Antigenic and biodegradable characteristics of the extracellular matrices from the pig derm

Introducere. Pansamentele moderne și inteligente devin din ce în ce mai căutate. Valorile lor constau în barieră de protecție, în mediu benefic, biocompatibilitate, autodizolvare, capacitate de absorbție a fluidelor, transfer de agenți terapeutici, implicare umană minimă, susțin vindecarea rănilor. Scopul lucrării. Scopul a fost evaluarea proprietăților antigenice și biodegradabile ale matricelor extracelulare obținute din dermul porcin. Material si metode. Examinarea probelor decelularizate s-a realizat prin examenul histologic cu hematoxilin-eozină, cuantificarea acizilor dezoxiribonucleici, testul de degradare a grefelor. În testul de absorbție a apei, a fost utilizat PBS cu pH 7,4. Greutatea probelor a fost de 87,9±3 mg pentru toate grupurile de studiu. Urmărirea dezorganizării in vitro a probelor s-a efectuat prin microscopie electronică cu scanare. Rezultate. Examenul histologic a evidențiat prezența a mai puține celule. Ca rezultat, am eliminat 80,5% din materialul genetic din structurile dermice porcine, demonstrat prin cuantificarea spectrofotometrică a ADN-ului. În studiul de degradare a grefei in vitro în soluție de PBS 0,01 M, am determinat o pierdere semnificativă (p < 0,05) a masei grefei cu 90,3% în pH 7,4 în ziua 28, 79,8% la pH 4,0 în ziua 21 și 74% în pH 10,0. în ziua 28 și 91,3% în PBS pH 7,4 combinat cu colagenază din Clostridium histolyticum la 35 de ore. În testul de absorbție am obținut o variabilă în funcție de timpul de expunere, respectiv probele înmuiate au ajuns să depășească de patru ori masa inițială de 87,9±3 mg la a 4-a oră de scufundare în lichid. Concluzii. Grefele acelulare din derma porcină pot juca un rol cheie în îngrijirea rănilor și în facilitarea strategiilor de inginerie tisulară, acționând ca o schelă acelulară și inertă imunologic, ca sursă de molecule bioactive cu trei proprietăți hidrofile și biodegradabile.Background. Modern and intelligent dressings are becoming increasingly sought after. Their values consist in a protective barrier, in beneficial environment, biocompatibility, self-dissolution, ability to absorb the fluids, transfer of therapeutic agents, minimal human involvement, and support the wound healing. Objective of the study. Purpose was to evaluate the antigenic and biodegradable properties of the extracellular matrices obtained from the porcine dermis. Material and methods. The examination of the decellularized samples was carried out by the histological examination with hematoxylin-eosin, quantification of deoxyribonucleic acids, and degradation test of the grafts. In the water absorption test, PBS with pH 7.4 was used. The weight of the samples was 87.9±3 mg for all study groups. In vitro disorganization of the samples followed by scanning electron microscopy. Results. Histological examination revealed the presence of fewer cells. As a result, we were able to remove 80.5% of the genetic material from the porcine dermal structures, demonstrated by spectrophotometric DNA quantification. In the in vitro graft degradation study in 0.01 M PBS solution, we determined a significant (p < 0.05) loss of graft mass by 90.3% in pH 7.4 at day 28, 79.8% at pH 4.0 at day 21 and 74% in pH 10.0 at day 28 and 91.3% in PBS pH 7.4 combined with collagenase from Clostridium histolyticum at 35 hours. In the absorption test, we obtained a variable depending on the exposure time, respectively the soaked samples ended up exceeding four times the initial mass of 87.9±3 mg at the 4th hour of immersion in the liquid. Conclusions. Acellular grafts from the porcine dermis can play a key role in the wound care and facilitating tissue-engineering strategies by the acting as an acellular and immunologically inert scaffold, as a source of the bioactive molecules with the hydrophilic and biodegradable properties

Efficiency of amniotic membrane transplantation in the management of limbal stem cell deficiency

State University of Medicine and Pharmacy “Nicolae Testemiţanu”, Department of Ophthalmology-Optometry, Chișinău, Republic of Moldova, State University of Medicine and Pharmacy “Nicolae Testemiţanu”, Department of Surgery no. 2, Chișinău, Republic of Moldova, State University of Medicine and Pharmacy “Nicolae Testemiţanu”, Department of Medical Rehabilitation, Physical Medicine and Manual Therapy, Chișinău, Republic of Moldova, Laboratory of Tissue Engineering and Cell Cultures, Chișinău, Republic of Moldova, Congresul consacrat aniversării a 75-a de la fondarea USMF ”Nicolae Testemițanu” 21-23 octombrie 2020Abstract. Objectives. This paper aims to examine the efficacy, safety, and long term outcomes of amniotic membrane transplantation for corneal surface reconstruction, in cases of limbal stem cell deficiency. Material and methods. A systematic literature search was performed on PubMed, for papers published up to February 2020, using the following combined search terms: "limbal stem cell deficiency", "amniotic membrane", "limbal transplant". Only clinical trials with human subjects were selected for analysis. We collected the data on amniotic membrane properties and mechanisms of action, processing, preservation and transplantation techniques, and clinical outcomes of different treatment methods. Results. The surgical approach for treating limbal stem cell deficiency depends on the extent of the disease. Isolated amniotic membrane transplantation appears to have a limited beneficial effect on limbal stem cells, whereas amniotic membrane transplantation, combined with certain types of limbal stem cell transplantation, provides long-term biological and mechanical support for the donor tissue explants. Combined with simple limbal epithelial transplantation, the amniotic membrane has shown excellent results in the surgical management of limbal stem cell deficiency. Conclusions. Preliminary results of amniotic membrane use in limbal transplantation show quite satisfactory data, but the lack of high-level randomized controlled studies makes it difficult to assess the comparative efficacy of amniotic membrane transplantation in limbal stem cell deficiency surgical management

Analysis of amniotic membrane processing in the Human Tissue and Cell Bank

Introduction. Since 2014, the amniotic membrane has been collected and preserved at the Human Tissue Bank (HTB) for use by ophthalmologists and combustologists. In recent years, the need for amniotic membrane grafts increased [1,2]. This has multiple applications in regenerative medicine. Its epithelial and mesenchymal cells are widely used in several components of modern medicine [3]. The amniotic membrane is a rich source of biologically active factors, promotes healing and acts as an effective wound dressing [4]. Materials and methods. The placenta is taken by caesarean section only after the informed consent has been signed by the donor. Serological tests for placenta sampling include human immunodeficiency virus, hepatitis B and C viruses, and syphilis, which must be negative. The amniotic membrane is prepared under sterile conditions, in the clean room. The primary method of processing the amniotic membrane (disinfection stage) includes washing with physiological solution, after which, for 24 hours, the amniotic membrane is kept in an antibiotic environment. In the second processing stage, the amniotic membrane is cut into pieces, depending on the purpose of use, and placed in culture medium and glycerol in a ratio of 1:1. The code, date, dimensions, and area of the graft are indicated on each kit. Amniotic membrane can be stored in a freezer at -80 degrees C for 5 years. Results. Starting from 2014, 102 placentas were taken for HTB, of which 3 were unvalidated (positive tests of the donor's serum). In total, 928 amniotic membrane grafts were obtained and preserved, of which 639 grafts for ophthalmological use and 289 for use in burn patients. In total, 855 amniotic grafts were released and used, of which 578 with an ophthalmological score and 277 with a combustiological purpose. Conclusions. The amniotic membrane can be preserved in different conditions, the cryopreservation method in glycerol or dimethyl sulfoxide or their mixture with culture medium is most often used. In the future, it is planned to manufacture and use decellularized and freeze-dried amniotic membrane, which will be processed and stored at HTB

3D printing in tissue engineering

Background. 3D bioprinting is an additive technology that uses bio-inks and biocompatible materials for three-dimensional tissue engineering. Bioprinting is an interdisciplinary field that combines medicine, engineering and materials science. Bioprinting can provide an alternative to autologous and allogeneic tissue implants, as well as replace animal testing for the study of diseases and the development of personalized treatments. Materials and methods. For the bioprinter, the frame from a Computer Numerical Control (CNC) machine was used, made of metal, for the axes there are trapezoidal screws T8 and linear guide MGN 12, as motors 42HSC4416-235N8-120 are used, DVR8825 drivers are used to control the motors, Arduino Mega and RAMPS 1.4 expansion board are used as control board, the user interface was made through LCD 2004 model, we used S-300-12 as power supply. The extrusion system is based on the piston action principle, we used 28H30H0604A2 stepper motor, with DVR8825 driver, the motor is connected to a T8 trapezoidal screw and MGN12 linear guide, some interconnection parts of the extrusion system were printed on the 3D printer Ultimaker 2+ extended, made of PLA plastic, they support the syringe and tubing from an infusion system, a G20 i/v cannula is used as a nozzle. Results. During the creation of the printer, some problems arose such as setting the extrusion speed depending on the density of the material used, the size of the nozzle and the diameter of the microperfusion system. This parameter is of great importance to achieve the desired accuracy. Also the precision of the axial movement of the extruder and the printing surface are important for creating the correct geometry. Conclusions. 3D printing and the great diversity of materials used in this process has revolutionized the medical field, especially in the manufacture of patient-specific implants and prostheses. Bioprinting has great potential in tissue engineering applications in the research phase and current in vitro and in vivo experiments and represents a near-future solution to the needs of modern transplant medicine

Managementul transplantului de cornee în banca de țesuturi și celule umane din Republica Moldova, pe parcursul anilor 2013-2019

Background. Tissue Bank serves with corneal grafts a population of 4 million people, which offers over 40 corneas per year for transplant in the Republic of Moldova. Objective of the study. Evaluation of the cornea transplant application rate in the Republic of Moldova the period of years 2013 - 2019. Material and Methods. Prospective study, electronic database of Tissue Bank for the period of 7 years 2013 - 2019 were analyzed for each year in terms of the donor number, the indications for the sampling, cause of death, interval from death to corneal preservation, storage methods, endothelial evaluation, bacteriological contamination and distribution. Results. During the study period, 306 corneas were taken from 153 donors (69,8% male, 30,2% female), with a mean age of donors 59,4 years (18,3 years SD) and between 18 and 91 years old. Donors were from forensic medicine (23,5%), public hospitals (67,6%) and multi-organ donors (7.1%). The most common causes of the donor deaths were the cardiovascular disease, trauma and the cerebrovascular diseases. The average storage time increased from 3,5 to 11,8 days, from when the culture medium replaced hypothermic storage. Invalidation of the corneas was in 22,8% of cases, of which were determined by serological infections ( HBsAg - positive, HCV positive, HIV / AIDS). Conclusion. The analysis of the Tisue Bank database provides valuable information about corneal transplantation in the Republic of Moldova. Introducere. Banca de țesuturi deservește cu grefe corneene o populație de 4 milioane de oameni, ce oferă peste 40 de cornee pe an pentru transplant în Republica Moldova. Scopul lucrării. Evaluarea ratei de solicitare a transplantului de cornee în Republica Moldova în anii 2013-2019. Material și Metode. Studiul prospectiv, înregistrările electronice ale BȚCU pentru perioada de 7 ani, 2013-2019, au fost analizate pentru fiecare an în ceea ce privește numărul donatorilor, indicațiile de prelevare, cauza morții, intervalul de la moarte până la conservarea corneei, metodele de stocare, evaluarea endotelială, contaminarea bacteriologică și distribuirea. Rezultate. În perioada de studiu, 306 de cornee au fost prelevate de la 153 de donatori (69,8% bărbați, 30,2% femei), cu vârsta medie a donatorilor 59,4 ani (18,3 ani SD) și între 18 și 91 de ani. Donatorii au fost de la medicina legală (23,5%), spitale publice (67,6%) și donatori multiorgan (7,1%). Cele mai frecvente cauze ale decesului donatorului au fost bolile cardiovasculare, traumatismele și bolile cerebrovasculare. Durata medie de depozitare a crescut de la 3,5 la 11,8 zile, de când mediul de cultură a înlocuit depozitarea hipotermică. Nevalidarea corneelor a fost în 22,8 % cazuri, din care din ei au fost determinate cu infecții serologice(AgHBs – pozitiv, HCV pozitiv, HIV/Sida). Concluzii. Analiza bazei de date a băncii de țesuturi oferă informații valoroase cu referire la transplantul de cornee în Republica Moldova. Rata de utilizare a corneei a crescut pe parcursul fiecărui an al studiului, reflectând ameliorări în toate domeniile de operare BȚCU, în special, depozitarea corneei

Acupoint embedding therapy

Laboratory of Tissue Engineering and Cell Cultures, Department of Rehabilitation Nicolae Testemitsanu State University of Medicine and Pharmacy, Chisinau, the Republic of MoldovaBackground: Peripheral nerve trauma remains a major cause of motor disability, at the same time functional restoration after treatment continues to show modest results. Acupoint embedding therapy is a type of acupuncture treatment in which different biodegradable materials are inserted into specific points for long-term stimulation. It has a good analgesic effect in chronic pain, and it is considered a cure for many diseases. Different biodegradable materials have been developed and widely used. Catgut has a good biodegradability and low price, but it could cause infections and having unstable chemical properties had been limited in clinical use. Such synthetic materials as polylactic acid and polyglycolic acid present low-cost, good biodegradability and biocompatibility compared with the catgut. However, their poor hydrophilicity and cell adhesion limited their therapeutic efficacy. The ideal embedding materials are required to be safe, non-toxic, biocompatible, and to have excellent swelling and biodegradation behaviors. Acupoint embedding therapy can be a promising treatment method of peripheral nerve disorders. Conclusions: Acupoint embedding therapy is an invasive treatment which can prolong point stimulation, reduces the frequencies of pain and psychological fear of patients. It seems to be a promising method of neuropathy treatment. The properties of the filaments for acupoint embedding therapy can be improved by surface modification technologies

OXYGEN CONSUMPTION DETERMINATION ADMINISTERING BENZITURONE

Pharmacology and Clinical Pharmacology Department, Nicolae Testemitanu State University of Medicine and Pharmacy, Chisinau, Republic of Moldova, The 6th International Medical Congress for Students and Young DoctorsIntroduction. Research of new isothiourea derivatives has reached significant proportions in recent years. Generally, they are known as effective vasoconstrictor substances possibly to be used in arterial hypotension. The last studies of these compounds undermined a substance with hypotensive effect chloride-S-benzilizotiourone (benziturone). Goals. Benziturone influence experimental elucidation of oxygen consumption in laboratory animals. Materials and methods. Oxygen consumption was determined within 3 min using S.V. Miropolski system at the time intervals: 1-3 min; 5-8 min; 15-18 min; 30-33 min; 60-63 min; 120-123 min. The experience included 2 groups of rats of the Wistar line, 10 in each, weighing 208-320g. The rats from the control group were administered 2 ml of saline solution intraperitoneally, those in the test group, benziturone in the dose of 2 mg / kg. Statistical study according to t-Student criterion. Results. In the time intervals 1-3 min; 5-8 min; 15-18 min; 30-33 min significant statistical differences of the mean value of oxygen consumption between the test group and control group were not determined. Conversely a difference in the mean value of the control group was observed: 19.61 ± 0.95 in 60-63 min; 17.54 ± 0.43 min in 120-123 and test group: 14.36 ± 1.33 in 60-63 min; 11.22 ± 1.55 in 120-123 min, where p = 0.004 for 60-63 min; and for 120-123 min p = 0.001 Conclusions: As a result of experiments a decrease in oxygen consumption was observed due to benziturone administration comparing with the control group. The decrease was significant starting with the minute 60

10 years of activity of the human tissue bank in the field of cornea sampling and processing from the Republic of Moldova

Introduction. The cornea is the window of the eye, allowing light to reach the sensory cells that allow us to see the world around us. In the TUB from the Republic of Moldova, the corneas with a long storage period are the lyophilized ones, with a period of 2 years, and those with a short period are kept in culture media (Tissu ,,C"), dehydration (Carry ,,C") and transport (Eusol ,,C") [1]. Material and methods. The conducted study presents the evaluation of cornea sampling, processing and validation in TUB over the 10-year period 2013 - 2022 for 395 corneas, from 202 donors (69.8% men, 30.2% women), with an average donor age of 59 .4 years (SD 18.3 years) and between 18 and 91 years. Donors were from forensic medicine (23.5%), public hospitals (67.6%) and multi-organ donors (7.1%). The most common causes of donor death were cardiovascular disease, trauma, and cerebrovascular disease. Invalidation of the cornea was in 25.4% of cases, of which they were determined by serological infections (HBsAg-positive, HCV-positive, HIV/AIDS) - 15%, and biological contamination occurred in 7.8% of the total donor cornea. In total (294 corneas), 74.6% of the processed corneal tissue was used for corneal transplantation (74.8% for penetrating keratoplasty, 2.1% for lamellar keratoplasty, and 1.3% for unspecified transplants) and 25, 4% (101 corneas) were destroyed [2]. Results.The corneas from TUB, during the period 2013-2022, were evaluated in a macro and microscopic study that determined 3 important groups: the first group (160 donors) up to 10 hours of sampling from death - the anterior surface of the cornea was most frequently determined cornea with edema of the epithelium, stroma absolutely "transparent, not thickened, rare short folds, very thin Descemetov membrane, endothelial layer is completely transparent, intact on the entire surface[5]. Areas with uniform redistribution of cells, preferentially at the edge of the cornea and the middle area. Density of endothelial cells being greater than 2800 cells / mm2, with moderate signs of polymegetism, cellular pleomorphism, being considered as indications for transfixing keratoplasty. The corneas from the second group (30 donors) - with the sampling period from 10 to 15 hours - the surface of the epithelium is slightly edematous, its integrity is not compromised (exception may be a minor mechanical desquamation). Stroma with initial signs of edema in the lower layers, not thickened, transparent. Descemet's membrane has a single smooth plica, located centro-radially; the endothelial layer is intact. The endothelial layer is arranged uniformly, with the persistence of the mosaic, slightly tumified, which counts 26 cells in a square that forms an average of 2600 cells per mm2 . The corneas from group III (12 donors) with the sampling period after 15 hours - edematous anterior epithelium, in some areas exfoliated with detachment of Bowman's membrane, sometimes mosaic desquamation is observed. The stroma is edematous throughout the layer, dull in color. Descemetov membrane has pronounced folds, the folds directed in different directions like "parquet floor" or "checkerboard". The endothelial layer is matte, interrupted along the contour of the envelopes that appear transparent. Microscopically, endothelial cells reach the figure of 2000 per mm2 [3]. Conclusions.1. The analysis of the clinical and socio-demographic factors of the donation process associated with the quality of the corneal tissue showed the importance of implementing TUB quality control programs, to promote the selection of good quality corneal tissues and guarantee a donation process with donor identification mechanisms, extraction, preservation and distribution of corneal tissue guided by best practices that aim to minimize the risk of compromising tissue quality.2. The quality of the corneal tissue is a fundamental factor for the success of transplantation and to guarantee good quality tissues, it is important that the time limits between death and enucleation, death and preservation, and enucleation and preservation are established by the TUB, in order to minimize the risks to which tissues are exposed due to chronological factors related to the sampling process. 3. The best quality of the cornea is that of group I, which had a sampling time of up to 10 hours, which defined the density of endothelial cells as 2800 cells / mm2 , with moderate signs of polymegetism, cellular pleomorphism, being considered as indications for transfixing keratoplasty

Structural and physical characteristics of the dermal decellularized structures evaluation

Introduction: Decellularized biomaterials derived from the biological tissues are ideal for tissue engineering applications because they mimic the biochemical composition of the native tissue. The physical and structural properties of the scaffold are important in the fields of tissue engineering and regenerative medicine. Material and methods: Study material was 20 decellularized dermal grafts. 10 samples were obtained from piglets slaughtered in the slaughterhouse. Other tissues (n=10) were received from the donor from the Human Tissue and Cell Bank of the Republic of Moldova. Extracellular matrices were obtained by decellularization with 0.5% sodium dodecyl sulfate/0.1% EDTA solution. The evaluation of the structural characteristics was carried out by the histological examination with hematoxylin and eosin, scanning electron microscopy and the quantification of the amount of deoxyribonucleic acids. Assessment of the physical characteristics included analysis of extracellular matrix volume porosity, density, and swelling rate. Results: Histological examination revealed fewer cells in decellularized tissues compared to non-decellularized ones. More than 80.5% of nucleic acids were removed from porcine matrix and 82.5% of genetic material – from decellularized human dermal structures. A mean correlation and inverse dependence of -0.43 was shown between porosity and swelling rate of decellularized dermis. Conclusions: The decellularization process significantly (P<0.05) removed the cellular components while preserving the connective three-dimensional structure of the dermal matrices clearly shown by quantification of the amount of DNA and microscopic examination of the structures

Morphological evaluation of the amniotic membrane decellularization

Background: Biological materials derived from decellularized tissues could be a good basis for progress in regenerative medicine while maintaining the main components of the extracellular matrix. A promising scaffold for tissue-engineered is the human amniotic membrane. It is one of the oldest biomaterials used for scaffolds. Material and methods: 3 placentas were obtained through Human Tissue Bank. Under sterile condition human amniotic membrane was collected. The human amniotic membrane was treated with 0.5% of sodium dodecyl sulphate (SDS), 1% Triton for 24 and 5 hours. Amniotic membrane decellularization was also carried out in combination with ultrasound bath for 20 minutes 3 times. For morphological and structure evaluation of human amniotic membrane the scanning electron microscopy of native amniotic membrane and histology of decellularized and native amniotic membrane were performed. Results: The human amniotic membrane decellularization process with 0.5% SDS solution and 1% Triton solution showed that decellularization for 24 hours is too aggressive for human amniotic membrane structure. The decellularization for 5h with 1% Triton solution was incomplete. Conclusions: The method of decellularization with 0.5% SDS solution is more suitable for amniotic membrane decellularization and can be performed in only 5 hours. The use of ultrasound bath did not have a significant effect on the obtained results