Downregulation of SOX9 expression in developing entheses adjacent to intramembranous bone.

Entheses are classified into three types: fibrocartilaginous, fibrous, and periosteal insertions. However, the mechanism behind the development of fibrous entheses and periosteal insertions remains unclear. Since both entheses are part of the temporomandibular joint (TMJ), this study analyzes the TMJ entheses. Here, we show that SOX9 expression is negatively regulated during TMJ enthesis development, unlike fibrocartilage entheses which are modularly formed by SCX and SOX9 positive progenitors. The TMJ entheses was adjacent to the intramembranous bone rather than cartilage. SOX9 expression was diminished during TMJ enthesis development. To clarify the functional role of Sox9 in the development of TMJ entheses, we examined these structures in TMJ using Wnt1Cre;Sox9flox/+ reporter mice. Wnt1Cre;Sox9flox/+ mice showed enthesial deformation at the TMJ. Next, we also observed a diminished SOX9 expression area at the enthesis in contact with the clavicle's membranous bone portion, similar to the TMJ entheses. Together, these findings reveal that the timing of SOX9 expression varies with the ossification development mode.

Transient contribution of the sphenoid ala major to the socket of the temporomandibular joint in near-term fetuses.

The temporomandibular joint (TMJ) is a complex structure that plays a vital role in the movement of the jaw. Some anatomy and dental textbooks show that, at the medial margin, the TMJ capsule attaches to a suture between the sphenoid ala major and the temporal bone squamosa. In near-term fetuses, the ala major extends posterolaterally to approach the TMJ. In this study, we aimed to investigate the contribution of the sphenoid ala major to the socket of the TMJ in near-term fetuses. We examined histological sections from 22 human fetuses (approximately 15-40 weeks). At midterm, the lateral and superior walls of the TMJ cavity were formed by the temporal bone squamosa, whereas the ala major was distant from the joint. However, at near-term, the ala major formed the medial wall of almost the entire part of the joint cavity. The top of the TMJ was attached to both the squamosa and ala major, with the condylar head consistently separated from the sphenoid by the joint disk. We observed a significant descent of the middle cranial fossa in near-term fetuses, which brought the ala major close to the TMJ. This transient position of the TMJ near the sphenoid is likely due to brain enlargement and posterolateral growth of the ala major. After birth, occlusion causes the anterior growth of the mandibular fossa of the squamosa, which moves the ala major away from the TMJ. Similarly, the lateral growth of the sphenoid toward the squamosa suture may also stop in children.

Mentalis nerve branches supplying the lower lip revisited: a study of human fetuses and donated elderly cadavers.

PURPOSE: Little information is known about the mentalis nerve course from the lower lip approximation margin (free margin) to the upper lip. Likewise, no difference in nerve distribution has been observed between the cutaneous and mucosal parts of the lip. Therefore, this study reexamined mentalis nerve morphology. METHODS: For macroscopic observations, three fresh cadavers were dissected (one male and two females; aged 78-93). We also evaluated histological sections obtained from five donated elderly cadavers (two males and three females, aged 82-96 years) and 15 human fetuses (11-40 weeks or crown-rump length 80-372 mm). Immunohistochemical analysis for S100 protein and tyrosine hydroxylase was performed. RESULTS: In both fetuses and adult cadavers, one to three nerve branches ran upward in the submucosal tissue from the mental foramen. Near the free margin of the lip, some branches passed through the orbicularis oris muscle layer toward the lip skin, whereas others followed a reversed J-shaped course along the free margin. Nerve twigs ran in parallel beneath the mucosa, whereas wavy nerve twigs attached to the basal lamina of the lip epidermis. The difference in nerve endings abruptly occurred at the skin-mucosal junction. Tyrosine hydroxylase-positive sympathetic nerve twigs surrounded arteries and formed a branch composed of S100-negative unmyelinated fibers. CONCLUSION: The lower lip skin was innervated by a perforating branch passing through the orbicularis oris muscle, that was different from the lip mucosa. A sudden change in the nerve ending configuration at the mucocutaneous junction seemed to develop postnatally.

Exploratory Study of Growth of Circumference of Mandibular Fossa Adjacent to Petrous Portion of Temporal Bone Using Dried Skulls.

The morphogenetic process of development of the circumference of the mandibular fossa during tooth eruption, which involves the replacement of deciduous teeth with permanent teeth, is strongly affected by occlusion. To the best of our knowledge, no studies have investigated the effect of occlusion on this process. This study investigated the morphogenetic process of development during tooth eruption using dried skulls harvested from Indian donors. The average distance between the ala-major-squamosa suture and the foramen ovale according to age group was as follows: 3.24 mm in the 8-month-old group and 8.92 mm in the adult group. The average distance between the ala-major-squamosa suture and the apex of the articular tubercle according to age groups was as follows: 10.38 mm in the 8-month-old group and 19.34 mm in the adult group. The average distance between the point of intersection of the petrosquamous fissure and petrotympanic fissure located on the perpendicular line drawn posteriorly from the shortest distance of the medio-lateral axis between the ala-major-squamosa suture and the apex of the articular tubercle according to age group was as follows: 9.68 mm in the 8-month-old group and 14.3 mm in the adult group. These results suggest that the mandibular fossa is strongly affected by load due to occlusion, unlike the growth of the neurocranium. This indicates that the effect of occlusion is a secondary element in the morphogenetic process of development of the circumference of the mandibular fossa.

Contrast-enhanced computed tomography of retromandibular vein position and course patterns relative to the mandible during sagittal split ramus osteotomy.

Background: Sagittal split ramus osteotomy is an established procedure used in oral and maxillofacial surgery. Major bleeding is representative intraoperative complication involves vessels such as the inferior alveolar, facial, and maxillary arteries and accompanying veins, in particular the retromandibular vein (RMV). We previously described the RMV course patterns using embalmed cadavers. However, owing to the possible influence of formalin fixation and unclear skeletal classification of the jaws, the present study aimed to use contrast-enhanced computed tomography to confirm the RMV in patients with defined jaw deformities. Methods: Twenty patients with orthognathic jaw deformities (40 sides) were included. The RMV and the lateral and posterior views of the mandible were evaluated using contrast-enhanced computed tomography. Course patterns and RMV positions were classified as previously reported. Results: Three patterns were identified in the lateral view. Type A: RMV posterior to the posterior border of the ramus (n=25; 62.5%). Type B: adjoining the posterior border of the ramus (n=12; 30.0%). Type C: anterior to the posterior border of the ramus (n=3; 7.5%). Five course patterns were identified in the posterior view. Pattern I: rectilinear course running medial to the posterior border of the ramus (n=3; 7.5%). Pattern II: diagonal course running medially from immediately posterior to posterior border of the ramus (n=11; 27.5%). Pattern III: rectilinear course running immediately posterior to the posterior border of the ramus (n=12; 30.0%). Pattern IV: diagonal course running from lateral to medial relative to the posterior border of the ramus (n=8; 20.0%). Pattern V: diagonal course running from lateral to immediately posterior relative to the posterior border of the ramus (n=6; 15.0%). In the no-course pattern group, the RMV inferior to the lingula was lateral to its position and superior to that of the lingula. In half of the cases, the left and right sides exhibited different running patterns. We observed no correlation between the skeletal patterns of jaw deformities and the course of the RMV. Conclusions: Type B/Pattern II that runs in a straight line bordering the posterior margin of the ramus requires the most attention during surgery. These findings suggest the possibility of predicting intraoperative bleeding risk.

Regeneration process of myotendinous junction injury induced by collagenase injection between Achilles tendon and soleus muscle in mice.

Recently, it has become clear that peri-muscular tissues play a significant role in the deterioration of muscle function. Understanding the function and regeneration of muscle, as well as its surrounding tissues, is crucial to determining the causes of muscular illnesses. However, the regeneration process of the myotendinous junction (MTJ), the most closely related peri-muscular tissue, is still unknown. Therefore, we generated a mouse model of MTJ injury by collagenase injection and searched for the process of regeneration of the MTJ and its adjacent regions. The MTJ region was damaged by collagenase injection, which greatly increased the tendon cross sectional area. Collagenase injections increased the proportion of myofibers with a central nucleus, which is a characteristic of regenerating muscle. The collagenase injection group had myofibers with central nuclei and expressing MTJ markers. Additionally, we measured the length of MTJs using serial cross sections of the soleus muscle and discovered that MTJs at 2 weeks after collagenase injection were shorter compared to the control group, with a propensity to progressively recover their length over time. The results showed that MTJs undergo morphological regeneration even when severely damaged, and that this regeneration occurs in conjunction with muscle regeneration. We anticipate that these findings will be valuable in upcoming research on motor unit regeneration.

Structural analysis of the mylohyoid muscle as a septum dividing the floor of the oral cavity for the purposes of dental implant surgery: variety of muscle attachment positions and ranges of distribution.

OBJECTIVES: The objective was to investigate the details of the attachments of the mylohyoid muscle to the mandible anterior to the hyoid and mylohyoid lines to understand the positional relationship between the sublingual space and the mylohyoid, knowledge that is essential for dental implant surgery in the incisal region, as well as the routes of communication between the sublingual space and other spaces. METHODS: While evaluating the presence or absence of an anterior mylohyoid muscle fiber attachment to the mandible, sublingual gland herniation, spaces between muscle fascicles were also recorded as sites of penetration. The mean muscle thickness in each of these areas was also calculated. RESULTS: In all specimens, the mylohyoid originated not only from the mylohyoid line but also from the lingual surface of the center of the mandibular body (the mandibular symphysis) below the mental spines. The mylohyoid muscle fascicles were thickest in the posterior region, and further anterior to this, they tended to become thinner. Sublingual gland herniations passing through the mylohyoid were noted in the anterior and central regions, but not in the posterior region. Penetration between the muscle fascicles was most common in the central region, and no such penetration was evident in the posterior region. CONCLUSIONS: These results suggest that the mylohyoid functions only incompletely as a septum, and that routes of communication from the sublingual space to the submandibular space may be present in both the anterior and central muscle fascicles of the mylohyoid. Therefore, bleeding complications during dental implant placement in the anterior mandible can be serious issues. There is a potential for sublingual hematoma that could compromise the airway by pressing the tongue against the soft palate into the pharynx.

Chronological Changes in the Expression and Localization of Sox9 between Achilles Tendon Injury and Functional Recovery in Mice.

Tendons help transmit forces from the skeletal muscles and bones. However, tendons have inferior regenerative ability compared to muscles. Despite studies on the regeneration of muscles and bone tissue, only a few have focused on tendinous tissue regeneration, especially tendon regeneration. Sex-determining region Y-box transcription factor 9 (Sox9) is an SRY-related transcription factor with a DNA-binding domain and is an important control factor for cartilage formation. Sox9 is critical to the early-to-middle stages of tendon development. However, how Sox9 participates in the healing process after tendon injury is unclear. We hypothesized that Sox9 is expressed in damaged tendons and is crucially involved in restoring tendon functions. We constructed a mouse model of an Achilles tendon injury by performing a 0.3 mm wide partial excision in the Achilles tendon of mice, and chronologically evaluated the function restoration and localization of the Sox9 expressed in the damaged sites. The results reveal that Sox9 was expressed simultaneously with the formation of the pre-structure of the epitenon, an essential part of the tendinous tissue, indicating that its expression is linked to the functional restoration of tendons. Lineage tracing for Sox9 expressed during tendon restoration revealed the tendon restoration involvement of cells that switched into Sox9-expressing cells after tendon injury. The stem cells involved in tendon regeneration may begin to express Sox9 after injury.

Optic nerve-associated connective tissue structures revisited: A histological study using human fetuses and adult cadavers.

Unlike the usual peripheral nerve, the optic nerve accompanies a thick "dural sheath," a thin "sheath of pia mater" (SPM), and multiple "septa," which divides the nerve fibers into fascicles. We collected specimens from 25 adult cadavers and 15 fetuses and revisited the histological architecture of the optic and oculomotor nerves. In the optic chiasma, the meningeal layer of the dura joins the pia to form a thick SPM, and the periosteum of the sphenoid is continuous with the dural sheath at the orbital exit of the bony optic canal. The septa appeared as a cluster of irregularly arrayed fibrous plates in the intracranial course near the chiasma. Thus, the septa were not derived from either the SPM or the dural sheath. In the orbit, the central artery of the retina accompanies collagenous fibers from the dural sheath and the SPM to provide the vascular sheath in the optic nerve. These connective tissue configurations were the same between adult and fetal specimens. At the optic disk, the dural sheath and SPM merged with the sclera, whereas the septa appeared to end at the lamina cribrosa. However, in fetuses without lamina cribrosa, the septa extend into the nerve fiber layer of the retina. The SPM and septa showed strong elastin immunoreactivity, in contrast to the absence of reactivity in the sheaths of the oculomotor nerve. Each S100 protein-positive Schwann sheath of the oculomotor nerve was surrounded by collagenous endoneurium. Glial fibrillary acidic protein-positive astrocytes showed a linear arrangement along the septa.

Tendinous annulus of Zinn for a common origin of the extraocular rectus muscles: a histological study of the orbital apex from donated elderly cadavers.

The medial, inferior, lateral, and superior rectus muscles (MR, IR, LR, SR), levator palpebrae superioris (LPS), and superior oblique muscle (SO) seem to originate from the tendinous annulus of Zinn, ring-like fibrous tissue crossing the bony orbital fissure. We observed the histological annulus structure using semi-serial histological sections of the orbital apex from 30 elderly donated cadavers. Nearly frontal sections demonstrated a ring-like fibrous structure (a candidate annulus) connecting or embedding four rectus muscles. The candidate annulus did not contain the LPS and SO, and, in the anterior side, the latter muscles originated from the optic canal opening. Far posterior to the annulus, there was a common tendon of the MR, IR, and LR attached to the infero-medial wall of the bony orbital fissure. At the superior part, the annulus is tightly attached to the optic nerve sheath and the periosteum. Sagittal (or Horizontal) sections clearly exhibited parts of the annulus at the MR (SR) origin. Both sagittal and horizontal sections displayed (1) the common origin of the three rectus muscles near the oculomotor nerve in the bony fissure and (2) an accessory, independent muscle bundle of the MR originating from the superomedial margin of the optic canal near the origins of the LPS or SO. Consequently, the so-called tendinous annulus appeared not to provide origins of all six muscles.

Muscle-bone relationship in temporomandibular joint disorders after partial discectomy.

OBJECTIVES: Temporomandibular joint osteoarthritis (TMJ-OA) causes degenerative changes in TMJ tissues. The inter-tissue crosstalk that exacerbates illness and organic changes in bone secondary to TMJ-OA potentially affects the muscles; therefore, patients with a muscular disease might also suffer from bone disease. However, knowledge gaps exist concerning muscle pathology at the onset of TMJ-OA. In this study, we documented the pathogeneses of the bone and muscle at the onset of TMJ-OA using a mouse model. METHODS: We performed a partial resection of the TMJ disk to establish a mouse model of TMJ-OA. After the onset of TMJ-OA, we performed various measurements at 8, 12, and 16 weeks post-surgery in the defined groups. RESULTS: The volume of the mandibular head in the TMJ-OA group was significantly greater than that in the control group. The temporal muscles in the TMJ-OA group were significantly deformed compared with those in the control group; however, between-group comparisons did not reveal significant differences in the mandibular head or temporal muscles after surgery. Therefore, we hypothesized that the degree of mandibular head hypertrophy would alter the temporal muscles. A subsequent analysis of the correlation between the bone and muscle confirmed that the deformity of the temporal muscle increased with increasing hypertrophy of the mandibular head. Temporal and masseter muscle contact was observed in 25% of surgical groups. CONCLUSIONS: This study demonstrates that TMJ-OA progressed when organic changes occurred in bones and muscles, supporting the symbiotic relationship between bones and muscles.

Development of the cartilaginous connecting apparatuses in the fetal sphenoid, with a focus on the alar process.

The human fetal sphenoid is reported to have a cartilaginous connecting apparatus known as the alar process (AP), which connects the ala temporalis (AT) (angle of the greater wing of the sphenoid) to the basisphenoid (anlage of the sphenoid body). However, how the AP develops in humans is unclear. In addition, although the AP is a common structure of the mammalian chondrocranium, little is known about whether it is really a fundamental feature in mammals. This study examined the histological sections of 20 human embryos and fetuses from 6 to 14 weeks of development, of 20 mouse embryos from embryonic days 12-18, and of 4 rats embryos form embryonic days 17 and 20. In addition, we reconsidered the definition of the AP by comparing humans and rats with mice. In humans, the AP was continuous with the basisphenoid but was separated from the AT by a thick perichondrium. Then, the AP-AT connection had a key-and-keyhole structure. Unlike a joint, no cavitation developed in this connection. In mice, there was no boundary between the AT and the basisphenoid, indicating the absence of the AP in the mouse chondrocranium. In rats, the AP was, however, separated from the AT by a thick perichondrium. Therefore, the AP can be defined as follows: the AP is temporally separated from the AT by a thick perichondrium or a key-and-keyhole structure during the fetal period. This is the first study that confirms the absence of the alar process in the mice skull, and its presence in other mammals skull should be further investigated.

Extraction of Maxillary Impacted Teeth with Simultaneous Immediate Full Mouth Loading Using Long Implant: A Case Report.

Here, we describe the provision of an implant-supported prosthesis in a patient with impacted teeth in the maxilla, which complicated implant placement and necessitated utilization of the extraction sockets of previously impacted teeth and residual submerged roots. The patient was a 63-year-old man who visited our clinic with the chief complaint of difficulty in mastication. Numerous residual roots were observed in the maxilla, and radiographic imaging revealed that the residual roots of teeth #13 and #16 were fully impacted. The patient complained of a strong sensation of a foreign body in the area of a denture support overlying these residual roots. Therefore, the impacted teeth were extracted, 5 implants placed, and a temporary prosthesis provided. Given the necessity of placing the implant through the extraction socket of the impacted canine (#13), favorable initial stability was achieved using a long (>20 mm) implant. Moreover, autogenous bone obtained by osteotomy was grafted onto the extracted impacted tooth socket. The clinical condition was stable at approximately 1 year after implant placement and so the final prosthesis was delivered, with periodic check-ups being performed every 3 months thereafter. After 4 years, the patient has reported no symptoms. Clinically, there are no signs of inflammation, and the postoperative condition is deemed to be very favorable.

Cavernous sinus and abducens nerve in human fetuses near term.

A long tortuous course of the abducens nerve (ABN) crossing a highly curved siphon of the internal carotid artery is of interest to neurosurgeons for cavernous sinus surgery. Although a "straight" intracavernous carotid artery in fetuses can change into an adult-like siphon in infants, there is no information on when or how the unique course of ABN is established. Histological observations of 18 near-term fetuses (12 specimens of frontal sections and 6 specimens of sagittal sections) demonstrated the following: (I) the ABN consistently took a straight course crossing the lateral side of an almost straight intracavernous carotid artery; (II) the straight course was maintained when sympathetic nerves joined; (III) few parasellar veins of the developing cavernous sinus separated the ABN from the ophthalmic nerve; and (IV) immediately before the developing tendinous annulus for a common origin of extraocular recti, the ABN bent laterally to avoid a passage of the thick oculomotor nerve. Since the present observations strongly suggested morphologies at birth and in infants, major angulations of the ABN as well as the well-known course independent of the other nerves in the cavernous sinus seemed to be established during childhood. In the human body, the ABN might be a limited example showing a drastic postnatal change in course. Consequently, it might be important to know the unique course of ABN before performing endovascular interventions and skull base surgery for petroclival and cavernous sinus lesions without causing inadvertent neurovascular injuries to neonates or infants.

Transient connection or origin of the inferior pharyngeal constrictor during fetal development: A study using human fetal sagittal sections.

The inferior pharyngeal constrictor (IPC) originates from the thyroid and cricoid cartilages and inserts to the pharyngeal raphe. In serial sagittal sections of 37 embryos and fetuses at 6-15 weeks (crown rump length 15-115mm), we found (1) the IPC connecting to the sternothyroideus and thyrohyoideus muscles (16 fetuses at 6-11 weeks) or (2) the cricothyroideus muscle (6 fetuses at 12-15 weeks) in addition to the usual cricoid origin. These aberrant connections were most likely to be transient origins of the IPC not from a hard tissue but nearby striated muscles. In four of the latter six specimens, a tendinous band from the IPC inferior end connected to the cricothyroideus muscle to provide a digastric muscle-like appearance. These aberrant connections with nearby muscles seemed to become separated by a growing protrusion of the thyroid cartilage. Therefore, these aberrant origins were, even if developed, most likely to be "corrected" to the adult morphology during midterm or late prenatal period. The aberrant or transient origin of the IPC seemed to result from a discrepancy in growth of the cartilage and muscles. Such a discrepancy in growth seems to resemble the IPC wrapping around the superior cornu of thyroid cartilage. In addition, a final or adult-like morphology was found in two of the present 37 fetal specimens. It seemed to suggest a significant redundancy in growth rate of the laryngeal structures.

Examination of the Topographical Anatomy and Fetal Development of the Tendinous Annulus of Zinn for a Common Origin of the Extraocular Recti.

Purpose: The aim was to clarify the topographical anatomy of the common tendinous ring for the four rectus muscles in both adults and fetuses. Methods: We histologically examined the annular ligament for a common origin of the extraocular rectus muscles using 10 specimens from elderly individuals and 31 embryonic and fetal specimens. Results: At 6 to 8 weeks, each rectus carried an independent long tendon, individually originating from the sphenoid. Notably, we found additional origins from the optic or oculomotor nerve sheath. At 12 to 15 weeks, the lateral, inferior, and medial recti muscles were united to provide a C-shaped musculofibrous mass that was separated from the superior rectus originating from the edge of the optic canal opening. Morphologic features at 31 to 38 weeks were almost the same as those at 12 to 15 weeks, but the long and thick common tendon of the three recti reached the sphenoid body in the parasellar area. In adults, a ring-like arrangement of the rectus muscles ended at a site 8.1 to 12.0 mm anterior to the optic canal opening and independent of the superior rectus origin, the lateral, inferior, and medial recti formed a C-shaped muscle mass. The united origins of the three recti changed to a fibrous band extending along the superomedial wall of the orbital fissure. Conclusions: Consequently, none of the specimens we examined exhibited an annular tendon representing a common origin of the four recti, suggesting that the common tendinous ring includes only medial, lateral, and inferior rectus muscles with the superior rectus taking its origin independently.

A temporary disc-like structure at the median atlanto-axial joint in human fetuses.

During observations of mid-term and late-stage fetuses, we found a joint disk-like structure at the anterior component of the median atlanto-axial joint. At mid-term, the disk-like structure was thick (0.1-0.15 mm) relative to the sizes of bones surrounding the joint. However, it did not completely separate the joint cavity, and was absent in the inferior and/or central part of the cavity. This morphology was similar to the so-called fibroadipose meniscoid of the lumbar zygapophysial joint that is usually seen in adults. In mid-term fetuses, there was evidence suggesting that a mesenchymal tissue plate was separated from a roof of the joint cavity. In late-stage fetuses, the thickness (less than 0.15 mm) was usually the same as, or less than that at mid-term, and the disk-like structure was often flexed, folded and fragmented. Therefore, in contrast to the zygapophysial meniscoid as a result of aging, the present disk-like structure was most likely a temporary product during the cavitation process. It seemed to be degenerated in late-stage fetuses and possibly also in newborns. Anomalies at the craniocervical junction such as Chiari malformations might accompany this disk-like structure at the median atlanto-axial joint even in childhood.

Developmental characteristics of secondary cartilage in the mandibular condyle and sphenoid bone in mice.

OBJECTIVE: Secondary cartilage develops from osteochondral progenitor cells. Hypertrophic chondrocytes in secondary cartilage increase within a very short time and then ossify rapidly. In the present study, we investigated the sequential development process of osteochondral progenitor cells, and the morphology and size of hypertrophic chondrocytes in secondary cartilage. DESIGN: ICR mice at embryonic days (E) 14.5-17.5 were used. The mandibular condyle and the medial pterygoid process of the sphenoid bone were observed as secondary cartilage, and the cranial base and the lateral pterygoid process of the sphenoid bone, which is primary cartilage, were observed as a control. Thin sections were subjected to immunostaining and alkaline phosphatase (ALP) staining. Using a confocal laser microscope, 3D stereoscopic reconstruction of hypertrophic cells was performed. To evaluate the size of hypertrophic chondrocytes objectively, the cell size was measured in each cartilage. RESULTS: Hypertrophic chondrocytes of secondary cartilage first expressed type X collagen (Col X) at E15.5. SRY-box 9 (Sox 9) and ALP were co-expressed in the fibroblastic/polymorphic tissue layer of secondary cartilage. This layer was very thick at E15.5, and then rapidly became thin. Hypertrophic cells in secondary cartilage were markedly smaller than those in primary cartilage. CONCLUSIONS: The small hypertrophic cells present in secondary cartilage may have been a characteristic acquired in order for the cartilage to smoothly promote a marked increase in hypertrophic cells and rapid calcification.