3D Printed Multi-Tissue Implants for Enhanced Bone Regeneration

Introduction: Our study aims to develop a 3D in vitro musculoskeletal tissue implant that simulates the synergistic relationship between bone, periosteum, and muscle tissues, focusing on their role in tissue regeneration. In vitro cultured musculoskeletal tissues have numerous critical applications, including tissue-engineered implants for musculoskeletal regeneration, disease modeling for pathological mechanistic studies, and preclinical drug testing platforms. The intricate architecture of the musculoskeletal system, consisting of bone, muscle, and connective tissues, plays a pivotal role in maintaining structural integrity and facilitating tissue regeneration. However, existing in vitro models often lack a comprehensive representation of these interconnected components, limiting their ability to recapitulate the dynamic cellular tissue regeneration processes observed in vivo. We aimed to develop a functional muscle-bone implant for treating damaged musculoskeletal tissues, utilizing bioprinting technology and a customized bioreactor system. These muscle-bone implants can also serve as an in vitro model for a comprehensive understanding of the growth and development mechanisms of musculoskeletal organs and for testing various drugs for treating musculoskeletal diseases and injuries.

Materials and

Methods: We developed three different bio-inks for the bone, periosteum, and muscle tissue parts, ensuring they supported cytocompatibility, printability, and post-fabrication structural integrity. We began by testing the bioinks' printability, simultaneously conducting cell retention and viability tests to determine whether each bioink composition supports cell viability during tissue maturation, followed by observations of matrix degradation over several weeks. Human mesenchymal stem cells (MSCs) were embedded in a 3D Gelatin methacrylate (GelMA) hydrogel matrix with bone minerals (calcium phosphate nanoparticles) to form the bone tissue component. A GelMA hydrogel layer with endothelial cells (ECs) and MSCs was added to simulate the periosteum. Additionally, GelMA hydrogel with human myoblasts was layered on top of the periosteum to create a multi-tissue structure of bone, periosteum, and muscle (Figure 1a). This multi-tissue structure was fabricated using 3D printing and layer-by-layer deposition techniques (Figure 1b-c). Culture media conditions were optimized to support the osteogenic differentiation of MSCs, myogenic differentiation of myoblasts, and vascular formation of ECs simultaneously. The engineered multi-tissue structures were cultured in this optimized media for 2-3 weeks under static culture conditions. They were then mechanically stimulated in custom bioreactors to mature into functional musculoskeletal systems. The matured tissues were then fixed and cryosectioned for immunohistostaining and imaging. The tissue structures were also implanted into mice with a drilled hole defect, and the regeneration of bone at the implant site was observed using microCT for 30 days post-surgery.

Results, Conclusions, and Discussions: We engineered in vitro musculoskeletal tissue structures with varying compositions to analyze the impact of multi-tissue interactions on the regeneration of defective bone. The 3D-printed multi-tissue structure consists of: i) bone tissue containing bone minerals (black dots observed in cryosectioned images in Figure 1d) and MSCs, ii) periosteum with a vascularized layer (red-labeled ECs forming a vascular network, MSCs labeled with green in a cryosection image of Figure 1e and top-view widefield image of Figure 1g), and iii) muscle tissue containing myoblasts (top-view widefield image of Figure 1f). Using optimized media and a bioreactor, the three tissue parts developed into their respective types with high cell viability. Osteogenic marker expressions increased in MSCs embedded in the bone part, vascularization occurred in the periosteum, and myogenic differentiation of myoblasts was observed in the muscle tissue. Adding periosteum tissue significantly enhanced osteogenic marker expressions in bone tissue, and this effect was further amplified by incorporating muscle tissues. Upon implantation in mouse bone defects, the multi-tissue implants showed enhanced bone healing after 28 days compared to the negative control (Figure 1h).
Our research presents an innovative 3D in vitro model simulating complex interactions among bone, periosteum, and muscle tissues. The advanced model enhances tissue regeneration capacity, outperforming that of single-type tissue models. This notable enhancement in tissue regeneration highlights the important role of interfacial interactions between different tissue types in the musculoskeletal system. This work demonstrated the capability to develop each tissue component to not only function independently but also coexist synergistically with other tissues, effectively mimicking the dynamic interplay involved in the regeneration of the musculoskeletal system. The control over individual elements within each tissue type enabled us to fine-tune the optimal conditions for co-culturing multiple tissues and effectively stimulate their maturation through interfacial interactions in vitro, similar to the musculoskeletal system. The model can serve as a valuable tool for advancing research in musculoskeletal tissue regeneration, disease modeling, and therapeutic drug testing.

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