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Study Design: The aim of this study was to develop a tissue engineering approach in regenerating the annulus fibrosus (AF) as part of an overall strategy to produce a tissue-engineered intervertebral disc (IVD) replacement.Objective: To determine whether a rehabilitative simulation regime on bone marrow–derived mesenchymal stem cell cell-sheet is able to aid the regeneration of the AF.Summary Of Background Data: No previous study has used bone marrow–derived mesenchymal stem cell cell-sheets simulated by a rehabilitative regime to regenerate the AF.Methods: The approach was to use bone marrow–derived stem cells to form cell-sheets and incorporating them onto silk scaffolds to simulate the native lamellae of the AF. The in vitro experimental model used to study the efficacy of such a system was made up of the tissue engineering AF construct wrapped around a silicone disc to form a simulated IVD-like assembly. The assembly was cultured within a custom-designed bioreactor that provided a compressive mechanical stimulation onto the silicone disc. The silicone nucleus pulposus would bulge radially and compress the simulated AF to mimic the physiological conditions. The simulated IVD-like assembly was compressed using a rehabilitative regime that lasted for 4 weeks at 0.25 Hz, for 15 minutes each day.Results: With the rehabilitative regime, the cell-sheets remained viable but showed a decrease in cell numbers and viability. Gene expression analysis showed significant upregulation of IVD-related genes and there was an increased ratio of collagen type II to collagen type I found within the extracellular matrix.Conclusion: The results suggested that a rehabilitative regime caused extensive remodeling to take place within the simulated IVD-like assembly, producing extracellular matrix similar to that found in the inner AF. [ABSTRACT FROM AUTHOR]

Study Design: Randomised double-blind placebo-controlled trial., Objectives: Multi-resistant organism (MRO) colonisation is common in people with SCI. We aimed to determine whether Lactobacillus reuteri RC-14 + Lactobacillus GR-1 (RC14-GR1) and/or Lactobacillus rhamnosus GG + Bifidobacterium BB-12 (LGG-BB12) are effective in preventing or clearing MRO colonisation., Setting: New South Wales, Australia., Methods: The 207 SCI participants were randomised to one of four arms: (i) RC14-GR1 + LGG-BB12, (ii) RC14-GR1 + placebo, (iii) LGG-BB12 + placebo or (iv) double placebos for 6 months. Microbiological samples of nose, groin, urine and bowel were taken at baseline, 3 and 6 months. Analysis was conducted for the presence of methicillin-resistant Staphylococcus aureus (MRSA), multi-resistant gram-negative organisms (MRGNs) and vancomycin-resistant enterococcus (VRE). The outcomes were clearance of, or new colonisation with MRSA, MRGN, VRE or MROs and whether participants remained free of MRSA, MRGN, VRE or MROs throughout the study. Risk factors associated with an outcome were adjusted for using nominal or binary logistic regression., Results: There was a significant reduction in new MRGN colonisation compared with placebo for participants treated with RC14-GR1 (OR 0.10, 95% CI, 0.01-0.88, P = 0.04), after allowing that inpatients were more likely to be newly colonised (OR 21.41, 95% CI, 3.98-115.13, P < 0.0001). Participants who intermittent self-catheterised (IMC) were more likely to remain MRO-free than those utilising SPC or IDCs (OR 2.80, 95% CI, 1.41-5.54, P = 0.009)., Conclusions: Probiotics are ineffective at clearing MROs in people with SCI. However, RC14-GR1 is effective at preventing new colonisation with MRGNs. The use of IMC significantly improves the chance of remaining MRO-free.

Study Design: Randomised double-blind factorial-design placebo-controlled trial., Objective: Urinary tract infections (UTIs) are common in people with spinal cord injury (SCI). UTIs are increasingly difficult to treat due to emergence of multi-resistant organisms. Probiotics are efficacious in preventing UTIs in post-menopausal women. We aimed to determine whether probiotic therapy with Lactobacillus reuteri RC-14+Lactobacillus GR-1 (RC14-GR1) and/or Lactobacillus rhamnosus GG+Bifidobacterium BB-12 (LGG-BB12) are effective in preventing UTI in people with SCI., Setting: Spinal units in New South Wales, Australia with their rural affiliations., Methods: We recruited 207 eligible participants with SCI and stable neurogenic bladder management. They were randomised to one of four arms: RC14-GR1+LGG-BB12, RC14-GR1+placebo, LGG-BB12+ placebo or double placebos for 6 months. Randomisation was stratified by bladder management type and inpatient or outpatient status. The primary outcome was time to occurrence of symptomatic UTI., Results: Analysis was based on intention to treat. Participants randomised to RC14-GR1 had a similar risk of UTI as those not on RC14-GR1 (HR 0.67; 95% CI: 0.39-1.18; P = 0.17) after allowing for pre-specified covariates. Participants randomised to LGG-BB12 also had a similar risk of UTI as those not on LGG-BB12 (HR 1.29; 95% CI: 0.74-2.25; P = 0.37). Multivariable post hoc survival analysis for RC14-GR1 only vs. the other three groups showed a potential protective effect (HR 0.46; 95% CI: 0.21-0.99; P = 0.03), but this result would need to be confirmed before clinical application., Conclusion: In this RCT, there was no effect of RC14-GR1 or LGG-BB12 in preventing UTI in people with SCI.

Background: Neuropathic or neurogenic bladder describes a process of dysfunctional voiding as the result of injury in the brain, spinal cord or nerves innervating the bladder. People with neuropathic bladder, such as from spinal cord injury (SCI), are at significant risk of morbidity from urinary tract infections (UTI). Effective methods to prevent UTI in people with SCI have been sought for many years. Probiotics (micro-organisms that exert beneficial health effects in the host) have been recommended for bacterial interference of the urological tract to reduce colonisation by uropathogen and to manage the dual problems of infection and antibiotic resistance., Objectives: This review looked at the benefits and harms of probiotics in preventing symptomatic UTI in people with neuropathic bladder compared with placebo, no therapy, or non-antibiotic prophylaxis (cranberry juice, methenamine hippurate, topical oestrogen)., Search Methods: We searched the Cochrane Kidney and Transplant Specialised Register up to 10 March 2017 through contact with the Information Specialist using search terms relevant to this review. Studies in the Specialised Register are identified through searches of CENTRAL, MEDLINE, and EMBASE, conference proceedings, the International Clinical Trials Register (ICTRP) Search Portal, and ClinicalTrials.gov., Selection Criteria: All randomised controlled trials (RCTs), quasi-RCTs and cross-over RCTs looking at the use of probiotics for the prophylaxis of UTI in people with neuropathic bladders was considered for inclusion. Men, women and children of all ages with neuropathic bladders from neurological injury such as suprapontine, supra sacral and sacral aetiologies was included. All bladder management types, including reflex voiding, time voiding, indwelling and intermittent catheterization were eligible for this review.Studies comparing probiotics to placebo, no treatment or other non-antibiotic prophylaxis was included. Studies comparing probiotics with antibiotics or in combination with antibiotics were excluded., Data Collection and Analysis: Summary estimates of effect were obtained using a random-effects model, and results were expressed as risk ratios (RR) and their 95% confidence intervals (CI) for dichotomous outcomes, and mean difference (MD) or standardised mean difference (SMD) and 95% CI were planned for continuous outcomes., Main Results: This review includes a total of three studies (one cross-over and two parallel RCTs) which involved 110 participants. All three studies looked at intravesical instillation of a low virulent Escherichia coli (E. coli) strain in reducing the risk of symptomatic UTI in participants with neuropathic bladder, predominantly from SCI. Two studies used the E. coli 83972 strain and one study used the E. coli HU2117 strain.We did not find any RCTs involving other probiotics or other routes of administration for preventing UTI in people with neuropathic bladder.There was consistency in definition of symptomatic UTI in all three studies. Symptoms that all studies considered were relevant to diagnose UTI were adequately defined. All three studies defined microbiological diagnosis of symptomatic UTI.Asymptomatic bacteriuria was not considered an outcome measure in any of the included studies; however it was defined in two studies to establish successful inoculation.It is uncertain if the risk of symptomatic UTI is reduced with bladder inoculation using E. coli because the certainty of the evidence is very low (3 studies, 110 participants: RR 0.32, 95% CI 0.08 to 1.19; I 2 = 82%).Two studies reported adverse events. One study reported one episode of autonomic dysreflexia. One study reported three symptomatic UTI occurring in two patients, and two studies mentioned the absence of septicaemia and pyelonephritis. Intravesical instillation was reported as "generally safe". One study reported high attrition rates in participants due to the need to adhere to strict instillation protocols.The overall quality of the studies was poor. All three studies had high risk of attrition bias due to failure of an intention-to-treat analysis which undermines the randomisation process and weakened the results of the studies. All three studies also had high risk of reporting bias., Authors' Conclusions: In this review, there were no studies identified addressing oral probiotics in preventing UTI in people with neuropathic bladder. It is uncertain if the risk of symptomatic UTI is reduced in people with neuropathic bladders via intravesical instillation of non-pathogenic E. coli as data were derived from small studies with high risk of bias.Although very minimal levels of harm was reported with this procedure, due to variable success rates, the need for strict adherence to instillation protocols together with high attrition rates in these studies, it is doubtful bladder instillation will be a widely accepted intervention in its current form.It is recommended that further appropriately powered RCTs with more robust methodological reporting be carried out.

Cell-sheet technology involves the recovery of cells with its secreted ECM and cell-cell junctions intact, and thereby harvesting them in a single contiguous layer. Temperature changes coupled with a thermoresponsive polymer grafted culture plate surface are typically used to induce detachment of this cell-matrix layer by controlling the hydrophobicity and hydrophilicity properties of the culture surface. This review article details the genesis and development of this technique as a critical tissue-engineering tool, with a comprehensive discussion on connective tissue applications. This includes applications in the myocardial, vascular, cartilage, bone, tendon/ligament, and periodontal areas among others discussed. In particular, further focus will be given to the use of stem cells-derived cell-sheets, such as those involving bone marrow-derived and adipose tissue-derived mesenchymal stem cells. In addition, some of the associated challenges faced by approaches using stem cells-derived cell-sheets will also be discussed. Finally, recent advances pertaining to technologies forming, detaching, and manipulating cell-sheets will be covered in view of the potential impact they will have on shaping the way cell-sheet technology will be utilized in the future as a tissue-engineering technique.

Recent studies have underlined the importance of matching scaffold properties to the biological milieu. Tissue, and thus scaffold, anisotropy is one such property that is important yet sometimes overlooked. Methods that have been used to achieve anisotropic scaffolds present challenges such as complicated fabrication steps, harsh processing conditions and toxic chemicals involved. In this study, unidirectional freezing was employed to fabricate anisotropic silk fibroin/gelatin scaffolds in a simple and mild manner. Morphological, mechanical, chemical and cellular compatibility properties were investigated, as well as the effect of the addition of gelatin to certain properties of the scaffold. It was shown that scaffold properties were suitable for cell proliferation and that mesenchymal stem cells were able to align themselves along the directed fibers. The fabricated scaffolds present a platform that can be used for anisotropic tissue engineering applications such as cardiac patches., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Anticancer drug discovery has been hampered by the lack of reliable preclinical models, which routinely use cells grown in two-dimensional (2D) culture systems. However, many of the characteristics of cells in 2D culture do not translate into the findings in animal xenografts. Three-dimensional (3D) growth may be responsible for some of these changes, and models using cells grown in 3D may form a more representative step in tumouricidal validation prior to animal implantation and human testing. For the 3D model, we cultured 143.98.2, SaOS2 or U2OS osteosarcoma cells seeded in porous Bombyx mori silk sponges. We conducted real-time PCR on cells grown in 2D culture and 3D scaffolds for the proliferation markers cyclin B1 and E2F1 and the actin regulator RhoA, and found a significant decrease in expression levels for the 3D tumour models (p = 0.02, < 0.001 and 0.008 for cyclin B1, E2F1 and RhoA for 143.98.2; p = 0.02, 0.002 and 0.02 for cyclin B1, E2F1 and RhoA for U2OS, respectively). In contrast, p21 was upregulated when SaOS2 and U2OS were cultured in the 3D scaffolds (p < 0.001) and there was no increase in DNA quantity during the culture period. We correspondingly observed G1 arrest when cell cycle analysis was conducted. Cytotoxicity results for cells treated with serial dilutions of doxorubicin and cisplatin showed that cells in 3D scaffolds were less sensitive to drug treatment than in 2D culture, and the difference was more pronounced for cell cycle specific agents. Copyright © 2013 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Cell-sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold-free constructs. Adipose tissue-derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow-derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC-CSs) is non-existent in the literature. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC-CSs development; and (b) the multipotentiality of ASC-CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid-supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC-CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT-PCR and histology at multiple time points. ASC-CSs remained healthy up to 3 weeks of hyperconfluent culture. One week-old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non-hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC-CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: Urinary tract infections [UTIs] are very common in people with Spinal Cord Injury [SCI]. UTIs are increasingly difficult and expensive to treat as the organisms that cause them become more antibiotic resistant. Among the SCI population, there is a high rate of multi-resistant organism [MRO] colonisation. Non-antibiotic prevention strategies are needed to prevent UTI without increasing resistance. Probiotics have been reported to be beneficial in preventing UTIs in post-menopausal women in several in vivo and in vitro studies. The main aim of this study is to determine whether probiotic therapy with combinations of Lactobacillus reuteri RC-14 + Lactobacillus rhamnosus GR-1 [RC14-GR1] and/or Lactobacillus rhamnosus GG + Bifidobacterium BB-12 [LGG-BB12] are effective in preventing UTI in people with SCI compared to placebo., Method: This is a multi-site randomised double-blind double-dummy placebo-controlled factorial design study conducted in New South Wales, Australia. All participants have a neurogenic bladder as a result of spinal injury. Recruitment started in April 2011. Participants are randomised to one of four arms, designed for factorial analysis of LGG-BB12 and/or RC14-GR1 v Placebo. This involves 24 weeks of daily oral treatment with RC14-GR1 + LGG-BB12, RC14-GR1 + placebo, LGG-BB12 + placebo or two placebo capsules. Randomisation is stratified by bladder management type and inpatient status. Participants are assessed at baseline, three months and six months for Short Form Health Survey [SF-36], microbiological swabs of rectum, nose and groin; urine culture and urinary catheters for subjects with indwelling catheters. A bowel questionnaire is administered at baseline and three months to assess effect of probiotics on bowel function. The primary outcome is time from randomisation to occurrence of symptomatic UTI. The secondary outcomes are change of MRO status and bowel function, quality of life and cost-effectiveness of probiotics in persons with SCI. The primary outcome will be analysed using survival analysis of factorial groups, with Cox regression modelling to test the effect of each treatment while allowing for the other, assuming no interaction effect. Hazard ratios and Kaplan-Meier survival curves will be used to summarise results., Discussion: If these probiotics are shown to be effective in preventing UTI and MRO colonisation, they would be a very attractive alternative for UTI prophylaxis and for combating the increasing rate of antibiotic resistance after SCI., Trial Registration: Australian New Zealand Clinical Trials Registry [ ACTRN 12610000512022 ]. Date of registration: 21 June 2010.

The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk-RADA peptide scaffold in a specially designed two-chambered co-culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two-chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold. We demonstrated that this co-culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage-like and subchondral bone-like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single-step approach that highlights the feasibility of rabbit BMSCs as a single-cell source for multilayered osteochondral construct generation in vitro., (Copyright © 2013 John Wiley & Sons, Ltd.)

Tissue engineering of a biological osteochondral multilayered construct with a cartilage-interface subchondral bone layer is a key challenge. This study presented a rabbit bone marrow stromal cell (BMSC)/silk fibroin scaffold-based co-culture approach to generate tissue-engineered osteochondral grafts with an interface. BMSC-seeded scaffolds were first cultured separately in osteogenic and chondrogenic stimulation media. The two differentiated pieces were then combined using an RADA self-assembling peptide and subsequently co-cultured. Gene expression, histological and biochemical analyses were used to evaluate the multilayered structure of the osteochondral graft. A complete osteochondral construct with a cartilage-subchondral bone interface was regenerated and BMSCs were used as the only cell source for the osteochondral construct and interface regeneration. Furthermore, in the intermediate region of co-cultured samples, hypertrophic chondrogenic gene markers type X collagen and MMP-13 were found on both chondrogenic and osteogenic section edges after co-culture. However, significant differences gene expression profile were found in distinct zones of the construct during co-culture and the section in the intermediate region had significantly higher hypertrophic chondrocyte gene expression. Biochemical analyses and histology results further supported this observation. This study showed that specific stimulation from osteogenic and chondrogenic BMSCs affected each other in this co-culture system and induced the formation of an osteochondral interface. Moreover, this system provided a possible approach for generating multilayered osteochondral constructs., (Copyright © 2013 John Wiley & Sons, Ltd.)

Silk is a versatile and established biomaterial for various tissue engineering purposes. However, it also exhibits strong autofluorescence signals-thereby hindering fluorescence imaging analysis of cells and proteins on silk-derived biomaterials. Sudan Black B (SB) is a lysochrome dye commonly used to stain lipids in histology. It has also been reported to be able to quench autofluorescence of tissues in histology and has been tested on artificial biomedical polymers in recent years. It was hypothesized that SB would exert similar quenching effects on silk, modulating the autofluorescence signals, and thereby enabling improved imaging analysis of cells and molecules of interests. The quenching effect of SB on the intrinsic fluorescence properties of silk and on commercial fluorescent dyes were first investigated in this study. SB was then incorporated into typical fluorescence-based staining protocols to study its effectiveness in improving fluorescence-based imaging of the cells and proteins residing with the silk-based biomaterials. Silk processed into various forms of biomaterials (e.g., films, sponges, fibers, and electrospun mats) was seeded with cells and cultured in vitro. At sacrificial time points, specimens were harvested, fixed, and prepared for fluorescence staining. SB, available commercially as a powder, was dissolved in 70% ethanol (0.3% [w/v]) to form staining solutions. SB treatment was introduced at the last step of typical immunofluorescence staining protocols for 15-120 min. For actin staining protocols by phalloidin toxin, SB staining solutions were added before and after permeabilization with Triton-X for 15-30 min. Results showed that ideal SB treatment duration is about 15 min. Apart from being able to suppress the autofluorescence of silk, this treatment duration was also not too long to adversely affect the fluorescent labeling probes used. The relative improvement brought about by SB treatment was most evident in the blue and green emission wavelengths compared with the red emission wavelength. This study has showed that the use of SB is a cost and time effective approach to enhance fluorescence-based imaging analyses of cell-seeded silk biomaterials, which otherwise would have been hindered by the unmodulated autofluorescence signals.

The interest in polymeric nanofibers has escalated over the past decade given its promise as tissue engineering scaffolds that can mimic the nanoscale structure of the native extracellular matrix. With functionalization of the polymeric nanofibers using bioactive molecules, localized signaling moieties can be established for the attached cells, to stimulate desired biological effects and direct cellular or tissue response. The inherently high surface area per unit mass of polymeric nanofibers can enhance cell adhesion, bioactive molecules loading and release efficiencies, and mass transfer properties. In this review article, the application of polymeric nanofibers for controlled bioactive molecules delivery will be discussed, with a focus on tendon and ligament tissue engineering. Various polymeric materials of different mechanical and degradation properties will be presented along with the nanofiber fabrication techniques explored. The bioactive molecules of interest for tendon and ligament tissue engineering, including growth factors and small molecules, will also be reviewed and compared in terms of their nanofiber incorporation strategies and release profiles. This article will also highlight and compare various innovative strategies to control the release of bioactive molecules spatiotemporally and explore an emerging tissue engineering strategy involving controlled multiple bioactive molecules sequential release. Finally, the review article concludes with challenges and future trends in the innovation and development of bioactive molecules delivery using polymeric nanofibers for tendon and ligament tissue engineering.

Poly (vinyl) alcohol (PVA) cryogels are reported in the literature for application in nucleus pulposus (NP) replacement strategies. However, these studies are mainly limited to acellular approaches-in part due to the high hydrophilicity of PVA gels that renders cellular adhesion difficult. Silk is a versatile biomaterial with excellent biocompatibility. We hypothesize that the incorporation of silk with PVA will (i) improve the cell-hosting abilities of PVA cryogels and (ii) allow better tailoring of physical properties of the composite cryogels for an NP tissue engineering purpose. 5% (wt/vol) PVA is blended with 5% silk fibroin (wt/vol) to investigate the effect of silk : PVA ratios on the cryogels' physical properties. Results show that the addition of silk results in composite cryogels that are able to swell to more than 10 times its original dry weight and rehydrate to at least 70% of its original wet weight. Adding at least 20% silk significantly improves surface hydrophobicity and is correlated with an improvement in cell-hosting abilities. Cell-seeded cryogels also display an increment in compressive modulus and hoop stress values. In all, adding silk to PVA creates cryogels that can be potentially used as NP replacements.

The induction of angiogenesis and the promotion of tumor growth and invasiveness are processes critical to metastasis, and are dependent on reciprocal interactions between tumor cells and their microenvironment. The formation of a clinically relevant tumor requires support from the surrounding stroma, and it is hypothesized that three-dimensional (3D) tumor coculture models offer a microenvironment that more closely resembles the physiological tumor microenvironment. In this study, we investigated the effects of tissue-engineered 3D architecture and tumor-stroma interaction on the angiogenic factor secretion profiles of U2OS osteosarcoma cells by coculturing the tumor cells with immortalized fibroblasts or human umbilical vein endothelial cells (HUVECs). We also carried out Transwell migration assays for U2OS cells grown in monoculture or fibroblast coculture systems to study the physiological effect of upregulated angiogenic factors on endothelial cell migration. Anti-IL-8 and anti-vascular endothelial growth factor (VEGF)-A therapies were tested out on these models to investigate the role of 3D culture and the coculture of tumor cells with immortalized fibroblasts on the efficacy of antiangiogenic treatments. The coculture of U2OS cells with immortalized fibroblasts led to the upregulation of IL-8 and VEGF-A, especially in 3D culture. Conversely, coculture with endothelial cells resulted in the downregulation of VEGF-A for cells seeded in 3D scaffolds. The migration of HUVECs through the Transwell polycarbonate inserts increased for the 3D and immortalized fibroblast coculture models, and the targeted inhibition of IL-8 greatly reduced HUVEC migration despite the presence of VEGF-A. A similar effect was not observed when anti-VEGF-A neutralizing antibody was used instead, suggesting that IL-8 plays a more critical role in endothelial cell migration than VEGF-A, with significant implications on the clinical utility of antiangiogenic therapy targeting VEGF-A.

In this article, low crystallinity hydroxyapatite (LHA) is developed and utilized to modify silk fibroin scaffolds which are applied to repair bone/ligament defects successfully. It can promote osteogenesis which is authenticated through in vitro and in vivo tests. The scaffold is an efficient carrier, supporting cell proliferation and differentiation. Meanwhile, cytocompatibility and osteoblastic gene expressions (RUNX2 and osteocalcin, for example) of rabbit's bone marrow derived mesenchymal stem cells (MSCs) are significantly boosted on LHA/silk scaffold. Further, for animal trial, almost 60% of bone volume and 80% of original mechanical strength are recovered after 4 months' bone/ligament regeneration in bone tunnel of rabbit model, where significant amount of bone tissue regeneration is also confirmed by data of histological evaluation and micro computed tomography (μ-CT). Hence, the invented scaffold is applicable for ligament/bone regeneration in future lager animal and clinical trials., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Topographical cell guidance has been utilized as a tissue-engineering technique to produce aligned cellular orientation in the regeneration of tendon- and ligament-like tissues. Other studies have investigated the effects of dynamic culture to achieve the same end. These works have, however, been limited to two-dimensional cultures, with focus given to the effects from the stimuli independently. The understanding of their combined effects in the tenogenic differentiation of mesenchymal stem cells (MSCs) has also been lacking. This study investigated the synergistic effects of mechanical stimulation on aligned MSCs in a three-dimensional (3D) aligned silk fibroin (SF) hybrid scaffold. Enhanced tenogenesis of seeded MSCs was observed in the scaffold group with aligned SF electrospun fibers (AL) under static culture conditions, as evidenced by the upregulation in expression and production of tendon/ligament-related proteins. The intensity and onset of these differentiative markers were increased and advanced, respectively, under dynamic culture conditions, indicative of an accelerated matrix deposition and remodeling process. Consequently, the tensile properties of dynamically cultured AL were significantly improved. We thus propose that the aligned hybrid SF scaffold facilitates mechanoactivity and tenogenic differentiation of MSCs by intensifying the positive effects of mechanical stimulation in a 3D environment.

Rehabilitation robots have direct physical interaction with human body. Ideally, actuators for rehabilitation robots should be compliant, force controllable, and back drivable due to safety and control considerations. Various designs of Series Elastic Actuators (SEA) have been developed for these applications. However, current SEA designs face a common performance limitation due to the compromise on the spring stiffness selection. This paper presents a novel compact compliant force control actuator design for portable rehabilitation robots to overcome the performance limitations in current SEAs. Our design consists of a servomotor, a ball screw, a torsional spring between the motor and the ball screw, and a set of translational springs between the ball screw nut and the external load. The soft translational springs are used to handle the low force operation and reduce output impedance, stiction, and external shock load. The torsional spring, being in the high speed range, has high effective stiffness and improves the system bandwidth in large force operation when the translational springs are fully compressed. This design is also more compact due to the smaller size of the springs. We explain the construction and the working principle of our new design, followed by the dynamic modeling and analysis of the actuator. We also show the preliminary testing results of a prototype actuator designed for a lower limb exoskeleton for gait rehabilitation.

Hybrid silk scaffolds combining knitted silk fibers and silk sponge have been recently developed for use as ligament-alone grafts. Incorporating an osteoinductive phase into the ends of a ligament scaffold may potentially generate an integrated "bone-ligament-bone" graft and improve graft osteointegration with host bone. To explore the possible application of hydroxyapatite (HA) coating in the fabrication of osteoinductive ends of silk-based scaffold, HA was coated on the hybrid silk scaffold and the effects to the bone-related cells were evaluated. HA could be coated in a uniform and controlled manner on the silk sponge, using an alternate soaking technology, with the amount deposited being dependent on the number of soaking cycles. HA coating also progressively reduced the hydrophobicity of silk surface (decreasing water contact angle from 87° to 42-76°, after 1-3 soaking cycles), making the HA-coated silk scaffold less favorable for initial cell attachments; but the attached cells showed viability and sustained proliferation on the HA-coated scaffold. As demonstrated by real-time polymerase chain reaction and alkaline phosphatase assay, the osteoinductivity of HA-coated silk scaffolds resulted in the osteogenic differentiation of bone marrow mesenchymal stem cells, and the osteoconductivity of HA-coated silk scaffolds supported osteoblasts growth and maintained the properties of mature osteoblasts. These properties of HA-coating demonstrated its possible application in fabricating osteoinductive ends of the silk-based ligament graft to potentially enhance graft-to-host bone integration., (Copyright © 2012 Wiley Periodicals, Inc.)

The ligament-bone interface is a complex structure that comprises ligament, fibrocartilage, and bone. We hypothesize that mesenchymal stem cells cocultured in between ligament and bone cells, on a hybrid silk scaffold with sections suitable for each cell type, would differentiate into fibrocartilage. The section of scaffold for osteoblast seeding was coated with hydroxyapatite. A trilineage coculture system (osteoblasts-BMSCs-fibroblasts) on a hybrid silk scaffold was established. RT-PCR results and immunohistochemistry results demonstrated that BMSCs cocultured between fibroblasts and osteoblasts had differentiated into the fibrocartilaginous lineage. The morphological change was also observed by SEM observation. A gradual transition from the uncalcified to the calcified region was formed in the cocultured BMSCs from the region that directly interacted with fibroblasts to the region that directly interacted with osteoblasts. The role of transforming growth factor β3 (TGF-β3) in this trilineage coculture model was also investigated by supplementing the coculture system with 10 ng/mL TGF-β3. The TGF-treated group showed similar results of fibrocartilaginous differentiation of BMSCs with coculture group without TGF-β3 supplement. However, no calcium deposition was found in the cocultured BMSCs in the TGF-treated group. This may indicate TGF-β3 delayed the mineralization process of chondrocytes.

Physiological osteochondral interface regeneration is a significant challenge. This study aims to investigate the effect of the coculture of chondrogenic rabbit bone marrow stromal cells (rBMSCs) with rabbit osteoblasts in a specially designed two-dimensional (2D)-three-dimensional (3D) co-interface culture to develop the intermediate osteochondral region in vitro. The 2D-3D coculture system was set up by first independently culturing chondrogenic rBMSCs on a scaffold and osteoblasts in cell culture plates, and subsequently placed in contact and cocultured. As control, samples not cocultured with osteoblasts were used. The regulatory effects exerted by osteoblasts on chondrogenic rBMSCs were quantified by real-time polymerase chain reaction. To study the effect of coculture on cells located in different parts of the scaffold, samples were separated into two parts and significantly different gene expression patterns were found between them. In comparison with the control group, a significant moderate downregulation of chondrogenic marker genes, such as Collagen II and Aggrecan was observed. However, the Sox-9 and Collagen I expression increased. More importantly, chondrogenic rBMSCs in the coculture system were shown to form the osteochondral interface layer by expressing calcified cartilage zone specific extracellular matrix marker Collagen X and the hypertrophic chondrocyte marker MMP-13, which were not observed in the control group. Specifically, only the chondrogenic rBMSC layer in contact with the osteoblasts expressed Collagen X and MMP-13, indicating the positive influence of the coculture upon interface formation. Biochemical analyses, histology results, and immunohistochemical staining further supported this observation. In conclusion, this study revealed that specific regulatory stimulations from osteoblasts in the 2D-3D interface coculture system could induce the formation of ostochondral interface for the purpose of osteochondral tissue engineering.

While silk-based microfibrous scaffolds possess excellent mechanical properties and have been used for ligament tissue-engineering applications, the microenvironment in these scaffolds is not biomimetic. We hypothesized that coating a hybrid silk scaffold with an extracellular matrix (ECM)-like network of self-assembling peptide nanofibers would provide a biomimetic three-dimensional nanofibrous microenvironment and enhance ligament tissue regeneration after bone marrow-derived mesenchymal stem cell (BMSC)-seeding. A novel scaffold possessing a triple structural hierarchy comprising macrofibrous knitted silk fibers, a silk microsponge, and a peptide nanofiber mesh was developed by coating self-assembled RADA16 peptide nanofibers on a silk microfiber-reinforced-sponge scaffold. Compared with the uncoated control, RADA-coated scaffolds showed enhanced BMSC proliferation, metabolism, and fibroblastic differentiation during the 3 weeks of culture. BMSC-seeded RADA-coated scaffolds showed an increasing temporal expression of key fibroblastic ECM proteins (collagen type I and III, tenascin-C), with a significantly higher tenascin-C expression compared with the controls. BMSC-seeded RADA-coated scaffolds also showed a temporal increase in total collagen and glycosaminoglycan production (the amount produced being higher than in control scaffolds) during 3 weeks of culture, and possessed 7% higher maximum tensile load compared with the BMSC-seeded control scaffolds. The results indicate that the BMSC-seeded RADA-coated hybrid silk scaffold system has the potential for use in ligament tissue-engineering applications.

Most studies on the intervertebral disc (IVD) focus on the regeneration of the nucleus pulposus (NP). However, without a proper strategy to regenerate the damaged annulus fibrosus (AF), the NP replacements are bound to fail. Therefore the objective of this study was to investigate whether the use of bone marrow-derived mesenchymal stem cells (BMSCs) to form cell sheets, and incorporating them onto silk scaffolds, has the potential to regenerate the annulus fibrosus. The BMSC cell sheets and silk scaffolds were wrapped around a silicone NP substitute to form a simulated IVD-like assembly. The simulated IVD-like assembly was cultured for 4 weeks in static conditions and it was shown that the BMSC cell sheets remained viable, with no significant change in cell numbers. Histological analysis showed that the BMSC cell sheets adhered well onto the silk scaffolds and glycosaminoglycans (GAGs) were detected within the extracellular matrix (ECM). The ratio of collagen type I to collagen type II within the ECM of the BMSC cell sheets also decreased significantly over the period of culture. The results suggested that extensive remodelling of the ECM occurred within the simulated IVD-like assembly, and it is suitable for the regeneration of the inner AF., (Copyright © 2011 John Wiley & Sons, Ltd.)

The lack of good preclinical models has hampered anticancer drug discovery. Standard preclinical protocols require the growth of cells in high throughput two-dimensional (2D) culture systems. However, such in vitro drug testing methods yield drug efficacy results that differ greatly from animal models. Conversely, it is much more difficult and expensive to use animal models for large-scale molecular biology research. It is conceivable that three-dimensional (3D) growth may be responsible for some of these changes. Porous silk sponges were fabricated through freeze drying and seeded with 143.98.2 osteosarcoma cells. Molecular profiles were obtained by carrying out real-time polymerase chain reaction for angiogenic growth factors and proliferation markers for osteosarcoma cells grown under 2D, 3D, and SCID mouse xenograft conditions. The angiogenic factor expression profiles for cells grown in 2D differed greatly from the 3D silk scaffold model (P < 0.05 for bFGF, HIF-1α, IL-8, and VEGF-A), whereas 3D tumor model profiles were found to be able to approximate that for the in vivo tumor better with no statistically different expression of HIF-1α and VEGF-A between the two. Immunohistochemistry staining for HIF-1α, VEGF-A, and VEGF receptor on osteosarcoma cells grown on the scaffolds validated the results obtained with the gene expression profiles. The results suggest that 3D tumor models could be used to bridge the gap between in vitro and in vivo tumor studies, and aid in the study of mechanisms activated during tumorigenesis for the development of novel targeted chemotherapy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The concept of contact guidance utilizes the phenomenon of anchorage dependence of cells on the topography of seeded surfaces. It has been shown in previous studies that cells were guided to align along the topographical alignment of the seeding substrate and produced enhanced amounts of oriented extracellular matrix (ECM). In this study, we aimed to apply this concept to a three-dimensional full silk fibroin (SF) hybrid scaffold system, which comprised of knitted SF and aligned SF electrospun fibers (SFEFs), for ligament tissue engineering applications. Specifically, knitted SF, which contributed to the mechanical robustness of the system, was integrated with highly aligned SFEF mesh, which acted as the initial ECM to provide environmental cues for positive cellular response. Mesenchymal stem cells seeded on the aligned hybrid scaffolds were shown to be proliferative and aligned along the integrated aligned SFEF, forming oriented spindle-shaped morphology and produced an aligned ECM network. Expression and production of ligament-related proteins were also increased as compared to hybrid SF scaffolds with randomly arranged SFEFs, indicating differentiative cues for ligament fibroblasts present in the aligned hybrid SF scaffolds. Consequently, the tensile properties of cultured aligned constructs were significantly improved and superior to the counterpart with randomly arranged SFEF. These results thus show that the aligned hybrid scaffold system is promising for enhancing cell proliferation, differentiation, and function for ligament tissue engineering applications.

Increasing incidence of musculoskeletal injuries coupled with limitations in the current treatment options have necessitated tissue engineering and regenerative medicine- based approaches. Moving forward from engineering isolated musculoskeletal tissues, research strategies are now being increasingly focused on repairing and regenerating the interfaces between dissimilar musculoskeletal tissues with the aim to achieve seamless integration of engineered musculoskeletal tissues. This article reviews the state-of-the-art in the tissue engineering of musculoskeletal tissue interfaces with a focus on Singapore's contribution in this emerging field. Various biomimetic scaffold and cellbased strategies, the use of growth factors, gene therapy and mechanical loading, as well as animal models for functional validation of the tissue engineering strategies are discussed.

In resonance with the NUS Mission, the aim of the Bioengineering undergraduate degree program is to produce engineers with a strong foundation in the relevant engineering, sciences and technology, who are able to contribute to the biomedical sciences through innovation, enterprise and leadership. Our educational program in Bioengineering is characterised by a strong emphasis on scientific and engineering fundamentals and a high degree of flexibility which can provide a wide diversity of educational experiences. By providing graduates with a combination of broad-based fundamentals and specialized knowledge, the Bioengineering program strives to graduate versatile engineers who would be best positioned to lead and be an integral part of the Bioengineering industries in the future. This paper describes the bioengineering program, both at undergraduate and postgraduate levels in the Division of Bioengineering at Faculty of Engineering in National University of Singapore.

A modified degumming technique, involving boiling in 0.25% Na2CO3 with addition of 1% sodium dodecyl sulphate and intermittent ultrasonic agitation, was developed for knitted silk scaffolds. Sericin was efficiently removed, while mechanical and structural properties of native silk fibroin were preserved. Biocompatible and mechanically robust hybrid nano-microscaffolds were fabricated by coating these degummed silk scaffolds with an intervening adhesive layer of silk solution followed by electrospun poly-lactic-co-glycolic acid (PLGA) nanofibers. Cell proliferation on the hybrid silk scaffolds was improved by seeding cells on both surfaces of the flat scaffolds. Rolling up and continued culture of the cell-seeded hybrid scaffolds yielded cylindrical constructs that permitted cell proliferation, extracellular matrix deposition, and generated ligament/tendon graft analogs. Although PLGA-based hybrid scaffolds have earlier been proposed for dense connective tissue engineering, rapid biodegradation of PLGA was a drawback. In contrast, the underlying strong and slowly-degrading microfibrous silk scaffold used in this study ensured that the hybrid scaffold maintained adequate mechanical properties for longer periods, which is vital for continued support to the injured ligament/tendon throughout its healing period.

Bio-electrospraying is fast becoming an attractive tool for in situ cell delivery into scaffolds for tissue engineering applications, with several cell types been successfully electrosprayed. Bone marrow derived mesenchymal progenitor/stem cells (BMSC), which are an important cell source for tissue engineering, have not been explored in detail and the effect of electrospraying on their "stemness" is not known. This study therefore investigates the effects of electrospraying on BMSC viability, proliferation, and multilineage differentiation potential. Electrospraying a BMSC suspension at flow rate of 6 mL/h and voltages of 7.5-15 kV could successfully generate a continuous, stable and linearly directed electrospray of cells. Morphological observation, trypan blue tests and alamar blue based metabolic assays revealed about 88% of these electrosprayed cells were viable, and proliferated at rates similar to native BMSCs. However, at higher voltages, electrospraying became unstable and reduced cell viability, possibly due to electrical or thermal damage to the cells. BMSCs electrosprayed at 7.5 kV also retained their multipotency and could be successfully differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating similar morphology and gene expression levels as induced native BMSCs. These results indicate that bio-electrospraying could be safely used as a progenitor/stem cell delivery technique for tissue engineering and regenerative medicine applications.

Tissue engineering scaffolds should ideally mimic the natural ECM in structure and function. Electrospun nanofibrous scaffolds are easily fabricated and possess a biomimetic nanostructure. Scaffolds can mimic ECM function by acting as a depot for sustained release of growth factors. bFGF, an important growth factor involved in tissue repair and mesenchymal stem cell proliferation and differentiation, is a suitable candidate for sustained delivery from scaffolds. In this study, we present two types of PLGA nanofibers incorporated with bFGF, fabricated using the facile technique of blending and electrospinning (Group I) and by the more complex technique of coaxial electrospinning (Group II). bFGF was randomly dispersed in Group I and distributed as a central core within Group II nanofibers; both scaffolds showed similar protein encapsulation efficiency and release over 1-2 weeks. Although both scaffold groups favored bone marrow stem cell attachment and subsequent proliferation, cells cultured on Group I scaffolds demonstrated increased collagen production and upregulated gene expression of specific ECM proteins, indicating fibroblastic differentiation. The study shows that the electrospinning technique could be used to prolong growth factor release from scaffolds and an appropriately sustained growth factor release profile in combination with a nanofibrous substrate could positively influence stem cell behavior and fate., ((c) 2009 Wiley Periodicals, Inc.)

In the process of removing sericin (degumming) from a raw silk scaffold, the fibroin structural integrity is often challenged, leading to mechanical depreciation. This study aims to identify the factors and conditions contributing to fibroin degradation during alkaline degumming and to perform an optimization study of the parameters involved to achieve preservation of fibro in structure and properties. The methodology involves degumming knitted silk scaffolds for various durations (5-90 min) and temperatures (60-100 ◦C). Mechanical agitation and use of the refreshed solution during degumming are included to investigate how these factors contribute to degumming efficiency and fibroin preservation. Characterizations of silk fibroin morphology, mechanical properties and protein components are determined by scanning electron microscopy (SEM), single fiber tensile tests and gel electrophoresis (SDS–PAGE),respectively. Sericin removal is ascertained via SEM imaging and a protein fractionation method involving SDS–PAGE. The results show that fibroin fibrillation, leading to reduced mechanical integrity, is mainly caused by prolonged degumming duration. Through a series of optimization, knitted scaffolds are observed to be optimally degummed and experience negligible mechanical and structural degradation when subjected to alkaline degumming with mechanical agitation for 30 min at 100 ◦C.

An ideal scaffold that provides a combination of suitable mechanical properties along with biological signals is required for successful ligament/tendon regeneration in mesenchymal stem cell-based tissue engineering strategies. Among the various fibre-based scaffolds that have been used, hybrid fibrous scaffolds comprising both microfibres and nanofibres have been recently shown to be particularly promising. This study developed a biohybrid fibrous scaffold system by coating bioactive bFGF-releasing ultrafine PLGA fibres over mechanically robust slowly-degrading degummed knitted microfibrous silk scaffolds. On the ECM-like biomimetic architecture of ultrafine fibres, sustained release of bFGF mimicked the ECM in function, initially stimulating mesenchymal progenitor cell (MPC) proliferation, and subsequently, their tenogeneic differentiation. The biohybrid scaffold system not only facilitated MPC attachment and promoted cell proliferation, with cells growing both on ultrafine PLGA fibres and silk microfibres, but also stimulated tenogeneic differentiation of seeded MPCs. Upregulated gene expression of ligament/tendon-specific ECM proteins and increased collagen production likely contributed to enhancing mechanical properties of the constructs, generating a ligament/tendon analogue that has the potential to be used to repair injured ligaments/tendons., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Bone-marrow-derived mesenchymal stem cells (BMSCs) are a promising source of cells for tissue engineering due to their multilineage mesenchymal differentiation potential. Their ability to proliferate and differentiate into the osteogenic, chondrogenic, and adipogenic lineage makes them an attractive cell source as compared to the terminally differentiated cells. In tissue engineering, use of cell sheet technology is gaining popularity. It is based on culturing cells until hyperconfluence, and it has resulted in the reduction of the number of cells lost when seeding onto scaffolds. Thus, formation of cell sheets with multipotent cells, such as BMSCs, would be a promising alternative to the conventional method of cell seeding, that is, single-cell suspension. However, the multilineage potential of BMSC cell sheets has yet to be verified. Therefore, the aim of this study was to characterize the formation of a hyperconfluent BMSC cell sheet as well as the effects of the hyperconfluent culture conditions on the multipotentiality of BMSCs. Our results showed that the BMSC cell sheets remained viable. The cell sheets were rich with type I collagen and were shown to have retained their multipotentiality. Hence, the use of BMSC cell sheets for tissue engineering application seems promising.

Background: Entheses are fibrocartilaginous organs that bridge ligament with bone at their interface and add significant insertional strength. To replace a severely damaged ligament, a tissue-engineered graft preinstalled with interfacial fibrocartilage, which is being regenerated from stem cells, appears to be more promising than ligament-alone graft. Such a concept can be realized by a biomimetic approach of establishing a dynamic communication of stem cells with bone cells and/or ligament fibroblasts in vitro., Aim: The current study has two objectives. The first objective is to demonstrate functional coculture of bone marrow-derived stem cells (BMSCs) with mature bone cells/ligament fibroblasts as evidenced by gap-junctional communication in vitro. The second objective is to investigate the role of BMSCs in the regeneration of fibrocartilage within the coculture., Materials & Methods: Rabbit bone/ligament fibroblasts were dual-stained with DiI-Red and calcein (gap-junction permeable dye), and cocultured with unlabeled BMSCs at fixed ratio (1:10). The functional gap junction was demonstrated by the transfer of calcein from donor to recipient cells that was confirmed and quantified by flow cytometry. Type 2 collagen (cartilage extracellular matrix-specific protein) expressed by the mixed cell lines in the cocultures were estimated by real-time reverse transcription PCR and compared with that of the ligament-bone coculture (control)., Results: Significant transfer of calcein into BMSCs was observed and flow cytometry analyses showed a gradual increase in the percentage of BMSCs acquiring calcein with time. Cocultures that included BMSCs expressed significantly more type 2 collagen compared with the control., Conclusion: The current study, for the first time, reported the expression of gap-junctional communication of BMSCs with two adherent cell lines of musculoskeletal system in vitro and also confirmed that incorporation of stem cells augments fibrocartilage regeneration. The results open up a path to envisage a composite graft preinstalled with enthesial fibrocartilage using a stem cell-based coculture system.

Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications., (2009 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Although in vivo studies in small animal model show the ligament regeneration by implanting mesenchymal stem cells (MSCs) and silk scaffold, large animal studies are still needed to evaluate the silk scaffold before starting a clinical trial. The aim of this study is to regenerate anterior cruciate ligament (ACL) in pig model. The micro-porous silk mesh was fabricated by incorporating silk sponges into knitted silk mesh with lyophilization. Then the scaffold was prepared by rolling the micro-porous silk mesh around a braided silk cord to produce a tightly wound shaft. In vitro study indicated that MSCs proliferated profusely on scaffold and differentiated into fibroblast-like cells by expressing collagen I, collagen III and tenascin-C genes in mRNA level. Then the MSCs-seeded scaffold was implanted in pig model to regenerate ACL. At 24 weeks postoperatively, the MSCs in regenerated ligament exhibited fibroblast morphology. The key ligament-specific extracellular matrix components were produced prominently and indirect ligament-bone insertion with three zones (bone, Sharpey's fibers and ligament) was observed. Although there was remarkable scaffold degradation, the maximum tensile load of regenerated ligament could be maintained after 24 weeks of implantation. In conclusion, the results imply that silk-based material has great potentials for clinical applications.

Aims: To characterise the mechanism of glycopeptide resistance, genetic relatedness, and pathogenicity factors in isolates of vancomycin-resistant enterococci (VRE) in Singapore., Methods: A total of 292 Enterococcus faecium and 17 Enterococcus faecalis were isolated from humans, and five E. faecium, two Enterococcus durans, two Enterococcus flavescens, one Enterococcus casseliflavus, and one Enterococcus gallinarum from chickens. The mechanism of glycopeptide resistance and pathogenicity factors were studied by PCR and the genetic relatedness determined by pulsed-field gel electrophoresis (PFGE), multi-locus variable number tandem repeat analysis (MLVA), and Tn1546 analysis., Results: There were five outbreak clones among the vancomycin-resistant E. faecium with one clone predominant. Four of the clones were vanB positive, and only one clone carried vanA. All outbreak clones were esp gene positive. Sporadic human isolates and chicken isolates were vanA positive and did not contain any pathogenicity genes. The situation was reversed in vancomycin-resistant E. faecalis where almost all isolates were vanA positive., Conclusions: Most VRE in Singapore is hospital associated with a small number of clones of esp-positive vanB E. faecium responsible for the majority of isolates.

Tissue engineering aims to regenerate tissues that can successfully take over the functions of the native tissue when it is damaged or diseased. In most tissues, collagen makes up the bulk component of the extracellular matrix, thus, there is great emphasis on its accurate quantification in tissue engineering. It has already been reported that pepsin digestion is able to solubilize the collagen deposited within the cell layer for accurate quantification of collagen content in cultures, but this method has drawbacks when cultured cells are hyperconfluent. In this condition, Pepsin digestion will result in fragments of the cell layers that cannot be completely resolved. These fragments of the undigested cell sheet are visible to the naked eye, which can bias the final results. To the best of our knowledge, there has been no reported method to accurately quantify the collagen content in hyperconfluent cell sheet. Therefore, this study aims to illustrate that sonication is able to aid pepsin digestion of hyperconfluent cell layers of fibroblasts and bone marrow mesenchymal stem cells, to solubilize all the collagen for accurate quantification purposes.

Campylobacter species are major enteric pathogens causing diarrhea illness in humans and animals. Immunological tests are needed for accurate and rapid identification of C. coli, in conjunction with the use of standard biochemical tests. We initiated the creation of monoclonal antibodies (MAbs) using whole C. coli cells as antigen. Four positive clones were identified, namely MAb2G6, MAb3B9, MAb4A10 and MAb5B9. Dot-blot assay and ELISA revealed that only MAb2G6 did not cross react with C. jejuni and other Campylobacter isolates. As demonstrated by dot-blot assay, MAb2G6 reacted with all 23 C. coli isolates tested but did not react with 29 isolates of C. jejuni, 3 other Campylobacter spp. isolates and 19 non-Campylobacter isolates, with the lowest detection limit was in the range of 10(3) to 10(4) bacteria. Western blots and dot blots showed that the antigen of MAb2G6 was a native protein, with immunoprecipitation assay showed that MAb2G6 bound to a protein band of approximately 43 kDa in size, corresponding to major outer membrane protein (MOMP) of C. coli revealed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). Immunofluorescence assay (IFA) showed that MOMP of C. coli was indeed the antigen of MAb2G6, with immunogold-electron microscopy demonstrated that MAb2G6 conjugated with immunogold particles bound to all over the surface of C. coli cells. MAb2G6 also showed potential usage in direct detection of C. coli in faecal samples.

Although most in vitro studies indicate that silk is a suitable biomaterial for ligament tissue engineering, in vivo studies of implanted silk scaffolds for ligament reconstruction are still lacking. The objective of this study is to investigate anterior cruciate ligament (ACL) regeneration using mesenchymal stem cells (MSCs) and silk scaffold. The scaffold was fabricated by incorporating microporous silk sponges into knitted silk mesh, which mimicked the structures of ligament extracellular matrix (ECM). In vitro culture demonstrated that MSCs on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of ligament-specific genes also increased with time. Then MSCs/scaffold was implanted to regenerate ACL in vivo. After 24 weeks, histology observation showed that MSCs were distributed throughout the regenerated ligament and exhibited fibroblast morphology. The key ligament ECM components including collagen I, collagen III, and tenascin-C were produced prominently. Furthermore, direct ligament-bone insertion with typical four zones (bone, mineralized fibrocartilage, fibrocartilage, ligament) was reconstructed, which resembled the native structure of ACL-bone insertion. The tensile strength of regenerated ligament also met the mechanical requirements. Moreover, its histological grading score was significantly higher than that of control. In conclusion, the results imply that silk scaffold has great potentials in future clinical applications.

The aim of this study was to compare the cellular responses of bone marrow-derived mesenchymal stem cells (BMSCs) and anterior cruciate ligament fibroblasts (ACLFs) on combined silk scaffolds for ligament tissue engineering application. Rabbit BMSCs and ACLFs were isolated and cultured in vitro for two weeks after seeding on the silk scaffolds. Samples were evaluated and compared for their cellular morphology, proliferation, gene and protein expression of tenascin-C, type I and type III collagen. In addition, the two cell types were transfected with green fluorescent protein (GFP) to trace their fate in the knee joints. Preliminary results comparing cell proliferation indicated that BMSCs proliferated faster than ACLFs. Gene expression of the phenotypic markers measured using real-time reverse transcription polymerase chain reaction (RT-PCR) indicated the transcript levels of BMSCs were significantly increased after two weeks of culture, whereas those of ACLFs had no significant difference. The protein levels and localization were determined by western blotting and immunohistochemical staining, the results showed more production of ligament-related extracellular matrix (ECM) by BMSCs as compared to ACLFs. Moreover, 4 weeks postoperatively, more fluorescent cells were presented in BMSC-loaded constructs than in ACLF-loaded constructs. Therefore, based on the cellular response in vitro and in vivo, BMSCs were found to be a better cell source than ACLFs for the further study of ACL tissue engineering.

The differentiation of mesenchymal stem cells (MSCs) towards fibroblasts is a crucial issue in ligament tissue engineering. This study aims to investigate the feasibility of using co-culture system to induce the differentiation of MSCs for constructing the tissue-engineered ligament in vitro. A kind of silk cable-reinforced gelatin/silk fibroin hybrid scaffold was used to provide three-dimensional (3-D) culture environments for MSCs. The 3-D co-culture system was set up by culturing MSCs/scaffold and ligament fibroblasts in the transwell insert and lower chamber, respectively. The regulatory effects of fibroblasts on MSCs were determined. After 2 weeks of co-culture the MSCs showed faster proliferation and higher DNA content compared with MSCs non-co-cultured. The MSCs were distributed uniformly throughout the scaffold and showed good viability. The collagen production also increased significantly with culture time. The MSCs in co-culture system were proved to differentiate into ligament fibroblasts by expressing ligament extra-cellular matrix (ECM)-specific genes including collagen I, collagen III, and tenascin-C in mRNA and protein level. The immunohistochemistry staining also confirmed the synthesis of key ligament ECM components. This study reveals that specific regulatory signals released from fibroblasts in 3-D co-culture system can enhance the differentiation of MSCs for ligament tissue engineering.

Cell seeding on knitted scaffolds often require a gel system, which was found to be practically unsuitable for anterior cruciate ligament (ACL) reconstruction as the cell-gel composite often gets dislodged from the scaffold in the in vivo dynamic situations. In order to solve this problem, we fabricated this combined silk scaffold with weblike microporous silk sponges formed in the openings of a knitted silk scaffold and subsequently combined with adult human bone marrow-derived mesenchymal stem cells (hMSCs) for in vitro ligament tissue engineering. Human MSCs adhered and grew well on the combined silk scaffolds. Moreover, in comparison with the knitted silk scaffolds seeded with hMSCs in fibroin gel the cellular function was more actively exhibited on the combined silk scaffolds, as evident by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ligament-related gene markers (e.g., type I, III collagen and tenascin-C), immunohistochemical and western blot evaluations of ligament-related extracellular matrix (ECM) components. While the knitted structure holds the microporous silk sponges together and provides the structural strength of the combined silk scaffold, the microporous structure of the silk sponges mimic the ECM which consequently promotes cell proliferation, function, and differentiation. This feature overcomes the limitation of knitted scaffold for ligament tissue engineering application.

Campylobacter species are important enteric pathogens causing disease in humans and animals. There is a lack of a good immunological test that can be used routinely to separate Campylobacter jejuni from other Campylobacter species. We produced monoclonal antibodies (MAbs) directed against the major outer membrane protein (MOMP) of C. jejuni using recombinant MOMP as the antigen. One MAb, designated MAb5C4 and of the immunoglobulin G1 isotype, was found to be potentially specific for C. jejuni. Dot blots demonstrated that MAb5C4 reacted with all 29 isolates of C. jejuni tested but did not react with 2 C. jejuni isolates, 26 other Campylobacter spp. isolates, and 19 non-Campylobacter isolates. Western blotting showed that MAb5C4 bound to a single protein band approximately 43 kDa in size, corresponding to the expected size of C. jejuni MOMP. The detection limit of MAb5C4 in a dot blot assay was determined to be about 5 x 10(3) bacteria. The epitope on the MOMP was mapped to a region six amino acids in length with the sequence 216GGQFNP221, which is 97% conserved among C. jejuni strains but divergent in other Campylobacter spp.; a GenBank search indicated that 95% of C. jejuni isolates will be able to be detected from non-Campylobacter spp. based on the highly specific and conserved region of the GGQFNP polypeptide. The epitope is predicted to be located in a region that is exposed to the periplasm. MAb5C4 is a potentially specific and sensitive MAb that can be used for the specific detection and identification of C. jejuni.

The objective of this study was to develop a silk cable-reinforced gelatin/silk fibroin hybrid scaffold for ligament tissue engineering. The scaffold was fabricated by lyophilizing the cross-linked gelatin and silk fibroin mixture with braided silk cables. Scanning electronic microscopy (SEM) observation showed that microporous gelatin/silk fibroin sponges formed around silk cables mimicked the microstructures of ligament extracellular matrix (ECM). The silk cables significantly increased the tensile strength of the scaffold to meet the mechanical requirements for ligament tissue engineering. The scaffold possessed good cell adhesion property, and when mesenchymal stem cells (MSCs) were seeded on it, cells proliferated profusely. After 2 weeks of culture, seeded MSCs were distributed uniformly throughout the scaffold and were highly viable. Occurrence of cell death during culture was not significant. Deposition of collagen on the scaffold was found to increase with time. Differentiation of MSCs into ligament fibroblasts was verified by expressions of ligament ECM specific genes including collagen type I, collagen type III, and tenascin-C in mRNA and protein level. Immunohistochemistry stains also confirmed the production of key ligament ECM components on the scaffold. The results demonstrate that silk cable-reinforced gelatin/silk fibroin scaffold possesses the appropriate mechanical properties and has enlarged surface area. It is also capable of supporting cell proliferation and differentiation for ligament tissue engineering.