Applications of electrospinning and supercritical carbon dioxide foaming techniques in controlled release and bone regeneration 3

Optimized Bone Regeneration Based on Sustained Release from Three-Dimensional Fibrous PLGA/HAp Composite Scaffolds Loaded with Bone Morphogenetic Protein-2... A number of different grow

in bone repair and regeneration Treatment of fractured bone with cells, growth factors and biomaterials are the three commonly used ways over the past decade, but it has been proved that none of the ways can achieve the clinical requirement Recent research focus has moved to the application of cells, growth factors and biomaterials in combination Using biodegradable, polymeric materials with known biocompatibility, several researchers have fabricated porous, bioresorbable scaffolds for bone regeneration

† This chapter highlights the work published in Y.C Fu, H Nie, M.L Ho, C.K Wang and C.H Wang Optimized Bone Regeneration Based on Sustained Release from Three-Dimensional Fibrous PLGA/HAp

Composite Scaffolds Loaded with Bone Morphogenetic Protein-2 Biotechnol Bioeng 99 (4), 996-1006

2008

(Coombes and Heckman, 1992; Devin et al., 1996; Thomson et al., 1995)

Over past years, many release dosage forms have been developed for drug or protein delivery, like nanoparticle and microsphere However, one common problem is the existence of a large burst over a narrow time period during the early stage of release Fibre has much lower release rate of drug or protein than microsphere because of its

smaller surface/volume ratio (Wei et al., 2006) Biodegradable

poly(lactide-co-glycolide)/hydroxyapatite (PLAGA/HAp) composites have been shown to support the

attachment, growth, and low cytotoxicity in vitro (Nie et al., 2008b) In addition to the

use of biodegradable scaffolds, cells, growth factors and other biological moieties can be added to the matrix to promote and expedite bone formation A number of different growth factors, including bone morphogenetic proteins (BMPs), transforming growth factor β, platelet-derived growth factor, fibroblast growth factor and insulin growth factor

have been shown to stimulate bone growth, collagen synthesis, and fracture repair both in vitro and in vivo (Jingushi et al., 1995; Nixon et al., 1998; Pfeilschifter et al., 1993;

Scherping et al., 1997; Thaller et al., 1993) In particular, BMPs are osteoinductive proteins originally identified in demineralized bone (Urist and Nogami, 1970) They are known to facilitate bone healing without transferring bone tissues Among this group of proteins, BMP-2 has been shown to induce healing in segmental bone defects Aebli and colleagues (Aebli et al., 2005) and Saito and colleagues (Saito et al., 2005) reported that

BMPs improve bone regeneration in vivo, and BMP-2 has been found to induce healing

of segmental bone defects

In a previous study, BMP-2 loaded PLGA/HAp composite scaffolds were successfully fabricated, and these scaffolds were found to be able to obtain integrity of BMP-2 encapsulated, enhance cell attachment and cause negligible cytotoxicity (Nie et al., 2008b) The main objective of this study is to examine whether the PLGA/HAp composite fibrous scaffolds loaded with BMP-2 through electrospinning can improve bone regeneration Our hypothesis is that different loading methods of BMP-2 and different

HAp contents in scaffolds can alternate the release profiles of BMP-2 in vivo, therefore

modify the performance of scaffolds in bone regeneration The current study will first check the mechanical strength of scaffolds and HAp distribution in scaffolds Next, the

bioactivity of the produced BMP-2 will be evaluated in vivo using a tibia bone defect

model (see Appendix A1)

4.2 Materials and methods

4.2.1 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 (MN, US) Poly(D,L-lactide-co-glycolide) (PLGA)

(L/G ratio 50:50, MW 40,000-75,000) and chitosan (medium molecular weight and 85% deacetylated), were procured from Sigma Aldrich (St Louis, MO, US) HAp nanocrystals with average diameter of 100nm, dichloromethane (DCM) (Cat No DR-0440), 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

75-4.2.2 Preparation of fibrous scaffolds

In all the experiments of the present work, the fibres were essentially fabricated from homogeneous emulsions formed from the sonication of organic and aqueous mixture The

compositons of 4 kinds of scaffold samples and their fabrication can be found in Section

3.2.2 (page 35)

4.3 Characterization of scaffolds

4.3.1 Physical characterization of fibrous scaffolds

Mechanical Property 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 fibres produced

in each experiment, while the quality of the fibres was determined by tensile strength testing The mechanical properties of all fibrous scaffolds (F1, F2, F3, and F4) prepared

in a sheet form (15mm wide x 20mm long x ~150µm thick) were evaluated by applying a tensile load Tensile tests of all fibrous scaffolds were conducted by Instron 5848 Microtester with 10 mm/min cross-head speed with a 30mm gauge length Tensile stress

of each sheet was calculated on the nominal cross-sectional area of the tensile specimens

Residual Solvent Content in Scaffolds

One of the concerns of pharmaceutical application is the residual solvent content in the scaffolds fabricated although this factor has seldom been addressed by other fibre

fabrication groups Before performing further characterizations in vivo, gas

chromatography was used in the present study to determine the residual amount of

Dichloromethane (DCM) remaining in the scaffolds To quantify the amount of DCM in the HAp or/and BMP-2 loaded scaffolds, standard solutions with 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 The calibration samples were run using gas chromatography with mass spectrometry detector (GC-MSD) in order to determine the peak areas and retention time for DCM A calibration curve was obtained for peak area of different concentrations of DCM in DMF To obtain the residual amount of DCM in the PLGA/HAp scaffolds obtained from the electrospinning method, 15mg of each sample of F1-F4 were weighed and dissolved in 5 mL of DMF solution to extract DCM after freeze drying for 3 days inside a Martin Christ freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Germany) Next, about 1 mL of the solution was filtered into standard GC bottles and analyzed using the GC-MSD with an auto-sampler together with the calibration samples

4.3.2 In vivo experiments

All procedures were performed in accordance to specifications in the Guidelines for Animal Experiments of Kaohsiung Medical University and approved by the Institutional Animal Care and Use Committee (IACUC) As explained in Appendix A1, nude mice were anesthetized by intraperitoneal injection (3.5 mg/20 g body weight) of Ketamine (Ketalar®, Parke-Davis, Taiwan) combined with local anesthesia (Xylocain®, AstraZeneca PLC in Taiwan) One-mm-long tibia bone on the right side of a mouse was cut out with saw The part of bone cut was frozen using liquid nitrogen for 5 min Next,

the fragment was reversed and put back to its original site in the tibia and fixed on both ends with the other parts of the tibia using an intramedullary needle perfectly (similar to the application of intramedullary nail in human patients) A scaffold was embedded around the bone fracture The wounds were then closed with 4-0 silk sutures A bone fragment treated with liquid nitrogen but without seeding any scaffold was used as a control Each experiment was performed for 3 nude mice independently unless mentioned otherwise

Soft X-ray Observation

After 1, 2, 4, and 6 weeks, the tibia bone fractures were radiographically examined by soft X-rays (SOFTEX, Model M-100, Japan) at 43 KVP and 2mA for 1.5s Appropriate magnification was applied throughout the observation and the resultant micrographs were compared among all scaffolds together with control

Semi-Quantification of Fragment’s Contact with Tibia

A semi-quantification method is utilized to compare the performances of all scaffolds at specific intervals The tibia bone is hollow and each bone fragment has two ends Henceforth, each bone fragment is checked whether all of the 4 corners of the fragment are connected with tibia bone, or just 1, 2, or 3 of them is (are) connected with it For quantification, a score 0, 1, 2, 3, or 4 is assigned to 0, 1, 2, 3, or 4 contact respectively As each scaffold is tested in triplicate, the final score for each scaffold at each time point is recorded with the arithmetic mean of the scores

Serum BMP-2 Concentration and ALP Activity Measurements

Serum was collected through cardiac puncture and taken out for biochemical assays 1, 2,

4, and 6 weeks after implantation of all scaffolds The serum BMP-2 concentration was determined by BMP-2 ELISA kit To analyze the osteogenic differentiation of bone, the

System (Cat No BP300, Applied Biosystems, MA, US), which incorporates Tropix CSPD® chemiluminescent substrate and EmeraldTM luminescence enhancer for high sensitivity and wide dynamic range Serum sample was diluted 2 times with dilution buffer before 50μL of the resultant sample was transferred into microplate well Subsequently 50μL of assay buffer and 50μL of CSPD® substrate were added into sample

in well one by one The incubation times for assay buffer and CSPD® are 5 min and 20 min respectively Finally the ALP activity was detected by microplate luminometer

Histological Analysis and Immunostain of Bone Tissue

Concurrently, histochemical and immunohistochemical analysis was employed to check the micro-changes of bone tissue, as a supplement to the X-ray observation Prior to H&E and IHC staining, all samples of bone tissue were decalcificated [0.5M EDTA-2H2O in DDW (186.1g/L)], followed by fixation with 4% paraformaldehyde The resultant samples were embedded into paraffin wax and 5-μm sections were prepared Sections were routinely stained with hematoxylin-eosin Under the magnification of 400X, all lacunae within the range of bone fractures were counted and the percentages of lacunae with cells encapsulated were calculated and compared among all samples and control Immunohistochemical staining for Von Willebrand factor (vWF) was performed as

follows Sections were treated for 9 min with 0.15 mg/L of trypsin in phosphate buffer at

a pH of 7.8 and then incubated overnight at 4 ºC with 1:300 dilution of polyclonal rabbit antihuman vWF antibody (CHEMICON International, Inc.) Goat antirabbit biotinylated immunoglobulin (DakoCytomation, Denmark) was used at 1:300 dilution as the secondary antibody for 60 min at 37 ºC An avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, California) was applied at 1:300 dilution for 60 min at 37 ºC Peroxidase activity was detected by 0.4 mg/L of 3, 3’-diaminobenzidine in phosphate buffer at a pH of 7.3, in the presence of 0.12 percent of H2O2 Then sections were counterstained with hematoxylin

4.3.3 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

4.4 Results and discussion

Scaffolds F1-F4 are all densely packed in a three-dimensional manner (Nie et al., 2007) Fabrication of such densely packed of thin micro- and nano-structured fibres 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) Mechanical strength testing was carried out to examine the effect of the addition of HAp on the mechanical property The stress-strain (S-S) curve of the samples was monitored, and representative examples were shown in Figure 4.1 All 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 scaffold (F1), PLGA/HAp fibrous scaffolds exhibited a higher initial slope and lower strain at failure, which was similar to the results obtained by Velayudhan and colleagues (Velayudhan et al., 2004) It was noted that, among F1-F3, F2 showed the highest tensile strength, suggesting that the encapsulation of a suitable amount (5%) of HAp in PLGA contributed to the enhancement of mechanical strength This was likely due to the fact that HAp nanoparticles integrated well with PLGA and formed a compact inorganic-organic composite structure commonly seen in natural bone system Under the same loading of HAp (5%), F2 showed much higher maximum tensile strength and lower strain than F4 This means that the addition of protein solution before (for the case of F2) and after (for the case of F4) scaffold fabrication affects the mechanical properties of scaffolds

Figure 4.1 Comparison of the typical stress-strain curves of fibrous scaffolds

Table 4.1 DCM residual contents in the four groups of fibrous scaffolds (F1-F4)

a much more compact structure, and this could have hindered the evaporation of DCM from the scaffolds during the fabrication and drying process

From Table 4.1, it was also observed that the DCM residual content decreases with increasing HAp content From 408ppm in scaffold F1, the DCM content dropped to 340-350ppm in scaffolds F2 and F4, and dropped even further to almost 332ppm in scaffold F3 The hydrophilicity of HAp could be the contributing factor because the DCM is hydrophobic and could not exist together well with HAp in scaffolds, and DCM inside scaffolds with HAp nanoparticles is easier to evaporate because the scaffolds with HAp

nanoparticles (F2, F3 and F4) are more rigid and the strong three dimensional frameworks give much less resistance to DCM evaporation than F1 As expected, scaffolds F2 and F4 have the same HAp content and their DCM residual contents are fairly equal

Figure 4.2 Time-course of serum BMP-2 concentrations over six weeks after

implantation of fibrous scaffolds F1-F4 (+p<0.05 and *p<0.05 by t-test comparison

between the samples)

Figure 4.2 shows the serum BMP-2 concentrations 1, 2, 4, and 6 week(s) after implantation of scaffolds F2, F3 and control experienced similar time profiles of serum BMP-2 concentration and showed their maximal concentrations after 4 weeks, while F4 got the highest serum BMP-2 concentration after 2 weeks and dropped dramatically in the following weeks F1 showed significant difference from control, F2, F3 and F4 over 6 weeks, and F4 demonstrated significant difference to all other groups over the initial 2 weeks As bone healing is a spontaneous process, it is not a surprise to detect BMP-2 in

the serum of the control group An interesting phenomenon is that F1 shows even lower serum BMP-2 concentrations than control over the observation period of 6 weeks This may be explained by the cytotoxicity of F1 For pure PLGA scaffold F1, its degradation and resultant acidic environment, combined with its much higher residual solvent compared with other scaffolds as shown in Table 4.1, may destroy peripheral cells and hinder the spontaneous secretion of BMP-2 The in-vitro release profiles of BMP-2 from the 4 scaffolds in PBS have been reported in our previous work (Nie et al., 2008b) As shown in Figure 3.6 (Chapter 3), the percentage release rate of BMP-2 is the highest for scaffold F4 at the early stage, with more than 96% of the protein being released within the first 15 days of the in-vitro release study Since protein was loaded after the fibrous scaffolds were fabricated, the protein molecules were essentially located outside the fibres and remained in the interstitial spaces within the 3D network Hence, it is easier for the protein molecules to diffuse into the release medium without requiring the fibres to undergo biodegradation before they can be released For F1-F3, the scaffolds with higher HAp contents release BMP-2 faster, but they share very similar release profiles over a 2-month period, close to a linear mode

Concurrently, ALP activity in serum 1, 2, 4, 6 week(s) after implantation was investigated As shown in Figure 4.3, different time profiles were observed for different samples Control showed a gradual increase of ALP activity over 6 weeks, which was a sign of spontaneous repair of bone defect Similar to the control group, bone defect in F1 group experienced spontaneous healing and the ALP activity increased with time, with the highest ALP activity being observed at week 6 The general trend of ALP for F4 is