Constructing Native 

Extracellular Matrix 

Scaffolds for Tissue 

Repair 

Sanjana Sureshbabu

Mentor: Minh Phan
Why Tissue Engineer?

- Biocompatible polymer is used for tissue repair and 

implantation, but cell communication may be 

inhibited.

- Native scaffolds from tissues have risks of disease, 

rejection, and is it hard to isolate.

- Cell derived ECM can be taken from human cell 

cultures, but ECM production can take months to 

occur.

- Goal: Engineer pre-scaffolds to significantly speed 

the process up.

Assunção, M., Dehghan-Baniani, D., Yiu, C. H. K., Später, T., Beyer, S., & Blocki, A. (2020, November 

10). Cell-derived extracellular matrix for tissue engineering and Regenerative Medicine. Frontiers. 2

Attach to 

template interact

ECM production
Pre-Scaffold Construct “The Bricks”

Collagen- main structural element, mechanical strength 

Elastin- elasticity of tissues (large arteries, heart valves, etc)

Fibronectin- promotes cell adhesion and migration 

Laminin- component of basement membrane, supports growth and differentiation of cells 

Pompili, Simona, et al., (22 Mar. 2021) The Charming World of the Extracellular 

Matrix Frontiers 3
Arranging Protein on Membrane

- Extracellular Matrix proteins tend to fold 

in native form, this will cause it to be 

inactive. 

- Utilizing lipid membranes can unfold 

proteins, and they can make it 

functional

What do we do to make this 

construction possible?

+

1. Individually

2. Combinations

a. Fibronectin and Collagen 

i. Accelerates the onset of fibril 

growth

b. Fibronectin and Laminin 

i. Fibrillogenesis occurs the 

same way in both proteins 

due to β-sheet structures

Paten, J. A., Martin, C. L., Wanis, J. T., Siadat, S. M., Figueroa-Navedo, A. M., & 

Ruberti, J. W. (2019, June 6). Molecular interactions between collagen and 

fibronectin: A reciprocal relationship that regulates de novo fibrillogenesis. 

Chem. 4

Ahn, Seungkuk et al. (2018) Formation of Multi-Component Extracellular 

Matrix Protein Fibers
Goals 

● To determine the membrane compositions that best supports 

the proteins and construct a scaffold on the lipid particles 

● The particles should be able to put on wounds to establish cell 

connection, tissue reconstruction, and healing

5
Langmuir Isotherms (X & Y Axes) and Adsorption Assays 

- Phase diagram that gives you insight of the 

two dimensional phase behavior of sample 

- To find the optimized pressure for the 

protein to be injected 

- Shows if protein is adsorbed at all; 

kinetics of the adsorption

ex) 

X-ray Reflectivity(Z Axis)

- How protein arranges under the membrane

- Measures thickness, density, roughness, etc.

Z

6
Experimental Design: Lipid Composition

DMPC: the skeleton of the membrane 

DMPE: regulates the non-lamellar structure in 

the membrane 

DMPS: negatively charged lipid, helps 

fibronectin and laminin binding 

Cholesterol: increases the lateral lipid 

headgroup separation on membrane surface, 

this promotes the association degree of 

collagen monomers

Phan, Minh D et.al Langmuir (2020) The Effect of Cholesterol on Membrane Binding and Self-Assembly

binding more uniform with 

collagen

7

collagen is 

bulky

no fibril 

formation DMPC: DMPE :DMPS: Chol

2: 1: 2-4: 1 ← mole ratio
Average of Trials With and Without Cholesterol

-3.37

-0.61

-2.65

8

-3.42

-0.66

- Plateau in the cholesterol 

curve vanishes; the film is 

almost all in one phase

- At lower pressures, 

cholesterol results in a more 

ordered membrane. It’s 

opposite at higher pressures

-2.72

-0.94

DMPC:DMPE:DMPS:Cholesterol

2:1:2:1

DMPC:DMPE:DMPS

2:1:2
X-ray Reflectivity Experiments

Measurement #1

- Bare membrane 

- Only the mixed 

lipid sample, no 

protein injected 

Measurement #2

- Initial injection of 

protein 

Measurement #3

- Equilibrium 

measurement, 

protein is at 

equilibrium 

9
Bare Membranes

(Before protein injection)

- Collagen and fibronectin 

graphs are very similar; 

reproducible 

- 3PS & 4PS graphs are 

shifted inward-- thicker

- Pressure decreases, film is 

thinner

Structure of film changes as PS and pressure are varied

10
Collagen XRR

- Uniform collagen 

assembly on membrane 

- Non-specific binding to 

membrane, not 

dependent on negative 

charge 

DMPC:DMPE:DMPS:Cholesterol

2:1:2:1

1

2

(lower 

Qz)

11
Laminin XRR

1

2

12
Fibronectin XRR

Shown that FN binding is dependent on 

PS, a negative charged lipid

Increase in PS and loosening 

packing density → no change

DMPC:DMPE:DMPS:Cholesterol

2:1:2:1

13
Elastin XRR

14
Conclusions

Lipid Sample Conclusion

- The presence of cholesterol results in 

the film being in mostly one phase 

- Raising the PS ratio in the lipid 

solution makes the bare membrane 

thicker. 

Protein Conclusions

- Successfully fabricated uniform 

collagen assembly on membrane

- Laminin shows signs of adsorption

- Fibronectin and elastin should 

bind better in neutral pH where 

the membrane has more effective 

negative charges for the proteins 

to interact with

15
Acknowledgments 

•Minh Phan -- Mentor, XRR procedure, etc.

•Sushil Satija-- Co mentor

•NCNR SURF Directors--

• Julie Borchers

• Susana Marujo Teixeira

• Leland Harriger

•Surabhi Singh -- Lab Partner

•NIST SURF Program