Abstract

   Platelet-rich plasma (PRP) is an extract enriched with growth factors
   that facilitate skin regeneration and rejuvenation. Here, the
   functionalities of PRP derived from various animal sources have been
   investigated and compared, focusing on its potential therapeutic
   applications in skin regeneration. Total antioxidant capacity, wound
   closure, and melanin content in cultured keratinocytes were used to
   evaluate the efficacy of different animal PRP sources. The PRP derived
   from deer exhibited the highest performance and was selected for
   subsequent proteomic and metabolomic analyses. Our findings indicate
   that deer blood is an optimal source of animal-derived PRP,
   demonstrating significant properties in promoting wound healing,
   anti-inflammatory responses, and skin regeneration. This identified PRP
   from deer sources can be developed as a safe and effective product for
   skin rejuvenation and regeneration.

   Keywords: deer blood, platelet-rich plasma, inflammatory, skin
   regeneration

1. Introduction

   Skin is essential for maintaining homeostasis by protecting the body
   from the external environment. Skin acts as a barrier that prevents
   harmful microbes and chemicals from entering the body and blocking
   harmful ultraviolet (UV) radiation from the sunlight [[34]1]. The
   stratum corneum serves as a heterogeneous barrier that protects against
   dryness and environmental damage while preserving moisture. When the
   skin barrier is compromised, it can lead to increased trans-epidermal
   water loss and decreased hydration, resulting in wounds and potentially
   skin carcinogenesis over time. Skin aging is also a major concern,
   occurring naturally and being exacerbated by environmental factors
   [[35]2,[36]3]. As a result, recent aesthetic research has aimed at
   finding ways to reduce visible signs of aging by addressing both
   internal and external factors [[37]4].

   Platelet-rich plasma (PRP) refers to a plasma fraction concentrate rich
   in platelets obtained after centrifugation of whole blood [[38]5]. It
   includes a variety of growth factors [[39]6,[40]7]. The elevated growth
   factors and fibrinogen can significantly enhance tissue regeneration
   and wound healing [[41]8,[42]9]. Recently, the application of PRP in
   dermatology has drawn great interest from industries [[43]10,[44]11].
   In facial rejuvenation, the injection of PRP has shown positive effects
   on wrinkles, elasticity, and striae dispense, as well as application in
   plastic surgery [[45]12,[46]13,[47]14]. Usage of PRP has also been
   reported in scar repair [[48]15] and melasma regression [[49]16].
   However, most of the works on PRP are focused on autologous PRP or
   allogeneic PRP of the same species, and there are few reports on
   heterologous PRP, which greatly limits the application in clinics.
   Indeed, heterologous dogs’ PRP facilitated wound healing in rabbits,
   and there were no side effects observed [[50]17].

   The family Cervidae, commonly known as the deer family, comprises 23
   genera and 47 species. The antlers from non-ossifying young male deer
   are being used as traditional Chinese medicine (TCM); the species
   includes Cervus nippon Temminck (red deer) and C. elaphus Linnaeus
   (sika deer). In addition, the blood of deer could also be used as TCM
   products. The earliest record of the medicinal properties of deer blood
   in China can be found in “Qianjin Yifang” by Sun Simiao in the Tang
   Dynasty (AD 581-682) [[51]18]. Similar to humans, deer blood is rich in
   various crucial growth factors that have demonstrated anti-inflammatory
   and anti-aging properties. The traditional usages of deer blood in TCM
   include the treatment of anemia, fatigue, and weakness [[52]19]. The
   usages of blood-derived products from deer, e.g., peptides, have been
   reported to have various therapeutic effects, including improving
   cognitive function and promoting wound healing [[53]18,[54]20,[55]21].

   PRP derived from animal sources may be highly effective in enhancing
   wound healing and facial rejuvenation. In searching for active
   non-human PRP, different animal blood sources were screened for their
   functionalities in anti-oxidation, wound healing, and melanin content
   in skin cells. The best source of PRP deriving from deer was
   identified, in accordance with its functions and safety. This research
   offers a fresh theoretical foundation for creating PRP from non-human
   sources as treatments for skin regeneration and rejuvenation, as well
   as in systematically determining the composition and function of
   deer-derived PRP, laying the foundation for the standardization and
   large-scale production of PRP.

2. Materials and Methods

2.1. Materials

   Dulbecco’s modified Eagle medium (DMEM, Cat# 11965092) and fetal bovine
   serum (FBS, Cat# 16000044) were procured from Thermo Fisher Scientific
   (Waltham, MA, USA). Antibodies targeting NF-κB p65 (Rabbit mAb #8242)
   and filaggrin (Mouse mAb #sc-66192) were acquired from Cell Signaling
   Technology (Danvers, MA, USA) and Santa Cruz Biotechnology (Santa Cruz,
   CA, USA), respectively. RNAzol^® RT reagent (R4533), dimethyl sulfoxide
   (DMSO, D2650), and protease inhibitors (P8340) were supplied by
   Sigma-Aldrich (St. Louis, MO, USA). Commercial ELISA kits for VEGF
   (ab100662), FGF (ab99979), IGF (ab100545), and EGF (ab100510) were
   obtained from Abcam (Cambridge, UK).

2.2. PRP Isolation and Processing

   Anticoagulated whole blood (3.2% sodium citrate) from unspecified
   animal donors was processed by centrifugation (2000× g, 15 min, 22 °C)
   to isolate PRP. The supernatant underwent three cycles of freezing (−80
   °C, 1 h) and thawing (37 °C, 10 min) to lyse platelets and liberate
   growth factors [[56]7]. UV-C irradiation (254 nm, 30 min, 15 W) ensured
   sterility prior to lyophilization. The resultant PRP extract was stored
   in light-protected vials at −20 °C.

2.3. Cell Culture

   Human keratinocytes (HaCaT, ATCC^® PCS-200-011), murine melanocytes
   (Melan-A, a gift from Prof. Mingfu Wang), and B16F10 melanoma cells
   (CRL-6475™) were propagated in DMEM containing 10% FBS, 100 U/mL
   penicillin, and 100 μg/mL streptomycin. Cultures were sustained at 37
   °C under 5% CO[2], with media replenished every 48 h.

2.4. Growth Factor Quantification

   ELISA-based detection of VEGF, FGF, IGF, and EGF in human and deer PRP
   followed established protocols [[57]22]. Samples (100 μL) were
   incubated in BSA-blocked 96-well plates (30 min, 25 °C) for 2 h,
   followed by sequential addition of biotinylated detection antibodies
   (1:1000, 2 h) and streptavidin-HRP (1:5000, 1 h). The reactions were
   terminated with 2 N H[2]SO[4], and absorbance at 450 nm was quantified
   (Multiskan™ FC).

2.5. Cell Viability Test

   To assess the cytotoxicity of PRP[deer] [[58]23], 1 × 10^5 cells/well
   (Melan-A, B16F10) were seeded in 96-well plates. After 24 h adhesion,
   the cells were exposed to PRP[deer] (0–50 mg/mL) in DMEM for 24 h.
   After that, 100 μL of MTT (5 mg/mL) was added per well, incubated for 4
   h at 37 °C, and formazan crystals dissolved in the same volume of DMSO.
   Absorbance at 570 nm was measured (SpectraMax^® i3x).

2.6. Melanogenesis Assay

   Cultured Melan-A and B16F10 cells (1 × 10⁵ cells/well, 2 mL) were
   seeded onto 6-well plates overnight and then treated with PRP[deer] (1,
   2, 5, 10, 15 mg/mL) or vitamin C (a positive control at 1 mM) for 48 h.
   Melanin content was spectrophotometrically quantified at 405 nm based
   on a published paper [[59]24].

2.7. AMES Bacterial Reverse Mutation Test

   The Ames test (OECD 471) was employed based on published paper [[60]25]
   to assess PRP[deer] genotoxicity using five Salmonella typhimurium
   strains (TA98, TA100, TA1535, TA1537, TA102) under metabolic activation
   (S9 mix) and non-activation conditions. Mutagenic potential was defined
   as ≥2-fold increase in revertant colonies versus controls (SGS Life
   Science Services, ON, Canada).

2.8. Xenogenic Immune Test

   Female BALB/c mice (6–8 weeks, 18–22 g) were randomized into five
   cohorts (n = 10): saline control, BSA (100 mg/kg), and PRP[deer]
   (50/100/200 mg/kg). The procedures were approved by the Animal Ethics
   Committee of the Hong Kong University of Science and Technology (HKUST)
   (Reference No. PRP/073/20FX). Subcutaneous injections (0.12 mL) were
   administered weekly for four weeks. Post-euthanasia serum IgG, IgM, and
   C3a levels were measured by ELISA.

2.9. Wound Closure Assay

   HaCaT cells at 3 × 10^5 cells/well were seeded onto 6-well plates, and
   serum-starved overnight. Confluent HaCaT monolayers were scratched with
   sterile pipette tips, and treated with PRP[deer] (1, 2, 5, 10, 15
   mg/mL) or VEGF (20 ng/mL) in serum-free medium. Wound closure was
   monitored at 0 h (A[t0]) and 20 h (A[t20])) using a Zeiss Axio Observer
   7 microscope. T-Scratch software (version 3.29.1) calculated recovery
   rates as follows: recovery (%) = (A[t0]–A[t20]) × 100%/A[t0].

2.10. DNA Transfection and Luciferase Assay

   Inflammatory stimulation was initiated by treating murine macrophage
   RAW264.7 cells with lipopolysaccharide (LPS, 100 ng/mL) for 20 h at 37
   °C in a humidified 5% CO[2] incubator. Parallel experiments utilizing
   human HaCaT keratinocytes (1 × 10^5 cells/well, 24-well plates)
   involved treatment with recombinant human TNF-α (20 ng/mL) for 6 h,
   with simultaneous administration of varying concentrations (1, 5, 10
   mg/mL) of PRP[deer.] Pharmacological controls included dexamethasone
   (10 nM) dissolved in DMSO (final concentration < 0.1%).

   Following treatments, cellular lysis was performed using lysis buffer.
   The homogenized lysates underwent centrifugation using a refrigerated
   Eppendorf 5424R microcentrifuge. Clarified supernatants (75 µL
   aliquots) were arrayed in black-walled 96-well microplates for
   luminescence analysis. Bioluminescent signals were quantified using a
   GloMax^® 96 Microplate Luminometer with 1s integration time following 5
   min thermal equilibration. Signal normalization employed total protein
   concentrations determined via micro-BCA assay (Waltham, MA, USA;
   catalog no. 23235).

   For pFLG2-eGFP transfected cell cohorts, fluorescence quantification
   was conducted using a microplate reader equipped with SoftMax^® Pro 7.1
   software. Excitation at 488 nm (5 nm bandwidth) and emission detection
   at 509 nm (8 nm bandwidth) were performed using optimal photomultiplier
   gain settings determined by auto-ranging controls. Protein
   normalization utilized the Bradford method. All experimental conditions
   included triplicate technical replicates with three biological repeats,
   randomized across plate quadrants to control for positional effects.

2.11. RNA Extraction and Reverse Transcription

   Inflammatory stimulation was initiated by treating murine macrophage
   RAW264.7 cells with lipopolysaccharide (LPS, 100 ng/mL) for 20 h in a
   cell incubator. Parallel experiments utilizing human HaCaT
   keratinocytes (1 × 10^5 cells/well, 24-well plates) involved treatment
   with recombinant human TNF-α (20 ng/mL) for 6 h, with simultaneous
   administration of varying concentrations (1, 5, 10 mg/mL) of PRP[deer.]
   Pharmacological controls included dexamethasone (10 nM) dissolved in
   DMSO (final concentration < 0.1%).

   Post-treatment, adherent cells were washed thrice with PBS and
   mechanically detached using cell scrapers. Total RNA isolation was
   performed using RNAzol^® RT reagent, with a modified monophasic
   phenol-guanidine isothiocyanate protocol. Cell lysates were homogenized
   in 500 μL RNAzol^® RT, followed by phase separation with 100 μL
   chloroform in nuclease-free polypropylene tubes.

   RNA precipitation was achieved by mixing the aqueous phase with 250 μL
   70% ethanol (v/v). Subsequent washing steps employed 75% ethanol (v/v)
   with vortex agitation at 1500 rpm for 10 s, followed by air-drying
   under laminar flow for 5 min. Purified RNA pellets were resuspended in
   20 μL nuclease-free water and quantified via NanoDrop 2000c
   spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) with
   dual-wavelength analysis (A260/A280: 1.8–2.1). Reverse transcription
   utilized the PrimeScript™ RT Master Mix in 10 μL reactions containing 5
   ng RNA template.

2.12. Immunofluorescent Staining

   HaCaT keratinocytes were cultured on #1.5 thickness glass coverslips
   pre-coated with poly-L-lysine (0.01% w/v) in 12-well plates at an
   initial density of 5 × 10^4 cells/mL. Post-treatment, cells underwent
   sequential fixation with freshly prepared 4% paraformaldehyde in PBS
   for 10 min at 22 °C, followed by three washes with Tris-buffered saline
   (TBS).

   Permeabilization and blocking were simultaneously performed using TBST
   buffer (TBS containing 5% BSA and 0.1% Triton X-100) for 120 min at 22
   °C under orbital shaking (50 rpm). Primary antibodies were applied in
   humidity chambers at specified dilutions: rabbit monoclonal anti-NF-κB
   p65 (Cell Signaling 8242S; 1:200) and mouse monoclonal anti-filaggrin
   (Santa Cruz sc-66192; 1:50), with overnight incubation at 4 °C on a
   thermoelectric cooling plate.

   Secondary detection employed spectrally distinct Alexa Fluor^®
   conjugates: donkey anti-mouse IgG-Alexa 488 (Abcam ab150105; 1:200) and
   donkey anti-rabbit IgG-Alexa 647 (Abcam ab150075; 1:200) dissolved in
   antibody stabilization buffer, incubated for 135 min at 22 °C under
   light-protected conditions. Nuclei were counterstained with ProLong™
   Gold Antifade Mountant containing DAPI, cured for 24 h at 4 °C before
   imaging.

   Confocal microscopy was performed on a Leica SP8 STED system (LAS X
   3.7.4) equipped with HyD detectors and 63× oil immersion objectives.
   Sequential acquisition parameters included the following: 405 nm diode
   (DAPI), 488 nm Argon (Alexa 488), and 638 nm HeNe (Alexa 647) lasers
   with 5% power, 0.8 Airy unit pinhole, and 4× line averaging. Z-stacks
   (0.3 μm steps) were processed using LAS X 3.7.4 software with
   deconvolution algorithms.

2.13. Proteomics and Untargeted Metabolomics Analyses

   The analyses of proteomics and untargeted metabolomics were performed
   by Shanghai Applied Protein Technology (Shanghai, China). The procedure
   of proteomics was consistent with a previous study [[61]26], and the
   metabolomics was following the protocol established by [[62]27].

2.14. Statistical Analysis

   GraphPad Prism 8.0.1 was used here to conduct the data analysis.
   Statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001) was
   determined via one-way ANOVA with Tukey’s test.

3. Results

3.1. The Non-Human Sources of PRP

   Different sources of PRP products, i.e., human, fish, chick, rabbit,
   pig, deer, and bovine, were chosen for comparison of their efficacies.
   Biochemical assays were developed and performed to test their
   activities relevant to the skincare functions. Firstly, antioxidant
   activity was compared as a screening indicator in these PRP samples.
   The results showed that PRP from deer blood (PRP[deer]) showed the best
   antioxidant capacity, with 30.9 nmol trolox equivalent ([63]Figure 1A).
   The human sample (PRP[human]) showed an antioxidant capacity of 23.2
   nmol trolox equivalent. Their ranking in terms of antioxidative
   activities was followed by bovine (PRP[bovine]), pig (PRP[pig]),
   chicken (PRP[chicken]), and fish and rabbit (PRP[fish]; PRP[rabbit]).
   The difference between PRP[deer] and PRP[rabbit] was over 20 nmol of
   trolox equivalent ([64]Figure 1A). Secondly, the ability of promoting
   wound healing of these PRPs was also evaluated in cultured
   keratinocytes in vitro. The results showed that the best performer was
   PRP[deer], which had a wound recovery rate of about 45.6% over the
   control group at 9.7%, followed by PRP[human] and PRP[bovine]
   ([65]Figure 1B). The other PRP sources, however, were low (less than
   33%) in wound-healing process. Application of VEGF served as a positive
   control here. Skin brightening efficacy is a very important metric when
   developing skincare products, and which is often evaluated by using
   melanin scavenging capacity. Here, we compared the melanin-reducing
   properties of different PRP samples in cultured melanocytes. PRP[deer]
   showed the best reduction in melanin content ([66]Figure 1C). The other
   sources of PRP did not show such an obvious reduction. Vitamin C served
   as a control to downregulate melanin content. In view of these
   functionalities, PRP[deer] was chosen for further analysis.

Figure 1.

   [67]Figure 1
   [68]Open in a new tab

   PRP[deer] shows better effectiveness than other sources of PRP samples.
   Different animal sources, all at 5 mg/mL of PRP, were subjected to
   various assays of skin function A–C. (A) The anti-oxidation ability of
   different PRPs was determined by a commercial kit. The data are
   expressed as trolox equivalents. (B) Confluent HaCaT monolayers were
   scratched with sterile pipette tips; images illustrating wound healing
   were taken at 0 h (A[t0]) and 20 h (A[t20]). Quantitation of migration
   was calibrated as the following formula: (A[t0]–A[t20]) × 100%/A[t0].
   (C) Different PRPs (5 mg/mL) were applied onto cultured melanocyte
   (Melan-A) cells for 48 h, and a melanogenesis assay was determined. (D)
   Different concentrations (0.5–50 mg/mL) of PRP[deer] were applied onto
   cultured HaCaT and Melan A cells for 48 h, and an MTT assay was
   determined. (E) Cultured HaCaT cells created a wound; PRP[deer] (1–15
   mg/mL) was applied for 20 h. Images illustrating wound healing were
   taken at 0 h (A[t0]) and 20 h (A[t20]). (F) Quantitation of migration
   was calibrated. The values in mean ± SEM., n = 8. * p < 0.05; ** p <
   0.01; *** p < 0.001 compared with corresponding human or control.

   To evaluate toxicity and identify the ideal concentration of PRP[deer]
   in cells, MTT assay was conducted in HaCaT keratinocytes and
   melanocytes. Experimental results showed that in the PRP samples,
   taking PRP[deer] as an example, significant cell death did not show
   when treated with PRP[deer] from 0.5–50 mg/mL. A robust proliferative
   effect was found at 50 mg/mL PRP[deer] in keratinocytes and 0.5 to 20
   mg/mL in melanocytes ([69]Figure 1D). High concentration of PRP[deer],
   however, achieved a decrease in cell number, in accordance with the
   human source of PRP [[70]22]. To avoid induction of cytotoxicity, the
   concentration of PRP[deer] 1–15 mg/mL in this study was applied. The
   migration ability of PRP[deer] in different concentrations was
   determined. The enhancement of wound healing did not show a significant
   increase at the lower concentrations of 1 and 2 mg/mL of PRP[deer].
   However, there was a significant increase from 25% at 5 mg/mL to 65% at
   15 mg/mL, which was better than that of the VEGF control, suggesting
   that PRP[deer] should have excellent wound-healing ability ([71]Figure
   1E,F).

   LPS-stimulated macrophages (RAW264.7 cells) were used as a model to
   mimic chronic inflammation. Treatment with LPS led to a significant
   increase in inflammatory markers’ expression, such as iNOS, IL-6, and
   IL-1β, ranging from 20 to 2000 times in cultured RAW264.7 cells
   ([72]Figure 2A). Dexamethasone effectively suppressed the expression of
   these inflammatory markers in LPS-treated cultures. Three different
   concentrations of PRP[deer] (0–10 mg/mL) were tested. Under the
   treatment of different concentrations of PRP[deer], the concentrations
   of iNOS, IL-1β, and IL-6 markers were markedly suppressed in cultured
   LPS-stimulated macrophages ([73]Figure 2A).

Figure 2.

   [74]Figure 2

   [75]Figure 2
   [76]Open in a new tab

   PRP[deer] reduces the induced inflammatory responses. (A) The mRNA
   levels of iNOS, IL-1β, and IL-6 were measured in inflammatory activated
   cultured RAW264.7 cells after being pretreated with PRP[deer] (1, 5, 10
   mg/mL) for 4 h. Then, the exposure of LPS (100 ng/mL) was 20 h.
   Dexamethasone at 10 nM was a positive control. (B) The mRNA levels of
   TNF-α, IL-1β, and IL-6 in cultured HaCaT cells after 4 h of
   pretreatment of dexamethasone (10 nM) and PRP[deer] (10 mg/mL), and 20
   h of TNF-α (20 ng/mL) stimulation. (C) The pNF-κB-Luc plasmid was
   transfected into RAW264.7 or HaCaT cells for 4 h, followed by
   pretreating with PRP[deer] (1, 5, 10 mg/mL) for 4 h, before the
   exposure to LPS (100 ng/mL) or TNF-α (20 ng/mL), respectively, for 20
   h. Dexamethasone (10 nM) served as a control. (D) The protein level of
   NF-κB p65 in cytosolic and nuclear cells was measured in HaCaT
   keratinocytes, in representative confocal images. (E) The
   quantifications of intensity percentage in (D). The values are
   expressed in the fold of change to the normalized basal activity set at
   1, in mean ± SEM., n = 8. * p < 0.05; ** p < 0.01; *** p < 0.001
   compared with LPS- or TNF-α-treated groups (Bkg).

   The effects of PRP[deer] samples on keratinocytes were evaluated using
   TNF-α treatment. After TNF-α treatment, the mRNA levels of TNF-α,
   IL-1β, and IL-6 increased significantly by 10–23-fold in keratinocytes
   ([77]Figure 2B). However, the treatment with PRP[deer] markedly reduced
   these mRNA expressions in keratinocytes. The maximal suppression
   observed for TNF-α expression was approximately 70% ([78]Figure 2B).

   A DNA construct, pNF-κB-Luc, was used for plasmid transfection in both
   RAW264.7 and keratinocyte cell models. In RAW264.7 cells, LPS induced
   the luciferase signal, indicating promoter activity, by more than
   2-fold, while TNF-α increased the signal by approximately 5.5-fold in
   HaCaT cells ([79]Figure 2C). Following co-treatment with PRP[deer],
   luciferase activity significantly decreased, showing reductions of 20%
   to 75%. Furthermore, the protein level of NF-κB p65 was evaluated in
   keratinocytes by immunostaining. When inflammation occurs, p65 protein
   migrates from the cytoplasm to the nucleus, so detection of the
   percentage of nuclear/cytosol of NF-κB p65 protein can reflect
   inflammation; the results revealed a significant decrease, showing
   about a 70% reduction after treatment with PRP[deer] ([80]Figure 2D,E).

3.2. PRP[deer] Potentiates Skin Rejuvenation

   To assess the moisturizing effects of PRP[deer] on skin keratinocytes,
   we evaluated its ability to regulate the mRNAs of filaggrin and
   filaggrin-2. Treatment with PRP[deer] showed a significant,
   dose-dependent rise in both filaggrin and filaggrin-2, with filaggrin-2
   exhibiting a notable sensitivity to the treatment, showing a maximum
   2-fold increase in expression ([81]Figure 3A). To demonstrate activated
   gene transcription, the filaggrin-2 promoter was tagged with an eGFP
   construct, i.e., pFLG2-eGFP. In keratinocytes transfected with
   pFLG2-eGFP, PRP[deer] application enhanced the fluorescent signal,
   indicating increased promoter activity, with a robust 2-fold increase
   in pFLG2-eGFP fluorescence, surpassing the positive control of
   dexamethasone ([82]Figure 3B). Filaggrin, a protein that is not
   associated with membranes and is in keratohyalin granules, was detected
   using immunochemical staining in cultured keratinocytes. Treatment with
   PRP[deer] induced a significant increase in overall cellular staining
   for filaggrin, consistent with the mRNA and promoter assay results
   ([83]Figure 3C,D).

Figure 3.

   [84]Figure 3
   [85]Open in a new tab

   PRP[deer] reverses the decrease in filaggrin and filaggrin-2
   expressions caused by TNF-α by promoting moisturization and
   differentiation. The cultured HaCaT keratinocytes were pretreated with
   dexamethasone (10 nM), PRP[deer] (1, 5, 10 mg/mL) for 4 h. Then, the
   exposure of TNF-α (20 ng/mL) was 20 h. (A) The mRNA levels of filaggrin
   (FLG) and filaggrin-2 (FLG-2) were evaluated by RT-PCR. (B) The
   pFLG2-eGFP plasmid was transfected into HaCaT cells for 4 h, followed
   by another 4 h pre-incubation of PRP[deer] before 20 h stimulation of
   TNF-α. Dexamethasone was a control. (C) HaCaT keratinocytes were
   treated with TNF-α, dexamethasone, PRP[deer] (10 mg/mL), as given in
   (A). The cultures were stained with FLG antibodies or DAPI.
   Representative fluorescence images are displayed. (D) The FLG intensity
   was quantified from (C). Values are normalized basal expression set at
   1, in mean ± SEM., n = 5. * p < 0.05; ** p < 0.01; *** p < 0.001
   compared with TNF-α-treated group (Bkg).

   To investigate PRP[deer] on melanogenesis, the productions of melanin
   in both Melan-A and B16F10 cells were conducted. The results showed
   that a higher concentration of PRP[deer] had a stronger ability to
   inhibit melanin production ([86]Figure 4A). When the PRP[deer] sample
   concentration was 1 mg/mL, the inhibition rate was only around 2%,
   while when PRP[deer] increased to 15 mg/mL, the melanin inhibition
   reached over 40% in Melan-A. The results were similar when the
   experiment was repeated on cultured B16F10 cells ([87]Figure 4A). The
   inhibition of melanin by PRP[deer] had an obvious dose-dependent
   relationship at ~10 mg/mL of PRP[deer].

Figure 4.

   [88]Figure 4
   [89]Open in a new tab

   PRP[deer] regulates the levels of melanin and collagen. (A) Cultured
   melanocytes (Melan-A) and melanoma (B16F10) were plated onto 6-well
   plates. PRP[deer] (1 to 15 mg/mL) was administered for 48 h, and the
   melanin content was assessed. Vitamin C (10 μM, positive control)
   demonstrates an approximate 40% reduction. (B) Cultures were treated as
   in (A), and collagen 2 (~141 kDa) was evaluated by Western blot assays;
   α-tubulin (~55 kDa) served as internal control for quantification (left
   panel). TGF-β1 (10 ng/mL, positive control). The quantification was
   conducted by ImageJ software (2.1.4.5) (right panel). Values are
   expressed as the percentage of decrease in the control or the fold of
   change relative to normalized basal expression set at 1, in mean ±
   SEM., n = 5. ** p < 0.01; *** p < 0.001 compared with the control.

   Collagen is a key factor in determining skin elasticity, and dermal
   fibroblasts are the primary cells responsible for collagen production
   in the skin. Therefore, the PRP[deer] was applied onto cultured mouse
   dermal fibroblasts. By using an antibody recognizing collagen type II,
   the result showed that PRP[deer] at a high dose could significantly
   induce collagen production by ~30-fold in cultured dermal fibroblasts
   ([90]Figure 4B), indicating that PRP[deer] could promote skin
   elasticity.

3.3. Proteomics Analysis of PRP[deer] and PRP[human]

   To evaluate the potential proteins of PRP[deer] in wound healing, a
   non-labeling proteomic analysis was performed, using PRP[human] protein
   as a control. Proteomic profiles were generated, and principal
   component analysis (PCA) was conducted to calculate scores for all
   samples. In the PCA plots, PRP[deer] subjects were distinctly separated
   from PRP[human] ([91]Figure 5A). The three independent subgroups showed
   significant overlap, indicating good stability and reliability in
   sample measurement and data analysis. The Venn diagram illustrated the
   number of identified proteins with significant quantitative
   similarities and differences among the three groups ([92]Figure 5B). A
   total of 558 proteins were identified in PRP[deer], while 426 proteins
   were identified in PRP[human]. Of these, 315 proteins were shared
   between the two groups, indicating a substantial overlap of 47.1%,
   which supports the robustness of the proteomic maps. Additionally, a
   volcano plot of overlapping proteins was generated ([93]Figure 5C). A
   total of 72 significant differential proteins were identified,
   representing 12.9% of all detected proteins, with 29 proteins showing
   significant upregulation and 43 proteins showing significant
   downregulation.

Figure 5.

   [94]Figure 5
   [95]Open in a new tab

   Proteomics analysis of PRP[deer] and PRP[human]. (A) PCA scores for
   proteomic analysis of PRP[deer] and PRP[human]. (B) Venn diagram
   showing the overlapping and unique proteins in PRP[deer] and
   PRP[human]. (C) Volcano plot analysis of proteomic changes in PRP[deer]
   and PRP[human]. (D) The amounts of VEGF, FGF, IGF, and EGF in PRP[deer]
   and PRP[human] were measured by ELISA. (E) GO analysis of proteins
   enriched in PRP[deer]. (F) KEGG displays the top 20 pathways analysis
   of PRP[deer] proteins.

   FGF, VEGF, EGF, and IGF are common ingredients in mammalian blood, as
   well as in PRP, and are well known to promote wound healing. The
   proteomic of PRP[deer] indicated the existence of the fragments of
   these proteins ([96]Table S1). Thus, the existence of growth factors in
   the prepared PRP from deer and humans was confirmed. Both PRP[deer] and
   PRP[human] contained similar amounts of VEGF, FGF, IGF, and EGF
   ([97]Figure 5D). IGF showed the largest concentration in PRP[deer] at
   3036 µg/g, slightly higher than that of PRP[human] at 2380 µg/g. Also,
   PRP[deer] and PRP[human] showed high levels of EGF and FGF, both over
   30 µg/g. VEGF was the lowest among these growth factors with a
   concentration of less than 5 µg/g.

   To further investigate the functions of proteins in PRP[deer], GO and
   KEGG pathway enrichment analyses were performed. GO analysis assessed
   cellular components (CCs), molecular functions (MFs), and biological
   processes (BPs) for PRP[deer] ([98]Figure 5E). The cellular component
   analysis indicated that 88.2% of the identified proteins were
   associated with cellular anatomical entities. Molecular function
   analysis revealed that binding (66.1%) was the predominant category for
   PRP[deer] proteins. Additionally, around 19, 13, and 12 proteins were
   linked to transporter activity, antioxidant activity, and ATP-dependent
   activity, respectively. Biological process analysis highlighted an
   enrichment of proteins involved in cellular processes (72.6%),
   metabolic processes (69.4%), and biological regulation (65.6%). Other
   notable categories included immune system processes (28.9%), cell
   proliferation (4.8%), and growth (4.5%). These proteins may be closely
   related to the antioxidant and wound-healing capabilities of PRP[deer].
   A comprehensive list of the identified proteins can be found in
   [99]Table S1. Subsequently, KEGG analysis identified the top 20
   pathways associated with PRP[deer] proteins. Key pathways included
   “Focal adhesion”, “PI3K-Akt signaling pathway”, “Platelet activation”,
   “HIF-1 signaling pathway”, and “MAPK signaling pathway”, all of which
   play significant roles in wound healing ([100]Figure 5F).

3.4. Metabolomics Analysis of PRP[deer] and PRP[human]

   Global metabolic profiles of PRP[deer] and PRP[human] were analyzed
   using UHPLC-MS/MS, and PCA analysis revealed a clear distinction
   between the two groups, confirming the reliability of the metabolomics
   data ([101]Figure 6A). After the annotation, 1425 metabolites were
   identified in PRP[deer] and 1372 in PRP[human] ([102]Figure 6B).
   Notably, 1345 metabolites were common to both groups, indicating a
   significant overlap of 92.6%, which supports the robustness of the
   metabolite maps. To further investigate the differences between
   PRP[deer] and PRP[human], statistical analysis was performed on the
   metabolic profiles, resulting in a volcano plot shown in [103]Figure
   6C. This analysis identified 209 significant differential molecules,
   representing 14.7% of all detected metabolites, with 101 showing
   significant upregulation and 108 downregulation. The extracted ion
   chromatogram (XIC) revealed thousands of mass spectral features
   primarily eluting in both positive and negative modes for PRP[deer]
   within the first 10 min ([104]Figure 6D). The metabolites were
   classified based on their chemical properties ([105]Figure 6E), with
   the majority being lipids and lipid-like molecules (29.2%), followed by
   organic acids and derivatives (17.6%), organo-heterocyclic components
   (13.5%), and benzenoids (12.6%). A comprehensive list of the identified
   metabolites can be found in [106]Table S2.

Figure 6.

   [107]Figure 6
   [108]Open in a new tab

   Metabolomics analysis of PRP[deer] and PRP[human]. (A) PCA scores for
   metabolomics analysis of PRP[deer] and PRP[human]. (B) Venn diagram
   showing the overlapping and unique metabolites in PRP[deer] and
   PRP[human]. (C) Volcano plot analysis of metabolite changes in
   PRP[deer]and PRP[human]. (D) Extracted ion chromatogram of PRP[deer]
   metabolomic. (E) Chemical categories of metabolites in PRP[deer]. (F)
   KEGG displays the top 20 pathways analysis of PRP[deer] metabolites.

   To further explore the metabolic disturbances in PRP[deer], pathway
   enrichment analysis was conducted, with the top 20 pathways illustrated
   in [109]Figure 6F. Most pathways were linked to metabolic processes,
   biosynthesis of secondary metabolites, and microbial metabolism in
   various environments. Additionally, 39 metabolites were associated with
   the biosynthesis of an amino acid’s pathway, which may be relevant to
   wound healing.

   The abundance of different proteins and metabolites in PRP[deer] varied
   greatly. The molecules related to skin rejuvenation are proposed for
   quantifying control on the isolated PRP. As listed in [110]Table 1,
   collagen type VI α3 chain and dexpanthenol, playing important roles in
   skin rejuvenation, are widely expressed in PRP[deer]. In addition,
   PRP[deer] contains excellent antioxidants, such as hemopexin,
   niacinamide, and glutathione oxidized. The PRP[deer] also contains a
   number of proteins and metabolites associated with wound healing, such
   as Ig-like domain-containing protein, apoptosis inhibitor, vitronectin,
   hydroxy arginine, 6-ketoprostaglandin E1, palmitic acid, and AMP; these
   metabolites are important in promoting cell migration. PRP[deer] also
   contains a variety of growth factors, such as IGF and EGF, which may be
   the main proteins involved in promoting angiogenesis, while the
   pathways related to angiogenesis in PRP[deer] include PI3K-Akt and the
   HIF-1 signaling pathway. PRP[deer] contains inflammatory-responsive
   molecules, such as complement C3, alpha-2-macroglobulin, plasma
   kallikrein, arachidonoyl serotonin, and docosatetraenoic acid.
   Arachidonoyl serotonin, formed by the conjugation of arachidonic acid
   and serotonin, and 6-ketoprostaglandin E1a produced during the
   metabolism of arachidonic acid are enriched in PRP[deer]. Thus,
   arachidonic acid could be a potential active substance in the promotion
   of wound healing by PRP; however, this remains to be verified at the
   molecular level.

Table 1.

   The main proteins and metabolites related to wound healing and skin
   rejuvenation in PRP[deer].
   Function Molecules Biological Process ^c
   Proteins ^a Metabolites ^b
   Skin rejuvenation COL6A3 Dexpanthenol Collagen trimer
   Hemopexin Niacinamide, Glutathione oxidized Antioxidant
   Wound healing Ig-like domain-containing protein, Apoptosis inhibitor,
   Vitronectin Hydroxyarginine, 6-Ketoprostaglandin E1, Palmitic acid, AMP
   Cell migration
   Fibronectin, IGF, EGF Arachidonic acid Angiogenesis
   Complement C3, Alpha-2-macroglobulin, Plasma kallikrein
   Arachidonoylserotonin, Docosatetraenoic acid Anti-inflammatory
   [111]Open in a new tab

   ^a The proteins from the results of proteomics list in [112]Table S1;
   ^b the metabolites from the results of metabolomics list in [113]Table
   S2; ^c biological process analysis based on the results of KEGG
   analysis of PRP[deer] proteins and metabolites.

3.5. Safety Parameters of PRP[deer]

   To test the safety of PRP[deer], the mice were injected by intravenous
   of the PRP. After a month of weekly injections, the concentrations of
   IgG, IgM, and C3a in the serum showed no significant differences among
   these three PRP groups. In comparison, the BSA group exhibited a
   notable increase when contrasted with the control group ([114]Figure
   7). Thus, the doses of PRP[deer] showed no immune activation even at
   high doses. The Ames Bacterial Reverse Mutation Test was used to test
   the mutagenic potential of PRP[deer]. In the test, TA97a, TA98, TA100,
   TA102, and TA1535—five Salmonella typhimurium strains—were employed.
   The number of colonies was calculated to determine the genotoxicity of
   PRP[deer]. Under the experimental conditions, compared with the solvent
   control, among the 20 samples of PRP[deer] that were tested, the number
   of revertant colonies had no significant difference ([115]Table 2).
   Thus, PRP[deer] had no mutagenic effect on Salmonella typhimurium.

Figure 7.

   [116]Figure 7
   [117]Open in a new tab

   Immunostimulatory analysis of PRP[deer]. After treated with bovine
   serum albumin (BSA, 1 mg/kg) or PRP[deer] (50, 100, and 200 mg/kg) for
   1 month with weekly intravenous injection, the amounts of IgG, IgM, and
   C3a in the serum of mice were measured by ELISA. The values are in mean
   ± SEM., n = 10. *** p < 0.001 compared with control.

Table 2.

   Genotoxicity analysis of PRP[deer] by bacterial reverse mutation Ames
   test.
   Strains ^a Treatment Number of Colonies Genotoxic
   No metabolic Activation Metabolic Activation
   TA97a Positive control 1052.7 ± 31.7 ^b 1145.7 ± 12.3 + ^c
   Solvent Group 163.7 ± 2.5 173.7 ± 4.9 −
   Untreated Group 162.3 ± 1.5 172.7 ± 2.1 −
   Test Group 162.7 ± 3.5 169.7 ± 3.5 −
   TA98 Positive control 455.7 ± 36.2 557.0 ± 25.9 +
   Solvent Group 44.3 ± 1.5 44.7 ± 1.5 −
   Untreated Group 44.7 ± 2.5 44.7 ± 2.5 −
   Test Group 44.0 ± 1.0 44.3 ± 1.5 −
   TA100 Positive control 1057.0 ± 28.5 1142.3 ± 15.8 +
   Solvent Group 164.7 ± 4.0 173.0 ± 3.0 −
   Untreated Group 162.7 ± 1.5 171.3 ± 3.2 −
   Test Group 142.7 ± 2.5 171.3 ± 4.2 −
   TA102 Positive control 958.7 ± 26.1 1058.3 ± 36.6 +
   Solvent Group 314.3 ± 3.5 325.0 ± 3.0 −
   Untreated Group 313.7 ± 5.7 323.3 ± 3.2 −
   Test Group 312.3 ± 3.2 324.9 ± 4.2 −
   TA1535 Positive control 95.3 ± 2.5 356.7 ± 28.4 +
   Solvent Group 27.0 ± 1 28.7 ± 2.1 −
   Untreated Group 28.0 ± 2.6 28.3 ± 3.1 −
   Test Group 28.3 ± 2.1 28.0 ± 3.0 −
   [118]Open in a new tab

   ^a S. typhimurium strains (TA97a, TA98, TA100, TA102, and TA1535) were
   used; ^b Values are in mean ± SEM., n = 20; ^c “+” indicates
   genotoxicity, “−” indicates non-genotoxicity.

4. Discussion

   Regenerative medicine is a developing area focused on replacing or
   restoring damaged tissues or diseased tissues and organs using
   biological materials and engineering approaches [[119]2]. PRP has
   become a promising therapeutic intervention in regenerative medicine
   because of its capacity to influence various cellular processes related
   to tissue repair and regeneration [[120]10,[121]15]. However, the
   availability of human blood donors may be limited in certain regions or
   populations, and human-derived PRP may be more expensive to prepare and
   require more stringent regulatory oversight. As a result, animals have
   become a popular source of PRP due to their ready availability and
   ability to provide large quantities of blood for preparation, which is
   particularly useful in research studies where large quantities of PRP
   are required for experimental purposes. It is important to note that
   PRP derived from different animal sources can vary greatly. The
   efficacy of PRP preparations is intrinsically linked to interspecies
   variations in hematological parameters, particularly the
   platelet-to-erythrocyte volume ratio. Empirical evidence confirms that
   centrifugal force and duration critically modulate PRP quality metrics,
   including mean platelet volume (MPV), platelet recovery efficiency, and
   functional integrity [[122]28]. Comparative studies in caprine and
   ovine models demonstrate that gradient centrifugation protocols
   employing reduced centrifugal forces (e.g., 200× g) with extended
   durations (15 min) enhance the platelet yield by 30–40% while
   concurrently elevating MPV [[123]29]. This phenomenon is attributed to
   the preferential sedimentation of erythrocytes under low-speed
   conditions, thereby minimizing platelet loss during phase separation.

   The choice of animal species affects the composition and activity of
   the PRP products, as well as the potential risks associated with that,
   including the risk of transmitting infectious diseases and the risk of
   immunogenic reactions. In a study, the authors extracted PRP from
   humans, rats, and goats, using rats as a model to compare the effects
   of PRP from these three different species on bone regeneration. The
   results showed that PRP derived from both rats and goats had no
   significant effect on bone formation. However, human PRP was found to
   improve the initial osteogenic response of human bone grafts [[124]30].
   In contrast, another study reported that PRP derived from dogs
   demonstrated bone regeneration efficacy [[125]31]. The studies suggest
   that the differences in functions among different animals may be
   related to variations in platelet content and types of growth factors
   in their PRPs. The average platelet count in rats is significantly
   higher than that in humans, and parallelly the average platelet volume
   in rats is significantly lower. Given the low platelet volume in rats
   and that growth factors are stored in the alpha granules of platelets,
   we can expect varied concentrations in growth factors between rat and
   human platelets. Quantitative analyses of growth factors in PRP
   prepared from rats, pigs, goats, and humans show that human PRP
   contains higher concentrations of TGF-β1 and PDGF αα, ββ, and αβ30 per
   platelet, as compared to rats, pigs, and goats [[126]32,[127]33]. Our
   results indicated that both deer PRP and human PRP contained similar
   amounts of VEGF, FGF, IGF, and EGF. It can be inferred that after
   platelet activation, deer PRP could release more growth factors
   compared to rat, pig, and goat PRPs, resulting in the more pronounced
   effect of deer PRP.

   Thus, selecting the right source of PRP for applications is essential
   to guarantee the product’s safety and effectiveness. To address this
   issue, we have conducted a study to screen several common animal types
   of PRP and evaluate them in terms of antioxidant activity,
   wound-healing ability, and inhibition of inflammation and
   melanogenesis. Our results indicated that the deer blood-derived PRP
   excelled in all indicators. Additionally, we performed genotoxicity
   testing and found that deer blood was non-toxic and non-mutagenic,
   suggesting that deer blood may be an ideal source of animal-derived PRP
   for certain applications. In addition, the farming of deer is very
   popular in China. The antler base from male C. nippon or C. elaphus has
   been extensively utilized in Chinese medicine to treat various
   ailments. Over 260,000 deer are raised mainly for velvet antler
   production in China. Blood withdrawal is a normal procedure to
   stimulate the growth of antlers, and this blood collection could be a
   means of recycling the waste products during deer farming.

   For a better medical application, PRP[deer] is necessary to be
   standardized by using established biomarkers before assessing the
   skincare-related biological activities of PRP[deer]. When selecting
   protein biomarkers in PRP, several factors must be considered,
   including their relevance to the target tissue or condition, their
   specificity and sensitivity, and their stability and reproducibility
   [[128]34,[129]35]. Here, we measured the proteins and metabolites of
   PRP[deer]. The proteins, including vitronectin, fibrinogen alpha chain,
   and plasma kallikrein, having important roles in the maintenance of
   platelet function and wound healing, were highly expressed in
   PRP[deer], and therefore, these proteins could be ideal candidates as
   biomarkers for quality control. Based on the same criteria, the
   non-protein components, e.g., arachidonic acid, glutathione oxidized,
   and palmitic acid, significantly enriched in PRP[deer] and having
   positive effects on wound healing, could be another biomarker. In
   addition, the high contents of growth factors, e.g., FGF, IGF, VEGF,
   and EGF, in PRP[deer], could also be included as biomarkers for quality
   control. A study compared the protein composition of human
   platelet-poor plasma and human platelet lysate, revealing that the
   majority of the proteins specific to human platelet lysate are
   implicated in the stress response and wound healing [[130]36]. This
   finding provides a reference for our subsequent research on standard
   markers from the deer source.

   Our study showed that PRP[deer] may enhance angiogenesis and encourage
   both cell proliferation and migration, and have anti-inflammatory
   effects, which can be particularly beneficial in the context of tissue
   injury and repair. The underlying mechanisms of therapeutic effects of
   PRP on tissue repair and regeneration are complex and should be
   multi-factorial. PRP is rich in growth factors that are known to
   promote angiogenesis as well as stimulate cell proliferation and
   migration [[131]11]. Furthermore, PRP can boost the accumulation of
   extracellular matrix components, like collagen and fibronectin, which
   are crucial for tissue repair and regeneration [[132]10]. Our
   proteomics analysis showed that many signaling proteins relating to the
   above functions were being enriched within PRP[deer], including MAPK
   signaling and PI3K/Akt signaling. Thus, animal-derived PRP is a
   promising alternative to human-derived PRP in regenerative therapy and
   tissue fabrication. However, the animal species are critical to
   guaranteeing the safety and effectiveness of the PRP product. Our study
   suggests that deer blood may be an ideal source of animal-derived PRP
   for clinical applications.

5. Conclusions

   Here, we provide compelling evidence of the superior skincare
   properties of PRP[deer], demonstrating its efficacy in rejuvenating
   skin, as well as surpassing the functionalities and contents of growth
   factors of other animal sources. Additionally, we conducted a thorough
   analysis of the protein composition and safety of PRP[deer]. These
   results suggest promising applications for PRP[deer] in both cosmetics
   and/or medical treatments.

Supplementary Materials

   The following supporting information can be downloaded at
   [133]https://www.mdpi.com/article/10.3390/cells14070526/s1. Table S1:
   the full list of the obtained proteins of PRPdeer; Table S2: the full
   list of the obtained metabolites of PRPdeer.
   [134]cells-14-00526-s001.zip^ (249.3KB, zip)

Author Contributions

   Conceptualization, Z.-Q.W. and K.W.-K.T.; methodology, Z.-Q.W.,
   Q.W.-S.L., X.G., Q.-Y.W., T.T.-X.D. and K.W.-K.T.; investigation,
   Z.-Q.W., Q.W.-S.L. and Q.-Y.W.; data curation, Z.-Q.W., Q.W.-S.L. and
   X.G.; writing—original draft preparation, Z.-Q.W.; writing—review and
   editing Q.W.-S.L., X.G., T.T.-X.D. and K.W.-K.T.; validation, Q.-Y.W.;
   resources, T.T.-X.D.; visualization, Z.-Q.W. and K.W.-K.T.;
   supervision, Q.W.-S.L., Q.-Y.W. and K.W.-K.T.; project administration,
   K.W.-K.T.; funding acquisition, T.T.-X.D. and K.W.-K.T. All authors
   have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

   The procedures were approved by the Animal Ethics Committee of the Hong
   Kong University of Science and Technology (HKUST) (Reference No.
   PRP/073/20FX; Approval date: 2 October 2020).

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The original contributions presented in this study are included in the
   article/[135]Supplementary Materials. Further inquiries can be directed
   to the corresponding author.

Conflicts of Interest

   The authors declare no conflicts of interest.

Funding Statement

   This research was funded by Guangdong-Hong Kong Technology Cooperation
   Funding Scheme (GHP/016/21SZ & GZSTI17SC02); The Key-Area Research and
   Development Program of Guangdong Province (2020B1111110006); GBA
   Institute of Collaborate Innovation (GICI-022); Hong Kong RGC-GRF
   (16100921); Hong Kong Innovation Technology Fund (PRP/073/20FX;
   ITCPD/17-9); Shenzhen Science and Technology Innovation Committee
   (ZDSYS201707281432317).

Footnotes

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References