Effect of Autologous Activated PRP Injection on Pattern Hair Loss_VCervelli

8/21/2019 Effect of Autologous Activated PRP Injection on Pattern Hair Loss_VCervelli

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Clinical Study The Effect of Autologous Activated PlateletRich Plasma (AA-PRP) Injection on Pattern Hair Loss:Clinical and Histomorphometric Evaluation

V. Cervelli,1 S. Garcovich,2 A. Bielli,3 G. Cervelli,4 B. C. Curcio,1 M. G. Scioli,3

A. Orlandi,3 and P. Gentile1,5

Plastic and Reconstructive Surgery Department, University of Rome or Vergata, Via Montpellier, No. , Rome, Italy Institute of Dermatology, Catholic University of the Sacred Heart, Rome, Italy Institute of Anatomic Pathology, University of Rome or Vergata, Via Montpellier, No. , Rome, Italy Science Education Department, University of Rome or Vergata, Via Montpellier, No. , Rome, Italy San Salvatore in Lauro Place, No. , Rome, Italy

Correspondence should be addressed to P. Gentile; [email protected]

Received November ; Revised January ; Accepted March ; Published May

Academic Editor: Garrett McGuinness

Copyright © V. Cervelli et al. Tis is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

o investigate the saety and clinical efficacy o AA-PRP injections or pattern hair loss. AA-PRP, prepared rom a small volumeo blood, was injected on hal o the selected patients’ scalps with pattern hair loss. Te other hal was treated with placebo. Treetreatments were given or each patient, with intervals o month. Te endpoints were hair re-growth, hair dystrophy as measuredby dermoscopy, burning or itching sensation, and cell prolieration as measured by Ki- evaluation. At the end o the cycles o treatment, the patients presented clinical improvement in the mean number o hairs, with a mean increase o . hairs in the targetarea, anda mean increase in totalhair density o . ( number o hairs/cm2) compared with baseline values. Microscopic evaluationshowedthe increase o epidermis thicknessand o the number o hair ollicles twoweeksafer the last AA-PRP treatment comparedto baseline value ( < 0.05). We also observed an increase o Ki+ keratinocytes o epidermis and o hair ollicular bulge cells anda slight increase o small blood vessels around hair ollicles in the treated skin compared to baseline ( < 0.05).

1. Introduction

Proponents o platelet-rich plasma (PRP) technology suggest

that its benets include an increase in hard- and sof-tissuewound healing. In addition, the role o PRP or the treatmento pattern hair loss has been demonstrated in recent reports[–]. In particular, Rinaldi described the use o PRP inalopecia areata (AA). Tis pilot study suggests that PRP may serve as a sae and effective treatment option in AA andcalls or more extensive controlled studies with this method[]. Uebel et al. showed that pretreatment o ollicular unitswith PRP beore transplantation resulted in improved hairgrowth and density []. Activated autologous PRP has beenreported to induce the prolieration o dermal papilla cellsby upregulating broblast growth actor (FGF-) and b-catenin as well as extracellular signal-related kinase (ERK)

and Akt signalling []. Anagen-associated angiogenesis hasbeen suggested as one o the important actors in activehair growth [], due to the secretion o vascular endothelial

growth actor (VEGF) by the keratinocytes o the outerroot sheath and broblasts o the dermal papilla [–].Increased secretion o VEGF inuences growth o normaland pathological dermal structures []. obin et al. reportedthat the hair ollicle mesenchyme exhibits signicant haircycle-associated plasticity. Modulation o these cell inter-changes is likely to be important during clinically importanthair ollicle transormations, or example, vellus-to-terminaland terminal-to-vellus transormations during androgeneticalopecia []. Injection o PRP has been demonstrated toimprove cutaneous ischemic conditions and to increase

vascular structures around hair ollicles [, ]. Many o thecurrent treatment modalities or pattern hair loss have been

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 760709, 9 pageshttp://dx.doi.org/10.1155/2014/760709

http://dx.doi.org/10.1155/2014/760709http://dx.doi.org/10.1155/2014/760709


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: Summary o patients’ characteristics.

Case AgeTe

Norwood-Hamiltonclassication stage

Injection site

IIa Frontal

IIa Frontal

III Parietal

III vertex Parietal

IIa Frontal

IV Parietal and vertex

III vertex Parietal

III Parietal

III Frontal

IIa Frontal

shown to modulate angiogenesisand enhance blood ow [].

Te aim is to evaluate the effects o AA-PRP obtained roma small volume o blood on active hair growth. Te data wereported proves the clinical efficacy o the treatment withAA-PRP; moreover, patients’ satisaction urther conrmsthe quality o the results. Afer studying this paper, thereader should be able to () prepare AA-PRP, () apply PRPintraoperatively, () evaluate the clinical effect o AA-PRP onhair growth, and () evaluate the histomorphometric effect o AA-PRP on the prolieration o dermal papilla cells.

2. Material and Methods

.. Patients. A total o male patients (age range: –)

with male pattern hair loss (MPHL) were treated. Te patientcharacteristics are summarized in able . Patients, who hadreceived topical (such as minoxidil, prostaglandin, analogues,retinoids, and corticosteroid) or systemic treatments orMPHL (such as nasteride, dutasteride, and antiandrogens)in the previous months were excluded. Patients witha propensity or keloids and patients who were immuno-suppressed were also excluded. In addition, the numberso platelets in PRP obtained rom all participants weremicroscopically counted. Tis was a randomized, richoScanevaluator blinded, placebo hal-head group study.

Te diagnosis o MPHL was established on the basis o clinical and trichoscopic eatures (more than % variability

in hair diameter between affected and uninvolved areas),while the extent and stage o MPHL were assessed accord-ing to the Norwood-Hamilton classication (as shown inable ).

All patients provided written inormed consent beoreparticipating in the study, which was perormed according tothe Declaration o Helsinki.

.. reatment Protocol. AA-PRP was prepared rom a small volume o blood ( cc) according to the method o Cascade-Selphyl-Esorax system, with modications [–]. Briey,to prepare PRP, blood was taken rom a peripheral veinusing sodium citrate as an anticoagulant. Te current systems

or preparing platelet concentrations use various centriuges(however in this case we used g or min). AA-PRPwas prepared in all cases with approval o the ransu-sional Service. Although the method o preparation was notselective and may include leukocytes, the nal aim is toobtain a platelet pellet. Growth actors are only secreted

once platelet activation begins, which in turn is stimulatedby Ca2+. o optimize the secretion process, the optimum

concentration o Ca2+ was previously determined [, ].Ten, autologous-PRP not activated (A-PRP) obtained afercentriugation ( mL) was switched into -mL tubes con-

taining Ca2+ extracted by Cascade-Selphyl-Esorax Kit. Tepatients’ scalp affected by hair loss was divided in ourhalves (Figure (a)) and cleansed with % alcohol, but localanaesthesia was not injected on the treated areas. Te AA-PRP was injected on selected areas o the scalp at the amount

o . mL/cm2 (Figure (d)). AA-PRP injections wereinjectedwith the AAPRP only on the rontal areas (Figures (b),(b), and (b)); the parietal area was treated with placebo

(Figure (b)). Te scalps o patients affected by hair loss weredivided, respectively, into our parts: rontal, parietal, vertex,and occipital parts. Patients with hair loss localized to therontal and parietal areas (Figures (a), (a), (a), and (a))were injected with the AA-PRP only on the rontal areas(Figure (b)); the parietal area was treated with placebo basedon the injection o physiological solution. Patients with hairloss in the parietal and vertex parts (Figures (a) and (a))were injected with the AA-PRP only in the parietal part o thescalp (Figure (b)); the vertex area was treated with placebobased on the injection o physiological solution. In detail theauthors repeat the same numbers o injections in the hal treated with PRP and in the hal treated with placebo. Te

analysis o the areas o the scalp treated with PRP and placebowas reported in Figures (a), (b), and (c).

.. Assessment Criteria. All patients were evaluated in ourstages: , beginning o study; in weeks; , months;and, months. Te effectso the treatment on hair growthwere assessed in all patients with the help o global photog-raphy, physician’s and patient’s global assessment scale, andstandardized phototrichograms.

Phototrichograms were perormed in all patientsby a trained evaluator by means o FotoFinder-video-epiluminescence microscopy in combination with therichoScan digital image analysis (Figure ). richoScan

is a digital sofware-supported epiluminescence techniqueor measuring hair count (number o hairs/. cm2), hair

density (number o hairs/cm2), hair diameter, anagen/telogenratio, and vellus hair/terminal hair ratio. o determine thequality o hair leading to an increased hair density, it isimportant to differentiate the number o terminal and vellushairs. In richoScan all hairs with a diameter > mare categorized as terminal hair, and all hairs with lesserdiameter are categorized as vellus hair. In all patients, in boththe treatment and control hal heads, two transitional areaso hair loss were dened and marked with a semipermanenttattoo or the subsequent trichogram. In the target areahairs were clipped and dyed with hair brown color or ten


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(a) (b)

F : A smoker -year-old male patient affected by hair loss. (a): preoperative situation o the rontal line. (b): postoperative situationo the rontal line afer two weeks rom the last treatment with increase o the hair count and hair density.

(a) (b)

F : (a): preoperativesituation o the scalp. (b):postoperative situation o the scalp twoweeksrom thelast treatment. Te picture showsa postoperative situation with increase o the hair count and hair density.

minutes in order to improve the hair contrast or the analyticsofware. richoScan analysis. Te evaluator o richoScananalysis was blinded regarding the treatment and controlareas o the scalp and not involved in administration o treatment.

.. Histological Evaluation. Incisional punch biopsies ( mmin diameter) o the hair skin were obtained (Figure (c))at baseline and afer two months rom the last AA-PRPtreatment and xed in buffered ormalin. Morphometricanalysis [] was perormed on Haematoxylin-Eosin-stainedparaffin serial sections (Figures and ) by evaluating the

thickness o epidermis and the number o ollicles per mm2,according to the method []. About the orientation o skinbiopsies, all samples were cut perpendicularly at the suraceand embedded making attention to the correct orientation.

.. Immunohistochemistry. Immunohistochemistry wasperormed using mouse monoclonal anti-Ki (DakoCyto-mation, Denmark) and anti-CD (DakoCytomation,

Denmark), with positive and negative controls [, ]. Tepercentage o Ki+ cells in basal layer o epidermis, in outerroot sheath o hair ollicles, and the number o vessels permm2 were calculated according to morphometric criteria[].

3. Results

.. Clinical Evaluation of AA-PRP Injection on Pattern Hair Loss. Te various hair growth parameters measured afer months o the rst treatment were compared with thebaseline study beore treatment (Figures (a), (a), (a), and(a)) and between both treatment and control areas. Meantotal hair counts, hair density, and terminal and vellus hairdensities or the treatment and control areas are listed inable . At baseline, there were no statistical differencesin hair count, hair density, and terminal and anagen hairdensities between the treatment and control area o the scalp.Te results o this study showed a signicant increase in themean hair count or the treatment area afer three months( months versus month), with a mean increase o .


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(a) (b)

(c) (d)

F : A nonsmoker -year-old male patient affected by hair loss. (a): preoperative situation o the scalp with hair loss localized to thetemporal and nuchal areas. (b): intraoperative injection with the AA-PRP at . mL/cm. (c): intraoperative incisional punch biopsies ( mmin diameter) o the hair skin xed in buffered ormalin. (d): intraoperative study design.

(a) (b)

F : A nonsmoker -year-old male patient affected by hair loss. (a): preoperative situation o the scalp with hair loss localized to theparietal and vertex areas. (b): postoperative situation o the scalp two weeks rom the last treatment with increase o the hair count and hairdensity.

hairs in the target area compared to baseline, while thecontrol area showed a mean decrease o , hairs (control

versus treatment; < 0.0001). Accordingly, in the treatmentarea, a mean increase in total hair density o . (number

o hairs/cm2) compared to baseline was observed afer months and the control area displayed a mean decrease o

. (number o hairs/cm2) in hair density at the same time(control versus treatment; < 0.0001). In addition, terminalhair density improved signicantly by 27.0±15.3 (number o

hairs/cm2) in the treatment area (Figures (b), (b), and (b))compared to baseline, while decreasing by 2.1±12. (number

o hairs/cm2) in the control area o the scalp (control versustreatment; = 0.0003). Tere were no statistically signicantdifferences in vellus hair density between the study and thecontrol area afer three months.

.. Histomorphometric Evaluation of AA-PRP Injectionon Pattern Hair Loss. Microscopic evaluation showed the


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Baseline

(a)

PRP treatment

(b)

Baseline

PRP treatment

0.2

0.1

0 E p i d e r m a l t h i c k n e s s ( m m )

∗

(c)

(d) (e)

N u

m b e r o f f o l l i c l e s ( m m

2 ) 2

1.5

1

0.5

0

Baseline

PRP treatment

∗

()

F : PRP treatment increases the thickness o epidermis and the number o ollicles o hair skin. (a) and (b): representativemicrophotographs o hair skin epidermis at baseline (a) and afer PRP treatment (b). (c): bar graph o epidermis thickness. (d) and (e):representative microphotographs o dermal hair ollicles at baseline (d) and afer PRP treatment (e). (): bar graph o the number o hairollicles/mm2 at baseline and afer PRP treatment; ∗ indicates < 0.05. Original magnication: (a) and (b): x and (d) and (e): x.

: Relevant hair growth parameters assessed by richoScan

analysis or the treatment and controlhal-head areas at baseline andafer weeks ().

reatment area Control area

Hair count (mean ± SD)

Baseline . ± . . ± .

. ± . . ± .

Hair density [/cm](mean ± SD)


. ± . . ± .

erminal hair density [/cm](mean ± SD)

Baseline . ± . . ± . . ± . . ± .

Vellus hair density [/cm](mean ± SD)


. ± . . ± .

increase o epidermis thickness (Figure (c); < 0.05)in PRP-treated hair skin (Figure (b); < 0.05) aferthree months rom the AA-PRP treatment compared tobaseline value (Figure (a)). wo-week PRP treatment

(Figure (e); < 0.05) was also accompanied by an increase

o the number o ollicles (Figure ( ); < 0.05) comparedto baseline value (Figure (d)). o better report the effectso PRP, we investigated the prolieration o epidermal andhair ollicular bulge cells (Figures (b) and (e); < 0.05).Afer two weeks rom the last treatment, we observed anincrease o Ki+ basal keratinocytes o epidermis and o hair ollicular bulge cells (Figures (c) and (); < 0.05)compared to baseline (Figures (a) and (d)). PRP treatment(Figure (h); < 0.05) also associated with a slight increaseo small blood vessels around hair ollicles in the skin treated(Figure (i); < 0.05) compared to baseline (Figure (g)).

4. Discussion

Current strategies or the treatment o pattern hair lossare mainly ocused on promoting cellular prolieration anddifferentiation during the hair growth cycle. It has beenpostulated that minoxidil prolongs anagen and increaseshair ollicle size through stimulation o potassium channelsand prostaglandin endoperoxide synthase-, which increaselevel o prostaglandin E (PGE) []. Minoxidil promotesthe survival o dermal papilla cells by increasing Bcl-/Baxratio and by activating ERK and Akt []. Oral nasteridealso induces the prolongation o anagen hairs, which resultsin gradual thickening and elongation o the hairs []. Inaddition, nasteride has been shown to reduce the pattern


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Baseline

(a)

PRP treatment

(b)

Baseline

PRP treatment

K i 6 7 +

e p i d e r m a l c e l l s ( % )

∗6

4

2

0

(c)

(d) (e)

Baseline

PRP treatment

K i 6 7 +

f o l l i c l e c e l l s ( % )

∗

6

4

2

0

()

(g) (h)

BaselinePRP treatment

C D 3 1 +

v e s s e l s ( m m

2 )

∗

3

2

1

0

(i)

F : PRP treatment increases prolieration o epidermis basal cells and hair ollicular bulge cells. (a) and (b): representativemicrophotographs o Ki+ prolierating cellsby immunohistochemistry o hairskin epidermis at baseline (a) andafer PRP treatment (b). (c):morphometric analysis o Ki+ cells o hair skin epidermisat baseline andafer PRPtreatment. (d) and(e): representativemicrophotographso Ki+ prolierating cells by immunohistochemistry o hair ollicles at baseline (d) and afer PRP treatment (e). ( ): morphometric analysiso the percentage o Ki+ nuclei in hair ollicles at baseline and afer PRP treatment. (g) and (h): representative microphotographs o CD+

small dermal vessels o hair skin at baseline (g) and afer PRP treatment (h). (i): morphometric analysis o CD+ small dermal vessels o hairskin at baseline and afer PRP treatment; ∗ indicates < 0.05. Original magnication: (a) and (b): x and (d), (e), (g), and (h): x.

hair loss associated with increased expression o caspases and

apoptosis inhibitors and thereore it is ultimately suggestedto activate anagen hair growth [, ]. Antiapoptotic effectso activated PRP have been suggested as one o the majorcontributing actors stimulating hair growth [, ]. PRP-induced activation o antiapoptotic regulators, such as theBcl- protein and Akt signalling, prolongs the survival o dermal papilla cells during the hair cycle [, ]. In addition,the upregulation o FGF-/b-catenin signalling pathwayswith PRP treatment is suggested to stimulate hair growthby inducing ollicular stem cell differentiation as well asprolonging the anagen phase o the hair growth cycle [, ].

Kang et al. [] reported the clinical efficacy o injectiono CD+ cell-containing PRP preparation or pattern hair

loss. In this study, at three months afer the rst treatment, the

patients presented clinical improvement in the mean numbero hairs, 20.5 ± 17.0%, mean hair thickness, 31.3 ± 30.1%,and mean two-point score, 84.4 ± 51.7%, compared withbaseline values. At months, the patients presented clinicalimprovement in mean hair count, 29.2 ± 17.8%, mean hairthickness, 46.4 ± 37.5%, and mean two-point score, 121.3 ±66.8%, compared with baseline.

In our study, AA-PRP was prepared rom a small volumeo blood ( cc) according to the method o Cascade-Selphyl-Esorax system [, ]. Te authors suggested that a sufficientnumber o platelets could be obtained in all patients by using an automated PRP preparation system. Giusti et al.demonstrated that the optimal platelet concentration or the


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(a) (b)

F : richoScan digital image analysis. (a) shows a preoperative hair count . hairs per cm2 and density . per cm2. (b) shows apostoperative hair count , hairs per cm2, and density . per cm2.

(a) (b) (c)

F : Photos demonstrating the division o the scalp in our halves: rontal, parietal, vertex, and occipital (a). Patients with hair losslocalized to the rontal and parietal areas were injected with the AA-PRP only on the rontal areas (b); the parietal area was treated withplacebo based on the injection o physiological solution. Patients with hair loss in the parietal and vertex parts were injected with the AA-PRP only in the parietal part o the scalp (c); the vertex area was treated with placebo based on the injection o physiological solution.

(a) (b)

F : A nonsmoker -year-old male patient affected by hair loss. (a) Preoperative situation o the scalp with hair loss localized to theparietal and rontal areas. (b) Postoperative situation o the scalp two weeks rom the last treatment with increase o the hair count and hairdensity.


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(a) (b)

F : A smoker -year-old male patientaffected by hair loss. (a) Preoperative situationo thescalpwith hair loss localizedto theparietaland rontal areas. (b) Postoperative situation o the scalp two weeks rom the last treatment with increase o the hair count and hair density.

induction o angiogenesis in human endothelial cells was,, platelets/L, whereas excessively high concentra-tions o platelets were suggested to decrease the angiogenicpotential []. In this study, a mean ,,. platelets/L inthe PRP preparation could effectively stimulate ollicular andperiollicular angiogenesis, which is suggestedto be oneo themajor actors in active hair growth [, ]. Our data suggestthat the injection o AA-PRP preparations has a positivetherapeutic effect on male and pattern hair loss without majorside effects.

Conflict of Interests

Te authors declare that there is no conict o interestsregarding the publication o this paper.

Authors’ Contribution

Pietro Gentile and Valerio Cervelli contributed to the ollow-ing: conception and design, paper writing, and nal approvalo the paper; Augusto Orlandi, Alessandra Bielli, and MariaGiovanna Scioli contributed to the ollowing: histomor-phometric evaluation o AA-PRP injection on pattern hairloss and immunohistochemistry analysis; Simone Garcovich

contributed to the ollowing: assessment criteria analysisand richoScan evaluation; Beniamino Cristiano Curcio andGiulio Cervelli contributed to the ollowing: English editing,collection and assembly o data, and data analysis.

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