Regeneration of Limb Joints in the Axolotl (Ambystoma mexicanum)

In spite of numerous investigations of regenerating salamander limbs, little attention has been paid to the details of how joints are reformed. An understanding of the process and mechanisms of joint regeneration in this model system for tetrapod limb regeneration would provide insights into developing novel therapies for inducing joint regeneration in humans. To this end, we have used the axolotl (Mexican Salamander) model of limb regeneration to describe the morphology and the expression patterns of marker genes during joint regeneration in response to limb amputation. These data are consistent with the hypothesis that the mechanisms of joint formation whether it be development or regeneration are conserved. We also have determined that defects in the epiphyseal region of both forelimbs and hind limbs in the axolotl are regenerated only when the defect is small. As is the case with defects in the diaphysis, there is a critical size above which the endogenous regenerative response is not sufficient to regenerate the joint. This non-regenerative response in an animal that has the ability to regenerate perfectly provides the opportunity to screen for the signaling pathways to induce regeneration of articular cartilage and joints.     

Evaluation of new porous β-tri-calcium phosphate ceramic as bone substitute in goat model

The present study was carried out to evaluate the porous β-tri-calcium phosphate (TCP) (prepared by aqueous solution combustion technique) as bone substitute and compared with normal healing in 12 adult Black Bengal goats on the basis of clinical and radiographic findings, histological studies, oxytetracycline labeling, angiography studies (on day 90). Bone defects created in the diaphysis of radius were left unfilled in control animals (group I); while in treated (group II) animals the defects were filled with porous TCP blocks. The three months study showed no marked acute inflammatory reactions in all animals, wound healing was uneventful and the implants were clinically stable in the bone. Radiological studies showed presence of unabsorbed implants which acted as a scaffold for new bone growth across the defect whereas in control animals the defect was more or less same except that the newly formed bony tissue was less organized. Histological section showed moderately differentiated lamellar bone in the cortical part with presence of woven bone at peripheral cortex whereas control animals showed moderate fibro-collagenisation and good amount of marrow material, fat cells and blood vessels. Oxytetracycline labeling study showed crossing over of new bony trabeculae along with presence of resorption cavities within the new osteoid tissues whereas in group I, the process of new bone formation was active from both the ends; the defect site appeared as a homogenous non-fluorescent area. Angiogram of the animals in control showed uniform angiogenesis in the defect site with establishment of trans transplant angiogenesis, whereas in group II there was complete trans transplant shunting of blood vessels communication. The results of this study pointed out that the porous TCP promoted extensive bone formation over the entire extension of the defect in comparison to control group, thus conforming their biological osteoconductive property.     

Evaluation of HTR polymer as a craniomaxillofacial graft material

An experimental animal study was undertaken to evaluate the potential use of HTR polymer as a craniomaxillofacial reconstructive material. Its properties of marked hydrophilicity, a negative surface charge, and extensive porosity combined with a calcium graft coating suggested favorable osteoconductive properties. Four sites of the rabbit craniomaxillofacial skeleton were evaluated by placement of both inlay and onlay implants. Postoperative assessment at 60, 120, and 240 days consisted of histologic evaluation by tetrachrome staining of calcified sections. At 60 days, extensive osseous ingrowth into the inlay implants had occurred extending up to 2000 microns. The onlay implants, however, exhibited more limited ingrowth extending only 100 to 200 microns into the material. Over the ensuing 120- and 240-day postoperative periods, the average depth of bony ingrowth did not progress in either the inlay or onlay specimens over that observed at 60 days. Differences also were noted between the inlayed skeletal sites, with increased marrow at the recipient site resulting in increased bony ingrowth. The material appears to be biocompatible, with no evidence of infection, inflammatory reaction, or bone resorption observed around any of the implants. The implants exhibited a significant osteoconductive capability that was most manifest when implants were exposed to bleeding cortical/marrow surfaces as inlay grafts.     

纵胞藻切段再生的初步研究

作者将纵胞藻切成1.5—3mm长的切段,用PES培养液,室内漫射光进行培养,成功地得到大量纵胞藻再生植株。通过实验探讨,作者认为纵胞藻的切段再生现象可能是该种海藻营养繁殖的一种形式。     

纵胞藻切段再生的初步研究

作者将纵胞藻切成1.5—3mm长的切段,用PES培养液,室内漫射光进行培养,成功地得到大量纵胞藻再生植株。通过实验探讨,作者认为纵胞藻的切段再生现象可能是该种海藻营养繁殖的一种形式。     

Analytical study of Xenopus hindlimb regenerate with special reference to muscle regeneration

Amputated hindlimbs of Xenopus laevis, develop various types of regenerates in relation with amputation level as well as stage development. The present experiments is an attempt to study the histological characteristics of Xenopus regenerations, i.e., rational changes of tissue components along the length of the regenerated part with special emphasis on the degree of muscle regeneration. Four types of regenerates were studied viz; a 4th toe obtained from a completely restored regenerated limb at 126 days after amputation of limb at base level in stage 51. An amputated limb with no external sign of regeneration of limb at thigh level in stage 60. A spike-shaped regenerate at 96 days after amputation of limb at shank level in stage 63. A spike-shaped regenerate at about 2 years after amputation of limb at shank level in stage 60. Cross sectional areas of muscle, skin gland, epidermis and cartilage in each of the four types of regenerates were measured with Image Analyzing Apparatus (VIP 121 CH, Olympus Co.). The relative area of each tissue was expressed as a percentage of the cross sectional area of the limb. The obtained values were plotted along the length of the regenerate. Digitiform regenerates were found to be more or less similar to the control limbs, i.e., provided joints and muscle, while the heteromorphic spike or rod shaped regenerates were simply provided with cartilaginous axial core without joint formation. Muscle area were reduced rapidly near the amputation area of these heteromorphic regenerates with no more continuation in the regenerated tissue. It is interesting to mention that percentage cartilage area of about 2 years old spike regenerate was higher than that of similar 96 days regenerate. In addition muscle regeneration was completely absent even in such an aged regenerate. The area showed fairly similar ratio irrespective of the external appearance of the regenerate. In 32 regenerates of which limbs were amputated at various developmental stages ranging between stage 51 and adult stage, the histological condition of muscle at the amputation site, were well observed. In all digitated types of regenerates even in those with reduced number of toes, muscles were found grown well in the regenerates. In heteromorphic regenerates without toe formation muscle did not usually regenerate. In few cases, however, a small mass of myoblastic like cells or small aggregation of differentiated muscle cells without any structural continuation with the stump muscles, were seen to develop in the midst of the regenerate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prospective fates and regulative capacities of fragments of the female genital disc of Drosophila melanogaster.

A fate map of the female genital disc of Drosophila melanogaster was established by examining the derivatives of fragments transplanted into host larvae for metamorphosis. The fate map is presented as a two-dimensional projection, but for several reasons it is proposed that the anal plates originate from the dorsal epithelial layer whereas the genitalia are produced from the ventral layer. Fragments produced by cuts parallel to the axis of symmetry of the disc undergo regeneration during culture in adult hosts if the fragments comprise more than half of the disc, or duplication if they comprise less than half. Most of the fragments generated by bilaterally symmetrical cuts across the line of symmetry of the disc undergo neither regeneration nor duplication during culture, but with some such fragments there is a low frequency of regeneration. It is argued that the usual lack of regeneration in these fragments results from wound healing which confronts identical positions from right and left sides, giving no growth stimulation. The fragments which regenerate might do so as a result of healing between dorsal and ventral surfaces, providing the discontinuity in positional information which is thought to be involved in growth stimulation.     

Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin

While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair.     

Lesion and regeneration in the medial cerebral cortex of lizards.

The cerebral cortex of Squamate reptiles (lizards and snakes) may be regarded as an archicortex or "reptilian hippocampus". In lizards, one cortical area, the medial cortex, may be considered as a true "fascia dentata" on grounds of its anatomy, connectivity and cyto- chemo-architectonics of its main zinc-rich axonal projection. Moreover, its late ontogenesis and postnatal development support this view. In normal conditions, it shows delayed postnatal neurogenesis and growth during the lizard's life span. Remnant neuroblasts in the medial cortical ependyma of adult lizards seasonally proliferate. The late-produced immature neurocytes migrate to the medial cortex cell layer where they differentiate and give off zinc-containing axons directed to the rest of cortical areas. This results in a continuous growth of the medial cortex and its zinc-rich axonal projection. Perhaps the most important characteristic of the lizard medial cortex is that it can regenerate after having been almost completely destroyed. Recent experiments in our laboratory have shown that chemical lesion of its neurons (up to 95%) results in a cascade of events; first, those related with massive neuronal death and axonal-dendritic retraction and, secondly, those related with a triggered neuroblast proliferation and subsequent neo-histogenesis, and the regeneration of an almost new medial cortex that shows itself undistinguishable from a normal undamaged one. This is the only report to our knowledge that an amniote central nervous centre may regenerate by new neuron production and neo-histogenesis. Perhaps the medial cortex of lizards may be used as a model for neuronal regeneration and/or transplant experiments in mammals or even in primates.     

The effect of the chemical nature of implant materials on regeneration processes in an implant site

The literature data on implant materials for recovering from osseous injuries and defects were reviewed. Hydroxyapatite and bioactive glass are the leading artificial implant materials. Chitosan, polylactide, adgelon, and salicylic acid have found application in this area as biocompatible surgical materials that also promote wound healing and regeneration. When using hydroxyapatite as an implant material, its active groups, such as phosphate, hydroxyl, and others provide contacts; cell migration and adhesion on the matrix surface, formation of an intermediate layer of osteoid type, and fusion of bone and implant then occur. In the case of bioactive glass, the silanol groups are involved in bond formation. The study of mechanisms of bond formation between biological tissue and implant material and search for new biocompatible materials are important tasks of medical research in the field of implantation and post-traumatic regeneration.     

Regeneration in youngBunodactis verrucosa Pennant (actinaria,anthozoa)

Summary YoungBunodactis verrucosa Pennant at the 12 tentacle stage are employed to test the applicability of the polar coordinate model to coelenterate regeneration. The animals are cut along every radius into fragments of 3 to 9 segments. Most fragments are patent 3–4 weeks later, but small fragments have a higher mortality rate than large fragments. Some fragments do not regenerate and occasionally tentacles fuse, thereby reducing the number of segments. Small fragments tend to regenerate more tentacies than large fragments, but large fragments may regenerate great numbers of supernumerary tentacles. Twenty-two percent of the fragments restore the missing number of tentacles, while 76% of all fragments produce an even number of tentacles.Fragments restoring the correct numbers of tentacles show a marked tendency to form the correct tentacles (regulative regeneration). Fragments regenerating two less than the number of tentacles already present show a marked tendency to reproduce tentacles of the types already present (miror image formation). Other fragments produce missing segments (forward regeneration), or those already present (reverse regeneration) at lower frequencies.No fragments beginning or ending with the number 1 directive tentacle fail to regenerate entirely, while first cycle segments maximally remote from segment 1 are associated with the absence of regeneration. No fragments beginning or ending with the number 4 directive tentacle fail to undergo forward regeneration, regulate or produce a mirror image when the appropriate number of segments are regenerated. In contrast, segment 4 is associated with a low frequency of reverse regeneration, and second cycle segments cut away from immediate contact with segment 4 show an increase in the frequency of reverse regeneration. Controls through morphogenic substances rather than polar coordinates seem to explain these results. Such substances would control the number and direction of tentacle regeneration.This work was performed while the author was on sabbatical leave from the University of Pittsburgh at the Stazione Zoologica di Napoli. The author gratefully acknowledges the assistance of Mr. Ciro Gargiulo and of Ms. Gisella Princivalli. This work was supported by a travel grant from the United States Italy Cooperative Science Program of the National Science Foundation. The paper is dedicated to Dr. Alberto Monroy whose generosity made it possible     

Simvastatin did not prevent nor restore ovariectomy-induced bone loss in adult rats

Current published results on whether statins have beneficial effects on bone metabolism have been conflicting so far. In order to further investigate if statins were promising candidates for the treatment for osteoporosis, we conducted a study in which rats were ovariectomized (OVX) at 6 months of age, allowed to lose bone for 60 days and followed by oral administration of simvastatin at the dose levels of 0.3-10 mg/kg/d for 60 days. PGE2 (6 mg/kg) was used as a positive control. Study endpoints included bone histomorphometry on the proximal tibial metaphysis (PTM) and the tibial diaphysis (TX), dual-energy X-ray absorptiometry on the right femur and micro computed tomography (ICT) on the 5th lumbar vertebra (LV). After 120 days of OVX, cancellous bone lost by 80% in the PTM and 18% in the LV accompanied by increased bone formation and resorption. Simvastatin at all dose levels did not affect bone volume, bone formation rate and bone erosion surface when compared to 120 day ovariectomized animals at all bone sites studied. By contrast, PGE2 restored cancellous and cortical bone area to sham control levels. In conclusion, this study demonstrated that unlike PGE2, oral administration of simvastatin did not have effects on cancellous or cortical bone formation and resorption; and consequently was not able to prevent further bone loss or restore bone mass in the osteopenic, OVX rats.     

O-phospho-L-serine: a modulator of bone healing in calcium-phosphate cements

Bone substitution materials are seen as an alternative to autogenous bone transplants in the reconstruction of human bone structures. The aim of the present animal study was to evaluate the clinical handling and the conditions of bone healing after the application of a phosphoserine and collagen-I-modified calcium-phosphate cement (Biozement D). The application of phosphoserine is supposed to influence the texture of the extracellular matrix. Standardised bone defects were created in the lower jaw of 10 adult minipigs. These defects were reconstructed with a pasty calcium-phosphate cement mixture. After a healing time of 4 months, the animals were sacrificed. The mandibles of all animals were resected and non-decalcified histological sections of the areas of interest were prepared. The experiment was evaluated by means of qualitative histology and histomorphometry. The hydroxyapatite cement entirely hardened intraoperatively. Modelling and handling of the cement was facile and the margin fit to the host bone was excellent. Histology showed that resorption started in the periphery and proceeded exceptionally fast. The bony substitution, especially in phosphoserine-endowed cements, was very promising. After a healing period of 4 months, phosphoserine cements showed a bone regeneration of nearly two-thirds of the defect sizes. In the applied animal experiment, the newly developed hydroxyapatite collagen-I cement is well suited for bone substitution due to its easy handling, its excellent integration and good resorption characteristics. The addition of phosphoserine is very promising in terms of influencing resorption features and bone regeneration.     

Bone regeneration within a coralline hydroxyapatite implant.

The hypothesis that incomplete resorption of osteons in an autogenous cortical bone graft may limit its replacement by new bone regeneration was explored by implanting a hydroxyapatite replica of a coral skeletal structure into bone gaps. This implant contained channels and interconnections similar to those in osteon-evacuated bone grafts. In 6 implanted mandibular defects in dogs, two of which were examined at two, 4, and 6 months, 11 percent, 46 percent, and 88 percent of the implant areas were filled with regenerated bone. The regenerated bone was a woven type at two months, but changed to a lamellar type by 6 months. In two implanted defects examined at 12 months, biodegradation of 29 percent of the implant had occurred. The bone regeneration was physiological, the implant was biocompatible, and the biodegradation began after the bone had regenerated.     

Homologies of positional information in thoracic imaginal discs ofDrosophila melanogaster

Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.     

Comparative effects of alendronate and alfacalcidol on cancellous and cortical bone mass and bone mechanical properties in ovariectomized rats.

The purpose of the present study was to compare the effects of alendronate and alfacalcidol on cancellous and cortical bone mass and bone mechanical properties in ovariectomized rats. Twenty-six female Sprague-Dawley rats, 7 months of age, were randomized by the stratified weight method into four groups: the sham-operated control (Sham) group and the three ovariectomy (OVX) groups, namely, OVX + vehicle, OVX + alendronate (2.5 mg/kg, p.o., daily), and OVX + alfacalcidol (0.5 mug/kg, p.o., daily). At the end of the 8-week experimental period, bone histomorphometric analyses of cancellous bone at the proximal tibial metaphysis and cortical bone at the tibial diaphysis were performed, and the mechanical properties of the femoral distal metaphysis and femoral diaphysis were evaluated. OVX decreased cancellous bone volume per total tissue volume (BV/TV), and the maximum load of the femoral distal metaphysis, as a result of increases in serum osteocalcin (OC) levels, and also the number of osteoclasts (N.Oc), osteoclast surface (OcS) and bone formation rate (BFR) per bone surface (BS), and BFR/BV, without any effect on cortical area (Ct Ar), or maximum load of the femoral diaphysis. Alendronate prevented this decrease in cancellous BV/TV by suppressing increases in N.Oc/BS, OcS/BS, BFR/BS, and BFR/BV, without any apparent effect on Ct Ar, or maximum load of the femoral distal metaphysis and femoral diaphysis. On the other hand, alfacalcidol increased cancellous BV/TV, Ct Ar, and the maximum load of the femoral distal metaphysis and femoral diaphysis, by mildly decreasing trabecular BFR/BV, maintaining trabecular mineral apposition rate and osteoblast surface per BS, increasing periosteal and endocortical BFR/BS, and preventing an increase in endocortical eroded surface per BS. The present study clearly showed the differential skeletal effects of alendronate and alfacalcidol in ovariectomized rats. Alendronate prevented OVX-induced cancellous bone loss by suppressing bone turnover, while alfacalcidol improved cancellous and cortical bone mass and bone strength by suppressing bone resorption and maintaining or even increasing bone formation.     

Temperature-Sensitive Mutations That Cause Stage-Specific Defects in Zebrafish Fin Regeneration

When amputated, the fins of adult zebrafish rapidly regenerate the missing tissue. Fin regeneration proceeds through several stages, including wound healing, establishment of the wound epithelium, recruitment of the blastema from mesenchymal cells underlying the wound epithelium, and differentiation and outgrowth of the regenerate. We screened for temperature-sensitive mutations that affect the regeneration of the fin. Seven mutations were identified, including five that fail to regenerate their fins, one that causes slow growth during regeneration, and one that causes dysmorphic bumps or tumors to develop in the regenerating fin. reg5 mutants fail to regenerate their caudal fins, whereas reg6 mutants develop dysmorphic bumps in their regenerates at the restrictive temperature. Temperature-shift experiments indicate that reg5 and reg6 affect different stages of regeneration. The critical period for reg5 occurs during the early stages of regeneration before or during establishment of the blastema, resulting in defects in subsequent growth of the blastema and failure to differentiate bone-forming cells. The critical period for reg6 occurs after the onset of bone differentiation and during early stages of regenerative outgrowth. Both reg5 and reg6 also show temperature-sensitive defects in embryonic development or in ontogenetic outgrowth of the juvenile fin.     

Alternative cells for regeneration

Normally, in fish fin regeneration, bone regenerates from bone. But what happens when there is no bone? Singh et al. (2012) show in this issue of Developmental Cell that the bony rays still regenerate from an alternative cell source.     

Evaluation of demineralised,freeze-dried,irradiated bone allografts in the treatment of osseous defects in the oral cavity

Demineralised, freeze-dried bone allografts (DFDBA) have been used extensively by dentists in the treatment of periodontal and periapical osseous defects resulting from inflammatory diseases. Their use in India however, is limited by the availability of quality allografts and the high cost of imported alternatives. A study was conducted to assess the osteogenic potential of DFDBA prepared for the first time in India by the Tata Memorial Hospital (TMH) Tissue Bank. The DFDBA was used in the treatment of osseous defects after removal of periapical lesions associated with devitalised teeth in 10 healthy patients. At the 6-month recall visit all the patients showed a remarkable decrease in the grades of mobility, and 9 out of the 10 patients showed radiographic evidence of complete healing of the osseous defects with evidence of normal bony trabaeculae. These findings indicate that the indigenously prepared DFDBA is a cost effective, biocompatible material with osteogenic potential that can be used effectively in treating osseous defects of periapical lesions associated with non vital teeth.     

Changes in the amount of mineral components in bone tissue during treatment of transcondylar fractures of the humerus

In 12 non-inbred 4-month-old puppies a closed brachial transcondylar fracture has been performed. By means of photonic absorbtiometry contents of mineral components have been estimated in the regenerate forming between the osseous broken pieces, in the osseous fragments and in the adjoining segment, depending on duration of the joint fixation during the treatment. Mineralization takes place more quickly in a mobile joint. Fixation for 5-7 days does not essentially affect the miniralization rate. Fixation for 14 days decreases intensity of the mineralization, produces deminiralization in the osseous pieces and in the adjoining segment of the bone, results in incompleteness of the brachial condyle rearrangement.