Tim C.R.1, Martignago C.C.2, Neves L.M.G.2, Assis L.1, Parizotto N.2, Renno A.C.1
1Federal University of São Paulo - UNIFESP, Santos, Brazil, 2Federal University of São Carlos - UFSCar, São Carlos, Brazil
Background: Osteoarthritis (OA) is a degenerative joint disease characterized by progressive destruction of articular cartilage and reactive new bone formation at the joint surface and surrounding areas. In general, patients with OA present joint stiffness, loss of mobility, and pain. Thus , it becomes important to develop technologies capable of treating and prevent the evolution of OA. Currently, low-level laser therapy (LLLT) and chitosan has demonstrated positive effects on cartilage metabolism. However, the mechanisms of action and the effects of the combination of both treatments still require elucidation.
Purpose: The aim of this study is to evaluate the viability of chitosan, the LLLT and the combination of both therapy, by evaluating the biological responses induced by the treatments.
Methods: Chondrocytes were obtained from collagenase-digested femoral growth plate cartilage of 7-week-old rats. Chitosan hydrogels were prepared using 1 mL chitosan solution was mixed with 0.67mL urease solution and 10 ul of the urea solution. Before gelation, 300 uL chitosan solution containing 10M urea and 30 U/mL urease were mixed with 30 mL proliferation medium containing 1.105 chondrocytes was prepared. After gelation chitosan, proliferation medium (1000 uL) was added and refreshed after 30 and 60 min to remove possible side products of the hydrogel formation. Cells were cultured for 3 days. Cell irradiation was performed using a Photon Laser III - DMC Equipament®, 808 nm, 50 mW, 30 J/cm2, 16 seconds, 0,8 J. The cells were irradiated 24 h after seeding, for three consecutive days, once every 24 h. Four experimental groups:
G1control group, which received no irradiation;
G2irradiated at 30 J/cm2;
G3irradiated at 30 J/cm2 + chitosan hydrogel;
G4 - Chitosan hydrogel.
The irradiation was performed with the laser pointer tip in direct contact with the plate. Cell metabolic activity was evaluated using Alamar Blue®, according to the manufacturers instructions. The alamarBlue® assay is based on the ability of metabolically active cells to convert the reagent into a fluorescent and colorimetric indicator.
Results: Results revealed that the cell metabolic activity was significantly lower for LLLT 30 J/cm2 + chitosan hydrogel (p=0,001) and chitosan hydrogel (p=0,001) compared to control group. Also, it possible to observed statics significantly higher for LLLT 30 J/cm2 (p=0,002) compared to LLLT 30 J/cm2 + chitosan hydrogel.
Conclusion(s): Based on the results of the present study, it has been shown that LLLT 30 J/cm2 within the parameters presented is capable of stimulating the Chondrocytes viability.
Implications: It is estimated that 10 % of the population in the world older than 60 years demonstrated signals of OA. Treatments of OA involve the administration of nonsteroidal anti-inflammatory drugs and physical therapy, such as muscle strengthening and stretching. However, these therapies are introduced only after the appearance of the first symptoms. In this context, due to the very limited cartilage regenerative capacity and consequently the limited efficacy of the standard treatments, it would be of great interest the investigation of strategic innovative approaches to prevent the development of the clinical condition of OA.
Funding acknowledgements: We would like to acknowledge the contributions of Brazilian funding agency Fapesp (2014/13702-6) for the financial support of this research.
Topic: Orthopaedics
Ethics approval: This study was approved by the Animal Care Committee guidelines at Federal University of São Paulo (CEUA N 2478130315).
All authors, affiliations and abstracts have been published as submitted.
