Introduction
Hyaluronic acid (HA) gels were first introduced in 1996 and since then, bovine collagen was replaced by HA gels for aesthetic procedures in daily practice. HA has an identical chemical structure in its native form among various species. In addition, HA has an exceptional physicochemical and viscoelastic characteristics, which makes it suitable for the development of various HA-based medical products [7]. However, endogenous HA has a short half-life and therefore, it is necessary to increase its half-life by the introduction of chemical modifications via a crosslinking process. This process alters the 3D structure of the chains of HA and leads to the development of gels with various characteristics. Two steps are basically used for HA crosslinking. At first, HA is dissolved and linearized in a medium with an alkaline pH. Then, a crosslinker is added to the medium with temperature control. The rheologic, mechanical, and pharmacological characteristics of HA gels vary in terms of their specific crosslinking technologies [6]. Differences in the final amount of crosslinked HA in the HA gels influence the behavior of the gels; therefore, the technique and depth of injection should be selected based on the HA gel. To evaluate the various characteristics of selected HA gels available in Iran, using simple, inexpensive, and reproducible experimental tests are necessary to better understand their appropriateness for specific indications.

Methods
In this study, the HA gels produced by various crosslinking technologies were evaluated by three experiments, including microscopic examination, cohesivity test, and resistance to stretch test. The gels subjected to these experiments in this study included Neuramis, Desirial, Belotero, Restylane, and Juvéderm. In the microscopic examination test, each gel was stained with toluidine blue and then the structure of the stained gel was examined under the microscope. In the cohesivity test, saline solution was mixed with Ecoline, and the mix was added to the HA gel. Then, ethanol was added; photos were captured before and after this addition to evaluating the cohesivity maintenance of the gel. Omnican syringes were used to examine the gels. In the resistance to stretch test, Adson’s plier was used to stretch the gel, and the length was measured. 

Results
Microscopic examination: All gels in this experiment showed differences in their viscosity during the slide preparation in the microscopic examination test. This difference was significant, especially during the spreading on the slide. Microscopic examination of the HA gels showed HA particles of varying distribution and size. Belotero and Desirial were the gels with no particles visible under the microscope. The microscopic structure of Neuramis was similar to Restylane. Neuramis was a biphasic gel. Desirial was shown to be a monophasic gel without any particles under the microscope. Belotero was similar to Desirial under the microscope. As a monophasic gel, Belotero was shown to be a network without particles. Microscopic examination of the HA gels showed HA particles of varying distribution and size. 
Cohesivity test: None of the gels were dispersed upon contact with the saline solution, and ethanol addition did not increase their dispersion. We found that cohesivity was different among the gels so that Desirial was the least cohesive and Belotero was the most cohesive gel. Other HA gels were observed as partially cohesive.
Resistance to stretch test: It was impossible to stretch Neuramis and Desirial gels more than 0.6 cm without breaking. These gels have lidocaine and antioxidant, which may influence their viscoelastic properties. Belotero had no lidocaine or antioxidant and was stretched more than 2.5 cm without breaking. The resistance to stretch test confirmed the cohesivity results and Belotero had the most significant resistance. 

Discussion 
Different crosslinking technologies are used for the production of HA gels. These technologies influence the viscoelastic properties of the final product. NASHA is the technology used for the production of Restylane gel. In this technology, 1, 4- butanediol diglycidyl ether (BDDE) is used as the crosslinking agent in the manufacturing process of NASHA technology. This agent leads to few crosslinks among the chains of polysaccharides. The final partially viscose matrix is then dried. Using filters, the final suitable particles are obtained for the product’s clinical indication [10]. The technology used for the production of Juvéderm gel is called Vycross. In this technique, the final product contains a high amount of low molecular weight HA compared to the high molecular weight HA. The low and high molecular weight molecules are mixed through a single step called crosslinking process [9]. CPM is the technology used for the production of the Belotero gel. In this technology, two more steps are performed in addition to the classic crosslinking process, which leads to a poly-dense monophasic product, in which the crosslinked HA is maintained within a cohesive matrix [11]. SHAPE is the technology used for the production of Neuramis gel. This technology involves two crosslinking steps and an additional purification step, which removes the non-crosslinked BDDEs [10]. Simple and inexpensive laboratory tests help to understand the way that they can affect the performance of a specific gel and may be beneficial for physicians to decide on the best product for a particular therapeutic indication.

Ethical Considerations

Compliance with ethical guidelines
This study was approved by the ethics committee of the Iran University of Medical Sciences (Code: IR.IUMS.REC.1399.1330)

Funding
The present study was funded by the Deputy for Research and Technology of Iran University of Medical Sciences.

Authors' contributions
Conceptualization, study design and drafting of the manuscript: Negar Mottaghi-Dastjerdi; Acquisition, analysis, and interpretation of data: Mehdi Aghsami; Critical revision of the manuscript for important intellectual content: Marjan Shariatpanahi and Hamed Montazeri; Study supervision: Negar Mottaghi-Dastjerdi and Marjan Shariatpanahi 

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
The authors would like to thank the Iran University of Medical Sciences.
 

1. Olenius M. The first clinical study using a new biodegradable implant for the treatment of lips, wrinkles, and folds. Aesthetic Plastic Surgery. 1998; 22(2):97-101. [Link]
2. Monheit GD, Coleman KM. Hyaluronic acid fillers. Dermatologic Therapy. 2006; 19(3):141-50. [DOI:10.1111/j.1529-8019.2006.00068.x] [PMID]
3. Micheels P. Human anti-hyaluronic acid antibodies: Is it possible? Dermatologic Surgery. 2001; 27(2):185-91. [DOI:10.1046/j.1524-4725.2001.00248.x]
4. De Boulle K, Glogau R, Kono T, Nathan M, Tezel A, Roca-Martinez JX, et al. A review of the metabolism of 1, 4-butanediol diglycidyl ether-crosslinked hyaluronic acid dermal fillers. Dermatologic Surgery. 2013; 39(12):1758-66. [DOI:10.1111/dsu.12301] [PMID] [PMCID]
5. Edwards PC, Fantasia JE. Review of long-term adverse effects associated with the use of chemically-modified animal and nonanimal source hyaluronic acid dermal fillers. Clinical Interventions in Aging. 2007; 2(4):509-19. [DOI:10.2147/CIA.S382] [PMID] [PMCID]
6. Tavsanli B, Okay O. Mechanically robust and stretchable silk/hyaluronic acid hydrogels. Carbohydrate Polymers. 2019; 208:413-20. [DOI:10.1016/j.carbpol.2018.12.088] [PMID]
7. Huang G, Chen J. Preparation and applications of hyaluronic acid and its derivatives. International Journal of Biological Macromolecules. 2019; 125:478-84. [DOI:10.1016/j.ijbiomac.2018.12.074] [PMID]
8. Aboutaleb E, Manoochehri S. [Bioequiwin: Design of a bioequivalence software (Persian)]. Journal of Guilan University of Medical Sciences. 2019; 27(108):35-44. [Link]
9. Micheels P, Sarazin D, Tran C, Salomon D. Effect of different crosslinking technologies on hyaluronic acid behavior: A visual and microscopic study of seven hyaluronic acid gels. Journal of Drugs in Dermatology. 2016; 15(5):600-6. [Link]
10. Edsman K, Nord LI , Öhrlund Å, Lärkner H, Kenne AH. Gel properties of hyaluronic acid dermal fillers. Dermatologic Surgery. 2012; 38(7):1170-9. [DOI:10.1111/j.1524-4725.2012.02472.x] [PMID]
11. Bezzola A, Micheels P. [Esthélis®, acide hyaluronique de conception Suisse. Première etude complète des caractéristiques physico-chimiques et essais cliniques (French)]. Journal de Médecine Esthétique et de Chirurgie Dermatologique. 2005. 32(125):11-20. [Link]
12. Flynn TC, Sarazin D, Bezzola A, Terrani C, Micheels P. Comparative histology of intradermal implantation of mono and biphasic hyaluronic acid fillers. Dermatologic Surgery. 2011; 37(5):637-43. [DOI:10.1111/j.1524-4725.2010.01852.x] [PMID]
13. Micheels P, Besse S, Flynn TC, Sarazin D, Elbaz Y. Superficial dermal injection of hyaluronic acid soft tissue fillers: Comparative ultrasound study. Dermatologic Surgery. 2012; 38(7pt2):1162-9. [DOI:10.1111/j.1524-4725.2012.02471.x] [PMID]
14. Lemperle G, Gauthier-Hazan N, Wolters M, Eisemann-Klein M, Zimmermann U, Duffy DM. Foreign body granulomas after all injectable dermal fillers: Part 1. Possible causes. Plastic and Reconstructive Surgery. 2009; 123(6):1842-63. [DOI:10.1097/PRS.0b013e31818236d7] [PMID]
15. Azizi Khereshki N, Aryafar S, Eftekhari H, Aboutaleb E. [Formulation and in vitro evaluation of dapsone topical gel 5% (Persian)]. Journal of Guilan University of Medical Sciences. 2020; 29(1):62-75. [Link]