Morphological and Molecular Evaluation of the Tissue Repair following Nasal Septum Biopsy in a Sheep Model

Abstract

Objective: Nasal septal pathologies requiring surgical intervention are common in the population. Additionally, nasal chondrocytes are becoming an important cell source in cartilage tissue engineering strategies for the repair of articular cartilage lesions. These procedures damage the nasal septal cartilage whose healing potential is limited due to its avascular, aneural, and alymphatic nature. Despite the high incidence of various surgical interventions that affect septum cartilage, limited nasal cartilage repair characterizations have been performed to date.

Methods: To evaluate the healing of the nasal septum cartilage perforation, a septal biopsy was performed in 14 sheep. Two and 6 months later, the tissue formed on the place of perforation was explanted and compared with the native tissue. Tissue morphology, protein and gene expression of explanted tissue was determined using histological, immunohistochemical and real-time quantitative polymerase chain reaction analysis.

Results: Tissue formed on the defect site, 2 and 6 months after the biopsy was characterized as mostly connective tissue with the presence of fibroblastic cells. This newly formed tissue contained no glycosaminoglycans and collagen type II but was positively stained for collagen type I. Cartilage-specific genes COL2, AGG, and COMP were significantly decreased in 2- and 6-month samples compared with the native nasal cartilage. Levels of COL1, COL4, and CRABP1 genes specific for perichondrium and connective tissue were higher in both test group samples in comparison with native cartilage.

Conclusions: Newly formed tissue was not cartilage but rather fibrous tissue suggesting the role of perichondrium and mucosa in tissue repair after nasal septum injury.

Keywords: biopsy; cartilage; healing; nasal chondrocytes; sheep.

Figure 1.

Nasal septum biopsy, tissue explantation, and preparation of samples for analysis. (A and B) Biopsy of nasal septum cartilage using an 8-mm skin biopsy puncher and forceps. (C) Harvested nasal septum cartilage with perichondrium layer. (D) Explantation of the nasal septum containing the place of the previous biopsy and surrounding tissue (2 and 6 months after the biopsy). (E) Half of the explanted tissue (with mucosa and submucosa layer) used for histological analysis. (F) Half of the explanted tissue (without surrounding layers) used for gene expression analysis. (G) Explanted half used for gene expression analysis divided into newly formed tissue (NFT) and native nasal cartilage (NC).

Figure 2.

Morphology of the tissue formed on the place of the defect site. (A) Morphology of the control healthy nasal cartilage (NC) samples revealed the presence of native hyaline nasal cartilage in the center, perichondrium by the sides of the cartilage and submucosal layer with blood vessels, and seromucous glands. (B and C) In 2- and 6-month newly formed tissue (NFT) samples, nasal cartilage tissue was discontinued and the place of incision was clearly visible. Tissue formed on the defect site was characterized as connective tissue with the presence of fibroblastic cells, blood vessels, and lymphocytes. (D and E) Newly formed cell aggrecations at edge of incised septal cartilage were visible in both NFT groups. Also, perichondrium cells overgrew the edges of native cartilage and were found on the site of perforation. Red dashed line indicates the place of incision, black asteriks indicate the presence of lymphocytes (*), newly formed cell aggregations are indicated with red asterisks (*). NC, native nasal cartilage; P, perichondrium; DCT, irregular dense connective tissue; B, blood vessels; G, seromucous glands. Hematoxylin-eosin (HE) staining. Scale on figures A, B, and C represents 1 mm. Scale on figures D and E represents 250 µm.

Figure 3.

Safranin O staining of the explanted tissue samples. (A) Control native nasal cartilage samples stained with safranin O revealed characteristic septal hyaline cartilage rich in glycosaminoglycans (GAGs). (B and C) In comparison, there were no GAGs in 2- and 6-month newly formed tissue (NFT) samples at the place of biopsy and the defect site was visible. Red stain indicates the presence of cartilage-specific proteoglycans. Scale represents 2.5 mm.

Figure 4.

Expression of collagen type II and I in tissue formed on the defect site. (A and B) Native nasal cartilage was positively stained for collagen type II and negatively for collagen type I in healthy native cartilage control samples. (C, D, E, and F) Collagen type I was abundantly present in the perichondrium, the mucosal and submucosal layers of both control and healing samples. Tissue formed on the place of the perforation was intensively stained brown indicating presence of collagen type I, while no positive staining for collagen type II was visible. Brown stain indicates positive staining. Scale bar represents 2.5 mm.

Figure 5.

Relative gene expression in native nasal cartilage, 2-, and 6-month group samples. (A) Cartilage-specific genes (COL II, AGG, and COMP) were significantly decreased in 2- and 6-month newly formed tissue (NFT) group samples compared with the healthy native nasal cartilage (NC). (B) Gene expression levels of COL I, COL IV, DKK3, and CRABP I were higher in 2- and 6-month group samples compared with the NC group but without significant differences. The exception was a higher expression of CRABP I in the 6-month group. Real-time quantitative polymerase chain reaction (qPCR) was performed with specific primers and results were expressed as ΔCt values. Data were presented as mean ± SD (NC, n = 14; 2-month samples, n = 7; 6-month samples, n = 7). COL II, collagen type II; AGG, aggrecan; COMP, cartilage oligomeric matrix protein; COL I, collagen type I; COL IV, collagen type IV; DKK3, Dickkopf WNT signaling pathway inhibitor 3; CRABPI, cellular retinoic acid binding protein-I. One-way analysis of variance and Tukey’s post hoc test, P > 0.0332 (*), P > 0.0002 (***), P > 0.0001 (****).