Micro Needling and PRP �  Define PRP

�  Define Growth Factor

�  Define Skin Needling

�  Treatment Protocol

Define PRP �  If you are using tubes you are not making PRP – this

is Plasma with some platelets in it.

�  Tubes only give you approx. 200,000 – 400,000/ul and in many cases far less. Results are partial and compromised.

�  Real PRP is made via a cell separating device and cannot be achieved in a single 8-10ml tube. A minimum of 40mls is required.

•  PRP means your platelet concentrate dose is 1,000,000/ul 1,000,000/ul is the dose required for mitogenesis of MSCs – this will regenerate connective tissue.

�  ( Haynesworth et al, 2002; Marx, 2004, Everts, 2007)

Haynesworth et al (1992)

�  Platelets release their GFs when the Platelet touches collagen in vivo.

�  In vitro, via a glass surface and/or when Calcium is added to the PRP.

�  You do not need to preactivate PRP when injecting.

�  Topically, you may want to pre activate. This is “easy” and does not need boiling, heating, freezing, complex mixing and so on.

�  Simply, add Calcium Gluconate or Chloride to the PRP in a specific glass petri dish

�  Within 15 minutes the platelets have released their GFs and the product can be applied topically.

Everts (2007)

�  Micro Needling will heal automatically via epithelialization. Topically applied EGF leads to accelerated epithelization.

�  In the beginning of the epithelization process, PDGF receptor genes were found. During the last phase of wound healing both FGF and PDGF increased contraction and remodeling time (Everts, 2007).

�  Exogenous application requires activation of the PRP, which will induce the release of PGF in a dose dependent fashion.

�  PGF increases the population of healing cells by mitogenesis. This action depends on the presence of further differentiated MSCs.

�  After PG has been applied to tissues and the clot has retracted and degranulated, PGF will be deposited in the ECM. During matrix degradation, GFs are released that interact and bind with the Platelet Tyrosine Kinase Receptor (TKR), present in the cell membranes of tissue cells.

�  The TKR extends into the cytoplasm, GFs interact to activate Signal Transduction which transfers into the cell nucleus and triggers DNA synthesis, transcription of messenger RNA, producing a biological response that initiates the cascades that induce tissue repair and regeneration.

�  GFs are capable of inducing effects on multiple cell types, and may therefore provoke a series of cellular functions in different tissues.

•  Classical wound is defined as a disruption to skin integrity •  Wounds by surgery or trauma that heal through secondary

intention rely on biological phases of healing – hemostasis, inflammation, proliferation and remodeling (HIPR)

•  One may assume that MN (200 needles) up to 1.5mm in depth will be a wound. However, this is not so.

•  Wound healing is curtailed and does not end in scar formation •  A less intense acute inflammation occurs and the mechanism of

action for MN is different. •  Let’s review the potential mechanisms by which microneedling of

the skin facilitates repair without scarring

Potential Mechanism of Microneedle Treatment of Normal Skin

•  Electro Taxis – enhanced cell communication and motility by endogenous electrical signals.

•  Skin cuts to a depth of 0.5-0.6mm close by electrical cell stimulation without any trace of scar tissue. Deeper cuts close by skin repair ultimately ending in scar formation

•  Leibl (2010) theorized that the mechanism for the main action of MN includes Trans Epithelial Potentials (TEPs) and the skin battery.

•  Electrodes were placed on the SC and inside the dermis, and measured a negative potential difference of the SC ranging from 10 – 60 mV, and averaging – 23.4V

�  Stainless steel, non traumatic needle enters the SC and is pushed into the intercellular space, the only reaction is a short circuit of the endogenous electric fields.

�  Non Traumatic needles penetrate for fractions of a second and have a tip radius of not more then 2- 3 um – do not create a classical bleed.

�  An ordinary hypodermic needle merely pushes cells aside. There is minimal to no bleeding since only capillaries are punctured. This is a mild inflammatory response due to bradykinins and histamine release from mast cells.

�  After soft tissue injury the NA/K – pump is activated to re establish the sodium and potassium pump. ATPase, a trans membrane protein delivers positively charged Na+ ions into the intercellular electrolyte and collects K+ ions and transports them into a cell.

�  Charging and discharging a cell takes 2 – 3 milliseconds only in the vicinity of the needle insertion (Figure 4)

�  The cells around the needle channels sense the re occurring penetrations as new repeated induced wound stimuli and therefore are in a permanent active state that leads to a polarized electro magnetic field ( EMF) in the inter cellular electrolyte. The EMF stimulates DNA expression of the surrounding cells.

�  This epigenetic DNA information by electro taxis leads to an enhanced motility of epithelial and endothelial cells in the wounded area and subsequently to gene expression of GFs that facilitate healing. (Figure 5)

�  MMPs play a vital role when Scar tissue is needled.

�  After MN, TGF –β3 controls collagen fiber integration into the skin’s matrix ( Figure 6)

�  MMPs are controlled by inhibitors ( TIMPS). They continue to be active to degrade excessive tissue until degradation of surplus tissue is complete (Figure 7).

�  Capillaries and fibroblasts migrate into the former scar tissue. Synthesized collagen fibers ( type III) integrate into the skin matrix.

�  Following MN hypo trophic scars raise to the skin level and former hypertrophic scars fall to the skin level ( Figure 8 and 9).

�  10 – 20 weeks for hypo trophic scars after 1 – 3 sessions

�  Several months for Hypertrophic scars and burn scars. Burns scars have a 30% failure rate.

Radio Frequency Micro Needling �  Heat Shock Proteins – diverse and essential

components of cell physiology.

�  HSPs are molecular chaperones and have a protective role.

�  Their expression is elevated in the cells exposed to several stress factors – heat shock

�  They provide a working environment for correct polypeptide folding, play a pivotal role in protein assembly, in repair and transportation of proteins.

�  A thermal stimulus that is applied by non ablative treatments induces a temperature increase which activates HSPs 27, 47, and 70.

(Dams, 2010)

�  HSP27 is anti apoptotic, inhibits release of cytochrome-c as a pro apoptotic: and downstream of mitochondria, by preventing caspase-3 and -9 activation, enzymes that play a central role in the executing phase of the cell.

�  Governs refolding of other proteins in the endoplasmic reticulum – collagen biosynthesis in skin fibroblasts by transporting procollagen from the RER to the Golgi system.

�  HSP47 is a constitutive protein, serves as a collagen type 1 specific molecular chaperone, and enables the correct three-dimensional conformation of procollagen chains and prevents their aggregation and precipitation

�  In response to stress, HSP70 binds to denatured proteins, preventing their intracellular aggregation and precipitation, whilst targeting them for appropriate environment for refolding or proteolysis.

�  HSP70 suppresses apoptosis of cells.

•  MN increases permeability •  Increases the trans epidermal delivery of ingredients

and drugs

In summary, �  PRP must be 1,000,000/ul platelets

�  PRP is activated in a glass petri dish designed for platelet activation – no need for special tricks, heating, freezing etc

�  Freezing cells diminishes their numbers and viability

�  Use and apply cells immediately

�  PRP is best injected into the skin

�  MN is electro magnetic taxis and mild acute wound healing

�  Topical application of ampoules is also recommended