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

Selective wrist arthrodesis: standard vs combined graft with stem cells. Experimental research

Introduction. Selective arthrodesis is a surgical procedure used to be done in advanced wrist joint arthritis. The aim of the surgery is to achieve a stable joint, by intercarpal ankylosis, which removes the pain syndrome and restores the strength of the hand, unfortunately with the risk of decreasing the range of motion in the wrist joint. Aim of study. Comparative evaluation of the method of selective wrist arthrodesis using standard vs combined graft with stem cells. Methods and materials. Our study is based on experimental research on 10 New Zealand rabbits. In the first study group, on 5 laboratory animals was performed standard arthrodesis of wrist joint. In the second study group, on other 5 laboratory animals, was developed the new treatment technique using the combined graft with stem cells, obtained by tissue engineering. Results. In all cases, an immediate postoperative radiograph was performed. The clinical and radiological evaluation, performed at 4, 8 and 12 weeks after the surgery. Computer tomography of each operated wrist was done 12 weeks postoperatively. Imagistic results showed us, that the group were performed arthrodesis using combined graft with stem cells, the ankylosis were achieved faster. Preliminary histological examinations of experiments indicate more active involvement in the process of osteogenesis in the use of combined stem cell transplantation. Conclusions. Our experimental research highlights an innovative method of surgical treatment - arthrodesis using the combined graft with stem cells. It turned out to be a harmless and safe method. A comparative study with the standard treatment method was performed, the imaging and preliminary histological results are encouraging. The final analysis of the results is still in process

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

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

Grefa combinată cu celule stem în artrodeza selectivă de pumn

Background. Selective arthrodesis is a rescue procedure for advanced arthritis of the wrist joint. The result of surgery is the formation of intercarpal ankylosis, which removes the pain syndrome and restores the strength of the hand at the cost of losing of some movements in the wrist joint. Objective of the study. Evaluation of the method of selective wrist arthrodesis using the combined graft with stem cells. Material and Methods. Was developed an experimental study with the application of a new treatment technique to laboratory animals. As methods of implementation of the study included: preparation of allogeneic demineralized bone grafts, bone marrow sampling and isolation of autologous stem cells. After obtaining the combined graft, it was implanted between the carpal bones and performed selective wrist arthrodesis. Results. For the initial stage, the methodology of obtaining the graft combined with stem cells was finalized. 8 osteo-cellular grafts were prepared. Subsequently, through the new treatment technique, the combined grafts were used in 4 surgeries. The clinical and radiological dynamic evaluation, performed for a period of 2 months postoperatively, allowed to obtain promising results. Conclusion. Surgical treatment by selective wrist arthrodesis using the combined graft with stem cells is a harmless method, but its peculiarities require further study. Introducere. Artrodeza selectivă este o procedură de salvare în cazul schimbărilor artrozice avansate ale articulației pumnului. Rezultatul intervenției chirurgicale este formarea anchilozei intercapiene, care înlătură sindromul algic și restabilește puterea mâinii cu prețul pierderii unui volum de mișcări. Scopul lucrării. Evaluarea metodei de artrodeză selectivă de pumn cu utilizarea grefei combinate cu celule stem. Material și Metode. S-a elaborat un studiu experimental cu aplicarea unei tehnici noi de tratament la animalele de laborator. Drept metode de implementare a studiului au inclus: prepararea grefelor osoase demineralizate alogene, prelevarea măduvei osoase și izolarea celulelor stem autologe. După obținerea grefei combinate, aceasta a fost implantată între oasele carpiene și efectuată artrodeza selectivă de pumn. Rezultate. Pentru etapa inițială s-a definitivat metodologia obținerii grefei combinate cu celule stem. S-au preparat 8 grefe osteo-celulare. Ulterior, prin tehnica nouă de tratament, grefele combinate s-au utilizat în 4 intervenții chirurgicale. Evaluarea clinică și radiologică în dinamică, efectuată pe o perioadă de 2 luni postoperator, a permis obținerea unor rezultate promițătoare. Concluzii. Tratamentul chirurgical prin artrodeza selectivă de pumn cu utilizarea grefei combinate de celule stem este o metodă inofensivă, dar particularitățile acesteia necesită studiere în continuare

The ethyology of the avascular necrosis of the femoral head

Laboratory of Tissue Engineering and Cells Culture, Nicolae Testemitanu State University of Medicine and Pharmacy of the Republic of MoldovaIntroduction. Avascular necrosis (AVN) is the disease characterized by a vascular insult to the blood supply of the femoral head, which can lead to necrosis of the spongiform bone followed by collapse of the femoral head with degenerative changes. It has been estimated that approximately 10 000 to 20 000 new cases are diagnosed in the USA each year and there are 300 000 – 600 000 people diagnosed with AVN. Aim of the study. To elucidate the actual status in etiology of AVN of femoral head. Material and methods. The following databases were used for articles search: Pubmed, Embrase, Hinary, Web of Science, Medline, Sciencedirect, for searching articles. We have selected and studied 74 articles containing the keywords: AVN of the femural head, etiology of AVN, genetic disorders in AVN. Results. Traumatic aseptic necrosis of the femoral head appears as results of mechanical disruption of blood flow to the femoral head. The non-traumatic causes of secondary AVN of the femoral head are: chronic alcohol consumption (20–40%), corticosteroid therapy (35–40%), after organ transplant, haematologic disease (anemia, polycythemia, hemophilia, thalassemia), clotting diseases, connective tissue disease, infiltrating diseases; some endocrine diseases (Cushing disease, hyperparathyroidism), metabolic diseases (gout, hyperuricemia, high cholesterol), congenital diseases (congenital sprain hip joint, Legg–Calvé–Perthes disease), Caisson disease, pancreatitis, chronic renal failure, hemodialysis, chronic liver disease, HIV infection, pregnancy, chemo- and radio- therapy, thrombophlebitis. Approximately 10 to 20% of cases do not have any identifiable risk factors and are therefore considered to be idiopathic in nature. It has been shown that some genes are involved in the pathogenesis of AVN: ADH2, ADH3, ALDH2 and P450E1. These genes are involved in the alcohol metabolism and polymorphisms of these genes have been associated with the risk of AVN. Jones et al. found that approximately 82% of patients in their study had at least one coagulation factor abnormality. Familial forms of AVN of the femoral head appear to be very rare, with only a few families reported in the medical literature. Liu et all. noted that a COL2A1 gene mutation in certain families predisposed to development of AVN of the femoral head by autosomal dominant transmission. Conclusions. 1. Avascular necrosis of the femoral head is especially common among young people, affecting mainly men. Often an underlying cause cannot be determined. 2. Aseptic necrosis of the femoral head is a disease whose etiology is not completely elucidated while the actual role of the genetic disorders in this pathology is to be determined

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

Background: 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