A 3D Biomimetic Scaffold using Electrospinning for Tissue Engineering Applications

Electrospinning holds great promise for designing functional 3D biomimetic scaffolds for tissue engineering applications. The technique allows for the reproducible fabrication of 3D scaffolds with control over the porosity and thickness. In this work, a novel method for the synthesis of a 3D electroactive scaffold using electrospinning from polycaprolactone (PCL), Polyvinylidene Fluoride (PVDF) and Polyaniline (PANI) is reported. Additional scaffolds involving different morphologies of PCL, PCL-PVDF and PCL-PANI-PVDF were also fabricated and evaluated. The scaffolds were characterized using electron microscopy to visualize the morphologies. Infrared spectroscopy was used to confirm the presence of polymers and their respective phases in the scaffolds, and the degree of crystallinity was calculated using data from X-ray diffraction. Mechanical properties of the scaffolds were studied and the data was used to predict the cell-scaffold response. The method of preparation of the PCL-PANI-PVDF scaffolds of nanofibrous morphology provided control over the architecture of the scaffold. The synthesis process involved the preparation of doped PCL-PANI dispersions which were used as the core polymer solution. A PVDF polymer solution was used as the sheath solution. The synthesized scaffolds had many layers of fibers and were aligned. The scaffolds were seeded with H9c2 cells derived from rat cardiomyoblasts to check the cell-scaffold interactions. The cell line was chosen among many others because of the membrane potential of the cells and mechanical stiffness of scaffold required. Immunofluorescent staining for the actin filaments were used to evaluate the cell response to the scaffold. The scaffolds seeded with cells were also imaged using electron microscopy to check for scaffold infiltration and cell-scaffold interaction. Among all the scaffolds, PCL-PANI-PVDF showed behavior of a true biomimetic scaffold with scaffold infiltration, cell alignment and cell proliferation. The scaffolds were used as fabricated after sterilization and no external treatment was required. This research can be used for the future fabrication of acellular scaffolds for different applications like organ engineering, neural interfaces and drug eluting scaffolds

ARTIFICIAL SYNTHETIC SCAFFOLDS FOR TISSUE ENGINEERING APPLICATION EMPHASIZING THE ROLE OF BIOPHYSICAL CUES

The mechanotransduction of cells is the intrinsic ability of cells to convert the mechanical signals provided by the surrounding matrix and other cells into biochemical signals that affect several distinct processes such as tumorigenesis, wound healing, and organ formation. The use of biomaterials as an artificial scaffold for cell attachment, differentiation and proliferation provides a tool to modulate and understand the mechanotransduction pathways, develop better in vitro models and clinical remedies. The effect of topographical cues and stiffness was investigated in fibroblasts using polycaprolactone (PCL)- Polyaniline (PANI) based scaffolds that were fabricated using a self-assembly method and electrospinning. Through this method, scaffolds of different topography and stiffness were fabricated with similar surface chemistries. The effect of scaffold morphologies on the cells were investigated. PCL scaffolds of three distinct morphologies- honeycomb, aligned and mesh were used with similar surface chemistry to investigate the changes in cell behavior of breast, renal, lung and bladder cancer to the physical cues. Selective adhesion and localization of cells to specific morphologies were determined. In order to demonstrate the scaffold as a source of biochemical signals, ManCou-H, capable of targeting the fructose-specific glucose transporter GLUT5 was electrospun with the scaffolds of different morphologies. The PCL scaffolds were used as the backbone to release ManCou-H and changes in protein expression and metabolic activity was characterized. The findings made available through this research will help in the design of better cell-specific in vitro model systems to better understand cellular responses to clinical therapies, assess cell response to specific mechanical and chemical cues

Multi-functional electrospun nanofibers from polymer blends for scaffold tissue engineering

Electrospinning and polymer blending have been the focus of research and the industry for their versatility, scalability, and potential applications across many different fields. In tissue engineering, nanofiber scaffolds composed of natural fibers, synthetic fibers, or a mixture of both have been reported. This review reports recent advances in polymer blended scaffolds for tissue engineering and the fabrication of functional scaffolds by electrospinning. A brief theory of electrospinning and the general setup as well as modifications used are presented. Polymer blends, including blends with natural polymers, synthetic polymers, mixture of natural and synthetic polymers, and nanofiller systems, are discussed in detail and reviewed

Self-assembly of 3D nanostructures in electrospun polycaprolactone-polyaniline fibers and their application as scaffolds for tissue engineering

The fabrication of synthetic scaffolds that mimic the microenvironment of cells is a crucial challenge in materials science. The honeycomb morphology is one such bio-mimicking structure that possesses unique physical properties and high packing efficiency in a 3-dimensional space. Here, we present a novel method for electrospinning polycaprolactone-polyaniline with continuous, self-assembled, uniform, interwoven nanofibers forming patterns without the use of templates or porogens. By using the approach presented here, unique architectures mimicking the natural mechanical anisotropy of extracellular matrix were created by varying the electric field. Adult human dermal fibroblasts (HDFa) cells were successfully cultured on the nanofiber scaffolds without any external growth factors or post-processing of the nanofibers and compared to a commercially available dermal template. Our data indicates that despite identical chemical composition, the physical properties impact cell attachment, alignment and penetration into the scaffold. The mechanical strength of the fibers also plays a role with a distinct preference to fibers with high stiffness and ultimate tensile strength. Thus, by tuning the electric field, fibers with different physical properties and patterns can be fabricated for different applications

Engineered three-dimensional scaffolds modulating fate of breast cancer cells using stiffness and morphology related cell adhesion

Goal: Artificially engineering the tumor microenvironment in vitro as a vital tool for understanding the mechanism of tumor progression. In this study, we developed three-dimensional cell scaffold systems with different topographical features and mechanical properties but similar surface chemistry. The cell behavior was modulated by the topography and mechanical properties of the scaffold. Adenocarcinoma (MCF7), triple-negative (MDA-MB-231) and premalignant (MCF10AneoT) breast cancer cells were seeded on the scaffold systems. The cell viability, cell-cell interaction and cell-matrix interactions were analyzed. The preferential growth and alignment of specific population of cells were demonstrated. Among the different scaffolds, triple-negative breast cancer cells preferred honeycomb scaffolds while adenocarcinoma cells favored mesh scaffolds and premalignant cells preferred the aligned scaffolds. The 3D model system developed here can be used to support growth of only specific cell populations or for the growth of tumors. This model can be used for understanding the topographical and mechanical features affecting tumorigenesis, cancer cell growth and migration behavior of malignant and metastatic cancer cells

Preclinical characterization of the efficacy and safety of biologic N‑001 as a novel pain analgesic for post‑operative acute pain treatment

Inhibition of actin remodeling in nerves modulates action potential propagation and therefore could be used to treat acute pain. N-001 is a novel protein analgesic engineered from several C. Botulinum toxins. N-001 targets sensory neurons through ganglioside GT1b binding and ADP-ribosylates G-actin reducing actin remodeling. The activity and efficacy of N-001 was evaluated previously in vitro and in a mouse inflammatory pain model. To assess the relevance of N-001 for treatment of acute post-surgical pain, the current study evaluated the efficacy of N-001 in a mouse hind-paw incision model by periincisional and popliteal nerve block administration combined with mechanical testing. N-001 provided relief of pain-like behavior over 3 days and 2 days longer than the conventional long-acting anesthetic bupivacaine. Preclinical safety studies of N-001 indicated the drug produced no toxic or adverse immunological reactions over multiple doses in mice. These results combined with past targeting results encourage further investigation of N-001 as an analgesic for post-operative pain management with the potential to function as a differential nociceptor-specific nerve block

Data for: Self-Assembly of 3D Nanostructures in Electrospun Polycaprolactone-Polyaniline Fibers and their Application as Scaffolds for Tissue Engineering

The current data set includes the processed data used in the manuscript

Topographical ues overlaid with fructose-like molecules to assess breast cancer recruitment in nanofiber scaffolds

Tumorigenesis is a complex process involving numerous cellular signaling cascades and environmental factors. While 2D cultures for stiffness measurements and more recently 3D cultures to demonstrate differences in cell structures have been reported, very little is known about the impact of sugars in cell recruitment. In this study, we report the fabrication of 3D-scaffolds with different morphologies obtained by electrospinning with fluorescent fructose-like molecular probes to study cancer cell proliferation and migration. Using a FDA approved, biocompatible and biodegradable polymer Polycaprolactone (PCL), we electrospun nanofiber scaffolds having random, aligned, and honeycomb morphologies. Scaffolds with similar morphology without the fructose mimicking analogs were fabricated and used as controls. The degradation rate of the scaffolds was also characterized to ensure long-term availability of probes and mechanical stability of PCL. Cell viability, cell morphology, formation of colonies with changes in morphology were investigated. The changes in biophysical properties of tumor microenvironment with change in morphology of the scaffolds were investigated. In vitro tests for proliferation, alignment and migration of normal breast epithelial cells (184B5), adenocarcinoma (MCF-7), pre-malignant (MCF10AneoT) and triple-negative (MDAMB231 on the scaffolds on days 1, 2, and 3 were carried out. The morphology of the scaffolds was characterized using FE-SEM; cell proliferation was assessed using CellTiter-Blue® Viability Assay; migration and cell-scaffold interactions were investigated using phalloidin for F-Actin and fructose response was determined by immunostaining for facilitative fructose transporter GLUT-5. Our data indicates that while topographical features affected cell adhesion and proliferation, cell lines that responded to the fructose-like probes tended to be more invasive. Furthermore, the preference to a specific scaffold was greatly altered by the presence of the probes with MDAMB231 showing least preference after 72 hours and pre-malignant AneoT showing highest preference. However, there was no significant difference in the cell numbers between scaffolds with probes and those without for the pre-malignant cells while this difference was noticeable in the control cell lines. Further investigation into specific response to glucose and fructose uptake through their major transporters GLUT2 and GLUT5 are currently ongoing

Mechanical Properties and Morphological Alterations in Fiber-Based Scaffolds Affecting Tissue Engineering Outcomes

Electrospinning is a versatile tool used to produce highly customizable nonwoven nanofiber mats of various fiber diameters, pore sizes, and alignment. It is possible to create electrospun mats from synthetic polymers, biobased polymers, and combinations thereof. The post-processing of the end products can occur in many ways, such as cross-linking, enzyme linking, and thermal curing, to achieve enhanced chemical and physical properties. Such multi-factor tunability is very promising in applications such as tissue engineering, 3D organs/organoids, and cell differentiation. While the established methods involve the use of soluble small molecules, growth factors, stereolithography, and micro-patterning, electrospinning involves an inexpensive, labor un-intensive, and highly scalable approach to using environmental cues, to promote and guide cell proliferation, migration, and differentiation. By influencing cell morphology, mechanosensing, and intracellular communication, nanofibers can affect the fate of cells in a multitude of ways. Ultimately, nanofibers may have the potential to precisely form whole organs for tissue engineering, regenerative medicine, and cellular agriculture, as well as to create in vitro microenvironments. In this review, the focus will be on the mechanical and physical characteristics such as porosity, fiber diameter, crystallinity, mechanical strength, alignment, and topography of the nanofiber scaffolds, and the impact on cell proliferation, migration, and differentiation

Electrospun acellular scaffolds for mimicking the natural anisotropy of the extracellular matrix

In tissue engineering, the use of scaffolds helps establish a synergistic relationship between the scaffolds and the tissues by improving cell–scaffold interaction. This interaction is enhanced when physiologically relevant biophysical cues are replicated in the artificial scaffolds. Here, we present a novel scaffold that mimics the natural anisotropy of the native extracellular matrix of tissues, fabricated by electrospinning a combination of three polymers: polycaprolactone (PCL), polyvinylidene fluoride (PVDF) and polyaniline (PANI). The scaffolds were characterized for their morphology, surface and mechanical properties. Rat cardiomyoblast (H9c2) cells, cultured on the PCL–PANI–PVDF scaffold, demonstrated cell alignment, penetration and proliferation across the entire surface area of the scaffold without any external chemical or physical stimuli. The PCL–PANI–PVDF scaffold, unlike other scaffolds, does not require post-processing or specific temperature conditions of storage, prior to use. These acellular scaffolds fabricated through polymer blending, open new avenues for research on functional acellular scaffolds for tissue engineering, based on synthetic materials