Improvement of intertrochanteric bone quality in osteoporotic female rats after injection of polylactic acid-polyglycolic acid copolymer/collagen type I microspheres combined with bone mesenchymal stem cells

Abstract

Purpose: Osteoporosis mainly involves cancellous bone, and the spine and hip, with their relatively high cancellous bone to cortical bone ratio, are severely affected. Studies of bone mesenchymal stem cells (BMSCs) from osteoporotic patients and animal models have revealed that osteoporosis is often associated with reduction of BMSCs' proliferation and osteogenic differentiation. Our aim was to test whether polylactic acid-polyglycolic acid copolymer(PLGA)/collagen type I(CoI) microspheres combined with BMSCs could be used as injectable scaffolds to improve bone quality in osteoporotic female rats.

Methods: PLGA microspheres were coated with CoI. BMSCs of the third passage and were cultured with PLGA/CoI microspheres for seven days. Forty three-month-old female non-pregnant SD rats were ovariectomized to establish osteoporotic animal models. Three months after being ovariectomized, the osteoporotic rats were randomly divided into five groups: SHAM group, PBS group, cell group, microsphere (MS) group, and cell+MS group. Varying materials were injected into the intertrochanters of each group's rats. Twenty rats were sacrificed at one month and three months post-op, respectively. The femora were harvested in order to measure the intertrochanteric bone mineral density (BMD) with DEXA and trabecular thickness (Tb.Th), percentage of trabecular area (%Tb.Ar), bone volume fraction (BV/TV) and trabecular spacing (Tb.Sp) with Micro CT. One-way ANOVA and Kruskal-Wallis tests were used.

Results: BMSCs seeded on PLGA/CoI microspheres had a nice adhesion and proliferation. At one month post-op, the BMD (0.33 ± 0.01 g/cm(2)), Tb.Th (459.65 ± 28.31 μm), %Tb.Ar (9.61 ± 0.29 %) and Tb.Sp (2645.81 ± 94.91 μm) of the cell+ MS group were better than those of the SHAM group and the cell group. At three months post-op, the BMD (0.32 ± 0.01 g/cm(2)), Tb.Th (372.81 ± 38.45 μm), %Tb.Ar (6.65 ± 0.25 %), BV/TV (6.62 ± 0.25 %) and Tb.Sp (1559.03 ± 57.06 μm) of the cell + MS group were also better than those of the SHAM group and the cell group.

Conclusion: The PLGA/CoI microspheres combined with BMSCs can repair bone defects more quickly. This means that PLGA/CoI microspheres combined with BMSCs can promote trabecular reconstruction and improve bone quality in osteoporotic rats. This scaffold can provide a promising minimally invasive surgical tool for enhancement of bone fracture healing or prevention of fracture occurrence which will in turn minimize complications endemic to patients with osteoporosis.

Fig. 1

Ovariectomy of rats. a Location of the ovary. b The ovaries at removal. c The incisions

Fig. 2

Bone mesenchymal stem cells (BMSCs) with osteogenic induction showed positive for collagen type I with brown cytoplasm (a), BMSCs without osteogenic induction showed negative (b) (immunohistochemistry staining × 400)

Graph 1

The diameter distribution of 224 microspheres that we used in our research

Fig. 3

Structure of PLGA microspheres (SEM). a, b Microspheres without CoI (magnification of a × 50, magnification of b × 500). c, d Microspheres coated by CoI (magnification of c × 50, magnification of d × 500); there was a lot of collagen coated on the surface of microspheres

Fig. 4

Bone mesenchymal stem cells (BMSCs) cultured with CoI in vitro for 7 days (SEM): magnification of a × 50, magnification of b × 1000, a large number of BMSCs (black arrows) combined with polylactic acid-polyglycolic acid copolymer (PLGA) microspheres

Graph 2

Intertrochanteric bone mineral density (BMD) in different groups at one month post-op and three months post-op. * Compared with SHAM group at 3 months post-op, P < 0.001. ** Compared with cell group at three months post-op, P = 0.008

Fig. 5

Rats’ intertrochanters at one month post-op (Micro CT) from the SHAM group (a), PBS group (b), cell group (c), MS group (d) and cell+ MS group (e). Standard ROI (region of interest) between the greater trochanter and the lesser trochanter was chosen (cancellous bone area between the two red parallel lines)

Graph 3

Intertrochanteric Tb.Th(μm) in different groups at one month post-op and three months post-op. * Compared with SHAM group at three months post-op, P < 0.001. ** Compared with cell group at three months post-op, P = 0.003

Graph 4

Intertrochanteric %Tb.Ar(%) in different groups at one month post-op and three months post-op. * Compared with cell group at 1 months post-op, P = 0.029. ** Compared with SHAM group at three months post-op, P = 0.009

Graph 5

Intertrochanteric BV/TV(%) in different groups at one month post-op and three months post-op. * Compared with SHAM group at three months post-op, P = 0.009

Graph 6

Intertrochanteric Tb.Sp(μm) in different groups at one month post-op and three months post-op