Differential expression of syndecans and glypicans in chronically inflamed synovium.

BACKGROUND: Membrane-bound heparan sulphate proteoglycans (HSPGs) act as co-receptors and presenters of cytokines and are involved in cell-matrix and cell-cell adhesion. AIM: To investigate which HSPGs are expressed in knee joint synovia from patients with different forms of arthritis and normal individuals. METHODS: Synovial samples were obtained from patients with early rheumatoid arthritis (n = 8), longstanding rheumatoid arthritis (n = 13), psoriatic arthritis (n = 7), osteoarthritis (n = 6) and normal joints (n = 12). Expression of syndecan-1, -2, -3 and -4 and glypican-1, -3 and -4 was analysed by immunohistochemistry and dual label immunofluorescence. RESULTS: The expression of HSPGs in chronically inflamed synovium exhibited a differential distribution. Syndecan-1 was present in the mononuclear infiltrates of synovia from patients with rheumatoid and psoriatic arthritis where it was expressed by plasma cells. Syndecan-2 was present mainly in blood vessels where it occurred on endothelial cells, pericytes and smooth muscle cells. Syndecan-3 stained intensely in endothelial cells but also occurred in sublining macrophages and the lining layer. Glypican-4 occurred in the lining layer and blood vessels. Increased expression of these HSPGs was apparent in rheumatoid and psoriatic compared to osteoarthritic and normal synovia. Little or no staining for syndecan-4, glypican-1 and glypican-3 was seen in all samples. DISCUSSION: Selected HSPGs, such as syndecan-1, -2 and -3 and glypican-4, could play a part in the pathophysiology of arthritis, such as the migration and retention of leukocytes and angiogenesis in the chronically inflamed synovium.

Successful treatment of refractory tibial nonunion using calcium sulphate and bone marrow stromal cell implantation.

Successful healing of a nine-year tibial nonunion resistant to six previous surgical procedures was achieved by tissue engineering. We used autologous bone marrow stromal cells (BMSCs) expanded to 5 x 10(6) cells after three weeks' tissue culture. Calcium sulphate (CaSO4) in pellet form was combined with these cells at operation. The nonunion was clinically and radiologically healed two months after implantation. This is the description of on healing of a long-standing tibial nonunion by tissue engineering. The successful combination of BMSCs and CaSO4 has not to our knowledge been reported in a clinical setting.

Chondrogenic and adipogenic potential of microvascular pericytes.

BACKGROUND: Previous studies have shown that pericytes can differentiate into osteoblasts and form bone. This study investigated whether pericytes can also differentiate into chondrocytes and adipocytes. METHODS AND RESULTS: Reverse transcription-polymerase chain reaction demonstrated that pericytes express mRNA for the chondrocyte markers Sox9, aggrecan, and type II collagen. Furthermore, when cultured at high density in the presence of a defined chondrogenic medium, pericytes formed well-defined pellets comprising cells embedded in an extracellular matrix rich in sulfated proteoglycans and type II collagen. In contrast, when endothelial cells were cultured under the same conditions, the pellets disintegrated after 48 hours. In the presence of adipogenic medium, pericytes but not endothelial cells expressed mRNA for peroxisome proliferator-activated receptor-gamma2 (an adipocyte-specific transcription factor) and incorporated lipid droplets that stained with oil red O. To confirm that pericytes can differentiate along the chondrocytic and adipocytic lineages in vivo, these cells were inoculated into diffusion chambers and implanted into athymic mice for 56 days. Accordingly, mineralized cartilage, fibrocartilage, and a nonmineralized cartilaginous matrix with lacunae containing chondrocytes were observed within these chambers. Small clusters of cells that morphologically resembled adipocytes were also identified. CONCLUSIONS: These data demonstrate that pericytes are multipotent cells that may contribute to growth, wound healing, repair, and/or the development and progression of various pathological states.

Vascular pericytes express osteogenic potential in vitro and in vivo.

At postconfluence, cultured bovine pericytes isolated from retinal capillaries form three-dimensional nodule-like structures that mineralize. Using a combination of Northern and Southern blotting, in situ hybridization, and immunofluorescence we have demonstrated that this process is associated with the stage-specific expression of markers of primitive clonogenic marrow stromal cells (STRO-1) and markers of cells of the osteoblast lineage (bone sialoprotein, osteocalcin, osteonectin, and osteopontin). To demonstrate that the formation of nodules and the expression of these proteins were indicative of true osteogenic potential, vascular pericytes were also inoculated into diffusion chambers and implanted into athymic mice. When recovered from the host, chambers containing pericytes were found reproducibly to contain a tissue comprised of cartilage and bone, as well as soft fibrous connective tissue and cells resembling adipocytes. This is the first study to provide direct evidence of the osteogenic potential of microvascular pericytes in vivo. Our results are also consistent with the possibility that the pericyte population in situ serves as a reservoir of primitive precursor cells capable of giving rise to cells of multiple lineages including osteoblasts, chondrocytes, adipocytes, and fibroblasts.

Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells.

To better understand the in vivo bone-inductive properties of recombinant human (rh) BMP-2, we examined the ability of the protein to alter the phenotype of a bone marrow stromal cell line. W-20-17. rhBMP-2 increased alkaline phosphatase activity in W-20-17 cells in a dose-responsive manner in the absence of an effect on proliferation. The induction of alkaline phosphatase activity was not apparent until 12 h after rhBMP-2 treatment had begun and was effectively eliminated by cotreatment with cycloheximide, suggesting a requirement for protein synthesis. Continued treatment of W-20-17 cells with rhBMP-2 for 8 days resulted in a significant increase, compared to control cultures, in the production of cellular cAMP in response to a PTH challenge. In addition, 4-day treatment with rhBMP-2 induced osteocalcin levels in W-20-17 cells. These results indicate that rhBMP-2 induces the expression of several markers associated with the osteoblast phenotype in W-20-17 cells and raises the possibility that BMP-2 may be involved in the differentiation of osteoblasts from progenitor cells resident in bone marrow.

Characterization of cells with high alkaline phosphatase activity derived from human bone and marrow: preliminary assessment of their osteogenicity.

Confluent cellular layers are reproducibly obtained (from 21 of 24 specimens) by outgrowth from composite pieces of human trabecular bone and marrow. The cells resemble fibroblasts in terms of morphology, esterase profile, and production of collagen type 1. However, the cells displayed some osteoblastlike features. Both the primary outgrowths and passaged cultures had high alkaline phosphatase activities (37 nmols min-1 X microgram DNA-1) in the range displayed by embryonic osteoblastlike cells. The cellular alkaline phosphatase activity, which showed similarity to the bone isoenzyme on kinetic criteria, was stimulated by 1,25-dihydroxyvitamin D3 but decreased by PTH (1-34). In addition, the cell preparations were shown to increase osteocalcin (bone Gla protein) production in response to 1,25-dihydroxyvitamin D3. The osteogenic potential of the bone and marrow-derived cells has been assessed in an in vivo diffusion chamber assay in which congenitally athymic (nude) mice were used as hosts. None of the 25 chambers examined showed evidence of osteogenesis, although the cells remained viable and fibroblastlike. The alkaline phosphatase activities decreased to less than 1% of the original, high in vitro values. The findings question the hypothesis that bone and marrow-derived cells are osteoblasts or osteoblastlike cells, rather than a mixture of cell lines of the bone and marrow stromal system.

A scanning electron microscopy study of the interface between ceramics and bone.

By use of scanning electron microscopy (SEM), together with energy dispersive chemical analysis, a study has been made of the comparison of an in vitro method of assessing interface reactions between bone and ceramic implants with the naturally occurring changes seen in the rat ear model. Interface reactions between bone and two ceramic materials were examined following 4 wk in culture and 4 wk implantation. In both cases a gradual chemical change occurred at the calcium silicate surface during the fibrous growth onto the ceramic material. Gradual mineralization of the connective fibres was found at the interface of the calcium silicate material, whereas, in the case of alumina ceramic a connective fibrous bond had formed with no associated chemical change at the ceramic surface.

Serum from patients anesthetized with opiates less effective in the support of chondrocyte growth in vitro.

Risk of viral and/or prion disease transmission associated with the use of fetal bovine serum in clinical cell culture has led to the increasing use of autologous human serum in tissue engineering. A relatively large volume of blood is needed and so, to decrease patient discomfort, we have investigated the feasibility of taking blood when the patient is anesthetized. Two serum samples were prepared from each of 22 patients: (1). from the awake patient (PRE) and (2). from the patient 5 min after induction of general anesthesia (PER). The sera were compared for their ability to support the in vitro proliferation of primary human chondrocytes, determined by cell counting. The effects of anesthetic agents on the PER/PRE cell number ratio were established by analysis of variance and stepwise multilinear regression analysis. The PER sample supported higher growth in 2 of 22 patients, equivalent growth in another 11, and significantly lower growth in the remaining 8. Only the opiate analgesics (fentanyl [Sublimaze], alfentanyl [Rapifen], and diamorphine) had a significant and inhibitory effect on chondrocyte proliferation. It is suggested that opiate analgesics be avoided when blood is taken to support the in vitro growth of human cells.

Phenotypic modulation of human articular chondrocytes by bistratene A.

Chondrocytes undergo phenotypic alterations following extended periods in monolayer culture, i.e., they become bipolar and flattened, proliferate, and synthesise type I as opposed to type II collagen. This process has been termed chondrocyte dedifferentiation. Bistratene A is a macrolide polyether that specifically activates the delta isoform of protein kinase C (PKCdelta) in some cell types. Here, we show that dedifferentiated human articular chondrocytes became rounded and underwent cell growth arrest after treatment with bistratene A. In addition, bistratene A-treated chondrocytes became more immunopositive for type II collagen, but less immunopositive for type I collagen. These phenotypic changes were associated with a prior and extensive disruption of actin microfilaments and translocation of PKCdelta to the nuclear membrane. Concurrent treatments of chondrocytes with a specific inhibitor of PKCdelta, rottlerin, partially blocked the morphological effects of bistratene A.

The collagens and glycosaminoglycans of the extracellular matrices secreted by bone marrow stromal cells cultured in vivo in diffusion chambers.

When rabbit bone marrow cells are cultured in a diffusion chamber implanted into the peritoneal cavity of an athymic mouse, the stromal cells proliferate, differentiate, and produce tissues that have the morphological features of loose fibrous tissue, woven or chondroid bone, and cartilage. The collagens synthesized during the development of the tissues from 7 to 28 days after implantation were identified using specific antibodies to rabbit types I, II, III, and V and rat type IX collagens, while the glycosaminoglycans were characterized histochemically using the dye, Alcian blue. Fibrous tissue forms in the first week and it contains types I, III, and V collagens and hyaluronan. Bone and cartilage develop within the fibrous tissue from about 12 days onwards. The bone matrix contains types I and V collagens, and chondroitin and keratan sulphates. The cartilaginous matrix contains types II and IX collagens, and chondroitin and keratan sulphates. Small amounts of type III collagen are found in the bone, and types I, III, and V collagens in the cartilage. These are thought to be the remnants of the fibrous matrix and decrease as the matrices mature. It is concluded that the tissue in diffusion chambers, formed by a small number of early precursor cells present in the soft tissues of the endosteum and marrow of young rabbits, contains extracellular matrix macromolecules similar to those found in bone and cartilage.

Morphological basis for back pain: the demonstration of nerve fibers and neuropeptides in the lumbar facet joint capsule but not in ligamentum flavum.

The innervation of lumbar facet capsule and ligamentum flavum was investigated using antisera to a general neuronal marker protein gene product (PGP) 9.5 and to peptide markers of sensory nerves (calcitonin gene-related peptide [CGRP] and substance P) and autonomic nerves (vasoactive intestinal polypeptide [VIP] and C-flanking peptide of neuropeptide Y [CPON]). In the facet capsule (n = 14), PGP 9.5 and CGRP-immunoreactive nerves occurred in 12 and five specimens, respectively, both around blood vessels and as free fibers in the stroma. Free fibers immunoreactive for substance P or VIP were noted in three and five specimens, whereas in nine specimens there were CPON-immunoreactive nerves located perivascularly. There was no immunoreactivity in the ligamentum flavum. This study provides further evidence that the facet capsule but not the ligamentum flavum has substantial innervation by sensory and autonomic nerve fibers and has a structural basis for pain perception.

Dexamethasone and retinoic acid differentially regulate growth and differentiation in an immortalised human clonal bone marrow stromal cell line with osteoblastic characteristics.

Clonogenic immortalised human pre-osteoblastic cell lines provide useful species-specific experimental tools for the study of the regulation of osteoblastic proliferation and differentiation. Steroid hormones are major regulators of bone formation. Although much is known about the effects of dexamethasone on osteoblastic growth and differentiation in vitro, there is less information on the effects of trans-retinoic acid (RA), particularly in human cultures. We have established a clonal adult human cell line (C1) derived from a bone marrow aspirate. The cell line appeared to be bi-potential. The cells were able to differentiate into an adipocytic phenotype under appropriate culture conditions. When grown in osteogenic medium, the cells expressed alkaline phosphatase (ALP) and osteocalcin mRNA. The C1 cells also expressed several other osteoblastic markers such as collagen type 1 (COL 1), PTH/PTH-rp receptor constitutively. Transcripts for the osteoblast transcription factor Cbfa1 was also detected under basal conditions. In addition treatment with 1,25(OH)(2)D(3) (10(-7)M) led to a marked increase in osteocalcin mRNA expression suggesting that this cell line represents a pre-osteoblastic population. We compared the effects of Dex and RA on osteoblastic function. For the assessment of PTH/PTH-rp receptor, osteocalcin and Cbfa1 mRNA expression and PTH-stimulated adenylate cyclase responsiveness, the cells were grown in the presence of Dex and RA and harvested on Days 1, 3, 7 and 14. RA (10(-7)M) had a mitogenic effect on the C1 cells. In contrast, Dex (10(-7)M) inhibited proliferation. A similar effect was observed with primary human bone marrow stromal cultures. Both Dex and RA inhibited COL 1 synthesis and decreased COL1 mRNA. Dex stimulated ALP activity and increased ALP mRNA expression whilst RA had an inhibitory effect. Dex treatment led to an increase in PTH/PTH-rp receptor mRNA and PTH-induced cAMP accumulation with a peak response at 24 h and this effect was sustained for up to 14 days. In contrast, long-term culture with RA resulted in a reduction in the cAMP response to PTH (Days 7 and 14) with no effect on PTH/PTH-rp receptor mRNA expression. Osteocalcin and Cbfa1 mRNA expression did not alter in the presence of Dex and RA at these time points. This study shows that Dex and RA have differential effects on the expression of the phenotypic markers and genes associated with osteoblast maturation. This homogeneous cell line can therefore be used further to elucidate the cellular and molecular mechanisms of action of Dex and RA at the different developmental stages of human osteoblastic differentiation. This cell line may thus provide a useful species-specific in vitro model for the evaluation of key genes and signalling molecules involved in osteogenesis. This would be of help in the design of 'in vivo' studies.

Human bone-cell proliferation in vitro decreases with human donor age.

We have measured the effect of age on the rate of outgrowth of cells from human trabecular bone, using a quantitative dye-binding technique. In cultures supplemented with autologous serum, there were significant negative correlations between the age of the donor and both the proportion of fragments from which outgrowths were seen after 7 days (r = -0.70; p < 0.001) and the total cell number after 14 days (r = -0.78; p < 0.005). The autologous serum supported greater cell proliferation than did fetal calf serum in all subjects regardless of age. Taken with previous observations that the in vitro growth kinetics of passaged human bone cells are independent of age, our results show that the number of proliferative precursor cells on trabecular-bone surfaces is higher in younger subjects. There is a marked decrease in precursor numbers in the second and third decades of life to a level which is maintained into old age.

Repair of human articular cartilage after implantation of autologous chondrocytes.

Tissue engineering is an increasingly popular method of addressing pathological disorders of cartilage. Recent studies have demonstrated its clinical efficacy, but there is little information on the structural organisation and biochemical composition of the repair tissue and its relation to the adjacent normal tissue. We therefore analysed by polarised light microscopy and immunohistochemistry biopsies of repair tissue which had been taken 12 months after implantation of autologous chondrocytes in two patients with defects of articular cartilage. Our findings showed zonal heterogeneity throughout the repair tissue. The deeper zone resembled hyaline-like articular cartilage whereas the upper zone was more fibrocartilaginous. The results indicate that within 12 months autologous chondrocyte implantation successfully produces replacement cartilage tissue, a major part of which resembles normal hyaline cartilage.

Development of a dietary supplement database.

Data describing the composition of dietary supplements are not readily available to the public health community. As a result, intake from dietary supplements is generally not considered in most dietary surveys and, hence, little is known about the significance of supplement intake in relation to total diet or disease risk. To enable a more comprehensive analysis of dietary data, a database of the composition of various dietary supplements has been compiled. Active ingredients of all dietary supplements sold in Australia are included in the Australian Register of Therapeutic Goods (ARTG), maintained by the Therapeutic Goods Administration. Products included in the database were restricted to those vitamin, mineral and other supplements identified in dietary data collected from studies conducted in southeast Queensland and New South Wales (850 supplements). Conversion factors from ingredients compounds to active elements were compiled from standard sources. No account has been made for bioavailability, consistent with current practice for food composition databases. The database can be queried by ARTG identification number, brand, product title, or a variety of other fields. Expected future developments include development of standard formulations for use when supplements are incompletely specified, and expansion of products included for more widespread use.

The dependency of expiratory airway collapse on pump system and flow rate in liquid ventilated rabbits.

PURPOSE: To evaluate the influence of pump system and flow pattern on expiratory airway collapse (EAC) in total perfluorocarbon ventilation. - METHODS: Prospective, controlled, randomized animal trial for determination of (1) post-mortem changes by repeated expiration procedures (EP) with a constant flow piston pump (PP) before and after sacrifice (n = 8 rabbits), (2) differences between pump systems by subjecting animals to both PP and roller pump (RP) circuits for expiration (n = 16 rabbits). EP were performed using a servo-controlled shut-off at airway pressures < 25 cm H subset 2O randomly with either pump at different flows. - RESULTS: Expired volumes before and after sacrifice were not significantly different. PP and RP revealed identical mean flows, while significantly more liquid was drained using PP (p<0.05). Increasing differences towards higher flow rates indicated profound flow pulsatility in RP. - CONCLUSIONS: (1) post-mortem changes in expired volumes are not significant, (2) EAC is related to flow rate and pump system; (3) relationship between expiratory flow rate and drainable liquid volume is linear inverse; (4) PP provides higher drainage than RP. - SUMMARY STATEMENT: Expiratory airway collapse is related to flow rate and pump system, post mortem changes in expirable volumes are not significant. Relationship between expiratory flow rate and drainable liquid volume is linear inverse, piston pump expiration provides higher drainage volumes than roller pump expiration.

The in vitro growth of human chondrocytes.

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects has been described by other workers, however, relatively few details of the in vitro growth of the cells have been published. Here we describe the release of cells from adult human articular cartilage and their growth characteristics in vitro.Cultures were successfully established from 29 of 30 biopsies taken from patients aged 20-72 year. No significant relationship was found between donor age and initial cell yield following cartilage digest, however, the time to primary confluence increased in direct proportion to age. Thereafter the kinetics of cell proliferation was independent of donor age.The proportion of apoptotic or necrotic cells in the cartilage digest was low and increased with time in culture only in those cells which remained non-adherent. Conversely, entry into cell cycle was restricted to those cells which had become adherent.These results illustrate that previously reported techniques for isolating and culturing chondrocytes are reproducible, that adherent chondrocytes have considerable proliferative potential, and that concern about cell growth and viability need not, in itself, limit the clinical application of ACI to younger patients.

The cell culture expansion of bone marrow stromal cells from humans with spinal cord injury: implications for future cell transplantation therapy.

STUDY DESIGN: Previous studies have shown that transplantation of bone marrow stromal cells (MSCs) in animal models of spinal cord injury (SCI) encourages functional recovery. Here, we have examined the growth in cell culture of MSCs isolated from individuals with SCI, compared with non-SCI donors. SETTING: Centre for Spinal Studies, Midland Centre for Spinal Injuries, RJAH Orthopaedic Hospital, Oswestry, UK. METHODS: Bone marrow was harvested from the iliac crest of donors with long-term SCI (>3 months, n=9) or from non-SCI donors (n=7). Mononuclear cells were plated out into tissue culture flasks and the adherent MSC population subsequently expanded in monolayer culture. MSC were passaged by trypsinization at 70% confluence and routinely seeded into new flasks at a density of 5 x 10(3) cells per cm(2). Expanded cell cultures were phenotypically characterized by CD-immunoprofiling and by their differentiation potential along chondrocyte, osteoblast and adipocyte lineages. The influence of cell-seeding density on the rate of cell culture expansion and degree of cell senescence was examined in separate experiments. RESULTS: In SCI, but not in non-SCI donors the number of adherent cells harvested at passage I was age-related. The proliferation rate (culture doubling times) between passages I and II was significantly greater in cultures from SCI donors with cervical lesions than in those with thoracic lesions. There was no significant difference, however, in either the overall cell harvests at passages I or II or in the culture doubling times between SCI and non-SCI donors. At passage II, more than 95% of cells were CD34-ve, CD45-ve and CD105+ve, which is characteristic of human MSC cultures. Furthermore, passage II cells differentiated along all three mesenchymal lineages tested. Seeding passage I-III cells at cell densities lower than 5 x 10(3) cells per cm(2) significantly reduced culture doubling times and significantly increased overall cell harvests while having no effect on cell senescence. CONCLUSION: MSCs from individuals with SCI can be successfully isolated and expanded in culture; this is encouraging for the future development of MSC transplantation therapies to treat SCI. Age, level of spinal injury and cell-seeding density were all found to relate to the growth kinetics of MSC cultures in vitro, albeit in a small sample group. Therefore, these factors should be considered if either the overall number or the timing of MSC transplantations post-injury is found to relate to functional recovery.