Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis.

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

Two Cases of Intraosseous Pseudomyogenic (Epithelioid Sarcoma-Like) Hemangioendothelioma With Unusual Features, Expanding the Clinicopathological Spectrum.

Pseudomyogenic (epithelioid sarcoma-like) hemangioendothelioma (PMHE) is a distinct vascular neoplasm mostly observed in the lower extremities of young adults with characteristic histopathological features. In this article, we present 2 unusual cases of PMHE. Case 1: A 28-year-old male presented with pain and stiffness in his left shoulder. Radiologic examination revealed an expansile, lytic, heterogeneously enhancing, destructive lesion in his left scapula, along with multiple avid marrow lesions in his other bones. Biopsy revealed a cellular tumor composed of plump spindly and epithelioid cells arranged in fascicles and focally, in clusters, admixed with neutrophils and histiocytes, with interspersed many osteoclast-like giant cells and reactive bone. Case 2: A 63-year-old male presented pain and swelling in his right middle finger, with no other lesion elsewhere in his body. Radiologic imaging disclosed a 1.5-cm-sized lobulated, expansile, lytic, destructive lesion in the distal end of the third metacarpal bone of his right hand. Biopsy examination revealed a cellular spindle cell tumor, composed of plump spindly cells, arranged in fascicles, including "tadpole-like" or "strap-like" cells and interspersed inflammatory cells. Immunohistochemically, tumor cells in both cases were positive for AE1/AE3, CD31, and Fli1, while negative for desmin and CD34. INI11 was retained. The presented cases of PMHE, occurring at unusual locations, in an older individual in the second case, along with variable histopathological features, noted in the first case, seem to expand the clinicopathological spectrum of these uncommon neoplasms. Differential diagnoses and review of similar cases are presented.

A cancellous bone matrix system with specific mineralisation degrees for mesenchymal stem cell differentiation and bone regeneration.

Bone regenerative therapies have been explored using various biomaterial systems. Notably, collagen biomineralisation is believed to be essential for promoting bone regeneration. However, ideal bone repair materials with an appropriate mineralised matrix, superior osteogenic activity with early vascularisation, and recellularisation properties are still needed. This study aimed to develop a method to subject the decellularised cancellous bone matrix (DCBM) to ultrasound to obtain specific demineralisation to investigate the effects of DCBM with different degrees of mineralisation on proliferation and osteogenic differentiation in bone marrow-derived mesenchymal stem cells (BMSCs) and in repairing femoral bone defects in rabbits. We established an optimised native DCBM mineralisation ECM scaffold for bone regeneration. Upon complete decellularisation of the cancellous bone matrix, DCBMs with specific degrees of mineralisation were obtained. We comprehensively evaluated their bioactive components, minimal immunogenicity, ultra-micro-structural mechanical properties, and degree of mineralisation. Furthermore, specific mineralised DCBMs (obtained by low-temperature rapid ultrasound for 4 and 8 h) had prominent effects in promoting the osteogenic differentiation of BMSCs in vitro. Moreover, more newly formed trabeculae, vessels, and endochondral bone were also detected in the aforementioned groups during early-stage bone repair in vivo. The underlying mechanism might be mineralisation-related regulation and ultra-micro-structural mechanical properties. Thus, the present study shows that specific demineralised DCBM obtained under optimal conditions had superior properties to those of unmineralised or completely demineralised DCBM by promoting MSC osteogenic differentiation and initiating endochondral bone formation and de novo osteogenesis.

The influence of a depressed scapular alignment on upper limb neural tissue mechanosensitivity and local pressure pain sensitivity.

BACKGROUND: A depressed scapular alignment could lead to prolonged and repetitive stress or compression of the brachial plexus, resulting in sensitization of neural tissue. However, no study has investigated the influence of alignment of the scapulae on sensitization of upper limb neural tissue in otherwise asymptomatic people. In this case-control study, we investigate the influence of a depressed scapular alignment on mechanosensitivity of the upper limb peripheral nervous system as well as pressure pain thresholds (PPT). METHODS: Asymptomatic individuals with neutral vertical scapular alignment (n = 25) or depressed scapular alignment (n = 25) participated. We measured the upper limb neurodynamic test (ULNT1), including assessment of symptom response and elbow range of motion (ROM), and PPT measured over upper limb peripheral nerve trunks, the upper trapezius muscle and overlying cervical zygapophyseal joints. RESULTS: Subjects with a depressed scapular reported significantly greater pain intensity (t = 5.7, p < 0.0001) and reduced elbow extension ROM (t = -2.7, p < 0.01) during the ULNT1 compared to those with a normal scapular orientation. Regardless of the location tested, the group presenting with a depressed scapular had significantly lower PPT compared to those with a normal scapular orientation (PPT averaged across all sites: normal orientation: 3.3 ± 0.6 kg/cm2, depressed scapular: 2.1 ± 0.5 kg/cm2, p < 0.00001). CONCLUSIONS: Despite being asymptomatic, people with a depressed scapular have greater neck and upper limb neural tissue mechanosensitivity when compared to people with a normal scapular orientation. This study offers insight into the potential development of neck-arm pain due to a depressed scapular position.

Commitment of chondrogenic precursors of the avian scapula takes place after epithelial-mesenchymal transition of the dermomyotome.

BACKGROUND: Cells of the epithelially organised dermomyotome are traditionally believed to give rise to skeletal muscle and dermis. We have previously shown that the dermomyotome can undergo epithelial-mesenchymal transition (EMT) and give rise to chondrogenic cells, which go on to form the scapula blade in birds. At present we have little understanding regarding the issue of when the chondrogenic fate of dermomyotomal cells is determined. Using quail-chick grafting experiments, we investigated whether scapula precursor cells are committed to a chondrogenic fate while in an epithelial state or whether commitment is established after EMT. RESULTS: We show that the hypaxial dermomyotome, which normally forms the scapula, does not generate cartilaginous tissue after it is grafted to the epaxial domain. In contrast engraftment of the epaxial dermomyotome to the hypaxial domain gives rise to scapula-like cartilage. However, the hypaxial sub-ectodermal mesenchyme (SEM), which originates from the hypaxial dermomyotome after EMT, generates cartilaginous elements in the epaxial domain, whereas in reciprocal grafting experiments, the epaxial SEM cannot form cartilage in the hypaxial domain. CONCLUSIONS: We suggest that the epithelial cells of the dermomyotome are not committed to the chondrogenic lineage. Commitment to this lineage occurs after it has undergone EMT to form the sub-ectodermal mesenchyme.

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Dual origin and segmental organisation of the avian scapula.

Bones of the postcranial skeleton of higher vertebrates originate from either somitic mesoderm or somatopleural layer of the lateral plate mesoderm. Controversy surrounds the origin of the scapula, a major component of the shoulder girdle, with both somitic and lateral plate origins being proposed. Abnormal scapular development has been described in the naturally occurring undulated series of mouse mutants, which has implicated Pax1 in the formation of this bone. Here we addressed the development of the scapula, firstly, by analysing the relationship between Pax1 expression and chondrogenesis and, secondly, by determining the developmental origin of the scapula using chick quail chimeric analysis. We show the following. (1) The scapula develops in a rostral-to-caudal direction and overt chondrification is preceded by an accumulation of Pax1-expressing cells. (2) The scapular head and neck are of lateral plate mesodermal origin. (3) In contrast, the scapular blade is composed of somitic cells. (4) Unlike the Pax1-positive cells of the vertebral column, which are of sclerotomal origin, the Pax1-positive cells of the scapular blade originate from the dermomyotome. (5) Finally, we show that cells of the scapular blade are organised into spatially restricted domains along its rostrocaudal axis in the same order as the somites from which they originated. Our results imply that the scapular blade is an ossifying muscular insertion rather than an original skeletal element, and that the scapular head and neck are homologous to the 'true coracoid' of higher vertebrates.

Isolation and characterization of an inhibitor of neovascularization from scapular chondrocytes.

An inhibitor of neovascularization from the conditioned media of scapular chondrocytes established and maintained in serum-free culture has been isolated and characterized. To determine whether this chondrocyte-derived inhibitor (ChDI) was capable of inhibiting neovascularization in vivo, this protein was assayed in the chick chorioallantoic membrane assay. ChDI was a potent inhibitor of angiogenesis in vivo (4 micrograms = 87% avascular zones). This inhibitor is also an inhibitor of fibroblast growth factor-stimulated capillary endothelial cell (EC) proliferation and migration, as well as being an inhibitor of mammalian collagenase. ChDI significantly suppressed capillary EC proliferation in a dose-dependent, reversible manner with an IC50 (the inhibitory concentration at which 50% inhibition is achieved) of 2.025 micrograms/ml. Inhibition by ChDI of growth factor-stimulated capillary EC migration was also observed using a modified Boyden chamber assay (IC50 = 255 ng/ml). SDS-PAGE analysis followed by silver staining of ChDI purified to apparent homogeneity revealed a single band having an M(r) of 35,550. Gel elution experiments demonstrated that only protein eluting at this molecular weight was anti-angiogenic. These studies are the first demonstration that chondrocytes in culture can produce a highly enriched, potent inhibitor of neovascularization which also inhibits collagenase.

Difference in size of bone islands formed by isolated bone cells transplanted intramuscularly under various conditions.

Bone cells isolated from the whole calvaria (2 x 10(6)) from either central or peripheral parts of parietal bones (1 x 10(6)) and from scapulas (2 x 10(6)) were allowed to adhere to devitalized calvarial bones in the number indicated in brackets and transplanted intramuscularly (supported transplants). Whole calvaria bone cells (2.4 or 8 x 10(6) cells per transplant) were also injected intramuscularly as free transplants. Calvarial cells produced solid bone islands with small intraosseous cavities, while bone formed by scapular cells contained large medullary spaces. The size of bone islands formed in transplants and the shortest distance between the neighbor islands were measured. The results of these measurements were similar in all groups of free transplants. The size of bone islands formed in supported transplants of cells from the whole calvaria or from central and peripheral parts of parietal bones was also roughly similar, but the shortest distance between islands was larger than in the free transplants. Furthermore, in these groups of transplants bone islands considerably larger than the largest islands in free transplants were present. Scapular bone cells formed islands much larger than those produced by calvarial cells. Bone islands formed by calvarial cells in free transplants were separated by bands of fibrous tissue which was absent in supported transplants. It appears that this tissue could limit growth and/or fusion of neighbor bone islands and in this manner influence their size. The population of transplanted scapular cells contained numerous stromal elements which could form an exclusion area inaccessible to local cells from the site of transplantation and thus favour formation of large bone islands within this area.

Difference in shape of bone formed by isolated calvarial and scapular osteoblasts transplanted under various conditions.

To study the influence of transplantation conditions on early stages of osteogenesis, isolated calvarial or scapular osteoblasts were injected into the leg or dorsal muscles (free transplants) or implanted after seeding on fragments of devitalized parietal bones (supported transplants) into dorsal muscles. The cross-sections of bone islands formed by calvarial osteoblasts in the different types of transplants were then compared according to their maximal breadth and length. Moreover, the same dimensions of pieces of bone formed by scapular osteoblasts in supported transplants were compared with those of bones formed in free transplants into leg muscles. Finally, comparison of the dimensions of cross-sections of supported transplants of calvarial and scapular osteoblasts was done. Calvarial osteoblasts in dorsal muscles produced a slightly higher percentage of wider and longer islands than those in leg muscles. In supported transplants of calvarial osteoblasts the percentage of narrow bone islands (breadth less than 100 microns) was considerably higher than in free transplants. Similarly, the percentage of narrow cross-sections in bones formed by scapular osteoblasts was higher in supported than in free transplants. In supported transplants of calvarial osteoblasts the percentage of narrow islands was higher than in similar transplants of scapular bone cells. It is suggested that the differences in shape of pieces of bone formed in supported and free transplants reflect the difference in mechanical conditions to which the bone cells were subjected. Furthermore, in supported transplants devitalized parietal bones could form a barrier for diffusion of nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)

Organization of extracellular matrix in epiphyseal growth plate.

Three cations of varying size and charge density, egg-white lysozyme, protamine and ruthenium red, were used to stain the extracellular matrix of epiphyseal cartilage growth plate. With these stains, it was possible to distinguish three types of proteoglycans or materials associated with them, which may well have as their major differences the type of cross linking to the tissue. One type was stained by ruthenium red and protamine but not by lysozyme, was extractable with 3 M guanidinium chloride and was relatively uniformly dispersed throughout the matrix of the growth plate. The other two types were stained by all three cations, were not extractable with 3 M guanidinium chloride and were intimately associated with fibrils. One of these was found on the collagen fibrils, was relatively scanty in the resting zone near the articular surface, relatively restricted to the extralacunar area in the columnar zones and appeared to diminish in amount in the hypertrophic zone. This material often had a 640-A periodic array on the surface of collagen fibrils. The third type also was stained by all three cations and was not extractable with 3 M guanidinium chloride. It was distinguished from the other class of lysozyme-reactive matrix components by the larger volume of distribution occupied by the stained material. It also had a different distribution in that it was widely dispersed in the resting zone, was restricted to the lacuna in the columnar zone and was absent in the hypertrophic zone. Thus, cartilage matrix as well as the chondrocytes undergo differentiation in the epiphyseal growth plate.