Beneficial immune-modulatory effects of the N-163 strain of Aureobasidium pullulans-produced 1,3-1,6 Beta glucans in Duchenne muscular dystrophy: Results of an open-label, prospective, exploratory case-control clinical study.

Background: This exploratory case-control study is to evaluate the effects of supplementation of Aureobasidium pullulans-N-163 strain produced 1,3-1,- 6 beta glucan in young patients with Duchenne muscular dystrophy (DMD). Methods: Twenty-seven male subjects aged 5-19 years with DMD were included, nine in the control arm and 18 in the treatment arm to receive N-163 beta glucan along with conventional therapies for 45 days. While performing the analysis, steroid usage was also taken into consideration, those not administered steroids (Steroid -ve) (Control, n = 5; treatment, n = 9), those administered steroids (Steroid +ve) (Control, n = 4; treatment, n = 9). Results: IL-6 showed a significant decrease in the treatment groups, especially the N-163 Steroid -ve group. IL-13 decreased in both treatment groups and TGF-ß levels showed a significant decrease in the treatment groups, especially the N-163 Steroid -ve group, (p < 0.05). Dystrophin levels increased by up to 32% in the treatment groups compared to the control. Medical research council (MRC) grading showed slight improvement in muscle strength improvement in 12 out of 18 patients (67%) in the treatment group and four out of nine (44%) subjects in the control group. Conclusion: Supplementation with the N-163 beta glucan food supplement produced beneficial effects: a significant decrease in inflammation and fibrosis markers, increase in serum dystrophin and slight improvement in muscle strength in DMD subjects over 45 days, thus making this a potential adjunct treatment for DMD after validation. Trial registration: The study was registered in Clinical trials registry of India, CTRI/2021/05/033346. Registered on 5th May, 2021.

Resolution of fibrosis in mdx dystrophic mouse after oral consumption of N-163 strain of Aureobasidium pullulans produced ß-glucan.

Recent advances in the management of Duchenne muscular dystrophy (DMD), such as exon skipping and gene therapy, though have reached a clinical stage, the outcome at its best is still considered suboptimal. In this study, we evaluated a novel N-163 strain of Aureobasidium pullulans produced ß-glucan (Neu-REFIX) for its potential as an adjuvant to slow down the progression of the disease by anti-inflammatory and anti-fibrotic effects. In this study, 45 mice in the three groups, 15 each in a group; Gr. 1 normal mice, Gr.2 mdx mice as vehicle, and Gr.3 mdx mice administered the N-163 ß-glucan for 45 days. The N-163 ß-glucan group showed a significant decrease in the plasma ALT, AST, and LDH levels (126 ± 69 U/l, 634 ± 371 U/l, 3335 ± 1258 U/l) compared with the vehicle group (177 ± 27 U/l, 912 ± 126 U/l, 4186 ± 398 U/l). Plasma TGF-ß levels increased, and plasma IL-13 levels decreased in the N-163 group. The inflammation score of HE-stained muscle sections in the N-163 group (1.5 ± 0.8) was lower than that in the vehicle group (2.0 ± 0.8). The N-163 strain ß-glucan group (24.22 ± 4.80) showed a significant decrease in the fibrosis area (Masson's Trichrome-positive area) compared with the vehicle group (36.78 ± 5.74). The percentage of centrally nucleated fibres evaluated by Masson's trichrome staining was 0 in the normal group, while it increased to 80% in the vehicle group but remained at 76.8% in the N-163 group. The N-163 ß-glucan group showed a significant decrease in the fibrosis area. Considering their safety and easy oral consumption, Neu-REFIX ß-glucan could be worth large multicentre clinical studies as adjuvant in slowing down the progress of DMD.

Re-examination of therapeutic management of muscular dystrophies using a vascular smooth muscle-centered approach.

In contrast to the long-standing focus on the pathophysiology of skeletal muscles in the hunt for a cure for Duchenne muscular dystrophy (DMD), we opine that the malfunctioning of dystrophin produced by vascular smooth muscle is a major contributor to the pathology of the illness. We believe that a biological response modifier glucan (BRMG), which has been shown in clinical studies of DMD to boost the expression of vascular smooth muscle dystrophin and provide anti-fibrotic and anti-inflammatory effects, may play a key role in reducing the pathogenesis of DMD. According to the evaluation of biomarkers, this BRMG, which is safe and side-effect-free, reduces the pathogenesis of DMD. We describe the possible mechanisms of action by which this BRMG helps in alleviating the symptoms of DMD by targeting smooth muscle dystrophin, in addition to its advantages over other therapeutic modalities, as well as how it can serve as a valuable adjunct to existing therapies. We suggest that using BRMG adjuncts that target smooth muscle dystrophin would be a potential therapeutic approach that prolongs the lifespan and extends the duration of ambulation from the onset of DMD. Further studies are needed to validate this hypothesis.

Engraftment and Proliferation of Thermoreversible-Gelation-Polymer-Encapsulated Human Corneal Limbal-Stem-Cells on Ocular Surface of a Cadaver Cornea.

PURPOSE: Corneal limbal stem cell (LSC) transplantation has been reported as a potential approach to treat the damaged corneal epithelium. Scaffolds such as human amniotic membrane (hAM) are commonly employed for the in vitro culture and as a carrier during in vivo transplantation. However, they carry the risk of biological contamination and donor to donor variability. To overcome these disadvantages, we herein report the capabilities of a synthetic thermoreversible gelation polymer (TGP) scaffold to serve as an encapsulation support during LSC transplantation and to enable engraftment for corneal regeneration. METHODS: Sixteen discarded human corneas were used to isolate the corneal epithelium which was cultured in TGP and hAM. The cell proliferation and characteristics between TGP and hAM culture methods were evaluated by microscopic observation, 3H Thymidine incorporation assay, immunoperoxidase and immunofluorescence staining. RESULTS: The 3H Thymidine assay's results showed that TGP allowed human-donor cornea-derived LSCs to proliferate well in vitro, compared to hAM and the cells encapsulated in TGP and transplanted ex vivo onto a human cadaver donor cornea denuded of its epithelium, migrated on the ocular surface, and proliferated to form a continuous layer in 25 days. Immunoperoxidase and Immunofluorescence staining of TGP-cultured cells were positive for LSC markers (p63, ABCG2, Connexin 43 and Integrin ß), proving that the TGP helps to preserve the limbal cells' stemness. CONCLUSION: TGP is found to be a multipurpose scaffold for (i) in vitro culture, (ii) ex vivo encapsulation, and in vivo transplantation (iii), enabling engraftment of LSCs in this study, with potentials to extend its application in cell-based therapies in several regenerative medicine approaches.

Benefits of Gut Microbiota Reconstitution by Beta 1,3-1,6 Glucans in Subjects with Autism Spectrum Disorder and Other Neurodegenerative Diseases.

BACKGROUND: Aureobasidium pullulans (black yeast) AFO-202 strain-produced beta glucan, Nichi Glucan, has been shown to improve the behavior and sleep pattern along with an increase in α-synuclein and melatonin in children with autism spectrum disorder (ASD). OBJECTIVE: In this randomized pilot clinical study, we have evaluated the gut microbiota of subjects with ASD after consumption of Nichi Glucan. METHODS: Eighteen subjects with ASD were randomly allocated: six subjects in the control group (Group 1): conventional treatment comprising remedial behavioral therapies and L-carnosine 500 mg per day, and 12 subjects (Group 2) underwent supplementation with Nichi Glucan 0.5 g twice daily along with the conventional treatment for 90 days. RESULTS: Whole genome metagenome (WGM) sequencing of the stool samples at baseline and after intervention showed that among genera of relevance, the abundance of Enterobacteriaceae was decreased almost to zero in Group 2 after intervention, whereas it increased from 0.36% to 0.85% in Group 1. The abundance of Bacteroides increased in Group 1, whereas it decreased in Group 2. The abundance of Prevotella increased while the abundance of Lactobacillus decreased in both Group 1 and Group 2. Among species, a decrease was seen in Escherichia coli, Akkermansia muciniphila CAG:154, Blautia spp., Coprobacillus sp., and Clostridium bolteae CAG:59, with an increase of Faecalibacterium prausnitzii and Prevotella copri, which are both beneficial. CONCLUSION: AFO-202 beta 1,3-1,6 glucan, in addition to balancing the gut microbiome in children with ASD and its role in effective control of curli-producing Enterobacteriaceae that leads to α-synuclein misfolding and accumulation, may have a prophylactic role in Parkinson's and Alzheimer's diseases as well.

Hepatoprotective Effects of Aureobasidium pullulans Derived ß 1,3-1,6 Glucans in a Murine Model of Non-alcoholic Steatohepatitis.

Background: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are highly prevalent conditions characterized by inflammation and fibrosis of the liver, which can progress to cirrhosis and hepatocellular carcinoma if left untreated. Conventional modalities are mainly symptomatic, with no definite solution. Beta-glucan-based biological response modifiers are a potential strategy in lieu of their beneficial metabolic effects. Aureobasidium pullulans strains AFO-202 and N-163 beta-glucans were evaluated for anti-fibrotic and anti-inflammatory hepatoprotective potentials in a NASH animal model in this study. Methods: In the STAM™ murine model of NASH, five groups were studied for 8 weeks: (1) vehicle (RO water), (2) AFO-202 beta-glucan; (3) N-163 beta-glucan, (4) AFO-202+N-163 beta-glucan, and (5) telmisartan (standard pharmacological intervention). Evaluation of biochemical parameters in plasma and hepatic histology including Sirius red staining and F4/80 immunostaining were performed. Results: AFO-202 beta-glucan significantly decreased inflammation-associated hepatic cell ballooning and steatosis. N-163 beta-glucan decreased fibrosis and inflammation significantly (P value < 0.05). The combination of AFO-202 with N-163 significantly decreased the NAFLD Activity Score (NAS) compared with other groups. Conclusion: This preclinical study supports the potential of N-163 and AFO-202 beta-glucans alone or in combination as potential preventive and therapeutic agent(s), for NASH.

Super-multifactorial survey YHAB revealed high prevalence of sleep apnoea syndrome in unaware older adults and potential combinatorial factors for its initial screening.

Study Objectives: Aging is a risk factor for sleep apnoea syndrome (SAS), which is associated with lower quality of life and sudden mortality. However, SAS is often overlooked in older adults without suspicions. Therefore, this study aimed to evaluate SAS incidence and 48 other general factors in older adults. Methods: This cross-sectional study included all non-caregiver-certified, healthy individuals (N = 32) who survived during the long-term cohort study and agreed to participate in apnoea-hypopnoea index (AHI) measurement (aged 83-95 years). AHI and 48 other general factors were evaluated, and simple linear regression analysis was used to identify potential AHI-related factors. Stepwise evaluation was further performed using multiple linear regression analyses. Results: Although no individuals were previously diagnosed with SAS, 30 (93.75%) participants had some degree of SAS (AHI > 5/h), and 22 (68.75%) had severe or moderate SAS (AHI > 15/h). Compared with typical single risk factors represented by body mass index, combining daily steps and other factors improved the fit to the multiple linear regression. Combining daily steps and body mass index improved the fit for males and combining daily steps and red blood cell count improved the fit for females. Conclusion: SAS was highly prevalent in unaware healthy Japanese older adults; combinations of daily steps and body mass index, and daily steps and red blood cell count may predict AHI in such individuals without the need for a specific AHI test.

Two unique biological response-modifier glucans beneficially regulating gut microbiota and faecal metabolome in a non-alcoholic steatohepatitis animal model, with potential applications in human health and disease.

OBJECTIVE: The gut microbiome and its metabolites are influenced by age and stress and reflect the metabolism and health of the immune system. We assessed the gut microbiota and faecal metabolome in a static animal model of non-alcoholic steatohepatitis (NASH). DESIGN: This model was subjected to the following treatments: reverse osmosis water, AFO-202, N-163, AFO-202+N-163 and telmisartan treatment. Faecal samples were collected at 6 and 9 weeks of age. The gut microbiome was analysed using 16S ribosomal RNA sequences acquired by next-generation sequencing, and the faecal metabolome was analysed using gas chromatography-mass spectrometry. RESULTS: Gut microbial diversity increased greatly in the AFO-202+N-163 group. Postintervention, the abundance of Firmicutes decreased, whereas that of Bacteroides increased and was the highest in the AFO-202+N-163 group. The decrease in the abundance of Enterobacteriaceae and other Firmicutes and the abundance of Turicibacter and Bilophila were the highest in the AFO-202 and N-163 groups, respectively. Lactobacillus abundance was highest in the AFO-202+N-163 group. The faecal metabolite spermidine, which is beneficial against inflammation and NASH, was significantly decreased (p=0.012) in the N-163 group. Succinic acid, which is beneficial in neurodevelopmental and neurodegenerative diseases, was increased in the AFO-202 group (p=0.06). The decrease in fructose was the highest in the N-163 group (p=0.0007). Isoleucine and Leucine decreased with statistical significance (p=0.004 and 0.012, respectively), and tryptophan also decreased (p=0.99), whereas ornithine, which is beneficial against chronic immune-metabolic-inflammatory pathologies, increased in the AFO-202+N-163 group. CONCLUSION: AFO-202 treatment in mice is beneficial against neurodevelopmental and neurodegenerative diseases, and has prophylactic potential against metabolic conditions. N-163 treatment exerts anti-inflammatory effects against organ fibrosis and neuroinflammation. In combination, these compounds exhibit anticancer activity.

Improvement of sleep and melatonin in children with autism spectrum disorder after ß-1,3/1,6-glucan consumption: An open-label prospective pilot clinical study.

INTRODUCTION: Poor sleep quality is a major problem in patients with autism spectrum disorder (ASD), and is attributed to low melatonin levels. Melatonin supplementation is recommended; however, its effectiveness varies. ß-Glucans have previously been shown to improve melatonin levels in animal studies. Herein, we examined the effectiveness of Aureobasidium pullulans (Nichi Glucan), a species of black yeast that contains beta-1,3/1,6-glucan, in a pilot study of children with ASD. METHODS: Thirteen children (age, 2.5-13 years) with ASD were recruited for the study. The control group consisted of four patients (Gr. 1), while nine patients were classified into the treatment group (Gr. 2). Gr. 2 received 1 g of Nichi Glucan along with conventional therapy, whereas the Gr. 1 (control) patients received conventional therapy alone for 90 days. Serum melatonin levels and sleep patterns, assessed using a subjective questionnaire, were evaluated before and after treatment. RESULTS: In Gr. 2, the average serum melatonin level increased from 238.85 ng/L preintervention to 394.72 ng/L postintervention. Eight of nine participants (88%) in Gr. 2 showed improvements in sleep pattern and quality, while no improvement was observed in the participants in Gr. 1. CONCLUSION: The consumption of Nichi Glucan for 90 days resulted in visible improvement in sleep quality, sleep pattern, and serum melatonin levels, which was reported for the first time by our study. A larger multicenter study is required to validate our findings.

Beneficial Immune Regulation by Biological Response Modifier Glucans in COVID-19 and Their Envisaged Potentials in the Management of Sepsis.

Sepsis is a life-threatening condition caused by an abnormal immune response induced by infection with no approved or specific therapeutic options. We present our perspectives for the therapeutic management of sepsis through a four-way approach: (1) infection control through immune enhancement; (2) immune suppression during the initial hyper-inflammatory phase; (3) balanced immune-modulation to counter the later immune-paralysis phase; and (4) advantageous effects on metabolic and coagulation parameters throughout. COVID-19 is a virus-triggered, accelerated sepsis-like reaction that is associated with the rapid progress of an inflammatory cascade involving a cytokine storm and multiorgan failure. Here, we discuss the potential of the biological response modifiers, ß-glucans (BRMGs), in the management of sepsis based on their beneficial effects on inflammatory-immune events in COVID-19 clinical studies. In COVID-19 patients, apart from metabolic regulation, BRMGs, derived from a black yeast, Aureobasidium pullulans strain AFO-202, have been reported to stimulate immune responses. BRMGs, produced by another strain (N-163) of A. pullulans, have been implicated in the beneficial regulation of inflammatory markers and immunity, namely IL-6, C-reactive protein (CRP), D-Dimer, ferritin, neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-C-reactive protein ratio (LCR), leucocyte-to-C-reactive protein ratio (LeCR), and leukocyte-to-IL-6 ratio (LeIR). Agents such as these ß-glucans, which are safe as they have been widely consumed by humans for decades, have potential as adjuncts for the prevention and management of sepsis as they exert their beneficial effects across the spectrum of processes and factors involved in sepsis pathology, including, but not limited to, metabolism, infection, inflammation, immune modulation, immune enhancement, and gut microbiota.

An efficient polymer cocktail-based transportation method for cartilage tissue, yielding chondrocytes with enhanced hyaline cartilage expression during in vitro culturing.

BACKGROUND: Chondrocytes are used in cell-based therapies such as autologous chondrocyte implantation (ACI) and matrix-associated cartilage implantation (MACI). To transport the cartilage tissue to the laboratory for in vitro culturing, phosphate-buffered saline (PBS), Euro-Collins solution (ECS) and Dulbecco's Modified Eagle's Medium (DMEM) are commonly employed at 4-8 °C. METHODS: In this study, eight samples of human cartilage biopsy tissues from elderly patients with severe osteoarthritis undergoing arthroscopy, which would otherwise have been discarded, were used. The cartilage tissue samples were compared to assess the cell yield between two transportation groups: i) a thermo-reversible gelation polymer (TGP) based method without cool preservation (∼25 °C) and ii) ECS transport at 4 °C. These samples were subjected to in vitro culture in a two-dimensional (2D) monolayer for two weeks and subsequently in a three-dimensional (3D) TGP scaffold for six weeks. RESULTS: The cell count obtained from the tissues transported in TGP was higher (0.2 million cells) than those transported in ECS (0.08 million cells) both after initial processing and after in vitro culturing for 2 weeks in 2D (18 million cells compared with 10 million cells). In addition, mRNA quantification demonstrated significantly higher expression of Col2a1 and SOX-9 in 3D-TGP cultured cells and lower expression of COL1a1 in RT-PCR, characteristic of the hyaline cartilage phenotype, than in 2D culture. CONCLUSION: This study confirms that the TGP cocktail is suitable for both the transport of human cartilage tissue and for in vitro culturing to yield better-quality cells for use in regenerative therapies.

Improvement of behavioural pattern and alpha-synuclein levels in autism spectrum disorder after consumption of a beta-glucan food supplement in a randomised, parallel-group pilot clinical study.

BACKGROUND: Autism spectrum disorders (ASDs) are a wide range of behavioural disabilities for which there are no definite interventional modalities available. Remedial therapies remain the only option but with varying outcomes. We have evaluated the Childhood Autism Rating Scale (CARS) and alpha-synuclein levels in this parallel-group, multiple-arm pilot clinical study after supplementation with a biological response modifier beta-glucan food supplement (Nichi Glucan). METHODS: Six subjects with ASD (n=6) Gr. 1 underwent conventional treatment comprising remedial behavioural therapies and L-carnosine 500 mg per day, and 12 subjects (n=12) Gr. 2 underwent supplementation with the Nichi Glucan 0.5 g two times per day along with the conventional treatment. RESULTS: There was a significant decrease in the CARS score in all of the children of the Nichi Glucan Gr.2 compared with the control (p=0.034517). Plasma levels of alpha-synuclein were significantly higher in Gr. 2 (Nichi Glucan) than in the control group Gr. 1 (p=0.091701). CONCLUSION: Improvement of the behavioural pattern CARS score and a correlating alpha-synuclein level, followed by a safe beta-glucan food supplement, warrants further research on other parameters, such as gut-microbiota evaluation, and relevant neuronal biomarkers which is likely to cast light on novel solutions.

ß­glucan vaccine adjuvant approach for cancer treatment through immune enhancement (B­VACCIEN) in specific immunocompromised populations (Review).

The incidence of cancer, which is the second leading cause of mortality globally, continues to increase, although continued efforts are being made to identify effective treatments with fewer side­effects. Previous studies have reported that chronic microinflammation, which occurs in diseases, including diabetes, along with weakened immune systems, may ultimately lead to cancer development. Chemotherapy, radiotherapy and surgery are the mainstream approaches to treatment; however, they all lead to immune system weakness, which in turn increases the metastatic spread. The aim of the present review was to provide evidence of a biological response modifier ß­glucan [ß­glucan vaccine adjuvant approach to treating cancer via immune enhancement (B­VACCIEN)] and its beneficial effects, including vaccine­adjuvant potential, balancing metabolic parameters (including blood glucose and lipid levels), increasing peripheral blood cell cytotoxicity against cancer and alleviating chemotherapy side effects in animal models. This suggests its value as a potential strategy to provide long­term prophylaxis in immunocompromised individuals or genetically prone to cancer.

Beneficial effects of novel aureobasidium pullulans strains produced beta-1,3-1,6 glucans on interleukin-6 and D-dimer levels in COVID-19 patients; results of a randomized multiple-arm pilot clinical study.

OBJECTIVE: In this pilot clinical study, we report the beneficial effects of beta glucans derived from two strains AFO-202 and N-163 of a black yeast Aureobasidium pullulans on the biomarkers for cytokine storm and coagulopathy in COVID-19 patients. METHODS: A total of 24 RT-PCR positive COVID-19 patients were recruited and randomly divided into three groups (Gr): Gr. 1 control (n = 8) - Standard treatment; Gr. 2: Standard treatment + AFO-202 beta glucan (n = 8); and Gr. 3, Standard treatment + combination of AFO-202 and N-163 beta glucans (n = 8) for 30 days. RESULTS: There was no mortality or requirement of ventilation of the subjects in any of the groups. There was a decrease in D-Dimer values (751 ng/ml to 143.89 ng/ml) and IL-6 values (7.395-3.16 pg/ml) in Gr. 1 in 15 days but the levels increased to abnormal levels on day 30 (D-Dimer: 202.5 ng/ml; IL-6 55.37 pg/ml); which steadily decreased up to day 30 in groups 2 (D-dimer: 560.99 ng/dl to 79.615; IL-6: 26.18-3.41 pg/ml) and 3 (D-dimer: 1614 ng/dl to 164.25 ng/dl; IL-6: 6.25-0.5 pg/ml). The same trend was observed with ESR. LCR and LeCR increased while NLR decreased significantly in Gr. 3. CD4 + and CD8 + T cell count showed relatively higher increase in Gr.3. There was no difference in CRP within the groups. CONCLUSION: As these beta glucans are well known food supplements with a track record for safety, larger multi-centric clinical studies are recommended to validate their use as an adjunct in the management of COVID-19 and the ensuing long COVID-19 syndrome.

In Vitro Culture Expansion and Characterization of Buccal Mucosal Epithelial Cells for Tissue Engineering Applications in Urethral Stricture After Transportation Using a Thermoreversible Gelation Polymer.

Introduction: The transportation of tissues from hospitals to clinical laboratories for cell therapy is an essential component of regenerative medicine. Previously, we used laboratory-cultured mucosal cells from buccal epithelium expanded and encapsulated using a scaffold-hybrid approach to the urethral stricture (BEES-HAUS) procedure. In this study, to improve the outcomes, we compared the thermoreversible gelation polymer (TGP) transportation procedure with conventional culture methods, and reported its advantages. Methods: Human buccal mucosal tissues in Phase I of the study were transported in Euro-Collins solution (ECS) and the cells obtained were cultured in two-dimensional (2D) Dulbecco's modified Eagle's medium (DMEM), CnT-Prime epithelial 2D differentiation medium (CnT-PR), and a three-dimensional (3D)-TGP scaffold. In Phase II, tissues were transported in a TGP cocktail and the ECS. The cells were cultured in 2D-DMEM and 3D-TGP, quantified, and characterized by immunohistochemistry. Results: The cells in 3D-TGP culture maintained epithelial morphology in a better manner compared with 2D-DMEM, in which they developed fibroblast-like morphology. The TGP-transported cells grew rapidly. Immunohistochemical analysis results for AE1/AE3, EGFR, integrin-ß1, p63, and p75 were intensely positive in 3D-TGP. Conclusion: The TGP-based cocktail used in human buccal tissue transportation yielded cells with better morphology maintenance. The TGP scaffold provides an optimal in vitro environment wherein epithelial cells better maintain their native phenotype compared to those cultured through conventional methods. These results suggest using TGP for the transportation and culture of human buccal tissues for clinical applications. In addition, the use of a TGP-based cocktail for the transport of other tissues for regenerative medicine applications is worth further analysis.

Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold.

Regenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-ßgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-ßgal and started degenerating at 25 days. SA-ßgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42-45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

Successful engraftment of epithelial cells derived from autologous rabbit buccal mucosal tissue, encapsulated in a polymer scaffold in a rabbit model of a urethral stricture, transplanted using the transurethral approach.

BACKGROUND: A pilot study reported an autologous buccal mucosal cell transplant in humans through the trans-urethral route using the buccal epithelium expanded and encapsulated in scaffold-hybrid approach to urethral stricture (BEES-HAUS), a minimally invasive approach to treat urethral stricture. Although successful outcomes were achieved in that study, for further validation, it is essential to prove that the transplanted buccal epithelium was engrafted over the urothelium through histological examination of the urethra, harvested post-transplant, which is infeasible in humans. Herein, we report the successful creation of an animal model of urethral stricture and the engraftment of epithelial cells derived from autologous buccal mucosal tissue, encapsulated in a thermo-reversible gelation polymer (TGP) scaffold, transplanted by trans-urethral route. METHODS: An animal model of urethral stricture was created in Japanese white male rabbits using electro-coagulation. Buccal tissue was harvested from the rabbits and subjected to enzyme digestion, followed by 5-7 days of in vitro culture in conventional two-dimensional (2D) culture and in a 3D platform of thermo-reversible gelation polymer (3D-TGP) culture. The cells harvested from the groups were mixed and encapsulated and transplanted with TGP, by transurethral catheterization. Fourteen days later, the urethra was harvested and subjected to histological examination. The buccal biopsy tissue, cells after digestion and cells post-culture were also subjected to histological examination. Urethrogram and endoscopy images were recorded at different time points. RESULTS: The stricture was successfully created, with the coagulated area markedly stenosed. Histological staining of the cells after in vitro processing showed that the cells grew with native epithelial and rounded cell morphology in 3D-TGP while they differentiated into fibroblast like-cells in 2D culture. Histological staining of the urethral tissue after transplantation revealed the engraftment of the transplanted buccal mucosal cells, with stratified squamous epithelium over the specialized stratified urothelium in the urethrotomy site. CONCLUSION: We used histology to prove the successful engraftment of TGP-encapsulated buccal mucosal epithelial cells in an animal model of urethral injury with healing of the injured tissue. The model of urethral stricture and cell therapy, using a transurethral approach, recapitulates the previously reported BEES-HAUS approach and lays the foundation for larger multi-centric translational clinical studies.