Manipulating Air-Gap Electrospinning to Create Aligned Polymer Nanoﬁber-Wrapped Glass Microﬁbers for Cortical Bone Tissue Engineering

bioengineering

Article Manipulating Air-Gap Electrospinning to Create Aligned Polymer Nanoﬁber-Wrapped Glass Microﬁbers for Cortical Bone Tissue Engineering

Houston R. Linder 1, Austin A. Glass 1, Delbert E. Day 2 and Scott A. Sell 1,* 1 Biomedical Engineering, Parks College of Engineering, Aviation, and Technology, Saint Louis University, Saint Louis, MI 63103, USA; [email protected] (H.R.L.); [email protected] (A.A.G.) 2 Material Science and Engineering, Missouri University of Science and Technology, Rolla, MI 65409, USA; [email protected] * Correspondence: [email protected]

Received: 20 November 2020; Accepted: 15 December 2020; Published: 20 December 2020

Abstract: Osteons are the repeating unit throughout cortical bone, consisting of canals filled with blood and nerve vessels surrounded by concentric lamella of hydroxyapatite-containing collagen fibers, providing mechanical strength. Creating a biodegradable scaffold that mimics the osteon structure is crucial for optimizing cellular infiltration and ultimately the replacement of the scaffold with native cortical bone. In this study, a modified air-gap electrospinning setup was exploited to continuously wrap highly aligned polycaprolactone polymer nanofibers around individual 1393 bioactive glass microfibers, resulting in a synthetic structure similar to osteons. By varying the parameters of the device, scaffolds with polymer fibers wrapped at angles between 5–20◦ to the glass fiber were chosen. The scaffold indicated increased cell migration by demonstrating unidirectional cell orientation along the fibers, similar to recent work regarding aligned nerve and muscle regeneration. The wrapping decreased the porosity from 90% to 80%, which was sufficient for glass conversion through ion exchange validated by inductively coupled plasma. Scaffold degradation was not cytotoxic. Encapsulating the glass with polymer nanofibers caused viscoelastic deformation during three-point bending, preventing typical brittle glass fracture, while maintaining cell migration. This scaffold design structurally mimics the osteon, with the intent to replace its material compositions for better regeneration.

Keywords: electrospinning; bioglass; 1393 bioactive glass; polycaprolactone; critical size bone defect; cortical bone; osteon; scaﬀold

1. Introduction Around 1.6 million people require bone grafts annually in the U.S. for degenerative diseases, injuries, tumors, and infections, accounting for approximately USD 244 billion [1,2]. Bone grafts are currently used when this bone injury is larger than what the natural bone healing process can mend, which is termed a critical size bone defect. This is clinically determined when the defect site is twice as large as the diameter of the injured bone [3]. The current gold standard for critical size bone defects involve the use of autologous bone grafts, but donor and receiving sites potentially experience pain, complications, limited donor bone volume, and increased risks of infection [4–7]. These current methods are lacking, whether it is due to the addition of an invasive surgery inducing another fracture, or risks immune rejection from another donor. Because of this, there has been a rise in interest over the last decade to create bone substitutes that mimic and replace the native bone. Bones are composed of the inner, spongy trabecular bone, which is surrounded by the outer, dense cortical bone. Cortical bone provides 80% of the bone’s mechanical strength and is made up

Bioengineering 2020, 7, 165; doi:10.3390/bioengineering7040165 www.mdpi.com/journal/bioengineering Bioengineering 2020, 7, 165 2 of 15 of repeating functional units, called the osteon. Osteons are hollow cylindrical structures of five to 20 concentric layers of bone tissue, surrounding the haversian canals, which house the blood and nerve vessels. While the haversian canals are approximately 50 µm in diameter, the varying number of lamellae cause different sizes of osteons, but are typically around 200 µm in diameter [8]. Osteogenic cells reside throughout the osteons, causing constant remodeling of the bone tissue, which is composed of collagen and hydroxyapatite (HA). Creating a biodegradable scaffold that mimics the osteon structure should further encourage osteon regeneration by guiding cell migration and promoting angiogenesis [9]. The design presented here utilizes the osteon structure to wrap aligned electrospun polymer nanofibers around bioactive glass microfibers, with the intent to promote directional cell migration and blood vessel infiltration. Electrospinning is a method of producing highly porous, nano-sized fibers by applying a high voltage to an extruding polymeric solution [10]. The high voltage causes instability and thus rapid spiraling motions from electrostatic repulsion to evaporate the solvent before depositing on the substrate. Typically, this method results in randomly aligned fibers that mimic the extracellular matrix, allowing for better cellular integration into the scaffold [11]. Altering the electrospinning parameters (polymer concentration, voltage, working distance, flow rate, solution conductivity, solvent, humidity, and temperature) changes the properties of the scaffold, such as the fiber diameter, porosity, and mechanical strength, creating a tailorable scaffold [10,12]. Aligning the fibers has been shown to increase directional cell migration, which has been utilized for different cell types such as nerve [13–15], blood vessel [16], muscle [17], bone [18], and cartilage regeneration [19]. The two most common methods for producing aligned nanofibers are through rapid rotation of the collecting mandrel or through manipulating the electric field [11]. By applying a negative voltage to the polymer solution and a positive voltage to two plates separated by a gap, the fibers are electro-statically attracted to both plates and so they stretch from one plate to the other. This method is termed "air gap electrospinning", and is primarily affected by the polymer concentration, plate distance, plate size, or voltage applied [15,20]. Air-gap electrospinning has a high potential for use in cortical bone, since the aligned electrospun fibers resemble the aligned collagen fibrous layers of the osteon. Furthermore, electrospun scaffolds have been shown to proliferate osteoblasts and mesenchymal stem cells [21,22], with aligned fibers demonstrating organized collagen deposition onto the fibers [23]. Despite these benefits, aligned fibers are limited in perpendicular tension and thus have yet to be used to their full potential. The increased cell migration from the aligned electrospun fibers will be limited by nutrient diffusion from the blood vessels to the osteogenic cells. Bioactive glass has been shown to promote blood vessel formation, as well as increasing bone formation quicker than HA alone [24]. This is due to bioactive glass converting to HA while releasing beneficial ions, with the conversion being a dynamic process [25]. In short, hydrolysis occurs on the outside of the glass onto the network modifiers (i.e., Na+ and Ca2+), ultimately increasing the pH in the surrounding liquid. For silica-based bioactive glasses, this causes the dissolution of silica, which then polymerizes onto the outside of the glass as porous silica-rich layers. As the glass continues to degrade from hydrolysis, the released Ca+ 3 and (PO4) − ions travel through the silica rich layer, and will deposit on the outside to form amorphous 2 calcium phosphate. After incorporating (OH)− and (CO3) − from the surrounding solution, it will form a HA-like layer, the primary mineral in bone. The result is a hollow HA structure that maintained the original glass structure. This rate of conversion can be quickened by replacing the silicate with borate [26], or by replacing network modifiers with an alkali ion of larger radius, such as replacing K2O with Na2O[27]. Certain bioactive glasses have been shown to be osteoconductive as well as osteoinductive, as opposed to HA, which may not promote osteoinduction [25]. This is due to the released calcium and silica during the glass conversion, which induces growth factor adsorption followed by the attachment, proliferation, and differentiation of osteoprogenitor cells [28]. These osteoblasts then secrete collagen onto the surface of the HA-converting bioglass, which the collagen begins to mineralize while Bioengineering 2020, 7, x FOR PEER REVIEW 3 of 16

elements [24,30–34]. The released ions during conversion to HA simultaneously induce angiogenic cellular responses and pro-osteogenic responses. Bioactive glass is capable of having strong physical bonds with bone [35], resorbed in 6 months Bioengineering 2020, 7, 165 3 of 15 with little inflammatory rate [36], and promotes neovascularization within two weeks [37,38]. However, the use of bioactive glass is limited due to its brittle nature, and so incorporation into a beingcomposite imbedded is necessary into the HA to compensate. layers [29]. Additionally, The novel design bioactive in this glasses paper haveutilizes been a modified proven to version have of angiogenicair gap potential, electrospinning as expected to wrap with the highly cellular aligned responses polymer of the nanofibers incorporated around elements a bioactive [24,30–34 glass]. Themicrofiber. released ions These during homologous conversion and to HAuniform simultaneously synthetic osteons induce angiogenic(HOUSteons) cellular can be responses placed together and pro-osteogenicand electrospun responses. around them in a similar manner, creating a HOUSteon bundle. The design presentedBioactive here glass mimics is capable the ofosteon having structure strong physicalusing aligned bonds electrospun with bone [ 35nanofibers], resorbed wrapped in 6 months around withbioactive little inflammatory glass microfibers, rate [36], to and promote promotes directional neovascularization cell migration within with two the weeks potential [37,38]. for However, increasing the useblood of vessel bioactive infiltration. glass is limited due to its brittle nature, and so incorporation into a composite is necessary to compensate. The novel design in this paper utilizes a modified version of air gap electrospinning2. Materials and to wrap Methods highly aligned polymer nanofibers around a bioactive glass microfiber. These homologous and uniform synthetic osteons (HOUSteons) can be placed together and electrospun around2.1. themDesign in and a similar Optimization manner, of creatingModified a Air-Gap HOUSteon Electrospinning bundle. The Apparatus design presented here mimics the osteon structureThe collecting using alignedair-gap design electrospun was primarily nanofibers composed wrapped of around 3D-printed bioactive polylactic glass microfibers,acid (PLA) and to promotealuminum directional foil, which cell was migration adhered with to the potentialair-gap mandrel for increasing for conduction. blood vessel The infiltration. final setup is shown using 3D design software (AutoCAD, Autodesk Inc., San Rafael, CA USA), in Figure 1. The 9V 2. Materials and Methods battery-powered 168-RPM motors were connected to gears in order to have a gear rotating around a 2.1.stationary Design and cylinder. Optimization The of rotating Modified gear Air-Gap was Electrospinning attached to a Apparatus metal bearing to simultaneously reduce friction and conduct electricity. This was attached to the conductive air-gap mandrel. The conductive wireThe was collecting attached air-gap to the design stationary was primarily part of the composed bearing, ofallowing 3D-printed electricity polylactic to conduct acid (PLA) through and the aluminummetal bearing foil, which to the was rotating adhered mandrel. to the air-gapThe mandrel mandrel had for a conduction.hole in the center The final for the setup stationary is shown glass usingholder 3D design so the glass software fiber (AutoCAD,could be placed Autodesk and held Inc., stationary San Rafael, with CA, tacky USA), glue. in Both Figure sides1. are The designed 9V battery-poweredthe same except 168-RPM the mandrels motors rotate were in connected opposite todirections gears in to order induce tohave fiber awrapping. gear rotating around a stationaryInitial cylinder. mandrel The testing rotating involved gear wastwo attachedmetal washers to a metal with bearingdiameters to of simultaneously 31 and 22 mm, reduce followed frictionby a and 4 mm conduct PLA cone-shaped electricity. This mandrel was attached coated toin thealuminum conductive foil. air-gapThe 4 mm mandrel. cone was The chosen conductive over its wireflat was counterpart attached to due the to stationary more consistent part of thescaffold bearing, fabrication. allowing The electricity mandrel to distance conduct had through no effect the on metalfiber bearing deposition, to the rotatingso the 8 cm mandrel. maximum The distance mandrel between had a hole the in mandrels the center of for this the setup stationary was selected. glass A holdervoltage so the regulator glass fiber was could used be placed to adjust and the held rotational stationary speed with tacky and glue. direction Both of sides the are mandrels. designed The the samestationary except glass the holder mandrels was rotate tested in within opposite the directions mandrel and to induce extended fiber from wrapping. the mandrel.

a b

FigureFigure 1. AutoCAD 1. AutoCAD images images of (a) of the (a final) the design final design of the modifiedof the modified air-gap air-gap electrospinning electrospinning with motor with setup,motor signifying setup, the stationarysignifying (green) the and stationary rotating (black) (green) parts. and ( b) rotating A simplified (black) setup demonstrating parts. (b) A the wrappingsimplified of setupelectrospun demonstrating fibers (blue) the aroundwrapping the of glass electrospun fiber (red). fibers (blue) around the glass fiber (red). Initial mandrel testing involved two metal washers with diameters of 31 and 22 mm, followed by a2.2. 4 mm Fabrication PLA cone-shaped of 1393 Glass mandrel Fibers coated in aluminum foil. The 4 mm cone was chosen over its

flat counterpartThe bioactive due to more1393 silicateconsistent glass sca (53ffold SiO fabrication.2‐20 CaO‐6 The Na mandrel2O‐4 P2O distance5‐12 K2O‐ had5 MgO no eff wt%)ect on was fiberprepared deposition, through so the standard 8 cm maximum procedures distance reported between elsewhere the mandrels [26]. In of short, this setup the powdered was selected. CaCO3, A voltageNa2CO regulator3, MgCO was3, K2 usedCO3, toSiO adjust2, and the CaHPO rotational4·2H speed2O (Fisher and directionScientific, of St. the Louis, mandrels. MO, TheUSA stationary) were mixed glassthoroughly holder was for tested 30 minutes. within the This mandrel batch was and melted extended in froma platinum the mandrel. crucible and an electric furnace at 1350 °C for 2 hours until homogeneously melted. Once melted, a silica rod was dipped into the Bioengineering 2020, 7, 165 4 of 15

2.2. Fabrication of 1393 Glass Fibers

The bioactive 1393 silicate glass (53 SiO2-20 CaO-6 Na2O-4 P2O5-12 K2O-5 MgO wt%) was prepared through standard procedures reported elsewhere [26]. In short, the powdered CaCO3, Na2CO3, MgCO3,K2CO3, SiO2, and CaHPO4 2H2O (Fisher Scientific, St. Louis, MO, USA) were mixed Bioengineering 2020, 7, x FOR PEER REVIEW · 4 of 16 thoroughly for 30 min. This batch was melted in a platinum crucible and an electric furnace at 1350 ◦C forsolution 2 h until and homogeneously raised to produce melted. initial Once fiber melted, formation. a silica These rod was fibers dipped were into pulled the solution by hand, and annealing raised toinstantly produce at initial room fiber temperature. formation. Fibers These that fibers were were less than pulled 150 by μm hand, without annealing bead formation instantly were at room used temperature.in this study. Fibers that were less than 150 µm without bead formation were used in this study. 2.3. Fabrication of Scaffold 2.3. Fabrication of Scaffold The randomly oriented electrospun scaffold was fabricated using a traditional stainless steel The randomly oriented electrospun scaffold was fabricated using a traditional stainless steel rectangular mandrel (9 5 2.5 cm3), rotating at 500 rpm. Polycaprolactone (PCL) with an average rectangular mandrel (9× × ×5 × 2.5 cm3), rotating at 500 rpm. Polycaprolactone (PCL) with an average molecular weight of 80,000 g/mol (Sigma Aldrich, Milwaukee, WI, USA) at 12 wt% was dissolved in molecular weight of 80,000 g/mol (Sigma Aldrich, Milwaukee, WI, USA) at 12 wt% was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) overnight on a shaker plate, and then 3 mL of this solution 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) overnight on a shaker plate, and then 3 mL of this solution was electrospun with a flow rate of 3 mL/hr. The voltage was 18 kV on the needle with the mandrel was electrospun with a flow rate of 3 mL/hr. The voltage was− −18 kV on the needle with the mandrel grounded, and the working distance was 15 cm. The scaffolds were stored in a desiccator. grounded, and the working distance was 15 cm. The scaffolds were stored in a desiccator. The HOUSteons were fabricated using the aforementioned 12% PCL solution and 1393 bioactive The HOUSteons were fabricated using the aforementioned 12% PCL solution and 1393 bioactive glass fibers. The glass fiber was first wiped with isopropyl alcohol three times before being placed in glass fibers. The glass fiber was first wiped with isopropyl alcohol three times before being placed in the stationary holder and the ends adhered with tacky glue for each experiment, with the setup shown the stationary holder and the ends adhered with tacky glue for each experiment, with the setup in Figure1. The working distance was approximately 11 cm, with the mandrel–mandrel distance shown in Figure 1. The working distance was approximately 11 cm, with the mandrel–mandrel of 8 cm and a rotation speed of 169 rpm in opposite directions. With 15 kV on the needle and distance of 8 cm and a rotation speed of 169 rpm in opposite directions.− With −15kV on the needle +15 kV on each of the mandrels, the PCL solution was extruded at 3 mL/hr. The deposited volume and +15kV on each of the mandrels, the PCL solution was extruded at 3 mL/hr. The deposited volume was approximately 0.25 mL per HOUSteon. The HOUSteon bundles were fabricated with similar was approximately 0.25 mL per HOUSteon. The HOUSteon bundles were fabricated with similar parameters, except 8 HOUSteon were placed on the glass holder instead of a glass fiber. The resultant parameters, except 8 HOUSteon were placed on the glass holder instead of a glass fiber. The resultant scaffolds are shown in Figure2. scaffolds are shown in Figure 2.

Glass HOUSteon Bundle

FigureFigure 2. 2.Macroscopic Macroscopic image image of of a glassa glass fiber, fiber, HOUSTeon, HOUSTeon, and and HOUSteon HOUSteon bundle. bundle. 2.4. SEM, FFT, Fiber Diameter 2.4. SEM, FFT, Fiber Diameter SEM was performed on the scanning electron microscope model EVO LS15 (Carl Zeiss Microscopy, SEM was performed on the scanning electron microscope model EVO LS15 (Carl Zeiss Cambridge, UK). The images were taken at 10–15 kV and 10–12 pA. Fast-Fourier transform (FFT) was Microscopy, Cambridge, UK). The images were taken at 10–15 kV and 10–12 pA. Fast-Fourier performed using image-processing software (ImageJ, LOCI, University of Washington, Seattle, WA, transform (FFT) was performed using image-processing software (ImageJ, LOCI, University of USA) after performing radial sum intensities using the Oval Profile Plot plugin (author, Bill O’Connell) to Washington, Seattle, WA, USA) after performing radial sum intensities using the "Oval Profile Plot" obtain alignment measurements. Manual angle measurements were performed using image-processing plugin (author, Bill O'Connell) to obtain alignment measurements. Manual angle measurements were software (ImageJ), validating the FFT angles. The diameters of 50 random fibers were manually performed using image-processing software (ImageJ), validating the FFT angles. The diameters of 50 measured throughout 2 images, by using image-processing software (ImageJ). random fibers were manually measured throughout 2 images, by using image-processing software 2.5.(ImageJ). Mechanical Testing-3pt Bending

2.5.Mechanical Mechanical Testing-3pt testing was Bending performed on an MTS Criterion model 42 (MTS Systems Corporation, Eden Prairie, MN, USA) using a 3-point bending apparatus with a support span of 1 cm. The strain rate usedMechanical was 0.01 mmtesting/s. was performed on an MTS Criterion model 42 (MTS Systems Corporation, Eden Prairie, MN, USA) using a 3-point bending apparatus with a support span of 1 cm. The strain rate used was 0.01 mm/s.

2.6. Porosity Porosity was determined through a version of the Archimedes method. A 10 mm biopsy punch was taken from the PCL control or a 2 cm long HOUSteon. The dry weight for each scaffold was measured, followed by a 5-second soak in 200 proof ethanol and then the wet weight was measured. The porosity (푛) was found using the following equation: Bioengineering 2020, 7, 165 5 of 15

2.6. Porosity Porosity was determined through a version of the Archimedes method. A 10 mm biopsy punch was taken from the PCL control or a 2 cm long HOUSteon. The dry weight for each scaﬀold was measured, followed by a 5-second soak in 200 proof ethanol and then the wet weight was measured. The porosity (n) was found using the following equation:

V n = eth 100 (1) (Veth + VPCL) × where Veth is the volume of ethanol intruded into the scaffold punches and VPCL is the volume of PCL fibers. The volume of ethanol intrusion was found by calculating the ratio of the observed change in mass between wet and dry weights, using the density of ethanol (0.789 g/cm3). The volume of PCL fibers was determined by taking the ratio of dry mass of the scaffolds and the density of PCL (1.145 g/cm3). To compensate for the non-porous glass fiber in the HOUSteons, the mass of the glass fiber with the same dimensions was subtracted from the dry mass. The variables Veth and VPCL were determined in the following manner, where ρ is the density: Mwet M M − dry dry Veth = VPCL = (2) ρeth ρPCL

2.7. Cell Cytotoxicity The PCL was punched out using a 10 mm diameter biopsy punch, whereas the glass and HOUSteon fibers were cut in 1 cm increments. All scaffolds were UV sterilized for 30 min, flipped, and UV sterilized for another 30 min. The scaffolds were placed in 48 well plates with cloning rings on top, and 400 µL of supplemented high glucose DMEM (1% penicillin–streptomycin and 10% fetal bovine serum) was added. Four glass and HOUSteon fibers were placed in each well. At days 1, 2, and 4, 200 µL of the media was removed, saved, and replaced with fresh media. Empty wells of media were used as controls. These timepoints of used media were added to 200 µL of fresh media containing 50,000 osteoblast-like cells from a passage 5 osteosarcoma cell line (MG63). After 3 days of culturing, a cell proliferation MTS assay (Celltiter 96 Aqueous Nonradioactive Cell Proliferation Assay, Promega, Madison, WI, USA) was performed to establish cell number. In short, the media were replaced with 200 µL of fresh media with 40 µL of 20:1 MTS:phenazine methosulfate (PMS) solution added. After an hour at 37 ◦C, 100 µL was removed from each well and placed in a 96 well plate, with the absorbance measured at 490 nm.

2.8. Cell Adhesion The PCL was punched out using a 10 mm diameter hole punch, whereas the glass and HOUSteon fibers were cut in 1 cm increments. The wells containing no scaffold were used as controls. The scaffolds were UV sterilized as before and placed into 48 well plates with cloning rings on top. Four glass and HOUSteon fibers were placed in each well. 200 µL of media containing 100,000 MG63 cells were added to each well, and allowed to culture for 3 h. Scaffolds were removed and placed in a new 48 well plate, where the aforementioned MTS/PMS assay was performed. When the HOUSteons and glass fibers were cut prior to the assay, pieces in between the cut were saved and imaged with the SEM. The measured diameter and length of those scaffolds were used to calculate and normalize to the surface areas, since the PCL was much larger (~25 mm2 for HOUSteon compared to the top surface area for the PCL of ~78 mm2).

2.9. Confocal Imaging All confocal images were taken using Olympus FV1000 laser scanning confocal microscope. All cells were stained with 40,6-diamidino-2-phenylindole (DAPI). Some cells were stained with Bioengineering 2020, 7, 165 6 of 15

Celltrace Far Red cell proliferation, which covalently binds to cellular amines and can indicate the extending protrusions of cells.

2.10. ICP-OES Glass Conversion The ﬁbers were cut into 1 cm long segments, approximately 1 mg of glass per sample. The samples were submerged in 1 mL PBS in 37 ◦C incubator for the 1–4 weeks, creating a 1 g/L ratio. The solutions were removed each week and stored in the 80 C freezer until use. The solutions were brought to − ◦ 10 mL with an end concentration of 2% HNO3 in deionized water prior to analysis. The samples were analyzed with an Optima 8300 inductively coupled plasma–optical emission spectrometer (ICP-OES, Perkin-Elmer, Waltham, MA, USA).

2.11. Statistical Analysis SPSS software (IBM, Armonk, NY, USA) was used to determine statistical signiﬁcance with an alpha value of 0.05. Independent sample t-tests were used to compare between two variables. One-way ANOVA with a Tukey post-hoc analysis was performed to evaluate signiﬁcance between multiple samples.

3. Results

3.1. Design and Optimization of the Modified Air-Gap Electrospinning Setup The design of the air-gap electrospinning apparatus is composed of both stationary and rotating parts, as shown in Figure1a. This design keeps the glass fiber stationary, allowing the electrospun fibers to wrap around it, which is demonstrated in Figure1b. When the glass fiber is not held in place, then typically the slower side of the mandrel entraps the glass fiber forcing it to rotate with that side. This ultimately ends up with half of the polymer fibers not wrapping around the glass fiber. Using this motor-gear setup allows for the mandrel to rotate around the stationary glass fiber, without the motors being shocked from the voltage applied to the mandrels. Furthermore, the flat-cone shaped mandrel was chosen over the flat plate stereotypical used for air gap electrospinning in order to decrease the effective diameter as much as possible. Decreasing the mandrel size causes a lower angle of fiber wrapping, making the polymer fibers more aligned to the glass fiber. The cone attracted the fibers more efficiently than its flat-plate equivalent, while only adhering to the tip of the cone. This allows for a significantly smaller effective diameter of the mandrel, such as the 4 mm cone-tip that was used in this design. One drawback with air gap electrospinning is that the fibers will deposit only on empty mandrel sites, preventing fibers from depositing when the mandrel is covered. Additional fibers will deposit on only one side of the mandrel, not deposit on either side, or randomly deposit onto the aligned fibers. When a fiber is deposited on this rotating mandrel, it will wrap around the glass fiber starting at the center and work its way towards the mandrels. With the current design involving the protruded stationary glass holder from the mandrel, the fibers will eventually wrap around the holder until enough tension pulls the fiber off the mandrel. This frees up space on the mandrel, allowing more fibers to deposit. This process can be repeated continuously until some other failure occurs, such as the glass fiber breaking. This allows for the number of layers of wrapped PCL to be controllable while not being limited by the mandrel not having available spots to deposit on. Initial designs had the stationary glass holder inside of the cone, but the polymer fibers pull the glass fiber out of its holder, halting electrospinning. To demonstrate the capability for this design to continuously electrospin, Figure3a shows a HOUSteon that was allowed to electrospin for longer, resulting in a diameter around 500 µm with a glass diameter around 150 µm. In Figure3b,c, eight typical HOUSteons with a diameter of ~200 µm were adhered together and then placed in the same modified air-gap electrospinning setup, undergoing the same electrospinning process to form a bundle of HOUSteons. In Figure3b, the outermost casing Bioengineering 2020, 7, 165 7 of 15 of electrospun fibers are shown, with the glass fibers exposed. In Figure3c, the outer casing was peeledBioengineering back, revealing 2020, 7, x FOR the PEER maintained REVIEW HOUSteon structure when incorporated into a bundle.7 of 16 Bioengineering 2020, 7, x FOR PEER REVIEW 7 of 16

a b a b

FigureFigure 3. SEMs 3. SEMs of of (a )(a a) a ~150 ~150 µμmm diameter glass glass fiber fiber wrapped wrapped in electrospun in electrospun nanofibers nanofibers to a total to of a total of ~500 µm diameter; (b) a bundle of eight ~200 µm diameter homologous and uniform synthetic ~500Figure μm diameter3. SEMs of; ( b(a) )a a bundle ~150 μm of eightdiameter ~200 glassμm diameter fiber wrapped homologous in electrospun and uniform nanofibers synthetic to osteonsa total of osteons (HOUSteons) wrapped together to a total diameter ~800 µm, with (c) the outer layer peeled (HOUSteons~500 μm diameter) wrapped; (b) together a bundle to of a eight total ~200diameter μm diameter ~800 μm, homologous with (c) the outer and uniform layer peeled synthetic back .osteons Scale back.bar Scale(HOUSteons is 100 bar μm. is) 100 wrappedµm. together to a total diameter ~800 μm, with (c) the outer layer peeled back. Scale bar is 100 μm. 3.2. SEM,3.2. SEM, FFT, FFT, Fiber Fiber Diameter Diameter To3.2. demonstrateTo SEM, demonstrate FFT, Fiber the the Diameter range range ofof angles that that the the polymer polymer fibers fibers can wrap can around wrap aroundthe glass thefiber, glass FFT fiber, FFT was performedTo demonstrate on on representative representative the range of angles images images that of the ofdifferent dipolymerfferent diameter fibers diameter can mandrels wrap mandrels around in Figure in the Figure glass 4. The fiber,4. The pixel FFT pixel intensityintensitywas plots performed plots showed showed on two representative two distinct distinct peaks, peaks, images which which of different correlates correlates diameter to to be be the mandrels the orientation orientation in Figure of the of 4.individual the The individual pixel polymerpointensitylymer fibers fibers plots as wellas showed well as asthe twothe orientation orientationdistinct peaks, ofof thethe which HOUSteon correlates as as a to awhole. whole.be the The orientation The difference difference of between thebetween individual these these peaks yields the angle of polymer fiber wrapping around the glass fiber. These values were validated peakspo yieldslymer the fibers angle as well of polymer as the orientation fiber wrapping of the HOUSteon around the asglass a whole. fiber. The These difference values between were validatedthese by using image-processing software (ImageJ) to measure the respective angles and comparing them by usingpeaks image-processing yields the angle of software polymer (ImageJ)fiber wrapping to measure around the the respective glass fiber. anglesThese values and comparing were validated them to toby the using FFT resultsimage-processing. software (ImageJ) to measure the respective angles and comparing them the FFT results. to the FFT results. a b 30 c

a b 30 c °

25 ° 25 Aligned 5 20 Aligned 5 200 90 180 0 90 180

d e 55 f

° d e f 5055

25 4550 Aligned 25 4045 Aligned 400 90 180 0 90 180

Figure 4. Cont. Bioengineering 2020, 7, 165 8 of 15 Bioengineering 2020, 7, x FOR PEER REVIEW 8 of 16

g h i 54 Bioengineering 2020, 7, x FOR PEER REVIEW 8 of 16

49 g h

Random i 54

Counts (x1000)

44 49 0 90 180 Angle (°)

Random

Counts (x1000) 44 FigureFigure 4.4. Fast Fourier Fourier transform transform (FFT) (FFT) of of representative representative scaffolds scaffolds to to demonstrate demonstrate the the tailorability tailorability in in fiber alignment. FFT was used on (a,d,g) SEM images in order0 to gain the90 respective (180b,e,h) FFT fiber alignment. FFT was used on (a,d,g) SEM images in order to gain the respectiveAngle (° ()b,e,h ) FFT output output images and (c,f,i) pixel intensity plots against the angle of acquisition. The alignment angle images and (c,f,i) pixel intensity plots against the angle of acquisition. The alignment angle was was determined through subtracting the peaks, resulting in (a,b,c) 5 ,(d,e,f) 25 , and (g,h,i) random. determinedFigure 4 through. Fast Fourier subtracting transform the (FFT) peaks, of representative resulting in ( a,b,cscaffolds) 5°, to(d,e,f demonstrate◦ ) 25°, and ◦the (g,h,i tailorability) random. in Scale Scale bar is 20 µm. bar isfiber 20 μalignment.m. FFT was used on (a,d,g) SEM images in order to gain the respective (b,e,h) FFT output images and (c,f,i) pixel intensity plots against the angle of acquisition. The alignment angle was The alignmentdetermined through angle ofsubtracting the polymer the peaks, primarily resulting depends in (a,b,c) 5° on, (d the,e,f) 25 diameter°, and (g,h,i of) therandom air-gap. Scale mandrels The alignment angle of the polymer primarily depends on the diameter of the air-gap mandrels and the distancebar is 20 fromμm. the mandrel, as summarized in Figure5 and Equation (3) Figure5b demonstrates thatand reducing the distance the diameter from the of themandrel, mandrel as is summarized the most eff ective in Figure way of 5 reducing and Equation the angle, (3) Figureas well as5b reducingdemonstratesThe the alignment angle that reducing variance angle of alongthe the diameter polymer the length primarilyof the of the mandrel depends HOUSteon. is on the the Thismost diameter not effective onlyof the increases airway-gap of mandrels reducing consistency the betweenangle,and as samples, the well distance as reducingbut from also thealong the ma angle thendrel, distance variance as summarized of along each the HOUSteon. in Figurelength 5 of andHence, the Eq HOUSteon.uation the 4 mm(3) Figure This cone-shaped not 5b only demonstrates that reducing the diameter of the mandrel is the most effective way of reducing the mandrelincreases was consistency chosen for between further samples, testing. but also along the distance of each HOUSteon. Hence, the 4 mm cone-shapedangle, as well mandrel as reducing was the chosen angle for variance further along testing. the length of the HOUSteon. This not only increases consistency between samples, but also along the distance of each HOUSteon. Hence, the 4 1 D D DD mm cone-shaped mandrel was chosenθ θ==Tan forTan −further−1 ( )ortesting. or x =x = ((3)3) 2x2x 2Tan2Tan(θ(θ)) D D θ = Tan−1 ( ) or x = 2x 2Tan(θ) (3) a 80 b 31 mm a 80 b 60 31 mm22 mm ) ° 60

40 22 mm4 mm ) ° 40 4 mm

Angle( 20

Angle ( Angle 20 0 0 0 2 4 6 0 2 4 6 Distance from Mandrel (cm) Distance from Mandrel (cm) Figure 5. (a) Diagram and Equation (3) of a theoretical polymer fiber wrapping around the glass FigureFigure 5. ( a 5). Diagram(a) Diagram and and Equation Equation (3) (3) ofof a a theoretical theoretical polymer polymer fiber fiber wrapping wrapping around around the glass the glass microfiber, where D is the diameter of the mandrel, θ is the angle the fiber wraps around the glass microfiber,microfiber, where where D is D the is diameterthe diameter of theof the mandrel, mandrel,θ is θ theis the angle angle the the fiber fiber wraps wraps around around thethe glassglass fiber, fiber,fiber, L is Lthe is thelength length between between mandrels, mandrels, andand x is thethe distance distance from from the the mandrel. mandrel. (b) Theoretical(b) Theoretical fiber fiber L is the length between mandrels, and x is the distance from the mandrel. (b) Theoretical fiber angles anglesangles with with mandrel mandrel diameters diameters of of 4, 4, 22, 22, and and 31 mm atat varying varying distances distances.. with mandrel diameters of 4, 22, and 31 mm at varying distances.

3.3.3.3. MechanicalMechanical3.3. Mechanical Testing-3ptTesting-3pt Testing-3pt BendingBending Bending andand and PorosityPorosity Porosity When comparing the PCL fibers in Figures 6a,b, the porosity decreases to 79% when the fibers WhenWhen comparingcomparing thethe PCL fibersfibers in FigureFigures6 a,b,6a,b, the the porosity porosity decreases decreases to to 79% 79% when when the the fibers fibers were aligned while still maintaining the same fiber diameter. Aligning and wrapping the fibers closes were aligned while still maintaining the same fiber diameter. Aligning and wrapping the fibers closes werethe aligned pores while by forcing still maintainingthe fibers to be the adjacent same fiber to one diameter. another, Aligningwhereas the and randomly wrapping oriented the fibers fibers closes thethe poresporesare allowe byby forcingforcingd to deposit thethe fibers fibersfreely. to toIn beFigurebe adjacentadjacent 6c, the toto addition oneone another,another, of the PCL whereaswhereas fibers hadthethe randomlynorandomly significant orientedoriented effect on fibersfibers areare allowedallowedthe peak toto stress depositdeposit applied freely.freely. to the InIn glass FigureFigure fibers.6 6c,c, the theAlthough addition addition they of of achieved the the PCL PCL similar fibers fibers hadpeak had no stresses,no significant significant the PCL e effectff inect onon thethe peakpeakthe HOUSteons stressstress appliedapplied maintained toto thethe glass glassthe load fibers.fibers. due AlthoughtoAlthough its tensile, theythey viscoelastic achievedachieved deformation similarsimilar peakpeak after stresses,stresses, glass fracture. thethe PCLPCL inin thethe HOUSteonsHOUSteonsThis is further maintainedmaintained demonstrated thethe in loadload Figure duedue 6 tod,to itswhereits tensile,tensile, bundles viscoelasticviscoelastic of eight HOUSteons deformationdeformation were after compared glass fracture. to ThisThis iseightis furtherfurther glass fibersdemonstrateddemonstrated bound at the inin ends. FigureFigure This6 d,6d, is where mostwhere evident bundles bundles with of ofthe eight eight sustained HOUSteons HOUSteons load after were were all of compared thecompared glass to to eighteightfibers glassglass fractured, fibersfibers boundbound but can atatthe thebe seen ends.ends. throughout ThisThis isis mostmost by the evidentevident increased withwith recovery thethe sustainedsustained after at loadloadleast afteroneafter of allall the ofof glass thethe glassglass fibersfibersfibers fractured,fractured, breaks ,but butindicated cancan bebe by seenseen vertical throughoutthroughout drops. byby thethe increasedincreased recoveryrecovery afterafter atat leastleast oneone ofof thethe glassglass fibersfibers breaks,breaks, indicatedindicated byby verticalvertical drops.drops. Bioengineering 2020, 7, 165 9 of 15

Bioengineering 2020, 7, x FOR PEER REVIEW 9 of 16

a 1.2 b 100 1 * 90 0.8 * HOUSteon HOUSteon 0.6 80 Random Random

0.4 Porosity (%) Porosity

Diameter (um) Diameter 70 0.2 0 60

c 2 d 0.6 0.5 HOUSteon 1.5 0.4 Bundle HOUSteon 0.3 Glass Bundle 1

Glass fiber 0.2

Load (N) Load (MPa) 0.1 0.5

0 Average Peak Stress Peak Average 0 0 1 2 3 Displacement (mm) Figure 6. PhysicalFigure 6. Physical characterization characterization of of ( a(a)) a averageverage PCL PCL fiber fiberdiameter diameter and (b) porosity and between (b) porosity the between HOUSTeon and randomly aligned PCL. The 3-point bending of (c) the peak stress between the the HOUSTeonHOUS andteon randomly and bare glass aligned at 0.01 mm/s. PCL. (d) A The representative 3-point 3 bending-point bending of (loadc) the-displacement peak stress curve between the HOUSteon andcomparing bare glass a bundle at of 0.01 eight mm HOUSteons/s. (d) A and representative 8 bare glass. 3-point bending load-displacement curve comparing a bundle of eight HOUSteons and 8 bare glass. * denotes significant difference (p < 0.05). 3.4. Cell Cytotoxicity and Adhesion 3.4. Cell CytotoxicityIn Figure and 7 Adhesiona, the released particles from all of the different scaffolds had no significant cytotoxic impact on the cells, as compared to the fresh media control. The decrease in cells of the glass- In Figurecontaining7a, the scaffolds released compared particles to PCL from can all be attributed of the di toff pHerent alkalization scaffolds during had glass no conversio significantn cytotoxic impact on theunder cells, these as comparedstatic conditions to [39] the. freshThis is mitigated media control. through dynamic The decrease cell culturing in cells conditions, of the which glass-containing are more representative of in vivo conditions, or through preconditioning the scaffolds. The cells scaffolds comparedadhered from to PCL Figure can 7b bewere attributed normalized toto the pH surface alkalization area available during to the glass cells and conversion to the cell under these static conditionscontrol [,39 resulting]. This in is Figure mitigated 7c. This throughwas to compare dynamic the number cell culturingof cells adhered conditions, to the estimated which are more representativeamount of in of vivo availableconditions, material, sinceor through the PCL preconditioning punches had around the three sca timesffolds. moreThe surface cells area adhered from exposed than the HOUSteons. After normalization, the amount of cells adhered is comparable to the Figure7b wererandomly normalized oriented PCL, to the indicating surface the areacells adhere available with similar to the affinity cells to andthe PCL to as the well cell as the control, PCL resulting in Figure7c. onThis the HOUSteons. was to compare It is worthwhile the number to note that of m cellsore accurate adhered results to can the be estimated obtained by increasing amount of available material, sincethe the amount PCL of punches HOUSteons had used around to make three it equivocal times moreto the PCL, surface but the area relative exposed amount than of cells the HOUSteons. adhered demonstrates its potential. After normalization, the amount of cells adhered is comparable to the randomly oriented PCL, indicating the cells adhere with similar affinity to the PCL as well as the PCL on the HOUSteons. It is worthwhile to note that more accurate results can be obtained by increasing the amount of HOUSteons used to make it equivocal to theBioengineering PCL, but 2020 the, 7, x FOR relative PEER REVIEW amount of cells adhered demonstrates its potential.10 of 16

a 170 160 HOUSteon 150 140 Glass 130 PCL 120 Media Cell Number (x1000) Number Cell 110 Day 1 Day 2 Day 4

b 120 HOUSteonComposite * c CompositeHOUSteon 100 Glass 0.3 Glass PCL 0.25 PCL 80 Cells 0.2 60 + 0.15 40 0.1

20 0.05

Cells adhered (x1000) adhered Cells #Cells/Surface Area Area #Cells/Surface 0 (x1000) Normalized 0

Figure 7. Cell assaysFigure 7.of Cell (a assays) in vitroof (a) incytotoxicity vitro cytotoxicity and and ( (b,cb,c) adherence) adherence.. (c) Cells (adheredc) Cells to the adhered scaffolds to the scaffolds were normalized to the available surface area on the scaffolds and to the cell control. were normalized to the available surface area on the scaffolds and to the cell control. * and + denote statistical significance3.5. Confocal Imaging (p < 0.05). The migration of the cells is sensitive to fiber alignment and responds to aberrant fibers. The cells are capable of compensating for defects in the scaffold, as demonstrated in Figure 8 when the cells used the bridging PCL to cross between the severed halves of the HOUSteons. This is beneficial for glass fractures, but can be inconvenient when aberrant fibers are deposited on the scaffold during manufacturing, such as in Figure 9a. The horizontal fiber hindered cell migration, forcing cells to either go around or cross over it. While not ideal, the cells were still capable of continued migration along the HOUSteon. Furthermore, scaffolds without any large defects in Figure 9b allowed cell migration to achieve a near confluent layer, signifying the ideal interaction between the cells and the PCL on the HOUSteon.

a b c

Figure 8. Confocal images of the (a) cytoplasm, (b) DAPI, and (c) composite image for MG63 cells on the HOUSteon. The HOUSteon fractured prior to cell adhesion, and cell migration is visible on the bridging PCL fibers (arrows). Scale Bar is 100 μm.

Bioengineering 2020, 7, x FOR PEER REVIEW 10 of 16

a 170 160 HOUSteon 150 140 Glass 130 PCL 120 Media Cell Number (x1000) Number Cell 110 Day 1 Day 2 Day 4

b 120 HOUSteonComposite * c CompositeHOUSteon 100 Glass 0.3 Glass PCL 0.25 PCL 80 Cells 0.2 60 + 0.15 40 0.1

20 0.05

Cells adhered (x1000) adhered Cells #Cells/Surface Area Area #Cells/Surface 0 (x1000) Normalized 0

BioengineeringFigure2020 7. Cell, 7, 165assays of (a) in vitro cytotoxicity and (b,c) adherence. (c) Cells adhered to the scaffolds10 of 15 were normalized to the available surface area on the scaffolds and to the cell control.

3.5.3.5. ConfocalConfocal ImagingImaging TheThe migrationmigration of of the the cells cells is sensitiveis sensitive to fiberto fiber alignment alignment and and responds responds to aberrant to aberrant fibers. fibers. The cells The arecells capable are capable of compensating of compensating for defects for defects in the in sca theffold, scaffold, as demonstrated as demonstrated in Figure in Figure8 when 8 thewhen cells the usedcells theused bridging the bridging PCL toPCL cross to cross between between the severed the severed halves halves of the of HOUSteons. the HOUSteons. This This is beneficial is beneficial for glassfor glass fractures, fractures, but but can can be inconvenientbe inconvenient when when aberrant aberrant fibers fibers are are deposited deposited on on the the sca scaffoldffold during during manufacturing,manufacturing, such such as as in in Figure Figure9a. 9 Thea. The horizontal horizontal fiber fiber hindered hindered cell migration, cell migration, forcing forcing cells to cells either to goeither around go around or cross or over cross it. over While it. notWhile ideal, not theideal, cells the were cells still were capable still capable of continued of continued migration migration along thealong HOUSteon. the HOUSteon. Furthermore, Furthermore, scaffolds scaffolds without withoutany large any defects large in defects Figure 9 inb allowed Figure 9 cellb allowed migration cell tomigration achieve ato near achieve confluent a near layer,confluent signifying layer, signifying the ideal interactionthe ideal interaction between thebetween cells andthe cells the PCLand onthe thePCL HOUSteon. on the HOUSteon.

a b c

FigureFigure 8.8. ConfocalConfocal imagesimages ofof thethe ((aa)) cytoplasm,cytoplasm, ((bb)) DAPI,DAPI, andand ((cc)) compositecomposite imageimage forfor MG63MG63 cellscells onon thethe HOUSteon.HOUSteon. TheThe HOUSteonHOUSteon fracturedfractured priorprior toto cellcell adhesion,adhesion, andand cellcell migrationmigration isis visiblevisible onon thethe Bioengineeringbridgingbridging 20 PCL20PCL, 7, ﬁbers xfibers FOR(arrows). PEER(arrows). REVIEW ScaleScale BarBar isis 100100µ μm.m. 11 of 16

a b

FigureFigure 9.9. RepresentativeRepresentative SEM SEM images images of aof HOUSteon a HOUSteon with with overlaid overlaid confocal confocal images images stained stained with DAPI with ofDAPI (a) an of aberrant(a) an aberrant ﬁber and fiber (b )and aligned (b) aligned ﬁbers. fibers.

3.6.3.6. ICP-OESICP-OES Glass Conversion IonIon releaserelease measurements measurements from from ICP-OES ICP-OE demonstrateS demonstrate that thatthe polymer the polymer wrapping wrapping does not does inhibit not theinhibit glass the conversion. glass conversion Similar. calcium Similar elutioncalcium was elution observed was betweenobserved sca betweenffolds in scaffolds Figure 10 ina, butFigure calcium 10a, isbut incorporated calcium is incorporated into HA. Magnesium into HA. Magnesium is an ion that is isan in ion neither that is PBS in neither nor in P normalBS nor HA,in normal with bothHA, scawithffolds both having scaffolds increased having magnesiumincreased magnesium elution at similarelution rates,at similar as shown rates, in as Figure shown 10 inb. Figure Therefore, 10b. theTherefore, eluted magnesiumthe eluted magnesium is indicative is ofindicative glass degradation. of glass degradation. Both scaff oldsBoth showedscaffolds similar showed trends similar of elutedtrends massof eluted forpotassium, mass for potassium, silicon, and silicon, carbon, and as carbon, shown as in shown Figure 10in c.Figure While 10 thec. While elution the of elution silicon of is indicativesilicon is indicative of glass degradation, of glass degradation, the conversion the conversion to HA utilizes to HA the porousutilizes silica the porous layers tosilica maintain layers the to originalmaintain structure the original of the structure phosphate-based of the phosphate glass. -based glass. 50 a Ca 15 b Mg 40 x 10 x 30 X * HOUSteon 20 * Glass 5 10 v v V

0 0

Cum. Ion Mass (ug) IonMass Cum. Cum. Ion Mass (ug) IonMass Cum. 0 2 4 6 0 2 4 6 Time (weeks) Time (weeks)

500 c 450 * 400 350 HOUSteon K 300 Glass K 250 HOUSteon Si 200 * Glass Si

150 Eluted Mass (ug) Mass Eluted 100 HOUSteon C V X o o x v x v x Glass C 50 TN T N TN 0 -50 1 2 3 4 Time (Weeks)

Figure 10. ICP-OES of the cumulative ion mass eluted of (a) calcium and (b) magnesium. (c) The eluted mass of potassium, silicon, and carbon. Strikethrough indicates parent significance.

Bioengineering 2020, 7, x FOR PEER REVIEW 11 of 16

a b

Figure 9. Representative SEM images of a HOUSteon with overlaid confocal images stained with DAPI of (a) an aberrant fiber and (b) aligned fibers. 3.6. ICP-OES Glass Conversion Ion release measurements from ICP-OES demonstrate that the polymer wrapping does not inhibit the glass conversion. Similar calcium elution was observed between scaffolds in Figure 10a, but calcium is incorporated into HA. Magnesium is an ion that is in neither PBS nor in normal HA, with both scaffolds having increased magnesium elution at similar rates, as shown in Figure 10b. Therefore, the eluted magnesium is indicative of glass degradation. Both scaffolds showed similar Bioengineeringtrends2020, of7, eluted 165 mass for potassium, silicon, and carbon, as shown in Figure 10c. While the elution of 11 of 15 silicon is indicative of glass degradation, the conversion to HA utilizes the porous silica layers to maintain the original structure of the phosphate-based glass.

50 a Ca 15 b Mg 40 x x 30 10 X * HOUSteon 20 * Glass 5 10 v v V 0 0 Cum. Ion Mass (ug) Ion Mass Cum. 0 2 4 6 (ug) Ion Mass Cum. 0 2 4 6 Time (weeks) Time (weeks)

500 c 450 * 400 350 HOUSteon K 300 Glass K 250 HOUSteon Si 200 * Glass Si 150 Eluted Mass (ug) Eluted 100 HOUSteon C V X o o x v x v x Glass C 50 TN T N TN 0 -50 1 2 3 4 Time (Weeks)

Figure 10. ICP-OES of the cumulative ion mass eluted of (a) calcium and (b) magnesium. (c) The eluted massFigure of potassium,10. ICP-OES of silicon, the cumulative and carbon. ion mass *,eluted V, X, of T,(a)N, calcium and and O denote (b) magnesium. statistical (c) The signiﬁcance, with strikethrougheluted mass indicating of potassium, parent silicon, signiﬁcanceand carbon. Strikethrough to respective indicates letters parent (p < significance.0.05).

4. Discussion The creation of a synthetic scaffold that is osteoconductive and osteoinductive, while promoting angiogenesis, is necessary to replace the current gold standard of autografts [40]. Most of the current designs for biomimetic scaffolds focus on the material aspect of bone, by using collagen derivatives and calcium phophate-based ceramics as a replacement for bone’s natural elasticity and mechanical strength [41]. Animal-extracted collagen and gelatin have high tissue regeneration potentials due to their resorbability, low antigenicity, and high cytocompatibility [42,43]. Bioactive ceramics, HA, calcium phosphates, and glasses are typically used to provide better mechanical strength and bone formation than collagen derived materials alone [44]. Incorporating well-characterized growth factors or cultured cells into these scaffolds have been shown to increase overall bone formation as well [44]. These composite scaffolds have a variety of structures, with some common forms being three-dimensional structures [42,45], hydrogels [46], and dry powder [47]. The formation of these scaffolds are typically through solvent [48], freeze-drying [49], gas-foaming, or electrospinning [50] followed by a crosslinking method for solidification of the polymer. Furthermore, the incorporation of the ceramics can be classified as either within the scaffold during fabrication or deposited onto the surface. Combining scaffold methods, adjusting the materials used, and incorporating additional materials have been shown to adjust the properties of the scaffolds [48]. Unfortunately, current scaffolds are not as efficient at bone healing as the autograft gold standard, one common problem with biomaterials being the slow implant integration [49]. Copying the structure of naturally occurring tissues is one strategy to improve integration with adjacent tissues [49]. The design in this study was focused on mimicking osteons with concentric polymer layers surrounding haversian canals while creating radial porosity for nutrient diffusion. The number of electrospun layers, which is analogous to the bone matrix lamellae, is contingent on the volume electrospun. This causes the HOUSteons to have dimensions comparable to native osteons [8]. The alignment of the electrospun fibers allows for increased cell migration along the length of the HOUSteon, whereas the degradation of the glass microfiber is designed to promote blood vessel Bioengineering 2020, 7, 165 12 of 15 ingrowths. Maintaining the high porosity from normal electrospinning is essential for not hindering the glass conversion using this setup, since the water must infiltrate through the electrospun layer, hydrolyze the glass, and begin its conversion process. The alignment of the polymer fibers is worth this slight decrease in porosity, since 80% porosity is sufficient for water infiltration and glass conversion, which is demonstrated by ICP. All of the eluted ions showed similar elution between the bare glass and the HOUSteon. This reduced porosity is expected to decrease the cellular infiltration through the thickness of the HOUSteon, but the aligned fibers increase directional cell migration along the length of the HOUSteon. Alternatives to increase porosity include increasing the fiber diameter or introducing sacrificial fibers. A common criticism of using ceramics or glass as an implant is that the breakage results in catastrophic failure, rendering the implant functionally useless afterwards. Although not comparable to native cortical bone of 100–150 MPa compressive strength or 20–40 MPa tensile strength [51], wrapping the glass with the polymer fibers holds the glass in place, maintaining some structural integrity after the glass breaks. This overcomes its catastrophic failure by instead having viscoelastic breakage, with no significant difference in peak stresses. More importantly, the cells were able to cross the polymer fibers after breakage, bridging the gap and progressing forward. This maintains the bioactivity of the scaffold even if it breaks. The initial degraded components of the HOUSteon were not significantly more cytotoxic to the cells than plain media. Although in vitro analysis of bioactive glass can be contradictory and cytotoxic at times due to static culturing conditions and different cytotoxicity methods, bioactive glass has positive results in vivo [24,52]. The degradation of the glass creates a nearby acidic environment, but the in vivo environment should dilute and remove the acidic byproducts [24,53]. Additionally, the normalized cell adherence of the HOUSteons was comparable to PCL. This is expected since the composition and diameters of the PCL fibers are the same, with the main superficial difference between them being the available surface area. One main component of this design is to increase cell migration due to the aligned fibers. Previous work has already demonstrated that aligned electrospun fibers enhance directional cell migration [13–19]. Initial cell seeding showed the cells extending parallel to the fibers, signifying unidirectional orientation and thus increased cell migration. Due to the weakness of a single HOUSteon as well as the expected mechanical strength decreasing during degradation, breakage of some HOUSteons is expected. Figure8 demonstrates that a broken HOUSteon is capable of cell migration along the stretched fibers between the broken pieces. This allows for continued cell activity and bone formation despite HOUSteon breakage. Additionally, the cells were affected by aberrant fibers, slowing the directional migration of cells when encountered. Because of this sensitivity though, the cells also elongate and respond to the normal, parallel fibers. These curved, aberrant fibers, which deviate from aligned fibers, were found to occur during electrospinning when the fiber is longer than the average fiber. This causes adjacent fibers to entrap it into the HOUSteon during wrapping. Guaranteeing there is an available spot on the mandrel by reducing the flow rate, or accelerating the fibers quicker to the mandrels by adjusting their charge, is expected to mitigate aberrant fibers. Since the purpose of the HOUSteon is to facilitate the migration and proliferation of the osteogenic cells, it is imperative that blood vessels infiltrate into the scaffold to provide the necessary nutrients. The HOUSteon design can easily change its composition to match well-researched materials and constituents relevant for bone and blood vessel growth. For example, incorporating already established pro-angiogenic ions into the glass, such as copper and cobalt, will induce blood vessel growth [54]. Adding calcium or pro-angiogenic growth factors into the electrospun fibers allows for a release rate separate from the bioactive glass. Furthermore, substituting borate for silicate [26] or increasing the ionic radius of network modifiers, such as replacing K2O for Na2O[27], will speed up the degradation rate to more closely match the ingrowths of blood vessels. Similarly, increasing the hydrophilicity of the electrospun fibers should help increase the influx of water towards the glass. Bioengineering 2020, 7, 165 13 of 15

Therefore, HOUSteons provide the structure similar to osteons, with the polymer and bioactive glass compositions capable of being easily altered to further promote regeneration.

5. Conclusions In this study, a synthetic scaffold was designed to mimic the structure and dimensions of an osteon, the individual unit of cortical bone. These aligned electrospun fibers held the bioactive glass fiber together, while not inhibiting glass conversion. Inversely, the degraded ions from the glass did not prevent cell adhesion or cause significant cell death. The versatility of this design allows for the glass and polymer fibers to be replaced with more osteogenic and angiogenic materials, with further incorporation of nanoparticles or growth factors to facilitate growth. Utilizing this biomimetic scaffold design with more bioactive materials, this synthetic scaffold aims to replace autografts to heal critical size bone defects.

Author Contributions: Conceptualization, H.R.L.; methodology, H.R.L.; software, H.R.L.; validation, H.R.L. and A.A.G.; formal analysis, H.R.L.; investigation, H.R.L. and A.A.G.; resources, D.E.D. and S.A.S.; data curation, H.R.L.; writing—original draft preparation, H.R.L.; writing—review and editing, H.R.L. and S.A.S.; visualization, H.R.L.; supervision, S.A.S.; project administration, H.R.L.; funding acquisition, S.A.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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