3D-Printed Collagen Scaffolds Promote Maintenance of Cryopreserved Patients-Derived Melanoma Explants.
ABSTRACT: The development of an in vitro three-dimensional (3D) culture system with cryopreserved biospecimens could accelerate experimental research screening anticancer drugs, potentially reducing costs and time bench-to-beside. However, minimal research has explored the application of 3D bioprinting-based in vitro cancer models to cryopreserved biospecimens derived from patients with advanced melanoma. We investigated whether 3D-printed collagen scaffolds enable the propagation and maintenance of patient-derived melanoma explants (PDMEs). 3D-printed collagen scaffolds were fabricated with a 3DX bioprinter. After thawing, fragments from cryopreserved PDMEs (approximately 1-2 mm) were seeded onto the 3D-printed collagen scaffolds, and incubated for 7 to 21 days. The survival rate was determined with MTT and live and dead assays. Western blot analysis and immunohistochemistry staining was used to express the function of cryopreserved PDMEs. The results show that 3D-printed collagen scaffolds could improve the maintenance and survival rate of cryopreserved PDME more than 2D culture. MITF, Mel A, and S100 are well-known melanoma biomarkers. In agreement with these observations, 3D-printed collagen scaffolds retained the expression of melanoma biomarkers in cryopreserved PDME for 21 days. Our findings provide insight into the application of 3D-printed collagen scaffolds for closely mimicking the 3D architecture of melanoma and its microenvironment using cryopreserved biospecimens.
Project description:Free from the limitations posed by exogenous scaffolds or extracellular matrix-based materials, scaffold-free engineered tissues have immense clinical potential. Biomaterials may produce adverse responses, interfere with cell-cell interaction, or affect the extracellular matrix integrity of cells. The scaffold-free Kenzan method can generate complex tissues using spheroids on an array of needles but could be inefficient in terms of time, as it moves and places only a single spheroid at a time. We aimed to design and construct a novel scaffold-free bioprinter that can print an entire layer of spheroids at once, effectively reducing the printing time. The bioprinter was designed using computer-aided design software and constructed from machined, 3D printed, and commercially available parts. The printing efficiency and the operating precision were examined using Zirconia and alginate beads, which mimic spheroids. In less than a minute, the printer could efficiently pick and transfer the beads to the printing surface and assemble them onto the 4 × 4 needles. The average overlap coefficient between layers was measured and found to be 0.997. As a proof of concept using human induced pluripotent stem cell-derived spheroids, we confirmed the ability of the bioprinter to place cellular spheroids onto the needles efficiently to print an entire layer of tissue. This novel layer-by-layer, scaffold-free bioprinter is efficient and precise in operation and can be easily scaled to print large tissues.
Project description:Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity with sufficient mechanical properties. This study tailored the phosphoric acid-based binder solution concentration to 8.75 wt% to maximize cytocompatibility and mechanical strength, with a supplementation of Tween 80 to improve printing. To further enhance the formulation, collagen was dissolved into the binder solution to fabricate collagen-calcium phosphate composites. Reducing the viscosity and surface tension through a physiologic heat treatment and Tween 80, respectively, enabled reliable thermal inkjet printing of the collagen solutions. Supplementing the binder solution with 1-2 wt% collagen significantly improved maximum flexural strength and cell viability. To assess the bone healing performance, we implanted 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks. The implants were confirmed to be osteoconductive, with new bone growth incorporating the degrading scaffold materials. In conclusion, this study demonstrates optimization of material parameters for 3D printed calcium phosphate scaffolds and enhancement of material properties by volumetric collagen incorporation via inkjet printing.
Project description:Repairing massive rotator cuff tendon defects remains a challenge due to the high retear rate after surgical intervention. 3D printing has emerged as a promising technique that enables the fabrication of engineered tissues with heterogeneous structures and mechanical properties, as well as controllable microenvironments for tendon regeneration. In this study, we developed a new strategy for rotator cuff tendon repair by combining a 3D printed scaffold of polylactic-co-glycolic acid (PLGA) with cell-laden collagen-fibrin hydrogels. We designed and fabricated two types of scaffolds: one featuring a separate layer-by-layer structure and another with a tri-layered structure as a whole. Uniaxial tensile tests showed that both types of scaffolds had improved mechanical properties compared to single-layered PLGA scaffolds. The printed scaffold with collagen-fibrin hydrogels effectively supported the growth, proliferation, and tenogenic differentiation of human adipose-derived mesenchymal stem cells. Subcutaneous implantation of the multilayered scaffolds demonstrated their excellent in vivo biocompatibility. This study demonstrates the feasibility of 3D printing multilayered scaffolds for application in rotator cuff tendon regeneration.
Project description:Keratin is a natural material that can be derived from the cortex of human hair. Our group had previously presented a method for the printed, sequential production of three-dimensional (3D) keratin scaffolds. Using a riboflavin-sodium persulfate-hydroquinone (initiator-catalyst-inhibitor) photosensitive solution, we produced 3D keratin-based constructs through ultraviolet crosslinking in a lithography-based 3D printer. In this study, we have used this bioink to produce a keratin-based construct that is capable of delivering small molecules, providing an environment conducive to healing of dermal burn wounds in vivo, and maintaining stability in customized packaging. We characterized the effects of manufacturing steps, such as lyophilization and gamma irradiation sterilization on the properties of 3D printed keratin scaffolds prepared for in vivo testing. Keratin hydrogels are viable for the uptake and release of contracture-inhibiting Halofuginone, a collagen synthesis inhibitor that has been shown to decrease collagen synthesis in fibrosis cases. This small-molecule delivery provides a mechanism to reduce scarring of severe burn wounds in vitro. In vivo data show that the Halofuginone-laden printed keratin is noninferior to other similar approaches reported in literature. This is indicative that the use of 3D printed keratin is not inhibiting the healing processes, and the inclusion of Halofuginone induces a more organized dermal healing after a burn; in other words, this treatment is slower but improves healing. These studies are indicative of the potential of Halofuginone-laden keratin dressings in dermal wound healing. We aim to keep increasing the complexity of the 3D printed constructs toward the production of complex scaffolds for the treatment and topographical reconstruction of severe burn wounds to the face.
Project description:In this study, we used an alginate-gelatin bioink to design and print 3D constructs with lattice, honeycomb and fibrous bundle patterns. These designs were printed using a small-scale laboratory printer, at first and later translated to a larger scale, high throughput-printing platform. A comparative analysis of the structures printed using two dissimilar platforms using gross morphologic evaluation, scanning electron microscopy and swelling assay confirmed our hypothesis that a design printed using a small-scale laboratory bioprinter for optimization of bioink composition and printing parameters can be successfully translated into a large scale-printing platform for high throughput printing of constructs. Since the designs for printing were implemented using a software which was common across both printers, this endpoint was feasible. The only difference in printing parameters resulted from variation in extrusion pressure which was due to a significant difference in barrel size used across both printers (3 ml versus 30 ml), while all other parameters stayed the same. Although the scaffolds were not bioprinted with cells, in future we will investigate how cell viability can be differentially regulated by the variation of extrusion pressure across both platforms.
Project description:Conventional 3D bioprinting allows fabrication of 3D scaffolds for biomedical applications. In this contribution we present a cryogenic 3D printing method able to produce stable 3D structures by utilising the liquid to solid phase change of a composite hydrogel (CH) ink. This is achieved by rapidly cooling the ink solution below its freezing point using solid carbon dioxide (CO2) in an isopropanol bath. The setup was able to successfully create 3D complex geometrical structures, with an average compressive stiffness of O(1) kPa (0.49?±?0.04 kPa stress at 30% compressive strain) and therefore mimics the mechanical properties of the softest tissues found in the human body (e.g. brain and lung). The method was further validated by showing that the 3D printed material was well matched to the cast-moulded equivalent in terms of mechanical properties and microstructure. A preliminary biological evaluation on the 3D printed material, coated with collagen type I, poly-L-lysine and gelatine, was performed by seeding human dermal fibroblasts. Cells showed good attachment and viability on the collagen-coated 3D printed CH. This greatly widens the range of applications for the cryogenically 3D printed CH structures, from soft tissue phantoms for surgical training and simulations to mechanobiology and tissue engineering.
Project description:Bioprinting is increasingly used to create complex tissue constructs for an array of research applications, and there are also increasing efforts to print tissues for transplantation. Bioprinting may also prove valuable in the context of drug screening for personalized medicine for treatment of diseases such as cancer. However, the rapidly expanding bioprinting research field is currently limited by access to bioprinters. To increase the availability of bioprinting technologies we present here an open source extrusion bioprinter based on the E3D motion system and tool changer to enable high-resolution multimaterial bioprinting. As proof of concept, the bioprinter is used to create collagen constructs using freeform reversible embedding of suspended hydrogels (FRESH) methodology, as well as multimaterial constructs composed of distinct sections of laminin and collagen. Data is presented demonstrating that the bioprinted constructs support growth of cells either seeded onto printed constructs or included in the bioink prior to bioprinting. This open source bioprinter is easily adapted for different bioprinting applications, and additional tools can be incorporated to increase the capabilities of the system.
Project description:Titanium (Ti) alloy implants can repair bone defects at load-bearing sites. However, they mechanically mismatch with the natural bone and lack customized adaption with the irregularly major-sized load-bearing bone defects, resulting in the failure of implant fixation. Mineralized collagen (MC), a building block in bone, can induce angiogenesis and osteogenesis, and 3D printing technology can be employed to prepare scaffolds with an overall shape customized to the bone defect. Hence, we induced the formation of MC, made of hydroxyapatite (HAp) nanocrystals and collagen fibers, in 3D-printed porous Ti6Al4V (PT) scaffolds through in situ biomimetic mineralization. The resultant MC/PT scaffolds exhibited a bone-like Young's modulus and were customized to the anatomical contour of actual bone defects of rabbit model. We found that the biocompatibility and osteogenic differentiation are best when the mass ratio between HAp nanocrystals and collagen fibers is 1 in MC. We then implanted the MC/PT scaffolds into the customized radius defect rabbit model and found that the MC/PT scaffolds significantly improved the vascularized bone tissue formation and integration between new bone and the implants. Therefore, a combination of 3D printing and biomimetic mineralization could lead to customized 3D PT scaffolds for enhanced angiogenesis, osteogenesis, and osteointegration. Such scaffolds represent novel patient-specific implants for precisely repairing irregular major-sized load-bearing bone defects.
Project description:Bone-tissue regeneration is a growing field, where nanostructured-bioactive materials are designed to replicate the natural properties of the target tissue, and then are processed with technologies such as 3D printing, into constructs that mimic its natural architecture. Type I bovine collagen formulations, containing functional nanoparticles (enriched with therapeutic ions or biomolecules) or nanohydroxyapatite, are considered highly promising, and can be printed using support baths. These baths ensure an accurate deposition of the material, nonetheless their full removal post-printing can be difficult, in addition to undesired reactions with the crosslinking agents often used to improve the final structural integrity of the scaffolds. Such issues lead to partial collapse of the printed constructs and loss of geometrical definition. To overcome these limitations, this work presents a new alternative approach, which consists of adding a suitable concentration of crosslinking agent to the printing formulations to promote the in-situ crosslinking of the constructs prior to the removal of the support bath. To this aim, genipin, chosen as crosslinking agent, was added (0.1 wt.%) to collagen-based biomaterial inks (containing either 38 wt.% mesoporous bioactive glasses or 65 wt.% nanohydroxyapatite), to trigger the crosslinking of collagen and improve the stability of the 3D printed scaffolds in the post-processing step. Moreover, to support the material deposition, a 15 wt.% alginic acid solution was used as a bath, which proved to sustain the printed structures and was also easily removable, allowing for the stable processing of high-resolution geometries.
Project description:Regenerative repair of craniomaxillofacial bone injuries is challenging due to both the large size and irregular shape of many defects. Mineralized collagen scaffolds have previously been shown to be a promising biomaterial implant to accelerate craniofacial bone regeneration in vivo. Here we describe inclusion of a 3D-printed polymer or ceramic-based mesh into a mineralized collagen scaffold to improve mechanical and biological activity. Mineralized collagen scaffolds were reinforced with 3D-printed Fluffy-PLG (ultraporous polylactide-co-glycolide co-polymer) or Hyperelastic Bone (90wt% calcium phosphate in PLG) meshes. We show degradation byproducts and acidic release from the printed structures have limited negative impact on the viability of mesenchymal stem cells. Further, inclusion of a mesh formed from Hyperelastic Bone generates a reinforced composite with significantly improved mechanical performance (elastic modulus, push-out strength). Composites formed from the mineralized collagen scaffold and either Hyperelastic Bone or Fluffy-PLG reinforcement both supported human bone-marrow derived mesenchymal stem cell osteogenesis and new bone formation. This was observed by increased mineral formation in Fluffy-PLG composites and increased cell viability and upregulation of RUNX2, Osterix, and COL1A2 genes in both composites. Strikingly, composites reinforced with Hyperelastic Bone mesh elicited significantly increased secretion of osteoprotegerin, a soluble glycoprotein and endogenous inhibitor of osteoclast activity. These results suggest that architectured meshes can be integrated into collagen scaffolds to boost mechanical performance and actively instruct cell processes that aid osteogenicity; specifically, secretion of a factor crucial to inhibiting osteoclast-mediated bone resorption. Future work will focus on further adapting the polymer mesh architecture to confer improved shape-fitting capacity as well as to investigate the role of polymer reinforcement on MSC-osteoclast interactions as a means to increase regenerative potential.