Human dental pulp stem cells expressing TGF{221}-3 transgene for cartilage-like tissue engineering

Abstract

A major challenge facing the tissue engineering discipline is cartilage tissue repair

and engineering, because of the highly specialized structure and limited repair

capacity that cartilage possesses. Dental pulp stem cells (DPSCs) were identified

about a decade ago as a potential candidate for cell based therapy and tissue

engineering applications. The present study aimed to utilize gene therapy with

isolated DPSCs to induce chondrogenic transgene expression and chondrogenic

lineage differentiation, with the ultimate goal of engineering cartilage tissue-like

constructs.

We isolated DPSCs from human teeth extracted for orthodontic treatment. We

further enriched the isolated population using immunomagnetic bead selection,

which increased stem cell markers: Stro-1 and CD146, compared to unselected

population.

The DPSCs showed the ability to differentiate into the chondrogenic lineage when

induced with recombinant hTGFβ-3 and when transduced with hTGFβ-3

transgene. We successfully constructed the recombinant adeno-associated viral

vector encoding the human TGFβ-3, and determined the best multiplicity of

infection for DPSCs. The transduced DPSCs highly expressed hTGFβ-3 for up to

60 days. Expression of chondrogenic markers; Collagen IIa1, Sox9, and aggrecan

was verified by immunohistochemistry and mRNA.

We successfully fabricated an electrospun nano-fiber scaffold upon which

morphology, proliferation and viability of the DPSCs were examined. DPSCs

attached and proliferated on nano-fiber scaffolds demonstrating better viability

compared to micro-fiber scaffolds.

Transduced cells expressed hTGFβ-3 protein up to 48 days. Cells seeded on nanofiber

scaffolds showed higher expression levels compared to micro-fiber scaffolds

or culture plate.

Scaffolds seeded with DPSCs were implanted in nude mice.

Immunohistochemistry for TGFβ-3 DPSCs constructs (n=5/group) showed

cartilage-like matrix formation with glucoseaminoglycans as shown by Alcian

blue. Immunostaining showed positivity for Collagen IIa1, Sox9 and aggrecan.

Semi-thin sections of the transduced DPSCs constructs examined by transmission

electron microscopy (TEM) showed chondrocytic cellular and intra-cellular

features, as well as extracellular matrix formation (n=2/group).

In vivo constructs with the TGFβ-3 DPSCs showed higher collagen type II and

Sox9 mRNA expression relative to non-transduced DPSCs constructs

(n=5/group). Western blot analysis confirmed this expression pattern on the

protein level (n=3/group).

Engineered constructs mechanical properties were examined and compared to

patellar bovine cartilage to assess functionality (n=5/group). TGFβ-3 transduced

DPSCs constructs showed a higher equilibrium elastic modulus compared to nontransduced

constructs. Micro-fiber scaffolds constructs showed a higher elastic

modulus (0.11 MPa, 18% of bovine cartilage), compared to nano-fiber constructs

modulus (0.032 MPa, 6% of bovine cartilage). Nano-fiber based constructs

showed a similar Poisson‘s ration to bovine cartilage, while that of micro-fiber

scaffolds was lower.

As an alternative gene delivery method, electroporation parameters for DPSCs

transfection were optimized, and compared to commonly used chemical

transfection methods. TGFβ-3 transfected DPSCs showed a significantly higher

relative TGFβ-3 mRNA and protein expression compared to non transfected

control and to eGFP transfected DPSCs. Transfected DPSCs showed increased

relative expression of chondrogenic markers; Collagen II, Sox9 and aggrecan,

compared to non transfected DPSCs.

Successful chondrogenic differentiation of DPSCs gene therapy with TGFβ-3

transgene, and seeding them on PLLA/PGA scaffolds makes it a potential

candidate for cartilage tissue engineering and cell based therapy.