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}{% \vskip8pt \textcolor{GJMediumGrey}{\rule{\textwidth}{2pt}} \vskip16pt } \usepackage[absolute,overlay]{textpos} \makeatother \usepackage{lineno} \linenumbers \begin{document} \author[1]{Araco Antonino, M.D.} \renewcommand\Authands{ and } \date{\small \em Received: 6 December 2019 Accepted: 3 January 2020 Published: 15 January 2020} \maketitle \begin{abstract} Introduction-Injectable fillers are used in aesthetic medicine to reduce visible signs of facial aging.It involves loss of volume in the skin, muscle and superficial and deep fat compartments, superficial wrinkles and deep folds (1-2).There are different products that vary on filling capacity, longevity, potentiality for causing allergic reaction, safety, indication.Physicians select the most suitable agent for each patient by considering the advantage and disadvantage.In fact, hyaluronic acid (HA) has the property to increase the skin's water-binding capacity (3-5).Calcium hydroxylapatite (6) and poly-L-lactic acid (7) have been proved to stimulate autologous collagen (8-9). \end{abstract} \keywords{Growth factors, PRP, regenerative medicine, face wrinkles, perioral wrinkles, dermal matrix.} \begin{textblock*}{18cm}(1cm,1cm) % {block width} (coords) \textcolor{GJBlue}{\LARGE Global Journals \LaTeX\ JournalKaleidoscope\texttrademark} \end{textblock*} \begin{textblock*}{18cm}(1.4cm,1.5cm) % {block width} (coords) \textcolor{GJBlue}{\footnotesize \\ Artificial Intelligence formulated this projection for compatibility purposes from the original article published at Global Journals. However, this technology is currently in beta. \emph{Therefore, kindly ignore odd layouts, missed formulae, text, tables, or figures.}} \end{textblock*} \let\tabcellsep& \section[{Introduction}]{Introduction}\par njectable fillers are used in aesthetic medicine to reduce visible signs of facial aging.\par It involves loss of volume in the skin, muscle and superficial and deep fat compartments, superficial wrinkles and deep folds \hyperref[b0]{(1)}\hyperref[b1]{(2)}.\par There are different products that vary on filling capacity, longevity, potentiality for causing allergic reaction, safety, indication.\par Physicians select the most suitable agent for each patient by considering the advantage and disadvantage.\par In fact, hyaluronic acid (HA) has the property to increase the skin's water-binding capacity \hyperref[b2]{(3)}\hyperref[b3]{(4)}\hyperref[b4]{(5)}.\par Calcium hydroxylapatite \hyperref[b5]{(6)} and poly-L-lactic acid \hyperref[b6]{(7)} have been proved to stimulate autologous collagen \hyperref[b7]{(8)}\hyperref[b8]{(9)}.\par Although fillers have lower immunogenicity and a good safety profile, several scientific articles have showed side effects \hyperref[b9]{(10)}.\par Those can vary from allergic reaction \hyperref[b10]{(11)}, inflammatory nodule formation \hyperref[b12]{(12)}, infection \hyperref[b13]{(13)}, vision loss \hyperref[b14]{(14)} and the limited use in human immunodeficiency \hyperref[b15]{(15)}.\par Platelet rich plasma (Prp) has proved to achieve regenerative capacity on human tissues \hyperref[b16]{(16)} and positive effect on facial dermal fibroblasts \hyperref[b17]{(17)}\hyperref[b18]{(18)}.\par Autologous plasma filler was first introduced by Krajcik et al. in 1999 \hyperref[b19]{(19)} and used for the treatment of different types and grades of facial wrinkles \hyperref[b20]{(20)}\hyperref[b21]{(21)} {\ref (22)}.\par The main issues of the clinical use of plasma filler is its poor filling effect and duration.\par In fact, the volume achieved just after the injection lasts only few hours because the plasma is reabsorbed.\par The goal of our study was the preparation of a new medical filler device able to fill soft tissues for a reasonable period of time and also containing the regenerative Prp property.\par Our first step was the formulation of a thermosensitive gelableto embed Prp, the second to evaluate the behavior of the formulation alone, in particular, its dispersibility and the homogeneity and the third the study of platelets and the growth factors behavior inside the formulation.\par After a detailed research on the scientific literature \hyperref[b22]{(23,}\hyperref[b23]{24)} poloxamer 407 was chosen for our purpose.\par Multiple reasons have corroborated this choice: first of all, the family of "poloxamer" polymers is listed in the US and European Pharmacopoeia \hyperref[b25]{(25)} and is approved by FDA for parenteral use in humans and this ensure safety, biocompatibility and tolerability required for parenchymal use.\par Furthermore, poloxamer 407, being a non-ionic block copolymer, does not negatively interact with the biomolecules embed in the gel, such as platelets and growth factors.\par Again, it allows to develop thermosensitive formulations without the need of excipients in preparations. \section[{II.}]{II.} \section[{Materials and Methods}]{Materials and Methods} \section[{Preparation of poloxamer formulation}]{Preparation of poloxamer formulation}\par Aqueous solutions of poloxamer 407, purchased from Sigma-Aldrich (Milano, Italy), were prepared by the so-called "cold method".\par Poloxamer powder was dissolved in bidistillate water at 277.15 K (4°C) under gentle stirring to facilitate the copolymer dissolution \hyperref[b26]{(26)}.\par Solutions at poloxamer concentration of 15\% (w/v) were prepared with this method and stored at 277.15 K (4 °C).\par Differential scanning calorimetry (DSC) was used to determine the micellization and the gelation point.\par Measurements were performed using a calorimeter Mettler 821 e (Mettler-Toledo, Greifensee, Switzerland) equipped with a cooling system with liquid nitrogen. Scans were recorded from 273.15 (0 °C) to 313.15 K (40 °C) at a heating rate of 5 K/min \hyperref[b27]{(27)}.\par Measurements were performed in duplicate and the results were expressed as the mean of the two measurements. \section[{Analysis of the Platelet}]{Analysis of the Platelet}\par The analysis of the platelet contained inside the formulation could not be address by normal hemochrome automatic analysis systems.\par For this reason, platelet numbers were firstly estimated by using a Burker's chamber and the Breker -Cronkite method.\par Observations were performed with a Nikon Eclipse 80i microscope provided of a digital camera Nikon Coolpix 8400.\par Breker -Cronkite solution was prepared adding 0,01 g of brilliant cresyl blue to 100 mL of 1\% (w/v) ammonium oxalate aqueous solution.\par Medical Device with Prp sample was diluted with Breker -Cronkite solution (dilution 1/100) in a Thoma pipette.\par After five minutes of pipette manual shaking, a drop of diluted solution was charged in the Burker's chamber and observed at microscope with magnification 40X.\par We counted platelets presents in a big square of 1 mm 2 (Figure \hyperref[fig_3]{1}).\par To obtain the number of platelets within 1 ?L of PRP, the average of platelets counts in three squares was corrected for the initial dilution (100) and for the height of chamber \hyperref[b9]{(10)} \hyperref[b28]{(28)}.\par For this reason, the final formula is: Platelet number counted X 1000.\par Prp platelet size and number were also determined using an AccusizerTM 770 Optical Particle Sizer (PSS Inc., Santa Barbara, CA, USA), using the technology "single particle optical counting", coupled with an auto-dilution system AccusizerTM 770A Autodiluter PAT (PSS Inc., Santa Barbara, CA, USA).\par 100 ?L of solution with Prp were injected in the system and measurements were performed for 30 minutes to allow the analysis of all the particles present in the sample. Saline (0.9\% NaCl) was used for the analysis and results were expressed as total particles size and mean volume diameter (Figure \hyperref[fig_4]{2}). \section[{HPLC analysis of growth factors}]{HPLC analysis of growth factors}\par We analyzed the Epidermal Growth Factor (EGF) which is centrally involved in the regulation of key processes of the epithelia.\par Calibration curve for RP-UHPLC analysis was obtained using an HPLC Jasco LC-2000 plus, equipped with the quaternary gradient pump PU 2089 Plus, the UV-vis detector diode array MD 2010 plus and injector Rheodyne.\par Controlled EGF was purchased from Sigma-Aldrich (Milano, Italy).\par The column used was a C18 Ascentis Express Peptide 150 × 4.6 mm (L × I.D.), 2.7 ?m and analysis were made combining two different mobile phases: The mobile phases were degassed prior to use; cell and column temperature at 313.15 K (40 °C).\par The calibration curve was performed in the concentration range of 0.086-0.4 mg/mL, with an injection volume of 5 ?L. \section[{Rheological analysis}]{Rheological analysis}\par Poloxamer rheological characterization was performed with the aim of better understanding the thermal behavior of poloxamer formulation.\par A Stresstech HR Rheometer (Rheologia Instruments AB, Milano, Italy), equipped with a Peltier device for temperature control, was used for the rheological characterization.\par Viscosity was measured with a cone-plate geometry (cone angle 1°, cone-plate diameter 40 mm).\par Poloxamer formulation was analyzed in duplicate at different temperature (278, 283, 288, 293, 298, 303 and 308 K corresponding to 5, 10, 15, 20, 25, 30 and 35 °C).\par For formulation in sol state, measurements were performed applying a stress range from 3.044?10 -3 to 1 Pa, while for temperatures at which the formulations were already at the gel state, measurements were carried out applying a stress range from 3.044?10 -3 to 10 Pa. 12. \section[{Spreadability}]{Spreadability}\par The gel was tested by two independent persons for the spreadability.\par The gels stored at 277.15 K (4 °C) were withdraw from the recipients and spread on the skin surface. \section[{Preparation of Prp}]{Preparation of Prp}\par For the preparation of the Prp, Plasma Active system (CE0373 -Medical Device s.r.l. via artigianato n.6, 52022 Meleto, Craviglia (AR) -Italy) was used.\par It is composed of two vacutainer tubes (BD Vacutainer® Brand SST II) of 9 mm each with separator gel and anti-coagulant for harvesting 18 ml of peripheral blood.\par Tubes were centrifugated for 5 minutes at 1800 rpm (Sorvall Legend XTR Centrifuge -Thermo Fisher Scientific Robert-Bosch-Straße 1 D -63505 Langenselbold Germany) and 8 ml of Prp were collected from the upper part of the tube in a sterile matter and placed inside the Medical Device. \section[{Preparation of poloxamer containing PRP}]{Preparation of poloxamer containing PRP}\par Five mL of Prp were added to 10 mL of thermosensitive gel. \section[{K}]{K}\par The ratio of Prp/gel was chosen by considering the future application of the thermosensitive gel and the size of the final device \hyperref[b30]{(30)}.\par Results have shown that 5 mL of Prp could be easily dispersed within 10 mL of the hydrogel by stirring the cold gel (4 °C) or by agitation of the closed container.\par III. \section[{Result}]{Result} \section[{Differential scanning calorimetry (DSC) results}]{Differential scanning calorimetry (DSC) results}\par DSC data show broad enthalpic transitions in the formulation due to poloxamer micellization.\par In fact, in aqueous solution, with increasing temperature, poloxamer aggregates in micelles to minimize the free energy of solution.\par Micellization temperatures decrease with increasing poloxamer concentration (Figure {\ref 3}). Also, micellization energy transition (?H mic ) per amount of poloxamer increases with increasing concentration.\par Data of micellization temperature and ?H mic of poloxamer preparation is reported (Table \hyperref[tab_0]{1}).\par Very interesting, in DSC thermograms is possible to see also a little enthalpic transition at temperature slightly higher than micellization point.\par This peak is more evident in solutions with higher poloxamer concentrations and it has been previously attributed to the gelation transition (Figure {\ref 4}).\par In agreement with this little peak, gelation energy (?H gel ) is very small (Table \hyperref[tab_1]{2}).\par Gel temperatures found from this approach agree with results reported in the literature \hyperref[b27]{(27)}.\par This DSC analysis give a first information about poloxamer solution thermal behavior.\par In fact, the decrease of micellization and gelation temperature with increasing poloxamer concentration clearly shows how formulations with high poloxamer concentration (e.g. 25-30\% w/v) pass to gel state at lower temperatures compared to the formulations with a low poloxamer concentration.\par Micellization and gelation temperatures are plotted in Figure \hyperref[fig_6]{5}.\par These results demonstrate that gelation point is strongly temperature and concentration dependent. \section[{Rheological analysis result}]{Rheological analysis result}\par In Figure {\ref 6} is shown the behavior of poloxamer formulation viscosities as a function of temperature.\par In the first part of curves, solution viscosity decreases slightly on warming but, reached a certain temperature, is possible to see a steep increase in viscosity.\par This viscosity increase can be attributed to the sol-to-gel transition.\par Very interesting, results obtained with rheological characterization agree with gel temperatures obtained from the DSC data (Figure {\ref 3}). \section[{Spreadability results}]{Spreadability results}\par The best spreading was individuated for the formulation containing 15\% of poloxamer that did not gelify immediately after contact with the skin.\par However, once the gel was spread it remained on surface as a thick gel. \section[{Result of poloxamer containing PRP}]{Result of poloxamer containing PRP}\par Prp dispersed homogeneously into the matrix without forming lipid globules but changing the appearance of the gel from completely transparent to a light-yellow color (Figure \hyperref[fig_7]{7A}).\par The formulation pH did not change with respect to the control formulation (blank gel).\par The smell of the formulation containing PRP was slightly different from the blank gel, but it was still pleasant.\par Prp loaded thermosensitive gel was stored at 4 °C for 1 month and its characteristics were evaluated 7 and 30 days after preparation.\par After 7 days of storage, the gel color and smell were unchanged and, more important, Prp was still homogeneously dispersed (Figure \hyperref[fig_7]{7B}).\par After 1 month of storage, the smell was unchanged while a slight phase separation was observed (Figure \hyperref[fig_7]{7C}).\par This phase separation was reversible by simpler agitation.\par In fact, by gently shaking the closed container, the gel assumed the same aspect and homogeneity of a fresh prepared formulation (Figure \hyperref[fig_7]{7D}). This finding is of particular interest for the final use and we store the formulation at: 2-8 °C andshake if before use. \section[{Platelet behaviour}]{Platelet behaviour}\par We analyzed 10 different samples of solution of poloxamer containing PRP after 12, 23, 73, 96, 120, 144, 168 hours from the preparation and expressed the results as mean.\par Platelet remained intact for 72 hours and then numbers dropped rapidly as shown on Figure {\ref 8}. \section[{HPLC results of growth factors}]{HPLC results of growth factors}\par Analysis made on the 10 different samples of solution of poloxamer containing Prp after 12, 23, 73, 96, 120, 144, 168 hours from the preparation showed that the EGF concentration increased progressively and reached his peak after 4 days and then decreased progressively.\par The Egf concentration remained above 2,5 µg/mL after 7 days (Figure {\ref 9}). \section[{IV.}]{IV.} \section[{Discussion}]{Discussion}\par Fillers are among the most performed cosmetic medicine procedures worldwide \hyperref[b31]{(31)}\hyperref[b32]{(32)}.\par They are used mostly on the face for the reduction of superficial and deep wrinkles \hyperref[b32]{(32)}\hyperref[b33]{(33)}\hyperref[b34]{(34)}, for increasing the volumes of lips \hyperref[b35]{(35)} and cheekbones \hyperref[b36]{(36)} and for the redefinition of the mandibular profile \hyperref[b37]{(37)}.\par There are different materials used for fillers including hyaluronic acid \hyperref[b38]{(38)}, collagen \hyperref[b39]{(39)}, calcium hydroxyapatite \hyperref[b40]{(40)}\hyperref[b41]{(41)}, and polycaprolactone \hyperref[b42]{(42)}, polymethylmethacrylate \hyperref[b43]{(43)}.\par They vary depending on the filling capacity, due to their ability of recalling water \hyperref[b44]{(44)}, or even for the ability to stimulate collagen \hyperref[b45]{(45)}.\par The advantages of commercial fillers are the simplicity of use, as they are supplied in single-use vials and the very low incidence of major complications as embolisms \hyperref[b46]{(46)}\hyperref[b47]{(47)}\hyperref[b48]{(48)}.\par The disadvantages are the high cost, since to obtain appreciable results, several vials are required, and because of the limited duration, the treatment must be repeated periodically \hyperref[b49]{(49)}\hyperref[b50]{(50)}.\par Again, possible side effects, although minor, create discomfort to the patients and stress to the doctors \hyperref[b51]{(51)}.\par And definitely, fillers do not have regenerative activities.\par Prp is used in dermatological clinic for its regeneration effects on dermal cells \hyperref[b17]{(17)}.\par Different studies have already shown the positive effects on the treatment of facial wrinkles.\par "Plasma filler" has always meant the simple injection of Prp into subcutaneous tissues \hyperref[b18]{(18,}\hyperref[b20]{20,}\hyperref[b21]{21)}.\par But the main limitation of plasma fillers is the fact that it does not generate filling effects, if not for a few hours.\par For this reason, we set up a new filler device able to combine volumetric and regenerative effects.\par We faced different problems: first of all, the Prp is a fluid and to be well mixed inside the new filler device it is necessary that this is also in the liquid state.\par Moreover, once injected into the tissues, this liquid would have to become a gel to guarantee the volumetric effect.\par Furthermore, since both the platelets and the Prp have a low half-life outside the plasma, we had to guarantee the new filler device a chemical and physical composition that allows the survival of platelet and growth factors for several days. So, we have chosen a polymer (poloxamer 407) that contains all these characteristics \hyperref[b22]{(23)}\hyperref[b23]{(24)}.\par In fact, at low temperatures (2-8 C °) it is found in the liquid phase and this allows the simple mixing of the Prp inside it.\par When the temperature reaches 32-37 ° C, the liquid passes to the gel state.\par This ensures either the volumizing effect and the progressive release of growth factors within the tissues.\par EGF is known to be a potent stimulator of cell proliferation of various cells including keratinocytes, fibroblasts and vascular endothelial cells.\par EGF stimulates the migration of keratinocytes and also stimulates fibroblasts and endothelial cells to promote the formation of granulation tissue \hyperref[b52]{(52)}\hyperref[b53]{(53)} {\ref (54)}.\par These measurements were also difficult because platelets and the EGF could not be measured with the common automatic measurement systems.\par The results found were encouraging for a clinical point.\par In fact, the platelets were maintained vitality until 72 hours and then their number felled by freeing the granules containing the growth factors.\par EGF, progressively increased its concentration with a peak after 72-96 hours and then slowly decreased until 7 days.\par This new filler device could open new scenarios in facial rejuvenation and revolutionize the treatment of face wrinkles.\par In fact, the advantages are evident: it would have volumizing and regenerative capacities.\par This would allow wrinkles and grooves to be filled and regenerated at each application.\par Furthermore, being autologous growth factors, there would be no side effects related to allergic reactions or inflammatory nodules.\par Again, since the immune system is also present inside the plasma, this would help prevent the infections.\par Finally, the cost of treatment would be cheaper than normal commercial filler, since for each session of treatment 8 ml of Prp are added to 16 ml of hydrogel to obtain a 24 ml filler. The disadvantage lies on the Prp preparation.\par V. \section[{Conclusions}]{Conclusions}\par Our study has shown that it is possible to obtain a new type of filler able to have both filling capacity, due to the gelling effect of the material used, and a regenerative effect due to the presence of Prp.\par Therefore, new clinical trials will be necessary to assess the duration of the volumizing effect in the different areas of the face. \begin{figure}[htbp] \noindent\textbf{}\includegraphics[]{image-2.png} \caption{\label{fig_0}}\end{figure} \begin{figure}[htbp] \noindent\textbf{}\includegraphics[]{image-3.png} \caption{\label{fig_2}}\end{figure} \begin{figure}[htbp] \noindent\textbf{1}\includegraphics[]{image-4.png} \caption{\label{fig_3}Figure 1 :A}\end{figure} \begin{figure}[htbp] \noindent\textbf{2}\includegraphics[]{image-5.png} \caption{\label{fig_4}Figure 2 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{3264}\includegraphics[]{image-6.png} \caption{\label{fig_5}Figure 3 : 26 AFigure 4 :A}\end{figure} \begin{figure}[htbp] \noindent\textbf{5}\includegraphics[]{image-7.png} \caption{\label{fig_6}Figure 5 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{7}\includegraphics[]{image-8.png} \caption{\label{fig_7}Figure 7 :A}\end{figure} \begin{figure}[htbp] \noindent\textbf{89}\includegraphics[]{image-9.png} \caption{\label{fig_8}Figure 8 :Figure 9 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{1} \par \begin{longtable}{} \end{longtable} \par \caption{\label{tab_0}Table 1 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{2} \par \begin{longtable}{} \end{longtable} \par \caption{\label{tab_1}Table 2 :}\end{figure} \footnote{© 2020 Global Journals} \footnote{A New Medical Device for Platelet Rich Plasma Filler} \backmatter \begin{bibitemlist}{1} \bibitem[Pasquanelli et al. ()]{b28}\label{b28} \textit{}, F Pasquanelli , Rosini Diagnostica E Tecniche Di Laboratorio , Editrice . 1981. \bibitem[Humbert et al. (2012)]{b1}\label{b1} \textit{}, P Humbert , C Viennet , K Legagneux , F Grandmottet , S Robin , T Oddos , P Muret . \textit{J Cosmet Dermatol} 2012 Mar. 11 (1) p. . (In the shadow of the wrinkle: theories) \bibitem[ Am J Clin Dermatol (2013)]{b13}\label{b13} \textit{}, \textit{Am J Clin Dermatol} 2013 Oct. 14 (5) p. . \bibitem[ Review (2014)]{b11}\label{b11} \textit{}, \xref{http://dx.doi.org/10.1055/s-0034-1396904}{10.1055/s-0034-1396904}. \textit{Review} 2014 Dec 23. 30 (6) p. . \bibitem[Choi et al. (2017)]{b21}\label{b21} ‘A clinical study on the usefulness of autologous plasma filler in the treatment of nasolabial fold wrinkles’. Yj1 Choi , Hs1 Kim , Jh1 Min , Jh1 Nam , Gy1 Lee , Ws1 Kim . \xref{http://dx.doi.org/10.1080/14764172.2016.1248443}{10.1080/14764172.2016.1248443}. \textit{J Cosmet Laser Ther} 2017 Jun. 2017 Feb 21. 19 (3) p. . \bibitem[Krajcik et al. ()]{b20}\label{b20} ‘A novel injectable autologous material for soft tissue augmentation’. R Krajcik , D S Orentreich , N Orentreich . \textit{J Aesthetic Dermatol and Cosmet Surg} 1999. 1 p. . \bibitem[Galadari et al. (2015)]{b42}\label{b42} ‘A randomized, prospective, blinded, splitface, single-center study comparing polycaprolactone to hyaluronic acid for treatment of nasolabial folds’. H Galadari , D Van Abel , Al Nuami , K , Al Faresi , F Galadari , I . \textit{J Cosmet Dermatol} 2015 Mar. 14 (1) p. . \bibitem[Yutskovskaya et al. (2014)]{b40}\label{b40} ‘A randomized, split-face, histomorphologic study comparing a volumetric calcium hydroxylapatite and a hyaluronic acid-based dermal filler’. Y Yutskovskaya , E Kogan , E Leshunov . \textit{J Drugs Dermatol} 2014 Sep. 13 (9) p. . \bibitem[Dumortier ()]{b24}\label{b24} ‘A review of Poloxamer 407 pharmaceutical and pharmacological characteristics’. G Dumortier . \textit{Pharm. Res} 2006. 23 p. . \bibitem[Emer and Sundaram (2013)]{b41}\label{b41} ‘Aesthetic applications of calcium hydroxylapatite volumizing filler: an evidence-based review and discussion of current concepts: (part 1 of 2)’. J Emer , H Sundaram . \textit{J DrugsDermatol} 2013 Dec. 12 (12) p. . (Review) \bibitem[Nkengne and Bertin (2013)]{b0}\label{b0} ‘Aging and facial changes--documenting clinical signs, part 1: clinical changes of the aging face’. A Nkengne , C Bertin . \textit{Skinmed} 2013 Sep-Oct. 11 (5) p. . \bibitem[Lynch and Bashir ()]{b22}\label{b22} ‘Applications of platelet-rich plasma in dermatology: A critical appraisal of the literature’. M D Lynch , S Bashir . \textit{J Dermatolog Treat} 2016. 27 (3) p. . \bibitem[Schmolka ()]{b26}\label{b26} ‘Artificial skin. I. Preparation and properties of pluronic F-127 gels for treatment of burns’. I R Schmolka . \textit{J. Biomed. Mater. Res} 1972. 6 p. . \bibitem[Shuck et al. (2013)]{b16}\label{b16} \textit{Autologous fat grafting and injectable dermal fillers for human immunodeficiency virus-associated facial lipodystrophy: a comparison of safety, efficacy, and long-term treatment outcomes. PlastReconstr Surg}, J Shuck , M L Iorio , R Hung , S P Davison . 2013 Mar. 131 p. . \bibitem[Cameli et al. (2017)]{b19}\label{b19} ‘Autologous Pure Platelet-Rich Plasma Dermal Injections for Facial Skin Rejuvenation: Clinical, Instrumental, and Flow Cytometry Assessment’. N Cameli , M Mariano , I Cordone , E Abril , S Masi , M L Foddai . \textit{Dermatol Surg} 2017 Jun. 43 (6) p. . \bibitem[Pierre et al. (2015)]{b31}\label{b31} ‘Basics of dermal filler rheology’. S Pierre , S Liew , A Bernardin . \textit{Dermatol Surg} 2015 Apr. 41 (1) p. . (Suppl) \bibitem[Ho and Jagdeo (2015)]{b38}\label{b38} ‘Biological properties of a new volumizing hyaluronic acid filler: a systematic review’. D Ho , J Jagdeo . \textit{J Drugs Dermatol} 2015 Jan. 14 (1) p. . (Review) \bibitem[Casabona (2015)]{b46}\label{b46} ‘Blood Aspiration Test for Cosmetic Fillers to Prevent Accidental Intravascular Injection in the Face’. G Casabona . \textit{Dermatol Surg} 2015 Jul. 41 (7) p. . \bibitem[Pavicict (2013)]{b5}\label{b5} ‘Calcium hydroxylapatite filler: an overview of safety and tolerability’. Pavicict . \textit{J Drugs Dermatol} 2013 Sep. 12 (9) p. . (Review) \bibitem[Kim et al. (2011)]{b18}\label{b18} ‘Can Platelet-rich Plasma Be Used for Skin Rejuvenation? Evaluation of Effects of Platelet-rich Plasma on’. D H Kim , Y J Je , C D Kim , Y H Lee , Y J Seo , J H Lee , Y Lee . \textit{Human Dermal Fibroblast.AnnDermatol} 2011 Nov. 23 (4) p. . \bibitem[Lucey and Goldberg (2014)]{b10}\label{b10} ‘Complications of collagen fillers’. P Lucey , D J Goldberg . \textit{Facial Plast Surg} 2014 Dec. \bibitem[Liu ()]{b30}\label{b30} ‘Controlled delivery of recombinant hirudin based on thermo-sensitive Pluronic® F127 hydrogel for subcutaneous administration: in vitro and in vivo characterization’. Y Liu . 387-395. 8. \textit{J. Biomed. Mater. Res} 2007. 1972. 117 p. . (J. Control. Release) \bibitem[Moradi and Watson (2015)]{b32}\label{b32} ‘Current Concepts in Filler Injection’. A Moradi , J Watson . \textit{Facial Plast Surg Clin North Am} 2015 Nov. 23 (4) p. . \bibitem[Callan et al. ()]{b49}\label{b49} ‘Efficacy and safety of a hyaluronic acid filler in subjects treated for correction of midface volume deficiency: a 24 month study’. P Callan , G J Goodman , I Carlisle . \textit{Clin CosmetInvestig Dermatol} 2013. 6 p. . \bibitem[Rhee Do et al. (2014)]{b34}\label{b34} ‘Efficacy and safety of a new monophasic hyaluronic acid filler in the correction of nasolabial folds: a randomized, evaluator-blinded, split-face study’. Y Rhee Do , C H Won , S E Chang , T K Noh , M S Kim , B J Kim , G H Park , J S An , M W Lee , J H Choi , K C Moon , S H Lim . \textit{J Dermatolog Treat} 2014 Oct. 25 (5) p. . \bibitem[Lee et al. (2014)]{b39}\label{b39} ‘Efficacy and safety of porcine collagen filler for nasolabial fold correction in Asians: a prospective multicenter, 12 months follow-up study’. J H Lee , Y S Choi , S M Kim , Y J Kim , J W Rhie , Y J Jun . \textit{J Korean Med Sci} 2014 Nov. 29 (3) p. . (Suppl) \bibitem[Farhi et al. ()]{b3}\label{b3} ‘Emervel French Survey Group. The Emervel French survey: A prospective real-practice descriptive study of 1,822 patients treated for facial rejuvenation with a new hyaluronic acid filler’. D Farhi , P Trevidic , P Kestemont . \textit{J Drugs Dermatol} 2013. 12 p. . \bibitem[Carpenter and Cohen ()]{b53}\label{b53} ‘Epidermal growth factor’. G Carpenter , S Cohen . \textit{Annu. Rev. Biochem} 1979. 48 p. . \bibitem[Carpenter and Cohen ()]{b52}\label{b52} ‘Human epidermal growth factor and the proliferation of human fibro-blasts’. G Carpenter , S Cohen . \textit{J. Cell. Physiol} 1976. 88 p. . \bibitem[Park et al. (2014)]{b48}\label{b48} ‘Iatrogenic occlusion of the ophthalmic artery after cosmetic facial filler injections: a national survey by the Korean Retina Society. Korean Retina Society’. K H Park , Y K Kim , S J Woo , S W Kang , W K Lee , K S Choi , H W Kwak , I H Yoon , K Huh , J W Kim . \textit{JAMA Ophthalmol} 2014 Jun. 132 (6) p. . \bibitem[Ruel-Gariepy ()]{b23}\label{b23} ‘In situ-forming hydrogels review of temperature-sensitive systems’. E Ruel-Gariepy . \textit{Eur. J. Pharm. Biopharm} 2004. 58 p. . \bibitem[Ledon et al.]{b12}\label{b12} \textit{Inflammatory nodules following soft tissue filler use: a review of causative agents, pathology and treatment options}, J A Ledon , J A Savas , S Yang , K Franca , I Camacho , K Nouri . \bibitem[Bass (2015)]{b33}\label{b33} ‘Injectable Filler Techniques for Facial Rejuvenation, Volumization, and Augmentation’. L S Bass . \textit{Facial Plast Surg Clin North Am} 2015 Nov. 23 (4) p. . \bibitem[Alam et al. (2011)]{b6}\label{b6} ‘Large particle calcium hydroxylapatite injection for correction of facial wrinkles and depressions’. M Alam , J Havey , N Pace , M Pongprutthipan , Yoos . \textit{J Am Acad Dermatol} 2011 Jul. 65 (1) p. . \bibitem[Rowe ()]{b25}\label{b25} \textit{Pharmaceutical handbook of pharmaceutical excipients, 5 th edn., Pharmaceutical, London UK and American Pharmaceutical Association}, R Rowe . 2005. Washington, USA. \bibitem[Lorenc et al. (2018)]{b45}\label{b45} ‘Physiochemical Characteristics of Calcium Hydroxylapatite (CaHA)’. Z P Lorenc , L M Bass , R Fitzgerald , D J Goldberg , M H Graivier . \textit{Aesthet Surg J} 2018 Apr 6. 38 (suppl\textunderscore 1) p. . \bibitem[Breithaupt et al. (2015)]{b7}\label{b7} ‘Poly-L-Lactic Acid and Calcium Hydroxyl Apatite’. A Breithaupt , R Fitzgerald , Collagen , Stimulators . \xref{http://dx.doi.org/10.1016/j.fsc.2015.07.007}{10.1016/j.fsc.2015.07.007}. \textit{FacialPlast Surg Clin North Am} 2015 Nov. 23 (4) p. . (Review) \bibitem[Loh et al. (2016)]{b15}\label{b15} ‘Prevention and management of vision loss relating to facial filler injections’. K T Loh , J J Chua , H M Lee , J T Lim , G Chuah , B Yim , B K Puah . \textit{Singapore Med J} 2016 Aug. 57 (8) p. . \bibitem[Schlesinger et al. (2013)]{b14}\label{b14} ‘Purpura and fillers: a review of pre-procedural, intraprocedural, and post-procedural considerations’. T E Schlesinger , J L Cohen , S Ellison . \textit{J Drugs Dermatol} 2013 Oct. 12 (10) p. . (Review) \bibitem[Lim et al. ()]{b2}\label{b2} ‘Rejuvenation effects of hyaluronic acid injection on nasojugal groove: Prospective randomized split face clinical controlled study’. H K Lim , D H Suh , S J Lee , M K Shin . \textit{J Cosmet Laser Ther} 2014. 16 p. . \bibitem[Lee et al. (2014)]{b43}\label{b43} ‘Safety and efficacy of a novel injectable filler in the treatment of nasolabial folds: polymethylmethacrylate and cross-linked dextran in hydroxypropyl methylcellulose’. Y B Lee , E J Song , S S Kim , J W Kim , D S Yu . \textit{J Cosmet Laser Ther} 2014 Aug. 16 (4) p. . \bibitem[Huang et al. ()]{b4}\label{b4} ‘Safety and efficacy of hyaluronic acid for the correction of nasolabial folds: A meta-analysis’. X Huang , Y Liang , Q Li . \textit{Eur J Dermatol} 2013. 23 p. . \bibitem[Few et al. (2015)]{b36}\label{b36} ‘Single-Blind Randomized, Controlled Study of a Volumizing Hyaluronic Acid Filler for Midface Volume Deficit: Patient-Reported Outcomes at 2 Years’. J Few , S E Cox , D Paradkar-Mitragotri , D K Murphy , Multicenter . \textit{Aesthet Surg J} 2015 Jul. 35 (5) p. . \bibitem[Paliwal et al. (2014)]{b44}\label{b44} ‘Skin extracellular matrix stimulation following injection of a hyaluronic acid-based dermal filler in a rat model’. S Paliwal , S Fagien , X Sun , T Holt , T Kim , C K Hee , D Van Epps , D J Messina . \textit{PlastReconstr Surg} 2014 Dec. 134 (6) p. . \bibitem[Cabana ()]{b27}\label{b27} ‘Study of the gelation process of polyethylene oxide -polypropylene oxidepolyethylene oxide copolymer (Poloxamer 407) aqueous solutions’. A Cabana . \textit{J. Colloid Interface Sci} 1997. 190 p. . \bibitem[San Miguel Moragas et al. (2015)]{b35}\label{b35} ‘Systematic review of "filling" procedures for lip augmentation regarding types of material, outcomes and complications’. J San Miguel Moragas , R R Reddy , Hernández Alfaro , F Mommaerts , MY . \textit{J Craniomaxillofac Surg} 2015 Jul. 43 (6) p. . \bibitem[Gilbert et al. (2012)]{b9}\label{b9} ‘The basic science of dermal fillers: past and present Part II: adverse effects’. E Gilbert , A Hui , S Meehan , H A Waldorf . \textit{J Drugs Dermatol} 2012 Sep. 11 (9) p. . (Review) \bibitem[Stein et al. (2015)]{b8}\label{b8} ‘The biological basis for poly-L-lactic acid-induced augmentation’. P Stein , O Vitavska , P Kind , W Hoppe , H Wieczorek , Schürerny . \textit{J Dermatol Sci} 2015 Apr. 78 (1) p. . \bibitem[Delorenzi et al. ()]{b50}\label{b50} ‘The long-term efficacy and safety of a subcutaneously injected large-particle stabilized hyaluronic acidbased gel of nonanimal origin in esthetic facial contouring’. C Delorenzi , M Weinberg , N Solish , A Swift . \textit{Dermatol Surg} 2009. 35 (1) p. . (Suppl) \bibitem[Belmontesi et al. (2006)]{b37}\label{b37} ‘Transdermal injection of Restylane SubQ for aesthetic contouring of the cheeks, chin, and mandible’. M Belmontesi , R Grover , A Verpaele . \textit{Aesthet Surg J} 2006 Jan-Feb;26. (1S) p. . \bibitem[Kim et al. (2014)]{b51}\label{b51} ‘Treatment algorithm of complications after filler injection: based on wound healing process’. J H Kim , D K Ahn , H S Jeong , I S Suh . \textit{J Korean Med Sci} 2014 Nov. 29 (3) p. . (Suppl) \bibitem[Yuksel et al. (2014)]{b17}\label{b17} ‘Turanli AYEvaluation of effects of platelet-rich plasma on human facial skin’. E P Yuksel , G Sahin , F Aydin , N Senturk . \textit{J Cosmet Laser Ther} 2014 Oct. 16 (5) p. . \bibitem[Burgess ()]{b29}\label{b29} ‘Two forms of murine epidermal growth factor: rapid separation by using reversephase HPLC’. A W Burgess . \textit{Proc. Natl. Acad. Sci. USA} 1982. 79 p. . \bibitem[Tansatit et al. (2015)]{b47}\label{b47} ‘Verification of Embolic Channel Causing Blindness Following Filler Injection’. T Tansatit , H J Moon , P Apinuntrum , T Phetudom . \textit{Aesthetic Plast Surg} 2015 Feb. 39 (1) p. . \end{bibitemlist} \end{document}