Human Osteopenic Bone‐Derived Osteoblasts: Essential Amino Acids Treatment Effects

Abstract

The development of in vitro cell culture methods has made it possible to study bone cell metabolism and growth and obtain a deeper insight into the pathophysiology of common orthopedic diseases such as osteoporosis. After analyzing the effect of two essential amino acids, L‐arginine (Arg) and L‐lysine (Lys), in previous in vitro and in vivo studies, the present authors investigated the administration of Arg and Lys in osteoblasts derived from human osteopenic bone. After isolation, osteoblasts were cultured in DMEM supplemented with either Arg (0.625 mg/ml/day, Arg Group) or Lys (0.587 mg/ml/day, Lys Group), or both of them (Arg‐Lys Group), whereas the Control Group was sham‐treated. After 7 days the following parameters were tested in all groups: MTT proliferation test, Alkaline Phosphatase (ALP), Nitric Oxide (NO), Calcium (Ca), Phosphorus (P), Osteocalcin (OC), C‐Terminal Procollagen type I (PICP), Interleukin‐6 (IL‐6), Transforming Growth Factor‐βeta 1 (TGF‐β 1), Platelet Derived Growth Factor (PDGF) and Insulin‐Like Growth Factor‐I (IGF‐I). Results were compared with those obtained from human healthy bone to verify the effect of the amino acids on osteoblasts derived from pathological tissue. In addition, a comparison was also made with the results obtained from rat osteopenic bone to assess reliability of the in vitro model.

The current results support previous findings and indicate that Arg and Lys stimulation has a positive effect on osteoblast proliferation, activation and differentiation. Therefore, administration of these amino acids may be useful in clinical treatment and prevention of osteoporosis.

• Osteoporosis
• Amino acids
• Osteoblast culture

Introduction

L‐Arginine (Arg) and L‐Lysine (Lys) have been shown to improve bone healing processes in fractures (Fini et al., [1999]; Torricelli et al., [2001a]) and bone status in metabolic bone diseases such as primary and secondary osteoporosis, in which the imbalance of osteoblast anabolic activity and osteoclast resorption is the cause of bone rarefaction (Visser and Hoekman, [1994]). Moreover, cytokine and growth factor impairment, and decline in mesenchymal stem cell repository are often associated with osteoporosis, in addition to bone mineralization and microarchitectural alterations (Bruder et al., [1998]; Jilka, [1998]). Finally, the inadequate protein intake, which appears to be involved in the pathogenesis of osteoporosis, determines low levels of essential amino acids and alters their role in mediating osteoblast growth and differentiation (Chevalley et al., [1998]; Conconi et al., [2001]).

A positive effect of Arg and Lys on osteoblast synthetic activity and proliferation has been observed in previous in vitro studies on healthy and osteopenic rat bone‐derived osteoblasts and healthy human bone‐derived osteoblasts (Fini et al., [2001]; Torricelli et al. [in press]). The present study was performed to provide reliable data for research, taking into account that the use of osteopenic bone‐derived osteoblasts could be the experimental condition which most closely mimics the clinical situation. Consequently, such condition is a necessary step for any experimental laboratory research investigating pathophysiological aspects and therapies to acquire greater knowledge and improve treatment of osteoporosis (Torricelli et al., [2000]). Human osteopenic bone‐derived osteoblasts were therefore used in the present study to test the effect of Arg and Lys on osteoblast proliferation, synthetic activity and differentiation.

Materials and Methods

Cell Cultures

Human osteoblasts were isolated sterilely from small specimens of trabecular bone derived from osteopenic (Human Osteopenic Bone, HOB) bone tissue. Bone specimens had been removed from the femoral head of four 78 ± 6‐year‐old women with low‐energy traumatic hip fractures who had undergone arthroplasty. Other metabolic bone diseases and secondary osteoporosis were excluded on the basis of biochemical testing, and women had not taken any drugs known to affect calcium metabolism. A lumbar DEXA performed in patients before surgery revealed a decrease in bone mineral density ≥2.5 SD. The patients, who also had evidence of fracture, were therefore classified as osteoporotic patients. Samples of trabecular bone were submitted to histomorphometric analysis that showed osteoporosis with a low bone turnover rate (unpublished data). Informed consent was obtained and procedures were approved by the Ethical Committee of the Rizzoli Orthopedic Institute.

Trabecular bone fragments were put in DMEM:F12 serum‐free culture medium and immediately processed to obtain primary cultured osteoblasts following a method previously described (Torricelli et al., [2000]). Briefly, bone fragments were washed with DMEM:F12 serum‐free medium and digested in F12 medium with 1 mg/ml collagenase for 90 min at 37°C. The enzymatic reaction was stopped by adding an equal volume of medium with 10% of FCS, and the supernatant containing the released cells was collected. Washing and collecting were repeated thrice. The cells obtained were pelletted by centrifugation, resuspended, seeded in culture flasks (75 ml), cultured in DMEM medium supplemented with ascorbic acid (50 μg/ml) and β‐glycerophosphate (10^{− 8} M), and incubated at 37°C in a humidified 95% air /5%CO₂ atmosphere. Cells were released at confluence with 0.05% (w/v) trypsin and 0.02% (w/v) EDTA, counted and used for the experiment in chamber slides. They were also seeded (1 × 10⁴ cells/ml) in 4‐well chamber slides for characterization, by adding 10^{− 9 M} 1,25(OH₂)D₃ to allow mineralization and expression of the osteoblast phenotype.

Cell Experiments

In the first passage, osteoblasts were seeded at the concentration of 1 × 10⁴ cells/ml in in four 24‐well plates. Six wells of each plate were sham‐treated and served as Controls, six were treated daily with Arg (Arg Group: 0.625 mg/ml/day, Sigma, St. Louis, MO, USA), another six were treated daily with Lys (Lys Group: 0.587 mg/ml/day, Sigma), and the remaining six were treated daily with Arg and Lys for 7 days at the concentrations mentioned above (Arg‐Lys Group). Amino acids were diluted with 100 μl of culture medium to be added daily, while the same volume of culture medium without amino acid was added daily to the control cell cultures. The dosages of amino acids were selected on the basis of recommendations for clinical use and the same dosages were also administered in previous in vitro studies by the current authors (Fini et al., [2001]; Torricelli et al. [in press]). Cultures were maintained in the same conditions as described above for seven days, and the medium was then replaced on days two and four. No bacterial or fungal contamination was found during the experiments.

At the end of the experiment, supernatant was centrifuged to remove particulates, if any, aliquoted, and partly stored at − 70°C. Alkaline Phosphatase activity (ALP, Sigma Kinetic method kit, St. Louis, MO, USA) Calcium (Ca, Sigma kit, St. Louis, MO, USA), Phosphorus (P, Sigma kit, St. Louis, MO, USA), Nitric Oxide (NO, Sigma colorimetric assay, St. Louis, MO, USA), Osteocalcin (OC, Novocalcin enzyme Immunoassay kit, Metra Biosystem, CA, USA) were immediately performed. C‐terminal procollagen type I (PICP, Prolagen‐C enzyme Immunoassay kit, Metra Biosystem, CA, USA), Interleukin‐6 (IL‐6, Human IL‐6 Immunoassay kit, Biosource International, CA, USA) and Transforming Growth factor‐β1 (TGF‐β1, Quantikine human TGF‐β1 Immunoassay, R&D Systems, MN, USA) were determined on the stored supernatant.

The MTT test (Sigma UK) was performed to assess cell proliferation: 80 μl of MTT solution (5 mg/ml in phosphate buffer) and 720 μl of medium were added to one half of the wells, and the plates were incubated at 37°C for another 4 h. After discarding supernatants, the dark blue crystals of formazan were dissolved by adding DMSO (800 μl) and quantified spectrophotometrically at 550 nm. Results were reported as optical density (OD). The cells in the remaining wells were formalin fixed. Immunostaining was performed for Platelet Derived Growth Factor (PDGF, anti‐human PDGF, Sigma, UK) and Insulin‐like Growth Factor I (IGF‐I, anti‐human IGF‐I, Sigma, UK).

Statistical Analysis

Statistical evaluation of data was performed using the software package SPSS/PC⁺ Statistics ^™ 7.5 (SPSS Inc., Chicago, IL USA). Data are reported as mean ± standard deviations (SD) at a significance level of p < 0.05. After having verified normal distribution and homogeneity of variance, a one‐way ANOVA was done for comparison among groups. Finally, Scheffé's post hoc multiple comparison tests were done to detect significant differences between groups. The unpaired Student's t test was used to compare ALP activity and OC production results between controls and 1.25(OH)₂D₃ stimulated human bone‐derived osteoblasts.

Results

Osteoblast Characterization

Characterization of osteoblasts according to the well‐established parameters of the osteoblast phenotype is shown in Table 1. A significant increase in ALP activity and OC production was highlighted when cultures were supplemented with 10^{− 9} mol/l 1,25(OH)₂D₃, as already reported by other authors (Wong et al., [1994]). Von Kossa staining confirmed these results and demonstrated that the cells had the capacity to differentiate and mineralize in vitro and thus showed osteoblastic behavior.

Table 1. Primary characterization of osteoblast cultures of human osteopenic bone‐derived cells (HOB) after administration of 10^{− 9}mol/l 1,25(OH)₂D₃ (Mean ± SD; no. = 4)

HOB cells	Control	1,25(OH)2D3	t, p
ALP (IU/l)	41.5 ± 1.4	67.3 ± 2.0	− 21.3, < 0.0001
OC (ng/ml)	22 ± 2	43 ± 3	− 12.1, < 0.0001
von Kossa	negative	positive

ALP: alkaline phosphatase; OC: osteocalcin.

Cell Experiment

After 7 days of cultivation the parameters listed above were tested to assess the effects of Arg and Lys on osteoblasts. Results are summarized in Tables 2 and 3, where means and SD with statistical significance, if any, are shown for each test and group.

Table 2. Biochemical results in human osteopenic bone‐derived osteoblasts (Mean ± SD, no. = 4)

Groups	MTT (OD at 550 nm)	ALP (IU/L)	NO (μM)	Ca (mg/dl)	P (mg/dl)	OC (ng/ml)
Control	0.53 ± 0.03	41.5 ± 1.9	3.6 ± 0.1	5.1 ± 0.4	2.5 ± 0.1	36.8 ± 1.0
Arg	0.46 ± 0.01a	53.9 ± 4.0c	3.9 ± 0.1	4.9 ± 0.4g	2.2 ± 0.1	38.0 ± 1.2
Lys	0.57 ± 0.01b	47.0 ± 1.2d	2.7 ± 0.3f	5.7 ± 0.2	2.8 ± 0.3h	37.0 ± 0.6
Arg‐Lys	0.55 ± 0.02	54.9 ± 1.0e	3.6 ± 0.1	5.0 ± 0.2	2.2 ± 0.1	38.5 ± 1.7
ANOVA F, p	26.49, < 0.0005	44.01, < 0.0005	46.50, < 0.0005	5.18, 0.016	24.04, < 0.0005	1.95, ns

MTT: tetrazolium salt; ALP: alkaline phosphatase; NO: nitric oxide; Ca: calcium; P: phosphorus; OC: osteocalcin.

Scheffé post hoc multiple comparison test:

^aMTT: Arg Group versus Control (p < 0.005), Lys and Arg‐Lys (p < 0.0001) Groups.

^bLys Group versus Control Group (p < 0.05).

^cALP: Arg Group versus Control (p < 0.0005) and Lys (p < 0.005) Groups.

^dLys versus Control (p < 0.05) and Arg‐Lys (p < 0.001) Groups.

^eArg‐Lys Group versus Control Group (p < 0.0001).

^fNO: Lys Group versus Control, Arg and Arg‐Lys (p < 0.0001) Groups.

^gCa: Arg Group versus Lys Group (p < 0.05).

^hP: Lys Group versus Control (p < 0.005), Arg and Arg‐Lys (p < 0.0001) Groups.

Table 3. Procollagen and cytokine levels in human osteopenic bone‐derived osteoblasts (Mean ± SD, no. = 4)

Groups	PICP (ng/ml)	IL‐6 (pg/ml)	TGFβ1 (pg/ml)
Control	23.0 ± 1.4	1238 ± 75	1267 ± 23
Arg	25.8 ± 1.0a	1168 ± 24	1162 ± 33
Lys	22.0 ± 1.4	1195 ± 33	728 ± 130c
Arg‐Lys	24.3 ± 1.0	1013 ± 48b	1120 ± 65
ANOVA F, p	7.2, 0.005	16.07, < 0.0005	39.22, < 0.0005

PICP: C‐terminal procollagen type I; IL‐6: Interleukin‐6; TGFβ1: Transforming Growth Factor‐β1.

Scheffé post hoc multiple comparison test:

^aPICP: Arg Group versus Lys Group (p < 0.01).

^bIL‐6: Arg‐Lys Group versus Control (p < 0.0001), Arg (p < 0.01) and Lys (p < 0.005) Groups.

^cTGFβ1: Lys Group versus Control, Arg, and Arg‐Lys Groups (p < 0.0001).

The MTT test revealed a significant increase in cell replication in the Lys Group when compared to the Control Group, while the Arg Group showed a significant decrement as compared to all of the other groups. Both single and combined administrations of amino acids significantly enhanced ALP production. No differences were observed among Arg, Arg‐Lys and Control Groups in terms of NO values, while a significantly lower value was detected in the Lys Group. The OC level was unchanged for all groups.

A significant difference in the PICP level was found only for the Arg Group (highest value) when compared to the Lys Group (lowest value), while the Lys and Arg‐Lys Groups did not differ from the Control Group.

The level of IL‐6 was unchanged in the Arg and Lys Groups as compared to the Control Group, while the level in the Arg‐Lys Group was significantly lower than in all of the other groups.

The expression of TGF‐β1 was not affected by the administration of Arg alone or in combination with Lys, and this growth factor decreased in the Lys Group when compared to the Control Group. The immunostaining intensity of PDGF was significantly higher in the Lys (+ 126%) and Arg‐Lys (+ 130%) Groups than in the Control Group (considered as 100), whereas it was slightly higher in the Arg Group than in the Control Group. The immunostaining intensity of IGF‐I was higher in the Arg and Arg‐Lys Groups (+ 19% and + 17%, respectively) and lower in the Lys group (− 21%) than in the Control Group.

Discussion

The possibility of culturing primary osteoblasts from the human skeleton has made it possible to conduct a great amount of research on osteoblasts cultured from osteoporotic patients and elucidate the different dysfunction patterns and responsiveness of cells to therapies (Katzburg et al., [1999]; Sasse et al., [1998]). Some of these studies have reported that no or little difference exists between healthy and osteoporotic bone‐derived osteoblasts in terms of proliferation, synthetic activity and morphology (Lomri and Marie, [1990]; Walsh et al., [2000]). On the contrary, other authors have highlighted differences in the reaction to differently applied stimuli, such as growth factors and biomaterials (Marie, [1999]; Torricelli et al., [2001b]). Thus, there is a strong rationale for the use of osteoporotic bone‐derived cells for the study of drugs to be potentially used in the clinical treatment of osteoporosis.

To the current authors' knowledge, no studies are available on the effect of essential amino acids on human osteoporotic bone‐derived cells. However, in vitro and in vivo studies on the effect of Arg and Lys on osteoblasts suggest that they may positively affect bone metabolism (Chevalley et al., [1998]; Clementi et al., [2001]; Conconi et al., [2001]; Fiore et al., [2000]).

Studies with primary human osteoblasts from osteoporotic donors are known to require a great number of donors because of the variability in the osteoblast/osteoclast dysfunction pattern (Thavarajah et al., [1993]). Some authors have observed that a great number of patients is required to reach significant data when human osteoblasts are used (Siggelkow et al., [1998]). However, in spite of the small number of osteoporotic patients in this series, significant, even if preliminary, results were obtained.

No differences in ALP production were observed under the baseline condition and after 1,25(OH)₂D₃ stimulation when compared to the characterization of osteoblasts derived from normal bone (Torricelli et al. [in press]). On the contrary, OC values were lower for osteoblasts cultered from osteoporotic bone than for normal bone cultures, even if the former responded positively to vitamin D stimulation.

The present study demonstrates that Lys has mainly a proliferative effect on osteoporotic bone‐derived cells when administered in vitro, whereas Arg is responsible for the improvement in synthetic activity (+ 29.8 % ALP; + 12.1 % PICP). The improvement in cell replication due to the Lys administration was accompanied by a general decrease in the synthetic activity of NO and TGFβ1, whereas OC and IL‐6 synthesis were unaffected. These findings are consistent with the previous data obtained using rat bone‐derived osteoblasts, with the exception of the NO levels observed in human osteopenic bone‐derived cells that did not change after Arg administration compared to the Control Group (Fini et al., [2001]). Such finding requires further comparative investigations on a larger number of osteoporotic patients. Interspecies differences should be taken into account, since they may lead to different results depending on the experimental model adopted.

Results obtained when measuring cytokine levels are of particular relevance, since cytokine impairment is gaining increasing popularity in the study of osteoporosis pathophysiology (Jilka, [1998]). IL‐6 mediates the effects of estrogen deficiency on osteoclast number, as it affects osteoclast progenitors by stimulating their replication and differentiation, and inhibiting apoptosis. Therefore, the ability of some estrogenic compounds to prevent bone loss secondary to ovariectomy is also due to the suppression of the highest circulating levels of IL‐6 that are associated with osteoporosis (Jilka, [1998]; Katzburg et al., [1999]). Consequently, the significant decrease in IL‐6 observed when Arg and Lys are administered in combination is of great interest to understand its role in the management of osteoporosis. On the contrary, the TGFβ1 exerts its inhibitory effect on osteoclastogenesis by inducing also apoptosis (Jilka, [1998]; Katzburg et al., [1999]). It is a mediator of normal bone remodeling and was found to be produced by cultured osteoblast‐like cells. Moreover, it can either stimulate or inhibit cellular proliferation, depending on the growth conditions and other growth factors in the cellular microenvironment (Bonewald and Dallas, [1994]; Centrella et al., [1994]; Kassem et al., [2000]). PDGF is highly mitogenic to cultures of osteoblastic cells and inhibits differentiated osteoblast function (Raisz and Rodan, [1998]). IGF‐I is an important regulator of osteoblast metabolism and differentiation, and enhances the synthesis of bone matrix proteins by the cultured osteoblastic cells (Tumber et al., [2000]).

In conclusion, the present study demonstrates that osteoporotic bone‐derived osteoblasts and healthy bone‐derived cells respond similarly to Arg and Lys administration (Torricelli et al. [in press]). In addition, this finding is supported by the majority of the parameters tested on osteoblast cultures from rat bone (Fini et al., [2001]). Treatment positively affects osteoblasts: they stimulated cell proliferation and production of marker of cellular activation and differentiation (ALP, NO, OC, PICP, PDGF, IGF‐I), as well as a lower IL‐6 level. TGF β1 does not appear to be particularly affected by treatment and should be further investigated.

Acknowledgments

Financial support for this research was partially provided by the Ministry of Health (Rome, Italy), special strategic project Fratture osteoporotiche, and the Istituti Ortopedici Rizzoli, research grants. The authors have appreciated the technical assistance of C. Dal Fiume, P. Di Denia, N. Corrado, F. Rambaldi, and P. Nini (Experimental Surgery Department, IOR).