Investigating cellulose derived glycosaminoglycan mimetic scaffolds for cartilage tissue engineering applications

Articular cartilage has a limited capacity to heal and, currently, no treatment exists that can restore normal hyaline cartilage. Creating tissue engineering scaffolds that more closely mimic the native extracellular matrix may be an attractive approach. Glycosaminoglycans, which are present in native cartilage tissue, provide signalling and structural cues to cells. This study evaluated the use of a glycosaminoglycan mimetic, derived from cellulose, as a potential scaffold for cartilage repair applications. Fully sulfated sodium cellulose sulfate (NaCS) was initially evaluated in soluble form as an additive to cell culture media. Human mesenchymal stem cell (MSC) chondrogenesis in pellet culture was enhanced with 0.01% NaCS added to induction media as demonstrated by significantly higher gene expression for type II collagen and aggrecan. NaCS was combined with gelatine to form fibrous scaffolds using the electrospinning technique. Scaffolds were characterized for fibre morphology, overall hydrolytic stability, protein/growth factor interaction and for supporting MSC chondrogenesis in vitro. Scaffolds immersed in phosphate buffered saline for up to 56 days had no changes in swelling and no dissolution of NaCS as compared to day 0. Increasing concentrations of the model protein lysozyme and transforming growth factor‐β3 were detected on scaffolds with increasing concentrations of NaCS (p < 0.05). MSC chondrogenesis was enhanced on the scaffold with the lowest NaCS concentration as seen with the highest collagen type II production, collagen type II immunostaining, and expression of cartilage‐specific genes. These studies demonstrate the feasibility of cellulose sulfate as a scaffolding material for cartilage tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.     

Human corneal fibroblast migration and extracellular matrix synthesis during stromal repair: Role played by platelet‐derived growth factor‐BB,basic fibroblast growth factor,and transforming growth factor‐β1

The development of treatments that modulate corneal wound healing to avoid fibrosis during tissue repair is important for the restoration of corneal transparency after an injury. To date, few studies have studied the influence of growth factors (GFs) on human corneal fibroblast (HCF) expression of extracellular matrix (ECM) proteins such as collagen types I and III, proteoglycans such as perlecan, or proteins implicated in cellular migration such as α5β1‐integrin and syndecan‐4. Using in vitro HCFs, a mechanical wound model was developed to study the influence of the GFs basic fibroblast GF (bFGF), platelet‐derived GF (PDGF‐BB) and transforming GF‐β1 (TGFβ1) on ECM protein production and cellular migration. Our results show that mechanical wounding provokes the autocrine release of bFGF and TGFβ1 at different time points during the wound closure. The HCF response to PDGF‐BB was a rapid closure due to fast cellular migration associated with a high focal adhesion replacement and a high expression of collagen and proteoglycans, producing nonfibrotic healing. bFGF stimulated nonfibrotic ECM production and limited the migration process. Finally, TGFβ1 induced expression of the fibrotic markers collagen type III and α5β1 integrin, and it inhibited cellular migration due to the formation of focal adhesions with a low turnover rate. The novel in vitro HCF mechanical wound model can be used to understand the role played by GFs in human corneal repair. The model can also be used to test the effects of different treatments aimed at improving the healing process. Copyright © 2016 John Wiley & Sons, Ltd.     

Repair of an osteochondral defect by sustained delivery of BMP‐2 or TGFβ1 from a bilayered alginate–PLGA scaffold

Regeneration of cartilage defects can be accelerated by localized delivery of appropriate growth factors (GFs) from scaffolds. In the present study we analysed the in vitro and in vivo release rates and delivery efficacies of transforming growth factor‐β1 (TGFβ1) and bone morphogenetic protein‐2 (BMP‐2) from a bilayered system, applied for osteochondral defect repair in a rabbit model. A bone‐orientated, porous PLGA cylinder was overlaid with GF containing PLGA microspheres, dispersed in an alginate matrix. Four microsphere formulations were incorporated: (a) blank ones; (b) microspheres containing 50 ng TGFβ1; (c) microspheres containing 2.5 µg BMP‐2; and (d) microspheres containing 5 µg BMP‐2. Release kinetics and tissue distributions were determined using iodinated (¹²⁵I) GFs. Bioactivity of in vitro released BMP‐2 and TGFβ1 was confirmed in cell‐based assays. In vivo release profiles indicated good GF release control. 20% of BMP‐2 and 15% of TGFβ1 were released during the first day. Virtually the total dose was delivered at the end of week 6. Significant histological differences were observed between untreated and GF‐treated specimens, there being especially relevant short‐term outcomes with 50 ng TGFβ1 and 5 µg BMP‐2. Although the evaluation scores for the newly formed cartilage did not differ significantly, 5 µg BMP‐2 gave rise to higher quality cartilage with improved surface regularity, tissue integration and increased collagen‐type II and aggrecan immunoreactivity 2 weeks post‐implantation. Hence, the bilayered system controlled GF release rates and led to preserved cartilage integrity from 12 weeks up to at least 24 weeks. Copyright © 2012 John Wiley & Sons, Ltd.     

Expression and related mechanisms of miR-330-3p and S100B in an animal model of cartilage injury

ObjectiveTo investigate the roles of and relationship between microRNA (miR)-330-3p and S100 calcium-binding protein B (S100B) in an animal model of cartilage injury.MethodsThis study included 30 New Zealand male rabbits randomly divided into three groups: an intervention group, a model group and a sham surgery control group. Modelling was performed in the intervention and model groups, but in the sham surgery group, only the skin was cut. After modelling, the intervention and model groups were injected with the miR-330-3p overexpression vector GV268-miR-330-3p or the control GV268-N-ODN vector, respectively, twice a week for 7 weeks.ResultsLevels of interleukin-1β and tumour necrosis factor-α in the synovial fluid were significantly higher in the model group than in the intervention and control groups. The level of miR-330-3p in the cartilage tissue was significantly higher in the control group than in the model group but it was significantly lower compared with the intervention group. Levels of S100B, fibroblast growth factor receptor 1 and fibroblast growth factor-2 in the cartilage tissue of rabbits in the model group were significantly higher compared with the control and intervention groups.ConclusionThese findings demonstrate that the upregulation of miR-330-3p can inhibit the expression of S100B.     

Transforming growth factor‐beta1 inhibits tissue engineering cartilage absorption via inducing the generation of regulatory T cells

The objective of the present study was to explore the mechanisms of transforming growth factor (TGF)‐β1 inhibiting the absorption of tissue engineering cartilage. We transfected TGF‐β1 gene into bone marrow mesenchymal stem cells (BMMSCs) and co‐cultured with interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α and CD4⁺CD25^? T lymphocytes. We then characterized the morphological changes, apoptosis and characterization of chondrogenic‐committed cells from TGF‐β1⁺BMMSCs and explored their mechanisms. Results showed that BMMSCs apoptosis and tissue engineering cartilage absorption in the group with added IFN‐γ and TNF‐α were greater than in the control group. In contrast, there was little BMMSC apoptosis and absorption by tissue engineering cartilage in the group with added CD4⁺CD25^? T lymphocytes; Foxp3⁺T cells and CD25⁺CD39⁺ T cells were found. In contrast, no type II collagen or Foxp3⁺T cells or CD25⁺CD39⁺ T cells was found in the TGF‐β1^–BMMSC group. The data suggest that IFN‐γ and TNF‐α induced BMMSCs apoptosis and absorption of tissue engineering cartilage, but the newborn regulatory T (Treg) cells inhibited the function of IFN‐γ and TNF‐α and protected BMMSCs and tissue engineering cartilage. TGF‐β1not only played a cartilage inductive role, but also inhibited the absorption of tissue engineering cartilage. The pathway proposed in our study may simulate the actual reaction procedure after implantation of BMMSCs and tissue engineering cartilage in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Controlled release of BMP‐2 from a sintered polymer scaffold enhances bone repair in a mouse calvarial defect model

Sustained and controlled delivery of growth factors, such as bone morphogenetic protein 2 (BMP‐2), from polymer scaffolds has excellent potential for enhancing bone regeneration. The present study investigated the use of novel sintered polymer scaffolds prepared using temperature‐sensitive PLGA/PEG particles. Growth factors can be incorporated into these scaffolds by mixing the reconstituted growth factor with the particles prior to sintering. The ability of the PLGA/PEG scaffolds to deliver BMP‐2 in a controlled and sustained manner was assessed and the osteogenic potential of these scaffolds was determined in a mouse calvarial defect model. BMP‐2 was released from the scaffolds in vitro over 3 weeks. On average, ca. 70% of the BMP‐2 loaded into the scaffolds was released by the end of this time period. The released BMP‐2 was shown to be active and to induce osteogenesis when used in a cell culture assay. A substantial increase in new bone volume of 55% was observed in a mouse calvarial defect model for BMP‐2‐loaded PLGA/PEG scaffolds compared to empty defect controls. An increase in new bone volume of 31% was observed for PLGA/PEG scaffolds without BMP‐2, compared to empty defect controls. These results demonstrate the potential of novel PLGA/PEG scaffolds for sustained BMP‐2 delivery for bone‐regeneration applications. Copyright © 2012 John Wiley & Sons, Ltd.     

Association between a TGFβ1 promoter polymorphism and the phenotype of aspirin‐intolerant chronic urticaria in a Korean population

Background: Chronic urticaria/angioedema is a common phenotype in patients with aspirin sensitivity; however, its genetic mechanism is not understood. Transforming growth factor (TGF)β1 is a key regulatory cytokine involved in allergic inflammation. Objective: We examined the association of a TGFβ1 genetic polymorphism with aspirin‐intolerant chronic urticaria (AICU) and aspirin‐tolerant chronic urticaria (ATCU) in a Korean population. Methods: A promoter polymorphism in the TGFβ1 gene, TGFβ1 ?509C>T, was analysed in 112 AICU patients, 153 ATCU patients and 457 normal controls (NC), and the frequency was compared among the groups. Serum TGFβ1 levels were measured by ELISA. Results: The minor allele frequency of the ?509C>T polymorphism was significantly higher in patients with AICU compared with the other two groups (P < 0·02 for AICU vs. NC; P < 0·05 for AICU vs. ATCU). Among the AICU patients, those with the T allele tended to have lower serum TGFβ1 levels. Conclusion: These findings suggest that the ?509C>T polymorphism in the TGFβ1 promoter may contribute to the development of the AICU phenotype.     

Withdrawal of inhaled steroids in children with non‐cystic fibrosis bronchiectasis

Background: To study the effects of inhaled steroid withdrawal on bronchial hyperreactivity, sputum inflammatory markers and neutrophilic apoptosis in children with non‐cystic fibrosis (non‐CF) bronchiectasis. Objectives: To evaluate the role of inhaled steroids in the treatment of children with non‐CF bronchiectasis with specific emphasis on the bronchial hyperreactivity and neutrophilic apoptosis. Methods: Twenty‐seven children with steady‐state non‐CF bronchiectasis were evaluated primarily with metacholine challenge tests and apoptotic neutrophil ratios in induced sputum and secondarily with symptom scores, pulmonary function tests and tumour necrosis factor‐alpha (TNF‐α), interleukin‐8 (IL‐8) levels and neutrophil ratios in induced sputum before and after 12‐week withdrawal of inhaled steroids. Results: There were 16 girls and 11 boys. Median (interquartile range) age was 11·4 (9·5–13·6) years, follow‐up duration was 3·5 (2–6·5) years. Symptom scores (4 vs. 3; P = 0·27), oxygen saturation (95% vs. 97%; P = 0·06), pulmonary function tests (FEV1: 82% predicted vs. 83% predicted; P = 0·73), sputum neutrophil ratios (29·9% vs. 46·8%; P = 0·20), TNF‐α (58 pg/mL vs. 44·5 pg/mL; P = 0·55) and IL‐8 (2·7 ng/mL vs. 2·4 ng/mL; P = 0·82) levels in induced sputum were similar before and after 12‐week withdrawal of inhaled steroids. However, the number of patients with bronchial hyperreactivity increased (37% vs. 63% of patients; P = 0·016) and neutrophilic apoptosis in induced sputum decreased (42·8% vs. 20·2%; P = 0·03) after withdrawal. Conclusion: In this study, 12 week‐withdrawal of inhaled steroid treatment resulted in a significant increase in bronchial hyperreactivity and decrease in neutrophil apoptosis, but no change in sputum inflammatory markers in children with non‐CF bronchiectasis was observed.     

Tubular open‐porous β‐tricalcium phosphate polycaprolactone scaffolds as guiding structure for segmental bone defect regeneration in a novel sheep model

Large segmental bone defect reconstruction with sufficient functional restoration is one of the most demanding challenges in orthopaedic surgery. Available regenerative treatment options, as the vascularized bone graft transfer, the Masquelet technique or the Ilizarov distraction osteogenesis, are associated with specific indications and distinct limitations. As an alternative, a hollow cylindrical ceramic‐polymer composite scaffold (β‐tricalcium phosphate and poly‐lactid co‐ε‐ caprolactone), facilitating a strong surface guiding effect for tissue ingrowth (group 1; n = 6) was investigated here. In combination with an additional autologous, cancellous bone graft filling, the scaffold's ability to work as an open‐porous membrane to improve the defect healing process was analysed (group 2; n = 6). A novel model of a critical size (40 mm) tibia osteotomy defect stabilized with an external hybrid‐ring fixator, was established in sheep. Segmental defect regeneration and tissue organization in relation to the scaffold were analysed radiologically, (immune‐) histologically, and with second‐harmonic generation imaging 12 weeks after surgery. The scaffold's tubular shape and open‐porous structure controlled the collagen fibre orientation within the bone defect and guided the following mineralization process along the scaffold surface. In combination with the osteoinductive stimulus, a unilateral bony bridging of the critically sized defect was achieved in one third of the animals. The external hybrid‐ring fixator was appropriate for large segmental defect stabilization in sheep.     

Chondrogenesis by bone marrow‐derived mesenchymal stem cells grown in chondrocyte‐conditioned medium for auricular reconstruction

Bone marrow‐derived mesenchymal stem cells (BMSCs) can be obtained by minimally invasive means and would be a favourable source for cell‐based cartilage regeneration. However, controlling the differentiation of the BMSCs towards the desired chondrogenic pathway has been a challenge hampering their application. The major aim of the present study was to determine if conditioned medium collected from cultured auricular chondrocytes could promote chondrogenic differentiation of BMSCs. Auricular chondrocytes were isolated and grown in BMSC standard culture medium (SM) that was collected and used as chondrocyte‐conditioned medium (CCM). The BMSCs were expanded in either CCM or SM for three passages. Cells were seeded onto fibrous collagen scaffolds and precultured for 2 weeks with or without transforming growth factor‐beta 3 (TGF‐β3). After preculture, constructs were implanted subcutaneously in nude mice for 6 and 12 weeks and evaluated with real‐time polymerase chain reaction, histology, immunohistochemistry and biochemistry. Real‐time polymerase chain reaction results showed upregulation of COL2A1 in the constructs cultured in CCM compared with those in SM. After 12 weeks in vivo, abundant neocartilage formation was observed in the implants that had been cultured in CCM, with or without TGF‐β3. In contrast, very little cartilage matrix formation was observed within the SM groups, regardless of the presence of TGF‐β3. Osteogenesis was only observed in the SM group with TGF‐β3. In conclusion, CCM even had a stronger influence on chondrogenesis than the supplementation of the standard culture medium with TGF‐β3, without signs of endochondral ossification. Efficient chondrogenic differentiation of BMSCs could provide a promising alternative cell population for auricular regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

Mathematical modelling of glycosaminoglycan production by stem cell aggregates incorporated with growth factor‐releasing polymer microspheres

Systems composed of high density cells incorporated with growth factor‐releasing polymer microspheres have recently been shown to promote chondrogenic differentiation and cartilage formation. Within these systems, the effects of spatial and temporal patterning of growth factor release on hyaline cartilage‐specific extracellular matrix production have been examined. However, at present, it is unclear which microsphere densities and growth factor delivery profiles are optimal for inducing human mesenchymal stem cell differentiation and glycosaminoglycan production. A mathematical model to describe glycosaminoglycan production as a function of initial microsphere loading and microsphere degradation rate over a period of 3 weeks is presented. Based on predictions generated by this model, it may be feasible to design a bioactive microsphere system with specific spatiotemporal growth factor presentation characteristics to promote glycosaminoglycan production at controllable rates. Copyright © 2014 John Wiley & Sons, Ltd.     

Stem cells display a donor dependent response to escalating levels of growth factor release from extracellular matrix‐derived scaffolds

Numerous growth factor delivery systems have been developed for tissue engineering. However, little is known about how the dose of a specific protein will influence tissue regeneration, or how different patients will respond to altered levels of growth factor presentation. The objective of the present study was to assess stem cell chondrogenesis within extracellular‐matrix (ECM)‐derived scaffolds loaded with escalating levels of transforming growth factor (TGF)‐β3. It was also sought to determine if stem cells display a donor‐dependent response to different doses of TGF‐β3, from low (5 ng) to high (200 ng), released from such scaffolds. It was found that ECM‐derived scaffolds possess the capacity to bind and release increasing amounts of TGF‐β3, with between 60% and 75% of this growth factor released into the media over the first 12 days of culture. After seeding these scaffolds with human infrapatellar fat pad‐derived stem cells (FPSCs), it was found that cartilage‐specific ECM accumulation was greatest for the higher levels of growth factor loading. Importantly, soak‐loading cartilage ECM‐derived scaffolds with high levels of TGF‐β3 always resulted in at least comparable levels of chondrogenesis to controls where this growth factor was continuously added to the culture media. Similar results were observed for FPSCs from all donors, although the absolute level of secreted matrix did vary from donor to donor. Therefore, while no single growth factor release profile will be optimal for all patients, the results of this study suggest that the combination of a highly porous cartilage ECM‐derived scaffold coupled with appropriate levels of TGF‐β3 can consistently drive chondrogenesis of adult stem cells. Copyright © 2016 John Wiley & Sons, Ltd.     

FGF,TGFβ and Wnt crosstalk: embryonic to in vitro cartilage development from mesenchymal stem cells

Articular cartilage is easily damaged, yet difficult to repair. Cartilage tissue engineering seems a promising therapeutic solution to restore articular cartilage structure and function, with mesenchymal stem cells (MSCs) receiving increasing attention for their promise to promote cartilage repair. It is known from embryology that members of the fibroblast growth factor (FGF), transforming growth factor‐β (TGFβ) and wingless‐type (Wnt) protein families are involved in controlling different differentiation stages during chondrogenesis. Individually, these pathways have been extensively studied but so far attempts to recapitulate embryonic development in in vitro MSC chondrogenesis have failed to produce stable and functioning articular cartilage; instead, transient hypertrophic cartilage is obtained. We believe a better understanding of the simultaneous integration of these factors will improve how we relate embryonic chondrogenesis to in vitro MSC chondrogenesis. This narrative review attempts to define current knowledge on the crosstalk between the FGF, TGFβ and Wnt signalling pathways during different stages of mesenchymal chondrogenesis. Connecting embryogenesis and in vitro differentiation of human MSCs might provide insights into how to improve and progress cartilage tissue engineering for the future. Copyright © 2013 John Wiley & Sons, Ltd.     

Amelioration of functional,biochemical and molecular deficits by epigallocatechin gallate in experimental model of alcoholic neuropathy

Long term alcohol consumption leads to decreased nociceptive threshold characterized by spontaneous burning pain, hyperalgesia and allodynia. The mechanism involved in this pain includes increased oxidative‐nitrosative stress, release of pro‐inflammatory cytokines and neuronal apoptosis. The present study was designed to explore the protective effect of epigallocatechin‐3‐gallate against alcoholic neuropathic pain in rats. Rats fed with alcohol (35%) for 10 weeks showed markedly decreased tail flick latency in tail‐immersion test (thermal hyperalgesia), vocalization threshold in Randall—Sellito test (mechanical hyperalgesia) and paw‐withdrawal threshold in von‐Frey hair test (mechanical allodynia) along with enhanced oxidative‐nitrosative stress and inflammatory mediators (TNF‐α, IL‐1β and TGF‐β1 levels). Co‐administration of epigallocatechin‐3‐gallate (25–100 mg/kg) significantly and dose‐dependently prevented functional, biochemical and molecular changes associated with alcoholic neuropathy. In conclusion, the current findings suggest the neuroprotective potential of epigallocatechin‐3‐gallate in attenuating the functional, biochemical and molecular alterations associated with alcoholic neuropathy through modulation of oxido‐inflammatory cascade.