Therapeutic application of decellularized porcine small intestinal submucosa scaffold in conjunctiva reconstruction.

The objective of this study was to investigate the biological feasibility and surgical applicability of decellularized porcine small intestinal submucosa (DSIS) in conjunctiva reconstruction. A total of 52 Balb/c mice were included in the study. We obtained the DSIS by decellularization, evaluated the physical and biological properties of DSIS in vitro, and further evaluated the effect of surgical transplantation of DSIS scaffold in vivo. The histopathology and ultrastructural analysis results showed that the scaffold retained the integrity of the fibrous morphology while removing cells. Biomechanical analysis showed that the elongation at break of the DSIS (239.00 ± 12.51%) were better than that of natural mouse conjunctiva (170.70 ± 9.41%, P < 0.05). Moreover, in vivo experiments confirmed the excellent biocompatibility of the decellularized scaffolds. In the DSIS group, partial epithelialization occurred at day-3 after operation, and the conjunctival injury healed at day-7, which was significantly faster than that in human amniotic membrane (AM) and sham surgery (SHAM) group (P < 0.05). The number and distribution of goblet cells of transplanted DSIS were significantly better than those of the AM and SHAM groups. Consequently, the DSIS scaffold shows excellent biological characteristics and surgical applicability in the mouse conjunctival defect model, and DSIS is expected to be an alternative scaffold for conjunctival reconstruction.

Crosslinked modified decellularized rabbit conjunctival stroma for reconstruction of tissue-engineered conjunctivain vitro.

Conjunctival reconstruction is an essential part of ocular surface restoration, especially in severe conjunctival disorders. Decellularized conjunctival tissues have been used in tissue engineering. In this study, we investigated the feasibility of constructing tissue-engineered conjunctiva using stem cell (human amniotic epithelial cells, hAECs), and cross-linked modified decellularized rabbit conjunctival stroma (DRCS-Asp-hEGF), and decellularized rabbit conjunctiva stroma (DRCS). With phospholipase A2 and sodium dodecyl, DRCS were nearly DNA-free, structurally intact and showed no cytotoxic effectsin vitro, as confirmed by DNA quantification, histology, and immunofluorescence. The results of Fourier transform infrared, Alcian blue staining and human epidermal growth factor (hEGF) release assays showed that DRCS-Asp-hEGF was successfully prepared via crosslinking with aspartic acid (Asp) and modified by hEGF at pH 7.7. The hAECs were positive for octamer-binding transcription factor-4 and ABCG2 cell markers. The hAECs were directly placed on the DRCS and DRCS-Asp-hEGF for five days respectively. Tissue-engineered conjunctiva was constructedin vitrofor five days, and the fluorescence staining results showed that hAECs grew in monolayers on DRCS-Asp-hEGF and DRCS. Flow cytometry results showed that compared with DRCS, the number of apoptotic cells stained in DRCS-Asp-hEGF was small, 86.70 ± 0.79% of the cells survived, and 87.59 ± 1.43% of the cells were in the G1 phase of DNA synthesis. Electron microscopy results showed that desmosome junction structures, which were similar to the native conjunctival tissue, were formed between cells and the matrix in the DRCS-Asp-hEGF.

A cocktail of small molecules maintains the stemness and differentiation potential of conjunctival epithelial cells.

PURPOSE: The conjunctival epithelial cells cultured with bovine serum or feeder cells were not suitable for clinical application. Therefore, we developed a novel serum-free and feeder cell-free culture system containing only a cocktail of three chemicals (3C) to expand the conjunctival epithelial cells. METHODS: The cell proliferative ability was evaluated by counting, crystal violet staining and Ki67 immunostaining. Co-staining of K7 and MUC5AC was performed to identify goblet cells. PAS staining was used to assess the ability of cells to synthesis and secrete glycoproteins. In vivo, eye drops containing 3C was administered to verify the role of 3C in the mouse conjunctival injury model. PAS, HE and immunofluorescence staining were performed to show conjunctival epithelial repair. RESULTS: Compared with other small molecule groups and the serum group, the cells in 3C group showed superior morphology and proliferative ability. Meanwhile, 3C maintained the well-proliferative capacity of cells even after fifth passage. The 3C group also exhibited more K7 and MUC5AC double positive cells, and the PAS staining positive areas were present in both the cytoplasm and extracellular matrix. The cell sheets treated with 3C in air-lifted culture were obviously stratified. In vivo, more goblet cells in the conjunctival epithelium were observed in the 3C group. CONCLUSION: Overall, our culture system can expand the conjunctival epithelial cells and retain their potential to differentiate into mature goblet cells, which provided a promising source of seed cells for conjunctival reconstruction. Furthermore, this system provides new insights for the clinical treatment of ocular surface diseases.

A thin film comprising silk peptide and cellulose nanofibrils implanting on the electrospun poly(lactic acid) fibrous scaffolds for biomedical reconstruction.

Conjunctival reconstruction using biocompatible polymers constitutes an effective treatment for conjunctival scarring and associated visual impairment. In this work, a thin film comprising silk peptide (SP), cellulose nanofibrils (CNF) and Ag nanoparticles (AgNPs) that implanted on the poly(lactic acid) (PLA) electrospun fibrous membranes (EFMs) was designed for biomedical reconstruction. SP and CNF as thin films can improve the surface hydrophilicity of the as-prepared scaffolds, which synergistically enhanced the biocompatibility. In in vivo experiments, the developed PLA EFMs modified with 3 wt% SP/CNF/AgNPs could be easily manipulated and transplanted onto conjunctival defects in rabbits, consequently accelerating the structural and functional restoration of the ocular surface in 12 days. Additionally, incorporation of 0.30 mg/g AgNPs efficiently reduced the topical application of antibiotics without causing infections. Thus, these resultant scaffolds could not only serve as useful alternatives for conjunctival engineering, but also prevent infections effectively with a very low content of AgNPs.

Decellularized Porcine Conjunctiva in Treating Severe Symblepharon.

This prospective study aimed to evaluate the effectiveness of decellularized porcine conjunctiva (DPC) in the management of severe symblepharon. Sixteen patients with severe symblepharon were enrolled in this study. After symblepharon lysis and Mitomycin C (MMC) application, tarsus defects were covered with residual autologous conjunctiva (AC), autologous oral mucosa (AOM), or DPC throughout the fornix, and DPC was used for all the exposed sclera. The outcomes were classified as complete success, partial success, or failure. Six symblepharon patients had chemical burns and ten had thermal burns. Tarsus defects were covered with DPC, AC, and AOM in two, three, and eleven cases, respectively. After an average follow-up of 20.0 ± 6 months, the anatomical outcomes observed were complete successes in twelve (three with AC+DPC, four with AC+AOM+DPC, and five with AOM+DPC) (75%) cases, partial successes in three (one with AOM+DPC and two with DPC+DPC) (18.75%) cases, and failure in one (with AOM+DPC) (6.25%) case. Before surgery, the depth of the narrowest part of the conjunctival sac was 0.59 ± 0.76 mm (range, 0-2 mm), tear fluid quantity (Schirmer II tests) was 12.5 ± 2.26 mm (range, 10-16 mm), and the distance of the eye rotation toward the opposite direction of the symblepharon was 3.75 ± 1.39 mm (range, 2-7 mm). The fornix depths increased to 7.53 ± 1.64 mm (range, 3-9 mm), eye movement was significantly improved, and the distance of eye movement reaching 6.56 ± 1.24 mm (range, 4-8 mm) 1 month after the operation; the postoperative Schirmer II test (12.06 ± 2.90 mm, range, 6-17 mm) was similar to that before surgery. Goblet cells were finally found in fifteen patients by conjunctival impression cytology in the transplantation area of DPC, except for one patient who failed. DPC could be considered an alternative for ocular surface reconstruction of severe symblepharon. Covering tarsal defects with autologous mucosa is necessary for extensive reconstruction of the ocular surface.

Influence of Storage Conditions on Decellularized Porcine Conjunctiva.

Porcine decellularized conjunctiva (PDC) represents a promising alternative source for conjunctival reconstruction. Methods of its re-epithelialization in vitro with primary human conjunctival epithelial cells (HCEC) have already been established. However, a long-term storage method is required for a simplified clinical use of PDC. This study investigates the influence of several storage variants on PDC. PDC were stored in (1) phosphate-buffered saline solution (PBS) at 4 °C, (2) in glycerol-containing epithelial cell medium (EM/gly) at -80 °C and (3) in dimethyl sulfoxide-containing epithelial cell medium (EM/DMSO) at -196 °C in liquid nitrogen for two and six months, respectively. Fresh PDC served as control. Histological structure, biomechanical parameters, the content of collagen and elastin and the potential of re-epithelialization with primary HCEC under cultivation for 14 days were compared (n = 4-10). In all groups, PDC showed a well-preserved extracellular matrix without structural disruptions and with comparable fiber density (p ≥ 0.74). Collagen and elastin content were not significantly different between the groups (p ≥ 0.18; p ≥ 0.13, respectively). With the exception of the significantly reduced tensile strength of PDC after storage at -196 °C in EM/DMSO for six months (0.46 ± 0.21 MPa, p = 0.02), no differences were seen regarding the elastic modulus, tensile strength and extensibility compared to control (0.87 ± 0.25 MPa; p ≥ 0.06). The mean values of the epithelialized PDC surface ranged from 51.9 ± 8.8% (-196 °C) to 78.3 ± 4.4% (-80 °C) and did not differ significantly (p ≥ 0.35). In conclusion, all examined storage methods were suitable for storing PDC for at least six months. All PDC were able to re-epithelialize, which rules out cytotoxic influences of the storage conditions and suggests preserved biocompatibility for in vivo application.

[Methods of surgical reconstruction of the conjunctiva]. / Metody khirurgicheskoi rekonstruktsii kon"yunktivy.

Reconstruction of the conjunctiva is required for restoration of damaged ocular surface and is an essential part of that process. Traumas, chemical and thermal burns, multiple surgical intervention can seriously damage the integrity of conjunctival tissue and promote the growth of fibrous tissue, scarring of contractures and their shortening, as well as other complications such as trichiasis, erosion and ulcers on the cornea. When a larger area is affected, there may not be enough donor tissue to replace the defect, in which case the tissue grafts are required to be large enough. Modern modifications of surgical techniques and the continued development of tissue engineering, as well as advancements in stem cell research offer promising novel alternatives for solution of those problems. This article reviews the existing surgical methods of conjunctival reconstruction.

Aspartic acid and epidermal growth factor modified decellularized rabbit conjunctiva for conjunctival reconstruction.

Conjunctival reconstruction is an indispensable part of ocular surface regeneration. Decellularized matrix has been considered as an ideal conjunctival substitution for conjunctival reconstruction. In the present study, we report the use of a decellularized rabbit conjunctiva (DRC) for conjunctival reconstruction in the rabbit surgical trauma model. Prepared by the phospholipase A2 decellularized method, the DRC was nearly DNA free while the collagen structure and natural extracellular matrix (ECM) were well preserved. In order to improve the performance of DRC, aspartic acid (Asp) was used as a spacer arm to crosslink epidermal growth factor (EGF) on the DRC to obtain DRC-Asp-EGF. The conjunctival epithelial cells cultured on the DRC-Asp-EGF showed a higher survival rates and a greater potential to differentiate into conjunctival goblet cells (CGCs) than those on the DRC. Finally, three groups were set to evaluate the transplantation effects in the rabbit surgical trauma model for 28 days: DRC-Asp-EGF group, amniotic membrane (AM) group, and ungrafted group. The DRC-Asp-EGF group was completely re-epithelized, and more CGCs were regenerated than the AM group, while no significant improvements were observed in the ungrafted group. Intact collagen structure, angiogenesis, and no scar formation were also observed in the DRC-Asp-EGF group. These results suggest that DRC-Asp-EGF is a feasible and effective transplant for conjunctival reconstruction and ocular surface regeneration.

Conjunctival calcinosis circumscripta in a dog: Treatment combining surgical resection and amniotic membrane grafting.

This report describes the clinical features, histopathology, and surgical treatment of a case of conjunctival calcification in a 5-month-old female English Setter, referred with a history of recurrent conjunctivitis in the right eye (OD). The ophthalmic findings were limited to multifocal white plaques embedded in a markedly inflamed conjunctiva of the eyelids and the anterior nictitating membrane OD. Calcification was suspected. The blood cell count, blood chemical profile, and urinalysis were within normal limits, and long-bone radiographs appeared normal. After removal of the affected area by means of a large conjunctivectomy, cryopreserved canine amniotic membrane (AM) was transplanted to fill in the defect. Multifocal ectopic calcium deposits in the conjunctival lamina propria were confirmed histopathologically. The postoperative healing was uneventful, and no recurrence was observed during a follow-up period of five years. Conjunctival mineralization is uncommon in canine ophthalmology, and the cause remained undetermined in the present case, for which AM transplantation was able to promote conjunctival healing after a large surgical excision.

Conjunctival reconstruction via enrichment of human conjunctival epithelial stem cells by p75 through the NGF-p75-SALL2 signaling axis.

Severe conjunctival diseases can cause significant conjunctival scarring, which seriously limits eye movement and affects patients' vision. Conjunctival reconstruction remains challenging due to the lack of efficient methods for stem cells enrichment. This study indicated that p75 positive conjunctival epithelial cells (CjECs) were mainly located in the basal layer of human conjunctival epithelium and showed an immature differentiation state in vivo. The p75 strongly positive (p75++) CjECs enriched by immuno-magnetic beads exhibited high expression of stem cell markers and low expression of differentiated keratins. During continuous cell passage cultivation, p75++ CjECs showed the strongest proliferation potential and were able to reconstruct the conjunctiva in vivo with the most complete structure and function. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs while proNGF played an opposite role. Altogether, p75++ CjECs present stem cell characteristics and exhibit the strongest proliferation potential so can be used as seed cells for conjunctival reconstruction, and NGF-p75-SALL2 signaling pathway was involved in regulating the differentiation of CjECs.

Ocular surface repair using decellularized porcine conjunctiva.

The primary functions of the conjunctiva embody ocular surface protection and the maintenance of the tear film equilibrium. Severe conjunctival defects such as symblepharon may impair the integrity of ocular surface and cause loss of visual functions. Here we report the use of a decellularized porcine conjunctiva (DPC) for conjunctival reconstruction in rabbit models and in clinic. Our results show that the major xenoantigens are efficiently removed, while abundant matrix components and integrated microstructures are well preserved in the DPC. These characteristics provide mechanical support and favorable histocompatibility for repairing damaged conjunctiva. The DPC application has demonstrated enhanced transplant stability and improved epithelial regeneration in severe ocular surface damage comparing to those of amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction nowadays. In order to test the DPC performance in clinic, three patients with pterygium and one patient with symblepharon underwent transplant with DPC. The grafts in all cases were completely re-epithelized and no graft melt or fibroplasia were observed. These results suggest that the strategy we developed is feasible and effective for conjunctival reconstruction and ocular surface repair. STATEMENT OF SIGNIFICANCE: In this study, we adopted an innovative approach to prepare decellularized porcine conjunctiva (DPC). The intricate conjunctiva-specific structures and abundant matrix components were preserved in DPC, which offers favorable mechanical properties for graft. DPC has shown positive effects in ocular surface repair, which has been proven particularly in a rabbit model with severe symblepharon. Reconstructed conjunctiva by DPC exhibited epithelial heterogeneity, extremely resembling that of native conjunctiva. In addition, results from clinical studies were encouraging for pterygium and symblepharon and clinical application of DPC is promising.

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

BACKGROUND: The aim of this study was to evaluate the surgical handling and clinical applicability of a specific 3D-printed membrane design fabricated using a gelatin, elastin and sodium hyaluronate blend for conjunctival reconstruction and compare it with amniotic membrane (AM), which is normally used in such surgeries. METHODS: 3D printing technique was employed to fabricate the membrane based on gradient design. Prior to printing, rheometry was employed to optimize the ink composition. The printed membranes were then fully characterized in terms of physical and mechanical properties. In vitro viability, proliferation and adhesion of human limbal epithelial cells were assessed using MTT assay and scanning electron microscopy (SEM), respectively. Prior to in vivo experiment, surgical handling of each membrane was evaluated by three surgeons. In vivo evaluation was conducted through implanting the gelatin-based membranes and AM on induced conjunctival defects in rabbits (n = 8). Clinical observations, including epithelialization, inflammation severity, scar tissue formation and presence of granulation tissue, were recorded from day 1 through day 28. Histological examination was performed on all enucleated eyes on day 28. In addition to H&E staining, specific stains including Periodic Acid Schiff staining, Masson's Trichrome staining and immuno-histochemical staining for α-SMA were further used to assess goblet cell proliferation, healed sub-epithelial stroma and scar tissue formation and the presence of myofibroblasts, respectively. RESULTS: Among all the examined compositions, a blend of 8% w/v gelatin, 2% w/v elastin and 0.5% w/v sodium hyaluronate was found to be appropriate for printing. The printed membranes had favorable optical characteristics (colorless and transparent), and the surgical handling was significantly easier compared to AM. Epithelial cells cultivated on the membranes indicated suitable viability and proliferation, and SEM images presented appropriate cell adhesion on the surface of the membranes. Clinical observations suggested similar epithelialization time (approximately 3 weeks) for both the membrane and AM grafted eyes but significantly lower levels of clinical inflammation in the membrane group from day 1 through day 28 (p = 0.01), which is a key advantage of using the printed membranes over the AM. Histological examination showed similar qualities in the healed epithelium in terms of cell morphology and cell layers. However, twice the density of goblet cells per 100 cells was observed in the gelatin-based membrane grafted group. Remnant of the degraded implant was seen in only 3 of the membranes, but in 7 of the AM grafted eyes. Inflammation and granulomatous reaction was significantly higher in sections containing the AM compared to membrane (p < 0.01 and p = 0.01, respectively). α-SMA staining was more evident, but not significantly different from the gelatin-based membrane, for the AM group (p = 0.25). CONCLUSION: The designed gelatin-based membrane offers the necessary physical and mechanical characteristics needed for successful ocular surface/conjunctival defect construction and may be considered a promising alternative to AM due to a more predictable degradation pattern, higher goblet cell density on the healed epithelium, less inflammation and reduced scar tissue formation.

Decellularised conjunctiva for ocular surface reconstruction.

Conjunctival reconstruction is an integral component of ocular surface restoration. Decellularised tissues are frequently used clinically for tissue engineering. This study identifies porcine decellularised conjunctiva (PDC) and human decellularised conjunctiva (HDC) as promising substitutes for conjunctival reconstruction. PDC and HDC were nearly DNA-free, structurally intact and showed no cytotoxic effects in vitro, which was confirmed by DNA quantification, histology, transmission electron microscopy, collagen quantification and cytotoxicity assay. Comparing the biomechanical properties to amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction today, the decellularised conjunctiva was more extensible and elastic but exhibited less tensile strength. The in vivo application in a rabbit model proofed significantly enhanced transplant stability and less suture losses comparing PDC and HDC to AM while none of the matrices induced considerable inflammation. Ten days after implantation, all PDC, 4 of 6 HDC but none of the AM transplants were completely integrated into the recipient conjunctiva with a partially multi-layered epithelium. Altogether, decellularised conjunctivas of porcine and human origin were superior to AM for conjunctival reconstruction after xenogeneic application in vivo. STATEMENT OF SIGNIFICANCE: Conjunctival integrity is essential for a healthy ocular surface and clear vision. Its reconstruction is required in case of immunological diseases, after trauma, chemical or thermal burns or surgery involving the conjunctiva. Due to limitations of currently used substitute tissues such as amniotic membrane, there is a need for the development of new matrices for conjunctival reconstruction. Decellularised tissues are frequently applied clinically for tissue engineering. The present study identifies porcine and human decellularised conjunctiva as biocompatible and well tolerated scaffolds with superior integration into the recipient conjunctiva compared to amniotic membrane. Decellularised conjunctiva depicts a promising substitute for conjunctival reconstruction in ophthalmology.

Promoting repair effects of chitosan/gelatin electrospinning membrane implantation on rabbit conjunctival injury / 中华实验眼科杂志

Objective To investigate the promoting repair effects of chitosan/gelatin electrospinning membrane implantation on conjunctival injury.Methods Chitosan/gelatin electrospinning membrane was prepared using electrospinning technique,and the general characteristics of the membrane was examined under the scanning electron microscope.Conjunctival injury models were established in the right eyes of 30 New Zealand white rabbits by surgical resection of 6 mm×8 mm bulbar conjunctiva,bums of weak acid at injury site and then randomized into model group,human amniotic membrane (AM) implanted group and electrospinning membrane implanted group.AM or electrospinning membrane was interlayerly implanted between conjunctiva and sclera during operation according to grouping.The inflammatory response and histopathological change at injury site were examined by slit lamp microscope and optical microscope in 4,8,14 and 28 days after surgery.The use and care of the animals followed by code of ethics for animal experiments at the animal experiment center of He University.Results Chitosan/gelatin electrospinning membrane presented with a crosslinked network structure like AM,with the nano-microstructure of 0.1-0.5 μm fibre.No obvious inflammatory symptom was seen in the experimental eyes of various groups in 4 days after operation.In 8 days after operation,conjunctival edema and erosion were found in the model group,and slight conjunctival congestion was exhibited in the eyes of AM implanted group and electrospinning membrane implanted group.In 14 to 28 days after operation,conjunctival edema and new blood vessels were seen in the model group,and the conjunctivas were well recovered in the AM implanted group and electrospinning membrane implanted group.Histopathological examination showed dissociation,attaching,thickening and epithelization of injuried conjunctivas gradually in the model group from 4 to 28 days after operation.In the AM implanted group,attaching of AM was in 4 days,melting of AM was in 8 days,epithelization of injuried conjunctivas was in 14 days and renovation of conjunctivas was in 24 days after operation,and a rapid repairing procedure was found in the electrospinning membrane implanted group under the optical microscope.Conclusions Compared with the self-repairing process of conjunctival injury,implantion of chitosan/gelatin electrospinning membrane or AM can speed up the renovation of injuried conjunctivas,and the effects of chitosan/gelatin electrospinning membrane are better than those of AM in promoting conjunctiva repair and inhibiting scarring.

Conjunctival structural and functional reconstruction using acellular bovine pericardium graft (Normal GEN®) in rabbits.

PURPOSE: To evaluate the effectiveness of acellular bovine pericardium grafts (Normal GEN®) used as scaffolds for conjunctival reconstruction. METHOD: The acellular bovine pericardium graft and the amnion graft were implanted into the bulbar conjunctival defects of adult rabbits. Conjunctival samples of implanted materials and blank defect controls were observed at day 3, 7, 14, 21, 28, and 56 postoperatively. Histological examination was observed at day 14, 28, and 56 of surgery, including hematoxylin-eosin staining, periodic acid-Schiff staining, and Masson's trichrome staining, while immunofluorescent microscopy was observed at 14 days and 28 days after surgery. Results were compared among the Normal GEN®, amnion, and blank defect controls. RESULTS: All three groups showed complete conjunctival reconstruction. Wounds that were not grafted closed by formation of conjunctival scar characterized by a linear array of densely packed collagen fibers in Tenon's capsule. Subepithelial tissue in the grafted groups comprised a loosely organized network of randomly oriented collagen that resembled that of the normal bulbar conjunctiva. However, there was a dense layer of aligned collagen between the conjunctival Tenon's capsule and the sclera in the NormalGEN® group, about 250 µm in thickness. CONCLUSIONS: Implantation of the NormalGEN® graft promoted the formation of conjunctiva as a kind of scaffold both in structure and in function. It had more advantageous mechanical properties than the amnion, strong and elastic, during the period of conjunctival reconstruction.