Morphogenetic Designs, and Disease Models in Central Nervous System Organoids.

Since the emergence of the first cerebral organoid (CO) in 2013, advancements have transformed central nervous system (CNS) research. Initial efforts focused on studying the morphogenesis of COs and creating reproducible models. Numerous methodologies have been proposed, enabling the design of the brain organoid to represent specific regions and spinal cord structures. CNS organoids now facilitate the study of a wide range of CNS diseases, from infections to tumors, which were previously difficult to investigate. We summarize the major advancements in CNS organoids, concerning morphogenetic designs and disease models. We examine the development of fabrication procedures and how these advancements have enabled the generation of region-specific brain organoids and spinal cord models. We highlight the application of these organoids in studying various CNS diseases, demonstrating the versatility and potential of organoid models in advancing our understanding of complex conditions. We discuss the current challenges in the field, including issues related to reproducibility, scalability, and the accurate recapitulation of the in vivo environment. We provide an outlook on prospective studies and future directions. This review aims to provide a comprehensive overview of the state-of-the-art CNS organoid research, highlighting key developments, current challenges, and prospects in the field.

Non-clinical, quality and environmental impact assessments of cell and gene therapy products: Report on the 5th Asia Partnership Conference of Regenerative Medicine - April 7, 2022.

The 5th Asia Partnership Conference of Regenerative Medicine (APACRM) was held online on April 7, 2022 to promote regulatory harmonization of regenerative medicine products throughout Asia. The recognition of domestic regulatory guidelines within each country and region and the underpinning rationales are important initial steps toward the harmonization of regulations. The 5th APACRM featured open dialog regarding non-clinical, quality and environmental impact assessment settings for cell and gene therapy products through presentations from the industry and panel discussions with regulatory agencies. The latest updates on regenerative medicine fields in each country and region were also introduced. This paper summarizes the proceedings of the 5th APACRM for public dissemination to foster future discussion.

An Injectable Engineered Cartilage Gel Improves Intervertebral Disc Repair in a Rat Nucleotomy Model.

Lower back pain is a major problem caused by intervertebral disc degeneration. A common surgical procedure is lumbar partial discectomy (excision of the herniated disc causing nerve root compression), which results in further disc degeneration, severe lower back pain, and disability after discectomy. Thus, the development of disc regenerative therapies for patients who require lumbar partial discectomy is crucial. Here, we investigated the effectiveness of an engineered cartilage gel utilizing human fetal cartilage-derived progenitor cells (hFCPCs) on intervertebral disc repair in a rat tail nucleotomy model. Eight-week-old female Sprague-Dawley rats were randomized into three groups to undergo intradiscal injection of (1) cartilage gel, (2) hFCPCs, or (3) decellularized extracellular matrix (ECM) (n = 10/each group). The treatment materials were injected immediately after nucleotomy of the coccygeal discs. The coccygeal discs were removed six weeks after implantation for radiologic and histological analysis. Implantation of the cartilage gel promoted degenerative disc repair compared to hFCPCs or hFCPC-derived ECM by increasing the cellularity and matrix integrity, promoting reconstruction of nucleus pulposus, restoring disc hydration, and downregulating inflammatory cytokines and pain. Our results demonstrate that cartilage gel has higher therapeutic potential than its cellular or ECM component alone, and support further translation to large animal models and human subjects.

Nonclinical and quality assessment of cell therapy products: Report on the 4th Asia Partnership Conference of Regenerative Medicine, April 15, 2021.

The 4th Asia Partnership Conference of Regenerative Medicine (APACRM) was held online on April 15, 2021, to promote regulatory harmonization of regenerative medicine products throughout Asia. Recognizing domestic regulatory guidelines within each country and region, and their underpinning rationales, is an important initial step toward a convergence of regulations. The 4th APACRM consisted of an open dialog with regulatory agencies regarding nonclinical and quality settings for cell therapy products (CTPs) through industry presentations and panel discussions with regulatory agencies. The latest updates on regenerative medicine fields in each country and region, and specific regulatory schematics in Japan, were also introduced. The objective of this paper is to summarize the proceedings of the 4th APACRM for public dissemination and to foster further discussion in the future.

Suppression of Osteoarthritis progression by post-natal Induction of Nkx3.2.

Osteoarthritis (OA) is an incurable joint disease affecting 240 million elderly population, and major unmet medical needs exist for better therapeutic options for OA. During skeletal development, Nkx3.2 has been shown to promote chondrocyte differentiation and survival, but to suppress cartilage hypertrophy and blood vessel invasion. Here we show that Nkx3.2 plays a key role in osteoarthritis (OA) pathogenesis. Marked reduction of Nkx3.2 expression was observed in three different murine OA models. Consistent with these findings, analyses of surgery-induced and age-driven OA models revealed that cartilage-specific post-natal induction of Nkx3.2 can suppress OA progression in mice. These results suggest that Nkx3.2 may serve as a promising target for OA drug development.

Effects of Human Placenta Extract (Laennec) on Ligament Healing in a Rodent Model.

Rich in bioactive substances such as amino acids and peptides, Laennec (human placenta hydrolysate) has been widely used to control various types of musculoskeletal pain. However, the effects of Laennec on tendon and ligament injuries are not clearly understood. In the present study, Laennec was tested to identify its in vivo effects on ligament injury in an animal model and its in vitro effects on tendon-derived fibrocytes. A total of 99 Sprague Dawley rats were divided into the negative control (normal) group (n = 11) and the ligament injury group (n = 88). The ligament injury group was subdivided into normal saline-treated group, Laennec-treated group, polydeoxyribonucleotide-treated group, and 20% dextrose-treated group. Ligaments were collected at 1 week and 4 weeks after treatment. Histologic and biomechanical properties were analyzed. In vitro effects of Laennec and polydeoxyribonucleotide on fibrocytes were also analyzed. Although all other treatment groups showed increased inflammatory cells, the Laennec-treated group maintained cell counts and activated macrophage levels that were similar to the normal group. Unlike the saline-treated group and dextrose-treated group, the Laennec-treated group had low levels of degenerative changes at 4 weeks after treatment. Supportively, in vitro results showed that the Laennec-treated group had increased collagen type I, scleraxis (Scx) and tenomodulin (Tnmd) expression (p < 0.05). Our study demonstrates that Laennec treatment enhances wound healing of damaged ligament by suppressing immune responses and reducing degenerative changes of damaged ligament. In addition, we found that Laennec induces the gene expression of type I collagen, Scx and Tnmd in fibrocytes, suggesting that Laennec may facilitate regeneration of damaged ligaments. Therefore, we expect that Laennec can be a useful drug to treat injured ligament.

Sizable Scaffold-Free Tissue-Engineered Articular Cartilage Construct for Cartilage Defect Repair.

This study introduces an implantable scaffold-free cartilage tissue construct (SF) that is composed of chondrocytes and their self-produced extracellular matrix (ECM). Chondrocytes were grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 21, and 35 days). For in vivo implantation, full-thickness defects (n = 5) were manually created on the trochlear groove of the both knees of rabbits (16-week old) and 3 week-cultured SF construct was implanted as an allograft for a month. The left knee defects were implanted with 1, 7, and 21 days in vitro cultured scaffold-free engineered cartilages. (group 2, 3, and 4, respectively). The maturity of the engineered cartilages was evaluated by histological, chemical and mechanical assays. The repair of damaged cartilages was also evaluated by gross images and histological observations at 4, 8, and 12 weeks postsurgery. Although defect of groups 1, 2, and 3 were repaired with fibrocartilage tissues, group 4 (21 days) showed hyaline cartilage in the histological observation. In particular, mature matrix and columnar organization of chondrocytes and highly expressed type II collagen were observed only in 21 days in vitro cultured SF cartilage (group 4) at 12 weeks. As a conclusion, cartilage repair with maturation was recapitulated when implanted the 21 day in vitro cultured scaffold-free engineered cartilage. When implanting tissue-engineered cartilage, the maturity of the cartilage tissue along with the cultivation period can affect the cartilage repair.

The effect of anti-TNF treatment on osteoblastogenesis in ankylosing spondylitis: the number of circulating osteoblast-lineage cells in peripheral blood decreased after infliximab therapy in patients with ankylosing spondylitis.

OBJECTIVES: The full effect of anti-TNF therapy on new bone formation is still in debate in spondylitis fields. We sought to obtain circulating osteoblast-lineage cells in peripheral blood from ankylosing spondylitis (AS) patients and healthy control subjects, and to evaluate the effect of before and after anti TNF-α therapy on osteoblastogenesis in patients with AS. METHODS: Sixteen male patients with AS slated for infliximab therapy and 19 controls were recruited. We cultured osteoblast-lineage cells from peripheral blood and measured the optical density of their Alizarin red S staining. We also measured serum P1NP (procollagen type 1 N-terminal propeptide) as an early osteoblast differentiation marker, osteocalcin as a late osteoblast differentiation marker, and inflammatory markers. RESULTS: There were significantly more circulating osteoblast-lineage cells in patients than in controls. The number of circulating osteoblast-lineage cells and optical density of Alizarin red S staining decreased 14 weeks after infliximab therapy (p=0.028); serum level of P1NP decreased, but that of osteocalcin increased (p=0.002 and 0.007, respectively). CONCLUSIONS: Our data reveals that first, the circulating osteoblast-lineage cells are recoverable and increased in AS patients, and also that they decrease after infliximab therapy; second, infliximab therapy resolves early inflammation, but allows mature osteoblast differentiation in late inflammation. The culture of osteoblast-lineage cells in peripheral blood may be a candidate for a new modality with which to study spondylitis and other autoimmune diseases.

Focal Adhesion Assembly Induces Phenotypic Changes and Dedifferentiation in Chondrocytes.

The expansion of autologous chondrocytes in vitro is used to generate sufficient populations for cell-based therapies. However, during monolayer culture, chondrocytes lose inherent characteristics and shift to fibroblast-like cells as passage number increase. Here, we investigated passage-dependent changes in cellular physiology, including cellular morphology, motility, and gene and protein expression, as well as the role of focal adhesion and cytoskeletal regulation in the dedifferentiation process. We found that the gene and protein expression levels of both the focal adhesion complex and small Rho GTPases are upregulated with increasing passage number and are closely linked to chondrocyte dedifferentiation. The inhibition of focal adhesion kinase (FAK) but not small Rho GTPases induced the loss of fibroblastic traits and the recovery of collagen type II, aggrecan, and SOX9 expression levels in dedifferentiated chondrocytes. Based on these findings, we propose a strategy to suppress chondrogenic dedifferentiation by inhibiting the identified FAK or Src pathways while maintaining the expansion capability of chondrocytes in a 2D environment. These results highlight a potential therapeutic target for the treatment of skeletal diseases and the generation of cartilage in tissue-engineering approaches. J. Cell. Physiol. 231: 1822-1831, 2016. © 2015 Wiley Periodicals, Inc.

Comparison of fetal cartilage-derived progenitor cells isolated at different developmental stages in a rat model.

Fetal cartilage-derived progenitor cells (FCPCs) could be a useful cell source in cell-based therapies for cartilage disorders. However, their characteristics can vary depending on the developmental stages. The aim of this study was to compare the characteristics of rat FCPCs from the hind limb on embryonic day 14 (E14), E16 and E20 regarding proliferation, pluripotency, and differentiation. Morphologically, rat fetal cartilage tissue showed an increase in cartilaginous differentiation features (Safranin-O, type II collagen) and decrease in pluripotency marker (Sox2) in the order of E14, E16 and E20. E14 FCPCs showed significantly higher doubling time compared to E16 and E20 FCPCs. While the E14 FCPCs expressed pluripotent genes (Sox2, Oct4, Nanog), the E16 and E20 FCPCs expressed chondrogenic markers (Sox9, Col2a1, Acan). E20 FCPCs showed the highest ability to both chondrogenic and adipogenic differentiation and E14 FCPCs showed relatively better activity in osteogenic differentiation. Further analysis showed that E20 FCPCs expressed both adipogenic (C/ebpß) and osteogenic (Runx2, Sp7, Taz) transcription factors as well as chondrogenic transcription factors. Our results show an inverse relationship overall between the expression of pluripotency genes and that of chondrogenic and lineage-specific genes in FCPCs under development. Due to its exceptional proliferation and chondrogenic differentiation ability, fetal cells from epiphyseal cartilage (E20 in rats) may be a suitable cell source for cartilage regeneration.

Low intensity ultrasound inhibits brain oedema formation in rats: potential action on AQP4 membrane localization.

AIMS: Brain oedema is a major contributing factor to the morbidity and mortality of a variety of brain disorders. Although there has been considerable progress in our understanding of pathophysiological and molecular mechanisms associated with brain oedema so far, more effective treatment is required and is still awaited. Here we intended to study the effects of low intensity ultrasound (LIUS) on brain oedema. METHODS: We prepared the rat hippocampal slice in vitro and acute water intoxication in vivo models of brain oedema. We applied LIUS stimulation in these models and studied the molecular mechanisms of LIUS action on brain oedema. RESULTS: We found that LIUS stimulation markedly inhibited the oedema formation in both of these models. LIUS stimulation significantly reduced brain water content and intracranial pressure resulting in increased survival of the rats. Here, we showed that the AQP4 localization was increased in the astrocytic foot processes in the oedematous hippocampal slices, while it was significantly reduced in the LIUS-stimulated hippocampal slices. In the in vivo model too, AQP4 expression was markedly increased in the microvessels of the cerebral cortex and hippocampus after water intoxication but was reduced in the LIUS-stimulated rats. CONCLUSIONS: These data show that LIUS has an inhibitory effect on cytotoxic brain oedema and suggest its therapeutic potential to treat brain oedema. We propose that LIUS reduces the AQP4 localization around the astrocytic foot processes thereby decreasing water permeability into the brain tissue.

The effect of dry needling and treadmill running on inducing pathological changes in rat Achilles tendon.

Achilles tendinopathy is a common degenerative condition without a definitive treatment. An adequate chronic animal model of Achilles tendinopathy has not yet been developed. The purpose of this study was to evaluate the individual and combined effects of dry needling and treadmill running on the Achilles tendon of rats. Percutaneous dry needling, designed to physically replicate microrupture of collagen fibers in overloaded tendons, was performed on the right Achilles tendon of 80 Sprague-Dawley rats. The rats were randomly divided into two groups: a treadmill group, which included rats that underwent daily uphill treadmill running (n = 40), and a cage group, which included rats that could move freely within their cages (n = 40). At the end of weeks 1 and 4, 20 rats from each group were sacrificed, and bilateral Achilles tendons were collected. The harvested tendons were subjected to mechanical testing and histological analysis. Dry needling induced histological and mechanical changes in the Achilles tendons at week 1, and the changes persisted at week 4. The needled Achilles tendons of the treadmill group tended to show more severe histological and mechanical changes than those of the cage group, although these differences were not statistically significant. Dry needling combined with free cage activity or treadmill running produced tendinopathy-like changes in rat Achilles tendons up to 4 weeks after injury. Dry needling is an easy procedure with a short induction period and a high success rate, suggesting it may have relevance in the design of an Achilles tendinopathy model.

Effects of low-intensity ultrasound on gramicidin D-induced erythrocyte edema.

OBJECTIVES: To determine whether low-intensity ultrasound (US) can reduce red blood cell (RBC) edema and, if so, whether the US activity is associated with aquaporin 1 (AQP-1), a water channel in the cell membrane. METHODS: Red blood cell edema was induced by gramicidin D treatment at 40 ng/mL for 20 minutes and evaluated by a hematocrit assay. Low-intensity continuous wave US at 1 MHz was applied to RBCs for the last 10 minutes of gramicidin D treatment. To determine whether US activity was associated with AQP-1, RBCs were treated with 40 µM mercuric chloride (HgCl(2)), an AQP-1 inhibitor, for 20 minutes at the time of gramicidin D treatment. Posttreatment morphologic changes in RBCs were observed by actin staining with phalloidin. RESULTS: Red blood cell edema increased significantly with gramicidin D at 20 (1.8%), 40 (6.7%), 60 (16.7%), and 80 (11.3%) ng/mL, reaching a peak at 60 ng/mL, compared to the control group (20 ng/mL, P = .019; 40, 60, and 80 ng/mL, P < .001). No significant RBC hemolysis was observed in any group. Edema induced by gramicidin D at 40 ng/mL was significantly reduced by US at 30 (3.4%; P = .003), 70 (4.4%; P = .001), and 100 (2.9%; P = .001) mW/cm(2). Subsequent experiments showed that edema reduction by US ranged from 7% to 10%. Cotreatment with HgCl(2) partially reversed the US effect and showed a significantly different level of edema compared to gramicidin D-alone and US-cotreated groups (P = .001). These results were confirmed by microscopic observation of RBC morphologic changes. CONCLUSIONS: Low-intensity US could reduce gramicidin D-induced RBC edema, and its effect appeared to at least partly involve regulation of AQP-1 activity. These results suggest that low-intensity US can be used as an alternative treatment to control edema and related disorders.

mTOR Plays an Important Role in the Stemness of Human Fetal Cartilage Progenitor Cells (hFCPCs).

BACKGROUND: Mammalian target of rapamycin (mTOR) is known to regulate self-renewal ability and potency of embryonic stem cells (ESCs) and adult stem cells in opposite manners. However, its effects vary even among adult stem cells and are not reported in fetal stem/progenitor cells. This study investigated the role of mTOR in the function of human fetal cartilage-derived progenitor cells (hFCPCs). METHODS: mTOR activity in hFCPCs was first examined via the level of phosphor-mTOR until passage 19, together with doubling time of cells and senescence-associated b-galactosidase (SA-bGal). Then, the effect of 100 nM rapamycin, the inhibitor of mTOR, was investigated on self-renewal ability, proliferation rate and osteogenic/adipogenic potential of hFCPCs in vitro. Expression of stemness genes (Oct-4, Sox2 and Nanog) and cell cycle regulators (CDK4 and Cyclin D1) was measured at mRNA or protein levels. RESULTS: mTOR activity was maintained constantly at high levels in hFCPCs until passage 19, while their proliferation rate was decreasing from 48 h at passage 13 to 70 h at passage 9 and senescent cells were observed at passage 18 (8.3 ± 1.2%) and 19 (15.6 ± 1.9%). Inhibition of mTOR in hFCPCs impaired their colony forming frequency (CFU-F) by 4 folds, while showing no change in their doubling time and expression of CDK4 and Cyclin D1. Upon mTOR inhibition, Oct4 expression decreased by 2 folds and 4 folds at the mRNA and protein levels, respectively, while that of Sox2 and Nanog did not change significantly. Finally, mTOR inhibition reduced osteogenic and adipogenic differentiation of hFCPCs in vitro. CONCLUSION: This study has shown that mTOR plays an important role in the self-renewal ability of hFCPCS but not in their proliferation, The effect of mTOR appears to be associated with Oct-4 expression and important in the osteogenic and adipogenic differentiation ability of hFCPCs.

Mechanical stimulation by ultrasound enhances chondrogenic differentiation of mesenchymal stem cells in a fibrin-hyaluronic acid hydrogel.

Chondrogenic differentiation and cartilage tissue formation derived from stem cells are highly dependent on both biological and mechanical factors. This study investigated whether or not fibrin-hyaluronic acid (HA) coupled with low-intensity ultrasound (LIUS), a mechanical stimulation, produces an additive or synergistic effect on the chondrogenesis of rabbit mesenchymal stem cells (MSCs) derived from bone marrow. For the purpose of comparison, rabbit MSCs were first cultured in fibrin-HA or alginate hydrogels, and then subjected to chondrogenic differentiation in chondrogenic-defined medium for 4 weeks in the presence of either transforming growth factor-beta3 (TGF-ß3) (10 ng/mL) or LIUS treatment (1.0 MHz and 200 mW/cm(2) ). The resulting samples were evaluated at 1 and 4 weeks by histological observation, chemical assays, and mechanical analysis. The fibrin-HA hydrogel was found to be more efficient than alginate in promoting chondrogenesis of the MSCs by producing a larger amount of sulfated glycosaminoglycans (GAGs) and collagen, and engineered constructs made with the hydrogel demonstrated higher mechanical strength. At 4 weeks of tissue culture, the chondrogenesis of the MSCs in fibrin-HA were shown to be further enhanced by treatment with LIUS, as observed by analyses for the amounts of GAGs and collagen, and mechanical strength testing. In contrast, TGF-ß3, a well-known chondrogenic inducer, showed a marginal additive effect in the amount of collagen only. These results revealed that LIUS further enhanced chondrogenesis of the MSCs cultured in fibrin-HA, in vitro, and suggested that the combination of fibrin-HA and LIUS is a useful tool in constructing high-quality cartilage tissues from MSCs.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) shows therapeutic effect on dimethylnitrosamine (DMN)-induced liver fibrosis in rats.

This study was undertaken to investigate the inhibitory effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Liver fibrosis was induced in Sprague-Dawley rats by injecting DMN intraperitoneally (at 10 mg/kg of body weight) daily for three consecutive days per week for 4 weeks. To investigate the effect of GM-CSF on disease onset, GM-CSF (50 µg/kg of body weight) was co-treated with DMN for 2 consecutive days per week for 4 weeks (4-week groups). To observe the effect of GM-CSF on the progression of liver fibrosis, GM-CSF was post-treated alone at 5-8 weeks after the 4 weeks of DMN injection (8-week groups). We found that DMN administration for 4 weeks produced molecular and pathological manifestations of liver fibrosis, that is, it increased the expressions of collagen type I, alpha-smooth muscle actin (α-SMA), and transforming growth factor-ß1 (TGF-ß1), and decreased peroxisome proliferator-activated receptor gamma (PPAR-γ) expression. In addition, elevated serum levels of aspartate aminotransferase (AST), total bilirubin level (TBIL), and decreased albumin level (ALB) were observed. In both the 4-week and 8-week groups, GM-CSF clearly improved the pathological liver conditions in the gross and histological observations, and significantly recovered DMN-induced increases in AST and TBIL and decreases in ALB serum levels to normal. GM-CSF also significantly decreased DMN-induced increases in collagen type I, α-SMA, and TGF-ß1 and increased DMN-induced decreases in PPAR-γ expression. In the DMN groups, survival decreased continuously for 8 weeks after DMN treatment for the first 4 weeks. GM-CSF showed a survival benefit when co-treated for the first 4 weeks but a marginal effect when post-treated for 5-8 weeks. In conclusion, co-treatment of GM-CSF showed therapeutic effects on DMN-induced liver fibrosis and survival rates in rats, while post-treatment efficiently blocked liver fibrosis.

Effects of Induction Culture on Osteogenesis of Scaffold-Free Engineered Tissue for Bone Regeneration Applications.

BACKGROUND: Restoration of the bone defects caused by infection or disease remains a challenge in orthopedic surgery. In recent studies, scaffold-free engineered tissue with a self-secreted extracellular matrix has been proposed as an alternative strategy for tissue regeneration and reconstruction. Our study aimed to engineer and fabricate self-assembled osteogenic and scaffold-free tissue for bone regeneration. METHODS: Osteogenic scaffold-free tissue was engineered and fabricated using fetal cartilage-derived progenitor cells, which are capable of osteogenic differentiation. They were cultured in osteogenic induction environments or using demineralized bone powder for differentiation. The fabricated tissue was subjected to real-time qPCR, biochemical, and histological analyses to estimate the degree of in vitro osteogenic differentiation. To demonstrate bone formation in an in vivo environment, scaffold-free tissue was transplanted into the dorsal subcutaneous site of nude mice. Bone development was monitored postoperatively over 8 weeks by the observation of calcium deposition in the matrix. RESULTS: In the in vitro experiments, engineered osteogenically induced scaffold-free tissue demonstrated three-dimensional morphological characteristics, and sufficient osteogenic differentiation was confirmed through the quantification of specific osteogenic gene markers expressed and calcium accumulation within the matrix. Following the evaluation of differentiation efficacy, in vivo experiments revealed distinct bone formation, and that blood vessels had penetrated the fabricated tissue. CONCLUSION: The novel engineering of scaffold-free tissue with osteogenic potential can be used as an optimal bone graft substitute for bone regeneration.

Human Fetal Cartilage-Derived Progenitor Cells Exhibit Anti-Inflammatory Effect on IL-1ß-Mediated Osteoarthritis Phenotypes In Vitro.

BACKGROUND: In this study, we have investigated whether human fetal cartilage progenitor cells (hFCPCs) have anti-inflammatory activity and can alleviate osteoarthritis (OA) phenotypes in vitro. METHODS: hFCPCs were stimulated with various cytokines and their combinations and expression of paracrine factors was examined to find an optimal priming factor. Human chondrocytes or SW982 synoviocytes were treated with interleukin-1ß (IL-1ß) to produce OA phenotype, and co-cultured with polyinosinic-polycytidylic acid (poly(I-C))-primed hFCPCs to address their anti-inflammatory effect by measuring the expression of OA-related genes. The effect of poly(I-C) on the surface marker expression and differentiation of hFCPCs into 3 mesodermal lineages was also examined. RESULTS: Among the priming factors tested, poly(I-C) (1 µg/mL) most significantly induced the expression of paracrine factors such as indoleamine 2,3-dioxygenase, histocompatibility antigen, class I, G, tumor necrosis factor- stimulated gene-6, leukemia inhibitory factor, transforming growth factor-ß1 and hepatocyte growth factor from hFCPCs. In the OA model in vitro, co-treatment of poly(I-C)-primed hFCPCs significantly alleviated IL-1ß-induced expression of inflammatory factors such as IL-6, monocyte chemoattractant protein-1 and IL-1ß, and matrix metalloproteinases in SW982, while it increased the expression of cartilage extracellular matrix such as aggrecan and collagen type II in human chondrocytes. We also found that treatment of poly(I-C) did not cause significant changes in the surface marker profile of hFCPCs, while showed some changes in the 3 lineages differentiation. CONCLUSION: These results suggest that poly(I-C)-primed hFCPCs have an ability to modulate inflammatory response and OA phenotypes in vitro and encourage further studies to apply them in animal models of OA in the future.

Anti-adhesive effect of chondrocyte-derived extracellular matrix surface-modified with poly-L-lysine.

After an injury, soft tissue structures in the body undergo a natural healing process through specific phases of healing. Adhesions occur as abnormal attachments between tissues and organs through the formation of blood vessels and/or fibrinous adhesions during the regenerative repair process. In this study, we developed an adhesion-preventing membrane with an improved physical protection function by modifying the surface of chondrocyte-derived extracellular matrices (CECM) with anti-adhesion function. We attempted to change the negative charge of the CECM surface to neutral using poly-L-lysine (PLL) and investigated whether it blocked fibroblast adhesion to it and showed an improved anti-adhesion effect in animal models of tissue adhesion. The surface of the membrane was modified with PLL coating (PLL 10), which neutralized the surface charge. We confirmed that the surface characteristics except for the potential difference were maintained after the modification and tested cell attachment in vitro. Adhesion inhibition was identified in a peritoneal adhesion animal model at 1 week and in a subcutaneous adhesion model for 4 weeks. Neutralized CECM (N-CECM) suppressed fibroblast and endothelial cell adhesion in vitro and inhibited abdominal adhesions in vivo. The CECM appeared to actively inhibit the infiltration of endothelial cells into the injured site, thereby suppressing adhesion formation, which differed from conventional adhesion barriers in the mode of action. Furthermore, the N-CECM remained intact without degradation for more than 4 weeks in vivo and exerted anti-adhesion effects for a long time. This study demonstrated that PLL10 surface modification rendered a neutral charge to the polymer on the extracellular matrix surface, thereby inhibiting cell and tissue adhesion. Furthermore, this study suggests a means to modify extracellular matrix surfaces to meet the specific requirements of the target tissue in preventing post-surgical adhesions.

Correction: Dhatwalia et al. Rubus ellipticus Sm. Fruit Extract Mediated Zinc Oxide Nanoparticles: A Green Approach for Dye Degradation and Biomedical Applications. Materials 2022, 15, 3470.

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