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FILLERS

Science of Hyaluronic Acid Beyond Filling:


Fibroblasts and Their Response to the
Extracellular Matrix
Marina Landau, MD
Steven Fagien, MD
Holon, Israel; and Boca Raton, Fla.

Summary: Loss of viscoelasticity is one of the primarily signs of skin aging,


followed by appearance of visible wrinkles. Hyaluronic acid (HA)-based fillers
are widely used to fill wrinkles and compensate for volume loss. Recent clinical observations demonstrate persistence of the filling effect longer than the
biological availability of the filler. Stimulation of new collagen by cross-linked
HA and up-regulation of elastin have been suggested as possible explanation
to this observation and have been supported experimentally. Cross-linked HA
substitutes for fragmented collagen in restoring extracellular matrix required
for normal activity of fibroblasts, such as collagen and elastin production.
To restore extracellular matrix efficiently, serial monthly treatments are required. Boosting of facial and nonfacial skin through fibroblast activation is
a new indication for HA-based products. Injectable HA has also been recently
registered in Europe as agents specific for the improvement of skin quality
(Restylane Skinboosters). Further explanation of the possible mechanisms
supported by long-term clinical examples is presented herein. (Plast. Reconstr.
Surg. 136: 188S, 2015.)

hanges in the mechanical properties of the


skin is one of the first measurable signs of
skin aging.1,2 In most cases, they are followed
by visible wrinkling. Dermal fillers are widely used
for skin rejuvenation for more than 20 years. Due
to their efficacy and safety, hyaluronic acid (HA)based fillers had become a gold standard and
the commonest type of filler currently used.3
HA-based fillers have been used primarily for
wrinkles and lines filling. Following better understanding of facial aging process, these products
started to be utilized for 3D volumization, compensating for age-associated volume loss.4 Clinical observations on patients repetitively treated
by HA-based fillers demonstrated some initially
unexpected discoveries, including the improvement in mechanical properties of the skin following field implantation of HA and persistence of
the results after retreatment longer than expected
from injected HA longevity.59 Restoration of
extracellular scaffold, stimulating formation of
From the Dermatology Unit, Edith Wolfson Medical Center;
and Private Practice.
Received for publication May 1, 2015; accepted July 28,
2015.
Copyright 2015 by the American Society of Plastic Surgeons
DOI: 10.1097/PRS.0000000000001823

188S

younger components of the skin by injectable


cross-linked HA, provides possible explanation
of the mechanism of this phenomena.1013 Skin
boosting is a new indication for clinical use of
injectable cross-linked HA gels.

SKIN AGING
Skin aging is a complex biological process
affected by both genetic and extrinsic factors. The
most important structural components of the dermis are collagen, elastin, and ground substance,
Disclosure: Dr. Landau consults Galderma, Merz
and Croma. She occasionally speaks and runs practical workshops on Skinboosters. Dr. Fagien is a consultant/investigator for Allergan, Galderma, Merz,
Kythera, and Aquavit.

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Volume 136, Number 5S Science of HA Beyond Filling


all produced mainly by fibroblasts. Each component plays a unique role in maintaining normal
function of human skin. Type I collagen is by far
the most abundant protein in the human skin
comprising about 90% of its dry weight. For successful production of connective tissue components, fibroblasts need a stable collagen scaffold
to which they can bind.14
This binding is carried out through specific
receptors on the fibroblasts surface, called integrins.15,16 Integrins attach to molecules of collagen
in the extracellular matrix and bridge them with
tubular cytoskeleton inside the cell. While intracellular microtubules pull fibroblast skeleton
inward, focal adhesions with extracellular stable
matrix push the cells to spread. This results in
increased mechanical tension inside the fibroblasts
and stimulates production of new collagen and
down-regulation of collagen degrading enzymes.
Due to the aging process, collagen fragmentation occurs.17,18 Fragmented collagen does not
provide sufficient mechanical support to the
fibroblasts required to counterbalance inward
pulling by the internal microtubular skeleton. As
a result, fibroblasts collapse. This collapse signals
to down-regulation of the production of type I collagen, possibly through stimulation in production
of prostaglandin E2.19 In addition, fibroblasts collapse up-regulates activity of collagen degrading
matrix metalloproteinases.

CLINICAL OBSERVATIONS
It has been shown that correction of nasolabial folds by Restylane lasts 18 months after a
single retreatment at 4.5 or 9 months.8 The same
was found for Juvederm line products. Treated
subjects sustained 1821 months correction with
a single repeat treatment.9
As these results do not correlate with the
expected, if degradation was the primary determinant of the effect duration, the authors wonder
whether another phenomenon is responsible for
the correction longevity. These phenomena could
be due to other biologic effects from injectable
HA including, but not limited to, new collagen
and elastin production.

NEOCOLLAGENESIS AND
NON-HA FILLERS
New collagen stimulation secondary to fillers
implantation is not a completely unknown phenomenon. Injectable silicon is well-known for its ability to
induce stimulation of collagen, through a process,

similar to foreign body reaction.20 Poly-l-lactic acid


induces slow and significant collagen stimulation
through usually controlled inflammatory process.21
Calcium hydroxylapatite stimulates collagen formation and dermal thickening.22 Restoration of extracellular matrix as a mechanism for rejuvenation
of dermal components and their function has been
shown for HA-injected skin exclusively.1013

FIBROBLASTS AND THEIR RESPONSE


TO EXTRACELLULAR MATRIX
Until 2007, it was an axiom that HA-based fillers act by directly adding dermal or subdermal
volume, which can be further augmented by intissue water attraction by HA.
The first report on intradermal changes
induced by injectable HA was published in 2007.10
Wang et al10 assessed the effect of relatively new
at that time nonanimal origin stabilized HA
(NASHA, Restylane) on human skin in vivo. They
injected 0.7mL of Restylane or normal saline into
photodamaged skin of the arm of 11 volunteers.
The injected sites were biopsied and studied 4
and 13 weeks after the injection. The investigators
found increased intracellular and extracellular
dermal staining for collagen I in NASHA-treated
samples, especially in the skin areas adjacent to
the filler. The increase in collagen production was
mediated by higher levels of transforming growth
factor-. In addition, in NASHA-treated skin, there
was increased expression of tissue inhibitors of
collagen degrading enzymes. As all the increased
compounds are produced by dermal fibroblasts,
the investigators looked closely at these cells. They
found that fibroblasts embedded in connective tissue surrounding NASHA appeared different morphologically from fibroblasts in saline-treated skin.
Adjacent to NASHA, fibroblasts were stretched
with abundant endoplasmic reticulum. Unlike
theoretically expected, no direct binding of fibroblasts to NASHA was demonstrated. Therefore, the
proposed mechanism of collagen stimulation was
not a direct effect of HA on fibroblasts but rather
indirect mechanical phenomenon. NASHA-mediated hydration of extracellular matrix induces
mechanical stretching of fibroblasts, thus, initiating production of connective tissue components,
such as collagen type I.
Young extracellular matrix maintains mechanical tension due to intact collagen fibers, providing stable scaffold. Fragmentation of the scaffold
causes fibroblasts to collapse and reduces their
procollagenic function. Clinically, impaired fibroblast function, coupled with reduced collagen

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Plastic and Reconstructive Surgery November Supplement 2015


synthesis, translates into atrophy and wrinkling of
aged skin. When a stiffer injected cross-linked HA
invades thinned skin, stable scaffold is restored
and fibroblasts become stretched and active,
enhancing skin appearance and function.
Quan et al11 investigated interaction of
NASHA with fibroblasts and extracellular matrix
in chronologically aged skin. Similar to Wang et
al,10 they found that enhancement of structural
support of the extracellular matrix by injectable
HA (Restylane) causes fibroblasts elongation with
subsequent increase in procollagen I and collagens I and III production for at least 12 weeks
after HA injection. This stimulation is mediated
by up-regulation of type II transforming growth
factor- receptor and connective tissue growth factor. Enhanced structural support of extracellular
matrix by injected HA was found to increase keratinocyte and fibroblast proliferation as well, resulting in epidermal thickening and expenditure of
dermal vasculature. These findings support the
importance of stable dermal scaffold for maintaining normal dermal and probably epidermal function and emphasize again the potency of injected
cross-linked HA on age-related cutaneous changes.
Stimulation of production of extracellular
matrix components, such as collagen and elastin,
by cross-linked injectable HA, other than NASHA,
has been demonstrated in human and animal
models in additional publications.12,13

SKIN-BOOSTING CONCEPT
The first product designed specifically for
skin-boosting purposes was Restylane Vital. The
product comprises small particles stabilized
smooth and relatively thin NASHA gel (20 mg/
mL). Restylane Vital Light skin booster is less concentrated (12 mg/mL) and injected in more delicate areas , such as a neck (Fig. 6) Both products
contain Lidocaine hydrochloride 3 mg/mL.
The idea of skin quality enhancement by
injectable HA, without targeting wrinkles or volume loss, was introduced by Kerscher et al,6 who
injected microdoses of small particles stabilized
HA (Restylane Vital) into the dermis of the lower

Fig. 1. A 62-year-old man presented for blepharoplasty. He was


not aware of deep horizontal forehead lines until after blepharoplasty. He was happy with surgery but now bothered by the forehead lines. A 0.6mL of reconstituted Juvederm (Juvederm Ultra
diluted with 1% lidocaine with epinephrine to a concentration

Fig. 1. (Continued). of 16mg/mL by adding 0.5mL anesthetic


to a 1-mL syringe) was injected directly into the depth of the
furrow in all areas at the approximate level of the mid-dermis
in a serial threading technique. Above, Before blepharoplasty
and reconstituted Juvederm to deep horizontal forehead lines.
Center, After upper and lower blepharoplasty with noted persistent forehead lines. Below, 1 year after reconstituted Juvederm
to deep horizontal forehead lines.

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Copyright 2015 American Society of Plastic Surgeons. Unauthorized reproduction of this article is prohibited.

Volume 136, Number 5S Science of HA Beyond Filling

Fig. 2. A 46-year-old woman before (left) and 1 month after (right) a single session of 2mL (1mL
per cheek) of Restylane Vital Skinbooster treatment. The product was injected using micropuncture techniques by smart-click system of the syringe.

Fig. 3. A 57-year-old woman before (left) and 1 month after (right) 2 monthly sessions of Restylane
Vital Skinbooster treatment (1mL per each cheek per treatment). The product was injected using
micropuncture technique. Visible improvement of cheek smile lines is noted due to better skin
elasticity.

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Plastic and Reconstructive Surgery November Supplement 2015

Fig. 4. A 56-year-old woman before (left) and after (right) 2 monthly sessions of Restylane Vital
Skinbooster 2mL per session.

Fig. 5. A 57-year-old woman before (left) and after (right) 3 monthly sessions of Restylane Vital
Skinbooster treatment (1mL per each cheek per treatment). The product was injected by using
micropuncture technique.

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Volume 136, Number 5S Science of HA Beyond Filling

Fig. 6. A 54-year-old woman with upper neck wrinkles before (left) and 1 month after (right) 3
monthly sessions of Restylane Vital Light Skinbooster treatment (total amount 4mL).

cheeks of 19 female patients.7 The procedure


has been carried out in 3 monthly sessions. The
investigators found that micropuncture injections of Restylane Vital significantly increased
skin elasticity and created positive impact on skin
surface roughness. Since greatest improvement
in these parameters was evident 24 weeks after
the last injection session, the authors concluded
that placement of HA into the dermis enhanced
biosynthesis of new dermal compounds and is
not only due to better skin hydration, as initially
expected.
Following our current better understanding of
the biological process induced by injected crosslinked HA, field treatment, as suggested by Kerscher et al,6 restores extracellular dermal scaffold
in a more diffused and homogenous fashion, than
localized injections for wrinkles or volume correction. Furthermore, to create sufficient stimulation,
a serial treatment is usually required. One monthly

treatment followed by 23 repetitions improves


skin quality in clinically evident fashion (Figs.15).
Additional studies and case reports substantiated skin-boosting concept as a novel treatment
for skin improvement by injectable cross-linked
HA.2327 Interestingly, noncross-linked HA
injected following the same principles (mesotherapy/biorevitalization therapy) demonstrate
controversial results, probably due to insufficient
or nonsustained effect on extracellular scaffold
stabilization.2830
Restylane Vital is currently packaged in
European countries in smart-click syringe.
The system doses the product and delivers
0.01 mL of it per each click. The procedure is
carried usually out in 3 monthly sessions. During each session, the targeted area is treated by
multiple intra/subdermal micropuncture injections of a dosed amount of Restylane Vital Skinbooster spaced approximately 1 cm from each

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Plastic and Reconstructive Surgery November Supplement 2015


patient consent

Patients provided written consent for the use of their


images.
REFERENCES

Video. Supplemental Digital Content 1, which demonstrates


the Restylane Skinbooster treatment technique, is available in
the Related Videos section of the full-text article on PRSJournal.com or, for Ovid users, at http://links.lww.com/PRS/B425.

other. The injections are either perpendicular


to the skin surface punctures or carried out as
linear repetitive clickings, leaving two to three
0.01-mL product deposits along the intradermal
channels. Restylane Vital Skinbooster syringe
can be also used in regular continuous mode
(without clicking) for skin defects correction
and not as a skinbooster. In the United States,
Restylane Vital has been recently (June 13, 2014)
registered as Restylane Silk without smart-click
delivery system. Detailed video clip demonstrating Restylane Skinbooster treatment technique
is attached. (See Video, Supplemental Digital
Content 1, which demonstrates Restylane Skinbooster treatment technique, available in the
Related Videos section of the full-text article on PRSJournal.com or, for Ovid users, at
http://links.lww.com/PRS/B425.)
In summary, improvement of skin quality by
injectable cross-linked HA has evolved in the last
years, as an additional indication (after wrinkles
filling and volumization) of HA-based dermal fillers use. The procedure hydrates the dermis and
creates stable extracellular matrix to support
intradermal fibroblasts structure that is essential
for their normal function. We postulate that multiple processes that improve the structural integrity and aesthetic appearance of aging skin are in
play, and further research will identify the relative
role of each component, so we can establish best
practices, products selection, and techniques for
optimal results.
Marina Landau, MD
Dermatology Unit
Edith Wolfson Medical Center
62 Halochamim Street
Holon 58100, Israel
mlandau@zahav.net.il

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