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PRP: SOUTH AFRICA

Bolandcell is the leading Cape Town loca ted biotechnological and clinical-based affiliation, through specialist surgeons and education ( CME accreditation) regarding the optimal medical application of Platelet-Rich Plasma ( PRP) and predicted outcomes in South Africa. Lead scientists in the laboratory at molecular and Tissue Culture (TC) level and clinicians trained in this biological field of medicine have made this possible and allowed benchmark autologous cell technology to be achieved and facilitated from the bench to the bedside during 2007. Bolandcell, thus base optimal patient care and treatment with PRP on a solid foundation of molecular laboratory research, unsurpassed in Africa, and biomedical science ex vivo, researching mechanism of action, quality assurance and safety, together with depth in the understanding of wound healing cascade through platelets, cytokines and secretory proteins. With clinical experience in the use of fibrin as sealant in the past (TISSEAL®), BOLANDCELL through strategic research management, ongoing R&D, top corporate scientific research leadership, prediction, publications in this scientific field, and clinical expertise in major surgery have emerged as the leading experts in the application of PRP biotechnology in South Africa.

BOLANDCELL BIOTECHNOLOGY AND PRP

PLATELET-RICH PLASMA ( PRP) AND TISSUE REGENERATION BY GROWTH FACTORS :

Basic scientists and clinicians have a vested interest in the field of soft tissue regeneration, rejuvenation by fibroblasts 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. These local effects are related to and orchestrated by the trophic and anabolic nature of platelet releasate following activation in the wound. 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. 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), . 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.

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 . 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. The plasma poor plasma (PPP) has potent sealant properties like Tisseel®. 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. Paying attention to fine detail, and avoiding contamination, is important during the generation of PRP. The authors prefer to do the centrifuging and plasma separation as aseptically as possible in a laminar flow hood. A mobile laboratory is also highly effective and practical and can be taken to the specialists rooms. 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 qualified medical personnel must be utilized. 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, medical technologist who assists the surgeon. Upholding aseptic principles during cell separation is mandatory to avoid contamination. 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. 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 MD IIA, 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. The layer containing the PRP, which can be differentiated from the plasma-poor 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. This is intentionally induced, shortly before use of the gel, and affected by addition of calcium chloride and or thrombin to the platelet concentrate. Use of these activators needs specialist medical supervision and control ( or by perfusionists) as endorsed by audited laborarotory code of conduct. Ongoing CME courses should be provided to familiarize specialists with this technology. Also, in the correct use of a specialized centrifuge, to prevent blood component cell fragmentation and contamination.The kit will be registered with medical aids and coded. The gel thus created, is available for soft tissue infiltration and augmentation or admixture with a bone graft. The SmartPRe2® and Regen® system renders good yields of PRP and trophic factors. However, there are numerous commercially available centrifuge machines, all of which yield slightly different concentrations in platelet and leucocyte cell counts. The addition of PRP is intended to improve and enhance bone grafting, by facilitation of greater and denser bone regeneration ( osteointegration) . 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 . The potential contamination of the PRP with pro-inflammatory leucocytes that result in inflammation needs to be taken into account by the specialist and is dealt with later.

From a wound healing perspective PRP contains important anabolic cytokines that have a trophic effect on cells.

From an electron microscopic point of view, PRP gel consists of two components: a fibrillar element and a cellular component that contains human platelet cells. This unique morphological structure is theoretically capable of acting as a vehicle for carrying of cells (of various lines) that is essential and complementary to soft/hard tissue regeneration. Single case studies and anecdotal reports indicate that PRP can favourably affect the outcome regarding wound healing and repair of chronic wounds, non-responsive to conventional treatment . Soft tissue regeneration and enhancement by PRP has been reported in the following clinical scenarios:

•  Surgical repair of torn Achilles tendons in man and other ligaments

•  Enhanced accelerated tissue repair in non-healing wounds of the lower equine limb

•  Treatment of diabetic foot ulcers

•  Face-lift surgery (rhytidectomy), because of the haemostatic properties of PRP

•  Cosmetic facial surgery induced alopecia (alopecia due to hair follicle loss in the side burn areas)

•  Enhancement and facilitation of skin sensory sensation return post-surgery. This is due to increased capillary in-growth, collagen synthesis which supports nerve regeneration

•  Reduction in wound healing complications after upper and lower eyelid blepharoplasty

•  Increased " take" of free dermal fat grafts destined for facial augmentation and lipoatrophy . This is supposedly due to increased lipoblast and lipocyte survival . It has also been proposed that capillary in-growth into the graft is enhanced

•  Periodontal disease (increased wound healing in periodontal tissue).

•  Repair of stretch marks

•  Enhanced healing of anterior cruciate ligaments in experimental porcine models

BIOSTIMULATORY EFFECTS OF PRP

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

  • Stimulation of cell proliferation from tendon explants in culture. Tendons have been cultured in explant fashion in PRP. In these studies, the cultured tendons showed enhanced expression of the matrix collagen molecules COL1, COL3A1 and COMP, with no increase in catabolic molecules MMP-3 and MMP-13. This reflects an anabolic effect of PRP on tendon metabolism, tendon matrix gene expression and matrix synthesis . Some studies recommend that tissue culture should be affected in PRP-enriched mediums of less than 40%. The monolayer of leucocytes may have to be extracted because of the presence of pro-inflammatory mediators, such as neutral proteases and acid hydrolases contained in white blood cells. On the other hand the leukocytes may be of benefit.
  • Stromal cell proliferation in culture
  • Increased epithelial regeneration, enhancement of dermal collagen deposition
  • Stimulation of fibroblasts in cell culture In BOLANDCELL experience, the enhanced stimulation of human dermal fibroblast proliferation ex vivo by 40% PRP differs from photo-light biomodulation of the same cell line.
  • Prevention of ecchymosis and haematomas
  • Enhanced proliferation of nucleus pulposis cells in culture
  • Prevention of surgically induced alopecia by increase in capillary in-growth and hair follicle survival
  • Improved haemostasis by the interaction of fibrin-fibronectin-vibronectin cell adhesion molecules due to PRP
  • Increase of capillaries, collagen and nerve in-growth from deep fascia
  • Improved angiogenesis

MOLECULAR BIOLOGY OF PRP, RELATED TO LIVING CELLS, TISSUE CULTURE AND PHOTO-LIGHT BIOMODULATION:

For facial rejuvenation, apart from the use of cosmoceuticals, and living cells ( Isolagen®), PRP and photo-light therapy are currently in use to treat fine-line wrinkles. The mechanism of action differs for the three treatment modalities. But, the common goals are to augment the face appearance, reduce laxicity and improve facial complexion due to a loss of collagen, extracellular matrix and elastin.

PRP as stand alone treatment for amelioration of facial rhytids : The physiological effect of PRP is based on the release of numerous anabolic growth factors from platelet alpha-granules . BOLANDCELL supports this view. Tissue culture studies show that PRP can enhance gene expression of matrix molecules such as collagen. PRP is also capable of stimulating fibroblasts ex vivo in experimental models and thereby increasing total protein synthesis . For the moment it is uncertain if PRP can enhance fibroblast generated elastin production and myofibroblast stimulation as seen after photo-light therapy. Compared to photo-therapy for facial rejuvenation, PRP differs in the biological mechanism of action on the dermis. PDGF is a mitogen present in activated PRP. Extracts of platelets can serve instead of serum, to stimulate fibroblast proliferation ex vivo. This has been confirmed by BOLANDCELL . This factor ( PDGF) plays a major role in stimulating cell division during wound healing, including fibroblasts . Also, TGF-beta1 can promote cell proliferation, survival, differentiation, and migration . Albert's et al (2002), have shown that mitogens (such as released by activated platelets), control the rate of cell division by acting in the G1 phase of the cell cycle. Possibly, mitogens can reverse the inhibitory effects of Cdk activity, thereby allowing the S-phase to begin. This is effected by binding to cell-surface receptors to initiate a complex array of intracellular signals that penetrate deep into the cytoplasm and nucleus .However, it still remains to be shown if the mitogen signalling in PRP can activate the small GTPase RAS , that leads to activation of the MAP KINASE cascade . If PRP mitogens can stimulate Myc , also needs to be quantified. Myc , is important, as it plays a major role in stimulating the transcription of genes that increase cell growth. When considering PRP as stand alone therapy for facial rejuvenation of wrinkles, these biochemical pathways need to be taken into account. Also, the drawbacks of PRP, such as release of pro-inflammatory proteins by the leucocytes residing in the buffy coat, and potential venous micro-thrombosis of the deep dermal capillary network, needs to be weighed up. These reactions may induce focal subdermal fat necrosis resulting in skin hardening and potential calcium deposition. The dermis is very thin on the sides of the face and tricky for the doctor to keep the injectate accurately in this layer. It is predicted that a substantial amount of the PRP injectate migrates during and after injection into the hypodermis that is rich in adipose tissue. This will inevitably result in PRP stimulated adipose-derived fibroblasts and not fibroblasts of dermal origin.

Tissue culture, cell dynamics and PRP :

BOLANDCELL concurs with Albert's et al (2002) that cell anchorage operates in G1 of the cell cycle. They confirm that confluent fibroblast monolayers, no longer proliferate. However, increased flow of diluted PRP (40%) strongly stimulates cell proliferation in the presence of monolayers ( keratinocytes, fibroblasts and skeletal myoblasts). For the moment it is not established if PRP will affect the differentiation and phenotype of the cultured cell lines. Possibly, the enhanced growth is related to replenishment of mitogens by the PRP, for which the cells compete .Of various cell lines, human dermal fibroblasts appear to be the most responsive to the culture enrichment of PRP. Strict adherence to aseptic technique is needed within a laminar flow hood when processing cultures with PRP. BOLANDCELL are in agreement with Albert's et al (2002) that extracellular growth factors that stimulate cell growth bind to receptors on the cell surface and activate intracellular signalling pathways . Also, one of the most important intracellular signalling pathways activated by growth factor receptors involves the enzyme PI3-kinase. The activation of PI3-kinase leads to the activation of several protein kinases, including S6 kinase . Protein synthesis therefore is increased following activation of phosphorylated S6 kinase, increase in mRNA translation which stimulates cell growth . Simulation of el4e also results in increased mRNA translation (See Albert's et al). Establishment of active intracellular signalling pathways is not established within a few days of cell dissociation and culture and may facilitate rapid phagocytosis or apoptosis of the new engrafted cells and a failure to imbed. BOLANDCELL are uncertain if PRP can facilitate cell engraftment under these circumstances. During the early phase of cell proliferation and growth, the cell cytoskeleton is established. In cell lines, the cytoskeleton consists of microtubules, actin filaments, and intermediate filaments and has been documented before. BOLANDCELL have not established if the enhanced cell growth by PRP interferes with the protein filaments of the cytoskeleton, and cytocavitary network.

Aesthetics: PRP in combination with photo-light therapy (PLT) for skin augmentation :

PRP outcomes relies on the mitogen stimulation on resident cells after dermal injection. In tissue culture, PRP can stimulate fibroblast proliferation and collagen release . PRP has been used in the clinic to ameliorate nasolabial lines and fine wrinkles . Compared to cell therapy with living fibroblasts, the effect may not be paracrine induced. Data is still needed to explain the effect of PRP on in vivo collagen synthesis, collagen contraction in the dermis and stimulation of myofibroblasts. Objective histological data is lacking to support the concept of " neocollagenesis". Currently, PRP is given as a single treatment, but maintenance treatment (needing further injections) may be imperative to sustain the initial transient results. However, few recipients are enthusiastic to undergo repeated facial dermal injections with modified blood. It may take months before facial regeneration becomes visible, definitive and measurable. For these reasons, aesthetic clinicians have been compelled to opt for complimentary, adjunctive, multimodal therapy, plus PRP, to ensure lasting results, in addition to local cosmoceutical application of creams. This is a very costly exercise especially if non-invasive skin stimulation by IPL and low level lasers is needed. Currently, PRP injections for facial wrinkles show no scientifically proven advantage in regenerative capacity over non-ablative lasers or radiofrequency devices. The molecular basis for the use of non-ablative photo-light therapy, differs from the application of PRP. The latter relies on cell stimulation by anabolic growth factor or mitogen stimulation, and the former by unique activation of photoacceptors, and oxidative respiration in the dermis. The regenerative efficacy of low energy photon therapy has been documented ( in vivo and ex vivo).

  • Enhanced wound healing
  • Activation of fibroblast proliferation in-culture
  • Up regulation of collagen production. This is mediated by up-regulating TGF-beta 1
  • Increase in myofibroblast stimulation
  • Increased expression of collagen and elastic fibres in certain experimental models

Because the clinical results, of anecdotal studies, suggest that a single dose of PRP for facial regeneration and solar damage is only transient, has compelled cosmetic physicians to combine PRP treatment with radiofrequency or low-level laser therapy. The two treatments are theoretically complimentary and possibly synergistic. However, low energy photon therapy has distinct advantages over PRP, in that the biomodulation can be repeated more often, increases elastic fibre expression and myofibroblast stimulation. The latter two elements play key roles in facial rejuvenation of the solar-aged face. Photo-biomodulation and photo-biostimulation differs from the biological action of PRP in that at cellular level, activation of mitochondrial respiratory chain components occurs together with cellular proliferation and cytoprotection. Quantification of the mechanism of action of PRP in facial rejuvenation is problematical and use of the Dermascan, Skin VisioT and dermatoscope at BOLANDCELL are difficult to interpret from a scientific point of view. There is no proof-of-science and therefore more research in this area is needed before clinical trials are commenced. It has been reported that photostimulation induces a cascade of signalling events initiated by the initial absorption by cytochrome oxidase. This results in an increase in oxidative metabolism, cell proliferation and enhanced healing . Other important molecular changes include improvement of vascularity, stimulation of collagen production, release of ATP and increased RNA and DNA synthesis. Therefore, compared to the use of PRP, the primary tissue response after laser light therapy is more fully understood by scientists. The primary response on the cytocavitary mitochondria, photon absorption by cytochromes, increase in ATP synthesis and energy are well documented.

PRP: addressing dermal elastic tissue loss in solar elastosis and cutaneous aging:

It is proposed that PRP and other biologicals can potentially shorten the "healing cascade" of the inflammatory process i.e. reduction in the stages of haemostasis, inflammation, tissue regeneration and tissue remodelling. This process may well be facilitated by the secretary proteins released by activated platelets in PRP. Five major steps in the biological regeneration process in which PRP is used are explained as follows by BOLANDCELL . Intradermal and hypodermal injection of autologous platelet- rich plasma acts theoretically as a bio-scaffold that comprises a "structure and signals" process including:

•  Formation of a three-dimensional fibrin network

•  Release of growth factors by thrombocytes and leucocytes

•  Chemo attraction of macrophages and resident stem cells

•  Stem cell proliferation (mitosis)

•  Stem cell differentiation

But the photo-aged facial skin has many and complex facets needing multimodal treatment to affect at best, a partial reversal that a sole modality cannot effect. For the moment there is no proof-of-science to confirm positive outcomes and complete reversal of the extra-cellular matrix (ECM) dysfunction induced by aging. Currently, there are no comparative studies showing a more efficacious effect of PRP over IPL / laser in improvement of facial fine-lines, solar elastosis and wrinkles. PRP, other biologicals or fillers are currently unable to convincingly reverse the following components of the photo-aged skin.

  • Restoration or improvement of photo-oxidative dermal damage, resulting in epidermal thinning
  • Photo-oxidative ECM matrix damage, resulting in elastotic and dermal changes
  • At molecular level, influence the ability of aged or " sluggish" fibroblasts and keratinocytes to respond to changes in their environment
  • Papillary dermal microfibrillar network remodelling observed in solar elastosis
  • Thinning of the stratum spinosum, flattening of the dermal-epidermal junction, and senescence of keratinocytes
  • Restoration of reduced oxytalan fibers by Fibulin-2
  • Reversal of the age-induced lyses of elaunic fibers, solar elastosis, elastolytic degeneration, and hypodermal atrophy resulting in less stretchability, resilience, more laxity of the skin, and proness to wrinkles
  • •  Restoration of the loss of dermal fibroblasts or activation of quiescent or senescent fibroblasts
  • Restimulation of wrinkle markers: filaggrin, keratohyalin granules and transglutaminase-1
  • Restoration of depleted collagen ( type I and III) in organized bundles at the level of the dermal-epidermal layer or the papillary dermis
  • Metalloproteinase activities of senescent dermal fibroblasts that contribute to the age-related atrophy of ECM architecture
  • Restimulation of reduced melanocyte density and Langerhan's numbers as seen in aging skin
  • CLINICAL AND SURGICAL APPLICATION OF PRP: FOR THE SPECIALIST

BOLANDCELL provides a list of indications for specialists in South Africa . Basically, PRP is a good alternative for dated fibrin sealants, because of the autologous nature of the technology, safety profile, solid base of translational and biotechnological research. Application thereof is based on solid wound healing principles. There are obviously, sceptics because of the lack of controlled studies and because much of the evidence is based on anecdotal reporting. They proclaim no proof of science. Nonetheless, PRP should be considered in select patients and with definitive indications. There is little to lose from the application of PRP and a lot to gain, because it is relatively inexpensive treatment. For medical-legal reasons, and in the interest of the public, regarding safety, specialists are urged to attend workshops directed by PRP-experts in the usage and application of PRP, which is generally safe to use because of autologous basis of treatment. Emphasis is placed again on informed consent, patients rights, allowing an informed decision to be made, redress, problems of litigation, patient safety, patient information, and need for surgeon education on an ongoing basis, especially with the application of new devices and biologicals. Discussion with anaesthetists, theatre nursing staff, hospital managers, who are part of the surgical team and decision making process, is also important. Clinical application include:

  1. Dental and craniofacial surgery: sinus lift grafting, ridge augmentation grafting, third molar sockets, periodontal surgery, soft tissue regeneration, implant surgery, mandible surgery, rhytidectomy, blepharoplasty, dermal fat grafts
  2. Major cardiovascular surgery, including heart bypass
  3. Major chest surgery: resections of lung
  4. Major abdominal surgery including liver resections
  5. Cosmetic surgery: face lifts, abdominoplasty, breast augmentation and reduction
  6. Spinal surgery: re-do and fusions
  7. Orthopaedic surgery: fractures, non-union, ligament surgery
  8. Burns, skin grafting
  9. Chronic wounds with wound healing deficiencies: ulcers, decubitus ulceration
  10. Neurosurgery: Skull base surgery, dural leaks
  11. Other: symptomatic dry eye ( www.perfusion.com )

REFERENCES AND BOLANDCELL INFORMATION RESOURCES ON PRP :

Marx R. E. Platelet-Rich Plasma: Evidence to support its use. J. Oral Maxillofac Surg 62:489-496, 2004.

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  9. Akeda K, An HS, Pichika R, Attavia M, Thonar EJ, Lenz ME, Uchida A. Platelet-rich plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and annulus fibrosus cells cultured in alignate beads. Spine 31:959-66, 2006.
  10. Okuda K, Kawase T, Momose M, Murata M, Saito Y, Suzuki H, Wolff LF. Platelet-rich plasma contains high levels of platelet-derived growth factors and transforming growth factor-beta and modulates the proliferation of periodontally related cells in vitro. J Periodontol 74: 849-57. 2003.
  11. Graziani F, Cei S, Ducci F, Giuci MR, Donos N, Gabriele M. In vitro effects of different concentration of PRP on primary bone and gingival cell lines. Preliminary results. Minerva Stomatol 54: 15-22, 2005.
  12. Grotendorst G. R, Rahmanie H, Duncan MR. Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation. FASEB J 18:469-79, 2004.
  13. Kawase T, Okuda K, Wolff L, Yoshie H. Plasma-rich plasma- derived fibrin clot formation stimulates collagen synthesis in periodontal ligament and osteoblastic cells in vitro. J Periodontal 74: 856-64, 2003.
  14. Lindeboom J. A, Mathura KR, Aartman IH, Kroon FH, Milstein DM. Influence of the application of platelet-enriched plasma in oral mucosal wound healing. Clin Oral Implants Res 18:133-9, 2007.
  15. Roukis T. S, Zgonis T, Tiernan B. Autologous Platelet-Rich Plasma for wound and osseus healing: A review of the literature and commercially available products. Advances in Therapy. 23, 218-237, 2006.
  16. Murray M. M, Spindler K. P, Abreu E, Mullwer J. A, Nedder A, Kelly M. Collagen-platelet-rich plasma hydrogel enhances primary repair of the porcine anterior cruciate ligament. J Orthop Res 25:81-91, 2007.
  17. Schnabel L.V, Mohammed H. O, Mc Dermott W. G, Miller B. J, Jacobson M. S, Santangelo K. S, Fortier L. A. Platelet-Rich Plasma (PRP) enhances anabolic gene expression patterns in flexor digitorum superficialis tendons. J Orthop Res 25:230-40, 2007.
  18. Carter C. A, Jolly D. G, Worde C. E, Hendren D. G, Kane C J. M. Platelet-rich plasma gel promotes differentiation and regeneration during equine wound healing. Exp Mol Pathol 74:244-55, 2003.
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