3D microfabricated scaffolds and microfluidic devices for ocular surface replacement: a review

In recent years, there has been increased research interest in generating corneal substitutes, either for use in the clinic or as in vitro corneal models. The advancement of 3D microfabrication technologies has allowed the reconstruction of the native microarchitecture that controls epithelial cell adhesion, migration and differentiation. In addition, such technology has allowed the inclusion of a dynamic fluid flow that better mimics the physiology of the native cornea. We review the latest innovative products in development in this field, from 3D microfabricated hydrogels to microfluidic devices

Cytocompatibility and Suitability of Protein-Based Biomaterials as Potential Candidates for Corneal Tissue Engineering

The vision impairments suffered by millions of people worldwide and the shortage of corneal donors show the need of substitutes that mimic native tissue to promote cell growth and subsequent tissue regeneration. The current study focused on the in vitro assessment of protein-based biomaterials that could be a potential source for corneal scaffolds. Collagen, soy protein isolate (SPI), and gelatin films cross-linked with lactose or citric acid were prepared and physicochemical, transmittance, and degradation measurements were carried out. In vitro cytotoxicity, cell adhesion, and migration studies were performed with human corneal epithelial (HCE) cells and 3T3 fibroblasts for the films’ cytocompatibility assessment. Transmittance values met the cornea’s needs, and the degradation profile revealed a progressive biomaterials’ decomposition in enzymatic and hydrolytic assays. Cell viability at 72 h was above 70% when exposed to SPI and gelatin films. Live/dead assays and scanning electron microscopy (SEM) analysis demonstrated the adhesion of both cell types to the films, with a similar arrangement to that observed in controls. Besides, both cell lines were able to proliferate and migrate over the films. Without ruling out any material, the appropriate optical and biological properties shown by lactose-crosslinked gelatin film highlight its potential for corneal bioengineering.This research study was supported by grants from the Department of Heath of the Basque Government (RIS3, 2019222049), the University of the Basque Country UPV/EHU-Instituto Clinico Quirurgico de Oftalmologia ICQO (US19/18), and MCI/AEI/FEDER, UE (grant number RTI2018-097100-B-C22). C.R.-V. was supported by a fellowship from the University of the Basque Country UPV/EHU

Mimicking the Hierarchical Organization of Natural Collagen: Toward the Development of Ideal Scaffolding Material for Tissue Regeneration

Biological materials found in living organisms, many of which are proteins, feature a complex hierarchical organization. Type I collagen, a fibrous structural protein ubiquitous in the mammalian body, provides a striking example of such a hierarchical material, with peculiar architectural features ranging from the amino acid sequence at the nanoscale (primary structure) up to the assembly of fibrils (quaternary structure) and fibers, with lengths of the order of microns. Collagen plays a dominant role in maintaining the biological and structural integrity of various tissues and organs, such as bone, skin, tendons, blood vessels, and cartilage. Thus, "artificial" collagen-based fibrous assemblies, endowed with appropriate structural properties, represent ideal substrates for the development of devices for tissue engineering applications. In recent years, with the ultimate goal of developing three-dimensional scaffolds with optimal bioactivity able to promote both regeneration and functional recovery of a damaged tissue, numerous studies focused on the capability to finely modulate the scaffold architecture at the microscale and the nanoscale in order to closely mimic the hierarchical features of the extracellular matrix and, in particular, the natural patterning of collagen. All of these studies clearly show that the accurate characterization of the collagen structure at the submolecular and supramolecular levels is pivotal to the understanding of the relationships between the nanostructural/microstructural properties of the fabricated scaffold and its macroscopic performance. Several studies also demonstrate that the selected processing, including any crosslinking and/or sterilization treatments, can strongly affect the architecture of collagen at various length scales. The aim of this review is to highlight the most recent findings on the development of collagen-based scaffolds with optimized properties for tissue engineering. The optimization of the scaffolds is particularly related to the modulation of the collagen architecture, which, in turn, impacts on the achieved bioactivity

Novel cornea-specific bioink for 3D bioprinting

Background and aims: Healthy cornea is transparent which is crucial for its role in light transmission and refraction. The corneal stroma (CS) constitutes a significant portion of cornea and its complex architecture, playing a major role in corneal transparency. The CS is populated by corneal keratocytes (CKs), which maintain the CS. As the cornea is the most anterior part of the eye it is susceptible to injuries and diseases, which disrupt the native corneal organization. This is associated with a decrease in corneal transparency and vision, referred to as corneal blindness. Currently, the choice of treatment for corneal blindness patients is corneal transplantation. However, there is a global shortage of donor corneas, thus only a fraction of patients can benefit from this treatment. Keratoprostheses (KPros) can be used instead, however these come with significant limitations. Previously, conventional corneal tissue engineering (TE) methods have been used to address this problem. However, it is not possible to mimic the corneal structure with these methods. Three-dimensional (3D) bioprinting has recently gained attention as a novel approach to generate transplantable corneal equivalents. The aim of this thesis was to develop a cornea-specific bioink for extrusion-based bioprinting (EBB) and to study the printability and biocompatibility of the bioink. Materials and methods: Human adipose tissue stem cells (hASCs) were differentiated towards CKs (hASC-CKs), which were used to produce cornea-specific cell-derived matrix (Co-CDM). The presence of corneal extracellular matrix proteins (ECM) in the Co-CDM was studied with immunofluorescence staining. Macromolecular crowding (MMC) conditions were used to study if the amount of produced Co-CDM can be increased. In order to incorporate the Co-CDM into the bioink it was decellularized, hence the name Co-dCDM, and processed. In this thesis the characteristics of Co-dCDM bioink were compared to a bioink which had similar composition, containing collagen I (Col I) instead of Co-dCDM. Then, the bioink compositions and printing parameters were optimized. Characterized bioink properties were printability, shape fidelity, swelling behavior and viscosity. In the final step of the thesis Co-dCDM bioink was printed with hASCs and biocompatibility was studied by measuring cell viability and proliferation. Results and conclusions: The immunofluorescence stainings of hASC-CKs showed expression of CK specific marker proteins in the Co-CDM. The studied MMC culture conditions did not increase the amount of produced Co-CDM, since it led to cell detachment. Decellularization of the produced Co-CDM resulted in a clear decrease in the DNA content, although the inclusion of DNase 1 did not improve decellularization efficacy compared to decellularization without DNase 1. The Co-dCDM bioink showed good printability and shape fidelity post-printing. Transparency of Co-dCDM was excellent, although it was greater for Col I bioink. In viscosity measurements Co-dCDM demonstrated the desirable shear-thinning property for EBB. The biocompatibility of Co-dCDM was excellent since hASCs were viable and proliferative in the bioink. Similarly, immunofluorescence staining’s showed expression of proliferation marker Ki-67 and elongated morphology of hASCs. This is the first study where a cornea-specific bioink, incorporating Co-dCDM instead of decellularized cornea, is developed and characterized. The Co-dCDM demonstrated beneficial properties and thus it should be studied more closely in the future to evaluate its potential for corneal 3D bioprinting applications.Tausta ja tavoitteet: Terve sarveiskalvo on läpinäkyvä, mikä on välttämätöntä sarveiskalvon roolille valon läpäisevyydessä ja taitossa. Sarveiskalvon strooma käsittää merkittävän osan sarveiskalvosta ja sen monimutkaisesta rakenteesta. Sarveiskalvon keratosyytit ylläpitävät tätä ja ovat merkittävässä roolissa sarveiskalvon läpinäkyvyyden ylläpitämisessä. Sarveiskalvo on altis vaurioille ja taudeille, koska se on silmän etummaisin osa. Nämä tuhoavat sarveiskalvon luontaisen rakenteen, mihin liittyy sarveiskalvon läpinäkyvyyden vähentyminen ja sokeutuminen. Sarveiskalvon siirto on yleisin hoitokeino potilaille, jotka kärsivät sarveiskalvon vaurioitumisesta aiheutuvasta sokeutumisesta. Kuitenkin maailmanlaajuisesta sarveiskalvosiirteiden pulasta johtuen vain pieni osa potilaista voidaan hoitaa. Keratoproteesit ovat vaihtoehtoinen hoitokeino sarveiskalvon siirrolle, mutta niihin liittyy merkittäviä rajoitteita. Aiemmin perinteisiä kudosteknologian menetelmiä on käytetty sarveiskalvopulan ratkaisemiseksi. Näillä menetelmillä ei kuitenkaan ole mahdollista jäljitellä sarveiskalvon luontaista rakennetta. Viime aikoina kolmiulotteinen (3D) biotulostus on saanut huomiota uudenlaisena lähestymistapana tuottaa sarveiskalvon kaltaisia siirrännäisiä. Tämän työn tavoite oli kehittää sarveiskalvospesifinen extruusio 3D biotulostettava biomuste ja tutkia sen tulostettavuutta ja bioyhteensopivuutta. Materiaalit ja menetelmät: Ihmisen rasvakudoksen kantasolut (human adipose tissue stem cells, hASCs) erilaistettiin keratosyyttien kaltaisiksi soluiksi, joita käytettiin sarveiskalvospesifisen soluperäisen matriisin (cornea-specific cell-derived matrix, Co-CDM) tuottamiseen. Immunofluoresenssi värjäyksillä tutkittiin sarveiskalvon soluväliaineen proteiinien olemassaoloa Co-CDM:sta. Makromolekulaarisella täytöllä (Macromolecular crowding, MMC) tutkittiin vaikutusta tuotetun Co-CDM määrään. Ennen Co-CDM:n sekoittamista biomusteeseen, se desellularisoitiin, josta tuli nimi Co-dCDM. Tässä työssä vertailtiin kahden biomusteen ominaisuuksia, joilla oli muuten sama koostumus, paitsi yksi sisälsi Co-dCDM:ta ja toinen kollageeni I:ta. Sitten biomusteiden koostumukset ja tulostusolosuhteet optimoitiin. Biomusteista tutkitut ominaisuudet olivat tulostettavuus, filamenttien muodon pysyvyyttä, turpoaminen ja leikkausohenevuus. Lopuksi työssä tulostettiin hASCs-soluja Co-dCDM-biomusteen kanssa. Biomusteen bioyhteensopivuutta tutkittiin mittaamalla solujen elinkelpoisuutta ja proliferaatiota. Tulokset ja päätelmät: Immunofluoresenssi värjäyksissä havaittiin, että hASC-CKs-solujen tuottamassa Co-CDM:ssa expressoitui keratosyyttispesifisiä proteiinimarkereita. Lisäksi MMC-olosuhteet eivät lisänneet tuotetun Co-CDM:n määrää, koska se johti solujen irtoamiseen. Desellularisaatio vähensi Co-CDM:n sisältämän DNA:n määrää, mutta DNase 1:n lisäys ei parantanut desellularisaatiota. Co-dCDM-biomusteen tulostettavuus ja filamenttien muodon pysyvyys tulostamisen jälkeen olivat hyvät. Lisäksi biomusteen läpinäkyvyys oli erinomainen, vaikkakin se oli matalampi kuin Col I-biomusteella. Viskositeettimittauksissa havaittiin, että Co-dCDM-biomuste oli leikkausohenevaa. Tämä on haluttu ominaisuus extruusio 3D biotulostuksessa käytettäville biomusteille. Myös Co-dCDM-biomusteen bioyhteensopivuus oli erinomainen, sillä hASCs olivat elinkelpoisia ja proliferatiivisia biomusteessa. Tämän lisäksi tulostettujen rakenteiden immunofluoresenssivärjäyksissä havaittiin proliferaatiomarkkerin Ki-67 expressio ja hASCs-solujen pitkänomainen morphologia. Tämä on ensimmäinen tutkimus, missä sarveiskalvospesifinen biomuste on kehitetty käyttämättä desellularisoituja sarveiskalvoja. Tutkitulla Co-dCDM-biomusteella oli lupaavia ominaisuuksia ja sitä tulisi tutkia lisää, jotta saadaan enemmän tietoa sen koostumuksesta ja potentiaalista 3D-biotulostuksessa

Oral mucosa keratinocytes and their exosomes for epithelial tissue regeneration

Early tumors, including high grade dysplasia and intramucosal invasive cancer, of the esophagus can today be removed using endoscopic resection, often using a technique called endoscopic submucosal dissection (ESD). This treatment is better tolerated and has considerably less mortality and morbidity compared to conventional, more invasive surgery, which usually entails esophagectomy. However, after larger endoscopic dissections, stricture formation is a common complication. Such strictures are usually treated with balloon dilatations, but the procedure often has to be repeated several times and is associated with risks such as perforations. In recent years, Japanese researchers developed a new method to reduce the risk of strictures. About two weeks before the treatment, an oral mucosa biopsy is taken from the patient from which epithelial cells are isolated and grown on special temperature-responsive polymer-coated surfaces. The polymer changes morphology and wettability properties depending on temperature, which enables non-enzymatic cell harvesting – the cells can be detached as contiguous sheets and with a large amount of extracellular matrix (ECM) maintained. After the ESD, cell sheets can be transplanted to the wound bed without the need for suture or other fixative, it is thought that the remaining ECM acts as a glue. Nine patients were treated in Tokyo and we subsequently transferred the technology to Stockholm where five additional patients were treated. Although one of nine patients in the Japanese cohort and three of five patients in the Swedish cohort still developed strictures, they appeared to be milder and easier to treat than expected. However, the aim of these projects was to evaluate safety and feasibility, further studies are needed to evaluate efficacy of the treatment. Since some patients still developed strictures despite the cell sheet transplantation, we next aimed to evaluate if exosomes from the cell culture media could be used as a pro-regenerative agent, perhaps in combination with cell sheet therapy. Media was collected from clinical- grade production of cell sheets from eight healthy donors. The media was concentrated by ultra-filtration and exosomes were isolated by size-exclusion chromatography. The exosomes were characterized by western blot (CD9+, Flotillin-1+, GRP94-), electron microscopy and nanoparticle tracking analysis (~125 nm). They reduced the proliferation of skin fibroblasts and stimulated upregulation of gene expression of growth factors relevant for wound healing. We studied the exosomes’ adhesion to esophageal wound bed by topical application to porcine esophageal wounds ex vivo and could detect signal after as little as one minute adhesion time. We also found that the exosomes stimulated wound healing of full-thickness skin wounds in immunocompetent rats, both at the 6th day and 17th day time point. In conclusion we found that exosomes could be isolated from cell sheet media and that they exhibited pro-regenerative properties even in a xenogeneic setting. Further studies are necessary to evaluate their potential to stimulate mucosal wound healing and reduce stricture formation of the esophagus

Development of an in-vitro animal model to evaluate novel pharmaceutical approaches to DED management

In the ophthalmic field, multifactorial pathologies such as Dry Eye Disease (DED) andcataract are largely studied in living animal models that can fail to precisely mirror thecomplexity of these conditions in humans. Recent advances in biomedical technologieshave improved the reliability of in-vitro/ex-vivo animal alternatives, and to date,the corneal and crystalline lens tissue have been independently maintained physiologicallystable for 10 days. This thesis details the development of a novel and complete ex-vivo anterior eye model,which is capable of sustaining both the cornea and crystalline lens in a physiologicallystable state in loco for 7 days. The platform is based on porcine eyes, which representa high quality and reliable human tissue source substitute, and being slaughterhousewaste, also perfectly align the project with the 3Rs principle of replacing, refining andreducing living animal experimentation. The model is modular and scalable, allowingfor the maximisation of experimental reliability, and the minimisation of wasteand energy use. In addition, the whole system is designed to be fitted in a laminarflow cabinet, avoiding external biological contamination, and is easily transportablebetween tissue engineering laboratories, maximising accessibility. The model was validated estimating cell viability over time. Stromal fibroblasts werefound to be viable up to the seventh day of culture, and corneal and crystalline lenstissue maintained their transparency over the culturing period. Dry Eye Disease wassuccessfully induced in the model by irrigating the ocular surface every 40s, and validatedusing impression cytology technique. Moreover, due to the unique presence inloco of the crystalline lens, the model was also used as a platform to perfect cataractsurgery and successfully implant intraocular lenses (IOLs). The novel and complete ex-vivo anterior eye model developed in this thesis providesfurther insights into pre-clinical anterior segment investigations in ophthalmology,taking a step forward toward bridging the existing gap between in-vitro and in-vivobiomedical technologies

Desarrollo de un modelo animal in vitro para evaluar nuevos fármacos para el tratamiento del ojo seco

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Óptica y Optometría, leída el 19/02/2019In the ophthalmic field, multifactorial pathologies such as Dry Eye Disease (DED) and cataract are largely studied in living animal models that can fail to precisely mirror the complexity of these conditions in humans. Recent advances in biomedical technologies have improved the reliability of in-vitro/ex-vivo animal alternatives, and to date, the corneal and crystalline lens tissue have been independently maintained physiologicallystable for 10 days. This thesis details the development of a novel and complete ex-vivo anterior eye model, which is capable of sustaining both the cornea and crystalline lens in a physiologically stable state in loco for 7 days. The platform is based on porcine eyes, which represent a high quality and reliable human tissue source substitute, and being slaughterhouse waste, also perfectly align the project with the 3Rs principle of replacing, refining and reducing living animal experimentation...Dentro del campo oftalmológico, patologías multifactoriales como Enfermedad del Ojo Seco (EOS) y Cataratas son ampliamente estudiadas a través de modelos animales que no reproducen con exactitud estas condiciones en humanos debido a su complejidad. Los recientes avances en la tecnología biomédica han mejorado la fiabilidad de modelos animales in-vitro/ex-vivo, y hasta el momento, el tejido de la córnea y cristalino se han mantenido fisiológicamente estables de forma independiente durante 10 días. Esta tesis describe el desarrollo de un nuevo y completo modelo de ojo anterior ex vivo, que es capaz de mantener tanto la córnea como el cristalino en un estado fisiológico estable durante 7 días. La plataforma se basa en ojos porcinos, que representan una fuente alternativa al tejido humano de alta calidad y fiable, y al tratarse de residuos de matadero, también alinea perfectamente el proyecto con el principio 3Rs de reemplazar, refinar y reducir la experimentación con animales vivos...Fac. de Óptica y OptometríaTRUEunpu

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels