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Polycaprolactone and Bovine Serum Albumin Based Nanofibers for Controlled Release of Nerve Growth Factor Chandra M. Valmikinathan, Steven Defroda, and Xiaojun Yu* Department of Chemistry, Chemical Biology, and Biomedical Engineering, Stevens Institute of Technology, Hoboken, New Jersey 07030 Received October 30, 2008; Revised Manuscript Received February 24, 2009

Tissue engineering approaches for peripheral nerve regeneration employ biodegradable scaffolds coupled with growth factors for improved performance in regeneration of large nerve injuries. Electrospun nanofibers provide a versatile platform for fabrication of scaffolds with extracellular matrix like architecture and increased surface area. Incorporation of growth factors in nanofibers have been previously demonstrated using oil in water emulsion techniques but are associated with burst release and loss of valuable growth factor. Here, we show a novel blend of polycaprolactone and bovine serum albumin (BSA) to form nanofibers containing nerve growth factors. The BSA helps in overcoming the most common drawbacks associated with hydrophobic polymers such as reduced loading efficiency, long degradation periods, and burst release. The controlled release of nerve growth factor (NGF) from the nanofibers was evaluated using enzyme linked immune sorbent assay (ELISA) and PC12 based bioassay over a 28 day time period. A sustained release of NGF was obtained for at least 28 days. PC12 bioassays confirmed the bioactivity of the NGF, and showed that the released NGF was sufficient to induce neurite outgrowth from PC12 cells throughout the period of release, therefore, demonstrating the successful incorporation and controlled release potential of PCL BSA scaffolds.

1. Introduction Nerve growth factor (NGF), the most prominent and the commonly used neurotrophic factors for peripheral nerve regeneration, plays a key role in providing a signal cascade for the influx of the Schwann cells to the site of the injury, process of myelination, and providing guidance for axons along the gap by creating a diffusional cue for a regrowth of the axons into the distal ends.1-3 Therapeutic application of NGF has been employed for regeneration of severed nerve gaps in the peripheral nervous system and also is known to influence some repair and regeneration in the central nervous system, including Alzheimer’s disease and traumatic spinal cord injury.4,5 For its therapeutic application, NGF is generally encapsulated into nerve grafts and released in a controllable fashion over 4 weeks for optimal nerve regeneration.6-8 Contemporary encapsulation strategies for NGF loading and release include microspheres,9 nanofibers,10,11 and phase separated scaffolds12 fabricated from slow degrading biodegradable materials. NGF delivery systems fabricated from hydrophobic polymers such as poly(lactide-co-glycotide) (PLGA), have been previously demonstrated and are often associated with an initial burst profile, releasing large quantities of the loaded protein as shown by their release profiles.6,12-14 A similar polymer, polycaprolactone (PCL), has also been employed for scaffold fabrication and controlled release of growth factors15,16 and is also associated with several drawbacks such as reduced loading efficiency, initial burst release, as well as extremely long degradation periods of around 2 years.17 To overcome the drawbacks associated with such polymers, several groups have synthesized novel polymer systems with PCL as the backbone and side groups containing other polymer moieties like ethyl * To whom correspondence should be addressed. Phone: 201 216 5256. Fax: 201 216 8306. E-mail: [email protected].

ethylene phosphate (EEP)18 and ethylene glycol19 to reduce hydrophobicity as well as hasten the degradation period. Similarly, blends of polycaprolactone20,21 with other natural or synthetic polymers like gelatin and ethylene glycol have been used for controlling the above-mentioned parameters. It has also been earlier reported that scaffolds generated from nonbiodegradable materials like ethyl vinyl acetate (EVA) were used for controlled release of NGF.22 To create the release from the nerve guidance channels, 20-30% of bovine serum albumin (BSA) was used during scaffold fabrication and the degradation of BSA would create the pores necessary for release of NGF around the site of injury.23 Also, BSA is added as a protectant to the NGF to prevent protein destruction under extremely hostile conditions involving an organic solvent during scaffold fabrication and protein encapsulation.24,25 Blends of a hydrophobic polymer, like PCL, with bovine serum albumin have not been employed for tissue engineering applications, especially in the peripheral nervous system regeneration for controlled release of NGF. The blends of PCL with BSA could alleviate some current drawbacks of PCL, aiding in controlled release of NGF over the desired time range. Electrospinning has drawn a lot of attention recently26,27 as an alternative method to fabricate scaffolds, owing to its increased surface areas, ability to mimic the extra cellular matrix, its versatility to create fibrous scaffolds from a wide range of starting materials, and as a carrier to deliver clinically relevant proteins like growth factors.28 Current electrospinning approaches for encapsulation of growth factors or proteins involve dispersion of the growth factor in preliminary prepared water in oil emulsion of the protein and the polymer in the organic solvent, leading to reduced loading efficiency, phase separation, and increased protein presence on the surface of the fibers rather than uniformly inside the fibers.29 The modified technique includes the modification of the electrospinning setup to produce

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Table 1. Weight Ratios of PCL-BSA Release Samples

BSA PCL ratio (wt/wt)

Figure 1. Release rates of BSA from various blends of PCL-BSA over a 28 day period.

core-shell structure,30,31 which lead to complicated assembly and does not modify the degradation rates of the polymer system. In the present study, we investigated electrospun scaffolds fabricated from blends of PCL and BSA as a potential carrier for nerve growth factor. Various blend ratios of PCL and BSA were created and tested for appropriate releasing rates, matching a typical nerve regeneration period of around 4 weeks. Assessment of the controlled release property revealed that BSA was released from the scaffold over 28 days. The sustained release potential of these scaffolds indicates potential application of such blends in peripheral nerve regeneration applications.

2. Materials and Methods 2.1. Electrospinning. 2.1.1. Releasing Rates of PCL-BSA Scaffolds. PCL-BSA was prepared in various ratios to understand the release of BSA from the scaffolds. PCL solution was made by dissolving 400 mg of PCL in 5 mL hexafluoro isopropanol (HFIP) to form 8% (wt/vol) solution of PCL. The BSA solution was prepared by dissolving 20 mg of BSA in 4 mL of HFIP solution (5 mg/mL), under constant stirring overnight. Three different ratios of blends of BSA to PCL (80:20, 50:50, and 20:80 (vol/vol)), namely, samples A, B, and C, respectively, were prepared by mixing PCL and BSA solutions (weight percentages as shown in Table 1). All electrospinning experiments were made using 0.6 mL/min, 10 cm distance, and a voltage of 12 kV. Fibers were collected on aluminum foils and 40 mg of fibers were transferred to 12-well plates containing 1 mL of sterile phosphate buffered saline (PBS). The PBS was replaced with fresh PBS every day and the collected supernatant was stored at -80 °C until they were analyzed. BSA release was analyzed using Bradford BSA assay kit (Biorad). All data was collected in triplicate and reported as mean ( standard deviation. 2.1.2. Electrospinning NGF Containing PCL and PCL-BSA Nanofibers. To prepare nanofibers for in vitro release and bioactivity studies, 1 mL of 10 µg/mL NGF in PBS was blended with 1 mL BSA (5 mg/mL in HFIP) and 4 mL of 8% (wt/vol) PCL solution in hexafluoro isopropanol (HFIP) under constant stirring overnight. A PCL to BSA ratio, corresponding to sample C in the previous study, was used owing to its matching controlled release properties of onedimensional tube like constructs under Fickian conditions as estimated by the degradation release rates (Figure 1). The polymer was transferred to a syringe with a blunt end for even distribution. A steady flow was established by using a syringe pump operating at 0.6 mL/h. A voltage of 12 kV was applied to the tip of the syringe, allowing the fibers to

sample A (mg)

sample B (mg)

sample C (mg)

4 16 0.25

2.5 40 0.06

1 64 0.01

be collected on a sheet of aluminum foil, grounded, and separated from the needle by 10 cm. As a control, 1 mL of NGF (10 µg/mL) in PBS was blended to form water in oil emulsion with 4 mL of 8% PCL (wt/vol) in HFIP was prepared under similar electrospinning conditions. Fibers were cut into 1 × 1 cm squares in triplicate and placed into 24-well plates for release studies. 2.2. Morphological Characterization of Nanofibers. The electrospinning process described above generated nanofibers, which were characterized using scanning electron microscopy and confocal microscopy, to analyze the fiber diameter, the uniformity of the fibers formed, and the encapsulation of nerve growth factor inside the fibers. 2.2.1. Scanning Electron Microscopy. The electrospun fiber meshes were transferred to loading stubs and were gold-coated at 40 amps for 30 s. The samples were loaded in a Leo 982 FEG-SEM and high and low magnification images were obtained at 2 kV and 7 mm working distance, to characterize the fiber morphologies. The fiber diameters of the PCL-BSA-NGF nanofibers were measured using ImagePro software. 2.2.2. Confocal Microscopy. The PCL-NGF and PCL-BSA-NGF fibers obtained in the previous steps were stained with primary rabbit antimouse NGF at a dilution of 1:1000 in phosphate buffered saline (PBS) and were allowed to bind to the NGF on the fibers overnight at 4 °C. The scaffolds were washed with PBS and were stained with Texas red anti mouse antibody for 2 h followed by three washes in PBS. Also, to confirm any nonspecific binding of the secondary antibody to the nanofibers, NGF containing PCL-BSA nanofibers were left in Texas red anti mouse antibody for 2 h and then thoroughly washed in PBS prior to imaging. The fibers were then imaged in a Nikon C1 confocal microscope to study the distribution of NGF in the fibers. 2.3. Encapsulation Efficiency of NGF in Nanofibers. The loading level and encapsulation efficiency of NGF in the PCL-BSA and PCL nanofibers were determined based on dissolution of PCL and PCLBSA scaffolds containing NGF in 1 mL 1:1 dichloromethane/PBS solution with 20 mg of scaffolds in triplicate. This would cause the phase transfer where the PCL would move to the organic phase and the BSA and NGF would move to the aqueous phase. The mixture was vortexed for 1 min followed by centrifugation at 6000-8000 rpm for 5 min. Post-separation, the supernatant (PBS solution containing NGF and BSA) was removed and analyzed using Biorad assay kit and NGF ELISA kit. All samples were tested in triplicate. The entrapment efficiency was calculated as the ratio of NGF loaded, estimated from the supernatant, to the total NGF added to the PCL or PCL-BSA during scaffold fabrication. 2.4. NGF Release. PCL-BSA NGF and PCL-NGF nanofibers were cut to a small pieces (1 × 1 cm) each and were placed in 1 mL of serum-free Roselle park Medical Institute (RPMI) medium supplemented with 0.01% fungizone and 1% penicillin streptomycin in 24well plates and placed in a water bath at 37 °C. Release samples were taken at the predetermined time points (days 1, 4, 7, 14, 21, and 28), replaced with fresh medium, and stored at -20 °C until they were needed for quantification using ELISA or PC12 bioassay as per Aebischer et.al.22,23 All samples collected were analyzed were evaluated for statistical significance and reported as mean ( standard deviation. 2.5. Circular Dichroism of Encapsulated and Released NGF and BSA. Protein dissolution in HFIP for controlled release applications has never been reported before, especially for nerve growth factor loading. To study the structural integrity of NGF and BSA in HFIP, circular dichroism was performed on the loaded NGF, extracted from the nanofibers. To obtain the NGF from the fibers, the PCL-BSA-NGF and the PCL-NGF fibers were left overnight in 2 mL of 50:50

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dichloromethane and PBS solution. The PCL broke down and moved to the DCM phase, while the BSA and NGF should be present in the PBS. The solution was vortexed twice and allowed to phase separate again to completely collect the released NGF and BSA. A 1 mg/mL BSA in PBS was used as control to determine structural significance of the data. All release protein samples were analyzed using JASCO spectra polarimeter from a wavelength of 200-300 nm. 2.6. NGF ELISA. The release samples that were collected over the 4-week time periods were assayed by a NGF ELISA kit (Chemicon) following the manufacturer’s protocol. Briefly, 96-well plates, precoated with mouse monoclonal IgG1 anti rabbit-NGF in PBS (100 µL/well), were used to trap the NGF present in the release samples. Once chemical binding happened, the nonspecific sites were blocked for 1 h using a solution of 1% BSA (w/v). The samples were then treated with biotinylated antibody and were incubated for 2 h, following which streptavidin-horseradish peroxidase was added and incubated for 20 min. The samples were then developed and the reaction was stopped by the addition of 100 µL of 0.1 mM HCl. Absorbance was read at 450 nm using a microplate reader (Biotek) and was analyzed for statistical significance. 2.7. PC12 Cell Differentiation Assay. To determine the effectiveness of NGF release as well as the bioactivity of the NGF after electrospinning, release samples from the respective time points were introduced into nondifferentiated PC12 cells. PC12 cells are known to respond to NGF by differentiating into neuronal phenotype by extension of neurites. PC12 cells were cultured and maintained in RPMI 1640 medium supplemented with 10% horse serum, 5% fetal bovine serum, and 1% penicillin streptomycin. Cells were maintained in a humid, 5% CO2 incubator, at 37 °C. The cells were primed with serum free medium supplemented with 50 ng/mL NGF for two days prior to experiments. PC12 cells were seeded at a density of 105 cells/well in a 24-well plate in 2 mL of serum free culture medium overnight. The medium was replaced with 500 µL of release medium from aliquots of NGF release collected over the 28 day time period. At least three samples were used per time point, per sample type. The positive control medium was a NGF-supplemented medium (50 ng/mL) and the negative control was a blank serum free medium without NGF. After 3 days, the cells were imaged using a Nikon phase contrast inverted microscope. At least five frames per well were imaged (each frame containing between 100-200 cells) and the percentage of neurite-bearing cells was determined by counting the cells from the images. By definition, neurite-bearing cells are the ones with processes (neurites) greater than or equal to the cell body diameter. Also, from the images, the neurite lengths were measured on the differentiated cells. 2.8. Statistical Analysis. All data presented in this study are represented as mean ( standard deviation. Statistical analyses were conducted on the collected data using students t test and data was considered statistically significant if p < 0.05.

3. Results and Discussions 3.1.1. Releasing Rates of PCL-BSA Nanofibers. The release rates of BSA from PCL-BSA blend scaffolds were studied up to 28 days to identify the most suitable scaffold for controlled release applications. From our data (Figure 1) we understood that there was a continuous and sustained release of BSA from all the PCL-BSA blends over 28 days. The diffusion mechanism from the fibers was estimated as per Leong et.al.,11 assuming one-dimensional release under perfect sink condition. The generalized equation that describes the release of drugs from one-dimensional nonswellable system can be expressed as

Mt /M∞ ) K × tn where Mt is the mass of drug release at time t and M∞ is amount

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Figure 2. Scanning electron microscopy images of electrospun PCLBSA-NGF nanofibers.

of drug released when time reaches infinity. K is a constant and n is the diffusion coefficient. For a one-dimensional system, as described above, the ideal diffusion coefficient was estimated to be 0.45-0.5 for the release of drugs from nanofibers. In our case, for the PCL-BSA nanofibers, the coefficients was estimated and sample C had a coefficient of 0.476, which is very close to the ideal value of 0.45 as shown by Leong et al.11 Therefore, we identified sample C (0.01% (wt/ wt)) blend of PCL to BSA) as the most suitable system for controlled release applications in tissue engineering and adopted this system for controlled release of Nerve growth factor for further experiments. 3.1.2. SEM Images of Nanofibers. Electrospinning based scaffold fabrication technique was used to synthesize growth factor carrying nanofibers for tissue engineering applications. Figure 2 shows scanning electron microscopy image of PCLBSA-NGF nanofibers. PCL-BSA-NGF nanofibers have a large nanofiber diameter distribution. The diameter of the nanofibers was 548.89 ( 214.37 nm as shown in the figure. We hypothesize that this variation in diameter happened due to the phase separation of PCL and the NGF during electrospinning, causing the NGF to bead in certain locations in the fiber. It can also be seen that the variations in diameter have no or less impact on the loading and release of the NGF inside the nanofibers, as indicated by the confocal images (Figure 3), which shows NGF uniformly loaded into the nanofibers. 3.1.2. Confocal Images Showing NGF Encapsulated Nanofibers. From the confocal images of the PCL nanofibers and the PCL-BSA-NGF nanofibers, it is evident that the NGF was loaded uniformly into the nanofibers. From Figure 3A it is evident that the PCL-NGF fibers had some phase separation owing to the presence of the water phase (NGF) and the organic phase (PCL in DCM) during electrospinning. This creates uneven distribution of the NGF in the nanofibers as shown by dark regions (where no NGF was present). In the case of PCLBSA-NGF nanofibers (Figure 3B), uniform distribution of the NGF was visible, owing to increased incorporation and dissolution of NGF in the solvent during electrospinning. Also, from Figure 3C, it is evident that no significant nonspecific binding was observed, therefore confirming the presence of NGF encapsulated in the nanofibers. 3.2. Circular Dichroism Showing Intact Structure of NGF after Electrospinning. An absorption spectra specific to BSA-NGF was obtained by exposing the loaded proteins to circularly polarized light. This data shown in Figure 4 indicates

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Figure 3. Confocal laser scanning microscope image of (A) PCL-NGF and (B) PCL-BSA-NGF. (C) Secondary antibody only staining of PCLBSA-NGF nanofibers.

Figure 4. Images of BSA and NGF CD spectra as compared to loaded BSA-NGF generated following exposure to HFIP and electrospinning.

the effect of interaction of the proteins to the solvent, HFIP. The peak at 220-250 nm arises from the BSA, as shown in the literature,33 and it is evident that no significant change in the peak width and intensity was visible, indicating no damage to the secondary structure was evident due to dissolution in HFIP as well as due to electrospinning. Also, from the peaks for NGF in the NGF control and the BSA-NGF HFIP-electrospinning, no significant change in peak intensity and width was visible from the peaks at data it is evident that the peak at 250 nm is from the NGF.34 Also, in the case of PCL-NGF nanofibers, a slight dip in the peak intensity at 243 nm was observed, probably owing to some denaturation that might have occurred because of unfavorable interaction of NGF with the solvent, in the absence of BSA as a protective agent.9,11 3.3. NGF Release Assay. Owing to its short duration of activity and an optimal quantity requirement for effectiveness, controlled release of NGF in close proximity to the injury is required. Therefore, a need for a controlled release mechanism is necessary. The release of NGF from PCL and PCL-BSA scaffolds was studied over 28 days at 37 °C. A burst release profile was visible in the case of PCL nanofibers (day 1) as shown in Figure 5 owing to increased hydrophobicity leading to accumulation of NGF near the surface rather than uniformly inside the polymer. Meanwhile, PCL-BSA nanofibers containing NGF showed reduced burst owing to increased hydrophilicity and improved and uniform loading. The encapsulation efficiency of the NGF in PCL scaffolds were found to be 26.3 ( 1.4%, and owing to reduced loading of NGF in PCL nanofibers and an initial burst release, the PCL nanofibers failed to produce a sufficient release over 28 days with reducing concentrations of NGF released as time progressed. In the case of PCL-BSA nanofibers, a higher encapsulation efficiency of 88.6 ( 4.7%

Figure 5. Cumulative release of NGF from PCL-BSA and PCL nanofibers as determined by the NGF-ELISA assay over 28 days. The percentages of release are reported as percentage of loaded NGF into the nanofibers, as determined by the loading efficiency. * indicates significant difference (p < 0.05) in NGF release between PCL-BSA and PCL nanofibers.

was observed and a more controlled release at all time points in the course of this study. On evaluating the power equation trend line for PCL-BSA approximately equal to n ) 0.523, which matches the Fickian diffusion for one-dimensional cylinders as described by Peppas et al.32 This confirms that there is a controlled release of BSA and NGF from the scaffolds over the entire duration of the study (28 days), from PCL-BSA nanofibers. Meanwhile, the release of NGF from PCL scaffolds does not match the Fickian diffusion equation with a diffusion coefficient of 0.203, which is less than 0.5, indicating nonuniform release kinetics and is therefore not applicable for controlled release applications. 3.4. Neurite Number Assay. It is important to evaluate the bioactivity of NGF released from the nanofibers. PC12 cells are known to differentiate to give out neurites in the presence of NGF. The PC12 cells were counted as differentiated if the neurite length was greater than the diameter of the cell body. To evaluate the bioactivity of the growth factor, percentage differentiation and length of neurites were quantified. For the negative control the cells appeared to be rounded with no clear evidence of processes arising from cells. The NGF released from PCL and PCL-BSA nanofibers at day 1 promoted processes extending from the cell bodies, with several processes greater than the cell body diameter itself (Figure 6A and B). The NGF released from the PCL-BSA fibers on day 28 significantly stimulated differentiation in PC12 cells (Figure 6C). However, from images corresponding to differentiation induced by NGF released from day 28 of PCL scaffolds (Figure 6D), a significantly smaller number of cells bearing neurites as well as rounded cell morphologies were observed, indicating insuf-

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Figure 6. Bioactivity of released NGF from PCL-BSA nanofibers for 28 days. Images indicate effect of controlled release over extended duration, causing PC12 cell differentiation, hence indicated by sprouting processes. (A) Represents neurites in the PCL-BSA nanofibers for day 1 of release; (B) neurite extension from PCL nanofibers releasing NGF for day 1; (C) day 28 of the release from PCL-BSA; (D) neurite extension from day 28 release from PCL; (E) neurite extension from negative control; (F) neurite extension from positive control.

Figure 7. Percentage differentiation of PC12 owing to the controlled release of NGF from nanofibers. Significant difference in the percentage of neurite producing cells between PCL and PCL-BSA was evident over days 14, 21, and 28. * Indicates significant difference (p < 0.05) between PCL-BSA and PCL nanofibers.

Figure 8. Neurite length from PC12 cells, induced by release of NGF from PCL and PCL-BSA scaffolds over various time points. * Indicates statistically significant (p < 0.05) increase in length of neurites from PCL-BSA scaffolds as compared to PCL.

ficient differentiation owing to reduced amounts of NGF available as indicated by the release profiles. Figure 6E shows that the negative control had no significant neurite outgrowth, meanwhile, Figure 6F shows a positive control sample, with a majority of cells bearing neurites due to exposure to NGF (50ng/mL). The data in Figure 7 indicates percentage differentiation of PC12 cells in the presence of NGF released from PCL-BSA scaffolds as compared to NGF released from PCL scaffolds at various time points. It is evident that PCL-BSA showed similar percentages of differentiation as compared to PCL from release as of day 1 to day 7 from the scaffolds. Meanwhile, the degree of differentiation began to drop off from various subsequent time points from the PCL scaffolds. However, in the case of PCL-BSA scaffolds, owing to sufficient amounts of NGF released, the percentage differentiation of PC12 cells remained stable. A significant difference in percentage differentiation between the two fiber types was observed at days 14, 21, and 28. 3.5. Neurite Length Assay. The mean length of PC12 neurite extension, as shown in Figure 8, was quantified using image

analysis as described earlier. The NGF released from PCL-BSA nanofibers significantly stimulated the increase in neurite lengths over those released from the PCL nanofibers at days 14, 21, and 28. In the PCL fiber group, a large initial growth of neurites followed by a steady decrease was observed, owing to insufficient loading and slower releasing rates, as expected, indicating the unsuitability of the material for controlled release applications. The consistent length of the neurites over days 14-28 are indicative of the uniform release of NGF from the nanofibers and the sustained bioactivity of the NGF release from the nanofibers, which is not seen in the case of PCL NGF nanofibers. In most cases, the PC12 based bioassay is used primarily as a quantitative tool and not as a qualitative tool to estimate the difference between the bioactivity of the available NGF, as indicated by Hadlock et al.35 Hadlock et al. discuss the loss of activity of NGF due to processing steps, especially exposure to organic solvents. Our results indicate that the encapsulation efficiency, percentage differentiation estimated by PC12 bioassay, and the release profiles estimated by ELISA match closely to the data shown by other researchers.10,36 We have, in this

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study, demonstrated the bioactivity of the loaded and released NGF over the 4 weeks, which is indicative of the stability of the NGF owing to the coencapsulation with BSA as indicated by Xu et.al.9

4. Conclusions Electrospinning has been successfully demonstrated as a practical way of administering NGF. The encapsulation and release of NGF in PCL fibers were proven to be more effective when BSA was incorporated into PCL. The ELISA assay proved an efficient release of NGF from PCL-BSA nanofibers over a 28 day period. By introducing the released NGF into the cultured PC12 cells, it was evident that the released NGF still kept the bioactivity for stimulating neurite outgrowth over a 28 day period. The electrospun PCL-BSA nanofibers could be used to deliver NGF and potentially serve as a platform for the delivery of other critical growth factors needed for tissue engineering applications.

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