Critical-sized Bone Defects Research Articles

Enhanced vascularity in gelatin scaffolds via copper-doped magnesium–calcium silicates incorporation: In-vitro and ex-ovo insights

Addressing a critical challenge in current tissue-engineering practices, this study aims to enhance vascularization in 3D porous scaffolds by incorporating bioceramics laden with pro-angiogenic ions. Specifically, freeze-dried gelatin-based scaffolds were infused with sol-gel-derived powders of Cu-doped akermanite (Ca2MgSi2O7) and bredigite (Ca7MgSi4O16) at various concentrations (10, 20, and 30 wt%). The scaffolds were initially characterized for their structural integrity, biodegradability, swelling behavior, impact on physiological pH, and cytocompatibility with human umbilical vein endothelial cells (HUVECs). The silicate incorporation effectiveness in promoting vascularity was then assessed through HUVEC attachment, capillary tube formation, and ex-ovo chick embryo chorioallantoic membrane assays. The findings revealed significant improvements in both in-vitro and ex-ovo vascularity of the gelatin scaffolds upon the addition of Cu-doped akermanite. The most effective concentrations were determined to be 10 and 20 %, which led to notable HUVEC metabolic activity, a well-spread morphology with extensive peripheral filopodia and lamellipodia at 10 % and a cobblestone phenotype indicative of in-vivo endothelium at 20 % during cell attachment, the formation of complex networks of tubular structures, and robust vascularization in chick embryo development. Moving forward, the incorporation of Cu-doped akermanite into tissue-engineering scaffolds shows great potential for addressing the limitations of vascularization, especially for critical-sized bone defects, by facilitating the controlled release of pro-angiogenic and pro-osteogenic ions.

Driving Osteocytogenesis from Mesenchymal Stem Cells in Osteon-like Biomimetic Nanofibrous Scaffolds.

The treatment of critical-sized bone defects caused by tumor removal, skeletal injuries, or infections continues to pose a major clinical challenge. A popular potential alternative solution to autologous bone grafts is a tissue-engineered approach that utilizes the combination of mesenchymal stromal/stem cells (MSCs) with synthetic biomaterial scaffolds. This approach aims to support new bone formation by mimicking many of the biochemical and biophysical cues present within native bone. Regrettably, osteocyte cells, crucial for bone maturation and homeostasis, are rarely produced within MSC-seeded scaffolds, thereby restricting the development of fully mature cortical bone from these synthetic implants. In this work, we have constructed a multimodal scaffold by combining electrospun poly(lactic-co-glycolic acid) (PLGA) fibrous scaffolds with poly(ethylene glycol) (PEG)-based hydrogels that mimic the functional unit of cortical bone, osteon (osteon-mimetic) scaffolds. These scaffolds were decorated with a novel bone morphogenic protein-6 (BMP6) peptide (BMP6p) after our findings revealed that the BMP6p drives higher levels of Smad signaling than the full-length protein counterpart, soluble or when bound to the PEG hydrogel backbone. We show that our osteon-mimetic scaffolds, in presenting concentric layers of BMP6p-PEG hydrogel overlaid on MSC-seeded PLGA nanofibers, promoted the rapid formation of osteocyte-like cells with a phenotypic dendritic morphology, producing early osteocyte markers, including E11/gp38 (E11). Maturation of these osteocyte-like cells was further confirmed by the observation of significant dentin matrix protein 1 (DMP1) throughout our bilayered scaffolds after 3 weeks, even when cultured in a medium without dexamethasone (DEX) or any other osteogenic supplements. These results demonstrate that these osteon-mimetic scaffolds, in presenting biochemical and topographical cues reminiscent of the forming osteon, can drive the formation of osteocyte-like cells in vitro from hBMSCs without the need for any osteogenic factor media supplementation.

Applying 3D-printed prostheses to reconstruct critical-sized bone defects of tibial diaphysis (> 10 cm) caused by osteomyelitis and aseptic non-union

BackgroundClinical repair of critical-sized bone defects (CBDs) in the tibial diaphysis presents numerous challenges, including inadequate soft tissue coverage, limited blood supply, high load-bearing demands, and potential deformities. This study aimed to investigate the clinical feasibility and efficacy of employing 3D-printed prostheses for repairing CBDs exceeding 10 cm in the tibial diaphysis.MethodsThis retrospective study included 14 patients (11 males and 3 females) with an average age of 46.0 years. The etiologies of CBDs comprised chronic osteomyelitis (10 cases) and aseptic non-union (4 cases), with an average defect length of 16.9 cm. All patients underwent a two-stage surgical approach: (1) debridement, osteotomy, and cement spacer implantation; and (2) insertion of 3D-printed prostheses. The interval between the two stages ranged from 8 to 12 weeks, during which the 3D-printed prostheses and induced membranes were meticulously prepared. Subsequent to surgery, patients engaged in weight-bearing and functional exercises under specialized supervision. Follow-up assessments, including gross observation, imaging examinations, and administration of the Lower Extremity Functional Scale (LEFS), were conducted at 3, 6, and 12 months postoperatively, followed by annual evaluations thereafter.ResultsThe mean postoperative follow-up duration was 28.4 months, with an average waiting period between prosthesis implantation and weight-bearing of 10.4 days. At the latest follow-up, all patients demonstrated autonomous ambulation without assistance, and their LEFS scores exhibited a significant improvement compared to preoperative values (30.7 vs. 53.1, P < 0.001). Imaging assessments revealed progressive bone regeneration at the defect site, with new bone formation extending along the prosthesis. Complications included interlocking screw breakage in two patients, interlocking screw loosening in one patient, and nail breakage in another.ConclusionsUtilization of 3D-printed prostheses facilitates prompt restoration of CBDs in the tibial diaphysis, enabling early initiation of weight-bearing activities and recovery of ambulatory function. This efficacious surgical approach holds promise for practical application.

Synergistic Interaction between Polysaccharide-Based Extracellular Matrix and Mineralized Osteoblast-Derived EVs Promotes Bone Regeneration via miRNA-mRNA Regulatory Axis.

Extracellular vesicles (EVs) derived from bone progenitor cells are advantageous as cell-free and non-immunogenic cargo delivery vehicles. In this study, EVs are isolated from MC3T3-E1 cells before (GM-EVs) and after mineralization for 7 and 14 days (DM-EVs). It was observed that DM-EVs accelerate the process of differentiation in recipient cells more prominently. The small RNA sequencing of EVs revealed that miR-204-5p, miR-221-3p, and miR-148a-3p are among the highly upregulated miRNAs that have an inhibitory effect on the function of mRNAs, Sox11, Timp3, and Ccna2 in host cells, which is probably responsible for enhancing the activity of osteoblastic genes. To enhance the bioavailability of EVs, they are encapsulated in a chitosan-collagen composite hydrogel that serves as a bioresorbable extracellular matrix (ECM). The EVs-integrated scaffold (DM-EVs + Scaffold) enhances bone regeneration in critical-sized calvarial bone defects in rats within 8 weeks of implantation by providing the ECM cues. The shelf life of DM-EVs + Scaffold indicates that the bioactivity of EVs and their cargo in the polymer matrix remains intact for up to 30 days. Integrating mineralized cell-derived EVs into an ECM represents a bioresorbable matrix with a cell-free method for promoting new bone formation through the miRNA-mRNA regulatory axis.

Mobilizing Endogenous Progenitor Cells Using pSDF1α-Activated Scaffolds Accelerates Angiogenesis and Bone Repair in Critical-Sized Bone Defects.

Mobilizing endogenous progenitor cells to repair damaged tissue in situ has the potential to revolutionize the field of regenerative medicine, while the early establishment of a vascular network will ensure survival of newly generated tissue. In this study, a gene-activated scaffold containing a stromal derived factor 1α plasmid (pSDF1α), a pro-angiogenic gene that is also thought to be involved in the recruitment of mesenchymal stromal cells (MSCs) to sites of injury is described. It is shown that over-expression of SDF1α protein enhanced MSC recruitment and induced vessel-like structure formation by endothelial cells in vitro. When implanted subcutaneously, transcriptomic analysis reveals that endogenous MSCs are recruited and significant angiogenesis is stimulated. Just 1-week after implantation into a calvarial critical-sized bone defect, pSDF1α-activated scaffolds are recruited MSCs and rapidly activate angiogenic and osteogenic programs, upregulating Runx2, Dlx5, and Sp7. At the same time-point, pVEGF-activated scaffolds are recruited a variety of cell types, activating endochondral ossification. The early response induced by both scaffolds leads to complete bridging of the critical-sized bone defects within 4-weeks. The versatile cell-free gene-activated scaffold described in this study is capable of harnessing and enhancing the body's own regenerative capacity and has immense potential in a myriad of applications.

Cellular Scale Curvature in Bioceramic Scaffolds Enhanced Bone Regeneration by Regulating Skeletal Stem Cells and Vascularization.

Critical-sized segmental bone defects cannot heal spontaneously, leading to disability and significant increase in mortality. However, current treatments utilizing bone grafts face a variety of challenges from donor availability to poor osseointegration. Drugs such as growth factors increase cancer risk and are very costly. Here, a porous bioceramic scaffold that promotes bone regeneration via solely mechanobiological design is reported. Two types of scaffolds with high versus low pore curvatures are created using high-precision 3D printing technology to fabricate pore curvatures radius in the 100s of micrometers. While both are able to support bone formation, the high-curvature pores induce higher ectopic bone formation and increased vessel invasion. Scaffolds with high-curvature pores also promote faster regeneration of critical-sized segmental bone defects by activating mechanosensitive pathways. High-curvature pore recruits skeletal stem cells and type H vessels from both the periosteum and the marrow during the early phase of repair. High-curvature pores have increased survival of transplanted GFP-labeled skeletal stem cells (SSCs) and recruit more host SSCs. Taken together, the bioceramic scaffolds with defined micrometer-scale pore curvatures demonstrate a mechanobiological approach for orthopedic scaffold design.

Polymer-ceramic composites for fused deposition modeling of biomimetic bone scaffolds

Current surgical treatments for segmental bone loss do not reliably result in healing, typically necessitate multiple surgeries, and may result in amputation. Although we have demonstrated that polybutylene terephthalate (PBT) scaffolds can regenerate large critical size bone defects, the material is non-resorbable and may lead to problems including acting as a nidus for infection. New strategies have emerged that use resorbable polymers, ceramics, and composite materials to produce patient specific scaffolds that stimulate rapid bone formation without retention of foreign material. This work describes the production of a composite polymer ceramic material composed of polylactic acid (PLA) and tricalcium phosphate (TCP) that can be used with additive manufacturing techniques to rapidly produce scaffolds with custom geometries. The technique we describe can be used to produce composite materials with a ratio of up to 50-50 PLA-TCP that is suitable for fused deposition modeling and does not necessitate the use of solvents. In this study the properties of PBT, PLA, 75-25 PLA-TCP, and 50-50 PLA-TCP materials are compared. PLA and PLA-TCP materials had a higher compressive elastic modulus compared to PBT, while composite PLA-TCP materials had a higher tensile elastic modulus compared to PLA and PBT. Cell culture experiments demonstrated that all materials were non-toxic to cells. Optimal printing parameters are provided for using pure polymer and composite materials for fused deposition modeling. The results of this study demonstrate that resorbable polymer ceramic materials with a high ceramic composition can be produced for additive manufacturing without necessitating solvents.

An Intact Periosteum is Required for Recombinant Human Jagged1 Guided Bone Regeneration in Calvaria Critical-size Defect Healing.

The need to promote calvaria bone healing as a consequence of injury or craniotomy is a major clinical issue. Previous reports tested recombinant human Jagged1 (rhJagged1) treatment for critical-size calvaria defects in the absence of periosteum, and this resulted in significant new bone formation. As the periosteum contributes to healing by serving as a source of progenitor cells, the present study aimed to examine whether significantly more bone is formed when the periosteum is intact for using rhJagged1 to treat critical-size parietal bone defects in mice. Fifteen healthy adult mice, 34 to 65 weeks of age, 26.9 to 48.2 g, were divided into different groups that compared the critical-size defects treated with either phosphate-buffered saline or rhJagged1 protein in either the presence or absence of periosteum. The results indicated that more bone was formed in the presence of periosteum when rhJagged1 is delivered [35% bone volume per tissue volume (BV/TV); P = 0.02] relative to nonperiosteum. Recombinant human Jagged1 protein delivered in the absence of periosteum had the next most new bone formed (25% BV/TV). Defects with phosphate-buffered saline delivered in the absence or presence of periosteum had the least new bone formed (15% and 18% BV/TV, respectively; P = 0.48). The results also show that rhJagged1 does not form ectopic or hypertrophic bone. The usage of rhJagged1 to treat critical-size defects in calvaria is promising clinically, but to maximize clinical efficacy it will require that the periosteum be intact on the noninjured portions of calvaria.

Effect of Local Simvastatin on the Healing of Surgically Created Critical-Sized Bone Defects: An experimental Study on sheep.

Background and objective: the role of simvastatin in lowering serum cholesterol levels is well described. However, recent findings suggest they have a role in bone formation as well. The study aims to determine the effect of local simvastatin application on bone defect healing and compare the amount of new bone produced by a simvastatin-treated defect with that produced by a bone graft (biphasic calcium phosphate) and non-treated defects (left empty) histologically. Methods: Forty-five critical-size defects were created (8mm in diameter and depth) in the iliac bone of 6 sheep. For the first three sheep (5 defects/ilium), the five defects on the right ilium were left empty as a Control group, while the five defects on the left ilium were filled with biphasic calcium phosphate as Test 1 group. For the other three sheep, 5 defects were created on the right ilium, the defects were filled with 10mg crushed simvastatin tablet with gelfoam (as a carrier) as Test 2 group. The animals were sacrificed over periods of 1, 2, and 3 months. Histopathological studies were done for all the samples. SPSS version 28 was used to analyze the results. The numerical variables were checked for normality using Smirnov – Kolmogorov test, then analyzed using ANOVA and unpaired t-test (p–values ≤ 0.05 were considered statistically significant). Results: All 6 adult male sheep passed the scheduled periods uneventfully. During the wound healing period, there was no complication such as infection, excessive hematoma, or wound dehiscence. All 45 standardized iliac bone defects were included in the final analysis (n= 45). The histologic results showed that Test 2 group (defect filled with simvastatin) in the 1st, 2nd, and 3rd months had significantly higher bone formation at the surface and depth of the defects than Test 1 and Control group with P values (&lt;0.0001) at all period intervals. Conclusion: Simvastatin enhances bone formation and accelerates the healing process of the bony defect. Keywords: Bone formation, Bone graft, Bone healing, Simvastatin, Statin.

Vascular restoration through local delivery of angiogenic factors stimulates bone regeneration in critical size defects

Critical size bone defects represent a significant challenge worldwide, often leading to persistent pain and physical disability that profoundly impact patients’ quality of life and mental well-being. To address the intricate and complex repair processes involved in these defects, we performed single-cell RNA sequencing and revealed notable shifts in cellular populations within regenerative tissue. Specifically, we observed a decrease in progenitor lineage cells and endothelial cells, coupled with an increase in fibrotic lineage cells and pro-inflammatory cells within regenerative tissue. Furthermore, our analysis of differentially expressed genes and associated signaling pathway at the single-cell level highlighted impaired angiogenesis as a central pathway in critical size bone defects, notably influenced by reduction of Spp1 and Cxcl12 expression. This deficiency was particularly pronounced in progenitor lineage cells and myeloid lineage cells, underscoring its significance in the regeneration process. In response to these findings, we developed an innovative approach to enhance bone regeneration in critical size bone defects. Our fabrication process involves the integration of electrospun PCL fibers with electrosprayed PLGA microspheres carrying Spp1 and Cxcl12. This design allows for the gradual release of Spp1 and Cxcl12 in vitro and in vivo. To evaluate the efficacy of our approach, we locally applied PCL scaffolds loaded with Spp1 and Cxcl12 in a murine model of critical size bone defects. Our results demonstrated restored angiogenesis, accelerated bone regeneration, alleviated pain responses and improved mobility in treated mice.

PH-Responsive Mesoporous Silica Nanoparticles Loaded with Naringin for Targeted Osteoclast Inhibition and Bone Regeneration.

It is well-established that osteoclast activity is significantly influenced by fluctuations in intracellular pH. Consequently, a pH-sensitive gated nano-drug delivery system represents a promising therapeutic approach to mitigate osteoclast overactivity. Our prior research indicated that naringin, a natural flavonoid, effectively mitigates osteoclast activity. However, naringin showed low oral availability and short half-life, which hinders its clinical application. We developed a drug delivery system wherein chitosan, as gatekeepers, coats mesoporous silica nanoparticles loaded with naringin (CS@MSNs-Naringin). However, the inhibitory effects of CS@MSNs-Naringin on osteoclasts and the underlying mechanisms remain unclear, warranting further research. First, we synthesized CS@MSNs-Naringin and conducted a comprehensive characterization. We also measured drug release rates in a pH gradient solution and verified its biosafety. Subsequently, we investigated the impact of CS@MSNs-Naringin on osteoclasts induced by bone marrow-derived macrophages, focusing on differentiation and bone resorption activity while exploring potential mechanisms. Finally, we established a rat model of bilateral critical-sized calvarial bone defects, in which CS@MSNs-Naringin was dispersed in GelMA hydrogel to achieve in situ drug delivery. We observed the ability of CS@MSNs-Naringin to promote bone regeneration and inhibit osteoclast activity in vivo. CS@MSNs-Naringin exhibited high uniformity and dispersity, low cytotoxicity (concentration≤120 μg/mL), and significant pH sensitivity. In vitro, compared to Naringin and MSNs-Naringin, CS@MSNs-Naringin more effectively inhibited the formation and bone resorption activity of osteoclasts. This effect was accompanied by decreased phosphorylation of key factors in the NF-κB and MAPK signaling pathways, increased apoptosis levels, and a subsequent reduction in the production of osteoclast-specific genes and proteins. In vivo, CS@MSNs-Naringin outperformed Naringin and MSNs-Naringin, promoting new bone formation while inhibiting osteoclast activity to a greater extent. Our research suggested that CS@MSNs-Naringin exhibited the strikingly ability to anti-osteoclasts in vitro and in vivo, moreover promoted bone regeneration in the calvarial bone defect.

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.

Biomimetic Hydrogels as the Inductive Endochondral Ossification Template for Promoting Bone Regeneration.

Repairing critical size bone defects (CSBD) is a major clinical challenge and requires effective intervention by biomaterial scaffolds. Inspired by the fact that the cartilaginous template-based endochondral ossification (ECO) process is crucial to bone healing and development, developing biomimetic biomaterials to promote ECO is recognized as a promising approach for repairing CSBD. With the unique highly hydrated 3D polymeric network, hydrogels can be designed to closely emulate the physiochemical properties of cartilage matrix to facilitate ECO. In this review, the various preparation methods of hydrogels possessing the specific physiochemical properties required for promoting ECO are introduced. The materiobiological impacts of the physicochemical properties of hydrogels, such as mechanical properties, topographical structures and chemical compositions on ECO, and the associated molecular mechanisms related to the BMP, Wnt, TGF-β, HIF-1α, FGF, and RhoA signaling pathways are further summarized. This review provides a detailed coverage on the materiobiological insights required for the design and preparation of hydrogel-based biomaterials to facilitate bone regeneration.

Experimental Study on Rats with Critical-Size Bone Defects Comparing Effects of Autologous Bone Graft, Equine Bone Substitute Bio-Gen® Alone or in Association with Platelet-Rich Fibrin (PRF).

A critical-sized bone defect (CsBD) is considered one that will not heal spontaneously and requires reconstruction. This study aims to compare the results of using different bone reconstructive techniques and to study the potential of platelet-rich fibrin (PRF) to enhance the healing properties of a bone substitute (BS). In this experimental study on rats, the treatment of critical-sized bone defects was carried out by analysing four groups: a control group in which the bone defect was left empty; a group treated with Bio-Gen®; another group in which the defect was treated with PRF in combination with Bio-Gen®; and the last that was treated with autologous bone graft (ABG). The defects were evaluated by microcomputed tomography (µCT) and then histomorphometrically. From both the histological and imagistic point of view, the best results were registered in the ABG group, followed by the group treated with Bio-Gen® with PRF, Bio-Gen® group, and control group, with statistically significant differences. A 5 mm defect in the rat radius can be considered critical. ABG showed the best results in treating the bone defect. PRF significantly enhanced the efficacy of Bio-Gen®.

Ultrasonic Coating of Poly(D,L-lactic acid)/Poly(lactic-co-glycolic acid) Electrospun Fibers with ZnO Nanoparticles to Increase Angiogenesis in the CAM Assay.

Critical-size bone defects necessitate bone void fillers that should be integrated well and be easily vascularized. One viable option is to use a biocompatible synthetic polymer and sonocoat it with zinc oxide (ZnO) nanoparticles (NPs). However, the ideal NP concentration and size must be assessed because a high dose of ZnO NPs may be toxic. Electrospun PDLLA/PLGA scaffolds were produced with different concentrations (0.5 or 1.0 s of sonocoating) and sizes of ZnO NPs (25 nm and 70 nm). They were characterized by SEM, EDX, ICP-OES, and the water contact angle. Vascularization and integration into the surrounding tissue were assessed with the CAM assay in the living chicken embryo. SEM, EDX, and ICP-OES confirmed the presence of ZnO NPs on polymer fibers. Sonocoated ZnO NPs lowered the WCA compared with the control. Smaller NPs were more pro-angiogenic exhibiting a higher vessel density than the larger NPs. At a lower concentration, less but larger vessels were visible in an environment with a lower cell density. Hence, the favored combination of smaller ZnO NPs at a lower concentration sonocoated on PDLLA/PLGA electrospun meshes leads to an advanced state of tissue integration and vascularization, providing a valuable synthetic bone graft to be used in clinics in the future.

Mussel inspired 3D elastomer enabled rapid calvarial bone regeneration through recruiting more osteoprogenitors from the dura mater.

Currently, the successful healing of critical-sized calvarial bone defects remains a considerable challenge. The immune response plays a key role in regulating bone regeneration after material grafting. Previous studies mainly focused on the relationship between macrophages and bone marrow mesenchymal stem cells (BMSCs), while dural cells were recently found to play a vital role in the calvarial bone healing. In this study, a series of 3D elastomers with different proportions of polycaprolactone (PCL) and poly(glycerol sebacate) (PGS) were fabricated, which were further supplemented with polydopamine (PDA) coating. The physicochemical properties of the PCL/PGS and PCL/PGS/PDA grafts were measured, and then they were implanted as filling materials for 8 mm calvarial bone defects. The results showed that a matched and effective PDA interface formed on a well-proportioned elastomer, which effectively modulated the polarization of M2 macrophages and promoted the recruitment of dural cells to achieve full-thickness bone repair through both intramembranous and endochondral ossification. Single-cell RNA sequencing analysis revealed the predominance of dural cells during bone healing and their close relationship with macrophages. The findings illustrated that the crosstalk between dural cells and macrophages determined the vertical full-thickness bone repair for the first time, which may be the new target for designing bone grafts for calvarial bone healing.

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.

Murine iPSC-Loaded Scaffold Grafts Improve Bone Regeneration in Critical-Size Bone Defects.

In certain situations, bones do not heal completely after fracturing. One of these situations is a critical-size bone defect where the bone cannot heal spontaneously. In such a case, complex fracture treatment over a long period of time is required, which carries a relevant risk of complications. The common methods used, such as autologous and allogeneic grafts, do not always lead to successful treatment results. Current approaches to increasing bone formation to bridge the gap include the application of stem cells on the fracture side. While most studies investigated the use of mesenchymal stromal cells, less evidence exists about induced pluripotent stem cells (iPSC). In this study, we investigated the potential of mouse iPSC-loaded scaffolds and decellularized scaffolds containing extracellular matrix from iPSCs for treating critical-size bone defects in a mouse model. In vitro differentiation followed by Alizarin Red staining and quantitative reverse transcription polymerase chain reaction confirmed the osteogenic differentiation potential of the iPSCs lines. Subsequently, an in vivo trial using a mouse model (n = 12) for critical-size bone defect was conducted, in which a PLGA/aCaP osteoconductive scaffold was transplanted into the bone defect for 9 weeks. Three groups (each n = 4) were defined as (1) osteoconductive scaffold only (control), (2) iPSC-derived extracellular matrix seeded on a scaffold and (3) iPSC seeded on a scaffold. Micro-CT and histological analysis show that iPSCs grafted onto an osteoconductive scaffold followed by induction of osteogenic differentiation resulted in significantly higher bone volume 9 weeks after implantation than an osteoconductive scaffold alone. Transplantation of iPSC-seeded PLGA/aCaP scaffolds may improve bone regeneration in critical-size bone defects in mice.

Sr-Incorporated Bioactive Glass Remodels the Immunological Microenvironment by Enhancing the Mitochondrial Function of Macrophage via the PI3K/AKT/mTOR Signaling Pathway.

The repair of critical-sized bone defects continues to pose a challenge in clinics. Strontium (Sr), recognized for its function in bone metabolism regulation, has shown potential in bone repair. However, the underlying mechanism through which Sr2+ guided favorable osteogenesis by modulating macrophages remains unclear, limiting their application in the design of bone biomaterials. Herein, Sr-incorporated bioactive glass (SrBG) was synthesized for further investigation. The release of Sr ions enhanced the immunomodulatory properties and osteogenic potential by modulating the polarization of macrophages toward the M2 phenotype. In vivo, a 3D-printed SrBG scaffold was fabricated and showed consistently improved bone regeneration by creating a prohealing immunological microenvironment. RNA sequencing was performed to explore the underlying mechanisms. It was found that Sr ions might enhance the mitochondrial function of macrophage by activating PI3K/AKT/mTOR signaling, thereby favoring osteogenesis. Our findings demonstrate the relationship between the immunomodulatory role of Sr ions and the mitochondrial function of macrophages. By focusing on the mitochondrial function of macrophages, Sr2+-mediated immunomodulation sheds light on the future design of biomaterials for tissue regenerative engineering.

Mechanically robust and personalized silk fibroin-magnesium composite scaffolds with water-responsive shape-memory for irregular bone regeneration

The regeneration of critical-size bone defects, especially those with irregular shapes, remains a clinical challenge. Various biomaterials have been developed to enhance bone regeneration, but the limitations on the shape-adaptive capacity, the complexity of clinical operation, and the unsatisfied osteogenic bioactivity have greatly restricted their clinical application. In this work, we construct a mechanically robust, tailorable and water-responsive shape-memory silk fibroin/magnesium (SF/MgO) composite scaffold, which is able to quickly match irregular defects by simple trimming, thus leading to good interface integration. We demonstrate that the SF/MgO scaffold exhibits excellent mechanical stability and structure retention during the degradative process with the potential for supporting ability in defective areas. This scaffold further promotes the proliferation, adhesion and migration of osteoblasts and the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. With suitable MgO content, the scaffold exhibits good histocompatibility, low foreign-body reactions (FBRs), significant ectopic mineralisation and angiogenesis. Skull defect experiments on male rats demonstrate that the cell-free SF/MgO scaffold markedly enhances bone regeneration of cranial defects. Taken together, the mechanically robust, personalised and bioactive scaffold with water-responsive shape-memory may be a promising biomaterial for clinical-size and irregular bone defect regeneration.