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SURFACE PROTEIN CHARACTERIZATION OF OSTEOBLASTS AND ADIPOSE DERIVED OSTEOBLASTS CELLS Vij  Senthilnathan
OBJECTIVE ,[object Object]
BONE MARROW STEM CELLS – NO!!! NO!!! ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
AN IDEAL SOURCE OF AUTOLOGOUS STEM CELLS ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Note:  App. 400,000 liposuction  in US/yr
BONE BIOLOGY Osteoblasts  (Support cells) Osteoclasts (Remodeling cells) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Secretion ALP BONE CELLS Osteocytes  (Support cells) MSCs BMP Differentiation Osteoprogenitor cells Activation by growth factors Cbfa1 Active osteoblast Bone mineralization facilitators: Osteocalcin, Collagen type 1 & Osteopontin
PROJECT OUTLINE Study the gene & protein expression profile ILIAC   CREST 1.TOTAL KNEE REPLACEMENT SURGERY 2.TOTAL HIP REPLACEMENT SURGERY Osteoblasts like cells Adipose tissue  Osteogenic conditions Bone samples from iliac crest Osteoblasts cells Bone media
PATIENTS DETAILS ADSCs: Adipose derived stem cells BMSCs: Bone marrow stem cells OB: Osteoblasts ADOBs: Adipose derived Osteoblasts cells Viability test n= 6 donors 81% 72% 28% OBs 81% 68% 30% BMSCs 84% 70% 28% ADSCs RL (30min) Saline (30min) PBS (30min) CELL TYPE
ISOLATION & CULTURE OF ADIPOSE TISSUES ,[object Object],[object Object],Fat tissue Removal of  vessels and fascia Collagenase digestion
ISOLATION & CULTURE OF OSTEOBLASTS ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
MORPHOLOGY OF THE CELLS ,[object Object],[object Object],[object Object],a. OB 10X LM b. ADOB 10X LM
ADIPOSE DERIVED OSTEOBLAST CELLS ,[object Object],[object Object],***
OSTEOBLASTS  ,[object Object],[object Object],***
HEMATOXYLIN & EOSIN STAINING ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Representative images of human OB & ADOB cells at P3. Representative of n=3 donors.Blue purple stains the nucleus & bright pink stains the cytoplasm
IMMUNOFLUORESCENCE  Osteocalcin & Nucleostemin Histochemical detection of protein expression by human OB & ADOB cells at P4. They were fixed and immunohistochemically  stained with Osteocalcin & Nucleostemin. Representative of n=5 donors ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION  ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
GENE EXPRESSION BY HUMAN OB FOR OSTEOCALCIN, ALP, CBFA1, GAPDH & COLLAGEN TYPE 1 n=5 ADOB & OB : Collagen type 1
GENE EXPRESSION BY HUMAN ADOB FOR OSTEOCALCIN, ALP, CBFA1, GAPDH n=5 ADOB: Osteocalcin & GAPDH ADOB: 1-7; OB: 8,9; Endothelial cells: 10 Osteocalcin & CBFA1 ADOB: ALP & Osteocalcin
[object Object],[object Object],OB : CD13 & GAPDH ADOB: CD10 & CD59 ADOB: CD44 & CD105 ADOB: CD13 & CD166
GENE EXPRESSION BY HUMAN OB & ADOB FOR NUCLEOSTEMIN   n=5 OB  ADOB
WESTERN IMMUNOBLOTTING ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],WESTERN BLOTTING Nucleostemin ADOB 1-4 OB 5-8 Nucleostemin ADOB 1-3  OB 4-6
DISCUSSION ,[object Object],[object Object],[object Object],[object Object]
DISCUSSION ,[object Object],[object Object],[object Object]
CONCLUSION ,[object Object]
Next phase … ,[object Object]
SEMINARS & JOURNALS ,[object Object],[object Object],[object Object],[object Object]
THANK YOU

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Adipose derived osteoblasts cells from fat tissues

  • 1. SURFACE PROTEIN CHARACTERIZATION OF OSTEOBLASTS AND ADIPOSE DERIVED OSTEOBLASTS CELLS Vij Senthilnathan
  • 2.
  • 3.
  • 4.
  • 5.
  • 6. PROJECT OUTLINE Study the gene & protein expression profile ILIAC CREST 1.TOTAL KNEE REPLACEMENT SURGERY 2.TOTAL HIP REPLACEMENT SURGERY Osteoblasts like cells Adipose tissue Osteogenic conditions Bone samples from iliac crest Osteoblasts cells Bone media
  • 7. PATIENTS DETAILS ADSCs: Adipose derived stem cells BMSCs: Bone marrow stem cells OB: Osteoblasts ADOBs: Adipose derived Osteoblasts cells Viability test n= 6 donors 81% 72% 28% OBs 81% 68% 30% BMSCs 84% 70% 28% ADSCs RL (30min) Saline (30min) PBS (30min) CELL TYPE
  • 8.
  • 9.
  • 10.
  • 11.
  • 12.
  • 13.
  • 14.
  • 15.
  • 16. GENE EXPRESSION BY HUMAN OB FOR OSTEOCALCIN, ALP, CBFA1, GAPDH & COLLAGEN TYPE 1 n=5 ADOB & OB : Collagen type 1
  • 17. GENE EXPRESSION BY HUMAN ADOB FOR OSTEOCALCIN, ALP, CBFA1, GAPDH n=5 ADOB: Osteocalcin & GAPDH ADOB: 1-7; OB: 8,9; Endothelial cells: 10 Osteocalcin & CBFA1 ADOB: ALP & Osteocalcin
  • 18.
  • 19. GENE EXPRESSION BY HUMAN OB & ADOB FOR NUCLEOSTEMIN n=5 OB ADOB
  • 20.
  • 21.
  • 22.
  • 23.
  • 24.
  • 25.
  • 26.