System and method for electrospun biodegradable scaffold for bone repair
10081794 ยท 2018-09-25
Assignee
Inventors
Cpc classification
A61L2300/602
HUMAN NECESSITIES
C08L67/04
CHEMISTRY; METALLURGY
A61L27/3834
HUMAN NECESSITIES
A61L2300/802
HUMAN NECESSITIES
C12N2537/00
CHEMISTRY; METALLURGY
A61L2430/02
HUMAN NECESSITIES
C08L67/04
CHEMISTRY; METALLURGY
C12N5/0654
CHEMISTRY; METALLURGY
A61L2300/252
HUMAN NECESSITIES
A61L27/54
HUMAN NECESSITIES
International classification
A61L27/54
HUMAN NECESSITIES
Abstract
This invention relates a structure and system for growth factor incorporation which can improve the osteogenic differentiation of hMSCs, for potential bone regeneration and bone growth applications or used alone for bone repair or growth applications. The system comprises a biodegradable polyester, a hydrophilic polymer, a growth factor and optionally a bioceramic.
Claims
1. An electrospun composite scaffold comprising a polyester, a hydrophilic polymer, a bioceramic that includes phosphate ions, a cationic surfactant that complexes with the phosphate ions of the bioceramic, and a growth factor or a protein, wherein complexing of the cationic surfactant with the phosphate ions of the bioceramic decreases electrostatic attraction of the growth factor or the protein to the bioceramic, thereby enhancing release of the growth factor or the protein from the scaffold relative to release from the same scaffold without a cationic surfactant.
2. The electrospun composite scaffold of claim 1, wherein the polyester is selected from the group consisting of polylactic acid, polyglycolic acid, polylactic co-glycolic acid copolymers and polycaprolactone.
3. The electrospun composite scaffold of claim 1, wherein the hydrophilic polymer is selected from the group consisting of polyethylene oxide, polyethylene glycol, polyvinyl alcohol, glycosaminoglycans, chitosan, sulfated dextran, sulfated cellulose, and heparin sulfate.
4. The electrospun composite scaffold of claim 1, wherein the growth factor is selected from the group consisting of Recombinant human Platelet Derived Growth Factor-BB (PDGF-BB), vascular endothelial factor (VEGF), transforming growth factor-beta (tgf-BETA) and Bone Morphogenetic Protein (BMP).
5. The electrospun composite scaffold of claim 1, wherein the protein is lysozyme.
6. The electrospun composite scaffold of claim 1, wherein the bioceramic is selected from the group consisting of hydroxyapatite, tricalcium phosphate, and biphasic calcium phosphate.
7. The electrospun composite scaffold of claim 1, wherein the cationic surfactant is cetyl trimethylammonium bromide (CTAB).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) So that those having ordinary skill in the art will have a better understanding of how to make and use the disclosed systems and methods, reference is made to the accompanying figures wherein:
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DETAILED DESCRIPTION OF THE INVENTION
(11) The electrospun composite scaffold of the invention most generally is comprised of polyester (poly alpha hydroxyl ester), a hydrophilic polymer and a growth factor or a protein.
(12) The polyester of the invention includes, for example, polylactic acid, polyglycolic acid and polylactic co-glyocolic acid copolymers, which are chosen, in part by the speed of degradation desired for a particular purpose. In certain embodiments of the invention polycaprolactone (PCL) is a preferred component.
(13) The hydrophilic polymers of the inventions are polymers such as, for example, polyethylene oxide, polyethylene glycol, polyvinyl alcohol, glycosaminoglycans, chitosan, sulfated dextran, sulfated cellulose and heparin sulfate. In certain embodiments of the invention polyethylene oxide (PEO) is a preferred component.
(14) Growth factors useful in the invention include, for example, bone-morphogenetic protein (BMP), platelet derived growth factor-BB (PDGF-BB), vascular endothelia growth factor (VEGF) and transforming growth factor-beta (TGF-beta).
(15) Optionally, the electrospun composite scaffold of the invention may further comprise a bioceramic, such as, for example, hydroxyapatite, tricalcium phosphate, biphasic calcium phosphates, calcium carbonate, calcium sulfate, and bioactive glass. Also included are biphasic bioceramics such as, for example, 20/80 Hydroxyapatite/-Tricalcium phosphate (HA/-TCP).
(16) Electrospun composite consisting of PCL and HA alone or beta-TCP alone can be formed using the same conditions as described for the PCL containing 20/80 HA/TCP. Since HA is a stable ceramic and beta-TCP is a more soluble/relatively fast-degrading ceramic, the use of either of these ceramics in the composite may alter the overall degradation and bioactivity of the composite as compared to the PCL containing 20/80 HA/TCP. Since HA is more stable than beta-TCP, a composite consisting of HA alone may be advantageous for producing a more stable composite material wherein the ceramic component remains in the composite long-term. For a composite containing beta-TCP alone, this will result in a fast-degrading ceramic component which may have an effect on the overall bioactivity and may enhance the formation of an apatite on the composite because the surrounding solution becomes saturated with calcium and phosphate ions.
(17) The scaffold may further comprise a non-ionic surfactant, such as, for example, Span 80, or a cationic surfactant, such as, for example, cetyl trimethylammonium bromide (CTAB).
(18) In a further embodiment of the invention the electrospun composite scaffold comprises a non-ionic surfactant such as, for example Span 80.
(19) In yet another embodiment of the invention, when the electrospun composite comprises a biphasic ceramic, it also may comprise a cationic surfactant such as, for example, cetyl trimethylammonium bromide (CTAB).
(20) Another embodiment of the invention relates to a process to support cell proliferation and osteogenic activity comprising culturing human mesenchymal stem cells (hMSCs) on an electrospun composite scaffold comprising from about 70% to about 100% PCL and from about 0% to about 30% PEO, which may further comprise PDGF-BB.
(21) Another embodiment of the invention relates to a process to support cell proliferation and osteogenic activity comprising culturing human mesenchymal stem cells (hMSCs) on an electrospun composite scaffold comprising about 9 wt % PCL+about 30% (w/w) 80/20 -TCP/HA.
(22) More particularly, the invention relates to an electrospun composite scaffold comprising polycaprolactone (PCL) and the biphasic ceramic: 80/20 -Tricalcium phosphate/Hydroxyapatite (-TCP/HA) can be used for the delivery of growth factors to enhance bone tissue growth. The composite scaffold consisting of PCL and the bioactive ceramic is described in U.S. patent application Ser. No. 12/141,340 for bone regeneration applications.
(23) For growth factor delivery, PCL was combined with the hydrophilic polymer polyethylene oxide (PEO) at a ratio of between about 0%-30% PEO to about 70%-100% PCL to enhance the incorporation of hydrophilic growth factors/proteins. Said growth factor delivery can be done with or without the addition of the ceramic. However, when added the ceramic used is a biphasic ceramic: 80/20 -TCP/HA was added to the polymer solutions of between about 9 wt %-17% wt PCL and about 9 wt %-about 17% wt 0-30/70-100 PEO/PCL at a concentration of 30% (w/w) with respect to polymer mass in chloroform. Recombinant human Platelet Derived Growth Factor-BB (PDGF-BB), a 25 kDa mitogenic growth factor, was incorporated into the solution of the polymers alone and in a dispersion of the polymers and ceramics. Initial in vitro release studies were conducted to predict the release kinetics and bioactivity of PDGF-BB using a model protein, lysozyme which has similar charge properties to PDGF-BB. Any growth factors in the class of transforming growth factors, including Bone Morphogenetic Protein could be utilized in lieu of PDGF-BB.
(24) Some embodiments of the present invention utilize the polymers alone, without the ceramic component.
(25) In studies of PDGF-BB release from electrospun scaffolds the quantity of PDGF-BB released from PCL over the 7 day study was nearly negligible compared to the PDGF-BB released from 30/70 PEO/PCL. It was further determined that for many applications requiring the release of PDGF-BB, the advantageous ration of PEO to PCL was about 30/70 PEO/PCL.
(26) It was further determined that for some embodiments of the present invention the addition of the non-ionic surfactant, Span 80, to the polymer solution followed by with or without ultrasonication prior to electrospinning sustained the release of PDGF-BB from the scaffold from 1 day without Span 80 to at least 4 days with Span 80 (
(27) For certain embodiments of the present invention, the initial in vitro release studies were conducted to predict the release kinetics and bioactivity of PDGF-BB using a model protein, lysozyme, which has similar charge properties to PDGF-BB.
(28) It was observed that the biphasic ceramic -TCP/HA had a significant effect on the release kinetics and bioactivity of the incorporated lysozyme from electrospun polymer/ceramic scaffolds compared to electrospun polymer scaffolds without -TCP/HA. Furthermore, the addition of CTAB altered the release kinetics and retained the activity of lysozyme released from the scaffolds as well as conserved some of the secondary structure conformation of lysozyme adsorbed onto the surface.
(29) Accordingly, in another embodiment of the present invention, PDGF-BB was incorporated in an electrospun polymer/ceramic composite comprising of polycaprolactone (PCL), and nanoparticles of biphasic ceramic, -tricalcium phosphate (-TCP) and hydroxyapatite (HA) and evaluated for release and bioactivity as determined by the growth and osteogenic differentiation of human MSCs. PDGF-BB was incorporated using phase separation by the addition of polyethylene oxide (PEO) and by examining a novel technique of incorporating a cationic surfactant.
(30) PDGF-BB has an isoelectric point of 11.1 which would have an electrostatic attraction to the negative phosphate ions in the ceramic and may impede its release from the composite. Therefore, the cationic surfactant, hexadecyltrimethylammonium bromide (CTAB) was added to form a complex with the phosphate ions in certain embodiments of the present invention which would, in turn, reduce the electrostatic attraction between PDGF-BB and the biphasic ceramic, leading to the release of PDGF-BB from the electrospun scaffolds.
(31) In embodiments of the present invention where PDGF-BB was incorporated into the polymer/ceramic electrospun scaffolds, it was discovered that significantly less PDGF-BB was released compared to embodiments prepared without ceramic (
(32) Embodiments of the present invention were further characterized for the ability to support cell proliferation and osteogenic activity using human mesenchymal stem cells (hMSCs). It was shown tha proliferation was higher on embodiments containing PDGF-BB incorporated scaffolds prepared with CTAB (
(33) Although it was determined that PDGF-BB was released at negligible quantities from the polymer/ceramic composites, the data suggests that the PDGF-BB was immobilized onto the scaffold of certain embodiments of the present invention by electrostatic interactions. Said immobilized PDGF-BB was bioactive as determined by the enhanced osteogenic activity of the hMSCs.
(34) In conclusion, a series of embodiments of the present invention comprising novel biodegradable electrospun scaffolds incorporated with PDGF-BB have been developed and implemented for use as possible scaffolds for bone regeneration. Said scaffolds prepared with PEO enhanced the release of PDGF-BB in certain exemplary embodiments of the present invention. Scaffolds containing CTAB significantly increased the quantity of PDGF-BB released from electrospun scaffolds prepared with polymers and ceramic and thus may have enhanced cell proliferation at early time-in certain exemplary embodiments and the most favorable for demonstrating osteogenic activity is the composite scaffold containing the growth factor without the use of PEO and CTAB in vitro conditions.
(35) Materials and Methods
(36) PDGF-BB purchased from Prospec Bio and Invitrogen. Serum starvation studies were then conducted to determine the bioactivity of PDGF-BB purchased from Prospec Bio and Invitrogen. Cell proliferation studies indicated that PDGF-BB from Invitrogen was more bioactive than PDGF-BB from Prospec Bio. Therefore, the PDGF-BB from Invitrogen was used for the subsequent studies.
(37) Direct Addition of PDGF-BB.
(38) The direct addition of PDGF-BB refers to the incorporation of PDGF-BB to polymer solutions that were not emulsified. PDGF-BB was incorporated into non-emulsified polymer solutions to determine the effect of PEO on the release of PDGF-BB from the electrospun mats. The polymer solutions were prepared in chloroform according to the formulations in Table 1. Lyophilized recombinant human PDGF-BB was reconstituted in 100 mM acetic acid for a 0.5 mg/mL solution. 2 L of the 0.5 mg/mL PDGF-BB solution was pipetted to each polymer solution for a total loading of 1 g of PDGF-BB. The polymers solutions were mixed on the magnetic stir plate for approximately 20 minutes prior to electro spinning.
(39) TABLE-US-00001 TABLE 1 Direct Addition of PDGF-BB Solution Preparation Conditions Polymer PEO/PCL PDGF-BB PDGF-BB PDGF-BB Concentration Ratio Vendor Concentration Loading 9 wt % 0/100 Prospec 0.5 mg/mL, 2 L 30/70 Bio diluted in 100 containing mM acetic acid 1 g of PDGF-BB 9 wt % 30/70 Prospec 0.5 mg/mL, 2 L 50/50 Bio diluted in 100 containing 70/30 mM acetic acid 1 g of PDGF-BB 9 wt % 30/70 Prospec 0.5 mg/mL, 2 L 50/50 Bio diluted in 100 containing 70/30 mM acetic acid 1 g of with 0.1% BSA PDGF-BB 9 wt % 30/70 Invitrogen 0.5 mg/mL 2 L diluted in 100 containing mM acetic acid 1 g of with 0.1% BSA PDGF-BB
(40) Emulsification by Ultrasonication.
(41) Various emulsion preparation conditions were investigated to determine the effect of Span 80 incorporation and sonication on the release of PDGF-BB from electrospun mats. It was determined that the bioactivity of lysozyme released at 24 hours from emulsions prepared from 9 wt % 30/70 PEO/PCL incorporated with 0.4% (v/v) Span 80 and sonicated, was statistically higher compared to emulsions prepared by the other conditions.
(42) Lyophilized recombinant human PDGF-BB (Invitrogen) was reconstituted in 100 mM acetic acid with 0.1% BSA for a 0.5 mg/mL solution. The 0.5 mg/mL PDGF-BB solution was further diluted in either 0.1% BSA in PBS or 0.1% BSA in DI water to a final concentration of 10 g/mL. These emulsions were prepared by pipetting Span 80 (for Span 80 incorporated conditions) into chloroform, followed by the addition of the PDGF-BB solution in a drop-wise manner into the chloroform, prior to the addition of the polymers according to the conditions in Table 2. After mixing on the magnetic stir plate for approximately one hour, the polymer solutions were either sonicated on ice with a probe ultrasonicator or left un-sonicated.
(43) Scaffolds for serum starvation studies were prepared with polymer solutions loaded with PDGF-BB at quantities that would theoretically induce cell proliferation upon release.
(44) For these scaffolds, PDGF-BB at a concentration of 100 g/mL in 0.1% BSA in DI water, was added to the polymer solutions at a loading of approximately 1.1 g of PDGF-BB per 1 gram of polymer solution or 10 g of PDGF-BB in 9 grams of polymer solution.
(45) TABLE-US-00002 TABLE 2 PDGF-BB Incorporated Emulsion Preparation Conditions Span 80 Concentration PDGF-BB (with respect to Polymer PEO/PCL Concentration PDGF-BB polymer solvent Concentration Ratio (Invitrogen) Loading volume) Sonication Conditions 9 wt % 30/70 10 g/mL, 100 L 0.4% 0% (v/v) Branson No diluted in 0.1% containing (v/v) for Ultrasonifier: sonication BSA in PBS or 1 g of conditions 2 minutes DI water PDGF-BB without pulse mode Span 80 at 20% amplitude 9 wt % 30/70 10 g/mL, 100 L 0.4% 0% (v/v) Branson No diluted in 0.1% containing (v/v) for Ultrasonifier: sonication BSA in DI 1 g of conditions 2 minutes water PDGF-BB without pulse mode Span 80 at 10% or 20% amplitude 9 wt % 30/70 10 g/mL, 100 L 1% 2% (v/v) Branson Ultrasonifier: 2 diluted in 0.1% containing (v/v) minutes pulse mode at BSA in DI 1 g of 10% amplitude water PDGF-BB 9 wt % 30/70 100 g/mL 200 L 0.4% Branson Ultrasonifier: 2 diluted in 0.1% containing (v/v) minutes pulse mode at BSA in DI 20 g of 10% amplitude water PDGF-BB 9 wt % 30/70 100 g/mL 100 L 0.4% Branson Ultrasonifier: 2 diluted in 0.1% containing (v/v) minutes pulse mode at BSA in DI 10 g of 10% water PDGF-BB
(46) Electrospinning.
(47) The polymer solutions/emulsions were electrospun in an environmental chamber at 21-23 C. and 17-26% relative humidity. Polymer solutions/emulsions were placed in a 10 mL syringe and passed through a blunt 20 gauge stainless needle at 3.0 mL/hour using a syringe pump (Harvard Apparatus). The applied voltage to the needle tip was 20 kV. The distance between the needle tip to the grounded collection plate was between 20-50 cm, depending upon the polymer solution. Electrospun mats were peeled off from the collector plate and placed in a desiccator until the initiation of the study.
(48) Some of the polymer/ceramic suspensions were electrospun in a non-enclosed electrospinning apparatus which did not have any humidity controls, because it was determined that electrospinning in the enclosed chamber produced undesirable fiber morphologies which may have been a result of residual solvent vapor.
(49) Polymer/ceramic suspensions were placed in a 10 mL syringe and passed through a blunt 20 gauge stainless steel needle at 3.0 mL/hour using a syringe pump (Harvard Apparatus). The applied voltage to the needle tip was 20 kV. The distance between the needle tip to the grounded collection plate was between 30-60 cm, depending upon the suspension.
(50) During the summer, when the bulk of the electrospinning was conducted, the high relative humidity (above 60%) resulted in unpredictable fiber and scaffold morphologies.
(51) In a modified electrospinning unit, the water vapor in the air was condensed into liquid and froze onto a cooling probe, leaving dehumidified air inside the electrospinning chamber. Furthermore, an exit diffuser compartment provided an outlet channel to reduce the solvent vapor inside the electrospinning chamber. The relative humidity inside the electrospinning chamber was maintained to below 50% which resulted in desirable fiber morphologies. The syringe pump rate, voltage and distance between needle tip and collector plate were similar to the conditions for electrospinning in the non-enclosed chamber. Electrospun mats were peeled off from the collector plate and placed in a desiccator until the initiation of the study.
(52) The lysozyme incorporated polymer/ceramic suspensions were electrospun in a non-enclosed electrospinning unit. Polymer/ceramic suspensions were placed in a 10 mL syringe and passed through a blunt 20 gauge stainless needle at 3.0 mL/hour using a syringe pump (Harvard Apparatus). The applied voltage to the needle tip was 20 kV. The distance between the needle tip to the grounded collection plate was between 30-60 cm, depending upon the suspension.
(53) PDGF-BB incorporated polymer/ceramic emulsions were electrospun in the modified enclosed electrospinning units with similar parameters outlined above. The PDGF-BB incorporated polymer/ceramic emulsions with CTAB were electrospun in a non-enclosed electrospinning unit with similar parameters outlined in the previous section.
(54) Post-Electrospun PDGF-BB Incorporation.
(55) Polymer solutions were prepared in chloroform and electrospun using the conditions described above. The electrospun mats were cut into 19 mm discs and placed into low attachment 24 well plates (Costar). A 40 g/mL solution of PDGF-BB (Invitrogen) was prepared in PBS. To each scaffold, 1 mL of the 40 g/mL PDGF-BB solution was added and then incubated overnight at 37 C., 5% CO2. Following the overnight incubation, the discs were rinsed three times with PBS and placed in a laminar air hood for approximately 7 hours to dry the discs until the initiation of the in vitro release study.
(56) In Vitro Release Studies.
(57) PDGF-BB incorporated electrospun scaffolds were cut into 19 mm discs (n=3) and placed in 24 well low-attachment plates. For some studies, the scaffolds were cut into 6 mm discs (n=3) and placed in 96 well polypropylene plates. At the initiation of the release study, the plates were sterilized with UV light prior to the addition of 0.5 mL of PBS in the 24 well plates and 150-300 L in the 96 well plates, and then placed in a 37 C., 5% CO2 incubator. At predetermined time-points during the study, the release medium was collected from each well which were replenished with fresh PBS. For some studies, the electrospun discs were dried under a laminar air hood, and dissolved in chloroform to which PBS was added to extract the residual PDGF-BB. For other studies, the residual PDGF-BB was extracted using a protocol described by Sakiyama-Elbert et al. Briefly, dried scaffolds were placed in a solution of 1% BSA, 2M of sodium chloride, and 0.01% Triton-X in PBS and placed in 4 C. for 72 hours, after which the supernatant was collected for the PDGF-BB ELISA.
(58) PDGF-BB Quantitation.
(59) The release media and residual PDGF-BB samples were stored at 20 C. until the PDGF-BB quantitation using a PDGF-BB ELISA (PeproTech). Briefly, 100 L of the release medium was added to an immunoplate coated with anti-PDGF-BB. The presence of PDGF-BB was detected through the color change of an enzymatic substrate which was detected at 410 nm with an absorbance plate reader. A calibration curve of known PDGF-BB concentrations was used to determine the unknown concentration in the release media.
(60) Percent Weight Change.
(61) The electrospun mats (n=3) were weighed prior to the incubation and placed in wells of a 24 well plate. After the in vitro release study, the electrospun mats were dried and then weighed. The percent weight loss was calculated using Equation 2.4, using the weight at the final time-point.
(62) Visualization of PDGF-BB or Lysozyme in Electrospun Fibers.
(63) Immunofluorescence was used to visualize PDGF-BB incorporated in electrospun mats. Briefly, electrospun scaffolds were cut into 6 mm discs and blocked with 1% BSA in PBS for approximately an hour. Following a rinsing step in PBS, 100 L of a 2 g/mL anti-PDGF-BB (R&D Systems) solution in 1% BSA in PBS was added to each mat for approximately an hour. Following another rinsing step, 100 L of 1 g/mL Fluorescein-conjugated Rabbit Antigoat secondary antibody (Pierce) in 1% BSA in PBS was added to each mat. For a negative control, electrospun mats were stained with only secondary antibody at 1 g/mL in 1% BSA in PBS to detect the presence of non-specific staining. The mats were viewed with the confocal microscope at 408 nm excitation/455 nm emission to view the autofluoresced electrospun fibers and 488 nm excitation/515 nm emission to view the secondary antibody stain.
(64) For lysozyme incorporated in electrospun mats, the electrospun scaffolds were stained with the 2 g/mL of anti-lysozyme primary antibody in 1% BSA in PBS and 1 g/mL of Alexa Fluor 488 Donkey anti-rabbit IgG secondary antibody in 1% BSA in PBS. For a negative control, designated electrospun mats were stained with the secondary antibody at 1 g/mL to detect the presence of non-specific staining. The mats were viewed with the confocal microscope at 408 nm excitation/455 nm emission to view the autofluoresced electrospun fibers and 488 nm excitation/515 nm emission to view the secondary antibody stain.
(65) Statistical Analysis.
(66) Quantitated data was represented as the meanstandard deviation. A student's t-test was conducted for the comparison of 2 groups at each timepoint, with significance set to p<0.05. One-way ANOVA was performed on data sets with more than 2 groups, collected at a single time-point. The Tukey post-hoc test was conducted to determine differences (p<0.05) between pairs. For the CTAB cytotoxicity study, TCPS was not included in the statistical analysis. For data represented as a function of time, a two-way repeated measures ANOVA was conducted to determine the effect of the material and PDGF-BB with time.
(67) Ceramic Preparation.
(68) The weight ratio of 80/20 -TCP to HA was determined as the ideal ratio to induce the osteogenic differentiation of mesenchymal stem cells. An 80/20 -TCP/HA suspension was prepared in chloroform and then placed in a water bath sonicator (VWR, Aquasonic 75T) for 2 minutes. The polymer solutions prepared in chloroform were added to the ceramic suspensions for final ceramic concentrations of 9% (w/w), 17% (w/w), 23% (w/w) or 30% (w/w) with respect to polymer mass.
(69) Native Lysozyme Incorporation Through Dissolution in DMSO.
(70) Native lysozyme was dissolved in DMSO and added to the polymer/ceramic suspension according to the conditions listed Table 3, then mixed on a magnetic stir plate overnight followed by electrospinning.
(71) TABLE-US-00003 TABLE 3 Polymer/Ceramic Solution Preparation Conditions for Pre- Electrospun Incorporated Lysozyme Dissolved in DMSO Concentration of 80/20 Lysozyme -TCP/HA Lysozyme Loading (with respect Concentra- (with respect Polymer PEO/PCL to polymer tion (in to polymer Concentration Ratio mass) DMSO) mass) 7.5 wt % 0/100 9% (w/w) 10 mg/mL 0.1% (w/w) 25/75 17% (w/w)
(72) Ultrasonic Emulsification.
(73) Solutions of native lysozyme were prepared in DI water. In Studies B and C, Span 80 and the aqueous lysozyme solutions were added to the polymer/ceramic suspensions according to the conditions listed in Table 4
(74) TABLE-US-00004 TABLE 4 Polymer/Ceramic Emulsion Preparation Conditions for Pre-Electrospun Lysozyme Dissolved in DI water Concentration Lysozyme Span 80 of 80/20 Loading Concentration -TCP/HA (with (with respect Sonication (with respect Lysozyme respect to to polymer Parameters Polymer PEO/PCL to polymer Concentration polymer solvent (Instrument- Concentration Ratio mass) (in DI water) mass) volume) Conditions) 7.5 wt % 0/100 9% (w/w) 10 mg/mL 0.1% (w/w) 0.002% (v/v) Omni-Rupter: 25/75 17% (w/w) 1 minute continuous at 20% amplitude 9 wt % 0/100 9% (w/w) 20 mg/mL 0.2% (w/w) 0.4% (v/v) Branson: 2 30/70 minutes pulse mode at 20% amplitude 9 wt % 0/100 23% (w/w) 20 mg/mL 0.2% (w/w) 0.4% (v/v) Branson: 2 30/70 minutes pulse mode at 20% amplitude
(75) Post-Electrospun Lysozyme Incorporated Polymer/Ceramic Scaffold Preparation.
(76) Prior to electrospinning, polymer/ceramic suspensions were sonicated with a probe ultrasonicator (Sonifier 5450-D, Branson) at 20% amplitude on pulse mode (1 second on, 1 second off) for 2 minutes, to disperse the ceramic nanoparticles. The electrospun mats were cut into 19 mm discs and placed into low attachment 24 well plates (Costar). The electrospun mats were sterilized with UV light for approximately 30 minutes prior to the addition of 500 L of a 0.5 mg/mL solution of lysozyme in PBS to each well containing the discs and then placed in a 37 C., 5% CO2 incubator overnight.
(77) Following the overnight incubation, the discs were rinsed three times with PBS and placed in a laminar air hood overnight to dry the discs until the initiation of the in vitro lysozyme release studies.
(78) Preparation of CTAB Modified Polymer/Ceramic Composite Scaffolds.
(79) Cetyl trimethylammonium bromide (CTAB, Sigma) was incorporated to correspond to a molecular ratio of 1 CTAB to 1 phosphate ions (PO4 3) in 80/20 -TCP/HA, and 1 CTAB to 6 PO4 3 ions in 80/20 -TCP/HA. The CTAB and 80/20 -TCP/HA were mixed in chloroform on a magnetic stir plate for approximately an hour prior to the addition of polymer for a final ceramic concentration of either 23% (w/w) -TCP/HA with 1:1 CTAB:PO4 3, renamed 23% (w/w) -TCP/HA+CTAB (high); and 23% (w/w) -TCP/HA with 1:6 CTAB:PO4 3, renamed 23% (w/w) -TCP/HA+CTAB (low).
(80) CTAB was incorporated to correspond to a molecular ratio of 1 CTAB to 6 PO4 3 in 80/20 -TCP/HA. The CTAB and -TCP/HA were mixed in chloroform on a magnetic stir plate for approximately an hour prior to the addition of polymer for a final ceramic concentration of 30% (w/w) 80/20 -TCP/HA with 1:6 CTAB:PO4 3, renamed 30% (w/w) -TCP/HA+CTAB.
(81) CTAB Cytotoxicity Study.
(82) The PicoGreen ds DNA quantitation assay (Invitrogen) was conducted to determine whether cells seeded on CTAB containing scaffolds maintained viability.
(83) Cell Culture Studies.
(84) Electrospun mats of PCL+23% (w/w) -TCP/HA+CTAB (high) and PCL+23% (w/w) -TCP/HA+CTAB (low) were cut into 6 mm discs (n=3) and placed in polypropylene 96 well plates. The scaffolds were compared to PCL, 30/70 PEO/PCL, PCL+23% (w/w) -TCP/HA, and 30/70 PEO/PCL+23% (w/w) -TCP/HA, and Tissue Culture Polystyrene (TCPS) which served as the control. All scaffolds were sterilized with UV light for 30 minutes. (Refer to section 5.1.1.1 for detailed instructions on hMSC isolation) One vial of hMSCs, Donor 7, passage 3 was thawed, and suspended in Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS). Cells were seeded at a density of 12,500 cells/cm2, which was 3500 cells per well. The cells were incubated at 37 C., 5% CO2 and harvested at Days 3 and 7 for the PicoGreen assay. Cell lysates were prepared by lysing cells on scaffolds and TCPS in 0.1% Triton X-100. The PicoGreen reagent fluorescently labels double stranded DNA which was correlated to cell number using a standard curve. The PicoGreen reagent was added to the cell lysates and the fluorescence was detected with a plate reader at 485 nm excitation/528 nm emission.
(85) Ceramic Preparation.
(86) A suspension of 80/20 -TCP/HA was prepared in chloroform and then placed in a water bath sonicator for 2 minutes. The polymer solutions prepared in chloroform were added to the ceramic suspensions for final ceramic concentrations of 30% (w/w) with respect to polymer mass.
(87) CTAB modified Ceramic Preparation. PDGF-BB Incorporation.
(88) The PDGF-BB incorporated polymer/ceramic scaffolds were prepared by ultrasonic emulsification according to the parameters in Table 5. Briefly, lyophilized recombinant human PDGF-BB (Invitrogen) was reconstituted in 100 mM acetic acid with 0.1% BSA for a 0.5 mg/mL solution. The 0.5 mg/mL PDGFBB solution was further diluted in 0.1% BSA in DI water to a final concentration of 10 g/mL for a loading of 1 g per mat or a concentration of 100 g/mL for the therapeutic loading of 10 g per mat (1.1 g of PDGF-BB/gram of polymer/ceramic emulsion).
(89) TABLE-US-00005 TABLE 5 Study Design for PDGF-BB Incorporated Polymer/Ceramic Electrospun Scaffolds Span 80 Concentration Concentration of 80/20 - (with respect TCP/HA (with PDGF-BB PDGF- to polymer Polymer PEO/PCL respect to CTAB Concentration BB solvent Sonication Concentration Ratio polymer mass) Incorporation (Invitrogen) Loading volume) Conditions 9 wt % 0/100 30% (w/w) None 10 g/mL in 10 L 0.4% (v/v) Branson 30/70 0.1% BSA in for a Ultrasonifier: DI water loading 2 minutes of 1 g pulse mode at 10% amplitude 9 wt % 0/100 30% (w/w) 1 CTAB: 6 10 g/mL in 10 L 0.4% (v/v) Branson 30/70 PO.sub.4.sup.3- 0.1% BSA in for a Ultrasonifier: DI water loading 2 minutes of 1 g pulse mode at 10% amplitude 9 wt % 0/100 30% (w/w) None, 100 g/mL in 100 L 0.4% (v/v) Branson 30/70 1 CTAB: 6 0.1% BSA in for a Ultrasonifier: PO4 DI water loading 2 minutes 3- of 10 g pulse mode at 10% amplitude
(90) In Vitro PDGF-BB Release Study.
(91) The PDGF-BB incorporated electrospun scaffolds were cut into 19 mm discs (n=3) and placed in 24 well low-attachment plates. For some studies, the electrospun mats were cut into 6 mm discs and placed in 96 well polypropylene plates (Nunc). At the initiation of the release study, the plates were sterilized with UV light prior to the addition of 0.5 mL of PBS for Studies A-B or 150 L of PBS for Study C and then placed in a 37 C., 5% CO2 incubator. At pre-determined time-points during the study, the release medium was collected from each well which were replenished with fresh PBS.
(92) Isolation and Culture of Human Mesenchymal Stem Cells.
(93) Human mesenchymal stem cells (hMSCs) were isolated from commercially obtained (Cambrex) bone marrow aspirates collected from the superior iliac crest of the pelvis of male donors. The isolation method described in detail by Haynesworth, proceeds with washing the marrow sample with PBS followed by centrifugation in a 70% density gradient solution at 13,000 g for 20 minutes. The hMSC fraction was collected and then plated into tissue culture polystyrene flasks (Nunc) with Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) containing 10% FBS (Hyclone) and 1% Antibiotic-Antimycotic (Invitrogen): 10% FBS DMEM, then placed in a 37 C., 5% CO2 humidified incubator. On average, confluency of hMSCs was achieved within 12-16 days. At the point of near confluency, cells were detached from the substrate with 0.25% Trypsin-EDTA (Invitrogen). Cells were resuspended in 10% FBS DMEM and centrifuged at 900 g for 5 minutes. The cells were then collected and placed into a new tissue culture flask. This procedure called serial passaging or subculturing was done up to a maximum of 4 passages. After each passage, cells were cryopreserved in freezing medium containing 90% FBS and 10% Dimethyl Sulfoxide (DMSO). The cryopreserved cells were stored in a liquid nitrogen tank until the initiation of the study.
(94) PDGF-BB and Cell Culture Media Preparation.
(95) At the initiation of this study, arbitrarily called Day 2, a vial of hMSCs was thawed, resuspended in 10% FBS DMEM, and seeded into 96 well TCPS plates at a seeding density of 12,500 cells/cm2 or 4000 cells/well. On Day 1, 24 hours following the initial cell seeding, the media in the wells was changed with DMEM prepared with various concentrations of FBS (Table 6). On Day 0, the media was supplemented with various concentrations of PDGF-BB (Prospec Bio or Invitrogen). PDGF-BB supplementation was done either once at Day 0, or continually for every 48-72 hours until Day 7.
(96) TABLE-US-00006 TABLE 6 Serum Starvation Study Conditions Table 5.1 Serum Starvation Study Conditions Concentration of FBS in 10% FBS 10% FBS 10% FBS DMEM at Day 2 Concentration of FBS in 2% FBS 5% FBS 10% FBS DMEM at Day 1 Concentration of PDGF-BB 2% FBS DMEM + 5% FBS DMEM + 10% FBS DMEM + supplemented to x% FBS in No PDGF-BB No PDGF-BB No PDGF-BB DMEM at Day 0 25 ng/mL PDGF-BB 25 ng/mL PDGF-BB 25 ng/mL PDGF-BB 50 ng/mL PDGF-BB 50 ng/mL PDGF-BB 50 ng/mL PDGF-BB 100 ng/mL PDGF-BB 100 ng/mL PDGF-BB 100 ng/mL PDGF-BB Frequency of PDGF-BB Day 0 only Day 0 only Day 0 only addition Continuous: Continuous: Continuous: Every 48-72 hours Every 48-72 hours Every 48-72 hours
(97) Cell Proliferation.
(98) At Days 4 and 7 following growth factor induction, cells were harvested for cell proliferation assay. Briefly, cell lysates (n=3 per media and PDGF-BB condition) were prepared by lysing cells in 0.1% Triton X-100. The PicoGreen reagent fluorescently labels double stranded DNA which was correlated to cell number using a standard curve. The PicoGreen reagent was added to the cell lysates and the fluorescence was detected with a plate reader at 485 nm excitation/528 nm emission.
(99) Alkaline Phosphatase Activity.
(100) The activity of Alkaline Phosphatase (AP) was determined by quantifying the conversion rate of para-nitrophenyl phosphate (p-Npp) to para-nitrophenol (p-Np). Standards of known p-Np concentrations were prepared in phosphatase buffer. Cell lysates prepared in 0.1% Triton X-100, and standards were incubated at 37 C. for 30 minutes in a water bath. The absorbance of the samples and standards was read at 405 nm with an absorbance plate reader. The AP activity was normalized to cell number determined from the PicoGreen assay (nmol of p-Np/min/cell).
(101) Cell Seeding on Transwell Membranes.
(102) The design of the transwell bioactivity studies was modeled after the serum starvation studies. At Day 2, a vial of hMSCs, Donor 7, passage 2, was thawed and resuspended in 10% FBS DMEM. The cells were seeded at a density of 12,500 cell/cm2 or 4000 cells/well in 100 L of media on 0.4 m transwell membrane inserts (Corning) which were placed in a low attachment 24 well plate. At Day 1, the media in designated wells was replaced with 5% FBS DMEM, other designated wells remained in 10% FBS DMEM for control. At Day 0, the electrospun scaffold: PEO/PCL incorporated with 1.1 g PDGF-BB/gram of polymer solution, was cut into 19 mm discs (n=3), and placed at the bottom of designated wells of which 500 L of 5% FBS DMEM was added to. The transwell membrane inserts were placed on top of the wells containing the scaffold. For positive control, 10 ng/mL of PDGF-BB was added to cells cultured in 5% FBS DMEM.
(103) At 4, 7 and 11 days following growth factor induction, the cells were harvested from the inserts with 0.1% Triton X-100 for the PicoGreen assay, following the methods described in the previous section.
(104) Cell Seeding.
(105) A list of electrospun scaffolds used in the cell/scaffold studies is listed in Table 7.
(106) TABLE-US-00007 TABLE 7 Electrospun Scaffolds used in the Cell/Scaffold Studies Scaffold Composition Ratio of PEO/PCL in blends was 30/70 Concentration of -TCP/HA was With PDGF-BB (1.1 g PDGF- 30% (w/w) 80/20 -TCP/HA BB/g polymer solution), CTAB concentration was (1:6 PO.sub.4.sup.3) Without PDGF-BB and 0.4% (v/v) Span 80 Polymer PCL PEO/PCL PEO/PCL Polymer/ceramic PCL + -TCP/HA PCL + -TCP/HA PEO/PCL + -TCP/HA PEO/PCL + -TCP/HA Polymer/ceramic + CTAB PCL + -TCP/HA + CTAB PCL + -TCP/HA + CTAB PEO/PCL + -TCP/HA + CTAB PEO/PCL + -TCP/HA + CTAB
(107) Electrospun scaffolds were cut into 6 mm discs and placed in 96 well polypropylene plates. At the initiation of the study, on Day 0, the scaffolds were sterilized with UV light for approximately 30 minutes. For Study A, cryopreserved hMSC, Donor 7, passage 2, was thawed and seeded onto the scaffolds at a density of 4000 cells/well in 10% FBS DMEM. After 24 hours of initial hMSC seeding, the media in the wells containing the scaffolds was replaced with either 5% FBS DMEM or 5% FBS OS DMEM which was 5% FBS DMEM supplemented with 10 mM beta glycerophosphate (Sigma), 50 M L-ascorbic acid phosphate (Wako) and 100 nM of dexamethasone (Sigma) to induce osteogenic differentiation.
(108) For other studies, cryopreserved hMSC was thawed and seeded onto scaffolds at a density of 4000 cells/well in 10% FBS DMEM. Instead of 5% FBS DMEM, the media in the wells containing the scaffolds were maintained in either 10% FBS DMEM or 10% FBS OS DMEM which was 10% FBS DMEM supplemented with osteogenic components. The cells were replenished with fresh media every 48-72 hours.
(109) For the control groups, hMSCs were seeded in 96 well TCPS plates and cultured in 10% FBS DMEM. After 24 hours of initial cell seeding, the media in the wells was replaced with either basal DMEM (5% FBS or 10% FBS) or the appropriate OS DMEM (5% FBS or 10% FBS). For a positive control group, hMSCs were seeded in 96 well TCPS plates and cultured in basal DMEM. After 24 hours of initial cell seeding, the media was replaced with either basal or OS DMEM supplemented with 10 ng/mL PDGFBB.
(110) In all studies, at Day 12, the media in the wells designated for OS DMEM was replaced with basal DMEM supplemented 10 nM Vitamin D3, 10 mM -GP and 5 M ascorbic acid for the duration of the 21 day study to induce osteocalcin production.
(111) Cell Proliferation.
(112) Cells were harvested from the scaffolds for the Picogreen DNA assay with 0.1% Triton X-100 at various time-points of the 21 day study at days 4, 7, 11, 14, and 21.
(113) Osteocalcin Assay.
(114) Cell lysates collected at Days 14 and 21 from Study B were assayed for the osteocalcin production with the human Osteocalcin ELISA kit (Invitrogen). Briefly, an aliquot of the cell lysates was added to the immunoplate. The osteocalcin was detected by the color change of the enzymatic substrate which was read by an absorbance plate reader at 450 nm. A calibration curve of known osteocalcin concentrations was used to determine the concentration of osteocalcin in the cell lysates.
(115) Gene Expression.
(116) For Studies B and C, the cells seeded on the scaffolds and TCPS were harvested for the gene expression studies. At days 0, 7 and 14, cells were harvested for RNA isolation using the RNeasy Micro kit (Qiagen, Valencia, Calif.). Briefly, RNA was eluted from the homogenized cells harvested from the substrates. For each target or housekeeping gene, a master-mix consisting of primers and reagents from the Sybr Green RT-PCR kit (Qiagen) was prepared according the manufacturer's instructions, and added to the isolated RNA. The reverse transcription of the RNA and polymerization of cDNA was performed on a thermal cycler (MX3000P, Stratagene) using the following conditions of: 30 minutes at 50 C., 15 minutes at 95 C., 40 cycles of 15 seconds at 94 C., 30 seconds at 55 C. and 72 C., followed by 1 minute at 95 C. and 41 cycles at 55 C. The target genes that investigated in this study were: Osteocalcin (OCN), Osteopontin (OPN), Collagen Type I (Col I), Sox2, Runx2. Relative quantification of a replicate of three samples per group, was obtained by normalizing the expression of each target gene to the expression of the housekeeping gene (large ribosomal protein, RPLPO) using the Q-gene software. Melting curves were used to evaluate the integrity of the amplified cDNA, and samples with compromised melting curve due to the presence of primer dimers were not used for analysis.
(117) Although the systems and methods of the present disclosure have been described with reference to exemplary embodiments thereof, the present disclosure is not limited thereby. Indeed, the exemplary embodiments are implementations of the disclosed systems and methods are provided for illustrative and non-limitative purposes. Changes, modifications, enhancements and/or refinements to the disclosed systems and methods may be made without departing from the spirit or scope of the present disclosure. Accordingly, such changes, modifications, enhancements and/or refinements are encompassed within the scope of the present invention.