A Composite Tissue Engineered Bone Material Consisting of Bone Mesenchymal Stem Cells, Bone Morphogenetic Protein 9 (BMP9) Gene Lentiviral Vector, and P3HB4HB Thermogel (BMSCs-LV-BMP9-P3HB4HB) Repairs Calvarial Skull Defects in Rats by Expression of Osteogenic Factors

Abstract

Background Bone tissue engineering has been proven to be an appropriate approach for treating bone defects. This study aimed to investigate the effects and mechanism of a composite tissue engineered bone material consisting of bone mesenchymal stem cells (BMSCs), bone morphogenetic protein (BMP9) gene lentiviral vector, and P3HB4HB thermogel (BMSCs-LV-BMP9-P3HB4HB) on calvarial skull defects in rats. Material/Methods LV-BMP9 viral vector was structured and infected to BMSCs-P3HB4HB composite scaffold, which was named as BMSCs-P3HB4HB composite bone repair material. Adipogenic differentiation was determined by oil-red O (ORO) and alkaline phosphatase (ALP) staining. Osteogenic differentiation was measured using Alizarin red staining. Cell viability was examined using Cell-Counting Kit-8 (CCK-8) assay. Protein expression of osteogenic factors, including BMP9, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN), and osterix (OSX), was detected with Western blot assay and immunohistochemistry. mRNA of these osteogenic factors was examined by RT-PCR. Histological changes were examined with hematoxylin and eosin (H&E) and Masson’s trichrome staining. Bone repair was measured using micro-computed tomography (micro-CT). Results BMSCs and LV-BMP9-infected BMSCs demonstrated adipogenic and osteogenic differentiation potential. BMSCs-P3HB4HB scaffold demonstrated good cell-tissue compatibility. BMSCs-LV-BMP9-P3HB4HB exhibited significantly higher osteogenic ability and cell viability of BMSCs compared to BMSCs-LV-P3HB4HB ( p <0.05). BMSCs-LV-BMP9-P3HB4HB significantly promoted osteogenic factors (RUNX2, OCN, OPN, and OSX) expression compared to the BMSCs-LV-P3HB4HB group ( p <0.05) in both BMSCs and in calvarial defect rats. BMSCs-LV-BMP9-P3HB4HB demonstrated stronger repair ability. BMSCs-LV-BMP9-P3HB4HB significantly alleviated pathological injury and increased collagen fiber production compared to the BMSCs-LV-P3HB4HB group ( p <0.05). Conclusions BMSCs-LV-BMP9-P3HB4HB composite bone repair material can effectively repair injured skull tissues of calvarial defect rats through triggering osteogenic factors expression. The present generated bone repair material may have applications in tissue engineering in regeneration of bone defects.

Clinical Application of Bone Marrow Mesenchymal Stem/Stromal Cells to Repair Skeletal Tissue

“…Certainly this later approach has shown some promise in rodent proof-of-concept preclinical cranial critical size defect models [ 178 , 179 ]. Where allogeneic BMSC pre-seeded onto specialized bio-compatible scaffolds containing BMPs, particularly BMP2, BMP6 and BMP9, augmented bone regeneration [ 178 , 180 ]. Furthermore, it was also established that CXCL12 in combination with BMP further enhanced bone repair [ 179 ].…”

Isolation and characterization of apical papilla cells from root end of human third molar and their differentiation into cementoblast cells: an in vitro study

“…The ability of osteoblasts to produce mineralized matrix and calcium deposition is known as extracellular matrix (ECM) mineralization [44,45], which can be identified as reddish-black spots using Alizarin red S staining [46]. Here, we used the Alizarin red S staining assay to evaluate the calcium deposition rate and formation of hydroxyapatite calcium in the cultured hSCAPs within 21 days in a differential microenvironment [29].…”

Immediate implantation of ultrafine fiber slow-release system based on cell electrospinning to induce osteogenesis of mesenchymal stem cells

“…Consequently, P34HB has garnered significant interest as a carrier for the sustained release of bioactive molecules or as a degradable implant material. Our preliminary investigations have confirmed its excellent biocompatibility, biodegradability, and promotive effect on the osteogenic differentiation of bone marrow mesenchymal stem cells [ 6 , 11 ]. In this study, we continue to employ P34HB for the development of CES fiber scaffolds for osteogenesis.…”