The extracellular matrix and the control of proliferation of corneal endothelial and lens epithelial cells

When rabbit or bovine corneal endothelial cells were plated at low cell density in the presence of high (10%) concentrations of serum, cells maintained on plastic proliferated slowly and after a few days enlarged considerably. If the cultures were exposed to FGF, the cells proliferated actively and, after a week, a confluent monolayer of closely apposed mononucleated cells was formed. In contrast to cells maintained on plastic, cells maintained on an extracellular matrix (ECM) produced by corneal endothelial cells proliferated even faster than cells maintained on plastic and exposed to FGF and no longer required the presence of FGF to reach confluence. Addition of FGF to such cultures did not decrease the mean doubling time, which was already at a minimum (16 hr), nor did it result in a higher final cell density, which was already at a maximum (700–1000 cells/mm²). It can therefore be concluded that, when the proliferation of corneal endothelial cells from two different species is compared, cells maintained on plastic proliferate poorly and FGF is needed in order for the cultures to become confluent. In contrast, when similar cultures are maintained on ECM, they proliferate actively and no longer require FGF in order to become confluent. Similar results were observed when lens epithelial cells were maintained on plastic versus an ECM. Although cells maintained on plastic hardly proliferated, yielding within a few days a population composed of large and binucleated cells, cells plated on an ECM proliferated actively, with an average doubling time for lens epithelial cells of 15 hr during their logarithmic growth phase.The ability of plasma vs. serum to sustain cell proliferation was analyzed using corneal endothelial cells maintained on plastic versus an ECM. It was observed that cultures maintained on plastic proliferate poorly when exposed to plasma. When exposed to serum they proliferate more actively. Nevertheless, in both cases cultures required the presence of FGF in order to become confluent. When similar cultures were maintained on an ECM, they proliferated equally well regardless of whether they were exposed to plasma or serum and no longer required FGF in order to become confluent. One can therefore conclude that the simple change of substrate from plastic to ECM will restore the sensitivity of these cells to agents present in plasma.     

Effect of growth factors on cell proliferation, matrix deposition, and morphology of human nasal septal chondrocytes cultured in monolayer

OBJECTIVES: Tissue engineering of septal cartilage provides ex vivo growth of cartilage from a patient's own septal chondrocytes for use in craniofacial reconstruction. To become clinically applicable, it is necessary to rapidly expand a limited population of donor chondrocytes and then stimulate the production of extracellular matrix on a biocompatible scaffold. The objective of this study was to determine favorable serum-free culture conditions for proliferation of human septal chondrocytes using various concentrations and combinations of four growth factors. STUDY DESIGN: Prospective, randomized, controlled study. METHODS: Nasal septal chondrocytes from six patient donors were isolated by enzymatic digestion and expanded in monolayer culture in both serum-free media (SFM) and 2% fetal bovine serum (FBS). Both of these groups were exposed to varying concentrations and combinations of transforming growth factor (TGF)-beta1, basic fibroblast growth factor (FGF)-2 both at 1, 5, and 25 ng/mL, and bone morphogenetic protein (BMP)-2 and insulin-like growth factor (IGF)-1, both at 5, 25, and 125 ng/mL in the medium during the expansion phase. Cell morphology was assessed throughout the culture duration. After 7 days of monolayer growth, cultures were assessed for cellularity and glycosaminoglycan (GAG) content. RESULTS: The addition of low-dose FBS in culture media consistently led to significantly greater cell proliferation and matrix deposition than the SFM cell cultures. FGF-2 and TGF-beta1 both alone and in combination led to the greatest proliferative effect compared with the other growth factors. In contrast, BMP-2 and IGF-1 led to the least cell proliferation although was most effective in retaining chondrocyte cell morphology. CONCLUSIONS: With the addition of TGF-beta1 and FGF-2 to culture media, the concentration of serum can be greatly decreased and possibly eliminated altogether without jeopardizing cell proliferation.     

Relationship between cytokine concentrations (FGF, sICAM-1 and SCF) in serum, follicular fluid and ICSI outcome

OBJECTIVE: The aim of this study was (i) to investigate the existence of fibroblast growth factor (FGF), soluble intracellular adhesion molecule-1 (sICAM-1), and stem cell factor (SCF) in serum and human follicular fluid (FF) of intracytoplasmic sperm injection (ICSI) patients, and (ii) to determine the relationship between these parameters and ICSI outcome. MATERIAL AND METHOD: Seventy-five patients undergoing controlled ovarian hyperstimulation with human menopausal gonadotropin (hMG) after down-regulation with GnRHa were included in this study. The concentrations of FGF, SCF, and sICAM-1 were measured by using commercially available enzyme-linked immunosorbent assay test kits. RESULTS: The FGF, sICAM-1, and SCF concentrations in the serum of women who become pregnant (group I) were 8.5 +/- 1.5 pg/mL, 235.8 +/- 81.1 ng/mL, and 597.7 +/- 139.9 pg/mL, and the corresponding concentrations of women who did not (group II) were 6.4 +/- 3.6 pg/mL, 230.6 +/- 66.5 ng/mL, and 569.6 +/- 91.4 pg/mL respectively. No significant difference was observed between the two investigated groups with regard to the number of hMG ampoules administered for controlled ovarian hyperstimulation, estradiol concentration on the day of human chorionic gonadotropin (hCG) injection, number of retrieved oocytes and fertilization rate. CONCLUSION: The concentration of FGF, sICAM-1, and SCF did not differ significantly between the two groups in serum or in FF. Besides, the ICSI outcome was not related to their concentrations in serum or FF. Therefore, these parameters could not be used as a prognostic factor in ICSI program.     

Expression and regulation of ROR-1 during early avian limb development

ROR-1 is a member of the ROR family of tyrosine kinase like orphan receptors and is highly conserved among various species. We have isolated the chick ROR-1 (cROR-1) and show that cROR-1 expression is high and restricted to the proximal limb region until HH-stage 25. At later stages, expression spreads towards the distal limb region. In order to determine the signals that control cROR-1 expression, factors known to be involved in limb patterning (FGFs, BMPs, SHH, retinoic acid) were applied to the developing limb. Whereas neither FGFs, BMPs, nor SHH affected cROR-1 expression, upregulation could be achieved by ectopic application of retinoic acid to the distal limb region. As retinoic acid also upregulated retinoic acid receptor beta (Rar-), we assume that cROR-1 upregulation is mediated by Rar-. We conclude that ROR-1 signaling is an independently regulated pathway, which is involved in late rather than early limb development.     

Vertebrate limb development: from Harrison's limb disk transplantations to targeted disruption of Hox genes

Various animal organs have long been used to investigate the cellular and molecular nature of embryonic growth and morphogenesis. Among those organs, the tetrapod limb has been preferentially used as a model system for elucidating general patterning mechanisms. At the appropriate time during the embryonic period, the limb territories are first determined at the right positions along the cephalocaudal axis of the animal body, and soon the limb buds grow out from the flanks as mesenchymal cell masses covered by simple ectoderm. The position, number, and identity of the limbs depend on the expression of specific Hox genes. Limb morphogenesis occurs along three axes, which become gradually fixed: first the anteroposterior axis, then the dorsoventral, and finally the proximodistal axis, along which the bulk of limb growth occurs. Growth of the limb in amniotes depends on the formation of the apical ectodermal ridge, which, by secreting many members of the fibroblast growth factors family, attracts lateral plate and somitic mesodermal cells, keeps these cells in the progress zone proliferating, and prevents their differentiation until an appropriate time period. Mutual interactions between mesoderm and ectoderm are important in the growth process, and signaling regions have been identified, such as the zone of polarizing activity, the dorsal limb ectoderm, and the apical ectodermal ridge. Several molecules have been found to play leading roles in various biological processes relevant to morphogenesis. Besides its intrinsic merit as a model for unraveling the mechanisms of development, the limb deserves considerable clinical interest because defects of limb development are the most common single category of congenital abnormalities.     

中医不同治法对成纤维细胞生长因子在大鼠肝癌中表达的影响

目的:观察成纤维细胞生长因子(FGF)在二乙基亚硝胺(DEN)诱发大鼠肝癌组织中的表达及不同中医治法的作用。方法:采用DEN诱发大鼠肝癌,随机分为正常组、模型组、喃氟啶组、抑癌扶正行气活血法组、清热解毒法组、行气活血法组、健脾扶正法组,应用Affymetrix Rat230A GeneChip及相关技术检测各组肝脏(含肝癌)FGF基因表达的差异。结果:Rat230A芯片中有24个FGFs、3个FGFRs、1个FGFBP,其中肝癌表达增加者15个,减少者13个。FGF13芯片读数(肝癌形成/正常肝脏)为10.8,FGF7芯片读数(正常肝脏/肝癌形成)为13.1。近10年有关FGFs文献857篇,涉及肝癌者仅49篇,仅3个基因(FGF1、FGF2及FGFR3)涉及。结论:FGFs基因在大鼠肝癌中的表达有一定意义,不同治疗方法对其表达水平存在差异。     

New bone formation by murine osteoprogenitor cells cultured on corticocancellous allograft bone

The gold standard for bone grafting in orthopedics is autograft, however autograft has a limited supply and is associated with significant morbidity at the harvest site. One alternative, allograft bone, provides an osteoconductive scaffold, is in less limited supply, and it does not require a harvest from the patient. However, allograft lacks both osteogenic cells and osteoinductive proteins that make autograft bone so advantageous. This study provides a model to investigate strategies for augmentation of corticocancellous allograft bone discs with bone marrow‐derived osteoprogenitor cells (OPCs) plus exogenous growth factors in vitro. In this model, allograft bone discs were created by cutting 1‐mm thick slices from the distal femur and proximal tibia of euthanized mice. The allografts were sterilized and scanned by micro‐computed tomography (µCT) to provide the pre‐culture graft volume and trabecular characteristics. The discs were then seeded with OPCs harvested from murine bone marrow. The seeded grafts were placed in organ culture until harvest, after which they were re‐scanned by µCT and the data compared to the corresponding pre‐culture data. In addition, bone morphogenetic protein‐7 (BMP‐7, also know as osteogenic protein‐1 or OP‐1), basic fibroblast growth factor (bFGF), and OP‐1 combined with bFGF were added on a daily basis to the cultures. After final µCT scanning, all grafts were sectioned and evaluated histologically after hematoxylin and eosin (H&E) staining. µCT scans of cultured allografts with cells at 3, 5, and 9 weeks showed a time‐dependent, statistically significant increase in bone volume. The trabecular thickness (Tb.Th.) of grafts, from both groups that were augmented with OP‐1, showed a statistically significant increase in trabecular thickness of allografts with OPCs. These data suggest that bone marrow‐derived OPCs adhere to, and produce, new bone on corticocancellous allograft in vitro. When exogenous OP‐1 is added to this model, an increase in the production of bone onto the corticocancellous allograft bone disc is seen. This model allows for the investigation of the effects of multiple growth factors, and other interventions, on OPCs seeded onto allograft bone in vitro. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res