Supercritical Carbon Dioxide Decellularized Bone Matrix Seeded with Adipose-Derived Mesenchymal Stem Cells Accelerated Bone Regeneration

Abstract

Large bone fractures with segmental defects are a vital phase to accelerate bone integration. The present study examined the role of supercritical carbon dioxide (scCO2) decellularized bone matrix (scDBM) seeded with allogeneic adipose-derived mesenchymal stem cells (ADSC) as bio-scaffold for bone regeneration. Bio-scaffold produced by seeding ADSC to scDBM was evaluated by scanning electron microscopy (SEM). Rat segmental femoral defect model was used as a non-union model to investigate the callus formation in vivo. Histological analysis and osteotomy gap closure in the defect area were analyzed at 12 and 24 weeks post-surgery. Immunohistochemical expression of Ki-67, BMP-2 and osteocalcin was evaluated to assess the ability of new bone formation scDBM. ADSC was found to attach firmly to scDBM bioscaffold as evidenced from SEM images in a dose-dependent manner. Callus formation was observed using X-ray bone imaging in the group with scDBM seeded with 2 × 106 and 5 × 106 ASCs group at the same time-periods. H&E staining revealed ASCs accelerated bone formation. IHC staining depicted the expression of Ki-67, BMP-2, and osteocalcin was elevated in scDBM seeded with 5 × 106 ASCs group at 12 weeks after surgery, relative to other experimental groups. To conclude, scDBM is an excellent scaffold that enhanced the attachment and recruitment of mesenchymal stem cells. scDBM seeded with ASCs accelerated new bone formation.

Current applications of adipose-derived mesenchymal stem cells in bone repair and regeneration: A review of cell experiments, animal models, and clinical trials

“…It is shown that PCL scaffolds with the transient release of hyperoxia enhance ASC-mediated skull regeneration in mice ( Farris et al, 2022 ). These findings provided biological clues for the application of scaffolds ( Liu et al, 2021 ). Nanomaterials have made new progress in ASCs contributing to bone formation.…”

“…Another animal experiment showed that the combination of ASCs improved the gap and surrounding ossification of the scaffold compared with the scaffold alone ( Lee et al, 2021 ). ASCs also display the ability to accelerate bone formation after using biological scaffolds ( Liu et al, 2021 ).…”

Criteria, Challenges, and Opportunities for Acellularized Allogeneic/Xenogeneic Bone Grafts in Bone Repairing

“…Although using in vivo and in vitro bioreactors can improve the adhesion coefficient and differentiation of cells loaded on ABGs, indicators such as cell migration or leaching, dedifferentiation, and apoptosis of stem cells in therapeutic processes simply affect treatment efficacy. In this regard, Liu et al 136 based on the SEM results showed that fat stem cells loaded on ABGs derived from porcine bone through physical approach (supercritical carbon dioxide with a pressure of 350 bar at 45 °C for 80 min) were effectively placed on the cavities. However, the regeneration time ranged between 12 and 24 weeks, indicating a delay in biological functions of such as cell migration, adhesion, and differentiation to osteogenic cells in vivo.…”

“…Despite an increase in bone mass and upregulation of K i -67, BMP-2, and OCN mRNA, which indicate promising cell differentiation, the slow rate of bone repair by ABGs can pose a challenge to therapeutic efficacy, particularly in diabetic, osteoporosis, and infectious disease patients. 136 Other agents, such as drugs or nanomaterials, appear to be effective in stimulating rapid proliferation and differentiation of stem cells in ABGs. For instance, Parmaksiz et al 137 by integrating 12−18 nm iron nanoparticles in the production process of ABGs molded from bovine tibia head granules using a chemical-enzymatic approach showed that the healing rate of rat calvaria bone tissue increases through osteoinductive amplification by low frequency-pulsed electromagnetic field.…”

Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering

“…Implantation of the DPB seeded ASCs stimulated endochondral ossification for substantial bone regeneration. The DPB seeded ASCs system is of clinical relevance for segmental defect bone regeneration [40].…”