ORIGINAL ARTICLE

Autologous Fibroblasts for Treatment
of Facial Rhytids and Dermal Depressions
A Pilot Study
Deborah Watson, MD; Gregory S. Keller, MD; Victor Lacombe, MD;
Peter B. Fodor, MD; Jeffrey Rawnsley, MD; Gary P. Lask, MD

Objective: To assess effectiveness of intradermal injec-

Main Outcome Measures: Subjective improvement

tions of autologous fibroblasts for the treatment offacial rhytids and dermal depressions.

scores were obtained by each patient and 2 clinicians at
every follow-up visit. Skin surface topographical features were evaluated with optical profilometry by comparing silicone molds before and after injection. Histological analysis was performed on a biopsy specimen of
the postauricular injection site.

Design: Six-month prospective pilot study. Photographs and silicone molds were taken of a prominent rhytid or dermal depression from each patient prior to treatment and at 6 months after treatment.

Results: Nine of 10 patients noted a 60% to 100% imSetting: Specialty clinic in academic medical center.
Patients: Ten adults (age range, 24-69 years) who eachexhibited a prominent rhytid or depressed facial scar.
Intervention: A 3-mm postauricular skin biopsy specimen from each participant was sent to Isolagen Technologies, Inc, laboratories, where a fibroblast cell line wasdeveloped. Three injection sessions were performed at
2-week intervals; target areas were the study site as well
as behind the ear.

I
From the Divisions of Head andNeck Surgery (Drs Watson,
Keller, Lacombe, andRawnsley), Plastic Surgery(Dr Fodor), and Dermatology(Dr Lask), University ofCalifornia, Los Angeles.
Dr Watson is now with theDivision of Head and Neck
Surgery, University ofCalifornia, San Diego.

provement with the treatment; clinicians made similar
observations. Size reduction of 10% up to 85% of the studysite was demonstrated by optical profilometry for every
patient. Microscopically, there was evidence of increased thickness and density of dermal-layer collagen.
Conclusions: Intradermal injection of autologous fibroblasts may be an effective treatment option for facialrhytids and depressed scars.

Arch Facial Plast Surg. 1999;1:165-170

NJECTABLE COLLAGEN has become a popular method for correcting soft tissue contour defects since its introduction in
1977.1 It has been used to fill superficial wrinkles around the eyes and lip
and deeper furrows often seen in the forehead, glabella, or nasolabial regions. Satisfying results have been observed whenused to correct acne and chickenpox depressions, iatrogenic-traumatic scars, andareas of dermal atrophy.
Purified bovine collagen is available
as Zyderm I (35 mg/mL of collagen), Zyderm II (65 mg/mL of collagen), andZyplast (35 mg/mL; collagen cross-linked
with glutaral) (Isolagen Technologies, Inc,
Paramus, NJ). Unfortunately, 1% to 6% of
healthy patients receiving collagen injections experience a localized hypersensitivity reaction.2-4 Usually this manifests as
temporary erythema, pruritus, induration, and swelling. Granulomatous foreign body reactions have been reported by

Overholt et al5 and Moscana et al.6 Others2,7 have documented the development oferythematous nodules at the injection sites,
which can take up to 3 years to subside.
Overholt et al5 indicated that the source of
the allergic response may be a reaction to
bovine antigen. Because of its heterogeneous nature, preliminary tolerance tests
to the collagen implant are necessary.8-12
The use of collagen is only a temporary corrective measure. Zyderm I is rapidly digested by tissue collagenases and isresorbed in several weeks. The higher concentration of collagen in Zyderm II delays resorption time to within 3 months.12
Zyplast has some advantage with its content of glutaral but still eventually disappears.
Soft tissue augmentation with autologous material theoretically circumvents
problems with resorption and allergic reactions. Fat, although autologous, tends
to have shortcomings as an injectable filler.
Surgical harvesting and processing are re-

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PATIENTS AND METHODSTen healthy adult patients (age range, 24-69 years) seeking improvement of facial rhytids or depressed facial scars
were referred to the study clinicians by the dermatology
or facial plastic surgery clinics in our institution. Any history of autoimmune disease, chronic skin disorders, disseminated cancer, or organ transplantation excluded a participant from the study. Each patient presented withprominent glabellar lines, perioral rhytids, nasolabial folds,
or depressed facial scars. A study site was chosen, and its
location was measured and recorded with respect to a nearby
facial anatomical landmark (eg, lateral canthus, oral commissure). All subjects were informed of the goals and methods of the study, the necessity for reliable follow-up, andthe potential risks associated with the use of injectable materials prior to signing consent forms. Patients were also
requested to refrain from any skin resurfacing-lifting procedure for the duration of the study.
Pretreatment photographs and a pair of identical silicone molds were taken of each participant’s study site. Acquiring silicone molds of the skin defect began with thorough cleaning of the target surface with 70% isopropyl
alcohol pads. A foam self-adhesive ring with a diameter of
19 mm (CuDerm Corp, Dallas, Tex) was carefully placed
over the study site so as not to distort the skin topography. Silicone elastomer (Silflo Silastic) replicating resin premixed with catalyst (CuDerm Corp) was spread over the
study site without applying pressure; an intentional 2- to
3-mm overlap was made on top of the ring. The resin polymerized in less than 3 minutes, at which time the silicone mold together with the ring was gently peeled off theskin surface and labeled.
A 3- to 4-mm punch biopsy specimen was taken fromthe postauricular crease for every subject using 1% lidocaine with 1:100 000 epinephrine for local anesthesia. The
skin defect was closed with a single 5-0 chromic suture. The
specimen was sent in a sterile, labeled transport media tube
on ice and in a thermos by overnight delivery to the Isolagenlaboratory. The cells were then expanded by proprietary tissue culture techniques during the next 6 weeks.17 At this time,
a 0.1-mL intradermal test injection of autologous cells was
performed using a 30-gauge needle on the volar aspect of theforearm for each participant. The patient was instructed torecord any evidence of an allergic skin reaction.
By the second week, a sterile 2-mL vial containing 1.3
to 1.5 mL of autologous fibroblasts suspended in 5% dextrose injection (5% dextrose-lactated Ringer’s solution;quired prior to its use, results are variable, and reapplication is necessary because of its tendency to undergo
resorption.13,14
Other autologous injectable materials include a dispersion of intact autologous collagen fibers that have been
derived from the patient’s dermal layer (Collagenesis Inc,
Beverly, Mass). One-year results demonstrating more than
75% correction after at least 3 injections have been reported.15 Unfortunately, this application has limited yield.
To obtain 1 cm2 of dispersed collagen fibers, up to 19.5
cm2 of skin is required for processing. Repeated applications to attain full correction necessitate additional skinexcision.

Baxter-Allegiance, Round Lake, Ill) was sent on ice in a thermos via overnight delivery to the clinic for each patient. To
ensure optimal viability of the cells, therapeutic injections
were always scheduled within 24 hours of shipping.16 No
preparation of the injectable material was required. The cells
were loaded into a 3-mL syringe and injected with a 2.2cm-long 30-gauge needle. Prior to injection, the target skinsurface was cleansed thoroughly with 70% isopropyl alcohol pads. Local anesthesia was preferred by most patientsand consisted of nerve blocks, topical injections of 1% lidocaine, or surface application of a mixture of 2.5 % lidocaine
and 2.5 % prilocaine cream. When topical injections wereused, care was taken to avoid distorting the skin defect. The
anesthetic was placed clear of the target site.
The injection technique required multiple passes into
the upper and middle dermal layers with the bevel of theneedle pointed down. It was necessary to fill in the skindefect with the injection and also to attempt to create blanching and tension on the skin surface during the process. This
ensured a maximal effect in the dermis each time
(Figure 1). All patients received injections to the study
site, a designated area behind the ear, and facial areas of
their choice. Ice packs were administered to the treated areas for the next 2 hours.
Therapeutic injection sessions were repeated at 2- to
3-week intervals for a total of 3 injection sessions for eachparticipant. Follow-up visits were made every 2 to 3 months
from the date of the last injection session. At each visit, theparticipant and 2 clinical observers (one of whom was D.W.)
were shown pretreatment photographs. Each individual was
asked to score any improvement using a 0% to 100% scale.
At the 6-month follow-up visit, photographs of the site
after the treatment and silicone molds were acquired. In
addition, a 3- to 4-mm punch biopsy specimen was taken
behind the ear at the site that had received all 3 injections.
This specimen was placed in formalin and processed for
microscopic analysis using hematoxylin-eosin staining.
Appropriately labeled silicone molds of each participant before and after the injection were sent to the Skin
Study Center (Broomall, Pa), where specific computerized optical profilometry measurement capability is available. The application involves a computerized digital image–processing system with specially designed imageprocessing hardware and software. A fiberoptic illuminator
at a fixed angle is used to enhance the surface features ofthe silicone mold so that an image profile can be created.
The image is then digitized into a pixel matrix to elicit
numeric data that corresponded to microtopographic features on the silicone mold.18

Ideal autologous, injectable material should provide
long-term correction, require negligible surgery for initial
tissue harvest, and have unlimited yield without the needfor additional tissue harvest. The soft tissue augmentation
system that aligns with these principles is available from Isolagen Technologies, Inc. The system requires a small skinsample from the patient to develop an autologous fibroblast
cell line. Living cells are then injected back into the patient
to correct skin contour defects. Once reintroduced into thedermis, the fibroblasts participate in a long-term protein repair process that helps to sustain the corrective effect.
Isolagen Technologies, Inc, has made this system
available for clinical use during the last 3 years. Long-

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Figure 1. Injection technique, using a 2.2-cm-long 30-gauge needle,
involved placing the cells into the upper and middle dermis. Blanching andsurface tension on the skin is observed during defect filling.

Figure 4. Pretreatment acne scar of patient 4 (arrow).

Figure 2. Pretreatment nasolabial fold of patient 1.

Figure 5. Acne scar after treatment of patient 4 at 6 months (arrow).

Figure 3. Nasolabial fold of patient after treatment at 6 months.

term correction of more than 2.5 years and absence ofallergic adverse effects (erythema, pruritus, or swelling)
have been reported.16 This pilot study was undertaken
to assess the effectiveness of the Isolagen system as wellas to substantiate these subjective findings with objective data.
RESULTS

Photographs before and after the injection are included
for patient 1, who demonstrated correction of nasolabial folds, and for patient 4, who revealed improvementof an acne scar over the malar region ( Figure 2 ,Figure 3, Figure 4, and Figure 5). There were noreports of infection or diffuse allergic reactions; however, patient 1 experienced self-limiting erythema at
her injection sites that persisted for 3 days without
sequelae.
Few of the participants noticed subjective improvement after the second injection session, but most reported changes subsequent to the third injection session. By the 3-month follow-up visit, 9 of the 10 patients
noted 40% to 100% subjective improvement; patient 6
described only a 5% improvement. The scores given by
the 2 clinicians (one of whom was D.W.) were averaged
for each subject and these demonstrated a 50% to 80%
subjective improvement for 8 of 10 patients. A score of
25% was given by the clinicians for patients 6 and 7(Figure 6). The patient scores correlated well with thosegiven by the clinicians (2-tailed t test, r = 0.92; P,.01).
Subjective improvement scores continued to increase by the 6-month follow-up visit. Nine of 10 patients reported 60% to 100% subjective improvement,
while patient 6 submitted a score of 20% improvement.
The averaged improvement scores by the clinicians ranged
from 60% to 100% for 8 of 10 subjects. Again, the lowest scores, 25% and 40%, were assigned to patients 6
and 7, respectively (Figure 7). The lowest scores ofsubjective improvement were consistently assigned topatients 6 and 7 during all follow-up visits. These
2 participants were the older individuals in our study(ages, 69 and 59 years, respectively).
Both pairs of silicone molds taken from each patient before treatment and 6 months after treatment were

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90

90

80

80

70

70

Percent Correction

100

Percent Correction

100

60
50
40
30

60
50
40
30

20

20

10

10

0

1

2

3

4

5

6

7

8

9

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1

2

3

Patients

4

5

6

7

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Patients

Figure 6. Subjective improvement scores at 3 months. Light-shaded bars
indicate patient scores and dark-shaded bars represent the averaged scoresof the 2 clinicians.

Figure 7. Subjective improvement scores at 6 months. Light-shaded bars
indicate patient scores and dark-shaded bars represent the averaged scoresof the 2 clinicians.

Figure 8. Representation (1:1) of a pretreatment silicone mold. Surface
detail demonstrates the negative impression of a skin rhytid. Diameter of
replica is 19 mm.

Figure 9. Representation (1:1) of a silicone mold after treatment taken at 6
months. Note the reduction in shadow effect due to the correction of therhytid.

sent to the Skin Study Center. The technicians at the center selected 1 replica of each pair with the greatest amountof skin topographic detail for optical profilometry measurements. For the purposes of the study, the portion ofdata analyzed involved the percentage of replica surface area
covered by a shadow that was cast by light against the skindefect. The shadow area percentages of pretreatment replicas were compared with those of molds made after treatment. Figure 8 and Figure 9 represent typical silicone
replicas before and after treatment, respectively.
The percent reduction of the replica shadow area wasdetermined after comparing the values before and after
treatment. Figure 10 illustrates the reduction percentages for all patients. Results from every patient revealed
shadow-area reduction, ie, all study sites became more
shallow. The least demonstrable effect is noted again withpatients 6 and 7.
A positive correlation was found between the objective measurement values and the subjective improvement scores given by the clinicians (one of whom was
D.W.) (2-tailed t test, r = 0.67; P,.05). However, a significant correlation was not present when the objective
measurements were analyzed with patient subjective improvement scores.
The histopathological features of the injected postauricular biopsy specimens were compared with thoseof healthy skin behind the ear. Figure 11 represents
normal epidermal and dermal layers (original magnification 3100) and Figure 12 is a photomicrograph of
an injected area at the identical magnification. The treatedskin specimen demonstrated a denser and thicker layerof collagen in the dermal region, absence of any inflammatory reaction, and viable fibroblasts throughout.
COMMENT

Treatment of superficial facial rhytids and small scar depressions with injectable materials is an alternative to implant products that are surgically inserted. The injection technique, however, offers precise control of quantity
delivered and depth of placement. Shortcomings withcurrent injectable materials include significant surgicaltissue harvest for material processing, reported inflammatory or immunologic reactions, and eventual resorption, thereby requiring numerous reapplications if continued correction is desired. Any autologous material thatis applied by injection technique does not require significant surgical harvest, provides limitless yield, demonstrates long-term correction by clinical reports, andrepresents the ideal augmentation material for facialrhytids and dermal depressions. Our pilot study suggests that the Isolagen system fulfilled these objectives.

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90

Percent Reduction of Shadow Areas

80
70
60
50
40
30
20
10
0

1

2

3

4

5

6

7

8

9

10

Patients

Figure 10. Percent reduction of the silicone replica shadow area for eachpatient was determined after comparing optical profilometry measurements
before and after treatment.

Figure 12. Six-month biopsy specimen of injected postauricular skin(hematoxylin-eosin, original magnification 3100). Note the increased density
and thickness of collagen in the dermal region as compared with Figure 11.

Figure 11. Biopsy specimen of untreated postauricular skin(hematoxylin-eosin, original magnification 3100).

Three injection sessions appeared to provide anadequate density of fibroblasts to maintain correction
beyond 6 months for most patients in our study. It is uncertain if patients 6 and 7, who demonstrated the least
amount of correction, would have had better success witha total of 4 or 5 injections. The age of these 2 participants (.59 years) could be a significant factor that affected the biological activity of the injected fibroblasts,
but it is difficult to draw any conclusions within our small
study group. Interestingly, poor results with patients older
than 65 years have been reported by other clinicians using the Isolagen system.16
A unique feature of the Isolagen system is its ability
to effect continual and gradual improvement of the skinsurface contour for months after the injection sessions havebeen completed. There have not been any reports of nodular hypertrophy or keloid formation since the initial useof Isolagen more than 3 years ago (oral communication,
Gregory Keller, MD, University of California, Los Angeles [UCLA] Medical Center, July 1997; oral communication, William Boss, Jr, MD, Hackensack University Medical Center, Hackensack, NJ, April 1998). Cell-to-cell contact
inhibition is postulated to play a role with regulating thesynthesis of collagen or the replication of fibroblasts.
Subjective improvement scores are useful indicators of changes with the study site. We found that scoresfrom the participants strongly correlated with those thatwere averaged from the 2 clinical observers (one of whom
was D.W.). Although the data from the optical profilometry measurements correlated strongly with thesubjective improvement scores from the clinicians, theeffect was not as pronounced with the patient scores. Perhaps greater objectivity by clinical observers provided
more accurate scoring. Moreover, results from opticalprofilometry were not as dramatic as the subjective improvement scores. A possible explanation for these more
subtle findings could be attributed to the viscosity of thesilicone resin. The moderately high viscosity of the resin
may have impeded the filling of the central crevice of arhytid; therefore, a more “shallow” mold was taken. Otherwise, the silicone replicas, once polymerized, reliably
maintained their contours for measurement and indefinite storage.19
With respect to the histological findings, fibroblasts appeared to be incorporated into the dermal architecture and either began nascent production ofcollagen or stimulated synthesis from the native cells,
subsequently, creating the thicker collagen layer.
Overall, there was a high index of participant satisfaction in this study. Patients and clinical observers did
not report any evidence of skin infection. Only 1 subject noted prolonged erythema at her injection sites; it
is unclear whether this might have been a reaction to theuse of lidocaine as the anesthetic.
Follow-up beyond 6 months is of great interest tous. A larger study at our institution is in progress, involving 38 participants with follow-up ranging from12 to 24 months. The Isolagen system holds numerous
advantages and negligible risks. Further evidence of its
long-term corrective ability would provide a significant
contribution to the practice of soft tissue augmentation.

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Accepted for publication May 17, 1999.
Presented at the fall meeting of the American Academy of Facial Plastic & Reconstructive Surgery, San Antonio, Tex, September 11, 1998.
We thank Isolagen Technologies, Inc, for processing
the skin biopsy specimens and providing the fibroblast injections; Mary Jo Grove, MSc, Skin Study Center, for the
optical profilometry measurements; Minu K. Patel, MSc, University of Illinois at Chicago, for the statistical analysis; Scott
Nelson, MD, UCLA Department of Pathology, for his interpretive assistance with the histological studies; and Sassan
Alavi, MD, and Kay Durairaj, MD, UCLA Division of Headand Neck Surgery, for their assistance with patient follow-up visits.
Corresponding author: Deborah Watson, MD, 4991
Concannon Ct, San Diego, CA 92130.
Reprints: Gregory Keller, MD, 2323 De La Vina, Suite
104, Santa Barbara, CA 93105 (e-mail: faclftr@aol.com).
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3. Cooperman LS, Mackinnon V, Bechler G, Pharriss BB. Injectable collagen: a six
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4. De Lustro F, Fries J, Kang A, et al. Immunity to injectable collagen and auto-

immune disease: a summary of current understanding. J Dermatol Surg Oncol.
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5. Overholt MA, Tscher JA, Font RL. Granulomatous reaction to collagen implant:
light and electron microscopic observations. Cutis. 1993;51:95-98.
6. Moscana RR, Bergman RB, Friedman-Birnbaum R. An unusual late reaction to
Zyderm I injections: a challenge for treatment. Plast Reconstr Surg. 1993;92:
331-334.
7. Clark DP, Hanke CW, Swanson NA. Dermal implants: safety of products injected
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8. Baral J. Collagen skin testing. J Dermatol Surg Oncol. 1990;16:70.
9. Barr RJ, Stegman SJ. Delayed skin testing reaction to injectable collagen implant (Zyderm). J Am Acad Dermatol. 1984;10:652-658.
10. Colton JJ, Tashjian RC. Injectable collagen materials in facial plastic surgery.
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11. Elson ML. The role of skin testing in the use of collagen injectable materials.
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13. Illouz YG. Present results of fat injection. Aesthetic Plast Surg. 1988;12:175-181.
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15. DeVore DP, Kelman CD, Fagien S, Casson P. Autologen: autologous, injectable dermal collagen. In: Bosniak S, ed. Principles and Practices of Ophthalmic Plastic andReconstructive Surgery. Philadelphia, Pa: WB Saunders Co; 1996:670-675.
16. Boss WK, Marko O. Isolagen. In: Klein AW, ed. Tissue Augmentation in Clinical
Practice. New York, NY: Marcel Dekker Inc; 1998:335-347.
17. Boss WK, inventor; Isolagen Technologies Inc, assignee. Use of autologous dermal fibroblasts for the repair of skin and soft tissue defects. US patent 5 660 850.
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18. Grove GL, Grove MJ, Leyden JJ. Optical profilometry: an objective method forquantification of facial wrinkles. J Am Acad Dermatol. 1989;21:631-637.
19. Gormley DE. Three-dimensional optical micrometric analysis of casts of cutaneous surfaces. Bioeng Skin. 1985;1:81-92.

Correction
Error in Figure Orientation. In the original article titled, “Nasal Tip Bossae in Rhinoplasty: Etiology, Predisposing Factors,
and Management Techniques,” published in the April-June 1999 issue of the ARCHIVES (1999;1:83-89), Figure 5, C and D,
on page 88 were transposed incorrectly during processing for publication. Figure 5 is reprinted correctly here.
A

B

C

D

Figure 5. Patient who underwent vertical dome division without suturing the medial elements together who developed bilateral bossae during a 2-year
period. Revised using delivery technique, shave excision, and suturing of medial crura together. Preoperative (A), 2-month postoperative (B), 2-year
postoperative (C), and 6-month postrevision (D) views.

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