Remodeling Osteoarthritic Articular Cartilage under Hypoxic Conditions.

Osteoarthritis (OA) is one of the leading joint diseases induced by abnormalities or inflammation in the synovial membrane and articular cartilage, causing severe pain and disability. Along with the cartilage malfunction, imbalanced oxygen uptake occurs, changing chondrocytes into type I collagen- and type X collagen-producing dedifferentiated cells, contributing to OA progression. However, mounting evidence suggests treating OA by inducing a hypoxic environment in the articular cartilage, targeting the inhibition of several OA-related pathways to bring chondrocytes into a normal state. This review discusses the implications of OA-diseased articular cartilage on chondrocyte phenotypes and turnover and debates the hypoxic mechanism of action. Furthermore, this review highlights the new understanding of OA, provided by tissue engineering and a regenerative medicine experimental design, modeling the disease into diverse 2D and 3D structures and investigating hypoxia and hypoxia-inducing biomolecules and potential cell therapies. This review also reports the mechanism of hypoxic regulation and highlights the importance of activating and stabilizing the hypoxia-inducible factor and related molecules to protect chondrocytes from mitochondrial dysfunction and apoptosis occurring under the influence of OA.

Skeletal microenvironment system utilising bovine bone scaffold co-cultured with human osteoblasts and osteoclast-like cells.

A three-dimensional ex vivo bone cell culture system mimicking the skeletal system is useful for bone tissue engineering and as drug discovery platforms. The present study aimed to establish a three-dimensional skeletal culture system using native bovine bone scaffolds and human bone cells. Bovine bone scaffolds were cultured with human foetal osteoblasts 1.19 and human peripheral blood mononuclear cells for 21 days under standard culture conditions. The following groups were established: Decalcified unseeded bone scaffold (DUBS) as baseline control, decalcified seeded bone scaffold (DSBS) to mimic osteoporosis condition and undecalcified seeded bone scaffold to mimic normal condition. The scaffold's porosity and cell attachment on the scaffolds were determined using scanning electron microscopy. Histological evaluation was used to examine changes in trabecular bone structure. Dual-energy X-ray absorptiometry analysis was performed to determine the bone mineral density (BMD) and bone mineral content (BMC) of the scaffolds. A compression test was performed to examine the total biomechanical strength of the scaffolds. The trabecular thickness and number increased, while the trabecular separationwas reduced slightly in DSBS than in DUBS (P>0.05). The BMD and BMC increased significantly (P<0.05), while the compressive strength only increased slightly in DSBS than in DUBS (P>0.05). In conclusion, the ex vivo skeletal microenvironment comprising native bovine bone scaffolds seeded with bone cells is structurally, functionally and mechanically comparable with natural bone. This system may be used as a platform to understand bone physiology and screen for potential drug candidates.

The Anti-Viral and Anti-Inflammatory Properties of Edible Bird's Nest in Influenza and Coronavirus Infections: From Pre-Clinical to Potential Clinical Application.

Edible bird's nest (BN) is a Chinese traditional medicine with innumerable health benefits, including anti-viral, anti-inflammatory, neuroprotective, and immunomodulatory effects. A small number of studies have reported the anti-viral effects of EBN against influenza infections using in vitro and in vivo models, highlighting the importance of sialic acid and thymol derivatives in their therapeutic effects. At present, studies have reported that EBN suppresses the replicated virus from exiting the host cells, reduces the viral replication, endosomal trafficking of the virus, intracellular viral autophagy process, secretion of pro-inflammatory cytokines, reorient the actin cytoskeleton of the infected cells, and increase the lysosomal degradation of viral materials. In other models of disease, EBN attenuates oxidative stress-induced cellular apoptosis, enhances proliferation and activation of B-cells and their antibody secretion. Given the sum of its therapeutic actions, EBN appears to be a candidate that is worth further exploring for its protective effects against diseases transmitted through air droplets. At present, anti-viral drugs are employed as the first-line defense against respiratory viral infections, unless vaccines are available for the specific pathogens. In patients with severe symptoms due to exacerbated cytokine secretion, anti-inflammatory agents are applied. Treatment efficacy varies across the patients, and in times of a pandemic like COVID-19, many of the drugs are still at the experimental stage. In this review, we present a comprehensive overview of anti-viral and anti-inflammatory effects of EBN, chemical constituents from various EBN preparation techniques, and drugs currently used to treat influenza and novel coronavirus infections. We also aim to review the pathogenesis of influenza A and coronavirus, and the potential of EBN in their clinical application. We also describe the current literature in human consumption of EBN, known allergenic or contaminant presence, and the focus of future direction on how these can be addressed to further improve EBN for potential clinical application.

Polycaprolactone-Based Scaffolds Facilitates Osteogenic Differentiation of Human Adipose-Derived Stem Cells in a Co-Culture System.

(1) Background: Stem cells in combination with scaffolds and bioactive molecules have made significant contributions to the regeneration of damaged bone tissues. A co-culture system can be effective in enhancing the proliferation rate and osteogenic differentiation of the stem cells. Hence, the aim of this study was to investigate the osteogenic differentiation of human adipose derived stem cells when co-cultured with human osteoblasts and seeded on polycaprolactone (PCL):hydroxyapatite (HA) scaffold; (2) Methods: Human adipose-derived stem cells (ASC) and human osteoblasts (HOB) were seeded in three different ratios of 1:2, 1:2 and 2:1 in the PCL-HA scaffolds. The osteogenic differentiation ability was evaluated based on cell morphology, proliferation rate, alkaline phosphatase (ALP) activity, calcium deposition and osteogenic genes expression levels using quantitative RT-PCR; (3) Results: The co-cultured of ASC/HOB in ratio 2:1 seeded on the PCL-HA scaffolds showed the most positive osteogenic differentiation as compared to other groups, which resulted in higher ALP activity, calcium deposition and osteogenic genes expression, particularly Runx, ALP and BSP. These genes indicate that the co-cultured ASC/HOB seeded on PCL-HA was at the early stage of osteogenic development; (4) Conclusions: The combination of co-culture system (ASC/HOB) and PCL-HA scaffolds promote osteogenic differentiation and early bone formation.

The role of Gelam honey in accelerating reepithelialization of ex vivo corneal abrasion model.

This study aimed to investigate the role of Gelam honey (GH) in accelerating reepithelialization of corneal abrasion. Corneal epithelial cells (CEC) isolated from New Zealand white rabbit corneas, were cultured and circular-shaped wounds were created onto them, representing the corneal abrasion model. These wounds were treated with basal (BM) and cornea media (CM) supplemented with GH. The percentage of wound closure was measured on day 0, 3, and 5. Expressions of cytokeratin 3 (CK3), cluster of differentiation 44 (CD 44), and connexin 43 (Cx43) were analyzed via qRT-PCR and immunocytochemistry. The results showed CEC cultured in GH-enriched media reepithelialized faster compared to control. Corneal abrasion treated with CM supplemented with GH closed completely on day 5. CK3, CD44, and Cx43 expressions correspond to the stages of reepithelialization. In conclusion, GH promotes the healing of the ex vivo corneal abrasion model. Further explorations of its potential as adjuvant therapy in treating corneal injuries are needed. PRACTICAL APPLICATIONS: Honey has been reported to have many medicinal properties including antibacterial, anti-inflammatory, and the ability to promote skin wound healing. However, the effects of honey on corneal wound healing have not been fully elucidated. In the present study, we aimed to determine the effects of Gelam honey (GH), well-known local honey obtained from the beehive of Gelam trees (Melaleuca spp.), on the ex vivo corneal abrasion model via cell migration study and analysis of genes and proteins during corneal epithelial wound healing. GH has proven to have accelerated effects on the corneal epithelial cell migration during the closure of the ex vivo corneal abrasion wound model. The expressions of the genes and proteins of the corneal epithelial wound healing markers were in accordance with the stages of healing. Therefore, GH has the potential to be developed as adjuvant therapy in the form of GH-based eye drop in treating corneal injuries.

Biosafety evaluation of culture-expanded human chondrocytes with growth factor cocktail: a preclinical study.

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor ß2 (TGF ß2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes' expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.

Gelam honey promotes ex vivo corneal fibroblasts wound healing.

This study evaluated the effects of Gelam honey (GH) on ex vivo corneal fibroblast ulcer model via wound healing assay, gene expression and immunocytochemistry. Corneal fibroblasts from New Zealand white rabbits were culture expanded. The corneal fibroblast wound healing capacity was observed by creating a circular wound onto confluent monolayer cells cultured in basal medium (BM), BM with GH, serum-enriched basal medium (BMS) and BMS with GH respectively. Wound healing assay and phenotypic characterization of the corneal fibroblast were performed at different stages of wound closure. Expression of aldehyde dehydrogenase (ALDH), vimentin, α-smooth muscle actin (α-SMA), lumican, collagen I and matrix metalloproteinase 12 (MMP 12) were measured at day 1, day 3 and complete wound closure day. Corneal fibroblast cultured in BMS with GH demonstrated the fastest wound closure, at day 5 post wounding. The gene expressions of ALDH and vimentin were higher than control groups while α-SMA expression was lower, in GH enriched media. The expressions of lumican, collagen I and MMP 12 were also higher in cells cultured in GH enriched media compared to the control groups. GH was shown to promote in vitro corneal fibroblast wound healing and may be a potential natural adjunct in the treatment of corneal wound.

Stichopus chloronotus aqueous extract as a chondroprotective agent for human chondrocytes isolated from osteoarthitis articular cartilage in vitro.

The proinflammatory cytokines, metalloproteinases family (MMPs), inflammatory mediators PGE2, COX-2 and NO are the most important group of compounds responsible for the loss of metabolic homeostasis of articular cartilage by promoting catabolic and destructive processes in the pathogenesis of osteoarthritis (OA). Stichopus chloronotus, a marine sea cucumber which is rich in n-3 PUFAs and phenolic compound, may exert a favorable influence on the course of the disease. The objective of this study was to investigate the regeneration and anti-inflammatory potential of S. chloronotus aqueous extract (SCAE) on human OA articular chondrocytes (HOC). METHODS: The HOC isolated from knee joint cartilage removed during surgery were cultured with SCAE for 7 days. The effect of SCAE on anabolic and catabolic gene expression was verified by real-time PCR. Monolayer chondrocytes were stained with toluidine blue whereas sGAG, NO and PGE2 production in medium were analyzed by ELISA. RESULTS: The HOC cultured in various SCAE have polygonal morphology maintaining their chondrocytes characteristic. SAE supplementation tested was found to be effective pro-chondrogenic, anti-inflammatory and anti-oxidative agents, as evidenced by upregulation of cartilage specific markers collagen type II, aggrecan core protein and sox-9 expression and downregulation of collagen type 1, IL-1, IL-6, IL-8, MMP-1, MMP-3, MMP-13, COX-2, iNOS and PAR-2 expression. The presence of SCAE in the culture was able to increase sGAG and reduce NO and PGE2 production significantly. CONCLUSIONS: These results suggested that SCAE demonstrated chondroprotective ability by suppressing catabolic activities, oxidative damage and effectively promoting chondrocytes growth.

Optimization of the Static Human Osteoblast/Osteoclast Co-culture System.

Osteoblasts (OBs) and osteoclasts (OCs) are 2 major groups of bone cells. Their cell-to-cell interactions are important to ensure the continuity of the bone-remodeling process. Therefore, the present study was carried out to optimize an OB/OC co-culture system utilizing the human OB cell line hFOB 1.19 and OCs extracted from peripheral blood mononuclear cells (PBMNCs). It was a 2-step procedure, involving the optimization of the OB culture and the co-culture of the OBs with PBMNCs at an optimum ratio. Firstly, pre-OBs were cultured to 90% confluency and the time required for differentiation was determined. OB differentiation was determined using the van Gieson staining to detect the presence of collagen and Alizarin Red for calcium. Secondly, OBs and OCs were co-cultured at the ratios of 1 OC: 1 OB, 1 OC: 4 OBs, 2 OCs: 1 OB, and 1 OC: 2 OBs. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the differentiation of the OCs. The results showed that collagen was present on day 1, whereas calcium was detected as early as day 3. Based on the result of TRAP staining, 1 OC: 2 OBs was taken as the most appropriate ratio. No macrophage colony-stimulating factor and receptor activator of the nuclear factor-κB ligand were added because they were provided by the OBs. In conclusion, these optimization processes are vital as they ensure the exact time point and ratio of the OB/OC co-culture in order to produce a reliable and reproducible co-culture system.

Protective Effects of Selected Botanical Agents on Bone.

Osteoporosis is a serious health problem affecting more than 200 million elderly people worldwide. The early symptoms of this disease are hardly detectable. It causes progressive bone loss, which ultimately renders the patients susceptible to fractures. Osteoporosis must be prevented because the associated fragility fractures result in high morbidity, mortality, and healthcare costs. Many plants used in herbal medicine contain bioactive compounds possessing skeletal protective effects. This paper explores the anti-osteoporotic properties of selected herbal plants, including their actions on osteoblasts (bone forming cells), osteoclasts (bone resorbing cells), and bone remodelling. Some of the herbal plant families included in this review are Berberidaceae, Fabaceae, Arecaceae, Labiatae, Simaroubaceaea, and Myrsinaceae. Their active constituents, mechanisms of action, and pharmaceutical applications were discussed. The literature shows that very few herbal plants have undergone human clinical trials to evaluate their pharmacological effects on bone to date. Therefore, more intensive research should be performed on these plants to validate their anti-osteoporotic properties so that they can complement the currently available conventional drugs in the battle against osteoporosis.

Human adipose-derived mesenchymal stem cells promote recovery of injured HepG2 cell line and show sign of early hepatogenic differentiation.

Currently, orthotopic liver transplantation is the gold standard therapy for liver failure. However, it is limited by the insufficient organ donor and risk of immune rejection. Stem cell therapy is a promising alternative treatment for liver failure. One of the most ideal sources of stem cells for regenerative medicine is adipose-derived stem cells (ADSCs). In this study, primary ADSCs seeded on cell culture insert were indirectly co-cultured with injured HepG2 to elucidate the role of ADSCs in promoting the recovery of injured HepG2 in non-contact manner. HepG2 recovery was determined by the surface area covered by cells and growth factor concentration was measured to identify the factors involved in regeneration. Besides, HepG2 were collected for q-PCR analysis of injury, hepatocyte functional and regenerative markers expression. For the ADSCs, expression of hepatogenic differentiation genes was analyzed. Results showed that non-contact co-culture with ADSCs helped the recovery of injured HepG2. ELISA quantification revealed that ADSCs secreted higher amount of HGF and VEGF to help the recovery of injured HepG2. Furthermore, HepG2 co-cultured with ADSCs expressed significantly lower injury markers as well as significantly higher regenerative and functional markers compared to the control HepG2. ADSCs co-cultured with injured HepG2 expressed significantly higher hepatic related genes compared to the control ADSCs. In conclusion, ADSCs promote recovery of injured HepG2 via secretion of HGF and VEGF. In addition, co-cultured ADSCs showed early sign of hepatogenic differentiation in response to the factors released or secreted by the injured HepG2.

Differential responses of lineages-committed hematopoietic progenitors and altered expression of self-renewal and differentiation-related genes in 1,4-benzoquinone (1,4-BQ) exposure.

Despite of reports on hematotoxic and leukemogenic evidences related to benzene exposure, the mechanism of benzene toxicity affecting the hematopoietic stem and progenitor cells (HSPCs) fate remains unclear. This study aims to elucidate the benzene's effect on the lineages-committed progenitors and genes-regulating self-renewal and differentiation of HSPCs. Isolated mouse bone marrow (BM) cells were exposed to the benzene metabolite, 1,4-benzoquinone (1,4-BQ) at 1.25, 2.5, and 5µM for 24h. The clonogenic potency of erythroid, myeloid, and Pre-B lymphoid progenitors was evaluated through colony-forming-cell assay. Quantitative real time-PCR was used to analyze the self-renewal (Bmi-1, HoxB4, and Wnt3) and differentiation (GATA1, GATA2, and GATA3)-related genes' expression levels. 1,4-BQ exposure significantly lowered the clonogenicity of the myeloid progenitor at 1.25 and 2.5µM (p<0.05), but affected neither the erythroid nor Pre-B lymphoid progenitors. Furthermore, significant upregulation of HoxB4 expression level was observed at all concentrations. GATA3 and Bmi-1 expressions were also significant upregulated at 2.5 and 5µM 1,4-BQ, respectively. In conclusion, 1,4-BQ could modulate the fate of HSPCs by altering the self-renewal and differentiation related genes. The definite role of lineages specificity and responsive genes in governing the hematotoxicity and leukemogenicity of 1,4-BQ should be further investigated.

Acanthamoeba-mediated cytopathic effect correlates with MBP and AhLBP mRNA expression.

BACKGROUND: In recent years, the concern of Acanthamoeba keratitis has increased since the infection is often associated with contact lens use. Partial 18S rRNA genotypic identification of Acanthamoeba isolates is important to correlate with pathophysiological properties in order to evaluate the degree of virulence. This is the first report of genotypic identification for clinical isolates of Acanthamoeba from corneal scrapings of keratitis in Malaysia. This study is also the first to correlate the mRNA expression of MBP and AhLBP as virulent markers for axenic strains of Acanthamoeba. RESULTS: In this study, ten clinical isolates were obtained from corneal scrapings. Rns genotype and intra-genotypic variation at the DF3 region of the isolates were identified. Results revealed that all clinical isolates belonged to the T4 genotype, with T4/6 (4 isolates), T4/2 (3 isolates), T4/16 (2 isolates) and one new genotype T4 sequence (T4/36), being determined. The axenic clinical isolates were cytopathogenic to rabbit corneal fibroblasts. MBP and AhLBP mRNA expression are directly correlated to Acanthamoeba cytopathic effect. CONCLUSIONS: All ten Malaysian clinical isolates were identified as genotype T4 which is predominantly associated with AK. Measuring the mRNA expression of Acanthamoeba virulent markers could be useful in the understanding of the pathogenesis of Acanthamoeba keratitis.

The Use of Engineered Bilayered Skin (MyDermTM) in the Management of Massive Skin Defect in Grade III Gustilo-Anderson Open Fracture.

Open fracture Gustilo-Anderson grade IIIC is associated with higher risk of infection and problems with soft tissue coverage. Various methods have been used for soft tissue coverage in open fractures with large skin defect. We report a case of a patient who had grade IIIC open fracture of the tibia with posterior tibial artery injury. The patient underwent external fixation and reduction. Because of potential compartment syndrome after vascular repair, fasciotomy of the posterior compartment was performed. This wound, however, became infected and because of further debridement, gave rise to a large skin defect. A tissue engineered skin construct, MyDermTM was employed to cover this large defect. Complete wound closure was achieved 35 days postimplantation. The patient then underwent plating of the tibia for nonunion with no adverse effect to the grafted site. The tibia eventually healed 5 months postplating, and the cosmetic appearance of the newly formed skin was satisfactory.

Comparison between tocotrienol and omeprazole on gastric growth factors in stress-exposed rats.

AIM: To investigate and compare the effects of tocotrienol and omeprazole on gastric growth factors in rats exposed to water-immersion restraint stress (WIRS). METHODS: Twenty-eight male Wistar rats were randomly assigned to four groups of seven rats. The two control groups were administered vitamin-free palm oil (vehicle) and the two treatment groups were given omeprazole (20 mg/kg) or tocotrienol (60 mg/kg) by oral gavage. After 28 d of treatment, rats from one control group and both treated groups were subjected to WIRS one time for 3.5 h. Gastric lesions were measured and gastric tissues were obtained to measure vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor-alpha (TGF-α) mRNA expression. RESULTS: Rats exposed to WIRS for 3.5 h demonstrated the presence of considerable ulcers in the form of gastric erosion. The lesion index in the stressed control (S) group was increased (P < 0.001) compared to the tocotrienol treated and omeprazole treated groups. Stress led to a decrease in gastric VEGF (P < 0.001), bFGF (P < 0.001) and TGF-α (P < 0.001) mRNA levels and caused an increase in EGF mRNA (P < 0.001) that was statistically significant compared to the non-stressed control group. Although both treatment agents exerted similar ulcer reducing ability, only treatment with tocotrienol led to increased expression of VEGF (P = 0.008), bFGF (P = 0.001) and TGF-α (P = 0.002) mRNA. CONCLUSION: Tocotrienol provides gastroprotective effects in WIRS-induced ulcers. Compared to omeprazole, tocotrienol exerts a similar protective effect, albeit through multiple mechanisms of protection, particularly through up-regulation of growth factors that assist in repair of gastric tissue injuries.

Piper betle L. Modulates Senescence-Associated Genes Expression in Replicative Senescent Human Diploid Fibroblasts.

Piper betle (PB) is a traditional medicine that is widely used to treat different diseases around Asian region. The leaf extracts contain various bioactive compounds, which were reported to have antidiabetic, antibacterial, anti-inflammatory, antioxidant, and anticancer effects. In this study, the effect of PB aqueous extracts on replicative senescent human diploid fibroblasts (HDFs) was investigated by determining the expressions of senescence-associated genes using quantitative PCR. Our results showed that PB extracts at 0.4 mg/ml can improve cell proliferation of young (143%), presenescent (127.3%), and senescent (157.3%) HDFs. Increased expressions of PRDX6, TP53, CDKN2A, PAK2, and MAPK14 were observed in senescent HDFs compared to young and/or presenescent HDFs. Treatment with PB extracts modulates the transcriptional profile changes in senescent HDFs. By contrast, expressions of SOD1 increased, whereas GPX1, PRDX6, TP53, CDKN2A, PAK2, and MAPK14 were decreased in PB-treated senescent HDFs compared to untreated senescent HDFs. In conclusion, this study indicates the modulation of PB extracts on senescence-associated genes expression of replicative senescent HDFs. Further studies warrant determining the mechanism of PB in modulating replicative senescence of HDFs through these signaling pathways.

Gamma-Tocotrienol prevents cell cycle arrest in aged human fibroblast cells through p16INK4a pathway

Human diploid fibroblasts (HDFs) proliferation in culture has been used as a model of aging at the cellular level. Growth arrest is one of the most important mechanisms responsible for replicative senescence. Recent researches have been focusing on the function of vitamin E in modulating cellular signaling and gene expression. Therefore, the aim of this study was to elucidate the effect of palm γ-tocotrienol (vitamin E) in modulating cellular aging through p16INK4a pathway in HDF cells. Primary culture of senescent HDFs was incubated with 70 μM of palm γ-tocotrienol for 24 hours. Silencing of p16INK4a was carried out by siRNA transfection. RNA was extracted from the different treatment groups and gene expression analysis was carried out by real-time reverse transcription polymerase chain reaction. Proteins that were regulated by p16INK4a were determined by western blot technique. The finding of this study showed that p16INK4a mRNA was overexpressed in senescent HDFs, and hypophosphorylated-pRb and cyclin D1 protein expressions were increased (p < 0.05). However, downregulation of p16INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions (p < 0.05) by γ-tocotrienol led to modulation of the cell cycle regulation during cellular aging. In conclusion, senescent HDFs showed change in biological process specifically in cell cycle regulation with elevated expression of genes and proteins which may contribute to cell cycle arrest. Palm γ-tocotrienol may delay cellular senescence of HDFs by regulating cell cycle through downregulation of p16INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions (AU)

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Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique.

CONCLUSION: In conclusion, these result showed HADSCs could differentiate into chondrocytes-like cells, dependent on signaling induced by TGF-ß3 and chondrocytes. This is a promising result and showed that HADSCs is a potential source for future microtia repair. The technique of co-culture is a positive way forward to assist the microtia tissue. OBJECTIVE: Reconstructive surgery for the repair of microtia still remains the greatest challenge among the surgeons. Its repair is associated with donor-site morbidity and the degree of infection is inevitable when using alloplastic prosthesis with uncertain long-term durability. Thus, human adipose derived stem cells (HADSCs) can be an alternative cell source for cartilage regeneration. This study aims to evaluate the chondrogenic potential of HADSCs cultured with transforming growth factor-beta (TGF-ß) and interaction of auricular chondrocytes with HADSCs for new cartilage generation. METHODS: Multi-lineages differentiation features of HADSCs were monitored by Alcian Blue, Alizarin Red, and Oil Red O staining for chondrogenic, adipogenic, and osteogenic differentiation capacity, respectively. Further, HADSCs alone were culture in medium added with TGF-ß3; and human auricular chondrocytes were interacted indirectly in the culture with and without TGF-ßs for up to 21 days, respectively. Cell morphology and chondrogenesis were monitored by inverted microscope. For cell viability, Alamar Blue assay was used to measure the cell viability and the changes in gene expression of auricular chondrocyte markers were determined by real-time polymerase chain reaction analysis. For the induction of chondrogenic differentiation, HADSCs showed a feature of aggregation and formed a dense matrix of proteoglycans. Staining results from Alizirin Red and Oil Red O indicated the HADSCs also successfully differentiated into adipogenic and osteogenic lineages after 21 days. RESULTS: According to a previous study, HADSCs were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen. The results showed HADSCs test groups (cultured with TGF-ß3) displayed chondrocytes-like cells morphology with typical lacunae structure compared to the control group without TGF-ß3 after 2 weeks. Additionally, the HADSCs test groups increased in cell viability; an increase in expression of chondrocytes-specific genes (collagen type II, aggrecan core protein, SOX 9 and elastin) compared to the control. This study found that human auricular chondrocytes cells and growth factor had a positive influence in inducing HADSCs chondrogenic effects, in terms of chondrogenic differentiate of feature, increase of cell viability, and up-regulated expression of chondrogenic genes.

Assessment of tumourigenic potential in long-term cryopreserved human adipose-derived stem cells.

Cryopreservation represents an efficient way to preserve human mesenchymal stem cells (hMSCs) at early culture/passage, and allows pooling of cells to achieve sufficient cells required for off-the-shelf use in clinical applications, e.g. cell-based therapies and regenerative medicine. To fully apply cryopreserved hMSCs in a clinical setting, it is necessary to evaluate their biosafety, e.g. chromosomal abnormality and tumourigenic potential. To date, many studies have demonstrated that cryopreserved hMSCs display no chromosomal abnormalities. However, the tumourigenic potential of cryopreserved hMSCs has not yet been evaluated. In the present study, we cryopreserved human adipose-derived mesenchymal stem cells (hASCs) for 3 months, using a slow freezing method with various cryoprotective agents (CPAs), followed by assessment of the tumourigenic potential of the cryopreserved hASCs after thawing and subculture. We found that long-term cryopreserved hASCs maintained normal levels of the tumour suppressor markers p53, p21, p16 and pRb, hTERT, telomerase activity and telomere length. Further, we did not observe significant DNA damage or signs of p53 mutation in cryopreserved hASCs. Our findings suggest that long-term cryopreserved hASCs are at low risk of tumourigenesis. These findings aid in establishing the biosafety profile of cryopreserved hASCs, and thus establishing low hazardous risk perception with the use of long-term cryopreserved hASCs for future clinical applications. Copyright © 2016 John Wiley & Sons, Ltd.

γ-Tocotrienol prevents cell cycle arrest in aged human fibroblast cells through p16INK4a pathway.

Human diploid fibroblasts (HDFs) proliferation in culture has been used as a model of aging at the cellular level. Growth arrest is one of the most important mechanisms responsible for replicative senescence. Recent researches have been focusing on the function of vitamin E in modulating cellular signaling and gene expression. Therefore, the aim of this study was to elucidate the effect of palm γ-tocotrienol (vitamin E) in modulating cellular aging through p16INK4a pathway in HDF cells. Primary culture of senescent HDFs was incubated with 70 µM of palm γ-tocotrienol for 24 hours. Silencing of p16INK4a was carried out by siRNA transfection. RNA was extracted from the different treatment groups and gene expression analysis was carried out by real-time reverse transcription polymerase chain reaction. Proteins that were regulated by p16INK4a were determined by western blot technique. The finding of this study showed that p16INK4a mRNA was overexpressed in senescent HDFs, and hypophosphorylated-pRb and cyclin D1 protein expressions were increased (p < 0.05). However, downregulation of p16INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions (p < 0.05) by γ-tocotrienol led to modulation of the cell cycle regulation during cellular aging. In conclusion, senescent HDFs showed change in biological process specifically in cell cycle regulation with elevated expression of genes and proteins which may contribute to cell cycle arrest. Palm γ-tocotrienol may delay cellular senescence of HDFs by regulating cell cycle through downregulation of p16INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions.