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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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

A biomimetic hyaluronic acid (HA)-based polymer scaffold was analysed in vitro for its characteristics and potential to support mineralization as carrier-vehicle. Biomimetic apatite crystal nucleation on the scaffold surface was obtained by a fine control of the pH level that increased ionic solubility thus controlling apatite formation kinetic. Different concentrations of human mesenchymal stromal cells (h-MSCs) were seeded on the scaffold, osteogenesis was induced in the presence or absence of fibroblast growth factor -2 and mineralization was analysed at different time points. We found that only at the highest h-MSCs concentration tested, the cells were uniformly distributed inside and outside the scaffold and proliferation started to decrease from day 7. Electron microscopy analysis evidenced that h-MSCs produced extracellular matrix but did not establish a direct contact with the scaffold. We found mineralized calcium-positive areas mainly present along the backbone of the scaffold starting from day 21 and increasing at day 35. FGF-2 treatment did not accelerate or increase mineralization. Non-biomimetic HA-based control scaffold showed immature mineralized areas only at day 35. Our data demonstrate that the biomimetic treatment of an HA-based scaffold promotes a faster mineralization process suggesting its possible use in clinics as a support for improving bone repair., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Mesenchymal stromal cells (MSCs) seem to be a good alternative to chondrocytes for cartilage regeneration. To obtain new information on the sequence of cellular and molecular events during in vitro chondrogenic differentiation we analysed MSCs on a widely used hyaluronic acid biomaterial (Hyaff-11). Cellular differentiation was induced using two different concentrations of TGFbeta1 (10 and 20 ng/ml) and the process was analysed at different time points (24 h, and 7, 14, 21 and 28 days) using techniques of light and electron microscopy, real-time PCR and immunohistochemistry. We found that without TGFbeta MSCs did not survive while in the presence of TGFbeta the cells significantly proliferated from day 7 until day 28. Light and electron microscopy showed that TGFbeta at 20 ng/ml better induced the formation of cartilage-like tissue. Real-time PCR showed an increased expression of collagen type II, IX and aggrecan associated to a down-regulation of collagen type I. Immunohistochemical analysis confirmed that collagen type I was down-modulated while collagen type II increased from day 14 to day 28. These data clearly showed that higher concentrations of TGFbeta (20 ng/ml) induce chondrogenesis of MSCs on Hyaff-11 scaffold better than 10 ng/ml of TGFbeta. This process is characterized by a sequence of cellular and molecular events that deal with the in vitro formation of a cartilage-like tissue.

Bone homeostasis is regulated by cells at different stages of maturation that are influenced by soluble factors. The modulatory function of two pro-inflammatory cytokines, IL-1beta and TNF-alpha, on the expression of CXCL13 chemokine was evaluated in osteoblasts (OB) and bone marrow stromal cells (BMSC) from osteoarthritis (OA) and post-traumatic (PT) patients. In basal condition, CXCL13 production by both BMSC and OB was significantly higher in OA than in PT patients. IL1beta, significantly induced CXCL13 production in differentiated OB, both from OA and PT patients, but not in BMSC from both either group. TNFalpha reduced CXCL13 production only in BMSC from OA patients. The combination of IL1beta and TNFalpha increased CXCL13 production only in OB in the same amount as for IL-1beta alone. OB from OA released a higher amount of CXCL13 compared to PT in all conditions tested. CD121a (IL1 receptor type I) was highly expressed only by OB. Moreover, in bone tissue biopsies CXCL13 was expressed by mesenchymal and mononuclear cells. This study demonstrates that cells at different stages of maturation (BMSC and OB) and derived from physiological (PT) or pathological conditions (OA) respond in different ways to inflammatory stimuli. These data may contribute to understand the basic maturation processes of bone cells in old patients.

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.

Osteogenesis of large segmental radius defects in a rat model was studied by implanting a biodegradable non-woven hyaluronic acid-based polymer scaffold (Hyaff 11) alone or in combination with bone marrow stromal cells (BMSCs). These cells had been previously grown in vitro in mineralising medium either supplemented with basic fibroblast growth factor (bFGF) or unsupplemented. The healing of bone defects was evaluated at 40, 80, 160 and 200 days and the repair process investigated by radiographic, histomorphometric (assessment of new bone growth and lamellar bone) and histological analyses (toluidine blue and von Kossa staining). Mineralisation of bone defects occurred in the presence of the Hyaff 11 scaffold alone or when combined with BMSCs grown with or without bFGF, but each process had a different timing. In particular, bFGF significantly induced mineralisation from day 40, whereas 160 days were necessary for direct evidence that a similar process was developing under the other two conditions tested (scaffold alone or with BMSCs). Radiographic score, new bone growth and lamellar bone percentage were highly correlated. The present outcomes were further confirmed by toluidine blue and von Kossa staining. According to these in vivo findings, the Hyaff 11 scaffold is an appropriate carrier vehicle for the repair of bone defects; additionally, it can significantly accelerate bone mineralisation in combination with BMSCs and bFGF.

A biodegradable non-woven hyaluronic acid polymer scaffold (Hyaff 11) was analysed in vitro as a carrier vehicle for differentiation and mineralization of rat bone marrow stromal cells (BMSC). BMSC were grown on Hyaff 11 in a mineralizing medium in the presence/absence of basic fibroblast growth factor (bFGF). Osteoblastic differentiation was investigated by light and electron microscopy analysing the expression of osteogenic markers: calcium, alkaline phosphatase (AP), osteopontin (OP), bone sialoprotein (BSP) and collagen type 1. We also measured proliferation, AP activity and mRNA expression of AP and osteocalcin (OC). Electron microscopy and Toluidine-blue staining demonstrated that bFGF accelerated (day 20 vs. day 40) and increased mineralization. With bFGF, calcium, OP and BSP were strongly enhanced at day 40, whereas AP decreased. Our in vitro results demonstrate that Hyaff 11 is a useful vehicle for growth, differentiation and mineralization of rat BMSC, and that it permits bone development.