Cell culture study of degenerative articular cartilage and its - PowerPoint PPT Presentation

Cell culture study of degenerative articular cartilage and its comparison with normal articular cartilage Dr. Rohit Prasad V MS Ortho PG Under the Guidance of Dr. B. K. Dinakar Rai: Department of Orthopaedics:

INTRODUCTION Study is aimed at finding the degenerative process Of cartilage STRUCTURE Composition : water > collagen > proteoglycan > noncollagenous protein > cells

AIM • To find out the difference between normal and degenerated cartilage using cell culture OBJECTIVES To optimize the in vitro articular cartilage cell culture • • To perform a comparative studies on degenerative and normal articular cartilage using cell counting, doubling time and extracellular matrix analysis in different age groups

INCLUSION CRITERIA • The patients of all ages and both sex who require interventional procedure of joints (young patients who are subjected to any arthroscopic procedures, and older patients subjected to joint replacement surgeries) EXCLUSION CRITERIA • Chronic inflammatory arthritis 1) Rheumatoid arthritis 2) Seronegative arthritis 3) Connective tissue disorders • Infective arthritis • Congenital skeletal anomalies • Malignancy Age < 20 years •

Composition of Articular cartilage Extracellular matrix • Water : makes up 65% to 80% of mass of the cartilage • Collagen : makes up 10 to 20% of total cartilage mass • type II collagen accounting for 90% to 95% of the total collagen content. • functions to provide cartilaginous framework and tensile strength • Proteoglycans : makes up 10 to 15% of cartilage • Functions to provide compressive strength and attract water • Aggrecan is most responsible for hydrophilic behavior • composed of GAG subunits • chondroitin sulfate • keratin sulfate

Importance of GAG in Articular cartilage • Glycosaminoglycans (GAGs) are one of the major constituents in the cartilage There are two types of the chondroitin sulphate (type 4 and type 6) . • Type 6 remains constant throughout life, whereas type 4 decreases with the age. Mourão PA. Distribution of chondroitin 4–sulfate and chondroitin 6–sulfate in human articular and growth cartilage. Arthritis & Rheumatology. 1988 Aug 1;31(8):1028-33.

Aging changes in Articular cartilage • Increases in: • chondrocytes size • protein content • stiffness (passive glycation leads to increased stiffness of collagen) • increase in ratio of proteoglycan keratin sulfate to chondroitin sulfate

• Decrease in: • absolute number of cells (individual chondrocytes - hypertrophy) • water content (in osteoarthritis - water content actually increases) • solubility • elasticity

METHODOLOGY ENZYMATIC COLLECTION OF DIGESTION USING ARTICULAR COLLAGENASE AND CARTILAGE SAMPLES TRYPSIN COMPARATIVE STUDY USING ISOLATION OF CELLS AND CELL COUNTING MAINTENANCE UNDER DOUBLING TIME STANDARD CONDITIONS GAG AND COLLAGEN (CELLS CULTURED USING ESTIMATION DMEM MEDIUM IN A CO2 INCUBATOR AT 37 °c )

In vitro cell culture studies Mince Tissue Enzym atic treatm ent Collect the cells Centrifugation Confluent cells viewed under Cell culture using DMEM Phase contrast m icroscope m edium +10 % FBS

DETAILS OF COLLECTED SAMPLES Samplе Coding Gеndеr Agе NI Malе 21 N2 Malе 23 N3 Malе 29 N4 Malе 33 N5 Fеmalе 36 N6 Malе 37 D1 Malе 53 D2 Malе 57 D3 Malе 59 D4 Fеmalе 62 D5 Malе 65 D6 Fеmalе 66 D7 Fеmalе 70 D8 Malе 75 D9 Malе 77 D10 Malе 79 D11 Malе 82 D12 Malе 83

Morphology of cells in vitro Detached cells from tissue Attached cells 4 days 12 days Floating tissue with live cells

Confluent cells in different passages (28 days)- Normal cartilage Passage 1 Passage 2 Passage 3 Passage 4 Sample: Male, 21 years

Confluent cells in different passages (28 days)- Degenerated cartilage Passage 1 Passage 2 Passage 3 Passage 4 Sample: Male, 79 years

NORMAL

DEGENERATED

Chondoitin 4 Sulfate IHC staining The staining intensities of chondroitin sulfate, a cartilage specific marker was observed to be increased in younger patients.

Observations • Doubling time - significantly less for the normal group • Cell count and Nuclear staining - higher in the normal group • GAG and collagen estimation - higher values in normal in all 4 passages. • Chondroitin-4 sulfate IHC staining – Higher concentration in young.

Conclusion • This study revealed that - prolifеration ability - dеcrеasеd in degenerated group Cartilagе еxtracеllular matrix synthеsis rеducеd Dеgеnеration of cartilagе .

• Isolatеd chondrocytеs with suitablе growth factors can rеgеnеratе cartilagе . • Advancements in these areas may help in identifying factors which could help in preventing and managing of osteoarthritis.

THANK YOU

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