Structural determinants of hydration, mechanics and fluid flow in freeze-dried collagen scaffolds.

UNLABELLED: Freeze-dried scaffolds provide regeneration templates for a wide range of tissues, due to their flexibility in physical and biological properties. Control of structure is crucial for tuning such properties, and therefore scaffold functionality. However, the common approach of modeling these scaffolds as open-cell foams does not fully account for their structural complexity. Here, the validity of the open-cell model is examined across a range of physical characteristics, rigorously linking morphology to hydration and mechanical properties. Collagen scaffolds with systematic changes in relative density were characterized using Scanning Electron Microscopy, X-ray Micro-Computed Tomography and spherical indentation analyzed in a time-dependent poroelastic framework. Morphologically, all scaffolds were mid-way between the open- and closed-cell models, approaching the closed-cell model as relative density increased. Although pore size remained constant, transport pathway diameter decreased. Larger collagen fractions also produced greater volume swelling on hydration, although the change in pore diameter was constant, and relatively small at ∼6%. Mechanically, the dry and hydrated scaffold moduli varied quadratically with relative density, as expected of open-cell materials. However, the increasing pore wall closure was found to determine the time-dependent nature of the hydrated scaffold response, with a decrease in permeability producing increasingly elastic rather than viscoelastic behavior. These results demonstrate that characterizing the deviation from the open-cell model is vital to gain a full understanding of scaffold biophysical properties, and provide a template for structural studies of other freeze-dried biomaterials. STATEMENT OF SIGNIFICANCE: Freeze-dried collagen sponges are three-dimensional microporous scaffolds that have been used for a number of exploratory tissue engineering applications. The characterization of the structure-properties relationships of these scaffolds is necessary to understand their biophysical behavior in vivo. In this work, the relationship between morphology and physical properties in the dry and hydrated states was investigated across a range of solid concentrations in the scaffolds. The quantitative results provided can aid the design of scaffolds with a target trade-off between mechanical properties and structural features important for their biological activity.Engineering and Physical Sciences Research Council CDT in Nanoscience and Nanotechnology (NanoDTC)This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.actbio.2016.05.02

Cartilage-like electrostatic stiffening of responsive cryogel scaffolds

Cartilage is a structural tissue with unique mechanical properties deriving from its electrically-charged porous structure. Traditional three-dimensional environments for the culture of cells fail to display the complex physical response displayed by the natural tissue. In this work, the reproduction of the charged environment found in cartilage is achieved using polyelectrolyte hydrogels based on polyvinyl alcohol and polyacrylic acid. The mechanical response and morphology of microporous physically-crosslinked cryogels are compared to those of heat-treated chemical gels made from the same polymers, as a result of pH-dependent swelling. In contrast to the heat-treated chemically-crosslinked gels, the elastic modulus of the physical cryogels was found to increase with charge activation and swelling, explained by the occurrence of electrostatic stiffening of the polymer chains at large charge densities. At the same time, the permeability of both materials to fluid flow was impaired by the presence of electric charges. This cartilage-like mechanical behavior displayed by responsive cryogels can be reproduced in other polyelectrolyte hydrogel systems to fabricate biomimetic cellular scaffolds for the repair of the tissue.G.S.O. and M.L.O. are grateful to the Nano Doctoral Training Centre (NanoDTC), University of Cambridge, and the EPSRC who supported this work through the EP/G037221/1 grant. I.M. and R.M.H. were supported by the Biotechnology and Biological Research Council, grant BB/J018236/1. P.J. was supported by Kidney Research UK. S.K.S. was supported by the European Research Council (ERC), grant EMATTER (#280078)

Raman Spectroscopy Reveals New Insights into the Zonal Organization of Native and Tissue-Engineered Articular Cartilage.

Tissue architecture is intimately linked with its functions, and loss of tissue organization is often associated with pathologies. The intricate depth-dependent extracellular matrix (ECM) arrangement in articular cartilage is critical to its biomechanical functions. In this study, we developed a Raman spectroscopic imaging approach to gain new insight into the depth-dependent arrangement of native and tissue-engineered articular cartilage using bovine tissues and cells. Our results revealed previously unreported tissue complexity into at least six zones above the tidemark based on a principal component analysis and k-means clustering analysis of the distribution and orientation of the main ECM components. Correlation of nanoindentation and Raman spectroscopic data suggested that the biomechanics across the tissue depth are influenced by ECM microstructure rather than composition. Further, Raman spectroscopy together with multivariate analysis revealed changes in the collagen, glycosaminoglycan, and water distributions in tissue-engineered constructs over time. These changes were assessed using simple metrics that promise to instruct efforts toward the regeneration of a broad range of tissues with native zonal complexity and functional performance.M.S.B., J.-P.S.-P., and M.M.S. acknowledge the support of the Medical Research Council, the Engineering and Physical Sciences Research Council, and the Biotechnology and Biological Sciences Research Council UK Regenerative Medicine Platform Hubs “Acellular Approaches for Therapeutic Delivery” (MR/K026682/1) and “A Hub for Engineering and Exploiting the Stem Cell Niche” (MR/K026666/1). J.-P.S.-P. and M.M.S. were also supported by the Medical Engineering Solutions in the Osteoarthritis Centre of Excellence, funded by the Wellcome Trust and the Engineering and Physical Sciences Research Council (088844). J.-P.S.-P. would like to acknowledge the Value in People Award from the Wellcome Trust Institutional Strategic Support Fund (097816/Z/11/A). M.M.S. also acknowledges the support from the ERC Seventh Framework Programme Consolidator grant “Naturale CG” under Grant Agreement No. 616417

Osteogenesis evaluation of duck’s feet derived collagen/hydroxyapatite sponges immersed in dexamethasone

Background: The aim of this study was to investigate the osteogenesis effects of DC and DC/HAp sponge immersed in without and with dexamethasone. Methods: The experimental groups in this study were DC and DC/HAp sponge immersed in without dexamethasone (Dex(â )DC and Dex(â )-DC/HAp group) and with dexamethasone (Dex(+)-DC and Dex(+)-DC/HAp group). We characterized DC and DC/HAp sponge using compressive strength, scanning electron microscopy (SEM). Also, osteogenic differentiation of BMSCs on sponge (Dex(â )DC, Dex(â )-DC/HAp, Dex(+)-DC and Dex(+)-DC/HAp group) was assessed by SEM, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay, alkaline phosphatase (ALP) activity assay and reverse transcription-PCR (RT-PCR). Results: In this study, we assessed osteogenic differentiation of BMSCs on Duckâ s feet-derived collagen (DC)/ HAp sponge immersed with dexamethasone Dex(+)-DC/HAp. These results showed that Dex(+)-DC/HAp group increased cell proliferation and osteogenic differentiation of BMSCs during 28 days. Conclusion: From these results, Dex(+)-DC/HAp can be envisioned as a potential biomaterial for bone regeneration applications.This work was supported by Technology Commercialization Support Program [grant number 814005-03-3-HD020], Ministry for Food, Agriculture, Forestry and Fisheries (MIFAFF).info:eu-repo/semantics/publishedVersio

Micromechanical study of the load transfer in a polycaprolactone-collagen hybrid scaffold when subjected to unconfined and confined compression

Scaffolds are used in diverse tissue engineering applications as hosts for cell proliferation and extracellular matrix formation. One of the most used tissue engineering materials is collagen, which is well known to be a natural biomaterial, also frequently used as cell substrate, given its natural abundance and intrinsic biocompatibility. This study aims to evaluate how the macroscopic biomechanical stimuli applied on a construct made of polycaprolactone scaffold embedded in a collagen substrate translate into microscopic stimuli at the cell level. Eight poro-hyperelastic finite element models of 3D printed hybrid scaffolds from the same batch were created, along with an equivalent model of the idealized geometry of that scaffold. When applying an 8% confined compression at the macroscopic level, local fluid flow of up to 20 [Formula: see text]m/s and octahedral strain levels mostly under 20% were calculated in the collagen substrate. Conversely unconfined compression induced fluid flow of up to 10 [Formula: see text]m/s and octahedral strain from 10 to 35%. No relevant differences were found amongst the scaffold-specific models. Following the mechanoregulation theory based on Prendergast et al. (J Biomech 30:539-548, 1997. https://doi.org/10.1016/S0021-9290(96)00140-6 ), those results suggest that mainly cartilage or fibrous tissue formation would be expected to occur under unconfined or confined compression, respectively. This in silico study helps to quantify the microscopic stimuli that are present within the collagen substrate and that will affect cell response under in vitro bioreactor mechanical stimulation or even after implantation

Nanofibrous hydrogel composites as mechanically robust tissue engineering scaffolds

Hydrogels closely resemble the extracellular matrix (ECM) and can support cell proliferation while new tissue is formed, making them materials of choice as tissue engineering scaffolds. However, their sometimes-poor mechanical properties can hinder their application. The addition of meshes of nanofibers embedded in their matrix forms a composite that draws from the advantages of both components. Given that these materials are still in the early stages of development, there is a lack of uniformity across methods for characterizing their mechanical properties. Here, we propose a simple metric to enable comparisons between materials. The fibrous constituent improves the mechanical properties of the hydrogel, while the biocompatibility and functionality of the gels are maintained or even improved

Structural determinants of hydration, mechanics and fluid flow in freeze-dried collagen scaffolds.

UNLABELLED: Freeze-dried scaffolds provide regeneration templates for a wide range of tissues, due to their flexibility in physical and biological properties. Control of structure is crucial for tuning such properties, and therefore scaffold functionality. However, the common approach of modeling these scaffolds as open-cell foams does not fully account for their structural complexity. Here, the validity of the open-cell model is examined across a range of physical characteristics, rigorously linking morphology to hydration and mechanical properties. Collagen scaffolds with systematic changes in relative density were characterized using Scanning Electron Microscopy, X-ray Micro-Computed Tomography and spherical indentation analyzed in a time-dependent poroelastic framework. Morphologically, all scaffolds were mid-way between the open- and closed-cell models, approaching the closed-cell model as relative density increased. Although pore size remained constant, transport pathway diameter decreased. Larger collagen fractions also produced greater volume swelling on hydration, although the change in pore diameter was constant, and relatively small at ∼6%. Mechanically, the dry and hydrated scaffold moduli varied quadratically with relative density, as expected of open-cell materials. However, the increasing pore wall closure was found to determine the time-dependent nature of the hydrated scaffold response, with a decrease in permeability producing increasingly elastic rather than viscoelastic behavior. These results demonstrate that characterizing the deviation from the open-cell model is vital to gain a full understanding of scaffold biophysical properties, and provide a template for structural studies of other freeze-dried biomaterials. STATEMENT OF SIGNIFICANCE: Freeze-dried collagen sponges are three-dimensional microporous scaffolds that have been used for a number of exploratory tissue engineering applications. The characterization of the structure-properties relationships of these scaffolds is necessary to understand their biophysical behavior in vivo. In this work, the relationship between morphology and physical properties in the dry and hydrated states was investigated across a range of solid concentrations in the scaffolds. The quantitative results provided can aid the design of scaffolds with a target trade-off between mechanical properties and structural features important for their biological activity

Multi-scale mechanical response of freeze-dried collagen scaffolds for tissue engineering applications

Tissue engineering has grown in the past two decades as a promising solution to unresolved clinical problems such as osteoarthritis. The mechanical response of tissue engineering scaffolds is one of the factors determining their use in applications such as cartilage and bone repair. The relationship between the structural and intrinsic mechanical properties of the scaffolds was the object of this study, with the ultimate aim of understanding the stiffness of the substrate that adhered cells experience, and its link to the bulk mechanical properties. Freeze-dried type I collagen porous scaffolds made with varying slurry concentrations and pore sizes were tested in a viscoelastic framework by macroindentation. Membranes made up of stacks of pore walls were indented using colloidal probe atomic force microscopy. It was found that the bulk scaffold mechanical response varied with collagen concentration in the slurry consistent with previous studies on these materials. Hydration of the scaffolds resulted in a more compliant response, yet lesser viscoelastic relaxation. Indentation of the membranes suggested that the material making up the pore walls remains unchanged between conditions, so that the stiffness of the scaffolds at the scale of seeded cells is unchanged; rather, it is suggested that thicker pore walls or more of these result in the increased moduli for the greater slurry concentration conditions

Stiffening by Osmotic Swelling Constraint in Cartilage-Like Cell Culture Scaffolds

Cartilage wounds result in chronic pain and degradation of the quality of life for millions of people. A synthetic cellular scaffold able to heal the damage by substituting the natural tissue is of great potential value. Here, it is shown for the first time that the unique interplay between the molecular components of cartilage can be reproduced in composite materials made of a polyelectrolyte hydrogel embedding a collagen scaffold. These composites possess a mechanical response determined by osmotic and electrostatic effects, comparable to articular cartilage in terms of elastic modulus, time-dependent response, and permeability to interstitial fluid flow. Made entirely from biocompatible materials, the cartilage-like composite materials developed permit 3D culture of chondrocyte-like cells through their microporosity. The biomimetic materials presented here constitute an entirely new class of osmotically stiffened composites, which may find use outside of biomedical applications

Investigation of the intrinsic permeability of ice-templated collagen scaffolds as a function of their structural and mechanical properties

Collagen scaffolds are widely used in a range of tissue engineering applications, both in vitro and in vivo, where their permeability to fluid flow greatly affects their mechanical and biological functionality. This paper reports new insights into the interrelationships between permeability, scaffold structure, fluid pressure and deformation in collagen scaffolds, focussing in particular on the degree of closure and the alignment of the pores. Isotropic and aligned scaffolds of different occlusivity were produced by ice templating, and were characterised in terms of their structure and mechanical properties. Permeability studies were conducted using two experimental set-ups to cover a wide range of applied fluid pressures. The permeability was found to be constant at low pressures for a given scaffold with more open structures and aligned structures being more permeable. The deformation of scaffolds under high pressure led to a decrease in permeability. The aligned structures were more responsive to deformation than their isotropic equivalents with their permeability falling more quickly at low strain. For isotropic samples, a broad (1 − ɛ)2 dependence for permeability was observed with the constant of proportionality varying with collagen fraction as the starting structures became more occluded. Aligned scaffolds did not follow the same behaviour, with the pores apparently closing more quickly in response to early deformation. These results highlight the importance of scaffold structure in determining permeability to interstitial fluid, and provide an understanding of scaffold behaviour within the complex mechanical environment of the body. Statement of significance: Collagen scaffolds are widely used in tissue engineering applications, for instance to contribute with wound healing. Their permeability to fluid flow, such as water and blood, is important to ensure they perform efficiently when inside the body. The present study reports new insights into the relationships between permeability, scaffold structure, fluid pressure and deformation in collagen scaffolds. It presents in particular the experimental setups used to measure these properties and the result of comparisons between collagen scaffolds with different structures: aligned and isotropic (non-aligned). It indicates quantitative differences in terms of permeability, and the effects of compression on such permeability. The results contribute to the development and understanding of collagen scaffolds and their applications