Validation of the 1,4‐butanediol thermoplastic polyurethane as a novel material for 3D bioprinting applications

Abstract Tissue engineering (TE) seeks to fabricate implants that mimic the mechanical strength, structure, and composition of native tissues. Cartilage TE requires the development of functional personalized implants with cartilage‐like mechanical properties capable of sustaining high load‐bearing environments to integrate into the surrounding tissue of the cartilage defect. In this study, we evaluated the novel 1,4‐butanediol thermoplastic polyurethane elastomer (b‐TPUe) derivative filament as a 3D bioprinting material with application in cartilage TE. The mechanical behavior of b‐TPUe in terms of friction and elasticity were examined and compared with human articular cartilage, PCL, and PLA. Moreover, infrapatellar fat pad‐derived human mesenchymal stem cells (MSCs) were bioprinted together with scaffolds. in vitro cytotoxicity, proliferative potential, cell viability, and chondrogenic differentiation were analyzed by Alamar blue assay, SEM, confocal microscopy, and RT‐qPCR. Moreover, in vivo biocompatibility and host integration were analyzed. b‐TPUe demonstrated a much closer compression and shear behavior to native cartilage than PCL and PLA, as well as closer tribological properties to cartilage. Moreover, b‐TPUe bioprinted scaffolds were able to maintain proper proliferative potential, cell viability, and supported MSCs chondrogenesis. Finally, in vivo studies revealed no toxic effects 21 days after scaffolds implantation, extracellular matrix deposition and integration within the surrounding tissue. This is the first study that validates the biocompatibility of b‐TPUe for 3D bioprinting. Our findings indicate that this biomaterial can be exploited for the automated biofabrication of artificial tissues with tailorable mechanical properties including the great potential for cartilage TE applications.


INTRODUCTION
In the last few years the 3D bioprinting technology has shown promising results in the biofabrication of artificial tissues for tissue engineering (TE) applications. 1 This emerging technology uses computer-aided design (CAD) and computer-aided manufacturing (CAM) techniques, which in combination with the layer-by-layer fabrication nature of 3D printing, allows to create structures with different geometries while controlling the spatial distribution of cells, biomaterials, and growth factors. 2,3 Furthermore, 3D bioprinting brings advantages to the clinical field such as shorter fabrication time, higher precision than conventional TE techniques, and tailored production. 4 Among 3D bioprinting techniques, extrusion-based is the most extended as it offers the possibility to print a wide variety of biomaterial viscosities and is the most adaptable technology to be transferred to the clinical field. 5,6 Additionally, there are several commercially available extrusion-based bioprinters, and they can also be adapted for testing novel biomaterials. Although this approach holds great promises for TE and regenerative medicine, as an emerging technology it also entails some bottlenecks. One of the main challenges is the restricted accessibility of materials necessary to produce constructs that can properly mimic the native tissue properties. The most common type of material used for this purpose are hydrogels, since they can offer a suitable 3D microenvironment that mimics the extracellular matrix (ECM) of natural tissues, promoting cell attachment and proliferation. 7 However, hydrogel scaffolds usually lack mechanical strength and structural integrity, therefore, their mechanical properties need to be tuned or combined with synthetic stiffer biomaterials to enhance its mechanical properties. 8 Several synthetic materials such as PLA, [9][10][11] or polylactic-co-glycolic acid (PLGA) [16][17][18][19] have been used to generate bioprinted scaffolds for TE applications. However, these materials do not easily achieve to mimic the native tissue mechanical characteristics. The stiffness of porous scaffolds produced using rigid biomaterials, such as PLA, 20 are in the MPa magnitude order comparable to those found in hard tissues such as porous bone. 21 Therefore, significant efforts are being made for engineering flexible tissues that suffer mechanical loading such as ligaments, tendons, cartilage, blood vessels, skin, or muscles. 22 In this sense, cartilage, as an avascular and stratified tissue, presents a limited capacity of repair, therefore, a severe damage will often require surgical intervention. However, the clinical surgical treatments such as ACI or MACI, which use a bilayer type I/III collagen membrane, lack long-term effectiveness. [23][24][25] Mosaicplasty, a treatment for focal chondral lesions, shows results that are relatively acceptable for the first 2 years but develops a sudden failure rate (approximately 55%) over the successive 2 years. 26 Currently, the strategies for cartilage repair are concentrated on the creation of a complex material that biologically mimics the native tissue and get close to its biomechanical properties. Hence, many biomaterials, such as fibrin, silk, hyaluronic acid, chitosan, PLA, or PCL 27 are being used to create scaffolds for cartilage TE, but, on one hand, natural-based materials do not show enough integrity, and on the other hand, synthetic-based materials do not have similar mechanical properties to cartilage such as friction and elasticity which limits their effectiveness and integration in the injury. 28,29 Polyurethane elastomers are a type of adaptable synthetic materials broadly applied to biomedical purposes because of their biocompatibility and good mechanical properties. [30][31][32] Recently, a novel elastic 3D printing filament consistent of a 1,4-butanediol thermoplastic polyurethane (b-TPUe) derivative shows a combination of mechanical properties that makes it a promising candidate for TE. 33 In this study, we evaluate, for the first time, the potential use of b-TPUe filament as a new 3D bioprinting material for biomedical applications. We carried out a rheological characterization to analyze their mechanical properties (in shear and compression) and a tribological study to evaluate the frictional behavior in synovial fluid-lubricated b-TPUe-cartilage tribopairs. Moreover, we compared in vitro and in vivo the biocompatibility of b-TPUe 3D printed scaffolds versus PCL and showed the potential application of this material for cartilage TE. Finally, we described the induced chondrogenic differentiation of MSCs isolated from infrapatellar fat pad when cultured in 3D bioprinted b-TPUe scaffolds. In conclusion we present a novel use of b-TPUe filament with potential to support the development of cartilage-like phenotype as a promising TE biomaterial.

Frictional test
The frictional behavior of the different plastics used in this work is exemplified in Figure 2a Figure 2b, a lower friction was measured for b-TPUe, with average friction coefficients (μ) under 0.1, followed by PCL and PLA, with average μ above 0.1.

Compression test
The

Effects of b-TPUe-conditioned medium on MSCs proliferation
We conducted a proliferation assay to evaluate if the exposure to b-TPUe could have a negative effect in the proliferative potential of MSCs. Results showed no adverse effects in the proliferative potential of MSCs cultured in b-TPUe-conditioned medium for 7 days when compared with MSCs cultured with control medium (Figure 3a).

Proliferation and viability of MSCs cultured in b-TPUe bioprinted scaffolds
Cell proliferation of MSCs cultured in b-TPUe bioprinted scaffolds was evaluated with an AlamarBlue® assay. PCL filament was used as a control material since it is a reference biomaterial used in cartilage bioprinting. [34][35][36][37] As can be observed in Figure 3b  Also, an enhanced cell growth that covered the pore spaces (Figure 4g, h) and over the filament surfaces was observed (Figure 4i).

In vivo assay
Biocompatibility of cell-free b-TPUe scaffolds was assessed in vivo by subcutaneous in situ implantation in the back of immunocompetent F I G U R E 3 In vitro biocompatibility of b-TPUe bioprinted scaffolds with MSCs. (a) Proliferative potential of MSCs cultured with control (DMEM 10% FBS, 1% P/S) or b-TPUeconditioned medium up to 7 days (**p < 0.01). (b) MSCs proliferation cultured in both b-TPUe and PCL bioprinted scaffolds up to 21 days with no significant differences between PCL and b-TPUe (no significance: ns). Significant cell growth was observed in both materials at day 7 of culture in both materials (**p < 0.01) (RFU: relative fluorescence units).
(c) Representative confocal images of MSCs grown in both b-TPUe and PCL bioprinted scaffolds at day 7 and 21. Live/ dead assay was employed, using calcein (green) and ethidium homodimer (red), live cells were stained green while dead cells were stained red. Scale bars: 500 μm. Graphs created using the GraphPad Prism 6.01 software CD-1 mice using PCL as control material (Figure 5a

DISCUSSION
The 3D bioprinting technology allows high precision, fabrication, and customized production, which are important features for biomedical F I G U R E 6 In vivo biocompatibility of b-TPUe bioprinted scaffolds with MSCs. (a) Macroscopic images for cellfree and cell-laden b-TPUe and PCL scaffolds fabricated by 3D bioprinting. Scaffolds were implanted in the dorsal region of 8 weeks old female NSG mice and resected 21 days after surgery procedure. (b) Histologic analysis of Toluidine blue and Masson's Trichrome staining of cell-free and cell-laden b-TPUe and PCL scaffolds 3 weeks postimplantation. Scale bars: 800 μm for black-labeled images, and 400 μm for red-labeled images applications. Traditional methods for scaffold manufacturing comprise phase separation, 38 electrospinning, 39 freeze-drying, 40 and gas forming. 41 Comparing this methods to 3D bioprinting, they lack a high precision control of the pore size and shape. 42 In this study, a polyurethane-based 3D printing material, b-TPUe, was successfully used to fabricate scaffolds by 3D bioprinting that were able to maintain cellular viability and growth. We selected the b-TPUe since it belongs to the polyurethane thermoplastics, an adaptable category of materials broadly used for biomedical purposes thanks to their biocompatibility, elasticity and strength. [43][44][45][46][47] There are other materials which are designed to fill and integrate irregular cartilage wounds, and are also already being tested in clinical trials, 48  A selected biomaterial for treating joint replacements is expected to preserve the remaining native cartilage from degradation while maintaining the frictional properties of the joint. 54 Analyzing the friction profile of the studied materials, b-TPUe showed to exert less friction toward the native cartilage surface than PLA and PCL, showing μ values closer to the cartilage-to-cartilage interaction. 55 Also, the mechanical properties of a scaffold are important for engineering tissues, especially for cartilage, which is subjected to cyclic mechanical forces. 56 Although scaffolds based on hydrogels mimic more adequately the mechanical properties found in native tissues, 57 their compressive modulus are typically an order of magnitude less than native cartilage tissue. 58,59 Otherwise, scaffolds produced with thermoplastics possess higher Young's modulus than those based on hydrogels. 60,61 The obtained results suggests that b-TPUe scaffold elasticity can be tailored, by changing the porosity, to achieve closer values to the natural cartilage Young's modulus than hydrogel scaffolds and synthetic polymers such as PCL or PLA, 57 thus exhibiting promising customizable mechanical properties. The viscoelastic modulus in scaffold-based TE is important to approximate and supply the unique properties of the normal articular cartilage that is trying to be replaced. 42 The ideal scaffold for cartilage regeneration is a material with viscoelastic and hydrodynamic properties that mimic the mechanical microenvironment of cartilage matrix, which could provide proper mechanical and biochemical signals for chondrocyte adhesion proliferation, differentiation, and ECM formation. 62 Moreover, this similarity to the natural viscoelastic properties and compliance with dynamic environments is important for the integration without damaging the surrounding tissue. In fact, recent researches noticed the importance of material viscoelasticity in cartilage TE, 63,64 since viscoelastic matrix with stress relaxation could mimic the mechanical microenvironment of soft tissues, and thus favor chondrogenic differentiation and a better integration with the cartilage. 65 Biocompatibility must be a priority when selecting biomaterials for TE. 66 Polyurethanes are considered to have good biocompatibility properties and are widely used for long-term medical implants, such as cardiac pacemakers and vascular grafts. 67 Since b-TPUe is a recently developed polyurethane-based 3D printing filament, no previous data concerning the possible cytotoxicity of this material on cell growth has been previously published. Results of the cytotoxicity, proliferation and viability assays showed no cytotoxic effects of b-TPUe, suggesting that it can provide an environment that supports MSCs proliferation in a same manner as PCL. 68 In fact, large spaces between the fibers allowed the adhered cells to start accommodating between the stacking fibers.
Regarding cartilage ECM production, expression of type II collagen and aggrecan, which are the main proteins of the hyaline cartilage ECM, 69  In the present study, we tried to evaluate qualitatively the macroscopic response to b-TPUe scaffolds in an in vivo environment. The lack of pain behavior, infection, edema, or macroscopic tissue inflammation during the in vivo assay with immunocompetent CD-1 mice, as well as the maintenance of shape and integrity of the scaffold, and its integration within the implantation surrounding tissue indicate the in vivo biocompatibility of b-TPUe as previously described for other 3D polyurethanes. 74 Similarly, when implanted in immunodeficient NSG mice, the deposition of collagenous fibers in both cell-free and cellladen scaffolds suggest that b-TPUe can allow in vivo GAGs and collagenous fiber production as well as PCL. Thus, it can be stated that b-TPUe polymer scaffolds showed good in vivo ECM deposition confirming the integration of b-TPUe within the host's tissue. 75

CONCLUSION
In this study, a novel elastic polyurethane-based 3D printing material, b-TPUe, was successfully used to fabricate 3D printed scaffolds with

Cell viability assay
Cell viability in the 3D printed scaffolds was determined on days 7 and 21 after bioprinting using Live/Dead™ Viability/Cytotoxicity Kit

In vitro cytotoxicity test
MSCs culture medium aliquots were conditioned with b-TPUe samples as previously described. 83

RNA isolation and real time-PCR analysis
Total cellular RNA was isolated using TriReagent (Sigma) and reverse transcribed using the Reverse Transcription System kit (Promega).
Real-time PCR was performed using the SYBR-Green PCR Master mix  Table 1.

Glycosaminoglycan quantification
The dimethylmethylene blue (DMMB) assay was used to study the glycosaminoglycans (GAGs) content as previously described. 84

Statistical analysis
Statistical calculations were performed using SPSS 13.0 software for Windows (SPSS, Chicago, IL, USA). All graphed data represent the mean ± SD from at least three replicas. Differences between treatments were tested using the two-tailed Student's t test.