RESOURCES

Abstract
The meniscus is characterised by an anisotropic collagen fibre network which is integral to its biomechanical functionality. The engineering of structurally organized meniscal grafts that mimic the anisotropy of the native tissue remains a significant challenge. In this study, inkjet bioprinting was used to deposit a cell-laden bioink into additively manufactured scaffolds of differing architectures to engineer fibrocartilage grafts with user defined collagen architectures. Polymeric scaffolds consisting of guiding fibre networks with varying aspect ratios (1:1; 1:4; 1:16) were produced using either fused deposition modelling (FDM) or melt electrowriting (MEW), resulting in scaffolds with different internal architectures and fibre diameters. Scaffold architecture was found to influence the spatial organization of the collagen network laid down by the jetted cells, with higher aspect ratios (1:4 and 1:16) supporting the formation of structurally anisotropic tissues. The MEW scaffolds supported the development of a fibrocartilaginous tissue with compressive mechanical properties similar to that of native meniscus, while the anisotropic tensile properties of these constructs could be tuned by altering the fibre network aspect ratio. This MEW framework was then used to generate scaffolds with spatially distinct fibre patterns, which in turn supported the development of heterogenous tissues consisting of isotropic and anisotropic collagen networks. Such bioprinted tissues could potentially form the basis of new treatment options for damaged and diseased meniscal tissue.
Statement of significance
This study describes a multiple tool biofabrication strategy which enables the engineering of spatially organized fibrocartilage tissues. The architecture of MEW scaffolds can be tailored to not only modulate the directionality of the collagen fibres laid down by cells, but also to tune the anisotropic tensile mechanical properties of the resulting constructs, thereby enabling the engineering of biomimetic meniscal-like tissues. Furthermore, the inherent flexibility of MEW enables the development of zonally defined and potentially patient-specific implants.

Abstract
Meniscus injuries are a common problem in orthopedic medicine and are associated with a significantly increased risk of developing osteoarthritis. While developments have been made in the field of meniscus regeneration, the engineering of cell-laden constructs that mimic the complex structure, composition and biomechanics of the native tissue remains a significant challenge. This can be linked to the use of cells that are not phenotypically representative of the different zones of the meniscus, and an inability to direct the spatial organization of engineered meniscal tissues. In this study we investigated the potential of zone-specific meniscus progenitor cells (MPCs) to generate functional meniscal tissue following their deposition into melt electrowritten (MEW) scaffolds. We first confirmed that fibronectin selected MPCs from the inner and outer regions of the meniscus maintain their differentiation capacity with prolonged monolayer expansion, opening their use within advanced biofabrication strategies. By depositing MPCs within MEW scaffolds with elongated pore shapes, which functioned as physical boundaries to direct cell growth and extracellular matrix production, we were able to bioprint anisotropic fibrocartilaginous tissues with preferentially aligned collagen networks. Furthermore, by using MPCs isolated from the inner (iMPCs) and outer (oMPCs) zone of the meniscus, we were able to bioprint phenotypically distinct constructs mimicking aspects of the native tissue. An iterative MEW process was then implemented to print scaffolds with a similar wedged-shaped profile to that of the native meniscus, into which we deposited iMPCs and oMPCs in a spatially controlled manner. This process allowed us to engineer sulfated glycosaminoglycan and collagen rich constructs mimicking the geometry of the meniscus, with MPCs generating a more fibrocartilage-like tissue compared to the mesenchymal stromal/stem cells. Taken together, these results demonstrate how the convergence of emerging biofabrication platforms with tissue-specific progenitor cells can enable the engineering of complex tissues such as the meniscus.

Abstract
Knee meniscus is the cushioning fibro-cartilage tissue present in between the femoral condyles and tibial plateau of knee joint. It is largely avascular in nature and suffers from a wide range of tears and injuries caused by accidents, trauma, active lifestyle of the populace and old age of individuals. Healing of meniscus is especially difficult due to their avascularity and hence requires invasive arthroscopic approaches such as surgical resection, suturing or implantation. Though various tissue engineering approaches are proposed for treatment of meniscus tears, 3D printing/bioprinting, injectable hydrogels and physical stimulation involving modalities are gaining forefront in the past decade. A plethora of new printing approaches such as direct light photopolymerization and volumetric printing, injectable biomaterials loaded with growth factors and physical stimulation such as low intensity ultrasound approaches are being added to the treatment portfolio along with the contemporary tear mitigation measures. This review discusses on the necessary design considerations, approaches for 3D modeling and design practices for meniscal tear treatments within the scope of tissue engineering and regeneration. Also, the suitable materials, cell sources, growth factors, mechanical stimulation approaches, 3D printing strategies and injectable hydrogels for meniscal tear management have been elaborated. We have also summarized potential technologies and the potential framework that could be the herald of future of meniscus tissue engineering and repair approaches.

Abstract
Decellularized extracellular matrix (dECM) has emerged as a promising biomaterial in the fields of tissue engineering and regenerative medicine due to its ability to provide specific biochemical and biophysical cues supportive of the regeneration of diverse tissue types. Such biomaterials have also been used to produce tissue-specific inks and bioinks for 3D printing applications. However, a major limitation associated with the use of such dECM materials is their poor mechanical properties, which limits their use in load-bearing applications such as meniscus regeneration. In this study, native porcine menisci were solubilized and decellularized using different methods to produce highly concentrated dECM inks of differing biochemical content and printability. All dECM inks displayed shear thinning and thixotropic properties, with increased viscosity and improved printability observed at higher pH levels, enabling the 3D printing of anatomically defined meniscal implants. With additional crosslinking of the dECM inks following thermal gelation at pH 11, it was possible to fabricate highly elastic meniscal tissue equivalents with compressive mechanical properties similar to the native tissue. These improved mechanical properties at higher pH correlated with the development of a denser network of smaller diameter collagen fibers. These constructs also displayed repeatable loading and unloading curves when subjected to long-term cyclic compression tests. Moreover, the printing of dECM inks at the appropriate pH promoted a preferential alignment of the collagen fibers. Altogether, these findings demonstrate the potential of 3D printing of highly concentrated meniscus dECM inks to produce mechanically functional and biocompatible implants for meniscal tissue regeneration. This approach could be applied to a wide variety of different biological tissues, enabling the 3D printing of tissue mimics with diverse applications from tissue engineering to surgical planning.

Abstract
The meniscus is a fibrocartilage tissue that is integral to the correct functioning of the knee joint. The tissue possesses a unique collagen fiber architecture that is integral to its biomechanical functionality. In particular, a network of circumferentially aligned collagen fibers function to bear the high tensile forces generated in the tissue during normal daily activities. The limited regenerative capacity of the meniscus has motivated increased interest in meniscus tissue engineering; however, the in vitro generation of structurally organized meniscal grafts with a collagen architecture mimetic of the native meniscus remains a significant challenge. Here we used melt electrowriting (MEW) to produce scaffolds with defined pore architectures to impose physical boundaries upon cell growth and extracellular matrix production. This enabled the bioprinting of anisotropic tissues with collagen fibers preferentially oriented parallel to the long axis of the scaffold pores. Furthermore, temporally removing glycosaminoglycans (sGAGs) during the early stages of in vitro tissue development using chondroitinase ABC (cABC) was found to positively impact collagen network maturation. Specially we found that temporal depletion of sGAGs is associated with an increase in collagen fiber diameter without any detrimental effect on the development of a meniscal tissue phenotype or subsequent extracellular matrix production. Moreover, temporal cABC treatment supported the development of engineered tissues with superior tensile mechanical properties compared to empty MEW scaffolds. These findings demonstrate the benefit of temporal enzymatic treatments when engineering structurally anisotropic tissues using emerging biofabrication technologies such as MEW and inkjet bioprinting.

Abstract
Objectives Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology. Methods A 3D model of bioengineered medial meniscus tissue was created, based on MRI scans of a human volunteer. The Digital Imaging and Communications in Medicine (DICOM) data from these MRI scans were processed using dedicated software, in order to obtain an STL model of the structure. The chosen 3D Discovery printing tool was a microvalve-based inkjet printhead. Primary mesenchymal stem cells (MSCs) were isolated from bone marrow and embedded in a collagen-based bio-ink before printing. LIVE/DEAD assay was performed on realized cell-laden constructs carrying MSCs in order to evaluate cell distribution and viability. Results This study involved the realization of a human cell-laden collagen meniscus using 3D bioprinting. The meniscus prototype showed the biological potential of this technology to provide an anatomically shaped, patient-specific construct with viable cells on a biocompatible material. Conclusion This paper reports the preliminary findings of the production of a custom-made, cell-laden, collagen-based human meniscus. The prototype described could act as the starting point for future developments of this collagen-based, tissue-engineered structure, which could aid the optimization of implants designed to replace damaged menisci. Cite this article: G. Filardo, M. Petretta, C. Cavallo, L. Roseti, S. Durante, U. Albisinni, B. Grigolo. Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold. Bone Joint Res 2019;8:101–106. DOI: 10.1302/2046-3758.82.BJR-2018-0134.R1.

Abstract
Abstract Injuries to the meniscus of the knee commonly lead to osteoarthritis. Current therapies for meniscus regeneration, including meniscectomies and scaffold implantation, fail to achieve complete functional regeneration of the tissue. This has led to increased interest in cell and gene therapies and tissue engineering approaches to meniscus regeneration. The implantation of a biomimetic implant, incorporating cells, growth factors, and extracellular matrix (ECM)‐derived proteins, represents a promising approach to functional meniscus regeneration. The objective of this study was to develop a range of ECM‐functionalised bioinks suitable for 3D bioprinting of meniscal tissue. To this end, alginate hydrogels were functionalised with ECM derived from the inner and outer regions of the meniscus and loaded with infrapatellar fat pad‐derived stem cells. In the absence of exogenously supplied growth factors, inner meniscus ECM promoted chondrogenesis of fat pad‐derived stem cells, whereas outer meniscus ECM promoted a more elongated cell morphology and the development of a more fibroblastic phenotype. With exogenous growth factors supplementation, a more fibrogenic phenotype was observed in outer ECM‐functionalised hydrogels supplemented with connective tissue growth factor, whereas inner ECM‐functionalised hydrogels supplemented with TGFβ3 supported the highest levels of Sox‐9 and type II collagen gene expression and sulfated glycosaminoglycans (sGAG) deposition. The final phase of the study demonstrated the printability of these ECM‐functionalised hydrogels, demonstrating that their codeposition with polycaprolactone microfibres dramatically improved the mechanical properties of the 3D bioprinted constructs with no noticeable loss in cell viability. These bioprinted constructs represent an exciting new approach to tissue engineering of functional meniscal grafts.