Chapter 111 CHAPTER 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). Chapter 112 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. Chapter 113 6.2 Materials and methods 6.2.1 Materials Poly(D,L-lactide-co-glycolide) (PLGA) (L/G ratio 50:50, MW 40,000-75,000) and 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 (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 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 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 Chapter 114 summarizes the composition of the emulsion of the groups (A, B and C) and samples (A1-A3, B1-B3 and C1-C3). The detailed fabrication procedures have been illustrated in Chapter (Section 5.2.4). Table 6.1 Compositions of different scaffold samples in the current study Group A Group B Group C HAp/PLGA (% w/w) HAp/PLGA (% w/w) HAp/PLGA (% w/w) A1 A2 A3 B1 B2 B3 C1 C2 C3 0/100 5/95 10/90 0/100 5/95 10/90 0/100 5/95 10/90 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 (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[...]... results, in which significant bone healing is observed for samples A1, B1, B3, and C1 Furthermore, this also confirms that during the progress of bone healing, tissue reabsorbing/remodeling, and the in growth of blood vessel are interactively together rather than being subsequent Chapter 6 124 Figure 6.6 Histological specimens from nude mice tibias after 2 and 4 weeks of implantation of A1, B1 and C1... into polymeric fibrous matrices in three different ways, and their individual performance was tested in nude mice Residual solvent in the scaffolds was well below the safety standard requirements Animal experimental results indicate that the bioactivity of BMP-2 plasmid released from Chapter 6 129 all the three kinds of scaffolds was well maintained, which helped improve the formation of new bone and. .. liquid nitrogen for 5 minutes, both bone cells and blood vessels in fragments will be totally destructed Consequently, the inactive bone will only become a physical support A successful treatment will be marked when newly formed bone cells and blood vessels are formed in bone fragments Von Willebrand factor is a large multimeric glycoprotein and produced constitutively in endothelium during blood vessel... 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... healthy bone tissue HAp is an osteoconductive material in nature For this reason, variable HAp nanoparticle contents are incorporated in the samples of each group (A, B, and C) to check their respective effect on osteoconduction and osteoinduction However, the scaffold was placed next to the bone defect, instead of exactly in the matrix of bone defect Furthermore, the detachment of HAp from scaffolds and. .. vitro release (naked plasmid or plasmid/chitosan nanoparticles) of groups A, B and C can last for 2, 4 and 8 weeks, respectively The bone defects investigated in the current study can heal spontaneously within six weeks, so effective and sustained release of plasmid DNA lasting for the initial four-week period becomes necessary for a successful treatment In this sense, group B samples are the best candidates... Figure 6 .5 Histological specimens from nude mice tibias after 2 and 4 weeks of implantation of different scaffolds (A1, A3, B1, B3, C1 and C3) along with control Blue arrows identify lacunae Original magnification is 100X for all Chapter 6 123 Besides H&E staining, IHC staining was also performed to check the formation of blood vessels in bone fractures After treatment of bone fragments in liquid... formation of new bone and the healing of segmental defects in vivo Groups A, B and C released DNA or DNA nanoparticles in different ways and time frames, thus, their performances in bone healing were not similar Scaffolds of group A, releasing plasmid the fastest over the initial two weeks, performed the best among all scaffolds over the first two weeks Interestingly, at four weeks of treatment, group B scaffolds... 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... bone cells can attach, migrate, grow, and divide In this way, the bone healing response is "conducted" through the graft site In this study, this function was served by the dead bone segment fixed by intra-medullary wire In contrast, osteoinduction refers to the capacity of several chemicals in the body responsible to stimulate primitive "stem cells" or immature bone cells to grow and mature, forming . 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 significant bone healing is observed for samples A1, B1, B3, and C1. Furthermore, this also confirms that during the progress of bone healing, tissue reabsorbing/remodeling, and the in growth of blood. 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 50 0 µL in order