Normoglycemia partially recovers the disrupted osteoblast differentiation of mesenchymal stem cells induced by type 1 but not type 2 diabetes mellitus.

Type 1 (T1DM) and type 2 (T2DM) diabetes mellitus are characterized by changes in glucose metabolism and cause bone damage via a variety of mechanisms, including effects on osteoblasts. We aimed to evaluate the osteoblast differentiation of mesenchymal stem cells (MSCs) from rats with T1DM or T2DM and the effects of removing the hyperglycemic stimulus on the osteogenic potential of these cells. MSCs from healthy rats were cultured in normoglycemic medium, whereas MSCs from rats with T1DM or T2DM were cultured in hyperglycemic or normoglycemic medium. T1DM and T2DM reduced osteoblast differentiation of MSCs grown in hyperglycemic media, with T1DM having a more pronounced effect, as evidenced by alkaline phosphatase activity, RUNX2 protein expression, and extracellular matrix mineralization, and modulated the gene expression of several components of the bone morphogenetic protein signaling pathway. The restoration of the normoglycemic environment partially recovers the osteogenic potential of MSCs from rats with T1DM but not with T2DM. Our findings highlight the need for specific therapies to treat T1DM- or T2DM-induced bone loss, as both disrupt osteoblast differentiation at distinct levels and likely through different mechanisms.

Mesenchymal stem cells overexpressing BMP-9 by CRISPR-Cas9 present high in vitro osteogenic potential and enhance in vivo bone formation.

Cell therapy is a valuable strategy for the replacement of bone grafts and repair bone defects, and mesenchymal stem cells (MSCs) are the most frequently used cells. This study was designed to genetically edit MSCs to overexpress bone morphogenetic protein 9 (BMP-9) using Clustered Regularly Interspaced Short Palindromic Repeats/associated nuclease Cas9 (CRISPR-Cas9) technique to generate iMSCs-VPRBMP-9+, followed by in vitro evaluation of osteogenic potential and in vivo enhancement of bone formation in rat calvaria defects. Overexpression of BMP-9 was confirmed by its gene expression and protein expression, as well as its targets Hey-1, Bmpr1a, and Bmpr1b, Dlx-5, and Runx2 and  protein expression of SMAD1/5/8 and pSMAD1/5/8. iMSCs-VPRBMP-9+ displayed significant changes in the expression of a panel of genes involved in TGF-ß/BMP signaling pathway. As expected, overexpression of BMP-9 increased the osteogenic potential of MSCs indicated by increased gene expression of osteoblastic markers Runx2, Sp7, Alp, and Oc, higher ALP activity, and matrix mineralization. Rat calvarial bone defects treated with injection of iMSCs-VPRBMP-9+ exhibited increased bone formation and bone mineral density when compared with iMSCs-VPR- and phosphate buffered saline (PBS)-injected defects. This is the first study to confirm that CRISPR-edited MSCs overexpressing BMP-9 effectively enhance bone formation, providing novel options for exploring the capability of genetically edited cells to repair bone defects.

Selection of reference genes for quantitative real-time polymerase chain reaction studies in rat osteoblasts.

Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR-106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty-one candidates for reference genes from a Taqman® array were assessed by qRT-PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh, were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR-106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2, Alp, and Oc. The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT-PCR studies in osteoblasts to generate precise and reliable data.

Potential of Osteoblastic Cells Derived from Bone Marrow and Adipose Tissue Associated with a Polymer/Ceramic Composite to Repair Bone Tissue.

One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.

Participation of extracellular signal-regulated kinases 1/2 in osteoblast and adipocyte differentiation of mesenchymal stem cells grown on titanium surfaces.

Osteoblasts and adipocytes coexist in the implantation site and affect the process of titanium (Ti) osseointegration. As extracellular signal-regulated kinases 1/2 (ERK1/2) are involved in osteogenesis and adipogenesis, the aim of our study was to investigate if the effects of Ti surface topography on osteoblast and adipocyte differentiation are modulated by ERK1/2. The experiments were conducted based on the effect of the ERK1/2 inhibitor, PD98059, on mesenchymal stem cells (MSCs) grown under osteogenic and adipogenic conditions on Ti with nanotopography (Ti-Nano) or on machined Ti (Ti-Machined). The results showed that, in general, ERK1/2 inhibition favored osteoblast and adipocyte differentiation of MSCs grown on Ti-Machined. In MSCs grown on Ti-Nano, ERK1/2 inhibition upregulated the expression of alkaline phosphatase and osteocalcin and reduced extracellular matrix mineralization. In terms of adipocyte differentiation, ERK1/2 inhibition elicited similar MSC responses to Ti-Nano and Ti-Machined, upregulating gene expression of adipocyte markers without affecting lipid accumulation. Our results indicate that, under osteogenic and adipogenic conditions, the responses of MSCs to Ti surface topography in terms of osteogenesis and adipogenesis are dependent on ERK1/2. Thus, a precise modulation of ERK1/2 expression and activity induced by surface topography could be a good strategy to drive the process of implant osseointegration.

Mesenchymal Stromal Cells Derived from Bone Marrow and Adipose Tissue: Isolation, Culture, Characterization and Differentiation.

Since their discovery, mesenchymal stromal cells (MSCs) have received a lot of attention, mainly due to their self-renewal potential and multilineage differentiation capacity. For these reasons, MSCs are a useful tool in cell biology and regenerative medicine. In this article, we describe protocols to isolate MSCs from bone marrow (BM-MSCs) and adipose tissues (AT-MSCs), and methods to culture, characterize, and differentiate MSCs into osteoblasts, adipocytes, and chondrocytes. After the harvesting of cells from bone marrow by flushing the femoral diaphysis and enzymatic digestion of abdominal and inguinal adipose tissues, MSCs are selected by their adherence to the plastic tissue culture dish. Within 7 days, MSCs reach 70% confluence and are ready to be used in subsequent experiments. The protocols described here are easy to perform, cost-efficient, require minimal time, and yield a cell population rich in MSCs.

The Wnt/ß-catenin signaling pathway is regulated by titanium with nanotopography to induce osteoblast differentiation.

Wnt/ß-catenin signal transduction is involved in the homeostatic control of bone mass. It is well established that a titanium surface with nanotopography (Ti-Nano) favors osteoblast differentiation by modulating different signaling pathways. However, few studies have investigated the participation of the Wnt/ß-catenin pathway in the osteogenic effect of nanoscale topographies. In this study, we aimed to determine whether the Wnt/ß-catenin signaling pathway is involved in the elevated osteogenic potential of Ti-Nano. MC3T3-E1 cells were cultured on Ti-Nano and machined Ti (Ti-Control) for evaluation of the expression of Wnt/ß-catenin signaling pathway-related genes. Based on the results to real-time PCR, the Wnt receptor Fzd4 was selected and silenced by CRISPRi. The resulting cells were cultured on both Ti surfaces, and several events involved in osteoblast differentiation were evaluated. The results revealed that Fzd4 gene silencing, corresponding to negative modulation of Wnt/ß-catenin, inhibits expression of the osteoblast phenotype. It is worthy of note that this inhibitory effect on osteoblast differentiation was more pronounced in cells grown on Ti-Nano compared with those grown on Ti-Control. By disrupting Fzd4 gene expression, we have shown that the elevated osteogenic potential of Ti-Nano is due to activation of the Wnt/ß-catenin signaling pathway, which reveals a new mechanism to explain osteoblast differentiation induced by nanotopography. Such an understanding of the intracellular machinery involved in surface guiding of osteoblast fate may contribute to the development of smart biomaterials to modulate the process of implant osseointegration.

Participation of integrin ß3 in osteoblast differentiation induced by titanium with nano or microtopography.

The major role of integrins is to mediate cell adhesion but some of them are involved in the osteoblasts-titanium (Ti) interactions. In this study, we investigated the participation of integrins in osteoblast differentiation induced by Ti with nanotopography (Ti-Nano) and with microtopography (Ti-Micro). By using a PCR array, we observed that, compared with Ti-Micro, Ti-Nano upregulated the expression of five integrins in mesenchymal stem cells, including integrin ß3, which increases osteoblast differentiation. Silencing integrin ß3, using CRISPR-Cas9, in MC3T3-E1 cells significantly reduced the osteoblast differentiation induced by Ti-Nano in contrast to the effect on T-Micro. Concomitantly, integrin ß3 silencing downregulated the expression of integrin αv, the parent chain that combines with other integrins and several components of the Wnt/ß-catenin and BMP/Smad signaling pathways, all involved in osteoblast differentiation, only in cells cultured on Ti-Nano. Taken together, our results showed the key role of integrin ß3 in the osteogenic potential of Ti-Nano but not of Ti-Micro. Additionally, we propose a novel mechanism to explain the higher osteoblast differentiation induced by Ti-Nano that involves an intricate regulatory network triggered by integrin ß3 upregulation, which activates the Wnt and BMP signal transductions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1303-1313, 2019.

Effect of cell therapy with osteoblasts differentiated from bone marrow or adipose tissue stromal cells on bone repair.

Aim: The aim of this study was to investigate the effect of local injection of osteoblasts differentiated from bone marrow (BM-OB) or adipose tissue (AT-OB) mesenchymal stromal cells on bone tissue formation. Materials & methods: Defects were created in rat calvaria and injected with BM-OB or AT-OB and phosphate-buffered saline without cells were injected as control. Bone formation was evaluated 4 weeks postinjection. Results: Injection of BM-OB or AT-OB resulted in higher bone formation than that obtained with control. The bone tissue induced by cell injections exhibited similar mechanical properties as those of pristine calvarial bone, and its molecular cues suggested the occurrence of a remodeling process. Conclusion: Results of this study demonstrated that cell therapy with osteoblasts induced significant bone formation that exhibited the same quality as that of pre-existent bone.

Cell Therapy: Effect of Locally Injected Mesenchymal Stromal Cells Derived from Bone Marrow or Adipose Tissue on Bone Regeneration of Rat Calvarial Defects.

Treatment of large bone defects is a challenging clinical situation that may be benefited from cell therapies based on regenerative medicine. This study was conducted to evaluate the effect of local injection of bone marrow-derived mesenchymal stromal cells (BM-MSCs) or adipose tissue-derived MSCs (AT-MSCs) on the regeneration of rat calvarial defects. BM-MSCs and AT-MSCs were characterized based on their expression of specific surface markers; cell viability was evaluated after injection with a 21-G needle. Defects measuring 5 mm that were created in rat calvaria were injected with BM-MSCs, AT-MSCs, or vehicle-phosphate-buffered saline (Control) 2 weeks post-defect creation. Cells were tracked by bioluminescence, and 4 weeks post-injection, the newly formed bone was evaluated by µCT, histology, nanoindentation, and gene expression of bone markers. BM-MSCs and AT-MSCs exhibited the characteristics of MSCs and maintained their viability after passing through the 21-G needle. Injection of both BM-MSCs and AT-MSCs resulted in increased bone formation compared to that in Control and with similar mechanical properties as those of native bone. The expression of genes associated with bone formation was higher in the newly formed bone induced by BM-MSCs, whereas the expression of genes involved in bone resorption was higher in the AT-MSC group. Cell therapy based on local injection of BM-MSCs or AT-MSCs is effective in delivering cells that induced a significant improvement in bone healing. Despite differences observed in molecular cues between BM-MSCs and AT-MSCs, both cells had the ability to induce bone tissue formation at comparable amounts and properties. These results may drive new cell therapy approaches toward complete bone regeneration.

Participation of MicroRNA-34a and RANKL on bone repair induced by poly(vinylidene-trifluoroethylene)/barium titanate membrane.

The poly(vinylidene-trifluoroethylene)/barium titanate (PVDF) membrane enhances in vitro osteoblast differentiation and in vivo bone repair. Here, we hypothesized that this higher bone repair could be also due to bone resorption inhibition mediated by a microRNA (miR)/RANKL circuit. To test our hypothesis, the large-scale miR expression of bone tissue grown on PVDF and polytetrafluoroethylene (PTFE) membranes was evaluated to identify potential RANKL-targeted miRs modulated by PVDF. The animal model used was rat calvarial defects implanted with either PVDF or PTFE. At 4 and 8 weeks, the bone tissue grown on membranes was submitted to a large-scale analysis of miRs by microarray. The expression of miR-34a and some of its targets, including RANKL, were evaluated by real-time polimerase chain reaction and osteoclast activity was detected by tartrate-resistant acid phosphatase (TRAP) staining. Among more than 250 miRs, twelve, including miR-34a, were simultaneously higher expressed (≥2 fold) at 4 and 8 weeks on PVDF. The higher expression of miR-34a was concomitant with a reduced expression of all its evaluated targets, including RANKL. Additionally, more TRAP-positive cells were observed in bone tissue grown on PTFE compared with PVDF in both time points. In conclusion, our results suggest that the higher bone formation induced by PVDF could be, at least in part, triggered by a miR-34a increase and RANKL decrease, which may inhibit osteoclast differentiation and activity, and bone resorption.

Effect of Surface Nanotopography on Bone Response to Titanium Implant.

Clinical success of implant therapy is directly related to titanium (Ti) surface properties and the quality of bone tissue. The treatment of Ti implants with H2SO4/H2O2 is a feasible, reproducible, and low-cost technique to create surface nanotopography (Ti-Nano). As this nanotopography induces osteoblast differentiation, we hypothesized that it may affect bone response to Ti. Thus, this study was designed to evaluate the bone response to a machined Ti implant treated with H2SO4/H2O2 to generate Ti-Nano and to compare it with a commercially available microtopographic Ti implant (Ti-Porous). Implants were placed in rabbit tibias and evaluated after 2 and 6 weeks, and the bone tissue formed around them was assessed by microtomography to record bone volume, bone surface, specific bone surface, trabecular number, trabecular thickness, and trabecular separation. Undecalcified histological sections were used to determine the percentages of bone-to-implant contact, bone area formed between threads, and bone area formed in the mirror area. At the end of 6 weeks, the removal torque was evaluated using a digital torque gauge. The results showed bone formation in close contact with both Ti-Nano and Ti-Porous implants without relevant morphological and morphometric differences, in addition to a similar removal torque irrespective of surface topography. In conclusion, our results have shown that a simple and low-cost method using H2SO4/H2O2 is highly efficient for creating nanotopography on Ti surfaces, which elicits a similar bone response compared with microtopography presented in a commercially available Ti implant.

MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms.

Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis.

Poly(vinylidene-trifluoroethylene)/barium titanate composite for in vivo support of bone formation.

In this study, we evaluated the effect of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane on in vivo bone formation. Rat calvarial bone defects were implanted with P(VDF-TrFE)/BT and polytetrafluoroethylene (PTFE) membranes, and at 4 and 8 weeks, histomorphometric and gene expression analyses were performed. A higher amount of bone formation was noticed on P(VDF-TrFE)/BT compared with PTFE. The gene expression of RUNX2, bone sialoprotein, osteocalcin, receptor activator of nuclear factor-kappa B ligand, and osteoprotegerin indicates that P(VDF-TrFE)/BT favored the osteoblast differentiation compared with PTFE. These results evidenced the benefits of using P(VDF-TrFE)/BT to promote new bone formation, which may represent a promising alternative to be employed in guided bone regeneration.