Osteogenic Gene Expression Research Articles

Mir-150-5p inhibits the osteogenic differentiation of bone marrow-derived mesenchymal stem cells by targeting irisin to regulate the p38/MAPK signaling pathway

PurposeTo study the effect of miR-150-5p on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and further explore the relationship between its regulatory mechanism and irisin.MethodsWe isolated mouse BMSCs, and induced osteogenic differentiation by osteogenic induction medium. Using qPCR to detect the expression of osteogenic differentiation-related genes, western blot to detect the expression of osteogenic differentiation-related proteins, and luciferase reporter system to verify that FNDC5 is the target of miR-150-5p. Irisin intraperitoneal injection to treat osteoporosis in mice constructed by subcutaneous injection of dexamethasone.ResultsUp-regulation of miR-150-5p inhibited the proliferation of BMSCs, and decreased the content of osteocalcin, ALP activity, calcium deposition, the expression of osteogenic differentiation genes (Runx2, OSX, OCN, OPN, ALP and BMP2) and protein (BMP2, OCN, and Runx2). And down-regulation of miR-150-5p plays the opposite role of up-regulation of miR-150-5p on osteogenic differentiation of BMSCs. Results of luciferase reporter gene assay showed that FNDC5 gene was the target gene of miR-150-5p, and miR-150-5p inhibited the expression of FNDC5 in mouse BMSCs. The expression of osteogenic differentiation genes and protein, the content of osteocalcin, ALP activity and calcium deposition in BMSCs co-overexpressed by miR-150-5p and FNDC5 was significantly higher than that of miR-150-5p overexpressed alone. In addition, the overexpression of FNDC5 reversed the blocked of p38/MAPK pathway by the overexpression of miR-150-5p in BMSCs. Irisin, a protein encoded by FNDC5 gene, improved symptoms in osteoporosis mice through intraperitoneal injection, while the inhibitor of p38/MAPK pathway weakened this function of irisin.ConclusionmiR-150-5p inhibits the osteogenic differentiation of BMSCs by targeting irisin to regulate the/p38/MAPK signaling pathway, and miR-150-5p/irisin/p38 pathway is a potential target for treating osteoporosis.

Three-Dimensional-Printed Spherical Hollow Structural Scaffolds for Guiding Critical-Sized Bone Regeneration.

The treatment of bone tissue defects continues to be a complex medical issue. Recently, three-dimensional (3D)-printed scaffold technology for bone tissue engineering (BTE) has emerged as an important therapeutic approach for bone defect repair. Despite the potential of BTE scaffolds to contribute to long-term bone reconstruction, there are certain challenges associated with it including the impediment of bone growth within the scaffolds and vascular infiltration. These difficulties can be resolved by using scaffold structural modification strategies that can effectively guide bone regeneration. This study involved the preparation of biphasic calcium phosphate spherical hollow structural scaffolds (SHSS) with varying pore sizes using 3D printing (photopolymerized via digital light processing). The chemical compositions, microscopic morphologies, mechanical properties, biocompatibilities, osteogenic properties, and impact on repairing critical-sized bone defects of SHSS were assessed through characterization analyses, in vitro cytological assays, and in vivo biological experiments. The results revealed the biomimetic properties of SHSS and their favorable biocompatibility. The scaffolds stimulated cell adhesion, proliferation, differentiation, and migration and facilitated the expression of osteogenic genes and proteins, including Col-1, OCN, and OPN. Furthermore, they could effectively repair a critical-sized bone defect in a rabbit femoral condyle by establishing an osteogenic platform and guiding bone regeneration in the defect region. This innovative strategy presents a novel therapeutic approach for assessing critical-sized bone defects.

In-depth investigation of FAM20A insufficiency effects on deciduous dental pulp cells: Altered behaviours, osteogenic differentiation, and inflammatory gene expression.

Loss-of-function mutations in FAM20A result in amelogenesis imperfecta IG (AI1G) or enamel-renal syndrome, characterized by hypoplastic enamel, ectopic calcification, and gingival hyperplasia, with some cases reporting spontaneous tooth infection. Despite previous reports on the consequence of FAM20A reduction in gingival fibroblasts and transcriptome analyses of AI1G pulp tissues, suggesting its involvement in mineralization and infection, its role in deciduous dental pulp cells (DDP) remains unreported. The aim of this study was to evaluate the properties of DDP obtained from an AI1G patient, providing additional insights into the effects of FAM20A on the mineralization of DDP. DDP were obtained from a FAM20A-AI1G patient (mutant cells) and three healthy individuals. Cellular behaviours were examined using flow cytometry, MTT, attachment and spreading, colony formation, and wound healing assays. Osteogenic induction was applied to DDP, followed by alizarin red S staining to assess their osteogenic differentiation. The expression of FAM20A-related genes, osteogenic genes, and inflammatory genes was analysed using real-time PCR, Western blot, and/or immunolocalization. Additionally, STRING analysis was performed to predict potential protein-protein interaction networks. The mutant cells exhibited a significant reduction in FAM20A mRNA and protein levels, as well as proliferation, migration, attachment, and colony formation. However, normal FAM20A subcellular localization was maintained. Additionally, osteogenic/odontogenic genes, OSX, OPN, RUNX2, BSP, and DSPP, were downregulated, along with upregulated ALP. STRING analysis suggested a potential correlation between FAM20A and these osteogenic genes. After osteogenic induction, the mutant cells demonstrated reduced mineral deposition and dysregulated expression of osteogenic genes. Remarkably, FAM20A, FAM20C, RUNX2, OPN, and OSX were significantly upregulated in the mutant cells, whilst ALP, and OCN was downregulated. Furthermore, the mutant cells exhibited a significant increase in inflammatory gene expression, that is, IL-1β and TGF-β1, whereas IL-6 and NFκB1 expression was significantly reduced. The reduction of FAM20A in mutant DDP is associated with various cellular deficiencies, including delayed proliferation, attachment, spreading, and migration as well as altered osteogenic and inflammatory responses. These findings provide novel insights into the biology of FAM20A in dental pulp cells and shed light on the molecular mechanisms underlying AI1G pathology.

Direct coupled electrical stimulation towards improved osteogenic differentiation of human mesenchymal stem/stromal cells: a comparative study of different protocols

Electrical stimulation (ES) has been described as a promising tool for bone tissue engineering, being known to promote vital cellular processes such as cell proliferation, migration, and differentiation. Despite the high variability of applied protocol parameters, direct coupled electric fields have been successfully applied to promote osteogenic and osteoinductive processes in vitro and in vivo. Our work aims to study the viability, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells when subjected to five different ES protocols. The protocols were specifically selected to understand the biological effects of different parts of the generated waveform for typical direct-coupled stimuli. In vitro culture studies evidenced variations in cell responses with different electric field magnitudes (numerically predicted) and exposure protocols, mainly regarding tissue mineralization (calcium contents) and osteogenic marker gene expression while maintaining high cell viability and regular morphology. Overall, our results highlight the importance of numerical guided experiments to optimize ES parameters towards improved in vitro osteogenesis protocols.

Polycaprolactone/graphene oxide/magnesium oxide as a novel composite scaffold for bone tissue engineering: Preparation and physical/biological assessment

Mechanically robust biocomposite scaffolds were fabricated by the electrospinning method for bone tissue engineering using a blend of polycaprolactone (PCL), and graphene oxide (GO) with magnesium oxide (MgO) nanoparticles. Physicochemical characteristics including morphology, tensile strength, swelling behavior, biodegradability, contact angle, cell viability, alkaline phosphatase (ALP) activity, mineralization ability, and osteogenic gene expression of the scaffolds were characterized. The addition of small amounts of GO and MgO nanoparticles significantly improved the morphological and mechanical properties of the PCL scaffold. The hydrophilicity, swelling ratio, and biodegradability of the developed composite scaffolds were improved. The PCL/GO/MgO scaffold demonstrated excellent biocompatibility and in-vitro biological performance with adipose-derived mesenchymal stem cells. Cell attachment, proliferation, ALP activity, mineral deposition, and osteogenesis-related gene expression were enhanced when compared to the pure PCL and PCL/GO scaffolds. Simultaneous incorporation of GO and MgO nanoparticles at a concentration of 2 wt% dramatically increased the differentiation of MSCs into osteoblasts. These findings may suggest that the hydrophilic properties and high protein adsorption of PCL/GO/MgO scaffold can stimulate cell proliferation, and nucleation to help improve bone mineralization.

Lanthanum Hydroxide and Chronic Kidney Disease Mineral and Bone Disorder: A Rat Model.

To investigate the pharmacological effects and molecular mechanisms of lanthanum hydroxide(LH) on ectopic mineralization of soft tissue and abnormal bone in rats with acute kidney injury(AKI). Wistar rats were modeled by 5/6 nephrectomy. After the operation, the rats were divided into different groups, the biochemical indexes of serum collected at different times. LH was administered by intragastric tube at doses of 0.4, 0.2, and 0.1g/kg, respectively. Rats were sacrificed in the 16th week after LH treatment. Observation of pathological changes in tissues were made by specific staining. Western Blot, Real-Time Quantitative PCR, and immunohistochemistry techniques were used to detect the impact on pathway-related proteins. Compared with the control group (no LH administered), the serum phosphate level of the LH group was significantly reduced (p<0.01), calcification of the thoracic aorta was reduced (p<0.05, p<0.01) (Serum biochemical tests before dosing and during drug treatment cycles), renal fibrosis was improved (p<0.01), nuclear entry of nuclear factor kappa-B (NF-κB) was reduced (p<0.01), and the expression of the smooth muscle protein 22α (SM22α) was significantly increased (p<0.01). The expression of osteogenic marker genes was decreased. In addition, compared with the controls, the receptor activator for nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio of the femur in the model group was increased (p<0.05). LH can inhibit the occurrence and development of vascular calcification and bone abnormalities in AKI rats by inhibiting the NF-κB and RANKL/OPG signaling pathways.

The Effect of Silk Fibroin/Poly(Lactic-Co-Glycolic Acid) Nanofiber Coating on Porous Calcium Phosphate-Based Composites for Osteoporosis Application

This study observed whether the silk fibroin/poly lactic-co-glycolic acid (SF/PLGA) nanofiber membrane three-dimensionally coated with porous calcium phosphate-based composite can promote the proliferation of osteoblasts, thereby improving osteoporosis. The SF/PLGA nanofiber membrane was used to three-dimensionally coated with the porous calcium phosphate-based composite to analyze proliferation of osteoblasts, hFOB1.19 expressions by Western blot, activity change of hFOB1.19 by CCK-8 assay. The morphology was observed with an electron microscope. Alizarin Red staining showed that the SF/PLGA nanofiber membrane three-dimensionally coated with porous calcium phosphatebased composite promoted osteogenic differentiation and upregulated the expression of osteogenic related genes. It inhibited adipogenic differentiation and the expression of adipogenic genes. SF/PLGA composite material promoted the viability of osteoblasts hFOB1.19, upregulatedGLUT3, downregulated the expression of various apoptosis-related proteins in hFOB1.19. SF/PLGA composite material can reduce the apoptosis efficiency of osteoblasts, promote the proliferation of osteoblasts, and inhibit apoptosis, thereby improving osteoporosis.

Evaluation of rabbit adipose derived stem cells fate in perfused multilayered silk fibroin composite scaffold for Osteochondral repair.

Development of osteochondral tissue engineering approaches using scaffolds seeded with stem cells in association with mechanical stimulations has been recently considered as a promising technique for the repair of this tissue. In this study, an integrated and biomimetic trilayered silk fibroin (SF) scaffold containing SF nanofibers in each layer was fabricated. The osteogenesis and chondrogenesis of stem cells seeded on the fabricated scaffolds were investigated under a perfusion flow. 3-Dimethylthiazol-2,5-diphenyltetrazolium bromide assay showed that the perfusion flow significantly enhanced cell viability and proliferation. Analysis of gene expression by stem cells revealed that perfusion flow had significantly upregulated the expression of osteogenic and chondrogenic genes in the bone and cartilage layers and downregulated the hypertrophic gene expression in the intermediate layer of the scaffold. In conclusion, applying flow perfusion on the prepared integrated trilayered SF-based scaffold can support osteogenic and chondrogenic differentiation for repairing osteochondral defects.

Injectable macroporous naturally-derived apatite bone cement as a potential trabecular bone substitute.

In this study, we have formulated a novel apatite bone cements derived from natural sources (i.e. eggshell and fishbone) with improved qualities that is, porosity, resorbability, biological activity, and so forth. The naturally-derived apatite bone cement (i.e. FBDEAp) was prepared by mixing hydroxyapatite (synthesized from fishbone) and tricalcium phosphate (synthesized from eggshell) as a solid phase with a liquid phase (a dilute acidic blend of cement binding accelerator and biopolymers like gelatin and chitosan) with polysorbate (as liquid porogen) to get a desired bone cement paste. The prepared cement paste sets within the clinically acceptable setting time (≤20 min), easily injectable (>85%) through hands and exhibits physiological pH stability (7.3-7.4). The pure apatite phased bone cement was confirmed by x-ray diffraction and Fourier transform infrared spectroscopy analyses. The FBDEAp bone cement possesses acceptable compressive strength (i.e. 5-7 MPa) within trabecular bone range and is resorbable up to 28% in simulated body fluid solution within 12 weeks of incubation at physiological conditions. The FBDEAp is macroporous in nature (average pore size ~50-400 μm) with interconnected pores verified by SEM and micro-CT analyses. The FBDEAp showed significantly increased MG63 cell viability (>125% after 72 h), cell adhesion, proliferation, and key osteogenic genes expression levels (up to 5-13 folds) compared to the synthetically derived, synthetic and eggshell derived as well as synthetic and fishbone derived bone cements. Thus, we strongly believe that our prepared FBDEAp bone cement can be used as potential trabecular bone substitute in orthopedics.

Mesenchymal stem cells in PRP and PRF containing poly(3-caprolactone)/gelatin Scaffold: a comparative in-vitro study.

Scaffold design is one of the three most essential parts of tissue engineering. Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) have been used in clinics and regenerative medicine for years. However, the temporal release of their growth factors limits their efficacy in tissue engineering. In the present study, we planned to synthesize nanofibrous scaffolds with the incorporation of PRP and PRF by electrospinning method to evaluate the effect of the release of PRP and PRF growth factors on osteogenic gene expression, calcification, proliferation, and cell adhesion of human bone marrow mesenchymal stem cell (h-BMSC) as they are part of scaffold structures. Therefore, we combined PRP/PRF, derived from the centrifugation of whole blood, with gelatin and Polycaprolactone (PCL) and produced nanofibrous electrospun PCL/Gel/PRP and PCL/Gel/PRF scaffolds. Three groups of scaffolds were fabricated, and h-BMSCs were seeded on them: (1) PCL/Gel; (2) PCL/Gel/PRP; (3) PCL/Gel/PRF. MTS assay was performed to assess cell proliferation and adhesion, and alizarin red staining confirmed the formation of bone minerals during the experiment. The result indicated that PCL/Gel did not have any better outcomes than the PRP and PRF group in any study variants after the first day of the experiment. PCL/gelatin/PRF was more successful regarding cell proliferation and adhesion. Although PCL/gelatin/PRP showed more promising results on the last day of the experiment in mineralization and osteogenic gene expression, except RUNX2, in which the difference with PCL/gelatin/PRF group was not significant.

Construction of a PEGDA/chitosan hydrogel incorporating mineralized copper-doped mesoporous silica nanospheres for accelerated bone regeneration

Hydrogels, integrating diverse biocompatible materials, have emerged as promising candidates for bone repair applications. This study presents a double network hydrogel designed for bone tissue engineering, combining poly(ethylene glycol) diacrylate (PEGDA) and chitosan (CS) crosslinked through UV polymerization and ionic crosslinking. Concurrently, copper-doped mesoporous silica nanospheres (Cu-MSNs) were synthesized using a one-pot method. Cu-MSNs underwent additional modification through in-situ biomineralization, resulting in the formation of an apatite layer. Polydopamine was employed to facilitate the deposition of Calcium (Ca) and Phosphate (P) ions on the surface of Cu-MSNs (Cu-MSNs/PDA@CaP). Composite hydrogels were created by integrating varied concentrations of Cu-MSNs/PDA@CaP (25, 50, 100, 150, 200 μg/mL). Characterization unveiled distinctive interconnected porous structures within the composite hydrogel, showcasing a notable 169.6 % enhancement in compressive stress (elevating from 89.01 to 240.19 kPa) compared to pure PEGDA. In vitro biocompatibility experiments illustrated that the composite hydrogel maintained elevated cell viability (up to 106.6 %) and facilitated rapid cell proliferation over 7 days. The hydrogel demonstrated a substantial 57.58 % rise in ALP expression and a surprising 235.27 % increase in ARS staining. Moreover, it significantly enhanced the expression of crucial osteogenic genes, such as run-related transcription factors 2 (RUNX2), collagen 1a1 (Col1a1), and secreted phosphoprotein 1 (Spp1), establishing it as a promising scaffold for bone regeneration. This study shows how Cu-MSNs/PDA@CaP were successfully integrated into a double network hydrogel, resulting in a composite material with good biological responses. Due to its improved characteristics, this composite hydrogel holds the potential for advancing bone regeneration procedures.

Biomimetic highly porous nanocellulose–nanohydroxyapatite scaffolds for bone tissue engineering

Wood-derived TEMPO-oxidized cellulose nanofibrils (CNFs) have potential as scaffolding for bone tissue engineering. Although biocompatible, the material lacks osteoconductive and appropriate mechanical properties. Incorporation of nano-hydroxyapatite (nHA) and modification of scaffold preparation methods could improve applicability. In this study, freeze-dried porous scaffolds were prepared using a range of nHA (0, 20, 33, 50%) and CNF compositions. Not only the microarchitecture but also the chemical composition of the scaffolds was studied. Osteoblast-like osteosarcoma derived cells (Saos-2) were cultured on the scaffolds and their responses (viability, attachment, proliferation, and osteogenic phenotype) to the different scaffolds were documented. The results show that incorporation of nHA influenced the microarchitecture, mechanical stiffness and surface properties of the scaffolds. Moreover, biological characterization demonstrated good cell viability in all the groups. However, the increase of nHA concentration beyond 20% does not offer further advantages. It is concluded that the incorporation of 20% nHA resulted in the widest and most biomimetic pore size distribution, increased surface roughness and improved protein adsorption. These changes in material properties enhanced cell spreading and the osteogenic gene expression of osteoblast-like cells seeded on the scaffolds. Moreover, 20% nHA warrants further investigation as a potential scaffolding material for bone tissue engineering.Graphical abstract

Cutaneous burn injury represents a major risk factor for the development of traumatic ectopic bone formation following blast-related extremity injury

Blast-related traumatic heterotopic ossification (tHO) impacts clinical outcomes in combat-injured patients, leading to delayed wound healing, inflammatory complications, and reduced quality of life. Blast injured patients often have significant burns. This study investigated whether a partial thickness thermal burn injury exacerbates blast-related tHO in a clinically relevant polytrauma animal model. Adult male Sprague Dawley rats were subjected to an established model involving a whole-body blast overpressure exposure (BOP), complex extremity trauma followed by hind limb amputation (CET) followed by the addition of a 10 % total body surface area (TBSA) second degree thermal burn (BU). Micro-CT scans on post-operative day 56 showed a significant increase in HO volume in the CET + BU as compared to the CET alone injury group (p < .0001; 22.83 ± 3.41 mm3 vs 4.84 ± 5.77 mm3). Additionally, CET + BU concomitant with BOP significantly increased HO (p < .0001; 34.95 ± 7.71 mm3) as compared to CET + BU alone, confirming BOP has a further synergistic effect. No HO was detectable in rats in the absence of CET. Serum analysis revealed similar significant elevated (p < .0001) levels of pro-inflammatory markers (Cxcl1 and Il6) at 6 h post-injury (hpi) in the CET + BU and BOP + CET + BU injury groups as compared to naïve baseline values. Real-time qPCR demonstrated similar levels of chondrogenic and osteogenic gene expression in muscle tissue at the site of injury at 168 hpi in both the CET + BU and BOP+CET + BU injury groups. These results support the hypothesis that a 10 % TBSA thermal burn markedly enhances tHO following acute musculoskeletal extremity injury in the presence and absence of blast overpressure. Furthermore, the influence of BOP on tHO cannot be accounted for either in regards to systemic inflammation induced from remote injury or inflammatory-osteo-chondrogenic expression changes local to the musculoskeletal trauma, suggesting that another mechanism beyond BOP and BU synergistic effects are at play. Therefore, these findings warrant future investigations to explore other mechanisms by which blast and burn influence tHO, and testing prophylactic measures to mitigate the local and systemic inflammatory effects of these injuries on development of HO.

Functionalized magnesium alloys obtained by superplastic forming process retain osteoinductive and antibacterial properties: An in-vitro study

ObjectivesThis study aimed to investigate the biocompatibility, osteogenic and antibacterial activity of biomedical devices based on Magnesium (Mg) Alloys manufactured by Superplastic Forming process (SPF) and subjected to Hydrothermal (HT) and Sol-Gel Treatment (Sol-Gel). MethodsMg-SPF devices subjected to Hydrothermal (Mg-SPF+HT) and Sol-Gel Treatment (Mg-SPF+Sol-Gel) were investigated. The biocompatibility of Mg-SPF+Sol-Gel and Mg-SPF+HT devices was observed by indirect and direct cytotoxicity assays, whereas the colonization of sample surfaces was assessed by confocal microscopy. qRT-PCR analysis and microbial growth curve analyses were employed to evaluate the osteogenic and antibacterial activity of both SPF-Mg treated devices, respectively. ResultsMg-SPF+HT and Mg-SPF+Sol-Gel showed a high degree of biocompatibility. Analysis of mRNA expression of osteogenic genes in cells cultured on Mg-treated devices revealed a significant upregulation of the expression levels of BMP2 and Runx-2. Furthermore, the bacterial growth in strains developed in contact with both the Mg-SPF+HT and Mg-SPF+Sol-Gel devices was lower than that observed in the control. SignificanceHydrothermal and Sol-Gel Treatments of Mg alloys obtained through the SPF process demonstrated bioactive, osteogenic and antibacterial activity, offering a promising alternative to conventional Mg-based devices. The obtained Mg-based materials may have the potential to enhance the tunability of temporary devices in maxillary reconstruction, eliminating the need for second surgeries, and ensuring a good bone reconstruction and a reduced implant failure rate due to bacterial infections.

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1β, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1β, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

Capturing cerium ions via hydrogel microspheres promotes vascularization for bone regeneration

The rational design of multifunctional biomaterials with hierarchical porous structure and on-demand biological activity is of great consequence for bone tissue engineering (BTE) in the contemporary world. The advanced combination of trace element cerium ions (Ce3+) with bone repair materials makes the composite material capable of promoting angiogenesis and enhancing osteoblast activity. Herein, a living and phosphorylated injectable porous hydrogel microsphere (P-GelMA-Ce@BMSCs) is constructed by microfluidic technology and coordination reaction with metal ion ligands while loaded with exogenous BMSCs. Exogenous stem cells can adhere to and proliferate on hydrogel microspheres, thus promoting cell-extracellular matrix (ECM) and cell-cell interactions. The active ingredient Ce3+ promotes the proliferation, osteogenic differentiation of rat BMSCs, and angiogenesis of endotheliocytes by promoting mineral deposition, osteogenic gene expression, and VEGF secretion. The enhancement of osteogenesis and improvement of angiogenesis of the P-GelMA-Ce scaffold is mainly associated with the activation of the Wnt/β-catenin pathway. This study could provide novel and meaningful insights for treating bone defects with biofunctional materials on the basis of metal ions.

Cryopreservation with DMSO affects the DNA integrity, apoptosis, cell cycle and function of human bone mesenchymal stem cells

Cryopreservation (CP) enables pooling and long-term banking of various types of cells, which is indispensable for the cell therapeutics. Dimethyl sulfoxide (DMSO) is universally used as a cryoprotectant in basic and clinical research. Although, the use of DMSO has been under serious debate due to significant clinical side effects correlated with infusions of cellular therapy products containing DMSO, the effect of CP with DMSO on the cell properties and functions remains unknown. Here, we experimentally found that the CP of human bone mesenchymal stem cells (hBMSCs) with 10 % DMSO results 10–15 % of cells apoptosis upon immediate freeze-thaw, ca. 3.8 times of DNA damage/repair relative to the fresh ones after post-thaw cultured in 48 h, and cell cycle arrests at G0/G1 after post-thaw cultured in 24 h. Moreover, CP with 10 % DMSO significantly increases the reactive oxygen species (ROS) level of the frozen-thawed MSCs which may be one of the causes impair cellular properties and functions. Indeed, we found that the differentiation and migration ability of post-thaw cultured hBMSCs decrease as the expression of adipogenic, osteogenic genes and F-actin reduces in the comparison with those of the fresh cells.

Extracellular Vesicle Clicking on Osteoimplants Through Biomimetic Molecular Adhesion Enables Immune‐Enhanced Osseointegration in Diabetics

AbstractExtracellular vesicles (EVs) derived from differentiating induced stem cells maintain their original stemness and differentiating trends and possess strong immunoregulatory capability. Biomaterials equipped with EVs are very promising in regenerative medicine. However, surface EV‐decoration on osteoimplants remains a challenge, due to the complexity of traditional molecular conjugations and the fragility of EVs. Here, mussel‐like molecular adhesion is combined with bioorthogonal click conjugation to introduce EVs on titanium (Ti) implants. The biomimetic adhesion and clickable molecular linkage allow mild and stable tethering of pre‐osteogenic mesenchymal stem cell (MSC)‐derived EVs on Ti implants. EV‐decorated implants exhibit significantly enhanced osseointegration on the bone‐implant surface under diabetic conditions, promoting increased expression of osteogenic genes. Modified surfaces impelled phenotypic alterations in macrophage polarization via multi‐pathway regulation, decreasing proinflammatory M1 macrophage formation, which can lead to the promotion of surface osteogenesis. On Ti rods implanted in a diabetic rat model, EV coating inhibited M1macrophages around the prosthesis, resulting in satisfactory long‐term osseointegration. This study offers a new perspective to represent a simple and effective means for surface EV decoration, providing an osteoimmunomodulatory effect to enhance the diabetic osseointegration of implants.

Synergistic Amelioration of Osseointegration and Osteoimmunomodulation with a Microarc Oxidation-Treated Three-Dimensionally Printed Ti-24Nb-4Zr-8Sn Scaffold via Surface Activity and Low Elastic Modulus.

Biomaterial scaffolds, including bone substitutes, have evolved from being primarily a biologically passive structural element to one in which material properties such as surface topography and chemistry actively direct bone regeneration by influencing stem cells and the immune microenvironment. Ti-6Al-4V(Ti6Al4V) implants, with a significantly higher elastic modulus than human bone, may lead to stress shielding, necessitating improved stability at the bone-titanium alloy implant interface. Ti-24Nb-4Zr-8Sn (Ti2448), a low elastic modulus β-type titanium alloy devoid of potentially toxic elements, was utilized in this study. We employed 3D printing technology to fabricate a porous scaffold structure to further decrease the structural stiffness of the implant to approximate that of cancellous bone. Microarc oxidation (MAO) surface modification technology is then employed to create a microporous structure and a hydrophilic oxide ceramic layer on the surface and interior of the scaffold. In vitro studies demonstrated that MAO treatment enhances the proliferation, adhesion, and osteogenesis capabilities on the scaffold surface. The chemical composition of the MAO-Ti2448 oxide layer is found to enhance the transcription and expression of osteogenic genes in bone mesenchymal stem cells (BMSCs), potentially related to the enrichment of Nb2O5 and SnO2 in the oxide layer. The MAO-Ti2448 scaffold, with its synergistic surface activity and low stiffness, significantly activates the anti-inflammatory macrophage phenotype, creating an immune microenvironment that promotes the osteogenic differentiation of BMSCs. In vivo experiments in a rabbit model demonstrated a significant improvement in the quantity and quality of the newly formed bone trabeculae within the scaffold under the contact osteogenesis pattern with a matched elastic modulus. These trabeculae exhibit robust connections to the external structure of the scaffold, accelerating the formation of an interlocking structure between the bone and implant and providing higher implantation stability. These findings suggest that the MAO-Ti2448 scaffold has significant potential as a bone defect repair material by regulating osteoimmunomodulation and osteogenesis to enhance osseointegration. This study demonstrates an optional strategy that combines the mechanism of reducing the elastic modulus with surface modification treatment, thereby extending the application scope of β-type titanium alloy.

Effect of lattice type on biomechanical and osseointegration properties of 3D-printed porous Ti6Al4V scaffolds

Porous structure is an efficient tool for optimizing the elastic modulus and osseointegration properties of titanium alloy materials. However, the investigations on pore shape remain scarce. In this study, we created porous Ti6Al4V scaffolds with a pore size of 600 &amp;mu;m but different lattices (cubic pentagon, diamond, cuboctahedron). The mechanical and biological properties of the scaffolds were investigated in static simulation analysis, in vitro mechanical compression test, computational fluid dynamics, as well as cell and animal experiments. The results demonstrated that the calculated yield strength difference between the three Ti6Al4V porous scaffolds was negligible, at approximately 140 MPa, allowing them to match the strength requirements of human bones. The diamond scaffold has the lowest calculated elastic modulus (11.6 GPa), which is conducive for preventing stress shielding. The shear stress was largely concentrated in the diamond scaffold, and the stress range of 120&amp;ndash;140 MPa accounted for the greatest share. The mouse MC3T3-E1 cells were found to attach to all three scaffolds, with the diamond scaffold displaying a higher degree of cell adherence. There was more proliferating cells on the diamond and cubic pentagon scaffolds than on the cuboctahedron scaffolds (P &lt; 0.05). The diamond scaffold exhibited the highest alkaline phosphatase activity and calcium salt accumulation in cell differentiation tests. Besides, the expression of osteogenic genes on the diamond scaffold was higher than that on the cuboctahedron scaffold, the cubic pentagon scaffold displaying the lowest expression. The in vivo studies revealed that all three scaffolds fused well with the surrounding bone and that there was no loosening or movement of the prosthesis. Micro-computed tomography, corroborated by the staining results of hard tissues, revealed that the level of new bone formation was the highest in the diamond scaffold, followed by the cuboctahedron scaffold (P &lt; 0.05). Taken together, the diamond scaffold is comparatively better at optimizing the elastic modulus and osseointegration properties of titanium alloy materials, and thus is a preferred choice for porous design.