Hygiene,health and environment: European directives

Summary The setting up of the 1993 single market aimed at promoting free movement within the European Community, requires the elaboration of documents intended for the State members, so that the latter may adapt their own national standards which, at the present time, often hinder this free circulation. As for the ?Construction products?, a Directive was passed in this respect on December 21, 1988. It defines six essential requirements applicable to both building and civil engineering works, among which the one entitled: ?Hygiene, Health and Environment?. Each of these requirements will give rise to the elaboration of an ?Interpretative Document? which is aimed at explaining the contents of each requirement, specifying the relevant terminology, allowing the requirements, to be transformed into levels (or classes) of performances to comply with, as well as stating precisely the standards and harmonized technical documents indispensable for both the expression and checking of these levels. With respect to the range of subjects pertinent to the ?Hygiene, Health and Environment? requirement, it should be possible to deal easily with certain aspects such as hydrological cycle, in so far as both regulations and means of verification have, for a long time, been determined in most EEC countries. On the other hand, it will be more difficult to treat other aspects such as: air quality, emission of pollutants or radiation, owing to an almost total absence of any regulations at the present time.     

Temporal regulation of hyaluronan and proteoglycan metabolism by human bone cells in vitro

Osteoblasts elaborate a dynamic extracellular matrix that is constructed and mineralized as bone is formed. This matrix is primarily composed of collagen, along with noncollagenous proteins which include glycoproteins and proteoglycans. After various times in culture, human bone cells were labeled with [35S]sulfate, [3H] leucine/proline, or [3H]glucosamine and the metabolism of hyaluronan and four distinct species of proteoglycans (PGs) was assayed in the medium, cell layer, and intracellular pools. These cells produce hyaluronan (Mr approximately 1,400,000; a chondroitin sulfate PG (CSPG), Mr approximately 600,000; a heparan sulfate PG (HSPG), Mr approximately 400,000; and two dermatan sulfate PGs with Mr approximately 270,000 (biglycan, PG I) and Mr approximately 135,000 (decorin, PG II) that distribute between the medium and cell layer. Two days following subculture, 12 h [35S]sulfate steady-state labeling yielded a composition of 24, 27, 31, and 18% for total CSPG, HSPG, biglycan, and decorin, respectively. While HSPG and decorin levels and distribution between medium and cell layer remained relatively constant during steady-state labeling at different times in culture, CSPG and biglycan levels increased dramatically at late stages of growth, and their distribution changed throughout culture. These results were independent of cell density, media depletion, and labeling pool effects. In contrast, hyaluronan synthesis was uncoupled from PG synthesis and apparently density-dependent. Pulse chase labeling at different stages of culture showed that the CSPG and decorin behaved as secretory PGs. Both HSPG and biglycan underwent catabolism, with HSPG possessing a t1/2 of 8 h and biglycan a t1/2 of 4 h. While the rate of HSPG turnover did not appreciably change between early and late culture, that of biglycan decreased. The mRNA for decorin was constant, while that of biglycan changed during culture. These results suggest that each PG possesses a distinct pattern of cellular and temporal distribution that may reflect specific stages in matrix formation and maturation.     

Review: Assessing fish welfare in research and aquaculture,with a focus on European directives

The number of farmed fish in the world has increased considerably. Aquaculture is a growing industry that will in the future provide a large portion of fishery products. Moreover, in recent years, the number of teleost fish used as animal models for scientific research in both biomedical and ecological fields has increased. Therefore, it is increasingly important to implement measures designed to enhance the welfare of these animals. Currently, a number of European rules exist as requirements for the establishment, care and accommodation of fish maintained for human purposes. As far as (teleost) fish are concerned, the fact that the number of extant species is much greater than that of all other vertebrates must be considered. Of further importance is that each species has its own specific physical and chemical requirements. These factors make it difficult to provide generalized recommendations or requirements for all fish species. An adequate knowledge is required of the physiology and ecology of each species bred. This paper integrates and discusses, in a single synthesis, the current issues related to fish welfare, considering that teleosts are target species for both aquaculture and experimental models in biological and biomedical research. We first focus on the practical aspects, which must be considered when assessing fish welfare in both research and aquaculture contexts. Next, we address husbandry and the care of fish housed in research laboratories and aquaculture facilities in relation to their physiological and behavioural requirements, as well as in reference to the suggestions provided by European regulations. Finally, to evaluate precisely which parameters described by Directive 2010/63/EU are reported in scientific papers, we analysed 82 articles published by European researchers in 2014 and 2015. This review found that there is a general lack of information related to the optimal environmental conditions that should be provided for the range of species covered by this directive.     

Bioceramic-collagen scaffolds loaded with human adipose-tissue derived stem cells for bone tissue engineering

The combination of bioceramics and stem cells has attracted the interest of research community for bone tissue engineering applications. In the present study, a combination of Bio-Oss^® and type 1 collagen gel as scaffold were loaded with human adipose-tissue derived mesenchymal stem cells (AT-MSCs) after isolation and characterization, and the capacity of them for bone regeneration was investigated in rat critical size defects using digital mammography, multi-slice spiral computed tomography imaging and histological analysis. 8 weeks after implantation, no mortality or sign of inflammation was observed in the site of defect. According to the results of imaging analysis, a higher level of bone regeneration was observed in the rats receiving Bio-Oss^®-Gel compared to untreated group. In addition, MSC-seeded Bio-Oss-Gel induced the highest bone reconstruction among all groups. Histological staining confirmed these findings and impressive osseointegration was observed in MSC-seeded Bio-Oss-Gel compared with Bio-Oss-Gel. On the whole, it was demonstrated that combination of AT-MSCs, Bio-Oss and Gel synergistically enhanced bone regeneration and reconstruction and also could serve as an appropriate structure to bone regenerative medicine and tissue engineering application.     

Characteristics of human mesenchymal stem cells isolated from bone marrow and adipose tissue

Populations of human mesenchymal stem cells were derived from bone marrow and adipose tissue. Here analysis of six individuals is represented. Cells were isolated, expanded and evaluated by the expression of surface antigens using flow cytometry. These cells displayed similar characteristics for many markers. Cells isolated from bone marrow and adipose tissue were found to be homogeneously positive for CD13, CD44, CD90, CD105, and negative for CD45, CD34, CD31 and CD117. Besides, differences in surface antigene CD10 expression between narrow and adipose tissue-derived cells were detected. All these findings indicate that both bone marrow and adipose tissue are important sources of mesenchymal stem cells, which could be used in cell therapy protocols.     

A study of MTT-hybrid assay using human bone and soft tissue tumor cells

We investigated a new chemosensitivity test, MTT-hybrid assay, which was a hybrid of MTT colorimetric assay and double-layered soft agar colony assay, using human bone and soft tissue tumor cells. MTT formazan crystals produced by viable cells in the soft agar medium were solubilized by SDS at 60 degrees C. The absorbance (560 nm) is directly proportional to the cell number over a wide range. The absorbance increased in proportion to colonial growth of osteosarcoma cells, while it decreased in a human diploid cell strain in a few days. Drug sensitivity of tumor cells is supposed to be assessed without contaminating normal cells by MTT-hybrid assay in primary tumor samples. Good correlation of IC50 was observed between MTT-hybrid assay and colony assay. The MTT-hybrid assay shows potential value as a rapid predictive test for chemotherapeutic agents in an individual patient.     

Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions

Current standard techniques for bone tissue engineering utilize ex vivo expanded osteogenic cells. However, ex vivo expansion requires serum, which may hinder clinical applications. Here, we report the feasibility and efficacy of bone tissue engineering with human bone marrow stromal cells (BMSCs) expanded in serum-free conditions. Bone marrow was aspirated from 4 healthy donors and adherent cells were cultured in either serum-free medium (STEMPRO^® MSC SFM) or conventional serum-containing medium (α-MEM supplemented with 10% serum). Efficacy of expansion was greater in serum-free medium. Phenotypically, serum-free expanded BMSCs were smaller in cell-size and showed expression of CD105⁺⁺ and CD146^dim. After osteogenic induction, serum-free expanded BMSCs showed lower alkaline phosphatase activity. However, they showed higher responsiveness to induction. In vivo bone-forming ability was also confirmed. In conclusion, bone tissue engineering with serum-free expanded BMSCs is feasible and as efficient as that obtained with BMSCs expanded in conventional serum-containing medium.     

Virus inactivation in bone tissue transplants (femoral heads) by moist heat with the 'Marburg bone bank system'

Several virus inactivation procedures like heat treatment, gamma irradiation and chemical sterilization are used to increase the safety of bone tissue transplants. In this study we present data on the virus-inactivating effect of heat disinfection on human femoral heads, using the Marburg bone bank system 'Lobator sd-2'. Three enveloped viruses (human immunodeficiency virus type 2 [HIV-2], bovine viral diarrhoea virus as a model for Hepatitis C virus [HCV], and the herpesvirus pseudorabies virus), and three non-enveloped viruses (hepatitis A virus, poliomyelitis virus, and bovine parvovirus) were investigated.In a model system the central part of human femoral heads was contaminated with the respective cell-free virus suspension, establishing a direct contact between virus and native bone tissue. The core temperature in the femoral heads during the sterilization process was determined in additional model experiments. A temperature of 82.5 degrees C, given by the manufacturer as the effective temperature for virus inactivation, was maintained for at least 15 min in decartilaged femoral heads with a diameter of < or = 56 mm. Heat treatment using the Lobator sd-2 inactivated all viruses in human femoral heads below the detection limit (at least by a factor of > or =4 log(10)).By combining a well-focussed anamnesis of the donors and serological testing for relevant infection markers (anti-HIV-1/2, HBsAg, anti-HBcore, anti-HCV, TPHA) with heat treatment of femoral heads in the Lobator sd-2 system, a high safety level is achieved. To further increase virus safety of cadaveric bone transplants, it is recommended that multi-organ donors are tested by nucleic acid testing (i.e. polymerase chain reaction) for HIV, HBV and HCV genome.     

Fetuin in human bone marrow: detection in foetal tissue and patients with mastocytosis

Fetuin, a foetal protein of unknown function, has been shown to be expressed in both the immune and nervous systems, especially during development. Here, we show for the first time, that fetuin is abundantly present in many cells of the foetal human bone marrow, but is restricted to cells of the monocytic lineage in the adult. Fetuin's immunoreactivity increased considerably in adult human bone marrow in some pathological conditions, particularly in mastocytosis and was also increased in bone marrows in some cases of acute leukaemias, especially in acute myeloid leukaemia. This increase in the presence of fetuin in neoplastic bone marrows is not reflected by an increased level of circulating fetuin. This last observation contradicts earlier suggestions that fetuin is specifically reduced in cancer patients. A consistent increase in fetuin immunoreactivity in bone marrow of most cases of mastocytosis, as demonstrated in this paper, could become a useful tool in the diagnosis of this disease.     

Human research tissue banks: the ATRA Project for establishing a human research tissue bank in switzerland

A large number of experiments in biomedical research are carried out on tissues, but, even though the results should be applicable to humans, these tissues are mainly of animal origin. The difficulty encountered in obtaining human organs and tissues is an acknowledged problem: not enough human tissues are available to meet research needs. We are introducing the ATRA Project, with the purpose of supporting progress in biomedical research in Switzerland through the establishment of one or more human tissue banks, which will be able to find, treat, preserve and supply human material. Where similar projects have already been launched, concerns have been expressed that donation for research purposes might compete with donation for transplantation, but most organs and tissues are in any case non-transplantable. Surplus surgical tissue is considered "sanitary waste", and must be treated according to specific regulations for collection, packaging, transport, treatment and disposal. A human tissue bank would not only abate the costs of treating sanitary waste, but would actually turn what is now considered waste into a resource which could be used to save human and animal lives.     

The response of human mesenchymal stem cells to osteogenic signals and its impact on bone tissue engineering

Bone tissue engineering using human mesenchymal stem cells (hMSCs) is a multidisciplinary field that aims to treat patients with trauma, spinal fusion and large bone defects. Cell-based bone tissue engineering encompasses the isolation of multipotent hMSCs from the bone marrow of the patient, in vitro expansion and seeding onto porous scaffold materials. In vitro pre-differentiation of hMSCs into the osteogenic lineage augments their in vivo bone forming capacity. Differentiation of hMSCs into bone forming osteoblasts is a multi-step process regulated by various molecular signaling pathways, which warrants a thorough understanding of these signaling cues for the efficient use of hMSCs in bone tissue engineering. Recently, there has been a surge of knowledge on the molecular cues regulating osteogenic differentiation but extrapolation to hMSC differentiation is not guaranteed, because of species- and cell-type specificity. In this review, we describe a number of key osteogenic signaling pathways, which directly or indirectly regulate osteogenic differentiation of hMSCs. We will discuss how and to what extent the process is different from that in other cell types with special emphasis on applications in bone tissue engineering.     

The utility of human dedifferentiated fat cells in bone tissue engineering in vitro

We compared the osteoblastic differentiation abilities of dedifferentiated fat cells (DFATs) and human bone marrow mesenchymal stem cells (hMSCs) as a cell source for bone regeneration therapies. In addition, the utility of DFATs in bone tissue engineering in vitro was assessed by an alpha-tricalcium phosphate (α-TCP)/collagen sponge (CS). Human DFATs were isolated from the submandibular of a patient by ceiling culture. DFATs and hMSCs at passage 3 were cultured in control medium or osteogenic medium (OM) for 14 days. Runx2 gene expression, alkaline phosphatase (ALP) activity, as well as osteocalcin (OCN) and calcium contents were analyzed to evaluate the osteoblastic differentiation ability of both cell types. DFATs seeded in a α-TCP/CS and cultured in OM for 14 days were analyzed by scanning electron microscopy (SEM) and histologically. Compared with hMSCs, DFATs cultured in OM generally underwent superior osteoblastogenesis by higher Runx2 gene expression at all days tested, as well as higher ALP activity at day 3 and 7, OCN expression at day 14, and calcium content at day 7. In SEM analyses, DFATs seeded in a α-TCP/CS were well spread and covered the α-TCP/CS by day 7. In addition, numerous spherical deposits were found to almost completely cover the α-TCP/CS on day 14. Von Kossa staining showed that DFATs differentiated into osteoblasts in the α-TCP/CS and formed cultured bone by deposition of a mineralized extracellular matrix. The combined use of DFATs and an α-TCP/CS may be an attractive option for bone tissue engineering.     

Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) have become the main cell source for bone tissue engineering. It has been reported that cryopreserved human MSCs can maintain their potential for proliferation and osteogenic differentiation in vitro. There are, however, no reports on osteogenesis with cryopreserved human MSCs in vivo. The aim of this study was to determine whether cryopreservation had an effect on the proliferation capability and osteogenic differentiation of human MSCs on scaffolds in vitro and in vivo. MSCs were isolated from human bone marrow, cultured in vitro until passage 2, and then frozen and stored at −196 °C in liquid nitrogen with 10% Me₂SO as cryoprotectant for 24 h. The cryopreserved MSCs were then thawed rapidly, seeded onto partially demineralized bone matrix (pDBM) scaffolds and cultured in osteogenic media containing 10 mM sodium β-glycerophosphate, 50 μM l-ascorbic acid, and 10 nM dexamethasone. Non-cryopreserved MSCs seeded onto the pDBM scaffolds were used as control groups. Scanning electronic microscopy (SEM) observation, DNA content assays, and measurements of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were applied, and the results showed that the proliferation potential and osteogenic differentiation of MSCs on pDBM in vitro were not affected by cryopreservation. After 2 weeks of subculture, the MSCs/pDBM composites were subcutaneously implanted into the athymic mice. The constructs were harvested at 4 and 8 weeks postimplantation, and histological examination showed tissue-engineered bone formation in the pDBM pores in both groups. Based on these results, it can be concluded that cryopreservation allows human MSCs to be available for potential therapeutic use to tissue-engineer bone.     

Analysis of sequences from human brain cDNA gene bank which are functionally active in nervous tissue and tumor cells

Construction of a human cortex cDNA bank is described as well as the isolation from this bank of pBH71 and pBH3 clones with preferential expression in nervous and in tumor cells. The clones can be included into the third class of cDNA according to Sutcliff's classification. The mRNA corresponding to this cDNA class is considered to play the key role in determination of specificity of nervous tissue. Expression of the pBH71 sequence was revealed in human cortex and in tissues of different genesis (from neuroblastoma to uterus myoma), a 2 kb mRNA which corresponds to one and the same cDNA chain having been found in all tissues under analysis. The nucleotide sequence of cDNA insertion into the pBH71 clone of 447 n.p. was determined, and particular features of cDNA nucleotide composition and possible schemes of its translation were analysed. Weak homology was found between the 3'-end of cDNA insertion of the pBH71 clone and the 3'-end region of human proopiomelanocortine. The cDNA of the pBH3 clone hybridizes with the 0.8 kb mRNA revealed in human cortex and neuroendocrine tumors of different nature. No homology was revealed between the cDNA sequence of the pBH3 clone and any genes deciphered.     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.     

Trichinellosis in the European union: epidemiology, ecology and economic impact

Trichinellosis, one of the most widespread helminthic zoonoses, is still endemic in most countries of the European Union. In the past few years, advanced biotechnology has been used to re-examine the taxonomy, epidemiology and life cycles of aetiological agents, providing additional information on the main factors contributing to the maintenance of these parasites in Nature. The old concept that pigs and rats are the main hosts of Trichinella spiralis, as still reported in many books, has been re-evaluated thoroughly. In this review, Edoardo Pozio summarizes the epidemiology and ecology of human and animal trichinellosis, quantifies the economic impact of this zoonosis and suggests methods of controlling this infection that would result in a great reduction in costs.