Effect of Use of Platelet-Rich Plasma (PRP) in Skin with Intrinsic Aging Process

Effect of Use of Platelet-Rich Plasma (PRP) in Skin with Intrinsic Aging Process Abstract Background In previous papers, we demonstrated that the treatment of human photoaged skin with stromal-vascular fraction-enriched fat or expanded adipose-derived stem cells showed a decrease of elastosis and the appearance of new oxytalan elastic fibers in dermis and an increase in the vascular network. The utilization of fat plus platelet-rich plasma (PRP) led to an increase in the vascular permeability and reactivity of the nervous component. Objectives The purpose of this study was to analyze the histologic and ultrastructural changes of human skin after the injection of only PRP in the retroauricular area that was not exposed to sun and did not present the photoaging process, in comparison with our previous results. Methods This study was performed in 13 patients who were candidates for facelift and whose ages ranged between 45 and 65 years. The PRP injection was performed in the mastoidea area. Fragments of skin were removed before and 3 months after treatment and analyzed by optical and electron microscopy. Results After the injection of PRP, we observed an increase of reticular dermis thickness because of the deposition of elastic fibers and collagen, with a fibrotic aspect. A modified pattern of adipose tissue was also found at the dermohypodermal junction. Significative regenerative aspects were not found at histologic and ultrastructural analysis. The presence of foci of moderate inflammation and microangiopathy were observed. Conclusions Treatment with PRP increased reticular dermis thickness with a fibrotic aspect. In the long term, the presence of inflammation and microangiopathy caused by PRP injection could lead to trophic alteration of the skin and the precocious aging process. Level of Evidence: 4 The aging process of the skin is a complex biological phenomenon that can be divided into intrinsic and extrinsic aging. The facial skin aging process is a degenerative process that affects the skin and deep structures of the face, resulting from the action of the intrinsic factor (age) associated with the extrinsic factor, particularly exposure to ultra-violet radiation. Intrinsic aging, which is largely genetically determined, affects the skin in a manner similar to that of most internal organs1 through a slow and partly reversible degeneration of connective tissue. Otherwise, extrinsic aging, more commonly termed photoaging, caused by environmental exposure, primarily ultraviolet radiation, leads to a premature aging phenotype even in young individuals.2 In sun-exposed areas, these two processes, intrinsic and extrinsic, are superimposed; there is evidence3 that they have at least in part overlapping, biological, biochemical, and molecular mechanisms.4 The connective tissue of the skin is composed mostly of collagen and elastin. Collagen makes up 70% to 80% of the dry weight of the skin and gives the dermis its mechanical and structural integrity. Elastin is a minor component of the dermis, but it has an important function in providing the elasticity of the skin, and elastin accounts for 2% to 4% of the extracellular matrix.5 In the photoaged skin, there is a progressive destruction of the entire network of elastin in the dermis. Elastic fibers become thickened, tangled, tortuous, degraded, and dysfunctional, with increased density of the elastic material, resulting in a cluster of amorphous and dystrophic elastotic materials throughout the dermis, setting up solar elastosis, the most important feature of photoaging.5 This is caused by the marked loss of collagen and the thickening of elastic fibers, causing a higher level of the elastic component in the elderly than the young.6 Regenerative medicine is an emerging field whose focus is on cell- and tissue-based therapies applicable to plastic surgery procedures, including the aging process of skin.6 The mechanism transforms undifferentiated stem cells into the differentiated cells that ultimately form the specialized tissues that are required for reconstruction, repair, and restoration, including the role of the surrounding environment.7 Moreover, a stem cell has paracrine effects and secretes a wide range of growth factors, cytokines, and bioactive molecules that can stimulate tissue regeneration.7 In this sense, platelet-rich plasma (PRP) has also been described as having a possible regenerative effect due to its growth factors and cytokines, however empirical, without scientific evidence until now.8-11 In a previous paper,6 we demonstrated that the treatment of human photoaged skin (preauricular area) with the autologous lipidic component plus stromal vascular fraction rich in adipose-derived stem cells (SVF-enriched fat) or expanded adipose-derived stem cells (ADSCs) showed a decrease in the elastic fiber network (elastosis) and the appearance of new oxytalan elastic fibers in papillary dermis. The ultrastructural examination showed a modified 3-dimensional architecture of the reticular dermis and the presence of a richer microvascular bed, representing a skin rejuvenation effect. Considering that expanded stem cells require a cell factor, we developed another study12 in which we wished to assess similar results replacing the ADSC by PRP. PRP is easier to obtain and could be utilized in an ambulatory environment. We aimed to determine if PRP injection could replace the cutaneous regenerative effect of ADSCs when injected together with fat in the preauricular area, with photoaging characteristics. We observed that the use of fat plus PRP led to the presence of more pronounced inflammatory infiltrates and a greater vascular reactivity, increasing in the vascular permeability and a certain reactivity of the nervous component. The addiction of PRP to fat did not improve the regenerative effect and did not present significant advantages over the use of expanded ADSC or SVF-enriched fat in skin rejuvenation.12 The purpose of this new article was to analyze the histologic and ultrastructural changes of skin after the injection of only PRP in the retroauricular area (mastoidea area) that normally suffered only the intrinsic aging process, because it was not exposed to sun and did not present the photoaging process. We observed the histologic alterations due to the intrinsic aging process and observed the action of PRP in this area. METHODS The study was executed in 13 patients, 11 females and 2 males, who were candidates for facial rejuvenation surgery (facelift), whose ages ranged between 45 and 65 years with no history of chronic viral, metabolic, ischemic, or autoimmune diseases, or other systemic pathologies. This study also excluded patients who reported a smoking habit. The study period ranged from May 2012 through May 2016. The patients were subject to the sampling of blood to prepare the PRP, as described below. The injection of PRP was performed in the retroauricular area. Prior to injection, with the patient under local anesthesia, a fragment of skin and subcutaneous tissue was removed from the mastoidea area and sent to the Section of Human Anatomy and Histology of Verona (Italy) and the Federal University of Rio de Janeiro (Brazil) for morphological analysis through optical and electron microscopy. At histology, the analysis of skin biopsies was performed through hematoxylin and eosin (HE) staining, picrosirius red staining (for visualization of collagen), and orcein staining (for visualization of elastic fibers). The morphological examination was repeated after 3 months, taking a piece of skin and subcutaneous tissue in the same area where the PRP was injected. After this second biopsy, the facelift was performed. The regenerative anti-aging activity was evaluated by comparing the morphology of the skin and subcutaneous area before and after treatment. The biopsy specimens, consisting of fragments of skin measuring 0.5 cm × 1 cm, were limited inside of the 1 cm2 retroauricular area (mastoid) and split into two equal parts of 0.5 cm × 0.5 cm. One half was sent in a plastic bottle immersed in 4% formaldehyde to the Institute of Biophysics Carlos Chagas Filho, Laboratory of Immunology, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil) for histologic study. The other half (0.5 cm × 0.5 cm) was sent in a plastic bottle with a solution of buffered 2% glutaraldehyde to the Section of Human Anatomy and Histology of Verona (Verona, Italy) to be subjected to histomorphometric analysis with electron microscopy. The skin biopsies (untreated and treated skin biopsies) were performed under local anesthesia with lidocaine 0.5% and 1:200.000 epinephrine, on an outpatient basis. All patients consented under approved guidelines set forth by the human clinical trial to the Brazilian investigation ethical committee board (protocol no. 28063) and the Brazilian Clinical Trials Registry (Universal Trial Number, U1111-1145-3081). Platelet-Rich Plasma Preparation Peripheral blood was collected in blood collection tubes containing 0.5 ml 3.2% sodium citrate solution. The PRP preparation procedure consisted of two centrifugation steps. All steps were performed in a refrigerated centrifuge (certified Jouan Br4i, Saint-Herblain, Loire-Atlantique, France). Whole blood was centrifuged at 300 g for 5 minutes at 18°C. After the first centrifugation, the whole plasma above the buffy coat was collected, separating platelets from red blood cells and leukocytes (PRP1). PRP1 was centrifuged at 700 g for 17 minutes at 18°C. After the second centrifugation step, the platelet pellet was suspended in 300 μl of platelet-poor plasma (PPP; new fraction named PRP2). Platelet activation was performed adding 20 mM CaCl2 (PRP2-Ca) and 25 IU/ml human plasma thrombin (PRP2-Thr, Ref: T6884; Sigma-Aldrich, St. Louis, Missouri, USA), incubating at 37°C for 1 hour and at 4ºC for 16 hours. To recover the activated PRP2, all of the treated samples were centrifuged at 3,000 × g for 20 minutes at 18°C. All samples were stored at –80°C.8 Different PRP preparations have been published, eliciting variable responses that cannot be compared, due to the lack of the final platelet quantification. This preparation, published by Amable et al,8 optimized PRP preparation and maximized platelet recovery, 46.9% to 69.5% of initial platelets, and the platelets were highly purified. The blood sample was collected in a 5-ml tube containing 3.2% sodium citrate. A blood sample for all 13 patients was stored and then analyzed in the laboratory before injection in the patient. It was injected in intradermal level of the retroauricular skin. Transmission Electron Microscopy (TEM) Different samples were fixed with glutaraldehyde 2% in a Sorensen buffer pH 7.4 for 2 hours, post-fixed in 1% osmium tetroxide in aqueous solution for 2 hours, dehydrated in graded concentrations of acetone, embedded in Epon-Araldite and cut with an Ultracut E ultramicrotome (Reichert, Wien, Austria). At the end of the dehydrating process, samples were positioned in a multi-well grid for electron microscopy and observed through the use of a TEM Morgagni 268D (FEI Philips). Scanning Electron Microscopy (SEM) The specimens were fixed in 2% glutaraldehyde in a phosphate buffer for 2 to 4 hours, post-fixed in 1% osmium tetroxide in the same buffer for 1 hour, and dehydrated in graded concentrations of acetone. The samples were then treated by critical point dryer (CPD 030, Balzers Vaduz, Liechtenstein), mounted on metallic specimen stubs, and sputter-coated with gold (MED 010 Balzers). SEM Imaging was performed with XL30 ESEM (FEI-Philips Eindhoven, Netherlands). RESULTS The present study was executed in 13 patients, 11 females and 2 males, age ranging between 45 and 65 years old (mean, 56.2 years), through the analysis of the retroauricular skin (mastoidea area) that normally is protected from the sun. No elastosis was observed before the use of PRP. Therefore, we can conclude that there is only an intrinsic aging process in this area (Figure 1). Figure 1. View largeDownload slide Light microscopy, orcein stain. The figure shows the structural differences between the preauricular skin (photoaging skin) and the retroauricular skin (protected skin). (A) Preauricular skin with the presence of elastic fibers stained in black (asterisk). (B) The retroauricular skin of the same patient appears poor in elastic fibers (lesser material stained in black). Scale bars: 150 µm. Figure 1. View largeDownload slide Light microscopy, orcein stain. The figure shows the structural differences between the preauricular skin (photoaging skin) and the retroauricular skin (protected skin). (A) Preauricular skin with the presence of elastic fibers stained in black (asterisk). (B) The retroauricular skin of the same patient appears poor in elastic fibers (lesser material stained in black). Scale bars: 150 µm. As described in the introduction, we wrote two previous papers pertaining to preauricular skin. This figure of preauricular skin was utilized in the present paper with the aim of comparing the photoaged skin (preauricular) with the protected skin, from the retroauricular area, of the same patient. Thus, this information is important to readers understand the cutaneous quality of the retroauricular area. The treatment with PRP induced modifications mainly in the reticular dermis. After the use of PRP, a great increase of the thickness of the reticular dermis, which resulted from the horizontal deposition of layers of mature elastic fibers and collagen, was observed (Figure 2). In this layer, activated fibroblasts were also visible. The great increase of elastic and collagen fibers and the morphology of the fibroblasts suggest that in some areas a fibrotic reaction occurs. Figure 2. View largeDownload slide Light microscopy. (A, C) Hematoxylin/eosin stain. (B, D) Orcein stain. The figure shows retroauricular skin before and after PRP injection. (A, B) Before PRP injection, adipocytes are located at the boundary of the dermis with the subcutaneous tissue organized in lobules, which are directed vertically toward the surface of the skin (stars). These lobules form adiposae papillae, which in their apical portion show a relationship with sebaceous or sweat glands. (C, D) After the use of PRP, fibrosis is apparent through the thickening of the reticular dermis (squares). Collagen and orcein-positive elastic fibers (asterisks) are increased. A small flogistic infiltrate is present (arrows). Scale bars: 300 µm. Figure 2. View largeDownload slide Light microscopy. (A, C) Hematoxylin/eosin stain. (B, D) Orcein stain. The figure shows retroauricular skin before and after PRP injection. (A, B) Before PRP injection, adipocytes are located at the boundary of the dermis with the subcutaneous tissue organized in lobules, which are directed vertically toward the surface of the skin (stars). These lobules form adiposae papillae, which in their apical portion show a relationship with sebaceous or sweat glands. (C, D) After the use of PRP, fibrosis is apparent through the thickening of the reticular dermis (squares). Collagen and orcein-positive elastic fibers (asterisks) are increased. A small flogistic infiltrate is present (arrows). Scale bars: 300 µm. At the dermohypodermal junction, before the treatment with PRP, the adipocytes located at the boundary of the dermis and the subcutaneous tissue were organized in lobules, and the lobules were often vertically directed toward the surface of the skin. These lobules formed adiposae papillae, in which a vascular peduncle was often seen. The papillae, in their apical portion, often showed a relationship with sebaceous or sweat glands. At this level, the adipocytes were covered by a dense basket of collagen strictly adherent to their plasma membrane. After the use of PRP, the relationship between subcutaneous fat and sebaceous or sweat glands appears less close, after the reticular dermis increases and forms a kind of barrier between these elements (Figures 2-4). At ultrastructural examination, after treatment with PRP, perivascular areas with mononuclear cell infiltration were visible in the reticular dermis and the dermohypodermal junction (Figure 3). Often the blood vessels (blood capillaries and arterioles) were surrounded by reduplicated and fragmented layers of basal lamina (Figure 5). The ultrastructural examination also confirmed the histologic data, revealing a large amount of elastic fibers and collagen mainly disposed parallel to the skin surface. Collagen fibers were disposed in compact bundles. Ultrastructural examination also revealed the presence of activated fibroblasts with a large amount of rough endoplasmic reticulum and large lysosomes. These findings suggest that PRP causes fibrosis in the area where it is injected (Figure 4 and 5). Figure 3. View largeDownload slide Light microscopy. (A, C) Hematoxylin/eosin stain. (B, D) Orcein stain. The figure shows retroauricular skin before and after PRP injection. In a different patient, the results are similar to those visible in the patient illustrated in Figure 2, indicating the reproducibility of the findings. (A, B) Before PRP injection, adipocytes are located at the boundary of the dermis, with the subcutaneous tissue organized in lobules (stars), which are vertically directed toward the surface of the skin. A small quantity of elastic fibers is present. (C, D) After the use of PRP, fibrosis is apparent through the thickening of the reticular dermis (squares). Collagen and orcein-positive elastic fibers (asterisks) are increased. Scale bars: 300 µm. Figure 3. View largeDownload slide Light microscopy. (A, C) Hematoxylin/eosin stain. (B, D) Orcein stain. The figure shows retroauricular skin before and after PRP injection. In a different patient, the results are similar to those visible in the patient illustrated in Figure 2, indicating the reproducibility of the findings. (A, B) Before PRP injection, adipocytes are located at the boundary of the dermis, with the subcutaneous tissue organized in lobules (stars), which are vertically directed toward the surface of the skin. A small quantity of elastic fibers is present. (C, D) After the use of PRP, fibrosis is apparent through the thickening of the reticular dermis (squares). Collagen and orcein-positive elastic fibers (asterisks) are increased. Scale bars: 300 µm. Figure 4. View largeDownload slide Scanning electron microscopy. (A) Retroauricular skin after treatment with PRP at low (A) enlargement. The dense dermis (square) is visible below the epidermis (e). (B) At medium enlargement, the deposition of elastic and collagen fibers parallel to the skin surface is visible. (C) The fibers are organized in compact bundles (asterisk). Scale bars: A, 500µ; B, 200µ; C, 5 µm. Figure 4. View largeDownload slide Scanning electron microscopy. (A) Retroauricular skin after treatment with PRP at low (A) enlargement. The dense dermis (square) is visible below the epidermis (e). (B) At medium enlargement, the deposition of elastic and collagen fibers parallel to the skin surface is visible. (C) The fibers are organized in compact bundles (asterisk). Scale bars: A, 500µ; B, 200µ; C, 5 µm. Figure 5. View largeDownload slide Transmission electron microscopy. Retroauricular skin after the use of PRP. (A) Duplication of the vascular basal membrane (d). (B) Activated fibroblasts (f) with a rich organular pattern surrounded by thick bundles of collagen fibers (c). Scale bars: A, 2 µm; B, 4 µm. Figure 5. View largeDownload slide Transmission electron microscopy. Retroauricular skin after the use of PRP. (A) Duplication of the vascular basal membrane (d). (B) Activated fibroblasts (f) with a rich organular pattern surrounded by thick bundles of collagen fibers (c). Scale bars: A, 2 µm; B, 4 µm. DISCUSSION The retroauricular skin has its own characteristics. It is a thin and non-hair-bearing skin and has skin-color matching. The retroauricular skin shows a thin stratum corneum, the dermo-epidermal junction is rather flat, and the connective papillae were poorly developed. The dermal papillae are also thin and showed a scarce cellularity. The reticular dermis is homogeneous and appeared as a regular network of collagen and elastin fibers disposed in thin bundles. Scanning microscopy showed the elastin fibers as thin ribbons with smooth surface. The subcutaneous adipose tissue is present in a variable amount and generally formed elongated lobules mainly disposed parallel to the external surface. The pilo-sebaceous structures, as well as the sweat glands, are scarce. In the male patients, a larger amount of sebaceous glands is evident and appeared larger in diameter than those visible in female patients.13,14 During aging, degenerative processes affecting the skin and deep structures of the face commonly occur. This aging process is the result of the action of intrinsic skin events associated with extrinsic agents, especially exposure to ultraviolet radiation. Normally, the retroauricular skin is protected from the sun and does not suffer the extrinsic aging process that leads to elastosis and actinic pathology. The age-related modifications extensively described in the elastic fibers of all organs and tissues may be largely interpreted as resulting from progressive degradation of a protein polymer that has been produced early in life.15-21 In previous papers,6,12 we demonstrated the regenerative effect of SVF-enriched fat and expanded ADSC when injected in preauricular skin with the photoaged process. When the fat plus PRP was injected in the same place, the presence of more pronounced inflammatory infiltrates and a greater vascular reactivity was observed, increasing in vascular permeability and a certain reactivity of the nervous component. The addiction of PRP to fat did not improve the regenerative effect and did not present significant advantages over the use of expanded ADSCs or SVF-enriched fat in skin rejuvenation.12 However, PRP has been described as having a possible regenerative effect due to its growth factors and cytokines, although the previous evidence is empirical.8-11,22-24 Considering the results, we observed that treatment with PRP induced modifications in the reticular dermis, due to the primarily horizontal deposition of layers of collagen and elastic fibers. The great increase of collagen suggests a fibrotic reaction, because there are activated fibroblasts in this layer. This fibrosis creates a kind of barrier between the sebaceous sweat glands and subcutaneous fat tissue. At the dermohypodermal junction, before treatment with PRP, the adipocytes located at the boundary of the dermis and the subcutaneous tissue were organized in lobules, which often were vertically directed toward the surface of the skin. These lobules formed adiposae papillae, in which a vascular peduncle was often seen. The papillae, in their apical portion, often showed a relationship with sebaceous or sweat glands. At this level, the adipocytes were covered by a dense basket of collagen strictly adherent to their plasma membrane. At ultrastructural examination, after treatment with PRP, perivascular areas with mononuclear cell infiltration were visible in the reticular dermis and the dermohypodermal junction. These areas showed blood capillaries and arterioles surrounded by mononuclear infiltrates. Often the blood vessels were surrounded by reduplicated and fragmented layers of basal lamina. Elastic fibers and collagen are horizontal and strongly attached, and the presence of lysosomes and activated fibroblasts suggests that PRP causes fibrosis in the area where it is injected. The limitation of this study is that PRP injections currently occur into photoaged skin, so this aspect, not investigated in this paper, could warrant new research. Otherwise, we published a previous paper12 in photoaged skin that was treated with fat plus PRP, in which we observed an inflammatory reaction and no regenerative effect as the ADSC demonstrated. It is not possible to do quantitative analysis in small fragments of skin tissue. Reproduction of the same morphology and the same phenomena in all patients treated with PRP allows us to justify our statements by commenting on the results obtained. CONCLUSIONS After the treatment with PRP, we observed an increase of reticular dermis thickness due to the deposition of elastic fibers and collagen, with a fibrotic aspect. A modified pattern of adipose tissue was also found at the dermohypodermal junction. Significative regenerative aspects were not found at histologic and ultrastructural analysis in our experimental conditions. The presence of long-term foci of moderate inflammation and microangiopathy could lead to trophic alteration of the skin and a precocious aging process, because an inflammatory process induces fibrosis which is an antithetic condition to the morphological integrity of each tissue and organ. This paper is a part of a series of human face skin studies that we are conducting during recent years. The interest in this approach arises from the fact that previous investigations have been done on animals or human skin of other districts, but not on the face. These extra face regions have been found to be sensitive to stem cells or adipose tissue. It is therefore of the utmost interest to know what changes are induced by PRP injected into the face. The present study provides important information on the biological effect of PRP on this district. The most obvious evidence is that we have not recorded very remarkable phenomena both in its regenerative or degenerative sense. However, the data derived from the ultrastructural examination reveal phenomena that seem to indicate a stimulation of connective fiber deposition together with modest inflammatory aspects. Our approach has obvious limits that arise from the analysis of small samples. In addition, on the one hand, the ultrastructural approach chosen gives an exact definition of the phenomena occurring at the dermal level, yet on the other hand, this results in a substantial impossibility to determine the exact amount of newly formed fibers. However, the comparison of samples from the same patient in the same area at different times on a significant population clearly leads to the ultimate conclusion that PRP injected in the facial skin for regenerative purposes induces modest inflammatory processes. In our view, this aspect has to be further investigated to determine, for example, whether these phenomena are lasting or temporary. And just considering these two possibilities, we must therefore investigate what could happen in both cases. In conclusion, we have verified these phenomena and we report them through a rigorous scientific approach. Disclosures The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding The authors received no financial support for the research, authorship, and publication of this article. Acknowledgments The authors would like to thank the Institute of Biophysics Carlos Chagas Filho, Laboratory of Immunology, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil) for institutional support. REFERENCES 1. Uitto J, Matsuoka LY, Kornberg RL. Elastic fibers in cutaneous elastoses. In: Rudolph R, ed. Problems in Aesthetic Surgery: Biological Causes and Clinical Solutions . St Louis, MO: Mosby; 1986: 307- 338. 2. Scharffetter-Kochanek K, Brenneisen P, Wenk Jet al.   Photoaging of the skin from phenotype to mechanisms. Exp Gerontol . 2000; 35( 3): 307- 316. Google Scholar CrossRef Search ADS PubMed  3. Oikarenen A. The aging of skin: chronoaging versus photoaging. Photodermatol Photoimmunol Photomed . 1990; 7( 1): 3- 4. 4. Fisher GJ, Kang S, Varani Jet al.   Mechanisms of photoaging and chronological skin aging. Arch Dermatol . 2002; 138( 11): 1462- 1470. Google Scholar CrossRef Search ADS PubMed  5. Uitto J. Biochemistry of the elastic fibers in normal connective tissues and its alterations in diseases. J Invest Dermatol . 1979; 72( 1): 1- 10. Google Scholar CrossRef Search ADS PubMed  6. Charles-de-Sá L, Gontijo-de-Amorim NF, Maeda Takiya Cet al.   Antiaging treatment of the facial skin by fat graft and adipose-derived stem cells. Plast Reconstr Surg . 2015; 135( 4): 999- 1009. Google Scholar CrossRef Search ADS PubMed  7. D’Amico RA, Rubin JP, Neumeister MWet al.  ; American Society of Plastic Surgeons/Plastic Surgery Foundation Regenerative Medicine Task Force. A report of the ASPS Task Force on regenerative medicine: opportunities for plastic surgery. Plast Reconstr Surg . 2013; 131( 2): 393- 399. Google Scholar CrossRef Search ADS PubMed  8. Amable PR, Carias RB, Teixeira MVet al.   Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors. Stem Cell Res Ther . 2013; 4( 3): 67. Google Scholar CrossRef Search ADS PubMed  9. Blanton MW, Hadad I, Johnstone BHet al.   Adipose stromal cells and platelet-rich plasma therapies synergistically increase revascularization during wound healing. Plast Reconstr Surg . 2009; 123( 2 Suppl): 56S- 64S. Google Scholar CrossRef Search ADS PubMed  10. Fukaya Y, Kuroda M, Aoyagi Yet al.   Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system. Exp Mol Med . 2012; 44( 5): 330- 339. Google Scholar CrossRef Search ADS PubMed  11. Zhu SJ, Choi BH, Jung JHet al.   A comparative histologic analysis of tissue-engineered bone using platelet-rich plasma and platelet-enriched fibrin glue. Oral Surg Oral Med Oral Pathol Oral Radiol Endod . 2006; 102( 2): 175- 179. Google Scholar CrossRef Search ADS PubMed  12. Rigotti G, Charles-de-Sá L, Gontijo-de-Amorim NFet al.   Expanded stem cells, stromal-vascular fraction, and platelet-rich plasma enriched fat: comparing results of different facial rejuvenation approaches in a clinical trial. Aesthet Surg J . 2016; 36( 3): 261- 270. Google Scholar CrossRef Search ADS PubMed  13. Urmacher C. Histology of normal skin. Am J Surg Pathol . 1990; 14( 7): 671- 686. Google Scholar CrossRef Search ADS PubMed  14. Bosset S, Barré P, Chalon Aet al.   Skin ageing: clinical and histopathologic study of permanent and reducible wrinkles. Eur J Dermatol . 2002; 12( 3): 247- 252. Google Scholar PubMed  15. Naylor EC, Watson RE, Sherratt MJ. Molecular aspects of skin ageing. Maturitas . 2011; 69( 3): 249- 256. Google Scholar CrossRef Search ADS PubMed  16. Bonta M, Daina L, Muţiu G. The process of ageing reflected by histological changes in the skin. Rom J Morphol Embryol . 2013; 54( 3 Suppl): 797- 804. Google Scholar PubMed  17. Mitchell RE. Chronic solar dermatosis: a light and electron microscopic study of the dermis. J Invest Dermatol . 1967; 48( 3): 203- 220. Google Scholar CrossRef Search ADS PubMed  18. Fisher GJ, Kang S, Varani Jet al.   Mechanisms of photoaging and chronological skin aging. Arch Dermatol . 2002; 138( 11): 1462- 1470. Google Scholar CrossRef Search ADS PubMed  19. Kadoya K, Sasaki T, Kostka Get al.   Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis. Br J Dermatol . 2005; 153( 3): 607- 612. Google Scholar CrossRef Search ADS PubMed  20. Cotta-Pereira G, Guerra Rodrigo F, Bittencourt-Sampaio S. Oxytalan, elaunin, and elastic fibers in the human skin. J Invest Dermatol . 1976; 66( 3): 143- 148. Google Scholar CrossRef Search ADS PubMed  21. Gimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy . 2003; 5( 5): 362- 369. Google Scholar CrossRef Search ADS PubMed  22. Cervelli V, Bocchini I, Di Pasquali Cet al.   P.R.L. platelet rich lipotransfert: our experience and current state of art in the combined use of fat and PRP. Biomed Res Int . 2013; 2013: 434191. Google Scholar CrossRef Search ADS PubMed  23. Gentile P, Orlandi A, Scioli MG, Di Pasquali C, Bocchini I, Cervelli V. Concise review: adipose-derived stromal vascular fraction cells and platelet-rich plasma: basic and clinical implications for tissue engineering therapies in regenerative surgery. Stem Cells Transl Med . 2012; 1( 3): 230- 236. Google Scholar CrossRef Search ADS PubMed  24. Tobita M, Tajima S, Mizuno H. Adipose tissue-derived mesenchymal stem cells and platelet-rich plasma: stem cell transplantation methods that enhance stemness. Stem Cell Res Ther . 2015; 6: 215. Google Scholar CrossRef Search ADS PubMed  © 2017 The American Society for Aesthetic Plastic Surgery, Inc. 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Abstract

Abstract Background In previous papers, we demonstrated that the treatment of human photoaged skin with stromal-vascular fraction-enriched fat or expanded adipose-derived stem cells showed a decrease of elastosis and the appearance of new oxytalan elastic fibers in dermis and an increase in the vascular network. The utilization of fat plus platelet-rich plasma (PRP) led to an increase in the vascular permeability and reactivity of the nervous component. Objectives The purpose of this study was to analyze the histologic and ultrastructural changes of human skin after the injection of only PRP in the retroauricular area that was not exposed to sun and did not present the photoaging process, in comparison with our previous results. Methods This study was performed in 13 patients who were candidates for facelift and whose ages ranged between 45 and 65 years. The PRP injection was performed in the mastoidea area. Fragments of skin were removed before and 3 months after treatment and analyzed by optical and electron microscopy. Results After the injection of PRP, we observed an increase of reticular dermis thickness because of the deposition of elastic fibers and collagen, with a fibrotic aspect. A modified pattern of adipose tissue was also found at the dermohypodermal junction. Significative regenerative aspects were not found at histologic and ultrastructural analysis. The presence of foci of moderate inflammation and microangiopathy were observed. Conclusions Treatment with PRP increased reticular dermis thickness with a fibrotic aspect. In the long term, the presence of inflammation and microangiopathy caused by PRP injection could lead to trophic alteration of the skin and the precocious aging process. Level of Evidence: 4 The aging process of the skin is a complex biological phenomenon that can be divided into intrinsic and extrinsic aging. The facial skin aging process is a degenerative process that affects the skin and deep structures of the face, resulting from the action of the intrinsic factor (age) associated with the extrinsic factor, particularly exposure to ultra-violet radiation. Intrinsic aging, which is largely genetically determined, affects the skin in a manner similar to that of most internal organs1 through a slow and partly reversible degeneration of connective tissue. Otherwise, extrinsic aging, more commonly termed photoaging, caused by environmental exposure, primarily ultraviolet radiation, leads to a premature aging phenotype even in young individuals.2 In sun-exposed areas, these two processes, intrinsic and extrinsic, are superimposed; there is evidence3 that they have at least in part overlapping, biological, biochemical, and molecular mechanisms.4 The connective tissue of the skin is composed mostly of collagen and elastin. Collagen makes up 70% to 80% of the dry weight of the skin and gives the dermis its mechanical and structural integrity. Elastin is a minor component of the dermis, but it has an important function in providing the elasticity of the skin, and elastin accounts for 2% to 4% of the extracellular matrix.5 In the photoaged skin, there is a progressive destruction of the entire network of elastin in the dermis. Elastic fibers become thickened, tangled, tortuous, degraded, and dysfunctional, with increased density of the elastic material, resulting in a cluster of amorphous and dystrophic elastotic materials throughout the dermis, setting up solar elastosis, the most important feature of photoaging.5 This is caused by the marked loss of collagen and the thickening of elastic fibers, causing a higher level of the elastic component in the elderly than the young.6 Regenerative medicine is an emerging field whose focus is on cell- and tissue-based therapies applicable to plastic surgery procedures, including the aging process of skin.6 The mechanism transforms undifferentiated stem cells into the differentiated cells that ultimately form the specialized tissues that are required for reconstruction, repair, and restoration, including the role of the surrounding environment.7 Moreover, a stem cell has paracrine effects and secretes a wide range of growth factors, cytokines, and bioactive molecules that can stimulate tissue regeneration.7 In this sense, platelet-rich plasma (PRP) has also been described as having a possible regenerative effect due to its growth factors and cytokines, however empirical, without scientific evidence until now.8-11 In a previous paper,6 we demonstrated that the treatment of human photoaged skin (preauricular area) with the autologous lipidic component plus stromal vascular fraction rich in adipose-derived stem cells (SVF-enriched fat) or expanded adipose-derived stem cells (ADSCs) showed a decrease in the elastic fiber network (elastosis) and the appearance of new oxytalan elastic fibers in papillary dermis. The ultrastructural examination showed a modified 3-dimensional architecture of the reticular dermis and the presence of a richer microvascular bed, representing a skin rejuvenation effect. Considering that expanded stem cells require a cell factor, we developed another study12 in which we wished to assess similar results replacing the ADSC by PRP. PRP is easier to obtain and could be utilized in an ambulatory environment. We aimed to determine if PRP injection could replace the cutaneous regenerative effect of ADSCs when injected together with fat in the preauricular area, with photoaging characteristics. We observed that the use of fat plus PRP led to the presence of more pronounced inflammatory infiltrates and a greater vascular reactivity, increasing in the vascular permeability and a certain reactivity of the nervous component. The addiction of PRP to fat did not improve the regenerative effect and did not present significant advantages over the use of expanded ADSC or SVF-enriched fat in skin rejuvenation.12 The purpose of this new article was to analyze the histologic and ultrastructural changes of skin after the injection of only PRP in the retroauricular area (mastoidea area) that normally suffered only the intrinsic aging process, because it was not exposed to sun and did not present the photoaging process. We observed the histologic alterations due to the intrinsic aging process and observed the action of PRP in this area. METHODS The study was executed in 13 patients, 11 females and 2 males, who were candidates for facial rejuvenation surgery (facelift), whose ages ranged between 45 and 65 years with no history of chronic viral, metabolic, ischemic, or autoimmune diseases, or other systemic pathologies. This study also excluded patients who reported a smoking habit. The study period ranged from May 2012 through May 2016. The patients were subject to the sampling of blood to prepare the PRP, as described below. The injection of PRP was performed in the retroauricular area. Prior to injection, with the patient under local anesthesia, a fragment of skin and subcutaneous tissue was removed from the mastoidea area and sent to the Section of Human Anatomy and Histology of Verona (Italy) and the Federal University of Rio de Janeiro (Brazil) for morphological analysis through optical and electron microscopy. At histology, the analysis of skin biopsies was performed through hematoxylin and eosin (HE) staining, picrosirius red staining (for visualization of collagen), and orcein staining (for visualization of elastic fibers). The morphological examination was repeated after 3 months, taking a piece of skin and subcutaneous tissue in the same area where the PRP was injected. After this second biopsy, the facelift was performed. The regenerative anti-aging activity was evaluated by comparing the morphology of the skin and subcutaneous area before and after treatment. The biopsy specimens, consisting of fragments of skin measuring 0.5 cm × 1 cm, were limited inside of the 1 cm2 retroauricular area (mastoid) and split into two equal parts of 0.5 cm × 0.5 cm. One half was sent in a plastic bottle immersed in 4% formaldehyde to the Institute of Biophysics Carlos Chagas Filho, Laboratory of Immunology, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil) for histologic study. The other half (0.5 cm × 0.5 cm) was sent in a plastic bottle with a solution of buffered 2% glutaraldehyde to the Section of Human Anatomy and Histology of Verona (Verona, Italy) to be subjected to histomorphometric analysis with electron microscopy. The skin biopsies (untreated and treated skin biopsies) were performed under local anesthesia with lidocaine 0.5% and 1:200.000 epinephrine, on an outpatient basis. All patients consented under approved guidelines set forth by the human clinical trial to the Brazilian investigation ethical committee board (protocol no. 28063) and the Brazilian Clinical Trials Registry (Universal Trial Number, U1111-1145-3081). Platelet-Rich Plasma Preparation Peripheral blood was collected in blood collection tubes containing 0.5 ml 3.2% sodium citrate solution. The PRP preparation procedure consisted of two centrifugation steps. All steps were performed in a refrigerated centrifuge (certified Jouan Br4i, Saint-Herblain, Loire-Atlantique, France). Whole blood was centrifuged at 300 g for 5 minutes at 18°C. After the first centrifugation, the whole plasma above the buffy coat was collected, separating platelets from red blood cells and leukocytes (PRP1). PRP1 was centrifuged at 700 g for 17 minutes at 18°C. After the second centrifugation step, the platelet pellet was suspended in 300 μl of platelet-poor plasma (PPP; new fraction named PRP2). Platelet activation was performed adding 20 mM CaCl2 (PRP2-Ca) and 25 IU/ml human plasma thrombin (PRP2-Thr, Ref: T6884; Sigma-Aldrich, St. Louis, Missouri, USA), incubating at 37°C for 1 hour and at 4ºC for 16 hours. To recover the activated PRP2, all of the treated samples were centrifuged at 3,000 × g for 20 minutes at 18°C. All samples were stored at –80°C.8 Different PRP preparations have been published, eliciting variable responses that cannot be compared, due to the lack of the final platelet quantification. This preparation, published by Amable et al,8 optimized PRP preparation and maximized platelet recovery, 46.9% to 69.5% of initial platelets, and the platelets were highly purified. The blood sample was collected in a 5-ml tube containing 3.2% sodium citrate. A blood sample for all 13 patients was stored and then analyzed in the laboratory before injection in the patient. It was injected in intradermal level of the retroauricular skin. Transmission Electron Microscopy (TEM) Different samples were fixed with glutaraldehyde 2% in a Sorensen buffer pH 7.4 for 2 hours, post-fixed in 1% osmium tetroxide in aqueous solution for 2 hours, dehydrated in graded concentrations of acetone, embedded in Epon-Araldite and cut with an Ultracut E ultramicrotome (Reichert, Wien, Austria). At the end of the dehydrating process, samples were positioned in a multi-well grid for electron microscopy and observed through the use of a TEM Morgagni 268D (FEI Philips). Scanning Electron Microscopy (SEM) The specimens were fixed in 2% glutaraldehyde in a phosphate buffer for 2 to 4 hours, post-fixed in 1% osmium tetroxide in the same buffer for 1 hour, and dehydrated in graded concentrations of acetone. The samples were then treated by critical point dryer (CPD 030, Balzers Vaduz, Liechtenstein), mounted on metallic specimen stubs, and sputter-coated with gold (MED 010 Balzers). SEM Imaging was performed with XL30 ESEM (FEI-Philips Eindhoven, Netherlands). RESULTS The present study was executed in 13 patients, 11 females and 2 males, age ranging between 45 and 65 years old (mean, 56.2 years), through the analysis of the retroauricular skin (mastoidea area) that normally is protected from the sun. No elastosis was observed before the use of PRP. Therefore, we can conclude that there is only an intrinsic aging process in this area (Figure 1). Figure 1. View largeDownload slide Light microscopy, orcein stain. The figure shows the structural differences between the preauricular skin (photoaging skin) and the retroauricular skin (protected skin). (A) Preauricular skin with the presence of elastic fibers stained in black (asterisk). (B) The retroauricular skin of the same patient appears poor in elastic fibers (lesser material stained in black). Scale bars: 150 µm. Figure 1. View largeDownload slide Light microscopy, orcein stain. The figure shows the structural differences between the preauricular skin (photoaging skin) and the retroauricular skin (protected skin). (A) Preauricular skin with the presence of elastic fibers stained in black (asterisk). (B) The retroauricular skin of the same patient appears poor in elastic fibers (lesser material stained in black). Scale bars: 150 µm. As described in the introduction, we wrote two previous papers pertaining to preauricular skin. This figure of preauricular skin was utilized in the present paper with the aim of comparing the photoaged skin (preauricular) with the protected skin, from the retroauricular area, of the same patient. Thus, this information is important to readers understand the cutaneous quality of the retroauricular area. The treatment with PRP induced modifications mainly in the reticular dermis. After the use of PRP, a great increase of the thickness of the reticular dermis, which resulted from the horizontal deposition of layers of mature elastic fibers and collagen, was observed (Figure 2). In this layer, activated fibroblasts were also visible. The great increase of elastic and collagen fibers and the morphology of the fibroblasts suggest that in some areas a fibrotic reaction occurs. Figure 2. View largeDownload slide Light microscopy. (A, C) Hematoxylin/eosin stain. (B, D) Orcein stain. The figure shows retroauricular skin before and after PRP injection. (A, B) Before PRP injection, adipocytes are located at the boundary of the dermis with the subcutaneous tissue organized in lobules, which are directed vertically toward the surface of the skin (stars). These lobules form adiposae papillae, which in their apical portion show a relationship with sebaceous or sweat glands. (C, D) After the use of PRP, fibrosis is apparent through the thickening of the reticular dermis (squares). Collagen and orcein-positive elastic fibers (asterisks) are increased. A small flogistic infiltrate is present (arrows). Scale bars: 300 µm. Figure 2. View largeDownload slide Light microscopy. (A, C) Hematoxylin/eosin stain. (B, D) Orcein stain. The figure shows retroauricular skin before and after PRP injection. (A, B) Before PRP injection, adipocytes are located at the boundary of the dermis with the subcutaneous tissue organized in lobules, which are directed vertically toward the surface of the skin (stars). These lobules form adiposae papillae, which in their apical portion show a relationship with sebaceous or sweat glands. (C, D) After the use of PRP, fibrosis is apparent through the thickening of the reticular dermis (squares). Collagen and orcein-positive elastic fibers (asterisks) are increased. A small flogistic infiltrate is present (arrows). Scale bars: 300 µm. At the dermohypodermal junction, before the treatment with PRP, the adipocytes located at the boundary of the dermis and the subcutaneous tissue were organized in lobules, and the lobules were often vertically directed toward the surface of the skin. These lobules formed adiposae papillae, in which a vascular peduncle was often seen. The papillae, in their apical portion, often showed a relationship with sebaceous or sweat glands. At this level, the adipocytes were covered by a dense basket of collagen strictly adherent to their plasma membrane. After the use of PRP, the relationship between subcutaneous fat and sebaceous or sweat glands appears less close, after the reticular dermis increases and forms a kind of barrier between these elements (Figures 2-4). At ultrastructural examination, after treatment with PRP, perivascular areas with mononuclear cell infiltration were visible in the reticular dermis and the dermohypodermal junction (Figure 3). Often the blood vessels (blood capillaries and arterioles) were surrounded by reduplicated and fragmented layers of basal lamina (Figure 5). The ultrastructural examination also confirmed the histologic data, revealing a large amount of elastic fibers and collagen mainly disposed parallel to the skin surface. Collagen fibers were disposed in compact bundles. Ultrastructural examination also revealed the presence of activated fibroblasts with a large amount of rough endoplasmic reticulum and large lysosomes. These findings suggest that PRP causes fibrosis in the area where it is injected (Figure 4 and 5). Figure 3. View largeDownload slide Light microscopy. (A, C) Hematoxylin/eosin stain. (B, D) Orcein stain. The figure shows retroauricular skin before and after PRP injection. In a different patient, the results are similar to those visible in the patient illustrated in Figure 2, indicating the reproducibility of the findings. (A, B) Before PRP injection, adipocytes are located at the boundary of the dermis, with the subcutaneous tissue organized in lobules (stars), which are vertically directed toward the surface of the skin. A small quantity of elastic fibers is present. (C, D) After the use of PRP, fibrosis is apparent through the thickening of the reticular dermis (squares). Collagen and orcein-positive elastic fibers (asterisks) are increased. Scale bars: 300 µm. Figure 3. View largeDownload slide Light microscopy. (A, C) Hematoxylin/eosin stain. (B, D) Orcein stain. The figure shows retroauricular skin before and after PRP injection. In a different patient, the results are similar to those visible in the patient illustrated in Figure 2, indicating the reproducibility of the findings. (A, B) Before PRP injection, adipocytes are located at the boundary of the dermis, with the subcutaneous tissue organized in lobules (stars), which are vertically directed toward the surface of the skin. A small quantity of elastic fibers is present. (C, D) After the use of PRP, fibrosis is apparent through the thickening of the reticular dermis (squares). Collagen and orcein-positive elastic fibers (asterisks) are increased. Scale bars: 300 µm. Figure 4. View largeDownload slide Scanning electron microscopy. (A) Retroauricular skin after treatment with PRP at low (A) enlargement. The dense dermis (square) is visible below the epidermis (e). (B) At medium enlargement, the deposition of elastic and collagen fibers parallel to the skin surface is visible. (C) The fibers are organized in compact bundles (asterisk). Scale bars: A, 500µ; B, 200µ; C, 5 µm. Figure 4. View largeDownload slide Scanning electron microscopy. (A) Retroauricular skin after treatment with PRP at low (A) enlargement. The dense dermis (square) is visible below the epidermis (e). (B) At medium enlargement, the deposition of elastic and collagen fibers parallel to the skin surface is visible. (C) The fibers are organized in compact bundles (asterisk). Scale bars: A, 500µ; B, 200µ; C, 5 µm. Figure 5. View largeDownload slide Transmission electron microscopy. Retroauricular skin after the use of PRP. (A) Duplication of the vascular basal membrane (d). (B) Activated fibroblasts (f) with a rich organular pattern surrounded by thick bundles of collagen fibers (c). Scale bars: A, 2 µm; B, 4 µm. Figure 5. View largeDownload slide Transmission electron microscopy. Retroauricular skin after the use of PRP. (A) Duplication of the vascular basal membrane (d). (B) Activated fibroblasts (f) with a rich organular pattern surrounded by thick bundles of collagen fibers (c). Scale bars: A, 2 µm; B, 4 µm. DISCUSSION The retroauricular skin has its own characteristics. It is a thin and non-hair-bearing skin and has skin-color matching. The retroauricular skin shows a thin stratum corneum, the dermo-epidermal junction is rather flat, and the connective papillae were poorly developed. The dermal papillae are also thin and showed a scarce cellularity. The reticular dermis is homogeneous and appeared as a regular network of collagen and elastin fibers disposed in thin bundles. Scanning microscopy showed the elastin fibers as thin ribbons with smooth surface. The subcutaneous adipose tissue is present in a variable amount and generally formed elongated lobules mainly disposed parallel to the external surface. The pilo-sebaceous structures, as well as the sweat glands, are scarce. In the male patients, a larger amount of sebaceous glands is evident and appeared larger in diameter than those visible in female patients.13,14 During aging, degenerative processes affecting the skin and deep structures of the face commonly occur. This aging process is the result of the action of intrinsic skin events associated with extrinsic agents, especially exposure to ultraviolet radiation. Normally, the retroauricular skin is protected from the sun and does not suffer the extrinsic aging process that leads to elastosis and actinic pathology. The age-related modifications extensively described in the elastic fibers of all organs and tissues may be largely interpreted as resulting from progressive degradation of a protein polymer that has been produced early in life.15-21 In previous papers,6,12 we demonstrated the regenerative effect of SVF-enriched fat and expanded ADSC when injected in preauricular skin with the photoaged process. When the fat plus PRP was injected in the same place, the presence of more pronounced inflammatory infiltrates and a greater vascular reactivity was observed, increasing in vascular permeability and a certain reactivity of the nervous component. The addiction of PRP to fat did not improve the regenerative effect and did not present significant advantages over the use of expanded ADSCs or SVF-enriched fat in skin rejuvenation.12 However, PRP has been described as having a possible regenerative effect due to its growth factors and cytokines, although the previous evidence is empirical.8-11,22-24 Considering the results, we observed that treatment with PRP induced modifications in the reticular dermis, due to the primarily horizontal deposition of layers of collagen and elastic fibers. The great increase of collagen suggests a fibrotic reaction, because there are activated fibroblasts in this layer. This fibrosis creates a kind of barrier between the sebaceous sweat glands and subcutaneous fat tissue. At the dermohypodermal junction, before treatment with PRP, the adipocytes located at the boundary of the dermis and the subcutaneous tissue were organized in lobules, which often were vertically directed toward the surface of the skin. These lobules formed adiposae papillae, in which a vascular peduncle was often seen. The papillae, in their apical portion, often showed a relationship with sebaceous or sweat glands. At this level, the adipocytes were covered by a dense basket of collagen strictly adherent to their plasma membrane. At ultrastructural examination, after treatment with PRP, perivascular areas with mononuclear cell infiltration were visible in the reticular dermis and the dermohypodermal junction. These areas showed blood capillaries and arterioles surrounded by mononuclear infiltrates. Often the blood vessels were surrounded by reduplicated and fragmented layers of basal lamina. Elastic fibers and collagen are horizontal and strongly attached, and the presence of lysosomes and activated fibroblasts suggests that PRP causes fibrosis in the area where it is injected. The limitation of this study is that PRP injections currently occur into photoaged skin, so this aspect, not investigated in this paper, could warrant new research. Otherwise, we published a previous paper12 in photoaged skin that was treated with fat plus PRP, in which we observed an inflammatory reaction and no regenerative effect as the ADSC demonstrated. It is not possible to do quantitative analysis in small fragments of skin tissue. Reproduction of the same morphology and the same phenomena in all patients treated with PRP allows us to justify our statements by commenting on the results obtained. CONCLUSIONS After the treatment with PRP, we observed an increase of reticular dermis thickness due to the deposition of elastic fibers and collagen, with a fibrotic aspect. A modified pattern of adipose tissue was also found at the dermohypodermal junction. Significative regenerative aspects were not found at histologic and ultrastructural analysis in our experimental conditions. The presence of long-term foci of moderate inflammation and microangiopathy could lead to trophic alteration of the skin and a precocious aging process, because an inflammatory process induces fibrosis which is an antithetic condition to the morphological integrity of each tissue and organ. This paper is a part of a series of human face skin studies that we are conducting during recent years. The interest in this approach arises from the fact that previous investigations have been done on animals or human skin of other districts, but not on the face. These extra face regions have been found to be sensitive to stem cells or adipose tissue. It is therefore of the utmost interest to know what changes are induced by PRP injected into the face. The present study provides important information on the biological effect of PRP on this district. The most obvious evidence is that we have not recorded very remarkable phenomena both in its regenerative or degenerative sense. However, the data derived from the ultrastructural examination reveal phenomena that seem to indicate a stimulation of connective fiber deposition together with modest inflammatory aspects. Our approach has obvious limits that arise from the analysis of small samples. In addition, on the one hand, the ultrastructural approach chosen gives an exact definition of the phenomena occurring at the dermal level, yet on the other hand, this results in a substantial impossibility to determine the exact amount of newly formed fibers. However, the comparison of samples from the same patient in the same area at different times on a significant population clearly leads to the ultimate conclusion that PRP injected in the facial skin for regenerative purposes induces modest inflammatory processes. In our view, this aspect has to be further investigated to determine, for example, whether these phenomena are lasting or temporary. And just considering these two possibilities, we must therefore investigate what could happen in both cases. In conclusion, we have verified these phenomena and we report them through a rigorous scientific approach. Disclosures The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding The authors received no financial support for the research, authorship, and publication of this article. Acknowledgments The authors would like to thank the Institute of Biophysics Carlos Chagas Filho, Laboratory of Immunology, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil) for institutional support. REFERENCES 1. Uitto J, Matsuoka LY, Kornberg RL. Elastic fibers in cutaneous elastoses. In: Rudolph R, ed. Problems in Aesthetic Surgery: Biological Causes and Clinical Solutions . St Louis, MO: Mosby; 1986: 307- 338. 2. Scharffetter-Kochanek K, Brenneisen P, Wenk Jet al.   Photoaging of the skin from phenotype to mechanisms. Exp Gerontol . 2000; 35( 3): 307- 316. Google Scholar CrossRef Search ADS PubMed  3. Oikarenen A. The aging of skin: chronoaging versus photoaging. Photodermatol Photoimmunol Photomed . 1990; 7( 1): 3- 4. 4. Fisher GJ, Kang S, Varani Jet al.   Mechanisms of photoaging and chronological skin aging. Arch Dermatol . 2002; 138( 11): 1462- 1470. Google Scholar CrossRef Search ADS PubMed  5. Uitto J. Biochemistry of the elastic fibers in normal connective tissues and its alterations in diseases. J Invest Dermatol . 1979; 72( 1): 1- 10. Google Scholar CrossRef Search ADS PubMed  6. Charles-de-Sá L, Gontijo-de-Amorim NF, Maeda Takiya Cet al.   Antiaging treatment of the facial skin by fat graft and adipose-derived stem cells. Plast Reconstr Surg . 2015; 135( 4): 999- 1009. Google Scholar CrossRef Search ADS PubMed  7. D’Amico RA, Rubin JP, Neumeister MWet al.  ; American Society of Plastic Surgeons/Plastic Surgery Foundation Regenerative Medicine Task Force. A report of the ASPS Task Force on regenerative medicine: opportunities for plastic surgery. Plast Reconstr Surg . 2013; 131( 2): 393- 399. Google Scholar CrossRef Search ADS PubMed  8. Amable PR, Carias RB, Teixeira MVet al.   Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors. Stem Cell Res Ther . 2013; 4( 3): 67. Google Scholar CrossRef Search ADS PubMed  9. Blanton MW, Hadad I, Johnstone BHet al.   Adipose stromal cells and platelet-rich plasma therapies synergistically increase revascularization during wound healing. Plast Reconstr Surg . 2009; 123( 2 Suppl): 56S- 64S. Google Scholar CrossRef Search ADS PubMed  10. Fukaya Y, Kuroda M, Aoyagi Yet al.   Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system. Exp Mol Med . 2012; 44( 5): 330- 339. Google Scholar CrossRef Search ADS PubMed  11. Zhu SJ, Choi BH, Jung JHet al.   A comparative histologic analysis of tissue-engineered bone using platelet-rich plasma and platelet-enriched fibrin glue. Oral Surg Oral Med Oral Pathol Oral Radiol Endod . 2006; 102( 2): 175- 179. Google Scholar CrossRef Search ADS PubMed  12. Rigotti G, Charles-de-Sá L, Gontijo-de-Amorim NFet al.   Expanded stem cells, stromal-vascular fraction, and platelet-rich plasma enriched fat: comparing results of different facial rejuvenation approaches in a clinical trial. Aesthet Surg J . 2016; 36( 3): 261- 270. Google Scholar CrossRef Search ADS PubMed  13. Urmacher C. Histology of normal skin. Am J Surg Pathol . 1990; 14( 7): 671- 686. Google Scholar CrossRef Search ADS PubMed  14. Bosset S, Barré P, Chalon Aet al.   Skin ageing: clinical and histopathologic study of permanent and reducible wrinkles. Eur J Dermatol . 2002; 12( 3): 247- 252. Google Scholar PubMed  15. Naylor EC, Watson RE, Sherratt MJ. Molecular aspects of skin ageing. Maturitas . 2011; 69( 3): 249- 256. Google Scholar CrossRef Search ADS PubMed  16. Bonta M, Daina L, Muţiu G. The process of ageing reflected by histological changes in the skin. Rom J Morphol Embryol . 2013; 54( 3 Suppl): 797- 804. Google Scholar PubMed  17. Mitchell RE. Chronic solar dermatosis: a light and electron microscopic study of the dermis. J Invest Dermatol . 1967; 48( 3): 203- 220. Google Scholar CrossRef Search ADS PubMed  18. Fisher GJ, Kang S, Varani Jet al.   Mechanisms of photoaging and chronological skin aging. Arch Dermatol . 2002; 138( 11): 1462- 1470. Google Scholar CrossRef Search ADS PubMed  19. Kadoya K, Sasaki T, Kostka Get al.   Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis. Br J Dermatol . 2005; 153( 3): 607- 612. Google Scholar CrossRef Search ADS PubMed  20. Cotta-Pereira G, Guerra Rodrigo F, Bittencourt-Sampaio S. Oxytalan, elaunin, and elastic fibers in the human skin. J Invest Dermatol . 1976; 66( 3): 143- 148. Google Scholar CrossRef Search ADS PubMed  21. Gimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy . 2003; 5( 5): 362- 369. Google Scholar CrossRef Search ADS PubMed  22. Cervelli V, Bocchini I, Di Pasquali Cet al.   P.R.L. platelet rich lipotransfert: our experience and current state of art in the combined use of fat and PRP. Biomed Res Int . 2013; 2013: 434191. Google Scholar CrossRef Search ADS PubMed  23. Gentile P, Orlandi A, Scioli MG, Di Pasquali C, Bocchini I, Cervelli V. Concise review: adipose-derived stromal vascular fraction cells and platelet-rich plasma: basic and clinical implications for tissue engineering therapies in regenerative surgery. Stem Cells Transl Med . 2012; 1( 3): 230- 236. Google Scholar CrossRef Search ADS PubMed  24. Tobita M, Tajima S, Mizuno H. Adipose tissue-derived mesenchymal stem cells and platelet-rich plasma: stem cell transplantation methods that enhance stemness. Stem Cell Res Ther . 2015; 6: 215. Google Scholar CrossRef Search ADS PubMed  © 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com

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Aesthetic Surgery JournalOxford University Press

Published: Mar 1, 2018

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