Electrical stimulation enhances sciatic nerve regeneration using a silk-based conductive scaffold beyond traditional nerve guide conduits.

Despite recent advancements in peripheral nerve regeneration, the creation of nerve conduits with chemical and physical cues to enhance glial cell function and support axonal growth remains challenging. This study aimed to assess the impact of electrical stimulation (ES) using a conductive nerve conduit on sciatic nerve regeneration in a rat model with transection injury. The study involved the fabrication of conductive nerve conduits using silk fibroin and Au nanoparticles (AuNPs). Collagen hydrogel loaded with green fluorescent protein (GFP)-positive adipose-derived mesenchymal stem cells (ADSCs) served as the filling for the conduit. Both conductive and non-conductive conduits were applied with and without ES in rat models. Locomotor recovery was assessed using walking track analysis. Histological evaluations were performed using H&E, luxol fast blue staining and immunohistochemistry. Moreover, TEM analysis was conducted to distinguish various ultrastructural aspects of sciatic tissue. In the ES + conductive conduit group, higher S100 (p < 0.0001) and neurofilament (p < 0.001) expression was seen after 6 weeks. Ultrastructural evaluations showed that conductive scaffolds with ES minimized Wallerian degeneration. Furthermore, the conductive conduit with ES group demonstrated significantly increased myelin sheet thickness and decreased G. ratio compared to the autograft. Immunofluorescent images confirmed the presence of GFP-positive ADSCs by the 6th week. Locomotor recovery assessments revealed improved function in the conductive conduit with ES group compared to the control group and groups without ES. These results show that a Silk/AuNPs conduit filled with ADSC-seeded collagen hydrogel can function as a nerve conduit, aiding in the restoration of substantial gaps in the sciatic nerve with ES. Histological and locomotor evaluations indicated that ES had a greater impact on functional recovery compared to using a conductive conduit alone, although the use of conductive conduits did enhance the effects of ES.

Effects of fresh bone marrow mononuclear cell therapy in rat model of retinopathy of prematurity.

Introduction: Retinopathy of prematurity (ROP) is a vasoproliferative disease that alters retinal vascular patterns in preterm neonates with immature retinal vasculature. This study was conducted to investigate the effects of cell therapy by bone marrow mononuclear cells (BMMNC) on neurological and vascular damages in a rat model of ROP. Methods: Ten newborn Wistar rats were divided randomly into the control and the oxygen-induced retinopathy (OIR) groups. Animals in the OIR group were incubated in an oxygen chamber to induce retinopathy. One eye of animals in the OIR group received BMMNC suspension (treated eyes), and the contralateral eye received the same volume of saline injection. Then, all animals underwent funduscopy, angiography, electroretinography, histopathology and immunohistochemical assessments. Results: Compared to the saline injection group, eyes treated with BMMNC had less vascular tortuosity while veins and arteries had relatively the same caliber, as revealed by fundus examinations. Eyes in the treatment group showed significantly elevated photopic and scotopic B waves amplitude. Neovascularization in the inner retinal layer and apoptosis of neural retina cells in the treatment group was significantly lower compared to untreated eyes. Also, BMMNC transplantation decreased glial cell activation and VEGF expression in ischemic retina. Conclusions: Our results indicate that intravitreal injection of BMMNC reduces neural and vascular damages and results in recovered retinal function in rat model of ROP. Ease of extraction without in vitro processing, besides the therapeutic effects of BMMNCs, make this source of cells as a new choice of therapy for ROP or other retinal ischemic diseases.

Evaluation of direct intramural injection to the bladder wall as a method for developing orthotopic tumor models.

BACKGROUND: Bladder cancer poses a great burden on society and its high rate of recurrence and treatment failure necessitates use of appropriate animal models to study its pathogenesis and test novel treatments. Orthotopic models are superior to other types since they provide a normal microenvironment. Four methods are described for developing bladder cancer models inside the animal's bladder. Direct intramural injection is one of these methods and is widely used. However, its efficacy in model development has not yet been studied. We aimed to evaluate the efficacy and success rate of the direct intramural injection method of developing an orthotopic model for the study of bladder cancer. METHOD: Tumor cell lines were prepared in four microtubes. Aliquots of 200 × 103 cells were injected through a 27 gauge needle into the ventral wall of the bladders of 4 male and 4 female BALB/c mice following a midline 1 cm laparotomy incision. In addition, 1 million cells from each microtube were injected into the flanks of control mice. To prevent infection and alleviate pain, 5 mg/kg enrofloxacin and 2.5 mg/kg flunixin meglumine, respectively, were injected subcutaneously. RESULTS: Tumors formed in all mice, resulting in 100% take rate and zero post-operation mortality. Surgery time was ≤15 min per mouse. In two mice, tumors were found in the peritoneal space as well. CONCLUSION: Direct intramural injection is a rapid, reliable, and reproducible method for developing orthotopic models of bladder cancer. It can be done on both male and female mice and only requires readily available surgical tools. However, needle track can result in cell spillage and peritoneal tumors.

Off-the-shelf acellular fetal skin scaffold as a novel alternative to buccal mucosa graft: the development and characterization of human tissue-engineered fetal matrix in rabbit model of hypospadiasis.

AIM: In this study, we aimed to develop a novel alternative to buccal mucosal graft from the acellular human fetal skin to manage hypospadias in a rabbit model. We optimized the decellularization protocol to develop and characterize the human tissue-engineered fetal dermal matrix as an "off-the-shelf" natural biomaterial. MATERIAL AND METHODS: Human fetal skin was obtained at 16-19 weeks gestational age with respect to a signed informed consent from parents under the university ethical committee approval. The dissected full-thickness fetal skin tissues were placed into SDS and Triton X-100 in different dosages to achieve the optimum decellularization protocol. Histopathology of the acellular fetal matrix was assessed by Hematoxylin & Eosin (H&E) and DAPI staining to confirm the removal of all cell materials, Masson's trichrome staining for collagen evaluation, DNA quantification for confirmation of DNA content, and scanning electron microscopy (SEM) for evaluation of scaffold microstructure. Immunohistochemistry (IHC) staining was used to detect specific dermal markers, namely vimentin, type I collagen, cytokeratin (CK)19. The prepared dermal scaffolds were then grafted on the 8 rabbit models of hypospadias. The rabbits underwent evaluations at 1, 2, 3, and 6 months postoperatively. RESULTS: H&E, Masson's trichrome, DAPI staining, and SEM confirmed the significant removal of cells; meanwhile, the ECM was completely preserved. At the time of biopsy, after 2, 4, and 6 months, no evidence of inflammation, fibrosis, necrosis, or rejection was observed. The grafted dermal scaffolds appeared histologically and anatomically normal. It was observed that the scaffolds were recellularized by circulating CD 34 + bone marrow stem cells (BMSCs) inside the body, implicating the body as a natural bioreactor. CONCLUSION: The application of acellular fetal skin (AFS) is a safe and feasible method that can decrease surgical time in a complex hypospadias reconstruction. Moreover, AFS demonstrated excellent angiogenesis characteristics and migration of the stem cells to the scaffold observed during the course of treatment. Novel natural AFS scaffold without cell seeding is an excellent alternative to buccal mucosal graft; hence, it can overcome the limitations concerning the graft size and prevent the creation of wounds in oral mucosal tissue.

Biomedical applications of silkworm (Bombyx Mori) proteins in regenerative medicine (a narrative review).

Silk worm (Bombyx Mori) protein, have been considered as potential materials for a variety of advanced engineering and biomedical applications for decades. Recently, silkworm silk has gained significant importance in research attention mainly because of its remarkable and exceptional mechanical properties. Silk has already been shown to have unique interactions with cells in tissues through bio-recognition units. The natural silk contains fibroin and sericin and has been used in various tissues of the human body (skin, bone, nerve, and so on). Besides, silk also still has anti-cancer, anti-tyrosinase, anti-coagulant, anti-oxidant, anti-bacterial, and anti-diabetic properties. This article is supposed to describe the diverse biomedical capabilities of B. Mori silk as the appropriate biomaterial among the assorted natural and artificial polymers that are presently accessible, and ideal for usage in regenerative medicine fields.

Evaluation and Comparison of the Effects of Mature Silkworm (Bombyx mori) and Silkworm Pupae Extracts on Schwann Cell Proliferation and Axon Growth: An In Vitro Study.

Background: Silkworm products were first used by physicians more than 8500 years ago, in the early Neolithic period. In Persian medicine, silkworm extract has several uses for treating and preventing neurological, cardiac, and liver diseases. Mature silkworms (Bombyx mori) and their pupae contain a variety of growth factors and proteins that can be used in many repair processes, including nerve regeneration. Objectives: The study aimed to evaluate the effects of mature silkworm (Bombyx mori), and silkworm pupae extract on Schwann cell proliferation and axon growth. Methods: Silkworm (Bombyx mori) and silkworm pupae extracts were prepared. Then, the concentration and type of amino acids and proteins in the extracts were evaluated by Bradford assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and liquid chromatograph-mass spectrometer (LC-MS/MS). Also, the regenerative potential of extracts for improving Schwann cell proliferation and axon growth was examined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, electron microscopy, and NeuroFilament-200 (NF-200) immunostaining. Results: According to the results of the Bradford test, the total protein content of pupae extract was almost twice that of mature worm extract. Also, SDS-PAGE analysis revealed numerous proteins and growth factors, such as bombyrin and laminin, in extracts that are involved in the repair of the nervous system. In accordance with Bradford's results, the evaluation of extracts using LC-MS/MS revealed that the number of amino acids in pupae extract was higher than in mature silkworm extract. It was found that the proliferation of Schwann cells at a concentration of 0.25 mg/mL in both extracts was higher than the concentrations of 0.01 and 0.05 mg/mL. When using both extracts on dorsal root ganglion (DRGs), an increase in length and number was observed in axons. Conclusions: The findings of this study demonstrated that extracts obtained from silkworms, especially pupae, can play an effective role in Schwann cell proliferation and axonal growth, which can be strong evidence for nerve regeneration, and, consequently, repairing peripheral nerve damage.