PROCESS TO CREATE 3D TISSUE SCAFFOLD USING ELECTROSPUN NANOFIBER MATRIX AND PHOTOSENSITIVE HYDROGEL

Abstract

A process providing a method to create 3D scaffolds using nano-scale fibers, comprising: deposition and alignment of a plurality of electrospun fiber layers on a substrate; application of a photosensitive biomedical polymer liquid to each fiber layer deposited on said substrate; deposition and cross-alignment of a plurality of electrospun fiber layers on said substrate; retaining said polymer liquid in place using said cross-aligned fiber layers; curing said polymer liquid on top of each fiber layer using UV light.

Claims

1. A process providing method to create 3D scaffolds using nano-scale fibers, comprising: deposition and alignment of a plurality of electrospun fiber in a first layer on a substrate; application of a photosensitive polymer liquid to said first fiber layer deposited on said substrate; deposition and cross-alignment of a plurality of electrospun fiber in a second layer on said substrate; retaining said polymer liquid in place using said cross-aligned fiber layers; curing said polymer liquid using ultra violet (UV) light.

2. The method of claim 1, wherein, said substrate is adapted to produce a cylindrical 3D scaffold.

3. The method of claim 1, wherein, said substrate is adapted to produce a 3D scaffold comprising at least two equal linear dimensions.

4. A process providing a method for fabrication of a PCL electrospun nanofiber-PEGDA 3D scaffold, comprising the steps of: depositing cross-aligned fibers on a substrate to produce a fiber matrix exhibiting a fiber-separation gap sufficient to prevent PEGDA gel from passing between the fibers in said fiber matrix; adjusting thickness of a PEGDA gel layer on said fiber matrix so that said PEGDA gel layer has uniform porosity; curing said PEGDA gel layer using ultraviolet light (UV) setting UV curing time to control and assure substantially uniform stiffness of said PEGDA gel layer; wherein, said steps are repeated to produce a plurality of fiber matrix and PEGDA layers, and wherein, the number of layers in said plurality of fiber matrix and PEGDA layers is increased to produce a specific thickness of said 3D scaffold.

5. The method of claim 4, wherein, said substrate is adapted to produce a cylindrical 3D scaffold.

6. The method of claim 4, wherein, said substrate is adapted to produce a 3D scaffold comprising at least two equal linear dimensions.

7. A process providing a method for producing cell-encapsulated hydrogels exhibiting complex three-dimensional (3D) structures using a PCL-ENF-PEGDA scaffold, comprising the steps of: creating a porous fiber membrane consisting of cross-directional fibers each of said fibers being separated from another by a gap distance; controlling porosity of said membrane by increasing or decreasing the number of cross-direction fibers to adjust the average gap distance between adjacent fibers; flowing biological cells in a medium through the PCL-ENF-PEGDA scaffold.

8. The method of claim 7, further comprising controlling variable porosity of said PCL-ENF-PEGDA scaffold by varying the number layers of PEGDA and varying its porosity by mixing PEGDA with osteo-conductive nanoparticles (e.g. chitin, chitosan, Hydroxyapatite).

9. The method of claim 8, further comprising controlling the number of PCL and PEGDA layers to produce the PCL-ENF-PEGDA scaffold.

10. The method of claim 7, further comprising controlling variable porosity of said PCL-ENF-PEGDA scaffold by varying the number layers of PCL and varying its porosity by changing the architecture of fibers (material, diameter, distribution, number of layers) to produce said membrane.

11. The method of claim 7, further comprising infusing nutrients into said PCL-ENF-PEGDA scaffold by mixing bone growth protein (collagen, fibronectin) with PCL fiber matrix before the construction of said PCL-ENF-PEGDA scaffold using said PCL fiber matrix.

12. The method of claim 11, further comprising mixing bone growth minerals (hydroxyapatite, MgO, CaO) with said PCL fiber matrix before the construction of PCL-ENF-PEGDA scaffold using said PCL fiber matrix.

13. The method of claim 11, further comprising mixing antibacterial agent (ZnO, silver) with said PCL fiber matrix before the construction of PCL-ENF-PEGDA scaffold using said PCL fiber matrix.

14. The method of claim 7, wherein said PCL-ENF-PEGDA scaffold is fabricated using PCL membranes exhibiting a specific porosity intended to encapsulate biological cells of a specific size.

15. The method of claim 14, wherein said biological cells with medium are flowed through the PCL-ENF-PEGDA scaffold multiple times.

16. The method of claim 15, wherein various biological cell types are encapsulated using PCL-ENF-PEGDA scaffold comprising PCL membranes in PCL-ENF-PEGDA adapted with differing porosity to encapsulate a specific size of cell.

17. The method of claim 16, wherein said biological cell types comprise cartilage cells.

18. The method of claim 16, wherein said biological cell types comprise skin cells.

19. The method of claim 16, wherein said biological cell types comprise organ cells.

20. The method of claim 16, wherein said biological cell types comprise plant cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is a non-limiting diagram showing a combined electrospin-UV photopolymerization unit with a robotic mechanism for automatic production of 3D scaffold of the present invention.

[0037] FIG. 2 is a non-limiting diagram showing a schematic view of the method of the present invention.

[0038] FIG. 3A is a non-limiting diagram showing equipments and accessories that may be used for fiber capture using the methods of the present invention.

[0039] FIG. 3B is a non-limiting diagram illustrating fiber matrix layered on a substrate used for fabrication of the 3D scaffold using the method of the present invention.

[0040] FIG. 3C is a non-limiting diagram showing UV curing station equipments and accessories used for fabrication of the 3D scaffold using the method of the present invention.

[0041] FIG. 3D is a non-limiting diagram comparing a PEGDA scaffold with a PCL-ENF-PEGDA scaffold produced using the methods of the present invention.

[0042] FIG. 4 is a non-limiting diagram showing SEM images of paraffin embedded and sectioned scaffolds: (a) PEGDA and (b) PCL-ENF-PEGDA.

[0043] FIG. 5 is a non-limiting diagram showing human Hepatocellular carcinoma cells attached to 12 layers of PCL nanofiber (Panel BF and CF) sandwiching the PEGDA scaffold.

[0044] FIG. 6 is a non-limiting diagram showing H&E staining of PCL-ENF-PEGDA scaffolds with Human Hepatocellular Carcinoma cells (Panel A) 0 day and (Panel B) after 7 days of incubation.

[0045] FIG. 7 is a non-limiting diagram showing human hepatocellular carcinoma cell proliferation after 14 days of culture on PCL-ENF-PEGDA scaffolds using Alamar Blue® assay.

[0046] FIG. 8 is a non-limiting diagram showing the process of the cells entrapment in PCL-ENF-PEGDA scaffold.

[0047] FIG. 9 depicts a table listing test results that show higher stiffness and elasticity of PCL-ENF-PEGDA composite scaffold compared to PEGDA scaffold.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0048] In brief:

[0049] FIG. 1 is a non-limiting diagram showing a combined electrospin-UV photopolymerization unit for automatic production of 3D scaffold of the present invention. The systems (electrospin and UV polymerization systems) used to produce 3D scaffold can be combined as shown. Using the automatic system as shown any number of Polyethylene Glycol Diacrylate (PEGDA) layer and PCL matrix layers can be used to produce any shape of 3D scaffold. Such scaffold can be used as an engineering scaffold for grafting of natural tissue such as liver, skin, bone etc.

[0050] FIG. 2 is a non-limiting diagram showing a schematic view of the method of the present invention. The invention uses a novel approach of controlled application of aligned electrospun fiber layer and photosensitive biomedical polymer to create 3D scaffolds. The process of creating 3 layers of fiber-polymer matrix is schematically represented. The number of cross aligned fiber layers holds the polymer liquid in place and then UV light is used to cure the liquid on top of fiber layer. Using this method, at least a circular 3D scaffold can be produced.

[0051] FIG. 3A is a non-limiting diagram showing equipments and accessories that may be used for fiber capture using the methods of the present invention. Using an electrospin setup, parallel fibers are collected between any two parallel collectors. The collectors can be dual disc (left) or two fixed parallel wires (middle) or two parallel conveyer belt (right).

[0052] FIG. 3B is a non-limiting diagram showing a substrate mold used to collect multiple layers of fiber. A mold may be used to touch the aligned fiber stream, then lowered and rotated 90° and the touch process repeated to collect another layer. The layers are coated with hydrogel where the hydrogel is held in place by the fibers. After fabrication of the two layers is complete, the mold with the fiber layers and hydrogel is positioned into a UV curing station to cure the 3D scaffold.

[0053] FIG. 3C is a non-limiting diagram showing UV curing station equipments and accessories used for fabrication of the 3D scaffold using the method of the present invention. Currently, in the laboratory setup the UV curing station is separated from the UV station. However, such a station can be incorporated into the chamber enclosure of the electrospining device as disclosed U.S. Pat. No. 9,359,694 and co-pending application Ser. No. 14/734,147.

[0054] FIG. 3D is a non-limiting diagram comparing a PEGDA scaffold with a PCL-ENF-PEGDA scaffold produced using the methods of the present invention. A novel electrospun polycaprolecton (PCL) nanofiber polyethylene glycol diacrylate (PEGDA) based 3D cell culture device (9.565 mm diameter×1.5 mm thickness) was successfully prepared as shown. The scaffold was made with 3 layers of PEGDA and 4 layer PCL nanofiber matrix.

[0055] FIG. 4 is a non-limiting diagram showing SEM images of top and longitudinally cut section views of a scaffold: (a) PEGDA and (b) PCL-ENF-PEGDA. PCL-ENF-PEGDA samples shows higher amounts of artifacts (void and presence of fibers) in comparison to PEGDA.

[0056] In detail:

[0057] Referring now to FIG. 1, a non-limiting diagram is shown illustrating a combined electrospin-UV photopolymerization unit for automatic production of 3D scaffold of the present invention. The systems (electrospin and UV polymerization systems) used to produce 3D scaffold can be combined as shown in FIG. 1. Using the automatic system as shown any number of polyethylene Glycol Diacrylate (PEGDA) layer and PCL matrix layers can be used to produce any shape of 3D scaffold. A substrate may be adapted to produce a 3D scaffold comprising at least two equal linear dimensions, or a circular shape. Such scaffold can be used as an engineering scaffold for grafting of natural tissue such as liver, skin, bone etc.

[0058] A syringe pump 10 is used to feed electrostatic polymer solution in to glass syringe 11 and flow through tube 12 to a metallic needle 13. The parallel metallic collectors 14 can be charged dual disks (30, FIG. 3A), where the disks are spun using speed controlled, direct current (DC) motors and gear mechanism. Alternatively, a pair of charged fixed wires aligned in opposing planes may be used to collect fiber (31, FIG. 3A). Another alternative is to use a conveyer system having two parallel wires that advance while collecting aligned fibers (32, FIG. 3A). The syringe needle 13 is electrically excited by applying a high-voltage in the range of (5 KVA to 15 KVA) produced by the power supply 15. This electrically charged syringe needle 13 for electrospinning synthetic polymer fiber streams is positioned above and substantially centered between the edges of parallel metallic collectors 14. This will realize an electrical potential difference between the needle tip and the collectors 14, positioning being adjustable by Z positon control stage 16. As a result, electromagnetic field propagates between the charged syringe needle 13 and the edge of the collectors 14 (e.g., rotating metallic discs). This enables capturing, depositing and aligning fiber between the collectors 14. To insure a homogenous polymer injection from the needle 13, the polymer solution flow by the syringe pump 10 may be controlled by a microcontroller 17 and a rack-and-pinion gear setup in the parallel collectors 18. The microcontroller 17 may also be used to optimize the distance between the parallel collectors 14, the distance between collectors 14 and the needle 13, and the distance from the collectors 14 and a base support (not shown). An anti-vibration Z positon control stage 16 may be incorporated to the base support to maximize the parallelism of the fiber lines captured by the collectors 14. A pair of gear boxes may be utilized when the collectors 14 are configured as rotating disks to help in changing the rotation direction between shafts to optimize the parameters.

[0059] A robotic arm mechanism 19 operating on a track 101 may be used to collect fiber from the collectors 14 to assemble layers on a substrate 106 and feed it to the curing station 105 without manually intervening in this process. In the last stage of the system 100, the robotic arm 19 may be integrated with the PEGDA developing process via interaction with the UV curing station, positioning substrate 106 in line with a spray/needle tip 103 supplied by second syringe pump 102 to deposit controlled amounts of PEGDA on the top of fiber matrix on the substrate 107. A mold or mask 104 can be used to cure any desired shape of PEGDA layer on the top of fiber matrix using a UV light 105.

[0060] Referring now to FIG. 2, a non-limiting diagram is shown schematically illustrating the method of the present invention for creating a composite scaffold made from three layers of PCL-ENF-PEGDA membranes. The embodiment shown in the diagram uses a novel approach of controlled application of aligned electrospun fiber layer and photosensitive biomedical polymer to create 3D scaffolds. A single layer of aligned unidirectional PCL ENF is intercepted on a substrate positioned between two collectors in the ENF machine 20, where PCL beads may be dissolved with acetone (15 wt %) to produce the fibers. The fibers may be harvested at 90° angles and stacked in layers to produce an ENF membrane 21 on a substrate. The ENF membrane 21 may be subsequently layered with a PEGDA hydrogel 22 that is cured by exposure to UV light (FIG. 3C), thereby creating a composite PCL-ENF-PEGDA scaffold 23. FIG. 3D shows 1.5 mm thickness PEGDA hydrogel 301. Multiple layers of ENF membranes were interspersed and bonded together by 0.3 mm PEGDA layer to produce a three dimensional (3D) PCL-ENF-PEGDA composite scaffold (FIG. 3D 302) shows produced 1.5 mm thickness PCL-ENF-PEGDA scaffold.

[0061] The fabrication of a PCL electrospun nanofiber-PEGDA 3D scaffold in the present invention requires the following unique features of the process: [0062] 1. Controlled deposition fiber collection to produce a fiber matrix having a porosity sized so that PEGDA gel does not go through the fiber matrix; [0063] 2. Thickness of PEGDA layers sized so that each layer has a substantially uniform porosity [0064] 3. UV curing time controlled to produce a substantially uniform stiffness of each PEGDA layer [0065] 4. Controlled number of fiber matrix and PEGDA layers to create a specific height scaffold.

[0066] Referring now to FIG. 3A through FIG. 3D, images are provided in FIG. 3A showing equipment and accessories used for fabrication of the 3D scaffold using the method of the present invention. The process of the present invention disclosed herein can be accomplished using a robotic arm mechanism (FIG. 1, 19). In a preliminary laboratory arrangement, such collection was made manually, for which a precise 90° rotation is almost impossible to achieve. To automate this step, a robotic arm (FIG. 1, 19) can be used to position a substrate (FIG. 1, 106) FIG. 3B 34 to collect the fiber layer from the collectors (FIG. 1, 14) FIG. 3A 30 or 31 or 32 and feed it to the curing station FIG. 3C without manually interfering in this step. Required numbers of layers of aligned fibers can be collected on a grounded custom made silicon mold chamber (FIG. 1, 106) FIG. 3B 34. After fabrication of the layers is complete, the mold chamber (FIG. 1, 106) FIG. 3B 35 with the fiber layers is fed into a UV curing station FIG. 3C to cure the 3D scaffold. The process is repeated to stack up layers of cross-aligned fibers on the mold chamber FIG. 3B 36. Currently, in the laboratory setup the UV curing station FIG. 3C is separated from the fiber capture components. However, such a station can be incorporated as an integrated step and located in the chamber enclosure of the electrospining device disclosed in U.S. Pat. No. 9,359,694 and co-pending application Ser. No. 14/734,147.

[0067] PEGDA is a UV cured polyethylene glycol diacrylate (PEGDA) hydrogel injected between the fiber layers to build the 3D fiber scaffold. A UV light source is exposed to the solution to completely cure of the PEGDA solution to a solidified state. The thickness of the PEGDA layer of the scaffold produced in our laboratory experiments was 0.5 mm. The forgoing PCL fiber mat and PEGDA steps were repeated 3 times to make 1.5 mm thick cylindrical 3D scaffold (FIG. 3D).

[0068] FIG. 4 is a non-limiting diagram showing SEM images of top and section views of a scaffold: (a) PEGDA and (b) PCL-ENF-PEGDA. Top surface of the produced samples was viewed without any embedding agent by SEM to get the top view of the scaffold. Each samples was paraffin embedded, then sectioned longitudinally and finally viewed in SEM images to produce the section views. PCL-ENF-PEGDA samples shows higher amounts of artifacts (void and presence of fibers) in compare to PEGDA.

[0069] FIG. 5 is a non-limiting diagram showing human Hepatocellular carcinoma cells attached to 12 layers of PCL nanofiber (Panel BF and CF) sandwiching the PEGDA scaffold. Panel BS and CS represent cells growing embedded in PEGDA scaffold and clearly show that our scaffolds support cell proliferation after 2 weeks of incubation. Panel AF and AS are control PCL-ENF-PEGDA scaffold without cells. White arrows point to cells. All the scaffolds were incubated for 2 weeks. Images were taken using Olympus light microscope at 100X total magnification.

[0070] FIG. 6 is a non-limiting diagram showing H&E staining of PCL-ENF-PEGDA scaffolds with Human Hepatocellular Carcinoma cells (Panel B) after 1 week of incubation. Black arrows in panel B point to cells. Our results clearly indicate that cells are able to migrate into PEGDA matrix. Panel A is control scaffold without any cells. Olympus light microscope at 400× magnification.

[0071] FIG. 7 is a non-limiting diagram showing human hepatocellular carcinoma cell proliferation after 14 days of culture on PCL-ENF-PEGDA scaffolds using Alamar Blue® assay. Our results clearly indicate that cells remain viable and actively proliferating on our scaffolds. Values are mean±SD of duplicates.

[0072] FIG. 8 is a non-limiting diagram showing cell entrapment in PCL-ENF-PEGDA scaffold. Entrapment of cells in the scaffolds during the fabrication process is advantageous because of their homogeneous distribution. Since the porosity and entrapment of cells or nutrients are directly related, therefore, higher number of cell entrapment is possible by having higher number of fiber layers in PCL ENF membrane. Increased addition of cells in the scaffold is possible by having higher number of fiber layers in ENF membrane. This may result in an increase in cell viability, proliferation, differentiation, and spreading in vitro and in vivo compared to pristine PEGDA hydrogels and PCL-ENF-PEGDA hydrogel made with less number of fibers. Such entrapment enables excellent spatial control that can potentially be used to recapitulate the complex 3D hierarchy of the tissue microenvironment. This may have significant impact on driving the development of in vitro 3D models toward broader applications, including those in tissue engineering, cell mechanics, and bio-hybrid artificial devices and machines. Applications may include growth or fabrication of biological material derived from all biological cell types including at least any of cartilage cells, skin cells, organ cells, and plant cells.

[0073] TABLE 1 in FIG. 9 is a non-limiting diagram comparing compression and viscoelastic properties between PEGDA and PCL-ENF-PEGDA scaffolds.

Experimental Methods

[0074] Two groups of samples were prepared for this innovation: PEGDA and PCL-ENF-PEGDA samples. Morphology, mechanical and cell viability properties were examined to compare the performances of the scaffolds in relation to the performance of functional tissue graft used for biomedical applications. Both samples have same diameters (9.56 mm). A novel electrospun polycaprolecton (PCL) nanofiber polyethylene glycol diacrylate (PEGDA) based 3D cell culture device (9.565 mm diameter×1.5 mm thickness) was successfully prepared as shown. The scaffold was made with 3 layers of PEGDA and 4 layer PCL nanofiber matrix. Lever cancerous cell were cultured in the 3D scaffold.

Material

[0075] Two solutions were combined to make the final PEGDA hydrogel solution mix. The first solution consisted of the liquid Polyethylene Glycol Diacrylate (PEGDA), M.sub.n=700 (mol), diluted with liquid Dulbecco's Phosphate Buffer Saline (PBS). The second solution consisted of a solute solid photo-initiator (PI) Alpha-alpha-dimethoxy-alpha-phenylacetophenone, M.sub.w=256.35 (g/mol); Sigma-Aldrich, that was dissolved in the liquid solvent 1-vinyl-2-pyrrolidone, M.sub.w=111.14 (g/mol). Two solutions were combined to make poly(ε-caprolactone) PCL fiber. PCL beads (pellet size .sup.˜3 mm, average M.sub.n 80,000) and acetone (laboratory reagent ≧99.5%) were mixed to prepare the PCL solution.

Specimen Preparation

[0076] PEGDA Samples:

[0077] The 20% PEGDA solution was produced by mixing 2 ml of PEGDA with 8 ml of DPBS. The PI solution was produced by mixing 0.3 (g) of PI powdered solid in 1 ml of the liquid vinyl solvent in a dark room to prevent premature cross-linked curing from light. The 0.2% PI volume concentration hydrogel solutions was produced by adding 4 μl of PI solution with 2 ml of PEGDA solution, respectively. The desired hydrogel mixtures were added to the cell pellet and vortexed to ensure thorough mixing. For curing, a 365 nanometer (nm) UV lamp was used to photo-polymerize. The UV-lamp was mounted in the electrospin chamber. The lamp was turned on 20 minutes before hydrogel curing to reach maximum UV light intensity. PEGDA was poured in to 10 mm diameter×1.5 mm thickness silicone mold and cured simultaneously in a dark room to prepare the PEGDA samples.

[0078] PCL-ENF-PEGDA Samples:

[0079] PCL solution was prepared by ultrasonic (Sonics & Materials, Inc., model # Vibra-cell VCX 130) mixing of 7.69 wt % of PCL pellets (pellet size.sup.˜3 mm, average M.sub.n 80,000) with acetone (laboratory reagent ≧99.5%). The sonication process was carried out at approximately 60° C. for an 30 minutes. The solution was poured into a glass syringe in an infusion pump (Harvard Apparatus, mode # PHD ULTRA) for fiber production. PCL fibers were ejected from the glass syringe via charged needle (23G blunt needle, aluminum hub, 1″ length, model # BX 25). The needle was charged by high voltage power source (Gamma High Voltage Research, Inc., model # ES 30 series).

[0080] The PCL fibers were harvested manually at approximately 90° angles and stacked in layers to produce an ENF membrane on the substrate. The PCL membranes were subsequently layered with PEGDA membranes cured by exposure to UV light, thereby creating a PCL-ENF-PEGDA scaffold. The process was repeated 3 times and finally coated by PCL membrane to create the PCL-ENF-PEGDA scaffolds

[0081] Morphological Difference Between PEGDA and PCL-ENF-PEGDA:

[0082] There is clear topographical difference observed between PCL and PCL-ENF-PEGDA samples (FIG. 4). PCL-ENF-PEGDA has more porosity in compare to PCL due to the present of PCL fibers. The existence of fibers was also visible.

[0083] Mechanical Tests on PEGDA and PCL-ENF-PEGDA Samples:

[0084] They were mounted between the holders in Evex mechanical test equipment. The samples were loaded up to 35 N. The load and the corresponding displacement of the scaffolds were directly recorded from Evex machine software. The slopes of the curves were used to compare the difference of stiffness between the samples. The test results (TABLE 1) showed that the higher surface artifacts of PCL-ENF-PEGDA composite scaffold compared to PEGDA scaffold. The average stiffness of PCL-ENF-PEGDA composite scaffold (5.36 N/mm) is approximately 2 times higher than that of PEGDA scaffold (3.00 N/mm). The results indicated that PCL-ENF-PEGDA composite scaffold strength was higher compared to PEGDA. The results confirm that PCL ENF membrane can reinforce the PEGDA scaffold. Further improvement of stiffness and other mechanical properties of PEGDA scaffold is possible by controlled deposition of PCL ENF membrane in the scaffold. Results showed that our developed scaffolds satisfied the minimum compressive modulus requirement for bone graft substitutes (>0.5 MPa). We have conducted cell viability studies on the scaffold to evaluate and confirm its biological compatibility.

[0085] Cell Viability on PCL-ENF-PEGDA Samples:

[0086] Biocompatibility of PCL-ENF-PEGDA composite scaffolds using human hepatoma cells at different time interval. The composite scaffold also facilitated the slow diffusion of oxygen and nutrients necessary for cell proliferation and differentiation (FIGS. 6A and 6B) The results show that PCL-PEGDA scaffold promotes highly desired cell arrangement with more hepatoma cells attachment at fiber junctions compared to PE (FIG. 6B). There is in increased number of cell growth in the PCL-ENF-PEGDA scaffold was observed with time (FIG. 7). These results prove that PEGDA-PCL scaffold promotes cell viability and proliferation.