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Stimulation and regeneration

Stimulation and regeneration

Dr Joseph Choukroun discusses platelet rich plasma and platelet rich fibrin

Choukroun_PRPGrowth factors are routinely used in reconstructive and regenerative therapies. They can be derived from human tissue, or recombinant DNA can be used, but the simplest method is to extract growth factors from the blood’s platelets.

Platelet concentrate technique uses centrifuge to separate the blood’s components, with the goal of increasing the platelet count. Clotting is essential because the platelets release their growth factors only after clotting, and it is from there that their use can be determined. It’s possible to use growth factors to simulate tissue stimulation undergoing regeneration, or to regenerate tissue. Stimulation needs only platelets, with or without leukocytes. Regeneration requires a scaffold, and then fibrin matrix, platelets and leukocytes.

PRP and PRF
Both PRP (platelet rich plasma), and PRF (platelet rich fibrin) can be extracted using centrifuge.

For PRF, there is no anticoagulant added during the spin, the clotting is physiological and the spin creates a clot, which contains platelets, white cells and fibrin. PRF is not injectable, it’s a gel.

The enrichment of platelets is not the same. For PRP, the platelets are enriched two to four times and there are few white cells. In the PRF, there are all platelets, 50% of white cells and all the fibrin of the blood. The role of the fibrin is very specific, it’s a recipient of growth factors and this recipient will allow very specific release of growth factors, very slowly and continuously over a time period of more than one week. It’s not the amount of growth factors that’s the most important, but it is released into the site. To treat severe infection, antibiotics can be injected drop by drop in an IV perfusion, ensuring that a stable concentration is maintained. This is the same in PRF biology—to obtain the most important function a stable concentration must be maintained. The best is a drop-by-drop infusion.

The clinical use of the PRF in dermatology and aesthetics
By using PRF as a matrix, it can be used to promote healing in an infected wound—for example in a diabetic foot, after amputation. The concept is to inject the fibrin, and this fibrin will release over a few days. By leaving the wound untouched for four to five days, a very fast vascularisation can be achieved without any anti-infectious threat. If vascularisation can be achieved on the surface, the healing is very easy, because the vessels are doing the job by slowly infusing the growth factors.

Ulcers in the diabetic foot can also be treated with the same technique. Fibrin is put on the wound, covered with a plastic sheet, or an aluminium sheet and then in four to five days fast healing is achievable.

To regenerate tissue we need a scaffold. We can use fat, but the most efficient scaffold in the body is fibrin. When I carry out cervico-facial lifting, I mix fat and the PRF, because fibrin provides an excellent scaffold to first get a new vascularisation, but then also new collagen. This fibrin is very important if we want to get the growth factors and with those factors we can achieve the new vascularisation and healing.

The healing always begins with formation of a provisional matrix. All the products are active, but in the beginning, we need the platelets and the white cells. The objective is to create homeostasis and inflammation. After five days granulation tissue begins to form and the matrix deposition becomes visible because the endothelial cells and the fibroblasts start to work, and after ten days collagen forms on this tissue. The provisional matrix is the most important, because when fibrin is introduced to into the site new vascularisation is immediately created.

Cell biology
Plasma protein is necessary to create collagen synthesis. White cells are also necessary, because inflammation is needed for the recovery. At an injury site, the endothelial cells immediately separate themselves and then the white cells squeeze through the gaps and then by chemotaxis they move through the injury site, and begin to release the pro-inflammatory interleukins. This is the sort of inflammation that causes activation of monocytes into macrophages.

The macrophages dominate the inflammatory phase, and then they start to release growth factors and BNPS. Stem cells are necessary alongside white cells in order to achieve a smart blood concentrate and regenerate new tissue.

White cells influence the quality of the PRP, so enriching the PRP in white cells creates greater tissue augmentation and a significantly higher proliferation of mesenchymal stem cells.

Spinning to create PRP, PRF, i-PRF and A-PRF
In PRF the white cells are in the bottom of the clot. If blood is spun at too high a G force, a lot of cells are lost, but with a reduced RPM (revolutions per minute) and lower G force, this can be altered.

The advanced PRF is the clot and the i-PRF is the injectable PRF, which is liquid. With the advanced PRF, the RCF (relative centrifugal force) is low : 200 G force—in order to get more white cells. This is not done in a lab—reducing G force simply increases white cells. An eight minute spin is needed to create A-PRF.

Increasing the white cells, increases both vascularisation and release of growth factors. By increasing vascularisation, the vessel density and the percentage of vascularisation can also be increased.

i-PRF is different. To obtain an injectable PRF, requires a very, very low G force—60 only—followed by the same protocol, with no additive and no anticoagulant. This is the concept of the i-PRF. There is a high concentration of white cells, plus fibrinogen, plasma protein and it clots spontaneously after the injection. Nothing additional needs adding into the wound to get the clot and to get the platelet to release growth factors.

Comparing the white cells between PRP and i-PRF, shows that i-PRF has over 20 times more white cells. Interestingly, the level of the platelets is the same in the three techniques; it seems that reducing the G force does not reduce the platelet enrichment.

Injectable-PRF can be created by spinning for only three minutes. Spinning for four minutes, decreases the quality a little, but is fine because more spinning creates more liquid. The best concentration of the stem cells is spinning only for three minutes. More blood can be used to create more liquid.

Research
In recent research, my objective was to try to extract stem cells from the blood. We did a flow cytometry to analyse the mesenchymal stem cells into the iPRF. Using information from the International Society for Cell Therapy, we knew we needed to find cells that are CD 34- and 45-, and CD44+, CD73+, CD90+ and CD105+ positive, since those are the characteristics of the mesenchymal stem cells. So we did an elimination of all the cells, and we found that in the iPRF we have from 1 to 2% of mesenchymal stem cells. When we analyse the IPRF we have a quantity of cells. 1-2% of these cells are mesenchymal stem cells.

It’s possible to produce many thousands of stem cells in just three minutes, by getting the blood in a tube and spinning with a low force. If we compare the concentration of the mesenchymal stem cells in the iPRF, in the PRP, we can see the difference of using a low spin speed. A separate specific manufactured device to produce mesenchymal stem cells isn’t needed. We need only to get blood, draw the blood and to spin it doing three minutes in the iPRF tube, without any additive, without any anticoagulant. It’s that simple.

Applications of iPRF
I’m not a dermatologist, I have my own pain clinic in Nice, and I have many patients with arthritis in the knee. I discovered it is possible to regenerate the cartilage with the stem cells from the iPRF, reduce inflammation and reduce pain.

Applying iPRF to the scalp with microneedling can improve the hair when the patient has alopecia. iPRF can also be used in the lips—we know that if we want to augment lips, we need to create new tissue, and for that, we need a fibrinogen to get collagen in the future. PRF can also be used to treat the mouth and regenerate the gingiva around the teeth.

Conclusion
The best results are the ones created the most simply. When we follow nature and our body’s natural biology it makes sense to be safe and avoid any manufactured additives. Using this method of using RCF and controlling the RPM, we not only get more cells, we can get a lot of stem cells very simply, naturally and at a low cost. I believe we are at the beginning of developments with the iPRF and that the future will be very exciting. I also believe we can use iPFR to improve the HA injection, because with the iPRF, we can create more collagen and create perfect stability.

Dr Joseph Choukroun is a MD, Anesthesiologist and Pain clinician

Author: bodylanguage

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