Science of Hyaluronic Acid Beyond Filling - Fibroblasts and Their Response to the Extracellular...

7/23/2019 Science of Hyaluronic Acid Beyond Filling - Fibroblasts and Their Response to the Extracellular Matrix

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

www.PRSJournal.com188S

Changes in the mechanical properties of theskin is one of the first measurable signs ofskin 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. Dueto their efficacy and safety, hyaluronic acid (HA)-based fillers had become a “gold” standard andthe commonest type of filler currently used.3

HA-based fillers have been used primarily for wrinkles and lines filling. Following better under-standing of facial aging process, these productsstarted to be utilized for 3D volumization, com-pensating for age-associated volume loss.4 Clini-cal observations on patients repetitively treatedby HA-based fillers demonstrated some initiallyunexpected discoveries, including the improve-

ment in mechanical properties of the skin follow-ing “field” implantation of HA and persistence ofthe results after retreatment longer than expectedfrom injected HA longevity.5–9 Restoration ofextracellular scaffold, stimulating formation of

younger components of the skin by injectablecross-linked HA, provides possible explanationof the mechanism of this phenomena.10–13 Skinboosting is a new indication for clinical use of

injectable cross-linked HA gels.

SKIN AGINGSkin aging is a complex biological process

affected by both genetic and extrinsic factors. Themost important structural components of the der-mis are collagen, elastin, and ground substance,

Disclosure: Dr. Landau consults Galderma, Merzand Croma. She occasionally speaks and runs prac- tical workshops on Skinboosters. Dr. Fagien is a con- sultant/investigator for Allergan, Galderma, Merz,Kythera, and Aquavit.

Copyright © 2015 by the American Society of Plastic Surgeons

DOI: 10.1097/PRS.0000000000001823

Marina Landau, MDSteven 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 fillersare widely used to fill wrinkles and compensate for volume loss. Recent clini-cal observations demonstrate persistence of the filling effect longer than thebiological availability of the filler. Stimulation of new collagen by cross-linkedHA and up-regulation of elastin have been suggested as possible explanationto this observation and have been supported experimentally. Cross-linked HAsubstitutes for fragmented collagen in restoring extracellular matrix requiredfor normal activity of fibroblasts, such as collagen and elastin production.To restore extracellular matrix efficiently, serial monthly treatments are re-

quired. Boosting of facial and nonfacial skin through fibroblast activation isa new indication for HA-based products. Injectable HA has also been recentlyregistered in Europe as agents specific for the improvement of skin quality(Restylane Skinboosters). Further explanation of the possible mechanismssupported by long-term clinical examples is presented herein. (Plast. Reconstr.Surg. 136: 188S, 2015.)

From the Dermatology Unit, Edith Wolfson Medical Center;and Private Practice.Received for publication May 1, 2015; accepted July 28,2015.

Science of Hyaluronic Acid Beyond Filling:Fibroblasts and Their Response to the

Extracellular Matrix

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FILLERS

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Volume 136, Number 5S • Science of HA Beyond Filling

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all produced mainly by fibroblasts. Each compo-nent plays a unique role in maintaining normalfunction of human skin. Type I collagen is by farthe most abundant protein in the human skincomprising about 90% of its dry weight. For suc-cessful production of connective tissue compo-nents, fibroblasts need a stable collagen scaffoldto which they can bind.14

This binding is carried out through specificreceptors on the fibroblasts surface, called integ-rins.15,16 Integrins attach to molecules of collagenin the extracellular matrix and bridge them withtubular cytoskeleton inside the cell. While intra-cellular microtubules “pull” fibroblast skeletoninward, focal adhesions with extracellular stablematrix push the cells to spread. This results inincreased mechanical tension inside the fibroblastsand stimulates production of new collagen anddown-regulation of collagen degrading enzymes.

Due to the aging process, collagen fragmen-tation occurs.17,18 Fragmented collagen does notprovide sufficient mechanical support to thefibroblasts required to counterbalance inwardpulling by the internal microtubular skeleton. Asa result, fibroblasts collapse. This collapse signalsto down-regulation of the production of type I col-lagen, possibly through stimulation in productionof prostaglandin E2.19 In addition, fibroblasts col-lapse up-regulates activity of collagen degradingmatrix metalloproteinases.

CLINICAL OBSERVATIONSIt has been shown that correction of naso-

labial folds by Restylane lasts 18 months after asingle retreatment at 4.5 or 9 months.8 The same was found for Juvederm line products. Treatedsubjects sustained 18–21 months correction witha single repeat treatment.9

As these results do not correlate with theexpected, if degradation was the primary determi-nant of the effect duration, the authors wonder

whether another phenomenon is responsible forthe correction longevity. These phenomena couldbe due to other biologic effects from injectableHA including, but not limited to, new collagenand elastin production.

NEOCOLLAGENESIS ANDNON-HA FILLERS

New collagen stimulation secondary to fillersimplantation is not a completely unknown phenom-enon. 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 acidinduces slow and significant collagen stimulationthrough usually controlled inflammatory process.21 Calcium hydroxylapatite stimulates collagen forma-tion and dermal thickening.22 Restoration of extra-cellular matrix as a mechanism for “rejuvenation”of dermal components and their function has beenshown for HA-injected skin exclusively.10–13

FIBROBLASTS AND THEIR RESPONSETO EXTRACELLULAR MATRIX

Until 2007, it was an axiom that HA-based fill-ers act by directly adding dermal or subdermal volume, which can be further augmented by in-tissue water attraction by HA.

The first report on intradermal changesinduced by injectable HA was published in 2007.10

Wang et al10 assessed the effect of relatively newat that time nonanimal origin stabilized HA(NASHA, Restylane) on human skin in vivo. Theyinjected 0.7 mL of Restylane or normal saline intophotodamaged skin of the arm of 11 volunteers.The injected sites were biopsied and studied 4and 13 weeks after the injection. The investigatorsfound increased intracellular and extracellulardermal staining for collagen I in NASHA-treatedsamples, especially in the skin areas adjacent tothe filler. The increase in collagen production wasmediated by higher levels of transforming growth

factor-β. In addition, in NASHA-treated skin, there was increased expression of tissue inhibitors ofcollagen degrading enzymes. As all the increasedcompounds are produced by dermal fibroblasts,the investigators looked closely at these cells. Theyfound that fibroblasts embedded in connective tis-sue surrounding NASHA appeared different mor-phologically from fibroblasts in saline-treated skin. Adjacent to NASHA, fibroblasts were stretched with abundant endoplasmic reticulum. Unliketheoretically expected, no direct binding of fibro-blasts to NASHA was demonstrated. Therefore, the

proposed mechanism of collagen stimulation wasnot a direct effect of HA on fibroblasts but ratherindirect mechanical phenomenon. NASHA-medi-ated hydration of extracellular matrix inducesmechanical stretching of fibroblasts, thus, initiat-ing production of connective tissue components,such as collagen type I.

Young extracellular matrix maintains mechan-ical tension due to intact collagen fibers, provid-ing stable scaffold. Fragmentation of the scaffoldcauses fibroblasts to collapse and reduces theirprocollagenic function. Clinically, impaired fibro-

blast function, coupled with reduced collagen




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Plastic and Reconstructive Surgery • November Supplement 2015

synthesis, translates into atrophy and wrinkling ofaged skin. When a stiffer injected cross-linked HA“invades” thinned skin, stable scaffold is restoredand fibroblasts become stretched and active,enhancing skin appearance and function.

Quan et al11 investigated interaction of

NASHA with fibroblasts and extracellular matrixin chronologically aged skin. Similar to Wang etal,10 they found that enhancement of structuralsupport of the extracellular matrix by injectableHA (Restylane) causes fibroblasts elongation withsubsequent increase in procollagen I and col-lagens I and III production for at least 12 weeksafter HA injection. This stimulation is mediatedby up-regulation of type II transforming growthfactor-β receptor and connective tissue growth fac-tor. Enhanced structural support of extracellularmatrix by injected HA was found to increase kera-

tinocyte and fibroblast proliferation as well, result-ing in epidermal thickening and expenditure ofdermal vasculature. These findings support theimportance of stable dermal scaffold for maintain-ing normal dermal and probably epidermal func-tion and emphasize again the potency of injectedcross-linked HA on age-related cutaneous changes.

Stimulation of production of extracellularmatrix components, such as collagen and elastin,by cross-linked injectable HA, other than NASHA,has been demonstrated in human and animalmodels in additional publications.12,13

SKIN-BOOSTING CONCEPTThe first product designed specifically for

skin-boosting purposes was Restylane Vital. Theproduct comprises small particles stabilizedsmooth and relatively thin NASHA gel (20 mg/mL). Restylane Vital Light skin booster is less con-centrated (12 mg/mL) and injected in more deli-cate areas , such as a neck (Fig. 6) Both productscontain Lidocaine hydrochloride 3 mg/mL.

The idea of skin quality enhancement byinjectable HA, without targeting wrinkles or vol-ume loss, was introduced by Kerscher et al,6 whoinjected microdoses of small particles stabilizedHA (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 blepharo-

plasty. He was happy with surgery but now bothered by the fore-

head lines. A 0.6 mL of reconstituted Juvederm (Juvederm Ultra

diluted with 1% lidocaine with epinephrine to a concentration

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

tent forehead lines. Below, 1 year after reconstituted Juvederm

to deep horizontal forehead lines.





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Fig. 2. A 46-year-old woman before (left ) and 1 month after (right ) a single session of 2 mL (1 mL

per cheek) of Restylane Vital Skinbooster treatment. The product was injected using micropunc-

ture 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 (1 mL 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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Fig. 4. A 56-year-old woman before (left ) and after (right ) 2 monthly sessions of Restylane Vital

Skinbooster 2 mL per session.

Fig. 5. A 57-year-old woman before (left ) and after (right ) 3 monthly sessions of Restylane Vital

Skinbooster treatment (1 mL per each cheek per treatment). The product was injected by using

micropuncture technique.





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cheeks of 19 female patients.7 The procedurehas been carried out in 3 monthly sessions. Theinvestigators found that micropuncture injec-tions of Restylane Vital significantly increasedskin elasticity and created positive impact on skinsurface roughness. Since greatest improvementin these parameters was evident 24 weeks after

the last injection session, the authors concludedthat placement of HA into the dermis enhancedbiosynthesis of new dermal compounds and isnot only due to better skin hydration, as initiallyexpected.

Following our current better understanding ofthe biological process induced by injected cross-linked HA, field treatment, as suggested by Ker-scher et al,6 restores extracellular dermal scaffoldin a more diffused and homogenous fashion, thanlocalized injections for wrinkles or volume correc-tion. Furthermore, to create sufficient stimulation,

a serial treatment is usually required. One monthly

treatment followed by 2–3 repetitions improvesskin quality in clinically evident fashion (Figs. 1–5).

Additional studies and case reports substanti-ated “skin-boosting” concept as a novel treatmentfor skin improvement by injectable cross-linkedHA.23–27 Interestingly, non–cross-linked HAinjected following the same principles (meso-

therapy/biorevitalization therapy) demonstratecontroversial results, probably due to insufficientor nonsustained effect on extracellular scaffoldstabilization.28–30

Restylane Vital is currently packaged inEuropean countries in “smart-click” syringe.The system doses the product and delivers0.01 mL of it per each click. The procedure iscarried usually out in 3 monthly sessions. Dur-ing each session, the targeted area is treated bymultiple intra/subdermal micropuncture injec-tions of a dosed amount of Restylane Vital Skin-

booster spaced approximately 1 cm from each

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 4 mL).




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other. The injections are either perpendicularto the skin surface punctures or carried out aslinear repetitive “clickings,” leaving two to three0.01-mL product deposits along the intradermalchannels. Restylane Vital Skinbooster syringecan be also used in regular continuous mode(without clicking) for skin defects correctionand not as a skinbooster. In the United States,Restylane Vital has been recently (June 13, 2014)registered as Restylane Silk without smart-clickdelivery system. Detailed video clip demonstrat-ing Restylane Skinbooster treatment technique

is attached. (See Video, Supplemental DigitalContent 1, which demonstrates Restylane Skin-booster treatment technique, available in the“Related Videos” section of the full-text arti-cle on PRSJournal.com or, for Ovid users, athttp://links.lww.com/PRS/B425 .)

In summary, improvement of skin quality byinjectable cross-linked HA has evolved in the last years, as an additional indication (after wrinklesfilling and volumization) of HA-based dermal fill-ers use. The procedure hydrates the dermis andcreates stable extracellular matrix to supportintradermal fibroblasts structure that is essentialfor their normal function. We postulate that mul-tiple processes that improve the structural integ-rity and aesthetic appearance of aging skin are inplay, and further research will identify the relativerole of each component, so we can establish bestpractices, products selection, and techniques foroptimal results.

Marina Landau, MD Dermatology Unit

Edith Wolfson Medical Center62 Halochamim Street

Holon 58100, Israel

[email protected]

PATIENT CONSENT

Patients provided written consent for the use of theirimages.

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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 PRSJour-

nal.com or, for Ovid users, at http://links.lww.com/PRS/B425 .

http://links.lww.com/PRS/B425







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