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Tuesday, 25 February 2014

Biomaterials and Scaffolds for tissue engineering – approaches to their mechanical testing

Surgeon's assistant in operating theatreEvery day thousands of surgical procedures are performed to replace or repair tissue that has been damaged through disease, injury or trauma. 

The developing field of tissue engineering aims to regenerate damaged tissues by combining cells from the body with highly porous scaffold biomaterials, which act as templates for tissue regeneration to guide the growth of new tissue.

Tissue engineering is the use of a combination of cells, engineering and materials methods, and suitable biochemical and physio-chemical factors to improve or replace biological functions. While it was once categorised as a sub-field of biomaterials, it has grown considerably in scope and importance and can be considered as a field in its own right.

While most definitions of tissue engineering cover a broad range of applications, in practice the term is closely associated with applications that repair or replace portions of or whole tissues (i.e., bone, cartilage, blood vessels, bladder, skin, muscle etc.). Often, the tissues involved require certain mechanical and structural properties for proper functioning.

Usually the term “scaffold” is associated primarily with its function as a temporary support for the culture of cells and tissues with the final aim of restoring lost tissue functionality, the so called “tissue engineering”.  In this context, a fundamental property of the scaffold resides in its biodegradable nature: as the tissue regeneration progresses, the scaffold should degrade at a comparable rate.  The pore architecture of scaffolds, including porosity, pore interconnectivity and average pore size are also critical in cell survival, proliferation and secretion of extracellular matrix.

Mechanical Property Requirements of Scaffolds
Numerous scaffolds produced from a variety of biomaterials and manufactured using a plethora of fabrication techniques have been used in the field in attempts to regenerate different tissues and organs in the body.

Regardless of the tissue type, a number of key considerations are important when designing or determining the suitability of a scaffold for use in tissue engineering. Biodegradability is often an essential factor since scaffolds should preferably be absorbed by the surrounding tissues without the necessity of a surgical removal. The rate at which degradation occurs has to coincide as much as possible with the rate of tissue formation: this means that while cells are fabricating their own natural matrix structure around themselves, the scaffold is able to provide structural integrity within the body and eventually it will break down leaving the neotissue, newly formed tissue which will take over the mechanical load.

Whilst biocompatibility and biodegradability are primary considerations, of equal importance are the mechanical properties. Ideally, the scaffold should have mechanical properties consistent with the anatomical site into which it is to be implanted and, from a practical perspective, it must be strong enough to allow surgical handling during implementation. While this is important in all tissues, it provides some challenges for cardiovascular and orthopaedic applications specifically.

Producing scaffolds with adequate mechanical properties is one of the great challenges in attempting to engineer bone or cartilage. For these tissues, the implanted scaffold must have sufficient mechanical integrity to function from the time of implantation to the completion of the remodelling process. A further challenge is that healing rates vary with age; for example, in young individuals, fractures normally heal to the point of weight-bearing in about six weeks, with complete mechanical integrity not returning until approximately one year after fracture, but in the elderly the rate of repair slows down. This too must be taken into account when designing scaffolds for orthopaedic applications.

However, as the field has evolved, it could be argued that too much focus has been placed on trying to develop scaffolds with mechanical properties similar to bone and cartilage. Many materials have been produced with good mechanical properties but to the detriment of retaining a high porosity and many materials, which have demonstrated potential in vitro have failed when implanted in vivo due to insufficient capacity for vascularisation.

It is clear that a balance between mechanical properties and porous architecture sufficient to allow cell infiltration and vascularisation is key to the success of any scaffold. Injectability is also important for clinical uses. Recent research on organ printing is showing how crucial a good control of the 3D environment is to ensure reproducibility of experiments and offer better results.

The main criterion for scaffolds in tissue engineering which all of the other criteria are dependent upon is the choice of biomaterial from which the scaffold should be fabricated.

Syringe image
Types of Scaffold
Many different materials (natural and synthetic, biodegradable and permanent) have been investigated. Most of these materials have been known in the medical field before the advent of tissue engineering. New biomaterials have been engineered to have ideal properties and functional customisation: injectability, synthetic manufacture, biocompatability, non-immunogenicity, transparency, nano-scale fibres, low concentration, resorption rates etc.

Typically, three individual groups of biomaterials; ceramics, synthetic polymers and natural polymers, are used in the fabrication of scaffolds for tissue engineering.

Ceramic Scaffolds

Although not generally used for soft tissue regeneration, there has been widespread use of ceramic scaffolds, such as hydroxyapatite and tri-calcium phosphate, for bone regeneration applications. Ceramic scaffolds are typically characterised by high mechanical stiffness (Young’s modulus), very low elasticity, and a hard brittle surface. From a bone perspective, they exhibit excellent biocompatibility due to their chemical and structural similarity to the mineral phase of native bone. The interactions of osteogenic cells with ceramics are important for bone regeneration, as ceramics are known to enhance osteoblast differentiation and proliferation. 

Various ceramics have been used in dental and orthopaedic surgery to fill bone defects and to coat metallic implant surfaces to improve implant integration with the host bone. However, their clinical applications for tissue engineering has been limited because of their brittleness, difficulty of shaping for implantation, and because new bone formed in a porous HA network cannot sustain the mechanical loading needed for remodelling. In addition, although HA is a primary constituent of bone and might seem ideal as a bone graft substitute, problems also exist in that it is difficult to control its degradation rate.

Synthetic Polymer Scaffolds
Numerous synthetic polymers have been used in the attempt to produce scaffolds including polystyrene, poly-l-lactic acid, polyglycolic acid and poly-dl-lactic-co-glycolic acid.  While these materials have shown much success as they can be fabricated with a tailored architecture, and their degradation characteristics controlled by varying the polymer itself or the composition of the individual polymer, they have drawbacks including the risk of rejection due to reduced bioactivity. In addition, concerns exist about the degradation process of PLLA and PGA as they degrade by hydrolysis, producing carbon dioxide and therefore lowering the local pH which can result in cell and tissue necrosis. 

Natural Polymer Scaffolds
The third commonly used approach is the use of biological materials as scaffold biomaterials.  Biological materials such as collagen, fibrin, various proteoglycans, alginate-based substrates and chitosan have all been used in the production of scaffolds for tissue engineering. 

Unlike synthetic polymer-based scaffolds, natural polymers are biologically active and typically promote excellent cell adhesion and growth. Furthermore, they are also biodegradable and so allow host cells, over time, to produce their own extracellular matrix and replace the degraded scaffold. However, fabricating scaffolds from biological materials with homogeneous and reproducible structures presents a challenge. In addition, the scaffolds generally have poor mechanical properties, which limits their use in, for example, load-bearing orthopaedic applications.  

Among organic polymers, poly (vinyl alcohol) (PVA) is one of the very few polymers soluble in water that has been studied intensively because of its attractive features for medical applications such as high hydrophilicity, good film forming ability and processability.  Applications of PVA hydrogels in the biomedical field include contact lenses, wound dressing, and coatings for sutures and catheters. Additionally, PVA hydrogels have been shown to be intrinsically cell and protein non-adhesive, thus providing a blank substrate.

Each of these individual biomaterial groups has specific advantages and, needless to say, disadvantages so the use of composite scaffolds comprised of different phases is becoming increasingly common. For example, a number of groups have attempted to introduce ceramics into polymer-base scaffolds while others have combined synthetic polymers with natural polymers in order to enhance their biological capacity. 

While scaffolds such as these have shown some promise, each consists of at least one phase which is not found naturally in the body and they all have associated problems with biocompatibility, biodegradability or both. A more typical approach is the use of collagen-based scaffolds, either alone or with an additional phase incorporated to enhance biological and/or mechanical properties.  

Testing mechanical suitability – how and where research is taking place using texture analysis
Researchers at STEM, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, have been performing a series of tests to assess the mechanical properties of fluid foamed poly(d,l-lactic acid) scaffolds. Throughout a series of published papers they have documented their use of t
he TA.XTplus texture analyser for the assessment of the scaffolds in a series of different approaches.

Tensile testing using pneumatic grips on a TA.HDplus texture analyser
Measuring Tensile Strength
Tensile tests of flexible polymer film scaffolds were conducted in triplicate on a TA.XTplus Texture Analyser with a load cell of 5 kg, using pneumatic clamps to secure the samples. Briefly, the sample (8 mm/54 mm/2.5 mm – length/width /thickness) was tested at four different rates: 18, 180, 300 and 500 mm/min. Strain, stress and the engineering Young’s modulus values were obtained The same texture analyser was used to evaluate in triplicate the fatigue of the porous material over 1000 cycles at 300 mm/min, 60% strain and at a frequency of 0.27 Hz.

Published work:
Cell adhesion and mechanical properties of a flexible scaffold for cardiac tissue engineering

Cylinder probe test on gel sample
Measuring Compressive Integrity
In this paper supercritical CO2 scaffolds were fabricated. This solvent free, low temperature process produces open cell, inter-connected foamed structures. Drug molecules and proteins can be encapsulated within these scaffolds as protein structure and activity are retained during processing. The fabrication process can produce scaffolds of divergent pore size and structure, hence, the study sought to elucidate the effects of processing conditions on the porosity, pore size distribution and mechanical properties of the scaffolds. Scaffolds were cut into uniform cubes prior to compression. The elastic collapse stress, Young’s modulus and ultimate stress to failure were measured.

Published work:
Mechanical and Morphological Studies of Supercritical Fluid Foamed Poly (D,L-Lactic Acid) Scaffolds

Meanwhile researchers at INSERM, U791, Laboratoire d'ingénierie ostéoarticulaire et dentaire, Faculté de chirurgie dentaire, Université de Nantes, France, determined the Young’s Modulus of all scaffolds measured in a standard compression testing mode. The compression speed of the top plate was set to 1mm/s and compression carried out until a maximum strain of 10% was achieved. A total of 6 of the scaffolds at each time point were tested and the resultant stress from an imposed strain measured. The initial modulus – i.e. the Young’s Modulus – is gained from the initial slope of the stress versus strain prior to plastic deformation.

Published work:
Rapid prototyped porous titanium coated with calcium phosphate as a scaffold for bone tissue engineering

Researchers at the Department of Chemical and Biomolecular Engineering, The University of Melbourne, Australia, used a similar approach in their work:
The influence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo

The in vitro and in vivo degradation properties of poly(lactic-co-glycolic acid) (PLGA) scaffolds produced by two different technologies-thermally induced phase separation (TIPS), and solvent casting and particulate leaching (SCPL) were compared. In order to understand the effect of changes in the internal scaffold architecture on the mechanical properties, the outside layer (a ‘skin’) was removed. The original cylindrical scaffolds were dipped into liquid nitrogen and a cubic sample was obtained using a surgical blade to remove the surrounding polymer without damage to the internal structure. Samples were further treated (see method details in publication) and left overnight to simulate in vivo conditions before testing. The initial stress (σ) and strain (τ) and Young’s modulus of the obtained cylindrical 3D PLGA scaffolds were determined using their TA.XT2 texture analyser.

A further publication (see link below) focuses on the selection of alternative solvents for the thermally-induced phase separation technique. The Young's moduli of the scaffolds under conditions of temperature, pH and ionic strength similar to those found in the body were tested and were found to be highly dependent on the architectures.
Systematic selection of solvents for the fabrication of 3D combined macro- and microporous polymeric scaffolds for soft tissue engineering

Researchers at the Norwegian University of Science and Technology, NOBIPOL, Department of Biotechnology, Norway, took a similar approach in their publication:
In situ gelation for cell immobilisation and culture in alginate foam scaffolds

The rigidity of alginate gels formed by in situ gelation was determined by compressing the gels with a TA.XTplus Texture Analyser equipped with a 5kg load cell and a 6.35mm diameter cylindrical probe  and measuring the Young’s modulus.  It was demonstrated that the mechanical properties of the gel could largely be varied through selection of type and concentration of the applied alginate and by immersing the already gelled discs in solutions providing additional gel forming ions.

Researchers at the Department of Biology and Biotechnology, “Sapienza” University of Rome, Italy, characterised their novel PVA 3D platforms via compression testing using their Texture Analyser in:
Synthesis and characterisation of a novel poly(vinyl alcohol) 3D platform for the evaluation of hepatocytes response to drug administration

They shaped their scaffolds into uniform cylinders of height 1.5cm and diameter of 2cm and applied compressive force to 80% deformation using a 10mm cylinder probe at 1mm/s.  Elastic (Young’s) moduli were derived from the regression of the linear portion of stress-strain curves.

Researchers at the University of Witwatersrand Department of Pharmacy & Pharmacology used their TA.XTplus to establish various stress-strain parameters of the polymeric scaffold.  Samples were assessed in both the hydrated and unhydrated states and the Matrix Resilience and Matrix Hardness were computed.  

Published work:
Design and pharmaceutical evalation of a nano-enabled crosslinked multipolymeric scaffold for prolonged intracranial release of Zidovudine

TA.XTplus texture analyser with syringe rig test Measuring Injectability/Syringeability
Researchers at the Department of Chemical Engineering and Department of Mechanical and Materials Engineering, Queen’s University, Canada, used their TA.XTplus Texture Analyser to measure injectability:

Injectable, high modulus and fatigue resistant, composite scaffold for load-bearing soft tissue regeneration

The syringe was secured vertically into a custom clamp to fit within the texture analyser and expelled at 15mm/s.

The TA.XTplus texture analyser is part of a family of texture analysis instruments and equipment from Stable Micro Systems. These specific examples of testing scaffolds are part of  a range of application possibilities for the testing of medical devices and controlled release products. Our technical experts can also custom design instrument fixtures according to individual specifications.

Watch our video about testing of materials  The Role of Texture Analysis in Pharmaceuticals

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