A short isoform of Col9a1 supports alveolar bone repair

Abstract

Bone wound created in intramembranous alveolar bone heals without the formation of cartilage precursor tissue. However, the expression of cartilage collagen mRNAs has been suggested. In this report, we examined the expression and the potential role of type IX collagen in bone restoration and remodeling. The sequence specific polymerase chain reaction demonstrated the exclusive expression of short transcriptional isoform of alpha1(IX) collagen (Col9a1) in alveolar bone wound healing, while the long isoform of Col9a1 transcript was absent. Type IX collagen was immunolocalized in the preliminary matrix organized in granulation tissue before trabecular bone formation in tooth extraction socket. In Col9a1-null mutant mice, there were considerable variations in alveolar bone wound healing with the absence of or abnormally organized trabecular bone. Occasionally, unusual apposition of cortical-bone-like layers in bone marrow space was observed. The Col9a1-null mice indicated no growth retardation, and their facial and long bones maintained the normal size and shape. However, the primary spongiosa region of adult Col9a1 mutant mice showed an abnormal trabecular bone structure associated with abnormal immunostaining with the hypertrophic cartilage specific type X collagen antibody. These data suggest that type IX collagen short transcriptional variant is involved in the restoration and remodeling processes of trabecular bone.

Figure 1.

A: Diagram of the α1(IX) collagen gene containing the upstream (UP-Pm) and downstream (DW-Pm) promoter/transcriptional start sites. The alternative use of these promoter/transcriptional start sites results in generation of long and short α1(IX) collagen transcripts differing in the amino-terminal NC4 domain encoded by exons 1–7. B: The nucleotide sequence of the rat α1(IX) collagen downstream promoter/transcriptional start site. Exon 6, the alternative exon 1*, and exon 7 are highlighted by overline with the translated amino acid sequence. The consensus DNA sequences, ie, TATA box, CAAT boxes, and intron donor and recipient sites, are underlined.

Figure 2.

Southern blot analysis of the PCR products depict the presence of short α1(IX) transcript (α1(IX)S) (A) and the absence of long α1(IX) transcript (α1(IX)L) (B). The RNA samples from the wound healing alveolar bone tissue were harvested immediately after tooth extraction (0) and at days 4 (4) and 7 (7) of healing periods. The RNA sample from adult rat tibia (T) was also used. When the root of a tooth (t) is removed from the alveolar bone (C), a sequential wound healing process is activated. During day 4 of the healing period, the granulation tissue gives rise to a preliminary matrix (*) as an extension from the residual alveolar bone (D, ab). The preliminary matrix is further organized to form trabecular bone in the extraction socket at day 7 after extraction (E, arrows).

Figure 3.

A: A day 4 postextraction socket showed the new trabecular bone (tb) that was formed after the preliminary matrix. B: In situ hybridization showed that cells synthesizing α1(IX) collagen mRNA (arrows) formed a cluster near the alveolar bone and appeared to associate with relatively unorganized preliminary matrix (*) (×80, hematoxylin counterstaining with a blue filter during microphotography). C: Immunohistology showed that the preliminary matrix (*) contained the type IX collagen epitope. The distribution of the type IX collagen was indicative of a pattern of trabecular bone (×100, hematoxylin counterstaining).

Figure 4.

A: The inactivation of the α1(IX) collagen gene alleles was achieved by inserting a neo gene in exon 8, which is located downstream of the alternative promoter. B: Wild-type mice (+/+) carried the 3.5-kb EcoRI restriction fragment of genomic DNA containing normal exon 8, whereas the neo gene homologous recombination resulted in the 3.0-kb EcoRI restriction fragment, which was seen in heterozygous (+/−) and homozygous (−/−) mutant mice. C: As compared to wild-type mice (+/+), homozygous Col9a1-null mice (−/−) exhibited no skeletal abnormalities in size and shape.

Figure 5.

A: In wild-type mice, trabecular bone (tb) pattern was established in a typical tooth extraction socket at day 7 (×40, H&E). B: At day 14, the extraction socket of wild-type mice was filled with trabecular bone (tb) (×40, H&E). C: Trabecular bone in the wild-type socket maintained the bone marrow space (×100, H&E). D: In homozygous Col9a1-null mutant mice, trabecular bone pattern was not well developed in the tooth extraction socket at day 7 (×40, H&E). E: Disorganized bone remodeling appeared in Col9a1-null mutant mice resulting in disturbed wound healing observed in an extraction socket at day 14 (×40, H&E). F: Col9a1-null mutant mice often exhibited a layer of cortical-type bone in the extraction socket, which was lined by osteoblast-like cells (arrowheads) (×100, H&E). G: Cross-section of the mandibular condyle from an adult wild type mouse, depicting the matured trabecular bone structure (×20, Alcian blue with H&E). H: Immunostaining with anti-type X collagen antibody was limited to hypertrophic cartilage zone (arrow) in the wild-type mandibular condyle. The primary spongiosa of trabecular bone was completely negative for the type X collagen immunostaining (×20). I: Mandibular condyle trabecular bone of a Col9a1-null mutant mouse was composed of unusual lamellar bones associated with unresorbed cartilage matrix stained with Alcian blue (×20, Alcian blue with H&E). J: The anti-type X collagen antibody stained not only the hypertrophic cartilage zone but also the entire area of primary spongiosa and trabecular bone (arrow, ×20).