Proliferation Properties and metabolic Activities of Chondrocytes are [PDF]

from sympathetic and sensory Nerve Fibers in vitro. +1,2Grässel, S; 1Opolka, A; 3Schubert, T; 4Straub, R H ... Sympathe

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Proliferation Properties and metabolic Activities of Chondrocytes are regulated by Neurotransmitters from sympathetic and sensory Nerve Fibers in vitro +1,2Grässel, S; 1Opolka, A; 3Schubert, T; 4Straub, R H; 1Grifka, J +1Orthopaedic Surgery, Experimental Orthopaedics, University of Regensburg, Germany, +2Centre for Medical Biotechnology, BioPark I, Regensburg, Germany 3 Institute of Pathology, University of Regensburg, Germany, 4 Dept. of Internal Medicine, University of Regensburg, Germany [email protected]

INTRODUCTION Fracture repair constitutes a sequential event following bone injury and recapitulates the steps of endochondral ossification observed during embryonic skeletal development and growth. Because of the different phases of fracture healing (inflammation, cartilage formation and callus remodeling) the fracture callus provides an excellent tool for analysis of cartilage and bone formation in adults. Sympathetic nerve fibers of the catecholaminergic phenotype and sensory nerve fibers of the nociceptive phenotype are known to innervate bone and fracture callus, however, little is known about their role in fracture healing and their influence on callus maturation and bone formation. The current research intends to understand the role of the peripheral nervous system for organization and differentiation of a cartilaginous callus and its impact on endochondral ossification. METHODS Animal model: Tibial fractures were set in 8 week old mice. Bone calli were dissected on days 1, 5, 9, 13, 16 and 20 after setting the fractures and parafomaldehyd-fixed paraffin-sections were stained with antibodies against specific cartilage components or neuroproteins. 3D culture: For 3D- pellet culture chondrocytes were isolated from rib cages of 1-3 days old C57Bl/6 mice. The rib cages were carefully dissected, pre-digested in pronase and collagenase (2 mg/ml), and after washing with PBS placed in a Petri dish for over night digestion in fresh collagenase (2 mg/ml). Chondrocyte pellets were formed by centrifugation at 500xg for 10 min. The culture medium was DMEM plus 50 µg/ml ascorbate, 1 mM cysteine, 1 mM pyruvate, and 1% penicillin/streptomycin. Chondrocyte pellets were stimulated with Substance P (SP; 10-9 M, 10-10 M, and 10-11 M) or norepinephrine (NE; 10-6 M, 10-7 M, and 10-8 M) for 7 days and the medium was changed every day throughout the culture period. 1, 4, and 7 days after stimulation pellets were collected for gene and protein expression analysis. Apoptosis was monitored by staining with the membrane impermeable dye propidium iodide (PI). Sections of PI stained and paraffin embedded pellets were treated with RNAse and counterstained with DAPI. Photographs of random fields were taken, and the number of dead and live cells was counted. A minimum of 1.000 cells were counted for each treatment and plotted as percent of cell death. 2D culture: For determination of cell proliferation and viability chondrocytes were kept in 2D- monolayer culture using DMEM (as in 3D- pellet culture set up) plus 10% FCS. Cell proliferation was quantified by ELISA based on the incorporation of BrdU. Primary mouse chondrocytes were seeded in 96well plates at a density of 7.500 cells/well and allowed to attach for 24 hours. Cells were stimulated with SP (10-8, 10−9, 10-10, 10-11, and 10-12 M) or NE (10-5, 10−6, 10-7, 10−8, and 10-9 M) and after 48 hours cell proliferation was assessed by adding a peroxidase coupled anti BrdUantibody (PBS served as negative control). After colour development the signal was monitored at 450/690 nm. A resazurin reduction assay was used as method for assessing cell viability. Primary mouse chondrocytes were seeded in 48-well plates at a density of 1×105 cells/cm2 and allowed to attach for 24 hours. Cells were stimulated with SP (10-8, 10−9, 10-10, 10-11, and 10-12 M) or NE (10-5, 10−6, 10-7, 10−8, and 10-9 M) and after 48 hours cell viability was assessed by adding resazurin in an amount equal to 10 % of the total culture medium volume (PBS served as negative control). After 3 hours at 37°C the fluorescent signal was monitored at 560/590 nm.

RESULTS In our animal fracture model we studied the time-dependent ingrowth of sympathetic and sensory nerve fibres into the fracture callus. Both, substance P - (sensory) and tyrosine hydroxylase - (sympathetic) positive nerves penetrate the callus in early stages of the healing process (day 1), mainly innervating blood vessels in the early granulation tissue. At later time points, when a cartilaginous matrix has been formed SPand TH - positive nerves retract towards the callus periphery. At day 5 and onwards they innervate the perichondrium and the periosteum. Additionally, TH - positive fibers innervate the blood vessels which penetrate the cartilage template during the course of endochondral bone formation. After day 9 of fracture setting both fibers could only be detected sparsely. Both, SP- and TH - positive nerve fibers do not seem to penetrate the cartilage tissue as well as the newly formed woven bone whereas chondrocytes originated from callus tissue express SP and its neurokinin 1 receptor (NK1-R). The expression starts at day 5 and prolongs until day 13. To further analyze the function of SP expressed by chondrocytes we have established an in vitro pellet culture system with chondrocytes from rib cages of newborn mice. Both primary chondrocytes from rib cages and chondrocyte pellets exhibit expression of SP and NK1-R at mRNA and protein level. Alcian blue staining and collagen IX immunohistochemistry revealed a regular cartilage-like extracellular matrix development in the proliferation phase of the chondrocytes which is unaffected by SP and NE. However, after 7 days of stimulation with NE Col1a1 and Col9a1 gene expression was suppressed compared to non-stimulated controls. In our pellet culture system, stimulation with SP and NE chondrocyte alters apoptosis in a dose-dependent manner. In comparison with the unstimulated control, the highest SP concentration (10-9 M) and all NE concentrations lead to an increase of the apoptosis rate after 7 days. NE at a concentration of 10-6 M exerted the strongest effect. In the resazurin assay cell viability is reduced by 40% after stimulation with SP and NE. Here, the most effective concentration is -10 10 M for SP and 10-7 M for NE. This supports our hypothesis, that SP and NE affect the cell viability in a negative way. In the presence of 10% FCS, the addition of SP promoted a dosedependent increase in cell number after 2 days of stimulation. Cell proliferation is increased by 75 % and the most effective concentration of SP is 10-9M. NE, on the other hand, did not induce alteration of cell proliferation in our monolayer culture system. CONCLUSIONS Our fracture model demonstrated a characteristic stage-specific appearance of TH- and SP - positive fibres during the healing progress. Both, TH - and SP - positive nerves penetrate the callus in early stages of the healing process, while at later time points when a cartilaginous matrix has been formed, they retract towards the callus periphery. TH - positive nerve fibres innervate blood vessels within the fracture callus, thereby regulating the blood-flow. Furthermore the release of neuroproteins from the cartilage periphery may affect chondrocyte differentiation and matrix formation in a direct way. Chondrocytes originated from callus tissue express SP and its receptor (NK1-R). This implicates yet unknown, trophic or anti-trophic functions of neuropeptides during musculoskeletal repair processes in adults. Preliminary results of our in vitro chondrocyte pellet culture system suggest opposite effects of SP and NE on chondrocyte proliferation and matrix formation.

Poster No. 848 • 56th Annual Meeting of the Orthopaedic Research Society

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