Biocompatibilidad del Chitosan

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InvivoBiocompatibilityofChitosanandCollagen–VitrigelMembranesforCornealScaffolding:aComparativeAnalysis

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DOI:10.2174/2211542004666151022204221

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In Vivo Biocompatibility of Chitosan and Collagen–vitrigel Membranes for Corneal Scaffolding: A Comparative Analysis

Valdez-García Jorge E.1,2,*, Mendoza Guillermo1,2, Zavala Judith1, Zavala-Pompa Angel3, Brito Gabriela4, Cortés-Ramírez Jorge A.4 and Elisseeff Jennifer5

1Tecnologico de Monterrey, School of Medicine, 3000 Morones Prieto Ave. Col. Los Doctores, Monterrey, N.L. Mexico, C.P. 6471, Mexico; 2Ophthalmology Institute, Tec Salud, Tecnologico de Monterrey, Monterrey, Mexico, 112 Batallon de San Patricio, Col. Real San Agustin, Monterrey, N.L. Mexico, C.P. 66278, Mexico; 3Medicina Diagnostica S.A. de C.V. Monterrey, Mexico, 1333 Simon Bolivar, Col. Mitras Centro, Monterrey, N.L. Mexico, C.P. 64460, Mexico; 4Centro de Innovación en Diseñoy Tecnología, Tecnologico de Monterrey, Monterrey, Mexico, 2501 Sur Eugenio Garza Sada Ave. Col. Tecnologico, Monterrey, N.L. C.P. 64849, Mexico; 5Department of Biomedical Engineering, Johns Hopkins University, Baltimore, USA, 5031 Smith Building, 400 N Broadway, Baltimore MD 21231, USA

Abstract: Purpose: To compare the biosafety of chitosan (CHM) and collagen–vitrigel biomembranes (CVM) when im-planted to the anterior chamber of an animal model to set an optimal scaffold for further corneal engineering research.

Methods: Four White New Zealand rabbits, 3 months old, were implanted with CHM in one eye, and other four rabbits were implanted with CVM membranes following cold burn damage on the corneal surface. The contralateral eye was used as the control. After 1 week, rabbits were sacrificed, and the obtained corneas were clinically evaluated and processed for histological analysis.

Results: Eyes implanted with CHM developed severe inflammation with 360° neovascularization, ciliary injection, optical opacity, and purulent exudate in the anterior chamber. Microscopically, CHM-implanted eyes showed severe exudative, inflammatory, and necrotic processes that were mainly composed of polymorphonuclear (PMN) leukocytes, cellular de-bris, and macrophages. Eyes implanted with CVM showed little or no signs of clinical inflammation. Histological analysis of the CVM and control eyes showed no signs of inflammation, except in places where corneal suture ports and closure with a suture were performed.

Conclusions: CHM are not biocompatible for ocular purposes. CVM are safe to be used for further in vivo research as cell scaffold in corneal engineering.

Keywords: Chitosan, collagen-vitrigel, cornea, corneal endothelium, tissue scaffold, rabbit.

1. INTRODUCTION

The cornea is an avascular connective tissue that forms, in conjunction with the sclera, the outer portion of the eye. It acts as the primary barrier against infection and mechanical damage to the internal ocular structures. It is organized into five layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane, and endothelium [1]. The epithelium constitutes a biodefense system against biological and chemical agents, the Bowman’s layer is an interface between the epithelium and the stroma, which consists mainly of collagen fibers and keratinocytes that provides strength and transparency to the cornea. The corneal endothelium (CE) is a monolayer of polygonal cells that are in contact with the aqueous humor. It

*Address correspondence to this author at the Morones Prieto Ave. # 3000. Col. Los Doctores. Monterrey, N.L. Mexico. C.P. 64710. Mexico; Tel: +52 (81) 88 88 20 66; E-mail: [email protected]

regulates the hydration state of the cornea through an active ATP and bicarbonate-dependent pump, which allows the cornea to keep its transparency. It is also important in the passage of nutrients and waste removal through simple diffu-sion, facilitated diffusion, and active transport mechanisms [2, 3]. CE cells (CECs) do not regenerate given that they possess limited mitotic ability in adults [4]. A major injury in this tissue can result in an irreversible damage affecting vis-ual function. Corneal opacity is one of the major causes of blindness worldwide along with cataract, glaucoma, and macular de-generation [5]. Dysfunction in the endothelium as a result of trauma, surgical complications or different pathologies (Fuch’s dystrophy and bullous pseudophakic dystrophy), also produce corneal opacity that can result in blindness [6]. Corneal transplant is currently the only treatment available for corneal blindness. However, this procedure faces a short-age of tissue donors and immunologic reject risk once the

Valdez-García Jorge E.

2 Current Tissue Engineering, 2016, Vol. 5, No. 1 Valdez-García et al.

graft has been placed; this is in addition to its associated high costs and increased rehabilitation time. Efforts that have been made in the development of alter-native therapies for corneal blindness are focused on tissue engineering. These strategies involve the use of cells, bio-molecules (growth factors) that enhance cell proliferation and assure proper biomarkers expression, and biomaterials to be used as cell scaffolds for transplantation [7]. The latter is of main importance given that it provides the physical me-dium for the graft to resemble de CE monolayer, since the solely injection of CECs into the anterior chamber has been reported as unsuccessful [8]. Given that the cornea is the principal refractive component of the eye, any artificial ele-ment used for the grafts needs to address similar corneal clarity. Additionally, they must offer mechanical strength for surgical manipulation and fit the corneal curvature, thus avoiding wrinkling after transplantation. Several biomaterials are being tested as CEC scaffolds; some of these include amniotic membranes, collagen sheets, the porcine acellular matrix, hydrogel, human corneal stroma, and chitosan-based membranes (CHM) [6, 9-13]. The ease of production, light transmittance (clarity), and biocompatibility are important characteristics that are being measured in order to consider these membranes for future clinical applications in CE transplants. In this research, we aimed to compare the biocompatibility of two different scaf-folds: CHM and collagen–vitrigel membranes (CVM). Chi-tosan has been extensively applied in tissue engineering be-cause of its compatibility, biodegradability, nonantigenic effects, wound-healing properties, and low costs [14]. It is known that protonated chitosan, which results from chitosan dissolution, can form a complex with negatively-charged molecules (growth factors, nucleic acids, cytokines, etc.), which is significant in the modulation of cell behavior during tissue regeneration [15, 16]. A number of topical use applica-tions in veterinary medicine have been reported, including bone repair, tendon healing, and antimicrobial effects [17]. In ophthalmic use, chitosan has demonstrated that it can in-crease the precorneal residence time of antibiotics when compared with commercial solutions [18]. Collagen bioma-terials have been widely used for tissue engineering, given that they function as an extracellular matrix that provides physical support to the cells; they also provide an environ-ment that is similar to physiological conditions, promoting cell adhesion and proliferation. Collagen scaffolds have been tested for several biomedical applications, including bone repair, wound and burn management, and they have been used in general surgery [19]. Different animal models are used for corneal tissue engi-neering, including cats, dogs, rats, and rabbits. From these animals, the rabbit possesses the advantage of being ease of manipulation and similarities with the human cornea, such as diameter (which allows the use of the same surgical instru-mentation and techniques as in humans), central thickness, composition, and repair mechanisms. Moreover, it has been demonstrated that parameters such as CE density, central corneal thickness, and corneal diameter decrease in rabbits similar to in humans [20, 21]. Our research group has dem-onstrated that age also affects the regeneration ability of CE after injury in rabbits [22] . In this study, 3 month old rabbits

were used in order to assess if the implanted biomembranes interfere with the ability to restore corneal clarity after in-jury. The objective of this study was to compare the biocom-patibility in terms of immune response developed following implantation of CHM and CVM in the ocular anterior cham-ber of young rabbits in order to set an optimal scaffold for further CECs transplants.

2. METHODS

2.1. Membrane Preparation

The CHM were prepared in the Department of Biomedi-cal Devices at the Tecnologico de Monterrey, Campus Mon-terrey according to their methodology [23]. The phase inver-sion technique was used, which involves casting of a poly-mer solution onto an inert support followed by immersion of the support with the cast film in a bath filled with a non-solvent for the polymer [24]. Briefly, a 2% w/v of chitosan (Sigma, St. Louis, MO) solution was diluted in 1% v/v lactic acid (Sigma) solution until homogeneity; 5 mL of this solu-tion were casted on a petri dish and mixed with 11.67 mL of 2% wt NaOH - 0.05% wt Na2CO3 (Sigma) solution, that was used as non-solvent. The mixture was dried on a microwave and the produced membrane was peeled carefully and washed several times with distilled water to neutralize it. The CVM were prepared at the Translational Tissue En-gineering Center of Johns Hopkins University, Baltimore, MA, USA according to their published methodology [25]. Briefly, a sterilized nylon membrane in a ring shape with an inner–outer diameter of 23–33 mm, was inserted into a poly-styrene culture dish with a diameter of 35 mm. Equal vol-umes of 0.5% type-I collagen solution and culture medium [DMEM 10% FBS, 20 mM HEPES, 100 units/mL penicillin, and 100 µg/mL streptomycin (all from Gibco, Grand Island, NY)] were uniformly mixed and 2.0 mL of the mixture was poured into the culture dish. The culture dish was incubated at 37°C to complete gelation of the collagen. The collagen gel was then aseptically dried for at least 2 weeks to convert the gel into a rigid glass-like material. This conversion proc-ess is known as vitrification. Finally, the vitrified rigid mate-rial was completely rehydrated with PBS.

2.2. Animal Model

This study was conducted under the consent of the Insti-tutional Committee for the Care and Use of Laboratory Ani-mals of Tecnologico de Monterrey (folio number 2012-Re-001). The animals that were used in accordance with the Guide for the Care and Use of Laboratory Animals. Eight New Zealand White rabbits, 3 months old and weighing be-tween 2 kg and 4 kg, were used for this study.

2.3. Surgical Procedure

One eye was used as the study eye (biomembrane im-planted), and the contralateral eye was used as the control. Under general anesthesia with intramuscular ketamine (30 mg/kg) (Pisa Farmaceutica, Guadalajara, Mexico) and topi-cal anesthesia with tetracaine (5 mg/mL) (Laboratorios Sophia S.A. de C.V., Zapopan, Mexico), a speculum was

In Vivo Biocompatibility of Chitosan and Collagen–vitrigel Current Tissue Engineering, 2016, Vol. 5, No. 1 3

placed and endothelial damage was achieved by endothelial transcorneal freezing with a stainless steel cryoprobe (with a 6 mm tip) placed in liquid nitrogen for 3 minutes to reach a temperature of about –80°C; the tip was subsequently posi-tioned over the center of the cornea for 30 seconds to achieve endothelial damage of approximately 50%, as has been dem-onstrated in previous publications [26, 27]. Subsequently, a peripheral corneal port was created with a 3.2 mm surgical knife, through which a folded 5 mm diameter disc of biomembrane was introduced with microforceps. Sterile CHM were implanted in four eyes of rabbits of the experimental group, and sterile CVM were implanted in the other four rabbits. In the control eyes, the same surgical procedure was performed, but no biomembrane implant was used. When it was necessary to provide volume and to form the anterior chamber in order to properly manipulate the biomembrane, viscoelastic material (Alcon Laboratories, Inc., Fort Worth, TX, USA) was used. Direct pressure was performed to locate the biomembrane disc in the previously damaged endothelial bed. Finally, the corneal port was su-tured with a single 10-0 nylon simple point. Then, 0.5% moxifloxacin ophthalmic drops (Alcon Laboratories, Inc.) were applied as a prophylactic antibiotic. Postoperatively, moxifloxacin 0.5% ophthalmic drops were applied every 4 hours and ophthalmic pranoprofen drops (SIFI, DF, Mexico) were applied every 6 hours.

2.4. Sacrificing of Animals and Tissue Processing

After 1 week, the rabbits were sacrificed via intracardiac injection of sodium pentobarbital at a lethal dose following the induction of general anesthesia with intramuscular keta-mine (30 mg/kg). The clinical macroscopic photographic record of both eyes was made to assess levels of inflamma-tion, neovascularization, corneal opacity, and membrane adhesiveness. A section was performed in the eyeball at the level of the eye equator for resection of the anterior segment, as well as the anterior part of the posterior region, and these were then fixed in 10% formalin for posterior preparation and staining with hematoxylin and eosin (H&E). Briefly, the

eyeballs were fixed in 10% buffered formalin solution by 24 hours, and then equatorial cut sections 2-3 mm in thickness were included for tissue processing (dehydration, clearing, and infiltration) with the paraffin method in an overnight schedule, using an automatic tissue processor. Tissue sec-tions of 4 µm in thickness were taken from the paraffin blocks, and then stained with H&E technique. The stained slides were reviewed in an Axiostar Zeiss microscope.

3. RESULTS

3.1. Macroscopic Analysis and Clinical Findings

One week after implantation, all CHM-implanted eyes showed severe inflammation with 360° neovascularization, ciliary injection, optical media opacity, and purulent exudate in the anterior chamber. Eyes implanted with CVM showed little or no signs of inflammation. Table 1 summarizes the clinical findings and the level of inflammation signs (neovascularization, ciliary injection, and exudate) found 1 week after implantation. Macroscopic findings were photo registered. Fig. (1) shows the clinical appearance of the implanted and control eyes 1 week after surgery. In one eye implanted with CHM, the wound became dehiscent, and purulent material was evi-dent throughout (Fig. 1D). None of the control eyes or those implanted with CVM exhibited this complication. All the CVM implanted eyes showed membrane attachment. In the CHM-implanted eyes it was not possible to assess the mac-roscopic membrane adhesiveness given the opacity levels registered.

3.2. Histopathological Analysis

Prior to histopathological processing, freshly sectioned implanted eyes showed abundant purulent exudate in the anterior chamber, which was adhered to the iris and cornea in the CHM eyes. The CVM eyes showed mild opacity, while the control eyes remained clear (Figs. 2A, B, and C). In all eyes implanted with CHM, severe exudative, inflam-matory, and necrotic processes mainly composed of poly-

Table 1. Clinical findings one week after implantation in eyes implanted with CVM and CHM. The left column indicates the membrane type used and the experimental eye. Levels: absent (0 +), very mild (1 +), mild (2 +), moderate (3 +), severe (4 +).

Corneal inflammation signs Eye

Neovascularization Exudate Ciliary injection Corneal opacity Biomembrane opacity Other findings

CVM 1 0+ 0+ 0+ 1+ Mild

CVM 2 0+ 0+ 1+ 1+ Mild

CVM 3 1+ 0+ 2+ 2+ Mild

CVM 4 1+ 0+ 2+ 2+ Mild

CHM 1 2+ 1+ 1+ 3+ Severe Purulent exudate

CHM 2 3+ 2+ 3+ 4+ Severe

CHM 3 4+ 3+ 4+ 4+ Severe Corneal port dehiscence

CHM 4 4+ 1+ 3+ 4+ Severe


morphonuclear (PMN) leukocytes, cellular debris, and macrophages were observed. The corneas were infiltrated by the inflammatory process, causing thickening and a loss normal histological architecture (Figs. 2E and H). Histologi-cal preparations of the corneas of the eyes implanted with CVM, as well as the control eyes, showed no signs of in-flammation, except in the places where corneal suture ports and closure with a suture were performed. In all CVM and control samples, surgical manipulation marks were found. The histological structure was preserved (Figs. 2D, F, G, and I).

4. DISCUSSION

CE bioengineering is currently aimed at the production of transplantable cell sheets in order to overcome the shortage of tissue donors in the treatment of corneal blindness. For this purpose, cultured CECs are placed in biocompatible scaffolds to replicate the monolayer of the CE. Several mate-rials have been used as CEC scaffolds, such as collagen, am-niotic membranes, biodegradable polymers, Descemet’s membrane, decellularized stroma, and hydrogel lenses. The ease of production, along with the ability of the cells to maintain their viability, morphology, density, and function when cultured on these scaffolds, will determine their reli-ability for in vitro use. In order to be used for in vivo pur-poses, scaffolds must be easy to surgically manipulate and they must produce reduced or no inflammatory signs, while allowing the graft to remain clear. In addition, CVM should allow CECs to form dense monolayers of a uniform cell size, while facilitating the ex-pression of specific functional markers [28]. In our experi-

ence, CHM allowed human CECs to adhere and proliferate maintaining their hexagonal morphology for at least two weeks (nonpublished data). In this study, we aimed to pro-vide an in vivo analysis of the compatibility of these two types of biomembranes for their future use in CEC scaffold-ing for transplantation. The CE engineering encompasses transplantation of CECs seeded in a biocompatible scaffold into a damaged CE of an animal model. Three month old rabbits possess ability to restore corneal clarity 48 h after injury. In order to assess the membrane biocompatibility, CE were injured in all eyes before membrane transplantation. In our study, even when CHM were suitable for cell cul-ture, manipulation with surgical instrumentation made it dif-ficult to implant them into the anterior chamber of the rab-bits. The CHM lacked the appropriate mechanical strength required for surgical handling. In this study, the CHM were prepared without additional compounds. Nevertheless, it has been reported that blending and polymerization of chitosan with different compounds, such as genipin [29], glutaralde-hyde [30], collagen [31], hydroxypropyl gelatin chondroitin sulfate [25], hydroxyethyl sulfate–gelatin [32], and polyca-prolactone [33, 34], enhances its mechanical and chemical properties. At 24 hours post-implantation, the CHM eyes developed inflammation and corneal opacity, which became severe af-ter 1 week. Previous studies have shown that chitosan en-hances the function of inflammatory cells, such as PMN leu-cocytes, macrophages, and fibroblasts, when used as a wound-healing accelerator in large, open wounds of animals, and when implanted into mouse skin [35, 36]. Additionally, it has been suggested that chito–oligosaccharides should be

Fig. (1). Clinical appearance 1 week after implantation of the biomembrane. Both the control eyes (A) and the CVM eye (B) retained corneal clarity and mild-to-moderate inflammation. Eyes implanted with CHM (C) showed severe inflammation with 360° limbal neovasculariza-tion, ciliary injection, corneal opacity, and purulent exudate in the anterior chamber. One eye implanted with CHM developed a corneal de-hiscence port (D).


used in functional foods for the prevention and alleviation of inflammatory diseases [37]. However, it has also been re-ported that chitosan causes lethal pneumonia in dogs that are given a high dose of chitosan, and that the intratumor injec-tion of chitosan in tumor-bearing mice increases the rate of metastasis and tumor growth [35]. These contradictory data are attributed to the purity, contaminant, and acetylation de-gree of chitosan [38]. In fact, it has been shown that the in-flammatory reaction to chitosan can be reduced by decreas-ing its degree of acetylation [39]. In order to improve the obtained results, chitosan should be characterized for the degree of acetylation and blended with other compounds. Collagen type I has been proposed for corneal matrix substitution [40, 41] and for the engineering of corneal scaf-folds [42-45]. In these studies, the membranes prepared with collagen I have been mixed with different molecules such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and poly N-isopropylacrylamide-co-acrylic acid-co-acryloxysuccinimide, and they have under-gone treatments with different concentrations of collagen I that may lead to differences in ocular clarity following im-

plantation. Collagen–vitrigel is a thin, transparent membrane that is prepared from collagen I, and it features enhanced gel strength that results from a three-stage process that includes gelation, vitrification, and rehydration [46]. Its use in corneal keratocytes and endothelial cells demonstrated efficacy in maintaining its morphology, adhesive structures, and mo-lecular markers [28]. Furthermore, optimal preparation con-ditions for collagen–vitrigel have been established in order to provide the best tensile strength and transmittance for its use in corneal applications [47]. In our study, no complications were detected when CVM was surgically manipulated. Injured eyes implanted with these membranes recovered their corneal clarity and exhib-ited minimal signs of inflammation. Microscopic analysis revealed signs of surgical manipulation and the preservation of microstructures. This was in accordance with the findings from a recent study conducted by our colleagues, which demonstrated the safety of CVM in combination with chon-droitin sulfate–polyethylen glycol in the treatment of ocular surface injuries [48]. Taken together, since CEC expansion was apparent in CVM, and given the biocompatibility dem-

Fig. (2). The fresh control eye sample (A) showed corneal clarity and integrity of structure. The fresh CHM-implanted eye (B) presented with abundant purulent exudate in the anterior chamber, which adhered to the iris and cornea, while the CVM-implanted (C) fresh eye revealed the anterior chamber and iris in its full state; the biomembrane exhibited mild-to-moderate opacity. Histology section with H&E staining of one control eye (50X) showed no evidence of inflammation (D). An eye im-planted with CHM (50X) showed exudative inflammatory and necrotic processes around the membrane, as seen in the pano-ramic photograph (E). Histology section of the CVM-implanted eye (F) (50X) showed no signs of inflammation. The magni-fied picture of a control eye (G) and a CVM eye (I) (250X) showed the integrity of the microstructure. The magnified picture of the CHM-implanted eye (H) (400X) exhibited cell detritus between the adjacent CHM and Descemet’s membrane, as well as complete loss of the endothelium.


onstrated in this study, it is clear that the use of CVM in cor-neal engineering for the reestablishment of corneal clarity could be beneficial.

CONCLUSION

To our knowledge, this is the first study to compare the biosafety of CHM and CVM in an animal model. CHM were not suitable for surgical manipulation and for in vivo grafts. Additional experiments with CHM blended with other com-pounds would determine if an improvement in the manipula-tion and decrease in the in vivo inflammatory response could be reached. CVM demonstrated to be ease for surgical ma-nipulation and allowed the cornea to reestablish clarity with minimal signs of inflammation. Further grafting of CVM seeded with CECs on CE-damaged eyes in animal models will determine the efficacy of this therapy as an alternative for corneal grafts in the treatment of corneal blindness.

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flict of interest.

ACKNOWLEDGEMENT

We want to thank to the Ophthalmology Institute, Tec Salud for facilitating the carrying out of this research.

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Received: April 09, 2015 Revised: July 22, 2015 Accepted: August 05, 2015

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