scientific and clinical s tudies...injection of dermal fillers is one of the most commonly performed...

S C I E N T I F I C A N D C L I N I C A L S T U D I E S

Background:

Injection of dermal fillers is one of

the most commonly performed

procedures in the cosmetic derma-

tology practice. The ever-expan-

ding range of dermal filler products

for aesthetic soft tissue augmenta-

tion is of benefit for patients and

physicians, but as indications and

the number of procedures perfor-

med increase, the number of com-

plications will likely also increase

due to the foreign body reaction

and presence of non-biodegra-

dable chemicals.

Objective:

To describe the biocompatibility,

safety and bio-degradability of an

Agarose gel injected in Human soft

tissue, with the purpose of evalua-

ting the interaction of this type of

filler with the tissues of the human

skin and evaluate the biocompati-

bility utilizing skin biopsies taken

from human volunteers

Material and Methods:

For this study, we have recruited

12 volunteers, healthy female sub-

jects, between the ages of 35 and

50 years old; the inclusion and ex-

clusion criteria were those normally

used for the clinical use of any ab-

sorbable filler.

A 1.5% agarose Gel filler (Algeness

HD) was used. The filler was injec-

ted into the hypodermis in a limited

zone of the superior-medial region

using a linear retrograde technique.

The protocol called for taking 4

biopsies, the first at T0 (prior to the

injection), the others at T1 (1 month

after the implant), T2 (after 3 mon-

ths) and T3 (after 6 months)

A punch for taking biopsies with a

diameter of 1.5 mm was used.

The biopsies obtained were fixed

in neutral formalin buffered at 10%

and set in paraffin.

The microtomic sections, 6 microns

thick, were stained with Hematoxy-

lin and Eosin, Mallory trichrome and

Weigert’s stain and then observed

by light microscopy.

Results:

The injections were completed wit-

hout any adverse events.

The biopsies at T1, T2, T3 shows a

good integration of the agarose gel

into the human tissue with a limited

physiological inflammatory reac-

tion, no granulomatous activity, a

degradation of the agarose gel over

time and an increase of fibroblastic

components, elastin and collagen

formation.

Conclusion: The results confirm the

safety and bio-compatibility of the

Agarose Gel filler with human skin.

Key words: Natural filler, Bio-com-

patibility, Bio-degradability

Agarose gel filler Histologic study of biocompatibility and interaction with human cutaneous structures.

Prof. Alessio Pirino, Dr. Giorgio Maullu.Department of Biomedical Sciences, CRISMENC, University of Sassari, Italy

IntroductionAgarose gels are used in different applications in various technological fields: for example, in microbiology, for culture media, in pharmaceuticals and phytotherapy applications as thickeners, as a laxative, a defense for the mu-cous membranes of the gastrointestinal tract, in the cooking and in the food industry to replace gelatin of animal origin. Due to its characteristics, Agarose gel also represents the bio-compatible vehicle of choice in numerous fields for pre-clinical applications: as a substrate in bio-compatibility, cytotoxicity, sensitization, genotoxicity and mutagenesis tests; in bio-engineering for three dimensional tissue growth; as a substrate for controlled released systems of pharmacological substances.

It is these important bio-compatibi-lity characteristics that for the past few years, Agarose gels have been widely used as fillers for soft tissue injections..

The important characteristics of this type of filler to point out are: • It is a hydrogel whose consistency is determined by hydrogen bonds between the linear chains and that it doesn’t contain harmful cross-linking chemical substances, which are present in hyaluronic acid fillers (for example, butanediol diglycidyl ether or BDDE); • It is not hydrophilic; therefore it doesn’t attract and retain water af-ter the injection, making the result of the treatment immediately ap-preciable. • Being natural absorbable filler, it is degraded by the action of macro-phages and not by an agarose en-zyme, which is not present in the human body. Agarose gel is not di-rectly metabolized via an enzyma-tic pathway and will remain in place for an extended period of time.

Until now, despite the widespread use of Agarose fillers and various clinical studies, a human histologic study for the purpose of evaluating the interaction of this type of filler with human tissues has not been published nor available in the pu-blic sector. There is in the published literature a couple of histologic works; howe-ver these studies were conducted on animals only. (Plast Reconstr Surg. 2007;120(5):1161-9, Connec-tive Tissue Research, 2012; 53(6): 548–554).

Method and results:The study was completed at the Center for Research and Deve-lopment of Aesthetic Medicine, Nutraceutic and Cosmetology (CRISMENC) at the University of Sassari.

We design a histological study to evaluate the biocompatibility and the interactions between Agarose Gel filler and human skin tissues utilizing skin biopsies taken from human volunteers.We have recruited 12 healthy fe-male subjects between the age of 35 and 50 ; the inclusion and exclu-sion criteria were those normally used for the clinical use of any ab-sorbable filler. A 1.5% agarose Gel filler (Algeness HD) was used. The filler was injec-ted into the hypodermis in a limited area of the superior-medial region using a linear retro-tracing tech-nique.The protocol called for taking 4 biopsies, the first at T0 (prior to the injection) as control, the others at T1 (1 month after the implant), T2 (after 3 months) and T3 (after 6 months)A punch for taking biopsies with a diameter of 1.5 mm was used.The biopsies obtained were fixed in neutral formalin buffered at 10% and set in paraffin.The microtomy sections, 6 microns thick, were stained with Hematoxy-lin and Eosin, Mallory Trichrome and Weigert’s stain and then obser-ved by light microscope.

Results:- At T1 the filler was well integrated in the hypodermis, with an inflam-matory reaction characterized by an increased presence of cellular elements mainly of a fibroblastic and macrophage nature, with some more or less voluminous multi-nucleate cells, while no significant lymphoid infiltrate was noted.

It also appears to be a greater accumulation of collagen fibers, which however does not form a thick fibrous capsule.

- At T2, an increase of the dermal collagen component in the hypo-

dermis is more evident and the fil-ler is incorporated into the confines of the tissue where the same cel-lular elements were observed at T1. It appears that there is also an increase in the elastic component when compared to the control (T0).

- At T3 we can still find a good quantity of filler in the hypodermis, while the connective and fibroblas-tic components of the tissues ap-pear to be more abundant than the control (T0).On the basis of these initial results obtained, a good integration of the filler into the hypodermis is evident, with a physiological inflammatory reaction, which has never given ori-gin to granulomatous formations, accompanied by an increase in col-lagen formation and more than li-kely elastin as well. In the continuation of the study beyond 6 month, using immunohis-tochemistry and immunofluores-cence methodologies, the produc-tion of collagen and elastin will be characterized with greater preci-sion, in particular, evaluating more precisely whether the newly for-med collagen is of type I or III. In addition, by means of in vitro expe-rimental studies, conducted on hu-man fibroblast cultures in the pre-sence of the filler, it will be possible to evaluate cellular vitality and pro-liferation compared to the controls without filler.

Conclusion: As the use of dermal fillers containing chemicals has in-creased, the number of cases re-porting adverse reactions has also increased. In the search of safe and biocompatible filler for soft tissue injection, Agarose gel seems to be a safe alternative confirmed by the results of this study proving the biocompatibility of the Agarose Gel filler with human skin.

________________________________________________________________________________

Biopsy at TO

Mallory Trichrome

Granulocyt neutrofilis

Hematoxylin-Eosin Mallory Trichrome


Collagen

Collagen

Fibroblast

Hypodermis T0

Biopsy at T1

Agarose gel filler (F)

Macrophages


At T1 the filler (F) was well integrated in the hypodermis, with an inflammatory reaction characterized by an increased presence of cellular elements mainly of a fibroblastic and macrophage nature, with some more or less voluminous multinucleate cells (MC), while no significant lymphoid infiltrate was noted.

Multinucleate cells


Hypodermis with filler at T2 Hypodermis with filler at T2

Elastic fibers at T0 Elastic fibers at T2 ________________________________________________________________________________

Biopsy at T3 Hypodermis at T3

Biopsy at T2

Biopsy at T3

There appears to be a greater accumulation of collagen fibers, which however does not form a thick fibrous capsule. At T2, an increase of the dermal collagen component in the hypodermis is more evident and the filler is incorporated into the confines of the tissue where the same cellular elements of T1 were observed. It appears that there is also an increase in the elastic component when compared to the control.

At T3 we can still find a good quantity of filler in the hypodermis (F), which proves the good persistence of Agarose gel in the tissue, while the connective and fibroblastic components of the tissues appear to be more abundant than the control. On the basis of these initial results obtained, it is possible to see a good integration of the filler into the hypodermis, with a physiological inflammatory reaction, which has never given origin to granulomatous formations, accompanied by an increase in collagen formation and more than likely elastin as well.



Background.

Many fillers have been used to aug-

ment lips. Agarose gel is a new and

absorbable filler indicated for the

correction of soft tissues and lip.

Objective.

This article reviews the results of 68

cases that have undergone lip aug-

mentation with this new filler in the

last 3 years.

Study design.

A total of 68 patients received aga-

rose gel for treatment for lip aug-

mentation in a 3-year period from

2005 to 2008. Each of the patients

signed an informed consent form.

The patients were between 35 and

70 years of age. Three patients

were male, and 65 were female. A

volume of 0.5-1.0 mL of agarose gel

was sufficient for each lip. A bigger

volume may result in a dense mass

and pain. All patients were success-

fully treated with injections of aga-

rose gel.

Results.

Clinical improvement was noted

immediately, and only mild bruising

was recorded. All of the the pa-

tients returned to the clinic 10 days

after treatment for follow-up, and

all felt that an excellent cosmetic

result was obtained. The patients

were told to return after an additio-

nal month for follow-up and pos-

sible reinjection. The results lasted

approximately 5 months with a

gradual decline to baseline. The

agarose gel was very well tolerated

with only a few mild adverse reac-

tions that resolved spontaneously.

Conclusion.

During 3 years of clinical use, aga-

rose gel proved to be a reliable and

predictable treatment for lip aug-

mentation. (Oral Surg Oral Med

Oral Pathol Oral Radiol Endod

2009;108:e11-e15)

Agarose gel is an absorbable filler

indicated for the correction of soft

tissues and for lip augmentation.1

It is made from a gel of injectable

agarose and is painless because it

is isotonic. It is a sterile, apyroge-

nic, viscoelastic, clear, colorless,

and transparent gel. Agarose gel is

a polysaccharide formed by repea-

ting units of 3,6-anhydrous L-ga-

lactose and D-galactose. There is

no specific enzyme in the human

organism which is able to degrade

agarose. Therefore the agarose is

first transported from the applica-

tion site by the action of macro-

phages and then subjected to en-

zymatic destruction of the polymer

by means of galactosidase. Aga-

rose is metabolized in the pentose

cycle at the level of the macro-

phages, platelets, and endothelial

reticulum. Agarose gel has been

chosen owing to its characteris-

tics as a biocompatible vehicle in

a wide range of preclinical and cli-

nical applications. Agarose is used

as the substrate of choice in nu-

merous biocompatible tests, such

as cytotoxity, genotoxicity,2 mu-

tagenesis,3 sensitivity,4 and sub-

cutaneous implants.5 Furthermore,

the gel is used in bioengineering for

3-dimensional tissue growth and as

a substrate for controlled release

of pharmacologic substances. Soft

tissue augmentation with injectable

filling agents in the perioral region

is rapidly increasing in popularity.6

In addition, the proven safety of

these products has also been a fac-

tor in their increased use. Although

only rarely, complications do oc-

cur.7 A novel use for filling agents

is to exploit their volume-occupy-

ing properties to replace lost sub-

dermal fat and remodeled maxillary

and mandibular bone. The “pushfill”

restores the youthful characteris-

tics of the face without “pull” sur-

gery, with less inconvenience and

few complications.8 It is used to

support the cosmetic application

of such agents and helps define the

adverse event profile.

Using fillers for facial rejuvenation

requires not only knowledge of

specific fillers but also insight into

the process and anatomy of aging.

The scientific support for the appli-

cation of a substance for soft tis-

sue augmentation is critical. One

of the central tenets of soft tissue

augmentation is the concept of the

3-dimensional face. A youthful face

has a soft, full appearance, as op-

posed to the flat, pulled, 2-dimen-

sional look often achieved by more

traditional surgical approaches. In-

jectable filling agents can augment

and even, at times, replace pulling.

Additionally, with the lip as the fo-

cal center of the lower face, subt-

le lip enhancement is here to stay,

and is, in fact, the primary indica-

tion for injectable fillers. Dermal fil-

lers are commonly categorized by

duration of effect: temporary, se-

mipermanent (duration is often 18

months but the exact time frame is

unknown), and permanent options.

Doctors today have a much larger

armamentarium of techniques and

materials with which to improve fa-

cial contours, ameliorate wrinkles,

and provide esthetic rejuvenation

to the face.

Lip augmentation with a new filler (agarose gel): a 3-year follow-up study

The present article briefly pre-

sents the first use of a temporary

alloplastic injectable soft tissue

filler, agarose gel, for lip augenta-

tion. The aim of this article was to

evaluate the results in 68 cases that

have undergone lip augmentation

in the last 3 years.

MATERIALS AND METHODS

A total of 68 patients received

agarose gel (Easy- Filler [Ghimas,

Casalecchio di Reno, Bologna, Ita-

lia] or Easy-Agarose [Sifarma, Mi-

lano, Italy]) for lip augmentation

in a 3-year period (2005-2008).

The agarose gel used in this study

contained 2.5% agarose and 97.5%

saline solution. Patients with acute

or chronic skin pathologies or direct

involvement in or around the area

to be treated were excluded. After

the patients read the brochure and

discussed the risks and benefits of

the procedure and the risks and

benefits of alternatives, and after

having all of their questions answe-

red, all of them signed the infor-

med consent form. In the consent

form the possible complications of

bruising, swelling, and granuloma

formation were outlined. To reduce

bruising, patients were reminded

not to take any salicylates, ibupro-

fen, or vitamin E for 2 weeks before

treatment. Patients were between

35 and 70 years of age. Median age

was 52 years. Three patients were

male, and the other 65 were female.

Superficial anesthetic was recom-

mended for the augmentation

of the upper and lower lips. In all

patients, both lips were treated.

To achieve a field block with der-

mal anesthetic cream (Emla; Astra,

Westborough, MA) or xylocaine

spray, 10% lidocaine solution was

applied on the mucosa and skin

of the upper and lower labiogingi-

val fold. In some cases infiltration

with local anesthesia was used. Af-

ter 10-15 minutes, the needle can

be directed into the correct plane

of the vermilion border. Generally,

30-gauge needles with a 13-mm

length should be used. The esthetic

evaluation was performed at 1, 2, 4,

6, and 12 months after injection.

Often, one-half of the “white roll”

can be implanted by withdrawing

the needle while injecting. Injec-

tions were repeated along the ver-

milion border until the procedure

was complete. In a few patients,

additional injections were occasio-

nally introduced into the lateral or-

bicularis oris muscle to attempt lip

eversion. A flattened philtrum can

be raised effectively by two vertical

injections of agarose gel starting

from below, e.g., from the two cor-

ners of the Cupid’s bow within the

white roll. Under no circumstances

should the microdroplet injection

technique be used in the lips. A

volume of 0.5-1.0 mL agarose gel

was sufficient for each lip. A bigger

volume may result in a dense mass

and pain; therefore, the lips should

always be augmented in stages. If

agarose gel was well tolerated and

lips were soft after 3 months, more

agarose-gel could be added to the

same pocket. Only rarely did the

implant dislodge into the surroun-

ding tissue during implantation. In

such a case, it became necessary to

mold the implant between 2 fingers

into the philtrum or the white roll.

Injection should be performed by

linear threading. Patients were told

to return after an additional month

for follow-up and possible reinjec-

tion to correct asymmetry or lack

of desired fullness. These touch-ups

were performed later, usually after 1

or 4 weeks, with 0.1-0.2 mL agarose

gel. Patients who received a further

injection (0.1 mL) 3-4 weeks after

the first injection only needed an

additional injection after 8-12 mon-

ths to maintain the desired results.

Finger compression of the angular

artery and direct compression with

cotton gauze were immediately ap-

plied to reduce bruising. Following

hemostasis, the area was com-

pressed with ice for 3-4 minutes

to reduce swelling. Patients were

warned not to drink hot tea or cof-

fee until anesthesia effects wore

off. Afterward, no special instruc-

tions were needed, and the patients

returned to work immediately. The

satisfaction or dissatisfaction ex-

perienced by the patients was eva-

luated through

RESULTS

All of the patients were success-

fully treated with injection of aga-

rose gel. Clinical improvement was

noted immediately, and only mild

bruising was recorded. All of the

patients returned to clinic 10 days

after treatment for follow-up, and

all of them felt to have obtained an

excellent cosmetic result (Figs. 1-7).

The mean score of satisfaction of

cosmetic result was 8-10 immedia-

tely after treatment, and the score

decreased after some months

(Table I). The results lasted 5 mon-

ths with a gradual decline to base-

line (Fig. 8). Agarose gel was very

well tolerated with only a few mild

adverse reactions which resolved

spontaneously. No major complica-

tion was observed.

No persistent ecchymosis, pain,

itching, outbreaks of herpes, in-

fectious processes, palpable im-

plants, uneven distribution, visible

implants, overcorrection, under-

correction, allergies, hypersensiti-

vity reactions, or nodularity (per-

manent or transient based on the

type of implant and its depth) were

observed.

InaResearcher,Dental School, University of Chieti-Pescara.bChief, Maxillofacial Surgery, University of Ferrara.cProfessor of Oral Pathology and Medicine, Dental School, Universityof Chieti-Pescara.Supported in part by the National Research Council (CNR), Rome,Italy, and by the Ministry of Education, University, and Research(M.I.U.R.), Rome, Italy.Received for publication Jan. 26, 2009; returned for revision Mar. 18,2009; accepted for publication Apr. 16, 2009.1079-2104/$ - see front matter© 2009 Published by Mosby, Inc.doi:10.1016/j.tripleo.2009.04.025

Antonio Scarano, DDS, MD, MS,a Francesco Carinci, DDS, MD,b and

Adriano Piattelli, MD, DDS,c Chieti and Ferrara, Italy

UNIVERSITY OF CHIETI-PESCARA AND UNIVERSITY OF FERRARA

Fig. 1. Before lip augmentation with agarose gel. The white cream at the upper left nasolabial fold is the dermal anesthetic cream Emla.

Fig. 6. The patient in Fig. 5 immediately after injection of agarose gel.

Fig. 3. A 58-year-old patient with the typical signs of an aging lip before treatment. This patient had a full lip with a vermilion curl that was lost with aging.

Fig. 7. Rejuvenation of lip with 1.5 mL agarse gel (same patient as in Fig. 5, immediately after injection). The results lasted approximately 4 months with a gradual decline to baseline.

Fig. 4. The patient in Fig. 3 immediately after injection of agarose gel.

Fig. 8. After 6 months of lip augmentation with 1.5 mL agarose gel. The results lasted approximately 6 months with a gradual decline to baseline.

Table I. Score of satisfaction of patients Score

Immediately after treatment 8-10

1 month 7-8

2 month 6-8

4 month 4-5

6 month 3-4

12 month 2-3

Table II. Esthetic indications for agarose gel

Lip augmentation

Nasolabial wrinklesMentolabial foldsFacial outlinesCorrection of facial depressionsZygomatic augmentationChin augmentation

Table III. Complications observed in litterature

Filler Adverse reactions/side effects Outcome

CollagenAutologous collagen harvested fromthe patient treatedHyaluronic acid L-Polylactic acidAgarose gel

Ecchymosis, possible infections; no allergies/hypersensitivity reaction Rare allergic/hypersensitivity reactions;acneiform eruptions, granulomas Hypersensitivity? Allergic reaction? Transitory bruising

Lasting up to 6 monthsLasting up to 6 months

Lasting up to 6 months

Lasting up to 6 monthsLasting up to 6 days

DISCUSSION

To our knowledge, this is the first

study reported in the literature of

the use of agarose gel for lip aug-

mentation. Decreased skin laxity,

along with habitual repeated

contraction of the underlying facial

muscles, result in wrinkles or rhy-

tids. In general, the aging process

of the face is a process of atrophy.

The ratio of type I to type III col-

lagen diminishes, and elastic fibers,

which maintain the pattern of col-

lagen bundles, become thin and

fragmented, resulting in an overall

reduction in the total amount of col-

lagen. In addition to aging, external

processes, such as actinic damage,

may accelerate this decline. Lips

are the foundation on which the

remainder of the perioral region is

centered. In modern esthetics, full

lips provide a youthful healthy ap-

pearance. Visually, an aging lip is

characterized by a decrease in the

vermilion show, blunting of the Cu-

pid’s bow, and an attenuated white

roll.9 Lip rejuvenation primarily for

the treatment of perioral rhytids is

a procedure commonly requested

by patients who are typically 50

years of age and who smoke or are

former smokers. Enhancement of

the vermilion border is one of the

most rewarding indications for aga-

rose gel. There is a natural pocket

between the vermilion border and

the orbicularis oris muscle that

should be filled. Agarose gel is an

excellent filler material to achieve

minimally invasive lasting impro-

vement of facial wrinkles, furrows,

and other soft tissue contour de-

ficiencies of similar size. There is

a broad spectrum of well defined

medical and esthetic indications

for the use of agarose gel outlined

in Table II. The viscosity of agarose

gel is lower than that of other fil-

lers, and a low and constant pres-

sure can be applied throughout the

injection procedure, depending on

the tissue and depth of placement.

This simple addition to the tech-

nique is easily and quickly mas-

tered. Agarose gel will give a

long-lasting lip augmentation if im-

planted correctly. It is important to

place it into the deep dermal plane

with slight overcorrection. Local

anesthetic might be not indicated,

because there is slightly more dis-

comfort during injection, because

the viscosity of agarose gel is lower

than that of other fillers, and be-

cause the patients tend to expe-

rience less postoperative pain.

Fig. 2. The patient (female) in Fig. 1 immediately after injection of 1.5 mL agarose gel.

Fig. 5. Female patient before injection. This patient never had a full lip.

W A K E F O R E S T I N S T I T U T E F O R R E G E N E R A T I V E M E D I C I N E

REFERENCES 1. Scarano A. Ringiovanimento dei tessuti molli periorali con agarose gel. Dent Clin 2009;2:5-13. 2. Cho YS, Hong ST, Choi KH, Chang YH, Chung AS. Chemopreventive activity of porphyrin derivatives against 6-sulfooxymethylbenzo[ a]pyrene mutagenicity. Asian Pac J Cancer Prev 2000;1:311-7. 3. Marczylo T, Arimoto-Kobayashi S, Hayatsu H. Protection against Trp-P-2 mutagenicity by purpurin: mechanism of in vitro antimutagenesis. Mutage-nesis 2000;15:223-8. 4. Naziruddin B, Durriya S, Phelan D, Duffy BF, Olack B, Smith D, et al. HLA antibodies present in the sera of sensitized patients awaiting renal transplant are also reactive to swine leukocyte antigens. Transplantation 1998;66:1074-80. 5. Gu Y, Tabata Y, Kawakami Y, Balamurugan AN, Hori H, Nagata N, et al. Development of a new method to induce angiogenesis at subcutaneous site of streptozotocin-induced diabetic rats for islet transplantation. Cell Transplant 2001;10: 453-7. 6. Andre P. Evaluation of the safety of a nonanimal stabilized hyaluronic acid (NASHAFQ-Medical, Sweden) in European countries: a retrospective study from 1997 to 2001. J Eur Acad Dermatol Venereol 2004;18:422-5. 7. Cohen JL. Understanding, avoiding, and managing dermal filler complications. Dermatol Surg 2008;34:S92-9. 8. Godin MS, Majmundar MV, Chrzanowski DS, Dodson KM. Use of Radiesse in combination with Restylane for facial augmentation. Arch Fac Plast Surg 2006;8:92-7. 9. Maloney BP. Aesthetic surgery of the lip. In: Papel ID, editor. Facial plastic and reconstructive surgery. 2nd ed. New York: Thieme Medical; 2002. p. 344-52. 10. Salles AG, Lotierzo PH, Gemperli R, Besteiro JM, Ishida LC, Gimenez RP, et al. Complications after polymethylmethacrylate injections: report of 32 cases. Plast Reconstr Surg 2008;121: 1811-20. 11. Duffy DM. Complications of fillers: overview. Dermatol Surg 2005;31:1626-33.

Moreover, agarose gel is injected

in a more economic way, because

there is no loose material during

implantation. Patient satisfaction

was very high. The rate of side ef-

fects and complications after aga-

rose gel treatment in this study

was lower compared with the com-

plications observed with other fil-

ler injections. 10 Agarose gel is not

broken down by any enzyme, and

therefore it causes no hypersensi-

tivity reactions or tissue hardening.

The only complication observed

in 3 patients in the present study

was a severe bruising that lasted

about 1 week after treatment. All 3

patients had forgotten to disconti-

nue aspirin before treatment. No

nodule ulceration or scarring was

observed. The complications ob-

served by other authors are listed in

Table III. 11 The present study confir-

med the good results obtained in a

previous study, where agarose gel

was used for rejuvenation of perio-

ral tissues and lip augmentation,

nasolabial wrinkles, mentolabial

folds, facial outlines, correction of

facial depressions, zygomatic aug-

mentation, and chin augmentation. 1 Furthermore, the molecule was

saturated with water, meaning that

there would be no additional water

attraction in the tissues.

In conclusion, during 3 years of

clinical use, agarose gel proved to

be a reliable and predictable treat-

ment for lip augmentation. The

authors express their thanks to Dr

Michela Marroni for linguistic assis-

tance and Dr. Raffaella Scarano for

the psychologic assistance of pa-

tients.

Introduction

This is a final report of the comparison

between the injection of Algeness®

and Juvederm® in a mouse model.

The objective of the study was to

compare the longevity/persistence

and adverse effects of the dermal

filler materials.

Methods

Female C57Bl/6 mice were used

in this study. Time points included

in the study were 1 week, 1 month,

3 months and 6 months post-in-

jection. Six mice were included

at each time point for a total of

24 mice. Each mouse received

one injection of Juvederm® Ultra

Plus (“Juvederm”), one injection

of Algeness®, and one injection of

phosphate-buffered saline (PBS)

subcutaneously. The injection vo-

lume was 0.25 ml for each material.

At each time point, three mice

received the Juvederm® injection in

the left craniodorsal quadrant, the

Algeness injection in the mid-late-

rodorsal quadrant, and PBS in the

left caudodorsal quadrant. The re-

maining three mice received the

Algeness® injection in the left cra-

niodorsal quadrant, the Juvederm

injection in the mid-laterodorsal

quadrant, and PBS in the left cau-

dodorsal quadrant. Changes in the

site of injections were performed to

reduce any site specific effects on

the injected material.

At the time of retrieval, the ma-

terial was measured using digital

calipers to determine the volume.

The volume was calculated using

the formula: V = ∏ ÷ 6 x L x W x H

where L is length, W is width and H

is the height of the recovered ma-

terial. The retrieved material was

also weighed. The material pliabi-

lity was scored on a sale of 1 to 4

with 1 being the softest and 4 being

the hardest. The pliability of the in-

jected material was determined by

the same individual for all samples

to maintain consistency during the

study.

After determining size, weight and

pliability, the harvested material

was processed for histology. Part

of the material was fixed in 10%

formalin and prepared for paraffin

embedding. These samples were

sectioned and stained with hema-

toxylin and eosin (H&E) to show

the overall appearance of the in-

jected material and with Masson’s

Trichrome to identify collagen de-

position. The remaining material

was processed for frozen sections

that were used for the CD68 and

Ly6g immunohistochemistry stai-

ning. CD68 is expressed by macro-

phages and Ly6g is expressed by

neutrophils.

Results

The injections were completed wi-

thout any adverse events. There

were adequate amounts of both

the Juvederm® and the Algeness®

filler material.

Algeness® filler had the consisten-

cy similar to petroleum jelly. This

consistency did not allow the use

of the supplied 27 gauge needle.

A 26 gauge needle was used that

allowed for better extrusion of the

material. The Algeness® syringes

did not have calibrations and re-

quired transfer to marked syringes

for appropriate measurement. There

was some product loss due to this

step. Additionally, this transfer and

the high viscosity of the material

caused some air entrapment in the

materials that reduced the uniformity

of the injection.

The Juvederm® was more smoo-

thly and consistently injected. The

calibrations on the syringe barrel

were helpful in determining the vo-

lume administered.

The PBS injection was rapidly ab-

sorbed and showed negligible vo-

lumetric change.

Generally, Juvederm® appeared

to migrate to a more extent than

Algeness, particularly at the later

time points. Because of this migra-

tion and spreading, it became more

difficult to locate the injected ma-

terial at the later time points. The-

refore, the measurements (volume

and weight) have increased error

associated with their values.

Physical Measures of the Injected

Material

At harvest, the volume of Juvederm®

and Algeness® decreased over time

(Figure 1). Juvederm® had a greater

volume initially but decreased to

similar values for Algeness®.

The weight of the harvested

Juvederm® material decreased

over time that matched the volume

measurements (Figure 2). However,

the weight of the harvested Algeness®

material maintained a consistent level

over time.

The pliability of the Juvederm®

decreased over time; whereas

the Algeness® maintained a more

consistent pliability score (Figure 3).

Evaluation of Algeness as a Dermal Filler MaterialNovember 2014Final Report

John D. Jackson, PhD, Associate Professor, Institute for Regenerative Medicine

Institute, Wake Forest Baptist Medical Center

Overall, these results suggest that Algeness® maintained

a more consistent weight and pliability than Juvederm®,

even though the volume of both materials decreased

over time. The injected Algeness® material did not

migrate post-injection to the extent that occurred with

Juvederm®.

Histological analysis

By week 1 post-injection, cellular infiltration is seen in

both materials that includes some immune cells (macro-

phages) and what appear to be stromal elements

(probably fibroblasts) (Figures 4 and 5). No collagen

deposition was seen (Figure 4).

At week 4 post-injection, a lattice work of cells can

be seen in the injected material from both Algeness®

and Juvederm® (Figure 6). These cells appear to be

fibroblasts since collagen deposition is seen (Figure 6).

Immune cells are present in both injected materials (Fi-

gure 7).

At week 12, Algeness® has maintained its lattice of stro-

mal cells; however, Juvederm® has become more

disorganized (Figure 8), potentially due to the in-

creased migration of Juvederm®. Collagen is also present

in both materials (Figure 8). Algeness® does not appear

to have an immune infiltration by week 12 (Figure 9). A

few CD68 positives cells can be seen in the Juvederm®

sample (Figure 9).

At week 24 post-injection, Algeness® has maintained

the same morphological appearance that was seen at

the earlier time points (Figure 10). Juvederm® appears

to have significant collagen deposition (Figure 10). No

immune cells can be seen in either of the materials at

this time point (Figure 11).

Volume of Dermal Filler

Weeks Post-Injection

Vo

lum

e (

mm

3)

F I G U R E 1

Weight of Dermal Filler


We

igh

t (g

)

F I G U R E 2

Material Pliability


Pliab

ilit

y R

ati

ng

(1t

o 4

)

F I G U R E 3

Trichrone Staining

F I G U R E 4

H&E and Masson’s Trichrome staining of paraffin embedded sections of Algeness and Juvederm at 1 week post-

injection (200x magnification).

H&E Staining

Algeness

Juvederm

F I G U R E 5

CD68 staining of frozen sections at 1 week post-injection. (200x magnification)

Algeness

Juvederm

Trichrone Staining

F I G U R E 6

H&E and Masson’s Trichrome staining of paraffin embedded sections of Algeness and Juvederm at 4 weeks post-


H&E Staining

Algeness

Juvederm

F I G U R E 7

CD68 and Ly6g staining of frozen sections at 4 weeks post-injection. (200x magnification)

Ly6g StainingCD68 Staining

Algeness

Juvederm

Trichrone Staining

F I G U R E 8



H&E Staining

Algeness

Juvederm

F I G U R E 9

CD68 and Ly6g staining of frozen sections at 12 weeks post-injection. (200x magnification)

Ly6g StainingCD68 Staining

Algeness

Juvederm

Trichrone Staining

F I G U R E 1 0



H&E Staining

Algeness

Juvederm

F I G U R E 1 1

Ly6g staining of frozen sections at 24 weeks post-injection. (200x magnification)

Algeness

Juvederm

Conclusion

Algeness® maintained a more consistent presence at the injection site. Although there was some migration of the

Algeness®; significantly more migration of Juvederm® was seen at the later time points. Juvederm® had a more

disorganized appearance at 12 and 24 weeks post-injection. Both materials had collagen deposition by week 4 post-

injection. Immune cells infiltrated both materials during the early time points, but no overt immune response was

seen at the later time points in either injected materials. No immune cells were detected by 24 weeks post-injection

in either material.

Algeness® is a product of Advanced Aesthetic Technologies, Inc., Suite 427, One Brookline Place, Brookline, MA, USA 02445Juvederm® is a product of Allergan Inc, Irvine, CA 92612 USA

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Plastic and Reconstructive Surgery • October 2007

1

Advances Aesthetic Technologies, Inc. –Final ReportFebruary 2017

Amanda Kern, Albert Robbat, Jr.Tufts University Sensory and Science Center200 Boston Avenue, Suite G700Medford MA 02155

Objective: To assess if Hylenex® recombinant (hyaluronidase human injection) degrades/reacts with Algeness® DF, an agarose-based dermal filler

Methods: First, pure agarose (0.2% and 1.5%) was exposed to Hylenex® recombinant (hyaluronidase human injection) and β-agarase. Liquid chromatography/mass spectrometry was used to detect the presence of oligosaccharides, which are known breakdown products of agarose. Finally, the effects of hyaluronidase and β-agarase on Algeness® DF were compared.

Results: Hylenex® recombinant (hyaluronidase human injection) is the so-called gold standard for treating over-injection of hyaluronic acid dermal fillers. Hyaluronidase cleaves the β-1,4 linkage of hyaluronic acid. While the structure of hyaluronic acid and agarose are very different,it appears hyaluronidase slowly hydrolyzes agarose at the β-1,4 bond. As expected, β-agarase cleaved agarose at the β-1,4 glyosidic linkages producing the dimer, tetramer and hexamer neoagarooligosaccharides for 0.2% agarose, and the dimer and tetramer for 1.5% agarose. The presence of the hexamer was also observed in both the 0.2% and 1.5% agarose samples exposed to Hylenex®. Similar results were observed in the enzymatic degradation of Algeness® DF when exposed to β-agarase and Hylenex®. The dimer and tetramer products were observed in the β-agarase/ Algeness® DF reaction, while the hexamer was observed in the Hylenex®/Algeness® DF reaction.

Conclusions: The enzymatic hydrolysis of pure agarose by β-agarase produced three compounds, neoagarobiose, neoagarotetraose and neoagarohexaose, which result from the cleavage of β-1,4 glycosidic bonds. The enzymatic hydrolysis of agarose by Hylenex® produced one product, neoagarohexaose in low amounts. Confirmation of these findings require additional experimentation however results are promising.

2

Introduction

Agarose, a linear polymer composed of repeating units of D-galactose and 3,6-anhydro-L-galactopyranose, linked by α-1,3 and β-1,4 glycosidic bonds (Figure 1A). β-agarase hydrolyzes agarose at the β-1,4 bond. The resulting reaction produces a series of neoagarooligosaccharideswith repeating disaccharide units (Figure 1B).1 Depending on the cleavage site, neoagarooligosaccharides of varying molecular mass are produced, including neoagarobiose (324.28 g/mol), neoagarotetraose (630.55 g/mol) and neoagarohexaose (936.82 g/mol).

Hylenex® recombinant (hyaluronidase human injection) is an FDA approved enzyme, and current standard for the off-label treatment to overcorrect hyaluronic acid (HA) based dermal fillers.2-4 Hyaluronidase hydrolyzes HA by cleaving the β-1,4 bond between the glucosamine and glucuronic acid (Figure 2).

The objective of this study was to determine if Hylenex® recombinant hydrolyzes the agarose-based dermal filler, Algeness® DF, at the β-1,4 linkage between D-galactose and 3,6-anhydro-L-

Figure 2. Hyaluronic acid (HA) is composed of repeating units of D-Glucuronic acid and N-Acetyl-D-Glucosamine, linked by β -1,3 and β-1,4 bonds. Hyaluronidase cleaves HA at the β-1,4linkage, producing a series of repeating disaccharide units. Image from: www.sigmaaldrich.com

Figure 1. A) Agarose is a polysaccharide that consists of repeating units of D-galactose and 3,6-anhydro-L-galactopyranose. B) β-agarase cleaves agarose at the β-1,4 bonds between D-galactose (G) and 3,6-anhydro-L-galactopyranose (A) producing a series of neoagarooligosaccharides consisting of repeating disaccharide units.

3

galactopyranose. Neoagarotetraose was used as a reference standard to confirm enzymatic activity in this study.Experimental

Control Samples – 0.2% Agarose Gel

Agarose powder (0.2g) was added to 100ml of water (99°C) and cooled in a water bath to 42°C.Three control samples were prepared using 1000µl of molten agarose (2mg). Samples were incubated at 42°C for 2 hr, and then either 1) centrifuged at 3000g for 5 min at 4°C, 2) centrifuged at 12000g for 5 min at 4°C or 3) heated at 90°C for 10 min (so-called “kill enzyme”step) and centrifuged at 12000g for 5 min at 4°C. The supernatant was filtered using 0.2 µm Nylon membrane filters. All samples were analyzed by LC/MS to determine if sample preparation methods contributed to the degradation of agarose gel. Additionally, the Molisch test for sugars was conducted on all samples.

Enzyme Degradation – 0.2% Agarose Gel

Agarose gel (0.2%) was exposed to Hylenex® recombinant (hyaluronidase human injection) and β-agarase to compare the effects of enzymatic degradation on the two samples. Briefly,Hylenex® (200µl – 30 units) was added to agarose (200µl – 0.4mg agarose) and incubated at37°C for 2 hr. Additionally, β-agarase (5µl – 5 units) was added to agarose (1000µl – 2mg agarose) and incubated at 37°C for 2 hr. Samples were centrifuged at 12000g for 5 min at 4°C.The supernatant was filtered using 0.2 µm Nylon membrane filters and analyzed by LC/MS. The Molisch test for sugars was conducted on all samples.

Enzyme Degradation – 1.5% Agarose Gel

Agarose gel (1.5%) was exposed to Hylenex® recombinant (hyaluronidase human injection) and β-agarase to compare the effects of enzymatic degradation. Briefly, Hylenex® (200µl – 30 units) was added to agarose (400µl – 14mg agarose) and incubated at 37°C for either 2 or 48 hr.Additionally, β-agarase (30µl – 30 units) was added to agarose (400µl – 14mg agarose) and incubated at 37°C for either 2 or 48 hr. Samples were centrifuged at 10000g for 5 min at 4°C.Supernatant was filtered using 0.2 µm Nylon membrane filters and analyzed by LC/MS.

Enzyme Degradation - Algeness® DF

Algeness® DF (3.5% agarose) was exposed to Hylenex® recombinant (hyaluronidase human injection) and β-agarase to compare the effects of enzymatic degradation. Briefly, enzyme was added to the dermal filler and incubated at 37°C for 48 hr. Table 1 shows the amount of dermal filler and enzyme used for each reaction. After incubation period, samples were heated at 90°Cfor 10 min (“kill enzyme”) and then centrifuged at 10000g for 5 min at 4°C. Supernatant was filtered using 0.2 µm Nylon membrane filters and analyzed by LC/MS.

Sample Vol. Algeness® Vol. Hylenex® Vol. β-agaraseAA 400µl – 14 mg Agarose - 30µl – 30 unitsAH 400µl – 14 mg Agarose 200µl – 30 units -

Liquid Chromatography/Mass Spectrometry

4

Separations were performed on an Agilent 1260 LC/MS equipped with a cooled autosampler tray (4°C) and temperature-controlled column compartment (20°C), which held a 2.1 x 100mm i.d., 3.5µm particle size C18 Zorbax Eclipse Plus column (Agilent, Santa Clara, CA). Sample injection volumes were 5 uL, except for the Algeness/Hylenex reaction which was 25uL. A gradient elution was employed with a mobile phase composition of 0.5mM ammonium acetate in 18Ω Millipore water (A) and acetonitrile (B), and flow rate of 0.4 mL/min. The gradient profile is shown in Table 2.

Time (min) % A % B0.5 100.0 0.05 2.0 98.06 2.0 98.07 100.0 0.08 100.0 0.0

The MS was operated in positive ionization mode for agarose and negative ionization mode for hyaluronic acid, with a fragmentation voltage of 100V. Spectra were recorded in full scan mode from 300 to 2000 m/z. Neoagarotetraose, a known degradation product of agarose when exposed to β-agarase, was used to confirm enzyme activity.

Results

Agarose Gel

The analysis of pure agarose and β-agarase and Hylenex® were carried out to determine whether the enzymes hydrolyzed the starting material. After incubation, samples were analyzed by LC/MS to determine the presence of neoagarooligosaccharides, thus confirming if the enzymeproduced the expected products. Neoagarotetraose was used as a reference standard. As expected, the analysis of the 0.2% agarose/β-agarase sample produced three breakdown products,neoagarobiose, neoagarotetraose and neoagarohexaose (Figure 3). In contrast, LC/MS analysis of the 0.2% agarose/Hylenex® sample revealed the hexasaccharide only (Figure 4). For the 1.5% agarose, neoagarobiose and neoagarotetraose were observed in both the 2 hr and 48 hr reactions with β-agarase. On the other hand, no products were observed in the 2 hr reaction with Hylenex®, while neoagarohexaose was the only product observed in the 48 hr reaction.

Although results are not quantitative, neoagarotetraose produced the highest signal in the agarose/β-agarase sample. The neoagarohexaose signal from the reaction of agarose/Hylenex® was buried in the sample matrix, but observable using spectral deconvolution data analysis software, at near instrument detection limits. Based on these results, Hylenex® hydrolyzesagarose, however, further studies should be conducted to determine reaction rate and amount.Note: all observations are based on one analysis. Additional experiments should be performed to confirm findings.

5

Figure 4 Neoagarohexaose was the only compound identified in the 0.2% agarose/ Hylenex®sample by LC/MS.

Figure 3. Ion Analytics Deconvolution Software (Robbat) confirmed the presence of neoagarooligosaccharides in the 0.2% agarose/β-agarase sample.

6

Algeness® DF

The Molisch test is a qualitative analysis indicating the presence of carbohydrates in a sample. Apositive result is indicated by a purple ring between the sample and the acid. The absence of sugars results in a clear solution. The presence of monosaccharides yields a faster reaction, while disaccharides and polysaccharides are slower to react. Immediately upon adding the Molisch reagent to the test tube, the Algeness®/β-agarase sample turned dark purple (Figure 5). The

control (Algeness®/water) formed a light purple ring at the interface of the sample and acid, presumably due to concentrated acid hydrolysis. After 1 hr, the Algeness®/β-agarase and the control remained the same, while the Algeness®/Hylenex® formed a light purple ring. The agarose/Hylenex® turned a darker shade of purple after 2 hr. Based on these observations, the Algeness®/β-agarase produces smaller chain sugars compared to Algeness®/Hylenex®.

For the LC/MS analyses, reactions were incubated for 48 hr at 37°C, then heated to kill the enzyme prior to analysis. Neoagarohexaose was the only product observed in the Algeness®/Hylenex® reaction. Similar to the pure agarose experiments, detection of the compound occurred at the detection limit of the instrument and hidden by other components in the sample (Figure 6). Overall, these results in combination with those observed for the pure agarose suggest that Hylenex® hydrolyzes agarose. However, further studies should be conducted to determine reaction rate and amount. Note: all observations are based on one analysis and should be repeated to confirm findings.

Figure 3. Molisch test results for Algeness®/ β-agarase, Algeness®/ Hylenex® and Algeness®/water (control). At time = 0 min (left), the Algeness®/ β-agarase reacted immediately, while the control formed a small purple ring. After 60 min (middle), the Algeness®/ Hylenex® began to form a purple ring. At 120 min (right), the Algeness®/Hylenex® had a strong positive signal.

7

References

1. Fu, X. T.; Kim, S. M., Agarase: Review of Major Sources, Categories, Purification Method, Enzyme Characteristics and Applications. Marine Drugs 2010, 8 (1), 200-218.

2. Brody, H. J., Use of Hyaluronidase in the Treatment of Granulomatous Hyaluronic Acid Reactions or Unwanted Hyaluronic Acid Misplacement. Dermatologic Surgery 2005, 31 (8), 893-897.

3. Buhren, B. A.; Schrumpf, H.; Hoff, N.-P.; Bölke, E.; Hilton, S.; Gerber, P. A., Hyaluronidase: from clinical applications to molecular and cellular mechanisms. European Journal of Medical Research 2016, 21 (1), 5.

4. Cavallini, M.; Gazzola, R.; Metalla, M.; Vaienti, L., The Role of Hyaluronidase in the Treatment of Complications From Hyaluronic Acid Dermal Fillers. Aesthetic Surgery Journal 2013, 33 (8), 1167-1174.

Figure 4. Neoagarohexaose was the only compound identified in the Algeness DF ®/Hylenex® sample by LC/MS. The ion signal was buried in the matrix noise

Aesthetic Medicine / Volume 3 / Nº 1 / January - March 2018 21

Agarose gel - high patient satisfactionof a full-facial volume augmentationArna Shab1, Catharina Shab2

1MD, Private Practice for Dermatology and Aesthetic Medicine, Frankfurt/Main, Germany2MD, Private Practice for Dermatology and Aesthetic Medicine, Frankfurt/Main, Germany

Review

AbstractAgarose gel is a new generation of dermal filler. It is a sterile, biodegradable, viscoelastic, isotonic, transparent injectable gel implant, which was already approved and used for more than a decade. It can be used for face reconstruction and face modelling. Facial aging consistently produces increasing prominence of the midface, nasolabial folds, cheeks, jowls, chin and nose.The aim of this article is to show agarose gel as at least equivalent to those products for full-facial treatment and non-surgical rhinoplasty.This material provides a non-surgical method for volume augmentation and restoration of the face and structures like the aging nose. Injecting agarose gel is like to inject a liquid implant. Attention must be paid to the injection technique and the post injection treatment. Visible improvements are noted immediately. Only mild bruising or hematoma are recorded as adverse events. The patients are highly pleased with the results and report that an excellent cosmetic result was obtained. The agarose gel was very well tolerated.Agarose gel is at least equivalent to other dermal fillers for full-facial treatments and non-surgical rhinoplasty. The utilization of these fillers by trained professionals provides an effective and safe therapy for the management of the aging face.

Keywords Agarose gel, dermal filler, full-facial, augmentation, restoration, aging face, wrinkles

Accepted for publication 25 January 2018 - © Salus Internazionale ECM srl - Provider ECM nº 763

Correspondence

Arna Shab, MD Address: Hanauer Landstrasse 151 - 153, Germany - 60314 Frankfurt/MainPhone: + 49 (0) 69 48 00 94 40E-mail: [email protected]

Agarose gel - high patient satisfaction of a full-facial volume augmentation

23Aesthetic Medicine / Volume 3 / Nº 1 / January - March 2018

involvement in or around the area to be treated were excluded. Pregnancy, lactation and hyaluronic acid treatment less than 3 months earlier were also excluded criteria. After discussing patient in-depth information and written consent, discussing the risks and benefits of the procedure, the risks and benefits of alternatives, and answering all questions, the written consent form outlined possible complications such as bruising, swelling and hematoma or pain. To reduce bruising, patients were asked not to take salicylates in the last 2 weeks before treatment.In total 27 patients were treated (14 non-surgical nose jobs, four augmentation of jaw angle and nine nasolabial folds). The patients were between 32 and 68 years old. All patients were female. Nobody had a treatment with permanent fillers before. 5 previously had an injection with hyaluronic acid in the area of the nasolabial fold.For a better comfort, a superficial anesthetic cream was applied. The agarose was mixed with 0.1 mL of lidocaine 0.1% to be as painless as possible during the injection. For the injection of the nose and nasolabial fold 30 gauge needles should be used with a length of 13 mm, for the jaw angle 27 gauge needles. The esthetic evaluation was done after 14 days, and 1, 3 and 6 months after the injection. While a volume of 1.4 mL agarose gel 2.5% was sufficient for the nasolabial fold (Figure. 1), 2.8 mL were necessary for the jaw angle of agarose gel 3.5% (Figure. 2). However, for the rhinoplasty, only 0.3 mL of 3.5% agarose gel was sufficient (Figure. 3). Direct finger compression with cotton gauze and mild cooling were used to reduce bruising and swelling. Further, no special instructions were required and the patients immediately returned to work. The only adverse events described were hematoma, redness, bruising and swelling. All adverse events lasted for a maximum of 4 days.Patients were asked to re-present 14 days and one month after injection for follow-up and possible reinjection to correct for asymmetry or lack of desired fullness. These repairs were usually made after 14 or 30 days, with 0.1 mL agarose in the area of the nose (by only three patients), 0.2-0.3 mL at the nasolabial folds (six patients), and a maximum of 0.4 mL at the jaw angle area (two patients). Such improvements were not necessary in these three patients (see below in Figures 1-3). Therefore, we asked for an additional follow-up after 3 and 6 months. All results remained after 6 months.

nasolabial fold is intensified. By increasing the cheek fat above the nasolabial fold this impression is emphasized.

Agarose gel as dermal filler

The cutaneous and subcutaneous shrinkage of fat or bone resorption can be temporarily compensated by appropriate injections of collagen, autologous fat, hyaluronic acid or even agarose gel6,16,17.Agarose is in principle not a completely new material in medicine. It has been used in the dental field for more than ten years6. The substance class is a polysaccharide of D-galactose and 3,6-anhydro L-galactose, which are glycosidically linked. Thus it represents a main component of the agar7. 100% based on natural polysaccharides, it is completely biocompatible and thus degradable. Agarose gel is a sterile, biodegradable, viscoelastic, isotonic, transparent injectable gel implant16,18.Agarose is broken down in the human organism. For this purpose, agarose is first transported by the action of macrophages from the site of application and then degraded enzymatically by means of galactosidase. Agarose is metabolized in the pentose cycle at macrophage, platelet and endothelial reticulum levels6,16.Because of its biocompatibility, agarose is widely used in clinical trials. Therefore, the substrate is used in biocompatible tests, for example with regard to cytotoxicity, genotoxicity19, mutagenesis20, sensitivity21, and subcutaneous implants22.In addition, the gel is used in biotechnology for three-dimensional tissue growth and as a controlled release substrate for pharmacological substances.

Application of agarose gel

For the preparation of the treatment a superficial anesthesia with a topical anesthetic is recommended. In some cases, a local anesthetic injection should also be considered. Treatment should be as painless as possible. In addition to topical anesthesia, the use of very thin cannulas also serves this purpose. It can also be mixed local anesthetic with the gel. Agarose itself is because of its isotonic properties, as mentioned above, an almost painless injectable. The injection should be done very slowly. Only when stretching out of the tissue does it burn. It is essential to have an extended massage of the injected area to disperse the imported substance with the surrounding tissue. Agarose gel transforms into a hydrocolloid after injection into the tissue. This creates a natural and harmonious look. According to the principle “What you see is what you get”, the result visible immediately after the injection is also the final result. An additional advantage is the use in patients who have demonstrated intolerances to hyaluronic acid or other ingredients in previous treatments.

Material and MethodsIn our practical everyday life we inject a variety of facial areas. The agarose gel used in this study contained 2.5% agarose and 97.5% saline solution for nasolabial fold (Figure. 1) and 3,5% agarose and 96,5% saline solution for rhinoplasty and jaw angle (Figure. 2 & 3). Patients with acute or chronic skin pathologies or direct

Figure 1 - Left before Injection. Right immediately after injection with 1.4

ml agarose gel 2,5% nasolabial fold and marionette-fold.



IntroductionIn recent years there has been a growing interest in non-surgical procedures for facial rejuvenation. Hyaluronic acid (HA) and calciumhydroxyapatite (CHA) are currently the most widely used dermal filler for full facial treatments1. These provide a high level of comfort in the treatment (both for the practitioner and for the patient) and a long-lasting effect. In addition, these are safe substances with regard to their compatibility and local resistance2,3,4. In addition, the proven safety of Agarose gel has also been a factor in their increased use. Although only rarely complications are reported5. Agarose is in principle not a completely new material in medicine. It has been used in the dental field for more than a decade6. The substance class is a neutral polysaccharide, it is completely biocompatible and thus degradable6. The aim of this article is to show agarose gel as at least equivalent to those for full-facial treatment.The following overview describes the possibilities of this treatment method.

General approach

Detailed knowledge of the facial anatomy with the knowledge of changes in the age are necessary to achieve balanced and natural results after the injection. It is also essential to be familiar with the character of the dermal filler to be used. Each face region has its own perfect dermal filler for its purpose. Complete biodegradability, local resistance and good tolerability as well as perfect biocompatibility are indispensable properties of a suitable dermal filler.The results of the filler injection are extremely technically and material dependent. It is necessary to place a 3-dimensional lattice of injected material below the surface of the skin to add volume, alter surface area and thicken skin or subcutaneous tissue. The degree of correction and the result depends heavily on the injection technique used and the material used hence the required volume7. In the past, surgical techniques dominated the field of facial rejuvenation. However, the importance of a three-dimensional volume restoration using dermal fillers has become increasingly recognized in recent years and has increasingly gained precedence over a two-dimensional lift by the scalpel8.

HA dermal fillers

Hyaluronic acid is very hydrophilic, therefore a change in the treated region after injection is expected. That makes the substance perfect for a more superficial correction. It also hydrates the skin, so that wrinkled skin is better glazed9. However, to correct for bone resorption at depth, a substance with less hydrophilic property is needed.

Anatomical changes of the face in old age

No part of the human organism reflects aging like the facial area. The face consists of skin, subcutaneous fat, muscle and bone structure. These individual components age independently, they have their own laws of aging.In old age, not only changes in the surface of the skin are noted. Aging processes also occur in various deep

structures of the human face especially in the area of the maxillary and mandibular; most notably fat loss and bone resorption. Striking is the sinking of soft tissue such as eyebrows and cheeks. Nasal manifestations of the nose result from degradation of the bony and cartilaginous framework. Therefore, the sinking of the tip of the nose and a shortening of the columella arise. Through this downward movement, the nose appears longer and larger. The nose is now larger and the nasolabial angle can be sharpened. In addition, the elderly patient usually has excessively inelastic skin, which makes an injection more difficult.However, there are differences, especially depending on race, genetic factors and individual aspects (some earlier, later)10.

Upper third of the face

The upper third of the face consists mainly of the forehead. Here, dominates the glabella and horizontal folds. These are usually dynamic wrinkles. Here, a treatment with botulinum toxin is recommended.

Middle face third

Therapeutically significant is the midface, especially with respect to the following changes e.g. slackening, sinking of the cheek fat, reduction of hypodermic fat, strengthening nasolabial folds, slackening of the lower eyelids with optical extension. In addition, there is an aging of the nose (extension of the nose, rarification of the nasal skeleton, loss of hypodermic fat in the nasal area, broadening of the nose and convex nose bridge).Overall, the midface in old age tends to change to hollow-cheeked, flat, empty, and narrow10,11.

The aging nose (as part of the middle face)

The nose as the central area in the middle face of the human being is one of the first visible structures for the opposite and the mirror image. It is a complex, three-dimensional, trapezoidal organ protruding from the face. Due to its three-dimensionality, however, the nose causes many people an increased aesthetic distress12.While wrinkles are often only perceived as two-dimensional disturbing strokes, the cosmetic problems and desires of the patients with regard to the appearance of the nose are much more complex. Surgical rhinoplasty is therefore one of the most common aesthetic surgical procedures. As with all surgical interventions, there is a certain amount of rhinoplastyrisk of complications with corresponding convalescence time and downtime. In addition, the operation is associated with high costs. Increasingly, therefore, many patients develop the desire to avoid surgical intervention. Still, most have never heard of a nonsurgical nose job13-15.

Lower third of the face

The lower third of the face extends from the subnasal area to the chin seat. Displacement of the cheek fat caudally. This also applies to the mimic muscles, the zygomatic muscle, major and minor, and the m. risorius.Bone atrophy of the maxilla in anterior-posterior direction and congenital low-grade retrognathia. Due to the sinking cheek fat, the impression of a deepened



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ConclusionThe agarose-based filler is a great new option for modeling and aesthetic correction in nonsurgical rhinoplasty and complete face treatment.Especially for patients who want to avoid surgery. But even for the practitioner, it offers a low-complication possibility of a nose correction with relatively little effort compared to an operative procedure. So far we have had several very good substances (such as hyaluron and calcium hydroxyapatite) available. Now, with agarose gel, another substance enriches the dermal filler range with an interesting option. This popularity of a substance such as agarose will continue to increase in the future as the aging population seeks viable options to correct the signs of aging without surgery. The utilization of these fillers by trained professionals provides an effective and safe therapy for the management of the aging face.

DisclosureThe authors declare that they have no conflicts ofinterest and have not received any contributions for thispublication.

Figure 2 - Left before Injection. Right after injection with 2.8 mL agarose

gel 3,5% jaw angle.

Figure 3 - Left before Injection. Right immediately after injection with 0.3

mL agarose gel 3,5% non-surgical rhinoplasty.

A d v a n c e d A e s t h e t i c Te c h n o l o g i e s , I n c

O n e B r o o k l i n e P l a c e , S u i t e 4 2 7 - B r o o k l i n e , M A 0 2 4 4 5

w w w . a l g e n e s s . c o m - i n f o @ a l g e n e s s . c o m

scientific and clinical s tudies...injection of dermal fillers is one of the most commonly performed...

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