Izolarea colagenului din complexul ombilico-placentar pentru utilizarea în ingineria tisulară

Background. Collagen-based bone substitutes are widely used in bone tissue regeneration in orthopedic, traumatic and oromaxillofacial surgery. The predominant sources of collagen extraction are from animals, but they can transmit zoonoses from animal to human. Thus, collagen from the umbilicalplacental complex is a particular interest in the use as a graft for bone regeneration. Objective of the study. Obtaining collagen from the umbilical-placental complex for use in tissue engineering. Material and Methods. Collagen was isolated from umbilical-placental complexes (n = 3) with a mass of 66,5± 0,06 g received from the Tissue Bank. Preliminarily from the material, the non-collagenous proteins were removed with 0.05 M Na2HPO4, pH 8.7-9.1. The extraction was performed with pepsin and СН3СООН 0,5М and 5 mМ EDTA. Statistical processing was performed in Excel 2003. Results. The collagen extracts that were obtained after precipitation with 0.9% NaCl initially settled to the bottom of the container in the form of collagen fibers. After centrifugation and decantation of the supernatant, white mucilaginous substances were obtained which were purified by dialysis. The collagen concentrations that were obtained constituted 5,86± 0,04 mg/ml and were determined on the basis of dry mass. Conclusion. The amount of collagen obtained from the placental umbilical complex is significant and indicates that the source of production is a safe one and the method of production is efficient. Introducere. Substituenții osoși pe bază de colagen sunt utilizați pe scară largă în regenerarea țesutului osos în chirurgia ortopedică, traumatică și oromaxilo-facială. Sursele de extragere a colagenului predominant sunt de la animale, însă acestea pot transmite de la animal la om zoonoze. Astfel, colagenul din complexul ombilico-placentar prezintă un interes deosebit în utilizare ca grefă pentru regenerarea osoasă. Scopul lucrării. Obținerea colagenului din complexul ombilico-placentar pentru utilizarea în ingineria tisulară. Material și Metode. Colagenul a fost izolat din complexele ombilico-placentare (n=3) cu masa 66,5± 0,06 g primite de la Banca de Țesuturi. Preliminar, din material au fost îndepărtate proteinele necolagenice cu 0,05М Na2HPО4, pH 8,7-9,1. Extracția a fost efectuată cu pepsină și СН3СООН 0,5М și 5 mМ EDTA. Prelucrarea statistică a fost efectuată în Excel 2003. Rezultate. Extractele colagenice care au fost obținute după precipitarea cu NaCl 0,9 % inițial s-au sedimentat la fundul recipientului având aspect de fibre de colagen. După centrifugare și decantarea supernatantului au fost obținute substanțe mucilaginoase de culoare albă care au fost purificate prin dializă. Concentrațiile de colagen care au fost obținute au constituit 5,86± 0,04 mg/ml și au fost determinate în baza masei uscate. Concluzii. Cantitatea de colagen obținut din complexul ombilico-placentar este semnificativă și denotă că sursa de obținere este una sigură, iar metoda de obținere este eficientă

The man in the world of advanced information technologies: theoretical-philosophical and social aspects

Catedra Filosofie şi Bioetică a USMF „Nicolae Testemiţanu”Developments in the information society are one of the main agents of change worldwide. The complexity of this problem requires a man to examine some aspects, highlighting in particular, the theoretical-philosophical and social aspect. The article examines the manner in which this development could contribute to improving the human condition, determined him on such a man to be able to confront the acute problems of the society and to understand just how long the role that it plays technologies performance information in daily activity. Evoluţia societăţii informaţionale este unul dintre principalii agenţi ai schimbării planetare. Această complexitate a problemei îl impune pe om de a precăuta unele laturi, evidenţiind în special aspectul teoretico-filosofic, cît şi cel social. În articol se examinează modalitatea în care această evoluţie ar putea contribui la îmbunătăţirea considerabilă a condiţiei umane, determinîndu-l astfel pe om de a putea face faţă problemelor stringente din societate, cît şi de a înţelege rolul pe care-l joacă tehnologiile informaţionale performante în activitatea cotidiană

Human intervention - alternative explanation of environmental consciousness: theoretical and methodological issues

Catedra Filosofie şi Bioetică, USMF „Nicolae Testemiţanu”Confronting the present century civilization with a range of global issues is a primary topic in terms of solving and involvement in the common effort of humanity to save globe’s world population. The article examines the evolution of human actions on nature for adequate answers and solutions of civilization concern about the dangers directed to human health. Confruntarea civilizaţiei secolului prezent cu o serie de probleme globale constituie un subiect primordial în ce priveşte soluţionarea şi implicarea în efortul comun al umanităţii de a salva populaţia globului pămîntesc. În articol se examinează evoluţia acţiunilor umane asupra naturii în vederea unor răspunsuri şi soluţii adecvate ale civilizaţiei cu privire la preocuparea de pericole direcţionate asupra sănătăţii umane

Environmental conception in the area of public health: theoretical and methodological configurations

Catedra Filosofie şi Bioetică, USMF „Nicolae Testemiţanu”The present civilization requires the need of a new configuration of the knowledge domain, where the branches of the future of science put in realization the sustainable way of human life by analysis of environmental conception, of innovation of ecological culture and of integration which is subject to technical control on the area of public health. The article examines the human intervention on nature to a high responsibility of humanity to contribute to creation of the conditions for a sustainable development and an adequate security of antroponoospheric society. Civilizaţia prezentă solicită necesitatea unei noi configuraţii a domeniului cunoaşterii, în care ramurile de viitor ale ştiinţei să pună în funcţiune modul durabil al vieţii umane prin intermediul analizei concepţiei invaironmentale, a inovaţiei culturii ecologice şi a integrării supuse controlului tehnicii asupra spaţiului sănătăţii publice. În articol se examinează intervenţia umană asupra naturii în vederea unei înalte responsabilităţi a umanităţii de a contribui la crearea condiţiilor pentru o dezvoltare durabilă şi o securizare adecvată a societăţii antroponoosferice

An efficient procedure of isolation, cultivation and identification of bone marrow mesenchymal stem cells

Tissue Engineering and Cells Cultures Laboratory, Nicolae Testemitsanu State University of Medicine and Pharmacy, Chisinau, the Republic of MoldovaBackground: Bone marrow mesenchymal stem cells (MSC) have a wide application in domain of Regenerative Medicine. Of a great importance is utilization of a suitable bone marrow extraction technique that can provide a sufficient number of MSC to perform laboratory tests without seriously affecting the health of the laboratory animal. At the same time, before using in researches and clinical application, the MSC needs to be identified. Material and methods: The study was conducted in rabbits (n = 9), in which, from one iliac bone, by aspiration were taken 3.39 ± 1.27 ml of bone marrow. The nucleated bone marrow cells were separated through centrifugation using concentration gradient. The specific for stem cells culture medium was used, and MSC were multiplied during 2 passages. From the obtained MSC, 1x106 cells were subject to differentiation by chondrocytes lineage for other 20 days. The obtained chondrocytes aggregates were morphologically examined by Hematoxylin–Eosin staining and specific cartilage staining with Safranin O and Toluidine blue/fast green. Results: There was a strong correlation between the volume of collected bone marrow and the time required to achieve a 70-80% of MSC confluence (p=0.01). Also, the MSC isolated from bone marrow extracted from rabbit iliac bone were differentiated successful on chondrocyte line in all cases, confirmed through the specific cartilage staining with Safranin O and Toluidine blue/fast green (p<0,001). Conclusions: The volume of 3.39 ± 1.27 ml of bone marrow, harvested from rabbit iliac bone is sufficient to obtain a large number of MSC for the laboratory tests in vitro and in vivo. As a standard method for MSC identification could be used just the capability of the cells to differentiate in the specialized cell, including chondrocytes

Hepatocytes isolation from adult rats for liver recellularization

Tissue Engineering and Cells Cultures Laboratory, Nicolae Testemitsanu State University of Medicine and Pharmacy, Chisinau, the Republic of MoldovaBackground: Currently hepatocytes obtaining is prerequisite to create the necessary conditions for medical research, because it is an important tool in developing of new strategies in tissue engineering domain, which represents obtaining functional organs in laboratory conditions. Material and methods: The study was made on adult Wistar rats liver with body weight 274.66± 2.52 g (n=3) which were used for hepatocytes extraction by perfusion through the upper cave vein with combination of type II collagenase and type I dispase and Hank’s 0.9 mM MgCl2, 0.5 mM EDTA and 25 mM HEPES (HiMedia, India). Results: The cells were counted with trypan blue 0.25% in hemocytometer and cultured in William’s E medium (HiMedia, India) with 2 mM L-glutamine, 5% fetal bovine serum (Lonza, Belgium), antibiotic antimycotic solution (HiMedia, India), 100 nM dexamethasone and 100 nM insulin, with 2.5 x 105 cells per well in 12-well plates. After isolation were obtained 324, 48 ± 1, 25 x 106 hepatocytes, with a viability of 94.7 ± 0.9 % which indicates a high yield of cells viability. Conclusions: The hepatocyte isolation method by liver perfusion with the combination of collagenase-dispase is feasible for obtaining a large amount of functional hepatocytes intended for the recellularization in vitro of decellularized liver scaffolds. The yield and viability of hepatic cells could be increased by enzymatic digestion of liver tissue using combination of collagenase/dispase solution due to the less cytotoxic effect

The cohesion between environmental ethics and biodiversity – the objective of protecting of the heritage of human existence

Catedra Filosofie şi Bioetică, USMF „Nicolae Testemiţanu” din RMThe article examines an analysis from the perspective of bioethics on the scientific findings that are confered to the problems with reference to biodiversity. It is attempting an awarness of the complexity of the concerns in the way that are interconnected the environmental ethics and biodiversity, given the case that the precarious situation of the present it makes the threats to the adress of biodiversity loss with ethical direct implications over the entire of the planet and that requires a mandatory requirement of protection of the environment not to damage irreversibly the health and the life of mankind. În articol se examinează o analiză din perspectivă bioetică asupra constatărilor ştiinţifice care se conferă problemelor cu referire la biodiversitate. Se încearcă o conştientizare a complexităţii dilemelor în modul în care sunt interconectate etica invaironmentală şi biodiversitatea, dat fiind cazul că situaţia precară din prezent condiţionează ameninţări la adresa pierderii biodiversităţii cu implicaţii etice directe asupra întregii planete şi care necesită o cerinţă imperativă a protejării mediului de a nu prejudicia ireversibil sănătatea şi viaţa omenirii

Purification of collagen from human umbilical-placental complex for bioengineering use

Introduction. Collagen is a structural protein found in large quantities in the animal kingdom and has vital functions in tissue formation, attachment and cell proliferation. As a biomaterial it has a wide use in medicine, namely in traumatology, ophthalmology, oncology, dentistry, combustiology, pharmacology, both in the form of a matrix and as a carrier of encapsulated substances. The obtaining of high purity collagen is an important condition for use in tissue engineering. Aim of study. Evaluation of the purity of collagen extracted from the umbilical-placental complex using different surfactants Methods and materials. Placenta and umbilical cord from the Tissue and Cell Bank were used. Protocols for the elimination of blood and non-collagenous proteins from the human umbilical-placental complex were tested using sodium dodecyl sulfate, sodium deoxycholate, CHAPS, Triton X-100, Chloramine T with hydrogen peroxide, EDTA, and the control group was treated with distilled water. Results. The purification of the collagen extracted from the umbilical-placental complex using different surfactants determined a differentiated purity. Non-ionic Triton X-100 detergent and CHAPS were most effective in removing non-collagenous proteins and blood. The lowest purity collagen is obtained using Chloramine T with hydrogen peroxide. Conclusion. Anionic surfactant Triton X - 100 is the most effective in obtaining of high purity collagen from the umbilical-placental complex and preserve its native structure

Obtaining of a suitable osteochondral graft for articular cartilage engineering

Laboratory of Tissue Engineering and Cells Cultures, Laboratory of genetics, Nicolae Testemitanu State University of Medicine and Pharmacy of the Republic of Moldova Department of Biomedical Sciences. Faculty of Medical Bioengineering “Grigore T.Popa" University of Medicine and Pharmacy, Iasi, RomaniaIntroduction. Chondral injuries are common following a knee trauma. There are numerous studies with different ways to obtain a suitable graft for articular cartilage regeneration, but without imposing results. Material and methods. From two freshly sacrificed rabbits the distal femurs were harvested and frozen at -84°C for one week. From each distal femur all tissues except cartilage and subcondral bone were removed and small pieces of normal osteochondral tissue (NOCT) were taken. The remaining osteochondral tissue has been demineralized in 0,6M HCl (Chem-Lab, Belgium) over night and again small pieces of demineralized osteochondral tissue (OCDT) were cutted with a scalpel and placed in a PBS solution for 24 hours. The remaining OCDT were separated in 4 groups. Two groups were decellularized in 0,5% and 1% SDS (Sigma, UK) and another two in 0,5% and 1% Triton X-100 (HiMedia, India). The decellularization lasted for 24 hours. At the next day the decellularized and demineralized osteochondral tissues (OCDDT) were washed with distilled water and PBS for 24 hours. All tissues were dessicated through centrifugation at 4000 rpm for 10 min (Hettich, Germany). From all types of OCT were cutted from three to nine pieces 20 mg each and quantification of DNA was performed with GeneJET Genomic DNA Purification Kit (Thermo Fisher, Lithuania). The results were read with spectrophotometer NanoDrop 2000c at wavelength of 260 nm (Thermo Fisher, USA). The best decellularized tissue and OCDT were tested for cytotoxicity with MTT test (ISO 10993-5) with mesenchymal stem cells and chondrocytes. Results. The average of DNA content in a rabbit NOCT is 36 ng/μl, in OCDT 4,23 ng/μl, OCDDT with 0,5% and 1% SDS is 3,23 ng/μl and 2,16 ng/μl respectively and in OCDDT with 0,5% and 1% TritonX-100 is 1,96 ng/μl and 0,96 ng/μl. At the MTT assay with mesenchymal stem cells and chondrocytes on the OCDT and OCDDT with 1% TritonX-100, we obtained a higher cell viability in both cases more than 80%. Conclusions. Obtaining a suitable osteochondral tissue for cartilaginous tissue engineering is very difficult because this process involves utilisation of a very toxic chemicals that harm this tissue. A shorter exposure period to chemical agents and preliminary modeling of the graft is mandatory. Also the OCDDT with 1% TritonX-100 shows the best results compared to others