Designer Self-assembling Peptide Nanofiber Research Articles

Designer self-assembling peptide nanofiber scaffolds containing link protein N-terminal peptide induce chondrogenesis of rabbit bone marrow stem cells.

Designer self-assembling peptide nanofiber hydrogel scaffolds have been considered as promising biomaterials for tissue engineering because of their excellent biocompatibility and biofunctionality. Our previous studies have shown that a novel designer functionalized self-assembling peptide nanofiber hydrogel scaffold (RLN/RADA16, LN-NS) containing N-terminal peptide sequence of link protein (link N) can promote nucleus pulposus cells (NPCs) adhesion and three-dimensional (3D) migration and stimulate biosynthesis of type II collagen and aggrecan by NPCs in vitro. The present study has extended these investigations to determine the effects of this functionalized LN-NS on bone marrow stem cells (BMSCs), a potential cell source for NP regeneration. Although the functionalized LN-NS cannot promote BMSCs proliferation, it significantly promotes BMSCs adhesion compared with that of the pure RADA16 hydrogel scaffold. Moreover, the functionalized LN-NS remarkably stimulates biosynthesis and deposition of type II collagen and aggrecan. These data demonstrate that the functionalized peptide nanofiber hydrogel scaffold containing link N peptide as a potential matrix substrate will be very useful in the NP tissue regeneration.

Biocompatibility and bioactivity of designer self‐assembling nanofiber scaffold containing FGL motif for rat dorsal root ganglion neurons

We report here a designer self-assembling peptide nanofiber scaffold developed specifically for nerve tissue engineering. We synthesized a peptide FGL-RADA containing FGL (EVYVVAENQQGKSKA), the motif of neural cell adhesion molecule (NCAM), and then attended to make a FGL nanofiber scaffold (FGL-NS) by assembling FGL-RADA with the peptide RADA-16 (AcN-RADARADARADARADA-CONH2). The microstructures of the scaffolds were tested using atomic force microscopy (AFM), and rheological properties of materials were accessed. Then we demonstrated the biocompatibility and bioactivity of FGL-NS for rat dorsal root ganglion neurons (DRGn). We found that the designer self-assembling peptide scaffold was noncytotoxic to neurons and able to promote adhesion and neurite sprouting of neurons. Our results indicate that the designer peptide scaffold containing FGL had excellent biocompatibility and bioactivity with adult sensory neurons and could be used for neuronal regeneration.

Significant Type I and Type III Collagen Production from Human Periodontal Ligament Fibroblasts in 3D Peptide Scaffolds without Extra Growth Factors

We here report the development of two peptide scaffolds designed for periodontal ligament fibroblasts. The scaffolds consist of one of the pure self-assembling peptide scaffolds RADA16 through direct coupling to short biologically active motifs. The motifs are 2-unit RGD binding sequence PRG (PRGDSGYRGDS) and laminin cell adhesion motif PDS (PDSGR). RGD and laminin have been previously shown to promote specific biological activities including periodontal ligament fibroblasts adhesion, proliferation and protein production. Compared to the pure RADA16 peptide scaffold, we here show that these designer peptide scaffolds significantly promote human periodontal ligament fibroblasts to proliferate and migrate into the scaffolds (for ∼300 µm/two weeks). Moreover these peptide scaffolds significantly stimulated periodontal ligament fibroblasts to produce extracellular matrix proteins without using extra additional growth factors. Immunofluorescent images clearly demonstrated that the peptide scaffolds were almost completely covered with type I and type III collagens which were main protein components of periodontal ligament. Our results suggest that these designer self-assembling peptide nanofiber scaffolds may be useful for promoting wound healing and especially periodontal ligament tissue regeneration.

Designer self-assembling peptide nanofiber biological materials

Scientists and bioengineers have dreamed of designing materials from the bottom up with the finest detail and ultimate control at the single molecular level. The discovery of a class of self-assembling peptides that spontaneously undergo self-organization into well-ordered structures opened a new avenue for molecular fabrication of biological materials. Since this discovery, diverse classes of short peptides have been invented with broad applications, including 3D tissue cell culture, reparative and regenerative medicine, tissue engineering, slow drug release and medical device development. Molecular design of new materials using short peptides is poised to become increasingly important in biomedical research, biomedical technology and medicine, and is covered in this tutorial review.

Controlled release of functional proteins through designer self-assembling peptide nanofiber hydrogel scaffold

The release kinetics for a variety of proteins of a wide range of molecular mass, hydrodynamic radii, and isoelectric points through a nanofiber hydrogel scaffold consisting of designer self-assembling peptides were studied by using single-molecule fluorescence correlation spectroscopy (FCS). In contrast to classical diffusion experiments, the single-molecule approach allowed for the direct determination of diffusion coefficients for lysozyme, trypsin inhibitor, BSA, and IgG both inside the hydrogel and after being released into the solution. The results of the FCS analyses and the calculated pristine in-gel diffusion coefficients were compared with the values obtained from the Stokes-Einstein equation, Fickian diffusion models, and the literature. The release kinetics suggested that protein diffusion through nanofiber hydrogels depended primarily on the size of the protein. Protein diffusivities decreased, with increasing hydrogel nanofiber density providing a means of controlling the release kinetics. Secondary and tertiary structure analyses and biological assays of the released proteins showed that encapsulation and release did not affect the protein conformation and functionality. Our results show that this biocompatible and injectable designer self-assembling peptide hydrogel system may be useful as a carrier for therapeutic proteins for sustained release applications.

Designer functionalized self-assembling peptide nanofiber scaffolds for growth, migration, and tubulogenesis of human umbilical vein endothelial cells

We previously reported a class of designer self-assembling peptide nanofiber scaffolds as a unique biological material for diverse applications including 3-D tissue cell culture, slow drug release, regenerative medicine, and tissue engineering. One of these peptide scaffolds, RADA16-I has been used in bone, cartilage, and neural regeneration studies that have shown great promises. We here report the development of two new functionalized self-assembling peptide nanofiber scaffolds designed specifically for angiogenesis study through directly coupling pure RADA16-I with short biologically angiogenic motifs. Angiogenesis is very important in regenerative medicine. An adequate blood vessel supply to the newly formed tissue and within the transplanted scaffold is essential in determining the success of new tissue regeneration. In our study, two designer functionalized peptides, KLT, Ac–(RADA)4G4KLTWQELYQLKYKGI–CONH2 and PRG, Ac–(RADA)4GPRGDSGYRGDS–CONH2 significantly enhanced endothelial cell survival, proliferation, migration, and morphological tubulogenesis compared with unmodified RADA16-I scaffold. We also showed in our clear-boundary sandwich culture without adding extract soluble growth factors that cells migrated uni-directionally from RADA16-I toward the functionalized scaffolds but not the reverse. Our results suggest that the functionalized designer peptide scaffolds will not only have great promise for promoting endothelial cell growth, migration, and tubulogenesis, but also may have widely potential applications for diverse tissue engineering and tissues regeneration.

Biological Designer Self-Assembling Peptide Nanofiber Scaffolds Significantly Enhance Osteoblast Proliferation, Differentiation and 3-D Migration

A class of self-assembling peptide nanofiber scaffolds has been shown to be an excellent biological material for 3-dimension cell culture and stimulating cell migration into the scaffold, as well as for repairing tissue defects in animals. We report here the development of several peptide nanofiber scaffolds designed specifically for osteoblasts. We designed one of the pure self-assembling peptide scaffolds RADA16-I through direct coupling to short biologically active motifs. The motifs included osteogenic growth peptide ALK (ALKRQGRTLYGF) bone-cell secreted-signal peptide, osteopontin cell adhesion motif DGR (DGRGDSVAYG) and 2-unit RGD binding sequence PGR (PRGDSGYRGDS). We made the new peptide scaffolds by mixing the pure RAD16 and designer-peptide solutions, and we examined the molecular integration of the mixed nanofiber scaffolds using AFM. Compared to pure RAD16 scaffold, we found that these designer peptide scaffolds significantly promoted mouse pre-osteoblast MC3T3-E1 cell proliferation. Moreover, alkaline phosphatase (ALP) activity and osteocalcin secretion, which are early and late markers for osteoblastic differentiation, were also significantly increased. We demonstrated that the designer, self-assembling peptide scaffolds promoted the proliferation and osteogenic differentiation of MC3T3-E1. Under the identical culture medium condition, confocal images unequivocally demonstrated that the designer PRG peptide scaffold stimulated cell migration into the 3-D scaffold. Our results suggest that these designer peptide scaffolds may be very useful for promoting bone tissue regeneration.

Citations

Citations

Systematic Studies of a Self-Assembling Peptide Nanofiber Scaffold with Other Scaffolds

A designer self-assembling peptide nanofiber scaffold has been systematically studied with 10 commonly used scaffolds in a several week study using neural stem cells (NSC), a potential therapeutic source for cellular transplantations in nervous system injuries. These cells not only provide a good in vitro model for the development and regeneration of the nervous system, but may also be helpful in testing for cytotoxicity, cellular adhesion, and differentiation properties of biological and synthetic scaffolds used in medical practices. We tested the self-assembling peptide nanofiber scaffold with the most commonly used scaffolds for tissue engineering and regenerative medicine including PLLA, PLGA, PCLA, collagen I, collagen IV, and Matrigel. Additionally, each scaffold was coated with laminin in order to evaluate the utility of this surface treatment. Each scaffold was evaluated by measuring cell viability, differentiation and maturation of the differentiated stem cell progeny (i.e. progenitor cells, astrocytes, oligodendrocytes, and neurons) over 4 weeks. The optimal scaffold should show high numbers of living and differentiated cells. In addition, it was demonstrated that the laminin surface treatment is capable of improving the overall scaffold performance. The designer self-assembling peptide RADA16 nanofiber scaffold represents a new class of biologically inspired material. The well-defined molecular structure with considerable potential for further functionalization and slow drug delivery makes the designer peptide scaffolds a very attractive class of biological material for a number of applications. The peptide nanofiber scaffold is comparable with the clinically approved synthetic scaffolds. The peptide scaffolds are not only pure, but also have the potential to be further designed at the molecular level, thus they promise to be useful for cell adhesion and differentiation studies as well as for future biomedical and clinical studies.

Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures

Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with β-Tubulin+, GFAP+ and Nestin+ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology.