Alginate in corneal tissue engineering.

Corneal blindness is the major cause of vision impairment and the fourth-largest leading cause of blindness worldwide. An allograft corneal transplant is the most routine treatment for visual loss. Further complications can occur, such as transplant rejection, astigmatism, glaucoma, uveitis, retinal detachment, corneal ulceration due to reopening of the surgical wounds, and infection. For patients with autoimmune disorders, allografting for chemical burns and infections is contraindicated because of the risk of disease transmission and further complications. Moreover, corrective eye surgery renders the corneas unsuitable for allografting, further increasing the gap between donor tissue demand and supply. Due to these challenges, other therapeutic strategies such as artificial alternatives to donor corneal tissue are being considered. This review focuses on the use of alginate as a building block of therapeutic drugs or cell delivery systems to enhance drug retention and encourage corneal regeneration. The similarity of alginate hydrogel water content to native corneal tissue makes it a promising support structure. Alginate possess desired drug carrier characteristics, such as mucoadhesiveness and penetration enhancing properties. Whilst alginates have been extensively studied for their application in tissue engineering (TE), with many reviews being published, no reviews exist to our knowledge directly looking at alginates for corneal applications. The role of alginate in drug delivery to the surface of the eye and as a support structure (bioinspired tissue scaffold) for corneal TE is discussed. Biofabrication techniques such as gel casting, electrospinning, and bioprinting to develop tissue precursors and substitutes are compared. Finally, cell and tissue encapsulation in alginate for storage and transport to expand the scope of cell-based therapy for corneal blindness is also discussed in the light of recent applications of alginate in maintaining the function of biofabricated constructs for storage and transport.

Storable Cell-Laden Alginate Based Bioinks for 3D Biofabrication.

Over the last decade, progress in three dimensional (3D) bioprinting has advanced considerably. The ability to fabricate complex 3D structures containing live cells for drug discovery and tissue engineering has huge potential. To realise successful clinical translation, biologistics need to be considered. Refinements in the storage and transportation process from sites of manufacture to the clinic will enhance the success of future clinical translation. One of the most important components for successful 3D printing is the 'bioink', the cell-laden biomaterial used to create the printed structure. Hydrogels are favoured bioinks used in extrusion-based bioprinting. Alginate, a natural biopolymer, has been widely used due to its biocompatibility, tunable properties, rapid gelation, low cost, and easy modification to direct cell behaviour. Alginate has previously demonstrated the ability to preserve cell viability and function during controlled room temperature (CRT) storage and shipment. The novelty of this research lies in the development of a simple and cost-effective hermetic system whereby alginate-encapsulated cells can be stored at CRT before being reformulated into an extrudable bioink for on-demand 3D bioprinting of cell-laden constructs. To our knowledge the use of the same biomaterial (alginate) for storage and on-demand 3D bio-printing of cells has not been previously investigated. A straightforward four-step process was used where crosslinked alginate containing human adipose-derived stem cells was stored at CRT before degelation and subsequent mixing with a second alginate. The printability of the resulting bioink, using an extrusion-based bioprinter, was found to be dependent upon the concentration of the second alginate, with 4 and 5% (w/v) being optimal. Following storage at 15 °C for one week, alginate-encapsulated human adipose-derived stem cells exhibited a high viable cell recovery of 88 ± 18%. Stored cells subsequently printed within 3D lattice constructs, exhibited excellent post-print viability and even distribution. This represents a simple, adaptable method by which room temperature storage and biofabrication can be integrated for on-demand bioprinting.

Hypothermically Stored Adipose-Derived Mesenchymal Stromal Cell Alginate Bandages Facilitate Use of Paracrine Molecules for Corneal Wound Healing.

Adipose-derived mesenchymal stromal cells (Ad-MSCs) may alleviate corneal injury through the secretion of therapeutic factors delivered at the injury site. We aimed to investigate the therapeutic factors secreted from hypothermically stored, alginate-encapsulated Ad-MSCs' bandages in in vitro and in vivo corneal wounds. Ad-MSCs were encapsulated in 1.2% w/v alginate gels to form bandages and stored at 15 °C for 72 h before assessing cell viability and co-culture with corneal scratch wounds. Genes of interest, including HGF, TSG-6, and IGF were identified by qPCR and a human cytokine array kit used to profile the therapeutic factors secreted. In vivo, bandages were applied to adult male mice corneas following epithelial debridement. Bandages were shown to maintain Ad-MSCs viability during storage and able to indirectly improve corneal wound healing in vivo. Soluble protein concentration and paracrine factors such as TSG-6, HGF, IL-8, and MCP-1 release were greatest following hypothermic storage. In vivo, Ad-MSCs bandages-treated groups reduced immune cell infiltration when compared to untreated groups. In conclusion, bandages were shown to maintain Ad-MSCs ability to produce a cocktail of key therapeutic factors following storage and that these soluble factors can improve in vitro and in vivo corneal wound healing.

Encapsulation of human limbus-derived stromal/mesenchymal stem cells for biological preservation and transportation in extreme Indian conditions for clinical use.

Human limbus-derived stromal/mesenchymal stem cells (hLMSC) can be one of the alternatives for the treatment of corneal scars. However, reliable methods of storing and transporting hLMSC remains a serious translational bottleneck. This study aimed to address these limitations by encapsulating hLMSC in alginate beads. Encapsulated hLMSC were kept in transit in a temperature-conditioned container at room temperature (RT) or stored at 4 °C for 3-5 days, which is the likely duration for transporting cells from bench-to-bedside. Non-encapsulated cells were used as controls. Post-storage, hLMSC were released from encapsulation, and viability-assessed cells were plated. After 48 and 96-hours in culture the survival, gene-expression and phenotypic characteristics of hLMSC were assessed. During transit, the container maintained an average temperature of 18.6 ± 1.8 °C, while the average ambient temperature was 31.4 ± 1.2 °C (p = 0.001). Encapsulated hLMSC under transit at RT were recovered with a higher viability (82.5 ± 0.9% and 76.9 ± 1.9%) after 3 (p = 0.0008) and 5-day storage (p = 0.0104) respectively as compared to 4 °C (65.2 ± 1.2% and 64.5 ± 0.8% respectively). Cells at RT also showed a trend towards greater survival-rates when cultured (74.3 ± 2.9% and 67.7 ± 9.8%) than cells stored at 4 °C (54.8 ± 9.04% and 52.4 ± 8.1%) after 3 and 5-days storage (p > 0.2). Non-encapsulated cells had negligible viability at RT and 4 °C. Encapsulated hLMSC (RT and 4 °C) maintained their characteristic phenotype (ABCG2, Pax6, CD90, p63-α, CD45, CD73, CD105, Vimentin and Collagen III). The findings of this study suggest that alginate encapsulation is an effective method of hLMSC preservation offering high cell viability over prolonged durations in transit at RT, therefore, potentially expanding the scope of cell-based therapy for corneal blindness.

Mesenchymal stromal cells for ocular surface repair.

INTRODUCTION: Cornea is a transparent, robust tissue that comprises highly organized cells. Disruption of this specialized tissue can lead to scarring and subsequent blindness, making corneal damage a considerable challenge worldwide. At present, the available medical treatments are unable to address the wide range of corneal diseases. Mesenchymal stem cells (MSCs) have increasingly been investigated for their regenerative effect on ocular surface injury due to their unique ability for growth factor production, anti-inflammatory activity, immunomodulatory capacity and differentiation into multiple cell lineages. AREAS COVERED: Within this review, we explore the pathogenesis of corneal disorders in response to injury and disease, and the potential for MSCs to modulate this process as a treatment. Through the review of over 25 animal studies, we investigate the common mechanisms of action by which MSCs have their effect and discuss their potential for treating and/or preventing corneal deterioration EXPERT OPINION: Depending on the environmental cues, MSCs can exert a potent effect on corneal wound healing through reducing opacity and vascularization, whilst promoting re-epithelialization. Whilst their mechanism is multifactorial, it seems clear that the anti-inflammatory/immunomodulatory factors they produce in response to damage are key to their control of cellular milieu and improving healing outcomes.

Alginate encapsulated multipotent adult progenitor cells promote corneal stromal cell activation via release of soluble factors.

To reduce the increasing need for corneal transplantation, attempts are currently aiming to restore corneal clarity, one potent source of cells are multipotent adult progenitor cells (MAPC®). These cells release a powerful cocktail of paracrine factors that can guide wound healing and tissue regeneration. However, their role in corneal regeneration has been overlooked. Thus, we sought to explore the potential of combining the cytoprotective storage feature of alginate, with MAPC to generate a storable cell-laden gel for corneal wound healing. 72 hours following hypothermic storage, alginate encapsulation was shown to maintain MAPC viability at either 4 or 15°C. Encapsulated MAPC (2 x106 cells/mL) stored at 15°C presented the optimum temperature that allowed for cell recovery. These cells had the ability to reattach to tissue culture plastic whilst exhibiting normal phenotype and this was maintained in serum-free and xenobiotic-free medium. Furthermore, corneal stromal cells presented a significant decrease in scratch-wounds in the presence of alginate encapsulated MAPC compared to a no-cell control (p = 0.018). This study shows that immobilization of MAPC within an alginate hydrogel does not hinder their ability to affect a secondary cell population via soluble factors and that these effects are successfully retained following hypothermic storage.

3D bioprinting of a corneal stroma equivalent.

Corneal transplantation constitutes one of the leading treatments for severe cases of loss of corneal function. Due to its limitations, a concerted effort has been made by tissue engineers to produce functional, synthetic corneal prostheses as an alternative recourse. However, successful translation of these therapies into the clinic has not yet been accomplished. 3D bioprinting is an emerging technology that can be harnessed for the fabrication of biological tissue for clinical applications. We applied this to the area of corneal tissue engineering in order to fabricate corneal structures that resembled the structure of the native human corneal stroma using an existing 3D digital human corneal model and a suitable support structure. These were 3D bioprinted from an in-house collagen-based bio-ink containing encapsulated corneal keratocytes. Keratocytes exhibited high cell viability both at day 1 post-printing (>90%) and at day 7 (83%). We established 3D bio-printing to be a feasible method by which artificial corneal structures can be engineered.

Alginate-Encapsulation for the Improved Hypothermic Preservation of Human Adipose-Derived Stem Cells.

Despite considerable progress within the cell therapy industry, unmet bioprocessing and logistical challenges associated with the storage and distribution of cells between sites of manufacture and the clinic exist. We examined whether hypothermic (4°C-23°C) preservation of human adipose-derived stem cells could be improved through their encapsulation in 1.2% calcium alginate. Alginate encapsulation improved the recovery of viable cells after 72 hours of storage. Viable cell recovery was highly temperature-dependent, with an optimum temperature of 15°C. At this temperature, alginate encapsulation preserved the ability for recovered cells to attach to tissue culture plastic on rewarming, further increasing its effect on total cell recovery. On attachment, the cells were phenotypically normal, displayed normal growth kinetics, and maintained their capacity for trilineage differentiation. The number of cells encapsulated (up to 2 × 10(6) cells per milliliter) did not affect viable cell recovery nor did storage of encapsulated cells in a xeno-free, serum-free,current Good Manufacturing Practice-grade medium. We present a simple, low-cost system capable of enhancing the preservation of human adipose-derived stem cells stored at hypothermic temperatures, while maintaining their normal function. The storage of cells in this manner has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state.

Defining Key Structural Determinants for the Pro-osteogenic Activity of Flavonoids.

Epidemiological studies suggest that fruits and vegetables may play a role in promoting bone growth and preventing age-related bone loss, attributable, at least in part, to phytochemicals such as flavonoids stimulating osteoblastogenesis. Through systematically screening the effect of flavonoids on the osteogenic differentiation of human mesenchymal stem cells in vitro and correlating activity with chemical structure using comparative molecular field analysis, we have successfully identified important structural features that relate to their activity, as well as reliably predicted the activity of compounds with unknown activity. Contour maps emphasized the importance of electronegativity, steric bulk, and a 2-C-3-C double bond at the flavonoid C-ring, as well as overall electropositivity and reduced steric bulk at the flavonoid B-ring. These results support a role for certain flavonoids in promoting osteogenic differentiation, thus their potential for preventing skeletal deterioration, as well as providing a foundation for the lead optimization of novel bone anabolics.