Meniscus ECM‐functionalised hydrogels containing infrapatellar fat pad‐derived stem cells for bioprinting of regionally defined meniscal tissue

Abstract

Injuries to the meniscus of the knee commonly lead to osteoarthritis. Current therapies for meniscus regeneration, including meniscectomies and scaffold implantation, fail to achieve complete functional regeneration of the tissue. This has led to increased interest in cell and gene therapies and tissue engineering approaches to meniscus regeneration. The implantation of a biomimetic implant, incorporating cells, growth factors, and extracellular matrix (ECM)-derived proteins, represents a promising approach to functional meniscus regeneration. The objective of this study was to develop a range of ECM-functionalised bioinks suitable for 3D bioprinting of meniscal tissue. To this end, alginate hydrogels were functionalised with ECM derived from the inner and outer regions of the meniscus and loaded with infrapatellar fat pad-derived stem cells. In the absence of exogenously supplied growth factors, inner meniscus ECM promoted chondrogenesis of fat pad-derived stem cells, whereas outer meniscus ECM promoted a more elongated cell morphology and the development of a more fibroblastic phenotype. With exogenous growth factors supplementation, a more fibrogenic phenotype was observed in outer ECM-functionalised hydrogels supplemented with connective tissue growth factor, whereas inner ECM-functionalised hydrogels supplemented with TGFβ3 supported the highest levels of Sox-9 and type II collagen gene expression and sulfated glycosaminoglycans (sGAG) deposition. The final phase of the study demonstrated the printability of these ECM-functionalised hydrogels, demonstrating that their codeposition with polycaprolactone microfibres dramatically improved the mechanical properties of the 3D bioprinted constructs with no noticeable loss in cell viability. These bioprinted constructs represent an exciting new approach to tissue engineering of functional meniscal grafts.

Fiber Reinforced Cartilage ECM Functionalized Bioinks for Functional Cartilage Tissue Engineering

“…Isolation, Expansion and Culture Conditions of Human or Porcine Bone Marrow MSCs : To extract the cECM, knee joints of 2–3 months old pigs were obtained from a local butcher and AC was aseptically harvested. Following a previously established protocol,, cECM was extracted from AC under sterile conditions. Briefly, the AC was finely diced into 1–3 mm pieces using a scalpel and subsequently solubilized in activated pepsin solution (1500 Units mg −1 of wet cartilage).…”

“…Biochemical Assays : Constructs were evaluated for their DNA, glycosaminoglycan (GAG), and total collagen content at day 0 (at the start of the experiment), 21 and 42 d of culture using previously established protocols . For study using hBMSCs, calcium content was also assessed .…”

“…Total RNA Isolation, cDNA Preparation, Real‐Time qPCR and Quantification : To assess gene expression, RNA isolation, cDNA preparation and RT‐PCR was performed following the previously established protocols . The KiCqStart SYBR Green Primers predesigned from Sigma‐Aldrich (Ireland) were used for all the genes.…”

“…3D‐Bioprinting has emerged as a promising strategy to engineer mechanically functional implants for orthopedic tissue engineering . An advantage of bioprinting is that soft, cell‐friendly bioinks can be mechanically reinforced with networks of stiffer polymers such as polycaprolactone (PCL) to engineer composites that are both mechanically functional and supportive of new tissue deposition by resident cells.…”

Three-Dimensional Bioprinting of Cartilage by the Use of Stem Cells: A Strategy to Improve Regeneration

“…Romanazzo et al were able to reproduce the two meniscal regions by bioprinting infrapatellar fat pad-derived stem cells with alginate hydrogels. They utilized two different growth factors in the two different zones: TGF-β3 to induce the synthesis of collagen type II and sulfated glycosaminoglycans, typical of the inner (avascular/aneural) region, and connective tissue growth factor (CTGF) to induce a more fibrogenic phenotype characteristic of the outer (vascular/neural) region [ 40 ].…”