82 Chapter CHAPTER Fabrication and Characterization of PLGA/HAp Composite Scaffolds for Delivery of BMP-2 Plasmid DNA † 5.1 Introduction Bone defects and fracture are common problems that affect as many as thousand patients around the world every year, and are difficult to heal using current therapies. It has been reported that bone morphogenetic protein-2 (BMP-2) has a very strong osteoinductive activity observed in many animal studies on the induction of bone formation by implantation of recombinant human BMP-2 (Fujimura et al., 1995; Kusumoto et al., 1998; Okubo et al., 2000; Boyne, 2001). However, the use of BMP-2 alone requires large amounts of protein because of its short half-life. Gene transfection is a powerful and promising alternative that involves the in vitro or in vivo incorporation of exogenous genes into cells for experimental and therapeutic purposes. Bone regeneration by gene transfer into human MSC has also been reported (Turgeman et al., 2001; Lieberman et al., 1999; Lou et al., 1999). These reports have mainly used a retrovirus, or adenovirus vector carrying human BMP-2, -4, or -7 as the therapeutic gene and these were effective in the formation of new bone. However, considering the immunological and safety issues of † This chapter highlights the work published in H. Nie and C.H. Wang. Fabrication and Characterization of PLGA/HAp Composite Scaffolds for Delivery of BMP-2 Plasmid DNA. J. Control. Release 120, 111-121. 2007. Chapter 83 viral vectors, necessity in the development of non-viral vector systems has been increasingly important (Hosseinkhani et al., 2006). In recent years, the potential of chitosan as a polycationic gene carrier has been explored in several research groups (Roy et al., 1999; Leong et al., 1998; MacLaughlin et al., 1998; Mao et al., 2001; Roy et al., 1997; Mao et al., 1996; Saito et al., 2005). Chitosan can condense DNA, which can ensure smaller diameter and easier entry into cells and nucleus. Moreover DNA/chitosan nanoparticles could partially protect the encapsulated DNA from nualease degradation. Hydroxylapatite (HAp), which is a major component of the bone, can be used as a subsidiary in the bone generation. HAp implants exhibit high mechanical strength and good biocompatibility. In addition, HAp has the added advantage of being able to bind directly to the bone since both of them have similar chemical structures. Over past years, many release dosage forms have been developed for drug and protein delivery, like nanoparticle and microparticle. However, one common problem with them is the burst release at very early stages together with a very short release course. Especially as for bone regeneration, a new kind of scaffold is needed because nanoparticles and microparticles are not suitable in view of their fluidity, and hence can’t be localized themselves and give new born bone enough support. Electrospun fibers are chosen in the present work as the release dosage form because of their release properties and morphology. We further explored the in vitro study of plasmid DNA by investigating the effects of HAp content and the different methods of DNA loading on the physical and Chapter 84 biological characteristics of the micro-fibers fabricated using the electrospinning method to explore an optimal DNA release system for bone regeneration. 5.2 Materials and methods 5.2.1 Materials Poly (DL-lactide-co-glycolide) (PLGA) (Lot Number W3066-603 with L/G ratio 50:50, IV 0.57 and MW 51000) used in the experiment was manufactured by Alkermes Controlled Therapeutics II, (OH, US) and purchased from Lakeshore Biomaterials (Birmingham, England). Chitosan (medium molecular weight and 75-85% deacetylated), chitosanase from Streptomyces griseus (lyophilized powder) and phosphate-buffered saline (PBS) containing 0.1 M sodium phosphate and 0.15 M sodium chloride, pH 7.4, used for in vitro release study were purchased from Sigma Aldrich (St. Louis, MO, US). HAp nanocrystals with average diameter 100nm were purchased from Berkeley Advanced biomaterials Inc. (Berkeley, CA, US). DCM (Cat. No. DR-0440) was purchased from Tedia Company Inc. (Fairfield, OH, U.S.A.). Human MSCs were purchased from Cambrex Bio Science (MN, US). PicoGreen dsDNA Quantitation kit was purchased from Invitrogen Corporation (MN, US) and PreMix WST-1 Cell Proliferation Assay System was purchased from Takara Bio Inc. (Otsu, Shiga, Japan). 5.2.2 Preparation of plasmid DNA A pT7T3D-PacI encoding BMP-2, purchased from ResGen, Invitrogen Corporation (clone identification number UI-R-E1-fb-c-11-0-UI; Ampicillin resistant, 50-200 µg/mL; RE_5': EcoRI and Re_3': NotI) was used in this study. The plasmid DNA was amplified Chapter 85 in a transformant of Escherichia coli bacteria and isolated from the bacteria by PureLinkTM HiPure Plasmid DNA Purification Kit-Maxiprep K2100-07 (Invitrogen Corporation, MN, US). The DNA concentration was identified by using a PicoGreen dsDNA Quantitation kit. 5.2.3 Preparation of DNA/chitosan nanoparticles In the present work, the DNA/chitosan nanoparticles were formed as a result of static attraction between DNA and chitosan. The size of DNA encapsulated particles is mainly determined by N/P ratio. From the previous works by Mao and coworkers (Mao et al., 2001; Roy et al., 1997; Mao et al., 1996), large aggregates formed at N/P ratios around and an N/P ratio below 0.75 and above yielded submicron size particles. Nanoparticles prepared with an N/P ratio between and tended to have higher thermal dynamic stability with an average size between 100 and 250 nm according to literature (Mao et al., 2001). A chitosan solution (0.02% in mM sodium acetate buffer, pH 5.0) and a DNA solution in 5-50 mM of sodium sulfate solution (100 µg/mL) were preheated to 50-55 °C separately. An equal volume of both solutions were quickly mixed together 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. 5.2.4 Fibers fabrication methods Biodegradable fibrous scaffolds fabricated using an electrospinning method can create a large surface area (Saito et al., 2005; Li et al., 2006; Gupta et al., 2005; Bottaro et al., Chapter 86 2002; Lazzeri et al., 2005). Another major advantage of using the electrospinning method is that the physical properties of fabricated fibers can be easily controlled by parameters like the composition of the emulsion and the voltage differences (Li et al., 2006). In all the experiments, the fibers were essentially fabricated from homogeneous emulsions formed from the sonication of organic and aqueous mixture. Table 5.1 summarizes the composition of the emulsion of the groups (A, B and C) and samples (A1-A3, B1-B3 and C1-C3) of scaffolds. Preparation of organic phase In each experimental case, a 30% wt/vol PLGA polymer solution using DCM as the solvent was prepared by dissolving 3g PLGA into 10 mL of DCM. The resultant mixture was agitated by applying vortex until a clear and homogeneous organic phase was formed. Preparation of aqueous phase In all experimental cases, the same weight of plasmid DNA was used, but using different loading methods for different groups. For groups A and B, as specified in Figure 5.1, DNA was not added into fabrication solution. Instead naked DNA (for group A) or DNA/chitosan nanoparticles (for group B) were added into scaffolds after the fabrication of scaffolds. Therefore, while preparing aqueous phase, only the specified weight of HAp was suspended in DI water and mixed well to form a homogeneous aqueous phase. For group C, after the fabrication of DNA/chitosan nanoparticles as specified in Section 5.2.3, the specified weight of HAp was added into DNA/chitosan nanoparticles suspension and mixed well to form a homogeneous aqueous phase. 87 Chapter Fabrication of fibrous scaffolds After adding the aqueous and organic phases together, the mixture was sonicated for about 60 seconds and the resultant emulsion was transferred to a 10mL glass syringe (MICRO-MATE interchangeable 10cc hypodermic syringe, Popper & Sons, Inc., New Hyde Park, NY. US) fitted with a 29-g needle and set up in the elecontrospinning apparatus. The flow rate of polymer solution from the syringe into the spinneret (diameter 340 mm) was controlled by a programmable syringe pump (KD Scientific, Holliston, MA, US). Scaffolds were electrospun at about a voltage difference of 10 kV with a solution flow rate of mL/h. The spinneret (anode) was fixed at about 15 cm above the aluminum-covered rotating collection drum (cathode). Table 5.1 Compositions and characteristics of different scaffold samples examined in the present work Sample compositions Group A Group B Group C 0%HAp 5%HAp 10%HAp 0%HAp 5%HAp 10%HAp 0%HAp 5%HAp 10%HAp A1 A2 A3 B1 B2 B3 C1 C2 C3 Tg (ºC) 48.50 49.83 50.50 48.50 49.83 50.50 49.33 48.67 46.17 Td (ºC) 344.33 367.33 375.83 344.33 367.33 375.83 355.83 373.50 375.83 DCM residual content (ppm) 365 243 277 249 195 201 297 252 133 ±50 ±38 ±39 ±29 ±21 ±16 ±57 ±28 ±24 100 100 100 100 100 100 65±5 78±9 87±4 Encapsulation Efficiency (%) Chapter Mode A Mode B Mode C Figure 5.1 Three DNA incorporation modes in the present work. 88 Chapter 89 5.3 Characterization of scaffolds 5.3.1 Physical characterization of fibrous scaffolds Morphology and mechanical properties of fibrous scaffolds Field emission scanning electron microscopy (FESEM, JSM-6700F, JEOL Technics Co. Ltd, Tokyo, Japan) was employed to study the surface morphology of the fibers produced in each experiment, while the mechanical quality of the fibers was determined by tensile strength testing. The mechanical properties of all fibrous scaffolds (A1, A2, A3, B3, and C3) prepared in a sheet form (15mm x 20mm x 150µm) were evaluated by applying a tensile load and then observed the corresponding strain. Differential scanning calorimetry (DSC) Differential scanning calorimetry (DSC) can be employed to determine the amount of crystalline structure within the microfibers as well as the effects of HAp concentration on the glass transition temperature and the decomposition temperature of PLGA. The sample was heated from 30 °C to 400 °C at a constant temperature increment of 10 °C/minute and purged with nitrogen gas at 30 mL/min. X-ray diffractrometry (XRD) The HAp nanoparticles or fiber sample were placed in a sample holder and the surface of the sample was flattened. Next, the sample was placed in the XRD equipment (SHIMADZU, Tokyo, Japan). A diffraction range of 10-35° (2θ) was selected and the XRD analysis was carried out at 2°/min. Chapter 90 Measurement of residual solvent content in scaffolds Gas Chromatography was used to determine the residual amount of Dichloromethane (DCM) remaining in the scaffolds. Standard solutions with the range of DCM concentrations in N, N Dimethyl Formamide (DMF) from 0.5 to 10 x 10-6 mL DCM per mL DMF were prepared and placed in the refrigerator before analysis to prevent evaporation of the volatile organic solvents. 5.3.2 In vitro release test and determination of encapsulation efficiency (EE) In vitro release test of plasmid DNA Approximately 25mg of microfiber samples made from each experiment were prepared and each of them is added to mL PBS, the release medium in the experiment. The resultant mixture was placed in an orbital shaker bath (GFL® 1092) at 37 °C, 120rpm. mL of sample mixture was extracted at specific intervals (1h, 4h, 16h, day1, 2, 3, 5, 7, 10, 12, 14, 16, 19, 23, 27, 30, 33, 36, 39, 42, 45, 50, 53, 56, 60, 63 and 66) from each test tube and the sample was stored at -20 °C to inhibit all DNA denaturation activities. mL of fresh PBS solution was then added to each mixture to make up mL again and all the mixtures were incubated in the orbital shaker bath again before the next set of sample mixtures were extracted. For the second and third DNA incorporation modes, the DNA encapsulated in chitosan nanoparticles is difficult to be released from the complex by common chemical methods. In this work, in order to quantify the concentration of plasmid DNA in each sample, chitosanase was utilized to degrade chitosan shell to release DNA for quantitative analysis. Briefly, chitosanase was dissolved in PBS to form 91 Chapter a working solution of mg/L. Subsequently, adequate chitosanase solution was applied to each sample to degrade chitosan. Encapsulation efficiency determination 5mg of each scaffold was dissolved in mL of DCM and mL of PBS (pH 7.4) then introduced to extract DNA. The resultant emulsion was then centrifuged using a centrifuge (Hettich Zentrifugen, Universal 32R, Andreas Hettich GmbH & Co KG, Tuttlingen, Germany) at 9000rpm and 20 °C for 20 to separate the water and oil phases. The water phase was then carefully collected and kept frozen at -20 °C until it was analyzed for DNA concentration using the PicoGreen dsDNA quantitation kit after the addition of chitosanase to degrade chitosan shell. The encapsulation efficiency can be obtained by the equation below: EE = C plasmid DNA × V water W sample × W plasmid DNA + W PLGA + W HAp W plasmid × 100 % (5.1) DNA Where Cplasmid DNA is the plasmid DNA concentration in the water phase of extraction; Vwater is the volume of water phase of extraction; Wsample is the weight of each scaffold sample used for EE analysis; Wplasmid DNA, WPLGA and WHAp are the weights of plasmid DNA, PLGA and HAp used in the scaffold fabrication process, respectively. 5.3.3 DNA integrity check by agarose DNA gel electrophoresis Agarose DNA gel electrophoresis was used to determine the integrity of plasmid DNA released out from scaffolds in vitro after day and 60 days. For groups B and C, DNA/chitosan nanoparticles before and after chitosanse digestion are both checked. DNA 95 Chapter throughout the present work and the resultant DNA/chitosan particles are not exactly spherical but all share about the same size of 100nm in diameter. 5.4.3 Fiber characteristics A1 (0% HAp) B1 (0% HAp) C1 (0% HAp) A2 (5% HAp) B2 (5% HAp) C2 (5% HAp) A3 (10% HAp) B3 (10% HAp) C3 (10% HAp) Figure 5.2 Field emission scanning electron micrographs for representative samples of groups A, B and C. In order to characterize the effects of HAp contents and DNA loading methods on scaffold characteristics more clearly, the scaffolds used in the present work are divided 96 Chapter into nine types based on their different compositions as shown in Table 5.1. Each of the subscript “1”, “2” and “3” for groups A, B, and C represents the different loadings of HAp 0%, 5%, and 10%, respectively. Figure 5.2 shows the micrographs of groups A, B and C fibers respectively with different contents of HAp nanoparticles (0%, 5% and 10%). It is shown that the addition of HAp or/and chitosan significantly affects the morphology of fiber. As specified in Section 5.2.4, HAp nanoparticles and DNA/chitosan nanoparticles were all suspended in DI water before mixing with 30% PLGA/DCM solution. The water/oil emulsion system is unstable especially at the later phase of electrospinning, so the fibers (loaded with 5% and 10% of HAp or loaded with chitosan nanoparticles) can not keep uniform diameter as fabricated in pure PLGA/DCM systems (A1 and B1). B1 C1 Figure 5.3 The morphology observed at the cross section of samples B1 and C1 [shown by dashed line in Figure 5.2]. This enlarged diagram illustrates clearly the encapsulated DNA/chitosan nanoparticles at the cut section of C1. FESEM pictures illustrating the cross sections of samples B1 and C1 are shown and compared in Figure 5.3. As indicated by white arrows, several particles with diameter of about 100 nm are found to be entrapped within the cross section of sample C1, while they Chapter 97 are absent in B1. No HAp nanoparticles were used in the samples for B1 and C1, therefore the 100nm-diameter particles observed within the cross-section of sample C1 must be DNA/chitosan nanoparticles. This comparison shows that in the group C, DNA/chitosan nanoparticles are encapsulated inside the fiber polymer matrix as designed. Mechanical strength testing was carried out to check the effect of the addition of HAp, and chitosan on the mechanical property. The stress-strain (S-S) curve of the samples was monitored, and representative examples were shown in Figure 5.4. All of the different types of fibrous scaffolds showed a similar S-S pattern, with an initial linear elastic regime, followed by subsequent failure. Compared to pure PLGA (A1), the HAp- PLGA fibrous scaffolds exhibited a higher initial slope and lower strain at failure. It was noted that, among A1, A2, and A3, A2 showed the highest tensile strength, suggesting that the encapsulation of a suitable amount (5%) of HAp in PLGA contributed to the mechanical strength. This was likely due to HAp nanoparticles integrating well with the PLGA, adopting an efficient composite structure of inorganic-organic system as observed in natural bone. The mechanical properties of the scaffolds fabricated using different loading methods were also determined. As shown in Figure 5.4, on the condition of same amount of HAp, C3 showed much higher tensile strength (more than times) than A3 and B3, which showed that the high viscosity of chitosan contributed to the tensile strength of fibrous scaffolds. 98 Chapter 2.8 C3 A2 2.0 B3 A3 1.6 1.2 A1 0.8 0.4 Tensile stress (MPa) Tensile stress (MPa) 2.4 0.0 Strain (%) Figure 5.4 Representative stress-strain curves of the fibrous scaffolds. The XRD pattern shows that there is no peak at 2θ = 28°and 32° which are the characteristic peaks of HAp nanoparticles (data not shown). Furthermore, no obvious peak exists at about 2θ = 20° which is the characteristic peak of chitosan. This observation shows that HAp nanoparticles and DNA loaded chitosan nanoparticles in fibers are both poorly crystallized. From the DSC endothermgram (data not shown) we reconfirm that HAp nanoparticles are poorly crystallized and the impregnation of HAp nanoparticles increases the decomposition temperature of PLGA fiber from 344.33 °C (without adding DNA/chitosan nanoparticles) and 355.83 °C (with adding DNA/chitosan nanoparticles) respectively to 375.83 °C. In contrast, the changes in glass transition are not so straightforward because after the addition of DNA/chitosan nanoparticles, the glass transition temperatures decrease from 49.33 °C to 46.17 °C with increasing HAp content Chapter 99 from 0% to 10%. This illustrates a different tendency from those cases without the addition of DNA/chitosan nanoparticles, as shown in Table 5.1. 5.4.4 Measurement of residual DCM content in scaffolds As shown in Table 5.1, the residual DCM content of the scaffolds fabricated using the electrospinning method was below the safety standards (600ppm) after freeze drying for days. The residual content is not very ideal compared with other dosage forms like nanoparticles or microparticles because fibers have very compacted network structure, which hinders the evaporation of DCM from scaffolds. 5.4.5 Determination of DNA encapsulation efficiency (EE) For groups A and B, the naked DNA solution or chitosan/DNA nanoparticles suspension was dripped into scaffolds after the fabrication of scaffolds; therefore it can be considered that full amount of DNA was adsorbed into scaffolds and the encapsulation efficiency (EE) is recoded as 100%. For group C, the encapsulation efficiency is well below 100% but with a very satisfactory EE value ranging between 65-87%, as shown in Table 5.1. The relationship between EE and HAp content shown clearly in Table 5.1 is that the incorporation of HAp can significantly enhance the encapsulation efficiency. This phenomenon may be explained by the hydrophilicity of HAp nanoparticle. In the emulsion solution of PLGA-DCM-HAp-DNA/nanoparticles, most of the DNA/chitosan nanoparticles would try their best to attach each other together with HAp nanoparticles in order to escape from the direct contact of DCM. As a result, in the process of electrospinning, DNA/chitosan nanoparticles are incorporated into fibers together with HAp nanoparticles. This means that more HAp nanoparticles enable higher encapsulation Chapter 100 efficiency of DNA. In contrast, for the case without HAp (sample C1), DNA/chitosan nanoparicles would try their best to be away from the emulsion solution of PLGA/DCM and therefore in the process of electrospinning, DNA/chitosan nanoparticles in the water phase go to the top layer of PLGA/DCM solution and phase separation occurs especially in the later phase of electrospinning process. As pure aqueous phase can not be electrosprayed due to its low viscosity, phase separation of PLGA/DCM and water can lead to the lower encapsulation efficiency of DNA. 5.4.6 In vitro release study of DNA from different scaffolds Figure 5.5 shows the in vitro profiles of plasmid DNA release from different scaffolds in PBS at 37 ºC. Irrespective of the release modes, the release rate of plasmid DNA increases with increasing loading of HAp nanoparticles, but the whole release courses are quite different for various release modes (groups A, B and C). For group A, an initial burst shows up suddenly from the starting point till days 7-9 for samples A1, A2 and A3. In contrast, when the cumulative DNA release reached 80-85%, the remaining 15-20% of DNA could be released within the following 4-5 days. This time scale of release is comparable to other dosage forms, like nanoparticles and microparticles. On the other hand, the release curves for group B are quite different: There are no obvious bursts of release and their release rates in the initial stage (cumulative release < 80%) are much lower than those of group A. Furthermore, the release curve shows better sustained release than group A. This may be explained by the flexibility of naked DNA molecules. In the process of diffusion into PBS buffer, naked DNA molecules can change their three-dimensional structure flexibly to avoid the obstruction of intercrossing fibers. In contrast, DNA loaded chitosan nanoparticles are rigid such that they meet more difficulty Chapter 101 for overcoming the hindrance of fibrous framework. For group C, their release curves are more linear than groups A and B and their sustained release characteristics are more obvious. This is because DNA/chitosan nanoparticles for group C face higher diffusion resistance due to the presence of fiber matrix as a dominant barrier until a significant proportion of PLGA has degraded. Compared with the release periods of groups A and B, group C scaffolds shows much longer release course and 95% cumulative release of DNA can be reached in 45-55 days, which is much longer than the 20-26 days for group B and 5-10 days for group A, respectively. FESEM picture of the sample C1 cross section (shown in Figure 5.3) proved that DNA/chitosan nanoparticles were encapsulated inside fibers. Moreover, from the linear release profile observed from group C (shown in Figure 5.5c), it could be deduced that DNA/chitosan nanoparticles were located throughout the fibrous scaffold in a random (uniform) form. They could be located on the surface of fibers, inside the fibers but near to the surface, or near the core of fibers. This is similar to the distribution of HAp particles within the fibers; otherwise a biphasic release of DNA/chitosan nanoparticles is expected. 102 Chapter 110 Cumulative Percentage (%) 100 90 80 70 60 50 40 A1 A2 A3 30 20 10 0 12 18 24 30 36 42 48 54 60 66 Time (days) (a) 110 Cumulative Percentage (%) 100 90 80 70 60 50 B1 B2 B3 40 30 20 10 0 12 18 24 30 36 42 48 54 60 66 Time (days) (b) 110 Cumulative Percantage (%) 100 90 80 70 60 50 C1 C2 C3 40 30 20 10 0 12 18 24 30 36 42 48 54 60 66 Time (days) (c) Figure 5.5 In vitro release curves of three groups of scaffolds (groups A, B and C). 103 Chapter Group A Group C Group B Group C60 Figure 5.6 Electrophoretic mobility analysis of naked DNA (group A) and DNA/chitosan nanoparticles (groups B, C and C60, here C60 refers to group C in vitro sample released after 60 days) following chitosanase digestion. All samples other than C60 were taken after days of in vitro release and run on a 0.7% agarose gel and stained with ethidium bromide. For group A, lane 1: native pT7T3D-PacI DNA; lane 2: DNA released from scaffold A1; lane 3: DNA released from scaffold A2; lane 4: DNA released from scaffold A3. For groups B and C, lane 1: native pT7T3D-PacI DNA; lane 2: DNA/chitosan nanoparticles released from scaffold B1/C1; lane 3: DNA/chitosan nanoparticles released from scaffold B1/C1 + chitosanase digestion; lane 4: DNA/chitosan nanoparticles released from scaffold B2/C2; lane 5: DNA/chitosan nanoparticles released from scaffold B2/C2 + chitosanase digestion; lane 6: DNA/chitosan nanoparticles released from scaffold B3/C3; lane 7: DNA/chitosan nanoparticles released from scaffold B3/C3 + chitosanase digestion. Chapter 104 5.4.7 Integrity study of plasmid DNA released from scaffolds Results from agarose gel electrophoresis demonstrate that the released DNA has retained its structural integrity as evidenced by the distinct bands present on the gel (Figure 5.6). In other words, the released DNA has survived both the electrospinning process and postprocessing conditions (handling of scaffold, incubations, and lyophilization). From the electrophoretic patterns of groups B and C, we can see that the chitosan encapsulation of DNA is very satisfactory because no free DNA is detected on lanes 2, and of group B and C and only some small dots are found to stay on these lanes. These dots may be very fine particles or just impurity. This phenomenon shows that “4” is a perfect N/P ratio, which can ensure DNA/chitosan particles smaller than 100nm and close to hundred percent encapsulation of plasmid DNA. Comparing the electrophoretic pattern of DNA released from DNA/chitosan nanoparticle by chitosanase digestion and native DNA, one can confirm that chitosan encapsulation posed no observable side effect on DNA integrity. 5.4.8 hMSC attachment ability and cell viability test on scaffolds Figure 5.7 shows the cell attachment results on each fibrous scaffold, using the plate well without scaffold as a control. Number of cells on scaffold = Cell count in control experiment - Cell count in well after the removal of scaffold (5.3) Attachment ability = Number of cells on scaffold / Cell count in control experiment (5.4) The results show that the relationship between the ability of scaffold adhering to cells and HAp content is not very straightforward in each group, but the attachment ability 105 Chapter difference among groups A, B and C is very clear. The attachment ability of group C is the highest while that of group A is the lowest. Considering the morphologies of scaffold groups A, B and C, the modified PLGA/HAp scaffolds (after adding naked DNA or DNA/chitosan nanoparticle) show more compacted morphology even after the complete water evaporation in freeze dryer. Group C scaffolds are porous after electrospinning and drying and have more space to hold cells upon competing with TCPS wells to “arrest” cells. Based on this explanation, it is not difficult to understand the highest normalized attachment factor for group C scaffolds. + 0.50 + 0.45 CA scaffold / CA blank TCPS 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 A1 A2 A3 B1 B2 B3 C1 C2 C3 Groups Figure 5.7 Cell attachment (4h after cell seeding) of hMSCs on all nine types of scaffolds (tissue culture polystyrene well as control) (+p[...]... 5 .4. 6 In vitro release study of DNA from different scaffolds Figure 5.5 shows the in vitro profiles of plasmid DNA release from different scaffolds in PBS at 37 ºC Irrespective of the release modes, the release rate of plasmid DNA increases with increasing loading of HAp nanoparticles, but the whole release courses are quite different for various release modes (groups A, B and C) For group A, an initial... Chapter 5 1 04 5 .4. 7 Integrity study of plasmid DNA released from scaffolds Results from agarose gel electrophoresis demonstrate that the released DNA has retained its structural integrity as evidenced by the distinct bands present on the gel (Figure 5.6) In other words, the released DNA has survived both the electrospinning process and postprocessing conditions (handling of scaffold, incubations, and lyophilization)... starting point till days 7-9 for samples A1, A2 and A3 In contrast, when the cumulative DNA release reached 80-85%, the remaining 15-20% of DNA could be released within the following 4- 5 days This time scale of release is comparable to other dosage forms, like nanoparticles and microparticles On the other hand, the release curves for group B are quite different: There are no obvious bursts of release and. .. solution of PLGA/DCM and therefore in the process of electrospinning, DNA/chitosan nanoparticles in the water phase go to the top layer of PLGA/DCM solution and phase separation occurs especially in the later phase of electrospinning process As pure aqueous phase can not be electrosprayed due to its low viscosity, phase separation of PLGA/DCM and water can lead to the lower encapsulation efficiency of DNA... C3 40 30 20 10 0 0 6 12 18 24 30 36 42 48 54 60 66 Time (days) (c) Figure 5.5 In vitro release curves of three groups of scaffolds (groups A, B and C) 103 Chapter 5 1 2 3 4 1 2 Group A 1 2 3 4 Group C 3 4 5 6 7 5 6 7 Group B 5 6 7 1 2 3 4 Group C60 Figure 5.6 Electrophoretic mobility analysis of naked DNA (group A) and DNA/chitosan nanoparticles (groups B, C and C60, here C60 refers to group C in. .. sterilized using gamma radiation and placed in the wells of 96-well plates About 200μL of hMSC suspension was added into each well and the well plates were incubated in a humid atmosphere at 37 °C and 5% CO2 (5.0 x 1 04 cells/well) For cell attachment test, Chapter 5 93 after incubation for 4 hours, all scaffolds were rinsed and moved from wells and the cell number inside wells was assessed and compared... their release rates in the initial stage (cumulative release < 80%) are much lower than those of group A Furthermore, the release curve shows better sustained release than group A This may be explained by the flexibility of naked DNA molecules In the process of diffusion into PBS buffer, naked DNA molecules can change their three-dimensional structure flexibly to avoid the obstruction of intercrossing... B2 and B3 respectively) and 16% (8, 16 and 24% for C1, C2 and C3 respectively) of the DNA is released after 3 days This finding means that the high transfection efficiency of groups A and B can not sustain very long while group C scaffolds just start its release at day 3 and its high transfection efficiency may go on to 60 days The electrophoretic mobility analysis of the DNA/chitosan particles released... results in Section 5 .4. 8 have indicated that group B scaffolds have the highest cytotoxicity as compared with groups A and C A similar trend is again confirmed in Figure 5.9(b) where total protein concentrations in wells with the group B scaffolds are the lowest Another important point to note is that from the in vitro release test, about 60% (40 , 60 and 85% for A1, A2 and A3 respectively), 30% (18, 31 and. .. Canada) and incubated at 37 °C and 5% CO2 humid atmosphere in 75cm2 cell culture flasks The cells were extracted with PBS solution containing 0.25wt% trypsin and 0.02wt% ethylenediaminetetraacetic (EDTA) acid The cells were normally sub-cultured at a density of 2 x 1 04 cells/cm2 Cell attachment and viability test Before cell testing, all scaffolds were punched into round sections with diameter of 6mm, . DNA release from different scaffolds in PBS at 37 ºC. Irrespective of the release modes, the release rate of plasmid DNA increases with increasing loading of HAp nanoparticles, but the whole release. C1 C2 C3 T g (ºC) 48 .50 49 .83 50.50 48 .50 49 .83 50.50 49 .33 48 .67 46 .17 T d (ºC) 344 .33 367.33 375.83 344 .33 367.33 375.83 355.83. evaporation of the volatile organic solvents. 5.3.2 In vitro release test and determination of encapsulation efficiency (EE) In vitro release test of plasmid DNA Approximately 25mg of microfiber