Method for repairing living tissue with a hollow fiber scaffold

Abstract

A scaffold of hollow fibers comprising a mixture of polylactic acid (PLA) and polybutylene succinate (PBS) and cellulose nanofibers (CNF), medical products made of these scaffolds and methods of using the scaffolds in regenerative medicine. A method for producing the scaffolds is also disclosed.

Claims

1. A method for repairing living tissue, comprising applying a hollow fiber scaffold on, in or around living tissue in need of repair, wherein the hollow fibers of the hollow fiber scaffold fibers have a hollow core and a peripheral shell, wherein the peripheral shell comprises a mixture of homogenously dispersed polylactic acid (PLA), polybutylene succinate (PBS) and cellulose nanofibers (CNF); wherein the hollow fibers have an average diameter ranging from 400 to 1,000 nm and the hollow core has an average diameter ranging from 300 to 900 nm.

2. The method of claim 1, wherein the living tissue in need of repair is a vascular tissue.

3. The method of claim 1, wherein the living tissue in need of repair is an epithelial tissue.

4. The method of claim 1, wherein the peripheral shell has pores ranging in size from 25 to 100 nm in diameter.

5. The method of claim 1, wherein the peripheral shell has pores ranging in size from 40 to 60 nm in diameter.

6. The method of claim 1, wherein the peripheral shell has uniformly distributed pores wherein an average size or numbers of pores in different areas of the peripheral shell varies by no more than 10%.

7. The method of claim 1, wherein the hollow fiber scaffold has an interconnected macroporous structure.

8. The method of claim 1, wherein the peripheral shell comprises micropores; and wherein the hollow core and the micropores of the peripheral shell provide enhanced diffusion of nutrients and oxygen to the cells grown on or with the scaffold as compared to cells grown on scaffold comprising solid fibers.

9. The method of claim 1, wherein the hollow fiber scaffold is a portion of a sterile or aseptic patch, packing, dressing or bandage applied to the tissue in need of repair, and wherein the hollow fiber scaffold comprises cells or one or more biologically active agents.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

(2) FIG. 1 schematically illustrates one embodiment of hollow fiber preparation and design based on a coaxial electrospinning method. Core solution 100 and shell solution 200 are fed to a spinneret 300 which comprises a spinneret fitting 310 and a double layer capillary 320, electric force 400 draws the materials out of the spinerette and produces a core-filled filament 510. The core is removed by evaporation step/evaporator 500 forming a hollow fiber 520.

(3) FIGS. 2A-2B. Cross-section by SEM of hollow fibrous scaffolds made of glycerol at a 1 ml/h shell feed rate and at a 0.03 ml/hr core feed rate.

(4) FIGS. 2C-2D. Cross-section by SEM of hollow fibrous scaffolds made of glycerol at a 1 ml/h shell feed rate and at a 0.05 ml/hr core feed rate.

(5) FIGS. 2E-2F. Cross-section by SEM of hollow fibrous scaffolds made of glycerol at a 1 ml/h shell feed rate and at a 0.08 ml/hr core feed rate.

(6) FIGS. 2G-2H. Cross-section by SEM of hollow fibrous scaffolds made of glycerol at a 1 ml/h shell feed rate and at a 0.10 ml/hr core feed rate.

(7) FIGS. 2I-2J. Cross-section by SEM of hollow fibrous scaffolds made of glycerol at a 1 ml/h shell feed rate and at a 0.12 ml/hr core feed rate.

(8) FIGS. 2K-2L. Cross-section by SEM of hollow fibrous scaffolds made of glycerol at a 1 ml/h shell feed rate and at a 0.15 ml/hr core feed rate.

(9) FIGS. 3A-3B. Cross-section by SEM of hollow fibrous scaffolds made of mineral oil at a 1 ml/h shell feed rate and at a 0.08 ml/hr core feed rate.

(10) FIGS. 3C-3D. Cross-section by SEM of hollow fibrous scaffolds made of mineral oil at a 1 ml/h shell feed rate and at a 0.10 ml/hr core feed rate.

(11) FIGS. 3E-3F. Cross-section by SEM of hollow fibrous scaffolds made of mineral oil at a 1 ml/h shell feed rate and at a 0.12 ml/hr core feed rate.

(12) FIGS. 3G-3H. Cross-section by SEM of hollow fibrous scaffolds made of mineral oil at a 1 ml/h shell feed rate and at a 0.15 ml/hr core feed rate.

(13) FIGS. 4A-4D. SEM images of hollow fibrous composite scaffolds: GC (FIG. 4A-4B) and OC (FIG. 4C-4D). See Table 1 for description of GC and OC scaffolds.

(14) FIGS. 5A-5F illustrate fiber size distributions of the solid scaffolds 50/50 (FIG. 5A) and C4 (FIG. 5B); and the hollow scaffolds GH (FIG. 5C), GC (FIG. 5D), OH (FIG. 5E) and OC (FIG. 5F). See Table 1 for description of 50/50, C4, GH, GC, OH, and OC scaffolds.

(15) FIGS. 6A-6D illustrate a high resolution SEM images of hollow fibrous scaffolds: GH (FIG. 6A), OH (FIG. 6B), GC (FIG. 6C), and OC (FIG. 6D).

(16) FIGS. 7A-7D. Deconvoluted XPS spectra of hollow fibrous scaffolds: GH (FIG. 7A), OH (FIG. 7B), GC (FIG. 7C), and OC (FIG. 7D).

(17) FIG. 8. Water contact angles of hollow fibrous scaffolds (GH, OH, GC, OC) compared to the solid fibrous scaffolds (50/50, C4).

(18) FIG. 9. Protein adsorption capacities of hollow fibrous scaffolds (GH, OH, GC, OC) compared to the solid fibrous scaffolds (50/50, C4). The Y-axis unit is “adsorbed protein in μg/mg and shows the amount of protein adsorbed in each mg of scaffold.

(19) FIGS. 10A-10D. GFP/DAPI stained images of GH (FIG. 10A), OH (FIG. 10B), GC (FIG. 10C) and OC (FIG. 10D) hollow fibrous scaffolds after 7 days of cell culturing.

(20) FIG. 11A. MTT optical density value for the hollow fibrous scaffold after one and two weeks of cell culturing.

(21) FIG. 11B. Viability comparison after one week between solid and hollow fibrous scaffolds.

(22) FIG. 12A. AFM surface topography of electrospun PLA/PBS single fiber.

(23) FIG. 12B. AFM surface topography of electrospun PLA/PBS/CNF composite single fiber.

DETAILED DESCRIPTION OF THE INVENTION

(24) The inventors recognized the significant disadvantages of using solid fibrous scaffolds for tissue engineering as these induce the formation of necrotic cores within a scaffold designed to induce recovery or regeneration in large tissue defects. This is because the boundary of the scaffold is often blocked by seeded cells, hence vital nutrients cannot be transported properly to cells in the central area of the scaffolds.

(25) In contrast, the microporous walls and hollowness in the hollow fiber scaffolds disclosed herein provide external channels for mass transport to ensure the cellular growth throughout the scaffold. Therefore, the cell viability, growth rate and tissue recovery rate are greatly enhanced.

(26) Surprisingly, compared to corresponding solid fiber scaffolds, the formation of hollow fiber scaffolds showed a significant decrease in the contact angle and wettability. In terms of contact angle and wettability, as shown herein, the morphological structure of the PLA/PBS/CNF hollow fiber scaffolds was determined to be more significant than their composition. It was found that both PLA and PBS exhibit hydrophobic surface properties, but their 50/50 wt. % combination reduced the water contact angle by more than 30°. Surprising it was also found that the incorporation of CNF into the PLA/PBS fiber also reduced the water contact angle; compare 50/50 with C4 in FIG. 8.

(27) Furthermore, protein adsorption results in FIG. 9 showed that the large surface area, which resulted from the use of microporous hollow fibrous scaffold, attracted more protein to the scaffold surface and that the incorporation of hydrophilic nano cellulose fibrils (CNF) further enhanced protein adsorption. Enhancement of protein adsorption to a scaffold can provide a microenvironment for sufficient cell-cell interaction, cell migration, proliferation and differentiation required for tissue repair or regeneration.

(28) Surprisingly, mechanical testing results showed that strength, elasticity and hardness of the hollow fibrous scaffolds were not significantly different than those of the solid fibrous scaffold.

(29) The flexibility and ductility of the hollow fibrous scaffolds was much lower when compared to a stiffer solid fibrous scaffold. This may be due to an increase in the number of interfaces in the hollow fibrous scaffolds which can act as crack initiation or stress concentration sites. The incorporation of cellulose nanofibrils into hollow fibers not only significantly increased the strength and elastic modulus of the scaffold but also improved the flexibility. In contrast for the solid fiber, ductility and flexibility of the scaffold were not significantly affected by the presence of cellulose nanofibrils.

(30) Polylactic acid (“PLA”), an aliphatic polyester, is a widely studied synthetic polymer for use in tissue engineering due to its renewability, biodegradability and cost effectiveness; O'Brien, F. J., Biomaterials & scaffolds for tissue engineering. Materials today, 2011. 14(3): p. 88-95; Gigli, M., et al., Poly (butylene succinate)-based polyesters for biomedical applications: A review. European Polymer Journal, 2016. 75: p. 431-460; and Hardiansyah, A., et al., Electrospinning and antibacterial activity of chitosan-blended poly(lactic acid) nanofibers. Journal of Polymer Research, 2015. 22(4): p. 59m each incorporated herein by reference in their entirety.

(31) Polybutylene succinate (“PBS”) is an aliphatic polyester polybutylene that is flexible, has a high degree of crystallinity, biodegradable, and has cell-friendly surface characteristics. The inventors have recognized that PBS, as a relatively soft polymer compared to PLA, can provide a plasticizing effect on the mechanically stiff but brittle PLA.

(32) As disclosed herein, it is possible to reinforce a PLA/PBS matrix using cellulose nanofibrils (CNF) which can be synthesized from the most abundantly found natural polymer cellulose; Benitez, A. and A. Walther, Cellulose nanofibril nanopapers and bioinspired nanocomposites: a review to understand the mechanical property space. Journal of Materials Chemistry A, 2017. 5(31): p. 16003-16024; and Hanif, Z., et al., Butanol-mediated oven-drying of nanocellulose with enhanced dehydration rate and aqueous re-dispersion. Journal of Polymer Research, 2017. 25(3): p. 191, each incorporated herein by reference in their entirety.

(33) In some embodiments, the cellulose nanofibrils or CNF will have lengths ranging from 1, 2, 3, or >3 μm, diameters ranging from <0.5, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or 30 nm as measured by TEM.

(34) In some embodiments, the ratio of length to diameter of a CNF may range from 200:1, 150:1 to 100:1. In some embodiments, CNF can exhibit a mechanical performance characterized by an elastic modulus in the range of 40-50 GPa, preferably about 46.6 GPa and a tensile strength in the range of 1000 to 1200 MPa, preferably about 1170 MPA. Physical properties of CNF are incorporated by reference to Clemons, C., Nanocellulose in spun continuous fibers: A review and future outlook. Journal of Renewable Materials, 2016. 4(5): p. 327-339. Typically, the CNF are ultrathin nanofibrils with large length to width aspect ratio. The average width of CNF is 50±10 nm and length is 2000±240 nm resulting in an aspect ratio of L/w=40±4. The average ratio of diameter to length is 0.025±0.002. During the electrospinning process single CNFs were uniformly dispersed into PLA/PBS matrix without agglomeration. The phenomenon can be identified from atomic force microscopy (AFM) images; see FIGS. 12A and 12B.

(35) The nanoscale characteristics of CNF are helpful in achieving high magnitude of orientation and high moisture adsorption capacity which are favorable for medical applications. Moreover, it is advantageous compared to other reinforcing agents such as carbon nanotube in terms of biocompatibility and bioactivity.

(36) An AFM analysis was performed on the CNF incorporated PLA/PBS and noticed that CNF is not only conjugated into the PLA/PBS matrix but also CNF orientation is exactly parallel to the electrospun fiber; see FIGS. 12A and 12B. This implies that the orientation of CNF can be tailored by electrical charge during the process of electrospinning.

(37) The moisture adsorption capacity of CNF ranges from about 13 to 24 g water/g CNF. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay test was performed on the electrospun scaffolds. The CNF contained scaffolds supported cell attachment and growth without showing any adverse effects. The cell growth rate on the CNF reinforced composite was much better than for the control reference. These results show that the CNF is 100% biocompatible making its suitable for biomedical applications.

(38) In view of these advantageous properties, it is not necessary to incorporate other types of fibers, fillers or particles non-CNF forms of cellulose, such as cellulose nanoballs or nanopellets, sisal or hemp fibers, or shorter cellulose nanowhiskers, cellulose nanocrystals, cellulose nanorods, which have needle-like or rod-like morphologies and have shorter length to diameter ratios than CNF; or other organic or inorganic fillers such as silver or other metals, or clays into the fibers as disclosed herein.

(39) Porosity. Scaffolds per se or the walls of the scaffold fibers may contain pores, preferably uniformly distributed pores where the number of pores in different areas of the fiber vary by no more than 5, 10, 15, or 20%. In most embodiments, the sizes of the pores in the walls of the scaffold fibers will be no more than 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm, preferably from about 40 to about 60 nm.

(40) In some embodiments, the scaffolds may contain pores ranging from >100, 200, 500 to 1,000 nm, or in scaffolds, at least 1, 2, 5 or 10 μm. Preferably, the pores in the scaffold are uniformly sized and distributed, for example, where the average size or numbers of pores in different areas of the scaffold or scaffold fiber walls differ by no more than 1, 2, 5, 10, or 20% in average area or average number. Apart from the porosity of the fiber walls, the scaffold itself has a porosity ranging from about 3, 4, 5, 6, 7, 8, 9, to 10 μm. The developed scaffold is an interconnected macroporous structure having average pore size of about 3.5 μm.

(41) Wettability of the scaffold or its fibers can be determined by measurement of the water contact angle. In some embodiments, the water contact angle of a scaffold as disclosed herein, ranges from not more than 10, 25, 30, 35, 40, 45, to 50° or any intermediate value within this range. Typically, the water contact angle of a scaffold of hollow fibers as disclosed herein will be less than that of a scaffold of compositionally similar solid, non-hollow fibers. As disclosed herein the inventors have also found that a combination of PLA and PBS (e.g. about a 40/60, 50/50 or 60/40 wt. % combination) reduces the water contact angle of the hollow fibers, for example by at least 10, 20, or 30 degrees when compared to a solid fiber

(42) A scaffold or mesh, such as a scaffold used for tissue engineering, is a structure made of artificial or natural substances that acts as a shape on which cells can grow. A scaffold can be inert and not interact with the cells growing on it, or it can actively help the cells to grow by releasing chemical signals and/or nutrients. A scaffold can be partially or completely biodegradable or non-biodegradable. Typically a scaffold as disclosed herein comprises hollow fibers made of PLA, PBS and CNF, is wettable, and has a surface roughness and porosity that permits it to bind to proteins and cells. The hollowness and porosity of the fibers permits enhanced transport of nutrients to cells growing on the scaffold. These features permit it to provide a suitable biocompatible microenvironment for the sufficient cell-cell interaction, cell migration, proliferation and differentiation. In preferred embodiments the scaffold as disclosed herein is biodegradable.

(43) The thickness of a flat scaffold or conformation of a shaped scaffold may be selected based on the type of tissue being repaired or regenerated. In some embodiments, the thickness of a scaffold as disclosed herein will range from <0.1, 0.1, 0.2. 0.5, 1, 2, 5, 10 or >10 mm. Preferably, a flat sheet scaffold has a thickness of 0.05, 0.1, 0.2, 0.5, 1.0 to 2 mm which is mainly dependent on electrospinning time.

(44) In some embodiments, the scaffold may be coated with or otherwise incorporate one or more therapeutic, prophylactic, or diagnostic agents, such as hemostatic agents, anti-infectives, growth factors, cytokines, stem cells, cultured cells such as induced stem cells, or other cells such as autologous or allogenic cells, anesthetics, vasoconstrictors, ECM components such as collagen (including Types 1, 2, 3, 4 and 5), fibrin, chitosan, glycosaminoglycan including hyaluronic acid, laminin, or fibronectin, antibodies or complement factors. These additional components may be coated or attached on the surfaces of, or in the hollow cores of, electrospun fibers.

(45) Electrospinning procedures. Electrospinning uses an electrical charge to draw very fine (typically on the micro or nanoscale) fibers from a liquid. One skilled in the art may select a commercially available electrospinning device equipped to feed the core and shell solutions into the inner and outer layers of spinnerets sized to produce the hollow fibers as disclosed herein. Core-shell fibers may be produced by using a co-axial nozzle for electrospinning as described by and incorporated by reference to Yarin A L, et al.. Evolution of core-shell structure: From emulsions to ultrafine emulsion electrospun fibers. J. Mater. Chem. 2007; 17: 2585. The co-axial nozzle comprises two cylinders with one cylinder situated within the core of a larger bore cylinder. Two different solutions are dispensed simultaneously through the inner and outer cylinders and charged in the same way as conventional single bore nozzle. There are several parameters that can be modified to control the size of the fibers and the volume ratio of the core and shell material. Fiber diameter can be controlled by the nozzle diameter while the volume ratio is by the feed-rate of the core and shell solution as described by and incorporated by reference to Chakraborty S, et al., Electrohydrodynamics: A facile technique to fabricate drug delivery systems. Adv Drug Deliv Rev 2009; 61: 1043. Those skilled in the art may select an appropriate electrospinning device, for example, from those which are commercially available. Such devices are available, and incorporated by reference to, the Electrospinning Device Catalogue hypertext transfer protocol://electrospintech.com/espincatalogue.html#.Xg4B de-WzIU (last accessed Jan. 2, 2020).

(46) Electrospinning stock solutions used to make the hollow fibers as disclosed herein typically comprise, for the core feed, glycerol, mineral oil, or another solvent that is immiscible with the stock solution for the shell. However, glycerol and mineral oil may be slightly soluble in some shell stock or precursor solutions as long as the degree of solubility does not significantly affect electrospinning of the core and shell.

(47) As shown herein electrospinning is a versatile technique to produce fibrous scaffold which mimics the native extracellular matrix (ECM) for vascular prosthesis because conventional solid fibrous scaffolds lack the ability of nutrient diffusion and have poor permeability resulting in the cell death and tissue necrosis. A hollow fibrous scaffold can greatly improve permeability and nutrient diffusion throughout the scaffold. The hollow fibers have vessel-like micro or nano structures which can serve as an excellent artificial vascular system.

(48) The inventors have developed scaffolds of hollow fibers comprising a unique combination of cellulose nanofibrils (CNF) reinforcing a PLA/PBS composite which are produced by a coaxial electrospinning technique forming a homogenous blended and hollow microporous fibrous structure. Glycerol and mineral oil were used as core templates which were removed by evaporation. Both templates allowed hollow fiber formation and uniform pore dispersion in the walls of the hollow fibers occurred when the mineral oil was used. The scaffold made by mineral oil template showed an enhanced wettability and protein adsorption capacity which may be due to the pore size distribution in the hollow fibers. These hollow fibrous scaffolds demonstrated superior ability to permit cell attachment and cellular proliferation as shown by a cell culture test and should provide a more favorable microenvironment for regeneration of tissues, such as to regenerate healthy vascular tissue.

Example

(49) Materials. Polylactic acid (PLA 2003D) was purchased from Nature Works, USA. Poly butylene succinate (PBS, commercial name is Bionolle) was obtained from Showa Denko, Japan. Cellulose Nanofibrils (CNF) was acquired from University of Maine, USA. Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Lonza, USA. Proteinase K solution (20 mg/ml), Dulbecco's phosphate-buffered saline and 10% fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific Inc., Fair Lawn, N.J. Glycerol was obtained from Loba Chemie Pvt Ltd, India. Mineral oil (P3) is from Pfeiffer Vacuum, Germany. Chloroform, ethanol, sodium dodecyl sulfate (SDS), Lysozyme, tris buffer solution (0.1 M, pH-7), calcium chloride (CaCl.sub.2) and sodium azide (NaN.sub.3) were from Sigma-Aldrich, St. Louis, Mo., USA.

(50) Preparation of hollow fibrous scaffold. A mixture of PLA and PBS at a weight ratio of 50/50 was dissolved in chloroform and ethanol with 3:1 volume ratio by stirring for 3 hours.

(51) In order to incorporate CNF into PLA/PBS, CNF/PBS composites with 4% of CNF concentrations were prepared first by melt extrusion. Then, the mixture of PLA and CNF/PBS composite in equal weight ratio was dissolved in the same solvent system overnight.

(52) A Nanon 101A electrospinning setup (NANON Supply, MECC, Fukuoka, Japan) was adapted for Coaxial Electrospinning. An ultra-thin coaxial spinneret (NANON Supply, MECC, Fukuoka, Japan) was used to create coaxial electrospun fibers. The PLA/PBS solution or the composite solution was delivered to outer layer of the spinneret by a system provided with a syringe pump using a Teflon® (Polytetrafluoroethylene)tube at 1 mL/h of feed rate.

(53) An extensional syringe pump (KDS 100, KD Scientific Inc., USA) connected with Teflon® tube was used to deliver the core materials into a 27-gauge blunt metallic needle.

(54) The double layer solution was electrically stretched at 20 kV of voltage over 12 cm of distance and finally collected on flat aluminum sheet.

(55) Two types of materials were selected for the core layers which were glycerol and mineral oil. Initial adjustments were performed on both materials using PLA/PBS solution as a shell layer.

(56) After electrospinning, the collected samples were placed in oven at 55° C. to evaporate the core solution. It was found that full evaporation of glycerol can be achieved in 24 hours while 5˜6 days were needed to evaporate the mineral oil from the sample. The prepared samples were named according to their core-shell composition before drying, as shown in Table 1.

(57) TABLE-US-00001 TABLE 1 Abbreviated name of hollow fiber, according to their core-shell composition Core Shell feed Scaffold Core feed rate rate Name material (mL/h) Shell material (mL/h) GH Glycerol 0.08 PLA/PBS 1.0 GC Glycerol 0.08 PLA/PBS/CNF 1.0 OH Mineral oil 0.1  PLA/PBS 1.0 OC Mineral oil 0.1  PLA/PBS/CNF 1.0 50/50 (solid) — — PLA/PBS 0.5 C4 (solid) — — PLA/PBS/CNF 0.5

(58) Characterization. The morphological features of the prepared fibers were observed using a Field emission scanning electron microscopy (FESEM, JEOL JSM 7600F, Tokyo, Japan). A customized image processing method was employed to calculate size distribution of the fibers. The SEM images were processed through adjustment, Gaussian smoothing, local thresholding and noise removal. Additionally, the fiber sizes were calculated by Canny Edge based Euclidian distance transform.

(59) The cross-section of the hollow fibrous scaffold was evaluated using SEM to confirm fiber hollowness and measure the inner diameter. The scaffold was frozen in liquid nitrogen and cut with a razor blade. The core removal was performed after cutting the sample and mounting it on the SEM specimen in order to avoid any deformation of the fiber cross section.

(60) The XPS measurements for the prepared scaffolds were carried out in an ultra-high vacuum multi-technique surface analysis system (SPECS GmbH, Germany). A standard dual anode X-ray source SPECS XR-50 with Mg-Kα, 1283.6 eV was used to irradiate the sample surface. The power of X-ray on the sample surface was 100 W and a take-off-angle for electrons relative to sample surface plane was 90°. The pressure in the analysis chamber was kept at 5×10.sup.−9 bar during the measurements. The wide scan survey spectra and high energy resolution narrow scan spectra were recorded at room temperature.

(61) A 180° hemispherical energy analyzer model PHOIBOS-150 and a set of nine channel electron multipliers MCD-9 was adopted for scanning. The analyzer was operated in Fixed Analyzer Transmission (FAT) and medium area lens modes at pass energy of 30 eV, step size of 1.0 eV for survey scans. The pass energy was set at 20 eV with step size of 0.025 eV and dwell times of 0.1 sec for narrow or high resolution scans.

(62) As the standard practice in XPS studies, the hydrocarbon C.sup.1s line (284.6 eV) corresponding to C—C bond has been used as binding energy reference for charge correction. The high energy resolution spectra were obtained under analysis conditions that would give a FWHM=0.85 eV from the Ag 3d.sub.5/2 signal of freshly argon ion etched silver (Ag) sample.

(63) The surface wettability of the fibrous mat was analyzed according to water contact angle measurement performed by drop shape analyzer (DSA 100). The BCCM was composed of 90% Dulbecco's Modified Eagle's Medium (DMEM) and 10% fetal bovine serum (“FBS”). A 2 ul of liquid was slowly dropped on the surface of the fiber mash then contact angle was determined as an angle between the drop contour and the projection of the surface (baseline).

(64) The protein attachment test was performed by the fibrous mats which were cleaned by DPBS (Dulbecco's phosphate-buffered saline). The fibrous mats were placed in 10% of FBS in DMEM for 24 hours. The samples were washed again by DPBS to remove unattached proteins. The mats were placed after washing in 2% of SDS (sodium dodecyl sulfate) for 3 hours to release the attached proteins. The protein concentration was measured according to the UV adsorption using the Nanodrop 2000. Also, the calculation was based on UV absorbance at 280 nm wavelength, where BSA (Bovine Serum Albumin) was used as reference.

(65) Biocompatibility test. The scaffolds collected on circular glass cover slips (diameter 124 mm) by two hours of electrospinning, were initially sterilized under UV for 1 h followed by PBS washes (thrice for 5 min each). The scaffolds were then washed with 100% ethanol for 10 min and again washed thrice with phosphate buffered saline. The scaffolds were then primed in the culture media and incubated at 37° C. incubator in a 5% CO.sub.2 in air atmosphere for 24 h to ensure no contamination. The scaffold thickness was about 0.2 mm. A preferred range for scaffold thickness is about 0.05 to 2 mm depending on the particular vascular tissue engineering application such as prosthesis of aorta, arteries and other smaller blood vessels.

(66) Subsequently, the media was removed and fresh medium containing dermal fibroblasts (2×10.sup.4 cells/well) of a 24 well tissue culture plate was added to the four different scaffolds and cultured at standard culture conditions of 37° C. in a 5% CO.sub.2 incubator with regular changes of media every 48 h.

(67) After culturing for 7 days, the scaffolds were stained using cell tracker fluorescent probe (CMFDA, c7025) to visualize the cell attachment on to the scaffolds. A fluorescent dye (5 μM) was added to cells in fresh media and incubated for 30 min under standard culture conditions. The media containing the dye was then removed and the scaffolds containing the cells washed once with PBS to remove any traces of the unbound dye and then the cell were fixed in 70% ice-cold ethanol for 10 min. After phosphate buffered saline washes the cells were incubated with 4′-6-Diamidino-2-phenylindole (DAPI) and green florescence protein (GFP) for 5 min at room temperature and washed with phosphate buffered saline again. The cells were then studied using fluorescent microscope (EVOS® FL Imaging System, Thermo Fisher Scientific).

(68) The cell proliferation assay was performed using an MTT reagent kit (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; Sigma). The 10 mL MTT reagent (final concentration of 0.5 mg/mL) was added to the medium in the culture dishes and the dishes were incubated for 4 h until a purple precipitate was visible. The medium was then removed and 100 mL of the detergent reagent was added into the dishes and incubation carried out in the dark for 2 h. The absorbance at 570 nm was spectrophotometrically measured using a microplate reader (SpectraMax i3, San Jose, USA) with a reference wavelength of 570 nm.

(69) FIG. 1 shows schematic representation of coaxial electrospinning setup for the development of hollow fiber. The setup consisted of two independent syringe pumps which allowed the control of feed rate for the core and shell solutions. The solutions were delivered through single metal needle tip with an 18 gauge outer needle and a 27 gauge inner needle. Furthermore, the inner and outer solutions experience the electrical field simultaneously during the electrospinning process. The glycerol and mineral oil were used as core solution to serve as templates and were removed after electrospinning the fibers.

(70) The process parameters were selected and carried out by initially setting the shell feed rate at 0.5 mL/h and core feed rate at 0.1 mL/h. During the process the formation of the beads occurred in the resultant fibers. The formation of beads happened may be attributed to the shell solution experiencing shear stress from both outer and inner wall resulting in insufficient feed rate for Tailor cone formation. Therefore the feed rate of the shell solution was increased to 1 mL/h to maintain the stable electrospinning process.

(71) Chloroform and acetone (3:1) were selected as solvents for the solid fiber preparation. It was noticed that the needle was blocked by quickly drying shell polymers in less than 15 seconds. The quick drying may be attributed to combined effects of needle configuration in coaxial electrospinning and the solvent system.

(72) Due to different capillary configuration in coaxial electrospinning, the solvent evaporation was slowed down and acetone was replaced with ethanol in the coaxial process to avoid unstable electrospinning conditions. In this stage, the inventors also tried to lower the voltage. However this produced an adverse effect on the fiber morphology.

(73) Subsequently, controlling the feed rate of the core solution was found to be detrimental for successful preparation of hollow fiber using coaxial electrospinning. The initial experiments were carried out by varying the core feed rate with increment of 0.1 from 0.1 mL/h to 0.3 mL/h. Among the different core feed rate, 0.1 mL/h showed acceptable result while higher feed rate caused formation of the beads. The formation of beads implies that the feed rate needs to be controlled more precisely. Based on this the inventors were able to determine advantageous conditions for suitable development of hollow fibers.

(74) The cross-sections of the hollow fibrous scaffolds prepared by using glycerol as a core template are shown by FIG. 2 and demonstrate that 0.05 mL/h of core feed rate is appropriate to develop hollow fiber but the fiber walls were found to be thick.

(75) Increasing the core feed to 0.08 mL/h resulted in larger opening of the hollow and relatively uniform fiber morphology. However, further increasing of the feed rate beyond 0.1 mL/h led to the formation of collapsed fiber and fiber fusion.

(76) This phenomenon may occur during the continuous fiber deposition process. During electrospinning the shell solution quickly dries during deposition. This leads to formation of complete solid phase in the shell of the fiber. However, the evaporation rate of glycerol was very low so it remained in liquid phase in the core throughout the electrospinning process. This might cause tearing of the shell layer and effluence of glycerol to create fiber fusion Therefore, a high core feed rate of glycerol can cause heavy fiber formation with easily deformable liquid phase fiber lumen. The initially deposited shell layer should be mechanically strong enough to bear upper pressure of the layers for continuous fiber deposition to avoid collapse.

(77) A similar observation was made in the case of mineral oil using a core feed rate of 0.1 mL/h (FIG. 3). The inventors believe that this may be due to the low viscosity of the mineral oil compared to glycerol. Additionally, it was found that the core feed needed to be controlled in a narrower range in order to maintain good fiber morphology without collapsing and fiber fusion. These results show that the feed rate of glycerol at 0.08 mL/h and feed rate of mineral oil at 0.1 mL/h may be selected to prepare hollow composite scaffolds. FIG. 4 shows that these conditions resulted in good hollow fiber morphology.

(78) FIG. 5 shows the fiber size distribution of the hollow and solid fibrous scaffolds. The average fiber size in the scaffolds was found to be twice higher in comparison to PLA/PBS (FIG. 5A) and PLA/PBS/CNF (FIG. 5B) scaffolds of solid fibrous. The difference in sizes may be due to the presence of a core layer during the fiber flight time which creates a resistivity against fiber thinning by the electrical field.

(79) Another potential reason is use of a different configuration of a Tailor cone during the coaxial electrospinning. In the solid fiber fabrication the polymer droplet forms a completely polarized Tailor cone which has influence on the size of the fiber. In case of coaxial electrospinning polarization of the Tailor cone is disturbed by the core solution. Therefore the size of fiber became larger in this case. It should be noted that this phenomena cannot be applied for the coaxial electrospinning in which both core and shell solutions involve spinning. The electric field influences both inner and outer solutions that control the overall fiber morphology and fiber size.

(80) The average diameter of the cores in the hollow fibrous scaffolds was measured using “Digimizer” software from 10 different places of SEM image. Table 2 shows that both core templates had a similar hollow size which was nearly 50% of average fiber size of the scaffolds. However, the core templates showed completely different results on the porosity their fiber walls.

(81) In the case of templates made using a glycerol core only few large pores could be observed and their dispersion was inconsistent as shown in FIG. 6. On the other hand, mineral oil generated uniformly dispersed pores in the fiber wall. This uniform dispersion may be due to the viscosity differences between glycerol and mineral oil. It was noted that both glycerol and mineral oil were slightly soluble with the shell solvent in the presence of ethanol.

(82) The high viscosity of the glycerol produces a stable one directional flow in the electrospinning. However, the less viscous mineral oil generated spray and partly mixed with the ethanol. This spraying and partial mixing encouraged more pore formation on the fiber wall.

(83) Both blended and composite scaffolds were produced having 50 nm pores. The pores in case of blended scaffold are more uniform and spherical as compared to the composite scaffolds. The geometry of the pores could be attributed to the presence of CNF in composite scaffold which preserves its shape and orientation.

(84) TABLE-US-00002 TABLE 2 Average diameter of the cores in the hollow fibrous scaffolds The scaffold name Fiber core diameter GH 475 ± 52 nm (PLA/PBS) OH 485 ± 66 nm (PLA/PBS) GC 327 ± 36 nm (PLA/PBS/CNF) OC 356 ± 47 nm (PLA/PBS/CNF)

(85) FIG. 7 illustrates the XPS spectra of carbon and oxygen in the hollow fibrous scaffolds. The PLA and PBS were formed by the covalent bonding of carbon, hydrogen and oxygen. Among them, hydrogen cannot be detected by XPS because it has no core electrons; see. Stojilovic, N., Why Can't We See Hydrogen in X-ray Photoelectron Spectroscopy? Journal of Chemical Education, 2012. 89(10): p. 1331-1332, incorporated herein by reference in its entirety.

(86) Except for hydrogen, each PLA monomer consisted of two C—C—C bonds, one C—C—O bond, one O═C—O bond, one O═C bond and one C—O—C bond. Similar bonds exist in each PBS monomer with double the ratio of the PLA monomer.

(87) The PLA/PBS were homogeneously blended at a 50/50 concentration resulting in a required ideal ratio of 33.33% C—C—C bonds and 16.67% other carbon and oxygen bonds, as shown in Table 2.

(88) From the XPS spectra, two main peaks were observed: the binding energy of the first peak was at 284.79 eV which corresponds to a carbon atom, and the binding energy of another peak was at 532.64 eV which relates to an oxygen atom; see Chastain, J., R. C. King, and J. Moulder, Handbook of X-ray photoelectron spectroscopy: a reference book of standard spectra for identification and interpretation of XPS data. 1995: Physical Electronics Eden Prairie, Minn., incorporated herein by reference in its entirety.

(89) As described above in the XPS results, when the PLA/PBS are homogeneously blended with 50/50 concentration, this results in a required ideal ratio of 33.33% C—C—C bonds and 16.67% of other chemical bonds (CCO, COO, OH/COC, —OC). In case of CNF incorporation OH concentration is supposed to be higher due to dominance of OH in the PLA/PBS/CNF composite. The chemical bond composition of each atom was calculated and the results are presented in the Table 2. The bond composition of the scaffold is similar to the ideal bond composition. For example, percentage of C—C—C bond is approximately 35% which is very close to ideal percentage 33.33%. For the PLA/PBS scaffold percentage of other bonds are near to 16.67%. While 25% of OH bond is observed for the PLA/PBS/CNF composite scaffold which highlights dominance of OH in the CNF. Therefore the result implies coherency of the individual material in the composite nanofiber.

(90) TABLE-US-00003 TABLE 2 Chemical bond composition and Binding Energy extracted from XPS peaks Binding Concentration (at. %) Name energy PLA/PBS PLA/PBS/CNF O 1 s 532.64 35.182 40.323 C 1 s 284.79 64.818 59.677 O 1 s_1_O═C 532.08 18.433 15.032 O 1 s_2_C—O—C/OH 533.48 16.749 25.291 C 1 s_1_C—C—C 284.8  35.414 35.192 C 1 s_2_C—C—O 286.59 15.396 12.824 C 1 s_3_O—C═O 288.82 14.008 11.662

(91) When compared to PLA/PBS blend scaffolds to the PLA/PBS/CNF composite scaffolds containing CNF exhibited higher oxygen concentration.

(92) In case of a PLA/PBS blend, the XPS characteristic of the hollow fibrous scaffolds was found to be similar to that of solid fibrous scaffold. When compared to PLA/PBS blend scaffolds, PLA/PBS/CNF composite scaffolds containing CNF exhibited higher oxygen concentration.

(93) Particularly, the intensity of O—H/O—C bond for the composite scaffolds significantly increased when compared to PLA/PBS scaffolds. The increase in intensity could be due to dominance of OH bonds in the CNF; see Krouit, M., J. Bras, and M. N. Belgacem, Cellulose surface grafting with polycaprolactone by heterogeneous click-chemistry. European Polymer Journal, 2008. 44(12): p. 4074-4081, incorporated herein by reference in its entirety.

(94) This result confirms that the materials were homogenously dispersed in the hollow fibrous scaffolds. Whereas overall intensity of the XPS spectra in case of oil templated hollow fibrous scaffolds is lower than the glycerol templated hollow fibrous scaffolds. The lower intensity could be result of high porosity of the fiber wall when oil is used as template.

(95) The developed hollow fibrous structure improves the wettability of the scaffold as shown by FIG. 8. For the PLA/PBS blend scaffolds, the water contact angle (WCA) was decreased by ˜30° in case of GH and by ˜40° in case of OH when compared to solid scaffold.

(96) Similar trends were observed for the composite scaffolds. The WCA of 41.6±2.1° is found in OH scaffold which is the lowest among the hollow fibrous scaffolds. This could be because of uniformly dispersed pores on the fiber wall. This result is favorable because the uniformity in the structure of pores is beneficial for better dispersion of liquids in a scaffold; see Francis, L., et al., PVDF hollow fiber and nanofiber membranes for fresh water reclamation using membrane distillation. Journal of Materials Science, 2014. 49(5): p. 2045-2053, incorporated herein by reference in its entirety.

(97) The hydrophilicity of the hollow fibrous scaffolds was found to be much higher than that of solid fibrous scaffolds within 40˜80° of WCA; see Arima, Y. et al., Effect of wettability and surface functional groups on protein adsorption and cell adhesion using well-defined mixed self-assembled monolayers. Biomaterials, 2007. 28(20): p. 3074-3082; and Wei, J., et al., Influence of surface wettability on competitive protein adsorption and initial attachment of osteoblasts. Biomedical Materials, 2009. 4(4): p. 045002, each incorporated herein by reference in their entirety. The improved mobility of liquids enables the interaction of the cells with the scaffolds and enhances the cell adhesion and proliferations.

(98) Moreover as shown by FIG. 9, hollow fibrous structures have an impact on protein adsorption capacity of the scaffolds. The mineral oil templated hollow fibrous scaffolds showed double protein adsorption capacity when compared to solid fibrous scaffolds. The enhanced adsorption capacity may be due to the increased surface area because of hollow conduits and micropore formation on the fiber wall. Also, higher hydrophilicity increases the protein adsorption capacity of the scaffolds.

(99) FIG. 10 depicts GFP/DAPI staining images of the hollow fibrous scaffolds after 7 days. All scaffolds supported better adhesion and dispersion of the cells through entire area. The mineral oil templated hollow scaffolds showed more occupied cell attachment compared to the glycerol templated hollow scaffolds.

(100) In the case of GH and GC scaffolds, produced using a glycerol core, a number of black spots can be observed from the staining image which implies that no cell attachment occurred in those areas.

(101) Meanwhile, OH and OC scaffolds, produced using a mineral oil core, showed completely overlapped cell attachment without any black spots. This could be strongly correlated with overlarge surface area of the microporous hollow fibrous scaffolds. The enhanced surface energy and enriched protein adsorption capacity of the scaffold are the contributing factors for the better cell adhesion performance of OH and OC scaffolds; Arima et al., Electrospun 1,6-diisocyanatohexane-extended poly(1,4-butylene succinate) fiber mats and their potential for use as bone scaffolds. Polymer, 2009. 50(6): p. 1548-1558; Garcia-Orue, I., et al., Chapter 2—Nanotechnology approaches for skin wound regeneration using drug-delivery systems A2—Grumezescu, Alexandru Mihai, in Nanobiomaterials in Soft Tissue Engineering. 2016, William Andrew Publishing. p. 31-55; and Tallawi, M., et al. Poly (glycerol sebacate)Poly (butylene succinate-dilinoleate) Blends as Candidate Materials for Cardiac Tissue Engineering. in Macromolecular Symposia. 2013. Wiley Online Library, each incorporated herein by reference in their entirety.

(102) The cell proliferation result for the hollow fibrous scaffolds at 7 and 14 days by MTT assay can be observed in FIG. 11A. Similarly, in the solid fibrous scaffolds the rapid increase in cell proliferation was achieved in the first week and followed by a slower increase in the second week. Whereas, the hollow fibrous scaffolds showed a higher cell proliferation rate compared to the solid fibrous scaffolds (FIG. 11B). The highest cell proliferation rate is found in case of OH scaffold which is even higher than that of OC scaffolds. The improved proliferation rate shows the importance of uniform pore formation on the fiber wall. The microporous hollow fibrous scaffolds can provide for better transport of nutrients, oxygen and other important biomolecules to the cells. The cells with the help of newly developed scaffold can be provided with an advantageous microenvironment for the growth, propagation and viability.

(103) As demonstrated herein, homogenously blended microporous hollow fibrous scaffolds were successfully synthesized from PLA/PBS blends and CNF reinforced composites using mineral oil as a core template. Among the coaxial electrospinning parameters core and shell feed rate were found to be the most determining parameters to generate uniform hollow fiber without fusion and beads. The mineral oil and glycerol both as core templates allowed hollow fiber formation form PLA/PBS blends and CNF reinforced composites in electrospinning. The mineral oil resulted in formation of uniformly dispersed microspores on the fiber wall. Also, the surface composition of the hollow fibrous scaffolds was not influenced by the core templates. The high porosity in the fiber wall was confirmed for the oil templated hollow fibrous scaffolds. The surface properties such as wettability and protein adsorption capacity were remarkably improved in the presence of microporous and hollow fibrous structure. Finally, the microporous hollow fibrous scaffolds showed enhanced cell adhesion and proliferation performance than the solid fibrous scaffolds.