3D Printed Multiphasic Scaffolds for Osteochondral Repair: Challenges and Opportunities.

Osteochondral (OC) defects are debilitating joint injuries characterized by the loss of full thickness articular cartilage along with the underlying calcified cartilage through to the subchondral bone. While current surgical treatments can provide some relief from pain, none can fully repair all the components of the OC unit and restore its native function. Engineering OC tissue is challenging due to the presence of the three distinct tissue regions. Recent advances in additive manufacturing provide unprecedented control over the internal microstructure of bioscaffolds, the patterning of growth factors and the encapsulation of potentially regenerative cells. These developments are ushering in a new paradigm of 'multiphasic' scaffold designs in which the optimal micro-environment for each tissue region is individually crafted. Although the adoption of these techniques provides new opportunities in OC research, it also introduces challenges, such as creating tissue interfaces, integrating multiple fabrication techniques and co-culturing different cells within the same construct. This review captures the considerations and capabilities in developing 3D printed OC scaffolds, including materials, fabrication techniques, mechanical function, biological components and design.

Design considerations when creating a high throughput screen-compatible in vitro model of osteogenesis.

Inducing osteogenic differentiation in vitro is useful for the identification and development of bone regeneration therapies as well as modelling bone disorders. To couple in vitro models with high throughput screening techniques retains the assay's relevance in research while increasing its therapeutic impact. Miniaturizing, automating and/or digitalizing in vitro assays will reduce the required quantity of cells, biologic stimulants, culture/output assay reagents, time and cost. This review highlights the design and workflow considerations for creating a high throughput screen-compatible model of osteogenesis, comparing and contrasting osteogenic cell type, assay fabrication and culture methodology, osteogenic induction approach and repurposing existing output techniques.

NEST3D printed bone-mimicking scaffolds: assessment of the effect of geometrical design on stiffness and angiogenic potential.

Tissue-engineered implants for bone regeneration require consideration regarding their mineralization and vascularization capacity. Different geometries, such as biomimetic designs and lattices, can influence the mechanical properties and the vascularization capacity of bone-mimicking implants. Negative Embodied Sacrificial Template 3D (NEST3D) printing is a versatile technique across a wide range of materials that enables the production of bone-mimicking scaffolds. In this study, different scaffold motifs (logpile, Voronoi, and trabecular bone) were fabricated via NEST3D printing in polycaprolactone to determine the effect of geometrical design on stiffness (10.44 ± 6.71, 12.61 ± 5.71, and 25.93 ± 4.16 MPa, respectively) and vascularization. The same designs, in a polycaprolactone scaffold only, or when combined with gelatin methacryloyl, were then assessed for their ability to allow the infiltration of blood vessels in a chick chorioallantoic membrane (CAM) assay, a cost-effective and time-efficient in ovo assay to assess vascularization. Our findings showed that gelatin methacrylolyl alone did not allow new chorioallantoic membrane tissue or blood vessels to infiltrate within its structure. However, polycaprolactone on its own or when combined with gelatin methacrylolyl allowed tissue and vessel infiltration in all scaffold designs. The trabecular bone design showed the greatest mineralized matrix production over the three designs tested. This reinforces our hypothesis that both biomaterial choice and scaffold motifs are crucial components for a bone-mimicking scaffold.

Negative Printing for the Reinforcement of In Situ Tissue-Engineered Cartilage.

In the realm of in situ cartilage engineering, the targeted delivery of both cells and hydrogel materials to the site of a defect serves to directly stimulate chondral repair. Although the in situ application of stem cell-laden soft hydrogels to tissue defects holds great promise for cartilage regeneration, a significant challenge lies in overcoming the inherent limitation of these soft hydrogels, which must attain mechanical properties akin to the native tissue to withstand physiological loading. We therefore developed a system where a gelatin methacryloyl hydrogel laden with human adipose-derived mesenchymal stem cells is combined with a secondary structure to provide bulk mechanical reinforcement. In this study, we used the negative embodied sacrificial template 3D printing technique to generate eight different lattice-based reinforcement structures made of polycaprolactone, which ranged in porosity from 80% to 90% with stiffnesses from 28 ± 5 kPa to 2853 ± 236 kPa. The most promising of these designs, the hex prism edge, was combined with the cellular hydrogel and retained a stable stiffness over 41 days of chondrogenic differentiation. There was no significant difference between the hydrogel-only and hydrogel scaffold group in the sulfated glycosaminoglycan production (340.46 ± 13.32 µg and 338.92 ± 47.33 µg, respectively) or Type II Collagen gene expression. As such, the use of negative printing represents a promising solution for the integration of bulk reinforcement without losing the ability to produce new chondrogenic matrix.

Within or Without You? A Perspective Comparing In Situ and Ex Situ Tissue Engineering Strategies for Articular Cartilage Repair.

Human articular cartilage has a poor ability to self-repair, meaning small injuries often lead to osteoarthritis, a painful and debilitating condition which is a major contributor to the global burden of disease. Existing clinical strategies generally do not regenerate hyaline type cartilage, motivating research toward tissue engineering solutions. Prospective cartilage tissue engineering therapies can be placed into two broad categories: i) Ex situ strategies, where cartilage tissue constructs are engineered in the lab prior to implantation and ii) in situ strategies, where cells and/or a bioscaffold are delivered to the defect site to stimulate chondral repair directly. While commonalities exist between these two approaches, the core point of distinction-whether chondrogenesis primarily occurs "within" or "without" (outside) the body-can dictate many aspects of the treatment. This difference influences decisions around cell selection, the biomaterials formulation and the surgical implantation procedure, the processes of tissue integration and maturation, as well as, the prospects for regulatory clearance and clinical translation. Here, ex situ and in situ cartilage engineering strategies are compared: Highlighting their respective challenges, opportunities, and prospects on their translational pathways toward long term human cartilage repair.

Characterization of Polycaprolactone Nanohydroxyapatite Composites with Tunable Degradability Suitable for Indirect Printing.

Degradable bone implants are designed to foster the complete regeneration of natural tissue after large-scale loss trauma. Polycaprolactone (PCL) and hydroxyapatite (HA) composites are promising scaffold materials with superior mechanical and osteoinductive properties compared to the single materials. However, producing three-dimensional (3D) structures with high HA content as well as tuneable degradability remains a challenge. To address this issue and create homogeneously distributed PCL-nanoHA (nHA) scaffolds with tuneable degradation rates through both PCL molecular weight and nHA concentration, we conducted a detailed characterisation and comparison of a range of PCL-nHA composites across three molecular weight PCLs (14, 45, and 80 kDa) and with nHA content up to 30% w/w. In general, the addition of nHA results in an increase of viscosity for the PCL-nHA composites but has little effect on their compressive modulus. Importantly, we observe that the addition of nHA increases the rate of degradation compared to PCL alone. We show that the 45 and 80 kDa PCL-nHA groups can be fabricated via indirect 3D printing and have homogenously distributed nHA even after fabrication. Finally, the cytocompatibility of the composite materials is evaluated for the 45 and 80 kDa groups, with the results showing no significant change in cell number compared to the control. In conclusion, our analyses unveil several features that are crucial for processing the composite material into a tissue engineered implant.