Preparation of PLCL/ECM nerve conduits by electrostatic spinning technique and evaluationin vitroandin vivo.

Objective. Artificial nerve scaffolds composed of polymers have attracted great attention as an alternative for autologous nerve grafts recently. Due to their poor bioactivity, satisfactory nerve repair could not be achieved. To solve this problem, we introduced extracellular matrix (ECM) to optimize the materials.Approach.In this study, the ECM extracted from porcine nerves was mixed with Poly(L-Lactide-co-ϵ-caprolactone) (PLCL), and the innovative PLCL/ECM nerve repair conduits were prepared by electrostatic spinning technology. The novel conduits were characterized by scanning electron microscopy (SEM), tensile properties, and suture retention strength test for micromorphology and mechanical strength. The biosafety and biocompatibility of PLCL/ECM nerve conduits were evaluated by cytotoxicity assay with Mouse fibroblast cells and cell adhesion assay with RSC 96 cells, and the effects of PLCL/ECM nerve conduits on the gene expression in Schwann cells was analyzed by real-time polymerase chain reaction (RT-PCR). Moreover, a 10 mm rat (Male Wistar rat) sciatic defect was bridged with a PLCL/ECM nerve conduit, and nerve regeneration was evaluated by walking track, mid-shank circumference, electrophysiology, and histomorphology analyses.Main results.The results showed that PLCL/ECM conduits have similar microstructure and mechanical strength compared with PLCL conduits. The cytotoxicity assay demonstrates better biosafety and biocompatibility of PLCL/ECM nerve conduits. And the cell adhesion assay further verifies that the addition of ECM is more beneficial to cell adhesion and proliferation. RT-PCR showed that the PLCL/ECM nerve conduit was more favorable to the gene expression of functional proteins of Schwann cells. Thein vivoresults indicated that PLCL/ECM nerve conduits possess excellent biocompatibility and exhibit a superior capacity to promote peripheral nerve repair.Significance.The addition of ECM significantly improved the biocompatibility and bioactivity of PLCL, while the PLCL/ECM nerve conduit gained the appropriate mechanical strength from PLCL, which has great potential for clinical repair of peripheral nerve injuries.

Sequential expression of miR-221-3p and miR-338-3p in Schwann cells as a therapeutic strategy to promote nerve regeneration and functional recovery.

The functional properties of endogenous Schwann cells (SCs) during nerve repair are dynamic. Optimizing the functional properties of SCs at different stages of nerve repair may have therapeutic benefit in improving the repair of damaged nerves. Previous studies showed that miR-221-3p promotes the proliferation and migration of SCs, and miR-338-3p promotes the myelination of SCs. In this study, we established rat models of sciatic nerve injury by bridging the transected sciatic nerve with a silicone tube. We injected a miR-221 lentiviral vector system together with a doxycycline-inducible Tet-On miR-338 lentiviral vector system into the cavity of nerve conduits of nerve stumps to sequentially regulate the biological function of endogenous SCs at different stages of nerve regeneration. We found that the biological function of SCs was sequentially regulated, the diameter and density of myelinated axons were increased, the expression levels of NF200 and myelin basic protein were increased, and the function of injured peripheral nerve was improved using this system. miRNA Target Prediction Database prediction, Nanopore whole transcriptome sequencing, quantitative PCR, and dual luciferase reporter gene assay results predicted and verified Cdkn1b and Nrp1 as target genes of miR-221-3p and miR-338-3p, respectively, and their regulatory effects on SCs were confirmed in vitro. In conclusion, here we established a new method to enhance nerve regeneration through sequential regulation of biological functions of endogenous SCs, which establishes a new concept and model for the treatment of peripheral nerve injury. The findings from this study will provide direct guiding significance for clinical treatment of sciatic nerve injury.

Nerve ECM and PLA-PCL based electrospun bilayer nerve conduit for nerve regeneration.

Introduction: The porcine nerve-derived extracellular matrix (ECM) fabricated as films has good performance in peripheral nerve regeneration. However, when constructed as conduits to bridge nerve defects, ECM lacks sufficient mechanical strength. Methods: In this study, a novel electrospun bilayer-structured nerve conduit (BNC) with outer poly (L-lactic acid-co-ε-caprolactone) (PLA-PCL) and inner ECM was fabricated for nerve regeneration. The composition, structure, and mechanical strength of BNC were characterized. Then BNC biosafety was evaluated by cytotoxicity, subcutaneous implantation, and cell affinity tests. Furthermore, BNC was used to bridge 10-mm rat sciatic nerve defect, and nerve functional recovery was assessed by walking track, electrophysiology, and histomorphology analyses. Results: Our results demonstrate that BNC has a network of nanofibers and retains some bioactive molecules, including collagen I, collagen IV, laminin, fibronectin, glycosaminoglycans, nerve growth factor, and brain-derived neurotrophic factor. Biomechanical analysis proves that PLA-PCL improves the BNC mechanical properties, compared with single ECM conduit (ENC). The functional evaluation of in vivo results indicated that BNC is more effective in nerve regeneration than PLA-PCL conduit or ENC. Discussion: In conclusion, BNC not only retains the good biocompatibility and bioactivity of ECM, but also obtains the appropriate mechanical strength from PLA-PCL, which has great potential for clinical repair of nerve defects.

Poly (ɛ-Caprolactone-co-l,l-Lactide) Vascular External Sheath Carrying Prednisone for Improving Patency Rate of the Vein Graft.

Coronary artery bypass graft (CABG) surgery is an impactful treatment for coronary heart disease. Intimal hyperplasia is the central reason for the restenosis of vein grafts (VGs) after CABG. The introduction of external vascular sheaths around VGs can effectively inhibit intimal hyperplasia and ensure the patency of VGs. In this study, the well-known biodegradable copolymer poly (ɛ-caprolactone-co-l,l-lactide) (PLCL) was electrospun into high porosity external sheaths. The prednisone loaded in the PLCL sheath was slowly released during the degradation process of PLCL. Under the combined effects of sheath and prednisone, intimal hyperplasia was inhibited. For the cell experiments, all sheaths show low cytotoxicity to L929 cells at different concentrations at different time intervals. The ultrasonography and histological results showed prominent dilation and intimal hyperplasia of VG without sheath after 2 months of surgery. But there was no dilation in PLCL and PLCLPrednisone groups. Of note, the prednisone-loaded sheath group exhibited efficacy in inhibiting intimal hyperplasia and ensured graft patency. Impact statement To inhibit intimal hyperplasia after coronary artery bypass graft, the use of external vascular sheaths can prevent vein graft (VG) dilatation, then reduce turbulent blood flow shear stress to vessel wall, and lower the stimulation of shear stress to smooth muscle cells (SMCs), so as to prevent the proliferation and migration of vascular SMC. We provide a biodegradable sheath electrospun by poly (ɛ-caprolactone-co-l,l-lactide) (PLCL) loading prednisone and utilize it around VG in animal models. Vascular ultrasound examinations show strong evidence of vascular patency. The histological alterations of VGs in PLCLPrednisone group gave a narrower intima layer owing to the inhibition effect of prednisone.

Tracing Carbon Nanotubes (CNTs) in Rat Peripheral Nerve Regenerated with Conductive Conduits Composed of Poly(lactide-co-glycolide) and Fluorescent CNTs.

Nerve regeneration can be promoted using nerve guide conduits (NGCs). Carbon nanotubes (CNTs) are often used to prepare conductive NGCs, however, the major concern for their applications is the final location of the implanted CNTs in vivo. Herein, photoluminescent multiwalled CNTs (MWCNTs) were prepared and electrospun with poly(lactide-co-glycolide) (PLGA), followed by shaping into multichannel NGCs for repairing of injured rat sciatic nerve, thereby the distribution of CNTs in vivo could be detected via bioimaging. Photoluminescent MWCNTs (MWCNT-FITC) were prepared by functionalization with poly(glycidyl methacrylate) (PGMA) and fluorescein-isothiocyanate-isomer I (FITC) subsequently. The conductivity of the PLGA/MWCNT-FITC fibers was approx. 10-4 S/cm at 3 wt % MWCNTs. Compared with PLGA fibers, Schwann cells on PLGA/MWCNT-FITC fibers matured at a faster rate, accordingly, nerve regeneration was promoted by the PLGA/MWCNT-FITC NGC. With a confocal laser scanning microscope and small-animal imaging system, the location of MWCNTs was detected. Alongside the degradation of PLGA, MWCNTs intended to aggregate and were entrapped in the regenerated nerve tissue without migrating into surrounding tissues and other organs (liver, kidneys, and spleen). This study provides a useful characterization method for MWCNTs and the guidance for in vivo applications of MWCNTs in tissue engineering.

In vitro and in vivo biocompatibility study on acellular sheep periosteum for guided bone regeneration.

This study addresses the fabrication of an extracellular matrix material of the acellular sheep periosteum and the systematic evaluation of its biocompatibility to explore its potential application in guided bone regeneration. Sheep periosteum was harvested and decellularized by a combined decellularization protocol. The effectiveness of cell removal was proved and residual α-Gal antigen was also quantitatively detected. Then, mouse MC3T3-E1 cells were seeded onto the acellular periosteum. A scanning electron microscope (SEM) was used to record the whole process of cell adhesion. The CCK-8 assay suggested that the acellular periosteum not only had zero toxic effect on pre-osteoblasts, but played a positive role in cell proliferation. We also tested whether the acellular periosteum possesses favorable osteogenesis induction activity using an alkaline phosphatase (ALP) assay and a quantitative real-time PCR (Col I, Runx2, OCN) assay. An in vivo study of a subcutaneous implantation test using Sprague Dawley (SD) rats was performed to detect the changes in IL-2, IFN-γ and IL-4 in serum and elucidate the host's local response to acellular periosteum through hematoxylin and eosin (HE) and immunohistochemical staining. The results show that acellular sheep periosteum did not elicit a severe immunogenic response via the Th1 pathway, unlike fresh sheep periosteum. In conclusion, acellular sheep periosteum possesses favorable biocompatibility to be employed for guided bone regeneration.

Preparation and evaluation of acellular sheep periostea for guided bone regeneration.

The objective of this study was to fabricate an acellular sheep periosteum and explore its potential application in guided bone regeneration. Sheep periosteum was collected and decellularized by a modified decellularization protocol. The effectiveness of cell removal was proved by hematoxylin and eosin and 4',6-diamidino-2-phenylindole staining, DNA quantitative test, and agarose gel electrophoresis. After decellularization, its microstructure was found to become more porous while the integrality of collagen fibers remained undamaged, and the contents of collagen and glycosaminoglycan were not decreased significantly. Biomechanical analysis showed that the elastic modulus was significantly declined, while the yield stress was not affected, probably due to the collagen integrality. In vitro study of CCK-8 assay demonstrated that the acellular periosteum not only had no toxic effect to the MC3T3-E1 cells, but benefited the cell proliferation to some degree. In vivo experiment of guided bone regeneration was performed using a rabbit cranial model. Micro-CT and histological results revealed that the acellular periosteum not only effectively prevented the ingrowth of fibrous connective tissues, but also potentially facilitated bone regeneration. In conclusion, acellular sheep periostea, with wider sources, less costs, and more convenient fabrication process, would have great potential in the employment for guided bone regeneration.

Fabrication and evaluation of an optimized acellular nerve allograft with multiple axial channels.

Acellular nerve allografts are promising alternatives to autologous nerve grafts, but still have many drawbacks which greatly limit their curative effects. Here, we developed an optimized acellular nerve allograft with multiple axial channels by a modified decellularization method. These allografts were confirmed to preserve more extracellular matrix components and factors, and remove cellular components effectively. Meanwhile, macrochannels and microchannels were introduced to optimize internal microstructure of allografts, which increases porosity and water absorption, without significant loss of mechanical strength. The in vitro experiments demonstrated that the multichannel allografts showed superior ability of facilitating proliferation and penetration of Schwann cells. Additionally, in the in vivo experiments, the multichannel allografts were used to bridge 10 mm rat sciatic nerve defects. They exhibited better capacity to guide regenerative nerve fibers through the defective segment and restore innervation of target organs, thus achieving better recovery of muscle and motor function, in comparison with conventional acellular allografts. These findings indicate that this multichannel acellular nerve allograft has great potential for clinical application and provides a new prospective for future investigations of nerve regeneration. STATEMENT OF SIGNIFICANCE: Acellular nerve allografts, with preservation of natural extracellular matrix, are officially approved to repair peripheral nerve injury in some countries. However, bioactive component loss and compact internal structure result in variable clinical effects of conventional acellular allografts. In the present study, we fabricated an optimized acellular nerve allograft with multiple axial channels, which could both enable decellularization to be easily accomplished and reduce the amount of detergents in the preparation process. Characterization of the multichannel acellular allografts was confirmed to have better preservation of ECM bioactive molecules and regenerative factors. Efficiency evaluation showed the multichannel allografts could facilitate Schwann cells to migrate inside them in vitro, and enhance regrowth and myelination of axons as well as recovery of muscle and motor function in vivo.