Rat Costal Cartilage Research Articles

Construction of tissue-engineered cartilage with different biological scaffolds in vitro

This study aims to find out which polymeric scaffolds were more appropriate for the construction of tissue engineering cartilage. Rat costal cartilage chondrocytes were cultured in vitro. The first generation of chondrocytes was used as seed cells and inoculated in chitosan/gelatin and bone matrix gelatin (BMG)/fibrin glue scaffolds. Haematoxylin and eosin staining, toluidine blue staining, safranine O, immunological detection, and scanning electron microscopy (SEM) observations were implemented. After2 weeks, all stains of BMG/fibrin glue showed that chondrocytes were uniformly distributed on the surface and inside of the material, and collagen and type II collagen staining was positive. There were slightly more cells on chitosan/gelatin than on BMG/fibrin glue. However, the number of internal cells was small with non-uniform distribution and the staining result was not as apparent as that of BMG/fibrin glue. As the culture time lengthened, results displayed a large quantity of expression of specific collagen and typed II collagen of chondrocytes, with chitosan/gelatin showing obvious advantages over BMG/fibrin glue material. The chitosan/gelatin contributed to maintaining chondrocyte phenotype and secretion of the extracellular matrix. Tissue engineering cartilage was successfully constructed in vitro. Moreover, such tissue growth, proliferation and matrix secretion of chondrocytes on chitosan/gelatin were superior to those on BMG/fibrin glue scaffold.

Utility of a rotation/revolution-type agitator for chondrocyte isolation during preparation of engineered cartilage

During the manufacture of cell- and tissue-based products, such as engineered cartilage for autologous chondrocyte implantation, maximizing the number of cells isolated from donor tissue substantially improves the productivity of these products. The method used for agitating tissues with digestive fluid and enzymes can considerably affect both the quality and quantity of isolated cells. This study aimed to investigate the effectiveness of a rotation/revolution-type agitator for chondrocyte isolation following the enzymatic digestion of rat costal cartilage. Cartilage tissue cut into 1mm3-thick sections was equally divided between two groups and placed in 50-mL conical tubes; sections in both groups were digested using 0.1mg/mL liberase TH (collagenase/thermolysin) at 37°C for 4h with either rotation/revolution or conventional orbital agitation method. Compared with using conventional orbital agitator, using the rotation/revolution-type agitator resulted in a significant (>two-fold) increase in the number of isolated cells. In subsequent primary cultures, chondrocytes obtained by rotation/revolution agitation showed superior initial attachment to tissue culture dish on day 1 and 2 compared with those obtained by conventional agitation; however, no differences in cell proliferation or cartilage-related molecule expression patterns were observed between cells derived from either method after 3 days of subculture. These findings suggested that there are no disadvantages to the proposed rotation/revolution agitation method. Rotation/revolution-type agitators are a promising apparatus for preparing chondrocytes for primary cultures and cartilage tissue engineering.

The N-Terminal Cleavage of Chondromodulin-I in Growth-Plate Cartilage at the Hypertrophic and Calcified Zones during Bone Development

Chondromodulin-I (ChM-I) is a 20–25 kDa anti-angiogenic glycoprotein in cartilage matrix. In the present study, we identified a novel 14-kDa species of ChM-I by immunoblotting, and purified it by immunoprecipitation with a newly raised monoclonal antibody against ChM-I. The N-terminal amino acid sequencing indicated that it was an N-terminal truncated form of ChM-I generated by the proteolytic cleavage at Asp37-Asp38. This 14-kDa ChM-I was shown by the modified Boyden chamber assay to have very little inhibitory activity on the VEGF-A-induced migration of vascular endothelial cells in contrast to the intact 20–25 kDa form of ChM-I (ID50 = 8 nM). Immunohistochemistry suggested that 20–25 kDa ChM-I was exclusively localized in the avascular zones, i.e. the resting, proliferating, and prehypertrophic zones, of the cartilaginous molds of developing long bone, whereas the 14-kDa form of ChM-I was found in hypertrophic and calcified zones. Immunoblotting demonstrated that mature growth-plate chondrocytes isolated from rat costal cartilage actively secrete ChM-I almost exclusively as the intact 20–25 kDa form into the medium in primary culture. Taken together, our results suggest that intact 20–25 kDa ChM-I is stored as a component of extracellular matrix in the avascular cartilage zones, but it is inactivated by a single N-terminal proteolytic cleavage in the hypertrophic zone of growth-plate cartilage.

Incomplete digestion preserves chondrocytes from dedifferentiating in long-termed culture on plastic substrate

Epiphyseal pieces from young rat's costal cartilage were predigested for 30 min by hyaluronidase then digested by collagenase for 1 h with gentle beating applied. Resulted grape-like chondrocytes connecting with the residual cartilage matrix were seeded in plastic culture dishes and 4 passages at about 12-days interval were carried out. Morphological observations were performed daily. Compared with completely isolated chondrocytes at the same passage, detection for collagen II, integrin-β 1 and focal adhesion kinase by immunochemistry staining, Western Blot and RT-PCR were performed to evaluate the preservation of chondrocytic phenotype and cellular functions. Primary chondrocytes isolated by complete enzymatic digestion served as control. Completely isolated chondrocytes in the monolayer culture were ready to lose the chondrocytic phenotype marked by the down-regulation of collagen II secretion and specific morphological alterations which were characterized as the cells gradually became long and spindle-like from their originally rounded shape. In case of the incompletely digested chondrocytes, the expression of collagen II was stable during the whole experiment while extensive cell–cell contacts and matrix–cell connections were observed. Transcription and expression of integrin-β 1 and FAK were active and paracrine of BMP-7 was positive. These results suggested stable chondrocytic phenotype. Conclusionly, by the incomplete digestion method, the requisite time for enzymatic isolation was reduced and chondrocytes with residual matrix were harvested instead of mono-cell suspension. Compared with the novel techniques, the incomplete digestion shortened the enzymatic procedure greatly and simplified the subculturing operations with less financial cost. Especially, as extracellular matrix was preserved, chondrocytes expressed stable phenotype in a rather long-termed culture.

Growth hormone axis overview--somatomedin hypothesis.

The possibility that the action of growth hormone (GH) on cartilage is mediated by a separate hormonal agent found in serum was suggested by incubation with hypophysectomized rat costal cartilage. The stability of this tissue permitted long incubations and the measurement of the uptake of 35S-sulfate provided a convenient index of growth stimulation. Under the conditions arbitrarily selected, normal rat serum, but not serum from hypophysectomized rats, induced a great stimulation of 35S uptake. In contrast, GH added directly to cartilage in these incubations was virtually inactive. It was suggested that a serum sulfation factor, now known as insulin-like growth factor-I (IGF-I), was a mediator of GH action. Recently it has been observed that addition of 35S-sulfate after 24 h of preincubation with GH permitted the direct effect of GH to be recognized. Other observations in intact hypophysectomized rats have established that GH can induce the expression of IGF-I in cartilage that acts in an autocrine-paracrine manner. The relative importance of the endocrine and autocrine-paracrine routes of IGF-I action on the growth of cartilage is in dispute. It is clearly established that serum IGF-I exerts a negative feedback on GH secretion by action on the hypothalamus and pituitary. Serum IGF-I concentrations reflect GH action in postnatal life. Measurement of serum IGF-I is the most-valuable index of GH hypersecretion in acromegaly and in conditions of growth impairment. GH receptor deficiency leads to a marked decrease in circulating IGF-I. Hypernutrition and hyperinsulinism of obesity directly promote hepatic IGF-I release and inhibit GH secretion by the pituitary. Differences in hepatic IGF-I synthesis in response to GH may contribute to physiological differences in stature.

The role of hepatocyte growth factor in growth plate cartilage

To investigate the physiological role of hepatocyte growth factor (HGF) in endochondral bone formation, we examined the expression of HGF and its receptor c-met and the effects of HGF on growth plate chondrocytes. HGF was highly expressed in the prehypertrophic zone and hypertrophic zone in rat costal growth plate cartilage. The expression of HGF increased in rabbit chondrocytes as they matured in culture. Conversely, c-met expression was down regulated along maturation of growth plate chondrocytes. HGF had weak stimulatory effects on DNA and proteoglycan synthesis of growth plate chondrocytes. However, HGF strongly inhibited expression of terminal differentiation-related phenotypes, such as type X collagen and alkaline phosphatase (APase) synthesis and cartilage matrix mineralization. When HGF was removed from the cultures, cells quickly expressed type X collagen and APase. Once chondrocytes differentiated to mature chondrocytes, HGF did not inhibit further differentiation of these cells. These results suggested that HGF is a negative regulator of terminal differentiation of growth plate chondrocytes..

Administration of growth hormone modulates the gene expression of basic fibroblast growth factor in rat costal cartilage, both in vivo and in vitro

We examined the effect of growth hormone on local growth factor mRNA expression in male Sprague-Dawley rats. Repetitive systemic administration of growth hormone (0.4 IU every 4 h) increased the expression of IGF-I mRNA up to 2.8-fold in costal cartilage tissue compared with controls. Basic FGF (bFGF) mRNA expression gradually increased up to 15.5-fold compared with pre-injection samples, where the mRNA expression was 5.3-times greater than vehicle-injected controls. TGF-β mRNA showed little changes. Moreover, one μg/ml of growth hormone enhanced the expression of bFGF mRNA in costal chondrocytes in culture. We conclude that growth hormone increased the local expression of bFGF, as well as that of IGF-I, in cartilage, and suggest that bFGF is directly regulated by growth hormone within a local area.

Effect of insulin on the altered production of proteoglycans in rib cartilage of experimentally diabetic rats

Costal cartilage from experimentally diabetic rats, labeled in vivo or in vitro with [ 35S]sulfate, was shown to incorporate less label into proteoglycans than cartilage from nondiabetic rats. Analyses of guanidine HCl cartilage extracts by gel chromatography on Sepharose CL-2B showed two major peaks at K av ∼ 0.4 and 0.8 (peaks I and II, respectively). Cartilage extracts from the diabetic rats contained predominantly peak II proteoglycans, while 60 and 55%, respectively, of the total 35S-labeled proteoglycans extracted from control cartilage labeled in vivo and in vitro with [ 35S]sulfate were present in peak I. After insulin treatment of the diabetic rats, the relative amount of peak I 35S-labeled proteoglycans synthesized in vivo was increased to 70%. The overall in vivo incorporation of [ 35S]sulfate into proteoglycans was also stimulated in diabetic rats treated with insulin to levels above those found for control rats. Thus, diabetes-induced changes in the biosynthesis of rat costal cartilage proteoglycans may be alleviated by normalization of the diabetic state by insulin treatment. However, addition of insulin (10 −5–10 −9 m) to the culture medium did not affect the amount of 35S-labeled proteoglycans synthesized in vitro or the relative amounts of peak I proteoglycans produced by control or diabetic cartilage, suggesting that insulin does not have a direct effect on proteoglycan production. Moreover, no decrease in the amount of 35S-labeled proteoglycans produced was found when glucose at high concentrations was present in the culture medium. However, the presence of rat serum resulted in an increase in the amount of 35S-labeled proteoglycans produced by both control and diabetic cartilage, demonstrating that the cartilage explants were metabolically responsive to stimulatory factors.

Hemoglobin as a direct inhibitor of cartilage growth in vitro.

Growth retardation is a feature of several diseases associated with chronic hemolysis (i.e., uremia and the hemoglobinopathies). Although the growth failure is undoubtedly multifactorial, circulating hemoglobin (Hb) may inhibit cartilage growth directly. We tested this hypothesis using the hypophysectomized rat costal cartilage sulfation bioassay and the embryonic chicken pelvic rudiment bioassay, both very sensitive to growth factors and growth inhibitors. In the rat bioassay, Hb produced a dose-dependent inhibition of both basal and normal rat serum (NRS)-stimulated 35SO4 uptake. In the chick bioassay, NRS stimulated cartilage growth as expected, but Hb severely inhibited both basal and NRS-stimulated growth. However, after the cartilages were preincubated with Hb for 2 days, subsequent exposure to NRS allowed them to resume growth at the same rate as cartilage exposed to NRS for the entire 5 days. The growth inhibition could be accounted for by the heme contained in Hb. We conclude that Hb produces a dose-dependent and reversible inhibition of cartilage growth and may contribute to the growth retardation associated with chronic hemolytic conditions.

Tetrapyrroles as inhibitors of normal cartilage metabolism: relative potency of different compounds.

The present study was designed to explore the role of different tetrapyrroles as inhibitors of cartilage metabolism. We studied the effects of tetrapyrroles on the incorporation of [35S]sulfate into proteoglycans, [14C]-leucine into protein, and [3H]uridine into the RNA of normal cartilage from two different vertebrate classes using the embryonic chicken pelvic rudiment bioassay and the hypophysectomized rat costal cartilage bioassay, both very sensitive to cartilage growth factors and growth inhibitors. We compared the relative potencies of the following compounds: both metalloporphyrins (heme and chlorophyllin), linear tetrapyrroles (bilirubin), and heme proteins (hemoglobin, myoglobin, and cytochrome c). Hemoglobin and heme were the most potent inhibitors of rat cartilage metabolism, and bilirubin was a far more potent inhibitor of embryonic chick cartilage metabolism. Chlorophyllin had moderate inhibitory activity, especially on chick cartilage, whereas cytochrome c was inactive in these bioassays. Surprisingly, myoglobin was relatively ineffective despite its close similarity to heme and hemoglobin. The bilirubin-induced inhibition of sulfate incorporation into chick cartilage was partially prevented when glutathione was included in the incubation medium, suggesting that a free-radical mechanism may be involved. There were significant differences in the sensitivity of the two cartilages studied, indicating there may be species-dependent sensitivity to different tetrapyrroles.

Absence of keratan sulphate from skeletal tissues of mouse and rat.

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.

Platelet-derived growth factor and multiplication-stimulating activity II, but not multiplication-stimulating activity III-2, stimulate [3H]thymidine and [35S]sulphate incorporation by fetal rat costal cartilage in vitro.

The actions of partially purified porcine platelet-derived growth factor (PDGF) and highly purified multiplication-stimulating activity (MSA) II and MSA III-2, which are somatomedins, were investigated on the incorporation of [3H]thymidine and [35S]sulphate by fetal rat costal cartilage in vitro. This was compared with their effects in the presence of 1% fetal calf serum (FCS) on the uptake of thymidine by growth-arrested fetal rat fibroblasts. Platelet-derived growth factor at concentrations of 0.21-21 micrograms/l enhanced the incorporation of both isotopes by fetal cartilage in the presence of 1% FCS, but had an inconsistent action on thymidine uptake and no significant action on sulphate uptake in serum-free medium. Platelet-derived growth factor promoted thymidine uptake by growth-arrested, isolated fetal rat fibroblasts. Multiplication-stimulating activity II (10-100 micrograms/l) stimulated the uptake of thymidine and sulphate by fetal cartilage in medium containing 1% FCS but had no consistent action in serum-free medium, although MSA II and PDGF had a synergistic effect on thymidine uptake in the absence of serum. Multiplication-stimulating activity III-2 had no consistent action on thymidine or sulphate incorporation by fetal cartilage in either serum-free or serum-supplemented medium. However, the same preparation of MSA III-2 stimulated the uptake of [3H]thymidine into fetal rat fibroblasts with a half-maximal response at a concentration of 5-10 micrograms/l. The results identify PDGF as a possible mitogenic agent for fetal rat connective tissues in vitro and show a differential sensitivity of fetal cartilage to MSA peptides.

Insulin-like growth factor characteristics of an acidic non-suppressible insulin-like activity.

The biological activities of an acidic form of non-suppressible insulin-like activity (ILA pI 4.8) have been studied. ILA pI 4.8 was isolated from Cohn fraction IV-1 of human serum by pH 5.5 ion-exchange chromatography on SP-Sephadex. Carrier-bound ILA was eluted at pH 9.7 and then sequentially gel chromatographed in 1% formic acid on Sephadex G-75 and Bio-Gel P-30. The low-Mr (7000) active material was subjected to flat bed isoelectric focusing. Overall recovery was 87 munit of insulin equivalents/100 g of Cohn fraction IV-1, with a specific activity in the range 4-10 munit/mg of protein, representing a purity of 1-6%. This material has been tested in a variety of insulin-like growth factor (IGF)/somatomedin assay systems. It stimulated, in a dose-related manner, [14C]glucose conversion into lipid by isolated rat adipocytes, 35SO4(2-) incorporation into weanling rat costal cartilage and [3H]thymidine incorporation into DNA of cultured human fibroblasts. Like IGF-I and -II, ILA pI 4.8 was able to inhibit degradation of 125I-insulin by crude homogenates of rat liver. In addition, the biological activity of ILA pI 4.8 was completely suppressible by a recently described inhibitor of IGF-I and IGF-II. ILA pI 4.8 was able to compete, in a parallel manner, with 125I-IGF-I and 125I-IGF-II and, at higher doses, with 125I-insulin in a placental radioreceptor assay. No cross-reactivity was seen in a radioimmunoassay for IGF-I and -II C-peptides, but at higher concentrations parallel displacement was observed in a somatomedin C/IGF-I radioimmunoassay using two different antisera. These data indicate that ILA pI 4.8 does possess many of the biological activities previously reported for the IGFs. Since ILA pI 4.8 does occur naturally in serum, it would appear reasonable to tentatively include it as one of the IGF/somatomedin family.

Somatomedin inhibitor in uremia.

In uremia, poor growth occurs despite normal to increased levels of insulin and GH. Since serum somatomedin levels measured by RIA and radioreceptor assay are normal in patients with renal failure, while serum somatomedin activity measured by bioassay is low but increased by dialysis, we asked if somatomedin activity could be decreased due to the presence of a low mol wt inhibitor(s). Serum was obtained from eight normal adults and eight uremic patients before hemodialysis treatment and was fractionated by gel filtration. Somatomedins and high mol wt inhibitors were separated on Sephadex G-50, pH 2.4, and high and low mol wt inhibitors were separated on Sephadex G-25, pH 7. Somatomedins were measured by stimulation of SO4 uptake by hypophysectomized rat costal cartilage in vitro, and inhibitor levels were determined by the blunting of stimulation produced by somatomedins in normal serum. Total biologically active somatomedin levels were comparable in uremic and normal sera. High mol wt somatomedin inhibitors (as found in malnutrition and diabetes) also were detected at similar levels in uremic and normal sera. In contrast, serum from uremic patients had increased levels of a low mol wt somatomedin inhibitor(s) [151 +/- 23% (mean +/- SEM) of serum stimulation inhibited vs. 47 +/- 9%; P less than 0.001]. Peak inhibitory activity was found at approximately 940 mol wt (range, 800-1100); an inhibitor of similar size was found in normal urine. Uremic serum fractions blunted cartilage sulfate uptake that was stimulated by whole serum, somatomedins (dissociated from serum carrier proteins), and insulin and lowered uridine and thymidine uptake that was stimulated by whole serum (all P less than 0.005). Lineweaver-Burk analysis indicated that somatomedin-inhibitor interactions on cartilage were noncompetitive, consistent with observations that direct exposure of cartilage to inhibitor decreased SO4 uptake to 30 +/- 3% below buffer levels (P less than 0.001). Despite these marked effects on cartilage, no alterations in basal or insulin-stimulated glucose oxidation occurred after addition of inhibitory serum fractions to adipose tissue incubations. Exposure of the inhibitor to proteolytic enzymes led to a significant decrease in inhibitory activity, indicating that the inhibitor may be a peptide. These studies suggest that decreased circulating somatomedin activity and impaired growth in uremia may reflect the accumulation of a circulating peptide inhibitor that would normally be cleared by the kidneys. Measurements of this factor may provide an index of growth potential in uremic children and help guide therapy of renal failure in both children and adults.

Cultured fetal rat myoblasts release peptide growth factors which are immunologically and biologically similar to somatomedin

The production of immunologically and biologically active somatomedin activity from isolated myoblasts and fibroblasts from fetal rats of 21 days gestational age was investigated. Myoblast-rich cell populations were derived from primary cultures of dispersed muscle cells by the tendency of myoblasts to become detached from the culture dish in the presence of cytochalasin B. Fibroblasts were obtained from fetal muscle. Culture medium conditioned by exposure to myoblasts for 48 hours produced an increased incorporation of both [35S]sulphate and [3H]thymidine by explants of fetal rat costal cartilage in vitro compared to fresh medium. Myoblast-conditioned medium also contained somatomedin-C-like immunoreactivity which diluted in parallel with partially purified human somatomedin-C (3,271 +/- 446 mU/mg cell protein; mean +/- SEM, seven experiments). Medium conditioned by exposure to fetal rat fibroblasts did not promote isotope uptake by fetal rat cartilage above control values, and contained only low levels of somatomedin-C-like immunoreactivity (343 +/- 89 mU/mg cell protein, three experiments). The release of both somatomedin bioactivity and immunoreactivity into conditioned medium was significantly reduced by the incubation of myoblasts in the presence of rat growth hormone (100 ng/ml and 500 ng/ml). We conclude that fetal rat myoblasts released growth factor activity during culture which exhibited biological and immunologic characteristics of somatomedin. Since the bioactivity was demonstrated on skeletal tissues from rat fetuses of the same gestational age as those that yielded myoblasts such growth factor release may be physiological.

Metabolic regulation of somatomedin activity in rat liver.

We have compared circulating and hepatic somatomedin (SM) activity in rats with diabetes or malnutrition of varying severity. Somatomedin activity was measured by the hypophysectomized rat costal cartilage bioassay. In both moderate diabetes and moderate malnutrition, mean serum SM activity was not significantly lower than normal (79 +/- 13% (S.E.M.) and 95 +/- 11% vs normal controls respectively). In contrast, liver perfusate SM activity was significantly reduced in both groups (51 +/- 12% for moderate diabetes and 44 +/- 12% for moderate malnutrition). Liver extract SM activity was also significantly decreased in both moderate diabetes and malnutrition (74 +/- 4% and 75 +/- 6% vs normal controls respectively). In severe diabetes and malnutrition, both liver and serum activities were low, consistent with previous reports. Our studies showed that liver SM activity fell in response to metabolic stress before a decrease in circulating levels occurred, supporting the concept that the liver regulates serum SM activity and growth in diabetes and malnutrition.

Nutrition and Somatomedin. XI. Studies of Somatomedin Inhibitors in Rats with Streptozotocin-induced Diabetes

Diabetics may have normal somatomedins by radioimmunoassay yet decreased somatomedin activity by bioassay. The discrepancy appears due to circulating inhibitory factors; inhibitors in whole diabetic serum antagonize the action of both somatomedins and insulin via noncompetitive interactions. Since little is known about the nature of the inhibitory activity, serum from streptozotocin-diabetic rats was fractionated, and inhibitory activity measured as the ability of fractions to blunt stimulation of SO4 uptake by hypophysectomized rat costal cartilage exposed in vitro to somatomedins in normal rat serum. After establishing that inhibitory activity was stable to pH and lyophilization, diabetic rat serum was gel filtered at pH 7 (somatomedins bound to carrier proteins) and 2.4 (somatomedins dissociated, mol wt approximately 8000). Using Sephadex and Sephacryl columns at neutral pH, inhibitors were detected at mol wt approximately 250,000, approximately 24,000, and approximately 940. Predominant activity was at approximately 24,000 and approximately 940. In contrast, Sephadex columns at acid pH revealed inhibitors only at mol wt approximately 21,000. Diabetic rat serum was also subjected to ion-exchange chromatography on CM-Sepharose. A single band of activity at pH 5-7 was found on elution with increasing pH, suggesting an isoelectric point(s) lower than that of the somatomedins. However, three areas of activity were seen on elution with increasing ionic strength at pH 5-at 0.02 M and 0.14-1.4 M and at 2.0 M pH 5.0-5.5. These studies indicate that reduced anabolism in diabetes may be due in part to three species of circulating somatomedin inhibitors, largely of mol wt approximately 21-24,000 and approximately 940, and also of mol wt approximately 250,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased thymidine incorporation into fetal rat cartilage in vitro in the presence of human somatomedin, epidermal growth factor and other growth factors.

The incorporation of [3H]thymidine by rat costal cartilage in vitro was studied at different fetal and postnatal ages and the effect of partially purified human somatomedin, mouse epidermal growth factor, platelet secretion products, insulin and growth hormone on thymidine uptake by fetal cartilage was examined. Thymidine uptake in plasma-free medium was many times greater in late fetal life than after birth. The incorporation of [3H]thymidine into costal cartilage from 21-day fetuses was significantly (P less than 0.05) increased above control values in the presence of 10 micrograms somatomedin/1, and when cartilage was incubated in medium containing somatomedin and diluted human plasma there was a synergistic action. Epidermal growth factor at a concentration of 1 ng/l was a potent stimulator of thymidine uptake. Secretion products from human platelets after their aggregation by thrombin stimulated [3H]thymidine uptake at a concentration of 2% (v/v), but were inhibitory at high concentrations. High concentrations of platelet secretion products stimulated the incorporation of [35S]sulphate by cartilage. A pharmacological concentration of 10 mu. insulin/ml stimulated [3H]thymidine uptake, but not concentrations of 1 or 100 mu./ml. Growth hormone had no effect. The results showed that fetal cartilage had a greater endogenous mitogenic activity than postnatal cartilage. While somatomedins may be important in the regulation of fetal body growth, other protein growth factors also stimulate fetal skeletal tissues.

Bovine growth hormone fragment (1-133) has in-vitro somatomedin-like activity.

Thrombin digestion of bovine growth hormone (1-191) resulted in cleavage of the peptide bond between amino acid residues 133 and 134. Native growth hormone and purified peptides (1-133) and (134-191) were assayed for somatomedin-like activity. Peptide (1-133), ranging in concentration from 0.15-15 nmol/l, stimulated in-vitro uptake of [3H]thymidine by rat costal cartilage. None of the other peptides was biologically active.

Stimulatory Effect of Phenylmethylsulfonyl Fluoride upon Sulfate Uptake by Costal Cartilage

Uptake of [35S]sulfate by segments of rat costal cartilage during culture was greatly stimulated when freshly prepared phenylmethylsulfonyl fluoride or diisopropylfluorophosphate was included in the incubation medium. By contrast, hydrolysed diisopropylfluorophosphate, sodium fluoride or soybean trypsin inhibitor did not stimulate [35S]sulfate uptake. Incorporation of four other radioactive precursors of cartilage synthesis was almost completely suppressed during cartilage incubation in the presence of phenylmethylsulfonyl fluoride. However, stimulation of [35S]sulfate binding by the latter was shown to occur at sites other than on glycosaminoglycan molecules and to a similar degree with both active and inactivated cartilage. These and other data indicate that the stimulatory effect of phenylmethylsulfonyl fluoride on [35S]sulfate uptake is independent of normal metabolic processes, and may involve the binding of phenylmethylsulfonyl fluoride to cartilage proteins.