A ANALYTICAL STUDY OF MOLECULAR BIOLOGICAL MARKER IL-6 IN SYNOVIAL FLUID IN KNEE JOINT BEFORE AND AFTER PRP INJECTION IN PATIENTS
WITH OSTEOARTHRITIS AND RHEUMATOID ARTHRITIS
Dissertation submitted to
THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY, CHENNAI,
In partial fulfilment of the requirements for the degree of MASTER OF SURGERY IN ORTHOPAEDICS
Under the Guidance of
Dr. B.K.DINAKAR RAI, M.S. (ORTHO) PROFESSOR & HOD
DEPARTMENT OF ORTHOPAEDICS,
PSG INSTITUTE OF MEDICAL SCIENCES AND RESEARCH COIMBATORE
MAY 2020
DECLARATION BY THE CANDIDATE
I hereby declare that this dissertation entitled “A ANALYTICAL STUDY OF MOLECULAR BIOLOGICAL MARKER IL-6 IN SYNOVIAL FLUID IN KNEE JOINT BEFORE AND AFTER PRP INJECTION IN PATIENTS WITH OSTEOARTHRITIS AND RHEUMATOID ARTHRITIS” is a bonafide and genuine research work carried by me under the guidance of DR.B.K.DINAKAR RAI, M.S Ortho, HOD & Professor, Department of Orthopaedics, PSGIMS&R, Coimbatore.
Place:
Date: Dr. V. ANAND BABU
CERTIFICATE BY THE GUIDE
This is to certify that the dissertation entitled “A ANALYTICAL STUDY OF MOLECULAR BIOLOGICAL MARKER IL-6 IN
SYNOVIAL FLUID IN KNEE JOINT BEFORE AND AFTER PRP INJECTION IN PATIENTS WITH OSTEOARTHRITIS AND
RHEUMATOID ARTHRITIS” is a bonafide work done by Dr. V.ANAND BABU in partial fulfilment of the requirement for the degree
of M.S. (Orthopaedics).
Place:
Date: Dr. B. K. DINAKAR RAI, Professor & HOD
Department of Orthopaedics, PSGIMS&R,
Coimbatore.
CERTIFICATE BY THE HOD/PRINCIPAL OF THE INSTITUTION
This is to certify that the dissertation entitled “A ANALYTICAL STUDY OF MOLECULAR BIOLOGICAL MARKER IL-6 IN
SYNOVIAL FLUID IN KNEE JOINT BEFORE AND AFTER PRP INJECTION IN PATIENTS WITH OSTEOARTHRITIS AND
RHEUMATOID ARTHRITIS” is a bonafide research work done by Dr.V.ANAND BABU under the guidance of Dr .B .K .DINAKAR RAI, M.S (Ortho), Professor & HOD, Department of Orthopaedics, PSGIMS&R, Coimbatore.
Dr. RAMALINGAM, Dr. B .K. DINAKAR RAI, Principal, Professor & HOD,
PSGIMSR& R, Department Of Orthopaedics, Coimbatore. PSGIMS& R
Coimbatore.
PLAGIARISM
PLAGIARISM CERTIFICATE
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SYNOVIAL FLUID IN KNEE JOINT BEFORE AND AFTER PRP INJECTION IN PATIENTS WITH OSTEOARTHRITIS AND
RHEUMATOID ARTHRITIS” of the candidate Dr.V.ANAND BABU with registration Number 221712451 for the award of Master of Surgery in the branch of Orthopaedics. I personally verified the urkund.com website for the purpose of plagiarism Check. I found that the uploaded thesis file contains from introduction to conclusion pages and result shows ONE percentage of plagiarism in the dissertation.
Guide & Supervisor sign with Seal.
ACKNOWLEDGEMENTS
I sincerely thank God for giving me the strength and will power to perform and complete this research.
Its indeed a great pleasure in thanking those who have helped me in the completion of this research and naming them all will be next to impossible. I have tried my best to name all those who have helped me various ways.
First of all I would like to offer my humble gratitude and sincere thanks to my guide, Dr. DINAKAR RAI B. K., Professor and head of the department of Orthopaedics at PSGIMS&R, Coimbatore for his valued guidance, motivation, comments, suggestions and assistance that he has given both during the period of study and also during the preparation of this dissertation.
I owe a great deal of respect and gratitude towards my professors DR. ARVIND KUMAR S. M. and DR. VENKATESH KUMAR N. for
their timely guidance and support towards the completion of this dissertation.
I thank my associate professors DR. PRASANNA C and DR.
SANDEEP M. M. R. for their support towards this study.
I also thank DR. SANKARGANESH JEYARAJ, PhD, Department of CMMT (Molecular Medicine), PSGIMS&R.
I thank my assistant professor DR. SATHYAMOORTHY and for his support towards this study.
I also thank the Assistant professors and Senior Residents of the Department of Orthopaedics PSGIMS&R, Coimbatore for their support and encouragement
I also thank the staff of the Department of Orthopaedics, the ward staff and the OT staff, PSGIMS&R, Coimbatore for their support.
I also offer my thanks to my Senoirs, Juniors and fellow post graduates for their continued support throughout the study.
I thank my parents Mr.Vadivelu and Mrs. Sargunam, my wife Dr. Shanthi for their whole hearted support and encouragement through the
course of the study without which this study could have not been possible.
TABLE OF CONTENTS
S NO. TITLE PAGE NO.
1. INTRODUCTION 1 - 3
2. AIM 4
3 OBJECTIVES 5
4. REVIEW OF LITERATURE 6 - 44
5. MATERIALS & METHODS 45-57
6. RESULTS 58 -
7. DISCUSSION CONCLUSION 8. LIMITATIONS
9 BIBLIOGRAPHY
10. ANNEXURES
LIST OF TABLES
SNO. TITLE PAGE
NO.
1. AGE DISTRIBUTUION IN OSTEOARTHRITIC
CASES. 59
2. GENDER DISTRIBUTUION IN OSTEOARTHRITIC
CASES. 60
3. BMI DISTRIBUTUION IN OSTEOARTHRITIC
CASES. 61
4 KELLEGREN LAWRENCE RADIOLOGICAL
GRADING OF OSTEOARTHRITIC CASES. 62 5
AGE DISTRIBUTUION IN RA CASES 64 6
GENDER DISTRIBUTUION IN RA CASES 65 7 COMPARISION OF PRE AND POST PRP LEVEL
OF IL-6 USING PAIRED T TEST IN
OSTEOARTHRITIS CASES. 66
8 COMPARISION OF PRE AND POST PRP LEVEL OF IL-6 USING PAIRED T TEST IN RHEUMATOID
ARTHRITIS CASES. 68
9 DIFFERENCE IN PAIN SCORE ACROSS FOLLOW-
UP IN OSTEOARTHRITIS CASES. 71
10 DIFFERENCE IN PAIN SCORE ACROSS FOLLOW-
UP IN RHEUMATOID ARTHRITIS CASES. 74
LIST OF FIGURES
S NO. TITLE PAGE NO.
1. COMPARISON OF NORMAL KNEE AND
OA KNEE. 8
2. VICIOUS CYCLE OF INNATE IMMUNE
ACTIVATION IN OSTEOARTHRITIS. 17
3. KL GRADING OF OA KNEE. 19
4 PATHOGENESIS OF RA INVOLVING
INNATE IMMUNITY 26
5 ROLE OF INFLAMMATORY MEDIATORS
IN RA 30
6 ACR CRITERIA FOR RA 31
7 MECHANISM OF ACTION OF IL-6 37 8 BLOOD COMPONENTS AFTER
CENTRIFUGATION 39
9 VARIOUS GROWTH FACTORS IN PRP. 42 10 SYNOVIAL FLUID ASPIRATION 49 11 PREPARATION OF PRP BY DOUBLE SPIN
METHOD. 50
12 PROCESS OF PRP INJECTION 51
13 HUMAN IL-6 ELISA KIT 52
14 CALCULATION OF OD VALUE AND IL-6
CONCENTRATON. 54
15 OPTICAL DENSITOMETRY MACHINE-
THERMOSTAT 55
16 SUMMARY OF IL-6 ASSAY
DETERMINATION 56
17 VISUAL ANALOG SCALE (VAS) 57 18 AGE OF OSTEOARTHRITIC CSES 59 19 GENDER OF OSTEOARTHRITIC CASES 60 20 GENDER OF OSTEOARTHRITIC CASES 61 21 KELLEGREN LAWRENCE
RADIOLOGICAL GRADING 62
22 AGE OF RHEUMATOID ARTHRITIS
CASES 64
23 GENDER OF RHEUMATOID ARTHRITIS
CASES 65
24
CHANGE IN THE IL-6 VALUES BEFORE AND AFTER PRP IN OSTEOARTHRITIC CASE (N=29)
67 25 CHANGE IN THE IL-6 VALUES BEFORE
AND AFTER PRP IN RA CASE (N=24) 69 26 CORRELATION BETWEEN CHANGE IN
IL-6 VALUES AND PAIN SCORE 75
ABBREVATIONS
• OA – OSTEOARTHRITIS
• RA – RHEUMATOID ARTHRITIS
• IL - INTERLEUKIN
• TNF-ALPHA – TUMOUR NECROSIS FACTOR ALPHA
• PRP – PLATELET RICH PLASMA
• EULAR – EUROPEAN LEAGUE AGAINST RHEUMATISM
• ECM – EXTRA CELLULAR MATRIX
• AGE – ADVANCED GLYCATION PRODUCTS
• MMP – MATRIX METALLOPROTEINASES
• SASP – SENESCENCE ASSOCIATED SECRETARY PHENOTYPE
• ROS – REACTIVE OXYGEN SPECIES
• PAMPS – PATHOGEN ASSOCIATED MOLECULAR PATTERNS
• DAMPS – DANGER ASSOCIATED MOLECULAR PATTERNS
• TLRS – TOLL LIKE RECEPTORS
• KL – KELLGREN AND LAWRENCE
• OARSI – OSTEOARTHRITIS RESEARCH SOCIETY INTERNATIONAL
• PEMFS – PULSED ELECTROMAGNETIC FIELDS
• ESW – EXTRA CORPOREAL SHOCKWAVE THERAPY
• LIPUS – LOW INTENSITY PULSED ULTRASOUND
• AAOS – AMERICAN ACADEMY OF ORTHOPAEDIC SURGEONS
• FLS – FIBROBLAST LIKE SYNOVIOCYTES
• DC – DENDRITIC CELLS
• RF – RHEUMATOID FACTOR
• ACR – AMERICAN COLLEGE OF RHEUMATOLOGY
• DMARDS – DISEASE MODIFYING ANTI RHEUMATIC DRUGS
• CTLA-4 – CYTOTOXIC T LYMPHOCYTE ASSOCIATED ANTIGEN 4
• ACD – ACID CITRATE DEXTROSE
• CIA – COLLAGEN INDUCED ARTHRITIS.
• VEGF – VASCULAR ENDOTHELIAL GROWTH FACTOR
• PDGF – PLATELET DERIVED GROWTH FACTOR
• HGF – HEPATOCYTE GROWTH FACTOR
• FGF- FIBROBLAST GROWTH FACTOR
• EGF- ENDOTHELIAL GROWTH FACTOR
• ELISA – ENZYME LINKED IMMUNE SORBENT ASSAY
• OD – OPTICAL DENSITOMETRY
• VAS – VISUAL ANALOGUE SCALE
• ANOVA – ANALYSIS OF VARIANCE
INTRODUCTION
INTRODUCTION
Rheumatic diseases are among the oldest diseases recognized worldwide. The classification of rheumatic diseases is hampered due to unknown causative factor and heterogeneity in their clinical presentation[1]. More than 100 different conditions are listed under rheumatic diseases, as it does not have a clear boundary including Osteoarthritis(OA), Rheumatoid arthritis(RA), autoimmune diseases like systemic lupus erythematosus, Osteoporosis, Gout, Fibromyalgia, back pain and so on[2].
Among the chronic rheumatic diseases, Osteoarthritis and Rheumatoid arthritis posses major public health problem, as is has an impact on individuals, societies and economic costs in all countries with the estimated global burden of 3.8% for OA knee[3] and 1-2% for RA[4] with a tendency more towards advancing age.
The patho physiology of both was initially thought to be a different entity, degenerative for OA and inflammatory for RA. Current concepts have changed the “wear and tear” pathology of OA to inflammatory basis[5]. Various researchers have come forth in the field of rheumatic medicine, to better understand the relationship of biomarkers and age related diseases.
With the progress in molecular biology, various group of cytokines, both pro and anti inflammatory were found to be involved in the pathogenesis of rheumatic diseases, of which the most important group of pro-inflammatory cytokines are interleukin-1𝛽 (IL-1 𝛽 ), tumor necrosis factor (TNF𝛼), interleukin-6 (IL-6), interleukin-15 (IL-15), interleukin-17 (IL-17) and interleukin-18 (IL-18)[6].
Although some of the causes of rheumatic diseases (genetic/aging/trauma) are not preventable, we can try to alter the disease course at the molecular level, through a better understanding of the sensitive cytokines. Hence in our study we focused on IL-6, a pro-inflammatory cytokine present in the individuals with confirmed clinical diagnosis of OA and RA at elevated levels in the synovial fluid[7].
From the basic science to surgery, there are lot of grey areas were we need to contribute to, so as to avoid total knee replacements for all cases of rheumatic diseases. Thus there is a need for new therapies to target inflammatory mediators without compromising innate immune responses.
Injective biologics like Platelet rich plasma (PRP) has recently seen as a promising therapeutic tool by researchers for diseases like OA and RA, for its ability to affect tissue regulation & in pain management[8].
Various studies have evaluated the effectiveness of PRP, but very few studies established a correlation between PRP and inflammatory mediators.
In an attempt to improve the usage of PRP for arthritic cartilage diseases, we should understand the strong implication of the same in down regulating the inflammatory mediators. Hence forth we undertook this study, to establish a strong role of IL-6 in OA & RA knee and the effect of PRP in altering the catabolic milieu of cytokines within the intra arthritic environment.
AIM
AIM
To assess the effect of PRP injection and its influence on molecular biological marker IL- 6 in the synovial fluid of knee joint in osteoarthritis and rheumatoid arthritis patients.
OBJECTIVES
OBJECTIVES
i. To compare the levels of cytokine IL-6 in the synovial fluid of OA and RA affected knee joints, pre and post PRP injections.
ii. To assess the effect of PRP on IL-6.
iii. To assess the effect of PRP on pain management using visual analog scale.
REVIEW OF LITERATURE
REVIEW OF LITERATURE
RHEUMATIC DISEASES
Ancient descriptions often referred to musculoskeletal pain and dysfunction as “rheumatism” [9]. Rheumatic diseases was first recognized by Hippocrates in 4th century BC. He refer atleast partially to joint diseases, in his eighteen published aphorisms. Flow of pain through the joints of the body - ‘rheuma’ , was first described in 4th century A.D[10]
History of osteoarthritis also very important as well, because it can help broaden our perspective on past and present controversies[11]. Term OA knee was first introduced by john.k.splender, but as a preferable term to rheumatoid arthritis.[9]
Modern usage of the term OA and its differentiation from RA , introduced by Garrod in 1907. However he was unable to make consistent distinctions between some of the pathology of OA and RA.[1]
Hence we undertook this study to review previous literatures in OA and RA and thereby conclude role of inflammation in both the diseases.
OSTEOARTHRITIS
Osteoarthritis is an enlightened chronic degenerative disorder of multifactorial etiology[12]. Broadly defined by joint symptoms and structural pathology. Joint pathology is diverse.[13] It consists of degradation of cartilage, inflammation of synovial membrane and tissues, formation of osteophyte,joint space narrowing and sclerosis of subchondral bone[12].Osteoarthritis has been seen as a prototypical non inflammatory arthropathy.[14]
OA is probably not a single disease but represents the final end result of various disorders leading to joint failure.[15] OA is analogous to organ failure like renal or cardiac system. pathological observations in the advance to disease are as much a product of attempted repair as of the primary insult or damage which contributed to initiation of the process.[13]
Unlike an automobile tire that wears thin over time, the tissues affected by OA contain living cells that respond to mechanical stimulation and function to maintain joint homeostasis.[16]
Figure – 1
COMPARISON OF NORMAL KNEE AND OA KNEE.
EPIDEMIOLOGY GLOBAL STATUS
OA ranks eighth in all diseases globally. Higher proliferative rate of OA is seen in India among the world.[17] OA seen as one of the most prevalent musculoskeletal disease among the world.[18]
Hinman RS et al stated that OA causes joint disability in approximately 100 million people among the world having age over 45 years, which is approximately 15% of all musculoskeletal disorders. More
than 50% of population over 65 years have radiographic confirmation of OA in any of the joints.
OSTEOARTHRITIS IN INDIA
Osteoarthritis is the second most common rheumatologic problem and it is the most common cause of locomotor disability in the elderly with a prevalence of 22% to 39% in India. Prevalence of OA knee in rural and urban India is 3.9% and 5.5%, respectively.[19]
Jain S et al , in a survey based study stated that “India is predicted as chronic disease capital by 2025 and expected to have 60 million people with arthritis”. Approximately 40% population of more than 70 years shows OA, in which nearly 2% have severe knee pain and disability.
Nearly, 45% of women over the age of 65 years have symptoms while radiological evidence is found in 70% of those over 65 years.[20] OA is more common in women than men, but the prevalence increases dramatically with age.
RISK FACTORS
▪ Old Age
▪ Instability of Joint causing hypermobility
▪ Obesity
▪ High energy loading sports injury
▪ Repetitive stress to knee joint while heavy weight lifting
▪ Post operative immobilisation
▪ Peripheral neuropathy
PATHOPHYSIOLOGY
OA is a slowly progressive disease of synovial joints characterized pathologically by focal destruction of the articular cartilage, a hypertrophic response in neighboring bone that results in osteophyte formation and subchondral sclerosis, variable degrees of synovial inflammation, a thickening of the joint capsule, and damage to soft tissue structures including ligaments and, in the knee, the meniscus[21].
Cartilage acts as a cushion between bones of joints and prevents rubbing of bones. Cartilage, bone, muscles, ligaments and other joint tissues and structures function as a biomechanical organ system that maintains
proper movement and prevents excessive joint loading[13]. Changes occurring in the tissue homeostasis of articular cartilage and subchondral bone, determine the predominance of destructive processes. A key role in the pathophysiology of articular cartilage is played by cell/extra-cellular matrix (ecm) interactions.[22]
One of the mechanisms of articular cartilage damage is stiffness of subchondral bone, if the bone becomes stiffer; it may be less able to absorb impact loads, which may in turn lead to increased stresses in the cartilage[23]
Chondrocytes present in cartilage, have very low metabolism activity with no ability to repair cartilage. Moreover, unlike all other tissues, articular cartilage, once damaged, cannot respond by a usual inflammatory response because it is non-vascularized and noninnervated.
OSTEOARTHRITIS - AN AGE RELATED DISEASE
Current conceptual framework shows that ageing has relationship with OA. Ageing increases the susceptibility to OA but alone does not cause it.
Factors like reduced proprioception and sarcopenia which occurs outside the joint and changes happening in the cell and matrix within the joint tissues contribute to the development of OA.[16]
Biomechanical properties of cartilage gets altered due to accumulation of advanced glycation end-products (AGE) and making it more “brittle” and susceptible to degeneration. Increased stiffness by AGE cross linking may contribute to the age-related failure of the collagen network in human articular cartilage to resist damage.[24]
FACTORS RESPONSIBLE FOR AGE RELATED CHANGES
1. Imbalance in the production and activity of pro-inflammatory and catabolic mediators to the activity of anabolic factors, including the growth factors
2. Overproduction of matrix degrading enzymes including the matrix metalloproteinases (MMP’s) and aggrecanases
3. Imbalance in chondrocyte signaling
4. Cellular (Chondrocyte) Senescence - senescence-associated secretory phenotype (SASP)
5. Increased levels of reactive oxygen species (ROS)
➢ CHONDROCYTE SENESCENCE
Chondrocyte is the one of the cell type present in articular cartilage and is responsible for both the synthesis and breakdown of the cartilaginous extracellular matrix.[25] Chondrocyte metabolic activity are regulated by Signals generated by cytokines, growth factors and the matrix.
Mechanism of senescence more likely caused due to oxidative damage, oncogenes and inflammation. Oxidative stress can promote cell senescence,especially chondrocyte senescence.[26] Senescent chondrocytes adopt a secretory phenotype which results in increased production of MMPs- MMP-3 and MMP-13 in cartilage with ageing.[27]Ageing cells can result in the senescent secretory phenotype.[28]
➢ OXIDATIVE STRESS
With ageing, Levels of reactive oxygen species (ROS) increase in cartilage and chondrocytes from older adults are more susceptible to ROS- mediated cell death.[29] Increased ROS production can damage both intracellular proteins and the extracellular matrix. Increased levels of ROS also results in DNA damage, which has been noted in OA cartilage[30]
Imbalance in catabolic and anabolic signaling in cartilage that results in progressive matrix destruction - central feature of OA. Many Studies providing evidence that age-related oxidative stress plays a key role in this catabolic-anabolic imbalance[31]. Aged chondrocytes respond poorly to growth factor stimulation and so are unable to maintain homeostasis in the articular cartilage. Ageing initiates and promotes a low grade pro inflammatory state.
Various inflammatory mediators found to be increased in OA, including IL-1, IL-6, IL-8, TNF-a and other cytokines.[30]
Stannus et al. concluded that Levels of systemic markers of inflammation have been shown to correlate with pain and function in older adults with knee OA.[32]
Franceschi et al, coined the term “INFLAMM-AGING” to describe the pro-inflammatory state that occurs with increasing age. It was originally proposed to be the result of an accumulation over time of an increased
“antigenic load” that resulted in immunosenescence.[33]. Morrisette-Thomas et al. examined levels of 19 inflammatory markers from an ongoing longitudinal study of older adults in Italy called the In CHIANTI study.
Study reported a significant age-related increase in pro-inflammatory markers, including hsCRP, IL-6, IL-15, IL-18.[34]
OA – AN INFLAMMATORY DISEASE
With the progress in molecular biology and invention of inflammatory mediators in 1990’s, this paradigm has dramatically changed. New concept of ‘Inflammatory theory’ has arrived. Studies have freed OA from its reputation as a noninflammatory, from ‘wear and tear’ arthritis.
➢ SYNOVITIS IN OA
Joint swelling is attributed to inflammation and it reflects the presence of synovitis due to thickening of synovium or to effusion.
Why the synovium getting inflamed in OA remains controversial.[35]
Once degradation of articular cartilage sets in, the minor cartilage fragments which tend to fall into the knee joint cavity and comes in to contact with the synovium. Synovial tissue Considers this small fragments as foreign body then the synovial cells recruit inflammatory cells as inflammatory response.
Inflammatory cells which are recruited in the synovial tissue stimulates the chondrocytes which results in the production of MMP’s and
increases the degradation of cartilage. Cartilage alteration which in turn amplifies synovial inflammation, thereby creating a vicious cycle.
➢ INNATE IMMUNITY- A TRIGGER FOR OA
Factors responsible for inducing catabolic / Inflammatory response in chondrocytes are:
• Pathogen-associated molecular patterns (PAMPs)[36]
• Danger-associated molecular patterns (DAMPs)[36]
• Toll-like receptors [TLRs] - basic signaling receptor[37]
➢ ADIPOSITY & OBESITY
Infrapatellar fat pad, an adipose tissue localized in the knee, was found to be a potential source of adipokines such as IL-6[38] Loeser et al. in an animal study concluded that Apart from aging fat, joint tissues including the meniscus and cartilage can be a source of pro-inflammatory mediators.[39]
Figure - 2
VICIOUS CYCLE OF INNATE IMMUNE ACTIVATION IN OSTEOARTHRITIS.
Interestingly, Recent epidemiological and clinical studies have highlighted that a metabolic syndrome rather than obesity itself has the greatest impact on the initiation and severity of OA.[40]
DIAGNOSTIC MODALITIES
Most commonly OA patients are evaluated by plain radiographs AP and Lateral views with weight bearing. Radiographic classification for OA were described by Kellgren and Lawrence in 1957 [41]
KL GRADING of OA knee.
Figure - 3
KL GRADING OF OA KNEE.
TREATMENT OPTIONS
Main aim in the management of OA is to control the pain, to improve the quality of life and functionality. OARSI guidelines states that Non- pharmacological treatment should always be the first line of treatment for knee OA[42]
NON-PHARMACOLOGICAL MANAGEMENT
• Light to moderate physical activity[43]
• Exercises - aerobic exercises, strenghthening exercises, stretching exercises[44]
• Aquatic therapies[43]
• Weight management – According to “The Framingham study”, a 50% risk reduction for knee OA if the patient weight loss of 12 lb.[45]
• Transcutaneous electrical nerve stimulation[46]
• Pulsed electromagnetic fields (PEMFs), Extracorporeal shock wave therapy (ESW), Low-intensity pulsed ultrasound
(LIPUS)[47]
PHARMACOLOGICAL TREATMENTS
In Early OA, Non-surgical treatments are the first approach for the management.
• Cyclooxygenase inhibitors (Acetaminophen and NSAIDS)[48]
• Topical NSAIDs[42]
• Opioids- Tramadol, a weak opioid[49]
• Gabapentinoids- for neuropathic pain in OA.[50]
• Glucosamine[47]
• Chondroitin sulphate[47]
• Diacerein[47]
• Bisphosphontes[51]
INJECTIVE TREATMENTS
• Corticosteroids
• Viscosupplementation
• Injective biologics
INTRA-ARTICULAR CORTICOSTEROID INJECTIONS
Intra-articular corticosteroid injection was first described by White and Norton in 1958 for OA Knee[52]
Indication for Intra-articular corticosteroid injection are mainly after failure of pharmacotherapy[53]. Zuber et al has suggested that injection can be repeated after six weeks, but not more frequently than every 6–8 weeks.[54]These are the commonly used intra articular injections for OA knee in practice - Hydrocortisone, methylprednisolone, Triamcinolone, Betamethasone.
INTRA-ARTICULAR VISCOSUPPLEMENTATION
Hyaluronic acid is the most commonly injected visco supplement drug to treat OA knee. However due to its inconclusive and conflicting current evidence regarding efficacy of this drug[55], AAOS doesn’t recommend Hyaluronic acid use.[48]
INJECTIVE BIOLOGICS
The ultimate Need for minimally invasive treatment for OA knee in early stages and to improve the status of the joint surface and a fast return to full activity led to the recent development of biological treatment.[56]
• Platelet Rich Plasma
• Mesenchymal Stem Cells
• These above said treatments reported to be safe and well tolerated by the patients and found to be superior in terms of pain relief.[57]
SURGICAL TREATMENT
Surgery plays an important role in the management of many OA patients with disease progress to advanced joint destruction.[58] Severe pain and deformity, impaired movement and (or) instability of the knee due to osteoarthritis are indications for total knee replacement.[59]
In these detailed existing literature, we want to bring about the role of inflammation in OA knee played by IL-6, and thereby create a strong evidence in the use of injectable biologics( PRP) against this inflammatory marker.
RHEUMATOID ARTHRITIS
Rheumatoid arthritis is an inflammatory rheumatic disease with course affecting articular and extra-articular structures which leads to erosive joint damage and functional impairment and resulting in pain, disability[60]Rheumatoid arthritis primarily affects the lining of the synovial joints.[61] Prevalence of rheumatoid arthritis is relatively constant in many populations, ranging from 0.5–1.0% regardless of ethnicity and race.[62]
ETIOLOGY FOR RHEUMATOID ARTHRITIS
Numerous works by researchers leads to a observation that underlying immuno reactivity antedates the onset of arthritis. Many mediators of inflammation playing an important role in initiating RA. They are listed below:
1. Small molecule mediators – Prostaglandin E2 and Arachidionic acid[63]
2. Autoantibodies[64]
3. Cytokines[65]
4. Growth factors[66]
5. Adhesion molecules[67]
6. Matrix metalloproteinases[68]
PATHOGENESIS OF RA
Rheumatoid arthritis is a complex autoimmune inflammatory disease that involves many cell types. It includes macrophages, T cells, B cells, chondrocytes, fibroblasts, and dendritic cells. Other factors which are contributing to etiopathogenesis of RA are:
1. Innate & Adaptive immunity[69]
2. Genes[70]
3. Autoimmunity[71]
4. Cytokine network
5. Environmental factors[72]
6. Pathogens[73]
ROLE OF INNATE & ADAPTIVE IMMUNITY
Adaptive and innate immune responses that occur in the synovium have been implicated in the pathogenesis of RA. Innate immunity is a primitive pattern-recognition system that can leads to rapid inflammatory responses. It “prepares” the joint for subsequent recruitment of immune cells and inflammatory cells.
In the early phase of RA, role of innate immunity comes to play in those individuals with underlying immune hyperreactivity as evidenced by the production of autoantibodies. Innate immunity activates fibroblast-like synoviocytes (FLS), dendritic cells (DC), and macrophages.
Mechanism and pathogenesis of RA involving Innate Immunity is depicted in the below figure:
FIGURE 4:
PATHOGENESIS OF RA INVOLVING INNATE IMMUNITY
ROLE OF GENES IN RA
Genetic influence have an association of around 40% with RA. Many researchers observed that HLA-DR4 occurred in 70% of RA and 4-5 times risk of developing RA in those who have strong association with HLA DR- 4.[70]
Class II MHC genes, especially those which containing specific 5 amino acid sequence in hypervariable region of HLA DR-4 have a prominent association. Other genes which are associated with RA are:
1. Polymorphism in PTPN22[74]
2. PAD14 GENE[74]
ROLE OF AUTOIMMUNITY IN RA
Many years before the onset of clinical arthritis, evidence of autoimmunity can be present in RA. Autoantibodies, such as Rhematoid Factor(RF) and Anticitrullinated protein antibodies are commonly associated with RA. Potential Autoantigens that are responsible for RA are:
1.Cartilage antigens
-Type II collagen -gp39
-Cartilage link protein
-Proteoglycans -Aggrecan 2. Citrullinated peptides
3. Glucose-6-phosphoisomerase 4. HLA-DR (QKRAA)
5.Heat-shock proteins
6. Heavy-chain binding protein (BiP) 7.hnRNP-A2
8. Immunoglobulins (IgG)
RHEUMATOID FACTOR
First direct evidence that autoimmunity might play a role in RA was the identification of RF as a self-antibody which binds to the Fc portion of IgG. RF able to fix the complement and activate it by the classic pathway.
Rheumatoid synovial tissue produces large quantities of IgG RF and form complexes. RF-containing immune complexes are readily detected in RA synovial tissue and the surface layers of cartilage.[75]
ANTICITRULLINATED PEPTIDE ANTIBODIES In 1970s , Researchers observed that antibodies directed against
keratin were detected in serum of RA patients. The primary antigen to which those antibodies are directed was filament-aggregating protein named filaggrin. These produced antibodies against keratin bind to epitopes on filaggrin that contain citrulline. Anticitrullinated peptide antibodies have been reported positive in serum samples of 80% to 90% of RA patients. It is more specific for RA than RF, with specificity around 90%.[76]
ROLEOF CYTOKINES
Cytokines have a important role in the pathophysiology of RA as pro- inflammatory cytokine like IL-1,TNFα, IL-6, IL-17 stimulates inflammation and causes degradation of bone and cartilage. There is an imbalance between the pro- and anti-inflammatory cytokine which leads toimmune complications.[77]
Anti-cytokine agents seem to evolve as potential therapies for treating RA. There is a need to develop those potential agents that target plenty of pathways which take part in the pathogenesis of RA.
In our study we decided tolook in to this IL-6, pro inflammatory cytokine in more detail and try to strongly establish its role of inflammation in rheumatoid patients.
FIGURE 5: ROLE OF INFLAMMATORY MEDIATORS IN RA
DIAGNOSIS
Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) provide the best information about the acute phase response in any inflammatory arthropathy[78] Auto antibodies such as anti-CCP and RF are helpful for the diagnosis of RA.
Mild juxtaarticular osteoporosis and Soft tissue swelling and may be visible as initial radiographic features in early - RA .Radiographic signs like erosions, joint space narrowing and subluxation can be seen at later stage of RA. [79] Before , 1987 ACR criteria was applied for the diagnosis of RA. Recently, new ACR 2010 criteria has been followed now.[80]
ACR 2010 CRITERIA (ALETAHA et al) [80]
FIGURE 6: ACR CRITERIA FOR RA
TREATMENT
Over the past three decades, treatment of RA has been revolutionized and pioneered by recent advances in understanding its pathologic mechanisms and development of therapies which target them. These newer modalities of treatment have shown great promise in improving disease outcomes.
PHARMACEUTICAL TREATMENT OF RA:
CONVENTIONAL DMARDs:
Disease Modifying Anti-Rheumatic Drugs (DMARDs) became the mainstay of RA treatment in the 1970s. Guidelines typically recommend starting with conventional DMARD treatment.
- Methotrexate[81]
- Hydroxychloroquine[82]
- Sulfasalazine[83]
- Leflunomide[84]
Other medications like azathioprine, minocycline, doxycycline and cyclosporine are used as adjunctive or substitute medications. But all have been shown beneficial effects on disease activity in RA.
BIOLOGIC DMARDs:
Biologic DMARDs, has revolutionized the treatment of RA by targetting specific abnormalities of immunesystem.
• Adulimumab, etanercept, infliximab ,certolizumab, golimumab - Tumor necrosis factor (TNF) blocking agent.[85]
• Rituximab is a chimeric anti-CD20 monoclonal antibody [86]
• Abatacept is a fusion protein of Cytotoxic T lymphocyte-associated Antigen 4 (CTLA-4) and t Fc portion of IgG1.[87]
• Anakinra, an IL-1 receptor antagonist [88]
• Tocilizumab - antibody that binds to the IL-6 receptor .It is recently approved biologic therapy for moderate to severe RA.[89]
Aggressive use of conventional DMARDs and biologic DMARDs has allowed patients to achieve a decreased joint destruction and improved function. These medications are not without side effects or long-term risks.
We focused on the therapy which targets a novel cytokine IL-6 other than toclizumab.
INTERLEUKIN-6 (IL-6)
- an historical cytokine in a new era.
❖ IL-6 is one of the major cytokine found in damaged articular cartilage, apart from the major cytokines IL-1ß and TNF[90]. Currently it serves both as a diagnostic and prognostic biomarker.
❖ The exact role of IL-6 in inflammation and OA is less clear compared with other cytokines.
❖ Hence a better understanding of the individual role of cytokine IL-6, is of utmost important in order to develop adequate anti cytokine therapies.
❖ BIOCHEMISTRY
IL-6 is a 184 aminoacid residue protein first identified in mid 1980’s.[91]
❖ SOURCES[91]
• Innate immune cells- macrophages, dendritic cells and mast cells
• B cells
• CD4 effecter T helper cells
• Non leucocytes- endothelial cells, fibroblasts, astrocytes, epithelial cell and several malignant cells.
IL-6 & IL-6 receptor
Two principal mechanisms by which IL-6 is known to interact with the target cells are[92]:
• CLASSICAL IL-6 SIGNALING
- IL-6 will exert its action via binding to membrane bound IL-6 receptor (mIL-6R) along with the signal transducer domain gp130.[93]
- Once after coupling, tyrosine kinase cascade will start, leading to activation of JAK-STAT pathways resulting in its function in various arthritic disorders.[94]
• TRANS IL-6 SIGNALING
- IL-6 exerts its action through soluble IL-6R (sIL-6R) along with the signal transducer domain gp130.
- 80% of sIL-6R produced by ectodomain shedding- proteolytic
cleavage of the mIL-6R via ADAM 17 and 20% by direct synthesis.[95]
sIL-6R is the critical component in IL-6 mediated signaling wherein the degree of signaling between the two pathways regulated the apparent dichotomy between pro & anti inflammatory pathways.[96]
❖ FUNCTIONS[97]
- Immune regulation - Inflammation - Oncogenesis
- Key mediator in the development of many chronic inflammation and autoimmune diseases.
- Dienz et al. 2009 , in his study, came to a observation and stated that IL-6 stimulates B cells and produces immunoglobulins by differentiating into plasma cells.[98] This observation also in accordance with a study published by Jogo et al..In a study conducted by Marin et al, he concluded that Trans-signalling via sIL-6R increases monocyte-specific chemokines which are secreted by endothelial cells.
FIGURE 7: MECHANISM OF ACTION OF IL-6
- The first solid evidence was from studies using IL-6 deficient mice.
Sasai et al in their study found that IL-6 deficient mice have either delayed onset or less severe form of disease in collagen induced arthritis (CIA).[99]
IL-6 & OA
A 15 year follow up study conducted by Livshits et al showed that higher serum levels of IL-6 associated with an increased chance of OA and hence supported IL-6 as a biomarker for early diagnosis of OA.[100]
A similar study conducted by Stannus et al included 172 OA patients, found that IL-6 levels was directly associated with joint space narrowing, knee cartilage loss and osteophytes formation.[32]
IL-6 & RA
- Walsh et al. 2005, observed that erosions in inflammatory arthritis are mediated by Osteoclasts. In a study by Yoshitake et al, concluded that IL-6 by acting on hematopoietic stem cells increases osteoclast recruitment.[101]
- Study by Murphy et al, showed that synovial lining cells, sublining fibroblasts and infiltrating leukocytes produces matrix
metalloproteinases (MMPs) in RA.[102]
- Roux-Lombard et al. in his work stated that in early RA, IL-6 and CRP levels correlating with proMMP-3 suggesting a link between IL-6 and proteinase activity[103].
PLATELET RICH PLASMA
It is an autologous blood having concentration of platelets above the normal value usually by 3-5 times[104]. It has been investigated in the recent years, for the treatment of variety of musculoskeletal disorders, because of its physiological role in the healing process in addition to homeostasis.
FIGURE: 8
BLOOD COMPONENTS AFTER CENTRIFUGATION.
➢ THE NEED FOR PRP
In rheumatic diseases ( OA & RA), articular cartilage is damaged primarily by an alteration of normal metabolism, which favors a decrease in anabolism and increase in catabolism. These changes occur simultaneously with the inflammation of synovium and synovial
fluid. In the era of increasing non surgical treatments, newer biologic products aim to slow the disease process, relieve pain and increase the life expectancy. It is cost effective and less reactive.
➢ IMPLICATION IN ORTHOPAEDICS
Till date it has been used in variety of orthopaedic settings to treat[105]-
✓ Achilles tendinopathy
✓ Anterior cruciate ligament taers
✓ Plantar fasciitis
✓ Epicondylitis
✓ To augment spinal fusion, bone healing, rotator cuff repair
✓ Arthroplasty
✓ Cartilage regeneration
✓ Non healing wounds
Of all these conditions, only in epicondylitis its use is proved by level-I clinical evidence[106]
➢ PRINCIPLES OF PRP PREPARATION
It is prepared by a process of differential centrifugation. Based on different specific gravity, various cellular constituents are adjusted to
sediment by using specific acceleration forces. Among the various ways of PRP preparation, the 2 most common and widely used methods are[107]:
-
PRP method
-
Buffy coat method
➢ COMPONENTS OF PRP
Each PRP preparations vary depending on the patient factors and the automated systems used to generate it. Various cellular and molecular components of platelets form the main stay of PRP. Platelet alpha- granules contain and on activation release numerous growth factors (GF)[108], including vascular endothelial GF(VEGF), platelet derived GF(PDGF), hepatocyte GF(HGF), fibroblast GF(FGF), epithelial GF(EGF) and TGF-ß, which could change the catabolic milieu in the environment.
FIGURE:9
VARIOUS GROWTH FACTORS IN PRP.
➢ MECHANISM AND ACTION OF PRP
Its main role in reestablishing the homeostasis of joint tissues is exerted by a wide range of actions.
- Anti inflammatory - Anti oxidative - Analgesic
- Chondroprotective
- Anabolic- trophic effects
ROLE OF PRP IN ARTHRITIC CARTILAGE BY VARIOUS STUDIES
The favorable clinical results of PRP injection in arthritic joints was attributed previously to the anabolic effects of growth factors on the articular cartilage, but now as a result of few in vivo studies, it was found that anti inflammatory mechanism plays an important role in the clinical improvement of arthritis[109].
o Dose dependant increase in the proliferation of chondrocytes o Induction of autophagy in chondrocytes
o Induces chondrocytes quiescence o Inhibit chondrocytes apoptosis
o Alteration in the inflammatory mediators
- Decrease in pro inflammatory mediators like MMP3, MMP13, ADAMDTS-5, IL-6 & COX-2.
- Increase in TGF ß3, aggregan, collagen, and anti- inflammatory cytokines IL-4, IL-10 & IL-13[110]. o Higher amounts of collagen II and prostaglandin synthesis[111]. o Increased hyaluronic acid secretion[112].
o Increased angiogenesis[113].
o Inhibition of nuclear factor-ĸß pathway[114].
VARIOUS STUDIES COMPARING THE EFFICACY OF PRP
• Cerza et al in their RCT of 120 patients, showed a significantly better clinical outcome with PRP than with HA treatment in arthritic patients, with sustained lower WOMAC scores[115].
• In a similar RCT studied by Kon et al in 150 patients he showed PRP has longer and higher efficacy than HA in reducing the symptoms and thereby recovering the articular physiology. He found a better results in younger and active patients than with older patients[116]. Study carried out but by Gormeli et al in 2015 and concluded that both PRP
& HA treated OA knee patients had better outcome than those treated with normal saline[117].
Hence forth we conducted this study to strengthen the existing evidence for anti inflammatory property of PRP by comparing the most important cytokine IL-6, pre and post PRP injections.
MATERIALS AND
METHODS
MATERIALS AND METHODS
STUDY DESIGN : Prospective ,interventional analytical study
STUDY POPULATION : Clinico-radiologically diagnosed case of osteoarthritis and rheumatoid arthritis
SAMPLE SIZE : 30 Osteoarthritis & 30 rheumatoid arthritis patients
STUDY AREA : Department of orthopaedics, PSGIMSR.
STUDY PERIOD : December 2017 – July 2019
PREREQUISITE : Approval from institutional ethics committee - December 2017
➢ INCLUSION CRITERIA
1. Diagnosed case of osteoarthritis and rheumatoid arthritis in adult patients.
2. Patients consenting for the study.
➢ EXCLUSION CRITERIA
1. Other local joint diseases/ systemic diseases –
• Septic arthritis
• Gouty arthritis
• Seronegative arthritis/connective tissue disorders
• Malignancy
• Adjacent osteomyelitis
• Hemearthrosis
• Impending joint replacement surgery
• Infectious arthritis, joint prosthesis
• Peri articular cellulitis
• Poorly controlled diabetes
• Coagulopathy.
2. Patients not consenting for the study.
3. Congenital skeletal anomalies.
➢ STUDY PROCEDURE
a. Study participants were classified based on the clinic- radiological findings of the knee.
b. After obtaining the informed consent, procedure was started in a systematic manner.
c. PRE PROCEDURE CHECKLIST o Patient demographic details o Weight, height & BMI o Detailed histories
- Presenting complaints - Past medical
- Past surgical - Treatment - Drug - Allergy
- Bleeding tendencies
o Radiological- X-ray AP & lateral view ( Kellegren Lawrence grading)
o Platelet count ( once in 4 patients)
o Informed written consent ( English & Tamil)
d. REQUIREMENTS
o Procedure room and table o Sterile draping
o Sterile gloves
o Syringes – 10ml o Needle – 18G o EDTA tubes
o PRP centrifuge machine o Freshly prepared PRP o Sterile dressing pad.
o Sterile containers o Deep freezer o IL-6 kit
e. PROCEDURE IN DETAIL
1. Pre PRP synovial fluid collection and storage 2. PRP preparation
3. PRP injection
4. Post PRP synovial fluid collection and storage
5. Bio marker analysis – IL-6 in pre and post PRP samples 6. Interpretation of the results
7. Assessment of pain score
Patients under study were given rescue medication (tab.paracetamol 1 gm) for OA.Patients who are rheumatoid were advised to continue DMARDs.
PRE PRP SYNOVIAL FLUID COLLECTION AND STORAGE
Under strict aseptic precautions, synovial fluid was aspirated from the affected knee joint using 10ml syringe. It was transported in a sterile container to the lab. After a process of centrifugation, a supernatant fluid was stored in the deep freezer for final analysis.
FIGURE 10: SYNOVIAL FLUID ASPIRATION PRP PREPARATION
• PRP was prepared by differential centrifugation method.
• First spin- Centrifuge the patients’ blood collected by venipuncture approximately 10ml in anticoagulant containers at 1500 rotations /min for 3 minutes.It gets separated into two layers. Top layer contains plasma and lower layer contains red blood cells.
• Second spin- Top layer is then separated and to do second centrifuge at 2500 rotations /min for 3 minutes. Again it gets separated in to 2 layers. Top layer contains platelet poor plasma and lower layer contains platelet rich plasma. Bottom layer is then collected and used.
FIGURE 11: PREPARATION OF PRP BY DOUBLE SPIN METHOD.
PRP INJECTION
The supernatant fluid after the centrifugation process was aspirated, and under sterile aseptic conditions, the freshly prepared PRP was injected the affected knee joint slowly.
FIGURE 12 : PROCESS OF PRP INJECTION
POST PRP SYNOVIAL FLUID COLLECTION AND STORAGE
After 4 weeks of post PRP injection, patient was reviewed.. After which, under strict aseptic precautions, synovial fluid was aspirated from the affected knee joint using 10ml syringe. It was transported in a sterile container to the lab. After a process of centrifugation, a supernatant fluid was stored in the deep freezer for final analysis.
BIO MARKER ANALYSIS–IL-6 IN PRE AND POST PRP SAMPLES
Interleukin-6 levels were measured using human InterLeukin-6 ELISA kit obtained from Bio Assay Technology Laboratory. The procedure was carried out in accordance with the laboratory manual.
FIGURE 13 : HUMAN IL-6 ELISA KIT
ASSAY PRINCIPLE
This kit is a Enzyme-Linked Immunosorbent Assay (ELISA). IL-6 is added to the wells pre-coated with IL-6 monoclonal antibody. After incubation a biotin-conjugated anti-human IL-6 antibody is added and binds to human IL-6. After incubation unbound biotin-conjugated anti-human IL-6 antibody is washed away during a washing step.
Streptavidin-HRP is added and binds to the biotin-conjugated anti- human IL-6 antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of human IL-6.
The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm using Optical Densitometry (OD) . OD value which is observed correlate to the Human IL-6R concentration. By comparing the OD value of the samples to the standard curve, can calculate Human IL-6R concentration.
CALCULATION OF RESULTS
Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph. These calculations can be best performed with computer-based curve-fitting software and the best fit line can be determined by regression analysis. If the standard have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
FIGURE 14: CALCULATION OF OD VALUE AND IL-6 CONCENTRATON.
FIGURE 15: OPTICAL DENSITOMETRY MACHINE- THERMOSTAT
SUMMARY
FIGURE 16: SUMMARY OF IL-6 ASSAY DETERMINATION
ASSESSMENT OF PAIN SCORE
All patients who received PRP injections were assessed at 1st, 3rd and 6th month post PRP Injection using Visual Analogue Scale (VAS).
Visual Analogue Scale (VAS) (with permission from Erdek, et. al)
FIGURE 17: VISUAL ANALOG SCALE (VAS)
RESULTS
RESULTS
This study was conducted among thirty five patients among whom seven had bilateral osteoarthritis of knee, fifteen had unilateral osteoarthritis of knee, eleven with bilateral rheumatoid arthritis of knee and two cases of unilateral rheumatoid arthritis of knee.
OSTEOARTHRITIS
In total 29 osteo-arthritic knee were studied. The general profiles of the cases are listed below.
TABLE - 1
AGE DISTRIBUTUION IN OSTEOARTHRITIC CASES.
Osteoarthritis of knee
Variable Category Number Percentage
Age
40-49 years 7 24.1%
50-59 years 11 38.0%
60-69 years 7 24.1%
70 years and above 4 13.8%
0 2 4 6 8 10 12
40-49 years 50-59 years 60-69 years 70 years and above
Fig 18: AGE of Osteoarthritic cases
Number
TABLE - 2
GENDER DISTRIBUTUION IN OSTEOARTHRITIC CASES.
Osteoarthritis of knee
Variable Category Number Percentage Gender
Male 7 24.1%
Female 22 75.9%
Male 24%
Female 76%
Fig 19: Gender of Osteoarthritic cases
TABLE – 3
BMI DISTRIBUTUION IN OSTEOARTHRITIC CASES.
Osteoarthritis of knee
Variable Category Number Percentage BMI
<18.5 0 0.0%
18.5 – 24.9 3 10.3%
25.0 - 29.9 17 58.7%
30.0 – 34.9 7 24.1%
≥ 35 2 6.9%
0 2 4 6 8 10 12 14 16 18
Normal (18.5 –24.9) Overweight (25.0 - 29.9)
Obese (30.0 –34.9) ≥ 35
Fig 20 : BMI of Osteoarthritic cases
TABLE - 4
KELLEGREN LAWRENCE RADIOLOGICAL GRADING OF OSTEOARTHRITIC CASES.
Osteoarthritis of knee
Variable Category Number Percentage Kellegren
Lawrence Radiological Grading
1 1 3.4%
2 8 27.6%
3 14 48.3%
4 6 20.7%
Grade 1 3%
Grade 2 28%
Grade 3 48%
Grade 4 21%
Fig 21: Kellegren Lawrence radiological
grading
The mean age of the cases ranging from 43 years to 78 years was 57.17 years with a standard error of 1.799 years. The median age was 55 years with inter-quartile range of 49.5 – 63.5 years. Among the cases 22 (75.9%) were females.
The BMI of the cases ranged between 23.2 to 35.7 . twenty six (89.7%) of them were either obese or overweight.
Kellegren Lawrence radiological grading 1-4 grade 3 was seen in 14 (48.3%).
RHEUMATOID ARTHRITIS(RA):
In total 24 rheumatoid arthritic knee were studied. The general profiles of the cases are listed below.
TABLE 5
AGE DISTRIBUTUION IN RA CASES.
RHEUMATOID ARTHRITIS OF KNEE
Variable Category Number Percentage Age Less than 40 years 9 37.5%
40-49 years 4 16.7%
50-59 years 6 25.0%
60-69 years 3 12.5%
70 years and above 2 8.3%
0 1 2 3 4 5 6 7 8 9 10
Less than 40 years
40-49 years 50-59 years 60-69 years 70 years and above
Fig 22: Age of Rheumatoid arthritis cases
TABLE - 6
GENDER DISTRIBUTUION IN RA CASES.
RHEUMATOID ARTHRITIS OF KNEE
Variable Category Number Percentage
Gender Male 1 4.2%
Female 23 95.8%
Change in interleukin-6 (IL-6) values Osteoarthritis
The pre PRP level of IL- 6 among the 29 cases was 90.83±25.66.
while the post PRP levels of IL-6 had reduced to 69.35±24.91. this difference was found to be statistically significant at level of p< 0.001 using the paired t test.
Male 4%
Female 96%
Fig 23: Gender of Rheumatoid arthritis
cases
TABLE – 7
COMPARISION OF PRE AND POST PRP LEVEL OF IL-6 USING PAIRED T TEST IN OSTEOARTHRITIS CASES.
IL-6 Values
n Mean SD Mean
difference ± SE
95%
Confidence Interval of Difference
p valve
Pre PRP 29 90.83 25.65 -21.47 ± 2.88
-27.36 to -15.58
<0.001 Post PRP 29 69.35 24.91
Fig 24: Change in the IL-6 values before and after PRP in osteoarthritic case (n=29)
15 8 16 28 27 13 11 9 20 22 14 7 5 26 19 10 6 29 23 21 3 2 24 1 4 12 18 25 17
44.67 44.85
54.57 59.9
66.38 74.73 74.78 75.47 76.4
81.92 84.36
85.32 87.42
89.36 89.49 91.64
92.68 92.89 93.58 96.03
96.97 103.26
104.88 108.76
110.6
132.8 138.96
140.48 140.85
41.08 42.36 39.42
43.91 38.58
43.09 47.81
62.83 49.38
55.62 69.31 69.38 67.12
82.76 86.29 76.81 63.04
74.38 68.78 68.21
79.99 71.54
101.5 72.2
66.62 53.01
127.73 131.65 116.85 PostPRP IL-6 PrePRP IL-6
