Application of hyperbaric oxygen in bone tissue engineering : effect of hyperbaric oxygen treatment on bone marrow stem cells

and non-union of bony fractures has been proposed since 1966, little has been known about the effect of HBOT on bone marrow stem cells (BMSC). The aim of this study is to investigate the effect of HBO treatment on osteogenetic differentiation of BMSC and potential application in bone tissue engineering. Adhesive stromal cells harvested from bone marrow were characterized by mesenchymal differentiation potential, cell surface markers and their proliferation capacity. Mesenchymal stem cells, which demonstrated osteogenic, chondrogenic and adipogenic differentiation potential and expressed positively for CD 29, CD 44, CD 73, CD 90, CD 105, CD 166 and negatively for CD34 and CD 45, were selected and treated in a laboratory-scale HBO chamber using different oxygen pressures and exposure times. No obvious effect of HBO treatment on BMSC proliferation was noticed. However, cytotoxic effects of HBO were considerably less pronounced when cells were cultured in medium supplemented with 10% FBS in comparison to medium supplemented with 2% FCS, as was evaluated by WST-1 assay. Under HBO treatment, bone nodules were formed in three days, which was clearly revealed by Von Kossa staining. In contrasts, without HBO treatment, bone nodules were not detected until 9-12 days using the same inducing culture media. Calcium deposition was also significantly increased after three days of HBO treatments compared to no HBO treatment. In addition it was also found that oxygen played a direct role in the enhancement of BMSC osteogenic differentiation, which was independent of the effect of air pressure

A multiangular approach towards biofabrication of an auricular cartilage implant

Cartilage tissue engineering opens new avenues for reconstruction of auricular deformities. Nevertheless, a number of challenges hinder the development of an auricular cartilage implant, including an appropriate cell source, nutrient limitation in large non-vascularized constructs, and maintenance of the complex auricular shape. This work uses a multiangular approach including biofabrication strategies to address these challenges. Firstly, we investigated the regenerative potential of novel auricular cartilage progenitor cells in 3D printable hydrogels. Furthermore, we proposed a modular construct to decrease the diffusion distance throughout the implant. In addition, the mechanical integrity of the developing construct is warranted by a polymer fiber-reinforced network integrated into a cell-laden hydrogel. Equine auricular cartilage progenitor cells (AuCPC) were encapsulated in 10% gelatin methacrylate (gelMA) hydrogel cylinders and chondrogenically differentiated up to 56 days in vitro. The neocartilage produced by AuCPC displayed GAG/DNA composition and mechanical integrity comparable to auricular chondrocytes (AuCH), and the production of cartilage-like extracellular matrix was confirmed by histology. Polycaprolactone (PCL) scaffolds for custom-designed modular parts of the auricle were fabricated using a Bioscaffolder and combined with gelMA to form hybrid constructs. Light microscopy confirmed homogenous distribution of the hydrogel through the reinforcing network, and the assembled modules displayed a convincing aesthetical appearance under a rubber skin. Bioprinted cell-laden constructs demonstrated homogenous cell distribution and good cell viability after printing up to 7 days of in vitro culture. These results indicate that a multi-faceted approach in creating large tissue constructs is a promising method that warrants further investigation

Application of different cell populations in hydrogel bioinks for zonal Cartilage biofabrication

Functional regeneration of articular cartilage is still a major challenge in human. Bioprinting permits to mimic the complex architecture of articular cartilage, by coordinating the deposition of multiple cell types and materials, termed bioinks. For this purpose, cells with high potential for zonal differentiation need to be encapsulated in bioinks that provide an instructive niche for extracellular matrix (ECM) synthesis. The recent identification of multipotent articular cartilage chondroprogenitor cells (ACPCs) represents a new opportunity to generate bioinks with defined zonal affinity. The aim of this work was to print zonal constructs using hydrogel bioinks encapsulating ACPCs, alone or in combination with other cell types, obtained from equine donors. Gelatin methacryloyl (gelMA)-based inks were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes (CHs) in casted gels. The expression of zonal markers and ECM molecules by each cell type was studied. Constructs composed of two adjacent regions, each containing a single cell type were also fabricated, as models for zonal co-culture of the possible MSCs, CHs, and ACPCs pairings. Finally, zonal constructs were printed using ACPC-laden gelMA as superficial zone-competent bioink, and a MSC-laden ink for the deeper zones, via bioink extrusion in a sacrificial poloxamer frame. The effect of printing on long-term cell performance was evaluated during 56 days of culture. GAG/DNA quantification, histological and qPCR analysis revealed that all cell types underwent chondrogenic differentiation in gelMA bioinks. Additionally, a differential expression of zonal markers was detected between MSCs and ACPCs, the latter significantly upregulating the superficial zone marker PRG4. Conversely, MSCs had higher expression of collagen type X, a marker for the calcified zone. Differential distribution of ECM molecules was preserved also in zonal co-cultures. These results pave the way to the biofabrication of multicellular, functional constructs with zone-mimicking composition to be used for cartilage regeneration or as in vitro tissue models

Convergence of printing technologies to engineer an interface between bone and cartilage

The combination of multiple three dimensional printing technologies can aid the generation of osteochondral grafts that display a strong interface between the cartilage and the bone compartment. In this study, the integration between bone biomimetic a three-dimensional (3D) printed calcium phosphate paste (PCP) and a gelatin methacryloyl (gelMA) hydrogel substrate for cartilage, was reinforced with a PCL mesh produced by melt electrospinning writing (MEW). Please download the file below for full content

Evaluation of bioink printability with quantitative methods to aid material development

During extrusion-based bioprinting, the deposited bioink filaments are subjected to deformations, such as collapse of overhanging filaments and fusion between adjacent filaments, which compromise shape fidelity of printed constructs. The degree of deformation of printed filaments could be used to quantitatively assess the printability of newly developed bioinks. This approach would be an alternative to current assessment through qualitative visual inspection after printing, which have been hampering any comparison between different bioinks. For this reason, we propose two quantitative printability tests based on the mentioned filament deformations: filament collapse of overhanging structures (Fig 1a) and filament fusion on parallel filaments (Fig 1b). Both printability tests were applied on two printable hydrogel platforms: poloxamer 407 and poly(ethylene glycol) blends (poloxamer/PEG), displaying a range of yield stress values. We also propose theoretical models for each test to predict printability from bioink yield stress. The results on poloxamer/PEG hydrogels show that as the yield stress decreases, the filament collapse is greater, decreasing the ability to maintain the shape of suspended filaments. Similarly, filament fusion occurs at bigger filament distances, decreasing resolution on the x-y plane. These results confirm that printability is largely dependent on yield stress. Our bioink printability testing is straightforward, assessible with any extrusion-based bioprinting system. The proposed method provides a quantitative evaluation based on physical deformation of printed filaments, potentially reducing long experimental trial-and-error printing with newly developed bioinks and allowing reproducible comparisons between different inks. Please click Additional Files below to see the full abstract

Bioprinting Neural Systems to Model Central Nervous System Diseases

n/

In situ forming IPN hydrogels of calcium alginate and dextran-HEMA for biomedical applications

AbstractIn situ forming hydrogels, which allow for the modulation of physico-chemical properties, and in which cell response can be tailored, are providing new opportunities for biomedical applications. Here, we describe interpenetrating polymer networks (IPNs) based on a physical network of calcium alginate (Alg-Ca), interpenetrated with a chemical one based on hydroxyethyl-methacrylate-derivatized dextran (dex-HEMA). IPNs with different concentration and degree of substitution of dex-HEMA were characterized and evaluated for protein release as well as for the behavior of embedded cells. The results demonstrated that the properties of the semi-IPNs, which are obtained by dissolution of dex-HEMA chains into the Alg-Ca hydrogels, would allow for injection of these hydrogels. Degradation times of the IPNs after photocross-linking could be tailored from 15 to 180days by the concentration and the degree of substitution of dex-HEMA. Further, after an initial burst release, bovine serum albumin was gradually released from the IPNs over approximately 15days. Encapsulation of expanded chondrocytes in the IPNs revealed that cells remained viable and, depending on the composition, were able to redifferentiate, as was demonstrated by the deposition of collagen type II. These results demonstrate that these IPNs are attractive materials for pharmaceutical and biomedical applications due to their tailorable mechanical and degradation characteristics, their release kinetics and biocompatibility

3D-microfibers improve the shear modulus of hydrogel composites

A major challenge in the field of biofabrication is to manufacture a construct soft enough to elicit optimal cell behavior while possessing the mechanical properties required to withstand the complex in-vivo mechanical environment [1]. Hydrogels that were reinforced with polycaprolactone (PCL) fibers arranged in box structures, obtained by melt electrospinning writing (MEW), showed a synergistic increase in the compressive Young’s modulus [2], however, collapsed in-vivo, possibly due to shear stress. Here, we used MEW to produce specifically designed PCL fibers to stabilize an existing structure and subsequently improve the shear modulus of hydrogel-fiber composites. Instrument parameters affecting fabrication of these fibers were studied and stabilizing fibers used for shear testing (fiber diameter = 13.16 ± 0.11 μm) were made with an amplitude of 500 μm, wavelength of 400 μm, and collector velocity of 400 mm/min, at a height of 20 layers (330 m) (Figure 1A). The stabilizing fibers were embedded in 5, 10, and 15 wt.% polyacrylamide and a frequency sweep test (0.05 – 500 rad/s, 0.01% strain, n = 5) was performed to measure the complex shear modulus of the hydrogel-fiber composites. To correspond the direction of the stabilizing fibers with the torque of the rheometer, stabilizing fibers were printed in a specific architecture (Figure 1B). Stabilizing fibers increased the complex shear modulus by 148%, 127%, and 165%, when embedded within a 5%, 10%, and 15% polyacrylamide hydrogel, respectively (Figure 1C). This study highlights the capacity of MEW to increase shear properties of matrix-fiber composites through inclusion of stabilizing fibers. Please click Additional Files below to see the full abstract

Orthotopic equine study confirms the pivotal importance of structural reinforcement over the pre-culture of cartilage implants

In articular cartilage (AC), the collagen arcades provide the tissue with its extraordinary mechanical properties. As these structures cannot be restored once damaged, functional restoration of AC defects remains a major challenge. We report that the use of a converged bioprinted, osteochondral implant, based on a gelatin methacryloyl cartilage phase, reinforced with precisely patterned melt electrowritten polycaprolactone micrometer-scale fibers in a zonal fashion, inspired by native collagen architecture, can provide long-term mechanically stable neo-tissue in an orthotopic large animal model. The design of this novel implant was achieved via state-of-the-art converging of extrusion-based ceramic printing, melt electrowriting, and extrusion-based bioprinting. Interestingly, the cell-free implants, used as a control in this study, showed abundant cell ingrowth and similar favorable results as the cell-containing implants. Our findings underscore the hypothesis that mechanical stability is more determining for the successful survival of the implant than the presence of cells and pre-cultured extracellular matrix. This observation is of great translational importance and highlights the aptness of advanced 3D (bio)fabrication technologies for functional tissue restoration in the harsh articular joint mechanical environment.</p