Natural bone tissue is composed of inorganic minerals and organic collagen. The collagen/HA complex gives the natural bone tissue piezoelectric properties. The application of piezoelectric materials to reconstruct the physiological electrical microenvironment at the defect site is an effective strategy to promote osteogenesis, especially in the initial stage of osteogenesis. Biomimetic scaffolds that mimic the composition and piezoelectric properties of natural bone provide a promising strategy for regenerative medicine; however, selecting suitable piezoelectric materials to meet the different needs during regeneration is also a challenge.
Based on this, The team of Yingjie Yu and Cai Qing from Beijing University of Chemical Technology developed a multifunctional bionic composite scaffold based on piezoelectric WH (PWH). As magnesium-doped calcium phosphate, annealed, the WH of also shows significant polarization due to domain transition above the phase transition temperature, which makes the PWH match well with the electroactive properties of natural bone, thereby enhancing osteogenesis. The research results related to were published in "Bioact. Mater." on November 29, 2022 under the title "A biomimetic piezoelectric scaffold with sustained Mg2+ release promotes neurogenic and angiogenic differentiation for enhanced bone regeneration".
Figure 1 Bone repair application of PWH piezoelectric composite scaffold
- Fabrication and characterization of 3D printed scaffold
First, the authors developed a bone repair scaffold with piezoelectricity and sustained release of bioactive ions through melt extrusion 3D printing technology. WH piezoelectric nanoparticles were synthesized using chemical precipitation method (Figure 2A). SEM showed that WH nanoparticles showed rhombic morphology and β-TCP nanoparticles were spherical; EDS confirmed the presence of calcium and phosphorus elements in WH and β-tricalcium phosphate nanoparticles, while magnesium was only found in WH nanoparticles (Figure 2B). Afterwards, the electrical activity of the material was characterized, demonstrating that piezoelectric PWH nanoparticles are more advantageous than WH nanoparticles and β-TCP nanoparticles in repairing the local endogenous electrical microenvironment (Figure 2C-D). Mechanical testing showed that the mechanical properties of scaffold were within the same range; in addition, the authors also monitored the release characteristics of Mg2+ and Ca2+ when the scaffold was immersed in PBS. The annealing treatment of and did not damage the ion release characteristics of the scaffold. The above results together demonstrate that the PWH scaffold displays excellent piezoelectricity and sustained release of bioactive ions, which is expected to benefit bone regeneration.
Figure 2 Preparation and characterization of PWH nanoparticles and PWH composite scaffolds Biocompatibility evaluation of
- scaffolds
Before using these scaffolds for cell seeding, the authors lightly etched them with NaOH solution at room temperature to increase surface hydrophilicity. The survival of the cells was characterized by using CCK-8 analysis and fluorescent staining. bone marrow mesenchymal stem cells grew well on various scaffolds, and all groups showed mainly green fluorescence, thus indicating that all scaffolds have excellent biocompatibility (Figure 3). Together these results demonstrate that piezoelectric PWH scaffolds behave similarly in cell proliferation compared to β-TCP and WH scaffolds.
Figure 3 Biocompatibility evaluation of different scaffolds
- Mg2+/ Synergistic effects of piezoelectricity on neurogenesis and angiogenesis
Bone healing is a complex signaling cascade process involving nerve regeneration and angiogenesis, which is an indispensable event for the formation of neurovascularized bone tissue. Therefore, the authors continue to study the piezoelectricity of the PWH stent and its impact on Mg2+ sustained release.. By seeding BMSCs directly on scaffolds, the neurogenic and vascular -derived differentiation of the cells was quantitatively and qualitatively assessed (Figure 4A). For neurogenic differentiation studies, real-time qPCR was applied to quantify three relevant genes for expression analysis. PWH scaffolds have stronger induction of neurogenic differentiation of bone marrow mesenchymal stem cells than WH scaffolds.In the PWH scaffold group, the expressions of , nestin, , TUBB3, and NEFL were 1.7, 1.3, and 1.6 times higher than those in the WH scaffold group, respectively. Obviously, this enhancement can be attributed to the synergistic effect of Mg2+/piezoelectric (Figure 4B-D) .
Figure 4 Neurogenic and vasogenic differentiation of bone marrow mesenchymal stem cells cultured on different scaffolds
In addition, the authors further used the CAM model to evaluate blood vessel formation (Figure 5A). As shown in Figure 5B, the most abundant vascularization could be observed in the PWH scaffold, followed by the WH scaffold, while only limited angiogenesis was observed in the β-TCP and PCL scaffolds, and the extracts from the PWH and WH scaffolds showed a stronger potential to induce angiogenesis than the β-TCP and PCL scaffold groups (Figure 5C-F). In summary: Mg2+ The synergistic piezoelectric effect can play a synergistic role in promoting neurogenesis and angiogenesis.
Figure 5 Using CAM model to detect angiogenesis of various scaffolds
- Effect of scaffolds on osteogenic differentiation of bone marrow mesenchymal stem cells
Subsequently, in order to verify the effect of soluble components in the scaffold (i.e., released ions) on osteogenesis, the authors cultured bone marrow mesenchymal stem cells on TCP for 7 or 14 days in the presence of various scaffolds. Then, the scaffold was removed and ALP staining was used to characterize the bone marrow mesenchymal stem cells on TCP. confirmed that the released Mg2+ entered the culture medium from the WH and PWH scaffolds to further promote the cell ALP activity (Figure 6A-B). At the same time, the expression of osteogenic activity also showed the best results .
Figure 6 Evaluation of osteogenic differentiation of bone marrow mesenchymal stem cells under different scaffold conditions
- Evaluation of in situ bone regeneration
Finally, The authors explored the ability of the PWH scaffold to induce bone regeneration in the rat calvarial defect model (Figure 7A) . The results showed that PCL and β-TCP scaffolds hardly induced the formation of new bone tissue, and pores were still displayed in the defect area after 8 W, without showing the presence of β-TCP scaffolds. However, both WH and PWH scaffolds induced significant bone formation in the central area of the defect, sprouted from the edge of the defect and grew into the scaffold framework. The most significant new bone formation could be observed in the PWH scaffold group, which was further supported by quantitative analysis of BV/TV and BMD.
Figure 7 Evaluation of in situ bone regeneration
Subsequently, authors used H&E and Masson's trichrome staining to illustrate the possible inflammatory response associated with scaffold implantation, as well as to show collagen synthesis and bone maturation (Figure 8). No obvious inflammation was observed in all groups of , indicating that the implanted scaffold containing various inorganic nanoparticles has good biocompatibility. New bone grew into the interior of the piezoelectric PWH scaffold, but not the PCL and β-TCP scaffolds. In addition, the WH and PWH scaffold groups showed stronger OCN and less TRAP expression than the PCL and β-TCP scaffold groups, with the PWH scaffold showing the strongest osteogenic induction ability.
Figure 8 Histology, immunohistology and immunofluorescence staining to evaluate bone regeneration
In summary, This paper designed a biomimetic scaffold that mimics the composition and piezoelectricity of natural bone by introducing electroactive PWH nanoparticles. The PWH scaffold showed significant piezoelectricity and sustained release of bioactive ions. Taking advantage of this synergistic effect, the PWH scaffold mimics the functions of natural bone, enhancing angiogenesis, promoting neuronal differentiation, inhibiting osteoclast activation, and ultimately increasing osteogenesis. These characteristics make the PWH scaffold highly capable of promoting neurovascularized bone regeneration compared with other scaffolds, which is supported by the rat calvarial defect model. Overall, , a bone-specific composite scaffold containing electroactive PWH, provides a comprehensive and effective strategy for ossification..
Article source: https://doi.org/10.1016/j.bioactmat.2022.11.004
Learn more
Focusing on the intersection of medicine and industry, EFL has established public accounts, related academic and industrial communities, search the "EngineeringForLife" public account to learn more~
