Amnion Cells in Tailored Hydrogels Deposit Human Amnion Native Extracellular Matrix

Fetal therapies regularly result in iatrogenic preterm premature rupture of the fetal membranes (iPPROM), which is associated with preterm birth. Biomaterials that promote the healing of traumatized fetal membranes (FMs) may prevent iPPROM-associated preterm births, addressing this unmet clinical need. Here, a fully defined synthetic poly(ethylene glycol) (PEG) hydrogel is developed to study the healing functions of human amnion-derived mesenchymal stromal cells (hAMCs) in 3D cultures. A pipeline to analyze extracellular matrix (ECM) proteins deposited by hAMCs in PEG hydrogels is established involving label-free quantification of mass-spectrometry data. Owing to the contaminant-free PEG hydrogels and a short fetal bovine serum (FBS)-free culture period, 128 ECM proteins, of which 97 are present in the native amnion, are identified. Upon stimulation with platelet-derived growth factor BB (PDGF-BB), a cell proliferation and migration inducing factor, hAMCs remodel their surroundings and deposit ECM proteins pericellularly. Among the most abundantly deposited amnion proteins, transforming growth factor β-induced protein ig-h3 (TGFβi), a very distinctive amnion protein that is involved in the wound healing cascade, is identified. These data support the potential of PDGF-BB to promote the repair of traumatized FMs and encourage its use for the engineering of biomaterials for FM healing, to ultimately prevent iPPROM

In Vitro and Ectopic In Vivo Studies toward the Utilization of Rapidly Isolated Human Nasal Chondrocytes for Single-Stage Arthroscopic Cartilage Regeneration Therapy

Nasal chondrocytes (NCs) have a higher and more reproducible chondrogenic capacity than articular chondrocytes, and the engineered cartilage tissue they generate in vitro has been demonstrated to be safe in clinical applications. Here, we aimed at determining the feasibility for a single-stage application of NCs for cartilage regeneration under minimally invasive settings. In particular, we assessed whether NCs isolated using a short collagenase digestion protocol retain their potential to proliferate and chondro-differentiate within an injectable, swiftly cross-linked and matrix-metalloproteinase (MMP)-degradable polyethylene glycol (PEG) gel enriched with human platelet lysate (hPL). NC-hPL-PEG gels were additionally tested for their capacity to generate cartilage tissue in vivo and to integrate into cartilage/bone compartments of human osteochondral plugs upon ectopic subcutaneous implantation into nude mice. NCs isolated with a rapid protocol and embedded in PEG gels with hPL at low cell density were capable of efficiently proliferating and of generating tissue rich in glycosaminoglycans and collagen II. NC-hPL-PEG gels developed into hyaline-like cartilage tissues upon ectopic in vivo implantation and integrated with surrounding native cartilage and bone tissues. The delivery of NCs in PEG gels containing hPL is a feasible strategy for cartilage repair and now requires further validation in orthotopic in vivo models. Keywords: cartilage regeneration; autologous chondrocyte implantation; nasal chondrocytes; single-stage; arthroscopy; tissue engineering; polyethylene glycol; hydrogel; platelet lysat

Extracellular Matrix Production by Mesenchymal Stromal Cells in Hydrogels Facilitates Cell Spreading and Is Inhibited by FGF‐2

In native tissues, the interaction between cells and the surrounding extracellular matrix (ECM) is reciprocal, as cells not only receive signals from the ECM but also actively remodel it through secretion of cell‐derived ECM. However, very little is known about the reciprocal interaction between cells and their secreted ECM within synthetic biomaterials that mimic the ECM for use in engineering of tissues for regenerative medicine or as tissue models. Here, poly(ethylene glycol) (PEG) hydrogels with fully defined biomaterial properties are used to investigate the emerging role of cell‐derived ECM on culture outcomes. It is shown that human mesenchymal stromal cells (MSCs) secrete ECM proteins into the pericellular space early after encapsulation and that, even in the absence of material‐presented cell adhesion motifs, cell‐derived fibronectin enables cell spreading. Then, it is investigated how different culture conditions influence MSC ECM expression in hydrogels. Most strikingly, it is found by RNA sequencing that the fibroblast growth factor 2 (FGF‐2) changes ECM gene expression and, in particular, decreases the expression of structural ECM components including fibrillar collagens. In summary, this work shows that cell‐derived ECM is a guiding cue in 3D hydrogels and that FGF‐2 is a potentially important ECM regulator within bioengineered cell and tissue systems

Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease-Related Cargo Absent in EVs from 2D Cultures

Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce clinically relevant cancer phenotypes in vitro. However, their effect on molecular cargo of secreted extracellular vesicles (EVs) has not yet been investigated. Here, the impact of hydrogel-based 3D engineered microtissues on EVs secreted by benign and malignant prostate cells is assessed. Compared to 2D cultures, yield of EVs per cell is significantly increased for cancer cells cultured in 3D. Whole transcriptome sequencing and proteomics of 2D-EV and 3D-EV samples reveal stark contrasts in molecular cargo. For one cell type in particular, LNCaP, enrichment is observed exclusively in 3D-EVs of GDF15, FASN, and TOP1, known drivers of prostate cancer progression. Using imaging flow cytometry in a novel approach to validate a putative EV biomarker, colocalization in single EVs of GDF15 with CD9, a universal EV marker, is demonstrated. Finally, in functional assays it is observed that only 3D-EVs, unlike 2D-EVs, confer increased invasiveness and chemoresistance to cells in 2D. Collectively, this study highlights the value of engineered 3D microtissue cultures for the study of bona fide EV cargoes and their potential to identify biomarkers that are not detectable in EVs secreted by cells cultured in standard 2D conditions

PEG/HA Hybrid Hydrogels for Biologically and Mechanically Tailorable Bone Marrow Organoids

Bone marrow (BM) organoids provide powerful tools to study the vital interplay between the BM microenvironment and resident cells. Current biomaterials, however, are limited in terms of versatility for independently studying the biochemical and biophysical properties that regulate BM function. Here, a transglutaminase (TG) crosslinked system that seamlessly incorporates poly(ethylene glycol) (PEG) and hyaluronic acid (HA) into hybrid hydrogels for the formation of BM analogues is presented. By combining features of PEG and HA, these novel biomaterials are tunable to optimize their physical and biological properties for BM organoid formation. Utility of the TG-PEG/HA hybrid hydrogels to maintain, expand, or differentiate human bone marrow-derived stromal cells and human hematopoietic stem and progenitor cells in vitro is demonstrated. Even more compelling, TG-PEG/HA hybrid hydrogels are superior to currently used natural biomaterials in forming humanized BM organoids in a xenograft model. Hybrid hydrogels in comparison to pure PEG or pure HA afford the ideal attributes of both regarding material handling, structural integrity, and minimizing macrophage infiltration in vivo. The engineered humanized BM organoids presented here may be effective tools for the study of this intricate organ

PEG/HA Hybrid Hydrogels for Biologically and Mechanically Tailorable Bone Marrow Organoids

Bone marrow (BM) organoids provide powerful tools to study the vital interplay between the BM microenvironment and resident cells. Current biomaterials, however, are limited in terms of versatility for independently studying the biochemical and biophysical properties that regulate BM function. Here, a transglutaminase (TG) crosslinked system that seam-lessly incorporates poly(ethylene glycol) (PEG) and hyaluronic acid (HA) into hybrid hydrogels for the formation of BM analogues is presented. By combining features of PEG and HA, these novel biomaterials are tunable to optimize their physical and biological properties for BM organoid for-mation. Utility of the TG-PEG/HA hybrid hydrogels to maintain, expand, or differentiate human bone marrow-derived stromal cells and human hematopoietic stem and progenitor cells in vitro is demonstrated. Even more compelling, TG-PEG/HA hybrid hydrogels are superior to currently used natural biomaterials in forming humanized BM organoids in a xenograft model. Hybrid hydrogels in comparison to pure PEG or pure HA afford the ideal attributes of both regarding material handling, structural integrity, and minimizing macrophage infiltration in vivo. The engineered humanized BM organoids presented here may be effective tools for the study of this intricate organ

Polyisocyanopeptide hydrogels: A novel thermo-responsive hydrogel supporting pre-vascularization and the development of organotypic structures

Contains fulltext : 191187.pdf (publisher's version ) (Open Access

Expanded skeletal stem and progenitor cells promote and participate in induced bone regeneration at subcritical BMP-2 dose

The regeneration of large bone defects remains an unsolved clinical problem, which could benefit from recent findings on the biology of skeletal stem and progenitor cells. The elucidation of conditions to specifically control their dynamic and function will likely enable the development of novel treatment strategies. In this study, we aimed at dissecting the role of osteogenic cues and skeletal stem (SSC) and progenitor cell (BCSP) recruitment during biomimetic hydrogel-assisted bone regeneration. To do so, we employed a biomimetic synthetic hydrogel based on poly (ethylene glycol) (PEG), highly controllable and enzymatically crosslinkable. We show that hydrogel-released bone morphogenetic protein-2 (BMP-2) dose-dependently promoted the enrichment of both SSCs and BCSPs within bone defects. Furthermore, we demonstrate that prospectively isolated neonatal bone-derived, as well as expanded SSCs and BCSPs, differentiate into osteogenic cells and enhance the healing of bone defects by low BMP-2 releasing biomaterials. These results indicate that growth factor releasing materials should be designed to first augment the number of SSCs and BCSPs, followed by their osteogenic differentiation to potentiate the healing of bone defects. Additionally, we demonstrate that expanded SSCs and BCSPs are easily accessible cell sources that allow the study of novel bone healing regimen under controlled in vitro and in vivo conditions

Extracellular Matrix Production by Mesenchymal Stromal Cells in Hydrogels Facilitates Cell Spreading and Is Inhibited by FGF-2

In native tissues, the interaction between cells and the surrounding extracellular matrix (ECM) is reciprocal, as cells not only receive signals from the ECM but also actively remodel it through secretion of cell-derived ECM. However, very little is known about the reciprocal interaction between cells and their secreted ECM within synthetic biomaterials that mimic the ECM for use in engineering of tissues for regenerative medicine or as tissue models. Here, poly(ethylene glycol) (PEG) hydrogels with fully defined biomaterial properties are used to investigate the emerging role of cell-derived ECM on culture outcomes. It is shown that human mesenchymal stromal cells (MSCs) secrete ECM proteins into the pericellular space early after encapsulation and that, even in the absence of material-presented cell adhesion motifs, cell-derived fibronectin enables cell spreading. Then, it is investigated how different culture conditions influence MSC ECM expression in hydrogels. Most strikingly, it is found by RNA sequencing that the fibroblast growth factor 2 (FGF-2) changes ECM gene expression and, in particular, decreases the expression of structural ECM components including fibrillar collagens. In summary, this work shows that cell-derived ECM is a guiding cue in 3D hydrogels and that FGF-2 is a potentially important ECM regulator within bioengineered cell and tissue systems