A Proof-of-Concept Preparation of Lipid-Plasmid DNA Particles Using Novel Extrusion-Based 3D-Printing Technology, SMART.

Gene therapy is a promising approach with delivery of mRNA, small interference RNA, and plasmid DNA to elicit a therapeutic action in vitro using cationic or ionizable lipid nanoparticles. In the present study, a novel extrusion-based Sprayed Multi Adsorbed-droplet Reposing Technology (SMART) developed in-house was employed for the preparation, characterization, and transfection abilities of the green fluorescence protein (GFP) plasmid DNA in cancer cells in vitro. The results showed 100% encapsulation of pDNA (GFP) in LNPs of around 150 nm (N/P 5) indicating that the processes developed using SMART technology are consistent and can be utilized for commercial applications.

Veering to a Continuous Platform of Fused Deposition Modeling Based 3D Printing for Pharmaceutical Dosage Forms: Understanding the Effect of Layer Orientation on Formulation Performance.

Three-dimensional (3D) printing of pharmaceuticals has been centered around the idea of personalized patient-based 'on-demand' medication. Fused deposition modeling (FDM)-based 3D printing processes provide the capability to create complex geometrical dosage forms. However, the current FDM-based processes are associated with printing lag time and manual interventions. The current study tried to resolve this issue by utilizing the dynamic z-axis to continuously print drug-loaded printlets. Fenofibrate (FNB) was formulated with hydroxypropyl methylcellulose (HPMC AS LG) into an amorphous solid dispersion using the hot-melt extrusion (HME) process. Thermal and solid-state analyses were used to confirm the amorphous state of the drug in both polymeric filaments and printlets. Printlets with a 25, 50, and 75% infill density were printed using the two printing systems, i.e., continuous, and conventional batch FDM printing methods. Differences between the two methods were observed in the breaking force required to break the printlets, and these differences reduced as the infill density went up. The effect on in vitro release was significant at lower infill densities but reduced at higher infill densities. The results obtained from this study can be used to understand the formulation and process control strategies when switching from conventional FDM to the continuous printing of 3D-printed dosage forms.

Multifunctional Three-Dimensional Printed Copper Loaded Calcium Phosphate Scaffolds for Bone Regeneration.

Bone regeneration using inorganic nanoparticles is a robust and safe approach. In this paper, copper nanoparticles (Cu NPs) loaded with calcium phosphate scaffolds were studied for their bone regeneration potential in vitro. The pneumatic extrusion method of 3D printing was employed to prepare calcium phosphate cement (CPC) and copper loaded CPC scaffolds with varying wt% of copper nanoparticles. A new aliphatic compound Kollisolv MCT 70 was used to ensure the uniform mixing of copper nanoparticles with CPC matrix. The printed scaffolds were studied for physico-chemical characterization for surface morphology, pore size, wettability, XRD, and FTIR. The copper ion release was studied in phosphate buffer saline at pH 7.4. The in vitro cell culture studies for the scaffolds were performed using human mesenchymal stem cells (hMSCs). The cell proliferation study in CPC-Cu scaffolds showed significant cell growth compared to CPC. The CPC-Cu scaffolds showed improved alkaline phosphatase activity and angiogenic potential compared to CPC. The CPC-Cu scaffolds showed significant concentration dependent antibacterial activity in Staphylococcus aureus. Overall, the CPC scaffolds loaded with 1 wt% Cu NPs showed improved activity compared to other CPC-Cu and CPC scaffolds. The results showed that copper has improved the osteogenic, angiogenic and antibacterial properties of CPC scaffolds, facilitating better bone regeneration in vitro.

HPLC-UV Method Validation for Amobarbital and Pharmaceutical Stability Evaluation When Dispersed in a Hyaluronic Acid Hydrogel: A New Concept for Post-Traumatic Osteoarthritis Prevention.

A mitochondrial electron transport chain member complex I inhibitor, amobarbital, can reduce oxidative damage and chondrocyte death, eventually preventing post-traumatic osteoarthritis (PTOA). Viscosupplementation using a crosslinked hyaluronic acid (HA) hydrogel is currently applied clinically for knee OA pain relief. In this work, we utilized the HA hydrogel as a drug delivery vehicle to improve the long-term efficacy of amobarbital. Here we evaluated the pharmaceutic stability of amobarbital when dispersed in a crosslinked HA hydrogel formulated in proportions intended for clinical use. We validated a high-performance liquid chromatography with an ultraviolet detector (HPLC-UV) method following International Conference for Harmonization Q2(R1) guidelines to ensure its suitability for amobarbital detection. The feasibility of this formulation's drug delivery capability was proven by measuring the release, solubility, and drug uniformity. The amobarbital/HA hydrogel showed comparable amobarbital stability in different biological fluids compared to amobarbital solution. In addition, the amobarbital/HA hydrogel imparted significantly greater drug stability when stored at 70°C for 24 hours. In conclusion, we confirmed the pharmaceutical stability of the amobarbital/HA hydrogel in various conditions and biological fluids using a validated HPLC-UV method. This data provides essential evidence in support of the use of this amobarbital/HA formulation in future clinical trials for PTOA treatment.

Applications of nanotechnology in 3D printed tissue engineering scaffolds.

Tissue engineering is an interdisciplinary field that aims to combine life sciences and engineering to create therapies that regenerate functional tissue. Early work in tissue engineering mostly used materials as inert scaffolding structures, but research has shown that constructing scaffolds from biologically active materials can help with regeneration by enabling cell-scaffold interactions or release of factors that aid in regeneration. Three-dimensional (3D) printing is a promising technique for the fabrication of structurally intricate and compositionally complex tissue engineering scaffolds. Such scaffolds can be functionalized with techniques developed by nanotechnology research to further enhance their ability to stimulate regeneration and interact with cells. Nanotechnological components, nanoscale textures, and microscale/nanoscale printing can all be incorporated into the manufacture of 3D printed scaffolds. This review discusses recent advancements in the merging of nanotechnology with 3D printed tissue engineering scaffolds, with a focus on applications of nanoscale components, nanoscale texture, and innovative printing techniques and the effects observed in vitro and in vivo.

Polydopamine functionalized VEGF gene-activated 3D printed scaffolds for bone regeneration.

Bone is a highly vascularized organ and the formation of new blood vessels is essential to regenerate large critical bone defects. In this study, polylactic acid (PLA) scaffolds of 20-80% infill were three-dimensionally (3D) printed using a fused deposition modeling based 3D printer. The PLA scaffolds were coated with polydopamine (PDA) and then were surface-functionalized with polyethyleneimine (PEI) and VEGF-encoding plasmid DNA (pVEGF) nanoplexes (PLA-PDA-PEI-pVEGF). The PLA-PDA-PEI-pVEGF scaffolds with 40% infill demonstrated higher encapsulation efficiency and sustained release of pVEGF than scaffolds with 20, 60 and 80% infill and were therefore used for in vitro and in vivo studies. The PLA-PDA-PEI-pVEGF increased the translation and secretion of VEGF and BMP-2. The PLA-PDA-PEI-pVEGF also yielded a 2- and 4.5-fold change in VEGF and osteocalcin gene expression in vitro, respectively. A tube formation assay using human umbilical vascular endothelial cells (HUVECs) showed a significant increase in tube length when exposed to the PLA-PDA-PEI-pVEGF scaffold, in comparison to PLA and PLA-PDA scaffolds. The PLA-PDA-PEI-pVEGF scaffold in an in vivo rat calvarial critical bone defect model demonstrated 1.6-fold higher new bone formation compared to the PLA-PDA scaffold. H&E and Masson's trichrome staining of bone sections also revealed that the PLA-PDA-PEI-pVEGF scaffold facilitated the formation of more blood vessels in the newly formed bone compared to the PLA and PLA-PDA scaffold groups. Thus, PLA-PDA-PEI-pVEGF might be a potential 3D printed gene activated scaffold for bone regeneration in clinical situations.

A proof of concept gene-activated titanium surface for oral implantology applications.

Dental implants are very successful medical devices, yet implant failures do occur due to biological and mechanical complications. Peri-implantitis is one such biological complication that is primarily caused by bacteria and their products at the implant soft tissue interface. Bacterial infiltration can be prevented by the formation of a reliable soft tissue seal encircling dental implants. Platelet-derived growth factor-BB (PDGF-BB) has significant chemotactic and proliferative effects on various mesenchymal cell types, including fibroblasts, and therefore can be an effective molecule to enhance the peri-implant soft tissue seal. To overcome the limitations of the recombinant protein form of PDGF-BB, such as cost and the need for supraphysiological doses, we have developed and characterized a titanium surface that is rendered bioactive by coating it with polyethylenimine-plasmid DNA (pDNA) nanoplexes in the presence of sucrose. Human embryonic kidney 293T (HEK293T) cells and human primary gingival fibroblasts (GFs) were successfully transfected in culture with enhanced green fluorescent protein (EGFP)-encoding pDNA or platelet-derived growth factor subunit B (PDGFB)-encoding pDNA loaded into nanoplexes and coated onto titanium disks in a dose-dependent manner. GFs were shown to secrete PDGF-BB for at least 7 days after transfection and displayed both minimal viability loss and increased integrin-α2 expression 4 days posttransfection.

Inhibition of BMP9 Induced Bone Formation by Salicylic-acid Polymer Capping.

This work focuses on the development of a system to control the formation of bone to complement developments that have enabled potent regeneration of bony tissue. Scaffolds were fabricated with chemically modified RNA encoding for bone morphogenetic protein-9 (cmBMP9) and capped with salicylic acid (SA)-containing polymer (SAPAE). The goal was to determine if SAPAE could inhibit the formation of bone in a pilot animal study since cmBMP9 has been demonstrated to be highly effective in regenerating bone in a rat calvarial defect model. The results indicated that cmBMP9 increased bone formation (30% increase in area covered compared to control) and that SAPAE trended toward reducing the bone formation. These results suggest SAPAE could be useful as a chemical agent in reducing unwanted bone formation in implants loaded with cmBMP9.

Tissue Engineering for the Temporomandibular Joint.

Tissue engineering potentially offers new treatments for disorders of the temporomandibular joint which frequently afflict patients. Damage or disease in this area adversely affects masticatory function and speaking, reducing patients' quality of life. Effective treatment options for patients suffering from severe temporomandibular joint disorders are in high demand because surgical options are restricted to removal of damaged tissue or complete replacement of the joint with prosthetics. Tissue engineering approaches for the temporomandibular joint are a promising alternative to the limited clinical treatment options. However, tissue engineering is still a developing field and only in its formative years for the temporomandibular joint. This review outlines the anatomical and physiological characteristics of the temporomandibular joint, clinical management of temporomandibular joint disorder, and current perspectives in the tissue engineering approach for the temporomandibular joint disorder. The tissue engineering perspectives have been categorized according to the primary structures of the temporomandibular joint: the disc, the mandibular condyle, and the glenoid fossa. In each section, contemporary approaches in cellularization, growth factor selection, and scaffold fabrication strategies are reviewed in detail along with their achievements and challenges.