In vivo performance of hierarchical HRP-crosslinked silk fibroin/β-TCP scaffolds for osteochondral tissue regeneration

Background: Osteochondral defects (OCD) can affect the articular cartilage and subchondral bone tissues, which requires superior therapies for the simultaneous and full restoration of such structurally and biologically different tissues. Methods: Tissue engineered OC grafts were prepared using a horseradish peroxidase (HRP) approach to crosslink silk fibroin (HRP-SF) as the articular cartilage-like layer and an underlying HRP-SF/ZnSrTCP subchondral bone-like layer (HRP-SF/dTCP), through salt-leaching/freeze-drying methodologies. In vivo OC regeneration was assessed by implantating the hierarchical scaffolds in rabbit critical size OC defects, during 8 weeks. A comparative analysis was performed using hierarchical OC grafts made of pure β-TCP (HRP-SF/TCP). Results: The hierarchical scaffolds showed good integration into the host tissue and no signs of acute inflammatory reaction, after 8 weeks of implantation. The histological analyses revealed positive collagen type II and glycosaminoglycansâ formation in the articular cartilage-like layer. New bone ingrowthâ s and blood vessels infiltration were detected in the subchondral bone-like layers. Conclusions: The proposed hierarchical scaffolds presented an adequate in vivo response with cartilage tissue regeneration and calcified tissue formation specially promoted by the ionic incorporation into the subchondral bone layer, confirming the hierarchical structures as suitable for OCD regeneration.Portuguese Foundation for Science and Technology for the Hierarchitech project (M-era-Net/0001/2014), for the fellowships (SFRH/BD/99555/2014) and (SFRH/BPD/101952/2014), and for the distinctions attributed to JMO (IF/01285/2015) and SP (CEECIND/03673/2017). Also, financial support from FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério Da Ciência, Tecnologia, e Ensino Superior) and fundo social europeu através do programa operacional do capital humano (FSE/POCH), PD/59/2013, PD/BD/113806/201

PLA short sub-micron fibers reinforcement of 3D bioprinted alginate constructs for cartilage regeneration

In this study, we present an innovative strategy to reinforce 3D printed hydrogel constructs for cartilage tissue engineering by formulating composite bioinks containing alginate and short submicron polylactide (PLA) fibers.Wedemonstrate that Young’s modulus obtained for pristine alginate constructs (6.9±1.7 kPa) can be increased threefold (up to 25.1±3.8 kPa) with the addition of PLA short fibers. Furthermore, to assess the performance of such materials in cartilage tissue engineering, we loaded the bioinks with human chondrocytes and cultured in vitro the bioprinted constructs for up to 14 days. Live/dead assays at day 0, 3, 7 and 14 of in vitro culture showed that human chondrocytes were retained and highly viable (∼80%) within the 3D deposited hydrogel filaments, thus confirming that the fabricated composites materials represent a valid solution for tissue engineering applications. Finally, we show that the embedded chondrocytes during all the in vitro culture maintain a round morphology, a key parameter for a proper deposition of neocartilage extra cellular matrix

3D Bioprinted hydrogel model incorporating β-tricalcium phosphate for calcified cartilage tissue engineering

One promising strategy to reconstruct osteochondral defects relies on 3D bioprinted three-zonal structures comprised of hyaline cartilage, calcified cartilage, and subchondral bone. So far, several studies have pursued the regeneration of either hyaline cartilage or bone in vitro while – despite its key role in the osteochondral region – only few of them have targeted the calcified layer. In this work, we present a 3D biomimetic hydrogel scaffold containing ß-tricalcium phosphate (TCP) for engineering calcified cartilage through a co-axial needle system implemented in extrusion-based bioprinting process. After a thorough bioink optimization, we showed that 0.5% w/v TCP is the optimal concentration forming stable scaffolds with high shape fidelity and endowed with biological properties relevant for the development of calcified cartilage. In particular, we investigate the effect induced by ceramic nano-particles over the differentiation capacity of bioprinted bone marrow-derived human mesenchymal stem cells (BM-hMSCs) in hydrogel scaffolds cultured up to 21 days in chondrogenic media. To confirm the potential of the presented approach to generate a functional in vitro model of calcified cartilage tissue, we evaluated quantitatively gene expression of relevant chondrogenic markers (COL1, COL2, COL10A1, ACAN, ALPL, BGLAP) by means of RT-qPCR and qualitatively by means of fluorescence immunocytochemistry

Photocurable Biopolymers for Coaxial Bioprinting

Thanks to their unique advantages, additive manufacturing technologies are revolutionizing almost all sectors of the industrial and academic worlds, including tissue engineering and regenerative medicine. In particular, 3D bioprinting is rapidly emerging as a first-choice approach for the fabrication—in one step—of advanced cell-laden hydrogel constructs to be used for in vitro and in vivo studies. This technique consists in the precise deposition layer-by-layer of sub-millimetric hydrogel strands in which living cells are embedded. A key factor of this process consists in the proper formulation of the hydrogel precursor solution, the so-called bioink. Ideal bioinks should be able, on the one side, to support cell growth and differentiation and, on the other, to allow the high-resolution deposition of cell-laden hydrogel strands. The latter feature requires the extruded solution to instantaneously undergo a sol-gel transition to avoid its collapse after deposition. To address this challenge, researchers are recently focusing their attention on the synthesis of several derivatives of natural biopolymers to enhance their printability. Here, we present an approach for the synthesis of photocurable derivatives of natural biopolymers—namely, gelatin methacrylate, hyaluronic acid methacrylate, chondroitin sulfate methacrylate, and PEGylated fibrinogen—that can be used to formulate tailored innovative bioinks for coaxial-based 3D bioprinting applications