Poly-ε-caprolactone/chitosan/Whitlockite Electrospun Bionic Membrane conjugated with an E7 peptide for bone regeneration

doi:10.21203/rs.3.rs-5266729/v1

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Research Article

Poly-ε-caprolactone/chitosan/Whitlockite Electrospun Bionic Membrane conjugated with an E7 peptide for bone regeneration

https://doi.org/10.21203/rs.3.rs-5266729/v1

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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 and FTIR. 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.

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.

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

Electrospinning

Angiogenesis

Whitlockite

Chitosan

bone regeneration

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.

Artificial periosteum should possess the unique requirements due to the difficulty of bone regeneration, including a suitable porosity for gas and nutrition exchange, the ability to build vascular networks and recruit endothelial cells and stem cells and the suitable mechanical properties to endure the strain of the operation process and tissue growth[4, 11–13]. Electrospinning is a potential technique for fabricating biomimetic membranes in the field of bone regeneration. Membranes built by this technology present large surface areas and high porosity with small inter-fibrous pore size[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].

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[3]. 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 [29]. 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 [30]. Shi et al. discovered that E7 peptide could enhance the chondrogenic differentiation of BMSCs and improves the efficiency of endogenous BMSC homing. [31]. E7 peptide has been shown to have the potential to promote the adhesion of mesenchymal stem cells and maintain their stemness.

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.

2.1. Preparation and Characterization of Electrospun Fiber Membranes

2.1.1 Preparation of Electrospun Fiber Membranes

WH nanoparticles were synthesized according to our previous study[3, 32] (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.

2.1.2. 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. One microliter of ultrapure water was added to the membrane, and the contact angle was measured using a contact angle test system (JC 2000C, Zhongchen, China).

2.2. 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 mins. 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).

2.3. In Vitro Experiments

2.3.1. Cell culture

BMSCs were isolated from Sprague Dawley (SD) rats according to our previous report[33]. 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.

Endothelial progenitor cells (EPCs) 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.

2.3.2 Cell viability and proliferation

Cell viability was evaluated by a LIVE/DEAD kit (Beyotime, China). Briefly, A total of 1 × 10⁴ cells (BMSCs and 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.

2.3.3. Cell Adhesion

The electrospun fiber membranes were cut into 1cm 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.

2.3.4. Mineralized nodule staining

Mineralized nodule formation in the BMSCs was detected by Alizarin red staining. 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) mixed with leach liquor of different membranes. 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. Ten percent cetylpyridinium chloride (Sinopharm Chemical Reagent Co. Ltd. China) solution was prepared to wash the stained nodules. The absorbance was detected at 562 nm.

2.3.5. Collagen staining

Collagen secretion in the BMSCs was detected by Sirius red staining. 1 ml of cell suspension containing 3 × 10⁴ cells was added to the 24-well plate. Using osteogenic medium with leach liquor of different membranes to culture cells after cells adherence, the mixed media was 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.

2.3.6. 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. A total of 1× 10⁴ EPCs were seeded onto matrigel after the gel solidified at 37°C, and cultured with leach liquor of different membranes. 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.

2.3.7. 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 EPCs 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 EPCs on the permeable film of the chamber after culturing for 24 h. After cultivating for 24 h, EPCs 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.

2.4. In Vivo Studies.

2.4.1 Subcutaneous implantation

In order to verify the angiogenic potential of the membranes in different groups in vivo, membranes 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 1.5cm 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.

2.4.2. 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 2cm 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 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) and bone volume/total volume (BV/TV), 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 osteocalcin (OCN), osteopontin (OPN) and CD31 respectively. The work has been reported in line with the ARRIVE guidelines 2.0

2.5. 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.

3.1 Characterization of nanofibrous membranes

In this study, we successfully fabricated PCL, PCL/CS, PCL/CS/WH, PCL/CS/WH/E7 bionic membrane by electrospin 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). The hydrophobicity of a biomaterial affects its biocompatibility[34]. The water contact Angle reflects the hydrophilicity or hydrophobicity of the biomaterials. 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 change in the water contact angle may be related to the hydrophilicity of CS [3, 35].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[36].

3.2 Ion release

It has been reported that the osteogenic differentiation of bone marrow mesenchymal stem cells is affected by the concentration of some ions[37, 38]. WH nanoparticles contain Ca²⁺ and Mg²⁺ions, which have a certain concentration dependence on the osteogenic induction of BMSCs[39]. 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. The accumulated Ca²⁺ and Mg²⁺ ions provided an environment that promoted osteogenic differentiation and angiogenesis[40, 41]. By comparing the results of PCL/CS/WH group and PCL/CS/WH/E7 group, it was shown that the coupling of E7 peptide did not affect the ion release of the membrane.

3.3 Membrane cytocompatibility

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. In this study, we first 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) 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 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). This is similar to the results of Li Q et al[42]. A similar proliferation trend was observed in EPCs (Fig. 2E), due to the release of WH and dissolution of CS by Mg^{2 +} to promote the proliferation of EPCs[43, 44]. In conclusion, these different membranes have good biocompatibility to BMSCs and EPCs.

3.4 Effect on angiogenic capacity in vitro

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[45, 46]. It has been reported that WH may promote EPCs differentiation and tube formation by activating Wnt/β-catenin signaling pathway[47]. E7 peptide may promote the overexpression of HIF-1/VEGFA by activating the Notch1/4 signaling pathway, thereby promoting angiogenesis[48]. 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), and thus they had better angiogenic properties.

3.5 Effect on cell migration ability

Appropriate cell colonization and vascularization of tissue engineering constructs after transplantation are critical steps for successful bone repair[49]. In order to reach the bone defect site and begin the repair process, cells must migrate to the target area. This migratory activity can be mediated by chemokines and is important in therapeutic approaches. 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. 3C). PCL/CS/WH/E7 membrane had the best effect with regard to promoting BMSCs migration (Fig. 3D). The same trend of promoting cell migration was observed in EPCs (Fig. 3E). PCL/CS/WH/E7 membrane also had the best effect with regard to promoting EPCs migration (Fig. 3F). In conclusion, PCL/CS/WH/E7 membranes may provide an excellent microenvironment for bone repair formation.

3.6 Effect on the ability to induce osteogenic differentiation in vitro

Chitosan may accelerate bone regeneration, although this activity was low compared to HA. Its modification involving the addition of bone morphogenetic proteins or growth factors leads to an improvement in the regenerative properties of chitosan relative to bone tissue[34]. WH, due to its negative surface charge, can stimulate osteogenic differentiation by electrostatic adsorption of positively charged osteoprotegerins[50]. 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. 4C). ARS staining was performed on the surface calcium nodules of each group at 10 days (Fig. 4B). 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(Fig. 4D). It is speculated that CS, WH and E7 peptide could play a synergistic role in promoting bone regeneration.

3.7 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).

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. Then, 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, 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).

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 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. 7B-C) confirmed the staining results. Immunofluorescence staining of OPN (an osteogenic marker) and quantitative analysis also showed the same trend (Fig. 7D-E).

In summary, we have successfully constructed a cytocompatible biomimetic periosteum that BMSCs and EPCs were promoted to migrate 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, doped CS and WH could persist for a long time 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.

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

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

VEGF: Vascular endothelial growth factor

BV: Bone volume

BV/TV: Bone volume/total volume

NB: New bone

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

Author 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.

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.

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

Competing interests

The authors declare no conflicts of interest.

Consent for publication

All authors agree to submission of the manuscript and agree to publication.

Author details

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

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

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

⁴Department of Orthopedics, People＇s Hospital, Wenchang , Hainan , P.R.China.

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

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

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Poly-ε-caprolactone/chitosan/Whitlockite Electrospun Bionic Membrane conjugated with an E7 peptide for bone regeneration

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Abstract

Figures

1. Introduction

2. Methods

2.1. Preparation and Characterization of Electrospun Fiber Membranes

2.1.1 Preparation of Electrospun Fiber Membranes

2.1.2. Characterization of the Electrospun Fiber Membranes

2.2. Ion Release

2.3. In Vitro Experiments

2.3.1. Cell culture

2.3.2 Cell viability and proliferation

2.3.3. Cell Adhesion

2.3.4. Mineralized nodule staining

2.3.5. Collagen staining

2.3.6. In Vitro Vascularization Assay

2.3.7. Cell Migration Assay

2.4. In Vivo Studies.

2.4.1 Subcutaneous implantation

2.4.2. Critical-sized calvarial defect repair

2.5. Statistical analysis

3. Results and discussion

3.1 Characterization of nanofibrous membranes

3.2 Ion release

3.3 Membrane cytocompatibility

3.4 Effect on angiogenic capacity in vitro

3.5 Effect on cell migration ability

3.6 Effect on the ability to induce osteogenic differentiation in vitro

3.7 In vivo osteogenesis and angiogenesis assessment

4. Conclusion

Abbreviations

Declarations

References

Supplementary Files

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