Biofabrication and Bone Tissue Regeneration: Cell Source, Approaches, and Challenges

The growing occurrence of bone disorders and the increase in aging population have resulted in the need for more effective therapies to meet this request. Bone tissue engineering strategies, by combining biomaterials, cells, and signaling factors, are seen as alternatives to conventional bone grafts for repairing or rebuilding bone defects. Indeed, skeletal tissue engineering has not yet achieved full translation into clinical practice because of several challenges. Bone biofabrication by additive manufacturing techniques may represent a possible solution, with its intrinsic capability for accuracy, reproducibility, and customization of scaffolds as well as cell and signaling molecule delivery. This review examines the existing research in bone biofabrication and the appropriate cells and factors selection for successful bone regeneration as well as limitations affecting these approaches. Challenges that need to be tackled with the highest priority are the obtainment of appropriate vascularized scaffolds with an accurate spatiotemporal biochemical and mechanical stimuli release, in order to improve osseointegration as well as osteogenesis

mRNAs and miRNAs profiling of Mesenchymal Stem Cells derived from amniotic fluid and skin

Mesenchymal Stem Cells (MSCs) may be isolated from different adult sources and even if the minimal criteria for defining MSCs have been reported, the scientific question about the potential distinctions among MSCs derived from different sources is still opened. In particular, it is debated if MSCs of different origin have the same grade of stemness or if the source affects their undifferentiated status. Here we report not only the isolation and the traditional characterization of MSCs derived from amniotic fluid (AF-MSCs) [1] and skin (S-MSCs) [2], but also a molecular characterization based on mRNAs and miRNAs profiling. Our results show that even if both AF- and S-MSCs are regulated by the same pathways (such as Wnt, MAPK and TGF-β), there is a fine and different control of them as suggested by altered levels of expression of some member of these pathways. In conclusion, it will be necessary to improve the knowledge about the role of each dysregulated miRs/gene because, actually, these differences may strengthen the question about the importance of tissue origin. This work was supported by grant FIRB-RBAP10MLK7_003 from Ministero dell’Istruzione, dell’Università e della Ricerca, Rome, Ital

Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque

BACKGROUND: The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. RESULTS: A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of ~42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. CONCLUSION: This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme

Expression of CD38 in human neuroblastoma SH-SY5Y cells.

Human CD38 antigen is a 42–45 kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular region. It is widely expressed in different cell types including thymocytes, activated T cells, and terminally differentiated B cells (plasma cells) and it is involved in cellular proliferation and adhesion. CD38 acts as an ectocyclase that converts NAD+ to the Ca2+-releasing second messenger cyclic ADP-ribose (cADPR). It has been also demonstrated that increased extracellular levels of NAD+ and cADPR are involved in inflammatory diseases and in cellular damage, such as ischemia. In the present study, we have characterized the expression of CD38 in human neuroblastoma SH-SY5Y cell line. All-trans-retinoic acid (ATRA) treatment was used to induce cell differentiation. Our results indicate that: a) even if SH-SY5Y cells have a negative phenotype express CD38 at nuclear level, ATRA treatment does not influence this pattern; b) CD38 localizing to the nucleus may co-localize with p80-coilin positive nuclear-coiled bodies; c) purified nuclei, by Western blot determinations using anti-CD38 antibodies, display a band with a molecular mass of −42 kDa; d) SH-SY5Y cells show nuclear ADP-ribosyl cyclase due to CD38 activity; e) the basal level of CD38 mRNA shows a time-dependent increase after treatment with ATRA. These results suggest that the presence of constitutive fully functional CD38 in the SH-SY5Y nucleus has some important implications for intracellular generation of cADP-ribose and subsequent nucleoplasmic calcium release

Isolation and characterization of Mesenchymal Stem Cells from pituitary tumours

In the past few years the introduction of the cancer stem cells (CSCs) notion opened new perspectives for the diagnosis and cure of solid tumors. According to this theory, CSCs originate from mutated stem cells, maintaining the self-renewal and differentiative abilities. Therefore, the development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients. Actually, no informations are available about stem cells and cancer stem cells on pituitary tumours. This work depicts some essential features of stem cells isolated from pituitary adenomas. Six tumour biopsies (3: GH-secreting; 3: non secreting) were collected and cultured with a specific culture medium. Cell growth and morphology were monitored and cells were subjected to analyses for stemness determination (immunophenotype, gene expression and differentiative potential) [1, 2] and GH secretion. Cells showed a stem-like immunophenotype, the expression of Oct-4, Sox-2, Nanog and Klf-4 and the ability to differentiate towards osteogenic, chondrogenic and adipogenic lineages. The hormone secretion ended after two weeks culturing. Even if further studies are needed for the fully comprehension of the specific nature of these cells and on their role on tumour onset and maintenance, this study opens to the possibility of isolation of stem cells from pituitary tumour, allowing a molecular targeting of it. This work was supported by grant FIRB-RBAP10MLK7_003 from Ministero dell’Istruzione, dell’Università e della Ricerca, Rome, Ital

The inflamed microenvironment: role on MSCs immunobiology and cancer

Inflammation and cancer are an inseparable binomial. The majority of cancers are triggered by somatic mutations and environmental factors with a common element: inflammation. Inflammation creates a microenvironment in which neoplastic cells can profit from the trophic factors secreted by inflammatory cells, useful to interfere with the anti-tumor response. Among the others, mesenchymal stem cells (MSCs) participate to microenvironment creation by a strong paracrine effect. The linkage between MSCs and inflammation is bidirectional: the inflamed microenvironment affects the complex MSCs immunobiology, but also MSCs can sustain inflammation. Here, we tried to clarify the influence of inflammation on the immunobiology of MSCs and deepen the paracrine effect of MSCs on tumor growth. MSCs were isolated from periprosthetic capsule caused by breast implant, affected by inflammation (I-MSCs). The contralateral part of the same patient, not inflamed, was used as control (C-MSCs). A panel of selected cytokines were analyzed by Real-Time PCR and ELISA. The cytokines expression was different in I-MSCs compared to C-MSCs, revealing that inflammation affects MSCs immunobiology. Then, C- and I-MSCs were indirectly co-cultured with MCF7 cells from breast adenocarcinoma. New analyses on proliferation rate and cytokines expression were performed. C- and I-MSCs gave almost the same results. The over-secretion of all the cytokines referred to the Th1 pathway and the decrease of those belonging to the Th2 pathway revealed the absence of a switch from Th1 to Th2 important to induce a chronic inflammation. The levels of TGF-β and G-CSF linked to the skill to damage the antigen-presenting cell function were decreased. In conclusion, even if MCF-7 proliferation increased after co-culture with I-MSCs, MSCs-derived paracrine effect does not sustain breast adenocarcinoma. These results absolve the breast implants from the insult to enhance adenocarcinoma onset

The role of the mesenchymal stem cells on breast cancer: friends or foes?

Mesenchymal stem cells (MSCs) have been the subject of an increased interest. Because of their ability to give rise to bone, cartilage, fat and muscle, their role in regenerative medicine has been extensively studied and the fact that they can be recruited at sites of inflammation and tissue repair has prompted their potential use as tissue regenerative cells. Contextually there has been a growing interest in the role of MSCs in cancer progression. The nature of the relationship between MSCs and tumor cells appears dual, with effects pro- as well as anti-tumorigenic. This paradox depends on the source and the degree of differentiation of MSCs and the tumor model used. Moreover, with the large range of cytokines and growth factors they produce, MSCs exert regulatory function on apoptosis, angiogenesis and an immunomodulatory role. Here we evaluate the interaction between MSCs derived from the periprosthetic capsule of mastectomyzed women and the breast cancer cell line MCF-7. Capsular tissue around breast implants is a normal inflammatory reaction versus a foreign body and it is rich of MSCs. To asses how MSCs interact with tumor cells, MCF-7 cells were incubated with medium previously conditioned by MSCs or directly co-coltured with MSCs; subsequently, we evaluated the proliferation and the expression of genes implicated in different pathways (angiogenesis, proliferation, anti-apoptosis, EMT transition). Our results showed that MCF-7 cells cultured together MSCs or using their conditioned medium have a more elevated proliferation rate but tumour cells seem less aggressive, like attested by a reduction of the expression of selected genes. The understanding of the mechanisms that control the interaction between MSCs and tumor cells is still at an early stage but recent literature confirm that MSCs and their progeny are not innocent bystanders in the tumor microenvironment. The study of these interactions is a critical area of future investigation that is needed to better define their role in cancer progression and their potential as therapeutic agents or targets

MSCs and inflammation: not only a guardian role

  The literature on the relation between mesenchymal stem cells (MSCs) and inflammation is continuing to expand at a rapid rate with over 600 entries in PubMed under “MSCs and inflammation” starting from 2002. Inflammation is an essential part of the malignant microenvironment. Chemokines, leukocyte infiltration and cytokines are crucial elements, which contribute to cancer-related inflammation. Attracted by chemokines, MSCs are recruited at injury sites. After exposure to inflammatory factors in the local microenvironment, MSCs secrete several cytokines and vascular endothelial growth factor, which promote immunosuppression, angiogenesis and tumor growth. Here we compare by RT-PCR the expression of selected genes, related to inflammation, on MSCs derived from control (C-MSCs) and inflamed tissues (I-MSCs). First of all, an immunohistochemistry using anti-CD43 antibody was performed to better test the status of inflammation at the moment of tissues’ collection. CD43 is known as marker of inflammation, since it is expressed by most T cells, activated B cells, basophils, macrophages, monocytes and NK cells. Its expression was absent in “control” tissues, while it was strong in the “inflamed”. Subsequently, RNA was extracted, retro-transcribed and used for quantitative PCR. The genes were selected according to their role in inflammation: IL6 and IL8 (known as pro-inflammatory interleukins), TNFα (involved in systemic inflammation), CXCL2 (secreted by monocytes and macrophages and is chemotactic for polymorphonuclear leukocytes), CCL20 (strongly chemotactic for lymphocytes, its expression is induced by inflammatory cytokines), IFNγ (an important activator of macrophages) and TGFβ1 (promoting immunosuppression). Quantification of mRNA expression was calculated with the 2−ΔΔCt method, where ΔCt = Ct (gene of interest) − Ct (control gene) and Δ(ΔCt) = ΔCt (I-MSCs) − ΔCt (C-MSCs). The results revealed that the expression of all tested genes was higher in MSCs derived from inflamed tissues than in MSCs from control tissues (expressed as 1). This study underlines how MSCs are not inert guardians on inflammation, but as they play an active role

Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials

Bone marrow is a useful cell source for skeletal tissue engineering approaches. In vitro differentiation of marrow mesenchymal stem cells (MSCs) to chondrocytes or osteoblasts can be induced by the addition of specific growth factors to the medium. The present study evaluated the behaviour of human MSCs cultured on various scaffolds to determine whether their differentiation can be induced by cell-matrix interactions. MSCs from bone marrow collected from the acetabulum during hip arthroplasty procedures were isolated by cell sorting, expanded and characterised by a flow cytometry system. Cells were grown on three different scaffolds (type I collagen, type I + II collagen and type I collagen + hydroxyapatite membranes) and analysed by histochemistry, immunohistochemistry and spectrophotometry (cell proliferation, alkaline phosphatase activity) at 15 and 30 days. Widely variable cell adhesion and proliferation was observed on the three scaffolds. MSCs grown on type I+II collagen differentiated to cells expressing chondrocyte markers, while those grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells. The study highlighted that human MSCs grown on different scaffold matrices may display different behaviours in terms of cell proliferation and phenotype expression without growth factor supplementation

The sign of psoriasis in mesenchymal stem cells of the skin

Psoriasis is a chronic inflammatory and immune-mediated skin disease, characterized by epidermal hyperproliferation, abnormal keratinocyte differentiation and angiogenesis, whose skin lesions are promoted by exogenous and endogenous factors. The cutaneous and systemic over-expression of several pro-inflammatory cytokines, observed in the initiation, maintenance and recurrence of skin lesions, is known to be caused also by reactive oxygen species (ROS). For this reason it has been postulated that ROS production and compromised function of antioxidant system may be involved in the pathogenesis of psoriasis. Some typical features of psoriasis, like growth rate, expression of VEGF and iNOS as well as the production of VEGF and nitric oxide (NO) and some antioxidant responses, have already been extensively evaluated in differentiated cells of psoriatic skin, but no indications are still available about, the mesenchymal stem, that may be isolated from skin (S-MSCs)