In response to the increasing request for minimally invasive non-surgical procedures, scientific research has led to new injectable materials with high efficacy and little or no side effects. To this extent, the ideal injection filler should be safe and effective, biocompatible, non-immunogenic, easy to distribute and store, and should not require allergy tests.


HA is a natural biopolymer which has unique and incomparable chemical-physical properties and is naturally present in soft connective tissues, extracellular tissues, skin, dermis, vitreous eye, hyaline cartilage, synovial articulation fluid, disk core and umbilical cord.

Thanks to its versatility, biocompatibility and biodegradability, along with its molecular features and its unique viscoelastic nature, HA has found diverse medical, pharmaceutical and cosmetic applications.


HA (as well as its sodium salt) is a linear bio-polysaccharide which belongs to the class of glycosaminoglycans and is composed of repeated disaccharide units, made up of Nacetyl-D-glucosamine and D-glucuronic acid, bound by a
β-1,4 glycosidic acid bond, while disaccharides are bound by β-1,3 glycosidic ties (►Figure 1).

Figure 1

The different molecular weights of the Hyaluronic Acid determine its chemical-physical and biological properties, allowing several applications both in pharmaceutical and cosmetics products.

Most HAFillers contain the high-molecular-weight form of this polymer (≥1MDa). In aqueous solutions HA forms an extended hydrogen-bonded system, both intramolecularly and intermolecularly; these H-bonds – along with the hydrophobic interactions which arise from HA hydrophobic patches – will cause the polymer to rearrange in a self-aggregate, physical cross-linked system. These intra- and inter-chain interactions are responsible for the formation of a temporary network structure, in the form of a reversible gel.

These are the basis of the gel shear-thinning and self-healing properties; the gel undergoes a reversible destructuration, upon a shear, of the transient polymer network. Upon cessation of the shear, the hydrogel is able to autonomously reassemble and recover its initial viscosity. The rheologic properties of HA solutions allow a simple application of the product and its rapid immobilization at the target site.


The main limitation of HA, when used in its free form (non cross-linked), is its poor durability in situ due to the rapid enzymatic degradation which will take place in 1 to 2 days after the treatment. In order to increase the viscosity, extend the in situ permanence and reduce the susceptibility to enzymatic degradation, HA is often subjected to cross-linking.  The cross linking agents are dysfunctional molecules of synthetic origin, such as divinyl sulfone and BDDE. The two main variables in HA cross-linked fillers are represented by the amount of HA cross-linked chains along with the cross-linking degree (% of cross-linking agent, meaning the number of cross-linking molecules for every 100 disaccharide monomeric units of HA). At a given concentration of Hyaluronic Acid, the lower the amount of free polymer chains, the higher are the viscosity (meaning the gel’s resistance to be deformed) and the gel cohesivity (meaning its tendency to stick together and hold its form or shape under stress). Cohesivity increases at higher HA concentrations, as well as with a more elevated cross-linking degree.


Parameters such as the elastic modulus G′ or viscosity, cross-linking degree, and cohesivity affect the extrusion force as the gel is subject to forces of injection through a fine needle. To this extent, an increase in viscosity may result in making injections more difficult.

However, a FILLER requires a cohesivity sufficiently high to resist to compression forces which, after its injection into the skin, could potentially provoke its migration.

Another parameter which affects the rheologic properties of the Filler is the gel particle size. After the cross-linking procedures, FILLERs are sized in order to allow injection into the skin. A filler produced with this technique is characterized by a high viscosity value and by particles of a well-defined average size.

The consistency also influences the degradation rate of HA: bigger HA chains offer limited total surface area for enzymes to degrade them, whereas smaller HA molecules offer more total surface area for the enzymes to break them down easily.


Usually, FILLERs are not responsible of relevant side effects: specifically, HA can cause allergic reactions under the form of transient erythema, edema, itch, or mild swelling. However, these non–immune-mediated adverse reactions tend to spontaneously regress within a few hours or, at most, after a few days.

The increasing success of cross-linked HA opens interesting perspectives in the field of functionalized HA, in consideration to the fact that these cross-polymers can be envisaged as multifunctional molecules being able to carry and release complementary active ingredients.

The most common cross-linking agents are, at present time Divinyl Sulfone (DVS) and BDDE ( ►Figure 2 and ►Figure 3).


The above figures show that DVS has a molecular mass of MW = 118, which is nearly one half times smaller in comparison to the molecular weight of BDDE (MW = 202). This implies a different behavior of the molecules when it comes to the cross linking reaction: a smaller mass will lead to crosslinking with the formation of smaller and, therefore, more stable lattices with a prolonged stereometry.

The DVS molecules, with only one SO2 sulfone which is highly reactive, while the molecular mass of BDDE, longer and more elongated, has a molecular weight of about twice the DVS. All this implies a diversity of use and particular advantages both in terms of safety and durability of the product itself.

Fig. 4 Fig.5



►Figure 6 . DViscosity /Dt HA crosslinked BDDE 1.5 %, HA crosslinked BDDE 2.0 %, HA crosslinked BDDE 2.5 %.

The above figures demonstrates that the degradation of the crosslinked HA compositions is essentially independent from the initial storage viscosity over this 160 hour period.

Product degradation tests have been performed to verify their durability over a period of 16 h.

To digestion with the hyaluronidase was evaluated. Hyaluronidase solution (15 μL of 0.15 mg / mL solution of hyaluronidase in 1.9 mM phosphate buffer in saline, ~ 2.5 units) was added and thoroughly mixed with 0.75 g of each product. 0.35 g of each mixture was then loaded onto a rheometer plate. Data accumulation started 5 minutes from the time of addition of the enzyme. Storage was at 20 ° C.



Figure 7 indicates the products viscosity over time demonstrating how the degradation of HA crosslinked with 3% DVS is slower in comparison to HA crosslinked with 2,5% BDDE. This is due to the closer ties and the difficulty of the enzyme to break the sulfur bridges.