Calvarial Bone Tissue Research Articles

Plasma-assisted multiscale topographic scaffolds for soft and hard tissue regeneration

The design of transplantable scaffolds for tissue regeneration requires gaining precise control of topographical properties. Here, we propose a methodology to fabricate hierarchical multiscale scaffolds with controlled hydrophilic and hydrophobic properties by employing capillary force lithography in combination with plasma modification. Using our method, we fabricated biodegradable biomaterial (i.e., polycaprolactone (PCL))-based nitrogen gas (N-FN) and oxygen gas plasma-assisted flexible multiscale nanotopographic (O-FMN) patches with natural extracellular matrix-like hierarchical structures along with flexible and controlled hydrophilic properties. In response to multiscale nanotopographic and chemically modified surface cues, the proliferation and osteogenic mineralization of cells were significantly promoted. Furthermore, the O-FMN patch enhanced regeneration of the mineralized fibrocartilage tissue of the tendon–bone interface and the calvarial bone tissue in vivo in rat models. Overall, the PCL-based O-FMN patches could accelerate soft- and hard-tissue regeneration. Thus, our proposed methodology was confirmed as an efficient approach for the design and manipulation of scaffolds having a multiscale topography with controlled hydrophilic property.

Pentamidine Inhibits Titanium Particle-Induced Osteolysis In Vivo and Receptor Activator of Nuclear Factor-κB Ligand-Mediated Osteoclast Differentiation In Vitro.

Wear debris-induced osteolysis leads to periprosthetic loosening and subsequent prosthetic failure. Since excessive osteoclast formation is closely implicated in periprosthetic osteolysis, identification of agents to suppress osteoclast formation and/or function is crucial for the treatment and prevention of wear particle-induced bone destruction. In this study, we examined the potential effect of pentamidine treatment on titanium (Ti) particle-induced osteolysis, and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. The effect of pentamidine treatment on bone destruction was examined in Ti particle-induced osteolysis mouse model. Ti particles were implanted onto mouse calvaria, and vehicle or pentamidine was administered for 10days. Then, calvarial bone tissue was analyzed using micro-computed tomography and histology. We performed in vitro osteoclastogenesis assay using bone marrow-derived macrophages (BMMs) to determine the effect of pentamidine on osteoclast formation. BMMs were treated with 20ng/mL RANKL and 10ng/mL macrophage colony-stimulating factor in the presence or absence of pentamidine. Osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining, real-time polymerase chain reaction, and immunofluorescence staining. Pentamidine administration decreased Ti particle-induced osteoclast formation significantly and prevented bone destruction compared to the Ti particle group in vivo. Pentamidine also suppressed RANKL-induced osteoclast differentiation and actin ring formation markedly, and inhibited the expression of nuclear factor of activated T cell c1 and osteoclast-specific genes in vitro. Additionally, pentamidine also attenuated RANKL-mediated phosphorylation of IκBα in BMMs. These results indicate that pentamidine is effective in inhibiting osteoclast formation and significantly attenuates wear debris-induced bone loss in mice.

Sutures Possess Strong Regenerative Capacity for Calvarial Bone Injury

Repair of calvarial bony defects remains challenging for craniofacial surgeons. Injury experiments on animal calvarial bones are widely used to study healing mechanisms and test tissue engineering approaches. Previously, we identified Gli1+ cells within the calvarial sutures as stem cells supporting calvarial bone turnover and injury repair. In this study, we tested the regenerative capacity of the suture region compared with other areas of calvarial bone. Injuries were made to mouse sagittal sutures or other areas of the calvarial bone at varying distances from the suture. Samples were collected at different time points after injury for evaluation. MicroCT and histological analyses were conducted. EdU incorporation analysis was performed to assay cell proliferation. Gli1-CreERT2;Tdtomatoflox mice were used to trace the fate of Gli1+ stem cells after injury. Calvarial sutures possess much stronger regeneration capability than the nonsuture bony areas of the calvaria. The healing rate of the calvarial bone is inversely proportional to the distance between the suture and injury site: injuries closer to the suture heal faster. After complete removal of the sagittal suture, regeneration and restoration of normal organization occur within 6 weeks. Gli1+ cells within the suture mesenchyme are the cellular source for injury repair and bone regeneration. These results demonstrate that calvarial bone healing is not an evenly distributed event on the calvarial surface. Sutures contain stem cells and are the origin of calvarial bone tissue regeneration. Therefore, current practice in calvarial surgery needs to be reevaluated and modified. These findings also necessitate the design of new approaches for repairing calvarial bony defects.

A Systematic Approach to Predicting Spring Force for Sagittal Craniosynostosis Surgery.

Spring-assisted surgery (SAS) can effectively treat scaphocephaly by reshaping crania with the appropriate spring force. However, it is difficult to accurately estimate spring force without considering biomechanical properties of tissues. This study presents and validates a reliable system to accurately predict the spring force for sagittal craniosynostosis surgery. The authors randomly chose 23 patients who underwent SAS and had been followed for at least 2 years. An elastic model was designed to characterize the biomechanical behavior of calvarial bone tissue for each individual. After simulating the contact force on accurate position of the skull strip with the springs, the finite element method was applied to calculating the stress of each tissue node based on the elastic model. A support vector regression approach was then used to model the relationships between biomechanical properties generated from spring force, bone thickness, and the change of cephalic index after surgery. Therefore, for a new patient, the optimal spring force can be predicted based on the learned model with virtual spring simulation and dynamic programming approach prior to SAS. Leave-one-out cross-validation was implemented to assess the accuracy of our prediction. As a result, the mean prediction accuracy of this model was 93.35%, demonstrating the great potential of this model as a useful adjunct for preoperative planning tool.

A Novel Porcine Graft for Regeneration of Bone Defects

Bone regeneration procedures require alternative graft biomaterials to those for autogenous bone. Therefore, we developed a novel porcine graft using particle sizes of 250–500 μm and 500–1000 μm in rabbit calvarial bone defects and compared the graft properties with those of commercial hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) over eight weeks. Surgery was performed in 20 adult male New Zealand white rabbits. During a standardized surgical procedure, four calvarial critical-size defects of 5 mm diameter and 3 mm depth were prepared. The defects were filled with HA/β-TCP, 250–500 μm or 500–1000 μm porcine graft, and control defects were not filled. The animals were grouped for sacrifice at 1, 2, 4, and 8 weeks post-surgery. Subsequently, sample blocks were prepared for micro-computed tomography (micro-CT) scanning and histological sectioning. Similar bone formations were observed in all three treatment groups, although the 250–500 μm porcine graft performed slightly better. Rabbit calvarial bone tissue positively responded to porcine grafts and commercial HA/β-TCP, structural analyses showed similar crystallinity and porosity of the porcine and HA/β-TCP grafts, which facilitated bone formation through osteoconduction. These porcine grafts can be considered as graft substitutes, although further development is required for clinical applications.

Poly (L-Lactic Acid) and Hydroxyapatite Scaffold for Bone Regeneration:<i> In Vivo</i> Study

In bone tissue engineering, synthetic scaffolds are commonly used and this should present the following requirements; (i) recapitulate the native three-dimensional (3D) hierarchical fibrous structure, (ii) possess biomimetic surface properties and (iii) demonstrate mechanical integrity. However, some methods of producing scaffolds do not achieve these requirements. The present study aims the application of a composite of poly (L-lactic acid) (PLLA) and Hydroxyapatite (HA) produced by rotary jet spinning, which can be used to obtain scaffolds that meet the above requirements with affordable costs (regarding materials and production). The morphology and thermal properties of the scaffolds were characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). For the in vivo tests, 20 Wistar rats, distributed into two groups, in which critical defects were performed in cranial calotte were used. Then scaffolds of PLLA/HA were implanted and compared with the control group that didn’t receive the implant. The results have shown that in the cases where only the defects in cranial caps were performed, bone healing did not occur. In cases where the scaffolds of PLLA/HA were used, rich neovascularization was noted, accompanied by foreign body type reaction and presence of reactive bone around the implants. The evaluation of PLLA/HA scaffolds used in the rat calvarial defect model, according to the criteria surveyed was favorable, showed the implants insurance and that they are suitable materials to be used as substitutes of calvarial bone tissue in these animals.

Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model

The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP-2 and protein extracted from Hevea brasiliensis (P-1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. Eighty-four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 μg of pure rhBMP-2, (2) 5 μg of rhBMP-2/monoolein gel, (3) pure monoolein gel, (4) 5 μg of pure P-1, (5) 5 μg of P-1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. The results showed an improvement in the bone healing process using the rhBMP-2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.

Human Circulating Peripheral Blood Mononuclear Cells for Calvarial Bone Tissue Engineering

Large-scale production of engineered tissues requires an adequate source of expandable cells. Current strategies that involve harvesting of cells from donor tissue or bone marrow for tissue engineering are invasive and unfeasible for obtaining large quantities of cells in a clinical setting. Peripheral blood has been reported to contain circulating hematopoietic cells as well as, in significantly smaller quantities, mesenchymal cells An adherent subset of CD14+ mononuclear cells was isolated from human peripheral venous blood and characterized in vitro by light microscopy, immunohistochemistry, flow cytometry, and quantitative differentiation assays. These cells were then evaluated for the purposes of tissue engineering in a rat calvarial defect model, in combination with biodegradable polymer matrices made from poly-e-caprolactone. Specimens were analyzed 6 weeks after implantation with histologic analysis, microcomputed tomography, and HLA immunostaining. CD14+ mononuclear cells were induced to differentiate into osteoblast-like cells in vitro, with areas of mineralization. In a rat calvarial defect model, tissue-engineered bone with evidence of mineralization was formed within 6 weeks. HLA immunohistochemistry demonstrated that de novo bone formation originated from the transplanted human cells. These findings show, for the first time, to our knowledge, the derivation of bone from human blood. They also demonstrate the utility of circulating mononuclear cells as a minimally invasive, potentially unlimited pool of cells for tissue engineering and organ regeneration.

On the role of cyclic AMP as a mediator of bone resorption: gamma-interferon completely inhibits cholera toxin- and forskolin-induced but only partially inhibits parathyroid hormone-stimulated 45Ca release from mouse calvarial bones.

The effects of gamma-interferon (gamma-IFN) on bone resorption and cyclic AMP formation stimulated by parathyroid hormone (PTH), forskolin, and cholera toxin have been studied in cultured neonatal mouse calvarial bones. Bone resorption was assessed by the release of 45Ca from prelabeled mouse calvarial bone fragments. Cyclic AMP formation was quantified by analyzing the amount of the nucleotide in calvarial bone tissue. gamma-IFN completely blocked the 45Ca release response to forskolin and cholera toxin in 96 h cultures. In contrast, the 45Ca release response to PTH was only partially inhibited, an effect that was seen over a wide range of PTH concentrations. The inhibitory effect of gamma-IFN was dose dependent, with a threshold for action at 10 U/ml. Forskolin-stimulated 45Ca release could only be inhibited when gamma-IFN was added simultaneously with forskolin; gamma-IFN added to bones prestimulated with forskolin had no effect. The inhibitory effect of gamma-IFN on PTH-stimulated 45Ca release was seen first after a time lag of 48 h. In contrast calcitonin caused an inhibition after only 3 h. PTH and cholera toxin stimulation of radioactive calcium release was also inhibited by gamma-IFN in bones treated with indomethacin. gamma-IFN inhibited forskolin-induced 45Ca release in bones treated with the mitotic inhibitor hydroxyurea. No effect of gamma-IFN on cyclic AMP formation induced by PTH, cholera toxin, or forskolin could be seen. These data show that gamma-IFN inhibits forskolin- and cholera toxin-induced bone resorption by a mechanism unrelated to prostaglandin production or mitotic activity.(ABSTRACT TRUNCATED AT 250 WORDS)