EVERYTHING YOU WANT TO KNOWIR

HYALURONIC ACID:

FROM THE ENDOGENOUS MOLECULE TO INJECTABLE GELS

                                   

Denis COUCHOUREL, PhD.  

Scientific and Medical Director of VIVACY Laboratories

Endogenous hyaluronic acid In 1934, Karl Meyer and John Palmer described a new polysaccharide initially isolated from of bovine vitreous humor. This substance contained a uronic acid and an amino sugar, so it was named hyaluronic acid (HA). During the following years, HA was isolated from many sources: the vitreous humor, the human umbilical cord and the cock's crest (Fuoss et al, 1948). In 1937, Kendall published an article describing the presence of AH in the capsule of the bacteria Streptococci of groups A and C (Kendall et al, 1937). This work was to revolutionize the use of hyaluronic acid many years later, since bacterial fermentation is still the most reliable and safest source of HA available to us today for the development of medical applications. During the same period, other observations allowed to highlight the existence of a molecule capable of drastically increasing the permeability of connective tissues by causing the depolymerization of HA.  (Duran-Reynalds, 1929). Thus, the study and use of hyaluronidases has provided a better understanding of the primary structure of HA. It was then the Hungarian scientist Dr. Endre Balazs, who definitely gave impetus to the applicative research on HA. In particular, he was the first to describe that the extracellular matrix of the articular synovial membrane contained enough viscous HA to replace intra-articular fluid (Balazs et al, 1943), thus paving the way for all subsequent work that led to the first robust medical application of hyaluronic acid injections viscosupplementation of joints. Since then, other avenues have been explored in ophthalmology, visceral surgery and, of course, in aesthetic medicine. These applications mainly take advantage of the particular rheological properties that can be obtained from HA gels Hyaluronic acid is a polymer composed of disaccharides, whose base unit repeated n times, is composed of a gluconic acid and an N-acetyl glucosamine (Figure 1)

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) .

Thus, the half-life of endogenous HA in extracellular matrices never exceeds a few weeks. In the skin specifically, the amount of HA measured is 0.5 mg/g in the dermis and 100 mg/g in the epidermis (Reed et al, 1988) with a half-life of 1.5 days (Tammi et al, 1991). It is synthesized by the "hyaluronan synthases". which form a group of 3 isozymes (HAS-1, HAS-2 and HAS-3). They function differently as they are able to synthesize molecules of different molecular weights.(Gupta, 2019) . HA is localized in the extracellular compartments mostly, and obviously has a critical role in the regulation of water homeostasis and hydration of these tissues. It should be noted in passing that although the epidermis is a specialized connective tissue made up of an abundant extracellular matrix and relatively few cells, it is in the epidermis (where keratinocytes are proportionately very numerous) that the concentration of HA is the highest. The skin is an organ of protection against stimuli such as light, UV, pollutants and other oxidative stresses. In this context, the HA of the epidermis could play a role of absorber of the free radicals induced by these external aggressions, thus protecting the structures which are adjacent to it (collagen and cellular membranes in particular). 7 to 8g (50% of the total amount) are specifically detected in the skin (Hascell et al, 1997). In addition to these intrinsic functions (hydration and anti-oxidative protection), it is important to emphasize that a certain number of HA-specific receptors have been characterized over the last 15 years. These molecules have been broadly referred to as hyaladherins. Here is the classification.
Figure 3 shows us that HA molecules are able to bind to about fifteen receptors, whether they are transmembrane, membrane (i.e. mobile on the cell surface), bound to proteins and GAGs of the extracellular matrix or dissolved in the interstitial fluid. Some of them, such as RHAMM, have been very well characterized and even cloned thanks to the work of Pr Eva Turley in 1992 (Turley E, 1992). Figure 4 summarizes the functions that we have been able to elucidate to date.

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.

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. Globally, 3 types of injections are currently performed in medical practices, regardless of the indication considered: very superficial injections (medium to superficial dermis) in order to improve the quality and appearance of the skin, dnjections average(deep dermis and superficial hypodermis) in order to correct wrinkles and fine lines, and des deep injections (hypodermis or deep fat) in order to create or restore volumes. It is therefore important to discuss some biomechanical aspects of these different target tissues in order to understand the rheological parameters. 1. The dermis The dermis is a dense, irregular, specialized connective tissue with a tight network of collagen and elastin fibers. (Leeds University Histology Guide). Collagen alone represents 75% of the dermis (dry weight). 28 types of collagens have been identifiedbut it is of course type I collagen which is the majority in the skin, types III, IV, VII, XIII and XIV are also present (Nemoto T et al, 2012). From a mechanical point of viewIt is also interesting to note that type XIV belongs to a subgroup of the collagen superfamily: the FACIT collagens (Fibril Associated Collagen with Interrupted Triple helixes) (Shaw LM et al, 1991). Their chemical nature is different from that of fibrillar collagens. On the other hand, by associating with type I molecules, they reinforce the mechanical strength of the matrices in which they are present (Nemoto T et al, 2012). More precisely, it is possible to measure this resistance by evaluating the Young's modulus. This calculation describes the capacity of a material to resist a deformation when a force is applied to it (McKee et al, 2011). Yet, in the case of the dermis, it is noted that resistance increases sharply as stress increases. This exceptional behavior is typical of collagen-rich content (Licup AJ et al, 2015). It should also be noted that more collagen fibers are organized and aligned, the greater the maximum tension value beyond which the fibers are irreparably damaged (Aziz J et al, 2016). Elastic fibers are a group of matrix proteins that are synthesized by the fibroblast through a complex process called elastogenesis based on a precursor, tropoelastin. In the same way that collagen is essential to give the skin its tensile strength, elastin gives it the property to expand and then return to normal without suffering irreversible mechanical damage (Rauscher S et al, 2012). This type of fiber is abundant in the dermis, and its importance is illustrated when studying, for example, the symptoms of patients with cutis laxa (a rare disease that affects the elastic fiber assembly system).  In addition to severe damage to the respiratory, digestive and vascular systems, the skin of these patients appears limp, unresponsive and prematurely aged (Andiran N et al, 2002). These data therefore illustrate the predominant role played by the elastin fiber network in the biomechanical properties of the skin. Collagen and elastin are the main elements of the extracellular matrix of the dermis. It can therefore be described as extremely dense (as illustrated in Figure 7). Thus, an injectable hyaluronic acid gel dedicated to this injection plan must necessarily be rheologically adapted to this particular environment. We will see later on how.
2. The hypodermis The subcutaneous adipose tissue is a connective tissue located between the dermis and the muscular aponeuroses. Histologically, it is a loose association of adipocytes filled with a lipid droplet (90 to 99% of triglycerides) and 2 to 3% of proteins. The diameter of these cells is important: 30 to 70µm (Avram AS et al, 2005). They are organized in fatty lobules separated from each other by connective tissue septa. Compared to the dermis, very few authors have devoted themselves to biomechanics of the adipose tissue, but a number of studies have shown that the Young's modulus of fatty tissue can vary from 0.5 to 25 KPa (Kilo Pascal) (Gefen A et al, 2007). It is interesting to compare these values to the 21 to 39 -MPa (Mega Pascal) measured on the dermis (McKee et al, 2011) since they are much lower. These results therefore imply an extremely different mechanical behavior of the dermis, which must necessarily be explained by fundamental structural differences in the extracellular matrix. When adipose tissue is observed by scanning electron microscopy, these differences become even more apparent. The Figure 8 is taken from an article (Panetttiere P et al, 2011) whose authors sought to highlight the ultra-structural characteristics of the trochanteric fat pad. Indeed, it is a potential source of interesting fat for autologous grafts, including at the facial level (Raskin BI, 2009). The fibrillar network supporting the adipocytes thus appears much less dense and provided than that which can be found in the dermis. The mechanical behavior of the hypodermis will therefore not only be very different from that of the dermis, but its structure will also impose an adaptation of the hyaluronic acid gels that will be injected there.

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.

Conclusion

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.

Aesthetic health based on scientific evidence

Sign up to view this latest issue and receive future issues of LM