rADSC-loaded tubular units composed of multilayer electrospun membranes promoted bone regeneration of critical-sized skull defects

doi: 10.1088/2752-5724/ad66ea

Huamin Jiang^{1, 2},
Zhaoyi Lin^{1, 2},
Jinze Li^{1, 2},
Ting Song^{1, 2},
Hongyun Zang^{1, 2},
Pengwen Li^{1, 2},
Jiarun Li³,
Wenyi Hou⁴,
Jianhua Zhou^{1, 2},
Yan Li^{1, 2
,
,}

1.
School of Biomedical Engineering, Shenzhen Campus of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China
2.
Guangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instrument, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China
3.
Hospital of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Institute of Stomatological Research, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou 510062, People’s Republic of China
4.
The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, People’s Republic of China

摘要

Abstract: AbstractAdipose-derived stem cells (ADSCs) have considerable potential for bone regeneration. However, their performance is limited by a lack of scaffolds that adequately mimic the hierarchical structure of bone to promote proliferation and osteogenic differentiation of ADSCs. In this study, nanofiber membranes composed of polycaprolactone, poly(lactide-co-glycolide), and hydroxyapatite (HAp) were prepared via electrospinning, and the membranes curled after responding to temperature stimuli in an aqueous solution. Transmission electron microscopy and scanning electron microscopy observations indicated that needle-like HAp nanoparticles with an average diameter of 57 ± 39 nm and a length-diameter ratio of 7.4 ± 1.56 were entrapped in the nanofiber matrix and did not affect the surface morphology of fibers. After cutting and deformation, the nanofibers changed from straight to bent, and the diameters increased; they were 1105 ± 200 nm for BPLG85-H and 1120 ± 199 nm for BPLG80-H. Additionally, tubular units with a single layer (BPLG-H(1.5)) or multiple layers (BPLG-H(3.5)) were obtained by controlling the initial shape and size of the membranes. rADSCs on the concave surface of BPLG-H(3.5) proliferated faster and exhibited better osteogenic activity than those on the convex side of BPLG-H(3.5) and both surfaces of BPLG-H(1.5), which was correlated with the higher expressions of vascular endothelial growth factor and bone morphogenetic protein 2. Additionally, rADSCs on both units maintained osteogenic activity after storage at -80 °C for 20 d. In rat skull defect (diameter of 8 mm) models, rADSC-loaded BPLG-H(3.5) units fixed using gelatin hydrogel (ADSC@BHM) exhibited 84.1 ± 6.6% BV/TV after implantation for 12 weeks, which was 155.6% higher than that of the Blank group. H&E and Masson’s staining results demonstrated that there was more bone regeneration at the defect center of ADSC@BHM than in the BHM and Blank groups. In conclusion, rADSC-loaded BPLG-H(3.5) with an osteon-mimic structure provides a potential strategy to repair bone defects.
- adipose-derived stem cells /
- electrospinning /
- composite fiber /
- bone regeneration
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Figure 1. Characterization of HAp and SL membranes with and without HAp. (a) SEM and TEM images, (b) XRD pattern (red lines represent peaks for HAp standard with PDF Card No. 9-432), and (c) XPS spectrum of HAp. (d) TEM and SEM images of electrospun nanofibers with and without HAp. (e) Shrinkage ratio and thickness (after shrinkage) of membranes with different components (data = mean ± SD, n = 3). (f) Scheme for the changes in the area and thickness of SL membranes after shrinkage (membranes remained flat; green, light blue, and dark blue represent PLG80-H, gelatin, and PLG85-H layers, respectively). (g) DSC spectra of as-spun (SPLG85-H, SPLG85, SPLG80, SPLG80-H) and after-shrinkage (SPLG85-H, SPLG80-H) membranes with different components.

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Figure 2. Optimization of fabrication parameters for BLCMs to obtain the lowest curl ratio and smallest gross size. (a) Light microscope images and (b) curl ratio and diameter after curling of BLCMs with different layer proportions at a fixed thickness and different thicknesses at a fixed layer proportion. (c) Light microscope images and (d) curl ratio and projected area after curling of BLCMs with different vertex angles and waist lengths. (e) Cross-sectional view of BPLG-H(3.5) and BPLG-H(1.5) obtained via OCT. (f) Scheme for the deformation process of BLCMs (red arrows represent directions of gelatin dissolution; green, dark blue, and light blue represent PLG80-H, PLG85-H, and gelatin layers, respectively).

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Figure 3. In vitro degradation properties of BLCMs. (a) SEM images of BLCMs after deformation (after-deform) and after in vitro degradation (after-degra) in PBS (pH 7.4, 37 °C) for 28 d. (b) Nanofiber diameters of as-spun, after-deform, and after-degra BLCMs. (c) XRD patterns of as-spun, after-deform, and after-degra BLCMs. ‘As-spun’ denotes freshly electrospun membranes without treatment.

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Figure 4. Proliferation and osteogenic differentiation behaviors of rADSCs on BLCMs. (a) Cells were cultured in a proliferation medium, and the CCK8 assay was performed. The concentrations of (b) VEGF and (c) BMP-2 in the proliferation medium on day 7 were quantified using the ELISA. (d) After osteogenic induction for 21 d and Alizarin Red staining, light microscope images were obtained. (e) 3D reconstruction of CLSM images of samples after DAPI staining. (f) Quantitative results for samples after Alizarin Red staining. Concentrations of (g) VEGF and (h) BMP-2 in the osteogenic medium on day 21. (Data = mean ± SD; n = 3; p < 0.05 compared with TCP group).

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Figure 5. Osteogenic differentiation of rADSCs on BLCMs under different culture conditions. (a) In the proliferation medium for 8 d, (b) in the osteogenic medium for 8 d, and (c) in the proliferation medium for 10 d and then the osteogenic medium for 8 d; light microscope images of the samples were captured after Alizarin Red staining. (d) Alizarin Red quantitative results and concentrations of (e) VEGF and (f) BMP-2 in the medium of samples under different culture conditions. (g) Experimental timeline for cryopreservation and osteogenic induction. (h) rADSC-loaded BLCMs were cryopreserved at -80 °C for 20 d. The CCK-8 assay was performed on the second day of recovery. (i) Light microscope images were obtained after 8 d of osteogenic induction followed by Alizarin Red staining and (j) quantitative results for the samples. (Data = mean ± standard deviation; n = 3; compared with the same shape and size fibrous membrane group on the same day, p < 0.05).

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Figure 6. In vivo bone regeneration of critical-sized skull defects after implantation of rADSC-loaded BLCMs for 12 weeks. (a) Digital images and micro-CT images of the Blank, BHM, and ADSC@BHM groups (red dashed circle represents the initial skull defect). (b) BV/TV results for each group. (Data = mean ± SD; n = 3; compared with the Blank group, p < 0.05). Representative histological images of bone tissue for the Blank, BHM, and ADSC@BHM groups after (c) H&E and (d) Masson’s staining (NB, new bone; CT, connective tissue; B, host bone).

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