With an increasing number of gel-based dermal fillers on the market, there is an increasing interest in correlating the different properties, such as rheological and cohesive properties, with the clinical effects and uses. The rheological properties of hyaluronan (HA) fillers have been explored for quite a long time, but more recently the discussions have been focused around the cohesive properties of HA fillers.
Cohesion is described as the force between particles of the same substance that acts to unite them. In other words, it is easy to separate the particles in a low-cohesive material but more difficult in a material with high cohesion. In IUPACs Golden book,1
The work of cohesion per unit area of a single pure liquid or solid phase α,wα c is the work done on the system when a column α of unit area is split, reversibly, normal to the axis of the column to form 2 new surfaces each of unit area in contact with the equilibrium gas phase.
Currently, there are no ready-made instruments designed to measure cohesion, or methods which could be easily adapted to gels. Several manufacturers of HA gels have investigated their cohesive properties using various different methods but as yet, unlike rheology, there is no standardized methodology that is acknowledged by the scientific community as a valid way of evaluating cohesion.
In a vast majority of articles where investigators state gels to be cohesive, no explanation is given on how this was concluded. It seems that the appearance and general behavior of the gel are often considered sufficient to give an estimate of this property. Because of this, it is of interest to include sensory analysis into comparison of methods for cohesivity measurement.
There have been suggestions that the different physicochemical properties of a gel relate to specific clinical effects, e.g., the effect of the gel strength, i.e., the elastic modulus and/or the complex modulus as measured by rheology, has been said to relate to lifting capacity and tissue integration. Several authors suggest that firm gels have a better ability to resist deformation,2–6 2–6 2–6 2–6 2–6 7
The suggested effects from cohesion are more diverse. Falcone and Berg8 6,9 6,9 10,11 10,11 9
To study and compare the possible clinical effects from the cohesive properties of a gel, it is necessary to standardize the definition and measurement of cohesion. The purpose of this study was to develop and evaluate methods for determining cohesion. Two different families of HA fillers (Emervel and Restylane [Galderma Aesthetics]) were used to evaluate the different methods.
Materials and Methods
Materials
Several batches of Restylane (RES), Restylane Perlane in US Restylane Lyft (PER), Restylane Vital in US Restylane Silk (RESV), Emervel Touch (EMET), Emervel Classic (EMEC), Emervel Lips (EMEL), Emervel Volume (EMEV), and Emervel Deep (EMED) were used. To replicate earlier published results from the compression force method Juvederm Ultra 2 (JUVU2) (batch X24L703701), Juvederm Ultra 3 (JUVU3) (batch X30L901494), and Juvederm Ultra 4 (JUVU4) (batch s30L804422) were used.
Rheology
The rheological properties were measured in a frequency sweep from 10 to 0.1 Hz at 0.1% strain (the strain is within the linear viscoelastic region). The measurements were made using an Anton Paar MCR 301 (Anton Paar, Graz, Austria) equipped with a PP-25 measuring system with a gap of 1 mm at 25°C. A 30-minute period was used for relaxation of the sample between loading and measuring. Two measurements were performed per sample.
Perceived Cohesion
To enable a correct judgement of cohesion by sensory analysis, it is important that the persons evaluating the properties are familiar with the concept. The evaluation was made in 2 groups, 1 consisting of 6 scientists and 1 consisting of 79 healthcare professionals (HCP). The 6 scientists were a group who had looked into cohesion and possible methods to measure cohesion over a longer period of time, and were thus well familiar with the concept. The 79 HCPs were first shown the definitions of cohesion and then asked to grade the 3 gels accordingly.
Perceived Cohesion 1
A test panel of 6 scientists performed a blinded sensory analysis of 8 different HA gels. The test was done by manually handling the gels and rating the gels on a scale of 1 to 5 (where 5 is the highest cohesion).
Perceived Cohesion 2
A panel of 79 HCPs performed a blinded sensory analysis of 3 different gels: (1) EMET, (2) EMED, and (3) PER. Before the test, the HCPs were presented with 3 different dictionary definitions of cohesion.
(1)The act or state of sticking together tightly12
(2)The sticking together of particles of the same substance13
(3)The molecular force between particles within a substance that acts to unite them14
The HCPs were asked to grade the gels on a scale of 1 to 5 (where 5 is the highest cohesion).
The results from the sensory analysis were used as a reference value for the other methods used in the study.
Compression Force
The measurements of the force during compression were made using an ARES G2 (TA-instruments, Newcastle, DE) equipped with a 25 mm plate–plate measuring geometry at a temperature of 25.0°C. A 1 mL sample was centered on the lower plate. After loading the sample, the upper plate was lowered to a position of 2.5 mm above the lower plate and the sample was allowed to relax for 120 seconds. The force was then measured every second while lowering the upper plate at a rate of 0.0133 mm/s to reach a 0.9 mm gap after 2 minutes.
Dispersion Time
The dispersion time methodology is based on that described by Sundaram.15
Each photograph was graded by 5 evaluators, who were blinded to the individual time points and samples shown in each photograph, on a scale of: 1 = fully dispersed; 2 = partly dispersed; 3 = partly dispersed/partly cohesive; 4 = partly cohesive; 5 = fully cohesive.
Drop Weight
The weight of an average fragment/drop of a HA gel that is pushed through a defined vertical orifice at a constant speed was determined. The gel was filled in a 1 mL BD glass syringe (Becton, Dickinson and Company) and the air was removed by centrifugation. An 18 G cannula with a plane orifice (Intramedic Luer-Stub Adapter 18 G; Becton, Dickinson and Company) was mounted on the syringe and with the use of a Zwick material tester, Zwick BTC-FR 2.5 TH.D09 (Zwick Roell), the gel was extruded at a constant speed of 7.5 mm per minute. When a constant force was achieved, 10 fragments/drops were collected and weighed and the average weight was calculated.
Results
Rheological Properties
The rheological properties of a gel are often described using the elastic modulus, G′, and the viscous modulus G″. Sometimes the complex modulus, G*, is also used, G* is a measure of the total resistance to deformation. A firmer gel has a higher elastic modulus meaning that the response to deformation is mainly elastic. Softer gels have lower elastic modulus and the ratio of elastic to viscous behavior is usually lower than for a firmer gel.
The gels used for evaluating the different cohesion methods are the Restylane and the Emervel families. To characterize the gels used in the study, the rheological properties were first measured. The results from the frequency sweeps are shown in Figure 1 . RES was the first HA gel on the market. RES and PER are firmer gels with a high elastic modulus. The Emervel family is a more recent family of HA fillers ranging from semifirm to soft with lower elastic modulus compared with RES and PER. The samples used for this investigation cover a large range in rheological properties with an elastic modulus from ∼25 to 800 Pa at 1 Hz.
Figure 1: Rheological properties of the samples used.
Perceived Cohesion
The results from the sensory evaluation are shown in Figure 2 . The Emervel family of products was perceived to be more cohesive than RES and PER. The perceived cohesion judged by the 79 HCPs was reasonably consistent with the judgements made by the 6 scientists.
Figure 2: Perceived cohesion; dark blue HCP perception (n = 79), light blue scientist perception (n = 6).
The results were unexpectedly consistent between the participants, given that no detailed instructions had been given on how to assess the level of cohesivity, apart from the definitions given in the methods section. Apparently sensory analysis, i.e., manual handling and observation of the gel, yields a reasonable estimate of the cohesivity of an HA filler. This may explain why many investigators state that products they have studied are cohesive without explaining how this was determined. The drawback of sensory analysis is the need for simultaneous comparison with other products, and the low resolution of scoring procedures compared with instrumental analysis.
Compression Force
It has been suggested that compression force is dependent on the cohesive properties of a material.6,9,16 6,9,16 6,9,16 6,9,16 6,9,16 6,9,16 Figure 3 .
Figure 3: Validation of compression force methodology from Bourdon and coworkers
16 (A) and Borrel and coworkers
6 (B).
The compression force used as a measure of the cohesion is the maximum force during the measurements or the area under the curve when plotting the force versus time. The results shown in Figure 3 are difficult to compare with those in the previously mentioned studies, as either the original methods were not described in sufficient detail6 16 Figure 3 suggest that the method used in this study produced similar results to those published.
The methodology was then repeated using the Emervel and Restylane products. The results are shown in Figure 4 . EMET and PER demonstrate almost the same compression force although EMET was perceived to be much more cohesive than PER in the sensory analysis (Figure 2 ) (see also Figure 5 for difference in behavior/cohesion between EMET and PER). When the compression force data are plotted against the perceived cohesion (Figure 6 ), it is obvious that the compression force method does not correlate with the perceived cohesion.
Figure 4: Compression force of study samples, n = 3, error bars show ±SD. The compression force data evaluated as the area under the force–time curve as suggested in Bourdon and coworkers
16 gives the following values EMET 15 ± 1.4, EMEC 30 ± 0.2, EMEL 30 ± 1.0, EMED 36 ± 0.4, EMEV 38 ± 1.1, RES 12 ± 1.0, and PER 17 ± 0.3.
Figure 5: The behavior of PER (left) and EMET (right) when trying to lift the gel using a glass rod.
Figure 6: Correlation between compression force and perceived cohesion.
Dispersion Time
The time taken to disperse a filler in water has been suggested as an indication of cohesivity.15 Figure 7 (only 3 gels are shown to illustrate the different behaviors). Two of the extremes were EMET and PER. PER was quickly dispersed and was judged to have a low cohesion at each time point. EMET was rated as the most cohesive gel at the beginning of the dispersion and the least cohesive gel after 10 minutes. As can be seen in Figure 7 , the change in the dispersion pattern of EMET was rapid and the gel was almost completely dispersed after 5 minutes. As EMET was perceived to be one of the most cohesive gels in the sensory analysis (Figure 2 ) it can be concluded that the longer dispersion times do not correspond to perceived cohesion. The relationship between the dispersion assessments at the shorter times of 15 and 95 seconds and perceived cohesion is shown in Figure 8 . There does seem to be a correlation at the shortest time but the correlation is not as good for products that are perceived to be low cohesive products.
Figure 7: Photographs of dispersion times.
Figure 8: Correlation between subjective evaluation of dispersion photographs at 15 and 95 seconds and perceived cohesion.
Drop Weight
The IUPAC definition of cohesion states that the work of cohesion is the work done when a column is split normal to the axis of the column to form 2 new surfaces, and the drop weight test would appear the best way to test this.
In the drop weight method a column of material is formed by extrusion through a cannula; when the weight of the extruded column is larger than the cohesion the column will break normal to the axis of the column, and 2 new surfaces will be formed (Figure 9 ). The weight of the detached fragment/drop is then proportional to the work of cohesion. The results from the analysis of drop weight test are presented in Table 1 and the correlation of the drop weight with perceived cohesion is shown in Figure 10 . The drop weight method correlates well with the perceived cohesion although EMET had a very high drop weight (Figure 10 ).
Figure 9: The drop weight method.
TABLE 1: Drop Weight
Figure 10: Correlation between drop weight and perceived cohesion.
Discussion
In relation to the different methods of assessment it is clear that, in this instance, the compression method did not reflect the cohesive properties of the gels as evaluated by sensory analysis.
With regard to the dispersion time, there were several drawbacks with this method. It was noted when performing the experiment that the gel could sometimes adhere to the beaker and was quite difficult to see in the photographs. This method relies on subjective assessment of photographs and it was difficult to determine whether there were well-dispersed large particles or crowds of small particles held together by cohesion. As the results are clearly time-dependent there is room for experimental error and difficulty with reproducibility of the experiment.
The advantages of the drop weight method are that it is not time dependent or subjective and is more easily reproducible. Furthermore, it would seem to more closely reflect the IUPAC definition of cohesion. In this study, the results from the drop weight test correlate well with the perceived cohesion.
In this study, the gels were characterized by their rheological properties. Even though there is no detailed knowledge of all properties of a gel that affect the rheological properties and to what extent, the knowledge about the factors that affect rheology is much greater than the knowledge about factors affecting cohesion. It is possible that one or several of those factors/properties are common to both cohesion and rheology. To investigate the correlation between cohesion and rheology, the results from the drop weight test and the sensory evaluation were plotted against the elastic modulus, see Figure 11 . There seems to be an inverse relationship between gel strength and cohesion and if future investigations support that relationship between rheological properties and cohesion there may be no need for a separate method to measure cohesion.
Figure 11: Correlation between elastic modulus and the drop weight (A) and elastic modulus and perceived cohesion (B).
The inverse relationship between gel strength and cohesion could perhaps explain why the more cohesive products are said to integrate better into the tissue,10,11 10,11
Conclusion
When discussing the clinical effect from cohesion or when looking for relationships between cohesion and clinical effects from different products, it is preferable to have a measure/a value for cohesion. Several methods to measure cohesion have been tested. The drop weight method which closely resembles the IUPAC definition of cohesion1
References
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