Critical-sized Calvarial Defects Research Articles

Micro/nanometer-sized porous structure of zinc phosphate incorporated Ti(HPO4)2 hydrate bioceramic induces osteogenic gene expression and enhances osteoporotic bone regeneration

In this work, we describe the synthesis of an ideal bioceramic to which cells can easily respond that has geometry and composition that are similar to those of bone-like apatite. The successful preparation of bone-inspired titanium hydrate phosphate with an optimized weight percentage (∼1.1 wt%) of zinc in (Ti(HPO4)2/Zn3(PO4)2·nH2O) bioceramic could represent a potential regenerative framework for bone defect repair and bone void filler that would be useful for various clinical approaches. We have for the first time demonstrated the detailed physicochemical and biological characterizations of the bioceramic required to achieve a biocompatible and porous architecture that is similar to hydroxyapatite. In vitro studies have shown that the phosphate-rich titanium and zinc nanocomposite promote bone regeneration after stimulating MC3T3 − E1 and hBM-MSCs cells activity. The metallic ions and phosphate had the effects of enhancing the cell viability and osteogenic differentiation of the cells, as confirmed by the expression of bone-related markers that included ALP, Col1a1, RUNX2, OPN/Spp1, and OCN through qRT-PCR, Western blotting, and immunocytochemistry. Moreover, the bioceramic has been shown to significantly improves new bone growth in critical size calvarial defect in rat model. The findings of the present work are the first that show the bone regeneration effect of bioceramic, which could be crucial for subsidies of Ca–P-based artificial bone implant materials. Our results provide insight into the Ti(HPO4)2/Zn3(PO4)2(0.1)·nH2O bioceramic in osteogenesis which can help to establish a therapeutic strategy in bone tissue regenerative medicine.

A cell-free difunctional demineralized bone matrix scaffold enhances the recruitment and osteogenesis of mesenchymal stem cells by promoting inflammation resolution.

The dialogue between host macrophages (Mφs) and endogenous mesenchymal stem cells (MSCs) promotes M2 Mφs polarization to resolve early-stage inflammation, thereby effectively guiding in situ bone regeneration. Once inflammation is unresolved/incontrollable, it will induce the impediment of MSCs homing at bone defect site, implying the seasonable resolution of inflammation in balancing bone homeostasis. Repeatedly, evidence elucidated that specialized pro-resolving mediators (SPMs) could conduce to proper resolve inflammation and promote the repairing of bone defect. A difunctional demineralized bone matrix (DBM) scaffold co-modified by maresin 1 (MaR1) and aptamer 19S (Apt19S) was fabricated to facilitate the osteogenesis of MSCs. To confirm the osteogenesis and immunomodulatory role of the difunctional DBM scaffold, the proliferation, recruitment, and osteogenic differentiation of MSCs and the Mφs M2 subtype polarization were evaluated in vitro. Then, the DBM scaffolds were implanted into mice model with critical size calvarial defect to evaluate bone repair efficiency. Finally, the specific resolution mechanism in Mφs cultured on the difunctional DBM scaffold was further in-depth investigated. This difunctional DBM scaffold exhibited an enhanced function on the recruitment, proliferation, migration, osteogenesis of MSCs and the resolution of inflammation, finally improved bone-scaffold integration. At the same time, MaR1 modified on the difunctional DBM scaffold increased the biosynthesis of 12-lipoxygenase (12-LOX) and 12S-hydroxy-eicosatetraenoic acid (12S-HETE), and also directly stimulated lipid droplets (LDs) biogenesis in Mφs, which suggested that MaR1 regulated Mφ lipid metabolism at bone repair site. Findings based on this synergy strategy demonstrated that Mφ lipid metabolism was essential in bone homeostasis, which might provide a theoretical direction for the treatment-associated application of MaR1 in inflammatory bone disease.

New use for old drug: Local delivery of puerarin facilitates critical-size defect repair in rats by promoting angiogenesis and osteogenesis.

ObjectivesLarge bone defect repair is a challenging clinical problem due to limited self-repair ability. A well-designed bone filling product should possess the ability to induce tissue in-growth and facilitate neovascularization and new bone formation. Puerarin has been used in clinics for a long time, and recently it was found to be able to promote osteogenesis. This study aimed to investigate a puerarin-based drug/delivery combination implant for promoting large bone defect repair.MethodsPuerarin was incorporated into the poly (lactic-co-glycolic acid)/β-calcium phosphate (PLGA/TCP, PT) to form a porous PLGA/TCP/Puerarin (PTP) composite scaffold by low-temperature rapid prototyping technology. Its structural and degradation were analyzed in vitro. Then we employed a rat calvarial critical size defect model to assess the potency of the PTP scaffold. MC3T3-E1 cells and EA. hy 926 ​cells were used to investigate the underlying mechanism.ResultsPTP scaffold inherited all advantages of PT scaffold in structural, mechanical, and biodegradation, meanwhile puerarin stably and continuously released from PTP scaffold and lasted for 5 months in vitro. At 8 weeks after implantation, the PTP scaffold triggered new bone formation in the macro-pores of the scaffold and inside the scaffold accompanied by the degrading materials. The underlying mechanism revealed that the PTP scaffold induced vascular infiltration and recruit repair cells through stimulating vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) expressions to promote angiogenesis and osteogenesis.ConclusionPuerarin-enriched porous PTP scaffold was a promising local delivery system with sustained release of puerarin for facilitating defect repair through getting synergistic angiogenic and osteogenic effects.The Translational Potential of this ArticleThe PTP scaffold presents a potential drug/device combination medical implant for large bone defect repair, which also provides a new and innovative application for the “old drug” puerarin.

Functionalized 3D-Printed ST2/Gelatin Methacryloyl/Polcaprolactone Scaffolds for Enhancing Bone Regeneration with Vascularization.

Growth factors were often used to improve the bioactivity of biomaterials in order to fabricate biofunctionalized bone grafts for bone defect repair. However, supraphysiological concentrations of growth factors for improving bioactivity could lead to serious side effects, such as ectopic bone formation, radiculitis, swelling of soft tissue in the neck, etc. Therefore, safely and effectively applying growth factors in bone repair biomaterials comes to be an urgent problem that needs to be addressed. In this study, an appropriate concentration (50 ng/mL) of Wnt3a was used to pretreat the 3D-bioprinting gelatin methacryloyl(GelMA)/polycaprolactone(PCL) scaffold loaded with bone marrow stromal cell line ST2 for 24 h. This pretreatment promoted the cell proliferation, osteogenic differentiation, and mineralization of ST2 in the scaffold in vitro, and enhanced angiogenesis and osteogenesis after being implanted in critical-sized mouse calvarial defects. On the contrary, the inhibition of Wnt/β-catenin signaling in ST2 cells reduced the bone repair effect of this scaffold. These results suggested that ST2/GelMA/PCL scaffolds pretreated with an appropriate concentration of Wnt3a in culture medium could effectively enhance the osteogenic and angiogenic activity of bone repair biomaterials both in vitro and in vivo. Moreover, it would avoid the side effects caused by the supraphysiological concentrations of growth factors. This functionalized scaffold with osteogenic and angiogenic activity might be used as an outstanding bone substitute for bone regeneration and repair.

Small extracellular vesicles derived from hypoxic mesenchymal stem cells promote vascularized bone regeneration through the miR-210-3p/EFNA3/PI3K pathway

Angiogenesis is closely coupled with osteogenesis and has equal importance. Thus, promoting angiogenesis during the bone repair process is vital for ideal bone regeneration. As important mediators of cell-cell communication and biological homeostasis, mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been proved to be highly involved in bone and vascular regeneration. Because hypoxia microenvironment promotes the proangiogenic activity of MSCs, in the present study, we investigate the effect and underlying molecular mechanisms of sEVs from hypoxia-preconditioned MSCs (hypo-sEVs) on angiogenesis and develop an effective strategy to promote vascularized bone regeneration. Compared to sEVs from normoxia MSCs (nor-sEVs), hypo-sEVs promoted the proliferation, migration, and angiogenesis of HUVECs and ultimately enhanced bone regeneration and new blood vessel reconstruction in a critical-size calvarial bone defect model. miRNA sequence and the verified results showed that miR-210-3p in hypo-sEVs was increased via HIF-1α under hypoxia. The upregulated miR-210-3p in hypo-sEVs promoted angiogenesis by downregulating EFNA3 expression and enhancing the phosphorylation of the PI3K/AKT pathway. Thus, this study suggests a successful strategy to enhance vascularized bone regeneration by utilizing hypo-sEVs via the miR-210-3p/EFNA3/PI3K/AKT pathway. Statement of significanceConsidering the significance of vascularization in ideal bone regeneration, strategies to promote angiogenesis during bone repair are required. Hypoxia microenvironment can promote the proangiogenic potential of mesenchymal stem cells (MSCs). Nonetheless, the therapeutic effect of small extracellular vesicles (sEVs) from hypoxia-preconditioned MSCs on cranio-maxillofacial bone defect remains unknown, and the underlying mechanism is poorly understood. This study shows that hypo-sEVs significantly enhance the proliferation, migration, and angiogenesis of HUVECs as well as promote vascularized bone formation. Moreover, this work indicates that HIF-1α can induce overexpression of miR-210-3p under hypoxia, and miR-210-3p can hinder EFNA3 expression and subsequently activate the PI3K/AKT pathway. The application of hypo-sEVs provides a facile and promising strategy to promote vascularized bone regeneration in a critical-size bone defect model.

Silicocarnotite: Novel Silicate Bioceramic With Osteogenic Property for Repairing Rat Cranial Critical-Sized Bone Defects

Critical-sized bone defects are an intractable orthopedic disease which often fails to regenerate spontaneously and requires additional intervention. Current therapies, including autografts and allografts, are not always satisfactory. Herein, the novel calcium phosphate bioceramic-containing silicon (CPS) with a carnotite structure was synthesized. In the present study, CPS was prepared for investigating the biocompatibility and bioactivity in vitro and in vivo in comparison to hydroxyapatite (HA). Our results showed that CPS bioceramics had favorable biocompatibility and rBMSCs could adhere on the surface well in vitro. Moreover, CPS could promote osteogenic differentiation of rBMSCs and the expression of osteogenic differentiation marker genes, including ALP, Runx-2, BSP, OCN, and OPN. In vivo, the results of micro-CT, histomorphometry, and histology analyses showed that CPS significantly enhanced critical-sized calvarial defects healing compared with HA. Overall, the present study demonstrated that CPS bioceramics had satisfactory bioactivities and osteogenic capacities, which could be a potential option for reconstructing critical-sized bone defects.

Bone regeneration of mouse critical-sized calvarial defects with human mesenchymal stem cell sheets co-expressing BMP2 and VEGF

Over-dependence on existing synthetic scaffolds and insufficient osteoinductive and vasculogenic growth factors have limited the development of bone regeneration. The study aimed to assess the feasibility of using marrow-derived mesenchymal stem cells (BMSCs) cell sheets co-expressing bone morphogenetic proteins 2 (BMP2) and vascular endothelial growth factor (VEGF) for repairing critical-sized calvarial defects. BMSCs cell sheets were genetically engineered to express BMP2/VEGF alone or together. Alterations in osteogenic markers were examined by quantitative real-time PCR (qRT-PCR) and western blotting. A critical-sized calvarial bone defect model was used to investigate the osteogenesis effects of BMP2/VEGF cell sheets alone or in combination. The efficacy was assessed with micro-computed tomography (micro-CT) and histology. Invitro, the expression of BMP2 and VEGF through lentiviral transduction was confirmed by qRT-PCR and western blotting against BMP2 and VEGF. Lentiviral delivery of BMP2 and VEGF resulted in the upregulation of osteogenic markers. Invivo, in a critical-sized calvarial bone defect model, 3D-reconstructed micro-CT images revealed that treatment of the calvarial defects with the BMP2/VEGF cell sheet resulted in significantly greater amounts of newly formed bone at 8 weeks after surgery than treatment with cell sheets with single gene transduction or vehicle controls. The results were confirmed by histological assessment by H&E staining and Masson staining. This study demonstrates that BMP2/VEGF co-expressing BMSCs sheets promote bone regeneration in critical-sized calvarial bone defects. The BMP2/VEGF cell sheets provide a functional bioactive scaffold for critical-size bone reconstruction.

Poster 107: The Use of Coacervate Sustained Release System to Identify the Most Potent BMP for Bone Regeneration

Poster 107: The Use of Coacervate Sustained Release System to Identify the Most Potent BMP for Bone Regeneration

Evaluation of the Effects of Topical Ellagic Acid and Graft Application on Bone Regeneration: an Experimental Study.

ABSTRACTObjectivesThe aim of this experimental animal study is to investigate the effect of bone graft and topical ellagic acid application on bone regeneration in rats with critical-sized calvarial bone defects.Material and MethodsA total of 24 male Wistar rats were divided into three groups, and 7 mm critical-sized calvarial bone defects were created surgically in them. In the first group, the created defect was left empty, and this acted as a control group. In the second group, only a bone graft was placed in the created defect. In the third group, in addition to placing a bone graft in the created defect, 0.325 mg/kg ellagic acid (EA) was applied topically to the defect.ResultsAs a result of semiquantitative scoring, osteoblast counts were 2 (SD 0.82) in the control group, 2.71 (SD 0.76) in the graft group, and 1.14 (SD 0.69) in the EA + graft group. The number of osteocytes was 2.29 (SD 0.76) in the control group, 2.71 (SD 1.11) in the graft group, and 1.43 (SD 0.54) in the EA + graft group. When inflammations were evaluated, values of 1.71 (SD 0.75), 1.14 (SD 0.69), and 3 (SD 0.82) were obtained in the control, graft, and EA + graft groups, respectively.ConclusionsTopical ellagic and graft applications show different effects at different doses under topical and systemic conditions. The dose amount of ellagic acid applied, especially in topical applications, has critical importance in bone healing.

The role of MEK1/2 and MEK5 in melatonin-mediated actions on osteoblastogenesis, osteoclastogenesis, bone microarchitecture, biomechanics, and bone formation.

Melatonin, the primary hormone involved in circadian entrainment, plays a significant role in bone physiology. This study aimed to assess the role of MEK1/2 and MEK5 in melatonin-mediated actions in mouse and human mesenchymal stem cells (MSCs) and on bone using small-molecule inhibitors and CRISPR/Cas9 knockout approaches. Consistent with in vitro studies performed in mMSCs and hMSCs, nightly (25 mg/kg, i.p., 45 days) injections with PD184352 (MEK1/2 inhibitor) or Bix02189 (MEK5 inhibitor) or SC-1-151 (MEK1/2/5 inhibitor) demonstrated that MEK1/2 and MEK5 were the primary drivers underlying melatonin's actions on bone density, microarchitecture (i.e., trabecular number, separation, and connectivity density), and bone mechanical properties (i.e., ultimate stress) through increases in osteogenic (RUNX2, BMP-2, FRA-1, OPG) expression and decreases in PPARγ. Furthermore, CRISPR/Cas9 knockout of MEK1 or MEK5 in mMSCs seeded on PLGA scaffolds and placed into critical-size calvarial defects in Balb(c) mice (male and female) revealed that treatment with melatonin (15 mg/L; p.o., nightly, 90 days) mediates sex-specific actions of MEK1 and MEK5 in new bone formation. This study is the first to demonstrate a role for MEK1/2 and MEK5 in modulating melatonin-mediated actions on bone formation in vivo and in a sex-specific manner.

Effect of Locally Administered Denosumab on Bone Graft Healing in Rabbit Critical-Size Calvarial Defects.

Denosumab is a human monoclonal immunoglobulin G2 antibody developed from the ovarian cells of Chinese hamsters. We aimed to histomorphometrically and radiologically evaluate the effects of xenografts used with local denosumab on the healing of defect sites using rabbit skulls. Two 10-mm diameter critical-size defects were created in 16 rabbits. The defect areas were filled with xenografts and xenograft + 3 mg denosumab in the control and denosumab groups (DEN), respectively. We evaluated new bone, residual graft, soft tissue areas, and bone volume in 4- and 8-week study groups. Histomorphometrically, there were no statistically significant differences between groups at both 4 and 8 weeks regarding residual graft, new bone, and soft tissue area (p > 0.05). The 4-week residual graft control group values were significantly higher than the 8-week values (p < 0.05). The soft tissue area was significantly greater in the 4-week compared with the 8-week DEN group (p < 0.05). The radiologically measured total bone volume was significantly greater in the 8-week specimens than in the 4-week specimens (p < 0.05). In this study, denosumab used locally with bone grafts, showed no direct effect on new and total bone volume.

Inhibition of Sympathetic Activation by Delivering Calcium Channel Blockers from a 3D Printed Scaffold to Promote Bone Defect Repair.

Enhancing osteogenesis by promoting neural network reconstruction and neuropeptide release is considered to be an attractive strategy for repairing of critical size bone defects. However, traumatic bone defects often activate the damaged sympathetic nervous system (SNS) in the defect area and release excessive catecholamine to hinder bone defect repair. Herein, a 3D printed scaffold loaded with the calcium channel blocker-nifedipine is proposed to reduce the concentration of catecholamine present in the bone defect region and to accelerate bone healing. To this end, nifedipine-loaded ethosome and laponite are added into a mixed solution containing sodium alginate, methacrylated gelatin, and bone mesenchymal stem cells (BMSCs) to prepare a cell-laden scaffold using 3D bioprinting. The released nifedipine is able to close the calcium channels of nerve cells, thereby blocking sympathetic activation and ultimately inhibiting the release of catecholamine by sympathetic nerve cells, which further promotes the osteogenic differentiation and migration of BMSCs, inhibits osteoclastogenesis in vitro, and effectively improves bone regeneration in a rat critical-size calvarial defect model. Therefore, the results suggest that sustained release of nifedipine from the scaffold can effectively block SNS activation, providing promising strategies for future treatment of bone defects.

The effect of experimental diabetes and membrane occlusiveness on guided bone regeneration: A proof of principle study.

To evaluate the effect of membrane occlusiveness and experimental diabetes on early and late healing following guided bone regeneration. A total of 30 Wistar rats were randomly allocated to three groups: healthy (H), uncontrolled diabetic (UD) and controlled diabetic (CD). A critical size calvarial defect (CSD) was created at the mid-portion of one parietal bone, and it was treated with a double layer of e-PTFE membrane presenting 0.5mm perforations. The animals were killed at 7 and 30days of healing, and qualitative and quantitative histological evaluations were performed. Data were compared with the ones previously obtained from other 30 animals (10H, 10UD, 10 CD), where two CSDs were randomly treated with a double-layer e-PTFE occlusive membrane or left empty. Following application of cell occlusive or cell permeable membranes, significant regeneration can be observed. However, at 30days in the H group occlusive compared to cell permeable membranes promoted enhanced bone regeneration (83.9 ± 7.3% vs. 52.5 ± 8.6%), while no significant differences were observed within the CD and UD groups. UD led to reduced regeneration compared to H when an occlusive barrier was applied, whereas comparable outcomes to H and CD were observed when placing perforated membranes. The application of cell permeable membranes may have masked the potentially adverse effect of experimental UD on bone regeneration. Membrane porosity might contribute to modulate the bone regenerative response in UD conditions. Future studies are needed to establish the degree of porosity associated with the best regenerative outcomesas well as the underlying molecular mechanisms.

Thermosensitive chitosan/poly(N-isopropyl acrylamide) nanoparticles embedded in aniline pentamer/silk fibroin/polyacrylamide as an electroactive injectable hydrogel for healing critical-sized calvarial bone defect in aging rat model

Thermosensitive nanoparticles with phase transition abilities have been considered as suitable materials in biomedical fields, especially drug delivery systems. Moreover, electroactive injectable hydrogels supporting bone regeneration of the elderly will highly be desired in bone tissue engineering applications. Herein, thermosensitive nanoparticles were fabricated using chitosan/poly(N-isopropyl acrylamide) for simvastatin acid delivery. The nanoparticles were incorporated into electroactive injectable hydrogels based on aniline pentamer/silk fibroin/polyacrylamide containing vitamin C. The nanoparticles had thermosensitive properties as simvastatin acid had higher release rates at 37 than 23 °C without significant burst release. The hydrogels also revealed an appropriate gelation time, stable mechanical and rheological characteristics, high water absorbency, and proper biodegradability. In vitro studies indicated that the hydrogel was biocompatible and nontoxic, especially those containing drugs. Implantation of the hydrogels containing both simvastatin acid and vitamin C into the critical calvarial bone defect of the aged rat also demonstrated significant enhancement of bone healing after 4 and 8 weeks post-implantation. We found that the electroactive injectable hydrogels containing thermosensitive nanoparticles exhibited great potential for treating bone defects in the elderly rats

Single-cell RNA sequencing unravels heterogeneity of skeletal progenitors and cell-cell interactions underlying the bone repair process.

IntroductionActivation of skeletal progenitors upon tissue injury and the subsequent cell fate specification are tightly coordinated in the bone repair process. Although known osteoimmunological signaling networks play important roles in the microenvironment of the bone defect sites, the molecular mechanism underlying the bone repair process has not been fully understood.MethodsTo better understand the behavior of the skeletal progenitors and the heterogeneity of the cells during bone repair at the microenvironmental level, we performed a combinatorial analysis consisting of lineage tracing for skeletal progenitors using the Sox9-CreERT2;R26RtdTomato mouse line followed by single-cell RNA sequencing (scRNA-seq) analysis using a mouse model of calvarial bone repair. To identify a therapeutic target for bone regeneration, further computational analysis was performed focusing on the identification of the cell–cell interactions, followed by pharmacological assessments with a critical-size calvarial bone defect mouse model.ResultsLineage tracing analysis showed that skeletal progenitors marked by Sox9 were activated upon bone injury and contributed to bone repair by differentiating into osteoblasts. The scRNA-seq analysis characterized heterogeneous cell populations at the bone defect sites; the computational analysis predicted a bifurcated lineage from skeletal progenitors toward osteogenic and adipogenic lineages. Chemokine C–C motif ligand 9 (Ccl9) was identified as a signaling molecule that regulates bone regeneration in the mouse model, possibly through the regulation of adipogenic differentiation at the bone defect site.ConclusionMultipotential skeletal progenitors and the direction of the cell differentiation were characterized at single cell resolution in a mouse bone repair model. The Ccl9 signaling pathway may be a key factor directing osteogenesis from the progenitors in the model and may be a therapeutic target for bone regeneration.

Fabrication and Effect of Strontium-Substituted Calcium Silicate/Silk Fibroin on Bone Regeneration In Vitro and In Vivo.

Achieving rapid osteogenesis and angiogenesis was the key factor for bone regeneration. In the present study, the strontium-substituted calcium silicate (SrCS)/silk fibroin (SF) composite materials have been constructed by combining the different functional component ratios of SrCS (12.5 wt%, 25 wt%) and SF. Then, the effects of SrCS/SF materials on proliferation, osteogenic differentiation, and angiogenic factor secretion of rat bone marrow-derived mesenchymal stromal cells (rBMSCs) were first evaluated in vitro. Moreover, the in vivo effect of osteogenesis was evaluated in a critical-sized rat calvarial defect model. In vitro studies showed that SrCS/SF significantly enhanced the cell proliferation, alkaline phosphatase (ALP) activity, and the expression of osteogenic and angiogenic factors of rBMSCs as compared with the SF and CS/SF, and the optimum proportion ratio was 25 wt%. Besides, the results also showed that CS/SF achieved enhanced effects on rBMSCs as compared with SF. The in vivo results showed that 25 wt% SrCS/SF could obviously promote new bone formation more than SF and CS/SF. The present study revealed that SrCS could significantly promote the osteogenic and angiogenic activities of SF, and SrCS/SF might be a good scaffold material for bone regeneration.

Comparison of rhBMP-2 in Combination with Different Biomaterials for Regeneration in Rat Calvaria Critical-Size Defects.

Regeneration of critical bone defects requires the use of biomaterials. The incorporation of osteoinductive agents, such as bone morphogenetic proteins (BMPs), improves bone formation. This study aimed to compare the efficacy of rhBMP-2 in combination with different materials for bone regeneration in critical-sized rat calvarial defects. This was an experimental animal study using 30 rats. In each rat, two 5-mm critical-size defects were made in the calvaria (60 bone defects in total) using a trephine. All rats were randomized to one of the six groups: control (C), autograft + rhBMP-2 (A), absorbable collagen sponge + rhBMP-2 (ACS), β-tricalcium phosphate + rhBMP-2 (B-TCP), bovine xenograft + rhBMP-2 (B), and hydroxyapatite + rhBMP-2 (HA). The outcome was assessed after 4 and 8 weeks using histological description and the histological bone healing scale. Statistical analysis was performed using the Kruskal-Wallis and Mann–Whitney U tests, with a p-value set at 0.05. The average bone healing scores per group were as follows: C group, 12.5; A group, 26.5; ACS group, 18.8; B-TCP group, 26.2; HA group, 20.9; and B group, 20.9. The C group showed a significant difference between weeks 4 and 8 (p = 0.032). Among the 4-week groups, the C group showed a significant difference compared to A (p = 0.001), ACS (p = 0.017), and B-TCP (p = 0.005) groups. The 8-week experimental group did not show any significant differences between the groups. The 5-mm critical size defect in rat calvaria requires the use of bone biomaterials to heal at 4 and 8 weeks. rhBMP-2, as applied in this study, showed no difference in new bone formation when combined with bovine, B-TCP, or HA biomaterials.

Osteoinductive hybrid hydrogel membranes for in situ bone regeneration in hyperglycemia

In hyperglycemia patients, suffering from insufficient vascularization and vascular network lesion, tissue regeneration, such as bone repair, is limited and maybe delayed by the secondary injury and hyperglycemic microenvironment. Typically, dental therapies involving guided bone regeneration is facing a difficult condition in the patients with diabetes. In this study, a hybrid membrane was endowed with biomimetic function to create an angiogenesis-inductive microenvironment by calcium ion release to overcome the limitations of bone tissue regeneration in diabetic patients. Biomineralized calcium resource was Janus-structured onto the surface of hybrid hydrogel by layer-by-layer technique to enhance vascularization and improve the bone regeneration in this study. The release of calcium ions from mineralized phases was controlled by the solubility of inorganic phases and the degradation of gels, promoting HIF-1α expression and creating a key role in angiogenesis stimulation. With highly enhanced calcium signaling and blood vessel formation, the hybrid hydrogel membranes improved the recruitment, proliferation and differentiation of mesenchymal stem cells and endothelial progenitors, confirmed by the enhancement of microvascular regeneration and new bone formation in the critical-sized calvarial defect in diabetic model in vivo. Our study demonstrates a translational potential of hybrid hydrogels engineered with inorganic minerals for orthopedic applications in hyperglycemia.

Alendronate crosslinked chitosan/polycaprolactone scaffold for bone defects repairing

Here, we evaluated osteogenic differentiation in vitro and new bone formation in vivo using an alendronate-loaded chitosan/polycaprolactone scaffold (CS/PCL) in rats with a critical-sized calvarial defect. Through the action of genipin, which has a crosslinking function, alendronate (AL) was anchored throughout the CS/PCL composite scaffold (CS/PCL@AL) to form an AL sustained release system. We demonstrated that CS/PCL@AL scaffolds significantly enhanced the osteogenic differentiation of ectomesenchymal stem cells (EMSCs) in vitro. Additionally, we explored the possible molecular mechanism of CS/PCL@AL scaffolds in the osteogenic differentiation of EMSCs. This composite scaffold exerted two positive effects on EMSC osteogenic differentiation: 1) the CS/PCL@AL scaffold enhanced EMSC osteogenic differentiation by upregulating bone morphogenetic protein 2, interleukin 10 and laminin expression; and 2) the CS/PCL@AL scaffold promoted the osteogenic differentiation of EMSCs by activating the yes-associated protein (YAP) signaling pathway. YAP and its downstream target transglutaminase are crucial mediators in the osteogenic differentiation of EMSCs. Finally, micro-computed tomography analyses and histology results suggested that the CS/PCL@AL scaffold exhibited a superior capacity to accelerate new and mature bone formation in skull bone defects in Sprague–Dawley rats. This simple and low-cost technology may represent a promising strategy to construct an efficient delivery system to repair bone defects.

Magnesium Oxide Nanoparticle Coordinated Phosphate-Functionalized Chitosan Injectable Hydrogel for Osteogenesis and Angiogenesis in Bone Regeneration.

Natural polysaccharide (NPH)-based injectable hydrogels have shown great potential for critical-sized bone defect repair. However, their osteogenic, angiogenic, and mechanical properties are insufficient. Here, MgO nanoparticles (NPs) were incorporated into a newly synthesized water-soluble phosphocreatine-functionalized chitosan (CSMP) water solution to form an injectable hydrogel (CSMP-MgO) via supramolecular combination between phosphate groups in CSMP and magnesium in MgO NPs to circumvent these drawbacks of chitosan-based injectable hydrogels. Water-soluble chitosan deviate CSMP was first synthesized by grafting methacrylic anhydride and phosphocreatine into a chitosan chain in a one-step lyophilization process. The phosphocreatine in this hydrogel not only provides sites to combine with MgO NPs to form supramolecular binding but also serves as the reservoir to control Mg2+ release. As a result, the lyophilized CSMP-MgO hydrogels presented a porous structure with some small holes in the pore wall, and the pore diameters ranged from 50 to 100 μm. The CSMP-MgO injectable hydrogels were restricted from swelling in DI water (lowest swelling ratio was 16.0 ± 1.1 g/g) and presented no brittle failure during compression even at a strain above 85% (maximum compressive strength was 195.0 kPa) versus the control groups (28.0 and 41.3 kPa for CSMP and CSMP-MgO (0.5) hydrogels), with regulated Mg2+ release in a stable and sustained manner. The CSMP-MgO injectable hydrogels promoted in vitro calcium phosphate (hydroxyapatite (HA) and tetracalcium phosphate (TTCP)) deposition in supersaturated calcium phosphate solution and presented no cytotoxicity to MC3T3-E1 cells; the CSMP-MgO hydrogel promoted MC3T3-E1 cell osteogenic differentiation with upregulation of BSP, OPN, and Osterix osteogenic gene expression and mineralization and HUVEC tube formation. Among them, CSMP-MgO (5) presented most of these properties. Moreover, this hydrogel (CSMP-MgO (5)) showed an excellent ability to promote new bone formation in critical-sized calvarial defects in rats. Thus, the CSMP-MgO injectable hydrogel shows great promise for bone regeneration.