Stump Tissue Research Articles

Thumb Metacarpal Lengthening

Over a period of 35 years, 92 patients have been treated: 70 adults and 22 children age 8 to 14. The metacarpal lengthening obtained varied from 2 to 4.5 cm with an average of 3.5 cm, or equal to the length of a thumb proximal phalanx. In one third of the adult patients, due to delayed spontaneous ossification, the interfragmental space was bridged by a bone graft taken from the iliac crest. Late follow-up of 21 patients made 10 to 14 years after the treatment showed good function of the thumb with a normal pattern of skin sensibility. The lack of trophic changes in the distal part of the reconstructed thumb is due to the interposition type of bone lengthening, that is, the new bone structure is placed into the bone middle and not superimposed on the bone stump end, as is done in other methods of thumb reconstruction. The metacarpal lengthening procedure has the following drawbacks: (1) the full treatment course lasts several months; (2) the reconstructed thumb has neither joint nor nail. Advantages of the method are: (1) reconstruction of the thumb is done by using the available stump tissue resources; (2) the reconstructed thumb is covered by its own, normally innervated skin.

Urodele spinal cord regeneration and related processes.

Urodele amphibians, newts and salamanders, can regenerate lesioned spinal cord at any stage of the life cycle and are the only tetrapod vertebrates that regenerate spinal cord completely as adults. The ependymal cells play a key role in this process in both gap replacement and caudal regeneration. The ependymal response helps to produce a different response to neural injury compared with mammalian neural injury. The regenerating urodele cord produces new neurons as well as supporting axonal regrowth. It is not yet clear to what extent urodele spinal cord regeneration recapitulates embryonic anteroposterior and dorsoventral patterning gene expression to achieve functional reconstruction. The source of axial patterning signals in regeneration would be substantially different from those in developing tissue, perhaps with signals propagated from the stump tissue. Examination of the effects of fibroblast growth factor and epidermal growth factor on ependymal cells in vivo and in vitro suggest a connection with neural stem cell behavior as described in developing and mature mammalian central nervous system. This review coordinates the urodele regeneration literature with axial patterning, stem cell, and neural injury literature from other systems to describe our current understanding and assess the gaps in our knowledge about urodele spinal cord regeneration.

Tissue specificity in rat peripheral nerve regeneration through combined skeletal muscle and vein conduit grafts.

Diffusible factors from the distal stumps of transected peripheral nerves exert a neurotropic effect on regenerating nerves in vivo (specificity). This morphological study was designed to investigate the existence of tissue specificity in peripheral nerve fiber regeneration through a graft of vein filled with fresh skeletal muscle. This tubulization technique demonstrated experimental and clinical results similar to those obtained with traditional autologous nerve grafts. Specifically, we used Y-shaped grafts to assess the orientation pattern of regenerating axons in the distal stump tissue. Animal models were divided into four experimental groups. The proximal part of the Y-shaped conduit was sutured to a severed tibial nerve in all experiments. The two distal stumps were sutured to different targets: group A to two intact nerves (tibial and peroneal), group B to an intact nerve and an unvascularized tendon, group C to an intact nerve and a vascularized tendon, and group D to a nerve graft and an unvascularized tendon. Morphological evaluation by light and electron microscopy was conducted in the distal forks of the Y-shaped tube. Data showed that almost all regenerating nerve fibers spontaneously oriented towards the nerve tissue (attached or not to the peripheral innervation field), showing a good morphological pattern of regeneration in both the early and late phases of regeneration. When the distal choice was represented by a tendon (vascularized or not), very few nerve fibers were detected in the corresponding distal fork of the Y-shaped graft. These results show that, using the muscle-vein-combined grafting technique, regenerating axons are able to correctly grow and orientate within the basement membranes of the graft guided by the neurotropic lure of the distal nerve stump.

Tissue specificity in rat peripheral nerve regeneration through combined skeletal muscle and vein conduit grafts

Diffusible factors from the distal stumps of transected peripheral nerves exert a neurotropic effect on regenerating nerves in vivo (specificity). This morphological study was designed to investigate the existence of tissue specificity in peripheral nerve fiber regeneration through a graft of vein filled with fresh skeletal muscle. This tubulization technique demonstrated experimental and clinical results similar to those obtained with traditional autologous nerve grafts. Specifically, we used Y-shaped grafts to assess the orientation pattern of regenerating axons in the distal stump tissue. Animal models were divided into four experimental groups. The proximal part of the Y-shaped conduit was sutured to a severed tibial nerve in all experiments. The two distal stumps were sutured to different targets: group A to two intact nerves (tibial and peroneal), group B to an intact nerve and an unvascularized tendon, group C to an intact nerve and a vascularized tendon, and group D to a nerve graft and an unvascularized tendon. Morphological evaluation by light and electron microscopy was conducted in the distal forks of the Y-shaped tube. Data showed that almost all regenerating nerve fibers spontaneously oriented towards the nerve tissue (attached or not to the peripheral innervation field), showing a good morphological pattern of regeneration in both the early and late phases of regeneration. When the distal choice was represented by a tendon (vascularized or not), very few nerve fibers were detected in the corresponding distal fork of the Y-shaped graft. These results show that, using the muscle-vein-combined grafting technique, regenerating axons are able to correctly grow and orientate within the basement membranes of the graft guided by the neurotropic lure of the distal nerve stump. © 2000 Wiley-Liss, Inc. MICROSURGERY 20:65–71 2000

An ultrasound indentation system for biomechanical properties assessment of soft tissues in-vivo.

An ultrasound indentation system for biomechanical assessment of soft tissues in vivo was developed. The pen-size, hand-held probe was composed of an ultrasound transducer and a load cell. The ultrasound transducer was at the tip of the probe serving also as the indentor. The thickness and deformation of the soft tissue layer were determined from the ultrasound echo. A compressive load cell was connected in series with the ultrasound transducer to record the force response. A validation experiment was performed on porcine tissues. Force and deformation acquired with the present system was in good comparison with those obtained from a Housfield material testing machine. Material constants were obtained via a curve-fitting procedure by predicting the force transient response from the deformation-time data using a quasilinear viscoelastic model. In addition, deformation in the fat and in the muscle could be differentiated. The potential applications of this type of indentation probes are many. The specific application of this current development is for stump tissue assessment in the design of prosthetics.

Clinical measurement of normal and shear stresses on a trans-tibial stump: characteristics of wave-form shapes during walking.

Stresses on the surface of a stump within a prosthetic socket during walking can potentially traumatize stump tissues. To gain insight into stresses and design parameters that affect them, normal and shear interface stresses were measured on three unilateral trans-tibial amputee subjects during walking trials. During stance phase repeated characteristics in wave-form shapes from different subjects were apparent. They included "loading delays", "high frequency events (HFE's)", "first peaks", "valleys", "second peaks", and "push-off". Characteristics did not necessarily occur at the same time from one step to the next but their timings matched well with events in shank force and moment data which were collected simultaneously. For "plantarflexion" and "dorsiflexion" alignment changes, the above wave-form characteristics were still present but their timings within the stance phase changed. The physical meaning and relevance of the characteristics to stump tissue mechanics are discussed.

Specificity in mammalian peripheral nerve regeneration at the level of the nerve trunk

Previous studies indicate that distal stumps of transected rat peripheral nerves secrete ‘tropic’ factors which can attract/support axonal regeneration over distances of several mm in vivo. The present study was undertaken in order to determine if there is specificity of neurotropic interaction at the level of the nerve trunk. Proximal stumps of transected peroneal or tibial nerves were inserted into the single inlet end of Y-shaped Silastic implants and offered alternative ‘lures’ at the paired outlet ends (specifically, grafts of peroneal vs tibial distal stump tissue). Several weeks later, the overwhelming majority of preparations showed exclusive growth of nerve fibers in implant forks attached to ‘native’ (originally associated) nerve stumps. Inversion of the distal stump grafts (such that the proximal stump was facing an analogous native distal stump, but a different region of it) diminished the frequency and extent of native preference. Taken together, data suggest the possibility that there can be a specificity nerve regeneration at the level of the nerve trunk.

Cellular contribution to supernumerary limbs in the axolotl, Ambystoma mexicanum.

Using the triploid cell marker, the cellular contribution from graft and stump to the supernumerary limbs which result from controlateral grafts of limb buds and regeneration blastemas in the axolotl has been analyzed. Grafts were made so as to appose anterior and posterior limb positions. Overall, the contribution from graft and stump tissue was found to be approximately equal although the position of the boundary between the two was variable from limb to limb. This result is consistent with models which suggest that intercalary regeneration is the driving force for patterning of the vertebrate limb. In addition, the pattern of cellular contribution to supernumerary limbs was consistently found to be asymmetrical in the dorsal-ventral axis. Hence, posterior limb tissue predominantly contributed cells to the posterior and dorsal part of the supernumerary limb whereas anterior limb tissue predominantly contributed cells to the anterior and ventral part of the supernumerary limb. The reason for this asymmetrical pattern remains unknown, but we suggest that it might result from a directional bias in intercalary regeneration, similar to that observed during intercalation in the proximal-distal axis of the urodele limb. Using the triploid cell marker in conjunction with a black/white pigmentation marker, the relationship between the cellular contribution boundary and the pigmentation boundary in supernumerary limbs has also been analyzed. It has been found that the positions of the two boundaries do not coincide, a result which suggests that the eventual location of pigment cells is not a good indicator of the location of nonpigment cells derived from graft and stump.

The role of plant hormones and carbohydrates in the growth and survival of coppiced Eucalyptus seedlings

Depending on the species, coppicing (decapitation) may promote vigorous growth (Eucalyptus camaldulensis Dehn), or cause rapid senescence and death (Eucalyptus obliqua L'Herit). In seedlings of the latter species, the presence of a small upwardly directed shoot on the decapitated stump prevents or delays decline. Coppiced seedlings of E. camaldulensis and E. obliqua, with and without a remaining shoot, were analyzed for starch and soluble sugars (with the anthrone method), gibberellin‐like substances (GAs) and cytokinin‐like substances (by bioassay), and ethylene (by gas‐liquid chromatography) before and after decapitation.Levels of soluble sugars declined similarly in both varieties of eucalypts, and starch reserves appeared adequate for sprouting, and did not diminish following decapitation of the susceptible species.Decapitation did not markedly alter the relatively high amounts of GAs in roots and shoots of E. obliqua, the susceptible species, although increased levels of Gas were observed in the stumps of seedlings left with 1 shoot after decapitation. The overall levels of GaS were relatively low in the roots and stems of the resistant E. camaldulensis, but higher in the shoots. Marked qualitative changes in GAs with decapitation were apparent in the shoots of E. camaldulensis. A single major GA peak occurred prior to decapitation but afer decapitation several additional peaks of GA‐like activity appeared.Cytokinin‐like activity was initially low in all tissues, but increased dramatically in stump and shoot tissue following decapitation. Increases ranged from approximately 5‐fold (stump tissue of either species, minus‐shoot treatment) to approximately 40‐fold (shoot tissue of the resistant E. camaldulensis seedlings left with 1 shoot). In both E. camaldulensis and E. obliqua ethylene production increased to a peak 7 days after decapitation provided a shoot had been retained. This ethylene peak precedes a marked upturning of the retained shoot, and was not present in the stumps of totally decapitated seedlings. For totally decapitated seedlings ethylene evolution in E. obliqua (the susceptible species), but not E. camaldulensis (the resistant species), had ceased by 15 days.

Organization of the Morphogenetic Field in Regenerating Amphibian Limbs

The amphibian limb is an example of a secondary embryonic field that can be reactivated during larval or adult life so that amputated parts are regenerated. Two major questions are: (1) what is the origin of the morphogenetic field of the regeneration blastema, and (2) what is the nature of this field and how does it specify the spatial pattern of blastemal redifferentiation? Evidence is analyzed here which leads to the following propositions: (1) the field is represented in latent form by properties of the mature limb cells, and these properties are activated and inherited by the blastemal cells after amputation and dedifferentiation. At the same time, the inherited field is sensitive to the mature stump tissues and its spatial organization can be altered by a stump pattern alien to the one from which it was derived. (2) The properties of the mesodermal limb tissues represent positional values that are arranged in gradients along the proximal-distal, anterior-posterior and dorsal-ventral axes. These properties allow dedifferentiated mesodermal cells to change their positional value to any value between their original one in the limb and the value of any neighboring cell after creation of a discontinuity. The direction of change is always from proximal to distal in the PD axis; it is uncertain as to whether change can take place only centripetally or both centripetally and centrifugally along the AP and DV axes. (3) Epidermal cells have the same positional value everywhere in the limb and act as the distal and circumferential boundaries up to which the mesodermal cells may change their positional values. The proximal boundary is represented by the level-specific properties of the mesodermal cells at the maximum extent of distal to proximal dedifferentiation. Normal regeneration can then be visualized as occurring in the following way. When deletions are made in the limb pattern, cells with widely different positional values are confronted. During regeneration, blastema cells increase in number and continually interact with their neighbors to adjust their positional values within the boundaries until discontinuities are eliminated. The multiple limbs resulting from rearrangement of stump tissue patterns can also be accounted for by using these propositions. It is suggested that positional information is encoded on the cell surface and/or in the extracellular matrix.

Injury requirement for initiation of regeneration of newt limbs which have whole skin grafts

THERE are three known requirements for salamander limb regeneration—injury, nerves, and wound epidermis1,2. During the past few years we have examined cellular events in amputated, regenerating limbs and compared these with non-regenerating limbs after either denervation3,4 or after whole skin grafts over the amputation surface5. Results of these experiments led to the formulation of an hypothesis6 which suggests unique roles for injury, nerves, and the wound epidermis during the initiation of regeneration : injury is important to cause dedifferentiation of limb stump tissue cells, entry of these cells into G1 of the cell cycle and DNA replication; nerves are important for one or more G2 events and thus indirectly for mitosis; and the wound epidermis maintains these cells in the cell cycle so that significant outgrowth (blastema formation) can occur. In the present study we have examined the extent and kinds of injury necessary to initiate regeneration in newt limbs which have had skin grafts for 5 weeks and externally show no regeneration. The results suggest that non-cycling dedifferentiated cells are present in 5-week skin graft limbs which immediately begin cycling when a wound epidermis forms after re-injury.

Evidence for polarizing zone in the limb buds of Xenopus laevis

To test for the presence of polarizing mesoderm in an amphibian, Xenopus laevis hindlimb bud tips were rotated 180° on the proximodistal axis and returned to the stump. Supernumerary outgrowths were induced in the preaxial stump and preaxial tip tissues, and the most postaxial digit always formed next to the grafted postaxial tissue. The occurrence of polarized supernumerary outgrowths indicated that the posterior limb border contained a polarizing zone. When the limb tip was cut at varying known lengths from the body wall, rotated, and grafted to the limb stump, the incidence of twinning along the proximodistal axis permitted insight into the distribution of the polarizing zone along the posterior border. The location of polarizing tissues was found to be similar to that in the chick wing bud at comparable stages. To confirm the posterior border stump influence on the rotated preaxial limb tip tissues, 180° tip rotations were made at the proximodistal level with the highest incidence of twinning. In these cases, the adjacent stump posterior border tissues (polarizing zone) were removed, leaving a substantial amount of the deeper postaxial stump tissue, however. The frequency of twinning from tip tissues was greatly reduced in these larvae compared to those with rotated limb tips on intact stumps. Cytological examination of supernumerary outgrowths resulting from grafts of two-nucleolate tips onto one-nucleolate stumps confirmed the preaxial source of the supernumerary outgrowths.

The effects of rotation and positional change of stump tissues upon morphogenesis of the regenerating axolotl limb

Rotation of a skin cuff 180° around the proximodistal axis of the upper arm in the axolotl results in the formation of multiple regenerates in about 80° of cases after amputation of the limb through the rotated skin. Rotation of the dermis or the flexor and extensor muscles folowed by amputation produced similar percentages of multiple regenerates. Rotated bone produced no abnormalities, and rotated stump epidermis was minimally effective in stimulating multiple regeneration. A thin strip of normally oriented skin interposed between a rotated skin cuff and the amputation surface blocks the morphogenetic effect of the rotated stump skin whereas removal of the normal skin between a rotated proximal skin cuff and the amputation surface allows the formation of a low percentage of multiple regenerates. Gross rotation of stump tissue components can be broken down into axial rotation per se and positional dislocation. Experiments conducted upon skin and muscle have shown that positional dislocation along the anteroposterior axis rather than axial rotation is the manipulation that leads to the formation of multiple regenerates. The first morphological indication of multiple regeneration is the appearance of a triaxial apical ridge on the blastema. Subsequently, digits form along the apical ridges.

Isocitrate lyase activity in the regenerating forelimb of the adult newt.

AbstractRegenerating and non‐regenerating limb tissues from the adult newt, Diemictylus viridescens, were assayed for isocitrate lyase activity. The enzyme assays were performed by micromodifications of existing procedures. In general, the whole homogenate, or a soluble fraction of the homogenate, was incubated with the substrate isocitrate. Isocitrate is cleaved by isocitrate lyase to glyoxylate and succinate. At the termination of the reaction, the 2,4‐dinitrophenylhydrazone derivative of glyoxylate was produced, extracted and quantitated spectrophotometrically. Isocitrate lyase activity was localized to bulb and two‐digit regenerates. The reaction product, glyoxylate (a monocarboxylic keto acid), was of special interest due to its role as a potent in vitro metabolic inhibitor. Therefore, the endogenous level of monocarboxylic keto acids, and the ability of regenerating tissue to produce and accumulate monocarboxylic keto acids, were investigated. Whole homogenates of regenerating tissue always contained more monocarboxylic keto acids than intact forearm or stump tissue. Also, after 30 minutes incubation with buffer alone, only regenerating tissue produced and accumulated additional monocarboxylic keto acids. In regenerating tissue, the isocitrate lyase reaction may be utilized in the metabolism of lipid. Since regenerating tissue can produce and accumulate monocarboxylic keto acids in vitro, glyoxylate produced by the isocitrate lyase reaction may accumulate in vivo and participate in metabolic regulation.

Coriphosphine O蛍光色素法を用いた胃粘膜病変の臨床的並びに組織学的研究

1. Before applying the fluorescence microscopic method with Coriphosphine O (CO) to the staining of gastric mucosal tissues. the type of solvent, PH and temperature for making most of the physicochemical properties of Coriphosphine O pigment were -examined, and the opitimal staining conditions were established.2. The CO fluorescence microscopic method was found to be capable of well indicating the tissue structure of the gastric mucosa histomorphologically. The method also enabled the visual identification of the individual cells, and could be used for histopathological diagnosis.3. The fluorescence microscopic method by use of CO fluorescent pigment is characterized by specific binding of the pigment with nucleic acid, as has been demonstrated by digestion with DNAse and RNAse.Dilutions in various concentrations of CO pigment with McIlvaine buffer, PH 5.4, were, when irradiated with ultraviolet rays in the vicinity of the wavelength 405μm, found to show a spectrum of color changes according to the pigment concentrations. These color changes were in agreement with those of the gastric mucosal epithelial cell eytoplasm in various gastric diseases under application of the CO fluorescence microscopic method.4. By comparing the color changes of gastric mucosal epithelial cell cytoplasm with concentrations in vitro, the average CO concentration of pathologic mucosal epithelial cell cytoplasm in various stomach diseases and RI (RNA Index) were calculated and comparatively stadied.5. The comparative study of the average CO concentration and RI, i.e., the fluorescent color of the epithelial cell cytoplasm alone was found to have littel usefulness for clinical purposes. This was pointed out to be an important matter in clinical cytodiagnosis.6. As far as the RNA level within the epithlial cell cytoplasm was concerned, the average CO concentration and RI. could be used as a yardstick of RNA level in the cell, only under a simple morphological provision that “the epithelial cell cytoplasm is stained diffusely.”7. As compared with normal mucosa, an increase in cell RNA was observed in the mucoosa of cancerous tissues of early as well as advanced cancer, in the rostral noncancerous stump tissue of advanced cancer, in a part of the noncancerous tissue adjacent to cancer, and in the regenerated epithlium. The clinical significance of these findings was discussed.8. The cells with remarkable mucous retention, i.e., goblet cells in the mucosal epithelium with intestinal metaplasia, signet-ring cells within mucus-producing cancerous tissues, and the mucous portion of lacunar epithlial cell cytoplasm in adenomatous polyps, were not stained.

Roentgenological study of certain stump-socket relationships in above-knee amputees with special regard to tissue proportions, socket fit and attachment stability.

Twenty-five healthy active male above-knee amputees were studied. They had been uniformly fitted with their prosthesis, which in all cases had a total-contact suction socket of laminated plastic, and their walking capacity with the prosthesis was good. Sixteen of the patients were studied with respect to tissue proportions in the stump and intact thigh, while all 25 underwent studies concerning socket fit and relative movement between femoral stump and socket.Mean total cross-sectional area of amputation stump 5–10 cm above end of femur was calculated to be 63% of that of intact thigh. Mean cross-sectional area of skin and subcutaneous tissue of the stump exceeded that of intact thigh by 12% (0.01 > P > 0.001). Muscle and bone tissues of stump were 55% (P P > 0.001) less, respectively, than those of intact thigh. In the stump, skin and subcutaneous tissue were calculated to occupy a mean of 41.8%, muscle 53.9% and bone 4.3% of the total cross-sectional area. Corresponding tissue p...

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