3D-printed Polylactic Acid Scaffolds Research Articles

Biomolecule-grafted GO enhanced the mechanical and biological properties of 3D printed PLA scaffolds with TPMS porous structure

Graphene oxide (GO) exhibits excellent mechanical strength and modulus. However, its effectiveness in mechanically reinforcing polymer materials is limited due to issues with interfacial bonding and dispersion arising from differences in the physicochemical properties between GO and polymers. Surface modification using coupling agents is an effective method to improve the bonding problem between polymer and GO, but there may be biocompatibility issues when used in the biomedical field. In this study, the biomolecule L-lysine, was applied to improve the interfacial bonding and dispersion of GO in polylactic acid (PLA) without compromising biocompatibility. The PLA/L-lysine-modified GO (PLA/L-GO) bone scaffold with triply periodic minimal surface (TPMS) structure was prepared using fused deposition modeling (FDM). The FTIR results revealed successful grafting of L-lysine onto GO through the reaction between their -COOH and -NH2 groups. The macroscopic and microscopic morphology characterization indicated that the PLA/L-GO scaffolds exhibited an characteristics of dynamic diameter changes, with good interlayer bonding. It was noteworthy that the L-lysine modification promoted the dispersion of GO and the interfacial bonding with the PLA matrix, as characterized by SEM. As a result, the PLA/0.1L-GO scaffold exhibited higher compressive strength (13.2 MPa) and elastic modulus (226.8 MPa) than PLA/0.1GO. Moreover, PLA/L-GO composite scaffold exhibited superior biomineralization capacity and cell response compared to PLA/GO. In summary, L-lysine not only improved the dispersion and interfacial bonding of GO with PLA, enhancing the mechanical properties, but also improved the biological properties. This study suggests that biomolecules like L-lysine may replace traditional modifiers as an innovative bio-modifier to improve the performance of polymer/inorganic composite biomaterials.

Fabrication of 3D-printed scaffolds loaded with gallium acetylacetonate for potential application in osteoclastic bone resorption

We recently reported the potential of a new gallium compound, gallium acetylacetonate (GaAcAc) in combating osteoclastic bone resorption through inhibition of osteoclast differentiation and function. Herein, we focused on 3D-printed polylactic acid scaffolds that were loaded with GaAcAc and investigated the impact of scaffold pretreatment with polydopamine (PDA) or sodium hydroxide (NaOH). We observed a remarkable increase in scaffold hydrophilicity with PDA or NaOH pretreatment while biocompatibility and in vitro degradation were not affected. NaOH-pretreated scaffolds showed the highest amount of GaAcAc loading when compared to other scaffolds (p < 0.05). NaOH-pretreated scaffolds with GaAcAc loading showed effective reduction of osteoclast counts and size. The trend was supported by suppression of key osteoclast differentiation markers such as NFAT2, c-Fos, TRAF6, & TRAP. All GaAcAc-loaded scaffolds, regardless of surface pretreatment, were effective in inhibiting osteoclast function as evidenced by reduction in the number of resorptive pits in bovine cortical bone slices (p < 0.01). The suppression of osteoclast function according to the type of scaffold followed the ranking: GaAcAc loading without surface pretreatment > GaAcAc loading with NaOH pretreatment > GaAcAc loading with PDA pretreatment. Additional studies will be needed to fully elucidate the impact of surface pretreatment on the efficacy and safety of GaAcAc-loaded 3D-printed scaffolds.

Effect of the filling pattern in 3D-printed polylactic acid scaffolds on mechanical properties and cell proliferation for bone tissue regeneration

3D printing technology can control the shape and filling pattern of the scaffold very well and has been widely used in the preparation of bone repair scaffolds. Modifying the filling pattern of scaffolds to alter their mechanical properties and cellular adhesion holds immense significance. In this study, 3D-printed polylactic acid (PLA) scaffolds with different fibre angles (15°-90°) were prepared. The 90° scaffold showed the best mechanical properties and was suitable for repairing long bones. The 60° scaffold has balanced mechanical properties and is suitable for maxillofacial bone repair. The scaffold with a 30° angle was the best for cell adhesion and proliferation and was suitable for cancellous bone repair.

Design, fabrication, and characterization of 3D-printed ABS and PLA scaffolds potentially for tissue engineering

Additive manufacturing technologies are getting attention as a prospective substitute for conventional medical implant production methods owing to their unique manufacturing properties. In addition, biomaterials are required for bone repair and tissue engineering (BTE) techniques to provide scaffolds with the necessary biological and structural properties and improved performance. In this study, widely available biocompatible materials acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) are utilized for the design, fabrication, simulation, and mechanical characterization of 3D-printed scaffolds with two lay-down patterns potentially for bone tissue engineering. Mechanical properties of the 3D-printed scaffolds are determined by compression test and finite element analysis (FEA). Moreover, scanning electron microscope (SEM) images are also analyzed to determine the printing accuracy of the scaffolds. The porosity of the scaffolds is estimated both theoretically and through the liquid displacement method. Finally, the comparative analysis between PLA and ABS scaffolds indicates that the PLA scaffold has a higher potential for bone tissue regeneration. This study provides a notable engineering contribution by using a cost-efficient and easily available biomaterial as an alternative to bone tissue.

Nuciferine-loaded chitosan hydrogel-integrated 3D-printed polylactic acid scaffolds for bone tissue engineering: A combinatorial approach

Critical-sized bone defects resulting from severe trauma and open fractures cannot spontaneously heal and require surgical intervention. Limitations of traditional bone grafting include immune rejection and demand-over-supply issues leading to the development of novel tissue-engineered scaffolds. Nuciferine (NF), a plant-derived alkaloid, has excellent therapeutic properties, but its osteogenic potential is yet to be reported. Furthermore, the bioavailability of NF is obstructed due to its hydrophobicity, requiring an efficient drug delivery system, such as chitosan (CS) hydrogel. We designed and fabricated polylactic acid (PLA) scaffolds via 3D printing and integrated them with NF-containing CS hydrogel to obtain the porous biocomposite scaffolds (PLA/CS-NF). The fabricated scaffolds were subjected to in vitro physicochemical characterization, cytotoxicity assays, and osteogenic evaluation studies. Scanning electron microscopic studies revealed uniform pore size distribution on PLA/CS-NF scaffolds. An in vitro drug release study showed a sustained and prolonged release of NF. The cyto-friendly nature of NF in PLA/CS-NF scaffolds towards mouse mesenchymal stem cells (mMSCs) was observed. Also, cellular and molecular level studies signified the osteogenic potential of NF in PLA/CS-NF scaffolds on mMSCs. These results indicate that the PLA/CS-NF scaffolds could promote new bone formation and have potential applications in bone tissue engineering.

Improving physio-mechanical and biological properties of 3D-printed PLA scaffolds via in-situ argon cold plasma treatment

As a bone tissue engineering material, polylactic acid (PLA) has received significant attention and interest due to its ease of processing and biocompatibility. However, its insufficient mechanical properties and poor wettability are two major drawbacks that limit its extensive use. For this purpose, the present study uses in-situ cold argon plasma treatment coupled with a fused deposition modeling printer to enhance the physio-mechanical and biological behavior of 3D-printed PLA scaffolds. Following plasma treatment, field emission scanning electron microscopy images indicated that the surface of the modified scaffold became rough, and the interlayer bonding was enhanced. This resulted in an improvement in the tensile properties of samples printed in the X, Y, and Z directions, with the enhancement being more significant in the Z direction. Additionally, the root mean square value of PLA scaffolds increased (up to 70-fold) after plasma treatment. X-ray photoelectron spectroscopy analysis demonstrated that the plasma technique increased the intensity of oxygen-containing bonds, thereby reducing the water contact angle from 92.5° to 42.1°. The in-vitro degradation study also demonstrated that argon plasma treatment resulted in a 77% increase in PLA scaffold degradation rate. Furthermore, the modified scaffold improved the viability, attachment, and proliferation of human adipose-derived stem cells. These findings suggest that in-situ argon plasma treatment may be a facile and effective method for improving the properties of 3D-printed parts for bone tissue engineering and other applications.

Degradation assessment of Mg-Incorporated 3D printed PLA scaffolds for biomedical applications

Polylactic acid (PLA)/Magnesium (Mg)-based composites exhibit great potential for applications in bone regeneration and tissue engineering. PLA is a biodegradable and biocompatible polymer, that has the ability to be easily shaped into diverse structures like scaffolds, films, and fibers. However, its inherent low biodegradability limits its applicability for tissue engineering. On the other hand, magnesium, a biocompatible metal known for its good biodegradability and osteoconductivity, is well-suited for bone tissue engineering. In this study, we fabricated and characterized a composite material of Mg/PLA with 5, 10, and 15 wt%Mg alloy (AZ61), which was subsequently 3D printed. The incorporation of Mg particles into PLA matrix offers a solution to overcome the low biodegradation limitations typically associated with the PLA. Moreover, it helps counteract the negative consequences related to the rapid degradation of Mg, such as alkalinization and excessive release of H2. Additionally, the change in pH values and changes in mass during in vitro degradation indicated that the addition of Mg effectively counteracted the acidic byproducts generated by PLA. Furthermore, X-Ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) spectroscopy were utilized to investigate the degradation of the scaffolds, while thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were used to compare and contrast the thermal properties of the composites. Our findings demonstrate that the addition of Mg significantly influences the thermal properties of PLA and notably accelerates its degradation, in addition to its noticeable influence on cell adhesion.

Calcium Carbonate Coating of 3D-Printed PLA Scaffolds Intended for Biomedical Applications.

The incorporation of ceramic additives is the most commonly used strategy to improve the biofunctionality of polymer-based scaffolds intended for bone regeneration. By embedding ceramic particles as a coating, the functionality improvement in the polymeric scaffolds can be concentrated on the cell-surface interface, thus creating a more favourable environment for the adhesion and proliferation of osteoblastic cells. In this work, a pressure-assisted and heat-induced method to coat polylactic acid (PLA) scaffolds with calcium carbonate (CaCO3) particles is presented for the first time. The coated scaffolds were evaluated by optical microscopy observations, a scanning electron microscopy analysis, water contact angle measurements, compression testing, and an enzymatic degradation study. The ceramic particles were evenly distributed, covered more than 60% of the surface, and represented around 7% of the coated scaffold weight. A strong bonding interface was achieved, and the thin layer of CaCO3 (~20 µm) provided a significant increase in the mechanical properties (with a compression modulus improvement up to 14%) while also enhancing the surface roughness and hydrophilicity. The results of the degradation study confirmed that the coated scaffolds were able to maintain the pH of the media during the test (~7.6 ± 0.1), in contrast to the pure PLA scaffolds, for which a value of 5.07 ± 0.1 was obtained. The ceramic-coated scaffolds developed showed potential for further evaluations in bone tissue engineering applications.

Personalized 3D-printed amniotic fornical ring for ocular surface reconstruction.

In the present work, we used three-dimensional (3D) printing technology to make a polylactic acid (PLA) amniotic fornical ring (AFR) for ocular surface reconstruction. This work is a retrospective and interventional case series of patients with ocular surface diseases who underwent either personalized 3D-printed AFR-assisted amniotic membrane transplantation (AMT) or sutured AMT (SAMT). Patient epidemiology, treatment, operative duration, epithelial healing time, retention time, vision changes, morbidity, and costs were analyzed. Thirty-one patients (40 eyes) and 19 patients (22 eyes) were enrolled in the 3D-printed AFR group and the SAMT group, respectively. The clinical indications of AFR and SAMT were similar, such as corneal and/or conjunctival epithelial defects due to chemical burns, thermal burns, Stevens-Johnson syndrome (SJS), or toxic epidermal necrolysis (TEN). The mean dissolution time was 15 ± 11 days in the AFR group, compared with 14 ± 7 days in the SAMT group. The percentage of healed corneal area was 90.91% (66.10%-100.00%) for AFR and 93.67% (60.23%-100.00%) for SAMT. The median time for corneal epithelial healing was 14 (7-75) days in the AFR group and 30 (14-55) days in the suture AMT group. There were no significant differences in the initial visual acuity, final visual acuity, or improvement in visual acuity between the two groups. The operation duration in the AFR group was significantly shorter than that in the SAMT group. Regarding the cost analysis, the average cost per eye in the AFR group was significantly lower than that in the SAMT group. Furthermore, 3D-printed and sterile AFR showed no obvious side effects on the eyes. Our results suggested that 3D-printed PLA scaffolds could be used as an AFR device for ocular surface disease. In addition, personalized 3D-printed AFR is superior to conventional AMT in operation duration and cost effectiveness, thereby reducing the financial burden on our health care system.

Physiologic Response Evaluation of Human Foetal Osteoblast Cells within Engineered 3D-Printed Polylactic Acid Scaffolds.

Large bone defect treatments have always been one of the important challenges in clinical practice and created a huge demand for more efficacious regenerative approaches. The bone tissue engineering (BTE) approach offered a new alternative to conventional bone grafts, addressing all clinical needs. Over the past years, BTE research is focused on the study and realisation of new biomaterials, including 3D-printed supports to improve mechanical, structural and biological properties. Among these, polylactic acid (PLA) scaffolds have been considered the most promising biomaterials due to their good biocompatibility, non-toxic biodegradability and bioresorbability. In this work, we evaluated the physiological response of human foetal osteoblast cells (hFOB), in terms of cell proliferation and osteogenic differentiation, within oxygen plasma treated 3D-printed PLA scaffolds, obtained by fused deposition modelling (FDM). A mechanical simulation to predict their behaviour to traction, flexural or torque solicitations was performed. We found that: 1. hFOB cells adhere and grow on scaffold surfaces; 2. hFOB grown on oxygen plasma treated PLA scaffolds (PLA_PT) show an improvement of cell adhesion and proliferation, compared to not-plasma treated scaffolds (PLA_NT); 3. Over time, hFOB penetrate along strands, differentiate, and form a fibrous matrix, tissue-like; 4. 3D-printed PLA scaffolds have good mechanical behaviour in each analysed configuration. These findings suggest that 3D-printed PLA scaffolds could represent promising biomaterials for medical implantable devices in the orthopaedic field.

3D printed polylactic acid/gelatin-nano-hydroxyapatite/platelet-rich plasma scaffold for critical-sized skull defect regeneration

BackgroundThree-dimensional (3D) printing is a capable approach for the fabrication of bone tissue scaffolds. Nevertheless, a purely made scaffold such as polylactic acid (PLA) may suffer from shortcomings and be restricted due to its biological behavior. Gelatin, hydroxyapatite and platelet-rich plasma (PRP) have been revealed to be of potential to enhance the osteogenic effect. In this study, it was tried to improve the properties of 3D-printed PLA scaffolds by infilling them with gelatin-nano-hydroxyapatite (PLA/G-nHA) and subsequent coating with PRP. For comparison, bare PLA and PLA/G-nHA scaffolds were also fabricated. The printing accuracy, the scaffold structural characterizations, mechanical properties, degradability behavior, cell adhesion, mineralization, systemic effect of the scaffolds on the liver enzymes, osteocalcin level in blood serum and in vivo bone regeneration capability in rat critical-sized calvaria defect were evaluated.ResultsHigh printing accuracy (printing error of < 11%) was obtained for all measured parameters including strut thickness, pore width, scaffold density and porosity%. The highest mean ultimate compression strength (UCS) was associated with PLA/G-nHA/PRP scaffolds, which was 10.95 MPa. A slow degradation rate was observed for all scaffolds. The PLA/G-nHA/PRP had slightly higher degradation rate, possibly due to PRP release, with burst release occurred at week 4. The MTT results showed that PLA/G-nHA/PRP provided the highest cell proliferation at all time points, and the serum biochemistry (ALT and AST level) results indicated no abnormal/toxic influence caused by scaffold biomaterials. Superior cell adhesion and mineralization were obtained for PLA/G-nHA/PRP. Furthermore, all the developed scaffolds showed bone repair capability. The PLA/G-nHA/PRP scaffolds could better support bone regeneration than bare PLA and PLA/G-nHA scaffolds.ConclusionThe PLA/G-nHA/PRP scaffolds can be considered as potential for hard tissue repair.

Biomimetic, mussel-inspired surface modification of 3D-printed biodegradable polylactic acid scaffolds with nano-hydroxyapatite for bone tissue engineering.

Polylactic acid (PLA) has been widely used as filaments for material extrusion additive manufacturing (AM) to develop patient-specific scaffolds in bone tissue engineering. Hydroxyapatite (HA), a major component of natural bone, has been extensively recognized as an osteoconductive biomolecule. Here, inspired by the mussel-adhesive phenomenon, in this study, polydopamine (PDA) coating was applied to the surface of 3D printed PLA scaffolds (PLA@PDA), acting as a versatile adhesive platform for immobilizing HA nanoparticles (nHA). Comprehensive analyses were performed to understand the physicochemical properties of the 3D-printed PLA scaffold functionalized with nHA and PDA for their potent clinical application as a bone regenerative substitute. Scanning electron microscopy (SEM) and element dispersive X-ray (EDX) confirmed a successful loading of nHA particles on the surface of PLA@PDA after 3 and 7 days of coating (PLA@PDA-HA3 and PLA@PDA-HA7), while the surface micromorphology and porosity remain unchanged after surface modification. The thermogravimetric analysis (TGA) showed that 7.7 % and 12.3% mass ratio of nHA were loaded on the PLA scaffold surface, respectively. The wettability test indicated that the hydrophilicity of nHA-coated scaffolds was greatly enhanced, while the mechanical properties remained uncompromised. The 3D laser scanning confocal microscope (3DLS) images revealed that the surface roughness was significantly increased, reaching Sa (arithmetic mean height) of 0.402 μm in PLA@PDA-HA7. Twenty-eight days of in-vitro degradation results showed that the introduction of nHA to the PLA surface enhances its degradation properties, as evidenced by the SEM images and weight loss test. Furthermore, a sustainable release of Ca2+ from PLA@PDA-HA3 and PLA@PDA-HA7 was recorded, during the degradation process. In contrast, the released hydroxyl group of nHA tends to neutralize the local acidic environments, which was more conducive to osteoblastic differentiation and extracellular mineralization. Taken together, this facile surface modification provides 3D printed PLA scaffolds with effective bone regenerative properties by depositing Ca2+ contents, improving surface hydrophilicity, and enhancing the in-vitro degradation rate.

Effects of ceramic additives and bioactive coatings on the degradation of polylactic acid-based bone scaffolds under hydrolytic conditions.

Polylactic acid (PLA) has been extensively used for the manufacturing of scaffolds in bone tissue engineering applications. Due to the low hydrophilicity and the acidic degradation process of this biomaterial, different strategies have been proposed to increase the biofunctionality of the support structure. The use of ceramic particles is a generally preferred option to increase the osteoconductivity of the base material, while acting as buffers to maintain the pH level of the surroundings tissues. Surface modification is another approach to overcome the limitations of PLA for tissue engineering applications. In this work, the degradation profile of 3D-printed PLA scaffolds containing beta-tricalcium phosphate (β-TCP) and calcium carbonate (CaCO3 ) particles has been studied under hydrolytic conditions. Composite samples treated with plasma and coated with Aloe vera extracts were also studied to evaluate the effect of this surface modification method. The characterization of the 3D structures included its morphological, calorimetric and mechanical evaluation. According to the results obtained, the proposed composite scaffolds allowed an adequate maintenance of the pH level of the surrounding medium, with no effects observed on the morphology and mechanical properties of these structures. Hence, these samples showed potential to be further investigated as candidates for bone tissue regeneration.

A tailored bioactive 3D porous poly(lactic-acid)-exosome scaffold with osteo-immunomodulatory and osteogenic differentiation properties

Polylactic acid (PLA) is a versatile and biodegradable scaffold widely used in biomedical fields to repair tissue defects. Exosomes derived from mesenchymal stem cells (MSCs) are nano-sized extracellular vesicles, which play an important role in tissue engineering in recent years. The primary focus of this study was to develop a bioactive 3D PLA scaffold using exosome-based strategy to improve its osteogenic and immunoregulatory potential. We firstly successfully isolated MSC-derived exosomes (MSC-Exo). Morphological analysis revealed that MSC-Exo exhibits a typical cup-shaped morphology with high expression of exosomal marker CD63. MSC-Exo internalization into recipient cells were also investigated using flow cytometry and confocal laser scanning microscopy. Porous 3D PLA scaffold coated MSC-Exo were used for immunoregulatory and osteogenic testing. Exosomes released from 3D PLA scaffold were validated in RAW264.7 and hBMSCs. The cell proliferation and live/dead assay indicated high biocompatibility for PLA-Exo scaffold. Additionally, PLA-Exo scaffold could reduce the pro-inflammatory marker expression and reactive oxygen species (ROS) production, indicating potential immunoregulatory potential. It is also confirmed that PLA-Exo scaffold could potentiate osteogenic differentiation in the osteogenesis assay. In conclusion, our results demonstrate this bioactive 3D-printed PLA scaffolds with MSC-Exo modification holds immunoregulatory potential and favor osteogenic differentiation, thus having potential applications in bone tissue regeneration.

Biological properties of human periodontal ligament cell spheroids cultivated on chitosan and polyvinyl alcohol membranes

Multicellular spheroid cultures have attracted increasing attention in the field of periodontal regeneration. However, very few studies have reported the periodontal ligament (PDL) cell spheroid formation via biomaterials-induced processes. This study investigated the biological characteristics of human PDL cell spheroids formed on two hydrophilic polymer-based biomaterials, namely chitosan and polyvinyl alcohol. The expressions of periostin, paxillin, hypoxia-inducible factor 1-α (HIF-1α), and vascular endothelial growth factor (VEGF) were analyzed. Cell migration ability was assessed using a scratch assay. Furthermore, PDL cell spheroids were cultured in 3D-printed polylactic acid scaffolds to evaluate mineralizing capability. Western blot analysis revealed increased expressions of periostin, HIF-1α, and VEGF in the 3D spheroids. After the spheroids were reseeded, the cells gradually migrated outward from the spheroids and time-dependent distribution of paxillin was observed. The cells migrating outward from the 3D spheroids demonstrated greater migration ability than that of 2D monolayer cells. Compared to the dissociated cells from a monolayer culture, the cell spheroids formed on the chitosan membrane exhibited elevated alkaline phosphatase activity and an increase in mineralized matrix deposition. The biomaterial-induced formation of PDL cell spheroids suggests a novel strategy for cell delivery in research and clinical applications of periodontal regeneration.

Degradation of 3D-Printed Porous Polylactic Acid Scaffolds Under Mechanical Stimulus.

Polylactic acid (PLA) is a biodegradable polymer commonly used as a scaffold material to repair tissue defects, and its degradation is associated with mechanical stimulus. In this study, the effect of mechanical stimulus on the degradation of 3D-printed PLA scaffolds was investigated by in vitro experiments and an author-developed numerical model. Forty-five samples with porosity 64.8% were printed to carry out the degradation experiment within 90 days. Statistical analyses of the mass, volume fraction, Young’s modulus, and number average molecular weight were made, and the in vitro experiments were further used to verify the proposed numerical model of the scaffold degradation. The results indicated that the mechanical stimulus accelerated the degradation of the PLA scaffold, and the higher mechanical stimulus led to a faster degradation of the scaffolds at the late stage of the degradation process. In addition, the Young’s modulus and the normalized number average molecular weight of the PLA scaffolds between the experiments and the numerical simulations were comparable, especially for the number average molecular weight. The present study could be helpful in the design of the biodegradable PLA scaffolds.

Chondrocyte-laden GelMA hydrogel combined with 3D printed PLA scaffolds for auricle regeneration

The current gold standard for auricular reconstruction after microtia or ear trauma is the autologous cartilage graft with an autologous skin flap overlay. Harvesting autologous cartilage requires an additional surgery that may result in donor area complications. In addition, autologous cartilage is limited and the auricular reconstruction requires complex sculpting, which requires excellent clinical skill and is very time consuming. This work explores the use of 3D printing technology to fabricate bioactive artificial auricular cartilage using chondrocyte-laden gelatin methacrylate (GelMA) and polylactic acid (PLA) for auricle reconstruction. In this study, chondrocytes were loaded within GelMA hydrogel and combined with the 3D-printed PLA scaffolds to biomimetic the biological mechanical properties and personalized shape. The printing accuracy personalized scaffolds, biomechanics and chondrocyte viability and biofunction of artificial auricle have been studied. It was found that chondrocytes were fixed in the PLA auricle scaffolds via GelMA hydrogels and exhibited good proliferative properties and cellular activity. In addition, new chondrocytes and chondrogenic matrix, as well as type II collagen were observed after 8 weeks of implantation. At the same time, the transplanted auricle complex kept full and delicate auricle shape. This study demonstrates the potential of using 3D printing technology to construct in vitro living auricle tissue. It shows a great prospect in the clinical application of auricle regeneration.

A Composite Lactide-Mineral 3D-Printed Scaffold for Bone Repair and Regeneration

Orthopedic tumor resection, trauma, or degenerative disease surgeries can result in large bone defects and often require bone grafting. However, standard autologous bone grafting has been associated with donor site morbidity and/or limited quantity. As an alternate, allografts with or without metallic or polyether-etherketone have been used as grafting substitutes. However, these may have drawbacks as well, including stress shielding, pseudarthrosis, disease-transmission, and infection. There is therefore a need for alternative bone substitutes, such as the use of mechanically compliant three-dimensional (3D)-printed scaffolds. Several off-the-shelf materials are available for low-cost fused deposition 3D printing such as polylactic acid (PLA) and polycaprolactone (PCL). We have previously described the feasibility of 3D-printed PLA scaffolds to support cell activity and extracellular matrix deposition. In this study, we investigate two medical-grade filaments consistent with specifications found in American Society for Testing and Materials (ASTM) standard for semi-crystalline polylactide polymers for surgical implants, a pure polymer (100M) and a copolymeric material (7415) for their cytocompatibility and suitability in bone tissue engineering. Moreover, we assessed the impact on osteo-inductive properties with the addition of beta-tricalcium phosphate (β-TCP) minerals and assessed their mechanical properties. 100M and 7415 scaffolds with the additive β-TCP demonstrated superior mesenchymal stem cells (MSCs) differentiation detected via increased alkaline phosphatase activity (6-fold and 1.5-fold, respectively) and mineralized matrix deposition (14-fold and 5-fold, respectively) in vitro. Furthermore, we evaluated in vivo compatibility, biosafety and bone repair potential in a rat femur window defect model. 100M+β-TCP implants displayed a positive biosafety profile and showed significantly enhanced new bone formation compared to 100M implants evidenced by μCT (39 versus 25% bone volume/tissue volume ratio) and histological analysis 6 weeks post-implantation. These scaffolds are encouraging composite biomaterials for repairing bone applications with a great potential for clinical translation. Further analyses are required with appropriate evaluation in a larger critical-sized defect animal model with long-term follow-up.

Comparison of the Biological Behaviour of Human Dermal Fibroblasts seeded on 3D Printed Polylactic acid, Polycaprolactone and Polyethylene Terephthalate Scaffolds in vitro

Regenerative medicine is a scientific field that improves and repairs diseased and injured tissues. Three-dimensional (3D) printing is an innovative technology that provides a new application field for regenerative medicine. 3D printed scaffolds by programming pore sizes and shapes serve as a temporary basis for cells until the natural extracellular matrix (ECM) is reconstructed. Dermal fibroblasts are mesenchymal cells located in the dermal skin layer that produce and organize ECM components. They play an essential role in skin wound healing and fibrosis. The aim of this study is to analyze the viability, adhesion, distribution, and collagen IV expression of human dermal fibroblasts (HDFs) seeded on 3D printed polylactic acid (PLA), polyethylene terephthalate (PET), and poly-ε-caprolactone (PCL) scaffolds in vitro. HDFs were seeded on scaffolds or tissue culture plastic plates as control and were cultured for 1 and 3 days. 3D PLA, PCL, and PET scaffolds were prepared using a custom made fused deposition modeling printer. The cell viability was measured by WST-1 assay on days 1 and 3. The cell adhesion was evaluated by scanning electron microscopy (SEM). The distribution was analyzed by hematoxylin and eosin (H&amp;E) staining. Collagen IV expression was analyzed by immunohistochemical (IHC) staining. On day 1, the viability of HDFs on the 3D PLA scaffolds was significantly higher than PCL scaffolds. On day 3, the viability of HDFs on 3D PLA and PET scaffolds was significantly higher than PCL scaffolds. SEM images showed that HDFs on 3D PLA scaffolds attached the surfaces, filled the interfiber gaps and maintained their tissue specific morphology on day 3 compared to PCL and PET scaffolds. Histological images stained with H&amp;E demonstrated that the distribution of HDFs on 3D PLA scaffolds was uniform on days 1 and 3. Collagen IV staining was more intense in HDFs on 3D PLA scaffolds on days 1 and 3. This study shows that 3D PLA scaffolds create a appropriate environment for cell viability, adhesion, distribution and may provide a high advantage in skin tissue regeneration.

Abstract 2637: The use of 3D-printed Polylactic acid scaffolds (PLA) as a substrate for growth of MCF7 human breast cancer cells

Abstract This study focuses on fabrication of large-scale tissue scaffolds from Polylactic acid (PLA) and their feasibility as a substrate for cell growth. The scaffolds were fabricated via combined additive manufacturing and microwave foaming. The benefit of this approach is the ability to generate scaffolds with specific architectures and controllable pore size and porosity. The resulting scaffolds have dual pore networks with porosity in excess of 80% which provide a pathway for both cell growth and circulation of culture media. We sought to determine the capability of these scaffolds to support attachment, growth and viability of the MCF7 human breast cancer cell line, as well as drug sensitivity. For these experiments the PLA scaffolds were sterilized in 24-well plates and 1 x 105 MCF7 cells were seeded into each well and cultured for up to 7 days. Scaffolds were then used for SEM imaging, hoescht-staining, or cell counting and viability measurements after trypsinization. Comparisons of cell numbers detached from the scaffolds vs. the remaining well suggest a proportionally small percentage of cells (less than 5%) attached to the PLA, which is not surprising since the scaffolds lack a solid bottom. However, our imaging data using hoescht-staining of PLA-attached cells pre- and post- trypsinization suggest that traditional trypsinization does not effectively detach cells from the PLA material, and therefore this method cannot be used to accurately determine the proportion of cells growing on the scaffolds. This also reveals a potential limitation for application. SEM imaging confirmed cell attachment, with cell growth in the form of sheets along the struts on the bottom layer, and clusters elsewhere with a size of around 50 micron. Analysis of viability using trypan blue exclusion shows no effect of the PLA material on cell viability. To determine the drug sensitivity of cells grown with the PLA material, we treated MCF7 cells grown in the presence or absence of oleuropein, the main phenolic compound found in olive leaves. Media was collected at 48 hours and 7 days and released LDH measured by a cytotoxicity assay. Our data showed that the PLA material itself did not increase cellular toxicity in the absence of oleuropein treatment. It also confirmed that treatment with 200 ug/ml oleuropein induces the expected cytotoxicity in MCF7 cells grown in the absence of PLA, with no significant difference in oleuropein-susceptibility for cells grown in the presence of PLA scaffolds. Together, our results suggest that PLA scaffolds can support MCF7 cell attachment and growth, with no effect on cell viability or drug-induced cytotoxicity. This provides support for the promise of PLA scaffolds for physiologically relevant cell and tissue culture work. Citation Format: Brenna McAllister, Mohammed Mustafa, Sriharsha Sundarram, Shelley A. Phelan. The use of 3D-printed Polylactic acid scaffolds (PLA) as a substrate for growth of MCF7 human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2637.