introduction
Bone defects lead to dyskinesia, loss of function, and even life-threatening. Due to the limited source of autologous bone and the rejection of allogeneic bone, bone repair and regeneration has always been a medical problem. Tissue engineering methods with stem cells, scaffolding materials and growth factors as the main constituent elements are the most promising solutions to this problem. Among them, regulating stem cells to differentiate into osteoblasts is the key to achieving new bone formation and bone tissue reconstruction. The method of regulating the differentiation of stem cells by using special proteins is difficult to be practical because of the low price of the protein, the high price, the easy inactivation, and the long control period. In recent years, it has been found that the use of nano-structures on the surface of materials can achieve precise, rapid, long-lasting and position-controlled control of stem cell differentiation. This physical regulation of stem cells provides an important approach for bone repair. However, so far, few people have used biodegradable materials to construct nanostructures to achieve nanostructure-mediated stem cell osteogenic differentiation and bone tissue repair processes.
Summary of results
Recently, Liu Hong and Liu Wei of Shandong University and Liu Chao (common communication) of Qilu Hospital of Shandong University used different types of porous anodized aluminum oxide (AAO) as templates to adopt simple and easy micro-imprint method. Nano-column arrays of different diameters were constructed on the surface of polylactic acid (PLA) films. By studying the differentiation behavior of human adipose-derived mesenchymal stem cells (hADSCs) on PLA nanocolumn arrays, it was found that hADSCs showed complete surface structure on different diameters of nano-pillars without any chemical or biological induction. Different differentiation capabilities. The study found that nano-column arrays with a diameter of 200 nm can best promote the differentiation of adipose-derived mesenchymal stem cells into osteoblasts. Animal experiments have also confirmed that this PLA nano-pillar array material can achieve ectopic osteogenesis. Related results are published on Nano Letters under the title "Polylactic Acid Nanopillar Array-Driven Osteogenic Differentiation of Human Adipose-Derived Stem Cells Determined by Pillar Diameter".
Graphic guide
Fig.1 Morphology and biocompatibility test of polylactic acid arrays with different nanocolumn diameters
Ac) the surface topography of the polylactic acid flat sheet;
Df,gh,jl) AAO template with diameters of 100 nm, 200 nm and 300 nm and corresponding PLA nanocolumn array morphology; mx. hADSCs staining of living cells on different nanocolumn diameter arrays;
Yz) Schematic diagram of hADSCs on different substrates and cck-8 test results.
Fig. 2 Spreading morphology of hADSCs on different nanocolumn diameter polylactic acid arrays
Af) the spread morphology of hADSCs on polylactic acid flat sheets;
Gl) the spread morphology of hADSCs on a nanometer column diameter of 100 nm;
Mr) the spread morphology of hADSCs on a nanocolumn diameter of 200 nm;
Sx) The spread morphology of hADSCs on a 300 nm substrate with a nanocolumn diameter.
Figure 3 q-PCR results of osteogenic related genes after hADSCs cultured on different nanocolumn diameter polylactic acid arrays for 21 days
a) ALP protein content of hADSCs on different culture substrates;
b) Runx2 gene expression of hADSCs on different culture substrates;
c) OPN gene expression of hADSCs on different culture substrates;
d) OCN gene expression of hADSCs on different culture substrates;
e) Alizarin red staining results of hADSCs on different culture substrates.
Figure 4 Fluorescent immunostaining results of osteogenic proteins after hADSCs cultured on different nanocolumn diameter polylactic acid arrays for 21 days
Aj) OPN immunofluorescence staining results of hADSCs on different culture substrates;
Kt) OCN immunofluorescence staining of hADSCs on different culture substrates.
Figure 5 ALP immunohistochemical staining and H&E staining results of ectopic osteogenic tissue sections
a) ALP immunohistochemical staining results of ectopic osteogenic tissue sections on PLA plain films;
b) ALP immunohistochemical staining results of ectopic osteogenic tissue sections on PLA nanocolumn arrays with a diameter of 200 nm;
c) H&E staining results of ectopic osteogenic tissue sections on PLA plain films;
d) H&E staining results of ectopic osteogenic tissue sections on a PLA nanocolumn array with a nanocolumn diameter of 200 nm.
summary
The study indicated that the three nanocolumn array materials (column diameters of 100 nm, 200 nm, and 300 nm, respectively) have different degrees of osteogenic induction, while PLA plain films have essentially no osteogenic induction. And only after 48 h of culture, the morphology of hADSCs on different substrates was very different. The stem cells on polylactic acid flat sheets and other substrates were still fusiform, while the PLA nanopillar array material with diameter of 200 nm was used. The cells on the surface have been characterized by a polygonal structure of osteoblasts. The results of gene and protein tests after 21 days of culture also showed that the PLA nanocolumn array material with diameter of 200 nm has the strongest osteogenic differentiation promoting effect, and can also enrich more osteogenesis-related proteins in ectopic osteogenesis. . Therefore, this study uses biodegradable materials to construct nanostructures, and promotes nanostructure-mediated stem cell osteogenic differentiation and bone tissue repair processes.
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