Patterned, organoid-based cartilaginous implants exhibit zone specific functionality forming osteochondral-like tissues in vivo

Abstract

Tissue engineered constructs have the potential to respond to the unmet medical need of treating deep osteochondral defects. However, current tissue engineering strategies struggle in the attempt to create patterned constructs with biologically distinct functionality. In this work, a developmentally-inspired modular approach is proposed, whereby distinct cartilaginous organoids are used as living building blocks. First, a hierarchical construct was created, composed of three layers of cartilaginous tissue intermediates derived from human periosteumderived cells: (i) early (SOX9), (ii) mature (COL2) and (iii) (pre)hypertrophic (IHH, COLX) phenotype. Subcutaneous implantation in nude mice generated a hybrid tissue containing one mineralized and one non-mineralized part. However, the non-mineralized part was represented by a collagen type I positive fibrocartilage-like tissue. To engineer a more stable articular cartilage part, iPSC-derived cartilage microtissues (SOX9, COL2; IHH neg) were generated.Subcutaneous implantation of assembled iPSC-derived cartilage microtissues resulted in a homogenous cartilaginous tissue positive for collagen type II but negative for osteocalcin.Finally, iPSC-derived cartilage microtissues in combination with the pre-hypertrophic cartilage organoids (IHH, COLX) could form dual tissues consisting of i) a cartilaginous safranin O positive and ii) a bony osteocalcin positive region upon subcutaneous implantation, corresponding to the pre-engineered zonal pattern. The assembly of functional building blocks, as presented in this work, opens possibilities for the production of complex tissue engineered implants by embedding zone-specific functionality through the use of pre-programmed living building blocks.

In vitro and in vivo evaluation of 3D constructs engineered with human iPSC‐derived chondrocytes in gelatin methacryloyl hydrogel

“…It seems that the human iPSC‐derived chondrocytes used in our study have enhanced matrix formation potential compared to GelMA‐embedded primary chondrocytes used in other reports, confirming the higher metabolic performance of these younger iPSC‐derived chondrocytes, which has also been described in pellet culture studies (Castro‐Viñuelas et al, 2018; Loeser, 2009). Our research group has recently compared human iPSC‐derived chondrocytes to adult human articular chondrocytes, showing that iPSC‐derived chondrocytes had increased mRNA expression of the early chondrogenic marker SOX9 and COL2A1 (Hall et al, 2021). An additional possible explanation for enhanced matrix formation in our setup, compared to other GelMA‐based studies, can be found in the origin of the gelatin used for GelMA, as Pahoff et al (2019) recently described that bovine gelatin is preferred over porcine gelatin for the development of hyaline cartilage‐like neotissue.…”

“…Human pluripotent stem cells that undergo mesoderm induction exhibit a decrease in pluripotency markers (Nanog, Oct3/4, and Sox2), while primitive streak markers (Brachyury, MIXL1, and KDR) are transiently upregulated. Next, upon chondrogenic differentiation, an increase in SOX9, Col2A1, and ACAN as well as Safranin O and collagen type II positive matrix is observed (Hall et al, 2021; Tam et al, 2021; Yamashita et al, 2015). Authors have shown the in vitro and in vivo cartilage‐producing potential of iPSC‐derived chondrocytes in a scaffold‐free approach, or in combination with hydrogels, such as alginate, chondroitin sulfate PEG, and nanofibrillated cellulose (Ko et al, 2014; J. Lee et al, 2015; Nakamura et al, 2021; Nguyen et al, 2017; Yamashita et al, 2015).…”

Application of Induced Pluripotent Stem Cells for Disease Modeling and 3D Model Construction: Focus on Osteoarthritis

“…Furthermore, as no tumors were formed after the transplantation in the articular defects, the 3D cultures were concluded to have relatively low tumorigenic risk. In a 2021 study performed by Hall et al, iPSC-derived cartilage organoids generated from the same protocol were implanted into osteochondral defect models [142]. Therefore, the hiPSC-derived scaffold-less cartilage model is expected to increase the clinical application of 3D cultures in treating cartilage conditions.…”

Microcarriers in application for cartilage tissue engineering: Recent progress and challenges

“…Third, implanted substitutes that can easily integrate into the host native tissues with immunocompatibility and biocompatibility. During the past decade, we have witnessed advanced progress in the field of cartilage TE, embodied by the following changes: (i) sufficient seed cell selection for applications (mesenchymal stem cells, induced pluripotent stem cells) [ [6] , [7] , [8] ]; (ii) precise patterning of biomaterials and novel biomaterials with advanced chemistries (more efficient and versatile biomaterial conjugations) [ 1 , 9 , 10 ]; (iii) active modulation of cellular biological functions and behaviors via structure and properties (e.g., stiffness, viscoelasticity, porosity and degradability) of biomaterials [ [11] , [12] , [13] ]; (iv) combination of biological drugs and factors are combined to improve bioavailability and bioactivity [ [14] , [15] , [16] ]; (v) rapid development of biofabrication technologies including programmed self-assembly and three-dimensional (3D) bioprinting [ [17] , [18] , [19] , [20] , [21] , [22] , [23] ].…”