Periodontal Ligament Cells Cultured Under Steady-Flow Environments Demonstrate Potential for Use in Heart Valve Tissue Engineering

Abstract

A major drawback of mechanical and prosthetic heart valves is their inability to permit somatic growth. By contrast, tissue-engineered pulmonary valves potentially have the capacity to remodel and integrate with the patient. For this purpose, adult stem cells may be suitable. Previously, human periodontal ligament cells (PDLs) have been explored as a reliable and robust progenitor cell source for cardiac muscle regeneration (Pelaez, D. Electronic Thesis and Dissertation Database, Coral Gables, FL, May 2011). Here, we investigate the potential of PDLs to support the valve lineage, specifically the concomitant differentiation to both endothelial cell (EC) and smooth muscle cell (SMC) types. We were able to successfully promote PDL differentiation to both SMC and EC phenotypes through a combination of stimulatory approaches using biochemical and mechanical flow conditioning (steady shear stress of 1 dyne/cm(2)), with flow-based mechanical conditioning having a predominant effect on PDL differentiation, particularly to ECs; in addition, strong expression of the marker FZD2 and an absence of the marker MLC1F point toward a unique manifestation of smooth muscle by PDLs after undergoing steady-flow mechanical conditioning alone, possible by only the heart valve and pericardium phenotypes. It was also determined that steady flow (which was performed using a physiologically relevant [for heart valves] magnitude of ~5-6 dynes/cm(2)) augmented the synthesis of the extracellular matrix collagen proteins. We conclude that under steady-flow dynamic culture environments, human PDLs can differentiate to heterogeneous cell populations that are relevant to heart valve tissue engineering. Further exploration of human PDLs for this purpose is thus warranted.

Movement Effects on the Flow Physics and Nutrient Delivery in Engineered Valvular Tissues

“…Importantly, Thy-1 has been shown to be present in undifferentiated stem cells (Martinez et al, 2013). Thus as a preliminary experimental finding, we have found here through increased Thy-1 expression from oscillatory flow conditions, that BMSCs remain in a less differentiated state.…”

“…The purpose of heart valve tissue engineering is to obtain an in vitro graft with mechanical properties and anatomical-like structures similar to those of native valves as well as to accommodate somatic growth (Martinez et al, 2013;Ramaswamy et al, 2010;Salinas et al, 2014a;Salinas et al, 2014b). This can be potentially achieved by seeding biomaterial scaffolds with autologous cells such as bone marrow derived stem cells (BMSCs) (Kadner et al, 2002;Leor et al, 2005;Perry et al, 2003;Ramaswamy et al, 2010;Salinas et al, 2014a;Salinas et al, 2014b) and subjecting them to mechanical conditioning paradigms until sufficient heart valve cells and tissue growth in vitro has been achieved .…”

“…Yet, after more than a decade of intensive TEHV investigations which included clinical studies (Dohmen et al, 2011), identification of a specific protocol for optimized construct development which can subsequently be clinically translated remains elusive. While potential cell sources and scaffold materials for heart valve tissue engineering have been identified (Chen and Wu, 2005;Fong et al, 2006;Frank et al, ;Hilfiker et al, 2011;Kadner et al, 2002;Leor et al, 2005;Lichtenberg et al, 2006;Martinez et al, 2013;Neuenschwander and P. Hoerstrup, 2004;Perry et al, 2003;Schmidt et al, 2006;Schmidt et al, 2010;Siepe et al, 2008;Simon et al, 2006;Weber et al, 2011a;Weber et al, 2011b), the biomechanical conditions needed to promote the heart valve phenotype still require further investigation. In addition, cellular responses to the applied mechanical stresses will undoubtedly depend on the cell type(s) being utilized; adult progenitor cells, such as bone marrow derived stem cells (BMSCs) are deemed likely candidates for utility in heart valve tissue engineering owing to their pluripotency, ease of access and lack of ethical concerns.…”

“…Tissue engineered heart valves (TEHVs) could potentially serve as a viable solution to congenital valve disease among pediatric patients who currently have extremely limited options (Fong et al, 2006;Kadner et al, 2002;Martinez et al, 2013;Schmidt et al, 2006). The primary appeal of TEHVs lies in their potential to integrate and grow with the patient, but in addition, TEHVs could potentially overcome current valve replacement barriers such as hemolysis, immune rejection, calcification, anticoagulation therapies and the need for multiple invasive surgeries (Chen et al, 2013;Hmiel, 2012;Leong et al, 2013;Munnelly et al, 2012;Nora et al, 2012;Syedain, 2009a).…”

Regulation of Bone Marrow Stem Cells through Oscillatory Shear Stresses - A Heart Valve Tissue Engineering Perspective

“…Quantitative real time-polymerase chain reaction (QRT-PCR) was performed as previously described [139], [140], [82], [141] . Engineered tissues were washed with DPBS and total RNA was isolated according to manufacturers protocol.…”