Abstract 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.
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
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.
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.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.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-β1) and 3 (TGF-β3) 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.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.
Abstract 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.
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.
Purpose With the increasing demand for corneal replacements and a shortage of suitable donors, tissue engineering and cell based therapies have emerged as a possible solution. Understanding the influence that biochemical factors have on corneal wound healing is needed to improve tissue regeneration. Our aim was to investigate the effect of several molecules on human corneal stromal cells. Methods Corneal fibroblasts were serum‐starved for 72 h in DMEM/F12 and then exposed to specific reagents for 7–14 days. The reagents examined in this study were ascorbic acid (AA), retinoic acid (RA), FGF‐2, PDGF‐BB, IGF, TGF‐ β 1, TGF‐ β 3, IBMX and insulin‐transferrin‐selenium (ITS). Proliferation was assessed by AlamarBlue assay, general morphology was monitored by bright field microscopy and the expression of keratocan, ALDH3A1 and smooth muscle actin ( α ‐SMA) at the mRNA level was quantified by qPCR. Immunohistochemistry and western blotting were also performed to assess protein expression. Results AA, IGF‐1, RA and ITS increased the expression of keratocyte markers such as keratocan and ALDH3A1, while not increasing fibrotic/myofibroblast marker α ‐SMA. FGF‐2 and PDGF‐BB decreased expression of keratocan and ALDH3A1 without up‐regulating α ‐SMA, suggesting a fibroblastic phenotype. TGF‐ β 1 and TGF‐ β 3 were shown to up‐regulate α ‐SMA expression but TGF‐ β 3 also up‐regulated keratocan. All reagents apart from AA resulted in an increase in cell proliferation by day 7. Conclusions This study demonstrates the influence different biochemical cues have on corneal stromal cells. These findings will be useful in developing the next generation of therapies for corneal diseases and injuries.
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.
Abstract Chronic kidney disease (CKD) leads to a gradual loss of kidney function, with fibrosis as pathological endpoint, which is characterized by extracellular matrix (ECM) deposition and remodeling. Traditionally, in vivo models are used to study interstitial fibrosis, through histological characterization of biopsy tissue. However, ethical considerations and the 3Rs (replacement, reduction, and refinement) regulations emphasizes the need for humanized 3D in vitro models. This study introduces a bioprinted in vitro model which combines primary human cells and decellularized and partially digested extracellular matrix (ddECM). A protocol was established to decellularize kidney pig tissue and the ddECM was used to encapsulate human renal cells. To investigate fibrosis progression, cells were treated with transforming growth factor beta 1 (TGF‐β1), and the mechanical properties of the ddECM hydrogel were modulated using vitamin B2 crosslinking. The bioprinting perfusable model replicates the renal tubulointerstitium. Results show an increased Young's modulus over time, together with the increase of ECM components and cell dedifferentiation toward myofibroblasts. Multiple fibrotic genes resulted upregulated, and the model closely resembled fibrotic human tissue in terms of collagen deposition. This 3D bioprinted model offers a more physiologically relevant platform for studying kidney fibrosis, potentially improving disease progression research and high‐throughput drug screening.
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