Non-clinical assessment of lubrication and free radical scavenging of an innovative non-animal carboxymethyl chitosan biomaterial for viscosupplementation: An in-vitro and ex-vivo study

Abstract

Objective: Lubrication and free radical scavenging are key features of biomaterials used for viscosupplementation (VS) of joints affected by osteoarthritis (OA). The objective of this study was to describe the non-clinical performance characterization of KiOmedine® CM-Chitosan, a non-animal carboxymethyl chitosan, in order to assess its intended action in VS and to compare it to existing viscosupplements based on crosslinked hyaluronan (HA) formulations.

Method: The lubrication capacity of the tested viscosupplements (VS) was evaluated in-vitro and ex-vivo. In-vitro, the coefficient of friction (COF) was measured using a novel tribological system. Meanwhile, an ex-vivo biomechanical model in ovine hindlimbs was developed to assess the recovery of join mobility after an intra-articular (IA) injection. Free radical scavenging capacity of HA and KiOmedine® CM-Chitosan formulations was evaluated using the Trolox Equivalent Antioxidant Capacity (TEAC) assay.

Results: In the in-vitro tribological model, KiOmedine® CM-Chitosan showed high lubrication capacity with a significant COF reduction than crosslinked HA formulations. In the ex-vivo model, the lubrication effect of KiOmedine® CM-Chitosan following an IA injection in the injured knee was proven again by a COF reduction. The recovery of joint motion was optimal with an IA injection of 3 ml of KiOmedine® CM-Chitosan, which was significantly better than the crosslinked HA formulation at the same volume. In the in-vitro TEAC assay, KiOmedine® CM-Chitosan showed a significantly higher free radical scavenging capacity than HA formulations.

Conclusion: Overall, the results provide a first insight into the mechanism of action in terms of lubrication and free radical scavenging for the use of KiOmedine® CM-Chitosan as a VS treatment of OA. KiOmedine® CM-Chitosan demonstrated a higher capacity to scavenge free radicals, and it showed a higher recovery of mobility after a knee lesion than crosslinked HA formulations. This difference could be explained by the difference in chemical structure between KiOmedine® CM-Chitosan and HA and their formulations.

Fig 1. Biomechanical ex vivo sheep model of 3D joint mobility.

The distal portion of the gastrocnemius and its tendon are tightened by a Colson ring (A). A metallic wire (B) is connected, via a pulley (C), to a metallic bar to which weight plates are loaded (D). The stereo-photogrammetric system (Optitrack) is composed of 10 cameras and 2 optical references frames (rigidly attached to the tibia and the femur) to record and measure the kinematics of a knee joint (E).

Fig 2. Analytical set-up of the ex-vivo biomechanical study.

The experimenter pulled the wire manually (passive test) to check the dispositive. Then the quadriceps was loaded with an initial weight of 0.5 kg and the motion of 3 flexion-extension cycles of the leg was recorded with increasing weight for up to 5 kg by 0.5 kg increment. Test item A = Hylan G-F 20 and test item B = KiOmedine® CM- chitosan.

Fig 3. Procedure of creation of cartilage default.

(1) The distal and cutting part was sawed from a biopsy punch (B). (2) After incision of the subcutis and joint capsule, the medial condyle (C) of the femur was exposed. Fat (F) was removed from the joint cavity. (3) The biopsy punch (B) was deeply entered into three adjacent sites of cartilage in the axial aspect of the medial condyle of the femur. The Volkman curette (V) was used to remove cartilage. Then the joint capsule was sutured and tests were performed. (4) Picture 4 illustrates the three cartilage defects on the medial condyle (MC) at gross anatomy dissection. LC = lateral condyle.

Fig 4. Schematic representation of force involved during the ex-vivo model.

Calculations were performed, with the assumption that the ovine hindlimb is a 2D hinge joint that is rotating around the O point, due to the quadriceps force (F), involving the following parameters:

1. F = quadriceps force (in the experimental analysis obtained as the force of the weight);

2. F_x = component of the force F along the direction X

3. F_y = component of the force F along the direction Y

4. m = mass of the limb;

5. mg = weight of the limb (g is the gravity, 9.81 m/s²);

6. F* = the contact force between the tibia and the femur;

7. f = friction coefficient (or COF);

8. fF* = the friction force;

9. r = radius of the femoral condyle;

10. a = distance between the tibial insertion point of the patellar tendon and the hinge centre;

11. b = distance between the barycentre of the lower leg and the hinge centre;

12. α (alpha) = flexion angle;

13. $\ddot{\alpha }$ = the second derivative (angular acceleration) of α with respect to the time

14. I₀ = the mass moment of inertia

15. β (beta) = angle from the patellar tendon and the tibial axis

.

Fig 5. COF measurements of KiOmedine® in the p-HEMA tribological model.

The lubrication capacity of KiOmedine® (N = 4) to reduce COF between the disks in rotating friction was compared to two commercial VSs biomaterial references composed of crosslinked hyaluronic acid, Hylan G-F 20 (N = 4) and NASHA(N = 3), the buffer control (N = 4) and synovial fluids punctured from osteoarthritic patients (N = 10). KiOmedine® exhibited similar or higher lubricating capacity compared to Hylan G-F 20 and NASHA (ANOVA, ***p < 0.0001).

Fig 6. Lubrication capacity of KiOmedine® CM-chitosan in the biomechanical ex-vivo sheep model of 3D joint mobility.

(A) A significant increase in COF is observed due to increased joint friction in damaged cartilage (paired T-test, **p<0.01); COF is then progressively significantly improved following the IA injection with KiOmedine® CM-chitosan in a volume-dependent manner (ANOVA, **p<0.01). (B) KiOmedine® CM-chitosan was compared to Hylan G-F 20 at 3 ml IA volume and showed significantly better recovery of joint motion loss on damaged cartilage (t-test, **p<0.01); the effect of the polymer-free buffer of KiOmedine® on the recovery of motion was low.

Fig 7. Free radical scavenging capacity using the TEAC assay.

KiOmedine® CM-chitosan was compared to Hylan G-F 20, NASHA, negative and positive controls respectively, the polymer-free buffer of KiOmedine® CM-chitosan and vitamin C. KiOmedine® CM-chitosan showed high free radical scavenging capacity in vitro as compared to crosslinked HA biomaterials, Hylan G-F 20, and NASHA (ANOVA, ***p<0.0001).