Effects of nanofibrous engineered matrix on osteoblast and odontoblast differentiation

Abstract

This study intended to investigate the effect of the morphological features of the extracellular matrix (ECM) and its nanofibrous structure, on the differentiation of osteoblasts and odontoblasts, and the applicability of a nanofibrous engineered matrix, to support bone and dentin regeneration. Collagen, a fibrous protein that accounts for most of the ECM proteins of hard tissues, plays a key role in osteoblast differentiation and hard tissue regeneration. Moreover, the fibrous structure of collagen, can enhance the stability of the Runx2 protein, which is known to promote osteoblast differentiation. In this context, polystyrene, a common tissue culture dish material, was chosen as a synthetic polymer to simulate the morphological characteristics of collagen for in-vitro tests. For in-vivo animal experiments, poly-ε-caprolactone, a highly biocompatible polymer, was tested. Each polymer was independently fabricated to a nanofibrous engineered matrix, with morphology similar to collagen, through electrospinning. As a natural polymer, autologous fibrin was used to prepare the fibrous engineered matrix and evaluated for its effect on osteoblastic differentiation and bone regeneration. In the in-vitro tests, electrospun polystyrene suppressed the degradation of the Runx2 protein, in a manner comparable to that of collagen. This was confirmed in both, MC3T3-E1 osteoblasts and Runx2 gene transfected C2C12 myoblasts. In the pulp capping model using beagle dogs, the fiber structured poly-ε-caprolactone membrane, promoted the formation of excellent dentin bridges and maintenance of healthy pulp. Through in-vitro tests, using MDPC-23 preodontoblasts, the electrospun poly-ε-caprolactone membrane, not only provided a fibrous ECM but also had the effect of blocking the cytotoxicity that occurs in the hydration stage of the mineral trioxide aggregate, one of the cement materials, used in the pulp capping procedure. The fibrin could be made to be morphologically similar to the collagen, and the selective adsorption of fibronectin, which is known to promote bone regeneration, was confirmed to be superior to collagen. In addition, MC3T3-E1 osteoblasts showed a higher proliferation rate on the fibrin than collagen, and fibrin was more favorable for bone differentiation and regeneration, at the level of the Runx2 protein, transcription activity, and alkaline phosphatase activity. It was verified that this property of fibrin could be controlled by the thrombin concentration treatment, used in the preparation of fibrin from fibrinogen. The selective adsorption of fibronectin, increased with the concentration of treated thrombin. In the in-vitro MC3T3-E1 osteoblast tests, the level of Runx2 protein increased, as the selective adsorption of fibronectin increased and the activity of integrins β1 and β3, reported to be associated with osteoblast differentiation, were jointly increased. These results suggest that the natural and synthetic polymer materials can stimulate differentiation of osteoblasts and odontoblasts by mimicking the fibrous morphological features of the ECM. Furthermore, the findings imply that a better performance may be expected, when suitable materials are selected, according to the medical purpose and applied as a nanofibrous engineered ECM which mimicks the morphological features of the hard tissue, required for regeneration.