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Morphologic Changes in the Dermis After the Single Administration of Autologous Fibroblastic Cells: A Preliminary Study
Morphologic Changes in the Dermis After the Single Administration of Autologous Fibroblastic Cells: A Preliminary Study skia,*, A. Brodkiewiczb, K. Szumilasa, D. Rogin skaa, M.P. Kawaa, I. Stecewicza, G. Trybekd, B. Machalin c d M. Marchlewicz , and B. Wiszniewska a
Department of General Pathology, bDepartment of Pediatrics, Nephrology with Dializotherapy and Management of Acute Poisoning, Department of Aesthetic Dermatology, Pomeranian Medical University, and dDepartment of Histology and Embryology, Pomeranian Medical University, Szczecin, Poland c
ABSTRACT Background. Aging is a multifactorial process defined by an accumulation of damage in all tissues and organs, including the skin, throughout the lifespan of an individual. The reduction of both cellular and extracellular matrix components of the dermis during the aging process is followed by the alteration of the morphology of the skin tissue. This study was conducted to assess skin morphology in men before and 3 months after the intradermal injection of autologous fibroblastic cells. Methods. Tissue biopsies were surgically obtained before and 3 months after the treatment with autogenously harvested fibroblasts expanded in vitro, as well as after injection of phosphate-buffered saline. The thickness of collagen fiber bundles and number of fibroblasts in the dermis were analyzed in morphometric studies. The morphologic evaluation, using different methods of staining has been performed to analyze of extracellular matrix proteins, including collagen and reticular fibers, fibrillin-1erich microfibrils, elastic fibers, and hyaluronic acid. Results. After administration of the cells, we found a noticeable increase in the number of fibroblasts within the dermis, a significant enlargement in diameter of the collagen fiber bundles, and an improvement in the density of reticular fibers, fibrillin-1erich microfibrils, and elastic fibers compared with the initial, steady-state condition. Conclusions. The administration of autogenous fibroblasts could be an effective and safe adjunctive therapy to conventional health care treatment to prevent and reduce the agerelated accumulation of dermal tissue damage.
A
GING is a multifactorial process defined by an accumulation of damage in all tissues and organs, including the skin, throughout the lifespan of an individual [1]. Typical histologic changes observed in chronological skin aging involve gradual atrophy of the epidermis and flattening of the dermaleepidermal junction [2]. However, the most pronounced changes occur in elements of the dermis [2,3], which is divided into the papillary layer and the reticular layer. Both layers are composed of connective tissue and contain the extracellular matrix (ECM), which is a major constituent of the tissue. The ECM consists of a different combination of protein fibers (collagen, reticular, and elastic fibers) and ground substance that fills the majority of the extracellular space within the tissue [4,5]. ECM ª 2016 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 48, 2833e2839 (2016)
is produced and secreted by fibroblasts, which are the main cell type in the dermis. Type I and III collagens are by far the most abundant proteins in the dermis [6,7]. These proteins are usually arranged in bundles, often oriented parallel to the epidermis, or are arranged into “basket-weave” bundles [7,8]. The elastic fiber system is an additional group This work was supported by the Polish National Centre for Research and Development grant STRATEGMED1/234261/ 2NCBR/2014 (to BM). ski, MD, PhD, *Address correspondence to Bogusław Machalin DSc, Department of General Pathology, Pomeranian Medical ców Wlkp. 72, 70e111 Szczecin, Poland. University, Powstan E-mail: [email protected] 0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.05.016
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of essential skin ECM proteins [9,10]. Adult elastic fibers are composed of an outer mantle of myofibrils (formed mainly by fibrillin-1) and an inner core of cross-linked elastin, both produced by dermal fibroblasts. They form a fine network extending vertically in dermal papillae and surround blood vessels, in the reticular dermis elastic fibers are thicker and run parallel to the skin surface, and surround the larger collagen fibers and skin adnexa [9,10]. Fibrillin-1erich microfibrils (oxytalan fibers) form a continuous network that extends vertically from the dermale epidermal junction into the deep dermis [11]. The predominant glycosaminoglycan in the ground substance of the dermis is hyaluronic acid, which constitutes >50% of total body hyaluronan [5]. It is widely accepted that damage to dermal connective tissue caused by the aging process is closely and continuously related to the alteration of its morphology. The histologic features associated with intrinsic (ie, related to nonesun-exposed tissue) and extrinsic (ie, related to sunexposed tissue) skin aging involves decreased fibroblast number, atrophy of the ECM, reduction and disintegration of collagen and elastic fibers [3], decreased hyaluronan content, and widespread structural modifications [12e14]. Given the important role that skin plays not only as a physiologic barrier for protection against environmental factors but also in interpersonal relations, novel technologies aimed at preventing skin aging in women and, more recently, in men are being extensively investigated in the field of aesthetic dermatology. However, despite the practical evidence, the clinical studies of autologous skin-derived fibroblasts transplantation remain insufficient. To further study the potential therapeutic use of fibroblast transplantation, it is necessary to fully characterize the biologic effects in the human skin setting. Therefore, we designed a pilot study to examine the skin morphology of men and fibrous components production before and 3 months after the intradermal injection of autologous skin-derived fibroblastic cells cultured ex vivo. The studies in vivo were performed with the following particular objectives: (1) to prove the survival and potential proliferation of cultured dermal fibroblasts after autologous transplantation by the cell number analysis of the skin biopsies and (2) to observe in vivo collagen and other fiber secretion activity of the fibroblasts after autologous transplantation by the histologic and immunohistochemical analyses of the skin biopsies. MATERIALS AND METHODS Patients Human skin samples were obtained from 3 volunteers male donors (47, 51, and 52 years of age) in accordance with the Declaration of Helsinki and with the approval of Local Ethics Committee of Pomeranian Medical University. Skin biopsies (0.8 0.6 cm) were taken from the postauricular area, which is one of the lower exposed to ultraviolet radiation areas of the head, both before (for fibroblastic cell harvest and for histologic examination) and 3 months after a single injection of in vitro-cultured autologous fibroblasts or phosphate-buffered saline (PBS) (for histologic
examination). The cell transplantation and second biopsy were performed near to the initial biopsy site. There were no differences in terms of regional skin specificity between the initial and second biopsy site, owing to the presence of the hair-bearing skin in both biopsy sites. Prior to obtaining the skin fragments, the voluntary patients were locally anesthetized via subcutaneous injection with 1 mL of lignocainum hydrochloricum 1% (Polfa Warszawa, Warsaw, Poland).
Cell Culture The skin specimens were transported to the laboratory in ice-cold Ca2þ/Mg2þ-free PBS containing 1:100 penicillin/streptomycin solution (Invitrogen; Life Technologies Warsaw, Poland) and 1 mg/mL Fungizone (both from Gibco, Life Technologies) and then processed immediately. The tissue samples were washed twice with cold Ca2þ/Mg2þ-free PBS, cut into smaller pieces and incubated in 0.6 U/mL Dispase II (Gibco, Life Technologies) for 1 to 2 hours at 37 C. The epidermis was manually removed from each tissue sample, and the dermis was cut into 1-mm3 pieces after enzymatic disaggregation with 0.62 Wünsch U/mL Liberase DH (Roche Applied Science, Penzberg, Germany) for 30 to 40 minutes at 37 C. Subsequently, tissue pieces were dissociated by vortexing and then passed through a 70-mm cell strainer (Becton Dickinson, Franklin Lakes, NJ). The dissociated cells were centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the pellet was suspended in Medium 199 (Life Technologies) containing 10% human serum isolated from the patients themselves and 0.5% penicillin and streptomycin (Invitrogen). To obtain human autologous serum, 10 mL of whole blood from each patient was collected into plastic tubes containing a serum separator gel with clot activator (Becton Dickinson). Serum separation was completed after centrifugation at 2,000 rpm for 10 minutes. The cells were cultured in a T25 tissue culture flask (Falcon, Becton Dickinson) at 37 C in 5% CO2 in a humidified atmosphere. The medium was changed 48 hours after plating and every 3 to 4 days thereafter. When the cultures reached 70% confluency, the cells were detached with Accutase (PAA Laboratories, Linz, Austria), washed with PBS and replated in complete medium at 4000 cells/cm2. The cell cultures were maintained until the fourth passage.
Cells Injection Cells expanded in vitro were detached with Accutase, washed with PBS, and passed through a 70-mm cell strainer to generate a singlecell suspension. The fibroblasts were diluted in 0.2 mL of PBS and intradermally injected into the right postauricular region of each patient. The series of injections was performed with a 1-mL syringe and a 30-G, 0.5-inch needle into a 1-cm2 area of superficial dermis using the puncture technique (1 puncture contained 0.05 mL of cell suspension per 0.25 cm2 of skin area tested). A total of 1.5 106 cells were transplanted. An analogous series of injections with PBS only was performed in left postauricular region as an additional control.
Histology Tissue biopsies were surgically obtained before and 3 months after treatment with autogenously harvested fibroblasts expanded in vitro, as well as after PBS injection. The samples were fixed in freshly prepared 4% paraformaldehyde and embedded in paraffin. For the morphologic analysis, serial slices (3e5 mm in thickness) of skin were mounted onto glass slides and stained with hematoxylin and eosin (H-E). To visualize collagen bundles, the slides were
MORPHOLOGIC CHANGES IN THE DERMIS stained with Sirius Red (Direct Red 80 Sigma Aldrich [St Louis, MO] 0.1% of Sirius Red in saturated aqueous picric acid), as previously described by Junqueira et al. [15]. Silver impregnation was performed to visualize reticular fibers (Bio-Optica Milano, Italy); elastic fibers were identified using Weigert’s method (Weigert’s for elastic fibers, Bio-Optica). Additionally, Alcian blue in buffers at pH 2.5 staining (IVD, Bio-Optica Milano) was performed for the histolocalization of hyaluronic acid in the dermis of each patient. All histochemical reactions were carried out according to protocols recommended by the manufacturers.
Morphometry Sections stained with H-E were analyzed to determine the number of fibroblasts using a 10x ocular lens with a square reticle. The number of fibroblasts in the dermis before and after the treatment (with cells or PBS only) was counted in 100 random squares (50 squares per slide). The evaluation was carried out on a Zeiss microscope equipped with a 40 objective. The surface of each square was 1.225 mm2 (35 35 mm) under the objective. Additionally, the diameter of the collagen fibers in the reticular layer of the dermis was measured. All morphometric measurements were obtained using the Axio Vision Rel. 4.6 program (Zeiss, Axioscope System, Jena, Germany).
Immunohistochemistry Immunohistochemistry was performed to identify fibrillin-1 in the dermis. The mouse antihuman fibrillin-1 (AbD Serotec, Biogenesis, Oxford, UK) monoclonal antibody (1:50) was used. The deparaffinized sections of skin were microwaved in citrate buffer (pH 6.0) for heat-induced epitope retrieval. After slow cooling to room temperature, the slides were washed twice in PBS for 5 minutes and then incubated for 60 minutes with primary mouse antihuman fibrillin-1 antibody (AbD Serotec MorphoSys AbD). Next, the sections were stained with an avidin-biotin-peroxidase system using diaminobenzidine as the chromogen (Dako LSABþ System-HRP: Code K0679 DakoCytomation, Glostrup, Denmark) in accordance with the staining procedure instruction suggested by the manufacturer. The sections were washed in distilled H2O and counterstained with hematoxylin. Negative control specimens were processed in the absence of primary antibody. Positive staining was defined microscopically by the visual identification of brown pigmentation.
Statistical Analysis Results are expressed as the median, the lower and upper quartiles (Q1-Q3) and the mean values standard deviation (X SD). The distribution of the results for individual variables was determined using the Shapiro-Wilk W test. Because most of the distributions deviated from the normal distribution, the nonparametric MannWhitney U test was used for further analyses. P < .05 was considered significant. Calculations were performed using the software package Statistica 6.1 (StatSoft, Tulsa, OK).
RESULTS
Injections with autogenously harvested and in vitroexpanded fibroblasts were well-tolerated and caused no side effects. Biopsies obtained from the patients before and 3 months after treatment with cells or PBS only were composed of the whole skin, and the histologic slides confirmed the proper structure of both the epidermis and
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the dermis, which was subdivided into the papillary and reticular layers. Morphometric Analysis of the Skin
Noticeable differences in the morphology of the dermis, in the cellular content and ECM components, before and after treatment in all patients were observed. Therefore, we performed a comprehensive morphometric analysis to assess the differences in the number/density of fibroblasts located between collagen bundles and the diameter of collagen bundles in the reticular layer of the dermis in samples obtained before treatment and after treatment with fibroblasts or with PBS. These results are presented in Table 1. In skin samples taken 3 months after the reimplantation of fibroblasts, both the number of fibroblasts in the dermis and the diameter of collagen bundles were significantly higher compared with before treatment and compared with post-PBS injection (Table 1). These results suggest that administered autogenous fibroblastic cells exerted a biological effect resulting in the rejuvenation of the collagen fibers in the dermis. Histology
We continued to characterize the effect of intradermal autologous fibroblast cells administration by analyzing the essential ECM elements of skin samples collected before and 3 months after fibroblasts reimplantation or PBS injection. Collagen Fibers
The skin slides were stained with H-E and picrosirius red. Skin sections stained with picrosirius red were analyzed by polarized microscopy. The larger collagen fibers that displayed yellow-red staining and strong birefringence were considered collagen type I, whereas fibers that displayed a greenish color and weak birefringence were considered collagen type III. The color and intensity of birefringence depend on differences in the pattern of physical aggregation and the thickness of the collagen fibers. Thin collagen fibers exhibit green to greenish-yellow polarizing colors, whereas thick fibers stain yellowish-orange, orange, and red [15]. In the papillary dermis of patients before the treatment and after the injection of PBS (H-E-stained sections), a loose network of thin bundles was observed (Fig 1A). The reticular dermis contained loosely woven collagen bundles of different thicknesses, which were frequently fragmented. Wide spaces separating the bundles were visible, with ovalshaped fibroblasts (Fig 1D). Similar structure of the collagen fibers was observed in the dermis after picrosirius red staining. In the papillary dermis, thin collagen bundles with typical organization and yellowish-orange birefringence were observed (Fig 1B). Collagen bundles in the reticular dermis appeared regularly ordered with multiple orientations with different birefringence intensities varying from yellowish-orange to
MACHALINSKI, BRODKIEWICZ, SZUMILAS ET AL
2836 Table 1. Fibroblast Number and Collagen Bundle Diameter in the Reticular Layer of the Dermis BT
Fibroblasts number (n ¼ 100) Median 1.0 Q1eQ3 0e1 X SD 0.58 0.63
AT
1.0 1e2 1.52 1.12 AT vs BT*** Diameter of collagen bundles, mm (n ¼ 100) Median 5.64 8.05 Q1eQ3 4.58e7.2 6.35e9.83 X SD 7.51 12.62 8.39 2.51 AT vs BT***
AI
1.0 0e1 0.62 0.69 AT vs AI*** 6.13 5.03e7.86 6.43 1.93 AT vs AI***
Asterisks indicate the statistically significant differences in the Mann-Whitney U test. ***P < .001. Abbreviations: AI, skin 3 months after the phosphate-buffered saline injection; AT, skin 3 months after the fibroblastic cells injection; BT, skin before treatment; Q1-Q3, upper quartile and lower quartile; X SD, arithmetic mean standard deviation.
yellowish-red. There were areas displayed green polarized color (Fig 1E). The dermis of patients after cells reimplantation was structurally similar. In H-Eestained slides, thin collagen bundles were organized in a loose network (Fig 1G). The reticular dermis was filled with thicker collagen bundles (Fig 1J) than it was observed in patients before the treatment (Fig 1D). There were no wide spaces between the collagen bundles, which were woven in a distinctive compact pattern (Fig 1J). Collagen bundles in the papillary dermis in slides stained with picrosirius red were thicker and revealed yellowish-red birefringence (Fig 1H). The reticular dermis was filled with
thick compacted collagen bundles with an enhanced intensity of birefringence from bright yellowish-red to red. Many areas with fibers displayed green polarized color were very well visible (Fig 1K). Reticular Fibers
Small amount of reticular fibers was detectable in both the papillary and reticular layers of the dermis from the patients before treatment and after PBS injection. Only few silverstained fibers were visible close to the epidermis (Fig 1C) and in the reticular layer of the dermis (Fig 1F). Additionally, these fibers were visible in the walls of blood vessels (Fig 1F). A similar arrangement was observed in slides stained with picrosirius red, a small amount of fibers with greenish polarized color was observed in the papillary dermis (Fig 1B) and reticular dermis (Fig 1E). In contrast, abundant reticular fibers were visible in both the papillary and reticular layers of the dermis in the patients after cells reimplantation. In the papillary layer, thin reticular fibers were accumulated near the basement membrane of the epidermis (Fig 1I), whereas in the reticular layer of the dermis, silver-stained reticular fibers were observed between and on the surface of collagen fiber bundles (Fig 1L). The fibers with greenish polarized color were also observed in sections stained with picrosirius red (Fig 1H, K). Fibrillin-1eRich Microfibrils
Immunohistochemical staining revealed only scant fibrillin1erich microfibrils in the papillary layer located near to stratum basale of the epidermis and in the elastic fibers of reticular dermis in patients before treatment and after PBS
Figure 1. Untreated and treated with fibroblastic cells skin. Staining: hematoxylin and eosin, picrosirius red, and silver impregnation.
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Figure 2. Untreated and treated with fibroblastic cells skin. Staining: immunohistochemistry and Weigert’s quick method.
injection (Fig 2A, C). After fibroblasts reimplantation, immunostaining showed a significant increase in the density of fibrillin-1 microfibrils in the papillary dermis and the reticular dermis (Fig 2E, G). Elastic Fibers
Elastic fibers were detected in the papillary and reticular layers of the dermis in the patients before treatment and after PBS injection. In the reticular layer, the fibers were located between collagenous bundles (Fig 2B, D). Elastic fibers were visible near sebaceous (Fig 2D) and sweat glands. In samples obtained after fibroblasts reimplantation, elastic fibers were more abundant and observed in both layers of the dermis. In the papillary layer, elastic fibers took the form of short, thin, single fibers localized close to the epidermis (Fig 2F), whereas in the reticular layer, elastic fibers were present between bundles of collagenous fibers (Fig 2H) and were condensed around hair follicles, sweat and sebaceous glands (Fig 2H). Hyaluronic Acid
Weakly blue-stained HA was visible in both the papillary and reticular layers of the dermis in patients both before and after the treatment. The results of histologic analyses were comparable between patients (data not shown). DISCUSSION
Skin aging progresses gradually and occurs owing to intrinsic factors as well as extrinsic factors, which together lead to the development of changes in the cellular and extracellular components of the skin. morphologic examination of aged skin typically reveals atrophy of the dermal ECM with the loss of fibers, predominantly type I and III collagens, and elastic fibers [16]. Recently, there has been increased interest in the prevention of skin aging, especially using nonablative procedures that enable skin rejuvenation with minimal downtime and complications [17e20].
In this pilot experimental study, we demonstrated for the first time that locally injected autogenous fibroblastic cells could successfully engraft and settle in the target tissue, resulting in effectively rejuvenated dermis in adult male patients. Several lines of evidence suggest that there are no differences in terms of particular skin aging symptoms in the old adults of age of 50 to 70 years, especially in the specific fibroblast subpopulation as reticular fibroblasts, which are mainly responsible for synthesizing extra-cellular matrix. Therefore, our obtained results can be extended to the elderly subjects as well. We showed in morphologic analysis evidence of the improvement of fibrous components in dermal connective tissue, which can reflect the rejuvenation process, that included the significant enlargement of collagen fiber diameters and increased density of reticular fibers, fibrillin-1erich microfibrils, and elastic fibers. Dermal fibroblasts, the main ECM-producing cells, are an essential component of the skin involved in the maintenance of dermal architecture and providing biomechanical properties of the skin [21e23]. Because they play a crucial role in skin physiology, their metabolism is strictly regulated by complex mechanisms, including cellecell and cellematrix interactions [21,22], what is confirmed by in vitro studies [22,24]. The interactions between fibroblasts and collagen fibers, relationship between mechanical tension on cells in vitro and biological cells response to stress has been well documented [25,26]. Chronologically aged skin is characterized by collagen fragmentation, reduction in total collagen, reduction in fibroblasts, and decreased celle collagen fiber interactions. The last results in the reduced mechanical tension on the cells and in this way, matrix production consequently falls, and matrix-degrading enzymes become activated [12,26]. Consistent with these reports, in the current study we revealed that injected fibroblastic cells were able to resume their basic functions and produce ECM proteins, what was visible as enlargement of thickness of collagen type I and density of collagen type III fibers. Fibroblastic cells are also known to naturally express an array of cytokines, growth factors, and proteases
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involved in the regulation of a variety of biological processes, including cell survival and proliferation, production and remodeling of ECM components, angiogenesis and epithelial morphogenesis. The ex vivo culture for clonal expansion of fibroblastic cells may augment the combination of such factors in the posttransplant milieu. After cultured ex vivo fibroblasts transplantation into the skin such factors may next promote fibroblast survival and engraftment and stimulate their ECM protein production on site. Importantly, the use of autologous blood serum enriched in different soluble growth factors may act as a specific fibroblast phenotype regulator during their culture ex vivo before transplantation, which might contribute to their hyperactive biological behavior after transplantation. Although collagen synthesis progressively decreases with passage of time [27,28], in our experiment we found a higher density of reticular fibers, composed of type III collagen, in the papillary layer and the coexistence of both type I and type III collagen fibers in the reticular layer of the dermis in patients after cells re-implantation in comparison with untreated skin. Thus, our results suggest that considerable improvement of dermis structure could occur after fibroblastic cells administration. The elastic fiber system, which is composed mainly of elastin, fibrillin, and elastin binding protein, is highly ordered in the skin [29]. The aging process broadly influences the structure and function of the elastic fiber system [29]. The degradation of existing fibers leads to accumulation of elastin-rich material in the upper and mid dermis, especially during the photoaging process [12,30]. In contrast, mildly photoaged skin is characterized histologically by loss of fibrillin microfibrils [29]. However, restoration of elastic fibers was observed in photoaged skin after topical retinoic acid treatment [11] and in response to hyaluronic acid injection [17,27]. Interestingly, in the present study we found increase in the density of the fibrillin-1erich microfibrils in the dermal papillae and the elastic fibers in the reticular dermis in mildly photoprotected skin of patients injected with the fibroblastic cells in comparison with the untreated skin. In contrast, we did not observe differences in the content of hyaluronic acid in the dermis before and after the injection of autogenous fibroblastic cells. These results are consistent with studies of other authors, which also confirmed that the total hyaluronic acid level in the dermis of skin undergoing intrinsic aging process remains stable; however, the epidermal HA diminishes markedly [5,27,31,32]. Despite improved scar quality when using acellular dermal substitutes for treatment, the optimal result of whole skin regeneration is still not achieved, when using acellular dermal substitutes. Cell-based therapy is one of the strategies that may further improve the whole skin regeneration. Particularly, the application of autologous dermal fibroblasts may improve the burn wound healing. It is expected that autologous fibroblasts may promote the whole skin wound healing owing to the fact that they have the capacity to further differentiate into myofibroblasts, which are
MACHALINSKI, BRODKIEWICZ, SZUMILAS ET AL
responsible for the wound contraction. This phenotype plasticity of the dermal fibroblasts makes them attractive candidate for cellular-based therapies of the wound healing, especially those which are clinically complicated or extended to greater areas. Therefore, autologous dermal fibroblasts might be used in the future as adjuvant regenerative therapy in such clinical problems as skin traumas, cutaneous burns, scaffolds production for minor tissue loss or congenital skin abnormalities in elderly patients. In conclusion, our preliminary findings, in addition to being relevant for the elucidation of cell-based regeneration process in aging skin, provide new insights regarding the role of autologous fibroblastic cells in this process. We also provide novel, compelling evidence for the efficacy of fibroblastic cell administration in improving dermal connective tissue structure. However, further experiments involving a larger number of subjects are necessary to confirm our results. REFERENCES [1] Giacomoni PU. Advancement in skin aging: the future cosmeceuticals. Clin Dermatol 2008;26:364e6. [2] Makrantonaki E, Zouloboulis CC. Molecular mechanisms of skin aging. State of the art. Ann N Y Acad Sci 2007;1119:40e50. [3] Zouloboulis CC, Makrantonaki E. Clinical aspects and molecular diagnostic of skin aging. Clin Dermatol 2011;29:3e14. [4] Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci 2010;123:4195e200. [5] Stern R, Maibach HI. Hyaluronan in skin: aspects of aging and its pharmacologic modulation. Clin Dermatol 2008;26:106e22. [6] Makrantonaki E, Zouloboulis CC. The skin as a mirror of the aging process in the human organism- state of the art and results of the aging research in the German National Genome Research Network 2 (NGFN-2). Exp Gerontol 2007;42:879e86. [7] Naylor EC, Watson RE, Sherratt MJ. Molecular aspects of skin ageing. Maturitas 2011;69:249e56. [8] Graham HK, Hodson NW, Hoyland JA, et al. In situ ultrastructural imaging of native biomolecules. Matrix Biol 2010;29: 254e60. [9] Doubal S, Klemera P. Visco-elastic response of human skin and aging. J Am Aging Assoc 2002;25:115e7. [10] Kielty CM, Sherratt MJ, Shuttleworth CA. Elastic fibres. J Cell Sci 2002;15:2817e28. [11] Lee JY, Kim YK, Seo JY, et al. Loss of elastic fibers causes skin wrinkles in sun-damaged human skin. J Dermatol Sci 2008;50: 99e107. [12] Humbert P, Viennet C, Legagneux K, et al. In the shadow of the wrinkle: experimental models. J Cosmet Dermatol 2012;11: 79e83. [13] Longas MO, Russell CS, He XY. Chemical alterations of hyaluronic acid and dermatan sulfate detected in aging human skin by infrared spectroscopy. Biochim Biophys Acta 1986;884:265e9. [14] Longas MO, Russell CS, He XY. Evidence for structural changes in dermatan sulfate and hyaluronic acid with aging. Carbohydr Res 1987;159:127e36. [15] Junqueira Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 1979;11:447e55. [16] Widgerow AD, Grekin SK. Effecting skin renewal: a multifaceted approach. J Cosmet Dermatol 2011;10:126e30. [17] Cattin TA. A single injection technique for midface rejuvenation. J Cosmet Dermatol 2010;9:256e9. [18] Draelos ZD. Creating an attractive aging face. J Cosmet Dermatol 2013;11:167e8.
MORPHOLOGIC CHANGES IN THE DERMIS [19] Seo KY, Kim DH, Lee SE, Yoon MS, Lee HJ. Skin rejuvenation by microneedle fractional radiofrequency and a human stem cell conditioned medium in Asian skin: a randomized controlled investigator blinded split-face study. J Cosmet Laser Ther 2013;15:25e33. [20] Seo KY, Yoon MS, Kim DH, Lee HJ. Skin rejuvenation by microneedle fractional radiofrequency treatment in Asian skin; clinical and histological analysis. Lasers Surg Med 2012;44:631e6. [21] McAnulty RJ. Fibroblasts and myofibroblasts: their source, function and role in disease. Int J Biochem Cell Biol 2007;39: 666e71. [22] Sorrell JM, Caplan AI. Fibroblast heterogeneity: more than skin deep. J Cell Sci 2004;117:667e75. [23] Vedrenne N, Coulomb B, Danigo A, et al. The complex dialogue between (myo)fibroblasts and the extracellular matrix during skin repair processes and ageing. Pathol Biol (Paris) 2012;60: 20e7. [24] Müller SA, van der Smissen A, von Feilitzsch M, et al. Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix. J Mater Sci Mater Med 2012;23:3053e65.
2839 [25] Grinnell F, Petroll WM. Cell motility and mechanics in three-dimensional collagen matrices. Annu Rev Cell Dev Biol 2010;26:335e61. [26] Varani J, Dame MK, Rittie L, et al. Decreased collagen production in chronologically aged skin: roles of age-dependent alteration in fibroblast function and defective mechanical stimulation. Am J Pathol 2006;168:1861e8. [27] Ganceviciene R, Liakou AI, Theodoridis A, et al. Skin antiaging strategies. Dermatoendocrinol 2012;4:308e19. [28] Levakov A, Vucovic N, Dolai M, et al. Age-related skin changes. Med Pregl 2012;65:191e5. [29] Sherratt MJ. Tissue elasticity and the ageing elastic fibers. Age (Dordr) 2009;31:305e25. [30] El-Domyati M, Medhat W, Abdel-Wahab HM, et al. Forehead wrinkles: a histological and immunohistochemical evaluation. J Cosmet Dermatol 2014;13:188e94. [31] Oh JH, Kim YK, Jung JY, et al. Changes in glycosaminoglycans and related proteoglycans in intrinsically aged human skin in vivo. Exp Dermatol 2011;20:454e6. [32] Papakonstantinou E, Roth M, Karakiulakis G. Hyaluronic acid: a key molecule in skin aging. Dermatoendocrinology 2012;4: 253e8.
Department of General Pathology, bDepartment of Pediatrics, Nephrology with Dializotherapy and Management of Acute Poisoning, Department of Aesthetic Dermatology, Pomeranian Medical University, and dDepartment of Histology and Embryology, Pomeranian Medical University, Szczecin, Poland c
ABSTRACT Background. Aging is a multifactorial process defined by an accumulation of damage in all tissues and organs, including the skin, throughout the lifespan of an individual. The reduction of both cellular and extracellular matrix components of the dermis during the aging process is followed by the alteration of the morphology of the skin tissue. This study was conducted to assess skin morphology in men before and 3 months after the intradermal injection of autologous fibroblastic cells. Methods. Tissue biopsies were surgically obtained before and 3 months after the treatment with autogenously harvested fibroblasts expanded in vitro, as well as after injection of phosphate-buffered saline. The thickness of collagen fiber bundles and number of fibroblasts in the dermis were analyzed in morphometric studies. The morphologic evaluation, using different methods of staining has been performed to analyze of extracellular matrix proteins, including collagen and reticular fibers, fibrillin-1erich microfibrils, elastic fibers, and hyaluronic acid. Results. After administration of the cells, we found a noticeable increase in the number of fibroblasts within the dermis, a significant enlargement in diameter of the collagen fiber bundles, and an improvement in the density of reticular fibers, fibrillin-1erich microfibrils, and elastic fibers compared with the initial, steady-state condition. Conclusions. The administration of autogenous fibroblasts could be an effective and safe adjunctive therapy to conventional health care treatment to prevent and reduce the agerelated accumulation of dermal tissue damage.
A
GING is a multifactorial process defined by an accumulation of damage in all tissues and organs, including the skin, throughout the lifespan of an individual [1]. Typical histologic changes observed in chronological skin aging involve gradual atrophy of the epidermis and flattening of the dermaleepidermal junction [2]. However, the most pronounced changes occur in elements of the dermis [2,3], which is divided into the papillary layer and the reticular layer. Both layers are composed of connective tissue and contain the extracellular matrix (ECM), which is a major constituent of the tissue. The ECM consists of a different combination of protein fibers (collagen, reticular, and elastic fibers) and ground substance that fills the majority of the extracellular space within the tissue [4,5]. ECM ª 2016 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 48, 2833e2839 (2016)
is produced and secreted by fibroblasts, which are the main cell type in the dermis. Type I and III collagens are by far the most abundant proteins in the dermis [6,7]. These proteins are usually arranged in bundles, often oriented parallel to the epidermis, or are arranged into “basket-weave” bundles [7,8]. The elastic fiber system is an additional group This work was supported by the Polish National Centre for Research and Development grant STRATEGMED1/234261/ 2NCBR/2014 (to BM). ski, MD, PhD, *Address correspondence to Bogusław Machalin DSc, Department of General Pathology, Pomeranian Medical ców Wlkp. 72, 70e111 Szczecin, Poland. University, Powstan E-mail: [email protected] 0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.05.016
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of essential skin ECM proteins [9,10]. Adult elastic fibers are composed of an outer mantle of myofibrils (formed mainly by fibrillin-1) and an inner core of cross-linked elastin, both produced by dermal fibroblasts. They form a fine network extending vertically in dermal papillae and surround blood vessels, in the reticular dermis elastic fibers are thicker and run parallel to the skin surface, and surround the larger collagen fibers and skin adnexa [9,10]. Fibrillin-1erich microfibrils (oxytalan fibers) form a continuous network that extends vertically from the dermale epidermal junction into the deep dermis [11]. The predominant glycosaminoglycan in the ground substance of the dermis is hyaluronic acid, which constitutes >50% of total body hyaluronan [5]. It is widely accepted that damage to dermal connective tissue caused by the aging process is closely and continuously related to the alteration of its morphology. The histologic features associated with intrinsic (ie, related to nonesun-exposed tissue) and extrinsic (ie, related to sunexposed tissue) skin aging involves decreased fibroblast number, atrophy of the ECM, reduction and disintegration of collagen and elastic fibers [3], decreased hyaluronan content, and widespread structural modifications [12e14]. Given the important role that skin plays not only as a physiologic barrier for protection against environmental factors but also in interpersonal relations, novel technologies aimed at preventing skin aging in women and, more recently, in men are being extensively investigated in the field of aesthetic dermatology. However, despite the practical evidence, the clinical studies of autologous skin-derived fibroblasts transplantation remain insufficient. To further study the potential therapeutic use of fibroblast transplantation, it is necessary to fully characterize the biologic effects in the human skin setting. Therefore, we designed a pilot study to examine the skin morphology of men and fibrous components production before and 3 months after the intradermal injection of autologous skin-derived fibroblastic cells cultured ex vivo. The studies in vivo were performed with the following particular objectives: (1) to prove the survival and potential proliferation of cultured dermal fibroblasts after autologous transplantation by the cell number analysis of the skin biopsies and (2) to observe in vivo collagen and other fiber secretion activity of the fibroblasts after autologous transplantation by the histologic and immunohistochemical analyses of the skin biopsies. MATERIALS AND METHODS Patients Human skin samples were obtained from 3 volunteers male donors (47, 51, and 52 years of age) in accordance with the Declaration of Helsinki and with the approval of Local Ethics Committee of Pomeranian Medical University. Skin biopsies (0.8 0.6 cm) were taken from the postauricular area, which is one of the lower exposed to ultraviolet radiation areas of the head, both before (for fibroblastic cell harvest and for histologic examination) and 3 months after a single injection of in vitro-cultured autologous fibroblasts or phosphate-buffered saline (PBS) (for histologic
examination). The cell transplantation and second biopsy were performed near to the initial biopsy site. There were no differences in terms of regional skin specificity between the initial and second biopsy site, owing to the presence of the hair-bearing skin in both biopsy sites. Prior to obtaining the skin fragments, the voluntary patients were locally anesthetized via subcutaneous injection with 1 mL of lignocainum hydrochloricum 1% (Polfa Warszawa, Warsaw, Poland).
Cell Culture The skin specimens were transported to the laboratory in ice-cold Ca2þ/Mg2þ-free PBS containing 1:100 penicillin/streptomycin solution (Invitrogen; Life Technologies Warsaw, Poland) and 1 mg/mL Fungizone (both from Gibco, Life Technologies) and then processed immediately. The tissue samples were washed twice with cold Ca2þ/Mg2þ-free PBS, cut into smaller pieces and incubated in 0.6 U/mL Dispase II (Gibco, Life Technologies) for 1 to 2 hours at 37 C. The epidermis was manually removed from each tissue sample, and the dermis was cut into 1-mm3 pieces after enzymatic disaggregation with 0.62 Wünsch U/mL Liberase DH (Roche Applied Science, Penzberg, Germany) for 30 to 40 minutes at 37 C. Subsequently, tissue pieces were dissociated by vortexing and then passed through a 70-mm cell strainer (Becton Dickinson, Franklin Lakes, NJ). The dissociated cells were centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the pellet was suspended in Medium 199 (Life Technologies) containing 10% human serum isolated from the patients themselves and 0.5% penicillin and streptomycin (Invitrogen). To obtain human autologous serum, 10 mL of whole blood from each patient was collected into plastic tubes containing a serum separator gel with clot activator (Becton Dickinson). Serum separation was completed after centrifugation at 2,000 rpm for 10 minutes. The cells were cultured in a T25 tissue culture flask (Falcon, Becton Dickinson) at 37 C in 5% CO2 in a humidified atmosphere. The medium was changed 48 hours after plating and every 3 to 4 days thereafter. When the cultures reached 70% confluency, the cells were detached with Accutase (PAA Laboratories, Linz, Austria), washed with PBS and replated in complete medium at 4000 cells/cm2. The cell cultures were maintained until the fourth passage.
Cells Injection Cells expanded in vitro were detached with Accutase, washed with PBS, and passed through a 70-mm cell strainer to generate a singlecell suspension. The fibroblasts were diluted in 0.2 mL of PBS and intradermally injected into the right postauricular region of each patient. The series of injections was performed with a 1-mL syringe and a 30-G, 0.5-inch needle into a 1-cm2 area of superficial dermis using the puncture technique (1 puncture contained 0.05 mL of cell suspension per 0.25 cm2 of skin area tested). A total of 1.5 106 cells were transplanted. An analogous series of injections with PBS only was performed in left postauricular region as an additional control.
Histology Tissue biopsies were surgically obtained before and 3 months after treatment with autogenously harvested fibroblasts expanded in vitro, as well as after PBS injection. The samples were fixed in freshly prepared 4% paraformaldehyde and embedded in paraffin. For the morphologic analysis, serial slices (3e5 mm in thickness) of skin were mounted onto glass slides and stained with hematoxylin and eosin (H-E). To visualize collagen bundles, the slides were
MORPHOLOGIC CHANGES IN THE DERMIS stained with Sirius Red (Direct Red 80 Sigma Aldrich [St Louis, MO] 0.1% of Sirius Red in saturated aqueous picric acid), as previously described by Junqueira et al. [15]. Silver impregnation was performed to visualize reticular fibers (Bio-Optica Milano, Italy); elastic fibers were identified using Weigert’s method (Weigert’s for elastic fibers, Bio-Optica). Additionally, Alcian blue in buffers at pH 2.5 staining (IVD, Bio-Optica Milano) was performed for the histolocalization of hyaluronic acid in the dermis of each patient. All histochemical reactions were carried out according to protocols recommended by the manufacturers.
Morphometry Sections stained with H-E were analyzed to determine the number of fibroblasts using a 10x ocular lens with a square reticle. The number of fibroblasts in the dermis before and after the treatment (with cells or PBS only) was counted in 100 random squares (50 squares per slide). The evaluation was carried out on a Zeiss microscope equipped with a 40 objective. The surface of each square was 1.225 mm2 (35 35 mm) under the objective. Additionally, the diameter of the collagen fibers in the reticular layer of the dermis was measured. All morphometric measurements were obtained using the Axio Vision Rel. 4.6 program (Zeiss, Axioscope System, Jena, Germany).
Immunohistochemistry Immunohistochemistry was performed to identify fibrillin-1 in the dermis. The mouse antihuman fibrillin-1 (AbD Serotec, Biogenesis, Oxford, UK) monoclonal antibody (1:50) was used. The deparaffinized sections of skin were microwaved in citrate buffer (pH 6.0) for heat-induced epitope retrieval. After slow cooling to room temperature, the slides were washed twice in PBS for 5 minutes and then incubated for 60 minutes with primary mouse antihuman fibrillin-1 antibody (AbD Serotec MorphoSys AbD). Next, the sections were stained with an avidin-biotin-peroxidase system using diaminobenzidine as the chromogen (Dako LSABþ System-HRP: Code K0679 DakoCytomation, Glostrup, Denmark) in accordance with the staining procedure instruction suggested by the manufacturer. The sections were washed in distilled H2O and counterstained with hematoxylin. Negative control specimens were processed in the absence of primary antibody. Positive staining was defined microscopically by the visual identification of brown pigmentation.
Statistical Analysis Results are expressed as the median, the lower and upper quartiles (Q1-Q3) and the mean values standard deviation (X SD). The distribution of the results for individual variables was determined using the Shapiro-Wilk W test. Because most of the distributions deviated from the normal distribution, the nonparametric MannWhitney U test was used for further analyses. P < .05 was considered significant. Calculations were performed using the software package Statistica 6.1 (StatSoft, Tulsa, OK).
RESULTS
Injections with autogenously harvested and in vitroexpanded fibroblasts were well-tolerated and caused no side effects. Biopsies obtained from the patients before and 3 months after treatment with cells or PBS only were composed of the whole skin, and the histologic slides confirmed the proper structure of both the epidermis and
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the dermis, which was subdivided into the papillary and reticular layers. Morphometric Analysis of the Skin
Noticeable differences in the morphology of the dermis, in the cellular content and ECM components, before and after treatment in all patients were observed. Therefore, we performed a comprehensive morphometric analysis to assess the differences in the number/density of fibroblasts located between collagen bundles and the diameter of collagen bundles in the reticular layer of the dermis in samples obtained before treatment and after treatment with fibroblasts or with PBS. These results are presented in Table 1. In skin samples taken 3 months after the reimplantation of fibroblasts, both the number of fibroblasts in the dermis and the diameter of collagen bundles were significantly higher compared with before treatment and compared with post-PBS injection (Table 1). These results suggest that administered autogenous fibroblastic cells exerted a biological effect resulting in the rejuvenation of the collagen fibers in the dermis. Histology
We continued to characterize the effect of intradermal autologous fibroblast cells administration by analyzing the essential ECM elements of skin samples collected before and 3 months after fibroblasts reimplantation or PBS injection. Collagen Fibers
The skin slides were stained with H-E and picrosirius red. Skin sections stained with picrosirius red were analyzed by polarized microscopy. The larger collagen fibers that displayed yellow-red staining and strong birefringence were considered collagen type I, whereas fibers that displayed a greenish color and weak birefringence were considered collagen type III. The color and intensity of birefringence depend on differences in the pattern of physical aggregation and the thickness of the collagen fibers. Thin collagen fibers exhibit green to greenish-yellow polarizing colors, whereas thick fibers stain yellowish-orange, orange, and red [15]. In the papillary dermis of patients before the treatment and after the injection of PBS (H-E-stained sections), a loose network of thin bundles was observed (Fig 1A). The reticular dermis contained loosely woven collagen bundles of different thicknesses, which were frequently fragmented. Wide spaces separating the bundles were visible, with ovalshaped fibroblasts (Fig 1D). Similar structure of the collagen fibers was observed in the dermis after picrosirius red staining. In the papillary dermis, thin collagen bundles with typical organization and yellowish-orange birefringence were observed (Fig 1B). Collagen bundles in the reticular dermis appeared regularly ordered with multiple orientations with different birefringence intensities varying from yellowish-orange to
MACHALINSKI, BRODKIEWICZ, SZUMILAS ET AL
2836 Table 1. Fibroblast Number and Collagen Bundle Diameter in the Reticular Layer of the Dermis BT
Fibroblasts number (n ¼ 100) Median 1.0 Q1eQ3 0e1 X SD 0.58 0.63
AT
1.0 1e2 1.52 1.12 AT vs BT*** Diameter of collagen bundles, mm (n ¼ 100) Median 5.64 8.05 Q1eQ3 4.58e7.2 6.35e9.83 X SD 7.51 12.62 8.39 2.51 AT vs BT***
AI
1.0 0e1 0.62 0.69 AT vs AI*** 6.13 5.03e7.86 6.43 1.93 AT vs AI***
Asterisks indicate the statistically significant differences in the Mann-Whitney U test. ***P < .001. Abbreviations: AI, skin 3 months after the phosphate-buffered saline injection; AT, skin 3 months after the fibroblastic cells injection; BT, skin before treatment; Q1-Q3, upper quartile and lower quartile; X SD, arithmetic mean standard deviation.
yellowish-red. There were areas displayed green polarized color (Fig 1E). The dermis of patients after cells reimplantation was structurally similar. In H-Eestained slides, thin collagen bundles were organized in a loose network (Fig 1G). The reticular dermis was filled with thicker collagen bundles (Fig 1J) than it was observed in patients before the treatment (Fig 1D). There were no wide spaces between the collagen bundles, which were woven in a distinctive compact pattern (Fig 1J). Collagen bundles in the papillary dermis in slides stained with picrosirius red were thicker and revealed yellowish-red birefringence (Fig 1H). The reticular dermis was filled with
thick compacted collagen bundles with an enhanced intensity of birefringence from bright yellowish-red to red. Many areas with fibers displayed green polarized color were very well visible (Fig 1K). Reticular Fibers
Small amount of reticular fibers was detectable in both the papillary and reticular layers of the dermis from the patients before treatment and after PBS injection. Only few silverstained fibers were visible close to the epidermis (Fig 1C) and in the reticular layer of the dermis (Fig 1F). Additionally, these fibers were visible in the walls of blood vessels (Fig 1F). A similar arrangement was observed in slides stained with picrosirius red, a small amount of fibers with greenish polarized color was observed in the papillary dermis (Fig 1B) and reticular dermis (Fig 1E). In contrast, abundant reticular fibers were visible in both the papillary and reticular layers of the dermis in the patients after cells reimplantation. In the papillary layer, thin reticular fibers were accumulated near the basement membrane of the epidermis (Fig 1I), whereas in the reticular layer of the dermis, silver-stained reticular fibers were observed between and on the surface of collagen fiber bundles (Fig 1L). The fibers with greenish polarized color were also observed in sections stained with picrosirius red (Fig 1H, K). Fibrillin-1eRich Microfibrils
Immunohistochemical staining revealed only scant fibrillin1erich microfibrils in the papillary layer located near to stratum basale of the epidermis and in the elastic fibers of reticular dermis in patients before treatment and after PBS
Figure 1. Untreated and treated with fibroblastic cells skin. Staining: hematoxylin and eosin, picrosirius red, and silver impregnation.
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Figure 2. Untreated and treated with fibroblastic cells skin. Staining: immunohistochemistry and Weigert’s quick method.
injection (Fig 2A, C). After fibroblasts reimplantation, immunostaining showed a significant increase in the density of fibrillin-1 microfibrils in the papillary dermis and the reticular dermis (Fig 2E, G). Elastic Fibers
Elastic fibers were detected in the papillary and reticular layers of the dermis in the patients before treatment and after PBS injection. In the reticular layer, the fibers were located between collagenous bundles (Fig 2B, D). Elastic fibers were visible near sebaceous (Fig 2D) and sweat glands. In samples obtained after fibroblasts reimplantation, elastic fibers were more abundant and observed in both layers of the dermis. In the papillary layer, elastic fibers took the form of short, thin, single fibers localized close to the epidermis (Fig 2F), whereas in the reticular layer, elastic fibers were present between bundles of collagenous fibers (Fig 2H) and were condensed around hair follicles, sweat and sebaceous glands (Fig 2H). Hyaluronic Acid
Weakly blue-stained HA was visible in both the papillary and reticular layers of the dermis in patients both before and after the treatment. The results of histologic analyses were comparable between patients (data not shown). DISCUSSION
Skin aging progresses gradually and occurs owing to intrinsic factors as well as extrinsic factors, which together lead to the development of changes in the cellular and extracellular components of the skin. morphologic examination of aged skin typically reveals atrophy of the dermal ECM with the loss of fibers, predominantly type I and III collagens, and elastic fibers [16]. Recently, there has been increased interest in the prevention of skin aging, especially using nonablative procedures that enable skin rejuvenation with minimal downtime and complications [17e20].
In this pilot experimental study, we demonstrated for the first time that locally injected autogenous fibroblastic cells could successfully engraft and settle in the target tissue, resulting in effectively rejuvenated dermis in adult male patients. Several lines of evidence suggest that there are no differences in terms of particular skin aging symptoms in the old adults of age of 50 to 70 years, especially in the specific fibroblast subpopulation as reticular fibroblasts, which are mainly responsible for synthesizing extra-cellular matrix. Therefore, our obtained results can be extended to the elderly subjects as well. We showed in morphologic analysis evidence of the improvement of fibrous components in dermal connective tissue, which can reflect the rejuvenation process, that included the significant enlargement of collagen fiber diameters and increased density of reticular fibers, fibrillin-1erich microfibrils, and elastic fibers. Dermal fibroblasts, the main ECM-producing cells, are an essential component of the skin involved in the maintenance of dermal architecture and providing biomechanical properties of the skin [21e23]. Because they play a crucial role in skin physiology, their metabolism is strictly regulated by complex mechanisms, including cellecell and cellematrix interactions [21,22], what is confirmed by in vitro studies [22,24]. The interactions between fibroblasts and collagen fibers, relationship between mechanical tension on cells in vitro and biological cells response to stress has been well documented [25,26]. Chronologically aged skin is characterized by collagen fragmentation, reduction in total collagen, reduction in fibroblasts, and decreased celle collagen fiber interactions. The last results in the reduced mechanical tension on the cells and in this way, matrix production consequently falls, and matrix-degrading enzymes become activated [12,26]. Consistent with these reports, in the current study we revealed that injected fibroblastic cells were able to resume their basic functions and produce ECM proteins, what was visible as enlargement of thickness of collagen type I and density of collagen type III fibers. Fibroblastic cells are also known to naturally express an array of cytokines, growth factors, and proteases
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involved in the regulation of a variety of biological processes, including cell survival and proliferation, production and remodeling of ECM components, angiogenesis and epithelial morphogenesis. The ex vivo culture for clonal expansion of fibroblastic cells may augment the combination of such factors in the posttransplant milieu. After cultured ex vivo fibroblasts transplantation into the skin such factors may next promote fibroblast survival and engraftment and stimulate their ECM protein production on site. Importantly, the use of autologous blood serum enriched in different soluble growth factors may act as a specific fibroblast phenotype regulator during their culture ex vivo before transplantation, which might contribute to their hyperactive biological behavior after transplantation. Although collagen synthesis progressively decreases with passage of time [27,28], in our experiment we found a higher density of reticular fibers, composed of type III collagen, in the papillary layer and the coexistence of both type I and type III collagen fibers in the reticular layer of the dermis in patients after cells re-implantation in comparison with untreated skin. Thus, our results suggest that considerable improvement of dermis structure could occur after fibroblastic cells administration. The elastic fiber system, which is composed mainly of elastin, fibrillin, and elastin binding protein, is highly ordered in the skin [29]. The aging process broadly influences the structure and function of the elastic fiber system [29]. The degradation of existing fibers leads to accumulation of elastin-rich material in the upper and mid dermis, especially during the photoaging process [12,30]. In contrast, mildly photoaged skin is characterized histologically by loss of fibrillin microfibrils [29]. However, restoration of elastic fibers was observed in photoaged skin after topical retinoic acid treatment [11] and in response to hyaluronic acid injection [17,27]. Interestingly, in the present study we found increase in the density of the fibrillin-1erich microfibrils in the dermal papillae and the elastic fibers in the reticular dermis in mildly photoprotected skin of patients injected with the fibroblastic cells in comparison with the untreated skin. In contrast, we did not observe differences in the content of hyaluronic acid in the dermis before and after the injection of autogenous fibroblastic cells. These results are consistent with studies of other authors, which also confirmed that the total hyaluronic acid level in the dermis of skin undergoing intrinsic aging process remains stable; however, the epidermal HA diminishes markedly [5,27,31,32]. Despite improved scar quality when using acellular dermal substitutes for treatment, the optimal result of whole skin regeneration is still not achieved, when using acellular dermal substitutes. Cell-based therapy is one of the strategies that may further improve the whole skin regeneration. Particularly, the application of autologous dermal fibroblasts may improve the burn wound healing. It is expected that autologous fibroblasts may promote the whole skin wound healing owing to the fact that they have the capacity to further differentiate into myofibroblasts, which are
MACHALINSKI, BRODKIEWICZ, SZUMILAS ET AL
responsible for the wound contraction. This phenotype plasticity of the dermal fibroblasts makes them attractive candidate for cellular-based therapies of the wound healing, especially those which are clinically complicated or extended to greater areas. Therefore, autologous dermal fibroblasts might be used in the future as adjuvant regenerative therapy in such clinical problems as skin traumas, cutaneous burns, scaffolds production for minor tissue loss or congenital skin abnormalities in elderly patients. In conclusion, our preliminary findings, in addition to being relevant for the elucidation of cell-based regeneration process in aging skin, provide new insights regarding the role of autologous fibroblastic cells in this process. We also provide novel, compelling evidence for the efficacy of fibroblastic cell administration in improving dermal connective tissue structure. However, further experiments involving a larger number of subjects are necessary to confirm our results. REFERENCES [1] Giacomoni PU. Advancement in skin aging: the future cosmeceuticals. Clin Dermatol 2008;26:364e6. [2] Makrantonaki E, Zouloboulis CC. Molecular mechanisms of skin aging. State of the art. Ann N Y Acad Sci 2007;1119:40e50. [3] Zouloboulis CC, Makrantonaki E. Clinical aspects and molecular diagnostic of skin aging. Clin Dermatol 2011;29:3e14. [4] Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci 2010;123:4195e200. [5] Stern R, Maibach HI. Hyaluronan in skin: aspects of aging and its pharmacologic modulation. Clin Dermatol 2008;26:106e22. [6] Makrantonaki E, Zouloboulis CC. The skin as a mirror of the aging process in the human organism- state of the art and results of the aging research in the German National Genome Research Network 2 (NGFN-2). Exp Gerontol 2007;42:879e86. [7] Naylor EC, Watson RE, Sherratt MJ. Molecular aspects of skin ageing. Maturitas 2011;69:249e56. [8] Graham HK, Hodson NW, Hoyland JA, et al. In situ ultrastructural imaging of native biomolecules. Matrix Biol 2010;29: 254e60. [9] Doubal S, Klemera P. Visco-elastic response of human skin and aging. J Am Aging Assoc 2002;25:115e7. [10] Kielty CM, Sherratt MJ, Shuttleworth CA. Elastic fibres. J Cell Sci 2002;15:2817e28. [11] Lee JY, Kim YK, Seo JY, et al. Loss of elastic fibers causes skin wrinkles in sun-damaged human skin. J Dermatol Sci 2008;50: 99e107. [12] Humbert P, Viennet C, Legagneux K, et al. In the shadow of the wrinkle: experimental models. J Cosmet Dermatol 2012;11: 79e83. [13] Longas MO, Russell CS, He XY. Chemical alterations of hyaluronic acid and dermatan sulfate detected in aging human skin by infrared spectroscopy. Biochim Biophys Acta 1986;884:265e9. [14] Longas MO, Russell CS, He XY. Evidence for structural changes in dermatan sulfate and hyaluronic acid with aging. Carbohydr Res 1987;159:127e36. [15] Junqueira Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 1979;11:447e55. [16] Widgerow AD, Grekin SK. Effecting skin renewal: a multifaceted approach. J Cosmet Dermatol 2011;10:126e30. [17] Cattin TA. A single injection technique for midface rejuvenation. J Cosmet Dermatol 2010;9:256e9. [18] Draelos ZD. Creating an attractive aging face. J Cosmet Dermatol 2013;11:167e8.
MORPHOLOGIC CHANGES IN THE DERMIS [19] Seo KY, Kim DH, Lee SE, Yoon MS, Lee HJ. Skin rejuvenation by microneedle fractional radiofrequency and a human stem cell conditioned medium in Asian skin: a randomized controlled investigator blinded split-face study. J Cosmet Laser Ther 2013;15:25e33. [20] Seo KY, Yoon MS, Kim DH, Lee HJ. Skin rejuvenation by microneedle fractional radiofrequency treatment in Asian skin; clinical and histological analysis. Lasers Surg Med 2012;44:631e6. [21] McAnulty RJ. Fibroblasts and myofibroblasts: their source, function and role in disease. Int J Biochem Cell Biol 2007;39: 666e71. [22] Sorrell JM, Caplan AI. Fibroblast heterogeneity: more than skin deep. J Cell Sci 2004;117:667e75. [23] Vedrenne N, Coulomb B, Danigo A, et al. The complex dialogue between (myo)fibroblasts and the extracellular matrix during skin repair processes and ageing. Pathol Biol (Paris) 2012;60: 20e7. [24] Müller SA, van der Smissen A, von Feilitzsch M, et al. Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix. J Mater Sci Mater Med 2012;23:3053e65.
2839 [25] Grinnell F, Petroll WM. Cell motility and mechanics in three-dimensional collagen matrices. Annu Rev Cell Dev Biol 2010;26:335e61. [26] Varani J, Dame MK, Rittie L, et al. Decreased collagen production in chronologically aged skin: roles of age-dependent alteration in fibroblast function and defective mechanical stimulation. Am J Pathol 2006;168:1861e8. [27] Ganceviciene R, Liakou AI, Theodoridis A, et al. Skin antiaging strategies. Dermatoendocrinol 2012;4:308e19. [28] Levakov A, Vucovic N, Dolai M, et al. Age-related skin changes. Med Pregl 2012;65:191e5. [29] Sherratt MJ. Tissue elasticity and the ageing elastic fibers. Age (Dordr) 2009;31:305e25. [30] El-Domyati M, Medhat W, Abdel-Wahab HM, et al. Forehead wrinkles: a histological and immunohistochemical evaluation. J Cosmet Dermatol 2014;13:188e94. [31] Oh JH, Kim YK, Jung JY, et al. Changes in glycosaminoglycans and related proteoglycans in intrinsically aged human skin in vivo. Exp Dermatol 2011;20:454e6. [32] Papakonstantinou E, Roth M, Karakiulakis G. Hyaluronic acid: a key molecule in skin aging. Dermatoendocrinology 2012;4: 253e8.