Deformation characteristics of hydrogel products based on cross‐linked hyaluronic acid

The cross‐linked hyaluronic acid (HA) fillers are the viscoelastic hydrogel with a dominant elasticity rather than a viscosity as a useful medical device in the soft tissue augmentation. These HA fillers undergo deformation to begin the biodegradation by the biochemical and physical environment of the body, and result of deformations are closely related to clinical performance.


| INTRODUC TI ON
Cross-linked hyaluronic acid (HA) gel is an alternative to implants of plastic surgery for soft tissue augmentation, which is most commonly used in medical aesthetic field to improve facial wrinkles and replace for the facial tissue volumization.
The market size of HA filler in the EU region is about USD 6181 M as of 2019.Increment of market is expected to be 8.6% of the annual average growth rate (GAGR 19-29) considering the negative growth in 2020 due to COVID-19. 1 About 160 products are distributed from more than 50 companies in globally licensed products such as Juvederm (Allergan, USA), Restylane (Galderma, Switzerland), and Teosyal (Teoxane, Switzerland) 2 as well as Yvoire (LG chemical) which was first released in Korea, The Chaeum (Hugel), and Neuramis (Medytox) from Korean manufacturers.The rheological properties of these products are variously composed by manufacturer.These diversities from each product could provide appropriate multiplicity for the characteristics of patients from the physician's point of view.Therefore, it is necessary to examine the rheological characteristics of the filler for the appropriate procedures.
Hyaluronate, also called HA or hyaluronan, is a main raw material of the cross-linked HA, which is colorless and transparent.HA is a high viscosity and high-molecular weight linear polysaccharide at 1-10 000 kDa, and consists of repeating with two alternating units −1,4-D-glucuronic acid and −1,3-N-acetyl-D-glucosamine.HA is widely distributed in dermis, vitreous humor, synovium, umbilical cord, rooster combs, and produced by bacteria such as Streptococcus genus. 3[6][7] The rheological properties of HA as a raw material for medicine and medical device for soft tissue regeneration are the viscousdominant gel which, viscous (G″, loss modulus, viscous modulus) is above elastic (G′, storage modulus, elastic modulus) (Figure 1).HA with this rheological characteristic is used in pharmaceutical products such as ophthalmic products and orthopedic joint injection.
However, HA with viscous dominant characteristic have difficulties to use as a medical device for aesthetic rejuvenation due to their disadvantages of low longevity and performance in their efficacy.][10] This elastic HA gel after rheological deformation has much higher storage modulus (G′) compared to loss modulus (G″) as shown in Figure 2. 11 The rheological deformation increases the persistence of an alternative for soft tissue augmentation and improves the performance for wrinkle and volumizing on the skin in plastic surgery.
In this study, deformation of five marked HA fillers was measured with rheological properties, and the factors maintaining rheological properties of implanted HA filler were defined by the comparison of the differences after and before of the deformation.These results suggest that the new physical properties of the HA filler could be used as a selection guide depending on the clinical purpose.

| Amplitude sweep test
Amplitude sweep test has generally been performed to measure changes in rheological properties while deformation occurred in the cross-linked HA gels using a rheometer (KINEXUS Pro+, Malvern instruments, UK) with 4/40 mm cone and plate geometry (disk type) at room temperature. 12However, measurement condition of a rheometer was carried out at 37°C mimicking the body condition.Storage modulus (elastic modulus, G′), loss modulus (viscous modulus, G″), and complex modulus (G*) that vary depending on strain (amplitude) were measured with maintaining a constant frequency.Frequency was 1 Hz, strain was set to 0.1%-1000%, and the gap between upper and bottom plate was set to 1 mm.All experiments were repeated three times and the results were expressed as mean or mean ± SD.

| Statistical analysis F I G U R E 1
Change in elastic modulus (G′) and loss modulus (G″) of hyaluronic acid raw material and cross-linked hyaluronic acid biomaterial.

| Optimization of the condition for the amplitude sweep test
The value of strain limitation and the temperature at the point with deformation of cross-linked HA fillers for amplitude sweep test were set.In this study, the measuring temperature was 37°C due to actual application to the human body.The cross-over point of storage modulus (G′) and loss modulus (G″) was intersected at 200% of strain, and storage and loss moduli value were reversed.This result indicate that the deformation and rearrangement of cross-linked HA gels was completed in the structure of the gels at 200% deformation.Since this process begins, deformed cross-linked HA gels could not be return to the natural properties of the before deformed cross-linked HA gels.Therefore, administration temperature was set at 37°C, and the stress value was set at 180 Pa for deformation point under 200% with constant stress value due to the stress value at the time of reaching the strain rate 200% varies from each filler for this study (Figure 2).

| Analysis of cross-over point and strain rate by amplitude sweep test
Among the tested cross-linked HA fillers, Juvederm > Neuramis > The Chaeum > Elravie > Restylane was the order of products that showed a rapid cross-over point.When the strain was increased, Juvederm was the product with the lowest resistance to defor- the increment in loss modulus and the decrement in storage modulus were rapidly changed before the cross-over of storage modulus and loss modulus.In Juvederm, the increment in loss modulus and the decrement in storage modulus were more rapidly changed than other products before the cross-over of storage modulus and loss modulus.This characteristic affects the change in viscoelasticity was observed rapid decrement of complex viscosity over 20% of shear strain (Figure 3).Restylane showed the highest composite viscosity when the same strain was reached, Juvederm presented the lowest complex viscosity, and Elavie and The Chaeum followed.

F I G U R E 2
Change of elastic and viscous moduli properties by increased strain in various cross-linked hyaluronic acid fillers.

| Difference rate of G′ and G″ between initial and after in deformation process for molding index power
The changes of the cross-over point, storage, and loss moduli of each filler were measured with the amplitude sweep test.The stress applied to each filler were observed at the strain 200%, and some fillers showed the intersection point in distant before the strain 200%.
Table 1 shows the analysis of complex modulus (G*) were measured on Elravie, and 179% on Juvederm, respectively.Molding index indicates the smaller the sum of the component values, the better the forming index power.Among the compared fillers, Restylane was the lowest value at 158.0, Juvederm was highest at 327.9, and Chaeum was 170.5 after Restylane.
In Table 2, the result of molding index power is confirmed by the Collins' equation. 13,14ere D is the constant and F is the energy loss from other than the material involved.G″/G* n is the loss compliance (n = 2 [if equal stress condition]).Loss compliance was related to the molding index power from the Collin's equation and confirmed the correlation between loss compliance and molding index power.Loss Compliance means that the smaller the value is the better like the molding index.Restylane was the lowest value at 1.6 × 10 −3 , Juvederm was highest at 8.0 × 10 −3 , and Chaeum was 1.9 × 10 −3 after Restylane.These loss compliance from each HA filler obtained correlative results that were same order with the molding index at Tables 1 and 2.

| CON CLUS IONS
Molding is "Using force to make something in a new shape" as a dictionary definition, which means having a new shape in aesthetic or plastic surgery.When the cross-linked HA biomaterials are subjected to maximum deformation without demolition of dynamic form networks, chemical bonding maintaining the physical structure of the cross-linked biomaterials is broken.The decomposing pattern of the bonding force is a decrement of viscoelasticity, and particularly the elastic modulus was decreased significantly, on the other side, viscous modulus was increased due to compensation force to maintain the structure.Therefore, the own properties of the viscoelastic structure disappear due to elasticity and viscosity of biomaterials at the cross-over point where the decrement of elastic modulus (G′) and the increment of viscous modulus (G″). 15) Energy loss of deformation process = DG �� ∕ G * n + F TA B L E 1 Rheological properties of five hyaluronic acid fillers deformed by amplitude sweep at the condition of stress 180 Pa.

Rheological property
The chaeum premium no.
TA B L E 2 Loss compliance deference of five hyaluronic acid fillers from the Collin's equation.

Rheological properties
The chaeum premium no. and strain rate by elasticity with rheological elements. 12,16These studies indicated that the elasticity is proportional to the crosslinking or modification rate, and refers to storage modulus (G′) as a rheological parameter resisting deformation by the external force.
Elasticity is commonly known to be resistant to deformation as a filler structure with high flexibility that endurable against the forces of various movements of human tissues.However, even though storage modulus (G′) acts a major role, loss modulus (G″) should be considered as a rheological characteristic for the cohesiveness of the filler structure.
Implanted filler should maintain the shape properly, but the dispersion of implanted filler is resulted from decreased viscosity, and the cohesion between the particles is also decreased.Moreover, foreign body sensation could be caused due to low viscosity and maintaining elasticity, and lead the physical tissue damage and inflammatory reactions in severe cases.Implanted filler is deformed by the temperature, enzyme, physical force in surrounding tissue compared to before implantation, and the deformation affect the changes of rheological properties.The decrement of elasticity and the increment of viscosity compensatively appear during deformation, overall viscoelasticity decreases, and the maintenance of the shape becomes advantageous by the increasing the cohesion between the particles of the implanted filler.
However, among the complex rheological elements, elasticity (G′) is not enough to suggest of the sufficient evidence for the explanation about the optimal biocompatible elasticity and rheological properties of the alternative to plastic surgery for soft tissue augmentation with the cohesion, flexibility, and firmness.
8][19] Molding index could be considered to depending on the amount of change in viscosity, rheological property based on cohesion, and molding index power with the least deformation is better.
In general, cross-linked HA biomaterials have higher storage modulus than loss modulus, and this loss modulus affect the changes of cohesion.Loss modulus is multiple increased compared to the decrease in the storage modulus value with deformation by the surrounding environment.Low value of strain % is preferable for the maintenance of the characteristics of the viscoelastic due to structurally stable and resistant for deformation, therefore, the molding index is proportional to the sum of the value of ∆G* and the strain rate.
This molding index power is expected that biological materials with low strain change the rheological properties by stress, and the mation, and Restylene showed high resistance.Strain of the cross-over point for Juvederm was at 40%, Neuramis at 158%, Chaeum at 200%, Elravie at 316%, and Restylane at 502%.As the strain over 20%, loss modulus was gradually increased except for Juvederm, and the tested products with the largest increment in loss modulus was Juvederm > Neuramis >Chaeum > Elravie > Rest ylane; however, the loss modulus value at strain 200% was shown in the order of Chaeum (102.7 Pa) > Neuramis (95.7 Pa) > Juvederm (87.7 Pa) > Restylane (71.8 Pa) > Elravie (43.1 Pa), and each stress to cross-linker was measured at 290 Pa, 260 Pa, 85 Pa, 312 Pa, and 297 Pa (Figure 2).Initial complex viscosity was Juvederm (60.3 Pa.s) > Neuramis (51.3 Pa.s) ≥ Chaeum (50.8 Pa.s) > Restylane (41.9 Pa.s) > Elravie (37.5 Pa.s).The values of complex viscosity in all samples were maintained up to 20% of strain; however, complex viscosity was gradually decreased in all tested products when the strain exceeded over 20%.The highest value at the strain 200% was measured as Restylane (24.9 Pa.s) > Chaeum (23.1 Pa.s) > Neuramis (20.7 Pa.s) > Elravie (19.4 Pa.s) > Juvederm (15.6 Pa.s).In Juvederm, at 180 Pa for the values of the stress applied to the deformation of the HA fillers.Molding index power was determined as the difference of G* value between initials stage value and after deformation value.This theory based on that the sum of the rheological values and deformation basis for cohesion affect to the biological materials with high resistance property in elasticity and maintenance of their shape properly.In brief, the degree of change in complex moduli initial and after deformation was ∆G* 61.3 Pa on Elravie, ∆G* 68.0 Pa on Restylane, F I G U R E 3 Change of complex viscosity by increased strain (A) and comparative of complex viscosity at the strain 200% (B) in various cross-linked hyaluronic acid fillers.∆G* 91.5 Pa on Chaeum, ∆G* 113.0 Pa on Neuramis, and ∆G* 148.9 Pa on Juvederm.In addition, at the stress of 180 Pa, the strains were 79% on Chaeum, 90% on Restylane, 101% on Neuramis, 110%