TNF-α-licensed exosome-integrated titaniumaccelerated T2D osseointegration by promoting autophagy-regulated M2 macrophage polarization.

Type 2 diabetes (T2D) is on a notable rise worldwide, which leads to unfavorable outcomes during implant treatments. Surface modification of implants and exosome treatment have been utilized to enhance osseointegration. However, there has been insufficient approach to improve adverse osseointegration in T2D conditions. In this study, we successfully loaded TNF-α-treated mesenchymal stem cell (MSC)-derived exosomes onto micro/nano-network titanium (Ti) surfaces. TNF-α-licensed exosome-integrated titanium (TNF-exo-Ti) effectively enhanced M2 macrophage polarization in hyperglycemic conditions, with increased secretion of anti-inflammatory cytokines and decreased secretion of pro-inflammatory cytokines. In addition, TNF-exo-Ti pretreated macrophage further enhanced angiogenesis and osteogenesis of endothelial cells and bone marrow MSCs. More importantly, TNF-exo-Ti markedly promoted osseointegration in T2D mice. Mechanistically, TNF-exo-Ti activated macrophage autophagy to promote M2 polarization through inhibition of the PI3K/AKT/mTOR pathway, which could be abolished by PI3K agonist. Thus, this study established TNF-α-licensed exosome-immobilized titanium surfaces that could rectify macrophage immune states and accelerate osseointegration in T2D conditions.

Micro/nano-modified titanium surfaces accelerate osseointegration via Rab7-dependent mitophagy.

To achieve rapid and successful osseointegration of titanium (Ti) implants, the underlying mechanisms of surface modification-mediated bone metabolism need to be clarified. Given that the microenvironment surrounding Ti implants may be altered after implant insertion, mitophagy as a key control system for cellular homeostasis is most likely to regulate osseointegration. Recent findings suggest that PTEN-induced putative kinase 1 (Pink1)/Parkin-mediated mitophagy plays a key role in bone metabolism. Since the micro/nano-modified surfaces of Ti implants have been widely appreciated for osseointegration acceleration, we used two common micro/nano-modified techniques and demonstrated elevations of both the osteo-differentiation potential and Pink1/Parkin pathway of osteoblasts. Moreover, the Pink1/Parkin pathway exhibited an upward trend during osteoblast differentiation. However, when osteoblasts were treated with CCCP, a Pink1/Parkin inducer, the osteo-differentiation potential decreased. Our further study showed that the small GTPase Rab7, which was inhibited by CCCP, was essential for the Pink1/Parkin pathway. Upon Pink1 or Rab7 knockdown, the pro-osteogenic effect of micro/nano-modified Ti surfaces was significantly weakened. The present results demonstrated that Rab7 activation was essential for active mitophagy and osteogenesis. In addition, Rab7 was confirmed to mediate the process of autophagosome formation. Our findings provide novel insights into new targets for osseointegration promotion, regardless of Ti surface characteristics.

Micro/nano topography of selective laser melting titanium inhibits osteoclastogenesis via mediation of macrophage polarization.

Selective laser melting (SLM) titanium (Ti) implants have shown good prospects for personalized clinical application, but further research is necessary to develop stabilized long-term properties. Since surface modification has been proven bioactive for osseointegration, conventional Ti surface treatment technologies, including sandblasting/acid-etching (SLA) and sandblasting/alkali-heating (SAH), were applied to construct micro and micro/nano surfaces. The SAH group with netlike nano-structure topography exhibited appropriate surface roughness and high hydrophilicity, and as expected, the osseointegration capacities in vivo of the three groups were in order of SAH > SLA > SLM. Besides, both in vivo and in vitro studies revealed that the SLA- and SAH-treated SLM Ti implants significantly inhibited osteoclast activity of peri-implants. Considering the close associations between osteoclasts and macrophages, the effects of Ti surface topography on macrophage polarization were detected. The results showed that the SLA- and SAH-treated SLM Ti implants, especially the latter, had the capacity to promote macrophage polarization to the M2 phenotype. Moreover, the cell culture supernatants of M2 macrophages and RAW264.7 cells seeded on SLA- and SAH-treated SLM Ti surfaces had an adverse effect on osteoclastogenesis. Collectively, this study demonstrated that micro/nano topographies of SLM Ti implants were effective for osseointegration promotion, and their inhibition of osteoclastogenesis might be attributed to macrophage polarization. Our findings shed some light on clinical application of SLM Ti implants and also prove a specific association between macrophage polarization and osteoclastogenesis.

Biomineral Precursor Formation Is Initiated by Transporting Calcium and Phosphorus Clusters from the Endoplasmic Reticulum to Mitochondria.

Mineral granules in the mitochondria of bone-forming cells are thought to be the origin of biomineral precursors, which are transported to extracellular matrices to initiate cell-mediated biomineralization. However, no evidence has revealed how mitochondrial granules form. This study indicates that mitochondrial granules are initiated by transporting calcium and phosphorus clusters from the endoplasmic reticulum (ER) to mitochondria based on detailed observations of the continuous process of mouse parietal bone development as well as in vitro biomineralization in bone-forming cells. Nanosized biomineral precursors (≈30 nm in diameter), which originate from mitochondrial granules, initiate intrafibrillar mineralization of collagen as early as embryonic day 14.5. Both in vivo and in vitro studies further reveal that formation of mitochondrial granules is induced by the ER. Elevated levels of intracellular calcium or phosphate ions, which can be induced by treatment with ionomycin and black phosphorus, respectively, accelerate formation of the calcium and phosphorus clusters on ER membranes and ultimately promote biomineralization. These findings provide a novel insight into biomineralization and bone formation.

Extracellular vesicles derived from the mid-to-late stage of osteoblast differentiation markedly enhance osteogenesis in vitro and in vivo.

Extracellular vesicles (EVs) play an important role in biological functions and may feature innate therapeutic potential for diseases. In the present study, EVs released by osteoblasts at different stages of the mineralization process were investigated for their potential ability to promote bone formation. Results showed that the characteristics of EVs of mineralizing osteoblasts changed with regularity. EVs derived from the mid-to-late differentiation stage remarkably promoted osteoblast differentiation of bone marrow-derived mesenchymal stem cells and improved osteoporosis in ovariectomized mice. The findings also revealed that the effect of EVs on osteogenesis was related with the maturity of matrix vesicles (MVs), a kind of EVs selectively released by mineralizing-related cells. Nevertheless, only the EVs from the mid-to-late stage showed osteoinductive properties, Synthetic cartilage lymph (SCL) treatment of EVs from the middle stage could promote MV maturation but showed no effect on osteoinduction. Additionally, EVs derived at the middle and mid-to-late stages showed innate bone-targeting potential. Collectively, this study demonstrated that EVs released by osteoblasts at the mid-to-late differentiation stage markedly enhance osteogenesis. Our findings present the prospective use of osteoblast-released EVs in bone tissue engineering.

Deproteinized bovine bone matrix induces osteoblast differentiation via macrophage polarization.

Bone grafts are widely used in bone regeneration to increase the speed and quality of new bone formation. While they are routinely characterized based on their biocompatible and bioactive properties, they also exert a profound impact on host immune responses, which in turn can display a significant effect on the healing and repair process. In this study, we investigated the role of macrophage behavior on deproteinized bovine bone matrix (DBBM, BioOss) to investigate their impact on creating either a pro- or anti-inflammatory microenvironment for tissue integration. RT-PCR and immunofluorescence staining results demonstrated the ability for RAW 264.7 cells to polarize toward M2 wound-healing macrophages in response to DBBM and positive control (IL-4). Interestingly, significantly higher expression of interleukin-10 and higher number of multinucleated giant cells (MNGCs) was observed in the DBBM group. Thereafter, conditioned media (CM) from macrophages cultured with DBBM seeded with MC3T3-E1 cells demonstrated a marked increase in osteoblast differentiation. Noteworthy, this effect was reversed by blocking IL10 with addition of IL10 antibody to CM from the DBBM macrophages. Furthermore, the use of dendritic cell specific transmembrane protein (DC-STAMP)-knockout to inhibit MNGC formation in the DBBM group resulted in a significant reduction in osteoblast differentiation, indication a pivotal role for MNGCs in biomaterials-induced osteogenesis. The results from this study indicate convincingly that the immune response of macrophages towards DBBM has a potent effect on osteoblast differentiation. Furthermore, DBBM promoted macrophage fusion and polarization towards an M2 wound-healing phenotype, further created a microenvironment favoring biomaterial-induced osteogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1236-1246, 2018.

Knockdown of Yes-Associated Protein Induces the Apoptosis While Inhibits the Proliferation of Human Periodontal Ligament Stem Cells through Crosstalk between Erk and Bcl-2 Signaling Pathways.

Objective: The purpose of this study was to provide an insight into the biological effects of knockdown Yes-associated protein (YAP) on the proliferation and apoptosis of human periodontal ligament stem cells (h-PDLSCs). Methods: Immunofluorescence and Western blot were used to evaluate Hippo-YAP signaling expression level. Enhanced green fluorescence protein lentiviral vector was constructed to down-regulate YAP in h-PDLSCs. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect the interfering efficiency of YAP expression. The proliferation activity was detected by EdU staining. Analysis of apoptosis in h-PDLSCs was done through Annexin V-APC staining, while cell cycle analysis was detected by flow cytometry. Cellular senescence was analyzed by ß-galactosidase activity detection. The expression of elements in signaling pathways related with proliferation and apoptosis was detected by Western blot. Results: YAP was located in nucleus and cytoplasm. After the lentivirus transfection, the expression of YAP mRNA and protein was significantly reduced (P<0.001). When YAP was knocked down, the proliferation activity of h-PDLSCs was inhibited; the early & late apoptosis rates increased; the proportion of cells in G1 phases increased (P<0.05), while that in G2 and S phase decreased (P<0.05); cellular senescence was accelerated (P<0.01); ERK and its target proteins P-P90RSK and P-MEK were reduced while Bcl-2 family members increased. Conclusion: Knockdown of YAP inhibits the proliferation activity and induces apoptosis of h-PDLSCs with the involvement of Hippo pathway and has a crosstalk between Erk and Bcl-2 signaling pathways.

Platelet-rich plasma enhanced umbilical cord mesenchymal stem cells-based bone tissue regeneration.

OBJECTIVES: To evaluate the effects of platelet-rich plasma (PRP) on the proliferation and differentiation of umbilical cord mesenchymal stem cells (UC-MSCs) and explore the possibility that PRP combined with UC-MSCs may be useful for bone tissue regeneration in vivo. METHODS: The proliferation potential of UC-MSCs was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The pluripotent differentiation capacity and alkaline phosphatase (ALP) expression were further determined by ALP staining. The expression of osteoblast-associated genes was evaluated by real-time PCR. In addition, rat critical-sized calvarial defects were examined to evaluate bone regeneration in vivo. RESULTS: PRP enhanced UC-MSC proliferation, and 10% PRP caused the strongest ALP and Alizarin red staining. At 7 days, the expression levels of ALP, Collagen 1 (COL-1) and Runt-related transcription factor 2 (RUNX2) in the PRP group were higher than those in the FBS group. Newly regenerated bone was observed in the defect areas, and PRP combined with UC-MSCs can accelerate bone regeneration at an early stage. CONCLUSIONS: Our current data suggest that UC-MSCs may be utilized in alternative stem cell-based approaches for the reconstruction and regeneration of bone defects, and PRP combined with UC-MSCs can enhance bone regeneration in vivo.