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Rheology measurement approaches for hyaluronic acid solutions and gels.

Hyaluronic acid, also known as hyaluronan, is a naturally occurring, high-molecular weight biopolymer that is found throughout the body.  Hyaluronan forms the main component of synovial fluid (along with lubricin) and is also found in cartilage and skin.  Hyaluronan rheology is of particular relevance as its viscosity and viscoelastic behaviour offers lubrication, shock absorbing and support properties.  What follows is a brief introduction to rheology and viscosity testing methods for hyaluronic acid formulations.

Typical rheology of hyaluronan

Any polymer in solution will impart non-Newtonian and viscoelastic behaviour.  A non-Newtonian liquid is a liquid whose viscosity is not a single fixed value but is dependent upon the shear applied.  The most common form of non-Newtonian behaviour is shear-thinning, where the viscosity decreases with increasing shear rate.  This is often a desirable characteristic in a material as it means the product can be moved around easily (pumped, spread, injected etc) but once it reaches its destination and comes to rest it returns to a high viscosity which helps it maintain its position.  However, this very behaviour presents a measurement dilemma because a simple single-point viscosity test, such as that typically performed using a Brookfield viscometer, will prove insufficient to fully characterise the material.  Instead, a viscosity/shear rate profile is more relevant, such as that shown in figure 1:

Viscosity/shear rate profile of hyaluronan solution
Figure 1: Viscosity/shear rate profile of hyaluronic acid solution

Here, shear rate is swept from low to high and viscosity is recorded throughout.  The shear thinning behaviour can be clearly seen with viscosity decreasing by a factor of over 20 times as shear rate is increased.  Also of interest here is that the sample tends to a Newtonian plateau with decreasing shear rate.  The plateau viscosity, known as the zero-shear viscosity, is a useful and important material attribute, signifying the effective viscosity in an at- rest condition.  The magnitude of the zero-shear viscosity and the shape of the low-shear portion of the flow curve are influenced by both molecular weight and weight distribution and hence can provide an indirect assessment of these factors for batch screening purposes.

Normal stress measurement and film-forming ability

The viscosity / shear rate profile performed here can also deliver a measurement of another useful property: normal stress.  When an elastic fluid is sheared a stress is generated normal (i.e. perpendicular) to the direction of shear.  The magnitude of this force often correlates well with film-forming ability, a pre-requisite for thick-film lubrication.  A good film-former typically generates a high normal stress compared to a poor one at a given shear rate. On a research rheometer this stress can be measured through a force transducer fitted underneath the measuring plate.  The normal stress growth with increasing shear rate for a hyaluronic acid solution can be clearly seen in figure 2:

Normal stress evolution with shear rate for HA solution
Figure 2: Normal stress evolution with shear rate for HA solution

Viscoelasticity characterisation of crosslinked and un-crosslinked Hyaluronic Acid products.

In addition to flow hyaluronic acid solutions and gels display elastic properties: when they are deformed they will store elastic strain energy to a degree and bounce back towards their original shape when the deforming stress is removed.  Elastic deformation and viscous flow combine as viscoelasticity.  The relative dominance of elastic or viscous behaviour is of obvious relevance for hyaluronic acid formulations, notably crosslinked materials, such as in dermal filler and scaffold applications, where compliance and suppleness must accompany creep resistance.  Weak gels and concentrated solutions also exhibit viscoelasticity however these materials will display a deformation timescale dependence where materials exhibit elastic storage of energy when the deformation timescale is short but relax into viscous flow over longer deformation timescales.  

Oscillation (or dynamic) rheological testing methods are an excellent tool for characterising the viscoelastic properties of hyaluronic acid solutions and gels.  These mechanical spectroscopy techniques work by imposing small-amplitude oscillatory (i.e. clockwise then counter clockwise) shear to the sample and observing the response.  By comparing storage (G’) and loss (G”) moduli, the relative amounts of energy stored elastically and dissipated viscously  throughout deformation are plotted as a function of deformation frequency to obtain a viscoelastic fingerprint of the material:

Viscoelastic fingerprint showing storage and loss moduli for HA solution
Figure 3: Viscoelastic fingerprint showing storage and loss moduli for HA solution

Figure 3 shows a viscoelastic fingerprint of a hyaluronic acid solution showing weak gel behaviour.  Molecular entanglements result in a clear time-dependent behaviour where elastic dominance (G'>G") is seen at high frequencies but viscous dominance (G">G') prevails at low frequencies.  The reciprocal of the crossover frequency is a relaxation time, the exponential time constant that quantifies the rate at which stresses will reduce towards zero when the sample is exposed to a defined deformation. The relaxation time calculated in this case is about 0.17s.  In very rough terms this means that on the imposition of a step strain deformation it would take approximately 0.17s for about 63% of the start stress to dissipate through viscous flow.

Figure 4 contrasts the viscoelastic properties of hyaluronic acid solution and crosslinked HA.  The ability for the crosslinked product to maintain elastic-dominance, in other words: not to relax, is clearly seen against the “relaxable” solution.  A crossover into viscous-dominant behaviour would eventually be found at much lower frequencies than those encompassed by this test, equating to a much longer relaxation time, as would be expected in the more "solid" material.

Comparison of HA solution and crosslinked HA
Figure 4: Comparison of HA solution and crosslinked HA

High performance rheometry techniques such as those demonstrated here are particularly relevant to the product formulator or quality controller working with hyaluronic acid-based products. 

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