Applications of electrospinning and supercritical carbon dioxide foaming techniques in controlled release and bone regeneration 2

CHAPTER 3 Three-Dimensional Fibrous PLGA/HAp Composite Scaffolds for Bone Morphogenetic Protein-2 BMP-2 Delivery †3.1 Introduction Bone fracture is a common form of injuries that bring

CHAPTER 3 Three-Dimensional Fibrous PLGA/HAp Composite Scaffolds for Bone Morphogenetic Protein-2 (BMP-2) Delivery †

3.1 Introduction

Bone fracture is a common form of injuries that bring great inconvenience to victims Bone grafting, as a common form of bone treatment, is done by transferring bone tissue from the donor site to the fractured site before adequate physiological treatment facilitates bone healing For autologous bone treatment, the bone tissues are usually transferred from the iliac crest of the patient’s pelvis (Swan and Goodarce, 2006) While the advantages of autologous bone treatment may include easy access of donor site without requiring the reposition of the patient as well as very low risks of infectious diseases, post-operation complications have been reported to be as high as 15% (Swan and Goodarce, 2006; Mischkowski et al., 2006) Furthermore, the donor site may not be able to provide sufficient bone tissue to the injury site (Huang et al., 2005) Other implications of grafting including severe pain, persistent aching, scarring and infection have also been reported (Swan and Goodarce, 2006; Huang et al., 2005)

Fortunately, there are less painful and risky alternatives to bone treatment A group of proteins, known as bone morphogenetic proteins (BMPs), are known to facilitate bone

† This chapter highlights the work published in H Nie, B.W Soh, Y.C Fu and C.H Wang Dimensional Fibrous PLGA/HAp Composite Scaffold for Bone Morphogenetic Protein-2 Delivery

Three-Biotechnol Bioeng 99 (1), 223-234 2008b

healing without transferring bone tissues By inducing marrow derived messenchymal stem cells (MSCs) to undergo chondroblastic and osteoblastic differentiation, BMPs can

induce bone regeneration in vivo (Saito et al., 2005) Among this group of proteins,

BMP-2 has been shown to induce healing in segmental bone defects Aebli et al (Aebli et al., 2005) and Saito et al (Saito et al., 2005) reported that BMPs improve bone regeneration

in vivo

Over past years, many release dosage forms have been developed for drug or protein delivery, like nanoparticle and microsphere However, one common problem is that the existence of a large burst over a narrow time period during the early stage of release In view of this problem, a new type of scaffold is needed urgently, especially for bone regeneration to overcome this challenge, because nanoparticles and microspheres are not suitable due to the non-ideal release profile and their fluidity as well which hinders them from localizing themselves and giving new bone tissues enough support Fiber is chosen

in the present study as the release dosage form because of its more favourable release properties and morphology Normally, a microsphere’s effectual release course can only sustain less than 30 days, which is far from enough for bone regeneration Fiber has much lower release rate of drug or protein than microsphere because of its smaller surface/volume ratio (Wei et al., 2006) Moreover, compared with microsphere, compacted fibrous scaffold can give cell stable three-dimensional growth environment and provide newly generated bone good support In this project, electrospinning (Kenawy

et al., 2003) is employed to fabricate fibers due to its flexibility of operations and the

fiber diameter can be easily controlled by changing operation parameters such as voltages, polymer concentrations and organic/aqueous mixture composition

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 bond directly to the bone since both of them have similar chemical structures Despite the above qualities, HAp is usually not used alone as its brittle nature creates difficulties in fabricating the transplant block to the exact shape of the bone defect configuration (Rebecca and Wozney, 2001) A study by Takaoka et al demonstrated that there is a lack

of bone healing when HAp is used alone as a carrier for BMP-2 (Takaoka et al., 1988)

The objective of the research project is to conduct an in vitro study of recombinant

BMP-2 encapsulated in fibrous scaffolds by investigating the effects of HAp content and the different methods of protein loading on the biological and physical characteristics of the micro-fibers fabricated using the electrospinning method The physical characteristics investigated are the surface morphology, thickness, crystallinity of HAp and residual DCM content The biological characteristics investigated are the cell attachment and cytotoxicity of the fibrous scaffolds Towards the end of this study, the protein

encapsulation efficiency, the in vitro release profile of the protein, the probability of

protein denaturation were also investigated

3.2 Materials and methods

3.2.1 Materials

Recombinant human bone morphogenetic protein-2 (rhBMP-2) (E coli expressed, Cat

No 355-BEC/CF) and its enzyme-linked immunosorbent assay (ELISA) kit were purchased from R&D Systems, Inc (Minneapolis, US) Poly (D,L-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 (AL, US) Dichloromethane (DCM) was manufactured in Tedia Company Inc (Fairfield, Ohio, US) Hydroxylapatite (HAp) nanoparticles of 100nm were purchased from Berkeley Advanced Biomaterials Inc

(Berkeley, CA, US) Phosphate Buffer Saline (PBS) buffer used for in vitro release study

was bought from Sigma Aldrich containing 0.1M sodium phosphate, 0.15M sodium chloride, pH 7.4 Dulbecco’s Modified Eagle Medium (DMEM), the cell culture medium

in the experiment, was 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, 25 mg/mL amphotericin B as Fungizone (Gibco) and 100mg/mL L-ascorbic acid from Sigma-Aldrich, Oakville (Ontario, Canada) and the cells were extracted using trypsin-EDTA Human marrow derived messenchymal stem cells (hMSCs) were purchased from Cambrex Bio Science Walkersville, Inc (Newington, NH, US) and the PreMix WST-1 Cell Proliferatiom Assay System was purchased from Takara Bio Inc (Otsu, Shiga, Japan)

3.2.2 Preparation of fibrous scaffolds

In all the experiments of the present work, the fibers were essentially fabricated from homogeneous emulsions formed from the sonication of organic and aqueous mixture Table 3.1 summarises the composition of the emulsion of the four different experimental cases 1-4 and the fibrous scaffolds fabricated are named F1-F4 respectively

Table 3.1 Compositions of emulsions for preparing different scaffolds F1-F4

electrospinning For F4, the BMP-2 solution was not added directly into the aqueous fabrication solution Instead, the protein was added to each fibrous scaffold sample of F4 after scaffold was fabricated and dried for 3 days using freeze dryer

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, homogeneous organic phase was formed In order to compare the effect of polymer concentration on fiber morphology, fibers using 10% and 20% PLGA/DCM solutions were also prepared

Preparation of aqueous phase

In the experimental cases F1-F4, 2 vials of BMP-2 of 10μg were dissolved in 90μL of 4mM hydrochloric acid (HCl) each and mixed well to produce a homogeneous BMP-2 solution In each of the experiments for case 1- case 3, 50μL BMP-2 solution containing 5μg of the protein was dissolved in deionised water and mixed well with specified weight

of HAp nanoparticles to give 800μL of homogeneous aqueous suspension

For experimental case 4, BMP-2 solution was not added directly into the aqueous solution Instead, the protein was loaded from a diluted BMP-2 solution (by 20 folds) using 50μL of the original BMP-2 solution with 750μL of deionised water All the solution would then be evenly added to the blank (meaning no encapsulation of BMP-2) F4 scaffold prepared beforehand To ensure that the viscosity of the emulsion is not affected by the organic-aqueous ratio, the volume of deionised water (HAp suspended without BMP-2) mixed with organic solution in experimental case 4 is 800μL to keep the same ratio 10:0.8 as in case 1-3

Fabrication of fibrous scaffolds

After adding the aqueous and organic phases together, the mixture was sonicated for

30-40 seconds and the resultant emulsion was transferred to a 10 mL glass syringe MATE interchangeable 10cc hypodermic syringe, Popper & Sons, Inc., New Hyde Park,

(MICRO-NY US) fitted with a 29-g needle and set up in the elecontrospinning apparatus The flow 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 10 kV with a solution flow rate

of 5mL /h The spinneret (anode) was fixed at about 15 cm above the aluminium-covered rotating collection drum (cathode) The syringe was loaded into the syringe pump and aluminium foil was wrapped around the spinning motor to collect the fiber samples

3.3 Characterization of scaffolds

3.3.1 Physical characterization

Morphology 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

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

3.3.2 Encapsulation efficiency (EE) determination

The encapsulation efficiency (EE) of the BMP-2 in the scaffolds is defined as the percentage of the actual BMP-2 loading to the total (theoretical) amount of BMP-2 loading In the EE analysis, about 5mg of each scaffold was dissolved in 1 mL of DCM and 5 mL of PBS (pH 7.4) was added subsequently The mixture was vortexed for 5 min

to extract BMP-2 Subsequently the system underwent centrifugation using a Hettich Zentrifugen system (Universal 32R, Andreas Hettich GmbH & Co KG, Tuttlingen, Germany) at 9000 rpm for 20 min to separate the oil and water phases At the same time, HAp nanoparticles settled to the bottom of tubes The water phase was then

concentration using the ELISA BMP-2 Immunoassay kit mentioned above The encapsulation efficiency of the BMP-2 in the fibers is the ratio of the actual amount of BMP-2 loaded into the fibers to the theoretical amount of BMP-2 loaded (Xie and Wang, 2005) by the following equation:

%100W

WW

WW

VC

EE

2 - BMP

HAp PLGA

2 - BMP sample

water 2

3.3.3 In vitro release studies

The in vitro release of BMP-2 was carried out over a period of 60 days and the

cumulative release curve can be plotted Approximately 25mg of microfiber samples made from each experiment were prepared and each of them was added to a tube with 5mL PBS, the release medium in the experiment The resultant mixture was placed in an

extracted at specific intervals (16h, days 1, 2, 3, 5, 7, 10, 12, 14, 16, 19, 23, 27, 30, 33, 36,

39, 42, 45, 48, 51, 54, 57 and 60) from each test tube 1 mL of 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 The ELISA kit was used to test the concentrations of BMP-2 inside the PBS solutions The optical density of each well was determined using the micro plate reader (Tecan Trading

AG, Switzerland), while setting the wavelength to 450nm with correction wavelength of 570nm

3.3.4 Protein integrity and secondary structure check

Continuous Native-polyacrylamide gel electrophoresis (continuous Native-PAGE)

To evaluate the effects of fabrication process on the molecular integrity and biological

activity of BMP-2, in vitro release sample was centrifuged and the supernatant was

analyzed by Native-PAGE to determine the integrity and conformation of BMP-2 In order to avoid stacking-induced aggregation, a continuous buffer system was used The electrophoresis buffer with pH 7.4 was prepared according to the MaLellan method (43

mM Histidine + 35mM HEPES) Each sample or native BMP-2 was diluted in sample

buffer [1.0 mL electrophoresis buffer, 3.0 mL glycerol, 0.2 mL 0.5% bromophenol blue, and 5.8 mL deionised water in each 10 mL sample buffer] before 10μl sample or native BMP-2 was loaded into each well of a 6% polyacrylamide gel and electrophoresis was conducted using a Bio-Rad Mini-PROTEAN 3 electrophoresis system (Cat No: 165-3301 and 165-3302) at a constant voltage difference (100V) Protein bands were detected by Coomassie G-250 staining using GelCode Blue Stain Reagent (24590, Pierce Biotechnology Inc., Rockford, IL, US) A Bioimaging system, Gene Genius (Syngene, Synoptics Ltd, Cambridge, United Kingdom) was used to image the gels

Fourier transform infrared Spectroscopy (FTIR)

FTIR spectroscopy, conducted using a Bio- Rad FTS3500 (Bio-Rad Laboratories, Cambridge, MA) was employed to explore the secondary structure of proteins in PBS solution A total of 32 scans at a resolution of 2 cm-1 were averaged for each sample To determine the secondary structure of protein, all spectra were analyzed by second derivatization in the amide I region (1700-1600cm-1) for their component composition and BMP-2 secondary structure quantified by Gaussian curve fitting after Fourier self-deconvolution of the corrected spectra by Peakfit 4.0 (SPSS Science) The area of each peak in the amide І region was calculated and used to determine the secondary structure

of the protein using procedures reported by Nahar and Riahi (Nahar and Riahi, 1996)

Tajmir-3.3.5 Cell culture, cell attachment and cytotoxicity studies

Cell culture

hMSCs 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 cytotoxicity test of scaffolds

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 (2.5 x 105 cells/mL) were added into each well and the well plates were incubated in a humid atmosphere at 37 °C and 5% CO2 For cell attachment test, after incubation for 4 hours, all scaffolds were rinsed and removed from wells and the cell number inside wells was assessed and compared with control to get the number of cell attached to each scaffold 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) For cytotoxicity testing, viable cell densities (after stained by MTT) in all wells were counted through microscope and compared after day 1, 2, 3, 4, 5,

6, 7 and 8, respectively

3.3.6 Statistical analysis

All the data were statistically analyzed to express the mean ± standard deviation (S.D.) and p<0.05 was accepted to be significant

3.4 Results and discussion

3.4.1 Physical characterization of fibrous scaffolds

Polymer concentration has crucial effect on fiber morphology, which can be proved by Figure 3.1 An overview of the SEM images shows relatively denser packed fibers of nano-sized range when increasing PLGA concentration In fact, the yield of fibers made from an emulsion with 10% PLGA is so low that SEM images could not be taken due to insufficiency of fibers formed Figure 3.1 shows a comparison between fibers made from emulsions with 10% PLGA (1A, 1B), 20% (2A, 2B) PLGA and 30% PLGA (3A, 3B) In terms of variations in polymer concentration, and hence the viscosity of resultant emulsion, the results obtained in this experiment is consistent with the results obtained by Berkland et al (Berkland et al., 2004)

Figure 3.2 shows the SEM images (magnified by 1000x) of the fibers produced in experimental cases 1-4, which are named as fiber samples F1, F2, F3 and F4 respectively All the fibers were produced from emulsion containing 30wt% PLGA and their thickness generally range from a few hundred nanometres to a few micrometers Furthermore, the fibers F1-F4 are densely packed in a three-dimensional manner Fabrication of such densely packed of thin micro- and nano-structured fibers creates potentially scaffold with

a large surface area for the release of BMP-2 as well as promoting cell interaction and growth (Lazzeri et al., 2005)

Figure 3.1 Comparison of the fibers formed from emulsions with different PLGA

concentrations 1A-1B: 10% PLGA, 2A-2B: 20% PLGA, 3A-3B: 30% PLGA, where A and B have different amplifications

Figure 3.2 SEM micrographs of fibrous scaffolds F1-F4 fabricated in experimental cases

1-4, respectively

Differential scanning calorimetry was performed to determine the physical state of HAp nanoparticles within the overall structure of the fabricated scaffolds In the DSC thermogram, as shown in Figures 3.3 and 3.4, all the fibrous samples including the pure PLGA fiber sample showed exothermic peaks at approximately 50 °C, which is the glass transition temperature of PLGA The glass transition temperature of PLGA obtained in this experiment is approximately consistent with the glass transition temperature for PLGA microspheres of about 50 °C obtained by Xie et al and about 40 ± 4 °C obtained

by Calis et al (Xie and Wang, 2006; Calis et al., 2002)