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Human Histology and Persistence of Various Injectable Filler
Substances for Soft Tissue Augmentation
Gottfried Lemperle, M.D., Ph.D.,
1
Vera Morhenn, M.D.,
2
and Ulrich Charrier, M.D
3
1,2
San Diego, CA, USA
3
Frankfurt, Germany
Abstract. An increasing number of soft tissue filler sub-
stances have been introduced to the beauty market outside
the U.S. which lack experimental and clinical data in sup-
port of their claim. Ten commercially available filler sub-
stances were examined for biocompatibility and durability:
0.1 cc of each substance was injected deep intradermally
into the volar forearm of one of the authors and observed
for clinical reaction and permanence. At 1, 3, 6, and 9
months the test sites were excised, histologically examined,
and graded according to foreign body reactions classifica-
tion. Collagen (Zyplast) was phagocytosed at 6 months and
hyaluronic acid (Restylane) at 9 months. PMMA micro-
spheres (Artecoll) had encapsulated with connective tissue,
macrophages, and sporadic giant cells. Silicone oil (PMS
350) was clinically inconspicuous but dissipated into the
tissue, causing a chronic foreign body reaction. Polylactic
acid microspheres (New-Fill) induced a mild inflammatory
response and had disappeared clinically at 4 months.
Dextran microspheres (Reviderm intra) induced a pro-
nounced foreign body reaction and had disappeared at 6
months. Polymethylacrylate particles (Dermalive) induced
the lowest cellular reaction but had disappeared clinically at
6 months. Polyacrylamide (Aquamid) was well tolerated
and remained palpable to a lessening degree over the entire
testing period. Histologically, it dissipated more slowly and
was kept in place through fine fibrous capsules. Polyvinyl-
hydroxide microspheres suspended in acrylamide (Evolu-
tion) were well tolerated, slowly diminishing over 9 months.
Calcium hydroxylapatite microspheres (Radiance FN) in-
duced almost no foreign body reaction but were absorbed
by the skin at 12 months.Host defense mechanisms react
differently to the various filler materials, but all substanc-
es—resorbable or nonresorbable—appeared to be clinically
and histologically safe, although all exhibit undesirable side
effects. Since the mechanism of late inflammation or gran-
uloma formation is still unknown, early histological find-
ings are not useful in predicting possible late reactions to
filler substances.
Key words: Dermal filler substances—Soft tissue augmen-
tation—Aquamid—Artecoll—Dermalive—Evolution—New-
Fill-Radiance FN—Restylane—Reviderm intra
In recent decades, dermal filler substances consisting
of highly viscous fluids [33,40,53] or polymer parti-
cle suspensions [21,37] have been injected beneath
wrinkles and acne scars [8,31]. These substances are
useful for the correction of congenital or traumatic
facial, bony, and soft tissue defects [10], and in pa-
tients suffering from scleroderma, Romberg’s disease,
facial wasting, or lipodystrophy following AIDS
treatment [2,11,57]. Additional indications are uni-
lateral paralysis of vocal cords [12,24,28], augmen-
tation of the lip and soft palate in cleft lip patients,
anophthalmic orbits [10], or enophthalmus. Other
potential applications as bulking agents are lower
esophageal sphincter in gastroesophageal reflux pa-
tients [22,48], and bladder neck or anal sphincter in
patients suffering from urinary [5] or fecal inconti-
nence [27,63].
Animal studies [38] and clinical trials [15,19] have
shown good acceptance and short- and long-term
Presented at the 33rd Annual Meeting of the Association of
German Plastic Surgeons in Heidelberg, Germany, Sep-
tember 21, 2002
Correspondence to Gottfried Lemperle, M.D., Ph.D.,
Division of Plastic Surgery, University of California, San
Diego; email: glemperle@aol.com
Aesth. Plast. Surg. 27:354–366, 2003
DOI: 10.1007/s00266-003-3022-1
efficacy in accordance with the chemical structure
and surface characteristics of the microparticles.
Resorbable materials such as collagen, hyaluronic
acid [19,36,49], polymethylacrylate (PMA) [6], dex-
tran [20], or polylactic acid [2] are removed by
phagocytosis over a period of 3–12 months de-
pending on the amount and type of bulking agent
implanted. Permanent fillers such as paraffin [33],
fluid silicone [11,17], Teflon [34], or silicone particles
[21] have an irregular surface and cannot be phag-
ocytosed but may eventually form foreign body
granulomas due to ‘‘frustrated macrophages’’ [20].
Microspheres below the size of 15 microns [44,61]
are generally phagocytosed and may be transported
to local lymph nodes. Larger microspheres from
nonresorbable polymers with a smooth surface
[35,38,51] are encapsulated with fibrous tissue and
escape phagocytosis. Clinically, all injected fluids
[45,59] and particles [54,56] have been shown to
cause foreign body granulomas in a small percent-
age of patients. Until the mechanism of granuloma
formation is fully understood, the chance of devel-
opment is not predictable.
The ideal soft tissue filler substance for wrinkles,
skin defects, and sphincter
is biocompatible and safe
is stable at the implantation site
keeps its volume and remains pliable
does not cause protrusion of the skin or mucosa
induces minimal foreign body reaction
will not be removed by phagocytosis
has no migration potential to distant locations
does not cause foreign body granuloma.
As a percentage of wet weight, the human skin is
composed of collagen (27–39%), elastin (0.2–0.6%),
glycosaminoglycans (0.03–0.3%), and 60–72%water.
The goal of this study was to confirm histocompati-
bility and permanence of various filler substances
under investigation. For some products there exist no
published scientific reports on biocompatibility, his-
tology, or clinical studies.
Materials and Methods
During the past 4 years, four 0.1-cc blebs of 10 dif-
ferent dermal filler substances have been injected deep
intradermally into the volar skin of a forearm, next to
an existing scar. The injection sites were inspected
weekly and clinical changes were recorded and pho-
tographed. Each raw of the different implants was
excised after 1, 3, 6, or 9 months. At least four sec-
tions were cut from each implantation site at different
levels for histological examination. The sections were
stained with hematoxilin–eosin or Masson trichrome
and evaluated in Frankfurt. An independent pathol-
ogist (U.C.) was blinded, receiving the histological
slices numbered only, and being unaware of the ma-
terial involved.
A classification of foreign body reactions, estab-
lished by Duranti et al. [19], was applied to each
histological slide. The grading was performed on at
least four slices of the same specimen.
Grade I: slight reaction with a few inflammatory
cells
Grade II: clear inflammatory reaction with one or
two giant cells
Grade III: fibrous tissue with inflammatory cells,
lymphocytes, and giant cells
Grade IV: granuloma with encapsulated implants
and clear foreign body reaction
The following commercially available filler mate-
rials were bought and injected in Europe:
1. Zyplast, a suspension of 3.5%crosslinked bovine
collagen, purchased from Collagen Aesthetics,
Inc., Fremont, CA
2. Restylane, a suspension of 2.0%crosslinked
hyaluronic acid (HA) produced biotechno-
logically from Streptococcus equi [36,47,50] in
saline, purchased from Q-Med AB, Uppsala,
Sweden
3. Artecoll, a suspension of 20%microspheres (40
lm) of polymethylmethacrylate (PMMA) in
3.5%bovine collagen solution [35,37], obtained
from Artes Medical Inc., San Diego, CA
4. PMS 350, medical grade silicone fluid (dimeth-
ylpolysiloxane) of 350 centistoke viscosity, pur-
chased from Vikomed, Meinerzhagen, Germany
5. New-Fill, which comes as a powder of poly-L-
lactic acid (L-PLA) microspheres (2–50 lm) to
prepare a 4.5%suspension in 2.7%methylcellu-
lose [2], purchased from Biotech Industry S.A.,
Luxembourg
6. Reviderm intra, a suspension of 2.5%dextran
microspheres (40 lm Sephadex) [20] in 2.0%hy-
aluronic acid (2.5 MDa) of bacterial origin
(Rofilan), obtained from Rofil Medical Interna-
tional N.V. (RMI), Breda, The Netherlands
7. Dermalive, a 240%suspension of hydroxyethyl-
methacrylate (HEMA) fragments in 1.14%
crosslinked hyaluronic acid of bacteriological
origin [6], purchased from Dermatech S.A., Paris,
France
8. Aquamid, a clear 5%crosslinked gel from poly-
acrylamide (PAAG) [42], purchased from Con-
tura International S.A., Montreux, Switzerland
9. Evolution, a suspension of 6%polyvinylhidrox-
ide (PVOH) microspheres (5–80 lm) in 2.5%
polyacrylamide gel, purchased from Laboratoires
ProCytech, Bordeaux, France
10. Radiance FN, a suspension of 30%calcium
hydroxylapatite microspheres (25-40 lm) in a
carboxymethyl-cellulose gel [9], purchased from
BioForm Inc., Franksville, WI
G. Lemperle et al. 355
Results
All four implant sites in the human forearm could
easily be identified after 1 and 3 months; however,
not all implants could be visualized after 6 and 9
months. In the latter cases, the whole area was ex-
cised and sectioned 5–10 times in order to identify
microscopic traces of the implant.
Zyplast
The tissue sections obtained from human skin
showed the presence of many macrophages and in-
vading capillaries at the circumference of the im-
plants at 1 month. This process goes along with slow
resorption of the crosslinked eosinophilic opaque
material with gradual and scarce infiltration of in-
flammatory cells, and little or no deposition of
structures, which resemble new collagen fibers at 3
months [32]. At 6 months, no residue of cell-free
Zyplast could be found in the human skin. The his-
tological reactions were evaluated according to the
Duranti scale (Fig. 1).
Restylane
The clear hyaluronic acid gel induced erythema and
swelling in the human skin for 2 days. Clinically,
Restylane and all other tested crosslinked hyaluronic
acid products disappeared from the skin within 4
months. Histologically, the blue stained hyaluronic
acid showed little foreign body reaction (Fig. 2) but
was slowly degraded by macrophages. Some macr-
ophages and rare giant cells were apparent in the
human skin at 3 months [19]. Clusters of these cells
could still be found after 6 months; however, no
residue could be identified at 9 months.
Artecoll
At 1 month, each individual microsphere was sepa-
rated from adjacent microspheres by a thin layer of
eosinophilic material representing collagen fibers
(Fig. 3). The implants were discrete and well cir-
cumscribed within the soft tissue. The peripheral re-
gions of these foci had infiltrated to a depth of 2–3
microspheres and contained macrophages (1 per ap-
proximately 15 beads) and few multinucleated giant
cells. The centers of the lesions were cell free and
separated through fibrin fibers only. At 3 and 6
months, isolated giant cells extended deeper into the
lesions (Fig. 4).
The implanted denatured collagen appeared to be
resorbed at 3 months. New collagen deposits, evident
at 1 month, increased the spaces between individual
microspheres. The number of inflammatory cells was
small at all times and indicates that collagen and
microspheres induce a minimal immunogenic re-
sponse.
The relative size of the palpable Artecoll lesions in
human skin remained unchanged over 9 months
(Fig. 5), suggesting that dissipation of microspheres
into the surrounding soft tissue or migration to
adjacent lymph nodes does not occur (Fig. 3).
PMS 350 Silicone Fluid
PMS 350 is a colorless oil with a low viscosity, 350
times that of water (1 centistoke = viscosity of wa-
ter), but with high chemical stability. Immediately
after injection, PMS 350 dispersed into the sur-
rounding tissue in form of millions of microdroplets
(Fig. 6). At 1 month, each microdroplet, about 20–
100 microns in size, was encapsulated by a monolayer
of fibroblasts and collagen fibers. Macrophages and
giant cells, which phagocytosed the foamy, translu-
cent, bifringent material, were found at 3 and 6
months. Asteroid bodies in the cytoplasma of macr-
ophages and giant cells were typically seen after
phagocytosis of the silicone fluid. At 9 months, gra-
nulomatous nodules in the dermis and subcutaneous
tissue were surrounded by strands of fibrous tissue.
New-Fill
The PLA implants were well palpable for the first 3
months but had disappeared from the human skin at
4 months. In the refrigerator, PLA microspheres with
diameters between 2 and 50 lm, were still recogniz-
able as microspheres 2 weeks after suspension, and
were partly hydrolyzed and deformed at 1 and 3
months.
Histologically, a fine capsule could be observed
around the implant throughout. At 3 months, the
microspheres had remained spherical and were sur-
rounded by macrophages and some lymphocytes
(Fig. 7). At 6 months, most microspheres showed a
porous surface structure, were fissured and sometimes
deformed, and were surrounded by macrophages and
small giant cells. Their pseudopodia infiltrated the
surface of some microspheres but did not degrade the
activity of these cells. The PLA was likely dissolved by
hydrolysis and extracellular enzymes [3,60] and sub-
sequently broken down by macrophages. At 9
months, the degradation of PLA microspheres was
completed. At 9 months, no remnant of cicatricial fi-
brosis was found after total disappearance of PLA
residues in the human skin. This finding illustrates the
excellent biocompatibility of PLA.
Reviderm intra
The injection of dextran beads caused swelling and
redness, which continued for 10 days—possibly due
356 Histology and Persistence of Filler Substances
Fig. 1. The Duranti-classification reflects the extent of
histological foreign body reaction at certain times. Class 2
is determined by a few giant cells.
Fig. 2. Restylane at 1 month. Minor cellular reaction of the
surrounding tissue, scattered macrophages and giant cells,
no immune response, and good biocompatibility. HE ·100.
Fig. 3. Artecoll at 1 month. The two strands of implants
here have a diameter of a 26 G needle and are still packed in
a fibrin network with some macrophages and giant cells.
Masson trichrome ·20.
Fig. 4. Artecoll at 6 month. All microspheres are encap-
sulated by connective tissue and some macrophages. Dur-
anti Grade II. Masson trichrome ·400.
Fig. 5. Artecoll at 10 years. The microspheres are still intact
and surrounded by collagen fibers, fibroblasts, some
macrophages, and isolated giant cells. Masson trichrome
·100.
Fig. 6. PMS 350 silicone gel at 1 month. The gel has dis-
sipated into millions of microdroplets, which are sur-
rounded by macrophages and lymphocytes. HE ·100.
G. Lemperle et al. 357
to the toxic effect of free dextranomers. Edematous
swelling of the implants continued for more than 3
months. The palpable deep dermal implant began to
disappear at 4 months and was no longer palpable at
6 months.
Histologically, the 40-lm dextran beads produced
the greatest amount of granulation tissue among all
injectables tested. At 1 month, the hydrophilic micr-
ospheres were swollen and measured up to 75 lmin
diameter (Fig. 8); some were broken apart and sur-
rounded by foamy macrophages and small giant cells.
The hyaluronic acid carrier had early separated from
the beads and was found in pools, surrounded by a
rim of giant cells (Fig. 9). At 3 months, only few
elastic fibers could be seen; instead there were great
numbers of macrophages and giant cells, which en-
veloped and tried to phagocytose the beads. The
surface of the dextran beads began to show irregu-
larities at 6 months and total disintegration at 9
months. It ranged to the top of the Duranti scale
among the resorbable implants (Fig. 10), possibly due
to the carrier from an undisclosed source of hyalu-
ronic acid (Rofilan), which has been crosslinked with
a plant extract.
Dermalive
The hydroxyethyl-methacrylate (HEMA) fragments
began to disappear in the human skin at 4 months
and were no longer palpable at 6 months.
Histologically, they showed the least cellular reac-
tion of all implants. The polygonal, translucent, and
irregular particles, 20–120 lm in size, which appeared
like clear broken glass gravel, were packed in clusters,
with minimal ingrowth of fibrous tissue, cells, and
blood vessels (Fig. 11). Only a fine network of elastic
fibers and occasionally macrophages were found, but
there were no apparent capillaries and no strong fi-
brous capsule. The hyaluronic acid was separated and
surrounded by macrophages, which had disappeared
at 3 months. At 9 months, only a few small clusters of
Dermalive with rounded corners and ridges, many
macrophages, and lymphoid cell clusters could be
detected. The few giant cells contained abundant as-
teroid bodies in their cytoplasma. Some pointed
particles had a tendency to irritate the surrounding
soft tissue, which showed clear evidence of low grade
inflammation. However, only about one tenth of the
implant volume consisted of cells and fibers (Fig. 11).
Aquamid
The clear gel of polyacrylamide was implanted into
human skin at four sites. Used in breast augmenta-
tion in Ukraine and China, it had a viscosity of 1045
centistoke. Since no anesthetic is added, the injections
into human skin caused a burning sensation for 20
sec. This was likely due to the cross-linked gel’s pH of
7.0 to 9.0. On examination, the implants revealed no
reaction and were still palpable at 9 months, but
decreasing in size.
Histologically, acrylamide gel was difficult to detect
at 1 month. The injected, non stainable transparent
gel produced only a fine fibrocellular capsule (Fig.
13), as expected from the literature [14,42]. At 3
months, no further histopathological reaction oc-
curred outside the implant site, such as foreign body
reaction. At 6 and 9 months, Aquamid had dispersed
into the skin and was surrounded by macrophages
and fibroblasts (Fig. 12). The histological reaction
resembled that of injected fluid silicone. In small
quantities, such as a slim strand beneath a wrinkle,
the gel appeared to be slowly absorbed, without vis-
ible foreign body reaction. Therefore, the manufac-
turer’s claim of ‘‘lifelong permanence’’ seems to be
dependent on the amount of implanted acrylamides.
Evolution
Clinically, the implant made of polyvinylhidroxide
microspheres suspended in acrylamide gel resembles
Artecoll. It was not painful during injection as was
Aquamid. Because of evaporation through the poly-
ethylene syringe, the water content of the material
was already diminished at the time of purchase. The
implants were well visible and diminishing palpably
over the whole course of 9 months. Histological ex-
amination showed the beads, most of them 30–40 lm
in diameter, within the clear acrylamide gel (Fig. 13)
surrounded by an almost invisible fibrous capsule.
Each droplet, 3–5 mm in size, was encapsulated with
a very fine layer of fibroblast and fibers without in-
growth into the implant. No foreign body reaction
was detectable. A few single microspheres outside the
implant site were covered with a fibrin layer or had
attached macrophages and fibroblasts. At 6 and 9
months, most of the carrier gel had been absorbed
and was replaced at the outer layers by granulation
tissue. At 9 months, the implant was totally infiltrated
by macrophages, fibroblasts, and giant cells (Fig. 14),
which resembled the tissue ingrowth of PLA at 3
months. The surface of the microspheres was still
intact after 9 months.
Radiance FN
Clinically, the subdermal implants in the forearm
were swollen for 3 days. Within 1 month, the palpable
implant diminished to half its prior size and became
whitish and shining through the skin. The hard
nodules diminished further in size and disappeared
clinically at 9 months from the skin.
Histologically, Radiance microspheres stimulate
almost no foreign body reaction. The 1-month sam-
ple had to be embedded in PMMA like bone tissue
because the implant could not be cut by conventional
358 Histology and Persistence of Filler Substances
Fig. 8. Reviderm at 1 month. Most dextran beads (its
empty vacuoles) are surrounded by macrophages and giant
cells with conspicuous foamy cytoplasm. The HA carrier is
separated from the beads and surrounded by a rim of giant
cells. Masson trichrome ·100.
Fig. 7. New-Fill at 3 months. Macrophages and giant cells
are surrounding the PLA microspheres and are filled with
phagocytosed PLA material. HE ·400.
Fig. 9. The HA carrier of Reviderm
TM
at 1 month has
separated early from the beads and is slowly phagocytosed
by a rim of multinucleated giant cells. Masson trichrome
·400.
Fig. 10. The Duranti classification reflects the extent of
histological foreign body reaction. Class 3 is defined by
infiltrating lymphocytes and giant cells. Zeraplast consists
of PMMA beads suspended in Rofilan. L40 are PLA beads
suspended in collagen.
Fig. 11. Dermalive at 3 months. The PMA particles are
packed and cause little foreign body reaction. Invading
macrophages and some giant cells gather at edges and
corners. Masson trichrome ·400.
Fig. 12. Aquamid at 6 month. The big droplets have been
dispersed into millions of mini-droplets, surrounded by fine
fibrous capsules with minimal foreign body reaction. This
picture resembles fluid silicone. HE ·100.
G. Lemperle et al. 359
methods. The beads were packed and surrounded by
some fibrin fibers but little cellular tissue. At 3
months, a method of rapid decalcification was ap-
plied prior to cutting and staining. The beads were
still packed (Fig. 15) and tissue ingrowth started from
the fine outer capsule of the implant. The main ‘‘in-
terstitium’’ still consists of fibrin fibers and few cel-
lular elements like fibroblasts and flattened
macrophages. No vascularity could be detected. At 6
months, the whole implant is surrounded by a fine
fibrous capsule and single microspheres are encap-
sulated by a thin fibroplastic stroma with flattened
cells. At 9 months, the voids are much smaller in
diameter and the microspheres deformed and slowly
adsorbed (Fig. 16). Since few macrophages were seen
it is suggested that calcium hydroxylapatite micro-
spheres are degraded by enzymatic breakdown rather
than phagocytosis.
Discussion
Collagen Gel
Bovine collagen is the ‘‘gold standard’’ for all
other newly introduced injectables. To date, Zyderm
Fig. 13. Evolution at 3 months. Most PVH microspheres
are still suspended in acrylamide gel, which results in a fine
fibrous capsule without foreign body reaction. Aquamid
induces the same inconspicuous histological reaction at that
time. Masson trichrome ·100.
Fig. 14. Evolution at 9 month. Macrophages and multi-
nucleated giant cells have phagocytosed most of the acryl-
amide carrier. At that stage, each single microsphere is
encapsulated with fibrin and some macrophages. Masson
trichrome ·400.
Fig. 15. Radiance at 3 months. The calcium microspheres are packed, the interstitium is filled with fibrin and scattered
fibroblasts and macrophages. Masson trichrome ·100.
360 Histology and Persistence of Filler Substances
and Zyplast have been the only FDA-approved
dermal filler substances in the U.S. for more than
20 years. The limited longevity of Zyderm and Zy-
plast, ease of use, low incidence of allergic reactions
(< 1 %), and safety are well established [32]. Late
granuloma formation occurs at a lower rate [26,45]
than with slowly resorbable gels and particulate
materials.
Hyaluronic Acid Fluids
Human hyaluronic acid, a polysaccharide of 4–5 kDa
molecular weight has a half-life of only 1–2 days. It
forms the cellular interstitium of the dermis and
creates volume by binding water. A human body
contains approximately 15 g HA. To avoid an early
breakdown, injectable hyaluronic acids have to be
crosslinked. The HA in Restylane has a molecular
weight of 1 ·10
6
Da, but 0.5%or every 200th amino
acid of the molecule is crosslinked (‘‘stabilized’’) with
a neighbor molecule. The company claims that it
contains a suspension of 1 ·10
5
HA particles of 40–
60 lm size in HA fluid. A similar product, Perlane, is
said to contain 8 ·10
3
gel particles/ml of approxi-
mately 100 lm in diameter, and Fine Lines approxi-
mately 2 ·10
5
gel particles/ml of 20–30 lmin
diameter. These gel particles, however, cannot been
seen under the microscope. Residues from the process
of fermentation of Streptococcus equi (>107 lg/ml
[41] may induce allergies to this bacterial protein in
certain patients. Acute and late inflammatory skin
reactions have been occasionally reported
[13,25,39,41].
PMMA Microspheres
These microspheres showed the most stable appear-
ance throughout the experiment. Once injected, the
PMMA microspheres cannot be broken down by
enzymes, since a methyl group in the alpha-position
stabilizes the molecule. Interestingly, the volume of
the injected collagen (80%) remained stable in the
implant over the years (Fig. 5). The microspheres act
merely as a scaffold and a stimulus for constant
connective tissue production. Here, the implant car-
rier is truly ‘‘replaced’’ by the body’s own tissue. In
contrast to other ‘‘dead’’ permanent filler substances
like acrylamide or Radiance, the ingrowth of con-
nective tissue creates a ‘‘living’’ implant.
Artecoll gives predictable results; however, at the
same time it is ‘‘non-forgiving’’ when mistakenly
implanted in an incorrect plane. To avoid technical
mistakes, introductory courses and a careful learning
curve are required. It may induce granuloma forma-
tion in very rare instances [35], as all other substances
will do in certain patients. Artecoll has a 10-year
history [35] and has been used in more than 200,000
patients worldwide outside the U.S.
Another injectable implant of PMMA micro-
spheres, 1–80 lm in diameter but suspended in Mg-
carboxy-gluconate (Metacrill), is produced and
distributed in Brazil [47].
Silicone Fluid
As we know from ruptured breast implants, silicone
gel causes the rarest foreign body reaction among the
Fig. 16. At 9 months, the Radiance microspheres appear deformed and partly degraded, probably by osteolytic enzymes and
not by phagocytosis. Masson trichrome ·100.
G. Lemperle et al. 361
filler materials. In most patients, silicone gel remains
very soft and is encapsulated by only a very thin layer
of fibroblasts. The lack of fibrous capsule formation
may lead to the displacement of larger quantities,
aided by gravity, which may migrate downwards
from the glabella to the cheeks and from the naso-
labial folds to beneath the chin. In time, the implant
can harden through ingrowth of connective tissue,
macrophages, and foreign body cells, which form a
granuloma [53].
The reputation of ‘‘medical grade’’ silicone fluid has
been damaged by five facts: (1) the use of large
quantities, e.g., in breast augmentation or facial dys-
trophies [17,18], has led to deformation; (2) the pos-
sibility of gravity induced ‘‘migration’’ in patients with
very lax skin and subcutaneous tissue; (3) the possi-
bility of late (5–20 years) granuloma (siliconoma)
formation; (4) the substitution of cheaper, non-medi-
cal-grade silicone fluids by nonprofessionals; and (5)
the lack of experience of most physicians in the
treatment of rare cases of late siliconoma. Though
difficult to remove surgically, siliconomas respond
favorably to multiple injections of corticosteroid [7] or
antimitotic agents [58]. The wrinkles and lips of
thousands of patients in Europe and Asia have been
treated successfully with the micro-droplet technique
with small amounts of medical-grade silicone fluid. On
the other hand, hundreds of women required total
mastectomy due to chronic inflammation of large
amounts of silicone oil injected directly into the breast.
As so often in medicine, success depends on the right
dosage, volume, and knowledge of side effects.
Polylactic Acid Microspheres
Polylactic acids do not occur naturally, but were first
synthesized by French chemists in 1954. PLA and
polyglycolic acid (PGA) have been used safely in
suture materials (Vicryl, Dexon), in resorbable plates
and screws, in guided bone regeneration, in ortho-
pedic, neuro-, and cranio-facial surgery, and as drug
delivery devices [60]. PLA does not stimulate the
natural production of collagen [2], but causes a for-
eign body reaction, characterized by macrophages,
giant cells, and some elastic fibers. The PLA polymer
New-Fill disappeared within 6 months, probably due
to extracellular hydrolysis, ester cleavage, and the
catalytic effect of hydrosoluble acid monomers
formed in the polymer matrix during degradation [3].
Whether the mild inflammatory response elicited by
PLA can be ascribed to degradation activity of
macrophages is not clear.
The degradation rate of PLA polymers in vivo is
said to be almost twice that in vitro [60]. Our experi-
ments with microspheres, however, have shown the
opposite. After refrigeration for 6 months, the micr-
ospheres in their fluid cellulose carrier had dissolved
totally, whereas the microspheres in living tissue were
still recognizable as round with irregular surface. The
degradation of different PLAs is affected dramatically
by the amount of glycolic units in the lactic acid chains
[60]. Degradation only appears when the molecular
weight of the PLA decreases below 20 ·10
3
Da. The
PLA in New-Fill has a molecular weight of 170 ·10
3
Da. Nine months after implantation, no polymer
residue or remnant cicatricial fibrosis were found,
confirming the good biocompatibility of PLA micro-
spheres. Since PLAs contain no animal proteins, al-
lergies are not expected; however, late granulomas
have been reported as with any other filler substance.
Dextran Microspheres
Dextrans are the substrate of chromatography col-
umns (Sephadex) used for the separation of proteins.
Dextran molecules of 40,000 and 80,000 Da are used
as plasma expanders, since dextran molecules
<20,000 Da will be filtered by the kidney. Dextran
beads of 100 lm in diameter were found intact 2 years
after implantation in the back skin of rats. Eppley et
al. [18] emphasized that the positive surface charges
of dextran beads apparently attracted macrophages.
The macrophages in turn release TGF-beta and
interleukins, which stimulate fibroblasts to produce
collagen fibers. After extensive resorption of the
dextran beads at 9 months, however, little or no
cicatricial residue could be detected at the implanta-
tion sites in these studies. Dextran beads of 100 lmin
diameter (Deflux, Q-Med, Uppsala, Sweden) are
currently used in clinical trials for the treatment of
stress urinary incontinence.
HEMA Fragments
Dermalive is a by-product of the manufacture of in-
traocular lenses and was introduced in the European
Fig. 17. Duranti classification reflects the extent of foreign
body reaction at certain times. Class 4 defines granuloma
formation. Bioplastique is a suspension of silicone particles
in PVP. Embosphere are soft microspheres of trisacryl-
gelatin used for tumor embolization [4].
362 Histology and Persistence of Filler Substances
market in 1998. Because of a rather high incidence of
granuloma formation, it is now used mainly in the
form of Dermadeep with HEMA fragments 80–110
lm in size for deep dermal and epiperiosteal im-
plantation. Inside the implant, the HEMA particles
were packed closely, probably due to diminished
viscosity of the carrier medium hyaluronic acid. This
carrier dissipated from the particles just after im-
plantation of Dermalive and was found outside of the
clusters of particles.
The great advantage of collagen as a suspension
medium for filler substances is its high viscosity,
which keeps the particles or microspheres apart
weeks after implantation. Since little host tissue for-
mation is stimulated, more Dermalive has to be in-
jected compared to other fillers. On the other hand,
HEMA has a free OH-group, which should stimulate
macrophage activity. Endogenous esterases in serum
and liver break down HEMA. Interestingly, the
amount of tissue reaction is no indication for the rate
of granuloma formation. In the studies described
here, Dermalive evoked the least new tissue forma-
tion but clinically causes a rather high rate of gran-
uloma formation [54].
Polyacrylamide Gel
Like dextran beads, polyacrylamide is used mainly
for protein separation by molecular biologists. The
use of polyacrylamides as injectable filler materials
was initiated in 1983, and they were used clinically in
the Ukraine and China as Interfall [14] or ‘‘Amazing
Gel’’ in thousands of patients. However, to date few
clinical and scientific data have been published in
Western literature [23]. Since Interfall’s patent ex-
pired, at least seven European companies are mar-
keting polyacrylamides as dermal filler substances
(Royamid, Argiform, Bioformacryl, OutLine, Aqu-
amid, Evolution, Kosmogel). So far, they have been
injected in large quantities for breast, buttock, and
calf augmentation, in facial lipodystrophy and con-
genital malformations [14], and have been called
‘‘endoprotheses’’ [42]. If overcorrection occurs, the
fluid can be withdrawn from the implant even after
years. Reportedly, it has a half-life in the human
body of >20 years. This may be true for large
quantities; however, the injection of 0.1 cc Aquamid
was resorbed in human skin within 9 months.
The concentration of acrylamide monomers that can
be toxic was reported to be <10 ppm or 0.04%.
Side effects were enlarged lymphnodes in 10%, mi-
gration of gel in 3%, and edema in 2%. The U.S.
Environmental Protection Agency classifies acryla-
mide as a medium-hazard probable human carcino-
gen. Even if there are few published reports [30],
granuloma formation after Interfall implantation is
well known in China since 1997 [14] and has to
be expected in certain patients as with all other
injectables.
Polyvinyl Gel Microspheres
The mixture of apparently slowly resorbable
P
olyvi-
nyl microspheres with a longer lasting polyacryla-
mide gel (Evolution) showed similar histological
reactions to Aquamid within the first 6 months, and
from then on was like Artecoll. Clinically and histo-
logically it showed the least reactions and remained
visible and palpable throughout the 9 months. Future
clinical experience has to show whether late side ef-
fects are as high as with acrylamides.
Calcium-Hydroxylapatite Microspheres
Ca-hydroxylapatite is the primary component of
bone and dents. In form of particles it was first used
as onlay grafts for bone regeneration in dentistry. Ca-
hydroxylapatite microspheres of 75-125 lm in dia-
meter (Coaptite) are injected as a bulking agent in
urinary incontinence. It is highly biocompatible,
causes little tissue reaction, well defined at the injec-
tion site, radio-opaque, and can therefore be used as
a tissue marker (Radiance FN). In the skin and es-
pecially in the lip, it does not ‘‘remain soft’’ but ex-
hibits a clear hardening of the implant, which resolves
over time. Since March 2002, Radiance FN can be
used ‘‘off-label’’ in the US for wrinkle treatment and
lip augmentation. Since it disappeared from the skin
within 12 months, it is a semi-permanent implant like
many others (Aquamid, Dermalive, New-Fill, Revi-
derm) and not a ‘‘near-permanent solution’’. While
Radiance is well tolerated beneath wrinkles, it should
not be recommended for lip augmentation. The
concomitant movement of the orbicularis muscle in
women during chewing compresses every injected
strand to a lump!
Histologically, the calcium hydroxylapatite micro-
spheres do not ‘‘provide a scaffold for tissue infil-
tration consistent with the form of the surrounding
tissue’’. Because of little tissue ingrowth [16, 43] and
absence of granulation tissue, triamcinolone (Kena-
log) injections into Radiance lumps will be ineffec-
tive and should be omitted. In some patients,
however, Radiance microspheres may induce a kind
of foreign body reaction - as demonstrated by one
histological picture in the company’s advertisement,
which of course will react to intralesional cortico-
steroids.
In general, the histological examination of all
samples showed distinctive morphological findings
for each type of micro-implant, which can help dis-
tinguish different injected substances. The differential
diagnosis could also be of medico-legal interest be-
cause adulterated or improper substances are some-
times injected fraudulently [54]. PMA-particles
(Dermalive) showed multiple sharp edges and points
taking on a ‘‘broken glass’’ appearance, whereas
microspheres are devoid of any jagged or sharp edges
that might serve as a continued source of irritation.
G. Lemperle et al. 363
Complications
Besides well-known allergies, displacement, and
granuloma formation, another potential complica-
tion of dermal filler substances was documented only
recently [62]. Localized fat atrophies in the cheeks,
similar to those seen in facial lipodystrophy after HIV
treatment [11,57], occurred 2–3 months after the im-
plantation of Restylane or New-Fill in the nasolabial
folds of healthy patients. At 9 months, the implants
could still be identified and were interspersed with
giant cells and granulomatous tissue. The residues
were encapsulated and surrounded by fat necroses
and vacuoles. No explanation has been found for this
side effect since there is no obvious link in the
chemical structure of these two filler substances and
HIV protease inhibitors.
Granulomas
All injectable filler materials cause normal foreign-
body-type reactions that may develop into a foreign
body granuloma in selected patients. Its cause is still
unknown and no predictions can be made. Some of
the patients reported a severe generalized viral or
bacterial infection [37,41,53], vaccination, or local
trauma some months before the appearance of the
granulomas.
Granulomas occur in patients at a rate of 0.01%to
1.0%according to the chemical composition, shape,
and surface structure of the particles [38]. They occur
significantly less often after implantation of micro-
spheres with a smooth surface (Artecoll, New-Fill,
Evolution) [37,38] than after implantation of particles
with irregular surface (Bioplastique, Dermalive)
[6,54]. As would be expected, they also occur less
frequently after injection of resorbable implants
(collagen [29,45], hyaluronic acid [13,25,39-41,59])
compared to long-lasting implants [53,54].
Legal Aspects
In contrast to the FDA, Notified Bodies of the Eu-
ropean Community do not require animal or clinical
studies for the registration and approval of injectable
filler substances or surgically introduced artificial
implants. The European product quality control
systems classify injectable dermal fillers as Medical
Devices Class IIa (resorbable substances), and Class
IIb (substances that cannot be reabsorbed). The FDA
has determined that collagen is a Class III.A device
and injected particles are a Class III.B device. In
November 1997, a new provision was added to the
Federal Food, Drug, and Cosmetic Act to allow any
legally marketed, FDA-approved product to be ad-
ministered for any condition within a doctor-patient
relationship. This is called ‘‘off-label use’’ of an FDA-
approved product.
The CE mark controls only Good Manufacturing
Practice (GMP) of an injectable agent but does not
guarantee a maximum of biological safety. Therefore,
a central office similar to the FDA should be estab-
lished in Europe to which all severe clinical side ef-
fects have to be reported. Some manufacturers report
the complication rates as a percentage of treatments
(Restylane, Hylaform, Dermalive)— most probably
of syringes sold—, whereas other manufacturers re-
port the side effects as percentage of treated patients
[35]. Only a careful statistical analysis of these data
will shed some light on the true incidence of side ef-
fects for each dermal filler substance.
Conclusions
The differences in histological reactions and in per-
manence lead to a classification of injectable filler
substances into five types:
1. Autologous fat rarely is permanent and its fate is
unpredictable. The mechanism for long-term
survival of fat or stem cells has yet to be un-
derstood.
2. Natural filler substances such as collagen and hy-
aluronic acids are phagocytosed slowly with min-
imal histological reaction.
3. Fluid filler substances, such as fluid silicone and
acrylamides, cause little fibrosis but can dislocate
larger volumes through muscle movement and
gravity; they are considered ‘‘dead implants.’’
4. Particulate materials like PMA gravel and PLA
microspheres are packed and induce minimal for-
eign body reaction and fibrosis. They are pure
fillers and are slowly resorbed.
5. Microspheres from non-resorbable PMMA or re-
sorbable dextran are stimulants for encapsulation
and scaffolds of permanent or temporary connec-
tive tissue formation, considered ‘‘living im-
plants.’’
Host defense mechanisms reacted differently to
each filler material; however, all substances—resorb-
able or nonresorbable—appeared to be clinically and
histologically safe. None of the tested substances is
without undesirable effects [1,46,52,55]. Since the
cause of late inflammation or granuloma formation is
not yet known, predictions can be not be made from
early histological results for possible late reactions of
the host to an individual filler substance.
Therefore, materials must be selected according to
the needs of the individual patient. The patient
should be informed and involved in the choice of
resorbable or long-lasting filler substance. Hyaluronic
acid and new collagen products such as Cymetra,
Fascian, or CosmoDerm could not be demonstrated
to last longer than bovine collagen. The development
of a possibly disease- and allergy-free human rec-
ombinant collagen from yeast (Fibrogen, San Fran-
364 Histology and Persistence of Filler Substances
cisco) or cow milk (Cohesion Technologies, Palo
Alto) has a long way to go. The search for the perfect
permanent injectable material with maximum safety
is ongoing. Time and a centralized registry of adverse
events—similar to the registries for silicone breast
implants—will bring improvement in efficacy and
safety of new generation filler substances.
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366 Histology and Persistence of Filler Substances
