Julie Glowacki

Hemangiomas and vascular malformations in infants and children: a classification based on endothelial characteristics.

… Those difficulties which have hitherto amused philosophers, and blocked up the way to knowledge, are entirely owing to ourselves. That we have first raids a dust and then complain we cannot see.

Age‐related intrinsic changes in human bone‐marrow‐derived mesenchymal stem cells and their differentiation to osteoblasts

In vivo and in vitro studies indicate that a subpopulation of human marrow‐derived stromal cells (MSCs, also known as mesenchymal stem cells) has potential to differentiate into multiple cell types, including osteoblasts. In this study, we tested the hypothesis that there are intrinsic effects of age in human MSCs (17–90 years). We tested the effect of age on senescence‐associated β‐galactosidase, proliferation, apoptosis, p53 pathway genes, and osteoblast differentiation in confluent monolayers by alkaline phosphatase activity and osteoblast gene expression analysis. There were fourfold more human bone MSCs (hMSCs) positive for senescence‐associated β‐galactosidase in samples from older than younger subjects (P < 0.001; n = 17). Doubling time of hMSCs was 1.7‐fold longer in cells from the older than the younger subjects, and was positively correlated with age (P = 0.002; n = 19). Novel age‐related changes were identified. With age, more cells were apoptotic (P = 0.016; n = 10). Further, there were age‐related increases in expression of p53 and its pathway genes, p21 and BAX. Consistent with other experiments, there was a significant age‐related decrease in generation of osteoblasts both in the STRO‐1+ cells (P = 0.047; n = 8) and in adherent MSCs (P < 0.001; n = 10). In sum, there is an age‐dependent decrease in proliferation and osteoblast differentiation, and an increase in senescence‐associated β‐galactosidase‐positive cells and apoptosis in hMSCs. Up‐regulation of the p53 pathway with age may have a critical role in mediating the reduction in both proliferation and osteoblastogenesis of hMSCs. These findings support the view that there are intrinsic alterations in human MSCs with aging that may contribute to the process of skeletal aging in humans.

Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes.

Immortalized human chondrocytes were established by transfection of primary cultures of juvenile costal chondrocytes with vectors encoding simian virus 40 large T antigen and selection in suspension culture over agarose. Stable cell lines were generated that exhibited chondrocyte morphology, continuous proliferative capacity (> 80 passages) in monolayer culture in serum-containing medium, and expression of mRNAs encoding chondrocyte-specific collagens II, IX, and XI and proteoglycans in an insulin-containing serum substitute. They did not express type X collagen or versican mRNA. These cells synthesized and secreted extracellular matrix molecules that were reactive with monoclonal antibodies against type II collagen, large proteoglycan (PG-H, aggrecan), and chondroitin-4- and chondroitin-6-sulfate. Interleukin-1 beta (IL-1 beta) decreased the levels of type II collagen mRNA and increased the levels of mRNAs for collagenase, stromelysin, and immediate early genes (egr-1, c-fos, c-jun, and jun-B). These cell lines also expressed reporter gene constructs containing regulatory sequences (-577/+3,428 bp) of the type II collagen gene (COL2A1) in transient transfection experiments, and IL-1 beta suppressed this expression by 50-80%. These results show that immortalized human chondrocytes displaying cartilage-specific modulation by IL-1 beta can be used as a model for studying normal and pathological repair mechanisms.

Expression of differentiated function by mineralizing cultures of chicken osteoblasts

This report documents osteoblast differentiation in vitro, as demonstrated by the 50-100X increase of proteins which are known markers of the osteoblast phenotype. Collagen type I and osteocalcin synthesis and accumulation, alkaline phosphatase activity, and matrix calcification show similar temporal relationships that are analogous to those seen during in vivo bone development. Chicken embryonic osteoblast progenitor cells were selected by initial growth at low densities in minimal medium. Upon subcultivation into nutrient-enriched medium at higher cell densities, near homogeneous populations of osteoblasts were obtained as demonstrated by the greater than 80% enrichment of cells positive for alkaline phosphatase activity. A comparison was made between cells grown in the presence or absence of 10 mM beta-glycerolphosphate (beta-GPO4), a chemical stimulant of matrix calcification, as a function of time. Cultures treated with beta-GPO4 showed visible calcification at Day 12 when culture monolayers became confluent. By Day 30, numerous large foci of calcification were visible and a 20-fold increase in calcium (Ca) content was observed. In contrast, untreated cultures had only a 3-fold increase in Ca content with many smaller diffuse areas of calcification. DNA, RNA, and total protein levels were nearly identical between the two cultures, indicating that beta-GPO4 had no marked effect on either cell proliferation or transcriptional activity. The major collagen type produced by either culture was type I, with no detectable type III as determined by CNBr peptide mapping and delayed reduction analysis. Alkaline phosphatase activity showed a rapid approximately 50-fold induction by Day 18 and remained elevated in control cultures. However, cultures treated with beta-GPO4 demonstrated a rapid 80% decline of enzyme activity after 18 days. In contrast, total osteocalcin levels showed a 100-fold induction by Day 18 and remained elevated in both control and beta-GPO4-treated cultures throughout the time period examined. While the overall levels of osteocalcin were the same in beta-GPO4-treated and untreated cultures, 2- to 5-fold more osteocalcin was associated with the more mineralized matrices of the beta-GPO4-treated cultures. In order to confirm the association of osteocalcin with areas of mineralization, co-localization of mineral to osteocalcin and collagen was carried out by combining vital labeling with tetracycline and immunofluorescent staining with anti-osteocalcin and anti-collagen antibodies. Both collagen and osteocalcin showed strong localization with areas of mineralization.(ABSTRACT TRUNCATED AT 400 WORDS)

Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges

Studies with human and animal culture systems indicate that a sub‐population of bone marrow stromal cells has the potential to differentiate into osteoblasts. There are conflicting reports on the effects of age on human marrow‐derived osteogenic cells. In this study, we used a three dimensional (3D) culture system and quantitative RT‐PCR methods to test the hypothesis that the osteogenic potential of human bone marrow stromal cells decreases with age. Marrow was obtained from 39 men aged 37 to 86 years, during the course of total hip arthroplasty. Low‐density mononuclear cells were seeded onto 3D collagen sponges and cultured for 3 weeks. Histological sections of sponges were stained for alkaline phosphatase activity and were scored as positive or negative. In the group ≤ 50 years, 7 of 11 samples (63%) were positive, whereas only 5 of 19 (26%) of the samples in the group  ≥ 60 years were positive (p = 0.0504). As revealed by RT‐PCR, there was no expression of alkaline phosphatase or collagen type I mRNA before culture, however there were strong signals after 3 weeks, an indication of osteoblast differentiation in vitro. We performed a quantitative, competitive RT‐PCR assay with 8 samples (age range 38–80) and showed that the group ≤ 50 years had 3‐fold more mRNA for alkaline phosphatase than the group ≥ 60 years (p = 0.021). There was a significant decrease with age (r = − 0.78, p = 0.028). These molecular and histoenzymatic data indicate that the osteogenic potential of human bone marrow cells decreases with age. J. Cell. Biochem. 82: 583–590, 2001.

Angiogenesis in fracture repair.

Fracture of bone disrupts its circulation and leads to necrosis and hypoxia of adjacent bone. Under normal circumstances, fractured bone undergoes the orderly regeneration of its component tissues with complete restoration of mechanical properties. Reestablishment of the circulation is an early event in fracture healing. Several experimental models of protracted, impaired, or compromised healing have been developed to evaluate the effects of angiogenic factors in accelerating or enhancing repair.

Cell Shape and Phenotypic Expression in Chondrocytes

Abstract The relationship between cell shape, proliferation, and phenotypic expression was studied in human chondrocytes. Shape was controlled independent of serum concentration, anchorage, and cell density by alteration of substratum adhesiveness with poly(2-hydroxyethyl methacrylate) (poly[HEMA]). Cells that were held rounded displayed features of the chondrocyte phenotype; i.e., they were round, proliferated slowly, incorporated low levels of [3H]thymidine into DNA, and incorporated large amounts of 35SO4 into glycosaminoglycans. In contrast, cells that were held flat were fibrolast-like: they exhibited flattened morphology, more rapid growth, greater incorporation of [3H]thymidine, and less incorporation of 35SO4. These studies suggest that cell shape may play an important role in phenotypic expression in chondrocytes.

Induced Osteogenesis for Repair and Construction in the Craniofacial Region

Solid and powdered forms of undemineralized and demineralized bone grafts were implanted in rat cranial defects. Demineralized calvarial discs healed the defects as well as did the fresh discs, as judged by histology and 45Ca incorporation. Gross and histologic evaluations demonstrated predictable endochondrial osteogenesis by demineralized bone powder (DBP). Undemineralized grafts, in contrast, showed poor and unpredictable bony healing. Construction of facial bones was achieved by implantation of demineralized bone powder within the soft tissues. The phenomenon of induced osteogenesis by demineralized implants was not species specific. These studies of osseous transformation provide insight into the mechanism of, and possible answers to, the problems of osseous transplantation.

Fate of mineralized and demineralized osseous implants in cranial defects.

SummaryWe have evaluated the fate of mineralized and demineralized osseous implants placed into cranial defects in rats. By 2 weeks, 100% of the defects that had been filled with demineralized bone powder (DBP, 75–250 µm) showed bony repair as judged by histomorphometric analysis and incorporation of45Ca. The DBP was not appreciably resorbed but rather was amalgamated within the new bone. Histomorphometric evaluation of osteogenesis induced by equal masses of demineralized bone powders of various particle sizes (<75, 75–250, 250–450, >450 µm) revealed that the smaller particles induced more bone per field than did the larger particles.In contrast, mineralized bone powder (BP) was completely resorbed by 3 weeks, without bony repair of the cranial defect. These specimens contained large multinucleated cells and connective tissue.Implants of bone minerals were also evaluated. Bone ash and deorganified bone powder were surrounded by multinucleated cells within 7 days and completely resorbed by 3 weeks.It is concluded that (a) demineralized bone powder predictably induces osteogenic healing of cranial defects, (b) demineralized bone powder is not appreciably resorbed prior to bone induction, (c) the extent of bone induction is a function of the surface area of the demineralized bone implant, and (d) mineralized bone powder undergoes obligatory resorption.

Cooperation Between TGF-β and Wnt Pathways During Chondrocyte and Adipocyte Differentiation of Human Marrow Stromal Cells†

Human marrow stromal cells have the potential to differentiate to chondrocytes or adipocytes. We show interactions between TGF‐β and Wnt signaling pathways during stimulation of chondrogenesis and inhibition of adipogenesis. Combining these signals may be useful in marrow stromal cell therapies.