Mouse Cranial Bone Research Articles

Anti-aging Metabolite-Based Polymeric Microparticles for Intracellular Drug Delivery and Bone Regeneration.

Alpha-ketoglutarate (AKG), a key component of the tricarboxylic acid (TCA) cycle, has attracted attention for its anti-aging properties. Our recent study indicates that locally delivered cell-permeable AKG significantly promotes osteogenic differentiation and mouse bone regeneration. However, the cytotoxicity and rapid hydrolysis of the metabolite limit its application. In this study, we synthesize novel AKG-based polymeric microparticles (PAKG MPs) for sustained release. In vitro data suggest that the chemical components, hydrophilicity, and size of the MPs can significantly affect their cytotoxicity and pro-osteogenic activity. Excitingly, these biodegradable PAKG MPs are highly phagocytosable for nonphagocytic pre-osteoblasts MC3T3-E1 and primary bone marrow mesenchymal stem cells (BMSCs), significantly promoting their osteoblastic differentiation. RNAseq data suggest that PAKG MPs strongly activate Wnt/β-catenin and PI3K-Akt pathways for osteogenic differentiation. Moreover, PAKG enables poly (L-lactic acid) and poly (lactic-co-glycolic acid) MPs (PLLA & PLGA MPs) for efficient phagocytosis. Our data indicate that PLGA-PAKG MPs-mediated intracellular drug delivery can significantly promote stronger osteoblastic differentiation compared to PLGA MPs-delivered phenamil. Notably, PAKG MPs significantly improve large bone regeneration in a mouse cranial bone defect model. Thus, the novel PAKG-based MPs show great promise to improve osteogenic differentiation, bone regeneration, and enable efficient intracellular drug delivery for broad regenerative medicine.

Oxysophocarpine attenuates inflammatory osteolysis by modulating the NF-κb pathway and the reactive oxygen species-related Nrf2 signaling pathway.

Inflammatory diseases often result in bone loss due to persistent inflammation, which activates osteoclasts and increases bone resorption. Oxysophocarpine (OSC), a bioalkaloid extracted from the roots of Sophora japonica and other leguminous plants, has neuroprotective and anti-tumor properties. However, it is still uncertain whether OSC can effectively inhibit the differentiation of osteoclasts and bone resorption. Therefore, this study explored the potential role of OSC in osteoclast formation and inflammatory osteolysis and its underlying mechanisms. This study involved inducing primary mouse bone marrow macrophages (BMMs) into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) and examined the effects of OSC on osteoclast (OC) differentiation, function, and intracellular reactive oxygen species (ROS) production. The impact of OSC on the expression of osteoclast-specific genes and inflammation-related factors was assessed using real-time quantitative PCR. Additionally, changes in oxidative stress-related factors, NF-κB, and MAPK signaling pathways were examined using western blotting. Finally, this study investigated the influence of OSC on a mouse cranial bone resorption model induced by titanium (Ti) particles in vivo. OSC inhibited OC differentiation and resorption and reduces intracellular ROS levels. Moreover, OSC suppressed IL-1β, TNF-α, IL-6, and osteoclast-specific gene transcription while increasing Nrf2 and HO-1 protein expression. Furthermore, OSC inhibited the expression and autoregulation of the NFATc1 gene, ultimately leading to a reduction in Ti particle-induced bone resorption in mice. OSC could be regarded as an innovative medication for the treatment of osteoclast-associated inflammatory osteolytic diseases.

Transcriptomic analysis reveals the role of SIX1 in mouse cranial neural crest patterning and bone development.

Genetic variants of the transcription factor SIX1 and its co-factor EYA1 underlie 50% of Branchio-oto-renal syndrome (BOR) cases. BOR is characterized by craniofacial defects, including malformed middle ear ossicles leading to conductive hearing loss. In this work, we expand our knowledge of the Six1 gene regulatory network by using a Six1-null mouse line to assess gene expression profiles of E10.5 mandibular arches, which give rise to the neural crest (NC)-derived middle ear ossicles and lower jaw, via bulk RNA sequencing. Our transcriptomic analysis led to the identification of 808 differentially expressed genes that are related to translation, NC cell differentiation, osteogenesis, and chondrogenesis including components of the WNT signaling pathway. As WNT signaling is a known contributor to bone development, we demonstrated that SIX1 is required for expression of the WNT antagonist Frzb in the mandibular arch, and determined that SIX1 expression results in repression of WNT signaling. Our results clarify the mechanisms by which SIX1 regulates the development of NC-derived craniofacial elements that are altered in SIX1-associated disorders. In addition, this work identifies novel genes that could be causative to this birth defect and establishes a link between SIX1 and WNT signaling during patterning of NC cells.

In-depth polarisation resolved SHG microscopy in biological tissues using iterative wavefront optimisation.

Polarised nonlinear microscopy has been extensively developed to study molecular organisation in biological tissues, quantifying the response of nonlinear signals to a varying incident linear polarisation. Polarisation Second harmonic Generation (PSHG) in particular is a powerful tool to decipher sub-microscopic modifications of fibrillar collagen organisation in type I and III collagen-rich tissues. The quality of SHG imaging is however limited to about one scattering mean free path in depth (typically 100 micrometres in biological tissues), due to the loss of focus quality, induced by wavefront aberrations and scattering at even larger depths. In this work, we study how optical depth penetration in biological tissues affects the quality of polarisation control, a crucial parameter for quantitative assessment of PSHG measurements. We apply wavefront shaping to correct for SHG signal quality in two regimes, adaptive optics for smooth aberration modes corrections at shallow depth, and wavefront shaping of higher spatial frequencies for optical focus correction at larger depths. Using nonlinear SHG active nanocrystals as guide stars, we quantify the capabilities of such optimisation methods to recover a high-quality linear polarisation and investigate how this approach can be applied to in-depth PSHG imaging in tissues, namely tendon and mouse cranial bone.

Long-term induction of endogenous BMPs growth factor from antibacterial dual network hydrogels for fast large bone defect repair

Osteoinductive, osteoconductive, and antibacterial properties of bone repair materials play important roles in regulating the successful bone regeneration. In the present work, we developed pH-sensitive gelatin methacryloyl (GelMA)-oxidized sodium alginate (OSA) hydrogels for dual-release of gentamicin sulfate (GS) and phenamil (Phe) to enhance the antibacterial activity and to promote large bone defect repair. Controlled release of GS was achieved through physical blending with GelMA-OSA solution before photo-polymeriaztion, while Phe was encapsulated into mesoporous silicate nanoparticles (MSN) within the hydrogels. In vitro antibacterial studies against Staphylococcus aureus and Escherichia coli indicated the broad-spectrum antibacterial property. Moreover, in vitro cell tests verified the synergistically enhanced osteogenic differentiation ability. Furthermore, in vivo studies revealed that the hydrogels significantly increased new bone formation in a critical-sized mouse cranial bone defect model. In summary, the novel dual-network hydrogels with both antibacterial and osteoinductive properties showed promising potential applications in bone tissue engineering.

Hypoxia-mimicking 3D bioglass-nanoclay scaffolds promote endogenous bone regeneration

Large bone defect repair requires biomaterials that promote angiogenesis and osteogenesis. In present work, a nanoclay (Laponite, XLS)-functionalized 3D bioglass (BG) scaffold with hypoxia mimicking property was prepared by foam replication coupled with UV photopolymerization methods. Our data revealed that the incorporation of XLS can significantly promote the mechanical property of the scaffold and the osteogenic differentiation of human adipose mesenchymal stem cells (ADSCs) compared to the properties of the neat BG scaffold. Desferoxamine, a hypoxia mimicking agent, encourages bone regeneration via activating hypoxia-inducible factor-1 alpha (HIF-1α)-mediated angiogenesis. GelMA-DFO immobilization onto BG-XLS scaffold achieved sustained DFO release and inhibited DFO degradation. Furthermore, in vitro data demonstrated increased HIF-1α and vascular endothelial growth factor (VEGF) expressions on human adipose mesenchymal stem cells (ADSCs). Moreover, BG-XLS/GelMA-DFO scaffolds also significantly promoted the osteogenic differentiation of ADSCs. Most importantly, our in vivo data indicated BG-XLS/GelMA-DFO scaffolds strongly increased bone healing in a critical-sized mouse cranial bone defect model. Therefore, we developed a novel BG-XLS/GelMA-DFO scaffold which can not only induce the expression of VEGF, but also promote osteogenic differentiation of ADSCs to promote endogenous bone regeneration.

Nanoclay promotes mouse cranial bone regeneration mainly through modulating drug binding and sustained release

Nanoclay (Nanosilicates, NS) is appearing as an intriguing 2D nanomaterial for bone tissue engineering with multiple proposed functions, e.g., intrinsic osteoinductivity, improving mechanical properties, and drug release capacity. However, the mechanism of NS for in vivo bone regeneration has been hardly defined so far. This knowledge gap will significantly affect the design/application of NS-based biomaterials. To determine the role of NS in osteoblastic differentiation and bone formation, we used mouse calvarial-derived pre-osteoblasts (MC3T3-E1) and a clinically-relevant mouse cranial bone defect model. Instead of a hydrogel, we prepared biomimetic 3D gelatin nanofibrous scaffolds (GF) and NS-blended composite scaffolds (GF/NS) to determine the essential role of NS in critical low-dose (0.5 µg per scaffold) of BMP2-induced cranial bone regeneration. In contrast to “osteoinductivity”, our data indicated that NS could enable single-dose of BMP2, promoting significant osteoblastic differentiation while multiple-dose of BMP2 (without NS) was required to achieve similar efficacy. Moreover, our release study revealed that direct binding to NS in GF scaffolds provided stronger protection to BMP2 and sustained release compared to GF/NS composite scaffolds. Consistently, our in vivo data indicated that only BMP2/NS direct binding treatment was able to repair the large mouse cranial bone defects after 6 weeks of transplantation while neither BMP2, NS alone, nor BMP2 released from GF/NS scaffolds was sufficient to induce significant cranial bone defect repair. Therefore, we concluded that direct nanoclay-drug binding enabled sustained release is the most critical contribution to the significantly improved bone regeneration compared to other possible mechanisms based on our study.

Cell behaviors on surface of pure tantalum with nano-dimpled structure

Tantalum exhibits excellent biocompatibility and good chemical stability, which has been employed in dental and orthopedic applications. The local microenvironment of the surface of implants influence determines the surrounding cell responses. In this work, nano-dimpled structure on the surface of pure tantalum was fabricated by electrochemical anodization. The surface morphology of the nano-dimpled structure was observed, and the dimensions of the nano-dimpled structure were measured by scanning electron microscopy (SEM). The surface roughness of the sample with nano-dimpled structure was measured by atomic force microscopy (AFM). The results show that the diameter of nano-dimpled ranges from 40 to 180 nm, and the higher the applied voltage, the larger the diameter. Moreover, nano-dimpled structure has good hydrophilicity. In vitro results show that nano-dimpled structure can promote the adhesion and proliferation of mouse cranial anterior bone (MC-3T3-E1) cells. The filopodia of MC-3T3-E1 cells can be fully extended on this nano-dimpled structure. On the surface of the nano-dimpled structure with a dimple size of 40 nm, the cells have the longest aspect ratio. The small dimple size of the nano-dimpled structure is beneficial to cell proliferation and osteogenic differentiation.

Characterization of wavefront errors in mouse cranial bone using second-harmonic generation.

Optical aberrations significantly affect the resolution and signal-to-noise ratio of deep tissue microscopy. As multiphoton microscopy is applied deeper into tissue, the loss of resolution and signal due to propagation of light in a medium with heterogeneous refractive index becomes more serious. Efforts in imaging through the intact skull of mice cannot typically reach past the bone marrow ( ? 150 ?? ? m of depth) and have limited resolution and penetration depth. Mechanical bone thinning or optical ablation of bone enables deeper imaging, but these methods are highly invasive and may impact tissue biology. Adaptive optics is a promising noninvasive alternative for restoring optical resolution. We characterize the aberrations present in bone using second-harmonic generation imaging of collagen. We simulate light propagation through highly scattering bone and evaluate the effect of aberrations on the point spread function. We then calculate the wavefront and expand it in Zernike orthogonal polynomials to determine the strength of different optical aberrations. We further compare the corrected wavefront and the residual wavefront error, and suggest a correction element with high number of elements or multiconjugate wavefront correction for this highly scattering environment.

524. Injectable BMP-2 Gene-Activated Scaffold for the Repair of Mouse Cranial Bone Defect

Introduction: A major requirement in bone tissue engineering is the persistent and robust promotion of osteogenesis, which requires sustained stimulation with osteoinductive factors, such as bone morphogenetic protein-2 (BMP-2). However, the direct application of these factors remains challenging due to their short half-life and rapid systemic clearance. Given the well demonstrated benefits of recombinant adeno-associated viral (rAAV) vector, including long-term gene transfer efficiency and relative safety, rAAV-based therapies have been developed in both basic and translational research. However, traditional ex vivo gene transduction process is time consuming and requires multiple steps of cell culture. In addition, rAAV shows inconsistent transfection efficacy in primary cells. In this study, we co-packed rAAV-BMP-2 and human bone marrow stem cells (hBMSCs) into a gelatin scaffolds through one-step visible light-based phtocorsslinking (PXL), and tested their application for bone regenerationi. We hypothesize that the concentrated and localized release of rAAV-BMP-2 from the scaffold matrix results in higher transfection efficacy and BMP-2 expression in hBMSCs seeded within the scaffolds, thus enhancing their osteogenic differentiation and bone formation upon implantation in bone defect. Methods: hBMSCs were obtained with IRB approval from total joint arthroplasty patient. The rAAV6-BMP-2 and rAAV6-GFP reporter (as control) vectors were designed and prepared using an established protocol in our laboratory. PXL was performed using 10% gelatin and 10×106 hBMSCs/ml with different amount of rAAV6-GFP. Transfection efficiency was determined by counting GFP-positive cells ratio as well as assessing cell viability. At an optimal ratio, rAAV-BMP-2 and hBMSCs were included into gelatin through PXL. At different time point, BMP-2 production and rAAV6 release kinetics were analyzed. The osteogenesis of hBMSCs was assessed at day 28 by real time PCR and histological staining. In animal study, a 0.5 cm diameter critical size of cranial bone defect model was generated in SCID mice and then the constructs described above were transplanted. Micro-CT imaging and histological staining were used to evaluate the skull bone healing process up to 6 weeks. Results: Both rAAV and hBMSCs survived during the scaffold fabrication process. 10×104 rAAV/hBMSCs was shown to be the optimal ratio in maintaining cell viability and achieving transduction efficiency. After in situ transfection within scaffolds, hBMSCs produced higher BMP-2 amount than those subjected to traditional ex vivo transduction process, and underwent robust osteogenesis as well. In animal study, micro-CT imaging results indicated that rAAV BMP-2 & hBMSCs-activated gelatin scaffolds effectively promoted bone regeneration in mice cranial bone defect, in a manner significantly superior to that of scaffolds loaded with hBMSCs/BMP-2 protein, and was remarkably compatible to scaffolds loaded with hBMSCs previously ex vivo transduced by rAAV-BMP-2. The histological staining further confirmed the efficacious bone formation from rAAV BMP-2 & hBMSCs loaded scaffold. The results shown here thus demonstrate the feasibility of combining gene and cell therapy with biomaterial engineering via a single-step fabrication and its application for the repair of bone defect.

Peptide‐based activation of alpha5 integrin for promoting osteogenesis

Promoting osteoblastogenesis remains a major challenge in disorders characterized by defective bone formation. We recently showed that the alpha 5 integrin subunit (ITGA5) is critically involved in human mesenchymal cell osteoblast differentiation. In this study, we determined the potential of pharmacological ITGA5 activation by a synthetic cyclic peptide (GA-CRRETAWAC-GA) on murine osteoblast differentiation and function in vitro and bone formation in vivo. Peptide-mediated activation of ITGA5 in murine C3H10T1/2 mesenchymal cells resulted in the generation of the integrin-mediated cell signals FAK and ERK1/2-MAPKs. In vitro, peptide-based activation of ITGA5 protected from cell apoptosis but did not affect cell adhesion or replication, while it enhanced the expression of the osteoblast marker genes Runx2 and type I collagen and increased extracellular matrix (ECM) mineralization as also found with bone morphogenetic protein-2 (BMP2), a standard bone anabolic factor. When injected on adult mouse cranial bone for 3 weeks, the peptide-mediated activation of ITGA5 increased bone thickness by twofold, an effect also induced by BMP2. Histomorphometric analysis showed that this anabolic effect resulted from decreased cell apoptosis and increased bone forming surfaces and bone formation rate (BFR). We conclude that pharmacological activation of ITGA5 in mesenchymal cells is effective in promoting de novo bone formation as a result of increased osteoprogenitor cell differentiation into osteoblasts and increased cell protection from apoptosis. This peptide-based approach could be used therapeutically to promote the osteogenic capacity of osteoblast progenitor cells and to induce de novo bone formation in conditions where osteoblastogenesis is compromised.

Dlx5 Specifically Regulates Runx2 Type II Expression by Binding to Homeodomain-response Elements in the Runx2 Distal Promoter

Two major isoforms of the Runx2 gene are expressed by alternative promoter usage: Runx2 type I (Runx2-I) is derived from the proximal promoter (P2), and Runx2 type II (Runx2-II) is produced by the distal promoter (P1). Our previous results indicate that Dlx5 mediates BMP-2-induced Runx2 expression and osteoblast differentiation (Lee, M.-H., Kim, Y-J., Kim, H-J., Park, H-D., Kang, A-R., Kyung, H.-M., Sung, J-H., Wozney, J. M., Kim, H-J., and Ryoo, H-M. (2003) J. Biol. Chem. 278, 34387-34394). However, little is known of the molecular mechanisms by which Dlx5 up-regulates Runx2 expression in BMP-2 signaling. Here, Runx2-II expression was found to be specifically stimulated by BMP-2 treatment or by Dlx5 overexpression. In addition, BMP-2, Dlx5, and Runx2-II were found to be expressed in osteogenic fronts and parietal bones of the developing cranial vault and Runx2-I and Msx2 in the sutural mesenchyme. Furthermore, Runx2 P1 promoter activity was strongly stimulated by Dlx5 overexpression, whereas Runx2 P2 promoter activity was not. Runx2 P1 promoter deletion analysis indicated that the Dlx5-specific response is due to sequences between -756 and -342 bp of the P1 promoter, where three Dlx5-response elements are located. Dlx5 responsiveness to these elements was confirmed by gel mobility shift assay and site-directed mutagenesis. Moreover, Msx2 specifically suppressed the Runx2 P1 promoter, and the responsible region overlaps with that recognized by Dlx5. In summary, Dlx5 specifically transactivates the Runx2 P1 promoter, and its action on the P1 promoter is antagonized by Msx2.

Identification of a 150-kDa membrane component which is modulated by parathyroid hormone.

Monoclonal antibodies have been produced against primary bone cells obtained from the collagenase digestion of mouse cranial bone. Antibodies were selected on the basis of their immunoglobulin class and those which were identified as IgG were further screened for their ability to inhibit cAMP accumulation in response to sub-maximal doses of the 1-34 amino-terminal peptide of bovine parathyroid hormone, bPTH(1-34). Nine hybridoma clones were subsequently characterized as inhibitory with respect to parathyroid hormone (PTH) responses in intact mouse cranial bone and which also identified a variety of membrane components from detergent extracts of surface-labeled primary bone cells. Five of these antibodies immunoprecipitated a membrane component with Mr of 80 000 that appeared to be a major component of the extract susceptible to surface-labeling with 125I. All nine monoclonal antibodies were shown to bind to a suspended-cell preparation of primary bone cells with 2-3 orders of magnitude greater binding than that of control antibodies. Using this assay, one clone, designated 3G12 IgG, was observed to exhibit desensitization effects at the binding level with a time course and dose dependency for PTH pre-incubation that was similar to the establishment of the refractory state in other systems. In addition, the desensitization effect occurred at 37 degrees C but not at 4 degrees C. This antibody was shown to bind saturably to both intact mouse cranial bone and primary bone cells with an apparent affinity constant (Ka) in the range of 10(9) M. Inhibition of bone cAMP accumulation in response to 2.5 nM bPTH(1-34) was directly correlated to the binding of 3G12 IgG to intact mouse calvariae. A maximum inhibition of approximately 85% was observed. 3G12 IgG immunoprecipitated a single membrane component, Mr 150 000, from NP-40 detergent extracts of 125I-labeled primary mouse bone cells. The molecular mass of this component was also 150 000 daltons when run on polyacrylamide gel slabs under non-reducing conditions. Control and PTH-pre-treated bone cells were surface-labeled, detergent-solubilized and immunoprecipitated with 3G12 IgG in order to investigate further the desensitization effect at the molecular level. Incubation of bone cells with 1 microgram/ml bPTH(1-34) for 45 min at 37 degrees C caused an increased susceptibility to surface-labeling with 125I that was approximately three-fold higher in specific activity than that of control cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a monoclonal antibody which interacts with the parathyroid hormone receptor-adenylate cyclase system in murine bone

We have produced monoclonal antibodies which bind specifically to mouse bone cells and then selected these monoclonal antibodies for their ability to inhibit parathyroid hormone (PTH) responses in mouse cranial bone treated with the (1–34) amino terminal peptide of bovine PTH [bPTH(1–34)]. One clone, designated 3–6, characterized as an IgM(k), significantly inhibited the accumulation of cAMP in response to bPTH(l-34) at concentrations of hormone between 10 −9 and 10 −7 M. This antibody was subsequently isolated by gel filtration and shown to bind to intact mouse calvariae, with saturation binding occurring at 3 μg/ml IgM. A maximal inhibition of approximately 70% of the cAMP accumulation produced in response to 2.5 × 10 −8M (100 ng/ml) bPTH(1–34) was obtained with 7 μg/ml of the purified 3–6 IgM. At this concentration of 3–6 IgM, the half-maximal dose of PTH for activation of cAMP accumulation was increased from 5 × 10 −9 M to 2 × 10 −8 M with no reduction in maximal levels of cAMP production. The utility of this antibody as an inhibitor was further tested by its ability to block the binding of an iodinated PTH analogue, 125I-[Nle 8,Nle 18,Tyr 34]-bPTH(l-34) to mouse cranial bone. The 3–6 IgM at a concentration of 5 × 10 −8 M inhibited 70% of the specific binding of the 125I-labeled analogue. In the absence of parathyroid hormone, 2 × 10 −8 M 3–6 IgM produced a 4-fold increase in cAMP above basal levels, as compared to 40-fold maximal increases observed with PTH, indicating a partial PTH agonist activity of this antibody. When tested for effects on other hormones, 3–6 IgM did not inhibit cAMP accumulation produced in response to salmon calcitonin, epinephrine, prostaglandin E 2 or cholera toxin. We propose that the 3–6 monoclonal IgM is specific for the PTH receptor or a component of the PTH receptor-adenylate cyclase system and that this or similar antibodies will serve as useful reagents for future molecular characterization of this receptor.