Research Article: 3D‐Bioprinted Osteoblast‐Laden Nanocomposite Hydrogel Constructs with Induced Microenvironments Promote Cell Viability, Differentiation, and Osteogenesis both In Vitro and In Vivo

Date Published: November 24, 2017

Publisher: John Wiley and Sons Inc.

Author(s): Xinyun Zhai, Changshun Ruan, Yufei Ma, Delin Cheng, Mingming Wu, Wenguang Liu, Xiaoli Zhao, Haobo Pan, William Weijia Lu.


An osteoblast‐laden nanocomposite hydrogel construct, based on polyethylene glycol diacrylate (PEGDA)/laponite XLG nanoclay ([Mg5.34Li0.66Si8O20(OH)4]Na0.66, clay)/hyaluronic acid sodium salt (HA) bio‐inks, is developed by a two‐channel 3D bioprinting method. The novel biodegradable bio‐ink A, comprised of a poly(ethylene glycol) (PEG)–clay nanocomposite crosslinked hydrogel, is used to facilitate 3D‐bioprinting and enables the efficient delivery of oxygen and nutrients to growing cells. HA with encapsulated primary rat osteoblasts (ROBs) is applied as bio‐ink B with a view to improving cell viability, distribution uniformity, and deposition efficiency. The cell‐laden PEG–clay constructs not only encapsulated osteoblasts with more than 95% viability in the short term but also exhibited excellent osteogenic ability in the long term, due to the release of bioactive ions (magnesium ions, Mg2+ and silicon ions, Si4+), which induces the suitable microenvironment to promote the differentiation of the loaded exogenous ROBs, both in vitro and in vivo. This 3D‐bioprinting method holds much promise for bone tissue regeneration in terms of cell engraftment, survival, and ultimately long‐term function.

Partial Text

Traditional strategies for bone tissue engineering are based on the facilitation of cell growth into engineered interconnecting scaffolds to generate a functional tissue construct for the reestablishment of structure and function in damaged bone tissues.1 However, the realization of the desired levels of cell deposition and cell distribution in 3D scaffolds remains a great challenge. Recent developments in the tempospatial specific 3D‐bioprinting of cells and inks show promise for a new approach in bone tissue engineering.2 With this technology, delicately tailored cell‐laden tissue constructs have been reportedly constructed to regenerate bone tissue. Zhang and co‐workers recently reported on bone mesenchymal stem cells (BMSCs)‐laden gelatin/sodium alginate/carboxymethyl chitosan hydrogel scaffolds prepared through a 3D‐bioprinting method. The prepared scaffolds exhibited good mechanical properties and favorable cytocompatibility, with cell viability of over 85% postprinting.3 Neufurth et al. encapsulated bone‐related SaOS‐2 cells into a biologically inert sodium alginate matrix and used a second layer containing polyP·Ca2+ to cover the bioprinted cell laden scaffold. The encapsulated cells exhibited a good proliferation rate and mineralization ability.4 These previous studies were mainly devoted to imitating the native structure of bone tissues and improving the viability of 3D‐bioprinted cells in the short term. However, whether or not the 3D‐bioprinted cells can realize the functional features of bone tissue after in vivo implantation still remains unknown. Indeed, the long‐term in vivo evaluation of most of the previously reported cell‐laden scaffolds for bone regeneration is deficient and limited, due to the lack of ideal bio‐inks for 3D‐bioprinting to favorably support cell growth and development both in the short and long terms.

In this study, ROB‐laden nanocomposite hydrogel constructs were fabricated by a two‐channel 3D‐bioprinting method by alternately extruding two bio‐inks (A and B). Bio‐ink A, PEG–Clay prehydrogel solution, offered a suitable viscosity for facilitating the process of 3D bioprinting, oxygen and nutrient delivery, and ultimately cell growth after crosslinking. The uniform distribution of the nanoclay in the PEG matrix not only enhanced the hydrogel mechanical properties but also rendered them more conducive to cell adhesion and proliferation than pure PEG hydrogels. Furthermore, the release of Mg2+ and Si4+ bioactive ions from the PEG–Clay scaffolds formed an induced microenvironment which stimulated the osteogenic differentiation of ROBs, to benefit bone regeneration. Simultaneously, bio‐ink B, based on HA, was applied as a vector to accurately and uniformly deposit an ROB load into the 3D‐printed scaffolds. The inclusion of HA not only guaranteed cell viability during 3D‐bioprinting with good distribution within the scaffolds, but its slow dissolution allowed for the gradual release of cells. Moreover, compared with the one‐channel method of 3D‐bioprinting, our two‐channel 3D‐bioprinting approach not only enhanced cell viability but also encouraged better cell spreading and proliferation.25

In summary, we successfully fabricated an osteoblast‐laden nanocomposite hydrogel construct via a two‐channel 3D‐bioprinting method. One channel carried bio‐ink A, PEG–Clay prehydrogel solution, which was suitably viscous to facilitate the 3D‐bioprinting process and was conducive to the delivery of oxygen and nutrients and cell growth after crosslinking. The other channel guided the accurate delivery of cells into the 3D scaffolds, using ROBs encapsulated in 20% HA solution. The HA component served to not only protect the ROBs from UV damage during the crosslinking process but also guaranteed uniform distribution and cell viability (more than 95% after 1 d). Furthermore, ROBs within the bioprinted scaffold showed better proliferation and differentiation than the same number of ROBs seeded on 3D PEG–Clay scaffolds. In tibia repair and ectopic osteoinduction experiments, ROB‐laden PEG–Clay scaffolds showed excellent osteogenic potential, due to the induced environment formed around the PEG–Clay scaffolds which was conducive to ROBs differentiation. This study offers a viable new approach for 3D‐bioprinting for the construction of bone substitutes in tissue regeneration.

Materials: PEG (Mw = 4K and 10K, Sigma‐Aldrich, St. Louis, USA), acryloyl chloride (98%, TCI, Shanghai, China), triethylamine (99%, TCI, Shanghai, China), diethyl ether (Lingfeng Chemical Reagent Company, Shanghai, China), 2‐hydroxy‐2‐methyl‐1‐phenyl‐1‐propanone (IRGACURE 1173, 98%, Sigma‐Aldrich, St. Louis, USA), Laponite XLG ([Mg5.34Li0.66Si8O20(OH)4]Na0.66; BKY, Wesel, Germany), and HA (Mw = 350K, TCI, Shanghai, China) were all used as received. All other chemicals and solvents were analytical reagents and were purchased from Lingfeng Chemical Reagent Company (Shanghai, China) and used as received.

The authors declare no conflict of interest.