Alejandro Rodríguez Ruiz

The objective of this study was to develop and characterize a novel hyaluronic acid (HA) 3D scaffold integrated with gelatin microparticles for sustained-delivery applications. To achieve this goal, the delivery microparticles were synthesized and thoroughly characterized, focusing on their crosslinking mechanisms (vanillin and genipin), degradation profiles, and release kinetics. Additionally, the cytotoxicity of the system was assessed, and its impact on the cell adhesion and distribution using mouse fibroblasts was examined. The combination of both biomaterials offers a novel platform for the gradual release of various factors encapsulated within the microparticles while simultaneously providing cell protection, support, and controlled factor dispersion due to the HA 3D scaffold matrix. Hence, this system offers a platform for addressing injure repair by continuously releasing specific encapsulated factors for optimal tissue regeneration. Additionally, by leveraging the properties of HA conjugates with small drug molecules, we can enhance the solubility, targeting capabilities, and cellular absorption, as well as prolong the system stability and half-life. As a result, this integrated approach presents a versatile strategy for therapeutic interventions aimed at promoting tissue repair and regeneration.

Introduction: Birt-Hogg-Dubé syndrome (BHD) is a rare disease caused by mutations of the FLCN gene which encodes for folliculin. A robust high expression of FLCN is observed in alveolar type II epithelial cells (AEC2), but it is unknown how this mutation affects alveolar epithelial cell biology. Aims and objectives: By introducing a mutation in FLCN in human induced pluripotent stem cells (hiPSCs), differentiating them into AEC2 organoids and exposing them to different triggers, we aim to understand the mechanisms governing alveolar epithelial cell dysfunction and the BHD phenotype in the lungs. Methods: A CRISPR/Cas9 knock out (KO) of the FLCN gene was generated in hiPSCs and differentiated into iAEC2 organoids. Primary BHD-AEC2 were cultured to allow validation of our hiPSC model. Formation of alveolar cysts was modelled by adding forskolin. Furthermore, primary BHD-AEC2 were seeded into a Lung-on-Chip system to mimic breathing-related stresses by stretch application. Results: Genome editing of the hiPSC line generated a FLCN KO line. In parallel, BHD-AEC2 organoids with a FLCN KO variant showed an increase in organoid lumen size upon forskolin addition compared to control organoids (1.97 times larger versus 1.1, respectively). BHD-AEC2 were seeded in the microfluidic system and stretched at 8% for three days. Conclusions: We have successfully generated a FLCN KO hiPSC line and derived it to iAEC2s. Parallelly, primary BHD-AEC2 displayed significant alveolar enlargement upon forskolin addition, when compared to alveolar organoids from a control. Further characterization of its phenotype is currently ongoing, by assessment of tight junctions, forskolin-induced swelling, and responses to stretch.

To study the mechanism by which the readthrough mutation in TNFRSF11B, encoding osteoprotegerin (OPG) with additional 19 amino acids at its C-terminus (OPG-XL), causes the characteristic bidirectional phenotype of subchondral bone turnover accompanied by cartilage mineralization in chondrocalcinosis patients. OPG-XL was studied by human induced pluripotent stem cells expressing OPG-XL and two isogenic CRISPR/Cas9-corrected controls in cartilage and bone organoids. Osteoclastogenesis was studied with monocytes from OPG-XL carriers and matched healthy controls followed by gene expression characterization. Dual energy X-ray absorptiometry scans and MRI analyses were used to characterize the phenotype of carriers and non-carriers of the mutation. Human OPG-XL carriers relative to sex- and age-matched controls showed, after an initial delay, large active osteoclasts with high number of nuclei. By employing hiPSCs expressing OPG-XL and isogenic CRISPR/Cas9-corrected controls to established cartilage and bone organoids, we demonstrated that expression of OPG-XL resulted in excessive fibrosis in cartilage and high mineralization in bone accompanied by marked downregulation of MGP, encoding matrix Gla protein, and upregulation of DIO2, encoding type 2 deiodinase, gene expression, respectively. The readthrough mutation at CCAL1 locus in TNFRSF11B identifies an unknown role for OPG-XL in subchondral bone turnover and cartilage mineralization in humans via DIO2 and MGP functions. Previously, OPG-XL was shown to affect binding between RANKL and heparan sulphate (HS) resulting in loss of immobilized OPG-XL. Therefore, effects may be triggered by deficiency in the immobilization of OPG-XL Since the characteristic bidirectional pathophysiology of articular cartilage calcification accompanied by low subchondral bone mineralization is also a hallmark of OA pathophysiology, our results are likely extrapolated to common arthropathies.

Objective: To explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele rs1052429-A on WWP2 expression in a human 3D in vitro model of cartilage. Methods: Co-expression behavior of WWP2 with genes expressed in lesioned OA articular cartilage (N=35 samples) was explored. By applying lentiviral particle mediated WWP2 upregulation in 3D in vitro pellet cultures of human primary chondrocytes (N=8 donors) the effects of upregulation on cartilage matrix deposition was evaluated. Finally, we transfected primary chondrocytes with miR-140 mimics to evaluate whether miR-140 and WWP2 are involved in similar pathways. Results: Upon performing Spearman correlations in lesioned OA cartilage, 98 highly correlating genes (|ρ|>0.7) were identified. Among these genes, we identified GJA1, GDF10, STC2, WDR1 , and WNK4 . Subsequent upregulation of WWP2 on 3D chondrocyte pellet cultures resulted in a decreased expression of COL2A1 and ACAN and an increase in EPAS1 expression. Additionally, we observed a decreased expression of GDF10, STC2 , and GJA1 . Proteomics analysis identified 42 proteins being differentially expressed with WWP2 upregulation, which were enriched for ubiquitin conjugating enzyme activity. Finally, upregulation of miR-140 in 2D chondrocytes resulted in significant upregulation of WWP2 and WDR1 . Conclusions: Mimicking the effect of OA risk allele rs1052429-A on WWP2 expression initiates detrimental processes in the cartilage shown by a response in hypoxia associated genes EPAS1,GDF10 , and GJA1 and a decrease in anabolic markers, COL2A1 and ACAN .

Fibronectin binding to collagen type II is crucial for articular chondrocytes to stay in a healthy steady state.