A bone substitute composed of polymethyl-methacrylate bone cement and Bio-Gene allogeneic bone promotes osteoblast viability,adhesion and differentiation

Abstract

BACKGROUND:

Increasing reports on new cement formulations that address the shortcomings of PMMA bone cements and various active components have been introduced to improve the biological activity of PMMA cement.

OBJECTIVE:

Evaluating the biological properties of PMMA cements reinforced with Bio-Gene allogeneic bone.

METHODS:

The MC3T3-E1 mouse osteoblast-like cells were utilized to determine the effects of Bio-Gene + PMMA on osteoblast viability, adhesion and differentiation.

RESULTS:

The combination of allogeneic bone and PMMA increased the number of adherent live cells compared to both control group and PMMA or Bio-Gene group. Scanning electron microscopy observed that the number of cells adhered to Bio-Gene + PMMA was larger than Bio-Gene and PMMA group. Compared with the control and PMMA or Bio-Gene group, the level of ALP and the number of calcium nodules after osteoinduction was remarkably enhanced in Bio-Gene + PMMA group. Additionally, the combination of Bio-Gene and PMMA induced the protein expression of osteocalcin, osterix and collagen I.

CONCLUSION:

The composition of PMMA and allogeneic bone could provide a more beneficial microenvironment for osteoblast proliferation, adhesion and differentiation. PMMA bone cement reinforced with Bio-Gene allogeneic bone may act as a novel bone substitute to improve the biological activity of PMMA cement.

Keywords

Allogeneic bone bone substitute osteoblast polymethyl-methacrylate bone cement

1. Introduction

Polymethyl methacrylate (PMMA) is the most commonly used bone defect filler in orthopedic surgery. It is an acrylic polymer composed of liquid MMA monomer and powdered MMA polymer [1]. As a bone cement, PMMA is the only material used to fix artificial substitutes on adjacent bones, and is widely used in osteoporotic vertebral fractures, bone metastases, and arthroplasty. PMMA bone cement not only relieves pain immediately, but also provides mechanical stability [2]. However, because PMMA bone cement is biologically inert and not suitable for bone growth, the biological activity of PMMA is poor, resulting in poor interaction between local bone and PMMA cement [3]. At the same time, only a mechanically filled interface is formed between bone and bone cement, so a large amount of PMMA particles will be produced after long-term wear in the body, and macrophages will be activated to cause osteolysis, which eventually results in aseptic loosening, limiting its long-term application [4]. Moreover, when PMMA polymerizes, it releases a lot of heat, and the local temperature can reach about 130 °C, which can cause death of surrounding tissue cells, and can also result in a series of adverse reactions such as allergies, decreased anti-infection ability of local tissues, and carcinogenesis [5].

In order to improve the biological activity of PMMA cement, it has been studied to incorporate various active components such as TiO₂, SiO₂, tricalcium phosphate (TCP), hydroxyapatite (HA) and bioceramics into PMMA cement [6–13]. The introduction of active components can reduce the heat generated during polymerization, promote the formation of a calcium-phosphorus deposition transition layer between bone cement and bone tissue, and endow the material with a certain biological activity [14]. For example, it has been reported that PMMA + TCP could have a synergic effect and be responsible for the improvement of the material colonization by bone cells, osteoblast activity, osteoinduction and osteoconduction processes as well as bone remodelling both in vitro and in vivo [15]. To provide an enhanced interfacial interaction and strong adhesion between the functionalized n–TiO₂ fibers and PMMA matrix could augment the mechanical properties of PMMA-based cement [16]. In addition, Si ions predominantly enhanced cell adhesion and viability of MC3T3-E1 mouse osteoblast-like cells [17].

Allogeneic transplantation refers to the transplantation between individuals of the same species while different genotypes, is the most common type of transplantation in clinic and the focus of transplant immunology research [18]. The emergence of allogeneic transplantation has effectively solved the problems including insufficient number of grafts, long operation time and large surgical wound area faced by auto-transplantation [19]. Human bones have self-regeneration and self-repair functions, which allows damaged organs to fully restore their composition, structure and function before injury. However, in pathological fractures or large bone defects, bone repair and reconstruction often fail, leading to delayed bone healing or nonunion. These patients often need bone transplantation [20]. Allogeneic bone transplantation is to take the bone of the allogeneic donor from the bone bank and implant it into the bone defect site of the patient. Allogeneic bone has a rich source and is not limited by shape and size, and has good biological activity and shows good bone tissue repair effect [21]. In contrast, allogeneic bone transplantation still has a high failure rate, mainly manifested as delayed union, nonunion, fatigue fracture, immune response and bone resorption [20,22]. However, the effects of allogeneic bone and PMMA composition on bone adhesion and bone formation have not been studied. In the present study, we aimed to investigate whether the composition of PMMA and Bio-Gene allogeneic bone could promote osteoblast viability, adhesion and differentiation.

2. Materials and methods

2.1. Cement preparation

The dry powder of PMMA material (Heraeu sostepalv, Germany) and Bio-Gene allogeneic bone material (Bio-Gene Technology Limited, Beijing, China) were each formulated into a thin paste with a solid-to-liquid ratio of 2 to 1, then poured into a cube mold, and freeze-dried to be made into cubes of 5 × 5 × 5 mm size, respectively. Then PMMA material or Bio-Gene allogeneic bone material or both (1:1) were soaked in the cell culture medium in an incubator of 37 °C for 48 hours. After removing the cement material, and storing in a 4 °C refrigerator, the cell culture medium (cement leach liquor) was used for the following cell experiments.

2.2. Cell culture and treatment

The MC3T3-E1 mouse osteoblast-like cells (Sigma-Aldrich, Germany) were cultured in DMEM medium (Hyclone, Thermo Fisher Scientific, USA) containing 10% of fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, USA), 1% of penicillin/streptomycin (Genview, Beijing, China) at 37 °C with 95% humidified air and 5% CO₂.

Cells were grown for 48 h to reach the logarithmic growth phase, before being harvested using Trypsin/EDTA (Genview, Beijing, China). Cells were counted by a hemocytometer (Roth, Germany) and diluted to a final concentration of 1 × 10⁵ cells/ml. Then cells were subjected to different culture medium: (i) Control: 10 μL 0.1% sterilized phenol; (ii) PMMA: 10 μL PMMA bone cement leach liquor; (iii) Bio-Gene: 10 μL Bio-gene bone leach liquor; (iv) Bio-Gene + PMMA: 10 μL composited bone cement leach liquor.

2.3. Cell counting kit-8 (CCK-8)

Cell viability was determined by using the CCK-8 assay (Sango Biotech, Shanghai, China), which contains water-soluble tetrazolium (WST) solution. For this purpose, after cultivating with different medium in 96-well plate for 72 h, the cell culture medium was removed, and samples were washed with PBS. Afterwards, 10 μL CCK-8 solution was added to each well and incubated for 2 h. Finally, the absorbance was measured using a microplate reader (ELx800; BioTek, USA) at 450 nm.

2.4. Calcein-AM/EthD-1 staining

The calcein-AM/EthD-1 staining (Beijing Baiolaibo Technology Co., Ltd, China) was used for observing dead and living cells at the same time. In short, after the preparation of dyeing working solution according to the manufacturer’s instruction, cells that incubated with sterilized phenol or cement leach liquor were washed with PBS and then exposed to 200 μL dyeing working solution at 4 °C in the dark for 15–30 min. After washing with PBS, cells were observed and photographed under a fluorescent microscope (the excitation wavelength is 490 ± 10 nm, the living cells were stained with yellow-green, whereas the dead cells were red).

2.5. Measurement of alkaline phosphatase (ALP)

The level of ALP was detected by ALP kit (Beyotime Biotechnology, China). In short, after incubating with sterilized phenol or cement leach liquor in a 96-well plate for 3 days, the culture mediums were changed to normal DMEM medium and cultured for 7 days and then the culture supernatants were collected for detecting ALP referring to the manufacture’s instruction. The absorbance was measured using a microplate reader (ELx800; BioTek, USA) at 405 nm.

2.6. Alizarin red staining

After incubating with sterilized phenol or cement leach liquor in a 6-well plate at a density of 2 × 10⁵ cells/well for 3 days, the culture mediums were changed to normal DMEM medium and cultured for 14 days. Then cells were subjected to Alizarin red staining (Cyagen Biotech, Guangzhou, China) according to the instruction. After removing the supernatant, cells were washed once with PBS, and fixed with 4% paraformaldehyde for 15 min. Followed by adding 1 mL of alizarin red staining solution to each group and staining for 5 min. Finally, removed the staining solution, washed cells with PBS, and took pictures under microscope (CKX41; Olympus, Japan).

2.7. Scanning electron microscope (SEM)

MC3T3-E1 cells were seeded on the surface of a cement with a diameter of 10 mm, a thickness of 3 mm, and a density of 5.0 × 10⁴ per hole. After culturing for 3 days, the surface of the cement sample was rinsed with PBS to remove non-adherent cells. The remaining adherent cells were fixed in cacodylate buffer with 2.5% glutaraldehyde and then washed with cacodylate buffer containing sucrose. After dehydration with gradient alcohol, the morphology and adhesion of cells were observed with SEM (SU8010; Hitachi, Japan).

2.8. Western blotting

After incubating with sterilized phenol or cement leach liquor in a 6-well plate at a density of 2 × 10⁵ cells/well for 3 days, the culture mediums were changed to normal DMEM medium and cultured for 14 days. Then total proteins were extracted from cells by RIPA lysis and extraction buffer (Beyotime Biotechnology, China) and the concentrations were determined by BCA kit (Thermo Fisher). Equal amounts (50 μg) of each sample was subjected to 6–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate the protein samples, and transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked with TBS-Tween-20 containing 5% skimmed milk at room temperature for 2 h and then incubated with the following primary antibodies overnight at 4 °C: osteocalcin, osterix, collagen I and GAPDH (Santa Cruz Biotechnology, Inc.). Finally, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h and visualized using an enhanced chemiluminescence kit (GE Healthcare).

2.9. Statistical analysis

All experiments were repeated at least three times and statistical analyses were performed using GraphPrism software (version 5.0; GraphPad, USA). Statistical analysis of the obtained results for each test were conducted using a one-way analysis of variance (ANOVA) followed by Tukey’s test, with significance denoted at P < 0.05.

3. Results

3.1. Effect of PMMA cements reinforced with Bio-Gene allogeneic bone on osteoblast viability

First of all, the cell viability of MC3T3-E1 cells that exposed to different bone cements was measured. As shown in Fig. 1A, the results from CCK-8 revealed that PMMA bone cement inhibited cell viability, but PMMA + Bio-Gene significantly increased cell viability. The same result was observed in Fig. 1B, in which the live cells were stained with green while dead cells were stained with red.

Fig. 1.

Effect of PMMA, Bio-Gene and PMMA + Bio-Gene composite cement on MC3T3-E1 osteoblast cell viability. A, cell viability was detected using CCK-8 assay after cultivating MC3T3-E1 with control, PMMA, Bio-Gene and PMMA + Bio-Gene composite cement medium. B, representative calcein-AM/EthD-1 staining images for observing MC3T3-E1 viability, the living cells were stained with yellow-green, while the dead cells were red. *P < 0.05 and ***P < 0.001 vs control; ^### P < 0.001 vs PMMA; ^Δ P < 0.05 vs Bio-Gene.

3.2. Effect of PMMA cements reinforced with Bio-Gene allogeneic bone on osteoblast adhesion

SEM was employed to observe the number of cells that adhered to different bone cements. As shown in Fig. 2, the structure of each bone cement and cells that adhered to the surface of bone cements, could be visualized clearly. We found that the surface structure of the PMMA and Bio-Gene changed after modification by mixing them. In addition, the number of cells that adhered to PMMA + Bio-Gene was much larger than that adhered to control, PMMA and Bio-Gene. Meanwhile, the structure of adhered cells was very different among each group.

Fig. 2.

The attachment of MC3T3-E1 osteoblast to different bone cements. After culturing MC3T3-E1 with control, PMMA, Bio-Gene and PMMA + Bio-Gene composite cement medium for 3 days, scanning electron microscope (SEM) was used to observe the attachment.

3.3. Effect of PMMA cements reinforced with Bio-Gene allogeneic bone on osteoblast differentiation

The alteration of osteoblast differentiation was also evaluated. ALP activity reflects the early differentiation ability of osteoblasts. As shown in Fig. 3A, PMMA or Bio-Gene treated separately reduced, while the combination of Bio-Gene and PMMA, significantly increased ALP activity.

Fig. 3.

Effect of PMMA, Bio-Gene and PMMA + Bio-Gene composite cement on MC3T3-E1 osteoblast differentiation. A, after culturing MC3T3-E1 with control, PMMA, Bio-Gene and PMMA + Bio-Gene composite cement medium for 7 days, the alkaline phosphatase (ALP) activity was measured. B, after culturing MC3T3-E1 with control, PMMA, Bio-Gene and PMMA + Bio-Gene composite cement medium for 14 days, Alizarin red staining was used to observe the formation of mineralized nodules. *P < 0.05 vs control; ^### P < 0.001 vs PMMA; ^ΔΔΔ P < 0.001 vs Bio-Gene.

Alizarin red staining was used to observe the number of calcium nodules and assess the mineralization ability of osteoblasts. Results from Fig. 3B revealed that Bio-Gene + PMMA culture medium obviously enhanced the number of calcium nodules in MC3T3-E1 cells.

Finally, the expression of proteins involved in osteoblasts differentiation including osteocalcin, osterix and collagen I was detected. As illustrated in Fig. 4, compared to control group, PMMA and bio-gene treatment significantly reduced the expression of these proteins. In contrary to PMMA and bio-gene, PMMA + Bio-Gene remarkably enhanced the expression of osteocalcin, osterix and collagen I.

Fig. 4.

Effect of PMMA, Bio-Gene and PMMA + Bio-Gene composite cement on the expression of proteins involved in MC3T3-E1 osteoblast differentiation. After culturing MC3T3-E1 with control, PMMA, Bio-Gene and PMMA + Bio-Gene composite cement medium for 14 days, the expression of osteocalcin, osterix and collagen I was determined using western blotting, GAPDH was used as a loading control. ***P < 0.001 vs control; ^### P < 0.001 vs PMMA; ^ΔΔΔ P < 0.001 vs Bio-Gene.

4. Discussion and conclusion

The differentiation, mineralization and adhesion capabilities of osteoblasts directly reflect the growth and development of bone tissue. In accordance with previous findings, our results showed that PMMA and Bio-Gene impaired the proliferation and osteoblastic differentiation of MC3T3-E1 osteoprogenitor cells [23,24]. Allogeneic bone has normal bone strength, large size range and accessibility, so it has the potential to be used as a bone replacement material in autograft [25]. In the present study, we showed that during the proliferation stage of osteoblasts, the composition of PMMA and Bio-Gene allogeneic bone leach liquor did not affect the viability of the MC3T3-E1 osteoblast. Compared with the blank control group, PMMA + Bio-Gene exhibited a certain promoting effect on cell viability and proliferation.

When observed by SEM, the combination of PMMA and Bio-Gene obviously changed the surface properties of PMMA and allogeneic bone. We found that the surface structure of PMMA + Bio-Gene became more rough and porous. When MC3T3-E1 cells were cultured in the leach liquor of PMMA + Bio-Gene group, a larger number of them adhered, compared to the single treatment group. Therefore, we believed that bone surface structure may play an important role in cell attachment and growth. Previous studies have shown that surface roughness may affect the differentiation and proliferation of osteoblasts [26].

Alkaline phosphatase (ALP) is an important marker for the early differentiation of osteoblasts. It is currently believed that the increased activity of ALP indicates that osteoblasts have an enhanced ability to form new bone [27]. During the stage of extracellular matrix maturation and differentiation, the expression of ALP is significantly enhanced at this time [28]. ALP activity illustrated that PMMA + Bio-Gene could increase the ALP activity of osteoblasts and promote early differentiation of osteoblasts. The formation of mineralized nodules is an important sign of the late differentiation and maturation of osteoblasts [29]. The results of Alizarin Red staining showed that compared with the control group, the mineralized nodules increased significantly after the action of PMMA + Bio-Gene. Osteoblasts have important roles in bone formation, involving the regulation on related proteins during differentiation such as osteocalcin, ALP, osterix and collagen I [23]. We also found that PMMA + Bio-Gene cement considerably enhanced the protein expression of osteocalcin, osterix and collagen I. All these data demonstrated that, in contrast to single treatment of PMMA and Bio-Gene, the incorporation of Bio-Gene allogeneic bone into PMMA could provide a more beneficial microenvironment for cell proliferation, attachment and differentiation and lead to a higher osteogenic capability by inhibiting the death and stimulating the differentiation of osteoblasts. However, there are some limitations in the current study. First of all, only mouse osteoblast-like cells was selected and analyzed. Secondly, our experiments only involved surface materials but lacked animal experiments. Finally, whether PMMA and Bio-Gene allogeneic bone were modified after mixing and how the chemical composition changed have not been explored.

In summary, the bioactive PMMA + Bio-Gene composite cements were developed by adding Bio-Gene allogeneic bone into PMMA cement as the reinforcement phase and bioactive filler. The PMMA + Bio-Gene composite cements provided a more beneficial microenvironment for the proliferation, attachment and differentiation of mouse osteoblast cell line MC3T3-E1. In this paper, the effects and mechanisms of PMMA + Bio-Gene composite cements on osteoblast attachment, differentiation and mineralization are investigated from the perspective of cell biology, which may provide a theoretical basis for animal experiments and clinical studies.

Conflict of interest

The authors declare no conflict of interest.

Funding

The Key Projects of Social Science and Technology Development in Dongguan City (2018507150241633) supported this work.

References

Song

Seta

Eichler

M.K.

Arts

J.J.

Boszczyk

B.M.

Markel

D.C.

, Comparison of in vitro biocompatibility of silicone and polymethyl methacrylate during the curing phase of polymerization, J Biomed Mater Res B Appl Biomater 106(7) (2018), 2693–2699.

Ridwan-Pramana

Idema

Te Slaa

Verver

Wolff

Forouzanfar

, Polymethyl methacrylate in patient-specific implants: Description of a new three-dimension technique, The Journal of Craniofacial Surgery 30(2) (2019), 408–411.

Wang

Lin

Shen

Tang

Han

and Chen

, Hydrophobic modification of polymethyl methacrylate as intraocular lenses material to improve the cytocompatibility, J Colloid Interface Sci 431 (2014), 1–7.

Hautamäki

Meretoja

V.V.

Mattila

R.H.

Aho

A.J.

and Vallittu

P.K.

, Osteoblast response to polymethyl methacrylate bioactive glass composite, J Mater Sci Mater Med 21(5) (2010), 1685–1692.

Lee

C.Y.

Chen

Y.T.

Lee

B.S.

and Chang

C.C.

, Suppressing antibacterial resistance: Chemical binding of monolayer quaternary ammonium salts to polymethyl methacrylate in an aqueous solution and its clinical efficacy, International Journal of Molecular Sciences 20(19) (2019).

Hesaraki

Safari

and Shokrgozar

M.A.

, Composite bone substitute materials based on beta-tricalcium phosphate and magnesium-containing sol-gel derived bioactive glass, J Mater Sci Mater Med 20(10) (2009), 2011–2017.

Le Ferrec

Mellier

Boukhechba

Le Corroller

Guenoun

Fayon

, Design and properties of a novel radiopaque injectable apatitic calcium phosphate cement, suitable for image-guided implantation, J Biomed Mater Res B Appl Biomater 106(8) (2018), 2786–2795.

Meng

H.S.

Chen

Yan

Z.R.

X.Y.

and Sheng

G.P.

, Co-doping polymethyl methacrylate and copper tailings to improve the performances of sludge-derived particle electrode, Water Research 165 (2019), 115016.

Harper

E.J.

, Bioactive bone cements, Proceedings of the Institution of Mechanical Engineers Part H, Journal of Engineering in Medicine 212(2) (1998), 113–120.

10.

Dalby

M.J.

Di Silvio

Harper

E.J.

and Bonfield

, In vitro adhesion and biocompatability of osteoblast-like cells to poly (methylmethacrylate) and poly(ethylmethacrylate) bone cements, Journal of Materials Science Materials in Medicine 13(3) (2002), 311–314.

11.

and Vazquez

, The effect of cross-linking agents on acrylic bone cements containing radiopacifiers, Biomaterials 22(15) (2001), 2177–2181.

12.

Miyazaki

Ohtsuki

Kyomoto

Tanihara

Mori

and Kuramoto

, Bioactive PMMA bone cement prepared by modification with methacryloxypropyltrimethoxysilane and calcium chloride, Journal of Biomedical Materials Research Part A 67(4) (2003), 1417–1423.

13.

Fukuda

Goto

Imamura

Neo

and Nakamura

, Bone bonding ability and handling properties of a titania-polymethylmethacrylate (PMMA) composite bioactive bone cement modified with a unique PMMA powder, Acta Biomaterialia 7(10) (2011), 3595–3600.

14.

Darwish

Huang

Knoernschild

Sukotjo

Campbell

Bishal

A.K.

, Improving polymethyl methacrylate resin using a novel titanium dioxide coating, Journal of Prosthodontics: Official Journal of the American College of Prosthodontists 28(9) (2019), 1011–1017.

15.

Fini

Giavaresi

Aldini

N.N.

Torricelli

Botter

Beruto

, A bone substitute composed of polymethylmethacrylate and alpha-tricalcium phosphate: Results in terms of osteoblast function and bone tissue formation, Biomaterials 23(23) (2002), 4523–4531.

16.

Khaled

S.M.

Charpentier

P.A.

and Rizkalla

A.S.

, Physical and mechanical properties of PMMA bone cement reinforced with nano-sized titania fibers, Journal of Biomaterials Applications 25(6) (2011), 515–537.

17.

Obata

Ogasawara

and Kasuga

, Combinatorial effects of inorganic ions on adhesion and proliferation of osteoblast-like cells, J Biomed Mater Res A 107(5) (2019), 1042–1051.

18.

Shono

and van den Brink

M.R.M.

, Gut microbiota injury in allogeneic haematopoietic stem cell transplantation, Nature Reviews Cancer 18(5) (2018), 283–295.

19.

Friedrichs

Stelljes

and Schmitz

, The role of allogeneic stem cell transplantation in T-cell lymphoma, Current Opinion in Oncology 30(5) (2018), 301–307.

20.

Bensinger

W.I.

, Allogeneic transplantation: Peripheral blood vs. bone marrow, Current Opinion in Oncology 24(2) (2012), 191–196.

21.

Waasdorp

and Reynolds

M.A.

, Allogeneic bone onlay grafts for alveolar ridge augmentation: A systematic review, Int J Oral Maxillofac Implants 25(3) (2010), 525–531.

22.

Meikle

M.C.

, On the transplantation, regeneration and induction of bone: The path to bone morphogenetic proteins and other skeletal growth factors, The Surgeon: Journal of the Royal Colleges of Surgeons of Edinburgh and Ireland 5(4) (2007), 232–243.

23.

Chiu

Smith

R.L.

and Goodman

S.B.

, Polymethylmethacrylate particles inhibit osteoblastic differentiation of MC3T3-E1 osteoprogenitor cells, J Orthop Res 26(7) (2008), 932–936.

24.

Devecioğlu

Tözüm

T.F.

Sengün

and Nohutcu

R.M.

, Biomaterials in periodontal regenerative surgery: Effects of cryopreserved bone, commercially available coral, demineralized freeze-dried dentin, and cementum on periodontal ligament fibroblasts and osteoblasts, Journal of Biomaterials Applications 19(2) (2004), 107–120.

25.

Huang

Cheng

Wang

Wei

Liu

Yan

, Surface properties and MC3T3-E1 cell response of cortical bone allografts modified with low-concentration phosphoric acid, Cell Physiol Biochem 41(4) (2017), 1572–1583.

26.

Martin

J.Y.

Schwartz

Hummert

T.W.

Schraub

D.M.

Simpson

Lankford

Jr. , Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63), J Biomed Mater Res 29(3) (1995), 389–401.

27.

Han

Lee

Y.L.

Yoon

Lee

Wang

S.E.

, Regulation of osteoblasts by alkaline phosphatase in ankylosing spondylitis, International Journal of Rheumatic Diseases 22(2) (2019), 252–261.

28.

Birmingham

Niebur

G.L.

McHugh

P.E.

Shaw

Barry

F.P.

and McNamara

L.M.

, Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche, European Cells & Materials 23 (2012), 13–27.

29.

X.N.

Zhou

B.F.

Zhen

H.P.

Shi

W.G.

, Icariin induces osteoblast differentiation and mineralization without dexamethasone in vitro, Planta Medica 79(16) (2013), 1501–1508.