EVERYTHING YOU WANT TO KNOWIR
FROM THE ENDOGENOUS MOLECULE TO INJECTABLE GELS
Denis COUCHOUREL, PhD.
Scientific and Medical Director of VIVACY Laboratories
Figure 1. hyaluronic acid monomer. The molecular weight of HA is therefore proportional to the number of disaccharides present. It is also a poly-anionic polymer, therefore very electrically charged.
It belongs to the group of glycosaminoglycans (GAG). Among these, HA is the only GAG not covalently associated with a protein, the only one not synthesized via the Golgi apparatus, and the only one that is not sulfated. Its molecular weight can reach 105 à 107 Daltons. In vivo, HA plays a crucial role in many processes such as: maintenance of viscoelasticity of biological fluids (synovial fluid, vitreous humor), control of tissue hydration, water transport, organization of proteoglycans in extracellular matrices, cell migration during embryonic development, development of inflammatory processes and wound healing. (Cowman et al, 2005). HA is detectable in many human tissues such as: the glazed mood, the cartilage, the synovial fluid, the skin, the mucous membranes (Almond A, 2007; Laurent UBG et al, 1991). From a quantitative point of view, in a 70 kg man we could find about 15 grams of HA, a third of which would be degraded (and therefore resynthesized) every day. It is thus a very fragile molecule with an intense and fast turnover (Figure 2) .
These functions can also vary according to the size (length) of the HA chain considered. A good illustration of this phenomenon has been demonstrated in the wound healing process: Figure 5: Differential role of HA based on its size in the wound healing process. Adapted from Garg HG and Hales HA, 2004. Figure 5 shows us that HA can have an inflammatory role characterized by an ability to promote the recruitment and migration of certain immune cells that are essential in the early phases of wound healing, or a function focused on the organization of extracellular matrices and their survival (vascularization, hydration)
Finally, HA is also a highly conserved molecule. During the evolution of species, the structure of hyaluronic acid has not changed. For example, it has been detected in the sub-branch of cephalochordates, which appeared more than 400 million years ago (Csoka et al, 2013). Because of this characteristic, HA molecules extracted from different animal sources are very well tolerated by the human body and do not provoke specific immune reactions.
However, this statement does not take into account the degree of purity of the HA used in the injectable preparations as well as any chemical modifications that the HA molecules may have undergone during the manufacture of these preparations. These are therefore key parameters that need to be very closely monitored by manufacturers of HA-containing drugs or medical devices, and will be discussed in the following paragraphs.
Exogenous hyaluronic acid: production.
1. Animal source.
Historically, the cock's crest is the first source of HA to have been really exploited at the industrial level. This organ contains up to 7.5mg of HA per gram of tissue. HA is mainly located in the subcutaneous fibrous zone of this organ. Prior to extraction, the ridges had to be washed with water and acetone until the solution became clear, to avoid enzymatic degradation and oxidation that would be deleterious to HA molecules. (Swann DA, 1968). Then, the tissues were crushed to enter the extraction phase. Different solvents were used such as distilled water at high temperature, saline solutions or mixtures containing organic solvents.
Yields of the order of 93% could be obtained when the temperature of the medium was brought to around 80 degrees Celsius. These conditions had the advantage of inactivating tissue hyaluronidases, but at the same time induced a partial depolymerization of HA since this molecule is temperature sensitive. It was therefore necessary to find the right compromise to obtain HA sizes compatible with the envisaged applications.
After extraction, the purification phase was obviously extremely important to ensure maximum biocompatibility. Indeed, proteins, peptides, nucleic acids, lipids and mucoplysaccharides were present in the extract. Although the methods used were essentially based on a series of precipitations (alcohol) / dissolutions (water), working from an animal source required in addition more complex techniques such as enzymatic hydrolysis or the use of organic solvents such as chloroform.Akasaka et al, 1985; Laurent TC, 1970These time-consuming, costly and less efficient protocols in terms of residual impurities led the industry to move towards production by biofermentation.
2. Bacterial source
In this system, HA is synthesized as an extracellular capsule by group A and C Streptococci bacteria. The cultures are carried out in a liquid medium, in reactors that allow for fine control of the growth conditions. The HA is therefore secreted into the culture medium in which the bacteria are immersed (Figure 6).
This production system is a sensitive process and a large number of parameters influence the size of the HA molecules synthesized. For example, dissolved oxygen level, substrate availability, mechanical stress due to agitation are all factors influencing the characteristics of the HA produced (Fallacara et al, 2018). Nevertheless, bacterial synthesis has many advantages over animal sources. First, the yield is much greater (Armstrong et al, 1997). Secondly, the quality of the material produced is far superior. From this point of view, 2 parameters are fundamental to consider: the molecular weight and the purity. Indeed, the size of the molecules will be an important determinant for the manufacturer of injectable gels since it is one of the parameters which will enable him to develop the rheology of his products. The purity must be understood as a prerequisite since we are talking about protein and nucleic residues resulting from the lysis of bacteria necessary for the manufacturing process. These are highly immunogenic molecules which, if they were to be found in too high a concentration in the injected gels, could be a source of intense inflammatory reactions.
The authorities, and in particular the EDQM (European Directorate for the Quality of Medicines & Healthcare) have, of course, set a number of standards that impose thresholds above which HA cannot be used for injectable products. In 2018, global bacterial production of hyaluronic acid reached 500 tons, including 230 tons of food-grade HA, 250 tons of cosmetic-grade HA, and only 20 tons of injection-grade HA. (https://cen.acs.org, 2018). These figures illustrate the technical difficulty of obtaining purity levels compatible with medical applications. 16 companies worldwide are currently certified to supply injectable manufacturers.
Manufacture and properties of injectable HA gels used in aesthetics
Between 2010 and 2017 the number of procedures that used HA injectable gels increased by 97% (https://www.isaps.org/). At the same time, manufacturers have worked to offer numerous technological innovations (Fagien et al, 2019).
Indeed, despite the fact that the products share the same injection sites, there are physicochemical and rheological differences that distinguish them, sometimes significantly. Rheology, the study of the behavior of a material when subjected to mechanical stress, is a direct consequence of the choices made by manufacturers of injectable gels in their manufacturing processes. More precisely, the concentration of HA, its molecular weight and the cross-linking intensity (as well as the technology used) are data that will directly impact the mechanical behavior of the gel. It is generally not easy to approach these topics from the rheological angle. Indeed, this science is complex and involves mathematical concepts that may not provide essential information to practitioners interested in the subject.
We will therefore first look at the needs and then draw a number of conclusions as to the properties necessary to determine whether or not an injectable product is suitable.
3. Adaptation of injectables to the injection plane
We have just described 2 characteristic environments in which the products will have to exert their effects throughout their 'active' life. It is now appropriate to recall that these gels are 'visco-elastic' materials. In other words, they all have a so-called viscous component (η) which allows them to spread out more or less, and an elastic component which allows them to resist an exogenous force and thus to return more easily to its original form (Heitmiller K et al, 2021). These 2 properties are illustrated by the figure 10.
Let us now transpose these 2 simple parameters to the descriptive data of the dermis and hypodermis developed above:
Dermis: a gel intended to be injected in this plane must have a low viscosity (η) given the high density of the extracellular matrix. This will ensure that the gel respects the histological structures and is minimally traumatic. It will have a strong tendency to spread. On the other hand, the dermis is by definition superficial. There will therefore be little pressure induced by the tissues between the gel and the skin surface. The product must therefore not be too elastic, otherwise it will cause over-correction, which is harmful to the expected aesthetic results.
Hypodermis: the reasoning is reversed. The gel is injected into a tissue with a much less dense extracellular matrix. Its viscosity (η) must then be high so that it can remain as much as possible in the area where the practitioner wishes to place it. Adipose tissue is also deeper than the dermis. The gel will therefore have to cope with much more compression and its elasticity must be adjusted accordingly. If this is not the case, the product will tend to crush under this force and will not be able to induce the desired volume creation on the surface.
A third parameter is important to consider: cohesiveness. This can be defined by the ability of the material to dissociate or not. In other words, depending on the technology used, HA molecules will have more or less affinity for their neighbors (Sundaram H. et al, 2015). This results in a different histological distribution in the tissues after injection.If the cohesivity is low, the product will tend to split into a myriad of micro-boluses separated from each other.Conversely, if the cohesivity is high, the product will appear to be placed in one piece in the target tissue, in a homogeneous manner. Cohesivity is therefore an important factor to consider in combination with viscosity with respect to respecting and preserving the intimate structures of the extracellular matrix (Tran C et al, 2014). Optimal biointegration must also logically constitute a key notion for product safety.
There are, of course, other constraints inherent in the movements caused by muscle contractions, making the definition of the ideal rheology for an injection plane more complex than the combination of elasticity, viscosity and cohesiveness (Gavard-Molliard S et al, 2018). Nevertheless, the reasoning developed above forms the basis for a coherent and logical range of injectable products.
4. How can the rheology of a gel be varied?
Three main parameters can be manipulated to achieve the desired rheology.
Cross-linking: The initial purpose of this gel manufacturing step is to increase its in vivo life span. Indeed, we have seen above that endogenous hyaluronic acid is a very fragile molecule with a low half-life. To increase its life span, the cross-linking reaction allows the HA chains to be brought closer together. To do this, it is necessary to use a reactive molecule, capable of creating covalent bonds between 2 HA molecules. The overwhelming majority of products available on the market use 1,4-butanediol diglycidyl ether (BDDE) for this reaction.
It is also possible to use other molecules, but these must necessarily present a strong chemical activity to be able to react with HA. This is for example the case of polyethylene glycol (PEG) which must be functionalized to become PEGDE (polyethylene glycol diglycidyl ether) before being used (Zerbinati et al, 2021). When cross-linking is performed, the space between HA molecules becomes small to the point of hindering the access of endogenous hyaluronidases. But these three-dimensional modifications also have a very strong impact on rheology. Indeed, the mesh size of the created network decreases as the cross-linking rate increases. The elasticity and viscosity are then strongly increased.
Focus: When HA is in aqueous solution at physiological pH, the carboxyl groups are dissociated and the polymer carries a very large number of negative charges. Consequently, it is able to attract most osmotically active cations (Sodium, potassium, calcium...), but also water molecules.
Thus, HA can bind via weak bonds more than 1000 times the weight of water in the considered HA chain (Khabarov et al, 2015). Because of this property, HA chains occupy an extremely large volume and are able to form gels even at low concentrations. If this concentration increases, then the viscosity of the solution increases, especially due to the formation of a three-dimensional network based on the formation of weak electrostatic bonds. It is therefore a simple way to act on the rheology of gels. It is therefore also a factor that will increase the hygroscopicity of the gel considered.
The molecular weight of HA molecules: Again, this parameter will influence the density of the network forming the gel. The larger the size of the HA chains, the more important their folding is, causing a drastic increase of the viscosity.
Gel manufacturers must therefore master the differential effects of these three parameters. For example, it is possible that a particular constraint imposes to decrease or increase one of these factors (the concentration for example, in order to modify the hygroscopy), but it will then be largely possible to maintain the initial rheological behavior by playing on the two other parameters. This is why it is not possible to base the behavior of a gel on the concentration value alone and to conclude, for example, that it is very volumizing and suitable for fatty tissue.
Hyaluronic acid is thus an endogenous molecule essential to extracellular matrices and with remarkable properties which are exploited by the organism in very precise compartments (vitreous humor, articular cavity?).
It is also a biologically active molecule involved in many signaling processes. It is highly conserved across species, which has allowed us to develop human applications from bacterial production systems, thus reliable and reproducible.
Among these uses, aesthetic medicine is at the forefront, since it is in this specialty that the challenges for manufacturers are the most demanding. Indeed, the injected tissues are histologically very diverse, the level of anatomical and physiological complexity of the face is important, and the tolerance to side effects is very low.
Thus, the rheological behavior of the gels had to be further developed and refined by the manufacturers to constantly strive for the best possible bio-integration while aiming at the best possible clinical performance.