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Due to anatomical and biological similarities with humans, pigs are increasingly used for inflammation- and immune-related studies in biomedical research, including the field of osteonecrosis and osteoimmunology. Here, we present a protocol for rib extraction, isolation of the bone marrow by centrifugation, and processing to obtain bone-marrow-derived macrophages (BMDMs). Then, we describe the procedures of in vitro experiments to evaluate the cell phenotype. For complete details on the use and execution of this protocol, please refer to Andre et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Formation of the skeletal structure in the human embryo has important consequences in terms of support, protection, and function of organs and other systems. We aimed to describe the formation of the rib cage during the embryonic period, in order to detect prominent features and identify the possible factors affecting rib cage morphology. We employed high-resolution digitized imaging data (n = 34) obtained in human embryos with Carnegie stage (CS) between 17 and 23. The rib cage became detectable as cartilage formation at CS17, expanding outward from the dorsal side of the chest-abdominal region. Ribs elongated progressively to surround the chest, differentiating into the upper and lower rib cage regions by CS20. The ends of corresponding ribs in the upper region elongated toward each other, leading to their joining and sternum formation between CS21 and CS23, while the lower region of the rib cage remained widely open. The rib cage area with the largest width shifted from the 5th rib pair at CS17 to the 9th pair at CS23. The depth of the rib cage was similar across the upper region at CS17, with the major portion remaining in the middle part after CS20. The heart was located beneath the rib pairs providing the largest depth, while the liver was located beneath the rib pairs providing the largest width. Formation of the sternum, development of spinal kyphosis, and organization of larger internal organs within the thoracic and abdominal cavity are possible factors affecting rib cage morphology. Anat Rec, 302:2211-2223, 2019. © 2019 American Association for Anatomy., (© 2019 American Association for Anatomy.)

During the manufacture of cell- and tissue-based products, such as engineered cartilage for autologous chondrocyte implantation, maximizing the number of cells isolated from donor tissue substantially improves the productivity of these products. The method used for agitating tissues with digestive fluid and enzymes can considerably affect both the quality and quantity of isolated cells. This study aimed to investigate the effectiveness of a rotation/revolution-type agitator for chondrocyte isolation following the enzymatic digestion of rat costal cartilage. Cartilage tissue cut into 1 mm 3 -thick sections was equally divided between two groups and placed in 50-mL conical tubes; sections in both groups were digested using 0.1 mg/mL liberase TH (collagenase/thermolysin) at 37 °C for 4 h with either rotation/revolution or conventional orbital agitation method. Compared with using conventional orbital agitator, using the rotation/revolution-type agitator resulted in a significant (>two-fold) increase in the number of isolated cells. In subsequent primary cultures, chondrocytes obtained by rotation/revolution agitation showed superior initial attachment to tissue culture dish on day 1 and 2 compared with those obtained by conventional agitation; however, no differences in cell proliferation or cartilage-related molecule expression patterns were observed between cells derived from either method after 3 days of subculture. These findings suggested that there are no disadvantages to the proposed rotation/revolution agitation method. Rotation/revolution-type agitators are a promising apparatus for preparing chondrocytes for primary cultures and cartilage tissue engineering., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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.

Smaller diaphragmatic motor unit potentials (MUPs) compared to MUPs of limb muscles lead to the hypothesis that diaphragmatic muscle fibers, being the generators of MUPs, might be also smaller. We compared autopsy samples of costal diaphragm and vastus lateralis of healthy men with respect to fibers' size and expression of slow myosin heavy chain isoform (MyHC-1) and fast 2A isoform (MyHC-2A). Diaphragmatic fibers were smaller than fibers in vastus lateralis with regard to the mean minimal fiber diameter of slow-twitch (46.8 versus 72.2  μ m, p < 0.001), fast-twitch (45.1 versus 62.4  μ m, p < 0.001), and hybrid fibers (47.3 versus 65.0  μ m, p < 0.01) as well as to the mean fiber cross-sectional areas of slow-twitch (2376.0 versus 5455.9  μ m 2 , p < 0.001), fast-twitch (2258.7 versus 4189.7  μ m 2 , p < 0.001), and hybrid fibers (2404.4 versus 4776.3  μ m 2 , p < 0.01). The numerical proportion of slow-twitch fibers was higher (50.2 versus 36.3%, p < 0.01) in costal diaphragm and the numerical proportion of fast-twitch fibers (47.2 versus 58.7%, p < 0.01) was lower. The numerical proportion of hybrid fibers did not differ. Muscle fibers of costal diaphragm have specific characteristics which support increased resistance of diaphragm to fatigue., Competing Interests: The authors declare that there are no competing interests regarding the publication of this paper.

Background and Objective: Magnetic nanoparticles with the ability to absorb laser radiation are the perspective agents for the early diagnostics and laser therapy of degenerative cartilage. The effect of starch stabilized magnetite nanoparticles (SSNPs) on the cartilage structure components has never been studied before. The aim of the work is to establish the Erbium:glass laser effect on costal and articular cartilage impregnated with SSNPs., Materials and Methods: Porcine articular and costal cartilage disks (2.0 mm in diameter and 1.5-2 mm in thickness) were impregnated with SSNPs and irradiated using a 1.56 μm laser in therapeutic laser setting. The one sample group underwent the second irradiation after the SSNPs impregnation. The samples were analyzed by the means of histology, histochemistry and transmission electron microscopy (TEM) to reveal the alterations of cells, glycosaminoglycans and collagen network., Results: The irradiated cartilage demonstrates the higher content of cell alterations than the intact one due to the heat and mechanical affection in the course of laser irradiation. However the alterations are localized at the areas near the irradiated surfaces and not dramatic. The impregnation of SSNPs does not cause any additional cell alterations. For both costal and articular cartilage the matrix alterations of irradiated samples are not critical: there is the slight decrease in acid proteoglycan content at the irradiated areas while the collagen network is not altered. Distribution and localization of impregnated SSNPs is described: agglomerates of 150-230 nm are observed located at the borders between matrix and cell lacunas of articular cartilage; SSNPs of 15-45 nm are found in the collagen network of costal cartilage., Conclusions: It was shown that SSNPs do not appreciably affect the structural components of both articular and costal cartilage and can be safely used for the laser diagnostics and therapy. The area of structural alterations is diffuse and local as the result of the mechanical and heat effect of laser impact. SSNPs reveal the areas of the structural alterations of cartilage matrix and give information about the size of the pores and defects., (© 2015 Wiley Periodicals, Inc.)

A simple scheme for multimodal coherent anti-Stokes Raman scattering (CARS) microscopy is based on the spectral focusing of ultrafast-oscillator-derived pump/probe light and synchronous photonic crystal fiber (PCF) fiber-generated broadband Stokes light. To date, such schemes allowed rapid hyperspectral imaging throughout the CH/OH high frequency region (2700-4000 cm(-1) ). Here we extend this approach to the middle (1640-3300 cm(-1) ) and fingerprint regions (850-1800 cm(-1) ) of the Raman spectrum. Our simple integrated approach to rapid hyperspectral CARS microscopy in the fingerprint region is demonstrated by applications to label-free multimodal imaging of cellulose and bulk bone, including use of the phosphate resonance at 960 cm(-1) ., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Primary cilia are present on most cell types including chondrocytes. Dysfunction of primary cilia results in pleiotropic symptoms including skeletal dysplasia. Previously, we showed that deletion of Ift88 and subsequent depletion of primary cilia from chondrocytes resulted in disorganized columnar structure and early loss of growth plate. To understand underlying mechanisms whereby Ift88 regulates growth plate function, we compared gene expression profiles in normal and Ift88 deleted growth plates. Pathway analysis indicated that Hedgehog (Hh) signaling was the most affected pathway in mutant growth plate. Expression of the Wnt antagonist, Sfrp5, was also down-regulated. In addition, Sfrp5 was up-regulated by Shh in rib chondrocytes and regulation of Sfrp5 by Shh was attenuated in mutant cells. This result suggests Sfrp5 is a downstream target of Hh and that Ift88 regulates its expression. Sfrp5 is an extracellular antagonist of Wnt signaling. We observed an increase in Wnt/β-catenin signaling specifically in flat columnar cells of the growth plate in Ift88 mutant mice as measured by increased expression of Axin2 and Lef1 as well as increased nuclear localization of β-catenin. We propose that Ift88 and primary cilia regulate expression of Sfrp5 and Wnt signaling pathways in growth plate via regulation of Ihh signaling., (Copyright © 2012 Orthopaedic Research Society.)

Introduction: Costochondral cells may be isolated with minimal donor site morbidity and are unaffected by pathologies of the diarthrodial joints. Identification of optimal exogenous stimuli will allow abundant and robust hyaline articular cartilage to be formed from this cell source., Methods: In a three factor, two level full factorial design, the effects of hydrostatic pressure (HP), transforming growth factor β1 (TGF-β1), and chondroitinase ABC (C-ABC), and all resulting combinations, were assessed in third passage expanded, redifferentiated costochondral cells. After 4 wks, the new cartilage was assessed for matrix content, superficial zone protein (SZP), and mechanical properties., Results: Hyaline articular cartilage was generated, demonstrating the presence of type II collagen and SZP, and the absence of type I collagen. TGF-β1 upregulated collagen synthesis by 175% and glycosaminoglycan synthesis by 75%, resulting in a nearly 200% increase in tensile and compressive moduli. C-ABC significantly increased collagen content, and fibril density and diameter, leading to a 125% increase in tensile modulus. Hydrostatic pressure increased fibril diameter by 30% and tensile modulus by 45%. Combining TGF-β1 with C-ABC synergistically increased collagen content by 300% and tensile strength by 320%, over control. No significant differences were observed between C-ABC/TGF-β1 dual treatment and HP/C-ABC/TGF-β1., Conclusions: Employing biochemical, biophysical, and mechanical stimuli generated robust hyaline articular cartilage with a tensile modulus of 2 MPa and a compressive instantaneous modulus of 650 kPa. Using expanded, redifferentiated costochondral cells in the self-assembling process allows for recapitulation of robust mechanical properties, and induced SZP expression, key characteristics of functional articular cartilage.

The aim of the present study was to investigate experimentally and numerically the influence of a fine threaded- against a roughened-cervical region of immediately loaded dental implants in combination with straight and 20°-angled abutments on the implant primary stability. A total of 30 implants were inserted in bovine rib-segments, 14 cervically roughened implants and 16 implants with fine cervical threads. Each implant system received two abutments, straight and 20°-angled. Implant displacements and rotations were measured using a biomechanical measurement system. Subsequently, eight samples were selected for geometrical reconstruction and numerical investigation of stress and strain distributions in the bone by means of the finite element method. Experimentally, both implant systems showed similar behaviour with the straight abutments concerning displacements and rotations. However, fine threaded implants showed much less displacement and rotation against roughened implants when angled abutments were considered. Numerically, stresses were within 35-45 MPa in the cortical bone for both implant systems. The strains showed highest values within the spongious bone with the roughened implants connected to angled abutments. The results indicate that implants with fine cervical threads could be recommended in particular with angled abutments. The outcomes of this study are currently confirmed by long-term clinical investigations., (Copyright © 2011 IPEM. Published by Elsevier Ltd. All rights reserved.)

In cartilage research bovine articular cartilage is used as an alternative to human tissue. However, animal material is subject to availability and primary cultures undergo senescence, limiting their use. Here we report the immortalization of primary bovine chondrocytes, which could be used as a surrogate for freshly isolated chondrocytes. Chondrocytes were isolated from cartilage explants and immortalized using 1.0 µg/ml benzo[alpha]pyrene. For 3-dimensional culture, chondrocytes were resuspended in 0.5% low-melt agarose at high density (HD) and cultured for 24 h prior to determining changes in expression profile and morphology. A13/BACii chondrocytes acquired a 'flat' irregular morphology and a foetal-like cell volume (1,509.59 ± 182.04 µm(3)). The human cell line C-20/A4 showed a statistically similar volume and length to A13/BACii. Two-dimensional-cultured A13/BACii expressed elevated levels of type I collagen (col1), reduced levels of type II collagen (col2) compared to freshly isolated chondrocytes and an overall col2 to col1 expression ratio (col2:col1) of 0.11 ± 0.01. Upon 3-dimensional encapsulation, there was a significant rise in col2 expression in both A13/BACii and C-20/A4, suggesting a capacity for redifferentiation in both cell lines with a return of col2:col1 values of A13/BACii to values previously observed in primary chondrocytes. A13/BACii chondrocytes expressed aggrecan, matrix metalloproteinase (MMP)-3, MMP-9 and MMP-13, further supporting indications of the differentiated phenotype. Here we report the creation of a novel chondrocytic cell line and demonstrate its strong potential for redifferentiation upon HD 3-dimensional encapsulation, providing an alternative to conventional dedifferentiated cell lines and primary culture., (Copyright © 2012 S. Karger AG, Basel.)

Background: Cartilage grafts are frequently used in nasal surgery for structural and/or aesthetic purposes. The literature holds contradictory reports concerning the effect of crushing on the viability of cartilage grafts., Methods: Nasal septal and costal cartilage grafts were harvested from 12 New Zealand rabbits. Each nasal septal and costal cartilage was divided into five equal pieces. One of the pieces was left intact and the remaining four were prepared as slightly, moderately, severely, or significantly crushed. The cartilage pieces were then autoimplanted into the paravertebral skin of the rabbits. The animals were euthanized 4 months later and the effect of crushing on cartilage grafts was assessed pathologically., Results: The viability of the chondrocytes was found to be decreased as the level of crushing increased. The mean chondrocyte viability rates for the intact, slightly crushed, moderately crushed, severely crushed, and significantly crushed cartilages were 88, 75, 51, 41, and 13 percent for the septal cartilages and 94, 83, 62, 32, and 26 percent for the costal cartilages, respectively. The differences between the mean viability rates of septal and costal cartilage groups were statistically not significant., Conclusions: The level of crushing determines the rate of viability for the crushed cartilage. Viability rates and the clinical properties of the slightly crushed cartilage grafts at long-term follow-up may be similar to those of the intact cartilage grafts. However, severe or significant crushing leads to a decrement in the viability of the chondrocytes and may cause unpredictable degrees of volume loss at long-term follow-up.

An important hypothesis is that the degree of infilling of secondary osteons (Haversian systems) is controlled by the inhibitory effect of osteocytes on osteoblasts, which might be mediated by sclerostin (a glycoprotein produced by osteocytes). Consequently, this inhibition could be proportional to cell number: relatively greater repression is exerted by progressively greater osteocyte density (increased osteocytes correlate with thinner osteon walls). This hypothesis has been examined, but only weakly supported, in sheep ulnae. We looked for this inverse relationship between osteon wall thickness (On.W.Th) and osteocyte lacuna density (Ot.Lc.N/B.Ar) in small and large osteons in human ribs, calcanei of sheep, deer, elk, and horses, and radii and third metacarpals of horses. Analyses involved: (1) all osteons, (2) smaller osteons, either ≤150 μm diameter or less than or equal to the mean diameter, and (3) larger osteons (>mean diameter). Significant, but weak, correlations between Ot.Lc.N/B.Ar and On.W.Th/On.Dm (On.Dm = osteon diameter) were found when considering all osteons in limb bones (r values -0.16 to -0.40, P < 0.01; resembling previous results in sheep ulnae: r = -0.39, P < 0.0001). In larger osteons, these relationships were either not significant (five/seven bone types) or very weak (two/seven bone types). In ribs, a negative relationship was only found in smaller osteons (r = -0.228, P < 0.01); this inverse relationship in smaller osteons did not occur in elk calcanei. These results do not provide clear or consistent support for the hypothesized inverse relationship. However, correlation analyses may fail to detect osteocyte-based repression of infilling if the signal is spatially nonuniform (e.g., increased near the central canal)., (Copyright © 2011 Wiley-Liss, Inc.)

The aim of this study was to determine if podoplanin was expressed by rudiment chondrocytes in human foetal cartilages. Podoplanin was immunolocalised in first trimester human foetal rib and knee joint rudiments to a sub-population of chondrocytes deep in the rib rudiments, tibial and femoral growth plates and cells associated with the cartilage canals of the foetal knee joint rudiments. Lymphatic vessels in the loose stromal tissues surrounding the developing rudiments were also demonstrated on the same histology slides using antipodoplanin (MAb D2-40) and anti-LYVE-1 and differentiated from CD-31 positive blood vessels confirming the discriminative capability of the antibody preparations used. The D2-40 positive rib and knee rudiment chondrocytes were not stained with antibodies to LYVE-1, CD-31 or CD-34 however perlecan was a prominent pericellular proteoglycan around these cells confirming their chondrogenic phenotype. Discernable differences were evident between the surface and deep rudiment chondrocytes in terms of their antigen reactivities detected with MAb D2-40 or antiperlecan antibodies. Binding of the cytoplasmic tail of PDPN to the ERM proteins ezrin, radixin and moeisin may result in changes in cytoskeletal organisation which alter the phenotype of this central population of rudiment cells. This may contribute to morphological changes in the rudiment cartilages which lead to establishment of the primary ossification centres and is consistent with their roles as transient developmental scaffolds during tissue development., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Multiple epiphyseal dysplasia (MED) can result from mutations in matrilin-3, a structural protein of the cartilage extracellular matrix. We have previously shown that in a mouse model of MED the tibia growth plates were normal at birth but developed a progressive dysplasia characterised by the intracellular retention of mutant matrilin-3 and abnormal chondrocyte morphology. By 3 weeks of age, mutant mice displayed a significant decrease in chondrocyte proliferation and dysregulated apoptosis. The aim of this current study was to identify the initial post-natal stages of the disease. We confirmed that the disease phenotype is seen in rib and xiphoid cartilage and, like tibia growth plate cartilage is characterised by the intracellular retention of mutant matrilin-3. Gene expression profiling showed a significant activation of classical unfolded protein response (UPR) genes in mutant chondrocytes at 5 days of age, which was still maintained by 21 days of age. Interestingly, we also noted the upregulation of arginine-rich, mutated in early stage of tumours (ARMET) and cysteine-rich with EGF-like domain protein 2 (CRELD2) are two genes that have only recently been implicated in the UPR. This endoplasmic reticulum (ER) stress and UPR did not lead to increased chondrocyte apoptosis in mutant cartilage by 5 days of age. In an attempt to alleviate ER stress, mutant mice were fed with a chemical chaperone, 4-sodium phenylbutyrate (SPB). SPB at the dosage used had no effect on chaperone expression at 5 days of age but modestly decreased levels of chaperone proteins at 3 weeks. However, this did not lead to increased secretion of mutant matrilin-3 and in the long term did not improve the disease phenotype. We performed similar studies with a mouse model of Schmid metaphyseal chondrodysplasia, but again this treatment did not improve the phenotype.

Semi-circular tracheal cartilage is a critical determinant of maintaining architectural integrity of the respiratory airway. The current effort to understand the morphogenesis of tracheal cartilage is challenged by the lack of appropriate model systems. Here we report an in vitro tracheal cartilage system using embryonic tracheal–lung explants to recapitulate in vivo tracheal cartilage developmental processes. With modifications of a current lung culture protocol, we report a consistent in vitro technique of culturing tracheal cartilage from primitive mouse embryonic foregut for the first time. This tracheal culture system not only induces the formation of tracheal cartilage from the mouse embryonic foregut but also allows for the proper patterning of the developed tracheal cartilage. Furthermore, we show that this culture technique can be applied to culturing other types of cartilage in vertebrae, limbs, and ribs. We believe that this novel application of our in vitro culture system will facilitate the manipulation of cartilage development under various conditions and thus enabling us to advance our current limited knowledge on cartilage biology and development.

This report evaluates the spatial profile of blood vessel fragments (BVFs) and CD34(+) and CD117(+) hematopoietic stem and progenitor cells (HSPCs) in human cancellous bone. Bone specimens were sectioned, immunostained (anti-CD34 and anti-CD117), and digitally imaged. Immunoreactive cells and vessels were then optically and morphometrically identified and labeled on the corresponding digital image. The distance of each BVF, or CD34(+) or CD117(+) HSPC to the nearest trabecular surface was measured and binned in 50-microm increments. The relative concentration of HSPCs and BVFs within cancellous marrow was observed to diminish with increasing distance in the marrow space. On average, 50% of the CD34(+) HSPC population, 60% of the CD117(+) HSPC population, and 72% of the BVFs were found within 100 microm of the bone surfaces. HSPCs were also found to exist in close proximity to BVFs, which supports the notion of a shared HSPC and vessel spatial niche.

Many tissues have a specific signal transduction system for endoplasmic reticulum (ER) dysfunction; however, the mechanisms underlying the ER stress response in cartilage remain unclear. BBF2H7 (BBF2 human homologue on chromosome 7), an ER-resident basic leucine zipper transcription factor, is activated in response to ER stress and is highly expressed in chondrocytes. In this study, we generated Bbf2h7(-/-) mice to assess the in vivo function of BBF2H7. The mice showed severe chondrodysplasia and died by suffocation shortly after birth because of an immature chest cavity. The cartilage showed a lack of typical columnar structure in the proliferating zone and a decrease in the size of the hypertrophic zone, resulting in a significant reduction of extracellular matrix proteins. Interestingly, proliferating chondrocytes showed abnormally expanded ER, containing aggregated type II collagen (Col2) and cartilage oligomeric matrix protein (COMP). We identified Sec23a, which encodes a coat protein complex II component responsible for protein transport from the ER to the Golgi, as a target of BBF2H7, which directly bound to a CRE-like sequence in the promoter region of Sec23a to activate its transcription. When Sec23a was introduced to Bbf2h7(-/-) chondrocytes, the impaired transport and secretion of cartilage matrix proteins was totally restored, indicating that by activating protein secretion the BBF2H7-Sec23a pathway has a crucial role in chondrogenesis. Our findings provide a new link by which ER stress is converted to signalling for the activation of ER-to-Golgi trafficking.

The study's objective was to investigate if transplanted chondrocyte or periosteal cell spheroids have influence on ingrowing bone marrow-derived cells in a novel cartilage repair approach in miniature pigs. Autologous rib chondrocytes or periosteal cells were cultured as spheroids and press-fitted into cavities that were milled into large, superficial chondral lesions of the patellar joint surface. Within the milled cavities, the subchondral bone plate was either penetrated or left intact (full-thickness or partial-thickness cavities). The transplantation of chondrocyte spheroids into full-thickness cavities induced the formation of additional secondary repair cartilage that exceeded the original volume of the transplanted spheroids. The resulting continuous tissue was rich in proteoglycans and stained positive for type II collagen. Cell labeling revealed that secondarily invading repair cells did not originate from transplanted spheroids, but rather from arroded bone marrow. However, secondary invasion of repair cells was less pronounced following transplantation of periosteal cells and absent in partial-thickness cavities. According to in vitro analyses, these observations could be ascribed to the ability of chondrocyte spheroids to secrete relevant amounts of bone morphogenetic protein-2, which was not detected for periosteal cells. Transplanted chondrocyte spheroids exert a dual function: they provide cells for the repair tissue and have a stimulatory paracrine activity, which promotes ingrowth and chondrogenesis of bone marrow-derived cells., ((c) 2009 Orthopaedic Research Society.)

Tissue-engineered fibrocartilage could become a feasible option for replacing tissues such as the knee meniscus or temporomandibular joint disc. This study employed five growth factors (insulin-like growth factor-I, transforming growth factor-beta1, epidermal growth factor, platelet-derived growth factor-BB, and basic fibroblast growth factor) in a scaffoldless approach with costal chondrocytes, attempting to improve biochemical and mechanical properties of engineered constructs. Samples were quantitatively assessed for total collagen, glycosaminoglycans, collagen type I, collagen type II, cells, compressive properties, and tensile properties at two time points. Most treated constructs had lower biomechanical and biochemical properties than the controls with no growth factors, suggesting a detrimental effect, but the treatment with insulin-like growth factor-I tended to improve the constructs. Additionally, the 6-week time point was consistently better than that at 3 weeks, with total collagen, glycosaminoglycans, and aggregate modulus doubling during this time. Further optimization of the time in culture and exogenous stimuli will be important in making a more functional replacement tissue.

Previous studies have demonstrated the functional expression, by osteoblasts, of N-methyl-D-aspartate (NMDA) receptors responsible for the promotion of cellular differentiation in bone. We have now evaluated the possible role of the endogenous co-agonist of NMDA receptors, glycine (Gly), in chondrogenesis. In ex vivo organotypic cultures of fetal mouse tibias, proximal and distal cartilaginous primordia were significantly increased in the presence of Gly, with the osteogenic center being unchanged. Exposure to Gly drastically increased mRNA expression of the calcified chondrocyte marker osteopontin, without markedly affecting that of a proliferating chondrocyte marker or a hypertrophic chondrocyte marker, as shown in organotypic cultures by in situ hybridization analysis. Gly significantly increased Ca2+ accumulation, osteopontin mRNA expression, and alkaline phosphatase activity in cultured rat costal chondrocytes, without significantly affecting those in cultured rat calvarial osteoblasts. The increase induced by Gly was significantly prevented by an NMDA receptor channel blocker and an antagonist at the Gly site on NMDA receptors, but not by an inhibitory Gly receptor antagonist or a Gly transporter inhibitor, in cultured chondrocytes. Constitutive mRNA expression was seen for NR1, NR2D, and NR3A subunits of NMDA receptors, but not for Gly receptors and transporters, in cultured chondrocytes. Corresponding immunoreactive proteins were detected for NR1 and NR2D subunits in cartilaginous zones of fetal mouse tibias. Thus, Gly might, at least in part, play a role as a trophic factor in the mechanisms associated with chondral calcification through the Gly site of NMDA receptors functionally expressed by chondrocytes in rodent cartilage.

Tissue-engineering of the temporomandibular joint (TMJ) disc aims to provide patients with TMJ disorders an option to replace diseased tissue with autologous, functional tissue. This study examined clinically relevant cell sources by comparing costal chondrocytes, dermal fibroblasts, a mixture of the two, and TMJ disc cells in a scaffoldless tissue-engineering approach. It was hypothesized that all constructs would produce matrix relevant to the TMJ disc, but the mixture constructs were expected to appear most like the TMJ disc constructs. Costal chondrocyte and mixture constructs were morphologically and biochemically superior to the TMJ disc and dermal fibroblast constructs, and their compressive properties were not significantly different. Costal chondrocyte constructs produced almost 40 times more collagen and 800 times more glycosaminoglycans than did TMJ constructs. This study demonstrates the ability of costal chondrocytes to produce extracellular matrix that may function in a TMJ disc replacement.

Bone morphogenetic proteins (BMPs) play pivotal roles in bone and cartilage growth and repair. Through phenotypes of short-ear (se) mice, which have BMP-5 mutations, a role for BMP-5 in some specific aspects of skeletogenesis and cartilage growth is known. This report examines BMP-5 expression in the growth plate and in differentiating cultures of primary chondrocytes, and the effects of addition of BMP-5 or its inhibition by anti-BMP-5 antibody in chondrocyte cultures. By laser capture microdissection and immunohistochemistry, we found that BMP-5 is expressed in proliferating zone (PZ) chondrocytes and that the expression increases sharply with hypertrophic differentiation. A similar pattern was observed in differentiating cultures of primary chondrocytes, with BMP-5 expression increasing as cells differentiated, in contrast to other BMPs. BMP-5 added to cultures increased cell proliferation early in the culture period and also stimulated cartilage matrix synthesis. Also, BMP-5 addition to the cultures activated phosphorylation of Smad 1/5/8 and p38 MAP kinase and caused increased nuclear accumulation of phospho-Smads. Anti-BMP-5 antibody inhibited the endogenous BMP-5, reducing cell proliferation and phospho-Smad nuclear accumulation. Together, the results demonstrate that BMP-5 is normally an important regulator of chondrocyte proliferation and differentiation. Whether other BMPs may compensate in BMP-5 loss-of-function mutations is discussed., ((c) 2007 Wiley-Liss, Inc.)

Costal cartilage has been proposed as an alternative donor of chondrocytes for articular-cartilage repair. In the present study we compared the initial cell yield of chondrocytes from rabbit costal cartilage and their cell expansion rates in monolayer culture with those of articular cartilage. Costal cartilage gave an approx. 2.6-fold higher cell yield than did articular cartilage. During in vitro culture, CCs (costal chondrocytes) grew faster and displayed approx. 3-fold more cell expansion up to P4 (passage 4) than did ACs (articular chondrocytes). In order to match the degree of dedifferentiation during serial cultivation with the cells' expansion rate, type II collagen expression and the emergence of fibroblastic morphology were monitored at each cell passage. Both ACs and CCs gradually lost their chondrocytic phenotype, changed to fibroblast-like cells and displayed a reduced expression of type II collagen. We then also evaluated the redifferentiation capacity of the expanded ACs and CCs by culturing them at high density in collagen gel. Almost fully dedifferentiated CCs at P4 were successfully redifferentiated into hyaline cartilage, which showed the expression of glycosaminoglycan and type II collagen as well as the formation of lacunae and a territorial matrix. In conclusion, costal cartilage may have advantages over articular cartilage as an alternative donor tissue for autologous chondrocytes on the basis of its higher cell yield, higher cell expansion and successful reversion into hyaline cartilage without ossification in vitro. However, although this experiment with a rabbit model gave a better insight into the problem than other experiments have done, it does not answer definitively the question as to which cells are most appropriate for articular cartilage repair in humans.

This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.

Hereditary multiple exostoses (HME) is a genetically heterogeneous human disease characterized by the development of bony outgrowths near the ends of long bones. HME results from mutations in EXT1 and EXT2, genes that encode glycosyltransferases that synthesize heparan sulfate chains. To study the relationship of the disease to mutations in these genes, we generated Ext2-null mice by gene targeting. Homozygous mutant embryos developed normally until embryonic day 6.0, when they became growth arrested and failed to gastrulate, pointing to the early essential role for heparan sulfate in developing embryos. Heterozygotes had a normal lifespan and were fertile; however, analysis of their skeletons showed that about one-third of the animals formed one or more ectopic bone growths (exostoses). Significantly, all of the mice showed multiple abnormalities in cartilage differentiation, including disorganization of chondrocytes in long bones and premature hypertrophy in costochondral cartilage. These changes were not attributable to a defect in hedgehog signaling, suggesting that they arise from deficiencies in other heparan sulfate-dependent pathways. The finding that haploinsufficiency triggers abnormal cartilage differentiation gives insight into the complex molecular mechanisms underlying the development of exostoses.

The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pump's constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henle's loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target.

Objective: To explore the method of fabricating tissue engineered laryngeal cartilage., Methods: The rib and articular cartilage of infant New Zealand white rabbits were harvested in sterile condition. The chondrocytes were separated by collagenase digestion and cultured in vitro for 3 passage. Serial steps of solution casting, extrusion molding and particulate leaching were used to make larynx-shaped biomaterial models with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHH). The chondrocytes were seeded onto PHBHH scaffolds to form cell-PHBHH composites, which were subsequently in vitro for one week. After that, the measure of filling inner space of cell-PHBHH composites together with wrapping total composites using either greater omentum (n = 9) or fascia flap and muscle (also n = 9) in experimental groups was taken to implant the larynx-shaped biomaterial models seeded with chondrocytes into the belly and the back of adult New Zealand white rabbits. Control groups (every group n = 3) were the same measure as experimental groups but without chondrocyte on PHBHH scaffolds. Finally, morphological observation, HE staining & special staining and immunohistochemical test were conducted to assess cartilage regeneration and its shape at different period following implantation., Results: The rate of viable cell in the final cell suspension was (93 +/- 2)% after well-controlled prolongation of digestion trypsin. Similar to that by traditional procedures (94 +/- 2)% (P > 0.05). The larynx-shaped PHBHH models with edges and corners of laryngeal cartilage made by us appeared to be hollow half-trumpet shape and its porosity was more than 90%. It showed that chondrocytes equally attached to the surface of porous PHBHH and filled within porousness with scanning electron microscopic examination. Tissue engineered larynx-shaped specimens could alternatively be harvested with the above mentioned two different implantation measures. The specimens presented to be similar to that before implantation in gross shape. It was demonstrated to be cartilaginous tissue through histological and immunohistochemical examination. Furthermore, There was nearly no difference between two kinds of tissue engineered laryngeal cartilage with two measures of implantation in morphology and histology., Conclusions: The regeneration of tissue engineered cartilage in vivo is not influenced by the chondrocytes harvested by improvement of well-controlled prolonged digestion with trypsin during in vitro cell culture. It seems that PHBHH may be used as scaffold in cartilage tissue engineering and wrapping together with filling method with either greater omentum or fascia flap and muscle is appropriate for fabricating tissue engineered laryngeal cartilage.

Human ear, nasal, and rib chondrocytes were compared with respect to their suitability to generate autologous cartilage grafts for nonarticular reconstructive surgery. Cells were expanded for two passages in medium containing 10% fetal bovine serum without (control) or with transforming growth factor beta(1) (TGF-beta(1)), fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor bb (PDGF-bb) (TFP). Expanded cells were cultured as three-dimensional pellets in chondrogenic serum-free medium containing insulin, dexamethasone, and TGF-beta(1). Chondrocytes from all three sources were successfully isolated, increased their proliferation rate in response to TFP, and dedifferentiated during passaging. Redifferentiation by ear and nasal, but not rib, chondrocytes was enhanced after TFP expansion, as assessed by the significant increase in glycosaminoglycan (GAG)/DNA content and collagen type II mRNA expression in the resulting pellets. TFP-expanded ear and nasal chondrocytes generated pellets of better quality than rib chondrocytes, as assessed by the significantly higher GAG/DNA content and collagen type II mRNA expression, and by the relative stain intensities for GAG and collagen types I and II. In conclusion, postexpansion cell yields suggest that all three sources investigated could be used to generate autologous grafts of clinically relevant size. However, ear and nasal chondrocytes, if expanded with TFP, display superior postexpansion chondrogenic potential and may be a preferred cell source for cartilage tissue engineering.

The culture of chondrocytes is one of the most powerful tool for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate to fibroblasts when they are subcultured, which is a major problem. This chapter describes several protocols for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes) from various species (humans, mice, rabbits, and cattle). All these protocols involve primary cultures in order to limit dedifferentiation. This chapter also describes a new protocol for culturing mouse articular chondrocytes.

Using a cell culture method, we histochemically and immunohistochemically investigated whether chondrocytes deriving from different origins, such as Meckel's or costal cartilages, express similar phenotypic characteristics. Chondrocytes isolated enzymatically from Meckel's and costal cartilages of 17-day embryonic mice both actively proliferated and formed cartilage nodules consisting of toluidine blue-positive proteoglycans and type II collagen. Both deposited calcified cartilaginous matrix as revealed by alkaline phosphatase (ALPase) activity and alizarin red staining throughout 3 weeks in culture. Immunostaining for osteopontin (OP), osteocalcin (OC), and osteonectin (ON) revealed that chondrocytes from both cartilages were positive for their proteins, but type I collagen was detected only in cells transforming from Meckel's chondrocytes late in the culture. Electron microscopy demonstrated that although costal and Meckel's chondrocytes had typical chondrocytic features during 2 weeks in culture, Meckel's chondrocytes transformed into osteocytic cells that produced thick, banded type I collagen fibrils. In contrast, costal chondrocytes maintained typical hypertrophic morphology throughout the final stage of culture. The present study suggests that Meckel's chondrocytes derived from neural crest-ectomesenchyme retain osteogenic potential, and differ from costal chondrocytes originating from mesoderm.

Background/purpose: This study was aimed at applying fetal tissue engineering to chest wall reconstruction., Methods: Fetal lambs underwent harvest of elastic and hyaline cartilage specimens. Once expanded in vitro, fetal chondrocytes were seeded onto synthetic scaffolds, which then were placed in a bioreactor. After birth, fetal cartilage constructs (n = 10) were implanted in autologous fashion into the ribs of all lambs (n = 6) along with identical, but acellular scaffolds, as controls (n = 6). Engineered and acellular specimens were harvested for analysis at 4 to 12 weeks postimplantation. Standard histology and matrix-specific staining were performed both before implantation and after harvest on all constructs., Results: Regardless of the source of chondrocytes, all fetal constructs resembled hyaline cartilage, both grossly and histologically, in vitro. In vivo, engineered implants retained hyaline characteristics for up to 10 weeks after implantation but remodeled into fibrocartilage by 12 weeks postoperatively. Mononuclear inflammatory infiltrates surrounding residual PGA/PLLA polymer fibers were noted in all specimens but most prominently in the acellular controls., Conclusions: Engineered fetal cartilage can provide structural replacement for at least up to 10 weeks after autologous, postnatal implantation in the chest wall. Fetal tissue engineering may prove useful for the treatment of severe congenital chest wall defects at birth.

Objective: The temporal and spatial behavior of transplanted hematopoietic stem cells (HSCs) within bones remains to be clarified. Our goal is to examine in vivo reconstitution processes and candidate niches in all bones in the mouse body using a new visualization method., Materials and Methods: Using bone marrow cells from green fluorescent protein (GFP) transgenic mice, the reconstitution processes of transplanted hematopoietic cells (HCs) under myeloablative or nonmyeloablative conditions were observed sequentially from outside the bones with a fluorescent stereomicroscope., Results: In case of myeloablative transplantation, GFP(+) spots were first detected at the epiphysis of femurs, and in some ribs and vertebrae among all intact bones. Thereafter, engrafted cells proliferated and spread into other bones. In case of nonmyeloablative transplantation with lin(-)Sca-1(+)c-kit(+) cells into W/Wv neonates, characterized by vacant niches because of stem cell defects, GFP(+) cells localized at the epiphysis of femurs and in some vertebrae and ribs, but not in all bones even 4 months after transplantation., Conclusion: Our findings show that transplanted HSCs or their immature progenies engraft preferentially at the epiphysis of the femurs or short and flat bones such as ribs and vertebrae. The transplanted cells remain quiescent for at least 4 months under nonmyloablative conditions, which implies the presence of stem cells in a niche. Our approach for the first time graphically demonstrates the kinetics of HCs in vivo and should facilitate analysis of HSC behavior in a three-dimensional mode.

There is no detailed information available concerning the variations in bone, the Haversian canal, and osteocyte populations in different-sized osteons. In this study a total of 398 secondary osteons were measured in archived rib sections from nine white men (20-25 years old). The sections were stained with basic fuchsin. The parameters included the osteon area (On.Ar), Haversian canal area (HC.Ar) and perimeter (HC.Pm), bone area (B.Ar), and osteocyte lacunar number (Lc.N). From these primary measurements the following indices were deduced: 1) lacunar number per bone area (Lc.N/B.Ar) and per osteon (Lc.N/On); 2) the ratio between Haversian canal perimeter and bone area (HC.Pm/B.Ar); and 3) the fraction of Haversian canal area (HC.Ar/On.Ar) and its complement, the fraction of bone area (B.Ar/On.Ar). The results showed that the osteons varied greatly in size, but very little in the fraction of bone area. Regression analyses showed that HC.Ar, HC.Pm, and Lc.N/On were positively associated with On.Ar (P < 0.001 for all). A significant negative correlation was found between On.Ar and Lc.N/B.Ar (P < 0.05) and HC.Pm/B.Ar (P < 0.0001). HC.Ar and HC.Pm increased significantly with increasing Lc.N/On (both P < 0.0001) rather than Lc.N/B.Ar. Lc.N/B.Ar had a significant positive correlation with HC.Ar/On.Ar (P < 0.05) and HC.Pm/B.Ar (P < 0.01). We conclude that: 1) the size of the osteon is determined by the quantum of bone removed by osteoclasts, 2) the osteon is well designed for molecular exchange, and 3) a well designed osteon may be produced via the regulation of bone apposition by osteocytes during the process of osteon refilling., (Copyright 2003 Wiley-Liss, Inc.)

Background: Hox genes encode transcription factors that are involved in pattern formation in the skeleton, and recent evidence suggests that they also play a role in the regulation of endochondral ossification. To analyze the role of Hoxc-8 in this process in more detail, we applied in vitro culture systems, using high density cultures of primary chondrocytes from neonatal mouse ribs., Results: Cultured cells were characterized on the basis of morphology (light microscopy) and production of cartilage-specific extracellular matrix (sulfated proteoglycans and type II Collagen). Hypertrophy was demonstrated by increase in cell size, alkaline phosphatase activity and type X Collagen immunohistochemistry. Proliferation was assessed by BrdU uptake and flow cytometry. Unexpectedly, chondrocytes from Hoxc-8 transgenic mice, which exhibit delayed cartilage maturation in vivo 1, were able to proliferate and differentiate normally in our culture systems. This was the case even though freshly isolated Hoxc-8 transgenic chondrocytes exhibited significant molecular differences as measured by real-time quantitative PCR., Conclusions: The results demonstrate that primary rib chondrocytes behave similar to published reports for chondrocytes from other sources, validating in vitro approaches for studies of Hox genes in the regulation of endochondral ossification. Our analysis of cartilage-producing cells from Hoxc-8 transgenic mice provides evidence that the cellular phenotype induced by Hoxc-8 overexpression in vivo is reversible in vitro.

The traditional view that all parts of the ribs originate from the sclerotome of the thoracic somites has recently been challenged by an alternative view suggesting that only the proximal rib derives from the sclerotome, while the distal rib arises from regions of the dermomyotome. In view of this continuing controversy and to learn more about the cell interactions during rib morphogenesis, this study aimed to reveal the precise contributions made by somitic cells to the ribs and associated tissues of the thoracic cage. A replication-deficient lacZ-encoding retrovirus was utilized to label cell populations within distinct regions of somites 19-26 in stage 13-18 chick embryos. Analysis of the subsequent contributions made by these cells revealed that the thoracic somites are the sole source of cells for the ribs. More precisely, it is the sclerotome compartment of the somites that contributes cells to both the proximal and distal elements of the ribs, confirming the traditional view of the origin of the ribs. Results also indicate that the precursor cells of the ribs and intercostal muscles are intimately associated within the somite, a relationship that may be essential for proper rib morphogenesis. Finally, the data from this study also show that the distal ribs are largely subject to resegmentation, although cell mixing may occur at the most sternal extremities.

Plasma concentrations of the main vitamin D(3) metabolites (i.e., 25(OH)D(3), 1,25(OH)(2)D(3), and 24,25(OH)(2)D(3)) were measured in 14 weeks old large- and small-breed dogs (adult body weight 60 kg vs. 6 kg), raised under the same conditions. Levels of 25(OH)D(3) (approx. 22 microg/l) and 1,25(OH)(2)D(3) (approx. 40 ng/l) were similar in both groups, whereas plasma 24,25(OH)(2)D(3) concentrations were lower in large-breed dogs (7 microg/l vs. 70 microg/l, large- vs. small-breed dogs, respectively). The lower plasma 24,25(OH)(2)D(3) concentrations could be explained by the higher plasma GH and IGF-I concentrations in the large- vs. small-breed dogs, and these hormones are known to suppress 24-hydroxylation. Plasma 24,25(OH)(2)D(3) concentrations increased during Ca supplementation in small-breed but not in large-breed dogs (100 microg/l vs. 7 microg/l, respectively). Hypophosphatemia induced by a high dietary Ca content was only seen together with increased plasma 1,25(OH)(2)D(3) concentrations in euparathyroid dogs and not in hypoparathyroid dogs. Hyperparathyroidism due to Ca deficiency was accompanied by increased plasma 1,25(OH)(2)D(3) concentrations and decreased plasma 24,25(OH)(2)D(3) concentrations in both large- and small-breed dogs, together with generalized osteoporosis. Large-breed pups fed on a standard diet supplemented with Ca and P had decreased plasma concentrations of both 25(OH)D(3) and 1,25(OH)(2)D(3), which may indicate an increased clearance of these metabolites; the low plasma concentrations of the di-hydroxylated vitamin D metabolites were considered responsible for the disturbance in cartilage maturation (i.e., osteochondrosis) in these dogs. Even lower concentrations of all vitamin D(3) metabolites were seen in young dogs raised on a vitamin D(3)-deficient diet, and led to disturbed osteoid and cartilage mineralization (i.e., rickets). These studies indicate that there is a hierarchy of factors regulating vitamin D(3) metabolism in dogs, i.e., GH and IGF-I suppress 24-hydroxylase more than hypercalcemia or hypophosphatemia does; 1,25(OH)(2)D(3) and 24,25(OH)(2)D(3) are only reciprocally related in hyperparathyroidism; excessive Ca and P intake increases the turnover of vitamin D(3) metabolites; and the synergism between parathyroid hormone and 1,25(OH)D(3) seems to play a role in skeletal mineralization. The low plasma 24,25(OH)(2)D(3) concentrations in large-breed dogs raised on standard dog food may play a role in the etiology of disturbances in endochondral ossification during the rapid growth phase.

Objective: Low-dose methotrexate (MTX) is often used in the treatment of rheumatoid arthritis (RA). To be effective, treatment must be long-term, and there are concerns that MTX may impair bone formation in a population already predisposed to osteoporosis. The purpose of this investigation was to determine the direct effects of MTX at clinically relevant doses on the growth and differentiation of human cells of the osteoblast (bone-forming) lineage., Methods: Cells derived from the marrow stroma (BMSC) and trabecular surfaces [human bone-derived cells (HBDC)] of adult ribs were cultured in the absence or presence of MTX (1-1000 nM). To promote the differentiation and further maturation of cells of the osteoblast lineage, one half of the cultures were treated additionally with 10 nM dexamethasone (Dx)., Results: In cultures of BMSC, treatment with MTX (+/-Dx) did not affect the total number of colonies that formed or the expression of the developmental markers STRO-1 and alkaline phosphatase (AP). At concentrations > or =10 nM, however, there was a statistically significant reduction in the number of cells harvested at the end of primary culture. In cultures of HBDC, treatment with MTX (in the presence of Dx) did not affect cell number or the expression of AP., Conclusions: At concentrations > or =10 nM, treatment with MTX inhibits the proliferation of primitive marrow stromal cells, but not their ability to undergo osteogenic differentiation. The proliferation and further maturation of cells of the osteoblast lineage is not affected by treatment with MTX. These findings are reassuring for clinicians using MTX in the treatment of RA.

During the differentiation cascade of growth plate chondrocytes, cells undergo as much as a 10-15-fold increase in volume. This volume increase, which occurs to different extents in growth plates growing at different rates, has been demonstrated to be the single most significant variable in understanding the quantitative aspects of the cellular kinetics of long bone growth. Our hypothesis is that this volume increase, which occurs through cell swelling by water imbibition, requires intracellular accumulation of osmolytes through activation or upregulation of membrane transport mechanisms. Significant intracellular accumulation of inorganic osmolytes, such as Na+, K+, and Cl-, is potentially disruptive to normal cellular metabolism, whereas intracellular accumulation of organic osmolytes is considered to be more compatible with metabolic function. Thus, we concentrated on determining the contributions of organic osmolytes--betaine, amino acids, inositol, and sorbitol--to volume increase. Pooled cryostat sections of young bovine growth plates were extracted followed by automated analysis for their content of amino acids. Analysis for betaine and the sugar alcohols was done by extraction and derivatization, followed by high-performance liquid chromatography (HPLC). Parallel stereological analyses correlated osmolyte changes to stages of chondrocytic differentiation, specifically comparing intracellular concentration and amount in proliferative vs. hypertrophic chondrocytes. Calculations demonstrated that, maximally, these organic osmolytes, in total, account for 6%-7% of the intracellular osmolytes required to sustain the volume increase, and that the most significant contribution is from betaine. This suggests that intracellular accumulation of organic osmolytes is not a primary strategy used by growth plate chondrocytes during volume increase of their terminal differentiation. The data also suggest that there is a differential regulation of transporters of these osmolytes such that intracellular concentrations are constantly modified as cells proceed through the differentiation cascade.

The OASIS gene, which encodes a novel CREB/ATF family member, was isolated from long-term cultured astrocytes that were employed as an in vitro gliosis model. In the present study, we examined the expression pattern of the OASIS gene in the developing mouse embryo by in situ hybridization histochemistry and compared it with the expression of osteogenesis markers. OASIS mRNA expression was most strongly detected in preosteoblasts of the outer bony cortex of the ribs. Alveolar bone also showed strong signals for OASIS gene expression. OASIS mRNA was also localized to the preodontoblast of tooth buds. Expression began at embryonic day 12 (D12.5), peaked around D14.5-16.5, and continued to D18.5. The pattern of expression was very similar to that of hXBP-1 mRNA, which encodes another CREB/ATF family member. Spatiotemporal patterns of OASIS partly overlapped that of osteopontin, osteonectin, and alpha1 type I procollagen genes. Among these, the time course of OASIS mRNA expression was most similar to that of osteopontin mRNA expression, suggesting that the OASIS protein is involved in the late phase of osteoblast differentiation, as compared to the Cbfa1 that regulates early phases of osteoblast differentiation.

Protein products of frizzled genes are cell membrane receptors for Wnt proteins that play multiple roles during development. We examined the expression of nine frizzled genes in mouse chondrocytes, and detected transcripts of six of the nine genes. We also cloned the entire cDNA of mouse frizzled-1 and compared its cDNA sequence and the cysteine-rich and transmembrane domains of its translated product to sequences of frizzled-1 from C. elegans, Drosophila, chicken and human. We used the T31 Mouse/Hamster radiation hybrid panel to map the mouse frizzled-1 to mouse chromosome 5 (5 cM from the centromere), and frizzled-9 to mouse chromosome 5 (74 cM from the centromere).

Stimulation of osteoblast survival signals may be an important mechanism of regulating bone anabolism. Protein kinase B (PKB/Akt), a serine-threonine protein kinase, is a critical regulator of normal cell growth, cell cycle progression, and cell survival. In this study we have investigated the signaling pathways activated by growth factors PDGF-BB, EGF, and FGF-2 and determined whether PDGF-BB, EGF, and FGF-2 activated Akt in human or mouse osteoblastic cells. The results demonstrated that both ERK1 and ERK2 were activated by FGF-2 and PDGF-BB. Activation of ERK1 and ERK2 by PDGF-BB and FGF-2 was inhibited by PD 098059 (100 microM), a specific inhibitor of MEK. Wortmannin (500 nM), a specific inhibitor of phosphatidylinositol 3-kinase ( PI 3-K), inhibited the activation of ERK1 and ERK2 by PDGF-BB but not by FGF-2 suggesting that PI 3-K mediated the activation of ERK MAPK pathway by PDGF-BB but not by FGF-2. Rapamycin, an inhibitor of p70 S6 protein kinase and a downstream target of ERK1/2 and PI 3-K, did not affect the activation of ERK1 and ERK2 by the growth factors. Furthermore, our results demonstrated that Akt, a downstream target of PI 3-K, was activated by PDGF-BB but not by FGF-2. Akt activation by PDGF-BB was inhibited by PI 3-kinase inhibitor LY294002. Rapamycin had no effect on Akt activation. Epidermal growth factor (EGF) also activated Akt in osteoblastic cells which was inhibited by LY294002 but not by rapamycin. Taken together, our data for the first time revealed that the activation of ERK1/2 by PDGF-BB is mediated by PI 3-K, and secondly, Akt is activated by PDGF-BB and EGF but not by FGF-2 in human and mouse osteoblastic cells. These results are of critical importance in understanding the role of these growth factors in apoptosis and cell survival. PDGF-BB and EGF but not FGF-2 may stimulate osteoblast cell survival., (Copyright 2001 Wiley-Liss, Inc.)

Late cartilage differentiation during endochondral bone formation is a multistep process. Chondrocytes transit through a differentiation cascade under the direction of environmental signals that either stimulate or repress progression from one step to the next. In human costal cartilage, chondrocytes reach very advanced stages of late differentiation and express collagen X. However, remodeling of the tissue into bone is strongly repressed. The second hypertrophy marker, alkaline phosphatase, is not expressed before puberty. Upon sexual maturity, both alkaline phosphatase and collagen X activity levels are increased and slow ossification takes place. Thus, the expression of the two hypertrophy markers is widely separated in time in costal cartilage. Progression of endochondral ossification in this tissue beyond the stage of hypertrophic cartilage appears to be associated with the expression of alkaline phosphatase activity. Costal chondrocytes in culture are stimulated by parathyroid hormone in a PTH/PTHrP receptor-mediated manner to express the fully differentiated hypertrophic phenotype. In addition, the hormone stimulates hypertrophic development even more powerfully through its carboxyterminal domain, presumably by interaction with receptors distinct from PTH/PTHrP receptors. Therefore, PTH can support late cartilage differentiation at very advanced stages, whereas the same signal negatively controls the process at earlier stages.

Somites are transient mesodermal structures giving rise to all skeletal muscles of the body, the axial skeleton and the dermis of the back. Somites arise from successive segmentation of the presomitic mesoderm (PSM). They appear first as epithelial spheres that rapidly differentiate into a ventral mesenchyme, the sclerotome, and a dorsal epithelial dermomyotome. The sclerotome gives rise to vertebrae and ribs while the dermomyotome is the source of all skeletal muscles and the dorsal dermis. Quail-chick fate mapping and diI-labeling experiments have demonstrated that the epithelial somite can be further subdivided into a medial and a lateral moiety. These two subdomains are derived from different regions of the primitive streak and give rise to different sets of muscles. The lateral somitic cells migrate to form the musculature of the limbs and body wall, known as the hypaxial muscles, while the medial somite gives rise to the vertebrae and the associated epaxial muscles. The respective contribution of the medial and lateral somitic compartments to the other somitic derivatives, namely the dermis and the ribs has not been addressed and therefore remains unknown. We have created quail-chick chimeras of either the medial or lateral part of the PSM to examine the origin of the dorsal dermis and the ribs. We demonstrate that the whole dorsal dermis and the proximal ribs exclusively originates from the medial somitic compartment, whereas the distal ribs derive from the lateral compartment.

Objective: To observe the effects of silks on attachment, shape and function of chondrocytes cultured in vitro., Methods: The silks from silk worm cocoons were digested by trypsin and coated with polylactic acid to from three dimensional scaffolds for rabbit rib chondrocyte culture. The growth and shape of chondrocytes were observed with phase contrast microscopy, scanning electron microscopy., Results: The chondrocytes were adhered to silks slowly after chondrocytes were seeded into silk scaffolds and cells fixed on silks well 1 or 2 days later. Cells began to proliferate after 3 days and multiplicative growth was observed on the 6th day. Microholes of silk scaffolds were filled with chondrocytes 2 weeks later. Scanning electron microscopy showed that there was a lot of extracellular matrix surrounding cells., Conclusion: Silks are ideal for attachment, growth and function maintenance of chondrocytes, and silks can be used as scaffolds for chondrocytes in three dimensional culture.

Objective: To investigate the technique of human tissue-engineered cartilage and to study the medical collagen membrane of guided tissue regeneration (GTR) as the carrier of in vitro chondrocytes culture., Methods: Tissue engineering technique was used to make human Tissue-engineered cartilage, which was examined histologically and functionally., Results: It was found that the chondrocytes seeded on the medical collagen membrane of GTR grew well. A layer of milk white and cartilage-like tissue grew on the surface of medical collagen membrane of GTR after 1 week. It was demonstrated that the cartilage-like tissue was strong enough to be transferred after being implanted for 8 weeks. The cartilage-like tissue was proved to be human tissue engineered cartilage by HE stain and Alcian blue-poncean S stain. The chondrocytes could secrete chondroitin sulfate as proved by Lev-Spicer stain., Conclusions: The medical collagen membrane of GTR has characteristics of three-dimensional structure and cell reticular function, and it has the possibility to be developed as a natural scaffold for tissue engineering. The results indicate that it is possible to make human tissue-engineered cartilage with tissue-engineering technique.

Background: Perichondrium is recognized as a tissue with chondrogenic potential yielding cells which can be used for osteochondral repair. Factors which influence the proliferative ability and chondrocytic phenotype of such cells include age and presence of specific growth factors, i.e. TGF-beta 1. The present in vitro study assessed proliferation and markers of chondrocytic phenotype in cells extracted from the rib perichondrium of four- to five-year-old aged rabbits, and assessed the effects of exogenously added TGF-beta 1 on those cells., Methods: Assays included 3H-thymidine incorporation (cell proliferation), 35S-sulfate incorporation (proteoglycan synthesis) and quantitative RT-PCR for determination of type II collagen gene expression., Results: The results demonstrated that addition of TGF-beta 1 to the culture media stimulated thymidine incorporation and proteoglycan synthesis up to four- and five-fold, respectively, in aged perichondrium-derived cells. Moreover, the exogenous addition of TGF-beta 1 to the culture media resulted in an upregulation of transcriptional expression of the type II collagen gene., Conclusions: In summary, the present study has demonstrated that exogenously added TGF-beta 1 can stimulate proliferation and chondrocytic phenotype in aged perichondrium-derived cells in vitro.

In contrast to articular cartilage the hyaline rib cartilage takes up a special position due to its size, shape and the kind of mechanical stress as well. These facts may influence the metabolism of rib cartilage. In our histological, histochemical and immunohistochemical investigations on pieces of rib cartilages of 34 persons at the age of fourth fetal month up to 60 years we could regularly demonstrate cartilage canals containing blood vessels without any spatial or temporal relationship to degenerative changes in cartilage tissue. Many of these cartilage canals are located in the center of the rib cartilage. Blood vessels as well as neuronal structures in the connective tissue of cartilage canals were detected by means of antibodies against components of the vessel wall (Von Willebrand factor) and nerve fibers (PGP 9.5). Nerves may have sensoric or vasomotoric functions as well, and they may influence cell differentiation and regeneration processes, respectively. Cartilage can not be regarded as vascularized like other tissues, but cartilage canals may have great functional importance for the metabolism of rib cartilage.

To date, many efforts to engineer cartilage have focused on matrix construction with the goal of producing a durable construct as cartilage replaces the resorbing matrix. However, the importance of matrix construction is at least matched by the challenge of efficient chondrocyte extraction, culture expansion, and prevention of dedifferentiation. This challenge is underscored by the large number of chondrocytes needed for a clinically significant construct such as an ear. Because human rib provides a large, readily available source of hyaline cartilage, the authors evaluated human rib chondrocyte extraction and found that maximum viable cell yield occurred after a 6-hour digestion. They also evaluated human microtic auricular remnant chondrocyte extraction and identified fibroblast contamination as a shortcoming of this potential source of chondrocytes. Initially, rib chondrocytes proliferated in vitro with a doubling time of approximately 1 week. As the cells were passaged, proliferation decreased such that the cells stopped proliferating and adopted a large, spindle-shaped morphology by passage 6. Interestingly, no increase in proliferation was noted when rib chondrocytes were stimulated with transforming growth factor beta 1, bone morphogenetic protein 2, and basic fibroblast growth factor. The major obstacles to the use of autologous rib chondrocytes in matrix construction are the low cell yield from a small piece of rib and the limited proliferation that these cells will undergo in vitro. Further investigation of culture systems and mitogenic cytokines may help resolve these limitations.