Biostimulation and biorevitalization: effects on human skin fibroblasts

Abstract Introduction Aesthetic medicine uses many injective techniques; biostimulation (BS) and biorevitalization (BR) are among these. The term BS indicates stimulation of the anabolic functions of dermal fibroblasts such as replication, protein synthesis and production of extracellular matrix components (ECM). BR instead uses the same injective technique but different medical devices. It is a direct supplementation of hyaluronic acid (HA) alone or added to other molecules (i.e. vitamins). This study discusses BS and BR and their effects on human skin fibroblasts. Materials and methods In order to verify the different metabolic effects of BS and BR fibroblast cell cultures, RNA extraction, cDNA synthesis and PCR were performed. Results BS and BR produce different metabolic effects in fibroblast cell cultures, thus showing that they are different therapies. For example, neutrophil elastine is activated by BS and to a lesser extent by BR, whereas hyaluronan synthase 1 is activated to a higher extent by BR using the medical device with the lowest HA content. Neutrophil elastase, responsible for the degradation of one of the fibrillar components of ECM, is activated to a lesser extent by BS. Conclusion Further experiments using more time points (i.e. not only 24 h of cell cultures but also 12, 48 and 72 h) are necessary to give additional insights on fibroblast behaviour after BS and BR. A better comprehension of fibroblast biology will result in a proper clinical application of BS and BR.


Introduction
The term biostimulation (BS) indicates stimulation of the anabolic functions of dermal fibroblasts such as replication, protein synthesis and production of extracellular matrix components (ECM). BS can be induced using chemical or physical devices. Two protocols are used to obtain chemical BS 1 : • Polydeoxyribonucleotide (PDRN) plus glucosamine sulphate (Gluc), which are delivered with multiple intradermal injections (0.05-0.1 cc each) of a solution of 5,623 mg (3 ml) of PDRN plus 400 mg (3 ml) of Gluc, 1 ml of lidocaine and 0.5-1 ml of sodium bicarbonate, to repeat every 7, 14, 21 and 28 days. The drugs used in PDRN plus Gluc have a common anti-inflammatory function. In fact, PDRN is indicated in wound healing, and its function is mediated by adenosine A2 receptors. Gluc is classified among antiinflammatory non-steroid drugs. The association of PDRN with Gluc is supported by the fact that wound healing is an essential homeostatic mechanism that depends on a series of overlapping phases: inflammation, angiogenesis, formation of new tissue and reorganization.
Bio-NAC has the aim to improve protein synthesis and simultaneously to give a precursor of glutathione (i.e. NAC), because it is the major anti-oxidant mechanism of our body.
Biorevitalization (BR) instead uses the same injective technique, but different medical devices. It is a direct supplementation of hyaluronic acid (HA) alone or added to other molecules (i.e. vitamins).
Since BS and BR have positive effects on dermal fibroblasts in different ways, an experimental study on fibroblasts cell culture is performed in order to get a new insight as regard differences in ECM synthesis and degradation as well as in metabolic signalling.

Materials and methods
This work conforms to the values laid down by the Declaration of Helsinki (1964). The protocol of this study has been approved by the relevant ethical committee related to our institution in which it was performed. All subjects gave full informed consent to participate in this study. Competing interests: none declared. Conflict of interests: none declared.

Primary human dermal fibroblast cell (HFb) culture
All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript. All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.
BS and less by BR ( Figure 1). On the other hand, hyaluronan synthase 1 (HAS1) is more activated by BR using the medical device with the lowest HA content. Figure 2 shows the effects on growth differentiation factor 6 (GDF6), insulin-like growth factor 1 (IGF1) and desmoplakin (DSP). GDF6 is more activated by the treatment with NAC/Aa and PDRN/Gluc. IGF1 is strong, inhibited by PDRN/ Gluc and very lightly stimulated in the other cases. DSP is always stimulated. Figure 3 shows the effects of activation on hyaluronidase 1 (HYAL1) given by all the treatments, whether neutrophil elastase (ELANE), responsible for degradation of one of the fibrillar components of ECM, is less activated by PDRN/Gluc. Figure 4 reports the effect on metallopeptidases (MMP). All the treatments enhanced the activity of MMP; only MMP13 is inhibited by PDRN/ Gluc and only lightly activated by NAC/Aa.

Discussion
The importance of PDRN in BS is due to its therapeutic indication in wound healing for the ability of purine nucleosides and deoxyribonucleotides, in micromolar concentration, to enhance cell proliferation and intracellular cAMP by increasing the extracellular concentration of adenosine 2 . Adenosine is generated from ATP catabolism and is a powerful regulator of cellular function. There are at least four different adenosine receptors on the cell surface. They are members of the family of 7-transmembrane spanning G protein-coupled receptors 3 . The subtype A2 is involved in many adaptive physiological processes 4 . The increase of deoxyribonucleotides and deoxyribonucleosides is reported to have a mitogenic effect in cultured fibroblasts, and this effect is mediated by the activation of purinergic receptors of the CO 2 at 37°C for 24 h. After the end of the exposure time, cells were trypsinized and lysed for RNA extraction.

RNA processing and real-time PCR
Reverse transcription to cDNA was performed directly from cultured cell lysate using the TaqMan Gene Expression Cells-to-Ct Kit (Ambion Inc., Austin, TX), following the manufacturer's instructions. Briefly, cultured cells were lysed with lysis buffer and RNA released in this solution. Cell lysate were reverse-transcribed to cDNA using the RT Enzyme Mix and appropriate RT buffer (Ambion Inc.).
Finally, the cDNA was amplified by real-time PCR. The amplification was performed by using Power SYBR ® Green PCR Master Mix (Applied Biosystems, Foster City, CA), and the specific assay was designed for the investigated genes. SYBER assay reactions were performed in a 20 µl volume using the ABI PRISM 7500 (Applied Biosystems). Each reaction contained 10 µl 2× Power SYBR ® Green PCR Master Mix (Applied Biosystems), 400 nM concentration of each primer and cDNA.
All experiments performed included non-template controls to exclude contamination of reagents. PCR was performed with two biological replicates.
Expression was quantified using real-time RT-PCR. The gene expression levels were normalized to the expression of the housekeeping gene Homo sapiens transferrin receptor protein 1 (TFRC). The expression was evaluated as fold changes relative to the expression of untreated HFb. Quantification was done with the delta/delta calculation method. Forward and reverse primers for the selected genes were designed using primer express software (Applied Biosystems), and are listed in Table 1.
Cells were incubated in a humidified atmosphere of 5% CO 2 at 37°C. Medium was changed the next day and twice a week. After 15 days, the pieces of dermal tissue were removed from the culture flask. Cells were harvested after 24 h of incubation.

Cell culture
For the investigation of BS HFb, at the second passage, they were seeded on a layer of: • PDRN 5,625 mg (Placentex integro, Mastelli, Sanremo, Italy) and Gluc 400 mg (Dona Rottapharm, Milan, Italy). • NAC (Almus s.r.l., Pomezia, Rome, Italy) and a solution of 8.5% Aa; electrolytes were used for intravenous nutrition. This product contains a series of essential amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) and non-essential amino acids (alanine, arginine, histidine, proline, serine and cysteine) (Freamine III Baxter S.p.A., Rome, Italy) at the concentration of 20 mg/ml as used the in vivo protocol.
For the study of BR HFb, at the second passage, they were seeded on a layer of three medical devices with different contents in HA: • Solution of HA 6.2 mg/ml with Aa and vitamins (Skinkò E Viscoderm Ibsa//Revitacare, Saint Ouen d' Aumone, France), • HA gel 10 mg/ml and polynucleotides (Newest Mastelli, Sanremo, Italy), • HA gel 20 mg/ml in saline solution (Restylane ® Vital, Uppsala, Sweden).
A set of untreated cells were used as control. The cells were maintained in a humidified atmosphere of 5% and enhance both cellular replication and protein synthesis. In fact, the nucleotides and nucleosides derived from PDRN degradation can be used as signalling transductors in the extracellular environment or can be internalized. In the intracellular compartment, they can provide purinic and pyrimidinic rings for nucleic acid synthesis via activation of the salvage ways 7 that permits a major speed and an energy spare with respect to the de novo metabolic ways.
Gluc is classified among antiinflammatory non-steroid drugs and used in knee arthrosis. Gluc is one of the major precursors in glucosaminoglycans synthesis. The glucosamine and acetic acid together produce N-acetylglucosamine, and its polymerization with glucuronic acid gives HA.
The reported result on BS shows an opposite activation of genes responsible for the formation and degradation of fibrillar and amorphous components of ECM (Figures 1  and 3). In fact, it showed less activation of HAS1 but double activation of HYAL1, strong activation of ELN and very light activation of ELANE. Thus, BS increases the synthesis of one of the fibrillar components of ECM and can enhance the degradation of amorphous component of ECM.
The second BS protocol (NAC/ Aa) has the aim to improve protein synthesis and simultaneously administer a precursor of glutathione; it is an indirect way to give an antioxidant without interfering with the homeostatic assessment. N-acetylcysteine is the precursor of an amino acid, cysteine, which is one of the three components of glutathione (gamma-glutamyl-cysteine-glycine) 8 . Glutathione is an antioxidant system that works either in intracellular or extracellular compartment. N-acetylcysteine is commonly used in acetaminophen poisoning and as a mucolytic 9  Competing interests: none declared. Conflict of interests: none declared.
All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript. All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.
and differentiation, regulation of the extracellular water content and protein homeostasis. HA is largely used in aesthetic medicine for its hydration capability, but its role is not only a filler. In fact, there are two different receptors: the cluster of differentiation 44 (CD44) located on the plasma membrane and the receptor for hyaluronan-mediated motility located in the cytoplasm.
The individualization of these receptors attributes to the role HA has in cellular motility, proliferation and angiogenesis 15 . For some authors, the receptor interaction is different on the basis of HA fragmentation 16,17 , but others have observed that hyaluronan supplementation to fibroblast culture results in inhibition of cell proliferation, and this is positively related to the concentration of HA but not related to molecular weight, probably because after interaction with receptor CD44 and internalization, HA is always fragmented 18 .
In these experiments, HA is present either as a delivered substance in BR or as a primer in the quality of HAS1 and HYAL1. It can be observed that the medical device with the lowest concentration of HA produced the major stimulation of HAS1 ( Figure 1). Moreover, there are evidences of stimulation of HYAL1 with all the tested products.
ECM degradation is an important factor in tissue repair. It is regulated by MMPs and by tissue inhibitors of metalloproteinases. In man, there are 24 different MMPs.
In our experiment, MMP3 is particularly activated by BR procedures, MMP2 is activated in a uniform way in all the cases and MMP13 is strongly inhibited by PDRN/Gluc and only very lightly activated by NAC/Aa ( Figure 2).

Conclusion
BS and BR produce different metabolic effects in 24 h fibroblast cell cultures, thus showing that they are BR uses the same injective technique, but the drugs are medical devices with different types and concentrations of HA. HA is a highmolecular mass polysaccharide of the extracellular matrix especially of soft connective tissues. It is synthesized in the plasma membrane of fibroblasts by addition of sugars to the reducing end, whereas the non-reducing end protrudes into the pericellular space. It is a polymer of dimeric units of N-acetylglucosamine and glucuronic acid 14 . Among its important biological functions, there are the modulation of cellular proliferation, migration other useful effects. In fact, it is an antioxidant, a hepatic protector, a booster of nitroglycerine, a promoter of glutathione synthesis and a depressor of synthesis of lipoproteins and homocysteine 10 . It can act as a modulator and protect neural cells from apoptosis 11 . The behaviour is very similar to BS performed with PDRN/Gluc, but with NAC/Aa, we did not see any strong inhibition of IGF1. The reason for this inhibition is not clear at the moment, even if some studies on animal models and on centenarians relate the reduction of GH or IGF1 to the length and quality of life 12,13 .   different therapies. Additional experiments using more time points (i.e. not only 24 h of cell cultures, but also 12, 48 and 72 h) are necessary to give additional insights as regard early stages of fibroblast response to BS and BR.
A better comprehension of fibroblast biology will result in a proper clinical application of BS and BR.