Establishment of a preclinical ovine model for tibial segmental bone defect repair by applying bone tissue engineering strategies

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. The field of tissue engineering has emerged as an important approach to bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. The subsequent gap between research and clinical translation, hence commercialization, is referred to as the ‘Valley of Death’ and describes a large number of projects and/or ventures that are ceased due to a lack of funding during the transition from product/technology development to regulatory approval and subsequently commercialization. One of the greatest difficulties in bridging the Valley of Death is to develop good manufacturing processes (GMP) and scalable designs and to apply these in pre-clinical studies. In this article, we describe part of the rationale and road map of how our multidisciplinary research team has approached the first steps to translate orthopaedic bone engineering from bench to bedside byestablishing a pre-clinical ovine critical-sized tibial segmental bone defect model and discuss our preliminary data relating to this decisive step.

Processing of polycaprolactone and polycaprolactone-based copolymers into 3D scaffolds and their cellular responses

Synthetic polymers have attracted much attention in tissue engineering due to their ability to modulate biomechanical properties. This study investigated the feasibility of processing poly(varepsilon-caprolactone) (PCL) homopolymer, PCL-poly(ethylene glycol) (PEG) diblock, and PCL-PEG-PCL triblock copolymers into three-dimensional porous scaffolds. Properties of the various polymers were investigated by dynamic thermal analysis. The scaffolds were manufactured using the desktop robot-based rapid prototyping technique. Gross morphology and internal three-dimensional structure of scaffolds were identified by scanning electron microscopy and micro-computed tomography, which showed excellent fusion at the filament junctions, high uniformity, and complete interconnectivity of pore networks. The influences of process parameters on scaffolds' morphological and mechanical characteristics were studied. Data confirmed that the process parameters directly influenced the pore size, porosity, and, consequently, the mechanical properties of the scaffolds. The in vitro cell culture study was performed to investigate the influence of polymer nature and scaffold architecture on the adhesion of the cells onto the scaffolds using rabbit smooth muscle cells. Light, scanning electron, and confocal laser microscopy showed cell adhesion, proliferation, and extracellular matrix formation on the surface as well as inside the structure of both scaffold groups. The completely interconnected and highly regular honeycomb-like pore morphology supported bridging of the pores via cell-to-cell contact as well as production of extracellular matrix at later time points. The results indicated that the incorporation of hydrophilic PEG into hydrophobic PCL enhanced the overall hydrophilicity and cell culture performance of PCL-PEG copolymer. However, the scaffold architecture did not significantly influence the cell culture performance in this study.

Vitreous cryopreservation of nanofibrous tissue-engineered constructs generated using mesenchymal stromal cells

Development of an effective preservation strategy to fulfill off-the-shelf availability of tissue-engineered constructs (TECs) is demanded for realizing their clinical potential. In this study, the feasibility of vitrification, ice-free cryopreservation, for precultured ready-to-use TECs was evaluated. To prepare the TECs, bone marrow-derived porcine mesenchymal stromal cells (MSCs) were seeded in polycaprolactone-gelatin nanofibrous scaffolds and cultured for 3 weeks before vitrification treatment. The vitrification strategy developed, which involved exposure of the TECs to low concentrations of cryoprotectants followed by a vitrification solution and sterile packaging in a pouch with its subsequent immersion directly into liquid nitrogen, was accomplished within 11min. Stepwise removal of cryoprotectants, after warming in a 38 degrees C water bath, enabled rapid restoration of the TECs. Vitrification did not impair microstructure of the scaffold or cell viability. No significant differences were found between the vitrified and control TECs in cellular metabolic activity and proliferation on matched days and in the trends during 5 weeks of continuous culture postvitrification. Osteogenic differentiation ability in vitrified and control groups was similar. In conclusion, we have developed a time- and cost-efficient cryopreservation method that maintains integrity of the TECs while preserving MSCs viability and metabolic activity, and their ability to differentiate.

Tissue engineering of articular cartilage with biomimetic zones

Articular cartilage damage is a persistent and increasing problem with the aging population, and treatments to achieve biological repair or restoration remain a challenge. Cartilage tissue engineering approaches have been investigated for over 20 years, but have yet to achieve the consistency and effectiveness for widespread clinical use. One of the potential reasons for this is that the engineered tissues do not have or establish the normal zonal organization of cells and extracellular matrix that appears critical for normal tissue function. A number of approaches are being taken currently to engineer tissue that more closely mimics the organization of native articular cartilage. This review focuses on the zonal organization of native articular cartilage, strategies being used to develop such organization, the reorganization that occurs after culture or implantation, and future prospects for the tissue engineering of articular cartilage with biomimetic zones.

Vitamin D in health and disease : an insight into traditional functions and new roles for the 'sunshine vitamin'

Vitamin D is unique among the vitamins in that humans can synthesize it via the action of UV radiation upon the skin. This combined with its ability to act on specific target tissues via Vitamin D Receptor’s (VDR) make its classification as a steroid hormone more appropriate. While Vitamin D deficiency is a recognized problem in some northern latitude countries, recent studies have shown even in sunny countries such as Australia, vitamin D deficiency may be more prevalent than first thought. Vitamin D is most well known for its role in bone health, however, the discovery of VDR’s on a wide variety of tissue types has also opened up roles for vitamin D far beyond traditional bone health. These include possible associations with autoimmune diseases such as multiple sclerosis and inflammatory bowel diseases, cancer, cardiovascular diseases and muscle strength. Firstly, this paper presents an overview of the two sources of vitamin D: exposure to ultraviolet-B radiation and food sources of vitamin D, with particular focus on both Australian and international studies on dietary vitamin D intake and national fortification strategies. Secondly, the paper reviews recent epidemiological and experimental evidence linking vitamin D and its role in health and disease for the major conditions linked to suboptimal vitamin D, while identifying significant gaps in the research and possible future directions for research.

Microassembly fabrication of tissue engineering scaffolds with customized design

Browse > Journals> Automation Science and Enginee ...> Volume: 5 Issue: 3 Microassembly Fabrication of Tissue Engineering Scaffolds With Customized Design 4468741 abstract Han Zhang; Burdet, E.; Poo, A.N.; Hutmacher, D.W.; GE Global Res. Center Ltd., Shanghai This paper appears in: Automation Science and Engineering, IEEE Transactions on Issue Date: July 2008 Volume: 5 Issue:3 On page(s): 446 - 456 ISSN: 1545-5955 Digital Object Identifier: 10.1109/TASE.2008.917011 Date of Current Version: 02 July 2008 Sponsored by: IEEE Robotics and Automation Society Abstract This paper presents a novel technique to fabricate scaffold/cell constructs for tissue engineering by robotic assembly of microscopic building blocks (of volume 0.5$,times,$0.5$,times,$0.2 ${hbox{mm}}^{3}$ and 60 $mu {hbox{m}}$ thickness). In this way, it becomes possible to build scaffolds with freedom in the design of architecture, surface morphology, and chemistry. Biocompatible microparts with complex 3-D shapes were first designed and mass produced using MEMS techniques. Semi-automatic assembly was then realized using a robotic workstation with four degrees of freedom integrating a dedicated microgripper and two optical microscopes. Coarse movement of the gripper is determined by pattern matching in the microscopes images, while the operator controls fine positioning and accurate insertion of the microparts. Successful microassembly was demonstrated using SU-8 and acrylic resin microparts. Taking advantage of parts distortion and adhesion forces, which dominate at micro-level, the parts cleave together after assembly. In contrast to many current scaffold fabrication techniques, no heat, pressure, electrical effect, or toxic chemical reaction is involved, a critical condition for creating scaffolds with biological agents.

In vitro and in vivo analysis of co-electrospun scaffolds made of medical grade poly(epsilon-caprolactone) and porcine collagen

In this study, a nanofiber mesh made by co-electrospinning medical grade poly(epsilon-caprolactone) and collagen (mPCL/Col) was fabricated and studied. Its mechanical properties and characteristics were analyzed and compared to mPCL meshes. mPCL/Col meshes showed a reduction in strength but an increase in ductility when compared to PCL meshes. In vitro assays revealed that mPCL/Col supported the attachment and proliferation of smooth muscle cells on both sides of the mesh. In vivo studies in the corpus cavernosa of rabbits revealed that the mPCL/Col scaffold used in conjunction with autologous smooth muscle cells resulted in better integration with host tissue when compared to cell free scaffolds. On a cellular level preseeded scaffolds showed a minimized foreign body reaction.

Can tissue engineering concepts advance tumor biology research?

Advances in tissue engineering have traditionally led to the design of scaffold- or matrix-based culture systems that better reflect the biological, physical and biochemical environment of the natural extracellular matrix. Although their clinical applications in regenerative medicine tend to receive most of the attention, it is obvious that other areas of biomedical research could be well served by the powerful tools that have already been developed in tissue engineering. In this article, we review the recent literature to demonstrate how tissue engineering platforms can enhance in vitro and in vivo models of tumorigenesis and thus hold great promise to contribute to future cancer research.

Mathematical modelling of muscle recruitment and function in the lumbar spine

Low back pain is an increasing problem in industrialised countries and although it is a major socio-economic problem in terms of medical costs and lost productivity, relatively little is known about the processes underlying the development of the condition. This is in part due to the complex interactions between bone, muscle, nerves and other soft tissues of the spine, and the fact that direct observation and/or measurement of the human spine is not possible using non-invasive techniques. Biomechanical models have been used extensively to estimate the forces and moments experienced by the spine. These models provide a means of estimating the internal parameters which can not be measured directly. However, application of most of the models currently available is restricted to tasks resembling those for which the model was designed due to the simplified representation of the anatomy. The aim of this research was to develop a biomechanical model to investigate the changes in forces and moments which are induced by muscle injury. In order to accurately simulate muscle injuries a detailed quasi-static three dimensional model representing the anatomy of the lumbar spine was developed. This model includes the nine major force generating muscles of the region (erector spinae, comprising the longissimus thoracis and iliocostalis lumborum; multifidus; quadratus lumborum; latissimus dorsi; transverse abdominis; internal oblique and external oblique), as well as the thoracolumbar fascia through which the transverse abdominis and parts of the internal oblique and latissimus dorsi muscles attach to the spine. The muscles included in the model have been represented using 170 muscle fascicles each having their own force generating characteristics and lines of action. Particular attention has been paid to ensuring the muscle lines of action are anatomically realistic, particularly for muscles which have broad attachments (e.g. internal and external obliques), muscles which attach to the spine via the thoracolumbar fascia (e.g. transverse abdominis), and muscles whose paths are altered by bony constraints such as the rib cage (e.g. iliocostalis lumborum pars thoracis and parts of the longissimus thoracis pars thoracis). In this endeavour, a separate sub-model which accounts for the shape of the torso by modelling it as a series of ellipses has been developed to model the lines of action of the oblique muscles. Likewise, a separate sub-model of the thoracolumbar fascia has also been developed which accounts for the middle and posterior layers of the fascia, and ensures that the line of action of the posterior layer is related to the size and shape of the erector spinae muscle. Published muscle activation data are used to enable the model to predict the maximum forces and moments that may be generated by the muscles. These predictions are validated against published experimental studies reporting maximum isometric moments for a variety of exertions. The model performs well for fiexion, extension and lateral bend exertions, but underpredicts the axial twist moments that may be developed. This discrepancy is most likely the result of differences between the experimental methodology and the modelled task. The application of the model is illustrated using examples of muscle injuries created by surgical procedures. The three examples used represent a posterior surgical approach to the spine, an anterior approach to the spine and uni-lateral total hip replacement surgery. Although the three examples simulate different muscle injuries, all demonstrate the production of significant asymmetrical moments and/or reduced joint compression following surgical intervention. This result has implications for patient rehabilitation and the potential for further injury to the spine. The development and application of the model has highlighted a number of areas where current knowledge is deficient. These include muscle activation levels for tasks in postures other than upright standing, changes in spinal kinematics following surgical procedures such as spinal fusion or fixation, and a general lack of understanding of how the body adjusts to muscle injuries with respect to muscle activation patterns and levels, rate of recovery from temporary injuries and compensatory actions by other muscles. Thus the comprehensive and innovative anatomical model which has been developed not only provides a tool to predict the forces and moments experienced by the intervertebral joints of the spine, but also highlights areas where further clinical research is required.

Enhancing in vivo vascularized bone formation by cobalt chloride-treated bone marrow stromal cells in a tissue engineered periosteum model

The periosteum plays an indispensable role in both bone formation and bone defect healing. In this study we constructed an artificial in vitro periosteum by incorporating osteogenic differentiated bone marrow stromal cells (BMSCs) and cobalt chloride (CoCl(2))-treated BMSCs. The engineered periostea were implanted both subcutaneously and into skull bone defects in SCID mice to investigate ectopic and orthotopic osteogenesis and vascularization. After two weeks in subcutaneous and four weeks in bone defect areas, the implanted constructs were assessed for ectopic and orthotopic osteogenesis and vascularization by micro-CT, histomorphometrical and immunohistochemical methods. The results showed that CoCl(2) pre-treated BMSCs induced higher degree of vascularization and enhanced osteogenesis within the implants in both ectopic and orthotopic areas. This study provided a novel approach using BMSCs sourced from the same patient for both osteogenic and pro-angiogenic purposes in constructing tissue engineered periosteum to enhance vascularized osteogenesis.

Eccentric calf muscle exercise produces a greater acute reduction in Achilles tendon thickness than concentric exercise

Objective: To investigate the acute effects of isolated eccentric and concentric calf muscle exercise on Achilles tendon sagittal thickness. ---------- Design: Within-subject, counterbalanced, mixed design. ---------- Setting: Institutional. ---------- Participants: 11 healthy, recreationally active male adults. ---------- Interventions: Participants performed an exercise protocol, which involved isolated eccentric loading of the Achilles tendon of a single limb and isolated concentric loading of the contralateral, both with the addition of 20% bodyweight. ---------- Main outcome measurements: Sagittal sonograms were acquired prior to, immediately following and 3, 6, 12 and 24 h after exercise. Tendon thickness was measured 2 cm proximal to the superior aspect of the calcaneus. ---------- Results: Both loading conditions resulted in an immediate decrease in normalised Achilles tendon thickness. Eccentric loading induced a significantly greater decrease than concentric loading despite a similar impulse (−0.21 vs −0.05, p<0.05). Post-exercise, eccentrically loaded tendons recovered exponentially, with a recovery time constant of 2.5 h. The same exponential function did not adequately model changes in tendon thickness resulting from concentric loading. Even so, recovery pathways subsequent to the 3 h time point were comparable. Regardless of the exercise protocol, full tendon thickness recovery was not observed until 24 h. ---------- Conclusions: Eccentric loading invokes a greater reduction in Achilles tendon thickness immediately after exercise but appears to recover fully in a similar time frame to concentric loading.

The osteogenic differentiation of adipose tissue-derived precursor cells in A 3D scaffold/matrix environment

Abstract: This paper details an in-vitro study using human adipose tissue-derived precursor/stem cells (ADSCs) in three-dimensional (3D) tissue culture systems. ADSCs from 3 donors were seeded onto NaOH-treated medical grade polycaprolactone-tricalcium phosphate (mPCL-TCP) scaffolds with two different matrix components; fibrin glue and lyophilized collagen. ADSCs within these scaffolds were then induced to differentiate along the osteogenic lineage for a 28-day period and various assays and imaging techniques were performed at Day 1, 7, 14, 21 and 28 to assess and compare the ADSC’s adhesion, viability, proliferation, metabolism and differentiation along the osteogenic lineage when cultured in the different scaffold/matrix systems. The ADSC cells were proliferative in both collagen and fibrin mPCL-TCP scaffold systems with a consistently higher cell number (by comparing DNA amounts) in the induced group over the non-induced groups for both scaffold systems. In response to osteogenic induction, these ADSCs expressed elevated osteocalcin, alkaline phosphatase and osteonectin levels. Cells were able to proliferate within the pores of the scaffolds and form dense cellular networks after 28 days of culture and induction. The successful cultivation of osteogenic by FDM process manufactured ADSCs within a 3D matrix comprising fibrin glue or collagen, immobilized within a robust synthetic scaffold is a promising technique which should enhance their potential usage in the regenerative medicine arena, such as bone tissue engineering.

Controversies of prophylactic hypothermia and the emerging use of brain tissue oxygen tension monitoring and decompressive craniectomy in traumatic brain-injured children

Background Despite being the leading cause of death and disability in the paediatric population, traumatic brain injury (TBI) in this group is largely understudied. Clinical practice within the paediatric intensive care unit (PICU) has been based upon adult guidelines however children are significantly different in terms of mechanism, pathophysiology and consequence of injury. Aim To review TBI management in the PICU and gain insight into potential management strategies. Method To conduct this review, a literature search was conducted using MEDLINE, PUBMED and The Cochrane Library using the following key words; traumatic brain injury; paediatric; hypothermia. There were no date restrictions applied to ensure that past studies, whose principles remain current were not excluded. Results Three areas were identified from the literature search and will be discussed against current acknowledged treatment strategies: Prophylactic hypothermia, brain tissue oxygen tension monitoring and decompressive craniectomy. Conclusion Previous literature has failed to fully address paediatric specific management protocols and we therefore have little evidence-based guidance. This review has shown that there is an emerging and ongoing trend towards paediatric specific TBI research in particular the area of moderate prophylactic hypothermia (MPH).