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Analysis of the role of hormones and growth factors in growth control and tissue differentiation using transplanted mammalian embryos and fetal structures.

Results obtained from our analyses of the role of hormones and growth factors in rat embryonic and fetal development are reviewed. Whole 10-day embryos or structures from 14-16-day fetuses (paws, intestines, reproductive tracts) were transplanted under the kidney capsule of syngeneic host rats of different age, sex, physiological state or level of nutrition. In intact hosts, fetal transplants grew almost as much as they would have if left in situ in the fetuses, and tissue differentiation in them was essentially normal. By contrast, growth of embryo transplants was severely depressed relative to their growth in utero, but tissue differentiation was only slightly retarded. Fetal paws grew equally well in female hosts widely diverging in age and growth rate. Thus, in such hosts growth of the fetal paw was highly independent of the growth rate of the hosts. In hosts in which growth was arrested or reduced by diabetes, hypophysectomy or food restriction, growth of paw transplants was impaired, but not as severely as that of the hosts themselves. Fetal skeletal structures were relatively independent of thyroid hormones (TH) for growth; TH dependence developed progressively postnatally. In pregnant hosts, fetal paw transplants grew as well during the first half of gestation as they did in virgin females. By contrast, during the second half of pregnancy and during both halves of the lactational period, growth of the transplants was significantly reduced. Host skeletal growth was also inhibited during late pregnancy and throughout lactation. The impaired growth during the second half of gestation was associated with a large reduction in serum IGF-I concentration and a resistance to the growth-promoting actions of GH. In the lactating hosts, serum IGF-I concentration returned almost to prepregnancy levels and the resistance to GH persisted, but at a reduced level. The direct effects of hormones, growth factors, and antibodies to them on growth and tissue differentiation in the transplants were assessed using infusion methods. Substances were infused into the renal artery of transplant-bearing kidneys via catheters attached to osmotic minipumps. The direct effects of insulin, GH, IGFs, basic FGF and EGF on transplant growth and tissue differentiation were evaluated. Insulin directly stimulated growth of transplants in diabetic hosts but GH did not have a direct effect in hypophysectomized rats. The growth-restorative effects of GH observed in hypophysectomized hosts were apparently mediated indirectly via IGF-I. Infused rat IGF-II (MSA) was more effective at stimulating growth of embryo transplants than was recombinant human IGF-I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

[Relationship between proliferative activity, and tissue differentiation and invasive mode in human oral squamous cell and colorectal carcinomas analysed by PCNA immunostaining].

The proliferative activity of carcinoma cells is generally considered to relate to the degree of the malignancy of carcinoma tissues. In this study, the proliferative activity at the tumor-stromal border was studied in 17 cases of oral squamous cell carcinoma (OSCC) and in 30 cases of colorectal adenocarcinoma (CAC) by means of proliferating cell nuclear antigen (PCNA) immunostaining, to evaluate the correlation between proliferative activity and tissue differentiation or invasive mode at the tumor-stromal border. No statistical difference was detected between the PCNA labelling index (PI) and the tissue differentiation of both OSCC and CAC. A significant difference was demonstrated between PI and invasive mode in OSCC, suggesting that the invasive mode at the tumor-stromal border relate to the degree of the malignancy of carcinoma tissues. However, no significance was found between PI and invasive mode of CAC. In addition, no difference of PI was demonstrated between tissue differentiation or invasive mode, and vascular invasion or lymph node metastasis. Therefore, it seems likely that the invasive mode at the tumor-stromal border in CAC also has no significance in deciding the degree of the malignancy of carcinoma tissues.

Adenocarcinoma↗

[Significance of echographic tissue differentiation for orbital surgery].

The high degree of accuracy of standardized echography in tissue differentiation of orbital tumors enables the ophthalmic surgeon to determine exactly whether or not orbitotomy is indicated. As a result of the echographic examination the ophthalmologist is optimally informed as to the location, extent, delimitation and nature of the tissue of the tumor, and therefore better able to perform the orbitotomy. Standardized A-scan echography is capable of demonstrating an orbital tumor with 99% accuracy. Its location, limits, and tissue differentiation decide whether conservative or surgical treatment is to be preferred. Orbital tumors in any location can be removed by Krönlein's temporal orbitotomy: the wide temporal approach enables even large tumors to be excised in toto. With appropriate care much of the orbital contents can be spared and an optimal functional and cosmetic result achieved.

Adenocarcinoma↗

Corroboration of mechanoregulatory algorithms for tissue differentiation during fracture healing: Comparison with in vivo results.

Several mechanoregulation algorithms proposed to control tissue differentiation during bone healing have been shown to accurately predict temporal and spatial tissue distributions during normal fracture healing. As these algorithms are different in nature and biophysical parameters, it raises the question of which reflects the actual mechanobiological processes the best. The aim of this study was to resolve this issue by corroborating the mechanoregulatory algorithms with more extensive in vivo bone healing data from animal experiments. A poroelastic three-dimensional finite element model of an ovine tibia with a 2.4 mm gap and external callus was used to simulate the course of tissue differentiation during fracture healing in an adaptive model. The mechanical conditions applied were similar to those used experimentally, with axial compression or torsional rotation as two distinct cases. Histological data at 4 and 8 weeks, and weekly radiographs, were used for comparison. By applying new mechanical conditions, torsional rotation, the predictions of the algorithms were distinguished successfully. In torsion, the algorithms regulated by strain and hydrostatic pressure failed to predict healing and bone formation as seen in experimental data. The algorithm regulated by deviatoric strain and fluid velocity predicted bridging and healing in torsion, as observed in vivo. The predictions of the algorithm regulated by deviatoric strain alone did not agree with in vivo data. None of the algorithms predicted patterns of healing entirely similar to those observed experimentally for both loading modes. However, patterns predicted by the algorithm based on deviatoric strain and fluid velocity was closest to experimental results. It was the only algorithm able to predict healing with torsional loading as seen in vivo.

Animals↗

Morules in endometrial carcinoma and benign endometrial lesions differ from squamous differentiation tissue and are not infected with human papillomavirus.

BACKGROUND: Squamous differentiation/squamous metaplasia is often associated with endometrial adenocarcinoma and benign lesions, such as endometrial hyperplasia and chronic endometritis. Morules have distinct histological characteristics, and are referred to as squamous metaplasia or squamoid metaplasia. AIM: To focus on the histological characteristics of morules and clarify the difference between morules and squamous differentiation. MATERIALS/METHODS: Twenty endometrioid carcinomas with morules or squamous differentiation, five adenosquamous carcinomas, and eight non-carcinomatous endometrial lesions with morules were investigated. Numerous antibodies for epithelial membrane antigen (EMA), involucrin, cytokeratins, neuropeptides, and oncofetal antigens were used for immunohistochemistry. In situ hybridisation and polymerase chain reaction were used to detect human papillomavirus (HPV). RESULTS: The morules observed were uniform cell clusters, with no squamous differentiation. They were immunonegative for epithelial antigens including involucrin, EMA, and cytokeratins, but were positive for neurone specific enolase. A few morules were immunopositive for acetylcholine esterase, and one case was positive for somatostatin; neither oncofetal nor proliferative cell markers, including blood group A, B, and AB, or other neuropeptides were demonstrated in the morules. HPV DNA was not found in either the morules in the carcinomas or in the benign lesions. However, true squamous differentiation tissue in four endometrioid carcinomas and two adenosquamous carcinomas was HPV positive using in situ hybridisation. CONCLUSION: Morules are histologically distinct from squamous metaplasia/squamous differentiation tissue. Morules are thought to be neuroectodermal-like cell clusters, and are not infected with HPV. In contrast, some of the true squamous differentiation tissue was associated with HPV infection.

Adenocarcinoma↗

Soft tissue differentiation using multiband signatures of high resolution ultrasonic transmission tomography.

In this paper, we are interested in soft tissue differentiation by multiband images obtained from the High-Resolution Ultrasonic Transmission Tomography (HUTT) system using a spectral target detection method based on constrained energy minimization (CEM). We have developed a new tissue differentiation method (called "CEM filter bank") consisting of multiple CEM filters specially designed for detecting multiple types of tissues. Statistical inference on the output of the CEM filter bank is used to make a decision based on the maximum statistical significance rather than the magnitude of each CEM filter output. We test and validate this method through three-dimensional interphantom/intraphantom soft tissue classification where target profiles obtained from an arbitrary single slice are used for differentiation over multiple other tomographic slices. The performance of the proposed classifier is assessed using receiver operating characteristic analysis. We also apply our method to classify tiny structures inside a bovine kidney and sheep kidneys. Using the proposed method we can detect physical objects and biological tissues such as styrofoam balls, chicken tissue, calyces, and vessel-duct successfully.

Algorithms↗

Cartilage tissue differentiation from mesenchymal cells derived from mature muscle in tissue culture.

Under the influence of biochemical components of bone matrix gelatin (BMG), cartilage differentiates in tissue culture from the connective tissue cell outgrowths of mature muscle. Proliferation and differentiation begin within 24 hr with synthesis of hyaluronate, continue with high levels of synthesis of DNA and hyaluronidase, and culminate in production of large quantities of chondroitin sulfate. The addition of hyaluronic acid to the culture medium during the first 48 hr of culture depresses, whereas chondroitin sulfate enhances, subsequent production of cartilage. These observations on the cell biosynthetic products prior to the appearance of mature cartilage suggest that the BMG--modified connective tissue outgrowths of mature muscle exhibit the developmental potential of embryonic axial mesenchyme. Whether muscle harbors embryonic cells in a programmed but not yet activated readiness (protodifferentiated state) to differentiate into cartilage, or simply contributes a population of temporarily dedifferentiated fibroblasts, is not known, but in any event, BMG switches the pathway of further development from fibrous connective tissue to cartilage.

Bone Matrix↗

Scaffold attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues.

Eukaryotic interphase chromatin is thought to be organized into topologically discrete, independent domains acting as units upon which differential patterns of gene expression are established. Sequences which attach chromatin to in vitro preparations of a nucleoprotein matrix (scaffold attachment regions [SARs]) may act as domain boundaries, but their role remains poorly defined compared with those of other elements such as locus control regions. We have produced mice homozygous for a transgene which is transcribed as early as the activation of the embryonic genome at the two-cell stage and which is expressed ubiquitously in a number of differentiated tissues. Transgenic lines were generated in the presence or absence of flanking SAR sequences, creating an original model which enabled us to examine the effects of these elements at different developmental stages. In the preimplantation mouse embryo, flanking SARs stimulated transgene expression in a copy-dependent manner. In contrast, in the differentiated tissues of newborn and adult mice, no significant SAR-dependent increase in transgene expression was found, correlation with copy number was lost, and position effects were observed. These results suggest a limited capacity of SARs to act as insulating elements but are consistent with a proposed model of SAR-mediated chromatin opening and closing.

Aging↗

Numerical simulation of tissue differentiation around loaded titanium implants in a bone chamber.

The application of a bone chamber provides a controlled environment for the study of tissue differentiation and bone adaptation. The influence of different mechanical and biological factors on the processes can be measured experimentally. The goal of the present work is to numerically model the process of peri-implant tissue differentiation inside a bone chamber, placed in a rabbit tibia. 2D and 3D models were created of the tissue inside the chamber. A number of loading conditions, corresponding to those applied in the rabbit experiments, were simulated. Fluid velocity and maximal distortional strain were considered as the stimuli that guide the differentiation process of mesenchymal cells into fibroblasts, chondrocytes and osteoblasts. Mesenchymal cells migrate through the chamber from the perforations in the chamber wall. This process is modelled by the diffusion equation. The predicted tissue phenotypes as well as the process of tissue ingrowth into the chamber show a qualitative agreement with the results of the rabbit experiments. Due to the limited number of animal experiments (four) and the observed inter-animal differences, no quantitative comparison could be made. These results however are a strong indication of the feasibility of the implemented theory to predict the mechano-regulation of the differentiation process inside the bone chamber.

Animals↗

Ci-Rga, a gene encoding an MtN3/saliva family transmembrane protein, is essential for tissue differentiation during embryogenesis of the ascidian Ciona intestinalis.

A novel gene (Ci-Rga) essential for tissue differentiation during embryogenesis of the ascidian Ciona intestinalis is reported here. This gene was identified through functional screening of Ciona genes required for development by translational inhibition experiments with morpholino antisense oligonucleotides. The deduced protein of Ci-Rga contains two copies of a domain with unknown function called the MtN3/saliva domain. Phylogenetic analysis showed that Ci-Rga belongs to the MtN3/saliva family of genes conserved among metazoans and plants, and is an ortholog of mouse Rga (Recombination-activating gene 1 gene activation). During Ciona embryogenesis, both maternal and zygotic transcripts of Ci-Rga were expressed. Translational inhibition of Ci-Rga with specific morpholino resulted in abnormal embryos in which the cleavage pattern became atypical and expression of marker genes for each of the six major tissues, namely the endoderm, muscle, mesenchyme, notochord, neural tissue, and epidermis, was lost or suppressed at the tailbud stage. Although differentiation of all the six major tissues was affected by Ci-Rga knock-down, the degree of abnormalities and the timing of appearance of abnormalities were different among tissues. Expression analysis of developmentally important genes involved in the fate specification, such as Ci-Bra, Ci-Twist-like1a, Ci-Otx, Ci-Fgf9/16/20, Ci-Lhx3, Ci-FoxD, and Ci-Tbx6b, showed that an initial step of the fate specification of notochord, mesenchyme, and neural tissue, but not of endoderm or muscle, is impaired in the knock-down embryo. These results showed that Ci-Rga is a multifunctional gene essential for tissue differentiation during embryogenesis, and is primarily required for the fate specification of notochord, mesenchyme, and neural tissue, and provide some insights into the function of this little-known group of genes.

Amino Acid Sequence↗

Effects of a p38 MAP kinase inhibitor on bone ingrowth and tissue differentiation in rabbit chambers.

The effects of an oral p38 mitogen-activated protein kinase (MAPK) inhibitor and polyethylene particles separately and together on tissue differentiation in the bone harvest chamber (BHC) in rabbits over a 3-week treatment period were investigated. The harvested tissue was analyzed histomorphometrically for markers of bone formation (percentage of bone area), osteoblasts (alkaline phosphatase staining), and osteoclasts (CD51, the alpha chain of the vitronectin receptor). Polyethylene particles decreased the percentage of bone ingrowth and staining for alkaline phosphatase. The p38 MAPK inhibitor alone decreased alkaline phosphatase staining. When the oral p38 MAPK inhibitor was given and the chamber contained polyethylene particles, there was a suppression of bone ingrowth and alkaline phosphatase staining. In contrast to oral non-steroidal anti-inflammatory drugs (NSAIDs) and local Interleukin-1 receptor antagonist (IL-1ra) administration, the oral p38 MAPK inhibitor alone did not suppress bone formation when given during the initial phase of tissue differentiation. Particle-induced inflammation and the foreign body reaction were not curtailed when the p38 MAPK inhibitor was given simultaneously with particles. Additional experiments are needed to establish the efficacy of p38 MAPK inhibitor administration on mitigating an established inflammatory and foreign body reaction that parallels the clinical situation more closely.

Administration, Oral↗

Assessment of mechanobiological models for the numerical simulation of tissue differentiation around immediately loaded implants.

Nowadays, there is a growing consensus on the impact of mechanical loading on bone biology. A bone chamber provides a mechanically isolated in vivo environment in which the influence of different parameters on the tissue response around loaded implants can be investigated. This also provides data to assess the feasibility of different mechanobiological models that mathematically describe the mechanoregulation of tissue differentiation. Before comparing numerical results to animal experimental results, it is necessary to investigate the influence of the different model parameters on the outcome of the simulations. A 2D finite element model of the tissue inside the bone chamber was created. The differentiation models developed by Prendergast, et al. ["Biophysical stimuli on cells during tissue differentiation at implant interfaces", Journal of Biomechanics, 30(6), (1997), 539-548], Huiskes et al. ["A biomechanical regulatory model for periprosthetic fibrous-tissue differentiation", Journal of Material Science: Materials in Medicine, 8 (1997) 785-788] and by Claes and Heigele ["Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing", Journal of Biomechanics, 32(3), (1999) 255-266] were implemented and integrated in the finite element code. The fluid component in the first model has an important effect on the predicted differentiation patterns. It has a direct effect on the predicted degree of maturation of bone and a substantial indirect effect on the simulated deformations and hence the predicted phenotypes of the tissue in the chamber. Finally, the presence of fluid also causes time-dependent behavior. Both models lead to qualitative and quantitative differences in predicted differentiation patterns. Because of the different nature of the tissue phenotypes used to describe the differentiation processes, it is however hard to compare both models in terms of their validity.

Animals↗

Role of mechanical environment and implant design on bone tissue differentiation: current knowledge and future contexts.

OBJECTIVES: To evaluate published evidence related to bone reactions to varying loading regimens and the impact of implant design on bone tissue differentiation. DATA AND SOURCES: The literature was searched for original research articles relating effect of mechanical environment on bone tissue and effects of implant design on bone biomechanics and marginal bone reactions using MEDLINE and manual tracing of references cited in key papers otherwise not elicited. STUDY SELECTION: Current literature on biomechanics of bone and dental implants as main focus and pertinent to key aspects of the review. CONCLUSIONS: Implant design influences force transmission characteristics in peri-implant bone, but not the time-dependent marginal bone reactions. Mechanical signals affect bone tissue differentiation. Therefore, it is essential to control biomechanical loads on implants to maintain osseointegration and/or to promote early bone-implant interface healing.

Animals↗

The influence of micro-motion on the tissue differentiation around immediately loaded cylindrical turned titanium implants.

OBJECTIVE: The aim of this study was to evaluate the effect of various degrees of implant displacement on the tissue differentiation around immediately loaded cylindrical turned titanium implants. DESIGN: The experiments were conducted in repeated sampling bone chambers placed in the tibia of 10 rabbits. Tissues could grow into the bone chambers via perforations. Due to its double structure, tissues inside the chamber could be harvested leaving the chamber intact. This allowed several experiments within the same animal. The chambers contained a cylindrical turned titanium implant that was loaded in a well-controlled manner. In each of the 10 chambers, four experiments were conducted with the following test conditions: immediate implant loading by inducing 0 (control), 30, 60 and 90 microm implant displacement, 800 cycles per day at a frequency of 1 Hz, twice a week during a period of 6 weeks. Histological and histomorphometrical analyses were performed on methylmethacrylate histological sections. An ANOVA was conducted on the dataset. RESULTS: The total tissue volume was significantly lowest in the unloaded control condition. The bone volume fraction on the other hand, was significantly larger in the unloaded and 90 microm implant displacement, compared to the 30 microm implant displacement. Bone density increased with increasing micro-motion with significantly higher values for the 60 microm- and 90 microm-test conditions compared to the unloaded situation. The chance to have bone-to-implant contact decreased in case of micro-motion at the tissues-implant interface. CONCLUSION: The magnitude of implant displacement had a statistically significant effect on the tissue differentiation around immediately loaded cylindrical turned titanium implants. Implant micro-motion had a detrimental effect on the bone-to-implant contact in an immediate loading regimen.

Animals↗

The fracture gap size influences the local vascularization and tissue differentiation in callus healing.

BACKGROUND: Revascularization of a fracture depends on fracture stability and fracture gap conditions. The aim of the study was to determine quantitatively the revascularization and tissue differentiation in an animal model with different fracture gaps and controlled biomechanical conditions. MATERIALS AND METHOD: The study was performed on ten sheep with an osteotomy on the right metatarsal. The fracture was stabilized by an external fixator that allowed adjustable axial interfragmentary movement. Two groups of five sheep each were adjusted to a medium sized gap (M, 2.1 mm) and a large gap (L, 5.7 mm) under comparable interfragmentary strain (30-32%). The animals were killed after 9 weeks, and the metatarsals were prepared for undecalcified histology and analysis of tissue differentiation and vessel distribution. RESULTS: Group M showed significantly more revascularization (M=1.62, L=0.85 vessels/mm2), more bone formation (M=37.2%, L=13.9%) and less fibrocartilage tissue (M=18.1%, L=39.1%) than group L. Larger vessels (>40 microm) were found mainly in the medullary channel, and smaller vessels (<20 microm) mainly in the peripheral callus. Histologically, group M showed partial bony bridging of the osteotomy gap, and the group L had delayed healing. CONCLUSION: A good reduction of a fracture with small interfragmentary gaps is important for its revascularization and healing.

Animals↗

The interaction of Kaposi's sarcoma with monoclonal antibodies to human sarcoma and connective tissue differentiation antigens.

Four monoclonal antibodies (McAbs) previously generated against human soft tissue sarcomas and reacting with connective tissue differentiation antigens were evaluated for their interaction with tissues obtained from patients with classic Kaposi's sarcoma. Biopsy was performed on active neoplastic lesions from the skin of 26 patients, frozen sections were prepared, and the binding of the McAbs was tested using the indirect immunofluorescence assay. Clinically uninvolved skin from the same patients as well as skin and muscle from eight non-cancer patients were treated similarly and served as controls. McAbs IXG11, 23H7, IIIE5, and 15G5 interacted strongly with the Kaposi's sarcoma lesions and weakly with the uninvolved skin in 22 of 26 (84%), 23 of 26 (88%), 12 of 14 (85%), and 1 of 6 (16%) of the patients, respectively. IXG11, 23H7, and IIIE5 interacted weakly with the skin of seven of eight non-cancer patients. McAb 15G5 was found to bind strongly to tumor lesions, to the respective uninvolved skin in four of five Kaposi's sarcoma patients, and also to skin and connective tissues of muscle from non-cancer patients. The mode of interaction was morphologically different for each McAb. It is suggested that McAbs IXG11, 23H7 and IIIE5 identify markers whose expression is markedly increased in Kaposi's sarcoma lesions as compared with uninvolved skin of the same patients. These markers may serve as immunologic probes for the investigation of this neoplastic process.

Adult↗

A repeated sampling bone chamber methodology for the evaluation of tissue differentiation and bone adaptation around titanium implants under controlled mechanical conditions.

A repeated sampling bone chamber methodology was developed for the study of the influence of the mechanical environment on skeletal tissue differentiation and bone adaptation around titanium implants. Via perforations, bone grows into the implanted outer bone chamber, containing an inner bone chamber with a central test implant. An actuator--easily mounted on the outer bone chamber--allows a controlled mechanical stimulation of the test implant. After each experiment, the inner bone chamber--with its content--can be harvested and analysed. A new inner bone chamber with a central implant can be inserted consecutively in the outer bone chamber and a new experiment can start. Pilot studies led to a reliable surgical protocol and showed the applicability of the methodology, offering the possibility to study skeletal tissue differentiation and adaptation around implants under well-controlled mechanical conditions, and this protected from external loading. Repeated sampling of the bone chamber allows conducting several experiments within the same animal at the same site, thereby excluding subject- and site-dependent variability and reducing the amount of experimental animals.

Adaptation, Physiological↗

Differential tissue distribution of diverse clones of Trypanosoma cruzi in infected mice.

Chagas disease, caused by the protozoan Trypanosoma cruzi, presents variable clinical course but the phenomena underlying this variability remain largely unknown. T. cruzi has a clonal population structure and infecting strains are often multiclonal. T. cruzi genetic variability could be a determinant of differential tissue tropism or distribution and consequently of the clinical forms of the disease. We tested this hypothesis by using low-stringency single specific primer polymerase chain reaction (LSSP-PCR) to type genetically the parasites in tissues of experimental infected mice. BALB/c mice were simultaneously inoculated with two different T. cruzi populations (JG strain and Coll.7G2 clone). Doubly infected animals showed clear differential tissue distribution for the two populations (chronic phase). Our results indicate a significant influence of the genetic polymorphism of infecting T. cruzi populations in the pathogenesis of chronic Chagas disease.

Acute Disease↗