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.

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.

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.

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.

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.

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.

Reduced platelet activation and thrombosis in extracorporeal circuits coated with nitric oxide release polymers.

OBJECTIVE: To determine whether the use of nitric oxide (NO)-releasing polymers coated onto the inner surface of extracorporeal circuits can reduce platelet consumption and activation in the absence of systemic heparinization using a rabbit model of venovenous extracorporeal circulation. DESIGN: Prospective, controlled trial. SETTING: Research laboratory at an academic medical institution. SUBJECTS: New Zealand White Rabbits. INTERVENTIONS: Anesthetized, tracheotomized, and ventilated New Zealand White rabbits were injected with freshly prepared, 111In(oxine)3 labeled single donor platelets through the external jugular vein. After baseline measurements, these animals were placed on venovenous extracorporeal circulation through a 1-m control circuit or NO test circuit for 4 hrs at a blood flow rate of 109-118 mL/min via roller pump. Four groups were studied: systemically heparinized control circuits, systemically heparinized NO test circuits, nonheparinized control circuits, and nonheparinized NO test circuits. Platelet counts, fibrinogen levels, and plasma free indium levels were measured hourly. Circuits were rinsed and retained for gamma counting after the 4-hr run or when the circuit clotted. Four animals, one from each group, did not receive radiolabeled platelets so that the circuits could be preserved for scanning electron microscopic examination after the 4-hr study. MEASUREMENTS AND MAIN RESULTS: Platelet consumption was significantly reduced in both the heparinized and nonheparinized NO test groups when compared with the controls (p < .0001 and p < .0004, respectively). Platelet adhesion to the extracorporeal circuits was significantly reduced in the nonheparinized test circuits when compared with the controls (p < .05). Scanning electron microscopic examination of the circuits revealed that in the absence of heparin and in the presence of a NO-releasing surface, platelets retained their spherical nonactivated shape. CONCLUSIONS: The incorporation of NO into the surface of extracorporeal circuits reduces platelet consumption and eliminates the need for systemic heparinization in a rabbit model of extracorporeal circulation.

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.

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.

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.

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.

Cellular immunity to cartilage aggrecan core protein in patients with rheumatoid arthritis and non-arthritic controls.

OBJECTIVE: To identify antigen(s) among purified deglycosylated aggrecan peptides spanning the chondroitin sulphate domain that may be responsible for the initiation or perpetuation of the autoimmune responses in rheumatoid arthritis (RA). METHODS: Aggrecan was purified from human articular cartilage and deglycosylated with either bacterial glycosidases or trifluoromethanesulphonic acid (TFMS). Twelve overlapping peptides (15 residues) spanning the chondroitin sulphate domain with N-terminal residues offset by three amino acids were synthesised. T cell responses to these antigens in RA patients and age matched controls were assessed in vitro by antigen specific T cell proliferation assays. RESULTS: Enzymically deglycosylated aggrecan (EDA) stimulated proliferation of T cells isolated from the peripheral blood in a greater proportion of patients with RA than controls. In a subset (12.5%) of RA patients, the magnitude of stimulation lay outside the control range. T cell proliferative responses to TFMS treated aggrecan were greater than, but well correlated with, responses to EDA. T cells from 15 patients were also stimulated with the pooled synthetic peptides. Four of seven patients who demonstrated T cell reactivity to EDA (seven of 15) also showed enhanced T cell proliferation to synthetic peptides. CONCLUSION: These data suggest that an autoantigenic T cell epitope may lie within the chondroitin sulphate domain of aggrecan.

Cellular immunity to cartilage link protein in patients with inflammatory arthritis and non-arthritic controls.

OBJECTIVES: To determine if increased T cell responses to articular cartilage link protein have any correlation with rheumatoid arthritis (RA), and if RA patients with increased responses to link protein also respond to a 17 amino acid peptide covering the 'arthritogenic' epitope in mycobacterial hsp65 which is homologous with link protein. METHODS: The reactivity of T cells from both peripheral blood and synovial fluid, to highly purified human cartilage link protein, hsp65, the 17 amino acid peptide, and bovine type II collagen was determined in patients with RA and nonarthritic controls, by measuring the rate of mononuclear cell proliferation in the presence and absence of antigen. RESULTS: Using peripheral blood mononuclear cells (PBMC), significant reactivity (stimulation index (SI) > 1.5) to link protein was found in 12 of 46 RA patients (26%), but in only four of 44 controls (9%). A greater proportion of RA patients (eight of 17:47%) were reactive to link protein when mononuclear cells from synovial fluid were tested. SI values, however, were generally low (0.5-3.1) and only one patient showed a PBMC response above a reference range of values calculated from the logarithmic values of the normal control population. No reactivity was observed against a 17 amino acid synthetic peptide including the arthritogenic epitope from the mycobacterial hsp65 to which T cell clones isolated from rats in the adjuvant arthritis model react. However, eight of nine RA patients and all of seven controls reacted to the intact hsp65. CONCLUSION: It remains unclear if T cell responses to link protein are involved in the pathogenesis of RA, but it is unlikely that T cells specific for the sequence homologous with the arthritogenic epitope in hsp65 are present in RA patients.

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.

[Comparative study of the in vitro effects of calcitonin, NaF and ipriflavone in cell culture]. / Calcitonin, NaF és Ipriflavon in vitro hatásának összehasonlító vizsgálata sejttenyészeten.

The aim of these in vitro series of experiments was to state whether the medicaments used in the treatment of loss of bone density the Calcitonin, NaF and Ipriflavon do have a direct effect on the preosteoblast cells. The results show that both the Calcitonin and Fluorid stimulated the development of the fibroblast colonies and the NaF had a role in the increase of the alkaline phosphatase too. In the concentration of Ipriflavon applied no effect could be demonstrated in any parameter.