Evaluation of the degradation,biocompatibility and osteogenesis behavior of lithium-doped calcium polyphosphate for bone tissue engineering

Abstract

BACKGROUND:

Calcium polyphosphate (CPP) is a commonly used biomaterial in bone tissue engineering, but CPP is insufficient in osteoinduction. This study aimed to fabricate lithium doped CPP (LiCPP) scaffolds and assess their characterization, degradation, biocompatibility and osteogenesis behavior for bone tissue engineering.

METHODS:

The novel scaffold was characterized by XRD, FTIR and SEM. The porosity, cell mediated degradation behavior and mechanical properties were also investigated. Meanwhile, cell proliferation activity and adhesion in vitro was exploited. Finally, osteogenesis the LiCPP scaffolds in vitro and in vivo was researched.

RESULTS:

The outcomes revealed that low-content Li doping had no significant influence on the structure of CPP. The results of cells mediated degradation experiments from the weight loss and the release of ions indicated that Li doped CPP improved biological degradation. The compressive strength of CPP with 66% porosity was improved to 7 MPa. Cells proliferation experiment and adhesion experiment demonstrated 2.0%LiCPP scaffold was most beneficial to cell growth and attachment. Furthermore, Li doped CPP up-regulated Wnt signal pathway when co-cultured with MG63 and increased osteogenic marker ALP expression and calcium phosphate deposition in vitro. At the same time, new bone formation in vivo was also enhanced by using LiCPP scaffolds and the 2.0%LiCPP scaffolds obtained best osteogenesis outcomes.

CONCLUSION:

The results obtained in our study suggest that 2.0%LiCPP scaffold could benefit from improving the osteogenesis behavior and is a promising biomaterial for bone repairing applications.

Keywords

Calcium polyphosphate lithium osteogenesis bone tissue engineering degradation

1. Introduction

Bone tissue engineering aims at providing excellent bone substitute to clinical practice. The ideal bone biomaterials should has similar composition to the bone and possess good controllable biocompatibility and bone inductivity [1–3]. Calcium polyphosphate (CPP) is a bioactive ceramic that possesses similar mineral components to human bones and teeth [4,5]. CPP has comparable physicochemical properties, controllable degradability, and excellent biocompatibility [6,7]. However, CPP scaffold also has shortcomings. For example, it is brittle, and insufficient in promoting osteogenesis [5,7]. In addressing these challenges, a lot of efforts have been put to modify the CPP, such as using optimizing porosity, improving the sintering temperature and synthesizing nano-particles, meanwhile doping microelement such as strontium, zinc, copper, et al into CPP to improve its physicochemical and biological properties in bone tissue engineering are also commonly recommended [5,8,9].

Lithium (Li) is a kind of microelement that important to human body. It has been reported that Li could play a role in activating the Wnt/GSK-3β signal pathway and promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) [10,11]. When Li was used in vivo study, it exerted significant effects on enhancing bone regeneration by improving osteogenesis and inhibiting the adipogenesis [12,13]. Therefore, Li is a good choice for modifying bone substitutes. According to previous studies [14,15], Li doped hydroxyapatite made good performance on the strength, toughness and degradation property and osteogenesis, but there was no study researched on lithium doped CPP.

In this study, we fabricated different content of LiCPP scaffolds and studied the optimal concentration of Li, which not only possessed good degradation properties, but also have excellent biological activities and bone inductivities.

2. Materials and methods

2.1. Scaffold fabrication and characterization

Porous LiCPP scaffolds (with a Ca/Li molar ratio of 100:0; 99.9:0.1; 99:1; 98:2 and 97:3) were prepared according to previously reported methods to fabricate the CPP and Li content [4,5,14,15]. The detailed methods are outlined in the supplementary materials. Finally, we obtained CPP; 0.1%LiCPP; 1.0%LiCPP; 2.0%LiCPP; 3.0%LiCPP scaffolds.

The FTIR spectra of the samples were obtained using a FTIR spectrophotometer. The scaffolds were tested by X-ray diffractometer (XRD). The morphology of obtained scaffolds was observed by scanning electron microscopy. The liquid displacement was generally employed to measure the porosity value of the scaffolds. The scaffolds for cells-culturing were sterilized by γ-rays (60Co) at a dose of 25 kGy. The detailed information is outlined in the supplementary materials.

3. In vitro studies

3.1. Degradation property

MG63 were used for the cell mediated degradation property (the detailed methods are outlined in the supplementary materials). Scaffolds weight was measured and the obsolete release medium was gathered for measuring ionized lithium/phosphate concentrations on the 5th, 10th, 15th, 20th, 25th and 30th day. The gathered supernatants were diluted by distilled water and the ion Li⁺, Ca²⁺ and PO₄ ³⁻ were analyzed by inductively coupled plasma mass spectrometry and the cumulative release amount of Ca²⁺ , Li⁺ and PO₄ ³⁻ were calculated. The compressive strengths of the dried scaffolds were measured using an universal testing machine at a crosshead speed of 0.1 mm/min. For each group, five samples were measured at the scheduled times.

3.2. Cell biocompatibility

MTT assay was conducted to evaluate the effect of the scaffolds on proliferation of MG63. The detailed methods are outlined in the supplementary materials.

Confocal laser scanning microscopy (CLSM) combined with fluorophore were used to observe cell distribution on the surface and inside their pores. Besides, the growth of cells on the scaffolds was observed on the 14th day by SEM to investigate cell attachment. The detailed methods are outlined in the supplementary materials.

3.3. In vitro osteogenesis evaluation

The slides of MG63 were prepared and cultured with CPP; 0.1%LiCPP; 1.0%LCPP; 2.0%LiCPP and 3.0%LiCPP scaffolds in 6-well plate. On the 7th day, cyto-immunofluorescence was performed to assess the Wnt signal pathway, and GSK-3β, β-catenin and Runx2 were detected. The detailed methods are outlined in the supplementary materials.

To measure the protein expression of phosphorylated GSK-3β, β-catenin and Runx2 we conducted western blot analysis. The detailed methods are outlined in the supplementary materials.

Fourteen days later, alkaline phosphatase (ALP) was detected, while mineralization was measured 21 days later. The detailed methods are outlined in the supplementary materials.

3.4. In vivo study evaluated the osteogenesis

To evaluate the osteogenic potential of the different content of LiCPP scaffolds in vivo, we implanted the scaffolds into the tibia bone defect. Twenty healthy adult male Japanese white rabbits weighing 2.6–3.8 kg were used to build bilateral tibia bone defect (5 mm in diameter and 3 mm in thickness).

After the bone defects were made, CPP (n = 8), 0.1%LiCPP (n = 8), 2.0%LiCPP (n = 8), 2.0%LiCPP (n = 8) and 3.0%LiCPP (n = 8) scaffolds were used to implant into the bone defects. All rabbits were killed at 4 weeks and 8 weeks, respectively. The detailed methods are outlined in the supplementary materials.

3.5. Statistical analysis

The SPSS19.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. The results were analyzed with nonparametric analysis and the two groups were compared with the Kruskal-wallis test. P < 0.05 indicates statistical significance.

4. Results

4.1. Identification of LiCPP

Figure 1a shows the FTIR spectra of 2.0%LiCPP scaffolds. It demonstrated typical peaks at 1283.05 cm⁻¹ and 770.3 cm⁻¹ , which means “O–P =O” and “O–P–O”, respectively. As shown in Fig. 1b, the characteristic peaks in the XRD profiles of LiCPP were in line with the XRD profiles of β-CPP [Ca(PO3)2, JCPDS #77-1953]. It suggested that there was no crystal system alteration in the incorporation of low-dose of Li into β-CPP and Li⁺ might have been perfectly doped into the structural frame of the β-CPP crystal.

Fig. 1.

Identification of the materials. (a): FTIR spectra of LiCPP and (b): XRD of LiCPP.

Figure 2a shows the SEM picture of LiCPP powder, which indicates that the powder was in the form of short acicular or polyhedron and the size was in nanoscale. Figure 2b shows the surface morphology of the 2.0%LiCPP and we can found that the pores with 200–400 μm were uniformly distributed. These scaffolds had porosity of 66.2 ± 6.4%. Figure 2c is the picture of the higher power lens in which a large amount of mesoporous can be seen.

Fig. 2.

SEM view of the LiCPP. a: LiCPP powder; b: the surface morphology of LiCPP scaffolds; c: high power field of LiCPP scaffolds.

4.2. Degradation and mechanical performance

Figure 3a–3d shows the weight loss and cumulative ions release for from different content of LiCPP in cells culture. During experimental session, the weight loss decreased along with the scaffolds’ Li content increasing (Fig. 3a). According to Fig. 3b and 3c, the concentration of PO₄ ³⁻ anion and Ca²⁺ in culture medium was highest in 2.0%LiCPP than other scaffolds, which indicates that a better effective degradability was achieved. Meanwhile, Li⁺ content in the medium was without significant difference in 2.0%LiCPP group and 3.0%LiCPP group, both of which was higher than other groups. Moreover, the release of these iron were continued with a relatively stable process 20 days later. Figure 4 shows the compression strength at different time during the degradation experiment, we find that the compression strength can be improved when the content of Li increased and can reached 7.5 Mpa. On the 15th day after the experiment, the LiCPP scaffolds can still achieve compression strength higher than 4 MPa, all of which were higher than pure CPP scaffolds (p < 0.05). When it came to the 30th day, the compression strength in LiCPP scaffolds were 2–3 MPa but the difference was without statistical significance (p > 0.05). The pure CPP scaffolds had a compression strength lower than 2 MPa on the 30th day and were inferior to the LiCPP scaffolds (p < 0.05).

Fig. 3.

Cells mediated degradation of different scaffolds.

Fig. 4.

Results of mechanical testing of the scaffolds in cell culture.

4.3. Cell biocompatibility

Figure 5 shows the cell proliferation activities of the MG63 that cultured with different LiCPP scaffolds. It indicates that CPP and 0.1%LiCPP scaffolds did not significantly improve the cell activities when compared with the blank control group (p > 0.05), but the cell activity was increased along with the Li content increasing and when Li content higher than 1.0%, the cell viability was significantly improved (P < 0.05). Meanwhile, 2.0%LiCPP group achieved best cell proliferation activities, while when he Li content came to 3.0%, the cell growth seems to be inhibited.

Fig. 5.

Cell viability when MG63 cultured with different scaffolds. ^∗means p < 0.05 between the two groups. ^∗∗means p < 0.01 between the two groups.

As exhibited in Fig. 6, cells grew well on the surface of scaffolds, and cells spanned between adjacent pores, yielding a high accumulation density of adherent cells. When Li content was increased from 0%–2%, cells approximately increased to spread over the surface and the adjacent pores spanned large number of cells. But the cells numbers seemed to be decreased when Li content bigger than 3%.

Fig. 6.

CLSM images of MG63 cultured with different scaffolds on the seventh day after co-culture.

4.4. LiCPP scaffolds improved osteogenesis in vitro

Figure 7, Fig. 8 and Fig. 9 show the cell immunofluorescence staining outcomes that presented the expression of GSK-3β, β-catenin and Runx2. The outcomes showed that doped Li into the CPP could decrease the expression of GSK-3β, while increase the β-catenin and Runx2 level. When Li content increased from 0 to 2.0%, the GSK-3β expression was further decreased, while β-catenin and Runx2 expression were gradually increased. As the western blot outcomes shown in Fig. 10, 2.0%LiCPP could obtain significant difference of GSK-3β, β-catenin and Runx2 expression when compared with the CPP scaffolds (p < 0.05).

Fig. 7.

Cell immunofluorescence analysis of GSK-3β expression. Red colour indicates cell nucleus. Yellow colour indicates GSK-3β positive expression.

Fig. 8.

Cell immunofluorescence analysis of β-catenin expression. Red colour indicates cell nucleus. Blue colour indicates β-catenin positive expression.

According to Figures 11–12, ALP expression and mineralized nodules were significantly increased by using LiCPP and the osteogenic effect seemed to be positively related to the Li content. However, when Li content reached 3.0%, the ALP expression and mineralized nodule seemed decreased. When quantitative analysis these two parameters, we can find that the ALP expression (Fig. 11f) and calcium deposition amount (Fig. 12f) were highest in 2.0%LiCPP group and the differences were statistically significant when compared with blank control group and CPP group (p < 0.05).

Fig. 9.

Cell immunofluorescence analysis of Runx2 expression. Red colour indicates cell nucleus. Green colour indicates Runx2 positive expression.

Fig. 10.

Western blot for quantitative analysis of the GSK-3β, β-catenin and Runx2 expression. ^∗means p < 0.05 when the two groups were compared. ^∗∗means p < 0.01 between the two groups.

Fig. 11.

ALP staining of MG63 cultured with different scaffolds for 14 days. (a): CPP; (b): 0.1%LiCPP; (c) 1.0%LiCPP; (d): 2.0%LiCPP and (e) 3.0%LiCPP. (f) shows quantitative outcomes of ALP amount in different scaffolds. *means p < 0.05 when the two groups were compared.

4.5. LiCPP scaffolds enhanced new bone formation in vivo

Outcomes of histological detection HE was used to evaluate the new bone formation. As can be seen in Fig. 13, there were no inflammatory cells in the peripheral region of the scaffolds and less fibrous tissues around materials were observed, but we can see some materials residual. In the bone defects with scaffolds, we found that new bone grew into the implants. The CPP group also showed some new immature bone matrix, but we can find mature new bone interconnected and formed irregular bone trabecula in the LiCPP groups. More importantly, along with Li content increasing, there was more new bone formed and 2.0%LiCPP seemed to achieve the largest number of new osteogenesis. From 4 weeks to 8 weeks, we found that the new bone areas in each group were increased and the trabeculae turned more mature.

Fig. 12.

Mineralized nodules after 21 days of MG63 cultured with different scaffolds. (a): CPP; (b): 0.1%LiCPP; (c): 1.0%LiCPP; (d): 2.0%LiCPP and (e) 3.0%LiCPP. (f) shows quantitative outcomes of mineralized nodules amount in different scaffolds. ^∗means p < 0.05 when the two groups were compared.

Fig. 13.

HE staining outcomes showing the new bone formation in different scaffolds.

Fig. 14.

Quantitative analysis of new bone formation. ^∗means p < 0.05 when the two groups were compared. ^∗∗means p < 0.01 between the two groups.

When doing quantitative analysis in the new bone area at 4 week and 8 week after surgery (Fig. 14), it was found that the 2.0%LiCPP and 1.0%LiCPP groups achieved significant better outcomes than the CPP group (p < 0.05, both). The 2.0%LiCPP group even obtained a larger new bone area in comparison to the 0.1%LiCPP group (p < 0.05).

5. Discussion

Incorporating low content Li ions into CPP did not change the standard FTIR spectra and XRD pattern. LiCPP scaffolds had typical CPP crystal form and doped proper content of Li did not lead to decomposition. In fact, the radius of Li is smaller than the Ca in the crystal grain, which makes it easy for Li to reach the clearance position of the lattice and replace or occupy the corresponding ion site [16,17]. The LiCPP scaffolds we established were with large number of micropore and mesoporous through the scaffolds, which were benefit to the cells’ attachment and in-growth. In this study, low dose of Li in CPP improved the brittleness of CPP, and we found that the LiCPP effective degradation was increased when co-cultured with MG63, which may be caused due to acidic metabolity or acidic enzyme secreted by cells which accelerate the degradation of scaffolds and Li⁺ increased the MG63 proliferation resulting in more acidic substances secretion [15,18]. Consequently, the optimal Li content in CPP could benefit to the effective degradation of CPP. The compressive strength of LiCPP scaffolds increased with Li content went up and can reach to more than was 7 MPa, which met the strength requirements of cancellous bone (2–12 MPa) [15,18,19]. It indicated that these LiCPP scaffolds could serve as a promising candidate for bone repairing. Meanwhile, we found that there were more cells attached on the scaffolds with Li content increasing and 2.0%LiCPP showed most cells adhesion, which indicated that doped Li into CPP may change the surface topography of original CPP and made LiCPP possess a more compact bulk, and the proper amount of Li⁺ released from LiCPP scaffolds could enhance the activity of MG63, all of which contributed to the scaffolds’ cell adherence and growth [20,21].

Li has been regarded as a Wnt signal pathway activator and reported to provided good performance in treating some bone disease, such as osteoporosis, osteonerosis, fracture, et al. [10,12,15,22]. In fact, activating of Wnt pathway could also improve cell viabilities and promote osteogenic differentiation [23–25]. In this study, we found that LiCPP scaffolds could decrease the GSK-3β, while increased β-catenin and Runx2 expression, which indicated the Wnt pathway was up-regulated and osteogenesis (Runx2) was improved. Consequently, when we evaluated the osteogenesis the ALP level and calcium deposition amount were all increased along with Li content in CPP went up, which indicated that Li could help to promote the osteogenesis and a proper content of LiCPP benefited to enhance the osteogenic potential. According to the outcomes, 2.0%LiCPP obtained best efficacy in promoting MG63 proliferation and osteogenesis differentiation.

New bone formation in vivo is a more meaningful and direct performance that shows the osteogenesis and it is also the ultimate goal of studying the bone substitute materials [26–28]. According to the in vivo outcome, LiCPP scaffolds increased the new bone formation and a higher Li content seemed to be associated with larger new bone area. The 2.0%LiCPP scaffolds achieved the best osteogenic effect, which was in accordance with in vitro studies. Consequently, the composite scaffolds containing Li release could activate Wnt signal pathway, which contributed to enhance osteogenesis in bone regeneration in vivo.

6. Conclusion

In this paper, a novel porous scaffold, LCPP, was successfully prepared, and its biological performances, degradation behavior and osteogenic behavior were investigated. The results suggested that low-content lithium doping showed no significant influence on the structure of CPP scaffolds. The cells mediated degradation of CPP was promoted and the LiCPP scaffolds could slow release of Li⁺, Ca²⁺ and PO₄ ³⁻. The compressive strength of CPP scaffolds was improved and Li content increased and reached 7 MPa. Meanwhile, doping Li into CPP promoted MG63 proliferation and adhesion. The slow release of Li form LiCPP scaffolds could up-regulate Wnt signal pathway in MG63 and promote the osteogenic differentiation. According to the outcomes, 2.0%LiCPP achieved best bioactivity in vitro. At the same time, LiCPP scaffolds enhanced new bone formation in vivo when used to repair the bone defect and the 2.0%LiCPP scaffolds also obtained the best outcomes. All results in our study contribute to the idea that 2.0%LiCPP scaffold is a promising biomaterial for bone repairing application. We believe that our research provides a way to improve the properties of CPP and extend the applications of bone tissue engineering.

Conflict of interest

The authors declare no potential conflict of interest with respect to the research, authorship, and/or publication of this article.

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