Evaluation of bone-protecting effects of palm carotene mixture in two- and three-dimensional osteoblast/osteoclast co-culture systems

Abstract

Background: Carotene exists naturally in a complex mixture consisting of alpha (α), beta (β), and gamma (γ)-isoforms. Previous studies investigated the effects of individual carotene isomers on bone rather than their actions in a mixture. Purpose: This study explored the bone-protective properties of palm carotene mixture using both two- and three-dimensional co-culture systems. Study design: The viability of human foetal osteoblasts (hFOB 1.19), viability of human monocytic cell line (THP-1), osteoblast differentiation, osteoclast maturation, bone quality and strength were assessed in two- and three-dimensional co-culture system after treatment of palm carotene mixture. Methods: The viability of hFOB 1.19 and THP-1 was determined on day 1, 3, and 6 following treatment of palm carotene mixture. The osteoblast-osteoclast co-culture (ratio of hFOB 1.19 to THP-1 = 2:1) was treated with palm carotene mixture as well as subjected to alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP) staining on day 21 to assess the osteoblast proliferation and osteoclast maturation. Dual-energy X-ray absorptiometry, micro-computed tomography, universal testing machine, and bone histomorphometry were used to assess the bone parameters of scaffolds co-cultured with osteoblasts and osteoclasts. Results: Palm carotene mixture (3.13 - 50 μg/mL) increased osteoblast viability. Monocyte viability decreased in lower concentration (3.13 - 12.5 μg/mL) but increased in higher concentration (25 - 50 μg/mL) of palm carotene mixture. Treatment with palm carotene mixture (12.5 µg/mL) demonstrated earlier peak for the ALP-positive area on day 14 but decreased total number of TRAP-positive multinucleated cells on day 21. Palm carotene mixture also increased bone volume and osteoblast number in the three-dimensional co-culture system. Conclusion: Palm carotene mixture potentially exhibits beneficial effects on bone by accelerating osteoblast proliferation and suppressing osteoclast maturation. The findings of current study serve as the basis for the further validation through animal experiments and human trials.

Keywords: bone cells; bone remodeling; osteoporosis; provitamin A; scaffold.

Figure 1

The viability of (A) hFOB 1.19 and (B) THP-1 cells treated with palm carotene mixture at different concentrations (0 - 50 µg/mL). Data are expressed as mean ± SEM. '+' indicates significant difference with day 1; '#' indicates significant difference with day 3; 'a' indicates significant difference with vehicle; 'b' indicates significant difference with 3.13 µg/mL palm carotene mixture; 'c' indicates significant difference with 6.25 µg/mL palm carotene mixture; 'd' indicates significant difference with 12.5 µg/mL palm carotene mixture; 'e' indicates significant difference with 25 µg/mL palm carotene mixture.

Figure 2

(A) ALP-positive area and (B) number of multinucleated osteoclasts in an area (μm²) in osteoblast/osteoclast co-culture treated with palm carotene mixture at different concentrations (0 - 12.5 μg/mL). Data are expressed as mean ± SEM. '+' indicates significant difference with day 7; '#' indicates significant difference with day 14; 'a' indicates significant difference with control; 'b' indicates significant difference with 3.13 µg/mL palm carotene mixture.

Figure 3

Representative images of ALP staining in osteoblast/osteoclast co-culture treated with palm carotene mixture at different concentrations (0 - 12.5 μg/mL). Microphotographs were taken at 100x magnification. Blue stain indicates ALP-positive site.

Figure 4

Representative images of TRAP staining in osteoblast/osteoclast co-culture treated with palm carotene mixture at different concentrations (0 - 50 μg/mL). Microphotographs were taken at 100x magnification. Green arrow indicates multinucleated osteoclasts.

Figure 5

Effects of palm carotene mixture (12.5 µg/mL) on (A) BMC and (B) BMD of bone scaffold co-cultured with osteoblasts and osteoclasts for 21 days. Data are expressed as mean ± SEM. Abbreviations: NB, native bone scaffold; OB, osteoporosis bone scaffold; NC, negative control bone scaffold; TB: 12.5 μg/mL palm carotene mixture-treated bone scaffold; PC, 10 nM alendronate-treated bone scaffold.

Figure 6

Effects of palm carotene mixture (12.5 µg/mL) on (A) BV/TV, (B) Tb.N, (C) Tb.Th, (D) Tb.Sp, and (E) porosity of bone scaffold co-cultured with osteoblasts and osteoclasts for 21 days using micro-CT. Data are expressed as mean ± SEM. '+' indicates significant difference with day 0; 'a' indicates significant difference with NB; 'b' indicates significant difference with OB. Abbreviations: NB, native bone scaffold; OB, osteoporosis bone scaffold; NC, negative control bone scaffold; TB: 12.5 μg/mL palm carotene mixture-treated bone scaffold; PC, 10 nM alendronate-treated bone scaffold.

Figure 7

Representative of three-dimensional images via micro-CT before decellularisation and demineralisation process as well as after treatment of 12.5 µg/mL palm carotene mixture or 10 nM alendronate for 21 days. Abbreviations: NB, native bone scaffold; OB, osteoporosis bone scaffold; NC, negative control bone scaffold; TB: 12.5 μg/mL palm carotene mixture-treated bone scaffold; PC, 10 nM alendronate-treated bone scaffold.

Figure 8

Effects of palm carotene mixture (12.5 µg/mL) on (A) load, (B) displacement, (C) stiffness, (D) stress, (E) strain, and (F) Young's modulus of bone scaffold co-cultured with osteoblasts and osteoclasts for 21 days using biomechanical strength test. Data are expressed as mean ± SEM. 'a' indicates significant difference with NB and 'b' indicates significant difference with OB. Abbreviations: NB, native bone scaffold; OB, osteoporosis bone scaffold; NC, negative control bone scaffold; TB: 12.5 μg/mL palm carotene mixture-treated bone scaffold; PC, 10 nM alendronate-treated bone scaffold.

Figure 9

Effects of palm carotene mixture (12.5 µg/mL) on (A) BV/TV, (B) Tb.N, (C) Tb.Sp, and (D) Tb.Th of bone scaffold co-cultured with osteoblasts and osteoclasts for 21 days after von Kossa staining. Data are expressed as mean ± SEM. 'a' indicates significant difference with NB; 'b' indicates significant difference with OB; 'c' indicates significant difference with NC. Abbreviations: NB, native bone scaffold; OB, osteoporosis bone scaffold; NC, negative control bone scaffold; TB: 12.5 μg/mL palm carotene mixture-treated bone scaffold; PC, 10 nM alendronate-treated bone scaffold.

Figure 10

Representative images of bone scaffolds after von Kossa staining before decellularisation and demineralisation process as well as after treatment of 12.5 µg/mL palm carotene mixture or 10 nM alendronate for 21 days. The black arrows indicate trabecular bone and blue arrows indicate intertrabecular space. Abbreviations: NB, native bone scaffold; OB, osteoporosis bone scaffold; NC, negative control bone scaffold; TB: 12.5 μg/mL palm carotene mixture-treated bone scaffold; PC, 10 nM alendronate-treated bone scaffold.

Figure 11

Effects of palm carotene mixture (12.5 µg/mL) on (A) Ob.N/BA, (B) Oc.N/BV, (C) ES/BS, (D) OS/BS, and (E) OV/BV of bone scaffold co-cultured with osteoblasts and osteoclasts for 21 days after H&E staining. Data are expressed as mean ± SEM. 'a' indicates significant difference with NB; 'b' indicates significant difference with OB; 'c' indicates significant difference with NC; 'd' indicates significant difference with TB. Abbreviations: NB, native bone scaffold; OB, osteoporosis bone scaffold; NC, negative control bone scaffold; TB: 12.5 μg/mL palm carotene mixture-treated bone scaffold; PC, 10 nM alendronate-treated bone scaffold.

Figure 12

Representative images of bone scaffold after H&E staining before decellularisation and demineralisation and after treatment of 21 days. The yellow arrows indicate osteoblasts and green arrows indicate osteoclasts. Abbreviations: NB, native bone scaffold; OB, osteoporosis bone scaffold; NC, negative control bone scaffold; TB: 12.5 μg/mL palm carotene mixture-treated bone scaffold; PC, 10 nM alendronate-treated bone scaffold; Ost, osteocyte; OS, osteoid surface; ES, eroded surface; EL, empty lacunae.