Critical-sized Bone Defect Model Research Articles

Modified and alternative bone cements can improve the induced membrane: Critical size bone defect model in rat femur

BackgroundAs a two-stage surgical procedure, Masquelet's technique has been used to care for critical-size bone defects (CSD). We aimed to determine the effects of modified and altered bone cement with biological or chemical enriching agents on the progression of Masquelet's induced membrane (IM) applied to a rat femur CSD model, and to compare the histopathological, biochemical, and immunohistochemical findings of these cements to enhance IM capacity. MethodsThirty-five male rats were included in five groups: plain polymethyl methacrylate (PMMA), estrogen-impregnated PMMA (E+PMMA), bone chip added PMMA (BC+PMMA), hydroxyapatite-coated PMMA (HA) and calcium phosphate cement (CPC). The levels of bone alkaline phosphatase (BALP), osteocalcin (OC), and tumor necrosis factor-alpha (TNF-α) were analyzed in intracardiac blood samples collected at the end of 4 weeks of the right femur CSD intervention. All IMs collected were fixed and prepared for histopathological scoring. The tissue levels of rat-specific Transforming Growth Factor-Beta (TGF-β), Runt-related Transcription Factor 2 (Runx2), and Vascular Endothelial Growth Factor (VEGF) were analyzed immunohistochemically. ResultsSerum levels of BALP and OC were significantly higher in E+PMMA and BC+PMMA groups than those of other groups (P = 0.0061 and 0.0019, respectively). In contrast, TNF-α levels of all groups with alternative bone cement significantly decreased compared to bare PMMA (P = 0.0116). Histopathological scores of E+PMMA, BC+PMMA, and CPC groups were 6.86 ± 1.57, 4.71 ± 0.76, and 6.57 ± 1.51, respectively, which were considerably higher than those of PMMA and HA groups (3.14 ± 0.70 and 1.86 ± 0.69, respectively) (P < 0.0001). Significant increases in TGF-β and VEGF expressions were observed in E+PMMA and CPC groups (P = 0.0001 and <0.0001, respectively) whereas Runx2 expression significantly increased only in the HA group compared to other groups (P < 0.0001). ConclusionsThe modified PMMA with E and BC, and CPC as an alternative spacer resulted in a well-differentiated IM and increased IM progression by elevating BALP and OC levels in serum and by mediating expressions of TGF-β and VEGF at the tissue level. Estrogen-supplemented cement spacer has yielded promising findings between modified and alternative bone cement.

Nano-laponite encapsulated coaxial fiber scaffold promotes endochondral osteogenesis.

Osteoinductive supplements without side effects stand out from the growth factors and drugs widely used in bone tissue engineering. Lithium magnesium sodium silicate hydrate (laponite) nanoflake is a promising bioactive component for bone regeneration, attributed to its inherent biosafety and effective osteoinductivity. Up to now, the in vivo osteogenic potential and mechanisms of laponite-encapsulated fibrous membranes remain largely unexplored. This study presents a unique method for homogeneously integrating high concentrations of laponite RDS into a polycaprolactone (PCL) matrix by dispersing laponite RDS sol into the polymer solution. Subsequently, a core-shell fibrous membrane (10RP-PG), embedding laponite-loaded PCL in its core, was crafted using coaxial electrospinning. The PCL core's slow degradation and the shell's gradient degradation enabled the sustained release of bioactive ions (Si and Mg) from laponite. In vivo studies on a critical-sized calvarial bone defect model demonstrated that the 10RP-PG membrane markedly enhanced bone formation and remodeling by accelerating the process of endochondral ossification. Further transcriptome analysis suggested that osteogenesis in the 10RP-PG membrane is driven by Mg and Si from endocytosed laponite, activating pathways related to ossification and endochondral ossification, including Hippo, Wnt and Notch. The fabricated nanocomposite fibrous membranes hold great promise in the fields of critical-sized bone defect repair.

Silicone rubber sealed channel induced self-healing of large bone defects: Where is the limit of self-healing of bone?

BackgroundLarge defects of long tubular bones due to severe trauma, bone tumor resection, or osteomyelitis debridement are challenging in orthopedics. Bone non-union and other complications often lead to serious consequences. At present, autologous bone graft is still the gold standard for the treatment of large bone defects. However, autologous bone graft sources are limited. Silicon rubber (SR) materials are widely used in biomedical fields, due to their safety and biocompatibility, and even shown to induce nerve regeneration. Materials and methodsWe extracted rat bone marrow mesenchymal stem cells (BMMSCs) in vitro and verified the biocompatibility of silicone rubber through cell experiments. Then we designed a rabbit radius critical sized bone defect model to verify the effect of silicone rubber sealed channel inducing bone repair in vivo. ResultsSR sealed channel could prevent the fibrous tissue from entering the fracture end and forming bone nonunion, thereby inducing self-healing of long tubular bone through endochondral osteogenesis. The hematoma tissue formed in the early stage was rich in osteogenesis and angiogenesis related proteins, and gradually turned into vascularization and endochondral osteogenesis, and finally realized bone regeneration. ConclusionsIn summary, our study proved that SR sealed channel could prevent the fibrous tissue from entering the fracture end and induce self-healing of long tubular bone through endochondral osteogenesis. In this process, the sealed environment provided by the SR channel was key, and this might indicate that the limit of self-healing of bone exceeded the previously thought. The translational potential of this articleThis study investigated a new concept to induce the self-healing of large bone defects. It could avoid trauma caused by autologous bone extraction and possible rejection reactions caused by bone graft materials. Further research based on this study, including the innovation of induction materials, might invent a new type of bone inducing production, which could bring convenience to patients. We believed that this study had significant meaning for the treatment of large bone defects in clinical practice.

Differences in macrophage expression in induced membranes by fixation method – Masquelet technique using a mouse's femur critical-sized bone defect model

IntroductionMasquelet's induced membrane technique (MIMT) is an emerging method for reconstructing critical-sized bone defects. However, an incomplete understanding of the underlying biological and physical processes hinders further optimization. This study investigated the effect of different bone-defect fixation methods on macrophage expression in an induced membrane using a novel mouse plate-fixed Masquelet model. MethodsMice were divided into Plate-fixed Masquelet (P-M), Intramedullary-fixed Masquelet (IM-M), Plate-fixed Control (P-C), and Back subfascial (B) groups. In the P-M and IM-M groups, a polymethylmethacrylate (PMMA) spacer was implanted into a 3 mm bone defect, while the defect in the P-C group remained unfilled. In group B, a spacer was inserted under the back fascia to examine membrane formation caused by a simple foreign body reaction. Tissues were collected at 1, 2, and 4 weeks postoperatively. Hematoxylin and eosin (H&E) staining and immunohistochemistry (CD68 and CD163: macrophage markers) were performed to assess macrophage expression within the membrane. qPCR was performed to measure the expression of CD68, CD163, and fibroblast growth factor 2 (FGF2). ResultsFour weeks post-operation, the P-M group presented with minimal callus growth, whereas the IM-M group exhibited vigorous growth. The P-M and IM-M groups displayed a tri-layered membrane structure, which is consistent with the results of previous studies. The IM-M group had significantly thicker membranes, whereas the P-M group exhibited higher expression levels of CD68, CD163, and FGF2. Group P-C showed no osteogenesis, whereas group B maintained a thin, cell-dense membrane structure. The P-M group consistently showed higher gene expression levels than the P-C and P-B groups. ConclusionThis study introduced a mouse plate fixation model for MIMT. The induced membranes could be adequately evaluated in this model. Induced membranes are formed by foreign body reactions to PMMA spacers; however, their properties are clearly different from those of simple foreign body reaction capsules and granulation tissues that infiltrate bone defects, suggesting that they are more complex tissues. The characteristics and expression of macrophages within these induced membranes varied according to the bone defect fixation method.

Nano artificial periosteum PCL/Ta/ZnO accelerates repair of periosteum via antibacterial, promoting vascularization and osteogenesis

The periosteum plays a critical role in bone development, shaping, remodeling, and fracture healing due to its abundance of osteoprogenitor cells, osteoblasts, and capillary network. However, the role of periosteum in bone injury healing has been underestimated, thus there is an urgent need to develop a multifunctional artificial periosteum that mimics the natural one. To tackle this issue, electrospinning technology was employed to fabricate an artificial periosteum composed of Poly-ε-caprolactone (PCL) doped with tantalum (Ta) and zinc oxide (ZnO) nanoparticles to enhance its antibacterial, osteogenic, and angiogenic properties. The in vitro cell experiments have demonstrated that the PCL/Ta/ZnO artificial periosteum exhibits excellent biocompatibility and can effectively facilitate osteogenic differentiation of BMSCs as well as angiogenic differentiation of EPCs. Antibacterial experiments have demonstrated the excellent bactericidal effects of PCL/Ta/ZnO artificial periosteum against both S. aureus and E. coli. The subcutaneous infection and critical-sized skull bone defect models have validated its in vivo properties of antibacterial activity, promotion of osteogenesis, and angiogenic potential. The PCL/Ta/ZnO artificial periosteum demonstrates remarkable efficacy in infection control and favorable immunomodulation, thereby achieving rapid vascularized bone repair. In conclusion, the utilization of PCL/Ta/ZnO tissue-engineered periosteum has been demonstrated to exhibit antibacterial properties, pro-vascularization effects, and promotion of osteogenesis at the site of bone defects. This promising approach could potentially offer effective treatment for bone defects.

In vivo evaluation of bone regeneration using ZIF8-modified polypropylene membrane in rat calvarium defects.

The profound potential of zeolitic imidazolate framework 8 (ZIF8) thin film for inducing osteogenesis has been previously established under in vitro conditions. As the next step towards the clinical application of ZIF8-modified substrates in periodontology, this in vivo study aimed to evaluate the ability of the ZIF8 crystalline layer to induce bone regeneration in an animal model defect. Following the mechanical characterization of the membranes and analysing the in vitro degradation of the ZIF8 layer, in vivo bone regeneration was evaluated in a critical-sized (5-mm) rat calvarial bone defect model. For each animal, one defect was randomly covered with either a polypropylene (PP) or a ZIF8-modified membrane (n = 7 per group), while the other defect was left untreated as a control. Eight weeks post surgery, bone formation was assessed by microcomputed tomography scanning, haematoxylin and eosin staining and immunohistochemical analysis. The ZIF8-modified membrane outperformed the PP membrane in terms of mechanical properties and revealed a trace Zn+2 release. Results of in vivo evaluation verified the superior barrier function of the ZIF8-coated membrane compared with pristine PP membrane. Compared with the limited marginal bone formation in the control and PP groups, the defect area was almost filled with mature bone in the ZIF8-coated membrane group. Our results support the effectiveness of the ZIF8-coated membrane as a promising material for improving clinical outcomes of guided bone regeneration procedures, without using biological components.

Early osteoimmunomodulation by mucin hydrogels augments the healing and revascularization of rat critical-size calvarial bone defects.

The design principle of osteogenic bone grafts has shifted from immunological inertness to limiting foreign body response to combined osteoimmunomodulatory activity to promote high-quality endogenous bone regeneration. Recently developed immunomodulatory mucin hydrogels have been shown to elicit very low complement activation and suppress macrophage release and activation after implantation in vivo. However, their immunoregulatory activity has not yet been studied in the context of tissue repair. Herein, we synthesized mucin-monetite composite materials and investigated their early osteoimmunomodulation using a critical-size rat bone defect model. We demonstrated that the composites can polarize macrophages towards the M2 phenotype at weeks 1 and 2. The early osteoimmunomodulation enhanced early osteogenesis and angiogenesis and ultimately promoted fracture healing and engraftment (revascularization of the host vasculature) at weeks 6 and 12. Overall, we demonstrated the applicability of mucin-based immunomodulatory biomaterials to enhance tissue repair in tissue engineering and regenerative medicine.

Biofunctionalization with Cissus quadrangularis phytobioactives accentuates Nano-Hydroxyapatite based ceramic Nano-Cement for Neo-Bone formation in critical sized bone defect

Developing biofunctionalized ceramic bone substitutes with phytobioactives for their sustained delivery is highly desired to enhance the osteo-active potential of ceramic bone substitutes, reduce the systemic toxicity of synthetic drugs, and increase the bioavailability of phytobioactives. The present work highlights the local delivery of phytobioactives of Cissus quadrangularis (CQ) through nano-hydroxyapatite (nHAP) based ceramic nano-cement. The phytoconstituent profiling represented the optimized CQ fraction to be rich in osteogenic polyphenols and flavonoids like quercetin, resveratrol, and their glucosides. Further, CQ phytobioactives-based formulation was biocompatible, increased bone formation, calcium deposition, proliferation, and migration of cells with simultaneous alleviation of cellular oxidative stress. In the in vivo critical-sized bone defect model, enhanced formation of highly mineralized tissue (BV mm3) in CQ phytobioactives functionalized nano-cement (10.5 ± 2 mm3) were observed compared to the control group (6.5 ± 1.2 mm3). Moreover, the addition of CQ phytobioactives to the bone nano-cement increased the fractional bone volume (BV/TV%) to 21 ± 4.2% compared to 13.1 ± 2.5% in non-functionalized nano-cement. The results demonstrated nHAP-based nano-cement as a carrier for phytobioactives which could be a promising approach for neo-bone formation in different bone defect conditions.

Multifunctional dendrimer@nanoceria engineered GelMA hydrogel accelerates bone regeneration through orchestrated cellular responses.

Bone defects in patients entail the microenvironment that needs to boost the functions of stem cells (e.g., proliferation, migration, and differentiation) while alleviating severe inflammation induced by high oxidative stress. Biomaterials can help to shift the microenvironment by regulating these multiple events. Here we report multifunctional composite hydrogels composed of photo-responsive Gelatin Methacryloyl (GelMA) and dendrimer (G3)-functionalized nanoceria (G3@nCe). Incorporation of G3@nCe into GelMA could enhance the mechanical properties of hydrogels and their enzymatic ability to clear reactive oxygen species (ROS). The G3@nCe/GelMA hydrogels supported the focal adhesion of mesenchymal stem cells (MSCs) and further increased their proliferation and migration ability (vs. pristine GelMA and nCe/GelMA). Moreover, the osteogenic differentiation of MSCs was significantly stimulated upon the G3@nCe/GelMA hydrogels. Importantly, the capacity of G3@nCe/GelMA hydrogels to scavenge extracellular ROS enabled MSCs to survive against H2O2-induced high oxidative stress. Transcriptome analysis by RNA sequencing identified the genes upregulated and the signalling pathways activated by G3@nCe/GelMA that are associated with cell growth, migration, osteogenesis, and ROS-metabolic process. When implanted subcutaneously, the hydrogels exhibited excellent tissue integration with a sign of material degradation while the inflammatory response was minimal. Furthermore, G3@nCe/GelMA hydrogels demonstrated effective bone regeneration capacity in a rat critical-sized bone defect model, possibly due to an orchestrated capacity of enhancing cell proliferation, motility and osteogenesis while alleviating oxidative stress.

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.

The optimal scaffold for silk sericin-based bone graft: collagen versus gelatin

BackgroundSilk sericin is an active ingredient in bone grafts. However, the optimal scaffold for silk sericin has yet to be identified.MethodA critical-sized bone defect model in rat calvaria was used to evaluate bone regeneration. Silk sericin from Yeonnokjam, Bombyx mori, was incorporated into gelatin (group G, n = 6) and collagen (group C, n = 6). Bone regeneration was evaluated using micro-computed tomography (mCT) and histology.ResultsGroup C showed a larger bone volume than group G in the mCT analysis (P = 0.001). Histological analysis showed a larger area of bony defects in group G than in group C. The bone regeneration area in group C was significantly larger than that in group G (P = 0.003).ConclusionCompared with gelatin, collagen shows better bone regeneration in silk sericin-based bone grafts.

SAXS imaging reveals optimized osseointegration properties of bioengineered oriented 3D-PLGA/aCaP scaffolds in a critical size bone defect model

Healing large bone defects remains challenging in orthopedic surgery and is often associated with poor outcomes and complications. A major issue with bioengineered constructs is achieving a continuous interface between host bone and graft to enhance biological processes and mechanical stability. In this study, we have developed a new bioengineering strategy to produce oriented biocompatible 3D PLGA/aCaP nanocomposites with enhanced osseointegration. Decellularized scaffolds -containing only extracellular matrix- or scaffolds seeded with adipose-derived mesenchymal stromal cells were tested in a mouse model for critical size bone defects. In parallel to micro-CT analysis, SAXS tensor tomography and 2D scanning SAXS were employed to determine the 3D arrangement and nanostructure within the critical-sized bone. Both newly developed scaffold types, seeded with cells or decellularized, showed high osseointegration, higher bone quality, increased alignment of collagen fibers and optimal alignment and size of hydroxyapatite minerals.

Aspirin synergizes with mineral particle-coated macroporous scaffolds for bone regeneration through immunomodulation.

Rationale: Mineral particles have been widely used in bone tissue engineering scaffolds due to their osteoconductive and osteoinductive properties. Despite their benefits, mineral particles can induce undesirable inflammation and subsequent bone resorption. Aspirin (Asp) is an inexpensive and widely used anti-inflammatory drug. The goal of this study is to assess the synergistic effect of Asp and optimized mineral particle coating in macroporous scaffolds to accelerate endogenous bone regeneration and reduce bone resorption in a critical-sized bone defect model. Methods: Four commonly used mineral particles with varying composition (hydroxyapatite v.s. tricalcium phosphate) and size (nano v.s. micro) were used. Mineral particles were coated onto gelatin microribbon (µRB) scaffolds. Macrophages (Mφ) were cultured on gelatin µRB scaffolds containing various particles, and Mφ polarization was assessed using PCR and ELISA. The effect of conditioned medium from Mφ on mesenchymal stem cell (MSC) osteogenesis was also evaluated in vitro. Scaffolds containing optimized mineral particles were then combined with varying dosages of Asp to assess the effect in inducing endogenous bone regeneration using a critical-sized cranial bone defect model. In vivo characterization and in vitro cell studies were performed to elucidate the effect of tuning Asp dosage on Mφ polarization, osteoclast (OC) activity, and MSC osteogenesis. Results: Micro-sized tricalcium phosphate (mTCP) particles were identified as optimal in promoting M2 Mφ polarization and rescuing MSC-based bone formation in the presence of conditioned medium from Mφ. When implanted in vivo, incorporating Asp with mTCP-coated µRB scaffolds significantly accelerated endogenous bone formation in a dose-dependent manner. Impressively, mTCP-coated µRB scaffolds containing 20 µg Asp led to almost complete bone healing of a critical-sized cranial bone defect as early as week 2 with no subsequent bone resorption. Asp enhanced M2 Mφ polarization, decreased OC activity, and promoted MSC osteogenesis in a dosage-dependent manner in vivo. These results were further validated using in vitro cell studies. Conclusions: Here, we demonstrate Asp and mineral particle-coated microribbon scaffold provides a promising therapy for repairing critical-sized cranial bone defects via immunomodulation. The leading formulation supports rapid endogenous bone regeneration without the need for exogenous cells or growth factors, making it attractive for translation. Our results also highlight the importance of optimizing mineral particles and Asp dosage to achieve robust bone healing while avoiding bone resorption by targeting Mφ and OCs.

Osteoimmunity-regulating nanosilicate-reinforced hydrogels for enhancing osseointegration.

Following the introduction of osteo-immunomodulation as a new and important strategy to enhance material osseointegration, achieving an appropriate immune response after biomaterial implantation has become a significant challenge for efficient bone repair. In this study, a nanosilicate-reinforced sodium alginate (SA) hydrogel was fabricated by introducing montmorillonite (MMT) nanoparticles. Meanwhile, an immunogenically bioactive agent, harmine (HM), was loaded and released to induce macrophage differentiation into the M2 type. The fabricated SA/MMT/HM (SMH) hydrogel exhibited improved mechanical stiffness and stability, which also efficiently promoted macrophage anti-inflammatory M2 phenotype polarization and enhanced the secretion of pro-tissue healing cytokines for inducing a favorable immunomodulatory microenvironment for the osteogenic differentiation of bone marrow stromal cells (BMSCs). Furthermore, a rat air-pouch model and a critical-size bone defect model were used and the results showed that the SMH hydrogel increased the proportion of M2 macrophages and markedly reduced local inflammation, while enhancing desirable new bone formation. Transcriptomic analysis revealed that the SMH hydrogel accelerated the M1-to-M2 transition of macrophages by inhibiting relevant inflammatory signaling pathways and activating the PI3K-AKT1 signaling pathway. Taken together, this high-intensity immunomodulatory hydrogel may be a promising biomaterial for bone regeneration and provide a valuable base and positive enlightenment for massive bone defect repair.

The osteogenesis and the biologic mechanism of thermo-responsive injectable hydrogel containing carboxymethyl chitosan/sodium alginate nanoparticles towards promoting osteal wound healing

With the development of minimally invasive orthopedics, injectable materials for bone repair are attracted more attention, especially for those wound with a small external mouth and sizeable internal cavity. In this work, the hydrogel with features of thermo-responsiveness, degradability and injectability was designed and fabricated. The hydrogel, named as FHCS, is composed of Pluronic F-127 (F127) loaded with carboxymethyl chitosan/sodium alginate nanoparticles (nCS) and nanohydroxyapatite (nHA). The hydrogel FHCS was non-toxic and good hemocompatible. It can enhance the ALP activity and extracellular matrix calcification of MC3T3-E1 due to the chitosan-based nanoparticle components (nCS). Moreover, FHCS-5 (containing 5 mg/mL nCS) showed relative high expression of osteogenic genes and protein markers. Osteal regeneration was observed treated by FHCS-5 hydrogel in a critical-size rat calvarial bone defect model. CT scanning showed that the whole defect was basically covered by new bone after FHCS-5 hydrogel. The results of H&E staining and Masson's trichrome staining on histological sections further confirmed that FHCS-5 hydrogel promoted new osteal formation and maturation, which up regulated the osteogenic related genes and proteins of ALP, OCN, OPN through BMP/Smad signaling pathway. Hence, this study suggests that FHCS-5 hydrogels have a promising application for non-loading bone regeneration.

A novel decellularized matrix of Wnt signaling-activated osteocytes accelerates the repair of critical-sized parietal bone defects with osteoclastogenesis, angiogenesis, and neurogenesis.

Cell source is the key to decellularized matrix (DM) strategy. This study compared 3 cell types, osteocytes with/without dominant active Wnt/β-catenin signaling (daCO and WTO) and bone marrow stromal cells (BMSCs) for their DMs in bone repair. Decellularization removes all organelles and >95% DNA, and retained >74% collagen and >71% GAG, maintains the integrity of cell basement membrane with dense boundaries showing oval and honeycomb structure in osteocytic DM and smooth but irregular shape in the BMSC-DM. DM produced higher cell survival rate (90%) and higher proliferative activity. In vitro, daCO-DM induces more and longer stress fibers in BMSCs, conducive to cell adhesion, spreading, and osteogenic differentiation. 8-wk after implantation of the critical-sized parietal bone defect model, daCO-DM formed tight structures, composed of a large number of densely-arranged type-I collagen under polarized light microscope, which is similar to and integrated with host bone. BV/TV (>54%) was 1.5, 2.9, and 3.5 times of WTO-DM, BMSC-DM, and none-DM groups, and N.Ob/T.Ar (3.2 × 102/mm2) was 1.7, 2.9, and 3.3 times. At 4-wk, daCO-DM induced osteoclastogenesis, 2.3 times higher than WTO-DM; but BMSC-DM or none-DM didn't. daCO-DM increased the expression of RANKL and MCSF, Vegfa and Angpt1, and Ngf in BMSCs, which contributes to osteoclastogenesis, angiogenesis, and neurogenesis, respectively. daCO-DM promoted H-type vessel formation and nerve markers β3-tubulin and NeuN expression. Conclusion: daCO-DM produces metabolic and neurovascularized organoid bone to accelerate the repair of bone defects. These features are expected to achieve the effect of autologous bone transplantation, suitable for transformation application.

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.

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.

Octacalcium Phosphate/Gelatin Composite (OCP/Gel) Enhances Bone Repair in a Critical-sized Transcortical Femoral Defect Rat Model.

Bone grafting is widely used to treat large bone defects. A porous composite of a bioactive octacalcium phosphate material with gelatin sponge (OCP/Gel) has been shown to biodegrade promptly and be replaced with new bone both in animal models of a membranous bone defect and a long bone defect. However, it is unclear whether OCP/Gel can regenerate bone in more severe bone defects, such as a critical-size transcortical defect. Using an in vivo rat femur model of a standardized, transcortical, critical-size bone defect, we asked: Compared with a Gel control, does OCP/Gel result in more newly formed bone as determined by (1) micro-CT evaluation, (2) histologic and histomorphometric measures, and (3) osteocalcin staining and tartrate-resistant acid phosphatase staining? Thirty-four 12-week-old male Sprague-Dawley rats (weight 356 ± 25.6 g) were used. Gel and OCP/Gel composites were prepared in our laboratory. Porous cylinders 3 mm in diameter and 4 mm in height were manufactured from both materials. The OCP/Gel and Gel cylinders were implanted into a 3-mm-diameter transcortical critical-size bone defect model in the left rat femur. The OCP/Gel and Gel were randomly assigned, and the cylinders were implanted. The biological responses of the defect regions were evaluated radiologically and histologically. At 4 and 8 weeks after implantation, CT evaluation, histological examination of decalcified samples, and immunostaining were quantitatively performed to evaluate new bone formation and remaining bone graft substitutes and activity of osteoblasts and osteoclast-like cells (n = 24). Qualitative histological evaluation was performed on undecalcified samples at 3 weeks postimplantation (n = 10). CT and decalcified tissue analysis was not performed blinded, but an analysis of undecalcified specimens was performed under blinded conditions. Radiologic analysis revealed that the OCP/Gel group showed radiopaque regions around the OCP granules and at the edge of the defect margin 4 weeks after implantation, suggesting that new bone formation occurred in two ways. In contrast, the rat femurs in the Gel group had a limited radiopaque zone at the edge of the defect region. The amount of new bone volume analyzed by micro-CT was higher in the OCP/Gel group than in the Gel group at 4 and 8 weeks after implantation (​​4 weeks after implantation: OCP/Gel versus Gel: 6.1 ± 1.6 mm 3 versus 3.4 ± 0.7 mm 3 , mean difference 2.7 [95% confidence interval (CI) 0.9 to 4.5]; p = 0.002; intraclass correlation coefficient [ICC] 0.72 [95% CI 0.29 to 0.91]; 8 weeks after implantation: OCP/Gel versus Gel: 3.9 ± 0.7 mm 3 versus 1.4 ± 1.1 mm 3 , mean difference 2.5 [95% CI 0.8 to 4.3]; p = 0.004; ICC 0.81 [95% CI 0.47 to 0.94]). Histologic evaluation also showed there was a higher percentage of new bone formation in the OCP/Gel group at 4 and 8 weeks after implantation (​​4 weeks after implantation: OCP/Gel versus Gel: 31.2% ± 5.3% versus 13.6% ± 4.0%, mean difference 17.6% [95% CI 14.2% to 29.2%]; p < 0.001; ICC 0.83 [95% CI 0.53 to 0.95]; 8 weeks after implantation: OCP/Gel versus Gel: 28.3% ± 6.2% versus 9.5% ± 1.9%, mean difference 18.8% [95% CI 11.3% to 26.3%]; p < 0.001; ICC 0.90 [95% CI 0.69 to 0.97]). Bridging of the defect area started earlier in the OCP/Gel group than in the Gel group at 4 weeks after implantation. Osteocalcin immunostaining showed that the number of mature osteoblasts was higher in the OCP/Gel group than in the Gel group at 4 weeks (OCP/Gel versus Gel: 42.1 ± 6.5/mm 2 versus 17.4 ± 5.4/mm 2 , mean difference 24.7 [95% CI 16.2 to 33.2]; p < 0.001; ICC 0.99 [95% CI 0.97 to 0.99]). At 4 weeks, the number of osteoclast-like cells was higher in the OCP/Gel composite group than in the Gel group (OCP/Gel versus Gel: 3.2 ± 0.6/mm 2 versus 0.9 ± 0.4/mm 2 , mean difference 2.3 [95% CI 1.3 to 3.5]; p < 0.001; ICC 0.79 [95% CI 0.35 to 0.94]). OCP/Gel composites induced early bone remodeling and cortical bone repair in less time than did the Gel control in a rat critical-size, transcortical femoral defect, suggesting that OCP/Gel could be used as a bone replacement material to treat severe bone defects. In a transcortical bone defect model of critical size in the rat femur, the OCP/Gel composite demonstrated successful bone regeneration. Several future studies are needed to evaluate the clinical application of this interesting bone graft substitute, including bone formation capacity in refractory fracture and spinal fusion models and the comparison of bone strength after repair with OCP/Gel composite to that of autologous bone.

Additively manufactured pure zinc porous scaffolds for critical-sized bone defects of rabbit femur.

Additive manufacturing has received attention for the fabrication of medical implants that have customized and complicated structures. Biodegradable Zn metals are revolutionary materials for orthopedic implants. In this study, pure Zn porous scaffolds with diamond structures were fabricated using customized laser powder bed fusion (L-PBF) technology. First, the mechanical properties, corrosion behavior, and biocompatibility of the pure Zn porous scaffolds were characterized in vitro. The scaffolds were then implanted into the rabbit femur critical-size bone defect model for 24 weeks. The results showed that the pure Zn porous scaffolds had compressive strength and rigidity comparable to those of cancellous bone, as well as relatively suitable degradation rates for bone regeneration. A benign host response was observed using hematoxylin and eosin (HE) staining of the heart, liver, spleen, lungs, and kidneys. Moreover, the pure Zn porous scaffold showed good biocompatibility and osteogenic promotion ability in vivo. This study showed that pure Zn porous scaffolds with customized structures fabricated using L-PBF represent a promising biodegradable solution for treating large bone defects.