Neocollagenesis after Injection of Calcium Hydroxylapatite

Composition in a Canine Model

KYLE M. COLEMAN, MD, ROBERT VOIGTS, MS,y DALE P. DEVORE, PHD,y PAUL TERMIN, DVM, PHD,z
y
AND WILLIAM P. COLEMAN, III, MD

Dale DeVore and Robert Voigts are employees of BioForm. This study was conducted at, and funded by,
BioForm Medical Inc.

T he ability of soft tissue fillers to induce neo-

collagenesis is a concept emerging in impor-
tance in predicting longevity of filler injections. Over
the past three decades, an increased demand for skin
rejuvenation and wrinkle correction has led to the
development of a diverse set of cosmetic skin fillers.
With recent evidence showing that hyaluronic acid
filler can lead to fibroblast cell changes indicative of
possible endogenous production of collagen, it is
important to see if similar responses are found with
different fillers. Investigators sought to determine if
injection of Radiesse (calcium hydroxylapatite;
BioForm Medical Inc., San Mateo, CA) can lead to
neocollagenesis over time in a canine model.
Radiesse was approved by the US Food and Drug
Administration (FDA) in December of 2006 for
correction of facial wrinkles and folds and for hu-
man immunodeficiency virus–associated facial
wasting.1 The product is composed of approximately
30% calcium hydroxylapatite and 70% carrier gel.2
The clinical effects of calcium hydroxylapatite in-
jections have been reported to last from 12 months
to greater than 3 years, and this material has been
used worldwide in greater than 100,000 patients.2–6
Six animals were obtained through a US Department
of Agriculture (USDA)-licensed supplier. Approval
for animal use in scientific investigation was ob-
tained through a divisional IACUC (International
Animal Care and Use Convention). The animals
were injected lateral to the lumbar spine. One side
on each animal was injected in a linear pattern
with 0.2 to 0.3 cm3 of calcium hydroxylapatite
subdermally; the opposite side was injected in a
similar manner but intradermally. Each animal was
randomly assigned to a study interval of 4, 16, 24,
32, 52, or 78 weeks. At the predetermined interval,
the animals were euthanized by intravenous injection
of sodium pentobarbital, and each injection site was
excised, fixed in 10% formalin, and submitted for
pathologic evaluation. At explant, each tissue sample
was grossly evaluated for the presence of capsule
formation prior to submission for histologic
analysis.
The pathologist was blinded to the time interval of
the sample during interpretations. Collagen content
was quantified using picrosirius red (PSR) staining
under polarized light with photometric analysis. PSR
was selected for the evaluation of collagen content as
routine formalin-fixed, paraffin-embedded tissues
stained with PSR has been shown to bind to collagen
and then exhibit a bright refractile appearance when
transilluminated with a plane polarized light and
viewed using crossed polarizers.7,8
PSR-stained sections were reviewed and appropriate
fields of view were selected based on the following
criteria:

Division of Dermatology, Tulane University Health Sciences Center, New Orleans, Louisiana; yBioForm Medical Inc.,
Franksville, Wisconsin; zLincoln Associates Inc., St. Paul, Minnesota; yTulane University Health Sciences Center,
New Orleans, Louisiana

& 2008 by the American Society for Dermatologic Surgery, Inc.
Published by Wiley Periodicals, Inc.

ISSN: 1076-0512
Dermatol Surg 2008;34:S53–S55
DOI: 10.1111/j.1524-4725.2008.34243.x

S53
 N E O C O L L A G E N E S I S A F T E R I N J E C T I O N O F C A L C I U M H Y D R O X Y L A PAT I T E

Figure 1. Picture representation of photometric analysis (Week 4 sample). (A) Histochemical stain with PSR. (B) Under
polarized light. (C) After photometric grayscale change. Original magnification,  40.

1. The study material filled the entire field of tive samples from each time interval after staining
view. with PSR.

2. Collagenous features including fascial tissues planes

were a minor constituent within the field of view.
Image analysis was standardized within the features
of Adobe PhotoShop 5.5 to convert the color pho-
tomicrograph into a posterized image comprised of
black or white pixels. The percentage of both black
and white pixels, with the white pixels being the
refractile collagen and the black pixels being all
other non–collagen-containing areas, was then
determined. Representative pictures of this technique
are seen in Figure 1.
All animals evaluated showed only minimal irrita-
tion at the injection sites. No capsule formation was
seen in any of the specimens obtained. After injection
of calcium hydroxylapatite, increased collagen was
seen over the time interval of 4 to 78 weeks. Results
can be seen in Figure 2. Figure 3 shows representa-
40.00
Intradermal
Subdermal
35.00
30.00
In this animal study, it was observed that skin spec-
imens injected with calcium hydroxylapatite dem-
onstrated an increased density of host collagen over
time. The intradermal injections led to a larger in-
crease in collagen content than subdermal injections
exhibited. Although these calculations are not
strictly quantitative, they are quantitative deriva-
tions of qualitative results. The Week 4 values mea-
sured were higher than those of the Week 16; this
initially high percentage of collagen may be due to
scar formation or tissue swelling. Collagen content
then increased in a more stepwise manner consistent
with neocollagenesis.
Many different mechanisms of increased collagen
production by tissues have been described including
fibroblast stretch, localized destruction, and in-
creased cytokine production such as transforming
growth factor-b.9–14 More study is needed as to
which mechanism predominates with use of soft
tissue fillers.

25.00 The limitations of this pilot study are the small

%collagen

number of animals, the use of a nonhuman model,

20.00
and the fact that no baseline collagen contents were
15.00 obtained and that only trends were observed. Be-
10.00 cause of the small size of the study sample, no sta-
5.00 tistical significance was able to be calculated.
Pretreatment assessments and larger sample size may
0.00
help to explain the relatively high collagen content at
11

18

25

46

53

74

81
32

39

60

67
4

weeks the Week 4 sampling compared to the following

Figure 2. Percentage of collagen present over observation
weeks. Although more studies are needed to deter-
intervals. mine if similar results occur in vivo in humans, in-

S54 D E R M AT O L O G I C S U R G E RY

Figure 3. Representative photographs of PSR-stained histologic specimens. (A) 4 weeks; (B) 16 weeks; (C) 24 weeks; (D) 32
weeks; (E) 52 weeks; (F) 78 weeks. Original magnification, A and B,  40; C-F,  60.
jections of calcium hydroxylapatite led to endoge-
nous collagen production in a canine model. This is
an important observation because local tissue atro-
phy and aging facial folds are partially caused by
decreased collagen density and increased collagen
fragmentation.15,16 The dermal injections produced
greater results than the subcutaneous injections did.
It is important to note that calcium hydroxylapatite
is indicated for injection at the dermal–subcutaneous
junction and that dermal injections may lead to
nodularity. Whether increased collagen content can
produce improved clinical results is not known.
However, we hypothesize that, due to neocollagen-
esis, calcium hydroxylapatite injections may lead to
longer lasting clinical results.
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Address correspondence and reprint requests to: Kyle M.
Coleman, MD, Resident, Division of Dermatology, Tulane
University Health Sciences Center, New Orleans, LA
70112, or e-mail: kmcoleman@hotmail.com
3 4 : S 1 : J A N U A RY 2 0 0 8 S55