Bone tissue content of TGF-β2 changes with time in human heterotopic ossification after total hip arthroplasty

Transforming growth factor beta isoforms (TGF-β₁, TGF-β₂, and TGF-β₃) most likely play a role in bone physiology, but little is known about their relative importance in normal as well as in heterotopic bone. This study focused on possible differences in the localization and relative content of different TGF beta isoforms in heterotopic ossifications (HO) by comparing HOs, which have developed less than 17 months (immature HOs) with those developed 3–9 years (mature HOs). The HOs were harvested after total hip arthroplasty (THA) during revision surgery. The HO samples were decalcified, embedded in paraffin and sectioned. Azan staining was used to evaluate histological structure of the ossifications and immunohistochemical analysis was performed to estimate the localization of three TGF beta isoforms in the HOs. Comparison of different TGF beta isoforms in the immature and the mature ossifications showed that the content of TGF-β₂ was decreased by almost three times in the mature HO as compared to the immature HO (p = 0.0064). The proportions of other isoforms in HOs did not differ significantly. This study shows that the relative importance of TGF betas change with HO development.     

Cross-sectional study of the neural ossification centers of vertebrae C1–S5 in the human fetus

Purpose

An understanding of the normal evolution of the spine is of great relevance in the prenatal detection of spinal abnormalities. This study was carried out to estimate the length, width, cross-sectional area and volume of the neural ossification centers of vertebrae C1–S5 in the human fetus.

Materials and methods

Using the methods of CT (Biograph mCT), digital-image analysis (Osirix 3.9) and statistics (the one-way ANOVA test for paired data, the Kolmogorov–Smirnov test, Levene’s test, Student’s t test, the one-way ANOVA test for unpaired data with post hoc RIR Tukey comparisons) the size for the neural ossification centers throughout the spine in 55 spontaneously aborted human fetuses (27 males, 28 females) at ages of 17–30 weeks was studied.

Results

The neural ossification centers were visualized in the whole pre-sacral spine, in 74.5 % for S1, in 61.8 % for S2, in 52.7 % for S3, and in 12.7 % for S4. Neither male–female nor right–left significant differences in the size of neural ossification centers were found. The neural ossification centers were the longest within the cervical spine. The maximum values referred to the axis on the right, and to C5 vertebra on the left. There was a gradual decrease in length for the neural ossification centers of T1–S4 vertebrae. The neural ossification centers were the widest within the proximal thoracic spine and narrowed bi-directionally. The growth dynamics for CSA of neural ossification centers were found to parallel that of volume. The largest CSAs and volumes of neural ossification centers were found in the C3 vertebra, and decreased in the distal direction.

Conclusions

The neural ossification centers show neither male–female nor right–left differences. The neural ossification centers are characterized by the maximum length for C2–C6 vertebrae, the maximum width for the proximal thoracic spine, and both the maximum cross-sectional area and volume for C3 vertebra. There is a sharp decrease in size of the neural ossification centers along the sacral spine. A decreasing sequence of values for neural ossification centers along the spine from cervical to sacral appears to parallel the same direction of the timing of ossification. The quantitative growth of the neural ossification centers is of potential relevance in the prenatal diagnosis and monitoring of achondrogenesis, caudal regression syndrome, diastematomyelia and spina bifida.     

New morphological evidence of the ‘fate’ of growth plate hypertrophic chondrocytes in the general context of endochondral ossification

The ‘fate’ of growth plate hypertrophic chondrocytes has been long debated with two opposing theories: cell apoptosis or survival with transformation into osteogenic cells. This study was carried out on the proximal tibial growth plate of rabbits using light microscopy, scanning and transmission electron microscopy. We focused particularly on the orientation of the specimens included in order to define the mineral deposition and the vascular invasion lines and obtain histological and ultrastructural images at the corresponding height of the plate. Chondrocyte morphology transformation through the maturation process (characterized by vesicles and then large cytoplasmic lacunae before condensation, fragmentation and disappearance of the nuclear chromatin) did not correspond to that observed in the ‘in vitro’ apoptosis models. These findings rather suggested the passage of free water from the cartilage matrix into a still live cell (swelling). The level of these changes suggested a close relationship with the mineral deposition line. Furthermore, the study provided evidence that the metaphyseal capillaries could advance inside the columns of stacked hypertrophic chondrocytes (delimited by the intercolumnar septa) without the need for calcified matrix resorption because the thin transverse septa between the stacked chondrocyte (below the mineral deposition line) were not calcified. The zonal distribution of cell types (hypertrophic chondrocytes, osteoblasts, osteoclasts and macrophages) did not reveal osteoclasts or chondroclasts at this level. Morphological and morphometric analysis recorded globular masses of an amorphous, necrotic material in a zone 0–70 μm below the vascular invasion line occasionally surrounded by a membrane (indicated as ‘hypertrophic chondrocyte ghosts’). These masses and the same material not bound by a membrane were surrounded by a large number of macrophages and other blood cell precursors, suggesting this could be the cause of macrophage recall and activation. The most recent hypotheses based on genetic and lineage tracing studies stating that hypertrophic chondrocytes can survive and transform into osteoblasts and osteocytes (trans-differentiation) were not confirmed by the ultrastructural morphology or by the zonal comparative counting and distribution of cell types below the vascular invasion line.     

Gender-specific haplotype association of collagen α2 (XI) gene in ossification of the posterior longitudinal ligament of the spine

Among Japanese, ossification of the posterior longitudinal ligament of the spine (OPLL) is a leading cause of myelopathy, showing ectopic bone formation in the paravertebral ligament. We have provided genetic evidence that the collagen α2 (XI) (COL11A2) locus of chromosome 6 constitutes susceptibility for OPLL. Five distinct single nucleotide polymorphisms (SNPs), identified in COL11A2, were combined to construct possible haplotypes by the use of a maximum likelihood program. Estimated haplotype frequency was compared in OPLL patients and non-OPLL controls. We report a gender-specific association of the COL11A2 haplotype with OPLL. The frequency of the most commonly observed haplotype was significantly higher in male patients (P = 0.0003) compared with controls, but not in female patients (P = 0.21). OPLL is predominantly observed in males, with a prevalence ratio of 2 : 1, and our gender-specific associations indicate that genetic factors involving COL11A2 play a specific role in the etiology of OPLL exclusively in males. Received: September 5, 2000 / Accepted: October 2, 2000     

Postnatal ossification sequences in Acrocephalus scirpaceus and Chroicocephalus ridibundus (Aves: Neognathae): The precocial–altricial spectrum and evolution of compound bones in birds

Although the development of the avian skeleton has attracted considerable attention, most of the studies have been concentrated on the embryonic period, while studies on the postnatal period are rare. We studied the postnatal development of the skeleton in two phylogenetically distant birds, an altricial passerine Acrocephalus scirpaceus and a semiprecocial charadriiform Chroicocephalus ridibundus. The neonates of the former, despite being altricial, have well-ossified skeleton—the degree of development approaches that of the semiprecocial gull. However, after hatching the limb bones (particularly those of the hind limb) ossify earlier in the gull which is probably related to faster acquisition of locomotor abilities. We have observed that, in contrast to previous reports from neognathous birds, in the ankle of the gull, the ascending process fuses with the astragalus rather than with the calcaneum. This type of development is present in palaeognaths and nonavian dinosaurs but has not yet been reported in neognaths. This indicates a greater diversity within Neognathae and suggests a more complex scenario for the evolution of the avian ankle. However, data from a greater number of species are needed to establish the developmental sequence ancestral for neognathous birds. Furthermore, the sequence of bone fusions in the wrist of Acrocephalus is similar to the fossil-documented evolutionary sequence observed in the phylogeny of early birds, with the semilunate carpal and major metacarpal fusing first, followed by the alular metacarpal fusing with the major metacarpal and then the major and minor metacarpal fusing proximally. These data underscore the importance of developmental studies for reconstructing the evolutionary history.     

Comparative study of traumatic heterotopic ossification in mice induced by Achilles tenotomy combined with skin burn injury and single Achilles tenotomy北大核心

BACKGROUND: At present, Achilles tenotomy model is an animal model mainly used for traumatic heterotopic ossification. However, this method requires a long time to form ectopic bone, and the size of the formed ectopic bone is always small. In addition, this method cannot accurately reproduce the systemic inflammatory state of most traumatic heterotopic ossification cases in clinic practice. OBJECTIVE: To verify the validity of the animal model of traumatic heterotopic ossification induced by Achilles tenotomy combined with skin burn injury, to compare this approach with single Achilles tenotomy, and to evaluate the practicability of the two methods. METHODS: Forty male C57BL/6 mice were randomly divided into two groups: Achilles tenotomy group (control group, n=20) and Achilles tenotomy+30% skin scald on the back group (experimental group, n=20). The survival rate and healing of surgical incisions of the mice in the two groups were recorded. Survival rate and wound healing in the two groups as well as skin recovery of burn injury in the experimental group were recorded. Micro-CT examination and Masson staining of the Achilles tendon was performed 8 weeks after surgery to observe the ectopic bone at the surgical site. Formation of ectopic bone was also observed in the two groups. RESULTS AND CONCLUSION: There was no death and wound infection in the two groups. The skin burn injury in the experimental group recovered well without ulceration. Micro-CT findings indicated that all mice in the experimental group developed traumatic heterotopic ossification, with obvious circular high-density shadow at the surgical site, and the volume of ectopic bone was (2.72±0.04) mm3. In contrast, only 17 mice developed traumatic heterotopic ossification in the control group, and the volume of ectopic bone was (0.65±0.08) mm3. There was a significant difference in the volume of ectopic bone between the two groups (P < 0.05). Masson staining showed that ectopic bone in both groups had bone trabecular and bone marrow structures, but the area of ectopic bone in the experimental group was significantly larger than that in the control group (P < 0.05). To conclude, Achilles tenotomy combined with skin burn injury can effectively induce traumatic heterotopic ossification earlier than single Achilles tenotomy in mice. This combination method has a higher successful rate and can produce larger size of ectopic bone, which can be an ideal method to establish an animal model of traumatic heterotopic ossification. © 2023, Publishing House of Chinese Journal of Tissue Engineering Research. All rights reserved.     

Cross-sectional study of the ossification center of the C1–S5 vertebral bodies

Purpose

Knowledge on the normative growth of the spine is relevant in the prenatal detection of its abnormalities. This study describes the size of the ossification center of C1–S5 vertebral bodies.

Materials and methods

Using CT, digital-image analysis, and statistics, the size of the ossification center of C1–S5 vertebral bodies in 55 spontaneously aborted human fetuses aged 17–30 weeks was examined.

Results

No sex significant differences were found. The body ossification centers were found within the entire presacral spine and in 85.5 % of S1, in 76.4 % of S2, in 67.3 % of S3, in 40.0 % of S4, and in 14.5 % of S5. All the values for the atlas were sharply smaller than for the axis. The mean transverse diameter of the body ossification center gradually increased from the axis to T12 vertebra, so as to stabilize through L1–L3 vertebrae, and finally was intensively decreasing to S5 vertebra. There was a gradual increase in sagittal diameter of the body ossification center from the axis to T5 vertebra and its stabilization for T6–T9 vertebrae. Afterward, an alternate progression was observed: a decrease in values for T10–T12 vertebrae, an increase in values for L1–L2 vertebrae, and finally a decrease in values for L3–S5 vertebrae. The values of cross-sectional area of ossification centers were gradually increasing from the axis to L2 vertebra and then started decreasing to S5 vertebra. The following cross-sectional areas were approximately equivalent to each other: for L5 and T3–T5, and for S4 and C1. The volumetric growth of the body ossification center gradually increased from the axis to L3 vertebra and then sharply decreased from L4 to S5.

Conclusions

No male–female differences are found in the size of the body ossification centers of the spine. The growth dynamics for morphometric parameters of the body ossification centers of the spine follow similarly with gestational age.     

Does the epiphyseal cartilage of the long bones have one or two ossification fronts?

Epiphyseal cartilage is hyaline cartilage tissue with a gelatinous texture, and it is responsible for the longitudinal growth of the long bones in birds and mammals. It is located between the epiphysis and the diaphysis. Epiphyseal cartilage also is called a growth plate or physis. It is protected by three bone components: the epiphysis, the bone bar of the perichondrial ring and the metaphysis. The epiphysis, which lies over the epiphyseal cartilage in the form a cupola, contains a juxtaposed bone plate that is near the epiphyseal cartilage and is in direct contact with the epiphyseal side of the epiphyseal cartilage. The germinal zone corresponds to a group of cells called chondrocytes. These chondrocytes belong to a group of chondral cells, which are distributed in rows and columns; this architecture is commonly known as a growth plate. The growth plate is responsible for endochondral bone growth. The aim of this study was to elucidate the causal relationship between the juxtaposed bone plate and epiphyseal cartilage in mammals. Our hypothesis is that cells from the germinal zone of the epiphyseal side of the epiphyseal cartilage are involved in forming a second ossification front that is responsible for the origin of the juxtaposed bone plate. We report the following: (a) The juxtaposed bone plate has a morphology and function that differs from that of the epiphyseal trabeculae; (b) on the epiphyseal edge of the epiphyseal cartilage, a new ossification front starts on the chondrocytes of the germinal area, which forms the juxtaposed bone plate. This ossification front is formed by chondrocytes from the germinal zone through a process of mineralisation and ossification, and (c) the process of mineralisation and ossification has a certain morphological analogy to the process of ossification in the metaphyseal cartilage of amphibians and differs from the endochondral ossification process in the metaphyseal side of the growth plate. The close relationship between the juxtaposed bone plate and the epiphyseal cartilage, in which the chondrocytes that migrate from the germinal area play an important role in the mineralisation and ossification process of the juxtaposed bone plate, supports the hypothesis of a new ossification front in the epiphyseal layer of the epiphyseal plate. This hypothesis has several implications: (a) epiphyseal cartilage is a morphological entity with two different ossification fronts and two different functions, (b) epiphyseal cartilage may be a morphological structure with three parts: perichondrial ring, metaphyseal ossification front or growth plate, and epiphyseal ossification front, (c) all disease (traumatic or dysplastic) that affects some of these parts can have an impact on the morphology of the epiphyseal region of the bone, (d) there is a certain analogy between metaphyseal cartilage in amphibians and mammalian epiphyseal cartilage, although the former is not responsible for bone growth, (e) comparative histological and anatomy studies are also warranted, to shed light on the phylogenetic study of epiphyseal cartilage throughout the changes that occur in the animal species.     

The crosstalk between transforming growth factor-β1 and delta like-1 mediates early chondrogenesis during embryonic endochondral ossification

Delta like-1 (Dlk1)/preadipocyte factor-1 (Pref-1)/fetal antigen-1 (FA1) is a novel surface marker for embryonic chondroprogenitor cells undergoing lineage progression from proliferation to prehypertrophic stages. However, mechanisms mediating control of its expression during chondrogenesis are not known. Thus, we examined the effect of a number of signaling molecules and their inhibitors on Dlk1 expression during in vitro chondrogenic differentiation in mouse embryonic limb bud mesenchymal micromass cultures and mouse embryonic fibroblast (MEF) pellet cultures. Dlk1/Pref-1 was initially expressed during mesenchymal condensation and chondrocyte proliferation, in parallel with expression of Sox9 and Col2a1, and was downregulated upon the expression of Col10a1 by hypertrophic chondrocytes. Among a number of molecules that affected chondrogenesis, transforming growth factor-β1 (TGF-β1)-induced proliferation of chondroprogenitors was associated with decreased Dlk1 expression. This effect was abolished by TGF-β signaling inhibitor SB431542, suggesting regulation of Dlk1/FA1 by TGF-β1 signaling in chondrogenesis. TGF-β1-induced Smad phosphorylation and chondrogenesis were significantly increased in Dlk1(-/-) MEF, while they were blocked in Dlk1 overexpressing MEF, in comparison with wild-type MEF. Furthermore, overexpression of Dlk1 or addition of its secreted form FA1 dramatically inhibited TGF-β1-induced Smad reporter activity. In conclusion, our data identified Dlk1/FA1 as a downstream target of TGF-β1 signaling molecule that mediates its function in embryonic chondrogenesis. The crosstalk between TGF-β1 and Dlk1/FA1 was shown to promote early chondrogenesis during the embryonic endochondral ossification process.     

Could heterotopic ossification be prevented by varying dietary n-3/n-6 polyunsaturated fatty acid ratio: A novel perspective to its treatment?

Heterotopic ossification (HO) is a common complication following with musculoskeletal trauma and surgical procedures. It usually decreases joint mobility and eventually causes loss of joint function. Despite nonsteroidal anti-inflammatory drugs (NSAIDs), the inhibitor of cyclooxygenase(COX), have been proven to prevent HO effectively via prostaglandin E2 synthesis regulation and modulation of tissue responsiveness to pro-inflammatory signaling, HO prevention is still a matter of debate for clinicians to avoid the side effect of NSAIDs. Interestingly, it is suggested that PGE2 production and pro-inflammatory microenvironment in body could be modified by varying the ratio of the precursor fatty acids in the diet. On account of the effect of dietary (n-6)/(n-3) PUFAs ratio on both COX metabolism and pro-inflammatory cytokines mediated biological responsiveness, we hypothesized lowering dietary (n-6)/(n-3) PUFAs ratio may not only directly reduce the substrate of COX-2 and COX-2 activity, but also partially ameliorate tissue inflammatory responsiveness to cytokines correlated with HO development,exerting an inhibitory effect on PGE2 synthesis to prevent HO formation. The negative role of lowering dietary (n-6)/(n-3) PUFAs ratio on angiogenesis, cytokines-induced apoptosis, inflammatory responsiveness and osteogenesis could also contribute to its action on HO development. If our hypothesis is proved to be corrected, it could be an innovative method to treat HO.     

Expression and regulation of αB-crystallin in the kidney in vivo and in vitro

αB-crystallin, a major component of the mammalian eye lens, is a small heat shock protein and molecular chaperone that is also abundant in the mammalian kidney. The present study aimed to characterize more closely the intrarenal expression and regulation of αB-crystallin in vivo and in vitro. In normal rat kidney, the expression of αB-crystallin mRNA and protein were both close to the detection limit in cortex, but increased steeply from the outer to the inner medulla where αB-crystallin constitutes approximately 2% of total tissue protein. Immunohistochemistry disclosed papillary collecting duct cells and thin limbs as the major sites for intrapapillary αB-crystallin immunoreactivity. In rats subjected to sucrose diuresis for 3 days, αB-crystallin mRNA expression was reduced by 27 and 46% in outer and inner medulla, respectively. In agreement with the results obtained in vivo, in Madine–Darby canine kidney cells, αB-crystallin mRNA and protein were induced significantly by elevating the medium osmolality to 500 mosm/kg H₂O by the addition of NaCl and raffinose, and also by urea. The NaCl-induced increase in αB-crystallin expression was concentration-dependently blunted by SP600125, a specific JNK inhibitor. Overexpression of αB-crystallin in 293 cells resulted in increased tolerance to acute osmotic stress. These results indicate that αB-crystallin may be regulated by papillary interstitial tonicity in a JNK-dependent process. Moreover, the high abundance of αB-crystallin in the renal medulla may be important for cell survival in an environment characterized by extreme interstitial solute concentrations as present during antidiuresis.     

Hepatitis E in liver transplant recipients in the Rhône-Alpes region in France

Purpose

In developed countries, hepatitis E virus (HEV) is considered an emerging pathogen, but prevalence seems highly variable according to previous European studies. As HEV can lead to chronic infections in immunosuppressed patients, it is thus essential to evaluate the prevalence and incidence of this infection.

Methods

We determined retrospectively, in a cohort of 206 pediatric and adult liver transplant recipients from the Rhône-Alpes region in France, pre-transplant anti-HEV-IgG prevalence and incidence of HEV infections during post-transplant follow-up (HEV IgG and IgM ± HEV-RNA).

Results

Transplantations were carried out between 2005 and 2012 and mean post-transplant follow-up was 32.8 months. Global pre-transplant prevalence of anti-HEV IgG was 29 %, increasing regularly with age from 7 % for children under 15 to 49 % for patients older than 60. From the 142 seronegative patients before transplant, 11 seroconversions (7.7 %) were observed during follow-up (incidence of 2.83 cases per 100 person-years). HEV RNA—tested at transaminases peak or randomly—was detected in only one case of seroconversion. For at least 2 HEV-seropositive patients, who had negative RNAemia before transplantation, viral RNA was detected chronically during follow-up, suggesting reinfection with HEV.

Conclusion

Acute infections were largely more frequent than chronic infections and were asymptomatic or misdiagnosed, suggesting that liver transplant patients may not be particularly prone to developing severe HEV hepatitis. In addition, the presence of IgG anti-HEV may not protect against re-infection. Serological testing, therefore, appears to be of limited interest for the diagnosis of HEV infections in liver transplant recipients.     

Changes in PaO_2 and pH in experimental hepatocarcinoma

Changes in PaO_2 and pH in experimental hepatocarcinoma     

Working Memory in Bees: Also in Flies?

Abstract: Decision making requires reference to both actual and remote information in the context of the requirements of the animal. Here, I explore a cognitive approach to decision making in honeybees and ask the question whether flies may share the faculties observed in bees. Examples are drawn primarily from natural behavior in bees, particularly from navigation and communication. I conclude that studies in Drosophila learning and memory in the tradition of Martin Heisenberg may gain from cognitive concepts, meaning that the “internal doing” of the brain should be included in the search for the neural basis of decision making.