Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion - a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with "built-in" perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.

Organ Printing: Tissue Spheroids as Building Blocks

During the resorbable-polymer-boom of the 1970s and 1980s, polycaprolactone (PCL) was used in the biomaterials field and a number of drug-delivery devices. Its popularity was soon superseded by faster resorbable polymers which had fewer perceived disadvantages associated with long term degradation (up to 3-4 years) and intracellular resorption pathways; consequently, PCL was almost forgotten for most of two decades. Recently, a resurgence of interest has propelled PCL back into the biomaterials-arena. The superior rheological and viscoelastic properties over many of its aliphatic polyester counterparts renders PCL easy to manufacture and manipulate into a large range of implants and devices. Coupled with relatively inexpensive production routes and FDA approval, this provides a promising platform for the production of longer-term degradable implants which may be manipulated physically, chemically and biologically to possess tailorable degradation kinetics to suit a specific anatomical site. This review will discuss the application of PCL as a biomaterial over the last two decades focusing on the advantages which have propagated its return into the spotlight with a particular focus on medical devices, drug delivery and tissue engineering.

The return of a forgotten polymer : Polycaprolactone in the 21st century

Articular cartilage is composed of chondrons within a territorial matrix surrounded by a highly organized extracellular matrix comprising collagen II fibrils, proteoglycans, glycosaminoglycans, and non-collagenous proteins. Damaged articular cartilage has a limited potential for healing and untreated defects often progress to osteoarthritis. High hopes have been pinned on regenerative medicine strategies to meet the challenge of preventing progress to late osteoarthritis. One such strategy, autologous chondrocyte implantation (ACI), was first reported in 1994 as a treatment for deep focal articular cartilage defects. ACI has since evolved to become a worldwide well-established surgical technique. For ACI, chondrocytes are harvested from the lesser weight bearing edge of the joint by arthroscopy, their numbers expanded in monolayer culture for at least four weeks, and then re-implanted in the damaged region under a natural or synthetic membrane via an open joint procedure. We consider the evolution of ACI to become an established cell therapy, its current limitations, and on-going strategies to improve its efficacy. The most promising developments involving cells and natural or synthetic biomaterials will be highlighted.

https://www.mdpi.com/2306-5354/6/1/22/pdf

Regenerative Medicine: A Review of the Evolution of Autologous Chondrocyte Implantation (ACI) Therapy.

Mesenchymal stem/stromal cell (MSC) exist within their in vivo niches as part of heterogeneous cell populations, exhibiting variable stemness potential and supportive functionalities. Conventional extensive 2D in vitro MSC expansion, aimed at obtaining clinically relevant therapeutic cell numbers, results in detrimental effects on both cellular characteristics (e.g., phenotypic changes and senescence) and functions (e.g., differentiation capacity and immunomodulatory effects). These deleterious effects, added to the inherent inter-donor variability, negatively affect the standardization and reproducibility of MSC therapeutic potential. The resulting manufacturing challenges that drive the qualitative variability of MSC-based products is evident in various clinical trials where MSC therapeutic efficacy is moderate or, in some cases, totally insufficient. To circumvent these limitations, various in vitro/ex vivo techniques have been applied to manufacturing protocols to induce specific features, attributes, and functions in expanding cells. Exposure to inflammatory cues (cell priming) is one of them, however, with untoward effects such as transient expression of HLA-DR preventing allogeneic therapeutic schemes. MSC functionalization can be also achieved by in vitro 3D culturing techniques, in an effort to more closely recapitulate the in vivo MSC niche. The resulting spheroid structures provide spatial cell organization with increased cell-cell interactions, stable, or even enhanced phenotypic profiles, and increased trophic and immunomodulatory functionalities. In that context, MSC 3D spheroids have shown enhanced "medicinal signaling" activities and increased homing and survival capacities upon transplantation in vivo. Importantly, MSC spheroids have been applied in various preclinical animal models including wound healing, bone and osteochondral defects, and cardiovascular diseases showing safety and efficacy in vivo. Therefore, the incorporation of 3D MSC culturing approach into cell-based therapy would significantly impact the field, as more reproducible clinical outcomes may be achieved without requiring ex vivo stimulatory regimes. In the present review, we discuss the MSC functionalization in 3D settings and how this strategy can contribute to an improved MSC-based product for safer and more effective therapeutic applications.

/pdf/increased-mesenchymal-stem-cell-functionalization-in-three-gtmjz4wde4.pdf

Increased Mesenchymal Stem Cell Functionalization in Three-Dimensional Manufacturing Settings for Enhanced Therapeutic Applications.

Biofabrication and bioprinting using cellular aggregates, microtissues and organoids for the engineering of musculoskeletal tissues.

To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 μm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.

https://biblio.ugent.be/publication/8663083/file/8663213.pdf

Sélina Fernandez

Papers

Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids.