Generation of stem cell-based bioartificial anterior cruciate ligament (ACL) grafts for effective ACL rupture repair

AbstractIn the present study, we combined stem cell technology with a non-absorbable biomaterial for the reconstruction of the ruptured ACL. Towards this purpose, multipotential stromal cells derived either from subcutaneous human adipose tissue (hAT-MSCs) or from induced pluripotent stem cells (iPSCs) generated from human foreskin fibroblasts (hiPSC-MSCs) were cultured on the biomaterial for 21days in vitro to generate a 3D bioartifical ACL graft. Stem cell differentiation towards bone and ligament at the ends and central part of the biomaterial was selectively induced using either BMP-2/FGF-2 or TGF-β/FGF-2 combinations, respectively. The bioartificial ACL graft was subsequently implanted in a swine ACL rupture model in place of the surgically removed normal ACL. Four months post-implantation, the tissue engineered ACL graft generated an ACL-like tissue exhibiting morphological and biochemical characteristics resembling those of normal ACL

Biocompatibility and immunogenicity of decellularised allogeneic aorta in the orthotopic rat model

Background and aim of the study: Peripheral arterial disease causes blood vessel dysfunction that requires surgical intervention. Current surgical interventions employ synthetic or allogeneic vascular grafts, which offer biocompatible materials solutions that are not able to regenerate or grow with the patient. Decellularised scaffolds have gained significant momentum in the past few years, since they have the potential to regenerate in the patient. The aim of this study was to investigate the effects of modified decellularisation protocol on the biocompatibility and immunogenicity of allogeneic rat abdominal aorta in an orthotopic rat model. Methods: Native syngeneic Wistar (W) and allogeneic Dark Agouti (DA) aortas, together with decellularised allogeneic DA aortas, were assessed histologically, immunohistochemically and biomechanically. The immunogenicity of the untreated and decellularized syngeneic and allogeneic grafts was assessed in W rats, implanted orthotopically. Following implantation for 6 weeks, the grafts were explanted and assessed for the presence of T cells and macrophages by immunohistochemistry, and for their biomechanical integrity and histoarchitecture. Results: No obvious histoarchitectural differences were observed between the native W and DA aortas, with both presenting similar three-layered structures. Histological analysis of decellularized DA aortas did not reveal any remaining cells. Explanted native DA allografts showed media necrosis, partial elastic fibre degradation and adventitia thickening, as well as infiltration by lymphocytes (CD3+, CD4+) and macrophages (CD68+) in the adventitia. The explanted decellularized DA allografts indicated reduced immune injury compared to the explanted native DA allografts. The explanted native W syngeneic grafts showed a mild immune response, with an intact media and no lymphocyte infiltration. The explanted native DA allografts showed significantly lower collagen phase slope than the decellularized DA allografts prior implantation, and significantly higher thickness than the explanted decellularized DA allografts. Conclusions: The results indicated that the modified decellularization protocol did not affect significantly the mechanical and histological properties of the native DA rat aorta. Overall, the immune response was improved by decellularization. Native DA allografts induced an adverse immune response in W rats, whereas syngeneic W grafts showed good tissue integration

Optimization of Decellularization Procedure in Rat Esophagus for Possible Development of a Tissue Engineered Construct

Background: Current esophageal treatment is associated with significant morbidity. The gold standard therapeutic strategies are stomach interposition or autografts derived from the jejunum and colon. However, severe adverse reactions, such as esophageal leakage, stenosis and infection, accompany the above treatments, which, most times, are life threating. The aim of this study was the optimization of a decellularization protocol in order to develop a proper esophageal tissue engineered construct. Methods: Rat esophagi were obtained from animals and were decellularized. The decellularization process involved the use of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) buffers for 6 h each, followed by incubation in a serum medium. The whole process involved two decellularization cycles. Then, a histological analysis was performed. In addition, the amounts of collagen, sulphated glycosaminoglycans and DNA content were quantified. Results: The histological analysis revealed that only the first decellularization cycle was enough to produce a cellular and nuclei free esophageal scaffold with a proper extracellular matrix orientation. These results were further confirmed by biochemical quantification. Conclusions: Based on the above results, the current decellularization protocol can be applied successfully in order to produce an esophageal tissue engineered construct

Improved Repopulation Efficacy of Decellularized Small Diameter Vascular Grafts Utilizing the Cord Blood Platelet Lysate

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

Evaluation of the Regenerative Potential of Platelet-Lysate and Platelet-Poor Plasma Derived from the Cord Blood Units in Corneal Wound Healing Applications: An In Vitro Comparative Study on Corneal Epithelial 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

The role of exercise training and the endocannabinoid system in atherosclerotic plaque burden and composition in Apo-E-deficient mice

Introduction: We investigated the effect of combining exercise training and treatment with an endocannabinoid receptor 1 inhibitor (Rimonabant) on atherosclerosis burden and composition. Methods: Forty-eight apolipoprotein E-deficient (ApoE-/-) mice were kept on a 16-week high-fat diet. Mice were then placed on a normal diet and were randomized to the following groups with n=12 mice for 6 more weeks: 1) Control (Co) - no intervention; 2) Exercise (Ex) - exercise training on treadmill; 3) Rimonabant (Ri) - oral administration of rimonabant (10 mg/kg/day); or 4) Rimonabant+Exercise (RiEx) - combination of Ri and Ex groups treatment. At the end, all animals were sacrificed, and blood samples, as well as aortic root specimens, were obtained for histomorphometric analysis and quantification of the serum and plaque content of matrix metalloproteinases (MMPs). Results: The mean plaque area was significantly smaller (RiEx: 43.18±1.72%, Ri: 44.66±3.1%, Ex: 49±4.10%, Co: 70.43±2.83%) in all active treatment groups relative to the Co group (p<0.01). Conversely, the relative concentrations of collagen and elastin were increased significantly across all treatment groups compared to Co (p<0.05). Immunohistochemical analysis revealed significantly reduced macrophage content within plaques after all interventions, with the most pronounced effect observed after combined treatment (RiEx: 9.4±3.92%, Ri: 15±2.45%, Ex: 19.78±2.79%, Co: 34.25±4.99%; p<0.05). Within plaques, the TIMP-1 concentration was significantly upregulated in exercise-treated groups. MMP-3 and MMP-9 concentrations were equivalently decreased in all three active treatment groups compared to controls (p<0.001). Discussion: Both exercise and rimonabant treatments induced plaque regression and promoted plaque stability. The combined treatment failed to show additive or synergistic benefits relative to either intervention alone

Insights into Biomechanical and Proteomic Characteristics of Small Diameter Vascular Grafts Utilizing the Human Umbilical Artery

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 &lt; 0.013) and strain (p &lt; 0.001). On the contrary, all parameters tested for circumferential direction exhibited significant differences (p &lt; 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

Investigation of the biomechanical integrity of decellularized rat abdominal aorta

Objectives. The loss or damage of an organ or tissue is one of the most common and devastating problems in healthcare today. Tissue engineering applies the principles of engineering and biology toward the development of functional biological replacements that are able to maintain, improve, or restore the function of pathological tissues. The aim of the overall project is to study an already existing method for the decellularization of homograft vascular grafts for use in vascular surgery. Materials and Methods. The biomechanical integrity of native and decellularized rat aortas was assessed under uniaxial tension tests. For this purpose, 36 male rats (12 Wistar and 24 Dark Agouti [DA]) were used to excise their abdominal aortas. Twelve of the aortas were tested fresh (Wistar and DA rats), within 24 hours from euthanasia, and the rest were decellularized using a modified protocol (DA rats only). Fresh and decellularized samples (n ¼ 12) were subjected to uniaxial tensile loading to failure, and the recorded stress-strain behaviour of each specimen was assessed in terms of 6 biomechanical parameters. Results. No statistically significant differences were found in any of the biomechanical parameters studied between the decellularized DA rat aorta group and both the native DA and Wistar rat aorta groups (P > .05). Also, no significant difference was shown between the native DA and native Wistar rat aorta groups. Conclusions. The results from this study have shown that the decellularization protocol did not affect the mechanical properties of the native rat aorta. In addition to this, both native Wistar and native/decellularized DA rat aorta groups shared similar mechanical properties

Toll-Like Receptors -2, -3, -4 and -7 Expression Patterns in the Liver of a CLP-Induced Sepsis Mouse Model

Objective: To investigate the expression of toll-like receptors (TLRs) in the liver of septic mouse model. Materials and methods: For this study seventy-two C57BL/6J mice were utilized. Sepsis was induced by cecal ligation and puncture (CLP) in the mice of the three septic (S) groups (euthanized at 24 hours, 48 hours and 72 hours). Sham (laparotomy)- operated mice constituted the control (C) groups (euthanized at 24, 48 and 72 hours). Blood samples were drawn and liver tissues were extracted and examined histologically. The expression of TLRs 2, 3, 4 and 7 was assessed via immunohistochemistry (IHC) and qrt-PCR (quantitative- Polymerase Chain Reaction). Results: Liver function tests were elevated in all S-groups in contrast to their time-equivalent control groups (S24 versus C24, S48 versus C48 and S72 versus C72) (p 0.05); whereas at 72 hours only TLR 4 levels were significantly elevated in the intestine (p < 0.05). Conclusion: TLRs seem to be expressed in significant levels in the livers of septic rodents, indicating that they have a possible role in the pathophysiology of liver damage in septic conditions