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Fibroblasts and Hyaluronic Acid Longevity

The Effect of Cultured Autologous Fibroblasts on Longevity of Cross-Linked Hyaluronic Acid Used as A Filler

Abstract

Background

Various kinds of biomaterials are being used for soft tissue augmentation in plastic surgery. Organic molecules are usually absorbed in a short amount of time. Inorganic molecules stay in the body for a longer period of time, but are prone to cause various reactions; therefore, none of them are ideal filler substances.

Objectives

This study was designed to examine the clinical and histologic effects of injection of cultured fibroblasts in hyaluronic acid as a filler material. The advantages, disadvantages, and side effects of the procedure were examined during the study.

Methods

Skin biopsies obtained from the backs of 30 Sprague Dawley rats were used in the study. Dermal fibroblasts obtained from these biopsies were cultured for 21 days and, after 3 weeks, autologous labeled cultured fibroblasts of the rats were injected intracutaneously alone and mixed with hyaluronic acid. Injections of culture medium and hyaluronic acid were also performed as control groups. At the end of the fourth and eighth months, skin biopsies were taken from the injection sites and normal skin and examined under light and electron microscopes.

Results

The injected fibroblasts, elastin, and collagen production were analyzed and found to be stable, long-lasting, and well tolerated. No complications were observed.

Conclusions

Cultured human dermal fibroblasts combined with hyaluronic acid can provide a suitable, biocompatible, and long-lasting material and should be regarded as a new method in dermal renovation even beyond their temporary filling effect.



For the elimination of facial wrinkles and skin contour defects, injectable filler substances composed of commercially prepared materials are now widely available. Injectable soft tissue substitutes provide an affordable, nonsurgical alternative for the correction of facial signs of aging. Younger patients with subcutaneous ptosis, atrophy without advanced skin laxity, and nasolabial folds are the best candidates for tissue fillers.

The search for an ideal augmentation material for facial soft tissue has been an ongoing effort for many years. The ideal soft tissue filler should be nonantigenic, noninflammatory, stable after injection, nonmigratory, nontoxic, noncarcinogenic, biologically inert, long-lasting but resorbable, and easy to apply.

There are 3 types of soft tissue fillers: autologous, allogenic, and synthetic. Good results have been reported with autologous fat transplants, although resorption rates are high, postoperative down time is long, and better results can be achieved by repeated sessions. Disappointing results have been documented with bovine collagen with respect to allergic reactions and lack of persistence.

Hyaluronic acid (HA) is an extremely large polymer made up of disaccharide repeats of N-acetylglucosamine and glucuronic acid, and constitutes the major part of the ground substance. Through its interactions with specific HA-binding proteins, HA is implicated in a variety of biologic cell processes, such as cell migration and proliferation. Furthermore, HA is a natural polymer with good biologic compatibility, and it has important structural functions in the extracellular matrix of all tissues. One of the shortcomings of HA was overcome by stabilizing the molecule, thereby enabling it to last longer in the dermal space. The intrinsic properties of the molecule of HA, including its ability to bind to water molecules and peptide growth factors and its high viscosity when stationary make it a good choice for etiologic therapy of the aging face. Non-animal (synthetic) stabilized HA compounds are widely used for soft tissue augmentation, and varying degrees of resorption rates are seen.

In an effort to obtain results that last for a longer amount of time, a combination of stabilized HA and cultured fibroblasts was created. The aim of this research study was to compare the longevity of the filling effect, in terms of resorption time, of cultured fibroblast suspensions, HA alone, and cultured fibroblasts in a HA matrix. Histologic analysis of each specimen was performed.

Methods

Skin biopsies obtained from 30 Sprague Dawley rats were used in the study. Dermal connective tissues were separated from the epidermis, minced into pieces, and treated with collagenase B (catalog no. 1 088 807; Roche Diagnostics GmbH, Mannheim, Germany) and DNAase (catalog no. 04 536 282 001; Roche Diagnostics GmbH) solution (1 mg/mL and 0.1 mg/mL, respectively) for 1 hour. The mixtures were centrifuged and pellets were passed from sieve for removal of debris. The filtrates were suspended with DMEM-F12 (catalog no. D-8900; Sigma Aldrich, St. Louis, MO) and 10% fetal calf serum and then cultured in 25 cm2 flasks at 37°C humidified 5% CO2 and air mixture. Fibroblasts were cultured for 3 weeks by 2 or 3 passages. At the end of this period, cells were detached from the surface of the culture plate with trypsine (catalog no. T-4799; Sigma Aldrich) solution (0.5%), and a homogenous suspension of cultured cells was obtained. The cells were washed in calcium magnesium phosphate buffered solution (catalog no. 042-04180 M; Gibco, Invitrogen, Carlsbad, CA) and the final pellets were dispersed by vortex for 10 seconds twice: once before mixing with HA and once during the preparation of the mixture of cells and HA. The density of the cells in mixture was approximately 30 × 106/mL. Injection material containing culture medium, matrix alone, culture medium containing cells, and the mixture of cells and matrix were administered intradermally into four distinct sites in each rat. Intradermal injections of the culture medium alone were administered to the left front leg (site 1) of each rat. HA matrix alone, the medium containing only fibroblastic cells, and the mixture of HA matrix and cells were injected intradermally to the right front leg (site 2), left back leg (site 3), and right back leg (site 4), respectively. The injection sites were peripherally signed via tattooing. Biopsy samples were taken from injection sites at the fourth and eighth months and were processed for light and electron microscopy.

Tissue samples were fixed with 10% neutral formalin and were embedded in paraffin blocks after dehydration. Ten 5-μm thick sections were cut and stained with hematoxylin–eosin and Masson's trichrome stains. Visual analyses were performed on these sections.

For electron microscopy tissue specimens, about 1 mm3 were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde solution in 0.1 M sodium cacodylate buffer for a night and then post-fixed with 0.1% osmium tetroxide (catalog no. 1.24505.0100; Merck & Co, Whitehouse Station, NJ) solution in the same buffer. After a routine dehydration process in graded alcohol series and propylene oxide, specimens were embedded in Epon-812 embedding media. Semi-thin (0.5-μm) and thin (0.1-μm) sections of tissue samples from the injection sites were cut by ultramicrotome (LKB, Bromma, Sweden). The thickness of the dermis and the number of visible vascular structures in dermal connective tissue were evaluated in paraffin sections, while the thicknesses of the collagen fibers (ultrastructural features of connective tissue constituents) were evaluated with a Jeol 1011 electron microscope (Jeol, Tokyo, Japan). Measurements were performed on digital images captured with a Mega View II digital camera (Soft Imaging Systems GmbH, Münster, Germany) attached to an electron microscope.

Quantitative evaluations including counting and measurements were performed via analySIS software (Soft Imaging Systems GmbH), and statistical significance was evaluated by paired sample t tests and performed with SPSS software (SPSS, Inc; Chicago, IL).

The histologic evaluations and qualitative counts were performed by systematic sampling in accordance with stereologic methods for two dimensions with unbiased counting frame because of the limitations of the sample size. The results were expressed as a numerical density for both cells and blood vessels.

Results

The dense and typical irregular arrangement of collagen bundles and areas containing amorphous substances were seen in all sites with light microscopy. Histologically, no difference was seen in site 1, where only the culture medium was injected. Inactive fibrocytes in fusiform shape were prevalent in the observed areas (Figure 1). HA bulks were observed in sites 2 and 4 at both the four- and eight-month periods. The amount of ground substance containing HA in site 4 was relatively abundant compared to site 2 in the eighth month. The diameters of collagen fibrils in site 4 measured slightly thinner than other sites, and the differences were not significant (Table 1). Other major components of connective tissue, elastin and microfibrils, were seen intact in all groups, and elaunin and oxytalan components were abundant in site 4 (Figure 2).

Table 1. Thickness of collagen fibrils in 4 administration sites

Site no. Thickness (μm) (mean ± SD
1 (medium) 48.3 ± 4.4
2 (hyaluronic acid alone) 53.2 ± 5.2
3 (fibroblast alone) 50.1 ± 4.7
4 (hyaluronic acid plus fibroblast) 45.3 ± 5.1

 

Figure 1. Electron micrograph of an inactive fibrocyte (F) in the culture media–injected site at the fourth month (original magnification, × 5000).

Figure 1. Electron micrograph of an inactive fibrocyte (F) in the culture media–injected site at the fourth month (original magnification, × 5000).

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Figure 2. Elastin material (E) amongst collagen fibrils (C) in the hyaluronic acid matrix and fibroblast mixture–injected site at the eighth month (original magnification, × 8000).

Figure 2. Elastin material (E) amongst collagen fibrils (C) in the hyaluronic acid matrix and fibroblast mixture–injected site at the eighth month (original magnification, × 8000).

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The average thickness of the dermis layer in site 4 (P < .05; Table 2Figure 3), and the distribution of vascular structures per 0.25 mm2 was significantly increased in sites 2 and 4 compared to other sites (P < .01 and P < .05, respectively; Table 3).

Table 2. Average thickness of dermis layer in 4 administration sites

Site no. Thickness (mm) (mean ± SD)
1 (medium) 2.63 ± 0.21
2 (hyaluronic acid alone) 2.56 ± 0.51
3 (fibroblast alone) 2.61 ± 0.24
4 (hyaluronic acid plus fibroblast) 3.23 ± 0.82a

a Significant difference compared to the results of the control (medium) group (P < .05).

Table 3. Numbers of vascular structures (arterioles, capillaries, and venules) in administration sites (per 0.25 mm2)

Site no. No. of vascular structures (mean ± SD)
1 (medium) 24.0 ± 1.7
2 (hyaluronic acid alone) 32.2 ± 2.3a
3 (fibroblast alone) 20.4 ± 1.4
4 (hyaluronic acid plus fibroblast) 28.3 ± 2.6b
 
a Significant difference compared to the results of the control (medium) group (P < .01).

b Significant difference compared to the results of the control (medium) group (P < .05).7

Figure 3. A, Micrograph from cross-sectioned skin of the medium-injected site at the eighth month. B, Micrograph from cross-sectioned skin of hyaluronic acid–injected site at the eighth month. C, Micrograph from cross-sectioned skin of fibroblast mixture–injected site at the eighth month. D, Micrograph from cross-sectioned skin of hyaluronic acid matrix and fibroblast mixture–injected site at the eighth month (original magnification, × 5000).

Figure 3. A, Micrograph from cross-sectioned skin of the medium-injected site at the eighth month. B, Micrograph from cross-sectioned skin of hyaluronic acid–injected site at the eighth month. C, Micrograph from cross-sectioned skin of fibroblast mixture–injected site at the eighth month. D, Micrograph from cross-sectioned skin of hyaluronic acid matrix and fibroblast mixture–injected site at the eighth month (original magnification, × 5000).

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Along with a significant increase in the number of blood vessels, colonization of macrophages associated with capillaries was observed in sites 2 and 4 as an electron microscopic finding (Figure 4). Ordinary fusiform-shaped fibroblasts were usually observed in sections of all sites, while enlarged, oval-shaped fibroblasts containing abundant granular endoplasmic reticulum and euchromatic nuclei were prevalent at the fourth month, and these cells were in close proximity to ground substance and collagen fibers (Figures 4 and 5).

Figure 4. Colonization (Col) of blood vessel–forming macrophages (M) in hyaluronic acid matrix–injected site at the fourth month (original magnification, ×5000). Ca, capillary; E, endothelial cell; F, fibroblast.

Figure 4. Colonization (Col) of blood vessel–forming macrophages (M) in hyaluronic acid matrix–injected site at the fourth month (original magnification, ×5000). Ca, capillary; E, endothelial cell; F, fibroblast.

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Figure 5. Electron micrograph of an enlarged, oval-shaped fibroblast containing granular endoplasmic reticulum cisterns (arrows) and cytoplasmic procollagen vesicles (asterisks) with tropocollagen-containing coves (arrowheads) in the hyaluronic acid matrix and fibroblast mixture–injected site at the eighth month. (Nu, nucleus; original magnification, × 5000).

Figure 5. Electron micrograph of an enlarged, oval-shaped fibroblast containing granular endoplasmic reticulum cisterns (arrows) and cytoplasmic procollagen vesicles (asterisks) with tropocollagen-containing coves (arrowheads) in the hyaluronic acid matrix and fibroblast mixture–injected site at the eighth month. (Nu, nucleus; original magnification, × 5000).

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No findings related to inflammation or granulation were observed in any group.

Discussion

The art of injecting fillers began in the beginning of the 20th century, with paraffin injection for augmentation after orchiectomy. The quest for more suitable filler materials continued over the years. A refined form of liquid silicone was introduced in the 1960s, and was widely used until countless complications arose and it was essentially banned. At the beginning of the 1980s, bovine collagen was introduced and it soon became the gold standard against other newly introduced materials. Allergic reactions seen after the application of bovine collagen and its limited longevity have sustained the search for a more ideal filler material.

Autologous fat, an abundant filler material, was popularized by Fournier in the 1970s. Resorption rates of 60% have been reported for autologous fat grafts. Newer injectable materials, such as autologous injectable human collagen (Autologen; Collagenesis Inc., Beverly, MA)—developed from surgically excised skin—and Dermalogen (Collagenesis, Inc.)—manufactured from cadaveric skin—have been introduced. Isologen (Isologen Technologies, Inc., Paramus, NJ) is cultured and expanded autologous fibroblasts with extracellular matrix, and it is used as a filler material for dermis and subcutaneous tissues that have lost collagen.

In recent years, HA derivatives have been used as injectable filler materials. Unlike collagen, HA is identical in all species; therefore, it is biocompatible and does not cause foreign body reaction. To maintain the longevity, cross-linked HA preparations are used. HA has the ability to bind water and form hydrated polymers of high viscosity. It has an isovolemic pattern of degradation; as the concentration in the dermis decreases, its ability to bind water increases.

Skin contains a relatively thick layer of dense, irregular connective tissue in the dermis called the reticular or deep layer of the dermis. Collagen and other non-cellular components (i.e., reticular and elastic fibers and ground substance) of connective tissue are synthesized by fibroblasts.

Tissue engineering has opened a new era for more permanent filler materials by enabling combinations of cell elements with biodegradable polymer scaffolds. In our study, cross-linked HA was used as a biodegradable polymer scaffold for cultured human fibroblasts, which are considered “biomaterials that heal.” Living autologous fibroblasts have the potential to produce collagen, especially when they exist in large quantities in the dermis. Boss et al. have shown that cultured fibroblasts can extend the longevity of bovine collagen for an indefinite period.

Our study was based on the hypothesis that a long-lasting correction can be achieved with HA combined with cultured human fibroblasts compared to HA alone. In the histologic examination, collagen fibrils in sites 3 and 4 were relatively thin compared to both other sites. This finding suggests active synthesis of collagen, because newly formed collagen fibrils are usually thin, and the thickness of these fibrils increases with the maturation of the bundle. Round-shaped fibroblasts with abundant granular endoplasmic reticulum in their cytoplasm and euchromatin in their nuclei in the sections of site 4, rather than fusiform cells in sites 1, 2, and 3, can also be regarded as a morphologic representation of proliferation and the active synthesis process that is associated with HA.

Another interesting finding is the observation of elaunin and oxytalan being more numerous in site 4, both in the fourth and eighth months of the experiment. These are precursors of elastin protein and appear early on in the synthesis of elastic fibers. The role of elastin degeneration in skin aging is a well known process. Being an indirect proof of elastin synthesis, this finding suggests a further role of cultured fibroblasts in the prevention of skin aging beyond their filling capacity. HA bulks surrounded by fibroblasts (Figure 1) suggest an interaction between hyaluronan and fibroblasts, which has been shown by other studies.

The significant increase in vascular structures in sites 2 and 4 correlates with the biologic activity of HA in vivo. The relatively high numbers of vessels counted in site 2, where HA was administered alone, compared with the HA plus fibroblast–administered site, could be related to the discrete angiogenic properties dependent on the molecular mass size of HA. Because the fragmented HA has an inducing effect on angiogenesis, we can suggest a degradation process of hyaluronan by the native cells found in the injection site which results in new vessel formation. Therefore, a relatively low number of blood vessels in the HA plus fibroblast–administered site can either be related to cultured fibroblasts' protective behavior on HA or inducing activity on native cells, or both.

There were no signs of apoptosis, inflammation, or necrosis in any site, which was expected because the cultured cells were autologous.

The results of our morphologic and morphometric analyses suggest that cross-linked HA combined with cultured autologous fibroblasts has greater longevity than solitary cross-linked HA, and leads to additional synthesis of extracellular components (ie, fibers and ground substance) in connective tissue. This supports Yoon's results, which showed that cross-linked HA combined with cultured human fibroblasts provides a longer-lasting effect compared with HA alone.

Conclusion

This study demonstrates that cultured human dermal fibroblasts combined with HA can be a suitable, biocompatible, and long-lasting material and should be regarded as a new method in dermal renovation even beyond its temporary filling effect.

Disclosures

The authors have no disclosures with respect to manufacturers of products mentioned in this article.

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