ASTM Scaffolds Workshop

05.21.13

Carl Simon

National Institute of Standards & Technology

NIST Reference Materials

Objective: Provide well-defined reference materials for use as calibrants and as standards during biomaterials characterization

Products:• SRM 2910: Calcium Hydroxylapatite, composition• RM 8456: Ultra High Molecular Weight Polyethylene,

modulus• RM 8457: Ultra High Molecular Weight Polyethylene,

crosslinks after sterilization• RM8011: 10 nm dia. gold nanoparticles• RM8012: 30 nm dia. gold nanoparticles• RM8013: 60 nm dia. gold nanoparticles

Partner:

RM 8456

RM 8457

RM 2910

RM 2910

RM 8011

Freeform Fabricated Reference Scaffolds Characterized for

Structure & Porosity

5 mm

RM 8395200 m Strut

Spacing

RM 8396300 m Strut

Spacing

RM 8397450 m Strut

Spacing

Reference Values

Strut Dia. (m)

Strut Spacing (m)

Porosity (%)

RM 8395 220 (9) 194 (19) 47 (2)

RM 8396 212 (9) 293 (15) 60 (3)

RM 8397 221 (8) 432 (10) 69 (1)

All values are mean (S.D.) and n = 10.

• For use as a standard/calibration when doing scaffold characterization to enable inter-lab comparison

• Deployed April 2009: 1 sale, 1 paper

• May 1: Price lowered from $591 to $252

• Goal: Use as standard when doing scaffold characterization, enables inter-lab comparison

• PCL: Widely studied for tissue engineering applications

• FFF: consistent structure, large features easy to characterize

• MC3T3-E1: well-characterized, ortho major target

• 1 Unit: 24 Scaffolds in a 96-Well Plate

Tissue Engineering Reference Scaffolds for Cell Culture

1d 7dMicroscopy 3 3

DNA Assay 6 6

(6 extra)

Deployed 05.01.13

Structural Measurements

Z-axis Strut Spacing

X-Y Plane Strut Diameter

Z-axis Strut Diameter

X-Y Plane Strut Spacing

1 d

500 mm 100 mm

7 d

500 mm 100 mm

Actin Nucleus

0

50

100

150

200

1 d 7 d

*

1.9 Fold Change(S.D. = 0.6, n = 3)

MC3T3-E1 Cell Culture

DN

A (n

g)

Picogreen DNA Assay

$252/unit

Future

• ASTM Standard Guide or Test Method for Using NIST Reference Scaffolds: New work item

• Round Robin: Barbara Boyan suggested (and volunteered to participate!)

• How many units? 6 participants = 6 units?

• Ruggedness testing: Design robust protocol

• Journal Publication: How to use NIST reference scaffolds (to publicize)

• Reference Scaffold Part 3: Nanofiber reference scaffold (?)

• Characterize Surface Chemistry: X-ray photoelectron spectroscopy (XPS), does it change over time?

Defining 3D Scaffold

3D scaffold can mean two things…

1) Scaffold itself has a 3D structure

• Fill defect, mechanical stability

• Increased surface area for cell expansion or delivery

2) 3D niche for cells

• Cells behave more physiologically

Scaffold Niche Dimensionality

Dimensionality of the scaffold niche:• Unclear which scaffolds provide 3D niche• Niche dimensionality metric (cell 3Dness)

3D cell niche • 3D cell morphology• 3D cell-matrix contacts (cells

polarized in 2D)

1 point“0D”

2 points“1D”

3 points“2D”

4 points“3D”

Scaffolds

Collagen Gel

PDLLA-NF (942 nm)

PCL-SC

PCL-FFF

PCL-SNF (492 nm)

PEGTMPCL-BNF (4151 nm)

Collagen Film

5 mm

50 m

• Seed hBMSCs into 3D scaffolds

• Image hBMSCs by 3D confocal microscopy

• Assess hBMSC shape for 3Dness

PCL-SNFCollagen Film PCL-SCCollagen Gel

Rod-Thin (1D)Sphere (3D)

PEGTMPCL-FFFPDLLA-NFPCL-BNF

x-y

x-z

y-z

Disk (2D)

Confocal imaging of actin-stained

hBMSCs

0.1 mm

Cell Shape Dimensionality

• Wide range of cell shapes seen

• Cells in 3D scaffolds are not very “3D”

Grid Divisions = 20 m3

PCL-BNF

• Tensor is an object that describes the relationships between a set of points

• Measures shape of point cloud of by fitting with an ellipsoid

Principal Moments of the Gyration Tensor

Principal Moments of the Gyration Tensor

PCL-BNF PDLLA-NF

PCL-SNF

PEGTM

Disk

PCL-FFF

Rod-ThinSphere

Collagen Gel PCL-SCCollagen Film

50 m

Cell Dimensionality Matrix

3D

1D

2DDisk

1, 16, 16

Sphere

16, 16, 16

Fatter Disk

8, 16, 16

Fat Disk

4, 16, 16

Really Fat Disk

12, 16, 16

Egg

12, 12, 16

Fried Egg

1, 12, 16

MostlyFlattened Egg

4, 12, 16

Lightly Flattened Egg

8, 12, 16

Football

8, 8, 16

Flat Football

1, 8, 16

Half-Flattened Football

4, 8, 16

Rod-Thick

4, 4, 16

Flat Cigar

1, 4, 16

Rod-Thin

1, 1, 16

Legend

XY ZY

XZ

X-axis

Y-ax

is

Z-axisY-

axis

X-axis

Z-ax

is

L10.5, L2

0.5, L30.5

Shape Name

0.333 Cutoff:• L1

0.5 < L20.5 < L3

0.5

• If L10.5/L3

0.5 > 0.333, then 3D• If L2

0.5/L30.5 > 0.333, then 2D

• Else 1D

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

L 20.

5 /L30.

5

L10.5/L3

0.5

PCL-SCCollagen FilmCollagen GelPCL-SNFPCL-BNFPDLLA-NFPCL-FFFPEGTMHorizontal 0.333Vertical 0.333

2D

1D

3D

Cell Dimensionality

Matrix

3D

1D

2D Disk SphereFatter DiskFat Disk Really Fat Disk

EggFried Egg MostlyFlattened Egg

Lightly Flattened Egg

FootballFlat Football Half-Flattened Football

Rod-ThickFlat Cigar

Rod-Thin

PCL-SC

Coll. Film

PCL-FFF

PEGTM

PCL-BNFColl.

Gel

PDLLA-NF

PEG

TMPC

L-FF

F

PDLL

A-N

FPC

L-B

NF

PCL-

SNF

Col

lage

n G

el

Col

lage

n Fi

lm

PCL-FFF

PDLLA-NFPCL-BNFPCL-SNF

Collagen Gel

Collagen Film

PCL-SC

L10.5/L3

0.5

PEG

TMPC

L-FF

F

PDLL

A-N

FPC

L-B

NF

PCL-

SNF

Col

lage

n G

el

Col

lage

n Fi

lm

PCL-FFF

PDLLA-NF

PCL-BNFPCL-SNF

Collagen Gel

Collagen Film

PCL-SC

L20.5/L3

0.5

1-Way ANOVA with Tukey’s

(P<0.5)

PCL-SNF

• Cell dimensionality dependent on scaffold

• Cells in 2D are 2D

• Cells in 3D are 1D, 2D or 3D

• Do nanofibers provide a 3D niche?

• Yes: Cells in nanofiber scaffolds were as “3D” as in collagen gel

• No: Cells 1D in nanofibers and 3D collagen gel

• Cells more 3D in larger diameter nanofibers

• Opens door to design experiments where scaffold structure is systematically varied to control cell dimensionality

Discussion for Cell Shape Dimensionality

In Progress:Dimensionality

Cell-Matrix Contacts

PDLLA Nanofibers (900 nm dia.)

Scan area is 123 m by 123 m

• Gyration tensor of cell-matrix contact area

Cell in Suspension Is Spherical (3D)

Cells on Non-Adhesive or Soft Surfaces Are

Spherical (3D)

PCL Film

Acknowledgements

Thank you!

Jeff Coles (Duke)Christopher Tison (Luna)

Sapun Parekh (Max Planck)Kaushik Chatterjee (IIT Bangalore)

Girish Kumar (FDA)Tanya Farooque (FDA)

Girish Kumar (FDA)Jirun Sun (ADA)

John Tesk (Consultant)Wei Sun (Drexel)

Lauren Shor (Drexel)Andrew Darling (Drexel)

Dana Schneider (Sligo School)Wing Lau (3D-Biotek)Qing Liu (3D-Biotek)Francis Wang (NIST)

Marcus Cicerone (NIST)Yuexin Liu (NIST)

Charles Camp (NIST)Steve Florczyk (NIST)

Subhadip Bodhak (NIST)Joy Dunkers (NIST)

Sumona Sarkar (NIST)Sai Sunkara (NIST)

Discussion & Questions?

RM Scaffolds

• ASTM Standard Guide for Using NIST Reference Scaffolds: New work item

• Round Robin: Barbara Boyan suggested (and volunteered to participate!)

• How many units for round robin? 6 participant labs required

• Ruggedness testing: To design robust protocol

• Journal Publication: How to use NIST reference scaffolds (to publicize)

• Reference Scaffold Part 3: Nanofiber reference scaffold (?)

• Characterize Surface Chemistry: X-ray photoelectron spectroscopy (XPS), does it change over time?

Define 3D Scaffolds: measurement for niche dimensionality

3D Shape Metrics for Scaffolds: confocal, NanoCT, computation

Future Goals for Standards & Reference Materials:

FDA Workshop: Developing Standards for Regenerative Medicine Products• Date: October 7, 2013• Location: FDA, White Oak, MD• Goal: Identify roles that standards can play in development

and use of stem cells

TERMIS Workshop: “Development of Standards in Tissue Engineering & Regenerative Medicine”• Date: Dec 2014• Location: Washington, DC (Convention Center?)

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