Hybrid Hydrogel Composed of Hyaluronic Acid, Gelatin, and Extracellular Cartilage Matrix for Perforated TM Repair

A novel series of composite hydrogels, built from the three components 1), hyaluronic acid methacryloyl (HAMA); 2), gelatin methacryloyl (GelMA), and 3), extracellular cartilage matrix (ECM), was prepared and studied regarding the possible utility in the surgical repair of damaged (perforated) tympanic membrane (TM). Noteworthy is component 3), which was harvested from the ribs of α-1,3-galactosidyltransferase-knockout (α-1,3 GalT-KO) pigs. The absence of α-1,3-galactosyl glycoprotein is hypothesized to prevent rejection due to foreign-body immunogenicity. The composite hydrogels were characterized by various aspects, using a variety of physicochemical techniques: aqueous swelling, structural degradation, behavior under compression, and morphology, e.g., in vitro biocompatibility was assessed by the CCK-8 and live–dead assays and through cytoskeleton staining/microscopy. Alcian blue staining and real-time PCR (RT-PCR) were performed to examine the chondrogenic induction potential of the hydrogels. Moreover, a rat TM defect model was used to evaluate the in vivo performance of the hydrogels in this particular application. Taken together, the results from this study are surprising and promising. Much further development work will be required to make the material ready for surgical use.

LncRNA NONHSAT030515 promotes the chondrogenic differentiation of human adipose-derived stem cells via regulating the miR-490-5p/BMPR2 axis

Background Chondrogenic differentiation of human adipose-derived stem cells (hADSCs) is important for cartilage generation and degradation. LncRNAs play an essential role in stem cell differentiation. However, the role and mechanism of lncRNA in hADSCs remain unclear. Our previous study showed that miR-490-5p was downregulated during chondrogenic differentiation of hADSCs. In this study, we investigated the effect and mechanism of lncRNA NONHSAT030515 interacting with miR-490-5p on chondrogenic differentiation of hADSCs. Methods Alcian blue staining was used to assess the deposition of chondromatrix proteins following chondrogenic differentiation of human adipose stem cells. Immunohistochemistry was used to evaluate the expression of collagenII. TargetScan, miRTarBase and miRDB database analyses were applied to find the miRNA and target genes of lncRNA NONHSAT030515. A dual luciferase experiment was conducted to identify the direct target of NONHSAT030515. pcDNA3.1- NONHSAT030515 transfection and sh- NONHSAT030515 treatment were conducted to verify the role of lncRNA NONHSAT030515 in chondrogenic differentiation. The levels of Aggrecan, SOX9 and COL2A1 were analyzed by qRT-PCR and Western blot assay. Results Alcian blue staining, immunocytochemical, qRT-PCR, and Western blot have determined that lncRNA NONHSAT030515 can promote the chondrogenic differentiation of hADSCs. MiR-490- 5p was the direct target of NONHSAT030515, while BMPR2 was the target gene. This result was confirmed by luciferase reporter assay. Up-regulation of NONHSAT030515 promoted BMPR2 protein expression and promoted chondrogenic differentiation, whereas down-regulation of NONHSAT030515 caused completely opposite results. Conclusion LncRNA NONHSAT030515 promotes the chondrogenic differentiation of hADSCs through increasing BMPR2 expression by regulating miR-490- 5p.

Altered Mucus Barrier Integrity and Increased Susceptibility to Colitis in Mice upon Loss of Telocyte Bone Morphogenetic Protein Signalling

FoxL1+-Telocytes (TCFoxL1+) are subepithelial cells that form a network underneath the epithelium. We have shown that without inflammatory stress, mice with loss of function in the BMP signalling pathway in TCFoxL1+ (BmpR1aΔFoxL1+) initiated colonic neoplasia. Although TCFoxL1+ are modulated in IBD patients, their specific role in this pathogenesis remains unclear. Thus, we investigated how the loss of BMP signalling in TCFoxL1+ influences the severity of inflammation and fosters epithelial recovery after inflammatory stress. BmpR1a was genetically ablated in mouse colonic TCFoxL1+. Experimental colitis was performed using a DSS challenge followed by recovery steps to assess wound healing. Physical barrier properties, including mucus composition and glycosylation, were assessed by alcian blue staining, immunofluorescences and RT-qPCR. We found that BmpR1aΔFoxL1+ mice had impaired mucus quality, and upon exposure to inflammatory challenges, they had increased susceptibility to experimental colitis and delayed healing. In addition, defective BMP signalling in TCFoxL1+ altered the functionality of goblet cells, thereby affecting mucosal structure and promoting bacterial invasion. Following inflammatory stress, TCFoxL1+ with impaired BMP signalling lose their homing signal for optimal distribution along the epithelium, which is critical in tissue regeneration after injury. Overall, our findings revealed key roles of BMP signalling in TCFoxL1+ in IBD pathogenesis.

Generation of a Biomimetic Substitute of the Corneal Limbus Using Decellularized Scaffolds

Patients with severe limbal damage and limbal stem cell deficiency are a therapeutic challenge. We evaluated four decellularization protocols applied to the full-thickness and half-thickness porcine limbus, and we used two cell types to recellularize the decellularized limbi. The results demonstrated that all protocols achieved efficient decellularization. However, the method that best preserved the transparency and composition of the limbus extracellular matrix was the use of 0.1% SDS applied to the half-thickness limbus. Recellularization with the limbal epithelial cell line SIRC and human adipose-derived mesenchymal stem cells (hADSCs) was able to generate a stratified epithelium able to express the limbal markers p63, pancytokeratin, and crystallin Z from day 7 in the case of SIRC and after 14–21 days of induction when hADSCs were used. Laminin and collagen IV expression was detected at the basal lamina of both cell types at days 14 and 21 of follow-up. Compared with control native limbi, tissues recellularized with SIRC showed adequate picrosirius red and alcian blue staining intensity, whereas limbi containing hADSCs showed normal collagen staining intensity. These preliminary results suggested that the limbal substitutes generated in this work share important similarities with the native limbus and could be potentially useful in the future.

Analysis of BMP3 Variants In The Causality of Ocular Coloboma

Coloboma, a congenital disorder characterized by gaps in ocular tissues, is caused when the choroid fissure fails to close during embryonic development. Several loci have been associated with coloboma, but these represent less than 40% of those that are involved with this disease. Here, we describe a novel coloboma-causing locus, BMP3. Whole exome sequencing and Sanger sequencing of patients with coloboma identified three variants in BMP3, two of which are predicted to be disease causing. Consistent with this, bmp3 mutant zebrafish have aberrant fissure closure. bmp3 is expressed in the ventral head mesenchyme and regulates phosphorylated Smad3 in a population of cells adjacent to the choroid fissure. Furthermore, mutations in bmp3 sensitize embryos to Smad3 inhibitor treatment resulting in open choroid fissures. Micro CT scans and Alcian blue staining of zebrafish demonstrate that mutations in bmp3 cause midface hypoplasia, suggesting that bmp3 regulates cranial neural crest cells. Consistent with this, we see active Smad3 in a population of periocular neural crest cells, and bmp3 mutant zebrafish have reduced neural crest cells in the choroid fissure. Taken together, this data suggests that Bmp3 controls Smad3 phosphorylation in neural crest cells to regulate early craniofacial and ocular development.

Analysis of BMP3 variants in the causality of ocular coloboma

Coloboma, a congenital disorder characterized by gaps in ocular tissues, is caused when the choroid fissure fails to close during embryonic development. Several loci have been associated with coloboma, but these represent less than 40% of those that are involved with this disease. Here, we describe a novel coloboma-causing locus, BMP3. Whole exome sequencing and Sanger sequencing of patients with coloboma identified three variants in BMP3, two of which are predicted to be disease causing. Consistent with this, bmp3 mutant zebrafish have aberrant fissure closure. bmp3 is expressed in the ventral head mesenchyme and regulates phosphorylated Smad3 in a population of cells adjacent to the choroid fissure. Furthermore, mutations in bmp3 sensitize embryos to Smad3 inhibitor treatment resulting in open choroid fissures. Micro CT scans and Alcian blue staining of zebrafish demonstrate that mutations in bmp3 cause midface hypoplasia, suggesting that bmp3 regulates cranial neural crest cells. Consistent with this, we see active Smad3 in a population of periocular neural crest cells, and bmp3 mutant zebrafish have reduced neural crest cells in the choroid fissure. Taken together, this data suggests that Bmp3 controls Smad3 phosphorylation in neural crest cells to regulate early craniofacial and ocular development.

Suramin attenuates intervertebral disc degeneration by inhibiting NF-κB signalling pathway

Aims Interleukin (IL)-1β is one of the major pathogenic regulators during the pathological development of intervertebral disc degeneration (IDD). However, effective treatment options for IDD are limited. Suramin is used to treat African sleeping sickness. This study aimed to investigate the pharmacological effects of suramin on mitigating IDD and to characterize the underlying mechanism. Methods Porcine nucleus pulposus (NP) cells were treated with vehicle, 10 ng/ml IL-1β, 10 μM suramin, or 10 μM suramin plus IL-1β. The expression levels of catabolic and anabolic proteins, proinflammatory cytokines, mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB-related signalling molecules were assessed by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence analysis. Flow cytometry was applied to detect apoptotic cells. The ex vivo effects of suramin were examined using IDD organ culture and differentiation was analyzed by Safranin O-Fast green and Alcian blue staining. Results Suramin inhibited IL-1β-induced apoptosis, downregulated matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5, and upregulated collagen 2A (Col2a1) and aggrecan in IL-1β-treated NP cells. IL-1β-induced inflammation, assessed by IL-1β, IL-8, and tumour necrosis factor α (TNF-α) upregulation, was alleviated by suramin treatment. Suramin suppressed IL-1β-mediated proteoglycan depletion and the induction of MMP-3, ADAMTS-4, and pro-inflammatory gene expression in ex vivo experiments. Conclusion Suramin administration represents a novel and effectively therapeutic approach, which could potentially alleviate IDD by reducing extracellular matrix (ECM) deposition and inhibiting apoptosis and inflammatory responses in the NP cells. Cite this article: Bone Joint Res 2021;10(8):498–513.

Human Platelet Lysate as an Alternative to Autologous Serum for Human Chondrocyte Clinical Use

Objective A pivotal aspect of cartilage tissue engineering resides in cell culture medium supplementation, in view of maximizing in vitro cell proliferation and preserving cellular functionality. Autologous human serum (aHS) is commonly used as an inducive supplement for safe human articular chondrocyte (HAC) proliferation prior to clinical implantation. However, practical clinical use of aHS is hindered by constraining manufacturing requirements and quality assurance-driven downstream processing. The present study investigated potential alternative use of commercial human platelet lysate (hPL) supplements in HAC manufacturing workflows related to clinical therapeutic pathways. Design Differential effects of hPL, aHS, and fetal bovine serum were assessed on primary cultured HAC parameters (viability, proliferative rates, and morphology) in 2-dimensional in vitro systems. A 3-dimensional HAC pellet model served for postexpansion assessment of cellular functionality, by visualizing proteoglycan production (Alcian blue staining), and by using qRT-PCR relative quantification of chondrogenic marker ( SOX9, COL2-A1, and ACAN) genetic expression. Results We found that monolayer HAC culture with hPL or aHS supplements presented similar characteristics (elongated cell morphology and nearly identical growth kinetics). Chondrogenic activity appeared as conserved in HACs expanded with human or bovine supplements, wherein histologic analysis indicated a progressive sGAG accumulation and SOX9, COL2-A1, ACAN gene expression was upregulated in 3-dimensional HAC pellet models. Conclusion This study therefore supports the use of hPL as a functional equivalent and alternative to aHS for cultured HAC batch preparation, with the potential to effectively alleviate pressure on clinical and manufacturing bottlenecks in cell therapy approaches for cartilage regeneration.

Morphological Peculiarities of Parasitic (Trichosomoides crassicauda) Infection in Rat Urinary Bladder

Trichosomoides crassicauda (T. crassicauda) is a parasite commonly localized in the urinary bladder (UB) of laboratory and wild rats. The presence of these helminths can influence the prediction of pathological changes in the UB. Therefore, the purpose of this research was to make a comprehensive study of the features of the morphological changes in the UB wall of white laboratory rats as a result of T. crassicauda infestation. The study was performed on male rats using histological (Hematoxyline-Eosin and Alcian Blue staining) and immunohistochemical (Ki-67, Hsp70, Hsp90α, CD3 and CD20) methods. T. crassicauda was detected in both urine and UB samples. Morphological changes were observed as disruption in urothelial cell stratification and insignificant proliferative and immune responses in the UB. Increased heat shock protein levels were observed which may suggest a natural body’s resistance to this parasite.

Treatment of Adult Metachromatic Leukodystrophy Model Mice Using Intrathecal Administration of Type 9 AAV Vector Encoding Arylsulfatase A

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by an arylsulfatase A (ASA) deficiency and characterized by severe neurological symptoms resulting from demyelination within the central and peripheral nervous systems. We investigated the feasibility and efficacy of intrathecal administration of a type 9 adeno-associated viral vector encoding ASA (AAV9/ASA) for the treatment of 6-week-old MLD model mice, which are presymptomatic, and 1-year-old mice, which exhibit neurological abnormalities. Immunohistochemical analysis following AAV9/ASA administration showed ASA expression within the brain, with highest activities in the cerebellum and olfactory bulbs. In mice treated at 1 year, alcian blue staining and quantitative analysis revealed significant decreases in stored sulfatide within the hindbrain but not forebrain. Behaviorally, mice treated at 1 year showed no improvement in their ability to traverse narrow balance beams as compared to untreated mice. By contrast, MLD mice treated at 6 weeks showed significant decreases in stored sulfatide throughout the entire brain and improved ability to traverse narrow balance beams. These findings suggest intrathecal administration of an AAV9/ASA vector is a promising approach to treating genetic diseases of the central nervous system, including MLD, though it may be essential to begin therapy before the onset of neurological symptoms.