Human adult, pediatric and microtia auricular cartilage harbor fibronectin-adhering progenitor cells with regenerative ear reconstruction potential

Remaining challenges in auricular cartilage tissue engineering include acquiring sufficient amounts of regeneration-competent cells and subsequent production of high-quality neocartilage. Progenitor cells are a resident subpopulation of native cartilage, displaying a high proliferative and cartilage-forming capacity, yet their potential for regenerative medicine is vastly understudied. In this study, human auricular cartilage progenitor cells were newly identified in healthy cartilage and, importantly, in microtia-impaired chondral remnants. Their cartilage repair potential was assessed via in vitro 3D culture upon encapsulation in a gelatin-based hydrogel, and subsequent biochemical, mechanical, and histological analyses. Auricular cartilage progenitor cells demonstrate a potent ability to proliferate without losing their multipotent differentiation ability and to produce cartilage-like matrix in 3D culture. As these cells can be easily obtained through a non-deforming biopsy of the healthy ear or from the otherwise redundant microtia remnant, they can provide an important solution for long-existing challenges in auricular cartilage tissue engineering

Small molecules to regulate the GH/IGF1 axis by inhibiting the growth hormone receptor synthesis

Growth hormone (GH) and insulin-like growth factor-1 (IGF1) play an important role in mammalian development, cell proliferation and lifespan. Especially in cases of tumor growth there is an urgent need to control the GH/IGF1 axis. In this study we screened a 38,480-compound library, and in two consecutive rounds of analogues selection, we identified active lead compounds based on the following criteria: inhibition the GH receptor (GHR) activity and its downstream effectors Jak2 and STAT5, and inhibition of growth of breast and colon cancer cells. The most active small molecule (BM001) inhibited both the GH/IGF1 axis and cell proliferation with an IC50 of 10-30 nM of human cancer cells. BM001 depleted GHR in human lymphoblasts. In preclinical xenografted experiments, BM001 showed a strong decrease in tumor volume in mice transplanted with MDA-MB-231 breast cancer cells. Mechanistically, the drug acts on the synthesis of the GHR. Our findings open the possibility to inhibit the GH/IGF1 axis with a small molecule

Bio-ink development for three-dimensional bioprinting of hetero-cellular cartilage constructs

Bioprinting is a promising tool to fabricate organized cartilage. This study aimed to investigate the printability of gelatin-methacryloyl/gellan gum (gelMA/gellan) hydrogels with and without methacrylated hyaluronic acid (HAMA), and to explore (zone-specific) chondrogenesis of chondrocytes, articular cartilage progenitor cells (ACPCs), and multipotent mesenchymal stromal cells (MSCs) embedded in these bio-inks. The incorporating of HAMA in gelMA/gellan bio-ink increased filament stability, as measured using a filament collapse assay, but did not influence (zone-specific) chondrogenesis of any of the cell types. Highest chondrogenic potential was observed for MSCs, followed by ACPCs, which displayed relatively high proteoglycan IV mRNA levels. Therefore, two-zone constructs were printed with gelMA/gellan/HAMA containing ACPCs in the superficial region and MSCs in the middle/deep region. Chondrogenic differentiation was confirmed, however, printing influence cellular differentiation. ACPC- and MSC-laden gelMA/gellan/HAMA hydrogels are of interest for the fabrication of cartilage constructs. Nevertheless, this study underscores the need for careful evaluation of the effects of printing on cellular differentiation

Differential production of cartilage ECM in 3D agarose constructs by equine articular cartilage progenitor cells and mesenchymal stromal cells

Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering

Differential Production of Cartilage ECM in 3D Agarose Constructs by Equine Articular Cartilage Progenitor Cells and Mesenchymal Stromal Cells

Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering

Differential production of cartilage ECM in 3D agarose constructs by equine articular cartilage progenitor cells and mesenchymal stromal cells

Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering

The bio in the ink : cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells

Cell-laden hydrogels are the primary building blocks for bioprinting, and, also termed bioinks, are the foundations for creating structures that can potentially recapitulate the architecture of articular cartilage. To be functional, hydrogel constructs need to unlock the regenerative capacity of encapsulated cells. The recent identification of multipotent articular cartilage-resident chondroprogenitor cells (ACPCs), which share important traits with adult stem cells, represents a new opportunity for cartilage regeneration. However, little is known about the suitability of ACPCs for tissue engineering, especially in combination with biomaterials. This study aimed to investigate the potential of ACPCs in hydrogels for cartilage regeneration and biofabrication, and to evaluate their ability for zone-specific matrix production. Gelatin methacryloyl (gelMA)-based hydrogels were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes, and as bioinks for printing. Our data shows ACPCs outperformed chondrocytes in terms of neo-cartilage production and unlike MSCs, ACPCs had the lowest gene expression levels of hypertrophy marker collagen type X, and the highest expression of PRG4, a key factor in joint lubrication. Co-cultures of the cell types in multi-compartment hydrogels allowed generating constructs with a layered distribution of collagens and glycosaminoglycans. By combining ACPC- and MSC-laden bioinks, a bioprinted model of articular cartilage was generated, consisting of defined superficial and deep regions, each with distinct cellular and extracellular matrix composition. Taken together, these results provide important information for the use of ACPC-laden hydrogels in regenerative medicine, and pave the way to the biofabrication of 3D constructs with multiple cell types for cartilage regeneration or in vitro tissue models. STATEMENT OF SIGNIFICANCE: Despite its limited ability to repair, articular cartilage harbors an endogenous population of progenitor cells (ACPCs), that to date, received limited attention in biomaterials and tissue engineering applications. Harnessing the potential of these cells in 3D hydrogels can open new avenues for biomaterial-based regenerative therapies, especially with advanced biofabrication technologies (e.g. bioprinting). This study highlights the potential of ACPCs to generate neo-cartilage in a gelatin-based hydrogel and bioink. The ACPC-laden hydrogel is a suitable substrate for chondrogenesis and data shows it has a bias in directing cells towards a superficial zone phenotype. For the first time, ACPC-hydrogels are evaluated both as alternative for and in combination with chondrocytes and MSCs, using co-cultures and bioprinting for cartilage regeneration in vitro. This study provides important cues on ACPCs, indicating they represent a promising cell source for the next generation of cartilage constructs with increased biomimicry

The bio in the ink : cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells

Cell-laden hydrogels are the primary building blocks for bioprinting, and, also termed bioinks, are the foundations for creating structures that can potentially recapitulate the architecture of articular cartilage. To be functional, hydrogel constructs need to unlock the regenerative capacity of encapsulated cells. The recent identification of multipotent articular cartilage-resident chondroprogenitor cells (ACPCs), which share important traits with adult stem cells, represents a new opportunity for cartilage regeneration. However, little is known about the suitability of ACPCs for tissue engineering, especially in combination with biomaterials. This study aimed to investigate the potential of ACPCs in hydrogels for cartilage regeneration and biofabrication, and to evaluate their ability for zone-specific matrix production. Gelatin methacryloyl (gelMA)-based hydrogels were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes, and as bioinks for printing. Our data shows ACPCs outperformed chondrocytes in terms of neo-cartilage production and unlike MSCs, ACPCs had the lowest gene expression levels of hypertrophy marker collagen type X, and the highest expression of PRG4, a key factor in joint lubrication. Co-cultures of the cell types in multi-compartment hydrogels allowed generating constructs with a layered distribution of collagens and glycosaminoglycans. By combining ACPC- and MSC-laden bioinks, a bioprinted model of articular cartilage was generated, consisting of defined superficial and deep regions, each with distinct cellular and extracellular matrix composition. Taken together, these results provide important information for the use of ACPC-laden hydrogels in regenerative medicine, and pave the way to the biofabrication of 3D constructs with multiple cell types for cartilage regeneration or in vitro tissue models. STATEMENT OF SIGNIFICANCE: Despite its limited ability to repair, articular cartilage harbors an endogenous population of progenitor cells (ACPCs), that to date, received limited attention in biomaterials and tissue engineering applications. Harnessing the potential of these cells in 3D hydrogels can open new avenues for biomaterial-based regenerative therapies, especially with advanced biofabrication technologies (e.g. bioprinting). This study highlights the potential of ACPCs to generate neo-cartilage in a gelatin-based hydrogel and bioink. The ACPC-laden hydrogel is a suitable substrate for chondrogenesis and data shows it has a bias in directing cells towards a superficial zone phenotype. For the first time, ACPC-hydrogels are evaluated both as alternative for and in combination with chondrocytes and MSCs, using co-cultures and bioprinting for cartilage regeneration in vitro. This study provides important cues on ACPCs, indicating they represent a promising cell source for the next generation of cartilage constructs with increased biomimicry

Bio-ink development for three-dimensional bioprinting of hetero-cellular cartilage constructs

Bioprinting is a promising tool to fabricate organized cartilage. This study aimed to investigate the printability of gelatin-methacryloyl/gellan gum (gelMA/gellan) hydrogels with and without methacrylated hyaluronic acid (HAMA), and to explore (zone-specific) chondrogenesis of chondrocytes, articular cartilage progenitor cells (ACPCs), and multipotent mesenchymal stromal cells (MSCs) embedded in these bio-inks. The incorporating of HAMA in gelMA/gellan bio-ink increased filament stability, as measured using a filament collapse assay, but did not influence (zone-specific) chondrogenesis of any of the cell types. Highest chondrogenic potential was observed for MSCs, followed by ACPCs, which displayed relatively high proteoglycan IV mRNA levels. Therefore, two-zone constructs were printed with gelMA/gellan/HAMA containing ACPCs in the superficial region and MSCs in the middle/deep region. Chondrogenic differentiation was confirmed, however, printing influence cellular differentiation. ACPC- and MSC-laden gelMA/gellan/HAMA hydrogels are of interest for the fabrication of cartilage constructs. Nevertheless, this study underscores the need for careful evaluation of the effects of printing on cellular differentiation