Mice Drawer System

The Mice Drawer System (MDS) is an Italian Space Agency (ASI) facility which is able to support mice onboard the International Space Station during long-duration exploration missions (from 100 to 150-days) by living space, food, water, ventilation and lighting. Mice can be accommodated either individually (maximum 6) or in groups (4 pairs). MDS is integrated in the Space Shuttle middeck during transportation (uploading and downloading) to the ISS and in an EXPRESS Rack in Destiny, the US Laboratory during experiment execution. Osteoporosis is a debilitating disease that afflicts millions of people worldwide. One of the physiological changes experienced by astronauts during space flight is the accelerated loss of bone mass due to the lack of gravitational loading on the skeleton. This bone loss experienced by astronauts is similar to osteoporosis in the elderly population. MDS will help investigate the effects of unloading on transgenic (foreign gene that has been inserted into its genome to exhibit a particular trait) mice with the Osteoblast Stimulating Factor-1, OSF-1, a growth and differentiation factor, and to study the genetic mechanisms underlying the bone mass pathophysiology. MDS will test the hypothesis that mice with an increased bone density are likely to be more protected from osteoporosis, when the increased bone mass is a direct effect of a gene involved in skeletogenesis (skeleton formation). Osteoporosis is a debilitating disease that afflicts millions worldwide. One of the physiological changes experienced by astronauts during space flight is the accelerated loss of bone mass due to the lack of gravitational loading on the skeleton, a loss that is similar to osteoporosis in the elderly population on Earth. Osteoblast Stimulating Factor-1 (OSF-1), also known as pleiotrophin (PTN) or Heparin-Binding Growth- Associated Molecule (HB-GAM) belongs to a family of secreted heparin binding proteins..OSF-1 is an extracellular matrix-associated growth and differentiation factor that is normally expressed in cartilage; it can stimulate the proliferation and differentiation of human osteoprogenitor cells (cell that differentiate into an osteoblast) in vitro. The Mice Drawer System will study the effects of microgravity on transgenic mouse bones in order to identify genetic mechanisms playing a role in the reduction of the bone mass observed in humans and animals as a consequence of long-duration (greater than 100 days) microgravity exposure. Onboard the ISS, MDS is relatively self-sufficient; a crewmember will check the health status of the rodents on a daily basis, by assessing them through the viewing window. Water levels will be assessed by the crew daily and refilled as needed. Replacement of the food bars and replacement of the waste filters will be conducted inflight by crewmembers every 20-days

The developmentally regulated avian Ch21 lipocalin is an extracellular fatty acid-binding protein.

Ch21, a developmentally regulated extracellular protein expressed in chick embryos and in cultured chondrocytes, was expressed in the baculovirus system, and the recombinant protein was purified to homogeneity by gel-filtration chromatography. Separation of two isoforms was achieved on an ion-exchange column. Previous work had shown that Ch21 belongs to the superfamily of lipocalins, which are transport proteins for small hydrophobic molecules. Studies were performed to identify the Ch21 ligand. By analysis of recombinant Ch21 on native polyacrylamide gel electrophoresis and by Lipidex assay, the binding of fatty acid to the protein was shown and a preferential binding of long-chain unsaturated fatty acids was observed. Both isoforms had the same behavior. The binding was saturable. Stoichiometry was about 0.7 mol of ligand/mol of protein. The protein binds the ligand in its monomeric form. Calculated dissociation constants were 2 X 10(-7) M for unsaturated fatty acids and 5 X 10(-7) M for stearic acid. The binding was specific; other hydrophobic molecules, as retinoic acid, progesterone, prostaglandins, and long-chain alcohols and aldehydes did not bind to the protein. Short-chain fatty acids did not bind to the protein. Ch21, also present in chicken serum, represents the first extracellular protein able to selectively bind and transport fatty acid in extracellular fluids and serum. We propose to rename the Ch21 protein as extracellular fatty acid-binding protein (Ex-FABP)

Platelet Lysate Inhibits NF-κB Activation and Induces Proliferation and an Alert State in Quiescent Human Umbilical Vein Endothelial Cells Retaining Their Differentiation Capability.

open6Injured blood vessel repair and blood circulation re-establishment are crucial events for tissue repair. We investigated in primary cultures of human umbilical vein endothelial cells (HUVEC), the eects of platelet lysate (PL), a cocktail of factors released by activated platelets following blood vessel disruption and involved in the wound-healing process triggering. PL exerted a protective eect on HUVEC in an inflammatory milieu by inhibiting IL-1-activated NF-B pathway and by inducing the secretion of PGE2, a pro-resolving molecule in the wound microenvironment. Moreover, PL enhanced HUVEC proliferation, without aecting their capability of forming tube-like structures on matrigel, and activated resting quiescent cells to re-enter cell cycle. In agreement with these findings, proliferation-related pathways Akt and ERK1/2 were activated. The expression of the cell-cycle activator Cyclin D1 was also enhanced, as well as the expression of the High Mobility Group Box-1 (HMGB1), a protein of the alarmin group involved in tissue homeostasis, repair, and remodeling. These in vitro data suggest a possible in vivo contribution of PL to new vessel formation after a wound by activation of cells resident in vessel walls. Our biochemical study provides a rationale for the clinical use of PL in the treatment of wound healing-related pathologies.openRomaldini A, Ulivi V, Nardini M, Mastrogiacomo M,Cancedda R, Descalzi FRomaldini, A; Ulivi, V; Nardini, M; Mastrogiacomo, M; Cancedda, R; Descalzi,

Encapsulation of human articular chondrocytes into 3D hydrogel : phenotype and genotype characterization

This chapter is intended to provide a summary of the current materials used in cell encapsulation technology as well as methods for evaluating the performance of cells encapsulated in a polymeric matrix. In particular, it describes the experimental procedure to prepare a hydrogel matrix based on natural polymers for encapsulating and culturing human articular chondrocytes with the interest in cartilage regeneration. Protocols to evaluate the viability, proliferation, differentiation, and matrix production of embedded cells are also described and include standard protocols such as the MTT and [3H] Thymidine assays, reverse transcription polymerase chain reaction (RT-PCR) technique, histology, and immunohistochemistry analysis. The assessment of cell distribution within the 3D hydrogel construct is also described using APoTome analysis.(undefined

Platelet lysate maintains chondrogenic potential and promotes cartilage regeneration

cartilage. We report the biological effect of the platelet lysate (PL), a PRP derivative, on primary human articular chondrocytes (HAC) cultured under both physiological and inflammatory condition. Added to the culture medium, PL induced a strong mitogenic response in the chondrocytes. The in vitro expanded cell population maintained a chondrogenic re-­‐differentiation potential as revealed by micromass culture in vitro as well as in vivo as demonstrated by ectopic cartilage formation in nude mice. Furthermore, in chondrocytes cultured in the presence of the pro-­‐inflammatory cytokine IL-­‐1α, the PL induced a drastic enhancement of the synthesis of the cytokines IL-­‐6 and IL-­‐8 and of NGAL, a lipocalin expressed in cells of the chondrogenic lineage. These events were controlled by the p38 MAP kinase and NF-­‐κΒ pathways. The pro-­‐inflammatory effect of the PL was a transient phenomenon. In fact, after an initial up regulation, we observed a significant reduction of the NF-­‐κΒ activity together with the repression of the inflammatory enzyme ciclooxygenase-­‐2 (COX-­‐2). Moreover, the medium of chondrocytes cultured in the contemporary presence of PL and IL-­‐1α, showed a significant enhancement of the chemoattractant activity versus untreated chondrocytes. On the whole, our findings support the concept that the platelet products have a direct beneficial effect on articular chondrocytes and at the same time could drive in sequence a trans

Platelet Lysate Activates Human Subcutaneous Adipose Tissue Cells by Promoting Cell Proliferation and Their Paracrine Activity Toward Epidermal Keratinocytes

Skin chronic wounds are non-healing ulcerative defects, which arise in association with a morbidity state, such as diabetes and vascular insufficiency or as the consequence of systemic factors including advanced age. Platelet Rich Plasma, a platelet-rich blood fraction, can significantly improve the healing of human skin chronic ulcers. Given that the subcutaneous adipose tissue is located beneath the skin and plays a role in the skin homeostasis, in this study, we investigated the in vitro response of human subcutaneous adipose tissue cells to platelet content in a model mimicking in vitro the in situ milieu of a deep skin injury. Considering that, at the wound site, plasma turn to serum, platelets are activated and inflammation occurs, human adipose-derived stromal cells (hASC) were cultured with Human Serum (HS) supplemented or not with Platelet Lysate (PL) and/or IL-1α. We observed that HS sustained hASC proliferation more efficiently than FBS and induced a spontaneous adipogenic differentiation in the cells. PL added to HS enhanced hASC proliferation, regardless the presence of IL-1α. In the presence of PL, hASC progressively lessened the adipogenic phenotype, possibly because the proliferation of less committed cells was induced. However, these cells resumed adipogenesis in permissive conditions. Accordingly, PL induced in quiescent cells activation of the proliferation-related pathways ERK, Akt, and STAT-3 and expression of Cyclin D1. Moreover, PL induced an early and transient increase of the pro-inflammatory response triggered by IL-1α, by inducing COX-2 expression and secretion of a large amount of PGE2, IL-6, and IL-8. Media conditioned by PL-stimulated hASC exerted a chemotactic activity on human keratinocytes and favored the healing of an in vitro scratch wound. In order to bridge the gap between in vitro results and possible in vivo events, the stimuli were also tested in ex vivo cultures of in toto human adipose tissue biopsies (hAT). PL induced cell proliferation in hAT and outgrowth of committed progenitor cells able to differentiate in permissive conditions. In conclusion, we report that the adipose tissue responds to the wound microenvironment by activating the proliferation of adipose tissue progenitor cells and promoting the release of factors favoring wound healing

New Small Nuclear RNA Gene-Like Transcriptional Units as Sources of Regulatory Transcripts

By means of a computer search for upstream promoter elements (distal sequence element and proximal sequence element) typical of small nuclear RNA genes, we have identified in the human genome a number of previously unrecognized, putative transcription units whose predicted products are novel noncoding RNAs with homology to protein-coding genes. By elucidating the function of one of them, we provide evidence for the existence of a sense/antisense-based gene-regulation network where part of the polymerase III transcriptome could control its polymerase II counterpart

A Modified Rabbit Ulna Defect Model for Evaluating Periosteal Substitutes in Bone Engineering: A Pilot Study

The present work defines a modified critical size rabbit ulna defect model for bone regeneration in which a non-resorbable barrier membrane was used to separate the radius from the ulna to create a valid model for evaluation of tissue-engineered periosteal substitutes. Eight rabbits divided into two groups were used. Critical defects (15 mm) were made in the ulna completely eliminating periosteum. For group I, defects were filled with a nanohydroxyapatite poly(ester urethane) scaffold soaked in PBS and left as such (group Ia) or wrapped with a tissue-engineered periosteal substitute (group Ib). For group II, an expanded-polytetrafluoroethylene (e-PTFE) (GORE-TEX\uae) membrane was inserted around the radius then the defects received either scaffold alone (group IIa) or scaffold wrapped with periosteal substitute (group IIb). Animals were euthanized after 12\u201316 weeks, and bone regeneration was evaluated by radiography, computed microtomography (\ub5CT), and histology. In the first group, we observed formation of radio-ulnar synostosis irrespective of the treatment. This was completely eliminated upon placement of the e-PTFE (GORETEX\uae) membrane in the second group of animals. In conclusion, modification of the model using a non-resorbable e-PTFE membrane to isolate the ulna from the radius was a valuable addition allowing for objective evaluation of the tissue-engineered periosteal substitut

Altered bone development and turnover in transgenic mice over-expressing lipocalin-2 in bone

Lipocalin-2 (LCN2) is a protein largely expressed in many tissues, associated with different biological phenomena such as cellular differentiation, inflammation and cancer acting as a survival/apoptotic signal. We found that LCN2 was expressed during osteoblast differentiation and we generated transgenic (Tg) mice over-expressing LCN2 in bone. Tg mice were smaller and presented bone microarchitectural changes in both endochondral and intramembranous bones. In particular, Tg bones displayed a thinner layer of cortical bone and a decreased trabecular number. Osteoblast bone matrix deposition was reduced and osteoblast differentiation was slowed-down. Differences were also observed in the growth plate of young transgenic mice where chondrocyte displayed a more immature phenotype and a lower proliferation rate. In bone marrow cell cultures from transgenic mice, the number of osteoclast progenitors was increased whereas in vivo it was increased the number of mature osteoclasts expressing tartrate-resistant acid phosphatase (TRAP). Finally, while osteoprotegerin (OPG) levels remained unchanged, the expression of the conventional receptor activator of nuclear factor-κB ligand (RANKL) and of the IL-6 was enhanced in Tg mice. In conclusion, we found that LCN2 plays a role in bone development and turnover having both a negative effect on bone formation, by affecting growth plate development and interfering with osteoblast differentiation, and a positive effect on bone resorption by enhancing osteoclast compartment