Long‐term in vivo integrity and safety of 3D‐bioprinted cartilaginous constructs

Abstract

Long-term stability and biological safety are crucial for translation of 3D-bioprinting technology into clinical applications. Here, we addressed the long-term safety and stability issues associated with 3D-bioprinted constructs comprising a cellulose scaffold and human cells (chondrocytes and stem cells) over a period of 10 months in nude mice. Our findings showed that increasing unconfined compression strength over time significantly improved the mechanical stability of the cell-containing constructs relative to cell-free scaffolds. Additionally, the cell-free constructs exhibited a mean compressive stress and stiffness (compressive modulus) of 0.04 ± 0.05 MPa and 0.14 ± 0.18 MPa, respectively, whereas these values for the cell-containing constructs were 0.11 ± 0.08 MPa (p = .019) and 0.53 ± 0.59 MPa (p = .012), respectively. Moreover, histomorphologic analysis revealed that cartilage formed from the cellcontaining constructs harbored an abundance of proliferating chondrocytes in clusters, and after 10 months, resembled native cartilage. Furthermore, extension of the experiment over the complete lifecycle of the animal model revealed no signs of ossification, fibrosis, necrosis, or implant-related tumor development in the 3D-bioprinted constructs. These findings confirm the in vivo biological safety and mechanical stability of 3D-bioprinted cartilaginous tissues and support their potential translation into clinical applications.

Adipose‐Derived Stromal Cells Preserve Pancreatic Islet Function in a Transplantable 3D Bioprinted Scaffold

“…[ 29,73 ] Also, we previously reported about the stability of the bioink material where cell‐free alginate/NFC scaffolds did not degrade, but stayed intact during 10 months of follow‐up period post‐transplantation in a mouse model. [ 60 ] Similarly, very low levels of biodegradation of alginate/NFC scaffolds have been reported after 10 months transplantation in an application with chondrocytes and chondrocytes together with MSCs in vivo. [ 20,54 ] Following up with this data, the measurement of the diffusion properties of alginate/NFC bioink using FRAP analysis reported that the used bioink in this study allows the diffusion of wide‐range of small molecules with the size from 3 to70 kDa.…”

“…An average of 1.2 × 10 6 ASCs per bioprinted scaffold (14 × 10 6 ASCs/1 mL bioink for about 12 scaffolds) was aimed since the previously published data showed good viability and function of cells with this density in the scaffolds. [20,60] Handpicked equally sized islets (100 human or mouse islets/scaffold) were added using PE-50 tubing and Hamilton threaded plunger syringe and gently mixed with bioink using a small spatula on a sterile petri dish. [60] Bioprinting was done using a pneumatic extrusion bioprinter INKREDIBLE+ (CELLINK AB) at a printing pressure of ≈5-12 kPa for all layers, which is tolerable for most cells types and the size of the nozzles suitable for the size of islets and ASCs is selected to avoid excessive shear stress on the embedded cells.…”

“…[20,60] Handpicked equally sized islets (100 human or mouse islets/scaffold) were added using PE-50 tubing and Hamilton threaded plunger syringe and gently mixed with bioink using a small spatula on a sterile petri dish. [60] Bioprinting was done using a pneumatic extrusion bioprinter INKREDIBLE+ (CELLINK AB) at a printing pressure of ≈5-12 kPa for all layers, which is tolerable for most cells types and the size of the nozzles suitable for the size of islets and ASCs is selected to avoid excessive shear stress on the embedded cells. [17,29] First, a solid ASC containing base layer was deposited using a blue standard conical 22 G (410 μm) nozzle.…”

Nanocellulose-Based Scaffolds for Chondrogenic Differentiation and Expansion

“…A further proliferative impact involving chondrocyte synthesis of GAGs and type II collagen was reported in constructions containing a mixture of chondrocytes and stem cells (Möller et al, 2017). The long-term (10-months study) outcomes on chondrogenesis associated with transplanted 3D-bioprinted CELLINK ® demonstated no adverse events, such as ossification, neoplasms, or necrosis (Apelgren et al, 2021). Also, a sulfated version of alginate that can bind FGF, TGF, and hepatocyte growth factors (HGF) was combined with NFC to form a printable bioink with better chondrogenic characteristics compared to inertia alginate-containing bioinks (Müller et al, 2017).…”

Biomaterial and biocompatibility evaluation of tunicate nanocellulose for tissue engineering

“…Polytetrafluoroethylene (PTFE) has been extensively used for medical purposes, including sutures, meshes in hernia-repair surgery, and vascular grafts/patches, and is known for its excellent mechanical properties and biocompatibility [19][20][21][22][23][24]. Other sources of nanocellulose (i.e., synthetic, bacterial, and wood nanocellulose) have been evaluated [1,5,12,[25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41] using human cells; however, their biological disadvantages include inflammatory reactions caused by endotoxins, such as contamination by lipopolysaccharides and cytotoxicity caused by silica particles [35], and lignin [42]. Some of these drawbacks have been reduced by employing different purifying steps and modifications.…”