Bioactive carbon dots direct the osteogenic differentiation of human bone marrow mesenchymal stem cells

https://doi.org/10.1016/j.colsurfb.2019.03.035Get rights and content

Highlights

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    Bioactive carbon dot is facilely fabricated from adenosine and aspirin.

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    AACDs demonstrate good cytocompatibility and biosafty.

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    AACDs show long-term fluorescent tracking of hBMSCs.

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    AACDs direct osteogenic differentiation of hBMSCs without osteoinductive factors.

Abstract

Bone marrow mesenchymal stem cells (BMSCs) have been the focus of bone regeneration due to their excellent osteogenic potential and abundant source. However, the high-cost and low-efficiency differentiation of BMSCs into functional osteoblasts limits their clinical application. It is desirable to develop bioactive materials to integrate efficient differentiation and traceable properties in a biocompatible manner for MSC-based therapy. In this study, a new kind of bioactive carbon dot (CD) was facilely fabricated through a one-step hydrothermal method from adenosine and aspirin. These bioactive CDs were cytocompatible and biosafe with the capability of long-term fluorescent tracking of human bone marrow mesenchymal stem cells (hBMSCs). Notably, the presence of bioactive CDs triggered and directed a series of events that followed the temporal pattern of osteogenic differentiation through the promotion of osteogenic transcription and enhancement of matrix mineralization. Moreover, cells with bioactive CDs exhibited more effective osteogenic differentiation behavior than cells treated with either adenosine or aspirin alone. Overall, these findings clearly showed that adenosine and aspirin-based CDs can direct osteogenic differentiation of hBMSCs in the absence of any external osteoinductive factors. The unique properties of bioactive CDs could provide insight into their potential for achieving efficient and safe MSC-based therapy.

Introduction

The management of bone loss, osteanabrosis, and other conditions with poor bone formation presents a formidable challenge in the field of regenerative medicine [1,2]. The crucial role of mesenchymal stem cells (MSCs) in the initial formation and maintenance of bone makes MSCs a promising cell source for generating new bone in the presence of osteoinductive cues [3,4]. In particular, bone marrow mesenchymal stem cells (BMSCs) have been the focus of most bone repair studies due to their excellent osteogenic potential and abundant source [5,6]. Despite these opportunities, the high-cost and low-efficiency differentiation of BMSCs into functional osteoblasts limits their application in the clinic [7]. Therefore, it is urgently necessary to develop reliable and effective approaches for directing and/or promoting osteogenic differentiation of BMSCs. Several small molecules have been widely explored for in vitro osteogenic differentiation with advantages including cost-effectiveness, non-immunogenicity, and easy manipulation and standardization [[8], [9], [10]]. To improve the efficacy of small molecules to direct osteogenic differentiation of BMSCs, an ideal strategy with combinational effects, higher retention, and traceable properties is desirable.

Carbon dots (CDs) are emerging fluorescent nanomaterials with superior biocompatibility for biological research considering their excellent optical properties, chemical stability, and renal clearance ability [[11], [12], [13], [14], [15]]. Benefitting from its effective endocytosis and ease of modification, CDs have extensively contributed to achieving targeted drug delivery and fluorescent tracking of drug distribution [[16], [17], [18]]. In addition to long-term monitoring of stem cell differentiation, our group has shown that citric acid-based CDs are capable of promoting the osteogenic differentiation of MSCs through the ROS-mediated MAPK pathway [19]. To further manipulate its physical characteristics and chemical compositions, multifunctional CDs have been designed and fabricated recently by the sophisticated introduction of bioactive precursors, including small moleculs, nucleic acids, and proteins, into a bottom-up synthetic strategy [20,21]. Compared with traditional citric acid-based CDs, these bioactive CDs could have distinct pharmacological activities for extending their applications against cancer and pathogens [22,23]. In a subsequent study, we also demonstrated that bioactive CDs exhibit improved anti-cancer performance compared with free small molecules in both in vitro and in vivo studies [17]. However, bioactive CDs obtained from dual-functional precursors have yet to be established.

In this study, we used adenosine and aspirin as co-precursors to fabricate multifunctional CDs with desired biofunctions and biocompatibility. In this system, adenosine, a metabolite of ATP, has been shown to directly convert hPSCs into functional osteoblasts through the involvement of the adenosine tri-phosphate (ATP) A2b receptor (A2bR) [24,25]. Aspirin, a nonsteroidal anti-inflammatory drug (NSAID) that has been well known for decades, has been explored for its immunomodulatory effect on MSCs and for preventing bone loss in vivo [26,27]. Considering these findings, we propose that adenosine- and aspirin-based CDs (AACDs) could be effective and safe osteoinduction agents for deriving osteoblasts from BMSCs. As a proof-of-concept, we characterized and compared human bone marrow mesenchymal stem cells (hBMSCs) cultured with these bioactive CDs or homologous molecular combinations and investigated the molecular mechanism for the combined effect on directing osteogenic differentiation.

Section snippets

Materials

Adenosine was obtained from Sigma-Aldrich (St Louis, MO, USA). Aspirin was purchased from Solarbio Company (Beijing, China). Dexamethasone, l-ascorbic acid, β-glycerophosphate and cetylpyridinium chloride were obtained from Sigma-Aldrich. DMEM/F-12 medium and fetal bovine serum (FBS, mesenchymal stem cell-qualified) were purchased from Gibco. LysoTracker Red, RIPA lysis buffer, a BCA assay kit, an alkaline phosphatase (ALP) staining kit and an ALP activity kit were achieved from the Beyotime

Results and discussion

CDs were fabricated using adenosine and aspirin as co-precursors through a facile and green one-pot hydrothermal method (Scheme 1). In addition to their positive functions during MSC-mediated bone regeneration, the structure of these two small molecules aligned with the principle of a bottom-up synthetic strategy due to the amino group and carboxyl group of adenosine and aspirin, respectively. The morphology and structure of the as-synthesized AACDs were first determined using a high-resolution

Conclusions

In summary, we prepared bioactive CDs through a facile hydrothermal treatment of adenosine and aspirin. The as-prepared AACDs were able to achieve tracking of hBMSCs through endocytosis without affecting cell viability, even at a dose of 100 μg/mL. Importantly, the presence of the AACDs not only facilitated the osteogenic differentiation efficiency of hBMSCs in the presence of osteogenic induction medium but also directed the osteogenic differentiation of hBMSCs in the absence of any external

Acknowledgements

This work is sponsored by the Jilin Science & Technology Development Plan (20190304029YY, 20180201025YY, 20170623062TC), and the Opening Project of the State Key Laboratory of Supramolecular Structure and Materials of Jilin University under Grant No. SKLSSM 201713 and 201832. This work was conducted in Nanomedicine Engineering Laboratory of Jilin Province, Preclinical Pharmacology R&D Center of Jilin Province, Key Lab of Traditional Medicine for Diabetes of Jilin Province, and Key Lab of

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