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. 2024 Feb 29;25:101015. doi: 10.1016/j.mtbio.2024.101015

Cascaded controlled delivering growth factors to build vascularized and osteogenic microenvironment for bone regeneration

Haifei Cao a,1, Shuangjun He d,1, Mingzhou Wu e,1, Lihui Hong b, Xiaoxiao Feng b,c, Xuzhu Gao b,c, Hongye Li b,c,⁎⁎⁎, Mingming Liu b,c,⁎⁎, Nanning Lv b,c,⁎
PMCID: PMC10945171  PMID: 38500557

Abstract

The process of bone regeneration is intricately regulated by various cytokines at distinct stages. The establishment of early and efficient vascularization, along with the maintenance of a sustained osteoinductive microenvironment, plays a crucial role in the successful utilization of bone repair materials. This study aimed to develop a composite hydrogel that would facilitate the creation of an osteogenic microenvironment for bone repair. This was achieved by incorporating an early rapid release of VEGF and a sustained slow release of BMP-2. Herein, the Schiff base was formed between VEGF and the composite hydrogel, and VEGF could be rapidly released to promote vascularization in response to the early acidic bone injury microenvironment. Furthermore, the encapsulation of BMP-2 within mesoporous silica nanoparticles enabled a controlled and sustained release, thereby facilitating the process of bone repair. Our developed composite hydrogel released more than 80% of VEGF and BMP-2 in the acidic medium, which was significantly higher than that in the neutral medium (about 60%). Moreover, the composite hydrogel demonstrated a significant improvement in the migratory capacity and tube formation ability of human umbilical vein endothelial cells (HUVECs). Furthermore, the composite hydrogel exhibited an augmented ability for osteogenesis, as confirmed by the utilization of ALP staining, alizarin red staining, and the upregulation of osteogenesis-related genes. Notably, the composite hydrogel displayed substantial osteoinductive properties, compared with other groups, the skull defect in the composite hydrogels combined with BMP-2 and VEGF was full of new bone, basically completely repaired, and the BV/TV value was greater than 80%. The outcomes of animal experiments demonstrated that the composite hydrogel effectively promoted bone regeneration in cranial defects of rats by leveraging the synergistic effect of an early rapid release of VEGF and a sustained slow release of BMP-2, thereby facilitating vascularized bone regeneration. In conclusion, our composite hydrogel has demonstrated promising potential for vascularized bone repair through the enhancement of angiogenesis and osteogenic microenvironment.

Keywords: Composite hydrogel, Vascularization, Drug delivery, Controlled release, Bone regeneration

Graphical abstract

Image 1

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1. Introduction

Bone defects are primarily attributed to trauma, infection, pathological fracture, tumor resection, and osteomyelitis debridement [1,2]. Despite the impressive regenerative capacity of bone, the restoration of bone defects remains a substantial hurdle in the field of orthopedic surgery. Autogenous and allogeneic bone transplantation are the principal approaches employed for addressing bone defects. Nevertheless, autologous bone transplantation may give rise to local complications, while the transplanted new bone may also undergo autolysis, leading to suboptimal repair outcomes [3]. Although the availability of material sources for bone allograft is extensive, the clinical applications are limited due to the risk of immune rejection and potential disease transmission [4]. In recent years, artificial materials for bone defects have garnered significant interest due to their advantageous features, including solid manufacturability, easy accessibility, and consistent quality [5]. Consequently, these materials have been progressively incorporated into clinical practice [6,7]. Nevertheless, numerous artificial materials continue to exhibit deficiencies in terms of biocompatibility, angiogenesis, and drug-loading capacity [8,9]. The delayed development of blood vessels following implantation is widely recognized as a substantial impediment in the field of bone tissue engineering. Consequently, considering the aspects of structure and functionality, it becomes imperative to devise a bone biomaterial possessing exceptional biocompatibility, prompt angiogenesis, and robust osteogenic capabilities for the effective treatment of bone defects.

When bone biomaterials are applied to treat bone defects, timely and abundant vascular regeneration is the basis to ensure successful repair of bone defects. The effect of blood flow reconstruction determines the speed and impact of bone regeneration at the bone defect site after implantation [10]. Neovascularization regeneration plays a crucial role in the initiation of bone repair during the process of bone healing. Neovascularization provides essential nutrients, osteoblasts, growth factors for bone repair, and a channel for repair cells to enter the damaged area [11]. Especially for large-area bone defects, if there is no complete blood vessel near the defect site, circulating stem cells in the blood vessels cannot migrate to the defect through chemotaxis, resulting in insufficient osteogenesis at the defect site and failure of integration of tissue-engineered bone [12]. To solve this problem, Chen et al. successfully constructed a dense microvascular system in a three-dimensional structure by using bioinks containing endothelial cells and mesenchymal stem cells [13]. Grellier et al. showed that ischemia in the center of a large area of bone defect in a hypoxic environment resulted in bone marrow mesenchymal stem cells (BMSCs) in tissue-engineered bone cannot be induced to differentiate into osteoblasts [14]. Therefore, improving the angiogenesis of bone biomaterials is of great significance for improving the success rate of bone regeneration [15,16].

Scholars have attempted to achieve early and rapid vascularization of bone substitute materials. For example, the angiogenic ability of biomaterials can be improved by loading vascularizing growth factors or doping metal ions. Cheng et al. constructed an injectable biomimetic hydrogel that regulated angiogenesis and bone regeneration by delivering deferoxamine (DFO) and bone morphogenetic protein-2 (BMP-2) [3]. Zhao et al. found that monodisperse bioactive glass microspheres containing strontium can control local inflammatory response by regulating macrophage phenotype, significantly promoting early angiogenesis [17]. Additionally, angiogenesis and bone regeneration are closely related in time and space [18]. Vascular endothelial growth factor (VEGF), one of the regulatory factors of angiogenesis and osteogenesis, is the most potent biological factor to induce angiogenesis. It plays an essential role in promoting angiogenesis by aggregating vascular endothelial cells towards bone defects and has good osteogenic properties by regulating the proliferation and differentiation of osteoblasts [19]. Street et al. proved that compared with other pro-angiogenic factors, VEGF not only has a strong ability to promote angiogenesis but also closely combines with angiogenesis with bone formation and has the effect of migrating osteoblasts, inhibiting osteoblast apoptosis and promoting osteoblast proliferation [20]. Therefore, constructing bone biomaterials that can sustainably release VEGF at the site of bone defect can more effectively repair bone tissue.

Although VEGF has functions of osteogenesis and angiogenesis, the delivery of VEGF alone cannot achieve good osteogenic effects [21], so the combined action of multiple factors is often required to construct the process of bone tissue angiogenesis. Bone morphogenetic protein (BMP) is the most famous osteogenic growth factor, showing strong osteoinductivity. Due to VEGF's excellent angiogenic ability and BMP's osteogenic properties, the most studied is the dual delivery of VEGF and BMP. From nanofibers and hydrogels to 3D-printed bone grafts [22], they have been shown to have excellent effects in promoting vascular-osteogenic coupling. In addition, the researchers found that the expression of the VEGF gene in the early stage of bone healing is earlier than the expression peak of BMP-2. Therefore, rapid early delivery of VEGF combined with slow and persistent delivery of BMP-2, simulating the natural bone healing microenvironment, can achieve better angiogenesis and osteogenesis effects.

In this study, we prepared mesoporous silica nanoparticles (MSN) loaded with BMP-2, called BMP-2@SiO2. In order to achieve a controlled release of VEGF and BMP-2 within the acidic environment of bone defects, a combination of VEGF and BMP-2@SiO2 was incorporated into aldehyde hyaluronic acid (HA-CHO) and gelatin methacryloyl (GelMA) hydrogel. This resulted in the formation of a BMP-2@SiO2-VEGF/HA-GelMA composite hydrogel (BVHG) through the photocrosslinking of GelMA. Furthermore, the interaction between VEGF and the hydrogel occurred via the Schiff base. In addition to characterizing the morphology and release behavior of VEGF and BMP-2, the angiogenic and osteoinductive activity of the composite hydrogel was also evaluated in vitro. Subsequently, the composite hydrogel was surgically introduced into the cranial defect of rats, followed by an assessment of its in vivo efficacy in stimulating angiogenesis and osteogenesis. (Fig. 1).

Fig. 1.

Fig. 1

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Schematic diagram of BVHG composite hydrogel promoting angiogenesis and enhancing osteogenesis. The composite hydrogel exhibited the capability to promptly release VEGF in the initial response to the acidic bone microenvironment, thereby facilitating early angiogenesis. Additionally, it demonstrated sustained release of BMP-2 over an extended period, thereby promoting osteoinductive characteristics.

2. Material and methods

2.1. Fabrication and characterization of materials

Fabrication of BMP-2@SiO2: A mixture comprising of 14 mL of ethanol, 2 mL of deionized water, and 500 μL of ammonia was subjected to magnetic stirring at a temperature of 50 °C for a duration of 5 min. Subsequently, 500 μL of tetraethoxysilane was introduced to the mixture, followed by thorough stirring to obtain SiO2. 1 μg BMP-2 was then added to the SiO2 solution (10 mg/mL) and incubated at 4 °C for 24 h, which was subsequently subjected to centrifugation at a speed of 25,000 g for a duration of 5 min to acquire SiO2 nanoparticles loaded with BMP-2 (BMP-2@SiO2).

Fabrication of HA-CHO: 1.5 g of HA (40–100 kDa) were dissolved in 150 mL of deionized water, followed by the addition of 802 mL of sodium periodate. The resulting mixture was stirred for a duration of 2 h. The reaction was ceased by introducing 200 μL of ethylene glycol, after which the solution was subjected to dialysis using deionized water. The resulting product, HA-CHO, was subsequently freeze-dried and stored at a temperature of 4 °C.

Fabrication of composite hydrogel: A solution was prepared by dissolving 1 g of GelMA, 100 mg of HA-CHO, and 50 mg of the photoinitiator lithium phenyl-2,4,6-trimethyl-benzoyl-phosphonic in 20 mL of deionized water at a temperature of 37 °C. Subsequently, 1 μg VEGF and 5 mg BMP-2@SiO2 were introduced into the aforementioned mixture. BMP-2@SiO2-VEGF/HA-GelMA (BVHG) composite hydrogels were obtained by photocrosslinking (wavelength 405 nm, 1 min).

Material characterizations: The morphology of SiO2 nanoparticles was examined by transmission electron microscopy (TEM: TECNAI G2 F20, FEI, USA). FTIR (Nicolet 6700, Thermo Fisher Scientific, USA) was used to detect the molecular structure of HA-CHO and HA. The cross-sectional morphology of the composite hydrogels was observed by scanning electron microscopy (SEM: Quanta 250, FEI, Hillsboro, OR, USA). In order to investigate the impact of pH on the release of BMP-2 and VEGF from composite hydrogels, the composite hydrogel specimens were subjected to immersion in buffer solutions with pH values of 7.2 and 5.5 at a temperature of 37 °C. The release profiles of BMP-2 and VEGF within the composite hydrogels were assessed at various time intervals. Subsequently, hydrogel samples with dimensions of 4.5 mm in diameter and 5 mm in height were immersed in buffer solutions with pH values of 7.2 and 5.5 at 37 °C for a duration of 24 h. The compressive strength and rheology of these samples was then determined using a universal ability tester manufactured by Shanghai Hengyi Precision Instrument Co., LTD., China, with a testing rate of 5 mm/min.

2.2. Cell morphology and viability

BMSCs were cultured in a mixed medium including α-MEM medium (Hyclone, Thermo Fisher Scientific, USA), 1% penicillin and streptomycin (Procell, China), and 10% fetal bovine serum (Gibco). BMSCs were cultured on hydrogel for two days. According to the different composition of hydrogel, it can be divided into HA-GelMA (HG), VEGF/HA-GelMA (VHG), BMP-2@SiO2/HA-GelMA (BHG), BMP-2@SiO2-VEGF/HA-GelMA (BVHG). These samples are used for cytoskeleton staining and SEM scanning. After fixation with 4% paraformaldehyde for 40 min, the cells were sealed and permeated with 0.3% Triton X-100. Subsequently, a 3% solution of bovine serum albumin was introduced to the cells, and the specimen was maintained at a temperature of 4 °C for a duration of 12 h. Following this, BMSCs were subjected to staining using cyclopeptide and 4′, 6-diamidino-2-phenylindole (DAPI). Images were acquired using an inverted fluorescence microscope. For SEM scanning, BMSCs were fixed using 4% paraformaldehyde and underwent dehydration through a gradient of ethanol. SEM was employed for observation subsequent to critical point drying.

Live/dead staining and CCK-8 assay were used to evaluate the effects of hydrogels on cell proliferation and biocompatibility. First, the cells were seeded on different hydrogels and incubated for 1, 3, or 5 days. The cells were incubated in a CCK-8 reagent for 2 h. Absorbance is then measured at a wavelength of 450 nm with a microplate reader (BioTek, Winooski, VT, USA). The same steps for live/dead staining.

2.3. Angiogenesis assessment

In the lower chamber of a 6-well transwell plate, a seeding density of 40,000 human umbilical vein endothelial cells (HUVECs) per well was utilized. Upon reaching a fusion level of 90%, hydrogels were introduced into the upper chamber, and scratches were generated using a 200 μL pipette tip.

HUVECs were implanted in the upper chamber of transwell plates (Sigma-Aldrich) at a density of 2 × 105/well and culture medium containing hydrogels were placed in the lower chamber. Following an 8-h incubation period, the cells were subjected to staining using crystal violet solution (Sigma-Aldrich) for 30 min, and subsequently examined under a microscope to capture images.

HUVECs were cultured on the surface of growth factor-reduced Matrigel in a 24-well plate to facilitate the process of tube formation. The HUVECs were observed with an optical microscope after 4 h culture in different hydrogel medium conditions, and the tube formation was quantified by ImageJ. In order to assess vascularization, a variety of hydrogels were implanted into the subcutaneous tissue of rats. Following a 10-day period, the samples were collected and preserved using a 4% paraformaldehyde solution. The in vivo vascularization of the hydrogels was evaluated through H&E staining. The resulting images were captured using an inverted microscope and subsequently analyzed using ImageJ.

The vasculogenic properties were assessed through the utilization of CD31 immunofluorescence staining. In brief, HUVECs were permeabilized with 0.2% Triton X-100 after fixation with 4% paraformaldehyde (Beyotime, China), and cellular closure was achieved with a 1% BSA solution. Following overnight incubation at 4 °C with CD31 (1:500, ABclone, China) antibody, the cells were subsequently incubated with Alexa Fluor coupled secondary antibody (1:500, ABclone, China) for 2 h, and DAPI (Sigma Aldrich, USA) for 10 min. Ultimately, the immunofluorescence images were observed utilizing an inverted microscope (ZEISS, Oberlich, Germany).

2.4. Osteogenic assessment

A quantity of 2 × 106 rat bone marrow-derived mesenchymal stem cells (BMSCs) were introduced into the lower chamber of a 24-well Transwell plate, while 300 μL of hydrogels (HG, VHG, BHG, and BVHG) were placed in the upper chamber. The cells were cultured using α-MEM complete medium, which was substituted with osteogenic medium once the degree of cell fusion reached 80%. After osteogenic induction for 7 days, the cells were fixed with 4% paraformaldehyde and incubated with an alkaline phosphatase (ALP) staining solution. Images were collected through an inverted microscope. ALP was quantified using the ALP assay kit. Following a 21-day period of osteogenic induction, alizarin red staining (ARS) was conducted to evaluate the mineralized matrix. The quantification of calcium nodules was accomplished by measuring the optical density at 420 nm subsequent to incubation with perchloric acid (10%).

A quantity of 2 × 106 rat BMSCs were introduced into the lower chamber of a 6-well transwell plate. Subsequently, 1 mL of hydrogels (HG, VHG, BHG, and BVHG) were introduced into the upper chamber after a 12-h period. The cells were then subjected to cultivation in an osteogenic differentiation medium. The expression levels of the characteristic osteogenic genes (Alpl, Runx2, Spp1, Col1a1, Bglap) were quantified using real-time PCR.

2.5. In vivo bone repair assessment

The Ethics Committee of Binzhou Medical University granted approval for all animal-related procedures (2023–302). To evaluate the in vivo osteogenic ability of the hydrogel, the hydrogels, namely HG, VHG, BHG, and BVHG, were surgically implanted into the cranial defect of male Sprague-Dawley rats at the age of 8 weeks. Skull specimens were collected after 4 and 8 weeks. The repair of bone defect was analyzed by Micro-CT. Bone volume ratio (BV/TV, %), and trabecular thickness (Tb.Th., mm) were used to evaluate bone microstructure.

The cranial specimens were collected and subjected to decalcification using an EDTA solution for 4 weeks. Subsequently, the specimens were embedded in paraffin blocks and sliced into sections measuring 5 μm in thickness. These sections were then subjected to staining using H&E and Masson's trichrome staining. The observation of the newly formed bone tissue was conducted through the utilization of Col1a1 and Runx2 immunohistochemical staining techniques. Furthermore, the evaluation of blood vessel formation was carried out via CD31 immunohistochemical staining.

2.6. Statistical analysis

SPSS 15.0 software was used for statistical analysis. Data were expressed as mean ± standard deviation. One-way analysis of variance followed by Tukey's multiple comparisons was used to evaluate differences between the groups. P < 0.05 indicated statistically significant differences between groups.

3. Results

3.1. Characterizations of the composite hydrogels

According to the findings presented in Fig. 2A, mesoporous silica nanoparticles exhibit a spherical shape and are evenly distributed, boasting an approximate diameter of 130 nm. Fourier transform infrared spectroscopy (FTIR) was used to measure whether HA-CHO was obtained successfully. Fig. 2B showed that HA-CHO had a new peak at 1731 cm−1 compared to HA, which is attributed to the C Created by potrace 1.16, written by Peter Selinger 2001-2019 O extension of HA-CHO. VEGF and BMP-2@SiO2 were added to HA-CHO and GelMA solution. BMP-2@SiO2-VEGF/HA-GelMA (BVHG) composite hydrogel was obtained after photocrosslinking. SEM results showed that the composite hydrogels had orderly and uniform interconnecting macropores (Fig. 2C). Moreover, the macroporous structure of the hydrogel did not change with the addition of VEGF or nanoparticles.

Fig. 2.

Fig. 2

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Characterizations of composite hydrogels. (A) TEM images of mesoporous silica nanoparticles. (B) FTIR spectra of HA and HA-CHO. (C) SEM images of composite hydrogels. (D) Release curve of VEGF and BMP-2 from the composite hydrogels under neutral and acidic solution, respectively. (E) Compressive strength of composite hydrogels under neutral and acidic solution, respectively.

The investigation focused on the release characteristics of VEGF and BMP-2 in the BVHG composite hydrogel. In order to replicate the acidic conditions found in bone defects, the release of VEGF and BMP-2 was examined separately in buffer solutions with pH levels of 5.5 and 7.2, respectively. As depicted in Fig. 2D, the composite hydrogel released more than 80% of VEGF and BMP-2 in the acidic medium, which was significantly higher than that in the neutral medium (about 60%). The faster release rate of VEGF and BMP-2 in an acidic solution indicated that the Schiff base bonds of the composite hydrogel were destroyed due to acidic conditions. More importantly, the release time of VEGF was only maintained for 2 weeks, while the release of BMP-2 could last for nearly 40 days. Moreover, the structure of the composite hydrogels was fragmentized in response to the acidic microenvironment, the compressive strength of HG decreased from 27.5 ± 1.3 to 15.7 ± 1.0 kPa, as well as BVHG from 22.9 ± 0.7 to 13.3 ± 0.2 kPa (Fig. 2E). Moreover, the compressive strength of composite hydrogels was decreased with time (Fig. S1). The rheology of composite hydrogels showed that the addition of VEGF could enhance the storage modulus (G′), demonstrating VEGF can form Schiff base bonds with HG composite hydrogel. However, the G’ of BHG and BVHG composite hydrogels was much sharply decreased with the addition of nanoparticles (Fig. S2).

3.2. Cell morphology and viability of the composite hydrogels

BMSCs were cultured on the composite hydrogel to evaluate the biocompatibility of the composite hydrogel. Cytoskeleton staining showed effective adhesion and diffusion of BMSCs on HG, VHG, BHG, and BVHG, exhibiting favorable biocompatibility characteristics. (Fig. 3A). The live/dead images of BMSCs seeded on the composite hydrogel were displayed in Fig. 3B. The vast majority of cells in all groups were alive (stained green), and only a few dead cells (stained red) were observed, which indicated the excellent cytocompatibility of composite hydrogel. Quantitative analysis showed that the living cells accounted for more than 90%, and there was no statistically significant disparity in cell proliferation between groups. As shown in Fig. 3C, cell morphology images captured by SEM showed that BMSCs spread on the hydrogels, demonstrating that the addition of BMP-2 and VEGF did not exert any adverse effects on its biocompatibility. The cell counting kit-8 (CCK-8) assay was used to evaluate cell proliferation on the composite hydrogel. The findings demonstrated a consistent increase in cell count across all groups over a span of five days, with no statistically significant variance observed in cell proliferation among the groups (Fig. 3D).

Fig. 3.

Fig. 3

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Biocompatibility of composite hydrogels. (A) Cytoskeleton staining of BMSCs seeded on the surface of composite hydrogels. (B) Live/dead staining of BMSCs seeded on the surface of composite hydrogels. (C) SEM images of cell morphology on the surface of composite hydrogels. (D) CCK-8 assay indicating the proliferation rate of BMSCs. (*p < 0.05). OD. optical density.

3.3. Angiogenesis of composite hydrogels in vitro

The recruitment of human umbilical vein endothelial cells (HUVECs) by composite hydrogels was evaluated using the scratch assay and cell migration assay. The findings from the scratch assay demonstrated that the healing area of the VHG and BVHG groups was significantly greater than that of the HG and BHG groups at both the 12-h and 24-h time points (Fig. 4A). However, there was no statistically significant difference in the healing areas between the VHG and BVHG groups at these time points (P < 0.05) (Fig. 4B). Furthermore, the cell migration assay demonstrated that both the VHG and BVHG groups significantly promoted the migration of HUVECs, whereas the VHG and BVHG groups had the least impact on cell migration (Fig. 4C&D).

Fig. 4.

Fig. 4

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Effect of composite hydrogels on cell migration. (A) Cell scratch assay for HUVECs migration at 0, 12, and 24 h. (B) Quantitative analysis of migration area, n = 3 (*p < 0.05). (C) Crystalline violet staining for cell migration assay. (D) Quantitative analysis of migrated cells. (*p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The evaluation of angiogenesis was conducted through a tube formation assay. After a duration of 4 h, the group treated with VEGF-loaded composite hydrogels (VHG and BVHG) exhibited the initiation of a primary vascular-like network structure, as depicted in Fig. 5A. Notably, the VHG and BVHG demonstrated a significantly greater total length of tubular structure formation compared to the HG and BHG, as illustrated in Fig. 5B. Furthermore, histological examination using H&E staining of hydrogels implanted in the subcutaneous tissue of rats revealed a significant promotion of angiogenesis in the VHG and BVHG, as shown in Fig. 5C. After 10 days of implantation, the neovascularization area in the VHG and BVHG groups was approximately 81.3% ± 4.6% and 73.0% ± 4.6%, respectively, which were higher than that in the HG group (23.3% ± 3.2%) and BHG group (24.9% ± 3.6%) (Fig. 5D). The immunofluorescence staining results revealed a substantial increase in the expression level of CD31 in both the VHG and BVHG groups, as compared to the HG and BHG groups (Fig. 5E).

Fig. 5.

Fig. 5

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Angiogenesis assay. (A) Tube formation assay. (B) Quantitation of tube length, n = 3. (*p < 0.05). (C) H&E staining of hydrogels implanted in rat subcutaneous tissue. Blood vessels are marked with yellow arrows. (D) Quantification of the area of blood vessels, n = 3. (*p < 0.05). (E) Immunofluorescence staining of CD31 in HUVECs. Green: CD31, blue: DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.4. Osteogenesis of composite hydrogels in vitro

To determine the effect of composite hydrogels on the osteogenic activity of BMSCs in vitro, ALP staining, ARS, and the expression of osteogenic genes were tested. The results showed that compared with the HG group, VHG group could slightly upregulate the expression of ALP (Fig. 6A). Moreover, hydrogels loaded with BMP-2@SiO2 (BHG and BVHG group) significantly promoted the expression of ALP (Fig. 6B). BMSCs were subjected to treatment with composite hydrogel and subsequently cultured for a duration of 21 days. The application of composite hydrogels resulted in the generation of a significant quantity of calcium nodules. (Fig. 6C). It should be noted that the BHG and BVHG groups had rich calcium deposition, more than that of the HG and VHG groups (Fig. 6D). Quantitative PCR results showed that VHG, BHG, and BVHG groups promoted the expression of osteogenic genes Alpl, Spp1, Runx2, Col1a1, and Bglap compared with the HG group (Fig. 6E). These results indicated that both VEGF and BMP-2@SiO2 promote the expression of osteogenic genes. In addition, BVHG groups containing VEGF and BMP-2@SiO2 significantly enhanced the expression of osteogenic genes compared with other groups.

Fig. 6.

Fig. 6

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Osteogenic differentiation. (A) ALP staining. (B) Quantification of ALP staining. (*p < 0.05). (C) Alizarin red staining. (D) Quantification of ARS, n = 3. (*p < 0.05). (E) Expression of osteogenesis-related genes, n = 3. (*p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.5. In vivo bone repair assessment

The composite hydrogels were surgically implanted into the cranial defects of rats for the purpose of investigating bone formation, as depicted in Fig. 7A. After a period of 4 weeks post-surgery, minimal bone formation was observed in the HG group, while the VHG and BHG groups exhibited some evidence of new bone tissue within the cranial defect, as illustrated in Fig. 7B. Moreover, most of the new bone formation was observed in the BVHG group, and the percentage of bone volume (BV/TV) showed that the new bone formation in the BVHG group reached 59.0% ± 3.0% (Fig. 7C), indicating that VEGF and BMP-2 had a significant synergistic effect on bone repair. After 8 weeks of implantation, varying amounts of new bone appeared in skull defects in all groups. In addition, compared with other groups, the skull defect in the BVHG group was full of new bone, basically completely repaired, and the BV/TV value was greater than 80% (Fig. 7C). The analysis of Tb.Th exhibited a comparable trend to that of BV/TV (Fig. 7C). The descending order of the Tb.Th analyses at 4 and 8 weeks were BVHG, BHG, VHG, and HG groups.

Fig. 7.

Fig. 7

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Characterizations of composite hydrogels for in vivo bone repair. (A) the critical-size bone defect model and implantation of composite hydrogels. (B) 3D reconstruction of defect areas at 4 and 8 weeks. (C) Quantification of BV/TV and Tb.Th in defect area, n = 3. (*p < 0.05).

The evaluation of bone defect repair was extended through the utilization of H&E and Masson's trichrome staining techniques. The outcomes obtained from H&E staining and Masson's trichrome staining were found to agree with the findings derived from Micro-CT reconstruction (Fig. 8A&B). The results indicated a noticeable inadequacy in bone regeneration within the HG group, where the defect region was predominantly filled with fibrous connective tissue. Conversely, the BVHG group exhibited a greater presence of newly generated bone tissue compared to the BHG and VHG groups (Fig. 8A&B).

Fig. 8.

Fig. 8

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Histological analysis. (A) H&E staining of the calvarial critical-sized defects repaired by composite hydrogels. (B) Masson's staining of calvarial critical-sized defects repaired by composite hydrogels.

Immunohistochemical staining revealed significant positive staining for Col1a1 and Runx2 in the BVHG group at both 4 and 8 weeks after surgery, suggesting a strong synthesis of the bone matrix at the site of the defect (Fig. 9A&C). Moreover, the BHG group exhibited a positive staining rate for Col1a1 and Runx2 that ranked second only to the BVHG group and surpassed that of the HG and VHG groups (Fig. 9B&D). To evaluate the impact of composite hydrogels on angiogenesis, immunohistochemical staining was employed to identify the presence of Vegf (Fig. 9E&F) and CD31 (Fig. S3). The results revealed that the BVHG group exhibited noticeable positive staining for Vegf (Fig. 9F) and CD31. These outcomes substantiated that the introduction of BVHG facilitated the formation of new osseous tissue and the regeneration of new capillaries, thereby promoting the repair process of critical-size bone defects.

Fig. 9.

Fig. 9

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Immunohistochemical analysis. (A) Immunohistochemical staining for Col1a1. (B) Quantitative analysis of Col1a1, n = 3. (*p < 0.05). (C) Immunohistochemical staining for Runx2. (D) Quantitative analysis of Runx2, n = 3. (*p < 0.05). (E) Immunohistochemical staining for Vegf. (F) Quantitative analysis of Vegf, n = 3. (*p < 0.05).

4. Discussion

The interdependence of bone formation and angiogenesis is evident in the process of bone regeneration [23]. Blood vessels play a crucial role in transporting minerals and growth factors to bone tissue, facilitating calcium salt deposition, and releasing paracrine signals that regulate the growth, differentiation, and regeneration of various cell types. Likewise, in biomaterial-induced bone tissue regeneration, the timely development of vascular networks is imperative for effective bone repair [24]. The insufficiency of blood supply during bone tissue regeneration presents a formidable obstacle in the repair of bone defects and reconstruction of bone tissue. Consequently, addressing the issue of promoting rapid vascularization of bone biomaterials after transplantation in vivo, as well as establishing a robust vascular network to facilitate sufficient nourishment for subsequent new bone formation, emerges as a pressing concern demanding resolution.

VEGF is one of the most critical regulatory factors of angiogenesis and also possesses a significant influence on bone regeneration through the regulation of osteogenic growth factors [25,26]. For instance, Li et al. developed a pH-responsive hydrogel microsphere that stimulates angiogenesis and bone regeneration by activating the HIF-1α/VEGF signaling pathway [27]. Nevertheless, the delivery of VEGF alone proves to be inadequate for effective bone tissue regeneration [28,29]. BMP-2 is widely acknowledged as the foremost and extensively employed osteoinductive agent, and scaffolds incorporating BMP-2 have demonstrated the capacity to augment in vivo bone regeneration. However, the sole administration of BMP-2 does not adequately stimulate the vascularization of scaffolds [30]. More importantly, combining angiogenesis and osteogenesis can achieve better bone repair [[31], [32], [33]]. However, as the primary and most potent modulators of angiogenesis and osteogenesis, respectively, the delivery of VEGF or BMP-2 alone does not always produce the desired results, indicating that they are indispensable in the bone repair process. In addition, VEGF is expressed in the early stages of natural bone repair [19,34], regulates the formation of vascular networks, and brings critical elements for subsequent bone formation, including oxygen, osteoblast progenitor cells, and nutrients. BMP-2 is observed subsequent to the formation of the fully developed vascular network [35,36] subsequently enhancing the expression of Runx2, which in turn stimulates the synthesis of bone sialoprotein (BSP), OCN, and OPN, thereby initiating the process of bone mineralization. Consequently, in order to effectively replicate the natural bone healing process, it is imperative for bone biomaterials to possess the capability of releasing multiple cytokines in a sustained and organized manner [37].

The bone microenvironment of the defect site is not wholly consistent with the physiological environment, which is related to the destruction and reconstruction of bone tissue structure and function. In the early stage after bone injury, the bone microenvironment is acidic due to carbon dioxide overflow from damaged cells, lactic acid production, and blood glucose transformation [11]. This acidic environment induces calcium salt absorption, resulting in bone resorption greater than bone formation [38]. Therefore, we used the objective factors of the acidic microenvironment in the early stage of bone injury to construct acid-responsive composite hydrogels. The Schiff base formed between HA-CHO and VEGF can break and release VEGF rapidly in response to acidic microenvironment, effectively promoting angiogenesis in the early stage and laying the foundation for rapid bone tissue repair. In addition, GelMA is a biocompatible, biodegradable, and photo-crosslinked hydrogel widely used in tissue engineering [6,39,40]. Therefore, the HG composite hydrogel prepared by combining HA-CHO and GelMA can be used as an excellent scaffold to intelligently release VEGF and promote angiogenesis in response to the acidic microenvironment at the site of bone injury. In this study, VEGF was released about 90 percent within 14 days and was released faster under acidic conditions, which meant that it could quickly promote angiogenesis in the early acidic bone microenvironment. The release of VEGF also recruits cells to assist in bone repair.

Researchers have been trying to develop various scaffolds for bone regeneration that can control the release of certain cytokines [41,42]. Still, most scaffolds have focused on the role of bone regeneration rather than the release performance. Some of these scaffolds have been reported to exhibit burst release rather than sustained release. Although the cytokine BMP-2 has good osteogenic properties, due to its rapid dispersion and degradation in vivo, its local concentration and therapeutic effect in the defect site are reduced, and the local sudden release is also easy to induce ectopic ossification. In recent years, mesoporous nanoparticles such as silica have attracted wide attention in bone tissue engineering due to their advantages of effectively improving the stability and solubility of loaded drugs, promoting transmembrane transport, and improving delivery efficiency [43,44]. Shi et al. found that europium-doped mesoporous silica nanoparticles could regulate the immune microenvironment, promoting osteogenesis and angiogenesis [45]. Therefore, loading the BMP-2 factor into mesoporous silica and delivering it continuously to the site of bone injury is an effective method to achieve bone repair. In this study, BMP-2@SiO2 was loaded into the composite hydrogel to delay the release of BMP-2 further. BMP-2 was released nearly 90% in the acidic microenvironment for 40 days, meaning it can participate in almost the entire process of bone repair and promote bone formation. Bone repair is a long-term process, so it is necessary to encapsulate BMP-2 in mesoporous silica for slower and sustainable release. Taken together, the developed composite hydrogel which combined with angiogenesis and osteogenesis may be acted as an excellent scaffold for bone repair and regeneration.

5. Conclusion

In this study, a composite hydrogel containing VEGF and BMP-2 was developed. The composite hydrogel effectively regulates the sequential release of VEGF during the initial phases and sustains a gradual release of BMP-2 in subsequent stages, thereby establishing an appropriate microenvironment conducive to both angiogenesis and bone formation. This composite hydrogel promoted tube formation in vitro and blood vessel formation in vivo. Furthermore, based on the outcomes of ALP staining, alizarin red staining, and the expression of osteogenic genes, it can be concluded that the hydrogel exhibited an enhanced osteogenic effect in vitro. The findings from animal experiments demonstrated that the composite hydrogel effectively enhanced bone formation in rats with skull defects, highlighting the synergistic impact of VEGF and BMP-2 on vascularized bone regeneration. Consequently, the augmentation of angiogenesis and the creation of an osteogenic microenvironment through our composite hydrogel hold significant potential for the field of vascularized bone repair.

CRediT authorship contribution statement

Haifei Cao: Writing – original draft, Methodology, Conceptualization. Shuangjun He: Formal analysis, Data curation. Mingzhou Wu: Writing – original draft, Funding acquisition. Lihui Hong: Visualization. Xiaoxiao Feng: Investigation. Xuzhu Gao: Software. Hongye Li: Visualization, Investigation. Mingming Liu: Writing – review & editing, Investigation, Funding acquisition. Nanning Lv: Writing – review & editing, Supervision, Project administration, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgement

This study was supported by the lianyungang key science and technology research and development plan (SF2206), Lianyungang City sixth "521 high-level talent training project" scientific research project (LYG06521202159), Lianyungang Traditional Chinese Medicine Science and Technology Development Program (ZD202210), the Basic Research Program of Medical Application in Suzhou (SKYD2023238), Research Project of Medical Innovative Application in Suzhou (SKYD2023071).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2024.101015.

Contributor Information

Haifei Cao, Email: chf1986@126.com.

Shuangjun He, Email: hsjdoctor@163.com.

Mingzhou Wu, Email: mzwu1993@163.com.

Lihui Hong, Email: honglihui1208@163.com.

Xiaoxiao Feng, Email: fxx20202021@163.com.

Xuzhu Gao, Email: alexgwan@163.com.

Hongye Li, Email: li19816269587@163.com.

Mingming Liu, Email: drliumingming@163.com.

Nanning Lv, Email: lvnanning123@163.com.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (1.2MB, docx)

Data availability

Data will be made available on request.

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Supplementary Materials

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Data Availability Statement

Data will be made available on request.

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