| BackgroundCalcium phosphate cement (CPC) has been developed as an alternative bone substitute material because of its good biocompatibility, excellent osteo-conductibility and easy shaping in complicated geometries. However, the drawbacks of CPC, including its inferior mechanical strength, poor injectability and lack of macroporosity for bone ingrowth, have restricted its clinical applications. The macroporous (average diameter larger than 100 urn) cements can facilitate the ingrowth of new bone, which is able to enhance the mechanical strength in CPC. However, there is still a general conflict between macroporosity and mechanical strength in CPC at early stage. Therefore, it is very important to improve the mechanical strength in CPC before new bone ingrowth.The objective of the present study was to develop an injectable CPC containing poly(lactide-co-glycolide) (PLGA) microspheres. The biodegradable PLGA microspheres were used to impart in situ macroporosity to the cement. The novel cement can offer optimal strength at early stage and macroporosity for bone ingrowth after microspheres degradation. In addition, Doxorubicin microspheres, instead of control microspheres, will be incorporated with CPC to obtain a drug delivery system. Further investigation will be made on the osteogenesis andantitumor effect of the composite.ObjectiveThe aim of the present study is to investigate the general properties, osteogenesis and antitumor effect of the novel CPC containing Doxorubicin PLGA microspheres. The ultimate purpose of the present study is to discuss the possibility of clinical application of the novel cement.Methods1 Biocompatibility of the cement incorporated with PLGA microspheres (1) Preparation of CPC powder: The CPC powder used in this study was prepared by mixing partially crystallized calcium phosphate (PCCP) and dicalcium phosphate anhydrous (DCPA) at a mass ratio of 1:1. (2) Preparation of PLGA microspheres: PLGA microspheres were made by a solvent evaporation method. (3) Preparation of control cement samples: sodium citrate solution was Sodium citrate solutions with concentrations of 15 wt.% were used as the liquid phase in this study. Then the CPC powder was homogeneously mixed with sodium citrate solution at a liquid to CPC ratio of 0.4 ml g-1. The cements with a pillar shape were made in a special mould.(4) Preparation of cement samples with PLGA microspheres: The PLGA microspheres were uniformly mixed with CPC powder at a PLGA to CPC weight ratio of 30/70. The cement samples incorporated with PLGA microspheres were thus made.(5) Grouping methods: Based on the difference of materials, 3 groups determined as Control Group, CPC Group and CPC/M Group were used for cytocompatibility study, and the latter two groups were divided into three subgroups respectively by different concentration or dose for in vivo biocompatibility.(6) Preparation for extracts: Samples were weighed and sterilized, then immersed into cell culture fluid at a weight volume ratio of 1g:10ml. Leaching liquid was obtained after the samples being immersed for 72h at 37℃, with 5 vol% CO2.(7) Cell proliferation curve: Human bone marrow stem cells (hBMSCs) suspension was seeded in 96 hole plate at a volume of 200μL per hole, cells then were incubated for 24 hours at 37℃in a incubator with 5 vol% CO2 . Cell culture fluid was poured, instead, isovolumic extracts were added into the holes. Cells were incubated again for 2, 4, 6, 8, 10 days respectively. Cell Counting Kit-8 was added into the holes, OD value of cells in each hole was measured under the wave length of 450 nm.(8) Observe cells under optical microscope and electron microscope: Cells incubated in different medium were observed under optical microscope and taken pictures. HBMSCs were seeded onto plaster samples and then observed under electron microscope.(9) Acute toxicity testing: 30 mice was divided into three groups as normal sodium (NS) Group, CPC Group and CPC/M Group, 5 female and 5 male mice was selected to each group, 1ml solution in each group was injected into mice by intratailvenous injection. The weight and behavior of each mouse were recorded 24h, 48h, 72h and 120h after injection.(10) Hemolysis test: Fresh human anticoagulated blood was diluted into suspension at a volume concentration of 2%. Cement samples were immersed into NS to regulate the concentration of suspension to 0.1 g·ml-1, 0.05g·ml-1 and 0.01g·ml-1. NS was used as negative group while double distilled water as positive group. 2 ml different liquor was mixed with 2 ml human blood and then accepted water bath at 37℃for 1 hour, centrifugalization at 1000 r·min-1 for 5 minutes. 4 ml 0.1vol% Na2CO3 solution was added into 1 ml supernatant fluid extracted from suspensions. OD value of was measured under the wave length of 540 nm and hemolytic ratio was then calculated by accordant values.(11) Micronucleus test: 80 healthy mice with half male and half female were randomly divided into eight groups: 60 mice in 6 experimental groups were injected in different doses (1, 2, 4 ml) of the material saline extracts, the other two groups were regarded as a negative control group (NS) and a positive control group (cyclophosphamide 40 mg·kg-1). All the animals were killed and bone marrow was extracted and mixed with calf serum, Rett's (Wright) staining was performed later. In the oil microscope count, micro-nucleus cells of 1000 polychromatic erythrocytes in each animal were recorded. The total micro-nucleus cells of 5000 polychromatic erythrocytes were calculated as the micro-nucleus ratio.(12) Pyrogen test: 6 healthy rabbits were selected and 5 ml suspensions of different samples were injected into each animal. The temperature of each rabbit was measured 3 times before and after injection, the difference was recorded as increased temperature.2 Characterization and preparation of calcium phosphate cements containing Doxorubicin PLGA microspheres(1) Grouping methods: Based on the difference of materials, 3 groups determined as Control Group, CPC/D Group and CPC/M/D Group.(2) Observation by eletron microscope: Surface structure of different samples was observed by eletron microscope. The average diameter of microspheres was measured by their images.(3)X-ray diffraction (XRD) analysis: Powder X-ray diffraction (XRD) analysis was used to estimate the extent of CPC and CPC/M/D samples.(4)Washout Resistance Test: shape the samples into spheres by hand, then put them immediately into NS or distilled water (37℃) under slightly shock for 24 h. The samples were considered to show good property of washout resistance if it did not visibly disintegrate in the liquor.(5) Setting time measurement: The initial and final setting time of cement samples in 3 different groups were measured at 25℃and 37℃respectively. 6 samples from each group were selected and measured. The average values of temperature were recorded.(6) Injectability: The injectability of different samples was tested with a syringe of 14.5 mm inner diameter, which was fitted with a needle of 1.6 mm inner diameter. After mixing different powder with sodium citrate solution of 15 wt.% for 1 min, the as-prepared paste was poured into the syringe. A 5 kg compressive load was then mounted vertically on the top of the plunger for 2 min. The injectability was calculated according to the percentage of mass expelled from the syringe to total mass before injection.(7) Interval porosity: Interval porosity was measured by dewatering method. The volume of samples and pores in samples were calculated. The volume ratio of pores to samples was recorded as interval porosity.(8) Maxium strength of compression (MSC): The compressive strength of the specimens was measured using a universal material testing machine at a crosshead speed of 0.5mm·min-1.3 In vitro drug release and degradation of calcium phosphate cement containing Doxorubicin PLGA microspheres(1) Drug load and encapsulation of Doxorubicin microspheres: Doxorubicin loading in microspheres was determined using a UV-visible spectrophotometer. Freeze-dried microspheres (5mg) were dispersed in dimethyl sulfoxide (DMSO). The concentrations of gentamicin releasing in vitro was calculated referring to the standard curve of realationship between the concentrations of Doxorubicin and their OD values. The experiment was repeated three times. (2) In vitro drug release from Doxorubicin microspheres: 48 bottles of microspheres with each weight of 100 mg were added into simulated body fluid and immersed for 1, 3, 5, 7, 10, 14, 21, 28 days respectively. Supernatants from 6 portions randomly selected each time were measured at 480 nm by UV-visible spectrophotometer.(3) In vitro drug release from different plasters: 2 different cement samples were added into simulated body fluid and immersed for 1, 3, 5, 7, 10, 14, 21, 28 days respectively. Supernatants from fluid at each interval were collected and measured at 480 nm by UV-visible spectrophotometer.(4) In vitro degradation of microspheres: 30 bottles of microspheres with each weight of 100 mg were added into simulated body fluid and immersed for 7, 14, 28, 42 and 56 days respectively. 6 portions were randomly selected each time, the supernatants were pored. Microspheres were weighed after being dried in vacuum for 2 days. The difference between the weight of microspheres before and after immersion was regarded as weight loss of microspheres.(5) In vitro degradation of plasters: 35 samples were selected and weighed before immersion in simulated body fluid for 7, 14, 28, 42 and 56 days respectively. 6 portions were randomly selected each time, the supernatants were pored. Samples were weighed after being dried in vacuum for 2 days. The difference between the weight of cement samples before and after immersion was regarded as weight loss of cement samples.4 Osteogenesis of calcium phosphate cement containing Doxorubicin PLGA microspheres(1) Animals and grouping method: 40 rabbits weighed from 400 to 450 gram were randomly divided into 20 pairs. The radius of rabbits were divided into four groups: Control group, CPC group, CPC/D Group and CPC/M/D group. (2) Preparation for animal models: Intravenous anesthesia was performed before radius being exposed. Radius defect with a length of 1.5cm was made bilaterally. Different samples were implanted into the defective bone.(3) General observation: Observe the appetite, behavior, sound situation and investigate the degradation of implants, osteogenesis of samples postoperatively.(4) X-ray examination: 5 pairs of rabbits were selected each time at 2, 4, 8 and 16 weeks postoperatively. X-ray examination was performed to learn bone growth and degradation of samples.(5) Mechanical strength: The samples of radius were obtained to measure mechanic strength after 5 rabbits being killed each 2, 4, 8 and 16 weeks after operation.(6) Residual calcium and phosphate in cement samples: XRD were performed at the 4th postoperative week to analyze the residual calcium and phosphate in cement samples.(7) Pathology of radius samples: Each radius was obtained after killing the rabbits 16 weeks after operation. The samples were fixed, embedded and sliced successively. Pathology of samples was observed in optical microspheres after hematoxylin-eosin staining.5 Antitumor effect of calcium phosphate cement incorporated with Doxorubicin PLGA microspheres(1) SaoS-2 cells culture: SaoS-2 cells were transferred to centrifuge tube to accept centrifugalization for 10min. Supernatant was poured and RPMI - 1640 nutritive medium containing 10 Ml 10vol% calf serum was added. Centrifuge for another 10 minutes and pour the supernatant again, add moderate nutritive medium into culture flask after cells being transferred. Cells were incubated at 37℃in a incubator with 5 vol% CO2. (2) Preparation for extracts: Samples were weighed and sterilized, then immersed into cell culture fluid at a weight volume ratio of 1g:20ml, 1g:10ml and 1g:5ml. Leaching liquid was obtained after the samples being immersed for 72h at 37℃, with 5 vol% CO2.(3) SaoS-2 cells inhibition: SaoS-2 cells suspension was seeded in 96 hole plate at a volume of 200μL per hole, cells then were incubated for 24 hours at 37℃in a incubator with 5 vol% CO2 . Cell culture fluid was poured, instead, isovolumic extracts were added into the holes. Cells were incubated again for 5 days. Cell Counting Kit-8 was added into the holes, OD value of cells in each hole was measured under the wave length of 490 nm.(4) Preparation of osteosarcoma models: Regulate the amount of SaoS-2 cells to 2×107ml-1 by the addition of NS. 0.2ml cells were injected into left buttock of each nude mouse.(5) Injection of cells with materials: Regulate the amount of SaoS-2 cells to 2 X 107ml-1 by the addition of NS. 0.1ml cells with 0.1ml cement supernatant with different concentration were injected into right buttock and both post aurems of each nude mouse.(6) General observation: Observe the appetite, behavior, injection area and growth of tumors postoperatively.(7) Tumor inhibition: Kill nude mice 12 days after cells injection, extract the total tumor tissues and weigh them one by one. Calculate the ratio of tumor inhibition by correspondent weight.(8) Pathology of tumor: Each tumor was obtained after killing the mice postoperatively. The samples were fixed, embedded and sliced successively. Pathology of samples was observed in optical microspheres after hematoxylin-eosin staining. 6 Statistical analysisThe software package SPSS 17.0 was applied for statistical analysis, including t test, x2 test, one-way ANOVA method of square-deviation (SD).Results1 Biocompatibility of the cement incorporated with PLGA microspheres(1) Observation in inverted microscope: Cells grew well in control group and materials groups with good activity and shape.(2) Cell proliferation: Calcium phosphate cement and the cement incorporated with microspheres nearly have no influence on the proliferation of cells.(3) Observation by electron microscope: Little cells could grow on the surface of solo-calcium phosphate cement and shrinkage of cells could be seen. Many cells could be seen growing well on the surface of calcium phosphate cement containing microspheres.(4) Acute toxicity test: The increase of weight in different materials groups was the same as that in NS group. Mice were all active without abnormal behavior.(5) Hemolysis test: Hemolysis did not appear in negative control group and different cement groups, but could be found clearly in positive control group. The difference between cement groups and negative control group was not significant, but the difference was significant compared to positive control group. The hemolysis ratio was less than 5% in each cement group.(6) Micronucleus test: The number of micronucleus in each cement group was less than 8‰. Compared with negative control group, no significant difference appeared in cement groups. However, the difference between cement groups and positive control group was significant.(7) Pyrogen test: Rectal temperature of rabbits increased a little after injection. the average increased temperature was 0.06℃, lower than 1.4℃. 2 Characterization and preparation of calcium phosphate cements containing Doxorubicin PLGA microspheres(1) Observation by scanning electron microscope: The average diameter of microspheres was between 100 and 150μm. Microspheres were globular and the surface was slick and sly. Calcium phosphate cement was made up of microparticles, the drug could not be found when mixed with cement powers. Microspheres with the average diameter between 100 and 150μm could be observed evenly adhering to calcium phosphate cement microparticles.(2) X-ray diffraction: All the X-ray diffraction pattern of three different sorts of cement revealed characteristic peaks of nano-HA and no secondary phases.(3) Washout resistance: Solo- calcium phosphate cement showed excellent washout resistance. It remained stable while immersed in NS, no obvious collapse phenomenon was observed. However, obvious collapsing appeared in both other two samples.(4) Setting time: The results showed that the addition of microspheres can significantly prolong the composite bone cement setting time, including both the initial and final time. The final setting time of calcium phosphate cement containg microspheres could delay to 45 minutes.(5) Injectability: The calcium phosphate cement incoporated with Doxrorubicin microspheres showed best property of injectability among the three kinds of cement.(6) Interval porosity: Interval porosity was up to 61.67%, which was the most in the calcium phosphate cement incoporated with Doxrorubicin microspheres.(7) Mechanical strength: The strength of calcium phosphate cement dramatically decreased while Doxrorubicin microspheres being incoporated. The decrease was not significant compared with the cement mixed with Doxorubicin powder.3 In vitro drug release and degradation of calcium phosphate cement containing Doxorubicin PLGA microspheres(1) Drug loading and encapsulation: Drug loading and encapsulation were 5.9±0.29(%) and 74.1±3.57(%) respectively.(2) Drug release from microspheres: Drug in microspheres released fast at the first week but lowered later.(3) Drug release from cement samples: Drug mixed with CPC powder directly released faster than that in the CPC incorporated with Doxorubicin microspheres. CPC containing Doxorubicin microspheres showed good property of slow-release.(4) Degradation of microspheres: Weight loss of control microspheres was less at the early stage than that after 4 weeks. The weight loss of Doxorubicin microspheres was fast at the first two weeks but became slow since then.(5) Degradation of cement samples: The strength of CPC decreased fastest wile Doxorubicin microspheres incorporated, and the acceleration of decrease began to be much faster than the other two samples since 4 weeks on.(6) Compressive strength of cement samples: The difference between 3 kinds of samples was not significant at the first 2 weeks. However, the difference became more and more significant between the cement containing Doxorubicin microspheres and the solo-cement since they were immersed for 4 weeks.4 Osteogenesis of calcium phosphate cement with Doxorubicin PLGA microspheres(1) General observation: Inflammatory reaction was not obvious in different materials groups and the skins were seemed the same as those in NS group. Mice were all active without abnormal behavior. All incisions cured as scheduled. CPC containing Doxorubicin microspheres degraded fastest in the three materials groups and new bone could be observed growing into the cement samples after implantation for 16 weeks. (2) X-ray examination: The shape of CPC containing Doxorubicin microspheres began to change at the 4th postoperative week, and the residual cement containing Doxorubicin microspheres disappeared after 16 weeks.(3) Mechanical strength: The strength of defected bone increased gradually after cement samples implantation. The strength of bones reconstructed by CPC containing Doxorubicin microspheres increased fast after implantation for 8 weeks.(4) Osteogenesis of cement samples: The cement containing Doxorubicin microspheres combined to bone compactly. New bone could be seen in the outer layer of CPC and CPC/Doxorubicin composites, but the ingrowth of new bone could be found in the center of CPC containing Doxorubicin microspheres.(5) Acceleration of cement degradation: CPC containing Doxorubicin microspheres degraded fastest among three sorts of cement samples. Degradation only could be observed at the bone-bone cement interface after implantation for 16 weeks, however, the new bone has grown into the CPC containing Doxorubicin microspheres.(6) Component analysis of residual cement samples: Content of calcium phosphate decreased dramatically at the 3rd month after implantation of CPC containing Doxorubicin microspheres, which indicated that the degradation of calcium salts played an important role in the degradation of CPC containing microspheres.5 Antitumor effect of calcium phosphate cement incorporated with Doxorubicin PLGA microspheres(1) Observe cells under optical microscope: Cells grew fast in control group and CPC group. Dead cells could be seen in CPC/M/D groups at the first two days after incubation, but the survival cells began to recover and grew fast two days later.(2) Cells proliferation: Inhibition of cells proliferation appeared in CPC/M/D groups after incubation in the extracts of CPC/M/D. The OD values in high dose group were lower than those in low dose group, which indicated that the inhibition effect was related to its dose.(3) General observation on mice: Nude mice seemed to be tired at the first two days after tumor cells injection. They became active after 3 days. Tumor could be observed growing fast after about 7 days.(4) Weight of tumor and tumor inhibition rate: The higher dose of CPC/M/D extract was injected, the smaller the tumor formed, which suggested that the tumor inhibition effect was positively related to its dose. The inhibition rate of high dose group could reach to 61.0%.(5) Pathology of tumor: Tumor necrosis could be found in high dose group, but nearly no necrosis appeared in low dose group.ConclusionsCPC containing PLGA microspheres exhibited good biocompatibility. When Doxorubicin microspheres were incorporated into CPC, the resultant production also showed good properties such as compressive strength, injectability, etc. The novel cement could degrade and release drug slowly in vitro. When the composites were implanted into defected bone, it could facilitate the ingrowth of new bone. The extracts of CPC containing Doxorubicin also could inhibit the Cells proliferation of tumor both in vitro and in vivo.
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