Micro-precise spatiotemporal delivery system embedded in 3D printing for complex tissue regeneration

Abstract

Three dimensional (3D) printing has emerged as an efficient tool for tissue engineering and regenerative medicine, given its advantages for constructing custom-designed scaffolds with tunable microstructure/physical properties. Here we developed a micro-precise spatiotemporal delivery system embedded in 3D printed scaffolds. PLGA microspheres (μS) were encapsulated with growth factors (GFs) and then embedded inside PCL microfibers that constitute custom-designed 3D scaffolds. Given the substantial difference in the melting points between PLGA and PCL and their low heat conductivity, μS were able to maintain its original structure while protecting GF's bioactivities. Micro-precise spatial control of multiple GFs was achieved by interchanging dispensing cartridges during a single printing process. Spatially controlled delivery of GFs, with a prolonged release, guided formation of multi-tissue interfaces from bone marrow derived mesenchymal stem/progenitor cells (MSCs). To investigate efficacy of the micro-precise delivery system embedded in 3D printed scaffold, temporomandibular joint (TMJ) disc scaffolds were fabricated with micro-precise spatiotemporal delivery of CTGF and TGFβ3, mimicking native-like multiphase fibrocartilage. In vitro, TMJ disc scaffolds spatially embedded with CTGF/TGFβ3-μS resulted in formation of multiphase fibrocartilaginous tissues from MSCs. In vivo, TMJ disc perforation was performed in rabbits, followed by implantation of CTGF/TGFβ3-μS-embedded scaffolds. After 4 wks, CTGF/TGFβ3-μS embedded scaffolds significantly improved healing of the perforated TMJ disc as compared to the degenerated TMJ disc in the control group with scaffold embedded with empty μS. In addition, CTGF/TGFβ3-μS embedded scaffolds significantly prevented arthritic changes on TMJ condyles. In conclusion, our micro-precise spatiotemporal delivery system embedded in 3D printing may serve as an efficient tool to regenerate complex and inhomogeneous tissues.

Effect of dose and release rate of CTGF and TGFβ3 on avascular meniscus healing

“…High (1,000 ng/ml) and low (100 ng/ml) doses of CTGF and 10 mg of fast (75:25, lactic and glycolic acids) and slow (85:15) TGFβ3‐encapsulated PLGA μS were applied. A single dose PLGA μS was applied to all the groups as optimized from our previous works in order to focus on the effect of release. Then, the meniscus explants were placed on top of monolayer‐cultured P2–3 synovial MSCs at 80– 90% confluence.…”

“…Then, the meniscus explants were placed on top of monolayer‐cultured P2–3 synovial MSCs at 80– 90% confluence. Supplements for fibrogenic and chondrogenic differentiation were applied as described in our recent publication . At 8 weeks, fibrocartilaginous tissue integration was evaluated by H&E and Saf‐O staining.…”

Spatiotemporal control of growth factors in three-dimensional printed scaffolds

“…When applied in vivo , 4-week TMJ disc explant samples displayed recovery of the defect site, being mechanically sound with a highly-organized fibrocartilage structure mirroring native tissue. (80) In a separate study, the authors also demonstrated that in addition to heterogeneous tissue formation after CTGF and TGF-β3 release, gene upregulation was highly dependent on the dose of growth factor used, with the 100mg microsphere/g scaffold dose yielded significantly increased collagen I and II and aggrecan expression than the 50mg/g dose. (81)…”

“…Tarafder et al showcased another multi-cartridge printing approach towards temporomandibular joint (TMJ) disc regeneration in which alternating strands of poly(ε-caprolactone) (PCL) were printed with poly(lactic-co-glycolic acid) (PLGA) microspheres containing either connective tissue growth factor (CTGF) or TGF-β3. (32) Other extrusion and inkjet techniques have utilized multi-printhead systems to great success for printing bioinks with distinct temperature, pneumatic pressure, and nozzle size requirements, such as the integrated tissue-organ printer system developed by Kang et al(33) Ultimately, single-printhead systems may be most suitable for the printing of bioinks with highly similar material compositions, while multi-printhead systems on the other hand will benefit from improvements to speed of printhead switching and the ability to maintain continuous printing when switching between bioinks with print conditions.…”

A review of in-vitro fibrocartilage tissue engineered therapies with a focus on the temporomandibular joint

“…Tarafder, et al in 2016 studied the use of micro-precise 3D printed spatiotemporal delivery systems embedded with PLGA microspheres and growth factors (Tarafder et al, 2016). Specifically, MSCs from either human bone marrow (100,000 cells/mL) or rabbit TMJ synovium (200,000 cells/mL) were seeded onto a 3D printed scaffold with a similar anatomical shape and contour to a native TMJ disc.…”