The authors have no acknowledgments.

The study protocol complied with the Declaration of Helsinki, and all patients provided written informed consent before participating in the study.
1. Non-activated A-PRP Preparation
<ol>
	<li>Harvest 55 mL of autologous blood from a peripheral vein of the arm using a tube with butterfly needle according to the local blood collection protocol.</li>
	<li>Add 7 mL of acid citrate dextrose (ACD) solution (112 mM of citrate) to the blood tube as an anticoagulant.</li>
	<li>Centrifuge the blood collected in the tube at 260 x g for 10 min. 
	Note: 23 mL of A-PRP + Autologous Platelet-Poor Plasma (A-PPP) and 20 mL of Red Blood Cells suspension (RBCs) were produced.</li>
	<li>Take out the tube containing A-PRP + A-PPP and RBCs from the centrifuge and insert it in a laser light selector device.</li>
	<li>Separate A-PRP + A-PPP in a sterile bag by mechanical suction performed by the laser selector in which the kit is placed. RBCs are automatically removed by mechanical suction. 
	Note: 14 mL of A-PPP was manually harvested and removed by the sterile bag from the kit. 9 mL of A-PRP was obtained.</li>
</ol>
2. Non-activated A-PRP Injections
<ol>
	<li>Divide the scalp of the patients affected by AGA into frontal, parietal, vertex, and occipital areas. 
	Caution: Do not perform anesthesia (local or systemic).</li>
	<li>Perform interfollicular A-PRP injections (0.20 mL x cm2) by the medical injector gun (see Table of Materials) to selected areas of the scalp.
	<ol>
		<li>In patients with hair loss localized to the frontal and parietal areas, perform A-PRP injections exclusively to the frontal area and use saline as placebo in the parietal area.</li>
		<li>In patients with hair loss localized to the parietal and vertex areas, inject A-PRP in the parietal area, and use saline as placebo in the vertex area.</li>
	</ol>
	</li>
	<li>Perform an equal number of A-PRP and placebo injections for each patient.</li>
	<li>Perform interfollicular A-PRP injections at a depth of 5 mm using the medical injector gun equipped with a 30G needle and 10 mL syringe.</li>
	<li>Perform three sessions spaced 30 days apart.</li>
</ol>

<ol><li>Rousso, D. E., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+medical+and+surgical+treatment+options+for+androgenetic+alopecia%5BTitle%5D)+AND+%22JAMA+Facial+Plast+Surg%22%5BJournal%5D)">A review of medical and surgical treatment options for androgenetic alopecia.</a> JAMA Facial Plast Surg. 16, 444-450 (2014).</li>
<li>Schweiger, E. S., Boychenko, O., Bernstein, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Update+on+the+pathogenesis%2C+genetics+and+medical+treatment+of+patterned+hair+loss%5BTitle%5D)+AND+%22J+Drugs+Dermatol%22%5BJournal%5D)">Update on the pathogenesis, genetics and medical treatment of patterned hair loss.</a> J Drugs Dermatol. 9, 1412-1419 (2010).</li>
<li>Price, V. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lack+of+efficacy+of+finasteride+in+postmenopausal+women+with+androgenetic+alopecia%5BTitle%5D)+AND+%22J+Am+Acad+Dermatol%22%5BJournal%5D)">Lack of efficacy of finasteride in postmenopausal women with androgenetic alopecia.</a> J Am Acad Dermatol. 43, 768-776 (2000).</li>
<li>Imperato-McGinley, J., Guerrero, L., Gautier, T., Peterson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Steroid+5alpha-reductase+deficiency+in+man%3A+An+inherited+form+of+male+pseudohermaphroditism%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Steroid 5alpha-reductase deficiency in man: An inherited form of male pseudohermaphroditism.</a> Science. 186, 1213-1215 (1974).</li>
<li>Drake, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+finasteride+on+scalp+skin+and+serum+androgen+levels+in+men+with+androgenetic+alopecia%5BTitle%5D)+AND+%22J+Am+Acad+Dermatol%22%5BJournal%5D)">The effects of finasteride on scalp skin and serum androgen levels in men with androgenetic alopecia.</a> J Am Acad Dermatol. 41, 550-554 (1999).</li>
<li>Van Neste, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Finasteride+increases+anagen+hair+in+men+with+androgenetic+alopecia%5BTitle%5D)+AND+%22Br+J+Dermatol%22%5BJournal%5D)">Finasteride increases anagen hair in men with androgenetic alopecia.</a> Br J Dermatol. 143, 804-810 (2000).</li>
<li>Kaufman, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Finasteride+in+the+treatment+of+men+with+androgenetic+alopecia%5BTitle%5D)+AND+%22J.+Am+Acad+Dermatol%22%5BJournal%5D)">Finasteride in the treatment of men with androgenetic alopecia.</a> J. Am Acad Dermatol. 39, 578-589 (1998).</li>
<li>Afifi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-level+laser+therapy+as+a+treatment+for+androgenetic+alopecia%5BTitle%5D)+AND+%22Lasers+Surg+Med%22%5BJournal%5D)">Low-level laser therapy as a treatment for androgenetic alopecia.</a> Lasers Surg Med. 49, 27-39 (2017).</li>
<li>Uebel, C. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+platelet+plasma+growth+factors+in+male+pattern+baldness+surgery%5BTitle%5D)+AND+%22Plast+Reconstr+Surg%22%5BJournal%5D)">The role of platelet plasma growth factors in male pattern baldness surgery.</a> Plast Reconstr Surg. 118, 1458-1466 (2006).</li>
<li>Cervelli, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+autologous+activated+platelet+rich+plasma+%28AA-PRP%29+injection+on+pattern+hair+loss%3A+Clinical+and+histomorphometric+evaluation%5BTitle%5D)+AND+%22BioMed+Res+Int%22%5BJournal%5D)">The effect of autologous activated platelet rich plasma (AA-PRP) injection on pattern hair loss: Clinical and histomorphometric evaluation.</a> BioMed Res Int. , 760709(2014).</li>
<li>Gkini, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+of+platelet-rich+plasma+injections+in+the+treatment+of+androgenetic+alopecia+through+an+one-year+period%5BTitle%5D)+AND+%22J+Cutan+Aesthet+Surg%22%5BJournal%5D)">Study of platelet-rich plasma injections in the treatment of androgenetic alopecia through an one-year period.</a> J Cutan Aesthet Surg. 7, 213-219 (2014).</li>
<li>Gentile, P., Garcovich, S., Bielli, A., Scioli, M. G., Orlandi, A., Cervelli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+platelet-rich+plasma+in+hair+regrowth%3A+A+randomized+placebo-controlled+trial%5BTitle%5D)+AND+%22Stem+Cells+Transl+Med%22%5BJournal%5D)">The effect of platelet-rich plasma in hair regrowth: A randomized placebo-controlled trial.</a> Stem Cells Transl Med. 4, 1317-1323 (2015).</li>
<li>Gentile, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+Not-Activated+and+Activated+PRP+in+Hair+Loss+Treatment%3A+Role+of+Growth+Factor+and+Cytokine+Concentrations+Obtained+by+Different+Collection+Systems%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Evaluation of Not-Activated and Activated PRP in Hair Loss Treatment: Role of Growth Factor and Cytokine Concentrations Obtained by Different Collection Systems.</a> Int J Mol Sci. 18 (2), 14(2017).</li>
</ol>

The authors have nothing to disclose.

The authors demonstrated that mechanical and controlled injections of A-PRP represent viable AGA treatment options, with reduction of pain during the procedure compared with manual injection performed by the hands of the surgeon. Patients treated with this protocol were found to have greater increases in hair count and total hair density than patients treated with manual injections.
The most important aspect to obtain an improvement of hair count and hair density is to pay attention to detail in various steps during the PRP preparation and the injection technique. For example, to obtain a good hair improvement, it is very helpful to use a sterilized closed system according to the transfusional service rules for PRP preparation and to perform mechanical injection of PRP. Before starting the injections, it is important to place the syringe with PRP in its definitive position, into the gun. After, it is necessary to use a 30G needle.
As previously described, we prefer to use a medical injector gun for mechanical and controlled injection of PRP; this provides the additional advantage of minimizing subjective bias produced by manual injections. In fact, the use of a mechanical device as a gun has the additional advantage of decreasing the probability of inadvertently injecting different amounts of PRP at different depths to the targeted area.
The authors observed that mechanical and controlled injections of PRP in the targeted area of the scalp using a gun or mechanical device, not only allows a better injection (more strict), but also minimizes the risk of undue over-injection or low-injection for each cm2.
Another important technical tip is to start the injection from the upper side and continuing to down the side observing 1 cm2 of space between each spot, in order to minimize the risk of overlapped injections.
If the amount of PRP obtained through centrifuge is very low compared to the targeted area to treat, it may be necessary to repeat a second time the procedure of blood harvesting and centrifugation using a new kit. In this way, a larger amount of PRP will be used.
Our experience of more than two years of using this kind of injection for clinical purposes strongly suggests that the mechanical and controlled injection of PRP using a gun or mechanical device is a reproducible model of PRP injection in the treatment of MPHL and FPHL. At the same time, our experience of more than four years of using this PRP preparation, suggests that the closed system for PRP preparation is a safe and reproducible model of PRP procedure.
Both can be easily incorporated into training courses, as they are good teaching and training models for trainees of aesthetic regenerative surgery and aesthetic regenerative medicine. In our experience, although technically challenging in the beginning for the novice in regenerative surgery, after some training, the PRP obtained can be successfully injected to the scalp of the patient with minimal to no subsequent pain in 90% of cases.
Numerous methods of activated and non-activated PRP preparations have been described in the literature10,11,12,13. Commercial systems seem to be the most commonly used for clinical, teaching, and research purposes. These procedures have been favored, due to their simplicity to use and EU safety marks. Patients treated with A-PRP were found to have greater increases in hair count and total hair density than patients treated with AA-PRP using an identical PRP collection device, indicating that PRP does not need to be activated when a specific system is employed13.
In addition, the use of mechanical and controlled injection of PRP has reduced the pain and the subjective bias during the injections by reducing the possibility overlap of injections from adjacent points.
The two main limitations of this model are the need for regenerative surgery skills in order to carry out the treatment and PRP preparation, and the necessity to obtain a mechanical device for injections. Another potential limitation of the model herein presented is the time of the procedure. In fact, the manual procedure is faster than the mechanical and controlled injection. The authors reported a mean of 25-45 min for each procedure of PRP injections when the mechanical device is used.
The gun to inject PRP can be used in male and female pattern hair loss treatment. Its use is particularly suitable for patients more sensitive to pain, without local or systemic anesthesia. In light of these results, the authors recommend more clinical trials based on the use of mechanical systems of injections.

The clinical injection of non-activated A-PRP has become an attractive resource for targeting hair growth. Here, the protocol of the A-PRP application in AGA is reported, using mechanical and controlled injections. Currently, only oral selective 5-&#945;-reductase inhibitor, topical diaminopirimidilpiperidin-N-ossido (2.0% and 5.0%), and low-level laser therapy (LLLT) are approved by the US Food and Drug Administration (FDA) to combat MPHL1,2.
Diaminopirimidilpiperidin-N-ossido 5% foam is also approved by the FDA for FPHL. The selective 5-&#945;-reductase inhibitor has proven largely ineffective in treating FPHL3, and, given that the drug may cause abnormalities in the external genitalia of male fetuses, is unsuitable for use by pre-menopausal women4. Conversely, daily treatment with 1 mg of selective 5-&#945;-reductase inhibitor has been shown to reduce serum dihydrotestosterone (DHT) levels by 70% and promotes hair growth of anagen hair leading to gradual increase in hair diameter and hair elongation in male AGA patients5,6, through significant improvements in hair density. The use of oral selective 5-&#945;-reductase inhibitor may require up to one year of treatment and users may develop loss of libido, which may persist after the medication is discontinued7.
Recently, the use of LLLT has been proposed to stimulate hair regrowth. Afifi et al.8 reviewed the existing research studies to determine whether LLLT was an effective therapy for AGA. The topical application of activated autologous platelet-rich plasma (AA-PRP) to harvested follicles prior to implantation has already been shown to increase their survival rate by 15% in patients affected by AGA9, as well as exhibit increased hair density 3 months post-surgery with terminal hair density increasing by 19% during that time10. These findings were confirmed in a study following AGA patients treated with calcium-activated PRP over the course of one year11.
Given the positive results of AA-PRP12 and A-PRP13 as an alopecia treatment, and the lack of data for the clinical injections system, the primary aim of this work is to show mechanical and controlled PRP injections protocol.
18 male and 5 female patients aged 21-70 years who displayed MPHL in Stage 2 to Stage 5 as determined by the Norwood-Hamilton classification scale, and FPHL in Stage 1 to Stage 2 as determined by the Ludwig classification scale, were treated with mechanical and controlled injections of A-PRP. AGA diagnoses were made by a medical history, trichoscopic analysis, clinical evaluation with negative hair pull test, laboratory tests, and urinalysis.
The stage of AGA was evaluated using the Norwood-Hamilton and Ludwig scale. All of the participants included in this study were assessed by a medical doctor specialized in dermatology and two plastic surgeon experts in regenerative plastic surgery and alopecia. The authors considered localized and systemic exclusion criteria for all patients. Systemic criteria were represented by blood and/or platelet disorders, anticoagulant and/or antiaggregation therapy, sepsis, immunosuppression, cancer, use of oral selective 5-&#945;-reductase inhibitor or antiandrogens in the previous 12 months.
Localized exclusion criteria were represented by use of diaminopirimidilpiperidin-N-ossido (2.0% and 5.0%) or corticosteroids for topical treatments in the previous 12 months.

<table><tbody><tr><td>C-PunT system</td><td>Biomed Device, Modena, Italy</td><td>www.biomeddevice.it</td><td>a closed system medical device to obtain autologous concentrated platelet-rich plasma from the blood</td></tr><tr><td>Ultim Gun</td><td>Anti-Aging Medical System</td><td>www.aamedicalsystems.fr</td><td>a gun medical device to perform a mechanical and controlled injections of autologous platelet-rich plasma in a selected area of the scalp</td></tr><tr><td>Trichoscan</td><td>Tricolog GmbH, Freiburg, Germany).</td><td>www.tricholog.de</td><td>trihoScan is a digital&lt;br/&gt; software-supported epiluminescence technique for measuring hair count (number of hairs per 0.65 cm2), hair density (number of hairs per cm2), hair diameter, anagen-to-telogen ratio, and vellus&lt;br/&gt; hair-to-terminal hair ratio.</td></tr><tr><td>sodium citrate ACD-A</td><td>S.A.L.F.</td><td>SAAS2205GPO</td><td>anticoagulant for PRP preparation</td></tr><tr><td>&amp;nbsp;30-Gauge needle&amp;nbsp;</td><td>Becton Dickinson&amp;nbsp;</td><td>Z192368</td><td>needle for injections</td></tr><tr><td>10 mL mL Luer-Lock syringe</td><td>Becton Dickinson&amp;nbsp;</td><td>BD309695</td><td>syringe for PRP collection</td></tr></tbody></table>

mechanical and controlled prp injections in patients affected by androgenetic alopecia

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the Norwood-Hamilton classification scale, and female pattern hair loss (FPHL) in Stage 1 to Stage 2 as determined by the Ludwig classification scale, were treated with non-activated autologous platelet-rich plasma (A-PRP). Autologous blood (55 mL) was harvested using sodium citrate as an anticoagulant. A-PRP (23 mL) was produced for all cases using a closed system according to the transfusion service protocol. Following centrifugation (260 x g for 10 min) the A-PRP was inserted in a laser light selector device, and after the centrifugation, 9 mL of A-PRP was collected.
The scalp of the patients affected by androgenetic alopecia (AGA) was divided into four areas (frontal, parietal, vertex, and occipital); local anesthesia was not performed. Interfollicular A-PRP injections (0.2 mL x cm2) were performed by controlled and mechanical injections scheduled at a depth of 5 mm using a medical injector gun. Treatment sessions were performed with a 30-day interval. For each patient, three treatment sessions were performed.
PRP was injected in the androgen-related areas of scalp affected by hair loss. Placebo (normal saline solution) was loaded in another syringe (10 mL) and injected on the adjacent side in a similar fashion.

The hair growth parameters measured three months after the third A-PRP injections by tricoscopic analysis were compared with the baseline measurements and between the A-PRP treatment area and the control area, which received physiological saline injections as placebo. Before the first treatment, at baseline, no statistical differences in hair count (122 &#177; 10) and hair density (218 &#177; 17) were reported between the targeted area and controlled area (126 &#177; 9 baseline hair count controlled area), (225 &#177; 15 baseline hair density controlled area).
The results obtained indicate that three months after the third A-PRP injections treatment with mechanical and controlled A-PRP injections, mean hair count increases significantly over baseline values in male patients. The targeted area showed an elevated hair count (158 &#177; 11) (36 &#177; 3 hairs compared to the baseline) and total hair density (282 &#177; 20) (65 &#177; 5 hair cm2 compared to the baseline), while the controlled area that received saline injection as placebo had negligible changes in hair count13. These values reflect a 31% &#177; 2% increase in hair density for the treatment group and less than a 1% increase in hair density for the placebo group.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56406/56406fig1.jpg" /> 
Figure 1. Interfollicular A-PRP injections by the medical injector gun equipped with a 30G needle, 10 mL syringe. Interfollicular A-PRP injections (0.2 mL x cm2) were performed at 5 mm of depth. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56406/56406fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hair Parameters</td>
			<td>Timing</td>
			<td>Control Area 
			(treated with saline)</td>
			<td>Targeted Area 
			(Treated with A-PRP)</td>
		</tr>
		<tr>
			<td rowspan="2">Hair density, 
			no. per cm2</td>
			<td>Baseline</td>
			<td>225 &#177; 15</td>
			<td>218 &#177; 17</td>
		</tr>
		<tr>
			<td>3 months later the third treatment</td>
			<td>227 &#177; 16</td>
			<td>282 &#177; 20</td>
		</tr>
		<tr>
			<td rowspan="2">Hair Count</td>
			<td>Baseline</td>
			<td>126 &#177; 9</td>
			<td>122 &#177; 10</td>
		</tr>
		<tr>
			<td>3 months later the third treatment</td>
			<td>127 &#177; 9</td>
			<td>158 &#177; 11</td>
		</tr>
		<tr>
			<td colspan="4">Data are presented as mean &#177; standard error.</td>
		</tr>
	</tbody>
</table>
Table 1. Relevant hair-growth parameters assessed by Trichoscan analysis for the A-PRP treatment and placebo negative control half-head areas at baseline and 3 months after the third treatment.

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Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

This paper aims to describe the interfollicular autologous platelet-rich plasma injections (0.20 mL x cm2) in the scalp of 23 patients affected by androgenetic alopecia at a depth of 5 mm using a medical injector gun equipped with a 30 gauge needle, in three sessions.

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the ...

mechanical-controlled-prp-injections-patients-affected-androgenetic

Nanyang Technological University

Research

JoVE Journal

Medicine

15.0K Views.  University of Rome Tor Vergata. Dit document heeft tot doel om te beschrijven de interfollicular autologe platelet-rich plasma injecties (0,20 mL x cm2) in de hoofdhuid van de 23 patiënten beïnvloed door alopecia androgenetica op een diepte van 5 mm met behulp van een medische injector pistool uitgerust met een naald van 30 meter, in drie sessies.

Video: Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Immunology and Infection

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of cultured cells, implying a possible use in regenerative medicine. Here, a double-spin method was used to prepare PRP from whole blood. PRP was further activated by autologous thrombin. The platelet count was measured in the activated PRP and the proliferation-stimulating effect in human adipose-derived stem cells (hASCs) was examined. The resulting platelet count was 11.5-times higher in PRP than in whole blood plasma. The proliferation of hASCs was markedly enhanced by incubation with 1% PRP. The described method can be used to reproducibly prepare PRP with a high concentration of platelets. PRP prepared by this method markedly promotes proliferation of hASCs.

In this report, we describe a double-spin method for preparing activated platelet-rich plasma (PRP). Using autologous thrombin, human adipose-derived stem cells (hASCs) were cultured. Activated PRP was shown to promote proliferation of hASCs.

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of ...

Developmental Biology

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

Bioengineering

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily.
Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This protocol describes the evaluation process of healing tendons in rats that have been injected with allogeneic platelet rich plasma (PRP) or saline solution after removing part of the Achilles tendon. The progress of tendon healing is evaluated at several time points using different types of analyses.

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Biology

Platelet-Rich Plasma Lysate for Treatment of Eye Surface Diseases

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite and growth factor content, which promotes eye surface regeneration. Blood-based eye drops can be prepared from different sources (i.e., whole blood or platelet apheresis donation), as well as with different protocols (e.g., different dilutions and freeze/thaw cycles). This variability hampers the standardization of clinical protocols and, consequently, the evaluation of their clinical efficacy. Detailing and sharing the methodological procedures may contribute to defining common guidelines. Over the last years, allogenic products have been diffusing as an alternative to the autologous treatments since they guarantee higher efficacy standards; among them, the platelet-rich plasma lysate (PRP-L) eye drops are prepared with simple manufacturing procedures. In the transfusion medicine unit at AUSL-IRCCS di Reggio Emilia, Italy, PRP-L is obtained from platelet-apheresis donation. This product is initially diluted to 0.3 x 109 platelets/mL (starting from an average concentration of 1 x 109 platelets/mL) in 0.9% NaCl. Diluted platelets are frozen/thawed and, subsequently, centrifuged to eliminate debris. The final volume is split into 1.45 mL aliquots and stored at &#8722;80 &#176;C. Before being dispensed to patients, eye drops are tested for sterility. Patients may store platelet lysates at &#8722;15 &#176;C for up to 1 month. The growth factor composition is also assessed from randomly selected aliquots, and the mean values are reported here.

Platelet lysates represent an emerging tool for the treatment of ocular surface diseases. Here, we propose a method for the preparation, dispensation, storage, and characterization of platelet lysate collected from platelet donors.

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite ...

RPNI Surgery for Treating Postamputation Pain

Regenerative Peripheral Nerve Interface: Surgical Protocol for a Randomized Controlled Trial in Postamputation Pain

Surgical procedures, including nerve reconstruction and end-organ muscle reinnervation, have become more prominent in the prosthetic field over the past decade. Primarily developed to increase the functionality of prosthetic limbs, these surgical procedures have also been found to reduce postamputation neuropathic pain. Today, some of these procedures are performed more frequently for the management and prevention of postamputation pain than for prosthetic fitting, indicating a significant need for effective solutions to postamputation pain. One notable emerging procedure in this context is the Regenerative Peripheral Nerve Interface (RPNI). RPNI surgery involves an operative approach that entails splitting the nerve end longitudinally into its main fascicles and implanting these fascicles within&#160;free denervated and devascularized muscle grafts. The RPNI procedure takes a proactive stance in addressing freshly cut nerve endings, facilitating painful neuroma prevention and treatment by enabling the&#160;nerve to regenerate and innervate an end organ, i.e., the free muscle graft. Retrospective studies have shown RPNI&#39;s effectiveness in alleviating postamputation pain and preventing the formation of painful neuromas. The increasing frequency of utilization of this approach has also given rise to variations in the technique. This article aims to provide a step-by-step description of the RPNI procedure, which will serve as the standardized procedure employed in an international, randomized controlled trial (ClinicalTrials.gov, NCT05009394). In this trial, RPNI is compared to two other surgical procedures for postamputation pain management, specifically, Targeted Muscle Reinnervation (TMR) and neuroma excision coupled with intra-muscular transposition and burying.

Author Spotlight: Regenerative Peripheral Nerve Interface (RPNI) Surgery in Postamputation Pain Management

Here, we describe the surgical procedure to perform Regenerative Peripheral Nerve Interface (RPNI) surgery for treating postamputation neuropathic pain in the context of an international, randomized controlled trial (RCT) (ClinicalTrials.gov, NCT05009394). The RCT compares RPNI with two other surgical techniques, namely, Targeted Muscle Reinnervation (TMR) and neuroma excision combined with intra-muscular transposition.

Surgical procedures, including nerve reconstruction and end-organ muscle reinnervation, have become more prominent in the prosthetic field over the past ...

Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

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