My sole motivation as a hair loss physician is to search for and deliver the very best hair restoration treatment for my patients. In continuing to do so, I changed my PRP treatment for hair restoration entirely. Therefore, out of all the PRP data I have shared over the past several years, the information presented in this blog may be the most influential for my patients.
In full disclosure, I do not receive money or benefits from a third-party and am not a company spokesperson, I am just a firm believer in the regenerative properties of PRP, and want to explain why I made changes and what this means for my patients. My thanks to Dr. John Cole and his group for a recently published article in Stem Cell Investigation from November 2017 on Alopecia and Platelet-Derived Therapies.
For the past five years, I have incorporated PRP into my hair restoration practice, and even for me, it has not been easy to find:
•Data on the levels of growth factors released from platelets
•Data on different methods of platelet activation
•Scientific information comparing one centrifuge system to another
In the era of regenerative medicine, there is a lot of information difficult for patients to access, specifically related to Platelet-Rich Plasma (PRP). I am not saying that companies and practices are putting out disinformation or misinformation. PRP treatment is considered an evolving component of different regenerative medical fields and I believe the information available is incomplete, specifically for hair restoration. So, when someone throws out the term “PRP,” I think there is an assumption by most people that all PRP used in ALL types of medicine is the same. This is SIMPLY NOT TRUE.
The focus of all of my PRP treatment is obtaining the maximum amount of growth factors from platelets, now, more importantly, like never before. PRP stands for platelet-rich-plasma, so let’s talk about the value of platelets both to heal wounds as well as promote hair restoration.
Figure 1 - Componets of blood
Platelets: Used to heal wounds
Under normal circumstances, platelets circulate in the bloodstream in an inactive form. Then when there is trauma to a blood vessel, the platelets receive signals to migrate to the site of injury. After the platelets stick to the collagen where the blood vessel is torn, they change their shape. Each platelet changes from a smooth disk to a ball shape with long arms that can reach out to other platelets. This is called platelet activation (we will talk more about OTHER activation methods in a minute). Activated platelets can stick to each other and to clotting proteins in the blood.
Inside each platelet are storage spaces called granules. Namely, three types:
1. Alpha granules: Contain clotting proteins and store growth factors like VEGF, PDGF, TGF-B, IGF-1, FGF, and others that are released in the healing & regeneration process.
2. Dense granules: Stores chemical structures such as:
• ATP: Molecule that stores energy
• ADP: A core component of ATP. This little molecule, ADP, is like a compressed spring, just waiting to spring into action and do work for our cells and body in general.
• Serotonin: Can trigger the release of substances in the blood vessels.
3. Liposomes: The liposome can be used as a vehicle for administration of nutrients and signal cells
Figure 2 - Contents of a platelet
When the platelet changes shape, chemicals inside the granules are pushed out into the bloodstream and signal other platelets to come and help. The chemicals allow the platelets to stick to each other as well as to the blood vessel, forming a clump that plugs the hole in the blood vessel.
Though it is a complex process, it normally only takes seconds. A platelet plug is only a temporary fix, and it can’t last long on its own. Proteins in the blood work together to make threads of a protein called fibrin. The fibrin threads weave over the platelet plug to form a strong fibrin clot, which is normally enough to stop the bleeding completely. The body then has time to heal the blood vessel, and when it is no longer needed, the body gets rid of the fibrin clot.
Platelets: Used for Hair Restoration
The initial enthusiasm for using platelets (PRP) for hair restoration was based on growth factors within the alpha granules in platelets. Activated platelets both release various growth factors (VEGF, PDGF, TGF-B, IGF-1, FGF, etc.) from alpha granules as well as frees up chemicals structures from dense granules. These work together to stop the bleeding, promote new blood vessel development, and ALSO stimulate hair growth. By definition, some say that for the product to be considered PRP the product must have an increase in platelet content over the level in blood and contain at least seven growth factors.
Figure 3 - Examples of growth factors in alpha granules of platelets
(But there are other bioactive factors contained in the dense granules of platelets and there is an emerging paradigm that more than just platelets are playing a role in PRP. So even as I write this blog, there is much left to learn as to the positive effects of PRP. Platelets release many other bioactive proteins responsible for attracting macrophages, mesenchymal stem cells, and osteoblasts.)
As I said before, the focus of all of my PRP treatment is obtaining the maximum amount of growth factors from platelets. So, as we measure levels of specific growth factors that are released by the platelets, I am making an assumption that the higher the level of specific growth factors, the greater the response with hair growth. You will see this demonstrated on the first graph in this blog.
Platelets: Release of growth factors
Three Types of Activation
In PRP, platelets are not active/are not ready to release growth factors from their Alpha-Granule, so platelets have to be activated. Activation helps hold platelets firmly at the site of injection thus growth factors are released at the desired area for a more extended period.
We know that activation releases growth factors.
How are platelets activated? Although PRP can be injected without prior activation, most hair restoration physicians who use PRP activate the platelets artificially, and there are three primary ways to do this:
1. Trauma: Involves injecting the PRP and then use micro needling or the trauma to the scalp to activate the platelets.
2. Thrombin: A liquid substance added to PRP right before injection. This substance a has its own potential side effects.
3. Calcium Gluconate or Calcium Chloride: Both are also liquid substances added to PRP right before injection. In fact, I have been using both of these Calcium compounds to achieve activation until very recently.
Which method of activation produces the highest level of growth factors? With any of these activation methods, a certain level of growth factors is released from the alpha granules. From all of the data I have seen, one activation method did not really release a more considerable amount than the other.
Then Dr. Cole’s study came out that introduced a completely different method to release growth factors that did not require any type of artificial activation, AND also produced the same or if not higher initial growth factor levels. This method is called sonication.
·Using Sonication to Create Platelet Lysate (PL)
Traditionally used in molecular biology, biochemistry, and cell biology laboratories, sonication uses high-intensity sounds waves to “disrupt” or physically break apart cell and platelet membranes. The technical term for breaking up a cell is lysis, so after lysing a cell, "lysate" is what remains.
Figure 4 - Lysis of a cell
Using sonication for PRP, there is no need to “activate” the platelet with trauma, thrombin, or calcium to release growth factors. After the centrifuge has separated components of the blood, PRP goes through sonication, and high-intensity sound waves destroy the platelet membrane which releases high levels of growth factors from alpha-granules into the plasma.
Figure 5 - Example of a Sonicator
Since this process destroys platelets, the product we are producing is not really “platelet-rich-plasma” anymore. Instead of PRP, what remains after sonication is platelet lysate or PL as Dr. Cole has accurately stated. PL has abundant independent growth factors, and we inject PL into the scalp.
Figure 6 - Platelet Lysate or PL before injection
As you will see later in the data from Dr. Cole’s study, there is an exponentially greater amount of growth factors (especially the larger growth factors like PDGF and VEGF) measured with the sonication.
Dr. Cole’s Study
There was not much published regarding sonication/PL and hair regeneration before Dr. John Cole’s recent article in Stem Cell Investigation from Nov 2017.
In his study, among other parts of analysis, he looked at which method (a type of activation or sonication) would release the most growth factors.
He compared: saline (unactivated) PRP, PRP activated by Calcium Gluconate, and PRP that was sonicated for 30 minutes (producing platelet lysate or PL). All of the samples were from the Arthrex centrifuge. From these methods he specifically compared the levels of growth factors IGF-1, TGF-B1, PDGF-BB, and VEGF each produced. Below is a graph of those results.
In terms of the growth factor measurements, the PL (platelet lysate) generated from the 30 minute sonication was found to have significantly higher levels of TGF-beta1, PDGF-BB, and VEGF over the Calcium Gluconate activated PRP or the PRP with no activation.
So, is there a difference in injecting the PRP vs PL as a stand alone medical treatment for follicular regeneration? Dr. Cole reported that when PL was injected alone the patient experienced a 50% increase in follicular unit density and a 122% increase in hair density 7 months post-injection.
You might also wonder, does this higher level of obtainable growth factors translate to better growth of hair follicles from PRP in a hair transplant? We again look at Dr. Cole’s study. The answer is YES.
When he measured follicular regeneration in transplanted grafts, the data showed superior regeneration using platelet lysate (PL) over PRP activated by Calcium Gluconate (AA-PRP) or saline (unactivated) PRP.
At 4 months post-op, 99%, 75% and 71% of follicle regeneration had occurred in the PL, Calcium Gluconate PRP, and saline treatment areas, respectively. Of course, we strive to achieve the highest level of surviving follicles with any hair transplant and the sonication method greatly improves the survival in his study.
There are many centrifuge systems on the market, and each has its own method (a certain amount of blood and number of spin cycles) to create PRP. Systems can use 11cc - 120cc of blood, but remember, platelets have growth factors that we want, and common sense says that 60cc of blood contains more platelets than 11cc of blood.
However, the amount of blood used doesn't always ensure a certain amount of platelets. The centrifuge system's efficiency separating the platelets is KEY. We want PRP to have a low RBC count and contain monocytes but minimal granulocytes and lymphocytes.
Over time I have used three different systems, with the most recent being the EmCyte system which can use 30cc or 60cc of blood and is a double spin system. To ensure I get more platelets, I draw 60cc of blood.
I wanted to share two pieces of data produced in 2015 and 2016 by Biosciences Research Associates comparing various centrifuge systems and their production of platelets and various centrifuge systems and their production of growth factors.
In both studies, thrombin was used to activate the platelets. (Remember, as has already discussed, platelets can also be activated by trauma, Calcium Chloride, Calcium Gluconate or injected without activation. Furthermore, platelets can be broken apart/lysed with sonication which is called platelet lysate or PL.)
Figure 7 - Study #1
Figure 8 - Study #2
Production of growth factors
Below is one more chart from Dr. Cole’s study that really shows the numerical improvement using sonication in PRP for hair restoration. The chart is comparing two different centrifuges, Regen and Arthrex, the various ways to release growth factors from PRP, and the resultant growth factor levels.
White Columns: The growth factor levels from the Regen Centrifuge Unactivated PRP and the Arthrex Unactivated PRP are similar, which we expect.
Yellow Columns: The yellow columns compare growth factor levels from Regen PRP that is more “traditionally activated” with Calcium Gluconate versus growth factors from Arthrex PRP exposed to sonication for 15 minutes.
As you can see in the row showing IGF-1 growth factor results, the levels from Regen (both Unactivated PRP and PRP activated with Calcium Gluconate) is similar to the Arthrex levels (including Unactivated PRP, and PRP exposed to 15 and 30 minutes of sonication) which may be related to the fact that there is a small protein and is easily released.
Then things start to get interesting.
After PRP was exposed to sonication, significantly higher levels of ALL growth factors (across all rows) were released (again, except IFG-1, which remained about the same). Furthermore, the growth factor levels were considerably greater after PRP was exposed to 30 minutes of sonication versus 15 minutes.
Final Take Away
After taking all of this knowledge into consideration, I made tremendous changes to the PRP treatment I offer, because as you can see from the data above, all PRP is not created equal. Again, if you believe that the level of growth factors is what is essential in stimulating the hair growth, the data very clearly shows that sonication releases immensely more growth factors than activation from thrombin or Calcium, as such I have also changed my practice to use sonication as the method to release growth factors. A quality centrifuge is also essential to obtain growth factors. I have begun using the EmCyte centrifuge system with 60cc of blood, although there is more than one top centrifuge on the market.
Countless centers offer PRP for hair restoration, but I believe, because of associated costs with a sonicator, and the time necessary to process PRP during an appointment, currently, there are very few centers preparing PRP to produce PL (platelet lysate). Even the treatment name has changed, as Dr. Cole recognizes. By using the sonication method, the treatment should be Platelet Lysate rather than Platelet-Rich-Plasma. Lastly, I also believe this updated form of PRP to PL is a powerful (maybe the most powerful) stand-alone treatment for hair regeneration, and I look forward to administering this treatment on all patients who could benefit.