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INTRODUCTION In round window vibroplasty the most efficient coupling technique for contact of the floating mass transducer (FMT) to the round window membrane (RWM) is yet to be determined. Various materials placed between the FMT and the RWM have been proposed to enable better stimulation of the cochlea. Collagenous scaffolds derived from decellularized extracellular cartilage matrices as a commercially available biomaterial are already used for other applications in ear surgery. We aimed to examine the coupling properties of collagen scaffolds compared with commonly used other materials (round window soft coupler, porcine perichondrium, and cartilage) as interponate between the FMT and the RWM in vibroplasty. METHODS A well-established in vitro temporal bone model was used. Volume velocities were measured with collagen scaffold compared with different interponates (round window soft coupler, porcine perichondrium, and cartilage) at 800, 1000, 1250, 1600, 2000, 2500, 3150, and 4000 Hz levels. RESULTS Statistical analysis revealed no superiority of commonly used materials compared with collagen scaffolds at all tested volume velocities (p > 0.05). DISCUSSION We could demonstrate that collagenous scaffolds of decellularized extracellular cartilage matrices have similar vibrational properties as conventional coupling materials of the FMT to the RWM in vibroplasty. Therefore, as a commercially available new material they display a suitable coupling option for round window vibroplasty.

Living primates show a complex trend in reduction of nasal cavity spaces and structures due to moderate to severe constraint on interorbital breadth. Here we describe the ontogeny of the posterior end of the primate cartilaginous nasal capsule, the thimble shaped posterior nasal cupula (PNC), which surrounds the hind end of the olfactory region. We used a histologically sectioned sample of strepsirrhine primates and two non-primates (Tupaia belangeri, Rousettus leschenaulti), and histochemical and immunohistochemical methods to study the PNC in a perinatal sample. At birth, most strepsirrhines possess only fragments of PNC, and these lack a perichondrium. Fetal specimens of several species reveal a more complete PNC, but the cartilage exhibits uneven or weak reactivity to type II collagen antibodies. Moreover, there is relatively less matrix than in the septal cartilage, resulting in clustering of chondrocytes, some of which are in direct contact with adjacent connective tissues. In one primate (Varecia spp.) and both non-primates, the PNC has a perichondrium at birth. In older, infant Varecia and Rousettus, the perichondrium of the PNC is absent, and PNC fragmentation at its posterior pole has occurred in the former. Loss of the perichondrium for the PNC appears to precede resorption of the posterior end of the nasal capsule. These results suggest that the consolidation of the basicranial and facial skeletons happens ontogenetically earlier in primates than other mammals. We hypothesize that early loss of cartilage at the sphenoethmoidal articulation limits chondral mechanisms for nasal complexity, such as interstitial expansion or endochondral ossification.

The nasal septum is a cartilaginous structure that serves as a pacemaker for the development of the midface. The septum is a hyaline cartilage which is surrounded by a perichondrium and epithelium. It remains cartilaginous anteriorly, but posteriorly it undergoes endochondral ossification to form the perpendicular plate of the ethmoid. Understanding of hyaline cartilage differentiation stems predominantly from investigations of growth plate cartilage. It is currently unclear if the morphological and molecular properties of the differentiating nasal septum align with what is known from the growth plate. In this study, we describe growth, molecular, and cellular characteristics of the nasal septum with reference to hyaline cartilage differentiation. The nasal septum grows asynchronous across its length with phases of rapid growth interrupted by more stagnant growth. Growth appears to be driven predominantly by acquisition of chondrocyte hypertrophy. Similarly, cellular differentiation is asynchronous, and differentiation observed in the anterior part precedes posterior differentiation. Overall, the nasal septum is structurally and molecularly heterogeneous. Early and extensive chondrocyte hypertrophy but no ossification is observed in the anterior septum. Onset of hypertrophic chondrocyte differentiation coincided with collagen fiber deposition along the perichondrium. Sox9, Col2, Col10, Mmp13, Sp7, and Runx2 expression was heterogeneous and did not always follow the expected pattern established from chondrocyte differentiation in the growth plate. The presence of hypertrophic chondrocytes expressing bone-related proteins early on in regions where the nasal septum does not ossify displays incongruities with current understanding of hyaline cartilage differentiation. Runx2, Collagen II, Collagen X, and Sp7 commonly used to mark distinct stages of chondrocyte maturation and early bone formation show wider expression than expected and do not align with expected cellular characteristics. Thus, the hyaline cartilage of the nasal septum is quite distinct from growth plate hyaline cartilage, and caution should be taken before assigning cartilage properties to less well-defined cartilage structures using these commonly used markers. Beyond the structural description of the nasal cartilage, this study also provides important information for cartilage tissue engineering when using nasal septal cartilage for tissue regeneration.

Background In remote thyroid surgery, a reliable intraoperative neuromonitoring (IONM) procedure is an important tool for reducing injury to recurrent laryngeal nerve (RLN). This study proposes an alternative or adjunct technique for performing full percutaneous (PC) IONM and confirms its feasibility in animal experiments. Methods This prospective porcine model study enrolled four piglets with eight nerve sides. Evoked electromyography (EMG) was stimulated from PC ball-tip probe, and recorded from EMG endotracheal tube (ETT) and from PC paired long-needle electrodes on the perichondrium of the lateral aspect of thyroid cartilage. Results In all RLNs and vagus nerves, typical laryngeal EMG waveforms were successfully evoked by PC probe stimulation and recorded by both ETT and PC needle electrodes. Conclusions This study confirms the feasibility of the full PC IONM techniques in porcine model. However, further clinical studies are needed to compare the practicality of different remote-access approaches for thyroid surgery.

Mandibular condylar cartilage is a representative secondary cartilage, differing from primary cartilage in various ways. Syndecan is a cell-surface heparan sulfate proteoglycan and speculated to be involved in chondrogenesis and osteogenesis. This study aimed to investigate the expression patterns of the syndecan family in the developing mouse mandibular condylar cartilage. At embryonic day (E)13.0 and E14.0, syndecan-1 and -2 mRNAs were expressed in the mesenchymal cell condensation of the condylar anlage. When condylar cartilage was formed at E15.0, syndecan-1 mRNA was expressed in the embryonic zone, wherein the mesenchymal cell condensation is located. Syndecan-2 mRNA was mainly expressed in the perichondrium. At E16.0, syndecan-1 was expressed from fibrous to flattened cell zones and syndecans-2 was expressed in the lower hypertrophic cell zone. Syndecan-3 mRNA was expressed in the condylar anlage at E13.0 and E13.5 but was not expressed in the condylar cartilage at E15.0. It was later expressed in the lower hypertrophic cell zone at E16.0. Syndecan-4 mRNA was expressed in the condylar anlage at E14.0 and the condylar cartilage at E15.0 and E16.0. These findings indicated that syndecans-1 and -2 could be involved in the formation from mesenchymal cell condensation to condylar cartilage. The different expression patterns of the syndecan family in the condylar and limb bud cartilage suggest the functional heterogeneity of chondrocytes in the primary and secondary cartilage.

Background The chest wall deformity is a well-known complication following costal cartilage harvest with the biomechanical factor considered to be a cause of this donor-site morbidity. Kawanabe-Nagata method is a widely-accepted approach to prevent the deformity. However, knowledge about the biomechanical properties of regenerated costal cartilage is limited, and the value of reimplantation of autologous costal cartilage blocks is not clear. Methods The fifth costal cartilage on both sides of six male, 8 weeks of age, New Zealand white rabbits were harvested with the perichondrium preserved intact in situ. The perichondrium was sutured to form a perichondrial pocket and part of the excised costal cartilage was cut into 0.5 mm cartilage blocks and returned to the perichondrial pocket of left side. The animals were sacrificed 16 weeks postoperatively and the regenerated and a piece native costal cartilage was harvested for morphological and three point bending test. Results There was no remarkable chest wall deformity in all animals, and there were no apparent differences in the appearance of the regenerated cartilage with and without reimplantation autologous cartilage blocks. The elastic modulus of native cartilage was significantly higher than the regenerated cartilage. The stiffness of regenerated cartilage without reimplantation was higher than that of with reimplantation, but this difference was not significant. Conclusions The stiffness of regenerated cartilage was significantly lower than the native cartilage. Reimplantation of autologous cartilage blocks was not superior to that without reimplantation in regard to restoring the volume defect and strengthening the regenerated cartilage.

Background Tacrolimus is used to prevent unaesthetic scars due to its action on fibroblast activity and collagen production modulation. Objectives To evaluate the action pathways, from the histopathological point of view and in cytokine control, of tacrolimus ointment in the prevention of hypertrophic scars. Methods Twenty-two rabbits were submitted to the excision of two 1-cm fragments in each ear, including the perichondrium. The right ear received 0.1% and 0.03% tacrolimus in ointment base twice a day in the upper wound and in the lower wound respectively. The left ear, used as the control, was treated with petrolatum. After 30 days, collagen fibers were evaluated using special staining, and immunohistochemistry analyses for smooth muscle actin, TGF-β and VEGF were performed. Results The wounds treated with 0.1% tacrolimus showed weak labeling and a lower percentage of labeling for smooth muscle actin, a higher proportion of mucin absence, weak staining, fine and organized fibers for Gomori's Trichrome, strong staining and organized fibers for Verhoeff when compared to controls. The wounds treated with 0.03% tacrolimus showed weak labeling for smooth muscle actin, a higher proportion of mucin absence, strong staining for Verhoeff when compared to the controls. There was absence of TGF-β and low VEGF expression. Study limitations The analysis was performed by a single pathologist. Second-harmonic imaging microscopy was performed in 2 sample areas of the scar. Conclusions Both drug concentrations were effective in suppressing TGF-β and smooth muscle actin, reducing mucin, improving the quality of collagen fibers, and the density of elastic fibers, but only the higher concentration influenced elastic fiber organization.

Objectives/hypothesis We evaluated the efficacy of a vocal fold reconstruction technique using a vascularized autologous pedicled fat (PEFA) flap in a rabbit model of vocal fold paralysis. Study design Animal model. Methods The study included 30 male New Zealand White rabbits; 20 received vocal fold reconstructions (PEFA group) and 10 served as normal controls (control group). The right recurrent laryngeal nerve was resected, and simultaneous PEFA flap reconstruction was performed. The PEFA flap, including a pre-epiglottic fat and thyroid perichondrium, was elevated and implanted through a window at the inferior border of the thyroid cartilage. Histological and high-speed video analyses of vocal fold vibration were performed 1 month after PEFA reconstruction. The maximum amplitude of vocal fold vibration and the dynamic glottal gap were used to assess vocal fold vibration. Results The histological findings showed that the lamina propria ratio (lamina propria pixels/total vocal fold pixels) and the total number of vocal fold pixels were similar between the PEFA and control groups. Vocal fold vibration analyses indicated that the maximum amplitude differences in the vibration were slightly lower in the PEFA group. However, the dynamic glottal gap of the vocal fold was not significantly different between the PEFA group and the control group. Conclusions The PEFA flap vocal fold reconstruction technique maintained the vocal fold area without a significant reduction in vocal fold vibration in a rabbit model. Level of evidence NA Laryngoscope, 130:1770-1774, 2020.

Immunolocalization studies have shown that fibrillin-1 is distributed ubiquitously in the connective tissue space from early embryonic times through old age. When mutated, the gene for fibrillin-1 (FBN1) causes the Marfan syndrome, a common inherited disorder of connective tissue. The multiple manifestations of the Marfan syndrome reflect the known distribution of fibrillin-1 in cardiovascular, musculoskeletal, ocular, and dermal tissues. In this study, a mouse model of Marfan syndrome in which fibrillin-1 is truncated and tagged with green fluorescence was used to estimate the relative abundance of fibrillin-1 in developing tissues. In embryonic tissues, the aorta was the only tissue in which fibrillin-1 green fluorescence was detectable. Other arteries gained detectable fibrillin-1 green fluorescence just after birth. Fibrillin-1 fluorescence was observed at later postnatal times in the lung, skin, perichondrium, tendon, and ocular tissues, while other tissues remained negative. These results indicated that tissues most affected in the Marfan syndrome are the tissues in which fibrillin-1 is most abundant. Focus was placed on the aorta, since aortic disease is life threatening in the Marfan syndrome and fibrillin-1 green fluorescence was most abundant in this tissue. Fibrillin-1 green fluorescence and immunostaining showed that fibrillin-1 is within aortic medial elastic lamellae. Endothelial-specific compared to smooth muscle-specific fibrillin-1 green fluorescence, together with light microscopic analyses of fragmentation of aortic elastic lamellae, demonstrated that smooth muscle cell mutated fibrillin-1 contributed most to progressive aortic fragmentation. However, these studies also indicated that other cells, possibly endothelial cells, also contribute to this aortic pathology. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.

The pellet formation has been regarded as a golden standard for in vitro chondrogenic differentiation. However, a spatially inhomogeneous chondrogenic microenvironment around a pellet resulted from the use of a traditional impermeable narrow tube, such as the conical tube, undermines the differentiation performance and therapeutic potential of differentiated cartilage pellet in defective articular cartilage treatment. To address this drawback, a perichondrium-inspired permeable nanofibrous tube (PINaT) well with a nanofibrous wall permeable to gas and soluble molecules is proposed. The PINaT well was fabricated with a micro deep drawing process where a flat thin nanofibrous membrane was transformed to a 3.5-mm-deep tube well with a ~ 50-µm-thick nanofibrous wall. Similar to in vivo perichondrium, the PINaT well was found to allow oxygen and growth factor diffusion required for chondrogenic differentiation across the entire nanofibrous wall. Analyses of gene expressions (COL2A1, COL10A1, ACAN, and SOX9), proteins (type II and X collagen), and glycosaminoglycans (GAGs) contents were conducted to assess the differentiation performance and clinical efficacy of differentiated cartilage pellet. The regulated spatially homogeneous chondrogenic microenvironment around the human induced pluripotent stem cell (hiPSC)-derived pellet (3 × 105cells per pellet) in the PINaT well remarkably improved the quality of the differentiated pellet toward a more hyaline-like cartilage pellet. Furthermore, an accelerated chondrogenic differentiation process of the pellet produced by the PINaT well was achieved for 14 days, demonstrating a hyaline cartilage-specific marker similar to the control pellet differentiated for 20 days. Finally, the enhanced clinical efficacy of the hyaline-like cartilage pellet was confirmed using an osteochondral defect rat model, with the repaired tissue resembling hyaline cartilage rather than fibrous cartilage after 8 weeks of regeneration.

Objective: Cartilage grafts are widely used in reconstructing nasal deformity for structural and aesthetic purposes. Despite being immunologically privileged, cartilage grafts are susceptible to volume loss with high risk of resorption over time. Therefore, experts opt for cartilage handling modification to resolve this issue through graft dicing, wrapping, or perichondrium preservation. This study will evaluate the effect cartilage graft preparations on graft viability. Design: Single-randomized post-test-only study design. Setting: Animal Hospital at Bogor Agricultural Institute. Participants: Six New Zealand, male, Hycole rabbits. Intervention: Conchal cartilage grafts were retrieved from 6 experimental rabbits and distributed into 3 treatment groups: diced cartilage graft (DC; control), one-sided perichondrium-attached scored cartilage (OPSC), and tube-shaped perichondrium-wrapped diced cartilage (TPDC). Main Outcome Measures: Macroscopic (weight and contour) and microscopic (chondroblast proliferation, graft thickness, apoptotic cells) evaluation through histological measures were recorded on week 12. Statistical analysis was done to compare between groups. Results: Diced cartilage and OPSC groups showed significant weight changes on week 12 ( P < .05) with OPSC presenting with the biggest difference. Diced cartilage and OPSC group showed moderate cell proliferation on week 12 while TPDC displayed most abundant apoptotic cells (5.8%; P < .05). Diced cartilage group had the highest cartilage thickness ratio ( P < .05). Discussion: Bare DC technique promoted graft thickness while perichondrium-attached scored cartilage showed the most abundant chondroblast proliferation and the least apoptotic cells. Perichondrium contributes to enhanced new cartilage formation. Conclusion: Diced cartilage graft is suitable for masking irregularity and volume augmentation, while perichondrium-attached cartilage graft is better for structural support in nasal reconstruction.

Chondrogenesis and angiogenesis drive endochondral ossification. Using the atmospheric scanning electron microscopy (ASEM) without decalcification and dehydration, we directly imaged angiogenesis-driven ossification at different developmental stages shortly after aldehyde fixation, using aqueous radical scavenger glucose solution to preserve water-rich structures. An embryonic day 15.5 mouse femur was fixed and stained with phosphotungstic acid (PTA), and blood vessel penetration into the hypertrophic chondrocyte zone was visualised. We observed a novel envelope between the perichondrium and proliferating chondrocytes, which was lined with spindle-shaped cells that could be borderline chondrocytes. At postnatal day (P)1, trabecular and cortical bone mineralisation was imaged without staining. Additional PTA staining visualised surrounding soft tissues; filamentous connections between osteoblast-like cells and osteocytes in cortical bone were interpreted as the osteocytic lacunar-canalicular system. By P10, resorption pits had formed on the tibial trabecular bone surface. The applicability of ASEM for pathological analysis was addressed using knockout mice of Keap1, an oxidative-stress sensor. In Keap1−/− femurs, we observed impaired calcification and angiogenesis of epiphyseal cartilage, suggesting impaired bone development. Overall, the quick ASEM method we developed revealed mineralisation and new structures in wet bone tissue at EM resolution and can be used to study mineralisation-associated phenomena of any hydrated tissue.

The expression profile of early B-cell factor (Ebf) genes and loss of function experiments denote a crucial role for these genes during the late stage of skeletogenesis. However, little is known regarding the expression and function of these genes during the early stage of skeletogenesis. Therefore, this study aimed to detail the spatiotemporal expression pattern of cEbf1, in comparison to cEbf2 and cEbf3, in chick limb buds and investigate its function during chondrogenesis. cEbf1-3 were co-expressed in the distal mesenchyme from a very early stage and later in the outer perichondrium and the surrounding noncartilaginous mesenchymal cells. Ebf1 loss of function through injection of RCASBP virus-carrying Ebf1 dominant-negative form (ΔEbf1) into the wing buds resulted in shortened skeletal elements with a clear defect in the chondrocyte differentiation program. In RCASBP-ΔEbf1 injected wing, the chondrogenesis was initiated normally but hindered at the maturation stage. Subsequently, the chondrocytes failed to become mature or hypertrophic and the long bone diaphysis was not properly developed. The final phenotype included shorter, thicker, and fused long bones. These phenotypic changes were associated with downregulation of the early [Sox9 and collagen type II (Col2a1)] and the late [alkaline phosphatase (AP)] chondrocytes differentiation markers in the limb buds. These results conclude that cEbf1 could be involved in a molecular cascade that promotes the terminal stages of chondrogenesis in the long bone anlagen.

Reconstruction of articular surfaces destroyed by infection or trauma is hampered by the lack of suitable graft tissues. Perichondrium autotransplants have been used for this purpose. However, the role of the transplanted perichondrium in the healing of resurfaced joints have not been investigated. Perichondrial and periosteal tissues were harvested from rats hemizygous for a ubiquitously expressed enhanced green fluorescent protein (EGFP) transgene and transplanted into full-thickness articular cartilage defects at the trochlear groove of distal femur in wild-type littermates. As an additional control, cartilage defects were left without a transplant (no transplant control). Distal femurs were collected 3, 14, 56, 112 days after surgery. Transplanted cells and their progenies were readily detected in the defects of perichondrium and periosteum transplanted animals but not in defects left without a transplant. Perichondrium transplants expressed SOX9 and with time differentiated into a hyaline cartilage that expanded and filled out the defects with Col2a1-positive chondrocytes and a matrix rich in proteoglycans. Interestingly, at later timepoints the cartilaginous perichondrium transplants were actively remodeled into bone at the transplant-bone interface and at post-surgery day 112 EGFP-positive perichondrium cells at the articular surface were positive for Prg4. In addition, both perichondrium and periosteum transplants contributed cells to the subchondral bone and bone marrow, suggesting differentiation into osteoblast/osteocytes as well as bone marrow cells. In summary, we found that perichondrium transplanted to articular cartilage defects develops into an articular-like, hyaline cartilage that integrates with the subchondral bone, and is maintained for an extended time. The findings indicate that perichondrium is a suitable tissue for repair and engineering of articular cartilage.

Mammalian articular cartilage is an avascular tissue with poor capacity for spontaneous repair. Here, we show that embryonic development of cartilage in the skate (Leucoraja erinacea) mirrors that of mammals, with developing chondrocytes co-expressing genes encoding the transcription factors Sox5, Sox6 and Sox9. However, in skate, transcriptional features of developing cartilage persist into adulthood, both in peripheral chondrocytes and in cells of the fibrous perichondrium that ensheaths the skeleton. Using pulse-chase label retention experiments and multiplexedin situhybridization, we identify a population of cyclingSox5/6/9+ perichondral progenitor cells that generate new cartilage during adult growth, and we show that persistence of chondrogenesis in adult skates correlates with ability to spontaneously repair cartilage injuries. Skates therefore offer a unique model for adult chondrogenesis and cartilage repair and may serve as inspiration for novel cell-based therapies for skeletal pathologies, such as osteoarthritis.

The growth plate is an essential component of endochondral bone development. Not surprisingly, the growth plate and its surrounding structure, the perichondrium, contain a wealth of skeletal stem cells (SSCs) and progenitor cells that robustly contribute to bone development. Recent in vivo lineage-tracing studies using mouse genetic models provide substantial insight into the diversity and versatility of these skeletal stem and progenitor cell populations, particularly shedding light on the importance of the transition from cartilage to bone. Chondrocytes and perichondrial cells are inseparable twins that develop from condensing undifferentiated mesenchymal cells during the fetal stage; although morphologically and functionally distinct, these cells ultimately serve for the same goal, that is, to make bone bigger and stronger. Even in the postnatal stage, a small subset of growth plate chondrocytes can transform into osteoblasts and marrow stromal cells; this is in part fueled by a unique type of SSCs maintained in the resting zone of the growth plate, which continue to self-renew for the long term. Here, we discuss diverse skeletal stem and progenitor cell populations in the growth plate and the perichondrium and their transition from cartilage to bone.

The aim of this study is to evaluate the histopathological effects of septoplasty techniques on the nasal septal mucosa of rabbits with light and electron microscope.The study was performed on 21 rabbits between August 2016 and February 2017. Rabbits were randomly divided into three groups. In Group-1, while preserving the L-strut structure of the septum, cartilage resection, was performed by open technique septoplasty. In Group-2, the same procedure was done except the resected cartilage was crushed and put back in place. No surgical procedure was performed on the Control group. Postoperative 2nd month; the specimens were histopathologically evaluated by light and electron microscope in terms of changes in the morphology of septum mucosa, perichondrial thickness, cilia and goblet cell deprivation, loss in glands, fibrosis and inflammatory cell infiltration in the lamina propria.The deprivation in cilia, goblet cells, serous gland and increase in the amount of collagen fibers were examined in both Group-1 and 2. The difference in Group-1 and Group-2 were statistically significant in terms of presence of cilia, number of goblet cells and glands and increase in collagen fibers when compared to control (p 0.001, p = 0.002, p = 0.020, p = 0.002, respectively). In terms of perichondrium thickness, statistically significant difference was found between the Control and Group-2 (p 0.001). CONCLUSıON: In this study, histopathological findings supported that the presence of cartilage in the septum is necessary to prevent the mucosal changes. Long-term studies are needed to observe whether changes in the morphology of epithelium and gland proceed more than 2 months follow-up.

BACKGROUND Limited information is available on the biological status of transplanted cartilage from which the perichondrium has been removed. This article describes the histological and three-dimensional structural picture of cartilage, using green fluorescent protein (GFP) transgenic rats and normal wild rats. METHODS Three sections of costal cartilage were harvested from 10-week-old wild rats. One section was used as a specimen while two were subcutaneously collected from the dorsal region of 10-week-old GFP rats at 4 and 8 weeks post-transplant. The experiment was performed in two randomized groups. The perichondrium was removed from transplanted cartilage in the first group and perichondrium of transplanted cartilage remained intact in the second group. Histology and focused ion beam/scanning electron microscope (FIB/SEM) tomography were used to evaluate the transplanted cartilage. RESULTS All 40 transplanted sections were harvested and no infections, exposure or qualitative change of cartilage matrix were seen following transplant. Histological analyses showed that the surface layer of the GFP-negative transplanted cartilage was replaced with GFP-positive chondrocytes 8 weeks post-transplant in the first group. A three-dimensional layer of perichondrium-like tissue reconstructed around the cartilage at 8 weeks was confirmed, resembling normal perichondrium. However, the GFP-positive chondrocytes were not replaced in the second group. CONCLUSIONS The cell renewal of chondrocytes is necessary for subcutaneously transplanted cartilage to maintain its tissue composition over a long period of time. The histological and ultrastructural analyses revealed that cells from recipient tissue generated new chondrocytes even when cartilage was implanted after removing the perichondrium.

Heparan sulfate (HS) is an essential component of cell surface and matrix proteoglycans (HS-PGs) that include syndecans and perlecan. Because of their unique structural features, the HS chains are able to specifically interact with signaling proteins -including bone morphogenetic proteins (BMPs)- via their HS-binding domain, regulating protein availability, distribution and action on target cells. Hereditary Multiple Exostoses (HME) is a rare pediatric disorder linked to germline heterozygous loss-of-function mutations in EXT1 or EXT2 that encode Golgi-resident glycosyltransferases responsible for HS synthesis, resulting in a systemic HS deficiency. HME is characterized by cartilaginous/bony tumors -called osteochondromas or exostoses- that form within perichondrium in long bones, ribs and other elements. This review examines most recent studies in HME, framing them in the context of classic studies. New findings show that the spectrum of EXT mutations is larger than previously realized and the clinical complications of HME extend beyond the skeleton. Osteochondroma development requires a somatic "second hit" that would complement the germline EXT mutation to further decrease HS production and/levels at perichondrial sites of osteochondroma induction. Cellular studies have shown that the steep decreases in local HS levels: derange the normal homeostatic signaling pathways keeping perichondrium mesenchymal; cause excessive BMP signaling; and provoke ectopic chondrogenesis and osteochondroma formation. Data from HME mouse models have revealed that systemic treatment with a BMP signaling antagonist markedly reduces osteochondroma formation. In sum, recent studies have provided major new insights into the molecular and cellular pathogenesis of HME and the roles played by HS deficiency. These new insights have led to the first ever proof-of-principle demonstration that osteochondroma formation is a druggable process, paving the way toward the creation of a clinically-relevant treatment.

The reconstruction of cartilage defects for cosmetic and/or functional reasons has become routine in plastic and reconstructive surgery. However, it remains challenging due to the slow turnover and low viability of cartilage grafts. Although autologous grafts can be used to determine the shape of the defect in cartilage-reconstruction surgeries, the effect of defect shape on cartilage healing has not been reported. Here, we present the first study aiming to investigate the influence of cartilage graft geometry on healing. Twelve New Zealand white rabbits were used in the study. Square-, rectangle-, sphere-, and fusiform-shaped cartilage defects were applied to both ears with 1-cm2 geometric templates that completely elevated the cartilage tissue without damaging the opposite perichondrium. As a control, the removed cartilage was sutured back to the right ear, whereas the left ear was sutured back without any graft. Histological examinations were made on samples taken during surgery and those taken four months post-surgery. Chondrocyte production and organisation, chondrocyte vacuolisation, collagen synthesis, proteoglycan levels, vascularisation, focal bleeding, and peripheral proliferation were scored independently by two histologists. There was no statistically significant difference in the growth rates of either the control or experimental cartilage tissues when compared with that of the initial cartilage tissue (p = 0.083). Histologic comparisons revealed better outcomes in the grafted cartilage groups compared to those receiving the donor cartilage, but this was not statistically significant. This study demonstrates that the geometric shape of the defect has no significant effect on cartilage healing. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The poor mechanical strength of tissue-engineered auricular cartilage which is possibly due to the lack of perichondrium limits its application in auricular reconstruction surgery. However, there is insufficient research and no reliable data to support this. This study aims to investigate the contribution of perichondrium to the mechanical strength of auricular cartilage under loading. Rabbit auricular cartilage was harvested and classified into two groups. The perichondrium was removed in Group A and was left intact in Group B. Young's modulus, stress relaxation slope, and relaxation amout were analyzed through tensile and compressive tests using a material testing machine. Group B exhibited significantly higher Young's modulus in both the tensile and compressive tests (p 0.05), lower relaxation slope (p 0.05 in tensile test, p = 0.65 in compressive test), and lower relaxation amout (p 0.05 in tensile test, p 0.01 in compressive test). Our results showed that the perichondrium has a definite contribution to the mechanical properties of ear cartilage. This study may provide new insights to researchers focusing on improving the mechanical strength of tissue-engineered auricular cartilage.

The aim of the study was to design a construct based on a nasal septal cartilage plate providing required cell differentiation in different layers to replace a deep osteochondral defect and develop an algorithm of chemical and physical effect sequence to create non-immunogenic two-layer porous structure with requisite elasto-mechanical properties. Materials and Methods The plates derived from porcine nasal septal hyaline cartilage covered by perichondrium were multi-stage treated including freezing, equilibrating in a hypotonic saline solution (type I specimens); trypsinization, point IR-laser effect, re-trypsinization (type II specimens); a stabilizing effect of crosslinking agents — glyceraldehyde/ribose in an acidic medium — washing (type III specimens). For all type specimens: there were established stability parameters (collagen denaturation temperature using a thermal analysis; and Young’s modulus using a mechanical analysis); there were determined morphological characteristics using light and polarization microscopy with classical staining and nonlinear optical microscopy in second-harmonic generation mode. Results Thermal, mechanical, and morphological properties in type I specimens slightly differed from those of the initial nasoseptal system. A considerable part of cells had destroyed membranes. In type II specimens, thermal stability of collagen frame was significantly lower; Young’s modulus decreased more than fourfold compared to type I specimens. Collagen structure of hyaline cartilage appeared to be disarranged, although the morphological differences of the hyaline part and perichondrium preserved. The construct matrix was almost completely decellularized. Successive exposure to laser radiation and trypsin resulted in the formation of partial holes in the matrix, ~100 μm in diameter. In type III specimens, both the thermal stability of the collagen frame and Young’s modulus (E) increased. Glyceraldehyde was more effective than ribose, E having reached the value typical for intact hyaline cartilage. Collagen fibers in type III specimens were thicker than in type I and II specimens. The morphological differences of the hyaline part and perichondrium and partial holes were preserved. Conclusion Due to sequential treatment by salts, trypsin, IR-laser radiation, and nontoxic crosslinking agents, nasal septal cartilage plate forms porous acellular construction consisting of two layers formed by type I (from perichondrium) and type II (from hyaline part) collagen fibers. In the present construction, stability, mechanical properties, and size of the partial holes can be assigned for cell colonization. It enables to use the construction to replace articular cartilage defects.

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.

Objective Kangaroo knee cartilages are robust tissues that can support knee flexion and endure high levels of compressive stress. This study aimed to develop a detailed understanding of the collagen architecture in kangaroo knee cartilages and thus obtain insights into the biophysical basis of their function. Design Cylindrical/square plugs from femoral and tibial hyaline cartilage and tibial fibrocartilage were excised from the knees of three adult red kangaroos. Multi-slice, multi-echo MR images were acquired at the sample orientations 0° and 55° (“magic angle”) with respect to the static magnetic field. Maps of the transverse relaxation rate constant (R2) and depth profiles of R2 and its anisotropic component (R2A) were constructed from the data. Results The R2A profiles confirmed the classic three-zone organisation of all cartilage samples. Femoral hyaline cartilage possessed a well-developed, thick superficial zone. Tibial hyaline cartilage possessed a very thick radial zone (80% relative thickness) that exhibited large R2A values consistent with highly ordered collagen. The R2A profile of tibial fibrocartilage exhibited a unique region near the bone (bottom 5-10%) consistent with elevated proteoglycan content (“attachment sub-zone”). Conclusions Our observations suggest that the well-developed superficial zone of femoral hyaline cartilage is suitable for supporting knee flexion; the thick and well-aligned radial zone of tibial hyaline cartilage is adapted to endure high compressive stress; while the innermost part of the radial zone of tibial fibrocartilage may facilitate anchoring of the collagen fibres to withstand high shear deformation. These findings may inspire new designs for cartilage tissue engineering.

Cartilage oligomeric matrix protein (COMP), an abundant cartilage extracellular matrix protein, plays an important role in mesenchymal chondrogenesis. To test our hypothesis that COMP could promote tissue engineering cartilage regeneration as well as improve cartilaginous mechanical properties, COMP gene transfected rabbit bone marrow mesenchymal stem cells (BMSCs) were used for chondrogenic differentiation in vitro and were implanted with biphasic scaffolds into osteochondral defects of New Zealand white rabbit trochlear grooves for cartilage regeneration in vivo. In vitro, over expressed COMP could enhance the chondrogenic differentiation and ECM secretion of BMSCs. After euthanasia at 12 weeks post implantation, macroscopic observation, histological staining, mechanical tests and micro-CT were performed for the assessment of repaired cartilage. Overexpression of COMP leads to more newly formed hyaline cartilage and significantly improved mechanical property. These results demonstrated the significant role of COMP in the cartilage regeneration in vivo and offered inspiring advantage of COMP in the application of tissue engineering.

The primary skeletal tissue in elasmobranchs -sharks, rays and relatives- is cartilage, forming both embryonic and adult endoskeletons. Only the skeletal surface calcifies, exhibiting mineralized tiles (tesserae) sandwiched between a cartilage core and overlying fibrous perichondrium. These two tissues are based on different collagens (Coll II and I, respectively), fueling a long-standing debate as to whether tesserae are more like calcified cartilage or bone (Coll 1-based) in their matrix composition. We demonstrate that stingray (Urobatis halleri) tesserae are bipartite, having an upper Coll I-based 'cap' that merges into a lower Coll II-based 'body' zone, although tesserae are surrounded by cartilage. We identify a 'supratesseral' unmineralized cartilage layer, between tesserae and perichondrium, distinguished from the cartilage core in containing Coll I and X (a common marker for mammalian mineralization), in addition to Coll II. Chondrocytes within tesserae appear intact and sit in lacunae filled with Coll II-based matrix, suggesting tesserae originate in cartilage, despite comprising a diversity of collagens. Intertesseral joints are also complex in their collagenous composition, being similar to supratesseral cartilage closer to the perichondrium, but containing unidentified fibrils nearer the cartilage core. Our results indicate a unique potential for tessellated cartilage in skeletal biology research, since it lacks features believed diagnostic for vertebrate cartilage mineralization (e.g. hypertrophic and apoptotic chondrocytes), while offering morphologies amenable for investigating the regulation of complex mineralized ultrastructure and tissues patterned on multiple collagens.

Purpose: Our previous study found that in the temporomandibular joint (TMJ) of the K14-cre; Ctnnb1ex3f mouse embryo, the morphogenesis of glenoid fossa was interrupted by the dislocated condyle. This observation suggested that the formation of the glenoid fossa required tissue interactions with condylar mesenchyme. The purpose of this study was to clarify if the interactions between other components are essential for TMJ morphogenesis.Materials and methods: We examined the gross morphology, histology, cell proliferation, and gene expression in the developing TMJ of K14-cre; Ctnnb1ex3f mice by whole-mount bone and cartilage staining, Azon staining, BrdU labeling, and in situ hybridization, respectively.Results: In K14-cre; Ctnnb1ex3f mice, the zygomatic arch was misconnected to the mandibular bone by ectopic bone formation, which disrupted the attachment of temporalis to the mandibular bone and joint capsule formation. Although the initiation and differentiation of the condylar cartilage were slightly impacted, the K14-cre; Ctnnb1ex3f TMJ lacked joint cavities and separated disc, suggesting that the tissue interactions between the joint capsule and the TMJ were indispensable for the cavity formation and disc separation. The ectopic activation of Gli2 in the cells occupying the cavities, and the enhanced PTHrP transcription in the condylar perichondrium of the K14-cre; Ctnnb1ex3f TMJ suggested that the disrupted interactions between the joint capsule and the TMJ impaired cavity formation and disc separation by altering Hh signaling.Conclusion: Joint capsule formation was essential for cavity formation and disc separation during TMJ development.

Diced cartilage is a significant alternative approach to cartilage grafting. However, the viability and biomechanical properties of diced cartilage grafts remain to be improved, and the role of perichondrium is largely neglected. This study aimed to evaluate the histological and biomechanical effects of perichondrium on custom-shaped diced cartilage grafts constructed via a high-density porous polyethylene mold. Seven New Zealand rabbits were used. Unilateral auricular cartilage was harvested and divided into 2 parts, with or without perichondrium, diced into 1 × 1 × 0.5 mm cubical pieces, and filled into high-density porous polyethylene molds. Three grafts with the perichondrium removed and 3 with the perichondrium preserved were implanted subcutaneously at the dorsum. The grafts underwent biomechanical and histological tests 4, 8, and 12 weeks after the implantation. The diced cartilage merged into integrated blocks without observable resorption in both groups at each time point. Additionally, the retention rate of weight was higher in the perichondrium-preserved group (P

This study aimed to evaluate the effect of a novel adjustable, closed-loop sewing machine lock stitching technique on cartilage shape. This study was performed on 18 rabbits that were divided into 3 groups to evaluate the short- (Group I), medium- (Group II), and long-term (Group III) effects of the technique on cartilage shape. Three cartilage grafts were obtained from the right and left ears of the rabbits and measured angularly. For the cartilage grafts obtained from the right ears, contours were corrected using the stitching technique proposed herein. The cartilage grafts obtained from the left ears were not stitched; these were maintained as the control group. Angular measurements were performed for the stitched cartilage grafts, and all the cartilages grafts obtained were finally embedded into the rabbits' backs. The rabbits in Groups I, II, and III were euthanized at week 2, 6, and 12, respectively, after which the cartilages were reevaluated for angular measurement and submitted for histopathological examination. A significant improvement from baseline was detected in the angular measurements of the stitched cartilage grafts obtained from the rabbits in each group. For the stitched cartilage grafts obtained from Group II and III rabbits, the angular measurements obtained immediately after stitching were found to be better than those obtained after euthanasia. In histopathological evaluation elevated collagen, perichondrium, and connective tissue levels were detected in grafts obtained from Group Ill rabbits. The novel stitching technique proposed herein appears to have beneficial long-term effects on the maintenance of cartilage contour.

The growth plate provides a substantial source of mesenchymal cells in the endosteal marrow space during endochondral ossification. The current model postulates that a group of chondrocytes in the hypertrophic zone can escape from apoptosis and transform into cells that eventually become osteoblasts in an area beneath the growth plate. The growth plate is composed of cells with various morphologies; particularly at the periphery of the growth plate immediately adjacent to the perichondrium are "borderline" chondrocytes, which align perpendicularly to other chondrocytes. However, in vivo cell fates of these special chondrocytes have not been revealed. Here we show that borderline chondrocytes in growth plates behave as transient mesenchymal precursor cells for osteoblasts and marrow stromal cells. A single-cell RNA-seq analysis revealed subpopulations of Col2a1-creER-marked neonatal chondrocytes and their cell type-specific markers. A tamoxifen pulse to Pthrp-creER mice in the neonatal stage (before the resting zone was formed) preferentially marked borderline chondrocytes. Following the chase, these cells marched into the nascent marrow space, expanded in the metaphyseal marrow, and became Col(2.3 kb)-GFP+ osteoblasts and Cxcl12-GFPhigh reticular stromal "CAR" cells. Interestingly, these borderline chondrocyte-derived marrow cells were short-lived, as they were significantly reduced during adulthood. These findings demonstrate based on in vivo lineage-tracing experiments that borderline chondrocytes in the peripheral growth plate are a particularly important route for producing osteoblasts and marrow stromal cells in growing murine endochondral bones. A special microenvironment neighboring the osteogenic perichondrium might endow these chondrocytes with an enhanced potential to differentiate into marrow mesenchymal cells. © 2019 American Society for Bone and Mineral Research.

BackgroundThrough precise implementation of distinct cell type specification programs, differentially regulated in both space and time, complex patterns emerge during organogenesis. Thanks to its easy experimental accessibility, the developing chicken limb has long served as a paradigm to study vertebrate pattern formation. Through decades’ worth of research, we now have a firm grasp on the molecular mechanisms driving limb formation at the tissue-level. However, to elucidate the dynamic interplay between transcriptional cell type specification programs and pattern formation at its relevant cellular scale, we lack appropriately resolved molecular data at the genome-wide level. Here, making use of droplet-based single-cell RNA-sequencing, we catalogue the developmental emergence of distinct tissue types and their transcriptome dynamics in the distal chicken limb, the so-called autopod, at cellular resolution.ResultsUsing single-cell RNA-sequencing technology, we sequenced a total of 17,628 cells coming from three key developmental stages of chicken autopod patterning. Overall, we identified 23 cell populations with distinct transcriptional profiles. Amongst them were small, albeit essential populations like the apical ectodermal ridge, demonstrating the ability to detect even rare cell types. Moreover, we uncovered the existence of molecularly distinct sub-populations within previously defined compartments of the developing limb, some of which have important signaling functions during autopod pattern formation. Finally, we inferred gene co-expression modules that coincide with distinct tissue types across developmental time, and used them to track patterning-relevant cell populations of the forming digits.ConclusionsWe provide a comprehensive functional genomics resource to study the molecular effectors of chicken limb patterning at cellular resolution. Our single-cell transcriptomic atlas captures all major cell populations of the developing autopod, and highlights the transcriptional complexity in many of its components. Finally, integrating our data-set with other single-cell transcriptomics resources will enable researchers to assess molecular similarities in orthologous cell types across the major tetrapod clades, and provide an extensive candidate gene list to functionally test cell-type-specific drivers of limb morphological diversification.

Accumulating evidence supports the idea that stem and progenitor cells play important roles in skeletal development. Over the last decade, the definition of skeletal stem and progenitor cells has evolved from cells simply defined by their in vitro behaviors to cells fully defined by a combination of sophisticated approaches, including serial transplantation assays and in vivo lineage-tracing experiments. These approaches have led to better identification of the characteristics of skeletal stem cells residing in multiple sites, including the perichondrium of the fetal bone, the resting zone of the postnatal growth plate, the bone marrow space and the periosteum in adulthood. These diverse groups of skeletal stem cells appear to closely collaborate and achieve a number of important biological functions of bones, including not only bone development and growth, but also bone maintenance and repair. Although these are important findings, we are only beginning to understand the diversity and the nature of skeletal stem and progenitor cells, and how they actually behave in vivo.

Active cell proliferation and turnover in the growth plate is essential for embryonic and postnatal bone growth. We performed a lineage tracing of Wnt/β-catenin signaling responsive cells (Wnt-responsive cells) using Axin2CreERT2 ;Rosa26ZsGreen mice and found a novel cell population that resides in the outermost layer of the growth plate facing the Ranvier's groove (RG; the perichondrium adjacent to growth plate). These Wnt-responsive cells rapidly expanded and contributed to formation of the outer growth plate from the neonatal to the growing stage but stopped expanding at the young adult stage when bone longitudinal growth ceases. In addition, a second Wnt-responsive sporadic cell population was localized within the resting zone of the central part of the growth plate during the postnatal growth phase. While it induced ectopic chondrogenesis in the RG, ablation of β-catenin in the Wnt-responsive cells strongly inhibited expansion of their descendants toward the growth plate. These findings indicate that the Wnt-responsive cell population in the outermost layer of the growth plate is a unique cell source of chondroprogenitors involving lateral growth of the growth plate and suggest that Wnt/β-catenin signaling regulates function of skeletal progenitors in a site- and stage-specific manner. © 2019 American Society for Bone and Mineral Research.

The degeneration of articular cartilage underscores the clinical pathology of temporomandibular joint osteoarthritis (TMJ-OA) and is promoted through dysfunctional biochemical or biophysical signaling. Transduction of these signals has a multifaceted regulation that includes important cell-matrix derived interactions. The matrix encapsulating the cells of the mandibular condylar cartilage (MCC) is rich in type VI collagen. Neuron/glia antigen 2 (NG2) is a type I transmembrane proteoglycan that binds with type VI collagen. This study defines the temporospatial dynamics of NG2-type VI collagen interactions during the progression of TMJ-OA. Membrane-bound NG2 is found to colocalize with pericellular type VI collagen in superficial layer cells in the MCC perichondrium but is present at high levels in the cytosol of chondroblastic and hypertrophic cells. When TMJ -OA is induced using a surgical instability model, localized disruptions of pericellular type VI collagen are observed on the central and medial MCC and are associated with significantly higher levels of cytosolic NG2. NG2 localized within the cytosol is found to be transported through clathrin and dynamin mediated endocytic pathways. These findings are consistent with NG2 behavior in other injury models and underscore the potential of NG2 as an entirely novel molecular mechanism of chondrocyte function contextually linked with TMJ-OA.

Yurekli, Aslan/0000-0003-2812-2133 WOS: 000473301300002 PubMed: 31233167 Introduction: The rabbit ear hypertrophic scar model is a preferred animal model of excessive scarring for investigating the scarring process and novel treatment modalities. In this model, surgical removal of perichondrium can be challenging, and it is often insufficient or damages the underlying cartilage. It is hypothesized that cryosurgery would offer a more efficient alternative to conventional surgery. The objective of this study was to compare structural changes in scar tissues in two groups of the hypertrophic scar model: cryosurgery compared to standard conventional surgery. Methods: We introduced a novel technique to remove perichondrium using cryosurgery. Hypertrophic scars obtained with conventional surgery and cryosurgery were studied in a left-right comparison method. Comparative parameters included the histological structure of the scars and structural changes in the cartilage just beneath the scarring. Results: Cryosurgery produced similar scars in comparison to conventional surgery. Although statistically not significant (p = 0.16), the histological findings of cartilage damage were lower in the cryosurgery group (six out of 21) compared to the established model (10 out of 20). Conclusions: This study suggests that cryotherapy can be used for removal of perichondrium. Gulhane School of Medicine Research Grant committee This study was financially supported by the Gulhane School of Medicine Research Grant committee and was conducted at the Animal Research Laboratory of the same institution.

Tendon and bone are attached by a transitional connective tissue that is morphologically graded from tendinous to osseous and develops from bipotent progenitors that co-express scleraxis (Scx) and Sox9 (Scx(+)/Sox9(+)). Scx(+)/Sox9(+) progenitors have the potential to differentiate into either tenocytes or chondrocytes, yet the developmental mechanism that spatially resolves their bipotency at the tendon-bone interface during embryogenesis remains unknown. Here, we demonstrate that development of Scx(+)/Sox9(+) progenitors within the mammalian lower jaw requires FGF signaling. We find that loss of Fgfr2 in the mouse tendon-bone interface reduces Scx expression in Scx(+)/Sox9(+) progenitors and induces their biased differentiation into Sox9(+) chondrocytes. This expansion of Sox9(+) chondrocytes, which is concomitant with decreased Notch2-Dll1 signaling, prevents formation of a mixed population of chondrocytes and tenocytes, and instead results in ectopic endochondral bone at tendon-bone attachment units. Our work shows that FGF signaling directs zonal patterning at the boundary between tendon and bone by regulating cell fate decisions through a mechanism that employs Notch signaling.

Tissue engineering based on cell seeding, bioscaffolds, and growth factors has been widely applied for the reconstruction of tissue defects. Recent progress has fueled in vivo tissue engineering techniques in becoming hot topics in regenerative medicine and reconstructive surgery. To improve the efficacy of tissue engineering, we here investigated the roles of cell seeding, bioscaffolds, growth factors, and in vivo microenvironment (IM) in tissue regeneration. Bone marrow-derived stem cells, allogeneic demineralized bone matrix as bioscaffold, and growth factor bone morphogenetic protein 2/transforming growth factor, and the IM of rib periosteum and perichondrium were used in different combinations for the generation of osteochondral composite tissue. Self-regenerated neocomposite tissue based on the IM alone exhibited excellent anatomical configuration, vascularization, biomechanical stability, and function similar to native controls. Our findings indicate that the IM is a crucial factor in biofunctional tissue generation. Further refinement and development of this technique may enable transfer to clinical application with broad spectrum of application.

Repair of total human ear loss or congenital lack of ears is one of the challenging issues in plastic and reconstructive surgery.The aim of the present study was 3D reconstruction of the human ear with cadaveric ear cartilages seeded with human mesenchymal stem cells.We used cadaveric ear cartilages with preserved perichondrium. The samples were divided into 2 groups: group A (cartilage alone) and group B (cartilage seeded with a mixture of fibrin powder and mesenchymal stem cell [1,000,000 cells/cm] used and implanted in back of 10 athymic rats). After 12 weeks, the cartilages were removed and shape, size, weight, flexibility, and chondrocyte viability were evaluated. P value0.05 was considered significant.In group A, size and weight of cartilages clearly reduced (P 0.05) and then shape and flexibility (torsion of cartilages in clockwise and counterclockwise directions) were evaluated, which were found to be significantly reduced (P 0.05). After staining with hematoxylin and eosin and performing microscopic examination, very few live chondrocytes were found in group A. In group B, size and weight of samples were not changed (P 0.05); the shape and flexibility of samples were well maintained (P 0.05) and on performing microscopic examination of cartilage samples, many live chondrocytes were found in cartilage (15-20 chondrocytes in each microscopic field).In samples with human stem cell, all variables (size, shape, weight, and flexibility) were significantly maintained and abundant live chondrocytes were found on performing microscopic examination. This method may be used for reconstruction of full defect of auricles in humans.

Hypothesis: The acoustic properties of scaffolds made from decellularized extracellular cartilage matrices of porcine origin are comparable to those of the human tympanic membrane. Background: Currently, the reconstruction of tympanic membrane in the context of chronic tympanic membrane defects is mostly performed using autologous fascia or cartilage. Autologous tissue may be associated with lack of graft material in revision patients and requires more invasive and longer operative time. Therefore, other materials are investigated for reconstruction. An increasingly important role could be played by scaffolds from different materials, which are known to induce constructive tissue remodeling. Methods: To analyze the acoustic properties, the vibrations of the scaffolds, cartilage, perichondrium and tympanic membrane were measured by laser scanning doppler vibrometry under different static pressures. Results: The analysis of volume velocities serves as an indicator for sound transmission. The results of the average volume velocities at atmospheric pressure show a similar frequency response of the tympanic membrane and the scaffolds with a peak at about 800 Hz. After changing the artificial ear-canal pressure from atmospheric pressure to negative pressure (-100, -200, and -300 daPa) the vibration characteristics of the different membranes remain fairly constant, whereas the results of the perichondrium show a decrease after changing the pressure into the negative range in the frequencies 1 to 3 kHz. Conclusion: The present study showed that the vibration characteristics of the scaffolds under atmospheric and negative pressure can be interpreted as similar to those of thin cartilage (

The cranial base is a component of the neurocranium and has a central role in the structural integration of the face, brain and vertebral column. Consequently, alteration in the shape of the human cranial base has been intimately linked with primate evolution and defective development is associated with numerous human facial abnormalities. Here we describe a novel recessive mutant mouse strain that presented with a domed head and fully penetrant cleft secondary palate coupled with defects in the formation of the underlying cranial base. Mapping and non-complementation studies revealed a specific mutation in Memo1 – a gene originally associated with cell migration. Expression analysis of Memo1 identified robust expression in the perichondrium and periosteum of the developing cranial base, but only modest expression in the palatal shelves. Fittingly, although the palatal shelves failed to elevate in Memo1 mutants, expression changes were modest within the shelves themselves. In contrast, the cranial base, which forms via endochondral ossification had major reductions in the expression of genes responsible for bone formation, notably matrix metalloproteinases and markers of the osteoblast lineage, mirrored by an increase in markers of cartilage and extracellular matrix development. Concomitant with these changes, mutant cranial bases showed an increased zone of hypertrophic chondrocytes accompanied by a reduction in both vascular invasion and mineralization. Finally, neural crest cell-specific deletion of Memo1 caused a failure of anterior cranial base ossification indicating a cell autonomous role for MEMO1 in the development of these neural crest cell derived structures. However, palate formation was largely normal in these conditional mutants, suggesting a non-autonomous role for MEMO1 in palatal closure. Overall, these findings assign a new function to MEMO1 in driving endochondral ossification in the cranium, and also link abnormal development of the cranial base with more widespread effects on craniofacial shape relevant to human craniofacial dysmorphology.

We recently developed a promising regenerative method based on the xenotransplantation of human cartilage progenitor cells, demonstrating self-renewing elastic cartilage reconstruction with expected long-term tissue restoration. However, it remains unclear whether autotransplantation of cartilage progenitors may work by a similar principle in immunocompetent individuals. We used a nonhuman primate (monkey) model to assess the safety and efficacy of our regenerative approach because the model shares characteristics with humans in terms of biological functions, including anatomical features. First, we identified the expandable and multipotent progenitor population from monkey ear perichondrium and succeeded in inducing chondrocyte differentiation in vitro. Second, in vivo transplanted progenitor cells were capable of reconstructing elastic cartilage by xenotransplantation into an immunodeficient mouse. Finally, the autologous monkey progenitor cells were transplanted into the subcutaneous region of a craniofacial section and developed mature elastic cartilage of their own 3 months after transplantation. Furthermore, we attempted to develop a clinically relevant, noninvasive monitoring method using magnetic resonance imaging (MRI). Collectively, this report shows that the autologous transplantation of cartilage progenitors is potentially effective for reconstructing elastic cartilage. This principle will be invaluable for repairing craniofacial injuries and abnormalities in the context of plastic and reconstructive surgery.

The musculoskeletal system is a striking example of how cell identity and position is coordinated across multiple tissues to ensure function. However, it is unclear upon tissue loss, such as complete loss of cells of a central musculoskeletal connecting tendon, whether neighboring tissues harbor progenitors capable of mediating regeneration. Here, using a zebrafish model, we genetically ablate all embryonic tendon cells and find complete regeneration of tendon structure and pattern. We identify two regenerative progenitor populations, sox10+ perichondrial cells surrounding cartilage and nkx2.5+ cells surrounding muscle. Surprisingly, laser ablation of sox10+ cells, but not nkx2.5+ cells, increases tendon progenitor number in the perichondrium, suggesting a mechanism to regulate attachment location. We find BMP signaling is active in regenerating progenitor cells and is necessary and sufficient for generating new scxa+ cells. Our work shows that muscle and cartilage connective tissues harbor progenitor cells capable of fully regenerating tendons, and this process is regulated by BMP signaling.

Development of mouse gonial bone and initial ossification process of malleus were investigated. Before the formation of the gonial bone, the osteogenic area expressing alkaline phosphatase and Runx2 mRNA was widely recognized inferior to Meckel's cartilage. The gonial bone was first formed within the perichondrium at E16.0 via intramembranous ossification, surrounded the lower part of Meckel's cartilage, and then continued to extend anteriorly and medially until postnatal day (P) 3.0. At P0, multinucleated chondroclasts started to resorb the mineralized cartilage matrix with ruffled borders at the initial ossification site of the malleus (most posterior part of Meckel's cartilage). Almost all CD31-positive capillaries did not run through the gonial bone but entered the cartilage through the site where the gonial bone was not attached, indicating the forms of the initial ossification site of the malleus are similar to those at the secondary ossification center rather than the primary ossification center in the long bone. Then, the reducing process of the posterior part of Meckel's cartilage with extending gonial bone was investigated. Numerous tartrate-resistant acid phosphatase-positive mononuclear cells invaded the reducing Meckel's cartilage, and the continuity between the malleus and Meckel's cartilage was completely lost by P3.5. Both the cartilage matrix and the perichondrium were degraded, and they seemed to be incorporated into the periosteum of the gonial bone. The tensor tympani and tensor veli palatini muscles were attached to the ligament extending from the gonial bone. These findings indicated that the gonial bone has multiple functions and plays important roles in cranial formation. Anat Rec, 302:1916-1933, 2019. © 2019 American Association for Anatomy.

Although Prx1 (also known as PRRX1)-expressing cells and their primary cilia are critical for embryonic development, they have yet to be studied in the context of postnatal skeletogenesis owing to the lethality of mouse models. A tamoxifen-inducible Prx1 model has been developed, and we determined that expression directed by this promoter is highly restricted to the cambium layers in the periosteum and perichondrium after birth. To determine the postnatal role of these cambium layer osteochondroprogenitors (CLOPs) and their primary cilia, we developed models to track the fate of CLOPs (Prx1CreER-GFP;Rosa26(tdTomato)) and selectively disrupt their cilia (Prx1CreER-GFP;Ift88(fl/fl)). Our tracking studies revealed that CLOPs populate cortical and trabecular bone, the growth plate and secondary ossification centers during the normal program of postnatal skeletogenesis. Furthermore, animals lacking CLOP cilia exhibit stunted limb growth due to disruptions in endochondral and intramembranous ossification. Histological examination indicates that growth is stunted due to limited differentiation, proliferation and/or abnormal hypertrophic differentiation in the growth plate. Collectively, our results suggest that CLOPs are programmed to rapidly populate distant tissues and produce bone via a primary cilium-mediated mechanism in the postnatal skeleton.

Septoclasts are mononuclear spindle-shaped phagocytes with their long processes in uncalcified cartilage matrices and locate adjacent to the capillary endothelium at the chondro-osseous junction of the growth plate. We have previously revealed a selective expression of epidermal-type fatty acid-binding protein (E-FABP/FABP5) in septoclasts. Although, pericytes are known to distribute along capillaries and directly surround their endothelial cells in a situation similar to septoclasts, no clear evidence is available on the relationship between septoclasts and pericytes. We investigated the chronological localization and morphological change of septoclasts during development of the tibia of mice to clarify the development of septoclasts and the immune-localization of pericyte markers in septoclasts to clarify the origin of septoclasts. E-FABP-immunoreactive septoclasts emerged at the perichondrium in the middle of the cartilaginous templates of the tibia in prenatal development. Septoclasts migrated to the surface of the cartilage adjacent to invading blood vessels. Processes of septoclasts became longer and their apexes attached to Von Kossa-negative uncalcified matrices during the formation process of the primary ossification center. Not only platelet-derived growth factor receptor beta, but also neuron-glial antigen 2 was localized in septoclasts of mice from E15 (embryonic day 15) to P6w (postnatal 6 week). Our results suggest that septoclasts are originated from pericytes and involved in the blood vessel invasion during formation of the primary ossification center.

Regeneration of cartilaginous tissues is limited in mammals but it occurs with variable extension in lizards (reptiles), including in their vertebrae. The ability of lizard vertebrae to regenerate cartilaginous tissue that is later replaced with bone has been analyzed using tritiated thymidine autoradiography and 5BrdU immunocytochemistry after single pulse or prolonged-pulse and chase experiments. The massive cartilage regeneration that can restore broad vertebral regions and gives rise to a long cartilaginous tube in the regenerating tail, depends from the permanence of some chondrogenic cells within adult vertebrae. Few cells that retain tritiated thymidine or 5-bromodeoxy-uridine for over 35 days are mainly localized in the inter-vertebral cartilage and in sparse chondrogenic regions of the neural arch of the vertebrae, suggesting that they are putative resident stem/progenitor cells. The study supports previous hypothesis indicating that the massive regeneration of the cartilaginous tissue in damaged vertebrae and in the regenerating tail of lizards derive from resident stem cells mainly present in the cartilaginous areas of the vertebrae including in the perichondrium that are retained in adult lizards as growing centers for most of their lifetime.

Cartilage canals have been shown to contain discontinuous blood vessels that enable circulating bacteria to bind to cartilage matrix, leading to vascular occlusion and associated pathological changes in pigs and chickens. It is also inconsistently reported that cartilage canals are surrounded by a cellular or acellular wall that may influence whether bacterial binding can occur. It is not known whether equine cartilage canals contain discontinuous endothelium or are surrounded by a wall. This study aimed to examine whether there were discontinuities in the endothelium of cartilage canal vessels, and whether canals had a cellular or acellular wall, in the epiphyseal growth cartilage of foals. Epiphyseal growth cartilage from the proximal third of the medial trochlear ridge of the distal femur from six healthy foals that were 1, 24, 35, 47, 118 and 122 days old and of different breeds and sexes was examined by light microscopy (LM), transmission electron microscopy (TEM) and immunohistochemistry. The majority of patent cartilage canals contained blood vessels that were lined by a thin layer of continuous endothelium. Fenestrations were found in two locations in one venule in a patent cartilage canal located deep in the growth cartilage and close to the ossification front in the 118‐day‐old foal. Chondrifying cartilage canals in all TEM‐examined foals contained degenerated endothelial cells that were detached from the basement membrane, resulting in gap formation. Thirty‐three percent of all canals were surrounded by a hypercellular rim that was interpreted as contribution of chondrocytes to growth cartilage. On LM, 69% of all cartilage canals were surrounded by a ring of matrix that stained intensely eosinophilic and consisted of collagen fibres on TEM that were confirmed to be collagen type I by immunohistochemistry. In summary, two types of discontinuity were observed in the endothelium of equine epiphyseal cartilage canal vessels: fenestrations were observed in a patent cartilage canal in the 118‐day‐old foal; and gaps were observed in chondrifying cartilage canals in all TEM‐examined foals. Canals were not surrounded by any cellular wall, but a large proportion was surrounded by an acellular wall consisting of collagen type I. Bacterial binding can therefore probably occur in horses by mechanisms that are similar to those previously demonstrated in pigs and chickens.

The aging process induces progressive and irreversible changes in the structural and functional organization of animals. The objective of this study was to evaluate the effects of aging on the structure and composition of the extracellular matrix of the arytenoid cartilage found in the larynx of male bullfrogs (Lithobates catesbeianus) kept in captivity for commercial purposes. Animals at 7, 180 and 1080 days post-metamorphosis (n=10/age) were euthanized and the cartilage was removed and processed for structural and biochemical analysis. For the structural analyses, cartilage sections were stained with picrosirius, toluidine blue, Weigert's resorcin-fuchsin and Von Kossa stain. The sections were also submitted to immunohistochemistry for detection of collagen types I and II. Other samples were processed for the ultrastructural and cytochemical analysis of proteoglycans. Histological sections were used to chondrocyte count. The number of positive stainings for proteoglycans was quantified by ultrastructural analysis. For quantification and analysis of glycosaminoglycans were used the dimethyl methylene blue and agarose gel electrophoresis methods. The chloramine T method was used for hydroxyproline quantification. At 7 days, basophilia was observed in the pericellular and territorial matrix, which decreased in the latter over the period studied. Collagen fibers were arranged perpendicular to the major axis of the cartilaginous plate and were thicker in older animals. Few calcification areas were observed at the periphery of the cartilage specimens in 1080-day-old animals. Type II collagen was present throughout the stroma at the different ages. Elastic fibers were found in the stroma and perichondrium and increased with age in the two regions. Proteoglycan staining significantly increased from 7 to 180 days and reduced at 1080 days. The amount of total glycosaminoglycans was higher in 180-day-old animals compared to the other ages, with marked presence of chondroitin- and dermatan-sulfate especially in this age. The content of hydroxyproline, which infers the total collagen concentration, was higher in 1080-day-old animals compared to the other ages. The results demonstrated the elastic nature of the arytenoid cartilage of L. catesbeianus and the occurrence of age-related changes in the structural organization and composition of the extracellular matrix. These changes may contribute to alter the function of the larynx in the animal during aging.