Preparation of Bone Marrow Mesenchymal Stem Cells Combined with

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Preparation of Bone Marrow Mesenchymal Stem Cells Combined with Hydroxyapatite/Poly (d, l-lactide) Porous Microspheres for Bone Regeneration in Calvarial Defects Qiuxia Ding, Ying Qu, Kun Shi, Xinye He, Zhengqiong Chen, Ying Yang, Xiangwei Wang, and Zhiyong Qian ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00312 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on September 1, 2018

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ACS Applied Bio Materials

Preparation of Bone Marrow Mesenchymal Stem Cells Combined with Hydroxyapatite/Poly (d, l-lactide) Porous Microspheres for Bone Regeneration in Calvarial Defects Qiuxia Ding1, Ying Qu2, Kun Shi2, Xinye He2, Zhengqiong Chen1, Ying Yang 1, Xiangwei Wang3, Zhiyong Qian2∗ 1

Department of Gynaecology and Obstetrics, Xinqiao Hospital, Third Military Medical University,

Chongqing, China. 2

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China

Medical School, Sichuan University, Chengdu, P.R. China. 3

Department of Urology, Center of Nephrology, General Hospital of Shenzhen University,

Shenzhen, China.

ABSTRACT A novel three-dimensional biomimetic porous microsphere was successfully designed in this study, which was composed of PDLLA, bone marrow mesenchymal stem cells (MSCs) and nano-hydroxyapatite (nHAp). nHAp/PDLLA/MSCs porous microspheres supposed to be a significant constituent of bone in vertebrate were prepared to act as biodegradable support materials. In addition, bone MSCs are act as seeding cells in bone defect repair. The microstructure of the obtained nHAp/PDLLA/MSCs porous microspheres was characterized. Scanning electronic microscopy showed that the composite

materials

exhibited

a

cross-linked

porous

structure.

In

vivo

biocompatibility was studied by way of implanting the nHAp/PDLLA porous microspheres in subcutaneous of rats for 4 and 8 weeks. In addition, the osteogenic capacity of the nHAp/PDLLA/MSCs porous microspheres was assessed by implanting in the 10 mm×10 mm×3 mm cranial defect of New Zealand White rabbits. In vivo studies confirmed that nHAp/PDLLA/MSCs porous microspheres had good biocompatibility and better in inducing bone regeneration than nHAp/PDLLA porous

∗

To whom should be corresponded. E-mail: [email protected] (Qian ZY). ACS Paragon Plus Environment

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microspheres and the self-healing process. All the results suggested that the nHAp/PDLLA/MSCs porous microspheres present a remarkable potential in calvarial defect repair and bone tissue engineering. KEYWORDS: Calvarial Defects; Porous Microspheres; Nano-hydroxyapatite (nHAp); Mesenchymal Stem Cells (MSCs); Bone Tissue Engineering

Introduction Bone defects regeneration remain as an unrealized challenge in contemporary medical research, along with an high incidence of morbidity and mortality particularly in patient with tumor, trauma and elderly1-2. The clinical use of autograft and allograft is limited by many disadvantages, such as insufficiency and immune rejection3-4. The new technologies of tissue engineering offered novel approaches to reconstruct the bone, which aims to develop new bone tissue substitutes from single component to multi-component biomaterials5-7. Therefore, the bone substitute with similar structure and function to bones has become a research hotspot and is urgently warranted. In order to manufacture suitable composite scaffold materials for tissue engineering applications and drug delivery, various kind of biomaterials such as hydrogels8-10, nanofibers11-13, nanoparticles14-17 and microspheres18-20, etc., have being developed to design. Three-dimensional scaffold materials with similar structure to natural extracellular matrix can provide not only support to cellular growth but also space to supply

nutrients21.

Porous

microspheres

with

good

biocompatibility,

osteoconductivity and osteoinductivity have become one of the most considered bone three-dimensional materials. In addition, as the important inorganic constituent of bone, hydroxyapatite can meet the needs of osteogenesis for the main components of calcium and phosphonium, have non-toxicity and favourable biocompatibility, so it is a recognized bioactive hard tissue implant materials in tissue engineering. Moreover, nano-hydroxyapatite has higher bioactivity and biocompatibility, outstanding mechanical property owning to its particular surface structure22-23. However, how hydroxyapatite is prepared into a three-dimensional porous configuration, which is conducive to the promotion of nutrient transport and the growth of cells, is the key ACS Paragon Plus Environment

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issue in the current bone tissue repair. In nowadays research, the seeding cells also play an important role in tissue regeneration. Bone MSCs are a promising tool for clinical therapy owing to their self-renewal characteristics, multiple differentiation potential, and easy accessibility 24-25

. Therefore, bone MSCs have been widely utilized in tissue engineering as seeding

cells which can remarkably enhance the bioactive and osteoinduction of scaffolds26-27. In Marcacci’s study, 4 patients with bone defects were treated by implanting a composite of autologous MSCs and macroporous scaffold, and the satisfactory long-term outcomes were received28. This research represented a typical protocol of the used MSCs-based therapy, which autologous MSCs are cultured in vitro, and the combination of expanded MSCs and scaffold is implanted in the defect sites in vivo. The application of seeding cells such as MSCs had dedicated significant progresses in tissue regeneration. Combination of seed cells and various 3-D biomaterials have continuously developed over the past few decades to improve the bone regeneration process 29-30. In this study, the nHAp/PDLLA porous microspheres were fabricated through the improved double emulsion solvent evaporation method reported in our previous studies 31, the microspheres combined with bone marrow MSCs through co-cultured in vitro. This nHAp/PDLLA/MSCs scaffold presented excellent biocompatibility with MSCs in vivo after transplanted into subcutaneous of rats. For further application in bone repair in vivo, the nHAp/PDLLA/MSCs composites were implanted into the complete calvarial defect model of New Zealand white rabbit to evaluate the repair effect through micro-computational tomography, Molybdenum target detection and Histological examination. All results indicated that the nHAp/PDLLA/MSCs porous microspheres scaffolds showed a potential application for calvarial defect repair in bone tissue engineering.

EXPERIMENTAL SECTION Materials and animals Poly-d,l-lactide

(Mw = 135 000)

was

purchased

from

ACS Paragon Plus Environment

Nature

Works

LLC


(Minneapolis,

MN),

nano-hydroxyapatite

(20-80nm),

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fluorescein

diacetate,

4,6-diamidino-2-phenylindole(DAPI) and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2, 4-tetrazolium bromide(MTT) were obtained from sigma, polyvinyl alcohol(PVA) was purchased from Aldrich, other chemical agents were obtained from KeLong Chemical(Chengdu ,China) and were belonged to analytical reagent grade. Female SD rats weigh 200g were purchased from Beijing HFK Bioscience (Beijing, China). Animals were housed separately at a controlled temperature of 20-22 ° C, 50-60% relative humidity and 12 hours light dark cycle. Allow free access to food and water. New Zealand White rabbits were purchased from the Experimental Animals Center of Sichuan Province (China). The rabbits were housed at controlled temperature of 20-22oC, relative humidity of 40-70% and fed with a standard laboratory diet. The studies were operated based on the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory. Animals and all operates were in accordance with the Animal Care and Use Committee of West China Hospital, Sichuan University.

Preparation and biomineralization of porous microspheres The fabricated of nHAp/PDLLA porous microspheres was followed the improved double emulsion solvent evaporation method. The 5% NH4HCO3 aqueous solution (6mL) was added into 6.25% nHAp/PDLLA dichloromethane solution (10% nHAp), the mixture was then stirred with T10 basic homogenizer at ice-water bath at the speed of 5000 rpm or 6000 rpm. The emulsion was slowly poured into 0.1% PVA aqueous solution and stirred overnight at room temperature with 500 rpm, then obtained nHAp/PDLLA porous microspheres. The microspheres with different size were filtrated by sieves. The nHAp/PDLLA microspheres were soaked into NaOH aqueous solution and stirred for 15-20 minutes, the microspheres after hydrolyzing were washed with ultrapure water in order to remove the remnant NaOH aqueous solution. At last the nano-hydroxyapatite/PDLLA porous microspheres were freeze-dried and then stored in air-tight bag before use. The biomineralization of porous microspheres were finished as follow. First, the ACS Paragon Plus Environment

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prepared of the mineralized solution was referred the this studies32-33, the PDLLA microspheres then added into excessive mineralized solution at 37oC and the solution changed every three days. The microspheres were taken out at different time, and washed with ultra-pure water to get rid of the remnant mineralized solution.

Surface morphplogy analysis of porous microspheres The surface morphology of the nHAp/PDLLA porous microspheres and nano-HAP/PDLLA/MSCs porous microspheres were observed by an electron scanning microscopy (SEM, JEM-100CX, Japan).

Element analysis of porous microspheres The element component of the HAP/PDLLA porous microspheres were analyzed by the energy dispersed spectroscopy (EscaLab220-IXL).

Isolation and culture of bone marrow mesanchymal stem cells and Cell Viability Assay The protocol of rabbit bone marrow mesanchymal stem cells isolation and culture was referred the Ni P.’s study34. After obtaining primary bone MSCs, the culture medium was changed every 3-4 days, and cells close to fusion after about 1 week. In this study, the third generation of MSCs was used for research in vitro and in vivo. After 2 weeks of initiating culture, the bone MSCs were washed and the cells lifted by incubation in 0.5ml of 0.25% trypsin/1 mM ethylenediaminete-traacetic acid for 2min at room temperature. Alizarin reds (ARS) and Alkaline phosphatase staining was used to identify the mineralization of bone MSCs. The cells were co-cultured with nHAp/PDLLA porous microspheres a density of 3×106 cells per well in 6-well plates and grown for different time points. Then, viability of cells only on nHAp/PDLLA porous microspheres was measured using the MTT method. Briefly, the mean percentage of cells survival was estimated from data of six individual experiments, and all data were expressed as the mean±S.D.

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Hemolytictest on nHAp/PDLLA porous microspheres In Vitro The hemolysis rate of HAP/PDLLA porous microspheres was evaluated in vitro according to the method reported by Wang C.

35

. In this study, 2.5 mL of

nHAp/PDLLA porous microspheres (10, 20, 40, 60, 80 mg/mL of nHAp/PDLLA porous microspheres) in normal saline were used to test.

Biocompatibility evaluation of porous microspheres in vitro and vivo The biocompatibility evaluation of porous microspheres was detected through co-cultured with MSCs and implanted into the subcutaneous of animals. The activity of cells on purous microspheres was observed by staining the cells though DAPI-FDA. At first, the sterilized microspheres were put into a six-well cell culture plate and a total of 3×106/ml bone MSCs were seeded on each well. The plate was taken out of the incubator for different time intervals. Then the cells were stained with DAPI and FDA. The fluorescence photographs were taken to analysis the activity of cells. In vivo biocompatibility of the nHAp/PDLLA porous microspheres was studied through the microspheres were implanted into the subcutaneous of the leg of SD rats. After 4 and 8 weeks, opened the surgery sites carefully and harvested the implants together with surrounding tissue (also contain cardiac muscle, liver, spleen, lung, and kidneys) for histological examination. The samples were stained by Heamatoxylin and Eosin (H&E).

Calvarial defect repair As shown in figure 7(A, B), calvarial defect was created by making two circular full-thickness defects (diameter: 10mm×10mm×3mm) in the cranium of New Zealand white rabbits. The defects of rabbits were treated with nHAp/PDLLA porous and scaffolds which were composed of nHAp/PDLLA porous microspheres combined with bone marrow mesanchymal stem cells (MSCs) (n=6), and the controlled group without any treatment. Skulls were collected at 6weeks, 12 weeks and 20 weeks after the operation and conducted for further examination. ACS Paragon Plus Environment

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Molybdenum target detection The samples were analyzed by using Mammary gland molybdenum target (Siemens, Germany), the setting were 25kV and 36mAS. The gray value represented bone mineral density of skull which could be used to analyze the effect of calvarial defect repair.

Micro-computational tomography (Micro-CT) As shown in figure 7(C-F), the samples taken at different times intervals were analyzed through Micro-CT detector (Y.Cheetah, YXLON International GmbH, Germany). The scan voltage and current were 55 kV and 90µA, the reconstruction area of bone: a circle with a radius of 5cm, and voxel resolution=20µm. The scans were then reconstructed to create 3D geometry using VG Studio Max. Three samples were analyzed at each time-point of harvest.

Histological examination of repair tissue The 10% neutral buffered formalin was used to fix sample and then 10% EDTA , PH=7.0 was used as decalcifying solution for 8 weeks. The bone defect repair samples were histological analyzed by H&E staining.

RESULTS AND DISCUSSION Surface morphology and element analysis of nHAp/PDLLA porous microspheres The surface morphology of nHAp/PDLLA porous microspheres at different synthesis condition was investigated by SEM. As shown in figure 1 a-g, the sizes of surface pore and inside pore in microspheres synthesized at 5000rpm stirring rate (A, C, E) were bigger compared with the microspheres at 6000rpm (B, D, F). Rapidly solidification of polymers in the interface of water and oil phase was the reason of a polymer film formed on the surface of prepared microspheres. We could remove the film by hydrolization in order to get microspheres with favourable pore structure. When treated with 0.1 M NaOH solution, the PDLLA polymer film was completely ACS Paragon Plus Environment


hydrolyzed and formed uniform connecting structure as we could see in figure 1(E, F). The width of hydroxyapatite scaffold was thicker after biomineralization as shown in figure 1(G). We could also get from figure 1 that the size of microspheres was between 200µm to 500µm, the width of scaffold was between 700nm to 1500nm and increased to 2um after biomineralization. The elementary compositon of hydroxyapatite on porous microspheres was measured by EDS. As shown in S1, the PDLLA porous microsphere (A) was composed of carbon and oxygen and nHAp/PDLLA porous microsphere (B) was made up of carbon, oxygen, phosphorus and calcium. When immerged in simulated body fluid (this was used to simulate the growth of mineral in natural skeleton), the content of calcium increased with the extra long steeping. The calcium content of PDLLA microsphere (A), nHAp/PDLLA microsphere (B) and nHAp/PDLLA microsphere which was immersed in simulated body fluid for 4 days (C) and 8days (D) were 0, 2.4%, 2.94% and 3.36% respectively. The result indicated that hydroxyapatite was embedded into porous microsphere and formed on the surface of porous microsphere. Therefore, the nHAp/PDLLA porous microspheres have bone osteoinductive functions and were promising in bone repair. From the results we could demonstrated that PDLLA porous microsphere scaffolds were successfully prepared and hydroxyapatite was embedded in PDLLA three-dimensional porous microspheres. This bone substitute materials should have excellent biocompatibility in vitro and vivo due to hydroxyapatite is covered on the surface and inside of the microspheres. Meanwhile, due to the three-dimensional and multi-space structure, the cells can gain space and scaffolds for adhesion growth, and nutrients can be effectively transported.

The extraction and activity analysis of bone MSCs cells The MSCs cell morphology was shown in figure 2A. The formation of minerazied node was measured to estimate the activity of bone MSCs by ALP and alizarin red staining. As shown in figure 2B-E, some minerazied node which were blue (B) and red (D) stained by ALP and alizarin red respectively were formed when the cells were ACS Paragon Plus Environment

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incubated for 14d after osteogenic induction, and the naive cells barely appeared blue (C) and red (E) node. Therefore, the extraction of bone MSCs is successful and had the osteogenic capability after osteogenic induction that meant it could be used as seeding cells in bone repair as other studies36-37.

Cell compatibility and hemolytic test of nHAp/PDLLA porous microspheres in vitro. A statistically increase of cell viability of MSCs cultured on the nHAp/PDLLA porous microspheres scaffolds was performed (figure 2F). The cells on the nHAp/PDLLA scaffolds proliferated faster than the pure PDLLA scaffolds. The higher cell viability for the nHAp/PDLLA porous microspheres displayed that the nHAp/PDLLA scaffolds can accelerate the attachment of the MSCs and promoted cells to proliferation on the composite, which is significant for the use of 3-D biomaterial scaffolds. FDA staining and DAPI staining were all used to observe the adhension and growth of cells cultured on PDLLA porous microsphere (figure 3A, B, E, F) and biomineralized nHAp/PDLLA porous microsphere ( figure 3C, D, G, H). We could see from the fluorescence micrographs that bone marrow MSCs were able to grow on the two microspheres, and more cells were on nHAp/PDLLA microspheres compared with PDLLA microspheres. This staining experiment indicated that the introduction of hydroxyapatite into PDLLA microspheres would promote cellular adhesion and growth. As shown in figure 4A-D, the MSCs interaction with the nHAp/PDLLA porous microsphere scaffolds was studied by SEM. The MSCs not only could adhere on the surface of the nHAp/PDLLA porous microspheres scaffolds, but also growth into the interior of the nHAp/PDLLA porous microspheres. Its morphology can be spherical or elongated along the scaffolds. Meanwhile, the seeded cells on the pure PDLLA scaffold were less, and adhered to a small area of the materials. The infiltration of cells into the interior part has significantly meaningful for the cell compatibility evaluation and very significant for application in vivo. ACS Paragon Plus Environment


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The results of hemolytic test shown in figure 5, the nHAp/PDLLA porous microsphere scaffolds at a high concentration did not cause any hemolysis. The results implied that nHAp/PDLLA porous microspheres scaffolds was safe enough for implanted or administration.

The histocompatibility compatibility evaluation of nHAp/PDLLA porous microspheres in vivo For served as an application in calvarial defect repair, bone repair material should have remarkable histocompatibility in vivo. Therefore, nHAp/PDLLA porous microspheres were injected subcutaneously of rat. Histocompatibility of microspheres was evaluated by H&E staining and Masson staining in figure 6. Some inflammatory cells were appeared after 4 weeks (A, B), revealing the normal stress response of organism to foreign matter. And only some slight inflammatory reaction could be seen at 8 weeks (C, D). At the same time, cells gradually grew in the surface and inside of microspheres as the nHAp/PDLLA porous microspheres degradation. Figure 6 E-H was the masson staining result and was used as a supplement to histocompability of microspheres. Those results obviously revealed that nHAp/PDLLA porous microspheres were safe as a biomaterial and had potential ability as support material in tissue engineering. Figures 6(a-j) is the result of the organ histocompatibility treated with nHAp/PDLLA porous microspheres and negative control, respectively. The tissue cells from cardiac muscle, liver, spleen, lung, and kidneys were clear and arranged in good order, and no necrosis, hemorrhage and inflammatory exudates were observed. Bone substitute materials should have excellent biocompatibility because need to be transplanted into the injured part. So the biocompatibility of nHAp/PDLLA porous microspheres in vivo and vitro were evaluated by transplanting the nHAp/PDLLA porous

microspheres

into

the

subcutaneously

of

rat

and

measured

the

histomorphology of surrounding tissue response to the nHAp/PDLLA porous microspheres and cell compatibility. We could see from figure 6 that some inflammatory response occurred 4 weeks after transplanted, and there were no ACS Paragon Plus Environment

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hematoma or purulence around the transplantation site. Besides, cells from rat gradually grew in the surface and inside of microspheres with the gradually degradation of microspheres, and tissue were newly formed in the site where microspheres existed originally. These amazing results revealed that our porous microspheres had favorable biocompatibility and promoted the regeneration of tissue.

X-ray, Molybdenum target, Micro-CT and Histological evaluation of calvarial defect regeneration in vivo Finally, the initial proof of principle for bone regeneration in a rabbit calvarial defect model in situ was demonstrated with X-ray, Molybdenum target, Micro-CT and histological analysis. Figure 8 showed the radiographic images of defect site in the cranium of New Zealand white rabbits at 6, 12, 20 weeks after surgery, and the degree of newly formed bone was evaluated through the grey-scale displayed by the imaging system. The dark grey-scale stand for host bone, and the pale grey-scale is little regeneration bone area. At 6 weeks (A, B, C), the grey-scale degree of cranial defects in nHAp/PDLLA/MSCs group (A) was higher than nHAp/PDLLA group (B), and control group (C) was the lowest, which demonstrate nHAp/PDLLA/MSCs composite had favourable bone regeneration effect at 6 weeks. At 12 weeks after surgery (D, E, F), the degree of grey-scale visible higher in the central defect area especially when treated with nHAp/PDLLA/MSCs composites (D), and the conformation of new bone was mainly in the periphery of defect area and lower increase in the center area than nHAp/PDLLA/MSCs group when treated with nHAp/PDLLA composites (E) and control group (F). The defect treated with nHAp/PDLLA/MSCs composites (G) had been completely covered with new bone when up to 20 weeks post-surgery, but there was still some defect in the other two groups (H, I). The results obtained by molybdenum target detection (figure 9A-I) also verified that nHAp/PDLLA/MSCs composite had remarkable bone regeneration effect. Micro-computational tomography was a substitutive approach for imagine bone in three dimensions38, and it consisted of a scout view, inspection volume selection, ACS Paragon Plus Environment


automatic positioning and measurement, offline reconstruction and evaluation39-40. The Micro-CT of defect site in the cranium of rabbits after surgery for 6, 12, 20 weeks was displayed and the data were exhibited in figure 10. At 6 weeks, the new bone were primary formed in the defects boundary in nHAp/PDLLA group (B) and control group (C), however, there were new bone formed both in the boundary and center of defects when treated with nHAp/PDLLA/MSCs composites (A). The defect sizes obviously diminished and the density of the new formed bone increased obviously in nHAp/PDLLA/MSCs group (D) compared with nHAp/PDLLA group (E) and control group (F) at 12 weeks after surgery. Up to 20 weeks after operation, the nHAp/PDLLA/MSCs group (G) had covered all the defected area, and the density of the new formed bone was uniform, yet the nHAp/PDLLA group (H) and control group (I) had some evident defect. The results obtained from Micro-CT were in consistence with X-R examination. All in all, the higher density and larger area of new bone and successful reconstruct of the defects demonstrated that the nHAp/PDLLA/MSCs scaffold materials presented excellent ability of osteoinduction. To further confirm the above conclusions, the decalcified samples were stained with H&E with the results shown in figure 11. At 6 weeks, the defect sites were filled with new immature osteons at the boundary part. The fibro-vascular tissues were gradually formed from the boundary to the center of defect site in the three groups (A, B, C), no obvious inflammatory response appeared between the microspheres and bone. At 12 weeks, in the defect area there are considerable amount of newly bone, some of which developed into mature cortical bone and part of them coalesced well with the host bone in nHAp/PDLLA/MSCs group (D). Meanwhile, the bone mineral density was much higher at the defect regions compared with the other two groups. We discovered that the nHAp/PDLLA/MSCs porous microspheres were co-existed with the newly formed bone both in nHAp/PDLLA/MSCs group (D) and nHAp/PDLLA group (E) that revealed the favorable biocompatibility of microspheres and could be part of the new bone. At 20 weeks, the defect region in nHAp/PDLLA/MSCs group (G) was completely covered with mature cortical bone which in agree with the Micro-CT results. Besides, microspheres were partly ACS Paragon Plus Environment

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degraded as a support material and gradually intruded by bone tissue. Some defect region still existed in nHAp/PDLLA group (H) and control group (I), and their width and density of new bone regenerated were all lower than nHAp/PDLLA/MSCs group (G). The percentage of newly formed bone area was calculated base on the H&E analysis, and the results were presented in figure 12. The amount of new bone in the nHAp/PDLLA/MSCs group was obviously higher than the other groups at 20 weeks. (nHAp/PDLLA/MSCs 64.2%, P

Preparation of Bone Marrow Mesenchymal Stem Cells Combined with

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