BACKGROUND: This study was conducted to examine the hypothesis that umbilical cord blood platelet lysate (UCB-PL) gel has a significant impact on the healing rate of DFU. Μethods: In this open-labeled, randomized controlled trial, 110 patients were randomized to treatment with UCB-PL gel (UCB-PL group, n = 52) every three days for one month or dressing with normal saline (control group, n = 58). All participants were followed up for 20 weeks post treatment. Ulcer surface area was assessed with the imitoMeasure application at two, four, and six weeks, and two, four and six months. This study's main outcome was the reduction in ulcer size over the six-month study period. RESULTS: The mean ulcer area at baseline was 4.1 cm2 in the UCB-PL group and 1.7 cm2 in the control group. At six months post treatment, patients on the UCB-PL treatment displayed a significant reduction in ulcer size compared to baseline 0.12 (0-8.16) in contrast to a more modest change in the control group 1.05 (0-24.7). The ulcer area was decreased at the end of the study in 40 patients (97.6%) in the UCB-PL group and 27 (73%) in the control group (Fisher's p = 0.002). CONCLUSIONS: The application of UCB-PL gel in DFU resulted in a significant reduction in ulcer size compared to regular saline dressing.
Human leukocyte antigens (HLA) represent one of the most polymorphic systems in humans, responsible for the identification of foreign antigens and the presentation of immune responses. Therefore, HLA is considered to play a major role in human disorders, donor-recipient matching and transplantation outcomes. This study aimed to determine the HLA class I and II alleles and haplotypes in the Greek population. Moreover, a comparative analysis of HLA alleles and haplotype frequencies found in Greek and pooled European populations was also performed to acquire a better knowledge about the HLA alleles distribution. A total number of 1896 healthy individuals were typed for their HLA alleles in the National Tissue Typing Center of Greece. High-resolution HLA typing for the HLA-A, -B, -C and -DR, -DQ, -DP with the use of the next-generation sequencing analysis was performed, followed by data analysis for establishing the HLA allele and haplotype differences. The results of this study showed that the most frequent alleles for the HLA-A were the A*02:01:01 (27.1%), *24:02:01 (14.4%), *01:01:01 (9.3%), for the HLA-B were the B*51:01:01 (15.3%), *18:01:01 (9.7%), *35:01:01 (6.8%) and for the HLA-C were the C*04:01:01 (15.4%), *07:01:01 (13.1%), *12:03:01 (9.6%). For the HLA class II, the most frequent alleles for the HLA-DRB1 were the DRB1*11:04:01 (16.4%), *16:01:01 (11.3%), *11:01:01 (9.5%), for the HLA-DQB1 were the DQB1*03:01:01 (30.5%), *05:02:01 (15.1%), *05:01:01 (10.6%) and for the HLA-DPB1 were the DPB1*04:01:01 (34.8%), *02:01:01 (11.6%), *04:02:01 (7.3%). Additionally, the most frequent haplotypes were the A*02:01:01â¼C*07:01:01-B*18:01:01â¼DRB1*11:04:01 (2.3%), followed by the A*01:01:01â¼C*07:01:01â¼B*08:01:01â¼DRB1*03:01:01 (2.2%), A*24:02:01â¼C*04:01:01â¼B*35:02:01â¼DRB1*11:04:01 (1.4%) and A*02:01:01â¼C*04:01:01â¼B*35:01:01-DRB1*14:01:01 (1.2%). The results herein were comparable to those obtained from the pooled European populations. Moreover, these results can be used for the improvement of the donor-recipient matching procedure and to understand better the disease association in Greece.
Alleles , Gene Frequency , Haplotypes , Registries , Tissue Donors , Humans , Greece , Histocompatibility Testing , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class II/genetics , Male , Female , Genetics, Population
Advanced Therapy Medicinal Products (ATMPs) comprise a heterogenous class of innovative medicinal products, which further require extensive preclinical and clinical assessments before their broader use in the general population [...].
COVID-19 , Humans , Cell- and Tissue-Based Therapy , Genetic Therapy
BACKGROUND: Functional restoration of abdominal wall defects represents one of the fundamental challenges of reconstructive surgery. Synthetic grafts or crosslinked animal-derived biological grafts are characterized by significant adverse reactions, which are mostly observed after their implantation. The aim of this study was to evaluate the efficacy of the decellularization protocol to produce a completely acellular full-thickness abdominal wall scaffold. METHODS: Full-thickness abdominal wall samples were harvested from Wistar rats and submitted to a three-cycle decellularization process. Histological, biochemical, and DNA quantification analyses were applied to evaluate the effect of the decellularization protocol. Mechanical testing and immunogenicity assessment were also performed. RESULTS: Histological, biochemical, and DNA analysis results showed efficient decellularization of the abdominal wall samples after the third cycle. Decellularized abdominal wall scaffolds were characterized by good biochemical and mechanical properties. CONCLUSION: The data presented herein confirm the effective production of a rat-derived full-thickness abdominal wall scaffold. Expanding this approach will allow the exploitation of the capacity of the proposed decellularization protocol in producing acellular abdominal wall scaffolds from larger animal models or human cadaveric donors. In this way, the utility of biological scaffolds with preserved in vivo remodeling properties may be one step closer to its application in clinical studies.
Since the first appearance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in December 2019, the disease has displayed a remarkable interindividual variability in the global population, resulting in different mortality and morbidity rates. Still, an effective cure against SARS-CoV-2 has not been developed, and therefore, alternative therapeutic protocols must also be evaluated. Considering that stem cells, especially Mesenchymal Stromal Cells (MSCs), are characterized by both regenerative and immunomodulatory properties and that their safety and tolerability have been investigated previously, these cells could potentially be applied against coronavirus disease 19 (COVID-19). In addition, an individual's genetic background is further related to disease pathogenesis, especially rare Inborn Errors of Immunity (IEIs), autoantibodies against Interferon type I, and the presence of different Human Leukocyte Antigens (HLA) alleles, which are actively associated with protection or susceptibility in relation to SARS-CoV-2. Herein, the use of MSCs as a potential stem cell therapy will require a deep understanding of their immunomodulatory properties associated with their HLA alleles. In such a way, HLA-restricted MSC lines can be developed and applied precisely, offering more solutions to clinicians in attenuating the mortality of SARS-CoV-2.
BACKGROUND: Most public cord blood (CB) banks currently discard more than 80% of umbilical CB units not suitable for hemopoietic stem cell transplant due to low stem cell count. Although CB platelets, plasma, and red blood cells have been used for experimental allogeneic applications in wound healing, corneal ulcer treatment, and neonatal transfusion, no standard procedures for their preparation have been defined internationally. MATERIALS AND METHODS: A network of 12 public CB banks in Spain, Italy, Greece, the UK, and Singapore developed a protocol to validate a procedure for the routine production of CB platelet concentrate (CB-PC), CB platelet-poor plasma (CB-PPP), and CB leukoreduced red blood cells (CB-LR-RBC) using locally available equipment and the commercial BioNest ABC and EF medical devices. CB units with >50 mL volume (excluding anticoagulant) and ≥150×109/L platelets were double centrifuged to obtain CB-PC, CB-PPP, and CB-RBC. The CB-RBC were diluted with saline-adenine-glucose-mannitol (SAGM), leukoreduced by filtration, stored at 2-6°C, and tested for hemolysis and potassium (K+) release over 15 days, with gamma irradiation performed on day 14. A set of acceptance criteria was pre-defined. This was for CB-PC: volume ≥5 mL and platelet count 800-1,200×109/L; for CB-PPP: platelet count <50×109/L; and for CB-LR-RBC: volume ≥20 mL, hematocrit 55-65%, residual leukocytes <0.2×106/unit, and hemolysis ≤0.8%. RESULTS: Eight CB banks completed the validation exercise. Compliance with acceptance criteria was 99% for minimum volume and 86.1% for platelet count in CB-PC, and 90% for platelet count in CB-PPP. Compliance in CB-LR-RBC was 85.7% for minimum volume, 98.9% for residual leukocytes, and 90% for hematocrit. Compliance for hemolysis ≤0.8% decreased from 89.0 to 63.2% from day 0 to 15. K+ release increased from 3.0±1.8 to 25.0±7.0 mmol/L from day 0 to 15, respectively. DISCUSSION: The MultiCord12 protocol was a useful tool to develop preliminary standardization of CB-PC, CB-PPP, and CB-LR-RBC.
Blood Banking , Hemolysis , Infant, Newborn , Humans , Erythrocytes , Blood Banks , Blood Platelets
Background: The intraovarian administration of autologous platelet-rich plasma (PRP) acts beneficially for the stimulation of follicle production in women presenting different forms of ovarian dysfunction. This pilot study aimed to evaluate and provide significant data regarding the efficacy of PRP to rejuvenate the ovaries. Methods: A total of 253 women aged 22-56 years, were divided into five groups, based on their status. All participants signed for informed consent for the current study. Blood sampling, preparation of PRP and intraovarian infusion of the latter were performed on all participants. The evaluation of PRP efficacy, a two-month follow-up detecting the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and anti-mullerian hormone (AMH), was performed for all participants. For women with advanced ages (>48 years), the restoration and regularity of the menstrual cycle were additionally evaluated. Results: After the two-month follow-up, the majority of the participants presented improvement in their hormonal profiles. Additionally, 17% of the women in this pilot study successfully conceived. The restoration of the menstrual cycle was detected in 15% of the women with advanced ages. Conclusions: Intraovarian infusion of autologous PRP exhibited remarkable evidence and promising results to restore ovarian insufficiency.
Major key features of stem cells' functions are self-renewal and their capacity for differentiation, allowing for maintain a proper stem cell reservoir as well as producing lineage-committed cells [...].
BACKGROUND: Cord blood platelet lysate (CB-PL) and cord blood platelet poor plasma (CB-PPP) have been applied with success in wound healing applications. Pathologies such as Sjogrens's Syndrome (SS) and chronic graft versus host disease (cGVHD) can lead to severe ophthalmology issues. The application of CB-PL and CB-PPP may be strongly considered for damaged cornea healing. This study aimed to the evaluation of the beneficial properties of CB-PL and CB-PPP in corneal wound healing applications. METHODS: Initially, the CB-PL and CB-PPP were produced from donated cord blood units (CBUs), followed by biochemical analysis. Corneal epithelial cells (CECs) were isolated from wistar rats and then cultured with medium containing 20% v/v either of CB-PL or CB-PPP. To define the impact of CB-PL and CB-PPP, biochemical, morphological analysis, scratch-wound assays, and immunoassays in CECs were performed. RESULTS: CB-PL and CB-PPP were characterized by good biochemical parameters, regarding their quality characteristics and biomolecule content. CECs' morphological features did not change after their cultivation with CB-PL or CB-PPP. A scratch wound assay and molecular analysis of CECs expanded with CB-PL indicated higher migratory capacity compared to those cultured with CB-PPP. CONCLUSION: CB-PL and CB-PPP exhibited good properties with respect to cell migration and proliferation, and could be considered an alternative source for eye drop production, to possibly be used in cornea wound healing applications.
BACKGROUND: The regenerative potential of platelet lysate (PL) and platelet gel (PG) is mediated by the release of platelets (PLTs) growth factors. The aim of this study was the evaluation of the PL production utilizing low volume single Cord Blood Units (CBUs) and the comparison of the biomolecule content between PLs obtained from intermediate and high volume CBUs. METHODS: CBUs (nâ¯=â¯90) with volumes greater than 50â¯ml and initial platelet count >â¯150â¯×â¯109/L were used. CBUs were classified into the following groups: group A (50-80â¯ml), group B (81-110â¯ml) and group C (111-150â¯ml). The CBUs were centrifuged twice for the production of the platelet concentrate (PC), which was stored at -â¯80⯰C for at least 48â¯h. Then, rapidly thawed and the biomolecule content was determined using commercial ELISA kits. The regenerative potential of PLs was evaluated using the scratch wound and in vitro angiogenesis assay. RESULTS: CBPL was produced from low volume single CBUs and contained 3.4 ± 0.3 ×109 PLTs. PL obtained from intermediate and high volume CBUs consisted of 10.2⯱â¯0.3 and 16.1⯱â¯0.4â¯×â¯109 PLTs. All PL groups were characterized by high biomolecule content. Gap closure was observed within 72â¯h after the wound assay initiation and the capillary tubes were formed in all study groups. CONCLUSION: This study provided significant evidence regarding the utilization of the low volume CBUs for the production of CB derivatives, thus can serve as healing mediators in regenerative medicine approaches.
Fetal Blood , Intercellular Signaling Peptides and Proteins , Humans , Blood Platelets , Platelet Count , Wound Healing
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic, which was initiated in December 2019. COVID-19 is characterized by a low mortality rate (< 6%); however, this percentage is higher in elderly people and patients with underlying disorders. COVID-19 is characterized by mild to severe outcomes. Currently, several therapeutic strategies are evaluated, such as the use of anti-viral drugs, prophylactic treatment, monoclonal antibodies, and vaccination. Advanced cellular therapies are also investigated, thus representing an additional therapeutic tool for clinicians. Mesenchymal stromal cells (MSCs), which are known for their immunoregulatory properties, may halt the induced cytokine release syndrome mediated by SARS-CoV-2, and can be considered as a potential stem cell therapy. AIM: To evaluate the immunoregulatory properties of MSCs, upon stimulation with COVID-19 patient serum. METHODS: MSCs derived from the human Wharton's Jelly (WJ) tissue and bone marrow (BM) were isolated, cryopreserved, expanded, and defined according to the criteria outlined by the International Society for Cellular Therapies. Then, WJ and BM-MSCs were stimulated with a culture medium containing 15% COVID-19 patient serum, 1% penicillin-streptomycin, and 1% L-glutamine for 48 h. The quantification of interleukin (IL)-1 receptor a (Ra), IL-6, IL-10, IL-13, transforming growth factor (TGF)-ß1, vascular endothelial growth factor (VEGF)-a, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and indoleamine-2,3-dioxygenase (IDO) was performed using commercial ELISA kits. The expression of HLA-G1, G5, and G7 was evaluated in unstimulated and stimulated WJ and BM-MSCs. Finally, the interactions between MSCs and patients' macrophages were established using co-culture experiments. RESULTS: Thawed WJ and BM-MSCs exhibited a spindle-shaped morphology, successfully differentiated to "osteocytes", "adipocytes", and "chondrocytes", and in flow cytometric analysis were characterized by positivity for CD73, CD90, and CD105 (> 95%) and negativity for CD34, CD45, and HLA-DR (< 2%). Moreover, stimulated WJ and BM-MSCs were characterized by increased cytoplasmic granulation, in comparison to unstimulated cells. The HLA-G isoforms (G1, G5, and G7) were successfully expressed by the unstimulated and stimulated WJ-MSCs. On the other hand, only weak expression of HLA-G1 was identified in BM-MSCs. Stimulated MSCs secreted high levels of IL-1Ra, IL-6, IL-10, IL-13, TGF-ß1, FGF, VEGF, PDGF, and IDO in comparison to unstimulated cells (P < 0.05) after 12 and 24 h. Finally, macrophages derived from COVID-19 patients successfully adapted the M2 phenotype after co-culturing with stimulated WJ and BM-MSCs. CONCLUSION: WJ and BM-MSCs successfully produced high levels of immunoregulatory agents, which may efficiently modulate the over-activated immune responses of critically ill COVID-19 patients.
Cardiovascular disease (CVD) currently represents one of the leading causes of death worldwide. It is estimated that more than 17.9 million people die each year due to CVD manifestations. Often, occlusion or stenosis of the vascular network occurs, either in large- or small-diameter blood vessels. Moreover, the obstruction of small vessels such as the coronary arteries may be related to more pronounced events, which can be life-threatening. The gold standard procedure utilizes the transplantation of secondary vessels or the use of synthetic vascular grafts. However, significant adverse reactions have accompanied the use of the above grafts. Therefore, modern therapeutic strategies must be evaluated for better disease administration. In the context of alternative therapies, advanced tissue-engineering approaches including the decellularization procedure and the 3D additive bioprinting methods, have been proposed. In this way the availability of bioengineered vascular grafts will be increased, covering the great demand that exists globally. In this Special Issue of Bioengineering, we tried to highlight the modern approaches which are focused on CVD therapeutics. This issue includes articles related to the efficient development of vascular grafts, 3D printing approaches and suitable atherosclerosis models.
BACKGROUND: The development of functional bioengineered small-diameter vascular grafts (SDVGs), represents a major challenge of tissue engineering. This study aimed to evaluate the repopulation efficacy of biological vessels, utilizing the cord blood platelet lysate (CBPL). METHODS: Human umbilical arteries (hUAs, n = 10) were submitted to decellularization. Then, an evaluation of decellularized hUAs, involving histological, biochemical and biomechanical analysis, was performed. Wharton's Jelly (WJ) Mesenchymal Stromal Cells (MSCs) were isolated and characterized for their properties. Then, WJ-MSCs (1.5 × 106 cells) were seeded on decellularized hUAs (n = 5) and cultivated with (Group A) or without the presence of the CBPL, (Group B) for 30 days. Histological analysis involving immunohistochemistry (against Ki67, for determination of cell proliferation) and indirect immunofluorescence (against activated MAP kinase, additional marker for cell growth and proliferation) was performed. RESULTS: The decellularized hUAs retained their initial vessel's properties, in terms of key-specific proteins, the biochemical and biomechanical characteristics were preserved. The evaluation of the repopulation process indicated a more uniform distribution of WJ-MSCs in group A compared to group B. The repopulated vascular grafts of group B were characterized by greater Ki67 and MAP kinase expression compared to group A. CONCLUSION: The results of this study indicated that the CBPL may improve the repopulation efficacy, thus bringing the biological SDVGs one step closer to clinical application.
BACKGROUND: Renal dysfunction remains a global issue, with chronic kidney disease being the 18th most leading cause of death, worldwide. The increased demands in kidney transplants, led the scientific society to seek alternative strategies, utilizing mostly the tissue engineering approaches. Unlike to perfusion decellularization of kidneys, we proposed alternative decellularization strategies to obtain acellular kidney scaffolds. The aim of this study was the evaluation of two different decellularization approaches for producing kidney bioscaffolds. METHODS: Rat kidneys from Wistar rats, were submitted to decellularization, followed two different strategies. The decellularization solutions used in both approaches were the same and involved the use of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate and sodium dodecyl sulfate buffers for 12 h each, followed by incubation in a serum medium. Both approaches involved 3 decellularization cycles. Histological analysis, biochemical and DNA quantification were performed. Cytotoxicity assay and repopulation of acellular kidneys were also applied. RESULTS: Histological, biochemical and DNA quantification confirmed that the 2nd approach had the best outcome regarding the kidney composition and cell elimination. Acellular kidneys from both approaches were successfully recellularized. CONCLUSION: Based on the above data, the production of kidney scaffolds with the proposed cost- effective decellularization approaches, was efficient.
Extracellular Matrix , Tissue Scaffolds , Animals , Kidney , Rats , Rats, Wistar , Tissue Engineering
This article provides additional knowledge for cord blood platelet gel (CBPG) production. Recently, it has been shown that CBPG exerts beneficial properties in wound healing applications. CBPG is produced after a two-step centrifugation process, following the addition of calcium gluconate. Clinical-grade CBPG can be produced in public cord blood banks, worldwide. However, standardization of the CBPG production process must be established in order to reduce discrepancies that occurred due to different platelet gel preparations. This article aims to provide an update regarding the selection criteria of cord blood units (CBUs), and to provide evidence for the improvement of the CBPG production process. (Comment on "Short Term Results of Fibrin Gel Obtained from Cord Blood Units: A Preliminary in Vitro Study" Bioengineering 2019, 6, 66).
The increased demands of small-diameter vascular grafts (SDVGs) globally has forced the scientific society to explore alternative strategies utilizing the tissue engineering approaches. Cardiovascular disease (CVD) comprises one of the most lethal groups of non-communicable disorders worldwide. It has been estimated that in Europe, the healthcare cost for the administration of CVD is more than 169 billion . Common manifestations involve the narrowing or occlusion of blood vessels. The replacement of damaged vessels with autologous grafts represents one of the applied therapeutic approaches in CVD. However, significant drawbacks are accompanying the above procedure; therefore, the exploration of alternative vessel sources must be performed. Engineered SDVGs can be produced through the utilization of non-degradable/degradable and naturally derived materials. Decellularized vessels represent also an alternative valuable source for the development of SDVGs. In this review, a great number of SDVG engineering approaches will be highlighted. Importantly, the state-of-the-art methodologies, which are currently employed, will be comprehensively presented. A discussion summarizing the key marks and the future perspectives of SDVG engineering will be included in this review. Taking into consideration the increased number of patients with CVD, SDVG engineering may assist significantly in cardiovascular reconstructive surgery and, therefore, the overall improvement of patients' life.
Severe acute respiratory syndrome coronavirus-2 and the related coronavirus disease-19 (COVID-19) is a worldwide emerging situation, which was initially reported in December 2019 in Wuhan, China. Currently, more than 7258842 new cases, and more than 411879 deaths have been reported globally. This new highly transmitted coronavirus is responsible for the development of severe acute respiratory distress syndrome. Due to this disorder, a great number of patients are hospitalized in the intensive care unit followed by connection to extracorporeal membrane oxygenation for breath supporting and survival. Severe acute respiratory distress syndrome is mostly accompanied by the secretion of proinflammatory cytokines, including interleukin (IL)-2, IL-6, IL-7, granulocyte colony-stimulating factor (GSCF), interferon-inducible protein 10 (IP10), monocyte chemotactic protein-1 (MCP1), macrophage inflammatory protein 1A (MIP1A), and tumor necrosis factor alpha (TNF-α), an event which is known as "cytokine storm". Further disease pathology involves a generalized modulation of immune responses, leading to fatal multiorgan failure. Currently, no specific treatment or vaccination against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been developed. Mesenchymal stromal cells (MSCs), which are known for their immunosuppressive actions, could be applied as an alternative co-therapy in critically-ill COVID-19 patients. Specifically, MSCs can regulate the immune responses through the conversion of Th1 to Th2, activation of M2 macrophages, and modulation of dendritic cells maturation. These key immunoregulatory properties of MSCs may be exerted either by produced soluble factors or by cell-cell contact interactions. To date, several clinical trials have been registered to assess the safety, efficacy, and therapeutic potential of MSCs in COVID-19. Moreover, MSC treatment may be effective for the reversion of ground-glass opacity of damaged lungs and reduce the tissue fibrosis. Taking into account the multifunctional properties of MSCs, the proposed stem-cell-based therapy may be proven significantly effective in critically-ill COVID-19 patients. The current therapeutic strategy may improve the patient's overall condition and in parallel may decrease the mortality rate of the current disease.
The gold standard vascular substitutes, used in cardiovascular surgery, are the Dacron or expanded polytetrafluoroethylene (ePTFE)-derived grafts. However, major adverse reactions accompany their use. For this purpose, decellularized human umbilical arteries (hUAs) may be proven as a significant source for the development of small diameter conduits. The aim of this study was the evaluation of a decellularization protocol in hUAs. To study the effect of the decellularization to the hUAs, histological analysis was performed. Then, native and decellularized hUAs were biochemically and biomechanically evaluated. Finally, broad proteomic analysis was applied. Histological analysis revealed the successful decellularization of the hUAs. Furthermore, a great amount of DNA was removed from the decellularized hUAs. Biomechanical analysis revealed statistically significant differences in longitudinal direction only in maximum stress (p < 0.013) and strain (p < 0.001). On the contrary, all parameters tested for circumferential direction exhibited significant differences (p < 0.05). Proteomic analysis showed the preservation of the extracellular matrix and cytoskeletal proteins in both groups. Proteomic data are available via ProteomeXchange with identifier PXD020187. The above results indicated that hUAs were efficiently decellularized. The tissue function properties of these conduits were well retained, making them ideal candidates for the development of small diameter vascular grafts.
BACKGROUND: The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations. Mesenchymal stromal cells (MSCs) derived from the Wharton's Jelly (WJ) tissue can be used as a source for obtaining vascular smooth muscle cells (VSMCs), while the human umbilical arteries (hUAs) can serve as a scaffold for blood vessel engineering. AIM: To develop VSMCs from WJ-MSCs utilizing umbilical cord blood platelet lysate. METHODS: WJ-MSCs were isolated and expanded until passage (P) 4. WJ-MSCs were properly defined according to the criteria of the International Society for Cell and Gene Therapy. Then, these cells were differentiated into VSMCs with the use of platelet lysate from umbilical cord blood in combination with ascorbic acid, followed by evaluation at the gene and protein levels. Specifically, gene expression profile analysis of VSMCs for ACTA2, MYH11, TGLN, MYOCD, SOX9, NANOG homeobox, OCT4 and GAPDH, was performed. In addition, immunofluorescence against ACTA2 and MYH11 in combination with DAPI staining was also performed in VSMCs. HUAs were decellularized and served as scaffolds for possible repopulation by VSMCs. Histological and biochemical analyses were performed in repopulated hUAs. RESULTS: WJ-MSCs exhibited fibroblastic morphology, successfully differentiating into "osteocytes", "adipocytes" and "chondrocytes", and were characterized by positive expression (> 90%) of CD90, CD73 and CD105. In addition, WJ-MSCs were successfully differentiated into VSMCs with the proposed differentiation protocol. VSMCs successfully expressed ACTA2, MYH11, MYOCD, TGLN and SOX9. Immunofluorescence results indicated the expression of ACTA2 and MYH11 in VSMCs. In order to determine the functionality of VSMCs, hUAs were isolated and decellularized. Based on histological analysis, decellularized hUAs were free of any cellular or nuclear materials, while their extracellular matrix retained intact. Then, repopulation of decellularized hUAs with VSMCs was performed for 3 wk. Decellularized hUAs were repopulated efficiently by the VSMCs. Biochemical analysis revealed the increase of total hydroyproline and sGAG contents in repopulated hUAs with VSMCs. Specifically, total hydroxyproline and sGAG content after the 1st, 2nd and 3rd wk was 71 ± 10, 74 ± 9 and 86 ± 8 µg hydroxyproline/mg of dry tissue weight and 2 ± 1, 3 ± 1 and 3 ± 1 µg sGAG/mg of dry tissue weight, respectively. Statistically significant differences were observed between all study groups (P < 0.05). CONCLUSION: VSMCs were successfully obtained from WJ-MSCs with the proposed differentiation protocol. Furthermore, hUAs were efficiently repopulated by VSMCs. Differentiated VSMCs from WJ-MSCs could provide an alternative source of cells for vascular tissue engineering.
BACKGROUND: The development of a biological based small diameter vascular graft (d < 6 mm), that can be properly stored over a long time period at - 196 °C, in order to directly be used to the patients, still remains a challenge. In this study the decellularized umbilical arteries (UAs) where vitrified, evaluated their composition and implanted to a porcine model, thus serving as vascular graft. METHODS: Human UAs were decellularized using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) detergents. Then, vitrified with vitrification solution 55 (VS55) solution, remained for 6 months in liquid nitrogen and their extracellular matrix composition was compared to conventionally cryopreserved UAs. Additionally, total hydroxyproline, sulphated glycosaminoglycan and DNA content were quantified in all samples. Finally, the vitrified umbilical arteries implanted as common carotid artery interposition graft to a porcine animal model. RESULTS: Decellularized and vitrified UAs characterized by proper preservation of extracellular matrix proteins and tissue architecture, whereas conventionally cryopreserved samples exhibited a disorganized structure. Total hydroxyproline content was preserved, although sulphated glycosaminoglycan and DNA contents presented significantly alterations in all samples. Implanted UAs successfully recellularized and remodeled as indicated by the histological analysis. CONCLUSION: Decellularized and vitrified UAs retained their structure function properties and can be possible used as an alternative source for readily accessible small diameter vascular grafts.
Tissue Engineering/methods , Umbilical Arteries/cytology , Vitrification , Animals , Arteries/cytology , Blood Vessel Prosthesis , Carotid Arteries , Carotid Artery, Common , Cryopreservation , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Humans , Sodium Dodecyl Sulfate , Swine , Tissue Scaffolds
