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Poly-ε-caprolactone/chitosan/whitlockite electrospun bionic membrane conjugated with an E7 peptide for bone regeneration

Stem Cell Research & Therapy volume 16, Article number: 212 (2025) Cite this article

Abstract

Background

Periosteum plays an important role in bone defect repair due to its rich vascular network and cells. However, natural periosteum is difficult to meet clinical requirements due to its low availability. Therefore, it is necessary to develop a tissue engineering strategy of biomimetic periosteum for bone defect repair.

Methods

Poly-ε-caprolactone/chitosan/Whitlockite electrospun bionic membrane (PCL/CS/WH) was prepared using electrospinning technology, then it was conjugated with an E7 peptide as PCL/CS/WH/E7 bionic membrane. The physical properties of the membranes were evaluated by TEM, FTIR and tensile strength testing. In vitro, LIVE/DEAD staining and Cell Counting Kit-8 assay of bone marrow mesenchymal stem cells (BMSCs) and Endothelial progenitor cells (EPCs) are used to assess the biocompatibility of bionic membranes. Matrigel was applied to evaluate the ability of the different composite nanofibers samples to promote angiogenesis. Mineralized nodule and collagen formation in the BMSCs was detected by alizarin red staining and sirius red staining respectively. The expression of osteogenesis related genes and angiogenesis associated genes were detected using quantitative real-time polymerase chain reaction (qRT-PCR). In vivo, the ability of PCL/CS/WH/E7 membrane to promote bone regeneration and angiogenesis was assessed by Micro-CT and associated staining.

Result

The addition of chitosan (CS) and E7 peptide (E7) enhanced the hydrophilicity and cytocompatibility of pure PCL membranes. Additionally, CS, E7 and Mg2+ released from Whitlockite (WH) had a synergistic effect to promote angiogenesis and osteogenic differentiation. Three weeks after implantation, the membrane successfully bridged the bone defect and significantly promoted the formation of new bone and blood vessels.

Conclusion

The PCL/CS/WH/E7 membrane to achieve efficient repair of bone tissue and enriches clinical solutions for the treatment of critical bone defects.

Introduction

One of the biggest problems in orthopedics today is the bone defect, caused by disease and trauma. However, the optimal treatment for the bone defects is an important unsolved issue [1]. To address this problem, previous studies focused on optimizing bone implant biomaterials, scaffolds structural designs, and growth factor delivery to promote bone repair [2] and overlooked the importance of the periosteum in segment bone repairs [3].

The periosteum is a dense and highly vascularized connective tissue membrane that covers most bone tissues [4]. It is rich in progenitor cells (mesenchymal stem cell or osteogenic progenitor cell) [5] and delivers essential blood, nutrition, and regenerative cells to the cambium layer of the bone cortex, which is deeply involved in the bone healing process [6, 7]. Although natural periosteum transplantation has been adopted clinically to accelerate bone healing [8]. Their therapeutic efficacy is significantly limited because of the limited healthy periosteum availability [9], the difficulties of periosteum isolating [10] and possible immune rejection. thus, constructing artificial periosteum as a substitute for the natural periosteum for bone defect repair has increasingly attracted the attention of researchers.

Electrospinning is a simple, cost-effective technique, which involves the formation of fibers from polymer solutions or polymer melting of natural and synthetic materials [11]. Its history goes back more than 260 years, with Bose using high electric potential to produce aerosols from liquid droplets in 1745 [12]. Nanofibers fabricated by electrospinning technology are widely used to manufacture tissue engineering scaffolds due to their high porosity and large specific surface area [13]. These features make electrospun fibers an effective substitute for constructing capillary systems in artificial periosteum [14]. Additionally, membranes produced through electrospinning may process a native extracellular matrix like physical structure, which makes membranes more similar to damaged tissue structure [15]. Therefore, the electrospinning technique is a promising method for the preparation of biomimetic periosteum.

The choices of materials used to fabricate electrospinning fibers also need to be carefully considered. In more recent years, composite electrospun membranes like poly-ε-caprolactone (PCL)/collagen-I (Col) /mineralized Col (MC) [16], PCL loaded with deferoxamine(DFO) [17], and PCL/gelatin [18] have been put forward to show PCL can be used as the basic material for artificial periosteum due to the excellent controllability over mechanical and degradation properties. Chitosan(CS), a polysaccharide and natural polymer, is biocompatible for numerous tissue engineering applications [19, 20] due to its biocompatibility [21], antibacterial activity [22] and degradability [23]. Moreover, it is easily processed into different surface coatings and scaffolds for tissue engineering. Romero et al. evaluated three types of coatings: N,N, N-trimethyl chitosan–heparin polyelectrolyte multilayers, freeze-dried porous chitosan foam and electrospun chitosan nanofibers, These coatings produce surfaces that are cytocompatible and ·maintain the phenotypic of osteoprogenitor cells [20]. Frohbergh et al. fabracted a biomimetic scaffold by co-electrospinning CS with hydroxyapatite, which can facilitate the proliferation, differentiation and maturation of osteoblast-like cells [24]. Therefore, CS is a promising candidate for artificial periosteum.

Whitlockite [WH: Ca18Mg2(HPO4)2(PO4)12] constitutes almost 25% of the human bone and is the second most abundant mineral component [25]. In particular, WH has higher mechanical compressive strength than that of HA and beta-tricalcium phosphate (β-TCP), which are commonly applied for bone tissue engineering. Its constant release of phosphate and magnesium ions also increases the expression of osteogenic genes and promotes the formation of new bone, which makes it a ideal material for bone regeneration [26, 27]. Cheng et al. developed a composite hydrogel scaffolds incorporating HA and WH nanoparticles with various ratios, WH improved the osteogenic capacity of the inorganic hybrid composite scaffold [28]. In our previous study, we prepared a PCL/WH electrospunfiber composite capable of promoting angiogenesis and osteogenic differentiation by simulating the periosteal microenvironment [29]. In a word, WH is advantageous for use in periosteal repair because of its capacity to stimulate the formation of new blood vessels and bone tissue.

Recently, it was reported that a novel peptide (E7 peptide, “EPLQLKM”) has a specific affinity for MSCs. This peptide could attract autologous MSC to synthetic polycaprolactone mesh effectively when it was chemically conjugated to PCL mesh [30]. Ge et al. reported that E7-modified substrates have an improving effect on proliferation and multilineage differentiation of the rat bone marrow-derived mesenchymal stem cells [31]. Shi et al. discovered that E7 peptide could enhance the chondrogenic differentiation of BMSCs and improves the efficiency of endogenous BMSC homing [32]. E7 peptide has been shown to have the potential to promote the adhesion of mesenchymal stem cells and maintain their stemness.

Our previous study only explored the osteogenic differentiation ability and angiogenesis ability of PCL/WH biomimetic membrane in vitro, verified the angiogenesis ability of PCL/WH biomimetic membrane in vivo, and did not further explore its bone defect repair effect in vivo. Compared with the previously constructed PCL/WH single biomimetic membrane, we hope to further improve its repair ability in the bone defect area by constructing a composite biomimetic periosteum, and verify its osteogenic induction and angiogenesis ability in vivo and in vitro experiments. In addition, the membrane surface was modified with peptides to further enhance its ability of chemotactic recruitment of stem cells and promoting cell proliferation, avoiding the process of seed cell culture in vitro and the occurrence of immune-derived diseases [33, 34]. Based on above, we designed present study. In this study, we constructed the PCL/CS/WH composite artificial periosteum by electrospinning PCL, CS and WH, the ratio of which is 74%, 11% and 15%. Afterwards, we conjugated the composition with E7 peptide, followed by evaluating its ability to promote the osteogenic differentiation of BMSCs and angiogenic properties of EPCs. The biocompatibility of the PCL/CS/WH/E7 composite artificial periosteum was detected by cell adhesion, proliferation and cell migration analyses. Additionally, a subcutaneous implantation model was established to verify the angiogenic properties of the PCL/CS/WH/E7 periosteum replacement in vivo. A critical-sized calvarial defect animal model was introduced to confirm that the artificial periosteum replacement could promote periosteum regeneration and vascularized bone formation in the bone defect areas.

Methods

Preparation and characterization of electrospun fiber membranes

Preparation of electrospun fiber membranes

WH nanoparticles were synthesized according to our previous study [29, 35] (all materials involved were purchased from Sinopharm Chemical Reagent Co. Ltd. (China) and were used as received without further purification). PCL (Sigma, USA) were dissolved in 2,2,2-trifluoroethanol as PCL solution. Next, CS (Shanghai Aladdin Biochemical Technology Co.Ltd. China) was dissolved in 90% acetic acid, then weighed poly ethylene oxide (PEO) (CS/PEO = 3/1; w/w) was weighed and added to the solution after 12 h of solution stirring, which was called CS solution. Finally, PCL and CS solutions were mixed at 8:1 (w: w) as PCL/CS solution. WH nanoparticles 15% (w/v) was added to the PCL/CS solution as PCL/CS/WH solution, then the mixture was sonicated for 30 min to evenly disperse the nanoparticles. Different composite electrospun fiber membranes were constructed by electrospin techology (Elite Series, Ucalery Co Ltd. Beijing), and the method was presented in the following steps. The PCL, PCL/CS and PCL/CS/WH solutions were drawn into a 10 mL plastic syringe through a 20-gauge needle respectively. The solution system in the syringe flowed in an electric field between the tip of the needle (+ 5kv) and the aluminum foil (-2 kV), 0.5 ml/h. Then, the pure PCL, PCL/CS, PCL/CS/WH without E7 membrane were fabricated as control groups respectively. The PCL/CS/WH was immersed in E7 peptide for 24 h as PCL/CS/WH/E7 membrane. In this study, the materials used in the biological experiments were sterilized with ethylene oxide. In the following experiments, they were grouped into PCL group, PCL/CS group, PCL/CS/WH group, and PCL/CS/WH/E7 group according to the membranes used.

Characterization of the electrospun fiber membranes

The research contents included surface morphology, crystal structure and surface hydrophilicity. The 2D morphology was observed by scanning electron microscopy (SEM, jeol, jsm-it200, Japan). Fourier transform infrared spectroscopy (FTIR) was used to characterize the chemical properties of the different membranes. An FTIR spectrometer (Varian 670, Agilent, Santa Clara, CA, USA) coupled with a plotting microscope (Varian 620-IR, Agilent, Santa Clara, CA, USA) was used to acquire data. These samples were examined within the 400–4000 cm− 1 range at 64 scans per minute and a total of 100 scans per spectrum. The surface hydrophilicity of the membranes was demonstrated by the water contact angle. The contact angle is the angle at the interface where water, air and solid meet. Low contact-angle values show a tendency for the surface to be hydrophilic, while high contact-angle values show a tendency for the surface to repel water [36]. One microliter of ultrapure water was added to the membrane, and the contact angle was measured using a contact angle test system (JC 2000 C, Zhongchen, China). To assess the mechanical properties of nanofiber membranes, tensile strength testing was conducted at a rate of 5 mm/min using an universal testing machine (Instron + MTS).

Ion release

The PCL/CS/WH composite electrospun fiber membrane was cut into 1 × 1 cm rectangles and immersed in 5 ml ultrapure water in a centrifuge tube at 37 °C. Then, these samples were placed in a shaker for 3,5,7,10 and 14 days. The ultrapure water was replaced every 4 days, and the solution was collected by centrifuging it at 5000 rpm for 10 min. The concentrations of calcium (Ca2+) and magnesium (Mg2+) ions in the solution were measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES, Leeman Labs, USA).

In vitro experiments

Cell culture

BMSCs were isolated from Sprague Dawley (SD) rats according to our previous report [37]. BMSCs were cultured in F-12 medium (Gibco, United States) containing 10% (v/v) fetal bovine serum (FBS, Gibco, United States) and 1% (v/v) penicillin/streptomycin (P/S, Absin, China). The BMSCs were passaged, and the cells were collected at passage 3 for further experiments.

Rat Endothelial progenitor cells (EPCs) purchased from Procell Life Science & Technology Co. Ltd. (China)were used for in vitro angiogenic experiments. The EPCs were cultured in M-199 medium (Gibco, United States). All cells were placed at 37 °C in 5% CO2 and nonadherent cells were removed during every passage. Passage 3 was used for subsequent experiments. Every in vitro experiment repeated at least 3 times with n = 3 technical replicates.

Cell viability and proliferation

Cell viability was evaluated by a LIVE/DEAD kit (Beyotime, China). Briefly, A total of 1 × 104 BMSCs or EPCs were seeded onto the PCL, PCL/CS, PCL/CS/WH and PCL/CS/WH/E7 membrane respectively. After 1 and 3 days of cultivation, the two cell types’ vitality on different membranes was assessed using a Calcein acetoxymethyl (Calcein AM) and propidium iodide (PI) staining. After removing the culture medium and washing the membranes with 1× assay buffer, the prepared working solution was added to the samples. Living cells were labeled as green by Calcein-AM probe, while dead cells were simultaneously stained as red by the PI probe. The cells were observed using an inverted fluorescencemicroscope (Leica DMC6200, Germany).

Cell proliferation on the membranes was accessed after 1, 3, and 5 days of incubation time for BMSCs and 1, 2, and 3 days of incubation time for EPCs, respectively. A total of 1 × 104 cells seeded on the different membranes were incubated with 10% Cell Counting Kit-8 (CCK-8, NCM Biotech, C6005, Suzhou, China) solution prepared in the cell complete medium for 4 h. 100 µL of the incubated solution was then transferred into a 96-well plate (Guangzhou Jet BioFiltration Co. Ltd., China). The absorbance of the samples was measured on a microplate reader (Multiskan GO, United States) at 450 nm.

Cell adhesion

The electrospun fiber membranes were cut into 1 cm diameter discs from different group. These samples were plated on 48-well plates. Subsequently, the cells were seeded onto each membrane at a density of 1 × 104 cells/well. After a three-day culture period, the cells’ nuclei and F-actin were stained with FITC phalloidin (Beyotime, China) and 4′,6-diamidino-2-phenylindole (DAPI) (Boster, China ) and examined using a confocal laser scanning microscope. Following the removal of the cell culture media, the samples were fixed with a 4% paraformaldehyde (PFA) solution and then cleaned with phosphate-buffered solution (PBS).

After permeabilizing the cells for ten minutes with a 0.5% Triton X-100 solution, the samples were again washed with PBS. A 1:200 ratio was used to dilute FITC phalloidin (Beyotime, China) with 1% bovine serum albumin (BSA) solution. The solution was then added to the cells and incubated at room temperature for 1 h. After three rounds of washing for 5 min, 100× diluted DAPI (Boster, China) solution was added to the samples for nuclear staining and incubated for 10 min. After cleaning the residual dye with PBS, the membranes were transferred to confocal dishes for observation.

Mineralized nodule staining

Mineralized nodule formation in the BMSCs was detected by Alizarin red staining. A total of 3 × 104 cells were seeded on different nanofiber membranes. After 24 h, the cell mediums were replaced with osteoblast-inducing conditioned media (αMEM medium supplemented with 10% FBS, 1% P/S, 10 nM dexamethasone, 50 µM ascorbic acid, and 10 mM ß-glycerin phosphate). which was replaced every 3 d. Alizarin red staining (ARS, Servicebio, China) was utilized to visualize the mineralized nodules on days 10 after induction, and images were captured as well. 10% cetylpyridinium chloride (Sinopharm Chemical Reagent Co. Ltd. China) solution was prepared to wash the stained nodules. The absorbance was detected at 562 nm.

Collagen staining

Collagen secretion in the BMSCs was detected by Sirius red staining. A total of 3 × 104 cells were seeded on different nanofiber membranes. After 24 h, the medias were replaced with osteoblast-inducing conditioned media, which were replaced every 3 d. After induction for 7 and 10 days, the cells on the membranes were fixed with 4% PFA, and picrosirius red staining solution (Phygene, China) was added for 18 h to stain collagen. The staining solution was washed off with 0.1 M acetic acid. A light microscope (Leica DMRBE, Germany) was used to obtain detailed images. To obtain semi-quantitative data, an eluent was prepared with 0.2 M NaOH and methanol at a ratio of 1:1 to elute the stained collagen. The absorbance was measured at a wavelength of 540 nm.

In vitro vascularization assay

Matrigel was applied to evaluate the ability of the different composite nanofibers samples to promote angiogenesis. After melted at 4°C, matrigel (100 µL) was added to the wells of a 96-well plate to induce tube formation. The different membranes were placed into 10 ml of medium for 24 h to obtain the leach liquor [38]. A total of 1 × 104 EPCs were seeded onto matrigel after the gel solidified at 37 °C, and cultured with medium (culture medium and leach liquor in a ratio of 1:1). The EPCs were imaged using optical microscopy after 4-hours culturing, and ImageJ software was used to quantify the overall length, number, and branches of tube development in five randomly selected fields.

Cell migration assay

The Transwell Migration Assay was utilized to evaluate the cell’s capacity to migrate after being treated with various nanofiber membranes. Firstly, samples of the various membranes were arranged at the bottom of the lower chamber of the Transwell chamber (NEST Biotechnology,701001, Wuxi, China), to which 400 µL of cell complete medium was added. Subsequently, a total of 5 × 104 cells were seeded on the polycarbonate film in the upper chamber and the same medium was added. PFA-fixed crystal violet staining was performed to visualize cells on the permeable film of the chamber after culturing for 24 h. After cultivating for 24 h, cells were visible on the permeable film of the chamber using PFA-fixed crystal violet staining. After three times PBS washes, cells that did not migrate were removed off the film’s upper side using a swab, and those that moved to the lower side were captured on camera. For counting purposes, three microphotographs were taken in each chamber using a light microscope (Leica, Germany), and each experimental group was repeated in three chambers. Cell numbers from a total of nine representative fields were obtained in each experimental group and the average number of migrating cells was used as a measure of migratory ability. Then, the GraphPad Prism 9.0 software was used for statistical analysis.

Quantitative real-time polymerase chain reaction (qRT-PCR)

To investigate the osteogenic induction and pro-angiogenic effect of different biofilms, BMSCs and EPCs were seeded on biofilms and cultured in respective conditioned media. After 7 d of culture for BMSCs and 3 d for EPCs, total RNA was extracted and purified using the FastPure Cell/Tissue Total RNA Isolation Kit V2(Vazyme, Nanjing, China). Then, the purity and concentration of the obtained RNA were analyzed using a microplate reader (BioTek, USA). Next, all RNA was reverse-transcribed into complementary DNA (cDNA) using the HiScript II cDNA Kit (Vazyme, Nanjing, China). Finally, the cDNA was then amplified and quantified using the RT-qPCR detection system. GAPDH serves as an internal control in all experiments. The primer sequences can be found in Table 1.

Table 1 Primer sequence used in the RT-qPCR experiment
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In vivo studies

Subcutaneous implantation

In order to verify the angiogenic potential of the membranes in different groups in vivo, membranes without the implantation of cells in different groups were implanted subcutaneously between the skin and muscle on the backs of SD rats. Briefly, rats were anesthetized with 3% w/v with pentobarbital sodium. Then, a skin incision of 1.5 cm in length was made at the selected implantation site on the back of the rat. Next, the subcutaneous tissue was bluntly separated, deep to the superficial fascial muscular layer, to form the subcutaneous sac. After that, the implant is carefully placed in the subcutaneous sac, and finally the incision is closed and the subcutaneous tissue and skin are sutured in layers. All in vivo experiments in this study had been approved by the Animal Care and Use Committee of Huazhong University of Science and Technology under Ethics approval Number: [2023] IACUC(3974). 24 male SD rats weighing 210–250 g (the Laboratory Animal Center of Tongji Hospital) were randomly divided into four groups (n = 6 rats per group): (i) PCL, (ii) PCL/CS, (iii) PCL/CS/WH and (iv) PCL/CS/WH/E7. The membranes used in each group were made into a round sample with a diameter of 1 cm and inserted into the each rat respectively. The rats were sacrificed using an over dose of pentobarbital sodium on day 14 after implantation. Together with the skin, the subcutaneous samples were removed and fixed with 4% PFA. For histological examination, the preserved specimens were subsequently cut and stained. Hematoxylin and eosin (H&E) and Masson’s trichome were used to stain the sections for general examination and collagen evaluation, respectively. Selected slices were also subjected to anti- vascular endothelial growth factor (VEGF) immunofluorescent and CD31 double labeling to directly observe neovascularization. The relative fluorescence intensities of CD31 were calculated using ImageJ software.

Critical-sized calvarial defect repair

A critical-sized calvarial defect model were conducted to verify whether the PCL/CS/WH/E7 could promote bone regeneration in vivo. 24 male SD rats weighing 210–250 g (the Laboratory Animal Center of Tongji Hospital) were randomly divided into four groups (n = 6 rats per group): (i) PCL, (ii) PCL/CS, (iii) PCL/CS/WH and (iv) PCL/CS/WH/E7. Each SD rat per group received critical bone defects. First, rats were anesthetized with 3% w/v with pentobarbital sodium. Then, the periosteum of the parietal bone was removed through a 2 cm longitudinal incision on the top of the head. Next, a 4 mm-diameter bone defect was created using a trephine on both sides of the sagittal suture. Various membranes without the implantation of cells were used to cover the defect in different groups. Finally, the incision was closed carefully. The rats were sacrificed using an over dose of with an overdose of sodium pentobarbital to obtain the skulls after 3 weeks. The harvested skulls were fixed in 10% paraformaldehyde for 2 days and then scanned by micro-CT (viva CT 40, Scanco 274 Medical, Switzerland). All 3D -reconstructed skull images were made by VGStudio software. The new bone formation induced by implants was detected by bone volume (BV), bone volume/total volume (BV/TV), trabecular thickness (Tb·Th) and trabecular number (Tb·N), which were quantitatively analyzed using CT Analyser software (v1.17). Afterwards, the specimens were decalcified and sectioned to observe histological bone formation using H&E and Masson’s trichome staining. In order to further characterize new bone formation and neovascularization in the defect areas, immunohistochemical analysis and immunofluorescent staining of the specimen slices was performed for OCN, osteopontin (OPN) and CD31 respectively. The work has been reported in line with the ARRIVE guidelines 2.0.

Statistical analysis

All data were expressed as the mean ± standard deviation (SD), which were analyzed by GraphPad Prism 9. A one-way analysis of variance (ANOVA) was performed to compare the differences among groups. The value of P<0.05 were considered statistically significant.

Results

Characterization of nanofibrous membranes

In this study, we successfully fabricated PCL, PCL/CS, PCL/CS/WH, PCL/CS/WH/E7 nanofibrous membranes by electrospinning technology. The surface morphology of different membranes was analyzed by SEM. Under different magnifications of SEM, the PCL bionic membrane exhibited nanofibers characterized by a smooth surface and the absence of particles, suggesting that optimal spinning conditions were achieved. Electrospinning technology enables the uniform doping of WH nanoparticles into the interior of the spun fibers (Fig. 1A ). The FTIR spectra of the different membranes exhibit bands for polymer components (Fig. 1C). In the pure PCL, the contact angles was 121.7 ± 0.9°. The contact angles of PCL/CS, PCL/CS/WH and PCL/CS/WH/E7 were 111.9 ± 1.3°, 111.1 ± 1.2° and 22.4 ± 1.2°, respectively (Fig. 1B). The results of tensile strength testing showed that the PCL/CS/WH nanofiber membrane had the strongest tensile strength and resistance to deformation among the four groups. The incorporation of CS and WH nanoparticles into pure PCL nanofibrous membrane increased the elongation at break. However, the tensile strength and resistance to deformation of the membranes decreased after coupling with the E7 peptide (Fig.S1-S2).

Fig. 1
figure 1

(A) Scanning electron microscopy (SEM) images depicted the surface morphology of the material, with black arrows indicating WH NPs. (B) The water contact angle was represented by each bionic membrane. (C) The FTIR of different bionic membranes. (D-E) Release curves of Ca2+ and Mg2+ions from the PCL/CS/WH/E7 membrane

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Ion release

We measured the ion release properties of different nanocomposite membranes in an aqueous medium by ICP-OES (Fig. 1D-E).The results showed that PCL/CS/WH/E7 membrane could release Ca2+ and Mg2+ ions for 2 weeks, and reached 80% of the release on day 10. The cumulative release amount of WH in 14 days was 87.5 µM, which was physiologically acceptable.

Membrane cytocompatibility

In this study, we evaluated the effect of different electrospun membranes on the activity and proliferation of BMSCs and EPCs. Phalloidin was used to delineate the cytoskeleton of BMSCs and EPCs cultured on different membranes for 3 days. The results showed that BMSCs and EPCs could adhere and spread on the surfaces of the four membranes (Fig. 2A). Live/dead cells were stained at day 1, 3 and 5 after culture, respectively. The living cells were dyed green with Calcein-AM and the dead cells were dyed red with PI to evaluate the cytotoxicity. Few dead BMSCs and EPCs were observed on the membranes in each group, and the number of living cells increased steadily (Fig. 2B-C). Furthermore, the number of BMSCs seemed not different between PCL, PCL/CS, PCL/CS/WH groups at the same time point, indicating that chitosan (CS) or Whitlockite (WH) nanoparticle incorporation did not cause cytotoxicity to BMSCs. Interestingly, the addition of E7 peptide seemed accelerate the proliferation of BMSCs on the membrane by day 5. For EPCs, the incorporation of CS, WH nanoparticles not only showed no cytotoxicity to EPCs on the biofilm, but also promoted its proliferation. These results were further confirmed by CCK-8, showing that there was no statistically significant difference in the number of BMSCs on different membranes during the first three days. However, the incorporation of E7 peptide promoted cell proliferation at day 5 (Fig. 2D). A similar proliferation trend was observed in EPCs (Fig. 2E). In conclusion, these different membranes have good biocompatibility to BMSCs and EPCs.

Fig. 2
figure 2

Biocompatibility of different bionic membranes. (A) Morphology of BMSC and EPCs on different membranes on day 3. (B) BMSCs and EPCs (C) were cultured on membranes for 1, 3, and 5 days before live/dead staining. (D-E) The proliferation of BMSC and EPC was detected by CCK-8 on day 1, 3 and 5. ( n = 3 *p < 0.05, **p < 0.01)

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Effect on angiogenic capacity in vitro

To explore the effect of addition of WH and E7 peptide on angiogenesis, we performed a tube formation assay. Figure 3A showed the formation of a microvascular network under a microscope, indicating that the WH and E7 peptided promote angiogenesis. To clarify the differences in vascular differentiation between the different groups, we also performed a statistical analysis of the vascular network to compare the differences in total length, number of nodes, and number of branches between the groups. The results showed that PCL/CS/WH and PCL/CS/WH/E7 membranes induced more small vascular networks at the early stage(P<0.05) (Fig. 3B-E), and thus they had better angiogenic properties. The results of qRT-PCR showed that the expression of Ang-1 and VEGF genes was up-regulated in the PCL/CS groups compared with the PCL group. Moreover, with the addition of CS, WH nanoparticles and E7 peptide in turn, the expression of Ang-1 and VEGF genes was gradually increased (Fig. 3F-G), and these differences were statistically significant(P<0.05).

Fig. 3
figure 3

To evaluate the angiogenesis and migration ability of EPCs and BMSCs on different membranes in vitro. (A) Representative bright-field images and (B-E) quantitative analysis of tube formation experiments. (F-G) The RT-qPCR results for angiogenesis-related genes Ang-1 and VEGF were obtained from the nanofibrous membranes. (H) Representative images and (J) quantitative analysis of the Transwell assay of EPCs on day 1. (K) Representative images and (J) quantitative analysis of the Transwell assay of BMSCs on day 1. (L) The mRNA levels of CXCR4 in different groups. ( n = 3 *p < 0.05, ***p ​< ​0.001, ****p ​< ​0.0001)

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Effect on cell migration ability

Transwell migration assays were performed to investigate the chemotactic response of BMSCs and EPCs to the co-culture medium with extracts from different membranes. The migration ability of BMSCs in PCL/CS, PCL/CS/WH, and PCL/CS/WH/E7 groups was higher than that in pure PCL group (P < 0.05) (Fig. 3J). PCL/CS/WH/E7 membrane had the best effect with regard to promoting BMSCs migration (Fig. 3K). The same trend of promoting cell migration was observed in EPCs (Fig. 3H). PCL/CS/WH/E7 membrane also had the best effect with regard to promoting EPCs migration (Fig. 3J). C-X-C motif chemokine receptor 4 (CXCR4) is one of the key molecules that promote the migration of BMSCs and EPCs to the damaged area, thus the mRNA levels of CXCR4 in different groups were detected by qRT-PCR. The results showed that the trend of CXCR4 gene expression levels in the four groups was similar to its angiogenesis related counterparts (Fig. 3L), and these differences were statistically significant(P<0.05). In conclusion, PCL/CS/WH/E7 membranes may provide an excellent microenvironment for bone repair formation.

Effect on the ability to induce osteogenic differentiation in vitro

To demonstrate the effect of PCL/CS/WH/E7 membranes on inducing osteogenic differentiation in vitro, BMSCs and extracts of different membranes were co-cultured and osteogenic induced. Sirius red and alizarin red were evaluated. Sirius red staining was performed on the surface collagen of each group at days 7 and 10 (Fig. 4A). The PCL/CS/WH/E7 groups had the highest staining intensity, while the PCL group had the lowest staining intensity. The quantitative results obtained after elution of the dye were consistent with the above staining results (Fig. 4B). ARS staining was performed on the surface calcium nodules of each group at 10 days (Fig. 4C). The PCL/CS/WH/E7 group showed the highest staining and the PCL group showed the lowest staining. The quantitative results obtained after dye elution were consistent with the above observations, and the differences between the groups were statistically significant (P < 0.05) (Fig. 4D). The expression levels of ALP, BMP-2, OCN, and RUNX2 genes in the four groups of BMSCs further confirmed the results of the above staining (Fig. 4E-H), and these differences were statistically significant(P<0.05). It is speculated that CS, WH nanoparticles and E7 peptide could play a synergistic role in promoting bone regeneration.

Fig. 4
figure 4

To evaluate the effect of different membranes on osteogenic differentiation of BMSCs in vitro. (A) Sirius staining was used to observe the collagen content of BMSC cultured with the extract of each nanofiber membrane was performed after 7 and 10 days of osteogenic induction. (B) The semi-quantitative analysis of Sirius staining. (C) After 10 days of osteogenic induction, alizarin red staining was used to observe the calcium nodule content in BMSCs cultured with different membranes extracts. (D) The semi-quantitative analysis of Alizarin red staining. (E-H) The RT-qPCR results for osteogenic genes ALP, BMP-2, OCN and RUNX2 were obtained from the nanofibrous membranes. ( n = 3 *p < 0.05, **p < 0.01)

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In vivo osteogenesis and angiogenesis assessment

We evaluated the angiogenic ability of different membranes in vivo by subcutaneous implant experiments (Fig. 5A). The effect of PCL/CS/WH/E7 membranes on angiogenesis in vivo was examined by HE staining of skin tissue after one week. In Fig. 5B, the black arrow indicates neovascularization, which was significantly promoted in the PCL/CS/WH/E7 group. From the immunofluorescence staining, it was found that the PCL/CS/WH/E7 group had the highest CD31 expression (Fig. 5C). Semi-quantitative analysis of CD31 fluorescence intensity was performed and a consistent trend was observed (Fig. 5D).

Fig. 5
figure 5

Subcutaneous implantation assay was used to evaluate the angiogenic ability of bionic membrane in vitro. (A) Schematic diagram of subcutaneous implantation experiment. (B) H&E staining was performed on day 10 (black arrows indicate neovascularization and M represents bionic membrane). (C) Representative images of immunofluorescence staining for CD31(red) and VEGF (green), and (D) the fluorescence intensity of CD31 in each field. (n = 6 **p < 0.01, ****p ​< ​0.0001)

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Critical size skull defect models were used to evaluate the effects of PCL, PCL/CS, PCL/CS/WH and PCL/CS/WH/E7 membranes on promoting periosteum formation and osteogenesis. The samples were collected and scanned by micro-CT, and the three-dimensional images were reconstructed (Fig. 6C). Quantitative analysis of BV and BV/TV showed that the PCL/CS/WH/E7 group had the best defect repair effect(Fig. 6D). The repair effect of pure PCL membrane was the worst, and the bone volume fraction was 14.36 ± 0.75%. The bone fraction volume of PCL/CS group was (29.07 ± 1.80%)%, which was better than that of PCL group. The PCL/CS/WH/E7 membrane had the strongest osteogenic ability, and the bone volume fraction was 41.76 ± 6.40%. The volume of new bone in the defect area had the same trend. What’more, the Tb·Th and Tb·N results were also similar to BV/TV, the results of Tb·Th and Tb·N in the PCL/CS/WH/E7 group were also better than those in the other groups(Fig. 6F-G). The above results indicated that the PCL/CS/WH/E7 membrane promotes osteogenesis and new bone stability. After that, samples were decalcified, and HE and Masson’s trichrome staining were performed after embedding sections to compare the histological changes of the defect area. The HE staining results showed that the repair of the defect area in the PCL group was limited, while the bone defect area in the PCL/CS, PCL/CS/WH, and PCL/CS/WH/E7 groups was significantly reduced compared to the PCL group. The Masson staining results showed that the PCL group had sparse collagen fibers in the bone defect area, and the abundance of collagen fibers doped with CS, WH nanoparticles, and E7 was higher (Fig. 6B).As for the new bone formation in the defect area, a small amount of new bone tissue was observed in the PCL alone group. The incorporation of WH and E7 promoted significant new bone formation, which confirmed the strong synergistic osteogenic ability of the two substances(Fig. 6B).

Fig. 6
figure 6

The osteogenic ability of different bionic membranes was evaluated in vivo. (A) Schematic illustration of the skull defect experiment. (B) H&E staining and masson staining were performed after 3 weeks to obtain representative histological images of the different membranes. (M: bionic membrane; NB: new bone). (C) 3D reconstruction of the skull defect area. (D-G) Statistical analysis of total bone volume (BV), bone volume fraction (BV/TV), trabecular thickness (Tb·Th) and trabecular number (Tb·N). (n = 6 *p < 0.05, **p < 0.01,***p ​< ​0.001)

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Immunohistochemical staining for CD31(angiogenesis marker) and OCN(osteogenesis marker) sections (Fig. 7A) showed that the number of OCN-positive cells and the number of CD31-positive cells gradually increased with the incorporation of CS, WH nanoparticles and E7, thus confirming the in vivo osteogenic and angiogenic properties of PCL/CS/WH/E7. Staining quantification of the intensity of both markers (Fig. 7C-D) confirmed the staining results. Immunofluorescence staining of OPN (an osteogenic marker) and quantitative analysis also showed the same trend (Fig. 7B, E).

Fig. 7
figure 7

The osteogenic ability of different bionic membranes was evaluated in vivo. (A) Representative images of immunohistochemical staining for CD31 and OCN. (B-C) Statistical quantitative assessment of CD31 and OCN. (D) Immunofluorescence staining of OPN in the defect area. (E) Quantitative evaluation for immunofluorescence staining of OPN. (n = 6 *p < 0.05, **p < 0.01, ****ps < 0.0001)

Full size image

Discussion

The periosteum plays a significant role in bone development and repair since it is not only rich in cells, including stem cells, but also offers cytokines that facilitate bone formation. Clinically, a consensus has been reached that the structural integrity of the periosteum should be maintained as far as possible to promote the repair of bone defects [39]. Given the challenges associated with acquiring natural periosteum and the inherent ethical concerns, biomimetic materials that replicate the structure and function of natural periosteum offer a promising alternative for repairing critical-sized bone defects [40]. Electrospinning technology has been widely used and studied in the construction of biomimetic periosteum. However, they cannot perfectly simulate the flexibility and extensibility of natural periosteum and need to be continuously optimized [41].

In this study, we first fabricated a WH-and CS-doped PCL composite nanofiber membrane by electrospinning and then coupled it with E7 peptides. Subsequently, it was compared with pure PCL nanofiber membrane, PCL nanofiber membrane doped only with CS and nanofiber membranes doped with CS and WH. Through SEM, it can be found that the addition of CS and WH and the coupling of E7 peptide will not affect the surface morphology of the nanofiber membrane (Fig. 1A), which meets the requirements of high area-volume ratio, high porosity and other characteristic structures of tissue engineered periosteum. The hydrophobicity of a biomaterial affects its biocompatibility [42]. The increased hydrophilicity of the material is beneficial to cell proliferation and adhesion [43]. The water contact Angle reflects the hydrophilicity or hydrophobicity of the biomaterials. The change in the water contact angle may be related to the hydrophilicity of CS [3, 44]. The water contact angle did not change after adding WH nano particles. In addition, the coupling of E7 peptide further improved the hydrophilicity of the bionic membrane [45]. The WH nanoparticles contained in PCL/CS/WH/E7 nanofiber membranes can release Ca2+ and Mg2+ ions, which can promote the osteogenic differentiation of BMSCs [46]. The results of ion release experiments showed that Ca2+ and Mg2+ could be released in a controlled manner within 14 days (Fig. 1D-E), the accumulated Ca2+ and Mg2+ ions provided an environment that promoted osteogenic differentiation and angiogenesis [47, 48]. In order to achieve the purpose of bone defect repair, biomaterials should have the ability to promote cell adhesion and proliferation to accelerate the repair process [34]. CCK8 assay found that the PCL/CS/WH/E7 nanofiber membrane effectively promoted the proliferation of BMSCs and EPCs compared with the other three groups. The results of Live/Dead staining and F-actin staining showed that the components of the PCL/CS/WH/E7 nanofiber membrane had no effect on the cell adhesion to the membrane and had no cytotoxicity (Fig. 2). The doping of WH nanoparticles and CS in the nanofiber films also leads to the enhancement of the tensile strength and the resistance to deformation of the membranes (Fig.S1-S2). However, introducing a certain amount of nanoparticles will enhance the mechanical properties, but subsequently leads to a decrease in the mechanical properties. This phenomenon can be attributed to the interaction between nanoparticles and segments within high polymer chains [49].

In recent years, the development of artificial and biomimetic periosteum aims to replicate the biological functions of natural periosteum to recruit bone stem cells and restore the structural integrity of bone tissue [50]. Wang et al. designed an artificial periosteum loaded with a filamentous bacteriophage clone named P11, which endowed the artificial periosteum with the capacity to recruit BMSCs for leading to improved efficiency in fracture repair [51]. PCL/CS/WH/E7 nanofiber membrane showed the best chemotaxis of BMSCs compared with the other three nanofiber membranes, according to the results of the migration assay (Fig. 3J-K). It was reported that the SDF-1 (CXCL12) /CXCR4 axis was involved in cell migration. SDF-1 is a key chemokine whose expression is significantly upregulated at the site of injury, attracting MSCs and EPCs expressing CXCR4 receptors to the damaged area [52,53,54]. Our results showed that PCL/CS/WH/E7 membranes could effectively stimulate the up-regulation of CXCR4 expression in BMSCs and EPCs to promote cells to migrate (Fig. 3L). In addition, osteogenic capacity is one of the key characteristics of artificial periosteum in promoting bone defect repair [55]. Mg2+released from WH nanoparticles could induce osteogenic differentiation by activating PI3K, Notch, ERK/c-Fos, BMP-4-related signaling pathways and TRPM7 protein channels [56]. By testing the secretion of collagen fibers, the deposition of calcium nodules, and the expression of osteogenic-related genes, it can be intuitively seen from the data that the expression of BMSCs cultured on PCL/CS/WH/E7 nanofibrous membranes resulted in a significant improvement in their osteogenic differentiation (Fig. 4). To further investigate the effect of PCL/CS/WH/E7 nanofiber membrane on the repair of critical-sized calvarial defect of SD rats. The results of Micro-CT, HE, Masson, immunofluorescence and histochemistry showed that PCL/CS/WH/E7 tissue-engineered periosteum was superior to the other three groups (Figs. 6 and 7).

Bone is a highly vascularized tissue, and its regeneration depends on blood vessels to transport nutrients and metabolic wastes, providing a suitable environment for the growth of osteoblasts [57, 58]. So, well-designed artificial periosteum should have the coupling effect of “osteogenesis-angiogenesis” [59]. It has been reported that WH may promote EPCs differentiation and tube formation by activating Wnt/β-catenin signaling pathway [60]. E7 peptide may promote the overexpression of HIF-1/VEGFA by activating the Notch1/4 signaling pathway, thereby promoting angiogenesis [61]. Therefore, the angiogenic potential of PCL/CS/WH/E7 nanofibrous membranes was also a focal point of our investigation. In the tube formation assay, four types of nanofibrous membrane extracts were extracted to examine the effects on the migration ability of EPCs, the ability to promote tube formation, and the expression of genes related to tube formation. PCL/CS/WH/E7 tissue-engineered periosteum had the best ability to promote EPCs migration to the site of injury, the best ability to promote tube formation of EPCs, and the strongest ability to promote the expression of angiogenesis-related genes (Fig. 3A-J). The same trend was found after sectioning and immunofluorescence staining of electrospun membranes implanted subcutaneously in rats (Fig. 5). This suggests that the PCL/CS/WH/E7 tissue engineered periosteum has a stable ability to promote angiogenesis.

Conclusion

In summary, we have successfully constructed a cytocompatible biomimetic periosteum that the migration of BMSCs and EPCs was promoted to the defect area and create a favorable microenvironment for bone formation during defect repair. Based on the degradable properties of PCL and the role of scaffolds, E7 peptide-modified CS and WH were shown to persist for an extended period to establish a high-density vascular network and provide a high-quality environment for bone defect repair. Our study confirmed that PCL/CS/WH/E7 biomimetic periosteum has a good effect on promoting bone repair and is expected to be used a potential therapeutic strategy for bone defect in clinic. However, our study also has some limitations, such as: the sample size is small, the experimental duration is short, making it impossible to predict long-term bone repair effects; the degradation dynamics of the materials have not been thoroughly studied, which could affect clinical translation; only Micro-CT and histological methods were used, without molecular biology techniques to validate the osteogenic and vascular mechanisms.

Data availability

All data generated during this study are included in this published article. The data that support the findings of this study are available on request from the corresponding author, upon reasonable request.

Abbreviations

BMSCs:

Bone marrow mesenchymal stem cells

EPCs:

Endothelial progenitor cells

CS:

Chitosan

E7:

E7 peptide

PCL:

Poly-ε-caprolactone

WH:

Whitlockite

OCN:

Osteocalcin

OPN:

Osteopontin

RUNX2:

Runt-related transcription factor 2

ALP:

Alkaline phosphatase

VEGF:

Vascular endothelial growth factor

ANG-1:

Angiopoietin 1

CXCR4:

C-X-C motif chemokine receptor 4

PEO:

Poly ethylene oxide

FTIR:

Fourier transform infrared spectroscopy

CCK-8:

Cell Counting Kit-8

SEM:

Scanning electron microscopy

DAPI:

4′,6-diamidino-2-phenylindole

PFA:

Paraformaldehyde solution

PBS:

Phosphate-buffered solution

BSA:

Bovine serum albumin solution

H&E:

Hematoxylin and eosin

BV:

Bone volume

BV/TV:

Bone volume/total volume

NB:

New bone

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Acknowledgements

This work was finically supported by National Natural Sciences Foundation of China (51877097) and Natural Science Foundation of Hainan Province (824MS165). The authors declare that they have not use AI-generated work in this manuscript. The authors appreciated the Experimental Medicine Research Center of Tongji Hospital, Huazhong University of Science and Technology, for providing the experimental instruments and equipment.

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Author notes

  1. Guangzi Chen and Tao Xu contributed equally to this work

Authors and Affiliations

  1. Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, P.R. China

    Guangzi Chen, Tao Xu, Xuan Fang, Gaohong Sheng, Hongqi Zhao & Chaoxu Liu

  2. Department of Pediatric Surgery, Hubei Geological Staff Hospital, Wuhan, P.R. China

    Weigang Li

  3. Department of Orthopedics, Xiangya Hospital of Central South University, Changsha, Hunan, P.R. China

    Wenbin Liu

  4. Department of Orthopedics, Peoples’s Hospital, Wenchang, Hainan, P.R. China

    Delu Zeng

  5. Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, P.R. China

    Ran Gao

  6. Department of Orthopedics, Shanxi Bethune Hospital, Third Hospital of Shanxi Medical University, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan, P.R. China

    Jian Li

Authors
  1. Guangzi Chen
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  2. Tao Xu
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  3. Ran Gao
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  4. Wenbin Liu
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  5. Weigang Li
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  6. Delu Zeng
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  7. Jian Li
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  8. Xuan Fang
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  9. Gaohong Sheng
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  10. Hongqi Zhao
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  11. Chaoxu Liu
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Contributions

GZ. C and T. X were responsible of conceptualization, original draft preparation, experiments and data analysis. WB. L, WG. L and R. G performed the data curation and visualization. DL. Z, GH. S and HQ. Z performed review editing and supervision. J. L and X. F did the animal experiment. CX. L was responsible of project administration and funding acquisition. GZ. C and T. X contributed equally to this manuscript.

Corresponding author

Correspondence to Chaoxu Liu.

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Ethics approval and consent to participate

Ethical approval was obtained from the Experimental Animal Ethics Committee of Huazhong University of Science and Technology prior to the commencement of the study. Title of the approved project: Explore the mechanism of bone regeneration promoted by polyε-caprolactone/chitosan/whitloite electrospun bionic membrane conjugated with an E7 peptide. Approval Number: [2023] IACUC(3974). Date of approval: 15th January,2023.

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Chen, G., Xu, T., Gao, R. et al. Poly-ε-caprolactone/chitosan/whitlockite electrospun bionic membrane conjugated with an E7 peptide for bone regeneration. Stem Cell Res Ther 16, 212 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04307-4

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04307-4

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512