Elsevier

Biomaterials

Volume 174, August 2018, Pages 95-112
Biomaterials

3D-Printed membrane as an alternative to amniotic membrane for ocular surface/conjunctival defect reconstruction: An in vitro & in vivo study

https://doi.org/10.1016/j.biomaterials.2018.05.013Get rights and content

Abstract

Background

The aim of this study was to evaluate the surgical handling and clinical applicability of a specific 3D-printed membrane design fabricated using a gelatin, elastin and sodium hyaluronate blend for conjunctival reconstruction and compare it with amniotic membrane (AM), which is normally used in such surgeries.

Methods

3D printing technique was employed to fabricate the membrane based on gradient design. Prior to printing, rheometry was employed to optimize the ink composition. The printed membranes were then fully characterized in terms of physical and mechanical properties. In vitro viability, proliferation and adhesion of human limbal epithelial cells were assessed using MTT assay and scanning electron microscopy (SEM), respectively. Prior to in vivo experiment, surgical handling of each membrane was evaluated by three surgeons. In vivo evaluation was conducted through implanting the gelatin-based membranes and AM on induced conjunctival defects in rabbits (n = 8). Clinical observations, including epithelialization, inflammation severity, scar tissue formation and presence of granulation tissue, were recorded from day 1 through day 28. Histological examination was performed on all enucleated eyes on day 28. In addition to H&E staining, specific stains including Periodic Acid Schiff staining, Masson's Trichrome staining and immuno-histochemical staining for α-SMA were further used to assess goblet cell proliferation, healed sub-epithelial stroma and scar tissue formation and the presence of myofibroblasts, respectively.

Results

Among all the examined compositions, a blend of 8% w/v gelatin, 2% w/v elastin and 0.5% w/v sodium hyaluronate was found to be appropriate for printing. The printed membranes had favorable optical characteristics (colorless and transparent), and the surgical handling was significantly easier compared to AM. Epithelial cells cultivated on the membranes indicated suitable viability and proliferation, and SEM images presented appropriate cell adhesion on the surface of the membranes. Clinical observations suggested similar epithelialization time (approximately 3 weeks) for both the membrane and AM grafted eyes but significantly lower levels of clinical inflammation in the membrane group from day 1 through day 28 (p = 0.01), which is a key advantage of using the printed membranes over the AM. Histological examination showed similar qualities in the healed epithelium in terms of cell morphology and cell layers. However, twice the density of goblet cells per 100 cells was observed in the gelatin-based membrane grafted group. Remnant of the degraded implant was seen in only 3 of the membranes, but in 7 of the AM grafted eyes. Inflammation and granulomatous reaction was significantly higher in sections containing the AM compared to membrane (p < 0.01 and p = 0.01, respectively). α-SMA staining was more evident, but not significantly different from the gelatin-based membrane, for the AM group (p = 0.25).

Conclusion

The designed gelatin-based membrane offers the necessary physical and mechanical characteristics needed for successful ocular surface/conjunctival defect construction and may be considered a promising alternative to AM due to a more predictable degradation pattern, higher goblet cell density on the healed epithelium, less inflammation and reduced scar tissue formation.

Introduction

Ocular surface damage can occur due to various reasons, such as ocular thermal/chemical burns [1], autoimmune diseases (Steven Johnson's, ocular cicatricial pemphigoid) [2,3], or due to iatrogenic causes during surgeries where conjunctival excision is necessary, such as Pterygium surgery [4] and conjunctival tumor removal [5]. Repair of the damaged or absent conjunctivae is of utmost importance, as conjunctivae is necessary for proper function of the ocular refractive surface and protection of the cornea. The latter is accomplished through equilibrium of the tear film composition by secreting aqueous, lipid and mucin [6]. The conjunctiva also plays a role in protecting the eye from microbial infections by acting as a mechanical barrier and containing lymphoid tissue (conjunctiva-associated lymphoid tissue―CALT) [7]. Although re-epithelization will occur spontaneously upon conjunctival injury [[8], [9], [10]], several limitations including reduced goblet cell density, scar tissue formation and contracture hinder the function and anatomy of the healed epithelium that may eventually lead to unfavorable results and even blindness. Goblet cell density and, consequently, the mucin layer thickness of the tear film, that is essential for maintaining the wettability of the ocular surface and clearing it of debris [11], decrease with injury in the healed epithelium [9,12]. On the other hand, conjunctival contracture and reduced overall surface may lead to fornix foreshortening and eventual symblepharon formation and entropion in the involved eye [13,14].

Despite favorable results [[14], [15], [16]], autologous tissues have some limitations to replace the absent conjunctiva or modulate the healing process, including unavailability of healthy conjunctiva in autoimmune diseases, cosmetic issues, and absence of goblets [7]. Therefore, allografts such as pericardium [10] and amniotic membrane (AM) have been introduced. AM currently serves amongst the most widespread grafts used in ocular surface reconstruction. It is currently used as a substitute for excised conjunctivae in pterygium surgery, symblepharon lysis, fornix reconstruction, conjunctivochalasis and in tumor resections [17]. AM has been shown to facilitate epithelialization by providing a basement membrane and stroma resembling that of the normal conjunctivae [2,14,18,19]. Studies have also mentioned anti-inflammatory and anti-fibrotic properties, along with lack of immunogenicity for AM [20,21]. The mentioned characteristics have made AM a favorable tissue for ophthalmic applications [22,23]. However, there are some studies that challenge the preferential use of AM. For example, histological examinations showed low levels of epithelial stratification (single) and low goblet density (sparse PAS positive cells) on the epithelium grown on cultured AM [24]. Despite favorable results on suppression of inflammation by AM reported in prior studies [1,2,14,18,21,[25], [26], [27]], there have been some evidence showing increased inflammatory response in histological sections of wounds grafted with AM [10,[28], [29], [30]] after conjunctival reconstruction with human AM. Moreover, considering that AM is an allogenic tissue, limitations in the number of diseases that it can be screened for remains a concern [22]. Another complication associated with AM is the considerable likelihood of microbial infections and unwanted host reactions due to lack of standardized and sterile tissue preparations. Post AM transplantation rate of infection (culture positive) has been reported to be 3.4% in a study on 326 patients [31]. Difficult surgical handling (notably suturing due to the thin structure of AM), prolonged presence in the AM graft site [13,28,29], and opacification of the site [32] are among other reported issues associated with AM. Such limitations necessitate development of efficient alternatives like bioengineered scaffolds that can have stroma-like structure similar to AM, promote epithelialization while not transmitting allogenic-related diseases. The results of animal study of several synthetic materials including collagen-glycosaminoglycan (CG) copolymer [9], poly (lactide-co-glycolide) (PLGA) [8], vitrified collagen [12], and cultured membranes [30,33] have been promising in terms of successful re-epithelialization, decreasing scar formation and fornix foreshortening [13,18,19]. However, they still have limitations like undesirable transparency, elasticity, and thickness [[8], [9], [10]]. Also, the ease of surgical handling and the goblet population have not been thoroughly discussed [8,9,34]. Among different biomaterials, biodegradability, biocompatibility, ease of processing, and relative inexpensiveness of gelatin-based ones have been the main drivers for further studies, albeit predominantly preclinical, on their ocular applications. Although gelatin-based constructs have been extensively investigated over the last few decades for a multitude of ocular applications [[35], [36], [37], [38], [39], [40], [41], [42]], conjunctival reconstruction has not been reported as a potential application for this group of biomaterials despite their favorable characteristics mentioned above.

This paper is aimed at the development and application of a 3D-printed gelatin/elastin/hyaluronic acid membrane for conjunctival reconstruction and evaluation of epithelialization (time to epithelialization and morphology of the healed epithelium), goblet cell morphology and density, degree and type of inflammation (clinical and histological assessment), quality of healed sub-epithelial stroma and scar tissue formation (gross tissue deformation, collagen deposition pattern from histological data, myofibroblasts by immune histochemical stationing for α-SMA). Additionally, results of the evaluation conducted on the fabricated membrane were compared with corresponding evaluation for the widely used AM graft. Prior to in vivo tests conducted on animal models, the membranes were tested for in vitro biocompatibility as well.

Section snippets

Ink formulation

The ink used in this study was composed of gelatin (Type A, from porcine skin, Bioreagent grade, Sigma, US), soluble elastin (MW 60 KDa, Elastin Products Company, Inc., USA) and sodium hyaluronate (Research Grade, 500 KDa-749 KDa, Lifecore Biomedical, USA). Gelatin was used as the main component of the ink, while elastin and sodium hyaluronate were employed as additives. Rheological measurements were used to characterize various ink formulations listed in Table 1.

The gelation temperature of

Ink formulation and 3D-Printing of the membranes

The ink employed in this study was composed of gelatin, elastin and sodium hyaluronate. Gelatin sol-gel transition plays an important role in the printing procedure as it might be both in favor of or against successful printing. While it assists the ink hardening on the platform and shape fidelity of the printed strands, it might cause obstruction of the dispensing needle and interruption of printing. The sol-gel transition temperature of each individual formulation was determined as the

Discussion

The gelatin-based membranes, in this study, offered ease of surgical handling, transparency and potentially geometrical conformity. Conformity is a particularly crucial factor in conjunctival reconstruction within the forniceal area, since placing anchoring sutures would be very difficult, if not impossible, if the membrane does not conform to the shape of the fornix. Conformity is of the utmost importance in diseases that cause forniceal shortening, like ocular burns and autoimmune diseases [13

Conclusions

In the current study, a 3D-printed membrane was fabricated using gelatin, elastin and sodium hyaluronate blend and was compared to amniotic membrane (AM) for conjunctival reconstruction. In-vivo experiments were conducted on induced conjunctival defects in rabbits. Clinical observations and histological examination suggested that the gelatin-based membranes is a promising synthetic substitute for AM in conjunctival reconstruction due to the following findings: 1) acceptable surgical handling,

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

Acknowledgements

This work was carried out with support from China Regenerative Medicine International Limited (CRMI). The stem cell preparation unit (SPU) at Farabi Hospital, particularly Mr. Zarrabi, Mrs. Aghajanpour and Mr. Mohammadnia were very instrumental in conducting the in-vitro and in-vivo tests.

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