magnitude. All formulations demonstrate a similar flow profile after network rupture, allowing the separation of initial mechanical properties from flow behavior during printing. Furthermore, the self-healing nature of hydrogels with high hydrazone content enables recyclability of printed structures. Last, a distinct threshold for cell spreading and morphology is observed within this hydrogel series, even in multi-material constructs. Simple cross-linker mixing enables fine control and is of general interest for bioink development, targeting viscoelastic properties of specific cellular niches, and as an accessible and flexible platform for designing dynamic networks.

Glycosaminoglycans: From Vascular Physiology to Tissue Engineering Applications.

Cardiovascular diseases represent the number one cause of death globally, with atherosclerosis a major contributor. Despite the clinical need for functional arterial substitutes, success has been limited to arterial replacements of large-caliber vessels (diameter > 6 mm), leaving the bulk of demand unmet. In this respect, one of the most challenging goals in tissue engineering is to design a "bioactive" resorbable scaffold, analogous to the natural extracellular matrix (ECM), able to guide the process of vascular tissue regeneration. Besides adequate mechanical properties to sustain the hemodynamic flow forces, scaffold's properties should include biocompatibility, controlled biodegradability with non-toxic products, low inflammatory/thrombotic potential, porosity, and a specific combination of molecular signals allowing vascular cells to attach, proliferate and synthesize their own ECM. Different fabrication methods, such as phase separation, self-assembly and electrospinning are currently used to obtain nanofibrous scaffolds with a well-organized architecture and mechanical properties suitable for vascular tissue regeneration. However, several studies have shown that naked scaffolds, although fabricated with biocompatible polymers, represent a poor substrate to be populated by vascular cells. In this respect, surface functionalization with bioactive natural molecules, such as collagen, elastin, fibrinogen, silk fibroin, alginate, chitosan, dextran, glycosaminoglycans (GAGs), and growth factors has proven to be effective. GAGs are complex anionic unbranched heteropolysaccharides that represent major structural and functional ECM components of connective tissues. GAGs are very heterogeneous in terms of type of repeating disaccharide unit, relative molecular mass, charge density, degree and pattern of sulfation, degree of epimerization and physicochemical properties. These molecules participate in a number of vascular events such as the regulation of vascular permeability, lipid metabolism, hemostasis, and thrombosis, but also interact with vascular cells, growth factors, and cytokines to modulate cell adhesion, migration, and proliferation. The primary goal of this review is to perform a critical analysis of the last twenty-years of literature in which GAGs have been used as molecular cues, able to guide the processes leading to correct endothelialization and neo-artery formation, as well as to provide readers with an overall picture of their potential as functional molecules for small-diameter vascular regeneration.

The effect of prior long-term recellularization with keratocytes of decellularized porcine corneas implanted in a rabbit anterior lamellar keratoplasty model.

Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 µm thick decellularized lenticule against one that had been recellularized with human stromal cells after serum-free culture. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. Since there was little difference between acellular and cell-seeded scaffolds in our in vivo study, future scaffold development should use acellular controls to determine if cells are necessary.

Multilayered Fabrication Assembly Technique to Engineer a Corneal Stromal Equivalent.

Tissue engineering has emerged as a strategy to combat the donor shortage of human corneas for transplantation. Synthetic corneal substitutes are currently unable to support the normal phenotype of human cells and so decellularized animal corneas have been deployed to more closely provide the topographical and biochemical cues to promote cell attachment and function. Although full thickness decellularized corneas can support corneal cells, the cells are slow to populate the scaffold and density declines from the surface. To avoid these problems, this protocol describes the stacking of alternate layers of decellularized porcine corneal sheets and cell-laden collagen hydrogel to produce a corneal construct. The sheets are obtained by cryosectioning porcine corneas, decellularizing them with detergents and nucleases and finally air drying for storage and ease of manufacture. Corneal stromal cells are then encapsulated in a collagen type I solution and cast between these sheets. This protocol presents a rapid method to ensure high cellularity throughout the construct using tissue-derived materials alone. Graphic abstract: Overview of main process to obtain corneal stromal equivalents.

Fabrication of Corneal Extracellular Matrix-Derived Hydrogels.

Hydrogels derived from corneal extracellular matrix (ECM) represent a promising biomaterial for corneal repair and regeneration. To fabricate these hydrogels, first corneas need to be decellularized using repeated freeze-thaw cycles and nucleases to remove all nuclear and cellular components. The remaining corneal ECM is lyophilized to remove all water and milled into a fine powder. The ECM powder is weighed and dissolved in pepsin solution at a concentration of 20 mg/mL. Hydrogels are formed by neutralizing the pH of the solution and maintaining it at 37 °C until fibrillogenesis has occurred. Corneal stromal cells may be suspended throughout the hydrogel solution prior to gelation to generate a corneal stromal substitute.

Development and Validation of a 3D In Vitro Model to Study the Chemotactic Behavior of Corneal Stromal Fibroblasts.

Chemotaxis plays a pivotal role in crucial biological phenomena including immune response, cancer metastasis, and wound healing. Although many chemotaxis assays have been developed to better understand these multicomplex biological mechanisms, most of them have serious limitations mainly due to the poor representation of native three-dimensional (3D) microenvironment. Here, we describe a method to develop and validate a novel 3D in vitro chemotaxis model to study the migration of corneal fibroblasts through a stromal equivalent. A hydrogel was used that contained gelatin microspheres loaded with platelet-derived growth factor-BB (PDGF-BB) in the inner section and corneal fibroblasts in the outer section. The cell migration toward the chemical stimuli over time can be monitored via confocal microscopy. The development of this in vitro model can be used for both qualitative and quantitative examinations of chemotaxis.

Engineering a Corneal Stromal Equivalent Using a Novel Multilayered Fabrication Assembly Technique.

To overcome the serious shortage of donor corneas for transplantation, alternatives based on tissue engineering need to be developed. Decellularized corneas are one potential alternative, but their densely packed collagen architecture inhibits recellularization in vitro. Therefore, a new rapid method of recellularizing these constructs to ensure high cellularity throughout the collagen scaffold is needed. In this study, we developed a novel method for fabricating corneal constructs by using decellularized porcine corneal sheets assembled using a bottom-up approach by layering multiple sheets between cell-laden collagen I hydrogel. Corneal lenticules were cut from porcine corneas by cryosectioning, then decellularized with detergents and air-dried for storage as sheets. Human corneal stromal cells were encapsulated in collagen I hydrogel and cast between the dried sheets. Constructs were cultured in serum-free medium supplemented with ascorbic acid and insulin for 2 weeks. Epithelial cells were then seeded on the surface and cultured for an additional week. Transparency, cell viability, and phenotype were analyzed by qPCR, histology, and immunofluorescence. Constructs without epithelial cells were sutured onto an ex vivo porcine cornea and cultured for 1 week. Lenticules were successfully decellularized, achieving dsDNA values of 13 ± 1.2 ng/mg dry tissue, and were more resistant to degradation than the collagen I hydrogels. Constructs maintained high cell viability with a keratocyte-like phenotype with upregulation of keratocan, decorin, lumican, collagen I, ALDH3A1, and CD34 and the corneal epithelial cells stratified with a cobblestone morphology. The construct was amenable to surgical handling and no tearing occurred during suturing. After 7 days ex vivo, constructs were covered by a neoepithelium from the host porcine cells and integration into the host stroma was observed. This study describes a novel approach toward fabricating anterior corneal substitutes in a simple and rapid manner, obtaining mature and suturable constructs using only tissue-derived materials.

Characterization of extracellular matrix modified poly(ε-caprolactone) electrospun scaffolds with differing fiber orientations for corneal stroma regeneration.

Alternatives to donor cornea transplantation based on tissue engineering are desirable to overcome the current severe donor tissue shortage. Many natural polymers have good biological properties but poor mechanical properties and degradation resistance; while synthetic polymers have good mechanical properties but do not contain biochemical molecules normally found in the real tissue. In addition, both fiber orientation and composition play a key role in dictating cell behavior within a scaffold. In this study, the effect on corneal stromal cells of adding decellularized corneal extracellular matrix (ECM) to an electrospun polymer with differing fiber organizations was explored. Electrospun matrices were generated using polycaprolactone (PCL) and PCL combined with ECM and electrospun into random, radial and perpendicularly aligned fiber scaffolds. Human corneal stromal cells were seeded onto these scaffolds and the effect of composition and orientation on the cells phenotype was assessed. Incorporation of ECM into PCL increased hydrophilicity of scaffolds without an adverse effect on Young's modulus. Cells seeded on these matrices adopted different morphologies that followed the orientation of the fibers. Keratocyte markers were increased in all types of scaffolds compared to tissue culture plastic. Scaffolds with radial and perpendicularly aligned fibers promoted enhanced cell migration. Aligned scaffolds with incorporated ECM show promise for their use as cell-free implants that promote endogenous repopulation by neighboring cells.

Decellularization and recellularization of cornea: Progress towards a donor alternative.

The global shortage of donor corneas for transplantation has led to corneal bioengineering being investigated as a method to generate transplantable tissues. Decellularized corneas are among the most promising materials for engineering corneal tissue since they replicate the complex structure and composition of real corneas. Decellularization is a process that aims to remove cells from organs or tissues resulting in a cell-free scaffold consisting of the tissues extracellular matrix. Here different decellularization techniques are described, including physical, chemical and biological methods. Analytical techniques to confirm decellularization efficiency are also discussed. Different cell sources for the recellularization of the three layers of the cornea, recellularization methods used in the literature and techniques used to assess the outcome of the implantation of such scaffolds are examined. Studies involving the application of decellularized corneas in animal models and human clinical studies are discussed. Finally, challenges for this technology are explored involving scalability, automatization and regulatory affairs.

Author Correction: The impact of decellularization methods on extracellular matrix derived hydrogels.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The impact of decellularization methods on extracellular matrix derived hydrogels.

Tissue-derived decellularized biomaterials are ideal for tissue engineering applications as they mimic the biochemical composition of the native tissue. These materials can be used as hydrogels for cell encapsulation and delivery. The decellularization process can alter the composition of the extracellular matrix (ECM) and thus influence the hydrogels characteristics. The aim of this study was to examine the impact of decellularization protocols in ECM-derived hydrogels obtained from porcine corneas. Porcine corneas were isolated and decellularized with SDS, Triton X-100 or by freeze-thaw cycles. All decellularization methods decreased DNA significantly when measured by PicoGreen and visually assessed by the absence of cell nuclei. Collagen and other ECM components were highly retained, as quantified by hydroxyproline content and sGAG, by histological analysis and by SDS-PAGE. Hydrogels obtained by freeze-thaw decellularization were the most transparent. The method of decellularization impacted gelation kinetics assessed by turbidimetric analysis. All hydrogels showed a fibrillary and porous structure determined by cryoSEM. Human corneal stromal cells were embedded in the hydrogels to assess cytotoxicity. SDS decellularization rendered cytotoxic hydrogels, while the other decellularization methods produced highly cytocompatible hydrogels. Freeze-thaw decellularization produced hydrogels with the overall best properties.

Potential for combined delivery of riboflavin and all-trans retinoic acid, from silk fibroin for corneal bioengineering.

Millions of people worldwide suffer from vision impairing conditions resulting from corneal injury or disease. Silk fibroin (SF) is an emerging biopolymer that has been used for several applications including the fabrication of bioengineered corneas and ocular prostheses. To improve the cell response to SF, riboflavin (RF) and all-trans retinoic acid (RA) were coupled onto SF matrices. RF is a photo-initiator that has previously been combined with ultraviolet light to crosslink corneal collagen while RA has been used to regulate the phenotype of corneal stromal cells and their extracellular matrix deposition. Different concentrations of RF and RA were respectively photo-crosslinked and covalently bound through carbodiimide coupling onto 2% SF matrices. The effect of incorporating these molecules on the physical, chemical and mechanical properties of the matrices was evaluated. The biological response of human corneal stromal cells to the matrices was examined using cellular adhesion assays, proliferation assays, cytoskeleton staining, gene expression analysis and immunocytochemical staining. RF and RA both led to changes in the surface nanostructure and hydrophilicity while just RF increased the material stiffness. Cells cultured on the matrices containing both biomolecules displayed improved cellular proliferation, increased GAG deposition and increased expression of keratocyte genes that are normally associated with healthy corneal stromal tissue. These in vitro studies serve as a starting point for the optimization of loading bioactive molecules on SF based matrices for formulating clinically relevant ocular implants.

Influence of Biochemical Cues in Human Corneal Stromal Cell Phenotype.

PURPOSE: To identify biochemical cues that could promote a keratocyte-like phenotype in human corneal stromal cells that had become fibroblastic when expanded in serum-supplemented media while also examining the effect on cell proliferation and migration. METHODS: Proliferation was assessed by PrestoBlue™, morphology was monitored by phase contrast microscopy, phenotype was analyzed by real-time polymerase chain reaction (qPCR), immunochemistry and flow cytometry, and migration was studied with a scratch assay. RESULTS: Ascorbic Acid (AA), Retinoic Acid (RA), Insulin-Transferrin-Selenium (ITS), Insulin-like Growth Factor 1 (IGF-1) and 3-isobutyl-1-methylxanthine (IBMX) promoted a dendritic morphology, increased the expression of keratocyte markers, such as keratocan, aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and CD34, and prevented myofibroblast differentiation, while in some cases increasing proliferation. Transforming Growth Factor beta 1 (TGF-ß1) and 3 (TGF-ß3) promoted the differentiation toward myofibroblasts, with increased expression of α-SMA. Fibroblast Growth Factor 2 (FGF-2) supported a fibroblastic phenotype while Platelet-Derived Growth Factor Homodimer B (PDGF-BB) induced a pro-migratory fibroblastic phenotype. A combination of all the pro-keratocyte factors was also compared to the serum-free only, which significantly increased CD34 and keratocan expression. CONCLUSIONS: Partially recovery towards a quiescent keratocyte-like phenotype was achieved by the removal of serum and the addition of AA, IGF-1, RA, ITS and IBMX to a basal medium. These findings can be used to develop cell-based corneal therapies and to study corneal diseases in vitro.

Limbal stromal cells derived from porcine tissue demonstrate mesenchymal characteristics in vitro.

Limbal stromal cells (LSCs) from the human ocular surface display mesenchymal stromal cell characteristics in vitro. In this study, we isolated cells from the porcine limbal stroma (pLSCs), characterised them, and evaluated their ability to support angiogenesis and the culture of porcine limbal epithelial stem cells (pLESCs). The isolated cells adhered to plastic and grew in monolayers in vitro using serum-supplemented or serum-free medium. The pLSCs demonstrated expression of CD29, and cross-reactivity with anti-human CD45, CD90, CD105, CD146, and HLA-ABC. However, expression of CD105, CD146 and HLA-ABC reduced when cultured in serum-free medium. PLSCs did not undergo adipogenic or osteogenic differentiation, but differentiated towards the chondrogenic lineage. Isolated cells were also co-cultured with human umbilical vein endothelial cells (HUVECs) in star-shaped Poly(ethylene glycol) (starPEG)-heparin hydrogels to assess their pericyte capacity which supported angiogenesis networks of HUVECs. PLSCs supported the three dimensional HUVEC network for 7 days. The isolated cells were further growth-arrested and evaluated as feeder cells for pLESC expansion on silk fibroin membranes, as a potential carrier material for transplantation. PLSCs supported the growth of pLESCs comparably to murine 3T3 cells. In conclusion, although pLSCs were not completely comparable to their human counterpart, they display several mesenchymal-like characteristics in vitro.

Aumento del costo directo de los pacientes con epilepsia y depresión / Increase in the direct cost of patients suffering from epilepsy and depression

Se realiza un estudio de costo por fármacos antiepilépticos (FAE) y fármacos antidepresivos (FAD) en una población de 110 pacientes con epilepsia. Se encontró que la depresión alcanzaba más del 70 por ciento de los estudiados, con un 40 por ciento que necesitaba medicación antidepresiva. Aparecía más frecuencia de crisis relacionada con la depresión de forma severa y moderada. El costo por FAE de los pacientes estudiados es totalmente bajo y solo llega a 0,25 centavos por paciente, en un año sería de 91,25 pesos. En los aproximadamente 80,000 pacientes con epilepsia que hay en todo el país tendríamos un costo anual 7 millones 300 mil pesos. El costo de la medicación por antidepresivos y antiepilépticos en cada paciente es de 0,47 centavos diarios, y en un año sería de 171,55 pesos por año. El costo actual por FAE es de 110,23 pesos en un año aproximadamenteSe hace un análisis de cómo disminuir el costo por la combinación de FAE y el uso de FAE asociados. Se sugiere el uso de medicina alternativa de forma priorizada para los pacientes que tiene aumento de la frecuencia de sus crisis de epilepsia y depresión