Polycaprolactone-based scaffold plus recombinant human bone morphogenetic protein (rhBMP-2) in an ovine model of anterior spinal fusion

Synthetic scaffolds combined with growth factors have the potential to replace allograft or autograft as a graft material for spinal interbody fusion. Such tissue engineering approaches may be useful in Adolescent Idiopathic Scoliosis (AIS) surgery, however there are no studies to date examining the use of such biodegradable implants in combination with biologics in a thoracic spine model. This in vivo study examines the use of biodegradable polycaprolactone (PCL) based scaffolds with rhBMP-2 as a bone graft substitute in a sheep thoracic fusion model, where an anterior approach is used to simulate minimally invasive surgical deformity correction in the setting of AIS.

Fabrication of a corneal-limbal tissue substitute using silk fibroin

Fibroin extracted from silkworm cocoon silk provides an intriguing and potentially important biomaterial for corneal reconstruction. In the present chapter we outline our methods for producing a composite of two fibroin-based materials that supports the co-cultivation of human limbal epithelial (HLE) cells and human limbal stromal (HLS) cells. The resulting tissue substitute consists of a stratified epithelium overlying a three-dimensional arrangement of extracellular matrix components (principally ‘degummed’ fibroin fibers) and mesenchymal stromal cells. This tissue substitute is currently being evaluated as a tool for reconstructing the corneal limbus and corneal epithelium.

Polycaprolactone-based scaffold plus recombinant human bone morphogenic protein rhBMP-2) in a sheep thoracic spine fusion model

Adolescent idiopathic scoliosis is a complex three dimensional deformity affecting 2-3% of the general population. The resulting spinal deformity consists of coronal curvature, hypokyphosis of the thoracic spine and vertebral rotation in the axial plane with posterior elements turned into the curve concavity. The potential for curve progression is heightened during the adolescent growth spurt. Success of scoliosis deformity correction depends on solid bony fusion between adjacent vertebrae after the intervertebral (IV) discs have been surgically cleared and the disc spaces filled with graft material. Recently a bioactive and resorbable scaffold fabricated from medical grade polycaprolactone has been developed for bone regeneration at load bearing sites. Combined with rhBMP-2, this has been shown to be successful in acting as a bone graft substitute in a porcine lumbar interbody fusion model when compared to autologous bone graft alone. The study aimed to establish a large animal thoracic spine interbody fusion model, develop spine biodegradable scaffolds (PCL) in combination with biologics (rhBMP-2) and to establish a platform for research into spine tissue engineering constructs. Preliminary results demonstrate higher grades of radiologically evident bony fusion across all levels when comparing fusion scores between the 3 and 6 month postop groups at the PCL CaP coated scaffold level, which is observed to be a similar grade to autograft, while no fusion is seen at the scaffold only level. Results to date suggest that the combination of rhBMP-2 and scaffold engineering actively promotes bone formation, laying the basis of a viable tissue engineered constructs.

A finite element model of seat cushion indentation with a soft tissue human occupant model

Effective digital human model (DHM) simulation of automotive driver packaging ergonomics, safety and comfort depends on accurate modelling of occupant posture, which is strongly related to the mechanical interaction between human body soft tissue and flexible seat components. This paper presents a finite-element study simulating the deflection of seat cushion foam and supportive seat structures, as well as human buttock and thigh soft tissue when seated. The three-dimensional data used for modelling thigh and buttock geometry were taken on one 95th percentile male subject, representing the bivariate percentiles of the combined hip breadth (seated) and buttock-to-knee length distributions of a selected Australian and US population. A thigh-buttock surface shell based on this data was generated for the analytic model. A 6mm neoprene layer was offset from the shell to account for the compression of body tissue expected through sitting in a seat. The thigh-buttock model is therefore made of two layers, covering thin to moderate thigh and buttock proportions, but not more fleshy sizes. To replicate the effects of skin and fat, the neoprene rubber layer was modelled as a hyperelastic material with viscoelastic behaviour in a Neo-Hookean material model. Finite element (FE) analysis was performed in ANSYS V13 WB (Canonsburg, USA). It is hypothesized that the presented FE simulation delivers a valid result, compared to a standard SAE physical test and the real phenomenon of human-seat indentation. The analytical model is based on the CAD assembly of a Ford Territory seat. The optimized seat frame, suspension and foam pad CAD data were transformed and meshed into FE models and indented by the two layer, soft surface human FE model. Converging results with the least computational effort were achieved for a bonded connection between cushion and seat base as well as cushion and suspension, no separation between neoprene and indenter shell and a frictional connection between cushion pad and neoprene. The result is compared to a previous simulation of an indentation with a hard shell human finite-element model of equal geometry, and to the physical indentation result, which is approached with very high fidelity. We conclude that (a) SAE composite buttock form indentation of a suspended seat cushion can be validly simulated in a FE model of merely similar geometry, but using a two-layer hard/soft structure. (b) Human-seat indentation of a suspended seat cushion can be validly simulated with a simplified human buttock-thigh model for a selected anthropomorphism.

Load bearing improves the limits of agreement for repeated in vivo measures of the speed of sound in human Achilles tendon

Axial acoustic wave propagation has been widely used in evaluating the mechanical properties of human bone in vivo. However, application of this technique to monitor soft tissues, such as tendon, has received comparatively little scientific attention. Laboratory-based research has established that axial acoustic wave transmission is not only related to the physical properties of equine tendon but is also proportional to tensile load to which it is exposed (Miles et al., 1996; Pourcelot et al., 2005). The reproducibility of the technique for in vivo measurements in human tendon, however, has not been established. The aim of this study was to evaluate the limits of agreement for repeated measures of the speed of sound (SoS) in human Achilles tendon in vivo. Methods: A custom built ultrasound device, consisting of an A-mode 1MHz emitter and two regularly spaced receivers, was used to measure the SoS in the mid-portion of the Achilles tendon in ten healthy males and ten females (mean age: 33.8 years, range 23-56 yrs; height: 1.73±0.08 m; weight: 68.4±15.3 kg). The emitter and receivers were held at fixed positions by a polyethylene frame and maintained in close contact with the skin overlying the tendon by means of elasticated straps. Repeated SoS measurements were taken with the subject prone (non-weightbearing and relaxed Achilles tendon) and during quiet bipedal and unipedal stance. In each instance, the device was detached and repositioned prior to measurement. Results: Limits of agreement for repeated SoS measures during non-weightbearing and bipedal and unipedal stance were ±53, ±28 and ±21 m/s, respectively. The average SoS in the non-weightbearing Achilles tendon was 1804±198 m/s. There was a significant increase in the average SoS during bilateral (2122±135 m/s) (P < 0.05) and unilateral (2221±79 m/s) stance (P < 0.05). Conclusions: Repeated SoS measures in human Achilles tendon were more reliable during stance than under non-weightbearing conditions. These findings are consistent with previous research in equine tendon in which lower variability in SoS was observed with increasing tensile load (Crevier-Denoix et al, 2009). Since the limits of agreement for Achilles tendon SoS are nearly 5% of the changes previously observed during walking and therapeutic heel raise exercises, acoustic wave transmission provides a promising new non-invasive method for determining tendon properties during sports and rehabilitation related activities.

Biological performance of a polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in an ovine thoracic interbody fusion model

Introduction. We develop a sheep thoracic spine interbody fusion model to study the suitability of polycaprolactone-based scaffold and recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within the thoracic spine. The surgical approach is a mini- open thoracotomy with relevance to minimally invasive deformity correction surgery for adolescent idiopathic scoliosis. To date there are no studies examining the use of this biodegradable implant in combination with biologics in a sheep thoracic spine model. Methods. In the present study, six sheep underwent a 3-level (T6/7, T8/9 and T10/11) discectomy with randomly allocated implantation of a different graft substitute at each of the three levels; (i) calcium phosphate (CaP) coated polycaprolactone based scaffold plus 0.54µg rhBMP-2, (ii) CaP coated PCL- based scaffold alone or (iii) autograft (mulched rib head). Fusion was assessed at six months post-surgery. Results. Computed Tomographic scanning demonstrated higher fusion grades in the rhBMP-2 plus PCL- based scaffold group in comparison to either PCL-based scaffold alone or autograft. These results were supported by histological evaluations of the respective groups. Biomechanical testing revealed significantly higher stiffness for the rhBMP-2 plus PCL- based scaffold group in all loading directions in comparison to the other two groups. Conclusions. The results of this study demonstrate that rhBMP-2 plus PCL-based scaffold is a viable bone graft substitute, providing an optimal environment for thoracic interbody spinal fusion in a large animal model.

Effects of whole body cryotherapy and cold water immersion on knee skin temperature

This study sought to a) compare and contrast the effect of 2 commonly used cryotherapy treatments, 4 min of − 110 °C whole body cryotherapy and 8 °C cold water immersion, on knee skin temperature and b) establish whether either protocol was capable of achieving a skin temperature ( < 13 °C) believed to be required for analgesic purposes. After ethics committee approval and written informed consent was obtained, 10 healthy males (26.5 ± 4.9 yr, 183.5 ± 6.0 cm, 90.7 ± 19.9 kg, 26.8 ± 5.0 kg/m 2 , 23.0 ± 9.3 % body fat; mean ± SD) participated in this randomised controlled crossover study. Skin temperature around the patellar region was assessed in both knees via non-contact, infrared thermal imaging and recorded pre-, immediately post-treatment and every 10 min thereafter for 60 min. Compared to baseline, average, minimum and maximum skin temperatures were significantly reduced (p < 0.001) immediately post-treatment and at 10, 20, 30, 40, 50 and 60 min after both cooling modalities. Average and minimum skin temperatures were lower (p < 0.05) immediately after whole body cryotherapy (19.0 ± 0.9 ° C) compared to cold water immersion (20.5 ± 0.6 ° C). However, from 10 to 60 min post, the average, minimum and maximum skin temperatures were lower (p < 0.05) following the cold water treatment. Finally, neither protocol achieved a skin temperature believed to be required to elicit an analgesic effect.

Enduring powers of attorney : promoting attorneys' accountability as substitute decision makers

Aim The misuse and abuse of Enduring Powers of Attorney (EPAs) by attorneys, particularly in relation to financial decision-making, is a growing concern. This paper explores the opportunities to enhance accountability of attorneys at the time of the execution of the document in Queensland. Method A four stage multi-method design comprised a critical reference group; semi-structured interviews with 32 principals or potential principals, attorneys and witnesses; two focus groups with service providers and a state wide survey of 76 principals, attorneys and witnesses. Results Across all methods and user groups, understanding the role and obligations of the attorney in an EPA was consistently identified as problematic. Conclusions Promoting accountability and understanding can be addressed by greater attention to the role of the attorney in the forms/ guidelines and in the structure and witnessing of the forms, increased direction about record keeping and access to appropriate advice and support.

The effect of MC1R variants and sunscreen on the response of human melanocytes in vivo to ultraviolet radiation and implications for melanoma.

We conducted a clinical trial to compare the molecular and cellular responses of human melanocytes and keratinocytes in vivo to solar-simulated ultraviolet radiation (SSUVR) in 57 Caucasian participants grouped according to MC1R genotype. We found that, on average, the density of epidermal melanocytes 14 days after exposure to 2 minimal erythemal dose (MED) SSUVR was twofold higher than baseline (unirradiated) skin. However, the change in epidermal melanocyte counts among people carrying germline MC1R variants (97% increase) was significantly less than those with wild-type MC1R (164% increase; P = 0.01). We also found that sunscreen applied to the skin before exposure to 2 MED SSUVR completely blocked the effects of DNA damage, p53 induction, and cellular proliferation in both melanocytes and keratinocytes.

Current status of pharmacogenomics testing for anti-tumor drug therapies : approaches to non-melanoma skin cancer

Skin cancer is one of the most commonly occurring cancer types, with substantial social, physical, and financial burdens on both individuals and societies. Although the role of UV light in initiating skin cancer development has been well characterized, genetic studies continue to show that predisposing factors can influence an individual's susceptibility to skin cancer and response to treatment. In the future, it is hoped that genetic profiles, comprising a number of genetic markers collectively involved in skin cancer susceptibility and response to treatment or prognosis, will aid in more accurately informing practitioners' choices of treatment. Individualized treatment based on these profiles has the potential to increase the efficacy of treatments, saving both time and money for the patient by avoiding the need for extensive or repeated treatment. Increased treatment responses may in turn prevent recurrence of skin cancers, reducing the burden of this disease on society. Currently existing pharmacogenomic tests, such as those that assess variation in the metabolism of the anticancer drug fluorouracil, have the potential to reduce the toxic effects of anti-tumor drugs used in the treatment of non-melanoma skin cancer (NMSC) by determining individualized appropriate dosage. If the savings generated by reducing adverse events negate the costs of developing these tests, pharmacogenomic testing may increasingly inform personalized NMSC treatment.

Characterisation of Polycaprolatone-Based Scaffold Plus Recombinant Human Morphogenetic Protein - 2 (RHBMP-2) in an Ovine Thoracic Spine Model

This thesis represents a step forward in the development of a pre-clinical model investigating a suitable substitute for host bone for use in human spinal fusion. By way of an animal model, it examines the biological performance of a novel bone graft substitute comprised of a combination of a custom-designed biodegradable material and biologics.

Tissue viability imaging of skin microcirculation following exposure to whole body cryotherapy (-110°C) and cold water immersion (8°C)

Cryotherapy is currently used in various clinical, rehabilitative, and sporting settings. However, very little is known regarding the impact of cooling on the microcirculatory response. Objectives: The present study sought to examine the influence of two commonly employed modalities of cryotherapy, whole body cryotherapy (WBC; -110°C) and cold water immersion(CWI; 8±1°C), on skin microcirculation in the mid- thigh region. Methods: The skin area examined was a 3 × 3 cm located between the most anterior aspect of the inguinal fold and the patella. Following 10 minutes of rest, 5 healthy, active males were exposed to either WBC for 3 minutes or CWI for 5 minutes in a randomised order. Volunteers lay supine for five minutes after treatment, in order to monitor the variation of red blood cell (RBC) concentration in the region of interest for a duration of 40 minutes. Microcirculation response was assessed using a non-invasive, portable instrument known as a Tissue Viability imaging system. After a minimum of seven days, the protocol was repeated. Subjective assessment of the volunteer’s thermal comfort and thermal sensation was also recorded. Results: RBC was altered following exposure to both WBC and CWI but appeared to stabilise approximately 35 minutes after treatments. Both WBC and CWI affected thermal sensation (p < 0.05); however no betweengroup differences in thermal comfort or sensation were recorded (p > 0.05). Conclusions: As both WBC and CWI altered RBC, further study is necessary to examine the mechanism for this alteration during whole body cooling.

Phenytoin metabolism by human cytochrome P450 : involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.