3D bioprinting for meniscus tissue engineering: a review of key components, recent developments and future opportunities

3D bioprinting has the potential to transform the field of regenerative medicine as it enables the precise spatial patterning of biomaterials, cells and biomolecules to produce engineered tissues. Although numerous tissue engineering strategies have been developed for meniscal repair, the field has yet to realize an implant capable of completely regenerating the tissue. This paper first summarized existing meniscal repair strategies, highlighting the importance of engineering biomimetic implants for successful meniscal regeneration. Next, we reviewed how developments in 3D (bio)printing are accelerating the engineering of functional meniscal tissues and the development of implants targeting damaged or diseased menisci. Some of the opportunities and challenges associated with use of 3D bioprinting for meniscal tissue engineering are identified. Finally, we discussed key emerging research areas with the capacity to enhance the bioprinting of meniscal grafts.

Three-Dimensional-Printed Scaffolds for Meniscus Tissue Engineering: Opportunity for the Future in the Orthopaedic World

The meniscus is a critical component of a healthy knee joint. It is a complex and vital fibrocartilaginous tissue that maintains appropriate biomechanics. Injuries of the meniscus, particularly in the inner region, rarely heal and usually progress into structural breakdown, followed by meniscus deterioration and initiation of osteoarthritis. Conventional therapies range from conservative treatment, to partial meniscectomy and even meniscus transplantation. All the above have high long-term failure rates, with recurrence of symptoms. This communication presents a brief account of in vitro and in vivo studies and describes recent developments in the field of 3D-printed scaffolds for meniscus tissue engineering. Current research in meniscal tissue engineering tries to combine polymeric biomaterials, cell-based therapy, growth factors, and 3D-printed scaffolds to promote the healing of meniscal defects. Today, 3D-printing technology represents a big opportunity in the orthopaedic world to create more specific implants, enabling the rapid production of meniscal scaffolds and changing the way that orthopaedic surgeons plan procedures. In the future, 3D-printed meniscal scaffolds are likely to be available and will also be suitable substitutes in clinical applications, in an attempt to imitate the complexity of the native meniscus.

Clinical Replacement Strategies for Meniscus Tissue Deficiency

Meniscus tissue deficiency resulting from primary meniscectomy or meniscectomy after failed repair is a clinical challenge because the meniscus has little to no capacity for regeneration. Loss of meniscus tissue has been associated with early-onset knee osteoarthritis due to an increase in joint contact pressures in meniscectomized knees. Clinically available replacement strategies range from allograft transplantation to synthetic implants, including the collagen meniscus implant, ACTIfit, and NUSurface. Although short-term efficacy has been demonstrated with some of these treatments, factors such as long-term durability, chondroprotective efficacy, and return to sport activities in young patients remain unpredictable. Investigations of cell-based and tissue-engineered strategies to treat meniscus tissue deficiency are ongoing.

Biomaterials and Meniscal Lesions: Current Concepts and Future Perspective

Menisci are crucial structures for knee homeostasis. After a meniscal lesion, the golden rule, now, is to save as much meniscus as possible; only the meniscus tissue that is identified as unrepairable should be excised, and meniscal sutures find more and more indications. Several different methods have been proposed to improve meniscal healing. They include very basic techniques, such as needling, abrasion, trephination and gluing, or more complex methods, such as synovial flaps, meniscal wrapping or the application of fibrin clots. Basic research of meniscal substitutes has also become very active in the last decades. The aim of this literature review is to analyze possible therapeutic and surgical options that go beyond traditional meniscal surgery: from scaffolds, which are made of different kind of polymers, such as natural, synthetic or hydrogel components, to new technologies, such as 3-D printing construct or hybrid biomaterials made of scaffolds and specific cells. These recent advances show that there is great interest in the development of new materials for meniscal reconstruction and that, with the development of new biomaterials, there will be the possibility of better management of meniscal injuries

Mechano-Hypoxia Conditioning of Engineered Human Meniscus

Meniscus fibrochondrocytes (MFCs) experience simultaneous hypoxia and mechanical loading in the knee joint. Experimental conditions based on these aspects of the native MFC environment may have promising applications in human meniscus tissue engineering. We hypothesized that in vitro “mechano-hypoxia conditioning” with mechanical loading such as dynamic compression (DC) and cyclic hydrostatic pressure (CHP) would enhance development of human meniscus fibrocartilage extracellular matrix in vitro. MFCs from inner human meniscus surgical discards were pre-cultured on porous type I collagen scaffolds with TGF-β3 supplementation to form baseline tissues with newly formed matrix that were used in a series of experiments. First, baseline tissues were treated with DC or CHP under hypoxia (HYP, 3% O2) for 5 days. DC was the more effective load regime in inducing gene expression changes, and combined HYP/DC enhanced gene expression of fibrocartilage precursors. The individual treatments of DC and HYP regulated thousands of genes, such as chondrogenic markers SOX5/6, in an overwhelmingly additive rather than synergistic manner. Similar baseline tissues were then treated with a short course of DC (5 vs 60 min, 10–20% vs 30–40% strain) with different pre-culture duration (3 vs 6 weeks). The longer course of loading (60 min) had diminishing returns in regulating mechano-sensitive and inflammatory genes such as c-FOS and PTGS2, suggesting that as few as 5 min of DC was adequate. There was a dose-effect in gene regulation by higher DC strains, whereas outcomes were inconsistent for different MFC donors in pre-culture durations. A final set of baseline tissues was then cultured for 3 weeks with mechano-hypoxia conditioning to assess mechanical and protein-level outcomes. There were 1.8–5.1-fold gains in the dynamic modulus relative to baseline in HYP/DC, but matrix outcomes were equal or inferior to static controls. Long-term mechano-hypoxia conditioning was effective in suppressing hypertrophic markers (e.g., COL10A1 10-fold suppression vs static/normoxia). Taken together, these results indicate that appropriately applied mechano-hypoxia conditioning can support meniscus fibrocartilage development in vitro and may be useful as a strategy for developing non-hypertrophic articular cartilage using mesenchymal stem cells.

Selection of Highly Proliferative and Multipotent Meniscus Progenitors through Differential Adhesion to Fibronectin: A Novel Approach in Meniscus Tissue Engineering

Meniscus injuries can be highly debilitating and lead to knee osteoarthritis. Progenitor cells from the meniscus could be a superior cell type for meniscus repair and tissue-engineering. The purpose of this study is to characterize meniscus progenitor cells isolated by differential adhesion to fibronectin (FN-prog). Human osteoarthritic menisci were digested, and FN-prog were selected by differential adhesion to fibronectin. Multilineage differentiation, population doubling time, colony formation, and MSC surface markers were assessed in the FN-prog and the total meniscus population (Men). Colony formation was compared between outer and inner zone meniscus digest. Chondrogenic pellet cultures were performed for redifferentiation. FN-prog demonstrated multipotency. The outer zone FN-prog formed more colonies than the inner zone FN-prog. FN-prog displayed more colony formation and a higher proliferation rate than Men. FN-prog redifferentiated in pellet culture and mostly adhered to the MSC surface marker profile, except for HLA-DR receptor expression. This is the first study that demonstrates differential adhesion to fibronectin for the isolation of a progenitor-like population from the meniscus. The high proliferation rates and ability to form meniscus extracellular matrix upon redifferentiation, together with the broad availability of osteoarthritis meniscus tissue, make FN-prog a promising cell type for clinical translation in meniscus tissue-engineering.