Out of 546 upper limbs (273 cadavers), supernumerary heads of the biceps brachii were found in 75 limbs (13.7%) of 58 cadavers (21.3%). The form, origin, and insertion of the supernumerary heads, and branching pattern of the musculocutaneus nerve were studied. In addition, the dimensions of the heads were measured. In many cases, the supernumerary head arose from the humerus, between the insertion of the coracobrachialis and the upper part of the origin of the brachialis, and/or from the medial intermuscular septum. In a few cases, a supernumerary head arose from the tendon of the pectoralis major or the deltoid, or from the articular capsule, or from the crest of the greater tubercle. The supernumerary heads typically joined the common belly, or the aponeurosis of the biceps brachii. Some heads joined the belly of the long head or that of the short head. In the examination of the branching pattern of the musculocutaneus nerve, communication between the musculocutaneus nerve and the median nerve was found in 43 out of the 75 limbs (57.3%). The communicating branch ran from the musculocutaneus nerve to the median nerve in 24, from the median nerve to the musculocutaneus nerve in 12, in both directions in 5, or in another type of pattern in 2 out of 43 limbs. Sometimes a branch of the musculocutaneus nerve ran around a supernumerary head and then fused with the present trunk. The presence of a supernumerary head seemed to affect the course and branching of the musculocutaneus nerve.

We investigated the immunohistochemical localisation of types II and X collagen as well as the cytochemical localisation of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible on d 14-16 of pregnancy. On d 14 of pregnancy, although no immunostaining for types II and X collagen was observed, alkaline phosphatase activity was detected in all cells in the anlage of the future condylar process. On d 15 of pregnancy, immunostaining for both collagen types was simultaneously detected in the primarily formed condylar cartilage. Alkaline phosphatase activity was also detected in chondrocytes at this stage. By d 16 of pregnancy, the hypertrophic cell zone rapidly increased in size. These findings strongly support a periosteal origin for the condylar cartilage of the fetal mouse mandible, and show that progenitor cells for condylar cartilage rapidly or directly differentiate into hypertrophic chondrocytes.

The aim of this study was to investigate the developmental characteristics of the mandibular condyle in sequential phases at the gene level using in situ hybridisation. At d 14.5 of gestation, although no expression of type II collagen mRNA was observed, aggrecan mRNA was detected with type I collagen mRNA in the posterior region of the mesenchymal cell aggregation continuous with the ossifying mandibular bone anlage prior to chondrogenesis. At d 15.0 of gestation, the first cartilaginous tissue appeared at the posterior edge of the ossifying mandibular bone anlage. The primarily formed chondrocytes in the cartilage matrix had already shown the appearance of hypertrophy and expressed types I, II and X collagens and aggrecan mRNAs simultaneously. At d 16.0 of gestation, the condylar cartilage increased in size due to accumulation of hypertrophic chondrocytes characterised by the expression of type X collagen mRNA, whereas the expression of type I collagen mRNA had been reduced in the hypertrophic chondrocytes and was confined to the periosteal osteogenic cells surrounding the cartilaginous tissue. At d 18.0 of gestation before birth, cartilage-characteristic gene expression had been reduced in the chondrocytes of the lower half of the hypertrophic cell layer. The present findings demonstrate that the initial chondrogenesis for the mandibular condyle starts continuous with the posterior edge of the mandibular periosteum and that chondroprogenitor cells for the condylar cartilage rapidly differentiate into hypertrophic chondrocytes. Further, it is indicated that sequential rapid changes and reductions of each mRNA might be closely related to the construction of the temporal mandibular ramus in the fetal stage.

Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.

Mandibular condylar cartilage is the principal secondary cartilage, differing from primary cartilage in its rapid differentiation from progenitor cells (preosteoblasts/skeletoblasts) to hypertrophic chondrocytes. The expression of three transcription factors related to bone and cartilage formation, namely Runx2, Osterix and Sox9, was investigated at the onset of mouse mandibular condylar cartilage formation by in situ hybridization. Messenger RNAs for these three molecules were expressed in the condylar anlage, consisting of preosteoblasts/skeletoblasts, at embryonic day (E)14. Hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. Runx2 mRNA was expressed in the embryonic zone at the posterior position of the newly formed cartilage, in the bone collar and in the newly formed cartilage, but expression intensity in the newly formed cartilage was slightly weaker.Osterix mRNA was also expressed in the embryonic zone and in the bone collar, but was at markedly lower levels in the newly formed cartilage. Sox9 mRNA was continuously expressed from the embryonic zone to the newly formed cartilage. At this stage, Sox5 mRNA was expressed only in the newly formed cartilage. These results suggest that reduced expression of Osterix in combination with Sox9-Sox5 expression is important for the onset of condylar (secondary) cartilage formation.

We investigated the expression pattern of versican, aggrecan, link protein and hyaluronan in the developing limb bud cartilage of the fetal mouse using in situ hybridization and/or immunohistochemistry. Versican mRNA and immunostaining were detected in the mesenchymal cell condensation of the future digital bone at E13. Versican mRNA expression rapidly disappeared from the tibial cartilage, as cartilage formation progressed during E13-15, but the immunostaining was gradually replaced by aggrecan immunostaining from the diaphysis. Immunostaining for both molecules thus had a 'nega-posi' pattern and consequently versican immunostaining was still detected at the epiphyseal end at E15. This result indicated that versican functions as a temporary framework in newly formed cartilage matrix. An aggrecan-positive region within the cartilage invariably had intense hyaluronan staining, whereas a versican-positive region also had affinity for hyaluronan within the cartilage, but not in the mesenchymal cell condensation. Therefore, the presence of versican aggregates was not confirmed in the developing limb bud cartilage. Furthermore, although link protein was more closely related with aggrecan than versican during limb bud cartilage formation, there was a discrepancy between the expression of aggrecan and link protein in tibial cartilage at E15. In particular, only a link protein-positive region was present in the marginal area of the metaphysis and the epiphysis at this stage. This finding may indicate a novel role for link protein.

To reveal the effect of compressive force on the mandibular condylar cartilage, an appliance was set on 8-week-old Wistar rats to load continuous compressive force. Immunohistochemical and histochemical analyses were performed using toluidine blue, antibodies, and probes for aggrecan, hyaluronan, type II collagen, type X collagen, and 5-bromo-2"-deoxyuridine (BrdU). Histomorphometry and statistical analyses were also performed for aggrecan and BrdU immunostaining. In toluidine blue staining, tissue metachromasia was observed in the transitional zone and the hypertrophic zone of the mandibular condylar cartilage. In histomorphometry and statistical analysis, thickness of the cartilage decreased significantly in all regions in the 3-day experimental group. However, the thickness of the cartilage in the anterior region showed recovery while it decreased continuously in the posterior region. Distributional changes of aggrecan, hyaluronan, type II collagen, and type X collagen in the experimental groups were similar to those for toluidine blue staining. The immunostained area of all these molecules decreased as a result of the decrement of the cartilage area. However, enhanced immunostaining for aggrecan in the proliferative zone was observed only in the 1-day experimental group. BrdU-positive cells, observed in the proliferating zone and the transitional zone, decreased significantly in the experimental group 3 days after force was applied. These results demonstrate that continuous compressive forces on the mandibular condylar cartilage decrease the proliferation of chondrocytes and the amount of extracellular matrices.

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