Therapeutic application of PRP in Regenerative medicine

basics of therapeutic application of PRP in regenerative medicine

1. Preparation of PRP for therapeutic uses Dr.Biplabendu Talukdar MBBS,MD,PhD,M.Phil,MBA Assistant Professor, Department of IHBT, MCH, Kolkata, Technical Resource Person, SBC & HCP,WB

2. About Platelets Giulio Bizzozero, the Italian physician in 1882 first described anucleate fragments of bone marrow derived from megakaryocytes circulating in the blood. Approximately 2 microns in diameter. Platelet contain more than 30 bioactive proteins.

3. Properties of Platelets Platelet contain more than 30 bioactive proteins Secretory molecules including –cytokines, Growth factors, coagulation factors, Chemokines and Integrins These are all packed in Macrophage vesicles as Granules, Lysosomes and core Dense granules. Growth factors are stored in zymogenic state known as α granules.

4. Dense storage Granules Dense storage granules contain 1.Adenosine triphosphate - - stored energy 2. Adenosine diphosphate– helps transfer energy between cells 3. Serotonin – natural relaxant that also relives pain 4. calcium – A mineral needed for the regeneration of cells

5. “ α ” storage granules Alpha storage granules assist in the healing , it is containing growth factors and cytokines that act as a messengers between the cells. Granules work to heal the injury in three stages: 1. Inflammation- blood rashes followed by swelling of injury site 2. Proliferation: The quick growth of new cells for healing 3. Remodeling : damaged tissues are replaced by regenerative cells.

6. Other components of PRP Vascular endothelial growth factors (VEGF) : helps to growth of inner cell surface of the blood vessels ( angiogenesis) Epidermal Growth Factors (EGF) : helps dermal tissue regeneration Transforming Growth factors (TGF) ; stimulates creation of normal cells. Fibroblast Growth factors (FGF): they helps the formation of collagen under the dermal tissue.

7. Continue Hepatocyte Growth factors (HGF) : helps rpaire the cells of liver Insulin like Growth factors (IGF) : helps in formation of tissues and bones.

8. Growth factors present in platelets Paques M et al (1999) demonstrated that 1 ml of platelets ( 10 9 platelets /ml ) contain growth factors given in table Weibrich et al (2001) Five times higher than the normal platelets has rich in growth factors except TGF levels Name of factor /cytokines quantity PDGF 115 ng/ TGF-β 106 ng/ FGF 20.8 ng PDEGF 0.8 ng

9. Difference between healing by scar and regeneration

10. Precautions before PRP applications Stop taking drugs and supplements, these includes : NSAIDs Black current seed oil Krill oil Borage oil Bromelain papain Fish oil wobenzyme Flax seed oil Evening primrose oil

11. After procedure Minimize the axctivity for 24-48 hours Apply ice pack for 15-20 minutes over the area Avoid major physical activity Not to take any drugs or food supplements that affect platelets for 2 weeks after the procedure. Assessment of response may take 6-8 weeks after the therapy.

12. History and investigations required before PRP procedure Complete Blood counts Blood sugar Any bleeding disorder Any haemato- onchological patients Haemoglobin < 10gm% Platelet count subnormal ( less than 1 lac/ cumm Any one who is unfit for repeated collection of blood, for any reasons

13. Advantages of PRP in healing Ease of preparation No ethical issue Safety- autologous Easily accseeible Low-cost On going clinical evidences : tendon healing, cartilage healing, wound healing, hair growth and other clinical conditions.

14. What is platelet rich plasma therapy (PRP)? Different synonyms used for PRP Autologous Platelet concentration (APC) Autologous Conditioned plasm (ACP) Platelet Rich Fibrin (ARF) Ehrenfest et al (2009) subdivided PRP/APC into 4 categories on the leucocyte and fibrin contents : Product name   Leukocyte content Low density fibrin High density fibrin Leukocyte rich PRP L-PRP) yes yes (after activation) NIL Leukocyte reduced PRP P-PRP Poor/nil yes nil Leukocyte platelet rich fibrin (L-PRF) L-PRF yes nil yes Pure Platelet ric Fibrin P-PRF nil nil yes

15. Therapeutic dose of platelets Whitman DH et al (1997) a platelet count 1000X 10 10 L, as measured in 5 ml plasma may be the therapeutic dose. Weibrich G et al (2004) suggested that 800-1200 X 10 9 platelets/ μl are necessary to obtain an effective PRP dose. Anitua et al (2004) stated that the platelet count of PRP should be more than 3,00,000/ μl Marx gave a platelet count of 10 lakh/ml in 5 ml of PRP as a working definition of PRP, based on the scientific proof on bone and soft tissue healing

16. Continue Rughetti et al : 1.studied the relationship between platelet concentration and change in functional activity of human endothelial cells. 2. He observed that proliferation peaks at- for endothelial cells at 1.25 X 10 6 for angiogenesis 1.5 X 10 6 platelets / ml

17. Various clinical implications of PRP

18. Cell proliferation and tissue differentiation Promote tenocytes and tenocyte stem cells proliferation in vitro Accelerate the proliferation rates of circulating stem cells such as bone marrow stem cells ( BMSCs) and adipose derived stem cells (ADSCs) Positively influence cell attachment and spreading on the fibrin scaffold and promoting cell proliferation. TSCs differentiation into tenocytes .

19. Anabolic effects Influence the metabolism of tendon cells Increase total collagen synthesis Specifically enhance the gene expression of collagen types I and III Enhance the expression of cartilage oligomeric matrix protein (COMP) , decorin and tenascin-C (tendon healing glycoprotein) Leukocyte in PRP may negatively affect the anabolic effect of PRP Lead to scar formation by increasing the collagen type III /collegen type I ration

20. Anti-Inflammatory Effects Induced the release of HGF- a major anti-inflammatory growth factor. VEGF and HGF expression in tendopathy tendons Reduce the levels of COX-1 and PGE2, pro-inflammatory cytokines, IL6 and its ligand,CXCL-6 and IL8 Increased anti-Inflammatory Cytokines-IL10 and TGF-β

21. Antibiotic Effects Active role in sepsis ( promoting innate immunity) Significantly decrese bacterial growth of ---- MRSA ---- P.acnes ---- S. epidermidis ---- S,aureas

22. Requirements list for PRP procedure 1. centrifuge machine 2.blood collecting tubes 8 ml size 3.Velcro, bandage, cotton, tape 4.Syringe and needle 5.Insulin syringe 6. PRP kits available in market 7.antiseptic lotions/ normal saline 8.USG machine - optional 9.PRP activator solution -optional

23. Process of PRP preparation Draw the patient's blood Transfer the blood in processing container Place those container in a centrifuge, which is FDA approved for PRP Spin the blood in the centrifuge, according to manufactures direction

24. PRP method Obtain WB by venipuncture in acid citrate dextrose (ACD) tubes Do not chill the blood at any time before or during platelet separation. Centrifuge the blood using a ‘soft’ spin. Transfer the supernatant plasma containing platelets into another sterile tube (without anticoagulant). Centrifuge tube at a higher speed (a hard spin) to obtain a platelet concentrate. The lower 1/3 rd  is PRP and upper 2/3 rd  is platelet-poor plasma (PPP). At the bottom of the tube, platelet pellets are formed. Remove PPP and suspend the platelet pellets in a minimum quantity of plasma (2-4 mL) by gently shaking the tube.

25. Buffy coat method WB should be stored at 20°C to 24°C before centrifugation. Centrifuge WB at a ‘high’ speed. Three layers are formed because of its density: The bottom layer consisting of RBCs, the middle layer consisting of platelets and WBCs and the top PPP layer. Remove supernatant plasma from the top of the container. Transfer the buffy-coat layer to another sterile tube. Centrifuge at low speed to separate WBCs or use leucocyte filtration filter.

26. Commercially available PRP kits

27. All of the following steps in the process can affect the final process

28. The size of blood draw needle A 19 gauge needle or larger seems to be ideal

29. The container Size of container – compatible for centrifuge buckets Material the container is made from- glass tubes polypropylene tubes polycarbonate tubes Shape of the container- test tube shaped conical cylindrical

30. The Rotor and Buckets Swinging buckets Fiver angle rotor usually at 15˚ angle Relative centrifugal force (CRF) (i)Amanda et al, showed best performance was 300g for 5 minutes or 240g for 8 minutes. (ii) RCF is determined by the diameter and speed in the rotor(RPM) (iii) Formula (RCF) – 0.000011118 X rotor radius X RPM

31. Association of PRP with age ? According to some studies, the growth factors in PRP progressively decrease with ageing , largest drop off are seen in PDGF-BB and IGF-1 Evason et al in 2014 studied among 71 males and 31 females and observed that female in all age group had higher concentration of all growth factors including EGF, HGF,IGF-1 and PDGF-BB. In the year of 2019 Japanese scientist shows the negative correlation for both PDGF and IGF between the age 20-50

32. Association of circulating stem cells with age Tenguchi et al, Journal of Exp Ortho in 2019 studied the circulating stem cell counts on to respond to an injury. The ratio of stem cells to regular cells from birth to 60 years of age is given below :

33. Indication of PRP

34. Dermatological Fine lines and wrinkles Dull, tired skin Decolletage Smokers lines Hands Suitable for atound the eyes Scars Strech marks Acne scar

35. Pain and orthopedic application Joint pain and tendonitis Tendon injury Frozen shoulder Tennis elbow Low back pain Osteoarthritis of knee joint Chronic pain

36. Obstetrics and gynecology Infertility Premature menopose Geriatric pregnancies Ovarian rejuvenation Stress incontinence Endometrium regeneration

37. Male sexual dysfunction Erectile dysfunction Peyronie’s disease Penis enlargement Sexual performance Urethral stricture – 1 st time used in India jointly by Prof. Dr. Sunirmal Chaudhury ( Department of Urology) and Dr. Biplabendu Talukdar (department of IHBT).

38. Dental uses Dental surgery Reducing dental bleeding Soft tissue healing Bone regeneration Jaw osteonecrosis

39. Risk of PRP injections Pain in the are of injection Infection Allergic reaction Bruising No improvement of symptoms

40. Contraindications of PRP Coagulopathy Haemodynamic instability Sever sepsis Unstable angina ITP,hemophilia Using blood thinners Pregnancy

41. Take home massage Platelet initiate tissue repair, regeneration and revivals is with the regenerative medicine. Platelet yields depend on process of preparation. Quality control and platelet is essential for better out come More research is need in future for better out come Common regenerative medicine products- i ) Stem cells – embryonic, Adult or bioengineered ii) Mesenchymal stem cells, iii) platelets

42. References 1. An illustrative Guide on Platelet rich Plasma- Dr. Sandeep and Deepti Shrivastav 2.Perfect PRP- Jeffrey Piccirillo,DO 3.Learn Plasma Rich Plasma Procedure-A complete practical guide: Maria LI 4.Principal of Regenerative Medicine (Third edition ) Lanza 5.Medical Embryology -12 th edition T.W Sadler

43. Choice of Regenerative medicine product as ideal solution The best chance for tissue repair, regeneration and revivals is with the regenerative medicine. It assisting the normal regenerative process. Common regenerative medicine products- i ) Stem cells – embryonic, Adult or bioengineered ii) Mesenchymal stem cells, iii) platelets

44. Introduction PRP falls under the category of regenerative medicine. The first mention of PRP in the literature of haematology was in 1971. Cardiovascular surgeons were one of the earliest specialists who begun studying the use of PRP in early 2004-2005 in their sternotomy and chest incisions. Maxillofacial surgeons prefer to use platelet rich fibrin (PRF) for bone growth during dental implants in poor quality bone, periodontal surgery , maxillofacial surgery, Dermatologist, plastic surgeons also shows interest for application in cosmetic surgery and skin grafting and hair implant The orthopedist showing interest in PRP during arthroscopy Dr. Charles Runels (2010) & Priapus uses PRP in the field of sexual health and increasing the orgasm in women.

45. Difference between healing by scar and regeneration

46. factors release from platelets during tissue injury

47. Functions of growth factors

48. Function of PDGF

49. Exclusion of patients for PRP therapy Any bleeding disorder Any haemato- onchological patients Haemoglobin < 10gm% Platelet count subnormal ( less than 1 lac/ cumm Any one who is unfit for repeated collection of blood, for any reasons.