Differential Expression Profiling of microRNAs in hPMSCs Co-culture With a Novel Porous Hydroxyapatite Scaffold

doi:10.21203/rs.3.rs-659704/v1

Download PDF

Research Article

Differential Expression Profiling of microRNAs in hPMSCs Co-culture With a Novel Porous Hydroxyapatite Scaffold

https://doi.org/10.21203/rs.3.rs-659704/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background and objective: Scaffold materials used for bone defect repair are often limited by the osteogenic efficacy. Meanwhile, microRNAs (miRNAs) have been shown to be involved in regulating the expression of osteogenic related genes. In previous studies, we have verified that the enhancement of osteogenesis using a novel porous hydroxyapatite scaffold (HAG). In this study, we analyzed the contribution of HAG to osteogenic differentiation of Human placenta-derived mesenchymal stem cells (hPMSCs) from the perspective of miRNA differential expression.

Methods: The properties of hPMSCs were identified by flow cytometry, including CD44, CD90 and CD45 surface marker. The expression of osteogenic differentiation related genes mRNA and protein were detected by quantitative real-time PCR (qRT-PCR) and western blotting. The mineral depositions were measured by Alizarin red S (ARS) staining. The miRNA profiles were performed by microarray assay, and then further summarized through target, gene ontology and pathway analysis. The expression of differential miRNA were verified by qRT-PCR.

Results: The results showed that HAG promoted the osteogenic differentiation of hPMSCs. Meanwhile, sequencing results showed that 16 miRNAs were significantly up-regulated and 29 miRNAs were down-regulated with HAG. In addition, bioinformatics analyses showed that the differentially expressed miRNAs are involved in a variety of biological processes including signal transduction, cell metabolic, cell junction, cell development and differentiation and connect to osteogenic differentiation through axon guidance, MAPK, and TGF-beta signaling pathway. Furthermore, multiple potential target genes of miRNA are closely related to osteogenic differentiation.

Conclusion: The work first confirmed that differential expression of miRNAs in the process of osteogenic differentiation promoted by HAG scaffold, which also lays the foundation for the further construction of bone scaffolds loaded with miRNAs.

Orthopedic Surgery

Osteogenic Differentiation

microRNA

hydroxyapatite scaffold

microarray assays

Bone is a mineralized mesenchymal tissue that resizes mechanical forces and regulates mineral homeostasis and energy metabolism¹. However, bone defects due to trauma, disease and other causes seriously impair people's quality of life and cause a huge social and economic burden. Although bone has a certain ability of regeneration, serious bone injuries such as large bone defects still need to be solved by bone grafting^2,3. Autografts is the most ideal way to avoid the occurrence of graft immune response, but it is limited by multiple surgeries and donor site injury. Therefore, bone tissue engineering, a scaffold material loaded seed cells and growth factors, has become a more promising method for clinical therapy. Importantly, biological scaffolds material is a key part of it. After the scaffold material is implanted in the affected area, the cells attach to the scaffold surface according to the properties of the material's surface including microstructure, charge, chemical composition, and so on^4,5. As a result, there is a growing demand for bone biomaterials with superior biological properties in recent years⁶.

MicroRNA (miRNA) is endogenous small noncoding ~ 22-nt RNA, which regulates a series of physiological and pathological processes including bone regeneration and metabolism by targeting mRNA sequences of related genes^7,8. For example, miR-335-5p has been shown to be highly expressed in mouse embryo osteogenesis⁹. Similarly, two consecutive studies demonstrated that both miR-2861 and miR-3960 are high expression in original mouse osteoblasts^10,11. Additionally, in different tissue sources, miR-140-5p and miR-140-3p are both confirmed to highly expressed, including human bone-marrow-derived stem cells (hBMSCs)¹². Li et al. showed that miR-29b expression peaks at the mineral deposition stage of MC3T3 cell osteogenic differentiation¹³. Bhushan and his colleagues found that miR-181a expression was upregulated during BMP6-induced osteogenic differentiation of MC3T3 cells¹⁴. Also, miR-210 was increased in ST2 cell line with BMP-4 dependent manner¹⁵. Furthermore, let-7, miR-20, miR-34a and miR-199 were confirmed to be increased during hBMSCs osteoblast differentiation¹⁶. As mentioned above, different miRNAs are involved in the complex process of osteogenic differentiation. However, the mechanism of scaffold material-induced specific osteogenic associated miRNAs has not been seen.

In this study, we first observed that the differential expression of miRNA in the process of cell osteogenic differentiation induced by HAG scaffolds in hPMSCs. Hydroxyapatite is the most commonly used scaffold material for bone regeneration, due to its mineral composition with natural bone and HAG is a porous hydroxyapatite scaffold with microchannel structure developed by our team, the osteogenic response of which had be verified both in vitro and vivo^17,18. Here, we compared the expression profiles of miRNAs in hPMSCs with or without HAG by microarray assays. Moreover, we further explore the biological functions that these differential miRNAs might be associated with osteogenesis through bioinformatics analysis and revealed that they are closely related to osteogenic differentiation related signaling pathways, including axon guidance, MAPK, and TGF-beta signaling pathway. The work may lay the foundation for the further construction of bone scaffolds loaded with miRNAs.

Cell culture and transfection

hPMSCs were cultured as before¹⁸. In details, cells were cultured in complete medium (DMEM with 10% FBS and 1% PS). When the cell density fusion was 80–90%, the digestive passage culture was conducted with 0.25% trypsin. The surface markers of hPMSCs were analyzed by flow cytometry, including CD44, CD90 and CD45. For cell transfection, 100pM miRNA mimics and NC were transfected to cells followed by the protocol of Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific, USA), when the cell density approached 80 percentage. The osteogenic differentiation of hPMSCs were cultured with conditional medium (DMEM with 5%FBS, 20 mmol/L β-glycerophosphate, 50 g/mL vitamin C and 10 mol/L dexamethasone). Alizarin Red staining was performed to detect calcium deposits after 21 days of induction culture. The GFP-mimics were synthesised from Genepharma. The DAPI (Sigma-Aldrich) were stained at 48 hours after transfection, then the pictures were captured by ZEISS AXIO Observer.A1.

Preparation of scaffolds

The HAG scaffold was produced and cleaned by referring to previous description [18]. The surface micromorphology of HAG was confirmed by scanning electron microscopy (SEM). The scaffolds were incubated overnight in DMEM prior to the cell experiment at 37°C.

Osteogenic differentiation of hPMSCs with HAG and quantitative real-time PCR (qRT-PCR)

20µL ell concentrate (4 × 10⁶ /mL) was seeded in a HAG. Then, the cells co-culture with the scaffold in incubator. After osteogenic induction for different time point, extracted the total RNAs (TRIzol™ Reagent, Invitrogen), and reversed to cDNAs (Thermo, USA). Quantitative fluorescence detection was performed using 2X ChamQ Universal SYBR Master Mix (Vazyme, China). ABI 7500 was applied for qRT-PCR. (5’-3’ALP: F-CTTGAAGTGTTGCATGGGC, R-CAAGTCTCAGGGTGGAAGG; OCN: F-CAGCGAGGTAGTGAAGAGAC, R-TGAAAGCCGATGTGGTCAG; BMP2: F-CTATCAGGACATGGTTGTGGAG, R-GGGAAATATTAAAGTGTCAACTGGG) Primers specific for human miRNAs and internal control U6 were designed and synthesized from Tsingke, China.

Western blotting

The proteins were isolated by 10% SDS-PAGE gel, which transferred with PVDF membranes (Millipore). After incubation with GAPDH, ALP, OCN (Huabio, China) primary antibodies overnight, the membranes were washed for 5 times with TBST (2‰ Tween). Then, the HRP- conjugated secondary antibody was incubated for 1h. Finally, an Super ECL kit (biosharp, China) was used to detect the protein bands. The Tanon-5200 was applied for exposure.

Microarray Assays

Total RNA were extracted using Trizol™ reagent and used to build a miRNA library. The library quality was determined by Agilent 2100 Bioanalyzer. It was eventually sequenced on a Illumina NextSeq 500 sequencer.

Bioinformatics Analysis

Edge R was used to analyze the differential expression of miRNA between groups. Based on the database of TargetScan7.1 and mirdbV6, target genes were screened for the Top 10 miRNA up and down regulated in significant difference. Then the function enrichment analysis of target genes was performed by Gene Ontology, including Molecular function, Cellular component and Biological process. The miRNA-mRNA networks were constructed using Cytoscape.

Statistical Analysis

Three independent repeated experiments were used to perform and analysis related results (mean ± SD). The T test was used to analyze differences between groups. P values < 0.05 were considered significant.

The osteogenic differentiation of hPMSC was promoted by HAG

Compared with autograft or allograft, biomaterials have become an indispensable bone material source in the field of bone regeneration due to their non-immunogenicity and accessibility. Among various bone materials, hydroxyapatite (HA) is the most commonly used one because of the following advantages, the same mineral composition as natural bone, good biocompatibility, bone conductivity and mechanical stability. However, the interaction between scaffolds and cells is influenced by various characteristics and morphology of scaffold surface, including chemical composition, electric charge, microstructure, and so on^4,5. In previous work, on the basis of porous hydroxyapatite scaffold, we prepared a new scaffold material HAG through integrates microchannel structure. Moreover, the HAG promoted the osteogenic response of hPMSCs than HA¹⁸. Consistently, in this study, we repeated the osteogenic effect of HAG in hPMSCs (Fig. 1). The properties of hPMSCs were detected by flow cytometry. The positive surface marker CD44 and CD90 were highly expressed (97.08%), while CD45 (0.10%) was hardly expressed in the cells (Fig. 1A). The porous structure (as the arrow shown, left) and a 25–30 µm groove structure (as the arrow shown, right) on HAG scaffolds surface as shown in Fig. 1B. After osteogenesis induced for 7, 14 days, the expression of ALP, BMP2, Ocn mRNA were quantified by qPCR, and the protein levels of ALP, OCN were detected at 7d (Fig. 1C,D) Alizarin Red staning was used to detect the mineralized nodules 3 weeks after induction (Fig. 1E). The results reproduced the positive effect of HAG scaffolds osteogenic differentiation in hPMSC.

The miRNA expression in HAG-hPMSCs and control hPMSCs were differential

In recent years, several studies revealed that miRNAs participated the process of osteogenic differentiation through targeting mRNA sequences of different genes related to osteogenesis^19–22. However, roles of miRNAs in bone formation by HAG scaffolds mediated remain unclear. Thus, in order to uncover that how HAG scaffolds promoted osteogenic differentiation of hPMSCs, microarray assay was used to analyse the miRNA expression profiles between HAG-hPMSCs and hPMSCs. We observed 45 mature miRNAs significantly differentially expressed in HAG-hPMSCs group (FC ≥ 2, Fig. 2A). Red represented highly expressed genes, while green represents the down-regulated genes, and the darker the color, the more distinct the expression difference. Compared with differentiated hPMSCs, the expressions of 16 miRNAs in HAG-hPMSCs were up-regulated and 29 were down-regulated, as shown in the volcano plot (FC ≥ 2, Fig. 2B).

Pathway and functional analysis of differential miRNAs

The differential expression of miRNAs in HAG-hPMSCs group may indicate that miRNA plays an important role in the process of osteogenic differentiation promoted by HAG. Therefore, the gene ontology enrichment analyses, including BP (biological process), CC (cellular component), and MF (molecular function) by cluster Profiler in R software, were performed to explore the biological functions may be regulated by differential miRNAs. The statistically significant results (p values < 0.05) showed that these miRNAs participated in the regulation of cell metabolic (p value = 2.58E-12), cell junction (p value = 6.45E-08), cell development (p value = 1.82E-11), cell differentiation (p value = 4.04E-07), and signal transduction (p value = 8.12E-12) (Fig. 3A). Importantly, the process of osteogenic differentiation is closely related to these functions.

Furthermore, KEGG pathway analysis was used to understand the signaling pathways in which these differentially expressed miRNAs were involved in regulation. The results indicated that genes in multiple signaling pathways, including axon guidance (p value = 5.02E-05), MAPK signaling (p value = 6.65E-07), and the TGF-β signaling pathway (p value = 5.7E-04), highly associated with osteogenic differentiation are potential target genes for these differentially expressed miRNAs (Fig. 3B). It suggested that HAG may achieve its osteogenic effect by promoting differential expression of these miRNAs.

qRT-PCR validation of miRNA expression and miRNA-mRNA network analysis

According to the results of microarray analyses, the 6 miRNAs (miR-210-3p;miR-146a-5p; miR-483-5p; miR-3615༛miR-125b-2-3p༛miR-145-5p), with the most significant expression differences were verified by qPCR. Consistently, the expression of miR-210-3p, miR-146a-5p, miR-483-5p were obviously increased, while miR-3615, miR-125b-2-3p and miR-145-5p were significantly decreased (FC > 2, p < 0.05) (Figu.4A). As known, miRAN achieve its regulatory effect by pairing with the complementary sequence of the target gene and degrading it or preventing its translation. More importantly, a miRNA can target multiple mRNA and the same mRNA can be regulated by multiple miRNAs²³. Thus, TargetScan7.1 and mirdbV6 were used to predict the target genes of the above 6 miRNAs. Moreover, the miRNA-mRNA networks were constructed by Cytoscape (Fig. 4B). As shown in Fig. 4B, the miRNAs can target hundreds of mRNA, while the mRNA of BRCC3 can be recognized by different miRNA. More importantly, a great quantity of target mRNA were to be involved in osteogenic differentiation related pathways. For instance, the Brain-Derived Neurotrophic Factor (BDNF), a well-known growth factor of the neurotrophin family, is involved in regulating the growth, survival of neurons and angiogenesis²⁴. Interestedly, Yamashiro et al. found that the mRNA and protein of BDNF were expressed in osteoblasts²⁵. More importantly, further studies showed that the osteogenic differentiation ability of hPMSCs were enhanced with the expression of BDNF^26,27.

Overexpression of miR146a-5p promoted the osteogenic differentiation of hPMSCs

To verify the effect of differentially expressed miRNA on osteoblastic differentiation, we transfected the miR146a-5p mimics into the hPMSCs. As shown in Fig. 5A and B, the mimics were transfected into cells successfully. Furthermore, we observed that the expression of osteogenic genes were increased when miR146a-5p were overexpressed (Fig. 5C). Meanwhile, following osteogenic differentiation, the hPMSCs transfected with miR-146a-5p mimics showed a higher intensity of ALP staining and formed a larger number of calcified nodules (Fig. 6D, E). Therefore, the differentially expressed miRNAs may play an important role in the process of HAG promoting osteogenic differentiation.

How to solve different conditions of bone defects is a major clinical challenge²⁸. Autograft is the ideal way to repair bone defects²⁹. However, due to the limited source, the high infection rate in donor sites, and increases the pain burden of patients made its clinical application rate low. With the rapid development of the field of biomaterials and the advantages of good biocompatibility and no immunogenicity, bone biomaterials are the most widely material for bone repair. Among them, hydroxyapatite, which has the same chemical composition as natural bone, long-term biocompatibility and interacts well with soft tissue or bone in vivo, is widely used to prepare various bone materials^30–32. Furthermore, the micros structure of the material surface is essential for cell adhesion, protein release, and interaction between the material and the cell³³. Therefore, hydroxyapatite scaffold materials with porous 750–900 µm and microchannel structure of 25–30 µm (HAG) were prepared in previous work. Importantly, the osteogenic capacity of HAG in vivo and vitro has been demonstrated¹⁸. However, the mechanism which the HAG promotes osteogenic differentiation remains unclear.

More and more studies have revealed that miRNAs expressed in the various stages of bone formation, including the differentiation of bone marrow stromal stem cells into osteogenic precursor and the further development of precursor cells into mature osteoblasts. During these processes, they may play either a positive or negative role, including miR-96, miR-124, and miR-199a and so on^34–38. However, the role of specific biological scaffold material-related miRNAs in bone formation remains unclear. In this work, we tried to explore the osteogenic mechanisms of HAG scaffolds, in particular the miRNA involved. Interestingly, we did detect differential expression of miRNAs in the HAG-hPMSCs group. Moreover, the integration of pathway analysis, functional analysis and miRNA-mRNA cross network analysis showed that the miRNAs differentially expressed in HAG scaffolds group were highly correlated with axon guidance, MAPK, and TGF-beta signaling pathway. These signaling pathways are closely related to osteogenic differentiation.

Consequently, our results indicated that the HAG scaffolds may promote the bone formation through regulating the differentially expressed of miRNAs. In details, we successfully uncovered the statistically significant miRNAs in HAG-hPMSCs and constructed the miRNA-mRNA network through different bioinformatics analysis. However, we did not elucidate the specific mechanism of action involved in osteogenic differentiation promoted by HAG. That's what we're working on right now. We believe that figuring out the key target miRNA for HAG promoting osteogenic differentiation will lay the foundation for the preparation of bone scaffolds loaded with miRNA. Therefore, in this study, we first analyzed the differential expression profile of miRNAs in the HAG-mediated osteogenic differentiation, which is an indispensable step to achieve the final goal.

microRNAs, miRNAs; a novel porous hydroxyapatite scaffold, HAG; Human placenta-derived mesenchymal stem cells, hPMSCs; quantitative real-time PCR, qRT-PCR; Alizarin red S, ARS staining; human bone-marrow-derived stem cells, hBMSCs; hydroxyapatite, HA; BP, biological process; CC, cellular component; MF, molecular function; KEGG, Kyoto Encyclopedia of Genes and Genomes pathway analysis; GO, gene ontology; FC, Fold change; Brain-Derived Neurotrophic Factor, BDNF.

Funding

National Natural Science Foundation of China (82071168) supported the work.

Ethics approval and consent to participate

All experimental protocols were approved by Medical Ethics Committee of Sichuan Provincial People's Hospital, Affiliated Hospital of UESTC.

Consent for publication

Not applicable.

Competing interests

There is no conflict of interest among the authors.

Lian, J.B.; Stein, G.S.; van Wijnen, A.J.; Stein, J.L.; Hassan, M.Q.; Gaur, T.; Zhang, Y. MicroRNA control of bone formation and homeostasis. Nat. Rev. Endocrinol. 2012, 8, 212–227.
Montazerolghaem M, Rasmusson A, Melhus H et al. Simvastatindoped pre-mixed calcium phosphate cement inhibits osteoclast differentiation and resorption. J Mater Sci Mater Med 2016; 27: 83.
Yang F, Wang J, Hou J et al. Bone regeneration using cell-mediated responsive degradable PEG-based scaffolds incorporating with rhBMP-2. Biomaterials 2013; 34: 1514–1528.
Bodhak, S. Bose, A. Bandyopadhyay, Role of surface charge and wettability on early stage mineralization and bone cell–materials interactions of polarized. hydroxyapatite, Acta Biomater. 5 (2009) 2178–2188.
Joy, D.M. Cohen, A. Luk, E. Animdanso, C. Chen, J. Kohn, Control of surface chemistry, substrate stiffness, and cell function in a novel terpolymer methacrylate library, Langmuir ACS J. Surf. Colloids 27 (2011) 1891–1899.
Shadjou N, Hasanzadeh M. Bone tissue engineering using silica-based mesoporous nanobiomaterials: recent progress. Mater Sci Eng C Mater Biol Appl 2015; 55: 401–409.
Liu, Q.; Paroo, Z. Biochemical principles of small RNA pathways. Annu. Rev. Biochem. 2010, 79, 295–319.
Ha, M.; Kim, V.N. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 2014, 15, 509–524.
Zhang, J.; Tu, Q.; Bonewald, L.F.; He, X.; Stein, G.; Lian, J.; Chen, J. Effects of miR-335-5p In modulating osteogenic differentiation by specifically downregulating Wnt antagonist DKK1. J. Bone Miner. Res. 2011, 26, 1953–1963.
Li, H.; Xie, H.; Liu, W.; Hu, R.; Huang, B.; Tan, Y.F.; Xu, K.; Sheng, Z.F.; Zhou, H.D.; Wu, X.P.; et al. A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans. J. Clin. Investig. 2009, 119, 3666–3677.
Hu, R.; Liu, W.; Li, H.; Yang, L.; Chen, C.; Xia, Z.Y.; Guo, L.J.; Xie, H.; Zhou, H.D.; Wu, X.P.;et al. A Runx2/miR-3960/miR-2861 regulatory feedback loop during mouse osteoblast differentiation. J. Biol. Chem. 2011, 286, 12328–12339
Hwang, S.; Park, S.K.; Lee, H.Y.; Kim, S.W.; Lee, J.S.; Choi, E.K.; You, D.; Kim, C.S.; Suh, N. miR-140-5p suppresses BMP2-mediated osteogenesis in undifferentiated human mesenchymal stem cells. FEBS Lett. 2014, 588, 2957–2963.
Li, Z.; Hassan, M.Q.; Jafferji, M.; Aqeilan, R.I.; Garzon, R.; Croce, C.M.; van Wijnen, A.J.; Stein, J.L.; Stein, G.S.; Lian, J.B. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J. Biol. Chem. 2009, 284, 15676–15684
Bhushan, R.; Grünhagen, J.; Becker, J.; Robinson, P.N.; Ott, C.E.; Knaus, P. miR-181a. promotes osteoblastic differentiation through repression of TGF-β signaling molecules. Int. J. Biochem. Cell Biol. 2013, 45, 696–705.
Mizuno, Y.; Tokuzawa, Y.; Ninomiya, Y.; Yagi, K.; Yatsuka-Kanesaki, Y.; Suda, T.; Fukuda, T.; Katagiri, T.; Kondoh, Y.; Amemiya, T.; et al. miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b. FEBS Lett. 2009, 583, 2263–2268.
Chen, L.; Holmstrøm, K.; Qiu, W.; Ditzel, N.; Shi, K.; Hokland, L.; Kassem, M. MicroRNA-34a inhibits osteoblast differentiation and in vivo bone formation of human stromal stem cells. Stem Cells 2014, 32, 902–912.
F.J. O'Brien, Biomaterials & scaffolds for tissue engineering, Mater. Today 14 (2011) 88–95.
Xiaohua R , Qiang T , Kun T , et al. Enhancement of osteogenesis using a novel porous. hydroxyapatite scaffold in vivo and vitro[J]. Ceramics International, 2018, 44: S0272884218322922-.
Li Cj, Cheng P, Liang MK, et al. MicroRNA-188 regulates age-related switch between. osteoblast and adipocyte differentiation [J]. J Clin Invest. 2015;125(4):1509-1522
Huang J, Zhao L, Xing L, Chen D. MicroRNA-204 regulates Runx2 protein expression. and mesenchymal progenitor cell differentiation. Stem Cells. 2010;28(2):357-364
Liu S, Liu D, Chen C, et al. MSC Transplantation Improves Osteopenia via Epigenetic. Regulation of Nptch Signaling in Lupus. Cell Metab. 2015;22(4):606-618
Tang Y, Zhang L, Tu T, et al. MicroRNA-99a is a novel regulator of KDM6B-mediated. osteogenic differentiation of BMSCs. J Cell Mol Med. 2018;22(4):2162-2176
S. Zhang, W. C. Chu, R. C. Lai, S. K. Lim, J. H. P. Hui, and W. S. Toh, “Exosomes. derived from human embryonic mesenchymal stem cells. promote osteochondral regeneration,” Osteoarthritis and Cartilage, vol. 24, no. 12, pp. 2135–2140, 2016.
Kermani, P.; Hempstead, B. Brain-derived neurotrophic factor: A newly described. mediator of angiogenesis. Trends Cardiovasc. Med. 2007, 17, 140–143.
Yamashiro, T.; Fukunaga, T.; Yamashita, K.; Kobashi, N.; Takano-Yamamoto, T. Gene. and protein expression of brain-derived neurotrophic factor and TrkB in bone and cartilage. Bone 2001, 28, 404–409.
Kauschke, V.; Gebert, A.; Calin, M.; Eckert, J.; Scheich, S.; Heiss, C.; Lips, K.S. Effects. of new beta-type Ti-40Nb implant materials, brain-derived neurotrophic factor, acetylcholine and nicotine on human mesenchymal stem cells of osteoporotic and non osteoporotic donors. PLoS ONE 2018, 13, e0193468.
Kauschke, V.; Schneider, M.; Jauch, A.; Schumacher, M.; Kampschulte, M.; Rohnke, M.; Henss, A.; Bamberg, C.; Trinkaus, K.; Gelinsky, M.; et al. Effects of a Pasty Bone Cement Containing Brain-Derived Neurotrophic Factor-Functionalized Mesoporous Bioactive Glass Particles on Metaphyseal Healing in a New Murine Osteoporotic Fracture Model. Int. J. Mol. Sci. 2018, 19, 3531.
S. Mosbahi, H. Oudadesse, E. Wers, M. Trigui, B. Lefeuvre, C. Roiland, H. Elfeki, A. Elfeki, T. Rebai, H. Keskes, Study of bioactive glass ceramic for use as bone biomaterial in vivo: investigation by nuclear magnetic resonance and histology, Ceram. Int. 42 (2016) 4827–4836.
M. Attal, J.L. Harousseau, A.M. Stoppa, J.J. Sotto, J.G. Fuzibet, J.F. Rossi, P. Casassus, H. Maisonneuve, T. Facon, N. Ifrah, A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome, N. Engl. J. Med. 335 (1996) 91–97.
H. Ghomi, M.H. Fathi, H. Edris, Preparation of nanostructure hydroxyapatite scaffold for tissue engineering applications, J. Sol-Gel Sci. Technol. 58 (2011) 642–650.
Q. Cao, X. Dai, Massive allograft for defects after bone malignant tumor resection, China J. Mod. Med. (2002).
F.Y. Lee, E.J. Hazan, M.C. Gebhardt, H.J. Mankin, Experimental model for allograft incorporation and allograft fracture repair, J. Orthop. Res. Off. Publ. Orthop. Res. Soc. 18 (2000) 303–306.
D. Nadeem, T. Sjostrom, A. Wilkinson, C.A. Smith, R.O. Oreffo, M.J. Dalby, B. Su, Embossing of micropatterned ceramics and their cellular response, J. Biomed. Mater. Res. Part A 101 (2013) 3247–3255.
Lian, J.B.; Stein, G.S.; van Wijnen, A.J.; Stein, J.L.; Hassan, M.Q.; Gaur, T.; Zhang, Y. MicroRNA control of bone formation and homeostasis. Nat. Rev. Endocrinol. 2012, 8, 212–227.
Kim, K.M.; Lim, S.K. Role of miRNAs in bone and their potential as therapeutic targets. Curr. Opin. Pharmacol. 2014, 16, 133–141.
Gámez, B.; Rodriguez-Carballo, E.; Ventura, F. MicroRNAs and post-transcriptional. regulation of skeletal development. J. Mol. Endocrinol. 2014, 52, R179–R197.
Taipaleenmäki, H.; Bjerre Hokland, L.; Chen, L.; Kauppinen, S.; Kassem, M. Mechanisms in endocrinology: Micro-RNAs: Targets for enhancing osteoblast differentiation and bone formation. Eur. J. Endocrinol. 2012, 166, 359–371.
Laine, S.K.; Alm, J.J.; Virtanen, S.P.; Aro, H.T.; Laitala-Leinonen, T.K. MicroRNAs miR-96, miR-124, and miR-199a regulate gene expression in human bone marrow-derived mesenchymal stem cells. J. Cell. Biochem. 2012, 113, 2687–2695.

Download PDF

Version 1

posted

You are reading this latest preprint version

Differential Expression Profiling of microRNAs in hPMSCs Co-culture With a Novel Porous Hydroxyapatite Scaffold

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Cell culture and transfection

Preparation of scaffolds

Osteogenic differentiation of hPMSCs with HAG and quantitative real-time PCR (qRT-PCR)

Western blotting

Microarray Assays

Bioinformatics Analysis

Statistical Analysis

Results

The osteogenic differentiation of hPMSC was promoted by HAG

The miRNA expression in HAG-hPMSCs and control hPMSCs were differential

Pathway and functional analysis of differential miRNAs

qRT-PCR validation of miRNA expression and miRNA-mRNA network analysis

Overexpression of miR146a-5p promoted the osteogenic differentiation of hPMSCs

Discussion And Conclusion

Abbreviations

Declarations

References

Status:

Version 1