Rat Cranial Defect Research Articles

Biomimetic Hydroxyapatite on 3D-Printed Nanoattapulgite/Polycaprolactone Scaffolds for Bone Regeneration of Rat Cranium Defects.

Nanoattapulgite (nano-ATP), a magnesium-aluminum silicate clay, can absorb substances and is a suitable material for bone repair and regeneration. In this study, using three-dimensional printing technology, a nano-ATP/polycaprolactone (PCL) scaffold was fabricated and modified using NaOH to form a rough surface. Biomimetic hydroxyapatite (HA) on nano-ATP/PCL scaffolds was fabricated using a biomineralized approach. The scaffold provided structural support through PCL and was modified with ATP and HA to improve hydrophilicity and promote the delivery of nutrients. The biocompatibility and osteogenic induction of scaffolds were assessed in vitro using mouse bone marrow mesenchymal stem cells. According to the in vitro study results, the nano-ATP/PCL/HA composite scaffold significantly boosted the expression levels of genes related to osteogenesis (p < 0.05), attributed to its superior alkaline phosphatase activity and calcium deposition capabilities. The outcomes of in vivo experimentation demonstrated an augmentation in bone growth at the rat cranial defect site when treated with the ATP/PCL/HA composite scaffold. It can be inferred from the results that the implementation of ATP and HA for the bone tissue engineering repair material displays encouraging prospects.

A Drug‐Mineralized Hydrogel Orchestrated by Spontaneous Dynamic Mineralization

AbstractFabrication of mineralized materials through the nature‐mimicking dynamic biomineralization process has attracted great interest but remains a huge challenge till now. Here, a drug‐mineralized hydrogel (DMH) with spontaneous dynamic mineralization behaviors is reported, which highly mimics the dynamic mineralization process of natural tissues. The hydrogel is facilely fabricated via directly mixing sodium hyaluronate (HA) with alendronate sodium (ADA) and CaCl2 in deionized water. The resultant hydrogel possesses a white‐to‐semi‐transparent transition and a soft‐to‐hard transition during incubation at 37 °C. Mechanism studies reveal that the spontaneous dynamic change of the hydrogel is ascribed to the HA‐regulated dynamic mineralization of ADA/Ca2+ complexes, where amorphous ADA/Ca2+ complexes will gradually transform into mineral crystals with the prolongation of incubation time. Owing to the dynamic transformation process, the DMH shows excellent injectability and moldability at the initial stage. More importantly, the DMH exhibits a sustained release behavior of ADA in vitro, superior adhesiveness with bone, biodegradability, and good biocompatibility. As a result, the developed DMH demonstrates promising uses for accelerating bone repair in a rat cranial defect model. Overall, the research provides a general strategy to prepare drug‐mineralized hydrogels with spontaneous dynamic mineralization behaviors, which may find potential uses in various biomedical fields.

In Vitro and In Vivo Evaluation of 3D Printed Poly(Ethylene Glycol) Dimethacrylate-Based Photocurable Hydrogel Platform for Bone Tissue Engineering.

This study focuses to develop a unique hybrid hydrogel bioink formulation that incorporates poly(ethylene glycol) dimethacrylate (PEGDMA), gelatin (Gel), and methylcellulose (MC). This formulation achieves the necessary viscosity for extrusion-based three-dimensional (3D) printing of scaffolds intended for bone regeneration. After thorough optimization of the hybrid bioink system with Gel, three distinct scaffold groups are investigated in vitro: 0%, 3%, and 6% (w/v) Gel. These scaffold groups are examined for their morphology, mechanical strength, biodegradation, in vitro cell proliferation and differentiation, and in vivo bone formation using a rat cranial defect model. Among these scaffold compositions, the 3% Gel scaffold exhibits the most favorable characteristics, prompting further evaluation as a rat mesenchymal stem cell (rMSC) carrier in a critical-size cranial defect within a Lewis rat model. The compressive strength of all three scaffold groups range between 1 and 2MPa. Notably, the inclusion of Gel in the scaffolds leads to enhanced bioactivity and cell adhesion. The Gel-containing scaffolds notably amplify osteogenic differentiation, as evidenced by alkaline phosphatase (ALP) and Western blot analyses. The in vivo results, as depicted by microcomputed tomography, showcase augmented osteogenesis within cell-seeded scaffolds, thus validating this innovative PEGDMA-based scaffold system as a promising candidate for cranial bone defect healing.

Engineering an injectable gellan gum-based hydrogel with osteogenesis and angiogenesis for bone regeneration

Injectable hydrogels are currently a topic of great interest in bone tissue engineering, which could fill irregular bone defects in a short time and avoid traditional major surgery. Herein, we developed an injectable gellan gum (GG)-based hydrogel for bone defect repair by blending nano-hydroxyapatite (nHA) and magnesium sulfate (MgSO4). In order to acquire an injectable GG-based hydrogel with superior osteogenesis, nHA were blended into GG solution with an optimized proportion. For the aim of endowing this hydrogel capable of angiogenesis, MgSO4 was also incorporated. Physicochemical evaluation revealed that GG-based hydrogel containing 5% nHA (w/v) and 2.5 mM MgSO4 (GG/5%nHA/MgSO4) had appropriate sol-gel transition time, showed a porosity-like structure, and could release magnesium ions for at least 14 days. Rheological studies showed that the GG/5%nHA/MgSO4 hydrogel had a stable structure and repeatable self-healing properties. In-vitro results determined that GG/5%nHA/MgSO4 hydrogel presented superior ability on stimulating bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteogenic linage and human umbilical vein endothelial cells (HUVECs) to generate vascularization. In-vivo, GG/5%nHA/MgSO4 hydrogel was evaluated via a rat cranial defect model, as shown by better new bone formation and more neovascularization invasion. Therefore, the study demonstrated that the new injectable hydrogel, is a favorable bioactive GG-based hydrogel, and provides potential strategies for robust therapeutic interventions to improve the repair of bone defect.

A novel adhesive dual-sensitive hydrogel for sustained release of exosomes derived from M2 macrophages promotes repair of bone defects

The repair of bone defects remains a huge clinical challenge. M2 macrophage-derived exosomes (M2-Exos) can act as immunomodulators to promote fracture healing; however, how to retain the sustained release of exosomes to the target area remains a challenge. Here, we report a composite hydrogel loaded with M2-Exos aiming to accelerate bone defect healing. It was verified that the F127/HA-NB hydrogel had a dense network structure, tissue adhesiveness, and dual sensitivity to temperature and light. F127/HA-NB loaded with M2-Exos (M2-Exos@F127/HA-NB) exhibited good biocompatibility and achieved sustained release of exosomes for up to two weeks. The study showed that both M0-Exos and M2-Exos@F127/HA-NB significantly promoted osteogenic differentiation of rat bone marrow mesenchymal stem cells. The mechanism study implied that M2-Exos activates the Wnt/β-catenin signaling pathway to promote osteogenic differentiation of BMSCs. Finally, we evaluated the osteogenetic effects of M2-Exos@F127/HA-NB in a rat cranial defect model, and the results showed that M2-Exos@F127/HA-NB had superior bone regeneration-promoting effects. This study provides a new strategy for cell-free treatment of bone defects.

A biomimetic in situ mineralization ECM composite scaffold to promote endogenous bone regeneration

Bone tissue engineering scaffolds constructed from single-component organic materials have inherent limitations. Inspired by the hierarchical structure of physiological natural bone hard tissues, our research explores the construction of organic-inorganic composite scaffold for bone regeneration. In this study, we used a natural and readily obtainable extracellular matrix (ECM) material, i.e., decellularized small intestinal submucosa (SIS), to build the organic component of a phosphorylated hydroxyapatite nanocrystal-containing composite scaffold (nHA@SIS). Guided by polymer-induced liquid-precursor theory, we introduced a soluble inorganic mineralization solution to achieve an inorganic component of nHA@SIS. Using in situ mineralization, we successfully formed inorganic component within SIS and constructed nHA@SIS composite scaffold. We analyzed the physicochemical properties and the osteogenic role of nHA@SIS via a series of in vitro and in vivo studies. Compared with SIS scaffold, the nHA@SIS possessed suitable physicochemical properties, maintained the excellent cell activity of SIS and better guided reorganization of the cell skeleton, thereby achieving superior osteoconductivity and maintaining osteoinductivity at the protein and gene levels. Furthermore, the rat cranial defect area in the nHA@SIS scaffold group was mostly repaired after 12 weeks of implantation, with a larger amount of higher-density new bone tissue being visible at the edge and center than SIS and blank control group. This significantly improved in vivo osteogenic ability indicated the great potential of nHA@SIS for bone tissue engineering applications.

Injectable periodontal ligament stem cell-metformin-calcium phosphate scaffold for bone regeneration and vascularization in rats.

Injectable and self-setting calcium phosphate cement scaffold (CPC) capable of encapsulating and delivering stem cells and bioactive agents would be highly beneficial for dental and craniofacial repairs. The objectives of this study were to: (1) develop a novel injectable CPC scaffold encapsulating human periodontal ligament stem cells (hPDLSCs) and metformin (Met) for bone engineering; (2) test bone regeneration efficacy in vitro and in vivo. hPDLSCs were encapsulated in degradable alginate fibers, which were then mixed into CPC paste. Five groups were tested: (1) CPC control; (2) CPC +hPDLSC-fibers +0% Met (CPC + hPDLSCs + 0%Met); (3) CPC +hPDLSC-fibers +0.1% Met (CPC + hPDLSCs + 0.1%Met); (4) CPC +hPDLSC-fibers +0.2% Met (CPC + hPDLSCs + 0.2%Met); (5) CPC +hPDLSC-fibers +0.4% Met (CPC + hPDLSCs + 0.4%Met). The injectability, mechanical properties, metformin release, and hPDLSC osteogenic differentiation and bone mineral were determined in vitro. A rat cranial defect model was used to evaluate new bone formation. The novel construct had good injectability and physical properties. Alginate fibers degraded in 7 days and released hPDLSCs, with 5-fold increase of proliferation (p＜0.05). The ALP activity and mineral synthesis of hPDLSCs were increased by Met delivery (p＜0.05). Among all groups, CPC+hPDLSCs+0.1%Met showed the greatest cell mineralization and osteogenesis, which were 1.5-10folds those without Met (p＜0.05). Compared to CPC control, CPC+hPDLSCs+0.1%Met enhanced bone regeneration in rats by 9 folds, and increased vascularization by 3 folds (p＜0.05). The novel injectable construct with hPDLSC and Met encapsulation demonstrated excellent efficacy for bone regeneration and vascularization in vivo in an animal model. CPC+hPDLSCs+0.1%Met is highly promising for dental and craniofacial applications.

Active components and mechanisms of total flavonoids from Rhizoma Drynariae in enhancing cranial bone regeneration: An investigation employing serum pharmacochemistry and network pharmacology approaches

Ethnopharmacological relevanceRhizoma Drynariae, as the dried rhizome of Drynaria fortunei (Kunze ex Mett.) J. Sm., is a traditional Chinese medicine for treating the injury and bone broken of falling and beating. Total flavonoids is considered as the major and effective compounds for the therapeutic efficacy of Rhizoma Drynariae. Aim of the studyTo explore the effect of total flavonoids from Rhizoma Drynariae (TFRD) on bone regeneration and the underlying mechanisms. Materials and methodsThe effect of TFRD in various doses on bone reconstruction in cranial bone defect rats was explored in vivo. The active ingredients in TFRD-medicated serum were characterized by serum pharmacochemistry and integrated by network pharmacology analysis and target prediction. To elucidate the underlying mechanism of TFRD on bone regeneration, experimental validation in vitro was executed to assess the influence of different concentrations of TFRD-medicated serum on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). ResultsMicro-CT, histological examination, immunohistochemical analysis, and ELSA demonstrated that administration of TFRD could promote bone reconstruction in a rat cranial defect model. We identified 27 active components of TFRD using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Results from CCK8, ALP, and Alizarin Red S staining revealed that TFRD-medicated serum notably enhanced BMSCs proliferation and osteogenic differentiation. qRT-PCR and Western blot harvested results consistent with those predicted by network pharmacology, providing further evidence that TFRD activated the TGF-β signaling pathway to benefit bone regeneration. ConclusionThe active components of TFRD modulate the TGF-β signaling pathway to facilitate osteogenesis, thereby repairing cranial bone defects.

Calvaria defect regeneration via human periodontal ligament stem cells and prevascularized scaffolds in athymic rats

BackgroundVascularization plays an important role in dental and craniofacial regenerations. Human periodontal ligament stem cells (hPDLSCs) are a promising cell source and, when co-cultured with human umbilical vein endothelial cells (hUVECs), could promote vascularization. The objectives of this study were to develop a novel prevascularized hPDLSC-hUVEC-calcium phosphate construct, and investigate the osteogenic and angiogenic efficacy of this construct with human platelet lysate (hPL) in cranial defects in rats for the first time. MethodshPDLSCs and hUVECs were co-cultured on calcium phosphate cement (CPC) scaffolds with hPL. Cell proliferation, angiogenic gene expression, angiogenesis, alkaline phosphatase activity, and cell-synthesized minerals were determined. Bone and vascular regenerations were investigated in rat critical-sized cranial defects in vivo. ResultshPDLSC-hUVEC-CPC-hPL group had 2-fold greater angiogenic expressions and cell-synthesized mineral synthesis than hPDLSC-hUVEC-CPC group (p < 0.05). Microcapillary-like structures were formed on scaffolds in vitro. hPDLSC-hUVEC-CPC-hPL group had more vessels than hPDLSC-hUVEC-CPC group (p < 0.05). In cranial defects in rats, hPDLSC-hUVEC-CPC-hPL group regenerated new bone amount that was 2.1 folds and 4.0 folds, respectively, that of hPDLSC-hUVEC-CPC group and CPC control (p < 0.05). New blood vessel density of hPDLSC-hUVEC-CPC-hPL group was 2 folds and 7.9 folds, respectively, that of hPDLSC-hUVEC-CPC group and CPC control (p < 0.05). ConclusionThe hPL pre-culture method is promising to enhance bone regeneration via prevascularized CPC. Novel hPDLSC-hUVEC-CPC-hPL prevascularized construct increased new bone formation and blood vessel density by 4–8 folds over CPC control. Clinical significanceNovel hPDLSC-hUVEC-hPL-CPC prevascularized construct greatly increased bone and vascular regeneration in vivo and hence is promising for a wide range of craniofacial applications.

Development of Gelatin Methacryloyl/Sodium Alginate Interpenetrating Polymer Network Hydrogels for Bone Regeneration by Activating the Wnt/β-Catenin Signaling Pathway via Lithium Release.

Hydrogels have gained significant attention as biomaterials due to their remarkable properties resembling those of the extracellular matrix (ECM). In the present investigation, we successfully synthesized interpenetrating polymer network (IPN) hydrogels using gelatin methacryloyl (GelMA) and sodium alginate (SA), incorporating various concentrations of lithium chloride (LiCl; 0, 5, and 10 mM), aiming to develop a hydrogel scaffold for bone regeneration. Notably, the compressive modulus of the IPN hydrogels remained largely unaffected upon the inclusion of LiCl. However, the hydrogel with the high concentration of LiCl exhibited reduced fragmentation after compression testing. Intriguingly, we observed a significant improvement in cellular biocompatibility, primarily attributed to activation of the Wnt/β-catenin signaling pathway induced by LiCl. Subsequently, we evaluated the efficacy of the newly developed IPN-Li hydrogels in a rat cranial defect model and found that they substantially enhanced bone regeneration. Nevertheless, it is important to note that the introduction of high concentrations of LiCl did not significantly promote osteogenesis. This outcome can be attributed to the excessive release of Li+ ions into the extracellular matrix, hindering the desired effect. Overall, the IPN-Li hydrogel developed in this study holds great promise as a biodegradable material for bone regeneration applications.

VEGF-modified PLA/HA nanocomposite fibrous membrane for cranial defect repair in rats.

A major obstacle to bone tissue repair is the difficulty in establishing a rapid blood supply areas of bone defects. Vascular endothelial growth factor (VEGF)-infused tissue-engineered scaffolds offer a possible therapeutic option for these types of injuries. Their role is to accelerate angiogenesis and improve bone healing. In this study, we used electrostatic spinning and biofactor binding to construct polylactic acid (PLA)/hydroxyapatite (HA)-VEGF scaffold materials and clarify their pro-vascular role in bone defect areas for efficient bone defect repair. PLA/HA nanocomposite fibrous membranes were manufactured by selecting suitable electrostatic spinning parameters. Heparin and VEGF were bound sequentially, and then the VEGF binding and release curves of the fiber membranes were calculated. A rat cranial defect model was constructed, and PLA/HA fiber membranes bound with VEGF and unbound with VEGF were placed for treatment. Finally, we compared bone volume recovery and vascular recovery in different fibrous membrane sites. A VEGF concentration of 2.5µg/mL achieved the maximum binding and uniform distribution of PLA/HA fibrous membranes. Extended-release experiments showed that VEGF release essentially peaked at 14days. In vivo studies showed that PLA/HA fibrous membranes bound with VEGF significantly increased the number of vessels at the site of cranial defects, bone mineral density, bone mineral content, bone bulk density, trabecular separation, trabecular thickness, and the number of trabeculae at the site of defects in rats compared with PLA/HA fibrous membranes not bound with VEGF. VEGF-bound PLA/HA fibrous membranes demonstrate the slow release of VEGF. The VEGF binding process does not disrupt the morphology and structure of the fibrous membranes. The fibrous membranes could stimulate both osteogenesis and angiogenesis. Taken together, this research provides a new strategy for critical-sized bone defects repairing.

Amyloid Fibril and Clay Nanosheet Dual-Nanoengineered DNA Dynamic Hydrogel for Vascularized Bone Regeneration.

Dynamic hydrogels have attracted enormous interest for bone tissue engineering as they demonstrate reversible mechanics to better mimic biophysical cues of natural extracellular matrix (ECM) compared to traditional static hydrogels. However, the facile development of therapeutic dynamic hydrogels that simultaneously recapitulate the filamentous architecture of the ECM of living tissues and induce both osteogenesis and angiogenesis to augment vascularized bone regeneration remains challenging. Herein, we report a dual nanoengineered DNA dynamic hydrogel developed through the supramolecular coassembly of amyloid fibrils and clay nanosheets with DNA strands. The nanoengineered ECM-like fibrillar hydrogel network is facilely formed without a complicated and tedious molecular synthesis. Amyloid fibrils together with clay nanosheets synergistically enhance the mechanical strength and stability of the dynamic hydrogel and, more remarkably, endow the matrix with an array of tunable features, including shear-thinning, injectability, self-healing, self-supporting, and 3D printable properties. The QK peptide is further chemically grafted onto amyloid fibrils, and its sustainable release from the hydrogel matrix stimulates the tube formation and migration with human umbilical vein endothelial cells. Meanwhile, the nanoengineered hydrogel matrix promotes osteogenic differentiation of bone marrow mesenchymal stem cells due to the sustainable release of Si4+ and Mg2+ derived from clay nanosheets. Furthermore, the manipulation of enhanced vascularized bone regeneration by the dynamic hydrogel is revealed in a rat cranial bone defect model. This dual nanoengineered strategy envisions great promise in developing therapeutic dynamic hydrogels for improved and customizable bone regeneration.

Optimized fabrication of DLP-based 3D printing calcium phosphate ceramics with high-precision and low-defect to induce calvarial defect regeneration

Acquiring high-performance calcium phosphate (CaP) ceramics via 3D printing is critical for their applications in bone regenerative repair. In current study, a complete process study of 3D printed CaP ceramics based on digital light processing (DLP) technology was performed to get the ideal implant for the regenerative repair of calvarial defects. The smaller particle size of initial powder would be more favorable to getting slurry with high solid content and low viscosity. CaP green body with high precision and high curing rate could be fabricated at the exposure energy density and exposure time of 6.20 mJ cm−2 and 2 s, respectively. Moreover, the post-curing treatment could eliminate the interlayer cracks and increase the curing rate from 69.78% to 93.91% of green body. After that, the mechanical strength of CaP ceramics increased by 46.34%. In-vitro cellular experiments showed that 3D printed CaP ceramics had good biocompatibility, and could promote the osteoblastic differentiation of BMSCs. In-vivo rat cranial defect implantation revealed 3D printed CaP ceramics exhibited better osteogenic ability than the commercial ones (BAM®), reaching the close level with the autografts. Overall, this study could provide a closed-loop solution for 3D printed CaP ceramics with high-precision and low-defect to induce bone regeneration.

Human nasal mucosa ectomesenchymal stem cells derived extracellular vesicles loaded omentum/chitosan composite scaffolds enhance skull defects regeneration

Engineered bone tissue that can promote osteogenic differentiation is considered an ideal substitute for materials to heal bone defects. Extracellular vesicle (EV)-based cell-free regenerative therapies represent an emerging promising alternative for bone tissue engineering. We hypothesized that EVs derived from human nasal mucosa-derived ectomesenchymal stem cells (hEMSCs) can promote bone tissue regeneration. Herein, hEMSCs were cultured with osteogenic induction medium or normal medium to generate two types of EVs. We first demonstrated that the two EVs exhibited strong potential to promote rat suture mesenchymal stem cell (SMSC) osteogenesis by transferring TG2 to SMSCs and regulating extracellular matrix (ECM) synthesis. Next, we developed a composite hydrogel made of porcine omentum and chitosan into which EVs were adsorbed to enable the effective delivery of EVs with sustained release kinetics. Implantation of the EV-loaded hydrogels in a critical-size rat cranial defect model significantly promoted bone regeneration. Therefore, we suggest that our hEMSC-derived EV-loading system can serve as a new therapeutic paradigm for promoting bone tissue regeneration in the clinic.

Seamlessly Adhesive Bionic Periosteum Patches Via Filling Microcracks for Defective Bone Healing.

Current artificial designs of the periosteum focus on osteogenic or angiogenic properties, while ignoring the filling and integration with bone microcracks, which trigger a prolonged excessive inflammatory reaction and lead to failure of bone regeneration. In this study, seamless adhesive biomimetic periosteum patches (HABP/Sr-PLA) were prepared to fill microcracks in defective bone via interfacial self-assembly induced by Sr ions mediated metal-ligand interactions among pamidronate disodium-modified hyaluronic acid (HAPD), black phosphorus (BP), and hydrophilic polylactic acid (PLA). In vitro, HABP/Sr-PLA exhibited excellent self-healing properties, seamlessly filled bone microcracks, and significantly enhanced osteogenesis and angiogenesis. Furthermore, in a rat cranial defect model, HABP/Sr-PLA was demonstrated to significantly promote the formation of blood vessels and new bone under mild 808 nm photothermal stimulation (42.8 °C), and the highest protein expression of CD31 and OPN was five times higher than that of the control group and other groups. Therefore, the proposed seamless microcrack-filled bionic periosteum patch is a promising clinical strategy for promoting bone repair.

Feather keratin-montmorillonite nanocomposite hydrogel promotes bone regeneration by stimulating the osteogenic differentiation of endogenous stem cells

Bone defects caused by bone trauma, infection, surgery, or other systemic diseases remain a severe challenge for the medical field. To address this clinical problem, different hydrogels were exploited to promote bone tissue regrowth and regeneration. Keratins are natural fibrous proteins found in wool, hair, horns, nails, and feather. Due to their unique characteristics of outstanding biocompatibility, great biodegradability, and hydrophilic, keratins have been widely applicated in different fields. In our study, the feather keratin-montmorillonite nanocomposite hydrogels that consist of keratin hydrogels serving as the scaffold support to accommodate endogenous stem cells and montmorillonite is synthesized. The introduction of montmorillonite greatly improves the osteogenic effect of the keratin hydrogels via bone morphogenetic protein 2 (BMP-2)/phosphorylated small mothers against decapentaplegic homolog 1/5/8 (p-SMAD 1/5/8)/runt-related transcription factor 2 (RUNX2) expression. Moreover, the incorporation of montmorillonite into hydrogels can improve the mechanical properties and bioactivity of the hydrogels. The morphology of feather keratin-montmorillonite nanocomposite hydrogels was shown by scanning electron microscopy (SEM) to have an interconnected porous structure. The incorporation of montmorillonite into the keratin hydrogels was confirmed by the energy dispersive spectrum (EDS). We prove that the feather keratin-montmorillonite nanocomposite hydrogels enhance the osteogenic differentiation of BMSCs. Furthermore, micro-CT and histological analysis of rat cranial bone defect demonstrated that feather keratin-montmorillonite nanocomposite hydrogels dramatically stimulated bone regeneration in vivo. Collectively, feather keratin-montmorillonite nanocomposite hydrogels can regulate BMP/SMAD signaling pathway to stimulate osteogenic differentiation of endogenous stem cells and promote bone defect healing, indicating their promising candidate in bone tissue engineering.

Bio-inspired Glycosylated Nano-hydroxyapatites Enhance Endogenous Bone Regeneration by Modulating Macrophage M2 Polarization.

A macrophage-associated immune response is vital in bone regeneration. Mannose receptor (MR), a macrophage pattern-recognition receptor, is crucial for the maintenance of immune homeostasis. Here, we designed MR-targeted glycosylated nano-hydroxyapatites (GHANPs) to reprogram macrophages into polarized M2s, promoting bone regeneration by improving the osteoimmune microenvironment. The prepared GHANPs induced macrophage M2 polarization, which then promoted osteoblastic differentiation of stem cells. Further, the mechanistic study showed that GHANPs might influence macrophage polarization by modulating cell metabolism, including enhancing mitochondrial oxidative phosphorylation and activating autophagy. Finally, a rat cranial defect model was used to verify the effect of GHANPs on endogenous bone regeneration in vivo, revealing that GHANPs promoted bone regeneration within the defect and increased the ratio of M2/M1 macrophages in early bone repair. Our results indicate that the MR-targeted macrophage M2 polarization strategy is promising in endogenous bone regeneration. STATEMENT OF SIGNIFICANCE: Macrophage is a pivotal immunity component for bone regeneration. A switch to M2 macrophage has been considered to contribute to osteogenesis. For inducing macrophage M2 polarization, an effective strategy to overcome off-target effects and insufficient specificity is a critical challenge. The mannose receptor on the surface of macrophages has been involved in regulating macrophage directional polarization. The glucomannan presented on the nano-hydroxyapatite rods acts as ligands targeting macrophage mannose receptors to promote their M2 polarization, improving the immunomicroenvironment and achieving bone regeneration. This approach has the advantage of easy preparation, specific regulation, and safety.

3D-Printed Bioactive Scaffold Loaded with GW9508 Promotes Critical-Size Bone Defect Repair by Regulating Intracellular Metabolism.

The process of bone regeneration is complicated, and it is still a major clinical challenge to regenerate critical-size bone defects caused by severe trauma, infection, and tumor resection. Intracellular metabolism has been found to play an important role in the cell fate decision of skeletal progenitor cells. GW9508, a potent agonist of the free fatty acid receptors GPR40 and GPR120, appears to have a dual effect of inhibiting osteoclastogenesis and promoting osteogenesis by regulating intracellular metabolism. Hence, in this study, GW9508 was loaded on a scaffold based on biomimetic construction principles to facilitate the bone regeneration process. Through 3D printing and ion crosslinking, hybrid inorganic-organic implantation scaffolds were obtained after integrating 3D-printed β-TCP/CaSiO3 scaffolds with a Col/Alg/HA hydrogel. The 3D-printed β-TCP/CaSiO3 scaffolds had an interconnected porous structure that simulated the porous structure and mineral microenvironment of bone, and the hydrogel network shared similar physicochemical properties with the extracellular matrix. The final osteogenic complex was obtained after GW9508 was loaded into the hybrid inorganic-organic scaffold. To investigate the biological effects of the obtained osteogenic complex, in vitro studies and a rat cranial critical-size bone defect model were utilized. Metabolomics analysis was conducted to explore the preliminary mechanism. The results showed that 50 μM GW9508 facilitated osteogenic differentiation by upregulating osteogenic genes, including Alp, Runx2, Osterix, and Spp1 in vitro. The GW9508-loaded osteogenic complex enhanced osteogenic protein secretion and facilitated new bone formation in vivo. Finally, the results from metabolomics analysis suggested that GW9508 promoted stem cell differentiation and bone formation through multiple intracellular metabolism pathways, including purine and pyrimidine metabolism, amino acid metabolism, glutathione metabolism, and taurine and hypotaurine metabolism. This study provides a new approach to address the challenge of critical-size bone defects.

BMP-2@PNH-TA hydrogel with osteogenic and antibacterial properties for integrated photothermal therapy

Progress toward developing next-generation biomaterials applied in complex bone defect is expected to effectively integrate osteogenic and anti-bacteria capability. However, current biomaterials often achieve the two functions separately. Inspired by photothermal effect, exogenous light-induced thermal variations can efficiently facilitate bone regeneration and bacteria eradication. Here, a bioactive hydrogel BMP-2@PNH-TA with simultaneously osteogenic and anti-bacterial capacities is presented, which is based on the near infrared (NIR) induced photothermal effect to perform integrated photothermal therapy (PTT). In detail, photothermal effect and photothermal-controlled release of osteogenic and anti-bacterial factors through sponge-like effect could exert integrated influence on bone regeneration under complex situation. The composite hydrogel can mediate pro-osteogenesis under mild temperature and controlled release of BMP-2 and tannin in response to NIR stimulation with low power, while efficiently eradicate bacteria via controlled release of O-HACC and thermal effect with high power. The customized PTT strategy significantly shortened the duration time of bone regeneration up to almost 50% in rat cranial bone defect. It is anticipated that the concept of integrated PTT for bone regeneration will cast new insights into developing high-performance therapeutic strategy with combined biological functions.

In vitro/ in vivo evaluation of double crosslinked bone glue with different degrees

ABSTRACT Successful bone fragment fixation is a crucial factor in bone fracture healing, the fixation of crushed bone fragments could hinder bone fracture healing. Thus, ideal bone glues to effectively adhere and splice comminuted bone fragments are needed in clinical. Herein, an osteoinductive and biodegradable double cross-linked bone glue (GelMA-oDex-AMBGN) was constructed through Schiff’s base reaction between commercial GelMA (with different substitution degrees of amino groups) and Odex mixed with amine-modified mesoporous bioactive glass nanoparticles (AMBGN), followed by crosslink with blue light irradiation. The GelMA-oDex-AMBGN bone glue successfully adhered and spliced the comminuted bone fragments of isolated rat skulls. GelMA-oDex-AMBGN promoted the proliferation of 3T3 cells and enhanced the expression of osteogenic proteins Runx2 and OCN in vitro. In rat cranial critical-sized defect models, GelMA-oDex-AMBGNs with different substitution degrees significantly increased the new bone contents at the fracture defect sites and promoted bone tissue regeneration in vivo. In conclusion, the double cross-linked bone glue (GelMA-oDex-AMBGN) was successfully constructed and can induce bone regeneration. Additionally, there was no significant difference in osteogenic activity among GelMA-oDex-AMBGNs with different substitution degrees and the equal content of AMBGN.