A sponge-like nanofiber melatonin-loaded scaffold accelerates vascularized bone regeneration via improving mitochondrial energy metabolism.

Electrospun nanofibers have been widely employed in bone tissue engineering for their ability to mimic the micro to nanometer scale network of the native bone extracellular matrix. However, the dense fibrous structure and limited mechanical support of these nanofibers pose challenges for the treatment of critical size bone defects. In this study, we propose a facile approach for creating a three-dimensional scaffold using interconnected electrospun nanofibers containing melatonin (Scaffold@MT). The hypothesis posited that the sponge-like Scaffold@MT could potentially enhance bone regeneration and angiogenesis by modulating mitochondrial energy metabolism. Melatonin-loaded gelatin and poly-lactic-acid nanofibers were fabricated using electrospinning, then fragmented into shorter fibers. The sponge-like Scaffold@MT was created through a process involving homogenization, low-temperature lyophilization, and chemical cross-linking, while maintaining the microstructure of the continuous nanofibers. The incorporation of short nanofibers led to a low release of melatonin and increased Young's modulus of the scaffold. Scaffold@MT demonstrated positive biocompatibility by promoting a 14.2 % increase in cell proliferation. In comparison to the control group, Scaffold@MT significantly enhanced matrix mineralization by 3.2-fold and upregulated the gene expression of osteoblast-specific markers, thereby facilitating osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). Significantly, Scaffold@MT led to a marked enhancement in the mitochondrial energy function of BMMSCs, evidenced by elevated adenosine triphosphate (ATP) production, mitochondrial membrane potential, and protein expression of respiratory chain factors. Furthermore, Scaffold@MT promoted the migration of human umbilical vein endothelial cells (HUVECs) and increased tube formation by 1.3 times compared to the control group, accompanied by an increase in vascular endothelial growth factor (VEGFA) expression. The results of in vivo experiments indicate that the implantation of Scaffold@MT significantly improved vascularized bone regeneration in a distal femur defect in rats. Micro-computed tomography analysis conducted 8 weeks post-surgery revealed that Scaffold@MT led to optimal development of new bone microarchitecture. Histological and immunohistochemical analyses demonstrated that Scaffold@MT facilitated bone matrix deposition and new blood vessel formation at the defect site. Overall, the utilization of melatonin-loaded nanofiber sponges exhibits significant promise as a scaffold that promotes bone growth and angiogenesis, making it a viable option for the repair of critical-sized bone defects.

Nanofiber-induced hierarchically-porous magnesium phosphate bone cements accelerate bone regeneration by inhibiting Notch signaling.

Magnesium phosphate bone cements (MPC) have been recognized as a viable alternative for bone defect repair due to their high mechanical strength and biodegradability. However, their poor porosity and permeability limit osteogenic cell ingrowth and vascularization, which is critical for bone regeneration. In the current study, we constructed a novel hierarchically-porous magnesium phosphate bone cement by incorporating extracellular matrix (ECM)-mimicking electrospun silk fibroin (SF) nanofibers. The SF-embedded MPC (SM) exhibited a heterogeneous and hierarchical structure, which effectively facilitated the rapid infiltration of oxygen and nutrients as well as cell ingrowth. Besides, the SF fibers improved the mechanical properties of MPC and neutralized the highly alkaline environment caused by excess magnesium oxide. Bone marrow stem cells (BMSCs) adhered excellently on SM, as illustrated by formation of more pseudopodia. CCK8 assay showed that SM promoted early proliferation of BMSCs. Our study also verified that SM increased the expression of OPN, RUNX2 and BMP2, suggesting enhanced osteogenic differentiation of BMSCs. We screened for osteogenesis-related pathways, including FAK signaing, Wnt signaling and Notch signaling, and found that SM aided in the process of bone regeneration by suppressing the Notch signaling pathway, proved by the downregulation of NICD1, Hes1 and Hey2. In addition, using a bone defect model of rat calvaria, the study revealed that SM exhibited enhanced osteogenesis, bone ingrowth and vascularization compared with MPC alone. No adverse effect was found after implantation of SM in vivo. Overall, our novel SM exhibited promising prospects for the treatment of critical-sized bone defects.

Towards Stem Cell Therapy for Critical-Sized Segmental Bone Defects: Current Trends and Challenges on the Path to Clinical Translation.

The management and reconstruction of critical-sized segmental bone defects remain a major clinical challenge for orthopaedic clinicians and surgeons. In particular, regenerative medicine approaches that involve incorporating stem cells within tissue engineering scaffolds have great promise for fracture management. This narrative review focuses on the primary components of bone tissue engineering-stem cells, scaffolds, the microenvironment, and vascularisation-addressing current advances and translational and regulatory challenges in the current landscape of stem cell therapy for critical-sized bone defects. To comprehensively explore this research area and offer insights for future treatment options in orthopaedic surgery, we have examined the latest developments and advancements in bone tissue engineering, focusing on those of clinical relevance in recent years. Finally, we present a forward-looking perspective on using stem cells in bone tissue engineering for critical-sized segmental bone defects.

Stem Cell Membrane-Coated Microribbon Scaffolds Induce Regenerative Innate and Adaptive Immune Responses in a Critical-Size Cranial Bone Defect Model.

Naturally-derived cell membranes have shown great promise in functionalizing nanoparticles to enhance biointerfacing functions for drug delivery applications. However, its potential for functionalizing macroporous scaffolds to enhance tissue regeneration in vivo remains unexplored. Engineering scaffolds with immunomodulatory functions represents an exciting strategy for tissue regeneration but is largely limited to soft tissues. Critical-sized bone defects cannot heal on their own, and the role of adaptive immune cells in scaffold-mediated healing of cranial bone defects remains largely unknown. Here, mensenchymal stem cell membrane (MSCM)-coated microribbon (µRB) scaffolds for treating critical size cranial bone defects via targeting immunomodulation are reported. Confocal imaging and proteomic analyses are used to confirm successful coating and characterize the compositions of cell membrane coating. It is demonstrated that MSCM coating promotes macrophage (Mφ) polarization toward regenerative phenotype, induces CD8+ T cell apoptosis, and enhances regulatory T cell differentiation in vitro and in vivo. When combined with a low dosage of BMP-2, MSCM coating further accelerates bone regeneration and suppresses inflammation. These results establish cell membrane-coated microribbon scaffolds as a promising strategy for treating critical size bone defects via immunomodulation. The platform may be broadly used with different cell membranes and scaffolds to enhance regeneration of multiple tissue types.

Bone Marrow Stem Cells with Tissue-Engineered Scaffolds for Large Bone Segmental Defects: A Systematic Review.

Critical-sized bone defects (CSBDs) represent a significant clinical challenge, stimulating researchers to seek new methods for successful bone reconstruction. The aim of this systematic review is to assess whether bone marrow stem cells (BMSCs) combined with tissue-engineered scaffolds have demonstrated improved bone regeneration in the treatment of CSBD in large preclinical animal models. A search of electronic databases (PubMed, Embase, Web of Science, and Cochrane Library) focused on in vivo large animal studies identified 10 articles according to the following inclusion criteria: (1) in vivo large animal models with segmental bone defects; (2) treatment with tissue-engineered scaffolds combined with BMSCs; (3) the presence of a control group; and (4) a minimum of a histological analysis outcome. Animal research: reporting of in Vivo Experiments guidelines were used for quality assessment, and Systematic Review Center for Laboratory animal Experimentation's risk of bias tool was used to define internal validity. The results demonstrated that tissue-engineered scaffolds, either from autografts or allografts, when combined with BMSCs provide improved bone mineralization and bone formation, including a critical role in the remodeling phase of bone healing. BMSC-seeded scaffolds showed improved biomechanical properties and microarchitecture properties of the regenerated bone when compared with untreated and scaffold-alone groups. This review highlights the efficacy of tissue engineering strategies for the repair of extensive bone defects in preclinical large-animal models. In particular, the use of mesenchymal stem cells, combined with bioscaffolds, seems to be a successful method in comparison to cell-free scaffolds.

Point-of-care treatment of geometrically complex midfacial critical-sized bone defects with 3D-Printed scaffolds and autologous stromal vascular fraction.

Critical-sized midfacial bone defects present a unique clinical challenge due to their complex three-dimensional shapes and intimate associations with sensory organs. To address this challenge, a point-of-care treatment strategy for functional, long-term regeneration of 2 cm full-thickness segmental defects in the zygomatic arches of Yucatan minipigs is evaluated. A digital workflow is used to 3D-print anatomically precise, porous, biodegradable scaffolds from clinical-grade poly-ε-caprolactone and decellularized bone composites. The autologous stromal vascular fraction of cells (SVF) is isolated from adipose tissue extracts and infused into the scaffolds that are implanted into the zygomatic ostectomies. Bone regeneration is assessed up to 52 weeks post-operatively in acellular (AC) and SVF groups (BV/DV = 0.64 ± 0.10 and 0.65 ± 0.10 respectively). In both treated groups, bone grows from the adjacent tissues and restores the native anatomy. Significantly higher torque is required to fracture the bone-scaffold interface in the SVF (7.11 ± 2.31 N m) compared to AC groups (2.83 ± 0.23 N m). Three-dimensional microcomputed tomography analysis reveals two distinct regenerative patterns: osteoconduction along the periphery of scaffolds to form dense lamellar bone and small islands of woven bone deposits growing along the struts in the scaffold interior. Overall, this study validates the efficacy of using 3D-printed bioactive scaffolds with autologous SVF to restore geometrically complex midfacial bone defects of clinically relevant sizes while also highlighting remaining challenges to be addressed prior to clinical translation.

Functional improvement of collagen-based bioscaffold to enhance periodontal-defect healing via combination with dietary antioxidant and COMP-angiopoietin 1.

Scaffolds combined with bioactive agents can enhance bone regeneration at therapeutic sites. We explore whether combined supplementation with coumaric acid and recombinant human-cartilage oligomeric matrix protein-angiopoietin 1 (rhCOMP-Ang1) is an ideal approach for bone tissue engineering. We developed coumaric acid-conjugated absorbable collagen scaffold (CA-ACS) and investigated whether implanting CA-ACS in combination with rhCOMP-Ang1 facilitates ACS- or CA-ACS-mediated bone formation using a rat model of critically sized mandible defects. We examined the mechanisms by which coumaric acid and rhCOMP-Ang1 regulate behaviors of human periodontal ligament fibroblasts (hPLFs). The CA-ACS exhibits greater anti-degradation and mechanical strength properties than does ACS alone. Implanting CA-ACS loaded with rhCOMP-Ang1 greatly enhances bone regeneration at the defect via the activation of angiogenic, osteogenic, and anti-osteoclastic responses compared with other rat groups implanted with an ACS alone or CA-ACS. Treatment with both rhCOMP-Ang1 and coumaric acid increases proliferation, mineralization, and migration of cultured hPLFs via activation of the Ang1/Tie2 signaling axis at a greater rate than treatment with either of them alone. Collectively, this study demonstrates that CA-ACS impregnated with rhCOMP-Ang1 enhances bone regeneration at therapeutic sites, and this enhancement is associated with a synergistic interaction between rhCOMP-Ang1-mediated angiogenesis and coumaric acid-related antioxidant responses.

Systemic and local innate immune responses to surgical co-transplantation of mesenchymal stromal cells and biphasic calcium phosphate for bone regeneration.

Bone regeneration from mesenchymal stromal cells (MSC) is attributed to comprehensive immune modulation mediated by the MSC. However, the temporal and spatial regulation of these immune responses has not yet been described. The aim of the present study was to assess the local and systemic innate immune responses to implantation of biphasic calcium phosphate biomaterial (BCP) alone, or with bone marrow derived MSC (BCP+MSC), in critical-sized calvarial bone defects of Lewis rats. Four weeks after implantation, flow cytometry analysis of innate immune cells revealed increased numbers of circulating classical monocyte-macrophages (MM) and decreased non-classical MM in the BCP+MSC group. At week 8, this differential systemic MM response was associated with an increased presence of local tissue anti-inflammatory macrophages expressing CD68 and CD163 markers (M2-like). In the BCP group without MSC, NK cells increased at weeks 1 and 4, and neutrophils increased in circulation at weeks 2 and 8. At week 8, the increase in number of neutrophils in circulation was associated with decreased local tissue neutrophils, in the BCP+MSC group. Gene expression analysis of tissue biopsies from defects implanted with BCP+MSC, in comparison to BCP alone, revealed upregulated expression of early osteogenesis genes along with macrophage differentiation-related genes at weeks 1 and 8 and neutrophil chemotaxis-related genes at week 1. This study is the first to demonstrate that surgical implantation of BCP or BCP+MSC grafts differentially regulate both systemic and local tissue innate immune responses which enhance bone formation. The results provide new insights into immune mechanisms underlying MSC-mediated bone regeneration. STATEMENT OF SIGNIFICANCE: The suitability of biphasic calcium phosphate and mesenchymal stromal cell construct (BCP+MSC) transplantation is evident from their progress in clinical trials for treating challenging maxillofacial bone defects. But less is known about the overall immune response generated by this surgical process and how it later impacts the bone formation. To this end, it is crucial to understand for both clinicians and researchers, the systemic immune response to transplanting MSC in patients for ensuring both the safety and efficacy of cell therapies. In this study, we used rat calvarial bone defect model and showed that both systemic and local innate immunes responses (monocyte-macrophages and neutrophils) are favorably directed towards enhanced bone formation in BCP+MSC implanted defects, as compared to BCP alone.

In vivo effectiveness of hybrid membranes with osteogenic growth peptide for bone regeneration.

Guided bone regeneration (GBR) technique helps to restore bone tissue through cellular selectivity principle. Currently no osteoinductive membrane exists on the market. Osteogenic growth peptide (OGP) acts as a hematopoietic stimulator. This association could improve the quality of bone formation, benefiting more than 2.2 million patients annually. The objective of this work was to develop membranes from ureasil-polyether materials containing OGP. The membranes were characterized by differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS). OGP was synthesized by the solid phase method. Sterilization results using gamma radiation at 24 kGy did not change the structure of the material, as confirmed by DSC. The SAXS technique revealed the structural homogeneity of the matrix. OGP was incorporated in 66.25 × 10-10  mol and release results showed that the ureasil-PPO400/PEO500 and ureasil-PPO400/PEO1900 membranes released 7% and 21%, respectively, after 48 h. In vivo results demonstrated that the amount and quality of bone tissue formed in the bone defects in the presence of ureasil-polyether membranes with OGP were similar to commercial collagen material with BMP. The results allow us to conclude that membranes with OGP have characteristics that make them potential candidates for the GBR.

The effect of enhanced bone marrow in conjunction with 3D-printed PLA-HA in the repair of critical-sized bone defects in a rabbit model.

BACKGROUND: Traditionally, the iliac crest has been the most common harvesting site for autologous bone grafts; however, it has some limitations, including poor bone availability and donor-site morbidity. This study sought to explore the effect of enhanced bone marrow (eBM) in conjunction with three-dimensional (3D)-printed polylactide-hydroxyapatite (PLA-HA) scaffolds in the repair of critical-sized bone defects in a rabbit model. METHODS: First, 3D-printed PLA-HA scaffolds were fabricated and evaluated using micro-computed tomography (µCT) and scanning electron microscopy (SEM). Twenty-seven New Zealand white rabbits were randomly divided into 3 groups (n=9 per group), and the defects were treated using 3D-printed PLA-HA scaffolds (the PLA-HA group) or eBM in conjunction with 3D-printed PLA-HA scaffolds (the PLA-HA/eBM group), or were left untreated (the control group). Radiographic, µCT, and histological analyses were performed to evaluate bone regeneration in the different groups. RESULTS: The 3D-printed PLA-HA scaffolds were cylindrical, and had a mean pore size of 500±47.1 µm and 60%±3.5% porosity. At 4 and 8 weeks, the lane-sandhu X-ray score in the PLA-HA/eBM group was significantly higher than that in the PLA-HA group and the control group (P<0.01). At 8 weeks, the µCT analysis showed that the bone volume (BV) and bone volume/tissue volume (BV/TV) in the PLA-HA/eBM group were significantly higher than those in the PLA-HA group and the control group (P<0.01). Hematoxylin and eosin staining indicated that the new bone area in the PLA-HA/eBM group was significantly higher than that in the PLA-HA group and the control group (P<0.01). CONCLUSIONS: The group that was treated with eBM in conjunction with 3D-printed PLA-HA showed enhanced bone repair compared to the other 2 groups. PLA-HA/eBM scaffolds represent a promising way to treat critical-sized bone defects.

Gingival mesenchymal stem cells as an alternative source to bone marrow mesenchymal stem cells in regeneration of bone defects: In vivo study.

Healing of critical sized bone defects represents a challenging issue in clinical and research fields. Current therapeutic techniques, such as bone grafts or bone grafts substitutes, still have limitations and drawbacks. Therefore, stem cell-based therapy provides a prospective approach to enhance bone regeneration. The present study aimed to assess the regenerative capacity of Gingival mesenchymal stem cells (GMSCs) as well as Bone marrow mesenchymal stem cells (BMSCs) loaded on NanoBone scaffold, in comparison to the unloaded one, in surgically created bone defects in rabbits' tibiae. To achieve this aim, critical sized bone defects, of 6-mm diameter each, were unilaterally created in tibiae of adult New Zeeland male white rabbits (n = 27). The rabbits were then divided randomly into three groups (9 each) and received the following: Group I: Unloaded NanoBone Scaffold, Group II: GMSCs Loaded on NanoBone Scaffold, and Group III: BMSCs Loaded on NanoBone Scaffold. Three rabbits from each group were then sacrificed at each time point (2, 4 and 6 weeks postoperatively), tibiae were dissected out to evaluate bone healing in the created bony defects; both histologically and histomorphometrically. The findings of this study indicate that both GMSCs and BMSCs exhibited fibroblast morphology and expressed phenotypic MSCs markers. Histologically, local application of GMSCs and BMSCs loaded on NanoBone scaffold showed enhanced the pattern of bone regeneration as compared to the unloaded scaffold. Histomorphometrically, there was astatistically insignificant difference in the new bone area % between the bony defects treated with GMSCs and BMSCs. Thus, GMSCs can be considered as a comparable alternative source to BMSCs in bone regeneration.

A Novel Bone Substitute with High Bioactivity, Strength, and Porosity for Repairing Large and Load-Bearing Bone Defects.

Achieving adequate healing in large or load-bearing bone defects is highly challenging even with surgical intervention. The clinical standard of repairing bone defects using autografts or allografts has many drawbacks. A bioactive ceramic scaffold, strontium-hardystonite-gahnite or "Sr-HT-Gahnite" (a multi-component, calcium silicate-based ceramic) is developed, which when 3D-printed combines high strength with outstanding bone regeneration ability. In this study, the performance of purely synthetic, 3D-printed Sr-HT-Gahnite scaffolds is assessed in repairing large and load-bearing bone defects. The scaffolds are implanted into critical-sized segmental defects in sheep tibia for 3 and 12 months, with bone autografts used for comparison. The scaffolds induce substantial bone formation and defect bridging after 12 months, as indicated by X-ray, micro-computed tomography, and histological and biomechanical analyses. Detailed analysis of the bone-scaffold interface using focused ion beam scanning electron microscopy and multiphoton microscopy shows scaffold degradation and maturation of the newly formed bone. In silico modeling of strain energy distribution in the scaffolds reveal the importance of surgical fixation and mechanical loading on long-term bone regeneration. The clinical application of 3D-printed Sr-HT-Gahnite scaffolds as a synthetic bone substitute can potentially improve the repair of challenging bone defects and overcome the limitations of bone graft transplantation.

Functional reconstruction of critical-sized load-bearing bone defects using a Sclerostin-targeting miR-210-3p-based construct to enhance osteogenic activity.

A considerable amount of research has focused on improving regenerative therapy strategies for repairing defects in load-bearing bones. The enhancement of tissue regeneration with microRNAs (miRNAs) is being developed because miRNAs can simultaneously regulate multiple signaling pathways in an endogenous manner. In this study, we developed a miR-210-based bone repair strategy. We identified a miRNA (miR-210-3p) that can simultaneously up-regulate the expression of multiple key osteogenic genes in vitro. This process resulted in enhanced bone formation in a subcutaneous mouse model with a miR-210-3p/poly-l-lactic acid (PLLA)/bone marrow-derived stem cell (BMSC) construct. Furthermore, we constructed a model of critical-sized load-bearing bone defects and implanted a miR-210-3p/ß-tricalcium phosphate (ß-TCP)/bone mesenchymal stem cell (BMSC) construct into the defect. We found that the load-bearing defect was almost fully repaired using the miR-210-3p construct. We also identified a new mechanism by which miR-210-3p regulates Sclerostin protein levels. This miRNA-based strategy may yield novel therapeutic methods for the treatment of regenerative defects in vital load-bearing bones by utilizing miRNA therapy for tissue engineering. STATEMENT OF SIGNIFICANCE: The destroyed maxillofacial bone reconstruction is still a real challenge for maxillofacial surgeon, due to that functional bone reconstruction involved load-bearing. Base on the above problem, this paper developed a novel miR-210-3p/ß-tricalcium phosphate (TCP)/bone marrow-derived stem cell (BMSC) construct (miR-210-3p/ß-TCP/BMSCs), which lead to functional reconstruction of critical-size mandible bone defect. We found that the load-bearing defect was almost fully repaired using the miR-210-3p construct. In addition, we also found the mechanism of how the delivered microRNA activated the signaling pathways of endogenous stem cells, leading to the defect regeneration. This miRNA-based strategy can be used to regenerate defects in vital load-bearing bones, thus addressing a critical challenge in regenerative medicine by utilizing miRNA therapy for tissue engineering.

Harnessing extracellular vesicles to direct endochondral repair of large bone defects.

Large bone defects remain a tremendous clinical challenge. There is growing evidence in support of treatment strategies that direct defect repair through an endochondral route, involving a cartilage intermediate. While culture-expanded stem/progenitor cells are being evaluated for this purpose, these cells would compete with endogenous repair cells for limited oxygen and nutrients within ischaemic defects. Alternatively, it may be possible to employ extracellular vesicles (EVs) secreted by culture-expanded cells for overcoming key bottlenecks to endochondral repair, such as defect vascularization, chondrogenesis, and osseous remodelling. While mesenchymal stromal/stem cells are a promising source of therapeutic EVs, other donor cells should also be considered. The efficacy of an EV-based therapeutic will likely depend on the design of companion scaffolds for controlled delivery to specific target cells. Ultimately, the knowledge gained from studies of EVs could one day inform the long-term development of synthetic, engineered nanovesicles. In the meantime, EVs harnessed from in vitro cell culture have near-term promise for use in bone regenerative medicine. This narrative review presents a rationale for using EVs to improve the repair of large bone defects, highlights promising cell sources and likely therapeutic targets for directing repair through an endochondral pathway, and discusses current barriers to clinical translation. Cite this article: E. Ferreira, R. M. Porter. Harnessing extracellular vesicles to direct endochondral repair of large bone defects. Bone Joint Res 2018;7:263-273. DOI: 10.1302/2046-3758.74.BJR-2018-0006.