STEM CELLS 2010

BOLAND CELL STEM CELL-PUBLICATIONS     
 TABLE OF CONTENTS: 2006-2010

• Platelet-rich plasma (PRP, gels, lysates, fibrin-platelet gels)
• Adipose-derived stem cells (ADSC): rapid automated technology

1. Du Toit DF et al. Regenerative cardiology: autologous human myoblast stem-cell based therapy for heart regeneration and heart failure. S.A Cardiology and Stroke. 2007, 1: 4-12
2. Du Toit, DF et al. Shoulder surgeon and autologous cellular regeneration (ACR). From bench to bed-the link between human fibroblast, connective tissue disorders and shoulder. International Journal of Shoulder Surgery: 2007, 2 :64-68.
3. Du Toit DF et al. New research developments and advances in the application of autologous cellular regeneration (ACR) and platelet-rich-plasma (PRP) in South Africa. The Specialist Forum 2007, 11:30-31.
4. Du Toit DF et al. New advances in biological wound-care and aesthetic-medicine. The Specialist Forum. 2008, 8: 15-23.
5. Du Toit DF et al. Stem cell science and regenerative medicine: latest trends in biological wound care. The Specialist Forum: 2009,9: 23-27
6. Du Toit DF et al. Advanced wound care: Vacuum-assisted closure (VAC) - Biomedical Science and Clinical Benefits: The Specialist Forum 2010, 2:46-52.
7. Du Toit DF. Stem cell Technologies: Biological Role In Wound-Healing and Regenerative Medicine. The Specialist Forum, 2010, 10: 22-28.
8. Du Toit DF. et al. Biotechnological anti-aging cell –therapy treatment of facial wrinkles with cultured human fibroblasts. The Specialist Forum.2005, 5: 38-46.
9. Du Toit DF et al. State–of-the-art rejuvenation and wound-healing with platelet-rich plasma growth factors-molecular cell biology and aesthetic perspective. The Specialist Forum. 2007: Part 1 and II, 15-20.
10. Du Toit DF. New developments in wound healing and care with autologous platelet-rich plasma. The Specialist Forum. 2008,8:39-41.
11. Du Toit DF. Soft and hard tissue augmentation with platelet-rich plasma; tissue culture dynamics, regeneration and molecular biology perspective. International Journal of Shoulder Surgery. 2007, 2: 64-73.
12. Du Toit DF et al. An in vitro tissue culture evaluation of the cell toxicity of platelet-rich plasma and silver dressings.US-China Jnl of Science: 2010 In Press.

SYNOPTIC PUBLICATION CONTENTS AND ABSTRACTS:
CENTRAL THEMES:

• Platelet-rich plasma technologies and use (gels, lysates, fibrin-matrix, platelet-rich fibrin-gels: PRP, ACR/PRP). Role with stem cells
• Adipose-derived stem cells (and rapid-delivery technology)
• Wound-care, wound-healing, ulcer-healing, aesthetic use of PRP for wrinkles, and cellular myoblast-progenitors for cardiac cellular-transplantation
• Bioequivalence-guidance and cell-biology testing and quantification

1. Du Toit DF. New developments in wound healing and care with autologous platelet-rich plasma. The Specialist Forum. 2008, 8: 39-41. Ulcer healing with Platelet-rich plasma, diabetic foot ulceration healing after application of PRP. Definition of PRP. Biometry assessment after ulcer healing with PRP. Safety-profile with the application of PRP onto open-wounds. Place of rhPDFGF-BB/Regranex®. Effect on wound-healing/contracture and re-epitheliazation. Competition with becaplermin and dangers of the chronic usage thereof.

2. Du Toit DF et al. New research developments and advances in the application of autologous cellular regeneration (ACR) and platelet-rich-plasma (PRP) in South Africa. The Specialist Forum 2007, 11:30-31.Contents of PRP and aspects on growth hormones released by activated platelets. Role of REGENKIT® PRP and manufacture in Switzerland. Indications in dry eye, thoracic surgery, leg ulceration and shoulder surgery. Use of PRP in tissue-culture of fibroblasts and other cell lines.

3. Du Toit DF et al. Biotechnological anti-aging cell–therapy treatment of facial wrinkles with cultured human-fibroblasts. The Specialist Forum.2005, 5: 38-46. Skin anti-aging using cultured human fibroblasts ex vivo. Place of facial rejuvenation by cell therapy. Tissue-culture of fibroblasts. Isolagen® process. What Isolagen® offers the patient. Patient selection and drawbacks. Clinical experience in the Cape Town area. Application of the Dermascan® and combination treatment with cosmeceuticals such as Beaute Pacifique®. Process of fibroblast proliferation ex vivo. Combination therapy approach with the use of the Dermascan®, specialised creams, dermal-fillers, Lasers and IPL. Costs, aspirations and when results become visible.

4. Du Toit DF et al. Stem cell science and regenerative-medicine: latest trends in biological wound-care. The Specialist Forum: 2009, 9: 23-27. Adult-derived stem cells and application. Stem cell tissue engineering. Use of bone marrow stem cells and in the treatment of chronic leg ulcers. Marrow-derived mesenchymal cells. Place of PRP and stem cells. Umbilical cord blood update. REGENLAB® PRP and Mycells®. Pluripotentiality, renewal differentiation. Embryonic and adult stem cells.

5. Du Toit DF et al. Stem cell science and regenerative-medicine: latest trends in biological wound care. The Specialist Forum: 2009, 9: 23-27. Negative-pressure concept. Granulations. Indications and contraindications. Effect on wound-closure. Comparison with topical-PRP.

6. Du Toit DF. Stem cell Technologies: Biological Role in Wound-Healing and Regenerative Medicine. The Specialist Forum, 2010, 10: 22-28.Chronic lower-leg ulceration. Available biological for wound-care: PRP, marrow mesenchymal-cells, adipose-derived stem cells/stromal cells. The role of each in the healing of chronic leg ulcers.

7. Du Toit DF et al. State–of-the-art rejuvenation and wound-healing with platelet-rich plasma growth factors-molecular cell-biology and aesthetic perspective. The Specialist Forum. 2007: Part 1 and II, 15-20. Growth-factors and proteins/cytokines released by the alpha-granules of platelets. Platelet-gel, platelet-fibrin clot, lyseate application. Regenlab® PRP efficacy and safety profile. Biostimulatory effects of PRP, relevant to clinical practice. Cell-biology, tissue-culture. Activated and non-activated PRP. Is calcium-activation needed to release cytokines, proteiens and GF from platelets in PRP? Is use of  non-activated PRP the possible cause of reported no response and failed rejuvenation in aesthetic medicine?

8. Du Toit DF et al. An in vitro tissue-culture evaluation of the cell toxicity of platelet-rich plasma and silver dressings.US-China Jnl of Science: 2010 In Press. Tissue culture study evaluating toxicity of silver and PRP against keratinocytes, dermal fibroblasts and ADSC. Diluted REGENPRP® shows good cyto-compatibility. Silver showed consistent toxicity.            

9. Du Toit, DF et al. Shoulder surgeon and autologous cellular regeneration (ACR). From bench to bed-the link between human fibroblast, connective tissue disorders and shoulder. International Journal of Shoulder Surgery: 2007,2: 64-68. Autologous cell regeneration (ACR). Tissue-regeneration and remodelling. Fibroblasts: types and function. Myofibroblasts. Healing of wounds by first-intention. Role of fibroblasts. Keratinocytes. Rotator-cuff repair with PRP augmentation. Tissue culture and cell engineering ex vivo.

10. Du Toit DF et al. New advances in biological wound care and aesthetic medicine. The Specialist Forum. 2008,8: 15-23. Biological role of PRP in aesthetic medicine. Amelioration of wrinkles and skin rejuvenation. No effect on hyper-pigmentation. Very modest rejuvenation is observed and far less than natural hyaluronic acid-fillers, IPL or lasers. No comparison with Botox. Application of platelet-rich plasma (PRP) in aesthetics, the clinic or operating room has rendered successes and failures so far, and many reports are mixed. Posed photographs before and after treatment are inconsistent, manipulated and modified. Because no commercial PRP bioequivalence-guidance exists. The haematological concept and safety of PRP is, however, evidenced-based. For use, autologous platelet concentrate (A-PRP) is suspended in a relative small quantity of blood, plasma or fibrin-gel (fibrin-platelet rich gel or lysate). Platelets contain various growth-factors (GF) that regulate wound-healing. PRP can be generated by automatic devices or prepared manually from a kit or syringe alone, for rapid clinical-application.  PRP may, depending on the technological capability of the kit, provide 3-8 fold increase in platelet-count, compared to base-line whole venous-blood. The injectate or product applied is popularly referred to as a “platelet injection”. Most PRP-generation technologies are FDA approved 510 (k) class II clearance and are CE marked, because the plasma-separation and end-biological product is similar. Platelet-activation status can be confirmed by analysis of platelet-receptor P Selectin. At the time of administration, the platelet-injection may contain activated or non-activated platelets, and the volume of PRP used may vary between 3 and 5ml. More than 20 clinical-trials are in progress and directed at the treatment of tendinopathies (orthopaedics), early arthritis, diabetic-foot ulceration, knee-ligament reconstruction, maxillary-sinus augmentation (dentistry), tennis-elbow, shoulder rotator-cuff disease, dry-eye, muscle-injuries, periodontal disease and cosmetic wrinkle-amelioration. Early provisional results (inclusive of anecdotal reports, level I and III evidence-based studies) show possible clinical benefit in two (10%). PRP as anti-aging and wrinkle-ameliorator has not stood up to robust-science; results vary, and are non-permanent requiring additional beauty treatments or manipulations. Outstanding issues include: when to give injections and how many are needed? In some conditions, PRP is still unproven therapy and not a standard of care. Strong PRP-competitors now on the market include Regen®, Mycells®, Plateltex®, Selphyl® and Arthrex®. The new Arthrex® technology show that blood-collection-tubes are not needed and that good quality PRP can still be generated in the syringe by centrifugation. In the future thus, there may be no need for blood-collection-tubes.

11. Du Toit DF et al. Regenerative Cardiology: Autologous human myoblast stem-cell based therapy for heart regeneration and heart failure. SA CARDIOLOGY AND STROKE: 2007, 2: 4-12. Causes of cardiac failure. Cell therapy for resurfacing the myocardium. Bone marrow versus skeletal muscle. Culture expansion, tissue engineering and transplantation of skeletal myoblasts. Autologous skeletal myoblast transplantation. Role of muscle-progenitors. Place for left ventricular assist device  to avoid dangerous arrhythmias. Fate of transplanted-myoblasts. MRI-changes to the myocardium after transplantation. Recovery and QOL score. Cell alternatives to myoblasts. 5 human myoblast cell-therapy transplants performed in Cape Town, South Africa ( aged 45-85 years).

12. General biological aspects regarding platelet-rich plasma (PRP): At the University School of Medicine, Dr. Brian Eppley pointed out the strategic importance of platelet and GF quantification in PRP. This group showed that it was possible to generate an 8-fold increase in platelet-count in A-PRP, compared to baseline whole blood. Also, that platelet-derived GF such as PDGF-BB, TGF-1, VEGF, EGF, were increased with increasing platelet-count, but important inter-patient variance was evident. The relevance of these findings to tissue-healing and regenerative-medicine has been emphasized by Marx and Karg from the Miami School of Medicine in 2005. Numerous studies are underway to assess the efficacy and application of PRP, platelet-activation, fibrin-gels and platelet-derived GF in therapeutic, aesthetic and regenerative-medicine. PRP is not viewed as stem cell therapy, but may act as a stimulant for enhanced wound-healing by stimulation of recipient mesenchymal-cells or other un-disclosed mechanism. Regarding aesthetic medicine, manufacturers and distributors should provide clinicians with further convincing evidence , regarding anti-aging or anti-wrinkle action, if A-PRP is indeed equal to or superior to non-invasive RF, fractional-RF, fractional-laser, peels, and IPL. The questions that remain are: “Does the PRP work and is it going to work in this patient?”.

BIOMEDICAL SCIENCE REGARDING AUTOLOGOUS PLATELET-RICH PLASMA (A-PRP): SHOULD WE USE LYSATE, PLASMA, GEL OR FIBRIN MATRIX ?

Platelets are anucleate blood-components and are cytoplasmic fragments of megakaryocytes . These cells are the progeny of the hemoblast and myeloid stem cell and their formation is regulated by thrombopoietin. Normal blood contains between 150,000 and 400,000 platelets. PRP generation –technology allows us to generate up to 5 times the number of platelets found in normal whole-blood . Relevant to platelet-plug formation and haemostasis, platelets, apart from GF release, also release serotonin, adenosine diphosphate (ADP) and thromboxane A-2. Centrifugation of anti-coagulated blood produces separation of the RBC’s and plasma in the blood-collection tube supplied in the kit . The platelets separate out just above the buffy-layer, on centrifugation of whole blood . Thus, the plasma consists of a platelet-rich (PRP) and platelet-poor (PPP) zones. About 3-5 ml of A-PRP is generated but depends on the volume of whole-blood used for centrifugation. Because most blood-collection-tubes contain ACD, the plasma is anti-coagulated and will not clot. Some kit-manufacturers avoid platelet-activation and fibrin-matrix precipitation by excluding the use of thrombin and calcium. In these cases the PRP is injected and coagulation occurs when the platelets and plasma make contact with the recipient’s collagen. A platelet-rich fibrin-mesh can be precipitated by addition of calcium and or thrombin to the centrifuged blood. If bovine thrombin is used, care must be exercised to detect unintentional lowering of Factor-V (proaccellerin). Plateltex® precipitates the fastest and makes the most consistent gel by the application of calcium-gluconate and batroxibin. This avoids platelet-activation and allows for a more controlled release of GF over hours compared to a more non-physiological bolus-release. All human PRP contains committed CD34+ progenitor cells in peripheral venous-blood (especially after exercise). CD34+ cells are of haemopoeitic origin and found in umbilical blood and bone-marrow. PRP can be prepared by automation or manually by the use of kits at the point-of-care (Regen®, MyCells®, Selphyl®, Arthrex®, Plateltex® and Fibrinet®). Others have reviewed physiological aspects of PRP. The Arthrex® system is unique and practical, because blood-collection tubes are not needed and the double-syringe is centrifuged instead of blood-collection tubes. In the case of Arthrex®, the PRP is generated in a double-syringe system and not in blood-collection tubes. The following A-PRP preparations are available  in 2010:

• Activated or non-activated PRP (no application of calcium or thrombin).
• Vortexed-A-PRP free of calcium or thrombin (MyCells®).
• PRP generation in the syringe with no need for blood-collection tubes (Arthrex®)
• Autologous Conditioned Plasma (Arthrex® ACP System®): same as point 3.
• Batroxibin-induced platelet-gel and fibrin-gel (Plateltex®).
• Platelet-rich fibrin matrix( PRFM): Selphyl® ( newly launched in the USA for aesthetic work) and solid fibrin-web (SFW: Fibrinet)®
• PRP for aesthetic work and wrinkle amelioration: Regen®, Selphyl®, Mycells®
• Pure platelet-rich plasma (P-PRP): Cell Separator PRP®, Vivostat PRF® and Anitua’s PRGF®
• Leukocyte-and platelet-rich plasma (L-PRP): Curasin®, Regen®, Plateltex®, SmartPrep®, Magellan or GPS PRP®
• Pure platelet-rich fibrin (P-PRF): Fibrinet®
• Leukocyte-and platelet-rich fibrin (L-PRF): Choukroun’s PRF®
• Platelet-rich fibrin matrix (P-PRFM): Selphyl® (PRFM, an injectable PRFM for the treatment of wrinkles and skin-depressions in the United-States. Sclafani et al have described the cosmetic facial applications of platelet-rich fibrin –matrix in facial plastic surgery. This entails rejuvenation of the face without tissue-manipulation as seen during surgical face-lift or modifications thereof.
• Autologous regenerative factor-rich plasma (A-RFRP): for ocular burns by subconjunctival infiltration of A-RFRP.

BIOLOGICAL ACTIVITY AND BIOSCIENCE OF PLATELET-RICH PLASMA: NEED FOR CLOT, PLASMA, OR FIBRIN MATRIX AT POINT-OF-CARE?

Current ex vivo research indicates that platelet-lysates can increase wound-healing by stimulating keratin migration through calcium and P38 dependent mechanisms. Furthermore, international ex vivo studies confirm that PRP as well as diluted-PRP in concentrations of 5-20%, can promote proliferation of human adipose-derived stem cells, keratinocytes, dermal fibroblasts and tenocytes . Safety as regarding the use of PRP in the clinic has been reported . The GF released by platelets in the PRP such as PDGF-AB and TGF-beta 1 are thought to be cell-proliferation drivers . These GF may upregulate proteoglycans and collagen synthesis and build ECM by enhanced cell-cell interactions. PRP and the GF may well facilitate recruitment of reparative cells, at the same time suppressing macrophage function in the early phase of inflammation. The GF effects may be more focused on the cell proliferation-phase of healing rather than the inflammatory-phase. Quality fibrin-platelet gel that promotes haemostasis and wound-healing can be induced by mixing PRP with calcium-chloride or gluconate and thrombin. Agonist priming of the platelets ex vivo in the PRP leads to release of PDGF and TGF. Batroxibin, in Plateltex®, gives a rapid onset platelet-rich fibrin-matrix and consistent gel-formation. This facilitates application of A-PRP for soft and hard tissue treatments. A recent French-publication showed that Plateltex® (a semi-solid-gel network of polymerized-fibrin) renders the highest concentration of platelets in the smallest volume available. Dr Piero Borzini, an undisputed expert in Italy, has played a central role in the application of Plateltex®. Regenlab PRP® also rendered good yields. No comparable data is available for other PRP-generation technologies such as Selphyl®, Fibrinet®, Mycells® and Arthrex®. Not much data is available regarding bioavailability of GF in tissue-healing, nor kinetics of GF-release from PRP-gels. But recent work from Alessandria, Italy shows that similar methods of platelet gel preparation are associated with different performances concerning GF recovery and GF kinetics during release from fibrin-platelet gels. Clearly this may well explain the variable results seen in the clinic in aesthetics and in therapeutic trials. Or, also explain and elucidate successes and failures that have occurred so far. Clearly there are different PRP products with differing biology .

NEED FOR PRP-BIOEQUIVALENCE GUIDANCE

PRP is a highly variable biological-product with a wide therapeutic-index. Currently, the bioequivalence acceptance range is unknown. Clearly, more research is needed to demonstrate if PRP is bioequivalent with other reference PRP-biologicals. Possibly, for highly variable biologicals such as PRP, scaled average bioequivalence may provide an alternative and practical approach for the comparisons of PRP-biologicals. This may facilitate researchers understanding of PRP-efficacy, when using activated and non-activated fractions, that may induce variable or no clinical results. Currently, there is no data regarding potential therapeutic-inequivalence of available PRP-generation products. We can measure the platelet concentration and GF content in the test-tube of the various PRP-kits, but cannot measure the tissue response after PRP-injection, because the outcomes are so different.

WEBSITE POSTING: 22 May 2010
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