Biology subjects

Riminucci, M.

Publications and source records attributed to Riminucci, M..

8 recordsLinked to original sources

bioRxiv2025

Inheritance entropy quantifies epigenetic regulation of cell-cycle exit in human bone marrow stromal cells

Human bone marrow stromal cells (BMSC) include skeletal stem cells with ground-breaking the-rapeutic potential. However, BMSC colonies have very heterogeneous in vivo behaviour, due to their different potency; this unpredictability is the greatest hurdle to the development of skeletal regeneration therapies. Colony-level heterogeneity urges a fundamental question: how is it possible that one colony as a collective unit behaves differently from another one? If cell-to-cell variability were just an uncorrelated random process, a million cells in a transplant-bound colony would be enough to yield statistical homogeneity, hence washing out any colony-level traits. A possible answer is that the differences between two originating cells are transmitted to their progenies and collectively persist through an hereditary mechanism. But non-genetic inheritance remains an elusive notion, both at the experimental and at the theoretical level. Here, we prove that heterogeneity in the lineage topology of BMSC clonal colonies is determined by heritable traits that regulate cell-cycle exit. The cornerstone of this result is the definition of a novel entropy of the colony, which measures the hereditary ramifications in the distribution of inactive cells across different branches of the proliferation tree. We measure the entropy in 32 clonal colonies, obtained from single-cell lineage tracing experiments, and show that in the greatest majority of clones this entropy is decisively smaller than that of the corresponding non-hereditary lineage. This result indicates that hereditary epigenetic factors play a major role in determining cycle exit of bone marrow stromal cells.

biophysics↗

bioRxiv2025

Effects of anti-RANKL, Zoledronate or combination therapy in a mouse model of Fibrous Dysplasia: a preclinical study

Bone fragility and pain are major clinical issues in fibrous dysplasia (FD) of bone, a genetic disorder characterized by increased bone resorption and lytic lesions. Both bisphosphonates (BPs) and denosumab are currently used to treat FD patients, although important concerns remain unsolved. BPs downregulate bone remodeling but their effects on FD lesions and pain are variable. Contrarily, denosumab converts FD tissue into mineralized bone and prevents disease progression, but disease rebound occurs upon treatment withdrawal. The combination of these two drugs may represent an effective and safe strategy for FD treatment. We used a FD mouse model (EF1-GsR201C) to assess whether zoledronate (ZOL) addition to anti-RANKL antibody (RANKL) treatment could preserve the effects of RANKL inhibition after treatment discontinuation. We show that RANKL treatment rapidly reduced bone turnover markers (BTMs) and increased bone mass in affected skeletal segments, but FD lesions recurred shortly after discontinuation. Importantly, RANKL+ZOL combination delayed disease rebound after RANKL withdrawal, as bone density was preserved, BTMs rise was prevented, and no new lesions were observed. ZOL monotreatment increased bone density and reduced BTMs but did not fully halt disease progression. Finally, both RANKL and RANKL+ZOL treatments reduced fracture incidence and ameliorated pain-like behavior in FD mice. These results demonstrate that combining zoledronate with denosumab may effectively treat FD. This strategy could particularly benefit patients with severe, rapidly progressive disease, in which RANKL inhibition would block lesion expansion and reduce bone turnover, while zoledronate would slow down the resumption of the disease and the rebound effect.

pathology↗

bioRxiv2025

Exploring the mechanism and pattern of bone formation during RANKL inhibition in a mouse model of fibrous dysplasia

Fibrous dysplasia (FD) of bone is a genetic fibro-osseous disorder with increased bone remodeling activity. Inhibition of RANKL modifies FD lesions by inducing the replacement of the fibrous tissue with bone. This effect was observed in FD murine models receiving anti-mouse RANKL antibodies or small molecule RANKL inhibitors and in FD patients treated with denosumab. However, in neither case the mechanism and pattern of deposition of the newly formed bone were clarified. We performed radiographic, morphological and molecular analyses on EF1-GsR201C (FD) mice receiving an anti-mouse RANKL antibody. We observed that RANKL inhibition caused a decrease in the expression of genes involved in osteogenesis, osteoclastogenesis, matrix remodeling and osteoblast-osteoclast cross-talk in affected skeletal segments. Nonetheless, intra-lesional bone surfaces were covered by a continuous layer of osteoid, indicating that bone formation was actively ongoing in the pathological tissue in spite of the treatment. Accordingly, all bone surfaces within FD lesions showed calcein labeling which was never detected in the fibrous tissue far from bone. These results indicate that in the absence of RANKL activity, bone formation in FD tissue does not occur diffusely or stochastically. In contrast, it is restricted to bone surfaces where osteoprogenitor cells are orderly recruited from the adjacent fibrosis, progressively converting it into bone. Clinically, this suggests that the volume of pre-treatment bone in FD lesions may be a determinant of the skeletal improvement that individual patients may achieve during the same denosumab treatment course. As a consequence, it may also be a determinant of the severity of the rebound effect that they can experience upon treatment discontinuation.

pathology↗

bioRxiv2025

Aqueous two-phase bioinks for discrete packing and compartmentalisation of 3D bioprinted cells

The unparalleled ability of aqueous two-phase systems (ATPS) to reproduce microscale cellular and biomaterial compartmentalisation to selectively modulate cell behaviour and functionality is ideal for tissue engineering and regenerative medicine (TERM) purposes. Herein, we introduce new ATPS biomaterial inks for 3D bioprinting of water-in-water (W/W) emulsions, enabling precise cellular crowding for tissue regeneration in vitro and ex vivo. Gelatin methacryloyl (GelMA) was hierarchically dispersed in an alginic acid phase depending on sodium chloride (NaCl) concentration (0-36 g/L). Emulsion droplet size (12.8{+/-}2.6 {micro}m to 52.4{+/-}11.4 {micro}m) influenced degradation and spatial cell localisation (A549, C2C12, MG63). A microfluidic-assisted 3D bioprinting approach allowed fine-tuning of fibre structure adjusting ATPS deposition by modulating flow rates and printing speed. Rheological properties supported the findings of the two-phase partitioning, aiding the selection of the ATPS ink formulation for functional cell-laden construct fabrication. Encapsulation of C2C12 cells revealed enhanced cytoskeletal remodelling at higher salt concentrations. Increased GelMA phase promoted human bone marrow stromal cells (HBMSCs) crowding, mineral deposition and skeletal differentiation. In ovo studies demonstrated degradation control and vascular infiltration via salt modulation. Altogether, ATPS bioinks offer a versatile platform for the assembling of complex, hierarchical tissues with microscale precision, expanding biofabrication strategies for TERM applications.

bioengineering↗

bioRxiv2025

Topology, Kinetics and Inheritance in Clonal Colonies of Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSC) - which include skeletal stem cells - are a promising tool in regenerative medicine. However, their heterogeneous and unpredictable in vivo behaviour remains a critical barrier preventing the development of standardized therapeutic approaches for skeletal tissue regeneration. Several studies have attempted to identify in vitro features that could correlate with the in vivo differentiation properties, yet the mechanisms ruling BMSC heterogeneity remain poorly understood. Here, using time-lapse imaging, we lineage-trace 32 single-cell-derived BMSC colonies through seven generations. We observe significant inter-colony and intra-colony heterogeneity in lineage topology (determined by the number of senescent or apoptotic cells) and in replicative kinetics (measured from proliferating cells only). Interestingly, topology and kinetics result strongly correlated, suggesting the existence of regulatory factors linking the non-dividing/apoptotic subpopulations with proliferating cells. Furthermore, BMSCs display highly synchronized cell cycles during early generations, indicating stage-specific regulatory mechanisms through which cells influence each other. By employing a non-interacting population growth model, we demonstrate that the observed synchronisation cannot be explained by an uncorrelated branching process; instead, cell-to-cell correlation of division times must exist. Our findings reveal fundamental mechanisms governing BMSC heterogeneity and growth dynamics that may inform strategies to control their regenerative potential.

biophysics↗

bioRxiv2025

Engineering a microfluidic-assisted 3D bioprinting approach for the hierarchical control deposition and compartmentalisation of graded bioinks

The advent of 3D bioprinting has revolutionised tissue engineering and regenerative medicine (TERM). Today, tissues of single cell type can be printed with extreme resolution and printing fidelity. However, the ultimate functionality of the desired tissue is limited, due to the absence of a multicellular population and diversity in micro-environment distribution. Currently, 3D bioprinting technologies are facing challenges in delivering multiple cells and biomaterials in a controlled fashion. The use of interchangeable syringe-based systems has often favoured the delamination between interfaces, greatly limiting the fabrication of interconnected tissue constructs. Microfluidic-assisted 3D bioprinting platforms have been found capable of rescuing the fabrication of tissue interfaces, but often fails to guarantee printing fidelity, cell density control and compatimentalisation. Herein, we present the convergence of microfluidic and 3D bioprinting platforms into a new deposition system capable of harnessing a microfluidic printhead for the continuous rapid fabrication of interconnected functional tissues. The use of flow-focusing and passive mixer printhead modules allowed for the rapid and dynamic modulation of fibre diameter and material composition, respectively. Cells were compartmentalised into discrete three-dimensional layers with defined density patterns, confirming the punctual control of the presented microfluidic platform in arranging cells and materials in 3D. In ovo and in vivo studies demonstrated the functionality of 3D bioprinted constructs with patterned vascular endothelial growth factor (VEGF) and transforming growth factor-{beta}1 (TGF-{beta}1), respectively. This, in turn, facilitated the simulation of diverse cellular environments and proliferation pathways within a single construct, which is currently unachievable with conventional 3D bioprinting techniques, offering new opportunities for the fabrication of functionally graded materials and physiologically-relevant skeletal tissue substitutes.

bioengineering↗

bioRxiv2025

Pde5a Deficiency Prevents Diet-Induced Obesity via Adipose cAMP-PKA Activation Enhancing Fat Browning

Cyclic nucleotides are critical regulators of adaptive thermogenesis and adipogenesis, with their intracellular levels finely tuned by phosphodiesterases. Phosphodiesterase type 5 (PDE5A) modulates cyclic guanosine monophosphate levels in adipocytes. While PDE5A inhibition has shown promise in patients with diabetes, its role in metabolism remains unclear. Using Pde5a knockout mouse models, we demonstrated that mice lacking Pde5a exhibit enhanced browning of white adipose tissue and reduced hepatic fat content. Following high-fat diet, Pde5a-/- mice are resistant to obesity, displaying improved glucose metabolism and enhanced thermogenesis. These protective effects stem from an early developmental knockdown of Pde5a, leading to a metabolic reprogramming driven by cAMP-PKA pathway activation. The convergence of cGMP and cAMP signaling orchestrates thermogenic and systemic metabolic adaptations. Our findings establish PDE5A as a novel regulator of energy homeostasis, suggesting its inhibition as a valuable adjuvant therapy for metabolic disorders.

cell biology↗

bioRxiv2024

Bone pain in Fibrous dysplasia does not rely on aberrant sensory nerve sprouting or neuroma formation

Bone pain is a major symptom of many skeletal disorders. Fibrous dysplasia (FD) is a genetic disease with mono or polyostotic skeletal phenotype due to the post-zygotic occurrence of the causative Gs mutation. Bone pain in FD often associates with skeletal deformities and fractures or nerve impingement by the pathological tissue. However, even in the absence of complications, FD patients often complain of a chronic pain that does not correlate with their disease burden. Multiple hypotheses have been made to explain this pain. However, its pathogenetic mechanisms remain, as yet, largely unexplored. In this study, we first demonstrate that the FD mouse model EF1-GsR201C develops a painful-like behavior and an altered response to nociceptive stimuli that, as in FD patients, do not correlate with the severity of their phenotype, thus providing a reliable model to study bone pain in FD. Then, we show that in EF1-GsR201C mice, the overall pattern of skeletal innervation is preserved and that within FD lesions, sensory fibers are variably and focally distributed, mainly at perivascular sites. Finally, we provide the first analysis of a series of human FD bone biopsies showing that sensory nerve fibers are rarely detected within the pathological tissue. These data confirm that bone pain is an intrinsic and reproducible feature of FD. They also show that, albeit sensory nerve fibers are found within FD lesions and may contribute to the unpleasant sensation that accompanies the disease, pathological sensory nerve sprouting or formation of neuromas are not detected in the Gs-mutated skeleton.

pathology↗