Fragment Viability Research Articles

Fragment viability, regenerative capacity and protoplast isolation of invasive Australian swamp stonecrop (Crassula helmsii)

Australian swamp stonecrop (Crassula helmsii) is an amphibious plant native to Australasia and highly invasive in Europe. Managing the spread and impacts of this invader is challenging due to its ability to regenerate from small vegetative plant fragments. In several infested water systems in the Netherlands, the dispersal of this species is currently being prevented using water filters with a mesh size as small as 1 mm2 in their outflows. However, it remains unclear whether these filters are truly capable of preventing the spread of small regenerative fragments. To identify potential shortcomings in the management of C. helmsii dispersal, we investigated the regeneration of detached vegetative fragments. A laboratory experiment was conducted to study the regenerative capacity of C. helmsii fragments of varying sizes (0.5–2 mm). We examined the growth performance (stem count, cumulative stem length) of fragments originating from nodes, leaves, shoots, roots, and meristem tissue cuts. All fragments originating from nodal tissue were able to successfully regenerate. Isolated meristem tissue of 0.5 mm was able to regenerate but exhibited malformed growth. Shoot tips demonstrated the best growth performance. While we successfully isolated protoplasts from C. helmsii node tissue, our cultures were compromised, and regeneration could not be assessed. Our research indicates regenerative potential from node fragments as small as a cluster of apical meristem cells. These findings suggest shortcomings in current measures for dispersal prevention of C. helmsii, as they do not prevent the dispersal of plant fragments with a length <2 mm. We recommend re-evaluating the effectiveness of various types of dispersal barriers currently applied in vulnerable natural areas to prevent the spread and subsequent regeneration of small vegetative fragments of C. helmsii.

Vegetative fragment production as a means of propagule dispersal for tropical seagrass meadows

Background and aimsLong distance dispersal (LDD) contributes to the replenishment and recovery of tropical seagrass habitats exposed to disturbance, such as cyclones and infrastructure development. However, our current knowledge regarding the physical attributes of seagrass fragments that influence LDD predominantly stems from temperate species and regions. The goal of this paper is to measure seagrass fragment density and viability in two tropical species, assessing various factors influencing their distribution. MethodsWe measured the density and viability of floating seagrass fragments for two tropical seagrass species (Zostera muelleri and Halodule uninervis) in two coastal seagrass meadows in the central Great Barrier Reef World Heritage Area, Australia. We assessed the effect of wind speed, wind direction, seagrass growing/senescent season, seagrass meadow density, meadow location and dugong foraging intensity on fragment density. We also measured seagrass fragment structure and fragment viability; i.e., potential to establish into a new plant. Key resultsWe found that seagrass meadow density, season, wind direction and wind speed influenced total fragment density, while season and wind speed influenced the density of viable fragments. Dugong foraging intensity did not influence fragment density. Our results indicate that wave action from winds combined with high seagrass meadow density increases seagrass fragment creation, and that more fragments are produced during the growing than the senescent season. Seagrass fragments classified as viable for Z. muelleri and H. uninervis had significantly more shoots and leaves than non-viable fragments. We collected 0.63 (±0.08 SE) floating viable fragments 100 m−2 in the growing season, and 0.13 (±0.03 SE) viable fragments 100 m−2 in the senescent season. Over a third (38%) of all fragments collected were viable. ConclusionThere is likely to be a large number of viable seagrass fragments available for long distance dispersal. This study's outputs can inform dispersal and connectivity models that are used to direct seagrass ecosystem management and conservation strategies.

Knee osteochondritis dissecans-treatment technical aspects

Purpose and objectiveCurrent treatments of different stages of knee osteochondritis Dissecans (OCD) are depending on the age of the patients and the stability of the diseased osteochondral area. The purpose of this paper was to summarize the treatment alternatives in order to simplify the choice for the treating surgeon. Background and principle resultsOsteochondritis dissecans (OCD) of the knee is an idiopathic and local osteochondral abnormality that affects mainly children and adolescents with risk of loosening of osteochondral fragments. A good clinical result can be expected when the physes are still open, when the osteochondritis is small and when the osteochondritis can be assessed as stable by MRI. Unstable OCD lesions most often need to be treated operatively by different fixation methods and when the osteochondral cannot be refixated, different local chondral and osteochondral repairs are available to fill up the defect area to congruity Summary and major conclusionsThe final choice of which treatment to use is depending on fragment viability and forms. Viable fragments are refixated while poor quality fragments are removed followed by a local biological osteochondral repair. Such osteochondral resurfacing may be single bone marrow stimulation with or without scaffold augmentation or different cell seeded grafts.

Hydrodynamic dissection of Stentor coeruleus in a microfluidic cross junction.

Stentor coeruleus, a single-cell ciliated protozoan, is a model organism for wound healing and regeneration studies. Despite Stentor's large size (up to 2 mm in extended state), microdissection of Stentor remains challenging. In this work, we describe a hydrodynamic cell splitter, consisting of a microfluidic cross junction, capable of splitting Stentor cells in a non-contact manner at a high throughput of ∼500 cells per minute under continuous operation. Introduction of asymmetry in the flow field at the cross junction leads to asymmetric splitting of the cells to generate cell fragments as small as ∼8.5 times the original cell size. Characterization of cell fragment viability shows reduced 5-day survival as fragment size decreases and as the extent of hydrodynamic stress imposed on the fragments increases. Our results suggest that cell fragment size and composition, as well as mechanical stress, play important roles in the long-term repair of Stentor cells and warrant further investigations. Nevertheless, the hydrodynamic splitter can be useful for studying phenomena immediately after cell splitting, such as the closure of wounds in the plasma membrane which occurs on the order of 100-1000 seconds in Stentor.

Identification of dispersal barriers for a colonising seagrass using seascape genetic analysis

Seagrasses are important habitats providing many ecological services. Most species have broad distributions with maximum dispersal distances of 100's of kms, however there is limited understanding of dispersal distances of colonising species like Halodule uninervis. It commonly grows in disturbed environments and could disperse to other meadows via clonal fragments. Effective conservation management requires greater understanding of genetic structure, dispersal barriers, and connectivity timescales to predict recovery following disturbance. Despite fragment viability of up to 28 days in a congenera, this theory remains untested in situ. Using 80 neutral single nucleotide polymorphisms, we investigated genetic diversity, gene flow patterns and structure among 15 populations of H. uninervis along 2000 km of Western Australian coastline. These data were combined with a multi-generational oceanographic dispersal model and a barrier dispersal analysis to identify dispersal barriers and determine which fragment dispersal duration (FDD) and timescale over which stepping-stone connectivity occurred, best matched the observed genetic structure. The 2-7 day FDD best matched the genetic structure with 4–12 clusters, with barriers to dispersal that persisted for up to 100 years. Modelling suggested greater fragmentation of metapopulations towards the southern edge of the species distribution, but genetic diversity did not decline. Several long-term boundaries were identified even with fragment viability of up to 28 days. This suggests H. uninervis dispersal is spatially limited by factors like oceanographic features and habitat continuity which may limit dispersal of this species. This study reiterates that potential dispersal does not equal realised dispersal, and management scales of 10's of kilometers are required to maintain existing meadows. Recruitment from distances further than this scale are unlikely to aid recovery after extreme disturbance events, particularly towards the range edge of H. uninervis distribution.

Chlorophyll fluorometry sheds light on the role of desiccation resistance for vegetative overland dispersal of aquatic plants

Abstract Submerged aquatic plant species predominantly spread through vegetative propagules, particularly shoot fragments. While it is known that the successful establishment of fragments in isolated water bodies is largely determined by retention of fragment viability during overland transport (i.e. desiccation resistance), detailed information on species‐specific desiccation resistance is still scarce and the underlying mechanisms remain uncharted. We combined measurements of chlorophyll a fluorescence (Fv/Fm: maximum quantum yield of photosystem II) with determination of water loss and post‐desiccation survival and regeneration to examine the desiccation resistance of shoot fragments without and with apical tips for six submerged aquatic plant species (Myriophyllum spicatum, Myriophyllum heterophyllum, Ceratophyllum demersum, Lagarosiphon major, Elodea canadensis, Hydrilla verticillata). Overall, the relationship between Fv/Fm and relative water loss was nonlinear, and a decrease in Fv/Fm was significantly related to reduced fragment survival and regeneration. We determined an overall critical minimum of 0.40 in Fv/Fm and a critical maximum of 84% in water loss for regeneration. Differences in the relationships between water loss and desiccation time and between Fv/Fm and water loss were species‐specific rather than fragment type‐specific. Plant fragments of M. spicatum (fragments without apices excluded), M. heterophyllum, and C. demersum maintained a high Fv/Fm even after losing a large proportion of the initial water content, while the Fv/Fm for L. major, E. canadensis, and H. verticillata decreased more rapidly with advancing water loss. Maintaining the function of the photosynthetic apparatus at high water loss can prolong fragment viability during overland transport. Our results suggest that aquatic plants have species‐specific mechanisms to cope with water deficit. Measurements of chlorophyll fluorescence can precisely predict the likelihood of fragment regeneration and constitute a useful tool to assess the spread potential of aquatic plants.

Don’t leave me behind: viability of vegetative propagules of the clonal invasive Carpobrotus edulis and implications for plant management

The capacity of succulent plants to create new invasive focus starting from disconnected plant fragments involves severe difficulties in the management of invaded areas. In the case of the succulent stoloniferous Carpobrotus edulis, clonality further difficults land restoration since management practices usually fragment and outspread vegetative propagules. In order to contribute to a better management of this highly invasive species, we explored fragment viability during the first month after disconnection. We hypothesized that period after separation from maternal clumps and propagule size, both influence the capacity of C. edulis to establish and develop new vegetative structures. With this objective, we separate apical fragments with two different sizes that were sown under greenhouse conditions after a sequence of storing time. We further examined morphological and physiological attributes as survival, length growth and biomass, FW/DW ratio, relative water content and leaf hydration, osmolarity and fluorescence. Biochemical parameters were also included through the measurement of phenol and tannin content together with photosynthetic pigments (chlorophyll a,b and carotenoids) and derived ratios. After 2 months, apical propagules did not show signs of morphological, physiological or biochemical deterioration with increasing storing periods. Propagules showed improved or at least similar performance after storing for longer periods, with slight or no differences between different sized propagules in morphological or physiological values as indicated by the absence of enhanced leaf water status, the maintenance of photosynthetic efficiency (high Fv/Fm values) and the stability in photosynthetic pigments content. Our results indicate that the growth of C. edulis propagules is not significantly constrained during the first month after disconnection, no matter the size of apical fragment considered. As a final remark, we consider that our results are important to ameliorate and efficiently monitor and manage invaded areas where C. edulis had been recently eliminated.

Extending the Timeframe for Rapid Response and Best Management Practices of Flood-Dispersed Japanese Knotweed (Fallopia japonica)

The objective of this article is to extend the reported period in which flood-distributed knotweed propagules may be successfully managed using only manual labor. During a second round of early detection and rapid response (EDRR) management along the Green River in Guilford, VT, we collected and measured all Japanese knotweed propagules that had been distributed by flooding approximately 21 mo earlier, in August 2011, at a single site. Our data suggest that knotweed s.l. prioritizes the growth of new stems over new rhizomes at the start of a growing season. Because the limiting factor for successful removal of new knotweed s.l. plants by hand is the size of the rhizome system, our findings support extending the time frame for EDRR management of flood-distributed knotweed s.l. into the second spring after its initial dispersal. Additionally, in November 2013, surveys of our work sites found no new knotweed s.l. plants in locations accessible to work crews. In addition to validating our EDRR management techniques, this implies that knotweed s.l. fragment viability does not extend past the second spring following its dispersal.

A boat hitchhiker’s guide to survival: Cabomba caroliniana desiccation resistance and survival ability

Cabomba caroliniana is a submersed macrophyte that has become a serious invader. Cabomba predominantly spreads by stem fragments, in particular through unintentional transport on boat trailers (‘hitch hiking’). Desiccation resistance affects the potential dispersal radius. Therefore, knowledge of maximum survival times allows predicting future dispersal. Experiments were conducted to assess desiccation resistance and survival ability of cabomba fragments under various environmental scenarios. Cabomba fragments were highly tolerant of desiccation. However, even relatively low wind speeds resulted in rapid mass loss, indicating a low survival rate of fragments exposed to air currents, such as fragments transported on a boat trailer. The experiments indicated that cabomba could survive at least 3 h of overland transport if exposed to wind. However, even small clumps of cabomba could potentially survive up to 42 h. Thus, targeting the transport of clumps of macrophytes should receive high priority in management. The high resilience of cabomba to desiccation demonstrates the risk of continuing spread. Because of the high probability of fragment viability on arrival, preventing fragment uptake on boat trailers is paramount to reduce the risk of further spread. These findings will assist improving models that predict the spread of aquatic invasive macrophytes.

Fragment reattachment, reproductive status, and health indicators of the invasive colonial tunicate Didemnum vexillum with implications for dispersal

The invasive colonial tunicate Didemnum vexillum is now widespread in coastal and offshore waters of New England, USA. D. vexillum can inflict ecological and economic damage through biofouling and habitat modification. Natural and anthropogenic processes that fragment colonies of D. vexillum may be accelerating the spread of this invader. Reattachment success and fragment viability were confirmed in the laboratory after four weeks of suspension in experimental aquaria. The shape of suspended D. vexillum fragments progressed from flattened to globular spheres and then flattened again after reattachment to the substrate. Reproductive activity, confirmed by the presence of eggs and larvae, was observed for fragments suspended up to 3 weeks suggesting that D. vexillum is capable of reproducing while in a fragmented, suspended state. An index of colony health was used to monitor change in D. vexillum health while in suspension. Overall, colony health declined with time in suspension although colonies that appeared dead (black and gray in overall color) still contained a substantial number of healthy live zooids. These results suggest that activities that cause fragmentation can significantly facilitate the spread of D. vexillum. Coastal managers should consider reducing or eliminating, when practical, activities that return fragmented colonies of D. vexillum to the water. In-water cleaning of biofouling and dredging are likely expediting the spread of this invasive species unless biofouling can be contained and removed from the water.

Viability of golden star tunicate fragments after high-pressure water treatment

High-pressure water spray is one of the mitigation strategies used by mussel growers against invasive colonial tunicates fouling aquaculture gear. However, this treatment leads to fragmentation of tunicate colonies and can potentially lead to increased recolonization of adjacent suspended aquaculture gear. We demonstrate that colony fragments of the golden star tunicate ( Botryllus schlosseri) are viable for at least 18 days following high-pressure water treatment. Therefore, future studies need to investigate the fate of colonial tunicate fragments after high-pressure washing to determine fragment viability under field conditions. Potentially, mitigation strategies against colonial tunicates need to incorporate techniques ensuring fragment mortality.

Invasive river plants from Portuguese floodplains: What can species attributes tell us?

Biological traits of aquatic and riparian plants were compared among invasive and non-invasive groups of species from both native and exotic taxa in Portuguese fluvial systems. Twenty-six riparian and aquatic species were selected and their frequency of occurrence and foliar percentage cover was analysed at 401 sampling sites on the main catchments of Central and Southern Portugal. Canonical correspondence analysis and a hierarchical clustering were applied to the species traits data set. No typical invader profile emerged from the analyses. However, some specific attributes were clearly related to the alien and native plant invasiveness, such as vegetative reproduction, fragment viability and life span. The results of the present work could help with the early detection of invasive plant species in Portuguese fluvial corridors.

The role of laser Doppler flowmetry in assessing the viability of bone fragments in an open fracture

The management of open fractures requires excision of all devitalised tissues, both bony and soft tissue, and failure to do so is likely to increase the risk of infection. This study evaluated the applicability of laser Doppler flowmetry for the objective evaluation of fracture fragment viability in an experimental open ballistic fracture over a period of 12 h. The results indicate that this technique could not be used to distinguish between vascularised and non-vascularised fragments at any time, and did not aid the surgeon in their decision making at the time of wound excision. Subjective evaluation, based upon the degree of soft tissue attachment of fragments, was a far better indicator of fragment vascularity, although it had a relatively low specificity. There remains the need for education and training for trauma surgeons in the evaluation of fragment viability to ensure adequate wound excision as part of fracture management.

OSTEOCHONDRITIS DISSECANS OF THE HUMERAL CAPITELLUM: Diagnosis and Treatment

Elbow pain seen in the at-risk athlete, such as a baseball player (in particular, a pitcher) or gymnast, should raise suspicion for OCD. OCD of the humeral capitellum remains a difficult problem to treat. Once radiographic changes are obvious, long-term studies suggest that half of affected individuals will be symptomatic. Currently, the key to successful treatment is early detection. Gymnasts, in general, fare worse in returning to sport. The reason is not entirely clear but is likely related to the amount of force directed across the elbow and the nature of the sport. Pitchers sometimes can be returned, but to another position. Radiographs remain the screening test of choice but can give the surgeon a false sense of security, because changes early in the disease process may not be obvious radiographically. Views at 45 degrees flexion and contralateral elbow views are helpful. The advent of MR imaging now allows the practicing orthopaedic surgeon to assess very early lesions effectively that might otherwise be underappreciated on radiographs. With more advanced lesions, radiographic findings are more obvious and demonstrate the more classic capitellar fragment with a surrounding zone of lucency. MR imaging is this setting is helpful in assessing the overlying articular cartilage and, hence, the stability of the fragment. In the absence of obvious loose bodies or mechanical symptoms, rest is the first step in treatment. If symptoms persist, then operative intervention is indicated. About half of these patients heal with nonoperative treatment. Pretreatment assessment of fragment viability has not traditionally been incorporated into the treatment algorithm. Recent anecdotal evidence suggests that stability and viability of a fragment can be assessed using intravenous contrast. Knowledge of the fragment viability could allow those lesions likely to heal without surgical intervention to be distinguished from those requiring surgical intervention. The procedure should be chosen based on the size of the lesion and the integrity of the subchondral bone. Subchondral drilling and microfracture can only resurface defects and cannot reconstitute subchondral bone. Autologous chondrocyte implantation has limited ability to address subchondral bone loss, whereas autograft and allograft osteochondral transplantation can restore subchondral bone. Most authors would agree that there is no role currently for reduction and fixation of long-standing, free loose bodies. No consensus exists regarding acute dislodging of an in situ loose fragment. Long-term results after radiographic changes are present suggest a degenerative course in about half the patients. Whether the newer techniques of cartilage resurfacing will significantly impact the natural history of this process remains to be seen.

Evaluation of a Short-term Rat Proximal Tubule Incubation System for the Detection of Nephrotoxicants.

Many nephrotoxic agents act primarily on proximal tubule cells. Accordingly, the optimal conditions for isolating rat proximal tubule fragments by a collagenase digestion technique and their short-term maintenance have been defined, and the viability of the preparation and its sensitivity to toxicants have been determined. Tubular fragment viability was maintained in incubation for up to 6 hours, as assessed by measuring lactate dehydrogenase leakage, levels of intracellular glutathione, and ATP content. In addition, tubular transport function was maintained, as determined by measuring the uptake of p-aminohippuric acid and α-methylglucose, which could be blocked by the selective inhibitors, probenecid and phloridzin, respectively. In this study, the toxicities of allyl alcohol, cephalosporins and cisplatin to proximal tubular fragments were investigated. Allyl alcohol toxicity was greater in tubular fragments isolated from female rats than in those from male rats. Cephaloridine was toxic, while cephalexin and cephalothin were not. These features were consistent with the known in vivo responses to these agents. Toxicity was evident after exposure to cisplatin, with an early reduction in tubular transport being noted. The results highlight the potential of the system described for the isolation and incubation of rat proximal tubule fragments to study xenobiotic-mediated nephrotoxicity. This system could be of benefit in the high-throughput toxicity screening of novel therapeutic agents.

Relation of necrosis to outcome and site of fracture in scaphoid pseudarthrosis

Usually, untreated scaphoid fractures lead to non-union and partial or complete avascular necrosis. The purpose of our study was to examine the influence of location and fracture line on the pattern of necrosis. Therefore, 85 patients with scaphoid non-union were examined by enhanced highfield MRI. Fractures were classified by location and fracture line. A staging system based on signal intensity in T1-weighted spin-echo sequences in enhanced MRI was used to evaluate extent and location of avascular necrosis, which was found in 81% of our patients. In our patients, viability of the scaphoid was independent of the fracture line without dominance of vertical oblique fractures (2%). Based on our staging system, signal intensity as a sign of fragment viability was reduced in the proximal fragment in 46 patients, whereas in eight patients the distal fragment showed reduced viability. In 26 patients, no difference was found between proximal and distal fragments. Fractures proximal to the transition of middle to distal third showed less viability of the proximal fragment, not dependent of how proximal the fracture was seen. In triple fractures (6%) signal intensity of scaphoid bone was more reduced in enhanced MRI.

Effect of various short-term storage methods on viability of cancellous bone fragments

Abstract Objective To determine effects of various storage methods on ex vivo viability of cancellous bone fragments. Sample Population Cancellous bone fragments obtained from 4 New Zealand White rabbits. Procedure Cancellous bone fragments were stored for 3 hours on ice in 1 of 5 preservation solutions or in 0.9% NaCI. Fragments were then reperfused (37 C) in oxygenated physiologic buffer solution for 1 hour. Cellular viability in fragments was assessed by ethidium monoazide labeling and fluorescence microscopy. Results Viability in fresh cancellous bone ranged from 85 to 100% (mean ± SEM, 88 ± 7%). Storage of fragments significantly reduced viability. Viability in bone fragments stored at 22 C in blood-soaked sponges or 0.9% NaCI solution was 63.8 ± 3 and 65.2 ± 7%, respectively. Use of cold 0.9% NaCI solution reduced viability to 53.6 ± 3%. Viability was significantly better for fragments stored in cold phosphate-buffered sucrose (70.4 ± 2%) and EuroCollins (71.4 ± 3%) solutions. After warm reperfusion, viability was best for fragments stored in cold phosphate-buffered sucrose (70.2 ± 4%), EuroCollins (72.6 ± 3%), or UW lactobionate solutions (69.6 ± 3%), compared with those stored in cold 0.9% NaCI (47.6 ± 3%) or hypertonic citrate (53.0 ± 3%) solutions or blood-soaked sponges (57.2 ± 3%). Conclusions Hypothermic storage in solutions designed to prevent temperature-dependent cell injury were best for maintaining cancellous bone fragment viability. Clinical Relevance Hypothermia may be advantageous for use in storing cancellous bone fragments during procedures that dictate a prolonged period between harvest and placement of graft fragments. (Am J Vet Res 1999;60:63–67)