Bruno Paiva Dos Santos

Stem cells from human exfoliated deciduous teeth (SHEDs) offer a promising alternative to bone marrow‐derived mesenchymal stem cells (BMSCs) for bone tissue engineering due to their accessibility, high proliferative potential, and multipotency. In this study, we compared the osteogenic and angiogenic potential of two mesenchymal stem cells subpopulations, SHEDs and BMSCs, when co‐cultured with human umbilical vein endothelial cells (HUVECs) into spheroids over a period of 28 days in porous pullulan/dextran scaffolds loaded with hydroxyapatite (HAp) particles as the sole osteoinductive cue. Spheroids were cultured under static and dynamic conditions, with the latter employing a perfusion flow bioreactor to enhance solute transport and oxygenation. Dynamic culture conditions significantly improved cell viability compared to static culture (85% vs. 54% at Day 28), maintained spheroid integrity, and promoted the expression of angiogenic markers, such as the cluster of differentiation 31 (CD31) and von Willebrand factor (vWF), which under static culture were largely confined to the spheroid periphery. Furthermore, alpha‐smooth muscle actin/neural‐glial‐antigen 2 (αSMA/NG2) and CD31/NG2 colocalization reflected close spatial associations between SHEDs and HUVECs, suggesting a supportive perivascular interaction under dynamic culture. In the presence of HUVECs, we found that HAp particles alone were insufficient to induce robust osteogenic differentiation in SHEDs. Weak alkaline phosphatase activity, minimal osteopontin and osteocalcin expression, and incomplete mineralization were observed under both static and dynamic conditions. In contrast, BMSC/HUVEC spheroids exhibited robust osteogenic differentiation and consistent mineral deposition. These results show intrinsic differences in the behavior of SHEDs and BMSCs when co‐cultured with endothelial cells; while BMSCs tend to favor osteogenesis, SHEDs appear to adopt a more perivascular or pericytic behavior.

Increasing evidences demonstrate the role of sensory innervation in bone metabolism, remodeling and repair, however neurovascular coupling in bone is rarely studied. Using microfluidic devices as an indirect co-culture model to mimic in vitro the physiological scenario of innervation, our group demonstrated that sensory neurons (SNs) were able to regulate the extracellular matrix remodeling by endothelial cells (ECs), in particular through sensory neuropeptides, i.e. calcitonin gene-related peptide (CGRP) and substance P (SP). Nonetheless, still little is known about the cell signaling pathways and mechanism of action in neurovascular coupling. Here, in order to characterize the communication between SNs and ECs at molecular level, we evaluated the effect of SNs and the neuropeptides CGRP and SP on ECs. We focused on different pathways known to play a role on endothelial functions: calcium signaling, p38 and Erk1/2; the control of signal propagation through Cx43; and endothelial functions through the production of nitric oxide (NO). The effect of SNs was evaluated on ECs Ca²⁺ influx, the expression of Cx43, endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, p38, ERK1/2 as well as their phosphorylated forms. In addition, the role of CGRP and SP were either analyzed using respective antagonists in the co-culture model, or by adding directly on the ECs monocultures. We show that capsaicin-stimulated SNs induce increased Ca²⁺ influx in ECs. SNs stimulate the increase of NO production in ECs, probably involving a decrease in the inhibitory eNOS T495 phosphorylation site. The neuropeptide CGRP, produced by SNs, seems to be one of the mediators of this effect in ECs since NO production is decreased in the presence of CGRP antagonist in the co-culture of ECs and SNs, and increased when ECs are stimulated with synthetic CGRP. Taken together, our results suggest that SNs play an important role in the control of the endothelial cell functions through CGRP production and NO signaling pathway.

Despite progress in bone tissue engineering, reconstruction of large bone defects remains an important clinical challenge. Here, a biomaterial designed to recruit bone cells, endothelial cells, and neuronal fibers within the same matrix is developed, enabling bone tissue regeneration. The bioactive matrix is based on modified elastin‐like polypeptides (ELPs) grafted with laminin‐derived adhesion peptides IKVAV and YIGSR, and the SNA15 peptide for retention of hydroxyapatite (HA) particles. The composite matrix shows suitable porosity, interconnectivity, biocompatibility for endothelial cells, and the ability to support neurites outgrowth by sensory neurons. Subcutaneous implantation leads to the formation of osteoid tissue, characterized by the presence of bone cells, vascular networks, and neuronal structures, while minimizing inflammation. Using a rat femoral condyle defect model, longitudinal micro‐CT analysis is performed, which demonstrates a significant increase in the volume of mineralized tissue when using the ELP‐based matrix compared to empty defects and a commercially available control (Collapat). Furthermore, visible blood vessel networks and nerve fibers are observed within the lesions after a period of two weeks. By incorporating multiple key components that support cell growth, mineralization, and tissue integration, this ELP‐based composite matrix provides a holistic and versatile solution to enhance bone tissue regeneration.

Mesenchymal stem cells present clinical potential to recover and regenerate injured tissues in diverse pathologies. The in vitro expansion and characterization of these cells contribute to elucidation of the mechanisms of senescence and strategies involving cell therapies. This study aimed to compare specific characteristics between initial and advanced passages of mesenchymal stem cells derived from adipose tissue and bone marrow. Both cell types were characterized according to immunophenotype, osteogenic differentiation, genomic instability, migration assay, doubling population time and colony forming ability. Our results demonstrated that both cell types were able to maintain an immunophenotypic profile typical of mesenchymal stem cells during increasing passages. Adipose stem cells at initial passage presented greater migration capacity compared to advanced passage cells, and advanced passage cells proliferated faster than initial passage cells. Bone marrow stem cells at early passages presented higher osteogenic potential than advanced. At advanced passages they presented higher colony forming capacity and genetic damage than those at initial passage. These results suggest that mesenchymal stem cells maintained in culture presented characteristics of senescence that should be monitored prior the use in regenerative medicine and cells derived from bone marrow at initial passage have better potential for therapeutic use in bone tissue engineering.