(A-33) Customized Robotic Arm In Vivo Bioprinter for Patient-Specific Reconstruction of Skeletal Muscle Defects

Introduction:: Introduction: Large muscle defects surpass tissue's innate regeneration capacity, so additional treatment is required for better healing outcomes. However, despite the available tissue-engineered products to replicate soft constructs, most are challenging to implant into the patient or fail to depict the hierarchical organization of skeletal muscle tissue. 3D-bioprinting is a fabrication method that could facilitate the reconstruction of soft tissue architecture and its implantation through direct in vivo bioprinting. Here, we upgraded an easy-to-operate robotic arm into an open-sourced bioprinter with customizable extruder morphologies and a temperature control system. Our system reduces the cost limitations in equipment, infrastructure, personnel, software requirements, and imaging tools of current bioprinting methods while providing an accurate, flexible, and real-time strategy for the automatic deposition of a wide variety of biomaterials aiding the opportune reconstruction of large skeletal muscle defects.


Materials and Methods:: Materials and Methods: A extrusion jacket for biomaterial temperature control was designed in CAD software and 3D printed in a Form 3B 3D printer. A copper pipe was included to improve heat transfer between the coolant system and the biomaterial. The temperature of the coolant was regulated using a Peltier plate adjunct to a copper cooling block. The extrusion rate was manipulated by varying the voltage of a micromotor that controlled a plush plate to move the biomaterial syringe plunger. Temperature range; time to achieve desired temperatures; temperature stability over time; and variation between set and actual temperature were measured to characterize the system. Resolution and printing speed were measured while the system was on to consider possible changes in the robotic arm performance due addition of the new extruder system. Rheological and filament extrusion properties of biomaterials like gelatin, GelMA, and Pluronic F-127 were evaluated to define optimal printing parameters like temperature, extrusion voltage, and printing speed. The system was linked to open-sourced software Ultimaker Cura to directly upload 3D models and generate STL files for spatial and temporal control of the biomaterials. A 3D construct from a bioink composed of 50% platelet-rich plasma (PRP), 4% GelMA, and 10 million cells/mL of C2C12 immortalized mouse myoblasts (CRL1772TM, ATCC) was printed with the system, and cell viability and differentiation were studied. To evaluate printing capabilities, a 3D scanner was used to create 3D models of simulated irregular defects in ex vivo tissue samples and assess in vivo printing of hydrogels.


Results, Conclusions, and Discussions:: Results and Discussion: The customized 3D-printing system was able to control the deposition of temperature-dependent biomaterials spatially and temporarily (Fig. 1). The coolant extruder jacket coupled with the copper pipe (Fig. 1B) allowed reliable heat transfer at different setting temperatures (Fig. 1C), making the system compatible with the extrusion temperatures of most bioinks. The addition of the customized extrusion jacket did not significantly affect the resolution capabilities of the robotic arm alone (Fig. 1D). The system was able to extrude gelatin-based materials (Fig. 1E-F) among other polymers (Fig. G-H), showing the effect of temperature in fiber uniformity and diameter. Once the ideal extrusion temperatures were selected, the optimum printing parameters were tested, such as extrusion voltage, printing speed, initial layer height, and z-stack (Fig. 1I-J). Both biomaterials demonstrated optimal printability under different infill ranges (Fig. 1K). The robotic arm system was able to reliably control the deposition and form complex 3D structures either designed in CAD software or from scanned defects (Fig. 1L). Cell viability and differentiation of 3D printed constructs were assessed to demonstrate the bioprinter's ability to deposit bioinks without significantly affecting the cells' capacity to survive, proliferate, and form aligned muscle fibers (Fig. 1M).



Conclusions: Our upgraded robotic system integrates a new translatable strategy for in vivo bioprinting of a broad range of temperature-dependent bioinks, capable of assembling elaborated architectures, such as thick tissue defects. The system is versatile, scalable, and adaptable to patient-specific needs shortening the gap between tissue-engineered products and clinical environments.


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