A Biomimetics Approach to Engineering Arterial Conduits for Congenital Heart Surgery

Timothy Martens, MD PhDCo-Director, Pediatric Heart Center

Loma Linda University Children’s Hospital

Congenital Heart Disease

• Affects roughly 40,000 births annually in the United States

• 25% of cases considered severe, requiring surgical intervention within the first year of life

• Currently 3.3 million cases of congenital heart disease in the United States alone

https://www.cdc.gov/ncbddd/heartdefects/data.html

Chapter 18, Cardiovascular System 3

External Heart: Anterior View

Figure 18.4b

Truncus Arteriosus

Ross Procedure - Biventricular Outflow Tract Reconstruction

Current Prosthetic and Conduit Options

Vascular Conduit Options

Valve Repair and Replacement

Holy Grail• Engineered replacement that is fully functional upon

implantation• Has the capacity to keep pace with somatic growth

and avoid reoperation• Composed of non-immunogenic materials / cells• Durable

Biomimetic Approach

Grayson et al, Semin Cell Dev Biol (2009)

Scaffold Cells

PerfusionBioreactor

Hypothesis: Mimicking the physiologic milieu through pulsatile perfusion of a cardiovascular progenitor cell populated scaffold will induce vascular smooth muscle differentiation to create an arterial conduit exhibiting native mechanical properties and hemodynamics.

Experimental Overview

Early CPC Selection

Early CPC Expansion

Experimental Overview

In Vitro Static Conditioning

In Vitro Pulsatile Conditioning

Early CPC Expansion

Optimized Detergent Decellularization

• 1% Sodium Dodecyl Sulfate (SDS) – 24 Hour Wash• Anionic surfactant• Lyses cells

• 1% Triton X-100 – 1 Hour Wash• Nonionic surfactant• Solubilizes SDS for removal

• Phosphate Buffered Saline (PBS) – 72 Hour Wash• Removes SDS, Triton X-100, and cellular contents from

decellularized scaffold

Decellularized Pulmonary and Aortic Roots

• Matrix proteins well conserved across species –no shortage of donor scaffolds

• Structural and mechanical properties well preserved• Retention of matrix components• Decellularized Products already in widespread clinical use

Flawed decellularization leaves nuclear tissue or damages the matrix

Nuclei – PurpleExtracellular Matrix – Pink

Scale Bar - 100μm

Inadequate SDS Exposure Excessive SDS Exposure

H&E staining shows complete removal of cell nuclei

Nuclei – PurpleExtracellular Matrix – Pink

Scale Bar - 100μm

Native Decellularized

Trichrome staining demonstrates collagen maintenance

Collagen - BlueCytoplasm – Red

Smooth Muscle - BrownScale Bar - 100μm

Native Decellularized

Elastin staining confirms elastin maintenance

Elastin – BlackCollagen - Pink

Scale Bar - 100μm

Native Decellularized

Scanning electron microscopy shows removal of cell bodies

Native Decellularized

Double-stranded DNA isolation showed significant removal of residual DNA

• 93% reduction in DNA content

• Acceptable for recellularization

• Biological activity of residual DNA unknown

• Will include DNase step for transplantation

Experimental Overview

Early CPC Selection

Early CPC Expansion

Cardiovascular Progenitor Lineages and Differentiation

Von Willebrand Factor (vWF) – EndothelialCardiac Troponin T (TropT) – CardiomyocyteSmooth Muscle Actin (SMA) – Smooth Muscle

Baio, J., npj Microgravity, 2018

Manual Recellularization with Tuberculin Syringe

• Cardiovascular progenitor cells (CPCs) suspended in 0.5mL of CPC growth media

• Injected into decellularized scaffolds with 27g tuberculin syringe• Eight linear tracts made with syringe and cells injected as the

needle was retracted• Static or pulsatile culture in CPC

growth media

Optimization of Manual Recellularization

Culturing Cells after Recellularization

CPC Injection

Static Culture Pulsatile Culture

One Week One WeekThree Week Three Week

Static Culture Conditions

• Seeded scaffold submerged in 20mLs of CPC growth media in T-25 flask

• Incubator maintained at 37°C with 5% CO2

• CPC growth media changed every other day

Pulsatile Perfusion Conditions

• Seeded scaffold submerged in 20mLs of CPC growth media in T-25 flask• Allow 24 hours for cell adherence

• Scaffold mounted in perfusion chamber after 24 hours and connected in circuit with centrifugal perfusion pump

• Scaffold perfused with CPC growth media

• Incubator maintained throughout entire process at 37°C with 5% CO2

Pulsatile Perfusion Conditions

Pulsatile Perfusion Conditions

Ex Vivo Pulsatile Perfusion

Desired Cell Lineages

Cardiovascular Progenitor Cell Markers

W

Cardiomyocyte Markers

Endothelial Cell Markers

Smooth Muscle Cell Phenotyping

Synthetic Contractile

ü Smooth Muscle ActinCalponinMyosin Heavy Chain 11

ü Smooth Muscle Actinü CalponinüMyosin Heavy Chain 11

Smooth Muscle Cell Markers

Smooth muscle actin present in recellularized scaffolds at three weeks

SMA – Red DAPI – Blue Scale Bar - 100μm

Positive Control Recellularized

Ki67 presence indicates seeded cells are proliferative at three weeks

Ki67 – Green DAPI – Blue Scale Bar - 100μm

Positive Control Recellularized

Endothelial cells absent in recellularized scaffolds at three weeks

Scale Bar - 200μm vWF – Red DAPI – Blue Scale Bar - 100μm

Positive Control Recellularized

Cardiomyocytes absent in recellularized scaffolds at three weeks

TropT – Red DAPI – Blue Scale Bar - 100μm

Positive Control Recellularized

Native pulmonary arteries express calponin ubiquitously

Calponin - Green DAPI – Blue Scale Bar - 300μm

Positive Control Negative Control

Calponin is present on 3 Week Pulse arterial scaffold.

Calponin - Green DAPI – Blue Scale Bar - 300μm

3 Week Static 3 Week Pulse

Smooth Muscle Cell Markers

Tissue bath contraction studies functionally confirm decellularization of our pulmonary arteries

Functional testing verified contractility of three week pulsatile scaffolds

All scaffolds were tensioned to 1.5 grams prior to contractility testing.

Conclusions

• Detergent decellularization completely removes cells while maintaining native extracellular matrix.

• The decellularized scaffold microenvironment directs mesodermal differentiation into synthetic smooth muscle cells.

• Pulsatile perfusion commits cardiovascular progenitor cells to a contractile smooth muscle fate.

• Pulsatile perfusion may also induce endothelial differentiation with longer duration.

Animal Study

3 month old juvenile sheep

Allogeneic Implant into mPA

Unseeded Control (n=3)vs.

3Week Pulsatile (n=3)

Two week survival

Repeat Echocadiography ans Sacrifice

Echo Data – Pulmonary Artery Diameter (cm)161G 164G 168G 176G 185G Mean p

PRE 1.55 1.14 1.51 1.61 1.81 1.55

POST 1.71 1.17 1.93 1.61 1.88 1.71 N.S.

EXPLANT 1.61 1.29 1.60 1.64 1.60 1.62 N.S.

No pulmonary valve insufficiency

No pulmonary artery stenosis

Normal RV function

Native Tissue Decellularized Graft Recellularized Graft

Smoo

th M

uscl

e Ac

tinCa

lpon

inVo

n W

illeb

rand

Fac

tor

Future Directions

• Longer term implants to study true growth potential

• Pulmonary Root Replacement (valved conduit)

• Allogeneic vs. Autologous

• Pericytes?

Acknowledgements

• Bright Field Microscopy – Jon Hough

• UCR Central Facility for Advanced Microscopy and Microanalysis• Michael Pidgeon

• Funding - 2017 Grants to Promote Collaborative and Translational Research (GCAT)

• Mary Kearns-Jonker• Keren Garcia• Aaron Deatherage• Larry Lopez• Sean Wilson• Monica Romero• Sam Murray• Arlin Blood• Gordana Vunjak-

Novakovic

Questions?