Another look

 

Skin regeneration with PRP-(HA) Swiss translational research projects, from in vitro and in vivo experiments to clinical evidence.

Dr. Ali Modaressi, MD (1)

Sarah Berndt, PhD (2)

Dr. Ali Modarressi MD and Dr. Sarah Berndt, PhD

1. Plastic, reconstructive and aesthetic surgery, Geneva (Switzerland)

2. Doctor of Biomedical and Pharmaceutical Sciences
Head of Cellular Therapy Research at Regen Lab SA (Switzerland)

1. Cell therapy research:

Cellular therapy aims to introduce new healthy cells into a patient's body to replace damaged cells.

  • This type of therapy is challenging because it requires a sufficient number of cells to be transplanted into the patient.
  • Indeed, specialized cells, such as fibroblasts or brain cells, are difficult to obtain from the human body.
  • In addition, specialized cells generally have a limited ability to multiply, which makes it difficult to produce sufficient numbers of cells for some cell therapies.

Some of these problems can be solved by the use of stem cells.

  • Stem cells are unspecialized cells that have the ability to develop into other functional cell types.
  • It is important to note that some types of stem cells can be grown outside the human bodyThis allows the production of a large number of cells necessary for the success of cell therapy applications in medicine.
  • When autologous cells are used, the patient owns their own therapy.

2. Demonstration of safety and efficacy in translational research projects: establishment of autologous 100% cell culture models.

In recent years, we initiated a translational research project that demonstrated the proliferative effect of PRP obtained from the patient's blood on adipose tissue-derived mesenchymal stem cells (ADSCs) and fibroblasts from the same patient [1-3] (Figure 1).

  • Our goal is to be totally autologous in order to offer personalized therapies.
  • This is why a range of medical devices, CuteCell, dedicated to the in vitro culture of human cells in research laboratories (GMP facilities) has been developed.
  • These devices allow the preparation of PRP (CuteCell-PRP), serum (CuteCell-serum) and PRP enriched with MNC (CuteCell-MNC).
  • These new products offer an autologous alternative to fetal bovine serum (FBS) in culture media for cell therapies [4].
CUTE CELL

2.1 Adipose tissue derived mesenchymal stem cells (ADSC)

PRP accelerates the expansion of adipose tissue-derived mesenchymal stem cells (ADSC).

  • To define an autologous system for ADSC proliferation, we evaluated the efficacy of autologous PRP on ADSC proliferation compared with conventional medium supplemented with FBS.
  • We studied the optimal concentration of PRP in culture media.
  • Culture media supplemented with 20% of PRP prepared from the patient's blood increased the in vitro proliferation of ADSCs by 14-fold compared with conventional culture medium prepared with 10% of FBS for 10 days [1] (Figure 2).

Figure 2: Culture medium supplemented with autologous PRP significantly increases in vitro proliferation compared with conventional culture medium containing 10% of FBS. Data from Atashi et al. Tissue Engineering. Part C. 2014 [1].

PRP does not alter the phenotype of ADSCs, their ability to differentiate, or their chromosomal status.

  • We verified that the intrinsic differentiation potential of ADSCs into adipocytes, osteocytes or chondrocytes was preserved intact, regardless of the supplements used for their proliferation.
  • We observed that PRP did not have a negative effect on the differentiation capacity of ADSCs.
  • The adipogenic, chondrogenic and osteogenic differentiation potentials were confirmed and were qualitatively comparable to those of FBS at 10% [1].
  • Cytogenetic analysis of cells cultured in 10% of FBS or 20% of PRP conditions did not show abnormal karyotype.
  • Both FBS and PRP culture conditions showed numerical and structural stability.

Thus, treatment of cells with PRP does not alter chromosomal stability [1].

2.2. Human dermal fibroblasts (NHDF):

Nowadays, the application of autologous fibroblasts for skin repair is of significant clinical interest.

In most cases, in vitro culture of skin cells is mandatory.

However, cell expansion using xenogeneic or allogeneic culture media has some disadvantages, such as the risk of infection transmission or slow cell expansion.

2.2.1. Improvement of fibroblast expansion with autologous PRP treatment.

In this study, we collected fibroblasts and blood from the same patients.

  • After 7 days of culture, fibroblasts supplemented with different concentrations of PRP showed a higher number of viable cells compared with media containing FBS (Figure 3).
  • This proliferative effect of PRP followed a dose-dependent bell-shaped curve.
  • The optimal culture condition was PRP 20% where the number of NHDFs was 7.7 times higher than FBS 10% [2,3].

Figure 3: Bright field optical photography of NHDF in the presence of FBS 10% or PRP (5-20%) after 7 days of culture. 10x magnification. Photos are representative of a single donor. Data from Berndt et al. Tissue Engineering. Part A. 2019 [3].

2.2.2. The optimal concentration of PRP is crucial for the maintenance of the fibroblast phenotype.
  • Fibroblasts grown in conventional culture medium supplemented with FBS showed a regular, flattened cell shape,
  • while those treated with PRP (10-50 %) were fusiform, a morphology more similar to 3D matrix cultures or the in vivo setting.

We investigated whether the morphological change occurring after 7 days of PRP treatment was related to a phenotypic change.

  • We first demonstrated a significant reorganization of F-actin from cortical actin localization (FBS 10 %) to thick cell-passing filaments (PRP 20 %).
  • We then assessed changes in alpha-SMA expression upon PRP treatment by flow cytometry and immunofluorescent analyses.
  • Alpha-SMA expression increased significantly with high concentration of PRP (40-50%), whereas cells treated with FBS-10% and PRP 5-10% showed basal perinuclear staining.
  • Immunofluorescence analysis showed an increase in vimentin staining in the presence of PRP at 20 %, but it was completely abolished at high PRP concentration (PRP at 50 %) [3].

These results underscore the importance of an optimal concentration of PRP. They confirm that a high concentration of platelets is not better than a moderate concentration and could even be harmful [5,6].

2.2.3. CuteCell PRP modulates metabolic activity, fibroblast adhesion and promotes migration.

Using an MTT metabolic assay, we demonstrated an increase in PRP-treated cells.

This directly reflects an increase in cell metabolic activity, peaking 3.12-fold in PRP 20%-treated cells compared with FBS 10%-treated cells after 48 h of treatment.

To further characterize the biological effects of PRP on fibroblast biology, we evaluated the effect of PRP treatment on cell adhesion to laminin and type I collagen.

  • PRP decreased the overall attachment of fibroblasts to laminin 4 h after seeding.
  • This effect occurred already after 15 min, with a 21% decrease in overall cell adhesion.
  • The same results were obtained for fibroblast attachment to collagen I matrix (41% of total cell adhesion after 15 min).

To investigate the migratory properties of fibroblasts exposed to PRP, we performed an in vitro scratch test (Figure 4).

  • Eight hours of treatment with 20% of PRP induced a 10% increase in the number of cells migrating from the scratch margin to the scratch area compared with cell cultures with FBS. This migration front was a collective cell migration.
  • Conversely, fibroblasts exposed to 10 % of FBS exhibited isolated cell migration characteristics [3].

Figure 4: Comparative cellular effects of 10% PRP treatment on cell migration in fibroblast cultures. (A) Migrating fibroblasts reduced the width of the scratch zone, as shown by phalloidin immunofluorescence staining after 8 hours. The cell migration front is evenly distributed along the scratch border in FBS 10%, whereas it is less homogeneous in PRP 10%-treated cells. (B) Zoomed in images showing isolated cell migration in FBS 10% cultures and collective cell migration in PRP 10% cultures. Data from Berndt et al. Tissue Engineering. Part A. 2019 [3].

2.2.4.Genome-wide analysis to demonstrate that CuteCell PRP is safe at the genomic level.

To document genetic stability during proliferation, we cultured NHDFs for 4 days with media supplemented with 10 % FBS or 10 % PRP.

Array CGH analysis of cells treated with the two different culture media did not show unbalanced chromosomal rearrangements.

The increase in proliferation rate in response to PRP treatment did not cause genomic instability [3].

These results are of primary importance because some studies claim that growth factors released by PRP may contribute to tumor progression [7].

3. Angiogenesis is differentially modulated by platelet-derived preparations

In the context of tissue regeneration, therapeutic angiogenesis aims to restore an appropriate vascular system through the administration of exogenous growth factors, cytokines and chemokines, among others.

Important angiogenic factors are:

  • VEGF,
  • angiopoietin,
  • the FGF,
  • the HGF,
  • the PDGF
  • and TGF
  • although a myriad of other proteins are also known to be involved in blood vessel formation.

The release of platelet-derived products, such as autologous growth factors, cytokines, and chemokines, can trigger therapeutic angiogenesis.

In an in vitro study, we evaluated and compared the ability of three platelet-derived preparations:

  • platelet-rich plasma (PRP),
  • PRP hyaluronic acid (PRP-HA)
  • and platelet lysates (PL) at different concentrations (5-40%) to modulate the biological effects of human umbilical vein endothelial cells (HUVEC) on metabolism,
  • viability,
  • senescence,
  • secretion of angiogenic factors
  • and angiogenic capacities in 2D (endothelial tube formation test or EFTA)
  • and in 3D (fibrin bead test or FBA) [8].
3.1. PRP and PRP-HA modulate the angiogenic activities of endothelial cells (HUVECs) in 3D.

In this work, we used a high-throughput in vitro 3D angiogenesis assay (the fibrin bead assay or FBA) to model the angiogenic effect of platelet-derived preparations used in tissue engineering studies or in the clinic (Figure 5A).

  • We tested a range of concentrations (5 to 40 %) of CuteCell's standardized PRP, CM-PRP-HA, obtained with the Cellular Matrix BCT-HA tube, and a commercial platelet lysate preparation. Blood samples were obtained from healthy donors.
  • The procedure was consistent with the principles of the Declaration of Helsinki and was approved by the Geneva Cantonal Ethics and Research Commission (ID 2017-00700, approved October 18, 2018).
  • FBA uses endothelial cell (EC) culture on the surface of ~200 μm Cytodex-3 microspheres embedded in a 3D bovine fibrin matrix, with normal human dermal fibroblasts (NHDF) used as feeder cells.
  • These stromal cells provide various angiogenic growth factors:
    • hepatocyte growth factor (HGF),
    • transforming growth factor alpha (TGF-α),
    • angiopoietin-1 (Ang-1),
    • as well as matrix molecules,
    • matrix-modifying proteins of matricellular proteins, e.g: 
      • procollagen C endopeptidase enhancer 1,
      • secreted acidic cysteine-rich protein (SPARC),
      • Ig-H3 protein induced by transforming growth factor-β (βIgH3).
      • and insulin-like growth factor binding protein 7 (IGFBP7)). [9].

Under control conditions, neovessel sprouting is apparent between days 2 and 3, and cultures are imaged on day 4.

  • For quantification of microvessel network sprouting, samples are automatically scanned with a high-throughput imager and microsphere image analysis is performed using a method we developed with ImageJ software (Figure 5B) [10,11].
  • This assay represents a significant improvement over conventional single-cell angiogenic assays, as the inclusion of multiple cell types more closely mimics the physiological environment [9].
  • The basic steps of sprouting angiogenesis include
    • the enzymatic degradation of the basal membrane of the capillaries,
    • endothelial cell (EC) proliferation, directed EC migration,
    • tubulogenesis (formation of tubes by EC),
    • the fusion of the vessels,
    • pruning of the vessels
    • and stabilization of pericytes.

 

  • Platelet-derived preparations elicit different angiogenic responses when tested in the BAF where they act on different steps of the angiogenic process.
  • Using the powerful automatic quantification method we developed [10], we can assess the modulation of morphometric parameters of neovessels formed in the fibrin matrix.
  • These include, for example, the total length of the microvascular network, anastomoses in the vessels (branches), the number of capillaries arising from the bulges (anchoring), and the number of vessel tips showing the complexity of the network (ends) (Figure 5B).
  • In our study, the most potent preparations were PRP and CM-PRP-HA, because the total length of the neovascular network, total length of branches, tips, and anchorage were strongly stimulated compared with control conditions (VEGF+/heparin treatment) (Figure 5C).

Platelet preparations stimulated all steps of the angiogenic process, with light microscopy showing massive sprouting of a branched network of microvessels (Figure 5A).

PRP was the most potent angiogenic preparation, significantly stimulating angiogenesis by 2 to 12-fold depending on the concentration of PRP used and the parameter of interest (Figure 5C).

  • Significant angiogenesis was already observed with the lowest concentration of PRP (5 %).
  • CM-PRP-HA also stimulated angiogenesis but to a lesser extent than PRP: this may be explained by the fact that the concentration of platelets in this preparation is reduced by half compared to PRP.
  • Platelet lysates had to be highly concentrated to elicit the same angiogenic response as PRP and CM-PRP-HA [8].

Figure 5: Platelet-derived products differentially modulate angiogenesis in the 3D fibrin bead assay.

  • (A) HUVEC 3D cultures were treated with platelet-derived products (PRP, PRP-HA, and PL) at different concentrations for 4 days. Representative images of a massive increase in angiogenesis (PRP 20, PRP-HA 20) or mild endothelial proliferation from EC-coated beads (PL20) compared with control conditions (control and heparin) at day 4. Scale bar: 150 μm.
  • (B) Representative images of enhanced angiogenesis. Cytodex microcarrier beads coated with HUVECs showing pseudocapillary growth after automatic analysis with a specific plugin developed for Image J software [11]; some of the morphometric parameters of interest are indicated. 
  • (C) Quantification of the morphometric parameters of the capillary network was performed by a computerized method (using the opensource software Image J) on photos taken on day 4.
  • Representative parameters measured were total length, total branch length, number of ends, and number of anchor junctions per sphere.

Data from Berndt et al. Biomedicines. 2021 [8].

3.2 Effect on HUVEC viability and senescence.

To assess cell viability, we added crystal violet as a dye to study the effect of platelet-derived products on cell viability (Figure 6C).

  • After 3 days of treatment, more viable purple cells were found in PRP and PRP-HA treated cultures.
  • PRP and PRP-HA (10-40 %) increased cell viability in a dose-dependent manner compared with control conditions.
  • LP also increased cell viability but to a lesser extent [8].
  • To assess the effect of platelet-derived preparations on HUVEC senescence, we induced aging in vitro by culturing the cells at low density [12] from passages 2 to 6.
    • In vitro aging induces an increase in cell size and nuclei, and more apoptotic cells appear [13].
    • In this assay, we tested 10% of FBS, 2IU/mL of heparin, 10% of PRP, 10% of PRP-HA, and 10% of PL.

Long-term serial culture in FBS or heparin media induced senescent cells, whereas no senescent cells were observed with platelet-derived treatments [8].

Figure 6:

A. Viability of HUVECs assessed by crystal violet staining in control and PRP, PRP-HA, and PL-treated cultures.

B. Quantification of the amount of dye released by measuring absorbance (590 nm).

C. Senescence-associated beta-galactosidase (SA-gal) staining of HUVECs (20x objectives) at passage 6 treated with FBS, heparin, 10 % of PRP, 10 % of PRP-HA, or 10 % of PL as shown in the figures.

Beta-gal positive cells appear blue in culture conditions with FBS and heparin.

4.Conclusions

Regenerative medicine encompasses a wide range of techniques aimed at repairing or even replacing damaged or aged tissue. Of these, autologous platelet-rich plasma is one of the simplest and most effective. This approach is based on the intrinsic ability of the human body to repair itself and the role of platelets in this process.

There is growing interest in the use of standardized PRP, alone or in combination, in regenerative medicine as a safe and natural treatment, and it has so far shown promising results in many therapeutic indications.

In clinical studies, PRP administration has shown significant improvement:

  • of wound healing [14],
  • quality and survival of fat grafts [15],
  • of hair restoration [16]
  • and skin quality with an anti-aging effect [17].

All these indications require an increase in

  • the proliferation of fibroblasts,
  • the production of collagen,
  • of stem cell stimulation
  • and the growth of angiogenesis.

Interestingly, our in vitro and in vivo experiments have scientifically demonstrated that PRP improves these key regenerative elements. These results support our daily regenerative treatment with PRP in plastic surgery and aesthetic medicine, and demonstrate the mechanism of action.

The CuteCell medical device has been designed and validated for in vitro cell culture under autologous conditions that meet GMP guidelines and regulatory agency standards.

CuteCell-PRP has been shown to be an effective, cost-effective and safe biological adjunct for fibroblast and adipose stem cell cultures, as well as a substitute for xenogeneic or allogeneic blood derivatives for the validation of future in vitro clinical cell expansion protocols.

We have shown that platelet-derived products are autologous biologics that stimulate angiogenesis in situ without requiring grafting of exogenous pre-vascularized material.

PRP and PRP-HA were the only preparations that allowed the entire angiogenic process to be performed.

References

  1. Atashi, F.; Jaconi, M.E.; Pittet-Cuenod, B.; Modarressi, A. Autologous platelet-rich plasma: a biological supplement to enhance adipose-derived mesenchymal stem cell expansion. Tissue Engineering. Part C, Methods 2015, 21, 253-262, doi:10.1089/ten.TEC.2014.0206.
  2. Berndt, S.; Turzi, A.; Modarressi, A. Production of autologous platelet-rich plasma to stimulate human fibroblast expansion in vitro. Journal of visualized experiments: JoVE 2021, doi:10.3791/60816.
  3. Berndt, S.; Turzi, A.; Pittet-Cuenod, B.; Modarressi, A. Autologous Platelet-Rich Plasma (CuteCell PRP) Safely Boosts In Vitro Human Fibroblast Expansion. Tissue Engineering. Part A 2019, 25, 1550-1563, doi:10.1089/ten.TEA.2018.0335.
  4. Anitua, E.; Zalduendo, M.; Troya, M.; Alkhraisat, M.H.; Blanco-Antona, L.A. Platelet-rich plasma as an alternative to xenogeneic sera in cell-based therapies: A Need for Standardization. International journal of molecular sciences 2022, 23, doi:10.3390/ijms23126552.
  5. Graziani, F.; Ivanovski, S.; Cei, S.; Ducci, F.; Tonetti, M.; Gabriele, M. The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts. Clinical oral implants research 2006, 17, 212-219, doi:10.1111/j.1600-0501.2005.01203.x.
  6. Yoshida, R.; Cheng, M.; Murray, M.M. Increasing platelet concentration in platelet-rich plasma inhibits anterior cruciate ligament cell function in three-dimensional culture. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 2014, 32, 291-295, doi:10.1002/jor.22493.
  7. Luzo, A.C.M.; Favaro, W.J.; Seabra, A.B.; Duran, N. What is the potential use of platelet-rich plasma (PRP) in cancer treatment? A mini review. Heliyon 2020, 6, e03660, doi:10.1016/j.heliyon.2020.e03660.
  8. Berndt, S.; Carpentier, G.; Turzi, A.; Borlat, F.; Cuendet, M.; Modarressi, A. Angiogenesis Is Differentially Modulated by Platelet-Derived Products. Biomedicines 2021, 9, 251.
  9. Nowak-Sliwinska, P.; Alitalo, K.; Allen, E.; Anisimov, A.; Aplin, A.C.; Auerbach, R.; Augustin, H.G.; Bates, D.O.; van Beijnum, J.R.; Bender, R.H.F.; et al. Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis 2018, 21, 425-532, doi:10.1007/s10456-018-9613-x.
  10. Carpentier, G.; Berndt, S.; Ferratge, S.; Rasband, W.; Cuendet, M.; Uzan, G.; Albanese, P. Angiogenesis Analyzer for ImageJ - A comparative morphometric analysis of "Endothelial Tube Formation Assay" and "Fibrin Bead Assay". Scientific reports 2020, 10, 11568, doi:10.1038/s41598-020-67289-8.
  11. Schneider, C.A.; Rasband, W.S.; Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nature methods 2012, 9, 671-675, doi:10.1038/nmeth.2089.
  12. Heng, B.C.; Bezerra, P.P.; Preiser, P.R.; Law, S.K.; Xia, Y.; Boey, F.; Venkatraman, S.S. Effect of cell-seeding density on the proliferation and gene expression profile of human umbilical venous endothelial cells within ex vivo culture. Cytotherapy 2011, 13, 606-617, doi:10.3109/14653249.2010.542455.
  13. Dimri, G.P.; Lee, X.; Basile, G.; Acosta, M.; Scott, G.; Roskelley, C.; Medrano, E.E.; Linskens, M.; Rubelj, I.; Pereira-Smith, O.; et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proceedings of the National Academy of Sciences of the United States of America 1995, 92, 9363-9367, doi:10.1073/pnas.92.20.9363.
  14. Oneto P, Etulain J. PRP in wound healing applications. Platelets. 2021 Feb 17;32(2):189-199. doi: 10.1080/09537104.2020.1849605. Epub 2020 Nov 29.
  15. Modarressi A.. Platelet-rich plasma (PRP) improves fat grafting outcomes. World J Plast Surg. 2013 Jan;2(1):6-13.
  16. Justicz N, Derakhshan A, Chen JX, Lee LN.Platelet-Rich Plasma for Hair Restoration. Facial Plast Surg Clin North Am. 2020 May;28(2):181-187. doi: 10.1016/j.fsc.2020.01.009. Epub 2020 Mar 2.
  17. Bajaj S, Orbuch D, Wang JV, Geronemus RG. Preparation and utility of platelet-rich plasma (PRP) for facial aging: A Comprehensive Review. Adv Ther. 2022 Sep;39(9):4021-4036. doi: 10.1007/s12325-022-02239-6. Epub 2022 Jul 23.

 

 

en_US

Aesthetic health based on scientific evidence

Sign up to view this latest issue on vivacy and receive future issues of LM.