Extracellular Matrix Derived From Dental Pulp Stem Cells Promotes Mineralization

Nunthawan Nowwarote; Stephane Petit; Francois Come Ferre; Florent Dingli; Victor Laigle; Damarys Loew; Thanaphum Osathanon; Benjamin P J Fournier

doi:10.3389/fbioe.2021.740712

Extracellular Matrix Derived From Dental Pulp Stem Cells Promotes Mineralization

Front Bioeng Biotechnol. 2022 Jan 27:9:740712. doi: 10.3389/fbioe.2021.740712. eCollection 2021.

Authors

Nunthawan Nowwarote^{1

2

3}, Stephane Petit², Francois Come Ferre², Florent Dingli⁴, Victor Laigle⁴, Damarys Loew⁴, Thanaphum Osathanon^{1

5}, Benjamin P J Fournier^{2

3}

Affiliations

¹ Dental Stem Cell Biology Research Unit, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
² Centre de Recherche des Cordeliers, INSERM UMRS 1138, Molecular Oral Pathophysiology, Université de Paris, Sorbonne Université, Paris, France.
³ Department of Oral Biology, Dental Faculty Garancière, Université de Paris, Paris, France.
⁴ Institut Curie, Centre de Recherche, Laboratoire de Spectrométrie de Masse Protéomique, PSL Research University, Paris, France.
⁵ Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.

Abstract

Background: Extracellular matrix (ECM) plays a pivotal role in many physiological processes. ECM macromolecules and associated factors differ according to tissues, impact cell differentiation, and tissue homeostasis. Dental pulp ECM may differ from other oral tissues and impact mineralization. Thus, the present study aimed to identify the matrisome of ECM proteins derived from human dental pulp stem cells (DPSCs) and its ability to regulate mineralization even in cells which do not respond to assaults by mineralization, the human gingival fibroblasts (GF). Methods: ECM were extracted from DPSCs cultured in normal growth medium supplemented with L-ascorbic acid (N-ECM) or in osteogenic induction medium (OM-ECM). ECM decellularization (dECM) was performed using 0.5% triton X-100 in 20 mM ammonium hydroxide after 21 days. Mass spectrometry and proteomic analysis identified and quantified matrisome proteins. Results: The dECM contained ECM proteins but lacked cellular components and mineralization. Interestingly, collagens (COL6A1, COL6A2, and COL6A3) and elastic fibers (FBN1, FBLN2, FN1, and HSPG2) were significantly represented in N-ECM, while annexins (ANXA1, ANXA4, ANXA5, ANXA6, ANXA7, and ANXA11) were significantly overdetected in OM-ECM. GF were reseeded on N-dECM and OM-dECM and cultured in normal or osteogenic medium. GF were able to attach and proliferate on N-dECM and OM-dECM. Both dECM enhanced mineralization of GF at day 14 compared to tissue culture plate (TCP). In addition, OM-dECM promoted higher mineralization of GF than N-dECM although cultured in growth medium. Conclusions: ECM derived from DPSCs proved to be osteoinductive, and this knowledge supported cell-derived ECM can be further utilized for tissue engineering of mineralized tissues.

Keywords: decellularization; dental pulp stem cells; extracellular matrix; gingival fibroblasts; osteogenic differentiation.