3-D Constructs--Molded vs. Printed: 

The differences from a cell based perspective

Kuan Che Fang, Miriam Rafailovich, Marcia Simon

Stony Brook University, Stony Brook, NY
Chung-Chue 

(Simon) Chang

Aneri

Kinariwalla

Garcia Research Scholar Program: Students from HS->Graduate work together with 

teachers and faculty in joint interdisciplinary research projects. 

Sneha

Subramanian

Daniel Ou-Yang Steven Wei
Jaw Case Study – 3D Titanium Implant (replacement)

• In 2012 an 83 year old woman 

with osteomyelitism –

requiring jaw removal 

• Patient had MRI so implant 

would be an anatomical match

• Jaw printed from titanium 

powder in a 2 day print

• Patient was eating, drinking, 

and speaking within 4 hours of 

surgery

• No FDA role

Liz Nickels, World's first patient-specific jaw implant, 

Metal Powder Report, Volume 67, Issue 2, March–April 

2012, Pages 12-14, 

This presentation does not pertain to FDA regulatory policy on 

Additive Manufacturing, and should not be construed as such. Courtesy of K. Vorvolakis, FDA
Digital Additive Manufacturing –

Individualizing bone augmentation

Control shape Control resorption

Alter materials and topography to control 

- Bone formation and turnover

- Angiogenesis

Potential for cell seeding

Protein coating (matrix/cytokine)

Images: Courtesy of M Abboud
3D Printing with PLA
• Solve Navier-Stokes equation with 

Advective component.

• Output temperature depends on flow 

rate. 

• Printer runs very close to Tm or Tg.

Tg~160

Tm~180

Temperature at nozzle
E Miller, JP Rothstein, Rheol Acta (2004) 44: 160–173

Well known stick/slip instability in flow of polymer filaments as they extrude 

through narrow nozzles. 

• Caused by surface interactions between entangled polymers and 

the nozzle wall. 

• Modulated by adjusting the extrusion rate and the nozzle 

temperature. 

Potential differences between 3D printed and molded surfaces

Surface roughness: Sharkskin effect in polymer fibers
Differential Scanning Calorimetry
Crystalinity: Kinetics of spherulite formation

• Provides strength

• Defects /faults crack propagation

• Uneven cooling warps

• Increased surface roughness

• Function of cooling rate

• Nucleation sites

• Time scale similar to thermal 

dissipation of printer
In-Situ SAXS WAXS

• Map crystallinity

• Map temperature

• Measure crystallization dynamics as a function of temperature
Topics that need further discussion

• Dynamics of crystallization under conditions 

far from equilibrium

• Effect of surface mechanics and roughness on 

protein adsorbtion, cell attachment, 

proliferation, and differentiation. 
Goal: Regenerate teeth

 To develop a 

biocompatible method 

for autologous dentin 

regeneration which 

combines printed 

scaffolds with stem 

cell differentiation.
Cells and their niche

Cell Substrates

• Mechanics

• Topography

• Chemistry

Soluble and cellular mediators

• Cytokines and growth factors

• Cell associated ligands and receptors

• Tissue fluid (media)

Scadden 2006, Nature 441:1075-79
CELL SYSTEM: DENTAL PULP DERIVED CELLS
Cell Source: Dental Pulp Stem 

Cells(DPSCs) 

Dental Pulp differentiate Osteoblasts

Stem Cells Odontoblasts

Adipocytes

Neuronal Cells

Muscle Cells

Cartilage (chondrocytes) 

DPSCs were chosen for the study because of

- Their easy accessibility 

- high efficiency of extraction procedure

- faster multiplication rate 

- their ability to self-renew in vivo 

For this study, DPSCs were isolated under IRB approval # 20076778. 
Induction: Dexamethasone(Dex /DXM)

• DPSC can be chemically induced with high doses of dexamethasone 

(glucocorticoid steroid) to differentiate into osteoblasts (in vitro) [1]

• Steroids (unnatural) have adverse side effects i.e. weakened immune 

system and hyperglycemia [2]

[1] “Dexamethasone stimulates differentiation of odontoblasts like cells in human pulp cultures”, Alliot-Licht et al., Cell Tissue 

Res (2005) 321. 

[2] “ Differential Effect of Glucocorticoids on Calcium Absorption and Bone Mass”, Gennari C. et al., Rheumatology 

32.2(1993):11-14, Oxford Jornals.

Special challenge in the tooth 

: 

• Confined space in the 

canal

• Difficult to localize soluble 

factors

• High degree of order

• Cells must sense very 

small changes in substrate
• Biocompatible 

• Similar to Polyisoprene 

• Used to obdurate root 

canal (same as tires). 

• Re-engineer for pulp 

regeneration rather 

than obduration. 

Polymeric substrate: 
Abstract: The differential adhesion hypothesis (DAH), advanced in the 

1960s, proposed that the liquid-like tissue-spreading and cell 

segregation phenomena of development arise from tissue surface 

tensions that in turn arise from differences in intercellular adhesiveness. 

......without exception, a cell aggregate of lower surface tension 

tends to envelop one of higher surface tension to which it adheres

Nobel Lecture, December 9, 1991: “SOFT MATTER”

A related (although more complex) system of this type is a red blood 

cell. For many years it had been known that, when observed under 

phase contrast, these cells flicker. - .... The essential property of 

insoluble bilayers is that they optimise their area at fixed surfactant 

number. Thus, the energy is stationary with respect to area: the 

surface tension vanishes.

“ Malcolm 

was My 

Biologist” 

Lecture at 

NYU Poly -

-2006
Embryonic Stem Cells Sense Mechanics of their Environment:

• Labeled outside shell and core cells of embryo

• Dissociated cells and allowed them to re-assemble

• Cells self assembled correctly into core-shell

• Shell cells had lower surface tension or number of cadherins

Davis, Phillips, and Steinberg
Elastomer: Why are they special?

d

•S=kT (d/R2) -> Loss due to 

confinement or cross linking.

• Confinement and surface 

interactions determine viscoelastic properties. 

• Radius of Gyration:

Kurt Binder website • We can control 

chemistry and 

mechanics separately. Rg

P.G. DeGennes Scaling concepts in polymer physics, Ithaca, N.Y, 

Cornell University Press (réimpr. 1985), 
free

“stragulated”

bound

air surface

Substrate

Two Fluid Model: 

D=Do Nc/Ne in a X-linked matrix. 

N 1⁄2 surface contacts~ Nc for surface

Predictions: (1) D~N 3/2 (2) N 1⁄2 ~Nc for 

Mw~4M (3) Surface effect propagated by 

entanglements in range; Ne<N<4x10 4

Predicts Scaling for Modulus: Resistance 

to Deformation and Viscous Flow

D~kT/η

Einstein relation
Influence of Radius of Gyration: Rg~sqrt (Mw)

• Gaussian Chains form N1/2 contact with surface

• If attached then they act as cross-linkers, 

D=Do (Nc/Ne)

• Ne=sqrt (N) for Mw~10 6 for PS 

• ONLY when Ne=Nc surface effect is removed. 
23

Two Phase

Lock-in AMP

Function

Generator

PC

3

2

1/ 2 



 





=

R

L h D

h: Tip indentation

E: Modulus of material

ν: Poisson ratio

R: Tip radius

L: Cantilever length

•At T=Tm sample melts and becomes 

soft. 

•Tip contact area increases.

•Indentation increases (h).

• ∆x increases sharply

• Friction increases

• Measure Tm

Flateral

Displacement

h

Indentation

Lnormal =25nN

∆x~h

E

1

4

3 D

2 − υ

= ,∆x~

Atomic Force Microscope
0 10 20 30 40 50 60 70 80 90

0

2

4

6

8

10

12

PB films

Response Amplitude (mV)

Driving Amplitude (mV)

 200A

 500A

 1500A

 2000A

 3000A

Y. Ji, B. Li, S.Ge, J.C. Sokolov, M. 

Rafailovich, Langmuir 2005, 22, 

1321-1328

G : shear 

modulus

∆X: lateral 

deflection

Hysitron

SMFM

• Modulus decreases with increasing 

film thickness. 

• Allows for variation of one order of 

magnitude. 
Is the modulus effect a 

function of Rg?

• Modulus decreases exponentially 

with increasing film thickness.

• Same functional form for all Mw-> 

Scales with Rg. 

• Confinement effect: Surface 

interaction influence on 

modulus for ~20 Rg’s

• Molecular weight: Another 

variable to increase flexibility 

of design. 

Molecular Weight Effect on 

Modulus: 
Polymer Film Processing 

PB or PI

Si

• Wafers were cut into 1cm2

squares 

• HF etched Si wafers 

• PB spun cast from toluene 

• Thickness of 200 to 3000 A were 

measured by ellipsometry

• annealed in Ultra High Vaccuum

(UHV) for 24 hours which 

prevents dewetting, removes 

toxic solvent, sterilizes 

substrates. 
Topography Lateral Modulation 260A

840A

840A

Do cells sense matrix modulus? Ingber, D.E., The Architecture of 

Life. Scientific American Jan 1998; 278:48-57

Donald Ingber : Cells redistribute stress to maintain

tensile integrity. 

• No tensile stress across cells surface. 

• No contrast with substrate

Bio-Tensegrity
2000A

Dying Cells

• Effects are observed after 40min in 

ambient T=23C

• Concentric rings are becoming disordered 

as cell loses its adhesion to the surface. 

•Modulus decreases (cells get softer). 

• Contrast with Pb/Si substrate increases. 

0 10 20 30 40 50 60 70 80 90

0

20

40

60

80

100

120

1

2

3

4

5

6

7

8 9

10 11

12 13

14 15 16 17

18

19

20 21 22 23

24 25

26

A

B

C

D

E

F

G

H

I

J K

L M

N

O

P Q R

S

T U

V W X Y Z

a

b

c

d

e

f

g

h

i

j

k

l m

n

o p

q

r s t u v w

x y z

Response Amplitude (mV)

Driving Amplitude (mV)

dead cells

healthy c

t= 40 min

t= 1 hr

t= 3 hrs
Thin PB Thick PB TCP

0

10

20

30

40

50

60

70

80

90

100

Doubling time (hr)

0 1 2 3 4 5 6 7 8

0

10000

20000

30000

40000

50000

60000

70000

80000

Cell number

Time (day)

 Thin PB

 Thick PB

 TCP

• Cells like PB. 

Proliferation without 

additional protein 

i.e. Fn) coating. 

• Doubling time is the 

same on thin PB as 

TCP.

• Cells adhere: Actin 

is well extended on 

TCP and thin PB

• PB similar to 

membrane? 

DPSC on PB: Influence on 

Proliferation

25μm

25μm

25μm
Dental pulp cells respond to surface mechanics:

Cell modulus follows surface modulus

Moduli decrease with film thickness Cell and surface moduli show 

the same functional form 

Cells respond to differentially small 

changes in surface modulus 

Evaluations using polybutadiene (PB) surfaces: 

PB (250K) spun cast out of toluene onto HF treated silicon

Film Modulus

Cell Modulus

0 500 1000 1500 2000 2500 3000

0

2

4

6

8

10

3000

TCP

Modulus (MPa)

Thickness of PB films (A)

 PB films

 DPSCs on PB films

 TCP

 DPSCs on TCP

Film Thickness(Å)

Modulus (MPa)
Polymer Film Processing 

PB or PI

Si

• Wafers were cut into 1cm2

squares 

• HF etched Si wafers 

• PB spun cast from toluene 

• Thickness of 200 to 3000 A were 

measured by ellipsometry

• annealed in Ultra High Vaccuum

(UHV) for 24 hours which 

prevents dewetting, removes 

toxic solvent, sterilizes 

substrates. 
SEM–EDAX of DPSCs on PB films 

after 21 day incubation

Non-Induced

Induced

200A 2000A

Calcium phosphate
Grazing Incidence X-ray Diffraction (GIXD) of DPSCs on PB films 

after 21 day incubation 

GIXD confirms SEM results; 

•Diffraction peak positions correspond to crystalline HA

• No crystalline deposits are formed on films 150nm or 

thicker. 

1.6 1.8 2.0 2.2 2.4

Intensity (a.u.)

q (A-1)

 Hydroxyapatite

 200A

 500A

 1500A

 2000A

 3000A

(+)

1.6 1.8 2.0 2.2 2.4

Intensity (a.u.)

q (A-1)

(-)

HA control sample

150nm
SEM of DPSCs on PB films after 21 days of incubation

Non-Induced

Induced

200A 500A 1500A 2000A 3000A

Above a film 

thickness of 

150nm (G <2.5 

MPa), no 

biomineralized 

deposits are 

observed.

Here 

Is 

The 

Boundary

8.25MPa 1.36MPa
Do cells sense Rg?

150 nm thick film: 

1 2 3 

• Cells moduli scale linearly with 

film moduli; not thickness.

• Cells sense film mechanics, 

not substrate.

• Confirm Rg scaling of 

modulus

200nm

150nm

20nm
Mineralization Markers involved in DPSCs 

differentiation

• Osteocalcin (OCN) – marker for bone formation, produced solely by mature

osteoblasts [1]

• Bone Sialoprotein (BSP) – specific maker for bone formation, is expressed in very

lower amounts ( ̴1%) in dentin and large amounts ( ̴8-12%) in bone [5]

• Dentinsialophosphoprotein (DSPP) – marker for odonotoblast differentiation [2,3]

and important for dentinogenesis and ECM mineralization [4].

• Alkaline Phosphatase (ALP) – enzyme involved in tissue mineralization, is

essential for deposition of minerals in bone and teeth

1. Rickard DJ, Sullivan TA, Shenker BJ, Leboy PS, Kazhdan I. Induction of rapid osteoblast differentiation in rat bone marrow 

stromal cell cultures by dexamethasone and BMP-2. Dev Biol 1994;161:218 –28.

2 Butler WT. Dentin specific proteins. Methods Enzymol 1987;145:290 –303.

3 Butler WT, Brunn JC, Qin C, McKee MD. Extracellular matrix proteins and the dynamics of dentin formation. Connect Tissue 

Res 2002;43:301–7. 

4 Yamada Y, Fujimoto A, Ito A, Yoshimi R, Ueda M. Cluster analysis and gene expression profiles: a cDNA microarray systembased comparison between human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs) for 

tissue engineering cell therapy. Biomaterials 2006;27:3766–81. 

5 Fujisawa R, Butler WT, Brunn JC, Zhou HY, Kuboki Y (1993).Differences in composition of cell-attachment sialoproteins 

between dentin and bone. J Dent Res 72:1222-1226.
Dental pulp cells respond to surface mechanics:

Mechanics controls osteocalcin expression (differentiation marker)

-Dex -Dex +Dex +Dex

200Å 2000Å 200Å 2000Å
RT-PCR: Surface induced gene expression DPSCs

Thin PB(-) Thick PB(-) Thin PB(+) Thick PB(+)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

attomole/ 20 ng RNA

 day7

 day14

 day21

 day28

Thin PB(-) Thick PB(-) Thin PB(+) Thick PB(+)

0.000

0.001

0.002

0.003

0.004

0.005

0.006

attomole/ 20 ng RNA

 day7

 day14

 day21

 day28

• ALP is elevated on all, except thick (-) PB. 

• Dex induces osteogenesis; OCN high .

• Thin (-) PB induces odontogenesis

(OCN decreases while DSPP increases @ 28 d)

• Thick (-) PB: No induction w/o dex.

Alkaline 

phosphatase ALP

Osteocalcin 

OCN Dentin sialophosphoprotein 

DSPP

Thin PB(-) Thick PB(-) Thin PB (+) Thick PB(+)

0

2

4

6

8

10

12

attomole/ 20 ng RNA

 day1

 day7

 day14

 day21
WHAT IS THE IMPACT OF MECHANICAL HETEROGENEITY?
cm Length Scale Differences in Mechanics 

Soluble factors limit response to substrate mechanics

Thick PB

0

1

2

3

4

5

tn(-)

tk(-)

contact-tn(-)

contact-tk(-)

non-contact-tn(-)

non-contact-tk(-)

Relative Modulus (a.u.)

No mineralization on thin PB (-Dex)

in co-culture with cells on thick PB

Cell moduli are responsive 

to substrate moduli , BUT

NO MINERALIZATION MINERALIZATION

NO MINERALIZATION
μm Length Scale differences in mechanics

(Identical chemically and topographically)

Topography Lateral Force 

50μm

Polymer film

Thin (hard) Thick (soft)

Pattern was produced by microlithography

PB was spun cast over the surface

PB Thickness (Å)
For μm length scales:

Cell moduli follow surface moduli but mineralization does not

Soluble factors limit response to substrate mechanics

-Dex

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

3-2 um 5-5 um 5-10 um

Relative Modulus (a.u.)

3-2 μm 5-5 μm 5-10 μm
nm Length Scale: cells feel multiple stimuli

Patterned area

Non-patterned area 

Thick PB

Nanopatterns were produced by imprinting from a phase separated polymer blend mask 

(PS/PMMA)

Sputter

Si wafer Si wafer Si wafer

Si wafer Si wafer

PB Spin cast 

PB

700C
25/75 50/50 thick PB

-Dex

+Dex 0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

25/75 pattern 50/50 pattern 25/75 nonpattern50/50 nonpattern

Relative Modulus (a.u.)

non-induced

induced

Cell moduli on patterns are high

(d7)

Mineralization on patterned surface

does not require dexamethasone (d28)

nm Length Scale Heterogeneity

Soluble factors do not limit cell responses to surface moduli

Patterns Thick PB
Length Scale: Regulation of Cell Function

When cells feel only one stimulus (cm to μm) -- soluble factors predominate 

Disconnect between modulus and mineralization 

When cells feel multiple stimuli (nm ) -- direct cell:substrate interactions can 

override soluble factor effects 

Increase in cell modulus and increase in mineralization 

Focal Adhesions 

• Signals from surface control cell mechanics and phenotype

• FA are as large as 1-5 μm with submicron to micron spacing

Choi et al, Trends Cell Bio 2010, 20(12)705
SEM of molded and 3D printed PLA

(Makerbot ReplicatorTM 2X, PLA filament)

Molded

3D Printed

1 um 10 um 100 um

2 um 20 um 100 um

Heterogeneity at multiple length scales
DPSC – plated on non-sterilized PLA

(24 hours post-plating)

3D Printed Molded
Adhesion and Proliferation

Topography Vs Ethylene oxide sterilization

3D Molded Tissue Culture Plastic

Day 1

Day 13

Ethylene oxide: representative pictures (n=3)

Day 28/3D
Fluorescent 

Microscopy

Day 1

Day 3

Day 5

Day 11

3D Mold

3D Mold

3D Mold

3D Mold

250μm 250μm

250μm 250μm

250μm 250μm

250μm 250μm

Plating density 9000/cm2
Cells and mineral deposits (28 d)

Molded 3D Printed

- Dexamethasone

+ Dexamethasone
Calcium phosphate: SEM/EDX

Molded

3D Printed 

(High resolution)

(- Dexamethasone)
PLA: Standard resolution -DEX

• Microns thick biomineralized 

layer.

• Nanoscale roughness induces 

cell differentiation.

• Flat and rough surface 

together—Boiomineraliztion 

overides micro-roughness in coculture 
Differentiation (RNA markers)

qRT-PCR

-Cells grown for 28 days (-dexamethasone) 

-mRNA isolated

-cDNA prepared 

-qPCR carried out with primers against 

alkaline phosphatase and osteocalcin

18S RNA was used to normalize RNA levels

Data shown is relative to β-glycerol phosphate negative control (TCP)
RT-PCR

The surface induced odontogenic differentiation in the

Absence of another cytokines. 
Conclusion

Cells respond to nanoscale surface structure. 

AM surfaces have roughness on multiple scales 

due multiple factors

To move AM forward we must have a better 

understanding of the underlying science. 

In-Situ Characterization of the polymer being 

printed makes this possible.
THANK YOU

This work was supported in part by NSF-Inspire award #1344267

and NYSTEM award #CO28096
Tissue culture plastic 

Day 1 post plating: Ethanol Vs Ethylene oxide

Ethanol molded Ethylene oxide molded
Day 1 post plating

Reduced cell plating efficiency on 3D printed surface

Ethylene oxide 3D Ethylene oxide molded

Tissue culture plastic 2/18/2015 experiment

?

Is there residual ethylene oxide

Or

Is topography impacting cell plating
13 Days post plating

Cells on 3D printed surface proliferate

Ethylene oxide 3D Ethylene oxide molded 

Tissue culture plastic
Cell morphology

3D printed (HR) Molded

Ethanol

Ethylene

oxide

DAY 7 POST-PLATING
Focal Adhesions: 

ROCK inhibition decreases cell moduli and mineralization

Inhibition of ROCK reduces cell moduli 

0

2

4

6

8

10

12

Relative Modulus (a.u)

day4

day7

-Dex,

Thin

-Dex

Thick

+Dex

Thin

+Dex

Thick

-Y2732 +Y2732
Is surface-induced phenotype reversible?

(Growth on PB without dexamethasone)

PB - 200Å

PB - 2000Å

Calcium phosphate deposited

Calcium phosphate Calcium carbonate
Additive Manufacturing of Bone Grafts

Repair of ameloblastoma Repair of traumatic injury

Images: Courtesy of M Abboud

Required to stabilize implants
TO QUESTIONS FACING MEDICINE TODAY:

TO WHAT EXTENT ARE 3-D PRINTED DEVICES EQUIVALENT TO TRADITIONAL 

MOLDED OR CAST ONES? 

Will cells respond similarly to printed and molded forms?

The Wake Forest Institute for Regenerative 

Medicine prints ear, nose and bone 

scaffolds that can be coated with cells to 

grow body parts. (Laurie Rubin)

Fabrication of PGA/PLA Scaffold

With the Shape of Human Nose

http://dx.doi.org/10.5772/55540

Cartilage replacement with porosity for cell penetration
For μm length scales:

Cell moduli follow surface moduli but mineralization does not

Soluble factors limit response to substrate mechanics

-Dex

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

3-2 um 5-5 um 5-10 um

Relative Modulus (a.u.)

+Dex

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

3-2 um 5-5 um 5-10 um

Relative Modulus (a.u.)

3-2 μm 5-5 μm 5-10 μm

3-2 μm 5-5 μm 5-10 μm