Order versus Disorder: in vivo bone formation within osteoconductive scaffolds

In modern biomaterial design the generation of an environment mimicking some of the extracellular matrix features is envisaged to support molecular cross-talk between cells and scaffolds during tissue formation/remodeling. In bone substitutes chemical biomimesis has been particularly exploited; conversely, the relevance of pre-determined scaffold architecture for regenerated bone outputs is still unclear. Thus we aimed to demonstrate that a different organization of collagen fibers within newly formed bone under unloading conditions can be generated by differently architectured scaffolds. An ordered and confined geometry of hydroxyapatite foams concentrated collagen fibers within the pores, and triggered their self-assembly in a cholesteric-banded pattern, resulting in compact lamellar bone. Conversely, when progenitor cells were loaded onto nanofibrous collagen-based sponges, new collagen fibers were distributed in a nematic phase, resulting mostly in woven isotropic bone. Thus specific biomaterial design relevantly contributes to properly drive collagen fibers assembly to target bone regeneration

Water-mediated structuring of bone apatite

International audienceIt is well known that organic molecules from the vertebrate extracellular matrix of calcifying tissues are essential in structuring the apatite mineral. Here, we show that water also plays a structuring role. By using solid-state nuclear magnetic resonance, wide-angle X-ray scattering and cryogenic transmission electron microscopy to characterize the structure and organization of crystalline and biomimetic apatite nanoparticles as well as intact bone samples, we demonstrate that water orients apatite crystals through an amorphous calcium phosphate-like layer that coats the crystalline core of bone apatite. This disordered layer is reminiscent of those found around the crystalline core of calcified biominerals in various natural composite materials in vivo. This work provides an extended local model of bone biomineralization

In vitro studies and preliminary in vivo evaluation of silicified concentrated collagen hydrogels

Hybrid and nanocomposite silicacollagen materials derived from concentrated collagen hydrogels were evaluated in vitro and in vivo to establish their potentialities for biological dressings. Silicification significantly improved the mechanical and thermal stability of the collagen network within the hybrid systems. Nanocomposites were found to favor the metabolic activity of immobilized human dermal fibroblastswhile decreasing the hydrogel contraction. Cell adhesion experiments suggested that in vitro cell behavior was dictated by mechanical properties and surface structure of the scaffold. First-to-date in vivo implantation of bulk hydrogels in subcutaneous sites of rats was performed over the vascular inflammatory period. These materials were colonized and vascularized without inducing strong inflammatory response. These data raise reasonable hope for the future application of silicacollagen biomaterials as biological dressings.Fil: Desimone, Martín Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Hélary, Christophe. Université Pierre et Marie Curie; FranciaFil: Quignard, Sandrine. Université Pierre et Marie Curie; FranciaFil: Rietveld, Ivo B. Universite de Paris; FranciaFil: Bataille, Clement. Université de Versailles Saint-quentin-en-yvelines.; FranciaFil: Copello, Guillermo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Mosser, Gervaise. Université Pierre et Marie Curie; FranciaFil: Giraud Guille, Marie-Madeleine. Université Pierre et Marie Curie; FranciaFil: Livage, Jacques. Université Pierre et Marie Curie; FranciaFil: Meddahi Pellé, Anne. Université de Versailles Saint-quentin-en-yvelines.; FranciaFil: Coradin, Thibaud. Université Pierre et Marie Curie; Franci

Multifunctional biomaterials from the sea: Assessing the effects of chitosan incorporation into collagen scaffolds on mechanical and biological functionality

Natural biomaterials such as collagen show promise in tissue engineering applications due to their inherent bioactivity. The main limitation of collagen is its low mechanical strength and somewhat unpredictable and rapid degradation rate; however, combining collagen with another material, such as chitosan, can reinforce the scaffold mechanically and may improve the rate of degradation. Additionally, the high cost and the risk of prion transmission associated with mammal-derived collagen has prompted research into alternative sources such as marine-origin collagen. In this context, the overall goal of this study was to determine if the incorporation of chitosan into collagen scaffolds could improve the mechanical and biological properties of the scaffold. In addition the study assessed if collagen, derived from salmon skin (marine), can provide an alternative to collagen derived from bovine tendon (mammal) for tissue engineering applications. Scaffold architecture and mechanical properties were assessed as well as their ability to support mesenchymal stem cell growth and differentiation. Overall, the addition of chitosan to bovine and salmon skin-derived collagen scaffolds improved the mechanical properties, increasing the compressive strength, swelling ratio and prolonged the degradation rate. Mesenchymal stem cell (MSC) attachment and proliferation was most improved on the bovine-derived collagen scaffold containing a 75:25 ratio of collagen:chitosan, and when MSC osteogenic and chondrogenic potential on the scaffold was assessed, a significant increase in calcium production (p < 0.001) and sulfated glycosaminoglycan (sGAG) production (p < 0.001) was observed respectively. Regardless of chitosan content, the bovine-derived collagen scaffolds out-performed the salmon skin-derived collagen scaffolds, displaying a larger pore size and higher percentage porosity, more regular architecture, higher compressive modulus, a greater capacity for water uptake and allowed for more MSC proliferation and differentiation. This versatile scaffold incorporating the marine biomaterial chitosan show great potential as appropriate platforms for promoting orthopaedic tissue repair while the use of salmon skin-derived collagen may be more suitable in the repair of soft tissues such as skin.This work was funded by Science Foundation Ireland (SFI) through the Research Frontiers Programme (Grant No. 11/RFP/ENM/3063) and by the European Regional Development Fund (ERDF) through INTERREG 2007-2013 Program (POCTEP project 0687_NOVOMAR_1_P). Bovine collagen materials were provided by Integra Life Sciences, Inc. through a Material Transfer Agreement. Salmon skins were kindly offered by Pingo Doce, Braga (Portugal)

Homogenized stiffness matrices for mineralized collagen fibrils and lamellar bone using unit cell finite element models

Mineralized collagen fibrils have been usually analyzed like a two phase composite material where crystals are considered as platelets that constitute the reinforcement phase. Different models have been used to describe the elastic behavior of the material. In this work, it is shown that, when Halpin-Tsai equations are applied to estimate elastic constants from typical constituent properties, not all crystal dimensions yield a model that satisfy thermodynamic restrictions. We provide the ranges of platelet dimensions that lead to positive definite stiffness matrices. On the other hand, a finite element model of a mineralized collagen fibril unit cell under periodic boundary conditions is analyzed. By applying six canonical load cases, homogenized stiffness matrices are numerically calculated. Results show a monoclinic behavior of the mineralized collagen fibril. In addition, a 5-layer lamellar structure is also considered where crystals rotate in adjacent layers of a lamella. The stiffness matrix of each layer is calculated applying Lekhnitskii transformations and a new finite lement model under periodic boundary conditions is analyzed to calculate the homogenized 3D anisotropic stiffness matrix of a unit cell of lamellar bone. Results are compared with the rule-of-mixtures showing in general good agreement.The authors acknowledge the Ministerio de Economia y Competitividad the financial support given through the project DPI2010-20990 and the Generalitat Valenciana through the Programme Prometeo 2012/023. The authors thank Ms. Carla Gonzalez Carrillo by her help in the development of some of the numerical models.Vercher Martínez, A.; Giner Maravilla, E.; Arango Villegas, C.; Tarancón Caro, JE.; Fuenmayor Fernández, FJ. (2014). Homogenized stiffness matrices for mineralized collagen fibrils and lamellar bone using unit cell finite element models. Biomechanics and Modeling in Mechanobiology. 13(2):1-21. https://doi.org/10.1007/s10237-013-0507-yS121132Akiva U, Wagner HD, Weiner S (1998) Modelling the three-dimensional elastic constants of parallel-fibred and lamellar bone. J Mater Sci 33:1497–1509Ascenzi A, Bonucci E (1967) The tensile properties of single osteons. Anat Rec 158:375–386Ascenzi A, Bonucci E (1968) The compressive properties of single osteons. Anat Rec 161:377–392Ashman RB, Cowin SC, van Buskirk WC, Rice JC (1984) A continuous wave technique for the measurement of the elastic properties of cortical bone. J Biomech 17:349–361Bar-On B, Wagner HD (2012) Elastic modulus of hard tissues. J Biomech 45:672–678Bondfield W, Li CH (1967) Anisotropy of nonelastic flow in bone. J Appl Phys 38:2450–2455Cowin SC (2001) Bone mechanics handbook, 2nd edn. CRC Press Boca Raton, FloridaCowin SC, van Buskirk WC (1986) Thermodynamic restrictions on the elastic constant of bone. J Biomech 19:85–86Currey JD (1962) Strength of bone. Nature 195:513Cusack S, Miller A (1979) Determination of the elastic constants of collagen by brillouin light scattering. J Mol Biol 135:39–51Doty S, Robinson RA, Schofield B (1976) Morphology of bone and histochemical staining characteristics of bone cells. In: Aurbach GD (ed) Handbook of physiology. American Physiology Soc, Washington, pp 3–23Erts D, Gathercole LJ, Atkins EDT (1994) Scanning probe microscopy of crystallites in calcified collagen. J Mater Sci Mater Med 5:200–206Faingold A, Sidney RC, Wagner HD (2012) Nanoindentation of osteonal bone lamellae. J Mech Biomech Materials 9:198–206Franzoso G, Zysset PK (2009) Elastic anisotropy of human cortical bone secondary osteons measured by nanoindentation. J Biomech Eng 131:021001Gebhardt W (1906) Über funktionell wichtige Anordnungsweisen der eineren und grösseren Bauelemente des Wirbeltierknochens. II. Spezieller Teil. Der Bau der Haversschen Lamellensysteme und seine funktionelle Bedeutung. Arch Entwickl Mech Org 20:187–322Gibson RF (1994) Principles of composite material mechanics. McGraw-Hill, New YorkGiraud-Guille M (1988) Twisted plywood architecture of collagen fibrils in human compact bone osteons. Calcif Tissue Int 42:167–180Gurtin ME (1972) The linear theory of elasticity. Handbuch der Physik VIa/ 2:1–296Halpin JC (1992) Primer on composite materials: analysis, 2nd edn. CRC Press, Taylor & Francis, Boca Raton, FloridaHassenkam T, Fantner GE, Cutroni JA, Weaver JC, Morse DE, Hanma PK (2004) High-resolution AFM imaging of intact and fractured trabecular bone. Bone 35:4–10Hohe J (2003) A direct homogenization approach for determination of the stiffness matrix for microheterogeneous plates with application to sandwich panels. Composites Part B 34:615–626Hulmes DJS, Wess TJ, Prockop DJ, Fratzl P (1995) Radial packing, order, and disorder in collagen fibrils. Biophys J 68:1661–1670Jäger I, Fratzl P (2000) Mineralized collagen fibrils: a mechanical model with a staggered arrangement of mineral particles. Biophys J 79:1737–1746Ji B, Gao H (2004) Mechanical properties of nanostructure of biological materials. J Mech Phy Sol 52:1963–1990Landis WJ, Hodgens KJ, Aerna J, Song MJ, McEwen BF (1996) Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography. Microsc Res Tech 33:192–202Lekhnitskii SG (1963) Theory of elasticity of an anisotropic elastic body. Holden-Day, San FranciscoLempriere BM (1968) Poisson’s ratio in orthotropic materials. Am Inst Aeronaut Astronaut J J6:2226–2227Lowenstam HA, Weiner S (1989) On biomineralization. Oxford University, New YorkLusis J, Woodhams RT, Xhantos M (1973) The effect of flake aspect ratio on flexural properties of mica reinforced plastics. Polym Eng Sci 13:139–145Martínez-Reina J, Domínguez J, García-Aznar JM (2011) Effect of porosity and mineral content on the elastic constants of cortical bone: a multiscale approach. Biomech Model Mechanobiol 10:309–322Orgel JPRO, Miller A, Irving TC, Fischetti RF, Hammersley AP, Wess TJ (2001) The in situ supermolecular structure of type I collagen. Structure 9:1061–1069Padawer GE, Beecher N (1970) On the strength and stiffness of planar reinforced plastic resins. Polym Eng Sci 10:185–192Pahr DH, Rammerstofer FG (2006) Buckling of honeycomb sandwiches: periodic finite element considerations. Comput Model Eng Sci 12:229–242Reisinger AG, Pahr DH, Zysset PK (2010) Sensitivity analysis and parametric study of elastic properties of an unidirectional mineralized bone fibril-array using mean field methods. Biomech Model Mechanobiol 9:499–510Reisinger AG, Pahr DH, Zysset PK (2011) Elastic anisotropy of bone lamellae as a function of fibril orientation pattern. Biomech Model Mechanobiol 10:67–77Rezkinov N, Almany-Magal R, Shahar R, Weiner S (2013) Three-dimensional imaging of collagen fibril organization in rat circumferential lamellar bone using a dual beam electron microscope reveals ordered and disordered sub-lamellar structures. Bone 52(2):676–683Rho JY, Kuhn-Spearing L, Zioupos P (1998) Mechanical properties and the hierarchical structure of bone. Med Eng Phys 20:92–102Rubin MA, Jasiuk I, Taylor J, Rubin J, Ganey T, Apkarian RP (2003) TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone. Bone 33:270–282Suquet P (1987) Lecture notes in physics-homogenization techniques for composite media. Chapter IV. Springer, BerlinWagermaier W, Gupta HS, Gourrier A, Burghammer M, Roschger P, Fratzl P (2006) Spiral twisting of fiber orientation inside bone lamellae. Biointerphases 1:1–5Wagner HD, Weiner S (1992) On the relationship between the microstructure of bone and its mechanical stiffness. J Biomech 25:1311–1320Weiner S, Wagner HD (1998) The material bone: structure-mechanical function relations. Annu Rev Mater Sci 28:271–298Weiner S, Traub W, Wagner H (1999) Lamellar bone: structure-function relations. J Struct Biol 126:241–255Yao H, Ouyang L, Ching W (2007) Ab initio calculation of elastic constants of ceramic crystals. J Am Ceram 90:3194–3204Yoon YJ, Cowin SC (2008b) The estimated elastic constants for a single bone osteonal lamella. Biomech Model Mechanobiol 7:1–11Yuan F, Stock SR, Haeffner DR, Almer JD, Dunand DC, Brinson LC (2011) A new model to simulate the elastic properties of mineralized collagen fibril. Biomech Model Mechanobiol 10:147–160Zhang Z, Zhang YWF, Gao H (2010) On optimal hierarchy of load-bearing biological materials. Proc R Soc B 278:519–525Zuo S, Wei Y (2007) Effective elastic modulus of bone-like hierarchical materials. Acta Mechanica Solida Sinica 20:198–20

Mechanical Strain Stabilizes Reconstituted Collagen Fibrils against Enzymatic Degradation by Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8)

Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain.The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded.In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen

Mapping Molecular Orientation with Phase Sensitive Vibrationally Resonant Sum-Frequency Generation Microscopy

We demonstrate a phase sensitive, vibrationally resonant sum-frequency generation (PSVR-SFG) microscope that combines high resolution, fast image acquisition speed, chemical selectivity, and phase sensitivity. Using the PSVR-SFG microscope, we generate amplitude and phase images of the second-order susceptibility of collagen I fibers in rat tail tendon tissue on resonance with the methylene vibrations of the protein. We find that the phase of the second-order susceptibility shows dependence on the effective polarity of the fibril bundles, revealing fibrous collagen domains of opposite orientations within the tissue. The presence of collagen microdomains in tendon tissue may have implications for the interpretation of the mechanical properties of the tissue. [Image: see text