Elsevier

Bioprinting

Volume 18, June 2020, e00083
Bioprinting

Novel bioinks from UV-responsive norbornene-functionalized carboxymethyl cellulose macromers

https://doi.org/10.1016/j.bprint.2020.e00083Get rights and content

Abstract

3D printing has significantly progressed in the past decade and become a potentially powerful biomanufacturing approach for tissue and organ printing. Availability of diverse hydrogel-based bioink formulations, particularly bioinks allowing biochemical functionalization, stimuli responsiveness, and control over mechanical and degradation properties are crucial for bioprinting to reach its full potential. In this study, we report two novel bioink platforms from norbornene modified cellulose-based macromers, either with an amide, norbornene CMC (NorCMC), or an ester linker, carbic (norbornene) functionalized CMC (cCMC). Both of the bioink formulations show autogelation in the absence of UV light, which allow us to adjust the viscosity of the ink formulation. Bioinks rapidly form cell-laden hydrogels when exposed to UV light due to photoinduced thiol-ene crosslinking mechanism. Bioink viscosity and printability as well as bioprinted construct mechanics are controlled by bioink concentration and thiol to norbornene (T:NB) ratio. Human mesenchymal stem cells (hMSCs), NIH 3T3 fibroblasts, and human umbilical vein endothelial cells (HUVECs) are successfully bioprinted using our novel bioink formulations. Considering the high abundance, low cost, ability to selectively tether molecules or control crosslinking properties, norbornene modified cellulose-based bioink platforms have a significant potential to enable 3D bioprinted constructs with increased complexity.

Introduction

Three-dimensional (3D) bioprinting is an emerging field with a significant potential to create custom-designed and patient-specific “living” constructs using a patient’s own medical images and cells [[1], [2], [3], [4]]. 3D bioprinting could potentially eliminate organ shortage [[5], [6], [7], [8]] and enable development of patient-specific tissue models for personalized drug screening [[9], [10], [11], [12], [13]]. A recent frontier is in situ bioprinting for reparative or regenerative therapy, in which a living tissue is printed directly at the site of an injury or a defect [[14], [15], [16]]. Despite the strong potential of bioprinting and recent advancements in the bioprinting technology, there is a notable lack of diversity in bioinks which significantly hinders the widespread use of bioprinting.

3D bioprinting enables layer-by-layer manufacturing of a living construct from bioinks, which are bioprintable formulations composed of cells that are usually supported with a hydrogel [17]. The requirement for live cell printing significantly limits the number of additive manufacturing technologies that are suitable for bioprinting [18]. Bioprinting technologies include extrusion-based direct ink writing (DIW), droplet-based inkjet printing, and light-based approaches, including projection stereolithography and laser-induced forward transfer (LIFT) [[18], [19], [20]]. DIW is the most commonly used technique due to its availability, affordability, and ease of use. In DIW, a bioink formulation is extruded through a blunt needle to form a self-supporting structure. In this process, the bioink should meet the basic requirements for extrusion-based bioprinting [[21], [22], [23]], such that it should (i) have a suitable viscosity, i.e., low enough for easy extrusion yet high enough for formation of self-supporting layers post-printing to minimize sagging, usually in the range of 30 to 6 ​× ​107 ​mPa ​s, and (ii) allow printing of living cells and support high viability (>90%) [17,21,22]. In addition, the bioink and its degradation products should be cytocompatible and should not induce an inflammatory response when implanted [22,24].

Most commonly used bioinks are formulated from cell-laden hydrogels due to their high water content and properties mimicking native tissue microenvironment [25,26]. A variety of hydrogel-based bioinks have been developed from synthetic (such as Pluronic [27,28] and poly(ethylene glycol) [29]), or natural (gelatin [[30], [31], [32]], hyaluronic acid [33,34], alginate [33,35], chitosan [36], collagen [37,38], fibrin [39], and silk [40,41]) polymers/macromers, or decellularized tissue materials (e.g., heart, bone, liver, pancreas, etc.) [42,43]. The building blocks of these formulations are usually modified to allow tunable viscosity and shape fidelity during printing process. Although innovative approaches have been developed to control printability including pre-crosslinking to control flow [29] or rapid crosslinking during or after-printing [44,45], or designing shear thinning formulations [34,46], novel bioink formulations are still needed to broaden the currently available bioink “library” and to develop stimuli responsive bioinks enabling control of bioprinted construct properties post-printing.

In this study, we focused on carboxymethyl cellulose (CMC), a commonly used cellulose derivative. Cellulose is one of the most abundant and renewable natural polymers [47,48]. As a natural polymer, cellulose is inherently bioactive, biodegradable, and biocompatible [47]. The hydroxyl groups on its backbone structure allows functionalization of cellulose to tune its properties [49]. When compared to cellulose, CMC is highly soluble in water due to its carboxyl groups [50] making it an attractive building block for hydrogels. CMC-based hydrogels have been developed utilizing a wide range of crosslink mechanisms including physical and chemical crosslinking [13]. For instance, Nie et al. reported CMC-based hydrogels by crosslinking sodium CMC with AlCl3, and studied the effects of crosslinker, CMC concentration and temperature on hydrogel stiffness and degradation [16]. Chemically crosslinked CMC-based hydrogels have been developed using irradiation-initiated [[17], [18], [19]], photo-initiated radical [20,21], enzymatic [22], and epoxide-opening reactions [23]. For instance, methacrylated CMC is synthesized to allow photo-initiated radical reaction to fabricate CMC-based hydrogels. These hydrogels were used to facilitate chondrogenic differentiation of encapsulated human mesenchymal stem cells (hMSCs) encapsulated within the hydrogels [21].

Cellulose has been used as a filler, or as a component, in ink formulations [[51], [52], [53], [54]]. Majority of the studies focused on cellulose/alginate based ink formulations, utilizing a range of cellulose derivatives (nanofibrillated cellulose, nanocellulose, and methylcellulose) and taking advantage of physical crosslinking ability of alginate with CaCl2 [[55], [56], [57], [58], [59], [60]]. For instance, nanocellulose-alginate based bioinks were developed for 3D bioprinting of human chondrocyte-laden hydrogels for cartilage regeneration [55,61]. Muller et al. developed alginate sulfate/nanocellulose bioinks but reported significantly compromised proliferation ability of chondrocytes during printing process [58]. Markstedt et al. developed bioinks from cellulose nanofibrils mixed with xylan for crosslinking [62]. Most recently, methylcellulose (MC)-based hydrogels were printed utilizing the sol-gel transition, or lower critical solution temperature (LCST), allowing printing of MC-based hydrogels at 21 ​°C with high cell survival (80%) post-printing [63]. Li et al. developed highly thixotropic inks from alginate/methylcellulose blend hydrogels, and showed that the treatment of the printed constructs with trisodium citrate (TSC) significantly enhanced the interfacial bonding between printed layers [64]. Finally, Lewis group developed hydrogel composite inks composed of soft acrylamide matrix supported with cellulose fibrils, and crosslinked with clay [51]. They were able to selectively align cellulose nanofibrils during the printing process to develop 3d printed structures with anisotropic stiffness, which led to shape change on immersion in water. In this study, we develop novel photocurable bioink formulations directly from carboxymethyl cellulose (CMC) eliminating the need for alginate or other additives/components.

Light-induced free radical polymerization of methacrylates or acrylates is a widely used approach in designing photoreactive bioinks, yet this reaction is not specific and leads to formation of a heterogenous network composed of kinetic chains. Thiol-norbornene photo-click chemistry is specific to norbornene and thiyl radicals (i.e., radicals from thiols) as compared to norbornene radicals (its own radicals) or nonradical thiols [65,66]. This is important to achieve selectivity in crosslinking (crosslinkers containing multi-thiols) and tethering of biomolecules (containing mono-thiols). This mechanism ensures a more homogeneous crosslinking in a controllable manner [65,67,68]. Natural (such as alginate [69], hyaluronic acid [68,70], and gelatin [71,72]) and synthetic polymers (such as poly(ethylene glycol) [67,72,73]) have been modified with norbornene group to fabricate photocurable, cell-laden hydrogels. Recently, CMC has been modified with norbornene groups [50,74] to develop renewable hydrogels. Gramlich group recently demonstrated high cell viability of encapsulated stem cells within norbornene functionalized CMC [75]. Motivated by these recent results, we focused on developing novel bioink formulations from norbornene functionalized CMC.

In this study, we report two novel stimuli responsive bioink platforms from CMC for extrusion based bioprinting. CMC is functionalized with thiol-ene reactive norbornene (Nor) with an amide, norbornene CMC (NorCMC), or an ester linker, carbic (norbornene) functionalized CMC (cCMC). CMC was chosen as the building block for both of our bioink platforms due to its high availability and low cost, and high solubility in water. Light-induced thiol-ene click chemistry enabling norbornene was selected as the functional group to achieve selective crosslinking and selective tethering of biomolecules. Printability of the bioink platforms was determined by the thiol-Nor ratio for each macromer concentration. CMC-based bioink platform allows tunable printability, stiffness and high viability of bioprinted cells, and broadens the range of currently available bioink platforms.

Section snippets

Polymer synthesis

The macromers, cCMC and NorCMC, were synthesized according to methods developed previously for cCMC [74] and NorCMC [75]. To synthesize cCMC, CMC (90KDa, 0.7 carboxymethyl groups per anhydroglucose unit, Sigma) was dissolved in reverse osmosis (RO) water at 1% (w/v). Then, 7.26 ​g of cis-endo-5-norbornene-2,3-dicarboxylic anhydride (carbic anhydride, TCI) was added to the CMC solution (per gram of CMC). The reaction was maintained for 2 ​h while the pH of the reaction was adjusted at the range

Bioink formulations

In this study, two distinct bioink formulations were developed from norbornene functionalized CMC (Fig. 1), either with an amide, NorCMC, or an ester linker, cCMC. 1H NMR results confirmed 30% and 20% functionalization for cCMC and NorCMC, respectively (Supplementary Fig. S1). The compositions of the bioink formulations are given in Table 1. Bioinks were formulated at 15% cCMC and 10% NorCMC, with thiol to norbornene ratio (T:NB) equal to (1:4), (1:2), and (1:1).

Rheological test results

The initial shear viscosities of

Discussion

Here, we report novel bioink formulations from norbornene modified, cellulose-based macromers for the first time. Cellulose-based materials are promising candidates as bioinks due to their inherent bioactivity, abundance and low cost. In this study, two distinct macromers were developed by functionalizing CMC with an amide (NorCMC) or an ester linker (cCMC) with 30% and 20% functionalization for cCMC and NorCMC, respectively. These degrees of functionalization were selected because hydrogels at

Conclusions

In conclusion, we report a two norbornene-modified cellulose-based macromers as novel bioink materials. Polymer concentration and thiol:norbornene ratio (T:NB) were optimized to prepare printable bioink formulations from cCMC (with (T:NB) = (1:2) and (1:4)) and NorCMC (with (T:NB) = (1:4)). All of the ink formulations were able to encapsulate cells (hMSCs, NIH 3T3 fibroblasts, and HUVECs), and to be printed as cell-laden scaffolds. We believe that these two cellulose-based macromers broaden the

CRediT authorship contribution statement

Shen Ji: Writing - original draft, Formal analysis, Data curation. Alperen Abaci: Formal analysis. Tessali Morrison: Formal analysis. William M. Gramlich: Supervision, Writing - review & editing. Murat Guvendiren: Supervision, Writing - review & editing, Data curation, Formal analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work is partially funded by the National Science Foundation Award Number DMR-1714882 (M.G.), and by the Faculty Seed Grant (M.G.) from the Center for Engineering MechanoBiology (CEMB), an NSF Science and Technology Center, under grant agreement CMMI: 15-48571. Use of the Bruker Avance NEO 500MHz nuclear magnetic resonance (NMR) spectrometer was supported by the National Science Foundation under grant CHE-1828408. Any opinions, findings, and conclusions or recommendations expressed in this

References (77)

  • J. Schurz

    A bright future for cellulose

    Prog. Polym. Sci.

    (1999)
  • C. Chang et al.

    Cellulose-based hydrogels: present status and application prospects

    Carbohydr. Polym.

    (2011)
  • A. Cataldi et al.

    Polyvinyl alcohol reinforced with crystalline nanocellulose for 3D printing application

    Mater. Today Commun.

    (2018)
  • L. Li et al.

    3D bioprinting of cellulose with controlled porous structures from NMMO

    Mater. Lett.

    (2018)
  • H. Martínez Ávila et al.

    3D bioprinting of human chondrocyte-laden nanocellulose hydrogels for patient-specific auricular cartilage regeneration

    Bioprinting

    (2016)
  • K. Markstedt et al.

    3D bioprinting human chondrocytes with nanocellulose-alginate bioink for cartilage tissue engineering applications

    Biomacromolecules

    (2015)
  • N. Contessi Negrini et al.

    3D printing of methylcellulose-based hydrogels

    Bioprinting

    (2018)
  • W.M. Gramlich et al.

    Synthesis and orthogonal photopatterning of hyaluronic acid hydrogels with thiol-norbornene chemistry

    Biomaterials

    (2013)
  • H.W. Ooi et al.

    Thiol-ene alginate hydrogels as versatile bioinks for bioprinting

    Biomacromolecules

    (2018)
  • B.D. Fairbanks et al.

    Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility

    Biomaterials

    (2009)
  • L. Archer

    3D printing with living inks

    Science

    (2017)
  • H. Cui et al.

    3D bioprinting for organ regeneration

    Adv. Healthc. Mater.

    (2016)
  • A.B. Dababneh et al.

    Bioprinting technology: a current State-of-the-Art review

    J. Manuf. Sci. Eng.

    (2014)
  • S.V. Murphy et al.

    3D bioprinting of tissues and organs

    Nat. Biotechnol.

    (2014)
  • H.W. Kang et al.

    A 3D bioprinting system to produce human-scale tissue constructs with structural integrity

    Nat. Biotechnol.

    (2016)
  • B. Grigoryan et al.

    Multivascular networks and functional intravascular topologies within biocompatible hydrogels

    Science

    (2019)
  • N. Noor et al.

    3D Printing of personalized thick and perfusable cardiac patches and hearts

    Adv. Sci.

    (2019)
  • C.Y. Liaw et al.

    Current and emerging applications of 3D printing in medicine

    Biofabrication

    (2017)
  • C.-Y. Liaw et al.

    Engineering 3D hydrogels for personalized in vitro human tissue models

    Adv. Healthc. Mater.

    (2018)
  • J. Jang et al.

    3D Printed tissue models: present and future

    ACS Biomater. Sci. Eng.

    (2016)
  • L.M. Norona et al.

    Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis

    PloS One

    (2019)
  • M. Albanna et al.

    In situ bioprinting of autologous skin cells accelerates wound healing of extensive excisional full-thickness wounds

    Sci. Rep.

    (2019)
  • N. Ashammakhi et al.

    In situ three-dimensional printing for reparative and regenerative therapy

    Biomed. Microdevices

    (2019)
  • V. Keriquel et al.

    In situ printing of mesenchymal stromal cells, by laser-assisted bioprinting, for in vivo bone regeneration applications

    Sci. Rep.

    (2017)
  • J. Groll et al.

    A definition of bioinks and their distinction from biomaterial inks

    Biofabrication

    (2018)
  • T.I. Ozbolat et al.

    Evaluation of bioprinter technologies

    Addit. Manuf.

    (2016)
  • S. Ji et al.

    Recent advances in bioink design for 3D bioprinting of tissues and organs

    Front. Bioeng. Biotechnol.

    (2017)
  • K. Dubbin et al.

    Quantitative criteria to benchmark new and existing bio-inks for cell compatibility

    Biofabrication

    (2017)
  • Cited by (26)

    • Recent advancements in 3D bioprinting technology of carboxymethyl cellulose-based hydrogels: Utilization in tissue engineering

      2021, Advances in Colloid and Interface Science
      Citation Excerpt :

      In this regard, hydrogel-based bioinks with significant antibacterial and antibiofilm properties in 3D printing technology have been introduced to replace various organs [92]. Guvendiren et al. investigated the application of two series of norbornene functionalized CMC-based hydrogels by the use of extrusion-based bioprinting and applied them in TE [93]. In this investigation, two diverse structures were designed based on CMC, which include the applying of norbornene functionalized (NorCMC) and carbic functionalized (cCMC) with 30% and 20% functionalization as linkers, respectively.

    View all citing articles on Scopus
    View full text