Development of artificial bone graft via in vitro endochondral ossification (ECO) strategy for bone repair

Abstract

Endochondral ossification (ECO) is a form of bone formation whereby the newly deposited bone replaces the cartilage template. A decellularized artificial cartilage graft (dLhCG), which is composed of hyaline cartilage matrixes, has been developed in our previous study. Herein, the osteogenesis of bone marrow-derived MSCs in the dLhCG through chondrogenic differentiation, chondrocyte hypertrophy, and subsequent transdifferentiation induction has been investigated by simulating the physiological processes of ECO for repairing critical-sized bone defects. The MSCs were recellularized into dLhCGs and subsequently allowed to undergo a 14-day proliferation period (mrLhCG). Following this, the mrLhCG constructs were subjected to two distinct differentiation induction protocols to achieve osteogenic differentiation: chondrogenic medium followed by chondrocytes culture medium with a high concentration of fetal bovine serum (CGCC group) and canonical osteogenesis inducing medium (OI group). The formation of a newly developed artificial bone graft, ossified dLhCG (OsLhCG), as well as its capability of aiding bone defect reconstruction were characterized by in vitro and in vivo trials, such as mRNA sequencing, quantitative real-time PCR (qPCR), immunohistochemistry, the greater omentum implantation in nude mice, and repair for the critical-sized femoral defects in rats. The results reveal that the differentiation induction of MSCs in the CGCC group can realize in vitro ECO through chondrogenic differentiation, hypertrophy, and transdifferentiation, while the MSCs in the OI group, as expected, realize ossification through direct osteogenic differentiation. The angiogenesis and osteogenesis of OsLhCG were proved by being implanted into the greater omentum of nude mice. Besides, the OsLhCG exhibits the capability to achieve the repair of critical-size femoral defects.

Keywords: Biomaterials; Endochondral ossification; Hypertrophic chondrocytes; Regeneration medicine; Scaffold; Tissue engineering; Transdifferentiation.

Graphical abstract

Fig. 1

Schematic illustrations. a) In vitro experiment of OsLhCG preparation, including MSC expansion, recellularization, and differentiation induction. b) The greater omentum implantation. c) The critical size defect implantation. d) Flow chart of in vitro and in vivo experiments.

Fig. 2

qPCR results of the CGCC group and the OI for genes that are involved in ECO. a) and b) are gene expressions of chondrogenesis-related genes. c) and d) are HCC gene expressions. e)-k) are gene expressions of osteogenesis-related genes.

Fig. 3

Statistics of the differentially expressed genes and heatmaps of the CGCC group and the OI group. Differentially expressed core genes related to each pathway, component, or biological process are listed. a) Volcano plot of differentially expressed genes. The threshold was defined as a P value < 0.05 and |log2FC|>1. The volcano plot shows that 844 genes were upregulated, 1177 genes of OsLhCG-CGCC were downregulated, and 14671 genes showed no significant difference. b) Heatmaps of genes that are involved in skeletal pathways, including ECO, collagen chain trimerization, and articular cartilage ECM. c) Heatmaps of genes that are involved in some general pathways, including integrin, PAK, and ERK. d) Heatmaps of genes that are involved in angiogenesis, ECM, and MMP.

Fig. 4

mRNA sequencing results, including KEGG pathway annotation of differentially expressed genes and differentially expressed gene GO annotation classification. Upregulation is defined as the gene expression of OsLhCG-CGCC being higher than OsLhCG-OI, while downregulation is defined as the gene expression of OsLhCG being higher. a) and b) are KEGG pathway classifications, and the top 35 KEGG pathways of the “upregulation” and “downregulation” groups are listed, respectively. c) and d) are Sankey-dot pathway enrichment plots of the “upregulation” and “downregulation” groups, where differentially expressed core genes involved in the major pathway are listed. The curve in the figure indicates the affiliation of genes to KEGG pathways, and the bubble plot reveals the significance, rich factor, and count number of each KEGG pathway. e) and f) are GO pathway enrichment circle diagrams. Each rectangle in the outermost layer of the circular plot represents a GO term, the middle layer represents the number of genes included in each GO term, and the inner layer represents the number of overlapping genes between the input genes and the genes included in each GO term. The bar chart in the center represents the rich factor of input genes in each GO term.

Fig. 4

mRNA sequencing results, including KEGG pathway annotation of differentially expressed genes and differentially expressed gene GO annotation classification. Upregulation is defined as the gene expression of OsLhCG-CGCC being higher than OsLhCG-OI, while downregulation is defined as the gene expression of OsLhCG being higher. a) and b) are KEGG pathway classifications, and the top 35 KEGG pathways of the “upregulation” and “downregulation” groups are listed, respectively. c) and d) are Sankey-dot pathway enrichment plots of the “upregulation” and “downregulation” groups, where differentially expressed core genes involved in the major pathway are listed. The curve in the figure indicates the affiliation of genes to KEGG pathways, and the bubble plot reveals the significance, rich factor, and count number of each KEGG pathway. e) and f) are GO pathway enrichment circle diagrams. Each rectangle in the outermost layer of the circular plot represents a GO term, the middle layer represents the number of genes included in each GO term, and the inner layer represents the number of overlapping genes between the input genes and the genes included in each GO term. The bar chart in the center represents the rich factor of input genes in each GO term.

Fig. 5

General analysis of samples collected at each time point. Figures a, b, c, and d demonstrate the cell number (per mg sample dry weight), glycosaminoglycans (μg/cell number), osteopontin concentration (pg/mL), and calcium concentration (mg/g), respectively. e) Alizarin Red S and Von Kossa staining. Positive expression of calcium deposition is represented in the figure by the sample being stained dark red in Alizarin Red S staining. In Von Kossa, calcium deposition is presented in brown, and cells are presented in pink. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Cell viability testing. a) The confocal microscope images of mrLhCG stained with live/dead dyes on Day 2, 6, 8, 10, and 14 after MSC implantation. The green regions in the images represent live cells, while the red regions represent dead cells. b) The results of the CCK-8 assay for mrLhCG on Day 2, 6, 8, 10, and 14 after MSC seeding. The vertical axis of the bar chart represents the relative cell viability, which was obtained by normalizing the absorbance values of mrLhCG at different time points with the absorbance value of a certain number of MSCs. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

In vitro samples staining and analyzing. a) In vitro samples characterization of H&E and immunohistochemistry staining (OCN, OPN, ON, type I collagen). In H&E staining, ECM is presented in light purple/red, and dark purple dots represent cells. In IHC staining, the brown area represents the positive expression of the related protein. b) The percentage of positive staining. (OCN, OPN, ON, Type I collagen). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

In vivo experiment sample demonstration and staining. (H&E, IHC). a) Images of the greater omentum, critical size defects, and samples obtained after implantation. b) H&E and IHC staining of OsLhCGang. Angiogenesis-related markers include CD31, HIF1-α, VEGF, and VWF. Osteogenesis-related genes include OCN, OPN, and ON. c) H&E and IHC staining of OsLhCGost. 40X photos give a general view of bone defect and newly deposited bone, while 100X photos reveal more detail of protein deposition, which is presented in brown color. (BT: bone tissue; NB: newly deposited bone; BM: bone marrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

Micro-CT Scanning results of untreated group and critical-size defects that implanted with decellularized OsLhCGost-CGCC and OsLhCGost-OI. a) 3D reconstructed micro-CT image and cross-sectional diagrams in three directions of control, OsLhCGost-CGCC, and OsLhCGost-OI. b)-d) General parameters that reflect bone volume, including bone volume/tissue volume ratio (BV/TV), bone surface/volume ratio (BS/BV), and bone surface density (BS/TV). e)-g) Parameters for trabecular bone microarchitecture, including trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N). h) Bone reparation score.