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The effect of prior long-term recellularization with keratocytes of decellularized porcine corneas implanted in a rabbit anterior lamellar keratoplasty model.

ABSTRACT: Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 μm thick decellularized lenticule against one that had been recellularized with human stromal cells after serum-free culture. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. Since there was little difference between acellular and cell-seeded scaffolds in our in vivo study, future scaffold development should use acellular controls to determine if cells are necessary.

SUBMITTER: Fernandez-Perez J

PROVIDER: S-EPMC8168847 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeTo evaluate difference in therapeutic outcomes between deep anterior lamellar keratoplasty (DALK) and penetrating keratoplasty (PKP) for the clinical treatment of keratoconus.MethodsA comprehensive search was conducted in Pubmed, EMBASE, Cochrane Library, and Web of science. Eligible studies should include at least one of the following factors: best corrected visual acuity (BCVA), postoperative spherical equivalent (SE), postoperative astigmatism and endothelial cell count (ECC), central corneal thickness (CCT), graft rejection and graft failure, of which BCVA, graft rejection and graft failure were used as the primary outcome measures, and postoperative SE, astigmatism, CCT and ECC as the secondary outcome measures. Given the lack of randomized clinical trials (RCTs), cohort studies and prospective studies were considered eligible.ResultsSixteen clinical trials involving 6625 eyes were included in this review, including 1185 eyes in DALK group, and 5440 eyes in PKP group. The outcomes were analyzed using Cochrane Review Manager (RevMan) version 5.0 software. The postoperative BCVA in DALK group was significantly better than that in PKP group (OR = 0.48; 95%CI 0.39 to 0.60; p<0.001). There were fewer cases of graft rejection in DALK group than those in PKP group (OR = 0.28; 95%CI 0.15 to 0.50; p<0.001). Nevertheless the rate of graft failure was similar between DALK and PKP groups (OR = 1.05; 95%CI 0.81 to 1.36; p = 0.73). There were no significant differences in the secondary outcomes of SE (p = 0.70), astigmatism (p = 0.14) and CCT (p = 0.58) between DALK and PKP groups. And ECC in DALK group was significantly higher than PKP group (p<0.001). The postoperative complications, high intraocular pressure (high-IOP) and cataract were analyzed, fewer cases of complications occurred in DALK group than those in PKP group (high-IOP, OR 0.22, 95% CI 0.11-0.44, P<0.001) (cataract, OR 0.22; 95% CI 0.08-0.61, P = 0.004). And no cases of expulsive hemorrhage and endophthalmitis were reported.ConclusionThe visual outcomes for DALK were not equivalent to PKP. The rate of graft failure was similar between DALK and PKP. Fewer postoperative complications occurred in DALK group, indicating that compared with PKP, DALK has lower efficacy but higher safety.

Project description:PurposeTo theoretically compare corneal displacement and the von Mises (VM) stress distribution of deep anterior lamellar keratoplasty (DALK) and penetrating keratoplasty (PK) for keratoconus (KC) and to evaluate the effects of residual stromal thickness (RST) and intraocular pressure (IOP) on postoperative corneal biomechanics.MethodsWe performed DALK and PK simulations using Ansys by employing anisotropic nonlinear hyperelastic corneal material properties. We analyzed corneal displacement and VM stress in DALK and PK models under IOPs of 10, 15, 20, and 25 mmHg. We established two DALK models: The ideal-type DALK ensured that postoperative central corneal thickness was constant at 560 µm and the corneal graft thickness varied with RST. The clinical-type DALK ensured that corneal grafts had the same thickness (500 µm) regardless of RST. Then we analyzed the effects of RST and IOP on postoperative corneal displacement and VM stress.ResultsCorneal displacement and VM stress were lower in the DALK than in the PK model. In the ideal-type DALK model, an increase in RST was associated with increased deformation and decreased VM stress in the healing zone, except for a RST of 0 µm. In the clinical-type DALK model, deformation and VM stress in the healing zone decreased with an increase in RST, except for a RST of 0 µm.ConclusionsDALK showed more stability than PK. For the ideal-type DALK model, an increase in RST resulted in decreased postoperative corneal biomechanics in the healing zone. For the clinical-type DALK model, corneal deformation and VM stress decreased with an increase in RST, which provides numerical evidence for the design of corneal transplantation for patients with KC.Translational relevanceIn this computational modeling study, we first theoretically compared corneal biomechanics between DALK and PK for KC. Then, the effects of RST and IOP on postoperative corneal biomechanics were investigated. Our findings provide novel insights into the optimal design for corneal transplantation for patients with KC.

Project description:To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH(4)OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.

Project description:BackgroudPrevious studies of internal graft-host malappositions have not dealt with the precise ways in which each malapposition affected post-penetrating keratoplasty (post-PK) visual outcomes. In this study, we reviewed our post-PK and post-deep anterior lamellar keratoplasty (post-DALK) keratoconic patients and used anterior segment optical coherence tomography (AS-OCT) to evaluate the associations between graft-host interface (GHI) characteristics and visual outcomes.MethodsNovel GHI metrics included: mean graft-host touch (GHT), total prevalence of malapposition proportion (Pm), frequency of apposition (F), size of malapposition (Sm), junctional graft thickness (Tg), junctional host thickness (Th) and the absolute value of difference between Tg and Th (|Tg-Th|). We connected the external and internal junction points of GHI (GHT) and drew a straight line through the central point, perpendicular to both sides of the cornea. Tg and Th were the thicknesses at cross-points 1?mm away from the meeting point on the external side of the graft and host, respectively. Linear regression analysis was used to describe associations between GHI metrics and postsurgical visual outcomes [logarithm of minimum angle of resolution best-corrected visual acuity (logMAR BCVA), spherical equivalent diopter (SE), diopter of spherical power (DS), diopter of cylindrical power (DC) and keratometric astigmatism (Astig value)].ResultsWe enrolled 22 post-PK and 23 post-DALK keratoconic patients. Compared with the regular-apposition results, GHT was decreased in step and gape patterns, and increased in hill and tag patterns. SE increased averagely by 6.851, 5.428 and 5.164 diopter per 1% increase in: F (step) [??=?6.851; 95% Confidence interval (CI)?=?2.975-10.727; P?=?0.001]; F (graft step) [??=?5.428; 95% CI?=?1.685-9.171; P?=?0.005]; and Pm [??=?5.164; 95%CI?=?0.913-9.146; P?=?0.018], respectively. SE increased averagely by 0.31 diopter per 10-?m increment in |Tg-Th| [??=?0.031; 95% CI?=?0.009-0.054; P?=?0.007]. LogMAR BCVA increased (on average) by 0.01 per 10-?m increment in both GHT [??=?0.001; 95% CI?=?0-0.002; P?=?0.030]. and Tg [??=?0.001; 95% CI?=?0.001-0.002; P?=?0.001]. Astig value increased on average by 0.17 diopter per 10-?m increment in Sm [??=?0.017; 95% CI?=?0-0.033; P?=?0.047].ConclusionThis investigation of GHI characteristics suggests explanations for varied ametropia in keratoconic eyes and has potential significance as a reference for promoting pre-surgical planning and technology for corneal transplantation.

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The effect of prior long-term recellularization with keratocytes of decellularized porcine corneas implanted in a rabbit anterior lamellar keratoplasty model.

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