Applications of electrospinning and supercritical carbon dioxide foaming techniques in controlled release and bone regeneration 4

CHAPTER 5 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 affec

CHAPTER 5

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

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

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

in a transformant of Escherichia coli bacteria and isolated from the bacteria by

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 1 and an N/P ratio below 0.75 and above 2 yielded submicron size particles Nanoparticles prepared with an N/P ratio between 3 and 8 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 5 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 4 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.,

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 3 groups (A, B and C) and 9 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

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 5 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

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

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 5 mL PBS, the release medium in the experiment The resultant mixture was placed in an orbital shaker bath (GFL® 1092) at 37 °C, 120rpm 1

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 1 mL

of fresh PBS solution was then added to each mixture to make up 5 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

a working solution of 1 mg/L Subsequently, adequate chitosanase solution was applied

to each sample to degrade chitosan

Encapsulation efficiency determination

5mg of each scaffold was dissolved in 1 mL of DCM and 5 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 min 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:

%100W

WW

WW

VC

EE

DNA plasmid

HAp PLGA

DNA plasmid sample

water 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 3 day and 60 days For groups B and C,

DNA/chitosan nanoparticles before and after chitosanse digestion are both checked DNA

samples were diluted sixfold in gel loading buffer [composition: 25mg bromophenol blue + 4g sucrose and with further addition of water to 10 mL] A 6 μL volume of loading buffer/sample was loaded into each well of a 0.7% agarose gel and electrophoresis was conducted using a Bio-Rad Mini-PROTEAN III electrophoresis system (Cat No: 165-

3301 and 165-3302, Bio-Rad Laboratories, CA, US) at a constant voltage (100V) for 50 minutes with native plasmid DNA as control

5.3.4 Culture of hMSC

Cell growth

Human MSCs were purchased from Cambrex Bio Science Walkersville, Inc (East Rutherford, NJ), cultured in DMEM supplemented with 4mM-glutamine, 4.5g/L glucose, 25mM HEPES buffer, 10% fetal bovine serum (Gibco), 10U/mL penicillin G sodium, 10 mg/mL streptomycin, and 25 mg/mL amphotericin B as Fungizone (Gibco), 100mg/mL L-ascorbic acid (Sigma-Aldrich, Oakville, Ontario, 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 104 cells/cm2

Cell attachment and viability test

Before cell testing, all scaffolds were punched into round sections with diameter of 6mm, 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 104 cells/well) For cell attachment test,

after incubation for 4 hours, all scaffolds were rinsed and moved from wells and the cell number inside wells was assessed and compared with control to get the number of cell attached to each scaffolds within the first 4 hours The cell number can be counted by using a cell proliferation assay (PreMix WST-1 Cell Proliferation Assay System, Takara Bio Inc, Shiga, Japan) A control without any scaffold was used in the cell culture experiment The process of assessing cell metabolic activity (cell number indirectly) was repeated at first day, second day and third day for cell viability test In this test, scaffolds

in wells were not removed before cells were treated by MTS They were removed from the wells just before the absorbance at 490 nm was determined in order to cover both the cells in wells and scaffolds The cell viability can be calculated by (Xie and Wang, 2005):

Cell viability (%) = (Abs test cells / Abs control cells) x 100% (5.2)

Where “Abs test cells” represents the amount of formazan determined for cells treated with the different formulations and “Abs control cells” represents the amount of formazan determined for untreated control cells

In vitro experiment of hMSC transfection by different scaffolds

To measure the level of gene transfection of hMSC cultured, the scaffolds collected were washed three times with PBS, cut up with a scissors, and homogenized in the lysis buffer (0.1M Tris-HCl, 2mM EDTA, 0.1% Triton X-100) The sample lysate (2 mL) was centrifuged at 12,000 rpm for 5 min at 4 °C, and the supernatant was carefully collected and kept in the ice To measure the expression level of BMP-2 gene, 50 µl of the supernatant was collected and the BMP-2 protein was determined by a human BMP-2

ELISA Kit (R&D Systems) The total protein concentration of the lysate was also

Chemical Company) Each experiment was carried out three times independently

5.3.5 Statistical analysis

All the data were statistically analyzed to express the mean ± the standard deviation (S.D.)

of the mean and p<0.05 was accepted to be significant

5.4 Results and discussion

5.4.1 pT7T3D-Pac purity and concentration

In order to ensure DNA purity isolated from Escherichia coli bacteria, the absorbance

ratio at the wavelength of 260-280nm has to be maintained between 1.8 and 2.0 (Hosseinkhani et al., 2006) The ratio for the pT7T3D-PacI after purification by

demonstrated that DNA purity was accorded with requirement Using the PicoGreen dsDNA quantitation kit, the DNA concentration was determined to be 400 µg/mL It should be diluted four times to 100µg/mL for fabrication of DNA/chitosan nanoparticles

5.4.2 Preparation and characterization of the DNA/chitosan nanoparticles

Particles fabricated by using higher N/P ratios, like 5 or 6, are not much smaller than at the N/P ratio of 4 However, too much chitosan can cause many problems in the analysis

of DNA concentration in the in vitro release tests Therefore, the N/P ratio of 4 was used

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) A2 (5% HAp) A3 (10% HAp)

B1 (0% HAp) B2 (5% HAp) B3 (10% HAp)

C1 (0% HAp) C2 (5% 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