Repair Of Alveolar Bone Defects Research Articles

Photodynamic and nitric oxide therapy-based synergistic antimicrobial nanoplatform: an advanced root canal irrigation system for endodontic bacterial infections

BackgroundThe main issues faced during the treatment of apical periodontitis are the management of bacterial infection and the facilitation of the repair of alveolar bone defects to shorten disease duration. Conventional root canal irrigants are limited in their efficacy and are associated with several side effects. This study introduces a synergistic therapy based on nitric oxide (NO) and antimicrobial photodynamic therapy (aPDT) for the treatment of apical periodontitis.ResultsThis research developed a multifunctional nanoparticle, CGP, utilizing guanidinylated poly (ethylene glycol)-poly (ε-Caprolactone) polymer as a carrier, internally loaded with the photosensitizer chlorin e6. During root canal irrigation, the guanidino groups on the surface of CGP enabled effective biofilm penetration. These groups undergo oxidation by hydrogen peroxide in the aPDT process, triggering the release of NO without hindering the production of singlet oxygen. The generated NO significantly enhanced the antimicrobial capability and biofilm eradication efficacy of aPDT. Furthermore, CGP not only outperforms conventional aPDT in eradicating biofilms but also effectively promotes the repair of alveolar bone defects post-eradication. Importantly, our findings reveal that CGP exhibits significantly higher biosafety compared to sodium hypochlorite, alongside superior therapeutic efficacy in a rat model of apical periodontitis.ConclusionsThis study demonstrates that CGP, an effective root irrigation system based on aPDT and NO, has a promising application in root canal therapy.

CoCl2-Induced hypoxia promotes hPDLSCs osteogenic differentiation through AKT/mTOR/4EBP-1/HIF-1α signaling and facilitates the repair of alveolar bone defects.

Bone defects are characterized by a hypoxic environment, which affects bone tissue repair. However, the role of hypoxia in the repair of alveolar bone defects remains unclear. Human periodontal ligament stem cells (hPDLSCs) are high-quality seed cells for repairing alveolar bone defects, whose behavior changes under hypoxia. However, their mechanism of action is not known and needs to be elucidated. We hypothesized that hypoxia might be beneficial to alveolar bone defect repair and the osteogenic differentiation of hPDLSCs. To test this hypothesis, cobalt chloride (CoCl2) was used to create a hypoxic environment, both in vitro and in vivo. In vitro study, the best osteogenic effect was observed after 48 h of hypoxia in hPDLSCs, and the AKT/mammalian target of rapamycin/eukaryotic translation initiation factor 4e-binding protein 1 (AKT/mTOR/4EBP-1) signaling pathway was significantly upregulated. Inhibition of the AKT/mTOR/4EBP-1 signaling pathway decreased the osteogenic ability of hPDLSCs under hypoxia and hypoxia-inducible factor 1 alpha (HIF-1α) expression. The inhibition of HIF-1α also decreased the osteogenic capacity of hPDLSCs under hypoxia without significantly affecting the level of phosphorylation of AKT/mTOR/4EBP-1. In vitro study, Micro-CT and tissue staining results show better bone regeneration in hypoxic group than control group. These results suggested that hypoxia promoted alveolar bone defect repair and osteogenic differentiation of hPDLSCs, probably through AKT/mTOR/4EBP-1/HIF-1α signaling. These findings provided important insights into the regulatory mechanism of hypoxia in hPDLSCs and elucidated the effect of hypoxia on the healing of alveolar bone defects. This study highlighted the importance of physiological oxygen conditions for tissue engineering.

Additive manufacturing of magnesium-doped calcium silicate/zirconia ceramic scaffolds with projection-based 3D printing: Sintering, mechanical and biological behavior

The lack of traditional bioactivity in zirconia (ZrO2) and limitations in the personalization capabilities of conventional manufacturing processes pose significant challenges for alveolar bone defect repair. This study aims to investigate whether ZrO2/CSi-Mgx scaffolds, produced by combining magnesium-doped calcium silicate (CSi–Mg) as a doping phase with ZrO2 as the matrix, using projection-based 3D printing (3DPP) technology, exhibit favorable biocompatibility and mechanical properties at low sintering temperatures. The effect of CSi–Mg content (x%), pore structure and heating temperature on the strength of scaffolds were investigated systematically. Incorporation of CSi–Mg could readily adjust the sintering properties of the ZrO2 scaffolds and the scaffolds with low (10–20 %) CSi–Mg possess much higher strength (74–92 MPa) after 1150 °C. Meanwhile, the ZrO2/CSi–Mg10 scaffolds with Triply periodic minimum surfaces (TPMS) pore structure had a compression strength of over 92 MPa and maintained a respectable strength (63 MPa) even after immersion in Tris buffer for 6 weeks. Concurrently, cellular experiments showed that incorporation of CSi–Mg could enhance cellular adhesion, proliferation, and migration of the ZrO2 scaffolds and also promote the osteogenic property of the scaffolds. In conclusion, the ZrO2/CSi–Mg10 scaffold with TPMS pore structure showcases remarkable mechanical performance and bioactivity, holding the potential to facilitate in-situ bone regeneration within the alveolar bone.

Bacteria-responsive, Cell-recruitable, and osteoinductive nanocomposite microcarriers for intelligent bacteriostasis and accelerated tissue regeneration

Repairing alveolar bone defects with chronic periodontitis is a major clinical challenge, since the periodontitis causes the intermittent bacteria invasion to irreversibly aggravates the degeneration of alveolar bones. To deal with the challenge, a kind of novel microcarriers with excellent abilities of responding to alkaline phosphatase (ALP) to intelligently eliminate the bacteria and promoting the recruitment and osteogenic differentiation of endogenous stem cells are developed. Mesoporous silica nanocarriers that chemically fix the polyethylene glycol-modified amoxicillin (PEG-AML) through the phosphoester groups and electrostatically adsorb the aspirin (ASP) are first prepared. The nanocarriers are then dispersed in a chitosan solution to prepare the bacteria-responsive, cell-recruitable, and osteoinductive nanocomposite microcarriers. When being stimulated by ALP that are secreted by the bacteria, phosphoester groups of microcarriers are cleaved, and PEG-AML are delivered on demand to well achieve the goal of intelligent bacteriostasis. The microcarriers slowly release the ASP to promote the recruitment and osteogenic differentiation of endogenous stem cells. At 8 weeks post-implantation, the microcarriers facilitate the repair of alveolar bone defects with periodontitis. The blood vessels in the neo-generated alveolar bones grow well. Due to the appropriate degradation time and excellent antibacterial ability of microcarriers, the microcarriers exhibit a better therapeutic efficacy than the standard clinical products (Geistlich Bio-Oss®), providing a new insight into the cure of alveolar bone defects with periodontitis.

PF127 Hydrogel-Based Delivery of Exosomal CTNNB1 from Mesenchymal Stem Cells Induces Osteogenic Differentiation during the Repair of Alveolar Bone Defects.

Pluronic F127 (PF127) hydrogel has been highlighted as a promising biomaterial for bone regeneration, but the specific molecular mechanism remains largely unknown. Herein, we addressed this issue in a temperature-responsive PF127 hydrogel loaded with bone marrow mesenchymal stem cells (BMSCs)-derived exosomes (Exos) (PF127 hydrogel@BMSC-Exos) during alveolar bone regeneration. Genes enriched in BMSC-Exos and upregulated during the osteogenic differentiation of BMSCs and their downstream regulators were predicted by bioinformatics analyses. CTNNB1 was predicted to be the key gene of BMSC-Exos in the osteogenic differentiation of BMSCs, during which miR-146a-5p, IRAK1, and TRAF6 might be the downstream factors. Osteogenic differentiation was induced in BMSCs, in which ectopic expression of CTNNB1 was introduced and from which Exos were isolated. The CTNNB1-enriched PF127 hydrogel@BMSC-Exos were constructed and implanted into in vivo rat models of alveolar bone defects. In vitro experiment data showed that PF127 hydrogel@BMSC-Exos efficiently delivered CTNNB1 to BMSCs, which subsequently promoted the osteogenic differentiation of BMSCs, as evidenced by enhanced ALP staining intensity and activity, extracellular matrix mineralization (p < 0.05), and upregulated RUNX2 and OCN expression (p < 0.05). Functional experiments were conducted to examine the relationships among CTNNB1, microRNA (miR)-146a-5p, and IRAK1 and TRAF6. Mechanistically, CTNNB1 activated miR-146a-5p transcription to downregulate IRAK1 and TRAF6 (p < 0.05), which induced the osteogenic differentiation of BMSCs and facilitated alveolar bone regeneration in rats (increased new bone formation and elevated BV/TV ratio and BMD, all with p < 0.05). Collectively, CTNNB1-containing PF127 hydrogel@BMSC-Exos promote the osteogenic differentiation of BMSCs by regulating the miR-146a-5p/IRAK1/TRAF6 axis, thus inducing the repair of alveolar bone defects in rats.

Mesenchymal Stem Cell-Derived Exosomes Enhance 3D-Printed Scaffold Functions and Promote Alveolar Bone Defect Repair by Enhancing Angiogenesis.

The reconstruction of severe alveolar bone defects remains a complex and challenging field for clinicians. Three-dimensional-printed scaffolds can adapt precisely to the complicated shape of the bone defects, which is an alternative solution to bone tissue engineering. Our previous study constructed an innovative low-temperature 3D-printed silk fibroin/collagen I/nano-hydroxyapatite (SF/COL-I/nHA) composite scaffold with a stable structure and remarkable biocompatibility. However, the clinical translation of most scaffolds is limited by insufficient angiogenesis and osteogenesis. In this study, we investigated the effects of human umbilical cord mesenchymal-stem-cell-derived exosomes (hUCMSC-Exos) on bone regeneration, especially from the perspective of inducing angiogenesis. HUCMSC-Exos were isolated and characterized. In vitro, the effect of hUCMSC-Exos on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) was examined. Moreover, the loading and release of hUCMSC-Exos on 3D-printed SF/COL-I/nHA scaffolds were evaluated. In vivo, hUCMSC-Exos and 3D-printed SF/COL-I/nHA scaffolds were implanted into alveolar bone defects, and bone regeneration and angiogenesis were investigated by micro-CT, HE staining, Masson staining, and immunohistochemical analysis. The results showed that hUCMSC-Exos stimulated HUVEC proliferation, migration, and tube formation in vitro, and the effect increased with increasing exosome concentrations. In vivo, the combination of hUCMSC-Exos and 3D-printed SF/COL-I/nHA scaffolds promoted alveolar bone defect repair by enhancing angiogenesis and osteogenesis. We constructed an elaborate cell-free bone-tissue-engineering system by combining hUCMSC-Exos with 3D-printed SF/COL-I/nHA scaffolds, potentially providing new ideas for treating alveolar bone defects.

Mechano-responsive microRNA-functionalized PDLSC exosomes as a novel therapeutic for inflammatory bone loss in periodontitis

Biomechanical stimulation is crucial for the regulation of osteogenesis in the alveolar bone. Periodontal Ligament Stem Cells (PDLSCs) are the sensor for mechanical stimulation during orthodontic tooth movement and regulators of alveolar bone remodeling through the paracrine effect. Our previous findings confirmed that tension stimulation on PDLSCs enhanced the osteogenic induction properties of exosomes (Exos). This study aims to explore the mechanism of tension-stimulated PDLSC Exos to regulate osteogenesis and develop a strategy to functionalize Exos for the treatment of bone loss in periodontitis. Here, by comparing the elevated miRNAs in the exosomes (Exos) secreted by the PDLSCs after tension stimulation, we found miR-200 family were mechano-responsive miRNAs in Exos. miR-200b/c carried by Exos secreted by strain Mechanical stimulated PDLSCs can be taken up by human mandibular BMSCs under tension, resulting in a bone-promoting effect. Through bioinformatics target prediction and verification through gain-and loss-of-function assays, we confirmed that miR-200b/c targeted Smurf1 to activate the BMP-Smad signaling pathway and promote osteogenic differentiation of mandibular BMSCs. Finally, a rat model of alveolar bone defect was applied to evaluate the therapeutic effect of functionalized Exos on bone formation in vivo. Histological analysis confirmed that miR-200b/c functionalized Exos could efficiently promote the repair of alveolar bone defects, suggesting a novel therapeutic for inflammatory bone loss in periodontitis.

Repair of Alveolar Bone Defects with Osteogenic Polypeptide Modified HA_CF_PEEK Dental Implants

The implant implanted into the body directly interacts with the bone tissue around the implant through its surface. The surface properties of the implant can affect the adhesion, proliferation, and differentiation of surrounding cells and ultimately determine the quality of osseointegration and the effect of implantation surgery. In this project, nano-hydroxyapatite (n-HA) and carbon fiber (CF) were added into the matrix, poly(ether-ether-ketone) (PEEK), forming the composite material HA/CF/PEEK. Since biologically active proteins and peptides can promote cell viability and proliferation, the osteogenic polypeptide sequence BEE1 was polymerized and grafted onto the surface of HA/CF/PEEK composites to generate HA/CF/PEEK-DPP and HA/CF/PEEK-DPP-BEE1 composites. The above materials were tested for physical characterization and in vitro cell activity by establishing a Beagle dog dentition defect model. HA/CF/PEEK type composites were selected for peri-implant bone defect repair and compared with titanium mesh alone or Bio-Gide membrane for alveolar bone defect repair. In the experiment, the HA/CF/PEEK material modified with osteogenic polypeptides changed in element composition, hydrophilicity, and surface morphology. in vitro cell assays detected that the surface of HA/CF/PEEK material modified with osteogenic polypeptides was more conducive to cell adhesion and proliferation and had no cytotoxicity. The peri-implant defect repair test showed that the HA/CF/PEEK-DPP-BEE1 composite material for alveolar bone repair in experimental dogs was better than that of titanium mesh or Bio-Gide membrane. Meanwhile, the mineralization speed and histomorphometry test at the bone defect further proved that this material could better promote bone neogenesis at the defect. Mineralization.

Construction of poly-(γ-benzyl‐l‐glutamate) composite microcarriers with osteogenic and angiogenic properties for the restoration of alveolar bone defects

The repair of alveolar bone defects is one of the urgent clinical problems to be solved. Injectable microcarriers show great potential for bone repair due to their ability to fill irregular bone defects. In this study, the ICA@nCP/nHA-g-PBLG composite microcarriers with osteogenic and angiogenic properties were fabricated via double emulsion (W/O/W) method. Firstly, poly-(γ-benzyl‐l‐glutamate) (PBLG) was linkaged onto the surface of nano copper hydroxyphosphate (nCP) and nano hydroxyapatite (nHA) to construct nCP-g-PBLG and nHA-g-PBLG nanohybrids by chemical grafting, respectively. Then, nCP/nHA-g-PBLG porous microcarriers were fabricated via double emulsion (W/O/W) method and immersed in icariin (ICA) solution to obtain ICA@nCP/nHA-g-PBLG composite microcarriers. The composite microcarriers were more conducive to the proliferation of bone marrow mesenchymal stem cells (BMSCs) when the content of nCP and ICA in the composite microcarriers were 0.5 wt% and 0.1 wt%, respectively. The release of Cu2+ in nCP could promote angiogenesis, while the release of ICA could promote the osteogenic differentiation of BMSCs. The ability of ICA@nCP/nHA-g-PBLG microcarriers to restore alveolar bone defects was proved in a rabbit model. In conclusion, the ICA@nCP/nHA-g-PBLG microcarriers exhibited two functions: osteogenesis and angiogenesis, which could be served as a promising implant for alveolar bone defects repair.

PLGA Containing Human Adipose-Derived Stem Cell-Derived Extracellular Vesicles Accelerates the Repair of Alveolar Bone Defects via Transfer of CGRP.

Calcitonin gene-related peptide (CGRP) is an important neuropeptide expressed in the nerve fibers during bone repair. Here, we aimed to pinpoint the role of CGRP in the osteogenic differentiation property of human periodontal ligament stem cells (hPDLSCs) and the resultant repair of alveolar bone defect. The key factor related to the osteogenic differentiation of hPDLSCs was retrieved from the GEO database. After extraction from hADSCs (hADSC-EVs) and identification, EVs were subjected to coculture with hPDLSCs, in which the expression patterns of CGRP and osteogenic differentiation marker proteins (ALP, RUNX2, and OCN), as well as ALP activity, were detected. A novel cell-free tissue-engineered bone (TEB) comprised of PLGA/pDA and hADSC-EVs was implanted into the rats with alveolar bone defects to evaluate the repair of alveolar bone defects. CGRP was enriched in hADSC-EVs. hADSCs delivered CGRP to hPDLSCs through EVs, thereby promoting the osteogenic differentiation potential of hPDLSCs. The PLGA/pDA-EV scaffold released EVs slowly, and its implantation into the rat alveolar bone defect area significantly induced bone defect repair, which was reversed by further knockdown of CGRP. In conclusion, our newly discovered cell-free system consisted of hADSC-EVs, and PLGA/pDA scaffold shows promising function in repairing alveolar bone defects.

Sequentially releasing self-healing hydrogel fabricated with TGFβ3-microspheres and bFGF to facilitate rat alveolar bone defect repair

Resorption and loss of alveolar bone leads to oral dysfunction and loss of natural or implant teeth. Biomimetic delivery of growth factors based on stem cell recruitment and osteogenic differentiation, as the key steps in natural alveolar bone regenerative process, has been an area of intense research in recent years. A mesoporous self-healing hydrogel (DFH) with basic fibroblast growth factor (bFGF) entrapment and transforming growth factor β3 (TGFβ3) - loaded chitosan microspheres (CMs) was developed. The formulation was optimized by multiple tests of self-healing, in-bottle inversion, SEM, rheological, swelling rate and in vitro degradation. In vitro tubule formation assays, cell migration assays, and osteogenic differentiation assays confirmed the ability of DFH to promote blood vessels, recruit stem cells, and promote osteogenic differentiation. The optimum DFH formula is 0.05 ml 4Arm-PEG-DF (20%) added to 1 ml CsGlu (2%) containing bFGF (80 ng) and TGFβ3-microspheres (5 mg). The results of in vitro release studied by Elisa kit, indicated an 95% release of bFGF in 7 d and long-term sustained release of TGFβ3. For alveolar defects rat models, the expression levels of CD29 and CD45, the bone volume fraction, trabecular number, and trabecular thickness of new bone monitored by Micro-CT in DFH treatment groups were significantly higher than others (*P < 0.05, vs Model). HE and Masson staining show the same results. In conclusion, DFH is a design of bionic alveolar remodelling microenvironment, that is in early time microvessels formed by bFGF provide nutritious to recruited endogenous stem cells, then TGFβ3 slowly released speed up the process of new bones formation to common facilitate rat alveolar defect repair. The DFH with higher regenerative efficiency dovetails nicely with great demand due to the requirement of complicated biological processes.

A Hierarchical-Structured Mineralized Nanofiber Scaffold with Osteoimmunomodulatory and Osteoinductive Functions for Enhanced Alveolar Bone Regeneration.

Alveolar bone resorption is a major cause of teeth loss and jeopardizes the osseointegration of dental implants, greatly affecting patient's quality of life and health. It is still a great challenge to completely regenerate the alveolar bone defect through traditional guided bone regeneration (GBR) membranes due to their limited bioactivity and regeneration potential. Herein, a new hierarchical-structured mineralized nanofiber (HMF) scaffold, which is combined with both anisotropic and isotropic nanofibrous surface topography and the mineralized particles, is fabricated via a simple template-assisted electrospinning technology and in situ mineralization method. This HMF scaffold can not only directly induce osteogenic differentiation of bone mesenchymal stem cells (osteoinduction), but also stimulate macrophage toward pro-healing (M2) phenotype-polarization with an elevated secretion of the pro-healing cytokines, eventually enhancing the osteogenesis (osteoimmunomodulation). The results of in vivo rat alveolar bone defect repair experiments demonstrate that as compared with the combination of commercial Bio-Gide and Bio-Oss, the single HMF scaffold shows comparable or even superior bone repair effect, with better tissue-integration and more suitable degradation time and accompanied by a simplified operation.

Effect of Attapulgite-Doped Electrospun Fibrous PLGA Scaffold on Pro-Osteogenesis and Barrier Function in the Application of Guided Bone Regeneration.

PurposeGuided bone regeneration (GBR) therapy, which is a widely used technique in clinical practice and is effective in improving the repair of alveolar bone defects or bone mass deficiency regeneration, requires the use of membrane materials with good biocompatibility, barrier function, rigidity matching the space maintenance ability, economic benefits and excellent clinical applicability. The aim of this study was to develop an electrospun attapulgite (ATT)-doped poly (lactic-co-glycolic acid) (PLGA) scaffold (PLGA/ATT scaffold) as a novel material for GBR applications.Methods and ResultsScanning electron microscopy (SEM) and X-ray diffraction (XRD) were used to determine the morphology and the crystalline structure of the PLGA/ATT scaffolds, respectively. Porosity and contact-angle measurements were also carried out to further characterize the physical properties of the PLGA/ATT scaffolds. The results of in vitro studies showed that bone marrow mesenchymal stem cells (BMSCs) attached more readily to and spread better over the PLGA/ATT scaffolds than the Bio-Gide membrane. Furthermore, in the in vitro osteoinductive experiments with BMSCs, the PLGA/ATT scaffolds were found to enhance the activity of alkaline phosphatase (ALP), promote the formation of mineralized bone nodules, and up-regulate the expression of several osteogenic markers—namely, runt-related transcription factor 2, alkaline phosphatase, osteopontin, and osteocalcin—which are similar to the effects of the Bio-Gide membrane. Further, in in vivo studies, the results of sequential fluorescent labeling, micro-computed tomography, and histological analysis suggest that using the PLGA/ATT scaffolds for repairing V-shaped buccal dehiscence on a dog’s tooth root improved bone regeneration, which is not only similar to the result obtained using the Bio-Gide membrane but also much better than that obtained using PLGA scaffolds and the negative control.ConclusionTo achieve satisfactory therapeutic results and to lower the cost of GBR treatment, this study provided a promising alternative material of bio-degradable membrane in clinical treatment.

EphrinB2 overexpression enhances osteogenic differentiation of dental pulp stem cells partially through ephrinB2-mediated reverse signaling

BackgroundAlveolar bone loss is a frequent occurrence. Dental pulp stem cells (DPSCs) which have invasive accessibility and high osteogenic potential is a promising source for cell-based bone regeneration. EphrinB2 is involved in bone homeostasis and osteogenesis. The aim of this study was to investigate the effect and mechanism of ephrinB2 overexpression on osteogenic differentiation of DPSCs and bone defect repair.MethodsEphrinB2 expression was analyzed during osteogenic induction of human DPSCs (hDPSCs). Endogenous ephrinB2 expression in hDPSCs was then upregulated using EfnB2 lentiviral vectors. The effect of ephrinB2 overexpression on osteogenic differentiation capacity of hDPSCs was investigated in vitro, and activation of ephrinB2-EphB4 bidirectional signaling in ephrinB2-overexpressing hDPSCs was detected. In vivo, a canine alveolar bone defect model was established and canine DPSCs (cDPSCs) were cultured, characterized, EfnB2-tranfected, and combined with a PuraMatrix scaffold. Micro-CT analysis was performed to evaluate the therapeutic effect of ephrinB2-overexpressing cDPSCs on bone defect repair.ResultsEphrinB2 was upregulated after osteogenic induction of hDPSCs. EphrinB2 overexpression enhanced osteogenic differentiation capacity of hDPSCs in vitro. Moreover, p-ephrinB2 instead of p-EphB4 was upregulated by ephrinB2 overexpression, and activation of ephrinB2-mediated reverse signaling promoted osteogenic differentiation of hDPSCs. In a canine bone defect model, ephrinB2 overexpression in cDPSCs significantly improved trabecular bone volume per tissue volume (BV/TV) and trabecular thickness, as demonstrated by radiographic analysis.ConclusionsEphrinB2 overexpression enhanced osteogenic potential of DPSCs partially via upregulation of ephrinB2-mediated reverse signaling and effectively promoted alveolar bone defect repair.

Investigating the repair of alveolar bone defects by gelatin methacrylate hydrogels-encapsulated human periodontal ligament stem cells.

Although various efforts have been made to develop effective treatments for alveolar bone defect, alveolar regeneration has been emerging as the one with the most potential Herein, we investigated the potential of gelatin methacrylate (GelMA) hydrogels-encapsulated human periodontal ligament stem cells (hPDLSCs) to regenerate alveolar bone. The easy, rapid, and cost-effective nature of GelMA hydrogels makes them a promising mode of stem cell-delivery for clinically relevant alveolar bone regeneration. More importantly, GelMA hydrogels provide an optimal niche for hPDLSCs proliferation, migration and osteogenic differentiation, which are critical for alveolar bone regeneration. In this study, we examined the microstructure of GelMA hydrogels, and identified a highly porous and interconnected network. Compressive test of GelMA hydrogels showed that the stress reached a maximum value of 13.67 ± 0.03 kPa when the strain reached 55%. The maximum values of swelling ratio were 700 ± 47% at the fifth hour. The proliferation rate of hPDLSCs in the GelMA hydrogels resembled that in 2D culture and gradually increased. We established a critical-sized rat model of alveolar bone defects, and applied Micro-CT to assess new bone formation. Compared to the control group, there was substantial bone regeneration in the GelMA + hPDLSCs group at both 4 and 8 weeks after the operation. Histological analysis results were consistent with Micro-CT results. Our study demonstrates that the GelMA hydrogels-encapsulated hPDLSCs have a significant alveolar regenerative potential, and may represent a new strategy for the therapy of alveolar bone defects.

Study of the timing of tooth movement after repair of alveolar bone defects by rabbit BMSCs combined with beta-TCP

To investigate the effect of tooth movement at different time after repair of alveolar bone defects with tissue engineering bone constructed by rabbit bone marrow mesenchymal stem cells (BMSCs) and beta-tricalcium phosphate (β-TCP). Alveolar bone defect (6 mm×4 mm×8 mm) was made on the right side of 40 New Zealand rabbits, which was filled with tissue engineering bone constructed by BMSCs and β-TCP as experimental sides. Tooth extraction on the other side was performed as control. The mandibular second molars in both sides were moved mesially 2, 4, 8 and 12 weeks after surgery. The specimens were taken 4 weeks after exertion. The distance of mandibular second molar moved was measured. The periodontal tissues were observed after H-E staining. TRAP staining was performed and osteoclasts were counted in the periodontal tissues on the pressure side. BMP-2 immunohistochemical staining was used to observe the average optical density of periodontal tissue on tension side. The results were analyzed with SAS 8.0 software package. The distance of tooth movement, the amount of TRAP positive cells and the optical density of BMP-2 in the experimental side of the 2-week and 4-week groups were all significantly lower than those in the control side, while there was no significant difference between the 8-week and 12-week groups. Eight weeks after repair of alveolar bone defect with rabbit BMSCs and β-TCP is an appropriate time for orthodontic tooth movement.

Exosomes Secreted by Stem Cells from Human Exfoliated Deciduous Teeth Promote Alveolar Bone Defect Repair through the Regulation of Angiogenesis and Osteogenesis

Exosomes are important mediators of intercellular communication and have a vital part in the diagnosis and treatment of various diseases in humans. Here, we investigated the benefits and underlying mechanism of exosomes secreted via stem cells from human exfoliated deciduous teeth (SHED-derived exosomes) in promoting alveolar bone regeneration, thus providing new insights into exosome-based therapy for periodontitis. SHED-derived exosomes were isolated by ultracentrifugation. The impacts of SHED-derived exosomes on the angiogenic ability of human umbilical vein endothelial cells (HUVECs) and the osteogenic capability of rat bone marrow mesenchymal stem cells (BMSCs) were evaluated in vitro. Compound C, a pharmacological blocker of adenosine monophosphate-activated protein kinase (AMPK), was used to examine the role of the AMPK signaling cascade in these processes. Periodontal defect rat models were established and treated with PBS, β-tricalcium phosphate (β-TCP), or a grouping of exosomes/β-TCP. Microcomputed tomography (micro-CT) scanning, hematoxylin and eosin (HE) staining, Masson staining, and immunofluorescence staining were done to inspect the impacts of the exosomes/β-TCP combination on periodontal bone regeneration. Our outcomes indicated that the expression of angiogenesis-related genes (KDR, SDF-1, and FGF2), osteogenesis-related genes (COL1, RUNX2, and OPN), and phosphorylated (p)-AMPK were upregulated after treatment with exosomes, while the positive impacts of SHED-derived exosomes on HUVECs and BMSCs were partially reversed by compound C. Micro-CT analysis demonstrated that the exosomes/β-TCP group exhibited better bone regeneration than either the β-TCP group or the control group. Additionally, the results of HE and Masson staining as well as immunofluorescence staining showed neovascularization and new bone formation in the exosomes/β-TCP group but only limited new bone formation in the other two groups. Thus, SHED-derived exosomes contribute to periodontal bone regeneration by promoting neovascularization and new bone formation, possibly through the AMPK signaling pathway.

COL4A2 in the tissue-specific extracellular matrix plays important role on osteogenic differentiation of periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) can repair alveolar bone defects in periodontitis in a microenvironment context-dependent manner. This study aimed to determine whether different extracellular matrices (ECMs) exert diverse effects on osteogenic differentiation of PDLSCs and accurately control alveolar bone defect repair.Methods: The characteristics of PDLSCs and bone marrow mesenchymal stem cells (BMSCs) with respect to surface markers and multi-differentiation ability were determined. Then, we prepared periodontal ligament cells (PDLCs)-derived and bone marrow cells (BMCs)-derived ECMs (P-ECM and B-ECM) and the related decellularized ECMs (dECMs). Transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and protein mass spectrometry were used to distinguish the ECMs. The expression of Type IV collagen A2 (COL4A2) in the ECMs was inhibited by siRNA or activated by lentiviral transduction of relevant cells. The stemness, proliferation, and differentiation of PDLSCs were determined in vitro in different dECMs. For the in vivo analysis, different dECMs under the regulation of COL4A2 mixed with PDLSCs and Bio-Oss bone powder were subcutaneously implanted into immunocompromised mice or in defects in rat alveolar bone. The repair effects were identified by histological or immunohistochemical staining and micro-CT.Results: B-dECM exhibited more compact fibers than P-dECM, as revealed by TEM, SEM, and AFM. Protein mass spectrometry showed that COL4A2 was significantly increased in B-dECM compared with P-dECM. PDLSCs displayed stronger proliferation, stemness, and osteogenic differentiation ability when cultured on B-dECM than P-dECM. Interestingly, B-dECM enhanced the osteogenic differentiation of PDLSCs to a greater extent than P-dECM both in vitro and in vivo, whereas downregulation of COL4A2 in B-dECM showed the opposite results. Furthermore, the classical Wnt/β-catenin pathway was found to play an important role in the negative regulation of osteogenesis through COL4A2, confirmed by experiments with the Wnt inhibitor DKK-1 and the Wnt activator Wnt3a.Conclusion: These findings indicate that COL4A2 in the ECM promotes osteogenic differentiation of PDLSCs through negative regulation of the Wnt/β-catenin pathway, which can be used as a potential therapeutic strategy to repair bone defects.

Experiment of GBR for repair of peri-implant alveolar defects in beagle dogs

To guide barrier membrane choice in the treatment of peri-implant alveolar bone defects, we evaluated guided bone regeneration (GBR) using titanium (Ti) mesh or Bio-Gide membrane, independently or in combination, for repair of alveolar bone defects in Beagle dogs. Six months after extraction of the mandibular premolars and first molars from three beagle dogs, we inserted implants assigned into 3 groups and covered with the following membrane combinations: Group A: Implant + Bio-Oss + Ti-mesh, Group B: Implant + Bio-Oss + Bio-Gide, and Group C: Implant + Bio-Oss + Ti-mesh + Bio-Gide. At 6 months, micro-CT revealed that bone volume/total volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) was significantly greater in Group C than the other two groups, while trabecular separation (Tb.Sp) was significantly lower, suggesting improved bone regeneration. The distance between bands of three fluorescent tracking dyes was significantly greater in Group C, indicating faster deposition of new bone. The Bio-Oss particles were ideally integrated with newly deposited bone and bone thickness was significantly larger in Group C. These findings suggest that combination of Bio-Gide membrane and titanium mesh can effectively repair peri-implant alveolar bone defects, achieving enhanced bone regeneration compared to titanium mesh or Bio-Gide alone, and therefore providing a novel treatment concept for clinical implant surgery.

Custom Repair of Mandibular Bone Defects with 3D Printed Bioceramic Scaffolds

Implanting artificial biomaterial implants into alveolar bone defects with individual shape and appropriate mechanical strength is still a challenge. In this study, bioceramic scaffolds, which can precisely match the mandibular defects in macro and micro, were manufactured by the 3-dimensional (3D) printing technique according to the computed tomography (CT) image. To evaluate the stimulatory effect of the material substrate on bone tissue regeneration in situ in a rabbit mandibular alveolar bone defect model, implants made with the newly developed, mechanically strong ~10% Mg-substituted wollastonite (Ca90%Mg10%SiO3; CSi-Mg10) were fabricated, implanted into the bone defects, and compared with implants made with the typical Ca-phosphate and Ca-silicate porous bioceramics, such as β-tricalcium phosphate (TCP), wollastonite (CaSiO3; CSi), and bredigite (Bred). The initial physicochemical tests indicated that although the CSi-Mg10 scaffolds had the largest pore dimension, they had the lowest porosity mainly due to the significant linear shrinkage of the scaffolds during sintering. Compared with the sparingly dissolvable TCP scaffolds (~2% weight loss) and superfast dissolvable (in Tris buffer within 6 wk) pure CSi and Bred scaffolds (~12% and ~14% weight loss, respectively), the CSi-Mg10 exhibited a mild in vitro biodissolution and moderate weight loss of ~7%. In addition, the CSi-Mg10 scaffolds showed a considerable initial flexural strength (31 MPa) and maintained very high flexural resistance during soaking in Tris buffer. The in vivo results revealed that the CSi-Mg10 scaffolds have markedly higher osteogenic capability than those on the TCP, CSi, and Bred scaffolds after 16 wk. These results suggest a promising potential application of customized CSi-Mg10 3D robocast scaffolds in the clinic, especially for repair of alveolar bone defects.