P18. Treatment of osteoporotic vertebral fractures/defect in rats by naringin/hydroxyapatite/silk fibroin composite scaffolds in regulation of osteogenic differentiation, angiogenesis and inflammation

Abstract

BACKGROUND CONTEXT Biomaterial scaffolds, as one of the three major elements of regenerative medicine, play an important role in bone remodeling and are therefore commonly used in the treatment of osteoporotic vertebral fractures (OVF). Among many biological materials, hydroxyapatite has attracted much attention in the research of bone defect regeneration due to its better bone conduction characteristics and mineralization processes and structures similar to bone tissue. Silk fibroin is a kind of natural biopolymer with high mechanical strength, controlled biodegradability, good biocompatibility and easy handling. The use of modern science and technology to make silk fibroin and hydroxyapatite into a composite scaffold can not only integrate the advantages of the two, but also avoid the disadvantages of low load bearing pressure, rejection reaction and non-degradability of traditional materials, which is an ideal, new tissue engineering material for OVF treatment. In recent years, traditional Chinese medicine has shown its unique advantages in the prevention and treatment of OVF. Traditional Chinese medicine has the effect of strengthening kidney and strengthening bone, promoting blood circulation and removing blood stasis. It is often used to promote fracture healing in clinic. Naringin is the main active ingredient of Rhizoma Drynariae, which can promote bone formation, angiogenesis, and inhibit inflammation. The combination of naringin, hydroxyapatite and silk fibroin as a new composite scaffold may have a synergistic effect and provide new insight on osteoporotic vertebral fractures/bone defect. PURPOSE To investigate the therapeutic effect and molecular mechanism of a new scaffold with naringin /hydroxyapatite/silk fibroin (NG/GMs/HA/SF) on vertebral fractures in osteoporotic rats. METHODS Loaded granules of naringin microspheres (NG/GMs), silk fibroin scaffolds (SF), hydroxyapatite/silk fibroin composite scaffolds (HA/SF), and granule naringin /hydroxyapatite/silk fibroin composite scaffold (NG//HA/SF) and loaded granule naringenin microsphere/hydroxyapatite/silk fibroin composite scaffold (NG/GMs/HA/SF) were conducted. The microscope was used to observe, calculate the volume of GMs and swelling ratio. X-ray diffraction (XRD) was used to analyse HA structure characterization; Fourier transform infrared spectroscopy was used to detect each scaffold structure. Scanning electron microscopy was used to analyse the surface structure, pore size and biocompatibility of each scaffold. Scanning calorimetry (DSC) was used to analyze the thermal properties and stability of each scaffold. UV spectrophotometer was used to detect the drug release rate in vitro. Trypan blue staining was used to detect the activity of MC3T3-E1 osteoblast cell line in the scaffold. We co-cultured bone marrow derived-mesenchymal stem cells (BMSCs) and scaffolds. And then ALP staining was used to detect the osteogenic differentiation potential of BMSCs, alizarin red staining used to detect mineralization ability, qRT-PCR used to detect the expression levels of osteogenic markers OCN, BMP-2 and Runx2 mRNA; AKP reagent used to detect osteoblast activity, Western-blot used to detect OCN, BMP-2, Runx2 protein expression. The rats experimented ovariectomize surgery to construct postmenopausal osteoporosis model, and next osteoporosis model was successfully used to construct lumbar vertebrae bone defect model, and then the scaffold was filled in bone Defects. X-ray was for the detection of bone defect formation, HE staining, Safranin solid staining and Masson staining for detection of bone defect morphological changes. Micro-CT examined three-dimensional structure and bone structure parameters of bone defect. Serum estrogen, CTSK, OCN/BGP, AKP levels were detected by ELISA. qRT-PCR, immunohistochemistry and Western-blot was used to detect osteoclast markers (OCN, Runx2, BMP2, CTSK), Collagen II), angiogenesis markers (VEGFA) and inflammatory markers (IK-6, TNF-a, NF-kB) mRNA and protein expression. RESULTS NG/GMs, SF, HA/SF, NG//HA/SF, NG/GMs/HA/SF scaffolds were successfully constructed, and the scaffolds had good biocompatibility and sustained drug release rate. The loaded granule naringenin microsphere/hydroxyapatite/silk fibroin composite scaffold can promote osteogenic differentiation and mineralization of BMSCs, and the scaffold can up-regulate the mRNA and protein expression of OCN, BMP-2 and Runx2. After 4 months, the X-ray and micro-CT three-dimensional reconstruction results showed that there was no obvious healing of the bone defect in the blank group, and the bone defect in the SF, HA/SF, NG/HA/SF group was partially healed, and the NG/GMs/HA/SF group has basically healed whose healing effect was better than that of SF, HA/SF, NG/HA/SF group. In the NG/GMs/HA/SF group, HE staining, Safranin solid staining and Masson staining showed more osteogenesis area. Futhermore, serological osteogenic indicators, molecular biology, osteogenesis, angiogenesis, inflammation-related genes and protein up-regulated. CONCLUSIONS The NG/GMs/HA/SF composite scaffolds may be used to treat osteoporotic vertebral fractures in rats by regulating the “osteogenesis differentiation-angiogenesis-inflammation” homeostasis. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.