PLATELET-RICH PLASMA (PRP)

- AND TISSUE REGENERATION BY GROWTH FACTORS: PUSHING OUT HORIZONS IN REGENERATIVE AND AESTHETIC MEDICINE WITH PERSPECTIVES FROM ASIA 2007.

BOLANDCELL provides an updated overview on PRP and REGEN-ACR , with illustrations and academic references: special reference for use in the clinic and in aesthetic medicine. For updated molecular and cell biology details, BOLANDCELL , recommend that the following academic article be consulted: " SOFT AND HARD TISSUE AUGMENTATION WITH PLATELET-RICH PLASMA: TISSUE CULTURE DYNAMICS, REGENERATION AND MOLECULAR BIOLOGY PERSPECTIVE: DU TOIT ET AL, International Journal of Shoulder Surgery, 2007, Vol 1, 64-73 ( Short cut: Google Search: PRP SHOULDER).

Aesthetic physicians, medical and dental clinicians have a vested interest in the field of soft tissue regeneration for the chronological and photoaged skin, rejuvenation by fibroblasts/keratinocytes and regarding the application of activated platelet-derived cytokines or growth factors in the clinic. Potential benefits to patients by the intraoperative application of PRP for specific indications during surgery, include reduced capillary bleeding and oozing in surgical flaps, reduced need for drains, reduced postoperative pain and swelling, accelerated post-operative recovery time and improved wound healing (Marx et al 2005). So, the benefits would be a sealant and trophic growth factors to enhance wound healing. These local effects are related to and orchestrated by the trophic and anabolic nature of platelet releasate following activation in the wound (Marx et al 2005). Seven anabolic and trophic factors identified in platelet-rich gel have now been described, the best known include platelet derived factor (PDGF) and transforming growth factor-beta 1 (Marx et al 2005) Details are reflected in Table 1. These factors are commercially available. Other important secretary proteins that influence wound healing and derived from alpha granules of activated platelets include, vascular endothelial growth factor ( VEGF), epidermal growth factor ( EGF), insulin-like growth factor (IGF), osteocalcin ( Oc), osteonectin (On), vitronectin ( Vn), fibronectin (Fn) and thrombospondin-1 ( TSP-1), (Marx et al 2005). These factors can supposedly increase the rate of collagen deposition, angiogenesis, fibroblast proliferation, extra cellular matrix synthesis relevant to wound healing and soft tissue regeneration (Marx et al 2005).

Platelet-Rich Plasma (PRP) can be defined as "an autologous concentration of human platelets in a small volume of plasma" ( Marx et al 2005 ). This concentrate contains the trophic growth factors that are released once the platelets therein are activated either by calcium chloride, thrombin or fibrinogen (Marx et al 2005). All seem equally effective in activating the platelets ex vivo. In the process a gel is created that can be used as a suitable carrier for bone chips that are destined to be used in cranio-facial interventions such as sinus augmentation or lifting (Marx et al 2005). The plasma poor plasma (PPP) has potent fibrin-sealant properties like Tisseel® (Marx et al 2005). The side-room preparation of PRP has been documented and has been simplified by the availability of table-top high speed centrifuges that are commercially available. But not all centrifuges render the same concentrate of PRP (Marx et al 2005). Paying attention to fine detail, and avoiding contamination, is important during the generation of PRP (Marx et al 2005). The static apparatus can be installed in an office side-room or adjacent to an operating theatre. In order to ensure quality assurance of the PRP, designated FDA or CE approved automated platelet concentrate systems and trained personnel must be utilized (Marx et al 2005). The surgeon needs to determine how much PRP he or she wants to generate for the particular procedure. Fifty milliliter venous blood can generate about 16 ml of PRP. This is possible in a 30 minute period and can easily be performed by a suitably qualified and board certified clinical, technologist who assists the surgeon. Less can be generated for smaller procedures, in which case, only 20 ml of venous blood is needed. Upholding aseptic principles during cell separation is mandatory to avoid contamination (Marx et al 2005). Briefly, 50 ml of venous blood is obtained from the patient by venesection of the median cubital forearm vein, shortly before the surgical procedure. Surgery activates the platelets, so timing of the venesection is important (Marx et al 2005). Informed consent is needed for venesection and details should be explained to the patient beforehand. The blood is collected and inserted into special designated tubes containing an anticoagulant. Storage of the blood should be avoided due to loss of platelet activity. Special cost effective venesection kits are available ( RegenkitT, Switzerland ), for blood collection and preparation of PRP. Medium speed centrifugation concentrates the platelets into a small volume without fragmentation. It is an easy task and the technology is available ( Marx et al 2005). The layer containing the PPP, which can be differentiated from the plasma-rich plasma, is aspirated into a sterile tube. A second spin is needed to obtain PRP. For the platelet growth factors to be released, platelets have to be activated ( Marx et al 2005). This is intentionally induced, shortly before use of the gel, and affected by addition of calcium chloride ( provided in the preparation kit) and or thrombin to the platelet concentrate ( Marx et al 2005). The gel thus created, is available for soft tissue infiltration and augmentation or admixture with a bone graft (Marx et al 2005). The blood clot is useful in maxillo-facial surgery and for placement in sockets or admixture with bone chips (Marx et al 2005). There are numerous commercially available machines, all of which yield slightly different concentrations in platelet and leucocyte cell counts (Marx et al 2005). Clear identification of the buffy coat is needed after centrifugation. The potential advantages of the biological approach of PRP are safety, quick release of platelet-derived growth factors, autologous nature of the preparation and avoidance of disease transmission (Marx et al 2005).

BIOSTIMULATORY EFFECTS OF PRP RELEVANT TO CLINICAL PRACTICE: UPDATE BY BOLANDCELL TECHNOLOGIES

PRP has many theoretical unique and biomedical mechanisms of actions, albeit not fully understood, some of which are enumerated below:

BASIC SCIENCES: CELL BIOLOGY, TISSUE CULTURE DYNAMICS AND PLASMA-RICH PLASMA RELEVANT TO CLINICAL PRACTICE..UPDATE BY BOLANDCELL TECHNOLOGIES .

In wound healing, whole cell movements and locomotion, are dependant on intracellular filament systems, structural polarity co-ordination of microtubules and actin filaments (Albert's 2002). During cell locomotion, cross-talk is needed between the cytoskeleton and cell adhesion as the cell moves through the ECM. This impacts strongly on the speed of cell crawling in the senior authors laboratory experience. Both dermal fibroblasts and skin keratinocytes, in our experience in the light laboratory, have rapidly progressive leading edges in tissue culture (TC), and with planar proliferation in 2D culture. Lamellipodium and filopodium activity promotes formation of new attachment sites and cell crawling in the culture dish, but initial cell polarization is important.

Fibroblast crawling can rapidly be established in 3D culture. By 4 weeks of TC, monolayers were tightly packed and cell growth exceeds those in serum starved control media. In culture, fibroblasts of the leading edge rapidly translocate from the 3D media and assume conventional phenotype expression on an adjacent fibronectin treated culture base, but cell proliferation becomes slower. Growth pattern of the fibroblasts in various media differ but are enhanced in PRP.

Regarding the maintenance of the integument, both keratinocytes and fibroblasts play major reparative roles in the epidermis and dermis. In recent years, cultured keratinocytes have played an adjunctive and supportive role in the treatment of partial-thickness burns in both adults and children.

Single and co-cultured keratinocytes/ fibroblasts can render established monolayers within 3 weeks. One and 5 million cells in G 2 phase, respectively are used for analysis for image phase contrast evaluation. Growth, locomotion and ruffling is greater in fibroblasts compared to keratinocytes presumably due to enhanced endowment of filopodia and lamellipodia with cytoskeletal actin projections. This can be confirmed by differential interference phase contrast imaging. H&E and Masson Trichrome staining at LM level confirm strong migration in the D&E junction region, also referred to as the space of Grenz. In PRP-enriched wells ( activated and non-activated), monolayers of both keratinocytes and fibroblasts are strongly in evidence. The presence of collagen is confirmed by the Masson Trichrome staining at light-microscopic level.

Fibroblasts and myofibroblasts are integral components of the extracellular matrix (ECM). Both are important for restoration, rejuvenation, wound healing and maintenance of the ECM , together with GAG, fibronectin, hyaluronic acid, mucopolysaccharides, collagen and elastin. Platelet-Rich Plasma releases 7 secretory proteins (growth factors) on activation and plays an important role in the inflammatory healing cascade including wound healing. Secretary proteins include VEGF, PDGF, TGF-beta-1, EGF, IGF, osteocalcin and nectin, VN, FN, FG and thrombospondin, all relevant to wound healing. ( Table 1).

Fibroblasts and myofibroblasts can be cultured in all mediums by static technology, but PRP enriched medium ( activated and non-activated) provides good results compared to planar spreading. Both activated (by Cacl 2 or thrombin) and non-activated work equally well. PRP ( REGENLABT) forms a fibrin matrix within 12 hours and changes the culture technology from 2 D to 3 D, eliminating partially the need for fibronectin. The presence of fibroblast branching, lamellipodia and filopodia indicate active cell locomotion. ECM network formation is clearly visible in monolayers within 3-4 weeks.

An early effect of platelet-rich-derived growth-factor (PDGF) on cultured human fibroblasts, is induction of dorsal plasmalemma or membrane ruffling from the forming face. This morphological outcome is due to the formation of motile cell surface protrusions facilitated by a meshwork of newly polymerized actin filaments. Rac , a small GTP-binding, protein is a key regulator of membrane ruffling. Our group is interested in PDGF-induced circular ruffle formation and on the dorsal surface because of the documented presence of cytokines in PRP ( REGENLABT). This membrane modulated ruffling is of interest because it is cytokine driven, and stimulated by protein kinase C (PKC)( See Albert's et al 2002).

Compared to 2 D culturing, both cells lines show dendritic cell extensions that contain microtubule cores (fibroblasts> keratinocytes), allowing cross talk between cells. Cell ruffling is more pronounced in cells exposed to platelet cytokines and 3 D culturing. These observations allow us to speculate, that the 3 D environment of platelet-derived fibrin, permits cell extensions to engage integrins on both dorsal and ventral cell surfaces simultaneously, thereby activating unique signaling mechanisms as proposed by Beningo et al 2004. Planar 2 D surfaces allow lamellipodial extensions, cell crawling and spread. A 3 D matrix derived from PRP, facilitates microtubule-dependent dendritic cell locomotion and motility. Because of the extensive dendritic cell formation and cell plasticity, mandates that more controlled laboratory study is warranted before PRP- 3D cultured fibroblasts are applied in man and especially in aesthetic medicine.

PRP AND RELEVANCE TO OPTHALMIC SURGERY PRACTICE: BOLANDCELL UPDATE

PRP has been shown to be very effective in the treatment of dormant corneal ulcers. It has now been possible to produce autologous PRP in topical eyedrops for the treatment of recalcitrant neurotrophic, herpetic and immunologically induced corneal ulceration ( Alio et al 2007). Treatment included chronic nonhealing ulcers in persons that had been unresponsive to conventional topical therapy. In these studies, measured outcomes included ulcer size, inflammation, healing, visual acuity and patients subjective symptoms. In all groups studied, improved healing was observed. Pain, is particularly improved in all patients. In conclusion autologous platelet-rich plasma promoted healing of dormant corneal ulcers in eyes threatened by corneal perforation and was accompanied by a reduction in pain and inflammation. The same group studied the effect of PRP in the treatment of symptomatic dry eye. Significant dry eye symptoms with autologous PRP proved to be very effective, improving both patient symptoms and major clinical signs ( Alio et al 2007). The same authors showed the usefulness of PRP in the treatment of symptomatic ocular surface syndrome following LASIK. Topical PRP eye-drops were prepared from total blood and enriched platelets by centrifugation. 80% of patients responded positively. Conclusions from this work show that autologous PRP was effective in the treatment of patients with ocular surface syndrome following LASIK with symptoms generally relieved and a positive effect on punctuate keratitis. These positive changes indicate that PRP will in future play an important strategy in these patients. The main focus of PRP will be on enhancing wound healing of the cornea by the bioavailability of GF derived from the platelet gel / clot.

AUTOLOGOUS PLATELET RICH PLASMA: APPLICATION IN CARDIOVASCULAR AND OPEN HEART SURGERY: BOLANDCELL UPDATE

The literature shows that it is possible to stimulate the bodies natural healing at cellular level through the application of platelet-derived growth factors ( Vang et al 2007). Recent work shows that it is now possible to apply activated-PRP ( by either calcium or thrombin) to surgical wound sites of patients undergoing cardiac surgery ( Vang et al 2007). Growth factor enriched PRP, in these studies has been directed at the sternum before and after re-approximation and before skin closure. The aims of this approach were to enhance osteointegration and facilitate sternal ( bony) healing thereby reducing the incidence of post-operative sternal disruption which is a documented complication of thoracic surgery. Apart from the wound healing properties of PRP, the platelet poor plasma component ( PPP component of centrifuged plasma) is also valuable as a sealant, and haemostatic agent ( via fibrin deposition), thereby reducing haematoma formation and fluid collections in dead space. Possibly deep and superficial sternal infections can be obviated by the application of PRP in the theatre to the wound edges shortly before closure. This approach adds no time to the operation as the PRP is rapidly deployed and sprayed or dripped on the edge. For the sternum and skin, 10 and 7 ml enriched PRP, are recommended ( Vang et al 2007; Mercy Medical Center , Sioux City , Iowa , USA ). Thirty-two ml venous blood is needed approximately to produce 16 ml PRP. Preliminary studies indicate that PRP may well confer beneficial effects on pain, bloodloss and bruising in the postoperative phase following cardiac surgery. Other benefits of the use of PRP transfusion during cardiopulmary bypass has been reported ( Crowther et al 2000; University of Aberdeen ). Hiramatsu et al 2002 have reported the benefits of PRP administration after open heart surgery in patients ( including paediatric cases) with non-cyanotic congenital heart disease. More than 10-years ago, cardiac surgeons from Japan showed that PRP intra-operatively could reduce the perioperative blood loss, the rate, and the volume of homologous blood transfusion ( Misumi et al 1995). Application of PRP to the leg harvest vein wounds may well also speed up skin healing and reduce postoperative swelling. PRP use is also indicated as a sealant in ascending aortic surgery.

APPLICATION OF PRP IN ORTHOPAEDIC AND SPINAL SURGERY: BOLANDCELL UPDATE

The wound healing and sealant properties of PRP have been documented in the orthopaedic literature. The following indications have appeared in the literature and relevant to clinical practice:

ENHANCED WOUND HEALING BY PRP: DIABETIC FOOT ULCER CARE: BOLANDCELL UPDATE

Recalcitrant diabetic foot and leg ulcer treatment is a huge challenge to ulcer clinics, diabetologists, and vascular surgeons. This stems from the underlying propensity to develop deep compartment sepsis ( because of defective chemotaxis and opsonization), reduced immunecompetency, calcified foot vessels, insensitive sole, forefoot ischaemia and neuropathy. The presence of an unstable and otherwise insensitive foot ( because of sensory, motory and autonomic fiber loss), " rocker-bottom deformity" of the sole and instep, autosympathectomy predispose the sole to decubitus and pressure ulcers that eventually become secondarily infected. The result is deep compartment abscess formation and gangrene that may necessitate fore-foot or below-knee amputation. Apart from the need for antibiotics, bacteriological cultures, dressings and creams ( L.MezitranT,Omnimed® ), off-loading footwear is imperative to facilitate healing of pressure sores. Amputation can be delayed if deep abscesses are drained properly ( optimal debridement and sloughectomy) and dead or necrotic tissue carefully debrided under general anaesthesia. Often conservative amputations can be performed and the patient kept mobile. In these cases special off-loading shoe-ware is necessary. Diabetic foot ulcers, are indeed difficult to treat because of poor wound healing, persistent sepsis and underlying neuropathy. Recently, the use of autologous PRP gel provide an ulcer management option that may avoid or prevent loss of a limb by major amputation ( Driver et al 2006). Together with L.MezitranT(Omnimed®) even better wound healing results may be achieved, thus enhancing good standards of ulcer care.

COSMETIC, RECONSTRUCTIVE AND BURN TREATMENT UTILIZING PRP: BOLANDCELL UPDATE RELEVANT TO CRITICAL CARE

In this surgical discipline, both the sealant and wound healing properties of PRP are utilized. The clinical application and indications are enumerated as follows, in order to facilitate wound healing, reduce pain, swelling, bruising and surgical down time.

DENTAL, CRANIOFACIAL AND NEUROSURGICAL APPLICATIONS OF PRP: BOLANDCELL UPDATE

Marx, of the University of Miami, Florida, USA, is the doyen, champion and pioneer regards the use of PRP in this discipline ( Marx et al, 2005, Quintessence Publishing Co, Inc). Indications for PRP in dental, maxillo-facial, oral and neurosurgery are as follows:

AESTHETIC MEDICINE: DERMAL REGENERATION FOR FACIAL WRINKLE AUGMENTATION WITH PRP (BIOLOGICAL FILLER) AND GROWTH FACTORS. BOLANDCELL PROVIDES HORIZONS UNLIMITED FOR 2008

Because of the safety of PRP ( by nature of the autologous blood component), activated platelet gels are now both applied in therapeutic and aesthetic medicine. In the latter discipline, the term " Autologous Cellular Regeneration" or ACR, has been coined. Simply, this means rejuvenation of the face, neck, arms and hands by multiple small dermal and hypodermal injections of biologically active PRP ( Du Toit et al 2007). The growth factors released by the activated platelet gel, supposedly leads to rejuvenation of the dermis, ECM, with the stimulation of fibroblasts and keratinocytes, and collagen production ( Du Toit et al 2007). For the moment, the conventional options used as anti-wrinkling treatments, with skin tightening, include thermal lasers, IPL, radiofrequency (RF), athermal LED( 633nm) together with moisturizing and anti-aging creams, some of which contain concentrations of vitamin-A ( Retin-A®., Renova®., Retacnyl®., Environ®, NeostrataT Bionic Serum). Results, regards amelioration of lines, wrinkles, sagging, mid face ptosis, crow feet, upperlip lines are very variable and are influenced by smoking and alcohol consumption habits, degree of solar damage or photo-aged face, chronological factors, dietary habits and genetic make-up. In the senior authors experience a synergic effect can be observed by a combination of miscodermabrasion and PRP given as a staged procedure together with anti-aging creams such as Neostrata® or Prevage®. Applying a skin barrier ( SPF 30-100) against the sun is critical if good results are to be achieved. The patients Fitzpatrick status and Glaucou classification also determine outcome of anti-aging treatment. Non invasive ultrasound assessment or diagnostics of the skin and dermis may also help with the prediction of treatment outcomes. PRP on its own is unlikely to reverse the aging process in all cases and will have to be given in addition to lasers, IPL, RF and LED. If PRP on its own is superior to lasers, IPL, and RF regards skin tightening remains to be seen. Multimodal and maintenance therapy is inevitable to reverse and combat further skin aging. A balanced diet is needed, rich in vegetables and fruit, and at least two liters of water should be drunk daily. If PRP is to be considered as part of an ACR program for facial rejuvenation, then client aspirations need to be taken seriously into account. Regardless of what treatments are applied, facial rejuvenation is gradual and a slow process. Not all treated clients show collagen regeneration, and in most cases it is impossible to reverse mid-face ptosis by non-surgical face-lift. In these cases, a face-lift or use of threads, gives far superior results. Dermal atrophy also cannot be reversed by non-invasive thermal light devices, some of which induce permanent dermal scarring, and dry up sebaceous glands. For persons with acne scarring, needling ( Environ®) plus vitamin-A creams ( NeostrataT Bionic Serum)or growth factors will render superior results than PRP alone. More details on PRP and ACR, in the context of the photoaged skin, can be obtained from the website link, www.bolandcell.co.za .

Conditions that preclude the application of PRP for REGEN-ACR of the face include, aspirin ingestion, vitamin-E containing medications, warfarin therapy, sepsis, acute and chronic infections, fibrinogen disorders, very low platelet counts and any coagulation disorders. Facial intradermal injection of REGEN-PRP is painful and there is a need for topical anaesthesia and even regional nerve blocks. Ice packs are needed because the swelling can be alarming in susceptible individuals. A purple discolouration can be anticipated. Haemorrhage into the skin may occur from tiny telangiectasis and staining for a few weeks can follow the deposition of haemosiderin. Bruising on the temple lines and below the eyes should be avoided especially in women with a thin skin. Careful, informative and skilled informed consent ( also allowing for redress) is needed that includes a discussion on the potential of dermal discoloration, nerve paresis, down-time, danger of inadvertent intravascular injection with a biological filler compared to other fillers, secondary infection, danger of cavernous sinus thrombosis and haematoma formation. Be careful in patients taking unspecified herbal products that could result in serious and transient hypertension. Contact the Medical Protection Society, in advance, to obtain the correct medical insurance rating ( in the event of a potential lawsuit, complaint, or patient dissatisfaction), if aesthetic or cosmetic work is done. Be comprehensive with informed consent. CME accreditation in the application of REGEN-PRP is sensible and supervised workshops are available at an academic institution in Cape Town , and directed by the senior author.

BOLANDCELL UPDATE ON IMCAS 2007, BANGKOK , THAILAND .COMMERCIAL ASPECTS

AUTOLOGOUS CELLULAR REGENERATION AND FACE RESURFACING: REGEN-ACR

Dr Kubota of Japan reported on the use of autologous cell rejuvenation ( REGEN-ACR ) with platelet-rich plasma (PRP) as a new and minimally invasive approach to skin rejuvenation for aging skin and acne scarring. He showed impressive results, with positive outcomes in the treatment of fine lines and wrinkles especially in the periocular area and nasolabial folds. He is the leading plastic surgeon in the world with extensive clinical experience in the biological regeneration of the skin. Prof du Toit, of Cape Town ( local doyen and pioneer of PRP and biological skin rejuvenation in South Africa via cells, fibroblasts and PRP, is also author of RIDE YOUR BEST ARGUS EVER, AND " DOKTER IN DIE HUIS, pub Tafelberg, Cape Town), has similar experience. He presented data on the application of PRP harvesting and preparation kit ( REGEN PRP KIT, REGENLAB SA, MOLLENS , SWITZERLAND ). He concluded that skin rejuvenation with PRP mediated ACR appears to be an effective, safe and well tolerated approach because autologous plasma is used. The explanation behind the positive clinical outcomes are based on the presence of growth factors in the PRP that enhance the wound healing cascade. Prof Don du Toit, of Cape Town , gave an academic overview on the topic and provided a live demonstration regarding the injection technique for facial rejuvenation using REGEN-PRP. He pointed out poor-technique, pitfalls and emphasized knowledge of facial anatomy, neuroanatomy and histology. Attendance at the live demonstration were prominent owners of SPA'S, aesthetic physicians from India , Chorea , Taiwan , Thailand and professors of dermatology. Professor du Toit, who has extensive clinical and molecular cell biology experience with the use of PRP and REGEN-ACR states that this is a promising rejuvenation option compared to minimally invasive, non-ablative fractional thermodermolysis. REGEN-ACR now refers to dermal regeneration and volumetric therapy via cell rejuvenation. From a biological point of view this is a five pronged approach according to REGEN-LAB:

The end-result or outcome of this cellular process at stem cell level, is to activate tissue regeneration and tissue remodelling thereby shifting the wound healing cascade to the left.

During the same innovative presentation, regarding new technologies, Prof du Toit provided further evidence for the clinical application of REGEN-ATS , especially in the dermatological and plastic surgery fields.

REGEN-ACR : This refers more specifically to dermal regeneration and volumetric therapy via " autologous cell rejuvenation". The purpose of this therapy are as follows:

Intradermal in jection of ACR plasma supposedly induces a biological scaffold that comprises a " structure and signals process".

REGEN KIT AND ACR ( PRP PROTOCOLS): REGEN KIT is CE marked and authorized for human use. Five different kits are available for the clinicians and his choices. The protocols are as follows:

• Selection of 1 of the 5 kits for specialist treatment ( ie mesotherapy, aesthetic, wound healing
• Venapuncture and collection of blood
• Centrifugation and separation of plasma from RBC
• Injection therapy ( ie facial rejuvenation), application of two syringe system and advanced cell culture and biotechnology

REGEN ATS FOR DERMATOLOGY AND PLASTIC SURGERY

• Chronic wounds: patients with chronic leg wounds have been studied and need multiple applications of PRP
• REGEN-PRP can be applied as a second layer at the grafted site
• To speed up donor site healing: REGEN PRP can be enriched with autologous keratinocytes. Compared to controls REGEN-PRP enriched with autologous keratinocytes can reduce healing time from 12 to 5 days
• REGENLAB also supplies PRP plus chondrocytes, fibroblasts and keratinocytes by special cellular technologies

REFERENCES AND FURTHER READING ON PRP PROVIDED BY BOLANDCELL .

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