Differentiating human NT2/D1 neurospheres as a versatile in vitro 3D model system for developmental neurotoxicity testing

Developmental neurotoxicity is a major issue in human health and may have lasting neurological implications. In this preliminary study we exposed differentiating Ntera2/clone D1 (NT2/D1) cell neurospheres to known human teratogens classed as non-embryotoxic (acrylamide), weakly embryotoxic (lithium, valproic acid) and strongly embryotoxic (hydroxyurea) as listed by European Centre for the Validation of Alternative Methods (ECVAM) and examined endpoints of cell viability and neuronal protein marker expression specific to the central nervous system, to identify developmental neurotoxins. Following induction of neuronal differentiation, valproic acid had the most significant effect on neurogenesis, in terms of reduced viability and decreased neuronal markers. Lithium had least effect on viability and did not significantly alter the expression of neuronal markers. Hydroxyurea significantly reduced cell viability but did not affect neuronal protein marker expression. Acrylamide reduced neurosphere viability but did not affect neuronal protein marker expression. Overall, this NT2/D1 -based neurosphere model of neurogenesis, may provide the basis for a model of developmental neurotoxicity in vitro.

Shortening cemented femoral implants. An in vitro investigation to quantify exeter femoral implant rotational stability vs simulated implant length

The Exeter stems vary in length from 90 to 150 mm. The shorter stems generally have lower offsets. The purpose of this study was to determine if length of stem, with fixed offset, affected rotational stability. Mechanical testing was carried out on 10 implant-cement constructs with 2 loading profiles, rising from chair and stair climbing, at different simulated implant lengths using purpose-built apparatus. This paper presents a mechanism for clinically observed rotational stability and explains the mechanical characteristics required for rotational stability in Exeter femoral stems. © 2012.

Computer-assisted measurements of coronal knee joint laxity in vitro are related to low-stress behavior rather than structural properties of the collateral ligaments

The relationship between coronal knee laxity and the restraining properties of the collateral ligaments remains unknown. This study investigated correlations between the structural properties of the collateral ligaments and stress angles used in computer-assisted total knee arthroplasty (TKA), measured with an optically based navigation system. Ten fresh-frozen cadaveric knees (mean age: 81 ± 11 years) were dissected to leave the menisci, cruciate ligaments, posterior joint capsule and collateral ligaments. The resected femur and tibia were rigidly secured within a test system which permitted kinematic registration of the knee using a commercially available image-free navigation system. Frontal plane knee alignment and varus-valgus stress angles were acquired. The force applied during varus-valgus testing was quantified. Medial and lateral bone-collateral ligament-bone specimens were then prepared, mounted within a uni-axial materials testing machine, and extended to failure. Force and displacement data were used to calculate the principal structural properties of the ligaments. The mean varus laxity was 4 ± 1° and the mean valgus laxity was 4 ± 2°. The corresponding mean manual force applied was 10 ± 3 N and 11 ± 4 N, respectively. While measures of knee laxity were independent of the ultimate tensile strength and stiffness of the collateral ligaments, there was a significant correlation between the force applied during stress testing and the instantaneous stiffness of the medial (r = 0.91, p = 0.001) and lateral (r = 0.68, p = 0.04) collateral ligaments. These findings suggest that clinicians may perceive a rate of change of ligament stiffness as the end-point during assessment of collateral knee laxity.

Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34NegCD31Pos LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34NegCD31Pos lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P < 0.0005 at 48 h, two-way ANOVA), increased migration (P < 0.001, two-way ANOVA) and formed fewer (P = 0.029, independent samples t test), shorter tubes (P = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel™-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplaninPos vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.

The stabilising role of the costovertebral joints and spinous processes on thoracic spine stability

Introduction The importance of in vitro biomechanical testing in today’s understanding of spinal pathology and treatment modalities cannot be stressed enough. Different studies have used differing levels of dissection of their spinal segments for their testing protocols[1, 2]. The aim of this study was to assess the impact of removing the costovertebral joints and partial resection of the spinous process sequentially, on the stiffness of the immature thoracic bovine spinal segment. Materials and Methods Thoracic spines from 6-8 week old calves were used. Each spine was dissected and divided into motion segments with 5cm of attached rib on each side and full spinous processes including levels T4-T11 (n=28). They were potted in polymethylemethacrylate. An Instron Biaxial materials testing machine with a custom made jig was used for testing. The segments were tested in flexion/extension, lateral bending and axial rotation at 37⁰C and 100% humidity, using moment control to a maximum 1.75 Nm with a loading rate of 0.3 Nm per second. They were first tested intact for ten load cycles with data collected from the tenth cycle. Progressive dissection was performed by removing first the attached ribs, followed by the spinous process at its base. Biomechanical testing was carried out after each level of dissection using the same protocol. Statistical analysis of the data was performed using repeated measures ANOVA. Results In combined flexion/extension there was a significant reduction in stiffness of 16% (p=0.002). This was mainly after resection of the ribs (14%, p=0.024) and mainly occurred in flexion where stiffness reduced by 22% (p=0.021). In extension, stiffness dropped by 13% (p=0.133). However there was no further significant change in stiffness on resection of the spinous process (<1%) (p=1.00). In lateral bending there was a significant decrease in stiffness of 13% (p<0.001). This comprised a drop of 11% on resection of the ribs (p=0.009) and a further 8% on resection of the spinous process (p=0.014). There was no difference between left and right bending. In axial rotation there was no significant change in stiffness after each stage of dissection (p=0.253). There was no difference between left and right rotation. Conclusion The costovertebral joints play a significant role in providing stability to the bovine thoracic spine in both flexion/extension and lateral bending, whereas the spinous processes play a minor role. Both elements have little effect on axial rotation stability.

In vitro microenvironments to study breast cancer bone colonisation

Bone metastasis occurs frequently in patients with advanced breast cancer and is a major cause of morbidity and mortality in these patients. In order to advance current therapies, the mechanisms leading to the formation of bone metastases and their pathophysiology have to be better understood. Several in vitro models have been developed for systematic studies of interactions between breast cancer cells and the bone microenvironment. Such models can provide insights into the molecular basis of bone metastatic colonisation and also may provide a useful platform to design more physiologically relevant drug testing assays. This review describes different in vitro approaches and discusses their advantages and disadvantages.

Multi-parametric hydrogels support 3D in vitro bioengineered microenvironment models of tumour angiogenesis

Tumour microenvironment greatly influences the development and metastasis of cancer progression. The development of three dimensional (3D) culture models which mimic that displayed in vivo can improve cancer biology studies and accelerate novel anticancer drug screening. Inspired by a systems biology approach, we have formed 3D in vitro bioengineered tumour angiogenesis microenvironments within a glycosaminoglycan-based hydrogel culture system. This microenvironment model can routinely recreate breast and prostate tumour vascularisation. The multiple cell types cultured within this model were less sensitive to chemotherapy when compared with two dimensional (2D) cultures, and displayed comparative tumour regression to that displayed in vivo. These features highlight the use of our in vitro culture model as a complementary testing platform in conjunction with animal models, addressing key reduction and replacement goals of the future. We anticipate that this biomimetic model will provide a platform for the in-depth analysis of cancer development and the discovery of novel therapeutic targets.