HUMAN OSTEOBLAST CULTURES FROM OSTEOPOROTIC AND HEALTHY BONE: BIOCHEMICAL MARKERS AND CYTOKINE EXPRESSION IN BASAL CONDITIONS AND IN RESPONSE TO 1,25(OH) ₂D ₃

Abstract

The development of methods to culture bone cells has enhanced in vitro studies and allowed researchers to investigate bone cell metabolism in healthy tissue and in various different bone diseases. Greater knowledge of cultures of pathologic bone tissue-derived osteoblasts may be helpful in performing in vitro experiments that test biomaterials and therapies to be used in the orthopedic field, since this kind of approach better reflects the conditions of clinical relevance to many patients.

In the present study primary cultures of human osteoblastic cells were isolated from donors with osteoporosis (HOB, Human Osteopenic Bone) and their respective controls (HNB, Human Normal Bone). They were then characterized in baseline conditions and after stimulation with 10⁻⁹ M 1,25(OH₂)D₃. Specific biochemical markers of bone cells and cytokines involved in bone turnover were evaluated to assess cell metabolism and any possible differences between osteoblasts derived from healthy and osteopenic bone tissue. Under baseline conditions, HNB and HOB in vitro cultures showed some differences in proliferation (MTT test), PICP, OC and IL-6. The HNB response to 1,25(OH₂)D₃ stimulation differed significantly from that of the HOB cultures but only with regard to the MTT test, and ALP and PICP levels; the other selected parameters showed a similar behavior for both cultures.

The current findings should be taken into account when cultures derived from human bone are used for in vitro experiments.

INTRODUCTION

The development of new methods to culture osteoblasts and bone marrow stromal cells has contributed to in vitro studies on bone, making it possible to investigate a number of aspects related to bone cell metabolism also in the presence of various different bone diseases.[1-4] The “pathological” primary osteoblasts in culture may also be used to test the material's cytotoxicity and investigate the surface-dependent responses of bone-forming cells.[[5]] This kind of approach to in vitro research may improve simulation of in vivo conditions, as the biomaterial behavior is tested in a microenvironment that is more similar to the clinical situation.

Animal studies on biocompatibility and osseointegration are usually performed on healthy models. However, researchers should take into account that an increasing number of implants are being applied in patients with altered bone mineralization.[6-10] The ageing population and longer life expectancy lead osteopenia and osteoporosis to be considered as major social health problems,[[11]] and bone rarefaction is one of the most negative factors predictive of implant failure.[[12]]

In previous investigations the present authors observed the differences between normal and osteopenic bone in basal conditions and provided a rationale for using both normal and osteopenic bone-derived cells for the in vitro evaluation of biomaterials. The materials were seen to behave differently when tested in normal and “pathological” cells. In these studies, bone cell cultures were derived from experimental osteopenic animals.[13-18] However, the behavior of human osteoblasts also seems to be relevant for the purposes of the present study. Obviously, human bone-derived cultures are associated with a greater number of variables compared to animal bone-derived cultures, both under normal conditions and in the presence of osteoporosis.[[19]] However, one of the relative advantages is a higher representation of the clinical applications.

The aim of the present study was to characterize the primary osteoblastic cell cultures derived from healthy and osteopenic human bone, both under basal conditions and in response to 1,25(OH)₂D₃ stimulation. The authors investigated if there were any significant differences between the two cultures after the first passage. These bone cultures are intended to be used in vitro for testing therapies and biomaterials.

MATERIALS AND METHODS

Cell Cultures

Human osteoblasts were isolated sterilely from small specimens of trabecular bone derived from healthy (Human Normal Bone, HNB) and osteopenic (Human Osteopenic Bone, HOB) bone tissue. Bone specimens had been removed from the femoral head of patients who had undergone arthroplasty after a traumatic fracture of the femoral neck. Informed consent had been obtained and procedures approved by the Ethical Committee of the Rizzoli Orthopedic Institute. No metabolic or systemic diseases were observed in the donors selected for the study on HNB cells, and they had taken no drugs affecting bone metabolism. Regarding the cells from HOB, a lumbar DEXA performed in the elderly patients before surgery revealed osteoporosis, and these individuals were therefore characterized as osteopenic patients. Trabecular bone fragments were put in DMEM:F12 serum-free culture medium and immediately processed to obtain primary cultured osteoblasts. Briefly, bone fragments were repeatedly washed with DMEM:F12 serum-free medium. Fragments were then seeded in culture flasks (75 ml), cultured in DMEM medium containing 10% FCS and antibiotics (penicillin 1000 U/ml, streptomycin 10 mg/ml), and incubated at 37°C in a humidified 95% air/5%CO₂ atmosphere. The cells outgrown from the trabecular fragments after a few days were fed every 3 days and released at 80% confluence with 0.05% (w/v) trypsin and 0.02% (w/v) EDTA. Moreover, cells were characterised to assess their osteoblast phenotype.

Cell Experiment

Cell suspensions were counted and plated in multi-well plates at the density of 5×10⁴ cells/ml in DMEM supplemented with 50 μg/ml ascorbic acid and 10⁻⁸ M β-glycerophosphate After 24 h in the incubator, the medium was removed, the cell monolayer was washed with sterile phosphate buffered saline (PBS), and fresh medium supplemented with 10⁻⁹ M 1,25(OH₂)D₃ was added to half of the wells. The remainder received the same medium without vitamin 1,25(OH₂)D₃. Cultures were maintained in the same conditions as described above for 48 hours. No bacterial or fungal contamination was found during this period.

Finally, samples of supernatant from all wells were centrifuged to remove particulates, if any, and aliquots were dispensed in Eppendorf tubes for storage at −70°C for assay for type I Collagen (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).

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), Alkaline Phosphatase activity (ALP, Sigma Kinetic method kit, St. Louis, MO, USA) and Osteocalcin (OC, Novocalcin enzyme Immunoassay kit, Metra Biosystem, CA, USA) were tested on supernatants immediately after collection.

Finally, 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 the cell monolayers, and the multi-well plates were incubated at 37°C for a further 4 h. After discarding supernatants, the dark blue crystals of formazan were dissolved by adding DMSO (800 μl), and they were quantified spectrophotometrically at 550 nm. Results were reported as optical density (OD).

Statistics

Statistical evaluation of data was performed using the software package SPSS/PC⁺ Statistics™ 10.1 (SPSS Inc., Chicago, IL USA). Data (result of 4 replications) are reported as mean±standard deviations (SD) at a significance level of p<0.05. After having verified normal distribution and homogeneity of variances, the non-parametric Mann–Whitney U test was used to compare data between groups and the Monte Carlo method to generate an unbiased estimate of the exact p value.

RESULTS

Osteoblasts from normal and osteopenic bone were characterized according to the well-established parameters of the osteoblast phenotype. Significant (p<0.05) increases in ALP activity and OC production were observed when cultures were supplemented with 10⁻⁹ mol/l 1,25(OH)₂D₃; Von Kossa staining confirmed these results. The cells presented the capacity to differentiate and mineralize in vitro, thus showing an osteoblastic behavior.

The results of each parameter tested are summarized in Table 1. Regarding MTT results, the Mann–Whitney U test showed that 1,25(OH)₂D₃ stimulation significantly reduced the osteoblast proliferation rate by about −7% and −14% compared with baseline in HNB and HOB cells, respectively. These reductions were significantly different.

Table 1. Characterization of Primary Osteoblast Cultures of Normal (HNB) and Osteopenic (HOB) Human Bone-Derived Cells; Basal Values at Confluence and After Administration of 10⁻⁹ mol/l 1,25(OH)₂D₃ (Mean±SD, no.=4)

	HNB			HOB
	Basal	1,25(OH)2D3	%Δ	Basal	1,25(OH)2D3	%Δ
MTT (OD550 nm)	0.65±0.02	0.61±0.011^a	−7.0±1.8	0.53±0.032^c	0.50±0.033^b	−14.1±2.74^d
ALP (UI/l)	42.9±1.4	64.2±1.71^a	49.8±1.2	41.5±1.4	67.3±2.03^b	64.8±7.64^d
OC (ng/ml)	27±1	53±21^a	98.0±9.4	22±12^c	43±33^b	96.6±7.4
PICP (ng/ml)	30.0±1.0	27.0±0.91^a	−10.0±2.1	27.5±0.72^c	23.0±0.93^b	−16.4±2.54^d
NO (μM)	3.3±0.3	3.3±0.2	17.9±6.2	3.6±0.1	3.5±0.2	14.3±2.7
IL-6 (pg/ml)	113±14	110±10	−1.1±19.4	182±352^c	185±34	6.3±37.1
TGF-β1 (pg/ml)	1386±120	1138±681^a	−17.6±8.1	1267±23	1066±583^b	−16.7±6.1

^aMann Whitney U test: HNB cells: 1,25(OH)₂D₃ Group versus Basal Group (p<0.05).

^bMann Whitney U test: HOB cells: 1,25(OH)₂D₃ Group versus Basal Group (p<0.05).

^cMann Whitney U test: Basal Groups: HOB cells versus HNB cells (p<0.05).

^dMann Whitney U test: %Δ: HOB cells versus HNB cells (p<0.05).

ALP baseline values were similar and increased in response to 1,25(OH)₂D₃ stimulation in both groups. However, the percentage increase for the HOB cells was significantly (p<0.05) higher than for the HNB cells. On the contrary, baseline values obtained for OC differed significantly (p<0.05) between the HNB and HOB cells, and the OC values of HOB cells were significantly (p<0.05) lower than those of HNB cells after 1,25(OH)₂D₃ stimulation (Table 1).

PICP basal values were found to be significantly higher in HNB versus HOB cultures, and this difference was still observable after 1,25(OH)₂D₃ stimulation in both groups. However, 1,25(OH)₂D₃ stimulation produced significant reductions of PICP for both groups, and the decrease observed for the HOB cells was significantly lower than for the HNB cells (Table 1).

NO values did not differ either at baseline or after 1,25(OH)₂D₃ stimulation.

IL-6 did not seem to be influenced by 1,25(OH)₂D₃ stimulation and HOB values were higher than HNB values. On the contrary, 1,25(OH)₂D₃ stimulation, significantly influenced TGF-β1, showing a decrease in TGF-β1 of about 17% for both types of cells.

DISCUSSION

Osteoblast activity, proliferation and differentiation strictly depend on interactions between hormones and cytokines, either produced locally or arriving via the circulation. In fact, their interaction and combined effects both influence and regulate bone metabolism. Since the activity of these factors is often interdependent, the function of each cytokine is not always easy to define without taking into account all the factors characterizing the cellular microenvironment. Some of the main parameters involved in osteoblast metabolism were therefore tested to obtain more information about the osteoblast activity in cultures both under baseline conditions and in response to 1,25(OH)₂D₃ stimulation.

Comparison between normal bone-derived cells and osteopenic bone-derived cells highlighted some basic differences: HOB cells proved to have a lower degree of proliferation, lower levels of PICP, OC, TGFβ1 production and a higher production of IL-6 compared to HNB cells. On the other hand, the other parameters tested (ALP, NO) were similar for the two groups of cells. The effects of 1,25(OH)₂D₃ stimulation on the behavior of HOB and HNB cells were comparable at the same interval of time. The differences observed between the two groups after stimulation appear to depend on the different baseline value and not on the altered response to the stimulus. The increase in ALP was the only response that was more marked than the others, and such finding is consistent with the results obtained by previous studies conducted on animal osteoblasts.[[13]]

Cell proliferation and osteoblast differentiation are influenced in the same way by 1,25(OH)₂D₃ in normal and osteopenic cells. Consequently, such hormonal stimulation does not involve interferences if cultures are used to study biocompatibility and osteointegration of new materials in vitro.

Some of the present findings in terms of cell proliferation and OC production differ from the previous results obtained by the current authors with animal cells (normal and ovariectomized rats and sheep), which had shown no differences between healthy and osteopenic bone under baseline conditions.[13-14] The differences observed in the present study may depend on the species used or on the mentioned variability typical of osteoporotic patients. A number of factors which are not encountered in experimental animals characterize the quality of bone tissue in humans, such as osteoporosis with lack of osteoblastic activity or increased osteoclastic activity, or both, and life style.[[19]]

For these reasons the current findings and the differences observed should be taken into account by researchers who use cellular cultures for studies on physiopathology and biocompatibility in osteoporotic bone.

ACKNOWLEDGMENTS

Financial support for this research was provided by the Rizzoli Orthopedic Institute, “Ricerca Corrente” and by the Project Fondazione Cassa di Risparmio, Bologna, “Clinica e Biologia delle gravi insufficienze d'organo.” The authors would like to thank C. Dalfiume, P. DiDenia, N. Corrado, F. Rambaldi, and P. Nini, of the Experimental Surgery Department, Rizzoli Orthopedic Institute, for their assistance.