Applications of electrospinning and supercritical carbon dioxide foaming techniques in controlled release and bone regeneration 5

CHAPTER 6 BMP-2 Plasmid Loaded PLGA/HAp Composite Scaffolds for Treatment of Bone Defects in Nude Mice † 6.1 Introduction Over past decades, varied controlled-release dosage forms have

CHAPTER 6

BMP-2 Plasmid Loaded PLGA/HAp Composite Scaffolds for

Treatment of Bone Defects in Nude Mice †

6.1 Introduction

Over past decades, varied controlled-release dosage forms have been developed for drug, protein and DNA delivery, for instance, nanoparticles and microspheres However, this type of devices is known to exhibit a large burst during the early period of release To tackle this drawback, electrospun fiber is chosen in the present study as the release dosage form because its moderate surface/volume ratio may produce a relatively constant controlled release of DNA with inhibited initial burst as compared to that of nanoparticles and microspheres (Wei et al., 2006; Wnek et al., 2003) Moreover, in comparison to microspheres, compact fibrous scaffolds give cell stable three-dimensional growth environments and may provide newly generated bone enough support On the other hand, hydroxyapatite (HAp), which is a major component of the bone, can be used as a subsidiary in the bone generation In addition, HAp has another advantage of being able

to bind directly to the bone since both of them have similar chemical structures (Li et al., 2006) Therefore, polymer/HAp composite scaffolds are promising as a substitute for

† This chapter highlights the work published in H Nie, M.L Ho, C.K Wang, C.H Wang, and Y.C Fu BMP-2 Plasmid Loaded PLGA/HAp Composite Scaffolds for Treatment of Bone Defects in Nude Mice

Biomaterials (in press)

bone graft In a previous study (Nie and Wang, 2007), PLGA/HAp composite scaffolds with different HAp contents (0%, 5% and 10%) were fabricated by electrospinning method and DNA was incorporated into the scaffolds in three ways (i.e coating of naked DNA or DNA/chitosan nanoparticles on scaffolds after fiber fabrication by dripping, and encapsulation of DNA/chitosan nanoparticles into scaffold by mixing them with PLGA/HAp solution before fiber fabrication) (see Figure 6.1) The results showed that BMP-2 plasmid loaded PLGA/HAp composite scaffolds could maintain the integrity of encapsulated BMP-2 plasmid, enhance cell attachment with negligible cytotoxicity In the present study, the main objective was to investigate the bone regeneration capability of

these PLGA/HAp composite fibrous scaffolds in vivo The hypothesis is that different

loading methods of BMP-2 plasmid and different HAp contents in scaffolds will alter the release profiles of BMP-2 plasmid, and consequently influence its performance in bone

regeneration in vivo

Figure 6.1 Illustration of three plasmid loading modes in the present work

6.2 Materials and methods

6.2.1 Materials

chitosan (medium molecular weight and 75-85% deacetylated), were procured from Sigma Aldrich (St Louis, MO, US) HAp nanocrystals with average diameter of 100nm, dichloromethane (DCM), Ketamine Ketalar® and Xylocain® were purchased from Berkeley Advanced biomaterials Inc (Berkeley, CA, US), Tedia Company Inc (Fairfield,

OH, US.), Parke-Davis Taiwan, and AstraZeneca PLC Taiwan, respectively

6.2.2 Preparation of DNA/chitosan nanoparticles

As described in Chapter 5 (Section 5.2.3), a pT7T3D-PacI plasmid encoding BMP-2 was

used throughout the present study and DNA/chitosan nanoparticles were formed as a result of static attraction between DNA and chitosan Chitosan solution (0.02% in 5 mM sodium acetate buffer, pH 5.0) and DNA solution of 100µg/mL in 5-50 mM of sodium sulfate solution were preheated to 50-55 °C separately An equal volume of both solutions were quickly mixed and vortexed for 15-30s The final volume of the mixture in each preparation was limited to below 500 µL in order to yield uniform nanoparticles In this way, nanoparticles with amino group to phosphate group ratio (N/P ratio) of 4 were obtained

6.2.3 Fibers fabrication methods

In all the experiments, fiber mats were essentially fabricated from homogeneous emulsions formed from the sonication of organic and aqueous mixture Table 6.1

summarizes the composition of the emulsion of the 3 groups (A, B and C) and 9 samples (A1-A3, B1-B3 and C1-C3) The detailed fabrication procedures have been illustrated in

Chapter 5 (Section 5.2.4)

Table 6.1 Compositions of different scaffold samples in the current study

Group A

HAp/PLGA (% w/w)

Group B

HAp/PLGA (% w/w)

Group C

HAp/PLGA (% w/w)

6.2.4 In vivo experiments

All procedures were performed in accordance to specifications in the Guidelines for Animal Experiments of Kaohsiung Medical University The animal model has been interpreted in our previous study (Fu et al., 2008) The detailed description of animal

model construction and subsequent characterizations can be found in Chapter 4 (Section

4.3.2)

6 2.5 Statistical analysis

All the data were statistically analyzed to express the mean ± standard deviation (S.D.) Student’s t-test was performed and p<0.05 was accepted to be significant

6.3 Results and discussion

6.3.1 Preparation and characterization of the DNA/chitosan nanoparticles

The N/P ratio of 4 was used throughout the present work and the resultant DNA/chitosan particles were not exactly spherical but all share about the same size of about 100nm in diameter

6.3.2 Fiber characteristics

As previously shown in Figure 5.2 (Chapter 5), PLGA/HAp composite fibers (loaded with 5 % or 10 % of HAp) or fibers loaded with chitosan nanoparticles could not maintain uniform diameter as compared with pure PLGA/DCM systems (A1 and B1) In addition, A1 and B1 have smaller diameters because a pure PLGA/DCM system has higher viscosity than emulsion Furthermore, the diameter of fibers becomes larger with the addition of more HAp nanopaticles (Nie and Wang, 2007) FESEM pictures illustrating the cross sections of samples B1 and C1 were compared (Nie and Wang, 2007) As displayed in Figure 5.3 (Chapter 5), particles with diameter of about 100 nm were found to be entrapped within the cross section of sample C1, while they were absent

in B1 This confirms that, in group C, DNA/chitosan nanoparticles are incorporated inside the fiber polymer matrix as intentionally designed

6.3.4 Animal experiments

Soft X-ray photographs clearly demonstrate the outcome of the treatments with different scaffolds Figure 6.2 shows the soft X-ray photographs of mice tibia fractures two and four weeks after implantation of scaffolds together with control

Figure 6.2 Radiographs of nude mice tibias after 2 and 4 weeks of implantation of

scaffolds Bone fragment without implantation of any scaffold is denoted as control and white arrows identify bone defects

The bone ends of the control samples were sharp and there was no significant bone regeneration after two and four weeks The delayed union of bone fractures was clearly shown by white arrows in micrographs In contrast, those from A1 and B1 showed wide and dull bone ends, indicating the new formation of bone after two and four weeks This was more evident due to the fact that the two disconnected sections on tibia formed new

bridges after four weeks These results demonstrate clearly that BMP-2 plasmid released from A1 and B1 within the observation period of four weeks, with the expression of BMP-2 proteins, helped the bone regeneration Similarly, other samples, including A2, A3, B2, B3, C1, C2 and C3, showed better treatments than control Amongst them, A1 performed the best, in which after only two weeks the two disconnected ends were fully joined Comparing those samples with 0% of HAp concentration (A1, B1 and C1) but different loading techniques of plasmid, A1 and B1 demonstrated the best performances and displayed best joints of two bone ends after just two weeks This observation suggests the advantages of groups A and B over group C in the early stage of bone healing (first two-week period)

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Figure 6.3 Comparison of serum BMP-2 concentrations in groups A1/A3, B1/B3 and

C1/C3 over the first 4 weeks along with control Values represent means ± S.D., n=3

(*p<0.05 as compared to control and +p<0.05 compared between samples)

Figure 6.3 shows the serum BMP-2 concentrations two and four weeks after the implantation of A1/A3, B1/B3 and C1/C3 scaffolds As bone healing is a spontaneous process, it is not strange to detect BMP-2 in the serum of the control sample An unexpected phenomenon was that BMP-2 concentrations detected in group A samples (A1 and A3) were lower than control over the testing period of four weeks The most important factor contributing to the phenomenon should be due to the serious cytotoxicity

of group A samples (Nie and Wang, 2007) The harsh environment due to cytotoxicity may interfere with signal transductions between cells and hinder the spontaneous secretion of BMP-2 As a result, lower serum BMP-2 concentration was detected in group A samples than control Another possible reason is the low transfection efficiency

of naked DNA, and thus the low expression of naked plasmid released from A1 and A3 Consequently, the serum BMP-2 levels of A1 and A3 did not improve significantly after four weeks, as compared to those levels after two weeks Similar to group A samples, group B samples also have cytotoxicity, but the much higher transfection efficiency of DNA/chitosan nanoparticles might offset the effect of cytotoxicity posed by them Consequently, group B samples demonstrated much higher BMP-2 concentrations than group A samples over the first four weeks Particularly, B1 showed the highest BMP-2 concentration among all samples, and the BMP-2 level was sustainable over the testing period For group C samples, serum BMP-2 concentration experienced a significant increase after four weeks, although the level after two weeks was lower than all other

samples These in vivo observations of serum BMP-2 concentration demonstrate different release properties of groups A, B and C, and are consistent with the in vitro release

profiles of BMP-2 plasmid from different scaffolds (Nie and Wang, 2007), As naked

DNA and DNA/chitosan nanoparticles are coated on the surface of fiber mats in groups A and B, respectively, they are more likely to detach from these scaffolds than those of group C Different transfection efficiency between naked plasmid and DNA/chitosan nanoparticle explains the difference in the serum BMP-2 concentration between groups A and B Group B samples displayed a more sustained serum BMP-2 profile due to its lower and more sustained release of DNA/chitosan nanoparticles from scaffolds, as compared to naked plasmid from group A On the other hand, in group C samples, DNA/chitosan nanoparticles were incorporated inside fibrous scaffolds and therefore their release were the lowest As a result, the BMP-2 concentration of group C at the end

of the second week was the lowest Interestingly, the concentration increased significantly in the subsequent two weeks, suggesting that the expression of DNA from group C samples was sustainable and lasting Nonetheless, these observations clearly show the advantages of groups B and C over group A for long-term performance For different samples in each group (A, B and C), the effects of HAp loading percentages on

serum BMP-2 concentration are not conclusive As observed from in vitro study (Figure 5.5), the incorporation of HAp helps enhance the in vitro release of DNA or DNA/chitosan nanoparticles from scaffolds This in vitro observation is consistent with the in vivo observation for groups A and C As shown in Figure 6.3, A1 and C1 showed

BMP-2 concentrations that are lower than A3 and C3, respectively However, over the period of four weeks, B1 exhibited significantly higher BMP-2 concentration than B3

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Figure 6.4 Comparison of serum ALP activity level in groups A1/A3, B1/B3 and C1/C3

over the first 4 weeks along with control Values represent means ± S.D., n=3 (*p<0.05

as compared to control, +p<0.05 compared between samples)

Concurrently, ALP activity level in serum was investigated after two and four weeks after the implantation As shown in Figure 6.4, the control group showed a low level of ALP activity but with a gradual increase over the period of four weeks, which was a sign of spontaneous repair of bone defect At the end of the second week, every sample demonstrated significantly higher ALP level than control, in which B3 showed the highest This observation confirms that the healing rate of bone defects in group B is the highest at the end of second week However, the situation drastically changed in the fourth week The increase in ALP level for group A was not obvious, but significant changes were observed for groups B and C, especially for the cases of B3 and C1 B3 showed the highest ALP level in the second week, but decreased significantly in the

fourth week, although the level was still comparable to other samples In contrast, C1 showed a steep increase before reaching a peak in the fourth week ALP activity is a good indicator for analyzing the activity of osteogenic differentiation of cytoclasts High ALP activity refers to high differentiation rate of cytoclasts and correspondingly high bone healing rate For example, B3 displayed the highest ALP level in the second week, implying that the cytoclast differentiation rate and bone healing rate in B3 were both higher than other samples Consequently, good healing result for B3 was detected in the fourth week Similarly, C1 displayed the highest ALP activity level in the fourth week and subsequently the fastest progress in bone healing was anticipated in the following weeks Similar to previous serum BMP-2 testing, the ALP results also suggest that groups B and C offer better performances over group A on a longer term

The X-ray micrographs of all samples after six weeks are not displayed in Figure 6.2 as all the fractures were physically “united” then, even including the control group However, structural integrity is only a basic requirement for bone healing More importantly, the functions of regenerated bone should be verified before any conclusion can be drawn One can examine the callus formation by the observation of bone fragments from H&E staining micrographs As shown in Figure 6.5, the fragmental defects of the control group at two and four weeks after implantation are circled in blue for easier identification of the defects From the micrographs, obvious callus formation around bone defects can be seen in all samples other than control After two weeks, some lacunae were detected in all the samples, but only low percentages of the bone graft were found However, after four weeks, the percentages increased Here, A1, B1 and C1