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Nanofiber-reinforced decellularized amniotic membrane improves limbal stem cell transplantation in a rabbit model of corneal epithelial defect
Acta Biomaterialia 97 (2019) 310–320
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Nanofiber-reinforced decellularized amniotic membrane improves limbal stem cell transplantation in a rabbit model of corneal epithelial defect Zhengbing Zhoua,b,c, Da Longd,e, Chih-Chien Hsue,f, Huanhuan Liub,g, Long Chenb,c,g, Benjamin Slaving, Hui Line, Xiaowei Lib,c,g,i, Juyu Tanga, Samuel Yiue, Sami Tuffahah,∗, Hai-Quan Maob,c,g,∗ a
Department of Hand and Microsurgery, Xiangya Hospital of Central South University, Changsha, Hunan Province 410008, PR China Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA c Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA d Department of Ophthalmology, The First Hospital of Hunan University of Chinese Traditional Medicine, Changsha, Hunan Province 410007, PR China e Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA f Department of Ophthalmology, Taipei Veterans General Hospital, National Yang-Ming University, No. 201, Shipai Rd. Sec. 2, Taipei, Taiwan g Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA h Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA i Mary and Dick Holland Regenerative Medicine Program, Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA b
a r t i c l e
i n f o
Article history: Received 19 March 2019 Revised 13 August 2019 Accepted 15 August 2019 Available online 19 August 2019 Keywords: Amniotic membrane Electrospinning Nanofibers Composite membrane Limbal stem cell deficiency Transplantation
a b s t r a c t Human amniotic membrane (AM) offers unique advantages as a matrix to support the transplantation of limbal stem cells (LSCs) due to its inherent pro-regenerative and anti-inflammatory properties. However, the widespread use of AM in clinical treatments of ocular surface disorders is limited by its weak mechanical strength and fast degradation, and high cost associated with preserving freshly isolated AM. Here we constructed a composite membrane consisting of an electrospun bioabsorbable poly(ε -caprolactone) (PCL) nanofiber mesh to significantly improve the ultimate tensile strength, toughness, and suture retention strength by 4–10-fold in comparison with decellularized AM sheet. The composite membrane showed extended stability and conferred longer-lasting coverage on wounded cornea surface compared with dAM. The composite membrane maintained the pro-regenerative and immunomodulatory properties of dAM, promoted LSC survival, retention, and organization, improved re-epithelialization of the defect area, and reduced inflammation and neovascularization. This study demonstrates the translational potential of our composite membrane for stem cell-based treatment of ocular surface damage. Statement of Significance Human decellularized amniotic membrane (dAM) has been widely shown as a biodegradable and bioactive matrix for regenerative tissue repair. However, the weak mechanical property has limited its widespread use in the clinic. Here we constructed a composite membrane using a layer of electrospun poly(ε -caprolactone) (PCL) nanofiber mesh to reinforce the dAM sheet through covalent interfacial bonding, while retaining the unique bioactivity of dAM. In a rabbit model of limbal stem cell (LSC) deficiency induced by alkaline burn, we demonstrated the superior property of this PCL-dAM composite membrane for repairing damaged cornea through promoting LSC transplantation, improving re-epithelialization, and reducing inflammation and neovascularization. This new composite membrane offers great translational potential in supporting stem cell-based treatment of ocular surface damage. © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
∗ Corresponding authors at: 720 Rutland Avenue, Russ 749D, Baltimore, MD 21205, USA (S. Tuffaha). 3400 N. Charles Street, Croft Hall 100, Baltimore, MD, 21218, USA (H.-Q. Mao).
https://doi.org/10.1016/j.actbio.2019.08.027 1742-7061/© 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
E-mail addresses: [email protected] (S. Tuffaha), [email protected] (H.-Q. Mao).
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1. Introduction The corneal epithelium depends upon limbal epithelial stem cells (LSCs) for continual maintenance and restoration of defects that would otherwise accumulate and compromise this critical barrier [1]. LSCs can be depleted by a number of pathologic processes that include chemical, thermal, infectious, and iatrogenic injury, as well as systemic autoimmune disorders such as Stevens-Johnson syndrome and bullous pemphigoid [2]. Conditions that significantly damage the LSCs can result in an invasion of conjunctival epithelium on to the corneal surface. The resulting syndrome, termed limbal stem cell deficiency (LSCD), is characterized by chronic inflammation of the corneal surface with overlying fibrovascular tissue and subsequent loss of vision [3]. Contemporary treatment options for LSCD depend upon pathological severity [2]. Conservative treatments such as supportive management and corneal scraping can be effective in restoring the epithelium in the setting of mild disease [4]. Amniotic membrane (AM) patching has also shown promise for partial LSCD, where residual native LSCs are available to populate the membrane. In contrast, effective treatment of severe LSCD requires both resurfacing of the damaged corneal surface and reconstitution of the missing LSC population [5]. Corneal grafts in these cases are prone to failure due to chronic inflammation of the graft bed and the inability to reconstitute LSCs, as they are not available within the grafts to begin with. The use of LSC-containing limbal rim autograft has demonstrated greater efficacy; but is limited by the morbidity associated with harvesting the graft from the contralateral eye [6]. More recently, transplantation of cultured, in vitro-expanded LSCs has been explored as a means of limiting donor site morbidity [7]. However, the cell transplantation approach requires a substrate to resurface the damaged corneal membrane and facilitate engraftment. Human decellularized amniotic membrane (dAM) has been used as a temporary biological wound dressing and a substrate for LSC transplantation in LSCD treatment [8]. Numerous clinical cases and animal studies have demonstrated the efficacy of either freshly isolated or cryopreserved dAM for this purpose [9,10]. In addition to its function as a cell carrier, the native growth factors and cytokines within dAM promote corneal repair and regeneration when used to treat chronic corneal diseases [11]. Due to the high cost associated with the preservation and delivery of fresh human dAM [12], decades of research efforts have focused on the optimization of processing, with the goal of elucidating methods of cost-effective storage and transportability. Currently available cryopreserved dAM products, including AmnioGraft® and NEOX®, are also limited by a lack of structural integrity, which hinders intraoperative handling and suturing, as well as a rapid degradation rate that introduces the potential need for re-application [13–15]. In order to address these limitations, we have developed a new composite membrane consisting of an electrospun, bioabsorbable polymer fiber mesh bonded to a human dAM sheet through interfacial conjugation. In vitro experiments have shown that interfacial bonding substantially improves the tensile properties and toughness of dAM, making the composite membrane better able to retain sutures and conform to the convex surface of the globe [16]. Additional benefits observed in vivo include maintenance of proliferation and biological activity of transplanted LSCs, as well as improved biocompatibility as seen by increased polarization of macrophages to an anti-inflammatory phenotype compared to other AM formulations. In this study, we tested the poly(ε -carprolactone) (PCL) nanofiber-dAM composite membrane in comparison to dAM alone, with and without cultured LSCs, in a rabbit LSCD corneal epithelial defect model. We hypothesized that the composite membrane would enhance the stability and durability of dAM alone while
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maintaining the pro-regenerative and immunomodulatory properties to thereby provide enhanced healing of corneal epithelial defects with reduced inflammation and vascularity in the setting of LSCD. 2. Materials and methods 2.1. Electrospun PCL nanofiber mesh preparation and activation of carboxyl groups on fiber surface An electrospun PCL nanofiber mesh was fabricated as previously described [17]. Briefly, the electrospinning procedure consisted of dissolving a 12 wt% PCL solution (viscosity average MW, 70–90 kDa; Sigma-Aldrich) in a mixture of dichloromethane and methanol (4:1, v/v). This mixture was then injected at a rate of 2.5 mL/h via a 27-gauge needle onto a grounded and stationary plate collector. There was a 12-kV positive potential between the needle and ground. The use of a grounded and stationary collector for deposition of fibers provided random fiber meshes. Modifying the collection time allowed for control of the thickness of the nanofiber mesh to approximately 40 μm. The PCL fiber meshes were air-dried in a laminar hood overnight and subjected to the following sequential steps: (i) treating with plasma in a radiofrequency plasma cleaner (Harrick Plasma) at a medium dose for 10 min, (ii) incubating the treated fiber mesh with 10% acrylic acid solution containing 0.5 mM NaIO4 , followed by exposure to ultraviolet light at an intensity of 30–50 mW/cm2 for 2 min in an ice bath (0–4 °C), (iii) removing un-grafted poly(acrylic acid) (PAAc) from the surface of the nanofiber mesh by washing with deionized (DI) water, (iv) measuring the concentration of the carboxyl groups on fiber mesh by the Toluidine Blue O (TBO) assay, (v) activating the surface carboxylic groups by reacting the treated mesh with N-hydroxysuccinimide (NHS, Sigma) and equal molar of N-(3-dimethylaminopropyl)-N-ethyl carbodiimide (EDC, Sigma) in 50% ethanol in a glass dish for 4 h at a molar ratio of 1:4:4 for COOH:NHS:EDC, (vi) removing excess reagents by thorough rinse with 70% ethanol for three times at 5 min/wash, and (vii) drying the modified mesh in a laminar flow hood. The modified nanofiber mesh was stored at −20 °C and rinsed with sterile phosphatebuffered saline (PBS) before use. The amounts of residual solvents (dichloromethane and methanol) were analyzed by gas chromatography and found to be below 200 ppm (Impact Analytical). These residue contents are significantly lower than the recommended levels (600 and 2000 ppm, respectively) by the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). 2.2. Preparation of decellularized human amniotic membrane Composite membrane preparation was carried out through the use of cryopreserved human AMs from a commercial supplier (AmnioGraft®, Bio-Tissue Inc.). The epithelial cell layer of AM was removed by dispase treatment. Once thawed, the sample of human AM was placed onto a membrane comprised of nitrocellulose. The epithelial cell layer was left exposed. A 2.5% (w/v) solution was made through the dissolution of dispase (Millipore) in a DMEM/F12 medium (Life Technology). The nitrocellulose membrane, along with the AM to which it was still adherent, was kept for 5 h at 4 °C in a 2.5% dispase solution and subsequently triple washed using sterile PBS. An Iris spatula (Fine Science Tools) was utilized with the aid of a stereoscope (Nikon SMZ800) in order to remove the layer of epithelial cells. Lastly, the decellularized AM (dAM) was triple washed with sterile PBS to ensure that it was ready for use. A DNA isolation kit (DNeasy Blood & Tissue Kit, QIAGEN) was employed in order to quantify the
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amount of DNA present as a measure to evaluate the effectiveness of removal of the epithelial cells. After the decellularization procedure, the total content of DNA in AM was 20.7 ± 13.5 ng/mg (n = 5 separate samples), which was <5% of the level in native AM (488.2 ± 140.7 ng/mg). These results confirm that the total level of DNA in the dAM is below 50 ng/mg, consistent with the FDA standard for a similar decellularized extracellular matrix products [18]. As an additional evidence, the absence of cell nuclei on the processed dAM was confirmed by examining the membranes under the fluorescence microscope after treatment. 2.3. Fabrication of a composite membrane via conjugation of the fiber mesh onto the dAM We have previously described the fabrication of a composite membrane with conjugation of the electrospun PCL nanofiber mesh to the dAM [16]. In brief, the dAM was placed on a sheet of polytetrafluoroethene (PTFE). The epithelial side of the dAM was oriented adjacently to the PTFE. Next, the surface-activated nanofiber mesh was placed on the stromal side of the dAM, with a second sheet of PTFE being positioned next to the nanofiber mesh. The assembled layers were subsequently placed between two metal plates, which applied 6 h of mechanical compression at a temperature of 4 °C in order to maintain total thickness of the composite at 20 μm. This technique ensured that there was sufficient contact between the dAM and nanofibers. A condensation reaction between the carboxyl groups found on the surface of the fibers and the amino groups of the protein constituents found on the dAM was used to complex the nanofibers to the dAM. Following removal of the metal plates and sheets of PTFE, a freeze dryer (Labconco Freezone 12L Cascade Freeze Dry System) was utilized in order to lyophilize the composite membrane, which was subsequently stored at −20 °C until needed for further utilization.
were transduced with an exogenous GFP gene. After one week, the cells covered the entire material. The cell side faced the cornea and covered the surface of the rabbit cornea when repairing LSCD. 2.6. Alkali Burn-Induced LSCD model Surgical and animal care procedures were conducted under a protocol approved by the Institutional Animal Care and Use Committee at Johns Hopkins University according to guidelines established by National Institutes of Health and American Association for the Accreditation of Laboratory Animal Care. Alkali burn-induced LSCD was performed as detailed in previous work [20,21]. In brief, ketamine (100 mg/kg) and xylazine (5 mg/kg) were injected intramuscularly into the gluteus maximus of the rabbits in order to achieve anesthetization. This study exclusively used the right eye of all rabbits. Ring-shaped sterile filter paper that had been saturated with 1 N NaOH was placed onto the cornea for 50 s. Following a rinse with 0.9% NaCl, steroids and antibiotic eye drops (0.5% moxifloxacin and 0.1% dexamethasone) (Vigadexa, Alcon) were applied to the rabbit eyes following an alkaline burn, twice daily for 2 weeks. Corneal epithelial defect (CED), opacity of the cornea, vascularization of the cornea, and conjunctivalization, when present, were counted as signs of LSCD. 2.7. Transplantation of LSCs on PCL Fiber-dAM composite membrane in a severe LSCD model
The degradation rate of the PCL-dAM composite was measured using the method described previously in the literature [19]. In brief, the dAM, PCL, and PCL-dAM membranes were placed in either the 1-U pancreatin buffer or PBS as a buffer control. To determine mass loss at different time points, samples were washed with deionized (DI) water 3 times, and lyophilized; and then mass loss was calculated accordingly for 1 week.
A total of 25 New Zealand white rabbits (8 weeks and 1.5 ± 0.2 kg) with LSCD model established in the right eye accordingly to the procedure described above were grouped randomly into 5 groups (5 rabbits per group), which received treatments with dAM, PCL membrane, PCL-dAM composite membrane, 3 × 106 LSC-seeded dAM (LSC-dAM), and LSC-seeded PCL-dAM composite membrane (LSC-PCL-dAM), respectively, in the damaged eye at 2 weeks after alkali burn to induce LSCD. Under general anesthesia, 0.2% fluorescein staining was performed to examine the repair of the corneal surface. After the eyes were washed with saline, a keratome was used to scrape the ocular surface scar tissue. A graft was then placed on the ocular surface and fixed to the sclera with a Nylon 10-O suture (Ethilon Nylon Suture, Johnson & Johnson Medical Device). An ointment combination of antibiotic and steroid was applied to the surgically modified eye twice per day for 5 consecutive days. After two weeks, the samples were collected.
2.5. Rabbit LSC isolation and culture
2.8. Immunohistochemistry and histology
Rabbit LSCs were isolated as previously described [16]. The limbus of fresh New Zealand white rabbit eyeballs (purchased from Pel-Freez Biologicals) was harvested with a scalpel under magnification and incubated with 2.5 mg/ml dispase for 12 h at a temperature of 4 °C. Single cells were obtained by further digestion with 0.25% trypsin/EDTA for a time of 5 min. After using a trypsin inhibitor for blocking, a centrifuge was utilized at 1200 rpm for a time of 5 min in order to collect the cells. The cells were then re-suspended in LSC culture medium containing a known keratinocyte-SFM basal medium (Invitrogen), known keratinocyte-SFM growth supplement (Invitrogen), 1% antibioticAntimycotic (Gibco), and a GFP Lentiviral Vector (ABM Inc.). The dAM or PCL-dAM membrane was trimmed into a circular shape with a diameter of 20 mm, and then wound into a polypropylene tube with an 18 mm diameter using a fine copper wire to allow for the preparation of a cell culture cup. After soaking for 3 h, the membranes were rinsed in PBS three times and then soaked for an additional hour. They were then placed into 16-well plates. Corneal limbal stem cells were subsequently cultivated at a density of 150 × 104 per culture cup, while the cells
Staining of frozen sections (8 μm) of cornea was done for rabbit anti-CD86 (1:500, Cell Signaling) anti-KRT12 (1:200, ABIN290007, Antibodies-online), anti-KRT13 (1:200, ABIN108618, Antibodiesonline) and anti-KRT3 (1:200, ABIN108617, Antibodies-online) as detailed previously. In brief, the frozen sections were desiccated at room temperature and post-fixed in chilled acetone for a time of 15 min. The sections were then washed with phosphate-buffered saline (PBS), followed by blocking with 8% normal donkey serum in PBS for a time of 1 h at room temperature. The samples were subsequently incubated overnight at a temperature of 4 °C with one of the primary antibodies mentioned above. Following triple washing with PBS/0.05% Tween-20, fluorescently-labeled secondary antibody of CyTM 5 AffiniPure Donkey Anti-Mouse IgG (1:200, 715– 175-151, Jackson ImmunoResearch) and CyTM 3 AffiniPure Donkey Anti-Goat IgG (1:200, 705–165-147, Jackson ImmunoResearch) were incubated along with the samples at room temperature for 1 h. Following an additional three washes with PBS, the sections were counterstained for a time of 3 min at room temperature using DAPI, a nuclear dye (1:1200; Sigma, St. Louis, MO). The slides were mounted using an Aquamount solution and fluorescence
2.4. Characterization of degradation rate
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microscopy (Axioskop; Carl Zeiss Meditec, Thornwood, NY, USA) was then utilized to visualize the samples. An identical exposure time was used for each sample, and the images were analyzed using Image-Pro Plus (Media Cybernetics, Silver Spring, MD). 2.9. RNA isolation and real-time polymerase chain reaction analysis Real-time polymerase chain reaction (PCR) analysis was used in order to assess the levels of mRNA of IL-6, IL-8, CCL2, and VEGF within rabbit cornea treated with different materials. The primers for IL-6 are GAA CAG AAA GGA GGC ACT GG (forward) and CTC CTG AAC TTG GCC TGA AG (reverse); the primers for IL-8 are CCA CAC CTT TCC ATC CCA AAT (forward) and CTT CTG CAC CCA CTT TTC CTT G (reverse); the primers for CCL2 are GTG AAT TCC CCA GTC ACC TG (forward) and TGT GTT CTT GGG TTG TGG AA (reverse); and the primers for VEGF are TTC AAC GTC ACC ATG CAG AT (forward) and AAA TGC TTT CTC CGC TCT GA (reverse). Isolation of total RNA was accomplished via RNeasy Mini Kits (QIAGEN). First-strand cDNA synthesis reactions were carried out though the use of a High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) with an accompanying light cycler component (Biometra TPERSONAL, German). Real-time PCR was carried out through the use of the TaqMan Gene Expression Master Mix (ThermoFisher Scientific) with a light cycler component (StepOnePlusTM Real-Time PCR System, Applied BiosystemsTM ). PCR cycling involved 40 cycles of amplification of the template DNA with primer annealing occurring at a temperature of 60 °C. The 2−Ct method was then used in order to calculate the relative expression level of each gene of interest. The amplification efficiencies of each primer pair were validated in order to allow for gene expression to be quantitatively compared. Only Taqman primers (ThermoFisher Scientific) were utilized. Every quantitative PCR was carried out three times on three different experimental replicates. The results were subsequently normalized to the value found using the reference gene. 2.10. Statistical analysis All quantitative data are expressed as a mean ± SD. Statistically significant differences between the results of various experimental groups were assessed via a student’s t-test. A p value of <0.05 is considered to be indicative of statistical significance. 3. Results 3.1. Composite membrane preparation and stability The composite membrane was prepared accordingly to our previously reported protocol [16]. Mechanical compression was applied to the tightly bonded dAM and PCL nanofiber mesh in order to facilitate the conjugation reaction between the PCL fiber surface and dAM components. The different layers that bonded together formed a composite membrane without delamination following lyophilization (Fig. 1). This interfacial bonding technique not only is important to improve the integrity of the composite membrane, but also improved the mechanical properties and stability of the membrane. The PCL-dAM composite membrane exhibited about 4-fold higher ultimate tensile strength (p < 0.01), 10-fold higher toughness (p < 0.01), and more than 10-fold higher suture retention strength (p < 0.01) in comparison with lyophilized dAM [16]. To determine whether this composite has the capacity to increase the stability of dAM when applied to the damaged corneal surface, we tested the degradation rate of dAM, PCL, and the PCLdAM composite membrane in a pancreatin buffer solution (1 U),
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using PBS as a buffer control. From Day 1–4, dAM showed significantly more rapid degradation than PCL or the PCL-dAM composite membrane. On Day 5, the PCL only membrane maintained its original shape and size in 1-U of pancreatin buffer, while the dAM only membrane completely dissolved (Fig. 1). The composite slows down the degradation rate by about 40%. These results demonstrate that PCL bonding has the potential to extend the stability of dAM in the presence of enzyme, thus improving its stability and lasting coverage on a wound surface. 3.2. Alkali Burn-Induced LSCD model We adopted an LSCD model using alkali burn. Following exposure to alkali and scraping of the epithelium, typical signs of corneal damage, including conjunctival congestion and epithelial damage, were observed within a few hours. Inflammation involving the anterior segment, such as soft tissue swelling of the operative eye and white dense exudation of the anterior chamber, developed within the first few weeks (Fig. 2). During the second week, exudation disappeared, and limbus neovascularization was indicative of chronic inflammation. H&E staining confirmed the loss of the central corneal epithelium and distortion of the limbal epithelium at 2 weeks post-exposure. 3.3. Assessing PCL-dAM composite membrane for corneal defect repair in the LSCD model 3.3.1. In vivo anti-inflammation of PCL-dAM composite membrane We demonstrated previously that the PCL-dAM composite membrane has similar immunomodulatory effects as the dAM and may promote the polarization of macrophages into the proregenerative M2 phenotype in vitro [16]. In order to investigate the immunomodulatory effects of PCL-dAM composite membrane in vivo, we transplanted the dAM only membrane or PCL-dAM composite membrane to the corneal epithelial defect site in a rabbit LSCD model. Corneal edema and inflammatory reactions gradually reduced from week 1 after transplantation in both groups. The PCL-dAM composite membrane was well-seated on the corneal surface without degradation at week 1, while the majority of grafts to which dAM alone had been applied demonstrated thinning and shifting (Fig. 3). Immunohistochemistry showed a high level of CD86-positive M1 macrophages in the control group compared with the dAM or PCL-dAM composite membrane groups. However, CD68, a marker of the M2 macrophage, showed negative staining in all groups. Furthermore, real time PCR revealed lower levels of CCL2, IL-8, and IL-6 mRNA expression in both the dAM and PCLdAM composite membrane groups compared with untreated controls. The high expression of IL-6 mRNA was consistent with an increased CD86 staining intensity in the control group. Reduced VEGF mRNA expression in treated corneas indicates that both dAM and PCL-dAM composite membrane are effective in preventing angiogenesis through inflammatory stimulation (Fig. 4). 3.3.2. PCL-dAM composite membrane promoting conjunctival epithelialization of cornea in LSCD Model. Conjunctivalisation is typically considered as a pathological process during corneal re-epithelialization in the setting of severe LSCD condition because neovascularization and inflammatory cell infiltration typically accompany conjunctivalization [22]. However, integrated conjunctival epithelialization can occur without neovascularization and inflammation and contribute to corneal repair, in which case it serves as a beneficial process to repair. In this study, degradation of the dAM was observed in the first week following transplantation. Conversely, the PCL and PCL-dAM exhibited excellent stability until 4 weeks post-transplantation. Immunohistochemistry demonstrated that K13-positive cells specific to the
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Fig. 1. General appearance and degradation profiles of the PCL-dAM composite membrane. (A) General appearance and (B) SEM images of dAM, PCL and the PCL-dAM composite membranes prepared from the PCL fiber mesh and dAM. (C, D) The degradation profiles of the three samples in PBS (C) and in the presence of enzyme (1 U of pancreatin) (D). ∗ p < 0.001 (in comparison with dAM or PCL mesh).
conjunctival epithelial cells continuously lined the corneal lesion site in the PCL-dAM composite membrane group but formed a much sparser dotting pattern in the dAM only group (Fig. 4). K12positive cells, considered a marker for basal corneal epithelium, were not identified in either group. This finding implies that PCLdAM composite membrane, when used as a protective dress for damaged cornea, can provide a stable environment for the proliferation of conjunctival epithelial cells and functional conjunctivalization, even in the absence of LSCs. 3.4. Transplantation of LSCs with dAM vs. PCL-dAM composite membrane for corneal defect repair in severe LSCD model LSCs were delivered to the corneal defects in combination with the membranes. GFP-labeled LSCs were cultured on either a dAM or PCL-dAM composite membrane for 1 week prior to implantation. Two weeks after implantation, conjunctival hyperemia and ocular irritation were noticeably relieved in both groups. In the PCL-dAM composite membrane group, the corneal epithelium gradually recovered with improved transparency and significantly reduced neovascularization. In contrast, the corneal epithelial defects
persisted in the dAM group and were gradually covered by the conjunctival epithelium, while the cornea became cloudy with extensive neovascularization. GFP-labeled LSCs were identified in the newly-formed epithelial layer in both groups received dAM + LSCs and CM + LSCs (Fig. 5). A higher number of LSCs were found in the group received CM + LSCs; and LSCs in this group spread over a greater area than those identified in the group with dAM + LSCs. The corneal epithelium is the outermost layer of the cornea. It is composed of a single layer of basal cells and four to five cell layers of nonkeratinized, stratified squamous epithelial cells. Immunohistochemistry of K12 and K3, which are cornea-specific keratins that compose the intermediate filament cytoskeleton of corneal epithelial cells, showed linear and spotted staining, respectively, in the “CM + LSCs” group. In contrast, the “dAM + LSCs” group demonstrated predominately K12 staining and minimal K3 staining. H&E staining of the sections at week 2 following composite membrane-supported LSC transplantation revealed restoration of corneal epithelial morphology and hierarchy. Specifically, highly cylindrical basal cells, the upper polygonal pterygium, and superficial flat cells were very similar to that of a normal corneal epithelium. Conversely, in the tissue sections from the “dAM + LSCs”
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Fig. 2. Alkali-burn induced LSCD model results in damage to corneal epithelium. (A) Procedure of LSCD model by alkali burn. Normal: The naive rabbit eye; NaOH: Ring-shaped sterile filter paper saturated with 1 N NaOH is placed onto the cornea for 50 s; Day 0-Rinse: the lesion site is then rinsed with 0.9% NaCl Day 0-Scraping: residual corneal epithelium is scraped; Day 0-Staining: corneal damage is confirmed with fluorescein staining; Week 2-LSCD: Cornea becomes neovascularization occurs at 2-weeks post-exposure. (B) H&E staining showing (left) normal corneal epithelial and (right) loss or distortion of limbal epithelium post-exposure on the LSCD model eye.
group, most epithelial cells were bulky, fusiform or polygonal, and the connections between cells were loose. In addition, cells were irregularly arranged, and ring-like goblet cells were observed in the control group. The presence of basal cells is the most important evidence of corneal epithelial normalization and conjunctivalization. Comparison of basal cell count in each 40 × field for the normal, control, dAM, PCL-dAM composite membrane, “dAM + LSCs”, and “CM + LSCs” groups showed eyes treated with CM + LSCs had basal cell counts closest to those of normal cornea. The cornea covered by PCL-dAM composite membrane without LSCs exhibited better epithelial morphological repair than dAM-covered cornea, however there was no significant difference in basal cell count in either group compared control (Figs. 5 and 6). 4. Discussion Ocular burn is one of the most common forms of LSCD. Experimental alkaline burns is typically used to mimic ocular burns,
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Day 0–1 N for 5 min; opaque as at 2-week
which causes severe depletion of LSCs and local inflammation, and leads to cornea opacity and neovascularzation [23]. A growing body of work has been reported to demonstrate the promise of LSC transplantation in treating alkaline burn-induced LSCD in animal models [24–26]. The success of this approach, however, requires a scaffold to resurface the damaged corneal epithelium and enhance LSC engraftment. AM is well-suited for this purpose, given its immunomodulatory and pro-regenerative properties. AMs have many intrinsic cytokines that promote wound healing by stimulating re-epithelialization and reducing local inflammation [27]. However, the unique microenvironment of the corneal surface, as well as shear forces from eyelid motion, make the grafted dAM more susceptible to deformation and degradation. Consequently, clinical LSC transplantation with dAM membranes must often be repeated due to a lack of stable coverage [28]. Additionally, the thin and fragile nature of AM makes it difficult to handle during the process of implantation to the ocular surface and susceptible to tear during suturing. Unlike approaches of chemical or enzymatic crosslinking
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Fig. 3. PCL-dAM composite membrane maintains structural integrity while reducing vascularization. PCL-dAM composite membrane (CM) remained intact without degradation while dAM had almost disappeared after 1 week. Two weeks after transplantation, conjunctival hyperemia and ocular irritation had been noticeably relieved in both groups. Compared with severe limbal neovascularization and corneal edema in control group, inflammatory reactions were reduced in all treatment groups. There was no limbal neovascularization seen with the group received CM + LSCs. Fluorescein staining at 2-week demonstrated that the majority of corneal epithelial surface had recovered, while higher percentages of the corneal surface stained positively in groups received dAM without LSCs, CM without LSCs, or dAM with LSCs.
that result in loss of bioactivities of the active growth factors and cytokines found in native AM [29,30], others have reported application of amniotic extracts in a gel form to retain more of the active compounds [31]. However, extracting soluble AM components will compromise the extracellular matrix structure, which has been shown to play a role in facilitating re-epithelialization [32,33]. To address these limitations, we developed a novel composite membrane that improves the stability and toughness of AM while maintaining its pro-regenerative and immunomodulatory
properties. The fabrication process involves interfacial bonding of biodegradable, electrospun, surface-carboxylated PCL nanofiber mesh to dehydrated amnion sheets. This avoids the loss of AM biological activity that results from stratification, shedding, and repeated chemical crosslinking used in other processing methods. Meanwhile, physical properties of the composite membrane, including toughness and stability, were enhanced as a result of effectively increasing the membrane thickness and slowing the rate of degradation. During the transplantation procedure, the
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Fig. 4. PCL-dAM composite membrane promotes conjunctival corneal epithelialization in the absence of LSCs the LSCD model. (A) Real time PCR of IL-8, IL-6, CCL2, VEGF mRNA expression. (n = 5, ∗ p < 0.05, ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001, ∗ ∗ ∗ ∗ p < 0.0 0 01). (B) Immunohistochemistry showing K13-positive cells continuously lining the corneal lesion site in PCL-dAM composite membrane (CM) group but forming a dotted pattern in dAM group. As expected, CD86-positive staining was observed more in the control group and dAM than the PCL-dAM composite membrane group. Scale bar: 100 μm.
resilient composite membrane, with a thickness of only 20 μm, largely improved operability over dAM alone, with the latter demonstrating a tendency to easily tear during application to a curved surface. In this study, the composite membrane demonstrated stable attachment to the cornea and resistance to tearing during suture placement and shifting from shear forces. The dAM membrane only and dAM-supported LSC transplantation group both have shown shifted and fully degraded membrane within one week. In contrast, the PCL-dAM composite membrane maintained its original shape, position and structural integrity for at least two weeks following implantation, at which point all membranes were explanted. We therefore speculate that the enhanced epithelial formation in the composite membrane group in vivo was due to the greater durability it conferred to dAM thereby providing a more stable environment for cellular attachment, migration and differentiation during the healing process.
In a typical rabbit LSCD model, a large number of neutrophils infiltrated the cornea during an acute inflammatory response. These neutrophils released proteolytic enzymes, including collagenase and other matrix metalloproteinases, causing progressive lysis of the corneal stroma as well as the release of activated polymorphonuclear leukocytes, and stimulation of the expression of inflammatory factors. The dAM membrane when used as a graft proved capable of secreting a variety of active growth factors and cytokines that promote the apoptosis of polymorphonuclear leukocytes, while inhibiting corneal neovascularization and mitigating the local inflammatory response [34,35]. In the present study, we used dAM membrane or PCL-dAM composite membrane to repair corneal epithelial defects in a rabbit model of LSCD. We observed greater epithelialization and reduced fluorescein staining, both indicators of improved corneal healing, in the PCL-dAM composite membrane group compared
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Fig. 5. PCL-dAM composite membrane improves LSC transplantation and the extent of corneal epithelialization in the LSCD model. Immunohistochemistry of K12 and K3 demonstrated linear and spotted staining in the PCL-dAM composite membrane (CM) supported LSC transplantation group (CM + LSCs) while K12 staining predominated over K3 in dAM supported LSC transplantation group (dAM + LSCs). Scale bar: 100 μm.
with the untreated control and dAM group. The control group developed the greatest degree of corneal opacity and neovascularization, while the ocular surface neovascularization and opacity were significantly decreased in the dAM and PCL-dAM composite membrane groups. PCR demonstrated diminished IL-8, IL-6, and VEGF mRNA in both the dAM and PCL-dAM composite membrane repaired corneas compared with the control group. Taken together, these findings support the hypothesis that the PCL-dAM composite membrane maintains the pro-regenerative and anti-inflammatory properties of the dAM components in vivo. It is worth noting that in the absence of transplanted LSCs, PCL-dAM composite membrane used in the setting of LSCD demonstrated a functional pattern of conjunctivalization without neovascularization or inflammation, which was not observed in dAM membrane group. Nonetheless, given that conjunctivalization has the potential to be pathological, long term studies are needed to fully characterize the repaired cornea structure and ensure the stability of the observed pattern of healing. The normal proliferation and differentiation of the epithelial cells of the ocular surface depends on the presence of a normal epithelial basement membrane [36]. In LSCD, where there is destruction of the epithelial basement membrane, AM provides an ideal substitute substrate capable of supporting LSC growth, given its tissue composition and micro-architecture that closely resembles that of the ocular epithelium basement membrane [37]. In order to assess the suitability of PCL-dAM composite membrane as a substrate for LSC transplantation in comparison with dAM, GFP-labeled LSCs were seeded on the respective membranes and tested in an LSCD corneal defect model. Both PCL-AM composite and dAM membranes allowed for successful engraftment and survival of transplanted LSCs. However, the labeled LSCs in the group received PCL-dAM composite membrane-supported LSCs were distributed in a more uniform band and covered a greater
surface area of the cornea in contrast to the punctuated epithelial fluorescence bands observed in the group received dAMsupported LSCs. H&E staining revealed that the composite membrane + LSCs group had better epithelial delaminated cell morphology than the AM + LSCs group. The corneal stromal collagen content of the composite membrane group was close to that of the normal control group, thus implying that the PCL-dAM composite membrane is more conducive to support LSC survival, retention, migration, and proliferation than dAM alone. Future studies will focus on identifying the long-term fate of the transplanted LSCs. It is also important to note that there were no differences in the number of basal cells observed in the PCL-dAM only and dAM only groups without LSC transplantation in comparison with the untreated control group, despite observed improvements in collagen organization and re-epithelialization, and inhibiting inflammation and neovascularization. Given that the presence of basal cells is the most important indicator of normal corneal healing, this finding reinforces the importance of cell transplantation as an adjunct to membrane resurfacing in the treatment of LSCD. Certainly, the improved mechanical property and extended stability of the PCL-dAM composite membrane further enhance the performance of the dAM as a supporting matrix to harness the full potential of LSC transplantation. Other cell types such as limbal epithelial cells have been tested as alternative to LSC transplantation. For example, the simple limbal epithelial transplantation technique has been shown to successfully repair damaged cornea in the case of unilateral severe LSCD, as an alternative to conjunctival limbal autograft or cultivated limbal epithelial transplantation [38]. Due to the rich component this PCL-dAM composite membrane would also likely to augment the treatment outcomes for transplantation of limbal epithelial cells.
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Fig. 6. PCL-dAM composite membrane group displays improved epithelial delaminated cell morphology. (A) H&E staining of the sections at week 2 following implantation of various membranes. Scale bar: 100 μm. (B) Different layers and cell types of corneal epithelium. Scale bar: 20 μm. (C) Comparison of basal epithelial cell count in each 40 × field for the normal, control, dAM without L SCs (dAM w/o L SCs), PCL-dAM composite membrane without LSCs (CM w/o LSCs), dAM with LSCs (dAM + LSCs), and PCLdAM composite membranes with LSCs (CM + LSCs) (n = 5).
Although non-essential, it may be ideal that the corneal repair membrane is optically transparent. As the PCL nanofiber mesh in the composite membrane is not transparent, the whole membrane is not transparent even after hydration. Nonetheless, we found that re-epithelialization occurs within the dAM sheet that interfaces with the corneal surface. Following integration of the LSC and reepithelialization and the degradation of the dAM components, the PCL mesh can be easily removed, so as not to obscure vision after healing. A transparent or translucent version of the composite membrane can be produced by using a transparent nanofiber sheet, which is currently ongoing.
provides a superior alternative to cryopreserved dAM with greater clinical utility and practicality. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgements This work was partially supported by Johns Hopkins University School of Engineering and a grant from Wuhan Kangchuang Technology Co. Ltd. to Johns Hopkins University.
5. Conclusions References The PCL-dAM composite membrane maintains and augments the beneficial pro-regenerative and immunomodulatory properties of dAM, with enhanced ability to support the survival, retention, migration and organization of LSCs, thus promoting the re-epithelialization and reducing inflammation and neovascularization in the repair site of a rabbit LSCD model. In addition to preserving the biological activity of dAM, the addition of the PCL nanofibers stabilizes and toughens the dAM to provide more durable and longer-lasting coverage of the defect site during the healing process. The PCL-dAM composite membrane therefore
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Full length article
Nanofiber-reinforced decellularized amniotic membrane improves limbal stem cell transplantation in a rabbit model of corneal epithelial defect Zhengbing Zhoua,b,c, Da Longd,e, Chih-Chien Hsue,f, Huanhuan Liub,g, Long Chenb,c,g, Benjamin Slaving, Hui Line, Xiaowei Lib,c,g,i, Juyu Tanga, Samuel Yiue, Sami Tuffahah,∗, Hai-Quan Maob,c,g,∗ a
Department of Hand and Microsurgery, Xiangya Hospital of Central South University, Changsha, Hunan Province 410008, PR China Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA c Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA d Department of Ophthalmology, The First Hospital of Hunan University of Chinese Traditional Medicine, Changsha, Hunan Province 410007, PR China e Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA f Department of Ophthalmology, Taipei Veterans General Hospital, National Yang-Ming University, No. 201, Shipai Rd. Sec. 2, Taipei, Taiwan g Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA h Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA i Mary and Dick Holland Regenerative Medicine Program, Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA b
a r t i c l e
i n f o
Article history: Received 19 March 2019 Revised 13 August 2019 Accepted 15 August 2019 Available online 19 August 2019 Keywords: Amniotic membrane Electrospinning Nanofibers Composite membrane Limbal stem cell deficiency Transplantation
a b s t r a c t Human amniotic membrane (AM) offers unique advantages as a matrix to support the transplantation of limbal stem cells (LSCs) due to its inherent pro-regenerative and anti-inflammatory properties. However, the widespread use of AM in clinical treatments of ocular surface disorders is limited by its weak mechanical strength and fast degradation, and high cost associated with preserving freshly isolated AM. Here we constructed a composite membrane consisting of an electrospun bioabsorbable poly(ε -caprolactone) (PCL) nanofiber mesh to significantly improve the ultimate tensile strength, toughness, and suture retention strength by 4–10-fold in comparison with decellularized AM sheet. The composite membrane showed extended stability and conferred longer-lasting coverage on wounded cornea surface compared with dAM. The composite membrane maintained the pro-regenerative and immunomodulatory properties of dAM, promoted LSC survival, retention, and organization, improved re-epithelialization of the defect area, and reduced inflammation and neovascularization. This study demonstrates the translational potential of our composite membrane for stem cell-based treatment of ocular surface damage. Statement of Significance Human decellularized amniotic membrane (dAM) has been widely shown as a biodegradable and bioactive matrix for regenerative tissue repair. However, the weak mechanical property has limited its widespread use in the clinic. Here we constructed a composite membrane using a layer of electrospun poly(ε -caprolactone) (PCL) nanofiber mesh to reinforce the dAM sheet through covalent interfacial bonding, while retaining the unique bioactivity of dAM. In a rabbit model of limbal stem cell (LSC) deficiency induced by alkaline burn, we demonstrated the superior property of this PCL-dAM composite membrane for repairing damaged cornea through promoting LSC transplantation, improving re-epithelialization, and reducing inflammation and neovascularization. This new composite membrane offers great translational potential in supporting stem cell-based treatment of ocular surface damage. © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
∗ Corresponding authors at: 720 Rutland Avenue, Russ 749D, Baltimore, MD 21205, USA (S. Tuffaha). 3400 N. Charles Street, Croft Hall 100, Baltimore, MD, 21218, USA (H.-Q. Mao).
https://doi.org/10.1016/j.actbio.2019.08.027 1742-7061/© 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
E-mail addresses: [email protected] (S. Tuffaha), [email protected] (H.-Q. Mao).
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1. Introduction The corneal epithelium depends upon limbal epithelial stem cells (LSCs) for continual maintenance and restoration of defects that would otherwise accumulate and compromise this critical barrier [1]. LSCs can be depleted by a number of pathologic processes that include chemical, thermal, infectious, and iatrogenic injury, as well as systemic autoimmune disorders such as Stevens-Johnson syndrome and bullous pemphigoid [2]. Conditions that significantly damage the LSCs can result in an invasion of conjunctival epithelium on to the corneal surface. The resulting syndrome, termed limbal stem cell deficiency (LSCD), is characterized by chronic inflammation of the corneal surface with overlying fibrovascular tissue and subsequent loss of vision [3]. Contemporary treatment options for LSCD depend upon pathological severity [2]. Conservative treatments such as supportive management and corneal scraping can be effective in restoring the epithelium in the setting of mild disease [4]. Amniotic membrane (AM) patching has also shown promise for partial LSCD, where residual native LSCs are available to populate the membrane. In contrast, effective treatment of severe LSCD requires both resurfacing of the damaged corneal surface and reconstitution of the missing LSC population [5]. Corneal grafts in these cases are prone to failure due to chronic inflammation of the graft bed and the inability to reconstitute LSCs, as they are not available within the grafts to begin with. The use of LSC-containing limbal rim autograft has demonstrated greater efficacy; but is limited by the morbidity associated with harvesting the graft from the contralateral eye [6]. More recently, transplantation of cultured, in vitro-expanded LSCs has been explored as a means of limiting donor site morbidity [7]. However, the cell transplantation approach requires a substrate to resurface the damaged corneal membrane and facilitate engraftment. Human decellularized amniotic membrane (dAM) has been used as a temporary biological wound dressing and a substrate for LSC transplantation in LSCD treatment [8]. Numerous clinical cases and animal studies have demonstrated the efficacy of either freshly isolated or cryopreserved dAM for this purpose [9,10]. In addition to its function as a cell carrier, the native growth factors and cytokines within dAM promote corneal repair and regeneration when used to treat chronic corneal diseases [11]. Due to the high cost associated with the preservation and delivery of fresh human dAM [12], decades of research efforts have focused on the optimization of processing, with the goal of elucidating methods of cost-effective storage and transportability. Currently available cryopreserved dAM products, including AmnioGraft® and NEOX®, are also limited by a lack of structural integrity, which hinders intraoperative handling and suturing, as well as a rapid degradation rate that introduces the potential need for re-application [13–15]. In order to address these limitations, we have developed a new composite membrane consisting of an electrospun, bioabsorbable polymer fiber mesh bonded to a human dAM sheet through interfacial conjugation. In vitro experiments have shown that interfacial bonding substantially improves the tensile properties and toughness of dAM, making the composite membrane better able to retain sutures and conform to the convex surface of the globe [16]. Additional benefits observed in vivo include maintenance of proliferation and biological activity of transplanted LSCs, as well as improved biocompatibility as seen by increased polarization of macrophages to an anti-inflammatory phenotype compared to other AM formulations. In this study, we tested the poly(ε -carprolactone) (PCL) nanofiber-dAM composite membrane in comparison to dAM alone, with and without cultured LSCs, in a rabbit LSCD corneal epithelial defect model. We hypothesized that the composite membrane would enhance the stability and durability of dAM alone while
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maintaining the pro-regenerative and immunomodulatory properties to thereby provide enhanced healing of corneal epithelial defects with reduced inflammation and vascularity in the setting of LSCD. 2. Materials and methods 2.1. Electrospun PCL nanofiber mesh preparation and activation of carboxyl groups on fiber surface An electrospun PCL nanofiber mesh was fabricated as previously described [17]. Briefly, the electrospinning procedure consisted of dissolving a 12 wt% PCL solution (viscosity average MW, 70–90 kDa; Sigma-Aldrich) in a mixture of dichloromethane and methanol (4:1, v/v). This mixture was then injected at a rate of 2.5 mL/h via a 27-gauge needle onto a grounded and stationary plate collector. There was a 12-kV positive potential between the needle and ground. The use of a grounded and stationary collector for deposition of fibers provided random fiber meshes. Modifying the collection time allowed for control of the thickness of the nanofiber mesh to approximately 40 μm. The PCL fiber meshes were air-dried in a laminar hood overnight and subjected to the following sequential steps: (i) treating with plasma in a radiofrequency plasma cleaner (Harrick Plasma) at a medium dose for 10 min, (ii) incubating the treated fiber mesh with 10% acrylic acid solution containing 0.5 mM NaIO4 , followed by exposure to ultraviolet light at an intensity of 30–50 mW/cm2 for 2 min in an ice bath (0–4 °C), (iii) removing un-grafted poly(acrylic acid) (PAAc) from the surface of the nanofiber mesh by washing with deionized (DI) water, (iv) measuring the concentration of the carboxyl groups on fiber mesh by the Toluidine Blue O (TBO) assay, (v) activating the surface carboxylic groups by reacting the treated mesh with N-hydroxysuccinimide (NHS, Sigma) and equal molar of N-(3-dimethylaminopropyl)-N-ethyl carbodiimide (EDC, Sigma) in 50% ethanol in a glass dish for 4 h at a molar ratio of 1:4:4 for COOH:NHS:EDC, (vi) removing excess reagents by thorough rinse with 70% ethanol for three times at 5 min/wash, and (vii) drying the modified mesh in a laminar flow hood. The modified nanofiber mesh was stored at −20 °C and rinsed with sterile phosphatebuffered saline (PBS) before use. The amounts of residual solvents (dichloromethane and methanol) were analyzed by gas chromatography and found to be below 200 ppm (Impact Analytical). These residue contents are significantly lower than the recommended levels (600 and 2000 ppm, respectively) by the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). 2.2. Preparation of decellularized human amniotic membrane Composite membrane preparation was carried out through the use of cryopreserved human AMs from a commercial supplier (AmnioGraft®, Bio-Tissue Inc.). The epithelial cell layer of AM was removed by dispase treatment. Once thawed, the sample of human AM was placed onto a membrane comprised of nitrocellulose. The epithelial cell layer was left exposed. A 2.5% (w/v) solution was made through the dissolution of dispase (Millipore) in a DMEM/F12 medium (Life Technology). The nitrocellulose membrane, along with the AM to which it was still adherent, was kept for 5 h at 4 °C in a 2.5% dispase solution and subsequently triple washed using sterile PBS. An Iris spatula (Fine Science Tools) was utilized with the aid of a stereoscope (Nikon SMZ800) in order to remove the layer of epithelial cells. Lastly, the decellularized AM (dAM) was triple washed with sterile PBS to ensure that it was ready for use. A DNA isolation kit (DNeasy Blood & Tissue Kit, QIAGEN) was employed in order to quantify the
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amount of DNA present as a measure to evaluate the effectiveness of removal of the epithelial cells. After the decellularization procedure, the total content of DNA in AM was 20.7 ± 13.5 ng/mg (n = 5 separate samples), which was <5% of the level in native AM (488.2 ± 140.7 ng/mg). These results confirm that the total level of DNA in the dAM is below 50 ng/mg, consistent with the FDA standard for a similar decellularized extracellular matrix products [18]. As an additional evidence, the absence of cell nuclei on the processed dAM was confirmed by examining the membranes under the fluorescence microscope after treatment. 2.3. Fabrication of a composite membrane via conjugation of the fiber mesh onto the dAM We have previously described the fabrication of a composite membrane with conjugation of the electrospun PCL nanofiber mesh to the dAM [16]. In brief, the dAM was placed on a sheet of polytetrafluoroethene (PTFE). The epithelial side of the dAM was oriented adjacently to the PTFE. Next, the surface-activated nanofiber mesh was placed on the stromal side of the dAM, with a second sheet of PTFE being positioned next to the nanofiber mesh. The assembled layers were subsequently placed between two metal plates, which applied 6 h of mechanical compression at a temperature of 4 °C in order to maintain total thickness of the composite at 20 μm. This technique ensured that there was sufficient contact between the dAM and nanofibers. A condensation reaction between the carboxyl groups found on the surface of the fibers and the amino groups of the protein constituents found on the dAM was used to complex the nanofibers to the dAM. Following removal of the metal plates and sheets of PTFE, a freeze dryer (Labconco Freezone 12L Cascade Freeze Dry System) was utilized in order to lyophilize the composite membrane, which was subsequently stored at −20 °C until needed for further utilization.
were transduced with an exogenous GFP gene. After one week, the cells covered the entire material. The cell side faced the cornea and covered the surface of the rabbit cornea when repairing LSCD. 2.6. Alkali Burn-Induced LSCD model Surgical and animal care procedures were conducted under a protocol approved by the Institutional Animal Care and Use Committee at Johns Hopkins University according to guidelines established by National Institutes of Health and American Association for the Accreditation of Laboratory Animal Care. Alkali burn-induced LSCD was performed as detailed in previous work [20,21]. In brief, ketamine (100 mg/kg) and xylazine (5 mg/kg) were injected intramuscularly into the gluteus maximus of the rabbits in order to achieve anesthetization. This study exclusively used the right eye of all rabbits. Ring-shaped sterile filter paper that had been saturated with 1 N NaOH was placed onto the cornea for 50 s. Following a rinse with 0.9% NaCl, steroids and antibiotic eye drops (0.5% moxifloxacin and 0.1% dexamethasone) (Vigadexa, Alcon) were applied to the rabbit eyes following an alkaline burn, twice daily for 2 weeks. Corneal epithelial defect (CED), opacity of the cornea, vascularization of the cornea, and conjunctivalization, when present, were counted as signs of LSCD. 2.7. Transplantation of LSCs on PCL Fiber-dAM composite membrane in a severe LSCD model
The degradation rate of the PCL-dAM composite was measured using the method described previously in the literature [19]. In brief, the dAM, PCL, and PCL-dAM membranes were placed in either the 1-U pancreatin buffer or PBS as a buffer control. To determine mass loss at different time points, samples were washed with deionized (DI) water 3 times, and lyophilized; and then mass loss was calculated accordingly for 1 week.
A total of 25 New Zealand white rabbits (8 weeks and 1.5 ± 0.2 kg) with LSCD model established in the right eye accordingly to the procedure described above were grouped randomly into 5 groups (5 rabbits per group), which received treatments with dAM, PCL membrane, PCL-dAM composite membrane, 3 × 106 LSC-seeded dAM (LSC-dAM), and LSC-seeded PCL-dAM composite membrane (LSC-PCL-dAM), respectively, in the damaged eye at 2 weeks after alkali burn to induce LSCD. Under general anesthesia, 0.2% fluorescein staining was performed to examine the repair of the corneal surface. After the eyes were washed with saline, a keratome was used to scrape the ocular surface scar tissue. A graft was then placed on the ocular surface and fixed to the sclera with a Nylon 10-O suture (Ethilon Nylon Suture, Johnson & Johnson Medical Device). An ointment combination of antibiotic and steroid was applied to the surgically modified eye twice per day for 5 consecutive days. After two weeks, the samples were collected.
2.5. Rabbit LSC isolation and culture
2.8. Immunohistochemistry and histology
Rabbit LSCs were isolated as previously described [16]. The limbus of fresh New Zealand white rabbit eyeballs (purchased from Pel-Freez Biologicals) was harvested with a scalpel under magnification and incubated with 2.5 mg/ml dispase for 12 h at a temperature of 4 °C. Single cells were obtained by further digestion with 0.25% trypsin/EDTA for a time of 5 min. After using a trypsin inhibitor for blocking, a centrifuge was utilized at 1200 rpm for a time of 5 min in order to collect the cells. The cells were then re-suspended in LSC culture medium containing a known keratinocyte-SFM basal medium (Invitrogen), known keratinocyte-SFM growth supplement (Invitrogen), 1% antibioticAntimycotic (Gibco), and a GFP Lentiviral Vector (ABM Inc.). The dAM or PCL-dAM membrane was trimmed into a circular shape with a diameter of 20 mm, and then wound into a polypropylene tube with an 18 mm diameter using a fine copper wire to allow for the preparation of a cell culture cup. After soaking for 3 h, the membranes were rinsed in PBS three times and then soaked for an additional hour. They were then placed into 16-well plates. Corneal limbal stem cells were subsequently cultivated at a density of 150 × 104 per culture cup, while the cells
Staining of frozen sections (8 μm) of cornea was done for rabbit anti-CD86 (1:500, Cell Signaling) anti-KRT12 (1:200, ABIN290007, Antibodies-online), anti-KRT13 (1:200, ABIN108618, Antibodiesonline) and anti-KRT3 (1:200, ABIN108617, Antibodies-online) as detailed previously. In brief, the frozen sections were desiccated at room temperature and post-fixed in chilled acetone for a time of 15 min. The sections were then washed with phosphate-buffered saline (PBS), followed by blocking with 8% normal donkey serum in PBS for a time of 1 h at room temperature. The samples were subsequently incubated overnight at a temperature of 4 °C with one of the primary antibodies mentioned above. Following triple washing with PBS/0.05% Tween-20, fluorescently-labeled secondary antibody of CyTM 5 AffiniPure Donkey Anti-Mouse IgG (1:200, 715– 175-151, Jackson ImmunoResearch) and CyTM 3 AffiniPure Donkey Anti-Goat IgG (1:200, 705–165-147, Jackson ImmunoResearch) were incubated along with the samples at room temperature for 1 h. Following an additional three washes with PBS, the sections were counterstained for a time of 3 min at room temperature using DAPI, a nuclear dye (1:1200; Sigma, St. Louis, MO). The slides were mounted using an Aquamount solution and fluorescence
2.4. Characterization of degradation rate
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microscopy (Axioskop; Carl Zeiss Meditec, Thornwood, NY, USA) was then utilized to visualize the samples. An identical exposure time was used for each sample, and the images were analyzed using Image-Pro Plus (Media Cybernetics, Silver Spring, MD). 2.9. RNA isolation and real-time polymerase chain reaction analysis Real-time polymerase chain reaction (PCR) analysis was used in order to assess the levels of mRNA of IL-6, IL-8, CCL2, and VEGF within rabbit cornea treated with different materials. The primers for IL-6 are GAA CAG AAA GGA GGC ACT GG (forward) and CTC CTG AAC TTG GCC TGA AG (reverse); the primers for IL-8 are CCA CAC CTT TCC ATC CCA AAT (forward) and CTT CTG CAC CCA CTT TTC CTT G (reverse); the primers for CCL2 are GTG AAT TCC CCA GTC ACC TG (forward) and TGT GTT CTT GGG TTG TGG AA (reverse); and the primers for VEGF are TTC AAC GTC ACC ATG CAG AT (forward) and AAA TGC TTT CTC CGC TCT GA (reverse). Isolation of total RNA was accomplished via RNeasy Mini Kits (QIAGEN). First-strand cDNA synthesis reactions were carried out though the use of a High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) with an accompanying light cycler component (Biometra TPERSONAL, German). Real-time PCR was carried out through the use of the TaqMan Gene Expression Master Mix (ThermoFisher Scientific) with a light cycler component (StepOnePlusTM Real-Time PCR System, Applied BiosystemsTM ). PCR cycling involved 40 cycles of amplification of the template DNA with primer annealing occurring at a temperature of 60 °C. The 2−Ct method was then used in order to calculate the relative expression level of each gene of interest. The amplification efficiencies of each primer pair were validated in order to allow for gene expression to be quantitatively compared. Only Taqman primers (ThermoFisher Scientific) were utilized. Every quantitative PCR was carried out three times on three different experimental replicates. The results were subsequently normalized to the value found using the reference gene. 2.10. Statistical analysis All quantitative data are expressed as a mean ± SD. Statistically significant differences between the results of various experimental groups were assessed via a student’s t-test. A p value of <0.05 is considered to be indicative of statistical significance. 3. Results 3.1. Composite membrane preparation and stability The composite membrane was prepared accordingly to our previously reported protocol [16]. Mechanical compression was applied to the tightly bonded dAM and PCL nanofiber mesh in order to facilitate the conjugation reaction between the PCL fiber surface and dAM components. The different layers that bonded together formed a composite membrane without delamination following lyophilization (Fig. 1). This interfacial bonding technique not only is important to improve the integrity of the composite membrane, but also improved the mechanical properties and stability of the membrane. The PCL-dAM composite membrane exhibited about 4-fold higher ultimate tensile strength (p < 0.01), 10-fold higher toughness (p < 0.01), and more than 10-fold higher suture retention strength (p < 0.01) in comparison with lyophilized dAM [16]. To determine whether this composite has the capacity to increase the stability of dAM when applied to the damaged corneal surface, we tested the degradation rate of dAM, PCL, and the PCLdAM composite membrane in a pancreatin buffer solution (1 U),
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using PBS as a buffer control. From Day 1–4, dAM showed significantly more rapid degradation than PCL or the PCL-dAM composite membrane. On Day 5, the PCL only membrane maintained its original shape and size in 1-U of pancreatin buffer, while the dAM only membrane completely dissolved (Fig. 1). The composite slows down the degradation rate by about 40%. These results demonstrate that PCL bonding has the potential to extend the stability of dAM in the presence of enzyme, thus improving its stability and lasting coverage on a wound surface. 3.2. Alkali Burn-Induced LSCD model We adopted an LSCD model using alkali burn. Following exposure to alkali and scraping of the epithelium, typical signs of corneal damage, including conjunctival congestion and epithelial damage, were observed within a few hours. Inflammation involving the anterior segment, such as soft tissue swelling of the operative eye and white dense exudation of the anterior chamber, developed within the first few weeks (Fig. 2). During the second week, exudation disappeared, and limbus neovascularization was indicative of chronic inflammation. H&E staining confirmed the loss of the central corneal epithelium and distortion of the limbal epithelium at 2 weeks post-exposure. 3.3. Assessing PCL-dAM composite membrane for corneal defect repair in the LSCD model 3.3.1. In vivo anti-inflammation of PCL-dAM composite membrane We demonstrated previously that the PCL-dAM composite membrane has similar immunomodulatory effects as the dAM and may promote the polarization of macrophages into the proregenerative M2 phenotype in vitro [16]. In order to investigate the immunomodulatory effects of PCL-dAM composite membrane in vivo, we transplanted the dAM only membrane or PCL-dAM composite membrane to the corneal epithelial defect site in a rabbit LSCD model. Corneal edema and inflammatory reactions gradually reduced from week 1 after transplantation in both groups. The PCL-dAM composite membrane was well-seated on the corneal surface without degradation at week 1, while the majority of grafts to which dAM alone had been applied demonstrated thinning and shifting (Fig. 3). Immunohistochemistry showed a high level of CD86-positive M1 macrophages in the control group compared with the dAM or PCL-dAM composite membrane groups. However, CD68, a marker of the M2 macrophage, showed negative staining in all groups. Furthermore, real time PCR revealed lower levels of CCL2, IL-8, and IL-6 mRNA expression in both the dAM and PCLdAM composite membrane groups compared with untreated controls. The high expression of IL-6 mRNA was consistent with an increased CD86 staining intensity in the control group. Reduced VEGF mRNA expression in treated corneas indicates that both dAM and PCL-dAM composite membrane are effective in preventing angiogenesis through inflammatory stimulation (Fig. 4). 3.3.2. PCL-dAM composite membrane promoting conjunctival epithelialization of cornea in LSCD Model. Conjunctivalisation is typically considered as a pathological process during corneal re-epithelialization in the setting of severe LSCD condition because neovascularization and inflammatory cell infiltration typically accompany conjunctivalization [22]. However, integrated conjunctival epithelialization can occur without neovascularization and inflammation and contribute to corneal repair, in which case it serves as a beneficial process to repair. In this study, degradation of the dAM was observed in the first week following transplantation. Conversely, the PCL and PCL-dAM exhibited excellent stability until 4 weeks post-transplantation. Immunohistochemistry demonstrated that K13-positive cells specific to the
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Fig. 1. General appearance and degradation profiles of the PCL-dAM composite membrane. (A) General appearance and (B) SEM images of dAM, PCL and the PCL-dAM composite membranes prepared from the PCL fiber mesh and dAM. (C, D) The degradation profiles of the three samples in PBS (C) and in the presence of enzyme (1 U of pancreatin) (D). ∗ p < 0.001 (in comparison with dAM or PCL mesh).
conjunctival epithelial cells continuously lined the corneal lesion site in the PCL-dAM composite membrane group but formed a much sparser dotting pattern in the dAM only group (Fig. 4). K12positive cells, considered a marker for basal corneal epithelium, were not identified in either group. This finding implies that PCLdAM composite membrane, when used as a protective dress for damaged cornea, can provide a stable environment for the proliferation of conjunctival epithelial cells and functional conjunctivalization, even in the absence of LSCs. 3.4. Transplantation of LSCs with dAM vs. PCL-dAM composite membrane for corneal defect repair in severe LSCD model LSCs were delivered to the corneal defects in combination with the membranes. GFP-labeled LSCs were cultured on either a dAM or PCL-dAM composite membrane for 1 week prior to implantation. Two weeks after implantation, conjunctival hyperemia and ocular irritation were noticeably relieved in both groups. In the PCL-dAM composite membrane group, the corneal epithelium gradually recovered with improved transparency and significantly reduced neovascularization. In contrast, the corneal epithelial defects
persisted in the dAM group and were gradually covered by the conjunctival epithelium, while the cornea became cloudy with extensive neovascularization. GFP-labeled LSCs were identified in the newly-formed epithelial layer in both groups received dAM + LSCs and CM + LSCs (Fig. 5). A higher number of LSCs were found in the group received CM + LSCs; and LSCs in this group spread over a greater area than those identified in the group with dAM + LSCs. The corneal epithelium is the outermost layer of the cornea. It is composed of a single layer of basal cells and four to five cell layers of nonkeratinized, stratified squamous epithelial cells. Immunohistochemistry of K12 and K3, which are cornea-specific keratins that compose the intermediate filament cytoskeleton of corneal epithelial cells, showed linear and spotted staining, respectively, in the “CM + LSCs” group. In contrast, the “dAM + LSCs” group demonstrated predominately K12 staining and minimal K3 staining. H&E staining of the sections at week 2 following composite membrane-supported LSC transplantation revealed restoration of corneal epithelial morphology and hierarchy. Specifically, highly cylindrical basal cells, the upper polygonal pterygium, and superficial flat cells were very similar to that of a normal corneal epithelium. Conversely, in the tissue sections from the “dAM + LSCs”
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Fig. 2. Alkali-burn induced LSCD model results in damage to corneal epithelium. (A) Procedure of LSCD model by alkali burn. Normal: The naive rabbit eye; NaOH: Ring-shaped sterile filter paper saturated with 1 N NaOH is placed onto the cornea for 50 s; Day 0-Rinse: the lesion site is then rinsed with 0.9% NaCl Day 0-Scraping: residual corneal epithelium is scraped; Day 0-Staining: corneal damage is confirmed with fluorescein staining; Week 2-LSCD: Cornea becomes neovascularization occurs at 2-weeks post-exposure. (B) H&E staining showing (left) normal corneal epithelial and (right) loss or distortion of limbal epithelium post-exposure on the LSCD model eye.
group, most epithelial cells were bulky, fusiform or polygonal, and the connections between cells were loose. In addition, cells were irregularly arranged, and ring-like goblet cells were observed in the control group. The presence of basal cells is the most important evidence of corneal epithelial normalization and conjunctivalization. Comparison of basal cell count in each 40 × field for the normal, control, dAM, PCL-dAM composite membrane, “dAM + LSCs”, and “CM + LSCs” groups showed eyes treated with CM + LSCs had basal cell counts closest to those of normal cornea. The cornea covered by PCL-dAM composite membrane without LSCs exhibited better epithelial morphological repair than dAM-covered cornea, however there was no significant difference in basal cell count in either group compared control (Figs. 5 and 6). 4. Discussion Ocular burn is one of the most common forms of LSCD. Experimental alkaline burns is typically used to mimic ocular burns,
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Day 0–1 N for 5 min; opaque as at 2-week
which causes severe depletion of LSCs and local inflammation, and leads to cornea opacity and neovascularzation [23]. A growing body of work has been reported to demonstrate the promise of LSC transplantation in treating alkaline burn-induced LSCD in animal models [24–26]. The success of this approach, however, requires a scaffold to resurface the damaged corneal epithelium and enhance LSC engraftment. AM is well-suited for this purpose, given its immunomodulatory and pro-regenerative properties. AMs have many intrinsic cytokines that promote wound healing by stimulating re-epithelialization and reducing local inflammation [27]. However, the unique microenvironment of the corneal surface, as well as shear forces from eyelid motion, make the grafted dAM more susceptible to deformation and degradation. Consequently, clinical LSC transplantation with dAM membranes must often be repeated due to a lack of stable coverage [28]. Additionally, the thin and fragile nature of AM makes it difficult to handle during the process of implantation to the ocular surface and susceptible to tear during suturing. Unlike approaches of chemical or enzymatic crosslinking
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Fig. 3. PCL-dAM composite membrane maintains structural integrity while reducing vascularization. PCL-dAM composite membrane (CM) remained intact without degradation while dAM had almost disappeared after 1 week. Two weeks after transplantation, conjunctival hyperemia and ocular irritation had been noticeably relieved in both groups. Compared with severe limbal neovascularization and corneal edema in control group, inflammatory reactions were reduced in all treatment groups. There was no limbal neovascularization seen with the group received CM + LSCs. Fluorescein staining at 2-week demonstrated that the majority of corneal epithelial surface had recovered, while higher percentages of the corneal surface stained positively in groups received dAM without LSCs, CM without LSCs, or dAM with LSCs.
that result in loss of bioactivities of the active growth factors and cytokines found in native AM [29,30], others have reported application of amniotic extracts in a gel form to retain more of the active compounds [31]. However, extracting soluble AM components will compromise the extracellular matrix structure, which has been shown to play a role in facilitating re-epithelialization [32,33]. To address these limitations, we developed a novel composite membrane that improves the stability and toughness of AM while maintaining its pro-regenerative and immunomodulatory
properties. The fabrication process involves interfacial bonding of biodegradable, electrospun, surface-carboxylated PCL nanofiber mesh to dehydrated amnion sheets. This avoids the loss of AM biological activity that results from stratification, shedding, and repeated chemical crosslinking used in other processing methods. Meanwhile, physical properties of the composite membrane, including toughness and stability, were enhanced as a result of effectively increasing the membrane thickness and slowing the rate of degradation. During the transplantation procedure, the
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Fig. 4. PCL-dAM composite membrane promotes conjunctival corneal epithelialization in the absence of LSCs the LSCD model. (A) Real time PCR of IL-8, IL-6, CCL2, VEGF mRNA expression. (n = 5, ∗ p < 0.05, ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001, ∗ ∗ ∗ ∗ p < 0.0 0 01). (B) Immunohistochemistry showing K13-positive cells continuously lining the corneal lesion site in PCL-dAM composite membrane (CM) group but forming a dotted pattern in dAM group. As expected, CD86-positive staining was observed more in the control group and dAM than the PCL-dAM composite membrane group. Scale bar: 100 μm.
resilient composite membrane, with a thickness of only 20 μm, largely improved operability over dAM alone, with the latter demonstrating a tendency to easily tear during application to a curved surface. In this study, the composite membrane demonstrated stable attachment to the cornea and resistance to tearing during suture placement and shifting from shear forces. The dAM membrane only and dAM-supported LSC transplantation group both have shown shifted and fully degraded membrane within one week. In contrast, the PCL-dAM composite membrane maintained its original shape, position and structural integrity for at least two weeks following implantation, at which point all membranes were explanted. We therefore speculate that the enhanced epithelial formation in the composite membrane group in vivo was due to the greater durability it conferred to dAM thereby providing a more stable environment for cellular attachment, migration and differentiation during the healing process.
In a typical rabbit LSCD model, a large number of neutrophils infiltrated the cornea during an acute inflammatory response. These neutrophils released proteolytic enzymes, including collagenase and other matrix metalloproteinases, causing progressive lysis of the corneal stroma as well as the release of activated polymorphonuclear leukocytes, and stimulation of the expression of inflammatory factors. The dAM membrane when used as a graft proved capable of secreting a variety of active growth factors and cytokines that promote the apoptosis of polymorphonuclear leukocytes, while inhibiting corneal neovascularization and mitigating the local inflammatory response [34,35]. In the present study, we used dAM membrane or PCL-dAM composite membrane to repair corneal epithelial defects in a rabbit model of LSCD. We observed greater epithelialization and reduced fluorescein staining, both indicators of improved corneal healing, in the PCL-dAM composite membrane group compared
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Fig. 5. PCL-dAM composite membrane improves LSC transplantation and the extent of corneal epithelialization in the LSCD model. Immunohistochemistry of K12 and K3 demonstrated linear and spotted staining in the PCL-dAM composite membrane (CM) supported LSC transplantation group (CM + LSCs) while K12 staining predominated over K3 in dAM supported LSC transplantation group (dAM + LSCs). Scale bar: 100 μm.
with the untreated control and dAM group. The control group developed the greatest degree of corneal opacity and neovascularization, while the ocular surface neovascularization and opacity were significantly decreased in the dAM and PCL-dAM composite membrane groups. PCR demonstrated diminished IL-8, IL-6, and VEGF mRNA in both the dAM and PCL-dAM composite membrane repaired corneas compared with the control group. Taken together, these findings support the hypothesis that the PCL-dAM composite membrane maintains the pro-regenerative and anti-inflammatory properties of the dAM components in vivo. It is worth noting that in the absence of transplanted LSCs, PCL-dAM composite membrane used in the setting of LSCD demonstrated a functional pattern of conjunctivalization without neovascularization or inflammation, which was not observed in dAM membrane group. Nonetheless, given that conjunctivalization has the potential to be pathological, long term studies are needed to fully characterize the repaired cornea structure and ensure the stability of the observed pattern of healing. The normal proliferation and differentiation of the epithelial cells of the ocular surface depends on the presence of a normal epithelial basement membrane [36]. In LSCD, where there is destruction of the epithelial basement membrane, AM provides an ideal substitute substrate capable of supporting LSC growth, given its tissue composition and micro-architecture that closely resembles that of the ocular epithelium basement membrane [37]. In order to assess the suitability of PCL-dAM composite membrane as a substrate for LSC transplantation in comparison with dAM, GFP-labeled LSCs were seeded on the respective membranes and tested in an LSCD corneal defect model. Both PCL-AM composite and dAM membranes allowed for successful engraftment and survival of transplanted LSCs. However, the labeled LSCs in the group received PCL-dAM composite membrane-supported LSCs were distributed in a more uniform band and covered a greater
surface area of the cornea in contrast to the punctuated epithelial fluorescence bands observed in the group received dAMsupported LSCs. H&E staining revealed that the composite membrane + LSCs group had better epithelial delaminated cell morphology than the AM + LSCs group. The corneal stromal collagen content of the composite membrane group was close to that of the normal control group, thus implying that the PCL-dAM composite membrane is more conducive to support LSC survival, retention, migration, and proliferation than dAM alone. Future studies will focus on identifying the long-term fate of the transplanted LSCs. It is also important to note that there were no differences in the number of basal cells observed in the PCL-dAM only and dAM only groups without LSC transplantation in comparison with the untreated control group, despite observed improvements in collagen organization and re-epithelialization, and inhibiting inflammation and neovascularization. Given that the presence of basal cells is the most important indicator of normal corneal healing, this finding reinforces the importance of cell transplantation as an adjunct to membrane resurfacing in the treatment of LSCD. Certainly, the improved mechanical property and extended stability of the PCL-dAM composite membrane further enhance the performance of the dAM as a supporting matrix to harness the full potential of LSC transplantation. Other cell types such as limbal epithelial cells have been tested as alternative to LSC transplantation. For example, the simple limbal epithelial transplantation technique has been shown to successfully repair damaged cornea in the case of unilateral severe LSCD, as an alternative to conjunctival limbal autograft or cultivated limbal epithelial transplantation [38]. Due to the rich component this PCL-dAM composite membrane would also likely to augment the treatment outcomes for transplantation of limbal epithelial cells.
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Fig. 6. PCL-dAM composite membrane group displays improved epithelial delaminated cell morphology. (A) H&E staining of the sections at week 2 following implantation of various membranes. Scale bar: 100 μm. (B) Different layers and cell types of corneal epithelium. Scale bar: 20 μm. (C) Comparison of basal epithelial cell count in each 40 × field for the normal, control, dAM without L SCs (dAM w/o L SCs), PCL-dAM composite membrane without LSCs (CM w/o LSCs), dAM with LSCs (dAM + LSCs), and PCLdAM composite membranes with LSCs (CM + LSCs) (n = 5).
Although non-essential, it may be ideal that the corneal repair membrane is optically transparent. As the PCL nanofiber mesh in the composite membrane is not transparent, the whole membrane is not transparent even after hydration. Nonetheless, we found that re-epithelialization occurs within the dAM sheet that interfaces with the corneal surface. Following integration of the LSC and reepithelialization and the degradation of the dAM components, the PCL mesh can be easily removed, so as not to obscure vision after healing. A transparent or translucent version of the composite membrane can be produced by using a transparent nanofiber sheet, which is currently ongoing.
provides a superior alternative to cryopreserved dAM with greater clinical utility and practicality. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgements This work was partially supported by Johns Hopkins University School of Engineering and a grant from Wuhan Kangchuang Technology Co. Ltd. to Johns Hopkins University.
5. Conclusions References The PCL-dAM composite membrane maintains and augments the beneficial pro-regenerative and immunomodulatory properties of dAM, with enhanced ability to support the survival, retention, migration and organization of LSCs, thus promoting the re-epithelialization and reducing inflammation and neovascularization in the repair site of a rabbit LSCD model. In addition to preserving the biological activity of dAM, the addition of the PCL nanofibers stabilizes and toughens the dAM to provide more durable and longer-lasting coverage of the defect site during the healing process. The PCL-dAM composite membrane therefore
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