Stereolithographic Rapid Prototyping of Clear, Foldable, Non-Refractive Intraocular Lens Designs: A Proof-of-Concept Study.

PURPOSE: A cataract is a cloudy area in the crystalline lens. Cataracts are the leading cause of blindness and the second cause of severe vision impairment worldwide. During cataract surgery, the clouded lens is extracted and replaced with an artificial intraocular lens, which restores the optical power. The fabrication of intraocular lenses using existing molding and lathing techniques is a complex and time-consuming process that limits the development of novel materials and designs. To overcome these limitations, we have developed a stereolithography-based process for producing models of clear lens designs without refractive function, serving as a proof of concept. This process has the potential to contribute toward new lens development, allowing for unlimited design iterations and an expanded range of materials for scientists to explore. METHODS: Lens-like 3D objects without refractive function were fabricated by using stereolithography. A photopolymerizable resin containing 2-phenoxyethyl acrylate, poly (ethylene glycol) dimethacrylate, and a suitable photoinitiator was developed for the production of lens-like 3D object prototypes. The morphology of the printed devices was characterized by scanning electron microscopy. The transparency and thermal properties were analyzed using spectrophotometry and differential scanning calorimetry, respectively. The biocompatibility of the devices was investigated in a cultured human lens cell line (FHL-124), using a standard lactate dehydrogenase assay, and the lenses were folded and implanted in the human capsular bag model. RESULTS: One-piece lens-like 3D objects without refractive function and with loop-haptic design were successfully fabricated using Stereolithography (SLA) technique. The resulting 3D objects were transparent, as determined by UV spectroscopy. The lactate dehydrogenase test demonstrated the tolerance of lens cells to the prototyping material, and apparent foldability and shape recovery was observed during direct injection into a human capsular bag model in vitro. CONCLUSIONS: This proof-of-principle study demonstrated the potential and significance of the rapid prototyping process for research and development of lens-like 3D object prototypes, such as intraocular lenses.

Synthesis and Formulation of Four-Arm PolyDMAEA-siRNA Polyplex for Transient Downregulation of Collagen Type III Gene Expression in TGF-ß1 Stimulated Tenocyte Culture.

The natural healing process for tendon repair is associated with high upregulation of collagen type III, leading to scar tissue and tendon adhesions with functionally deficient tendons. Gene delivery systems are widely reported as potential nanotherapeutics to treat diseases, providing a promising approach to modulate collagen type III synthesis. This work investigates a proof-of-concept four-arm cationic polymer-siRNA polyplex to mediate a transient downregulation of collagen type III expression in a tendon cell culture system. The tendon culture system was first supplemented with TGF-ß1 to stimulate the upregulation of collagen type III prior to silencing experiments. The four-arm poly[2-(dimethylamino) ethyl acrylate] (PDMAEA) polymer was successfully synthesized via RAFT polymerization and then mixed with siRNA to formulate the PDMAEA-siRNA polyplexes. The formation of the polyplex was optimized for the N:P ratio (10:1) and confirmed by agarose gel electrophoresis. The size and solution behavior of the polyplex were analyzed by dynamic light scattering and zeta potential, showing a hydrodynamic diameter of 155 ± 21 nm and overall positive charge of +30 mV at physiological pH. All the polyplex concentrations used had a minimal effect on the metabolic activity of cultured cells, indicating good biocompatibility. The dose and time effects of the TGF-ß1 on collagen type III gene expressions were analyzed by qPCR, showing an optimal dose of 10 ng mL-1 TGF-ß1 and 3-fold increase of COL3α1 expression at 48 h in cultured tenocytes. The PDMAEA-siRNA polyplex concept observed a limited yet successful and promising efficiency in silencing collagen type III at 48 h compared to PEI-siRNA. Therefore, this concept is a promising approach to reduce tissue scarring and adhesion following injuries.

Induction of Tendon-Specific Markers in Adipose-Derived Stem Cells in Serum-Free Culture Conditions.

IMPACT STATEMENT: Herein, we describe the tenogenic effect of bone morphogenetic protein-12 and transforming growth factor-ß1 in cultured adipose-derived stem cells (ADSCs) in serum-free conditions. This culture system provides an insight into serum-free culture conditions in stem cell differentiation protocols. A positive response of the ADSCs to the tenogenic induction was observed. In particular, the different growth factors used in this study displayed notable differences both on the gene and on the protein expression of the tendon-specific markers. The results underline the positive outcome of the serum removal in tenogenic differentiation protocols, contributing to the development of future cell-based therapies for tendon regeneration and repair.

Biocompatible Three-Dimensional Printed Thermoplastic Scaffold for Osteoblast Differentiation of Equine Induced Pluripotent Stem Cells.

Horses, like humans, can experience bone fractures and due to their large size and the need to bear weight on all limbs during the recovery period, they can be difficult to treat. Surgical techniques to improve fracture repair are improving, but to date, regenerative medicine technologies to aid fracture healing are not commonly applied in horses. We have previously demonstrated that equine induced pluripotent stem cells (iPSCs) can be differentiated into bone forming osteoblasts in 2D culture. In this study, we report on the use of a thermoplastic, 3D-printed polymer to provide a scaffold for successful, in vitro osteoblast differentiation of equine iPSCs. The scaffold provides a transparent, cost-effective solution to allow the analysis of osteoblast differentiation using live-cell imaging, immunohistochemistry, and quantitative polymerase chain reaction. This in vitro study demonstrates the future feasibility of generating 3D bone constructs through the cell seeding of scaffolds to use in regenerative medicine strategies to improve fracture repair in a relevant, large animal model.

Synthesis and Fabrication of Surface-Active Microparticles Using a Membrane Emulsion Technique and Conjugation of Model Protein via Strain-Promoted Azide-Alkyne Click Chemistry in Physiological Conditions.

The rapid surface immobilization of protein on monodispersed polyester microcarriers is reported. A model protein, functionalized with a dibenzocyclooctyne core, immobilizes on the surface of azide-terminal polycaprolactone microcarriers within 10 min compared to 12 h for other conjugation techniques, and it is conducted in physiological conditions and in the absence of coupling reagents.

A Clinical, Biological, and Biomaterials Perspective into Tendon Injuries and Regeneration.

Tendon injury is common and debilitating, and it is associated with long-term pain and ineffective healing. It is estimated to afflict 25% of the adult population and is often a career-ending disease in athletes and racehorses. Tendon injury is associated with high morbidity, pain, and long-term suffering for the patient. Due to the low cellularity and vascularity of tendon tissue, once damage has occurred, the repair process is slow and inefficient, resulting in mechanically, structurally, and functionally inferior tissue. Current treatment options focus on pain management, often being palliative and temporary and ending in reduced function. Most treatments available do not address the underlying cause of the disease and, as such, are often ineffective with variable results. The need for an advanced therapeutic that addresses the underlying pathology is evident. Tissue engineering and regenerative medicine is an emerging field that is aimed at stimulating the body's own repair system to produce de novo tissue through the use of factors such as cells, proteins, and genes that are delivered by a biomaterial scaffold. Successful tissue engineering strategies for tendon regeneration should be built on a foundation of understanding of the molecular and cellular composition of healthy compared with damaged tendon, and the inherent differences seen in the tissue after disease. This article presents a comprehensive clinical, biological, and biomaterials insight into tendon tissue engineering and regeneration toward more advanced therapeutics.

A thermoresponsive and magnetic colloid for 3D cell expansion and reconfiguration.

A dual thermoresponsive and magnetic colloidal gel matrix is described for enhanced stem-cell culture. The combined properties of the material allow enzyme-free passaging and expansion of mesenchymal stem cells, as well as isolation of cells postculture by the simple process of lowering the temperature and applying an external magnetic field. The colloidal gel can be reconfigured with thermal and magnetic stimuli to allow patterning of cells in discrete zones and to control movement of cells within the porous matrix during culture.

Evaluation of a thermoresponsive polycaprolactone scaffold for in vitro three-dimensional stem cell differentiation.

Tissue engineering (TE) strategies aim at imitating the natural process of regeneration by using bioresorbable scaffolds that support cellular attachment, migration, proliferation, and differentiation. Based on the idea of combining a fully degradable polymer [poly(ɛ-caprolactone)] with a thermoresponsive polymer (polyethylene glycol methacrylate), a scaffold was developed, which liquefies below 20°C and solidifies at 37°C. In this study, this scaffold was evaluated for its ability to support C2C12 cells and human adipose-derived stem cells (ASCs) to generate an expandable three-dimensional (3D) construct for soft or bone TE. As a first step, biomaterial seeding was optimized and cellular attachment, survival, distribution, and persistence within the 3D material were characterized. C2C12 cells were differentiated toward the osteogenic as well as myogenic lineage, while ASCs were cultured in control, adipogenic, or osteogenic differentiation media. Differentiation was examined using quantitative real-time PCR for the expression of osteogenic, myogenic, and adipogenic markers and by enzyme activity and immunoassays. Both cell types attached and were found evenly distributed within the material. C2C12 cells and ASCs demonstrated the potential to differentiate in all tested lineages under 2D conditions. Under 3D osteogenic conditions for C2C12 cells, only osteocalcin expression (fold induction: 16.3±0.2) and alkaline phosphatase (ALP) activity (p<0.001) were increased compared with the control C2C12 cells. Three-dimensional osteogenic differentiation of ASC was limited and donor dependent. Only one donor showed an increase in the osteogenic markers osteocalcin (p=0.027) and osteopontin (p=0.038). In contrast, differentiation toward the myogenic or adipogenic lineage showed expression of specific markers in 3D, at least at the level of the 2D culture. In 3D culture, strong induction of myogenin (p<0.001) as well as myoD (p<0.001) was found in C2C12 cells. The adipogenic differentiation of one donor showed greater expression of peroxisome proliferative-activated receptor gamma (PPARγ) (p=0.004), fatty acid binding protein 4 (FABP4) (p=0.008), and adiponectin (p=0.045) in 3D compared with 2D culture. Leptin levels in the supernatant of the ASC cultures were elevated in the 3D cultures in both donors at day 14 and 21. In conclusion, the thermoresponsive scaffold was found suitable for 3D in vitro differentiation toward soft tissue.

Programmable polymer-DNA hydrogels with dual input and multiscale responses.

Combination switchable polymer-DNA hydrogels have been synthesized to respond to both a specific oligonucleotide recognition signal and a non-specific but biorelevant environmental trigger. The hydrogels exhibit rheological properties that can be modulated through interaction with complementary DNA strands and/or reduction. Furthermore, individual and combined oligonucleotide recognition and reduction responses allow control over pore sizes in the gel, enabling programmable release and transport of objects ranging from the nano- to micro-scale.

Gelatin embedding for the preparation of thermoreversible or delicate scaffolds for histological analysis.

Thermoreversible hydrogels for tissue engineering (TE) purposes have gained increased attention in recent years as they can be combined with cells and drugs and directly injected into the body. Following the fate of transplanted cells in situ is essential in characterizing their distribution and survival, as well as the expression of specific markers or cell-matrix interactions. Existing histological embedding methods, such as paraffin wax embedding, can mechanically damage some biomaterials during processing. In this study, we describe a broadly applicable preparation protocol that allows the handling of delicate, thermoreversible scaffolds for histological sectioning. The gelatin solution permits the embedding of samples at 37 °C, which suits the solid phase of most TE scaffolds. A thermoreversible scaffold of polycaprolactone microparticles, combined with poly(polyethylene glycol methacrylate ethyl ether) and containing human adipose-derived stem cells, was prepared for histology by an initial gelatin embedding step in addition to the standard cryosectioning and paraffin processing protocols. Sections were evaluated by hematoxylin eosin staining and immunostaining for human vimentin. The gelatin embedding retained the scaffold particles and permitted the complete transfer of the construct. After rapid cooling, the solid gelatin blocks could be cryosectioned and paraffin infiltrated. In contrast to direct cryosectioning or paraffin infiltration, the extended protocol preserved the scaffold structure as well as the relevant cell epitopes, which subsequently allowed for immunostaining of human cells within the material. The gelatin embedding method proposed is a generalizable alternative to standard preparations for histological examination of a variety of delicate samples.

"One-step" preparation of thiol-ene clickable PEG-based thermoresponsive hyperbranched copolymer for in situ crosslinking hybrid hydrogel.

A well-defined poly(ethylene glycol) based hyperbranched thermoresponsive copolymer with high content of acrylate vinyl groups was synthesized via a "one-pot and one-step" deactivation enhanced atom transfer radical polymerization approach, which provided an injectable and in situ crosslinkable system via Michael-type thiol-ene reaction with a thiol-modified hyaluronan biopolymer. The hyperbranched structure, molecular weight, and percentage of vinyl content of the copolymer were characterized by gel permeation chromatography and (1)H NMR. The lower critical solution temperature of this copolymer is close to body temperature, which can result in a rapid thermal gelation at 37 °C. The scanning electron microscopy analysis of crosslinked hydrogel showed the network formation with porous structure, and 3D cell culture study demonstrated the good cell viability after the cells were embedded inside the hydrogel. This injectable and in situ crosslinking hybrid hydrogel system offers great promise as a new class of hybrid biomaterials for tissue engineering.

Thermoresponsive hyperbranched copolymer with multi acrylate functionality for in situ cross-linkable hyaluronic acid composite semi-IPN hydrogel.

Thermoresponsive polymers have been widely used for in situ formed hydrogels in drug delivery and tissue engineering as they are easy to handle and their shape can easily conform to tissue defects. However, non-covalent bonding and mechanical weakness of these hydrogels limit their applications. In this study, a physically and chemically in situ cross-linkable hydrogel system was developed from a novel thermoresponsive hyperbranched PEG based copolymer with multi acrylate functionality, which was synthesized via an 'one pot and one step' in situ deactivation enhanced atom transfer radical co-polymerization of poly(ethylene glycol) diacrylate (PEGDA, M(n) = 258 g mol(-1)), poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, M(n )= 475 g mol(-1)) and (2-methoxyethoxy) ethyl methacrylate (MEO(2)MA). This hyperbranched copolymer was tailored to have the lower critical solution temperature to form physical gelation around 37°C. Meanwhile, with high level of acrylate functionalities, a chemically cross-linked gel was formed from this copolymer using thiol functional cross-linker of pentaerythritol tetrakis (3-mercaptopropionate) (QT) via thiol-ene Michael addition reaction. Furthermore, a semi-interpenetrated polymer networks (semi-IPN) structure was developed by combining this polymer with hyaluronic acid (HA), leading to an in situ cross-linkable hydrogel with significantly increased porosity, enhanced swelling behavior and improved cell adhesion and viability both in 2D and 3D cell culture models.

The reverse of polymer degradation: in situ crosslinked gel formation through disulfide cleavage.

In contrast to the usual inclusion of disulfide bonds within a polymer structure to facilitate bio-degradation, we have designed a self-curing "one pot" hyperbranched polymer capable of forming crosslinked gels under reducing conditions abundant in tumoural tissue.

Modular construction of multifunctional bioresponsive cell-targeted nanoparticles for gene delivery.

Multifunctional and modular block copolymers prepared from biocompatible monomers and linked by a bioreducible disulfide linkage have been prepared using a combination of ring-opening and atom-transfer radical polymerizations (ATRP). The presence of terminal functionality via ATRP allowed cell-targeting folic acid groups to be attached in a controllable manner, while the block copolymer architecture enabled well-defined nanoparticles to be prepared by a water-oil-water double emulsion procedure to encapsulate DNA with high efficiency. Gene delivery assays in a Calu-3 cell line indicated specific folate-receptor-mediated uptake of the nanoparticles, and triggered release of the DNA payload via cleavage of the disulfide link resulted in enhanced transgene expression compared to nonbioreducible analogues. These materials offer a promising and generic means to deliver a wide variety of therapeutic payloads to cells in a selective and tunable way.

Facile synthesis of responsive nanoparticles with reversible, tunable and rapid thermal transitions from biocompatible constituents.

Responsive polymeric nanoparticles composed of hybrid block co-polymers were prepared from biocompatible components that displayed rapid, tunable and multiply reversible transitions in response to change of temperature.

Ion-sensitive "isothermal" responsive polymers prepared in water.

Ion-sensitive responsive polymers are prepared under fully aqueous conditions using controlled radical polymerization. Variations in comonomer content and sequence lead to temperature and salt-dependent solution behavior, with cloud-points ranging by +/-40 degrees C following addition of Hofmeister series salts. A "hybrid" block copolymer, composed of a statistical sequence of monomers tipped with a hydrophilic block, formed stable micelle-like assemblies that exhibited burst release of an encapsulated model drug in response to addition of a kosmotrope, Na2SO4, at room temperature.