Large volume bone defects that do not spontaneously heal despite surgical stabilization (“critical-sized” defects) remain a challenge to treat clinically. Recent research investigating bone regenerative implants made from 3D printed materials have shown promise as a potential alternative to current treatment methods, such as autografting, allografting, and multi-step surgical interventions. Recent work has shown that implanting 3D printed calcium phosphate cement (CPC) scaffolds loaded with bone morphogenetic protein-2 (BMP-2) can provide a one-step surgical intervention that has similar bone healing outcomes to a popular two-step intervention: the Masquelet technique. The aim of this study was to investigate whether a 3D printed CPC scaffold loaded with a lyophilized polyplex gene-delivery formulation could serve as an alternative to loading BMP-2 protein onto such scaffolds. We 3D printed CPC scaffolds, hardened them with multiple methods, and explored the impact of these hardening methods on surface texture, mechanical strength, osteogenic differentiation, and ion flux. We then gene-activated these materials with cationic polyplexes containing plasmid DNA encoding reporter genes to investigate transfection from the gene-activated scaffolds. We found that incubating CPC scaffolds in aqueous solutions after initial hardening in a humid environment could enhance scaffold mechanical strength (compressive strength of 21.28 MPa vs. 6.54 MPa) and osteogenic differentiation. We also found that when we increased the total surface area of the CPC material exposed to polyplex solutions, there was a reduction in transfection via adsorption of polyplexes to the CPC surface. This study shows that 3D printed, gene-activated CPC scaffolds are a promising avenue for future exploration in the field of bone regeneration, though the level of gene expression induced by the scaffolds must be improved.

Titanium is the metal of choice for dental implants because of its biocompatibility and ability to merge with human bone tissue. Despite the great success rate of dental implants, early and late complications occur. Coating titanium dental implant surfaces with polyethyleneimine (PEI)-plasmid DNA (pDNA) polyplexes improve osseointegration by generating therapeutic protein expression at the implantation site. Lyophilization is an approach for stabilizing polyplexes and extending their shelf life; however, most lyoprotectants are sugars that can aid bacterial growth in the peri-implant environment. In our research, we coated titanium surfaces with polyplex solutions containing varying amounts of lyoprotectants. We used two common lyoprotectants (sucrose and polyvinylpyrrolidone K30) and showed for the first time that sucralose (a sucrose derivative used as an artificial sweetener) might act as a lyoprotectant for polyplex solutions. Human embryonic kidney (HEK) 293T cells were used to quantify the transfection efficiency and cytotoxicity of the polyplex/lyoprotectant formulations coating titanium surfaces. Polyplexes that were lyophilized in the presence of a lyoprotectant displayed both preserved particle size and high transfection efficiencies. Polyplexes lyophilized in 2% sucralose have maintained transfection efficacy for three years. These findings suggest that modifying dental implants with lyophilized polyplexes might improve their success rate in the clinic.

Choline and geranic acid (CAGE) ionic liquids have recently been shown to have applications in the delivery of macromolecules and poorly soluble drugs across epithelial barriers and in bacterial growth inhibition. Ionic liquids are known to denature proteins by the disruption of forces that guide natural protein folding, and the inflammatory enzyme elastase was recently shown to be inhibited by a variety of ionic liquids other than CAGE. Inhibition of collagenolytic enzymes, including elastase, has been shown to improve outcomes in cases of periodontitis via amelioration of periodontal inflammation and alveolar bone resorption. In this study, we investigated whether CAGE prepared with varying stoichiometries was able to inhibit elastase at varying concentrations and whether these CAGE formulations could inhibit the growth of key pathogenic bacterial species associated with oral health conditions. We found that CAGE was capable of inhibiting both porcine elastase and human neutrophil elastase at concentrations as low as 5 mM, and that CAGE formulations were effective at inhibiting the growth of all tested pathogenic oral bacteria. The inhibition of elastase by CAGE may be a mechanism by which CAGE can improve outcomes in periodontitis independent from CAGE's known antibacterial properties.

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. Threedimensional (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.

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.

The aim of tissue engineering as a field is to use cells and scaffolding materials to replicate native tissue structure and function. Tissue engineering scaffolds have been generated through a variety of manufacturing techniques including salt leaching, foaming, and electrospinning. Three-dimensional (3D) printing is a newer technology that allows the fabrication of complex structures, uniform pore sizes, and multiple materials incorporated in specific spatial ways. Nanoscale parameters of tissue engineering scaffolds have also been shown to influence the regenerative potential of scaffolds. Mimicry of native tissue microenvironments and modulation of nanotexture of scaffold material have been demonstrated to improve regenerative outcomes in vitro and in vivo, and research has demonstrated similar capabilities with 3D printed (3DP) scaffolds as well. Nanotechnological components have been incorporated into the printable materials, nanotextured surfaces have been crafted on printed scaffolds, and microscale to nanoscale printing techniques have been developed. All of these approaches have been demonstrated to influence the regenerative capacity of produced scaffolds and have differing impacts on cell interactions with the resultant scaffolds. Looking forward, more complex and specific nanotechnological modification techniques should be investigated. To date, most research has focused on the effects of single modifications to 3DP scaffolds. Future studies could incorporate multiple modifications demonstrated to have specific effects to enact improved regeneration to investigate more complex modulation of cellular responses. Techniques to manufacture nanoscale 3DP scaffolds must also be improved to yield improved cell–material interactions and meet the larger size scale required for clinical applications.

Large bone defects are usually managed by replacing lost bone with non-biological prostheses or with bone grafts that come from the patient or a donor. Bone tissue engineering, as a field, offers the potential to regenerate bone within these large defects without the need for grafts or prosthetics. Such therapies could provide improved long- and short-term outcomes in patients with critical-sized bone defects. Bone tissue engineering has long relied on the administration of growth factors in protein form to stimulate bone regeneration, though clinical applications have shown that using such proteins as therapeutics can lead to concerning off-target effects due to the large amounts required for prolonged therapeutic action. Gene-based therapies offer an alternative to protein-based therapeutics where the genetic material encoding the desired protein is used and thus loading large doses of protein into the scaffolds is avoided. Gene- and RNAi-activated scaffolds are tissue engineering devices loaded with nucleic acids aimed at promoting local tissue repair. A variety of different approaches to formulating gene- and RNAi-activated scaffolds for bone tissue engineering have been explored, and include the activation of scaffolds with plasmid DNA, viruses, RNA transcripts, or interfering RNAs. This review will discuss recent progress in the field of bone tissue engineering, with specific focus on the different approaches employed by researchers to implement gene-activated scaffolds as a means of facilitating bone tissue repair.

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.

3D printing offers unique solutions for bone tissue regeneration with an opportunity to engineer scaffolds with the desired structure, shape, and porosity. Scaffold’s surface-to-cell interaction is critical for better tissue integration and regeneration. To achieve this, we have surface modified the 3D printed scaffolds with polydopamine (PDA) to evaluate the potential of the surface modification for bone regeneration.

Research in bone tissue engineering aims to design materials that are effective at generating bone without causing significant side effects. The osteogenic potential of combining matrices and protein growth factors has been well documented, however, improvements are necessary to achieve optimal therapeutic benefits upon clinical translation. In this article, rat calvarial defects were treated with gene-activated matrices (GAMs). The GAMs used were collagen sponges mineralized with a simulated body fluid (SBF) containing a nonviral gene delivery system. Both in vitro and in vivo studies were performed to determine the optimal mode of gene delivery. After 6 weeks, the defects were extracted to assess bone formation and tissue quality through histological and microcomputed tomography analyses. The optimal GAM consisted of a collagen sponge with polyethylenimine plasmid DNA (PEI-pDNA) complexes embedded in a calcium phosphate coating produced by SBF, which increased total bone formation by 39% compared with 19% for control samples. A follow-up in vivo study was performed to optimize the ratio of growth factors included in the GAM. The optimal ratio for supporting bone formation after 6 weeks of implantation was five parts of pBMP-2 to three parts pFGF-2. These studies demonstrated that collagen matrices biomimetically mineralized and activated with plasmids encoding fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) can optimally improve bone regeneration outcomes. Impact statement Bone tissue engineering has explored both nonviral gene delivery and the concept of biomimetic mineralization. In this study, we combined these two concepts to further enhance bone regeneration outcomes. We demonstrated that embedding polyethylenimine (PEI)-based gene delivery within a mineral layer formed from simulated body fluid (SBF) immersion can increase bone formation rates. We also demonstrated that the ratio of growth factors utilized for matrix fabrication can impact the amount of bone formed in the defect site. This research highlights a combined approach using SBF and nonviral gene delivery both in vitro and in vivo and prepares the way for future optimization of synthetic gene activated matrices.

BACKGROUND:Calcium ions (Ca2+ ) influence natural bone healing, and calcium is frequently used in bone tissue engineering scaffolds and cements. Scaffolds can also incorporate gene delivery systems to further promote osteoblast differentiation. Thus, our goal was to identify if Ca2+ concentration affects the transfection of bone marrow stromal cells because these cells play a major role in bone healing and can infiltrate gene-activated scaffolds designed to promote bone growth.METHODS:Bone marrow-derived mesenchymal stem cells (BMSCs) were cultured in media with Ca2+ concentrations ranging from 0 to 20 mM and transfected with polyethyleneimine-plasmid DNA (PEI-pDNA) complexes. Cell viability and transfection efficiency were determined using MTS assays and flow cytometry, respectively. PEI-pDNA complex localization in BMSCs was assessed using fluorescence microscopy. To determine BMSC differentiation, messenger RNA (mRNA) for osteocalcin and CBFA1 was quantified using real time-polymerase chain reaction (PCR). Calcium deposition was qualitatively assessed after three and 14 days using Alizarin Red staining.RESULT:Our results indicate that Ca2+ levels between 8 and 12 mM positively impacted transfection of BMSCs with PEI-pDNA complexes in terms of cell viability and transfection efficiency. A Ca2+ concentration of 10 mM also increased the expression of an osteogenic gene, osteocalcin, when the cells were transfected with plasmid DNA encoding bone morphogenetic protein 2 (BMP-2).CONCLUSION:Ca2+ at a 10 mM concentration can significantly reduce toxicity and enhance transfection efficiency when combined with PEI-pDNA complexes, and this combination can be specifically applied to further enhance the differentiation of BMSCs by using the combination of polyethyleneimine-plasmid bone morphogenetic protein 2 (PEI-pBMP-2) and 10 mM Ca2+ as compared with PEI-pBMP-2 alone.

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.