Impact of isolation procedures on the development of a preclinical synovial fibroblasts/macrophages in an in vitro model of osteoarthritis

There is a lack of in vitro models able to properly represent osteoarthritis (OA) synovial tissue (ST). We aimed to characterize OA ST and to investigate whether a mechanical or enzymatic digestion procedures influence synovial cell functional heterogeneity in vitro. Procedures using mechanical nondigested fragments (NDF), synovial digested fragments (SDF), and filtrated synovial digested cells (SDC) were compared. An immunophenotypic profile was performed to distinguish synovial fibroblasts (CD55, CD73, CD90, CD106), macrophages (CD14, CD68), M1-like (CD80, CD86), and M2-like (CD163, CD206) synovial macrophages. Pro-inflammatory (interleukin 6 IL6), tumor necrosis factor alpha (TNFα), chemokine C-C motif ligand 3 (CCL3/MIP1α), C-X- motif chemokine ligand 10 (CXCL10/IP10) and anti-inflammatory (interleukin 10 (IL10)), transforming growth factor beta 1 (TGFβ1), C-C motif chemokine ligand 18 (CCL18) cytokines were evaluated. CD68 and CD163 markers were higher in NDF and SDF compared to the SDC procedure, while CD80, CD86, and CD206 were higher only in NDF compared to the SDC procedure. Synovial fibroblast markers showed similar percentages. TNFα, CCL3/MIP1α, CXCL10/IP10, and CCL18 were higher in NDF compared to SDC, but not compared to SDF. IL10 and TGFβ1 were higher in NDF than SDC at the molecular level, while IL6 did not show differences among procedures. We demonstrated that NDF isolation procedures better preserved the heterogeneity of specific OA synovial populations (fibroblasts, macrophages), fostering their use for testing new cell therapies or drugs for OA, reducing or avoiding the use of animal models

Evidence for redifferentiation of human chondrocytes grown on a hyaluronan-based biomaterial (HYAFF11): Molecular, Immunohistochemical and ultrastructural analysis.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. Such scaffolds should provide a preformed three-dimensional shape and prevent cells from escaping into the articular cavity. Furthermore, these constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and their phenotype redifferentiation. The aim of this study was to verify the ability of HYAFF®11, a recently developed hyaluronic-acid-based biodegradable polymer, to support the growth of human chondrocytes and to maintain their original phenotype. This capability was assessed by the evaluation of collagen types I, II and aggrecan mRNA expression. Immunohistochemical analyses were also performed to evaluate collagen types I, II and proteoglycans synthesis. A field emission in lens scanning microscopy was utilized to verify the interactions between the cells and the biomaterial. Our data indicate that human chondrocytes seeded on HYAFF®11 express and produce collagen type II and aggrecan and downregulate the production of collagen type I. These results provide an in vitro demonstration for the therapeutic potential of HYAFF®11 as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects

Mesenchymal stromal cells from a progressive pseudorheumatoid dysplasia patient show altered osteogenic differentiation

Background: Progressive pseudorheumatoid dysplasia (PPRD) is a rare autosomal recessive non-inflammatory skeletal disease with childhood onset and is characterized by a progressive chondropathy in multiple joints, and skeletal abnormalities. To date, the etiopathological relationship between biological modification occurring in PPRD and genetic mutation remains an open issue, partially due to the limited availability of biological samples obtained from PPRD patients for experimental studies. Case presentation: We describe the clinical features of a PPRD patient and experimental results obtained from the biological characterization of PPRD mesenchymal stromal cells (MSCs) and osteoblasts (OBs) compared to normal cell populations. Phenotypic profile modifications were found in PPRD compared to normal subjects, essentially ascribed to decreased expression of CD146, osteocalcin (OC) and bone sialoprotein in PPRD MSCs and enhanced CD146, OC and collagen type I expression in PPRD OBs. Gene expression of Dickkopf-1, a master inhibitor of WNT signaling, was remarkably increased in PPRD MSCs compared to normal expression range, whereas PPRD OBs essentially exhibited higher OC gene expression levels. PPRD MSCs failed to efficiently differentiate into mature OBs, so showing a greatly impaired osteogenic potential. Conclusions: Since all regenerative processes require stem cell reservoirs, compromised functionality of MSCs may lead to an imbalance in bone homeostasis, suggesting a potential role of MSCs in the pathological mechanisms of PPRD caused by WNT1-inducible signaling pathway protein-3 (WISP3) mutations. In consideration of the lack of compounds with proven efficacy in such a rare disease, these data might contribute to better identify new specific and effective therapeutic approaches

Co-culture of hematopoietic stem/progenitor cells with human osteblasts favours mono/macrophage differentiation at the expense of the erythroid lineage

Hematopoietic stem cells (HSCs) are located in the bone marrow in a specific microenvironment referred as the hematopoietic stem cell niche, where HSCs interact with a variety of stromal cells. Though several components of the stem cell niche have been identified, the regulatory mechanisms through which such components regulate the stem cell fate are still unknown. In order to address this issue, we investigated how osteoblasts (OBs) can affect the molecular and functional phenotype of Hematopoietic Stem/Progenitor Cells (HSPCs) and vice versa. For this purpose, human CD34+ cells were cultured in direct contact with primary human OBs. Our data showed that CD34+ cells cultured with OBs give rise to higher total cell numbers, produce more CFUs and maintain a higher percentage of CD34+CD38- cells compared to control culture. Moreover, clonogenic assay and long-term culture results showed that co-culture with OBs induces a strong increase in mono/macrophage precursors coupled to a decrease in the erythroid ones. Finally, gene expression profiling (GEP) allowed us to study which signalling pathways were activated in the hematopoietic cell fraction and in the stromal cell compartment after coculture. Such analysis allowed us to identify several cytokine-receptor networks, such as WNT pathway, and transcription factors, as TWIST1 and FOXC1, that could be activated by co-culture with OBs and could be responsible for the biological effects reported above. Altogether our results indicate that OBs are able to affect HPSCs on 2 different levels: on one side, they increase the immature progenitor pool in vitro, on the other side, they favor the expansion of the mono/macrophage precursors at the expense of the erythroid lineage

Development and validation of low-intensity pulsed ultrasound systems for highly controlled in vitro cell stimulation

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500 kHz to 5 MHz) and for a low- frequency value (38 kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3 min. Time-domain acoustic simula- tions allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the trans- ducer’s input voltage. Biological in vitro tests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven day

Development and validation of low-intensity pulsed ultrasound systems for highly controlled in vitro cell stimulation

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500 kHz to 5 MHz) and for a low-frequency value (38 kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3 min. Time-domain acoustic simulations allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the transducer's input voltage. Biological in vitro tests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven days

Ultrasound Stimulation of Piezoelectric Nanocomposite Hydrogels Boosts Chondrogenic Differentiation in Vitro, in Both a Normal and Inflammatory Milieu

The use of piezoelectric nanomaterials combined with ultrasound stimulation is emerging as a promising approach for wirelessly triggering the regeneration of different tissue types. However, it has never been explored for boosting chondrogenesis. Furthermore, the ultrasound stimulation parameters used are often not adequately controlled. In this study, we show that adipose-tissue-derived mesenchymal stromal cells embedded in a nanocomposite hydrogel containing piezoelectric barium titanate nanoparticles and graphene oxide nanoflakes and stimulated with ultrasound waves with precisely controlled parameters (1 MHz and 250 mW/cm2, for 5 min once every 2 days for 10 days) dramatically boost chondrogenic cell commitment in vitro. Moreover, fibrotic and catabolic factors are strongly down-modulated: proteomic analyses reveal that such stimulation influences biological processes involved in cytoskeleton and extracellular matrix organization, collagen fibril organization, and metabolic processes. The optimal stimulation regimen also has a considerable anti-inflammatory effect and keeps its ability to boost chondrogenesis in vitro, even in an inflammatory milieu. An analytical model to predict the voltage generated by piezoelectric nanoparticles invested by ultrasound waves is proposed, together with a computational tool that takes into consideration nanoparticle clustering within the cell vacuoles and predicts the electric field streamline distribution in the cell cytoplasm. The proposed nanocomposite hydrogel shows good injectability and adhesion to the cartilage tissue ex vivo, as well as excellent biocompatibility in vivo, according to ISO 10993. Future perspectives will involve preclinical testing of this paradigm for cartilage regeneration