60 Chapter CHAPTER Optimized Bone Regeneration Based on Sustained Release of Bone Morphogenetic Protein-2 from Three-Dimensional Fibrous PLGA/HAp Composite Scaffolds† 4.1 Introduction Bone is one of the most commonly repaired organs of the body (Langer and Vacanti, 1993; Laurencin et al., 2000; Laurencin et al., 2001). Currently, treatment of fracture and bone loss associated with trauma, cancer and revision total joint arthroplasty remains a significant challenge in the field of orthopaedic surgery (Calvert et al., 2003; Crane et al., 1995; Goldstein et al., 1999; Huang et al., 2005; Constantz et al., 1995; Petite et al., 2000; Vacanti and Upton, 1994). Due to limitations associated with bone grafts, engineered biomaterials combined with growth factors have emerged as a new treatment alternative 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. Chapter 61 (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-coglycolide)/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. Chapter 62 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 7585% deacetylated), were procured from Sigma Aldrich (St. Louis, MO, US). HAp nanocrystals with average diameter of 100nm, dichloromethane (DCM) (Cat. No. DR0440), Ketamine Ketalar® and Xylocain® were purchased from Berkeley Advanced biomaterials Inc. (Berkeley, CA, US), Tedia Company Inc. (Fairfield, OH, US.), ParkeDavis Taiwan, and AstraZeneca PLC Taiwan, respectively. 63 Chapter 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 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 64 Chapter 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 mL of DMF solution to extract DCM after freeze drying for days inside a Martin Christ freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Germany). Next, about 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 min. Next, Chapter 65 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 nude mice independently unless mentioned otherwise. Soft X-ray Observation After 1, 2, 4, and 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 corners of the fragment are connected with tibia bone, or just 1, 2, or of them is (are) connected with it. For quantification, a score 0, 1, 2, 3, or is assigned to 0, 1, 2, 3, or 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. Chapter 66 Serum BMP-2 Concentration and ALP Activity Measurements Serum was collected through cardiac puncture and taken out for biochemical assays 1, 2, 4, and 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 placental alkaline phosphatase (ALP) activity was determined by Phospha-LightTM 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 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 and 20 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 Chapter 67 follows. Sections were treated for with 0.15 mg/L of trypsin in phosphate buffer at a pH of 7.8 and then incubated overnight at 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 at 37 ºC. An avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, California) was applied at 1:300 dilution for 60 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[...]... 2, 4, and 6 week(s) of implantation of scaffolds F1-F4 Bone fragment without implantation of any scaffold is taken as the control White arrows identify bone defects 75 Chapter 4 Table 4 .3 Comparison of performance scores for all samples at specific intervals* Control F1 F2 F3 F4 Week 1 0.00 0.00 0 .33 0.67 1 .33 Week 2 1.67 2.00 1.00 2.00 2.00 Week 4 2.67 3. 33 2.67 3. 33 3 .33 Week 6 3. 67 4.00 4.00 3. 67... motivation to design the release profile of scaffolds This study investigated two methods to load BMP-2 into three dimensional fibrous scaffolds using an electrospinning method, including encapsulating into fibres or coating on fibre surface, and the results revealed that BMP-2 encapsulated into fibres retained its biological activity in vitro and in vivo The addition of suitable amount of HAp nanoparticles... 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... in bone fragments after some treatment Von Willebrand factor is a large multimeric glycoprotein and produced constitutively in endothelium during blood vessel formation After treatment of IHC staining, vWF can be stained as brown color Therefore brown color staining detected in bone fragments can be seen as the formation and growth of new blood vessels H&E staining analysis above has shown bone healing... placed in the defects In this study, we focused on osteoinductive role of BMP-2 and HAp nanoparticles were acting as a regulator of BMP-2 release rate Higher amount of HAp can enhance BMP-2 release from scaffolds, as shown in the in vitro release profiles (Figure 3. 6) Our previous study shows that BMP-2 loaded pure PLGA fibrous scaffold (F1) can release BMP-2 in a sustained mode and the corresponding... staining can show the formation of blood vessels in bone fractures After treatment of bone fragments in liquid nitrogen for 5 minutes, cells and vessels in fragments should Chapter 4 78 have both been destroyed, which just represents the osteoconductivity of bone graft On the other hand, it can be seen as a good sign of bone regeneration if newly formed bone cells and blood vessels can be detected in. .. color staining is observed for control and F1, but F2-F4 show more of brown staining The observations demonstrate that the cells in bone fragments treated by F2-F4 are refreshing and growing, accompanied by neovascularization The neovasculization represents the new vessels ingrowth and this process can bring more bone marrow stromal cells to facilitate bone healing and substitute the dead bone more... strength and decrease residual solvent content Animal experiments demonstrated that BMP-2 loaded pure PLGA scaffold (F1) can’t keep the bioactivity of BMP-2 in vivo and has no effect on bone healing The bioactivity of BMP2 released from F2-F4, where BMP-2 was encapsulated inside fibres (F2 and F3) or just coated on the surface of fibres (F4), was well maintained in vivo and better performance of bone healing... indirect indicator for the rate of new bone formation The highest ALP level for the F4 group (against F1, F2 and F3) in the first 2 weeks verifies the most active new bone formation during this time period F3 releases BMP-2 in a sustained mode till week 6 because of HAp effect, but its effect on bone growth in vivo is not obvious at all It is postulated that the corresponding level of BMP-2 concentration did... well maintained in vitro, and their in vivo performances on defect healing are also better than F1 This observation proves that Chapter 4 81 F2, F3 and F4 can keep BMP-2 activity better than F1 4.5 Conclusions BMP-2 is easy to be digested by enzyme once it is exposed to serum in vivo Sustained release provides the best strategy to maintain high level of BMP-2 in the local area and that is the main motivation . Burlingame, California) was applied at 1 :30 0 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. 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. 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