EFFECT OF PLATELET RICH PLASMA ON CHONDROCYTE NUMBER IN CHEMICALLY INDUCED ANIMAL MODEL OF OSTEOARTHRITIS.
Keywords: Chondrocyte, Mono iodoacetate, Osteoarthritis, Platelet rich plasma.
Osteoarthritis (OA) is the most common joint disorder affecting hip, knee and other joints of the body. Knee OA affects 28% of the urban and 25% of the Pakistani population with women of middle age being the prime target1. OA is a multifactorial disease affected by various risk factors like age, gender, obesity, ethnicity and activity. OA is characterized by classical features at histopathological level including cartilage degradation, reduced bone mineral density, osteophyte formation, synovitis and capsule thickening2. Articular cartilage is a stiff and load bearing connective tissue which covers the bone ends making the smooth and frictionless movements possible at joints. Articular cartilage has low metabolic activity making it difficult to repair. It is devoid of perichondrium and has no vascular, neural and lymphatic supply. The structure of articular cartilage changes as we move deeper from the joint surface.
On the basis of morphology of chondrocytes and matrix, articular cartilage structure can be divided into three zones; superficial (tangential), middle (transitional) and deep zone. These areas respectively constitute 10-20%, 40-60% and 30% of the articular cartilage volume. Middle zone of the articular cartilage acts as first line of defence against the compressive forces acting on joint surface. As we move from superficial to deep zones the arrangement of collagen fibres changes from parallel to perpendicular with reference to the surface with an increase in thickness of fibres. Chondrocyte count is most abundant in superficial zone with the cells being flat which become spherical and less dense in middle zone. In the middle zone chondrocytes are arranged in isogenous groups but their arrangement becomes perpendicular to joint surface once they enter the deep zone. Proteoglycan content is highest in the deep zone which provides maximum resistance against compressive forces.
Tide mark separates deep zone from calcified cartilage which contains few hypertrophic chondrocytes3. Animal models are used to produce OA and can be classified as spontaneous or induced models. Osteoarthritic changes can be induced by surgical, chemical, mechanical and genetic modifications. Monosodium iodoacetate (MIA), papain, collagenase and carrageenan are the most commonly used agents for producing changes in knee joint. MIA, a derivative of acetic acid, causes chondrocyte apoptosis by inhibiting glyceraldehyde-3-phosphate dehydrogenase and is considered the most appropriate method for inducing OA changes4. MIA injected in a dose of 0.5mg in the knee joint showed apoptotic changes on the 1st post administration day with chondrocyte shrinkage, nuclear condensation, vacuolar degeneration and apoptotic bodies5. Teeple et al, noticed that MIA injection stimulates intra articular damage, matrix degradation and chondrocyte toxicity6.
The effects produced by MIA are strongly related to its concentration with cell count decreasing in the central regions of the cartilage suggesting that the chondrocytes in the central region are more sensitive to MIA7. Various treatment modalities are used for managing OA symptoms which include pain killers, anti inflammatory drugs, osteotomy, intra articular knee injections and ultimately knee replacement surgery. At present, platelet rich plasma (PRP) is the new approach being used for treating OA and has been reported with relief of symptoms and repair of injured tissues. PRP is defined as a concentrate of autogenous growth factors derived from blood which influences healing of musculoskeletal system by promoting cell multiplication, collagen synthesis and new vessel formation. American Red Cross society declared that plasma sample having platelet count two fold or more above the baseline or greater than 1.1 x 106 platelets/ul is labelled as PRP8.
Marx et al, proposed that platelet count of 10 lakh/ml in 5ml of the plasma promoted bone and soft tissue healing and could be labelled as PRP however Rughetti et al, tossed the working definition of PRP with platelet count of 1million/ml9. PRP prepared from differential centrifugation of blood contains growth factors contained in [alpha] granules of platelets and promotes angiogenesis, collagen synthesis and cell proliferation10. Fortier et al, found that chondrocytes and mesenchymal stem cells (MSCs) exposed to PRP showed increase in cell count along with proteoglycan and collagen type-II synthesis8. The aim of this study was to establish the histopathological effect of PRP on chondrocyte count in articular cartilage of MIA induced osteoarthritic rat knee model.
MATERIAL AND METHODS
This study was approved by Ethical Review Committee of Army Medical College Rawalpindi and was conducted from March 2018 to May 2018 in the department of Anatomy, Army Medical College Rawalpindi, in collaboration with National Institute of Health (NIH), Islamabad. Thirty two male Sprague Dawley rats with average weight of 200-300 gm were taken, housed in separate well ventilated cages. Twelve hours light and dark cycles were maintained at standard room temperature of 20-26AdegC. Standardized laboratory diet and water ad libitum was provided to all animals. Both MIA and PRP were administered using insulin syringes. MIA was used for inducing osteoarthritic changes in knee joint. Solution was prepared by dissolving 80 mg of the chemical in 2 ml of 0.9% saline yielding 2 mg of MIA in 50 ul. PRP was prepared by double centrifugation method proposed by Zhang et al11, using hettich EBA 20 benchtop centrifuge machine.
3 ml of venous blood was collected through intracardiac route and 0.05 ml of 0.1M sodium citrate solution was added as anticoagulant11. First centrifugation was carried out at 500g for ten minutes. Clear serum and buffy coat were removed using 20 gauge lumbar puncture needle and subjected to second centrifugation at 2200g for 10 minutes. Platelet poor plasma was removed and PRP was collected and activated using 0.05 ml of 10% CaCl212. Animals were divided into control group A (n = 8) and experimental group B (n = 24). Group B was subdivided into groups B1, B2 and B3 (8 animals per group). All animals in group B were injected with 50 ul of MIA solution in the right knee. Group B1 along with group A was sacrificed after two weeks for establishing presence of osteoarthritic changes. Group B2 was injected with PRP while group B3 was left as such.
All animals in group B2 and B3 were sacrificed four weeks after the administration of PRP. Gross parameters were noted and specimens obtained were fixed in 10% Neutral Buffered Formalin. Decalcification was done using 5% nitric acid solution and processed for paraffin embedding. Sections of 5um thickness were obtained using rotary microtome and hematoxylin and eosin stains were used for routine histological study. For quantitative analysis, ocular micrometer was calibrated with stage micrometer. Four random fields were selected and chondrocytes count was done at 40 x magnification in 0.06mm2 area excluding the calcified zone. Chondrocyte lacunae with absent nuclei were not counted. Data were analysed by using SPSS (Statistical Package for Social Sciences) version 21. Chondrocyte count was expressed as mean +- standard deviation.
Statistical significance was calculated between the groups using independent sample t-test. A p-value a$?0.05 was considered significant and confidence intervals were kept at 95%.
Table: Comparison of mean values of chondrocyte count between control group A and experimental groups B1, B2 and B3.
###Group A###Group B1###Group B2###Group B3###Group###Group
Parameter###Mean +- SD###Mean +- SD###Mean +- SD###Mean +- SD###A vs B1###B2 vs B3
Chondrocyte count 63.12 +- 12.80###39.25 +- 10.36###38.12 +- 8.611###18.90 +- 13.80###0.001###0.006
Histologically, control group showed normal articular cartilage (A), however osteoarthritic changes like surface irregularities and decreased number of chondrocyte were noted among group B1 (B1). Chondrocyte count was done in control group A and experimental groups B1, B2 and B3. The mean count +- SD were calculated. The mean chondrocyte count +- SD in groups B2 and B3 was found to be 38.12 +- 8.6 and 18.90 +- 13.8 respectively. The p-value between the experimental groups B2 and B3 was found to be 0.006 which was statistically significant.
In the present study, PRP prevented apoptosis of articular chondrocytes thus providing the chondroprotective effect. Extracellular matrix changes and decrease in chondrocyte number is the hall mark of OA. MIA is an alkylating agent which reacts with proteins in cysteine residues and causes inhibition of glycolytic pathway ultimately leading to death of chondrocytes. Miyamato et al, showed that MIA induced osteoarthritic knee models in rats and rabbits exhibited chondrocyte death and loss of proteoglycan which progressed to cartilage destruction, subchondral bone exposure and functional impairment similar to humans13. Morais et al, induced osteoarthritic changes in rat knee using two mg of MIA and showed decrease in chondrocyte number due to death and degeneration of chondrocytes along with osteophyte formation and bone degradation which was similar to our study14.
Udo et al, induced osteoarthritis using different doses of MIA and concluded that induction of OA is both dose and time dependant15. Ferreira et al, showed that 1mg and 2mg did not induced bone destruction even after 4 weeks which was different than our findings16. All these researches showed use of wistar rats which were younger than rats used in this study. Miyamato et al, showed that 2mg of MIA injected into rat hip joint induced articular cartilage degeneration which was evident at day 14 which was similar to ours13. Guzman et al17, observed that 1 mg produced extensive chondrocyte degeneration at day 1 with moderate collapse and loss of chondrocyte details by day 5 or 7 which was opposed to our research.
Cell/extracellular matrix interactions play a vital role in pathophysiology of OA. Chondrocytes are the only cells regulating the breakdown and synthesis of cartilage matrix. Cytokines; a family of proteins involved in immune responses along with growth factors maintain the balance between catabolic and anabolic processes within the matrix. Interleukins (IL) being pro inflammatory cytokines interfere with activity of growth factors and decrease the synthesis of aggrecan which is a prime component of matrix. Lipid peroxidation and radical oxygen species (ROS) induced by interleukins have also been found to cause matrix degradation18. These are believed to cause disruption of mitochondrial function by damaging mitochondrial DNA, lipids and proteins. Tumour Necrosis Factor (TNF)-and IL-1 increase nitricoxide (NO) and Reactive Oxygen Species (ROS) in OA chondrocytes causing mitochondrial dysfunction, ultimately leading to apoptosis19.
Inhibition of chondrocyte apoptosis serves as a therapeutic strategy for treatment of OA. Moussa et al, concluded that PRP inhibits apoptosis of chondrocytes which coincided with our findings. PRP therapy has been found to induce proliferation of chondrocytes, decrease apoptosis and increased autophagy among chondrocytes20. PRP prevents aging of chondrocytes by increasing autophagy which prevents chondrocytes from excessive inflammation and nutritional deficiency. PRP isolated from autologous blood contains multiple growth factors which have been proved to promote chondrocyte proliferation and extracellular matrix secretion. PRP promotes expression of anabolic genes and reduces inflammatory stress ultimately leading to attenuation of post traumatic cartilage degene-ration. This study was also supported by Knut et al, who carried out a research on chondrocyte viability and concluded that PRP increased chondrocyte and tenocyte viability in vitro21.
The present study has some limitations. First, MIA induced OA is a chemically induced model. To confirm the beneficial effects of PRP, this study should be conducted with other non chemically induced models like immobilization and partial menisectomy which involves removal of all or some part of meniscus. Literature review suggested that MIA is used widely for induction of OA and demonstrates the characteristics of progressive inflammatory arthritis resulting in rapid destruction of cartilage structure14. The absence of proliferative effect noted in this study can be attributed to the fact that MIA continued to have its effect even after the administration of PRP. Second, this study was conducted with a single dose of PRP injection. Literature search suggested that chondrocyte proliferation increased in a dose dependant manner with increasing concentrations of platelets however our research was carried out using a single injection of PRP.
This proposes that if chondrocyte count was done after two or three doses of PRP, increase in chondrocyte count would have been noticed.
Platelet rich plasma infusion prevented chondrocytes apoptosis in the treated group as compared to the untreated group.
We are highly indebted to Army Medical College, Rawalpindi for providing us with immense help and opportunities for carrying out our research. We are also grateful to Dr. Hussain Ali, Scientific Officer, National Institute of Health, Islamabad for his immense help in animal handling and housing of animals. We thank Dr. Humera Mehmood, Assistant Professor, Department of Public Health, AFPGMI, Rawalpindi for her helpful discussion regarding methodology and statistical analysis of the result and Dr. Asifa Majeed, Assistant Professor of Biochemistry, Cream Lab for her help in establishing protocol for PRP preparation. Grant for this project was provided by National University of Medical Sciences (NUMS).
CONFLICT OF INTEREST
This study has no conflict of interest to be declared by any authors.
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|Publication:||Pakistan Armed Forces Medical Journal|
|Date:||Jun 30, 2019|
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