Repopulation of decellularised articular cartilage by laser-based matrix engraving

Abstract

Background In spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage. Methods Human articular cartilage matrix was engraved using a CO 2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold. Findings Crossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix. Interpretation Due to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment. Funding Austrian Research Promotion Agency FFG (“CartiScaff” #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08)

Sequential Enzymatic Digestion of Different Cartilage Tissues: A Rapid and High-Efficiency Protocol for Chondrocyte Isolation, and Its Application in Cartilage Tissue Engineering

“…The enzymatic digestion protocol is based on degrading the cartilaginous collagen network, cleaving the core protein of proteoglycans or their connecting element of hyaluronidase, and releasing chondrocytes from the matrix. In this study, collagenase NB4, a widely used collagenase with a balanced mix of collagenolytic and additional enzymatic activities, 20,21 was used for chondrocyte dissociation. Incomplete digestion and low cell-yield efficiency are frequently observed in chondrocyte isolation, 9 although the digestion periods are usually prolonged to 10 to 22 hours.…”

Bioactive Inks Development for Osteochondral Tissue Engineering: A Mini-Review

“…According to observations, the matrix could support proliferation and chondrogenic differentiation as a function of decellularized ECM addition besides the good mechanical properties and degradation rate. In another recent study, a 3D structure was produced using engraving decellularized cartilage, called CartiScaff, derived through a CO 2 laser of human articular cartilage [ 58 ]. Implantation of the scaffold in articular cartilage defect of mice osteochondral plugs illustrated increased cellular interaction and improved cartilage regeneration.…”

Current Advances in the Regeneration of Degenerated Articular Cartilage: A Literature Review on Tissue Engineering and Its Recent Clinical Translation

“…On the other hand, researchers are studying the application of innovative strategies, such as laser technology, to improve the scaffolds used in cartilage tissue engineering. Nürnberger et al (2021) focused on developing a scaffold with a defined architecture that properly guides collagen alignment during articular cartilage regeneration. To achieve their goal, they first engraved human articular cartilage ECM using a CO 2 laser in three patterns (hole, line, and grid), then decellularized (with subsequent glycosaminoglycan (GAG) depletion) or devitalized the matrix, and finally seeded it with different types of cells for analysis in vitro and in vivo (subcutaneous implantation of bovine osteochondral cylinders into female athymic NMRI nude mice).…”

“…Human articular cartilage ECM + AD-MSCs/chondrocytes [27] HA/collagen/fibrinogen + SMSCs + rhTG-4 [28] Human placenta + BM-MSCs ± PRP [30] Alginate spheres + chondrocytes/chondrons [36] 02 Scaffold-Free Strategies Human freeze-dried cancellous bone + human chondrocyte sheets [43] Human chondrocytes ± human synoviocytes [44] hAMSCs cell sheets + cartilage particles [46] 03 Injectables AD-MSCs ± HA [49] Open-porous PLGA microspheres + BM-MSCs [50] MSCs + chondrocytes [51] PEG/ApoPep-1/TCO + chondrocytes [52] E7-Exo + SF-MSCs + KGN [53] 04…”

“…To achieve their goal, they first engraved human articular cartilage ECM using a CO 2 laser in three patterns (hole, line, and grid), then decellularized (with subsequent glycosaminoglycan (GAG) depletion) or devitalized the matrix, and finally seeded it with different types of cells for analysis in vitro and in vivo (subcutaneous implantation of bovine osteochondral cylinders into female athymic NMRI nude mice). The matrix with grid patterns was selected for its preferable properties, and the decellularized GAG-depleted one exhibited superior ability to guide the newly synthesized matrix towards a vertical alignment, avoiding an undesired random deposition, and thus, improving the neo-cartilage regeneration [ 28 ].…”