Percutaneous vertebroplasty (PVP) is widely used in the treatment of painful osteoporotic vertebral compression fractures with the injection of PMMA cement, and the controversy for PMMA damage to the osteoporotic bone tissue and to affect the fractures repairing never stops. 72 old female rabbits, each age 3.0~3.5 y, rabbits were assigned randomly to two groups of thirty-six each; PMMA cement were injected into vertebral body in rabbits via mimic PVP, sacrificed at 1 h, 24 h, 3 d, 7 d, 4 w, and 12 w. The expression VEGF and collagen type I, the tissue response, and repair reaction in the interface between PMMA and bone tissue were observed dynamically with RT-PCR and western blot technique; the osteocalcin expression were studied by immunohistochemistry. Compared with the control group, the expression of collagen I increased at 1 hour and was higher from 24 h to 3 d. From 4 weeks to 12 weeks after injection of PMMA. The expression of VEGF decreased at 1 hour and 24 hours, significantly increased at 3 days, decreased once again at 7 days, then increased significantly at 4–12 weeks. The osteocalcin expression continued to increase during 4 to 12 week. PMMA would not cause local bone permanent necrosis, and interface injury repairing cycle could be prolonged in a vertebroplasty.
Percutaneous vertebroplasty (PVP), a persistent developing procedure, is nowadays widely used in the treatment of painful osteoporotic vertebral compression fractures with the injection of polymethylmethacrylate (PMMA) cement, which is accepted most in clinical surgery contributing for its effect of immediate pain relief, biomechanical function reconstruction, and low price, whereas PMMA was also denounced of goodish bone compatibility and combination [
Seventy-two old New Zealand female rabbits (provided by KunMing Medical College, China), each aged from 3.0 to 3.5 years, weighed between 3 and 4.2 kg, were used in this study. All the rabbits were demonstrated as osteoporosis by DXA (Lunar, USA). Preoperative frontal and lateral position X-ray film demonstrates that all these rabbits had normal lumbar vertebrae sequences and bone architectures. Rabbits were assigned randomly to two groups of thirty-six each. All groups were treated with the same surgical procedures and evaluations but were sacrificed at different postoperative times at 1 h, 24 h, 3 d, 7 d, 4 w, and 12 w. All animals received general anesthesia using 4% of pentobarbital sodium (30 mg/kg) followed by helix veinintravenous injection. Considering elimination of biological variance caused by different level of vertebrae, injection (powder and liquid mixed at 20 g : 5 mL) of modified PMMA (Tianjin Synthetic Material Research Institute, China) applied to randomly selected lumbar vertebrae in the experimental group. The control group received operation alone, without PMMA injection.
A skin incision was made in the median of the back at the level of vertebral L7 (top of hip); the subcutaneous tissue and masseter muscle were divided to expose the anterior half of L4, L5, and L6. Referred to clinical PVP surgical procedures [
Each group of animals, respectively, took lateral X-ray film to inspect the presence, and morphologic change of PMMA before the animals was sacrificed at their predetermined time.
Each group of animals was sacrificed at their predetermined time; the vertebraes (L2, L4) were dissected and removed out the PMMA and cut into blocks containing the interface between bone cement and bone tissue, part of them for total tissues RNA extraction and the others for histology.
Total RNA extraction of tissues [
Primer sequences are used in the experiment as follows: VEGF-L 5′-GAC ATC TTC CAG GAG TAC CC-3′ 157 bp VEGF-R 5′-TGA GGT TTG ATC CGC ATG AT-3′ actin-L TGG CTC TAA CAG TCC GCC TAG 295 bp.
Total protein extraction was manipulated on ice and preserved at −70°C following the instruction of BCA-100 protein quantitation kit (Shanghai Biocolor BioScience & Technology Company).
The first antibody for 18 h was incubated under 4°C after diluted to 1 : 100 (VEGF) and 1 : 1000 (collagen), subsequently went through TBST rinshing for 4 × 10 min. The second antibody was put under ambient temperature for 1 h and TBST rinshing for 4 × 15 min. The PVDF film which was prior incubated in ECL reagent for 5 min was placed into the chromogenic film cassette. Finally in dark room carried out the exposure and developing with X-ray film according to an optimal fixing time.
The immunohistochemical procedure has been described previously reports [
RT-PCR result computation: firstly, sample Ct-intraparameter Ct = ΔCt, to compute ΔΔCt we set a sample ΔCt (the control group as usual) as reference. Computing method was ΔCt (sample) − ΔCt (reference). Outcome illustrated the mathematics relation between samples was 2 − (−ΔΔCt)th power.
Protein relative amount computation: place the X-ray film into gel Image Processing System (Tanon) to detect intensity and area of the target band. Relative amount = intensity × area.
The data were represented in the form of mean ± standard deviation (X ± S) and analyzed utilizing the one-way ANOVA. The means of sample from both groups were compared using
Bone trabecula was full of PMMA cement that is tightly connecting to bone tissues and well distributed. PMMA was covered with considerable soft tissues and a few new born bone tissues in 12 w. In the control, soft tissues coverage and bone defect about 0.5 cm were visible. The bone defect whose volume kept invariableness was filled with hematoma and some granulation tissues but without new osteogenesis.
All injected cement did not appear of any defluxion, crack, or loosening. The boundary between PMMA and bone became indistinct with new osteogenesis in subgroup 12 w.
Compared with that of control, the samples were tightly combined with PMMA cement. The interface between bone tissues and PMMA was infiltrated with inflammatory cells and fibrous tissues. Inflammatory cell infiltration was obvious in 24 h, developed to a peak in 3 d, alleviated comparatively in 7 d. Chondrocytes were found growing in cluster and differentiating to woven bone at the interface without conspicuous inflammatory cells in 4 w; massive lamellar bone formed and occasional haematopoietic bone marrow could be observed without any inflammation in 12 w (Figures
(a) Normal bone trabecula of vertebral in control, HE ×50. (b) Bone trabecula apparently reduced with inflammatory cells infiltration after PMMA injected 24 h, HE ×50.
(a) Inflammatory cells infiltration after PMMA injected 3 d, HE ×50. (b) Inflammatory reaction lightened after PMMA injected 7 d, HE ×50.
(a) Large quantity of chondrocytes growing in cluster after PMMA injected 4 w, HE ×100. (b) Chondrocytes in cluster without conspicuous inflammation after PMMA injected 4 w, HE ×100.
VEGF was amplified to the target band of 157 bp molecular weight, taking actin as intraparameter. It confirmed the target band on coincidence to the gene fragment in the literature according to intraparameter.
The VEGF mRNA expression was lower (
(a) VEGF mRNA expression was lower in 1 h and 24 h (
VEGF protein expression was increased a little at 1 h and 24 h. It was increased a lot in 3 d while decreased in 7 d. After that the expression was transparently elevated and higher than that of control (
Collagen type I mRNA expression was increased from 1 h. A persistent high level formed from postoperative 24 h to 3 d. Despite of being slightly descended in 7 d, it kept a comparative high expression from 4 w to 12 w which was remarkably higher than that of control (
(a) Collagen type I mRNA expression was increased in 1 h. A persistent high level was formed in postoperative 24 h to 3 d. Despite being slightly descended in 7 d, it kept a comparative high expression from 4 w to 12 w which was remarkably higher than that of control (
Compared with normal bone tissue, after 1 h, 24 h, and 3 d, the OC expression had no significant increase in both groups (
(a) OC expression slightly increased in PMMA-bone interface in 1 week, 20 × 10SP + HE. (b) OC expression in PMMA-bone interface in 4 weeks, when chondrocytes were found to be growing in cluster and differentiating to woven bone, 20 × 10SP + HE.
Several inherent advantages to PMMA include familiarity for orthopedic surgeons, ease of handling, good biomechanical strength and stiffness, and cost effectiveness [
Lieberman et al. identified particles consistent with cement and/or barium sulfate in vascular spaces in human vertebrae obtained from surgical excision and autopsy cases [
The result of our research on VEGF illustrated in the 1 h and 24 h was lower than the corresponding of control (
Meanwhile, in our studies, collagen type I at 1 h was multiple amplified, and a persistent high level formed from postoperative 24 h to 3 d. PMMA has the effect of promoting collagen information. Viateau et al. [
In the process of fracture healing, collagen type I is the characteristic marker representing bone formation and molding. Its peak expression occurred between 3 w and 5 w after fracture which is the state from intracellular ossification of chondrocyte to bone information, being related to vasiformation. From the result of our study, PMMA cement does harm to bone trabecula at a certain degree in PVP. The major influence on osteoporotic fracture repair is time delay. Repair mechanism coincides with normal fracture healing, whereas the time is delayed for 4 weeks around. It is thus clear that, the PMMA injected in PVP influenced the function of local bone tissue and cell, but it did not induce irreversible damage.
In conclusion, interface injury caused by PVP injection of domestic advanced PMMA bone cement could be repaired and mineralized undergoing a process similar to normal fracture healing. However, the expression of VEGF, collagen type I, and OC delayed about 4 weeks. Bone cement would not cause local bone permanently necrosis; interface injury repairing cycle could be prolonged in a vertebroplasty.
Percutaneous vertebroplasty
Polymethylmethacrylate
Vascular endothelial growth factor.
The authors report no conflict of interests. The authors alone are responsible for the content and writing of the paper.
Dr. Gang Zhao and LiJun Wang also can be considered as the first authors.
This work was supported in part by the Scientific Research Foundation of Chongqing Government.