Scaffolds for Dental Pulp Tissue Engineering

For tissue engineering strategies, the choice of an appropriate scaffold is the first and certainly a crucial step. A vast variety of biomaterials is available: natural or synthetic polymers, extracellular matrix, self-assembling systems, hydrogels, or bioceramics. Each material offers a unique chemistry, composition and structure, degradation profile, and possibility for modification. The role of the scaffold has changed from passive carrier toward a bioactive matrix, which can induce a desired cellular behavior. Tailor-made materials for specific applications can be created. Recent approaches to generate dental pulp rely on established materials, such as collagen, polyester, chitosan, or hydroxyapatite. Results after transplantation show soft connective tissue formation and newly generated dentin. For dentinpulp- complex engineering, aspects including vascularization, cell-matrix interactions, growth-factor incorporation, matrix degradation, mineralization, and contamination control should be considered. Self-assembling peptide hydrogels are an example of a smart material that can be modified to create customized matrices. Rational design of the peptide sequence allows for control of material stiffness, induction of mineral nucleation, or introduction of antibacterial activity. Cellular responses can be evoked by the incorporation of cell adhesion motifs, enzymecleavable sites, and suitable growth factors. The combination of inductive scaffold materials with stem cells might optimize the approaches for dentin-pulp complex regeneration

LINAC based stereotactic radiosurgery for multiple brain metastases: guidance for clinical implementation

Introduction: Stereotactic radiosurgery (SRS) is a promising treatment option for patients with multiple brain metastases (BM). Recent technical advances have made LINAC based SRS a patient friendly technique, allowing for accurate patient positioning and a short treatment time. Since SRS is increasingly being used for patients with multiple BM, it remains essential that SRS be performed with the highest achievable quality in order to prevent unnecessary complications such as radionecrosis. The purpo

Stabilization of Peptide Vesicles by Introducing Inter-Peptide Disulfide Bonds

PURPOSE: Previously, we have shown that the amphiphilic oligopeptide SA2 (Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu-Glu-COOH) spontaneously self-assemble into nano-sized vesicles in aqueous environment. Relative weak individual intermolecular interactions dominate such oligopeptide assemblies. In this study we aimed at improving the stability of such peptide vesicles by covalently crosslinking the oligopeptide vesicles using disulfide bonds. Two and three cysteines were introduced in the SA2 peptide sequence to allow crosslinking (Ac-Ala-Cys-Val-Cys-Leu-(Leu/Cys)-Leu-Trp-Glu-Glu-COOH). RESULTS: Upon disulfide formation the crosslinked vesicles remained stable under conditions that disrupted the non-crosslinked peptide vesicles. The stabilized vesicles were more closely examined in terms of particle size (distribution) using atomic force microscopy, cryogenic electron microscopy, as well as dynamic light scattering analysis, showing an average particle radius in number between 15 and 20 nm. Using entrapment of calcein it was shown that intermolecular crosslinking of peptides within the vesicles did not affect the permeability for calcein. CONCLUSION: Introduction of cysteines into the hydrophobic domain of the SA2 amphiphilic oligopeptides is a feasible strategy for crosslinking the peptide vesicles. Such small crosslinked oligopeptide vesicles may hold promise for drug delivery applications

Interaction between acrylic substrates and RAD16-I peptide in its self-assembling

[EN] Self-assembling peptides (SAP) are widely used as scaffolds themselves, and recently as fillers of microporous scaffolds, where the former provides a cell-friendly nanoenvironment and the latter improves its mechanical properties. The characterization of the interaction between these short peptides and the scaffold material is crucial to assess the potential of such a combined system. In this work, the interaction between poly(ethyl acrylate) (PEA) and 90/10 ethyl acrylate-acrylic acid copolymer P(EAcoAAc) with the SAP RAD16-I has been followed using a bidimensional simplified model. By means of the techniques of choice (congo red staining, atomic force microscopy (AFM), and contact angle measurements) the interaction and self-assembly of the peptide has proven to be very sensitive to the wettability and electro-negativity of the polymeric substrate.The authors acknowledge funding through the European Commission FP7 project RECATABI (NMP3-SL-2009-229239), and from the Spanish Ministerio de Ciencia e Innovacion through projects MAT2011-28791-C03-02 and -03. This work was also supported by the Spanish Ministerio de Educacion through M. Arnal-Pastor FPU 2009-1870 grant. The authors acknowledge the assistance and advice of Electron Microscopy Service of the UPV.Arnal Pastor, MP.; González-Mora, D.; García-Torres, F.; Monleón Pradas, M.; Vallés Lluch, A. (2016). Interaction between acrylic substrates and RAD16-I peptide in its self-assembling. Journal of Polymer Research. 23(9):173-184. https://doi.org/10.1007/s10965-016-1069-3S173184239Davis ME, Motion JP, Narmoneva DA, Takahashi T, Hakuno D, Kamm RD, Zhang S, Lee RT (2005) Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells. Circulation 111(4):442–450Zhang S, Lockshin C, Cook R, Rich A (1994) Unusually stable beta-sheet formation in an ionic self-complementary oligopeptide. Biopolymers 34:663–672Zhang S, Altman M (1999) Peptide self-assembly in functional polymer science and engineering. Reac Func Polym 41:91–102Zhang S, Gelain F, Zhao X (2005) Designer self-assembling peptide nanofiber scaffolds for 3D tissue cell cultures. Semin Cancer Biol 15(5):413–420Zhang S, Zhao X, Spirio L, PuraMatrix (2005) Self-assembling peptide nanofiber scaffolds. In: Ma PX, Elisseeff J (eds) Scaffolding in tissue Engineering. CRC Press, Boca Raton, FL, pp. 217–238Sieminski AL, Semino CE, Gong H, Kamm RD (2008) Primary sequence of ionic self-assembling peptide gels affects endothelial cell adhesion and capillary morphogenesis. J Biomed Mater Res A 87(2):494–504Quintana L, Fernández Muiños T, Genove E, Del Mar Olmos M, Borrós S, Semino CE (2009) Early tissue patterning recreated by mouse embryonic fibroblasts in a three-dimensional environment. Tissue Eng Part A 15(1):45–54Garreta E, Genové E, Borrós S, Semino CE (2006) Osteogenic differentiation of mouse embryonic stem cells and mouse embryonic fibroblasts in a three-dimensional self-assembling peptide scaffold. Tissue Eng 12(8):2215–2227Semino CE, Merok JR, Crane GG, Panagiotakos G, Zhang S (2003) Functional differentiation of hepatocyte-like spheroid structures from putative liver progenitor cells in three-dimensional peptide scaffolds. Differentiation 71:262–270Thonhoff JR, Lou DI, Jordan PM, Zhao X, Compatibility WP (2008) Of human fetal neural stem cells with hydrogel biomaterials in vitro. Brain Res 1187:42–51Tokunaga M, Liu ML, Nagai T, Iwanaga K, Matsuura K, Takahashi T, Kanda M, Kondo N, Wang P, Naito AT, Komuro I (2010) Implantation of cardiac progenitor cells using self-assembling peptide improves cardiac function after myocardial infarction. J Mol Cell Cardiol 49(6):972–983Takei J (2006) 3-Dimensional cell culture scaffold for everyone: drug screening. Tissue engineering and cancer biology. AATEX 11(3):170–176McGrath AM, Novikova LN, Novikov LN, Wiberg MBD (2010) ™ PuraMatrix™ peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration. Brain Res Bull 83(5):207–213Wang W, Itoh S, Matsuda A, Aizawa T, Demura M, Ichinose S, Shinomiya K, Tanaka J (2008) Enhanced nerve regeneration through a bilayered chitosan tube: The effect ofintroduction of glycine spacer into the CYIGSR sequence. J Biomed Mater Res Part A 85:919–928Sargeant TD, Guler MO, Oppenheimer SM, Mata A, Satcher RL, Dunand DC, Stupp SI (2008) Hybrid bone implants: self-assembly of peptide amphiphile nanofibers within porous titanium. Biomaterials 29(2):161–171Vallés-Lluch A, Arnal-Pastor M, Martínez-Ramos C, Vilariño-Feltrer G, Vikingsson L, Castells-Sala C, Semino CE, Monleón Pradas M (2013) Combining self-assembling peptide gels with three-dimensional elastomer scaffolds. Acta Biomater 9(12):9451–9460Valles-Lluch A, Arnal-Pastor M, Martinez-Ramos C, Vilarino-Feltrer G, Vikingsson L, Monleon Pradas M (2013) Grid polymeric scaffolds with polypeptide gel filling as patches for infarcted tissue regeneration. Conf Proc IEEE Eng Med Biol Soc 2013:6961–6964Soler-Botija C, Bagó JR, Llucià-Valldeperas A, Vallés-Lluch A, Castells-Sala C, Martínez-Ramos C, Fernández-Muiños T, Chachques JC, Monleón Pradas M, Semino CE, Bayes-Genis A (2014) Engineered 3D bioimplants using elastomeric scaffold, self-assembling peptide hydrogel, and adipose tissue-derived progenitor cells for cardiac regeneration. Am J Transl Res 6(3):291–301Martínez-Ramos M, Arnal-Pastor M, Vallés-Lluch A, Monleón Pradas M (2015) Peptide gel in a scaffold as a composite matrix for endothelial cells. J Biomed Mater Res Part A 103 A:3293–3302Rico P, Rodríguez Hernández JC, Moratal D, Altankov G, Monleón Pradas M, Salmerón-Sánchez M (2009) Substrate-induced assembly of fibronectin into networks: influence of surface chemistry and effect on osteoblast adhesion. Tissue Eng Part A 15(11):3271–3281Gugutkov D, Altankov G, Rodríguez Hernández JC, Monleón Pradas M, Salmerón Sánchez M (2010) Fibronectin activity on substrates with controlled -OH density. J Biomed Mater Res A 92(1):322–331Rodríguez Hernández JC, Salmerón Sánchez M, Soria JM, Gómez Ribelles JL, Monleón Pradas M (2007) Substrate chemistry-dependent conformations of single laminin molecules on polymer surfaces are revealed by the phase signal of atomic force microscopy. Biophys J 93(1):202–207Cantini M, Rico P, Moratal D, Salmerón-Sánchez M (2012) Controlled wettability, same chemistry: biological activity of plasma-polymerized coatings. Soft Matter 8:5575–5584Anselme K, Ponche A, Bigerelle M (2010) Relative influence of surface topography and surface chemistry on cell response to bone implant materials. Part 2: biological aspects. Proc Inst Mech Eng H J Eng Med 224:1487–1507Hartgerink JD, Beniash E, Stupp SI (2002) Peptide-amphiphile nanofibers: a versatile scaffold for the preparation of self-assembling materials. Proc Natl Acad Sci U S A 99(8):5133–5138Busscher HJ, Vanpelt AWJ, Deboer P, Dejong HP, Arends J (1984) The effect of surface roughening of polymers on measured contact angles of liquids. Colloids Surf 9:319–331Birdi, KS. (1997) Surface tension of polymers. In: Yildrim Erbil H, ed. Handbook of surface and colloid chemistry CRC Press, Boca Raton, p. 292.Collier JH (2003) MessersmithPB.Enzymatic modification of self-assembled peptide structures with tissue transglutaminase. Bioconjug Chem 14(4):748–755Kakiuchi Y, Hirohashi N, Murakami-Murofushi K (2013) The macroscopic structure of RADA16 peptide hydrogel stimulates monocyte/macrophage differentiation in HL60 cells via cholesterol synthesis. BiochemBiophys Res Commun 433(3):298–304Pérez-Garnes M, González-García C, Moratal D, Rico P, Salmerón-Sánchez M (2011) Fibronectin distribution on demixednanoscale topographies. Int J Artif Organs 34(1):54–63Salmerón-Sánchez M, Rico P, Moratal D, Lee TT, Schwarzbauer JE, García AJ (2011) Role of material-driven fibronectin fibrillogenesis in cell differentiation. Biomaterials 32(8):2099–2105Ye Z, Zhang H, Luo H, Wang S, Zhou Q, DU X, et al. (2008) Temperature and pH effects on biophysical and morphological properties of self-assembling peptide RADA16-I. J Pept Sci 14:152–162Keselowsky BG, Collard DM, García AJ (2004) Surface chemistry modulates focal adhesion composition and signaling through changes in integrin binding. Biomaterials 25:5947–5954Scotchford CA, Gilmore CP, Cooper E, Leggett GJ, Downes S (2002) Protein adsorption and human osteoblast-like cell attachment and growth on alkylthiol on gold self-assembled monolayers. J Biomed Mater Res 59:84–99Coelho NM, González-García C, Planell JA, Salmerón-Sánchez M, Altankov G (2010) Different assembly of type IV collagen on hydrophilic and hydrophobic substrata alters endothelial cells interaction. Eur Cell Mater 19:262–272Briz N, Antolinos-Turpin CM, Alió J, Garagorri N, Gómez Ribelles JL, Gómez-Tejedor JA (2013) Fibronectin fixation on poly(ethyl acrylate)-based copolymers. J Biomed Mater Res B Appl Biomater 101(6):991–997Owens DK, Wendt RC (1969) Estimation of the surface free energy of polymers. J Appl Polym Sci 13(8):1741–1747Soria JM, Martínez Ramos C, Bahamonde O, García Cruz DM, Salmerón Sánchez M, García Esparza MA, Casas C, Guzmán M, Navarro X, Gómez Ribelles JL, García Verdugo JM, Monleón Pradas M, Barcia JA (2007) Influence of the substrate's hydrophilicity on the in vitro Schwann cells viability. J Biomed Mater Res A 83(2):463–470Van Krevelen, DW. (1997) Properties of polymers. Chapter 13 mechanical properties of solid polymers. Elsevier, pp. 367–43

Intermolecular channels direct crystal orientation in mineralized collagen

The mineralized collagen fibril is the basic building block of bone, and is commonly pictured as a parallel array of ultrathin carbonated hydroxyapatite (HAp) platelets distributed throughout the collagen. This orientation is often attributed to an epitaxial relationship between the HAp and collagen molecules inside 2D voids within the fibril. Although recent studies have questioned this model, the structural relationship between the collagen matrix and HAp, and the mechanisms by which collagen directs mineralization remain unclear. Here, we use XRD to reveal that the voids in the collagen are in fact cylindrical pores with diameters of ~2 nm, while electron microscopy shows that the HAp crystals in bone are only uniaxially oriented with respect to the collagen. From in vitro mineralization studies with HAp, CaCO3 and γ-FeOOH we conclude that confinement within these pores, together with the anisotropic growth of HAp, dictates the orientation of HAp crystals within the collagen fibril

Identification of Genes Directly Involved in Shell Formation and Their Functions in Pearl Oyster, Pinctada fucata

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the ‘aragonitic line’. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P.fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the ‘aragonitic line’, and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth