Osteoporosis result from age-related decline in the number of osteoblast progenitors in the bone marrow. Probiotics have beneficial effects on the host, when administered in appropriate amounts. This study investigated the effects of probiotics expressing specific genes, especially the effects of genetically modified bone morphogenetic protein (BMP)-2-expressing
Twenty-eight female Wistar rats (250–300 g, 12 weeks old) were divided into four groups : the sham (control), the ovariectomy (OVX)-induced osteoporosis group (OVX), the OVX and LP (OVX/LP), OVX and genetically modified BMP-2-expressing LP (OVX/LP with BMP) groups. The three groups underwent bilateral OVX and two of these groups were administered two different types of LP via oral gavage daily. At 16 weeks post-OVX, blood was collected from the heart and the bilateral tibiae were extracted and were scanned by
The 3D-micro-computed tomography images showed that the trabecular structure in the OVX/LP with BMP group was maintained compared with OVX and OVX/LP groups. No significant differences were detected in trabecular thickness (Tb.Th) between control and OVX/LP with BMP groups (
Our results showed that the expression of genetically modified BMP-2 showed inhibition effect for bone loss in a rat model of osteoporosis.
Osteoporosis is an important health concern. It is associated with abnormal bone metabolism in which bone resorption is greater than bone formation [
Probiotics are living microorganisms [
Recent reports suggest that gene modification for the synthesis of antioxidant enzymes or cytokines in probiotics may facilitate the development of beneficial strains that can be used in the treatment of various diseases. Furthermore, the design of efficient systems of gene expression and appropriate protein synthesis in probiotics allow application of these microorganisms in the production and secretion of various cytokines or proteins [
Therefore, the object of this study was to develop and evaluate the effects of genetically modified bone morphogenetic protein (BMP)-2-expressing
This experiment was approved by the Institutional Animal Care and Use Committee of Asan Institute for Life Sciences Seoul, Korea (2017-14-228).
To construct a secretion system expressing BMP-2 gene in lactic acid bacteria, a promoter and secretion signal sequence of the bifidobacterial α-amylase gene was used [
Using computerized random numbers, this study was performed with 28 Wistar rat (Orient Bio, Seongnam, Korea) (250–300 g, aged 12 weeks), which were divided into four groups, seven for each group, which is the minimal number for the statistical analysis : sham (control), ovariectomy (OVX)-induced osteoporosis group (OVX), OVX and LP feeding group (OVX/LP), and OVX and genetically modified BMP-2-expressing LP feeding group (OVX/LP with BMP). Three groups underwent bilateral OVX and two of these groups were administered two different types of LP via oral gavage daily.
The animals were sedated with 5% isoflurane. Ovariectomy was performed via bilateral dorsal skin incision (4 cm) in the middle. The peritoneal cavity was opened to reveal the ovary surrounded by fat. After blood vessel ligation, the site between the Fallopian tube and the uterine horn was cut and the ovary was removed. The incision required suturing. An intramuscular injection of antibiotic (cefazolin 10 mg/kg, ketoprofen 5 mg/kg) was administered for three postoperative days. Body weight was monitored once weekly for 20 weeks. All animal was treated in accordance with the Guide for the Care and Use of Laboratory Animals.
Probiotic treatment was started 28 days after OVX surgery. During 16 weeks, rats in the OVX/LP group were treated with a single LP and genetically modified BMP-2 expressing LP was administered to the OVX/LP with BMP group at a volume of 400 µL (approximately 108 CFU).
Twenty weeks after OVX, all rat were sedated with 5% isoflurane. Blood was drawn via cardiac puncture and were euthanized. Bilateral tibiae were removed and analyzed by high-resolution micro-CT using a Skyscan 1172 micro-CT apparatus (Skyscan NV, Kontich, Belgium). The volume of interest (VOI) was started at a distance of 2.5 mm distally from the growth plate of the proximal tibia and extended towards the diaphysis for 100 slices. In each transverse slice of the VOI, the region of interest was defined as the entire trabecular compartment. The acquisition settings entailed an X-ray source voltage of 49 kV and a current of 200 µA. The pixel size was 19.97 mm. The exposure time was 670 ms and a rotation of 0.4° with complete rotation was achieved. Image reconstruction and analysis were performed using Skyscan software (Skyscan NV). The parameters evaluated included trabecular bone volume (BV/TV; %), trabecular number (Tb.N; /mm), trabecular separation (Tb.Sp; mm), trabecular thickness (Tb.Th; mm), and bone mineral density (BMD; g/cm2).
After decalcification, the samples were fixed in paraffin and cut longitudinally at 3 µm thickness using a microtome (mode : RM2255; Leica Microsystems, Wetzlar, Germany). Each sample was mounted on a slide and exposed to a temperature of 55°C. And then, the samples were deparaffinized in four 5-minute changes of xylene and dehydrated in 2-minute changes in decreasing alcohol solutions (100%, 95%, 80%, and 70%) sequentially. The slides were washed in flowing purified water for 1 minute and engrossed in Harris Hematoxylin-I (YD-Diagnostics, Yongin, Korea) for 5 minutes, followed by a 1-minute wash under flowing tap water. Sections were immersed in ammonia water for 30 seconds and then washed under tap water for 3 minutes. The sections were stained with Eosin Y (Sigma-Aldrich, St. Louis, MO, USA) for 2 minutes 30 seconds and dried out from 95% to 100% ethanol. Any residual stain washed away with distilled water. Each section was mounted (Shandon Synthetic Mountant; Thermo Fisher Scientific, Waltham, MA, USA).
After deparaffinization and hydration, slices were treated with a mordant comprising 5% iron alum solution at 56°C dry oven for 30 minutes. Sections were washed in running tap water to remove picric acid for 5 to 6 minutes. Sections were stained with Weigert’s iron hematoxylin solution for 10 minutes, and rinsed with purified water. They were then stained with Biebrich scarlet-acid fuchsin solution (Sigma-Aldrich) for 5 minutes and washed with purified water. The sections were placed in phosphomolybdic-phosphotungstic acid solution for 5 minutes. The solution was discarded and the sections were washed with distilled water. Samples were differentiated in 1% glacial acetic acid solution for 3 minutes. After discarding the solution, the residual stain was washed off with distilled water.
Blood was drawn via cardiac puncture and then the animals were euthanized. The blood samples were centrifuged at 3000 rpm for 10 minutes at 4° to separate serum. The serum was removed and stored at -80°C until analysis. Subsequently, the serum levels of osteocalcin (OC), rat C-telopeptide of type I collagen (CTX-I), BMP-2, and receptor activator of nuclear factor-ĸB ligand (RANKL) were measured. OC was determined with a Rat-Mid OC ELISA kit (IDS, Fareham, UK), CTX-I with a Rat CTX-I ELISA Kit (Cusabio, Houston, TX, USA), BMP-2 with a Quantikine BMP-2 Immunoassay (Cusabio), and RANKL using a Rat RANK ELISA kit (Cusabio). The serum parameters were based on their absorbance using a microplate absorbance reader (model Sunrise; TECAN, Seestrasse, Männedorf, Switzerland) according to the manufacturer’s instruction.
Data are expressed as means±standard error of mean or standard deviations. Two-way analysis of variance (ANOVA) with Bonferroni post-test correction for body weight and Mann-Whitney U test for micro-CT measurements were conducted to determine whether the parameters differed significantly between groups. A
PSBMP2, in which a promoter and secretion signal sequence of the bifidobacterial α-amylase gene was linked to a murine BMP-2 gene cDNA, was successfully amplified by PCR and cloned into the pUC18 plasmid vector. The nucleotide sequence of the PSBMP2 was analyzed using the universal primers of pUC18 vector to confirm that the promoter and secretion signal sequence was correctly ligated with the BMP-2 gene cDNA (data not shown). The PCR-amplified PSBMP2 was cloned into pLR5cat, an
All animals tolerated the procedure well. There was no wound dehiscence or local infection. Furthermore, there were no complications associated with implanted devices during the study period. The mean body weight of rats in all groups increased over a period of 20 weeks after ovariectomy. Weights of the rats in the OVX, OVX/LP, OVX/LP with BMP groups were significantly higher than in the control group (
Using 3D-micro-CT, osteoporotic changes of bone were observed in rats in the OVX, OVX/LP, and OVX/LP with BMP groups; however, the trabecular structure in the OVX/LP with BMP group was maintained compared with OVX and OVX/LP groups (
During the 20 weeks post-ovariectomy, H&E and Masson’s trichrome-stained sections of the tibia showed a decrease of bony trabeculae and increased adipose tissue infiltration in the bone marrow of rats in OVX, OVX/LP and the OVX/LP with BMP groups when compared with those of the control group. However, the trabecular formation in the OVX/LP with BMP group was retained compared with the OVX and OVX/LP groups (
The serum level of BMP-2 was higher in the control group compared with that of the other three groups, with the lowest level observed in the OVX group (
Multiple animal models were designed to study postmenopausal osteoporosis [
It is theoretically possible that treatment with genetically modified probiotics is associated with health benefits. Due to the availability of genomic data, genetic manipulation is easy and various genetic tools have been developed. A few
Osteoporosis is due to an age-linked decline in the number of osteoblast progenitors in the bone marrow as well as decreasing estrogen levels [
BMP is a sub-family of transforming growth factor-β first identified by Urist [
Bone undergoes remodeling by replacing old and damaged tissue with new bone. Osteoclasts and osteoblasts work consecutively in the same bone remodeling unit. Bone remodeling is most prominent on the cancellous bone surfaces, where osteoclasts remove the eroded surface in the old and damaged bone [
Serum OC is one of the bone formation marker secreted by osteoblasts and osteocytes [
This study had limitations that could be debated. First, this research only measured the amplification of PSBMP2 from the recombinant probiotics by PCR and did not evaluate Western blots to confirm the expression of the corresponding protein [
Our results showed that the expression of genetically modified BMP-2 showed inhibition effect for bone loss in a rat model of osteoporosis.
No potential conflict of interest relevant to this article was reported.
This type of study does not require informed consent.
Conceptualization : ESJ, GSM, JHJ
Data curation : JMY, JYK
Formal analysis : JYK, JKM
Funding acquisition : JHJ, GSM
Methodology : JSK, JKM
Project administration : JHJ, GSM
Visualization : SRJ, KHC
Writing - original draft : ESJ, GSM, JHJ
Writing - review & editing : ESJ, JYK, JMY, JSK, JKM, SRJ, KHC, GSM, JHJ
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (grant no. NRF-2017R1A2B1005327), and this work was supported by the Soonchunhyang University Research Fund.
This document has been checked by at least two professional editors, both of whomare native English speakers.
Confirmation of recombinant
Graphic analysis of body weight. In all groups, the mean body weight increased over 20 weeks after ovariectomy-induced osteoporosis group (OVX). Weights of rats in the OVX, OVX and
A representative micro-computed tomography image. In spite of osteoporotic changes in rats in the ovariectomy-induced osteoporosis group (OVX), OVX and
Graphic analysis of micro-computed tomography evaluation. The control group (sham group) had the highest values of bone mineral density (BMD), trabecular bone volume (BV/TV), trabecular thickness (Tb. Th), and trabecular number (Tb.N) and the lowest values of trabecular spacing (Tb.Sp). The differences in Tb.Th between control and ovariectomy (OVX) and genetically modified bone morphogenetic protein (BMP)-2-expressing
Images of (A) hematoxylin and eosin (H&E) and (B) Masson’s trichrome staining of tibia. The images show loss of bony trabeculae and increased adipose tissue in the bone marrow of rats in the ovariectomy-induced osteoporosis group (OVX), OVX and
Changes in the levels of serum markers. A : The serum level of bone morphogenetic protein (BMP)-2 was higher in the control group (sham group) than in the other three groups, with the lowest level observed in the ovariectomy-induced osteoporosis group (OVX) group (