Gu Q et al / Acta Pharmacol Sin 2002 Sep; 23 (9): 808-812
Apoptosis of rat osteoblasts in process of calcification in vitro
GU Qi1, ZHU Han-Min
Shanghai Institute of Gerontology, Huadong Hospital, Shanghai 200040, China
ZHANG Xue-Jun1
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
1 Correspondence to Prof GU Qi for osteoblast biology. Phn 86-6248-3180, ext 60702. Fax 86-21-6248-4981. E-mail gu_qi00@hotmail.com
to Dr ZHANG Xue-Jun for apoptosis. Phn 86-6431-5030, ext 2098. Fax 86-21-6433-1090. E-mail zhang_XJ@sunm.shcnc.ac.cn
Received 2002-02-25 Accepted 2002-05-28
KEY WORDS cultured cells; skull; osteoblasts; physiologic calcification; alkaline phosphatase; apoptosis
ABSTRACT
AIM: To establish a cell model of osteoblasts retaining their differentiated phenotype in culture and observe the apoptosis of osteoblasts in the process of calcification using a novel acetylcholinesterase (AChE) staining method. METHODS: Osteoblasts were isolated enzymatically from skull of newborn SD rats; alkaline phosphatase (AKP) activity was determined by reformed cobalt method and azo dye method; mineral deposition was assessed with Von Kossa staining and Fluo-3 staining; a novel AChE staining method was used to assay cellular apoptosis based on the higher expression of AChE in apoptotic cells. RESULTS: During the 44 d of cells cultured, primary rat skull-derived osteoblasts progressively developed into a bone-like tissue of multi-layered nodules of cells with mineralized extra-cellular matrix and the apoptotic cells increased while the matrix calcificated. CONCLUSION: The phenotype of developmental sequence of rat skull-derived osteoblasts can reflect the maturation of osteoblasts in vitro. It is a convenient model for the research of osteoblasts biology.
INTRODUCTION
Osteoblast, a highly biosynthetic cell type capable of complex process of matrix fibrillogenesis, as well as directing many of the activities of osteoclasts, plays an important role in bone deposition and remodeling[1]. Researches on osteoblastic differentiation have been done in the recent years[2,3]. McCarthy et al[2] investigated the possible presence of advanced glycation end product-binding proteins on osteoblast like cells, and found that rat and mouse osteoblast-like cells expressed specific advanced glycation endproduct-binding sites, with an affinity constant depending on the stage of osteoblastic differentiation. It suggested that the biological characteristics of osteoblast varied at different stages of differentiation, and might have clinical meaning.
Apoptosis is recognized as an important component of growth during embryogenesis, organogenesis, and tissue morphogenesis as well as in the maintenance of homeostasis in many adult tissues. The primary function of AChE is to hydrolyse ACh and thus terminates cholinergic neurotransmission[4]. However, recent studies have shown that this gene is also expressed in other cells when they are undergoing apoptosis, and postulated that AChE was induced and played an important role in apoptosis[5]. The AChE protein was found in the cytoplasma at the initiation of apoptosis and then accumulated in the nucleus. Because it is easy, cheap, rapid, and reliable, AChE cytochemical staining is employed to detect apoptotic cells which never express this enzyme while they are in viable condition[6,7].
To isolate osteoblasts that will retain in culture their differentiated phenotype, we derived cells from new born rat skull by sequentially timed-digestions with enzymes. The later digested cell populations express parameters having osteoblasts phenotype[8].
In this study, we studied the incidence of apoptosis in this cultured rat skull osteoblasts, with this novel staining method, based on the high expression of AChE in apoptoic cells. The purpose of this study was to confirm the role of this cell model in osteoblast research.
MATERIALS AND METHODS
Cell culture Osteoblasts were isolated mechanically from newborn rat skull as previously described[9,10]. Briefly, skull (frontal and perietal bones) were dissected from Sprague-Dawley (SD) rats of 24-h old (Grade II, provided by Shanghai Research Center of Life Science, qualified certificate No 005, released from Animal Administration Committee of Chinese Academy of Sciences), endosteum and periosteum were stripped off, and the bone was cut into approximately 1-2 mm2 pieces and digested with trypsin (2.5 g/L, 1:250, Gibco) and collagenase A (2.0 g/L, Sigma). The initial digestion including 20 min and 40 min digests was discarded. The third digestion lasted for another 60 min, and the cells were collected and cultured in small bottles containing a 8 mm×24mm cover glass with an initial seeding at a cell density of 1×107/L. The culture medium was changed every 3 d in all the experiments. After culture for 5-7 d in Dulbecco's modified Eagle's medium (DMEM, Gibco) with 10 % calf serum (Gibco), benzylpenicillin (1×105 U/L), and streptomycin (100 mg/L), cells reached confluence. The cultures were then supplemented with sodium ¦Â-glycerophosphate (10 mmol/L, Sigma) and ascorbate acid (50 mg/L, Shanghai Xinyi Pharmaceuticals Factory, China).
Histochemical analysis Cells attached to cover glasses were washed twice with phosphate buffer solution (PBS) on ice, fixed for 10 min with 2 % paraformaldehyde. Mineral deposition was assessed by staining cells with 3 % AgNO3 for 30 min under bright light (Von Kossa staining)[11]. For alkaline phosphatase (AKP) detection, we used reformed cobalt method[12] and azo dye method (Shanghai Hongxiao Medical Reagents Institution Kits, China).
Fluo-3 staining Fluo-3/AM (Molecular Probes, 10 ¦Ìmol/L) dissolved in Me2SO was added to the cultures. After incubation for 30 min at 37 ºC, cells were washed 3 times by serum-free medium, and observed under the fluorescent microscope.
Identification of apoptotic cells A novel approach of apoptotic cell detection was utilized in the present study. Cells of 10 d, 20 d, and 40 d were first fixed (4 % paraformaldehyde in PBS, pH 7.4). Then they were stained by hematoxylin and observed under light microscope (Nikon, Japan). AChE cytochemical staining was performed according to the method of Karnovsky and Roots[13]. AChE activity was determined as described previously[14].
RESULTS
Morphology From d 2 to d 4 after primary culture, cells were polymorphological and most of them were in dividing phase. After 10-d culture, proliferation was decreased, regions of multilayered cells were apparent, and cells were surrounded by the extra-cellular matrix. By d 21, there were a lot of cell nodules in culture. Histochemical assays showed that there were very few AKP positive cells in the 2-4 d cultures (Fig 1A). However, since d 8 after isolation, cells exhibited heavier staining of AKP activity (Fig 1B, 1C). After d 15, Fluo-3 and Von Kossa staining of the minerized nodules showed hydroxyapatite deposition (Fig 2, 3).
Fig 1. Osteoblasts isolated from rat skull. A) Three days after primary culture. B) Twelve days after primary culture. C) Eighteen days after primary culture. Azo dye staining, ×320.
Fig 2. Osteoblasts isolated from rat skull 25 d after primary culture. Cobalt staining, ×320.
Fig 3. Osteoblasts isolated from rat skull 30 d after primary culture. Fluo-3 staining, ×100.
Apoptosis Apoptosis is a naturally occurring suicide process exhibited in a variety of cell types. The differentiation of rat skull-derived osteoblasts in vitro progressed through three successive stages. During the proliferative stage, a few individual apoptotic cells were observed by AChE staining. Instead, we could find many cells undergoing mitotic division. As cell nodules were formed, proliferation was slowing down. With progressive development of the nodules and calcification of the extra-cellular matrix, a marked increase in the proportion of cells heavily expressed AChE was found (Fig 4A, 4B, 4C).
Fig 4. Osteoblasts isolated from rat skull 40 days after primary culture. A) Early stage of apoptosis, showing high AChE activity in cytoplasma (AChE staining, ×400). B) Mid stage of apoptosis, showing nucleus condensation (AChE staining, ×400). C) Final stage of apoptosis, showing cell debris and apoptotic bodies (AChE staining, ×320).
DISCUSSION
During a 44-d period, primary culture of rat skull-derived osteoblasts progressively developed into a bone tissue-like organization consisting of multi-layered nodules of cells with mineralized extra-cellular matrix. We found that there were a few AKP positive cells in 2-4 d after primary culture. Then the number of AKP positive cells increased, reflecting the enhanced AKP activity during the process of osteoblast maturation. Thus AKP positive cells appeared very early and existed throughout the process of osteoblast development. We have determined the AKP activity by BM/Keysys analyzer (unpublished data). The results showed that the activity of AKP remained elevated and reached the peak value during the stage of extra-cellular matrix formation (d 14-16), and decreased markedly after d 26. The AKP data taken together with the results of the histochemical staining, we suggested that AKP should be regarded as an early marker of osteoblast differentiation.
Once osteoblasts have completed their bone-forming function, they are either entrapped in bone matrix, and become osteocytes or remain on the surface as lining cells. Nonetheless, 50 %-70 % of the osteoblasts initially present at the remodeling site can not be accounted for after enumeration of lining cells and osteocytes[15]. We hypothesize that the missing osteoblasts undergo apoptosis and our results supported this hypothesis with AChE staining method.
With enzymatic removel of extra-cellular matrix which was synthesized in vivo, we established an osteoblast model, in which the extra-cellular matrix that controlled the differentiation process of the surrounding cells, supported the reinitiation of the developmental sequence of proliferation and extra-cellular matrix maturation, mineralization, and apoptosis. This kind of culture could be maintained for up to 140 d with the cells and extra-cellular matrix retaining bone-like features. Therefore this rat skull derived osteoblast model described here is convenient for the research of osteoblast biology.