Efficient electroporation of peptides into adherent cells: investigation of the role of mechano-growth factor in chondrocyte culture

Peptide therapeutics are of increasing interest due to their biological specificity. We used a simple technique to study the efficacy of inducing peptides into adherent chondrocytes by transiently permeabilizing the membrane with electric pulses (in situ electroporation). Mechano-growth factor (MGF) was selected as a model peptide. FITC-labeled MGF was added to cultures of adherent primary chondrocytes grown on ITO coated glass slides. Cells were subjected to 3-9pulses of 175-275V and evaluated by flow cytometry. Under optimal conditions, an electroporation efficiency of close to 50% could be achieved. This technique can be used to study the functional domains of intracellular peptides, peptide inhibition of signal transduction and intracrine-mediated effects of peptides in adherent cell

Electrochemically switchable platform for the micro-patterning and release of heterotypic cell sheets

This article describes a dynamic platform in which the biointerfacial properties of micro-patterned domains can be switched electrochemically through the spatio-temporally controlled dissolution and adsorption of polyelectrolyte coatings. Insulating SU-8 micro-patterns created on a transparent indium tin oxide electrode by photolithography allowed for the local control over the electrochemical dissolution of polyelectrolyte mono- and multilayers, with polyelectrolytes shielded from the electrochemical treatment by the underlying photoresist stencil. The platform allowed for the creation of micro-patterned cell co-cultures through the electrochemical removal of a non-fouling polyelectrolyte coating and the localized adsorption of a cell adhesive one after attachment of the first cell population. In addition, the use of weak adhesive polyelectrolyte coatings on the photoresist domains allowed for the detachment of a contiguous heterotypic cell sheet upon electrochemical trigger. Cells grown on the ITO domains peeled off upon electrochemical dissolution of the sacrificial polyelectrolyte substrate, whereas adjacent cell areas on the insulated weakly adhesive substrate easily detached through the contractile force generated by neighboring cells. This electrochemical strategy for the micro-patterning and detachment of heterotypic cell sheets combines simplicity, precision and versatility, and presents great prospects for the creation of cellular constructs which mimic the cellular complexity of native tissue

Zwitterionic Granular Hydrogel for Cartilage Tissue Engineering

Zwitterionic hydrogels have high potential for cartilage tissue engineering due to their ultra-hydrophilicity, nonimmunogenicity, and superior antifouling properties. However, their application in this field has been limited so far, due to the lack of injectable zwitterionic hydrogels that allow for encapsulation of cells in a biocompatible manner. Herein, a novel strategy is developed to engineer cartilage employing zwitterionic granular hydrogels that are injectable, self-healing, in situ crosslinkable and allow for direct encapsulation of cells with biocompatibility. The granular hydrogel is produced by mechanical fragmentation of bulk photocrosslinked hydrogels made of zwitterionic carboxybetaine acrylamide (CBAA), or a mixture of CBAA and zwitterionic sulfobetaine methacrylate (SBMA). The produced microgels are enzymatically crosslinkable using horseradish peroxidase, to quickly stabilize the construct, resulting in a microporous hydrogel. Encapsulated human primary chondrocytes are highly viable and able to proliferate, migrate, and produce cartilaginous extracellular matrix (ECM) in the zwitterionic granular hydrogel. It is also shown that by increasing hydrogel porosity and incorporation of SBMA, cell proliferation and ECM secretion are further improved. This strategy is a simple and scalable method, which has high potential for expanding the versatility and application of zwitterionic hydrogels for diverse tissue engineering applications

Enzymatically Crosslinked Collagen as a Versatile Matrix for In Vitro and In Vivo Co‐Engineering of Blood and Lymphatic Vasculature

Adequate vascularization is required for the successful translation of many in vitro engineered tissues. This study presents a novel collagen derivative that harbors multiple recognition peptides for orthogonal enzymatic crosslinking based on sortase A (SrtA) and Factor XIII (FXIII). SrtA-mediated crosslinking enables the rapid co-engineering of human blood and lymphatic microcapillaries and mesoscale capillaries in bulk hydrogels. Whereas tuning of gel stiffness determines the extent of neovascularization, the relative number of blood and lymphatic capillaries recapitulates the ratio of blood and lymphatic endothelial cells originally seeded into the hydrogel. Bioengineered capillaries readily form luminal structures and exhibit typical maturation markers both in vitro and in vivo. The secondary crosslinking enzyme Factor XIII is used for in situ tethering of the VEGF mimetic QK peptide to collagen. This approach supports the formation of blood and lymphatic capillaries in the absence of exogenous VEGF. Orthogonal enzymatic crosslinking is further used to bioengineer hydrogels with spatially defined polymer compositions with pro- and anti-angiogenic properties. Finally, macroporous scaffolds based on secondary crosslinking of microgels enable vascularization independent from supporting fibroblasts. Overall, this work demonstrates for the first time the co-engineering of mature micro- and meso-sized blood and lymphatic capillaries using a highly versatile collagen derivative

Factor XIII Cross-Linked Hyaluronan Hydrogels for Cartilage Tissue Engineering

ISSN:2373-987

Combination of a Collagen Scaffold and an Adhesive Hyaluronan-Based Hydrogel for Cartilage Regeneration: A Proof of Concept in an Ovine Model

Objective Hyaluronic acid–transglutaminase (HA-TG) is an enzymatically crosslinkable adhesive hydrogel with chondrogenic properties demonstrated in vitro and in an ectopic mouse model. In this study, we investigated the feasibility of using HA-TG in a collagen scaffold to treat chondral lesions in an ovine model, to evaluate cartilage regeneration in a mechanically and biologically challenging joint environment, and the influence of the surgical procedure on the repair process. Design Chondral defects of 6-mm diameter were created in the stifle joint of skeletally mature sheep. In a 3-month study, 6 defects were treated with HA-TG in a collagen scaffold to test the stability and biocompatibility of the defect filling. In a 6-month study, 6 sheep had 12 defects treated with HA-TG and collagen and 2 sheep had 4 untreated defects. Histologically observed quality of repair tissue and adjacent cartilage was semiquantitatively assessed. Results HA-TG adhered to the native tissue and did not cause any detectable negative reaction in the surrounding tissue. HA-TG in a collagen scaffold supported infiltration and chondrogenic differentiation of mesenchymal cells, which migrated from the subchondral bone through the calcified cartilage layer. Additionally, HA-TG and collagen treatment led to better adjacent cartilage preservation compared with empty defects ( P < 0.05). Conclusions This study demonstrates that the adhesive HA-TG hydrogel in a collagen scaffold shows good biocompatibility, supports in situ cartilage regeneration and preserves the surrounding cartilage. This proof-of-concept study shows the potential of this approach, which should be further considered in the treatment of cartilage lesions using a single-step procedure

Guidelines for standardization of bioprinting: a systematic study of process parameters and their effect on bioprinted structures

Biofabrication techniques including three-dimensional bioprinting could be used one day to fabricate living, patient-specific tissues and organs for use in regenerative medicine. Compared to traditional casting and molding methods, bioprinted structures can be much more complex, containing for example multiple materials and cell types in controlled spatial arrangement, engineered porosity, reinforcement structures and gradients in mechanical properties. With this complexity and increased function, however, comes the necessity to develop guidelines to standardize the bioprinting process, so printed grafts can safely enter the clinics. The bioink material must firstly fulfil requirements for biocompatibility and flow. Secondly, it is important to understand how process parameters affect the final mechanical properties of the printed graft. Using a gellan-alginate physically crosslinked bioink as an example, we show shear thinning and shear recovery properties which allow good printing resolution. Printed tensile specimens were used to systematically assess effect of line spacing, printing direction and crosslinking conditions. This standardized testing allowed direct comparison between this bioink and three commercially-available products. Bioprinting is a promising, yet complex fabrication method whose outcome is sensitive to a range of process parameters. This study provides the foundation for highly needed best practice guidelines for reproducible and safe bioprinted grafts

Hypoxia regulates RhoA and Wnt/β-catenin signaling in a context-dependent way to control re-differentiation of chondrocytes

Cartilage tissue is avascular and hypoxic which regulates chondrocyte phenotype via stabilization of HIFs. Here, we investigated the role of hypoxia and HIFs in regulation of Rho and canonical Wnt signaling in chondrocytes. Our data demonstrates that hypoxia controls the expression of RhoA in chondrocytes in a context-dependent manner on the culturing conditions. Within a 3D microenvironment, hypoxia suppresses RhoA on which hypoxia-driven expression of chondrogenic markers depends. Conversely, hypoxia leads to upregulation of RhoA in chondrocytes on 2D with a failure in re-expression of chondrogenic markers. Similarly to RhoA, hypoxic regulation of Wnt/β-catenin signaling depends on the microenvironment. Hypoxia downregulates β-catenin within 3D hydrogels whereas it causes a potent increase on 2D. Hypoxia-induced suppression of canonical Wnt signaling in 3D contributes to the promotion of chondrogenic phenotype as induction of Wnt signaling abrogates the hypoxic re-differentiation of chondrocytes. Inhibiting Wnt/β-catenin signaling via stabilization of Axin2 leads to a synergistic enhancement of hypoxia-induced expression of chondrogenic markers. The effects of hypoxia on Rho and Wnt/β-catenin signaling are HIF-dependent as stabilizing HIFs under normoxia revealed similar effects on chondrocytes. The study reveals important insights on hypoxic signaling of chondrocytes and how hypoxia regulates cellular mechanisms depending on the cellular microenvironment