The one-step fabrication of porous hASC-laden GelMa constructs using a handheld printing system

Tissue engineering requires highly porous, biocompatible, biodegradable, and mechanically stable substitutes. Cell-encapsulation methods like 3D bioprinting offer efficient cell positioning and microenvironment control. However, non-porous cell-laden struts in 3D-bioprinted constructs cause cell necrosis due to poor nutrient and oxygen transport. Existing methods for creating porous cell-laden constructs, such as using sacrificial beads or whipping techniques, have limitations in porosity, homogeneity, or complexity. This study aimed to develop a simple, one-step method to fabricate highly porous (above 90%) human adipose stem cell (hASC)-laden constructs using a handheld 3D printer equipped with a novel air-bubbling system. This system avoids the need for separately prepared porous hydrogel solutions and allows for simultaneous mixing of cells and air during printing. The use of a lightweight handheld printer facilitates in situ fabrication and implantation, offering advantages over conventional 3D printers. The study evaluated the impact of various processing parameters (mesh filter size and number, air-bioink volume ratio, and flow rate) on air bubble formation, pore size, and foamability. In vitro cellular activities (cell viability, proliferation, F-actin activity, and myogenic differentiation) were assessed using hASCs. The feasibility of the method for muscle regeneration was evaluated in vivo using a mouse volumetric muscle loss (VML) model.

The literature extensively covers bioprinting cell-laden structures for tissue engineering applications. However, limitations exist due to relatively poor metabolic activity in non-porous constructs. High porosity is crucial for successful vascularization and nutrient/waste transport. Existing methods for creating porous cell-laden constructs include using non-toxic sacrificial beads (e.g., gelatin beads), which have limited porosity (≈70%). Protein-based bioinks with a whipping method offer improved cellular activity and vascularization but require surfactants and complex multi-step processes. Air injection methods simplify the process but lack precise pore size control. Aqueous two-phase emulsion (ATPE), gas foaming, and microfluidic systems have also been employed, but these techniques present complexities in the process or necessitate sacrificing materials. This paper proposes a novel method to overcome these challenges, offering a simpler and more effective approach for fabricating highly porous cell-laden constructs.

Gelatin methacryloyl (GelMa) was synthesized by methacrylation of gelatin, followed by dialysis and lyophilization. GelMa solutions of varying concentrations (5, 10, and 15% w/v) were prepared with lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photoinitiator. Rheological properties were measured using a rotational rheometer to determine complex viscosity. A custom-built handheld 3D printer was designed, consisting of two channels for air and GelMa bioink, and several microscale mesh filters to create microbubbles. The printer's parameters, including mesh filter size (three sizes, FS-1 to FS-3), filter number, air-bioink volume ratio, and flow rate were systematically altered to optimize the porous structure. The hASCs (or C2C12 cells for certain tests) were mixed into the GelMa bioink. Porous GelMa constructs were fabricated, and their pore size, foamability, and cell viability were assessed using optical microscopy and scanning electron microscopy (SEM). For in vitro evaluation, hASCs-laden porous GelMa constructs and non-porous controls were cultured in a polydimethylsiloxane (PDMS) mold. Cell proliferation was measured using the MTT assay. Cell viability was assessed with live/dead staining and confocal microscopy. Cell morphology and F-actin activity were assessed using phalloidin staining and confocal microscopy. Myogenic activity was evaluated using MHC immunofluorescent staining. For in vivo evaluation, a volumetric muscle loss (VML) model was created in mice by excising 40% of the tibialis anterior (TA) muscle. The porous hASC-laden GelMa constructs were directly printed into the defect region using the handheld 3D printer and then exposed to UV light for crosslinking. Control groups included a sham group, a defect group (no treatment), an acellular foam group, and an hASC-printed group (conventionally bioprinted hASC-laden GelMa). Muscle function was evaluated using grip strength and latency to fall tests over four weeks. Histological staining (H&E and Masson's Trichrome) and immunofluorescent staining (MHC, MRPL11, HLA-A, LAMA1) were performed on harvested TA muscles to analyze muscle regeneration and fiber formation.

The study successfully demonstrated the fabrication of highly porous (≈97%) hASC-laden GelMa constructs using a handheld 3D printer. The processing parameters significantly affected the pore size and foamability of the constructs. Specifically, smaller filter sizes and increased filter numbers led to smaller pore sizes and higher foamability. A bioink-to-air ratio of 1:3 yielded optimal foamability (≈93%). Increasing the flow rate beyond 3 mL/s reduced cell viability. In vitro studies demonstrated high cell viability (≈98%) in the porous constructs, with significantly higher cell proliferation compared to non-porous controls. The porous structure promoted better nutrient transport, as shown by higher cell viability in the bottom region compared to non-porous constructs. Cells in porous constructs exhibited more active F-actin cytoskeletal expansion and higher myogenic differentiation (MHC expression) compared to controls. In vivo experiments showed significantly improved muscle function (grip strength and latency to fall) and muscle regeneration in mice with VML treated with the porous hASC-laden GelMa constructs, comparable to the sham group. The hASC-laden constructs demonstrated higher myofiber area, smaller fibrotic area, and increased expression of MRPL11, HLA-A, and LAMA1, indicating successful hASC differentiation into myofibers.

The findings demonstrate that the novel handheld 3D printing system successfully fabricates highly porous, cell-laden GelMa constructs with a high degree of cell viability and efficient muscle regeneration. The improved nutrient and oxygen transport in the porous structure promotes cell survival and proliferation, highlighting its advantage over non-porous constructs. The enhanced myogenic differentiation and improved muscle function in the VML model validate the constructs' potential for muscle tissue engineering. The simplicity and portability of the handheld 3D printer offer significant advantages for in situ tissue regeneration, particularly in time-sensitive clinical settings. The ability to precisely control pore size and foamability through manipulation of processing parameters enables tailoring the constructs to specific tissue engineering applications.

This study presents a novel and efficient method for fabricating highly porous, cell-laden GelMa constructs using a handheld 3D printer. The method enables in situ fabrication, simplifies the process compared to existing techniques, and yields constructs with superior cell viability and muscle regeneration capabilities. Future research could focus on further optimizing the printing parameters for even finer control over pore architecture and exploring the use of this technology for regenerating other tissue types.

While the study demonstrated promising results, some limitations should be noted. The pore size distribution in the fabricated constructs was not perfectly uniform, although it was within an acceptable range. The in vivo study utilized a mouse model, and further studies in larger animal models are needed to validate the results before clinical translation. Long-term studies are also needed to assess the constructs’ long-term biocompatibility and efficacy.