Gene regulatory and gene editing tools and their applications for retinal diseases and neuroprotection: From proof-of-concept to clinical trial.

Gene editing and gene regulatory fields are continuously developing new and safer tools that move beyond the initial CRISPR/Cas9 technology. As more advanced applications are emerging, it becomes crucial to understand and establish more complex gene regulatory and editing tools for efficient gene therapy applications. Ophthalmology is one of the leading fields in gene therapy applications with more than 90 clinical trials and numerous proof-of-concept studies. The majority of clinical trials are gene replacement therapies that are ideal for monogenic diseases. Despite Luxturna's clinical success, there are still several limitations to gene replacement therapies including the size of the target gene, the choice of the promoter as well as the pathogenic alleles. Therefore, further attempts to employ novel gene regulatory and gene editing applications are crucial to targeting retinal diseases that have not been possible with the existing approaches. CRISPR-Cas9 technology opened up the door for corrective gene therapies with its gene editing properties. Advancements in CRISPR-Cas9-associated tools including base modifiers and prime editing already improved the efficiency and safety profile of base editing approaches. While base editing is a highly promising effort, gene regulatory approaches that do not interfere with genomic changes are also becoming available as safer alternatives. Antisense oligonucleotides are one of the most commonly used approaches for correcting splicing defects or eliminating mutant mRNA. More complex gene regulatory methodologies like artificial transcription factors are also another developing field that allows targeting haploinsufficiency conditions, functionally equivalent genes, and multiplex gene regulation. In this review, we summarized the novel gene editing and gene regulatory technologies and highlighted recent translational progress, potential applications, and limitations with a focus on retinal diseases

3D printing of silver-doped polycaprolactone-poly(propylene succinate) composite scaffolds for skin tissue engineering

Scaffold-based tissue engineering approaches have been commonly used for skin regeneration or wound healings caused by diseases or trauma. For an ideal complete healing process, scaffold structures need to meet the criteria of biocompatibility, biodegradability, and antimicrobial properties, as well as to provide geometrical necessities for the regeneration of damaged tissue. In this study, design, synthesis and characterization of a three dimensional (3D) printable copolymer based on polycaprolactone-block-poly(1,3-propylene succinate) (PCL-PPSu) including anti-microbial silver particles is presented. 3D printing of PCL-PPSu copolymers provided a lower processing temperature compared to neat PCL, hence, inclusion of temperature-sensitive bioactive reagents into the developed copolymer could be realized. In addition, 3D printed block copolymer showed an enhanced hydrolytic and enzymatic degradation behavior. Cell viability and cytotoxicity of the developed copolymer were evaluated by using human dermal fibroblast (HDF) cells. The addition of silver nitrate within the polymer matrix resulted in a significant decrease in the adhesion of different types of microorganisms on the scaffold without inducing any cytotoxicity on HDF cells in vitro. The results suggested that 3D printed PCL-PPSu scaffolds containing anti-microbial silver particles could be considered as a promising biomaterial for emerging skin regenerative therapies, in the light of its adaptability to 3D printing technology, low-processing temperature, enhanced degradation behavior and antimicrobial properties.</p

Hybrid 3D bioprinting of functionalized structures for tissue engineering

Tissue engineering is an interdisciplinary field of research aiming at developing methods and technologies for regenerating damaged tissues. It relies on a combinatory platform of biomaterials with cells and bioactive molecules to resemble the human microenvironment to stimulate tissue constructs. Hence, numerous factors, including biochemical, biophysical, and mechanical aspects of the host tissue, have to be taken into account for developing a successful tissue replacement. Skin replacements caused by traumas, injuries, and burns are a burden to the healthcare system globally. The human body cannot fully regenerate the tissue with all the functionalities and features in severe wounds or skin loss. Poor mechanical properties, scarring, delayed cell and biomolecules infiltration, and non/poor vascularization are the main challenges yet to be addressed. Three-dimensional (3D) bioprinting, also known as additive manufacturing (AM), a layer-by-layer fabrication method, is regarded as a gold standard technique with the ability of controlled deposition of biomaterials in the desired geometry by using computer-aided design (CAD) models. Together with the development of biomaterials and architecture design, 3D bioprinting could ease the long and complicated journey towards functional tissue regeneration. In this context, the fabrication of small fibers mimicking natural extracellular matrix (ECM), selection of functional material with good mechanical and biochemical properties, the inclusion of bioactive molecules to enhance functionality, and printability are prerequisite factors of successful scaffold fabrication. In this work, novel hybrid 3D bioprinting approaches have been developed for functionalized structures, mainly for skin tissue engineering. Within this framework, we first optimized the effect of printing parameters on fiber diameter for Melt Electrospinning Writing (MEW), a special 3D printing process, using response surface methodology (RSM) as a predictive tool. Then we copolymerized polycaprolactone (PCL) with polypropylene succinate to improve its degradation rate and hydrophilicity and functionalized it with silver nitrate to induce antibacterial properties, and finally, it was 3Dprinted using an extrusion-based printer. For preparing hybrid 3D bioprinting, we used a composite support-bath system based on Pluronic PF127 was formulated with the inclusion of Laponite RDS and calcium chloride as rheological modifier and stabilizer, respectively. The rheological characterization of support-bath showed thixotropic behavior with a high degree of recoverability which facilitated bioprinting of complex hydrogel structures within the support-bath through an extrusion system. Then, we fabricated a polymer-hydrogel construction using MEW-casting for skin tissue substitute. In this context, we first investigated the geometrical effect of melt electrowritten scaffolds on cord-like structure formation for pre-vascularization. Mesh scaffolds with 0-90and 60-120 degree orientations and honeycomb shape were explored and cell-laden gelatin hydrogels were infiltrated inside those PCL scaffolds, and the results suggested the potential of honeycomb structure for better mechanical and invitro properties. In the final stage, a functionalized hybrid MEW-hydrogel scaffold for wound healing was fabricated. A functionalized mesh structure of PCL-bioactive glass was created via MEW, and a gelatin hydrogel comprising basic fibroblast and vascular endothelial growth factors was cast within the mesh scaffold. In vivo implantation of hybrid scaffolds showed promising results for accelerating and functionality of the healed parts according to wound closure and histological evaluation

Preparation and characterization of nanoclay-hydrogel composite support-bath for bioprinting of complex structures

Three-dimensional bioprinting of cell-laden hydrogels in a sacrificial support-bath has recently emerged as a potential solution for fabricating complex biological structures. Physical properties of the support-bath strongly influence the bioprinting process and the outcome of the fabricated constructs. In this study, we reported the application of a composite Pluronic-nanoclay support-bath including calcium ions as the crosslinking agent for bioprinting of cell-laden alginate-based hydrogels. By tuning the rheological properties, a shear-thinning composite support-bath with fast self-recovery behavior was yielded, which allowed continuous printing of complex and large-scale structures. The printed structures were easily and efficiently harvested from the support-bath without disturbing their shape fidelity. Moreover, the results showed that support-bath assisted bioprinting process did not influence the viability of cells encapsulated within hydrogel. This study demonstrates that Pluronic-nanoclay support-bath can be utilized for bioprinting of complex, cell-laden constructs for vascular and other tissue engineering applications

Biomimicry in bio-manufacturing: developments in melt electrospinning writing technology towards hybrid biomanufacturing

Melt electrospinning writing has been emerged as a promising technique in the field of tissue engineering, with the capability of fabricating controllable and highly ordered complex three-dimensional geometries from a wide range of polymers. This three-dimensional (3D) printing method can be used to fabricate scaffolds biomimicking extracellular matrix of replaced tissue with the required mechanical properties. However, controlled and homogeneous cell attachment on melt electrospun fibers is a challenge. The combination of melt electrospinning writing with other tissue engineering approaches, called hybrid biomanufacturing, has introduced new perspectives and increased its potential applications in tissue engineering. In this review, principles and key parameters, challenges, and opportunities of melt electrospinning writing, and particularly, recent approaches and materials in this field are introduced. Subsequently, hybrid biomanufacturing strategies are presented for improved biological and mechanical properties of the manufactured porous structures. An overview of the possible hybrid setups and applications, future perspective of hybrid processes, guidelines, and opportunities in different areas of tissue/organ engineering are also highlighted

Effect of zinc-doped hydroxyapatite/graphene nanocomposite on the physicochemical properties and osteogenesis differentiation of 3D-printed polycaprolactone scaffolds for bone tissue engineering

Processing and composition can significantly affect the mechanobiology, biodegradability, and cellular behavior of polymer-based bone scaffolds to replace damaged bone tissue. In this research, hydroxyapatite (HA), zinc-doped HA (ZnHA), and ZnHA-graphene (ZnHA-rGO) nanoparticles are composed in a polycaprolactone (PCL) matrix. After compositing PCL with nanoparticles, 3D bone scaffolds were built by a custom-built 3D printing system. The characterization of nanoparticles was extensively investigated by TEM, EDX-MAP, XRD, and ATR-FTIR. Simultaneously, 3D-printed scaffolds with different compositions were studied in terms of structure, morphology, thermogravimetry, biodegradability, and mechanical behaviors. The FE-SEM images of the scaffolds showed a highly regular structure and good printability of the developed material system. Moreover, the stiffness modulus of the samples increased due to the presence of the nanoparticles, especially in the ZnHA-rGO nanocomposite. In vitro cell assessment of 3D bone scaffolds was investigated by cell viability tests, cell attachment, and alizarin red staining via mesenchymal stem cells (MSCs). For differentiation capacity of the developed scaffolds, stem cell osteogenesis differentiation was studied by RT-PCR to analyze the ALP, RUNX2, BMP2, TGFβ, and OCN genes. The cellular assessments revealed an increase in PCL scaffold's cell osteogenesis due to the HA nanoparticles in the scaffold matrix. Zinc doping in the HA nanoparticles and rGO addition significantly increased the osteogenesis of MSCs. In particular, the nanocomposite of ZnHA-rGO in PCL scaffold matrix significantly improved the osteogenic differentiation and, thus, it is a viable option for effective regeneration of damaged bone tissue

Embedded 3D printing of cryogel-based scaffolds

Cryogel-based scaffolds have attracted great attention in tissue engineering due to their interconnected macroporous structures. However, three-dimensional (3D) printing of cryogels with a high degree of precision and complexity is a challenge, since the synthesis of cryogels occurs under cryogenic conditions. In this study, we demonstrated the fabrication of cryogel-based scaffolds for the first time by using an embedded printing technique. A photo-cross-linkable gelatin methacryloyl (GelMA)-based ink composition, including alginate and photoinitiator, was printed into a nanoclay-based support bath. The layer-by-layer extruded ink was held in complex and overhanging structures with the help of pre-cross-linking of alginate with Ca2+ present in the support bath. The printed 3D structures in the support bath were frozen, and then GelMA was cross-linked at a subzero temperature under UV light. The printed and cross-linked structures were successfully recovered from the support bath with an integrated shape complexity. SEM images showed the formation of a 3D printed scaffold where porous GelMA cryogel was integrated between the cross-linked alginate hydrogels. In addition, they showed excellent shape recovery under uniaxial compression cycles of up to 80% strain. In vitro studies showed that the human fibroblast cells attached to the 3D printed scaffold and displayed spread morphology with a high proliferation rate. The results revealed that the embedded 3D printing technique enables the fabrication of cytocompatible cryogel based scaffolds with desired morphology and mechanical behavior using photo-cross-linkable bioink composition. The properties of the cryogels can be modified by varying the GelMA concentration, whereby various shapes of scaffolds can be fabricated to meet the specific requirements of tissue engineering applications

Design and bioprinting for tissue interfaces

Tissue interfaces include complex gradient structures formed by transitioning of biochemical and mechanical properties in micro-scale. This characteristic allows the communication and synchronistic functioning of two adjacent but distinct tissues. It is particularly challenging to restore the function of these complex structures by transplantation of scaffolds exclusively produced by conventional tissue engineering methods. Three-dimensional (3D) bioprinting technology has opened an unprecedented approach for precise and graded patterning of chemical, biological and mechanical cues in a single construct mimicking natural tissue interfaces. This paper reviews and highlights biochemical and biomechanical design for 3D bioprinting of various tissue interfaces, including cartilage-bone, muscle-tendon, tendon/ligament-bone, skin, and neuro-vascular/muscular interfaces. Future directions and translational challenges are also provided at the end of the paper

Design and 3D printing of personalized hybrid and gradient structures for critical size bone defects

Treating critical-size bone defects with autografts, allografts, or standardized implants is challenging since the healing of the defect area necessitates patient-specific grafts with mechanically and physiologically relevant structures. Three-dimensional (3D) printing using computer-aided design (CAD) is a promising approach for bone tissue engineering applications by producing constructs with customized designs and biomechanical compositions. In this study, we propose 3D printing of personalized and implantable hybrid active scaffolds with a unique architecture and biomaterial composition for critical-size bone defects. The proposed 3D hybrid construct was designed to have a gradient cell-laden poly(ethylene glycol) (PEG) hydrogel, which was surrounded by a porous polycaprolactone (PCL) cage structure to recapitulate the anatomical structure of the defective area. The optimized PCL cage design not only provides improved mechanical properties but also allows the diffusion of nutrients and medium through the scaffold. Three different designs including zigzag, zigzag/spiral, and zigzag/spiral with shifting the zigzag layers were evaluated to find an optimal architecture from a mechanical point of view and permeability that can provide the necessary mechanical strength and oxygen/nutrient diffusion, respectively. Mechanical properties were investigated experimentally and analytically using finite element analysis (FEA), and computational fluid dynamics (CFD) simulation was used to determine the permeability of the structures. A hybrid scaffold was fabricated via 3D printing of the PCL cage structure and a PEG-based bioink comprising a varying number of human bone marrow mesenchymal stem cells (hBMSCs). The gradient bioink was deposited inside the PCL cage through a microcapillary extrusion to generate a mineralized gradient structure. The zigzag/spiral design for the PCL cage was found to be mechanically strong with sufficient and optimum nutrient/gas axial and radial diffusion while the PEG-based hydrogel provided a biocompatible environment for hBMSC viability, differentiation, and mineralization. This study promises the production of personalized constructs for critical-size bone defects by printing different biomaterials and gradient cells with a hybrid design depending on the need for a donor site for implantation

3D Bioprinting of molecularly engineered PEG-based hydrogels utilizing gelatin fragments

Three-dimensional (3D) bioprinting is an additive manufacturing process in which the combination of biomaterials and living cells, referred to a bioink, are deposited layer-by-layer to form biologically active 3D tissue constructs. Recent advancements in the field show that the success of this technology highly depends on the development of novel biomaterials or the improvement of existing bioinks for bioprinting. Polyethylene glycol (PEG) is one of the well-known synthetic biomaterials and has been commonly used as a photocrosslinkable bioink for bioprinting. In this work, we proposed a novel micro-capillary based approach for bioprinting of molecularly engineered PEG-based bioink. PEG was firstly functionalized with cell-adhesive RGD ligands, which was then cross-linked using protease-sensitive peptides via Michael-type addition reaction inside the micro-capillary before bioprinting processes. Low molecular weight gelatin fragments (LMWG) were supplemented into the bioink to extrude smooth cylindrical strands of the hydrogel for shape fidelity. The cells encapsulated in the bioprinted PEG-based bioink showed high viability and continued to proliferate over time in culture in well-defined cell-morphology. In summary, the presented micro-capillary based bioprinting process for a PEG-based hydrogel system can be promising to construct the complex 3D structures with spatiotemporal variations without using any cytotoxic photoinitiator, UV light, or polymer support