Quantification of Thrombocyte Growth Factors in Platelet Concentrates Produced by Discontinuous Cell Separation

Platelet concentrates (PC) are increasingly used to increase bone regeneration in pre-prosthetic surgery. Although it is generally appreciated that certain growth factors (PDGF, TGF, EGF, and ECGF) are present in thrombocyte preparations, relatively little is known about these components in quantitative terms. The study reported here analysed the amounts of growth factors in PC produced under standard conditions from healthy volunteers. All the blood samples (237 in total) were analysed using Quantikine ELISA kits (R and D). The mean &#45 SD platelet count in whole blood from these donors was 262,000 &#45 58,000/ &#119 l, while in PC produced by discontinuous cell separation it was 1,419,000 &#45 333,000/ &#119 l. The mean growth factor concentrations in PC preparations in ng/ml were as follows: PDGF-AB 125 &#45 55 ng/ml; TGF- &#103 1 221 &#45 92 ng/ml; IGF-I 85 &#45 25 ng/ml; PDGF-BB 14 &#45 9 ng/ml; TGF- &#103 2 0.4 &#45 0.3 ng/ml. These growth factor concentrations typically covered a 3-10 fold range: PDGF-AB 29-277 ng/ml; PDGF-BB 2-33 ng/ml; TGF- &#103 1 32-397 ng/ml; TGF- &#103 2 0.1-1.2 ng/ml; IGF-I 40-138 ng/ml. Platelet counts in PC were slightly higher for women (Mann-Whitney Test all p <0.001 ) than for men, while the concentrations of growth factors in PC exhibited no gender-related difference of any statistical significance.     

The effect of growth factor treatment on meniscal chondrocyte proliferation and differentiation on polyglycolic acid scaffolds

The aim of this investigation was to determine the effect of growth factor treatment on ovine meniscal chondrocyte (OMC) proliferation in vitro and on the production of matrix proteins by OMCs grown within a polyglycolic acid (PGA) scaffold. Analysis of 72-h monolayer cultures using the mean transit time (MTT) assay revealed a greater increase in OMC numbers in the presence of platelet-derived growth factor (PDGF)-AB, PDGF-BB, insulin-like growth factor (IGF)-I, transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) than in untreated controls. In contrast, IGF-II and bone morphogenetic protein-2 had no effect on OMC proliferation at the concentrations tested. The growth factors that elicited the greatest proliferative response (PDGF-AB, PDGF-BB, TGF-beta1, and IGF-I) were subsequently tested for their ability to enhance OMC proliferation and differentiation within PGA scaffolds. Biochemical analysis revealed less glycosaminoglycan (GAG) production in the presence of all growth factors tested compared to untreated control samples. In contrast, all of the growth factors increased collagen type I production by OMCs within the scaffolds at day 20, and all except PDGF-BB resulted in an increase at day 39, when compared to appropriate control samples. With the exception of IGF-I, none of the growth factors tested had any significant effect on collagen type II production. Histological staining of sections from OMC-PGA scaffolds did not reveal any difference in GAG or collagen production between the treatment groups. However, immunohistochemical analysis demonstrated an increase in collagen type I expression and a decrease in collagen type II at day 39 in all growth factortreated constructs, concomitant with a high infiltration of cells. This suggests that PDGF-AB, PDGF-BB, TGF-beta1, and IGF-1 may be useful in future tissue engineering studies for promoting meniscal cell proliferation and differentiation within scaffolds.     

Effects of calcium and thrombin on growth factor release from platelet concentrates: kinetics and regulation of endothelial cell proliferation

Platelet concentrates (PCs) constitute new biological mediators used in osseous reconstructive surgery. In this study, we assessed (i) the effects of various concentrations of calcium and thrombin on the kinetics of platelet-derived growth factor (PDGF-BB), transforming growth factor-beta1(TGF-beta 1), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) release by PCs and (ii) the contribution of PC supernatants to endothelial cell proliferation. Our results indicate that high concentrations of calcium (Ca) and thrombin (Thr) trigger an immediate and significant increase in bFGF, TGF-beta 1 and PDGF-BB concentrations. Thereafter, PDGF-BB, VEGF and TGF-beta 1 levels remained generally constant over a 6-day period while a decrease in bFGF concentrations was noted after 24h. Lower Ca and Thr concentrations tended to reduce and delay growth factors release from PCs. Endothelial cell proliferation was greatly enhanced with PC supernatants (mean: 20-fold increase). This was especially evident when endothelial cells were treated with supernatants harvested early after PC treatment with high concentrations of Ca and Thr or later after PC treatment with low Ca and Thr concentrations. Additional research aiming to measure the effects of Ca and Thr on bone formation in vivo is needed.     

Association of serum insulin-like growth factor-I and erythropoiesis in relation to body iron status

This study investigated the associations between serum insulin-like growth factor-I (IGF-I) concentrations and erythropoietic activities in relation to body iron status. Serum IGF-I concentrations, free erythrocyte protoporphyrin (FEP), hemograms, and serum iron markers were measured in 71 female adolescents, age 14 to 17 yr. No significant differences were observed in hemograms, iron parameters, or FEP between the subjects with IGF-I <681.2 ng/ml and IGF-I 681.2 ng/ml. However, blood hemoglobin and serum iron concentrations averaged 13.4 +/- 0.8 g/dl and 93.7 +/- 41.2 microg/dl in the subjects with IGF-I >809 ng/ml, which were above the values in those with IGF-I <523 ng/ml (12.3 +/- 0.9 g/dl and 50.5 +/- 30.8 microg/dl, p < 0.05, respectively). On the other hand, FEP was significantly lower in the adolescents with IGF-I >809 ng/ml than in those with IGF-I <523 ng/ml (38.9 +/- 16.2 microg/dl vs 63.4 +/- 23.1 microg/dl, p <0.05). Prevalences of iron deficiency or iron deficiency anemia were 3- or 5-fold higher in the subjects with IGF-I <523 ng/ml, compared to those with IGF-I >809 ng/ml. Serum IGF-I correlated significantly with FEP (r = -0.45, p <0.05) and serum iron concentrations (r = 0.40, p <0.05) in iron deficient subjects. In summary, IGF-I seems to have an important relationship to iron metabolism and protoporphyrin synthesis in adolescents.     

Differential proliferation of rat lung fibroblasts induced by the platelet-derived growth factor-AA, -AB, and -BB isoforms secreted by rat alveolar macrophages

Platelet-derived growth factor (PDGF)-like molecules secreted by alveolar macrophages have been postulated to be mediators of lung fibrogenesis since these cytokines stimulate the proliferation and chemotaxis of lung fibroblasts. We are studying the biology and biochemistry of rat macrophage-derived PDGF and have identified for the first time the specific isoforms of PDGF (-AA, -AB, and -BB) that these macrophages secreted in vitro following activation with either chrysotile asbestos or carbonyl iron spheres. Subsequently, the proliferative response of rat lung fibroblasts (RLF) to the different PDGF isoforms was established. Using several antibodies raised against the distinct isoforms, we established that two different PDGF-like factors with molecular masses of 30 to 34 kD and 16 to 18 kD were contained in alveolar macrophage-conditioned medium. Within each of these molecular mass regions was a mixture of all three PDGF isoforms. We estimated that the 30- to 34-kD PDGF was mainly PDGF-BB (approximately 50%), while the remaining consisted of PDGF-AA (approximately 13%) and PDGF-AB (approximately 37%). Purified recombinant PDGF isoforms were tested for their ability to stimulate the growth of early-passage RLF and Swiss 3T3 cells in a 3-day cell proliferation assay. PDGF-BB and PDGF-AB were the most potent inducers of RLF proliferation and stimulated growth half-maximally at approximately 1 ng/ml and approximately 7 ng/ml, respectively. While these two B-chain-containing dimers stimulated lung fibroblast growth by as much as 150% above control medium, the PDGF-AA homodimer stimulated lung fibroblast proliferation less than 25% above control medium at the highest concentrations tested (20 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Migration of Human Colonic Myofibroblasts

Background and Aim: The migration of mesenchymal cells to areas of mucosal or submucosal tissue damage is an essential factor for wound healing in the intestine. Thus far, neither migration inducing factors nor signal transduction cascades involved in the migration of colonic myofibroblasts (CMF) have been studied in detail. Methods: Primary CMF were isolated from the mucosa of surgical specimens or endoscopic biopsies. Migration assays of CMF were performed in the modified 48-well Boyden chamber. Secreted growth factors were quantified by ELISA. Results: CMF secrete autocrine or paracrine migration stimulating factors. Culture supernatant of CMF collected after 24, 48, and 72 h (=conditioned media) stimulated the migration of CMF ( 48.9 &#45 4.5; 60.3 &#45 5.3 and 67.8 &#45 6.4 cells/hpf, respectively). Heating of conditioned media to 95°C or addition of cycloheximide during the conditioning period abolished migration. Addition of PDGF-AB (2.5-50 ng/ml) or IGF-I (10-300 ng/ml) to CMF conditioned media further increased the migration of CMF to a maximum of 177 and 160%, respectively, when compared to the migration induced by conditioned medium alone. Addition of EGF (2.5-50 ng/ml) or TGF- &#103 1 (1-50 pg/ml) caused an increased CMF migration up to 139 and 128%, respectively. MCP-1 (5-50 ng/ml) and bFGF (10-200 ng/ml) had no effect on CMF migration. Conclusion: The growth factors PDGF-AB, IGF-I, EGF and TGF- &#103 1 stimulate the migration of CMF. However, factors secreted by CMF are essential for their ability to migrate in response to these growth factors. The identification of physiologically relevant migration inducing factors may help to elucidate the network of interactions and the complex mechanisms involved in intestinal wound healing or fibrosis.     

Paracrine changes in the peritoneal environment of women with endometriosis

During the past decade, macrophage-derived substances such as prostanoids, cytokines, growth factors and angiogenic factors have been detected in the peritoneal fluid of women with endometriosis. In particular, growth-promoting and angiogenic factors are considered to be substantially involved in the pathogenesis of endometriosis. In this study, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta) and intercellular adhesion molecule 1 (ICAM-1), substances recently detected in the peritoneal fluid of women with endometriosis, were assessed with regard to their concentrations in different stages of endometriosis and changes of the peritoneal paracrine activity after medical treatment with a gonadotrophin releasing hormone agonist (GnRHa). Peritoneal fluid was obtained from patients with endometriosis during laparoscopy before and after a 4-month treatment with a GnRHa. VEGF, TGF-beta and ICAM-1 could be detected in all women presenting with various stages of active endometriosis. After GnRHa therapy, all patients showed significant decreases in mean concentrations of VEGF (194+/-77 pg/ml), TGF-beta (902+/-273 pg/ml) and ICAM-1 (157+/-52 ng/ml). Patients with stage III and IV endometriosis (according to the rAFS score) had much higher concentrations of VEGF and TGF-beta before treatment compared with those patients with mild endometriosis (rAFS stages I and II). The most striking decrease in concentration was for TGF-beta, from 902 pg/ml before to 273 pg/ml after therapy. These results indicate an important role for paracrine activity in the establishment and maintenance of endometriosis. Indeed, treatment with a GnRHa may reduce paracrine activity in the peritoneal cavity via hypo-oestrogenism and provide proof of successful therapy.     

Assessment of growth factor effects on post-thaw development of cryopreserved mouse morulae to the blastocyst stage

The objective of this study was to assess the influence of specific factors on post-thaw development of mouse cryopreserved morulae. Thawed morulae (n = 206) were randomly distributed between 10 treatment groups: medium alone control (CT), Vero (VR) cells, leukaemia inhibitory factor (1 ng/ml), interleukin-6 (1 ng/ml), transforming growth factor (TGF) alpha (2 ng/ml), epidermal growth factor (EGF) (4 ng/ml), platelet-derived growth factor (1 ng/ml), insulin-like growth factor (IGF)-I (30 ng/ml), IGF-II (1 ng/ml) and TGFbeta (2 ng/ml). At 4, 8, 20, 30 and 48 h, a digitized image of each thawed embryo was captured and stored for later analysis. The following parameters were examined: blastocoel formation, blastocyst expansion, zona thickness and hatching. At termination of the experiment, cell number per embryo was determined by bisbenzimide staining. When contrasted to the medium alone control, co-culture consistently accelerated the development of frozen-thawed morulae to the hatched blastocyst stage, allowing embryos to recover rapidly from any damage sustained during the cryopreservation process. While no single growth factor/cytokine was able to completely mimic the results achieved with co-culture, all of the growth factors impacted positively on at least one of the morphological parameters studied. Cell proliferation was significantly stimulated by just 48 h exposure to growth factors, either through co-culture or by direct media supplementation. Co-culture again yielded the best results with a mean cell count of 217 +/- 76 cells per blastocyst as compared with 131 +/- 36 in control medium alone. Amongst the factors tested, IGF-I, IGF-II and EGF had the greatest impact, with mean cell counts of 172 +/- 50, 168 +/- 50 and 179 +/- 55 respectively. Whereas only 5% of CT embryos developed to blastocysts with > 200 cells, 51% of thawed embryos placed on co-culture monolayers and 25-32% of embryos cultured with IGF-I, IGF-II or EGF had > 200 cells. This study for the first time systematically describes the effect of culture regimen and growth factor additives on the post-thaw development of cryopreserved embryos.     

Critically low hormone and catecholamine concentrations in the primed extracorporeal life support circuit

The first hours of extracorporeal life support (ECLS) are commonly marked by new hemodynamic instability without a known etiology. We measured hormone and catecholamine concentrations in six ECLS primed circuits immediately before joining the patient's circulation to assess a potential role of these agents in this condition. The following hormones were significantly below the lower end of the normal range for the first week of life (data are presented as mean +/- SEM): cortisol 1.95 +/- 0.15 microg/dl (p < 0.001), aldosterone 3.73 +/- 0.74 ng/dl (p < 0.05), free thyroxine 1.2 +/- 0.1 ng/dl (p < 0.05), free triiodothyronine 0.53 +/- 0.03 pg/ml (p < 0.001), thyroid stimulating hormone 0.31 +/- 0.05 microU/ml (p < 0.001), growth hormone (GH) 0.09 +/- 0.01 ng/ml (p < 0.001), estradiol 38.3 +/- 3.72 pg/ml (p < 0.001), IGF-BP1 0.95 +/- 0.1 ng/ml (p < 0.001), glucagon 26 +/- 1.2 pg/ml (p < 0.001), epinephrine 17.3 +/- 3.7 pg/ml (p < 0.001), and norepinephrine 127 +/- 27 pg/ml (p < 0.05). No dopamine was detected. Normal hormone concentrations included IGF-I, IGF-BP3, insulin, parathyroid hormone, leptin, and testosterone. Critically low concentrations of cortisol, thyroid hormones, GH, IGF-BP1, glucagon and catecholamines were measured in the ECLS circuit even though it was primed with fresh frozen plasma. These concentrations may cause significant and precipitous dilutional reductions in the patient's circulating levels immediately after connection to the ECLS circuit and hence contribute to hemodynamic instability.     

Thyroxine effects on serum insulin-like growth factor I levels, anthropometric measures, and body composition in patients after thyroidectomy

Thyroxine is an important hormone related to growth. To study the effects of thyroxine on serum levels of insulin-like growth factor I (IGF-I), anthropometric measures, and body composition, we studied 28 patients who were receiving thyroxine therapy after thyroidectomy for well-differentiated thyroid cancer. Serum IGF-I levels decreased from 38.3 +/- 19.0 ng/ml (mean +/- SD) to 26.8 +/- 12.8 ng/ml (p <0.001) after withdrawal of thyroxine and increased to 43.8 +/- 20.2 ng/ml (p <0.001) after thyroxine was resumed. The serum levels of TSH, T3, and thyroxine changed accordingly after withdrawal and resumption of thyroxine therapy. Skinfold thickness increased from 21.8 +/- 6.5 mm to 23.7 +/- 6.4 mm (p <0.001) after withdrawal of thyroxine and decreased to 21.5 +/- 6.9 mm after resumption of thyroxine. Similar changes occurred in the circumferences of the waist and hip (p <0.01), but no significant change occurred in arm muscle circumference (18.7 +/- 2.3 cm vs 18.7 +/- 2.3 cm vs 19.0 +/- 2.4 cm). Body composition analysis by bioelectrical impedance showed no significant changes in the percentages of body fat and lean mass. In conclusion, withdrawal of thyroxine from patients with thyroid cancer was associated with an immediate decrease in serum IGF-I levels. However, lean mass did not respond to the changes in serum IGF-I levels.     

Systemic inflammation alters the inflammatory response in experimental lipopolysaccharide-induced meningitis

Experiments to evaluate the effect of the level and duration of endotoxaemia on the meningeal inflammatory response were performed in order to determine if systemic inflammation alters meningitis. Rabbits received either saline or Escherichia coli O111:B4 lipopolysacharide (LPS) intravenously at various doses (1, 3 or 10 microg) and times (-8, -2 or 0 h) before an intracisternal injection of 20 ng LPS. An intracisternal LPS injection together with saline intravenously produced a peak cerebrospinal fluid (CSF) tumour necrosis factor (TNF) level (95 +/- 26 ng/ml) at 2 h and peak leucocyte level (5413 +/- 764 cells/microl) at 4 h post-injection. Blood leucocytes were slightly elevated (12 000 +/- 500/microl at 0 h; 16 900 +/- 280/microl at 8 h) but plasma TNF was always undetectable (< 0.05 ng/ml). Conversely, intravenous injection of 3 or 10 microg LPS 2 h prior to intracisternal LPS injection impaired pleocytosis (peak < 220 cells/microl) and delayed ( approximately 4 h) and reduced peak CSF TNF levels (3 microg LPS 5.0 +/- 1.2 ng/ml; 10 microg LPS 6.9 +/- 1.9; P < 0.05). Intravenous administration of 1 microg LPS was less inhibitory to CSF inflammation, but delayed onset (peak 1100 +/- 60 leucocytes/microl CSF at 8 h; 6.3 +/- 0.3 ng TNF/ml CSF at 4 h; both P < 0.05). Neutropenia nadirs were dependent on LPS dose (1 microg, 4500 +/- 1700; 3 microg, 1900 +/- 60; 10 microg, 1100 +/- 100 all at 4 h post-intravenous dose). Peak plasma TNF levels were not dose-dependent (> 8 ng/ml), but plasma TNF was always detectable (> 0.2 ng/ml at 10 h post-intravenous dose). Intravenous LPS administration at 0 h also blocked pleocytosis, but the inhibitory effect was lost when administration at -8 h. In conclusion, the degree and duration of endotoxaemia affect the meningeal inflammatory response to LPS in experimental meningitis.     

Insulin-like growth factor I and pulmonary hypertension induced by continuous air embolization in sheep.

Chronic pulmonary hypertension is associated with arterial structural remodeling. Insulin-like growth factor I (IGF-I) has been proposed as one of the mediators of vascular change because of its ability to stimulate proliferation in, and elastin production by, cultured vascular smooth muscle cells. We have shown previously that 12 days of continuous air embolization into the pulmonary arterial circulation of sheep results in the functional and structural changes of chronic pulmonary hypertension. In the present study, measurements of IGF-I (by radioimmunoassay) and IGF-I binding protein activity in sheep lung lymph and plasma were made before and during the 12 days of air embolization in six sheep. Two untreated animals served as controls. Baseline lung lymph contained 23.5 +/- 3.6 ng/ml (mean +/- SEM) of IGF-I, and there was a slight increase to 36.7 +/- 9.8 on day 3, but by day 6 levels were back to baseline. The flux of IGF-I from the lung (concentration times lymph flow) increased significantly by day 2 embolization and remained elevated through day 12 (baseline = 37.2 +/- 11.1 ng/15 min; day 2 = 237.7 +/- 55.8; day 5 = 190.2 +/- 53.4; day 6 = 82.6 +/- 21.9; day 12 = 78.7 +/- 12.5). IGF-I binding protein activity was also present in lung lymph at baseline (29.6 +/- 3.0%) and was unchanged during air embolization. Plasma levels of IGF-I and plasma binding protein activity remained at baseline throughout the 12 days of embolization (71.51 +/- 34.48 ng/ml and 36.4 +/- 3.5%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effectiveness of autologous preparation rich in growth factors for the treatment of chronic cutaneous ulcers

Autologous Preparation Rich in Growth Factors (PRGF), a small volume of plasma enriched in platelets, is a novel therapeutic strategy for the acceleration of the wound healing of a wide range of tissues because of the continuous release of multiple growth factors, including PDGF-AB, TGF-beta1, IGF-I, HGF, VEGF-A, and EGF. In this article, we have characterized the PRGF preparation and designed a randomized open-label controlled pilot trial to evaluate the effectiveness of PRGF in the treatment of chronic cutaneous ulcers. Results showed that at 8 weeks, the mean percentage of surface healed in the PRGF group was 72.94% +/- 22.25% whereas it was 21.48% +/- 33.56% in the control group (p < 0.05). These results, with the limitations of a pilot study, suggest that topical application of PRGF is more effective than standard therapy in helping a chronic ulcer to heal.     

Platelet and growth factor concentrations in activated platelet-rich plasma: a comparison of seven commercial separation systems

Platelet-rich plasma (PRP) is blood plasma that has been enriched with platelets. It holds promise for clinical use in areas such as wound healing and regenerative medicine, including bone regeneration. This study characterized the composition of PRP produced by seven commercially available separation systems (JP200, GLO PRP, Magellan Autologous Platelet Separator System, KYOCERA Medical PRP Kit, SELPHYL, MyCells, and Dr. Shin’s System THROMBO KIT) to evaluate the platelet, white blood cell, red blood cell, and growth factor concentrations, as well as platelet-derived growth factor-AB (PDGF-AB), transforming growth factor beta-1 (TGF-β1), and vascular endothelial growth factor (VEGF) concentrations. PRP prepared using the Magellan Autologous Platelet Separator System and the KYOCERA Medical PRP Kit contained the highest platelet concentrations. The mean PDGF-AB concentration of activated PRP was the highest from JP200, followed by the KYOCERA Medical PRP Kit, Magellan Autologous Platelet Separator System, MyCells, and GLO PRP. TGF-β1 and VEGF concentrations varied greatly among individual samples, and there was almost no significant difference among the different systems, unlike for PDGF. The SELPHYL system produced PRP with low concentrations of both platelets and growth factors. Commercial PRP separation systems vary widely, and familiarity with their individual advantages is important to extend their clinical application to a wide variety of conditions.     

Differential effects of growth factors on tissue-engineered cartilage

The effects of four regulatory factors on tissue-engineered cartilage were examined with specific focus on the ability to increase construct growth rate and concentrations of glycosaminoglycans (GAG) and collagen, the major extracellular matrix (ECM) components. Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured in medium with or without supplemental insulin-like growth factor (IGF-I), interleukin-4 (IL-4), transforming growth factor-beta1 (TGF-beta1) or platelet-derived growth factor (PDGF). IGF-I, IL-4, and TGF-beta1 increased construct wet weights by 1.5-2.9-fold over 4 weeks of culture and increased amounts of cartilaginous ECM components. IGF-I (10-300 ng/mL) maintained wet weight fractions of GAG in constructs seeded at high cell density and increased by up to fivefold GAG fractions in constructs seeded at lower cell density. TGF-beta1 (30 ng/mL) increased wet weight fractions of total collagen by up to 1.4-fold while maintaining a high fraction of type II collagen (79 plus minus 11% of the total collagen). IL-4 (1-100 ng/mL) minimized the thickness of the GAG-depleted region at the construct surfaces. PDGF (1-100 ng/mL) decreased construct growth rate and ECM fractions. Different regulatory factors thus elicit significantly different chondrogenic responses and can be used to selectively control the growth rate and improve the composition of engineered cartilage.     

Serum insulin-like growth factors (IGFs) and IGF binding protein (IGFBP)-3 in patients with gastric cancer: IGFBP-3 protease activity induced by surgery.

Recent findings have indicated that insulin-like growth factors (IGF-I and IGF-II) may play a role in neoplasia. Alteration of serum IGFs or IGF Binding Proteins (IGFBPs) have been reported in some tumors. In this study, we measured serum IGF-I, IGF-II and IGFBPs profile in gastric cancer by radioimmunoassay and Western ligand blots. The serum IGF-I level in gastric cancer was significantly lower than in control subjects (65.2 +/- 26.5 vs 148.4 +/- 55.2 ng/ml, p < 0.01) and was further decreased to 45.5 +/- 20.9 ng/ml after surgery. The serum IGF-II level was slightly higher than that in control subjects (826.3 +/- 360.2 vs 735.7 +/- 154.6 ng/ml) but it was significantly decreased after surgery (525.7 +/- 220.1 ng/ml, p < 0.05). The serum IGFBP-3 level was not significantly different from those in control subjects. However, we observed a decreased level of serum IGFBP-3 after surgery, and incubation of postoperative serum with control serum resulted in a significant reduction of IGFBP-3 level. The reduction of IGFBP-3 in postoperative serum was mainly due to surgery associated IGFBP-3 protease activity. This protease activity was totally inhibited by aprotinin, EDTA and PMSF but not by pepstatin and leupeptin. This inhibition pattern is consistant with cation dependent serine protease. We speculate that proteolysis of IGFBP-3 may contribute to increase the bioavailability of IGFs.     

Transforming growth factor-beta synthesis by human peritoneal mesothelial cells. Induction by interleukin-1.

Peritoneal mesothelial cells are uniquely located to regulate cellular events in the peritoneal cavity and are a potentially important source for various cytokines. The present study was designed to elucidate the capacity of human peritoneal mesothelial cells (HPMCs) to synthesize and secrete the transforming growth factor (TGF)-beta isoforms 1, 2, and 3 and to characterize their regulation by inflammatory cytokines. HPMCs constitutively released appreciable amounts of TGF-beta 1 and low amounts of TGF-beta 2 as detected by specific immunoassays. TGF-beta 1 levels secreted within 48 hours (45 +/- 8.9 pg/10(5) cells) were 60-fold higher than TGF-beta 2 levels (0.9 +/- 0.1 pg/10(5) cells), respectively. Treatment of HPMCs with interleukin (IL)-1 beta (10 ng/ml) resulted in a significant increase of both TGF-beta 1 (mean, 5-fold; P < 0.001) and TGF-beta 2 (mean, 6-fold; P < 0.01) generation. After 48 hours of IL-1 beta treatment the levels were 185 +/- 17.1 pg/10(5) cells for TGF-beta 1 and 5.3 +/- 1.5 pg/10(5) cells for TGF-beta 2, respectively. Neither tumor necrosis factor (TNF)-alpha nor interferon (IFN)-gamma (both 10 ng/ml) affected TGF-beta 1 or TGF-beta 2 synthesis by HPMCs. TGF-beta 3 could not be detected in any of the supernatant media. Stimulation of HPMCs with IL-1 beta increased steady-state levels of TGF-beta 1- and TGF-beta 2-specific mRNA. Western blot analysis of supernatants revealed the presence of an immunoreactive band at 25 kd. Indirect competition assays confirmed receptor-binding activity of HPMC-derived TGF-beta. Appreciable amounts of TGF-beta were present in a bioactive form. Our results demonstrate that HPMCs synthesize the TGF-beta isoforms 1 and 2 and that the levels of mRNA and protein release can be up-regulated by the proinflammatory cytokine IL-1 beta.     

Progesterone production by human granulosa cells cultured in serum free medium: effects of gonadotrophins and insulin-like growth factor I (IGF-I).

There is evidence that insulin-like growth factor I (IGF-I) is a potent regulator of oestradiol synthesis by human granulosa and luteal cells; however, the question of whether IGF-I regulates progesterone synthesis by these cell types has yet to be answered. As a first step towards this goal, we have compared the effects of IGF-I, follicle stimulating hormone (FSH), and human chorionic gonadotrophin (HCG) on progesterone production by human granulosa cells obtained from individual dominant and cohort follicles, and granulosa luteal cells from preovulatory follicles of patients undergoing in-vitro fertilization (IVF). Granulosa cells from normal, unstimulated follicles cultured in serum-free medium as controls (no additions) produced some progesterone spontaneously. In all cases, FSH stimulated basal progesterone levels (10-fold average increase) and the effect was dose-dependent (ED50 of FSH = 9.1 +/- 3.9 ng/ml). Similar effects were observed when granulosa cells from large follicles were incubated with HCG (ED50 of HCG = 6.9 +/- 2.8 ng/ml). By comparison, the effects of IGF-I on progesterone production were not marked, being absent in 80% of the follicles tested. However, granulosa cells from healthy follicles co-incubated with IGF-I and FSH or HCG produced more progesterone compared with cells treated with the gonadotrophins alone; this effect of IGF-I was dose dependent (ED50 of IGF-I = 10 ng/ml). When the effect of each agonist was tested on IVF granulosa luteal cells, HCG but not FSH or IGF-I stimulated basal progesterone levels but the HCG effect required a two-day lag phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comprehensive analysis of chemotactic factors for bone marrow mesenchymal stem cells

To understand which growth factors/cytokines can affect migration of mesenchymal stem cells (MSCs) to injured tissues, we compared the effects of many (26) growth factors/cytokines on the migration activity of rabbit and human MSCs using a microchemotaxis chamber. Among them, platelet-derived growth factor (PDGF)-BB, PDGF-AB, epidermal growth factor (EGF), HB-EGF, transforming growth factor (TGF-alpha), insulin growth factor (IGF-I), hepatocyte growth factor (HGF), fibroblast growth factor (FGF-2), and thrombin consistently enhanced the migration of rabbit and human MSCs at appropriate concentrations. PDGF-BB showed the greatest effect on migration. Various combinations of these factors further enhanced the migration of MSCs, whereas combinations of factors that shared common cell-surface receptors did not induce the additive stimulation. On the other hand, some combinations, including that of FGF-2 or thrombin with PDGF-BB, suppressed the migration activity of MSCs. These findings suggest that combinations of growth factors are important to eliciting the maximal chemotactic effect. The factors that induced the migration of MSCs also enhanced their proliferation, suggesting that migration and proliferation can take place simultaneously. The above factors were also effective in stimulating the migration of fibroblasts, but thrombin alone selectively enhanced the migration of MSCs, suggesting that thrombin is useful to stimulate migration of MSCs without migration of fibroblasts.     

Platelet-rich plasma: quantification of growth factor levels and the effect on growth and differentiation of rat bone marrow cells

Platelet-rich plasma (PRP) is a new application of tissue engineering and a developing area for clinicians and researchers. It is a storage vehicle of growth factors (GFs) such as platelet-derived growth factor (PDGF)- AA, -BB, -AB; transforming growth factor (TGF)-beta1 and -2; platelet-derived epidermal growth factor (PDEGF); platelet-derived angiogenesis factor (PDAF); insulin growth factor-1 (IGF-1); and platelet factor- 4 (PF-4), which are known to influence bone regeneration. However, animal and clinical studies reveal different results with the use of PRP and its effect on bone healing. This could be due to the differences between species, that is, differences between species in GF concentrations or variation in presence of GFs between the various PRPs. In this study, rat bone marrow cells were cultured in PRP-coated wells or in uncoated wells for 16 days in osteogenic medium, and analyzed on cell growth (DNA content) and cell differentiation (alkaline phosphatase [ALP] activity, calcium content, scanning electron microscopy, and QPCR). The concentrations of TGF-beta1, PDGF-AA, PDGF-AB, and PDGF-BB in rat, goat, and human PRP were subsequently determined. The results showed that PRP stimulated initial cell growth and had no effect on ALP activity. The calcium measurements showed a significant increase in calcium at days 8, 12, and 16. The real-time PCR results showed that PRP upregulated osteocalcin at day 1 and collagen type I at day 8. Overall, the immunoassays revealed that human PRP contained higher concentrations of growth factors per platelet compared to rat and goat PRP. Goat PRP showed higher concentrations of growth factors per platelet as compared to rat PRP except for PDGF-BB, which had a higher concentration in rat PRP. TGF-beta1 was the most abundant growth factor in all 3 PRPs. On the basis of our results, we conclude that platelet-rich plasma contains osteo-inductive growth factors, which are probably species related. However, we cannot generalize the results because of large intraspecies variations. Further, we conclude that rat PRP gel stimulates initial growth and differentiation of rat bone marrow cells in vitro.