High Cell Population Research Articles

Muscle regeneration is not impaired by ablation of UCP-1-expressing cells

INTRODUCTION: Recently, cells expressing uncoupling protein 1 (UCP-1) have been proposed to impact muscle physiology. First, transplant of brown adipose tissue (which has high expression of UCP-1) enhanced regeneration in the injured mouse rotator cuff. Second, isolated UCP-1+ muscle fibroadipogenic progenitors (FAPs) treated to enhance UCP-1 expression improved injured mouse rotator cuff muscle pathology. However, it remains unknown whether endogenous UCP-1-expressing cells contribute to muscle regeneration. Therefore, the objective of this study was to assess whether mice that lack UCP-1-expressing cells demonstrate impaired muscle regeneration following toxin administration. METHODS: Experiments were performed using tibialis anterior (TA) and 5th toe extensor digitorum longus (EDL) muscle of wild type C57BL/6J (WT; n=22), and uncoupling protein 1 diphtheria Toxin A (UCP1-DTA; n=18) mice, which lack all UCP-1 expressing cells. All procedures were performed in accordance with the National Institute of Health’s Guide for the Use and Care of Laboratory Animals and were approved by Washington University School of Medicine IACUC. Muscle injury was induced using intramuscular injection of 50%v/v solution of glycerol. Regeneration was assessed at “early” (7 days post-injury (dpi)), “intermediate” (14 dpi) and “late” (21 dpi) stages. Outcome measures were cell population dynamics by flow cytometry, ex-vivo physiology testing and histological scoring as appropriate for each timepoint. RESULTS: Our preliminary data in early regeneration show that UCP1-DTA mice have significantly greater percentage of FAPs compared to WT mice (29.84 ± 1.24% higher cell population, p<0.0134), however no differences in muscle satellite (stem) cells between genotypes. Physiological and histological outcomes at intermediate and late-stage regeneration find a baseline deficit in muscle mass (23.69 ± 2.3% smaller, p<0.0001), type 2B fiber cross-sectional area (32.56 ± 4.8% smaller, p<0.0001) and normalized muscle active force generation (16.2 ± 3.3% lower force, p<0.0154) in UCP1-DTA compared with WT. However, mass and contractile forces recover to a similar extent between the genotypes following glycerol injection suggesting the phenotypic drivers of the reduced mass and contractile function do not affect muscle regeneration. CONCLUSIONS: Taken together, the data from this study point to a baseline growth deficit in mice lacking UCP-1-expressing cells. However, in two separate timepoints of regeneration (14 and 21dpi), we see that UCP1-DTA mice have similar recovery of mass and normalized contractile force to baseline values suggesting regeneration can proceed normally without UCP-1 expressing cells. However, further data collection will be needed at early-stage regeneration to help discern whether mice lacking UCP-1-expressing cells have initial changes in cell population dynamics given the increased FAP population at 7dpi. ACKNOWLEDGMENTS: This work was supported by R01AR075773 and R21AR071582 to GAM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract LB421: Entinostat alters cell cycle in ovarian cancer cells

Abstract Introduction: We have reported that Class I/II HDACi sensitize PARPi resistant tumors, creating a possibility to expand the use of PARPi beyond homologous recombination deficient tumors. However, the underlying mechanism is still unknown. Here, we report leads obtained from differential gene expression in ovarian cancer cells treated with entinostat and olaparib. Methods: OVCAR3 cells were treated with 0.5 µM entinostat and 10 µM olaparib (alone and combined) for 6h, followed by RNA Seq. Cell cycle was analyzed by flow cytometry in SKOV-3 cells treated with olaparib and entinostat (alone and combined) for 24h, 48h and 72h. Results: RNA-Seq analysis showed reduced expression of genes that regulate G2/M checkpoint progression with both entinostat and olaparib. This correlated with flow cytometry results in SKOV-3 cells, where we observed accumulation of G2/M phase cells when treated with entinostat and olaparib. At 24h post treatment, olaparib and entinostat treated cells had higher population of cells in G2/M phase compared to control (18.5% and 16.7% respectively compared to 14.7% in control). Olaparib combined with entinostat further increased G2/M population to 21.7%. At 48h olaparib treated cells had 17.4% cells in G2/M compared to 14% in control. Entinostat treated cells had 18% G2/M cells. Combination treated cells had 19.7% G2/M cells. At 72h, olaparib and control had similar number of G2/M cells (9.2% and 9.7% respectively). Entinostat treated cells had 12% G2/M cells and combination treated cells had 13.6% cells at G2/M cells. This indicates that entinostat and olaparib disrupt progression of cells from G2/M phase, potentially leading to cell death before they can enter mitosis/cell division. Cell cycle analysis in OVCAR3 cells is in progress. Conclusion: Entinostat and olaparib cause G2/M cell cycle arrest in ovarian cancer cells. Entinostat maintains G2/M cell cycle arrest longer than olaparib, potentially extending the window of efficacy of the combination therapy. Validation of G2/M markers and other gene targets/pathways is in progress. Citation Format: Vijayalaxmi G. Gupta, Heng Liu, Rugang Zhang, Michael Onken, Andrew Wilson, Fiona Yull, Marta Crispens, Mary Mullen, Dineo Khabele. Entinostat alters cell cycle in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB421.

Pterostilbene loaded poly(vinyl alcohol)-gelatin cryogels as potential bioactive wound dressing material.

Cryogels are support materials which are good at mimicking extracellular matrix due to their excellent hydrophilicity, biocompatibility, and macroporous structure, thus they are useful in facilitating cell activities during healing process. In this study, polyvinyl alcohol-gelatin (PVA-Gel) based cryogel membranes loaded with pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene; PTS) (PVA-Gel/PTS) was synthesized as wound dressing materials. PVA-Gel and PVA-Gel/PTS were synthesized with the polymerization yields of 96% ± 0.23% and 98% ± 0.18%, respectively, and characterized by swelling tests, Brunauer-Emmett-Teller (BET) and scanning electron microscopy (SEM) analysis. The swelling ratios were calculated as 98.6% ± 4.93% and 102% ± 5.1%, macroporosities were determined as 85% ± 2.13% and 88% ± 2.2%, for PVA-Gel and PVA-Gel/PTS, respectively. It was determined that PVA-Gel and PVA-Gel/PTS have 17 m2 /g ± 0.76 m2 /g and 20 m2 /g ± 0.92 m2 /g surface areas, respectively. SEM studies were demonstrated that they have ~100 μm pore sizes. According to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue exclusion and live-dead assay results, it was observed that cell proliferation, cell number and cell viability were higher in PVA-Gel/PTS cryogel at 24, 48, and 72 h compared to PVA-Gel. A strong and transparent fluorescent light intensity was observed indicating higher cell population in PVA-Gel/PTS in comparison with PVA-Gel, according to 4',6-diamidino-2-phenylindole (DAPI) staining. SEM, F-Actin, Giemsa staining and inverted-phase microscope image of fibroblasts in PVA-Gel/PTS cryogels revealed that dense fibroblast proliferation and spindle-shaped morphology of cells were preserved. Moreover, DNA agarose gel data demonstrated that PVA-Gel/PTS cryogels had no effect on DNA integrity. Consequently, produced PVA-Gel/PTS cryogel can be used as wound dressing material to promote wound therapies, inducing cell viability and proliferation.

Abstract P205: Novel 1,1-diarylethylene compounds degrade FOXM1 and selectively and potently reduce survival of high-grade serous ovarian cancer cells

Abstract Background: Forkhead box M1 (FOXM1) is a master transcriptional regulator and multifunctional oncoprotein that is widely overexpressed in ovarian cancer and associated with poor prognosis. Due to its promotion of numerous oncogenic phenotypes in ovarian cancer, FOXM1 has emerged as an attractive therapeutic target for ovarian cancer treatment. Recently, we reported that 1,1-diarylethylene compounds, NB-55, NB-73, and NB-115 (NB compounds), potently inhibit FOXM1 in breast cancer cells and mediate anti-cancer effects. Given the high molecular similarity between triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC), we characterized this new class of FOXM1 inhibitors in HGSC cells in the present study and compared their effects to other reported FOXM1 inhibitors. Methods: We conducted dose-response curves of NB compounds and other FOXM1 inhibitors in HGSC cells (CAOV3 and OVCAR4) and normal control (FT282 C11) cells. Western blot analyses probed for FOXM1, p-FOXM1, CCNB1, FOXA1, and FOXO3A in HGSC cells treated with NB compounds. We used the proteosome inhibitor MG132 to investigate the mechanism of FOXM1 inhibition by NB-73 and NB-115. We investigated FOXM1 transcriptional activity, colony formation, cell cycle, and apoptosis in HGSC cells treated with NB-73 and NB-115. Results: Dose-response curves in HGSC cells revealed IC50 values in the low nM for monensin; mid-nM for NB-73 and NB-115; high nM for thiostrepton; and low µM for NB-55, FDI-6, and n-phenylphenanthren-9-amine. RCM-1 did not affect HGSC cell proliferation. Dose-response curves in normal control fallopian tube epithelial (FTE) cells revealed IC50 values of high nM for thiostrepton and low µM for NB compounds and FDI-6. HGSC cells treated with NB compounds demonstrated decreased levels of FOXM1 and p-FOXM1 but not FOXA1 and FOXO3A. NB-73 and NB-115 decreased FOXM1 promoter activity and gene target expression but were no longer able to decrease FOXM1 levels when combined with MG132 in HGSC cells. Clonogenic assays of HGSC cells treated with NB-73 and NB-115 revealed decreased colony formation at low nM concentrations. HGSC cells treated with NB-73 and NB-115 have higher cell populations in G2/M and undergo higher rates of apoptosis compared to non-treated HGSC cells. Conclusions: NB-73 and NB-115 promote proteasomal degradation of FOXM1 and are more potent FOXM1 inhibitors of cell proliferation than previously characterized FOXM1 inhibitors in HGSC cells. NB-73 and NB-115 potently reduce HGSC survival and selectively inhibit HGSC cells as compared to normal control FTE cells. Our data encourage further assessment of NB compounds as a novel therapeutic strategy in the vast majority of HGSC that overexpress FOXM1. Support: NIH T32CA009476(CL), research grants 083(BSK) and 084(JAK) from the Breast Cancer Research Foundation, the Landfield Cancer Research Fund(BSK), NIH R03CA224339(ARK), The Marsha Rivkin Center(ARK), The Betty J. and Charles D. McKinsey Ovarian Cancer Research Fund(ARK), and The Fred & Pamela Buffett Cancer Center (NCI P30CA036727)(ARK). Citation Format: Cassie Liu, Catalina Muñoz-Trujillo, John A. Katzenellenbogen, Benita S. Katzenellenbogen, Adam R. Karpf. Novel 1,1-diarylethylene compounds degrade FOXM1 and selectively and potently reduce survival of high-grade serous ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P205.

ISOLATION AND DEGRADATION STUDY ON CARBOSULFAN CONTAINING PESTICIDE OF BIOSUFACTANT-PRODUCING BACTERIA FROM BELAWAN SUMATERA

Uncontrol use of synthetic pesticide causes environmental pollution and leaves its residue in soil and water. The objective of this research was to obtain bacterial isolates capable of producing biosurfactant from Belawan North Sumatera and to observe their biosurfactant activities as well as their potential in degrading of carbosulfan pesticide. Bacteria were isolated using selective medium, Bushnell-Hass Agar (BHA) containing 2% of pesticide with carbosulfan as the active compound. The result showed that all bacterial isolates were able to grow on the medium and consumed carbosulfan as the carbon sourc. The highest cell population was shown by isolate Sp.1 with total count was 7.6×109 CFU/ml. The highest biosurfactant activity was shown by isolate Sp.6 with the volume of emulsion was 5.627 cm3, meanwhile the highest production of biosurfactant shown by Sp.7 with concentration was 54.6 ppm. Two isolates, Sp.1 and Sp.6 were selected for further test to study their abilities in carbosulfan degradation. Both isolates were able to degrade carbosulfan completely after 21 days of incubation.

Thermal and non-thermal treatment effects on Staphylococcus aureus biofilms formed at different temperatures and maturation periods

The objective of this study was to investigate the effect of temperature and maturation period on the resistance of Staphylococcus aureus biofilms to thermal and non-thermal treatments. First, biofilm development was compared at three different temperatures (15, 25, and 37°C) for 5 days. The cell population at 15 and 25°C remained relatively consistent approximately at 6.3 log CFU/cm2, whereas 37°C resulted in the highest cell population on day 1 (7.6 log CFU/cm2) followed by a continual decline. Then, biofilm resistance to steam and sodium hypochlorite (NaOCl) treatments was evaluated. Obtained results highlighted that biofilms had different resistance to both treatments depending on development conditions. Specifically, steam treatment of 10 s eliminated 4.1 log CFU/cm2 of the biofilm formed at 25°C for 5 days. The same treatment inactivated over 5 log population of biofilms developed in other temperature and maturation period conditions. Treatment with NaOCl reduced approximately 1 log CFU/cm2 of biofilm cells developed at 25°C for 5 days. However, inactivation was found to be over 2 log CFU/cm2 under other development conditions. An extracellular polymeric substances (EPS) quantification using 96-well plates and stainless steel coupons was conducted. In the 96-well plate experiment, it was found that the highest amount of polysaccharide was secreted at 25°C (p < 0.05), while total biomass and protein contents were greatest at 37°C (p < 0.05). No significant difference in EPS content was observed for stainless steel, but the results displayed a similar trend to the 96-well plate. In particular, biofilms developed at 25°C tended to secret the highest amount of polysaccharide, which aligned with the current literature. This finding indicated that polysaccharide was the main contribution to the enhanced resistance of S. aureus biofilms. Overall, it was shown that biofilms formed at 25°C for 5 days exhibited the greatest resistance to thermal and nonthermal treatments due to the elevated exopolysaccharide secretion. This study demonstrates that temperature and maturation period significantly affect the resistance of S. aureus biofilms to thermal and non-thermal treatments.

Chondrogenic differentiation of mesenchymal stem/stromal cells on 3D porous poly (ε-caprolactone) scaffolds: Effects of material alkaline treatment and chondroitin sulfate supplementation

Cartilage defects resultant from trauma or degenerative diseases (e.g., osteoarthritis) can potentially be repaired using tissue engineering (TE) strategies combining progenitor cells, biomaterial scaffolds and bio-physical/chemical cues. This work examines promoting chondrogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs) by combining the effects of modified poly (ε-caprolactone) (PCL) scaffolds hydrophilicity and chondroitin sulfate (CS) supplementation in a hypoxic 5% oxygen atmosphere. 3D-extruded PCL scaffolds, characterized by μCT, featured a 21mm-1 surface area to volume ratio, 390μm pore size and approximately 100% pore interconnectivity. Scaffold immersion in sodium hydroxide solutions for different periods of time had major effects in scaffold surface morphology, wettability and mechanical properties, but without improvements on cell adhesion. In-situ chondrogenic differentiation of BM-MSC seeded in 3D-extruded PCL scaffolds resulted in higher cell populations and ECM deposition along all scaffold structure, when chondrogenesis was preceded by an expansion phase. Additionally, CS supplementation during BM-MSC expansion was crucial to enhance aggrecan gene expression, known as a hallmark of chondrogenesis. Overall, this study presents an approach to tailor the wettability and mechanical properties of PCL scaffolds and supports the use of CS-supplementation as a biochemical cue in integrated TE strategies for cartilage regeneration.

Novel surface modification of three-dimensional bacterial nanocellulose with cell-derived adhesion proteins for soft tissue engineering

Bacterial nanocellulose (BNC) is a natural polymer composed of glucose units with an important application as a two and three-dimensional scaffold for tissue engineering. However, as a polysaccharide, BNC does not have the biological signals of protein biomaterials. Therefore, this paper aims to develop a novel methodology to biomimic soft extracellular matrix (ECM) chemistry on to 3D BNC using the bioengineering of fibroblasts (the cells responsible for producing and regenerating the ECM) to immobilise adhesion proteins such as collagen and fibronectin. Modified 3D BNC (Mod-BNC) biomaterials were morphologically, thermally, and chemically characterised, and furthermore, the cell response was analysed by adhesion studies using atomic force microscopy (AFM), XTT assay, and confocal microscopy.Cell-derived proteins were deposited on the BNC nanoribbon network to modify its surface. The contact angle was increased from 40° to 60°, reducing the wettability of the biomaterial, and during thermogravimetry, the proteins in Mod-BNC exhibited an enhanced thermal stability because of the interactions between themselves and BNC. Chemical and immunocytochemistry analyses confirmed the presence of collagen type I and fibronectin on 3D BNC. These proteins activate integrin adhesion pathways that generate stronger cell adhesions. AFM experiments showed higher forces and energies on modified biomaterials, and moreover, the cells that adhered on to Mod-BNC exhibited higher mitochondrial activity and higher cell populations per cubic millimetre than non-modified surfaces (NMod-BNC). Accordingly, it was established that this novel methodology is robust and able to biomimic the chemical surface of soft ECM and immobilise cell-derived adhesion proteins from fibroblast; moreover, the Mod-BNC exhibited better cell response than NMod-BNC because of the biological signals in 3D BNC.

Sustained Depolarization of the Resting Membrane Potential Regulates Muscle Progenitor Cell Growth and Maintains Stem Cell Properties In Vitro.

It is important to maintain the myogenic properties of muscle progenitor cells (MPCs) during in vitro expansion for stem cell therapies and tissue engineering applications. Controlling cell fate for biomedical interventions will require insight on all aspects that influence cellular properties. The resting membrane potential (Vmem) has proven to be a key parameter involved in cell proliferation, migration and differentiation. This current work is focused on elucidating the impact of sustained depolarization on MPC growth and differentiation in vitro. Cultures were treated with either potassium gluconate or the sodium-potassium pump blocker ouabain and evaluated for proliferation, DNA content using propidum iodide staining, and differentiation. Cell proliferation measurements showed a modest stimulatory effect at certain concentration ranges for each agent, but higher concentrations of potassium gluconate strongly inhibited growth in a dose dependent manner. Cell cycle analysis with flow cytometry demonstrated an increase in the number of cells in S phase, but increasing concentrations of potassium gluconate arrested cells at G1. Immunostaining, Western blot analysis and light microscopy revealed that potassium gluconate exposure delayed cell fusion and maintained a higher population of cells expressing the muscle stem cell marker Pax7. The impairment on cell fusion was transient and myotube formation recovered after the treatments were removed. Taken together, this work suggests that transmembrane voltage gradients can be used as a powerful regulator of MPC properties in vitro. Examination of how these physiological parameters modulate cell behavior will reveal a new set of tools that can be capitalized on in tissue engineering and regenerative medicine.

Spirulina platensis Geitler sebagai Fikoremediator Logam Berat Pb Skala Laboratorium

Industrial waste contributes the heavy metals pollution in the environment, especially in the water. One of the heavy metals that pollute the environment is lead (Pb). Lead is a substance that is harmful to organisms, especially humans, both adults and children. Solutions to tackle Pb pollution isby remediation process. Physical and chemical remediation methods usually costly and ineffective. One of the alternative methods which is used is the bioremediation uses microalgae or called as phycoremediation. One of microalgae which is used as agents of phycoremediation is Spirulina platensis Geitler because it’s easily cultured and it’s abundant in nature. This study aims to assess the population growth and the percentage reduction of Pb after inoculated with the use of Spirulina platensis Geitler. The method which is to cultivate S. platensis Geitler in brackish watermedia with a salinity of 15 ppt which have been added Pb with a concentration of 1 mg, 3 mg, and 5 mg for 8 days and observed the population and the percentage reduction of Pb heavy metals. Environmental factors such as temperature, salinity, pH, light intensity are conditioned to remain stable. After 8 days of research, study was able to reduce Pb. Each treatment has different concentrations decrease. Treatment Pb 1 mg of 0.949 into 0.603 mg / L; Pb 3 mg of 2.894 to 2.46 mg / L; and Pb 5 mg of 4.88 to 4.31 mg / L, with each percentage decline of 36%, 15% and 12%. The Pb 3 mg treatment has higher cell population (129.367 cell / ml) than control (106.600 cell/ml) while 1 mg and 5 mg treatment has 93.700 cell / ml and 93.500 cell / ml. Keywords: Spirulina platensis, phycoremediation, waste, lead.

Assessing the Effects of Transforming Growth Factor-β1 on Bladder Smooth Muscle Cell Phenotype. II. Modulation of Collagen Organization

Pathological alterations in the relationship between cells and the extracellular matrix have a profound effect on tissue morphology and function. Transforming growth factor-beta1 is thought to have a role in bladder pathology by modulating the bladder smooth muscle cell phenotype and, thus, interactions with the extracellular matrix. We investigated the effects of transforming growth factor-beta1 on the organization of an in vitro extracellular matrix by bladder smooth muscle cells. Rat bladder smooth muscle cells were seeded at different densities (5 x 10(4), 1 x 10(5) and 2.5 x 10(5) cells) on anchored collagen gels and allowed to contract for 18 or 24 hours. Transforming growth factor-beta1 effects on collagen organization were assessed by analyzing collagen fibril orientation using small angle light scattering. Phase contrast microscopy was used to correlate changes in bladder smooth muscle cell morphology to areas of high fibril orientation. Bladder smooth muscle cells were trypsinized from the gels to confirm altered collagen architecture. Transforming growth factor-beta1 altered collagen fibril organization locally but this was only significant in the highest cell population. Transforming growth factor-beta1 induced a population dependent effect, in which bladder smooth muscle cells formed bundles or aggregates. These aggregates corresponded with local areas of high collagen fibril alignment. These changes in collagen architecture were maintained macroscopically after removing the bladder smooth muscle cells. Changes in collagen architecture organization in response to transforming growth factor-beta1 indicate changes in the bladder smooth muscle cell phenotype, resulting in altered cell/extracellular matrix interactions. Changes in this relationship at the microscopic level could be an important component of tissue remodeling and subsequent dysfunction, and indicate a possible role for transforming growth factor-beta1 in bladder pathology cases.

Effective Isolation and Culture of Endothelial Cells in Embryoid Body Differentiated from Human Embryonic Stem Cells

We describe the isolation of endothelial cells from the center region of the attached embryoid body (EB) by a two-step enzyme treatment. The isolated cells from the center and outgrowth region of the EB were characterized separately. As human embryonic stem (hES) cells differentiated in EB, they lost expression of the undifferentiated marker Oct-4, whereas expression levels of endothelial-specific markers were increased. Using RT-PCR, fluorescence-activated cell sorting (FACS) analysis, and immunofluorescence, we have shown that various endothelial cell markers, including platelet/endothelial cell adhesion molecule (PECAM), von Willebrand factor (vWF), Flk-1, and Tie2, were expressed on the attached EB. Compared with the outgrowth region of EB, the center region had a higher population of cells with these endothelial cell markers. Once isolated by the twostep enzyme treatment, cells from the center region continued to differentiate into endothelial cell lineage while expression level of endothelial cell markers in cells from the outgrowth region decreased in subcultures. This study has demonstrated that the isolation of EB by a two-step enzyme treatment is a useful technique to obtain endothelial cell marker-positive cells with high yield. Furthermore, a similar approach can be taken to identify the location and distribution of specific cell types in EB and thereby allow us to isolate and expand specific cell types.

Attachment and proliferation of human periodontal ligament fibroblasts on bioactive glass modified ceramics

In this study, six groups of modified ceramic specimens were constructed and were studied comparatively with dental porcelain (P:control) for their ability to support human periodontal ligament fibroblasts attachment and proliferation. The dental porcelain was initially coated with bioactive glass (PCB) or with a mixture of porcelain and bioactive glass (PCBP) and then calcium-phosphate rich (Ca-P) or hydroxy-carbonate apatite (HCAp) layers were bio-mimetically developed on both surfaces (PCB and PCBP) after immersion in simulated body fluid. The development and characterization of Ca-P and HCAp layers on PCBCa-P, PCBHCAp, PCBPCa-P, PCBPHCAp specimens' surfaces were evaluated by Scanning Electron Microscopy (SEM) and further confirmed by Fourier Transform Infrared Spectroscopy (FTIR). The modified ceramics differed from their controls concerning their surface morphology as evaluated by SEM, and their surface chemical composition (Al, P, Si, Ca, Na and K) as evaluated by Energy Dispersive Spectroscopy (EDS). Almost all modified specimens supported cell attachment, spreading and proliferation at higher extent than the control porcelain specimens. The additional layers of Ca-P or HCAp on PCBP and PCB specimens were found to positively affect cell attachment and proliferation. The highest cell population, of all specimens tested, was observed on PCBPCa-P and PCBPHCAp. The Ca-P particles present on all Ca-P and HCAp coated specimens seemed to be involved in cell adhesion.

Wavy-Walled Bioreactor Supports Increased Cell Proliferation and Matrix Deposition in Engineered Cartilage Constructs

Hydrodynamic forces in bioreactors can decisively influence extracellular matrix deposition in engineered cartilage constructs. In the present study, the reduced fluid shear, high-axial mixing environment provided by a wavy-walled bioreactor was exploited in the cultivation of cartilage constructs using polyglycolic acid scaffolds seeded with bovine articular chondrocytes. Increased growth as defined by weight, cell proliferation and extracellular matrix deposition was observed in cartilage constructs from wavy-walled bioreactors in comparison with those from spinner flasks cultured under the same conditions. The wet weight composition of 4-week constructs from the wavy-walled bioreactor was similar to that of spinner flask constructs, but the former were 60% heavier due to equally higher incorporation of extracellular matrix and 30% higher cell population. It is most likely that increased construct matrix incorporation was a result of increased mitotic activity of chondrocytes cultured in the environment of the wavy-walled bioreactor. A layer of elongated cells embedded in type I collagen formed at the periphery of wavy-walled bioreactor and spinner flask constructs, possibly as a response to local shear forces. On the basis of the robustness and reproducibility of the extracellular matrix composition of cartilage constructs, the wavy-walled bioreactor demonstrated promise as an experimental cartilage tissue-engineering vessel. Increased construct growth in the wavy-walled bioreactor may lead to enhanced mechanical properties and expedited in vitro cultivation.

Growth and production of cholesterol oxidase by alginate-immobilized cells of Rhodococcus equi No. 23.

Rhodococcus equi No. 23 was immobilized in calcium alginate. No detrimental effect on the viability of the test organism was observed during the immobilization procedure. Approx. 98% of the cell population originally present in the alginate solution were immobilized in the gel beads. When the cells of an equal volume of the culture, obtained respectively at exponential phase (12 h preculture), late-exponential phase (20 h preculture) or stationary phase (36 h preculture) were immobilized, the gel beads prepared with the stationary-phase culture were found to contain the highest cell population [about 10(8) colony-forming units (CFU)/g of beads]. In addition, gel beads, prepared with late-exponential-phase culture, exhibited the highest production of cholesterol oxidase (CholOx) after 48 h of incubation. Increasing the bead mass from 3.5 to 14.0 g/100 ml of medium increased CholOx production. However, further increasing the bead mass resulted in a reduction of CholOx production. Furthermore, on the basis of a similar initial cell population, the alginate-immobilized cells of R. equi No. 23 produced a significantly higher amount of CholOx (P<0.05) than did the free cells.

Bone formation induced by calcium phosphate ceramics in soft tissue of dogs: a comparative study between porous alpha-TCP and beta-TCP.

Two kinds of tri-calcium phosphate ceramics (Ca/P = 1.50), alpha-TCP and beta-TCP, which has the same macrostructure and microstructure, but different phase composition, were implanted in dorsal muscles of dogs. The samples were retrieved at 30, 45 and 150 days, respectively, after implantation, and were analyzed histologically. There were critically different tissue responses between alpha-TCP ceramic and beta-TCP ceramic. Higher cell populations were observed inside the pores of beta-TCP than those of alpha-TCP, bone tissue was found in beta-TCP at 45 and 150 days, but no bone formation could be detected in any alpha-TCP implants in this study. On the other hand, the bone tissue in beta-TCP seemed to degenerate at 150 days. The results indicate that porous beta-TCP can induce bone formation in soft tissues of dogs; while the rapid dissolution of the ceramic and the higher local Ca2+, PO(4)3- concentration due to the rapid dissolution of alpha-TCP may resist bone formation in alpha-TCP and the less rapid dissolution of beta-TCP may be detrimental to already formed bone in beta-TCP.

Clinical Studies with D-Trp6-Luteinizing Hormone-Releasing Hormone in Men with Hypogonadotropic Hypogonadism

Three male patients, diagnosed to have hypogonadotopic hypogonadism, were treated with intramuscular injections of 10 microgram of D-Trp6-luteinizing hormone-releasing hormone (D-Trp6-LH-RH) every 8 hours for more than 90 days (13 to 17 weeks). The gonadotropin and testosterone concentrations reached levels twice as great during the treatment as those measured previously and on one occasion increased 5-fold. Before treatment, the patients were clinically in grade 1 on Tanner's scale (Growth at Adolescence, Second Edition. Oxford, Blackwell Scientific Publications, 1962) but after treatment they advanced to grades 2 to 4. Testicular sizes before treatment were between 2 and 2 according to the scales of Waaler et al. (Acta Paediatr Scand [Suppl]249:1, 1974) and afterwards between 4 and 10. The penis of one of the patients grew 3 cm. The testicular biopsy after treatment showed significant increases in the number of Leydig and Sertoli cells. After treatment, spermatogonia were present in increased number, together with a much higher population of cells corresponding to additional stages of development (spermatids). We are of the opinion that therapy with D-Trp6-LH-RH may be beneficial for such patients.

Sizes and numbers of nuclei in the cortex of thymus glands of red-billed weavers Quelea quelea.

The cortex of the thymus glands of embryos, chicks, juveniles, fledglings and adults from several colonies of Quelea quelea were studied using an image analyser (Quantimet 720) to determine cell populations and nuclear sizes. Just prior to hatching the lobes showed a high level of mitosis and consisted of predominantly small lymphocytes. The larger glands of chicks and juveniles had higher cell populations; pyknotic cells and erythrocytes occurred free in the cortex. The lobes of adults were more variable but in general mitosis occurred in enlarging glands of adults from colonies with eggs; most lobes contained pyknotic cells but not in such high numbers as in lobes from chicks and fledglings. Erythrocytes were common, occurring in large numbers in the cortex in some birds. The factors affecting the interpretation of these data are discussed in detail.