Rat Calvarial Bone Research Articles

3D magnetic nanocomposite scaffolds enhanced the osteogenic capacities of rat bone mesenchymal stem cells in vitro and in a rat calvarial bone defect model by promoting cell adhesion.

Magnetic scaffolds incorporated with iron oxide nanoparticles (IONPs) are biocompatible and present excellent osteogenic properties. However, the underlying mechanism is unclear. In this study, 3D-printed poly(lactic-co-glycolic acid) scaffolds were coated with IONPs using layer-by-layer assembly (Fe-scaffold) to prepare magnetic scaffolds. The effects of this modification on osteogenesis were investigated by comparison with untreated scaffolds (Uncoated-scaffold). The results showed that the proliferation of rat bone mesenchymal stem cells (rBMSCs) on the Fe-scaffold was enhanced compared with those on the Uncoated-scaffold (p < 0.05). The alkaline phosphatase activity and expression levels of osteogenic-related genes of cells on the Fe-scaffold were higher than those on the Uncoated-scaffold (p < 0.05). Fe-scaffold was found to promote the cell adhesion compared with Uncoated-scaffold, including increasing the adhered cell number, promoting cell spreading and upregulating the expression levels of adhesion-related genes integrin α1 and β1 and their downstream signaling molecules FAK and ERK1/2 (p < 0.05). Moreover, the amount of new bone formed in rat calvarial defects at 8 weeks decreased in the order: Fe-scaffold > Uncoated-scaffold > Blank-control (samples whose defects were left empty) (p < 0.05). Therefore, 3D magnetic nanocomposite scaffolds enhanced the osteogenic capacities of rBMSCs in vitro and in a rat calvarial bone defect model by promoting cell adhesion. The mechanisms were attributed to the alteration in its hydrophilicity, surface roughness, and chemical composition.

In Vitro Prevascularization of Self-Assembled Human Bone-Like Tissues and Preclinical Assessment Using a Rat Calvarial Bone Defect Model.

In vitro prevascularization has the potential to address the challenge of maintaining cell viability at the core of engineered constructs, such as bone substitutes, and to improve the survival of tissue grafts by allowing quicker anastomosis to the host microvasculature. The self-assembly approach of tissue engineering allows the production of biomimetic bone-like tissue constructs including extracellular matrix and living human adipose-derived stromal/stem cells (hASCs) induced towards osteogenic differentiation. We hypothesized that the addition of endothelial cells could improve osteogenesis and biomineralization during the production of self-assembled human bone-like tissues using hASCs. Additionally, we postulated that these prevascularized constructs would consequently improve graft survival and bone repair of rat calvarial bone defects. This study shows that a dense capillary network spontaneously formed in vitro during tissue biofabrication after two weeks of maturation. Despite reductions in osteocalcin levels and hydroxyapatite formation in vitro in prevascularized bone-like tissues (35 days of culture), in vivo imaging of prevascularized constructs showed an improvement in cell survival without impeding bone healing after 12 weeks of implantation in a calvarial bone defect model (immunocompromised male rats), compared to their stromal counterparts. Globally, these findings establish our ability to engineer prevascularized bone-like tissues with improved functional properties.

Efficacy of extracellular vesicles from dental pulp stem cells for bone regeneration in rat calvarial bone defects

BackgroundExtracellular vesicles (EVs) are known to be secreted by various cells. In particular, mesenchymal stem cell (MSC)-derived EVs (MSC-EVs) have tissue repair capacity and anti-inflammatory properties. Dental pulp stem cells (DPSCs), which are MSCs isolated from pulp tissue, are less invasive to the body than other MSCs and can be collected from young individuals. In this study, we investigated the efficacy of EVs secreted by DPSCs (DPSC-EVs) for bone formation.MethodsDPSC-EVs were isolated from the cell culture medium of DPSCs. DPSC-EVs were unilaterally injected along with collagen (COL), beta-tricalcium phosphate (β-TCP) or hydroxyapatite (HA) into rat calvarial bone defects. The effects of DPSC-EVs were analyzed by micro-computed tomography (micro-CT) and histological observation.ResultsMicro-CT showed that administration of DPSC-EVs with the abovementioned scaffolds resulted in bone formation in the periphery of the defects. DPSC-EVs/COL specifically resulted in bone formation in the center of the defects. Histological observation revealed that DPSC-EVs/COL promoted new bone formation. Administration of DPSC-EVs/COL had almost the same effect on the bone defect site as transplantation of DPSCs/COL.ConclusionsThese results suggest that DPSC-EVs may be effective tools for bone tissue regeneration.

ALLOGRAFT BONE APPLICATION IN THE RAT CALVARIAL BONE DEFECT MODEL: A HISTOPATHOLOGICAL STUDY

Trauma, neoplasms, infections, and congenital anomalies may be the reason for the calvarial bone defects. For eliminating bone defects in the cranial region to stimulate bone regeneration different graft types have been tried. In our study, we aimed to investigate the effects of allograft application in the rat calvarial bone defect model. For this purpose, 14 Wistar male rats were determined; defect (n=7) and defect + graft (n=7) groups. . The frontal bone was opened and a circular full thickness bone defect (5 mm) was created in the midline. Allograft material was placed in the defect area. All animals were sacriced after 28 days and the calvarial bones were followed up for routine histologic preparations. Sections were stained with H-E and scoring for histopathological parameters (inammation, brosis, osteoclast number, osteoblast number, osteocyte number, matrix formation, new bone trabecular diameter). In our study, inammation, brosis and osteoclast numbers decreased in the defect + graft group compared to the defect group, and osteocyte, osteoblast, matrix formation and bone trabecular diameter has increased signicantly. Histopathological evaluation revealed scar tissue, increased mononuclear cell inltration, and necrosis in the defect group. In the defect + graft group, an increase in collagen ber, a decrease in inammatory cells, an increase in osteoblast cells and bone matrix were observed. As a result, allograft application has been found to support new bone formation in the calvarial defect model by creating an osteoinductive and osteoconductive effect.

Non-invasive in vivo monitoring of transplanted stem cells in 3D-bioprinted constructs using near-infrared fluorescent imaging.

Cell‐based tissue engineering strategies have been widely established. However, the contributions of the transplanted cells within the tissue‐engineered scaffolds to the process of tissue regeneration remain poorly understood. Near‐infrared (NIR) fluorescence imaging systems have great potential to non‐invasively monitor the transplanted cell‐based tissue constructs. In this study, labeling mesenchymal stem cells (MSCs) using a lipophilic pentamethine indocyanine (CTNF127, emission at 700 nm) as a NIR fluorophore was optimized, and the CTNF127‐labeled MSCs (NIR‐MSCs) were printed embedding in gelatin methacryloyl bioink. The NIR‐MSCs‐loaded bioink showed excellent printability. In addition, NIR‐MSCs in the 3D constructs showed high cell viability and signal stability for an extended period in vitro. Finally, we were able to non‐invasively monitor the NIR‐MSCs in constructs after implantation in a rat calvarial bone defect model, and the transplanted cells contributed to tissue formation without specific staining. This NIR‐based imaging system for non‐invasive cell monitoring in vivo could play an active role in validating the cell fate in cell‐based tissue engineering applications.

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 Runx2and 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-β/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.

A novel vertical aligned mesoporous silica coated nanohydroxyapatite particle as efficient dexamethasone carrier for potential application in osteogenesis

Bone defect is a common problem and inducing osteoblasts differentiation is the key process for the regenerative repair. Recently, the mesoporous silica (MS) coated nanohydroxyapatite (nHA) particles (nHA-MS) has shown enhanced intrinsic potency for bone regeneration, whereas whether the osteogenesis potency can be further enhanced after drug delivery has not been investigated. In this study, the nHA-MS was fabricated by a novel biphase stratification growth way. The cytotoxicity in MC3T3-E1 was validated by MTT assay, apoptosis analysis and cell cycle examination. The cell uptake was observed by confocal laser scanning microscope and transmission electron microscope respectively. After adsorption with dexamethasone (DEX), the osteogenic differentiation was determined both in vitro and in vivo. The synthesized nHA-MS showed a core–shell structure that the nanorod-like nHA was coated by a porous MS shell (∼5 nm pores diameter, ∼50 nm thickness). A dose-dependent cytotoxicity was observed and below 10 µg ml−1 was a safe concentration. The nHA-MS also showed efficient cell uptake efficiency and more efficient in DEX loading and release. After DEX adsorption, the nanoparticles exhibited enhanced osteogenic induction in MC3T3-E1 and rat calvarial bone defect regeneration. In conclusion, the nHA-MS is a favorable platform for drug delivery to obtain more enhanced osteogenesis capabilities.

Biosilicated collagen/\u03b2-tricalcium phosphate composites as a BMP-2-delivering bone\u2010graft substitute for accelerated craniofacial bone regeneration

BackgroundBioceramic β-tricalcium phosphate (β-TCP) is used as a bone-grafting material and a therapeutic drug carrier for treatment of bone defects in the oral and maxillofacial regions due to the osteoconductivity and biocompatibility. However, the low mechanical strength and limited osteoinductivity of β-TCP agglomerate restrict bone regenerating performance in clinical settings.MethodsHerein, a biomimetic composite is proposed as a bone morphogenetic protein-2 (BMP-2)-delivering bone graft substitute to achieve a robust bone grafting and augmented bone regeneration.ResultsThe sequential processes of brown algae-inspired biosilicification and collagen coating on the surface of β-TCP enable the effective incorporation of BMP-2 into the coating layer without losing its bioactivity. The sustained delivery of BMP-2 from the biosilicated collagen and β-TCP composites promoted in vitro osteogenic behaviors of pre-osteoblasts and remarkedly accelerated in vivo bone regeneration within a rat calvarial bone defect.ConclusionsOur multicomposite bone substitutes can be practically applied to improve bone tissue growth in bone grafting applications with further expansion to general bone tissue engineering.

Safety evaluation of atmospheric pressure plasma jets in in vitro and in vivo experiments.

PurposeThe atmospheric pressure plasma jet (APPJ) has been introduced as an effective disinfection method for titanium surfaces due to their massive radical generation at low temperatures. Helium (He) has been widely applied as a discharge gas in APPJ due to its bactericidal effects and was proven to be effective in our previous study. This study aimed to evaluate the safety and effects of He-APPJ application at both the cell and tissue levels.MethodsCellular-level responses were examined using human gingival fibroblasts and osteoblasts (MC3T3-E1 cells). He-APPJ was administered to the cells in the experimental group, while the control group received only He-gas treatment. Immediate cell responses and recovery after He-APPJ treatment were examined in both cell groups. The effect of He-APPJ on osteogenic differentiation was evaluated via an alkaline phosphatase activity assay. In vivo, He-APPJ treatment was administered to rat calvarial bone and the adjacent periosteum, and samples were harvested for histological examination.ResultsHe-APPJ treatment for 5 minutes induced irreversible effects in both human gingival fibroblasts and osteoblasts in vitro. Immediate cell detachment of human gingival fibroblasts and osteoblasts was shown regardless of treatment time. However, the detached areas in the groups treated for 1 or 3 minutes were completely repopulated within 7 days. Alkaline phosphatase activity was not influenced by 1 or 3 minutes of plasma treatment, but was significantly lower in the 5 minute-treated group (P=0.002). In vivo, He-APPJ treatment was administered to rat calvaria and periosteum for 1 or 3 minutes. No pathogenic changes occurred at 7 days after He-APPJ treatment in the He-APPJ-treated group compared to the control group (He gas only).ConclusionsDirect He-APPJ treatment for up to 3 minutes showed no harmful effects at either the cell or tissue level.

Mechanical, Histological, and Scanning Electron Microscopy Study of the Effect of Mixed-Acid and Heat Treatment on Additive-Manufactured Titanium Plates on Bonding to the Bone Surface.

The additive manufacturing (AM) technique has attracted attention as one of the fully customizable medical material technologies. In addition, the development of new surface treatments has been investigated to improve the osteogenic ability of the AM titanium (Ti) plate. The purpose of this study was to evaluate the osteogenic activity of the AM Ti with mixed-acid and heat (MAH) treatment. Fully customized AM Ti plates were created with a curvature suitable for rat calvarial bone, and they were examined in a group implanted with the MAH-treated Ti in comparison with the untreated (UN) group. The AM Ti plates were fixed to the surface of rat calvarial bone, followed by extraction of the calvarial bone 1, 4, 8, and 12 weeks after implantation. The bonding between the bone and Ti was evaluated mechanically. In addition, AM Ti plates removed from the bone were examined histologically by electron microscopy and Villanueva–Goldner stain. The mechanical evaluation showed significantly stronger bone-bonding in the MAH group than in the UN group. In addition, active bone formation was seen histologically in the MAH group. Therefore, these findings indicate that MAH resulted in rapid and strong bonding between cortical bone and Ti.

Biodegradable metal-derived magnesium and sodium enhances bone regeneration by angiogenesis aided osteogenesis and regulated biological apatite formation

Biodegradable metals have great attraction to become orthopaedic implants. Here, we demonstrated a biodegradable magnesium alloy incorporated with essential element sodium through Sn-Na master alloying technique. The designed MgSnZnNa alloy presented better hardness and corrosion resistance due to the uniform distribution of Na in Mg2Sn second phase and solid soluble Zn in Mg matrix. The co-release of Mg and Na ions resulted in advanced upregulation of osterix and osteocalcin expression in adipose derived stem cells in vitro. It significantly promoted the rat calvarial defect bone regeneration through osteogenesis and angiogenesis, attributed to the co-release of Na and Mg ions, by increasing the expression of calcitonin gene-related peptide, osteocalcin as well as vascular endothelial growth factor. The current study provided an innovative approach by using master alloy to incorporate essential elements (such as Na or K) for fabricating biodegradable Mg alloys with reduced galvanic corrosion and enhanced biological functions.

Rat Calvarial Bone Regeneration by 3D-Printed Beta-Tricalcium Phosphate Incorporating MicroRNA-200c

Advanced fabrication methods of bone grafts designed to match defect sites that combine biodegradable, osteoconductive materials with potent, osteoinductive biologics would significantly impact the clinical treatment of large bone defects. In this study, we engineered synthetic bone grafts using a hybrid material approach that combined 3D-printed biodegradable, osteoconductive β-tricalcium phosphate (β-TCP) with osteoinductive microRNA(miR)-200c. 3D-printed β-TCP scaffolds were fabricated utilizing suspension-enclosing projection-stereolithography (SEPS) process to produce constructs with reproducible microarchitectures that enhanced the osteoconductive properties of β-TCP. 3D-printed β-TCP scaffolds coated with miR-200c incorporated collagen increased the transfection efficiency of miR-200c and osteogenic differentiation of hBMSCs in vitro. Furthermore, these hybrid material, miR-incorporated scaffolds significantly enhanced bone regeneration in critical-sized rat calvarial defects. These results strongly indicate that through use of SEPS 3D-printing technology, hybrid scaffolds combined by osteoconductive biomaterials and osteoinductive biologics can be used as superior bone substitutes for the clinical treatment of large bone defects.

Behavior of Osteoclast Cells Response on Dicalcium Phosphate Dihydrate Layer-Coated β-Tricalcium Phosphate Granular

The aim of this study is to investigate the behavior of osteoclast cells response on dicalcium phosphate dihydrate (DCPD) layer-coated β-TCP granules. β-TCP granules with 300-600 μm were exposed to acidic calcium phosphate solution for 30 mins in order to get 10 mol% DCPD layer-coated β-TCP granular. DCPD free-coated β-TCP granular had used as control specimen. Both specimens were implant in 9 mm of rat calvarial bone defect for 4 weeks. After 4 weeks, the block section of rat calvarial containing specimen were removed for Tatrate-Resistance Acid Phosphatase (TRAP) analysis. Results of TRAP staining reveal that the number of osteoclast cells attached on 10 mol% layer-coated β-TCP granular is higher than DCPD free-coated β-TCP granular. Since remodeling of new bone formation involved simultaneous osteoclast and osteoblast cells response, therefore, the results obtained in this study indicated that the presence of DCPD layer-coated on β-TCP granular helps to improve osteoclast cells response that contribute in stimulating new bone formation.

Transmission Electron Microscopic Examination of the Healing Power Effect of Milled Teeth Versus Beta- Tri Calcium Phosphate Grafted in Calvarial Bone Defect in Rats.

Objectives: to evaluate the effect of milled teeth versus beta tricalcium phosphate bone graft material on new bone formation at the cell level using transmission electron microscope Materials and methods: Forty-five adult male albino rats, were divided into three main groups (n=15); control and two experimental groups. Under general anesthesia, 1.5 cm incision was done exposing the cranial bone. Then two noncritical size bone defects were created one in each parital sides of the rat calvarial bone. In Group I (control group): the bilateral calvarial bony defects were left to heal spontaneously. In Group II the bilateral calvarial bony defects completely filled with β-TCP. In Group III: the bilateral calvarial bony defects completely filled with tooth ash. Five rats from each group were sacrificed after 7, 15, and 30 days postoperatively. Results: tooth ash group: showed more new bone formation. Different types of cells appeared more active: multiple lymphocytes, fibroblast, osteoprogenitor cells, osteoblasts. Multinucleated osteoclasts with prominent vacuolated cytoplasm and ruffled border in Howship’s lacunae were clearly seen. At day 30, disappearing of the tooth ash and infiltration of different types of cells; osteoprogenitor cells, bone lining cells, and osteoblasts while β-TCP particles were still seen in the sections. Conclusion: Bone activity, formation and maturity were ahead in tooth ash filled calvarial bony defects. Tooth ash had a role in the stimulation of osteoblastic activity in matrix formation and its mineralization.

2D titanium carbide(MXene) nanosheets and 1D hydroxyapatite nanowires into free standing nanocomposite membrane: in vitro and in vivo evaluations for bone regeneration.

Bone loss or insufficiency remains a great challenge for implant integrated and subsequently functional loading, where developing biomaterials to augment bone quantity and regenerate alveolar bone defects at implant site is vitally necessary. Recently, MXene, as a large new family of 2D materials, exhibits a great prospect in biomedical applications owing to its ultrathin structure and morphology with a range of extraordinary properties such as chemical, electronic, optical and biological properties etc. Besides, hydroxyapatite is a favorable biomaterial with outstanding bioactivity and osteogenic capacity. In this study, we prepared free standing UHAPNWs/MXene nanocomposite membranes via introducing ultralong hydroxyapatite nanowires (UHAPNWs) with different weight ratios into MXene to explore their potential in bone regeneration. SEM, XPS, FTIR, XRD, tensile strength, Young's modulus and water contact angles were used to characterize the morphology, chemical composition, surface properties, mechanical properties and hydrophilicity of the materials. Subsequently, in vitro studies like cell adhesion, proliferation and osteogenic differentiation of MC3T3-E1 were evaluated. The incorporation of UHAPNWs improved mechanical properties and hydrophilicity with an enhancement in cell adhesion, proliferation, and osteogenic differentiation. More importantly, 10wt% UHAPNWs/MXene exhibited the optimal mechanical properties while biological improvement was more pronounced along with the addition of UHAPNWs when the weight fraction of UHAPNWs was from 0 to 30wt%. Furthermore, in vivo experiments the UHAPNWs/MXene nanocomposite membranes effectively enhanced bone tissue formation quantitatively and qualitatively in a rat calvarial bone defect. Therefore, an appropriate amount of UHAPNWs into MXene plays a positive and evident role in enhancing mechanical properties, biocompatibility and osteoinductivity, leading a novel inorganic composite material for regeneration of bone tissue.

NIR fluorescence for monitoring in vivo scaffold degradation along with stem cell tracking in bone tissue engineering

Stem cell-based tissue engineering has the potential to use as an alternative for autologous tissue grafts; however, the contribution of the scaffold degradation along with the transplanted stem cells to in vivo tissue regeneration remains poorly understood. Near-infrared (NIR) fluorescence imaging has great potential to monitor implants while avoiding autofluorescence from the adjacent host tissue. To utilize NIR imaging for in vivo monitoring of scaffold degradation and cell tracking, we synthesized 800-nm emitting NIR-conjugated PCL-ran-PLLA-ran-PGA (ZW-PCLG) copolymers with three different degradation rates and labeled 700-nm emitting lipophilic pentamethine (CTNF127) on the human placental stem cells (CT-PSCs). The 3D bioprinted hybrid constructs containing the CT-PSC-laden hydrogel together with the ZW-PCLG scaffolds demonstrate that NIR fluorescent imaging enables tracking of in vivo scaffold degradation and stem cell fate for bone regeneration in a rat calvarial bone defect model. This NIR-based monitoring system can be effectively utilized to study cell-based tissue engineering applications.

Concentration-dependent effects of latex F1-protein fraction incorporated into deproteinized bovine bone and biphasic calcium phosphate on the repair of critical-size bone defects.

F1-protein fraction (F1) is a natural bioactive compound extracted from the rubber tree, Hevea brasiliensis, and has been recently studied for its therapeutic potential in wound healing. In this study, we investigated the concentration-dependent effects of F1 (0.01%, 0.025%, 0.05%, and 0.1%) incorporated into deproteinized bovine bone (DBB) and porous biphasic calcium phosphate (pBCP), on the repair of rat calvarial critical-size bone defects (CSBD). The defects were analyzed by 3D-microtomography and 2D-histomorphometry at 12 weeks postsurgery. The binding efficiency of F1 to pBCP (96.3 ± 1.4%) was higher than that to DBB (67.7 ± 3.3%). In vivo analysis showed a higher bone volume (BV) gain in all defects treated with DBB (except in 0.1% of F1) and pBCP (except in 0.05% and 0.1% of F1) compared to the CSBD without treatment/control group (9.96 ± 2.8 mm3 ). DBB plus 0.025% F1 promoted the highest BV gain (29.7 ± 2.2 mm3 , p < .0001) compared to DBB without F1 and DBB plus 0.01% and 0.1% of F1. In the pBCP group, incorporation of F1 did not promote bone gain when compared to pBCP without F1 (15.9 ± 4.2 mm3 , p > .05). Additionally, a small BV occurred in defects treated with pBCP plus 0.1% F1 (10.4 ± 1.4 mm3, p < .05). In conclusion, F1 showed a higher bone formation potential in combination with DBB than with pBCP, in a concentration-dependent manner. Incorporation of 0.25% F1 into DBB showed the best results with respect to bone formation/repair in CSBD. These results suggest that DBB plus 0.25% F1 can be used as a promising bioactive material for application in bone tissue engineering.

Size-Optimized Microspace Culture Facilitates Differentiation of Mouse Induced Pluripotent Stem Cells into Osteoid-Rich Bone Constructs.

Microspace culture is promising for self-organization of induced pluripotent stem cells (iPSCs). However, the optimal size of microspaces for osteogenic differentiation is unclear. We hypothesized that a specific microspace size could facilitate self-organizing iPSC differentiation to form bone-like tissue in vitro. The objectives of this study were to investigate such effects of microspace size and to evaluate bone regeneration upon transplantation of the resulting osteogenic constructs. Dissociated mouse gingival fibroblast-derived iPSCs were plated in ultra-low-attachment microspace culture wells containing hundreds of U-bottom-shaped microwell spots per well to form cell aggregates in growth medium. The microwells had different aperture diameters/depths (400/560 μm (Elp400), 500/700 μm (Elp500), and 900/700 μm (Elp900)) (Kuraray; Elplasia). After 5 days of aggregation, cells were maintained in osteogenic induction medium for 35 days. Only cells in the Elp500 condition tightly aggregated and maintained high viability during osteogenic induction. After 10 days of induction, Elp500 cell constructs showed significantly higher gene expression of Runx2, Osterix, Collagen 1a1, Osteocalcin, Bone sialoprotein, and Osteopontin compared to constructs in Elp400 and Elp900. In methylene blue-counterstained von Kossa staining and Movat's pentachrome staining, only Elp500 constructs showed robust osteoid formation on day 35, with high expression of type I collagen (a major osteoid component) and osteocalcin proteins. Cell constructs were transplanted into rat calvarial bone defects, and micro-CT analysis after 3 weeks showed better bone repair with significantly higher bone mineral density in the Elp500 group compared to the Elp900 group. These results suggest that microspace size affects self-organized osteogenic differentiation of iPSCs. Elp500 microspace culture specifically induces mouse iPSCs into osteoid-rich bone-like tissue possessing high bone regeneration capacity.

Induction of osteogenic differentiation in a rat calvarial bone defect model using an In situ forming graphene oxide incorporated glycol chitosan/oxidized hyaluronic acid injectable hydrogel

Graphene oxide (GO) incorporated hydrogels have received great attention and have shown excellent potential for use in the field of bone tissue engineering due to their unique osteogenic functionalities. However, current hydrogel systems are limited in their ability to provide an appropriate amount of GO to the lesion area. To overcome this issue, we designed a GO incorporated glycol chitosan (gC)-hyaluronic acid (HA) injectable hydrogel system via a simple oxidation technique. Through oxidation, aldehyde groups were introduced onto the HA. Blending this with gC allowed for the formation of an aqueous hydrogel matrix. Physico-chemical characterization demonstrated that the gC/oHA/GO hydrogel matrix exhibited robust mechanical properties and stability. The in vitro and in vivo results showed that GO incorporated injectable hydrogels enhanced bone tissue regeneration as compared to control injectable hydrogels. Therefore, our results indicate that our injectable hydrogel system could be used for delivering GO. This material may serve as an excellent tissue scaffold for use in treating bone defects.

Silymarin effect on experimental bone defect repair in rat following implantation of the electrospun PLA/carbon nanotubes scaffold associated with Wharton's jelly mesenchymal stem cells.

In this study, the ability of silymarin to heal rat calvarial bone critical defects with mesenchymal stem cells isolated from human Wharton's jelly (HWJMSC) cultured on the electrospun scaffold of poly (lactic acid)/carbon nanotube (PLA/CNT) has been examined. In this study, 20 adult male Wistar rats were divided into four groups of five each. Under general anesthesia, 8 mm defects were created in the calvarial bone of the rats. Then, study groups were defined as no treatment group, the scaffold alone, the scaffold and HWJMSCs, and the scaffold/cells plus oral silymarin, respectively. The histomorphometric study was performed using H&E staining and Goldner's Masson trichrome as specific staining. The results of this study showed that the electrospun PLA/CNT scaffold is a biocompatible scaffold and HWJMSCs can considerably attach and proliferate on this scaffold, and the scaffold itself is also a suitable option for improving the bone repair process. The results of the histomorphometric analysis also showed a significantly higher amount of recently formed bone in the silymarin group plus scaffold/cells compared to the scaffold and cell group alone (p < .05). Utilizing silymarin plus HWJMSCs cultured on PLA/CNT scaffold can be used as a suitable method for the process of osteogenesis and bone repair.