Hyaluronidase — Harmful for the skin?
Hyaluronidase—Can it destroy your skin?
The use of hyaluronidase (HYAL) is of utmost importance during inadvertent vascular occlusion from hyaluronic acid (HA)-based dermal fillers. Its use has demonstrated significant improvement to impending necrosis and facilitates re-perfusion following arterial occlusion. In addition, non-vascular occlusive adverse events of inappropriate filler placement, such as over-filling, superficial placement (Tyndall effect), filler migration, and nodules, may necessitate elective filler dissolution with HYAL. One of the main advantages of HA-based fillers is their ability to be reversed with the use of HYAL. While effective, precise placement of minimal effective doses of HYAL may not dramatically change tissue quality and structure. However, repetitive, excessive use with high doses of HYAL may have unintended, less well-known side effects on the treated tissue. The dogma of HYAL use is that endogenous HA is replenished within a few days, without a long-lasting effect on total HA skin content. However, this does not have robust scientific support (at least that I could find), so more studies are needed to look at the long-lasting effects of high-dose HYAL. In addition, HYAL seems to affect other components of the skin rather than just HA, at least with high doses.
To the keen physician-observer, and from patient reports, repetitive use of HYAL may change skin quality and underlying tissue structure. At Clinic5C, we have appreciated this effect in many patients during consultation for facial plastic surgery. Dr. Ben Talei describes his experience with patients undergoing lip lift procedures with a history of lip filler dissolution – noting hyaluronidase’s effects on the SMAS. In addition, a consensus group paper on the use of HYAL in aesthetic medicine revealed that 1% of respondents (3/264) noted destruction to deep tissues from HYAL. A brief exploration of the potential etiologies of these effects is highlighted below as this closer look dives into the potential negative effects of HYAL.
Hyaluronic Acid
Before we examine HYAL and its effects in detail, it is important to understand the structure and function of HA. HA is a glycosaminoglycan comprised of repeating disaccharide units. At physiological pH, the carboxyl group binds to Na+ cations and is termed "Hyaluronan." HA is one of the most hygroscopic (ability to bind to water) molecules found in nature, with a binding capacity of 1000x its body weight. HA provides viscoelasticity, hydration and lubrication for tissues. HA is the principal glycosaminoglycan in the hypodermis and 50% of total body HA is found in the skin. About one-third of HA content is turned over daily. It has a half-life of less than 2 days in the skin. HA is synthesized by hyaluronan synthases, of which there are 3 principal enzymes. HAS2 is the principal HAS isoform of the skin and the primary enzyme responsible for generating HA from fibroblasts. Fibroblasts are the primary cell that synthesizes HA; however, keratinocytes also synthesize HA, but to a lesser degree.
Endogenous HA catabolism occurs via enzymatic depolymerization with HYAL or via chemical degradation via reactive oxygen species into smaller degradative byproducts. In its high-molecular form (HMW-HA), HA is a space-filling molecule that provides cushion and hydration for tissues while also being anti-inflammatory, and anti-angiogenic. Low-molecular-weight HA (LMW-HA) has opposing functions - pro-inflammatory, angiogenic, and immunostimulatory.
When evaluating the effects of HYAL on native tissues, it is important to consider the dose of HYAL, the cell/tissue type, and size of HA-degradation byproducts as it influences cell and tissue responses in extracellular matrix (ECM) regulation. However, this is certainly not an exhaustive list of HA-HYAL biomechanics and physiology. HYAL has a half-life of 2 minutes in tissue (some studies suggest less than 5 min, in others less than 30 min), but the duration of effect is 24-48 hours given its high potency. The effects are immediate within the tissue, and traditional thinking often describes HA content being replenished within 24 hours. However, the reconstitution of baseline HA may be more complex, and the effects of HYAL go beyond HA dissolution.
Potential Negative Effects of HYAL
HA has an important role during wound healing. It increases during the initial phases of wound healing and infection to promote cell migration and proliferation of leukocytes and is degraded to LMW-HA during wound healing progression. In an in vitro mice study, HYAL accelerated the wound healing process, in a dose-dependent manner via increasing migration and proliferation of fibroblasts. Early on during wound healing, there is an increase of collagen vs. control (non-HYAL). However, at 21 days post-wounding and HYAL, there was a significant decrease in collagen content relative to the control.
In addition, a recent 2024 study assessed the effects of HA vs. HA + HYAL vs. HYAL on rat skin. They injected 0.2 mL of HA and used 30 units of HYAL, and assessed the response at different time points, up to 14 days. They found that HYAL alone significantly reduced dermal thickness, collagen density, and extracellular matrix density up to at least the 14-day mark relative to the control group. So, we do not know when or if baseline levels are restored. And, some of these effects were more pronounced in the group with HA + HYAL, which may be due to HA breakdown.
However, an ex vivo study examined the effects of HYAL on peri-ocular skin structure and found no difference in skin structure, elastic fibers, and collagen fibers following 300 units of HYAL. But, they did find increased amorphous extracellular deposits in the dermis. These deposits were not glycosaminoglycans and the authors were uncertain of their origin or significance.
In vitro treated normal human dermal fibroblasts with HYAL increase HA synthases (HAS). However, what is interesting is that HAS expression increased as the concentration of HYAL decreased. Low-dose HYAL (0.15 U/mL) increased HA concentration, while moderate-to-high dose HYAL (1.5-15 U/mL) decreased HA content relative to control, and the 15 U/mL dose effectively eliminated HA content altogether. At all doses, HYAL decreased HAS and HA in keratinocytes. Higher-dose HYAL decreased dermal HA content in human skin samples (fat was removed) even at the 24-hour mark. High-dose HYAL may overcome fibroblast compensatory mechanisms to replenish HA content via leading to the total breakdown of all available HA so there is no positive feedback to increase HA synthesis. The amount of newly synthesized HA was highest in cells treated with low doses of HYAL, whereas high doses of HYAL led to downmodulation of HA in dermal fibroblasts. This is important because, in this study, high doses were much lower than what we use during emergent and elective dissolution.
HYAL may limit fibrosis and its use has been shown to improve post-traumatic fibrosis and lung fibrosis. Depending on the study, HYAL has been shown to inhibit or increase fibroblast proliferation, likely a dose-dependent effect. High-dose HYAL may degrade too much HA to the point it disrupts the environment in which fibroblast activation and subsequent collagen deposition may occur. A study looking at rat periodontal ligament fibroblast found that low doses of HYAL did not affect cell proliferation, but at high doses cell proliferation was inhibited. They demonstrated that HYAL inhibits TGF-B-induced collagen matrix formation and myofibroblast transformation.
HYAL and Fat Tissue
Studies have shown that the effects of HYAL are not limited to HA, directly or indirectly. HA is necessary for adipocyte maturation. HYAL dose-dependently decreases morphologic differentiation of fat cells, intracellular lipid accumulation, and adipocyte-specific gene expression. In mice, injecting HYAL into the tail vein reduced fat mass and total HA content vs. controls.
In mice, HYAL has also been shown to inhibit reactive adipogenesis in the skin. Reactive adipogenesis is the local expansion of pre-adipocytes and dermal adipocytes that provides a host defense by releasing antimicrobial peptides. HYAL results in increased bacterial translocation across the epithelial barrier, however, there is less tissue injury from inflammation suppression. HYAL use decreases fat cell differentiation markers to mature fat cells. In addition, in mice, HYAL-induced medium-sized HA fragments inhibited adipogenesis and adipocyte differentiation. Furthermore, in human adipose tissue samples and cultured fat cells, the main response of HA removal was downregulation of adipogenesis-related gene expression, further supporting HYAL-mediated inhibition of fat cell differentiation. No acute effect was seen on mature adipocytes.
However, some advantages of HYAL have been demonstrated in fat retention. Despite the pre-clinical work that highlights HYAL effects on pre-adipocytes, a study demonstrated increased fat graft retention in mice following pretreating the recipient site with 100 U/mL of HYAL. At 8 weeks post-fat grafting, volume retention was 78% in the HYAL group vs. 68% in the control group. Without HYAL, the control group had more fibrosis within the graft, while the HYAL group demonstrated increased blood vessels, suggesting HYAL increased neoangiogenesis to the graft. This suggests that while HYAL may affect pre-adipocyte differentiation to mature adipocytes, the advantages of increased neoangiogenesis may outweigh its effects on adipocyte differentiation.
Conclusion
The effects of HYAL and HA-degradation fragments are dose-dependent and size-dependent, respectively. While certainly, the use of HYAL in aesthetic medicine has many advantages and utility, caution should be exercised from excessive use of HYAL. As demonstrated above, the effects of HYAL go beyond HA dissolution and should be considered when using HYAL for elective dissolution of filler. The lowest effective dose should be used when able (will be discussed in detail in a future post), and the use of ultrasound guidance may aid in minimizing the total dose required via precise placement of HYAL to minimize off-target effects. Overall, more human studies are needed to determine the toxicity and long-lasting effects of high-dose HYAL; however, based on limited animal and human tissue models, potential deleterious effects of repeated, excessive use of HYAL use include:
Decrease HA content
Decrease collagen content
Decrease dermal thickness
Decreased extracellular matrix density
Increase amorphous extracellular deposits of unknown significance
Inhibit fibroblast proliferation and myofibroblast transformation
Inhibit pre-adipocyte differentiation to mature adipocytes
Decrease adipogenesis
Increase neoangiogenesis
These effects, coupled with the effects of persisting LMW-HA byproducts, may explain the skin quality (skin thinning, red hue, which may be due to increased neoangiogenesis or the reflectance of light from muscle closer to the surface) and atrophic changes following repeated filler placement and dissolution cycles.
Cheers,
Kelm
PMID’s
- 38262634
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