Role of miR-214 in biomaterial transplantation therapy for osteonecrosis

Abstract

BACKGROUND:

The effectiveness and availability of conservative therapies for osteonecrosis of the femoral head (ONFH) are limited. Transplantation of bone marrow mesenchymal stem cells (BMSCs) combined with Bio-Oss, which is a good bone scaffold biomaterial for cell proliferation and differentiation, is a new potential therapy. Of note, the expression of miRNAs was significantly modified in cells cultured with Bio-Oss, and MiR-214 was correlated positively with osteonecrosis. Furthermore, miR-214 was upregulated in cells exposed to Bio-Oss.

OBJECTIVE:

To investigate whether targeting miR-214 further improves the transplantation effect.

METHODS:

We treated BMSCs with agomiR-214 (a miR-214 agonist), antagomiR-214 (a miR-214 inhibitor), or vehicle, followed by their transplantation into ONFH model rats.

RESULTS:

Histological and histomorphometric data showed that bone formation was significantly increased in the experimental groups (Bio-Oss and BMSCs treated with antagomiR-214) compared with other groups.

CONCLUSIONS:

miR-214 participates in the inhibition of osteoblastic bone formation, and the inhibition of miR-214 to bone formation during transplantation therapy with Bio-Oss combined with BMSCs for ONFH.

Keywords

Bio-Oss BMSCs miR-214 bone regeneration transplantation therapy

1. Introduction

Osteonecrosis of the femoral head (ONFH) is a refractory disease with progressive osteocyte and bone marrow necrosis [1–5]. In contemporary societies, alcohol abuse and long-term high-dose corticosteroid treatment increase the risk of ONFH [6–8]. The management of ONFH is evolving but remains challenging. Patients who have necrosis in the non-weight-bearing areas and the area <15%, can be actively treated with anticoagulants and vasodilator drugs and avoidance of impingement and impact-loading activities [9–12]. When ONFH progresses rapidly and nonsurgical treatments are not effective, most patients require surgical treatment. The surgical methods can be sorted into two categories: (1) repair and reconstruction methods, which primarily preserve the patient’s own femoral head, and (2) hip joint replacement method; (1) includes osteotomy and core decompression alone or in combination with stem cell transplantation [13–16]. If this approach is effective, hip joint replacement can be avoided or delayed. Hip joint arthroplasty may lead to many associated problems [17].

The application of bone marrow mesenchymal stem cell (BMSC) transplantation to the treatment of ONFH has led to the development of strategies that preserve the patient’s own femoral head [18–20]. However, several studies reported an absence of significant differences between stem cell implantation and the traditional methods [21,22], probably because of the lack of a good bone scaffold material for the implantation of stem cells. Bio-Oss is composed of organic bovine bone, which has excellent osteoconductive properties, high biocompatibility, and a low biodegradation rate, and has been widely used in several bone regeneration procedures during oral surgery [23–25]. However, the manner in which the biomaterials alter osteoblast activity to promote bone formation is poorly understood. It is noteworthy that the expression of miRNAs was significantly modified in osteoblast-like cells cultured with Bio-Oss [26,27].

MicroRNAs (miRNAs) are short, single-stranded, endogenous noncoding RNAs that modulate target mRNA transcription and translation through binding to their untranslated region [28–30]. Previous studies have demonstrated that miRNAs play important roles in bone formation and remodeling [31–34] and in the development of ONFH [35–37]. miR-214 participates in the inhibition of osteoblast differentiation and the acceleration of osteoclast activity in skeletal disorders [38,39]. Furthermore, miR-214 was upregulated in an osteoblast-like cell line exposed to Bio-Oss [26,27]. To investigate whether targeting miR-214 further improves the transplantation effect, we designed experimental groups to evaluate the function of miR-214 in the transplantation of BMSCs combined with Bio-Oss in a rat ONFH disease model. We found that miR-214 promoted osteoblast activity but did not inhibit osteoclast activity in vivo. These results suggest that miR-214 is a useful therapeutic target for preventing ONFH.

2. Materials and methods

2.1. Human bone preparation

All clinical procedures were approved by the Committee of Clinical Ethics of the Affiliated Hospital of HangZhou Normal University, HangZhou, China. Bone tissue samples were obtained between May 2010 and July 2021. The non-traumatic ONFH samples (n = 12) were derived from the ONFH patients who underwent hip arthroplasty. We used patients with hemiarthroplasty for displaced femoral neck traumatic fractures (n = 8) as the healthy controls (HC). The age distributions were similar between the two groups, and the influence of age was excluded. The clinical information of the patients is shown (Table 1). Among the 12 patients with non-traumatic ONFH, seven patients were treated with steroids, four patients suffered from alcohol abuse, and one patient was diagnosed with congenital osteonecrosis.

Table 1
Characteristics of patients with osteoarthritis and osteonecrosis

No. of patients Age (years) Gender (F/M)

HC 8 47 ± 12 3/5

ONFH

Steroid 7 49 ± 13 5/2

Alcohol 4 51 ± 11 0/4

Idiopathic 1 53 1/0

	No. of patients	Age (years)	Gender (F/M)
HC	8	47 ± 12	3/5
ONFH
Steroid	7	49 ± 13	5/2
Alcohol	4	51 ± 11	0/4
Idiopathic	1	53	1/0

Abbreviations: F, female; M, male; HC, osteoarthritis as the healthy control; ONFH, osteonecrosis of the femoral head.

2.2. Isolation of BMSCs

Specific pathogen-free (SPF) 6-week-old male Sprague Dawley rats were purchased from the Hangzhou Normal University Experimental Animal Research Center. Rats were fed a standard diet and allowed to adapt to the environment for 1 week before initiating the experiments. The isolation methods were as follows. Anesthetized rats were sacrificed by cervical dislocation and placed on the operation table. After alcohol immersion and disinfection, the bilateral femurs and tibias were separated, the two ends of the bone were cut off, and the bone marrow cavity was exposed. Serum-free α-MEM was drawn into the port of the bone cavity, to wash the bone marrow. The tissue was placed in a 50 mL centrifuge tube; tissue fragments were then filtered and transferred to a new tube before centrifugation at 1,200 revolutions per minute (rpm) for 5 min. After resuspension in α-MEM containing 10% FBS and 1% penicillin and streptomycin (basal culture medium), the cells were seeded in culture flasks in proportion. Bone marrow cells from two rats were inoculated into one flask and placed in an incubator at 37 °C with 5% CO₂. The fluid was replaced after 3 days and non-adhered cells were discarded. When the cells reached 70% confluence, they were digested with 0.25% trypsin and passaged every 2 or 3 days.

2.3. Induction of BMSCs

Cells were passaged three times to obtain sufficient BMSCs. BMSCs were seeded at a density of 1 × 10⁵ cells/well in six-well dishes. For osteoblast differentiation, 10 nM dexamethasone, 50 μg/ml ascorbic acid and 10 mM β-glycerol phosphate were added to basal culture medium, and staining for alkaline phosphatase (ALP) activity and mineralization was performed as described previously [40]. For osteoclast differentiation, cells were cultured in medium containing 30 ng/mL macrophage colony-stimulating factor (M-CSF) and 50 ng/mL receptor activator of nuclear factor kB ligand (NFkB), and TRAP staining was performed according to the manufacturer’s instructions (Sigma-Aldrich).

2.4. Co-culture of BMSCs and Bio-Oss

BMSCs were seeded at a density of 5 × 10⁴ cells/mL in 12-well plates. There were four groups: Group 1, BMSCs, as control group; Group 2, Bio-Oss and BMSCs; Group 3, Bio-Oss and BMSCs treated with agomiR-214; and Group 4, Bio-Oss and BMSCs treated with antagomiR-214. For group 2, 3 and 4, Bio-Oss was added to one set of wells at a concentration of 10 mg/mL. For group 3 and 4, the medium was separately supplemented with 200 μM agomiR-214 or 200 μM antagomiR-214 (Guangzhou RiboBio) and changed every two days. On day 7, the cells were harvested to detect the expression of miR-214 using quantitative real-time PCR.

2.5. Establishment of a rat model of femoral head necrosis and transplantation

Ten weeks old healthy SPF female SD rats (about 320–370 g) were used in our study. Rats were fed a standard diet and allowed to adapt to the environment for 1 week before the experiments. They were then randomly and equally assigned to four groups (n = 8/group): Group 1, control group; Group 2, ONFH group; Group 3, Bio-Oss and BMSCs treated with agomiR-214 transplantation group; and Group 4, Bio-Oss and BMSCs treated with antagomiR-214 transplantation group.

The ONFH model was established via an improved liquid nitrogen freezing protocol [41–43]. Rats were anesthetized with 45 mg/kg pentobarbital sodium. A skin incision was made on the left hip joint. Following, the subcutaneous muscles were separated. Both sides of the femur neck were exposed without dislocation of hip joint. The osteonecrosis model was generated by the application of a cotton ball dipped in liquid nitrogen to the femoral head area for 2 min. After rewarming, a 2 mm drill was used to drill into the femoral head to a depth of 2 mm, from the posterior side of the femoral neck to the epiphyseal line.

The femoral heads in Groups 3 and 4 were resealed with bone wax after being filled with the compound of Bio-Oss and BMSCs, the compound treated with agomiR-214, and the compound treated with antagomiR-214, respectively (10⁷ cells/mL). The femoral heads in Group 2 were filled with an equal volume of phosphate-buffered saline and then cemented. The hip joints were reset and the muscles and skin were sutured. The rats were then injected with 2 mL of 8 IU/ml gentamicin sulfate (Aodong, Jilin) for 5 days, to prevent infection in the first postoperative week. Four weeks after transplantation, the rats were sacrificed. The muscle, connective tissue, and periosteum surrounding the bilateral femurs were removed as far as possible. The femurs were prepared for immunohistological, histomorphological, and radiography analysis.

All experimental and animal care procedures were approved by the Animal Research Ethics Committee of Hangzhou Normal University and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

2.6. Quantitative real-time PCR

Total RNA was extracted from cells or tissues using the TRIzol reagent (Invitrogen). Quantitative real-time PCR was performed using the SYBR Green PCR Master Mix and a 7900HT thermocycler (Applied Biosystems). Data were analyzed using the SDS Relative Quantification Software (Applied Biosystems). The expression of the U6 gene was used as a normalized value. The results are presented as the comparative threshold cycle for gene. The reaction system was followed as the instruction. The primers used here were described by Juan et al. [44]. Each PCR was performed in triplicate.

2.7. Analysis of bone tissue

For histological analysis, the specimens were fixed immediately in 4% paraformaldehyde (pH = 7.4) for 2 days and then decalcified by placement in 8% EDTA-Tris for 21 days. The decalcified fluid was replaced every 2 days until a large pillow was easily pierced. Once decalcification was complete, the specimens were placed on an automatic dewatering instrument, dehydrated in 70%–100% ethanol, cleaned with xylene until transparent, and embedded in paraffin. The tissue pieces were trimmed along the coronal slices and cut into 4 μm sections. Immunohistochemistry for collagen I and ALP was performed using an anti-collagen I antibody (PA1-26204, Thermo Fisher Scientific Inc) and an anti-ALP antibody (PA5-106391, Thermo Fisher Scientific Inc), respectively. TRAP staining of bone tissue was performed as described previously [45]. Images were captured using an optical microscope (Leica Microsystems).

For radiographic and histomorphometric analyses, the femurs were fixed immediately in 70% ethanol. For histomorphometric analysis, bone histomorphometric analyses were performed as previously described [45]. The nomenclature, symbols, and units used here were as recommended by the Nomenclature Committee of the American Society for Bone and Mineral Research [46,47].

2.8. Statistical analysis

Data are shown as the mean ± standard deviation (SD). Mann–Whitney U-test was used for statistical comparison of the data between two groups. For four groups, one-way analysis of variance (one-way ANOVA) was firstly used to determine the existence of difference, and then Scheffé’s multiple-comparison test was used for statistical significance analysis. Statistical analyses were carried out using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). The effects were considered statistically significant at P values < 0.05. All experiments were repeated three times.

Fig. 1.

miR-214 level is up-regulated in patients with ONFH patients and cell co-culture groups. (A) miR-214 levels were determined by quantitative real-time PCR in ONFH patients compared to healthy controls (HC). miR-214 levels were normalized to U6. (B) Expression of miR-214 were determined in BMSCs or coculture with Bio-Oss. miR-214 levels were normalized to U6. All data are presented as mean ± standard error of the mean (SEM). $^{**}P< 0.01$ .

3. Results

3.1. Upregulated expression of miR-214

Bone samples from patients with non-traumatic ONFH (ONFH group) and patients with hemiarthroplasty (HC group) were analyzed via quantitative real-time PCR. The results revealed the upregulation of miR-214 in the ONFH group compared with the HC group (Fig. 1A). This finding was consistent with previous reports by Wang et al. [48]. The expression of miR-214 was significantly upregulated in the BMSCs co-cultured with Bio-Oss compared with the BMSC single-culture group (Fig. 1B). This result was consistent with previous reports by Palmieri et al. [27].

3.2. miR-214 inhibits osteoblast differentiation in vitro

The number of TRAP-positive multinucleated osteoclasts was not significantly different among the four groups (Fig. 2A–D). However, regarding osteoblastogenesis, Group 4 (BMSCs treated with antagomir-214 and Bio-Oss) showed enhanced ALP staining (Fig. 2H) compared with other groups (Fig. 2F, 2G). Concordantly, significantly greater Kossa staining was detected in the antagomir-214 treatment group (Fig. 2L) vs. the other groups (Fig. 2J, 2K). These results suggest that miR-214 inhibits osteoblastogenesis and matrix mineralization in vitro.

Fig. 2.

miR-214 inhibits osteoblastgenesis and matrix mineralization in vitro. (A–D) Representative images of TRAP staining of BMSCs after treatment with CTL(A), Bio-Oss(B), agomir-214 and Bio-Oss (C), antagomir-214 and Bio-Oss (D) for 4 days. (E-H) Representative images of ALP staining of BMSCs after treatment with CTL(E), Bio-Oss(F), agomir-214 and Bio-Oss (G), antagomir-214 and Bio-Oss (H) for 2 days. (I–L) Representative images of Kossa staining of BMSCs after treatment with CTL(I), Bio-Oss(J), agomir-214 and Bio-Oss (K), antagomir-214 and Bio-Oss (L) for 14 days.

3.3. Expression of miR-214 in a transplantation rat model

The levels of miR-214 in the control, ONFH, and two other transplantation groups were measured using real-time PCR. The expression of miR-214 was significantly enhanced in the ONFH and agomiR-214 transplantation groups, whereas it markedly decreased in the anatgomiR-214 transplantation group (Fig. 3). ONFH substantially upregulated the intracellular miR-214 levels compared with the control group. Compared with the ONFH group, the intracellular miR-214 levels were markedly downregulated by antagomiR-214 treatment.

Fig. 3.

The expression of miR-214 in bone samples. The use of antagomir-214 suppressed the expression of miR-214. miR-214 levels were normalized to U6. All data are presented as mean ± standard error of the mean (SEM). ^∗ P < 0.05, ^∗∗ P < 0.01.

3.4. Histological analysis of bone tissue sections

Many TRAP-positive cells were observed in the sagittal suture and the surrounding bone surface in the ONFH group (Fig. 4B). This phenomenon did not change after the transplantation of Bio-Oss and BMSCs treated with antagomiR-214 in Group 4 (Fig. 4D). However, immunohistochemical staining of ALP and collagen I showed that the number of immunoreactive cells was significantly higher in Group 4 (Fig. 4H, 4L) compared with Group 2 (Fig. 4F, 4J) and Group 3 (Fig. 4G, 4K). ALP-positive and collagen I-positive osteoblasts were arranged on the surface of the newly formed bone. These results suggest that the inhibition of miR-214 is beneficial for osteoblast activity, indicating that miR-214 participates in the osteoblastic bone formation that occurs after transplantation therapy.

Fig. 4.

Histological analysis of bone tissue sections. (A–D) Representative images of TRAP staining of bone tissue in group 1 (A), group 2 (B), group 3 (C), group 4 (D). Scale bar, 50 um. (E–H) Representative images of immunostaining (ALP) in group 1 (E), group 2 (F), group 3 (G), group 4 (H). ALP-positive osteoblasts are arranged on the surface of the newly formed bone. Scale bar, 30 um. (I–L) Representative images of immunostaining (collagen I) in group 1 (I), group 2 (J), group 3 (K), group 4 (L). Collagen-positive osteoblasts are arranged on the surface of the newly formed bone. Scale bar, 30 um. For both ALP and collagen I, the number of immunoreactive cells in group 4 was significantly higher than those in group 2 and group 3. Group 1, control group; Group 2, ONFH group; Group 3, Bio-Oss and BMSCs treated by agomiR-214 transplantation group; Group 4, Bio-Oss and BMSCs treated by antagomiR-214 transplantation group.

Fig. 4

(Continued).

3.5. Histomorphometric analysis of bone tissue sections

Histomorphometric data revealed no significant differences in group 4 in the bone volume/total volume ratio (BV/TV), osteoid surface (OS/BS), number of osteoclasts (Mu.N.Oc/B.Pm), or osteoclast surface (Oc.S/BS) (Fig. 5A, 5B, 5E, and 5F), compared with group 2 and group 3. However, the number of osteoblasts (N.Ob/B.Pm) and the osteoblast surface (Ob.S/BS) were significantly increased in the femur sections of group 4 compared with the group 2 and group 3 (Fig. 5C, 5D). A histomorphometric analysis indicated no significant differences in bone resorption between these groups; however, there was a significant increase in the number and surface of osteoblasts in Group 4 (P < 0.01).

Fig. 5.

Histomorphometric analysis of bone tissue sections. (A) Trabecular bone volume (bone volume/tissue volume, BV/TV). (B) Osteoid surface/bone surface (OS/BS). (C) Number of osteoblasts/bone resorption parameters (N.Ob/B.Pm). (D) Osteoblast surface (Ob.S)/BS. (E) Number of multinucleated osteoclasts (Mu.N.Oc)/B.Pm. (F) Osteoclast surface (Oc.S)/BS. Data represent the mean ± SD of eight mice. ^∗ P < 0.05, ^∗∗ P < 0.01.

3.6. Radiographic analysis

Radiographic data suggested that there was no significant differences in group 4 (Fig. 6A), compared with group 2 and group 3. This implies that the integration of these materials into the bone may require a longer time.

Fig. 6.

Representative radiography images of the femoral head in the control group (A), ONFH group (B), agomiR-214 transplantation group (C), antagomiR-214 transplantation group (D). Scale bar, 1 mm.

4. Discussion

ONFH, which is characterized by progressive osteocyte and bone marrow necrosis, is an increasing health problem worldwide. miR-214 is highly expressed in patients with bone fractures, primary osteoporotic and necroptotic ones [49–51]. These studies suggested that mir-214 serves important roles in the regulation of cell growth and apoptosis, and that apoptotic processes may be involved in the pathogenesis of ONFH. Here, we demonstrated that miR-214 was correlated positively with osteonecrosis (Fig. 1).

Cell-based bone tissue engineering techniques have been developed to promote bone formation. Bio-Oss provides a good bone scaffold material for the implantation of simple cells. However, a recent clinical study revealed a lower survival rate of implants in the BMSCs/Bio-Oss group compared with the autogenous group [52]. The expression of miR-214 was elevated in the co-culture combined with Bio-Oss (Fig. 1). To investigate the functional effects of miR-214 on the co-culture of BMSCs and Bio-Oss, we treated BMSCs with either agomiR-214 (a miR-214 agonist) or antagomiR-214 (a miR-214 inhibitor). The intracellular miR-214 levels were substantially upregulated by agomiR-214 treatment and markedly downregulated by antagomiR-214 treatment (Fig. 3).

miR-214 negatively regulates osteogenic differentiation [53,54]. The levels of collagen I and ALP were significantly increased in cells of the osteoblast cell line treated with a miR-214 agonist [38]. In contrast, miR-214 protected osteoblasts from H₂O₂-induced apoptosis [55]. In our study, enhanced ALP staining and greater Kossa staining was detected in antagomir-214 treatment group in vitro (Fig. 2H, 2L). Futhermore, to investigate the role of miR-214 in the regulation of osteoblast differentiation and activity in vivo, we transplanted cells treated with agomiR-214 or antagomiR-214 into the ONFH rat model. The number of osteoblasts was significantly higher in antagomir-214 treatment group in vivo (Fig. 4H, 4L, Fig. 5C, 5D). Unfortunately, we did not detect macroscopic changes in bone volume in the antagomir-214 treatment group compared with the ONFH group in vivo (Fig. 5A). Our results suggest that miR-214 participates in the inhibition of osteoblast differentiation and osteoblastic bone formation. Previous studies suggested that miR-214 directly targeted ATF4 to modulate osteoblastic differentiation and inhibit osteoblast activity in MC3T3-E1 cells, bone marrow cells and valvular interstitial cells [38,48,56]. ATF4 has been confirmed to be an important regulatory factor in the classical signaling pathway of endoplasmic reticulum stress, which may determine the fate of cells under various adverse conditions. Whether miR-214 also acts directly on ATF4 in the current transplantation system remains to be further verified.

5. Conclusion

In this study we reported that the inhibition of miR-214 contributed to bone formation in transplantation therapy consisting of Bio-Oss combined with BMSCs for ONFH. Our results demonstrated a therapeutic potential for miR-214 in ONFH healing. Further investigations are underway in our laboratory to clarify the mechanisms that contribute to the upregulation of miR-214 induced by Bio-Oss in cell culture and those underlying the inhibition of bone formation by miR-214.

Footnotes

Acknowledgements

This work was supported by the Natural Science Foundation of Zhejiang Province (Grant number: LY20H060003, LY20H090007) and National Natural Science Foundation of China (Grant number: 81401811).

Conflict of interest

None to report.

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Role of miR-214 in biomaterial transplantation therapy for osteonecrosis

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1. Human bone preparation

Table 1 Characteristics of patients with osteoarthritis and osteonecrosis No. of patients Age (years) Gender (F/M) HC 8 47 ± 12 3/5 ONFH Steroid 7 49 ± 13 5/2 Alcohol 4 51 ± 11 0/4 Idiopathic 1 53 1/0

2.3. Induction of BMSCs

2.4. Co-culture of BMSCs and Bio-Oss

2.5. Establishment of a rat model of femoral head necrosis and transplantation

2.6. Quantitative real-time PCR

2.7. Analysis of bone tissue

2.8. Statistical analysis

3.1. Upregulated expression of miR-214

3.2. miR-214 inhibits osteoblast differentiation in vitro

5. Conclusion

Footnotes

Acknowledgements

Conflict of interest

References

Table 1
Characteristics of patients with osteoarthritis and osteonecrosis

No. of patients Age (years) Gender (F/M)

HC 8 47 ± 12 3/5

ONFH

Steroid 7 49 ± 13 5/2

Alcohol 4 51 ± 11 0/4

Idiopathic 1 53 1/0