PRP is an autologous biologically active product prepared from fresh blood of patients using one or two stage centrifugation. It is also called as platelet rich concentrate or platelet releasate or plasma gel. It has platelet concentration of around 1-1.5 million/microlitre which is 4-5 times the normal platelet concentration in blood.
PRP is a good source of growth factors such as IGF-1 , PDGF, VEGF, EGF, cytokines and plasma proteins. The alpha granules in platelet degranulate to release growth factors in high concentration on activation. The supra-physiological concentration of biologically active substances modulate inflammation and tissue repair. It stimulates cell survival, proliferation and differentiation. It promotes vascularisation and angiogenesis. It increases differentiation of fibroblasts promoting collagen synthesis and remodelling. It also increases myofibroblasts in wound promoting contraction. PRP with its regenerative and wound healing properties has wide application in plastic surgery. PRP being autologous carries decreased risk of hypersenstivity , immunogenic reaction and disease transmission.
INDICATIONS OF PRP USE :
- Hair Restoration in Androgenic Alopecia
- Facial and skin rejuvenation in patients with acne scars, atrophic scars, wrinkles, striae distensae
- With fat grafting to increase fat survival
- Chronic wounds like diabetic ulcers, venous ulcers to promote healing
- To promote flap survival
- To promote bone graft survival and healing
Preparation of PRP
Steps of PRP prepearation are as follows:
- Patient history: Platelet medications such as aspirin and statins affect platelet function. Any history of platelet function disorder, bleeding disorder, anticoagulation therapy, thrombocytopenia, hepatitis , local infection, hemodynamic instability and tendency for keloid formation are contra-indications for PRP therapy.
- Blood Collection: Around 20-60 ml of fresh blood sample is drawn preferably in single venepuncture using wide bore needle to avoid trauma to platelets, degranulation and loss of growth factors.
- Centrifugation: The force, time duration and number of cycles of centrifugation affect the concentration of PRP. Longer and more forceful centrifugation push platelets further down in sediment layer and potentially affect growth factors and cellular integrity. There is no standard method of centrifugation with some proposing single step centrifugation and others two step process i.e slow centrifugation followed by fast centrifugation. There is no protocol set for speed and duration of centrifugation and hence a lot of variation is seen with regard to this. This has led to lack of standardisation of PRP preparation and quality control.
The initial slow centrifugation at 1500 rpm for 15 min separates the erythrocytes at bottom, from lighter plasma with buffy coat at interface in middle and top layer of platelet poor plasma. Plasma and buffy coat are aspirated and centrifuged at high speed (3200 rpm for 10 min) when platelet separate as pellet with platelet poor plasma at top. Platelet pellet is resuspended in small volume of plasma for final product. In case of single step centrifugation at 3200 rpm for 15 min, out of the three layers the middle layer is used for PRP after removal of supernatant.
Commercial kits are also available for preparing PRP.
4. Anticogulation: Some may use the final product immediately. Others may add anticoagulants like sodium citrate, trisodium extract, acid citrate dextrose, heparin to prevent platelet activation and its conversion to fibrin matrix.
5. Activation: Calcium chloride or thrombin is added to reverse anticoagulation and activate platelets before use. 70% growth factors are released within 10 minutes and nearly all growth factors within 1 hour.
6. Injection: usually given in area of pathology in subcutaneous or intradermal plane using 26G to 30G needle.
Types of PRP
Based on platelet concentration and activation of platelets, it can be further classified as platelet rich plasma (PRP) and Platelet rich fibrin (PRF). PRP is liquid platelet suspension which needs activation step to release growth factors either by addition of exogenous factors or may be simply activated by trauma of injection. PRF is in the form of gel. It is activated fibrin matrix and has low platelet concentration. Further both can be subclassified based on leucocytes concentration as leucocyte rich or poor as follows:
- P-PRP: It is leucocyte poor. It has small volume and minimal fibrin polymerisation.
- L-PRP: It is leucocyte rich. It has small volume and minimal fibrin polymerisation.
- P-PRF: It is leucocyte poor. It has larger volume and dense fibrin polymerisation.
- L-PRF: It is leucocyte rich. It has larger volume and dense fibrin polymerisation.
Leucocytes in PRP are expected to help with their anti-infection properties which is beneficial. Practically we are unable to determine leucocyte content in routine and hence usually general term PRP is used. Usually we get L-PRP and cell separation is needed to process out leucocytes to form P-PRP.
PRP enriched with leucocytes in addition to concentrated plasma proteins has shown good results in mild and moderate male as well as female patterned baldness. PRP promotes growth of hair follicles and significantly shortens time of hair formation. PRP promotes angiogenesis in scalp. PRP increases proliferation of dermal papilla cells and induces faster telogen to anagen transition. Bulge cells, inducible stem cells found along the shaft of hair follicle have been found to repopulate hair follicle epithelium and are fundamental to progression of hair cycling. It is these hair follicle stem cells that contain growth factor receptors responsible for hair growth manipulation and molecular pathway regulation. PRP when combined with CD34+ cells also shows good results. Only slight improvement is seen with non activated PRP.
Usually 3 sessions of PRP over 3 months, followed by two session over year has shown satisfactory results. If no improvement is seen in first three months, further attempts of injection are futile. PRP can be injected in areas of thinning i.e usually frontal, vertex and parietal areas. Interfollicular injections are given of about 2ml to 12ml volume i.e 0.1 to 0.2 ml/cm2 of scalp. It is usually given in intradermal plane but can be given in subcutaneous plane after topical local anaesthesia application on scalp for numbing.
Hair traction test, hair density index ,phototrichoscan and scalp biopsy are various methods of gauzing the improvements after PRP therapy. Increased hair density, number of hair follicles and decreased hair on traction test are seen. Increased thickness of epidermis of scalp, vascularisation of scalp between follicle and epidermis and increased ki-67 proliferation index of bulge stem cells and cells of basal layer of epidermis is seen on biopsy. Oil secretion is also improved post PRP treatment. Results are assessed 4 months after last session.
Facial and skin rejuvenation
PRP promotes collagen synthesis and remodelling improving skin elasticity. PRP alone or in combination with fat is injected intradermally in infra-orbital area, nasolabial folds, crow’s feet area, forehead/malar region and pre-auricular region. Also, dermaroller/microneedling/fractional laser followed by application of PRP is used, popular as vampire facials. The pores produced acts as route for penetration of PRP. It leads to improved collagen synthesis and hence skin texture with decreased erythema, edema and post-inflammatory hyperpigmentation. Improved results are seen in patients of acne scars and atrophic scars. Around 2ml of PRP is used at 2 – 4 weeks interval for 4 sessions.
PRF has postulated to yield better results than PRP as it releases growth factors over longer period of time. PRF has been used to treat crow’s feet, wrinkles, tear troughs, suborbital hollows, glabellar furrows, malar augmentation, zygomatic arch enhancement, correction of nasolabial folds and marionette folds as well as acne scars.
PRP with its pro-angiogenic, anti-apoptotic and anti-inflammatory properties has been seen to prevent fat atrophy after fat grafting in face. It also improves volume. PRP promotes proliferation and differentiation of stem cells and pre-adipocytes into mature adipocytes. PRP acts as scaffold for adipocytes and adipose derived stem cells(ADSC) retaining them at graft site for longer duration. Fibrin scaffold reduce apoptosis of differntiated adipocytes. When in fibrin clot, ADSC show higher secretion of VEGF and FGF. In vitro, when ADSCs were grown in scaffold of fibrin with adhesion molecules and growth factors, they differentiated into keratinocytes enhancing wound healing. Activated PRP is used for injection. 0.1 to 0.5 ml of PRP is used per ml of fat graft. The adequate concentration of PRP needed for optimal growth of ADSC is still uncertain with several studies stating 5-15% being optimal. Higher concentration of 40-50% PRP can have negative regulatory effect of platelets on fat. In breast fat grafting, no benefit of PRP is seen but higher rate of fat necrosis is seen.
Healing of Chronic Wounds
PRP encourages ADSC to differentiate into fibroblasts and keratinocytes that are crucial cells in wound healing process. PRP also encourages migration of fibroblasts to wound site. Direct infusion of PRP to wound bed and topical application of PDGF have shown positive results in diabetic foot ulcer healing.
PRP enhances skin flap survival rate. It enhances angiogenesis and reduce the inflammation response to skin flap transplantation. Release of growth factors, platelets, immune activating factors and fibrin help in skin flap survival. Plateletsand fibrin accelerate coagulation and provide a scaffold for skin flap.
PRP enriched bone grafts have higher bone augmentation, shorter time to bone regeneration, decreased post-operative pain, lower rates of haematoma and edema. An overall superior outcome is seen.
In conclusion, PRP with its wound healing properties, ease of preparation, great safety profile, being minimally invasive in application and showing satisfactory results, is becoming increasingly popular modality of treatment for improved outcomes in plastic surgery.