DZNep promotes mouse bone defect healing via enhancing both osteogenesis and osteoclastogenesis

Background Enhancer of zeste homolog 2 (EZH2) is a novel oncogene that can specifically trimethylate the histone H3 lysine 27 (H3K27me3) to transcriptionally inhibit the expression of downstream tumor-suppressing genes. As a small molecular inhibitor of EZH2, 3-Deazaneplanocin (DZNep) has been widely studied due to the role of tumor suppression. With the roles of epigenetic regulation of bone cells emerged in past decades, the property and molecular mechanism of DZNep on enhancing osteogenesis had been reported and attracted a great deal of attention recently. This study aims to elucidate the role of DZNep on EZH2-H3K27me3 axis and downstream factors during both osteoclasts and osteoblasts formation and the therapeutic possibility of DZNep on bone defect healing. Methods Bone marrow-derived macrophages (BMMs) cells were cultured, and their responsiveness to DZNep was evaluated by cell counting kit-8, TRAP staining assay, bone resorption assay, podosome actin belt. Bone marrow-derived mesenchymal stem cells (BMSC) were cultured and their responsiveness to DZNep was evaluated by cell counting kit-8, ALP and AR staining assay. The expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), Wnt signaling pathway was determined by qPCR and western blotting. Mouse bone defect models were created, rescued by DZNep injection, and the effectiveness was evaluated by X-ray and micro-CT and histological staining. Results Consistent with the previous study that DZNep enhances osteogenesis via Wnt family member 1(Wnt1), Wnt6, and Wnt10a, our results showed that DZNep also promotes osteoblasts differentiation and mineralization through the EZH2-H3K27me3-Wnt4 axis. Furthermore, we identified that DZNep promoted the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation via facilitating the phosphorylation of IKKα/β, IκB, and subsequently NF-κB nuclear translocation, which credit to the EZH2-H3K27me3-Foxc1 axis. More importantly, the enhanced osteogenesis and osteoclastogenesis result in accelerated mice bone defect healing in vivo. Conclusion DZNep targeting EZH2-H3K27me3 axis facilitated the healing of mice bone defect via simultaneously enhancing osteoclastic bone resorption and promoting osteoblastic bone formation.

The Healing of Bone Defects by Cell-Free and Stem Cell-Seeded 3D Printed PLA Tissue Engineered Scaffolds

One of the main issues in bone tissue engineering is to realize the response of the host to the engineered scaffolds. In this paper, the in-vivo healing of critical-sized bony defects by cell-free and stem cell-seeded 3D printed PLA scaffolds was studied in rat calvaria bone. First, the scaffolds were 3D printed based on a designed computer model and half of them were seeded by with bone marrow-derived mesenchymal stem cells (BMSCs). The SEM images of the surfaces of PLA and PLA+Cell scaffolds were taken for morphological analysis. All the scaffolds were implanted in the defect sites of rat calvaria bones and histological analysis was conducted after 8 and 12 weeks. The results showed that both cell-free and stem cell-seeded scaffolds exhibited superb healing compared with the empty defect controls. The histological observation revealed the formation of both new bone and connective tissues in the healing site after 8 and 12 weeks, postoperatively. The bone cells including osteoblasts and osteocytes with lacuna were also observed. The higher filled area and the higher bone formation and bone maturation were observed after 12 weeks and in particular for PLA+Cell scaffolds. Furthermore, the systemic toxicity evaluation of the scaffolds using ALT and AST tests reject any toxicity for both cell-free and stem cell-seeded scaffolds. It can be concluded that the 3D printed PLA scaffold with BMSCs seeding has well osteogenic potential to be used for bone defect healing.

Osteoclast-Derived Extracellular miR-106a-5p Promotes Osteogenic Differentiation and Facilitates Bone Defect Healing

Small extracellular vesicles (sEVs) are considered to play critical roles in intercellular communications during normal and pathological processes since they are enriched with miRNAs and other signal molecules. In bone remodeling, osteoclasts generate large amounts of sEVs. However, there is very little research about whether and how osteoclast-derived sEVs (OC-sEVs) affect surrounding cells. In our study, microarray analysis identified miR-106a-5p highly enriched in OC-sEV. Further experiments confirmed that OC-sEVs inhibited Fam134a through miR-106a-5p and significantly promoted bone mesenchymal stem cell (BMSC) osteogenic mineralization in vitro. Next, we prepared sEV-modified demineralized bone matrix (DBM) as a repair scaffold, and used a calvarial defect mouse model to evaluate the pro-osteogenic activities of the scaffold. In vivo result indicated DBM modified with miR-106a-5p-sEVs showed an enhanced capacity of bone regeneration. This important finding further emphasizes that sEV-mediated miR-106a-5p transfer play critical roles in osteogenesis and indicate a novel communication mode between osteoclasts and BMSCs.

A Review on the Enhancement of Calcium Phosphate Cement with Biological Materials in Bone Defect Healing

Calcium phosphate cement (CPC) is a promising material used in the treatment of bone defects due to its profitable features of self-setting capability, osteoconductivity, injectability, mouldability, and biocompatibility. However, the major limitations of CPC, such as the brittleness, lack of osteogenic property, and poor washout resistance, remain to be resolved. Thus, significant research effort has been committed to modify and reinforce CPC. The mixture of CPC with various biological materials, defined as the materials produced by living organisms, have been fabricated by researchers and their characteristics have been investigated in vitro and in vivo. This present review aimed to provide a comprehensive overview enabling the readers to compare the physical, mechanical, and biological properties of CPC upon the incorporation of different biological materials. By mixing the bone-related transcription factors, proteins, and/or polysaccharides with CPC, researchers have demonstrated that these combinations not only resolved the lack of mechanical strength and osteogenic effects of CPC but also further improve its own functional properties. However, exceptions were seen in CPC incorporated with certain proteins (such as elastin-like polypeptide and calcitonin gene-related peptide) as well as blood components. In conclusion, the addition of biological materials potentially improves CPC features, which vary depending on the types of materials embedded into it. The significant enhancement of CPC seen in vitro and in vivo requires further verification in human trials for its clinical application.

DZNep Promotes Mouse Bone Defect Healing via Enhancing Both Osteogenesis and Osteoclastogenesis

Background: EZH2 (Enhancer of zeste homolog 2) is a novel oncogene that can specifically trimethylate the histone H3 lysine 27 (H3K27me3) to transcriptionally inhibit the expression of downstream tumor-suppressing genes. As a small molecular inhibitor of EZH2, 3-Deazaneplanocin (DZNep) has been widely studied due to the role of tumor suppression. With the roles of epigenetic regulation of bone cells emerged in past decades, the property and molecular mechanism of DZNep on enhancing osteogenesis had been reported and attracted a great deal of attention recently. this study aims to elucidate the role of DZNep on EZH2-H3K27me3 axis and downstream factors during both osteoclasts and osteoblasts formation and the therapeutic possibility of DZNep on bone defect healing.Methods: Bone marrow drived macrophages (BMMs) cells were cultured and their responsiveness to DZNep was evaluated by Cell Counting Kit-8, TRAP staining assay, Bone Resorption Assay, Podosome Actin Belt. Bone marrow drived mesenchymal stem cells (BMSC) were cultured and their responsiveness to DZNep was evaluated by Cell Counting Kit-8, ALP and AR staining assay. The expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), Wnt signaling pathway was determined by qPCR and western blotting. Mouse bone defect models were created, rescued by DZNep injection and the effectiveness was evaluated by X-ray and Micro-CT and Histological staining.Results: Consistent with the previous study that DZNep enhances osteogenesis via Wnt family member 1(Wnt1), Wnt6, and Wnt10a, our results showed that DZNep also promotes osteoblasts differentiation and mineralization through the EZH2-H3K27me3-Wnt4 axis. Furthermore, we identified that DZNep promoted the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation via facilitating the phosphorylation of IKKα/β, IκB, and subsequently NF-κB nuclear translocation, which credit to the EZH2-H3K27me3-Foxc1 axis. More importantly, the enhanced osteogenesis and osteoclastogenesis result in accelerated mice bone defect healing in vivo.Conclusion: DZNep targeting EZH2-H3K27me3 axis facilitated the healing of mice bone defect via simultaneously enhancing osteoclastic bone resorption and promoting osteoblastic bone formation.

Application of graphene oxide-based hydrogels in bone tissue engineering

: This paper provides a review on the applications of some graphene oxide-based hydrogels in bone tissue engineering. Hydrogels used in bone tissue engineering usually require suitable biocompatibility, porosity, and mechanical strength. By adding GO, under the original hydrogel condition, the various indexes of the composite hydrogel can reach more suitable conditions for inducing bone defect healing. This article also introduces some high-performance graphene oxide-based hydrogels, which are likely to play an essential role in bone tissue engineering in the future.

Bone morphogenetic protein 2-induced cellular chemotaxis drives tissue patterning during critical-sized bone defect healing: an in silico study

Critical-sized bone defects are critical healing conditions that, if left untreated, often lead to non-unions. To reduce the risk, critical-sized bone defects are often treated with recombinant human BMP-2. Although enhanced bone tissue formation is observed when BMP-2 is administered locally to the defect, spatial and temporal distribution of callus tissue often differs from that found during regular bone healing or in defects treated differently. How this altered tissue patterning due to BMP-2 treatment is linked to mechano-biological principles at the cellular scale remains largely unknown. In this study, the mechano-biological regulation of BMP-2-treated critical-sized bone defect healing was investigated using a multiphysics multiscale in silico approach. Finite element and agent-based modeling techniques were combined to simulate healing within a critical-sized bone defect (5 mm) in a rat femur. Computer model predictions were compared to in vivo microCT data outcome of bone tissue patterning at 2, 4, and 6 weeks postoperation. In vivo, BMP-2 treatment led to complete healing through periosteal bone bridging already after 2 weeks postoperation. Computer model simulations showed that the BMP-2 specific tissue patterning can be explained by the migration of mesenchymal stromal cells to regions with a specific concentration of BMP-2 (chemotaxis). This study shows how computational modeling can help us to further understand the mechanisms behind treatment effects on compromised healing conditions as well as to optimize future treatment strategies.

Use of Amniotic Membrane and Its Derived Products for Bone Regeneration: A Systematic Review

Thanks to their biological properties, amniotic membrane (AM), and its derivatives are considered as an attractive reservoir of stem cells and biological scaffolds for bone regenerative medicine. The objective of this systematic review was to assess the benefit of using AM and amniotic membrane-derived products for bone regeneration. An electronic search of the MEDLINE—Pubmed database and the Scopus database was carried out and the selection of articles was performed following PRISMA guidelines. This systematic review included 42 articles taking into consideration the studies in which AM, amniotic-derived epithelial cells (AECs), and amniotic mesenchymal stromal cells (AMSCs) show promising results for bone regeneration in animal models. Moreover, this review also presents some commercialized products derived from AM and discusses their application modalities. Finally, AM therapeutic benefit is highlighted in the reported clinical studies. This study is the first one to systematically review the therapeutic benefits of AM and amniotic membrane-derived products for bone defect healing. The AM is a promising alternative to the commercially available membranes used for guided bone regeneration. Additionally, AECs and AMSCs associated with an appropriate scaffold may also be ideal candidates for tissue engineering strategies applied to bone healing. Here, we summarized these findings and highlighted the relevance of these different products for bone regeneration.