Name of growth factor
Action of growth factor
Platelet-Derived Growth Factor (PDGF) [48]
Angiogenesis and blood vessel repair
Cell growth and collagen production
Vascular Endothelial Growth Factor (VEGF)
Growth and de novo generation of vascular endothelial cells
Insulin Growth Factor 1 (IGF-1)
Triggers platelet aggregation
Transforming Growth Factor
Growth and de novo production of vascular endothelial and epithelial cells
Epidermal Growth Factor
Angiogenesis and promotion of wound healing
Three groups of granules have been described, according to structure, as visualised with the use of an electron microscope [10]:
- (i)
Dense, deriving their name from their opaque “dense” core, which promotes aggregation
- (ii)
Alpha, the largest in both size and number, with a role in adhesion and repair
- (iii)
Lysosomal granules, which aid in the degradation of the platelet aggregate [10]
Alpha granules give rise to the therapeutic potential of PRP. The phenomenon of activation (also known as degranulation) describes the release of the growth factors to a bioactive state via the addition of histones and carbohydrate side chains. The process occurs within minutes of exposure to either clotting cascade factors or basement membrane and leads to the formation of a mature clot. The growth factors bind to transmembrane receptors on mesenchymal cells, osteoblasts and others, in turn stimulating cellular proliferation, collagen synthesis and matrix formation promoting tissue repair [11, 12].
4.2.3 Preparation of PRP
The first stage in preparation of PRP is the collection of autologous whole blood from the patient. This is collected via a large bore cannula to avoid trauma to the blood constituents whilst also preventing the activation of platelets [13]. Anticoagulant, usually citrate dextrose, is used to prevent activation and the onset of clotting [11–13]. Other anticoagulants that have been used include ethylene-diamine-tetraacetic acid (EDTA); however, EDTA has been noted to cause structural and functional damage to platelets [3, 14] and its use is not recommended by the United States Food and Drug Administration [15].
These include:
- (i)
Gravitational platelet sequestration technique
- (ii)
Cell saver/separator technique
- (iii)
Selective filtration technology or plateletpheresis
- (i)
Gravitational platelet sequestration techniques are among the most common techniques used. Three distinct layers, according to specific gravity, are created via the process of centrifugation. Plasma forms the top layer with a specific gravity of 1.03 with the middle layer composed of platelets and white blood cells, also known as the buffy coat. The heaviest layer is composed of red blood cells. The yield of PRP is approximately 10 % of the whole blood obtained [3, 11]. Gravitational platelet sequestration can be further classified according to the buffy coat method or the PRP method. In the buffy coat method, whole blood is chilled and then undergoes high speed, “hard” centrifugation. After the three layers have been created, the PRP layer is further refined, by being separated from the plasma and RBCs. The PRP then undergoes a second bout of “soft”, slower centrifugation. The white cells are further separated from the PRP or a leucocyte filtration filter is employed [11]. In the PRP method, differences include the whole blood being drawn into acid citrate dextrose tubes whilst the timing of the two types of centrifugation is reversed. The method forms platelet poor plasma (PPP) in the upper 2/3 of the test tube whilst the lower 1/3 is the platelet-rich plasma concentration. Platelet pellets precipitate at the bottom of the tube. After discarding the PPP, the platelet pellets are suspended in plasma [11].
The PRP method is falling out of favour. The reasons for this are twofold [17]. Firstly, the activation, which occurs both during the precipitation of platelet pellets and during storage is higher than that of the buffy coat method (although it is not clear whether this in vitro phenomenon affects biological performance in the body [17]). Secondly, the process of storage also appears to increase activation. Metcalf measured the change in a series of platelet surface markers, using antibodies to the surface markers after 8 days of storage. The changes were attributed to the component separation, rather than venesection [18]. The activation is a cause for concern, as it leads to the release of cytokines, which can later lead to febrile non hemolytic transfusion reactions at the point of transfusion [17].
- (ii)
Standard cell separators use a whole unit of blood for PRP manufacture. The manufacture involves two steps with the first being a separation technique using a continuous flow centrifuge bowl or a continuous disc separation technique. This is followed by a fast (hard) centrifugation with a later slow (soft) centrifugation. One benefit includes the ability to transfuse red blood cells and PPP back to the donor patient [3].
- (iii)
The process of plateletpheresis involves the filtration of platelets alone, from a unit of whole blood, with the use of a disposable single filter. Centrifugation does not play a role in this method [3].
4.2.4 Classification
There has been much confusion regarding the terminology governing PRP. PRP can be considered an umbrella term, encompassing a number of different platelet concentrates with a multitude of different constituents. Dohan Ehrenfest and colleagues [19] propose a clarification of the term, based upon:
- (i)
The presence of cellular (i.e. leukocyte) contents
- (ii)
Type of fibrin architecture
The clarification, in turn, permits classification into four main groups [19] (Table 4.2).
Table 4.2
A current general classification of platelet concentrates
Type of preparation | Appearance after activation | Form | Clinical application |
|---|---|---|---|
Pure Platelet-Rich Plasma (P-PRP) | Preparations without leukocytes and with low density fibrin network | Liquid solutions, gel form | Ability to be used as injectable in sports medicine and orthopaedics |
Leukocyte and Platelet-Rich Plasma (L-PRP) | Preparations with leukocytes and presence of low density fibrin network | Liquid solutions, gel form | Can improve skin healing |
Pure Platelet-Rich Fibrin (P-PRF) | With leukocytes and high density fibrin network | Solid, gel | Treatment of ulcer wounds. Not suitable for orthopaedic application |
Leukocyte and Platelet-Rich Fibrin (L-PRF) | Preparations with leukocytes and high density fibrin network | Solid, gel | Maxillofacial surgery for use as filling or interposition material. High strength and slow release of growth factors |
The method of preparation, and therefore of resultant cell concentrations alters the clinical outcome. A study by Sundman et al. aimed to define what biological effect the inclusion of growth factors and leukocytes would have [20]. Two commercial systems were examined, one offering a PRP solution, which is leukocyte deplete, whilst the other offered a leukocyte rich solution. The presence of growth factors was measured after preparation, with the use of an ELISA assay. In the leukocyte-deplete PRP group, increased levels of PDGF-AB and TGF-ß1 were noted whilst MMP 9 and IL1-beta were noted in the leukocyte rich PRP group.
The presence of these growth factors in the leukocyte deplete group is advantageous. There is evidence to suggest that both factors hasten wound healing. TGF-ß1 improves collagen synthesis and deposition whilst PDGF-AB is chemotactic to fibroblasts and encourages deposition of glycosaminoglycan and fibronectin. The presence of white cells was postulated to increase tissue breakdown and encourage leukocyte activation. One of the benefits of TGF-ß1 is its immunomodulatory function, with an ability to retard IL1-beta activity whilst also synthesizing an IL1-ß antagonist. In the presence of a leukocyte rich preparation, this may be hindered [20]. Anitua also describes reservations with the use of leukocyte rich preparations, highlighting the creation of a proinflammatory environment, which is unfavourable to successful wound healing. The study also noted a detrimental effect to the mechanical properties of fibrin scaffolds [21].
4.2.5 Applications
4.2.5.1 Osteoarthritis
Osteoarthritis (OA) of the knee and knee arthroplasty are forecast to increase in frequency until at least 2030 [22]. With some evidence suggesting sporting participation will increase in all age groups, sporting injuries are also likely to escalate [23–25]. Sporting injuries are more likely to occur in the lower limb with the knee particularly prone [26], and can lead to post-traumatic OA.
PRP has been suggested as a possible adjunct to conventional treatment in OA, occupying a therapeutic middle ground between conservative management and surgery. In the example of sporting injuries, there is some evidence to suggest a role for PRP in pain reduction and the improvement of function in cartilage damage, whilst PRP may also facilitate earlier recovery in tendinopathy [27].
A systematic review and meta-analysis carried out by Laudy and colleagues, sought to assess the role of platelet-rich plasma in decreasing pain, improving function and preventing the progression of radiographic changes in knee OA [28]. Ten studies were analysed, of which six were randomised controlled trials (RCTs) and the others non-randomised with a high degree of bias. Eight studies compared PRP with hyaluronic acid, one compared PRP to placebo whilst the final study compared single versus double spinning. The review concluded that PRP may have a beneficial effect on both pain and function at both 6 months and a year in comparison to placebo and hyaluronic acid. The study also concludes that the application of platelet-rich plasma for knee osteoarthritis may be safe although one study by Patel et al., recorded a number of systemic adverse effects including syncope, tachycardia and dizziness in 17 from 78 patients. The symptoms were recorded in those who were subject to a higher quantity of platelets and were limited, arising in the first 30 min after injection. The symptoms were ascribed to either the higher quantity of platelets or the use of calcium chloride as an activating agent [29].
4.2.5.2 Tendinopathy
Overuse activities such as jumping or running can be the trigger for patellar tendinopathy, producing inferior pole tenderness of the patella [30, 31]. Whilst its etiology was previously ascribed to inflammation, current thought suggests that neural, mechanical or vascular triggers lead to a failed healing response. In an effort to establish the mechanism underlying the impaired response, Andia and colleagues compared healthy semitendinosus cells to tendinopathic rotator cuff cells [32]. The study concluded that PRP influences tendon healing by immunomodulatory and pain reduction pathways. At present there is no gold standard treatment for the problem but PRP’s ability to release growth factors has been advocated to encourage the healing process.
Dragoo and colleagues performed an RCT comparing PRP to dry needling in a population of patient who had failed conservative treatment [31]. From 23 patients, 13 were allocated to the dry needling group whilst 10 were allocated to the PRP group. Outcome measures were collected at 12 and 26 weeks. Results demonstrated a significant improvement in outcome scores at 12 weeks for PRP compared to dry needling p = 0.02. However, at 26 weeks, the difference between the two groups was not significant. The study concluded that PRP might have a role to play but time weakens its therapeutic value [31].
These findings have been supported by a recent systematic review of studies relating to PRP in patellar tendinopathy [33]. This review, including two RCTs, one non-randomised comparative study and eight case series, concluded that PRP appears to be a safe intervention, and that all non-comparative studies that were included demonstrated improvement after its administration. However, comparative studies did not demonstrate a consistent effect of PRP compared to other treatments such as physiotherapy.
4.2.5.3 Meniscus
PRP may enhance regeneration of meniscal tissue. In animal models, the use of PRP in conjunction with gelatin hydrogel led to an improved healing with the healed tissue demonstrating a structural integrity comparable to the inner part of the meniscus [34]. These findings were contradicted by Zellner et al., who did not describe any improvement upon the use of PRP with hyaluronan-collagen composite matrix [35].
A case control study examined the use of PRP in addition to meniscal repair. Outcome measures included the need for subsequent menisectomy and patient outcome scores at 2 years [36]. No difference was found between the two cohorts in terms of either reoperation or patient related outcome measures, return to work or athletic activity. The study was underpowered, did not allow for subgroup analysis of the different types of meniscal tears and the two groups varied in important factors such as age and body mass index. Pujol et al., examining the use of PRP in the open repair of horizontal meniscal tears, suggest an improvement with PRP. In those patients to whom PRP was administered, a statistically significant improvement in both KOOS score and the normalisation of MRI appearances was seen [37].
4.2.6 Remarks on PRP
The use of PRP promises many potential theoretical benefits in the treatment of knee pathologies. However the evidence collected to date has not supported these hopes. Part of the problem lies in the lack of a standardised preparation of PRP. The preparation and activation of the therapy, as well as the inclusion of fibrin or leukocytes, also vary from study to study.
The pool of evidence for treatment is small whilst the studies are not robustly designed to definitively support the use of PRP. In the case of the use of PRP in osteoarthritis, there are six RCTS to draw upon, with multiple sources of bias, whilst the evidence for PRP in tendinopathy is similarly limited [33]. There is also marked variability of the types of outcome measures used to quantify function between studies.
4.3 Mesenchymal Stem Cells (MSCs)
4.3.1 Introduction
Stem cell research began with the work of the German pathologist Julius Cohnheim in the late nineteenth century. By labelling inflammatory cells, Cohnheim discovered serendipitously associated dye labelled cells derived from bone marrow migrating to sites of injury. The idea that cells derived from blood and consequently bone marrow permitted tissue regeneration became known as the Cohnheim theory. A year later, in 1868, Emile Goujon, a French physiologist, furthered this work. Osteogenesis was demonstrated by the ectopic implantation of autologous bone marrow into the bones of rabbits and chickens. After the flurry of early advances, progress was arrested for almost a century.
Tavassoli and Crosby confirmed bone marrow’s ability to undergo de novo osteogenesis. Alexander Friedenstein extended this work and realised that this osteogenesis was attributable to a subset of bone marrow cells of fibroblast like-appearance. This appearance suggested that the cells precursors were of the stromal compartment of the bone marrow. Friedenstein demonstrated that with the ectopic implantation of an aggregation of bone marrow stromal cells, at clonal density, led to the formation of a variety of mesenchymal tissues. This led Friedenstein to christen these cells CFU F (colony forming unit Fibroblastic). However the cells are better known, somewhat misleadingly, as mesenchymal stem cells, owing to Arnold Caplan’s work coinciding with the isolation of human embryonic stem cells [38, 39].
4.3.2 Definition
Stem cells can be defined as those cells with:
The ability to self-renew
The quality of multilineage differentiation
The property of arising from a single cell
Stem cells can be classified on the basis of their origin (embryonic stem cells, adult stem cells and cancer stem cells [40, 41]), the degree of plasticity they exhibit, or the ability to form different tissues.
Embryonic stem cells are derived from the morula, the inner cell mass of the blastocyst. These cells have the ability to form cell types from all three germ cell layers of the embryo. It is this ability of embryonic stem cells which forms an important avenue of research, as they can be used to understand pathophysiology, and to trial the efficacy and safety of medications and other treatments of a multitude of diseases.
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