Strategy in developing cell based therapy for large bone

Regeneration of osseous defects by tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. The concept of in vitro cultured osteoblasts having an ability to induce new bone formation has been demonstrated in the critical size defects using small animal models. The bone derived cells can be incorporated into bioengineered scaffolds and synthesize bone matrix, which on implantation can induce new bone formation. In search of optimal cell delivery materials, the extracellular matrix as cell carriers for the repair and regeneration of tissues is receiving increased attention. We have investigated extracellular matrix formed by osteoblasts in vitro as a scaffold for osteoblasts transplantation and found a mineralized matrix, formed by human osteoblasts in vitro, can initiate bone formation by activating endogenous mesenchymal cells. To repair the large bone defects, osteogenic or stem cells need to be prefabricated in a large three dimensional scaffold usually made of synthetic biomaterials, which have inadequate interaction with cells and lead to in vivo foreign body reactions. The interstitial extracellular matrix has been applied to modify biomaterials surface and identified vitronectin, which binds the heparin domain and RGD (Arg-Gly-Asp) sequence can modulate cell spreading, migration and matrix formation on biomaterials. We also synthesized a tri-block copolymer, methoxy-terminated poly(ethylene glycol)(MPEG)-polyL-lactide(PLLA)-polylysine(PLL) for human osteoblasts delivery. We identified osteogenic activity can be regulated by the molecular weight and composition of the triblock copolymers. Due to the sequential loss of lineage differentiation potential during the culture of bone marrow stromal cells that hinderers their potential clinical application, we have developed a clonal culture system and established several stem cell clones with fast growing and multi-differentiation properties. Using proteomics and subtractive immunization, several differential proteins have been identified and verified their potential application in stem cell characterization and tissue regeneration

Biological significance of dead biomass retention trait in Mediterranean Basin species: an analysis between different successional niches and regeneration strategies as functional groups

Standing dead biomass retention is considered one of the most relevant fuel structural traits to affect plant flammability. However, very little is known about the biological significance of this trait and its distribution between different functional groups. Our aim was to analyse how the proportion of dead biomass produced in Mediterranean species is related to the successional niche of species (early-, mid- and late-successional stages) and the regeneration strategy of species (seeders and resprouters). We evaluated biomass distribution by size classes and standing dead biomass retention in nine dominant species from the Mediterranean Basin in different development stages (5, 9, 14 and 26 years since the last fire). The results revealed significant differences in the standing dead biomass retention of species that presented a distinct successional niche or regeneration strategy. These differences were restricted to the oldest ages studied (>9 years). Tree and small tree resprouters, typical in late-successional stages, presented slight variations with age and a less marked trend to retain dead biomass, while seeder shrubs and dwarf shrubs, characteristic of early-successional stages, showed high dead biomass loads. Our results suggest that the species that tend to retain more dead branches are colonising species that may promote fire in early-successional stages.

中国暖温带辽东栎林主要树种共存机制的研究

物种共存是植物群落生态学的一个重要问题。对物种共存的研究可以促进人们对生态系统结构与功能的认识，并为生物多样性保护及恢复提供理论指导。在本论文中，通过对辽东栎－棘皮桦林主要树种空间格局及空间关系、竞争作用和繁殖策略的研究，初步确定本地区辽东栎林主要树种共存的机制。 空间点格局分析表明辽东栎、棘皮桦和五角枫的幼苗、幼树和大树均呈聚集分布。辽东栎幼苗和幼树的分布与同种大树个体正相关，与棘皮桦的大树个体负相关，而与死树个体无显著的相关关系。这些结果说明种子散播和萌生繁殖过程影响辽东栎树木个体的空间格局。棘皮桦幼苗和幼树的分布在小的空间尺度上与同种活树和死树个体正相关，而与其它树种的活树及死树个体的分布呈负相关，说明萌生是该物种繁殖的一个重要途径。五角枫的幼苗和幼树呈聚集分布，并与同种大树个体的分布有关，说明其种子有限的散播范围影响其分布。辽东栎大树个体的分布与棘皮桦大树个体在小尺度上呈负相关，表明这两个树种之间可能存在竞争关系。 离散模型（the diffusion model）和生长动态模型（the growth dynamics model）分析表明在一些树种之间存在单向的种间竞争，例如，黄花柳对白桦和棘皮桦的竞争作用，棘皮桦对辽东栎，辽东栎对五角枫，和五角枫对山杨的竞争作用。这些单向竞争主要是林冠层的大树个体对林下层小个体的竞争。在这些树种中没有发现种内竞争。但是，各树种的生长率（G (t, x)）与生长率方差（ ）呈直线相关，而且样地中树木的死亡率较低，说明树种间的竞争强度很小。因此我们推测非对称的种间竞争在塑造该森林群落结构和物种共存过程中的作用不大。 五角枫、辽东栎、糠椴和山杨的幼苗和幼树在森林的林下层占优势，密度和出现频率均较高，可以形成稳定的幼苗库。五角枫、辽东栎和糠椴的幼苗大小级结构呈倒J形，山杨幼苗的大小结构呈单峰形。棘皮桦幼苗很少，但幼树相对较多，说明该树种可以形成幼树库。辽东栎幼苗在林窗中密度较大，而幼树的密度与林窗的出现与否无明显的相关关系，说明林窗的形成对新更新苗有显著影响，而林窗内的幼树可能是由林窗形成前已经建立的幼苗发展而来。林冠下的辽东栎幼苗多数为萌生起源，寿命较长，一旦上层大树死亡，可以迅速生长，进入林冠层。因此，林冠层下的幼苗库和林窗更新对辽东栎种群动态至关重要。五角枫幼苗和幼树的耐荫性或避荫性较强，可以形成巨大的长期存在的幼苗库和幼树库，以维持其种群的更新。糠椴和山杨的幼苗，主要起源于根的萌蘖。由于母树提供营养，幼苗生长速度较其它三个树种快。但它们对土壤湿度和营养的要求较高，这限制了它们很难占据其它生境。 虽然，在所研究的处于不同演替阶段的群落中，树种的组成有所不同，但辽东栎、大叶白腊和五角枫的幼苗均非常丰富，可以形成幼苗库。辽东栎的幼苗库大部分为萌生苗，而大叶白腊的幼苗库主要由实生苗构成。大叶白腊的幼苗种群中小个体丰富，它们生长速度很慢，在林下采取一种“坐－等”（sit-and-wait）的策略。在三个树种中，五角枫幼苗主枝的伸长生长率最高，而且生长率与幼苗的基径和生境的光照强度正相关。 通过以上研究，我们初步得出结论：竞争作用在研究的辽东栎－棘皮桦林主要树种共存中起的作用很小;形成稳定长久的幼苗库是这些树种的主要繁殖策略。因此，理解这些树种共存机制的关键在于这些树种幼苗库或幼树库的不同起源，及幼苗和幼树对生境因子的不同生态响应所创造的丰富的更新生态位。

Eco-physiological aspects of melaleuca seed germination

The invasive behavior of melaleuca (Melaleuca quinquenervia) plants in wetlands is due to its aggressive regeneration strategy, which is based on its seeds germination performance. Understanding of the eco-physiological aspects of the seed germination in melaleuca plants may significantly contribute for the development of management strategies. The objective of this research was to learn how the germination of M. quinquenervia seeds are affected by light and temperature. Melaleuca seeds were placed on filter paper moistened with 12 ml of distilled water at temperatures between 10 and 45°C. Germination was evaluated in dark and light conditions. Seed germination, first count of seed germination (seven days), germination speed index and germination mean time were determined up to 40 days after seeding, when germination had ceased in most of the treatments. After that period, the seeds were transferred to conditions of 30°C and light, which was found to be ideal in the previous phase. Seed germination was daily evaluated up to 63 days when it was again observed no longer to occur. The treatment repetitions were distributed in the growth-chamber according to a completely randomized design in a factorial scheme (eight temperatures x two light conditions) and four repetitions. The data were submitted to analysis of variance with the F test and the means were adjusted to polynomial and non linear regression models. The highest seed germination performance was observed to take place under conditions of 27.3°C with light. The temperatures of 35 and 40°C in the dark induced thermal inhibition of seed germination. The temperature of 45°C was lethal to the seeds.

Armazenamento de sementes pré-germinadas de Inga vera Willd

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modelling seed germination in forest tree species through survival analysis. The Pinus pinea L. case study

The direct application of existing models for seed germination may often be inadequate in the context of ecology and forestry germination experiments. This is because basic model assumptions are violated and variables available to forest managers are rarely used. In this paper, we present a method which addresses the aforementioned shortcomings. The approach is illustrated through a case study of Pinus pinea L. Our findings will also shed light on the role of germination in the general failure of natural regeneration in managed forests of this species. The presented technique consists of a mixed regression model based on survival analysis. Climate and stand covariates were tested. Data for fitting the model were gathered from a 5-year germination experiment in a mature, managed P. pinea stand in the Northern Plateau of Spain in which two different stand densities can be found. The model predictions proved to be unbiased and highly accurate when compared with the training data. Germination in P. pinea was controlled through thermal variables at stand level. At microsite level, low densities negatively affected the probability of germination. A time-lag in the response was also detected. Overall, the proposed technique provides a reliable alternative to germination modelling in ecology/forestry studies by using accessible/ suitable variables. The P. pinea case study highlights the importance of producing unbiased predictions. In this species, the occurrence and timing of germination suggest a very different regeneration strategy from that understood by forest managers until now, which may explain the high failure rate of natural regeneration in managed stands. In addition, these findings provide valuable information for the management of P. pinea under climate-change conditions.

Cartilage regeneration using zonal chondrocyte subpopulations : a promising approach or an overcomplicated strategy?

Cartilage defects heal imperfectly and osteoarthritic changes develop frequently as a result. Although the existence of specific behaviours of chondrocytes derived from various depth-related zones in vitro has been known for over 20 years, only a relatively small body of in vitro studies has been performed with zonal chondrocytes and current clinical treatment strategies do not reflect these native depth-dependent (zonal) differences. This is surprising since mimicking the zonal organization of articular cartilage in neo-tissue by the use of zonal chondrocyte subpopulations could enhance the functionality of the graft. Although some research groups including our own have made considerable progress in tailoring culture conditions using specific growth factors and biomechanical loading protocols, we conclude that an optimal regime has not yet been determined. Other unmet challenges include the lack of specific zonal cell sorting protocols and limited amounts of cells harvested per zone. As a result, the engineering of functional tissue has not yet been realized and no long-term in vivo studies using zonal chondrocytes have been described. This paper critically reviews the research performed to date and outlines our view of the potential future significance of zonal chondrocyte populations in regenerative approaches for the treatment of cartilage defects. Secondly, we briefly discuss the capabilities of additive manufacturing technologies that can not only create patient-specific grafts directly from medical imaging data sets but could also more accurately reproduce the complex 3D zonal extracellular matrix architecture using techniques such as hydrogel-based cell printing.

From the margin to the centre : towards an art strategy for the ethnic enclaves in the city

Sunnybank represents a distinctly Australian take on the classic ‘Chinatown’ – or indeed other ethic community enclaves such as ‘little Italy’, ‘little Bombay’, ‘little Athens’ and so on. In the Northern Hemisphere these tended to grow up in the dense working class neighbourhoods of industrial cities, especially in port cities like Liverpool, London, New York and San Francisco. The existing Chinatowns of Sydney and Melbourne, and to some extent Brisbane’s Fortitude Valley, are of this variety. In the late 1970s, with the growth of suburbanisation and the de-industrialisation and consequent dereliction of the ‘inner city’, these ethnic communities were one of the few signs of life in the city. Apart from the daily commute into the CBD, business with the city council or a trip to the big shopping streets these areas were one of the few reasons for visiting city centres stigmatised by urban decay and petty crime.

A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex

This study describes the design of a biphasic scaffold composed of a Fused Deposition Modeling scaffold (bone compartment) and an electrospun membrane (periodontal compartment) for periodontal regeneration. In order to achieve simultaneous alveolar bone and periodontal ligament regeneration a cell-based strategy was carried out by combining osteoblast culture in the bone compartment and placement of multiple periodontal ligament (PDL) cell sheets on the electrospun membrane. In vitro data showed that the osteoblasts formed mineralized matrix in the bone compartment after 21 days in culture and that the PDL cell sheet harvesting did not induce significant cell death. The cell-seeded biphasic scaffolds were placed onto a dentin block and implanted for 8 weeks in an athymic rat subcutaneous model. The scaffolds were analyzed by μCT, immunohistochemistry and histology. In the bone compartment, a more intense ALP staining was obtained following seeding with osteoblasts, confirming the μCT results which showed higher mineralization density for these scaffolds. A thin mineralized cementum-like tissue was deposited on the dentin surface for the scaffolds incorporating the multiple PDL cell sheets, as observed by H&E and Azan staining. These scaffolds also demonstrated better attachment onto the dentin surface compared to no attachment when no cell sheets were used. In addition, immunohistochemistry revealed the presence of CEMP1 protein at the interface with the dentine. These results demonstrated that the combination of multiple PDL cell sheets and a biphasic scaffold allows the simultaneous delivery of the cells necessary for in vivo regeneration of alveolar bone, periodontal ligament and cementum. © 2012 Elsevier Ltd.

A bi-lineage conducive scaffold for osteochondral defect regeneration

Because cartilage and bone tissues have different lineage-specific biological properties, it is challenging to fabricate a single type of scaffold that can biologically fulfill the requirements for regeneration of these two lineages simultaneously within osteochondral defects. To overcome this challenge, a lithium-containing mesoporous bioglass (Li-MBG) scaffold is developed. The efficacy and mechanism of Li-MBG for regeneration of osteochondral defects are systematically investigated. Histological and micro-CT results show that Li-MBG scaffolds significantly enhance the regeneration of subchondral bone and hyaline cartilage-like tissues as compared to pure MBG scaffolds, upon implantation in rabbit osteochondral defects for 8 and 16 weeks. Further investigation demonstrates that the released Li+ ions from the Li-MBG scaffolds may play a key role in stimulating the regeneration of osteochondral defects. The corresponding mechanistic pathways involve Li+ ions enhancing the proliferation and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) through activation of the Wnt signalling pathway, as well as Li+ ions protecting chondrocytes and cartilage tissues from the inflammatory osteoarthritis (OA) environment through activation of autophagy. These findings suggest that the incorporation of Li+ ions into bioactive MBG scaffolds is a viable strategy for fabricating bi-lineage conducive scaffolds that enhance regeneration of osteochondral defects.

Bone regeneration based on tissue engineering conceptions – a 21st century perspective

The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteo- conductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineer- ing and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental “origin” require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.

Long-term repeated burning reduces Lantana camara regeneration in a dry eucalypt forest

Previous short-term studies predict that the use of fire to manage lantana (Lantana camara) may promote its abundance. We tested this prediction by examining long-term recruitment patterns of lantana in a dry eucalypt forest in Australia from 1959 to 2007 in three fire frequency treatments: repeated annual burning, repeated triennial burning and long unburnt. The dataset was divided into two periods (1959–1972, 1974–2007) due to logging that occurred at the study site between 1972 and 1974 and the establishment of the triennial burn treatment in 1973. Our results showed that repeated burning decreased lantana regeneration under an annual burn regime in the pre- and post-logging periods and maintained low levels of regeneration in the triennial burn compartment during the post-logging period. In the absence of fire, lantana recruitment exhibited a dome-shaped response over time, with the total population peaking in 1982 before declining to 2007. In addition to fire regime, soil pH and carbon to nitrogen ratio, the density of taller conspecifics and the interaction between rainfall and fire regime were found to influence lantana regeneration change over time. The results suggest that the reported positive association between fire disturbance and abundance of lantana does not hold for all forest types and that fire should be considered as part of an integrated weed management strategy for lantana in more fire-tolerant ecosystems.

Morphological Adaptations to Drought and Reproductive Strategy of the Moss Syntrichia caninervis in the Gurbantunggut Desert, China

The biological soil crusts (BSCs) in the Gurbantunggut Desert, the largest fixed and semi-fixed desert in China, feature moss-dominated BSCs, which play an indispensable role in sand fixation. Syntrichia caninervis Mitt. (S. caninervis) serves as one of the most common species in BSCs in the desert. In this study we examined the morphological structure of S. caninervis from leafy gametophyte to protonema using light and scanning electron microscopy (SEM). We also examined the relationships between the morphological structure of S. caninervis and environmental factors. We found that: (1) this moss species is commonly tufted on the sand surface, and its leaves are folded upwards and twisted around the stem under dry conditions; (2) the cells on both upper and lower leaf surfaces have C-shaped dark papillae, which may reflect sunlight to reduce the damage from high temperature; (3) the leaf costa is excurrent, forming an awn with forked teeth; and (4) the protonema cells are small and thickset with thick cell walls and the cytoplasm is highly concentrated with a small vacuole. In addition, we also found that the protonema cells always form pouches on the tip of the mother cells during the process of cell polarization. Our results suggest that S. caninervis has, through its life cycle, several morphological and structural characteristics to adapt to dry environmental conditions. These morphological features of S. caninervis may also be found in other deserts in the world due to the world-wide distribution of the species.