When the dental pulp is infected by the bacteria of carious or traumatic origin, inflammation and tissue necrosis occur, eventually leading to loss of functions [1, 2]. Traditional clinical treatments of pulp diseases such as root canal therapy or pulp revascularization are unable or difficult to regenerate the pulp tissue [3, 4]. Recently, regenerative endodontic treatment (RET) is emerging as an effective strategy, replacing damaged pulp tissue to restore the vitality of the tooth and promote the development of the root [4], [5], [6]. The main goals of RET include the promotion of root development, regeneration of the pulp-dentin complex, and restoration of pulp functions [7]. The difficulties of the pulp regeneration research include the following: i) the root canal system is complex, with multiple and narrow root canals, and the presence of intercanal traffic [8, 9]; ii) the structure and components of the pulp tissue are complex and contain nerves, blood vessels, lymphatic vessels, and connective tissues [10]. To regenerate functional dental pulp, dental pulp regeneration medicine has elicited widespread interest [11, 12]. However, the available bioscaffolds are limited. In-situ crosslinked injectable bulk hydrogels are suitable for the root canal system, but are subject to inadequate blood supply and nutrition in the central region with the dense structure [13, 14] and eventually cause ischemic necrosis. Preformed or rigid scaffold materials, such as decellularized dental pulp are fixed in shapes and therefore difficult to stuff into narrow and curved root canals [9, 15]. Therefore, there is still an imminent need to develop a more effective engineering approach for RET.

Hydrogel microspheres can serve as an ideal bioscaffold. First, they are small in size (1-1000 μm in diameter) and can be injected through a needle, making them suitable for complex root canal systems. Second, microspheres still provide 3D microenvironments that are similar to those of the natural extracellular matrice (ECM). Also, compared with traditional bulk hydrogels, due to better oxygen and nutrition conditions, the microsphere systems oftentimes exhibit enhanced cell viability and functions [13]. Third, the use of hydrogel microspheres for delivering cells is a reliable and protective engineering strategy for avoiding the low survival rate, cell damage, faster cell death, and rapid dislocation caused by direct cell injection [15]. Another advantage of utilizing hydrogel microspheres is that they are easily functionalized by multiple bioactive factors to develop multifunctional microspheres for steering stem cell fate [16, 17].

Researchers have developed pure microspheres or microspheres functionalized with platelet lysate or simvastatin for RET [8, 18, 19]. All the microspheres modified with only simple functions, such as pro-angiogenic or pro-odontogenic attributes, are difficult to achieve multifunctional dental pulp tissue regeneration. To this end, the natural and multiple biochemical components of decellularized ECM are extremely similar to the native tissue from which it is derived [20, 21]. To further utilize the active components of the decellularized dental pulp matrix, it can be chemically transformed into solution that contains abundant odontogenic-related bioactive factors, which have been confirmed to induce the multidirectional differentiation of hDPSCs in vitro [22, 23]. Biomimetic engineering strategies indicate that decellularized dental pulp matrix solution is a suitable candidate for dental pulp regeneration due to its bioactive factors that can regulate cellular behaviors [9].

Based on these premises, to construct an injectable dental pulp-regeneration material adapted to the complex root canal system, we combined hydrogel microspheres with decellularized dental pulp-derived bioactive factors. The study design is shown in Scheme 1. A 3D dental pulp-specific microenvironment was achieved by surface modification of the hydrogel microspheres using dental pulp-derived bioactive factors. As such, the 3D dental pulp-specific microenvironment would act as a dental pulp stem cell ecological niche, regulating stem cell self-renewal, proliferation, and differentiation. We analyzed the biocompatibility and multidirectional differentiation ability of hDPSCs cultured on the functionalized microspheres. The effect of in vivo dental pulp regeneration was assessed by implanting the cell-populated microspheres in a semi-orthotopic model of immunodeficient nude mice.