R. M. Leach

Development of a serum-free system to study the effect of growth hormone and insulinlike growth factor-I on cultured postembryonic growth plate chondrocytes

SummaryWe have developed a serum-free system to culture postembryonic growth plate chondrocytes while maintaining some important phenotypic characteristics of their tissue of origin. This serum-free medium was as effective as medium containing 10% newborn bovine serum (NBS) for recovering the cells from enzymatic isolation. Surface secretory activity of chondrocytes cultured in monolayer, assessed through scanning electron microscopy, was also comparable to cells grown in medium containing serum. The effects of growth hormone (GH) and insulinlike growth factor-I (IGF-I) were also studied using the serum-free medium. GH had no effect on cell density and morphology of the cells compared to the control without the hormone. In contrast, chondrocytes grown in medium containing IGF-I had a marked increase in cell density after 3 days and presented similar morphologic characteristics to cells grown in the presence of NBS. The growth factors required for proliferation of chondrocytes cultured in the serum-free medium are IGF-I and fibroblast growth factor (100 ng/ml, respectively). Addition of ascorbic acid to the serum-free medium (0 to 50 µg/ml) produced a dose-dependant decrease in cell proliferation. This medium should provide a useful tool for studying the effects of different growth factors/hormones in the regulation of longitudinal bone growth and their interactions.

Development of avian tibial dyschondroplasia: gene expression and protein synthesis.

Abstract. Age-dependent gene expression and protein synthesis associated with chondrocyte differentiation were evaluated in the epiphyseal growth plates of normal and tibial dyschondroplasia (TD)-afflicted chickens. In the normal growth plate, collagen type II gene is expressed mainly by chondrocytes at the upper zone of the growth plate and by the chondrocytes in the articular cartilage. Collagen type X and osteopontin (OPN) genes are expressed in the lower zone of the growth plate and in the zone of cartilage-to-bone transition. No age-dependent changes in the pattern of OPN and collagen type II or X gene expression were observed up to 20 days of age. In the TD-afflicted growth plates, the lesion is enlarged with age, and chondrocytes expressing the collagen type II gene were observed in the hypertrophic zone as early as 8 days posthatching. Abnormal expression of OPN and collagen type X genes was also observed starting at 13 days of age. At day 20, the entire TD lesion—which was significantly enlarged—was surrounded by collagen type II, collagen type X, and OPN expressing cells. The level of OPN in TD was reduced with increasing age, and at 20 days almost no OPN could be detected in either the upper or the lower hypertrophic zones. The level of bone sialoprotein (BSP) also diminished with increasing age in the TD growth plates. In contrast to OPN, the age-dependent reduction in BSP levels was mainly in the lower hypertrophic zone (LHZ), and at 20 days of age, BSP was barely detected in the LHZ, whereas in the upper hypertrophic zone, the levels of BSP were similar to those in normal growth plate. In summary, our results suggest that the primary event of the TD lesion occurs in cells of proliferative phenotype within the hypertrophic zone. These cells divide and form the TD lesion, which consists of cells that do not express the genes associated with hypertrophy.

Effects of strontium on bone strength, density, volume, and microarchitecture in laying hens.

Strontium has been reported to have beneficial effects on bone. Treatment of laying hens, which are susceptible to osteoporosis and bone fracture, with strontium increased DXA measurements of BMD and BMC and μCT measurements of bone volume and microarchitecture and improved the mechanical performance of whole bone, but had no effect on the estimated material properties of the bone tissue.

Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation

It has been demonstrated that fibroblast growth factor receptors are key regulators of endochondral bone growth. However, it has not been determined what fibroblast growth factor ligand(s) (FGFs) are important in this process. This study sought to determine whether FGFs 1, 2, 4, 5, 6, 7, 8, 9, and 10 were capable of stimulating avian chondrocyte proliferation in vitro. We have found that FGFs 2, 4, and 9 strongly stimulate avian chondrocyte proliferation while FGFs 6 and 8 stimulate proliferation to a lesser extent. RT‐PCR indicates that FGF‐2 and FGF‐4 are expressed in the postnatal avian epiphyseal growth plate (EGP) while FGF‐8 and FGF‐9 are not. Thus, FGF‐2 and FGF‐4 stimulate chondrocyte proliferation and are both present in the EGP. This suggests that FGF‐2 and FGF‐4 may be important ligands, in vivo, for the regulation of endochondral bone growth. These observations coupled with our observation that multiple avian FGF receptors (Cek1, Cek2, Cek3, and FREK) are expressed in proliferative chondrocytes highlights the complexity of FGF signaling pathways in postnatal endochondral bone growth. J. Cell. Biochem. 84: 359–366, 2002.

Basic fibroblast growth factor: An autocrine growth factor for epiphyseal growth plate chondrocytes

Basic fibroblast growth factor (bFGF) is a permissive mitogen for cultured chondrocytes and has been localized in the specific zones of the epiphyseal growth plate. In this study, we demonstrate that bFGF present in cartilage originates from within the cellular constituents of this tissue. Utilizing reverse transcription coupled to the polymerase chain reaction (PCR), bFGF mRNA was found in extracts of cartilage tissue. Immunocytochemical studies revealed that bFGF was present intracellularly in freshly isolated proliferative chondrocytes and in the extracellular matrix (ECM) after 24 h of culture. Western blot analysis of protein extracts from isolated proliferative chondrocytes identified a bFGF immunoreactive species with a molecular weight of approximately 18 kDa. In situ hybridization confirmed the presence of bFGF mRNA in freshly isolated proliferative chondrocytes. The bFGF in the ECM seemed to be sequestered and not available for biological activity, since these cells still required exogenous bFGF for cell proliferation. This sequestered bFGF could be released to stimulate cell proliferation when cultures were treated with plasmin, a proteolytic enzyme. These data support the hypothesis that bFGF is synthesized by chondrocytes and functions as an autocrine/paracrine mitogen via its deposition into the ECM with subsequent release from the ECM of cartilage being a critical step in biological activity. In addition, the study provides further evidence that locally produced bFGF plays an important role in normal growth and development of cartilage tissue.

Parathyroid hormone‐related peptide expression in the epiphyseal growth plate of the juvenile chicken: Evidence for the origin of the parathyroid hormone‐related peptide found in the epiphyseal growth plate

Parathyroid hormone‐related peptide (PTHrP) has been shown to be essential for normal endochondral bone formation. Along with Indian hedgehog (Ihh), it forms a paracrine regulatory loop that governs the pace of chondrocyte differentiation. However, the source of PTHrP for this regulatory loop is not clear. While one hypothesis has suggested the periarticular perichondrium as the source of PTHrP for growth plate regulation, other data utilizing immunohistochemistry and in situ hybridization would indicate that growth plate chondrocytes themselves are the source of this peptide. The data described in this report supports the view that postnatal growth plate chondrocytes have the ability to synthesize this important regulatory peptide. Immunohistochemistry of tissue sections showed that PTHrP protein was evident throughout the chick epiphysis. PTHrP was seen in chondrocytes in the periarticular perichondrium, the perichondrium adjacent to the growth plate, the prehypertrophic zone of the growth plate, and the hypertrophic zone of the growth plate. However, cells in the proliferative zone, as well as some chondrocytes in the deeper layers of articular cartilage were predominantly negative for PTHrP. PTHrP was detected by Western blotting as a band of 16,400 Da in extracts from hypertrophic chondrocytes, but not from proliferative cells. RT‐PCR detected PTHrP mRNA in both proliferative and hypertrophic growth plate chondrocytes, as well as in articular chondrocytes. PTH/PTHrP receptor mRNA was detected by Northern blotting in growth plate, but not articular chondrocytes. Thus, we conclude that most of the PTHrP present in the epiphyseal growth plate of the juvenile chick originates in the growth plate itself. Furthermore, the presence of large amounts of PTHrP protein in the hypertrophic zone supports the concept that PTHrP has other functions in addition to regulating chondrocyte differentiation. J. Cell. Biochem. 80:504–511, 2001.

Osteopontin gene expression and alkaline phosphatase activity in avian tibial dyschondroplasia

Osteopontin (OPN) gene expression and alkaline phosphatase activity were evaluated in the epiphyseal growth plates of normal chickens and in diet-induced tibial dyschdroplasia (TD)-afflicted chickens. In the normal growth plate, OPN gene was expressed by a) cells of the subperichondrial zone surrounding the articular cartilage, b) a narrow layer of hypertrophic chondrocytes at the hypertrophic zone, and c) lower hypertrophic chondrocytes at the zone of matrix calcification and endochondral bone formation. The latter two layers were separated by OPN-negative chondrocytes. Osteopontin gene was not expressed throughout the zone of articular cartilage in the nonhypertrophic or upper hypertrophic portions of the growth plate cartilage. Only at sites of calcification of the lower hypertrophic zone was the expression of the OPN gene associated with alkaline phosphatase activity. In all TD lesions, regardless of the induction procedure, the layer of chondrocytes of the lower hypertrophic zone expressing the OPN gene and the layer of OPN-negative cells separating the two areas of OPN-expressing cells were grossly enlarged. This resulted in a wide discontinuity between the chondrocytes of the lower hypertrophic zone expressing the OPN gene and the cells expressing the OPN gene that are associated with mineralization. In TD, no alkaline phosphatase activity was detected within the growth plate cartilage, but normal OPN gene expression was observed at the subperichondrium zone and at the zone of endochondral bone formation. The results of this study suggest that in the epiphyseal growth plate, OPN expression is not restricted to sites of bone calcification.

PARATHYROID RECEPTOR GENE EXPRESSION BY EPIPHYSEAL GROWTH PLATES IN RICKETS AND TIBIAL DYSCHONDROPLASIA

PTH/PTHrP receptor gene expression was evaluated in situ in avian epiphyseal growth plates taken from normal, rachitic and tibial dyschondroplasia (TD) afflicted chicks induced by thiram or by genetic selection. In the normal growth plates, PTH/PTHrP receptor gene expression was localized to the maturation zone as demonstrated by the expression of collagen type II (col II), osteopontin (OPN) genes and alkaline phosphatase activity (AP). In TD, either induced by thiram or by genetic selection, normal levels of PTH/PTHrP receptor gene expression were observed up to 21 days post-hatch. In rickets, on the other hand, no PTH/PTHrP receptor gene expression was observed in the growth plate from day 8 of a vitamin D-deficient diet. In cultured chondrocytes, PTH caused time-dependent down-regulation of its own receptor. These results suggest that alterations in the PTH/PTHrP receptor gene expression are associated with rickets but not with TD. The reduction in the PTH/PTHrP receptor gene expression in rickets may be due to the high plasma levels of PTH.

Comparative assessment of bone among wild-type, restricted ovulator and out-of-production hens

1. The aim of this study was to assess bone characteristics in restricted ovulator (RO) hens. These hens generally are unable to ovulate due to a point mutation in the oocyte VLDL receptor gene whose protein product mediates the uptake of yolk precursors. Because these hens do not have the cyclic calcium (Ca) metabolism associated with egg formation, they could be a useful model for studying bone metabolism. 2. RO hens had greater humerus, femur and tibia ash concentrations than wild-type (WT) and out-of-production (OP) hens. Bone mineral content and density obtained with dual-energy X-ray absorptiometry (DXA) were highly correlated with the results of conventional bone assays. 3. Gross and histological examination of the femurs confirmed the presence of extremely dense medullary bone deposition in the RO hens. However, the composition of non-collagenous protein extracts of medullary bone was similar for the two genotypes. 4. Analysis of medullary bone extracts for glycosaminoglycans (GAG) confirmed the presence of large amounts of keratan sulphate (KS) in the matrix of medullary bone. 5. Plasma Ca, total GAG and KS concentrations of RO hens were markedly higher than WT and OP hens. The changes in plasma calcium and keratan sulphate are probably a reflection of elevated Ca-binding yolk precursor molecules and intensive medullary bone formation in response to increased plasma oestrogen observed by others in RO hens.

Ovocleidin (OC 116) Is Present in Avian Skeletal Tissues

Ovocleidin (OC-116), a protein identified in eggshell matrix, was found to be expressed in avian growth plate chondrocytes. Because OC-116 has been reported to be a member of a family of related phosphoprotein genes clustered on avian chromosome 4, we expanded our search to other skeletal tissues. Using Western blotting, we found OC-116 in the matrix of chick cortical bone and laying hen medullary bone as well as in hypertrophic chondrocyte lysates. Furthermore, other members of this family (bone sialoprotein, dental matrix protein-1, and osteopontin) were also present in the eggshell matrix. Reverse transcription-PCR was used to confirm the presence of the OC-116 gene in bone tissues as well as the expression of bone sialoprotein and dental matrix protein-1 in uterine tissue. These results, in combination with those of other laboratories, show that this family of phosphoproteins is found in a wide variety of avian mineralized tissues.