A novel artificial nerve graft for repairing longdistance sciatic nerve defects:a self-assembling peptide nanofiber scaffold-containing poly (lactic-co-glycolic acid) conduit

In this study, we developed a novel artificial nerve graft termed self-assembling peptide nanofiber scaffold （SAPNS）-containing poly（lactic-co-glycolic acid） （PLGA） conduit （SPC） and used it to bridge a 10-mm-long sciatic nerve defect in the rat. Retrograde tracing, behavioral testing and histomorphometric analyses showed that compared with the empty PLGA conduit implantation group, the SPC implantation group had a larger number of growing and extending axons, a markedly increased diameter of regenerated axons and a greater thickness of the myelin sheath in the conduit. Furthermore, there was an increase in the size of the neuromuscular junction and myofiber diameter in the target muscle. These findings suggest that the novel artificial SPC nerve graft can promote axonal regeneration and remyelination in the transected peripheral nerve and can be used for repairing peripheral nerve injury.

Chondrogenesis of Precartilaginous Stem Cells in KLD-12 Self-assembling Peptide Nanofiber Scaffold Loading TGF-β3 Gene

The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells （PSCs） into chondrocytes was studied. KLD-12 was synthesized by solid-state method. After TGF-β3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold, DNA release ability was investigated. PSCs and hTGF-β3 gene were loaded into KLD-12 3-D scaffold, and MTT assay was performed to investigate the cell proliferation, and ELASA assay was used to investigate the expression of TGF-β3. Specific cartilage matrix was examined by quantitative real-time PCR, immunohistochemistry and Alcian Blue staining. Compared with control group, DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, and maintained this high level within 2 weeks. MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group （P〈0.05）. Quantitative real-time PCR suggested that the percentage of TGF-β3 positive PSCs in experimental group was higher than that in control group （P〈0.01）. ELISA assay showed that the TGF-β3 protein level increased in supernatant of experimental group＇s PSCs, reached the peak after 72 h and then declined a little to the plateau phase. Compared with the control group, the specific gene of chondrocyte typical extracellular matrix significantly up-regulated （P〈0.01）. The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, which provided a fresh approach to cartilage tissue engineering.

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 （HC1）, 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 PCI2 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/cm^2 to 15.6 μg/cm^2）. 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 PCI2 cells adherence is dosage-dependent within a certain range of densities.

A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

Vascular endothelial growth factor and its mimic peptide KLTWQELYQLKYKGI(QK)are widely used as the most potent angiogenic factors for the treatment of multiple ischemic diseases.However,conventional topical drug delivery often results in a burst release of the drug,leading to transient retention(inefficacy)and undesirable diffusion(toxicity)in vivo.Therefore,a drug delivery system that responds to changes in the microenvironment of tissue regeneration and controls vascular endothelial growth factor release is crucial to improve the treatment of ischemic stroke.Matrix metalloproteinase-2(MMP-2)is gradually upregulated after cerebral ischemia.Herein,vascular endothelial growth factor mimic peptide QK was self-assembled with MMP-2-cleaved peptide PLGLAG(TIMP)and customizable peptide amphiphilic(PA)molecules to construct nanofiber hydrogel PA-TIMP-QK.PA-TIMP-QK was found to control the delivery of QK by MMP-2 upregulation after cerebral ischemia/reperfusion and had a similar biological activity with vascular endothelial growth factor in vitro.The results indicated that PA-TIMP-QK promoted neuronal survival,restored local blood circulation,reduced blood-brain barrier permeability,and restored motor function.These findings suggest that the self-assembling nanofiber hydrogel PA-TIMP-QK may provide an intelligent drug delivery system that responds to the microenvironment and promotes regeneration and repair after cerebral ischemia/reperfusion injury.

Experimental Study on Self-assembly of KLD-12 Peptide Hydrogel and 3-D Culture of MSC Encapsulated within Hydrogel In Vitro

To synthesize KLD-12 peptide with sequence of AcN-KLDLKLDLKLDL-CNH2 and trigger its self-assembly in vitro, to encapsulate rabbit MSCs within peptide hydrogel for 3-D culture and to evaluate the feasibility of using it as injectable scaffold for tissue engineering of IVD. KLD-12 peptide was purified and tested with high performance liquid chromatography （HPLC） and mass spectroscopy （MS）. KLD-12 peptide solutions with concentrations of 5 g/L, 2.5 g/L and 1 g/L were triggered to self-assembly with 1 xPBS in vitro, and the self-assembled peptide hydrogel was morphologically observed. Atomic force microscope （AFM） was employed to examine the inner structure of self-assembled peptide hydrogel. Mesenchymal stem cells （MSCs） were encapsulated within peptide hydrogel for 3-D culture for 2 weeks. Calcein-AM/PI fluorescence staining was used to detect living and dead cells. Cell viability was observed to evaluate the bioactivity of MSCs in KLD-12 peptide hydrogel. The results of HPLC and MS showed that the relative molecular mass of KLD-12 peptide was 1467.83, with a purity quotient of 95.36%. KLD-12 peptide at 5 g/L could self-assemble to produce a hydrogel, which was structurally integral and homogeneous and was able to provide sufficient cohesion to retain the shape of hydrogel. AFM demonstrated that the self-assembly of KLD-12 peptide hydrogel was successful and the assembled material was composed of a kind of nano-fiber with a diameter of 3040 nm and a length of hundreds of nm. Calcein-AM/PI fluorescence staining revealed that MSCs in KLD-12 peptide hydrogel grew well. Cell activity detection exhibited that the A value increased over the culture time. It is concluded that KLD-12 peptide was synthesized successfully and was able to self-assemble to produce nano-fiber hydrogel in vitro. MSCs in KLD-12 peptide hydrogel grew well and proliferated with the culture time. KLD-12 peptide hydrogel can serve as an excellent injectable material of biological scaffolds in tissue engineering of IVD.

Aligned fibrin/functionalized self-assembling peptide interpenetrating nanofiber hydrogel presenting multi-cues promotes peripheral nerve functional recovery

Nerve guidance conduits with hollow lumen fail to regenerate critical-sized peripheral nerve defects(15 mm in rats and 25 mm in humans),which can be improved by a beneficial intraluminal microenvironment.However,individual cues provided by intraluminal filling materials are inadequate to eliminate the functional gap between regenerated nerves and normal nerves.Herein,an aligned fibrin/functionalized self-assembling peptide(AFG/fSAP)interpenetrating nanofiber hydrogel that exerting synergistic topographical and biochemical cues for peripheral nerve regeneration is constructed via electrospinning and molecular self-assembly.The hydrogel possesses an aligned structure,high water content,appropriate mechanical properties and suitable biodegradation capabilities for nerve repair,which enhances the alignment and neurotrophin secretion of primary Schwann cells(SCs)in vitro,and successfully bridges a 15-mm sciatic nerve gap in rats in vivo.The rats transplanted with the AFG/fSAP hydrogel exhibit satisfactory morphological and functional recovery in myelinated nerve fibers and innervated muscles.The motor function recovery facilitated by the AFG/fSAP hydrogel is comparable with that of autografts.Moreover,the AFG/fSAP hydrogel upregulates the regeneration-associated gene expression and activates the PI3K/Akt and MAPK signaling pathways in the regenerated nerve.Altogether,the AFG/fSAP hydrogel represents a promising approach for peripheral nerve repair through an integration of structural guidance and biochemical stimulation.

Kinetically Controlled Self-Assembly of Phthalocyanine–Peptide Conjugate Nanofibrils Enabling Superlarge Redshifted Absorption

Supramolecular assembly could in principle lead to redshifted absorption through J-aggregation of chromophores,which would be a highly promising method for achieving near-infrared materials with improved functionality and flexibility.To effectively enhance the material functionalities,one of the great challenges remaining is to achieve an aggregation statewith a redshift larger than 100 nm.

Crosstalk between PC 12 cells and endothelial cells in an artificial neurovascular niche constructed by a dual-functionalized selfassembling peptide nanofiber hydrogel

The coordination between neurogenesis and angiogenesis plays an important role in nerve tissue development and regeneration.Recently,using bioactive materials to drive neurogenic and angiogenic responses has gained increasing attention.Understanding the neurovascular link between regulatory cues offers valuable insight into the mechanisms underlying nerve regeneration and the design of new bioactive materials.In this study,we utilized a dual-functionalized peptide nanofiber hydrogel presenting the brain-derived neurotrophic factor and vascular endothelial growth factor mimetic peptides RGIDKRHWNSQ(RGI)and KLTWQELYQLKYKGI(KLT)to construct an artificial neurovascular microenvironment.The dual-functionalized peptide nanofiber hydrogel enhanced the neurite outgrowth of pheochromocytoma(PC12)cells and tube-like structures formation of human umbilical vein endothelial cells(HUVECs)in vitro,and promoted rapid lesion infiltration of neural and vascular cells in a rat brain injury model.Using indirect co-culture models,we found that the dual-functionalized peptide hydrogel effectively mediated neurovascular crosstalk by regulating secretion of paracrine factors from PC12 cells and HUVECs.When the two cells types were directly co-cultured on the dua卜functionalized peptide hydrogel,the efficiency of cell-cell communication was enhanced,which further accelerated the differentiation and maturation of PC12 cells with an increased number of pseudopodia and spread morphology,and HUVECs tube-like structure formation.In summary,the dual-functionalized peptide nanofiber hydrogel successfully formed an artificial neurovascular niche to directly regulate the behaviors of neural and vascular cells and promote their neurovascular crosstalk through paracrine signaling and direct cell-cell contact.

A parathyroid hormone related supramolecular peptide for multi-functionalized osteoregeneration

Supramolecular peptide nanofiber hydrogels are emerging biomaterials for tissue engineering,but it is difficult to fabricate multi-functional systems by simply mixing several short-motif-modified supramolecular peptides because relatively abundant motifs generally hinder nanofiber cross-linking or the formation of long nanofiber.Coupling bioactive factors to the assembling backbone is an ideal strategy to design multi-functional supramolecular peptides in spite of challenging synthesis and purification.Herein,a multi-functional supramolecular peptide,P1R16,is developed by coupling a bioactive factor,parathyroid hormone related peptide 1(PTHrP-1),to the basic supramolecular peptide RADA16-I via solid-phase synthesis.It is found that P1R16 self-assembles into long nanofibers and co-assembles with RADA16-I to form nanofiber hydrogels,thus coupling PTHrP-1 to hydrogel matrix.P1R16 nanofiber retains osteoinductive activity in a dose-dependent manner,and P1R16/RADA16-I nanofiber hydrogels promote osteogenesis,angiogenesis and osteoclastogenesis in vitro and induce multi-functionalized osteoregeneration by intramembranous ossification and bone remodeling in vivo when loaded to collagen(Col)scaffolds.Abundant red blood marrow formation,ideal osteointegration and adapted degradation are observed in the 50%P1R16/Col scaffold group.Therefore,this study provides a promising strategy to develop multi-functional supramolecular peptides and a new method to topically administrate parathyroid hormone or parathyroid hormone related peptides for non-healing bone defects.

Selectively enhancing radiosensitivity of cancer cells via in situ enzyme-instructed peptide self-assembly

The radiotherapy modulators used in clinic have disadvantages of high toxicity and low selectivity.For the first time,we used the in situ enzyme-instructed self-assembly(EISA)of a peptide derivative(Nap-GDFDFpYSV)to selectively enhance the sensitivity of cancer cells with high alkaline phosphatase(ALP)expression to ionizing radiation(IR).Compared with the in vitro pre-assembled control formed by the same molecule,assemblies formed by in situ EISA in cells greatly sensitized the ALPhigh-expressing cancer cells to y-rays,with a remarkable sensitizer enhancement ratio.Our results indicated that the enhancement was a result of fixing DNA damage,arresting cell cycles and inducing cell apoptosis.Interestingly,in vitro pre-formed assemblies mainly localized in the lysosomes after incubating with cells,while the assemblies formed via in situ EISA scattered in the cell cytosol.The accumulation of these molecules in cells could not be inhibited by endocytosis inhibitors.We believed that this molecule entered cancer cells by diffusion and then in situ self-assembled to form nanofibers under the catalysis of endogenous ALP.This study provides a successful example to utilize intracellular in situ EISA of small molecules to develop selective tumor radiosensitizers.

自组装多肽支架材料的研究进展

多肽分子可以利用氢键、疏水性作用、π-π堆积作用等非共价键力自组装形成形态与结构特异的多肽分子聚集体,如分子积木、分子开关、分子涂料及纳米纤维等,由于多肽分子良好的生物相容性和可调控的降解性,自组装多肽在组织工程、药物缓释等方面表现出巨大的应用潜力。本文总体介绍了多肽自组装的几种结构模型及自组装的设计原则,重点叙述了自组装多肽作为组织工程支架材料的研究进展。

自组装短肽系统及其材料学应用

模仿天然蛋白质的氨基酸序列设计自组装短肽作为纳米材料近年来引起了人们极大的兴趣。本文介绍了几个发展较为成熟的自组装短肽材料以及它们在纳米医学和纳米生物技术等领域的应用,阐述了自组装短肽作为新型纳米材料的巨大潜力。

由D型氨基酸设计自组装短肽D-EAK16构建新型三维纳米纤维支架材料

采用D型氨基酸设计自组装短肽D-EAK16,运用圆二色仪及原子力显微镜等仪器和细胞三维培养,发现短肽D-EAK16在30℃时具有稳定的二级结构β-sheet,在一定浓度下D-EAK16可形成由纳米纤维构成的透明水凝胶,含水量高达99%,可在细胞培养基(如PBS,DMEM)中形成支架.细胞三维培养显示,该水凝胶对细胞HO-8910和SPC-A-1的生长未见毒性.比较D型氨基酸纳米支架和L型氨基酸纳米支架,细胞的毒性未发现显著性差异.采用D型氨基酸构建的自组装短肽,可提供一个三维基质培养系统,期望能广泛应用于生物医学工程等领域.

功能化自组装多肽水凝胶支架促进小鼠骨髓间充质干细胞的粘附、增殖及成骨

目的探讨功能化自组装纳米多肽DGEAmx水凝胶对小鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)的粘附、增殖和成骨分化的影响。方法原子力显微镜观察功能化自组装多肽形成的纳米级结构特征;用倒置显微镜对贴壁细胞拍照计数,对比细胞粘附情况;用CCK-8法检测细胞增殖;激光共聚焦显微镜观察细胞在材料表面形貌;检测碱性磷酸酶(alkaline phosphatase,ALP)活性、成骨分化特异性基因Ⅰ型胶原(ICAM-1)和骨钙蛋白(osteocalcin,OCN)的表达来评价成骨效果。结果原子力显微镜观察功能化自组装多肽DGEAmx可以形成纳米纤维网状结构;倒置显微镜对贴壁细胞拍照计数结果显示在30、60、90 min时DGEAmx组对比RADA16-Ⅰ组粘附细胞数差异有统计学意义(P<0.05);CCK-8法检测结果显示在培养第3、5、7天,DGEAmx组MSCs增殖较RADA16-Ⅰ组明显增高(P<0.05);DGEAmx组成骨诱导3、7、14 d后,ALP活性高于RADA16-Ⅰ组(P<0.05);Real-time PCR结果显示成骨诱导3、7、14 d后DGEAmx组较RADA16-Ⅰ组有更高的ICAM-1和OCN的表达(P<0.05)。结论功能化自组装多肽DGEAmx纳米纤维水凝胶支架能促进MSCs粘附、增殖和成骨分化,作为骨组织工程支架材料有良好的应用潜力。

自组装多肽纳米纤维支架的结构特点及应用优势

背景:3D自组装肽纳米纤维凝胶支架能很好模拟体内的微环境,提供一种能促进细胞生长、改善细胞功能、具有合理构成细胞外基质的结构模式。目的:综述自组装多肽纳米纤维支架的基础研究及其在神经组织工程中的研究现状。方法:检索2000至2013年PubMed数据库、维普数据库有关自组装多肽纳米纤维支架研究进展的文献,关键词为"自组装多肽,纳米纤维支架,神经组织工程,神经干细胞;self-assembling peptide,nanofiber scaffold,RADA16,Nerve tissue engineering,Neural stem cell"。结果与结论:自组装多肽纳米纤维支架作为新型组织工程支架材料不仅解决了材料与细胞相容性差的问题,而且在维持材料的三维特性、促进细胞活性、模仿细胞外基质等方面均优于其他支架材料,是一种理想的组织工程材料,为神经损伤修复研究提供了新的方法。但自组装多肽领域内仍面临一些挑战,短期的问题包括自组装多肽与新型生物高分子的整合,以及与相对成熟传统移植物的整合;长期问题涉及如何更好地应对体内免疫系统,如何对细胞内的靶点进行治疗,以及如何预测未来高整合支架的发展方向等。

自组装肽纳米纤维支架及其在组织工程的应用

自组装肽纳米纤维支架(SAPNS)是近年来被广泛研究的一种生物支架材料,其在三维细胞培养、组织修复与再生等领域得到了越来越多的应用。本综述在介绍SAPNS研究现状的基础上,按外、中、内3个胚层来源组织的顺序阐述了SAPNS在组织工程中的应用进展、存在问题及展望。

Carboxylated-xyloglucan and peptide amphiphile co-assembly in wound healing

Hydrogel wound dressings can play critical roles in wound healing protecting the wound from trauma or contamination and providing an ideal environment to support the growth of endogenous cells and promote wound closure.This work presents a self-assembling hydrogel dressing that can assist the wound repair process mimicking the hierarchical structure of skin extracellular matrix.To this aim,the co-assembly behaviour of a carboxylated variant of xyloglucan(CXG)with a peptide amphiphile(PA-H3)has been investigated to generate hierarchical constructs with tuneable molecular composition,structure,and properties.Transmission electron microscopy and circular dichroism at a low concentration shows that CXG and PA-H3 co-assemble into nanofibres by hydrophobic and electrostatic interactions and further aggregate into nanofibre bundles and networks.At a higher concentration,CXG and PA-H3 yield hydrogels that have been characterized for their morphology by scanning electron microscopy and for the mechanical properties by smallamplitude oscillatory shear rheological measurements and compression tests at different CXG/PAH3 ratios.A preliminary biological evaluation has been carried out both in vitro with HaCat cells and in vivo in a mouse model.

Imaging cellular distribution of fluorescent supramolecular nanofibers

In this study, we used the 4-nitro-2,1,3-benzoxadiazole （NBD） as an aromatic capping group for a peptide to construct the su- pramolecular nanofibers. Taking the advantage of the fluorescence property of NBD, we could directly observe the cellular distribution of the self-assembled nanofibers. We found that the distributions of the nanofibers of NBD-FFETIGGY are dif- ferent in four mammalian cells and two plant cells. The nanofibers are mainly located at the surface of two mammalian cells and one plant cell, while in the intracellular space of other cells. Different distributions of nanofibers lead to different protein binding patterns o1! the nanofibers in two different cell lines. We believe that a useful and versatile platform has been offered to the image cellular distribution of nanofibers, which can provide useful information to the biological functions of the self-assembled nanostructures.

Antibiofilm and immunomodulatory resorbable nanofibrous filing for dental pulp regenerative procedures

Multifunctional scaffolds with host defense peptides designed for regenerative endodontics are desirable nanobiotechnological tools for dentistry.Here,different scaffolds were tested for use during the pulp revascularization process,including poly(vinyl alcohol)-PVA hydrogels or resins,collagen hydrogels and poly(vinyl alcohol)PVA/Chitosan(PVA/CS)nanofibers.Based on time to degradation(21 days),nanofibers were chosen to be incorporated with ciprofloxacin and IDR-1002(each at 50 mg/g).Nanofibers containing ciprofloxacin and IDR-1002 had anti-biofilm activity against Enterococcus faecalis,Staphylococcus aureus and a multispecies oral biofilm,besides anti-inflammatory activities.The in vivo subcutaneous tissue response to tooth fragments filled with nanofibers demonstrated a pulp-like tissue formation,when compared to empty teeth fragments.Thus,we designed a strong antimicrobial,immunomodulatory and regenerative candidate for pulp revascularization and regeneration procedures.

自组装多肽纳米纤维材料在神经组织工程中的应用

目的综述自组装多肽纳米纤维材料(self-assembling peptide nanofiber scaffold,SAPNS)的基础研究及其在神经组织工程中的研究现状。方法广泛查阅近期国内外SAPNS研究的相关文献,并进行回顾和综合分析。结果SAPNS可促进神经干细胞的黏附、增殖和分化以及神经元轴突向外生长和延伸,促进ECM的合成并能抑制神经胶质细胞的黏附和分化,很好地模拟了细胞在体内的生长环境。结论SAPNS是一种理想的基质材料,为损伤神经组织的修复提供了新的方法。