*P* < 0.05, when compared to the empty control at corresponding time (Scheffe test for multiple comparison).

**Table 2.** Results of the histological grading scale

(SOX9). Namely, this approach could lead to the preparation of hyaline cartilage directly from skin without generating iPS cells. Recently, Yamashita et al. [36] reported that hyaline cartilage was generated from human iPS cells in immunodeficiency rats and immunosuppressed mini-

Table 1. Histological grading scale for cartilage defect\*

A. Cell morphology and Matrix staining

B. Surface regularity†

C. Integration of donor with host adjacent cartilage

D. Filling of defect

E. Reconstitution of subchondral bone and osseous connection

Category II

Category I

196 Osteoarthritis - Progress in Basic Research and Treatment

Points

 Hyaline cartilage Mostly hyaline cartilage Moderately hyaline cartilage Partly hyaline cartilage

 Fibrous Smooth (>3/4) Moderate (>1/2–3/4) Irregular (>1/4–1/2) Severely irregular (<1/4) Both edges integrated One edge integrated Neither edge integrated

 ~100% ~75% ~50% 3 ~25% 4 0% Yes Almost Partly Not close

(Subtotal 13)

(Subtotal 7)

1

Total maximum 20

Total smooth area of the reparative cartilage compared with the entire area of the cartilage defect.

†

**Table 1.** Histological grading scale for cartilage defect\*

†

the cartilage defect.

\*Modified from the scale described by Pineda et al. [46] and Wakitani et al. [47].

\*Modified from the scale described by Pineda et al. [46] and Wakitani et al. [47].

Total smooth area of the reparative cartilage compared with the entire area of

pigs.

#### **2.5. Multilineage-differentiating stress enduring (Muse) cell**

As a novel type of pluripotent stem cells, Muse cells were recently reported as adult human MSCs without introducing exogenous genes, and they are present in mesenchymal tissues such as the bone marrow, adipose tissue, dermis, and connective tissue of organs [37–40]. In particular, Muse cells have been detected more abundant in adipose tissues than in other organizations [41]. Also, Muse cells have a low tumor-forming ability compared with embry‐ onic stem (ES) cells and a high efficiency of change to iPS cells by Yamanaka gene introduction [42]. They can migrate to damaged tissues by intravenous injection *in vivo*, spontaneously differentiate into cells compatible with the targeted tissue, and contribute to tissue repair. Thus, Muse cells will be expected to play important role in regenerative therapy by further studies.

#### **3. MSCs in synovial fluid of human OA**

2 In 2004, Jones et al. [43, 44] reported that the MSCs in synovial fluid in the inflammatory and degenerative arthritis, including OA, possessed high proliferative potential and could differentiate into several mesenchymal lineages. Aspiration of synovial fluid in the cases of hydrarthrosis caused by OA has the following great advantages: extremely simple technique, feasible during routine practice in outpatients, no need for local or general anesthesia for cell harvest, and effective usage of synovial fluid supposed to be discarded in the cases of hy‐ drarthrosis.

#### **3.1. Potential of chondrogenic differentiation of synovial fluid cells**

We investigated the possibility of chondrogenic differentiation of the cells derived from synovial fluid and compared with the BMMCs previously performed in human OA [45]. Synovial fluid was aspirated from 26 knee joints of outpatients with OA and those of six patients just before skin incision at TKA. Bone marrow was obtained from the femur before the insertion of the femur rod at the time of TKA. Each aspirated fluid was diluted in αmodified Eagle's minimum essential medium (αMEM), and mononuclear cells using Ficoll-Paque PLUS (GE Healthcare) were harvested and cultured. Primary passage cells were used for flow cytometry assay and for chondrogenic assay, total RNA was prepared from each pellet of cultured cells, and pellets were used for immunohistochemical staining.

**Figure 2.** A, B) Phase-contrast photomicrographs of cultured synovial fluid cells on day 6 (A) and day 28 (B) showing fibroblast-like morphology. On day 28, the culture dish in subconfluency. (C–E) Multipotency of the cultured synovial fluid cells. (C) Osteogenesis was shown by alkaline phosphatase staining and the expression of osteopontin messenger (m) RNA (*OPN*). (D) Chondrogenesis was shown by toluidine blue staining and the expression of type II collagen mRNA (*Col2a1*). (E) Adipogenesis was shown by oil red-O staining and the expression of PPAR*γ* mRNA (*PPARγ*).

In the results, the morphology of the cultured synovial fluid cells was fibroblastic, similar to that of BMMCs. Also, the synovial fluid cells had an ability to differentiate into osteoblasts, chondrocytes and adipocytes (Figure 2). The cultured synovial fluid cells strongly expressed CD13, CD44, and CD105 but lacked CD10, CD14, and CD45 in flow cytometry analysis. Both mRNA expression of aggrecan and type II collagen had an increasing tendency at day 21 compared with day 7. Also, the cell pellets derived from synovial fluid showed intense toluidine blue staining, indicating chondrogenic differentiation.

#### **3.2. Potential of cartilage regeneration of synovial fluid cells**

**3.1. Potential of chondrogenic differentiation of synovial fluid cells**

198 Osteoarthritis - Progress in Basic Research and Treatment

of cultured cells, and pellets were used for immunohistochemical staining.

We investigated the possibility of chondrogenic differentiation of the cells derived from synovial fluid and compared with the BMMCs previously performed in human OA [45]. Synovial fluid was aspirated from 26 knee joints of outpatients with OA and those of six patients just before skin incision at TKA. Bone marrow was obtained from the femur before the insertion of the femur rod at the time of TKA. Each aspirated fluid was diluted in αmodified Eagle's minimum essential medium (αMEM), and mononuclear cells using Ficoll-Paque PLUS (GE Healthcare) were harvested and cultured. Primary passage cells were used for flow cytometry assay and for chondrogenic assay, total RNA was prepared from each pellet

**Figure 2.** A, B) Phase-contrast photomicrographs of cultured synovial fluid cells on day 6 (A) and day 28 (B) showing fibroblast-like morphology. On day 28, the culture dish in subconfluency. (C–E) Multipotency of the cultured synovial fluid cells. (C) Osteogenesis was shown by alkaline phosphatase staining and the expression of osteopontin messenger (m) RNA (*OPN*). (D) Chondrogenesis was shown by toluidine blue staining and the expression of type II collagen mRNA (*Col2a1*). (E) Adipogenesis was shown by oil red-O staining and the expression of PPAR*γ* mRNA (*PPARγ*).

In the results, the morphology of the cultured synovial fluid cells was fibroblastic, similar to that of BMMCs. Also, the synovial fluid cells had an ability to differentiate into osteoblasts, chondrocytes and adipocytes (Figure 2). The cultured synovial fluid cells strongly expressed Synovial fluid was aspirated from the OA knees before 1 month of TKA and cultured *in vitro*. Degenerative OA cartilage was obtained at the time of TKA. Approximately 5 × 105 autologous synovial fluid cells were labeled with Cell Tracker Green (CTG) (Invitrogen) in 200-µL αMEM and were transplanted gently on macroscopic degenerative tissues of OA cartilage. After10 min, medium was removed and changed into chondrogenic medium. One week later, the tissues were observed under a fluorescent microscope.

**Figure 3.** *Ex vivo* study using synovial fluid cells. (A) The formation of repaired tissue was shown by hematoxylin and eosin (HE) staining (Black arrows). (B) Magnified feature of A. (C) Repaired tissues and the surroundings of CTGstained cells were weakly positive by safranin-O staining. (D) Fluorescent microscopy showed that CTG-labeled syno‐ vial fluid cells existed in repaired cartilage. (E) Magnified feature of D. (F) CTG-positive cells had a tendency to infiltrate into the original degenerative cartilage (White arrows). N.C.: Negative Control without CTG-labeled cells.

Histopathologically, degenerative tissues with transplanted CTG-labeled cells were weakly positive by safranin-O staining, which indicated that they were toward cartilage tissues (Figure 3). Fluorescent microscopy showed that CTG-labeled synovial fluid cells existed in the repaired tissues, which indicated that the tissues were constructed of autologous transplanted cells and the synovial fluid cells had a tendency to adhere to the degenerative cartilage. Furthermore, they seemed to infiltrate into the original degenerative cartilage of OA.

#### **3.3. Cell-based therapy using synovial fluid cells**

From previous and our current study, it has been recognized that synovial fluid in OA knee joints contain the adherent cells, and these cells have a potential of cell proliferation and chondrogenic differentiation *in vitro*. The primary culture of the human synovial fluid cells showed the formation of colonies of fibroblast-like cells, similar to those of BMMCs in both flow cytometry and real-time PCR analysis [34]. The infiltration of synovial fluid cells into the degenerative cartilage indicates that the possibility of attachment to OA cartilage in humans may promote the production of extracellular matrix and regeneration of hyaline cartilage. Further studies are need, but we expect the benefits of synovial fluid cells on OA cartilage tissues.

#### **4. Conclusions**

Many studies using various cell types for OA treatment are being performed. These short-term results appear mostly satisfactory, but there remains a problem that repaired tissues become fibrocartilage thereafter. Fibrocartilage leads to different biomechanical characteristics compared with hyaline cartilage and progresses to OA. MSCs based on self-repair and multilineage potentials provide to hyaline cartilage regeneration. In particular, bone marrowderived MSCs are the most commonly used cell type for cartilage regeneration, but harvesting of the bone marrow is a painful procedure and has the risk of wound infection and sepsis. Alternatively, synovial fluid cells have great advantages and cartilage regeneration potential similar to bone marrow-derived MSCs. Naturally, long-term studies are needed whether repaired tissues are durable within the joint, but the use of synovial fluid cells may be expected to cartilage regeneration for OA therapy.

#### **Author details**

Rie Kurose1\* and Takashi Sawai2

\*Address all correspondence to: riekuro@hirosaki-u.ac.jp

1 Department of Orthopaedic Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Japan

2 Department of Histopathology, Tohoku University Graduate School of Medicine, Sendai, Japan

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tissues, which indicated that the tissues were constructed of autologous transplanted cells and the synovial fluid cells had a tendency to adhere to the degenerative cartilage. Furthermore,

From previous and our current study, it has been recognized that synovial fluid in OA knee joints contain the adherent cells, and these cells have a potential of cell proliferation and chondrogenic differentiation *in vitro*. The primary culture of the human synovial fluid cells showed the formation of colonies of fibroblast-like cells, similar to those of BMMCs in both flow cytometry and real-time PCR analysis [34]. The infiltration of synovial fluid cells into the degenerative cartilage indicates that the possibility of attachment to OA cartilage in humans may promote the production of extracellular matrix and regeneration of hyaline cartilage. Further studies are need, but we expect the benefits of synovial fluid cells on OA cartilage

Many studies using various cell types for OA treatment are being performed. These short-term results appear mostly satisfactory, but there remains a problem that repaired tissues become fibrocartilage thereafter. Fibrocartilage leads to different biomechanical characteristics compared with hyaline cartilage and progresses to OA. MSCs based on self-repair and multilineage potentials provide to hyaline cartilage regeneration. In particular, bone marrowderived MSCs are the most commonly used cell type for cartilage regeneration, but harvesting of the bone marrow is a painful procedure and has the risk of wound infection and sepsis. Alternatively, synovial fluid cells have great advantages and cartilage regeneration potential similar to bone marrow-derived MSCs. Naturally, long-term studies are needed whether repaired tissues are durable within the joint, but the use of synovial fluid cells may be expected

1 Department of Orthopaedic Surgery, Hirosaki University Graduate School of Medicine,

2 Department of Histopathology, Tohoku University Graduate School of Medicine, Sendai,

they seemed to infiltrate into the original degenerative cartilage of OA.

**3.3. Cell-based therapy using synovial fluid cells**

200 Osteoarthritis - Progress in Basic Research and Treatment

tissues.

**4. Conclusions**

**Author details**

Hirosaki, Japan

Japan

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\*Address all correspondence to: riekuro@hirosaki-u.ac.jp

Rie Kurose1\* and Takashi Sawai2


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### **Local Therapies for Osteoarthritis — An Update and a Review of the Literature**

Sarah Karrar and Charles Mackworth-Young

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60557

#### **1. Introduction**

Osteoarthritis (OA) is the most common chronic joint condition affecting an estimated 8 million people in the UK alone. It manifests as localised joint pain, stiffness and occasionally swelling. Osteoarthritis can be secondary to pre-existing joint damage - commonly inflammatory arthropathy or previous injury - or primary with no known pre-existing damage. Risk factors for primary OA include old age, female sex and family history and obesity.

The disease can be restricted to a particular joint or generalised, affecting multiple joints. In severe cases, it can be progressive eventually leading to loss of function and deformity.

Treatment has mainly focused on symptomatic relief from pain, physical approaches such as rehabilitation and physiotherapy, disease modifying treatment (such as hydroxychloroquine) and surgery. Pain relief with systemic drugs has drawbacks. In particular the use of nonsteroidal anti-inflammatories (NSAIDs) has been associated with significant adverse events including gastritis and increased risk of cardiovascular disease. In view of this, there has been increasedinterestinlocalisedtreatments forOA.-i.e.therapies that are administeredto the joint itself, or in the region of the joint. These can be divided into topical treatment, such as antiinflammatory gels and creams and thermotherapy, and more invasive local treatment includ‐ ing joint aspiration, and intra-articular joint injection with corticosteroid and hyaluronans.

#### **2. Topical treatments**

#### **2.1. Thermotherapy**

Thermotherapy refers to the application of either heat or cold (cryotherapy) to affected joints in an attempt to improve pain, stiffness and swelling.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ice massage and ice packs application have both been studied in knee osteoarthritis [5-10]. In one review [7], cryotherapy was found to reduce pain, stiffness and oedema. Regular ice massage, given 5 times a week, was found to have a clinically significant effect on all three symptoms as well as function (11% improvement relative difference), strength (29% improve‐ ment) and range of movement (8% relative difference) over a short period of time [8]. However, these improvements were not replicated with less frequent applications (3 times per week) [9] and there are no data to indicate a more long term effect of cold therapy on osteoarthritis as these studies looked at short term results. It is likely that most of the effects of cryotherapy are related to the induction of local vasoconstriction and the reduction of local blood flow resulting in reduced swelling.

Common methods of superficial heat administration are electrical heating pads, application of hot packs, towels or wax, or immersion in warm water or wax baths. In some early trials, heat application was not found to improve function or symptoms [8,9]. In recent years, however, there has been an explosion of studies looking at different modalities of local heat therapy[10-13]. These include the application of heat packs [12], ultrasound [13 11] and diathermy. The application of local heat packs has been found to provide short-lived benefit in terms of pain relief [12, 14]; and in particular, wet heat has been found to be better than dry heat [15] for symptomatic improvement. In one study [12] 18 patients were treated with either steam generating heat sheets for 6 hours daily or with quadriceps strengthening exercises only for a total of 12 weeks. At the end of the study, patients in the heat treated group reported statistically significant improvements in symptoms as well as the Timed Up and Go time (a measure of function). The mechanism of heat therapy in osteoarthritis is unclear, although *ex vivo* studies of cartilage [15, 16] have indicated raising chondrocyte temperature might increase their metabolism and production of proteoglycans. This in part, maybe secondary to increased blood flow to the chondrocytes.

On the whole, the data suggest that thermotherapy maybe useful as an adjunct in the treatment of osteoarthritis although long-term benefits have not been established.

#### **2.2. Local ultrasound therapy**

The role of ultrasound (US) in diagnosis of musculoskeletal problems has been well establish‐ ed. Its popularity in large part due to the low cost and non-invasive nature of the modality. In recent years, there has been growing interest in its application for therapeutic purposes [13,18-20]. In theory direct treatment with US leads to local heating of the tissue at depths not achieved by applying heat packs. There are two methods for doing this: continuous US which leads to a rise in temperature of the treated tissues; and pulsed wave treatment which harnesses other properties of US. *In vitro* and animal studies [18, 19] have suggested that pulsed wave US can increase collagen production and reduce expression of membrane metallo-proteinase, suggesting a protective role. This has failed to translate to clinical benefit as recent randomised controlled studies [13, 20] comparing continuous, pulsed and sham US on knee osteoarthritis symptoms and joint function, have shown no difference in pain scores nor 15m-walk time. In general, the safety of US has been established but evidence is scarce for any therapeutic advantage [13,20].

#### **2.3. Laser therapy**

Ice massage and ice packs application have both been studied in knee osteoarthritis [5-10]. In one review [7], cryotherapy was found to reduce pain, stiffness and oedema. Regular ice massage, given 5 times a week, was found to have a clinically significant effect on all three symptoms as well as function (11% improvement relative difference), strength (29% improve‐ ment) and range of movement (8% relative difference) over a short period of time [8]. However, these improvements were not replicated with less frequent applications (3 times per week) [9] and there are no data to indicate a more long term effect of cold therapy on osteoarthritis as these studies looked at short term results. It is likely that most of the effects of cryotherapy are related to the induction of local vasoconstriction and the reduction of local blood flow resulting

Common methods of superficial heat administration are electrical heating pads, application of hot packs, towels or wax, or immersion in warm water or wax baths. In some early trials, heat application was not found to improve function or symptoms [8,9]. In recent years, however, there has been an explosion of studies looking at different modalities of local heat therapy[10-13]. These include the application of heat packs [12], ultrasound [13 11] and diathermy. The application of local heat packs has been found to provide short-lived benefit in terms of pain relief [12, 14]; and in particular, wet heat has been found to be better than dry heat [15] for symptomatic improvement. In one study [12] 18 patients were treated with either steam generating heat sheets for 6 hours daily or with quadriceps strengthening exercises only for a total of 12 weeks. At the end of the study, patients in the heat treated group reported statistically significant improvements in symptoms as well as the Timed Up and Go time (a measure of function). The mechanism of heat therapy in osteoarthritis is unclear, although *ex vivo* studies of cartilage [15, 16] have indicated raising chondrocyte temperature might increase their metabolism and production of proteoglycans. This in part, maybe secondary to increased

On the whole, the data suggest that thermotherapy maybe useful as an adjunct in the treatment

The role of ultrasound (US) in diagnosis of musculoskeletal problems has been well establish‐ ed. Its popularity in large part due to the low cost and non-invasive nature of the modality. In recent years, there has been growing interest in its application for therapeutic purposes [13,18-20]. In theory direct treatment with US leads to local heating of the tissue at depths not achieved by applying heat packs. There are two methods for doing this: continuous US which leads to a rise in temperature of the treated tissues; and pulsed wave treatment which harnesses other properties of US. *In vitro* and animal studies [18, 19] have suggested that pulsed wave US can increase collagen production and reduce expression of membrane metallo-proteinase, suggesting a protective role. This has failed to translate to clinical benefit as recent randomised controlled studies [13, 20] comparing continuous, pulsed and sham US on knee osteoarthritis symptoms and joint function, have shown no difference in pain scores nor 15m-walk time. In general, the safety of US has been established but evidence is scarce for any therapeutic

of osteoarthritis although long-term benefits have not been established.

in reduced swelling.

208 Osteoarthritis - Progress in Basic Research and Treatment

blood flow to the chondrocytes.

**2.2. Local ultrasound therapy**

advantage [13,20].

Laser beam therapy directs intense light to treated tissue. Two types of laser therapy have been trialled in osteoarthritis: low-level and high intensity. Low- level laser therapy uses red and infra-red light whilst high intensity laser therapy uses higher wavelengths of radiation for deeper tissue penetration. Low level laser therapy has been found to reduce pain, possibly by modulating the local inflammatory process In a rat model of osteoarthritis, laser therapy caused a reduction in neutrophil migration, oxidative stress, altered levels of cyclo-oxygen‐ ase-2 and other pro-inflammatory mediators) [21]. Other than providing symptomatic relief, there is also some evidence that laser promotes fibroblast proliferation, collagen synthesis and bone regeneration [22-26]. In a rabbit model of osteoarthritis, six weeks of treatment with laser therapy not only resulted in improved pain but also histological evidence of reduced inflam‐ mation as well as a reduction in cartilage damage [27]. This suggests that laser therapy could have disease-modifying as well as symptomatic benefits.

So far, the results of early clinical trials have been mixed [28]. More recent studies have tended to be more positive with those treated with laser therapy and exercise doing better than those treated with exercise alone on pain measurements as well as function [29, 30]. These studies suggest that laser in combination with standard physiotherapy could have advantages over standard therapy. We have little evidence regarding long term effect and whether the cellular effects noted results in halting disease progression. The use of low-level laser therapy has now been approved by the US Food and Drug Agency (FDA) and so we are likely to see an expansion in its use in the coming years.

#### **2.4. Topical non-steroidal anti-inflammatory drugs**

Non-steroidal anti-inflammatory drugs (NSAIDS) work by blocking the action of cyclooxygenases responsible for prostaglandin synthesis, the latter being known mediators of inflammation [31]. Locally this reduces pain, swelling and heat. NSAIDs also have central analgesic actions, possibly by reducing brain prostaglandin synthesis although alternative mechanisms include the induction of endogenous opioid peptides and blockade of serotonin release. From this, it can be seen why systemic NSAIDs have long been used for osteoarthritis. However, significant side effects including gastritis, renal impairment and increased risk of cardiovascular disease has meant that their long-term use has been limited. It is on this background that topical NSAID use has been promoted, theoretically providing analgesic and anti-inflammatory benefits without systemic adverse effects.

There are many types of topical NSAID. There are preparations containing diclofenac, ibuprofen, piroxicam, ketoprofen or felbinac as the active ingredient. Some include a penetra‐ tion enhancer such as menthol or dimethylsulfoxide (DMSO). Gels and sprays tend to be more penetrative than cream preparations. Once applied, a topical NSAID must be absorbed by the underlying tissue or enter the local blood stream. Studies have shown that the absorption of NSAIDs into the underlying tissue gives rise to therapeutic local concentrations of the drug without significant systemic absorption [32,33]. An estimated 3-7% of the applied dose is thought to be absorbed systemically [33] with plasma concentrations being approximately 5% of those achieved with oral administration [33].

The skin seems to act as a reservoir from which the drug disseminates to the deeper tissue. Peak concentrations in the skin are achieved 2 hours after application with a further spike at about 19 hours later, likely secondary to systemic absorption. Further proof of their local action is the absence of analgesic effect at joints distant to the point of application [34].

There have been many studies looking into the effectiveness of topical NSAIDs in treating osteoarthritis [35-41]. These on the whole have found that topical NSAIDs were superior to placebo in the treatment of chronic pain. Most of the initial studies found no benefit beyond two weeks of treatment [35-41] but larger randomised controlled trials found long term benefit for up to 3 months when compared to placebo [42-43].

When compared to oral NSAID use, the results have been variable. A meta-analysis in 2004 [41] found that topical NSAIDs were less effective than systemic NSAIDs. Since then, however, there have been several studies showing comparable effectiveness. Two studies comparing oral diclofenac with a topical preparation of the drug [44,45] found that there was no difference in pain scores or physical function. They also found that those in the topical treatment arm had a much lower incidence of severe gastrointestinal side effects, deranged liver function tests and abnormal creatinine clearance [44,45]. These results were replicated in another study comparing oral and topical treatment with ibuprofen for knee osteoarthritis, which also found no difference in pain and function scores between the two arms [43].

On the whole, topical NSAID use is associated with fewer systemic adverse events [35, 39, 44, 45] as compared to oral preparations. The main adverse event associated with topical NSAID use is local skin irritation, which has been reported in up to 39.3% of patients [46]. However, these skin reactions occur with placebo gel application with equal frequency indicating that it may not be related to the drug itself [39]. Other studies also suggest that skin reactions may be more common with solutions containing DMSO than DSG [37]. There is some contradictory evidence regarding their safety in older patients as some studies have found the rate of GI side effects in the over 50s to be as high as 15% [46].

Overall the data suggest that topical NSAIDs may be considered as first line therapy for osteoarthritis as they appear to be efficacious and associated with fewer adverse events. There should be caution about their long term use in the elderly as these patients may be more prone to adverse events.

#### **2.5. Other topical treatments**

Topical capsaicin cream has been used to treat a multitude of different painful conditions including osteoarthritis, inflammatory arthritis and neuropathic pain. Derived from chilli peppers, capsaicin is a lipophilic alkaloid that acts as a local irritant. It activates local pain receptors (c-nociceptors) leading to the release of substance P [47]. This in turn causes local irritation in the initial phase of treatment. With repeated use, however, levels of substance P are depleted, leading to desensitisation of the pain fibres and hypoalgesia [48]. In clinical practice, capsaicin is better than placebo for the treatment of chronic pain but compares less favourably with other treatments. In a meta-analysis comparing capsaicin with plaster, capsaicin was found to be only marginally effective [49]. Other drawbacks include the need to apply the cream four times a day for maximum benefit, as well as local irritation and intense burning sensation (occurring in uto 40% of patients) [50]. These problems lead 10% of patients to discontinue treatment [49]. In view of this, topical capsaicin should be used in conjunction with more traditional treatments.

Other topical treatments include the use of salicylate or nicotine esters, which acts as a local counter-irritant or rubefacient. These cause localised vasodilatation and reddening of the skin. This results in a local sensation of warmth, which often palliates pain. Theories of mechanisms of action include irritation of the sensory nerve endings in underlying muscle and tissue [51] as well as activation of the transient receptor ion channels involved in relaying thermal and pain sensation [52,53]. Clinical studies have shown modest benefits with regular use [54,55]. Compared to placebo, 16% achieved ≥50% improvement in pain scores at 2 weeks [54]. However, when compared to topical NSAIDs, counter-irritants performed poorly [55]. On the whole, counter-irritants are well tolerated and may be useful as adjuvants to standard therapy or patients in whom standard analgesics are contra-indicated [55]. There are no data to support their long term use and they are not recommended as continuing therapy.

#### **3. Local injections**

The skin seems to act as a reservoir from which the drug disseminates to the deeper tissue. Peak concentrations in the skin are achieved 2 hours after application with a further spike at about 19 hours later, likely secondary to systemic absorption. Further proof of their local action

There have been many studies looking into the effectiveness of topical NSAIDs in treating osteoarthritis [35-41]. These on the whole have found that topical NSAIDs were superior to placebo in the treatment of chronic pain. Most of the initial studies found no benefit beyond two weeks of treatment [35-41] but larger randomised controlled trials found long term benefit

When compared to oral NSAID use, the results have been variable. A meta-analysis in 2004 [41] found that topical NSAIDs were less effective than systemic NSAIDs. Since then, however, there have been several studies showing comparable effectiveness. Two studies comparing oral diclofenac with a topical preparation of the drug [44,45] found that there was no difference in pain scores or physical function. They also found that those in the topical treatment arm had a much lower incidence of severe gastrointestinal side effects, deranged liver function tests and abnormal creatinine clearance [44,45]. These results were replicated in another study comparing oral and topical treatment with ibuprofen for knee osteoarthritis, which also found

On the whole, topical NSAID use is associated with fewer systemic adverse events [35, 39, 44, 45] as compared to oral preparations. The main adverse event associated with topical NSAID use is local skin irritation, which has been reported in up to 39.3% of patients [46]. However, these skin reactions occur with placebo gel application with equal frequency indicating that it may not be related to the drug itself [39]. Other studies also suggest that skin reactions may be more common with solutions containing DMSO than DSG [37]. There is some contradictory evidence regarding their safety in older patients as some studies have found the rate of GI side

Overall the data suggest that topical NSAIDs may be considered as first line therapy for osteoarthritis as they appear to be efficacious and associated with fewer adverse events. There should be caution about their long term use in the elderly as these patients may be more prone

Topical capsaicin cream has been used to treat a multitude of different painful conditions including osteoarthritis, inflammatory arthritis and neuropathic pain. Derived from chilli peppers, capsaicin is a lipophilic alkaloid that acts as a local irritant. It activates local pain receptors (c-nociceptors) leading to the release of substance P [47]. This in turn causes local irritation in the initial phase of treatment. With repeated use, however, levels of substance P are depleted, leading to desensitisation of the pain fibres and hypoalgesia [48]. In clinical practice, capsaicin is better than placebo for the treatment of chronic pain but compares less favourably with other treatments. In a meta-analysis comparing capsaicin with plaster, capsaicin was found to be only marginally effective [49]. Other drawbacks include the need to

is the absence of analgesic effect at joints distant to the point of application [34].

for up to 3 months when compared to placebo [42-43].

210 Osteoarthritis - Progress in Basic Research and Treatment

effects in the over 50s to be as high as 15% [46].

to adverse events.

**2.5. Other topical treatments**

no difference in pain and function scores between the two arms [43].

#### **3.1. Intra-articular corticosteroids**

Intra-articular (IA) corticosteroid injections are frequently used to treat osteoarthritis. In common practice, they are diluted in local anaesthetic to provide immediate relief, ensure accurate drug delivery and allow even dispersal of the drug within the joint due to the larger volume [57]. Commonly used corticosteroids in IA injections include hydrocortisone acetate (HCA), methylprednisolone acetate (MPA) and triamcinolone acetonide (TCA). These vary in solubility with the former being more soluble than the latter. Less soluble preparations are longer acting and theoretically provide more long term relief. In one randomised control trial comparing MPA (more soluble and shorter acting) and TCA in knee osteoarthritis, greater improvement in pain scores was found in the TCA group at 3 weeks compared to MPA [56]. Interestingly, there was no difference between the 2 groups at 8 weeks despite TCA being longer acting. There was also no significant difference in functional scores [56].

Several studies have looked into whether intra-articular steroid injections have symptomatic or functional benefit in knee osteoarthritis [58-61]. These have shown short term (lasting between 1-4 weeks) improvement in pain but not function in these patients following injections when compared to placebo. Follow up beyond 4 weeks did not show longer lasting benefits as compared to placebo. These results were further corroborated in a Cochrane systematic review [62]. This suggests that IA steroid injections should be used as a short term bridging treatment to resolve acute painful flares pending further intervention such as surgery or physiotherapy. Trials looking IA injections in the hip echo the results of the studies done in the knee: patients gained rapid and short lived pain relief following injection but that these benefits were not maintained beyond 1 month [63,64].

Other studies, looking at 1st carpo-metacarpal joint (CMC) injections found more mixed results in terms of long term relief. In one study of 40 patients, no benefit was observed between steroid injection when compared to placebo [65]. Patients less likely to have sustained long term benefits were those with worse radiographic appearances (increased number of osteophytes and more advanced joint space narrowing) [66]. In patients with less advanced disease, IA 1st CMC joint injection could provide symptomatic relief up to 18 months following injection and splinting [66].

IA steroid injections work locally via anti-inflammatory effects, inhibiting the inflammatory cascade at multiple points. Local injection avoids the systemic problems associated with steroid use and allows delivery of high doses to the affected tissue. Response to IA injection, however, does not appear to be dependent on inflammation in the affected joint itself [61]. Further studies looking at whether inflammation detected on ultrasound predicted clinical response found that those without inflammatory change fared better in response to IA injection than those with evidence of inflammation. The presence of synovial thickening, synovial fluid volume and white cell count did not predict better response to IA injection [60, 61]. In knee OA, joint aspiration prior to IA injection appears to provide greater symptomatic benefit [60]. This is partly due to confirmation of correct position by prior aspiration and more concentrated drug delivery due to a lower volume [67].

Although IA injections avoid the toxic side effects of systemic steroids, they are not without risks themselves. All patients undergoing IA injection should be consented for the risk of infection, although this is a rare event (incidence reported between 1 in 3,000 to 1 in 50,000) [68] and may be clinically difficult to differentiate from an injection-induced crystal arthritis which can occur in 2-6% of patients [58, 60]. In general, septic arthritis following IA injection occurs 3 to 4 days post procedure. There is a risk of lipoatrophy at the site of injection (estimated 0.6% of patients) [69]. The risk of this is reduced by using shorter-acting preparations and doing imaging-guided injections where possible. Other serious local adverse events include tendon rupture, muscle wasting and local depigmentation. The risk of these can also be minimised by using guided injections where possible.

Systemic adverse events are rare with local corticosteroid injections. The most common is flushing (up to 40%) [69]. There have been reported incidents of unstable diabetic glycaemic control post injection but this tends to be minor and usually settles [70]. There is evidence for systemic absorption of intra-articular steroids [71]. Studies looking at the endocrine axis in patients who had received intra-articular steroid injections found that serum cortisol dipped 24-48 hours after IA injection and took up to 4 weeks to return to baseline [71]. Major compli‐ cations, such as steroid induced osteoporosis, have not been observed however [72].

Studies in animals have suggested that intra-articular steroids can induce chondrocyte degeneration [73] but prospective clinical trials where patients were receiving regular IA injections have failed to demonstrate increased rate of cartilage loss [74]. There are also limited data to support significant increased risk of osteonecrosis in injected joints. Repeated IA injections offer no long-term benefit [67] and should generally be avoided but short-term use may provide rapid pain relief and can be used as a bridging treatment pending further intervention.

#### **3.2. Intra-articular hyaluronic acid/hyaluronan**

Other studies, looking at 1st carpo-metacarpal joint (CMC) injections found more mixed results in terms of long term relief. In one study of 40 patients, no benefit was observed between steroid injection when compared to placebo [65]. Patients less likely to have sustained long term benefits were those with worse radiographic appearances (increased number of osteophytes and more advanced joint space narrowing) [66]. In patients with less advanced disease, IA 1st CMC joint injection could provide symptomatic relief up to 18 months following injection

IA steroid injections work locally via anti-inflammatory effects, inhibiting the inflammatory cascade at multiple points. Local injection avoids the systemic problems associated with steroid use and allows delivery of high doses to the affected tissue. Response to IA injection, however, does not appear to be dependent on inflammation in the affected joint itself [61]. Further studies looking at whether inflammation detected on ultrasound predicted clinical response found that those without inflammatory change fared better in response to IA injection than those with evidence of inflammation. The presence of synovial thickening, synovial fluid volume and white cell count did not predict better response to IA injection [60, 61]. In knee OA, joint aspiration prior to IA injection appears to provide greater symptomatic benefit [60]. This is partly due to confirmation of correct position by prior aspiration and more concentrated

Although IA injections avoid the toxic side effects of systemic steroids, they are not without risks themselves. All patients undergoing IA injection should be consented for the risk of infection, although this is a rare event (incidence reported between 1 in 3,000 to 1 in 50,000) [68] and may be clinically difficult to differentiate from an injection-induced crystal arthritis which can occur in 2-6% of patients [58, 60]. In general, septic arthritis following IA injection occurs 3 to 4 days post procedure. There is a risk of lipoatrophy at the site of injection (estimated 0.6% of patients) [69]. The risk of this is reduced by using shorter-acting preparations and doing imaging-guided injections where possible. Other serious local adverse events include tendon rupture, muscle wasting and local depigmentation. The risk of these can also be minimised by

Systemic adverse events are rare with local corticosteroid injections. The most common is flushing (up to 40%) [69]. There have been reported incidents of unstable diabetic glycaemic control post injection but this tends to be minor and usually settles [70]. There is evidence for systemic absorption of intra-articular steroids [71]. Studies looking at the endocrine axis in patients who had received intra-articular steroid injections found that serum cortisol dipped 24-48 hours after IA injection and took up to 4 weeks to return to baseline [71]. Major compli‐

Studies in animals have suggested that intra-articular steroids can induce chondrocyte degeneration [73] but prospective clinical trials where patients were receiving regular IA injections have failed to demonstrate increased rate of cartilage loss [74]. There are also limited data to support significant increased risk of osteonecrosis in injected joints. Repeated IA injections offer no long-term benefit [67] and should generally be avoided but short-term use may provide rapid pain relief and can be used as a bridging treatment pending further

cations, such as steroid induced osteoporosis, have not been observed however [72].

and splinting [66].

drug delivery due to a lower volume [67].

212 Osteoarthritis - Progress in Basic Research and Treatment

using guided injections where possible.

intervention.

Hyaluronic acid is a large glycosamino-glycan molecule found in synovial and cartilage extra cellular matrix (ECM). It is produced by synoviovytes, chondrocytes and fibroblasts and functions as both lubricant as well as a means to maintain hydration within the joint [75]. Studies have shown that osteoarthritic joints have decreased hyaluronan content in the synovial fluid [76] and therefore IA injection with a synthetic analogue was developed to restore the function in degenerative joints.

Synthetic preparations of hyaluronic acid closely mimic endogenous molecules. Later prepa‐ rations contain cross linked hyaluronin in order to achieve greater elasticity and viscosity. In theory this confers greater intra-articular durability of the solution. Preparations with a higher molecular weight also seem to be more beneficial than those with a lower weight [78]. This may be related to the difference in volume required for injection as well as the number of injections required and the intra-articular durability of the solution.

Multiple studies have been conducted into the effectiveness of IA injections of hyaluronans in osteoarthritis, mostly of the knee. These have found mixed evidence to recommend their use. In general, hyaluronans appear to be better than placebo in improving pain scores, function and patient global assessment when used in knee osteoarthritis [77]. The greatest clinical benefit is achieved at week 5-13 after a course of treatment of several injections. Part of the problem with interpreting the data is wide variability in trial design, frequency of injections and molecular weight of the synthetic product being used. In hip OA, hyaluronan injections were not superior to placebo nor corticosteroid injections in reducing pain or improving function [79]. These results were echoed in studies looking at its use in hand OA [80].

These injections are relatively safe and tend to provide longer term relief than corticosteroid injections. Its use, however, is restricted by the relatively high cost of the treatment [75]. IA hyaluronan injection is generally reserved for knee osteoarthritis and is offered either as a holding measure until more definitive treatment can be undertaken (e.g. surgery), or in patients for whom such treatment is inappropriate.

#### **3.3. Subcutaneous and soft tissue injections**

Trigger points are localised areas of tenderness and thickening in the soft tissues. They are typically found proximal to an inflamed or painful joint such as the rectus femoris in patients with knee OA and paraspinal regions in the cervical and lumbar spine [81]. They have also been described as interstitial fibrositis, myofasciitis and myofascial trigger points [82-84]. The aetiology and pathogenesis of these trigger points is unknown.

Trigger point injection has been used as a way of alleviating pain and discomfort associated with these areas of thickening. This can be direct injection of a substance (e.g. local anaesthetic or corticosteroid) into the point or indirect needling of the soft tissue in that area. The trigger point is identified as the maximal area of tenderness in the muscle and the point is then isolated by the thumb and forefinger to prevent movement in the underlying muscle. A small sterile needle is then introduced into the area and the substance injected directly into it (or alterna‐ tively it can be dry needled). If the injection is performed correctly, there is usually an acute worsening of pain associated with muscle spasm [85].

A systematic review of trigger point injection in the management of chronic musculoskeletal pain found an improvement in symptoms when used exclusively [86]. This was irrespective of the injectant used [86]. The addition of a local anaesthetic, however, has been found to reduce the pain and irritation of the caused by the procedure [84].

There are limited data on the efficacy of trigger point injection in the treatment of osteoarthritis. One study found that trigger point injection in conjunction with IA corticosteroid was more effective than IA injection alone in both pain and functional scores [87]. Other studies have looked at trigger point injections as sole treatment and this does not reflect clinical practice. Overall, trigger point injections are safe and can be used as additional therapy in OA.

Drugs used for trigger point injections have included local anaesthetic, corticosteroids, antiinflammatories such as acetylsalicylate and ketorolac as well as saline and water [84, 88-92]. There have also been several studies looking at the use of subcutaneous salicylate therapy for OA. In one trial 40 patients with OA of the 1st CMC joint [93] were randomised into either sham injection or subcutaneous injection with salicylate into trigger points. Patients were assessed blind at 3, 7 and 13 weeks. Pain scores and tenderness were significantly lower in those treated with salicylate compared to sham injections [93].

The mechanism of action of subcutaneous salicylate injections is unclear, particularly as the site of injection is distant from the affected joint. One theory is that salicylate may alter central sensitisation and this is supported by the immediate relief patients report following injection. An alternative model would be that the local effect of salicylate modifies the neurogenic control of inflammation, which may be abnormal in diseases that affect musculoskeletal structures [94, 95]. Changes in the expression and transport of neurogenic peptides might be induced by the local irritant effect of salicylate [96]. Systemic anti-inflammatory effects are unlikely, since the benefits are not observed in distant sites [93].

There is, however, a degree of overlap with acupuncture in that the injection sites are standard acupuncture locations. Acupuncture involves the insertion of fine filiform needles at or near the painful site, or sometimes at distant acupuncture "points". In a variation of this, the needles are sometimes stimulated electronically or with heat. Patients typically receive six or more sessions for a complete course of treatment. A systematic review of 393 patients with osteoar‐ thritis found that acupuncture significantly improved pain but not function when compared to sham acupuncture [97-104]. In addition, it was not better than standard treatment with physiotherapy or being on a waiting list to receive acupuncture [97,100]. There was also no additional benefit of including acupuncture to standard therapy with exercise and advice [103]. Moreover, there is little evidence for long term benefit following treatment with improvements in symptoms lasting up to 12 weeks only [97,100]. Acupuncture is relatively safe, however, with minimal risks of serious side effects [101-104].

#### **4. Splinting/support**

tively it can be dry needled). If the injection is performed correctly, there is usually an acute

A systematic review of trigger point injection in the management of chronic musculoskeletal pain found an improvement in symptoms when used exclusively [86]. This was irrespective of the injectant used [86]. The addition of a local anaesthetic, however, has been found to reduce

There are limited data on the efficacy of trigger point injection in the treatment of osteoarthritis. One study found that trigger point injection in conjunction with IA corticosteroid was more effective than IA injection alone in both pain and functional scores [87]. Other studies have looked at trigger point injections as sole treatment and this does not reflect clinical practice.

Drugs used for trigger point injections have included local anaesthetic, corticosteroids, antiinflammatories such as acetylsalicylate and ketorolac as well as saline and water [84, 88-92]. There have also been several studies looking at the use of subcutaneous salicylate therapy for OA. In one trial 40 patients with OA of the 1st CMC joint [93] were randomised into either sham injection or subcutaneous injection with salicylate into trigger points. Patients were assessed blind at 3, 7 and 13 weeks. Pain scores and tenderness were significantly lower in those treated

The mechanism of action of subcutaneous salicylate injections is unclear, particularly as the site of injection is distant from the affected joint. One theory is that salicylate may alter central sensitisation and this is supported by the immediate relief patients report following injection. An alternative model would be that the local effect of salicylate modifies the neurogenic control of inflammation, which may be abnormal in diseases that affect musculoskeletal structures [94, 95]. Changes in the expression and transport of neurogenic peptides might be induced by the local irritant effect of salicylate [96]. Systemic anti-inflammatory effects are unlikely, since the

There is, however, a degree of overlap with acupuncture in that the injection sites are standard acupuncture locations. Acupuncture involves the insertion of fine filiform needles at or near the painful site, or sometimes at distant acupuncture "points". In a variation of this, the needles are sometimes stimulated electronically or with heat. Patients typically receive six or more sessions for a complete course of treatment. A systematic review of 393 patients with osteoar‐ thritis found that acupuncture significantly improved pain but not function when compared to sham acupuncture [97-104]. In addition, it was not better than standard treatment with physiotherapy or being on a waiting list to receive acupuncture [97,100]. There was also no additional benefit of including acupuncture to standard therapy with exercise and advice [103]. Moreover, there is little evidence for long term benefit following treatment with improvements in symptoms lasting up to 12 weeks only [97,100]. Acupuncture is relatively

Overall, trigger point injections are safe and can be used as additional therapy in OA.

worsening of pain associated with muscle spasm [85].

214 Osteoarthritis - Progress in Basic Research and Treatment

the pain and irritation of the caused by the procedure [84].

with salicylate compared to sham injections [93].

benefits are not observed in distant sites [93].

safe, however, with minimal risks of serious side effects [101-104].

Osteoarthritic joints may be helped by various forms of external support. Benefit can be obtained by adjusting alignment, modifying stress or load, providing shock absorption, or simply resting the joint. Orthoses (including braces, splints and elasticated sleeves) are frequently used in OA of the hand and knee and hand.. For hand OA they include thumb and wrist splints; for the knee they include rest orthoses, knee sleeves and unloading braces. Medial patellar strapping may be specifically helpful for patellar maltracking [105]. Shoe insoles may be particularly helpful for OA affecting the ankle and knee, and can sometimes alleviate symptoms from OA of the hip: they include cushioned or neutral insoles, which act as a shock absorbers; and wedged insoles, which also modulate mechanical stress.

For OA of the knee and ankle the main purpose of orthoses and insoles is to support joint that is unstable, and to help correct alignment [106]. They can modify load bearing and contribute to pain reduction, and they often improve physical function. There is also evidence that they can improve proprioception [107] and that they may slow disease progression [108]. They are especially useful for mild or moderate uni-compartmental knee OA [109-110, 42] where there may be varying degrees of instability and mal-alignment. Unloading knee braces are designed to reduce the load transmitted to the diseased compartment by applying an external valgus or varus force. Symptomatic relief is achieved by stabilizing the joint, increased joint opening and reduced local muscle contraction [108]. One study [111] demonstrated that patients treated with unloading knee braces had better functional and symptomatic outcomes at 6 months with medial compartment knee OA. These results were not replicated in other studies [112] although there is evidence that they can improve quadriceps strength and gait symmetry [113]. The main disadvantage of these braces is poor tolerability due to the weight and heat of the device. In one study, 41% of patients complained of skin irritation [114] and up to 20% of patients discontinue use within 6 months [115].

Splinting of the thumb carpometacarpal (CMC) joint has also been found to be helpful in improving function and pain [116]. CMC joint OA contributes more to pain and disability than inter-phalangeal joint OA [117] and thus splinting of the CMC joint makes sense. In a system‐ atic review in 2010, CMC splinting was found to improve function and grip strength [116]. Further RCT data has corroborated this finding and showed sustained benefit at 12 months [118]. However, these splints are inevitably somewhat cumbersome to wear, and inhibit many day-today functions of the hand.

In general, splinting might be useful for symptomatic relief and may even improve function with prolonged use in appropriately selected patients.

#### **5. Conclusion**

There are a number of different local treatments for osteoarthritis which focus on symptomatic relief. Choice of treatment should, therefore, be guided by patient response and personal preference. Most local therapies are safe, avoiding any major systemic side effects. In general, these therapies should be used as adjuncts to physiotherapy and systemic analgesia. Although some of these treatments are well established and have been used in clinical practice for many years (e.g. intra-articular injections and orthoses), newer approaches are being developed such as local laser therapy and subcutaneous sodium salicylate injections. There is limited data to show any benefit for long term outcome for any of these local therapies and further studies are required to establish this.

#### **Author details**

Sarah Karrar and Charles Mackworth-Young\*

\*Address all correspondence to: c.mackworth-young@imperial.ac.uk

Department of Rheumatology, Charing Cross Hospital, Fulham Palace Road, London, UK

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Department of Rheumatology, Charing Cross Hospital, Fulham Palace Road, London, UK

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## **The Conservative Management of Osteoarthritis — Hyaluronic Acid, Platelet Rich Plasma or the Combination?**

Michele Abate, Isabel Andia and Vincenzo Salini

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60538

#### **1. Introduction**

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224 Osteoarthritis - Progress in Basic Research and Treatment

539–48

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Osteoarthritis (OA) is a progressively degenerating joint disease. The prevailing paradigm suggests that OA results from an impaired regeneration ability of the damaged cartilage due to biomechanical and biochemical changes. There is an increasing recognition that OA is a disease of the joint as an organ involving all intraarticular tissue i..e. subchondral bone, synovium, cartilage, menisci, ligaments. Therefore, changes as increased metabolism and sclerosis of the subchondral bone, chondrocyte death and extracellular matrix (ECM) catabo‐ lism, as well as primary or secondary changes in the synovium (including endothelial cell proliferation, macrophage infiltration and inflammation) will induce alterations in the molecular composition of the synovial fluid. The clinical signs associated with these changes include pain, rigidity and decreased functionality, which may compromise overall health and quality of life [1].

The prevalence of OA is very high, expected to affect more than 50 million subjects in the US by 2020, and will increase with the ageing of the population [2]. According to the American College of Rheumatology, nearly 70% of people over age 70 have X-ray evidence of OA, although only half ever develop symptoms [3].

The barriers to treatment development include the insufficient understanding of the pathology and the fact that the OA physiopathology may not be identical for all patients. Several pharmaceutical approaches (analgesics, non steroidal anti-inflammatory drugs, COX-2 inhibitors and steroids) have been proposed, with the aim of reducing pain and maintaining and/or improving function [4]. However, none of these options has shown to delay the progression of the disease or reverse joint damage. In addition, the incidence of adverse

reactions to these drugs increases with age. Data from epidemiological studies consistently show that the risk of cardio-vascular and gastro-intestinal complications is very high and largely dose-dependent [5]. It is well known that non steroidal anti-inflammatory drugs, as well as selective COX-2 inhibitors, may cause renal failure, hypertension and water retention and have a thrombotic potential, especially for high doses and long term treatments [6]. On the other hand, corticosteroids are burdened with relevant side effects, when given systemi‐ cally, and therefore are usually administered by intra-articular (IA) injection in patients who fail to respond to other conservative measures; in particular, patients with joint effusions and local tenderness may have greater benefit from this option. Although it has been established that corticosteroid injections are relatively safe, there are concerns regarding their possible adverse events following repeated treatments. These effects include local tissue atrophy, particularly when small joints are injected, long-term joint damage, due to reduced bone formation, and risk of infection, due to suppression of adrenocortical function [7].

Considering the limits of therapies at present available, drugs with minimal side effects, and which can stop the progression of the disease, are therefore warranted. Research in Regener‐ ative Medicine in the last decades, has shaped new investigational biological preparations that can be injected within the joints. These include mesenchymal stem cells (BMSCs [Bone Marrow Mesenchymal Stem Cells] and AdSCs [Adipose Mesenchymal Stem Cells]). The initial idea of MSC therapies was to replace damaged or death cells but moved towards using MSCs as a tool to modify the tissue environment. The concept is based on the paracrine actions of MSCs, hence major biological mechanisms to be targeted by these therapies are inflammation, angiogenesis or modifications of the catabolic environment. Injectable MSCs therapies are not capable of structurally modifying the OA joint. Due to the costs, and lack of evidences of the regenerative potential of these cellular therapies, other less expensive and easier to implement approaches are being investigated. These include Platelet Rich Plasma (PRP) injections or combination products such as PRP + Hyaluronic acid (HA).

Both PRP and HA have been extensively used to improve lubrication, modulate inflammation and modify the catabolic micro-environment. In doing so, these conservative treatments not only aim to reduce clinical symptoms, but interfere with OA progression.

In this article we seek to summarize the current pre-clinical and clinical knowledge on this topic, reporting comparative studies between HA and PRP injections, and suggesting the possibility of the combined use of these therapeutic agents.

#### **2. Hyaluronic acid**

#### **2.1. Basic concepts and experimental research**

Hyaluronan is a polyanionic, unbranched glycosaminoglycan polymer composed of disac‐ charide subunits of N-acetyl-D-glucosamine and D-glucuronic acid. It was isolated for the first time in 1934 by Karl Meyer and John Palmer from the vitreous of bovine eyes [8]. These scientists proposed the name "hyaluronic acid" from hyaloid (vitreous) and uronic acid*.* Although the term HA is often used in the literature, the correct name reflecting the configu‐ ration of this molecule in vivo is hyaluronan. Indeed, *in vivo*, at physiological pH, it exists as a polyanion and not in the protonated acid form [9,10]. In this chapter, for convenience, we will use the term HA all through the text. HA is present in many tissues, but the larger amount of hyaluronan resides in the dermis and epidermis. It is also an essential component of the hyaline cartilage (1mg/g wet weight) where it organizes the ECM by creating specific interac‐ tions with aggrecan, a large chondroitin sulfate proteoglycan, present at higher concentration (25-50 mg/g wet weight). Aggrecan and type II collagen are the main macromolecules in the cartilage ECM. The spatial configuration of aggrecan and type II collagen are responsible of mechanical properties of the hyaline articular cartilage.

reactions to these drugs increases with age. Data from epidemiological studies consistently show that the risk of cardio-vascular and gastro-intestinal complications is very high and largely dose-dependent [5]. It is well known that non steroidal anti-inflammatory drugs, as well as selective COX-2 inhibitors, may cause renal failure, hypertension and water retention and have a thrombotic potential, especially for high doses and long term treatments [6]. On the other hand, corticosteroids are burdened with relevant side effects, when given systemi‐ cally, and therefore are usually administered by intra-articular (IA) injection in patients who fail to respond to other conservative measures; in particular, patients with joint effusions and local tenderness may have greater benefit from this option. Although it has been established that corticosteroid injections are relatively safe, there are concerns regarding their possible adverse events following repeated treatments. These effects include local tissue atrophy, particularly when small joints are injected, long-term joint damage, due to reduced bone

formation, and risk of infection, due to suppression of adrenocortical function [7].

combination products such as PRP + Hyaluronic acid (HA).

226 Osteoarthritis - Progress in Basic Research and Treatment

possibility of the combined use of these therapeutic agents.

**2.1. Basic concepts and experimental research**

**2. Hyaluronic acid**

only aim to reduce clinical symptoms, but interfere with OA progression.

Considering the limits of therapies at present available, drugs with minimal side effects, and which can stop the progression of the disease, are therefore warranted. Research in Regener‐ ative Medicine in the last decades, has shaped new investigational biological preparations that can be injected within the joints. These include mesenchymal stem cells (BMSCs [Bone Marrow Mesenchymal Stem Cells] and AdSCs [Adipose Mesenchymal Stem Cells]). The initial idea of MSC therapies was to replace damaged or death cells but moved towards using MSCs as a tool to modify the tissue environment. The concept is based on the paracrine actions of MSCs, hence major biological mechanisms to be targeted by these therapies are inflammation, angiogenesis or modifications of the catabolic environment. Injectable MSCs therapies are not capable of structurally modifying the OA joint. Due to the costs, and lack of evidences of the regenerative potential of these cellular therapies, other less expensive and easier to implement approaches are being investigated. These include Platelet Rich Plasma (PRP) injections or

Both PRP and HA have been extensively used to improve lubrication, modulate inflammation and modify the catabolic micro-environment. In doing so, these conservative treatments not

In this article we seek to summarize the current pre-clinical and clinical knowledge on this topic, reporting comparative studies between HA and PRP injections, and suggesting the

Hyaluronan is a polyanionic, unbranched glycosaminoglycan polymer composed of disac‐ charide subunits of N-acetyl-D-glucosamine and D-glucuronic acid. It was isolated for the first time in 1934 by Karl Meyer and John Palmer from the vitreous of bovine eyes [8]. These scientists proposed the name "hyaluronic acid" from hyaloid (vitreous) and uronic acid*.* High molecular weight (HMW) HA is also the main component of synovial fluid; it is synthe‐ sized and extruded within the synovial fluid by the lining fibroblastic cells of the joint capsule named type B synoviocytes [10]. The volume of synovial fluid in the knee is 3-4 ml. Its role, in addition to lubrication, is to provide nutrients to the hyaline cartilage and create a hypoxic environment on the cartilage surface.

The physiologic turnover (i.e. catabolism balanced by production) of HA within the joint is very dynamic; the half-life of HA is estimated in 12 hours [11]. Importantly, HA turnover, in both the cartilage ECM and the synovial fluid, is fundamental in the maintenance of joint homeostasis. Reticulo-endothelial cells lining the lymphatic vessels actively remove about 90% of the HA. HA catabolism can occur through hyalurodinase actions and/or peroxidative cleavage. It has been hypothesized that peroxidative cleavage, a process that consumes O2 and helps to maintain the hypoxic environment within the joint, is required for normal synovial function and joint homeostasis [12].

HA polymers are synthesized by three different synthases termed HAS (HAS-1, -2 and -3). HMW HA has anti-inflammatory, anti-angiogenic and anti-immunogenic properties [13-16]. The high viscosity of HMW HA grants viscoelastic properties to the fluid, and along with lubricin contributes to the boundary lubrication that is necessary for low friction levels on the articular surface [17]. Thus, it has a shielding effect on cartilage surfaces and other joint components.

Beside HA role in viscosupplementation and major functions in the biophysical and homeo‐ static conditions of the joint, an important aspect of HA biology to be considered is its influence on cell behavior.

Basic research, performed in OA models in animals (rats, rabbits, dogs and sheep), has shown that HA has several pleiotropic signaling properties (biosupplementation), such as immuno‐ suppressive, anti-inflammatory, anti-apoptotic, anti-angiogenic and anti-fibrotic effects, with normalization of endogenous HA synthesis, and chondroprotection [16]. Actually, HA binds to a number of cell membrane receptors termed hyaladherins. The predominant and more widely expressed is CD44, a membrane glycoprotein made of ten stable exons and ten variable exons inserted in different combinations at a particular extra-membrane site [16].

HA can be pro-inflammatory, however immune cells only bind HA when activated by an inflammatory agent. Actually, CD44 is expressed in the membrane of immune cells, therefore HA can participate in the recruitment of neutrophils, macrophages and lymphocytes [13]. Indeed, CD44 decrease in articular cartilage is related to progression of knee OA [16].

Paradoxically, HA-CD44 binding is involved in the resolution of inflammation. Besides, both CD44 and RHAMM (CD168) (the HA receptor for HA-mediated motility) are involved in the regulation of growth factor (GF) signaling.

A recent study has been performed in an experimental model of murine OA (TGF-β1 injection and treadmill running), which displays many OA-like changes, including synovial activation. HA injection, 24 hours after TGF-β1 injection, hinders neovascularization and fibrosis of the synovium, and keeps in good condition articular cartilage in wild-type, but not in CD44 knockout mice. This finding suggests that the injected HA enhances the synthesis of chondro‐ genic proteins, and blocks that of fibrogenic/degradative proteins in both the cartilage and subchondral bone [18].

A further research, performed in patients with knee or hip OA, has demonstrated the presence of activated T cells in the synovial fluid, so confirming that OA is a disease with a immuno‐ logical/inflammatory involvement. In these patients, HA injections decreased the levels of activated T cells, and so regulated the articular milieu [19].

The analgesic properties of HA, besides to the activities previously described, could be also attributed to a specific activity on opioid receptors [20]. Pain in OA is likely to have multiple sources, including subchondral bone marrow lesions, synovium and the periostium as well as soft tissues surrounding the joint, including extra-articular bursae and fat pad. Pain in OA is classified as nociceptive, based on the presence of opioid receptors in the synovial lining and sublining cells. Various molecules present in extracellular space modulate nociceptor sensi‐ tivity by targeting different receptor types. The biological mechanisms responsible for HA analgesic activity have been partially elucidated by Zavan *et al*. [20] in experiments performed with Chinese Hamster Ovary cells that express a panel of opioid receptors. The results demonstrated that HA stimulates the k receptor (KOP), also expressed on fibroblast-like synoviocytes, in a concentration dependent manner, but not the DOP, MOP and NOP receptors. This selective activity could be due to the singular conformational structures of HA compared to morphine, more closely related to dynorphin organization. The pain threshold also increases, due to the direct analgesia through inhibition of pain receptors, and by a direct action on synovial nerve endings and stimulation of synovial lining cells.

At present, HA compounds with different molecular weight (MW) are commercially available. The enhanced diffusion of LMW preparations (0.5-1.5 millions Dalton) through the ECM of the synovium makes possible the interaction with synovial cells [21]. However, because of the modest elastoviscosity of these compounds, compared to native hyaluronan in the synovial fluid, HA preparations with HMW (6-7 millions Dalton), have been developed. These formu‐ lations retain higher amounts of fluid in the articular space using their hydrophilic properties, and also have a greater anti-inflammatory activity, as shown by reduced prostaglandin E 2 and bradykinin concentration attributed to a reduced migration of inflammatory cells [22].

Currently, many types of particulate carriers have been investigated aiming to increase the retention time of the therapeutic agents within the joint capsule. Among them, cationic polymeric nanoparticles form ionically connected filamentous arrangements ("ionically crosslinked hydrogels") linked with local hyaluronan [23]. After intra-articular (IA) injection in rat knees about 70% of these hydrogels are retained into the joint for 1 week. Thus, cationic polymeric nanoparticles increase HA retention into joints and are suitable for therapeutic use. Another medical device combines chondroitin sulphate and HA [24]. The role of chondroitin sulfate is twofold: a) to create specific interactions designed to optimize HA rheological behavior; and b) to regulate cartilage metabolism by performing as a substrate for polysulph‐ ated glycosaminoglycans synthesis, as well as inhibiting the synthesis of catabolic cytokines and metalloproteinases.

To ameliorate OA treatment while avoiding adverse effects, a mixture of celecoxib-loaded liposomes embedded in HA gel has been formulated [25]. Celecoxib is a COX-2 selective inhibitor with analgesic and anti-inflammatory properties. Liposomes are good candidates for local delivery of therapeutic agents because they are derived from naturally occurring biodegradable and nontoxic lipids. The combination of the two drugs, both efficient in the treatment of OA, but with different mechanisms, injected into the joints, is expected to have synergistic effect. Indeed, in a rabbit knee OA model, the liposomal combination was more effective than a single drug in pain control and cartilage protection, as shown by histopatho‐ logical studies [26].

Preliminary studies suggest positive results using an innovative viscosupplement (SynolisV-A) produced from high a concentration of HA, combined with a high concentration of sorbitol as a free radical scavenger (single injection of 4 ml in total), in subjects with symptomatic hip OA [27].

These pharmaceutical studies taken together show how intense is nowadays the research aiming to ameliorate the therapeutic efficacy of HA. Open-label, non controlled studies have proven the clinical efficacy of some of these products. However, high quality clinical studies proving their superiority towards the available preparations of HA are still lacking.

#### **2.2. Clinical trials**

#### *2.2.1. Knee*

HA can participate in the recruitment of neutrophils, macrophages and lymphocytes [13].

Paradoxically, HA-CD44 binding is involved in the resolution of inflammation. Besides, both CD44 and RHAMM (CD168) (the HA receptor for HA-mediated motility) are involved in the

A recent study has been performed in an experimental model of murine OA (TGF-β1 injection and treadmill running), which displays many OA-like changes, including synovial activation. HA injection, 24 hours after TGF-β1 injection, hinders neovascularization and fibrosis of the synovium, and keeps in good condition articular cartilage in wild-type, but not in CD44 knockout mice. This finding suggests that the injected HA enhances the synthesis of chondro‐ genic proteins, and blocks that of fibrogenic/degradative proteins in both the cartilage and

A further research, performed in patients with knee or hip OA, has demonstrated the presence of activated T cells in the synovial fluid, so confirming that OA is a disease with a immuno‐ logical/inflammatory involvement. In these patients, HA injections decreased the levels of

The analgesic properties of HA, besides to the activities previously described, could be also attributed to a specific activity on opioid receptors [20]. Pain in OA is likely to have multiple sources, including subchondral bone marrow lesions, synovium and the periostium as well as soft tissues surrounding the joint, including extra-articular bursae and fat pad. Pain in OA is classified as nociceptive, based on the presence of opioid receptors in the synovial lining and sublining cells. Various molecules present in extracellular space modulate nociceptor sensi‐ tivity by targeting different receptor types. The biological mechanisms responsible for HA analgesic activity have been partially elucidated by Zavan *et al*. [20] in experiments performed with Chinese Hamster Ovary cells that express a panel of opioid receptors. The results demonstrated that HA stimulates the k receptor (KOP), also expressed on fibroblast-like synoviocytes, in a concentration dependent manner, but not the DOP, MOP and NOP receptors. This selective activity could be due to the singular conformational structures of HA compared to morphine, more closely related to dynorphin organization. The pain threshold also increases, due to the direct analgesia through inhibition of pain receptors, and by a direct

At present, HA compounds with different molecular weight (MW) are commercially available. The enhanced diffusion of LMW preparations (0.5-1.5 millions Dalton) through the ECM of the synovium makes possible the interaction with synovial cells [21]. However, because of the modest elastoviscosity of these compounds, compared to native hyaluronan in the synovial fluid, HA preparations with HMW (6-7 millions Dalton), have been developed. These formu‐ lations retain higher amounts of fluid in the articular space using their hydrophilic properties, and also have a greater anti-inflammatory activity, as shown by reduced prostaglandin E 2 and bradykinin concentration attributed to a reduced migration of inflammatory cells [22]. Currently, many types of particulate carriers have been investigated aiming to increase the retention time of the therapeutic agents within the joint capsule. Among them, cationic

Indeed, CD44 decrease in articular cartilage is related to progression of knee OA [16].

regulation of growth factor (GF) signaling.

228 Osteoarthritis - Progress in Basic Research and Treatment

activated T cells, and so regulated the articular milieu [19].

action on synovial nerve endings and stimulation of synovial lining cells.

subchondral bone [18].

Viscosupplementation with HA in knee OA has been approved by the FDA and is recom‐ mended by OARSI for non-severe OA. Guidelines are based on a meta-analysis of randomized saline-controlled trials, including a total of 29 studies representing 4866 unique subjects (IA HA: 2673; IA saline: 2193) [28].

Prospective single or double-blind trials have been done using different types of HA. The number of injections varied from 3 to 5 weekly, with a maximum of 11 in 23 weeks, the doses ranged from 15 to 60 mg, and follow-up periods ranged from 4 weeks to 18 months. Pain outcomes were followed using the Visual Analogic Scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). A minor number of studies evaluated the functional outcomes (WOMAC, Lequesne Index, Range of motion [ROM]), the subjective global assessment and the quality of life of the patients.

HA injection resulted in very large treatment effects between 4 and 26 weeks for knee pain and function compared to pre-injection values. Compared to saline controls, standardized mean difference values with IA HA ranged from -0.38-0.43 for knee pain and +0.32-0.34 for knee function. Clinical changes were similar in the trials where LMW or HMW HA was used. However, the number of injections was lower for HMW preparations, and this is an important advantage for the patients and the clinician. There were no statistically significant differences between IA HA and saline controls in any safety outcomes, including serious adverse events, and study withdrawal. The authors concluded that IA HA injections are safe and effective in patients with symptomatic knee OA [28].

Another meta-analysis [29], comparing the time course of symptoms (the "therapeutic trajectory") in HA and corticosteroid treated patients, highlights that, from baseline to week 4, IA corticosteroids were more effective than HA. By week 4, the two approaches had equal efficacy, but beyond week 8 HA was superior.

It must be noted that, in all trials, some of the patients were non-responders to therapy. Currently, the characteristics of responders have not been clearly identified, but a greater benefit was observed in patients with low grade OA (Kellgren and Lawrence [KL] grade I-II) [9]. This is indirectly confirmed by changes of the serum levels of specific OA biomarkers (Coll2-1 and Coll2-1 NO2) after viscosupplementation. The serum concentrations of these biomarkers were significantly higher in KL grade III/IV patients compared to KL grade I/II patients, and were significantly lower at baseline in responders than in non-responders [30]. Therefore, a rapid decrease of type II collagen degradation and joint inflammation after HA injection supports the utility of serum biomarkers as predictive factors for response to treatment. The role of age in predicting the therapeutic response is debated: some authors report no significant difference among subjects of different ages, whereas others claim that IA joint HA injections are effective in both young and old patients with regard to pain and functional status over a short-term period, but that, in the long term (12 months), the benefit declines rapidly in the elderly subjects [15]. Essentially, advanced OA may be a non-return back irreversible condition.

#### *2.2.2. Hip*

The number of studies about viscosupplementation of hip OA is limited when compared with knee OA. The reasons can be the deeper localization of this joint, and the proximity of femoral vessels and nerves, which makes mandatory performing the injections under imaging control [31]. Moreover, the level of evidence for most of these trials is low, because they are cohort studies and lack of a reference group [9].

In these studies, several HA compounds were used. The number of injections ranged from 1 to 3 for each patients, and only in few cases 4 or 5 injections were performed. In general, the injections number was lower for HMW preparations. The length of treatments and the outcome measures were similar to those used in knee randomized controlled trials. All the trials have shown a reduction of pain, which, in general, becomes evident within 3 months and persists in the following 6, 12 and 18 months. Besides the reduction of pain, also the articular function (Harris Hip Score, WOMAC score, Lequesne Index, and American Academy of Orthopaedic Association Lower Limb Core Scale) was improved [9,15]. Moreover, an improvement of kinematic and kinetic parameters in walking pattern at 6 months (higher cadence, stride length, significant increase for the pelvic tilt at heel contact and for hip flexion-extension moment at loading response sub-phases of gait cycle) has been shown by a recent study [32]. Interestingly, the IA injection of both LMW and HMW HA has been proven efficacious in delaying the total hip replacement in patients affected by symptomatic hip OA [33,34].

A further observation, which confirms the previous data, is the reduction of non steroidal antiinflammatory drugs consumption [35].

#### *2.2.3. Ankle*

HA injection resulted in very large treatment effects between 4 and 26 weeks for knee pain and function compared to pre-injection values. Compared to saline controls, standardized mean difference values with IA HA ranged from -0.38-0.43 for knee pain and +0.32-0.34 for knee function. Clinical changes were similar in the trials where LMW or HMW HA was used. However, the number of injections was lower for HMW preparations, and this is an important advantage for the patients and the clinician. There were no statistically significant differences between IA HA and saline controls in any safety outcomes, including serious adverse events, and study withdrawal. The authors concluded that IA HA injections are safe and effective in

Another meta-analysis [29], comparing the time course of symptoms (the "therapeutic trajectory") in HA and corticosteroid treated patients, highlights that, from baseline to week 4, IA corticosteroids were more effective than HA. By week 4, the two approaches had equal

It must be noted that, in all trials, some of the patients were non-responders to therapy. Currently, the characteristics of responders have not been clearly identified, but a greater benefit was observed in patients with low grade OA (Kellgren and Lawrence [KL] grade I-II) [9]. This is indirectly confirmed by changes of the serum levels of specific OA biomarkers (Coll2-1 and Coll2-1 NO2) after viscosupplementation. The serum concentrations of these biomarkers were significantly higher in KL grade III/IV patients compared to KL grade I/II patients, and were significantly lower at baseline in responders than in non-responders [30]. Therefore, a rapid decrease of type II collagen degradation and joint inflammation after HA injection supports the utility of serum biomarkers as predictive factors for response to treatment. The role of age in predicting the therapeutic response is debated: some authors report no significant difference among subjects of different ages, whereas others claim that IA joint HA injections are effective in both young and old patients with regard to pain and functional status over a short-term period, but that, in the long term (12 months), the benefit declines rapidly in the elderly subjects [15]. Essentially, advanced OA may be a non-return

The number of studies about viscosupplementation of hip OA is limited when compared with knee OA. The reasons can be the deeper localization of this joint, and the proximity of femoral vessels and nerves, which makes mandatory performing the injections under imaging control [31]. Moreover, the level of evidence for most of these trials is low, because they are cohort

In these studies, several HA compounds were used. The number of injections ranged from 1 to 3 for each patients, and only in few cases 4 or 5 injections were performed. In general, the injections number was lower for HMW preparations. The length of treatments and the outcome measures were similar to those used in knee randomized controlled trials. All the trials have shown a reduction of pain, which, in general, becomes evident within 3 months and persists in the following 6, 12 and 18 months. Besides the reduction of pain, also the articular function (Harris Hip Score, WOMAC score, Lequesne Index, and American Academy of Orthopaedic

patients with symptomatic knee OA [28].

230 Osteoarthritis - Progress in Basic Research and Treatment

efficacy, but beyond week 8 HA was superior.

back irreversible condition.

studies and lack of a reference group [9].

*2.2.2. Hip*

In the few studies performed in ankle OA, patients suffering from post-traumatic KL grade II-IV ankle OA were enrolled. Different HA preparations were used, and patients received 1 up to 5 injections. Clinical benefit was evaluated by means of different scales (VAS, Ankle Osteoarthritis Scale, American Orthopaedic Foot and Ankle Society, Short Form-12, Short Form-36, WOMAC), and the follow-up period varied from 6 to 18 months [36].

An improvement in all the outcome measures was reported, with the effect lasting for 18 months. However, it is not clear from reports whether the pain reduction was clinically significant, or could be ascribed only to a placebo effect. In addition, the lack of controls does not allow definitive conclusions on the efficacy of HA.

The level 1 evidence studies are more qualified to assess the therapeutic efficacy, but also these trials show several limitations (e.g., no information on the actual number of potential patients, no clear randomization, imbalance of baseline characteristics between intervention and control groups, statistical weakness), and therefore have to be considered as low quality. The patients treated with HA showed a significant decrease in pain and disability at 6 months, with the effects lasting 12-13 months [37, 38].

Besides the reduction of these parameters, an improvement in ankle sagittal ROMs, and gait quality was observed. However, it must be noted that in any study the authors found difference between HA and controls groups. In particular, in the studies performed by Salk [38], Cohen [39] and DeGroot [37], the patients, treated with a 1-2.5 ml phosphate-buffered saline solution injection, reported a similar improvement in all parameters evaluated. Analogously, positive results were observed in patients, who followed a 6 weeks exercise therapy (muscle strength‐ ening and ankle ROM exercises) [40], and after arthroscopic lavage of OA ankle joint [41].

Recent observations aimed to identify the baseline prognostic factors of outcome have shown that early stage disease and duration of pain less than 1 year are independent predictors associated with higher satisfaction at 3 and 6 months after treatment [42]. However, this assumption has been challenged by Lucas et al. [43], who have observed that viscosupple‐ mentation had a significant positive effect after a very long observation period (45.5 months), and that neither etiology nor severity of OA was predictive of the response.

On the basis of these observations, the efficacy of HA in reducing pain and improving function in ankle OA is still debated. Several factors can explain these discrepancies. Ankle joint, anatomically and functionally, is more complex than other joints, which are usually treated with positive results with HA (hip, knee) [36].

Finally, it must be considered that the majority of studies has been performed blindly and only few under imaging guidance [36]. This can be a valid explanation of several unsatisfactory results, because there is evidence that about one third of IA injections are not delivered into the IA cavity, when performed without a visual aid [44]. At this regard, ankle joint presents many technical difficulties of injecting IA, due to its complex anatomy, further complicated from the OA joint changes.

#### *2.2.4. Shoulder*

HA is effective and well tolerated for the treatment of OA and persistent shoulder pain refractory to other standard non operative interventions. Both 3 and 5 weekly IA injections of LMW HA provide significant improvement in terms of shoulder pain (VAS score on move‐ ment), with the effects lasting 7-26 weeks [9].

Similarly, in a 6 months follow-up study, a significant reduction in VAS pain score was also provided with 3 weekly IA HMW HA (Hylan G-F 20) injections [45]. In addition, most of the patients experienced an improvement in the shoulder function scores (Oxford Shoulder Score and Constant-Murley Score) and in the activities of daily living.

Studies comparing Hylan G-F 20 *versus* 6 methylprednisolone acetate [46] or versus physiotherapy [45] show that HA is effective in reducing pain for up to 6 months, whereas the positive results observed after corticosteroid or physiotherapy have a shorter efficacy and decline after 1 or 2 months. However, these positive results are challenged by a doubleblind, randomized, controlled multicenter trial, where patients who received 3 weekly HA injections did not report any significant difference in term of pain reduction at 26 weeks when compared with placebo [47].

#### *2.2.5. Other joints*

The efficacy of HA has been investigated in the treatment of carpo-metacarpal OA, and positive results have been reported by most of the authors [9]. In particular, an early improvement in VAS score was observed after 2 weeks post treatment, with the effects lasting until 1-3 months. The long term effects of HA were demonstrated only in few studies, in which the pain relief was reported at 6 months [48]. Beside pain reduction, also grip strength improved significantly, although this effect was achieved slowly, with better results observed at 6 months. Moreover, the local inflammation measured by means of Power Doppler exam significantly decreased after 2 weeks of treatment [49].

Similar positive results have been reported in the treatment of temporo-mandibular OA (with or without effusion). The superiority of HA injections (associated or not with arthrocentesis) was shown only against placebo saline injections, whereas outcomes were comparable with those achieved with corticosteroid injections [50-53]. Interestingly, in an experimental model of arthropatic temporo-mandibular joint, El-Hakim and Elyamani [54] found, after repeated IA injections, an increase in the thickness of the cartilagineous layer, suggesting that HA can inhibit the progression of OA changes. A recent study, aiming to identify predictors for treatment efficacy, has shown that only unilateral temporo-mandibular joint OA predicts better the benefit [55], while sex, age, pain duration are not provided of predictive power.

In the treatment of elbow OA the results are inconclusive [9,15]. Positive effects have been observed only in two small studies, while, in a larger study (18 patients), IA HA was not effective in the treatment of post-traumatic OA of the elbow.

Controversial results have been observed also in the treatment of spine OA. Fuchs et al. [56] reported significant pain relief and improved quality of life, also in the long term, in patients affected from facet joints OA with chronic non-radicular pain in the lumbar spine. However, these results are not in agreement with a recent study by Cleary et al. [57], who have not shown any benefit of viscosupplementation in the management of symptomatic lumbar facet OA.

Finally, it is worth of note that the side effects of HA are negligible. In quite all the clinical trials, no general side effects were observed, and only few patients reported a sensation of heaviness and pain in their joint after injection [9]. These effects were more frequent in studies performed in blind conditions compared to those performed under imaging guidance. No differences were observed in relation to HA preparation used or to the number of injections [9]. Side effects usually disappeared after 2-7 days without any therapeutic intervention and did not limit basic or instrumental activities of daily living. Vascular or nervous complications were never reported, neither gout, chondrocalcinosis, sometimes observed after viscosupple‐ mentation of the knee [15]. Septic arthritis or aseptic synovial effusion occurred in a very limited number of cases [58].

#### **3. Platelet rich plasma**

anatomically and functionally, is more complex than other joints, which are usually treated

Finally, it must be considered that the majority of studies has been performed blindly and only few under imaging guidance [36]. This can be a valid explanation of several unsatisfactory results, because there is evidence that about one third of IA injections are not delivered into the IA cavity, when performed without a visual aid [44]. At this regard, ankle joint presents many technical difficulties of injecting IA, due to its complex anatomy, further complicated

HA is effective and well tolerated for the treatment of OA and persistent shoulder pain refractory to other standard non operative interventions. Both 3 and 5 weekly IA injections of LMW HA provide significant improvement in terms of shoulder pain (VAS score on move‐

Similarly, in a 6 months follow-up study, a significant reduction in VAS pain score was also provided with 3 weekly IA HMW HA (Hylan G-F 20) injections [45]. In addition, most of the patients experienced an improvement in the shoulder function scores (Oxford Shoulder Score

Studies comparing Hylan G-F 20 *versus* 6 methylprednisolone acetate [46] or versus physiotherapy [45] show that HA is effective in reducing pain for up to 6 months, whereas the positive results observed after corticosteroid or physiotherapy have a shorter efficacy and decline after 1 or 2 months. However, these positive results are challenged by a doubleblind, randomized, controlled multicenter trial, where patients who received 3 weekly HA injections did not report any significant difference in term of pain reduction at 26 weeks

The efficacy of HA has been investigated in the treatment of carpo-metacarpal OA, and positive results have been reported by most of the authors [9]. In particular, an early improvement in VAS score was observed after 2 weeks post treatment, with the effects lasting until 1-3 months. The long term effects of HA were demonstrated only in few studies, in which the pain relief was reported at 6 months [48]. Beside pain reduction, also grip strength improved significantly, although this effect was achieved slowly, with better results observed at 6 months. Moreover, the local inflammation measured by means of Power Doppler exam significantly decreased

Similar positive results have been reported in the treatment of temporo-mandibular OA (with or without effusion). The superiority of HA injections (associated or not with arthrocentesis) was shown only against placebo saline injections, whereas outcomes were comparable with those achieved with corticosteroid injections [50-53]. Interestingly, in an experimental model of arthropatic temporo-mandibular joint, El-Hakim and Elyamani [54] found, after repeated

with positive results with HA (hip, knee) [36].

232 Osteoarthritis - Progress in Basic Research and Treatment

ment), with the effects lasting 7-26 weeks [9].

when compared with placebo [47].

after 2 weeks of treatment [49].

*2.2.5. Other joints*

and Constant-Murley Score) and in the activities of daily living.

from the OA joint changes.

*2.2.4. Shoulder*

Assuming that tissue repair involves the sequential signaling of multiple factors, and therefore the delivery of a single type of molecule is insufficient, the use of PRP is gaining ground focusing on the concept that the co-delivery of various proteins can break the vicious circle based on failure of the repair process that progressively leads to OA.

Indeed, the PRP therapeutic activity is to release a collection of signaling proteins, including growth factors (GFs) and chemokines, among other proteins, to the joint environment, thereby inducing tissue regeneration mechanisms [59-61]. Briefly, regulatory proteins released from PRP must be capable of interfering with the catabolic microenvironment in OA joints while modulating the inflammatory response, inducing cell migration and proliferation, and regulating angiogenesis and cell differentiation. Since PRPs can have a broad range of functions, it is difficult to decide which function or aspect is the most relevant for OA outcome. A full description of signaling proteins released from PRP and their role in modulating inflammation and vascular pathology have been recently appraised in a personal review [59]. Here we focus on describing the properties of PRP on mechanisms that help in building the rationale for creating a combined HA/PRP treatment.

#### **3.1. Precursor cell migration, proliferation and differentiation**

Precursor cell migration, proliferation and differentiation are intended biological effects theoretically related to the PRP clinical response. Cartilage is composed of post-mitotic cells incapable of proliferation. Therefore, regeneration may be based on migration of mitotic stem cells or their progeny (precursor cells) to the cartilage surface, followed by differentiation and the synthesis of ECM components. However, avascular nature of cartilage hinders migration of circulating stem cells, thus precursor cells identified in other joint tissues such as the synovium or the Hoffa fat, although with distinct differentiation potency, are candidates to repair cartilage defects; alternatively cells can home cartilage lesions by migrating from the subchondral bone when arthroscopic drilling is performed [62].

#### *3.1.1. Cell migration*

PRP exploits the ability of cells to migrate. Actually, by inducing changes in the cell microen‐ vironment, PRP facilitates the motility of BMSCs, Adipose-Derived Stem Cells (ADSCs) and chondrocytes. The ability of PRP to create gradients of GFs and chemokines is based on three central features: first, on the kinetics of release of chemokines from α-granules in platelets, second on the structural and chemical properties of the fibrin scaffold, and finally on the plasmin degradation of the fibrin [63,64].

Platelets release upon activation a huge repertoire of chemokines, GFs and other cytokines prestored in their α-granules. In parallel upon PRP activation, a specialized provisional fibrin network is formed. Fibrin binds several plasma proteins including vitronectin, fibronectin, Von Willebrand factor (vWF), and thrombospondin. Recent research has identified fibronectin as a major factor in human serum to recruit subchondral progenitor cells [65]. Additionally, these proteins within the fibrin bind GFs and form molecular complexes that can dramatically enhance the potency of GFs.

Besides these effects PRP stimulates HA synthesis, as shown by *in vitro* experiments performed on synovial fibroblasts isolated from the synovium of patients with OA undergoing prosthetic surgery, and the newly synthesized HA may help to improve cell motility [66,67].

#### *3.1.2. Cell proliferation*

PRP also supports other mechanisms necessary for cartilage repair (i.e. proliferation) [68]. In fact, PRP has been deeply tested and shown to be a safe and suitable supplement to achieve large scale expansion of MSCs for cell therapy purposes. Moreover, PRP not only supports MSCs proliferation, but it is safer and more effective than fetal bovine serum (the typical serum supplement used to expand cells). However, there is no consensus on which PRP formulation is more proliferative: the PRP releasate or the platelet lysate. The former is the supernatant extruded after PRP coagulation, and it may be considered as a PRP serum; the activation method to achieve granule secretion may introduce variability between products [59,69,70]. Alternatively, the platelet lysate is obtained after several freeze/thaw cycles of either PRP or Leukocyte-PRP (L-PRP) [71-73]*.*

Importantly, studies demonstrate that MSCs expanded in PRP derived formulations maintain pluripotency along the passages [71].

#### *3.1.3. Chondrogenic differentiation*

**3.1. Precursor cell migration, proliferation and differentiation**

234 Osteoarthritis - Progress in Basic Research and Treatment

subchondral bone when arthroscopic drilling is performed [62].

*3.1.1. Cell migration*

plasmin degradation of the fibrin [63,64].

enhance the potency of GFs.

Leukocyte-PRP (L-PRP) [71-73]*.*

*3.1.2. Cell proliferation*

Precursor cell migration, proliferation and differentiation are intended biological effects theoretically related to the PRP clinical response. Cartilage is composed of post-mitotic cells incapable of proliferation. Therefore, regeneration may be based on migration of mitotic stem cells or their progeny (precursor cells) to the cartilage surface, followed by differentiation and the synthesis of ECM components. However, avascular nature of cartilage hinders migration of circulating stem cells, thus precursor cells identified in other joint tissues such as the synovium or the Hoffa fat, although with distinct differentiation potency, are candidates to repair cartilage defects; alternatively cells can home cartilage lesions by migrating from the

PRP exploits the ability of cells to migrate. Actually, by inducing changes in the cell microen‐ vironment, PRP facilitates the motility of BMSCs, Adipose-Derived Stem Cells (ADSCs) and chondrocytes. The ability of PRP to create gradients of GFs and chemokines is based on three central features: first, on the kinetics of release of chemokines from α-granules in platelets, second on the structural and chemical properties of the fibrin scaffold, and finally on the

Platelets release upon activation a huge repertoire of chemokines, GFs and other cytokines prestored in their α-granules. In parallel upon PRP activation, a specialized provisional fibrin network is formed. Fibrin binds several plasma proteins including vitronectin, fibronectin, Von Willebrand factor (vWF), and thrombospondin. Recent research has identified fibronectin as a major factor in human serum to recruit subchondral progenitor cells [65]. Additionally, these proteins within the fibrin bind GFs and form molecular complexes that can dramatically

Besides these effects PRP stimulates HA synthesis, as shown by *in vitro* experiments performed on synovial fibroblasts isolated from the synovium of patients with OA undergoing prosthetic

PRP also supports other mechanisms necessary for cartilage repair (i.e. proliferation) [68]. In fact, PRP has been deeply tested and shown to be a safe and suitable supplement to achieve large scale expansion of MSCs for cell therapy purposes. Moreover, PRP not only supports MSCs proliferation, but it is safer and more effective than fetal bovine serum (the typical serum supplement used to expand cells). However, there is no consensus on which PRP formulation is more proliferative: the PRP releasate or the platelet lysate. The former is the supernatant extruded after PRP coagulation, and it may be considered as a PRP serum; the activation method to achieve granule secretion may introduce variability between products [59,69,70]. Alternatively, the platelet lysate is obtained after several freeze/thaw cycles of either PRP or

surgery, and the newly synthesized HA may help to improve cell motility [66,67].

Differentiation is of paramount importance to cartilage regeneration. Differentiated cells synthesize specific molecules unique to hyaline cartilage ECM, such as type 2 collagen and aggrecans, in adequate proportion conferring exceptional biomechanical properties to the ECM. Noteworthy, an inflammatory synovial fluid hinders the differentiation of human subchondral progenitor cells, decreasing the expression of aggrecan, type 2 collagen and cartilage oligomeric matrix protein [74]. However, PRP stimulates migration and differentia‐ tion of human subchondral cells into chondrocytes highlighting the consequences of manip‐ ulating the biological milieu.

Whether PRP drives the cell to chondrogenic differentiation *in vitro* depends on the precursor cell characteristics along with the culture system (i.e. monolayer culture, micromass, 3-D scaffolds).

Proteomic studies demonstrated that chondrocytes cultured with PRP in either mono- or 3D conditions maintained the chondrocyte phenotype or at least de-differentiation was inhibited after several days in culture [75].

#### **3.2. Clinical trials**

Conservative management of musculoskeletal conditions with PRP is becoming increasingly popular; however, clinical evidence is preliminary and limited. At present, PRP is an investi‐ gational product. However, to advance PRP the International Cellular Medical Society (ICMS) has developed guidelines for the handling and delivery of PRP and safety recommendations. The final goal is to assist physicians in performing safe therapies. Absolute contraindications for PRP administration include platelet dysfunction syndrome, critical thrombocytopenia, hemodynamic instability, septicemia, local infection at the site of the procedure and patient unwilling to accept risks.

OA is one of the most common indications for PRP injections. Most studies focus on knee OA [76-78], but there are two case series on hip OA and a prospective cohort on osteochondral lesions in the ankle [59]. These studies, merely aiming to demonstrate a clinically meaningful result (e.g., pain relief and functional improvement), have used patient-reported outcomes as end points (WOMAC, KOOS, IKDC and VAS). Importantly, clinical studies performed thus far have strongly supported the safety of PRP (no infections, worsened outcomes, or serious complication have been reported). The fact that PRP is not a potentially harmful experimental treatment is corroborated by clinical studies in other conditions and medical fields [60].

Two systematic reviews with quantitative synthesis [79] and meta-analysis [80] have evaluated the efficacy of PRP in the treatment of symptomatic knee OA [79] and the effectiveness of PRP injections for treating knee joint cartilage degenerative pathology [80]. The former study included six controlled studies (level I and II) including a total of 577 patients, 264 patients in the experimental PRP treatment. Pooled data using the WOMAC showed that PRP was better than HA injections. Similarly, pooled IKDC scores favored PRP treatment. The authors concluded that as compared with HA, multiple sequential PRP injections are beneficial for patients with mild to moderate OA at six months follow-up.

The latter study [80] was less restrictive in the inclusion criteria and included eight single-arm studies, three controlled cohorts and five randomized controlled trials with a total of 1543 patients. They compared the PRP group pooled values with the pre-treatment values and the control groups treated with HA or placebo. PRP injections showed continual efficacy up to 12 months. The benefits of PRP lasted more than HA. Injection doses equal or less than 2 lead to uncertainty in the therapeutic effects. Regarding knee severity, stratified analysis showed better results at six months in the degenerative chondropathy group (effect size: 3.90, 95%; CI: 2.54-5.26) compared with advanced OA (effect size: 1.59, 95%; CI: 0.85-2.32).

Similar findings were reported in young active patients with osteochondral lesions in the talus [59], while a comparative study in hip OA failed to show any difference between PRP and HA, after 12 months follow-up [81]. Unfortunately, a comparison between these studies is difficult due to different affected joints, differences between products, protocols and outcome meas‐ ures. Indeed, as pointed out in a recent excellent review [76], several variables can interfere, such as the preparation method, the needle gauge for blood harvest and injection, the platelet concentration, storage, pre-activation and granule secretion, the leukocyte concentration, the anticoagulant and local anesthetic use, the blind or ultrasound-controlled injection, the injection volume and frequency, the disease type and severity, and patient-specific factors. For example, pure PRP and L-PRP formulations are not comparable with each other in terms of leukocyte content, platelet count and plasma volume. Moreover, the platelet and leukocyte concentration of the final L-PRP product can vary by as much as 100% [59]. Whether the differences in the clinical results are secondary to the differences in the formulation requires clarification. Similar improvements in pain and function have been observed in patients treated both with PRP and L-PRP, albeit L-PRP caused more swelling and post-injection pain [60]. It is worth mentioning that in these studies, storage of L-PRP introduces additional variability of the final product [59,60]. Regarding administration procedures, the volume and number of injections is empirically determined for each study. Although most studies involve three injections, the period between the injections is variable, ranging from 1 to 4 weeks. In knee OA, PRP is generally injected into the femoro-tibial compartment, although Sampson [82] injected PRP into the suprapatellar bursa and reported increased cartilage thickness in 6 out of 13 patients (46%). Theoretically, PRP application would be much more efficacious in patients with early post-traumatic OA before the radiographic signs become severe, but this needs further confirmation. In patients with significant irreversible bone and cartilage damage, the effect of PRPs would most likely be less impressive, even so, PRP therapy probably would still improve the patients' quality of life. Whether frequent PRP administration can delay OA progression and replacement surgery in patients with advanced OA may be a plausible hypothesis, but long-term studies using surrogate end points such as WOMAC reduction and refined imaging and biochemical markers potentially predictive of the delay of OA progression are required.

### **4. Hyaluronic acid and platelet rich plasma association**

#### **4.1. PRP+HA: A pericellular bioactive scaffold**

the experimental PRP treatment. Pooled data using the WOMAC showed that PRP was better than HA injections. Similarly, pooled IKDC scores favored PRP treatment. The authors concluded that as compared with HA, multiple sequential PRP injections are beneficial for

The latter study [80] was less restrictive in the inclusion criteria and included eight single-arm studies, three controlled cohorts and five randomized controlled trials with a total of 1543 patients. They compared the PRP group pooled values with the pre-treatment values and the control groups treated with HA or placebo. PRP injections showed continual efficacy up to 12 months. The benefits of PRP lasted more than HA. Injection doses equal or less than 2 lead to uncertainty in the therapeutic effects. Regarding knee severity, stratified analysis showed better results at six months in the degenerative chondropathy group (effect size: 3.90, 95%; CI:

Similar findings were reported in young active patients with osteochondral lesions in the talus [59], while a comparative study in hip OA failed to show any difference between PRP and HA, after 12 months follow-up [81]. Unfortunately, a comparison between these studies is difficult due to different affected joints, differences between products, protocols and outcome meas‐ ures. Indeed, as pointed out in a recent excellent review [76], several variables can interfere, such as the preparation method, the needle gauge for blood harvest and injection, the platelet concentration, storage, pre-activation and granule secretion, the leukocyte concentration, the anticoagulant and local anesthetic use, the blind or ultrasound-controlled injection, the injection volume and frequency, the disease type and severity, and patient-specific factors. For example, pure PRP and L-PRP formulations are not comparable with each other in terms of leukocyte content, platelet count and plasma volume. Moreover, the platelet and leukocyte concentration of the final L-PRP product can vary by as much as 100% [59]. Whether the differences in the clinical results are secondary to the differences in the formulation requires clarification. Similar improvements in pain and function have been observed in patients treated both with PRP and L-PRP, albeit L-PRP caused more swelling and post-injection pain [60]. It is worth mentioning that in these studies, storage of L-PRP introduces additional variability of the final product [59,60]. Regarding administration procedures, the volume and number of injections is empirically determined for each study. Although most studies involve three injections, the period between the injections is variable, ranging from 1 to 4 weeks. In knee OA, PRP is generally injected into the femoro-tibial compartment, although Sampson [82] injected PRP into the suprapatellar bursa and reported increased cartilage thickness in 6 out of 13 patients (46%). Theoretically, PRP application would be much more efficacious in patients with early post-traumatic OA before the radiographic signs become severe, but this needs further confirmation. In patients with significant irreversible bone and cartilage damage, the effect of PRPs would most likely be less impressive, even so, PRP therapy probably would still improve the patients' quality of life. Whether frequent PRP administration can delay OA progression and replacement surgery in patients with advanced OA may be a plausible hypothesis, but long-term studies using surrogate end points such as WOMAC reduction and refined imaging and biochemical markers potentially predictive of the delay of OA progression

2.54-5.26) compared with advanced OA (effect size: 1.59, 95%; CI: 0.85-2.32).

patients with mild to moderate OA at six months follow-up.

236 Osteoarthritis - Progress in Basic Research and Treatment

are required.

It is conceivable that HA facilitates the molecular pool released from PRP to reach the target cells by creating a pericellular bioactive scaffold around the cells. This pericellular matrix may facilitate molecular diffusion and also adequate presentation of cytokines to their transmem‐ brane receptors located in the cytoplasmic membrane of the target competent cell. Moreover, because the synovium is more cellular than the cartilage or the meniscus, and the ECM is looser, the signaling molecules will diffuse and reach the synovial cells easier. In doing so the molecular signaling pool released from PRP will reach mainly synovial cells before being degraded. Therefore molecules released from PRP will reach primarily the synovial cells and change their secretory pattern. The latter are responsible for creating the biological conditions within the joint.

Instead, cartilage has a tightly packed ECM. Although signaling molecules such as cytokines and GFs have low molecular weight, are water-soluble and diffuse easily, the number of cell targeted may be much lower than in the synovium.

Another advantage of HA+PRP is facilitation of cells division and migration as this is a common hallmark of matrices that are rich in HA. This is relevant since MSCs have been identified in the synovium and in the synovial fluid.

Assuming that we target synovial cells and MSCs from the synovial niche, whether HMW or LMW is optimal to be mixed with PRP should be investigated in the laboratory.

#### **4.2. How PRP+HA injections may work?**

Recent basic research supports the idea that HA and PRP treatments can be advantageously associated without altering the original relevant characteristics of both products. Recently, in a co-culture model, involving synovial cells and chondrocytes, Sundman et al. [83] compared the effects of PRP or HA on inflammation, as measured by TNF-α, IL-6 and IL-8 proteins; they found that, although both treatments decreased TNF-α production, IL-6 was decreased only in HA cultures, but not in PRP treated cells, suggesting that both treatments influence inflammation through different mechanisms. The expression of catabolic enzymes such as metalloproteinases was reduced in synoviocytes and chondrocytes treated with PRP but not in cells treated with HA. Moreover, the synoviocytes treated with PRP but not those treated with HA expressed HAS-2. Thus, separately, HA and PRP are beneficial for joint cells although they function through different mechanisms. Therefore, we may infer that their advantages might be additive when both products are injected. Anitua et al. [66] evaluated the potential of pure PRP to induce tendon cells and synovial fibroblasts migration and examined whether the combination of PRP with HA improves their motility *in vitro*. Human fibroblast cells were isolated from synovium and tendon biopsies and cultured by standard procedures. Therefore, PRP was obtained from a young healthy donor and added to the culture medium at different doses. Finally, the migratory capacity induced by PRP, HA, and both in association were tested. PRP stimulated the migration of fibroblasts, as well as HA, but this effect was more prominent when HA was combined with PRP. Indeed, an increase of 335% in motility was observed in the case of HA+PRP treatment compared with HA. Therefore, this *in vitro* study definitely proves that PRP improves the biological properties of HA. CD44 has been implicated in the migratory signal transduction, as well as receptor for HA-mediated motility in several cell lineages. Plasma derived GFs increase the CD44 expression, and this favors cell migration though the interaction of this receptor with extracellular HA. In another study [84], the outgrowth of rabbit chondrocytes from cartilage fragments, loaded onto a composite scaffold made of a HMW HA and autologous PRP, was evaluated. Defects were created by means of a medial arthrotomy, in rabbit knees, and cartilage fragments were collected. Therefore, membrane scaffolds with HA were prepared and cartilage fragments were loaded into the membrane. Finally, the *in vivo* defects were filled with cartilage fragment load HA scaffolds alone, or adding PRP. A histological evaluation at 6 months showed that this latter group had better results, being filled by a repair tissue with some features of hyaline cartilage. This repair tissue was better quality than that of the lesions treated with scaffold only and untreated lesions.

The idea of positive interactions between HA and PRP is supported by an elegant experiment where HA was added to GFs present in platelets. Bone morphogenetic protein (BMP) 2 plays a critical role in the embryologic development of normal cartilage, thus it may enhance reparative processes by setting in motion morphogenetic processes, including the formation of ECM. Unfortunately, the high chondrogenic potency of BMP2 is hampered by its short half life and rapid degradation in vivo. Perlecan/HSPG2, a heparan sulfate proteoglycan, represents an essential component of cartilage ECM. Due to specific receptors Perlecan can act as a depot for BMP2 storage and controlled kinetics, protecting BMP2 from proteolytic cleavage. To avoid its own diffusion and susceptibility to degradation, PlnD1 was immobilized through conju‐ gation to a larger biocompatible carrier forming HA-based microgels (PlnD1-HA) in order to preserve BMP2 activities [85]. The efficiency of this system was tested using an experimental OA model in mice. It was observed that knees treated with PlnD1-HA/BMP2 had lesser damage compared to control knees [86]. Moreover, they had, in comparison to controls treated with Perlecan+HA, higher mRNA levels of type II collagen, proteoglycans, and xylosyltrans‐ ferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycans. In conclusion, this study shows that HA can favor the stabilization of some GFs, enabling their therapeutic potential.

A synergistic anabolic actions of HA and PRP has been also demonstrated in a 3D arthritic neo-cartilage and ACLT-OA model. Indeed, the combination of HA+PRP can synergistically promote cartilage regeneration and inhibit OA inflammation [87].

Currently, no clinical studies support this basic research. There are reports, which claim excellent results of the HA+PRP association in Morton neuroma surgery [88] and in the healing of pressure ulcers and surgical wounds [89,90]. However, these findings need confirmation by controlled trials, because only one study [90] has assessed the superiority of the composite PRP/HA in the treatment of pressure ulcers, in comparison with HA or PRP, used alone.

The ability to understand and control the factors that play a role in the therapeutic effect of HA+PRP shall guide the optimization and design of the combination i.e. optimal ratio, molecular weight of HA, optimal PRP formulation.

#### **5. Conclusions**

when HA was combined with PRP. Indeed, an increase of 335% in motility was observed in the case of HA+PRP treatment compared with HA. Therefore, this *in vitro* study definitely proves that PRP improves the biological properties of HA. CD44 has been implicated in the migratory signal transduction, as well as receptor for HA-mediated motility in several cell lineages. Plasma derived GFs increase the CD44 expression, and this favors cell migration though the interaction of this receptor with extracellular HA. In another study [84], the outgrowth of rabbit chondrocytes from cartilage fragments, loaded onto a composite scaffold made of a HMW HA and autologous PRP, was evaluated. Defects were created by means of a medial arthrotomy, in rabbit knees, and cartilage fragments were collected. Therefore, membrane scaffolds with HA were prepared and cartilage fragments were loaded into the membrane. Finally, the *in vivo* defects were filled with cartilage fragment load HA scaffolds alone, or adding PRP. A histological evaluation at 6 months showed that this latter group had better results, being filled by a repair tissue with some features of hyaline cartilage. This repair tissue was better quality than that of the lesions treated with scaffold only and untreated

The idea of positive interactions between HA and PRP is supported by an elegant experiment where HA was added to GFs present in platelets. Bone morphogenetic protein (BMP) 2 plays a critical role in the embryologic development of normal cartilage, thus it may enhance reparative processes by setting in motion morphogenetic processes, including the formation of ECM. Unfortunately, the high chondrogenic potency of BMP2 is hampered by its short half life and rapid degradation in vivo. Perlecan/HSPG2, a heparan sulfate proteoglycan, represents an essential component of cartilage ECM. Due to specific receptors Perlecan can act as a depot for BMP2 storage and controlled kinetics, protecting BMP2 from proteolytic cleavage. To avoid its own diffusion and susceptibility to degradation, PlnD1 was immobilized through conju‐ gation to a larger biocompatible carrier forming HA-based microgels (PlnD1-HA) in order to preserve BMP2 activities [85]. The efficiency of this system was tested using an experimental OA model in mice. It was observed that knees treated with PlnD1-HA/BMP2 had lesser damage compared to control knees [86]. Moreover, they had, in comparison to controls treated with Perlecan+HA, higher mRNA levels of type II collagen, proteoglycans, and xylosyltrans‐ ferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycans. In conclusion, this study shows that HA can favor the stabilization of some GFs, enabling their

A synergistic anabolic actions of HA and PRP has been also demonstrated in a 3D arthritic neo-cartilage and ACLT-OA model. Indeed, the combination of HA+PRP can synergistically

Currently, no clinical studies support this basic research. There are reports, which claim excellent results of the HA+PRP association in Morton neuroma surgery [88] and in the healing of pressure ulcers and surgical wounds [89,90]. However, these findings need confirmation by controlled trials, because only one study [90] has assessed the superiority of the composite PRP/HA in the treatment of pressure ulcers, in comparison with HA or PRP, used alone.

promote cartilage regeneration and inhibit OA inflammation [87].

lesions.

238 Osteoarthritis - Progress in Basic Research and Treatment

therapeutic potential.

OA is characterized by an impaired regeneration ability mainly attributed to an aberrant cycle of cartilage degradation inducing synovial inflammation and protease activities that in turn induce further joint catabolism. PRP+HA IA injections may break this vicious cycle skewing the milieu towards anti-inflammatory and chondroprotective functions.

It is definitely proved that both HA or PRP alleviate symptoms in patients with mild-moderate OA. Both treatment schedules most often involve repetitive injections, and are comparable in terms of the route of administration and safety.

The concept behind HA application is to mimic the properties of synovial fluid, that is to lubricate the hinge joint. Nevertheless, more recently, the idea that in homeostasis the synovial fluid not only lubricates the joint but provides a positive biological micro-environment has prompted research on HA signaling through hyaladherins located in the cell membrane. Unfortunately, in OA, a deleterious fluidic micro-environment is already established, with the presence of HA fragments, catabolic enzymes, and inflammatory molecules. In this context, the central concept underlying IA injection is to modify this vicious circle. Alternatively, the joint micro-environment can be modified with PRP injections that deliver multiple factors that modulate angiogenesis and inflammation as well as cell anabolism. In fact, the relationships between joint tissues (meniscus, synovium, ligaments, articular surface) and the synovial fluid are bidirectional meaning that they both (the tissues and the synovial fluid) modify and are modified. So, by injecting PRP and HA we aim to replace the ill-fluid with an "engineered fluid" providing lubrication and able to control the delivery and presentation of signaling molecules.

HA and PRP may improve OA symptoms through dissimilar biological mechanisms. Since HA and PRP are not mechanical but biological approaches, the ability of PRP+HA to change the biological status of the joint and promote tissue healing will be particularly critical during the initial stages of OA, before the onset of structural changes. Although mixing HA and PRP involves minimal manipulation, studies to verify critical aspects of the character and perform‐ ance of the composite are mandatory. Several key aspects such as the molecular weight of HA and the concentration to be mixed with PRP should be analyzed. Ideally, HA tertiary structure should let spaces through which molecules can diffuse, and approach the cell membrane to interact closely with their specific receptors. Whether or not HA may help to retain PRP in the joint cavity by exerting osmotic pressure on the joint surface calls for exploration. Considering that the HA chains are constantly moving in solution, and that effective pores in this meshwork will depend on HA concentration and MW, molecules may reach the articular surfaces with different kinetics, depending on their size and hydrodynamic volumes. The future impact of HA+PRP would depend on the capacity of delivering molecules that meet the requirements of the injured joint. An efficient IA therapy would be achieved if modulatory proteins released from PRP+HA are capable of interfering with the catabolic micro-environment while modu‐ lating the inflammatory response, enhancing cell migration and proliferation, and controlling the angiogenic status as well as cell differentiation. A new device capable to mix PRP with HA in prefixed amounts is on the market. Un-published preliminary data suggest that this combination is useful in the treatment of different forms of OA but only prospective random‐ ized double blind studies, preferably using both HA and PRP as comparators (three armed), and a selected stage of OA severity, preferable early OA, will provide sound information about the impact of this novel approach.

#### **Author details**

Michele Abate1 , Isabel Andia2 and Vincenzo Salini1

\*Address all correspondence to: m.abate@unich.it

1 Department of Medicine and Science of Aging, University G. d'Annunzio, Chieti, Italy

2 BioCruces/Cruces University Hospital, Barakaldo, Spain

#### **References**


[6] Roughead EE, Ramsay E, Pratt N, Gilbert, AL. NSAID use in individuals at risk of renal adverse events: an observational study to investigate trends in Australian vet‐ erans. Drug Saf. 2008;31(11) 997-1003.

of the injured joint. An efficient IA therapy would be achieved if modulatory proteins released from PRP+HA are capable of interfering with the catabolic micro-environment while modu‐ lating the inflammatory response, enhancing cell migration and proliferation, and controlling the angiogenic status as well as cell differentiation. A new device capable to mix PRP with HA in prefixed amounts is on the market. Un-published preliminary data suggest that this combination is useful in the treatment of different forms of OA but only prospective random‐ ized double blind studies, preferably using both HA and PRP as comparators (three armed), and a selected stage of OA severity, preferable early OA, will provide sound information about

and Vincenzo Salini1

1 Department of Medicine and Science of Aging, University G. d'Annunzio, Chieti, Italy

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**Chapter 13**

### **Superficial Heat and Cold Applications in the Treatment of Knee Osteoarthritis**

Çalışkan Nurcan and Mevlüde Karadağ

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60534

#### **1. Introduction**

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248 Osteoarthritis - Progress in Basic Research and Treatment

There is information related to the need to perform heat and cold applications as nonpharmacological methods for the pain control in patients with knee osteoarthritis available in the literature. However, the basis of scientific data supporting the therapeutic effect of the superficial heat and cold application in knee osteoarthritis is weak. The purpose of this study is to consider the basic information about the evidence for the superficial heat and cold application in the treatment of knee osteoarthritis.

#### **2. Body**

Heat and cold applications for the purpose of treatment are applied to a part or whole of the body and cause local or systemic effects [1]. In general, the physiological effects of heat are vasodilatation, increased capillary permeability, acceleration of cell metabolism, muscle relaxation, acceleration of inflammation, pain reduction by relaxing muscles, sedative effect, and reducing the viscosity of the synovial fluid to decrease joint stiffness. The physiologi‐ cal effects of cold are generally the opposite of warm effects. The effects of cold are vasoconstriction, a slowdown in cell metabolism, local anesthesia, decrease in blood flow, reduction of the arrival of oxygen and metabolites to the area and the reduction of residuum removal (Table 1) [1-4].

When applying heat to a local and large area of the body, low blood pressure can be seen due to the excessive peripheral vasodilation. This reduction in blood pressure can cause

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

fainting if it is serious. This effect of heat application in individuals with heart, lung or circulatory system diseases, such as arteriosclerosis, develops more frequently than healthy individuals.


**Table 1.** The effects of heat and cold application [4]

The prolonged cold application and vasoconstriction may cause an increase in the blood pressure. Because, the way of blood flow changes towards the internal blood vessels from the surface (cutaneous) due to the vasoconstriction. Shivering is another general effect of long stay in the cold and a response of the body to warm itself [5].

#### **3. Physiological tolerance to heat and cold**

The tolerations to the heat and cold applications of individuals are quite different (See Table 2). In some cases, when performing the heat and cold applications, precautions are required to reduce the risk of injury. Conditions that increase the risk of injury are given in Table 3.


**Table 2.** Factors affecting physiological tolerance to heat and cold [5]

fainting if it is serious. This effect of heat application in individuals with heart, lung or circulatory system diseases, such as arteriosclerosis, develops more frequently than healthy

Vasodilatation Accelerates the transport of nutrients and the removal of

Decrease in blood viscosity. Accelerates the transport of leucocyte and antibody to the

Decrease in blood spasm Reduces the pain caused by muscle relaxation, muscle

Increase in capillary permeability Transition of nutrients and residuum increases

Slowdown in cell metabolism Decreases the oxygen requirement of the tissues

Increase of blood viscosity Increases blood coagulation in injured area

Increase in tissue metabolism Blood flow increases due to the increased local temperature

Vasoconstriction Blood flow to the injured area decreases, edema formation

The prolonged cold application and vasoconstriction may cause an increase in the blood pressure. Because, the way of blood flow changes towards the internal blood vessels from the surface (cutaneous) due to the vasoconstriction. Shivering is another general effect of long stay

The tolerations to the heat and cold applications of individuals are quite different (See Table 2). In some cases, when performing the heat and cold applications, precautions are required to reduce the risk of injury. Conditions that increase the risk of injury are given in Table 3.

the region.

injured area

spasm or stiffness

is prevented, inflammation reduces

the residuum by increasing blood flow to the injured area of the body. It reduces the accumulation of venous blood in

**Heat Application Effects Therapeutic Benefits**

250 Osteoarthritis - Progress in Basic Research and Treatment

**Cold Application Effects Therapeutic Benefits**

Local anesthesia Reduces the localized pain

Decrease the blood spasm Reduce the pain

in the cold and a response of the body to warm itself [5].

**3. Physiological tolerance to heat and cold**

**Table 1.** The effects of heat and cold application [4]

individuals.


**Table 3.** Situations enhancing the ınjury risk in heat and cold applications [4]

#### **4. The adaptation of heat and cold receptors**

Heat and cold receptors (thermo receptors) are attuned to the changes of temperatures. Receptors are initially and strongly stimulated when exposed to sudden temperature changes. This stimulation is strong at the first few seconds, and stimulation continues more slowly during the following half an hour. Thus, receptors are adapted to the new temperature. This adaptation explains why an individual feels very cold when suddenly comes out of a cold environment to a warm room, or vice a versa, why the individual feels so hot when suddenly comes out of a warm environment to a cold room.

An understanding of this adaptation mechanism is very important when heat and cold applications are performed. Patients cannot evaluate the temperature of the heat or cold applications developed right after adaptation. When heat applications are performed, serious burns after performing adaptation can develop. On the contrary, when the cold application is performed, this may result in pain, or blood circulation may be impaired in the area of application [5].

#### **4.1. Rebound phenomenon**

Rebound phenomenon is a situation that is developed by exceeding the maximum time of therapeutic effect in heat and cold applications, and in which an opposite effect of the practice other than the desired effect occurs. The heat generates vasodilation maximum 20-30 minutes. If the application continues more than 30-45 minutes, the congestion occurs in tissue, and the blood vessels react to this case by constricting. The reason of this reaction is unknown. If heat applications further extend, burn risks are likely to occur, since the constricted blood vessels cannot deliver the heat application to the other parts of the body by circulation enough [5].

In cold applications, when the skin temperature falls to 15 o C (60 o F), vasoconstriction gets the maximum level, and vasodilatation starts below 15 o C. In normal conditions, this is a protective mechanism that prevents the body tissues such as the nasal and ear from freezing when exposed to cold. This is due to this mechanism that a person's skin becomes red when walking at cold weather. Heat and cold applications must be completed before a rebound phenomenon initiates [5].

#### **5. Heat and cold applications**

The use of hot and cold applications in medicine history goes back to the ancient times. For example, Hippocrates, in his book titled "Management of Acute Disease ", recommended the application of the hot water -filled caps made of clay or metal for the pain in the costal joints, and to place a soft material between skin and cap to prevent the burns. In addition, he mentioned the dry and heat applications that consisted of a heated corn in the blanket made of wool [6].Different types of materials related to general and local heat and cold applications have been produced by the medical technology since Hippocrates.

Patients should be considered before performing heat or cold applications. The application area should be checked for the tolerance of application, skin integrity of the patient, bleeding, and circulatory disorder, and the information about the application to the patient should be provided. The conditions in which heat application must be avoided are shown in Box 1.

#### **Box 1. The conditions in which heat and cold applications must be avoided:**

#### **The conditions in which heat applications must be avoided [5]:**

**4. The adaptation of heat and cold receptors**

252 Osteoarthritis - Progress in Basic Research and Treatment

comes out of a warm environment to a cold room.

In cold applications, when the skin temperature falls to 15 o

have been produced by the medical technology since Hippocrates.

maximum level, and vasodilatation starts below 15 o

**5. Heat and cold applications**

application [5].

initiates [5].

**4.1. Rebound phenomenon**

Heat and cold receptors (thermo receptors) are attuned to the changes of temperatures. Receptors are initially and strongly stimulated when exposed to sudden temperature changes. This stimulation is strong at the first few seconds, and stimulation continues more slowly during the following half an hour. Thus, receptors are adapted to the new temperature. This adaptation explains why an individual feels very cold when suddenly comes out of a cold environment to a warm room, or vice a versa, why the individual feels so hot when suddenly

An understanding of this adaptation mechanism is very important when heat and cold applications are performed. Patients cannot evaluate the temperature of the heat or cold applications developed right after adaptation. When heat applications are performed, serious burns after performing adaptation can develop. On the contrary, when the cold application is performed, this may result in pain, or blood circulation may be impaired in the area of

Rebound phenomenon is a situation that is developed by exceeding the maximum time of therapeutic effect in heat and cold applications, and in which an opposite effect of the practice other than the desired effect occurs. The heat generates vasodilation maximum 20-30 minutes. If the application continues more than 30-45 minutes, the congestion occurs in tissue, and the blood vessels react to this case by constricting. The reason of this reaction is unknown. If heat applications further extend, burn risks are likely to occur, since the constricted blood vessels cannot deliver the heat application to the other parts of the body by circulation enough [5].

mechanism that prevents the body tissues such as the nasal and ear from freezing when exposed to cold. This is due to this mechanism that a person's skin becomes red when walking at cold weather. Heat and cold applications must be completed before a rebound phenomenon

The use of hot and cold applications in medicine history goes back to the ancient times. For example, Hippocrates, in his book titled "Management of Acute Disease ", recommended the application of the hot water -filled caps made of clay or metal for the pain in the costal joints, and to place a soft material between skin and cap to prevent the burns. In addition, he mentioned the dry and heat applications that consisted of a heated corn in the blanket made of wool [6].Different types of materials related to general and local heat and cold applications

C (60 o

F), vasoconstriction gets the

C. In normal conditions, this is a protective


#### **The conditions in which cold applications must be avoided:**


#### **6. Selecting wet or dry applications**

Heat and cold applications can be applied in two ways: dry and wet. The type of wound or injury, the part of the body that application will be carried out, and the presence of inflam‐ mation are the factors affecting the choice of dry or wet application (Table 4).

Multiple treatment options are available for patients with OA of the knee including the use of superficial heat or cold, transcutaneous electrical nerve stimulation (TENS), oral medications, the injection of hyaluronic acid or a corticosteroid, or ultimately knee joint replacement surgery. Common ways of superficial heat-cold application are thermoforms, electrical heating pads, aquathermia pads, hot-cold packs, hot water bath, ice bags or collars, and warm – cold soaks, combined cold compression system [1, 3 -5, 7-10].


**Table 4.** Selecting Wet or Dry Application[4]

#### **6.1. Thermophore**

Thermophore (hot water bag) is widely used, especially for dry heat application at home. It is cheap and easy to use, but there are some disadvantages. Leaking of the bag of hot water or using the bag in appropriate circumstances can cause burns. The temperature of the water in adults and children 2 years of age should be 46-52 °C (115-125 o F). The temperature of the water in unconscious, weak persons and the children under the age of 2 should be 40.5 - 46 °C (105-115 o F) [5, 9].

#### **6.2. Aquathermic pad (Pillow)**

Aquatermic or aquamatic (also named K-pad) pads consist of tubes that provide water circulation inside (See Figure 1). Some pads are water-resistant, and some pads have absorbent surface that allows applying wet application. There are various sizes of pads depending on the body parts to be treated. Pads should be applied to the skin in a cover. If pads are directly applied to the skin, they can cause burns. Patients should never lie down on the pad and should be observed for the signs of skin burns during application. While aquatermic pad is applied, the temperature should be 40.5 - 43 ° C (105-110 o F) for adults, and the application period is usually 20 - 30 minutes [4, 5].

#### **6.3. Electric heating pad**

Electric Heating Pads are utilized for the purpose of local heat application. They are easy to use, relatively reliable, and provide warmth of the same value and can easily get shape of the body part they are applied to. There are a variety of sizes depending on the body area to be treated. There are water resistant covers to make application to the wet dressing in some models [1, 5].

**Figure 1.** Aquatermic pad [1]

**Advantages Disadvantages**

*Wet applications*

term application.

heat applications.

*Dry applications*

Thermophore (hot water bag) is widely used, especially for dry heat application at home. It is cheap and easy to use, but there are some disadvantages. Leaking of the bag of hot water or using the bag in appropriate circumstances can cause burns. The temperature of

temperature of the water in unconscious, weak persons and the children under the age of

Aquatermic or aquamatic (also named K-pad) pads consist of tubes that provide water circulation inside (See Figure 1). Some pads are water-resistant, and some pads have absorbent surface that allows applying wet application. There are various sizes of pads depending on the body parts to be treated. Pads should be applied to the skin in a cover. If pads are directly applied to the skin, they can cause burns. Patients should never lie down on the pad and should be observed for the signs of skin burns during application. While aquatermic pad is applied,

Electric Heating Pads are utilized for the purpose of local heat application. They are easy to use, relatively reliable, and provide warmth of the same value and can easily get shape of the body part they are applied to. There are a variety of sizes depending on the body area to be treated. There are water resistant covers to make application to the wet dressing in some

the water in adults and children 2 years of age should be 46-52 °C (115-125 o

F) [5, 9].

Softening of the skin (maceration) occurs in the long-

Quick cooling appears due to the evaporation in wet

Burn risk is high because moisture transmits the

Dry heat increases fluid loss by perspiration. Dry application is not effective in deep tissues. Dry heat can cause drying of the skin.

F) for adults, and the application period is

F). The

temperature in wet heat applications.

Wet applicationprevents drying of the skin and softens the

Wet compresses are suitable for many part of the body.

254 Osteoarthritis - Progress in Basic Research and Treatment

Slightly wet and heat application does not increase the

Dry heat application has less risk of burns than Wet heat

Dry application does not cause the skin softening (maceration). Temperature is retained longer because there is no evaporation

Wet heat applications affect the deep tissues.

perspiration and insensible fluid loss.

of water in dry heat application.

**6.1. Thermophore**

**Table 4.** Selecting Wet or Dry Application[4]

2 should be 40.5 - 46 °C (105-115 o

the temperature should be 40.5 - 43 ° C (105-110 o

**6.2. Aquathermic pad (Pillow)**

usually 20 - 30 minutes [4, 5].

**6.3. Electric heating pad**

models [1, 5].

*Wet applications*

wound exudates.

*Dry applications*

application.

Electric heating pads provide as much temperature as the heat packs with silicondioxyte (hot packs) do. They do not require reheating between the applications, and can be used more than 20 minutes. Furthermore, it has been emphasized that electric heating pads are more suitable for the home use [11].

There are some rules that should be attended by the practitioner when electric heating pad application is performed, otherwise serious burns can occur. These rules are;


The utilization of electric heating pad is limited in hospitals due to the security problems caused by not paying attention to these rules in crowded places such as hospitals [1, 12].

#### **6.4. Hot – Cold pack**

Hot - cold packs are used for heat or cold applications and are the bags filled with the silicate gel (Figure 2). The use and preparation of hot and cold packs varies according to the manu‐ facturing company. Therefore, the preparation of the pack and the application time must done as specified in the user guide. The pack should be prepared by waiting in hot water or microwave if it will be used for the purpose of heat application. Otherwise, the pack should be prepared by waiting in the fridge at least 1.5 hours if it will be used for the purpose of cold application. When disposable quick hot or cold packs are pressurized, the chemical mixture in the package and the heat are released and warming / cooling begins. These packages cannot be used again. These packages should not be tightened, and should be protected from kneaded and shock until the time of use [4, 5, 13].

Hot packs are used for the purpose of dry heat applications, however there are some varieties that are used for wet heat applications. These packages are made of impermeable fabric, and there are special heating units to heat (Figure 3). Silica gel in the fabric bag swells by absorbing large amounts of heat and water. Therefore, the extra water must be filtered; dry towel should be wrapped to the package, and the package should not be placed under the patients, but should be placed over the patients [10, 14].

Hot - cold packs are also utilized by wrapping in a wet towel. Water-resistant treatment cloth or plastic wrap should be used during application to prevent the loss of temperature as in the compressed application [5]. Steps for the application of hot packs:

**Figure 2.** Hot Packs A. Commercial Hot pack B. Single Use Hot pack

**Figure 3.** Hot Pack (used for wet heat applications)[13]

#### **6.5. Hot compress**

**6.4. Hot – Cold pack**

and shock until the time of use [4, 5, 13].

256 Osteoarthritis - Progress in Basic Research and Treatment

should be placed over the patients [10, 14].

compressed application [5]. Steps for the application of hot packs:

**Figure 2.** Hot Packs A. Commercial Hot pack B. Single Use Hot pack

Hot - cold packs are used for heat or cold applications and are the bags filled with the silicate gel (Figure 2). The use and preparation of hot and cold packs varies according to the manu‐ facturing company. Therefore, the preparation of the pack and the application time must done as specified in the user guide. The pack should be prepared by waiting in hot water or microwave if it will be used for the purpose of heat application. Otherwise, the pack should be prepared by waiting in the fridge at least 1.5 hours if it will be used for the purpose of cold application. When disposable quick hot or cold packs are pressurized, the chemical mixture in the package and the heat are released and warming / cooling begins. These packages cannot be used again. These packages should not be tightened, and should be protected from kneaded

Hot packs are used for the purpose of dry heat applications, however there are some varieties that are used for wet heat applications. These packages are made of impermeable fabric, and there are special heating units to heat (Figure 3). Silica gel in the fabric bag swells by absorbing large amounts of heat and water. Therefore, the extra water must be filtered; dry towel should be wrapped to the package, and the package should not be placed under the patients, but

Hot - cold packs are also utilized by wrapping in a wet towel. Water-resistant treatment cloth or plastic wrap should be used during application to prevent the loss of temperature as in the

(a) (b)

Sterile wet hot compress improves circulation in open wounds, helps to solve the edema, provides drainage and prevents the spread of infections. The temperature of the water in the hot compress applications varies according to the purpose, and it is sufficient to be 40.5 - 43 ° C (105-110 o F). During the applications heat disperses quickly. Compresses should be changed frequently for the purpose of keeping the temperature at the same level, and compresses should be wrapped with a plastic cover or a dry towel. Wetness causes Vasodilatation and evaporates the heat from the skin's surface; therefore the patient may feel cold. The room temperature should be controlled [4, 5, 9].

#### **6.6. Hot immersion bath**

Hot immersion bath is performed by immersing a part of the body in a heated solution. Hot immersion bath accelerates the circulation, decreases the edema, and provides muscle relaxation. Immersion bath can also be applied to an area that is closed by dressing, and the bath is available with a heated solution. The position of the patient should be comfortable during the application and the solution should be heated up to 40.5 – 43 o C (105 - 110 o F) [4]. Surgical asepsis principles must be complied if the bath is applied with an open wound [5].

#### **6.7. Sitz bath**

Sitz bath is an operation by immersing the pelvic part into hot water or in some cases cold water. Sitz bath is useful in patients with painful hemorrhoids, vaginal inflammation, a history of rectal surgery, and opened episiotomy (incision made between the vagina and rectum to facilitate the birth and to prevent the tearing of the vagina) during childbirth. Application should take 15 - 20 minutes and the temperature of the water should be 40.5 - 43 ° C (105-110 o F). However, the time and temperature vary depending on the patient's health status. The patient sits in a tub or a special chair. A large part of the body is exposed to heat during the sitz bath so that the patient should be evaluated in terms of changes in consciousness, nausea, pallor in face, and increase in patient pulse rate [4, 5].

#### **6.8. Cold compress**

The application should take 20 minutes to reduce the edema and inflammation and the temperature should be 15 o C (59 o F). Surgical aseptic technique should be used for open wounds. When a cold compress is applied, some unwanted side effects such as burning or numbness, mottling of the skin, redness, extreme pallor, and bluish - purple mottled appear‐ ance on the skin can be observed. The procedure for the application of cold compress are the same as hot compress applications [4].

#### **6.9. Cold immersion bath**

The procedure for application of cold immersion bath are the same as hot immersion bath applications. The application should take 20 minutes and the temperature should be 15 o C (59 o F) [4].

#### **6.10. Ice bag**

Ice bag is made of plastic or rubber, and its mouth is wide enough to contain ice. The ice bag is used for sprains, localized bleeding or hematoma, preventing edema after dental surgery, controlling the bleeding, and creating a local anesthetic effect in the area [4]. An ice bag must be applied by wrapping with a towel or blanket [5].

#### **6.11. Combined cold compress system**

Combined Cold Compress System is capable of performing both compression and cold application. It consists of cuffs in the appropriate size of each body region, a refrigerator that is placed in into ice-water, a connecting pipe that provides ice water flows to cuff (Figure 4). Compression allows conductance by increasing the contact between the skin and ice water and reduces blood flow. Thus, it has been indicated that the co-administration of cold with compression in the therapy is more effective than only implementing cold. Combined Cold Compress System is preferred in the control of postoperative pain and the swelling after acute trauma [15].

**Figure 4.** Combined Cold Compress System

**6.7. Sitz bath**


**6.8. Cold compress**

temperature should be 15 o

**6.9. Cold immersion bath**

(59 o

F) [4].

**6.10. Ice bag**

trauma [15].

same as hot compress applications [4].

258 Osteoarthritis - Progress in Basic Research and Treatment

be applied by wrapping with a towel or blanket [5].

**6.11. Combined cold compress system**

Sitz bath is an operation by immersing the pelvic part into hot water or in some cases cold water. Sitz bath is useful in patients with painful hemorrhoids, vaginal inflammation, a history of rectal surgery, and opened episiotomy (incision made between the vagina and rectum to facilitate the birth and to prevent the tearing of the vagina) during childbirth. Application should take 15 - 20 minutes and the temperature of the water should be 40.5

health status. The patient sits in a tub or a special chair. A large part of the body is exposed to heat during the sitz bath so that the patient should be evaluated in terms of changes in

The application should take 20 minutes to reduce the edema and inflammation and the

wounds. When a cold compress is applied, some unwanted side effects such as burning or numbness, mottling of the skin, redness, extreme pallor, and bluish - purple mottled appear‐ ance on the skin can be observed. The procedure for the application of cold compress are the

The procedure for application of cold immersion bath are the same as hot immersion bath applications. The application should take 20 minutes and the temperature should be 15 o

Ice bag is made of plastic or rubber, and its mouth is wide enough to contain ice. The ice bag is used for sprains, localized bleeding or hematoma, preventing edema after dental surgery, controlling the bleeding, and creating a local anesthetic effect in the area [4]. An ice bag must

Combined Cold Compress System is capable of performing both compression and cold application. It consists of cuffs in the appropriate size of each body region, a refrigerator that is placed in into ice-water, a connecting pipe that provides ice water flows to cuff (Figure 4). Compression allows conductance by increasing the contact between the skin and ice water and reduces blood flow. Thus, it has been indicated that the co-administration of cold with compression in the therapy is more effective than only implementing cold. Combined Cold Compress System is preferred in the control of postoperative pain and the swelling after acute

consciousness, nausea, pallor in face, and increase in patient pulse rate [4, 5].

C (59 o

F). However, the time and temperature vary depending on the patient's

F). Surgical aseptic technique should be used for open

C

### **7. Superficial heat and cold application in the treatment of knee osteoarthritis: Is the evidence enough?**

Rakel and Barr (2003) have stated that nurses/physicians, traditionally apply heat and cold applications and some massages, thus they should be informed about the strength of the evidence for the efficiency of these applications. The authors have expressed that the meth‐ odological evidence for the effect of heat and cold applications on chronic pain is limited [16]. Wright and Sluka (2001) have claimed that the information about the effect of superficial heat application on depressing the pain or improving the physical function are contradictory, and that there are different studies stating that heat application increased, decreased or did not change arthritis pain and other symptoms [17].

Brosseau et al. (2003) investigated the literature in The Cochrane Library for the purpose of determining the effectiveness of heat and cold applications in knee OA, and they found 3 randomized controlled trial involving 179 patients. In one of these studies, ice massage did not have a significant impact on the increase the quadriceps muscle strength (29% relative difference) for 20 minutes for 5 times a week, totally for three weeks compared with the clinical control group. However, ice massage in patients with knee OA was statistically significant different compared with the control group in physical function in and ROM. The role of ice in reducing the pain is uncertain. In another study, ice packs were applied to three days a week for three weeks and there was no positive effect of the treatment compared with the control group. It was suggested that cold packs could be used for reducing knee edema. Heat application had no significant effect compared with the control group or alternative application. Hot pack had no effect on edema compared with placebo or cold packs. The authors stated that a greater number of participants and wellplanned researches should be required to determine heat and cold applications in the treatment of knee OA [18]. Jamtvedt et al. (2008) indicated that the above-mentioned studies had small samples and low quality of the work. Furthermore, the authors concluded that the effects of heat and cold applications were unclear [19].

Yıldırım et al. (2010) studied the effect of superficial local heat application that was performed with digital moist heating pad for 20 minutes every other day for 4 weeks. The results showed that heat application reduce pain and increase physical function in patients with knee OA, however it had no effect on stiffness. In this study, patients were followed for routine treatment plan suggested by the physician [20].

Mazzuca et al. (2004) investigated the comparison of warm maintaining kneepad and an elastic kneepad made of cotton. The difference between them was insignificant. The patients in this study continued to pharmacological treatment [21].

Shereif and Hassa (2011) determined that when therapeutic exercise and heat application performed together, pain and stiffness reduced, and physical function improved. However, objective measurement criteria were not used in the evaluation. The patients in this study continued to pharmacological treatment, as well [22].

According to Brandt (1998), although the effect of heat or cold application was not wellresearched, approximately 60% of patients diagnosed with rheumatoid arthritis (RA) and OA preferred heat application on their aching joints, while 20% of the patients preferred cold application [23]. Veitiene & Tamulaitiene (2005) studied the applications preferred by the patients diagnosed with OA and RA and benefits obtained. They found that patients with OA specified exercise (55 7%), the use of assistive devices like walking sticks and walkers (29 6%), and heat application (25 9%) as the most effective; splint (3 7%), resting (3 7%) and joint protection (3 7%) as the least effective; and cold application (0%), not effective at all [24].

Davis and Atwood (1996) stated that the current knowledge on the therapeutic benefits of heat application is insufficient and the expected benefit from heat application is low in conditions of severe pain, but in their study with 82 patients with RA and OA, 70% expressed that they applied heat application [25].

Chandler et al. (2002) have found limited scientific evidence for the analgesic effects of heat although heat has been used to relieve pain for years. They attributed this to the lack of wellorganized studies [12]. Similarly, Öneş et al. (2006) have mentioned that although heat application has been used extensively on outpatient treatment, the randomized controlled studies were limited in number and were not scientifically conducted [26].

As a result of the meta-analysis study by Philadelphia Panel (2001), which was randomized controlled and depended on the findings of observation studies; it was found that no evidence was present neither for the usage / not usage nor for the clinical usefulness of the physical rehabilitation applications like heat, cold, ultrasound, and massage in practical life [27].

The responsibility for providing better quality care requires decisions based on evidence. Evidence-based applications are important for the results such as improving quality of care and care outcomes, making a difference in the results in clinical practices and patient care results, and standardizing the care [28]. Application guidance is directive for the evidencebased practice. Osteoarthritis Research Society International (OARSI), which is the most comprehensive guide published on hip and knee OA treatment so far, has referred to the study by Brosseau et al. in 2003 as the first systematic review published regarding heat and cold applications [18]. Kirazlı (2011) stated that research evidences supporting the treatment of localized heat and cold applications were less, however this treatment method was widely used by the patients with OA, and it was recommended as a simple and reliable method for the relief of pain in most guidelines [29].

#### **8. Conclusion**

Brosseau et al. (2003) investigated the literature in The Cochrane Library for the purpose of determining the effectiveness of heat and cold applications in knee OA, and they found 3 randomized controlled trial involving 179 patients. In one of these studies, ice massage did not have a significant impact on the increase the quadriceps muscle strength (29% relative difference) for 20 minutes for 5 times a week, totally for three weeks compared with the clinical control group. However, ice massage in patients with knee OA was statistically significant different compared with the control group in physical function in and ROM. The role of ice in reducing the pain is uncertain. In another study, ice packs were applied to three days a week for three weeks and there was no positive effect of the treatment compared with the control group. It was suggested that cold packs could be used for reducing knee edema. Heat application had no significant effect compared with the control group or alternative application. Hot pack had no effect on edema compared with placebo or cold packs. The authors stated that a greater number of participants and wellplanned researches should be required to determine heat and cold applications in the treatment of knee OA [18]. Jamtvedt et al. (2008) indicated that the above-mentioned studies had small samples and low quality of the work. Furthermore, the authors concluded that

Yıldırım et al. (2010) studied the effect of superficial local heat application that was performed with digital moist heating pad for 20 minutes every other day for 4 weeks. The results showed that heat application reduce pain and increase physical function in patients with knee OA, however it had no effect on stiffness. In this study, patients were followed for routine treatment

Mazzuca et al. (2004) investigated the comparison of warm maintaining kneepad and an elastic kneepad made of cotton. The difference between them was insignificant. The patients in this

Shereif and Hassa (2011) determined that when therapeutic exercise and heat application performed together, pain and stiffness reduced, and physical function improved. However, objective measurement criteria were not used in the evaluation. The patients in this study

According to Brandt (1998), although the effect of heat or cold application was not wellresearched, approximately 60% of patients diagnosed with rheumatoid arthritis (RA) and OA preferred heat application on their aching joints, while 20% of the patients preferred cold application [23]. Veitiene & Tamulaitiene (2005) studied the applications preferred by the patients diagnosed with OA and RA and benefits obtained. They found that patients with OA specified exercise (55 7%), the use of assistive devices like walking sticks and walkers (29 6%), and heat application (25 9%) as the most effective; splint (3 7%), resting (3 7%) and joint protection (3 7%) as the least effective; and cold application (0%), not effective at all [24].

Davis and Atwood (1996) stated that the current knowledge on the therapeutic benefits of heat application is insufficient and the expected benefit from heat application is low in conditions of severe pain, but in their study with 82 patients with RA and OA, 70% expressed that they

the effects of heat and cold applications were unclear [19].

plan suggested by the physician [20].

260 Osteoarthritis - Progress in Basic Research and Treatment

applied heat application [25].

study continued to pharmacological treatment [21].

continued to pharmacological treatment, as well [22].

As discussed above, there is some information in the literature that heat and cold applications should be superficially performed in patients with knee OA as a non-pharmacological method for controlling pain. However, the basis of scientific data that supports heat and cold applica‐ tions could be effective on chronic pains such as the knee OA is weak [16, 30]. Today, although the need for quality care based on evidence exists, the existing studies on the effectiveness of heat and cold applications on knee OA are not sufficient to cover it. As a result, in spite of the need for further research to determine the effectiveness of the superficial heat and cold applications, superficial heat and cold applications are available to help treatment due to the presence of very few side effects, ease of implementation, and their non-invasiveness [31].

#### **Author details**

Çalışkan Nurcan\* and Mevlüde Karadağ

\*Address all correspondence to: nurcany@gazi.edu.tr

Gazi University, Faculty of Health Sciences, Nursing Department, Ankara, Turkey

#### **References**


[14] Delisa J.A., (2007) Terapötik Fiziksel Ajanlar, Fiziksel Tıp ve Rehabilitasyon İlkeler ve Uygulamalar. In:T. Arasıl (Ed.) Ankara:Güneş Tıp Kitabevleri: In Tech;2007.

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262 Osteoarthritis - Progress in Basic Research and Treatment

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[2] Berman A., Synder S., Kozier B., Erb G. Heat And Cold Measures techniques, Kozier and Erb's Techniques in Clinical Nursing. (5th ed.), New Jersey: Prentice Hall: In

[3] Çalışkan N. Sıcak – Soğuk Uygulamalar In: Atabek Aştı T., Karadağ A. (ed.) Hemşir‐ elik Esasları Hemşirelik Bilimi ve Sanatı. Akademi Basın ve Yayıncılık, İstanbul: In

[4] Potter PA., Perry AG. Fundamentals of Nursing. (7th ed.), St. Louis: Mosby Elsevier:

[5] Berman A., Synder S., Kozier B., Erb G. Kozier and Erb's Fundamentals of Nursing.

[6] Smith RP. Heat therapy: the next hot topic. The Female Patient, 2003;27:25–31, http:// www.femalepatient.com/html/arc/sel/march03/028\_03\_030.asp (accessed 10 August

[7] McHugh GA., Silman AJ., Luker KA. Quality of care for people with osteoarthritis: a

[8] Kozier B., Erb G., Berman AJ., Snyder SJ. Fundamentals of Nursing Concepts, Proc‐ ess, and Practice, seventh edition, New Jersey, Prentice Hall Health: In Tech;2004.

[9] Evans-Smith P. Taylor's Clinical Nursing Skills. Philadelphia: Lippincott Williams &

[10] Öztürk C., Akşit R. Tedavide Sıcak ve Soğuk. Tıbbi Rehabilitasyon. In: Oğuz H., Dur‐

[11] Wood C., Dry and moist heat application and the subsequent rise in tissue tempera‐ tures. https://orca.byu.edu/content/jug/2002 reports/ \_hhp /wood.pdf (accessed 01

[12] Chandler A., Preece J., Lister, S. Using heat therapy for pain management, Nursing

[13] Rosdahl C., MaKowalski MT. Heat and Cold Applications. Textbook of Basic Nurs‐ ing, (9th ed.), Philadelphia: Lippincott Williams & Wilkins: In Tech;2008. http:// books.google.com.tr/books?id=tB1YiYj\_kdkC&printsec=frontcover&dq=Textbook+of +Basic+Nursing+book&source=bl&ots=xBPJxx6NLx&sig=UYqzZ3aRbT-1YCnpN‐ XuBJCo71tk&hl=tr&ei=FXBqTJu5K9fPjAfV6bl2&sa=X&oi=book\_result&ct=result&re‐ snum=3&ved=0CCwQ6AEwAg#v=onepage&q&f=false (accessed 01 October 2010).

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[30] Hanada EY. Efficacy of rehabilitative therapy in regional musculoskeletal conditions.

[31] Çalışkan N. Diz Osteoartriti Tedavisinde Yüzeysel Sıcak Soğuk Uygulama: Kanıtlar

Yeterli mi? Journal of Contemporary Medicine 2013;3(2):144-147.

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264 Osteoarthritis - Progress in Basic Research and Treatment

### *Edited by Qian Chen*

The most common form of arthritis is osteoarthritis (OA), which most often affects the hip, knee, foot and hand. The degeneration of joint cartilage and changes in underlying bone and supporting tissues such as ligament leads to pain, stiffness, movement problems and activity limitations. This book, containing three major sections in OA research and therapy, is an update of the book Osteoarthritis - Diagnosis, Treatment and Surgery published by InTech in 2012. The authors are experts in the osteoarthritis field, which include biologists, bioengineers, clinicians, and health professionals. The scientific content of the book will be beneficial to patients, students, researchers, educators, physicians, and health care providers who are interested in the recent progress in osteoarthritis research and therapy.

Osteoarthritis - Progress in Basic Research and Treatment

Osteoarthritis

Progress in Basic Research and Treatment

*Edited by Qian Chen*

Photo by stockdevil / iStock