**7. Application of PRP in the treatment of chronic wounds**

The process of healing of skin wounds is very intense and includes a number of phenomena, such as hemostasis, inflammation, formation of granulation tissue, epithelialization, neovas‐ cularization, collagen synthesis, and wound contraction. Several studies demonstrate that the growth factors are depleted in chronic wounds. Platelets have a tendency to aggregate when activated, having a leading role in skin healing. Activated platelets release growth factors, adhesion molecules, and lipids through which migration, proliferation, and the function of keratinocytes, fibroblasts, and endothelial cells are regulated [47].

ished of platelets. The macrophages clean the area through phagocytosis and secrete factors responsible for the formation of granulation tissue from the fibroblasts. During this phase, angiogenesis also begins under the influence of growth factors and thrombin. With the recovery of vascular endothelial cells new vessels also develop. Endothelial cells are activated by thrombin, while a process of interaction is also ensured, which restricts the intensity of the formation of new vessels [40]. In this phase mesenchymal stem cells also emerge and their differentiation is performed in specific tissues such as bone, cartilage, or vascular tissues being

At the time of the remodeling phase, contraction of the collagen and convergence of the edges of the wound is observed. Cell density and vascularization is reduced, excess matrix is removed, and the collagen fibrils are aligned along the stress lines, which increases the strength of the newly-formed tissue [10]. Accumulated granulation tissue remodels or slowly trans‐

A number of growth factors are located in platelet alpha-granules. In order to be released into the injured tissue monocytes, neutrophils, fibroblasts, mesenchymal stem cells and osteoblasts need the chemotactic effect of an available growth factor. Platelet-derived growth factor (PDGF) produces such effect. This growth factor influences mitogenesis of fibroblasts and smooth muscle cells. PDGF improves the formation of fibrous tissue, assists in the three phases of the healing cascade, and also significantly affects angiogenesis and re-epithelialization. Another important growth factor is transforming growth factor beta (TGF-b). It impacts the connection between fibronectins. It influences cell migration, proliferation and replication, and

Chronic wounds' and endochondral ossification's healing processes are highly influenced by vascular endothelial growth factor (VEGF). It is active in angiogenesis, too [13,42]. The next factor taking part in the recovery of chronic wounds is epidermal growth factor (EGF). It also influences the mitogenesis of endothelial cells, fibroblasts, and keratinocytes [42]. Hepatocite growth factor (HGF) is located in various tissues (several types of epithelium, liver, lung, kidney and tumor). It affects tissue regeneration and possesses morphogenic, mitogenic,

The process of healing of skin wounds is very intense and includes a number of phenomena, such as hemostasis, inflammation, formation of granulation tissue, epithelialization, neovas‐ cularization, collagen synthesis, and wound contraction. Several studies demonstrate that the growth factors are depleted in chronic wounds. Platelets have a tendency to aggregate when activated, having a leading role in skin healing. Activated platelets release growth factors,

determined by chemical signals [36,38,39].

is present during inflammation processes [41].

antiapoptotic, and neurotrophic qualities [38,43-46].

**7. Application of PRP in the treatment of chronic wounds**

**6. Platelet growth factors**

178 Immunopathology and Immunomodulation

forms into a specific tissue such as skin or bone [36,38,39].

Skin wounds are defined as the absence of tissue, where epidermis and dermis are affected, while sometimes can reach the adipose tissue and muscle fascia. In this process, no natural regeneration is observed and tissue lesion is transformed into fibrotic scarring [48,49]. Skin wounds have a frequency of 0.78% and huge funts are spent for their treatment. Annually, 2% of the budget is used for health in the European Union (EU), £ 40 million in the UK, \$-1.3 billion are used for the treatment of decubitus in the US [48].

According to Zhu et al., chronic cutaneous wounds are quite frequent in developing countries. They are difficult to treat due to insufficient amount of growth factors. Sometimes wound infections are observed. Conventional treatment with various known techniques and methods is not enough due to the insufficiency of growth factors. Dressing and surgical debridement is used for these patients, but these strategies do not lead to significant outcomes [50].

For the development of skin wounds facilitate various etiological factors such as: chronic venous disease, peripheral arterial disease, neuropathy, arterial hypertension, physical injury, hematologic disorders, skin infection, inflammatory diseases, neoplasms, iatrogenic changes, and those associated with nutrition [48,49,51].

PRP gel has been used for wound healing stimulation on molecular and cellular levels for the last two decades [48,52]. Martinez-Zapata et al. showed tissue regeneration in maxillofacial surgery, chronic ulcers, and surgical wounds in their systematic review [53]. Weglein et al. assessed studies dealing with diabetic wound healing and another study reviewed the healing of chronic wounds [24]. Carter et al. reviewed wound healing when PRP gel is applied and wound healing of control groups (conventional wound care) and reported on a number of indicators [48,52].

With these studies, it was observed that PRPs have effectively stimulated healing in wounds with delayed healing. In a study, Martinez-Zapata et al. reported that the percentage of full healing of wounds treated with PRP has increased compared to that of control wounds. Carter et al. performed a meta-analysis of articles for chronic wounds and support the idea that the use of platelet rich plasma favors full healing compared to control groups [9,19,43]. In a study by Villela et al., they reached similar conclusions, with one of their conclusions reaching so far as to conclude that PRP is the method of choice for the treatment of wounds [48,21].

Difficult to heal wounds are considered those that do not heal after the fourth week of their appearance after being treated with standard methods for the particular pathology. Chronic is a wound that is not healing for a period of three months. The evaluation of each wound is very important. For the purpose, a number of point scales suggested by Cancela et al. based on clinical and anatomical criteria for the size of the wound are used, while a patient variable is also taken into account. The points are randomly defined using clinical experience in wound healing. Common parameters are assessed such as the presence or absence of erythema and edema surrounding the wound, purulent discharge, presence of fibrin, swelling in pressure, swelling with ocher dermatitis, and granulation [48].

Anatomical features are noted and graded such as open bone or tendon, location of the wound, and quality of the pulse of artery dorsalis pedis and artery tibialis posterior (when related to wound location) [48]. Wounds are measured to determine their overall surface, depth, and distribution of undermining of the wound edges. The duration of the wound is determined by the history [48].

There are many studies relating to the pathophysiology of wounds. Skin wound healing is an intensive process and in it are observed a number of events such as hemostasis, inflammation, formation of granulation tissue, formation of new vessels, collagen synthesis, epithelialization, and contraction of the wound. The process of reduction of growth factors in chronic wounds is demonstrated in numerous reports as a result of their reduced production and release, sequestration, or degradation. These mechanisms can be combined [48,54].

Platelet aggregation has a clearly expressed effect in the process of skin wound healing. In this process, growth factors are released and, adhesion of molecules and lipids is observed, while they are responsible for the regulation of cellular migration, proliferation and function of keratinocytes, fibroblasts, and endothelial cells [11,55]. Platelets secrete several antimicrobial peptides when activated by thrombin. PRP has an important antimicrobial and immunoregu‐ latory activity obtained from leukocytes [56,57]. A review of studies using different products rich in platelets demonstrates significant improvement in the treatment of chronic wounds.

Anitua et al. presented an open and randomized study to evaluate the effect of PRP in chronic ulcers of 14 patients. They reported good healing response in 80% of the cases after 8 weeks of treatment, compared with 20% in the control group. Leukocytes were not detected in the analyzed products. Their explanation for the good results lies in the high concentrations of growth factors [48,54].

Crovetti et al. monitored the evolution of chronic skin wounds in 24 patients treated with autologous or homologous gel (depending on the case) and observed a complete healing in 9 of them after an average of 10 applications, with a reduction of pain in all cases [48,49].

Marté-Mestre et al. reported the recovery of vascular chronic ulcers with the use of PRP in 12 of 14 patients for an average treatment period of 2.93 months (average of 0.5 - 7 months) [48,58].

Dellinger and Britton applied autologous platelet gel and reported positive results, with no complications and acceleration of the healing process (5 - 8 weeks) regardless of Pan size. A reduction of the risk of amputation and improvement of quality of life was observed [22,48]. PRP has the advantage of having no adverse effects [59].

Margolis et al. found in a retrospective group study with neuropathic wounds in diabetic feet greater efficiency in the use of PRP against the conventional therapies, with a more obvious effect in severe wounds [48,60].

PRP may be used for treatment of various chronic cutaneous wounds, especially when standard conventional therapy is not good enough and surgical treatment is not possible. It reduces the duration, cost of treatment and the hospital stay. These procedures may be performed in outpatient clinics. There is reduction of wound pain after starting the treatment, reduced risk of blood-borne disease transmission, wound healing is restored, and local immunity is activated [61,62].

#### **8. PRP in osteoarthritis**

Anatomical features are noted and graded such as open bone or tendon, location of the wound, and quality of the pulse of artery dorsalis pedis and artery tibialis posterior (when related to wound location) [48]. Wounds are measured to determine their overall surface, depth, and distribution of undermining of the wound edges. The duration of the wound is determined

There are many studies relating to the pathophysiology of wounds. Skin wound healing is an intensive process and in it are observed a number of events such as hemostasis, inflammation, formation of granulation tissue, formation of new vessels, collagen synthesis, epithelialization, and contraction of the wound. The process of reduction of growth factors in chronic wounds is demonstrated in numerous reports as a result of their reduced production and release,

Platelet aggregation has a clearly expressed effect in the process of skin wound healing. In this process, growth factors are released and, adhesion of molecules and lipids is observed, while they are responsible for the regulation of cellular migration, proliferation and function of keratinocytes, fibroblasts, and endothelial cells [11,55]. Platelets secrete several antimicrobial peptides when activated by thrombin. PRP has an important antimicrobial and immunoregu‐ latory activity obtained from leukocytes [56,57]. A review of studies using different products rich in platelets demonstrates significant improvement in the treatment of chronic wounds.

Anitua et al. presented an open and randomized study to evaluate the effect of PRP in chronic ulcers of 14 patients. They reported good healing response in 80% of the cases after 8 weeks of treatment, compared with 20% in the control group. Leukocytes were not detected in the analyzed products. Their explanation for the good results lies in the high concentrations of

Crovetti et al. monitored the evolution of chronic skin wounds in 24 patients treated with autologous or homologous gel (depending on the case) and observed a complete healing in 9 of them after an average of 10 applications, with a reduction of pain in all cases [48,49].

Marté-Mestre et al. reported the recovery of vascular chronic ulcers with the use of PRP in 12 of 14 patients for an average treatment period of 2.93 months (average of 0.5 - 7 months) [48,58].

Dellinger and Britton applied autologous platelet gel and reported positive results, with no complications and acceleration of the healing process (5 - 8 weeks) regardless of Pan size. A reduction of the risk of amputation and improvement of quality of life was observed [22,48].

Margolis et al. found in a retrospective group study with neuropathic wounds in diabetic feet greater efficiency in the use of PRP against the conventional therapies, with a more obvious

PRP may be used for treatment of various chronic cutaneous wounds, especially when standard conventional therapy is not good enough and surgical treatment is not possible. It reduces the duration, cost of treatment and the hospital stay. These procedures may be performed in outpatient clinics. There is reduction of wound pain after starting the treatment,

PRP has the advantage of having no adverse effects [59].

sequestration, or degradation. These mechanisms can be combined [48,54].

by the history [48].

180 Immunopathology and Immunomodulation

growth factors [48,54].

effect in severe wounds [48,60].

Cartilage defects and the most common joint disease, osteoarthritis(OA), are characterized by degeneration of the articular cartilage that ultimately leads to joint destruction [50]. Several treatments for OA exist, including exercise, weight control, bracing, nonsteroidal antiinflammatories, cortisone shots, and viscosupplementation. The biochemical environment of the disease process should be influenced in order to achieve better control of the symptoms. There is an imbalance between various cytokines resulting in the increased quantity of proteolytic enzymes leading to cartilage destruction [25,63 - 65]. The repair of damaged articular cartilage and bone defects represents a great challenge for the orthopedist because of the regenerative limitations of cartilage and the difficulty in obtaining an adequate bone substitute. Damages to the cartilage havess been treated by microfracture, debridement, and grafting procedures [42]. The results obtained by these techniques are so far unsatisfactory, and in most cases, these techniques result in deposition of fibrous connective tissue with low mechanical strength at the defect location [56,66]. Broad application of PRP is typical for oral, maxillofacial, and plastic surgery, while in the last few years there have been papers showing administrations in orthopedics and traumatology. When obtaining PRP, high concentration of growth factors in a volume unit is observed and acceleration of the regeneration process in the arthritic cartilage. Consecutively, PRP stimulates local cell activity. This technique is lowpriced and has no side-effects or dangers for the patient [33,67].

The presence of concentrated growth factors modulates the phenotypic expression of chon‐ drocytes. This improves their recovery. TGF-b plays a major role in the differentiation of stem cells into chondrocytes. Synthesis of cartilaginous matrix is improved, as well. PDGF and IGF are both key parts of the recovery process, as the first one stimulates proliferation of chondro‐ cytes while the second one increases the synthesis of proteoglycan - macromolecules. Thus, the strength of cartilage is improved [30,56]. Not only does PRP stimulate cellular activity and the process of regeneration and repair, but it also improves bone and cartilage recovery when biomaterials are applied [68,69]. In order to recover cartilage defects, not only PRP is needed but also an adequate biomaterial that has similar characteristics as the natural tissue and promotes cellular adhesion, proliferation, and differentiation [56].

According to Hunziker et al., TGF-b increases chondrogenesis [24,70], while Kon et al. documented PRP amplification of chondrocyte proliferation with convincing clinical effects on degenerative knee OA [24,25,70,71]. Results of the Kon et al. publication on 115 arthritic knees treated with PRP showed improvement in the functional status and pain scores "which remained positive at 6 months, with only mild degradation of scores at 1 year". It was also mentioned that lower grades of arthritis and younger patients had better outcomes [25,72].

Qi et al. shared results on the treatment of osteochondral defects in rabbits with PRP and biodegradable collagen scaffolds. PRP was put into collagen scaffolds (0.05 ml PRP/structure) and then implanted in osteochondral defects (4 x 3 mm) produced in the patellar sulcus of rabbit femurs. Evaluation of tissues was completed after 6 and 12 weeks by histological analysis and a mechanical indentation test. The results in both follow-up times were improved and showed that PRP possesses the capacity to stimulate the regeneration of critical defects in cartilage [56,73].

According to Patel et al. in a study with level 1 evidence, PRP has a superior effect compared to placebo in patients with varying degrees of knee OA. They tested 78 patients (156 knees) and observed that PRP alleviated the symptoms of OA when compared with a saline injection used as control. Follow-up period was up to 6 months. The results showed that with the help of PRP, the homeostasis and function of the knee joint can be improved [74,75].

Wei et al. reported the effect of PRP on the regeneration of cartilage in vivo. Auricular cartilage chondrocytes from rabbits were added to PRP (5 x 107 cells/ml PRP), injected subcutaneously in the dorsum of the donor animal, and after 60 days the tissue was evaluated by magnetic resonance, histological, and histochemical analysis. The results exhibited cartilaginous tissue formation in those groups that were injected with chondrocytes and PRP. In the groups in which only PRP was injected, cartilaginous tissue formation was not

observed. The authors inferred that PRP is effective in stimulating regeneration of cartilage when associated with viable cells [56,76].

A level 1 study published in 2012 in the Journal of Arthroscopy by Sanchez et al. used PRP type 4A to observe the results of PRP injection compared with hyaluronic acid. In this study 176 patients, were randomized to receive three weekly applications of PRP (8 ml at weekly intervals) or hyaluronic acid injections. The study was well-conducted, but the results only focused on the short term (until 6 months of follow up). The study was awarded as the best randomized clinical trial (level 1) for 2012, which gives useful fundamental guidelines on how to study the effects of PRP [74,77,78].

In the light of orthopedic and trauma critical literature, there is no standardization of treatment methods, such that most of the works use 3–5 applications of PRP in intervals ranging from 1–3 weeks with no standardization of the amount of volume to be applied and the ideal platelet concentration method [74].

Important factors for results of PRP treatment are the amount of platelets, grade of chondrocyte damage, patient's physical activity, and medical history combined with psychosocial factors. It is good to investigate whether PRP balances anabolism-catabolism, and if there is, what effect of does the PRP have on the synovium. Given the patient's condition, appropriate PRP treatment in OA should use more anti-inflammatory cytokines instead of pro-inflammatory cytokines. The future of OA research aims at isolating cytokines contained in PRP termed as "autologous conditioned serum" to specifically inhibit IL-1 and TNF alpha and down regulate MMP-13 [25,79].

Early OA can be treated by implementing intra-articular PRP. Worse outcome is observed with patients of advanced age and disease process. Khoshbin et al. compared intra-articular administration of PRP to hyaluronic acid and non-setoroid injections and reported that sequential intra-articular PRP injections may have beneficial effects on adult patients with mild to moderate osteoarthritis for a period of 6 months [80].

Zhu et al. reported the fact that PRP contains growth factors, which may have a negative impact on the treatment of a joint with osteoarthritis. That is why those growth factors should be isolated. The following years may be focused on finding suitable and harmless growth factors to improve PRP outcome [50]. Dold et al. paid attention to the fact that there is insufficient data on PRP implementation on post-traumatic osteochondral defects. Further studies with longterm follow-up are necessary in order to illustrate the benefits of isolated PRP application or application in combination with surgical treatment when treating cartilage pathology [81].
