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Biomedical subjects

B Johnstone

Publications and source records attributed to B Johnstone.

At least 19 recordsLinked to original sources
PubMed2006

Ascorbate-enhanced chondrogenesis of ATDC5 cells.

The ATDC5 cell line exhibits the multistep chondrogenic differentiation observed during endochondral bone formation. However, it takes up to two months to complete the process of cell expansion, insulin addition to promote differentiation and further changes in culture conditions effectively to induce hypertrophy. We sought to produce consistent chondrogenesis with significant hypertrophic differentiation with simpler conditions in a more practical time period. By adding ascorbate, the prechondrogenic proliferation phase was shortened from 21 to 7 days, with production of cartilaginous nodules during the chondrogenic phase, after insulin addition, that were greater in number and larger in size. Immunohistochemistry indicated much greater matrix elaboration and the mRNA expression of sox9, aggrecan and collagen type II were all significantly increased earlier and to a much higher degree when compared with controls. Moreover, there was a robust induction of hypertrophy: Col10a1, Runx2 and Mmp13 were all induced within 7-10 days. In conclusion, addition of ascorbate to ATDC5 cultures shortened the prechondrogenic proliferation phase, produced earlier chondrogenic differentiation, heightened gene expression and robust hypertrophic differentiation, abrogating the need for extended culture times and the changes in culture conditions. This simple modification considerably enhances the practicality of this cell line for studies of chondrogenesis.

Aggrecans↗
PubMed2003

Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro.

Much attention has been given to the influences of bioactive factors on mesenchymal progenitor cell differentiation and proliferation, but few studies have examined the effect of mechanical factors on these cells. This study examined the effects of cyclic hydrostatic pressure on human bone marrow-derived mesenchymal progenitor cells undergoing chondrogenic differentiation. Aggregates of bone marrow-derived mesenchymal progenitor cells were cultured in a defined chondrogenic medium and were subjected to cyclic hydrostatic pressure. Aggregates were loaded at various time points: single (day 1 or 3) or multiple (days 1-7). At 14 and 28 days, aggregates were harvested for histology, immunohistochemistry, and quantitative DNA and matrix macromolecule analysis. The aggregates loaded for a single day did not demonstrate significant changes in proteoglycan and collagen contents compared with the non-loaded controls. In contrast, for the multi-day loaded aggregates, statistically significant increases in proteoglycan and collagen contents were found on both day 14 and day 28. Aggregates loaded for seven days were larger and histological staining indicated a greater matrix/cell ratio. This study indicates that hydrostatic pressure enhances the cartilaginous matrix formation of mesenchymal progenitor cells differentiated in vitro, and suggests that mechanical forces may play an important role in cartilage repair and regeneration in vivo.

Bone Marrow Cells↗
PubMed2001

BMP-2 induction and TGF-beta 1 modulation of rat periosteal cell chondrogenesis.

Periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondrocytes during normal bone growth and fracture healing. TGF-beta 1 and BMP-2 have been implicated in the regulation of the chondrogenic differentiation of these cells, but their roles are not fully defined. This study was undertaken to investigate the chondrogenic effects of TGF-beta 1 and BMP-2 on rat periosteum-derived cells during in vitro chondrogenesis in a three-dimensional aggregate culture. RT-PCR analyses for gene expression of cartilage-specific matrix proteins revealed that treatment with BMP-2 alone and combined treatment with TGF-beta 1 and BMP-2 induced time-dependent mRNA expression of aggrecan core protein and type II collagen. At later times in culture, the aggregates treated with BMP-2 exhibited expression of type X collagen and osteocalcin mRNA, which are markers of chondrocyte hypertrophy. Aggregates incubated with both TGF-beta 1 and BMP-2 showed no such expression. Treatment with TGF-beta 1 alone did not lead to the expression of type II or X collagen mRNA, indicating that this factor itself did not independently induce chondrogenesis in rat periosteal cells. These data were consistent with histological and immunohistochemical results. After 14 days in culture, BMP-2-treated aggregates consisted of many hypertrophic chondrocytes within a metachromatic matrix, which was immunoreactive with anti-type II and type X collagen antibodies. In contrast, at 14 days, TGF-beta 1 + BMP-2-treated aggregates did not contain any morphologically identifiable hypertrophic chondrocytes and their abundant extracellular matrix was not immunoreactive to the anti-type X collagen antibody. Expression of BMPR-IA, TGF-beta RI, and TGF-beta RII receptors was detected at all times in each culture condition, indicating that the distinct responses of aggregates to BMP-2, TGF-beta 1 and TGF-beta 1 + BMP-2 were not due to overt differences in receptor expression. Collectively, our results suggest that BMP-2 induces neochondrogenesis of rat periosteum-derived cells, and that TGF-beta 1 modulates the terminal differentiation in BMP-2 induced chondrogenesis.

Activin Receptors, Type I↗
PubMed2001

Application of a short form of the Category Test for individuals with a traumatic brain injury: a cautionary note.

The Category Test, notable for its length, has been the subject of many proposed short-form versions. In our present managed care era, the pressure to utilize short forms of various tests with clinical populations has increased, yet the appropriateness of using short forms with various populations must be demonstrated not assumed. The present study examined the appropriateness of applying the short form of the Category Test proposed by Charter and colleagues (1997) with a sample of 100 outpatients with traumatic brain injuries referred for neuropsychological testing. Both variance accounted for by the short-form Category Test and the residuals or errors of prediction resulting from the use of the short-form Category Test were examined. While the short form of the Category Test accounted for 93% of the variance of full-form Category Test scores, examination of residuals indicated clinically significant (approximately 10 points or greater) estimation errors for 25% of the subjects. In light of the clinical significance of such extreme estimation errors, it was not recommended that this short form of the Category Test be used for individuals with a traumatic brain injury.

Adolescent↗
PubMed2001

Mesenchymal cell transfer for articular cartilage repair.

Mature articular cartilage has a poor reparative response to injury and its irreparable breakdown is the common feature of degenerative joint diseases. If articular cartilage lesions become symptomatic, the orthopaedic surgeon must decide on a treatment option. The treatment options include conversion of chondral lesions to osteochondral lesions, which facilitates migration of cells from the marrow space to effect repair. In recent years, a greater emphasis has been placed on tissue engineering strategies and thus several new treatment options have been introduced, including the use of cell transplantation. Several tissue sources and cell types can potentially be used for this type of therapy. These include autologous or allograft chondrocytes and mesenchymal progenitor cells from various tissues. These cells may be delivered to articular cartilage lesions by a variety of methods including direct cell injection to the lesion or seeding in a biodegradable scaffold prior to implantation. In this review, the potential of cell transplantation for articular cartilage repair and regeneration will be discussed. The authors will focus on the available technologies and the present limitations of cell-based therapies.

Cartilage, Articular↗
PubMed2000

Synthetic zinc finger transcription factor action at an endogenous chromosomal site. Activation of the human erythropoietin gene.

We have targeted the activation of an endogenous chromosomal locus including the human erythropoietin gene using synthetic transcription factors. These transcription factors are targeted to particular DNA sequences in the 5'-flanking region of the erythropoietin gene through engineering of a zinc finger DNA binding domain. The DNA binding domain is linked to a VP16 transcriptional activation domain. We find that these synthetic transcription factors invariably activate transiently transfected templates in which sequences within the 5' flank of the erythropoietin gene are fused to a luciferase reporter. The efficiency of activation under these circumstances at a defined site is dependent on DNA binding affinity. In contrast, only a subset of these same zinc finger proteins is able to activate the endogenous chromosomal locus. The activity of these proteins is influenced by their capacity to gain access to their recognition elements within the chromatin infrastructure. Zinc finger transcription factors will provide a powerful tool to probe the determinants of chromatin accessibility and remodeling within endogenous chromosomal loci.

Cells, Cultured↗
PubMed2000

Morphologic considerations of C2 isthmus dimensions for the placement of transarticular screws.

STUDY DESIGN: This study examines the C2 vertebrae using both direct anatomic and computed tomographic measurements. OBJECTIVE: To define the relation of the C2 vertebrae bony elements to the vertebral artery and the spinal canal, to determine individuals at risk for vertebral artery injury during C1-C2 transarticular screw placement. SUMMARY OF BACKGROUND DATA: Recent literature assessing the safety of upper cervical spine transarticular screws has concentrated on technique, including the optimal point of entry and path projection of the screw. The actual dimensions of the C2 isthmus of the pars interarticularis has not been examined in a large number of specimens. METHODS: C2 isthmus width and height measurements were made on 205 human cadaveric C2 vertebrae. Vertebrae predicted to be at risk for vertebral arterial injury were imaged by computed tomography. RESULTS: There were 102 female and 103 male specimens with mean isthmus widths of 8.2 +/- 1.5 mm and 7.2 +/- 1.3 mm, respectively. Five specimens (2.4%) had an isthmus width less than 5 mm. The mean isthmus heights were 8.6 +/- 2.0 mm and 6.9 +/- 1.5 mm for male and female specimens, respectively. In twenty-four specimens (11.7%), one or both isthmi had a height of less than 5 mm. Six of these specimens were male and 18 were female. The right C2 isthmus was generally smaller than the left. Computed tomographic measurements closely approximated those of the actual dimensions of the isthmi. CONCLUSIONS: Placing a 3.5 mm screw in a patient with narrow C2 isthmus dimensions (smaller than 5 mm in either the height or width) is technically difficult. Because of narrow C2 isthmus width and/or height, approximately 10% of patients may be at risk for a vertebral artery injury with placement of C1-C2 transarticular screws.

Adolescent↗
PubMed2000

Involvement of caspase-3 in epigallocatechin-3-gallate-mediated apoptosis of human chondrosarcoma cells.

Green tea polyphenol-(-)epigallocatechin-3-gallate (EGCG)-is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis. In this study we describe a novel observation that EGCG displayed strong inhibitory effects on the proliferation and viability of HTB-94 human chondrosarcoma cells in a dose-dependent manner and induced apoptosis. Investigation of the mechanism of EGCG-induced apoptosis revealed that treatment with EGCG resulted in DNA fragmentation, induction of caspase-3/CPP32 activity, and cleavage of the death substrate poly(ADP-ribose)polymerase (PARP). Pretreatment of cells with a synthetic pan-caspase inhibitor (Z-VAD-FMK) and a caspase-3-specific inhibitor (DEVD-CHO) prevented EGCG-induced PARP cleavage. The induction of apoptosis by EGCG via activation of caspase-3/CPP32-like proteases may provide a mechanistic explanation for its antitumor effects.

Antineoplastic Agents↗
PubMed2000

Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1.

OBJECTIVE: To determine whether articular chondrocytes express growth factor genes delivered by adenoviral vectors and whether expression of these genes influences matrix synthesis in the presence and absence of interleukin-1 (IL-1). METHODS: Monolayer cultures of rabbit articular chondrocytes were infected with recombinant adenovirus carrying genes encoding the following growth factors: insulin-like growth factor 1 (IGF-1), transforming growth factor beta1 (TGFbeta1), and bone morphogenetic protein 2 (BMP-2). As a control, cells were transduced with the lac Z gene. Cultures were also treated with each growth factor supplied as a protein. Levels of gene expression were noted, and the synthesis of proteoglycan, collagen, and noncollagenous proteins was measured by radiolabeling. Collagen was typed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The effects of growth factor gene transfer on proteoglycan synthesis in the presence of IL-1 were also measured. RESULTS: The expression of all transgenes was high following adenoviral transduction. Proteoglycan synthesis was stimulated approximately 8-fold by the BMP-2 gene and 2-3-fold by the IGF-1 gene. The effects of BMP-2 and IGF-1 genes were additive upon cotransduction. Synthesis of collagen and noncollagenous proteins, in contrast, was most strongly stimulated by the IGF-1 gene. In each case, collagen typing confirmed the synthesis of type II collagen. IL-1 suppressed proteoglycan synthesis by 50-60%. IGF-1 and TGFbeta genes restored proteoglycan synthesis to control levels in the presence of IL-1. The BMP-2 gene, in contrast, elevated proteoglycan synthesis beyond control levels in the presence of IL-1. CONCLUSION: Transfer of growth factor genes to articular chondrocytes can greatly increase matrix synthesis in vitro, even in the presence of the inflammatory cytokine IL-1. This result encourages the further development of gene therapy for the repair of damaged cartilage.

Animals↗
PubMed2000

Effect of age and sampling site on the chondro-osteogenic potential of rabbit marrow-derived mesenchymal progenitor cells.

Four sequential bone-marrow aspirations from the ipsilateral tibia and iliac crest of New Zealand White rabbits, aged 4 months or 1, 2, or 3 years, were assayed in vitro and in vivo for their chondro-osteogenic potential. Nonhematopoietic cells from the samples of bone marrow were isolated and expanded in culture; their colony-forming efficiency was determined, and second-passage marrow-derived cells, referred to as mesenchymal progenitor cells, were loaded into porous calcium-phosphate ceramic cubes as carrier vehicles for an in vivo cartilage and bone-formation assay. The cubes were placed subcutaneously in nude BALB/C mice for 3 and 6 weeks. On histological examination, the cubes were scored for the presence of bone and cartilage in their pores, and average values for age groups and location were compared. At aspiration, the samples from the iliac crest exhibited an overall reduction in cell concentration with increasing age, and at the first harvest time, they showed a decrease in colony-forming efficiency and cube score with increasing age. This study demonstrated that repeated bone-marrow aspirations may be performed and may have an enhancing effect on the osteochondral progenitor cells of older animals.

Age Factors↗
PubMed2000

Increased matrix synthesis following adenoviral transfer of a transforming growth factor beta1 gene into articular chondrocytes.

Monolayer cultures of lapine articular chondrocytes were transduced with first-generation adenoviral vectors carrying lacZ or transforming growth factor beta1 genes under the transcriptional control of the human cytomegalovirus early promoter. High concentrations of transforming growth factor beta1 were produced by chondrocytes following transfer of the transforming growth factor beta1 gene but not the lacZ gene. Transduced chondrocytes responded to the elevated endogenous production of transforming growth factor beta1 by increasing their synthesis of proteoglycan, collagen, and noncollagenous proteins in a dose-dependent fashion. The increases in collagen synthesis were not accompanied by alterations in the collagen phenotype; type-II collagen remained the predominant collagen. Transforming growth factor beta1 could not, however, rescue the collagen phenotype of cells that had undergone phenotypic modulation as a result of serial passaging. These data demonstrate that chondrocytes can be genetically manipulated to produce and respond to the potentially therapeutic cytokine transforming growth factor beta1. This technology has a number of experimental and therapeutic applications, including those related to the study and treatment of arthritis and cartilage repair.

Adenoviridae↗
PubMed2000

Neuropsychological predictors of distress following traumatic brain injury.

Emotional and behavioural difficulties are one of the most common difficulties following traumatic brain injury (TBI), although it is not clear which individuals with TBI become most distressed. Numerous factors contribute to adjustment following TBI, and the current study examined degree of cognitive decline as one potential contributor to distress following TBI. The relationship between cognitive functioning and distress may be conceptualized as being related to (a) an individual's absolute level of cognitive ability following TBI, or (b) relative degree of decline following TBI (i.e. the greater the decline, the greater the distress, regardless of absolute level of ability). The current study tested these hypotheses by comparing a measure of global emotional distress with measures of absolute level of neuropsychological functioning and indices of cognitive decline. In contrast to hypotheses, regression analyses indicated that estimated pre-morbid ability accounted for more variance in distress following TBI than either absolute level of functioning or indices of cognitive decline, with individuals with higher estimated pre-morbid abilities reporting lower levels of distress. Treatment implications are discussed.

Adolescent↗
PubMed2000

Chondrogenitor cells and gene therapy.

The development of isolation and culture techniques for mesenchymal progenitor cells from various tissues has promoted interest in the use of these cells for repair and regeneration of musculoskeletal tissues. The chondrogenic differentiation of these pluripotential cells seems to be mediated by numerous cytokines most of which belong to the transforming growth factor-beta superfamily. Strategies to repair articular cartilage have focused on delivery of these cytokines or progenitor cells to the area of damage. More recently, with the development of gene transfer techniques, these cells have become the target of in vivo gene therapy, which involves direct injection of viral and nonviral vectors carrying transgenes. Furthermore, they are viewed as potential carriers of the transgenes for ex vivo gene therapy, in which the gene transfer is done in vitro with culture-expanded cells that then are implanted or injected. In vitro data suggest that the chondrogenic potential of these cells is maintained with virally mediated ex vivo gene transfer. By transducing these cells with chondroinductive factors, the bioactive factors and the target cells are delivered to the repair site.

Animals↗
PubMed2000

Neuropsychological deficit profiles in systemic lupus erythematosus.

Although neuropsychological deficits have been reported in several cognitive domains in patients with systemic lupus erythematosus (SLE), there is considerable variability in the literature about which neuropsychological domains are most affected. Similar to studies that demonstrated that specific profiles of neuropsychological deficits exist for those with traumatic brain injury (TBI; Johnstone, Hexum, & Ashkanazi, 1995), this study examined whether a specific pattern of deficits is present in SLE. By comparing reading scores (as estimates of premorbid ability) to tests of concurrent cognitive abilities (i.e., memory, attention, etc.), it was determined that SLE presents a profile distinct from TBI, with the most significant impairments noted in expressive language (Zdiff = -1.39), attention (Zdiff = -0.41), and speed of processing (Zdiff = -0.40). In contrast to TBI, no impairment was noted in intelligence, memory, or cognitive flexibility. Results suggest that memory problems reported by individuals with SLE may be related to inattention. Clinical implications are discussed.

Adult↗
PubMed1999

Augmentation of spinal arthrodesis with autologous bone marrow in a rabbit posterolateral spine fusion model.

STUDY DESIGN: Posterolateral spinal fusion with autologous bone marrow aspirate in addition to autograft iliac crest bone graft in a rabbit model. OBJECTIVE: To demonstrate that the addition of autologous bone marrow can have positive effects on bone formation and spinal fusion. SUMMARY OF BACKGROUND DATA: Bone marrow has been shown to contain osteoprogenitor cells. A number of studies have demonstrated that bone formation is possible with autologous marrow injection into orthotopic sites such as that performed in femur fracture models. METHODS: A bone paucity model of posterolateral spine fusion was developed. The control animals received 0.8 g of morselized autogenous iliac crest bone graft harvested from a single iliac crest. The graft was mixed with 2 mL of clotted peripheral blood. In the experimental group, 2 mL of bone marrow aspirated from the opposite iliac crest was substituted for the peripheral blood clot. All rabbits were killed at 12 weeks, and the specimens were subjected to evaluation by posteroanterior radiography for the presence of fusion, computed tomography for bone volume, and biomechanical testing for stiffness. RESULTS: Successful fusion was achieved in 61% of the animals in the experimental group versus 25% in the control group (P < 0.05). The fusion mass in the experimental group had a mean volume of 919 +/- 387 mm3 versus 667 +/- 512 mm3 for the control group, as measured from computed tomography images. The results of the biomechanical testing validated the radiographic scoring system. The stiffness in specimens, graded as having a radiographic score of 4, was significantly greater than in specimens with radiographic scores of 1 and 2. CONCLUSION: In cases for which an adequate quantity of autogenous bone graft is not available, addition of bone marrow may facilitate greater bone formation and successful fusion.

Animals↗
PubMed1999

Chondroprogenitor cells of synovial tissue.

OBJECTIVE: To assess the chondrogenic potential of cells within the synovium. METHODS: Explants of synovium taken from various sites in the joint were embedded in agarose and cultured with transforming growth factor beta1 (TGFbeta1) to assess their chondrogenic potential. Isolated synovial cells were also tested for their chondrogenic potential by culturing them as aggregates in a chemically defined medium with TGFbeta1. Cartilage formation was determined with histologic staining and immunohistochemistry. The osteochondral potential of the isolated cells was also assessed after subcutaneous implantation of the cells, loaded into porous calcium phosphate ceramic cubes, in athymic mice. RESULTS: A total of 48 synovial explants were cultured in agarose with TGFbeta1. The formation of cartilage was observed in the outer region of 21 explants, and type II collagen was localized in that region by immunohistochemistry. A larger percentage of TGFbeta1+ explants from the inner synovium sites formed cartilage compared with those from the outer synovium sites. Chondrogenesis occurred in aggregates incubated with TGFbeta1 as early as day 7, and by day 14, all TGFbeta1+ aggregates demonstrated chondrogenesis. In contrast with the results of the in vitro aggregate assay for chondrogenesis, no formation of cartilage or bone was evident in any section containing synovial cell-loaded ceramic cubes that were harvested at either 3 or 6 weeks after implantation subcutaneously in athymic mice. CONCLUSION: Synovial explants and isolated synovial cells will undergo chondrogenesis when cultured in the presence of TGFbeta1. The data indicate a possible synovial origin for the chondrocytic cells found in rheumatoid pannus. Furthermore, these data are consistent with the clinical findings of synovial chondrogenesis leading to synovial chondromatosis.

Animals↗
PubMed1999

CACP, encoding a secreted proteoglycan, is mutated in camptodactyly-arthropathy-coxa vara-pericarditis syndrome.

Altered growth and function of synoviocytes, the intimal cells which line joint cavities and tendon sheaths, occur in a number of skeletal diseases. Hyperplasia of synoviocytes is found in both rheumatoid arthritis and osteoarthritis, despite differences in the underlying aetiologies of the two disorders. We have studied the autosomal recessive disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP; MIM 208250) to identify biological pathways that lead to synoviocyte hyperplasia, the principal pathological feature of this syndrome. Using a positional-candidate approach, we identified mutations in a gene (CACP) encoding a secreted proteoglycan as the cause of CACP. The CACP protein, which has previously been identified as both 'megakaryocyte stimulating factor precursor' and 'superficial zone protein', contains domains that have homology to somatomedin B, heparin-binding proteins, mucins and haemopexins. In addition to expression in joint synovium and cartilage, CACP is expressed in non-skeletal tissues including liver and pericardium. The similarity of CACP sequence to that of other protein families and the expression of CACP in non-skeletal tissues suggest it may have diverse biological activities.

Amino Acid Sequence↗