Electrospun scaffolds provide a dense framework of nanofibers with pore sizes and fiber diameters that closely resemble the architecture of native extracellular matrix. However, it generates limited three-dimensional structures of relevant physiological thicknesses. 3D printing allows digitally controlled fabrication of three-dimensional single/multimaterial constructs with precisely ordered fiber and pore architecture in a single build. However, this approach generally lacks the ability to achieve submicron resolution features to mimic native tissue. The goal of this study was to fabricate and evaluate 3D printed, electrospun, and combination of 3D printed/electrospun scaffolds to mimic the native architecture of heterogeneous tissue. We assessed their ability to support viability and proliferation of human adipose derived stem cells (hASC). Cells had increased proliferation and high viability over 21 days on all scaffolds. We further tested implantation of stacked-electrospun scaffold versus combined electrospun/3D scaffold on a cadaveric pig knee model and found that stacked-electrospun scaffold easily delaminated during implantation while the combined scaffold was easier to implant. Our approach combining these two commonly used scaffold fabrication technologies allows for the creation of a scaffold with more close resemblance to heterogeneous tissue architecture, holding great potential for tissue engineering and regenerative medicine applications of osteochondral tissue and other heterogeneous tissues.
Tissue engineering is a growing field that aims to create living biological substitutes to restore, repair, or regenerate native tissue or organ function that may be affected by disease or injury. The main components of engineered tissues include cells, scaffolds, and chemical and/or mechanical cues to replicate or mimic the physiological conditions of the target tissue [
The most commonly used strategies in tissue engineering involve seeding a uniform or homogenous scaffold with a single cell type. But, in reality, most tissues are composed of several cell types and a diverse and heterogenic extracellular matrix (ECM) framework [
The goal of this study was to combine two commonly used fabrication techniques—electrospinning and 3D printing—to develop a simple and reproducible scaffold that incorporates both nano- and microscale fibrous architecture and more closely mimic heterogenous tissues. We evaluated a combined 3D printed/electrospun scaffold architecture mimicking heterogeneous tissues such as the osteochondral complex, in comparison to solely 3D printed microfibrous or solely electrospun nanofibrous scaffolds, for their ability to support viability and proliferation of human adipose-derived stem cells (hASC). Further, we also tested and compared the feasibility and efficacy of implanting full-thickness (6 mm) combined 3D printed/electrospun versus stacked electrospun scaffolds in an ex vivo porcine model using a clinically relevant procedure for osteochondral defect repair. The results show the ability to successfully engineer a scaffold that resembles the physiological thickness as well as a multiscale heterogeneous fibrous architecture of osteochondral tissue. This combined 3D printing/electrospinning approach could be extended to other tissues with heterogenous ECM framework and/or transitional tissues like ligament and tendon bone insertions in the future.
Thin disc-shaped scaffolds (Ø 14.5 mm × 2 mm) (Figure
Fabrication of combined micro- and nanofibrous scaffold by sandwiching an electrospun layer between 3D-bioplotted layers. (a) Schematic of technical approach. (b) 3D-bioplotted scaffold with and without an electrospun layer (scale bars top = 2 mm; bottom = 500
PCL was dissolved in chloroform and dimethylformamide (Sigma) at a ratio of 3 : 1 to create an 11% solution. The solution was mixed continuously at 80°C for at least 4 hours. The PCL solution was electrospun using an internal nozzle diameter of 0.508 mm on a static collector covered with aluminum for 3 hours immediately after preparation at a feed rate of 0.7
Stacked scaffolds (6 mm thick) used for implantation were generated by stacking together multiple electrospun layers using collagen type I gel in between the layers, at a concentration of 3 mg/mL (Vitrogen, Angiotech BioMaterials Corporation, Palo Alto, CA) [
The integrated micro- and nanofibrous PCL scaffolds were fabricated using a combination of 3D-bioplotting and electrospinning. The electrospun layers were cut into 14.5 mm diameter circles to match the size of the 3D scaffolds. First, a 2 mm basal section of 3D-bioplotted scaffold was printed as mentioned above. We then placed the circular electrospun layer directly over the basal layer and continued printing another 2 mm section on top of the electrospun layer to generate a final 4 mm thick scaffold with an electrospun layer in the middle (Figure
Excess adipose tissue was collected from five female premenopausal donors (ages 24 to 36) in accordance with an approved IRB protocol at UNC Chapel Hill (IRB 04-1622) [
The 3D-bioplotted, electrospun, and combined bioplotted/electrospun disc scaffolds (Ø 14.5 mm) were designed and fabricated to fit in 24-well plates (well Ø 15.6 mm), limiting any space between the walls of the well and the periphery of the scaffold where cells could potentially migrate towards the bottom of the wells. Prior to seeding, scaffolds were sterilized for 30 minutes in 70% ethanol, rinsed three times with sterile phosphate buffered saline (PBS) and once with CGM. Due to the difference in thickness between the scaffold designs (electrospun = 200
Seeded scaffolds were cultured in CGM for 21 days to promote growth and proliferation. After 21 days of culture, a LIVE/DEAD viability assay (Life Technologies) was performed per the manufacturer’s instructions on all scaffolds to assess hASC viability within the scaffolds. Briefly, hASC-seeded scaffolds were gently washed with sterile PBS three times, and then 500
Cell proliferation was assessed (
Cadaveric porcine knees were utilized to create a suitable ex vivo environment in a large animal model that resembles the human knee. This model has been used extensively in vivo to evaluate articular cartilage repair techniques [
Statistical analysis was performed using Prism (version 6.07, GraphPad Software). Bar graphs are represented as mean ± SEM. Differences were determined using a one-way ANOVA with Tukey post hoc test. A level of
Scanning electron microscopy (SEM) images show the different fiber size and arrangement between 3D-bioplotted and electrospun scaffolds (Figures
Scanning electron microscopy of (a) 3D-bioplotted scaffold; (b) electrospun nanofibers; (c) combined 3D-bioplotted and electrospun scaffolds (electrospun layer in middle); (d) cells growing on 3D-bioplotted scaffold; (e) cells growing on electrospun nanofibers; and (f) cells growing on combined 3D and electrospun scaffold (scale bars (a), (c), (d) = 500
To ensure cells could grow and proliferate throughout individual nano- and microfibrous scaffolds, we first measured and compared hASC proliferation and viability after 21 days in culture in both electrospun and 3D-bioplotted scaffolds. Cells were able to adhere and proliferate in both micro- and nanofibrous scaffolds, with minimal dead cells observed after 21 days in culture. However, the hASC exhibited higher proliferation and more uniform spreading in electrospun scaffolds when compared to 3D-bioplotted scaffolds (Figure
Cell viability and proliferation of human adipose-derived stem cells seeded on (a, c) 3D-bioplotted scaffolds and (b, d) electrospun scaffolds (green = live cells; red = dead cells).
We then compared cell proliferation and migration in all three scaffolds (Figure
Cell spreading and proliferation of human adipose-derived stem cells throughout scaffolds. Cells were cultured for 21 days in 3D-bioplotted, electrospun, or combination of 3D-bioplotted/electrospun scaffolds, fixed, and stained (actin = red; nuclei = blue). Superficial and cross-sectional views show cells present both on the surfaces of the scaffolds (superficial) and throughout the centers of the scaffolds on the 3D-bioplotted and combined scaffolds (cross-section). Human ASC exhibited steady proliferation over 21 days of culture on all scaffold types as indicated by AlamarBlue (% AB reduction). Bars indicate mean ± SEM (
Standard human operative techniques were used to implant both stacked nanofibrous scaffolds and combination of 3D-bioplotted/electrospun scaffolds into a cadaveric porcine knee model, to determine the translational applicability of these scaffolds in a relevant in vivo model for osteochondral tissue engineering. Due to the limited thickness of each electrospun layer (approximately 200
Implantation technique of 3D-bioplotted, electrospun, and combined 3D-bioplotted/electrospun scaffolds into a cadaveric porcine knee. (a) A power reamer was used to create an osteochondral defect to a depth of 8 mm and 8 mm diameter. (b) The COR system was used to implant the scaffolds into the osteochondral defect. (c) View of 3D-bioplotted scaffold (left) and electrospun scaffold (right) after implantation. (d) Combined 3D-bioplotted/electrospun scaffold prior to implantation (black arrows indicate electrospun layer, scale bar = 1 mm). (e) Combined 3D-bioplotted/electrospun scaffold inserted into the COR system for implantation (bracket and arrow pointing at scaffold inside the device). (f) Combined 3D-bioplotted/electrospun scaffold after implantation.
Electrospinning is a commonly used technique in tissue engineering allowing for production of a dense framework of fibers with pore sizes and fiber diameters that closely resemble the architecture of native ECM [
Previous investigators have also attempted to combine micro- and nanofibrous architecture into a single scaffold for different tissue engineering applications. For example, Yeo and Kim created cell-laden hierarchical scaffolds that incorporated microsized fibers for support, combined with electrospun nanofibers to enhance cell proliferation and distribution [
In this study, we present a facile and reproducible technique to develop an integrated approach combining both electrospun nanofibers and 3D-plotted microfibers to recapitulate the heterogenous architecture of native tissue. In the first approach, we electrospun nanofibers over a 3D-bioplotted scaffold. Although we successfully coated the 3D-bioplotted scaffold with electrospun nanofibers, the two layers delaminated during culture of the combined micro/nanofibrous scaffold in cell culture medium (data not shown). Such delamination would likely be a greater problem in vivo; therefore we believe this technique is not appropriate for generating a combined nano/microfibrous scaffold.
We therefore evaluated an alternative approach by 3D-bioplotting directly on an electrospun scaffold. An electrospun nanofibrous layer was placed directly over a freshly printed 3D scaffold, and then another 3D scaffold was printed on top of this electrospun layer (Figure
Cell proliferation was significantly increased over time in both 3D-bioplotted and electrospun scaffolds. Although proliferation did not significantly increase over time in the combined scaffolds, we still observed a steady proliferation with no decline over time. Although cell proliferation and viability were higher in the electrospun scaffolds, this technique has thickness and 3-dimensional limitations, typically resulting in creation of a ~200-micron scaffold after hours of electrospinning using conventional electrospinning systems. In addition, stacking several electrospun layers to create a thicker scaffold not only is time consuming but can also limit the migration of cells throughout the layers (data not shown). We first tested hASC cell viability and migration throughout micro- and nanofibers by seeding cells on scaffolds with either the electrospun layer on top or underneath the 3D-bioplotted layer. We observed that when cells were seeded on the 3D-bioplotted scaffold, the cells proliferated and migrated down to the electrospun layer. However, if the cells were seeded over the electrospun layer, the nanofiber acted as a barrier and prevented cells from migrating down into the 3D-bioplotted layer.
With our technique, the electrospun membrane can be used to separate layers that require different cell types in heterogenous tissues, like bone and cartilage in osteochondral tissue. This way, the chondrocytes, for instance, will not migrate into the underlying subchondral bone layer or vice versa, while providing a natural framework that resembles the tight collagen network in that area.
Ex vivo handling of 3D-bioplotted, stacked electrospun, and combined 3D-bioplotted/electrospun constructs confirmed that they morphologically approximated the current human tissue utilized for autologous osteochondral transfer within human joints. Using clinically relevant surgical techniques and commercially available hardware, acellular scaffolds comprised of all three designs were successfully implanted in situ using a porcine cadaveric knee. However, the stacked electrospun nanofibrous scaffolds generated by collagen binding of multiple single electrospun layers easily delaminated when implanted in the porcine knee. These stacked scaffolds needed to be frozen prior to implantation in order to prevent delamination. This could be a critical factor when dealing with seeded scaffolds and can affect cell viability. The 3D-bioplotted and combined 3D-bioplotted/electrospun scaffolds allowed for creation of clinically relevant thicknesses (5–7 mm) and were easily implanted using standard surgical procedures without delamination or breakage. This suggests a facile implementation of current autograft human osteochondral techniques to implant such multiphasic osteochondral scaffolds, indicating the immediate potential clinical translatability of our proposed combined micro- and nanofibrous scaffolds.
Creation and utilization of appropriate scaffold architecture are a critical step towards generation of an engineered tissue construct that mimics complex native tissue. Electrospun nanofibrous scaffolds, with their dense framework, pore sizes, and fiber diameters, have limitations for creation of three-dimensional structures of relevant physiological thicknesses. We compared these nanofibrous scaffolds to 3D-bioplotted scaffolds, constructs with better dimensional control and reproducibility but thicker fibers and larger pore sizes. We combined these two fabrication approaches, with results indicating that a combination of 3D-bioplotted and electrospun scaffolds could provide an excellent alternative for full heterogenous tissue regeneration. We tested our scaffolds in a relevant implantation model for osteochondral tissue and showed that although electrospun scaffolds yield higher cell proliferation, they are hard to manipulate during a clinically relevant osteochondral transplantation technique. Our combined scaffold was easily implanted using common surgical procedures into an osteochondral defect, without delamination or breaking of the scaffold.
This is one of the first studies to combine two commonly used scaffold fabrication technologies into a simple scaffold to more closely match thicker tissues with heterogenous matrix architecture. Such approaches may hold great potential for tissue engineering and regenerative medicine applications.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Special thanks are due to Saahil Mehendale for support on scaffold design. This work was supported in part by the Orthopaedic Research and Education Foundation 15-059 (Jeffrey Spang), NIH/NIBIB 1R03EB008790 (Elizabeth G. Loboa), NIH/CTSA 550KR71418 (Elizabeth G. Loboa), NIH/CTSA 550KR61325 (Elizabeth G. Loboa), and NSF/CBET 1133427 (Elizabeth G. Loboa). The 3D-printing aspect of this study was supported by a grant from NCSU Faculty Research and Professional Development (FRPD) program (Rohan A. Shirwaiker).