Applications of Both Physicochemical Properties and Cell Biological Functions of Hyaluronan

Cure Arthritis Naturally

Cure Arthritis Naturally

Get Instant Access

A. Osteoarthritis

A large number of osteoarthritis (OA) patients have already been treated with HA preparations after it was put into clinical practice around the world. The use of HA for the treatment of OA is based on the concept of Balazs and Denlinger (22). Several studies have shown that HA suppresses cartilage degeneration and reduces the release of proteoglycans from the extracellular matrix in cartilage tissue. It also protects the surface of articular cartilage (23,24), normalizes the properties of synovial fluid (25) and reduces pain perception (26,27).

HA influences physical properties such as viscoelasticity and/or lubrication of the stroma. Therefore, HA of a higher molecular weight is considered to be more beneficial for OA treatment because the efficacy of HA as a shock absorber to protect joint tissues is dependent on its average molecular weight. Kikuchi et al. (28) have shown that higher average molecular weight HA (2000 X 103) suppressed cartilage damage in a rabbit anterior cruciate ligament transection (ACLT) model for OA better than a lower average molecular weight preparation (900 X 103). In contrast, Ghosh et al. (29) have shown that the lower average molecular weight HA better protected articular cartilage from damage in a sheep medial meniscectomy model than did HA with the higher average weight. Taken together, the molecular weight-dependent effects of HA vary among experimental models, and the effects of HA on articular cartilage cannot be explained only by molecular weight, i.e., viscoelasticity.

The effects of an HA preparation HA84 (average MW 840 X 103) were compared to HA230 (average MW 2300 X 103) using a canine ACLT model for OA (25). In this model, proliferation and degeneration of synovial cells and increased synovial fluid volume were observed. However, cartilage damage was not detected. These synovial changes were more significantly suppressed in the group treated with HA84 than in those treated with HA230. The exposure of HA to synovial cells was determined by examining the distribution of fluorescein-labeled HA84 or HA230. Many fluorescein particles were scattered in the synovial lining layers, in most cases in the HA84-treated group. In contrast, there were only a few granules of fluorescein in the same location for the HA230-treated group. Thus, HA84 was exposed to synovial cells more effectively than HA230. In addition, we examined the distribution of fluorescein-labeled HA230 or HA90 (average MW 900 X 10 ) in synovial tissues of 12-month-old guinea pigs 3, 12, 24 h, 3 and 7 days after intra-articular injections of them. It is well known that OA naturally occurs in old Hartley strain guinea pigs (30). Laser-scan microscopy showed that the fluorescein-labeled HA90 was more widely distributed than the fluorescein-labeled HA230 in the synovial tissues at all time points (Fig. 3). HA230 cannot immediately permeate the synovial lining layers, probably because of strong entanglement of the HA230 molecules as well as its larger molecular size. This may be due to the network formation of HA depending on the molecular size (31). These events described above suggest that HA access to synovial cells is inversely dependent on molecular size in vivo (Fig. 4).

Prostaglandin E2 (PGE2) production is up-regulated, and proliferation of synovial cells occurs in the synovium of patients with OA and rheumatoid arthritis. HA suppresses PGE2 production (32) and proliferation of synovial cells (33) in a molecular size-dependent manner, the larger sizes being more effective in vitro. In contrast, we have shown that HA84 suppresses both the increase in PGE2 concentration in synovial fluid and the proliferation of synovial cells more significantly than HA230 using the canine ACLT model described earlier (Table 1) (25). It has also been shown that HA84, but not HA of 3600 X 103 average molecular weight, suppressed proliferation of synovial cells in a rabbit ACLT model (34). These also indicate that the molecular size dependency of the effects of HA found in vitro could be reversed in vivo (Fig. 4). Moreover, Yamashita et al. (35) showed that HA90 suppressed bradykinin-induced knee joint pain in rats better than HA230. Therefore, the molecular size dependency of the effects of HA found in vitro does not necessarily coincide with the in vivo data (Fig. 4).

PGE2 is implicated in the progression of arthritis (36). Therefore, PGE2 suppression by HA treatment leads to improvement of arthritis. Because vascular permeability is enhanced by PGE2, increased vascular leak may be down-regulated by the suppressive effect of HA on PGE2 production. Further, the pain relief after intra-articular administration of HA (26,27) may be due to reduced PGE2 production, since PGE2 enhances nociceptive activity.

Homandberg et al. (37) showed that HA, of 900 X 103 average molecular weight, suppressed both the alteration in proteoglycan synthesis induced by fibronectin fragments and subsequent cartilage degradation. In addition, HA suppressed the production of free radicals and reversed proteoglycan synthesis induced by interleukin-1 b (IL-1 b) in cultured bovine articular chondrocytes (23). Further, HA84 suppressed IL-1b production in cultured synovial cells from rheumatoid arthritis patients (38). Takahashi et al. (39) have found that the

Time after the intraarticular-injection of hyaluronan

Figure 3 (A) Time course of distribution of fluorescein-labeled HA90 or HA230 in synovial tissues of guinea pigs that were intra-articularly injected. a-e, HA90; f-j, HA230. (B) Depth of fluorecein penetration from synovial surface into synovial tissues at several time points after the injection of fluorescein-labeled HA90 or HA230. 3d, 3 days; 7d, 7 days.

Time after the intraarticular-injection of hyaluronan

Figure 3 (A) Time course of distribution of fluorescein-labeled HA90 or HA230 in synovial tissues of guinea pigs that were intra-articularly injected. a-e, HA90; f-j, HA230. (B) Depth of fluorecein penetration from synovial surface into synovial tissues at several time points after the injection of fluorescein-labeled HA90 or HA230. 3d, 3 days; 7d, 7 days.

expressions of IL-1b and MMP-3 were suppressed in synovium but not in cartilage tissue of the rabbit ACLT model by treatment with HA84. The roles of PGE2, MMP-3 and IL-1b in OA, whose production was suppressed by HA treatment, are summarized in Table 2.

In the canine ACLT model described above, treatment with HA84 suppressed the degeneration of synovial cells (25). In this study, we found that heat shock protein 72, which can suppress cell death and degeneration of cells, is up-regulated in synovial cells of the group treated with HA84. However, HA84 did not rescue the rat pheochromocytoma cell line (PC12 cells) from apoptosis

Physicochemical Properties Cell

Figure 4 Proposed mechanism of molecular size dependency in the effects of hyaluronate preparations on synovial cells in vitro and in vivo. In vitro, higher molecular weight of HA, HA230 (2300 kDa), forms capping of HA receptors more densely than lower molecular weight of HA, HA84 (840 kDa) (a, b). In vivo, HA230 does not easily access synovial cells because of its large molecular sizes and entanglement among these molecules (square) (d), whereas HA84 can penetrate within the synovial tissue and adhere to the synovial cells (c). The sizes of arrows indicate the intensity of effects induced by the HA preparations (a, b). The effects by HA230 are more intense than those by HA84 in vitro (a, b). However, in vivo, the effects of HA230 are less than those of HA84, because accessibility of HA230 to the synovial cells is lower than that of HA84 in vivo (c, d). Molecular size dependency of the effects of HA in vitro is reversed in vivo.

Figure 4 Proposed mechanism of molecular size dependency in the effects of hyaluronate preparations on synovial cells in vitro and in vivo. In vitro, higher molecular weight of HA, HA230 (2300 kDa), forms capping of HA receptors more densely than lower molecular weight of HA, HA84 (840 kDa) (a, b). In vivo, HA230 does not easily access synovial cells because of its large molecular sizes and entanglement among these molecules (square) (d), whereas HA84 can penetrate within the synovial tissue and adhere to the synovial cells (c). The sizes of arrows indicate the intensity of effects induced by the HA preparations (a, b). The effects by HA230 are more intense than those by HA84 in vitro (a, b). However, in vivo, the effects of HA230 are less than those of HA84, because accessibility of HA230 to the synovial cells is lower than that of HA84 in vivo (c, d). Molecular size dependency of the effects of HA in vitro is reversed in vivo.

induced by serum deprivation (3). We then examined many sizes of HA to PC12 cells and, as a result, found that HA 4-mers, and not the other sizes of HA, suppress apoptosis (3). Moreover, only HA4 up-regulated mRNA and protein expression of heat shock protein 72 (Hsp72) in human leukemic cell line K562 cells exposed to

Table 1 Effects of HA on Prostaglandin E2 Concentration in Synovial Fluids and Proliferation of Synovial Cells of the Canine ACLT Model

Group

Non-operated

PBS

HA84

HA230

PGE2 (pg/ml) Thickness

39.8 ± 4.97 13.2 ± 0.84

89.8 ± 19.5* 31.0 ± 1.22**

± 1 57***

95.2 ± 10.6** 26.4 ± 1.54

Mean ± standard error; PGE2, prostaglandin E2; Non-operated, non-operated non-injected group; *P < 0.05 versus non-operated group; **P < 0.01 versus non-operated group; ***P < 0.05 versus PBS group.

Mean ± standard error; PGE2, prostaglandin E2; Non-operated, non-operated non-injected group; *P < 0.05 versus non-operated group; **P < 0.01 versus non-operated group; ***P < 0.05 versus PBS group.

Table 2 Roles of PGE2, MMP-3 and IL-lbeta in Osteoarthritis

PGE2

MMP-3

IL-lbeta

Increase in vascular permeability, synovial fluid and pain perception Degradation of collagen and proteoglycans Inflammation, reduction of proteoglycans synthesis hyperthermia (3). Collectively, these data are consistent with the hypothesis that, in the synovial tissue of the canine ACLT model, HA84 is degraded into HA oligosaccharides that up-regulate Hsp72 expression of synovial cells (25).

In hydroarthrosis, vascular permeability is increased in synovial tissue (40) due to neuropeptides released from fine nerve terminals in neurogenic inflammation, and the concentration of neuropeptides, e.g., of substance P, in joint fluid is increased in arthritis. These events suggest that neurogenic inflammation is involved in arthritis. Kato et al. (personal communication) have shown the suppressive effect of HA90 on Evans blue extravasation and substance P release in the skin of a neurogenic inflammation model induced by electrical stimulation of sensory nerves of the skin. After intravenous injection of HA or PBS in rats, Evans blue was intravenously administered to evaluate extravasation of plasma. The saphenous nerve was electrically stimulated using bipolar electrodes 5 min after Evans blue injection. The amount of dye released into the skin extract was measured, as was the substance P content in the skin extract with an enzyme immunoassay method. Treatment with HA significantly suppressed Evans blue extravasation and substance P release. When HA (10 mg/kg) was injected, the amount of Evans blue extravasation and substance P release was only 37 and 31% of that in the PBS group, respectively. However, further studies are required to elucidate the precise mechanism. It seems that the suppressive effect of HA on neurogenic inflammation is involved in the therapeutic effects of HA on arthritis, in particular, in alteration of pain and hydroarthrosis.

Viscoelasticity of HA preparations is an important factor in the efficacy of HA for OA. In addition, many cell biological functions of HA, e.g., suppression of PGE2, cytokine release, MMP production, and of substance P release are implicated in the efficacy of HA for OA.

B. Tissue Engineering

HA is abundant in fetal or young tissue extracellular matrix (41), and HA provides a fetal-like environment to cultured cells, stimulating regeneration. Moreover, during embryonic development, tissue regeneration and wound healing, the extracellular matrix surrounding migrating and proliferating cells is rich in HA (42). HA is implicated in morphogenesis (41,42) and plays a role in tissue organization (43). These characteristics of HA provide applications of HA for either tissue engineering or wound healing. In other words, the application of HA to wound healing is closely associated with that for tissue engineering.

Several studies have used HA in tissue engineering. An HA-based scaffold, consisting of chemically modified HA, has been used to support the growth of human chondrocytes (44). Human chondrocytes can proliferate and produce aggrecan and type II collagen but not type I collagen, indicating that the HA-based scaffold maintains the phenotype of chondrocytes. Therefore, the HA-based scaffold may also be used for the repair of articular cartilage defects.

Keratinocytes cultured on the HA-based scaffold form sheets, mimicking epidermis (45). On the other hand, fibroblasts can be cultured in an HA-based scaffold to form a three-dimensional dermis (46). Taken together, an HA-based scaffold can be used to form a skin equivalent.

Chemically modified HA has also been formed into a gel, film, sheet, tube or sponge. These designed forms of HA can be applied for growth or differentiation of many kinds of cells or tissues according to their form. In addition to its physicochemical property as a scaffold, interaction of HA with HA receptors, e.g., CD44 or RHAMM, influences cell behavior (47). Therefore, the application of HA to tissue engineering or wound healing is based on both its physicochemical properties and cell biological functions.

C. Wound Healing

1. Cystitis

Interstitial cystitis is characterized by prolonged pollakiuria and pain in the suprapubic region when the bladder is full of urine (48). For the treatment of interstitial cystitis, sulfated exogenous polysaccharides, sodium pentosanpoly-sulfate (49), DMSO (50), heparin (5l) and other agents are used (52-54). Interstitial cystitis may be related to a primary defect in glycosaminoglycans (GAGs), i.e., HA, heparan sulfate, dermatan sulfate and keratan sulfate in the vesical mucosa (55). A GAG coating of the bladder urothelium may protect the bladder tissue from injury by stimulants, e.g., by microorganisms, crystals and other toxic substances (56). It has been reported that intravesical injection of HA is useful for interstitial cystitis (57,58).

Intravesical HA has been used to treat interstitial cystitis, since this may possibly replenish bladder GAGs. Takahashi et al. (59) have evaluated the effect of 0.1-0.4% HA (average MW 890 X 103) on epithelial healing of the vesical mucosa and vesical fibrosis. They injected three concentrations of HA intravesically into rabbits with cystitis induced by instillation of 5% acetic acid solution. The effect of HA on cystitis was evaluated 7 days after injection. An increase in the capacity of the bladder was observed in HA-treated groups, and the area of the epithelial defect was reduced. Moreover, treatment with HA accelerated epithelial healing of the vesical mucosa and suppressed vesical fibrosis. Treatment with HA alleviated the model of cystitis in a concentration-dependent manner. Further studies are required to elucidate the mechanism of the effects of HA on this model of cystitis. It seems that the injected HA coats the injured area of the epithelium to protect the bladder tissue from injury, in a manner similar to that reported by Erickson et al. (60) for endogenous HA. In addition, the acceleration of epithelial wound healing by treatment with HA may be due to the interaction between HA and epithelial cell CD44, as shown in corneal epithelial wound healing (61). Further, Boucher et al. (62) have shown that immobilization stress induces bladder mast cell activation and the secretion of proinflammatory mediators, histamine and IL-6, which are inhibited by HA. These data suggest that the effect of HA on cystitis is in part due to its cell biological functions.

2. Corneal Epithelial Wound Healing

HA has already been developed as a preparation for corneal wound healing in dry eyes. In this case, it seems that HA protects the cornea from dryness through its high capacity for holding water. In addition, interaction between HA and epithelial cell CD44 is observed during wound healing of the corneal epithelium. In a rabbit corneal epithelial wound-healing model induced by n-heptanol, we examined the expression of HA and CD44 (61). HA concentration in the cornea gradually increased until day 14 after injury, and then decreased, returning to a normal level by 56 days. Staining of CD44 and HA was examined in corneal tissues by immunohistochemical and histochem-ical techniques, respectively. HA staining in corneal tissues correlated synchronously with the level of HA determined by biochemical analysis in corneal tissues. Both HA and CD44 were detected in the corneal epithelium and stroma. During the process of epithelial wound healing, a single layer of epithelium first extended to the damaged area 3 days after injury. When the epithelial layer completely covered the damaged area, HA staining was enhanced in both the epithelial layer and the underlying stroma. The enhanced staining of HA in the epithelium gradually decreased until it reached normal thickness. Immunoreactivity for CD44 changed almost concomitantly with that for HA after injury. HA expression returned to a normal level by day 28. This investigation indicates that HA and CD44 synergistically play important roles in corneal epithelial wound healing, and that epithelial cells produce HA as a scaffold for their proliferation and migration. In support of these studies, Miyazaki et al. (63) have reported that treatment with HA enhanced the growth of cultured corneal epithelial cells. Moreover, Miyauchi et al. (64) have shown that the topical administration of HA accelerates corneal wound healing in the rabbit by enhancing the growth of corneal epithelial cells.

D. Postoperative Adhesions

Adhesions remain a significant postoperative complication of abdominal surgery. A sodium hyaluronate and carboxymethylcellulose bioresorbable membrane (65) as well as a cross-linked hyaluronate hydrogel (66) have been shown to reduce postoperative adhesions. These HA derivatives were used as physical barriers in these studies.

Theoretically, it seems possible to reduce adhesion formation by taking the following steps: [1] reducing the initial inflammatory reaction and subsequent exudate release; [2] inhibiting coagulation of this exudate; [3] promoting the removal of fibrin deposition; [4] mechanically separating fibrin-covered surfaces; and [5] inhibiting fibroblast proliferation (67). Weigel et al. (68) have shown that HA binds fibrin and fibrinogen. This suggests that the effects of HA derivatives on the adhesion protection may be due to the binding of HA to fibrin and fibrinogen preventing fibrin from acting as a glue in adhesion formation.

It has been reported that halofuginone, an inhibitor of collagen type I synthesis, prevents postoperative adhesion formation in the rat uterine horn model (69). This indicates that collagen is implicated in postoperative adhesion. Coleman (70) has shown that HA chains have a major organizational role within the collagen bundle. Exogenous HA may prevent the formation of collagen bundles in postoperative adhesions.

Collagen is produced by fibroblasts. It is well known that HA suppresses the proliferation of fibroblasts (33). Therefore, the prevention of adhesions may involve the suppressive effect of HA on the proliferation of fibroblasts. Moreover, HA suppresses inflammation by preventing cytokine production (5). Thus, HA may prevent several steps in postoperative adhesions.

These events described above suggest that the effects of HA on postoperative adhesions are due to the role of HA as a physical barrier based on its physicochemical properties and also due to cell biological or physiological function of HA.

Was this article helpful?

0 0
Natural Arthritis Pain Remedies

Natural Arthritis Pain Remedies

It's time for a change. Finally A Way to Get Pain Relief for Your Arthritis Without Possibly Risking Your Health in the Process. You may not be aware of this, but taking prescription drugs to get relief for your Arthritis Pain is not the only solution. There are alternative pain relief treatments available.

Get My Free Ebook


Post a comment