Smurf1‐targeting microRNA‐136‐5p‐modified bone marrow mesenchymal stem cells combined with 3D‐printed β‐tricalcium phosphate scaffolds strengthen osteogenic activity and alleviate bone defects

Suitable biomaterials with seed cells have promising potential to repair bone defects. However, bone marrow mesenchymal stem cells (BMSCs), one of the most common seed cells used in tissue engineering, cannot differentiate efficiently and accurately into functional osteoblasts. In view of this, a new tissue engineering technique combined with BMSCs and scaffolds is a major task for bone defect repair. Lentiviruses interfering with miR‐136‐5p or Smurf1 expression were transfected into BMSCs. The effects of miR‐136‐5p or Smurf1 on the osteogenic differentiation (OD) of BMSCs were evaluated by measuring alkaline phosphatase activity and calcium deposition. Then, the targeting relationship between miR‐136‐5p and Smurf1 was verified by bioinformatics website analysis and dual luciferase reporter assay. Then, a rabbit femoral condyle bone defect model was established. miR‐136‐5p/BMSCs/β‐TCP scaffold was implanted into the defect, and the repair of the bone defect was detected by Micro‐CT and HE staining. Elevating miR‐136‐5p‐3p or suppressing Smurf1 could stimulate OD of BMSCs. miR‐136‐5p negatively regulated Smurf1 expression. Overexpressing Smurf1 reduced the promoting effect of miR‐136‐5p on the OD of BMSCs. miR‐136‐5p/BMSCs/β‐TCP could strengthen bone density in the defected area and accelerate bone repair. SmurF1‐targeting miR‐136‐5p‐modified BMSCs combined with 3D‐printed β‐TCP scaffolds can strengthen osteogenic activity and alleviate bone defects.

as well as patients with initial joint replacement and revision, which may cause serious damage to the normal musculoskeletal system and psychology of the human body, and usually requires surgical bone grafting to repair the lesions. 1,2However, due to the shortcomings of clinical transplantation materials, the repair of bone defects presents a complex process, so the reconstruction of bone shape and function is still faced with great challenges, especially the repair of large-volume bone defects, which is the difficulty of clinical treatment at present.Therefore, the search for the repair method for bone defects has been the subject of continuous in-depth research.
3D bioprinting technique can directly, accurately, and quickly design and produce personalized materials with high precision, complex structure, and certain biological activity by using appropriate bioprinters and materials under the control of computer software and preset data. 3,4β-c (β-TCP) has a chemical structure similar to natural bone 5 and is the most promising biological material due to its bone transduction, immune response deficiencies, and unlimited availability. 6,7However, β-TCP faces a challenge in being applied in bone defect repair because of the lack of osteogenic induction properties.Tissue engineering is a perspective method to repair bone defects by a combination of biomaterial science, cell biology, and biomedical engineering. 8,9Significantly, modifying osteogenic genes in bone marrow mesenchymal stem cells (BMSCs) has become a promising therapeutic approach.Currently, functional gene molecules such as DNA plasmid, 10,11 siRNA, 12 and miRNA 13 have been employed to bone tissue engineering.In contrast to pDNA method, 14 ncRNAs can modify gene expression when delivered to the cytoplasm.Furthermore, ncRNAs as a bioactive factor in bone tissue engineering have become a new star in bone defect repair. 15iquitination can translate and modify proteins as well as degrade endogenous proteins. 16As the most concerned ubiquitin enzyme in bone biology, 17 Smurf1 is a key mediator in bone formation.For example, Smurf1 mediates Runx2 degradation and inhibits osteoblast differentiation and bone formation. 18Inhibition of Smurf1 expression can stimulate osteogenic differentiation (OD) of MSCs and bone formation. 19Smurf1 has also been considered to mediate Smad1/5 degradation, thus inhibiting osteoblast differentiation. 20,21is study found that miR-136-5p may be a miRNA regulating Smurf1 through the bioinformatics website starBase.Therefore, this study aims to clarify the mechanism by which miR-136-5p inhibits Smurf1 expression, providing a bone tissue engineering method through miR-136-5p-modified BMSCs with 3D printed β-TCP scaffolds.

| Preparation of 3D printed β-TCP scaffolds
Commercial β-TCP scaffolds (average particle size: 550 nm) were purchased from Berkeley Advanced Biomaterials (CA, USA).The scaffolds (7 mm in diameter and 10 mm in height) were developed in a CAD file and fed into a 3D printer (ProMetal, USA).The 500 μm connected holes were square and distributed in X, Y, and Z directions.In addition to the optimized layer thickness of 20 μm, 3D printing process parameters such as drying time, binder drop volume, and saturation may be optimized. 22The scaffolds were treated by a blast blower, solidified at 175 C for 1.5 h, sintered at 1250 C by a muffle furnace for 2 h, and cooled to room temperature.

| Characteristics of β-TCP scaffolds
The compression strength of the β-TCP scaffolds was evaluated using a universal testing machine (AG-IS, Shimadzu, Japan) with a constant crosshead velocity of 0.33 mm/min.The compression strength is calculated from the maximum bearing load and the size of the sintered support.The scaffolds were observed by electric field emission scanning electron microscope (FEI Inc., OR, USA).The porosity of the scaffolds was calculated using the ethanol replacement method, 23 according to the following formula: Porosity (%) = (V1 À V3)/ (V2 À V3) Â 100%, where V1 is the total volume after adding absolute ethanol to the graduated cylinder and immersing the scaffold sample in it.V2 is the total volume recorded after the air in the pores of the material was evacuated by a vacuum desiccator, and the pores were filled with ethanol.V3 is the total volume of the material after the graduated cylinder was removed.

| Induction of osteoblast differentiation
BMSCs were plated into 6-well plates (1 Â 10 5 cells/well) and cultured in an osteoblast-specific induction medium containing 0.05 mM ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone (Cyagen, China) to induce OD.The medium was changed every 2 days for 14 days.The cells were then collected and analyzed on a specified day.

| RT-qPCR
TRIzol reagent (Invitrogen; Thermo Fisher Scientific) was employed for total RNA extraction.cDNA was obtained through reverse transcription from 2 μg RNA using a Biochemical kit (Takara, Japan), amplified using SYBR GREEN PCR Master mix (Takara), and analyzed in the ABI7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific).Data were analyzed by 2 ÀΔΔCt .miR-136-5p, and Smurf1 levels were normalized to U6 and GAPDH, respectively.The cycle conditions of qPCR were: predenaturation at 95 C for 10 min, denaturation at 95 C for 10 s, annealing at 58 C for 20 s, and extension at 72 C for 10 s, 40 cycles in total.Table 1 presents the primer sequences.

| Western blot
Total proteins were extracted by radioimmunoprecipitation test buffer (Sigma-Aldrich) containing a protease inhibitor (Thermo Fisher Scientific) and quantified using the protein detection kit (Qcbio Science Technologies, Shanghai, China).The total protein (30 μg) was separated by 10% SDS-PAGE electrophoresis and transferred to a PVFD (Millipore, USA) membrane, which was then sealed with 5% skim milk and mixed with primary antibodies Smurf1 (ab236081; Abcam), Runx2 (ab76956; Abcam), OCN (ab13420; Abcam) or GAPDH (1:2000; Santa Cruz Biotechnique) overnight at 4 C.The membrane was cleaned and incubated with a secondary antibody (1:5000) coupled with horseradish peroxidase for 2 h.ECL assay was performed using the ECL substrate kit (Thermo Fisher Scientific).

| Alkaline phosphatase activity determination
Seven days after osteogenic induction, cells were fixed with 4% paraformaldehyde for 30 min and washed with PBS three times.Alkaline phosphatase (ALP) staining was performed using the ALP kit (Beyotime, China) and observed under an optical microscope (Olympus, Japan).

| Calcium deposition determination
After 14 days of osteogenic induction, cells were stained with 0.2% Alizarin Red S staining solution (Sigma-Aldrich; Merck KGaA), rinsed with PBS, and observed under an optical microscope (Olympus).

| Formation of BMSCs/β-TCP scaffolds
The average pore size of β-TCP scaffolds is 360 μm ± 40 μm, with a porosity of 65%.BMSCs (1.0 Â 10 7 cells/mL) containing lentivirus was established on the femoral condyle using dental burr.The scaffolds were then implanted into the bone defect area.Finally, the muscle and skin was repaired in layers.After surgery, all rabbits received intramuscular injections of antibiotics for 3 days.The rabbits were kept freely in cages and fed and watered according to traditional eating habits.Animals were euthanized 3 months after implantation.

| Micro-CT
The

| HE staining
After Micro-CT examination, the femur tissue was thoroughly cleaned with saline, then fixed in 4% paraformaldehyde, followed by decalcification in 10% EDTA (PH 7.0) for 5 weeks.After dehydration and complete decalcification, the sample was embedded in paraffin wax and prepared into 5 μm vertical continuous sections.HE staining was then conducted, and images were observed by an optical microscope (Olympus).

| Statistical analysis
All data were expressed as mean ± standard deviation and compared by unpaired Student's t test.GraphPad Prism software (version 8.1.2) was employed for all statistical analyses, with a statistical significance of p < 0.05.

| Characterization of a 3D printed β-TCP scaffold
The 3D-printed β-TCP scaffolds were made from CAD files using a 3D printer, and were designed to have porous channels from the top to the sides of each scaffold, forming a lattice network through interconnected pores.The 3D printed β-TCP scaffolds were cylindrical, with an average diameter of 7 mm and an average height of 10 mm, white in color, with a rough surface and uniform texture (Figure 1A).
The microstructure of the PCL/β-TCP/CS composite scaffold is shown in Figure 1B.The β-TCP scaffolds were filled with interlocking pore structures inside, the pores were interconnected, and the average diameter of the micropores was 360 μm ± 40 μm.In addition, the porosity of β-TCP scaffold was 65%, the density was 0.87 ± 0.03 g/cm 3 , and the compression strength was 5.48 ± 0.04 MPa.

| Characteristics of rabbit BMSCs
Rabbit BMSCs were rounded at passage 1 and fusiform at passage 3 (Figure 2A).BMSC surface markers detected by flow cytometry showed positive expressions of CD29 and CD44, while negative expressions of CD34 (Figure 2B).It was proved that the cultured cells were rabbit BMSCs, which could be used for follow-up experiments.
starBase found that there was a targeted binding site between miR-136-5p and Smurf1 (Figure 4C), and dual luciferase reporter assay results showed that after co-transfection of Smurf1-WT and miR-136-5p, the luciferase activity significantly decreased (Figure 4D).Micro-CT analysis of femur samples showed that no significant new bone formation was observed in the Control group and the β-TCP group, but new bone formation was observed in the BMSCs/β-TCP group, miR-NC/BMSCs/β-TCP group, and miR-136-5p/BMSCs/β-TCP group.miR-136-5p/BMSCs/β-TCP group completed bone defect repair (Figure 6A).It should be considered that the scaffold material was mostly degraded after 3 months so it cannot be shown by micro-CT scans.This phenomenon suggests that miR-136-5p-modified BMSCs combined with β-TCP scaffolds accelerate bone defect repair.Quantitative analysis showed that BMD, BV/TV, TbTh, and TbN levels in miR-136-5p/BMSCs/β-TCP group were highest (Figure 6B-E).To confirm the Micro-CT findings, HE staining was performed on the femur tissue sections.miR-136-5p/BMSCs/β-TCP group had repaired most of the bone defect areas, but in other groups, there were a large number of unrepaired areas, and repair degree was relatively low (Figure 6F).conductivity, and customizable biodegradability. 28,29However, β-TCP is limited by its inherent osteogenic deficiency. 30,31The three main elements of tissue engineering are seed cells, scaffold materials, and growth factors.Finding suitable seed cells is an important factor in tissue engineering.BMSCs are stem cells with multi-differentiation potential in bone marrow, and can differentiate into various cell lines under specific induction conditions. 24BMSCs have become ideal seed cells for tissue engineering, cell engineering, and gene engineering due to their advantages such as convenient sampling, easy culture and amplification, and weak immunogenicity. 32Tissue engineering methods using BMSCs have attracted people's attention in the reconstruction of large bone defects. 33Several studies have demonstrated the osteogenic ability of implants prepared with BMSCs to achieve bone defect regeneration. 34,35The results showed that BMSCs combined with β-TCP scaffolds implanted in the defect area of rabbit femur strengthened bone density in the defect area and accelerated bone defect repair.
newly formed bone around the scaffold was evaluated based on GE eXplore Locus SP Micro-CT.The bones were scanned by GEHC MicroView software (GE Healthcare BioSciences, UK) at 80 kV and 80 μA, with exposure time of 3000 ms and resolution of 15 μm.Region of interest (ROI) was selected according to the anatomical location of the bone defect.First, the CT value of the new bone tissue was used to identify and select the new bone tissue in the ROI.Then, the scaffold in the ROI was identified and selected by using the CT value of the scaffold.By removing the selected portion of the scaffold from the selected portion of the newly formed bone tissue, the actual portion of the newly formed bone tissue in the scaffold could be obtained.Bone mineral density (BMD), trabecular thickness (TbTh), bone volume fraction (bone volume/total volume [BV/TV]), and trabecular number (TbN) were calculated.
osteoblast maturation.At the same time, alizarin red staining was used to detect calcium deposition on the cell surface to verify the successful differentiation of BMSCs into osteoblasts.ALP and alizarin red staining results showed that ALP activity and calcium deposition were increased after overexpressing miR-136-5p or reducing Smurf1 (Figure3F,G).Osteogenic genes Runx2 and OCN were detected, and results manifested that overexpressing miR-136-5p or silencing Smurf1 significantly promoted their expression levels (Figure3H,I).

F I G U R E 1
Characterization of 3D printed β-TCP scaffold.(A) The mean diameter and height of the β-TCP scaffold.(B) SEM images of the surface topography of β-TCP scaffold.F I G U R E 2 Characteristics of rabbit bone marrow mesenchymal stem cells (BMSCs).(A) The morphology of rabbit BMSCs.(B) Flow cytometry analysis of cell surface markers.

3. 6 |
miR-136-5p/BMSCs/β-TCP can strengthen bone density and accelerate bone defect repair A rabbit femur defect model was established.BMSCs containing lentivirus transduction of miR-NC or miR-136-5p were loaded onto β-TCP scaffolds, and then implanted into the femoral defect area of rabbit.

3. 7 |
miR-136-5p-modified BMSCs targeting Smurf1 combined with β-TCP strengthen bone density in the defect area and accelerate bone defect repairThe bone defect repair effect of miR-136-5p + oe-Smurf1/BMSCs/ β-TCP group was significantly lower than that of miR-136-5p + oe-NC/BMSCs/β-TCP group (Figure7A-F).This phenomenon suggests that miR-136-5p/BMSCs can accelerate bone repair by targeting Smurf1.F I G U R E 3 Up-regulating miR-136-5p or down-regulating Smurf1 can stimulate OD of bone marrow mesenchymal stem cells (BMSCs).(A and B) RT-qPCR tested miR-136-5p and Smurf1 during OD of BMSCs.(C-E) RT-qPCR or Western blot verified successful transfection.(F) ALP staining, arrows indicate ALP-positively stained cells.(G) Alizarin red staining, arrows indicate alizarin red positively stained cells.(H and I) RT-qPCR and Western blot analysis of Runx2 and OCN.*p < 0.05 versus miR-NC/BMSCs; # p < 0.05 versus sh-NC/BMSCs.According to the type and severity of bone defect, the repair methods vary greatly.Autologous bone is widely recognized as the best replacement material in bone transplantation, with the effects of bone induction, conduction, and genesis.After transplantation, the activities of osteoblasts can be maintained to a large extent.Meanwhile, the bone matrix contains a variety of bioactive substances, which can accelerate the differentiation of osteoblasts, and rapidly integrate with the surrounding bone tissue for growth.25However, it is limited byF I G U R E 4 Smurf1expression is negatively regulated by targeting miR-136-5p.(A and B) RT-qPCR and Western blot analysis of Smurf1 after up-regulation of miR-136-5p.(C) Bioinformatics website starBase found the presence of targeted binding sites between miR-136-5p and Smurf1.(D) Dual luciferase reporter assay to verify the targeting relationship between miR-136-5p and Smurf1.F I G U R E 5 Promoting Smurf1 expression can weaken the effect of miR-136-5p up-regulation on OD of bone marrow mesenchymal stem cells.(A and B) RT-qPCR and Western blot analysis of Smurf1.(C) ALP staining, arrows indicate ALP-positively stained cells.(D) Alizarin red staining, arrows indicate alizarin red positively stained cells.(E and F) RT-qPCR and Western blot analysis of Runx2 and OCN.*p < 0.05 versus miR-136-5p + oe-NC.insufficientfactors such as limited supply, extended trauma, increased secondary operation, and spread of infection.26The clinical application of allograft bone is also limited due to the possibility of immune rejection and disease transmission, as well as economic and ethical reasons.1 Therefore, it is necessary to seek a new method of bone defect repair.Materials that mimic the functional and structural features of natural tissues remain a challenge.Over the past few decades, bioceramics have emerged as viable candidates for orthopedic, maxillofacial, and dental applications.27Calcium phosphate materials, especially β-TCP, have been widely used in non-low-load orthopedic implants due to their similar composition to natural bone, inherent bone F I G U R E 6 miR-136-5p/BMSCs/β-TCP can strengthen bone density in the defect area and accelerate bone defect repair.(A) Micro-CT analysis of new bone formation.(B-E) Quantitative analysis of BMD, BV/TV, TbTh, and TbN levels.(F) HE staining, arrows indicate new bone formation.*p < 0.05 versus β-TCP; # p < 0.05 versus miR-NC/BMSCs/β-TCP.F I G U R E 7 miR-136-5p/BMSCs/β-TCP can strengthen bone density in the defect area and accelerate bone defect repair.(A) Micro-CT analysis of new bone formation.(B-E) Quantitative analysis of BMD, BV/TV, TbTh, and TbN levels.(F) HE staining, arrows indicate new bone formation.*p < 0.05 versus miR-136-5p + oe-NC/BMSCs/β-TCP.

Finally, we demonstrated
the repair effect of miR-136-5pmodified BMSCs combined with β-TCP scaffolds in bone defects.The rabbit femoral defect model was established, showing that the miR-136-5p/BMSCs/β-TCP composite scaffold strengthened the bone density in the defect area and induced bone defect repair.5 | CONCLUSION In summary, (1) miR-136-5p positively regulates OD of BMSCs based on negatively regulating Smurf1.(2) miR-136-5p-modified BMSCs combined with β-TCP scaffolds can significantly strengthen bone density and accelerate bone defect repair.(3) A novel miR-136-5p/ Smurf1 pathway may contribute to OD of BMSCs, thus providing new and deeper insights into the regulatory mechanisms of miR-136-5p on OD. (4) Tissue engineering scaffolds combined with miR-136-5p-modified BMSCs and β-TCP scaffolds are a promising and effective strategy for repairing bone defects.