C-kit+VEGFR-2+ Mesenchymal Stem Cells Differentiate into Cardiovascular Cells and Repair Infarcted Myocardium after Transplantation

Background: Resent preclinical studies and clinical trails prove that transplantation of mesenchymal stem cells (MSCs) is a promised therapy for ischemic diseases. However, the properties of c-kit+ cells in MSCs remain unclear. We investigated the differential potential of c-kit+VEGFR-2+ MSCs and evaluated their effects on repairing the infarcted myocardium after transplantation. Methods: c-kit+VEGFR-2+ MSCs were isolated from rat bone marrow. Gene expression profile of the cells was examined with RNA-sequencing. Differential potential of the cells was determined after induction with VEGF, TGF-β and BMP-2 for 2 weeks. Improvement of cardiac function and repair of the infarcted myocardium were assessed at 4 weeks after transplantation of the cells preconditioned with hypoxia and serum deprivation. Results: Gene expression profile revealed that the upregulated genes are enrichment of genes related to immune process and cell differentiation. The cells represented a potential of differentiation towards endothelial cell, smooth muscle cells and cardiamyocytes. In hypoxic condition, secretion of VEGF, SCF and SDF-1α from the cells was increased. VEGF and SCF promoted proliferation and migration of the cells. VEGF could induce the cells to incorporate to the microvessels. After transplantation of the preconditioned cells into the infarcted myocardium, cardiac function was improved, scar size of the infarcted myocardium was decreased, and angiogensis and myocardium repair were enhanced significantly. With preconditioning and delivery by fibrin gel, survival of the cells in the ischemic tissue was augmented. Conclusion: These results suggest that c-kit+VEGFR-2+ MSCs have a potential of differentiation towards cardiovascular cells. SCF/c-kit and VEGF/VEGFR-2 singnalling pathways regulate proliferation, migration and differentiation of the cells. Transplantation of c-kit+VEGFR-2+ MSCs may enhance repair of the infarcted myocardium effectively.

Comparative Evaluation of Fibrin Gel and Platelet-Rich Fibrin on Stem Cells of the Apical Papilla Fate Using a Dentin-Based Model

Background: PRF as one of the favorable scaffolds in Regenerative Endodontic Treatment (RET), has several limitations such as the need for blood sampling and special equipment. High available commercial scaffolds such as fibrin are able to meet all the necessary requirements of dentin tissue engineering. The present study was designed to evaluate the effect of PRF and fibrin gel, with and without the presence of EDTA-treated radicular dentin segments on SCAP viability, proliferation, migration, and differentiation.Methods: Radicular dentin were prepared from extracted teeth and treated by EDTA 17% .The samples were divided into 6 groups: Dentin/PRF/Cell, Dentin/Fibrin/Cell, Dentin/Cell, PRF/Cell, Fibrin/Cell and Cell (Control). SCAP viability was assessed using MTT assay. Gene expression levels of odontogenic markers [Dentin sialophosphoprotein (DSPP), Dentin matrix protein 1(DMP1), Collagen type I Alpha 1(COL 1A1) and Alkaline phosphatase (ALP) were assessed using qrt-PCR. Cell migration were also evaluated by means of scratch test. Results: The results of MTT assay at showed that the viability of SCAP significantly increased after 7 days for both groups containing fibrin (P <0.05). The viability of SCAP seeded on Dentin/PRF and PRF significantly decreased after 7 days (P <0.001). The odontogenic markers were significantly expressed for both scaffolds in the presence of dentin segment (p<0.05). Significant decrease in scratch area was seen in Fibrin/Dentin group (p < 0.001)Conclusions:Fibrin beside EDTA-treated dentin showed great ability in survival, proliferation, differentiation, and migration of SCAP rather than PRF.

Inhibition of Post-Surgery Tumour Recurrence via a Sprayable Chemoimmunotherapy Gel Releasing PD-L1 Antibody and Platelet-Derived Exosomes

Background: Melanoma is the most serious type of skin cancer, and surgery is an effective method to treat melanoma. Unfortunately, local residual micro-infiltrated tumour cells and systemic circulating tumour cells (CTCs) are significant causes of treatment failure, leading to tumour recurrence and metastasis. Methods: Exosomes were isolated from platelets by differential centrifugation, and exosome-loaded doxorubicin (PexD) was prepared by mixing exosomes with doxorubicin (DOX). PexD and an anti-PD-L1 monoclonal antibody (aPD-L1) were coencapsulated in fibrin gel. The synergistic antitumour efficacy of the gel containing PexD and aPD-L1 was assessed both in vitro and in vivo. Results: Herein, we developed an in situ-formed bioresponsive gel combined with chemoimmunotherapeutic agents as a drug reservoir that could effectively inhibit both local tumour recurrence and tumour metastasis. In comparison with a DOX solution, PexD could better bind to tumour cells, induce more tumour immunogenic cell death (ICD) and promote a stronger antitumour immune response. PexD could enter the blood circulation through damaged blood vessels to track and eliminate CTCs. The concurrent release of aPD-L1 at the tumour site could impair the PD-1/PD-L1 pathway and restore the tumour-killing effect of cytotoxic T cells. This chemoimmunotherapeutic strategy triggered relatively strong T cell immune responses, significantly improving the tumour immune microenvironment. Conclusion: Our findings indicated that the immunotherapeutic fibrin gel could “awaken” the host innate immune system to inhibit both local tumour recurrence postsurgery and metastatic potential, thus, it could serve as a promising approach to prevent tumour recurrence.

Potential of Melt Electrowritten Scaffolds Seeded with Meniscus Cells and Mesenchymal Stromal Cells

Meniscus injury and meniscectomy are strongly related to osteoarthritis, thus there is a clinical need for meniscus replacement. The purpose of this study is to create a meniscus scaffold with micro-scale circumferential and radial fibres suitable for a one-stage cell-based treatment. Poly-caprolactone-based scaffolds with three different architectures were made using melt electrowriting (MEW) technology and their in vitro performance was compared with scaffolds made using fused-deposition modelling (FDM) and with the clinically used Collagen Meniscus Implants® (CMI®). The scaffolds were seeded with meniscus and mesenchymal stromal cells (MSCs) in fibrin gel and cultured for 28 d. A basal level of proteoglycan production was demonstrated in MEW scaffolds, the CMI®, and fibrin gel control, yet within the FDM scaffolds less proteoglycan production was observed. Compressive properties were assessed under uniaxial confined compression after 1 and 28 d of culture. The MEW scaffolds showed a higher Young’s modulus when compared to the CMI® scaffolds and a higher yield point compared to FDM scaffolds. This study demonstrates the feasibility of creating a wedge-shaped meniscus scaffold with MEW using medical-grade materials and seeding the scaffold with a clinically-feasible cell number and -type for potential translation as a one-stage treatment.

In Vitro Models for the Development of Peripheral Nerve Conduits, Part I: Design of a Fibrin Gel-Based Non-Contact Test

Current clinical strategies to repair peripheral nerve injuries draw on different approaches depending on the extent of lost tissue. Nerve guidance conduits (NGCs) are considered to be a promising, off-the-shelf alternative to autografts when modest gaps need to be repaired. Unfortunately, to date, the implantation of an NGC prevents the sacrifice of a healthy nerve at the price of suboptimal clinical performance. Despite the significant number of materials and fabrication strategies proposed, an ideal combination has not been yet identified. Validation and comparison of NGCs ultimately requires in vivo animal testing due to the lack of alternative models, but in the spirit of the 3R principles, a reliable in vitro model for preliminary screening is highly desirable. Nevertheless, more traditional in vitro tests, and direct cell seeding on the material in particular, are not representative of the actual regeneration scenario. Thus, we have designed a very simple set-up in the attempt to appreciate the relevant features of NGCs through in vitro testing, and we have verified its applicability using electrospun NGCs. To this aim, neural cells were encapsulated in a loose fibrin gel and enclosed within the NGC membrane. Different thicknesses and porosity values of two popular polymers (namely gelatin and polycaprolactone) were compared. Results indicate that, with specific implementation, the system might represent a useful tool to characterize crucial NGC design aspects.

Establishment of a Pre-vascularized 3D Lung Cancer Model in Fibrin Gel—Influence of Hypoxia and Cancer-Specific Therapeutics

Lung cancer is the most frequently diagnosed cancer worldwide and the one that causes the highest mortality. In order to understand the disease and to develop new treatments, in vitro human lung cancer model systems which imitate the physiological conditions is of high significance. In this study, a human 3D lung cancer model was established that features the organization of a tumor with focus on tumor angiogenesis. Vascular networks were formed by co-culture of human umbilical vein endothelial cells and adipose tissue-derived mesenchymal stem cells (ASC) for 14 days in fibrin. A part of the pre-vascularized fibrin gel was replaced by fibrin gel containing lung cancer cells (A549) to form tri-cultures. This 3D cancer model system was cultured under different culture conditions and its behaviour after treatment with different concentrations of tumor-specific therapeutics was evaluated. The evaluation was performed by measurement of metabolic activity, viability, quantification of two-photon laser scanning microscopy and measurement of the proangiogenic factor vascular endothelial growth factor in the supernatant. Hypoxic conditions promoted vascularization compared to normoxic cultured controls in co- and tri-cultures as shown by significantly increased vascular structures, longer structures with a higher area and volume, and secretion of vascular endothelial growth factor. Cancer cells also promoted vascularization. Treatment with 50 µM gefitinib or 50 nM paclitaxel decreased the vascularization significantly. VEGF secretion was only reduced after treatment with gefitinib, while in contrast secretion remained constant during medication with paclitaxel. The findings suggest that the herein described 3D lung cancer model provides a novel platform to investigate the angiogenic potential of cancer cells and its responses to therapeutics. Thus, it can serve as a promising approach for the development and patient-specific pre-selection of anticancer treatment.