Physiological dynamic strain amplitudes increase human osteoblast proliferation but decrease osteocalcin synthesis in vitro

DESIGN OF IN VITRO HEMOTOXICITY TESTS FOR POTBNTIAL POLYMBRIC DRUG CARRIERS E. Momau’, A. Drochon’, P. Chapon’, D. Domurado*. M. Vet? ‘UTC UMR CNRS 6600 60205 Compiegne ‘C.R.B.A. URA 1465 CNRS 34060 Montpellier INTRODUCTION: A copolymer of the partially quatemizcd poly(tertiary amine)-type, Q-P(TDAE)x, has been proposed for molecular encapsulation and pHinduced release of hydrophobic drugs [l]. With decreasing pH, this polymer changes its conformation from globular to extended, then releasing the entrapped drugs. Unfortunately, intravenous injection to small animals caused cardiovascular distress [2]. In order to understand the mechanism of the polymer toxicity, interactions of this polycation with red blood cells (RBC) and plasma proteins were studied. Our goal is to design in vitro hemotoxicity tests in order to select innocuous polycationic drug carrier systems. METHODS: The experimental in vitro conditions were determined from the in vivo conditions in terms of temperature, polymer concentration and volume of polymer added to RBC suspensions (hematocrit 40%). The effect of plasma proteins on polymer toxicity was studied by suspending RBC in media containing variable amounts of plasma proteins: plasma, mixtures of plasma and serum, serum, albumin buffer, and Tris buffer. In order to investigate electrostatic interactions between polycationic polymer and negatively charged RBC, cells charge was reduced by a neuraminidase treatment. The action of polymer was followed as a function of time, and its effect on RBC membrane mechanical resistance was also tested. Polymer-induced aggregation was followed by microscopy. RBC hemolysis was assessed by measuring hemoglobin and potassium release. RESULTS AND DISCUSSION: Polymer induces instantaneous aggregation and hemolysis (which is a consequence of aggregation). These phenomena increase with polymer concentration. The membrane mechanical properties of non aggregated RBC are not altered. RBC aggregation and hemolysis increase when fibrinogen concentration decreases and they are maximum when no plasma protein is present in suspending medium. This can be due to a competitive interaction of the polymer with plasma proteins or RBC since both are negatively charged. Furthermore, a precipitate formed in a plasma-polymer mixture may be caused through polyanion-polycation complexation. Other physico-chemical properties of proteins could also be involved in this phenomenon. Consequently, plasma proteins have a protective effect against polymer toxicity. The removal of RBC surface charge reduces RBC aggregation and hemolysis when cells are suspended in plasma or serum. This confirms the role of the polymer cationicity in its toxicity. CONCLUSION: Further investigation is needed to understand the behaviour of the polymer on the RBC membrane and its interactions with plasma proteins. The in vitro hemotoxicity test thus developpcd will then be tested with other polycationic macromolecules. REFERENCES: 1. Huguet et al., I. Controlled Release, 1. 217-224, 1985. 2. Vallin et al., Polymer J., 12, 113-124, 1980. CORRESPONDENCE: Elisabeth Moreau UTC UMR CNRS 6600,60205 Compiegne, France. [email protected]

I I’” Conference

PHYSIOLOGICAL DYNAMIC STRAIN AMPLITUDES INCREASE HUMAN OSTBOBLAST PROLIFERATION BUT DECREASE OSTROCALCIN SYNTHES IS IN VITRO D. Kaspar, W. Seidl, C. Neidlinger-Wilke. L. Claes Abteilung Unfallchirurgische Forschung und Biomeehanik, Universimt Ulm, Germany INTRODUCTION: The responsiveness of bone derived cells to mechanical strain has been confirmed by several in vifro investigations. However, cellular reactions of human osteoblasts to low mechanical strain have not yet been proved. For this purpose we determined cell activity of human bone cell cultures after mechanical stimulation by cyclic, uniaxial strain (500-3OOOpstrain)“) We studied the following questions: Does there exist a proliferative or metabolic responsiveness of human osteoblasts at low strain levels? Is there a relationship between strain level and extent of cellular reaction? METHODS: Human osteoblasts were isolated from trabecular or/and cortical bone biopsies and incubated in DMEM on deformable culture dishes”’ for 3 days prior to initiating cell stretching at 1Hz for 1800 cycles/d with 500. 1000, and 3000 ustrain. 24 h after the second stimulation cycle cells were harvested and cell number was determined with a Coulter Counter. Cell bound alkaline phosphatase activity was analyzed by a colotimetric assay, osteocalcin production by an ELISA. For each condition three parallel cultures were tested. Statistics: Wilcoxon signed rank test. RESULTS: Within the studied amplitude range the proliferative response of osteoblasts increased significantly compared to the unstimulated controls (fig. 1: percentages of cell number after cell seeding = 0%) (~~0.05, n=5). No significant difference was observed with regard to alkaline phosphatase activity. Osteocalcin synthesis was significantly reduced compared to unstimulated controls (p-cO.05).

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Pig.1 DISCUSSION: The results indicate an inverse relationship between cell proliferation and osteocalcin synthesis after mechanical strain application. No relationship exists between extent of cellular response and strum magnitude. This observation excludes a dose-dependent cell response within the tested range. CONCLUSION: Our results may contribute to the explanation of mechanisms playing a role during functional bone tissue adaptation to certain mechanical situations. REFERENCES: “Bottlang, M. et al., Biomed. Technik, submitted, 1997. *‘Neidlinger-Wilke, C. et al., JOR, 12.70-78, 1994. ACKNOWLEDGEMBNTS: Supported in pat? by the Deutsche Forschungsgemeinschaft. CORRESPONDENCE: Dip].-Biol. Daniela Kaspar, Abt. Unfallchiturgische Forschung & Biomechanik University of Ulm, Helmholtzstra8e 14,89081 Ulm, Germany. Phone: +49-73 l-5023487, Fax: +49-73 l-5023498 [email protected]

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