Enhancing neurite growth and neural functions on polymeric nerve conduit with BMSC-derived ECM coating.

The therapy of large defects in peripheral nerve injury (PNI) suffers from several drawbacks, especially the lack of autologous nerve donors. Nerve conduits are considered as a solution for nerve injury treatment, but biocompatibility improvements is still required for conduits prepared with synthetic materials. Cell-derived extracellular matrix (ECM) has drawn attention due to its lower risk of immunogenic response and independence from donor availability. The goal of this study is to coat bone mesenchymal stem cell-derived ECMs on poly(lactic-co-glycolic) acid (PLGA) conduits to enhance their ability to support neural growth and neurite extensions. The ECM-coated conduits have better hydrophilic properties than the pure PLGA conduits. A marked increase on PC12 and RSC96 cells' viability, proliferation and dorsal root ganglion neurite extension was observed. Quantitative PCR analysis exhibited a significant increase in markers for cell proliferation (GAP43), neurite extension (NF-H, MAP2, andßIII-tubulin) and neural function (TREK-1). These results show the potential of ECM-coated PLGA conduits in PNI therapy.

Dual-layer conduit containing VEGF-A - Transfected Schwann cells promotes peripheral nerve regeneration via angiogenesis.

Peripheral nerve injuries (PNIs) can cause neuropathies and significantly affect the patient's quality of life. Autograft transplantation is the gold standard for conventional treatment; however, its application is limited by nerve unavailability, size mismatch, and local tissue adhesion. Tissue engineering, such as nerve guidance conduits, is an alternative and promising strategy to guide nerve regeneration for peripheral nerve repair; however, only a few conduits could reach the high repair efficiency of autografts. The healing process of PNI is frequently accompanied by not only axonal and myelination regeneration but also angiogenesis, which initializes nerve regeneration through vascular endothelial growth factor A (VEGF-A). In this study, a composite nerve conduit with a poly (lactic-co-glycolic acid) (PLGA) hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with VEGF-A transfected Schwann cells (SCs) as the inner layer was established to evaluate its promising ability for peripheral nerve repair. A rat model of peripheral nerve defect was used to examine the efficiency of PLGA/GelMA-SC (VA) conduits, whereas autograft, PLGA, PLGA/GelMA, and PLGA/GelMA-SC (NC) were used as controls. VEGF-A-transfected SCs can provide a stable source for VEGF-A secretion. Furthermore, encapsulation in GelMA cannot only promote proliferation and tube formation of human umbilical vein endothelial cells but also enhance dorsal root ganglia and neuronal cell extension. Previous animal studies have demonstrated that the regenerative effects of PLGA/GelMA-SC (VA) nerve conduit were similar to those of autografts and much better than those of other conduits. These findings indicate that combination of VEGF-A-overexpressing SCs and PLGA/GelMA conduit-guided peripheral nerve repair provides a promising method that enhances angiogenesis and regeneration during nerve repair. STATEMENT OF SIGNIFICANCE: Nerve guidance conduits shows promise for peripheral nerve repair, while achieving the repair efficiency of autografts remains a challenge. In this study, a composite nerve conduit with a PLGA hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with vascular endothelial growth factor A (VEGF-A)-transfected Schwann cells (SCs) as the inner layer was established to evaluate its potential ability for peripheral nerve repair. This approach preserves growth factor bioactivity and enhances material properties. GelMA insertion promotes Schwann cell proliferation and morphology extension. Moreover, transfected SCs serve as a stable VEGF-A source and fostering angiogenesis. This study offers a method preserving growth factor efficacy and safeguarding SCs, providing a comprehensive solution for enhanced angiogenesis and nerve regeneration.

3D bioprinting of multi-cellular tumor microenvironment for prostate cancer metastasis.

Prostate cancer (PCa) is one of the most lethal cancers in men worldwide. The tumor microenvironment (TME) plays an important role in PCa development, which consists of tumor cells, fibroblasts, endothelial cells, and extracellular matrix (ECM). Hyaluronic acid (HA) and cancer-associated fibroblasts (CAFs) are the major components in the TME and are correlated with PCa proliferation and metastasis, while the underlying mechanism is still not fully understood due to the lack of biomimetic ECM components and coculture models. In this study, gelatin methacryloyl/chondroitin sulfate-based hydrogels were physically crosslinked with HA to develop a novel bioink for the three-dimensional bioprinting of a coculture model that can be used to investigate the effect of HA on PCa behaviors and the mechanism underlying PCa-fibroblasts interaction. PCa cells demonstrated distinct transcriptional profiles under HA stimulation, where cytokine secretion, angiogenesis, and epithelial to mesenchymal transition were significantly upregulated. Further coculture of PCa with normal fibroblasts activated CAF transformation, which could be induced by the upregulated cytokine secretion of PCa cells. These results suggested HA could not only promote PCa metastasis individually but also induce PCa cells to activate CAF transformation and form HA-CAF coupling effects to further promote PCa drug resistance and metastasis.

Evaluation of the Effect of Fibroblasts on Melanoma Metastasis Using a Biomimetic Co-Culture Model.

Melanoma is a highly malignant tumor originating from melanocytes. The 5-year survival rate of primary melanoma is 98%, whereas the survival rate of metastatic melanoma is only 10%, which can be attributed to the insensitivity to existing treatments. Fibroblasts are the primary cells in the dermis that promote melanoma metastasis; however, the molecular mechanism underlying the fibroblast-melanoma interaction is yet to be completely understood. Herein, gelatin methacryloyl (GelMA) was used to construct a co-culture model for melanoma cells (A375) and fibroblasts. GelMA retains the good biological properties of collagen, which has been identified as the primary component of the melanoma tumor microenvironment. Fibroblasts were encapsulated in GelMA, whereas A375 cells were cultured on the GelMA surface, which realistically mimics the macrostructure of melanoma. A375 cells co-cultured with fibroblasts demonstrated a higher cellular proliferation rate, potentials of neoneurogenesis, overexpression of epithelial mesenchymal transition markers, and a faster migration rate compared with A375 cells cultured alone, which could be due to the cancer-associated fibroblast activation and the overexpression of transforming growth factor ß1 and fibroblast growth factor-2 by fibroblasts. Overall, this study revealed the possible mechanisms of fibroblast-melanoma interaction and suggested that this co-culture model could be potentially further developed as a platform for screening chemotherapies in the future.

Cells, growth factors and biomaterials used in tissue engineering for hair follicles regeneration.

Alopecia is a common and distressing medical condition that has affected a majority of people worldwide, which leads to great effects on the quality of life and self-esteem. Numerous treatments had been used to cure alopecia, including hair growth stimulants, herbal products, and hair transplantation. However, these treatments have their side effects, such as hypertrichosis, edema, and even cardiovascular adverse effects, which lead to the urgent requirement to explore a new hair-follicle (HF) regeneration approach. Tissue engineering could be the potential way for HF regeneration by simulating the epithelial-mesenchymal interaction and cell-extracellular matrix interactions. This review summarized the potential cells that are used in tissue engineering, commonly used tissue engineering techniques, and most importantly, the biomaterials that have been applied for in vitro three-dimensional cell culture or in vivo co-transplantation in HF regeneration. The literature shows that advances in this field toward functional HF development have progressively increased. Although the clinical application of biomaterial co-transplantation for HF regeneration still faces various challenges, numerous studies have proved that this is a promising direction that could be achieved in the future.

In vitro study of decellularized rat tissues for nerve regeneration.

Peripheral nerve injuries cause an absence or destruction of nerves. Decellularized nerves, acting as a replacement for autografts, have been investigated in the promotion of nerve repair and regeneration, always being incorporated with stem cells or growth factors. However, such a strategy is limited by size availability. The potential application in heterotopic transplantation of other decellularized tissues needs to be further explored. In this study, rat decellularized kidney (dK) was selected to be compared with decellularized peripheral nerve (dN), since dK has aboundant ECM components and growth factors. The PC-12 cells were cultured on dK and dN scaffolds, as shown in the similar behaviors of cell metabolism and viability, but have a more regular arrangement on dN compared to dK, indicating that the natural structure plays an important role in guiding cell extension. However, we found significant upregulation of axon-growth-associated genes and proteins of PC-12 cells in the dK group compared to the dN group by qRT-PCR, immunofluorescence, and western blotting. Furthermore, various neurotrophic factors and growth factors of acellular kidney and nerve were evaluated by ELISA assay. The lower expression of neurotrophic factors but higher expression of growth factors such as VEGF and HGF from dK suggests that axon growth and extension for PC-12 cells may be partially mediated by VEGF and HGF expression from decellularized kidney, which further points to a potential application in nerve repair and regeneration.

The application of 3D bioprinting in urological diseases.

Urologic diseases are commonly diagnosed health problems affecting people around the world. More than 26 million people suffer from urologic diseases and the annual expenditure was more than 11 billion US dollars. The urologic cancers, like bladder cancer, prostate cancer and kidney cancer are always the leading causes of death worldwide, which account for approximately 22% and 10% of the new cancer cases and death, respectively. Organ transplantation is one of the major clinical treatments for urological diseases like end-stage renal disease and urethral stricture, albeit strongly limited by the availability of matching donor organs. Tissue engineering has been recognized as a highly promising strategy to solve the problems of organ donor shortage by the fabrication of artificial organs/tissue. This includes the prospective technology of three-dimensional (3D) bioprinting, which has been adapted to various cell types and biomaterials to replicate the heterogeneity of urological organs for the investigation of organ transplantation and disease progression. This review discusses various types of 3D bioprinting methodologies and commonly used biomaterials for urological diseases. The literature shows that advances in this field toward the development of functional urological organs or disease models have progressively increased. Although numerous challenges still need to be tackled, like the technical difficulties of replicating the heterogeneity of urologic organs and the limited biomaterial choices to recapitulate the complicated extracellular matrix components, it has been proved by numerous studies that 3D bioprinting has the potential to fabricate functional urological organs for clinical transplantation and in vitro disease models.

Promotion of Adrenal Pheochromocytoma (PC-12) Cell Proliferation and Outgrowth Using Schwann Cell-Laden Gelatin Methacrylate Substrate.

Peripheral nerve injuries cause different degrees of nerve palsy and function loss. Due to the limitations of autografts, nerve tissue engineering (TE) scaffolds incorporated with various neurotrophic factors and cells have been investigated to promote nerve regeneration. However, the molecular mechanism is still poorly understood. In this study, we co-cultured Schwann cells (SCs) and rat adrenal pheochromocytoma (PC-12) cells on 50% degrees of methacryloyl substitution gelatin methacrylate (GelMA) scaffold. The SCs were encapsulated within the GelMA, and PC-12 cells were on the surface. A 5% GelMA was used as the co-culture scaffold since it better supports SCs proliferation, viability, and myelination and promotes higher neurotrophic factors secretion than 10% GelMA. In the co-culture, PC-12 cells demonstrated a higher cell proliferation rate and axonal extension than culturing without SCs, indicating that the secretion of neurotrophic factors from SCs can stimulate PC-12 growth and axonal outgrowth. The mRNA level for neurotrophic factors of SCs in 5% GelMA was further evaluated. We found significant upregulation when compared with a 2D culture, which suggested that this co-culture system could be a potential scaffold to investigate the mechanism of how SCs affect neuronal behaviors.

Effect of Electrical and Electromechanical Stimulation on PC12 Cell Proliferation and Axon Outgrowth.

Peripheral nerve injuries have become a common clinical disease with poor prognosis and complicated treatments. The development of tissue engineering pointed a promising direction to produce nerve conduits for nerve regeneration. Electrical and mechanical stimulations have been incorporated with tissue engineering, since such external stimulations could promote nerve cell proliferation, migration and differentiation. However, the combination of electrical and mechanical stimulations (electromechanical stimulation) and its effects on neuron proliferation and axon outgrowth have been rarely investigated. Herein, silver nanowires (AgNWs) embedded polydimethylsiloxane (PDMS) electrodes were developed to study the effects of electromechanical stimulation on rat pheochromocytoma cells (PC12 cells) behaviors. AgNWs/PDMS electrodes demonstrated good biocompatibility and established a stable electric field during mechanical stretching. PC12 cells showed enhanced proliferation rate and axon outgrowth under electrical stimulation alone, and the cell number significantly increased with higher electrical stimulation intensity. The involvement of mechanical stretching in electrical stimulation reduced the cell proliferation rate and axon outgrowth, compared with the case of electrical stimulation alone. Interestingly, the cellular axons outgrowth was found to depend on the stretching direction, where the axons prefer to align perpendicularly to the stretch direction. These results suggested that AgNWs/PDMS electrodes provide an in vitro platform to investigate the effects of electromechanical stimulation on nerve cell behaviors and can be potentially used for nerve regeneration in the future.

Evaluation of the effect of 3D porous Chitosan-alginate scaffold stiffness on breast cancer proliferation and migration.

Breast cancer (BCa) is one of the most common cancers for women and metastatic BCa causes the majority of deaths. The extracellular matrix (ECM) stiffens during cancer progression and provides biophysical signals to modulate proliferation, morphology, and metastasis. Cells utilize mechanotransduction and integrins to sense and respond to ECM stiffness. Chitosan-alginate (CA) scaffolds have been used for 3D culture, but lack integrin binding ligands, resulting in round cell morphology and limited cell-material interaction. In this study, 2, 4, and 6 wt% CA scaffolds were produced to mimic the stages of BCa progression and evaluate the BCa response to CA scaffold stiffness. All three CA scaffold compositions highly porous with interconnected pores and scaffold stiffness increased with increasing polymer concentration. MDA-MB-231 (231) cells were cultured in CA scaffolds and 2D cultures for 7 d. All CA scaffold cultures had similar cell numbers at 7 d and the 231 cells formed clusters that increased in size during the culture. The 2 wt% CA had the largest clusters throughout the 7 d culture compared with the 4 and 6 wt% CA. The 231 cell migration was evaluated on 2D surfaces after 7 d culture. The 6 wt% CA cultured cells had the greatest migration speed, followed by 4 wt% CA, 2D cultures, and 2 wt% CA. These results suggest that 231 cells sensed the stiffness of CA scaffolds without the presence of focal adhesions. This indicates that a non-integrin-based mechanism may explain the observed mechanotransduction response.

3D porous chitosan-chondroitin sulfate scaffolds promote epithelial to mesenchymal transition in prostate cancer cells.

Prostate cancer (PCa) is a common cancer in men that is curable prior to metastasis, when its prognosis worsens. Chondroitin sulfate (CS) is found in the extracellular matrix of normal prostate tissue and PCa, with greater content in metastatic PCa. Biomaterial scaffolds containing CS have yet to be evaluated for tumor microenvironment applications. Three-dimensional porous chitosan-CS (C-CS) scaffolds were developed and evaluated for PCa culture. Three C-CS scaffold compositions were prepared with 4 w/v% chitosan and 0.1, 0.5, and 1.0 w/v% CS and named 4-0.1, 4-0.5, and 4-1, respectively. The C-CS scaffolds had 90-95% porosity, average pore sizes between 143 and 166 µm, and no significant difference in scaffold stiffness. PC-3 and 22Rv1 PCa cells were cultured on the C-CS scaffolds to study the effect of CS on PCa growth and epithelial to mesenchymal transition (EMT). All C-CS scaffold compositions supported PCa growth and the 4-1 scaffolds had the greatest cell numbers for both PC-3 and 22Rv1. The C-CS scaffolds promoted upregulated EMT marker expression compared to 2D cultures with the greatest EMT marker expression in 4-1 scaffolds. Increasing CS concentration promoted upregulated vimentin expression in PC-3 cultures and N-cadherin and MMP-2 expression in 22Rv1 cultures. C-CS scaffolds promoted docetaxel drug resistance in PC-3 and 22Rv1 cultures and the 4-1 scaffold cultures had the greatest drug resistance. These results indicate that C-CS scaffolds are a promising in vitro platform for PCa.

3D porous chitosan-alginate scaffold stiffness promotes differential responses in prostate cancer cell lines.

Prostate cancer (PCa) is a leading cause of death for men worldwide. Most PCa patients die from metastasis and bone is the most common metastatic site. Three dimensional (3D) porous chitosan-alginate (CA) scaffolds were developed for bone tissue engineering and demonstrated for culture of cancer cells and enrichment of cancer stem cells. However, only a single scaffold composition was studied. Three compositions of 3D porous CA scaffolds (2, 4, and 6 wt%) were used to investigate the effect of scaffold stiffness on PCa cell response with PC-3, C4-2B, and 22Rv1 cell lines. The PC-3 cells formed cell clusters while the C4-2B and 22Rv1 cells formed multicellular spheroids. The three cell lines demonstrated stiffness independent cell growth and expressed phenotypic PCa biomarkers. The osteoblastic PCa lines C4-2B and 22Rv1 mineralized in basal media, while the osteolytic PC-3 line did not, demonstrating that CA scaffold cultures revealed differences in PCa phenotypes. The CA scaffolds are a 3D culture platform that supports PCa growth and phenotypic expression with adjustable scaffold stiffness to mimic stages of metastatic progression. Further investigation of the scaffolds for co-culture of PCa cells with fibroblasts and primary PCa cell culture should be conducted to develop a platform for screening chemotherapies.