Quantitative architectural description of tissue engineering scaffolds

Arguably one of the most specialised subtopics in porous materials research is that of tissue engineering scaffolds. The porous architecture of these scaffolds is a key variable in determining biological response. However, techniques for characterising these materials tend to vary widely in the literature. There is a need for a set of transferable and effective methods for architectural characterisation. In this review, four key areas of importance are addressed. First, the definition and interpretation of pore size are considered in relation to fluid transport properties, by analogy with filtration research. Second, the definition of interconnectivity is discussed using insight obtained from cement and concrete research. Third, the issue of data scalability is addressed by consideration of percolation theory, as implemented for the study of geological materials. Finally, emerging techniques such as confocal and multiphoton microscopy are discussed. These methods allow the three-dimensional observation of pore strut arrangement, as well holding great potential for understanding changes in pore architecture under dynamic conditions

The effects of scaffold architecture and fibrin gel addition on tendon cell phenotype.

This is the preprint version. The final version is available from Springer via http://dx.doi.org/10.1007/s10856-014-5349-3Development of tissue engineering scaffolds relies on careful selection of pore architecture and chemistry of the cellular environment. Repair of skeletal soft tissue, such as tendon, is particularly challenging, since these tissues have a relatively poor healing response. When removed from their native environment, tendon cells (tenocytes) lose their characteristic morphology and the expression of phenotypic markers. To stimulate tendon cells to recreate a healthy extracellular matrix, both architectural cues and fibrin gels have been used in the past, however, their relative effects have not been studied systematically. Within this study, a combination of collagen scaffold architecture, axial and isotropic, and fibrin gel addition was assessed, using ovine tendon-derived cells to determine the optimal strategy for controlling the proliferation and protein expression. Scaffold architecture and fibrin gel addition influenced tendon cell behavior independently in vitro. Addition of fibrin gel within a scaffold doubled cell number and increased matrix production for all architectures studied. However, scaffold architecture dictated the type of matrix produced by cells, regardless of fibrin addition. Axial scaffolds, mimicking native tendon, promoted a mature matrix, with increased tenomodulin, a marker for mature tendon cells, and decreased scleraxis, an early transcription factor for connective tissue. This study demonstrated that both architectural cues and fibrin gel addition alter cell behavior and that the combination of these signals could improve clinical performance of current tissue engineering constructs

Scaffold architecture and fibrin gels promote meniscal cell proliferation

Stability of the knee relies on the meniscus, a complex connective tissue with poor healing ability. Current meniscal tissue engineering is inadequate, as the signals for increasing meniscal cell proliferation have not been established. In this study, collagen scaffold structure, isotropic or aligned, and fibrin gel addition were tested. Metabolic activity was promoted by fibrin addition. Cellular proliferation, however, was significantly increased by both aligned architectures and fibrin addition. None of the constructs impaired collagen type I production or triggered adverse inflammatory responses. It was demonstrated that both fibrin gel addition and optimized scaffold architecture effectively promote meniscal cell proliferation.The authors gratefully acknowledge the financial support of the Gates Cambridge Trust, the ERC Advanced Grant No. 320598 3D-E, and the Technology Strategy Board UKThis is the final published version which appears at http://dx.doi.org/10.1063/1.490088

Ice-templated structures for biomedical tissue repair: From physics to final scaffolds

Ice-templating techniques, including freeze-drying and freeze casting, are extremely versatile and can be used with a variety of materials systems. The process relies on the freezing of a water based solution. During freezing, ice nucleates within the solution and concentrates the solute in the regions between the growing crystals. Once the ice is removed via sublimation, the solute remains in a porous structure, which is a negative of the ice. As the final structure of the ice relies on the freezing of the solution, the variables which influence ice nucleation and growth alter the structure of ice-templated scaffolds. Nucleation, the initial step of freezing, can be altered by the type and concentration of solutes within the solution, as well as the set cooling rate before freezing. After nucleation, crystal growth and annealing processes, such as Ostwald ripening, determine the features of the final scaffold. Both crystal growth and annealing are sensitive to many factors including the set freezing temperature and solutes. The porous structures created using ice-templating allow scaffolds to be used for many diverse applications, from microfluidics to biomedical tissue engineering. Within the field of tissue engineering, scaffold structure can influence cellular behavior, and is thus critical for determining the biological stimulus supplied by the scaffold. The research focusing on controlling the ice-templated structure serves as a model for how other ice-templating systems might be tailored, to expand the applications of ice-templated structures to their full potential.The authors gratefully acknowledge the financial support of the Gates Cambridge Trust, the Newton Trust, and ERC Advanced Grant No. 320598 3D-E. A.H. holds a Daphne Jackson Fellowship funded by the University of Cambridge.Copyright 2014 American Institute of Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics. The following article appeared in Applied Physics Reviews,1, 021301(2014) and may be found at: http://scitation.aip.org/content/aip/journal/apr2/1/2/10.1063/1.4871083

Understanding anisotropy and architecture in ice-templated biopolymer scaffolds.

Biopolymer scaffolds have great therapeutic potential within tissue engineering due to their large interconnected porosity and biocompatibility. Using an ice-templated technique, where collagen is concentrated into a porous network by ice nucleation and growth, scaffolds with anisotropic pore architecture can be created, mimicking natural tissues like cardiac muscle and bone. This paper describes a systematic set of experiments undertaken to understand the effect of local temperatures on architecture in ice-templated biopolymer scaffolds. The scaffolds within this study were at least 10mm in all dimensions, making them applicable to critical sized defects for biomedical applications. It was found that monitoring the local freezing behavior within the slurry was critical to predicting scaffold structure. Aligned porosity was produced only in parts of the slurry volume which were above the equilibrium freezing temperature (0°C) at the time when nucleation first occurs in the sample as a whole. Thus, to create anisotropic scaffolds, local slurry cooling rates must be sufficiently different to ensure that the equilibrium freezing temperature is not reached throughout the slurry at nucleation. This principal was valid over a range of collagen slurries, demonstrating that by monitoring the temperature within slurry during freezing, scaffold anisotropy with ice-templated scaffolds can be predicted.The authors gratefully acknowledge the financial supp ort of the Gates Cambridge Trust, the Newton Trust, and ERC Advanced Grant 320598 3D-E. A.H. holds a Daphne Jackson Fellowship funded by the University of Cambridge.This is a pre-print of an article which received final publication in Materials Science and Engineering: C Volume 37, 1 April 2014, Pages 141–147. The version offered here does not reflect changes resulting from peer-review. The version of record is available at http://www.sciencedirect.com/science/article/pii/S0928493114000101

Ionic solutes impact collagen scaffold bioactivity.

The structure of ice-templated collagen scaffolds is sensitive to many factors. By adding 0.5 wt% of sodium chloride or sucrose to collagen slurries, scaffold structure could be tuned through changes in ice growth kinetics and interactions of the solute and collagen. With ionic solutes (sodium chloride) the entanglements of the collagen molecule decreased, leading to fibrous scaffolds with increased pore size and decreased attachment of chondrocytes. With non-ionic solutes (sucrose) ice growth was slowed, leading to significantly reduced pore size and up-regulated cell attachment. This highlights the large changes in structure and biological function stimulated by solutes in ice-templating systems.The authors gratefully acknowledge the financial support of the Gates Cambridge Trust, the Newton Trust, NIHR, and ERC Advanced Grant 320598 3D-E. A.H. holds a Daphne Jackson Fellowship funded by the University of Cambridge. Also, the authors thank Dr. S. Butler for help with the rheological measurements.This is the accepted manuscript. The final publication is available at Springer via http://dx.doi.org/10.1007/s10856-015-5457-8

Non-linear dissolution mechanisms of sodium calcium phosphate glasses as a function of pH in various aqueous media

© 2020 Elsevier Ltd Phosphate glasses for bioresorbable implants display dissolution rates that vary significantly with composition, however currently their mechanisms of dissolution are not well understood. Based on this systematic study we present new insights into these mechanisms. Two-stage dissolution was observed, with time dependence initially parabolic and later linear, and a two-stage model was developed to describe this behaviour. Dissolution was accelerated by lower Ca concentration in the glass, and lower pH in the dissolution medium. A new dissolution mechanism is proposed, involving an initial stage where diffusion-controlled formation of a conversion layer occurs. Once the conversion layer is stabilised, layer dissolution reactions become rate-limiting. Under this mechanism the transition time is sensitive to the nature of the conversion layer and solution conditions. These results reveal the dependence of P2O5–CaO–Na2O glass dissolution on solution pH, and provide new insight into the dissolution mechanisms, particularly regarding the transition between the two dissolution stages

Collagen scaffolds as a tool for understanding the biological effect of silicates

Dietary silicon is essential in the maintenance of bone and cartilage. However, the mechanism by which silicon, in the form of silicates, triggers a biological response has never been uncovered. Here we demonstrate the incorporation of orthosilicic acid (Si(OH)4), the form of silicon in the body, within collagen scaffolds for use as an in vitro platform to identify key genes affected by silicates. Ice-templated collagen–silicate scaffolds, containing 0.21 wt% silicon, were validated by examining the mRNA levels for an array of genes in human osteoblasts and mesenchymal stromal cells (MSC) after 48 h in culture. Several novel genes, such as tumor necrosis factor alpha (TNF), were identified as having potential links to orthosilicic acid, verifying that collagen–silicate scaffolds are a versatile platform for identifying novel mechanisms in which silicates regulate musculoskeletal tissue.The authors gratefully acknowledge the financial support of the Gates Cambridge Trust , ERC Advanced Grant 320598 3D-E and from the National Institute for Health Research. RJ is supported by the Medical Research Council (Grant number MC_US_A090_0008/Unit Programme number U1059).This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0167577X15300203#

Effect of fiber crosslinking on collagen-fiber reinforced collagen-chondroitin-6-sulfate materials for regenerating load-bearing soft tissues.

Porous collagen-glycosaminoglycan structures are bioactive and exhibit a pore architecture favorable for both cellular infiltration and attachment; however, their inferior mechanical properties limit use, particularly in load-bearing situations. Reinforcement with collagen fibers may be a feasible route for enhancing the mechanical characteristics of these materials, providing potential for composites used for the repair and regeneration of soft tissue such as tendon, ligaments, and cartilage. Therefore, this study investigates the reinforcement of collagen-chondroitin-6-sulfate (C6S) porous structures with bundles of extruded, reconstituted type I collagen fibers. Fiber bundles were produced through extrusion and then, where applicable, crosslinked using a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide. Fibers were then submerged in the collagen-C6S matrix slurry before being lyophilized. A second 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide crosslinking process was then applied to the composite material before a secondary lyophilization cycle. Where bundles had been previously crosslinked, composites withstood a load of approximately 60 N before failure, the reinforcing fibers remained dense and a favorable matrix pore structure resulted, with good interaction between fiber and matrix. Fibers that had not been crosslinked before lyophilization showed significant internal porosity and a channel existed between them and the matrix. Mechanical properties were significantly reduced, but the additional porosity could prove favorable for cell migration and has potential for directing aligned tissue growth.This is the pre-peer reviewed version of the following article: J.H. Shepherd, S. Ghose, A. Moavenian, S.J. Kew, S.M. Best and R.E. Cameron. “Effect of fibre Cross-linking on Collagen-fibre reinforced Collagen-chondroitin-6-sulphate materials for regenerating load-bearing soft tissues”. Journal of Biomedical Materials Research: Part A, 2013;101(1):176-84., which has been published in final form at http://dx.doi.org/10.1002/jbm.a.34317

The process of EDC-NHS Cross-linking of reconstituted collagen fibres increases collagen fibrillar order and alignment.

We describe the production of collagen fibre bundles through a multi-strand, semi-continuous extrusion process. Cross-linking using an EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide) combination was considered. Atomic Force Microscopy (AFM) and Raman spectroscopy focused on how cross-linking affected the collagen fibrillar structure. In the cross-linked fibres, a clear fibrillar structure comparable to native collagen was observed which was not observed in the non-cross-linked fibre. The amide III doublet in the Raman spectra provided additional evidence of alignment in the cross-linked fibres. Raman spectroscopy also indicated no residual polyethylene glycol (from the fibre forming buffer) or water in any of the fibres.The authors would like to acknowledge the support of the Engineering and Physical Sciences Research Council (EPSRC), UK through a Knowledge Transfer Secondment (KTS) (to JHS), The National Institute for Health Research (NIHR) through their i4i grant to Tigenix Ltd and the TSB grant TP/8/BIO/6/I/Q0052.This is the accepted manuscript. The final version is available from AIP at http://scitation.aip.org/content/aip/journal/aplmater/3/1/10.1063/1.4900887