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

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#

Collagen: a network for regenerative medicine.

The basic building block of the extra-cellular matrix in native tissue is collagen. As a structural protein, collagen has an inherent biocompatibility making it an ideal material for regenerative medicine. Cellular response, mediated by integrins, is dictated by the structure and chemistry of the collagen fibers. Fiber formation, via fibrillogenesis, can be controlled in vitro by several factors: pH, ionic strength, and collagen structure. After formation, fibers are stabilized via cross-linking. The final bioactivity of collagen scaffolds is a result of both processes. By considering each step of fabrication, scaffolds can be tailored for the specific needs of each tissue, improving their therapeutic potential.REC acknowledges financial assistance from European Research Council (ERC) Advanced Grant 320598 3D-E.This is the final version of the article. It first appeared from the Royal Society of Chemistry via http://dx.doi.org/10.1039/c6tb00807

Altering crystal growth and annealing in ice-templated scaffolds.

The potential applications of ice-templating porous materials are constantly expanding, especially as scaffolds for tissue engineering. Ice-templating, a process utilizing ice nucleation and growth within an aqueous solution, consists of a cooling stage (before ice nucleation) and a freezing stage (during ice formation). While heat release during cooling can change scaffold isotropy, the freezing stage, where ice crystals grow and anneal, determines the final size of scaffold features. To investigate the path of heat flow within collagen slurries during solidification, a series of ice-templating molds were designed with varying the contact area with the heat sink, in the form of the freeze drier shelf. Contact with the heat sink was found to be critical in determining the efficiency of the release of latent heat within the perspex molds. Isotropic collagen scaffolds were produced with pores which ranged from 90 μm up to 180 μm as the contact area decreased. In addition, low-temperature ice annealing was observed within the structures. After 20 h at -30 °C, conditions which mimic storage prior to lyophilization, scaffold architecture was observed to coarsen significantly. In future, ice-templating molds should consider not only heat conduction during the cooling phase of solidification, but the effects of heat flow during ice growth and annealing.The authors gratefully acknowledge the financial support of the Gates Cambridge Trust, the Newton Trust, and ERC Advanced Grant 320598 3D-E. A.H. held a Daphne Jackson Fellowship funded by the University of Cambridge.This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s10853-015-9343-

Respiratory Infections by HMPV and RSV Are Clinically Indistinguishable but Induce Different Host Response in Aged Individuals

Background: Human metapneumovirus and respiratory syncytial virus can cause severe respiratory diseases, especially in infants, young children, and the elderly. So far it remains unclear why infections in the elderly become life threatening despite the presence of neutralizing antibodies in the serum, and to which extent double infections worsen the clinical course. Methods: Young and aged BALB/c-mice were infected with RSV or/and HMPV. Appearance of the mice was observed during course of infection. On day 5 p.i. animals were dispatched by cervical dislocation and levels of TNF-a and NF-kB were determined. Results: The observation of activity, weight and appearance of the different mice showed no differences among the tested groups. Despite this, the immunologic response depends on the animals ’ age and the virus they were infected with. In young animals, NF-kB levels were elevated if infected with HMPV and HMPV/RSV but remained low in RSV infections, whereas in aged animals the opposite was observed: solely RSV-infected animals showed elevated levels of NF-kB. TNF-a was slightly elevated in HMPV-infected young and old animals, but only in young animals this elevation was significant. Conclusions: Contrary to other studies, no weight loss or change in activity despite productive lung infection with the different viruses were observed. This may be due to the weaker anaesthesia or the lesser volume of virus solution used