Type II collagen scaffolds repair critical-sized osteochondral defects under induced conditions of osteoarthritis in rat knee joints via inhibiting TGF-β-Smad1/5/8 signaling pathway

Abstract

The bidirectional relationship between osteochondral defects (OCD) and osteoarthritis (OA), with each condition exacerbating the other, makes OCD regeneration in the presence of OA challenging. Type II collagen (Col2) is important in OCD regeneration and the management of OA, but its potential applications in cartilage tissue engineering are significantly limited. This study investigated the regeneration capacity of Col2 scaffolds in critical-sized OCDs under surgically induced OA conditions and explored the underlying mechanisms that promoted OCD regeneration. Furthermore, the repair potential of Col2 scaffolds was validated in over critical-sized OCD models. After 90 days or 150 days since scaffold implantation, complete healing was observed histologically in critical-sized OCD, evidenced by the excellent integration with surrounding native tissues. The newly formed tissue biochemically resembled adjacent natural tissue and exhibited comparable biomechanical properties. The regenerated OA tissue demonstrated lower expression of genes associated with cartilage degradation than native OA tissue but comparable expression of genes related to osteochondral anabolism compared with normal tissue. Additionally, transcriptome and proteome analysis revealed the hindrance of TGF-β-Smad1/5/8 in regenerated OA tissue. In conclusion, the engrafting of Col2 scaffolds led to the successful regeneration of critical-sized OCDs under surgically induced OA conditions by inhibiting the TGF-β-Smad1/5/8 signaling pathway.

Keywords: Osteoarthritis; Osteochondral defect; Tissue engineering; Type II collagen.

Graphical abstract

Fig. 1

Schematic illustration of study design. Day −30: anterior cruciate ligament transection (ACLT) was performed on one hind limb (OA legs), leaving the other unoperated (non-OA legs). Day 0: osteoarthritis (OA) was induced; osteochondral defects (OCDs), measuring either 1.5 mm (critical-sized) or 2 mm (over critical-sized) in diameter, were created in both hind limbs in the non-weight-bearing area of the trochlear groove, followed by the engraftment of Col2 scaffolds; the 1.5 mm or 2 mm OCDs without scaffold implantation in the OA legs and non-OA legs served as negative control, referred to as OA dLhCG (−) and non-OA dLhCG (−), respectively. Day 90: 1.5 mm OCDs with dLhCG implantation were regenerated in OA and non-OA legs, while the 1.5 mm OCDs without dLhCG implantation were filled with fibrous tissue. Day 150: 2 mm OCDs treated with dLhCG were regenerated in OA and non-OA legs, while the 2 mm OCDs without dLhCG implantation were filled with fibrous tissue. In the OA legs, the osteochondral tissue regenerated from surgical sites and surrounding native tissue was referred to as 'OA regenerated' (OR) and 'OA native' (ON), respectively. In the non-OA legs, the osteochondral tissue regenerated from surgical sites and surrounding native tissue was referred to as 'non-OA regenerated' (NOR) and 'non-OA native' (NON), respectively.

Fig. 2

The histological staining of critical-sized osteochondral defects on day 90 revealed that non-OA and OA legs implanted with Col2 scaffolds exhibited complete osteochondral defect regeneration. In contrast, untreated samples demonstrated limited tissue regeneration, with either no regeneration or the most defective area filled with random fibrous tissue. Scale bar: 200 μm.

Fig. 3

Microcomputed tomography. (a, b) Micro-CT images and bone morphological analysis of critical-sized OCDs on day 90. (c, d) Micro-CT images and bone morphological analysis of over critical-scale OCDs on day 150. Red arrow, untreated defect; yellow arrow, untreated native bone; green arrow, regenerated bone. OCD, osteochondral defects; BV/TV, bone volume fraction; Tb·N, trabecular number; Tb·Th, trabecular thickness; Tb.Sp, trabecular separation; Tt.Ar, total cross-sectional area inside the periosteal envelope; Ct.Ar, average cortical area; Ct.Ar/Tt.Ar, cortical area fraction; Ct.Th, average cortical thickness. NON, non-OA native; NOR, non-OA regenerated; ON, OA native; OR, OA regenerated. Scale bar: 1 mm.

Fig. 4

(a–c) The quantification results of DNA, glycosaminoglycans (GAGs), and hydroxyproline for critical-sized osteochondral defects (OCDs) on day 90. (d–f) The quantification results of DNA, GAGs, and hydroxyproline for over critical-sized OCDs on day 150. (g, h) Biomechanical analysis of critical-sized OCDs on day 90. (i) Biomechanical analysis of over critical-sized OCDs on day 150. NON, non-OA native; NOR, non-OA regenerated; ON, OA native; OR, OA regenerated.

Fig. 5

The histological staining for over critical-sized osteochondral defects on day 150 revealed that the samples engrafted with dLhCG exhibited satisfactory osteochondral repair, albeit with minor imperfections. In contrast, non-OA dLhCG (−) and OA dLhCG (−) samples exhibited sparse and random fibrous tissue infiltration. Scale bar: 200 μm.

Fig. 6

Gene expression analysis of the critical-sized osteochondral defects on day 90. (a) Principal component analysis. (b) Relative expression level of key genes. (c, d, e) Volcano plot, gene enrichment analysis, and GSEA of TGF-β signaling pathway for ON vs. NON. (f, g, h) Volcano plot, gene enrichment analysis, and GSEA of TGF-β signaling pathway for NOR vs. NON. (i, j, k) Volcano plot, gene enrichment analysis, and GSEA of TGF-β signaling pathway for OR vs. ON. GSEA, gene set enrichment analysis; NES, normalized enrichment score; NON, non-OA native; NOR, non-OA regenerated; ON, OA native; OR, OA regenerated.

Fig. 7

The proteome analysis of the critical-sized osteochondral defects on day 90. (a) Venn diagram for proteins in proteome and genes in the transcriptome. (b) Relative protein quantification across the tissue samples. (c, d) The integration analysis of transcriptome and proteome. (e) Gene set enrichment analysis of TGF-β signaling pathway for OR vs. ON. NES, normalized enrichment score; NON, non-OA native; NOR, non-OA regenerated; ON, OA native; OR, OA regenerated.

Fig. 8

Gene expression analysis of the over critical-sized osteochondral defects on day 150. (a) Principal component analysis. (b) Relative expression level of key genes. (c) GSEA of TGF-β signaling pathway for ON vs. NON. (d) GSEA of TGF-β signaling pathway for OR vs. ON. (e) Volcano plot for NOR vs. NON. GSEA, gene set enrichment analysis; NES, normalized enrichment score; NON, non-OA native; NOR, non-OA regenerated; ON, OA native; OR, OA regenerated.