Functional recovery of injured cavernous nerves achieved through endogenous nerve growth factor-containing bioactive fibrous membrane.

Radical prostatectomy is a highly successful treatment for prostate cancer, among the most prevalent manifestations of the illness. Damage of the cavernous nerve (CN) during prostatectomy is the main cause of postoperative erectile dysfunction (ED). In this study, the capability of a personalized bioactive fibrous membrane to regenerate injured CN was investigated. The fibrous membrane bioactivity is conferred by the selectively bound nerve growth factor (NGF) present in the rat urine. In a rat model of bilateral CN crush, the implanted bioactive fibrous membrane induces CN regeneration and restoration of erectile function, showing a significantly increased number of smooth muscle cells and content of endothelial and neuronal nitric oxide synthases (eNOS; nNOS). In addition, the bioactive fibrous membrane promotes nerve regeneration by increasing the number of myelinated axons and nNOS-positive cells, therefore reversing the CN fibrosis found in untreated rats or rats treated with a bare fibrous membrane. Therefore, this personalized regenerative strategy could overcome the recognized drawbacks of currently available treatments for CN injuries. It may constitute an effective treatment for prostate cancer patients suffering from ED after being subject to radical prostatectomy. STATEMENT OF SIGNIFICANCE: The present work introduces a unique strategy to address post-surgical ED resulting from CN injury during pelvic surgery (e.g., radical prostatectomy, radical cystoprostatectomy, abdominoperineal resection). It comprises a bioactive and cell-free fibrous implant, customized to enhance CN recovery. Pre-clinical results in a rat model of bilateral CN crush demonstrated that the bioactive fibrous implant can effectively heal injured CN, and restore penile structure and function. This implant selectively binds NGF from patient fluids (i.e. urine) due to its functionalized surface and high surface area. Moreover, its local implantation reduces adverse side effects. This tailored regenerative approach has the potential to revolutionize the treatment of ED in prostate cancer patients following radical prostatectomy, overcoming current treatment limitations.

Chondrogenic differentiation induced by extracellular vesicles bound to a nanofibrous substrate.

Extracellular vesicles (EVs) are being increasingly studied owing to its regenerative potential, namely EVs derived from human bone marrow mesenchymal stem cells (hBM-MSCs). Those can be used for controlling inflammation, repairing injury, and enhancing tissue regeneration. Differently, the potential of EVs derived from human articular chondrocytes (hACs) to promote cartilage regeneration has not been thoroughly investigated. This work aims to develop an EVs immobilization system capable of selectively bind EVs present in conditioned medium obtained from cultures of hACs or hBM-MSC. For that, an anti-CD63 antibody was immobilized at the surface of an activated and functionalized electrospun nanofibrous mesh. The chondrogenic potential of bound EVs was further assessed by culturing hBM-MSCs during 28 days under basal conditions. EVs derived from hACs cultured under differentiation medium or from chondrogenically committed hBM-MSCs induced a chondrogenic phenotype characterized by marked induction of SOX9, COMP, Aggrecan and Collagen type II, and matrix glycosaminoglycans synthesis. Indeed, both EVs immobilization systems outperformed the currently used chondroinductive strategies. These data show that naturally secreted EVs can guide the chondrogenic commitment of hBM-MSCs in the absence of any other chemical or genetic chondrogenic inductors based in medium supplementation.

Stimulation of Neurite Outgrowth Using Autologous NGF Bound at the Surface of a Fibrous Substrate.

Peripheral nerve injury still remains a major clinical challenge, since the available solutions lead to dysfunctional nerve regeneration. Even though autologous nerve grafts are the gold standard, tissue engineered nerve guidance grafts are valid alternatives. Nerve growth factor (NGF) is the most potent neurotrophic factor. The development of a nerve guidance graft able to locally potentiate the interaction between injured neurons and autologous NGF would be a safer and more effective alternative to grafts that just release NGF. Herein, a biofunctional electrospun fibrous mesh (eFM) was developed through the selective retrieval of NGF from rat blood plasma. The neurite outgrowth induced by the eFM-NGF systems was assessed by culturing rat pheochromocytoma (PC12) cells for 7 days, without medium supplementation. The biological results showed that this NGF delivery system stimulates neuronal differentiation, enhancing the neurite growth more than the control condition.

Spatial immobilization of endogenous growth factors to control vascularization in bone tissue engineering.

The intimate crosstalk between endothelial and bony cells is essential for the reconstruction of bone tissue defects. Indeed, a successful bone repair is greatly dependent on the formation of new blood vessels, to ensure the supply of nutrients and gases, as well as the removal of metabolites. Bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) are involved on cells differentiation and bone vascularization aiming to develop viable bone tissue. Herein it is hypothesized that endogenous BMP-2 and VEGF bound in a parallel arrangement over a single nanofibrous substrate (NFM) can lead to a successful osteogenic and angiogenic differentiation of mesenchymal stem cells. For that, an engineered biofunctional system was developed comprising anti-BMP-2 and anti-VEGF antibodies, immobilized over an electrospun NFMs in a parallel pattern design, with the attempt to recreate the vasculature of bone tissue. The osteogenic and angiogenic potential of this engineered biofunctional system was demonstrated by culturing human bone marrow-derived mesenchymal stem cells (hBM-MSCs) during 21 days without exogenous induction. A chick chorioallantoic membrane (CAM) assay showed that the engineered biofunctional system, comprising bound endogenous BMP-2 and VEGF, is able to induce an increased angiogenic response. The angiogenic ability of this system, together with the osteogenic inductor BMP-2, enable obtaining an effective vascularized bone tissue engineering approach.

Fibronectin Bound to a Fibrous Substrate Has Chondrogenic Induction Properties.

Articular cartilage is an avascular tissue characterized by a dense and specific extracellular matrix (ECM). Fibronectin (FN) is a key constituent of the pericellular ECM, assembled into a fibrillar matrix through a cell-mediated process, being implicated in chondrogenic events. In this study, we evaluate the chondrogenic potential of FN bound to the surface of an electrospun nanofibrous mesh (NFM). For that, an anti-FN antibody was immobilized at the surface of NFMs, rendering them capable of selectively binding endogenous FN (eFN) from blood plasma. The chondrogenic potential of bound eFN was further assessed by culturing human bone marrow-derived mesenchymal stem cells (hBM-MSCs) for 28 days, in a basal growth medium. The biological results indicate that NFMs functionalized with eFN were able to successfully induce the chondrogenesis of hBM-MSCs, as demonstrated by the high expression of SOX9, Aggrecan, and Collagen type II. Therefore, biofunctionalized nanofibrous substrates comprising eFN significantly enhance the efficacy of a cartilage tissue-engineering strategy.

The Role of Diet Related Short-Chain Fatty Acids in Colorectal Cancer Metabolism and Survival: Prevention and Therapeutic Implications.

Colorectal Cancer (CRC) is a major cause of cancer-related death worldwide. CRC increased risk has been associated with alterations in the intestinal microbiota, with decreased production of Short Chain Fatty Acids (SCFAs). SCFAs produced in the human colon are the major products of bacterial fermentation of undigested dietary fiber and starch. While colonocytes use the three major SCFAs, namely acetate, propionate and butyrate, as energy sources, transformed CRC cells primarily undergo aerobic glycolysis. Compared to normal colonocytes, CRC cells exhibit increased sensitivity to SCFAs, thus indicating they play an important role in cell homeostasis. Manipulation of SCFA levels in the intestine, through changes in microbiota, has therefore emerged as a potential preventive/therapeutic strategy for CRC. Interest in understanding SCFAs mechanism of action in CRC cells has increased in the last years. Several SCFA transporters like SMCT-1, MCT-1 and aquaporins have been identified as the main transmembrane transporters in intestinal cells. Recently, it was shown that acetate promotes plasma membrane re-localization of MCT-1 and triggers changes in the glucose metabolism. SCFAs induce apoptotic cell death in CRC cells, and further mechanisms have been discovered, including the involvement of lysosomal membrane permeabilization, associated with mitochondria dysfunction and degradation. In this review, we will discuss the current knowledge on the transport of SCFAs by CRC cells and their effects on CRC metabolism and survival. The impact of increasing SCFA production by manipulation of colon microbiota on the prevention/therapy of CRC will also be addressed.

Chondrogenesis-inductive nanofibrous substrate using both biological fluids and mesenchymal stem cells from an autologous source.

During the last decade, many cartilage tissue engineering strategies have been developed, being the stem cell-based approach one of the most promising. Transforming Growth Factor-ß3 (TGF-ß3) and Insulin-like Growth Factor-I (IGF-I) are key proteins involved in the regulation of chondrogenic differentiation. Therefore, these two growth factors (GFs) were immobilized at the surface of a single electrospun nanofibrous mesh (NFM) aiming to differentiate human Bone Marrow-derived Mesenchymal Stem Cells (hBM-MSCs). The immobilization of defined antibodies (i.e. anti-TGF-ß3 and anti-IGF-I) allows the selective retrieval of the abovementioned GFs from human platelet lysates (PL). Biochemical assays, involving hBM-MSCs cultured on biofunctional nanofibrous substrates under basal culture medium during 28 days, confirm the biological activity of bound TGF-ß3 and IGF-I. Specifically, the typical spherical morphology of chondrocytes and the immunolocalization of collagen type II confirmed the formation of a cartilaginous ECM. Therefore, the proposed biofunctional nanofibrous substrate is able to promote chondrogenesis.

Colorectal Cancer Cells Increase the Production of Short Chain Fatty Acids by Propionibacterium freudenreichii Impacting on Cancer Cells Survival.

Propionibacterium freudenreichii is a commercially relevant bacterium with probiotic potential. This bacterium can exert protective effects particularly against colorectal cancer (CRC), via the production of short chain fatty acids (SCFA), namely acetate and propionate. In this work, we aimed to evaluate the performance and adaptation capacity of P. freudenreichii to a simulated digestive stress using different culture media, namely YEL, Basal medium, Mimicking the Content of the Human Colon medium (MCHC) and DMEM. The effect of the fermented culture broth on CRC cells survival and of CRC cells conditioned media on the bacteria performance was also evaluated. Basal medium was found to be the best for P. freudenreichii to produce SCFA. MCHC medium, despite being the medium in which lower amounts of acetate and propionate were produced, showed higher acetate and propionate yields as compared to other media. We also observed that the presence of lactate in CRC cells conditioned growth medium resulting from cell metabolism, leads to an increased production of SCFA by the bacteria. The bacterial fermented broth successfully inhibited CRC cells proliferation and increased cell death. Our results showed for the first time that P. freudenreichii performance might be stimulated by extracellular lactate produced by CRC metabolic switch also known as "Warburg effect," where cancer cells "ferment" glucose into lactate. Additionally, our results suggest that P. freudenreichii could be potentially used as a probiotic in CRC prevention at early stages of the carcinogenesis process and might help in CRC therapeutic approaches.

The Use of Electrospinning Technique on Osteochondral Tissue Engineering.

Electrospinning, an electrostatic fiber fabrication technique, has attracted significant interest in recent years due to its versatility and ability to produce highly tunable nanofibrous meshes. These nanofibrous meshes have been investigated as promising tissue engineering scaffolds since they mimic the scale and morphology of the native extracellular matrix. The sub-micron diameter of fibers produced by this process presents various advantages like the high surface area to volume ratio, tunable porosity, and the ability to manipulate the nanofiber composition in order to get desired properties and functionality. Electrospun fibers can be oriented or arranged randomly, giving control over both mechanical properties and the biological response to the fibrous scaffold. Moreover, bioactive molecules can be integrated with the electrospun nanofibrous scaffolds in order to improve the cellular response. This chapter presents an overview of the developments on electrospun polymer nanofibers including processing, structure, and their applications in the field of osteochondral tissue engineering.