UPDATE OF THE SKIN REGENERATION PROTOCOL

 

Maurizio Ceccarelli M.D., Sc.D., Cl.Path.S.

Director International Centre for Study and Research in Aesthetic and Physiological Medicine

Rome, Italy

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Abstract

Regenerative medicine today is an important area in improving the biological state of the skin.

In this treatment, growth factors derived from platelet degranulation are mainly used. The clinical outcome depends on the correct concentration of growth factors and the presence of young fibroblasts capable of forming a new juvenile dermis.

Fundamental research and clinical studies have made it possible to formulate the correct regeneration protocol for the skin.

This requires:

- differentiation of dermic stem cells into new fibroblasts

- the separation of platelet growth factors

- proliferative and metabolic stimulation of neoformed fibroblasts

- optimization of skin regeneration

Introduction

Growth factors are small protein fragments belonging to the cytokine group, produced by various types of cells and tissues, capable of activating or inhibiting cellular functions.

Growth factors were discovered by Professor Rita Levi Montalcini in collaboration with Professor Cohen in 1954. The two scientists subsequently codified the biological mechanisms induced by growth factors.

Growth factors, for their action, bind to particular receptors, said tyrosine kinase receptors, and this binding results in a series of intracellular activations leading to biological response. Tyrosin kinase receptors are so called because they have, in their intracellular portion, tyrosine residues that are phosphorylate once activated by the receptor.

The phosphorylation of tyrosine residues triggers the cell response. These responses begin with the activation of Growth Factor Receptor-Bound Protein 2, followed the activation of RAS and the formation of Mitogen Activated Protein Kinase. This induces the cell multiplication.

MAPK also activates the AP-1 Transcription Factor, necessary for DNA reading. The AP-1 Transcription Factor, with Jun and Fos genes, allows the RNA-polymerase attack on DNA to allow, after transcription, protein synthesis.

All this results allow an increase in the number of the cells and an increase in the synthesis of the cellular products. The final response of these intracellular mechanisms determines both cellular multiplication and metabolic function increase.

The importance of the discovery of the Nerve Growth Factor and its biological functions led in 1986 to receive the Nobel Prize for Medicine at Prof. Rita Levi Montalcini. The importance of the discovery of the Epidermal Growth Factor and its biological functions led in 1986 to receive the Nobel Prize for Medicine at prof. Stanley Cohen.

In 1971, it began to talk about biologically active factors in the blood. And, in 1974, it is stated that biologically active factors released from platelets act on cell proliferation.

In 1999, Dr. Eduardo Anitua, in Spain, starts using PDGF to improve repair processes. In these work, platelet growth factors were applied above the lesion and allow a faster repair of the lesion. For a correct clinical response, in topical application, it is used a platelet concentration increased by 5 to 10 times the normal (PRP).

In 2001, Prof. Victor Garcia began the biostimulation of the skin with platelet growth factors, introducing them directly into the dermis and inducing regeneration of the skin tissue.

We recall that with the term regeneration we mean a physiological process that leads to the continuous reconstruction of labile tissues. That is, in regeneration, we have a normalization of the amount of functional tissue.

It is very important to differentiate the concept of regeneration from repair. In regeneration we normalize the amount of tissue diminished by aging, stimulating the new formation of new tissue equal to that lost, rich in reticular collagen. We get a quantitative normalization of functional tissue.

By the term repair, however, we mean a pathophysiological process that leads to compensation for a tissue damage by the neoformation of a new tissue (fibrotic tissue), rich in I type (cicatricial) collagen, non-functional.

Activating the formation of reticular collagen is essential for the skin rejuvenation. In fact, the increase in type I collagen characterizes an aged skin, while the rise of type III collagen characterizes a young skin.

The initial study

For the formulation of a proper skin regeneration protocol, prof. Victor Garcia with prof. Maurizio Ceccarelli make a bibliographic study useful for deepening the biological effects of the platelet growth factors.

Growth factors are contained inside the platelets in specific granules (alpha granules). Platelets contain various types of granules. Alpha granules that release PDGF, PF4, Fatt. V, Fatt. XIII, fibrinogen, delta granules (or dense bodies) that release serotonin, ADP and calcium.

Around the platelets there are numerous other growth factors that contribute to the biological functions of tissue regeneration. Among them, the highest concentration is Insulin Like Growth Factor.

Platelet-released growth factors (mainly PDGF and IGF) have a specific function in regenerating the skin and, in particular, on the synthesis of the dermal matrix and on the fibroblast activation.

A work published on J.Biol Chem in July 2008 tells us that:

- with only 5 ng/ml of platelet growth factors, there is a strong induction of fibroblastic proliferation.

A work published on Blood in August 1984 tells us that:

- the concentration of PDGF, in healthy human serum, after platelet degranulation is about 20 ng/ml.

Each platelet releases, when activated and degranulated, a quantity of 7.5 x 10^-8 nanograms of PDGF.

From the above, there are a presence of platelet growth factors, in the whole plasma, four times that required to stimulate cellular biological functions, we use, in our regeneration treatment, all the plasma collected after centrifugation of the blood.

PDGFs are released from platelets linked to a heparinoid. Detached from this, in dimeric form, they bind to tyrosine kinase receptors with the cell activation.

A work published on Blood in August 1984 tells us that the half-life of the PDGF, released from the eparan sulphate, is very short, about 2 minutes. After this time we have not a biological effect.

For this reason we do not activate platelet degranulation by adding calcium, but we, directly, introduce plasma into the dermis to allow the platelet binding to the connective collagen, inducing their activation and degranulation, with the release of growth factors, directly in the  action field.

In addition, a 2008 work published in Communications & Integrative Biology writes that:

- After 6-8 hours from a fibroblastic stimulation with PDGF, we have a recruitment of receptors on the cell surface, with their number increase.

From this, after 6-8 hours from a first stimulation of the fibroblast with PDGF, in the particularly damaged areas, we perform a second phase using a greater concentration of growth factor (PRP).

The professor. Victor Garcia, in collaboration with Dr. González Nicolás Albandea J. Antonio, evaluated histologically, in a paper, published in the International Journal of Cosmetic Medicine and Surgery, the dermal response to the introduction of PDGF.

After 7 days of biostimulation with PDGF we have the maximal angiogenesis, that is, a new vases production and an improved microcirculation in the stimulated tissue.

After 30 days of dermal biostimulation with growth factors we have a high concentration of activated fibroblasts (proliferation). After two months we have the neoformation of a juvenile matrix rich in type III collagen (real rejuvenation).

On the scientific basis, the correct skin regeneration protocol was created with the intradermal introduction of PDGF. Garcia and Ceccarelli's studies are then advanced to obtain the final regeneration protocol for all face tissues, called Full Face Medical Regeneration. The final protocol was published in 2010 on The Physiological Medical Letter.

With Full Face Medical Regeneration we can regenerate:

• The epidermis
• The dermis
• The hypodermis
• The bone

For this we use the term Medical Face Lifting.

To achieve this, we use all autologous products obtained by the patient and, in particular:

• Autologous Platelets Derived Growth Factors
• Autologous Platelets Rich Plasma
• Autologous Plasmatic Fibrin
• Autologous Adult Fat Stem Cells
• Autologous Biological Tissue Support + Tricalcium Phosphate

The actuality

On the basis of the clinical responses, obtained in a lot of years of regenerative skin treatment, prof. Maurizio Ceccarelli has recently investigated the causes of the different results obtained in young patients and in elderly patients: studying the solution for the poor response that can be obtained on elderly patients.

The old fibroblast has two negative characteristics: reduced number of receptors with reduced response to stimulation, and low capacity to the production for reticular collagen and proteoglycans compared to fibrotic collagen production.

In addition, the old fibroblast has already reached a high number of mitoses and the proliferative stimulation of PDGF facilitates the achievement of the Hayflick Limit with the cell death.

The solution to this problem requires the formation of young fibroblasts.

Fibroblasts, like all mesoderm derived cells, require the differentiation of mesenchymal stem cells inserted into the environment suitable for fibroblastic neoformation. From this, it would be necessary, before, the intradermal introduction of mesenchymal stem cells and, subsequently, the application of platelet growth factors.

This causes a problem in the implementation of regenerative skin treatment.

In fact, in order to obtain this protocol, we must pick up the stromal vascular fraction of the adipose tissue, because the immunohistochemical and immunofluorescence studies have shown that the stem cells are found in the perivascular connective zone mixed with pericytes. Once the adipose tissue is extracted, the lipoaspirate, first, must be fragmented, mechanically, to obtain small tissue particles that are more easily attackable by the collagenase. Then, we must treat the fragmented fat with collagenase to free stem cells.

Once the connective is solubilized, we must perform a number of washings to eliminate the collagenase traces and to obtain the stem cells to be injected.

As described, it greatly limits our ability to use skin regeneration. In fact, the fat aspiration requires a surgical outpatient and the separation and purification process of stem cells requires a sterile and protected environment.

All this, limit the possibility of performing skin regeneration in a normal medical outpatient clinic. We have, therefore, deepened our studies to solve this problem.

Scientific literature tells us that stem cells are present in all tissues.

These cells have the function of regenerating a tissue subjected to mild damage. A major damage, however, results in a repairing (cicatrical) process of lost cell volumes, for apoptosis or necrosis.

There is also a pool of stem cells circulating continuously in the bloodstream. When a tissue suffers a slight damage, it recalls these cells that move for diapedesis until to reach the tissue and regenerate it. The recall and the stimulus to differentiate the mesenchymal cells is given by the inflammation that follows the tissue damage. Specifically, by the release of the transforming growth factor beta.

The latent stem cells are also present in the skin.

From all this, we do not think it is necessary to introduce new stem cells into the skin to form young fibroblasts, but simply stimulate the differentiation of those already present, in a quiescent phase.

Scientific literature tells us that the stimulus for the quiescent stem cell differentiation is made by the release of small amounts of ROS (free radicals of oxygen).

All this confirms that the ROS are intracellular mediators that regulate the cell functions. In small amounts induce the differentiation of quiescent stem cells, while in high amounts induce the cellular apoptosis.

In conclusion, in our tissues, and in the skin, there are quiescent stem cells. These, subjected to a small damage (ROS), are activated and differentiate by regenerating the tissue.

From these scientific findings, in November 2015, at the Center for Experimental Dermatology at the Madrid University's Ramón y Cajal Hospital, a skin regeneration work was set up using photodynamic therapy at low dosage.

In the work it is proposed to apply a low concentration of ALA (aminolevulinic acid), stimulated with a red light at 630 nm at low energy. The release of a small amount of ROS induce, not producing apoptosis or necrosis, but the differentiation of quiescent stem cells.

Scientific literature tells us that a low concentration of ROS (0.1-0.5 mMol) stimulates the quiescent stem cell to the differentiation, while higher concentrations (greater than 1.0 mMoles) stimulate cellular apoptosis.

From this, releasing ROS in a concentration above 1.0 mMol induces cell death for apoptosis, while lower concentrations, ranging from 0.1 to 0.5 mM, stimulate the differentiation of quiescent stem cells.

It seemed difficult to calculate the correct amount of ROS useful to differentiate stem cells without risking apoptosis by applying ALA and light at 630 nm. That is why we have sought a possibility that would allow us to produce a correct stechiometric calculation of the ROSs to be produced.

In 2009, we began to use the ROS release in a concentration of 5 mMoles to obtain apoptosis of adipocytes. A dilution of this solution could allow us to activate the quiescent stem cells.

The ascorbic acid, in the presence of ferric ions, activates Fenton's reaction with ROS release. On the chemical reaction described, it is possible to calculate the stechiometric amount of ROS released from a given amount of ascorbic acid.

For the apoptosis we use a concentration of 5 mM ascorbic acid in ferric solution, dilution of this can make us get the right concentration to activate the quiescent stem cells.

Diluting to 6 mg per liter of ascorbic acid we obtain a ROS concentration of 0.34 mM, useful and sufficient to trigger the differentiation of quiescent stem cells.

Intradermal injection of the prepared solution allows the activation and the differentiation of quiescent stem cells both at the dermis and at the epidermis level, without risking apoptosis. That is why we have sought a possibility that would allow us to produce a correct stechiometric calculation of the ROSs to be produced.

The cell differentiation process is completed within 21 days. This allows the obtaining of new young fibroblasts capable of building a young matrix.

After 21 days we activate the proliferation and metabolic activity of neoformed fibroblasts with the introduction, into the dermis, of platelet-derived growth factors. All this allows us to achieve a real skin rejuvenation.

The ROS induce the differentiation of quiescent stem cells into new fibroblasts. The PDGF activate the proliferation and the metabolic activity of these. The dermis regeneration is obtained for reticular collagen formation.

The final skin regeneration protocol therefore provides for the inictial activation of quiescent stem cells and subsequently the proliferative and metabolic stimulation of these. All this without risking apoptosis, by applying ALA and light at 630 nm.

The separation of platelet growth factors

We now see the proper separation and use of growth factors.

First we need to check the separation tubes. We need to select tubes containing a gel capable of separating red blood cells and polymorphonucleates from plasma with platelets (and lymphocytes). The test tube must contain an anticoagulant and, in particular, sodium citrate at 3.8%. This salt, in the presence of calcium, forms a more stable salt, the calcium citrate. Calcium sequestration leads to the impossibility of trasforming prothrombin in thrombin and thus prevents blood clotting.

Commercially we find tubes containing heparin as anticoagulant. Heparin binds to a blood factor, the antithrombin III, activating it. AT-III activated, inactivates thrombin preventing blood clotting. These tubes should not be used because the heparin damages platelets. Scientific literature shows that heparin induces platelet aggregation, resulting in degranulation and loss of PDGF. In addition, literary references show that heparin decreases the number of active platelets containing PDGF and their biological function.

When we buy a platelet separation tube, we must first check the number of platelets we have after centrifugation. This value may vary based on the type of separator gel used, the type of centrifuge, the number of rotations per minute and the centrifugation time.

We therefore carry out a centrifugation which, with our centrifuge, allows us to separate the red and white corpusculated part from plasma with platelets. We take the plasma with the platelets and compare, by means of a contaglobules, the amount of platelets obtained, with the one present in the whole blood. By comparing the two values we have the Platelet Increase Factor we use to evaluate how much blood we need to take.

We, then, evaluate the amount of plasma needed for our treatment.

Normally, skin regeneration is done at the level of the face, neck, decollete and hands. By developing the volume of tissue we have to deal we get an average value of 60 cubic centimeters representing 60 milliliters.

Literature tells us that to activate the cells contained in one milliliter of tissue we need 5 nanograms of PDGF. 5 nanograms multiplied by 60 milliliters give us 300 nanograms. This is the amount of growth factors needed for the regeneration treatment of all the areas described.

Always, the literature tells us that in normal plasma (non-concentrate in platelets) we have, after the degranulation, 20 nanograms per milliliter of PDGF. We know that each platelet is capable of releasing 7.5 x 10^-8 nanograms of PDGF.

With this value we can calculate how many platelets are needed to obtain 300 nanograms of PDGF.

We divide 20 nanograms per 7.5 x 10^-8 and we get the platelets needed to release 20 nanograms of PDGF, equal to 300 million per milliliter. 300 million per milliliter are equivalent to 300,000 per cubic millimeter.

Now we calculate the amount of blood to be taken.

Dividing the 300 nanograms per milliliter needed for the amount of PDGF present in one milliliter, we obtain the required volume of plasma (15 ml).

We consider, for example, that our tube allows us to have a platelet increase factor of 0.86.

We multiply the volume for the Platelet Increase Factor and get the required plasma volume.

We must now report the volume of plasma to the actual amount of platelets of our patient. We have seen that to have 20 ng /ml of PDGF we need to have 300,000 platelets per cubic millimeter. The platelet value of the patient may be higher or lower, this requires a variation in the volume to be used. We therefore calculate a correction factor by dividing the theoretical value for that of the patient.

Now we calculate the amount of blood to be taken. Considering the value of the patient's hematocrit we can trace back to the volume of blood that we have to take.

A hematocrit of 0.4 tells us that after blood centrifugation we will get 60% plasma and 40% corpusculous part. We see that to get 13 ml of plasma from blood with a hematocrit of 0.4 and a platelet value of 300.000 (factor 1), we have to withdraw 21.7 ml of whole blood.

Now we see how to separate the plasma, after centrifugation, to obtain both the whole plasma and platelet rich plasma (PRP).

After centrifuging the blood, we get a separation with the red and polymorphonuclear cells under the gel, and the mononucleates and platelets placed over the gel.

We rotate the tube to suspend the platelets inside the plasma and, with the inverted tube, we puncture the rubber stopper and extract all the plasma (Plasma Intero).

For PRP, after centrifuging the blood, we get a separation with red and polymorphonuclear cells under the gel, and mononucleates and platelets placed over the gel.

We put a needle inside the tube and aspirate the two-thirds higher than the plasma.

We rotate the tube by inversion to suspend the platelets within the reduced plasma volume and, with the inverted tube, perforate the rubber stopper and extract the PRP.

By separating the blood with the gel, we obtain platelets mixed with mononuclear leukocytes. We answer the question: "The presence of white blood cells in the plasma is negative?"

It is said that while platelets regenerate the tissue, lymphocytes and polymorphonucleates cause damage to the matrix for the release of metalloproteinases and inflammatory interleukins.

But literature is controversial in this regard and we find works that indicate a better clinical outcome with the use of platelets and leukocytes.

To better understand, we carry out a biological evaluation of what happens when the platelets and leukocytes are in the dermis.

Platelets in the dermis are activated by joining the connective collagen. This process follows degranulation with the release of growth factors.

Polymorphonucleates to release active substances must be in an inflammatory environment. While lymphocytes release inflammatory cytokines only in the presence of heterologous antigens.

It follows that the presence of leukocytes in our treatment has no positive or negative results.

Separation of Autologous Fibrin

Let's now look at how to separate patient plasma to get autologous fibrin (APF). We must make a blood sampling in a tube that does not contain either anticoagulant or activator of the coagulation. This allows us, from the moment of withdrawal, a working time of about 12 minutes (physiological time of plasma coagulation).

The purpose of the intradermal infiltration of the APF is to create a scaffold where the fibroblasts migrate and obtain, in that area, a preferential regeneration of the tissue. To keep fibrin scaffolds longer, we need to slow down the clot retraction followed by plasminogen transformation into plasmin destroying the clot.

We therefore carry out a high speed spin cycle to remove the platelets and avoid the release of the thrombostin (reactozyme) responsible for the retraction of the clot. After centrifugation, we take all the plasma already under coagulation. Before infiltrating it, we can add tranexamic acid (capronic amino acid) to prevent plasminogen from plasmin (a drop is enough for 10 ml).

Update on Cutaneous Regeneration

 

Now we see, in practice, the regeneration procedure of the skin.

At 0 (beginning of treatment) we stimulate the formation of young fibroblasts by injecting 0.34 mM ROS in the dermis with a ferrous iron ascorbic acid solution.

We inject 10 ml of physiological solution with 6 mg of ascorbic acid in iron and sterile irons, corresponding to 0.34 mM ROS.

When we talk about millimoles we talk about milligrams of a substance divided by the molecular weight of this (176.12 in the case of ascorbic acid) dissolved in a liter of solution.

By calculating, in our case, we see that to obtain a concentration of 0.34 mMol we need to solubilize about 60 mg of ascorbic acid in a liter of solution. 60 milligrams per liter are 0,6 mg in 10 milliliters.

Virtually, we prepare the solution for apoptosis, which contains 30 milligrams per milliliter of ascorbic acid. Take 0.2 ml of this containing 6 milligrams and solubilize them in 10 ml of physiological solution, obtaining the solution of 0.34 mmoles.

This initial treatment allows us to stimulate stem cell differentiation within 21 days. We get new young fibroblasts capable of building a young matrix.

We will prescribe to our patient the platelet count and the hematocritic value to determine the blood to be taken at the second stage of the treatment.

After 21 days, the patient returns for the second phase of treatment and we take the amount of blood needed to treat all areas with full plasma. This allows proliferative and metabolic stimulation of young neoformed fibroblasts.

We use full plasma because the amount of PDGF present per milliliter is four times that required to stimulate cells contained in the same volume.

We use test tubes containing a gel capable of separating red blood cells and plasma polymorphonucleates with platelets (and lymphocytes) and sodium citrate as anticoagulants.

Based on the hemochemical values of the patient, we make the corrections necessary to calculate the right amount of blood to be taken.

We centrifuge the blood (on average at 2500 rpm for 5 min) (560 g) to obtain a separation between red and polymorphonuclear globules under the gel and mononucleates and platelets placed over the gel.

We rotate the tube to suspend platelets inside the plasma and, with an inverted tube, we puncture the rubber stopper and we take all the plasma.

We perform dermal biostimulation on face, neck, décolleté and hands, infiltrating the plasma with a 4 mm 30G needle.

We guarantee the intradermal introduction by looking at the formation of the papula. This leads the platelets in contact with the dermal collagen, their activation, and the subsequent degranulation.

The PDGF is released by platelets bound to a heparinoid. Detached from this, in dimeric form, it binds to tyrosine kinase receptors with cell activation.

Both the PDGF and the heparinoid vector have an important function to regenerate the skin. PDGF activates tyrosine kinase receptors and heparinoids act as antithrombotic agents.

The microcirculation of the dermal plexus is particularly slow, both for the small size of the capillaries and their shape. This slowing down of the flow favors the formation of small thrombi. The heparinoid, with its antithrombotic function, solubilizes the microtrombies by facilitating microcirculation of the skin and skin metabolism.

We can verify the platelet degranulation process and PDGF action by simultaneous degranulation of dense bodies or delta granules. The degranulation of these, which accompanies degranulation of alpha granules (with release of PDGF), releases serotonin. Thus, the safety of platelet degranulation has been observed through the effects of serotonin release from dense granules. Serotonin on the skin causes redness, warmth and pruritus to the patient, indicating also the release of growth factors.

In subjects where signs of skin aging are more apparent, we can perform a second biostimulation after 6-8 hours to improve the biological response.

The second biostimulation is performed after this time because after 6-8 hours we have a greater number of receptors on the fibroblast surface and therefore a second biostimulation with greater amount of PDGF induces a higher biological response.

From this, in the second phase, we use Platelets Rich Plasma (PRP).

In this case, separate the plasma from the centrifugation, but before taking platelets, we take two-thirds of the upper plasma. We homogenize the platelet concentration by inversion of the tube and obtain a triple concentration of the platelets.

We do biostimulation only in the most damaged areas of the face, where we need a greater metabolic response, always introducing platelets into the dermis to allow contact with collagen and degranulation.

Skin regeneration stimulates fibroblastic proliferation (increased number) and subsequent neoformation of the juvenile matrix rich in type III collagen (rejuvenation).

Proliferation takes place within 30 days of the first PDGF treatment, with a maximum peak of activated fibroblasts.

It is useful to direct the work of these cells to the most desirable points and to optimize their work by administering biological precursors. Even the normalization of the environmental pH is important to form a juvenile matrix.

From this, after 30 days, we carry out a new biostimulation with aminoacids precursors of the dermis components and the sodium bicarbonate. First, we will infiltrate the fibrin to direct the work of fibroblasts at particular points.

Autologous plasma fibrin (APF) forms a scaffold where we need preferential regeneration as a basis for the movement and function of fibroblasts. In particular we will infiltrate surface wrinkles, depressions and hands. In these points we get a more evident regeneration, resulting in aesthetic problems reduction.

The introduction of precursor amino acids allows the formation of, according to the principles of endomodulation, the right concentration of the matrix components. Adjusting the pH to the physiological value of 7.4 allows the formation of reticular collagen. In fact, in acidic environment rich in positive charges, we have easier secretion of carboxy-terminal fragments (with negative charge) and better formation of I-type collagen.

We do this last dermal biostimulation, at carpet, on face, neck, decolleté and hands.

The complete cycle of skin regeneration is repeated twice a year.

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