BONE BIOLOGY Osteoblasts (Support cells) Osteoclasts (Remodeling cells)
Arise from osteoprogenitor cells
Bone matrix synthesis & mineralization
Derived from hematopoietic lineage
These are osteoblasts incorporated in the newly formed osteoid
Transduce messages to the osteoblastic cells
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
Human subcutaneous adipose tissue:Transported in anticoagulant or EDTA within 2h post-surgery. Minced & digested in digestion buffer:1-2 hrs at 37’c.
Stromal vascular fraction incubated in Osteogenic media: αMEM, supplemented with 10% FBS, 10mM ß-glycerophosphate, 0.1 uM dexamethasone, 1% antibiotics/antimycotic solutions, 50 uM ascorbate-2-phosphate (Zuk et al 2001)
Fat tissue Removal of vessels and fascia Collagenase digestion
Bone pieces plated in T-25 flasks containing in αMEM, 10% FBS, 250ul Vitamin C,1% antibiotic / antimycotic solutions
Non-adherent cells were removed after 12-24 hrs
MORPHOLOGY OF THE CELLS
Representative images of human OB & ADOB cells at P4. The cells at each passage were examined under light microscopy
Figure a: 100% confluent osteoblasts (OB) at day 10;
Figure b: 100% confluent adipose derived osteoblasts cells (ADOB) at day 7
a. OB 10X LM b. ADOB 10X LM
ADIPOSE DERIVED OSTEOBLAST CELLS
The proliferation of adipose tissue in response to the osteogenic media. Data expressed as mean +/- SEM (n=5). Statistical analysis was performed using a repeated measure ANOVA followed by Newman – Keuls. (Prism 4.0C for Word, Graphpad, CA, USA)
*** P value <0.05.
The proliferation of primary osteoblasts. Data expressed as mean +/- SEM (n=5). Statistical analysis was performed using a repeated measure ANOVA followed by Newman – Keuls. (Prism 4.0C for Word, Graphpad, CA, USA)
*** P value <0.05
HEMATOXYLIN & EOSIN STAINING
150,000 cells/PD of cells were grown in petridishes overnight
Extensive washing with 1xPBS
Fixed with 2% PFA
Stain in Harris Hematoxylin solution
Differentiate in 1% acid alcohol solution
Treat in 0.2% ammonia water solution
Counter stain in eosin solution
Dry O/N & view under light microscope
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
GENE EXPRESSION BY HUMAN OB & ADOB FOR NUCLEOSTEMIN n=5 OB ADOB
Protein isolation : 50ul RIPA (Rapid Immunoprecipitation Assay) buffer
50 mM TrisHCl pH7.4,
150 mM NaCl
2 mM EDTA
1% NP-40 (Nonidet P-40)
Centrifugation at 1200rpm for 15minutes at 4’c and supernatant containing proteins were stored at –20’c until use
Protein concentration determined by Lowry’s method using BSA as standard
25ug proteins were loaded to each well and separated using NuPAGE 4-12% Bis-Tris gel and transferred to nitrocellulose paper
Blocking with skimmed milk solution
Washing and O/N primary (Nucleostemin, Osteocalcin and GAPDH) at 4’c
Peroxidase conjugated secondary antibody for 2 hrs at RT
Blots visualized using ECL Chemiluminescence kit and auto radiographic exposure
Immunoblot detection of proteins expressed by human OB & ADOB cells. Total cell lysates from donors were electrophoresed on 4-12% Bis-Tris polyacrylamide gels, transferred and immunoblotted with antibodies directed against following antigens: Nucleostemin ( 62kDa) & GAPDH (37kDa). Representative of n= 4.
Antibody source: Santa Cruz Biotechnology,Inc. Switzerland
WESTERN BLOTTING Nucleostemin ADOB 1-4 OB 5-8 Nucleostemin ADOB 1-3 OB 4-6
In this study, we defined the gene and protein expression profile of human osteoblasts cells and adipose derived osteoblasts cells in vitro . The expression pattern of various primers and antibodies were studied in both the cell types by Immunohistochemistry, RT-PCR and Western blotting.
Type I collagen and ALP are early markers of osteoblasts and the expression of these proteins by OB & ADOB cells have confirmed the osteogenic differentiation in our study.
Osteocalcin has been identified as the only osteoblasts specific gene which regulates the function of fully differentiated osteoblasts. Osteocalcin gene is regulated by its transcription factor called Cbfa1, belonging to the Runt family . Cbfa1 and Osteocalcin are the best suited osteogenic markers. Cbfa1 is strongly expressed by both the cell types but the expression of Osteocalcin is significantly less in ADOB in comparison to OB cells.
RT-PCR analysis revealed that the non-differentiated ADSCs contained different types of stromal cells with a large variety of CD marker expression.The pattern of expression of ADOB for all of the listed stem cell markers except CD13 were significantly positive.
Nucleostemin is a p53 binding protein which plays a significant role in proliferation of stem cells and cancer cells. Although nucleostemin has been associated for its cell proliferation regulation in adult stem cells, embryonic stem cells and cancer cells, we believe that our data supports a role other than in proliferation. Previous reports has demonstrated that nucleostemin expression was persistent over a long period of time in human BMSCs culture.
The expression of nucleostemin in OB and ADOB may be due to the fact that the cells are undergoing proliferation and have not been fully differentiated. But recent work also shows that cells proliferate after nucleostemin expression is lost, which further questions the role of nucleostemin in proliferation. This suggest that nucleostemin plays a role other than proliferation in differentiated cells and that may be the possible reason for its expression in OB and ADOB cells in our study.
Our results are a striking contrast to other studies and thus begins a new debate for the possible reason for its expression in cells other than adult stem cells and cancer cells.
We believe that the adipose tissue may represent as an another source for multipotent cells and will be a promising approach for future tissue engineering strategies and cell based therapies, due to its general availability and low donor morbidity. The current findings now identify the adipose tissue as another site containing stromal cells.However, future studies are necessary to establish the role of permanent differentiation of these multilineage potential cells.
Next phase …
The honeycomb collagen was found to be an effective substrate for tissue engineering applications, and is very useful in the advancing field of stem cell technology and cell-based therapy. The differentiation of osteoblasts like cells from adipose tissue in honey-comb collagen scaffolds will be next step of this project.
SEMINARS & JOURNALS
Results accepted at 10th European Society of Trauma and Emergency Surgery (ESTES) 2009, Antalya, Turkey
Results accepted at NJ stem cell symposium 2009, New Jersey, USA
Results presented at the National Symposium of Trauma and Emergency Surgery, MIOT Hospitals, India
Paper submitted for European Journal of Trauma and Emergency Surgery