Howard L. Hosick

Development of controlled porosity polymer-ceramic composite scaffolds via fused deposition modeling

This research is focused on development and fabrication of controlled porosity polymer-ceramic composite scaffolds, with 3-D interconnectivity designed to promote richer supply of blood, oxygen and nutrients for healthy in-growth of bone cells. Particulate-reinforced polymer-ceramic composites were developed by high shear mixing of polypropylene (PP) polymer and tricalcium phosphate (TCP) ceramic. Processing aids were used to improve plasticity and processibility to the composites. Controlled porosity scaffolds were fabricated via the fused deposition process, one of the commercially available rapid prototyping (RP) techniques. These porous scaffolds were characterized for their use as bone grafts in terms of physical, mechanical and biological properties. Hg-porosimetry was performed to determine pore size and their distribution. Scaffolds with different complex internal architectures were also fabricated using this composite material. Tensile properties of neat PP (as received), PP with processing aids (without TCP) and PP-TCP composite (with processing aids) were evaluated and compared using standard dog bone samples. Uniaxial compression tests were performed on cylindrical porous samples with an average pore size of 160 μm and varying vol.% porosity (36%, 48% and 52%). Samples with 36 vol.% porosity showed the best compressive strength of 12.7 MPa. Cytotoxicity and cell proliferation studies were conducted with a modified human osteoblast cell-line (HOB). Results showed that these samples were non-toxic with excellent cell growth during the first two weeks of in vitro testing.

Pore size and pore volume effects on alumina and TCP ceramic scaffolds

Abstract Controlled porosity alumina and β-tricalcium phosphate ceramic scaffolds with pore sizes in the range of 300–500 μm and pore volumes in the range of 25–45% were processed using the indirect fused deposition process. Samples having different pore sizes with constant volume fraction porosity and different volume fractions porosity with a constant pore size were fabricated to understand the influence of porosity parameters on mechanical and biological properties. In vitro cell proliferation studies were carried out with OPC1 human osteoblast cell line for 28 days with different scaffolds. Variation in pore size did not show any conclusive differences, but samples with higher volume fraction porosity showed some evidence of increased cell growth. Volume fraction porosity also showed a stronger influence on the mechanical properties under uniaxial compression loading. Compression strength dropped significantly for samples with higher volume fraction porosity, but changed marginally when only the pore size was varied.

Processing and characterization of porous alumina scaffolds

Bioceramic materials are used for the reconstruction or replacement of the damaged parts of the human body. In this study an improved procedure is described for producing ceramic scaffolds with controlled porosity. Bioinert alumina ceramic was used to make porous scaffolds by using indirect fused deposition modeling (FDM), a commercially available rapid prototyping (RP) technique. Porous alumina samples were coated with hydroxyapatite (HAp) to increase the biocompatibility of the scaffolds. Initial biological responses of the porous alumina scaffolds were assessed in vitro using rat pituitary tumor cells (PR1). Both porous alumina and HAp coated alumina ceramics provided favorable sites for cell attachments in a physiological solution at 37 °C, which suggests that these materials would promote good bonding while used as bone implants in vivo. Based on these preliminary studies, similar tests were performed with human osteosarcoma cells. Cell proliferation studies show that both the ceramic materials can potentially provide a non-toxic surface for bone bonding when implanted in vivo.

Identification and characterization of a novel angiotensin binding site in cultured vascular smooth muscle cells that is specific for the hexapeptide (3–8) fragment of angiotensin II, angiotensin IV

This study demonstrates the existence of a previously unrecognized class of angiotensin binding sites on vascular smooth muscle that exhibit high affinity and specificity for the hexapeptide (3-8) fragment of angiotensin II (AngIV). Binding of [125I]AngIV is saturable, reversible and describes a pharmacologic profile that is distinct and separate from the classic AT1 or AT2 angiotensin receptors. Saturation binding studies utilizing cultured vascular smooth muscle cells obtained from bovine aorta (BVSM) revealed that [125I]AngIV bound to a single high affinity site with an associated Hill coefficient of 0.99 +/- 0.003, exhibiting a KD = 1.85 +/- 0.45 nM and a corresponding Bmax = 960 +/- 100 fmol mg-1 protein. Competition binding curves in BVSM demonstrated the following rank order effectiveness: AngIV > AngII(3-7) >> AngIII > Sar1,Ile8 AngII > AngII > AngII(1-7) > AngII(4-8), DuP 753, PD123177. The presence of the non-hydrolyzable GTP analog GTP gamma S, had no effect on [125I]AngIV binding affinity in BVSM. The presence of this novel angiotensin binding site on smooth muscle in high concentration suggests the possibility that this system may play an important, yet unrecognized role in vascular control.

Establishment and Characterization of a Human Ovarian Serous Cystadenocarcinoma Cell Line That Produces the Tumor Markers CA-125 and Tissue Polypeptide Antigen

A cultured cell line (SHIN-3) derived from a human ovarian serous cystadenocarcinoma which consistently produces two tumor markers, CA-125 and tissue polypeptide antigen (TPA) was established. After 1 week of culture of 1 x 10(5) cells, high levels of tumor marker were observed (the total CA-125 release was 1,500 U and the total TPA release was 37.5 U). Expression of CA-125 and TPA was also demonstrated in cultured cells immunohistochemically. The volume of CA-125 release per cell was highest just before the start of the logarithmic growth phase. TPA production was increased in the logarithmic growth phase, but its relationship to the total number of cells was not clear.

New cell line. Epithelial cell line and subline established from premalignant mouse mammary tissue.

SummaryA cell line and subline with epithelial characteristics were established from mouse mammary hyperplastic alveolar nodules (HAN). The cells do not grow in suspension cultures in vitro or form tumors in vivo. The cells do produce significant amounts of C-type and A-type virus and low amounts of plasminogen activator.

Expression of epidermal growth factor‐related proteins in the aged adult mouse mammary gland and their relationship to tumorigenesis

Mammary glands from female BALB/c mice of different ages and parity were screened for production of three epidermal growth factor (EGF) related transforming growth factors and their corresponding mRNAs. Glands were obtained from 2–26‐month‐old nulliparous, 4–26‐month‐old parous, and 2–8‐month‐old midpregnant mice. Reverse‐transcribed polymerase chain reaction (RT‐PCR) was used to screen for mRNA from the transforming growth factor alpha (TGFα), cripto‐1 (CR‐1), and amphiregulin (AR) genes in extracts of whole mammary glands. TGFα, CR‐1, and AR transcripts were detected in all of the mammary glands assayed. In situ hybridization was then used to localize these mRNAs among various cell types in sections of glands. TGFα mRNA levels were low in the mammary epithelium from young nulliparous mice, high in the stroma of midpregnant mammary glands, and highest in luminal epithelium of the aged glands. AR mRNA levels were high and remained unchanged in all developmental stages. CR‐1 mRNA level increased with age and was detected primarily in epithelium, with some scattered expression in adjacent stroma. Finally, TGFα, CR‐1, and AR proteins were immunolocalized in histological sections of mammary glands from the various developmental stages. TGFα was detected sporadically in midpregnant mice, with more conspicuous reactivity seen in 18–26‐month‐old mice (38% of mice). CR‐1 immunoreactivity was detected in 100% of the 18–26‐month‐old glands but not in any other age groups. Strong AR immunoreactivity was observed in in all glands, including 100% of the 18–26‐month‐old glands. Staining for all three of these growth factors was observed primarily in the epithelium, with some reactivity detected in the periductal fibroblasts. No significant difference was discerned between glands from nulliparous and parous animals. We also found intense CR‐1 and AR mRNA expression and strong immunoreactivity in seven different carcinogen‐induced and eight spontaneous mammary tumors. Our results demonstrate that these growth factors accumulate in significant amounts in the old gland of both nulliparous and parous mice. The observations suggest that these growth factors are positioned to contribute to abnormal development in the older mammary gland, predisposing them to tumorigenesis. J Cell Physiol 170:47–56, 1997

Isolation and characterization of clonal vascular smooth muscle cell lines from spontaneously hypertensive and normotensive rat aortas

SummaryVascular smooth muscle cells were isolated from the aortas of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats by use of the explant method on collagen gels. Clonal cell lines derived from these enriched populations possessed ultrastructural characteristics of vascular smooth muscle cells in culture; they grew in hill and valley configuration, immunostained with the muscle actin antibody HHF35, and failed to react with von Willebrand Factor VIII antibody. Fourteen clonal cell lines were characterized for growth and ligand binding characteristics. Large variations in growth rate and cell density at saturation were exhibited by clones of both strains. Similar variability was noted for specific binding of endothelial 1 and Sar1,Ile8-angiotensin II to their receptors, indicating considerable phenotypic heterogeneity among the clonal cell lines. Six selected clones were further characterized for angiotensin II receptor linkage to G proteins. Cells of both strains exhibited comparable affinity shifts in the presence of GTPγS. These clonal cell lines should be useful for a variety of analyses of the comparative biology of aortic cells. It is possible that the diversity of phenotypic traits exhibited by these clones reflects the heterogeneity of vascular smooth muscle tissue found in vivo.

Metastatic Potential of Hyperplastic Alveolar Nodule Derived Mouse Mammary Tumor Cells Following Intravenous Inoculation

Abstract A comparison was made of the in vivo growth potential of tumor cells derived from mouse mammary adenocarcinomas arising in premalignant outgrowth lines D1 and D2. Twenty-one tumors were dissociated and cultured as cell monolayers in vitro. Three to four days later 50,000–100,000 cells were inoculated intravenously into syngeneic female BALBlc mice. Primary cultures of tumor cells which arose in D2 HAN outgrowths varied considerably in their ability to form tumor nodules in the lungs of syngeneic mice 4 weeks post-inoculation. An increase in either the number of cells inoculated or in the length of in vivo incubation influenced the extent of tumor growth observed in the lungs. Primary cultures of tumor cells which arose in D1 HAN outgrowths inoculated at the same cell number rarely formed tumor nodules in the lungs 4 weeks post-injection. If, however, twice as many cells were introduced, a small number of large nodules were observed within 4 weeks. An epithelial cell line (WAZ-2T) isolated from D1 tumor tissue produced small lung nodules by 4 weeks, but larger more numerous nodules arose when in vivo incubation was extended an additional 3 weeks. This study indicates that primary D2 tumor cells have a variable but higher degree of metastatic potential than primary D1 tumor cells following intravenous inoculation. Tumors which develop in the D2 and D1 premalignant lines may be useful for studies of chemotherapy and cell latency of tumor metastases and the identification of the specific cell subpopulations involved in these events.

Heregulin-? is especially potent in activating phosphatidylinositol 3-kinase in nontransformed human mammary epithelial cells

The neu differentiation factors/heregulins (HRGs) comprise a family of polypeptide growth factors that activate p185erbB‐2 through direct binding to either erbB‐3 or erbB‐4 receptor tyrosine kinases. We have previously shown that HRG‐β is mitogenic for various human mammary epithelial cell lines that coexpress c‐erbB‐2 and c‐erbB‐3. Phosphatidylinositol 3‐kinase (PI3K) is activated by p185erbB‐2 /erbB‐3 heterodimers in cells stimulated by HRG, and PI3K is constitutively activated by p185erbB‐2 /erbB‐3 in breast carcinoma cells that overexpress c‐erbB‐2. To better understand the relative abilities of HRGs, epidermal growth factor (EGF), or insulin to activate PI3K under normal physiological conditions, we compared the levels of recruitment of the 85‐kDa regulatory subunit of PI3K when activated by the type I (erbB) or type II [insulin‐like growth factor (IGF)] receptor tyrosine kinases in two different nontransformed human mammary epithelial cell lines. The nontransformed H16N‐2 cells isolated from normal tissue express EGFR, p185erbB‐2 , and erbB‐3, and are highly responsive to the mitogenic effects of HRG‐β as well as to the combination of EGF and insulin in serum‐free culture. We measured the stoichiometry of p85 recruited by tyrosine‐phosphorylated proteins induced in H16N‐2 cells by either the α or the β isoform of HRG. HRG‐β was greater than 10‐fold more potent in inducing p85 recruitment than was the less biologically active HRG‐α isoform. HRG‐β was also a more potent inducer of p85 recruited by tyrosine‐phosphorylated proteins than was either EGF, insulin, or EGF and insulin combined. Furthermore, erbB‐3 principally mediated the direct recruitment of p85 in cells stimulated by HRG or EGF, indicating that, in addition to the high‐level activation of PI3K by p185erbB‐2 / erbB‐3, EGFR/erbB‐3 heterodimer interaction is essential for the weak but significant level of PI3K activated by EGF in cells that express normal EGFR levels. Studies using the PI3K inhibitor wortmannin also indicated that PI3K activation was required for the proliferation of H16N‐2 cells induced by either HRG‐β or EGF and insulin in serum‐free culture. Finally, HRG‐β was also an especially potent inducer of PI3K in the nontransformed MCF‐10A cells, which were derived spontaneously from normal reduction mammoplasty tissue. These data show, for the first time, a side‐by‐side quantitative comparison of the relative degree of PI3K activated by different growth factors in nontransformed growth factor–dependent cells under precisely defined conditions in culture. J. Cell. Physiol. 183:301–313, 2000.