Calcium/Cobalt Alginate Beads as Functional Scaffolds for Cartilage Tissue Engineering

Articular cartilage is a highly organized tissue with complex biomechanical properties. However, injuries to the cartilage usually lead to numerous health concerns and often culminate in disabling symptoms, due to the poor intrinsic capacity of this tissue for self-healing. Although various approaches are proposed for the regeneration of cartilage, its repair still represents an enormous challenge for orthopedic surgeons. The field of tissue engineering currently offers some of the most promising strategies for cartilage restoration, in which assorted biomaterials and cell-based therapies are combined to develop new therapeutic regimens for tissue replacement. The current study describes the in vitro behavior of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated within calcium/cobalt (Ca/Co) alginate beads. These novel chondrogenesis-promoting scaffolds take advantage of the synergy between the alginate matrix and Co+2 ions, without employing costly growth factors (e.g., transforming growth factor betas (TGF-βs) or bone morphogenetic proteins (BMPs)) to direct hADSC differentiation into cartilage-producing chondrocytes

Implication of Cellular Senescence in Osteoarthritis: A Study on Equine Synovial Fluid Mesenchymal Stromal Cells

: Osteoarthritis (OA) is described as a chronic degenerative disease characterized by the loss of articular cartilage. Senescence is a natural cellular response to stressors. Beneficial in certain conditions, the accumulation of senescent cells has been implicated in the pathophysiology of many diseases associated with aging. Recently, it has been demonstrated that mesenchymal stem/stromal cells isolated from OA patients contain many senescent cells that inhibit cartilage regeneration. However, the link between cellular senescence in MSCs and OA progression is still debated. In this study, we aim to characterize and compare synovial fluid MSCs (sf-MSCs), isolated from OA joints, with healthy sf-MSCs, investigating the senescence hallmarks and how this state could affect cartilage repair. Sf-MSCs were isolated from tibiotarsal joints of healthy and diseased horses with an established diagnosis of OA with an age ranging from 8 to 14 years. Cells were cultured in vitro and characterized for cell proliferation assay, cell cycle analysis, ROS detection assay, ultrastructure analysis, and the expression of senescent markers. To evaluate the influence of senescence on chondrogenic differentiation, OA sf-MSCs were stimulated in vitro for up to 21 days with chondrogenic factors, and the expression of chondrogenic markers was compared with healthy sf-MSCs. Our findings demonstrated the presence of senescent sf-MSCs in OA joints with impaired chondrogenic differentiation abilities, which could have a potential influence on OA progression

The hypoxia-mimetic agent cobalt chloride differently affects human mesenchymal stem cells in their chondrogenic potential

Adult stem cells are a promising cell source for cartilage regeneration. They resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Cobalt chloride (CoCl2) imitates hypoxia in vitro by stabilizing hypoxia-inducible factor-alpha (HIF-1\ue1), which is the master regulator in the cellular adaptive response to hypoxia. In this study, the influence of CoCl2 on the chondrogenic potential of human MSCs, isolated from dental pulp, umbilical cord, and adipose tissue, was investigated. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 \uecM. Cell viability, HIF-1\ue1 protein synthesis, and the expression of the chondrogenic markers were analyzed. The results showed that the CoCl2 supplementation had no effect on cell viability, while the upregulation of chondrogenic markers such as SOX9, COL2A1, VCAN, and ACAN was dependent on the cellular source. This study shows that hypoxia, induced by CoCl2 treatment, can differently influence the behavior of MSCs, isolated from different sources, in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies

The Hypoxia-Mimetic Agent Cobalt Chloride Differently Affects Human Mesenchymal Stem Cells in Their Chondrogenic Potential

Adult stem cells are a promising cell source for cartilage regeneration. They resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Cobalt chloride (CoCl2) imitates hypoxia in vitro by stabilizing hypoxia-inducible factor-alpha (HIF-1α), which is the master regulator in the cellular adaptive response to hypoxia. In this study, the influence of CoCl2 on the chondrogenic potential of human MSCs, isolated from dental pulp, umbilical cord, and adipose tissue, was investigated. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 μM. Cell viability, HIF-1α protein synthesis, and the expression of the chondrogenic markers were analyzed. The results showed that the CoCl2 supplementation had no effect on cell viability, while the upregulation of chondrogenic markers such as SOX9, COL2A1, VCAN, and ACAN was dependent on the cellular source. This study shows that hypoxia, induced by CoCl2 treatment, can differently influence the behavior of MSCs, isolated from different sources, in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies

Synovium-derived stromal cell-induced osteoclastogenesis: a potential osteoarthritis trigger

none7noPurpose: To shed light on the idea that mesenchymal stem/stromal cells (MSCs) recruited in synovium (SM) (i.e. Synovium-Derived Stromal Cells, SDSCs) could be involved in Osteoarthritis (OA) pathophysiology. Attention was also paid to a further stromal cell type with a peculiar ultrastructure called telocytes (TCs), whose role is far from clarified. Methods: In the present in vitro study, we compared SDSCs isolated from healthy and OA subjects in terms of phenotype, morphology and differentiation potential as well as in their capability to activate normal Peripheral Blood Mononuclear Cells (PBMCs). Histological, immunohistochemical and ultrastructural analyses were integrated by qRT-PCR and functional resorbing assays. Results: Our data demonstrated that both SDSC populations stimulated the formation of osteoclasts from PBMCs: the osteoclast-like cells generated by healthy-SDSCs via transwell co-cultures were inactive, while OA-derived SDSCs have a much greater effectiveness. Moreover, the presence of TCs was more evident in cultures obtained from OA subjects and suggests a possible involvement of these cells in OA. Conclusions: Osteoclastogenic differentiation capability of PBMCs from OA subjects, also induced by B synoviocytes has been already documented. Here we hypothesized that SDSCs, generally considered for their regenerative potential in cartilage lesions, have also a role in the onset/maintenance of OA. Clinical relevance: Our observations may represent an interesting opportunity for the development of a holistic approach for OA treatment, that considers the multifaceted capability of MSCs in relation to the environment.embargoed_20210517Dicarlo, Manuela; Teti, Gabriella; Cerqueni, Giorgia; Iezzi, Iolanda; Gigante, Antonio; Falconi, Mirella; Mattioli-Belmonte, MonicaDicarlo, Manuela; Teti, Gabriella; Cerqueni, Giorgia; Iezzi, Iolanda; Gigante, Antonio; Falconi, Mirella; Mattioli-Belmonte, Monic

The open body: a “new” book

At the beginning of the ‘300, Mondino de’ Liuzzi, a physician from Bologna, was the first anatomist who started again the dissection of human body neglected from the III century. He hinted at the existence of the conflict between book and body, between “auctoritas” and the direct observation of the human body . The Mondino’s masterwork “Anothomia” remained the key book up to the middle of the sixth century, when Andrea Vesalio wrote “De Umani Corporis Fabrica,” in which the body (cadaver) eventually became the main player of the book . During the years, the technologic evolution led to the wrong conviction that dissection could be dismissed, albeit, still in our day, doctors in training feel the need to associate the direct experience on the cadaver with the very valuable digital means and the modern imaging technologies even in 3D. Thinking to Anatomy as an already fully well known discipline is a mistake. The most advanced methodologies for surgical access, namely the minimally invasive surgery, require the evolution of the traditional anatomical knowledge. The Human Anatomy Institute of the University of Bologna, among the first in Italy, has recognized this need. Thanks to the generosity of the people enrolled in the Body donation programme for research and teaching, our Institute allows medical students to practice dissection on cadavers, beginning as Freshman, then Sophomore, Junior and Senior. The sharing of Bologna’s experience could be the chance to think about the perspectives offered by the dissection of the corpse: a wide range of possibilities spanning from research projects to advanced training courses in collaboration with clinicians and surgeons belonging to different branches. Moreover the practice of corpse dissection is extremely important for the recruitment of young graduates in Medicine which, by means of the experience vested acting as “tutor of anatomy”, acquire interest in the field of research of morphological sciences, spanning from macroscopic up to the cellular and molecular level. Hic mors gaudet succurrere vitae: the motto, reported in dissection room of most of the Italian anatomical institutes, represents the synthesis of the experience of an ancient discipline which, nowadays , has the chance to rewrite a new chapter dedicated to modern frontiers of scientific research and medical education

Increased MG-63s invasion potential mediated by HFs

During a malignant transformation, the crosstalk between the stroma and the cancer cells is described as a growing network of physical and paracrine signals, and it seems to have a direct influence on the phenotypic, genetic and epigenetic changes that affect the cells (1). In order to invade and metastasize to distant tissues, cancer cells transform themselves via ECM, induce tumor angiogenesis as well as undergo proliferation, detachment, migration, and invasion through secretion of various tumor derived factors (2). In this study we decided to analyze morphological and molecular aspects due to the coexistence between tumor cells MG-63s and fibroblasts HFs, verifying in particular the ability of MG-63s of invasion and microenvironment modulation. Monolayers of co-cultured cells were morphologically analyzed in time-laps by HR-SEM microscopy and a trans-well migration assay was performed over 24 h, 48 h, 72 h, and 96 h. The expression of several proteins, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis (TNF alpha, IL-6, YKL-40, MMP-1, MMP-9, and VEGF) was validated by Western blotting analysis. The images in time-laps for HR - SEM showed that fibroblasts in contact with MG-63 lost their spatial orientation, while the MG-63 quickly reached the confluence advancing towards HF cells, invading their space and overlying them. The increased MG-63s invasion mediated by the coexistence with HFs was confirmed by invasion assays in transwell co-culture. The protein levels of TNF-alpha, IL-6, YKL-40 and VEGF confirmed that tumor cells can regulate the development of a “tumor-stroma” via the aberrant expression of growth factors in the stromal compartment. Our results showed how tumor-stroma interactions play a significant role in tumor development and progression

A synergic effect of alginate and hypoxia-inducing ions on chondrogenic differentiation in adipose derived mesenchymal stem cells

Cartilage is a highly organized tissue with complex biomechanical properties, but since it has a poor intrinsic capacity of self-healing, injuries at this site usually lead to several problems, often ending in disabling symptoms. Although, different approach- es have been proposed, even now cartilage repair represents a great challenge for orthopaedic surgeons (1, 2). One of the promising approach is given from tissue engineering, employing the combination of biomaterials and cell therapy to develop new therapeutic strategies. In this paper, we describe the behaviour of human adipose derived mesenchymal stem cells encapsulated into Ca/Co alginate beads as potential chondrogenic inducing biomaterial tacking advance on the synergy between alginate matrix and Co+2 ions without employing other expensive growth factors such as TGFbs or BMPs. Cells were cultured up to 3 weeks into alginate beads at different Ca/Co ratio, Calcein/Ethidium assay was performed to evaluate cell viability, light, and transmis- sion electron microscopy were carried out to check the cells behaviour. The expression of chondrogenic markers such as sox9, collagen type II, and versican was investigated by Real Time PCR. The expression of hif1mRNA was investigated to check the capability of Co+2 ions to induce a chemical hypoxia. Results showed an high cell viability at high Ca/Co ratio value of alginate beads. Real Time PCR data reveal a different cells behaviour on chondrogenic marker expression. In conclusion, the synergic effect of alginate and Co+2 ions can represent a valid strategy for chondrogenic differentiation of stem cells

Assessment of the structural and functional characteristics of human mesenchymal stem cells associated with a prolonged exposure of morphine

The discovery of the expression of opioid receptors in the skin and their role in orchestrating the process of tissue repair gave rise to questions regarding the potential effects of clinical morphine treatment in wound healing. Although short term treatment was reported to improve tissue regeneration, in vivo chronic administration was associated to an impairment of the physiological healing process and systemic fibrosis. Human mesenchymal stem cells (hMSCs) play a fundamental role in tissue regeneration. In this regard, acute morphine exposition was recently reported to impact negatively on the functional characteristics of hMSCs, but little is currently known about its long-term effects. To determine how a prolonged treatment could impair their functional characteristics, we exposed hMSCs to increasing morphine concentrations respectively for nine and eighteen days, evaluating in particular the fibrogenic potential exerted by the long-term exposition. Our results showed a time dependent cell viability decline, and conditions compatible with a cellular senescent state. Ultrastructural and protein expression analysis were indicative of increased autophagy, suggesting a relation to a detoxification activity. In addition, the enhanced transcription observed for the genes involved in the synthesis and regulation of type I collagen suggested the possibility that a prolonged morphine treatment might exert its fibrotic potential risk, even involving the hMSCs