Manufacturing of self-standing multi-layered 3D-bioprinted alginate-hyaluronate constructs by controlling the cross-linking mechanisms for tissue engineering applications.

Three-dimensional (3D) bioprinting of self-supporting stable tissue and organ structure is critically important in extrusion-based bioprinting system, especially for tissue engineering and regenerative medicine applications. However, the development of self-standing bioinks with desired crosslinking density, biocompatibility, tunable mechanical strength and other properties like self-healing,in situgelation, drug or protein incorporation is still a challenge. In this study, we report a hydrogel bioink prepared from alginate (Alg) and hyaluronic acid (HA) crosslinked through multiple crosslinking mechanisms, i.e. acyl-hydrazone, hydrazide interactions and calcium ions. These Alg-HA gels were highly dynamic and shear-thinning with exceptional biocompatibility and tunable mechanical properties. The increased dynamic nature of the gels is mainly chemically attributed to the presence of acyl-hydrazone bonds formed between the amine groups of the acyl-hydrazide of alginate and the monoaldehyde of the HA. Among the different combinations of Alg-HA gel compositions prepared, the A5H5 (Alginate-acyl-hydrazide:HA-monoaldehyde, ratio 50:50) gel showed a gelation time of ∼60 s, viscosity of ∼400 Pa s (at zero shear rate), high stability in various pH solutions and increased degradation time (>50 days) than the other samples. The A5H5 gels showed high printability with increased post-printing stability as observed from the 3D printed structures (e.g. hollow tube (∼100 layers), porous cube (∼50 layers), star, heart-in, meniscus and lattice). The scanning electron microscopy analysis of the 3D constructs and hydrogels showed the interconnected pores (∼181µm) and crosslinked networks. Further, the gels showed sustained release of 5-amino salicylic acid and bovine serum albumin. Also, the mechanical properties were tuned by secondary crosslinking via different calcium concentrations.In vitroassays confirmed the cytocompatibility of these gels, where the 3D bioprinted lattice and tubular (∼70 layers) constructs demonstrated high cell viability under fluorescence analysis. Inin vivostudies, Alg-HA gel showed high biocompatibility (>90%) and increased angiogenesis (threefolds) and reduced macrophage infiltration (twofold decrease), demonstrating the promising potential of these hydrogels in 3D bioprinting applications for tissue engineering and regenerative medicine with tunable properties.

Self-crosslinking hyaluronic acid-carboxymethylcellulose hydrogel enhances multilayered 3D-printed construct shape integrity and mechanical stability for soft tissue engineering.

One of the primary challenges in extrusion-based 3D bioprinting is the ability to print self-supported multilayered constructs with biocompatible hydrogels. The bioinks should have sufficient post-printing mechanical stability for soft tissue and organ regeneration. Here, we report on the synthesis, characterization and 3D printability of hyaluronic acid (HA)-carboxymethylcellulose (CMC) hydrogels cross-linked through N-acyl-hydrazone bonding. The hydrogel's hydrolytic stability was acquired by the effects of both the prevention of the oxidation of the six-membered rings of HA, and the stabilization of acyl-hydrazone bonds. The shear-thinning and self-healing properties of the hydrogel allowed us to print different 3D constructs (lattice, cubic and tube) of up to 50 layers with superior precision and high post-printing stability without support materials or post-processing depending on their compositions (H7:C3, H5:C5 and H3:C7). Morphological analyses of different zones of the 3D-printed constructs were undertaken for verification of the interconnection of pores. Texture profile analysis (TPA) (hardness (strength), elastic recovery, etc) and cyclic compression studies of the 3D-printed constructs demonstrated exceptional elastic properties and fast recovery after 50% strain, respectively, which have been attributed to the addition of CMC into HA. A model drug quercetin was released in a sustained manner from hydrogels and 3D constructs. In vitro cytotoxicity studies confirmed the excellent cyto-compatibility of these gels. In vivo mice studies prove that these biocompatible hydrogels enhance angiogenesis. The results indicate that controlling the key properties (e.g. self-crosslinking capacity, composition) can lead to the generation of multilayered constructs from 3D-bioprintable HA-CMC hydrogels capable of being leveraged for soft tissue engineering applications.

Directing human embryonic stem cells towards functional endothelial cells easily and without purification.

Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.

Evaluation of the biocompatibility of a coating material for an implantable bladder volume sensor.

As the applications for implantable medical devices have increased, the need for biocompatible packaging materials has become important. Recently, we reported an implantable sensor for real-time monitoring of the changes in bladder volume, which necessitated finding a safe coating material for use in bladder tissue. At present, materials like polyethylene glycol (PEG), polydimethylsiloxane (PDMS) and parylene-C are used in biomedical devices or as coating materials, owing to their excellent safety in various medical fields. However, few studies have assessed their safety in bladder tissue, therefore, we evaluated the biocompatibility of PEG, PDMS and parylene-C in the bladder. All three materials turned out to be safe in in vitro tests of live/dead staining and cell viability. In vivo tests with hematoxylin and eosin and immunofluorescence staining with MAC387 showed no persistent inflammation. Therefore, we consider that the three materials are biocompatible in bladder tissue. Despite this safety, however, PEG has biodegradable characteristics and thus is not suitable for use as packaging. We suggest that PDMS and parylene-C can be used as safe coating materials for the implantable bladder volume sensor reported previously.