Stefania Pagani

Malnutrition, anorexia and cachexia in cancer patients: A mini-review on pathogenesis and treatment

Malnutrition, anorexia and cachexia are a common finding in cancer patients. They become more evident with tumor growth and spread. However, the mechanisms by which they are sustained often arise early in the history of cancer. For malnutrition, these mechanisms can involve primary tumor or damage by specific treatment such as anticancer therapies (surgery, chemotherapy, radiotherapy) also in cancers that usually are not directly responsible for nutritional and metabolic status alterations (i.e. bone tumors). For anorexia, meal-related neural or hormonal signals and humoral signals related to body fat or energy storage and the interaction of these signals with the hypothalamus or the hypothalamic inappropriate response play a pathogenetic role. Some cytokines are probably involved in these mechanisms. For cachexia, the production of proinflammatory cytokines by tumour cells is the initial mechanism; the main biochemical mechanisms involved include the ubiquitine proteasome-dependent proteolysis and heat shock proteins. Treatment includes pharmaceutical and nutritional interventions.

In vitro tenocyte metabolism in aging and oestrogen deficiency

Little is known about tendons and tenocyte biological behaviour during aging and, especially, oestrogen deficiency. The aim of this study was to evaluate in vitro the proliferation and metabolism of tenocytes isolated from the Achilles tendons of ovariectomised (OVX), middle-aged (OLD) and young (YOUNG) rats. An in vitro model of micro-wound healing was also used to assess age and oestrogen deficiency differences in tendon healing. In standard culture condition, OLD and OVX tenocytes showed a significantly lower proliferation rate, collagen I, aggrecan and elastin than YOUNG ones. In OVX group, fibronectin and elastin significantly decreased in comparison to YOUNG and OLD groups, respectively, whereas vascular endothelial growth factor and metalloproteinases-13 increased than those of both YOUNG and OLD groups. In the micro-wound healing model, tenocytes from both OVX and OLD showed a significantly lower healing rate, proliferation rate, collagen I and nitrix oxide in comparison to YOUNG. OVX elastin value was significantly lower than YOUNG one and OVX healing rate and cell migration speed, proliferation rate and fibronectin results were lower, whereas collagen III and metalloproteinase-13 higher in comparison to both YOUNG and OLD groups. These results highlighted how aging and, more significantly, oestrogen deficiency negatively affect tendon metabolism and healing. Our work improves the body of knowledge on the effects of senescence and oestrogen deficiency on tenocyte behaviour and allows further studies to find solution for the prevention of tendon injuries in aging and menopause.

In vitro mutual interaction between tenocytes and adipose-derived mesenchymal stromal cells

BACKGROUND AIMS Tendon is a complex tissue with a reduced regenerative ability. Nowadays, little or nothing is known about the regenerative effect of adipose-derived mesenchymal stromal cells (ADSCs) on tendons. METHODS The study aimed to evaluate the in vitro mutual interaction of ADSCs and tenocytes in standard culture conditions and a microwound healing model. Tenocyte viability, microwound recovery and the expression of genes encoding for the main extracellular matrix components and ADSC viability, differentiation and growth factor gene expression were evaluated. RESULTS The effects of ADSCs on tenocytes were observed more in the microwound healing model, in which the rate of microwound healing and the expression of decorin, tenascin and collagens were significantly increased. The influence of tenocytes on ADSCs was also found in standard culture conditions: ADSCs were directed toward a tenogenic lineage, and growth factor expression increased. CONCLUSIONS This study clarifies some aspects of the mutual interaction of ADSCs and tenocytes and provides in vitro evidence for a possible future application of ADSCs as a therapeutic strategy for tendon repair.

Antiresorption implant coatings based on calcium alendronate and octacalcium phosphate deposited by matrix assisted pulsed laser evaporation

The integration of an implant material with bone tissue depends on the chemistry and physics of the implant surface. In this study we applied matrix assisted pulsed laser evaporation (MAPLE) in order to synthesize calcium alendronate monohydrate (a bisphosphonate obtained by calcium sequestration from octacalcium phosphate by alendronate) and calcium alendronate monohydrate/octacalcium phosphate composite thin films on titanium substrates. Octacalcium phosphate coatings were prepared as reference material. The powders, which were synthesized in aqueous medium, were suspended in deionised water, frozen at liquid nitrogen temperature and used as targets for MAPLE experiments. The transfer was conducted with a KrF* excimer laser source (λ = 248 nm, τFWHM ≤ 25 ns) in mild conditions of temperature and pressure. XRD, FTIR and SEM analyses confirmed that the coatings contain the same crystalline phases as the as-prepared powder samples. Osteoblast derived from stem cells and osteoclast derived from monocytes of osteoporotic subjects were co-cultured on the coatings up to 14 days. Osteoclast displayed significantly reduced proliferation and differentiation in the presence of calcium alendronate monohydrate, pointing to a clear role of the coatings containing this bisphosphonate on inhibiting excessive bone resorption. At variance, osteoblast production of alkaline phosphatase and type I pro-collagen were promoted by the presence of bisphosphonate, which also decreased the production of interleukin 6. The positive influence towards osteoblast differentiation was even more enhanced in the composite coatings, thanks to the presence of octacalcium phosphate.

Response of human chondrocytes and mesenchymal stromal cells to a decellularized human dermis

BackgroundAlthough progress has been made in the treatment of articular cartilage lesions, they are still a major challenge because current techniques do not provide satisfactory long-term outcomes. Tissue engineering and the use of functional biomaterials might be an alternative regenerative strategy and fulfill clinical needs. Decellularized extracellular matrices have generated interest as functional biologic scaffolds, but there are few studies on cartilage regeneration. The aim of this study was to evaluate in vitro the biological influence of a newly developed decellularized human dermal extracellular matrix on two human primary cultures.MethodsNormal human articular chondrocytes (NHAC-kn) and human mesenchymal stromal cells (hMSC) from healthy donors were seeded in polystyrene wells as controls (CTR), and on decellularized human dermis batches (HDM_derm) for 7 and 14 days. Cellular proliferation and differentiation, and anabolic and catabolic synthetic activity were quantified at each experimental time. Histology and scanning electron microscopy were used to evaluate morphology and ultrastructure.ResultsBoth cell cultures had a similar proliferation rate that increased significantly (p < 0.0005) at 14 days. In comparison with CTR, at 14 days NHAC-kn enhanced procollagen type II (CPII, p < 0.05) and aggrecan synthesis (p < 0.0005), whereas hMSC significantly enhanced aggrecan synthesis (p < 0.0005) and transforming growth factor-beta1 release (TGF-β1, p < 0.0005) at both experimental times. Neither inflammatory stimulus nor catabolic activity induction was observed. By comparing data of the two primary cells, NHAC-kn synthesized significantly more CPII than did hMSC at both experimental times (p < 0.005), whereas hMSC synthesized more aggrecan at 7 days (p < 0.005) and TGF-β1 at both experimental times than did NHAC-kn (p < 0.005).ConclusionsThe results obtained showed that in in vitro conditions HDM_derm behaves as a suitable scaffold for the growth of both well-differentiated chondrocytes and undifferentiated mesenchymal cells, thus ensuring a biocompatible and bioactive substrate. Further studies are mandatory to test the use of HDM_derm with tissue engineering to assess its therapeutic and functional effectiveness in cartilage regeneration.

Increased Chondrogenic Potential of Mesenchymal Cells From Adipose Tissue Versus Bone Marrow-Derived Cells in Osteoarthritic In Vitro Models.

Primarily, to compare the behavior of human mesenchymal stem cells (MSCs) derived from bone marrow (hBMSCs) and adipose tissue (hADSCs) in an osteoarthritic (OA) microenvironment; secondly, to investigate the reaction of these cell types in two alternative in vitro culture systems, obtained by using TNFα and/or IL1β as inflammation mediators, or by using synovial fluid harvested by OA patients (OSF) to simulate the complex inflamed knee microenvironment. 3D micromass cultures of hBMSCs or hADSCs were grown in chondrogenic medium (CTR), in the presence of TNFα and/or IL1β, or synovial fluid from OA patients. After 1 month of culture, the chondrogenic differentiation of micromasses was evaluated by gene expression, matrix composition, and organization. Both hMSCs types formed mature micromasses in CTR, but a better response of hADSCs to the inflammatory environment was documented by micromass area and Bern score evaluations. The addition of OSF elicited a milder reaction than with TNFα and/or IL1β by both cell types, probably due to the presence of both catabolic and protective factors. In particular, SOX9 and ACAN gene expression and GAG synthesis were more abundant in hADSCs than hBMSCs when cultured in OSF. The expression of MMP1 was increased for both hMSCs in inflammatory conditions, but in particular by hBMSCs. hADSCs showed an increased chondrogenic potential in inflammatory culture systems, suggesting a better response of hADSCs in the OA environment, thus underlining the importance of appropriate in vitro models to study MSCs and potential advantages of using these cells for future clinical applications. J. Cell. Physiol. 232: 1478–1488, 2017.

Effect of adipose-derived mesenchymal stromal cells on tendon healing in aging and estrogen deficiency: an in vitro co-culture model.

BACKGROUND AIMS Aging and estrogen deficiency play a pivotal role in reducing tenocyte proliferation, collagen turnover and extracellular matrix remodeling. Mesenchymal stromal cells are being studied as an alternative for tendon regeneration, but little is known about the molecular events of adipose-derived mesenchymal stromal cells (ADSCs) on tenocytes in tendons compromised by aging and estrogen deficiency. The present in vitro study aims to compare the potential therapeutic effects of ADSCs, harvested from healthy young (sham) and aged estrogen-deficient (OVX) subjects, for tendon healing. METHODS An indirect co-culture system was set up with ADSCs, isolated from OVX or sham rats, and tenocytes from OVX rats. Cell proliferation, healing rate and gene expression were evaluated in both a standard culture condition and a microwound-healing model. RESULTS It was observed that tenocyte proliferation, healing rate and collagen expression improved after the addition of sham ADSCs in both culture situations. OVX ADSCs also increased tenocyte proliferation and healing rate but less compared with sham ADSCs. Decorin and Tenascin C expression increased in the presence of OVX ADSCs. CONCLUSIONS Findings suggest that ADSCs might be a promising treatment for tendon regeneration in advanced age and estrogen deficiency. However, some differences between allogenic and autologous cells were found and should be investigated in further in vivo studies. It appears that allogenic ADSCs improve tenocyte proliferation, collagen expression and the healing rate more than autologous cells. Autologous cells increase collagen expression only in the absence of an injury and increase Decorin and Tenascin C more than allogenic cells.

Three-dimensional cellular distribution in polymeric scaffolds for bone regeneration: a microCT analysis compared to SEM, CLSM and DNA content

In orthopaedic surgery the tissues damaged by injury or disease could be replaced using constructs based on biocompatible materials, cells and growth factors. Scaffold design, porosity and early colonization are key components for the implant success. From biological point of view, attention may be also given to the number, type and size of seeded cells, as well as the seeding technique and cell morphological and volumetric alterations. This paper describes the use of the microCT approach (to date used principally for mineralized matrix quantification) to observe construct colonization in terms of cell localization, and make a direct comparison of the microtomographic sections with scanning electron microscopy images and confocal laser scanning microscope analysis. Briefly, polycaprolactone scaffolds were seeded at different cell densities with MG63 osteoblastic‐like cells. Two different endpoints, 1 and 2 weeks, were selected for the three‐dimensional colonization and proliferation analysis of the cells. By observing all images obtained, in addition to a more extensive distribution of cells on scaffolds surfaces than in the deeper layers, cell volume increased at 2 weeks compared to 1 week after seeding. Combining the cell number quantification by deoxyribonucleic acid analysis and the single cell volume changes by confocal laser scanning microscope, we validated the microCT segmentation method by finding no statistical differences in the evaluation of the cell volume fraction of the scaffold. Furthermore, the morphological results of this study suggest that an effective scaffold colonization requires a precise balance between different factors, such as number, type and size of seeded cells in addition to scaffold porosity.

An innovative co-axial system to electrospin in situ crosslinked gelatin nanofibers

Crosslinking of gelatin nanofibers maintaining a fibrous morphology after exposure to an aqueous solution is still a challenge. In this work, we developed an innovative method based on the use of an ad hoc designed co-axial needle to fabricate gelatin mats crosslinked with a very small amount of genipin and still able to retain their morphology when immersed in aqueous solution. Genipin-containing gelatin nanofibers are obtained by allowing mixing of the two solutions just within the needle. Genipin content of the electrospun mats can be modulated by varying feeding rates of the inner and outer solutions and their relative concentration. A subsequent thermal treatment of the mats, performed at 55 °C or 37 °C for 1 or 3 days and followed by rapid rinsing in ethanol and then in PB, allows one to obtain highly crosslinked gelatin nanofibers that perfectly maintain their morphology after immersion in an aqueous solution, display improved mechanical properties and enhanced stability. This new approach allows us to achieve gelatin mat stabilization using a very small amount of genipin with respect to other methods and to avoid post-treatment of the mats with the crosslinking agent, with a consequent significant reduction of the final cost of the materials. Moreover, in vitro tests demonstrate that the crosslinked mats support normal human primary chondrocyte culture, promoting their differentiation.

The active role of osteoporosis in the interaction between osteoblasts and bone metastases

INTRODUCTION To minimize the severity of bone metastases and to delay their onset, it is important to analyze the underlying biological mechanisms. The present study focused on the link between OP and metastatic cells, with particular attention to osteoblast behavior. METHODS Osteoblasts (OB) were isolated from the trabecular bone of iliac crest of healthy (SHAM) and ovariectomized (OVX) adult female rats and co-cultured with MRMT-1 rat breast carcinoma cells as conditioned medium (CM) or alone (CTR) for 24h, 7 and 14 days and tested for cell viability, morphology and synthetic activity, i.e. C-terminal procollagen type I, alkaline phosphatase, osteoprotegerin, receptor activator for nuclear factor KB ligand and interleukin-8. RESULTS Osteoblast morphology showed a reduced organization in the OVX group, in particular in the CM condition. Conversely, the analysis of cell viability revealed significantly higher values in the OVXCM group with respect to the SHAMCM group at all experimental times, whereas the OVXCTR group had significantly lower values at 7 and 14 days in comparison to those of the SHAM group. ALP release was significantly lower in the CM condition than that of CTR at all timepoints, and so was procollagen type I at 7 and 14 days. The RANKL/OPG ratio showed significantly higher values in OVX osteoblasts in comparison with those of the SHAM group, both in CTR and in CM conditions at each experimental time. Finally, OVXCM showed significantly higher values of IL-8 than those of SHAMCM at 7 and 14 days. CONCLUSIONS The results clearly indicate an influence of the metastatic cells on the osteoblastic physiology at different levels: morphology, viability, release of typical proteins, and also IL-8 as a proinflammatory cytokine, especially marked by osteoporosis. Further investigations might highlight the relationship between osteoblasts and breast cancer cells, which might be useful to improve common drugs used against osteoporosis and bone metastases, by enhancing the bone deposition/tumor progression ratio.