Poly-ε-caprolactone/Whitlockite Electrospun Bionic Membrane

Poly-ε-caprolactone/Whitlockite Electrospun Bionic Membrane with an Osteogenic–Angiogenic Coupling Effect for Periosteal Regeneration

The periosteum is rich in vascular networks, osteoprogenitor cells, and stem cells and plays an important role in bone defect repair. However, existing artificial periosteum materials still have difficulty in meeting clinical requirements, such as good mechanical properties and bionic structure construction, osteogenic differentiation, and vascularization capabilities. Here, a poly-ε-caprolactone (PCL)/whitlockite artificial periosteum with different doping amounts was prepared by electrospinning technology. According to the results of in vitro mineralization experiments, the rapid ion release from WH promotes the deposition of mineralized hydroxyapatite. Inductively coupled plasma-optical emission spectroscopy, in vitro angiogenesis, and cell migration experiments showed that the bionic periosteum of the 15% WH group had the best release rate of Mg2+ and the best ability to promote the human umbilical vein endothelial cell angiogenesis and migration. In addition, this group promoted collagen formation and calcium deposition. Finally, the subcutaneous implantation model was used to verify the biocompatibility and angiogenesis ability of the proposed membrane in vivo. Overall, this biomimetic PCL/WH nanofiber membrane combines the positive osteogenic differentiation ability and angiogenic ability of calcium phosphate materials and thus has good application prospects in the field of periosteal repair in the future.

The treatment of segmental bone defects is still a challenge for clinicians because of the lack of a unified treatment method.Previous studies focused on optimizing bone implant materials,structural designs, and drug delivery to promote bone repair and overlooked the importance of the periosteum in segment bone repairs.The periosteum is a highly vascularized tissue found around the cortical bone, and it provides cell support and nutrition for a new bone. The periosteum contains cells with multidirectional differentiation potential that can rapidly proliferate and differentiate into a variety of bone cells and thus promote the growth of new bones. Moreover, the abundant blood vessels in the periosteum provide oxygen and nutrients necessary for the growth of new bones and can also participate in the metabolic exchange between blood and tissue fluids, thereby accelerating the formation of new bones. The periosteum has attracted increasing attention from researchers because of its important role in staged bone defect repair.

Morphological characterization and elemental analysis of different WH doping nanocomposite fiber membranes. (A) SEM micrographs, scale: 10 μm. (B) 3D surface morphology micrographs. (C) Elemental analysis micrographs. (D) TEM micrographs, scale: 100 nm.

The characteristics of bone regeneration require artificial periostea to have certain unique requirements, including an appropriate flexibility to allow for combination with the complex surface of bone grafts, an appropriate porosity for the exchange of gas and nutrients, suitable mechanical properties to withstand the stress of the operation process and tissue growth, and the ability to induce vascular growth and recruit endothelial cells and stem cells to imitate the natural periosteum and induce bone regeneration Electrospinning techniques have shown broad prospects in biomimetic reconstruction and can be used to prepare three-dimensional porous fiber membranes with large specific surface areas to simulate the structure and biological function of natural extracellular matrices.Moreover, the fiber membrane can also provide a certain mechanical strength to support the early separation of surrounding soft tissues and prevent scar tissue formation. Therefore, the electrospinning technology has unique advantages in the preparation of periosteal repair materials.

(A) XRD diffraction of WH nanoparticles and nanocomposite fiber membranes. (B) TGA curves and (C) representative stress–strain curves of the nanocomposite fiber membranes. Ion release from nanocomposite fiber membranes: (D) Mg2+, (E) Ca2+, and (F) PO43–.

Various materials have recently been applied in electrospinning technology, including natural polymers (such as collagen,chitosan, silk fibroin,and fibrinand synthetic polymers [such as poly-ε-caprolactone (PCL),polylactic acid, and polyglycolic acid.To meet the unique requirements for artificial periosteum, PCL, a kind of synthetic polyester material approved by the Food and Drug Administration (FDA), has attracted extensive attention because of its good biocompatibility and processability and moderate degradation velocity for long-term usage in vivo.However, the lack of inherent osteoinductive ability limits its application for bone tissue engineering. To improve its osteogenic activity, studies have introduced calcium phosphate nanoparticles (CaPs) into the spinning membrane system because they possess a good osteoinductive ability. For example, Liu et al. combined the prepared CaPs with gelatin–methacryloyl (GelMA) by electrospinning to prepare composite hydrogel fibers that showed the potential to promote osteogenesis. Wang et al. prepared a nanofiber membrane by doping octacalcium phosphate, which provided the membrane with bone induction ability. These studies showed that the addition of CaPs can improve the osteoinductive properties of polymers; however, their ability to promote angiogenesis is weak, which hinders their application for periosteal regeneration.

(A) SEM analysis showing the in vitro mineralization of the nanocomposite fiber membranes after incubation in SBF at day 7. (B) By utilizing a focused ion beam, flower-shaped bone minerals were separated and placed on a TEM grid for TEM observation. (C) Diffraction pattern of the bone mineral, which matched well with the crystal structure data of hydroxyapatite (JCDPS 73-1731).

Whitlockite [Ca18Mg2(HPO4)2(PO4)12, WH] is the second most abundant calcium phosphate in natural bone tissues, accounting for approximately 20%. Its content in adolescent human bones is generally higher than at other ages and its content in the load-bearing bone is higher than that in other parts.

Compared to CaPs, the appropriate amount of Mg ions in WH inhibits osteoclast activity and promotes angiogenesis without obstructing the formation of hydroxyapatite crystals in vivo.Jang et al. evaluated the bioactive properties of WH, hydroxyapatite (HAP), and β-tricalcium phosphate (β-TCP) through in vitro and in vivo testing. Compared to HAP and β-TCP composite scaffolds, WH composite scaffolds have been shown to facilitate bone-specific differentiation and induce a better bone regeneration in calvarial defects in a rat model. The presence of Ca2+ and PO43– in WH can upregulate the BMP-2 gene expression, stimulate BMP-2 protein secretion, and then act as an autocrine/paracrine bone induction pathway to induce osteogenic differentiation. Yegappan et al.reported that Mg2+ in WH can further promote angiogenesis by activating endothelial cells to secrete angiogenic factors. In conclusion, WH is feasible for applications in periosteal repair due to its ability to promote bone and angiogenesis and induce cell migration.

Biocompatibility of different WH-doped nanocomposite fiber membranes. (A) CCK-8 assay of BMSC proliferation after 1, 3, and 5 days. (B) BMSC morphologies at 1, 3, and 5 days.

In this study, they constructed PCL/WH composites with different ratios of artificial periosteum by electrospinning technology. They compared the morphology, chemical composition, and thermal behavior of different membranes. The mechanical strength and stability of the membranes with different dopants were compared by tensile testing. The biocompatibility of the PCL/WH nanofiber membrane was evaluated by cell adhesion, proliferation, osteogenic differentiation, angiogenesis, and cell migration analyses. Furthermore, the angiogenesis ability of the nanofiber membrane in vivo was also studied.

Angiogenic activity stimulated by the ions released from nanocomposite fiber membranes on HUVECs cultured in conditioned media. (A) Representative images and (C–E) quantitative analysis of the tube formation assay. (B) Representative images and (F) quantitative analysis of the Transwell assays (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Evaluation of the osteogenic differentiation of BMSCs in different nanocomposite fiber membranes. (A) Representative images of Sirius red staining at days 7 and 14 and (B) ARS staining at days 14 and 21. (C) Sirius red activity and (D) alizarin red staining. (E) Expression levels of osteogenesis-specific genes in BMSCs at 7 days. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

In vivo subcutaneous biocompatibility evaluation of the composites. (A) Histological images of different nanocomposite fiber membranes with H&E staining for 7 and 14 days. (Red arrows: boundaries of materials and skin; black arrows: blood vessel). (B) Immunofluorescence staining of CD31 and α-SMA on days 7 and 14 for different nanocomposite fiber composites. (C) Relative flux intensity was calculated using ImageJ system software (****P < 0.0001).

  1. Poly-ε-caprolactone/Whitlockite Electrospun Bionic Membrane with an Osteogenic–Angiogenic Coupling Effect for Periosteal Regeneration Xiangke Zhang, Wenbin Liu, Jiawei Liu, Yihe Hu, and Honglian Dai ACS Biomaterials Science & Engineering Article ASAP DOI: 10.1021/acsbiomaterials.1c00426