The evaluation of bone composition and carcass quality traits on back fracture in pigs stunned using electrical system.

2016

Efficient genetic transformation and regeneration system from hairy root of Origanum vulgare.

2016

Vitrification as a prospect for cryopreservation of tissue-engineered constructs

Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation

Endochondral ossification in vitro is influenced by mechanical bending

Bone development is influenced by the local mechanical environment. Experimental evidence suggests that altered loading can change cell proliferation and differentiation in chondro- and osteogenesis during endochondral ossification. This study investigated the effects of three-point bending of murine fetal metatarsal bone anlagen in vitro on cartilage differentiation, matrix mineralization and bone collar formation. This is of special interest because endochondral ossification is also an important process in bone healing and regeneration. Metatarsal preparations of 15 mouse fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days. After 3 days in culture to allow adherence they were stimulated 4 days for 20 min twice daily by a controlled bending of approximately 1000-1500 microstrain at 1 Hz. The paraffin-embedded bone sections were analyzed using histological and histomorphometrical techniques. The stimulated group showed an elongated periosteal bone collar while the total bone length was not different from controls. The region of interest (ROI), comprising the two hypertrophic zones and the intermediate calcifying diaphyseal zone, was greater in the stimulated group. The mineralized fraction of the ROI was smaller in the stimulated group, while the absolute amount of mineralized area was not different. These results demonstrate that a new device developed to apply three-point bending to a mouse metatarsal bone culture model caused an elongation of the periosteal bone collar, but did not lead to a modification in cartilage differentiation and matrix mineralization. The results corroborate the influence of biophysical stimulation during endochondral bone development in vitro. Further experiments with an altered loading regime may lead to more pronounced effects on the process of endochondral ossification and may provide further insights into the underlying mechanisms of mechanoregulation which also play a role in bone regeneration.

Endogenous musculoskeletal tissue engineering : a focussed perspective

Two major difficulties facing widespread clinical implementation of existing Tissue Engineering (TE) strategies for the treatment of musculoskeletal disorders are (1) the cost, space and time required for ex vivo culture of a patient’s autologous cells prior to re-implantation as part of a TE construct, and (2) the potential risks and availability constraints associated with transplanting exogenous (foreign) cells. These hurdles have led to recent interest in endogenous TE strategies, in which the regenerative potential of a patient’s own cells is harnessed to promote tissue regrowth without ex vivo cell culture. This article provides a focused perspective on key issues in the development of endogenous TE strategies, progress to date, and suggested future research directions toward endogenous repair and regeneration of musculoskeletal tissues and organs.

A case for optimising fracture healing through inverse dynamization

The mechanical conditions in the repair tissues are known to influence the outcome of fracture healing. These mechanical conditions are determined by the stiffness of fixation and limb loading. Experimental studies have shown that there is a range of beneficial fixation stiffness for timely healing and that fixation stiffness that is either too flexible or too stiff impairs callus healing. However, much less is known about how mechanical conditions influence the biological processes that make up the sequence of bone repair and if indeed mechanical stimulation is required at all stages of repair. Secondary bone healing occurs through a sequence of events broadly characterised by inflammation, proliferation, consolidation and remodelling. It is our hypothesis that a change in fixation stiffness from very flexible to stiff can shorten the time to healing relative to constant fixation stiffness. Flexible fixation has the benefit of promoting greater callus formation and needs to be applied during the proliferative stage of repair. The greater callus size helps to stabilize the fragments earlier allowing mineralization to occur faster. Together with stable/rigid fixation applied during the latter stage of repair to ensure mineralization of the callus. The predicted benefits of inverse dynamization are shortened healing in comparison to very flexible fixation and healing time comparable or faster than stable fixation with greater callus stiffness.

Characterisation of a human skin equivalent model to study the effects of ultraviolet B radiation on keratinocytes

The incidences of skin cancers resulting from chronic ultraviolet radiation (UVR) exposure are on the incline both in Australia and globally. Hence, the cellular and molecular pathways associated with UVR-induced photocarcinogenesis urgently need to be elucidated, in order to develop more robust preventative and treatment strategies against skin cancers. In vitro investigations into the effects of UVR (in particular the highly-mutagenic UVB wavelength) have, to date, mainly involved the use of cell culture and animal models. However, these models possess biological disparities to native skin, which to some extent have limited their relevance to the in vivo situation. To address this, we characterised a 3-dimensional, tissue-engineered human skin equivalent (HSE) model (consisting of primary human keratinocytes cultured on a dermal-derived scaffold) as a representation of a more physiologically-relevant platform to study keratinocyte responses to UVB. Significantly, we demonstrate that this model retains several important epidermal properties of native skin. Moreover, UVB-irradiation of the HSE constructs was shown to induce key markers of photodamage in the HSE keratinocytes, including the formation of cyclobutane pyrimidine dimers, the activation of apoptotic pathways, the accumulation of p53 and the secretion of inflammatory cytokines. Importantly, we also demonstrate that the UVB-exposed HSE constructs retain the capacity for epidermal repair and regeneration following photodamage. Together, our results demonstrate the potential of this skin equivalent model as a tool to study various aspects of the acute responses of human keratinocytes to UVB radiation damage.

Collagen in the scarless fetal skin wound : detection with picrosirius-polarization

Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a "response" to burn injury.

Deep dermal burn injury results in scarless wound healing in the ovine fetus

Early to mid-term fetuses heal cutaneous incisional wounds without scars; however, fetal response to burn injury has not been ascertained. We present a fetal model of thermal injury and subsequent analysis of fetal and lamb response to burn injury. A reproducible deep dermal burn injury was created in the fetus by application of water at 66 degrees C for 7 seconds, and at 82 degrees C for 10 seconds to the lamb. Macroscopically, the area of fetal scald was undetectable from day 7 post injury, while all lamb scalds were readily identified and eventually healed with scarring. Using a five-point histopathology scoring system for alteration in tissue morphology, differences were detected between control and scalded skin at all stages in lamb postburn, but no difference was detected in the fetal model after day 7. There were also large differences in content of alpha-smooth muscle actin and transforming growth factor-beta1 between control and scalded lamb and these differences were statistically significant at day 14 (P < 0.01). This novel model of fetal and lamb response to deep dermal injury indicates that the fetus heals a deep burn injury in a scarless fashion. Further elucidation of this specific fetal process of burn injury repair may lead to improved outcome for patients with burn injury.

Conservative surgical debridement as a burn treatment : supporting evidence from a porcine burn model

In thermal deep-dermal burns, surgical debridement is normally used in conjunction with skin grafting or skin substitutes and debridement alone as a burn treatment is not usually practiced. The current study addresses whether or not debridement alone would enhance burn wound healing on small deep-dermal-partial thickness burns. This was a prospective and blinded experimental trial using a porcine deep-dermal-partial thickness burn model. Four burns, approximately 50 cm(2) in size, were created on each of eight pigs. Two burns from each pig were immediately surgically debrided and the other two were not debrided as the internal control. Hydrate gel together with paraffin gauze were used to cover the burns for four pigs and silver dressings for the other four. Clinical assessment of wound healing was conducted over a 6-week period. Skin samples were collected at the end of the experiment and histopathological evaluation was performed. The results show thinner scar formation and lower scar height in the debrided compared with nondebrided wounds in the hydrate gel/paraffin gauze groups. There were no statistically significant differences in wound healing assessment between the debrided and nondebrided wounds dressed with silver dressings. This study provides supporting evidence that immediate debridement with an appropriate dressing and without skin grafting may promote wound healing, suggesting its potential benefit for clinical patients.

Disparate companions : tissue engineering meets cancer research

Recreating an environment that supports and promotes fundamental homeostatic mechanisms is a significant challenge in tissue engineering. Optimizing cell survival, proliferation, differentiation, apoptosis and angiogenesis, and providing suitable stromal support and signalling cues are keys to successfully generating clinically useful tissues. Interestingly, those components are often subverted in the cancer setting, where aberrant angiogenesis, cellular proliferation, cell signalling and resistance to apoptosis drive malignant growth. In contrast to tissue engineering, identifying and inhibiting those pathways is a major challenge in cancer research. The recent discovery of adult tissue-specific stem cells has had a major impact on both tissue engineering and cancer research. The unique properties of these cells and their role in tissue and organ repair and regeneration hold great potential for engineering tissue-specific constructs. The emerging body of evidence implicating stem cells and progenitor cells as the source of oncogenic transformation prompts caution when using these cells for tissue-engineering purposes. While tissue engineering and cancer research may be considered as opposed fields of research with regard to their proclaimed goals, the compelling overlap in fundamental pathways underlying these processes suggests that cross-disciplinary research will benefit both fields. In this review article, tissue engineering and cancer research are brought together and explored with regard to discoveries that may be of mutual benefit.

In vitro investigations on the effect of dermal fibroblasts on keratinocyte responses to ultraviolet B radiation

Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280-320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB-induced damage. To investigate these processes, established two and three-dimensional culture models were utilized to study the impact of fibroblast-keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase-3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast-produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation-induced damage.

Les cellules stromales multipotentes accélèrent la guérison de plaies cutanées chez les souris irradiées via la sécrétion de la chimiokine SDF-1α

Le traitement du cancer à l’aide d’une exposition aux radiations ionisantes (RI) peut mener au développement de plusieurs effets secondaires importants, dont un retard de réparation et de régénération des tissus. Les mécanismes responsables de ces effets demeurent largement inconnus encore aujourd’hui, ce qui a pour effet de limiter le développement d’approches thérapeutiques. À l’aide d’un modèle de guérison de plaie cutanée chez la souris, nous avons cherché à déterminer les mécanismes par lesquels l’exposition aux RI limite la régénération de la peau. Nos résultats démontrent que l’induction de la "stromal-derived growth factor 1α" (SDF-1α), une cytokine normalement surexprimée dans les tissus hypoxiques, est sévèrement diminuée dans les plaies de souris irradiées versus non-irradiées. Ce défaut corrèle avec un retard de guérison des plaies et est encore évident plusieurs mois suivant l’exposition aux RI, suggérant qu’il y a une altération permanente de la capacité de la peau à se réparer. Parce que SDF-1α est secrété principalement par les fibroblastes du derme, nous avons évalué le potentiel des cellules stromales multipotentes (MSCs), qui sont reconnues pour secréter des niveaux élevés de SDF-1α, à accélérer la régénération de la peau chez les souris irradiées. L’injection de MSCs en périphéries des plaies a mené à une accélération remarquable de la guérison de la peau chez les souris exposées aux RI. Les actions des MSCs étaient principalement paracrines, dû au fait que les cellules n’ont pas migré à l’extérieur de leur site d’injection et ne se sont pas différentiées en kératinocytes. L’inhibition spécifique de l’expression de SDF-1α a mené à une réduction drastique de l’efficacité des MSCs à accélérer la fermeture de plaie indiquant que la sécrétion de SDF-1α par les MSCs est largement responsable de leur effet bénéfique. Nous avons découvert aussi qu’un des mécanismes par lequel SDF-1α accélère la guérison de plaie implique l’augmentation de la vascularisation au niveau de la peau blessée. Les résultats présentés dans ce mémoire démontrent collectivement que SDF-1α est une importante cytokine dérégulée au niveau des plaies cutanées irradiées, et que le déclin du potentiel de régénération des tissus qui est observé suivant une exposition au RI peut être renversé, s’il est possible de restaurer le microenvironnement de la blessure avec un support stromal adéquat.

L’effet des différents facteurs de croissance sur la viabilité et la prolifération des ostéoblastes scoliotiques

Résumé: La Scoliose Idiopathique de l’Adolescent (SIA) est une condition débilitante qui peut avoir comme résultat une douleur importante, une altération du fonctionnement quotidien et une détérioration de la qualité de vie. Pour les patients qui ne répondent pas au traitement conservateur, la fusion vertébrale, en utilisant des greffes osseuses, est devenue un traitement de choix pour stabiliser la colonne. Des connaissances plus pointues à propos des facteurs impliqués dans l’ostéogénèse et la formation de l’os peuvent raccourcir le processus de guérison et permettre aux patients de réintégrer leurs activités dans un laps de temps plus court. Les plaquettes peuvent jouer un rôle important dans la première étape de la guérison des fractures car elles sont une source autologue de plusieurs facteurs de croissance qui soutiennent la prolifération et la différenciation des ostéoblastes in vivo et in vitro. Au cours des dernières années, plusieurs tentatives ont été réalisées afin de trouver des traitements additionnels pour : 1) Raccourcir le temps de guérison des fractures relativement long ; 2) Obtenir une plus courte période de convalescence pour les patients qui ont besoin de prothèses ; 3) Corriger plus facilement plusieurs maladies congénitales; 4) Améliorer le processus de fusion vertébrale et 5) Développer de nouvelles approches thérapeutiques, notamment au niveau des processus régularisant le remodelage osseux et la régénération des tissus osseux. Dans le cadre de la présente étude, j’ai étudié la contribution possible du facteur de croissance de l’insuline (IGF) et du facteur vasculaire endothélial de croissance (VEGF) sur la maturation de l’ostéoblaste scoliotique dans des cultures cellulaires in vitro et j’ai comparé les résultats avec celles obtenues dans les mêmes conditions mais en stimulant les ostéoblastes avec de la mélatonine. Cette étude préliminaire a été réalisée sur des échantillons d’os récoltés de quatre patients atteints par la Scoliose Idiopathique de l‘Adolescent (SIA), ainsi que sur des échantillons d’os issus de quatre sujets témoins (cas traumatiques). Les résultats montrent que l’IGFs et le VEGFs possèdent une action d’inhibition sur la prolifération d’ostéoblastes scoliotiques et non scoliotiques, et que cette action est proportionnelle à la concentration de ces facteurs. Les ostéoblastes scoliotiques tendent à avoir une prolifération cellulaire plus rapide et plus élevée que les témoins non scoliotiques. De façon générale les ostéoblastes provenant de patients scoliotiques ont une ostéogénèse in vitro plus accélérée que le sujet non scoliotique. De plus, il semble que la mélatonine joue un rôle physiologique dans la différenciation de l’ostéoblaste scoliotique et elle semble aider à avoir une différenciation plus précoce que chez les non traités. Les ostéoblastes scoliotiques expriment un défaut d’expression de l’IGF 1 et d’IGF 1R en présence de la mélatonine. En conclusion, le VEGF A et l’IGF 1 peuvent également promouvoir la différenciation et la prolifération des ostéoblastes humains scoliotiques en culture primaire.