Low dose native type II collagen prevents pain in a rat osteoarthritis model

Osteoarthritis pathophysiology involves the whole joint in a disease process that includes focal and progressive loss of hyaline articular cartilage. Concomitant changes in the bone underneath the cartilage involve formation of osteophytes and bony sclerosis, as well as alterations in the synovium and joint capsule [1]. Cartilage loss or degeneration may be a result of natural aging, obesity, repeated trauma or hormone disorders. The mechanical stress on the damaged joint irritates and inflames the cartilage causing joint pain and swelling [2, 3].

An integrative treatment of osteoarthritis, or rheumatoid arthritis, must consider a supplementation with collagen since it is the most prevalent component of the solid phase of articular cartilage [4]. The three major groups of collagen derivatives clinically used for arthritis treatment are based on the various degrees of hydrolysis of collagen: gelatin, collagen hydrolysate and native undenatured collagen [5]. Analogous working mechanisms has been described for gelatin and collagen hydrolysate: after oral administration peptides can be used as building blocks for the cartilage [6‐8]. Moreover, it is hypothesized that collagen hydrolysate also influences bone metabolism [9, 10] or the vascular system involved in the atheromatous disease of the subchondral bone [11, 12]. For these purposes collagen hydrolysate is dosed in grams per day (usually 10 g) [13‐15]. On the contrary, undenatured collagen has been reported as beneficial for articular pain when per os administered in the order of milligrams [16, 17]. Undenatured collagen was preclinically and clinically studied mainly in rheumatoid arthritis [16, 17]; the autoimmune component of this pathology suggests a mechanism called oral tolerance, the usual response of the gut-associated lymphoid tissue (GALT) to harmless gut antigens inducing local and systemic immunological tolerance [18‐20]. The knowledge about the relevance of low doses of undenatured collagen in osteoarthritis are more limited [21] and the absence of an immune component in the pathology of osteoarthritis make difficult to assume the oral tolerance as possible mechanism of collagen action.

In order to verify the efficacy of low doses of porcine native type II collagen as pain reliever and cartilage protector, we determined its pharmacological profile in a rat unilateral osteoarthritis induced by sodium monoiodoacetate (MIA).

The pharmacological activity of low dosages of type II collagen was evaluated in the rat unilateral osteoarthritis model induced by MIA. 14 days after intra-articular MIA injection the mechanical withdrawal threshold to a noxious stimulus was measured by Paw pressure test (Figure 1). The weight tolerated on the ipsilateral paw was significantly reduced (42.2 ± 2.0 g) compared to the contralateral (77.0 ± 4.1 g). 1 mg kg^-1 collagen administered daily p.o. for 13 days (starting from the day of MIA injection) increased the withdrawal threshold of the ipsilateral paw up to 67.5 ± 3.1 g. The doses of 3 and 10 mg kg^-1 were significantly active but progressively less effective. Figure 2 shows the response to a non-noxious mechanical stimulus evaluated by the Von Frey test. On day 14 pain threshold of the ipsilateral paw (MIA + vehicle group) was decreased to 14.1 ± 2.0 g as compared to the contralateral (31.4 ± 4.2 g). Animals treated with 1 mg kg^-1 collagen showed an ipsilateral threshold of 25.3 ± 4.0 g; the groups treated with higher dosage tolerated a stimulus of about 21 g. In both Paw pressure and Von Frey tests the pain sensitivity of the contralateral paw of MIA + vehicle or MIA + collagen groups was not different with respect to the control (vehicle + vehicle, data not shown). Articular pain was evaluated by PAM test, in Figure 3 the difference (Δ Force) between the force tolerated directly on the knee joint contralateral to the injury and the force tolerated on the ipsilateral one was described. Δ Force in control rats (vehicle + vehicle, 36.1 ± 21.1 gf) was dramatically increased on the day 14 in MIA + vehicle group (129.5 ± 10.1 gf). Collagen treatment prevented articular pain in a manner inversely proportional to the dose, reaching 15.8 ± 2.7 gf Δ Force in animals treated with 1 mg kg^-1.

Unilateral pain was also able to induce hind limb weight bearing alterations (Incapacitance test): the difference between the weight burdened on the contralateral and the ipsilateral limb was significantly increased in MIA + vehicle (54.1 ± 5.3 g) with respect to vehicle + vehicle (4.3 ± 3.5). The protective effect of collagen was shown in Figure 4. Moreover, the Animex test showed a collagen-dependent improvement of motor activity increasing the number of movements by about 50% (Figure 5).

Biochemical analysis of biological fluids performed 14 days after MIA injection, allowed to observe a 4 fold increase of CTX-II in plasma of MIA + vehicle rats compared to the control group (vehicle + vehicle). The plasmatic CTX-II increase was reduced by 53% in the rats treated with 1 mg kg^-1 collagen and by 40% in those treated with the higher doses (Figure 6). The same parameter was increased by MIA also in urine (2.6 fold with respect to the control); 3 mg kg^-1 collagen induced the higher protective effect (75% inhibition compared to MIA + vehicle; Figure 7). 1, 3 and 10 mg kg^-1 collagen daily administered p.o. for 14 days, in the absence of articular damage, did not alter CTX-II levels in plasma and urine (data not shown). In Table 1 are shown the plasmatic levels of the collagen synthesis marker CPII. MIA-induced articular damage evoked an increase of CPII that is unaltered by collagen repeated treatments.

Osteoarthritis, also called degenerative joint disease, is a chronic pathology frequently seen in knee, hip, spine and hand causing pain, stiffness, decreased range of motion, and reduced quality of life for million people throughout the world [5]. It is by far the most widespread joint-affecting disease. According to the World Health Organization, osteoarthritis is the sixth-leading cause of disability in the world [30], being comparable to that of asthma [31, 32]. It is estimated that it affects over 12% of the total population in the USA [33], compared with 0.6% for rheumatoid arthritis [34]. The prevalence of osteoarthritis increases with age because the condition is not spontaneously reversible [35]. Almost 9.6% of men and 18.0% of women ages 60 years in the world are thought to have symptomatic osteoarthritis [36]. Given the increasing incidence of osteoarthritis with age, the extended life expectancy observed in the Western world (for example 20% of the Italian population is age > 65 years; [37]) is expected to result in a progressively higher number of people affected by this pathology.

The usual management of patients with hip or knee osteoarthritis requires a combination of non-pharmacological and pharmacological modalities of therapy. Pharmacological treatments include acetaminophen, cyclooxygenase-2 (COX-2) non-selective and selective oral non-steroidal anti-inflammatory drugs (NSAIDs), topical NSAIDs and capsaicin, intra-articular injections of corticosteroids and hyaluronates, glucosamine and/or chondroitin sulphate for symptom relief. Glucosamine sulphate, chondroitin sulphate and diacerein have possible structure-modifying effects [38]. The characteristic of chronicity determines the need of new active disease modifying drugs.

In the present research osteoarthritis was mimicked in rats injecting MIA in the knee joint. The intra-articular injection of MIA induces necrosis of condrocytes with decrease of cartilage thickness and osteolysis [23], in the presence of a relevant component of oxidative stress [39]. Kobayashi et al. [40] showed that MIA is able to disorganize condrocytes and to promote cartilage erosion. These alterations are comparable with joint damages typical of humans affected by osteoarthritis [22, 41, 42].

In our experiments behavioral and biochemical features were evaluated 14 days after MIA injection, when pain as well as the degenerative articular process are overt [42, 43]. At this time, CTX-II levels were strongly increased as measured in urine and plasma. CTX-II is a C-terminal peptide generated by the concerted action of matrix metalloproteinase (MMPs) on the fibrillar type II collagen, and it is considered a biomarker of cartilage degradation [44, 45]. Its level was found to correlate with cartilage loss in animal models of osteoarthritis [46]. In agreement, clinical studies showed increased CTX-II levels in patients with osteoathritis compared with controls [47, 48].

Type II collagen is the principal molecular component of mammalian cartilages [4]: the present work is focused on the study of this fibrous protein as preventive of MIA-induced articular damage. Different dosages of native type II collagen were daily administered per os for 14 days starting from the day of MIA injection. 1-10 mg dose range was chosen on the bases of the efficacy demonstrated in rheumatoid arthritis models [17]. Lower doses, in particular 1 mg kg^-1, was able to strongly prevent pain behavior when evaluated both as an increase on suprathreshold stimulation (hyperalgesia-related measure) or as pain threshold decrease (allodynia-related measure). Collagen was able to reduce pain on the paw ipsilateral to the MIA injection. Since articular pain and joint tenderness are the most frequent and disabling symptoms [5] we evaluated also the animal responses to a direct stimulation of knee joint. The efficacy of collagen on reducing articular pain progressively increased lowering in the dose. Moreover, collagen-dependent pain relief allowed also to reduce postural unbalance, a feature of disease progression [26], as measured by hind limb weight bearing alterations. A general improvement of motor activity was observed.

The behavioral positive effects of collagen may be related to a prevention of articular damage since CTX-II levels were reduced in urine and in plasma of collagen-treated rats. On the other hand, low dosed collagen did not promote the synthesis of new collagen given that the plasmatic levels of CPII, the carboxyl propeptide of type II procollagen [28, 29], was not altered by 14 days of collagen treatment.

The higher efficacy of the lower dose and, in general, the low dosages administered in the present work do not justify a mechanism founded on cartilage structure supplementation, as confirmed by the lack of CPII increase. Moreover, cartilage has limited repair capabilities and cartilage damage is difficult to heal since chondrocytes are bound in lacunae and they cannot migrate to damaged areas; hyaline cartilage does not have a blood supply and the deposition of new matrix is slow [49]. Other working mechanisms remain to be explored.

In rheumatoid arthritis the induction of oral tolerance was suggested as mechanism for the beneficial effect on pain evoked by low doses of native collagen [17, 50]. The relevance of oral tolerance has been described for pathologies related to immune dysregulations and for autoimmune diseases [51], indeed a complex immune mechanism contribute to the pathology of rheumatoid arthritis [16, 52]. The proposed mechanism for collagen-induced oral tolerance is that dendritic cells in the GALT take up the collagen and present it to T cells to generate regulatory T cells. Regulatory T cells control the immune response inducing several inhibitory cytokines, such as transforming growth factor β and interleukin 10, while decreasing pro-inflammatory cytokines [17, 53].

To the actual knowledge osteoarthritis has not immune characteristic, but shares with rheumatoid arthritis cartilage degradation and the consequent inflammatory response. The role of oral tolerance in the management of osteoarthritis remains to be investigated. However, future research focused on the pharmacodynamic study of collagen therapeutic effects could offer new insights in osteoarthritis pathophysiology.

Low dose collagen decrease pain induced by the intrarticular injection of MIA, a rat model of articular damage that mimics osteoarthritis alteration. Treated animals showed a reduced postural unbalance and a general improvement of motor activity. The decrease of CTX-II levels in urine and plasma suggests a protective effect on cartilage. This evidence highlights the interest for further investigation about the mechanism of low dose collagen and its relevance in osteoarthritis therapy.