Received: 2 January 2023 | Revised: 6 March 2023 | Accepted: 30 March 2023

DOI: 10.1111/jocd.15770

C L I N I C A L C O M M E N TA R Y

Histological effects of a combined collagen stimulation

procedure consisting of microfocused ultrasound, soft tissue
filler, and Ca-­HA injections

Gabriela Casabona MD1 | Kai Kaye MD1 | Sebastian Cotofana MD, PhD, PhD2 |
Kristina Davidovic MD3 | Michael Alfertshofer MD4 | Lysander Freytag MD5

1
Ocean Clinic, Marbella, Spain
2
Department of Clinical Anatomy, Mayo
Abstract
Clinic College of Medicine and Science, Background: An increasing demand for in-­office aesthetic procedures aiming to im-
Rochester, Minnesota, USA
3
prove collagen stimulation has been reported.
Center for Radiology and Magnetic
Resonance Imaging, University Clinical Objective: To analyze the effects of combinations of different aesthetic collagen stim-
Center of Serbia, Belgrade, Serbia ulation procedures using histological analysis.
4
Division of Hand-­, Plastic and Aesthetic
Methods: Histological analysis was performed on skin samples of the excess skin of a
Surgery, Ludwig-­Maximilians-­University
Munich, Munich, Germany 60-­year-­old patient after sub-­superficial musculo-­aponeurotic system facelift surgery.
5
Department of Plastic Surgery, The excess skin before facelift surgery was divided into three areas per hemiface.
Community Hospital Havelhöhe Berlin,
Berlin, Germany Each area—­with exception of area A as a control—­received an isolated or combined
treatment of microfocused ultrasound (MFU), injections of calcium-­hydroxylapatite/
Correspondence
Gabriela Casabona, Ocean Clinic, Av hyaluronic-­acid-­based soft tissue filler, and microneedling. The effect of the treat-
Ramon y Cajal 7, Marbella 29601, Spain. ments was analyzed using histological analysis with H&E coloration and PAS staining.
Email: grcasabona@gmail.com
Results: A threefold increase of epidermal and dermal thickness was achieved by
treating the skin with the proposed combined treatment comprising MFU and injec-
tions of calcium-­hydroxylapatite-­and hyaluronic-­acid-­based soft tissue fillers.
Conclusion: The investigated treatments act synergistically in the production of col-
lagen and combining these treatments allows for an increase in collagen production.

KEYWORDS
calcium hydroxyapatite, microfocused ultrasound, neocollagenesis, skin tightening, SMAS

1 | I NTRO D U C TI O N of Plastic Surgery (ASPS) an increase of 312% in the injection of soft

tissue fillers between the years 2000 and 2017 was reported.4 This
Due to collagen's contributing role to a youthful appearance, the in- indicates an increased acceptance of in-­office aesthetic procedures
terest in collagen enhancement in the skin and deeper tissues such to address the demand for a more youthful appearance. Considering
as the superficial musculo-­aponeurotic system (SMAS) through the the less invasive nature of collagen-­stimulating treatments that are
use of in-­office aesthetic procedures has grown in recent years.1–­3 also capable to achieve a youthful appearance, 5–­7 we see the need
According to the annual statistics released by the American Society for systematic examination of the efficiency of these treatments.
[Correction added on 18th May 2023, after first online publication: The Figures has been updated in this version.]

Michael Alfertshofer and Lysander Freytag contributed equally.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2023 The Authors. Journal of Cosmetic Dermatology published by Wiley Periodicals LLC.

1724 | 
wileyonlinelibrary.com/journal/jocd J Cosmet Dermatol. 2023;22:1724–1730.
CASABONA et al.
Facial aging is a multifactorial process where not only the skin is
8,9
involved, but also the deeper tissues. Aging is also related to dif-
ferent layers of the facial structure such as bone,10–­12 ligaments,8,13,14
15 16–­19 20,21
muscles, fat, and skin. The complex interplay of age-­related
changes in these different tissues all add to the typical characteristics
of an aged face. Especially, the specific changes to the superficial and
deep fat compartments contribute to the aged appearance of the face
through loss of tension and volume in the soft tissues. This tension is
responsible for a youthful appearance and loss thereof results in an
aged appearance.18 Collagen stimulation procedures are widely used
and well-­
known amongst practitioners and consumers and recent
studies showed that transcutaneous microfocused ultrasound (MFU)
is beneficial for skin tightening, collagen regeneration, and lower face
22–­24
contour improvement. MFU lifting is the only FDA-­approved lift-
25
ing technology that acts solely on the skin and the SMAS. MFU lifting
can be combined with an injection of calcium hydroxylapatite (Ca-­HA),
and multiple studies in recent years indicate an increase in the stimu-
lation of collagen in the skin.26–­28 However, to date there is no study
showing this procedure's real histological effect on the SMAS.
We hypothesize that combining MFU lifting with the injection
of Ca-­HA and hyaluronic acid (HA)–­based soft tissue filler injections
yields better collagen stimulation in the skin and the SMAS com-
pared with the sole use of either treatment, which could optimize
current clinical practice. We test this hybrid procedure's effective-
ness in a case study by analyzing its histological effects on the skin
and the SMAS on the face of a patient undergoing facelift surgery.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Patient
In this case study, a 60-­year-­old patient who had undergone sub-­SMAS
facelift surgery was examined. The patient had no previous history of
injections or aesthetic treatments in the treatment area, nor a history
of autoimmune diseases, cancer, pregnancy, or allergies. All proce-
dures inducing collagen production were carried out within 180 days
of the facelift surgery and no later than 150 before the surgery.
2.2 | Treatment protocol
The treatment protocol consisted of four individual treatments: MFU
(Ultherapy®, Ulthera Inc), injection of calcium-­
hydroxyalapatite-­
based soft tissue fillers (Radiesse®, Merz Aesthetics), injection of
hyaluronic-­acid-­based soft tissue fillers (Belotero® Revive, Merz
Aesthetics) and microneedling (Dermapen 4™, Dermapenworld).
Ultherapy® is a high-­focused ultrasound treatment that stimulates
collagen production and skin regeneration by targeting the deep der-
mal and subdermal layers. This treatment is especially attractive, as it
does not penetrate the skin itself. The subdermal soft tissues were tar-
geted at three different depths: 1.5, 3.0, and 4.5 mm. Each depth was
treated with five rounds of ultrasound. Radiesse® is a biocompatible,
| 1725
biodegradable, biostimulatory calcium hydroxyapatite-­based (CaHA)
injectable filler that is known for its collagen-­
stimulating and vo-
lumizing properties.29 A dilution at a ratio of 1:1 with 1.5 cc of lido-
caine without a vasoconstrictor was used in this study after mixing it
12 times with a 5 cc counter-­syringe. The solution was injected using
a 22 G cannula with subdermal traces of 0.1 cc. Belotero® Revive is
an injectable filler with a hyaluronic acid-­glycerol combination, used
to improve hydration, as well as to reduce redness and roughness of
the skin.30 This was injected in the deep dermal layer with an ultra-­
thin needle at 2.5 mm depth. Lastly, we also used microneedling in the
treatment of the patient. Microneedling is the process of stimulating
the dermis through the use of microneedles, which penetrate the skin
causing micro wounds. The skin reacts to these wounds with its natural
healing process and therefore with the production of collagen.31 In this
case, 20 passes were made at a depth of 2.5 mm.
To investigate the effectiveness of currently used collagen-­
stimulating treatments compared with our proposed combined treat-
ment, we divided the pre-­auricular excess skin prior to the facelift into
six areas, with three areas per hemiface (Figures 1 and 2). The patient's
right hemiface was divided into areas A, B, and C. Area A was defined
as the area of the excess skin superior to the tragus, and no treat-
ment was performed. Thus, area A constituted the control (Figure 3).
Area B describes the area at the level of the tragus and was treated
with both Ultherapy® and Radiesse®. Ultherapy® was administered
immediately before the injection of Radiesse®. Area C was defined
as the area of excess skin inferior to the tragus and was also treated
with both Ultherapy® and Radiesse®. However, the Radiesse® in-
jection was administered first followed by the Ultherapy® after
1 month. The patient's left hemiface was divided into areas D, E, and F.
Correspondingly, area D was defined as the area of the excess skin su-
perior to the tragus and received same-­day treatment of Ultherapy®
immediately before the injection of Radiesse®, as well as injection of
Belotero® Revive intradermally. Area E was defined as the area of
excess skin at the level of the tragus and only received Ultherapy®.
F I G U R E 1 Photograph of the right hemifacial side showing the
excess facial skin divided into three areas: Area A superior to the
tragus, area B at the level of the tragus, and area C inferior to the
tragus.
1726 | CASABONA et al.
Similar to area C, area F was defined as the area caudal to the tragus
and received only microneedling therapy.
2.3 | Skin sample analysis
Intraoperatively, we collected samples of the previously treated skin
and SMAS for histological analysis. For this, the degree of collagen
production in all layers of each of the studied areas using H&E col-
oration and PAS-­staining was analyzed.
3 | R E S U LT S
3.1 | Area B (Ultherapy® immediately before
Radiesse®)
Compared with control area A, the thickness of the epidermal and
the dermal layers doubled in Area B. Also, the septae connecting
F I G U R E 2 Photograph of the left hemifacial side showing the
excess facial skin divided into three areas: Area D superior to the
tragus, area E at the level of the tragus and area F inferior to the
tragus.
the superficial fascia to the skin thickened. The histological analysis
with PAS staining showed a parallel organization of collagen fibers.
Closer inspection showed some particles of Radiesse present along
the fibrous septae (Figure 4).
3.2 | Area C (Radiesse® first and Ultherapy® after
1 month)
Compared with control area A, area C showed a thickening of the ep-
idermal and dermal layers. The connective septae here also showed
signs of thickening and had furthermore become more visible. Also
here, the collagen fibers showed a parallel organization. Compared
with area B, a smaller degree of thickening of the epidermal and der-
mal layers could be observed (Figure 5).
3.3 | Area D (Ultherapy® immediately before
Radiesse® and Belotero® Revive)
Compared with control area A, area D showed a thickening of
the epidermal and dermal layers by a factor of three. The con-
nective septae from the skin to the superficial fascia thickened,
and the collagen fibers were organized in a parallel manner. Some
Radiesse-­p articles were found along the fibrous septae, but no
presence of hyaluronic acid gel was found in the dermal layer
(Figure 6).
3.4 | Area E (Ultherapy®)
Area E showed a slight thickening of the epidermal and the der-
mal layers—­especially in the papillary dermis, and the connective
septae also thickened and became more visible. Again, the histo-
logical analysis showed a parallel organization of the collagen fibers
(Figure 7).
F I G U R E 3 HE-­colored and PAS-­stained
histological analysis of area A, which
served as a control and did not receive
any treatment.
CASABONA et al. | 1727

F I G U R E 4 HE-­colored and PAS-­stained

histological analysis of area B, which was
treated with microfocused ultrasound
(Ultherapy®) immediately before injection
with calcium-­hydroxylapatite-­based soft
tissue filler (Radiesse®).

F I G U R E 5 HE-­colored and PAS-­

stained histological analysis of area C
which was treated with the injection
of calcium-­hydroxylapatite-­based soft
tissue filler (Radiesse®) first followed by
microfocused ultrasound (Ultherapy®)
after 1 month.

F I G U R E 6 HE-­colored and PAS-­stained

histological analysis of area D, which was
treated with microfocused ultrasound
(Ultherapy®) immediately before
injections of calcium-­hydroxylapatite-­
based (Radiesse®) and hyaluronic-­acid-­
based soft tissue fillers (Belotero®
Revive).

3.5 | Area F (Microneedling) to have thickened. However, there were no changes in the
­connective septae between the skin and superficial fascia. The
The epidermal and the dermal layers of area F likewise showed collagen fibers were found to be organized in a parallel structure
slight thickening, and the epidermis around follicles was shown (Figure 8).
1728 | CASABONA et al.
The comparison of all five areas with the control area shows that
Area D had the biggest change in skin thickening, as well as SMAS
thickening, followed by areas B and C. Area E showed a modest im-
provement in thickness of SMAS and a slight improvement of skin
thickness. Area F showed improvement mainly in the papillary der-
mis with no changes in the fibrous septae.
4 | DISCUSSION
The results of this interventional prospective study revealed that
changes to the dermis and subdermal connective tissue architec-
ture can be induced by applied energy, injection of biostimulators
and soft tissue fillers, and by microneedling. The methodology
of the study design allowed for the histologic investigation of six
different facial regions following tissue removal during facelift
surgery.
To date, many procedures are available that have been shown to
increase collagen stimulation individually; however, only few investi-
gations have aimed to quantify the effect of combination therapies.
Furthermore, there has been a paucity of data available for hybrid
treatments combining various collagen-­stimulating procedures. Our
results showed that the combination of Ultherapy® immediately
before Radiesse® and Belotero® Revive injections was the most
F I G U R E 7 HE-­colored and PAS-­stained
histological analysis of area E, which was
treated with microfocused ultrasound
(Ultherapy®).
F I G U R E 8 HE-­colored and PAS-­stained
histological analysis of area E, which was
treated with the microneedling device.
effective combination for thickening of the epidermal and the der-
mal layers as well as the SMAS, indicating the highest level of neocol-
lagenesis. However, an induction of neocollagenesis was observed in
all areas treated, even though the effect was not as strong as the
combination of Ultherapy®, Radiesse®, and Belotero® Revive.
Clinically the results of the study indicate that applied energy in
combination with a biostimulator and HA-­based soft tissue filler are
most efficient to induce fibroblast activation. The activated fibro-
blasts are able to induce collagen production, which was visible in
the present study as thickening of the SMAS layer and in an increase
of retinacula cutis, a crucial component of the superficial fascial
system. These collagenous components of the subdermal three-­
dimensional architecture provide stability for the overlying skin and
form a tighter interaction between the parallel layers of the lateral
face. This increased stability allows in return for a reduced soft tissue
descend and for a better adhesion of the dermis to the subdermis.
This presents clinically as reduced skin laxity, greater skin firmness,
and in reduced amounts of facial sagging. The latter is known to re-
sult in the formation of jowls and loss of jawline contouring.
The results of this study also provide guidance for the sequence
in which such combination therapy should be best applied due to
the performed study design. Interestingly, increased thickness of
SMAS and dermal layers was observed in Area B (MFU and Ca-­HA
injections on the same day) compared with Area C (MFU and Ca-­HA
CASABONA et al.
injections 1 month apart) indicating a possible synergistic mecha-
nism of both treatments when applied in a short time interval. This is
novel and, therefore, the results provide guidance for practitioners
for their daily clinical practice. Several aspects of the present study
contribute to its advantage. Holding physiology and anatomy con-
stant when testing the efficiency of individual treatments and com-
binations of treatments is advantageous, as direct effects are easier
to identify, and the results of the analysis become more valid. This
was achieved in the present study by treating the same face in dif-
ferent areas. Additionally, the examined patient was a “clean slate,”
meaning they had had no previous treatment in the face that could
have influenced the results of the collagen-­stimulating treatments.
This further increased the validity of the study. Furthermore, high
reliability was achieved in this study through the use of histopatho-
logical analysis, as it constitutes an objective measure of anatomical
change.
While the case study has had its advantages, there are also dis-
advantages related to such a method. A case study cannot yield gen-
eralizable results. As the effects of the employed treatments could
vary from person to person, statistical analysis, and thus a larger-­
scale study, ought to be conducted in the future. The examination of
fibrous collagen through histopathological analysis is also not with-
out fault. The single samples extracted from each observation area
on our patient's face do not necessarily reflect the actual structure
throughout the treated areas. The sample extracted may have been
taken from an area whose collagen synthesis was caused by some
other undetected and unintended factor. Nonetheless, the inclusion
of a control area in this study reduces the risk of overestimating the
effect caused by the employed treatments. Lastly, the free will of the
patient makes it difficult to exclude effects from other treatments
the patient may have undergone within the study period. To mini-
mize this risk, we asked the patient to sign waiver of similar treat-
ments outside of the study during the study period. In addition, it
has to be noted that no adverse events (nodules, pain, inflammation,
vascular incidents) were observed during the study period, which
can potentially be attributed to the safety profile of the procedures
applied.
5 | CO N C LU S I O N
The study was conducted to investigate both the individual and
combined effects of multiple treatments with collagen-­stimulating
properties, such as MFU (Ultherapy®), injection of Ca-­HA-­based,
hyaluronic-­acid-­based soft tissue fillers, and microneedling. While
a combination of Ultherapy® immediately before Radiesse® and
Belotero® Revive injections proved to be the most effective pattern
of treatment regarding thickening of the epidermal and the dermal
layers as well as the SMAS, similar tendencies were observed to a
lesser degree in all the areas treated. These treatments act syner-
gistically for the production of collagen and combining these treat-
ments allows for an increase of collagen production consecutively
leading to clinical improvement of skin quality.
| 1729
AU T H O R C O N T R I B U T I O N S
Gabriela Casabona, Kai Kaye, Sebastian Cotofana, Kristina
Davidovic, Michael Alfertshofer, Lysander Freytag have made sub-
stantial contributions to conception and design, or acquisition of
data, or analysis and interpretation of data. Gabriela Casabona, Kai
Kaye, Sebastian Cotofana, Kristina Davidovic, Michael Alfertshofer,
Lysander Freytag have been involved in drafting the manuscript or
revising it critically for important intellectual content and given final
approval of the version to be published. Each author has participated
sufficiently in the work to take public responsibility for appropriate
portions of the content and agreed to be accountable for all aspects
of the work in ensuring that questions related to the accuracy or
integrity of any part of the work are appropriately investigated and
resolved.
F U N D I N G I N FO R M AT I O N
The authors received no financial support for the research, author-
ship, and publication of this article. The products used in this study
were donated by the injectors for the purposes of this study.
C O N FL I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no conflicts of interest to
disclose.
DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from
the corresponding author upon reasonable request.
E T H I C S S TAT E M E N T
This article was accepted by ethical comitee UB under number
8N11S85AP2234/2022.
I N FO R M E D C O N S E N T
Patient signed all inform consents for the study according to the
Declaration of Helsink.
ORCID
Gabriela Casabona https://orcid.org/0000-0002-3183-0432
Sebastian Cotofana https://orcid.org/0000-0001-7210-6566
Michael Alfertshofer https://orcid.org/0000-0002-4892-2376
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How to cite this article: Casabona G, Kaye K, Cotofana S,
Davidovic K, Alfertshofer M, Freytag L. Histological effects of
a combined collagen stimulation procedure consisting of
microfocused ultrasound, soft tissue filler, and Ca-­HA
injections. J Cosmet Dermatol. 2023;22:1724-1730.
doi:10.1111/jocd.15770