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The effect of retinyl palmitate on skin composition and rnorphornetry

  • STI Pharma, LLC
j. Soc. Cosmet. Chem., 39, 235-240 (July/August 1988)
The effect of retinyl palmitate on skin composition
and rnorphornetry
and A. G. WICH, The Lilly Research Laboratories, Eli Lilly and
Company, Lilly Corporate Center, Indianapolis, IN 46285.
Received January 21, 1988.
Topical administration of increasing doses (0.1%-5% (w/w)) of retinyl palmirate (RP) for 14 days, in a
suitable cosmetic vehicle, caused significant dose-related changes in skin composition and morphometry of
the hairless mouse. There was a maximum 32% increase of protein per unit of skin surface area and a
maximum of 128% increase of collagen per unit of skin surface area in response to RP administration when
compared to control vehicle. In addition, there was an increase of DNA content in response to RP adminis-
tration. There was significant thickening of the epidermis in response to the increasing dose of RP. Al-
though the total thickness of skin was not significantly increased by RP application, the total skin thick-
ness was greater than the untreated control or the control-vehicle-treated animals at every dose of RP tested.
These results indicate that the topical application of RP (in an active form) can alter the composition and
morphometry of the skin in the hairless mouse.
Vitamin A or retinol is essential for normal skin development. Vitamin A is an impor-
tant regulator of keratinocyte terminal differentiation (1). An excess of vitamin A in-
hibits keratinization (1), whereas a deficiency results in squamous metaplasia and kera-
tinization of epithelial tissue (2). Thus, epidermal development is, in part, regulated by
vitamin A. It is also known that vitamin A can alter or modulate total collagen syn-
thesis (3-5). Also, retinoic acid has been demonstrated to alter the type of collagen
synthesized (6).
Retino! has the potential to alter the expression of protein molecules in both the epi-
dermis and dermis. Although retinoic acid can alter both keratinocyte as well as fibro-
blast (7) protein metabolism, the precise effect of retino! on intact skin composition has
not been determined. Several cosmetic formulations contain vitamin A. The present
study was undertaken to determine whether or not vitamin A, if present in an active
form in cosmetic formulations, can alter skin composition and morphology.
Female hairless mice (HRS/J) (six to eight weeks old) were obtained from Jackson Labo-
ratories (Bar Harbor, Maine) and maintained for one week prior to all studies. The mice
were housed five animals per cage in standard "shoe box"-type cages (6 in x 10 in x 5
in) and given food and water ad libitum. Each animal received 0.1 ml of a test cosmetic
formulation applied to the dorsal skin surface and rubbed into the skin (approximately
5-10 sec), using a gloved finger as an applicator. This treatment was continued each
day for 13 additional days.
On Day 15, the animals were terminated by cervical dislocation and the skin removed
from the dorsal, treated surface of the animals. One section of the skin was taken for
histological evaluation and a second piece was scraped free of subcutaneous fat. Two
circular punch biopsies (6 mm in diameter) were taken from the latter, blotted dry, and
weighed. These biopsies were then pulverized in liquid nitrogen and homogenized in
phosphate-buffered saline using the Polytron-ST homogenization system. This homoge-
nate was used to determine the protein content, DNA content, and collagen content (as
measured by proteinaceous hydroxyproline content of the tissue) (8). An equal volume
of 10% (w/v) trichloroacetic acid (TCA) was added to the homogenate, and the suspen-
sion mixed and allowed to equilibrate for five minutes (3-5øC). The suspension was
centrifuged at 10,000 x g for ten minutes. The pellet was suspended in 0.5 ml of 5%
(w/v) TCA and centrifuged at 10,000 x g for ten minutes. The pellet was suspended in
0.2 ml of 10% (w/v) TCA and heated at 90øC for 20 minutes. The suspension was then
set in an ice bath for 30 minutes. The suspension was then centrifuged at 10,000 x g
for three minutes. The supernatant was analyzed for DNA as described by Schneider
(9). The precipitate was suspended in 0.5 ml of 0.5M NaOH, heated for one hour at
55øC, and an aliquot was removed for protein determination (10). The remainder of the
sample was sealed in a glass ampule in vacuuo with an equal volume of 12 N HC1 and
hydrolyzed for 24 hours at ! !0øC. After hydrolysis the sample was dried in a desiccator
and the dried material was assayed for hydroxyproline (! 1).
The skin samples taken for micromorphometric analyses were fixed in buffered neutral
formalin solution, dehydrated, and embedded in paraffin blocks which were then cut
into 7-1•m-thick sections. These sections were stained with hematoxylin and eosin for
later examination. Thickness measurements were based on the cross-sectional thickness
of the skin. Several (six to eight) sections were stained and used for evaluation per
animal. Thickness measurements (eight to ten) per animal were made by selecting the
measurement site at random. The average of these measurements was considered as the
measurement for one animal. Total skin thickness was considered the thickness of the
skin from the outer stratum corneum to the panniculus carnosus. Dermal thickness was
taken as the distance from the dermal epidermal junction to the top of the dermal
adipose deposits.
All cosmetic formulations used in this study were oil-in-water emulsions and were
manufactured under a nitrogen blanket. The water phase of the emulsion systems
ranged from 79-$4%, the oil phase was !6%, and the retinyl palmitate (Roche) con-
centration ranged from 0-5 %. Water was removed from the emulsion to accommodate
the additional retinyl palmitate. The concentration of the retinyl palmitate in each
formulation was determined by HPLC on a Lichrosorb Si 60/5 micron column (25 cm
X 4.5 mm id) (Alltech Assoc.) using 0.4% (v/v) methanol / 0.4% (v/v) isopropanol in
99.2% (v/v) isooctane as the mobile phase after separation and saponification according
to a modified USP XXI procedure (12). The determinations of the retinyl palmitate
concentration were made both before and after the animal treatment period. Results
indicated no significant degradation of the retinyl palmitate throughout the two-week
treatment period and were found to vary by less than 20% of the theoretical amount of
retinyl palmitate.
Skin tissue per surface area, as determined by punch biopsy weight, did not change
appreciably (Table I). This finding correlates well with the little or no change in total
skin thickness determined by micromorphometry (Table II). Although there was a 21%
increase in total skin thickness in animals treated with 0.1% retinyl palmitate when
compared to untreated control mouse skin, this change was not noted at any of the
other doses of retinyl palmitate. This increase was noted to be due to changes in both
the dermal thickness (Table II) and the adipose layer in the dermis. Although there was
a dramatic and dose-related increase in skin collagen content when up to 1.5 % retinyl
palmitate (Table I) was administered, there was no concomitant increase in dermal
thickness (Table II).
The most dramatic change in skin composition was noted in the collagen content. The
Table I
Effect of Treatment With Increasing Doses of Retinyl Palmitate in Visible Difference on Skin Composition
Punch Protein DNA content
Treatment weight content content (nmole HYP)
group (mg/punch) (mg/punch) (Ixg/punch) (punch)
Control 11.8 - 0.5 1.28 _ 0.09 44.2 - 3.7 82 _ 20
(no treatment) (8) (8) (8) (7)
Vehicle q- 12.1 - 0.5 1.37 - 0.07 51.3 - 2.3 67 - 8
0% retinyl palmitate (8) (8) (8) (8)
Vehicle q- 12.7 - 0.4 1.71 -+ 0.13"** 62.1 ñ 4.3*'** 126 - 18'*
0.1% retinyl palmitate (8) (8) (8) (8)
Vehicle q- 13.1 ñ 0.5 1.71 - 0.10"** 57.7 ñ 3.2* 135 ñ 27**
0.5 % retinyl palmitate (8) (7) (8) (8)
Vehicle + 12.5 - 0.5 1.81 - 0.10"** 54.2 ñ 4.4 153 - 15"**
1.5% retinyl palmitate (8) (8) (8) (8)
Vehicle q- 12.9 ñ 0.4 1.70 ñ 0.08*'** 66.5 ñ 3.4"** 134 ñ 14"**
5.0% retinyl palmitate (8) (8) (8) (8)
Values represent the mean ñ S.E.M. of the number of animals in parentheses. The diameter of each punch
was 6 mm.
* Indicates significantly different from untreated control at p 0.05.
** Indicates significantly different from the control-vehicle-treated group at p 0.05.
Table II
Effect of Treatment With Increasing Doses of Retinyl Palmitate in Visible Difference on
Skin Morphometry
Epidermal Dermal Total
Treatment thickness thickness thickness
group (}xm) (}xm) (}xm)
Control 27.9 --- 1.8 171 --- 17 377 -+ 28
(no treatment) (8) (8) (8)
Vehicle + 42.5 - 4.3* 180 --- 15 411 --- 25
0% retinyl palmirate (8) (8) (8)
Vehicle + 63.5 --- 2.9"** 206 ___ 24 458 --- 25*
O. 1% retinyl palmirate (8) (8) (8)
Vehicle + 71.7 - 2.6*'** 201 --- 14 431 --- 15
O. 5% retinyl palmirate (8) (8) (8)
Vehicle + 74.7 - 3.7"** 209 + 19 430 --- 28
1.5% retinyl palmitate (8) (8) (8)
Vehicle + 86.0 --- 4.9"** 286 --- 13"** 424 - 17
5.0% retinyl palmirate (8) (8) (8)
Values represent the mean --- S.E.M. of the number of animals in parentheses.
* Indicates significantly different from untreated control at p •< 0.05.
** Indicates significantly different from the control-vehicle-treated group at p •< O. 05.
lowest administered retinyl palmitate dose maximally increased the amount of protein
present per unit of surface area. The DNA content was near maximal at 0.1% retinyl
palmitate and decreased to near normal at 1.5% retinyl palmitate but increased dramat-
ically at 5% retinyl palmitate.
The photomicrographs in Figure 1 demonstrate the dose-related increase in epidermal
thickness (Table II). Although there is an inflammatory cell infiltrate into the dermal
NT 0% RP 0.1% RP 0.5% RP 1.5% RP 5% RP
Figure 1. Cross section of skin following 14 days of application of 0.1 ml of cosmetic vehicle. The
concentration of retinyl palmitate in each cosmetic vehicle is indicated under each skin cross section. E
indicates the epidermal tissue, DEJ indicates the dermal/epidermal junction, and D indicates the dermal
tissue. Magnification is 80 X.
tissue, note that changes in skin composition and the epidermal proliferative changes
are also apparent.
Cosmetic materials are generally accepted to have a minimal effect on skin biology. The
proven benefits of the application of cosmetic skin treatment products have usually been
attributed to a moisturization effect. One particular material used in cosmetic formula-
tions, vitamin A, is known to have systemic physiological and biochemical effects
(7,13). All of the physiological functions of retinoids (such as the requirement for
normal vision and reproduction) cannot be satisfied by retinoic acid (7). Indeed, these
functions seem to be adequately satisfied by retinol. However, the role of retinoids in
the regulation of skin development seems to be best satisfied by retinoic acid. Thus,
part of the actions of retinyl palmitate in skin may depend on its conversion to retinoic
acid (13). This conversion depends on the enzymatic cleavage of the ester bond in the
retinyl palmitate. Nonspecific esterase enzyme activity exists within skin (14). In addi-
tion, the skin must be able to oxidize the retinol to retinoic acid. It has been demon-
strated that skin preparations can indeed convert retinyl to retinoic acid (15). Although
it has been known that retinyl palmitate has the potential to effect a change of skin
composition and physiology, this potential has never been examined.
Retinoids, in general, prevent connective tissue atrophy. This prevention of atrophy is
probably mediated by an inhibition of collagenolytic activity (16). In addition, dermal
repair in response to UV damage, which is accelerated by retinoic acid administration,
appears to involve a zone of regenerating connective tissue (17). Although retinoids
inhibit collagen synthesis (3,5,18), they do promote glycosaminoglycan synthesis
(5,19,20). Therefore, some of the effects of retinoids in terms of altering dermal com-
position can be thought of as due to an inhibition of connective tissue breakdown and a
promotion of the synthesis of glycosaminoglycans. This hypothesis is .supported by the
observed changes in skin composition following treatment with retinyl palmitate.
The increase of DNA content within the skin can be partially explained by the influx of
inflammatory cells into the skin. Although there was an increase of skin DNA content
following treatment with 0.1% retinyl palmitate, this was the maximal response. There
is then a decrease of DNA content with increasing concentrations of retinyl palmitate.
This may be due to the inhibitory effect of high concentrations of retinoids on both cell
mobility and cellular proliferative capacity (18,21). However, at the highest dose ad-
ministered (5 %), there was a tremendous increase in DNA content. This may be due to
an irritant/inflammatory response to the application of the retinoid. Of interest in this
study is that even with irritation present, there was little or no change in the punch
biopsy weight. This suggests that the response is not significantly mediated by an
edematous response. Finally, retinyl palmitate administration causes an accumulation of
collagen within the dermis. These changes indicate that although there is an irritation
response in the skin treated with RP, there is also a significant effect on the collagen
content within the dermis. This alteration of the collagen content is unlikely to be due
to an irritation response.
Thus, cosmetic products which contain retinyl palmitate, in an active form, may indeed
bring about biochemical changes with both the epidermis and the dermis.
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... It can be safely applied around the eyes with better skin penetration and higher stability [9]. A study by Counts et al [10]. showed that topical application of RP in rats for 14 days resulted in epidermal thickening and enhanced protein and collagen stimulation. ...
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Gravi-A nanoparticles, composed of retinyl propionate (RP) and hydroxypinacolone retinoate (HPR), were prepared by encapsulating the two using the high-pressure homogenization technique. The nanoparticles are effective in anti-wrinkle treatment with high stability and low irritation. We evaluated the effect of different process parameters on nanoparticle preparation. Supramolecular technology effectively produced nanoparticles with spherical shapes with an average size of 101.1 nm. The encapsulation efficiency was in the 97.98–98.35% range. The system showed a sustained release profile for reducing the irritation caused by Gravi-A nanoparticles. Furthermore, applying lipid nanoparticle encapsulation technology improved the transdermal efficiency of the nanoparticles, thereby allowing these to penetrate deep into the dermis layer to achieve precise and sustained release of active ingredients. Gravi-A nanoparticles can be extensively and conveniently used in cosmetics and other related formulations by direct application.
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The specific tissue changes which follow the deprivation of fat-soluble vitamin A in albino white rats and in the human concerns epithelial tissues. This effect is the substitution of stratified keratinizing epithelium for normal epithelium in various parts of the respiratory tract, alimentary tract, eyes, and paraocular glands and the genitourinary tract. We have described the morphological sequences which clearly show that the replacement of epithelium arises from focal proliferation of cells arising from the original epithelium and not by differentiation or change of preexisting cells. Young rats respond to the deficiency more promptly than adults. Growth activity of epithelium is not diminished. On the contrary, there is convincing evidence that it is greatly augmented. In a few of our animals the behavior of the replacing epithelium in respect to numbers of mitotic figures and response on the part of connective tissue and blood vessels suggests the acquisition of neoplastic properties. While the epitheliums which are the seats of these changes are largely of covering types, glandular epithelium is involved, specifically in the paraocular glands and salivary glands. It is highly probable also that the epithelium of gland ducts, respiratory mucosa, and genitourinary tract have secretory functions so that we conclude that the deficiency results in loss of specific (chemical) functions of the epitheliums concerned, while the power of growth becomes augmented. Explanation for the substitution of a chemically inactive (nonsecretory) epithelium, common in type for all locations, remains a matter of speculation. We can only speculate also in regard to the absence of change in the epithelium of such organ as the liver, parenchyma of the kidney, stomach, and intestines. The significance of the order or sequence in which different organs exhibit this change has not been determined. In general the respiratory mucosa in nares, trachea, and bronchi respond first, then the salivary glands, eye, genitourinary tract, then paraocular glands and pancreas, although as has been noted there are exceptions to this order. Our studies show that the mitochondrial apparatus is not primarily affected. Study of individual cells indicates that the first morphological evidence of avitaminosis will be found in the nucleus. We have not devoted sufficient study to be certain, but an increase of chromatin and in some instances in size of nucleoli are early morphological manifestations. Other important effects of fat-soluble A deficiency are atrophy of glandular organs, emaciation, localized edema of testes, submaxillary gland, and connective tissue structures of the lungs and focal myocardial lesions. From our own limited experience with rats fed on a water-soluble B deficient diet and from work by Cramer, Drew, and Mottram, the loss of fat in water-soluble B deficiency is as great, if not greater than in vitamin A deuciency, so that tor tne present we assume that this is not a specific manifestation of any one avitaminosis. The same applies to glandular atrophy. Both of these effects probably concern the nutrition as a whole and may be ascribed to inanition. The occurrence of transient edema in testes and salivary gland coinciding with a period of maximum atrophic change, suggests the hypothesis that this edema is the result of failure of epithelium to utilize transported material, which leads further to the hypothesis that the capillaries of these organs are differentiated in regard to permeability to the respective materials utilized by the cells. It would seem that in the case of the testis we have a unique instance of complete atrophy producible at will without impairment of circulation and supporting tissues. This phenomenon may possibly be followed with advantage in the study of the mechanism of edema. Vascularization of the cornea, as we have shown it to be independent of infection, must be a physiological response to the increased demands of the rapidly growing epithelium which has replaced the corneal epithelium. We have assumed throughout this work that the diet on which we kept our animals was deficient in respect to a single substance or group of substances having similar physiological properties, designated by the term fat-soluble vitamin A. Whether or not more than one so called vitamin or accessory substance was missing in the diet we employed does not affect the theoretical importance of the morphological results. Work by Evans and Bishop would indicate that other factors affecting fertility in addition to the so called antixerophthalmic or vitamin A factor may have been missing. Our own experience leads us to believe the specific effects we have described upon epithelial tissues were in all probability due to withdrawal of a single factor. We have shown how these effects, that is the replacement of uterine epithelium by keratinizing epithelium can account for sterility in the female. Whether or not the atrophy of the testis is due to the same factor remains to be proved, but presumptive evidence is strong that this is the case. The study of the reverse changes that follow in the rapid amelioration when the rats are restored to an adequate diet has been begun and will be reported later. We have shown that the substitution of keratinizing epithelium in all locations is not secondary to infections, and presumably is a primary effect of the withdrawal of factors essential for the chemical activities or maintenance of differentiation of the epitheliums concerned. It is, of course, possible that the phenomenon is produced in a roundabout way in that it may be secondary to the effects of the avitaminosis upon the metabolism of tissue-sustaining substances. This possibility is supported by the cessation of growth of the skeleton and teeth, although we know that other avitaminoses produce retardation of growth.
The procedure for the determination of nucleic acids described in this chapter is based on the finding that nucleic acids can be separated from other tissue compounds by their preferential solubility in hot trichloroacetie acid. The isolated nucleic acids are then quantitated by means of colorimetric reactions involving the pentose components of the nucleic acids. The determination of nucleic acids in tissues is largely a problem in identification. By means of the extraction procedures described in the chapter and the colorimetric reactions of peptide nucleic acid and DNA, a considerable degree of specificity is placed on the determination of these compounds. Occasionally however, false results will be obtained, owing to the presence of materials in the nucleic acid extracts that interfere with the pentose reactions. It is emphasized that the extraction methods described were developed for nucleic acid determinations by spectrophotometric methods. Although it was at first thought that these procedures might be directly applicable to isotopic work, it has become quite clear that the separations are not sufficiently refined for such studies. The methods have served, however, as starting points for other separation procedures more suitable for isotopic work.
The Ehlers-Danlos syndromes (ED-S) are a group of connective tissue diseases which occur in humans and other mammals. Mink inherit an autosomal dominant form of ED-S which is characterized by laxity and decreased tensile strength of the skin. We wish to report some of the biochemical changes in the skin of affected mink as compared to age-matched, nonaffected mink. There was a 39% increase in acetic acid extractable collagen per wet weight of tissue of the skins of the affected mink. This was accompanied by a 260% increase in prolyl hydroxylase specific activity, a 179% increase in lysyl hydroxylase specific activity, and a 118% increase in lysyl oxidase specific activity in the skins of affected mink. [3H1-Hydroxyproline formation was increased 133% when skin tissue minces were incubated with [3H]-proline. This was accompanied by a 77% increase of [3H]-proline incorporation into protein and a 93% increase of [14C]-glycine incorporation into protein. Noncollagen protein synthesis, evaluated by measuring [3H]-tryptophane incorporation into protein, revealed a 40% increase in noncollagen protein synthesis. The increased collagen synthesis rate in the skins of the ED-S affected mink may represent either the absence of the control of collagen metabolism which contributes to the molecular defect of the ED-S in the affected mink, or a response to the damaged skin caused by the ED-S in the affected mink, or both.
This study elucidates the biochemical response of rabbit corneal keratocytes (fibroblasts) to retinol and retinoic acid in their production of collagen, fibronectin, sulfated glycosaminoglycans, collagenase, and [3H]thymidine incorporation. The morphologic appearance of cultured keratocytes was not altered by retinoid treatment. Collagen production and [3H]thymidine incorporation demonstrated a parallel decline in response to retinoids. Collagen type was unaffected as was collagenase activity. Retinoids had minimal effect on cell layer-associated 35S-labeled glycosaminoglycans, however medium-soluble 35S-glycosaminoglycans were increased. The most dramatic effect was in fibronectin synthesis which was increased 2-3-fold. These data demonstrate that rabbit keratocytes alter their biosynthesis of extracellular matrices in response to retinoids. This may be significant in corneal pathology, since the delicate balance of these extracellular macromolecules is responsible for corneal integrity and stability.
Retinoic acid, unlike the naturally occurring vitamin A (retinol), is a minor component of the human diet. It is formed in vivo from retinol and has many metabolites. The biological activity of the metabolites is not higher than that of retinoic acid itself, indicating that the metabolites must be products of retinoic acid catabolism. Little is known about the enzymatic systems responsible for forming retinoic acid or about how it enters the cell. Discovering the molecular mechanism(s) of retinoic acid activity in cellular metabolism is important to understanding its physiologic role. The pharmacologic effects of high doses of retinoic acid may be caused by its action on cellular membranes. Conversely, low concentrations appear to produce physiologic effects. Results of experiments with animals and with cell cultures indicate that the primary physiologic role of retinoic acid is in cellular differentiation. Retinoic acid influences genomic expression, inducing the appearance of some proteins while suppressing the expression of others. The existence of an intracellular retinoic acid-binding protein suggests that it may mediate the physiologic effects of retinoic acid on cellular differentiation.
All-trans-retinoic acid formation from topically applied retinol has been demonstrated in the skin of skh/hr1 (hairless) mice. The all-trans-retinoic acid was identified on the basis of its chromatographic properties on HPLC at various pH values, its photoisomerization to reaction products identical to those formed from authentic all-trans-retinoic acid, and its co-chromatography with methyl retinoate after methylation with diazomethane. Topically applied retinol is about 2-fold less potent at inducing epidermal hyperplasia and 7-fold less potent at inhibiting the induction of epidermal ornithine decarboxylase by phorbol esters than all-trans-retinoic acid in this strain of mice. To elucidate the possible role all-trans-retinoic acid formation from retinol may have in these pharmacological activities, the epidermal and dermal all-trans-retinoic acid levels were compared in mice treated topically with retinol or [11-3H]-all-trans-retinoic acid. The levels of all-trans-retinoic acid found after retinol treatment were several orders of magnitude lower than those found after [11-3H]-all-trans-retinoic acid treatment, and they were insufficient to account for the difference in potencies between all-trans-retinoic acid and retinol. Retinol was eliminated from the epidermis at a rate similar to that of all-trans-retinoic acid after topical administration, but the initial tissue levels achieved were lower. These results suggest that the lower potencies of retinol may simply reflect lower tissue uptake.
The differentiated phenotype of rabbit articular chondrocytes can be characterized by the synthesis of high levels of cartilage specific proteoglycan and collagen (type II). Treatment of these cells in primary monolayer culture for periods of up to 18 days with 0.03 to 3.0 micrograms/ml retinoic acid (RA) resulted in suppression of colony formation, altered morphology, and decreased (eightfold) proteoglycan and collagen synthesis. With the exception of collagen synthesis, these changes were complete with all doses after 4 days of treatment. Collagen synthesis declined more slowly; it was dose dependent after 4 days and maximally inhibited by all doses by 9 days. Detailed analysis of the collagen phenotype was performed using SDS-PAGE of intact chains and 2-D CNBr peptide analysis. RA caused cessation of type II synthesis, and transient stimulation of type III and type I trimer collagen synthesis, without induction of type I collagen. Essentially identical results were obtained with retinol. The resultant collagen phenotype differed significantly from the type I-containing phenotype induced by subculture. Thus, suppression of this differentiated program did not elicit a common modulated phenotype. The results are discussed in the context of direct and indirect mechanisms of RA-dependent modulation of chondrocyte gene expression.