Single-cell transcriptomics reveals maturation of transplanted stem cell-derived retinal pigment epithelial cells toward native state.

Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched in vitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all in vitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (FOS, JUND, and MAFF) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.

Surgical management of mucosal contact headache.

BACKGROUND: Most research on mucosal contact headache has focused on mucosal contact between the nasal septum and middle or inferior turbinate. However, rarely have any studies explored how headache is related to the only one contact point between superior turbinate and nasal septum. OBJECTIVE: To explore how headache is related to the only one contact point between superior turbinate and nasal septum. METHODS: 80 patients with headache were selected. The mucosal contact between superior turbinate and nasal septum was removed to study the relationship between the contact point and headache, with a follow-up of 12 months. RESULTS: Headache symptoms in 56 cases disappeared entirely. Significant relief was observed in 20 patients, and unsatisfactory results in only 4 patients, with the success rate being 95%. CONCLUSION: Some patients with headaches who had intranasal mucosal contact areas benefitted from the surgery. Satisfactory results were achieved by endonasal surgery in 95% of our patients in whom intranasal contact points were believed to be the cause of their headaches who had a mucosal contact point between the superior turbinate and the septum.

Surgical Approaches for Cell Therapeutics Delivery to the Retinal Pigment Epithelium and Retina.

Developing successful surgical strategies to deliver cell therapeutics to the back of the eye is an essential pillar to success for stem cell-based applications in blinding retinal diseases. Within this chapter, we have attempted to gather all key considerations during preclinical animal trials.Guidance is provided for choices on animal models, options for immunosuppression, as well as anesthesia. Subsequently we cover surgical strategies for RPE graft delivery, both as suspension as well as in monolayers in small rodents, rabbits, pigs, and nonhuman primate. A detailed account is given in particular on animal variations in vitrectomy and subretinal surgery, which requires a considerable learning curve, when transiting from human to animal. In turn, however, many essential subretinal implantation techniques in large-eyed animals are directly transferrable to human clinical trial protocols.A dedicated subchapter on photoreceptor replacement provides insights on preparation of suspension as well as sheet grafts, to subsequently outline the basics of subretinal delivery via both the transscleral and transvitreal route. In closing, a future outlook on vision restoration through retinal cell-based therapeutics is presented.

Geometry, penetration force, and cutting profile of different 23-gauge trocars systems for pars plana vitrectomy.

BACKGROUND: To investigate the geometry, penetration force, and cutting profile of 23-gauge trocar systems for pars plana vitrectomy based on their grinding methods in a standardized laboratory setting. METHODS: In this experimental study, Eleven different commercially available 23-gauge sclerotomy trocar systems were divided into 4 groups according to their needle grinding and deburring: "back" bevel, "spear" bevel, "lancet" bevel, and "spatula" bevel. The normative geometrical data of the trocar systems were systematically analyzed according to nomenclature ISO 7864 and ISO 9626. Force to penetrate a 0.4-mm thick polyurethane foil was measured by a Penetrometer, when the trocar needle was piercing, cutting, and sliding through the foil at different defined loading phases and plotted as a load-displacement diagram. Magnified images of the consecutive cut were taken under a microscope after the entire penetration through the foil. Three physicians used all trocar systems in a masked fashion on human sclera to evaluate the manual penetration force in 30° and 90°. RESULTS: The mean outer diameter of the trocar systems was 0.630 ± 0.009 mm, and the mean outer diameter of the trocars was 0.750 ± 0.013 mm. The mean point length was 3.11 ± 0.49 mm, and the mean length of the bevel was 1.46 ± 0.23 mm. The primary bevel angle was 10.75 ± 0.41°, and the secondary bevel angle was 65.9 ± 42.56°. The piercing forces of the back bevel and spear-pointed trocars/needles were at the same level (0.087 ± 0.028 N). The lancet-pointed needle had remarkable low piercing and cutting forces with 0.41 N (range, 0.35-0.47 N). The spatula bevel tip showed the highest penetration piercing force with 1.6 N (range, 1.59-1.73 N). The back bevel systems induced frequently triangular-shaped incisions, with two nearly rectangular cuts of short length. The spear bevels produced a regular characteristic linear cut. Especially, the lancet blade created straight cut with a linear wound apposition. Spatula trocar systems caused usually an arched accurate incision. The manual force to penetrate the human sclera in an angled and rectangular angle appeared in the surgeons hand lower with a back bevel, lancet, or spear tip, whereas higher with spatula bevel systems. CONCLUSION: Lancet and back bevel systems show less penetration force of inner needles than the spatula systems. The results of the penetration force experiments correlated well with the manual force on sclera.

Lutein Modulates Cellular Functionalities and Regulates NLRP3 Inflammasome in a H2O2-Challenged Three-Dimensional Retinal Pigment Epithelium Model.

Excessive hydrogen peroxide (H2O2) generated during retinal cell metabolic activity could lead to oxidative degeneration of retinal pigment epithelium (RPE) tissue, a specific pathological process implicated in various retinal diseases resulting in blindness, which can be mitigated by taking dietary antioxidants to prevent inflammation and impaired cellular dysfunction. This study tested the hypothesis that damages induced by oxidative stresses can be mitigated by lutein in a H2O2-challenged model, which was based on an ARPE-19 cell monolayer cultured on three-dimensional (3D)-printed fibrous scaffolds. Pretreating these models with lutein (0.5 µM) for 24 h can significantly lower the oxidative stress and maintain phagocytosis and barrier function. Moreover, lutein can modulate the NLRP3 inflammasome, leading to a ∼40% decrease in the pro-inflammatory cytokine (IL-1ß and IL-18) levels. Collectively, this study suggests that the 3D RPE model is an effective tool to examine the capability of lutein to modulate cellular functionalities and regulate NLRP3 inflammation.

Matrix stiffening promotes chondrocyte senescence and the osteoarthritis development through downregulating HDAC3.

Extracellular matrix (ECM) stiffening is a typical characteristic of cartilage aging, which is a quintessential feature of knee osteoarthritis (KOA). However, little is known about how ECM stiffening affects chondrocytes and other molecules downstream. This study mimicked the physiological and pathological stiffness of human cartilage using polydimethylsiloxane (PDMS) substrates. It demonstrated that epigenetic Parkin regulation by histone deacetylase 3 (HDAC3) represents a new mechanosensitive mechanism by which the stiffness matrix affected chondrocyte physiology. We found that ECM stiffening accelerated cultured chondrocyte senescence in vitro, while the stiffness ECM downregulated HDAC3, prompting Parkin acetylation to activate excessive mitophagy and accelerating chondrocyte senescence and osteoarthritis (OA) in mice. Contrarily, intra-articular injection with an HDAC3-expressing adeno-associated virus restored the young phenotype of the aged chondrocytes stimulated by ECM stiffening and alleviated OA in mice. The findings indicated that changes in the mechanical ECM properties initiated pathogenic mechanotransduction signals, promoted the Parkin acetylation and hyperactivated mitophagy, and damaged chondrocyte health. These results may provide new insights into chondrocyte regulation by the mechanical properties of ECM, suggesting that the modification of the physical ECM properties may be a potential OA treatment strategy.

Effect of novel vital dyes on outer blood-retina barrier function in cultured human retinal pigment epithelium.

PURPOSE: To assess tight junction (TJ) integrity in cultured human fetal retinal pigment epithelium (HFRPE) after exposure to clinically relevant novel vital dyes. METHODS: HFRPE floater cells were harvested from RPE primary cultures of 4 donor eyes and seeded on polyester Transwell® for 4-6 weeks. The apical compartments of well-differentiated cultures were exposed to 0.005 mg/ml Coomassie violet R200 (CVR), methyl 2B (M2B) or Orange II. Periods of 30-300 s were chosen to mimic surgical exposure times, while 3 h was used for toxicity assays, with subsequent washout. Cell-cell junctions were studied by immunofluorescence (zonula occludens-1, ZO-1). Transepithelial electrical resistance (TER) was measured regarding blood-retina barrier (BRB) function. RESULTS: At 4-6 weeks after confluence, HFRPE had grown into pigmented hexagonal monolayers with stable TER values (451-1,520 Ω·cm(2)). After 300-second dye treatments, a continuous ZO-1 signal was detected in all vital dye-treated groups 1.5 h after exposure, whereas trypsin controls showed patchy loss of the TJ stain. TER of CVR-, M2B- and Orange-II-treated groups had dropped 1.5 h after exposure to 148 ± 58.4, 162 ± 23.7 and 164 ± 18.5 Ω·cm(2), respectively, compared to 73 ± 44.9 Ω·cm(2) in positive controls. After 3 h of exposure to 0.005 mg/ml vital dyes in thick drops, TER maintained similar levels to those prior to exposure (90.8 ± 4.7% of the original values, 93.8 ± 6.5 and 91.9 ± 3.6%, respectively), together with no difference from the vehicle controls (94.8 ± 6.6%). TER values recovered in all groups to prior levels within 3 days. CONCLUSION: Novel vital dyes (CVR, M2B and Orange II) caused no outer BRB function alteration.

[Extranodal NK/T cell lymphoma, nasal type involving the larynx and digestive tract: a case report and literature review].

Extranodal NK/T cell lymphoma, nasal type（ENKTL） is a highly aggressive malignant tumor derived from NK cells. This article reports a case of ENKTL invading the larynx and digestive tract. The clinical clinical manifestations include hoarseness and intranasal masses.

Release and velocity of micronized dexamethasone implants with an intravitreal drug delivery system: kinematic analysis with a high-speed camera.

PURPOSE: Ozurdex, a novel dexamethasone (DEX) implant, is released by a drug delivery system into the vitreous cavity. We analyzed the mechanical release aperture of the novel applicator, obtained real-time recordings using a high-speed camera system and performed kinematic analysis of the DEX application. DESIGN: Experimental study. METHODS: : The application of intravitreal DEX implants (6 mm length, 0.46 mm diameter; 700 µg DEX mass, 0.0012 g total implant mass) was recorded by a high-speed camera (500 frames per second) in water (Group A: n = 7) or vitreous (Group B: n = 7) filled tanks. Kinematic analysis calculated the initial muzzle velocity as well as the impact on the retinal surface at approximately 15 mm of the injected drug delivery system implant in both groups. A series of drug delivery system implant positions was obtained and graphically plotted over time. RESULTS: High-speed real-time recordings revealed that the entire movement of the DEX implant lasted between 28 milliseconds and 55 milliseconds in Group A and 1 millisecond and 7 milliseconds in Group B. The implants moved with a mean muzzle velocity of 820 ± 350 mm/s (±SD, range, 326-1,349 mm/s) in Group A and 817 ± 307 mm/s (±SD, range, 373-1,185 mm/s) in Group B. In both groups, the implant gradually decelerated because of drag force. With greater distances, the velocity of the DEX implant decreased exponentially to a complete stop at 13.9 mm to 24.7 mm in Group A and at 6.4 mm to 8.0 mm in Group B. Five DEX implants in Group A reached a total distance of more than 15 mm, and their calculated mean velocity at a retinal impact of 15 mm was 408 ± 145 mm/s (±SD, range, 322-667 mm/s), and the consecutive normalized energy was 0.55 ± 0.44 J/m (±SD). In Group B, none of the DEX implants reached a total distance of 6 mm or more. An accidental application at an angle of 30 grade and consecutively reduced distance of approximately 6 mm may result in a mean velocity of 844 and mean normalized energy of 0.15 J/m (SD ± 0.47) in a water-filled eye. CONCLUSION: The muzzle velocity of DEX implants is approximately 0.8 m/s and decreases exponentially over distance. The drag over time in vitreous is faster than in water. The calculated retinal impact energy does not reach reported damage levels for direct foreign bodies or other projectiles.

Polymeric biomaterials in the treatment of posterior segment diseases.

Polymeric biomaterials are biological or synthetic substances which can be engineered to interact with biological systems for the diagnosis or treatment of diseases. These biomaterials have immense potential for treating eyes diseases, particularly the retina-a site of many inherited and acquired diseases. Polymeric biomaterials can be engineered to function both as an endotamponade agent and to prevent intraocular scarring in retinal detachment repair surgeries. They can also be designed as a drug delivery platform for treatment of retinal diseases. Finally, they can be used as scaffolds for cellular products and provide non-viral gene delivery solutions to the retina. This perspective article explains the role of polymeric biomaterials in the treatment of retinal conditions by highlighting recent advances being translated to clinical practice. The article will also identify potential hurdles to clinical translation as future research directions in the field.

Clinical Strategies for Enhancing the Efficacy of CAR T-Cell Therapy for Hematological Malignancies.

Chimeric antigen receptor (CAR) T cells have been successfully used for hematological malignancies, especially for relapsed/refractory B-cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma. Patients who have undergone conventional chemo-immunotherapy and have relapsed can achieve complete remission for several months with the infusion of CAR T-cells. However, side effects and short duration of response are still major barriers to further CAR T-cell therapy. To improve the efficacy, multiple targets, the discovery of new target antigens, and CAR T-cell optimization have been extensively studied. Nevertheless, the fact that the determination of the efficacy of CAR T-cell therapy is inseparable from the discussion of clinical application strategies has rarely been discussed. In this review, we will discuss some clinical application strategies, including lymphodepletion regimens, dosing strategies, combination treatment, and side effect management, which are closely related to augmenting and maximizing the efficacy of CAR T-cell therapy.

[Superficial angiomyxoma in nasal vestibule: a case report].

This paper reported a case of superficial angiomyxoma in the region of the nasal vestibule. The clinical manifestation was swelling of the left nasal vestibular skin, while paranasal sinus CT showed swell soft tissue in the anterior and superior region to the left maxilla. Under general anesthesia, the left nasal vestibular mass was resected under nasal endoscopy. The postoperative pathological diagnosis was superficial angiomyxoma. The patient underwent a CT scan of the paranasal sinuses 4 months after the operation, and there was no recurrence of the tumor.

[Scanning electron microscope of the human nasal septum].

Objective:Explore the significance of ultrastructural differences in tissue engineering, 3D printing, and rhinoplasty. Methods: 32 specimens （8 vomers, 8 perpendicular plates of ethmoid bone, 8 maxillary nasal crests, and 8 septal cartilage） of the nasal septum from patients with a nasal deviated septum and chronic sinusitis undergoing septoplasty were selected and examined using scanning electron microscopy. Results: The nasal septum of patients of different ages behaves similarly under the scanning electron microscope, and the bones of different parts of the nasal septum have similarities and differences. Conclusion:By observing the scanning electron micrograph of the nasal septum and analyzing the surface ultrastructure, it provides important information for the development of tissue engineering, assists in the refined modeling of 3D printing technology, and provides more ideal restoration materials for clinical operations.

A giant pyogenic granuloma gravidarum in the nasal cavity: A case report.

Pyogenic granuloma (PG) is a benign fibrovascular proliferative lesion on the skin and mucous membranes, but its pathogenesis remains unclear. PG usually occurs on the head and neck region, fingers and toes. The oral gingiva is the most common location for pregnant patients, while it is rarely found in the nasal cavity. This case is notable not only for its uncommon site and size but also for its gradual growth after delivery. Endoscopic surgery can achieve the desired cosmetic effect and a satisfactory airway. A rapidly growing hemorrhagic lesion in the nasal cavity should be considered as a differential diagnosis.

Verification of an iPSC line (LZUi002-A) from a patient with a novel mutation in the TBL1X gene.

More than 120 genes have been reported to be associated with deafness, and deletion of the TBL1X gene may cause deafness in humans. In this study, we generated an induced pluripotent stem cell (iPSC) line from dermal fibroblasts of a 34-year-old deaf person with a novel variant c.342_343insGCGGCG in the TBL1X gene. The induced patient-specific iPSC line with a normal karyotype and expressed pluripotent markers, it also shows differentiation totipotency and tridermogenesis in vivo. It may be a good model for studying hearing loss in vitro and it will benefit to the development of new therapies for deafness.

PKIB involved in the metastasis and survival of osteosarcoma.

Osteosarcoma is frequently metastasized at the time of diagnosis in patients. However, the underlying mechanism of osteosarcoma metastasis remains poorly understood. In this study, we evaluated DNA methylation profiles combined with gene expression profiles of 21 patients with metastatic osteosarcoma and 64 patients with non-metastatic osteosarcoma from TARGET database and identified PKIB and AIM2 as hub genes related to the metastasis of osteosarcoma. To verify the effects of PKIB on migration and invasion of osteosarcoma, we performed wound-healing assay and transwell assay. The results showed that PKIB significantly inhibited the migration and invasion of osteosarcoma cells, and the Western blot experiments showed that the protein level of E-cad was upregulated and of VIM was downregulated in 143-B cell recombinant expression PKIB. These results indicate that PKIB inhibit the metastasis of osteosarcoma. CCK-8 assay results showed that PKIB promote the proliferation of osteosarcoma. In addition, the Western blot results showed that the phosphorylation level of Akt was upregulated in 143-B cells overexpressing PKIB, indicating that PKIB promotes the proliferation of osteosarcoma probably through signaling pathway that Akt involved in. These results give us clues that PKIB was a potential target for osteosarcoma therapy. Furthermore, combined clinical profiles analysis showed that the expression of AIM2- and PKIB- related risk scores was significantly related to the overall survival of patients with osteosarcoma. Thus, we constructed a nomogram based on AIM2 and PKIB expression-related risk scores for osteosarcoma prognostic assessment to predict the 1-, 2-, 3-, and 5-year overall survival rate of patients with metastatic osteosarcoma, assisting clinicians in the diagnosis and treatment of metastatic osteosarcoma.

Endoscopic Management of Recurrent Maxillary Sinus Ameloblastoma in a Child With Autism Spectrum Disorder: A Case Report.

Ameloblastomas are slow-growing, aggressive odontogenic epithelial tumors that originate from the jawbone. One of the most easily relapsing maxillofacial tumors, ameloblastomas mainly occur in the mandibular molar area and ascending branch, although they can occasionally occur in the nasal cavity and paranasal sinuses. A 14-year-old child with autism spectrum disorder underwent sinus computed tomography (CT) under anesthesia. A swollen tumor had grown in the left maxillary sinus, and the bone of the maxillary sinus was damaged. Nine months after the first operation, recurrence was observed in the left maxillary sinus. The pathological diagnosis was ameloblastoma. Due to the child's inability to communicate and cooperate with the treatment normally, he underwent endoscopic surgery again combined with low-temperature plasma treatment. No tumor recurrence was found on reexamination 6 months after surgery.

Injectable PTHF-based thermogelling polyurethane implants for long-term intraocular application.

BACKGROUND: Hydrogels show great potential to be used for intraocular applications due to their high-water content and similarity to the native vitreous. Injectable thermosensitive hydrogels through a small-bore needle can be used as a delivery system for drugs or a tamponading substitute to treat posterior eye diseases with clear clinical potential. However, none of the currently available thermosensitive hydrogels can provide intraocular support for up to 3 months or more. METHOD: In this study, an injectable polytetrahydrofuran (PTHF)-based thermosensitive hydrogel was synthesized by polyurethane reaction. We examined the injectability, rheological properties, microstructure, cytotoxicity, and in vivo compatibility and stability of the hydrogels in rabbit eyes. RESULTS: We found that the PTHF block type and PTHF component ratio could modulate thermogelation properties of the polyurethane polymers. The PTHF-based hydrogel implants retained normal retinal structure and function. Incorporating bioinert PTHF generated highly biocompatible and more stable thermogels in the vitreous cavity, with gel networks and the presence of polymer still observed after 3 months when other thermogels would have been completely cleared. Moreover, despite lacking hydrolytically cleavable linkages, the polymers could be most naturally removed from the native vitreous by bio-erosion without additional surgical interventions. CONCLUSION: Our findings suggest the potential of incorporating hydrophobic bioinert blocks to enhance the in vivo stability of supramolecularly associated hydrogels for long-term intraocular applications.

Electrohydrodynamic Jet-Printed Ultrathin Polycaprolactone Scaffolds Mimicking Bruch's Membrane for Retinal Pigment Epithelial Tissue Engineering.

Age-related macular degeneration (AMD) is the leading cause of visual loss and affects millions of people worldwide. Dysfunction of the retinal pigment epithelium (RPE) is associated with the pathogenesis of AMD. The purpose of this work is to build and evaluate the performance of ultrathin scaffolds with an electrohydrodynamic jet (EHDJ) printing method for RPE cell culture. We printed two types of ultrathin (around 7 µm) polycaprolactone scaffolds with 20 µm and 50 µm pores, which possess mechanical properties resembling that of native human Bruch's membrane and are biodegradable. Light microscopy and cell proliferation assay showed that adult human retinal pigment epithelial (ARPE-19) cells adhered and proliferated to form a monolayer on the scaffolds. The progress of culture matured on the scaffolds was demonstrated by immunofluorescence (actin, ZO-1, and Na+/K+-ATPase) and Western blot analysis of the respective proteins. The RPE cells cultured on EHDJ-printed scaffolds with 20 µm pores presented higher permeability, higher transepithelial potential difference, and higher expression level of Na+/K+-ATPase than those cultured on Transwell inserts. These findings suggest that the EHDJ printing can fabricate scaffolds that mimic Bruch's membrane by promoting maturation of RPE cells to form a polarized and functional monolayered epithelium with potential as an in vitro model for studying retinal diseases and treatment methods.

Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources.

Retinal pigment epithelial (RPE) cell dysfunction and death are characteristics of age-related macular degeneration. A promising therapeutic option is RPE cell transplantation. Development of clinical grade stem-cell derived RPE requires efficient in vitro differentiation and purification methods. Enzymatic purification of RPE relies on the relative adherence of RPE and non-RPE cells to the culture plate. However, morphology and adherence of non-RPE cells differ for different stem cell sources. In cases whereby the non-RPE adhered as strongly as RPE cells to the culture plate, enzymatic method of purification is unsuitable. Thus, we hypothesized the need to customize purification strategies for RPE derived from different stem cell sources. We systematically compared five different RPE purification methods, including manual, enzymatic, flow cytometry-based sorting or combinations thereof for parameters including cell throughput, yield, purity and functionality. Flow cytometry-based approach was suitable for RPE isolation from heterogeneous cultures with highly adherent non-RPE cells, albeit with lower yield. Although all five purification methods generated pure and functional RPE, there were significant differences in yield and processing times. Based on the high purity of the resulting RPE and relatively short processing time, we conclude that a combination of enzymatic and manual purification is ideal for clinical applications.