Final Exam

1. Osteoinduction of Human Adipose Stem
Cells (hASCs) on Poly (L-lactic acid)
Scaffolds Prepared by Thermal Control
Harish Chinnasami
Mechanical Engineering,
Louisiana State University,
Baton Rouge, Louisiana, USA 70803
Bioengineering Laboratory
Doctoral Final Exam (April 28, 2017)

2. Outline of Talk
1. Stem Cell Harvesting and multi-potency of hASCs
2. Scaffold Choice
• Material (PLLA)
• Fabrication (TIPS, Cooling Rate)
3. Characterization (Scaffolds)
• SEM, Compression strength, Porosity and Pore-size
4. Cell culture: DNA proliferation, viability and 3D growth.
5. Osteogenic study: Alizarin Red S
6. Results and Discussion
7. Conclusion

3. Bone Tissue Engineering Paradigm
1. Harvest Fat Tissue 2. Isolate Stem Cells
3. Scaffold TE products 4. Cryopreserve TEs
5. Thaw and Implant
There are about 1.5 million cases of skeletal defects a year
that require bone-graft procedures to achieve union*
*OPTN/SRTR 2015 Annual Data Report

4. Scaffold Based Tissue Engineering
1. Engineer Fully Viable Biological Bone Grafts
a. Osteoprogenitor Cells (Adult Stem Cells)
b. Scaffolds (3-D structure)
2. Scaffold Requirements
a. Mechanical Support
b. Osteoinductive: induce bone formation
c. Osteoconductive: facilitate “in vivo integration”
d. Osteogenic: forms bone
3. Common Scaffold Materials
a. Numerous combinations in the literature
b. Commonly HA/TCP, PLLA, Akeramite, etc..
c. Other: Hydrogels, Cell Sheets, etc..

5. Stem Cells
2 major types
embryonic stem cells
adult autologous stem cells
(bone marrow (stromal and hematopoietic) and adipose tissue)
Ethical issues*
*Lee et al., Biochem & Biophy Res Comm 2008; **Zuk et al., Mol. Biol. Cell 2002
**Bone sialoprotein
expressed at 2 weeks
after osteogenic
differentiation on hASCs

6. LIPOSUCTION PROCESS
(Baton Rouge Hospitals)
Pennington Biomedical
Research Center (PBRC)
Stem Cell Laboratory
Source: Adipose (fat) tissue
Human Fat Tissue Adult Stem Cells (hASCs)
Adipose Tissue
Explants
Saline, blood
other medication
hASCs
SVF, Passages: 0, 1, 2, 3, 4
Differentiation
Adipocytes
Chondrocytes
Osteoblasts
Neuronal
Undifferentiated
hASCs
Growth Factors Chemical Stimuli
LSU ERB #E9119 – NIH Exemption #4

7. Choice of Scaffold Material
Ceramics
(inorganic, brittle,
highly crystalline)
Polymers
(organic, thermoplastic,
both crystalline and amorphous)
Hydroxyapatite (HA) /
Tricalcium phosphate (TCP)
• Non-toxic, high mechanical strength,
• 30-60% porosity from 3D printing[4]
• Pore size 300-1000μm*
• Osteoinductivity of Ca-P is debated*
• Compressive modulus of HA ~670MPa**
• Calcium support-tissue binding
• bone substitutes in clinics
*Will et al., 2008; **Pathiraja et al., 2003; ***Gina et al., 2010; Garlotta 2001;
****Ishaug-Riley et al., 1999; Therin et al., 1992; ****Williams et al., 1977
Poly (l-lactic acid) (PLLA) /
Poly (lactic-co-glycolic acid) (PLGA)
• Polymerization of l,l-lactide →PLLA
• 37% crystalline
• Soluble in chlorinated solvents, hot
benzene, dioxane***
• Hydrophobicity × Cell adhesion****
• Hydrolytic degradation of ester
bonds to lactic acid (present in the
body)*****

8. Methods of Scaffold Fabrication
Solvent-Casting and Particulate
Leaching Technique*
Gas-Foaming Process**
Electrospinning Technique***
Thermally Induced Phase
Separation****
*Ma PX et al., 1998; Mikos AG et al., 1994; Thomson RC et al., 1995;
** Cooper AI et al., 2000; Harris LD et al., 1977; Mooney DJ et al., 1996;
***Boland ED et al., 2001; Huang L et al., 2001; Matthews JA et al., 2002; Reneker DH et al., 1996; Yoshimoto H et al., 2003;
****Zhang R et al., 2001; Lo H et al., 1996; Nam YS et al., 1996; Schugens C et al., 1996; Ma PX et al., 2001;

9. Cooling PLLA/dioxane with liquid nitrogen
vapors (instruments)

10. In situ thermal profile

11. Characterization of resulting scaffolds

12. TIPS: Thermally Induced Phase Separation
• Dissolve PLLA into a solvent (1, 4 Dioxane) at 323 K
• 3, 7 or 10 (wt/vol) %
• Controllably “Freeze” PLLA-Dioxane solution
• Cylindrical capsules (13 mm diameter, 5 mL)
• Cooling Protocol used in CRF:
• Room temperature to 10 ˚C at 10°C/min
• Hold at 10 ˚C for 1 min to nucleate dioxane
• Cool from 10 ˚C to -60 ˚C @ 1, 10 or 40 ˚C/min
• Lyophilize Frozen PLLA-Dioxane
• 48 hrs (0.037bar & -50°C)
• Sublimate Dioxane, create porous scaffolds

13. Scaffold Characterizaton
• SEM Images (JEOL JSM 6610LV)
• Analyze Microstructure
• Pore Size and Distribution
• Porosity (Void Fraction Measurement)
• Measure Scaffold Density: Gravimetric (weight)
• Porosity = 1 - {scaffold density / polymer density}
• Mechanical Properties
• Scaffolds 12 mm in diameter and 10 mm in height
• Compressive Strength (Instron 5900) - 20% along axis
• Compressive Modulus (E)
E = {load/area}/strain
height

14. Testing Devices
From top left
clockwise:
•Control Rate Freezer
(CRF) (Planer Series
Kryo 560-16)
•Scanning Electron
Microscopy (JEOL
JSM-6610LV)
•Freeze dryer
(FreeZone Plus 2.5
Liter Cascade
Control)
•Compression Tester
(INSTRON 5869
Series)

15. SEM Images
7 (wt/vol) %3 (wt/vol) % 10 (wt/vol) %
200μm
1
10
40
Cooling
Rate
(°C/min)

16. Pore Size and Porosity
• No significant changes in pore-sizes with increasing PLLA (wt/vol) %
• Pore size distribution decreases from 3 to 10% (more consistency)
• Effective pore diameters larger than 100μm were reported to be adequate
to induce osteoconductivity*.
• % Porosity decreases with increasing PLLA (wt/vol) %
*Dorozhkin S 2007

17. Compressive Stress Vs Strain
Increasing
Cooling Rate

18. Compressive Modulus (E)
• Calculated using “linear regression”
• E increases as PLLA (wt/vol) % increases
• E increases as “cooling rate” is increased
• “E” factor of 10 lower than in vivo cortical bone!

19. Porosity (%) vs Compressive Modulus (E)
Significant effect of imposed cooling rate on the compressive
modulus of PLLA scaffolds.

20. PLLA in Dioxane:Ethanol (0.85:0.15)
90
95
100
0.5 5 50
%Porosity
Cooling rate -(°C/min)
3% (wt/v)
7% (wt/v)
10% (wt/v)

21. hASCs Culture
• Cryo-preserved cells were thawed and cultured in
traditional Stromal Medium (SM):
• 90%(v/v) DMEM/F-12 (Gibco)
• 10%(v/v) Fetal Bovine Serum (Gibco)
• Triple antibiotics (Penicillin - 90 IU/mL , Streptomycin
– 90 μg/mL and Amphotericin B – 225 ng/mL)
• After 2 Passages, cells were trypsinized and loaded on
the selected PLLA scaffolds (7 and 10(wt/v)%).
• Equal no. of cells were cultured in 6-well plates as
control.

22. Instruments used
From top left
clockwise:
•Thermo Forma Series
II H2O jacketed CO2
incubator
•Nikon Eclipse E600
fluorescence
microscope with
CoolSNAP color
camera
•SANYO MLS-3780
autoclave
•Dounce tissue grinder
(7mL) with large
clearance pestle.
•Perkin Elmer Wallac
Victor 2 V Multi-label
Counter 1420

23. Loading efficiency using bioreactor
No. of scaffolds
Cell count before
loading
Cell count after
loading
Efficiency
9 1,900,000 1,055,070 44.47%
6 2,325,000 1,320,135 43.22%
12 4,260,000 2,525,000 40.73%
• Dynamic loading method uses a stirrer
with hASCs in 120 mL of SM.
• Efficiency was too low (hASCs
wastage of ~55%)
• About 30% dead cells were observed
in the cell counting post loading.
• Inefficient method of loading.

24. Viability Assay
• Equal no. of cells (2 × 105) were
loaded and cultured on all scaffolds
and control.
• Staining solution (2 µM Calcein AM
(live cells) and 4 µM Ethidium
homodimer-1 (EthD-1) (dead cells)).
• Calcein (Ex/Em: 495 / 515 nm);
EthD-1 (Ex/Em: 528 / 617 nm).
• Few dead cells in control and in
PLLA scaffolds (>90% viable).
• Observing cells on the surface of
scaffolds suggest 3D penetrative
growth of cells.

25. 7% 1°C/min 10% 10°C/min
Days
7% 10%
control
1° C/min 10° C/min 40° C/min 1° C/min 10° C/min 40° C/min
3 96.64 % 93.09 % 96.23 % 97.95 % 93.79 % 96.78 % 99.78 %
21 98.10 % 96.76 % 98.90 % 99.58 % 96.36 % 98.72 % 99.96 %
42 99.75 % 99.36 % 99.11 % 99.78 % 99.97 % 99.36 % 99.98 %
Quantification of viability

26. • DNA quantity increased by 11.6 fold (~500ng →1500ng) in 10% PLLA scaffolds
cooled at 10°C/min from 1 to 21 days.
• Control samples were confluent by day 7 and hence constant DNA values.
• Good proliferation is implied by quantitative increase in DNA
DNA Quantification
•Equal no. of cells (1 – 2 ×
105) were cultured on
scaffolds and control. (n
= 6)
•DNA extraction: Treated
with lysis buffer
(0.2mg/mL Sodium
Dodecyl Sulfate and
0.2mg/mL Proteinase K)
at 37°C for 20 mins.
* Weak statistical significance ( 0.1 > p > 0.05). ** No statistical significance ( p > 0.1)

27. Calibration of thickness for cell culture
*
*
* Statistically not significant (p>0.05)

28. 3 days 21 days
1
°C/min
10
°C/min
40
°C/min
Three dimensional cell growth on 7% PLLA scaffolds
• About 1 × 106 cells
were loaded on all
scaffolds and cultured
in SM.
• Fixed with 70% ethanol
(20 min incubation at
RT), at 3 and 21 days
• Stained using 0.1 %
(wt/v) osmium tetroxide
in ddH2O for 12 hrs at
RT.
• Stained samples were
air dried for 3 days

29. 3 days 21 days
1
°C/min
10
°C/min
40
°C/min
Three dimensional cell growth on 10% PLLA scaffolds
• Scanned with SKYSCAN
1074 portable X-ray
microtomography in
Radioisotope Imaging
Laboratory, Physics
Department, LSU.
• 400 scans in 360°
rotation of the sample.
• Density difference in the
resulting X-ray scan
was used to distinguish
Os-stained cells and
PLLA.

30. hASCs preferring peripheral region on scaffolds
10% 40°C/min after 21 days

31. Osteogenesis
• Cryo-preserved cells were thawed and cultured in
traditional Stromal Medium (SM).
• After 2 Passages, cells were trypsinized and loaded on
the selected PLLA scaffold (10(wt/v)% cooled at
10°C/min) with stromal and osteogenic medium (stromal
medium, 50 µg/ml ascorbic acid, 10 mM β-
glycerophosphate and 10 nM dexamethasone) (OM).
• Equal no. of cells (2 × 105) were cultured in 6-well plates
in SM and OM as negative and positive controls
respectively.

32. Alizarin Red S (ARS) Staining
• Fixed with 70% ethanol at 4°C
for 20 mins
• Stained with 40mM of ARS at
RT for 20 mins
• Nikon Eclipse E600 to capture
images in bright field

33. ARS Quantification
• 10% (wt/v) cetylpyridinium chloride for elution
• Absorbance at 531nm using Wallac Victor2 Plate reader.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
14 21 28
Calcificationperunitsurfacearea(µM/cm2)
Days
negative control
scaffolds in SM
scaffolds in OM
positive control
*
*

34. Conclusion
• Related cooling rate and the (wt/v) percentage of PLLA to:
• The structural properties (SEM Images)
• Pore size, Porosity
• Compressive Strength
• PLLA found to be highly bio-compatible by viability and DNA
quantification studies:
• DNA quantification
• Viability assay (scaffold > control)
• Optimal Scaffolds prepared with 10% (wt/v) and cooled at 10°C/min
was chosen for osteogenic study:
• Increased calcification on scaffolds in osteogenic media.
• Calcification (> +ve control) was observed in PLLA + hASCs
cultured in SM. Thus PLLA is osteo-inductive by nature.

35. Future scope: “Off the Shelf” Product
• Cryopreserve scaffolds loaded with ASCs
• Day 0 Load cells, Freeze/Store/Thaw/Differentiate,
At day 21: Viability/ARS/uCT
Check Mechanical Properties
• Day 21 Freeze/store/thaw
Post-thaw check: Viability/ARS/uCT
Check Mechanical Properties
• Thermal profile variables
• Cryopreservation cooling rate (1, 5, 10 and 40 ˚C/min)
• Choice of Cryo-protective Agent (10% PVP and Serum Free)*
*Thirumala S et al., J Tissue Eng Regen Med 2010

36. Hydroxyapatite Scaffold Freezing Results

37. Before Freezing
After Freezing to -
60°C
After thawing to
37°C
1°C/min
10°C/min
40°C/min
10% 10°C/min scaffolds freezing results

38. Acknowledgements
Dr. Ram Devireddy, Dr. Todd Monroe,
Dr. Ingmar Schoegl, Dr. Manas Gartia and
Dr. Dennis Landin (DR)
Prof. J. Gimble, Tulane University and LaCell LLC,
New Orleans.
Lab Members: Shahensha, Anoosha, Colton
Funding: LSU Economic Development Fellowship
LSU IRB (E9119) under NIH Exemption #4.

39. Questions?

40. Literature Review

41. RNA isolation calibration
• PureLink® RNA Mini Kit was used to extract RNA from 1mm thick scaffolds
(106 hASCs)
• Scaffolds were homogenized with 1 mL Lysis buffer and a centrifugation step
(Eppendorf Minispin Plus) was added before following manufacturer’s protocol.
• Variables for centrifugation were time and speed of centrifugation.
• The results were qualified by the absorbance values (A260/280 and A260/230).
Speed
(rpm)
Time (min) A260/280 A260/230 ng/µL
- (Lysis buffer added on top) 1.71 0.54 10.9
2,000 5 1.90 0.83 26.0
3,000 5 2.06 1.42 20.6
5,000 5 1.99 1.97 26.4
8,000 5 1.83 0.09 3.96
10,000 5 2.06 0.75 33.1
5,000 3 2.19 1.41 38.7

42. qPCR efficiency
• 5000 rpm was calibrated as the ideal speed.
• A260/280 values were good (~2.0) for both times (5 and 3 min).
• The effect of difference in the A260/230 values was evaluated by running a qPCR
efficiency run with serial dilution (2.5 times) of both the samples. (cyclophilin B)
5 min 3 min
cDNA (ng) Ct values Efficiency cDNA (ng) Ct values Efficiency
95.65 17.9194773
89.96 %
82 18.029702
83.64 %
38.26 19.1452127 32.8 19.017316
15.3 20.518727 13.12 20.337962
6.12 21.8269623 5.25 21.7644223
2.45 23.696718 2.1 24.1723327