Calvarial Bone Defects Research Articles

MicroRNA-200c Release from Gelatin-Coated 3D-Printed PCL Scaffolds Enhances Bone Regeneration.

The fabrication of clinically relevant synthetic bone grafts relies on combining multiple biodegradable biomaterials to create a structure that supports the regeneration of defects while delivering osteogenic biomolecules that enhance regeneration. MicroRNA-200c (miR-200c) functions as a potent osteoinductive biomolecule to enhance osteogenic differentiation and bone formation; however, synthetic tissue-engineered bone grafts that sustain the delivery of miR-200c for bone regeneration have not yet been evaluated. In this study, we created novel, multimaterial, synthetic bone grafts from gelatin-coated 3D-printed polycaprolactone (PCL) scaffolds. We attempted to optimize the release of pDNA encoding miR-200c by varying gelatin types, concentrations, and polymer crosslinking materials to improve its functions for bone regeneration. We revealed that by modulating gelatin type, coating material concentration, and polymer crosslinking, we effectively altered the release rates of pDNA encoding miR-200c, which promoted osteogenic differentiation in vitro and bone regeneration in a critical-sized calvarial bone defect animal model. We also demonstrated that crosslinking the gelatin coatings on the PCL scaffolds with low-concentration glutaraldehyde was biocompatible and increased cell attachment. These results strongly indicate the potential use of gelatin-based systems for pDNA encoding microRNA delivery in gene therapy and further demonstrate the effectiveness of miR-200c for enhancing bone regeneration from synthetic bone grafts.

In Vivo Evaluation of Regenerative Osteogenic Potential Using a Human Demineralized Dentin Matrix for Dental Application.

The use of a demineralized dentin matrix (DDM) has garnered substantial importance in dentistry. This study was carried out to evaluate the osteoinductive performance of DDM in comparison to nano-hydroxyapatite (n-HA) on calvarial critical-sized bone defect. Two critical-sized defects (CSDs) were bilaterally trephined in the calvarium of sixteen healthy white rabbits. The rabbits were categorized into four groups: in group 1, the defect was left empty; in group 2, defects were filled with sodium alginate (SA) hydrogel as a sole material; in group 3, defects were treated with nano-hydroxyapatite hydrogel (NHH); in group 4, defects were treated using demineralized dentin matrix hydrogel (DDMH). Histological and immunohistochemical analyses were carried out to evaluate the total areas of newly formed bone. The DDMH group showed that new woven bone tissue progressively bridged the defect area while there was no bone in the control group. Collagen expression was significantly different in the DDMH- and NHH-treated groups compared to in the SA group at 4 and 8 weeks (p < 0.01). OCN expression was significantly higher in the DDMH group in comparison to in the NHH or SA groups at 8 weeks (p < 0.01). The DDMH group exhibited significantly higher levels of new bone formation compared to the NHH group at both 4 and 8 weeks post-surgically.

3D Stem Cell Spheroids with 2D Hetero-Nanostructures for In Vivo Osteogenic and Immunologic Modulated Bone Repair.

3D stem cell spheroids have immense potential for various tissue engineering applications. However, current spheroid fabrication techniques encounter cell viability issues due to limited oxygen access for cells trapped within the core, as well as nonspecific differentiation issues due to the complicated environment following transplantation. In this study, functional 3D spheroids are developed using mesenchymal stem cells with 2D hetero-nanostructures (HNSs) composed of single-stranded DNA (ssDNA) binding carbon nanotubes (sdCNTs) and gelatin-bind black phosphorus nanosheets (gBPNSs). An osteogenic molecule, dexamethasone (DEX), is further loaded to fabricate an sdCNTgBP-DEX HNS. This approach aims to establish a multifunctional cell-inductive 3D spheroid with improved oxygen transportation through hollow nanotubes, stimulated stem cell growth by phosphate ions supplied from BP oxidation, in situ immunoregulation, and osteogenesis induction by DEX molecules after implantation. Initial transplantation of the 3D spheroids in rat calvarial bone defect shows in vivo macrophage shifts to an M2 phenotype, leading to a pro-healing microenvironment for regeneration. Prolonged implantation demonstrates outstanding in vivo neovascularization, osteointegration, and new bone regeneration. Therefore, these engineered 3D spheroids hold great promise for bone repair as they allow for stem cell delivery and provide immunoregulative and osteogenic signals within an all-in-one construct.

Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model.

There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

Osteogenically committed hUCMSCs-derived exosomes promote the recovery of critical-sized bone defects with enhanced osteogenic properties.

Low viability of seed cells and the concern about biosafety restrict the application of cell-based tissue-engineered bone (TEB). Exosomes that bear similar bioactivities to donor cells display strong stability and low immunogenicity. Human umbilical cord mesenchymal stem cells-derived exosomes (hUCMSCs-Exos) show therapeutic efficacy in various diseases. However, little is known whether hUCMSCs-Exos can be used to construct TEB to repair bone defects. Herein, PM-Exos and OM-Exos were separately harvested from hUCMSCs which were cultured in proliferation medium (PM) or osteogenic induction medium (OM). A series of in-vitro studies were performed to evaluate the bioactivities of human bone marrow mesenchymal stem cells (hBMSCs) when co-cultured with PM-Exos or OM-Exos. Differential microRNAs (miRNAs) between PM-Exos and OM-Exos were sequenced and analyzed. Furthermore, PM-Exos and OM-Exos were incorporated in 3D printed tricalcium phosphate scaffolds to build TEBs for the repair of critical-sized calvarial bone defects in rats. Results showed that PM-Exos and OM-Exos bore similar morphology and size. They expressed representative surface markers of exosomes and could be internalized by hBMSCs to promote cellular migration and proliferation. OM-Exos outweighed PM-Exos in accelerating the osteogenic differentiation of hBMSCs, which might be attributed to the differentially expressed miRNAs. Furthermore, OM-Exos sustainably released from the scaffolds, and the resultant TEB showed a better reparative outcome than that of the PM-Exos group. Our study found that exosomes isolated from osteogenically committed hUCMSCs prominently facilitated the osteogenic differentiation of hBMSCs. TEB grafts functionalized by OM-Exos bear a promising application potential for the repair of large bone defects.

In Situ Photo-Crosslinkable Protein Bioadhesive for Bone Graft Fixation

Bone grafting is a fundamental dental surgery procedure widely used for implant placement and periodontal disease management treatments. Despite its broad applications, vertical bone augmentation presents unique challenges, including the risk of graft displacement due to gravitational and masticatory forces. Traditional physical stabilization methods introduce additional complexities and risks, underscoring the need for innovative fixation technologies. This study aimed to develop an in situ photo-crosslinkable bioadhesive hydrogel (iPBAH) as a multifunctional bone graft binder to enhance the process of bone reconstruction. The bioadhesive is composed of mussel-derived adhesive protein (MAP) fused with the cell-adhesive peptide RGD. The numerous tyrosine residues in MAP facilitate rapid photo-crosslinking, enabling efficient hydrogel formation using visible blue light. Subsequently, iPBAH underwent comprehensive characterization to evaluate its suitability as a multifunctional bone graft binder. iPBAH efficiently underwent in situ crosslinking through harmless exposure to visible light within minutes and displayed several exceptional properties, including a microporous structure, underwater adhesion, extended durability, high compressive strength, and biocompatibility. In vivo assessments, using male Sprague-Dawley rats, demonstrated that iPBAH binder significantly enhanced bone regeneration in a rat calvarial bone defect model. The in situ crosslinking of the iPBAH binder during bone graft transplantation can effectively fill irregular and complex defect shapes while simultaneously preventing graft material leakage. The improved physical attributes of the bound graft material can enhance its resistance to external forces, thereby ensuring sustained retention over time. Moreover, the interaction between iPBAH and surrounding tissues promotes adhesion and integration of the graft material with host tissues in the defect area. In addition, the included RGD peptide in iPBAH can augment inherent cell recruitment, adhesion, and growth, consequently expediting osteogenesis.

Copper-doped magnesium phosphate nanopowders for critical size calvarial bone defect intervention.

Calvarial defects of bone present difficult clinical situations, and their restoration using biocompatible materials requires special treatments that enable bone regeneration. Magnesium phosphate (MgP) is known as an osteoinductive biomaterial because it contains Mg2+ ions and P ions that enhance the activity of osteoplast cells and help in bone regeneration. In this study, MgP and CuO-doped MgP were fabricated and characterized for their physicomechanical properties, particle size, morphology, surface area, antibacterial test, and in vitro bioactivity evaluation using the following techniques: X-rays diffraction, Fourier-transformer infrared, TEM, and Brunauer, Emmett and Teller (BET) surface area, X-rays photoelectron spectroscopy (XPS), and Scanning electron microscopy (SEM). Furthermore, these nanopowders were implanted in adult inbred male Wistar rats and studied after two periods (28 and 56 days). The results demonstrated that the obtained semiamorphous powders are in nanoscale (≤ 50 nm). XPS analysis ensured the preparation of MgP as mono MgP and CuO were incorporated in the structure as Cu2+ . The bioactivity was supported by the observation of calcium phosphate layer on the nanopowders' surface. The in vivo study demonstrated success of MgP nanopowders especially those doped with CuO in restoration of calvarial defect bone. Therefore, fabricated biomaterials are of great potential in restoration of bone calvarial defects.

Effect of Bone Marrow Mesenchymal Stem Cells derived Exosomes loaded in Collagen Hydrogel on Bone Regeneration in male Rat Calvarial Critical-Size Bone Defect

Effect of Bone Marrow Mesenchymal Stem Cells derived Exosomes loaded in Collagen Hydrogel on Bone Regeneration in male Rat Calvarial Critical-Size Bone Defect

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.

A Case Report of an Isolated Calvarial Defect in the Region of the Lambdoid Suture: A Rare Bone Manifestation of Neurofibromatosis Type 1

The Calvarial bone defects in the lambdoid suture region are a rare bone manifestation of neurofibromatosis type 1. They may be isolated or associated with an adjacent structural lesion, such as a dural ectasia or plexiform neurofibroma. The underlying pathophysiology for the development of these types of skull defects is still unclear. We present the case of a Calvarial defect in the region of the lambdoid suture in a 7-year-old boy with a medical history of neurofibromatosis type 1 who was referred for a brain scan to explore a palpable skull defect in right parieto-occipital region , noticed by his doctor.

Impact of Specialized Pro-Resolving Lipid Mediators on Craniofacial and Alveolar Bone Regeneration: Scoping Review.

Craniofacial bone defects caused by tumors, trauma, long-term tooth loss, or periodontal disease are a major challenge in the field of tissue engineering. In periodontitis and peri-implantitis, reconstructive therapy is also a major challenge for the dental surgeon. Lipoxins, resolvins, protectins, and maresins, known as specialized pro-resolving lipid mediators (SPMs), have been widely studied in the field of dental, oral, and craniofacial research for bone regeneration for their actions in restoring tissue homeostasis and promoting tissue healing and regeneration. Therefore, this study focuses on a survey of the use of SPMs for craniofacial and alveolar bone regeneration. Thus, electronic searches of five databases were performed to identify pre-clinical studies that evaluated the actions of SMPs on craniofacial and alveolar bone regeneration. Of the 523 articles retrieved from the electronic databases, 19 were included in the analysis. Resolvin (Rv) E1 was the mostly assessed SPM (n=8), followed by maresins (Ma) R1 (n=3), lipoxins (Lx) A4 (n=3), RvD1 (n=3), RvD2 (n=1), LxB4 (n=1), and maresin (M)-CTR3 (n=1). Meta-analysis showed that SPMs increased the newly formed bone by 14.85% compared to the control group (p<0.00001), decreased the area of the remaining defect by 0.35 mm2 (p<0.00001), and decreased the linear distance between the defect to the bone crest by 0.53 mm (p<0.00001). RvE1 reduced inflammatory bone resorption in periodontal defects and calvarial osteolysis and enhanced bone regeneration when RvE1 was combined with a bovine bone graft. RvD2 induced active resolution of inflammation and tissue regeneration in periapical lesions, while RvD1 controlled the inflammatory microenvironment in calvarial defects in rats, promoting bone healing and angiogenesis. MaR1 induced the proliferation and migration of mesenchymal stem cells, osteogenesis, and angiogenesis in calvarial defects, and benzo (b)-LxA4 and LxA4 promoted bone regeneration calvarial and alveolar bone defects in rats, inducing regeneration under inflammatory conditions. In summary, SPMs have emerged as pivotal contributors to the resolution of inflammation and the facilitation of bone neoformation within craniofacial and alveolar bone defects. These results are based on pre-clinical studies, in vivo and in vitro, and provide an updated review regarding the impact of SPMs in tissue engineering.

Timely delivery of bone marrow mesenchymal stem cells based on the inflammatory pattern of bone injury environment to promote the repair of calvarial bone defects in rats: An optimized strategy for bone tissue engineering

Stem cell-based therapy plays a significant role in the repair of bone defects. However, traditional stem cell transplantation strategies in bone tissue engineering are characterized by low survival rates and unstable treatment outcomes. In this study, we propose a timely delivery strategy for inflammatory changes in the setting of bone injury to improve the survival rate of transplanted cells and bone repair. The results of cell tracing in vivo showed that this strategy could effectively improve the survival rate of low-dose exogenous transplanted cells in bone defect areas, and CD31 immunofluorescence and histological sections suggested that this strategy effectively promoted vascularization and new bone formation in the calvarial defect area. Subsequently, we analyzed the mechanism of action of the “Two-step” strategy from the perspective of inflammatory microenvironment regulation, and the results suggested that the first batch transplanted stem cells caused localized and transient increases in tissue apoptosis levels and inflammatory factors, and recruited macrophage chemotaxis, and the second batch of cells may promote pro-inflammatory - anti-inflammatory transformation of the tissue. Finally, mRNA sequencing results suggest that the first batch cells in the “Two-step” strategy are important initiators in bone repair, which not only actively regulate the immune microenvironment at the bone defect, but also guide richer cellular activity and more positive biochemical responses. Therefore, the “Two-step” strategy leads to efficient inflammatory environment regulation and superior bone repair effects, which may provide an alternative option for the treatment of bone defects in the future.

Simulation of the bone remodelling microenvironment by calcium compound-loaded hydrogel fibrous membranes for in situ bone regeneration.

The endowment of guided bone regeneration (GBR) membranes with the ability to activate the endogenous regenerative capability of bone to regenerate bone defects is of clinical significance. Herein we explored the preparation of the calcium compound (CC) (calcium sulfate (CaSL), calcium hydrophosphate (CaHP), or tricalcium phosphate (TCaP)) loaded ultrathin silk fibroin (SF)/gelatin (G) fibre membranes via electrospinning as the GBR membranes to regenerate the calvarial bone defects. The in vitro experiments demonstrated that the CaSL-loaded ultrathin fibrous membranes could simulate optimally the bone remodelling microenvironment in comparison with the CaHP- and TCaP-loaded fibrous membranes, displaying the highest activity to regulate the migration, proliferation, and differentiation of mesenchymal stem cells (MSCs). Also, the in vivo experiments demonstrated that the CaSL-loaded fibrous membranes presented the highest intrinsic osteoinduction to guide in situ regeneration of bone. Furthermore, the in vivo experiments demonstrated that the as-prepared composite fibrous membranes possessed good degradability. In summary, our results suggested that the CaSL-loaded fibrous membranes with high intrinsic osteoinduction and good degradability have potential to translate into clinical practice.

Graphene Oxide Quantum Dots-Preactivated Dental Pulp Stem Cells/GelMA Facilitates Mitophagy-Regulated Bone Regeneration.

In bone tissue engineering (BTE), cell-laden scaffolds offer a promising strategy for repairing bone defects, particularly when host cell regeneration is insufficient due to age or disease. Exogenous stem cell-based BTE requires bioactive factors to activate these cells. Graphene oxide quantum dots (GOQDs), zero-dimensional derivatives of graphene oxide, have emerged as potential osteogenic nanomedicines. However, constructing biological scaffolds with GOQDs and elucidating their biological mechanisms remain critical challenges. We utilized GOQDs with a particle size of 10 nm, characterized by a surface rich in C-O-H and C-O-C functional groups. We developed a gelatin methacryloyl (GelMA) hydrogel incorporated with GOQDs-treated dental pulp stem cells (DPSCs). These constructs were transplanted into rat calvarial bone defects to estimate the effectiveness of GOQDs-induced DPSCs in repairing bone defects while also investigating the molecular mechanism underlying GOQDs-induced osteogenesis in DPSCs. GOQDs at 5μg/mL significantly enhanced the osteogenic differentiation of DPSCs without toxicity. The GOQDs-induced DPSCs showed active osteogenic potential in three-dimensional cell culture system. In vivo, transplantation of GOQDs-preactivated DPSCs/GelMA composite effectively facilitated calvarial bone regeneration. Mechanistically, GOQDs stimulated mitophagy flux through the phosphatase-and-tensin homolog-induced putative kinase 1 (PINK1)/Parkin E3 ubiquitin ligase (PRKN) pathway. Notably, inhibiting mitophagy with cyclosporin A prevented the osteogenic activity of GOQDs. This research presents a well-designed bionic GOQDs/DPSCs/GelMA composite scaffold and demonstrated its ability to promote bone regeneration by enhancing mitophagy. These findings highlight the significant potential of this composite for application in BTE and underscore the crucial role of mitophagy in promoting the osteogenic differentiation of GOQDs-induced stem cells.

Harnessing 3D printed highly porous Ti-6Al-4V scaffolds coated with graphene oxide to promote osteogenesis.

Bone tissue engineering (BTE) strategies have been developed to address challenges in orthopedic and dental therapy by expediting osseointegration and new bone formation. In this study, we developed irregular porous Ti-6Al-4V scaffolds coated with reduced graphene oxide (rGO), specifically rGO-pTi, and investigated their ability to stimulate osseointegration in vivo. The rGO-pTi scaffolds exhibited unique irregular micropores and high hydrophilicity, facilitating protein adsorption and cell growth. In vitro assays revealed that the rGO-pTi scaffolds increased alkaline phosphatase (ALP) activity, mineralization nodule formation, and osteogenic gene upregulation in MC3T3-E1 preosteoblasts. Moreover, in vivo transplantation of rGO-pTi scaffolds in rabbit calvarial bone defects showed improved bone matrix formation and osseointegration without hemorrhage. These findings highlight the potential of combining rGO with irregular micropores as a promising BTE scaffold for bone regeneration.

A bilayer hydrogel mimicking the periosteum-bone structure for innervated bone regeneration.

In bone tissue, nerves are primarily located in the periosteum and play an indispensable role in bone defect repair. However, most bone tissue engineering approaches ignored the reconstruction of the nerve network. Herein, we aimed to develop a bilayer hydrogel simulating periosteum-bone structure to induce innervated bone regeneration. The bottom "bone" layer consisted of gelatin methacryloyl (GelMA), poly(ethylene glycol) diacrylate (PEGDA), and nano-hydroxyapatite (nHA), whereas the upper "periosteum" layer consisted of GelMA, sodium alginate (SA) and MgCl2. The mechanical properties of the upper and bottom hydrogels were designed to be suitable for neurogenesis and osteogenesis, respectively. Besides, Mg2+ from the "periosteum" layer released at the early stage (within 7 d), which aligned with the optimal time window for nerve regeneration and osteogenic related neuropeptide release. Simultaneously, the prevention of long-term Mg2+ release (after 7 d) could avoid osteogenic inhibition caused by prolonged Mg2+ exposure. Additionally, the incorporation of nHA in the bottom "bone" layer supported the long-term osteogenesis due to its osteoconductivity and slow degradation. In vitro biological experiments revealed that the bilayer hydrogel (GS@Mg/GP@nHA) promoted neurite growth and calcitonin gene-related peptide (CGRP) expression in rat dorsal root ganglion (DRG) neurons, as well as the osteogenesis of rat bone-derived mesenchymal stem cells (BMSCs). Moreover, the in vivo experiments demonstrated that the GS@Mg/GP@nHA hydrogel efficiently promoted nerve network reconstruction and bone regeneration of rat calvarial bone defects. Altogether, the bilayer hydrogel GS@Mg/GP@nHA could promote innervated bone regeneration, providing new insights for biomaterial design for bone tissue engineering.

Histopathological Evaluation of Eggshell and DBM Combination on the Repair of Critical Size Experimental Calvarial Bone Defects in Rats.

Fracture repair is a constant clinical challenge, and finding a method to promote and improve restoration is a primary goal for researchers. This is examined from various perspectives, such as fewer complications, increased speed, and cost-effectiveness. The present study aimed to investigate the effectiveness of eggshell powder, compared to the commercial form of demineralized bone matrix (DBM), in critical-size defects in rat calvarial bone. In this study, 40 adult male Wistar rats were selected and randomly divided into four groups of 10. The first group was the control group (C), the second was the eggshell powder group (E), the third was the DBM group (D), and the fourth was the one simultaneously receiving eggshell powder and DBM (DE). In these groups, a 5 mm diameter defect was created in the calvaria using a trephine. All animals received the appropriate treatment for their group. Each group was then divided into two subgroups of five. On days 30 and 60 post-surgery, these subgroups were euthanized, followed by sampling and histopathology examinations. After evaluating the repair percentage using Quick Photo software, the DE group had the highest repair percentage on days 30 and 60. Groups E and D had similar recovery percentages, with group D having a slightly higher one. There was a significant difference between all three groups and the control group. In conclusion, eggshell powder may potentially serve as a suitable substitute for some transplants.

Radiographic Evaluation of the Effect of Vitamin D3 Supplementation on Regeneration of Calvarial Bone Defects in Rats

Aim: The present study was aimed to evaluate radiographically the effect of orally administered vitamin D3 on guided bone regeneration in calvarial critical size defects (CSD) in rats.&#x0D; Methods: Two calvarial CSD were created in 12 male Sprague-Dawley rats. One of the defects was left empty (E defect), while the other one was treated with deproteinized bovine bone graft and collagen-based resorbable membrane (GM-filled defect). Following surgical intervention, rats were randomly assigned into two groups; the control group was administered distilled water, and the test group was treated with 2 µg /kg vitamin D3 by gavage once a day for 8 weeks. Radiological images were obtained from rats on 4th and 8th weeks. The area fraction of newly formed osteoid was determined using Image Fiji Analysis Software. &#x0D; Results: The percentages of area fraction in the GM-filled defects were statistically higher than the E defects in both study groups at 4th and 8th weeks (p

Enhanced Calvarial Bone Repair Using ASCs Engineered with RNA-Guided Split dCas12a System that Co-Activates Sox 5, Sox6, and Long Non-Coding RNA H19.

Healing of large calvarial bone defects remains challenging. An RNA-guided Split dCas12a system is previously harnessed to activate long non-coding RNA H19 (lncRNA H19, referred to as H19 thereafter) in bone marrow-derived mesenchymal stemcells (BMSCs). H19 activation in BMSCs induces chondrogenic differentiation, switches bone healing pathways, and improves calvarial bone repair. Since adipose-derived stem cells (ASCs) can be harvested more easily in large quantity, here it is aimed to use ASCs as an alternative cell source. However, H19 activation alone using the Split dCas12a system in ASCs failed to elicit evident chondrogenesis. Therefore, split dCas12a activators are designed more to co-activate other chondroinductive transcription factors (Sox5, Sox6, and Sox9) to synergistically potentiate differentiation. It is found that co-activation of H19/Sox5/Sox6 in ASCs elicited more potent chondrogenic differentiation than activation of Sox5/Sox6/Sox9 or H19 alone. Co-activating H19/Sox5/Sox6 in ASCs significantly augmented in vitro cartilage formation and in vivo calvarial bone healing. These data altogether implicated the potentials of the Split dCas12a system to trigger multiplexed gene activation in ASCs for differentiation pathway reprogramming and tissue regeneration.

Bifunctional tissue-engineered composite construct for bone regeneration: The role of copper and fibrin.

Bifunctional tissue engineering constructs promoting osteogenesis and angiogenesis are essential for bone regeneration. Metal ion-incorporated scaffolds and fibrin encapsulation attract much attention due to low cost, nontoxicity, and tunable control over ion and growth factor release. Herein, we investigated the effect of Cu.nHA/Cs/Gel scaffold and fibrin encapsulation on osteogenic and angiogenic differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs) in vitro and in vivo. Cu-laden scaffolds were synthesized using salt leaching/freeze drying and were characterized using standard techniques. WJMSCs were isolated from the human umbilical cord and characterized. WJMSCs with or without encapsulating in fibrin were seeded onto scaffolds, followed by differentiating into the osteogenic lineage for 7 and 21 days. Osteogenic and angiogenic differentiation were evaluated using real-time polymerase chain reaction, western blot, and Alizarin red staining. Then, scaffolds were implanted into critical-sized calvarial bone defects in rats and histological assessments were performed using hematoxylin/eosin, Masson's trichrome, and CD31 immunohistochemical staining at 4 and 12 weeks. The scaffolds had good physicochemical and biological characteristics suitable for cell attachment and growth. Cu and fibrin increased the expression of ALP, RUNX2, OCN, COLI, VEGF, and HIF1α in differentiated WJMSCs. Implanted scaffolds were also biocompatible and were integrated well with the host tissue. Increased collagen condensation, mineralization, and blood vessel formation were observed in Cu-laden scaffolds. The fibrin-encapsulated groups showed the highest collagen and cell densities, immune cell infiltration, and bone trabeculae. CD31-positive cell population increased with fibrin encapsulation and seeding onto Cu-laden scaffolds. Adding Cu to scaffolds and encapsulating cells in fibrin are promising methods that guide osteogenesis and angiogenesis cellular signaling, leading to better bone regeneration.