The human cornea as a model tissue for additive biomanufacturing: a review

© 2016 Published by Elsevier B.V. The recent development and clinical success of molded recombinant collagen corneal substitutes offers a tantalizing prospect for the application of additive biomanufacturing in corneal tissue engineering and regenerative medicine (TERM). The cornea has a number of advantages over other tissues for additive biomanufacturing in that it is relatively homogeneous and completely avascular and has low metabolic demand. Additive biomanufacturing could bring a number of advantages over molding to corneal TERM, including increased visual acuity and mechanical strength. We have developed a method for producing thin collagen films, which are suitable for the culturing of corneal mesenchymal stem cells (MSCs), using additive manufacturing techniques, and we hope to use this technique to produce functional corneal substitutes.status: publishe

Additive manufacturing of recombinant collagen type III (RHC III) corneal substitute for tissue engineering

Introduction: The advent of clinically successful moulded RHC III corneal substitutes has paved a possible path for additive manufacturing to expand into ophthalmology. The cornea has a number of advantages over other tissues for additive biomanufacturing, being a relatively homogeneous and avascular tissue. However, collagen has had limited success in additive manufacturing, and is used most often in composite inks alongside other hydrogels with better gelation kinetics. In this work, we present an additive manufacturing as a technique to produce thin RHC III films and explore its potential for mimicking corneal tissue. Materials and Methods: RHC III was deposited in multiple layers by nebulizing a 3.4mg/ml RHCIII solution via sonication and accelerating it through a small nozzle of 100µm -300µm diameter towards a substrate with an inert gas. The print direction imparted a pattern on the sample, which was exploited to lay orthogonal layers with small rotations in order to achieve isotropy. The samples were subsequently crosslinked using EDC (1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide methiodide) and NHS (N-hydroxysuccinimide). Gel electrophoresis was used to investigate the effect of the nebulization process on the material. Human corneal mesenchymal stem cells (MSCs) were seeded at a density of 1000 cells per 6.5 mm diameter sample and imaged using an IncuCyte ZOOM Live cell imaging system. Results: Thin RHC III films were produced using this method with resolution as low as 10µm, a layer thickness under 1µm and different printed patterns (see Fig.1a-b-c). Gel electrophoresis showed that the α-1 chains of the RHC III stayed intact throughout sonication. The RHC III films showed improved stability and handling after crosslinking. Human corneal MSCs attached to the RHC III films two hours after seeding and did so in an aligned fashion, following the print lines (see Fig1d). Discussion: Despite the limited use of collagen as stand-alone structural material, we demonstrate the use of combining nebulizing additive manufacturing techniques and cross-linking to produce thin RHC III films with customized patterns. No denaturation of the collagen was observed due to the nebulization process. Early following seeding, we observed that the MSCs preferentially attached to the surface and proliferated following the RHCIII print pattern. At this stage, MSCs could potentially be induced to differentiate into keratocytes influencing them to deposit their extracellular matrix in an aligned manner. Further experiments are required to substantiate this. Conclusion: These results show promise for the combined use of printing techniques and cross-linking procedures in the fabrication of RHC III corneal scaffold which can be seeded with mesenchymal stem cells to produce a tissue engineered corneal substitute.status: publishe

High resolution 3D printing of collagen for corneal tissue engineering

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Xeno-Free Cultivation of Mesenchymal Stem Cells From the Corneal Stroma

PURPOSE. The human cornea has recently been described as a source of corneal stromaderived mesenchymal stem cells (hMSCs). In vitro expansion of these cells involves basal medium supplemented with fetal bovine serum (FBS). As animal-derived serum can confer a risk of disease transmission and can be subject to considerable lot-to-lot variability, it does not comply with newer Good Manufacturing Practice (GMP)-required animal component-free culture protocols for clinical translation. METHODS. This study investigated animal-free alternatives to FBS for cultivation of human corneal stromal MSCs. Proliferative capacity was studied for cultures supplemented with different concentrations (2.5%, 5%, and 10%) of FBS, human AB serum, human platelet lysate (HPL), and XerumFree. Unsupplemented basal medium was used as a control. The expression of specific hMSC markers À , and HLA-DR À ) and trilineage differentiation (adipogenesis, osteogenesis, and chondrogenesis) were compared for the two outperforming supplements: 10% FBS and HPL. RESULTS. HPL is the only consistent non-xeno supplement where hMSC cultures show significantly higher proliferation than the 10% FBS-supplemented cultures. Both FBS-and HPLsupplemented hMSC cultures showed plastic adherence and trilineage differentiation, and no significant differences were found in the expression of the hMSC marker panel. No significant differences in stemness were detected between FBS and HPL cultures. CONCLUSIONS. We conclude that HPL is the best supplement for expansion of human corneal stromal MSCs. HPL significantly outperforms human AB serum, the chemically defined XerumFree, and even the gold standard, FBS. The xeno-free nature of HPL additionally confers preferred standing for use in GMP-regulated clinical trials using human corneal stromal MSCs

3D printed recombinant collagen type III (RHC III) scaffolds and their potential for corneal tissue engineering

Introduction: Corneal visual impairment is the 4th cause of blindness globally (5.1%), according to the WHO1, and currently the only treatment is surgical intervention using a human donor cornea, a procedure known as penetrating keratoplasty. The greatest challenge facing the treatment of corneal visual impairment is the global lack of donor corneas. The use of 3D printing in tissue engineering (TE) is a way of producing customized scaffolds, offering a tantalising prospect in the treatment of corneal visual impairment. This work studies the potential of 3D printing for producing recombinant collagen type III (RHC III) scaffolds for corneal tissue engineering. Methods: RHC III scaffolds were formed by nebulizing a 3.4mg/ml RHC III solution in 10mM HCL and accelerating it onto a substrate using nitrogen gas. Layers were formed with assumed thickness of approximately 3µm and repeated 2, 10, or 29 times to achieve thicknesses of up to 90µm. Via this method, patterns were imparted on the sample surface in the direction of printing, and spiral patterns were used. Collagen scaffolds were then crosslinked using EDC [1-ethyl-3-(3-diaminopropyl)carbodiimide] and NHS (N-hydroxysuccinimide) at a molar ratio of 1:1. The thickness of 10- and 29-layer samples was measured using Optical Coherence Tomography (OCT). Corneal MSCs were isolated through collagenase digestion (4 hours) of the stroma. Cells were cultivated in DMEM + 10% FBS. First passage MSCs were seeded onto the collagen scaffold samples consisting of 2 layers (n=4), 10 layers (n=4) and 29 layers (n=4) of RHC III at a density of 100 cells/sample. For controls, cells were seeded on glass (n=3) and plastic (n=3). At day 23, scaffolds were processed for immunocytochemistry (n=9) and inspected via Scanning Electron Microscopy (SEM) (n=3). Results & Discussion: OCT thickness measurements showed a mean thickness of 28 ± 9µm for 10-layer samples and 87 ± 23µm for 29-layer samples. Light microscopy showed that corneal MSCs proliferated on all printed RHC III scaffolds, regardless of the number of layers. SEM showed the circular print pattern of the RHC III. With immunocytochemistry, we observed collagen type III, and MSCs were observed throughout the samples, indicating penetration into the RHCIII along with superficial growth, which was also observed. The MSCs cytoskeleton stained positive for phalloidin, and we observed alignment of the superficial cells following the direction of the collagen in the SEM images. Conclusions: These results demonstrate that 3D printing can be used to produce RHC III scaffolds which are suitable as a substrate for cultivating corneal MSCs. 1 World Health Organization, Causes of Blindness and Visual Impairment, http://www.who.int/blindness/causes/en/, accessed 30/11/2016.status: publishe

High resolution printing of collagen

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Magnetron discharge volt-ampere characteristic investigation at thin film coating process

© Published under licence by IOP Publishing Ltd. Magnetron discharge at reactive and working gases mixture atmosphere current-voltage characteristic (I-U) for different sputtering parameters is investigated. It is shown, that form of volt-ampere characteristic doesn't depend on gas supply scheme at vacuum chamber pressure 4- 6.10-2 Pa. Reactive gas (oxygen) flow increasing leads to making I-U transition part wider and amplification of difference between top and bottom parts of hysteresis loop I-U. Discharge voltage is less at oxygen and argon gases mixture atmosphere than at argon atmosphere