Biomedical subjects

Makarand V Risbud

Publications and source records attributed to Makarand V Risbud.

At least 19 recordsLinked to original sources

PubMed2007

Galectin-3 expression in the intervertebral disc: a useful marker of the notochord phenotype?

STUDY DESIGN: Galectin-3 expression in rat intervertebral disc at different stages in postnatal life is evaluated. OBJECTIVE: To determine if galectin-3 expression is confined to cells of the nucleus pulposus in the postnatal rat intervertebral disc. SUMMARY OF BACKGROUND DATA: During embryonic development, the anulus fibrosus is derived from the sclerotome, whereas the nucleus pulposus is notochordal. Many authorities opine that in the postnatal disc, notochordal cells play a central role in controlling the development of degenerative disc disease. Surprisingly, unequivocal evidence supporting the existence of notochordal cells in the nucleus pulposus in postnatal life has yet to be demonstrated. Since the expression of galectin-3 is commonly used to identify notochordal cells, we evaluated its expression in tissues of the rat disc and in cultured cells. METHODS: Galectin-3 expression was studied in the nucleus pulposus and anulus fibrosus tissue of rat discs (2 days, 9 weeks, and 10 months old), and cultured cells using different biochemical and molecular biology methods. Rat sternal cartilage and cultured sternal chondrocytes were used as controls. RESULTS: Immunohistochemical studies indicated that galectin-3 was present in the nucleus pulposus and anulus fibrosus. In both discal tissues and cultured cells, studies confirmed that there was a robust expression of galectin-3 messenger ribonucleic acid and protein. Protein expression patterns were similar in neonatal, young, and mature rats. There was also evidence of intracellular and membrane expression of galectin-3 in the cultured disc cells. Finally, significant levels of galectin-3 were evident in rat sternal cartilage and cultured sternal chondrocytes. CONCLUSIONS: Results of the study indicate that galectin-3 is expressed in the neonatal, young, and mature rat disc, and its expression is not restricted to the cells of the nucleus pulposus. Because of its ubiquitous expression, this protein cannot be used as a marker of notochordal cells in the postnatal rat disc.

Aging↗

PubMed2006

TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc.

The nucleus pulposus is an aggrecan-rich hydrated tissue that permits the intervertebral disc to resist compressive loads. Adaptation to loading is achieved through an elevation in disc osmolarity mediated by the numerous charged glycosoaminoglycan side chains of the aggrecan molecule. The goal of this investigation was to determine the functional role of the osmo-regulatory protein, TonEBP, in cells of the nucleus pulposus. We found that TonEBP and its downstream target genes were robustly expressed in the tissues of the disc. Above 330 mosmol/kg, cultured nucleus pulposus cells up-regulated target genes TauT, BGT-1, and SMIT; above 450 mosmol/kg, there was raised expression of HSP-70. In hypertonic media there was activation of TauT and heat shock protein-70 (HSP-70) reporter activity and increased binding of TonEBP to the TonE motif. When cells were transfected with the dominant-negative form of TonEBP (DN-TonEBP) there was suppression of TauT and HSP-70 reporter gene expression; pTonEBP enhanced reporter gene expression. Moreover, in hypertonic media, forced expression of DN-TonEBP induced apoptosis. We suppressed TonEBP using small interfering RNA technique and noted a decrease in TauT reporter activity in isotonic as well as hyperosmolar media. Finally, we report that the aggrecan promoter contains two conserved TonE motifs. To evaluate the importance of these motifs, we overexpressed DN-TonEBP and partially silenced TonEBP using small interfering RNA. Both approaches resulted in suppression of aggrecan promoter activity. It is concluded that TonEBP permits the disc cells to adapt to the hyperosmotic milieu while autoregulating the expression of molecules that generate the unique extracellular environment.

Amino Acid Motifs↗

PubMed2006

Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment.

Nucleus pulposus (NP) cells of the intervertebral disc reside in an environment that has a limited vascular supply and generate energy through anaerobic glycolysis. The goal of the present study was to examine the expression and regulation of HIF-1alpha, a transcription factor that regulates oxidative metabolism in nucleus pulposus cells. Nucleus pulposus cells were isolated from rat, human, and sheep disc and maintained at either 21% or 2% oxygen for various time periods. Cells were also treated with desferrioxamine (Dfx), a compound that mimics the effects of hypoxia (Hx). Expression and function of HIF-1alpha were assessed by immunofluorescence microscopy, Western blot analysis, gel shift assays, and luciferase reporter assays. In normoxia (Nx), rat, sheep, and human nucleus pulposus cells consistently expressed the HIF-1alpha subunit. Unlike other skeletal cells, when maintained under low oxygen tension, the nucleus pulposus cells exhibited a minimal induction in HIF-1alpha protein levels. Electromobility shift assays confirmed the functional binding of normoxic HIF-1alpha protein to its putative DNA binding motif. A dual luciferase reporter assay showed increased HIF-1alpha transcriptional activity under hypoxia compared to normoxic level, although this induction was small when compared to HeLa and other cell types. These results indicate that normoxic stabilization of HIF-1alpha is a metabolic adaptation of nucleus pulposus cells to a unique oxygen-limited microenvironment. The study confirmed that HIF-1alpha can be used as a phenotypic marker of nucleus pulposus cells.

Adaptation, Physiological↗

PubMed2006

Toward an optimum system for intervertebral disc organ culture: TGF-beta 3 enhances nucleus pulposus and anulus fibrosus survival and function through modulation of TGF-beta-R expression and ERK signaling.

STUDY DESIGN: Rat lumbar discs comprising nucleus pulposus, annulus fibrosus, and cartilaginous endplates were cultured for 1 week in a specialized media containing either TGF-beta1 or TGF-beta3. Role of TGF-beta isoforms on cell function was evaluated. OBJECTIVE: To develop an in vitro organ culture of rat intervertebral disc and evaluate effects of TGF-beta3 on disc cell function. SUMMARY OF BACKGROUND DATA: An in vitro model system is of considerable value in understanding the cell biology of the intervertebral disc. Development of a useful organ culture model would enhance understanding of disc function in health and disease. MATERIALS AND METHODS: Rat lumbar intervertebral discs were maintained in organ culture in media supplemented with TGF-beta3 or TGF-beta1 for 1 week. Tissue morphology was studied using routine histologic, histochemical and immunohistochemical techniques. Cell function was assessed by gene expression, sulfate incorporation, and Western blot analysis. RESULTS: After 1 week in culture with TGF-beta3 and TGF-beta1, the gross morphology and tissue architecture of the disc were preserved. TUNEL analysis indicated that there was no evidence of cell death in the nucleus pulposus or the anulus fibrosus. The level of Alcian blue staining in the nucleus pulposus was similar to that of the freshly isolated disc. However, when compared with TGF-beta1, TGF-beta3 elevated the expression of critical matrix genes, enhanced [S] incorporation into proteoglycans, preserved the expression of TGF-beta receptors, and decreased aggrecan turnover. There was also increased activation (phosphorylation) of ERK, a critical signaling protein. Moreover, inhibition of ERK activity, in the presence TGF-beta3, resulted in suppression of collagen Type II, aggrecan, TGF-beta-RI, TGF-beta-RII and TGF-beta-RIII mRNA expression. CONCLUSIONS: TGF-beta3 maintains the phenotype of disc cells in organ culture. It exerts this effect, in part, by elevating the levels of activated ERK1/2, which in turn regulates the expression of TGF-beta-RI and TGF-beta-RII.

Activin Receptors, Type I↗

PubMed2006

Osteogenic potential of adult human stem cells of the lumbar vertebral body and the iliac crest.

STUDY DESIGN: Marrow was aspirated from the vertebral body (VB) and iliac crest (IC) of patients undergoing lumbar spinal surgery, following an approved protocol. Progenitor cells were isolated using standard culture conditions and their osteogenic potential evaluated. OBJECTIVE: To evaluate the osteogenic potential of mesenchymal stem cells (MSCs) isolated from the bone marrow of the human VB. SUMMARY OF BACKGROUND DATA: IC marrow grafting during cervical discectomy and fusion procedure is associated with donor site morbidity. Since the VB contains marrow cells, it may be possible to circumvent this problem by using this tissue for osseous graft supplementation. However, there is paucity of information concerning the osteogenic potential of non-IC-derived progenitor cells. Herein, we address this issue. METHODS: Marrow samples from VB of patients undergoing lumbar spinal surgery were collected; marrow was also harvested from the IC. Progenitor cells were isolated and the number of colony forming unit-fibroblastic (CFU-F) determined. The osteogenic potential of the cells was characterized using biochemical and molecular biology techniques. RESULTS: Both the VB and IC marrow generated small, medium, and large sized CFU-F. Higher numbers of CFU-F were obtained from the VB marrow than the IC (P < 0.05). Progenitor cells from both anatomic sites expressed comparable levels of CD166, CD105, CD49a, and CD63. Moreover, progenitor cells from the VB exhibited an increased level of alkaline phosphatase activity. MSCs of the VB and the IC displayed similar levels of expression of Runx-2, collagen Type I, CD44, ALCAM, and ostecalcin. The level of expression of bone sialoprotein was higher in MSC from the IC than the VB. VB and IC cells mineralized their extracellular matrix to a similar extent. CONCLUSIONS: Our studies show that CFU-F frequency is higher in the marrow of the VB than the IC. Progenitor cells isolated from both sites respond in a similar manner to an osteogenic stimulus and express common immunophenotypes. Based on these findings, we propose that progenitor cells from the lumbar vertebral marrow would be suitable candidate for osseous graft supplementation in spinal fusion procedures. Studies must now be conducted using animal models to ascertain if cells of the VB are as effective as those of the IC for the fusion applications.

Bone Marrow Cells↗

PubMed2005

Hypoxia activates MAPK activity in rat nucleus pulposus cells: regulation of integrin expression and cell survival.

OBJECTIVE: The aim of the present study was to investigate whether activation of MAPK subtypes ERK and p38 influences integrin expression and promotes nucleus pulposus cell survival in hypoxia. SUMMARY OF BACKGROUND DATA: We have recently shown that in a low oxygen environment, rat nucleus pulposus cells activate phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways. However, the role of these signaling pathways in regulating cell matrix interactions is not known. METHODS: Rat nucleus pulposus cells were cultured in hypoxia (2% O2) or normoxia (20% O2). Western blotting and kinase assay were used to analyze expression of MAPK signaling molecules. Cell attachment to collagen was studied using an adhesion assay, whereas flow cytometry and RT-PCR were performed to quantify integrin receptor expression. RESULTS: In a hypoxic environment (2% O2), rat nucleus pulposus cells showed a persistent phosphorylation of p38 and ERK proteins; pERK catalyzed the phosphorylation of Elk1-Gst fusion protein. When ERK activity was blocked, cell adhesion to Type II collagen, one of the major extracellular matrix proteins in the nucleus pulposus tissue, was impaired. A similar inhibitory effect on collagen adhesion was observed when nucleus pulposus cells were treated with an antibody to alpha2 integrin. Furthermore, when ERK activity was inhibited, there was a decrease in alpha2 integrin mRNA expression. In contrast to ERK, inhibition of p38 activity did not modulate alpha2 integrin subunit mRNA expression. Likewise, inhibition of ERK, but not p38, resulted in downregulation of nucleus pulposus alpha2 integrin protein levels and blocked cell survival in hypoxia. CONCLUSIONS: Hypoxia activated MAPK signaling pathway activity in nucleus pulposus cells. ERK, but not p38, regulated alpha2 integrin expression and cell survival.

Animals↗

PubMed2005

Nucleus pulposus cells upregulate PI3K/Akt and MEK/ERK signaling pathways under hypoxic conditions and resist apoptosis induced by serum withdrawal.

STUDY DESIGN: To examine the impact of hypoxia, rat nucleus pulposus cells were maintained in monolayer culture in 2% O2 and survival and signal transduction pathways identified. OBJECTIVE: To elucidate the signaling pathways that allow nucleus pulposus cells to adapt to low oxygen environment. SUMMARY OF BACKGROUND DATA: Mammalian cell function is critically dependent on a continuous supply of oxygen. Interestingly, some specialized cell types that include nucleus pulposus cells of the intervertebral disc reside in a hypoxic environment. However, the mechanism of their adaptation to this low oxygen environment is not known. METHODS: Rat nucleus pulposus cells were harvested from explant cultures and grown to confluence in monolayer. Cells from passage 3-7 were maintained under hypoxia (2% O2) and normoxia (20% O2) for various time periods in complete or serum-free medium. Cells were also treated with pharmacologic agents that block PI3K and MAPK signaling pathways. Cell survival was assessed by MTT assay, annexinV-PI dual-color flow cytometry, and the TUNEL procedure. Expression of signaling proteins was evaluated by Western blot analysis. Cell phenotype was studied by semiquantitative RT-PCR. RESULTS: Under hypoxic conditions, rat nucleus pulposus cells were resistant to apoptosis induced by serum starvation. Protection was also observed after treatment of the nucleus cells by desferrioxamine, a compound that mimics many of the effects of hypoxia. Cell survival in hypoxia was related to activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways. Induction of Akt activation and ERK1/2 activationunder hypoxic condition was detected at 12 hours and correlated with inactivation of glycogen synthase kinase-3beta (GSK-3beta), an effector protein involved in regulation of apoptosis. Finally, inhibition of PI3K/Akt and MEK/ERK pathway using the inhibitors LY294002 and PD98059, respectively, impaired cell survival. CONCLUSION: It is concluded that under hypoxic conditions, rat nucleus pulposus cells are adapted for survival by regulation of expression of critical genes, downregulation of apoptosis through activation of the PI3K/Akt and MAPK survival pathways.

Animals↗

PubMed2005

Chitosan: a versatile biopolymer for orthopaedic tissue-engineering.

Current tissue engineering strategies are focused on the restoration of pathologically altered tissue architecture by transplantation of cells in combination with supportive scaffolds and biomolecules. In recent years, considerable attention has been given to chitosan (CS)-based materials and their applications in the field of orthopedic tissue engineering. Interesting characteristics that render chitosan suitable for this purpose are a minimal foreign body reaction, an intrinsic antibacterial nature, and the ability to be molded in various geometries and forms such as porous structures, suitable for cell ingrowth and osteoconduction. Due to its favorable gelling properties chitosan can deliver morphogenic factors and pharmaceutical agents in a controlled fashion. Its cationic nature allows it to complex DNA molecules making it an ideal candidate for gene delivery strategies. The ability to manipulate and reconstitute tissue structure and function using this material has tremendous clinical implications and is likely to play a key role in cell and gene therapies in coming years. In this paper we will review the current applications and future directions of CS in articular cartilage, intervertebral disk and bone tissue engineering.

Animals↗

PubMed2004

Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: implications for cell-based transplantation therapy.

OBJECTIVE: Because mesenchymal stem cells can differentiate into chondrocyte-like cells, we ask the question, can mesenchymal stem cells commit to the nucleus pulposus phenotype? BACKGROUND: Back pain, a significant source of morbidity in our society, is linked to degenerative changes of the intervertebral disc. Absence of suitable graft tissue limits therapeutic approaches for repair of disc tissue. For this reason, there is considerable interest in developing "tissue engineering" strategies for the regeneration of the nucleus pulposus. METHODS: Rat mesenchymal stem cells were immobilized in 3-dimensional alginate hydrogels and cultured in a medium containing transforming growth factor-beta1 under hypoxia (2% O2) and normoxia (20% O2). Mesenchymal stem cells were examined by confocal microscopy to evaluate their viability and metabolic status after labeling with Celltracker green, a thiol sensitive dye, and Mitotracker red, a dye sensitive to the mitochondrial membrane potential. Flow cytometry, semiquantitative reverse transcription polymerase chain reaction and Western blot analysis were carried out to evaluate phenotypic and biosynthetic activities and the signaling pathways involved in the differentiation process. RESULTS: Under hypoxic conditions, mesenchymal stem cells formed large aggregates and exhibited positive Celltracker and Mitotracker signals. Glucose transporter-3, matrix metalloproteinase-2, collagen type II and type XI, and aggrecan mRNA and protein expression was upregulated, whereas there was no change in the levels of decorin, biglycan, fibromodulin, and lumican. Hypoxia maintained the expression of CD44 (hyaluronan receptor), ALCAM (CD166), and endoglin (transforming growth factor-beta receptor). Likewise, expression of beta3 and alpha2 integrin was upregulated. Transforming growth factor-beta treatment increased MAPK activity and Sox-9, aggrecan, and collagen type II gene expression. Basal levels of the phosphorylated MAPK isoform ERK1/2, but not p38, were higher under hypoxic conditions than normoxia, and its activation was further augmented by treatment of cells with transforming growth factor-beta. In hypoxia, transforming growth factor-beta sustained phosphorylated p38 expression for an extended time period. Pharmacological inhibition of ERK1/2 and p38 enzymatic activity resulted in a decrease in Sox-9, aggrecan, and collagen type II mRNA levels. CONCLUSIONS: Our results indicate that hypoxia and transforming growth factor-beta drive mesenchymal stem cell differentiation towards a phenotype consistent with that of the nucleus pulposus. Measurement of selected signaling molecules and response to specific inhibitors suggest involvement of MAPK signaling pathways. It is concluded that mesenchymal stem cells could be used to repopulate the damaged or degenerate intervertebral disc.

Animals↗

PubMed2004

Current strategies for cell delivery in cartilage and bone regeneration.

Several cell-based tissue-engineering therapies are emerging to regenerate damaged tissues. These strategies use autologous cells in combination with bioresorbable delivery materials. Major functions of a delivery scaffold are to provide initial mechanical stability, homogenous three-dimensional cell distribution, improved tissue differentiation, suitable handling and properties for delivery and fixation into patients. Delivery of cells can be achieved using injectable matrices, soft scaffolds, membranes, solid load-bearing scaffolds or immunoprotective macroencapsulation. Thus, to expand the clinical potential, next generation therapies will depend on smart delivery concepts that make use of the regenerative potential of stem cells, morphogenetic growth factors and biomimetic materials.

Animals↗

PubMed2004

Scaffold-based tissue engineering: rationale for computer-aided design and solid free-form fabrication systems.

One of the milestones in tissue engineering has been the development of 3D scaffolds that guide cells to form functional tissue. Recently, mouldless manufacturing techniques, known as solid free-form fabrication (SFF), or rapid prototyping, have been successfully used to fabricate complex scaffolds. Similarly, to achieve simultaneous addition of cells during the scaffold fabrication, novel robotic assembly and automated 3D cell encapsulation techniques are being developed. As a result of these technologies, tissue-engineered constructs can be prepared that contain a controlled spatial distribution of cells and growth factors, as well as engineered gradients of scaffold materials with a predicted microstructure. Here, we review the application, advancement and future directions of SFF techniques in the design and creation of scaffolds for use in clinically driven tissue engineering.

Biocompatible Materials↗

PubMed2003

An organ culture system for the study of the nucleus pulposus: description of the system and evaluation of the cells.

STUDY DESIGN: The goal of this study was to develop a methodology to maintain intervertebral discs in organ culture, thereby preserving tissue architecture and metabolic function in a three-dimensional environment. METHODS: Using a microdissection technique, intervertebral discs were removed from rat lumbar vertebrae. The discs were maintained in organ culture, and cell viability was evaluated histochemically and using probes that measured mitochondrial function and thiol status. The biosynthetic activity of the cells was evaluated by Western blot and RT-PCR analysis. RESULTS: The in vitro organ culture system maintained the vitality of the nucleus pulposus cells. Cells exhibited a high membrane potential for 1 week. When cells were exposed to carbonyl cyanide 4-trifluoromethoxy phenylhydrazone, a known protonophore, the fluorescence was lost, indicating that the staining was specific for viable cells. In many cells, Celltracker Green, probe for reduced thiols, colocalized with the membrane potential. Histologic studies revealed that in culture for 1 week, normal nucleus pulposus structure was maintained; after this time period, alterations were observed. We evaluated the two tissues for characteristic phenotypic markers HIF-1alpha and MMP-2. We noted that the nucleus pulposus expressed these proteins. The RT-PCR profile at 7 days indicated that the cells also expressed collagen type II, aggrecan, and decorin. DISCUSSION: Three factors contributed to success in maintaining the vitality of the nucleus pulposus in vitro. First, the cells were confined within the disc itself; second, the medium was hyperosmotic; third, the medium was supplemented with transforming growth factor-beta. The fluorescence measurement provided a rapid method for evaluation of the status of nucleus pulposus cells. Histologic analysis confirmed that the cells remained viable for at least 1 week. Viability in terms of biosynthetic activity was further confirmed using RT-PCR and Western blot analysis. We conclude that short-term intervertebral disc organ culture can be used as a suitable in vitro model to study effects of environmental factors linked to disc degeneration and/or regeneration.

Animals↗

PubMed2003

In vivo biocompatibility evaluation of cellulose macrocapsules for islet immunoisolation: Implications of low molecular weight cut-off.

Earlier we showed the in vitro suitability of cellulose molecular dialysis membrane of low cut-off for islet immunoisolation. Using a syngenic islet transplantation model, here we report the in vivo biocompatibility status and discuss implications of the low molecular weight cut-off of this membrane. Streptozotocin diabetic mice were transplanted ip with islet grafts in cellulose macrocapsules and monitored for 6 weeks for their blood glucose profiles. Membrane biocompatibility was evaluated by assessing islet graft morphometry, viability, functionality, and tissue reaction against the capsules. Animals (n = 10) attained normoglycemia after 1 week of transplantation and remained nondiabetic throughout a follow-up of 6 weeks. Animals subjected to a glucose tolerance test at the end of the study showed delayed glycemic control, indicating a delayed insulin response. Grafts retrieved after the 15(th) day and after 6 weeks showed viable islets, between 70-80% and approximately 40%, respectively, with morphometric parameters similar to freshly isolated islets. Graft retrievals resulted in a recurrence of hyperglycemia, indicating a functional tissue mass. A mild tissue reaction with a few immunocytes and a very thin fibrous capsular reaction were seen against the macrocapsule membrane. This study points out the in vivo suitability of cellulose membrane for islet immunoisolation. However, applicability of this membrane is limited due to its low cut-off value. Membranes with a higher molecular weight cut-off may render better glycemic control and higher graft viability.

Animals↗

PubMed2003

Hydrogel-coated textile scaffolds as candidate in liver tissue engineering: II. Evaluation of spheroid formation and viability of hepatocytes.

In this study we evaluate the performance of primary rat hepatocytes and HepG2 cells on chitosan-collagen hydrogel-coated textile scaffolds. Light microscopy and electron microscopic observations showed attachment and aggregate formation tendency of hepatocytes on the scaffolds. As tested by the tetrazolium reduction (MTT) assay it was evident that cells had preserved mitochondrial functionality. It was also observed that pure collagen and collagen blended scaffolds allowed higher cell growth than pure chitosan scaffold. Fluorescent live/dead staining showed a metabolically active, viable cell population on all scaffold compositions with occurrence of few dead cells. Cell functionality was confirmed by secretion of albumin, which was maintained throughout culture period. Take collectively our results suggests that hydrogel-coated textile scaffolds could be promising for tissue-engineering applications, as they allow favorable hepatocyte attachment, spheroid formation and maintenance of function. These scaffolds could be useful for co-culturing hepatocytes and non-parenchymal endothelial cells in bioartificial liver support systems.

Animals↗

PubMed2002

Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2.

Attempts to study the biology of the nucleus pulposus have been limited in scope due to the low rates of cell proliferation, difficulties in maintaining viable disc cells in culture and the absence of a clearly defined phenotype. The major objective of this communication is to construct a phenotypic signature for cells of the nucleus pulposus that is based on the hypothesis that in response to restriction on oxygen and nutrient flux, there is expression of HIF-1, GLUT-1 and MMP-2. Nucleus pulposus, as well as annulus fibrosus and cartilage of the vertebral end plates, was collected from rat spinal units. Western blot analysis and immunohistochemistry clearly showed that there was a significant level of expression of the HIF-1 beta isoform in the nucleus pulposus; HIF-1 beta was present at lower levels in cells of the annulus and the end plate. In contrast to HIF-1 beta, HIF-1 alpha was expressed only in the nucleus pulposus. This isoform was absent from both the cartilage end plate and annulus. We detected HIF-1 alpha immunohistochemically in the nucleus pulposus; however, the staining was light and diffuse. Cells of the nucleus pulposus expressed GLUT-1; in contrast, when probed by Western blot analysis the annulus and cartilage were negative for this protein. Western blot analysis also showed that in the nucleus pulposus the level of MMP-2 was high when compared to the adjacent tissues. We suggest that the differential expression of the two HIF isoforms, and GLUT-1 and MMP-2, provides a phenotypic signature that permits cells of the nucleus pulposus to be distinguished from neighboring tissues. Moreover, the presence of these isoforms provides evidence that cells of the disc respond to hypoxia and nutrient stress by upregulating stress-responsive genes.

Animals↗

PubMed2002

Immunocytochemical localization of growth hormone-releasing hormone-like peptide in the brain of the tiger frog, Rana tigrina.

Using antisera directed against carp growth hormone-releasing hormone (cGHRH), we found more extensive brain distribution of GHRH-like immunoreactive (ir) neurons in the tiger frog, Rana tigrina, than reported in previous studies, which employed mammalian GHRH antibodies. In the telencephalon, GHRH-ir perikarya were present in the pallium dorsale, pallium laterale (pars dorsalis and pars ventralis), pallium mediale, nucleus entopeduncularis, amygdala pars medialis, and in the ventral portion of the lateral preoptic area. Most GHRH somata were present in the nucleus infundibularis ventralis located around the third ventricle, extending from the region posterior to the optic chiasma to the caudal end of infundibulum. In the thalamic region, GHRH-ir perikarya occurred in the area ventrolateralis thalami, the nuclei posterocentralis thalami, and the posterolateralis thalami. The ir cell bodies in the nucleus posteroventralis tegmenti mesencephali represented the caudal-most brain GHRH perikarya. Extensive GHRH-ir fibers occurred around the nonreactive cells in the ventral preoptic area and ventral area of the infundibulum. GHRH-ir fibers were present in the outer layers of the median eminence, but not in the neural lobe or pars distalis of the pituitary gland. This wider neuroanatomical distribution of GHRH-like peptide in the brain of R. tigrina should, provide the basis for future studies to establish the exact role of GHRH-like peptides in anuran brain.

Animals↗

PubMed2002

Tissue engineering: advances in in vitro cartilage generation.

Damaged or diseased articular cartilage frequently leads to progressive debilitation resulting in a marked decrease in the quality of life. Tissue engineering, a budding field in modern biomedical sciences, promises creation of viable substitutes for failing organs or tissues. It represents the amalgamation of rapid developments in cellular and molecular biology on the one hand and material, chemical and mechanical engineering on the other. Current tissue engineering approaches are mainly focused on the restoration of pathologically altered tissue structure based on the transplantation of cells in combination with supportive matrices and biomolecules. The ability to manipulate and reconstitute tissue structure and function in vitro has tremendous clinical implications and is likely to have a key role in cell and gene therapies in coming years.

Biocompatible Materials↗

PubMed2002

Models of pancreatic regeneration in diabetes.

The number of functionally intact beta cells in the islet organ is of decisive importance for the development, course and outcome of diabetes mellitus. Generally speaking, the total beta-cell mass reflects the balance between the renewal and loss of these cells. Assuming that virtually all forms of diabetes mellitus are characterized by an insufficient extent of beta cell replication needed to compensate for the loss or dysfunction of beta cells occurring in diabetes, elucidation of the regenerating potential in experimentally induced diabetic animal would be of interest as alternative therapy for diabetes. Here we have attempted to take a stock of different models developed in the last few years, which permit investigation of regenerative process from various angles. The review focuses on factors responsible for induction of islet neogenesis in the diabetic pancreas, ultimately leading to pancreatic regeneration and possible reversal of diabetes. On the whole the study of these models will enhance our understanding of regenerative potential of diabetic pancreas and factors necessary to trigger stem cells' population within the pancreas so as to suggest an alternative therapeutic approach for the control and/or cure of diabetes.

Animals↗