Magnesium Ammonium Phosphate Composite Cell-Laden Hydrogel Promotes Osteogenesis and Angiogenesis In Vitro.

Injectable hydrogels provide an effective strategy for minimally invasive treatment on irregular bony defects in the maxillofacial region. To improve the osteoinduction of gelatin methacrylate (GelMA), we fabricated a three-dimensional (3D) culture system based on the incorporation of magnesium ammonium phosphate hexahydrate (struvite) into GelMA. The optimal concentration of struvite was investigated using the struvite extracts, and 500 μg mL-1 was found to be the most suitable concentration for the osteogenesis of dental pulp stem cells (DPSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs). We prepared the GelMA composite (MgP) with 500 μg mL-1 struvite. Struvite did not affect the cross-linking of GelMA and released Mg2+ during degradation. The cell delivery system using MgP improved the laden-cell viability, upregulated the expression of osteogenic and angiogenic-differentiation-related genes, and promoted cell migration. Overall, the modifications made to the GelMA in this study improved osteoinduction and demonstrated great potential for application in vascularized bone tissue regeneration.

Introduction

Due to the additional trauma and insufficient sources of traditional autologous bone for grafting, there is a huge demand for artificial bone regeneration materials.[1,2] Irregular bone defect regeneration in the oral and maxillofacial region is still a major challenge. Although traditional bone substitute materials based on calcium phosphates such as β-tricalcium phosphate (β-TCP) or hydroxyapatite (HAp) have been clinically applied for filling irregular bone defects, there are still downsides to the use of these materials, such as the complicated operation and the resulting large wounds.[3] The development of injectable bone regeneration materials for minimally invasive surgery can be an effective strategy to overcome these obstacles.[4]

Gelatin methacrylate (GelMA) has proven to be a promising bioink with outstanding cell viability and has been widely used to print three-dimensional (3D) scaffolds encapsulated with stem cells.[5,6] The 3D architecture can reflect a more realistic extracellular microenvironment and enhance the interaction between cells and the scaffold matrix, thereby affecting the fate of the cells.[6−9] Moreover, the 3D cell delivery system can significantly improve the transplanted cell survival.[10] The superior fluidity and biocompatibility make GelMA an appropriate filling material for irregular bony cavities. However, GelMA provides only osteoconduction but not osteoinduction, restricting its application of bone regeneration.[11] It has been proven that the combination of various bioactive ions such as strontium (Sr), copper (Cu), lithium (Li), and magnesium (Mg) is an effective way to improve the bioactivity of the current biomaterials.[12] For instance, strontium ion (Sr2+) has been proved to promote bone regeneration by enhancing the bone formation ability of osteoblasts and weakening the bone resorption activity of osteoclasts.[13,14] Besides, copper ion (Cu2+) induces vascularization by upregulating the expression of angiogenic growth factors.[15] The released Cu2+ significantly facilitated the ingrowth of new bone into the scaffolds.[16,17] These results indicate that the osteoinductive properties of GelMA can be enhanced by the incorporation of certain metal ions.

Mg2+ is currently one of the most popular biologically active ions. It has been proven that the incorporation of Mg2+ into bone substitutes promotes cell migration and osteogenic differentiation in vitro and accelerates osseointegration as well as the formation of new vascularized bone in vivo.[18,19] However, the implanted magnesium or magnesium alloy would release hydrogen and thereby produce an alkaline environment, which may lead to severe bone resorption.[16] The use of magnesium phosphate is increasing in the field of biomaterials due to the significance of Mg2+ to cell growth, proliferation, and differentiation. Magnesium ammonium phosphate hexahydrate (MgNH4PO4·6H2O), which is also called struvite, is a magnesium-based bioceramic whose degradation releases Mg2+, which is conducive to a safe microenvironment for bone formation.[20,21] As the most common setting material in magnesium phosphate cements (MPCs), struvite can serve as an ideal component to improve the osteoinduction of GelMA.[22,23]

In this work, we developed a novel injectable composite hydrogel for vascularized bone regeneration by introducing struvite and stem cells into GelMA. GelMA provided a microenvironment that mimicked the extracellular matrix and the ionic products released by struvite promoted vascularized bone formation by laden-dental pulp stem cells (DPSCs).[24] The cell biocompatibility and osteogenic and angiogenic differentiation potential were evaluated in vitro.

Results and Discussion

Osteogenic Induction Capacity of the Struvite Extracts

Human dental pulp stem cells (DPSCs) are mesenchymal stem cells that are easy to obtain from extracted teeth without ethical concerns.[25] The high self-renewal ability, proliferation ability, and potential to differentiate into multiple tissue types, including dentin, bone, and blood vessels make DPSCs not only favored in the field of pulp regeneration but also widely used in vascularized bone regeneration.[26,27] Moreover, it is believed that DPSCs have greater cloning and proliferation potential than bone marrow stem cells (BMSCs).[28,29] Therefore, DPSCs were chosen for the following research. DPSCs were isolated and expanded from the extracted teeth (Figure S1a–d). The isolated DPSCs were identified by flow cytometry and showed high expression of mesenchymal stem cell markers (CD29, CD44, and CD73) but almost no expression of hematopoietic stem cell markers (CD45) (Figure S1e).

To determine the suitable concentration for osteogenesis, the ionic products from both the struvite groups and the TCP group were collected. After 7 days of culture, the cells still had good viability and showed no detectable differences among all groups, indicating that struvite has excellent biocompatibility (Figure a). Alkaline phosphatase (ALP) staining and ALP activity evaluation showed that the osteogenic differentiation of DPSCs was enhanced with increasing concentrations of the extracts, reached a peak in the MgP-500 group, and was significantly higher than that in the control groups (Figure b,c). In addition, the osteogenic-differentiation-related genes ALP (Figure d), osteocalcin (OCN) (Figure e), collagen-1 (Col-1) (Figure f), bone morphogenetic protein 2 (BMP-2) (Figure g), and runt-related transcription factor 2 (Runx2) (Figure h) were all upregulated in the struvite groups, especially the MgP-500 group. In comparison, the dissolution products of struvite resulted in better osteogenic differentiation ability than that observed in the blank control group and β-TCP group and the MgP-500 group was proved to be the best. Struvite is a magnesium ammonium phosphate composite. Mg2+ and PO43– may be the active components that exert osteoinductive effects. Previous studies indicated that an appropriate concentration of Mg2+ promotes the expression of osteogenic-related genes in bone marrow mesenchymal stem cells (BMSCs).[30] Extracellular PO43– uptake plays an important role in inducing osteogenic differentiation by upregulating the synthesis of ATP, which improves the osteogenesis of MSCs through adenosine signaling.[31,32] Thus, it is speculated that PO43– may have a synergistic effect with Mg2+ on the osteogenic differentiation of DPSCs.

Figure 1

Osteogenic effect of the struvite extracts. (a) Proliferation of human DPSCs incubated with different concentrations of struvite extract at different time points as detected by a cell counting kit-8 (CCK-8) assay (n = 3). (b) To observe the osteogenic induction capacity of struvite in human DPSCs, ALP staining was performed after incubation with extracts from struvite and TCP for 7 days. The scale bar is 400 μm. (c) ALP phosphatase activity was detected after 14 days (n = 3). The expression of the osteogenic-differentiation-related genes (d) ALP, (e) OCN, (f) Col-1, (g) BMP-2, and (h) Runx2 was also detected by reverse transcription-polymerase chain reaction (RT-PCR) assay (n = 3) (*p < 0.05, **p < 0.01).

Angiogenic Effect of the Struvite Extracts

Apart from the promotion of osteogenic differentiation, Mg2+ was proven to induce the migration, proliferation, and angiogenesis of vascular endothelial cells and accelerate early blood vessel formation in vitro.[18,33] The migration of vascular endothelial cells is one of the initial processes of angiogenesis.[34] To further investigate the migration effect of struvite, an in vitro Transwell migration assay was performed. The struvite extracts induced a greater number of human umbilical vein endothelial cells (HUVECs) to penetrate the Transwell membranes compared to the control group, especially in the MgP-500 group and the MgP-1000 group (Figure a,b). To further evaluate the influence of the dissolution products on angiogenesis, we carried out a tube formation assay, which indicated that the number of branches in the MgP-500 group was significantly higher than that in the control groups (Figure c,d). Additionally, the angiogenesis-related genes, vascular endothelial growth factor (VEGF) (Figure e), angiotensin-2 (Ang-2) (Figure f), and hypoxia-inducible factor-1α (HIF-1α) (Figure g) were upregulated in the struvite groups, especially the MgP-500 and MgP-1000 groups. These results showed that the ion products of the struvite extract not only induced the osteogenic differentiation of DPSCs but also regulated the migration of HUVECs. Considering the concentration-related osteogenic and angiogenic differentiation effects on DPSCs, 500 μg mL–1 struvite was used for the subsequent experiments.

Figure 2

Angiogenic effect of the struvite extracts. (a) Migration assay of HUVECs in response to serial concentrations of struvite and TCP extracts after 12 h. The scale bars are 200 μm. (b) Statistical results for the percentage of HUVECs penetrating the Transwell membranes compared to the control group (n = 5). (c) Tube formation assay of HUVECs seeded on the gel basement and cultured with the struvite and TCP extracts after 6 h. The scale bars are 200 μm. (d) Statistical results for the percentage of HUVEC branch points compared to that in the control group (n = 5). The expression of the angiogenesis-related genes (e) VEGF, (f) Ang-2, and (g) HIF-1α was detected by an RT-PCR assay (n = 3) (*p < 0.05, **p < 0.01).

Characterization of the Composite Hydrogel

GelMA with or without struvite was successfully prepared and gelled after exposure to blue light for 30 s (Figure a). The surface morphologies of the hydrogels with or without struvite were analyzed by scanning electron microscope (SEM) after lyophilization (Figure b). The struvite was observed by SEM separately (Figure S2). The MgP group was observed to have particles distributed in the matrix. The elemental composition of part of both hydrogels was further determined by energy-dispersive spectroscopy (EDS) (Figure c,d). EDS analysis indicated distinct Mg signals in the MgP group, while no Mg2+ distributed within the Gel group. Oxygen, carbon, and nitrogen were the main elements on the surface of both groups.

Figure 3

Morphologies and elemental composition of hydrogels. (a) Photos of GelMA before and after cross-linking. (b) SEM images. (c) EDS spectrum and amount of element present (wt %). (d) Line scan analysis.

A uniaxial compression test was performed to quantify the effects of struvite on mechanical strength (Figure a). The addition of a small amount of struvite resulted in a significant increase in the compressive modulus (Figure b). A shear rate scan was also performed to analyze the viscosity and injectability of the samples. Both types of hydrogel showed the characteristics of a pseudoplastic fluid with shear thinning, while the shear-thinning ability of the MgP group was slightly improved, indicating that both materials had good injectability (Figure c). After that, we performed the frequency-sweep test. The storage modulus (G′) was higher than the loss modulus (G″) in both groups, and the phase angle (δ) was relatively stable, which means that both groups exhibited good cross-linking stability (Figure d). In brief, the composite hydrogel presented good mechanical strength, stability, and injectability, which met the needs for the repair of irregular bone defects.

Figure 4

Mechanical properties of hydrogels. (a) Both hydrogels were subjected to compression up to a strain of 0.9. (b) From the stress–strain curves, the compressive modulus of the hydrogels was calculated for a strain of 0.10–0.20 (toe region) (n = 5) (*p < 0.05, **p < 0.01). (c) Shear-thinning behaviors of hydrogels with or without struvite. (d) Time-sweep oscillatory rheometry to confirm the hydrogel formation and stability in response to dynamic forces.

The hydrophilicity of the hydrogel is essential for regulating the penetration of nutrients and metabolites.[35] After immersion in phosphate-buffered saline (PBS), both hydrogels showed rapid water swelling performance in the first 2 h and typically reached equilibrium after 4 h (Figure a). The addition of struvite improved the swelling properties of GelMA, which may improve its ability to fill irregular bony defects and better fit the bone surface. In addition to good swelling properties, hydrogels should also show biodegradability, which plays an important role in the fate of stem cells.[36] Scaffolds tend to act as temporary bridges for cell migration and need to be degraded to allow stem cells to remodel their microenvironment.[37] The addition of struvite had no significant effect on the biodegradability of GelMA since both gels degraded over time (Figure b). As mentioned above, the appropriate amount of Mg2+ has a positive effect on osteogenesis and angiogenesis, and PO43– may have a synergistic effect with Mg2+. Therefore, the released concentrations of both ions wrapped in GelMA in PBS were further investigated (Figure c,d). The particles encapsulated in GelMA realized the sustained release of Mg2+, which is essential for bone formation and biological safety.

Figure 5

Swelling, degradability, and ion release of hydrogels. Swelling test (a) and weight loss analysis (b) of GelMA with or without struvite. Cumulative release of magnesium (c) and phosphorus (d) from the hydrogels.

Composite Hydrogels Provide a Microenvironment Conducive to the Survival and Differentiation of DPSCs

Based on the previous results, we constructed a 3D culture system with optimized concentrations of struvite and GelMA to provide a Mg-enriched microenvironment for bone regeneration. Cell survival is the primary consideration for this system and is the basis of osteogenic differentiation. The cell delivery capacity of both coculture systems was investigated by live/dead staining. After 1, 3, and 7 days (Figure a), the encapsulated DPSCs diffused within the material and presented high cytocompatibility in both materials, while the MgP group presented increased cell viability after being cultured in vitro for 7 days, indicating that the addition of struvite improved the transplanted cell survival (Figure b).

Figure 6

Cell viability of the laden cells was detected by the live/dead assay. (a) Live/dead detection of cell viability in hydrogels with or without struvite cultured in vitro for 1, 3, and 7 days. The scale bar is 250 μm. (b) Percentages of live cells and statistical analysis based on the live/dead double-staining results (n = 3) (*p < 0.05, **p < 0.01).

As mentioned before, the Mg2+ released from magnesium-incorporating bone substitutes promoted cell osteogenic differentiation in vitro and accelerated osseointegration and new bone formation in vivo, suggesting that the extracellular Mg2+ microenvironment had a positive effect on bone regeneration.[17,18] The osteogenic potential of the composite hydrogels was subsequently investigated. The osteogenic-differentiation-related genes ALP (Figure a), OCN (Figure b), Col-1 (Figure c), BMP-2 (Figure d), Runx2 (Figure e), and osteopontin (OPN) (Figure f) were all upregulated in the MgP group. In addition, the level of osteopontin (OPN) protein expression, an early marker of osteogenesis, was observed to evaluate the osteogenic differentiation of DPSCs embedded in GelMA with or without struvite, which was consistent with the results at the genetic level (Figure g). The MgP group presented better osteoinductive effects than the Gel group in both experiments, thus indicating that the microenvironment formed by the improved 3D system with struvite induced the osteogenic differentiation of the laden DPSCs.

Figure 7

Osteogenic effect of the cell-laden hydrogel. The expression of the osteogenic-differentiation-related genes (a) ALP, (b) OCN, (c) Col-1, (d) BMP-2, (e) Runx2, and (f) OPN was detected by RT-PCR assay (n = 3) (* p < 0.05, ** p < 0.01). (g) OPN expression after 7 days in cells cultured in GelMA with or without struvite. The scale bar is 100 μm.

Similarly, the angiogenesis-related genes, Ang-2 (Figure a), VEGF (Figure b), and HIF-1α (Figure c) were upregulated in the MgP group. The expression of VEGF, which encodes a protein important for driving cell migration and promoting angiogenesis, was also detected by immunofluorescence staining and proved to be enhanced in the MgP group (Figure d).[38] Transwell assays were performed to further investigate the impact of the composite hydrogels on the migration of HUVECs (Figure e). The fewest migrating cells were observed in the Gel group, followed by the MgP group, while the MgP-DPSC group presented the largest number of migrating cells (Figure f). Previous studies showed that Mg2+ promoted the formation of blood vessels not only by inducing nitric oxide production in endothelial cells but also by upregulating the secretion of angiogenic proteins such as VEGF and Arp 2/3.[39−41] Thus, we speculated that the migration of HUVECs was stimulated not only directly by ionic components in struvite but also indirectly by upregulation of the expression of VEGF in the encapsulated DPSCs. Taken together, the results showed that the composite hydrogel provided a microenvironment conducive to the survival and differentiation of DPSCs.

Figure 8

Angiogenic effect of the cell-laden hydrogel. The expressions of angiogenesis-related genes (a) Ang-2, (b) VEGF, and (c) HIF-1α were detected by RT-PCR assay (n = 3). (d) VEGF expression after 7 days in cells cultured in GelMA with or without struvite. The scale bar is 100 μm. (e) Migration assay of HUVECs in response to Gel, MgP, and DPSC-laden MgP after 12 h. The scale bar is 200 μm. (f) Statistical analysis of the number of cells penetrating the Transwell membranes compared to that in the Gel group (n = 5) (* p < 0.05, ** p < 0.01).

In view of the excellent biological properties and injectability, the composite hydrogel is expected to be applied to the regeneration of other tissues, such as dental pulp tissue. The key to dental pulp regeneration is the establishment of an effective vascular network and the reconstruction of dentin–pulp complex, which is similar to vascularized bone regeneration.[42] In general, the three-dimensional coculture system constructed by encapsulating DPSCs in GelMA with or without struvite provided an ideal microenvironment to support DPSC maturation, osteogenesis, and angiogenesis, suggesting promising applications in tissue engineering.

Conclusions

In this study, we fabricated a novel DPSC-laden composite hydrogel consisting of struvite for vascularized tissue regeneration. The ionic components released by struvite promote the osteogenic differentiation of DPSCs and enhance the chemoattraction of HUVECs to increase angiogenesis in vitro. Thus, the composite hydrogel based on struvite and GelMA not only retains the remarkable fluidity, stability, and degradability of GelMA but also promotes osteogenesis and angiogenesis, which meets the need to fill irregular bone defects via minimally invasive methods in the maxillofacial region.

Materials and Methods

Preparation of the Extract Solution of Struvite

The extract solution used in this experiment was prepared by adding the required amount of aseptic struvite powder to the high-dose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) or endothelial basal medium (EBM; AllCells, Shanghai, China) containing 10% fetal bovine serum (FBS; Gibco) and 2% penicillin–streptomycin (Gibco) to prepare a 5000 μg mL–1 struvite suspension. After incubating for 24 h at 37 °C, the solution was centrifuged at 1000 rpm for 5 min and ionic products were collected and designed as the MgP-5000 group. Then, an adequate amount of DMEM was added to the extract solution to prepare the serial concentrations (MgP-2000, MgP-1000, MgP-500, MgP-250, MgP-125). Also, the β-TCP group was prepared as the positive control group at the concentration of 500 μg mL–1 using the same protocol.

Cell Culture and Characterization

Human dental pulp stem cells (DPSCs) were derived from complete premolars or the third molars extracted for orthodontic reasons from patients aged under 16 years old. The experiment was approved by the Ethics Committee of the Ninth People’s Hospital (Shanghai, China) (SH9H-2019-TK11-1). The pulp tissue was immediately separated as soon as the teeth were extracted. After rinsing three times in phosphate-buffered saline (PBS), the pulp was cut into blocks smaller than 1 cm3 and then pressed with cover glass in a Petri dish (Corning, NY) and cultured with DMEM containing 10% fetal bovine serum and 2% penicillin–streptomycin at 37 °C in a 5% CO2 atmosphere. DPSCs were accumulated by collecting multiple colonies and the third to fifth passages were selected for further experiments. Cell phenotype analysis was detected by flow cytometric analysis for CD45-phycoerythrin (PE), CD73-PE, CD29-PE, and CD44-PE (BD Biosciences) following the manufacturer’s instructions.[43]

CCK-8 Assay

DPSCs were seeded in 96-well plates (Corning) and treated with different concentrations of extract solution for 1, 3, and 7 days. The cell counting kit (CCK-8; Dojindo, Japan) assay was used to measure cell proliferation and viability as described by the manufacturer’s protocol. The optical density (OD) value of each sample was measured at a wavelength of 450 nm (OD450).

Alkaline Phosphatase (ALP) Staining and ALP Phosphatase Activity

After DPSCs were treated with different concentrations of struvite extraction for 7 days, the cells were fixed with 4% paraformaldehyde and then stained with the dye solution prepared according to the BCIP/NBT alkaline phosphatase assay kit (Beyotime, China) instructions at 37 °C for 30 min.

After being treated for 10 days, DPSCs were collected with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) and centrifuged at 4 °C and 8000 rpm for 15 min. The triplicates of the supernatant fluid were mixed with 5 mM para-nitrophenyl phosphate (pNPP) in a 96-well plate according to the alkaline phosphatase assay kit (Beyotime, China). After 1 h of incubation, the absorbance was detected at 405 nm. The ALP activity was obtained by molecular weight and incubation time.

Real-Time PCR

The total RNA from DPSCs cultured with extracts or wrapped with hydrogels was harvested with Trizol (Takara Bio Inc., Japan) and PrimeScript RT Master Mix (Takara) was used for the reverse transcription of the samples to cDNA. Real-time PCR was performed with TB Green (Takara) using LightCycler 480 (Roche, Switzerland). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as a housekeeping gene. Primer sequences for ALP, osteocalcin (OCN), bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), collagen-1 (Col-1), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF-1α), and angiotensin-2 (Ang-2) are listed in Table S1.

Transwell Migration Assay

Human umbilical vein endothelial cells (HUVECs; eahy926) were purchased from AllCells and cultured in EBM. They were seeded in the upper chamber, while the lower chambers were filled with extracts or the 3D culture system. After 12 h of incubation, the cells that penetrated the Transwell (Corning) membranes were fixed and stained with crystal violet (Sigma-Aldrich, MO) at RT for 15 min and observed under a light microscope. The experiment was repeated three times. The cell number was counted with ImageJ.

Tube Formation Assay

After incubating Matrigel (Corning) basement at 37 °C for 30 min, the HUVECs were seeded and cultured with different concentrations of the extracts. The cells were incubated for 12 h and observed under a light microscope. The experiment was repeated three times. The number of branch points was counted with ImageJ.

Preparation of the Hydrogels

The lyophilized gelatin methacryloyl (GelMA) and lithiumphenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were bought from Engineering for Life, Zhejiang, China. GelMA (7% w/v) and LAP (0.25% w/v) were dissolved in PBS at 60 °C for 30 min and stored at 4 °C after sterilization with 0.22 μm filters and designed as Gel group. Struvite powder (500 μg mL–1) mixed with GelMA solution was designed as the MgP group. The samples were exposed to the blue light for 30 s to solidify and washed three times in PBS to remove the uncross-linked components. Injecting 800 μL of GelMA solution with (MgP) or without (Gel) struvite into cylindrical molds and fabricating into samples with a diameter of 10 mm and a height of 6 mm after light curing.

Characterization of Composite Hydrogels

The samples were flash-frozen in liquid nitrogen for 10 min and then lyophilized for 8 h. After that, a scanning electron microscope (SEM; S-4800; Hitachi, Japan) was used to visualize the distribution of the struvite in hydrogels. The elemental composition of hydrogels was determined by energy-dispersive spectroscopy (EDS; Quantax 400-30; Burker, Germany).

The mechanical properties of both the gel and MgP groups were determined using a mechanical tester (HY-0230; Hengyi, China). Hydrogels were subjected to unconfined compression up to 0.9 strain at the speed of 15 mm min–1. The tests were repeated three times. From the stress–strain curves, the compressive modulus of the hydrogels was calculated from the slope in the toe region corresponding to the 0.10–0.20 strain.

The viscosity of the hydrogels was evaluated using a HAAKE MARS III rheometer (Thermo Scientific, Germany) equipped with a 40 mm parallel-plate and a gap of 1 mm, and the shear rate scanning range of viscosity was between 0.01 and 20 s–1 at 1 Hz. The strain test was also performed and the linear viscoelastic region was in the range of 1% strain and 0.1–100 Hz frequency. The tests were repeated three times and the storage modulus (G′) and loss modulus (G″) were recorded.

The swelling ratio and weight loss ratio of the hydrogels was assessed by a conventional gravimetric method. In brief, the samples were fabricated as mentioned above and weighed before being immersed into PBS and incubated at 37 °C in a shaking incubator for 20 days. The samples were weighed at different time points. The swelling ratio and the weight loss ratio of both samples at different time points were calculated.

The samples were incubated in 10 mL of deionized water at 37 °C in a shaking incubator and the solution was collected and refreshed at days 1, 3, 5, 10, 15, and 20. The concentrations of the P and Mg ions were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) (Varian Co.).

Laden-Cell Viability Assessment

The cells were encapsulated in hydrogels instead of seeding onto the hydrogel to construct a three-dimensional coculture system. DPSCs were collected and resuspended at the concentration of 3 × 106/mL with hydrogels with or without struvite before gelling. The samples were prepared into 10 mm diameter and 4 mm thickness and cultured with full culture medium.

The viability of encapsulated DPSCs in the hydrogels was evaluated using a calcein-AM/PI double stain kit (BestBio, China). After 1, 3, and 7 days, the samples were washed and incubated with prepared live/dead assay reagents according to the instructions for 30 min at 37 °C. The images from the middle layer of the hydrogels were acquired by confocal laser scanning microscopy (CLSM, Leica SP8, Germany). The experiment was repeated three times. The live and dead cells were counted with ImageJ.

Cell Differentiation in Hydrogels

To further evaluate the impact of struvite on osteogenesis and angiogenesis, immunofluorescence staining was performed. The cell-laden hydrogels were fabricated as described above and cultured for 7 days. After being fixed and blocked, the samples were incubated overnight at 4 °C with the primary antibodies against OCN and VEGF (Abcam). After that, the samples were washed with PBS three times and incubated with secondary antibodies (Alexa Fluor 594, Abcam) for 1 h. The cell nuclei were stained with 4,6-diamidino-2-phyindole dilactate (DAPI) for 5 min. After being washed three times, the samples were observed by confocal laser scanning microscopy.

Statistical Analysis

All data are displayed as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS 22 statistical software package. The results were analyzed for significance by two-tailed paired Student’s t-test for two groups or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons.