Calcif Tissue Int (1992) 51:356-362
Calcified Tissue International 9 1992 Springer-Verlag New York Inc.
Isolation and Characterization of Human Embryonic Osteoblasts Adriana Oliva, 1 Giovanni Marrone, 2 Fulvio Della Ragione, 1 Vincenzo Riccio, 2 Rosanna Palumbo, 1 Fabio Rossano, 3 and Vincenzo Zappia 1 Institutes of 1Biochemistry of Macromolecules, 2pediatric Orthopedics, and 3Microbiology, First Medical School, University of Naples, Federico II, Via Costantinopoli, 16-80138 Napoli, Italy Received January 31, 1992, and in revised form April 22, 1992
Summary. Human osteoblasts were obtained by migration and proliferation of cells from embryonic membranous bone on glass fragments. Light and electron microscopy analyses revealed a typical osteoblast-like appearance with high protein synthesis activity. The cells showed high alkaline phosphatase activity that was associated with plasma membranes and matrix vesicles and was 1,25-dihydroxyvitamin D 3 [1,25(OH)2D3] responsive. In contrast to the adult osteoblasts, embryonic cells could not produce detectable levels of osteocalcin, not even in the presence of 1,25(OH)2D 3. Osteoblasts grown in multilayers produced a thick extracellular matrix, mainly composed of type I collagen, that mineralized in the presence of 10 mM [3-glycerophosphate. Because of their intrinsic osteogenic capacity, embryonic ost e o b l a s t s r e p r e s e n t a valuable model for studying the mineralization process in vitro. In addition, the embryonic origin of these cells renders them a precious experimental system for the elucidation of mechanisms at the basis of differentiation of osteoblastic lineage. Key words: Human embryonic osteoblasts - Alkaline phosphatase - Mineralization - Matrix vesicles - Osteocalcin.
teoblastic activity is provided by osteogenic capacity expressed by in vitro mineralization of the extracellular matrix [151. This paper represents the first report, as far as we know, of the isolation of human embryonic osteoblasts. The cells have been partially characterized by both biochemical markers and light and electron microscopy. Furthermore, the osteoblastic phenotype of the isolated cells has been conclusively demonstrated by their capacity to form a calcified matrix in culture.
Materials and Methods Materials All tissue culture biochemicals were obtained from Flow Laboratories (Irvine, Scotland, UK). Plasticware was from A/S Nunc (Denmark). 1,25(OH)zD3 was a generous gift from Hoffman La Roche (Basel, Switzerland). Anti-human type I and III collagen antibodies were from Institut Pasteur (Lyon, France). All chemicals were obtained from Sigma (St. Louis, MO, USA) and were of the highest grade commercially available. Cell Isolation and Culture
Studies on the molecular mechanisms underlying bone metabolism and regulation have been greatly facilitated by the development of methods for the isolation and culture of bone cells. Since the pioneer studies performed by Peck et al. [1], many attempts have been carried out, essentially using embryonic bone tissues from mouse, rat, or chicken, or employing malignant cells [2-6]. The first report of human bone cell cultures was described by Mills et al. in 1979 [7]. These cultures, derived from patients with Paget's disease, were a mixture of cell types of bone and marrow origin. Subsequently, several methods were developed to isolate human cells with osteoblastic properties as pure as possible. F o r this purpose, both enzymatic digestion [8] and explant procedures [9, 10] have been used. The osteoblast phenotype is generally assessed on the basis of a number of biochemical features, such as high levels of alkaline phosphatase activity (AP) [11], the synthesis of large amounts of type I collagen [9], the production of specific noncollagenous proteins such as osteocalcin [12], and the responsiveness to calcitropic hormones, that is, parathyroid hormone (PTH) and 1,25 dihydroxyvitamin D 3 [I,25(OH)2D3] [13, 14]. However, the best evidence of os-
The isolation technique is based on the ability of osteoblasts to migrate from bone onto glass fragments [15, 16]. Membranous bone (calvariae and scapulae) from an 8-10-week-old human embryo was removed aseptically, accurately stripped of mesenchimal layers, and rinsed several times in phosphate-buffered saline (PBS). The bone was then minced and plated in 100 mm dishes containing DMEM with 10% fetal calf serum (FCS), sodium ascorbate (50 p~g/ml),penicillin (100 U/ml), and streptomycin (100 p.g/ml), in the presence of glass chips. The cultures were incubated at 37~ in a 5% CO2 humidified atmosphere. After 2 weeks of culture, ceils on glass fragments were trypsinized and plated in the same medium at a density of 5 • 103/cm2. The medium was changed every 3-4 days until the cells reached confluence. Cultures between the 3rd and the 10th passage were used in our experiments. In some experiments, FCS was replaced with bovine serum albumin (BSA) at a concentration of 10 mg/ml. Light Microscopy After being thoroughly washed with Tris-buffered saline (TBS) to remove free phosphorus, cell cultures were stained for calcium phosphate salts using the von Kossa methods. Electron Microscopy
Offprint requests to: A. Oliva
Cultures were fixed in situ with 2.5% glutaraldehyde in 0.13 M so-
A. Oliva et al.: Human Embryonic Osteoblasts
357
Table 1. Effect of time in culture on alkaline phosphatase activity of human embryonic osteoblasts.
Table 2. Osteocalcin levels in the incubation medium of confluent cultures of embryonic osteoblasts.
Culture period (day)
Experimental conditions
1
4 7 10
Alkaline phosphatase activity (U • 103/mg protein) 2 0 +-
1
25 -+3 50-+5 70 -+ 5
dium cacodylate buffer, pH 7.4 for 1 hour. After being rinsed several times in the same buffer, the cells were postfixed in 1% osmium tetraoxide, dehydrated in graded ethanol, and embedded in EponAraldite. Ultrathin sections were stained with uranyl-acetate and lead citrate and examined with a Zeiss EM 109 electron microscope.
Non-calcified osteoblasts a Non-calcified osteoblasts plus 10 nM 1,25(OH)2D3 a Non-calcified osteoblasts b Calcified osteoblasts b
Osteocalcin (ng/ml)
Calcified Tissue International 9 1992 Springer-Verlag New York Inc.
Isolation and Characterization of Human Embryonic Osteoblasts Adriana Oliva, 1 Giovanni Marrone, 2 Fulvio Della Ragione, 1 Vincenzo Riccio, 2 Rosanna Palumbo, 1 Fabio Rossano, 3 and Vincenzo Zappia 1 Institutes of 1Biochemistry of Macromolecules, 2pediatric Orthopedics, and 3Microbiology, First Medical School, University of Naples, Federico II, Via Costantinopoli, 16-80138 Napoli, Italy Received January 31, 1992, and in revised form April 22, 1992
Summary. Human osteoblasts were obtained by migration and proliferation of cells from embryonic membranous bone on glass fragments. Light and electron microscopy analyses revealed a typical osteoblast-like appearance with high protein synthesis activity. The cells showed high alkaline phosphatase activity that was associated with plasma membranes and matrix vesicles and was 1,25-dihydroxyvitamin D 3 [1,25(OH)2D3] responsive. In contrast to the adult osteoblasts, embryonic cells could not produce detectable levels of osteocalcin, not even in the presence of 1,25(OH)2D 3. Osteoblasts grown in multilayers produced a thick extracellular matrix, mainly composed of type I collagen, that mineralized in the presence of 10 mM [3-glycerophosphate. Because of their intrinsic osteogenic capacity, embryonic ost e o b l a s t s r e p r e s e n t a valuable model for studying the mineralization process in vitro. In addition, the embryonic origin of these cells renders them a precious experimental system for the elucidation of mechanisms at the basis of differentiation of osteoblastic lineage. Key words: Human embryonic osteoblasts - Alkaline phosphatase - Mineralization - Matrix vesicles - Osteocalcin.
teoblastic activity is provided by osteogenic capacity expressed by in vitro mineralization of the extracellular matrix [151. This paper represents the first report, as far as we know, of the isolation of human embryonic osteoblasts. The cells have been partially characterized by both biochemical markers and light and electron microscopy. Furthermore, the osteoblastic phenotype of the isolated cells has been conclusively demonstrated by their capacity to form a calcified matrix in culture.
Materials and Methods Materials All tissue culture biochemicals were obtained from Flow Laboratories (Irvine, Scotland, UK). Plasticware was from A/S Nunc (Denmark). 1,25(OH)zD3 was a generous gift from Hoffman La Roche (Basel, Switzerland). Anti-human type I and III collagen antibodies were from Institut Pasteur (Lyon, France). All chemicals were obtained from Sigma (St. Louis, MO, USA) and were of the highest grade commercially available. Cell Isolation and Culture
Studies on the molecular mechanisms underlying bone metabolism and regulation have been greatly facilitated by the development of methods for the isolation and culture of bone cells. Since the pioneer studies performed by Peck et al. [1], many attempts have been carried out, essentially using embryonic bone tissues from mouse, rat, or chicken, or employing malignant cells [2-6]. The first report of human bone cell cultures was described by Mills et al. in 1979 [7]. These cultures, derived from patients with Paget's disease, were a mixture of cell types of bone and marrow origin. Subsequently, several methods were developed to isolate human cells with osteoblastic properties as pure as possible. F o r this purpose, both enzymatic digestion [8] and explant procedures [9, 10] have been used. The osteoblast phenotype is generally assessed on the basis of a number of biochemical features, such as high levels of alkaline phosphatase activity (AP) [11], the synthesis of large amounts of type I collagen [9], the production of specific noncollagenous proteins such as osteocalcin [12], and the responsiveness to calcitropic hormones, that is, parathyroid hormone (PTH) and 1,25 dihydroxyvitamin D 3 [I,25(OH)2D3] [13, 14]. However, the best evidence of os-
The isolation technique is based on the ability of osteoblasts to migrate from bone onto glass fragments [15, 16]. Membranous bone (calvariae and scapulae) from an 8-10-week-old human embryo was removed aseptically, accurately stripped of mesenchimal layers, and rinsed several times in phosphate-buffered saline (PBS). The bone was then minced and plated in 100 mm dishes containing DMEM with 10% fetal calf serum (FCS), sodium ascorbate (50 p~g/ml),penicillin (100 U/ml), and streptomycin (100 p.g/ml), in the presence of glass chips. The cultures were incubated at 37~ in a 5% CO2 humidified atmosphere. After 2 weeks of culture, ceils on glass fragments were trypsinized and plated in the same medium at a density of 5 • 103/cm2. The medium was changed every 3-4 days until the cells reached confluence. Cultures between the 3rd and the 10th passage were used in our experiments. In some experiments, FCS was replaced with bovine serum albumin (BSA) at a concentration of 10 mg/ml. Light Microscopy After being thoroughly washed with Tris-buffered saline (TBS) to remove free phosphorus, cell cultures were stained for calcium phosphate salts using the von Kossa methods. Electron Microscopy
Offprint requests to: A. Oliva
Cultures were fixed in situ with 2.5% glutaraldehyde in 0.13 M so-
A. Oliva et al.: Human Embryonic Osteoblasts
357
Table 1. Effect of time in culture on alkaline phosphatase activity of human embryonic osteoblasts.
Table 2. Osteocalcin levels in the incubation medium of confluent cultures of embryonic osteoblasts.
Culture period (day)
Experimental conditions
1
4 7 10
Alkaline phosphatase activity (U • 103/mg protein) 2 0 +-
1
25 -+3 50-+5 70 -+ 5
dium cacodylate buffer, pH 7.4 for 1 hour. After being rinsed several times in the same buffer, the cells were postfixed in 1% osmium tetraoxide, dehydrated in graded ethanol, and embedded in EponAraldite. Ultrathin sections were stained with uranyl-acetate and lead citrate and examined with a Zeiss EM 109 electron microscope.
Non-calcified osteoblasts a Non-calcified osteoblasts plus 10 nM 1,25(OH)2D3 a Non-calcified osteoblasts b Calcified osteoblasts b
Osteocalcin (ng/ml)
