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\title{A New Medical Device for Platelet Rich Plasma Filler}
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             \author[1]{Araco Antonino,  M.D.}

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\date{\small \em Received: 6 December 2019 Accepted: 3 January 2020 Published: 15 January 2020}

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\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.}

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\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}
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\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}
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\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}
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\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}
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\end{bibitemlist}
 			 		 	 
\end{document}
