Sage Journals: Discover world-class research

Abstract

Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs + proteins mixture containing the major components found in platelet extracts [platelet-derived growth factor-BB, transforming growth factor (TGF)-β1, TGF-β2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red–stained nodular areas were detected in cultures treated with GFs + proteins. Results were confirmed in the rat calvaria–derived osteogenic cell culture model. The addition of bone morphogenetic protein 7 or growth and differentiation factor 5 to treated cultures upregulated Runx2 and ALP mRNA expression, but surprisingly, ALP activity was not restored. These results showed that a mixture of GFs + proteins affects the development of the osteogenic phenotype both in human and rat cultures, leading to an increase in the number of cells, but expressed a less differentiated state.

Keywords

cell culture osteoblasts growth factors cell proliferation alkaline phosphatase mineralization

The term growth factor (GF) refers to a class of signaling proteins that stimulate the processes of cell proliferation and/or differentiation in target cells. The induced cellular effects depend on several parameters, which include the (a) concentration of GFs in the extracellular milieu, (b) time and duration of exposure, (c) target cell type and its differentiation state, (d) the expression of specific cell receptors, and (e) the presence of other GFs, which can promote synergistic or antagonistic effects (Giannobile et al. 1997; Schmidmaier et al. 2003; Chaudhary et al. 2004; Huang et al. 2005).

Because of the known effects of individual GFs on cell activities of different cell types, such proteins/peptides have been used in a series of strategies to promote tissue healing, usually in conjunction with delivery systems to control the speed, time, and quantity of release (reviewed in Lieberman et al. 2002; Luginbuehl et al. 2004; Rose et al. 2004; Anitua et al. 2007a; Lee and Shin 2007). In bone tissue, therapies with GFs have been applied to promote, and in some cases to induce, desirable amounts of new bone formation in bone defects, in sites of fracture healing, and adjacent to metal implant devices (Bessho et al. 1999; Ramoshebi et al. 2002; Schliephake 2002; Kloen et al. 2003). Considering that in the body, cells are exposed to diverse GFs simultaneously, ideally any therapy should involve a mixture of GFs with similarities to those implicated in bone tissue healing (Cheifetz et al. 1996; Roethy et al. 2001; Raiche and Puleo 2004; Arosarena and Puleo 2007). Indeed, a well-known therapy with GFs for bone defects is the application of autologous platelet-rich plasma (PRP) (Marx et al. 1998; Dugrillon et al. 2002; reviewed in Anitua et al. 2007b), a concentrate of platelets in plasma. These platelets liberate their content whose major components are platelet-derived growth factor (PDGF) and transforming growth factor β (TGF-β) (Gruber et al. 2003; Lucarelli et al. 2003; Graziani et al. 2006; van den Dolder et al. 2006). Although beneficial clinical results have been reported, some studies have shown that the use of PRP does not promote new bone formation (Ferreira et al. 2005; Hokugo et al. 2005; Gerard et al. 2006; Graziani et al., 2006; Sarkar et al. 2006; Hokugo et al. 2007; Mooren et al. 2007; Ranly et al. 2007; Roussy et al. 2007). These divergent outcomes have been attributed to intra- and interspecies variations in the relative proportions of PRP components (Lacoste et al. 2003; van den Dolder et al. 2006).

The objective of our study was to determine the effects of a PRP-like mixture of GFs and proteins (hereafter referred to as GFs + proteins) on human alveolar bone osteogenic cell cultures. To avoid the inherent variations in PRP preparations and results, we opted to experiment with a well-defined mixture formulated to contain the major components found in PRP extracts. Applications of these standardized mixtures show that they have inhibitory effects on in vitro osteogenic events, ultimately resulting in a complete lack of bone-like nodule formation.

Materials and Methods

Culture of Osteogenic Cells Derived From Human Alveolar Bone

Human alveolar bone fragments were obtained from adult healthy donors, using the research protocols approved by the Committee of Ethics in Research of the School of Dentistry of Ribeirão Preto of the University of São Paulo. Osteogenic cells were isolated by enzymatic digestion of the explants using type II collagenase (Gibco–Life Technologies; Grand Island, NY) as previously described (Mailhot and Borke 1998; Beloti et al. 2006). They were cultured in osteogenic medium, which was composed of Gibco α-MEM (Invitrogen; Carlsbad, CA), supplemented with 10% FBS (Gibco), 50 μg/ml gentamicin (Gibco), 0.3 μg/ml Fungizone (Gibco), 10^-7 M dexamethasone (Sigma; St. Louis, MO), 5 μg/ml ascorbic acid (Gibco), and 7 mM β-glycerophosphate (Sigma). Subconfluent cells in primary cultures were harvested after treatment with 1 mM EDTA (Gibco) and 0.25% trypsin (Gibco) and subcultured on Thermanox coverslips (Nunc; Rochester, NY) in 24-well polystyrene plates (Falcon; Franklin Lakes, NJ) at a density of 2 × 10⁴ cells/well. Cells were grown at 37C in a humidified atmosphere of 5% CO₂ and 95% air, and the medium was changed every 3 or 4 days.

Exposure of Osteogenic Cultures to a Mixture of GFs and Proteins

The mixture of GFs and proteins tested was diluted in osteogenic medium and contained 27 ng/ml recombinant human PDGF-BB, 22 ng/ml recombinant human TGF-β1, 15 ng/ml recombinant human TGF-β2, 3.7 μg/ml serum-derived human albumin, 2 μg/ml plasma-derived human fibronectin, and 0.5 μg/ml platelet-derived thrombospondin, all purchased from Sigma. The GFs and proteins of the mixture were selected from major components found in porcine (Venne et al. 1999) and human (Lacoste et al. 2003; Martineau et al. 2004) platelet preparations. Treated cultures were exposed during the first 7 days to the GFs + proteins mixture, which was added at time 0 and at day 3 and to osteogenic medium alone thereafter. Control cultures were only exposed to the osteogenic medium. To evaluate the influence of known osteogenic factors on cells exposed to GFs + proteins, 100 ng/ml recombinant human bone morphogenetic protein 7 (BMP-7; Sigma) or 20 ng/ml recombinant human growth and differentiation factor 5 [GDF-5 or cartilage-derived morphogenetic protein-1 (CDMP-1); Pepro Tech, Rocky Hill, NJ) as added to GFs + proteins–treated cultures at time 0 and day 3 or at days 7 and 10. Cultures exposed to BMP-7 and GDF-5 were evaluated at day 14 in terms of runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) mRNA expression and ALP activity.

Fluorescence Labeling

At 1, 4, 7, and 14 days, cells were fixed for 10 min at room temperature with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.2. After washing in PB, they were processed for indirect immunofluorescence labeling (de Oliveira et al. 2007) for detection of ALP (Eghbali-Fatourechi et al. 2005) and the nuclear antigen Ki-67 (Robertson et al. 2005). In addition, cell morphology was evaluated by direct fluorescence with fluorophore-conjugated probes. Briefly, cells were permeabilized with 0.5% Triton X-100 in PB for 10 min, followed by blocking with 5% skimmed milk in PB for 30 min. Primary antibodies to human bone ALP (monoclonal B4-78, 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA) and human Ki-67 (polyclonal, 1:70; Diagnostic Biosystems, Pleasanton, CA) were used, followed by a mixture of Alexa Fluor 594 (red fluorescence)–conjugated goat anti-mouse or goat anti-rabbit secondary antibody (1:200; Molecular Probes, Eugene, OR) and Alexa Fluor 488 (green fluorescence)–conjugated phalloidin (1:200; Molecular Probes), which labels the actin cytoskeleton. Replacement of the primary antibodies with PB was used as control. All antibody incubations were performed in a humidified environment for 60 min at room temperature. Between each incubation step, the samples were washed three times (5 min each) in PB. Before mounting for microscope observation, cell nuclei were stained with 300 nM 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI; Molecular Probes) for 5 min and samples were briefly washed with dH₂O. Thermanox coverslips were placed face up on glass slides and mounted with a Fisherbrand 12-mm-round glass coverslip (Fisher Scientific; Suwanee, GA) using an antifade mounting medium (Vectashield; Vector Laboratories, Burlingame, CA). The samples were examined under epifluorescence using a Leica DMLB light microscope, with N Plan (×10/0.25, ×20/0.40) and HCX PL Fluotar (×40/0.75) objectives, outfitted with a Leica DC 300F digital camera (Leica; Bensheim, Germany). The acquired digital images were processed with Adobe Photoshop software (version 7.0; Adobe Systems, San Jose, CA).

Growth Curve and Cell Viability

Cells grown for periods of 1, 4, 7, and 10 days were enzymatically detached from Thermanox coverslips using 1 mM EDTA, 1.3 mg/ml collagenase, and 0.25% trypsin solution (Gibco; Invitrogen). Total number of cells/well and percentage of viable and non-viable cells were determined after Trypan blue (Sigma) staining using a hemacytometer.

Proportion of Cycling Cells

The proportion of cycling cells at days 1 and 4 was determined by means of double Ki-67/DAPI labeling. The total number of nuclei (DAPI stained) and of cycling cells (Ki-67 positive; Scholzen and Gerdes 2000) on control and treated cultures was calculated by light microscope counting under epifluorescence at ×40 objective. A minimum of 200 total cells were counted on three Thermanox coverslips at each time point.

Total Protein Content

Total protein content was calculated at days 4, 7, 10, and 14, according to the method described by Lowry et al. (1951). The wells were filled with 2 ml of deionized water. After five cycles of thermal-shock (alternating temperature between 15 min at 37C and 20 min at −20C), 1 ml of the sample from each well was mixed with 1 ml of Lowry solution (Sigma) and left for 20 min at room temperature. Subsequently, 0.5 ml of phenol reagent of Folin and Ciocalteau (Sigma) was added. This was left for 30 min at room temperature, after which absorbance was measured using a spectrophotometer (CE3021; Cecil, Cambridge, UK) at 680 nm. Total protein content was calculated from a standard curve using BSA (Sigma), giving a range of 25–400 μg protein/ml, and data were expressed as micrograms of protein per milliliter.

ALP Activity

For cultures at days 4, 7, 10, and 14, aliquots of the same wells used for calculating total protein content were assayed to measure ALP activity. p-Nitrophenyl-phosphatase (PNPPase) activity was assayed discontinuously at 37C in a spectrophotometer (Spectronic Genesys 20; Thermo Electron, Madison, WI) by following the liberation of p-nitrophenolate ion (∊ = 1 M, pH 13 = 17,600 M^-1 cm^-1) at 410 nm. Standard conditions were 50 mM AMPOL buffer, pH 10.0, containing 2 mM MgCl₂ and 10 mM PNPP in a final volume of 1.0 ml as previously described (Simão et al. 2007). The reaction was stopped by adding 1 ml of 1M NaOH. All determinations were carried out in duplicate, and the initial velocities were constant for at least 90 min provided that <5% of substrate was hydrolyzed. Controls without added enzyme were included in each experiment to allow the determination of non-enzymatic hydrolysis of substrate. Data were expressed as nmoles p-nitrophenyl per microgram protein per minute. Cultures were also stained with Fast red for in situ histochemical detection of ALP activity (Majors et al. 1997) at days 10 and 14.

RNA Extraction and Quantitative Real-time RT-PCR

The primer sequences, the predicted amplicon sizes, and the annealing and melting temperatures, which were designed using the PrimerExpress software (Applied Biosystems; Foster City, CA), are depicted in Table 1.

Total RNA from cultures at day 14 was extracted using the Promega RNA extraction kit (Promega; Madison, WI), according to the manufacturer's instructions. The concentration of RNA was determined by optical density at a wavelength of 260 nm, using the GeneQuant (Amersham Biosciences; Piscataway, NJ). cDNA was synthesized using 2 μg of RNA through a reverse transcription reaction (M-MLV reverse transcriptase; Promega). Real-time PCR quantitative mRNA analyses were performed in an ABI Prism 7000 Sequence Detection System using the SybrGreen system (Applied Biosystems). SybrGreen PCR Master-Mix (Applied Biosystems), specific primers, and 2.5 ng of cDNA were used in each reaction. The standard PCR conditions were 95C (10 min) and 40 cycles of 94C (1 min), 56C (1 min), and 72C (2 min), followed by the standard denaturation curve. For mRNA analyses, the relative level of gene expression was calculated in reference to β-actin expression and the control group using the cycle threshold (Ct) method (Livak and Schmittgen 2001).

Table 1

Primers sequences and reaction properties

Target	Sense and antisense sequences	At (C)	Mt (C)	Product length (bp)
β-actin	ATGTTTGAGACCTTCAACA	56	75	495
	CACGTCAGACTTCATGATGG
ALP	ACGTGGCTAAGAATGTCATC	60	86	475
	CTGGTAGGCGATGTCCTTA
Runx2	TATGGCACTTCGTCAGGATCC	61	83	110
	AATAGCGTGCTGCCATTCG

At, annealing temperature; Mt, melting temperature.

Figures 1–4

Figure 1

Epifluorescence to detect actin cytoskeleton (green fluorescence) and cell nuclei (blue fluorescence) of control (A–D) and growth factors (GFs) + proteins–treated (E–H) human alveolar bone–derived osteoblastic cell cultures grown on Thermanox coverslips. Whereas the progression of control cultures resulted in the formation of focal, calcified nodular areas at day 14 (D, red fluorescence), GFs + proteins–treated cultures showed a significantly higher number of cells from day 4 onward (F), cells generally oriented in the same direction (G), and no bonelike nodule formation at day 14 (H). Bars: A, B, D, E, F, H = 100 μm; C, G = 50 μm.

Figure 2

Epifluorescence to detect actin cytoskeleton (green fluorescence) and cell nuclei (blue fluorescence) at day 1 of control (A) and GFs + proteins–treated (B) human alveolar bone–derived osteoblastic cell cultures grown on Thermanox coverslips. A higher proportion of treated cells exhibited relatively longer cytoplasmic extensions (compare B with A). Bar = 50 μm.

Figure 3

Double labeling Ki-67/4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (red and blue fluorescence, respectively) to determine the proportions of cycling cells at days 1 (A, C) and 4 (B, D) of control (A, B) and GFs + proteins–treated (C, D) human alveolar bone–derived osteoblastic cell cultures grown on Thermanox coverslips. A significant enhancement in the proportion of cycling cells of at least 40% was observed for GFs + proteins–treated cultures. Bars: A, C = 100 μm; B, D = 50 μm.

Figure 4

Double labeling alkaline phosphatase (ALP; red fluorescence)/DAPI (A, B, D, E) and in situ histochemical detection of ALP activity (Fast red staining; C, F) of control (A–D) and GFs + proteins–treated (E–H) human alveolar bone–derived osteoblastic cell cultures grown on Thermanox coverslips at days 4, 7, and 14. Cultures exposed to GFs + proteins exhibited significantly reduced amounts of ALP. Bars: A, D = 100 μm; B, E = 50 μm; C, F = 200 μm.

Table 2

Quantitative analysis (mean ± SD, n) of total cell number, cell viability, cycling cells, total protein content, ALP activity, number of calcified nodular areas of human alveolar bone–derived osteoblastic cell cultures and proportion of Alizarin red–stained areas of rat calvaria osteogenic cell cultures in control and GFs + proteins–treated cultures grown on Thermanox coverslips

Parameters	Time points (days)	Control	GFs + proteins	Mann-Whitney test
Total cell number (×10⁴)	1	1.9 ± 0.2 (5)	2.1 ± 0.4 (5)	NS a
	4	2.5 ± 0.4 (5)	9.2 ± 1.4 (5)	S b
	7	5.9 ± 1.2 (5)	44.6 ± 6 (5)	S b
	10	10.4 ± 1.5 (5)	30.9 ± 4 (5)	S b
Cell viability (%)	4	100 ± 0 (5)	98.4 ± 2.6 (5)	NS a
	7	97.4 ± 2.7 (5)	99.3 ± 0.1 (5)	NS a
	10	97.7 ± 0.8 (5)	99.1 ± 0.4 (5)	S b
Cycling cells (% Ki-67–positive cells)	1	56.5 ± 11.3 (3)	80 ± 6.8 (3)	S c
	4	59.6 ± 8.3 (3)	86.7 ± 4.9 (3)	S c
Total protein content (μg/ml)	4	11.6 ± 2.3 (5)	27.4 ± 2.4 (5)	S b
	7	20.6 ± 1.8 (5)	78.1 ± 6.7 (5)	S b
	10	33.4 ± 4.4 (5)	79.5 ± 5.9 (5)	S b
	14	40.1 ± 6.7 (5)	73.4 ± 16.7 (5)	S b
ALP activity (nmol p-nitrophenyl/μg protein/min)	4	0.2 ± 0.02 (5)	0.06 ± 0.004 (5)	S b
	7	0.19 ± 0.04 (5)	0.01 ± 0.002 (5)	S b
	10	0.39 ± 0.04 (5)	0.007 ± 0.006 (5)	S b
	14	0.75 ± 0.15 (5)	0.06 ± 0.008 (5)	S b
Number of calcified nodular areas	14	2.6 ± 1.7 (5)	0.0 ± 0.0 (5)	S b
Percent of mineralized bone-like nodules (rat calvarial cells)	14	18.4 ± 1.8 (5)	0.0 ± 0.0 (5)	S b

^a Non-significant (>0.05).

^b Significant (p<0.01).

^c Significant (p = 0.05).

ALP, alkaline phosphatase; GF, growth factor.

Mineralized Bone-like Nodule Formation

At day 14, cultures were washed in Hanks' balanced salt solution (Sigma) and fixed in 70% ethanol for 60 min at 4C. The samples were washed in PBS and dH₂O and stained with 2% Alizarin red (Sigma), pH 4.2, for 15 min at room temperature, followed by permeabilization with 0.5% Triton X-100 and DAPI staining for epifluorescence microscopic detection. To validate the results obtained with human alveolar osteogenic cells, the effect of the GFs + proteins mixture was also tested on the rat calvaria osteogenic cell culture system (Nanci et al. 1996; de Oliveira et al. 2007). Cells were grown under the same experimental conditions, and mineralized bone-like nodule formation was evaluated at day 14. Alizarin red–stained calvarial cultures were photographed with a high-resolution digital camera (Canon EOS Digital Rebel Camera, 6.3 Megapixel CMOS sensor, with a Canon EF 100 mm f/2.8 macro lens; Canon, Lake Success, NY), and the percentage of the substrate area occupied by Alizarin red–stained nodules was determined using Image Tool software (University of Texas Health Science Center; San Antonio, TX).

Statistical Analysis

Data are presented as mean ± SD. The χ² test was used to determine normality of data sets. Comparisons were carried out using the non-parametrical Mann-Whitney test for two independent samples or Kruskal-Wallis test for multiple comparisons, followed by the Fischer test based on rank (level of significance: 5%), where appropriate. The results described below are representative of a minimum of two sets of human and rat osteogenic cell cultures. The real-time PCR analysis was carried out in triplicate.

Figure 5

Bone-like nodule formation in control (A–C) and GFs + proteins–treated (D–F) rat calvarial cell cultures grown on Thermanox coverslips. The complete lack of bone-like nodule formation was clearly noticed for the GFs + proteins–treated cultures at days 10 (A,D, conventional light; insets, epifluorescence of DAPI staining for DNA) and 14 (B, E, conventional light of Alizarin red staining for calcium deposits; C, F, macroscopic observation). Bars: A, D = 600 μm; A, D insets and B, E = 800 μm; C, F = 2.4 mm.

Results

Epifluorescence showed marked differences in cell morphology and tissue architecture during the progression of the cultures (Figure 1). At day 1, although both control and treated cells were polygonal in shape, cytoplasmic extensions were observed in a higher proportion of treated cells (Figures 1A, 1E, and 2). At day 4, whereas treated cultures were confluent with areas of cell multilayering and abundant mitotic figures, control ones formed discrete cell aggregates (Figures 1B and 1F). At day 7, control cells formed a confluent layer and were oriented randomly, whereas in the GFs + proteins–treated group, cells generally oriented in the same direction (Figures 1C and 1G). Noticeably, no areas of calcified matrix were detected in treated cultures at day 14, whereas control cultures exhibited occasional Alizarin red–stained areas (Figures 1D and 1H).

Growth analyses indicated that there were significantly more cells in treated cultures from day 4 onward (Table 2). Significantly higher values in cell viability were detected for treated cultures only at day 10 (Table 2). The proportion of cycling cells was significantly higher for treated cultures both at days 1 and 4 (Figure 3; Table 2) compared with the control. Indeed, an increase of at least 40% in the population of Ki-67–positive cells was detected during the first 7 days of treatment with GFs + proteins.

Total protein content was significantly higher in treated cultures at all time points (Table 2) and the amount of ALP, as judged by activity measurements, Fast red staining and immunolabeling of the protein was reduced (Figure 4; Table 2).

Both control human and rat cell cultures exhibited an osteogenic phenotype as determined by qualitative and quantitative analysis of mineralized nodules (Figures 1D and 5A–5C, respectively; Table 2). Conversely, treated cultures showed no bone-like nodule formation at days 10 (Figure 5D) and 14 (Figures 1H, 5E, and 5F; Table 2).

Expression of Runx2 mRNA was not affected by the exposure of cultures to the mixture of GFs + proteins (Figure 6A; Table 3). However, exposure of treated cultures to BMP-7 or GDF-5 generally resulted in a significant increase in Runx2 mRNA above levels found in untreated and GFs + proteins cultures (Figure 6A; Table 3). Strikingly, treatment with GFs + proteins resulted in the downregulation of ALP mRNA expression (Figure 6B; Table 3). Such inhibitory effect was reverted by the exposure of treated cultures to BMP-7 (0–7 days), GDF-5 (0–7 days), or GDF-5 (7–14 days) (Figure 6B; Table 3). However, ALP activity remained at significantly reduced levels for all groups compared with the control [control, 0.6 ± 0.14; GFs + proteins, 0.03 ± 0.03; BMP-7 (0–7 days), 0.009 ± 0.01; GDF-5 (0–7 days), 0.01 ± 0.006; BMP-7 (7–14 days), 0.05 ± 0.05; GDF-5 (7–14 days), 0.02 ± 0.01; Table 3).

Discussion

The results of this study showed that a mixture of GFs + proteins containing the major components found in platelet extracts promotes a significant increase in cycling cells and cell number but dramatically reduces the osteogenic potential of both human alveolar bone and rat calvaria–derived cell cultures. There was a reduction in ALP activity and mRNA expression and inhibition of bone-like nodule formation. In addition, we showed that, whereas the inhibitory effects on ALP mRNA expression can be reverted by exposure of cells to BMP-7 or GDF-5, ALP activity is not restored.

During the processes of bone formation and repair, a complex sequence of signaling events takes place, which includes interaction of a series of GFs and other matrix proteins with receptors in various target cells at different stages of differentiation. The ensuing cell signaling events will promote the development of the osteoblastic phenotype, which ultimately results in bone tissue formation and remodeling. Because bone repair is a multifactorial process, molecular therapies for the local delivery of mixtures of GFs have been devised, such as found in PRP extracts (Marx et al. 1998; Dugrillon et al. 2002; reviewed in Anitua et al. 2007b). These, however, have been reported to show significant intra- and interspecies variations in relative proportions of components, to which differences in the quality and quantity of new tissue formation and in clinical outcomes have been attributed (van den Dolder et al. 2006). To avoid this variability, we prepared from recombinant human GFs and proteins a well-defined mixture that contained the major components of PRP. Addition of our standardized mixture of GFs + proteins allowed obtaining consistent results throughout the various sets of experiments. Interestingly, despite interspecies variations, inhibition of bone-like nodule formation occurred in both human alveolar bone and rat calvaria osteogenic cell cultures. Although FBS in the culture medium contains GFs and proteins, the results obtained clearly correlate with the addition of our PRP-like mixture.

Figure 6

Effects of the exposure of GFs + proteins–treated cultures to bone morphogenetic protein (BMP)-7 or growth and differentiation factor (GDF)-5 (0–7 or 7–14 days) on Runx2 (A) and ALP (B) mRNA relative expression at day 14 using real-time PCR in triplicate. The results are presented as the expression of the target mRNAs normalized to β-actin and to the control group (mean ± SD).

Table 3

Comparative analysis (Fischer posttest) of the effect of BMP-7 or GDF-5 on the expression of Runx2 and ALP mRNA (Kruskal-Wallis test, p<0.01) and ALP activity (Kruskal-Wallis test, p<0.05) in GFs + proteins–treated human alveolar bone–derived osteoblastic cell cultures at day 14 on Thermanox coverslips

Comparisons (Fischer posttest)	Runx2 mRNA	ALP mRNA	ALP activity
Control × GFs + proteins	NS a	0.1%	1%
Control × BMP-7 (0–7)	0.1%	5%	0.1%
Control × GDF-5 (0–7)	5%	NS a	1%
Control × BMP-7 (7–14)	0.1%	0.1%	1%
Control × GDF-5 (7–14)	0.1%	NS a	1%
GFs + proteins × BMP-7 (0–7)	0.1%	1%	0.1%
GFs + proteins × GDF-5 (0–7)	NS a	0.1%	NS a
GFs + proteins × BMP-7 (7–14)	1.0%	NS a	NS a
GFs + proteins × GDF-5 (7–14)	0.1%	0.1%	NS a
BMP-7 (0–7) × GDF-5 (0–7)	0.1%	1%	NS a
BMP-7 (0–7) × BMP-7 (7–14)	1%	NS a	NS a
BMP-7 (0–7) × GDF-5 (7–14)	NS a	0.1%	NS a
GDF-5 (0–7) × BMP-7 (7–14)	5%	0.1%	NS a
GDF-5 (0–7) × GDF-5 (7–14)	0.1%	NS a	NS a
BMP-7 (7–14) × GDF-5 (7–14)	5%	0.1%	NS a

^a Non-significant (p>0.05).

BMP-7, bone morphogenetic protein 7; GDF-5, growth and differentiation factor 5; ALP, alkaline phosphatase; GF, growth factor.

Inhibition of ALP activity and mRNA expression by GFs + proteins and no changes in Runx2 mRNA levels compared with control indicate that the expanding cell population was made up of cells with a less differentiated osteoblastic phenotype and thereby decreased capacity to lay down an osteogenic matrix and/or nucleate biological apatite. The lack of calvarial cell multilayering typically observed during development of bone-like nodules lends additional support to the interpretation that the treated osteogenic cells did not achieve a fully differentiated state. It has been proposed that the role of tissue nonspecific ALP (TNAP) in the bone matrix is to generate the Pi needed for hydroxyapatite crystallization (Majeska and Wuthier 1975; McComb et al. 1979; Fallon et al. 1980; reviewed in Bonucci 2007) and that such enzyme is crucial for the initiation, but not for the progression/maintenance, of the matrix mineralization process (Bellows et al. 1991). TNAP may also hydrolyze the mineralization inhibitor PPi to facilitate mineral precipitation and growth (Moss et al. 1967; McComb et al. 1979; Rezende et al. 1998; Millán 2006; Addison et al. 2007; Bonucci 2007). In addition, it has been shown that a specific level of TNAP activity has to be reached for the initiation of apatite nucleation and that a moderate reduction in TNAP expression levels and enzyme activity could be sufficient to impair the mineralization process (Wennberg et al. 2000). Therefore, in this study, the absence of bone-like nodule formation in GFs + proteins–treated cultures could be caused not only by an effect on the progression of osteoblastic differentiation (cells not reaching a fully differentiated state) but also by the reduced ALP levels they exhibited. Such an inhibitory effect has also been described after long-term exposure of calvarial cells to platelet extracts (Soffer et al. 2004).

In an attempt to rescue the osteogenic phenotype, treated cultures were exposed to BMP-7 or GDF-5, two osteogenic GFs known to promote development of the osteoblastic phenotype (King et al. 1996; Wozney 2002; Kuniyasu et al. 2003). Both BMP-7 and GDF-5 upregulated Runx2 mRNA expression and reverted downregulation of ALP mRNA expression. Surprisingly, despite upregulation of mRNA, ALP activity was not restored. These results indicate that monitoring of mRNA expression levels alone should be interpreted with caution and support the concept that the relation between transcription and translation is complex (Mehra et al. 2003). Indeed, the lack of correlation between mRNA and protein expression has been reported elsewhere (Lee et al. 2003).

In conclusion, this study showed that exposure of osteogenic cells to a well-defined mixture of GFs + proteins, such as found in platelet preparations, causes downregulation of the osteogenic phenotype. The data are consistent with previous reports showing that PRP preparations do not promote bone formation under certain conditions. Intriguingly, the inhibitory effect of GFs + proteins was expressed even in presence of BMP-7 and GDF-5. However, ALP mRNA levels were restored in cultures first treated with GFs + proteins followed by GDF-5. Our results support the concept that the timing and duration of multiple component molecular therapies play an important role in their outcome and indicate the need for more comprehensive studies using combinations of GFs and proteins aiming to enhance bone formation particularly under unfavorable/adverse conditions.

Footnotes

Acknowledgements

This study was supported by the State of São Paulo Research Foundation (FAPESP; Brazil) and by the National Council of Scientific and Technological Development (CNPq; Brazil). A.N. wishes to acknowledge support from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. M.A.O. is the recipient of a Masters scholarship from FAPESP. W.M.A.M. and K.E.V.S. were recipients of internship scholarships from FAPESP and CNPq, respectively. G.E.C. is grateful for a postdoctoral fellowship from FAPESP. The mouse monoclonal anti-human bone ALP antibody (B4-78), developed by Jerry A. Katzmann, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biological Sciences of the University of Iowa (Iowa City, IA).

We thank Roger Rodrigo Fernandes and Júnia Ramos for invaluable technical assistance.

References

Addison

Azari

Sørensen

Kaartinen

McKee

(2007) Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity. J Biol Chem 282:15872–15883

Anitua

Sánchez

Orive

Andia

(2007a) Delivering growth factors for therapeutics. Trends Pharmacol Sci 29:37–41

Anitua

Sánchez

Orive

Andia

(2007b) The potential impact of the preparation rich in growth factors (PRGF) in different medical fields. Biomaterials 28:4551–4560

Arosarena

Puleo

(2007) In vitro effects of combined and sequential bone morphogenetic protein administration. Arch Facial Plast Surg 9:242–247

Bellows

Aubin

Heersche

(1991) Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone Miner 14:27–40

Beloti

Oliveira

Gimenes

Zaghete

Bertolini

Rosa

(2006) In vitro biocompatibility of a novel membrane of the composite poly (vinylidene-trifluoroethylene)/barium titanate. J Biomed Mater Res A 79:282–288

Bessho

Carnes

Cavin

Chen

Ong

(1999) BMP stimulation of bone response adjacent to titanium implants in vivo. Clin Oral Implants Res 10:212–218

Bonucci

(2007) Main suggested calcification mechanisms: cells. In: Biological Calcification. Normal and Pathological Processes in the Early Stages. Berlin-Heidelberg, Springer, 491–506

Chaudhary

Hofmeister

Hruska

(2004) Differential growth factor control of bone formation through osteoprogenitor differentiation. Bone 34:402–411

10.

Cheifetz

McCulloch

Sampath

Sodek

(1996) Influence of osteogenic protein-1 (OP-1; BMP-7) and transforming growth factor-β 1 on bone formation in vitro. Connect Tissue Res 35:71–78

11.

de Oliveira

Zalzal

Beloti

Rosa

Nanci

(2007) Enhancement of in vitro osteogenesis on titanium by chemically produced nanotopography. J Biomed Mater Res A 80:554–564

12.

Dugrillon

Eichler

Kern

Kluter

(2002) Autologous concentrated platelet-rich plasma (cPRP) for local application in bone regeneration. Int J Oral Maxillofac Surg 31:615–619

13.

Eghbali-Fatourechi

Lamsam

Fraser

Nagel

Riggs

Khosla

(2005) Circulating osteoblast-lineage cells in humans. N Engl J Med 352:1959–1966

14.

Fallon

Whyte

Teitelbaum

(1980) Stereospecific inhibition of alkaline phosphatase by L-tetramisole prevents in vitro cartilage calcification. Lab Invest 43:489–494

15.

Ferreira

Gomes

MCC

Filho

Granjeiro

Simões

CMO

Magini

(2005) Platelet-rich plasma influence on human osteoblasts growth. Clin Oral Implants Res 16:456–460

16.

Gerard

Carlson

Gotcher

Jacobs

(2006) Effects of platelet-rich plasma on the healing of autologous bone grafted mandibular defects in dogs. J Oral Maxillofac Surg 64:443–451

17.

Giannobile

Whitson

Lynch

(1997) Non-coordinate control of bone formation displayed by growth factor combinations with IGF-I. J Dent Res 76:1569–1578

18.

Graziani

Ivanovski

Cei

Ducci

Tonetti

Gabriele

(2006) The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts. Clin Oral Implants Res 17:212–219

19.

Gruber

Karreth

Frommlet

Fischer

Watzek

(2003) Platelets are mitogenic for periosteum-derived cells. J Orthop Res 21:941–948

20.

Hokugo

Ozeki

Kawakami

Sugimoto

Mushimoto

Morita

Tabata

(2005) Augmented bone regeneration activity of platelet-rich plasma by biodegradable gelatin hydrogel. Tissue Eng 11:1224–1233

21.

Hokugo

Sawada

Hokugo

Iwamura

Kobuchi

Kambara

Morita

. (2007) Controlled release of platelet growth factors enhances bone regeneration at rabbit calvaria. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 104:44–48

22.

Huang

Kaigler

Rice

Krebsbach

Mooney

(2005) Combined angiogenic and osteogenic factor delivery enhances bone marrow stromal cell-driven bone regeneration. J Bone Miner Res 20:848–857

23.

King

Storm

Marker

Dileone

Kingsley

(1996) The role of BMPs and GDFs in development of region-specific skeletal structures. Ann NY Acad Sci 785:70–79

24.

Kloen

Di Paola

Borens

Richmond

Perino

Helfet

Goumans

(2003) BMP signaling components are expressed in human fracture callus. Bone 33:362–371

25.

Kuniyasu

Hirose

Ochi

Yajima

Sakaguchi

Murata

Pohl

(2003) Bone augmentation using rhGDF-5-collagen composite. Clin Oral Implants Res 14:490–499

26.

Lacoste

Martineau

Gagnon

(2003) Platelet concentrates: effects of calcium and thrombin on endothelial cell proliferation and growth factor release. J Periodontol 74:1498–1507

27.

Lee

Shaw

Choe

Mehra

Hatzimanikatis

Lee

(2003) Insights into the relation between mRNA and protein expression patterns: II. Experimental observations in Escherichia coli. Biotechnol Bioeng 84:834–841

28.

Lee

Shin

(2007) Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering. Adv Drug Deliv Rev 59:339–359

29.

Lieberman

Daluiski

Einhorn

(2002) The role of growth factors in the repair of bone. Biology and clinical applications. J Bone Joint Surg Am 84:1032–1044

30.

Livak

Schmittgen

(2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-^ΔδCT method. Methods 25:402–408

31.

Lowry

Rosebrough

Farr

Randall

(1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

32.

Lucarelli

Beccheroni

Donati

Sangiorgi

Cenacchi

Del Vento

Meotti

. (2003) Platelet-derived growth factors enhance proliferation of human stromal stem cells. Biomaterials 24:3095–3100

33.

Luginbuehl

Meinel

Merkle

Gander

(2004) Localized delivery of growth factors for bone repair. Eur J Pharm Biopharm 58:197–208

34.

Mailhot

Borke

(1998) An isolation and in vitro culturing method for human intraoral bone cells derived from dental implant preparation sites. Clin Oral Implants Res 9:43–50

35.

Majeska

Wuthier

(1975) Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase. Biochim Biophys Acta 391:51–60

36.

Majors

Boehm

Nitto

Midura

Muschler

(1997) Characterization of human bone marrow stromal cells with respect to osteoblastic differentiation. J Orthop Res 15:546–557

37.

Martineau

Lacoste

Gagnon

(2004) Effects of calcium and thrombin on growth factor release from platelet concentrates: kinetics and regulation of endothelial cell proliferation. Biomaterials 25:4489–4502

38.

Marx

Carlson

Eichstaedt

Schimmele

Strauss

Georgeff

(1998) Platelet-rich plasma: growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:638–646

39.

McComb

Bowers

Posen

(1979) Alkaline Phosphatase. New York, Plenum

40.

Mehra

Lee

Hatzimanikatis

(2003) Insights into the relation between mRNA and protein expression patterns: I. theoretical considerations. Biotechnol Bioeng 84:822–833

41.

Millán

(2006) Mammalian Alkaline Phosphatases: From Biology to Applications in Medicine and Biotechnology. Weinhein, Wiley

42.

Mooren

Merkx

Bronkhorst

Jansen

Stoelinga

(2007) The effect of platelet-rich plasma on early and late bone healing: an experimental study in goats. Int J Oral Maxillofac Surg 36:626–631

43.

Moss

Eaton

Smith

Whitby

(1967) Association of inorganic-pyrophosphatase activity with human alkaline-phosphatase preparations. Biochem J 102:53–57

44.

Nanci

Zalzal

Gotoh

McKee

(1996) Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary cultures. Microsc Res Tech 33:214–231

45.

Raiche

Puleo

(2004) In vitro effects of combined and sequential delivery of two bone growth factors. Biomaterials 25:677–685

46.

Ramoshebi

Matsaba

Teare

Renton

Patton

Ripamonti

(2002) Tissue engineering: TGF-β superfamily members and delivery systems in bone regeneration. Expert Rev Mol Med 4:1–11

47.

Ranly

Lohmann

Andreacchio

Boyan

Schwartz

(2007) Platelet-rich plasma inhibits demineralized bone matrix-induced bone formation in nude mice. J Bone Joint Surg Am 89:139–147

48.

Rezende

Pizauro

Ciancaglini

Leone

(1998) Inorganic pyrophosphate-phosphohydrolytic activity associated with rat osseous plate alkaline phosphatase. Cell Mol Biol 44:293–302

49.

Robertson

Fisher

Pearce

Shay

Wright

Cavanagh

. (2005) Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line. Invest Ophthalmol Vis Sci 46:470–478

50.

Roethy

Fiehn

Suehiro

Shimizu

Wang

. (2001) A growth factor mixture that significantly enhances angiogenesis in vivo. J Pharmacol Exp Ther 299:494–500

51.

Rose

Hou

Oreffo

(2004) Delivery systems for bone growth factors: the new players in skeletal regeneration. J Pharm Pharmacol 56:415–427

52.

Roussy

Duchesne

MPB

Gagnon

(2007) Activation of human platelet-rich plasmas: effect on growth factors release, cell division and in vivo bone formation. Clin Oral Implants Res 18:639–648

53.

Sarkar

Augat

Shefelbine

Schorlemmer

Huber-Lang

Claes

Kinzl

. (2006) Bone formation in a long bone defect model using a platelet-rich plasma-loaded collagen scaffold. Biomaterials 27:1817–1823

54.

Schliephake

(2002) Bone growth factors in maxillofacial skeletal reconstruction. Int J Oral Maxillofac Surg 31:469–484

55.

Schmidmaier

Wildemann

Lubberstedt

Haas

Raschke

(2003) IGF-I and TGF-β 1 incorporated in a poly (D, L-lactide) implant coating stimulates osteoblast differentiation and collagen-1 production but reduces osteoblast proliferation in cell culture. J Biomed Mater Res 65B:157–162

56.

Scholzen

Gerdes

(2000) The Ki-67 protein: from the known and the unknown. J Cell Physiol 182:311–322

57.

Simão

Beloti

Cezarino

Rosa

Pizauro

Ciancaglini

(2007) Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture. Comp Biochem Physiol A Mol Integr Physiol 146:679–687

58.

Soffer

Ouhayoun

Dosquet

Meunier

Anagnostou

(2004) Effects of platelet lysates on select bone cell functions. Clin Oral Implants Res 15:581–588

59.

van den Dolder

Mooren

Vloon

Stoelinga

Jansen

(2006) Platelet-rich plasma: quantification of growth factor levels and the effect on growth and differentiation of rat bone marrow cells. Tissue Eng 12:3067–3073

60.

Venne

Raymond

Allas

Roy

Leclerc

Boushira

Brazeau

(1999) Healing of experimental aneurysms. II: Platelet extracts can increase the thickness of the neointima at the neck of treated aneurysms. J Neuroradiol 26:92–100

61.

Wennberg

Hessle

Lundberg

Mauro

Narisawa

Lerner

Millán

(2000) Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice. J Bone Miner Res 15:1879–1888

62.

Wozney

(2002) Overview of bone morphogenetic proteins. Spine 27:S2–S8

Effects of a Mixture of Growth Factors and Proteins on the Development of the Osteogenic Phenotype in Human Alveolar Bone Cell Cultures

Abstract

Keywords

Materials and Methods

Culture of Osteogenic Cells Derived From Human Alveolar Bone

Exposure of Osteogenic Cultures to a Mixture of GFs and Proteins

Fluorescence Labeling

Growth Curve and Cell Viability

Proportion of Cycling Cells

Total Protein Content

ALP Activity

RNA Extraction and Quantitative Real-time RT-PCR

Mineralized Bone-like Nodule Formation

Statistical Analysis

Results

Discussion

Footnotes

Acknowledgements

References