A LETTER FROM: BARCELONA, SPAIN

– Written by Eduardo Mauri, Montserrat Garcia-Balletbo and Ramon Cugat, Spain.

Biological therapies and the abundance of literature for and against their use, evoke both interest and controversy. In the 1940s Levi Montalcini discovered nerve growth factor and in the 1950s Stanley Cohen discovered epidermal growth factor. When we talk about Growth Factors (GFs) it is also important to mention the work of Prof Marshall R Uris, who in 1965 discovered a substance in the extracellular bone matrix which had the capacity to induce bone formation, subsequently called bone morphogenetic protein¹. Another scientist who studied and isolated regenerative substances was Harry Antoniades, who in 1981 identified platelet derived growth factors I and II, obtained from platelets. In 1970, Matras used a fibrin preparation for re-implantation of rat skin and in 1994 Tayaponsak used autologous fibrin adhesive in mandibular reconstruction for the first time. Subsequently, Fung proposed the name ‘tissue engineering’ to describe the developing field, mixing biology and engineering². In 1997, Whitman developed and used a platelet gel, an autologous alternative to fibrin gel and finally, in 1998, Marx was the first to use platelet rich plasma.

Since 1995, a multidisciplinary group lead by oral surgeon Eduardo Anitua, who founded the Biotechnology Institute, has made significant contributions to both the clinical and scientific understanding of platelet function and its therapeutic applications. Since then, many other authors have established research in this area^3-9.

REGENERATIVE BIOLOGY

The objective of tissue regeneration is to create restored tissue with properties indistinguishable from the original. The goal is not to repair but to regenerate, reconstruct the form and restore function by stimulating artificial or natural bio-substances, cell migration, proliferation and differentiation.

GROWTH FACTORS: FUNCTIONS AND TYPES

GFs are multi-functional; for example on one hand they stimulate proliferation of certain types of cells, on the other hand they inhibit the proliferation of others. GFs are found in and derived from the extracellular matrix, the platelets-megakaryocytes and plasma, among others.

Platelets have two functions: interrupting bleeding where there has been a vascular injury and stimulating cell proliferation and tissue scarring when they release GFs from their alpha-granules^8-12.

GFs include:

·         platelet-derived growth factor,

·         vascular endothelial growth factor,

·         transforming growth factor-beta,

·         acidic and basic fibroblastic growth factors,

·         insulin-like growth factor I and II and

·         epidermal growth factor.

·         The biological effects of GFs include:

·         haemostasis,

·         stimulation of angiogenesis,

·         promotion of cell proliferation,

·         acceleration of tissue remodelling,

·         pain reduction and

·         antimicrobial action.

GROWTH FACTOR THERAPY

The aim of GF therapy is to use platelets to mimic the physiological process when a tissue is damaged: platelets deliver GFs to the injured area¹². Currently, various techniques are being used all over the world and as a result, it is not possible to compare between sites because each uses different techniques and applications, with different elements and/or substances being injected.

Our group has vast experience using Eduardo Anitua’s Technique (Platelet Rich Growth Factor®-Endoret)   a plasma enriched in platelets 2 to 2.5 times the peripheral blood platelet concentration plus 5% CaCl2. This technique does not use bovine thrombin to coagulate and does not include leukocytes in the preparation. Leukocytes reduce fibrin stability and they express matrix metalloproteinases that contribute to extra cellular matrix degradation¹³. Using this technique we can obtain a range of preparations including non-liquid-activated, liquid-activated, coagulant/clot, fibrin membrane and supernatant. PRGF®-Endoret can be applied to injuries of all types of connective tissue such as cartilage, bone, ligament, tendon, muscle and skin¹⁴.

Cartilage: the aim of treating chondral injuries with PRGF®-Endoret is to re-fill defects with new chondral tissue. There are numerous research studies that support the treatment^15-18 and some publications about clinical applications^19-22.

Bone: in-vitro studies have demonstrated that platelet-derived growth factor stimulates the proliferation of human trabecular bone cells and osteoblast-like cells. Initial in-vivo studies were reported in the fields of oral-maxillofacial surgery and dentistry, focusing on the effects of isolated platelet rich plasma.

Sanchez et al published results obtained in non-union fractures treated with PRGF®²³.

Ligament: many studies have been published in recent years on the study and clinical use of the bone morphogenetic protein and GFs. The goal is to identify:

·         the optimum combination of these proteins,

·         the most effective therapeutic dosages and

·         the right ways to release them^24-35.

·          

Animal studies have found that transforming growth factor-beta 1 and epidermal growth factor, as well as GFs obtained from autologous platelet concentrate can be used in anterior cruciate ligament (ACL) graft remodeling to:

·         increase collagen synthesis,

·         increase fibroblast synthesis,

·         improve scarring speed,

·         increase tension strength resistance and

·         increase maturing speed^36-39.

·          

The use of PRGF®- Endoret in ACL surgery has two goals: to prevent anterior knee pain and to achieve a quicker fixation and maturity of the graft. Sánchez et al reported that compared to a control group, 50 patients who underwent ACL repair surgery and were treated with PRGF®-Endoret had:

·         minimised haematomas,

·         reduced postoperative signs of inflammation,

·         reduced pain,

·         a reduced recovery period,

·         accelerated integration of the graft and

·         reduced the probability of laxity post-surgery.

·          

Radice et al also reported the that the application of PRGF®-Endoret in ACL repair surgery in a group of 25 patients significantly reduced the biological maturing time of the graft (by at least 49%), compared with a control group^40,41.

An animal study by Kuroda et al found that GF release in reconstructed ACL ligaments peaks between the 3rd and 6th week and almost completely ceases at 12 weeks⁴².

Yoshikawa et al showed up-regulated expression of vascular endothelial growth factor is a potent stimulator of angiogenesis   at 2 to 3 weeks post-reconstruction. Recent studies found that exogenous application of vascular endothelial growth factor enhanced cell infiltration and fibroblast expression during the proliferation phase of healing, but this also induced significant deterioration of the mechanical properties of the graft. These findings support the reports of numerous other studies that all found the mechanical properties to be at a minimum around the proliferation phase of healing at 6 to 8 weeks⁴³.

In the early phase, Kawamura et al and Kuroda et al reported that graft necrosis leads to a release source of interleukins that trigger a cascade of GF expression, resulting in cell migration and proliferation as well as extra cell matrix synthesis and revascularisation⁴⁴.

Scheffler, Unterhauser & Weiler share the same results with the studies of Kuroda Kawamura and Yoshikawa⁴⁵.

Tendon: the goal is to obtain new healthy tendon tissue⁴⁶. Sanchez et al reported good results in Achilles tendon repair injuries⁴⁷.

Muscle: GFs shorten injury recovery time. However one must control the creation of fibrosis or, in extreme cases, calcification.

Skin: GFs facilitate healing with a regeneration-repair process.

THE FUTURE

The literature includes evidence for both positive, neutral, and negative results from the clinical utilisation of GFs. For the last 8 years the number of randomised controlled clinical trials has progressively increased. In 2006 there was just one trial considered as level 1 evidence, there are now 47 randomised controlled clinical trials with level 1 evidence. However, among these works there is a variety of collection methods, applied substances (different cellular and chemical characteristics) volumes, number of doses and clinical follow-up (some late, some early). These ‘small details’ directly influence a positive or negative result and must be considered when critically reviewing the evidence.

Furthermore, both ongoing basic studies and clinical trials are required, paying special attention to the ‘small details’. A Consensus is required. Our group’s experience began in 2002 and ranges from laboratory research, clinical practice both with animals and humans. The results are satisfactory if the methodology of obtaining the substance is respected and when the diagnosis indicates the treatment.

References:

1.Urist M. Bone: formation by autoinduction. Science 1965; 150:893-899.

2.Woo S. Tissue engineering: use of scaffolds for ligament and tendon healing and regeneration. Knee Surg Sports Traumatol Arthros 2009; 17:559-560.

3.Balk S. Calcium as a regulator of the proliferation of normal, but not of transformed, chicken fibroblasts in a plasma-containing medium. Proc Natl Acad Sci 1971; 68A: 271-275.

4.Kohler N, Lipton A. Platelets as a source of fibroblast growth-promoting activity. Exp Cell Res 1974; 87:297-301.

5.Ross R, Glomset J, Kariya B, Harker L. A platelet dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc Natl Acad Sci 1974; 71: 1207-1210.

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13.Bramondo D, Richmond J, Weitzel P, Kaplan D, Altman G. Matrix metalloproteases and their clinical applications in orthopaedics. Clin Orthop Rel Res 2005; 4:434-439.

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15.Cugat R, Carrillo J, Sopena J, Tarrago A, Soler C, Serra I. Treatment results of chondral lesions using plasma rich in growth factors and other substances. Electronical Poster in ISAKOS Bi-Annual Congress. Hollywood, FL, US, 2005.

16.Cugat R, Carrollo J, Serra C, Soler C. Articular cartilage defects reconstruction by plasma rich growth factors. In: Basic Science, Clinical Repair and Reconstruction of Articular Cartilage Defects: Current Status and Prospects. Edited by Timeo 2006. P. 801-807.

17.Serra C. Análisis Biomecánico e Histológico del Tejido de Reparación en Defectos Condrales de Espesor Completo tras la Aplicación de Plasma Rico en Plaquetas Autólogo [Biomechanical and histological analysis of tissue repair full thickness chondral defects after the application of autologous platelet rich plasma]. Estudio Experimental [Experimental study]. Doctoral Thesis. Universidad Cardenal Herrera CEU. Valencia, Spain, 2006.

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19.Wang-Saegusa A.  Infiltración de PRGF (PRP) en OA de Rodilla. Efecto-Repercusión en la Calidad de Vida y Función Física [Infiltration of PRGF (PRP ) in Knee OA . Effect - Impact on Quality of Life and Physical Function]. Tesina para Máster en Medicina Cosmética y Antienvejecimiento [Thesis for Masters in Cosmetic and Anti-Aging Medicine]. UAB, Barcelona, Spain 2008.

20.Sanchez M, Azofra J, Anitua E, Andia I, Padilla S, Santisteban J et al. Plasma rich in growth factors to treat an articular cartilage avulsion: A case report. Med Sci Sports Exerc 2003; 35:1648-1652.

21.Cugat R, Garcia-Balletbo M. Treatment of chondral lesions with Plasma-Rich Growth Factors. Oral Presentation at the Japan Knee Society 29th Annual Meeting. Hiroshima, Japan, 2004.

22.Fu F, Musahl V. The treatment of focal articular cartilage lesions of the knee future trends and technologies. Sports Med Arthro 2003; 11:202-212.

23.Sanchez M, Anitua E, Cugat R, Azofra J, Guadilla J, Seijas R, Andia I. Nonunions treated with autologous preparation rich in growth factors. J Orthop Trauma 2009; 23: 52-9.

24.Uludag F, Gao T, Porter T, Friess W, Wozney J. Delivery systems for BMPs: factors contributing to protein retention at an application site. J Bone Joint Surg 2001; 83A:128-135.

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27.Howell H, Fiorellini J, Paqutte D, Offenbacher S, Giannobile W, Lynch S. A phase I/II clinical trial to evaluate a combination of recombinant human platelet-derived growth factor-BB and recombinant human insulin-like growth factor-I in patients with periodontal disease. J Periodontol 1997. 68:1186-1193.

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29.Schmidt C, George H, Koh C, Blomstrom G, Engle C, Larkin L, Ecan C et al. Effect of growth factors on the proliferation of fibroblasts from the medial collateral and anterior cruciate ligaments. J Orthop Res 1995; 3:184-190.

30.Azuma H, Yasuda K, Tohyama H, Sakai T, Majima T, Aoki Y et al. Timing of administration of transforming growth factor-beta and epidermal growth factor influences the effect on material properties of the in situ frozen-thawed anterior cruciate ligament. J Biomech 2003; 36:373-381.

31.Weiler A, Forster C, Hunt P, Falk R, Jung T, Unterhauser F, et al. The influence of locally applied Platelet-Derived Growth Factor–BB on free tendon graft remodelling after anterior cruciate ligament reconstruction. Am J Sports Med 2004; 32:881-891.

32.Howell S, Knox K, Farley T, Taylor M. Revascularization of a human anterior cruciate ligament graft during the first two years of implantation. Am J Sports Med 1995. 23:42-49.

33.Steiner M, Murray M, Rodeo S. Strategies to improve anterior cruciate ligament healing and graft placement. Am J Sports Med 2008; 36:176-189.

34.Unterhauser F, Bail H, Hoher J, Haas N, Weiler A.  Endoligamentous revascularization of an anterior cruciate ligament graft. Clin Orthop Relat Res 2003; 414:276-288.

35.Yoshikawa T, Tohyama H, Enomoto H, Matsumoto H, Toyama Y, Yasuda K et al. Temporal changes in relationships between fibroblast repopulation, VEGF expression, and angiogenesis in the patellar tendon graft after anterior cruciate ligament reconstruction. Trans Orthop Relat Res Soc 2003; 29:236.

36.Yasuda K, Tomita F, Yamazaki S, Minami A, Tohyama H. The effect of growth factors on biomechanical properties of the bone–patellar tendon–bone graft after anterior cruciate ligament reconstruction. A canine model study. Am J Sports Med 2004; 32:870-880.

37.Sakai T, Yasuda K, Tohyama H, Azuma H, Nagumo A, Majima T, et al. Effects of combined administration of transforming growth factor-beta 1 and epidermal growth factor on properties of the in situ frozen anterior cruciate ligament in rabbits J Orthop Res 2002; 20:1345-1351.

38.Yamazaki S, Yasuda K, Tomita F, Tohyama H, Minami A. The effect of Transforming growth factor- 1 on intraosseous healing of flexor tendon autograft replacement of anterior cruciate ligament in dogs. Arthroscopy 2005; 21: 1034-1041.

39.Ju Y, Tohyama H, Kondo E, Yoshikawa T, Muneta T, Shinomiya K, et al. Effects of local administration of vascular endothelial growth factor on properties of the in situ frozenthawed anterior cruciate ligament in rabbits. Am J Sports Med 2006. 34:84-91.

40.Sanchez M, Azofra J, Aizpurua B, Elorriaga R, Anitua E, Andia I.  Aplicación de Plasma Autólogo Rico en Factores de Crecimiento en Cirugía Artroscópica [Application of Autologous Plasma Rich in Growth Factors in Arthroscopic Surgery].  Cuadernos de Artroscopia[Journal of Arthroscopy] 2003; 10:12–19.

41.Radice, F, Yánez, R, Gutiérrez, V, Pinedo, M, Rosales, J, Coda, S (2008) Uso de Concentrado Autólogo Rico en Factores de Crecimiento en la Reconstrucción del LCA [Using Autologous Concentrate Rich in Growth Factors in ACL Reconstruction]. Argent, Artroscopia. 15: pp. 31-40

42.Kuroda R, Kurosaka M, Yoshiya S, Mizuno K. Localization of growth factors in the reconstructed anterior cruciate ligament: immunohistological study in dogs. Knee Surg Sports Traumatol Arthrosc 2000; 8:120-126.

43.Yoshikawa T, Tohyama H, Katsura T, Kondo E, Yasuda K et al. Effects of local administration of vascular endothelial growth factor on mechanical characteristics of the semitendinosus tendon graft after anterior cruciate ligament reconstruction in sheep. Am J Sports Med 2006; 36:1918-1925.

44.Kawamura S, Yin L, Kim H, Dynybil C, Rodeo S. Macrophages accumulate in the early phase of tendon-bone healing. J Orthop Res 2005; 23:1425-1432.

45.Scheffler S, Unterhauser F, Weiler A. Graft remodeling and  ligamentization after CL reconstruction. Knee Surg Sports Traumatol Arthrosc 2008; 16: 834-42.

46.Tohyama H, Yasuda K. Extrinsic cell infiltration and revascularization accelerate mechanical deterioration of the patellar tendon after fibroblast necrosis. J Biomech Eng 2000; 122:594-599.

47.Sanchez M, Anitua, Azofra J, Andia I, Padilla S, Mujika I. Comparison of Surgically Repaired Achilles Tendon Tears Using Platelet-Rich Fibrin Matrices. Am J Sports Med 2007; 35:245-251.

Eduardo Mauri Montero M.D.

Sports Medicine Physician

Sports Medicine Unit

Montserrat Garcia-Balletbo

Director of the Regenerative Medicine Department

Hospital Quiron

Barcelona, Spain

Ramon Cugat M.D., Ph.D.

President of Medical Staff

Catalan Soccer Delegation's Health Insurance Company, Spanish Soccer Federation

  Director of Orthopaedic Surgery and Sports Medicine Department

Hospital Quiron

Barcelona, Spain

Contact: 26177emm@comb.cat

Image via Moyan Brenn