Self-assembling Peptide Scaffolds Research Articles

Biomimetic Electrospun Self-Assembling Peptide Scaffolds for Neural Stem Cell Transplantation in Neural Tissue Engineering.

Spinal cord regeneration using stem cell transplantation is a promising strategy for regenerative therapy. Stem cells transplanted onto scaffolds that can mimic natural extracellular matrix (ECM) have the potential to significantly improve outcomes. In this study, we strived to develop a cell carrier by culturing neural stem cells (NSCs) onto electrospun 2D and 3D constructs made up of specific crosslinked functionalized self-assembling peptides (SAPs) featuring enhanced biomimetic and biomechanical properties. Morphology, architecture, and secondary structures of electrospun scaffolds in the solid-state and electrospinning solution were studied step by step. Morphological studies showed the benefit of mixed peptides and surfactants as additives to form thinner, uniform, and defect-free fibers. It has been observed that β-sheet conformation as evidence of self-assembling has been predominant throughout the process except for the electrospinning solution. In vitro NSCs seeded on electrospun SAP scaffolds in 2D and 3D conditions displayed desirable proliferation, viability, and differentiation in comparison to the gold standard. In vivo biocompatibility assay confirmed the permissibility of implanted fibrous channels by foreign body reaction. The results of this study demonstrated that fibrous 2D/3D electrospun SAP scaffolds, when shaped as micro-channels, can be suitable to support NSC transplantation for regeneration following spinal cord injury.

Long-term cultures of human pancreatic islets in self-assembling peptides hydrogels.

Human pancreatic islets transplantation is an experimental therapeutic treatment for Type I Diabetes. Limited islets lifespan in culture remains the main drawback, due to the absence of native extracellular matrix as mechanical support after their enzymatic and mechanical isolation procedure. Extending the limited islets lifespan by creating a long-term in vitro culture remains a challenge. In this study, three biomimetic self-assembling peptides were proposed as potential candidates to recreate in vitro a pancreatic extracellular matrix, with the aim to mechanically and biologically support human pancreatic islets, by creating a three-dimensional culture system. The embedded human islets were analyzed for morphology and functionality in long-term cultures (14-and 28-days), by evaluating β-cells content, endocrine component, and extracellular matrix constituents. The three-dimensional support provided by HYDROSAP scaffold, and cultured into MIAMI medium, displayed a preserved islets functionality, a maintained rounded islets morphology and an invariable islets diameter up to 4 weeks, with results analogues to freshly-isolated islets. In vivo efficacy studies of the in vitro 3D cell culture system are ongoing; however, preliminary data suggest that human pancreatic islets pre-cultured for 2weeks in HYDROSAP hydrogels and transplanted under subrenal capsule may restore normoglycemia in diabetic mice. Therefore, engineered self-assembling peptide scaffolds may provide a useful platform for long-term maintenance and preservation of functional human pancreatic islets in vitro.

Pericardial Grafting of Cardiac Progenitor Cells in Self-Assembling Peptide Scaffold Improves Cardiac Function After Myocardial Infarction.

Many studies have explored cardiac progenitor cell (CPC) therapy for heart disease. However, optimal scaffolds are needed to ensure the engraftment of transplanted cells. We produced a three-dimensional hydrogel scaffold (CPC-PRGmx) in which high-viability CPCs were cultured for up to 8 weeks. CPC-PRGmx contained an RGD peptide-conjugated self-assembling peptide with insulin-like growth factor-1 (IGF-1). Immediately after creating myocardial infarction (MI), we transplanted CPC-PRGmx into the pericardial space on to the surface of the MI area. Four weeks after transplantation, red fluorescent protein-expressing CPCs and in situ hybridization analysis in sex-mismatched transplantations revealed the engraftment of CPCs in the transplanted scaffold (which was cellularized with host cells). The average scar area of the CPC-PRGmx-treated group was significantly smaller than that of the non-treated group (CPC-PRGmx-treated group = 46 ± 5.1%, non-treated MI group = 59 ± 4.5%; p < 0.05). Echocardiography showed that the transplantation of CPC-PRGmx improved cardiac function and attenuated cardiac remodeling after MI. The transplantation of CPCs-PRGmx promoted angiogenesis and inhibited apoptosis, compared to the untreated MI group. CPCs-PRGmx secreted more vascular endothelial growth factor than CPCs cultured on two-dimensional dishes. Genetic fate mapping revealed that CPC-PRGmx-treated mice had more regenerated cardiomyocytes than non-treated mice in the MI area (CPC-PRGmx-treated group = 0.98 ± 0.25%, non-treated MI group = 0.25 ± 0.04%; p < 0.05). Our findings reveal the therapeutic potential of epicardial-transplanted CPC-PRGmx. Its beneficial effects may be mediated by sustainable cell viability, paracrine function, and the enhancement of de novo cardiomyogenesis.

Increased Stiffness Downregulates Focal Adhesion Kinase Expression in Pancreatic Cancer Cells Cultured in 3D Self-Assembling Peptide Scaffolds.

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that participates in integrin-mediated signal transduction and contributes to different biological processes, such as cell migration, survival, proliferation and angiogenesis. Moreover, FAK can be activated by autophosphorylation at position Y397 and trigger different signaling pathways in response to increased extracellular matrix stiffness. In addition, FAK is overexpressed and/or hyperactivated in many epithelial cancers, and its expression correlates with tumor malignancy and invasion potential. One of the characteristics of solid tumors is an over deposition of ECM components, which generates a stiff microenvironment that promotes, among other features, sustained cell proliferation and survival. Researchers are, therefore, increasingly developing cell culture models to mimic the increased stiffness associated with these kinds of tumors. In the present work, we have developed a new 3D in vitro model to study the effect of matrix stiffness in pancreatic ductal adenocarcinoma (PDAC) cells as this kind of tumor is characterized by a desmoplastic stroma and an increased stiffness compared to its normal counterpart. For that, we have used a synthetic self-assembling peptide nanofiber matrix, RAD16-I, which does not suffer a significant degradation in vitro, thus allowing to maintain the same local stiffness along culture time. We show that increased matrix stiffness in synthetic 3D RAD16-I gels, but not in collagen type I scaffolds, promotes FAK downregulation at a protein level in all the cell lines analyzed. Moreover, even though it has classically been described that stiff 3D matrices promote an increase in pFAKY397/FAK proteins, we found that this ratio in soft and stiff RAD16-I gels is cell-type-dependent. This study highlights how cell response to increased matrix stiffness greatly depends on the nature of the matrix used for 3D culture.

Xeno-free self-assembling peptide scaffolds for building 3D organotypic skin cultures.

Organotypic skin cultures represent in vitro models of skin which can be used for disease modeling, tissue engineering, and screening applications. Non‐human collagen is currently the gold standard material used for the construction of the supporting matrix, however, its clinical applications are limited due to its xenogeneic origin. We have developed a novel peptide hydrogel‐based skin construct that shows a pluristratified epidermis, basement membrane, and dermal compartment after 3 weeks of in vitro culture. Peptide‐based constructs were compared to collagen‐based constructs and stratification marker expression was histologically higher in peptide constructs than in collagen constructs. Transepithelial electrical resistance also showed mature barrier function in peptide constructs. This study presents a novel application of the self‐assembling peptide hydrogel in a defined xeno‐free in vitro system.

β-Sheet to Random Coil Transition in Self-Assembling Peptide Scaffolds Promotes Proteolytic Degradation.

One of the most desirable properties that biomaterials designed for tissue engineering or drug delivery applications should fulfill is biodegradation and resorption without toxicity. Therefore, there is an increasing interest in the development of biomaterials able to be enzymatically degraded once implanted at the injury site or once delivered to the target organ. In this paper, we demonstrate the protease sensitivity of self-assembling amphiphilic peptides, in particular, RAD16-I (AcN-RADARADARADARADA-CONH2), which contains four potential cleavage sites for trypsin. We detected that when subjected to thermal denaturation, the peptide secondary structure suffers a transition from β-sheet to random coil. We also used Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) to detect the proteolytic breakdown products of samples subjected to incubation with trypsin as well as atomic force microscopy (AFM) to visualize the effect of the degradation on the nanofiber scaffold. Interestingly, thermally treated samples had a higher extent of degradation than non-denatured samples, suggesting that the transition from β-sheet to random coil leaves the cleavage sites accessible and susceptible to protease degradation. These results indicate that the self-assembling peptide can be reduced to short peptide sequences and, subsequently, degraded to single amino acids, constituting a group of naturally biodegradable materials optimal for their application in tissue engineering and regenerative medicine.

Erlotinib Promotes Ligand-Induced EGFR Degradation in 3D but Not 2D Cultures of Pancreatic Ductal Adenocarcinoma Cells.

Simple SummaryThe EGFR is a tyrosine kinase receptor that responds to different stresses such as UV irradiation, hypoxia and drug treatment by internalizing into endosomal compartments. Receptor trafficking and degradation due to tyrosine kinase inhibitors has been widely studied in two- dimensional (2D) cell culture systems, but little is known about how cells respond to these types of drugs in more physiologically relevant models such as three-dimensional (3D) cultures, whose nanostructured properties allow cells to grow, proliferate, migrate and extend cellular processes in their 3D space. In this study, we show that EGFR suffers degradation in response to erlotinib treatment in 3D-cultured cancer cells but not in classic 2D culture systems, demonstrating that dimensionality strongly affects cell drug response. This 3D model may pave the way for the development of more physiological culture platforms to obtain mechanistic insights into how cells respond to chemotherapy.The epithelial growth factor receptor (EGFR) is a tyrosine kinase receptor that participates in many biological processes such as cell proliferation. In addition, EGFR is overexpressed in many epithelial cancers and therefore is a target for cancer therapy. Moreover, EGFR responds to lots of stimuli by internalizing into endosomes from where it can be recycled to the membrane or further sorted into lysosomes where it undergoes degradation. Two-dimensional cell cultures have been classically used to study EGFR trafficking mechanisms in cancer cells. However, it has been widely demonstrated that in 2D cultures cells are exposed to a non-physiological environment as compared to 3D cultures that provide the normal cellular conformation, matrix dimensionality and stiffness, as well as molecular gradients. Therefore, the microenvironment of solid tumors is better recreated in 3D culture models, and this is why they are becoming a more physiological alternative to study cancer physiology. Here, we develop a new model of EGFR internalization and degradation upon erlotinib treatment in pancreatic ductal adenocarcinoma (PDAC) cells cultured in a 3D self-assembling peptide scaffold. In this work, we show that treatment with the tyrosine kinase inhibitor erlotinib promotes EGFR degradation in 3D cultures of PDAC cell lines but not in 2D cultures. We also show that this receptor degradation does not occur in normal fibroblast cells, regardless of culture dimensionality. In conclusion, we demonstrate not only that erlotinib has a distinct effect on tumor and normal cells but also that pancreatic ductal adenocarcinoma cells respond differently to drug treatment when cultured in a 3D microenvironment. This study highlights the importance of culture systems that can more accurately mimic the in vivo tumor physiology.

Improvement of Rat Spinal Cord Injury Following Lentiviral Vector-Transduced Neural Stem/Progenitor Cells Derived from Human Epileptic Brain Tissue Transplantation with a Self-assembling Peptide Scaffold

Spinal cord injury (SCI) is a disabling neurological disorder that causes neural circuit dysfunction. Although various therapies have been applied to improve the neurological outcomes of SCI, little clinical progress has been achieved. Stem cell–based therapy aimed at restoring the lost cells and supporting micromilieu at the site of the injury has become a conceptually attractive option for tissue repair following SCI. Adult human neural stem/progenitor cells (hNS/PCs) were obtained from the epileptic human brain specimens. Induction of SCI was followed by the application of lentiviral vector-mediated green fluorescent protein–labeled hNS/PCs seeded in PuraMatrix peptide hydrogel (PM). The co-application of hNS/PCs and PM at the SCI injury site significantly enhanced cell survival and differentiation, reduced the lesion volume, and improved neurological functions compared to the control groups. Besides, the transplanted hNS/PCs seeded in PM revealed significantly higher migration abilities into the lesion site and the healthy host tissue as well as a greater differentiation into astrocytes and neurons in the vicinity of the lesion as well as in the host tissue. Our data suggest that the transplantation of hNS/PCs seeded in PM could be a promising approach to restore the damaged tissues and improve neurological functions after SCI.

Self-Assembling Peptide EAK16 and RADA16 Nanofiber Scaffold Hydrogel.

Short peptides are ubiquitous in nature. They are found as hormones, pheromones, antibacterial and antifungal agents in innate immunity systems, toxins, and pesticides. But no one seriously considered that peptides could be useful as scaffold hydrogel materials. There has been a significant change since 1990 after the discovery of an ionic self-complementary peptide as a very interesting repeating segment in a yeast protein. It is now recognized that self-assembling peptides made from 20 natural amino acids have real material properties. Currently, many diverse applications have been developed from these simple and designer self-assembling peptide scaffold hydrogels and are commercially available. Examples include: (1) real 3D tissue cell cultures of diverse tissue cells and various stem cells, (2) reparative and regenerative medicine as well as tissue engineering, (3) 3D tissue printing, (4) sustained releases of small molecules, growth factors, and monoclonal antibodies, and (5) accelerated wound healings of skin and diabetic ulcers as well as instant hemostatic applications in surgery. Self-assembling peptide nanobiotechnology will likely continue to expand in many directions in the coming years.

Enhanced osteogenesis of human mesenchymal stem cells by self-assembled peptide hydrogel functionalized with glutamic acid templated peptides.

Self-assembling peptide (SAP) hydrogel has been shown to be an excellent biological material for three-dimensional cell culture and stimulatie cell migration and differentiation into the scaffold, as well as for repairing bone tissue defects. Herein, we designed one of the SAP scaffolds KLD (KLDLKLDLKLDL) through direct coupling to short bioactive motif O1 (EEGGC) and O2 (EEEEE) of which bioactivity on osteogenic differentiation was previously demonstrated and self-assembled in different concentrations (0.5%, 1%, and 2%). Our aim was to enhance osteogenesis and biomineralization of injectable SAP hydrogels with controlled mechanical properties so that the peptide hydrogel also becomes capable of being injected to bone defects. The molecular integration of the nanofibrous peptide scaffolds was observed using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The rheological properties and degradation profile of SAP hydrogels were evaluated to ensure stability of SAPs. Compared with pure KLD scaffold, we found that these designed bioactive peptide scaffolds significantly promoted hMSCs proliferation depicted by biochemical analysis of alkaline phosphatase (ALP) activity, total calcium deposition. Moreover, key osteogenic markers of ALP activity, collagen type I (COL-1), osteopontin (OP), and osteocalcin (OCN) expression levels determined by real-time polymerase chain reaction (PCR) and immunofluorescence analysis were also significantly increased with the addition of glutamic acid residues to KLD. We demonstrated that the designed SAP scaffolds promoted the proliferation and osteogenic differentiation of hMSCs. Our results suggest that these designed bioactive peptide scaffolds may be useful for promoting bone tissue regeneration.

Self-Assembling Peptide Scaffold Carrying Neural-Cell Adhesion Molecule-Derived Mimetic-Peptide Transplantation Promotes Proliferation and Stimulates Neurite Extension by Modulating Tau Phosphorylation and Calpain/Glycogen Synthase Kinase 3 beta (GSK-3β) in Neurons.

BackgroundSelf-assembling peptide scaffolds have been extensively applied in tissue engineering. Many investigations have modified self-assembling peptide scaffolds by integrating functional motifs, with promising applications. This study aimed to generate a novel RADA16 self-assembling peptide scaffold integrating a neural-cell adhesion molecule-derived mimetic-peptide (SIDRVEPYSSTAQ) and evaluated the effects on neuron proliferation.Material/MethodsA 37-amino-acids peptide of RADA16-activation motif containing neural-cell adhesion molecule-derived mimetic-peptide (SIDRVEPYSSTAQ) was synthesized and self-assembled into a scaffold. Dorsal root ganglion (DRG) and spinal cord motor neurons (SCMN) were primarily isolated and identified. Neurons (DRG and SCMN) were divided into FRM, FRM-MP, and FRM-MP-LiCl groups. The adherence ability of neurons was evaluated using toluidine blue staining. Proliferation and apoptosis of neurons were assessed using CCK-8 and flow cytometry assay, respectively. Immunofluorescence assay was used to measure neurite extension. Western blot assay was used to assess GSK-3β/p-GSK-3β, Tau/p-Tau, and calpain expression in neurons.ResultsFRM-MP-LiCl released multiple-peptide with higher efficiency. FRM-MP-LiCl significantly enhanced proliferation and inhibited apoptosis compared to FRM and FRM-MP groups (p<0.05). FRM-MP-LiCl incubation increased adherent ability compared to the FRM and FRM-MP groups (p<0.05). FRM-MP-LiCl remarkably increased neurite length (p<0.05), numbers (p<0.05), and branches. FRM-MP-LiCl significantly reduced GSK-3β phosphorylation (p-GSK-3β/GSK-3β) and Tau phosphorylation (p-Tau/Tau) compared to the FRM and FRM-MP groups (p<0.05). FRM-MP-LiCl significantly downregulated calpain compared to the FRM and FRM-MP groups (p<0.05).ConclusionsThe generated self-assembling peptide scaffold carrying FRM motif (SIDRVEPYSSTAQ) promoted neuron proliferation and adherent ability, inhibited apoptosis, and stimulated neurite extension, by reducing Tau protein phosphorylation through the calpain/GSK-3β signaling pathway.

Culture and Differentiation of Human Hair Follicle Dermal Papilla Cells in a Soft 3D Self-Assembling Peptide Scaffold

Hair follicle dermal papilla cells (HFDPC) are a specialized cell population located in the bulge of the hair follicle with unique characteristics such as aggregative behavior and the ability to induce new hair follicle formation. However, when expanded in conventional 2D monolayer culture, their hair inductive potency is rapidly lost. Different 3D culture techniques, including cell spheroid formation, have been described to restore, at least partially, their original phenotype, and therefore, their hair inductive ability once transplanted into a recipient skin. Moreover, hair follicle dermal papilla cells have been shown to differentiate into all mesenchymal lineages, but their differentiation potential has only been tested in 2D cultures. In the present work, we have cultured HFDPC in the 3D self-assembling peptide scaffold RAD16-I to test two different tissue engineering scenarios: restoration of HFDPC original phenotype after cell expansion and osteogenic and adipogenic differentiation. Experimental results showed that the 3D environment provided by RAD16-I allowed the restoration of HFDPC signature markers such as alkaline phosphatase, versican and corin. Moreover, RAD16-I supported, in the presence of chemical inductors, three-dimensional osteogenic and adipogenic differentiation. Altogether, this study suggests a potential 3D culture platform based on RAD16-I suitable for the culture, original phenotype recovery and differentiation of HFDPC.

Controlled release of basic fibroblast growth factor from a peptide biomaterial for bone regeneration.

Self-assembled peptide scaffolds based on D-RADA16 are an important matrix for controlled drug release and three-dimensional cell culture. In this work, D-RADA16 peptide hydrogels were coated on artificial bone composed of nano-hydroxyapatite/polyamide 66 (nHA/PA66) to obtain a porous drug-releasing structure for treating bone defects. The developed materials were characterized via transmission electron microscopy and scanning electron microscopy. The proliferation and adhesion of bone mesenchymal stem cells (BMSCs) were examined by confocal laser microscopy and CCK-8 experiments. The osteogenic ability of the porous materials towards bone BMSCs was examined in vitro by staining with Alizarin Red S and alkaline phosphatase, and bioactivity was evaluated in vivo. The results revealed that nHA/PA66/D-RADA16/bFGF reduces the degradation rate of D-RADA16 hydrogels and prolongs sustained release of bFGF, which would promote BMSCs proliferation, adhesion and osteogenesis in vitro and bone repair in vivo. Thus, it deserves more attention and is worthy of further research.

Vascularization of self-assembled peptide scaffolds for spinal cord injury repair

The disruption of the blood-spinal cord barrier (BSCB) following spinal cord injury contributes to inflammation and glial scarring that inhibits axon growth and diminishes the effectiveness of conduits transplanted to the injury site to promote this growth. The purpose of this study is to evaluate whether scaffolds containing microvessels that exhibit BSCB integrity reduce inflammation and scar formation at the injury site and lead to increased axon growth. For these studies, a self-assembling peptide scaffold, RADA-16I, is used due to its established permissiveness to axon growth and ability to support vascularization. Immunocytochemistry and permeability transport assays verify the formation of tight-junction containing microvessels within the scaffold. Peptide scaffolds seeded with different concentrations of microvascular cells are then injected into a spinal contusion injury in rats to evaluate how microvessels affect axon growth and neurovascular interaction. The effect of the vascularized scaffold on inflammation and scar formation is evaluated by quantifying histological sections stained with ED-1 and GFAP, respectively. Our results indicate that the peptide scaffolds containing microvessels reduce inflammation and glial scar formation and increase the density of axons growing into the injury/transplant site. These results demonstrate the potential benefit of scaffold vascularization to treat spinal cord injury. Statement of SignificanceThis study evaluates the benefit of transplanting microvascular cells within a self-assembling peptide scaffold, RADA-16I, that has shown promise for facilitating regeneration in the central nervous system in previous studies. Our results indicate that vasculature featuring tight junctions that give rise to the blood-spinal cord barrier can be formed within the peptide scaffold both in vitro and in a rat model of a subacute contusion spinal cord injury. Histological analysis indicates that the presence of the microvessels encourages axon infiltration into the site of injury and reduces the area of astrocyte activation and inflammation. Overall, these results demonstrate the potential of vascularizing scaffolds for the repair of spinal cord injury.

Angiogenic Self-Assembling Peptide Scaffolds for Functional Tissue Regeneration

Implantation of acellular biomimetic scaffolds with proangiogenic motifs may have exciting clinical utility for the treatment of ischemic pathologies such as myocardial infarction. Although direct delivery of angiogenic proteins is a possible treatment option, smaller synthetic peptide-based nanostructured alternatives are being investigated due to favorable factors, such as sustained efficacy and high-density epitope presentation of functional moieties. These peptides may be implanted in vivo at the site of ischemia, bypassing the first-pass metabolism and enabling long-term retention and sustained efficacy. Mimics of angiogenic proteins show tremendous potential for clinical use. We discuss possible approaches to integrate the functionality of such angiogenic peptide mimics into self-assembled peptide scaffolds for application in functional tissue regeneration.

Development of a 3D Co-Culture System as a Cancer Model Using a Self-Assembling Peptide Scaffold.

Cancer research has traditionally relied on two-dimensional (2D) cell culture, focusing mainly on cancer cells and their abnormal genetics. However, over the past decade, tumors have been accepted as complex tissues rather than a homogenous mass of proliferating cells. Consequently, cancer cells’ behavior can only be deciphered considering the contribution of the cells existing in the tumor stroma as well as its complex microenvironment. Since the tumor microenvironment plays a critical role in tumorigenesis, it is widely accepted that culturing cells in three-dimensional (3D) scaffolds, which mimic the extracellular matrix, represents a more realistic scenario. In the present work, an in vitro 3D co-culture system based on the self-assembling peptide scaffold RAD16-I (SAPS RAD16-I) was developed as a cancer model. For that, PANC-1 cells were injected into a RAD16-I peptide scaffold containing fibroblasts, resulting in a 3D system where cancer cells were localized in a defined area within a stromal cells matrix. With this system, we were able to study the effect of three well-known pharmaceutical drugs (Gemcitabine, 5-Fluorouracil (5-FU), and 4-Methylumbelliferone (4-MU)) in a 3D context in terms of cell proliferation and survival. Moreover, we have demonstrated that the anti-cancer effect of the tested compounds can be qualitatively and quantitatively evaluated on the developed 3D co-culture system. Experimental results showed that Gemcitabine and 5-FU prevented PANC-1 cell proliferation but had a high cytotoxic effect on fibroblasts as well. 4-MU had a subtle effect on PANC-1 cells but caused high cell death on fibroblasts.

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Human Neural Stem/Progenitor Cells Derived From Epileptic Human Brain in a Self-Assembling Peptide Nanoscaffold Improve Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) is a disruption in the brain functions following a head trauma. Cell therapy may provide a promising treatment for TBI. Among different cell types, human neural stem cells cultured in self-assembling peptide scaffolds have been suggested as a potential novel method for cell replacement treatment after TBI. In the present study, we accessed the effects of human neural stem/progenitor cells (hNS/PCs) derived from epileptic human brain and human adipose-derived stromal/stem cells (hADSCs) seeded in PuraMatrix hydrogel (PM) on brain function after TBI in an animal model of brain injury. hNS/PCs were isolated from patients with medically intractable epilepsy undergone epilepsy surgery. hNS/PCs and hADSCs have the potential for proliferation and differentiation into both neuronal and glial lineages. Assessment of the growth characteristics of hNS/PCs and hADSCs revealed that the hNS/PCs doubling time was significantly longer and the growth rate was lower than hADSCs. Transplantation of hNS/PCs and hADSCs seeded in PM improved functional recovery, decreased lesion volume, inhibited neuroinflammation, and reduced the reactive gliosis at the injury site. The data suggest the transplantation of hNS/PCs or hADSCs cultured in PM as a promising treatment option for cell replacement therapy in TBI.

Cross-linked self-assembling peptide scaffolds

Self-assembling peptides (SAPs) are synthetic bioinspired biomaterials that can be feasibly multi-functionalized for cell transplantation and/or drug delivery therapies. Despite their superior biocompatibility and ease of scaling-up for production, they are unfortunately hampered by weak mechanical properties due to transient non-covalent interactions among and within the self-assembled peptide chains, thus limiting their potential applications as fillers, hemostat solutions, and fragile scaffolds for soft tissues. Here, we have developed and characterized a cross-linking strategy that increases both the stiffness and the tailorability of SAP hydrogels, enabling the preparation of transparent flexible threads, discs, channels, and hemispherical constructs. Empirical and computational results, in close agreement with each other, confirmed that the cross-linking reaction does not affect the previously self-assembled secondary structures. In vitro tests also provided a first hint of satisfactory biocompatibility by favoring viability and differentiation of human neural stem cells. This work could bring self-assembling peptide technology to many applications that have been precluded so far, especially in regenerative medicine.