Neuroactive Peptide Nanofibers for Regeneration of Spinal Cord after Injury

The highly complex nature of spinal cord injuries (SCIs) requires design of novel biomaterials that can stimulate cellular regeneration and functional recovery. Promising SCI treatments use biomaterial scaffolds, which provide bioactive cues to the cells in order to trigger neural regeneration in the spinal cord. In this work, the use of peptide nanofibers is demonstrated, presenting protein binding and cellular adhesion epitopes in a rat model of SCI. The self‐assembling peptide molecules are designed to form nanofibers, which display heparan sulfate mimetic and laminin mimetic epitopes to the cells in the spinal cord. These neuroactive nanofibers are found to support adhesion and viability of dorsal root ganglion neurons as well as neurite outgrowth in vitro and enhance tissue integrity after 6 weeks of injury in vivo. Treatment with the peptide nanofiber scaffolds also show significant behavioral improvement. These results demonstrate that it is possible to facilitate regeneration especially in the white matter of the spinal cord, which is usually damaged during the accidents using bioactive 3D nanostructures displaying high densities of laminin and heparan sulfate‐mimetic epitopes on their surfaces.     

Phosphorus-containing compounds regulate mineralization

Phosphorus is the main constitutive element of minerals and fat in the body. The process of mineral formation is defined as mineralization. The minerals in the body are mainly apatite, which is the inorganic phase that composes bones and teeth. It is worth noting that people with high fat content tend to cause excessive bone mineralization, which leads us to believe that different phosphorus-containing compounds in the body are mutually transformed and can regulate mineralization in different ways. The conversion and regulation of different phosphorus-containing compounds on the mineralization are essential for formation of a complex hierarchical structure and adaptation of the bone to various mechanical environments. Therefore, this review introduces the natural phosphorus-containing compounds in the body, introduces the hierarchical structure of the bone, and summarizes recent studies on different phosphorus-containing compounds (inorganic, organic, and phosphorus-containing proteases) involved in the biomineralization. We also discuss potential research directions of the biomineralization, offering the basis for future investigation of advanced bone substitute materials.     

含骨脱细胞基质电纺纤维的成骨性能

通过脱细胞技术制备了猪骨脱细胞基质(DBM), 用胃蛋白酶消化DBM使其变为可溶形式, 采用静电纺丝技术制备了含有DBM的左旋聚乳酸(PLLA)电纺纤维(PLLA/DBM), 并对PLLA/DBM的形貌、 亲水性、 细胞相容性、 成骨性能和体外矿化能力进行评价. 研究结果表明, 脱细胞处理能够有效去除骨组织中的细胞成分, 使DNA含量显著下降. DBM经胃蛋白酶处理后溶于六氟异丙醇(HFIP), 可进行静电纺丝, 制备的PLLA/DBM[m(PLLA)∶m(DBM)=10∶0, 9∶1, 7∶3, 5∶5]电纺纤维具有良好的亲水性, 且无细胞毒性, 对骨髓间充质干细胞的黏附及成骨分化有明显的诱导促进作用, 体外生物矿化效果优良.     

A Multi‐Functional Silicon Nanoparticle Designed for Enhanced Osteoblast Calcification and Related Combination Therapy

Implant materials applied in bone defect commonly focus on the inducement of bone regeneration and neglect to cure complications including bacterial infection and inflammation, which may result in delayed unions or even amputation. In this study, a microporous silica nanoparticle‐poly(N‐isopropylacrylamide‐b‐(2‐(dimethylamino)ethyl methacrylate) is synthesized for loading DXMS and the ECM‐derived peptide (Sequence: Succinic acid‐GTPGPQGIAGQRGVV) in order to enhance the osteoblast calcification and relieve related symptoms. Positively charged PDMA blocks endow the nanoparticle with the antimicrobial property. Moreover, the combination of DXMS makes it have the ability of anti‐inflammation and promoting calcification formation. Furthermore, incorporation of the peptide leads to a significant improvement of mineralization and alkaline phosphatase expression in the preosteoblast. After intramuscular implantation in mice for four weeks, the results indicate the composite nanoparticle can promote ectopic bone formation. These combined properties make the composite silicon nanoparticle a promising osteogenic drug appropriate for further study in bone repair and related combination therapy.     

Fabrication of Bioactive Polyoxymethylene Nanocomposites for Bone Tissue Replacement

Summary: Stearic acid modified nano hydroxyapatite (n-SHA) filled polyoxymethylene (POM) nanocomposites were prepared by melt mixing method for bone tissue replacement and regeneration applications. Contact angle measurements of POM nanocomposites were carried out to understand the effect of n-SHA addition on the hydrophobicity of nanocomposites. The mechanical properties like tensile strength, Young's modulus and elongation at break were found to be increased significantly by the incorporation of n-SHA into the POM matrix. The bone-bonding ability of the nanocomposites was evaluated by examining the apatite formation on their surface after soaking in simulated body fluid (SBF) and apatite formation was studied by atomic force microscopy (AFM). The protein adhesion studies revealed the enhanced biocompatibility of the nanocomposites due to the presence of n-SHA nanofillers on the surface and it provides favorable binding sites for protein adsorption. The significant improvement in the biocompatibility as well as mechanical, thermal and hydrophobic properties of the POM nanocomposites makes it a potential future material for bone implantation.     

Bioactive Glasses—Structure and Properties

Bioactive glasses were the first synthetic materials to show bonding to bone, and they are successfully used for bone regeneration. They can degrade in the body at a rate matching that of bone formation, and through a combination of apatite crystallization on their surface and ion release they stimulate bone cell proliferation, which results in the formation of new bone. Despite their excellent properties and although they have been in clinical use for nearly thirty years, their current range of clinical applications is still small. Latest research focuses on developing new compositions to address clinical needs, including glasses for treating osteoporosis, with antibacterial properties, or for the sintering of scaffolds with improved mechanical stability. This Review discusses how the glass structure controls the properties, and shows how a structure‐based design may pave the way towards new bioactive glass implants for bone regeneration.     

Polylysine coated bacterial cellulose nanofibers as novel templates for bone-like apatite deposition

The study on bacterial cellulose (BC) nanofibers used as templates for hydroxylapatite (HAp) deposition has been investigated by our group and many other researchers. However, BC is only microscopically similar to natural collagen but not molecular structure. If protein could be introduced to the surfaces of BC nanofibers, the BC nanofibers could mimic the natural collagen fibers in terms of both shape and molecular structure. In this work, our latest results concerning the preparation of polylysine (PLL) coated BC nanofibers are reported. It is found that the ε-polylysine (PLL), a natural coming peptide, was introduced to the surfaces of BC nanofibers via crosslinking method by using procyanidins as crosslinker. The bioactivity of PLL coated BC nanofibers was demonstrated by the bone-like HAp deposition throughout the scaffold in a simulated body fluid (SBF). To initiate mineralization the PLL coated BC nanofibers were immersed in 1.5 times simulated body fluids (1.5 SBF) at 37 °C for 7 days. The deposited minerals on the nanofiber surfaces were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), and fourier transformed infrared spectroscopy (FTIR). These PLL coated BC nanofibers were proved to act as nano templates to induce the formation of nano-sized platelet-like, calcium-deficient, B-type carbonated HAp of which the features was closed to those of biological apatite.     

Bioactive Seed Layer for Surface‐Confined Self‐Assembly of Peptides

The design and control of molecular systems that self‐assemble spontaneously and exclusively at or near an interface represents a real scientific challenge. We present here a new concept, an active seed layer that allows to overcome this challenge. It is based on enzyme‐assisted self‐assembly. An enzyme, alkaline phosphatase, which transforms an original peptide, Fmoc‐FFY(PO₄^2?), into an efficient gelation agent by dephosphorylation, is embedded in a polyelectrolyte multilayer and constitutes the “reaction motor”. A seed layer composed of a polyelectrolyte covalently modified by anchoring hydrogelator peptides constitutes the top of the multilayer. This layer is the nucleation site for the Fmoc‐FFY peptide self‐assembly. When such a film is brought in contact with a Fmoc‐FFY(PO₄^2?) solution, a nanofiber network starts to form almost instantaneously which extents up to several micrometers into the solution after several hours. We demonstrate that the active seed layer allows convenient control over the self‐assembly kinetics and the geometric features of the fiber network simply by changing its peptide density.     

Bioinspired growth of crystalline carbonate apatite on biodegradable polymer substrata

Mineralization in biological systems is a widespread, yet incompletely understood phenomenon involving complex interactions at the biomacromolecule-mineral nucleus interface. This study was aimed at understanding and controlling mineral formation in a poly(alpha-hydroxy ester) model system, to gain insight into biological mineralization processes and to develop biomaterials for orthopaedic tissue regeneration. We specifically hypothesized that providing a high surface density of anionic functional groups would enhance nucleation and growth of bonelike mineral following exposure to simulated body fluids (SBF). Polymer surface functionalization was achieved via hydrolysis of 85:15 poly(lactide-co-glycolide) (PLG) films. This treatment led to an increase in surface carboxylic acid and hydroxyl groups, resulting in a substantial increase in polymer surface energy from 42 to 49 dynes/cm2. Treated polymers exhibited a 3-fold increase in heterogeneous mineral grown and growth of a continuous mineral film on the polymer surface. The mineral grown on PLG surfaces is a carbonate apatite, the major mineral component of vertebrate bone tissue. Mineral crystal size and morphology were dependent on the solution characteristics but unaffected by the degree of surface prehydrolysis. The mechanism of heterogeneous carbonate apatite growth was examined via ion binding assays, which indicated that calcium binding is mediated independently by the presence of soluble phosphate counterions and surface functional groups. These findings indicate that poly(alpha-hydroxy ester) materials can be readily mineralized using a biomimetic process, and that the impetus for mineral nucleation in this system appears more complicated than the simple electrostatic interactions proposed in previous biomineralization theory.     

磷灰石晶体构型及其与生物分子相互作用的计算模拟研究

生物磷灰石是动物和人体骨骼及牙釉质的主要无机矿物成分,磷灰石矿物晶体的组成和结构影响了骨及牙釉质的机械强度和生理功能。羟基磷灰石空间群的确定一直存在争议,其中羟基存在两种不同排列方式,使得其具有六方和单斜两种晶相。另外,磷灰石晶体结构中的类质同象替换,影响了其结构、物理和化学特性。本文综述了计算机模拟方法在原子及分子水平上对磷灰石晶体的空间群确定、磷灰石替代机制、小分子及生物大分子相互作用的研究,对磷灰石晶体化学、界面化学及开发生物材料的深入研究具有一定的科学意义和较强的应用价值。     

Electrostatic effects on nanofiber formation of self-assembling peptide amphiphiles

Self-assembling peptide amphiphile molecules have been of interest to various tissue engineering studies. These molecules self-assemble into nanofibers which organize into three-dimensional networks to form hydrocolloid systems mimicking the extracellular matrix. The formation of nanofibers is affected by the electrostatic interactions among the peptides. In this work, we studied the effect of charged groups on the peptides on nanofiber formation. The self-assembly process was studied by pH and zeta potential measurements, FT-IR, circular dichroism, rheology, atomic force microscopy, scanning electron microscopy and transmission electron microscopy. The aggregation of the peptides was triggered upon neutralization of the charged residues by pH change or addition of electrolyte or biomacromolecules. Understanding the controlled formation of the hydrocolloid gels composed of peptide amphiphile nanofibers can lead us to develop in situ gel forming bioactive collagen mimetic nanofibers for various tissue engineering studies including bioactive surface coatings.     

Bone‐guided regeneration: from inert biomaterials to bioactive polymer (nano)composites

Natural bone is a unique nanostructured material made of collagen fibre matrix and hydroxyapatite (HA) nanocrystals, providing mechanical support and protection from the vertebrate skeleton. However, in severe cases like bone‐deficiencies, bone needs to be “externally” repaired. Initially, different biological solutions were developed in bone‐guided regeneration. However, due to the limitations with the existing biological grafts, a lot of researches have been devoted toward biomaterials including metals, ceramics, and polymers. On the basis of the interface reactions between the implant and the surrounding tissues, these biomaterials may be classified as “nearly inert” or bioactive. Interestingly, the bioactive materials exhibit a specific biological response, leading to the formation of a natural bonding junction between the bone and the implant during bone regeneration. Recently, a special attention has been paid to a new generation of bioactive materials, i.e. (nano)structured biomaterials composed of a bioresorbable polymer matrix reinforced with bioactive inorganic compounds. While (bio)ceramic component provides the bioactivity, these materials can be readily engineered in such a way that their resorption rate in the body match the formation rate of the new tissue. This review hence reports the different biological and non‐biological solutions developed in bone‐guided regeneration, with a special emphasis on polymer‐based materials, and our recent results obtained in osseointegration The bone physiology, and its natural regeneration are also described. Copyright © 2011 John Wiley & Sons, Ltd.     

Orientation of mineral crystals by collagen fibers during in vivo bone engineering: An X-ray diffraction imaging study

The mechanism of mineralized bone matrix deposition was investigated taking advantage of a tissue engineering approach in which bone tissue is formed when porous ceramic scaffold is loaded with bone marrow stromal cells and implanted in vivo. The aim of our study is to point out the interaction between the newly formed mineral crystals and the scaffold imposing the three-dimensional desired architecture to the growing bone. High spatial resolution Small Angle X-ray Scattering measurements obtained using synchrotron radiation and X-ray waveguide as optical element allowed a local structural study at the bone–scaffold interface. Using an original methodology for data analysis, we obtained a two-dimensional microscopic map of the mineralization degree, the collagen presence and the mineral orientation degree around the scaffold pore.     

Evolutionary screening of collagen-like peptides that nucleate hydroxyapatite crystals

The biogenesis of inorganic/organic composite materials such as bone typically involves the process of templated mineralization. Biomimetic synthesis of bone-like materials therefore requires the development of organic scaffolds that mediate mineralization of hydroxyapatite (HAP), the major inorganic component of bone. Using phage display, we identified a 12-residue peptide that bound to single-crystal HAP and templated the nucleation and growth of crystalline HAP mineral in a sequence- and composition-dependent manner. The sequence responsible for the mineralizing activity resembled the tripeptide repeat (Gly-Pro-Hyp) of type I collagen, a major component of bone extracellular matrix. Using a panel of synthetic peptides, we defined the structural features required for mineralizing activity. The results support a model for the cooperative noncovalent interaction of the peptide with HAP and suggest that native collagen may have a mineral-templating function in vivo. We expect this short HAP-binding peptide to be useful in the synthesis of three-dimensional bone-like materials.     

Hydrolysis and biomineralization of porous PLA microspheres and their influence on cell growth

Poly(lactic acid) (PLA) microspheres have great potential in bone tissue engineering. However, their applications have been limited by surface and bulk properties such as hydrophobicity, lack of cell recognition sites and acidic degradation products. Apatite is a mineral which can effectively promote the adhesion and growth of bone cells. In this study, the bonelike mineral, carbonate apatite, was successfully used to functionalize porous PLA microspheres by a biomimetic mineralization method. To improve apatite formation, porous PLA microspheres were first selectively hydrolyzed in NaOH solution to increase the density of polar anionic groups on the surface, and then immersed in simulated body fluid for biomineralization. The morphology, composition, and phase structure of bioactive mineral grown on the original and hydrolyzed PLA microspheres were analyzed and compared quantitatively. The results showed that the hydrolysis which took place on the PLA microspheres enhanced the nucleation and growth of apatite. MG-63 cells attached well and spread actively on the mineralized PLA microspheres, indicating their strong potential in bone tissue engineering.     

Enzymatic Mineralization of Hydrogels for Bone Tissue Engineering by Incorporation of Alkaline Phosphatase

Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These are collagen type I, a mussel‐protein‐inspired adhesive consisting of PEG substituted with catechol groups, cPEG, and the PEG/fumaric acid copolymer OPF. After incubation in Ca‐GP solution, FTIR, EDS, SEM, XRD, SAED, ICP‐OES, and von Kossa staining confirm CaP formation. The amount of mineral formed decreases in the order cPEG > collagen > OPF. The mineral:polymer ratio decreases in the order collagen > cPEG > OPF. Mineralization increases Young's modulus, most profoundly for cPEG. Such enzymatically mineralized hydrogel/CaP composites may find application as bone regeneration materials.

     

Effect of grafting RGD and BMP-2 protein-derived peptides to a hydrogel substrate on osteogenic differentiation of marrow stromal cells

Osteogenic differentiation and mineralization of bone marrow stromal (BMS) cells depends on the cells' interactions with bioactive peptides associated with the matrix proteins. The RGD peptides of ECM proteins interact with BMS cells through integrin surface receptors to facilitate cell spreading and adhesion. The BMP peptide corresponding to residues 73-92 of bone morphogenetic protein-2 promotes differentiation and mineralization of BMS cells. The objective of this work was to investigate the effects of RGD and BMP peptides, grafted to a hydrogel substrate, on osteogenic differentiation and mineralization of BMS cells. RGD peptide was acrylamide-terminated by reacting acrylic acid with the N-terminal amine group of the peptide to produce the functionalized Ac-GRGD peptide. The PEGylated BMP peptide was reacted with 4-carboxybenzenesulfonazide to produce an azide functionalized Az-mPEG-BMP peptide. Poly (lactide-co-ethylene oxide- co-fumarate) (PLEOF) macromer was cross-linked with Ac-GRGD peptide and propargyl acrylate to produce an RGD conjugated hydrogel. Az-mPEG-BMP peptide was grafted to the hydrogel by "click chemistry". The RGD and BMP peptide density on the hydrogel surface was 1.62+/-0.37 and 5.2+/-0.6 pmol/cm2, respectively. BMS cells were seeded on the hydrogels and the effect of RGD and BMP peptides on osteogenesis was evaluated by measuring ALPase activity and calcium content with incubation time. BMS cells cultured on RGD conjugated, BMP peptide grafted, and RGD+BMP peptide modified hydrogels showed 3, 2.5, and 5-fold increase in ALPase activity after 14 days incubation. BMS cells seeded on RGD+BMP peptides modified hydrogel showed 4.9- and 11.8-fold increase in calcium content after 14 and 21 days, respectively, which was significantly higher than RGD conjugated or BMP grafted hydrogels. These results demonstrate that RGD and BMP peptides, grafted to a hydrogel substrate, act synergistically to enhance osteogenic differentiation and mineralization of BMS cells. These findings are potentially useful in developing engineered scaffolds for bone regeneration.     

Homogeneous organic/inorganic hybrid scaffolds with high osteoinductive activity for bone tissue engineering

Composite scaffolds of polymers/β-tricalcium phosphate (TCP) have been widely used for bone regeneration due to the combination of osteoinductivity of TCP and mechanical properties of the polymers. However, the difference in surface properties of the two material causes composite has poor uniformity and weak two-phase interaction, resulting in poor TCP release and weak new bone-forming ability. In this research, a TCP sol was developed to replace traditional TCP nanoparticles for the preparation of homogeneous polycaprolactone (PCL)/TCP sol nanofibrous scaffolds. It was found that compared with TCP nanoparticles, TCP sol homogeneously distributed in PCL nanofibers, and greatly improved the hydrophilicity, biodegradability, and mechanical properties of the scaffolds. It is also confirmed that loading TCP sol promoted the formation of bone-like apatite on the surface of the scaffolds. Biological experiments showed that all scaffolds supported rat bone marrow mesenchymal stem cells (rBMSCs) proliferation, especially scaffolds loaded with TCP sol. The increase in alkaline phosphatase activity and collagen production, enhanced calcium deposition, and up-regulation of osteocalcin expression demonstrated that the loading TCP sol expanded an advantage of scaffolds in promoting rBMSCs osteogenic differentiation, suggesting it dramatically improved the osteoinductive activity of PCL/TCP hybrid system and had a great potential application in bone regeneration.     

The low level laser therapy effect on the remodeling of bone extracellular matrix

The low level laser therapy (LLLT) has been used as an option to accelerate the regeneration of bone tissue. In this study, both femurs of male Wistar rats (30 animals) were injured with a drill and the effect of LLLT using a laser diode (100 mW at 660 nm) in the bone matrix on the left paw measured. LLLT effect on the healing bone tissue matrix was evaluated by a combination of immunohistochemical histomorphometry, confocal immunofluorescence microscopy and isolation and characterization of glycosaminoglycans. Histomorphometric analysis showed that LLLT increased bone matrix and showing more organized. Alcian Blue and PAS staining seems to suggest differential glycosaminoglycans and glycoproteins. The data showed increased expression of chondroitin sulfate and hyaluronic acid, after reduction as the LLLT and mature bone, resembling the expression of osteonectin and biglycan. The difference in expression of siblings (DMP‐1, OPN and BSP) is in accordance with the repair accelerated bone formation after the application of LLLT as compared with control. The expression of osteonectin and osteocalcin supports their role in bone mineralization protein, indicating that LLLT accelerates this process. The overall data show that LLLT bone changes dynamic array, shortening the time period involved in the bone repair.     

Molecular Mechanisms of Biomineralization in the Formation of Calcified Shells

Biological calcification processes (calcification, biomineralization) occur in microorganisms, in plants, and in the animal kingdom. Under physiological conditions, the results of mineral deposition in biological systems can be seen in the formation of bones, teeth, mollusc shells, egg shells, pearls, and corals. There are, however, also pathological aspects of biomineralization, including the formation of kidney stones (renal calculus), gallstones (biliary calculus), intravasal depositions (atherosclerosis, calcinosis), gallstones (biliary calculus), intravasal depositions (atherosclerosis, calcinosis). Abnormal mineralization processes (demineralization) can be found, e.g., in bone resorption (osteoporosis) and caries. A detailed knowledge of the molecular mechanisms of biomineralization could help considerably toward solving some of the problems encountered in orthopedics, urology, cardiovascular science, dentistry and veterinary medicine. Research in biomineralization is always interdisciplinary. In this article the typical interaction between mineral phase and organic matrix will be demonstrated using two examples—mollusc shells and egg shells.