Héctor Martínez Ávila

3D Bioprinting Human Chondrocytes with Nanocellulose–Alginate Bioink for Cartilage Tissue Engineering Applications

The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs.

Mechanical evaluation of bacterial nanocellulose as an implant material for ear cartilage replacement

Bacterial nanocellulose (BNC) is a novel non-degradable biocompatible material that promotes chondrocyte adhesion and proliferation. In this work, its potential use in ear cartilage tissue engineering (TE) is investigated. Firstly, the mechanical properties of native ear cartilage are measured in order to set a preliminary benchmark for ear cartilage replacement materials. Secondly, the capacity of BNC to match these requirements is assessed. Finally, a biofabrication process to produce patient-specific BNC auricular implants is demonstrated. BNC samples (n=78) with varying cellulose content (2.5-15%) were compared using stress-relaxation indentation with human ear cartilage (n=17, from 4 males, aged 49-93 years old). Additionally, an auricle from a volunteer was scanned using a 3T MRI with a spoiled gradient-echo sequence. A negative ear mold was produced from the MRI data in order to investigate if an ear-shaped BNC prototype could be produced from this mold. The results show that the instantaneous modulus Ein, equilibrium modulus Eeq, and maximum stress σmax of the BNC samples are correlated to effective cellulose content. Despite significantly different relaxation kinetics, the Ein, Eeq and σmax of BNC at 14% effective cellulose content reached values equivalent to ear cartilage (for Eeq, BNC: 2.4±0.4MPa and ear cartilage: 3.3±1.3MPa). Additionally, this work shows that BNC can be fabricated into patient-specific auricular shapes. In conclusion, BNC has the capability to reach mechanical properties of relevance for ear cartilage replacement, and can be produced in patient-specific ear shapes.

Novel bilayer bacterial nanocellulose scaffold supports neocartilage formation in vitro and in vivo

Tissue engineering provides a promising alternative therapy to the complex surgical reconstruction of auricular cartilage by using ear-shaped autologous costal cartilage. Bacterial nanocellulose (BNC) is proposed as a promising scaffold material for auricular cartilage reconstruction, as it exhibits excellent biocompatibility and secures tissue integration. Thus, this study evaluates a novel bilayer BNC scaffold for auricular cartilage tissue engineering. Bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, were seeded with human nasoseptal chondrocytes (NC) and cultured in vitro for up to 6 weeks. To scale up for clinical translation, bilayer BNC scaffolds were seeded with a low number of freshly isolated (uncultured) human NCs combined with freshly isolated human mononuclear cells (MNC) from bone marrow in alginate and subcutaneously implanted in nude mice for 8 weeks. 3D morphometric analysis showed that bilayer BNC scaffolds have a porosity of 75% and mean pore size of 50 ± 25 μm. Furthermore, endotoxin analysis and in vitro cytotoxicity testing revealed that the produced bilayer BNC scaffolds were non-pyrogenic (0.15 ± 0.09 EU/ml) and non-cytotoxic (cell viability: 97.8 ± 4.7%). This study demonstrates that bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds provide a suitable environment for culture-expanded NCs as well as a combination of freshly isolated NCs and MNCs to form cartilage in vitro and in vivo as demonstrated by immunohistochemistry, biochemical and biomechanical analyses.

Bioprinting of cartilage and skin tissue analogs utilizing a novel passive mixing unit technique for bioink precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Developing staining protocols for visualization of tissue-engineering scaffolds using micro computed tomography in native wet state

BNC-alginate and silk fibroin tissue-engineering scaffolds were stained with X-ray contrast agents in order to visualize internal microstructure in the native wet state with microcomputed tomography. A successful protocol employing amphiphilic contrast agents (CAs) dissolved in a water-based staining solution was used. The CAs were then fixed to the scaffold by neutralizing their charged functional groups, increasing their hydrophobicity and retention on the scaffold surface in water. While some unresolved issues concerning homogeneous staining and strength of contrast remain, these first successes constitute an important milestone by identifying good contrast agent candidates and staining protocols for longitudinal monitoring of tissue-engineering studies.