PROLOTHERAPY FOR MENISCAL PATHOLOGY
In order to understand how growth factors affect the treatment of meniscus injuries, it is first important to understand the role that they play in the natural process of healing. The preliminary steps of healing begin with the attraction of blood cells to the site of an injured tissue. When a tissue is injured, bleeding will naturally occur in that area. A specialized type of blood cell called platelets, rush to the area to cause coagulation, or the clotting of blood cells, to prevent excessive bleeding from an injury. In addition, platelets also release growth factors which are an integral part of the healing process. Each platelet is made up of an alpha granule and a dense granule which contain a number of proteins and growth factors; the growth factors contained in the alpha-granule are an especially important component to healing. When activated by an injury, the platelets will change shape and develop branches to spread over injured tissue to help stop the bleeding in a process called aggregation, and then release growth factors, primarily from the alpha granules.
At this point, the healing process then proceeds in three simple stages: inflammatory, fibroblastic, and maturation. After growth factors are released from the platelets, they stimulate the inflammatory stage, each growth factor playing a key role. (See Figure 14.) This stage is marked by the appearance of monocytes which are white blood cells that respond quickly to inflammatory signals and elicit an immune response. Growth factor production is at its highest level immediately following the inflammatory stage. Fibroblasts begin to enter the site within the first 48 hours after an injury and become the most abundant cells in that area by the seventh day. The fibroblasts deposit collagen, the main material of tissues such as the meniscus, for up to many weeks afterward. The maturation of collagen may then continue for up to one to two years after the initial inflammatory event.
It is important to understand that each of these stages stimulates the next. If the inflammatory stage does not occur, neither will the fibroblastic stage, and so on. If there is not a significant enough immune response to completely regenerate the damaged tissue in any of these stages, the injury will be unable to heal completely, leaving the person with a chronic degenerated knee.
In the case of the injured meniscus, it is clear that the damaged tissue can not repair itself. Healing in the meniscus depends on the having enough of a blood supply and/or growth factors at the site of the injury. Since less than 20% of the meniscus is vascularized by the time a person reaches the age of 40 years, meniscal healing is generally incomplete.117 Once torn, the menisci, because of its low cellularity and incomplete healing response, is unable to fully repair itself.118, 119 In one study, upon a five-year follow up after meniscal allograft transplantation, transplanted menisci were found to have decreased growth factor production indicating decreased biological function. Furthermore, the transplanted menisci were repopulated with fewer cells than even an untreated torn meniscus.120, 121 It has also been shown that the number of cells in the meniscus decreases with age.122
| Figure 14. Various growth factors found in platelets and their actions. |
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IN VIVO AND IN VITRO STUDIES ON GROWTH FACTORS IN STIMULATING MENISCAL REPAIR
Because growth factors are known to be a basic component of healing, the adjunct use of growth factors to stimulate connective tissue repair has been studied as a potential for the treatment of injured soft tissues, including the meniscus. Direct exposure of connective tissues to fibroblastic growth factors can indeed cause new cell growth and formation of collagen. Therefore, injecting growth factors at the site of a soft tissue injury allows the damaged tissue to heal itself.
Before any treatment is tested on humans, it is common practice to investigate the effect of that treatment, in this case growth factors, on cells (see side bar), as well as on animal models with similar pathology to humans. The primary objective of these studies is to determine if and how a poorly vascularized tissue, such as the meniscus, can be stimulated for reliable cellular and tissue repair. In such studies, growth factors, such as the ones extracted and secreted from the platelets are incubated with meniscal cells and then injected into injured meniscal tissue to see if cellular repair and regeneration occurs. Many studies demonstrate that injection of various growth factors can increase meniscal cell activity and stimulate repair, in this tissue and other connective tissues.123-140 As with other tissues that have a poor blood supply, like cartilage, meniscal cells are sparse. They are best categorized as fibrochondrocytes, as they have cellular characteristics of chondrocytes, cartilage cells, and fibroblasts that synthesize connective tissues such as ligaments.141 The meniscal cells are responsible for maintaining the extracellular matrix. The ideal mode of treatment for meniscal tears and degeneration would stimulate the production of meniscal fibrochondrocytes and its synthesis of extracellular matrix (ECM). Increased ECM synthesis would render the generated meniscal tissue more able to withstand the forces placed on the knee. For it is the collagen, proteoglycans and glycoproteins in the ECM which give the meniscus its compressive properties to withstand tensile loads.142
| in vivo - experimentation done on live isolated cells. |
| in vitro - experimentation done in an artificial environment outside the living organism. |
Platelet-derived growth factor (PDGF) is one growth factor commonly used in animal meniscus studies. One recent study measured both cell proliferation and extracellular collagen matrix formation in each of the inner, middle, and outer regions of sheep menisci, in the presence of PDGF-AB. After one week, meniscal cell proliferation was apparent in all three meniscal zones, reaching an 800% increase in the inner vascular zone compared to control. The formation of the collagen matrix had increased by 450% in the middle zone and by 300% in the outer zone. (See Figure 15.) An increase in the production of glycosaminoglycans, a main component of synovial fluid, in each of the three zones was observed.132 Meniscal cell migration was also stimulated. A similar in vitro study found that cell production of sheep menisci increased with proportion to the increased concentration of PDGF-AB used. This study observed a 2.5-fold increase in cell production.133 Another in vitro study placed bovine meniscal cells in different solutions containing cytokines and measured the effect of each on the synthesis of new cells in each of the three meniscal zones. The authors reported that significant DNA synthesis occurred in meniscal cells treated with PDGF-AB, hepatocye growth factor, and bone morphogenic protein-2, in all three regions.134 Similar results were found when analyzing the effect of basic fibroblastic growth factor (bFGF) on meniscal cells from sheep. When cultured in the bFGF, the formation of DNA increased by as much as sevenfold, and protein synthesis increased by as much as 15- fold in the inner (avascular) zone of the meniscus. The results of the outer and middle zones likewise yielded statistically significant cell growth.135, 138 The synthesis of proteoglycans, the principle component of the extracellular collagen matrix, was specifically measured in another study on sheep menisci. In all meniscal zones, transforming growth factor beta (TGF-β) stimulated proteoglycan production by up to 100% and the proteoglycans were larger than controls. TGF-β also stimulated cell division in the fibrochondrocyte cultures.137 Other authors have also confirmed that meniscal fibrochondrocytes from all three zones, including the avascular zone, can proliferate and generate new extracellular matrix given the proper stimuli.128, 129, 139, 140 Such findings have been the basis of the integration of growth factors in the treatment of meniscal pathology.
| Figure 15. Effect of platelet-derived growth factor-AB on DNA synthesis in cells from the three zones of the meniscus. Results are mean + SEM (N = 12). Platelet-derived growth factor at 1 and 10 ng/ml increased DNA synthesis (in both the middle and inner zones) by over 400% compared with control. Source: Bhargava MM, et al. The effect of cytokines on the proliferation and migration of bovine meniscal cells. Am J Sports Med. 1999;27:636-643. |
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PROLOTHERAPY STIMULATES GROWTH FACTOR FORMATION
The primary objective of Prolotherapy injections is to initiate or recreate the inflammatory stage of the healing process. It does so by raising the levels of growth factors to resume or initiate a repair sequence that has prematurely aborted or never started.145 Cells in the area of exposure, such as fibroblasts, chondrocytes and fibrochondrocytes, can also be expected to respond if the growth factors are those that proliferate such cells.146 By triggering this cascade of anabolic events, Prolotherapy stimulates the new growth of cells and is indirectly responsible for rebuilding depleted tissues.
Typical Prolotherapy solutions use a hypertonic solution of dextrose (glucose) as its base. Studies have shown that even a brief exposure to small amounts of glucose molecules causes an elevation in growth factors such as IGF-1, TGF-β, TFG-β, bFGF, and PDGF-B.145-150 Another substance used in Prolotherapy, especially for degenerative knee conditions, is human growth hormone (HGH).151 HGH stimulates the production of IGF-1 in the liver, but it can also have an important role in the localized treatment of degenerate cartilage cells. Circulating and locally produced IGF-1 can stimulate DNA synthesis, cell replication, and proteoglycan and glycosaminoglycan synthesis in articular chondocytes.152 Additional studies show that HGH and IGF-1 have both been shown to cause growth and repair of articular cartilage cells.153-155 One reason for this cartilage growth can be that cartilage cells have HGH receptors.156 Anecdotal radiographic evidence of the regeneration of articular cartilage has been seen with and without the use of HGH with Prolotherapy to the knee.157, 158 Other published studies have documented symptomatic improvement in patients with degenerative knee arthritis with Prolotherapy.159-161
Another emerging technique in the field of Prolotherapy is Platelet Rich Plasma Prolotherapy (PRPP), which utilizes the injection of human autologous blood components to facilitate healing of degenerative tissue injuries. In this technique a small amount of whole blood is drawn and is separated into platelet poor plasma and platelet rich plasma. The latter is used in PRPP and consists of plasma, which is the liquid component of blood, containing a high concentration of platelets. Because platelets are the storehouses of growth factors, platelet rich plasma is abundant in growth factors. PRPP though not only provides a higher concentration of growth factors to the tissue than is provided by normal blood supply, it stimulates the injured tissues to increase their own innate growth factor production.162-164 PRPP has shown in one controlled study to give statistically significantly better results for knee osteoarthritis then hyaluronan injection.165
In actual clinical practice, there are a host of solutions available to the doctor performing Prolotherapy. Although there have been no studies to date directly addressing the use of Prolotherapy for meniscal injuries, Prolotherapy has a long history of treating degenerative knee conditions including meniscal pathology.166-169 For seventeen years the primary author (R.H.) has treated meniscal pathology successfully with Prolotherapy. This retrospective study was done to document the degree of improvement in meniscal tears and degeneration with Prolotherapy.
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