Additive biofabrication (3D bioprinting) makes it possible to create scaffolds with precise geometries, control over pore interconnectivity and architectures that are not possible with conventional techniques. Inclusion of cells within the ink to form a "bio-ink" presents the potential to print 3D structures that can be implanted into damaged/diseased tissue to promote highly controlled cell-based regeneration and repair. The properties of an 'ink' are defined by its formulation and critically influence the delivery and integrity of structure formed. Importantly, the ink properties need to conform to biological requirements necessary for the cell system that they are intended to support and it is often challenging to find conditions for printing that facilitate this critical aspect of tissue bioengineering. In this study, alginate (Alg) was selected as the major component of the 'bio-ink' formulations for extrusion printing of cells. The rheological properties of alginate-gelatin (AlgGel) blends were compared with pre-crosslinked alginate and alginate solution to establish their printability whilst maintaining their ability to support optimal cell growth. Pre-crosslinked alginate on its own was liquidlike during printing. However, by controlling the temperature, Alg-Gel formulations had higher viscosity, storage modulus and consistency which facilitated higher print resolution/precision. Compression and indentation testing were used to examine the mechanical properties of alginate compared to Alg-Gel. Both types of gels yielded similar results with modulus increasing with alginate concentration. Decay in mechanical properties over time suggests that Alg-Gel slowly degrades in cell culture media with more than 60% decrease in initial modulus over 7 days. The viability of primary myoblasts delivered as a myoblast/Alg-Gel bio-ink was not affected by the printing process, indicating that the Alg-Gel matrix provides a potential means to print 3D constructs that may find application in myoregenerative applications. control over pore interconnectivity and architectures that are not possible with conventional techniques. Inclusion of cells within the ink to form a "bio-ink" presents the potential to print 3D structures that can be implanted into damaged/diseased tissue to promote highly controlled cell-based regeneration and repair. The properties of an 'ink' are defined by its formulation and critically influence the delivery and integrity of structure formed. Importantly, the ink properties need to conform to biological requirements 10 necessary for the cell system that they are intended to support and it is often challenging to find conditions for printing that facilitate this critical aspect of tissue bioengineering. In this study, alginate (Alg) was selected as the major component of the 'bio-ink' formulations for extrusion printing of cells. The rheological properties of alginate-gelatin (Alg-Gel) blends were compared with pre-crosslinked alginate and alginate solution to establish their printability whil...

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.

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.