Biology subjects

Mazzini, G.

Publications and source records attributed to Mazzini, G..

7 recordsLinked to original sources

bioRxiv2026

Dynamic suspension culture enhances scalable maturation of hiPSC-derived cartilage organoids for regenerative medicine

BackgroundCartilage tissue engineering requires scalable culture strategies to produce high-quality organoids. Human induced pluripotent stem cells (hiPSCs) provide a renewable source of chondrogenic cells. However, conventional static 3D culture limits tissue maturation, reproducibility, and scalability. Dynamic culture systems may help overcome these limitations, although their application for hiPSC-derived cartilage maturation remains poorly explored. MethodsIn this study, we established and validated a dynamic suspension bioreactor culture platform (CERO, OLS) for scalable maturation of hiPSC-derived chondroprogenitor cells (hiCPCs) into cartilage organoids populated by biomimetic human induced chondrocytes (hiCHOs). Key culture parameters, including aggregate preparation strategy, agitation speed, and maturation duration, were systematically evaluated. Cartilage maturation under dynamic and conventional static culture conditions was assessed by histology and immunohistochemistry, biochemical assays, organoid size measurements, and gene expression (RT-qPCR). In addition, the functional integration of optimized organoids was evaluated in a human osteochondral explant model. ResultsPre-formed manually picked hiCPC aggregates showed improved cartilage formation compared with single-cell seeding or pelleted aggregates in the bioreactor. Dynamic suspension culture promoted increased construct growth, enhanced ECM deposition, and a more favourable cartilage-associated molecular phenotype compared with static culture. HiCHO organoids matured under dynamic suspension conditions displayed increased sulphated glycosaminoglycan and proteoglycan deposition together with higher expression of cartilage-associated genes ACAN, COMP, MGP, and COL2A1. Although prolonged static maturation alone supported continued cartilage development, introducing dynamic suspension culture during later maturation stages further reinforced favourable molecular and matrix-associated features. Importantly, hiCHO organoids generated under optimized dynamic culture conditions successfully filled human cartilage defects and established matrix continuity with surrounding native tissue in a human osteochondral ex vivo explant model. ConclusionsThis study shows that dynamic suspension culture is an effective and scalable strategy for maturation of hiPSC-derived cartilage organoids. Consequently, this approach supports reproducible neo-cartilage production and allows functional testing in human tissue models. These findings support the use of dynamic culture systems for cartilage repair and in vitro/ex vivo cartilage research.

Cell Biology↗

bioRxiv2026

Nuclear Factor I genes drive chondrogenic cell-fate commitment

Human induced pluripotent stem cells (hiPSCs) offer a powerful platform to model chondrogenesis and enable regenerative strategies, yet regulation of cell-fate commitment remains elusive. Here, we systematically mapped cell-fate trajectories from 7 time points during a 49-day chondrogenic hiPSC differentiation protocol using single-nucleus multimodal transcriptomic and chromatin accessibility profiling (scRNA-seq and scATAC-seq). Integrative analysis of dynamics revealed branching differentiation trajectories with clear bifurcation points and distinct cell-fates. Notably, the chondrogenic trajectory originated at day 6 as a neurogenic development and branched off at day 21 to a chondrogenic cell-fate. Through transcription factor activity analysis (TFAA) and cis-co-accessibility networks, we found that NFIA and NFIB drove chondrogenic distinction, exhibited in both modalities as directly targeting chondrogenic genes such as COMP, FIBIN, VIM. This was then confirmed by experimental validation as modulation of NFIA expression at this point further enhanced chondrocyte formation. Together, our study provides a high-resolution multimodal atlas of chondrogenic differentiation and identified critical transcriptional regulators that can now be leveraged to implement regenerative cartilage therapies from hiPSCs.

cell biology↗

bioRxiv2025

Evaluating the Therapeutic Efficacy of Iopanoic Acid in a DMM-Induced Osteoarthritis Mouse Model and Osteochondral Lesioned Human Explants

ObjectiveTo evaluate the therapeutic potential of iopanoic acid (IOP), a thyroid hormone pathway inhibitor, in preserving cartilage and bone integrity in osteoarthritis (OA), using in vivo and ex vivo tissue models. DesignIn the DMM mouse model, IOP was administered through intra-articular (i.a.) injection, either alone or combined with a thermosensitive hydrogel to enable sustained release. Histological analyses included Safranin O/Fast Green staining and OARSI scoring. Immunohistochemistry was performed for COL2, MMP13, and CCDC80 to evaluate anabolic, catabolic, and hypertrophic markers. Micro-CT assessed subchondral bone changes. In the ex vivo studies, IOP was applied to lesioned human osteochondral OA explants. Matrix degradation and repair were evaluated by sulfated glycosaminoglycan (sGAG) release, Mankin histology scores, and RT-qPCR for cartilage matrix genes. ResultsAdministration of IOP significantly reduced cartilage degeneration in DMM mice (P [≤] 1.0x10-4), characterized by increased COL2, and decreased MMP13 and CCDC80 expression. Notably, IOP also prevented pathological subchondral bone thickening. In human explants, IOP treatment led to a significant reduction in sGAG release compared to untreated explants on day 6 of the IOP treatment. Moreover, Mankin scores were significantly improved in IOP-treated compared to untreated explants, indicating reduced cartilage degradation. ConclusionIOP demonstrates strong chondroprotective effects, reducing cartilage degradation and promoting repair in OA models. Its combination with a thermosensitive hydrogel amplifies therapeutic potential, offering a promising strategy for OA treatment. Next steps are to optimize delivery and validate early molecular effects.

molecular biology↗

bioRxiv2023

Nanobodies counteract the toxicity of an amyloidogenic light chain by stabilizing a partially open dimeric conformation

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 VL domain and stabilise an unexpected partially open LC dimer in which the two VL domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms.

biophysics↗

bioRxiv2022

Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter

AA amyloidosis is a systemic disease characterized by deposition of misfolded serum amyloid A protein (SAA) into cross-{beta} amyloid in multiple organs in humans and animals. AA amyloidosis occurs at high SAA serum levels during chronic inflammation. The disease can be transmitted horizontally, likely facilitated by prion-like mechanism, in captive animals leading to extreme disease prevalence, e.g. 70% in captive cheetah and 57-73% in domestic short hair (DSH) cats kept in shelters. Herein, we present the 3.3 [A] cryo-EM structure of an AA amyloid extracted post-mortem from the kidney of a DSH cat with renal failure. The structure reveals a cross-{beta} architecture assembled from two 76-residue long proto-filaments. Despite >70% sequence homology to mouse and human SAA, the cat SAA variant adopts a distinct amyloid fold. Based on shared disease profiles and almost identical protein sequences, we propose a similar amyloid fold of deposits identified previously in captive cheetah.

biophysics↗

bioRxiv2022

AA-amyloidosis in cats (Felis catus) housed in shelters

Systemic AA-amyloidosis is a protein-misfolding disease that is characterized by fibril deposition of serum amyloid-A protein (SAA) in several organs in humans and many animal species. Fibril deposits originate from abnormally high serum levels of SAA during chronic inflammation. In domestic short-hair cats, AA-amyloidosis has only been anecdotally reported and is considered a rare disease. Here we report that an astonishing 57-73% of early deceased short-hair cats kept in three independent shelters suffer from amyloid deposition in the liver, spleen, or kidney. Histopathology and mass spectrometry of post-mortem extracted deposits identified SAA as the major protein source. The duration of stay in the shelters was positively associated with a histological score of AA-amyloidosis (B=0.026, CI95%=0.007-0.046; p=0.010). Presence of SAA fragments in bile secretions raises the possibility of fecal-oral transmission of the disease.

pathology↗

bioRxiv2022

Mind the gap: Nodes of Ranvier are remodeled by chronic psychosocial stress and neuronal activity

Differential expression of myelin-related genes and changes in myelin thickness have been demonstrated in mice after chronic psychosocial stress, a risk factor for anxiety disorders. To determine whether and how stress affects structural remodeling of nodes of Ranvier, another form of myelin plasticity, we developed a 3D reconstruction analysis of node morphology in C57BL/6NCrl and DBA/2NCrl mice. We identified strain-dependent effects of chronic stress on node morphology, including elongation of paranodes in the medial prefrontal cortex (mPFC) in DBA/2NCrl mice. Furthermore, chronic chemogenetic activation of the ventral hippocampus-to-mPFC pathway resulted in increased risk assessment behavior and shortened paranodes specifically in stimulated axons, providing a direct link between anxiety-like behavior and remodeling of the nodes. Altogether, our data demonstrate genetic regulation of nodal remodeling in stress and suggest an activity-dependent regulation of paranodes in anxiety-related circuits. Nodal remodeling may thus contribute to the aberrant circuit function associated with anxiety disorders.

neuroscience↗