Yongchao Wu

Preparation and ectopic osteogenesis in vivo of scaffold based on mineralized recombinant human-like collagen loaded with synthetic BMP-2-derived peptide.

The ideal bone graft material must be biocompatible, biodegradable, osteoconductive and osteoinductive. In this study, a new biomimetic scaffold based on mineralized recombinant collagen, nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) (nHA/RHLC/PLA), was prepared and the synthetic P24 peptide derived from BMP-2 was introduced into the porous nHA/RHLC/PLA scaffold to improve its osteoinductive property. The nHA/RHLC/PLA implants loaded with 3 mg, 2 mg, 1 mg and 0 mg P24 peptide were implanted subcutaneously into rats. At the 4th, 8th and 12th weeks after implantation, the rats were sacrificed in batch and the samples were harvested. Their osteogenic capability was detected by CT scan and histological observation. The results indicated that the osteogenic capability of 3 mg, 2 mg and 1 mg of the P24 peptide was superior to the implants without the P24 peptide. There was no significant difference between implants with 3 mg and 2 mg P24 peptide, but the osteogenic capability of the two dosage groups was significantly better than that of the 1 mg group. It was concluded that BMP-2-derived peptide can increase the osteoinduction of nHA/RHLC/PLA scaffold and the P24 peptide induced new bone formation in a dose-dependent manner. The nHA/RHLC/PLA scaffold loaded with the synthetic BMP-2-derived peptide is a kind of ideal scaffold material for bone tissue engineering.

Functionalized self-assembling peptide nanofiber hydrogel as a scaffold for rabbit nucleus pulposus cells.

In this study, a new functionalized peptide RLN was designed containing the bioactive motif link N, the amino terminal peptide of link protein. A link N nanofiber scaffold (LN-NS) was self-assembled by mixing peptide solution of RLN and RADA16. The characterization of LN-NS was tested using atomic force microscopy (AFM). The biocompatibility and bioactivity of this nanofiber scaffold for rabbit nucleus pulposus cells (NPCs) were also evaluated. This designer functionalized nanofiber scaffold exhibited little cytotoxicity and promoted NPCs adhesion obviously. In three-dimensional cell culture experiments, confocal reconstructed images testified that the functionalized LN-NS-guided NPCs migration from the surface into the hydrogel considerably, in which the RADA16 scaffold did not. Moreover, the functionalized LN-NS significantly stimulated the biosynthesis of extracelluar matrices (ECM) by NPCs. Our findings demonstrate that the functionalized nanofiber scaffold containing link N had excellent biocompatibility and bioactivity with rabbit NPCs and could be useful in the nucleus pulposus regeneration.

Neural Progenitor Cells Survival and Neuronal Differentiation in Peptide-Based Hydrogels

We designed nanofibrous hydrogels as 2-D and 3-D scaffolds for anchorage-dependent cells. The IKVAV-containing peptide amphiphile molecules spontaneously self-assembled into higher-order nanofiber hydrogels under cell-containing media. Neural progenitor cells (NPCs) were incubated in peptide-based hydrogels. Effects of self-assembling hydrogels on survival and neural differentiation of NPCs were observed. Peptide was synthesized using a solid-phase method. TEM study of the hydrogel revealed a network of nanofibers. Phase-contrast light micrographs showed that the described hydrogel had no observable cytotoxicity to NPCs. Additionally this hydrogel could induce cells to differentiate into neuron-like cells and glial-like cells. Moreover, the cells encapsulated within hydrogel had a higher neuronal differentiation rate than in the surface of the hydrogel. This self-assembled hydrogel might serve as nerve tissue-engineering scaffold.

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.

Self-assembled IKVAV Peptide Nanofibers Promote Adherence of PC12 Cells

Lack of biocompatibility and bioactivity is a big problem for the synthetic materials that have been generated for neural tissue engineering. To get around the problem and generate better scaffold for neural tissue repair, we intended to generate nano-fibers by self-assembly of polypeptide IKVAV. Bioactive IKVAV Peptide-Amphiphile (IKVAV-PA) was first synthesized and purified, the property of which was analyzed and determined by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Then, by addition of hydrogen chloride (HCl), self-assembly of IKVAV-PA was induced in vitro and nano-fibers formed as shown by transmission electron microscopy (TEM). The effect of IKVAV nanofibers on adherence of PC12 cells was assayed in cell culture and the results showed that the rates of adherence of PC12 increased significantly when the density of IKVAV was within a certain range (0.58 µg/cm2 to 15.6 µg/cm2). However, its effect on the rates of adherence did not significantly alter with time, whether after 1 hour or 3 hours of culture. In general, we showed that IKVAV-PA can successfully self-assemble to form nanofiber, and promote rapid and stable adherence of PC12 cells, and the effect of the self-assembled IKVAV to promote PC12 cells adherence is dosage-dependent within a certain range of densities.

FGL-functionalized self-assembling nanofiber hydrogel as a scaffold for spinal cord-derived neural stem cells.

A class of designed self-assembling peptide nanofiber scaffolds has been shown to be a good biomimetic material in tissue engineering. Here, we specifically made a new peptide hydrogel scaffold FGLmx by mixing the pure RADA16 and designer functional peptide RADA16-FGL solution, and we analyzed the physiochemical properties of each peptide with atomic force microscopy (AFM) and circular dichroism (CD). In addition, we examined the biocompatibility and bioactivity of FGLmx as well as RADA16 scaffold on spinal cord-derived neural stem cells (SC-NSCs) isolated from neonatal rats. Our results showed that RADA16-FGL displayed a weaker β-sheet structure and FGLmx could self-assemble into nanofibrous morphology. Moreover, we found that FGLmx was not only noncytotoxic to SC-NSCs but also promoted SC-NSC proliferation and migration into the three-dimensional (3-D) scaffold, meanwhile, the adhesion and lineage differentiation of SC-NSCs on FGLmx were similar to that on RADA16. Our results indicated that the FGL-functionalized peptide scaffold might be very beneficial for tissue engineering and suggested its further application for spinal cord injury (SCI) repair.

Biocompatibility of KLD-12 Peptide Hydrogel as a Scaffold in Tissue Engineering of Intervertebral Discs in Rabbits *

KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH2 was synthesized and its biocompatibility was assessed in animals. Rabbit MSCs were cultured in the hydrogel for 2 weeks. Live cells were counted by using Calcein-AM/PI fluorescence staining. MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%, 0.03%, and 0.05%. Hemolysis test, skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues. Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape. Cell survival rate was 92.15% (mean: 92.15%±1.17%) at the 7th day and there was no difference in survival rate between day 7 and day 14. Cell proliferation test showed that the A value of the KLD-12 solutions was not significantly different from that of control groups (complete culture media) (P>0.05) at the 24th and 48th h. The hemolysis rate of KLD-12 solution was 0.112%. Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0. The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes. It is concluded that KLD-12 peptide hydrogel had a good biocompatibility with host rabbit and MSCs, and KLD-12 peptide hydrogel can provide an appropriate microenvironment for MSCs.SummaryKLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH2 was synthesized and its biocompatibility was assessed in animals. Rabbit MSCs were cultured in the hydrogel for 2 weeks. Live cells were counted by using Calcein-AM/PI fluorescence staining. MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%, 0.03%, and 0.05%. Hemolysis test, skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues. Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape. Cell survival rate was 92.15% (mean: 92.15%±1.17%) at the 7th day and there was no difference in survival rate between day 7 and day 14. Cell proliferation test showed that the A value of the KLD-12 solutions was not significantly different from that of control groups (complete culture media) (P>0.05) at the 24th and 48th h. The hemolysis rate of KLD-12 solution was 0.112%. Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0. The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes. It is concluded that KLD-12 peptide hydrogel had a good biocompatibility with host rabbit and MSCs, and KLD-12 peptide hydrogel can provide an appropriate microenvironment for MSCs.

Biocompatibility of functionalized designer self‐assembling nanofiber scaffolds containing FRM motif for neural stem cells

Peptide self-assembling scaffolds have been widely used in tissue engineering. Much work has been focused on modifying the self-assembling scaffolds with functional motifs for desired biological activities. We report here the development of a biological material designed specifically for neural tissue engineering (NTE). Using RADA-16 (AcN-RADARADARADARADA-CONH2) as a base scaffold, we synthesized a 31 amino acid peptide RADA-FRM (AcN-RADARADARADARADAGGSIDRVEPYSSTAQ-CONH2) containing the neural cell adhesion molecule (NCAM)-derived mimetic peptide FRM (SIDRVEPYSSTAQ), which could undergo self-assembly into a nanofiber scaffold. We tested the characterization of the nanofiber scaffold using atomic force microscopy (AFM) and accessed the rheological properties of FRM-containing nanofiber scaffold (FRM-NS). Then we examined its biocompatibility on neural stem cells (NSCs) from neonatal rats. Regrettably, we found that FRM-NS had no effect on differentiation of NSCs. However, we tested that FRM-NS was noncytotoxic. Furthermore, compared to pure RADA-16 scaffold, we found that the designer self-assembling peptide scaffold containing FRM motif could significantly promote NSCs proliferation and stimulate NSCs migration into the three-dimensional scaffold. Our results indicate that the novel designer peptide scaffold containing FRM had excellent biocompatibility with NSCs and may be useful for central nervous tissue repair.

Polydopamine-assisted BMP-2-derived peptides immobilization on biomimetic copolymer scaffold for enhanced bone induction in vitro and in vivo

In this study, a polydopamine (pDA)-coated PLGA-[Asp-PEG]n scaffold was developed for sustained delivery of bone morphogenetic protein-2 (BMP-2)-derived peptide (designated as P24), and then used to address the hypothesis that P24 peptides delivered from the scaffolds could enhance bone induction in vitro and in vivo. We found pDA coating as compared with physical adsorption could more efficiently mediate the grafting of peptides onto polymer surfaces, and the release of P24 peptides from PLGA-[Asp-PEG]n-pDA-P24 was sustained for about 21 days, while a burst P24 release was observed in initial 4h and almost all peptides were released within 24h in physisorbed PLGA-[Asp-PEG]n-P24 group. In vitro, significantly greater ALP activity and mRNA expressions of osteo-specific markers of rat-derived mesenchymal stem cells (rMSCs) were observed in the sustained delivery system than those in physisorbed PLGA-[Asp-PEG]n-P24 and unmodified PLGA-[Asp-PEG]n groups. In vivo, ectopic bone formation studies showed that the sustained delivery system could induce bone formation to a much greater extent than physisorbed PLGA-[Asp-PEG]n-P24. Meanwhile, there were no evidences of bone formation in non-P24-loaded PLGA-[Asp-PEG]n. It is concluded that PLGA-[Asp-PEG]n-pDA-P24 biomaterial can delivery bioactive P24 peptides in a sustained manner, which can more efficiently promote osteogenic differentiation of rMSCs in vitro and induce ectopic bone formation in vivo, as compared with PLGA-[Asp-PEG]n-P24 delivering P24 in a burst manner. This pDA-coated PLGA-[Asp-PEG]n-pDA-P24 composite promises to be an excellent biomaterial for inducing bone regeneration. Moreover, pDA-mediated catechol functionalization can be an effective, simple technique for developing sustained delivery systems.

Effect of IKVAV Peptide Nanofiber on Proliferation, Adhesion and Differentiation into Neurocytes of Bone Marrow Stromal Cells *

SummaryThis study examined the effect of IKVAV peptide nanofiber on proliferation, adhesion and differentiation into neurocytes of bone marrow stromal cells (BMSCs). IKVAV Peptide-amphiphile was synthesized and purified. Then, hydrogen chloride was added to the diluted aqueous solutions of PA to induce spontaneous formation of nanofiber in vitro. The resultant samples was observed under transmission electron microscope. BMSCs were cultured with IKVAV peptide nanofiber. The effect of IKVAV nanofiber on the proliferation, adhesion and induction differentiation of BMSCs was observed by inverted microscopy, calcein-AM/PI staining, cell counting and immunofluorescence staining. The results demonstrated that IKVAV peptide-amphiphile could self-assemble to form nanofiber gel. BMSCs cultured in combination with IKVAV peptide nanofiber gel grew well and the percentage of live cells was over 90%. IKVAV peptide nanofiber gel exerted no influence on the proliferation of BMSCs and could promote the adhesion of BMSCs and raise the ratio of neurons when BMSCs were induced to differentiate into neurocytes. It is concluded that BMSCs could proliferate and adhere well and yield more neurons during when induced to differente into neurocytes on IKVAV peptide nanofiber gel.This study examined the effect of IKVAV peptide nanofiber on proliferation, adhesion and differentiation into neurocytes of bone marrow stromal cells (BMSCs). IKVAV Peptide-amphiphile was synthesized and purified. Then, hydrogen chloride was added to the diluted aqueous solutions of PA to induce spontaneous formation of nanofiber in vitro. The resultant samples was observed under transmission electron microscope. BMSCs were cultured with IKVAV peptide nanofiber. The effect of IKVAV nanofiber on the proliferation, adhesion and induction differentiation of BMSCs was observed by inverted microscopy, calcein-AM/PI staining, cell counting and immunofluorescence staining. The results demonstrated that IKVAV peptide-amphiphile could self-assemble to form nanofiber gel. BMSCs cultured in combination with IKVAV peptide nanofiber gel grew well and the percentage of live cells was over 90%. IKVAV peptide nanofiber gel exerted no influence on the proliferation of BMSCs and could promote the adhesion of BMSCs and raise the ratio of neurons when BMSCs were induced to differentiate into neurocytes. It is concluded that BMSCs could proliferate and adhere well and yield more neurons during when induced to differente into neurocytes on IKVAV peptide nanofiber gel.