Advanced tissue engineering scaffold design for regeneration of the complex hierarchical periodontal structure

AIM: This study investigated the ability of an osteoconductive biphasic scaffold to simultaneously regenerate alveolar bone, periodontal ligament and cementum. MATERIALS AND METHODS: A biphasic scaffold was built by attaching a fused deposition modelled bone compartment to a melt electrospun periodontal compartment. The bone compartment was coated with a calcium phosphate (CaP) layer for increasing osteoconductivity, seeded with osteoblasts and cultured in vitro for 6 weeks. The resulting constructs were then complemented with the placement of PDL cell sheets on the periodontal compartment, attached to a dentin block and subcutaneously implanted into athymic rats for 8 weeks. Scanning electron microscopy, X-ray diffraction, alkaline phosphatase and DNA content quantification, confocal laser microscopy, micro computerized tomography and histological analysis were employed to evaluate the scaffold's performance. RESULTS: The in vitro study showed that alkaline phosphatase activity was significantly increased in the CaP-coated samples and they also displayed enhanced mineralization. In the in vivo study, significantly more bone formation was observed in the coated scaffolds. Histological analysis revealed that the large pore size of the periodontal compartment permitted vascularization of the cell sheets, and periodontal attachment was achieved at the dentin interface. CONCLUSIONS: This work demonstrates that the combination of cell sheet technology together with an osteoconductive biphasic scaffold could be utilized to address the limitations of current periodontal regeneration techniques.

Biofabrication of customized bone grafts by combination of additive manufacturing and bioreactor knowhow

This study reports on an original concept of additive manufacturing for the fabrication of tissue engineered constructs (TEC), offering the possibility of concomitantly manufacturing a customized scaffold and a bioreactor chamber to any size and shape. As a proof of concept towards the development of anatomically relevant TECs, this concept was utilized for the design and fabrication of a highly porous sheep tibia scaffold around which a bioreactor chamber of similar shape was simultaneously built. The morphology of the bioreactor/scaffold device was investigated by micro-computed tomography and scanning electron microscopy confirming the porous architecture of the sheep tibiae as opposed to the non-porous nature of the bioreactor chamber. Additionally, this study demonstrates that both the shape, as well as the inner architecture of the device can significantly impact the perfusion of fluid within the scaffold architecture. Indeed, fluid flow modelling revealed that this was of significant importance for controlling the nutrition flow pattern within the scaffold and the bioreactor chamber, avoiding the formation of stagnant flow regions detrimental for in vitro tissue development. The bioreactor/scaffold device was dynamically seeded with human primary osteoblasts and cultured under bi-directional perfusion for two and six weeks. Primary human osteoblasts were observed homogenously distributed throughout the scaffold, and were viable for the six week culture period. This work demonstrates a novel application for additive manufacturing in the development of scaffolds and bioreactors. Given the intrinsic flexibility of the additive manufacturing technology platform developed, more complex culture systems can be fabricated which would contribute to the advances in customized and patient-specific tissue engineering strategies for a wide range of applications.

Vaccination of koalas (Phascolarctos cinereus) with a recombinant chlamydial major outer membrane protein adjuvanted with poly I:C, a host defense peptide and polyphosphazine, elicits strong and long lasting cellular and humoral immune responses

Chlamydial infections are wide spread in koalas across their range and a solution to this debilitating disease has been sought for over a decade. Antibiotics are the currently accepted therapeutic measure, but are not an effective treatment due to the asymptomatic nature of some infections and a low efficacy rate. Thus, a vaccine would be an ideal way to address this infectious disease threat in the wild. Previous vaccine trials have used a three-dose regimen; however this is very difficult to apply in the field as it would require multiple capture events, which are stressful and invasive processes for the koala. In addition, it requires skilled koala handlers and a significant monetary investment. To overcome these challenges, in this study we utilized a polyphosphazine based poly I:C and a host defense peptide adjuvant combined with recombinant chlamydial major outer membrane protein (rMOMP) antigen to induce long lasting (54 weeks) cellular and humoral immunity in female koalas with a novel single immunizing dose. Immunized koalas produced a strong IgG response in plasma, as well as at mucosal sites. Moreover, they showed high levels of C. pecorum specific neutralizing antibodies in the plasma as well as vaginal and conjunctival secretions. Lastly, Chlamydia-specific lymphocyte proliferation responses were produced against both whole chlamydial elementary bodies and rMOMP protein, over the 12-month period. The results of this study suggest that a single dose rMOMP vaccine incorporating a poly I:C, host defense peptide and polyphosphazine adjuvant is able to stimulate both arms of the immune system in koalas, thereby providing an alternative to antibiotic treatment and/or a three-dose vaccine regime.

Does failed chronic wet cough response to antibiotics predict bronchiectasis?

Aim: To determine whether a child with chronic wet cough and poor response to at least 4 weeks of oral antibiotics is more likely to have bronchiectasis. Methods: All chest multi-detector computerised tomography (MDCT) scans at a single paediatric tertiary hospital from April 2010 to August 2012 were reviewed retrospectively so as to identify those ordered by respiratory physicians for assessment of children with a chronic wet cough. Information regarding age, sex, ethnicity, indication for imaging and the response to at least 4 weeks of antibiotics before having the scan were recorded from their charts. The data were analysed using simple and multiple logistic regression. Results: Of the 144 (87 males) eligible children, 106 (65 males, 30 Indigenous) aged 10–199 months had MDCT scan evidence of bronchiectasis. Antibiotic data were available for 129 children. Among the 105 children with persistent cough despite at least 4 weeks of antibiotics, 88 (83.8%) had bronchiectasis, while of the 24 children whose cough resolved after antibiotics, only six (25.0%) received this diagnosis (adjusted OR 20.9; 95% CI 5.36 to 81.8). Being Indigenous was also independently associated with radiographic evidence of bronchiectasis (adjusted OR 5.86; 95% CI 1.20 to 28.5). Conclusions: Further investigations including a MDCT scan should be considered in a child with a chronic wet cough that persists following 4 weeks of oral antibiotics. However, while reducing the likelihood of underlying bronchiectasis, responding well to a single prolonged course of antibiotics does not exclude this diagnosis completely.

Molecular and pathological insights into Chlamydia pecorum-associated sporadic bovine encephalomyelitis (SBE) in Western Australia

Background Despite its global recognition as a ruminant pathogen, cases of Chlamydia pecorum infection in Australian livestock are poorly documented. In this report, a C. pecorum specific Multi Locus Sequence Analysis scheme was used to characterise the C. pecorum strains implicated in two cases of sporadic bovine encephalomyelitis confirmed by necropsy, histopathology and immunohistochemistry. This report provides the first molecular evidence for the presence of mixed infections of C. pecorum strains in Australian cattle. Case presentation Affected animals were two markedly depressed, dehydrated and blind calves, 12 and 16 weeks old. The calves were euthanized and necropsied. In one calf, a severe fibrinous polyserositis was noted with excess joint fluid in all joints whereas in the other, no significant lesions were seen. No gross abnormalities were noted in the brain of either calf. Histopathological lesions seen in both calves included: multifocal, severe, subacute meningoencephalitis with vasculitis, fibrinocellular thrombosis and malacia; diffuse, mild, acute interstitial pneumonia; and diffuse, subacute epicarditis, severe in the calf with gross serositis. Immunohistochemical labelling of chlamydial antigen in brain, spleen and lung from the two affected calves and brain from two archived cases, localised the antigen to the cytoplasm of endothelium, mesothelium and macrophages. C. pecorum specific qPCR, showed dissemination of the pathogen to multiple organs. Phylogenetic comparisons with other C. pecorum bovine strains from Australia, Europe and the USA revealed the presence of two genetically distinct sequence types (ST). The predominant ST detected in the brain, heart, lung and liver of both calves was identical to the C. pecorum ST previously described in cases of SBE. A second ST detected in an ileal tissue sample from one of the calves, clustered with previously typed faecal bovine isolates. Conclusion This report provides the first data to suggest that identical C. pecorum STs may be associated with SBE in geographically separated countries and that these may be distinct from those found in the gastrointestinal tract. This report provides a platform for further investigations into SBE and for understanding the genetic relationships that exist between C. pecorum strains detected in association with other infectious diseases in livestock.

Simulated microgravity affects chondrogenesis and hypertrophy of human mesenchymal stem cells

Purpose During in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) hypertrophy is an inadvertent event associated with cell differentiation toward the osteogenic lineage. Up to now, there is no stringent experimental control mechanism to prevent hypertrophy of MSCs. Microgravity is known to have an impact on osteogenesis. In this study, the influence of simulated microgravity (SMG) on both chondrogenesis and hypertrophy of hMSCs was evaluated. Methods A bioreactor using a rotating wall vessel was constructed to simulate microgravity. Pellet cultures formed from hMSCs (P5) were supplemented with human transforming growth factor-β3 (TGF-β3). The hMSC pellet cultures treated with TGF-β3 were either kept in SMG or in a control system. After three weeks of culture, the chondrogenic differentiation status and level of hypertrophy were examined by safranin-O staining, immunohistochemistry and quantitative real-time PCR. Results SMG reduced the staining for safranin-O and collagen type II. The expression of collagen type X α1 chain (COL10A1) and collagen type II α1 chain (COL2A1) were both significantly reduced. There was a higher decrease in COL2A1 than in COL10A1 expression, resulting in a low COL2A1/COL10A1 ratio. Conclusions SMG reduced hypertrophy of hMSCs during chondrogenic differentiation. However, the expression of COL2A1 was likewise reduced. Even more, the COL2A1/COL10A1 ratio decreased under SMG conditions. We therefore assume that SMG has a significant impact on the chondrogenic differentiation of hMSCs. However, due to the high COL2A1 suppression under SMG, this culture system does not yet seem to be suitable for a potential application in cartilage repair.

Validation of a Spanish version of the Spine Functional Index

Background The Spine Functional Index (SFI) is a recently published, robust and clinimetrically valid patient reported outcome measure. Objectives The purpose of this study was the adaptation and validation of a Spanish-version (SFI-Sp) with cultural and linguistic equivalence. Methods A two stage observational study was conducted. The SFI was cross-culturally adapted to Spanish through double forward and backward translation then validated for its psychometric characteristics. Participants (n = 226) with various spine conditions of >12 weeks duration completed the SFI-Sp and a region specific measure: for the back, the Roland Morris Questionnaire (RMQ) and Backache Index (BADIX); for the neck, the Neck Disability Index (NDI); for general health the EQ-5D and SF-12. The full sample was employed to determine internal consistency, concurrent criterion validity by region and health, construct validity and factor structure. A subgroup (n = 51) was used to determine reliability at seven days. Results The SFI-Sp demonstrated high internal consistency (α = 0.85) and reliability (r = 0.96). The factor structure was one-dimensional and supported construct validity. Criterion specific validity for function was high with the RMQ (r = 0.79), moderate with the BADIX (r = 0.59) and low with the NDI (r = 0.46). For general health it was low with the EQ-5D and inversely correlated (r = −0.42) and fair with the Physical and Mental Components of the SF-12 and inversely correlated (r = −0.56 and r = −0.48), respectively. The study limitations included the lack of longitudinal data regarding other psychometric properties, specifically responsiveness. Conclusions The SFI-Sp was demonstrated as a valid and reliable spine-regional outcome measure. The psychometric properties were comparable to and supported those of the English-version, however further longitudinal investigations are required.

Histopathological features of bone regeneration in a canine segmental ulnar defect model

Background Today, finding an ideal biomaterial to treat the large bone defects, delayed unions and non-unions remains a challenge for orthopaedic surgeions and researchers. Several studies have been carried out on the subject of bone regeneration, each having its own advantages. The present study has been designed in vivo to evaluate the effects of cellular auto-transplantation of tail vertebrae on healing of experimental critical bone defect in a dog model. Methods Six indigenous breeds of dog with 32 ± 3.6 kg average weight from both sexes (5 males and 1 female) received bilateral critical-sized ulnar segmental defects. After determining the health condition, divided to 2 groups: The Group I were kept as control I (n = 1) while in Group II (experimental group; n = 5) bioactive bone implants were inserted. The defects were implanted with either autogeneic coccygeal bone grafts in dogs with 3-4 cm diaphyseal defects in the ulna. Defects were stabilized with internal plate fixation, and the control defects were not stabilized. Animals were euthanized at 16 weeks and analyzed by histopathology. Results Histological evaluation of this new bone at sixteen weeks postoperatively revealed primarily lamellar bone, with the formation of new cortices and normal-appearing marrow elements. And also reformation cortical compartment and reconstitution of marrow space were observed at the graft-host interface together with graft resorption and necrosis responses. Finally, our data were consistent with the osteoconducting function of the tail autograft. Conclusions Our results suggested that the tail vertebrae autograft seemed to be a new source of autogenous cortical bone in order to supporting segmental long bone defects in dogs. Furthermore, cellular autotransplantation was found to be a successful replacement for the tail vertebrae allograft bone at 3-4 cm segmental defects in the canine mid- ulna. Clinical application using graft expanders or bone autotransplantation should be used carefully and requires further investigation.

The effect of β-alanine and NaHCO3 co-ingestion on buffering capacity and exercise performance with high-intensity exercise in healthy males

Introduction β-alanine (BAl) and NaHCO3 (SB) ingestion may provide performance benefits by enhancing concentrations of their respective physiochemical buffer counterparts, muscle carnosine and blood bicarbonate, counteracting acidosis during intense exercise. This study examined the effect of BAl and SB co-supplementation as an ergogenic strategy during high-intensity exercise. Methods Eight healthy males ingested either BAl (4.8 g day−1 for 4 weeks, increased to 6.4 g day−1 for 2 weeks) or placebo (Pl) (CaCO3) for 6 weeks, in a crossover design (6-week washout between supplements). After each chronic supplementation period participants performed two trials, each consisting of two intense exercise tests performed over consecutive days. Trials were separated by 1 week and consisted of a repeated sprint ability (RSA) test and cycling capacity test at 110 % Wmax (CCT110 %). Placebo (Pl) or SB (300 mg kgbw−1) was ingested prior to exercise in a crossover design to creating four supplement conditions (BAl-Pl, BAl-SB, Pl–Pl, Pl-SB). Results Carnosine increased in the gastrocnemius (n = 5) (p = 0.03) and soleus (n = 5) (p = 0.02) following BAl supplementation, and Pl-SB and BAl-SB ingestion elevated blood HCO3 − concentrations (p < 0.01). Although buffering capacity was elevated following both BAl and SB ingestion, performance improvement was only observed with BAl-Pl and BAl-SB increasing time to exhaustion of the CCT110 % test 14 and 16 %, respectively, compared to Pl–Pl (p < 0.01). Conclusion Supplementation of BAl and SB elevated buffering potential by increasing muscle carnosine and blood bicarbonate levels, respectively. BAl ingestion improved performance during the CCT110 %, with no aggregating effect of SB supplementation (p > 0.05). Performance was not different between treatments during the RSA test.

Altered nociceptive, endocrine, and dorsal horn neuron responses in rats following a neonatal immune challenge

Summary The neonatal period is characterized by significant plasticity where the immune, endocrine, and nociceptive systems undergo fine-tuning and maturation. Painful experiences during this period can result in long-term alterations in the neurocircuitry underlying nociception, including increased sensitivity to mechanical or thermal stimuli. Less is known about the impact of neonatal exposure to mild inflammatory stimuli, such as lipopolysaccharide (LPS), on subsequent inflammatory pain responses. Here we examine the impact of neonatal LPS exposure on inflammatory pain sensitivity and HPA axis activity during the first three postnatal weeks. Wistar rats were injected with LPS (0.05 mg/kg IP, Salmonella enteritidis) or saline on postnatal days (PNDs) 3 and 5 and later subjected to the formalin test at PNDs 7, 13, and 22. One hour after formalin injection, blood was collected to assess corticosterone responses. Transverse spinal cord slices were also prepared for whole-cell patch clamp recording from lumbar superficial dorsal horn neurons (SDH). Brains were obtained at PND 22 and the hypothalamus was isolated to measure glucocorticoid (GR) and mineralocorticoid receptor (MR) transcript expression using qRT-PCR. Behavioural analyses indicate that at PND 7, no significant differences were observed between saline- or LPS-challenged rats. At PND 13, LPS-challenged rats exhibited enhanced licking (p < .01), and at PND 22, increased flinching in response to formalin injection (p < .05). LPS-challenged rats also displayed increased plasma corticosterone at PND 7 and PND 22 (p < .001) but not at PND 13 following formalin administration. Furthermore, at PND 22 neonatal LPS exposure induced decreased levels of GR mRNA and increased levels of MR mRNA in the hypothalamus. The intrinsic properties of SDH neurons were similar at PND 7 and PND 13. However, at PND 22, ipsilateral SDH neurons in LPS-challenged rats had a lower input resistance compared to their saline-challenged counterparts (p < .05). These data suggest neonatal LPS exposure produces developmentally regulated changes in formalin-induced behavioural responses, corticosterone levels, and dorsal horn neuron properties following noxious stimulation later in life. These findings highlight the importance of immune activation during the neonatal period in shaping pain sensitivity later in life. This programming involves both spinal cord neurons and the HPA axis.

Implementing a context-based environmental science unit in the middle years: Teaching and learning at the creek

Engaging middle-school students in science continues to be a challenge in Australian schools. One initiative that has been tried in the senior years but is a more recent development in the middle years is the context-based approach. In this ethnographic study, we researched the teaching and learning transactions that occurred in one 9th grade science class studying a context-based Environmental Science unit that included visits to the local creek for 11 weeks. Data were derived from field notes, audio and video recorded conversations, interviews, student journals and classroom documents with a particular focus on two selected groups of students. This paper presents two assertions that highlight pedagogical approaches that contributed to learning. Firstly, spontaneous teaching episodes created opportunities for in-the-moment questioning by the teacher that led to students’ awareness of environmental issues and the scientific method; secondly, group work using flip cameras afforded opportunities for students to connect the science concepts with the context. Furthermore, students reported positively about the unit and expressed their appreciation for the opportunity to visit the creek frequently. This findings from this study should encourage teachers to take students into the real-world field for valuable teaching and learning experiences that are not available in the formal classroom.

Changes in lycopene and beta carotene contents in aril and oil of gac fruit during storage

Gac fruits were physically measured and stored under ambient conditions for up to 2 weeks to observe changes in carotenoid contents (lycopene and beta carotene) in its aril. Initial concentrations in the aril of lycopene were from 2.378 mg/g fresh weight (FW) to 3.728 mg/g FW and those of beta carotene were from 0.257 to 0.379 mg/g FW. Carotenoid concentrations in the aril remained stable after 1 week but sharply declined after 2 weeks of storage. Gac oil, pressed from gac aril, has similar concentrations of lycopene and beta carotene (2.436 and 2.592 mg/g, respectively). Oil was treated with 0.02% of butylated hydroxytoluene, or with a stream of nitrogen or untreated then stored in the dark for up to 15 or 19 weeks under different temperatures (5 °C, ambient, 45 and 60 °C). Lycopene and beta carotene in control gac oil degraded following the first-order kinetic model. The degradation rate of lycopene and beta carotene in the treated oil samples were lower than that in the control oil but the first-order kinetic was not always followed. However, both lycopene and beta carotene degraded quickly in gac oil with the first-order kinetic under high temperature conditions (45 and 60 °C) regardless of the treatments used. © 2009 Elsevier Ltd. All rights reserved.

Current management of transitional feeding issues in preterm neonates born in Queensland, Australia

Background: Many preterm neonates display difficulty establishing suck-feeding competence in the weeks following birth. Ineffective management of transitional feeding issues may cause patient complications, and can contribute to increased length of stay. Aims: Given that many neonatal nurseries appear to vary in their neonatal feeding management practices, the aim of this study was to investigate and document the routine level of support and intervention currently provided for preterm neonates with transitional feeding issues across the various level II (special care) nurseries (SCNs) in Queensland, Australia. Methods: A questionnaire was mailed to all Queensland SCNs in 2005 (n = 36). The questionnaire contained a series of closed-choice and short-answer questions designed to obtain information from each SCN regarding their current practices for managing transitional feeding issues in preterm neonates. Results were confirmed during a follow-up phone call. Results: Responses were obtained from 29 SCNs (80.6%). None of these nurseries reported having any formal, written policies regarding the management of transitional feeding issues in preterm neonates. Wide variations were reported in relation to the suck-feeding assessments and interventions used by staff within the various SCNs. Of the 29 nurseries, 4 (13.8%) reported using checklists or assessments to judge readiness for suck-feeds, and 5 (17.2%) reported using pulse oximetry to judge tolerance of suck-feeding attempts. Eighteen SCNs (62.1%) reported offering some form of active intervention to assist neonates with transitional feeding issues, with the most common intervention techniques reported being non-nutritive sucking during tube feeds, pre-feeding oral stimulation, and actively pacing suck-feeds. Twenty-two SCNs (75.4%) reported having access to a lactation consultant to assist mothers with breastfeeding issues. Conclusions: Differences were reported in the routine management of transitional feeding issues in preterm neonates across the various SCNs in Queensland. It is suggested that evidence based guidelines need to be developed, and that, in order to do this, further research studies are required to determine current best practice, as well as to answer remaining questions. © 2008 Elsevier Ireland Ltd. All rights reserved.

Attainment of early feeding milestones in preterm neonates

Objective: This study aimed to document the ages at which preterm neonates commence suckle-feeds and attain exclusive suckle-feeding, as well as the time taken to transition from commencement of suckle-feeds to exclusive suckle-feeding. It was hypothesized that gestational age (GA) at birth and degree of neonatal morbidity would influence the timing of these early feeding milestones. Study Design: A chart review was conducted for all neonates born <37;0 weeks GA admitted to a tertiary level perinatal facility over a 12-month period (n=735). Complete data relating to attainment of feeding milestones were available on 472 neonates. Results: Correlation analysis indicated that both a low GA at birth and a high neonatal morbidity rating were statistically significantly correlated with an increased transition time from commencement of suckle-feeds to exclusive suckle-feeding. Cox regression indicated that both of these variables were statistically significant risk factors for a delayed GA at attainment of exclusive suckle-feeding. Conclusion: Preterm neonates who were less mature at birth and/or who displayed a greater degree of neonatal morbidity took longer to transition from starting suckle-feeds to achieving independent suckle-feeding, and were more mature at attainment of independent suckle-feeding.

Tissue engineered humanized bone supports human hematopoiesis in vivo

Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches.