Introduction

Despite the development of biomaterials with different properties and new ways to biofabricate scaffolds, the repair of cartilage lesions remains a challenge. Cell-material interactions determine the final result, in cell-free as well as cell containing procedures, but we do not fully understand these interactions and how they influence cartilage regeneration. We have evaluated several materials for their effects on cell migration, cartilage tissue formation as well as inflammation using in vitro and in vivo assays.

Content

METHODS:

Different hydrogels and scaffolds and modifications of their physical and chemical properties were used. In vitro assays were performed to study migration of human Bone marrow-derived Mesenchymal Stromal Cells (BMSCs) and the capacity of the materials to support cartilage tissue formation. For in vivo evaluation bovine osteochondral tissue biopsies were harvested in which a defect was created that we filled with the materials and implanted subcutaneously in athymic mice. uCT and histology were performed to analyse cell ingrowth, tissue repair and inflammatory reaction.

RESULTS:

Migration of BMSC into a hydrogel in vitro depended on stiffness and composition of the hydrogel and could be stimulated by addition of chemokines such as PDGF-BB. Inflammatory cytokines as well as factors secreted by macrophages also stimulated migration of BMSCs in vitro.

The formation of cartilage tissue was also hydrogel composition dependent. In the semi-orthotopic osteochondral defect model in vivo, migration of cells from the bone (marrow) side was obvious and depended on the biomaterial used. Induction of an inflammatory process by the material is not necessarily bad for the repair response, as long as the inflammation resolves since inflammatory factors impair chondrogenesis. Loading factors such as PDGF-BB, BMP-2 or antiMiR221 in the material could stimulate the repair process.

It remains a challenge to prevent ossification of the newly generated cartilage in an osteochondral defect using a cell-free procedure. We have demonstrated that cartilage, even mature articular cartilage, secretes angiogenic factors and the integrity of the cartilage matrix was demonstrated to be key for prevention of vessel ingrowth in a bone environment.

DISCUSSION & CONCLUSIONS:

The in vitro and in vivo models are useful to study materials for capacity to stimulate cartilage repair. More fundamental knowledge of how the physical properties and the composition of materials influence cell behaviour is important to improve cartilage regeneration. To regenerate a functional osteochondral unit choices related to embedding the right cells or attract the right cells at the right moment, preventing vessels from entering the cartilage part and rebuilding a functional interface between bone and cartilage will be the challenges for the future.

References

Presented work will be based on previously published work of the group:

Fahy N et al. Human osteoarthritic synovium impacts chondrogenic differentiation of mesenchymal stem cells via macrophage polarisation state. Osteoarthritis Cartilage. 2014;22(8):1167

Sivasubramaniyan K. et al. Bone Marrow-Harvesting Technique Influences Functional Heterogeneity of Mesenchymal Stem/Stromal Cells and Cartilage Regeneration. Am J Sports Med. 2018;46(14):3521

Lolli A et al. Hydrogel-based delivery of antimiR-221 enhances cartilage regeneration by endogenous cells. J Control Release. 2019;309:220

Vainieri ML, et al. Evaluation of biomimetic hyaluronic-based hydrogels with enhanced endogenous cell recruitment and cartilage matrix formation. Acta Biomater. 2020;101:293

Nossin Y et al. The Releasate of Avascular Cartilage Demonstrates Inherent Pro-Angiogenic Properties In Vitro and In Vivo. Cartilage. 2021 Dec;13(2_suppl):559S-570

Andres Sastre et al. A new semi-orthotopic bone defect model for cell and biomaterial testing in regenerative medicine. Biomaterials 2021 Dec;279:121187

Muntz I et al, The role of cell-matrix interactions in connective tissue mechanics. Phys Biol. 2021- epub

Acknowledgments

TargetCaRe (HORIZON2020-MSCA-2014-ITN nr 642414); CarBon (HORIZON2020-MSCA-2016-ITN nr 721432); Hunter (NWO Perspectief P15-23); NanoScores (Eurnanomed3 JTC-2017 nr ENMIII 077-2); AO Foundation (AO-OCD consortium TA1711481); Medical Delta Regenerative Medicine 4D programme; Convergence Health and Technology Impuls program

Introduction

The regeneration of musculoskeletal tissues such as articular cartilage and meniscus remains a significant challenge. This can be attributed, at least in part, to our inability to engineer grafts that mimic the spatial structure and composition of the native tissue. Additive (bio)manufacturing strategies are impacting on diverse areas of healthcare, including the development of regenerative scaffolds and the in vitro engineering of biological tissues. In particular, the ability to spatially and temporally pattern cells, regulatory factors and supporting biomaterials using emerging bioprinting platforms is transforming our ability to engineer functional cartilaginous tissues. Not only can bioprinting enable the engineering of anatomically complex grafts, but also the biofabrication of tissues mimicking the spatial composition of articular cartilage and meniscus. Using multiple-tool biofabrication strategies, it is also possible to reinforce such biological grafts to produce composites with mechanical properties approaching that of native tissues. Alternatively, 3D printing can be used to produce structures to help guide the development of engineered grafts, enabling the fabrication of tissues with biomimetic collagen networks critical to the biomechanical functionality of soft tissues. This talk will describe two examples from our lab where different biofabrication and 3D (bio)printing strategies are combined to engineer functional cartilage and osteochondral grafts.

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The first example will describe how we have used melt-electrowriting (MEW) to produce arrays of polymeric structures whose function is to orient the growth of cellular aggregates spontaneously generated within these structures, and to provide tensile reinforcement to the resulting tissues. Inkjeting was used to deposit defined numbers of cells into MEW structures, which self-assembled into an organized array of spheroids within hours, ultimately generating a hybrid tissue that was hyaline-like in composition. Structurally, the engineered cartilage mimicked the histotypical organization observed in skeletally immature synovial joints. This biofabrication framework was then used to generate scaled-up (6mm × 6mm) cartilage implants containing over 3,500 cellular aggregates in under 15 minutes. After 8 weeks in culture, a 50-fold increase in the compressive properties of these MEW reinforced tissues were observed, while the tensile properties were still dominated by the polymer network, resulting in a composite construct demonstrating tension-compression nonlinearity mimetic of the native tissue. Helium ion microscopy further demonstrated the development of an arcading collagen network within the engineered tissue. This hybrid bioprinting strategy provides a versatile and scalable approach to engineer cartilage biomimetic grafts for biological joint resurfacing.

Modular biofabrication strategies that use microtissues or organoids as biological building blocks represent another promising approach for engineering replacement tissues and organs at scale. The second part of this talk will describe the development of such a biofabrication strategy to engineer osteochondral tissues by spatially localising phenotypically distinct cartilage microtissue populations within an instructive 3D printed polymer framework. We first identified that immature cartilage microtissues could spontaneously fuse to form a homogenous macrotissue, and that the fusion and quality of this macrotissue was a function of microtissue cellular density. Next, 3D printed polymeric frameworks were used to further guide microtissue fusion and the subsequent self-organisation process, resulting in the development of a macroscale tissue with a zonal collagen organisation analogous to the structure seen in native articular cartilage. Hypertrophic cartilage microtissues were also engineered as bone precursor tissues, which were then spatially localised below phenotypically stable cartilage microtissues to engineer osteochondral grafts. Implantation of these engineered grafts into critically-sized caprine osteochondral defects resulted in effective stabilisation of the defect and aided in restoring a more normal articular surface after 6 months. This work presents an effective method for engineering developmentally inspired osteochondral implants at a clinically relevant scale.

Acknowledgments

This publication was developed with the financial support of Science Foundation Ireland (SFI) under grant number 12/RC/2278 and 17/SP/4721. This research is co-funded by the European Regional Development Fund and SFI under Ireland’s European Structural and Investment Fund. This research has been co-funded by Johnson & Johnson 3D Printing Innovation & Customer Solutions, Johnson & Johnson Services Inc.