Alkali-extracted proteins from the tooth dentin matrix as a mixture of bioactive molecules for cartilage repair and regeneration.

ABSTRACT

Introduction: Dentin matrix extracted protein (DMEP) is a mixture of proteins extracted from the organic matrix of a natural demineralized dentin matrix that is rich in a variety of growth factors. However, the effect of DMEP on cartilage regeneration is unclear. The aim of this study was to investigate the efficacy of DMEP extracted via a novel alkali conditioning method in promoting cartilage regeneration. Methods: Alkali-extracted DMEP (a-DMEP) was obtained from human dentin fragments using pH 10 bicarbonate buffer. The concentration of chondrogenesis-related growth factors in a-DMEP was measured via enzyme-linked immunosorbent assay (ELISA). Human bone marrow mesenchymal stem cells (hBMMSCs) in pellet form were induced with a-DMEP. Alcian blue and Safranin O staining were performed to detect cartilage matrix formation, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess chondrogenic-related gene expression in the pellets. Rabbit articular osteochondral defects were implanted with collagen and a-DMEP. Cartilage regeneration was assessed with histological staining 4 weeks after surgery. Results: Compared with traditional neutral-extracted DMEP, a-DMEP significantly increased the levels of transforming growth factor beta 1(TGF-ß1), insulin-like growth factor-1(IGF-1) and basic fibroblast growth factor (bFGF). After coculture with hBMMSC pellets, a-DMEP significantly promoted the expression of the collagen type II alpha 1(COL2A1) and aggrecan (ACAN) genes and the formation of cartilage extracellular matrix in cell pellets. Moreover, compared with equivalent amounts of exogenous human recombinant TGF-ß1, a-DMEP had a stronger chondrogenic ability. In vivo, a-DMEP induced osteochondral regeneration with hyaline cartilage-like structures. Conclusions: Our results showed that a-DMEP, a compound of various proteins derived from natural tissues, is a promising material for cartilage repair and regeneration.