Bone defects lead by traumatic injuries, congenital malformations and other surgical rescissions rises the immediate need for a more evolved and safer approaches in tissue repair at alarming rates for the downgrading issues with existing strategies which needs to be addressed. Currently practiced treatment methods addressing the issue with bone defects are invasive, traumatic and are not cost effective. Yet, issues of immune rejection either immediately or in the later stages have been reported claiming its ineffectiveness in some selective case studies. Previous work by researchers carried out the "Biomimetic" approach to provide the cells with the microenvironment and in situ conditions for the cells seeded into the 3D Osteogenic scaffolds enriched with growth supplments. Here, we address the issue of non-availability of therapeutic cells - a major problem with current translational medicine by proposing the use of Human Embryonic Stem Cells in generating strong and structurally rigid bone tissue. Inducing the production of Mesenchymal Progenitor cells from Human Embryonic Stem cells in Serum supplemented expansion medium and elimination of bone Fibroblast growth factor produced high quality MPCs which were induced in osteogenic medium to result in bone differentiating cells. Culturing these MPCs produced from three different protocols into 3D Scaffold and 3D-Endoret Osteogenic Scaffold produced tissue constructs which are analysed both biochemically and Histologically to check for the Bone tissue differentiation parameters such as Bone sialoprotein deposition, Osteopontin accumulation and Collagen deposition. Matrix mineralization in these constructs were studied by uCT imaging and safety studies were conducted by studying Orthotopic implantation models in SCID mouse. And the results are expected to be optimistically affirmative which shall lay a new foundation and pioneer a whole new approach in the field of Tissue Engineering.

1. Engineering Bone
Tissue from Human
Embryonic Stem Cells
Balaganesh Kuruba
Department of Biological Chemical and Physical sciences
Illinois Institute of Technology

2. Bone defects from
Congenital and
trauma
malformations.
Metatarsus adductus
Hammer Toe
Bunion’s feet

3. Current strategies in
treating bone defects
Illizarov frame: To treat bone deformations or fractures.
Osteobridge:
Stabilization
of bone defects

4. Problems with conventional methods
• Extremely Traumatic .
• Not so economical use of mechanical and structurally
competent scaffolds.
• Requirement of synergistic development of vascular
supply and bone to maintain viability.
Alternative
Use of Human Embryonic Stem cells in
engineering bone tissue custom made for individuals
thus easing the process in avoiding graft rejection
related issues.
Safe to use and time saving by
generating the bone tissue on an increased time scale.

5. Hypothesis
Cultivation of hESC-derived Mesenchymal
Progenitor Cells (MPCs) on 3D Osteoconductive
scaffolds in perfusion reactor system leads to
formation of large and compact bone constructs.

6. Bone Tissue engineering protocol
and timeline
Fig 1: Bone Engineering protocol and time line.

7. Specific aims
• Aim 1: Derivation of Mesenchymal
progenitors from hESCs.
• Aim 2: Culturing MPCs on 3D scaffolds in
perfusion bioreactor system.
• Aim 3: Effects of Reactor cultivation on MPCs
in tissue development.
• Aim 4: In vivo Safety and stability analysis of
the engineered bone construct.

8. Aim 1: Derivation of Mesenchymal
progenitors from hESCs.
• Differentiation is
induced in two hESC
Cell lines –
H9 and H13.
• Mesenchymal
Differentiation
potential was
analysed and suitable
cell line was selected
for further analysis.
Differentiation of hESCs
into different cell types.

9. • H9 and H13
cultures were
found to show
continuous
growth.
• Mesenchymal
surface antigens
(Blue box) were
expressed on
progenitors.
• Further analysis
on the cell lines
were carried out.
Fig S1C: Surface antigen expression of hESC
progenitors

10. Fig : Mesenchymal differentiation potential screened in monolayer cultures.

11. Fig : Mesenchymal differentiation potential screened in monolayer cultures.

12. • From the tests performed H9 cultures showed
– Lesser or zero adipogenic potential
– Higher chondrogenic potential
– Higher activity of Alkaline phospatase and
– Increased matrix mineralization
In comparison with H13 cultures and Bone
Marrow derived mesenchymal stem cells (BMSCs)
as positive control.
Based on which, H9 cultures are selected for
the long run.

13. Aim 2: Culturing MPCs on 3D scaffolds in
perfusion bioreactor system.
Compact Bone structure
3D
Scaffold Structure

14. Perfusion Bioreactor system
Criteria:
Interstital media flow
Concept:
Channeling media into scaffolds
Design:
Final End product

15. Fig 2
Aim 3: Effects of Reactor cultivation
on MPCs in tissue development.

16. Fig 3: uCT analysis revealing bone mineralization and
maturation during in vitro and invivo conditions.

17. Aim4: In vivo Safety and stability analysis of
the engineered bone construct.
Fig 4

18. Fig 4B

19. And what am I proposing..
• Analysis of Bone-tissue characteristics of hESCmesenchymal progenitors generated from
different tissue engineering protocols
– Suggesting addition of Beta glycerol phosphate
– And Ascorbic acid – 2 - phosphate.
• Determination of influence of PRGF- Endoret
implanted 3D scaffold system on the rate of
development of MPCs into bone tissue.
• Long term safety studies in orthotopic
implantation models.

20. Thank you