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

• Dr. Rohit Prasad V

MS Ortho PG Under the Guidance of

• Dr. B. K. Dinakar Rai:

Department of Orthopaedics:

Cell culture study of degenerative articular cartilage and its comparison with normal articular cartilage

INTRODUCTION

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

STRUCTURE

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
• f 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

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

Composition of 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.

Importance of GAG in Articular cartilage

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

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

In vitro cell culture studies

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

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 Floating tissue with live cells

4 days 12 days

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.

• This study revealed that - prolifеration ability - dеcrеasеd in

degenerated group

Cartilagе еxtracеllular matrix synthеsis rеducеd Dеgеnеration of cartilagе.

Conclusion

• 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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