Protocols for Culturing and Imaging a Human Ex Vivo Osteochondral Model for Cartilage Biomanufacturing Applications.

Cartilage defects and diseases remain major clinical issues in orthopaedics. Biomanufacturing is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. However, several limitations of in vitro and experimental animal models pose serious challenges to the translation of preclinical findings into clinical practice. Ex vivo models are of great value for translating in vitro tissue engineered approaches into clinically relevant conditions. Our aim is to obtain a viable human osteochondral (OC) model to test hydrogel-based materials for cartilage repair. Here we describe a detailed step-by-step framework for the generation of human OC plugs, their culture in a perfusion device and the processing procedures for histological and advanced microscopy imaging. Our ex vivo OC model fulfils the following requirements: the model is metabolically stable for a relevant culture period of 4 weeks in a perfusion bioreactor, the processing procedures allowed for the analysis of 3 different tissues or materials (cartilage, bone and hydrogel) without compromising their integrity. We determined a protocol and the settings for a non-linear microscopy technique on label free sections. Furthermore, we established a clearing protocol to perform light sheet-based observations on the cartilage layer without the need for tedious and destructive histological procedures. Finally, we showed that our OC system is a clinically relevant in terms of cartilage regeneration potential. In conclusion, this OC model represents a valuable preclinical ex vivo tool for studying cartilage therapies, such as hydrogel-based bioscaffolds, and we envision it will reduce the number of animals needed for in vivo testing.

Design, Fabrication and Validation of a Precursor Pulsed Electromagnetic Field Device for Bone Fracture Repair.

Pulsed electromagnetic field (PEMF) stimulation has been utilized in the medical field since the early 20th century. A number of therapeutic devices have been developed for the treatment of bone fractures and other medical applications. Most of these devices are backed by limited quantitative evidence. In this paper we present the development of a PEMF device for the purposes of determining, through in vitro experimentation, the exposure parameters required to give the most optimal fracture repair. Following electromagnetic field characterization, the device was shown to match well with computational field simulations. The exposure system has been validated through adipose-derived stem cell viability studies with an exposure frequency of 5 Hz and an intensity of 1.1 mT, for a duration of seven days at 30 minutes per day. Under the specific field characteristics chosen, the fatty-tissue derived stem cell proliferation was not hindered and in fact was stimulated $( 0. 025 < P < 0.01)$ by the PEMF exposure. With continued experimentation of numerous exposure conditions at the cellular scale, it will be possible to quantitatively determine the optimal exposure conditions required to produce the most rapid fresh fracture repair. Following this, there is significant potential for development of an optimized wearable device suitable for enhancing repair of all types of bone fractures.

In situ handheld three-dimensional bioprinting for cartilage regeneration.

Articular cartilage injuries experienced at an early age can lead to the development of osteoarthritis later in life. In situ three-dimensional (3D) printing is an exciting and innovative biofabrication technology that enables the surgeon to deliver tissue-engineering techniques at the time and location of need. We have created a hand-held 3D printing device (biopen) that allows the simultaneous coaxial extrusion of bioscaffold and cultured cells directly into the cartilage defect in vivo in a single-session surgery. This pilot study assessed the ability of the biopen to repair a full-thickness chondral defect and the early outcomes in cartilage regeneration, and compared these results with other treatments in a large animal model. A standardized critical-sized full-thickness chondral defect was created in the weight-bearing surface of the lateral and medial condyles of both femurs of six sheep. Each defect was treated with one of the following treatments: (i) hand-held in situ 3D printed bioscaffold using the biopen (HH group), (ii) preconstructed bench-based printed bioscaffolds (BB group), (iii) microfractures (MF group) or (iv) untreated (control, C group). At 8 weeks after surgery, macroscopic, microscopic and biomechanical tests were performed. Surgical 3D bioprinting was performed in all animals without any intra- or postoperative complication. The HH biopen allowed early cartilage regeneration. The results of this study show that real-time, in vivo bioprinting with cells and scaffold is a feasible means of delivering a regenerative medicine strategy in a large animal model to regenerate articular cartilage.

Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair.

Three-dimensional (3D) bioprinting is driving major innovations in the area of cartilage tissue engineering. Extrusion-based 3D bioprinting necessitates a phase change from a liquid bioink to a semi-solid crosslinked network achieved by a photo-initiated free radical polymerization reaction that is known to be cytotoxic. Therefore, the choice of the photocuring conditions has to be carefully addressed to generate a structure stiff enough to withstand the forces phisiologically applied on articular cartilage, while ensuring adequate cell survival for functional chondral repair. We recently developed a handheld 3D printer called "Biopen". To progress towards translating this freeform biofabrication tool into clinical practice, we aimed to define the ideal bioprinting conditions that would deliver a scaffold with high cell viability and structural stiffness relevant for chondral repair. To fulfill those criteria, free radical cytotoxicity was confined by a co-axial Core/Shell separation. This system allowed the generation of Core/Shell GelMa/HAMa bioscaffolds with stiffness of 200KPa, achieved after only 10 seconds of exposure to 700 mW/cm2 of 365 nm UV-A, containing >90% viable stem cells that retained proliferative capacity. Overall, the Core/Shell handheld 3D bioprinting strategy enabled rapid generation of high modulus bioscaffolds with high cell viability, with potential for in situ surgical cartilage engineering.

Development of the Biopen: a handheld device for surgical printing of adipose stem cells at a chondral wound site.

We present a new approach which aims to translate freeform biofabrication into the surgical field, while staying true to the practical constraints of the operating theatre. Herein we describe the development of a handheld biofabrication tool, dubbed the 'biopen', which enables the deposition of living cells and biomaterials in a manual, direct-write fashion. A gelatin-methacrylamide/hyaluronic acid-methacrylate (GelMa/HAMa) hydrogel was printed and UV crosslinked during the deposition process to generate surgically sculpted 3D structures. Custom titanium nozzles were fabricated to allow printing of multiple ink formulations in a collinear (side-by-side) geometry. Independently applied extrusion pressure for both chambers allows for geometric control of the printed structure and for the creation of compositional gradients. In vitro experiments demonstrated that human adipose stem cells maintain high viability (>97%) one week after biopen printing in GelMa/HAMa hydrogels. The biopen described in this study paves the way for the use of 3D bioprinting during the surgical process. The ability to directly control the deposition of regenerative scaffolds with or without the presence of live cells during the surgical process presents an exciting advance not only in the fields of cartilage and bone regeneration but also in other fields where tissue regeneration and replacement are critical.

Genomic organisation and nervous system expression of radial spoke protein 3.

Following their generation in the germinal zones, young neurons of the neocortex, hippocampus and cerebellum undergo long-distance migration to reach their final destinations. This locomotive activity depends on active deployment of cytoskeletal elements including the microtubule apparatus. In this study, we report the identification and expression of radial spoke protein 3 (RSP3), a member of a protein cluster responsible for anchoring and modifying dynein motor activity known to be crucial to microtubule sliding. The mouse RSP3 gene consists of eight exons and seven introns and spans over 230 kb. The genomic organisations of the human and rat RSP3 genes are similar although they span approximately 23 and 53 kb, respectively. This is in contrast to the Chlamydomonas RSP3 gene, where RSP3 was first isolated, which consists of four exons and three introns and spans approximately 2.7 kb. Despite these differences, the nucleotide and amino acid sequences upstream of, and throughout the RPII-binding domain of RSP3 are highly conserved between all the above-mentioned species. Mouse RSP3 mRNA was restricted to the developing neocortex, hippocampus and cerebellum during the stages when these structures are known to contain large numbers of migratory neurons. Gene expression studies suggest that RSP3 function is consistent with a locomotory role for this protein in migrating young neurons. In addition, expression of RSP3 mRNA in adult neurons point to additional, though still unknown functions. Our data provides the first evidence for the expression of radial spoke proteins in higher eukaryotes, and provides a biological framework for how these proteins may participate in microtubule sliding and neuronal migration in the embryonic brain.

Region-specific expression of the helix-loop-helix gene BETA3 in developing and adult brains.

Members of the basic helix-loop-helix (bHLH) transcription factor family are crucial regulators of neuronal cell generation and cell fate. A number of bHLH genes are expressed in the developing cerebral cortex, including MASH-1, neurogenin2 and NeuroD implying the existence of a regulatory and possibly redundant network of family members. BETA3 is a novel member originally cloned from pancreatic cells but we report here highly restricted expression patterns in developing forebrain structures that are highly stage-specific. We show that BETA3 mRNA is found in both neocortex and archicortex, mainly in cells that have reached their migratory destinations but is largely absent from proliferative zones. These expression data would suggest that BETA3 function is linked to the establishment rather than the initiation of neuronal fates.