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Sequential BMP-2/BMP-7 delivery from polyester nanocapsules Article in Journal of Biomedical Materials Research Part A · November 2009 DOI: 10.1002/jbm.a.32520 · Source: PubMed
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Sequential BMP-2/BMP-7 delivery from polyester nanocapsules P. Yilgor,1 N. Hasirci,1,2,3 V. Hasirci1,3,4 1 Department of Biotechnology, METU, BIOMAT, 06531 Ankara, Turkey 2 Department of Chemistry, METU, BIOMAT, 06531 Ankara, Turkey 3 Department of Biomedical Engineering, METU, BIOMAT, 06531 Ankara, Turkey 4 Biotechnology Research Unit, Department of Biological Sciences, METU, BIOMAT, 06531 Ankara, Turkey Received 18 September 2008; revised 5 January 2009; accepted 27 March 2009 Published online 7 July 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32520 Abstract: The aim of this study was to develop a nanosized, controlled growth factor release system to incorporate into tissue engineering scaffolds and thus activate the cells seeded in the scaffold. Nanocapsules of poly(lactic acid-co-glycolic acid) (PLGA) and poly(3-hydroxybutyrateco-3-hydroxyvalerate) (PHBV) were loaded with the bone morphogenetic proteins BMP-2 and BMP-7, respectively, and with bovine serum albumin (BSA), the model protein. BSA-loading efficiency and release kinetics were used to determine the most appropriate nanocapsule pair to achieve the delivery of growth factors in a sequential manner, as occurs in natural processes. BSA-encapsulation efficiency was highest when the polymer concentration used in the preparation of PLGA and PHBV nanocapsules was 10% (w/v) (84.75% and 16.72%, respectively). Release of BSA was faster from PLGA than it was from PHBV. Based on the encapsulation efficiency and release data, 10%
INTRODUCTION Bone morphogenetic proteins (BMPs) are members of a family of proteins that promote the formation of cartilage and bone by inducing mesenchymal stem cells (MSCs) toward chondroblastic and osteoblastic differentiation and causing them to proliferate in vivo.1–3 They are members of the transforming growth factor-beta (TGF-b) superfamily, and several of them, BMP-2 to BMP-18, were identified in humans. BMP-2, 4, 6, 7, and 9 are known to induce complete bone morphogenesis4; BMP-2 and 7 possess a strong ability to induce bone formation and Correspondence to: V. Hasirci; e-mail:
[email protected]. tr Contract grant sponsor: EU FP6 NoE Project Expertissues; contract grant number: NMP3-CT-2004-500283 Contract grant sponsor: Scientific and Technical Research Council of Turkey (TUBITAK) Project METUNANOBIOMAT; contract grant number: TBAG 105T508
Ó 2009 Wiley Periodicals, Inc.
PLGA and 10% PHBV nanocapsules were chosen to provide the early BMP-2 and later BMP-7 release, respectively. Simultaneous, sequential delivery and individual release of the BMPs were studied for 7, 14, and 21 days, using rat bone marrow mesenchymal stem cells. Individual BMP-2 release suppressed cell proliferation while providing higher alkaline phosphatase activity with respect to BMP7. The sequential delivery of BMP-2 and BMP-7 provided slightly lower proliferation than did simultaneous delivery, but the highest alkaline phosphatase activity of all indicated a synergistic effect on the osteogenic differentiation of mesenchymal stem cells caused by the use of the two growth factors in a sequential fashion. Ó 2009 Wiley Periodicals, Inc. J Biomed Mater Res 93A: 528–536, 2010 Key words: BMP; sequential delivery; nanocapsule; PLGA; PHBV
are the only ones that recently received the US Food and Drug Administration approval for use within collagen carriers for the applications of spinal fusion and sinus lift.5,6 Several BMP molecules participate in a cascade that regulates the fracture healing process by following different paths.7,8 It was shown in mice that7 in the absence of both BMP-2 and BMP-4, there was a severe decrease in the development of bone tissue, even though chondrogenic differentiation was taking place. In other in vivo studies, BMPs naturally secreted by the tissues were shown to appear sequentially to influence different aspects of skeletal development.9 In particular, BMP-2 was reported to be an early factor, peaking at day 1 after fracture, whereas BMP-14 peaks at day 7 during cartilage formation, and BMP-3, 4, 7, and 8 are expressed mainly after 2 weeks.10 Therefore, delivering BMPs in combination, particularly in a sequential manner by controlling their release time and duration, may have great advantage over the conventional strategies for bone tissue engineering applications.
SEQUENTIAL BMP-2/BMP-7 DELIVERY FROM POLYESTER NANOCAPSULES
After the realization of their potential of BMPs to induce bone regeneration in 1965 by Urist,3 numerous delivery strategies have been developed for their sustained release, as well as release of other growth factors, especially over the last decade. Both nano- and microsized delivery vehicles, mainly made of synthetic materials,11–14 natural polymers,4,15–18 and hydroxyapatite-based particles19,20 have been reported. Poly(lactic acid-co-glycolic acid) (PLGA) microspheres were used to deliver BMP-2 for over a 70-day period and increased alkaline phosphatase (ALP) activity was observed in both an in vitro and an in vivo rat model.11 Several other models that have been developed using BMP-delivering micro and nanoparticles of PLGA showed enhanced bone production when implanted in rat hind limb muscle pocket,12 rat femur,13 and calvarial defects in rabbits.14 rhBMP-7, encapsulated within PLGA nanoparticles which were then loaded into nanoporous poly(L-lactic acid) (PLLA) scaffolds, induced bone healing.21 Gelatin microparticles were used in a mouse model for rhBMP-2 delivery, and a lower burst release was observed when compared with PLGA particles.15 Particles made of other natural polymers, such as collagen,16 chitosan-alginate,4 and dextran,17 were also successfully used for the delivery of BMPs. Hydroxyapatite particles were produced at different temperatures, and the absorbability of BMP molecules on these particles were shown.19 Although copying nature could have been effective in bone healing, there are not many reports in the literature on mimicking the sequential delivery of growth factors. The effect of combined and sequential delivery of insulin-like growth factor-1 (IGF-1) and BMP-2 with two-layered, heterogeneously loaded, and crosslinked gelatin coatings was studied in vitro.22 The early delivery of BMP-2, followed by increased release of BMP-2 and IGF-1 after 5 days, resulted in the largest, and the earliest, elevation of ALP activity and mineralized matrix formation. Other studies involving the use of multiple growth factors included combinations of BMP-7/ IGF-1 or BMP-7/interleukin-6 (IL-6) which revealed increased ALP activity and bone nodule formation in osteoblastic cell cultures.23 Combined delivery of IGF-1 and TGF-b1 from biodegradable implant coatings has been shown to improve maximum load and torsional stiffness after improved bone formation.24 Sequential delivery of IGF-1 and TGF-b1 from PLGA microspheres was also studied, showing an ability to preserve bioactivity over a 70-day period.25 However, there are no reports in the literature on the controlled sequential delivery of BMP-2 and BMP-7 and of their synergetic effects for bone tissue engineering applications, except a recent study from our research group in which sequential delivery of BMP2 and BMP-7 was studied from complexed micro-
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spheres that were embedded in porous PLGA scaffolds.26 The positive effect of the coadministration of BMP-2 and BMP-7 on osteogenic differentiation was shown. The aim of the present study was to develop a nanoscale controlled delivery system that enables the sequential release of BMP-2 and BMP-7. A nanolevel delivery system is a powerful tool for tissue engineering applications, as such a system could easily be incorporated into various types of scaffolds, giving the scaffold the ability to release the active agent and generating a construct with direct control on the cells that are carried within. The delivery system was constructed using PLGA and PHBV nanoparticles with different degradation rates, therefore suspending the delivery of one of the growth factors while presenting the other one. The encapsulation efficiency, release kinetics, size distribution, degradation behavior, and BMP-release characteristics of the delivery pair were studied. The effect of single, simultaneous, and sequential BMP-2/BMP-7 delivery was illustrated in vitro on rat bone marrow origined MSCs though the application of the loaded nanocapsules on the MSC cultures.
MATERIALS AND METHODS Materials PLGA (50:50) (Resomer, RG503H) (i.v. 0.32–0.44 dL/g, 0.1% in chloroform, 258C) was purchased from Boehringer Ingelheim (Germany). PHBV (HV content 8% [w/w]), dexamethasone, b-glycerophosphate disodium salt, and L-ascorbic acid were bought from Sigma-Aldrich (Germany). Bovine serum albumin (BSA) and polyvinyl alcohol (PVA) (molecular weight [MW] 15,000) were obtained from Fluka. BMP-2 from InductOs kit (Medtronic, USA) and recombinant human BMP-7 from RayBiotech were used. For the determination of BMP-2 and BMP-7, the Quantikine BMP-2 immunoassay from R&D Systems and the human BMP-7 Elisa kit from Ray Biotech, respectively, were used. Dulbecco’s modified Eagle’s medium (DMEM; high glucose), fetal bovine serum (FBS) were obtained from Hyclone. NucleoCounter reagents were supplied by Chemometec (Denmark) and Alamar Blue cell proliferation assay was from USBiological. For the assessment of cell differentiation, alkaline phosphatase kit (Randox) was used.
Preparation of loaded nanocapsules Nanocapsules encapsulating model protein and growth factors were prepared by the double emulsion solvent evaporation technique.27 Briefly, an aqueous solution of the agent was emulsified in dichloromethane (0.6 mL) containing PLGA or PHBV (30, 60, and 120 mg, creating 5, 10, and 20% [w/v]) by probe sonication for 15 s (ultrasonic homogenizer, 4710 series; Cole-Parmer Instruments) at an Journal of Biomedical Materials Research Part A
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output of 50 W. The first emulsion (w1/o) was added into an aqueous solution of PVA (2 mL, 4% [w/v]) to form the second emulsion by sonication (w1/o/w2). The double emulsion was then added into PVA (50 mL, 0.3% [w/v]), and the organic solvent was evaporated overnight by vigorous stirring. Nanocapsules were collected by centrifugation (15,000 g, 10 min) and washed twice with Tris-HCl (pH 7.4). The nanocapsules were then resuspended in the buffer and lyophilized (2808C, 6 3 1022 mbar).
Scanning electron microscopy The structure of the polymeric nanocapsules was investigated by scanning electron microscopy (SEM) after sputter coating with gold (QUANTA 400F Field Emission SEM; The Netherlands).
Particle size distribution analysis Size distribution of nanocapsules was determined using a Zeta Potential and Mobility Measurement System (MALVERN Nano ZS90; UK).
Particle degradation For the assessment of the extent of in situ degradation, 5 mg of BSA-loaded nanocapsules (both PLGA and PHBV) were placed in 1 mL of sterilized phosphate-buffered saline (PBS; pH 7.4) in duplicate and incubated at 378C on a shaking plate. At 3, 10, 15, and 21 days, samples were centrifuged, washed twice with distilled water, and lyophilized before SEM examination.
In situ release studies Release kinetics and encapsulation efficiencies of the nanocapsules were studied using BSA-loaded particles, where BSA served as a model protein. The amount of released protein was determined using Coomassie Plus– The Better Bradford Assay (Pierce). Encapsulation efficiency was determined by disrupting the particles with dichloromethane, followed by repeated extraction with water. Released protein was then quantified using the assay. After optimization of the loading efficiency and release behavior, BMP-2 and BMP-7 were encapsulated in nanocapsules, and their release kinetics were determined with corresponding ELISA kits.
Cell culture Bone marrow MSCs were isolated from 6-week-old male Sprague–Dawley rats. The rats were euthanized, and their femurs and tibia were excised and washed with DMEM containing 1000 U/mL penicillin and 1000 lg/mL streptomycin under aseptic conditions. The marrow in the midshaft was flushed out with DMEM containing 20% FBS, 100 U/mL penicillin, and 100 lg/mL streptomycin, the cells were cenJournal of Biomedical Materials Research Part A
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trifuged at 500 g for 5 min, and the resulting cell pellet was resuspended and added to T-75 flasks. These primary cultures were incubated for 2 days. The hematopoietic and other unattached cells were removed from the flasks through repeated washes with PBS (10 mM, pH 7.4), and the medium in the flasks was renewed every other day until confluence. These primary cultures were then stored in liquid nitrogen until use. Incubation was performed at 378C and 5% CO2 in DMEM supplemented with 10% FBS, 10 mM b-glycerophosphate, 50 lg/mL L-ascorbic acid, 10 nM dexamethasone, and penicillin/streptomycin/amphotericin B. Cells (50,000) were seeded on tissue culture polystyrene and 3 h after seeding, UV-sterilized BMP-loaded nanocapsules were suspended in the growth medium and supplemented to the wells containing the cells. The amount of nanoparticles carrying 40 ng BMP was calculated from the encapsulation efficiency and introduced to individual wells. This necessitated the use of 2 mg of PLGA and 10 mg of PHBV nanoparticles per well. In the simultaneous and sequential cases, 40 ng of each BMP type were used, whereas in the single BMP cases, only 40 ng of the specific BMP was introduced. Medium from the wells was centrifuged; the collected particles were resuspended in fresh medium and then put onto the cells. Viable cell number was assessed with the Alamar Blue assay. The ALP activity was determined using Randox kit. The absorbance of the p-nitrophenol formed from p-nitrophenyl phosphate was determined at 405 nm, and the enzyme amount was calculated as described by the manufacturer. Experiments were carried out in triplicate.
Statistical analysis MSC proliferation and differentiation assays were carried out in triplicate. Data were analyzed with statistically significant values defined as p < 0.05, based on one-way analysis of variance (ANOVA), followed by Tukey’s test for the determination of the significance of difference among groups (p 0.05).
RESULTS AND DISCUSSION Particle structure and size In order to construct a sequential growth factor delivery system, two populations of nanoparticles were used; one to provide a fast release and the other, a relatively slower release, of the content. The biodegradable and biocompatible polymers PLGA and PHBV have both been successfully used earlier as nanoparticulate carriers of bioactive agents.11,21,25,28–30 The differences in release rates for these particles were due to the differences in their hydrophilicity, crystallinity, and degradation rates. These parameters have been earlier used in controlling release rates.31 Additional parameters are MW and the characteristics such as particle size and loading.32–34 PLGA degrades significantly more rapidly in vivo as compared to
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Figure 1. BSA-loaded capsules: (a) PLGA: 5% (33000); (b) PHBV: 5% (33000).
PHBV (99% vs. 43% in 6 months), although they have similar chemical structures.35 When all the other properties are comparable (MW, crystallinity), PLGA is expected to degrade faster. Therefore, in this study, the rapid release component of the sequential delivery system was constructed using PLGA particles, and the slower release component was prepared using PHBV particles. The production of PLGA and PHBV capsules was carried out using 5, 10, and 20% (w/v) polymer solutions. The use of 5% polymer solutions led to the formation of aggregate structures for both PLGA and PHBV and did not lead to the formation of
spherical capsules, possibly because of the very low viscosity (Fig. 1). Proper capsular structures were obtained using higher concentrations of both polymers. The SEM micrographs revealed that increasing the concentration of the polymer improved the shape of the nanoparticle and increased the capsule’s diameter (Fig. 2). An increase in polymer concentration from 10% to 20% led to an almost 5-fold (from ca. 300 to 1500 nm) increase in capsule diameter for the PLGA capsules and about 10-fold (from ca. 400 to 4000nm) for PHBV capsules. The capsules produced using 10% PLGA and PHBV were in the submicron range, whereas the 20% capsules were larger. The
Figure 2. BSA-loaded capsules: (a) PLGA: 10% (350,000; inset shows wall thickness of 220 nm); (b) PLGA: 20% (35000); (c) PHBV: 10% (320,000); (d) PHBV: 20% (31000). Journal of Biomedical Materials Research Part A
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Figure 3. Particle size distribution of 10% PLGA and 10% PHBV capsules.
Figure 4. Change in the pH of the medium during degradation of PLGA and PHBV nanocapsules.
wall thickness of the capsules was also observed to be about 200 nm for 10% PLGA particles [Fig. 2(a) inset]. The particle size distribution of the submicron range particles, 10% PLGA and 10% PHBV, were determined. PLGA capsules were found to have an average diameter of 327 nm, with the particle size in the 190–615 nm range. PHBV capsules had a larger mean diameter of 438 nm and a particle size range of 255–712 nm (Fig. 3). These measurements indi-
cated that both capsules were in the nanometer range and had smooth surface structures. Degradation of loaded PLGA and PHBV nanocapsules Because the delivery of the bioactive agent from the carrier depends partially on the degradation of the nanocapsules, their degradation behavior was investigated in situ. A change in the pH of the degra-
Figure 5. Degradation of BSA-loaded nanocapsules at 378C in sterile PBS (pH 5 7.4): (a) PLGA, day 15 (350,000); (b) PLGA, day 21 (350,000); (c) PHBV, day 15 (310,000); (d) PHBV, day 21 (310,000). Journal of Biomedical Materials Research Part A
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TABLE I BSA Encapsulation Efficiency of PLGA and PHBV Nanoparticles Polymer Concentration (%, w/v)
PLGA
PHBV
5 10 20
74.30 6 2.33 84.75 6 1.47 70.66 6 1.34
12.55 6 0.12 16.72 6 1.06 12.06 6 0.53
dation medium was recorded during 21 days of incubation for both PLGA and PHBV nanocapsules (Fig. 4). A decrease in the pH of the medium was observed with time for both particle types as a result of the hydrolytic degradation that produces acidic degradation products. The rate of decrease of the pH of the medium was faster at the beginning of the incubation period (first 3 days) for PLGA nanocapsules, as was expected based on the higher degradation rate of PLGA. The change in pH followed a similar pattern for the rest of the incubation period. The SEM analysis was conducted after the in situ degradation of the PLGA and PHBV nanocapsules. The loss in capsule integrity was more evident for PLGA capsules than for PHBV counterparts during the 21-day incubation period (Fig. 5), which indicates the potential of PLGA nanocapsules to constitute the early stage component of the sequential delivery system by providing a high release rate upon degradation. Encapsulation efficiency and release kinetics Encapsulation efficiency and release rates for the 5, 10, and 20% (w/v) PLGA and PHBV capsules were investigated initially using BSA as a model for BMPs and later with BMP-2 and BMP-7. For both PLGA and PHBV capsules, it was observed that the 10% polymer concentration was optimal for achieving maximum encapsulation of BSA (Table I). The encapsulation efficiency of PLGA capsules was almost 5-fold higher than that of their PHBV counterparts (84.75 6 1.47 vs. 16.72 6 1.06). The percent encapsulation values of the model protein with both PLGA and PHBV nanocapsules were in agreement with the literature: 78–81% of BMP-7 in PLGA nanospheres21 and 24% of L-asparaginase in PHBV nanocapsules.30 The release results of the model protein BSA from PLGA and PHBV capsules are presented in Figure 6(a,b) and Table II. For both capsule populations, sizes and release rates increased with increasing polymer concentration. This increase in release rate could be due to thinner capsule walls. The kinetics of release were investigated by fitting the data to rate equations, and the best fit was obtained with the Higuchi model36 (Table II), which indicates a dif-
Figure 6. BSA release from (a) PLGA capsules and (b) PHBV capsules with varying polymer contents.
fusion-based release from spherical matrix systems. The Higuchi rate constant k calculated from the earlier part of the release profile showed that with an increase in polymer concentration, the release rate also increases. After considering the release rates, encapsulation efficiency, and size distribution of the particles, the 10% (w/v) PLGA capsules were selected as the rapid release element and the 10% (w/v) PHBV capsules as the slower release element of the sequential delivery system. The BMP-2 release profile was similar to that of BSA, but slightly faster. On the other hand, BMP-7 TABLE II Kinetic Analysis of BSA, BMP-2, and BMP-7 Release from PLGA and PHBV Nanocapsules According to Higuchi Diffusion Model Polymer Type and Concentration (w/v) PLGA, PLGA, PLGA, PHBV, PHBV, PHBV, PLGA, PHBV,
5% 10% 20% 5% 10% 20% 10% 10%
Capsule Content
kH
r2
BSA BSA BSA BSA BSA BSA BMP-2 BMP-7
0.0306 0.0908 0.0924 0.0754 0.0789 0.1081 0.0987 0.0636
0.9454 0.9842 0.9141 0.9834 0.9869 0.9289 0.9525 0.9941
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Figure 7. BMP release from polymeric nanocapsules (BMP-2 from 10% PLGA nanocapsules; BMP-7 from 10% PHBV nanocapsules) (inset graph represents the kinetic analysis of BMP release according to Higuchi diffusion model).
release rate was slower than that of BSA (Fig. 7 and Table II). It was observed that combined together, they would form the sequential delivery system for the two growth factors. Influence of sequential BMP-2/BMP-7 delivery on MSC proliferation and differentiation After preparation and characterization of the sequential delivery system, the BMP-loaded nanoparticles were tested using rat bone marrow origined MSCs in order to assess their suitability for use in bone tissue engineering applications. The effect of single, sequential, and simultaneous delivery of BMP-2 and BMP-7 on cell proliferation and differentiation was studied. The conditions tested were: (1) single BMP-2 delivery (BMP-2-loaded PLGA nanocapsules), (2) single BMP-7 delivery (BMP-7-loaded PHBV nanocapsules), (3) sequential BMP-2 and BMP-7 delivery (BMP-2loaded PLGA nanocapsules and BMP-7-loaded PHBV nanocapsules), (4) simultaneous BMP-2 and BMP-7 delivery (BMP-2-loaded PLGA nanocapsules and BMP-7-loaded PLGA nanocapsules). The difference observed in cell proliferation and differentiation through different administration routes of BMPs suggests that PLGA and/or PHBV nanocapsules released the growth factors in a bioactive form for an extended period. The Alamar Blue test was used to quantify the cell proliferation in the presence of BMP-loaded particles (Fig. 8). The change in cell numbers was statistically significant (p < 0.001) among days 7, 14, and 21 for single delivery of BMP-7 and sequential and simultaneous delivery of BMP-2 and BMP-7. For single delivery of BMP-2, cell proliferation was not statistically significant (p > 0.05) among days 7, 14, and 21; however, there appeared to be a distinct increase in Journal of Biomedical Materials Research Part A
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Figure 8. Effect of single, simultaneous, and sequential delivery of BMP-2 and BMP-7 on MSC proliferation.
the proliferation with time. Generally, it was observed that the presence of growth factors improved cell proliferation. The increasing order of effectiveness was BMP-2 < BMP-7 < BMP-2/BMP-7 sequential delivery < BMP-2/BMP-7 simultaneous delivery for days 7, 14, and 21. In Figure 9, BMP-2 was more effective in inducing differentiation than BMP-7, and sequential delivery was better than the simultaneous delivery. In the literature, it is reported that proliferation and differentiation follow and counteract each other.37 In this study, we observed the same trend. The cell proliferation observed with simultaneous delivery of BMP-2/BMP-7 was higher than with sequential delivery, and higher differentiation was observed with sequential delivery, as expected. A similar comparison can be made for the BMP-2 and the BMP-7 samples. Simultaneous application of BMP-2 and BMP-7 by encapsulating them both into PLGA nanocapsules (BMP-2/BMP-7 simultaneous) enabled the release of contents in the very first days of incubation and led to increased cell proliferation. Sequential delivery of these two bone growth factors, on the other hand, led to the highest ALP activity. If the ALP production efficiency is plotted as activity per cell, the BMP-2 value becomes the highest; however, we are concerned with bone
Figure 9. Effect of single, simultaneous, and sequential delivery of BMP-2 and BMP-7 on MSC differentiation into osteoblastic cells.
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healing and the highest amount of ALP in the given population is more important than that per cell. The highest value obtained with sequential delivery indicates the importance of mimicking natural conditions; BMP-2 was shown to appear in the first days after fracture, whereas BMP-7 peaked after 2 weeks.10 The increase in ALP activity was statistically significant (p < 0.001) among days 7, 14, and 21 for every delivery condition tested. This improvement was achieved through the developed sequential delivery system.
CONCLUSION Conventional strategy in bone tissue engineering involves the use of cells with biodegradable scaffolds and growth factors to form viable three-dimensional constructs. However, in such systems, various growth factors are provided at the beginning and simultaneously, unlike in nature, where the process is gradual and sequential. To provide this time pattern and also to increase the stability of the growth factors in the cell culture medium, BMP-2 and BMP7 were encapsulated in nanosized biodegradable and biocompatible capsules. Sequential delivery was achieved using populations of nanocapsules (different encapsulation efficiency, release rate, different growth factor) with different chemistry. As predicted, the sequentially provided growth factors led to the highest level of differentiation, implying that mimicking nature in providing the growth factors was effective. Because the nanosized sequential delivery system can be incorporated into the scaffold structures without affecting the structural and mechanical properties of the construct, our newly developed system could constitute an in-built growth factor delivery system for the engineering of many different tissues.
References 1. Wang EA, Rosen V, D’Alessandro JS, Bauduy M, Cordes P, Harada T, Israel DI, Hewick RM, Kerns KM, LaPan P, Luxenberg DP, McQuaid D, Moutsatsos IK, Nove J, Wozney JM. Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci USA 1990;87:2220–2224. 2. Hammonds RG, Schwall R, Dudlev A, Berkemeier L, Lai C, Lee J, Cunningham N, Reddi AH, Wood WI, Mason AJ. The bone inducing activity of recombinant BMP-2A and BMP-2B and the purification and characterization of mature BMP-2B from an hybrid BMP-2A/2B precursor. Mol Endocrinol 1990;4:149–155. 3. Urist MR. Bone: Formation by autoinduction. Science 1965; 150:893–899. 4. Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: The road from the laboratory to the clinic, part I (basic concepts). J Tissue Eng Regen Med 2008;2:1–13.
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5. White AP, Vaccaro AR, Hall JA, Whang PG, Friel BC, McKee MD. Clinical applications of BMP-7/OP-1 in fractures, nonunions and spinal fusion. Int Orthop 2007;31:735–741. 6. McKay WF, Peckham SM Badura JM. A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE1 Bone Graft). Int Orthop 2007;31:729–734. 7. Bandyopadhyay A, Tsuji K, Cox K, Harfe BD, Rosen V, Tabin CJ. Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet 2006; 2:e216. 8. Saito N, Murakami N, Takahashi J, Horiuchi H, Ota H, Kato H, Okada T, Nozaki K, Takaoka K. Synthetic biodegradable polymers as drug delivery systems for bone morphogenetic proteins. Adv Drug Deliv Rev 2005;57:1037–1048. 9. Hogan BL. Bone morphogenetic proteins: Multifunctional regulators of vertebrate development. Genes Dev 1996;10: 1580–1594. 10. Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J Bone Miner Res 2002;17:513–520. 11. Kempen DHR, Lu L, Hefferan TE, Creemers LB, Maran A, Classic KL, Dhert WJA, Yaszemski MJ. Retention of in vitro and in vivo BMP-2 bioactivities in sustained delivery vehicles for bone tissue engineering. Biomaterials 2008;29:3245–3252. 12. Jeon O, Song SJ, Yang HS, Bhang SH, Kang SW, Sung MA, Lee JH, Kim BS. Long-term delivery enhances in vivo osteogenic efficacy of bone morphogenetic protein-2 compared to short-term delivery. Biochem Biophys Res Commun 2008; 369:774–780. 13. Lee SC, Shea M, Battle MA, Kozitza K, Ron E, Turek T, Schaub RG, Hayes WC. Healing of large segmental defects in rat femurs is aided by RhBMP-2 in PLGA matrix. J Biomed Mater Res 1994;28:1149–1156. 14. Schrier JA, Fink BF, Rodgers JB, Vasconez HC, DeLuca PP. Effect of a freeze-dried CMC/PLGA microsphere matrix of rhBMP-2 on bone healing. AAPS PharmSciTech 2001;2: article 18. 15. Patel ZS, Yamamoto M, Ueda H, Tabata Y, Mikos AG. Biodegradable gelatin microparticles as delivery systems for the controlled release of bone morphogenetic protein-2. Acta Biomater 2008;4:1126–1138. 16. Wang YJ, Lin FH, Sun JS, Huang YC, Chueh SC, Hsu FY. Collagen-hydroxyapatite microspheres as carriers for bone morphogenic protein-4. Artif Organs 2003;27:162–168. 17. Chen FM, Zhao YM, Zhang R, Jin T, Sun HH, Wu ZF, Jin Y. Periodontal regeneration using novel glycidyl methacrylated dextran (Dex-GMA)/gelatin scaffolds containing microspheres loaded with bone morphogenetic proteins. J Controlled Release 2007;121:81–90. 18. Tabata Y, Yamada K, Miyamoto S, Nagata I, Kikuchi H, Aoyama I, Tamura M, Ikada Y. Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels. Biomaterials 1998;19:807–815. 19. Matsumoto T, Okazaki M, Inoue M, Yamaguchi S, Kusunose T, Toyonaga T, Hamada Y, Takahashi J. Hydroxyapatite particles as a controlled release carrier of protein. Biomaterials 2004;25:3807–3812. 20. Akazawa T, Murata M, Sasaki T, Tazaki J, Kobayashi M, Kanno T, Nakamura K, Arisue M. Biodegradation and bioabsorption innovation of the functionally graded bovine boneoriginated apatite with blood permeability. J Biomed Mater Res A 2006;76:44–51. 21. Wei G, Jin Q, Giannobile WV, Ma PX. The enhancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMP-7 nanospheres. Biomaterials 2007;28:2087–2096. 22. Raiche AT, Puleo DA. In vitro effects of combined and sequential delivery of two bone growth factors. Biomaterials 2004;25:677–685.
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23. Yeh LC, Zavala MC, Lee JC. Osteogenic protein-1 and interleukin-6 with its soluble receptor synergistically stimulate rat osteoblastic cell differentiation. J Cell Physiol 2002;190:322– 331. 24. Raschke M, Wildemann B, Inden P, Bail H, Flyvbjerg A, Hoffmann J, Haas NP, Schmidmaier G. Insulin-like growth factor-1 and transforming growth factor-b1 accelerates osteotomy healing using polylactide-coated implants as a delivery system: A biomechanical and histological study in minipigs. Bone 2002;30:144–151. 25. Jaklenec A, Hinckfuss A, Bilgen B, Ciombor DM, Aaron R, Mathiowitz E. Sequential release of bioactive IGF-I and TGFb1 from PLGA microsphere-based scaffolds. Biomaterials 2008;29:1518–1525. 26. Basmanav FB, Kose GT, Hasirci V. Sequential growth factor delivery from complexed microspheres for bone tissue engineering. Biomaterials 2008;29:4195–4204. 27. Baran ET, Ozer N, Hasirci V. Poly(hydroxybutyrate-cohydroxyvalerate) nanocapsules as enzyme carriers for cancer therapy: An in vitro study. J Microencapsul 2002;19: 363–376. 28. Kang SW, Lim HW, Seo SW, Jeon O, Lee M, Kim BS. Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs. Biomaterials 2008;29:1109–1117. 29. Kim HK, Chung HJ, Park TG. Biodegradable polymeric microspheres with ‘‘open/closed’’ pores for sustained release of human growth hormone. J Controlled Release 2006;112:167– 174.
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YILGOR, HASIRCI, AND HASIRCI
30. Baran ET, Ozer N, Hasirci V. In vivo half life of nanoencapsulated L-asparaginase. J Mater Sci: Mater Med 2002;13:1113– 1121. 31. Burgess DJ, Hickey AJ. Microsphere technology and applications. In: Swarbrick J, Boylan JC, editors. Encyclopedia of Pharmaceutical Technology. New York: Marcel Dekker; 1994. p 1–29. 32. Lemaire V, Belair J, Hildgen P. Structural modeling of drug release from biodegradable porous matrices based on a combined diffusion/erosion process. Int J Pharm 2003;258:95–107. 33. Zolnik BS, Leary PE, Burgess DJ. Elevated temperature accelerated release testing of PLGA microspheres. J Controlled Release 2006;112:293–300. 34. Cui F, Cun D, Tao A, Yang M, Shi K, Zhao M, Guan Y. Preparation and characterization of melittin-loaded poly(DL-lactic acid) or poly(DL-lactic-co-glycolic acid) microspheres made by the double emulsion method. J Controlled Release 2005;107:310–319. 35. Kok F, Hasirci V. Polyhydroxybutyrate and its copolymers: Applications in the medical field. In: Yaszemski MJ, Trantolo DJ, Lewandrovski KU, Hasirci V, Altobelli DE, Wise DL, editors. Tissue Engineering and Novel Delivery Systems. New York: CRC Press; 2003. p 543–562. 36. Higuchi T. Rate of release of medicaments from ointment bases containing drugs in suspensions. J Pharm Sci 1961;50: 874–875. 37. Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev 1993;14: 424–442.