Architecture of implanted bone matrix gelatin influences heterotopic calcification and new bone formation

Heterotopic bone formation in skeletal muscle induced by compacted demineralized bone matrix gelatin (BMG) was studied histologically and biochemically. BMG was obtained by dehydrating diaphyseal shafts of femora and tibiae of 4-week-old male Sprague-Dawley rats, cutting the bone into chips, and demineralizing and extracting the chips with various solutions. The BMG was treated with 4 M guanidine-HCl, and compacted BMG was prepared by centrifugation. The compacted BMG was implanted into the rectus abdominis muscle of 5-week-old male Sprague-Dawley rats. The resulting specimens were examined histologically, and their alkaline phosphatase activity and the calcium content of the tissues were measured 3, 5, 7, 10, and 15 days after implantation. The BMG (separated BMG) with 75- to 500-microns particle sizes were implanted into control rats. The results showed that calcification, alkaline phosphatase activity, and bone formation were suppressed by implantation of the compacted BMG and that scarcely any vascularization occurred. Calcification, vascularization, and alkaline phosphatase activity were related and were indispensable for bone formation. In the control group, bone formation was observed at sites of high activity of alkaline phosphatase and well-developed vascularization. These results suggested that compacting of BMG suppressed vascularization, decreased calcification, and consequently reduced the induction of bone formation.     

Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody

A novel monoclonal antibody, SM/C-2.6, specific for mouse muscle satellite cells was established. SM/C-2.6 detects mononucleated cells beneath the basal lamina of skeletal muscle, and the cells co-express M-cadherin. Single fiber analyses revealed that M-cadherin+ mononucleated cells attaching to muscle fibers are stained with SM/C-2.6. SM/C-2.6+ cells, which were freshly purified by FACS from mouse skeletal muscle, became MyoD+ in vitro in proliferating medium, and the cells differentiated into desmin+ and nuclear-MyoD+ myofibers in vitro when placed under differentiation conditions. When the sorted cells were injected into mdx mouse muscles, donor cells differentiated into muscle fibers. Flow cytometric analyses of SM/C-2.6+ cells showed that the quiescent satellite cells were c-kit-, Sca-1-, CD34+, and CD45-. More, SM/C-2.6+ cells were barely included in the side population but in the main population of cells in Hoechst dye efflux assay. These results suggest that SM/C-2.6 identifies and enriches quiescent satellite cells from adult mouse muscle, and that the antibody will be useful as a powerful tool for the characterization of cellular and molecular mechanisms of satellite cell activation and proliferation.     

Demonstration of bone resorbing cells in elasmobranchs: Comparison with osteoclasts

We have previously shown that Elasmobranchs-characterized by a partially calcified cartilaginous endoskeleton-presented a bony vertebral arch containing osteoblasts, osteocytes and resorbing cells. The aim of this study is to test the ability of Elasmobranchs to resorb bone tissue. The subcutaneous implantation in dogfish (Scyliorhinus canicula) of devitalized mineral-containing bone particles, obtained from a bony fish (the eel, Anguilla anguilla) resulted, after 21 2 months, in the formation of mononucleated as well as multinucleated cells around and between the bone fragments. By light microscopy, the multinucleated giant cells presented the general aspect of osteoclastic cells whereas, by transmission electron microscopy they never showed ruffled borders which are considered as the typical features of osteoclasts. Except for this character, the mononucleated and multinucleated cells exhibited the typical ultrastructural aspects leading us to say that these cells are involved in the resorption of the bone fragments. This study shows that Elasmobranchs are able to resorb implanted bone.     

Proliferation of muscle satellite cells on intact myofibers in culture

Muscle satellite cells are quiescent myogenic stem cells situated between the basal lamina and plasmalemma of mature skeletal muscle fibers. Injury to the fiber triggers the activation and proliferation of satellite cells whose progeny subsequently fuse to form new myotubes during regeneration. In this paper we report the proliferation of satellite cells on single muscle fibers isolated from adult rats and placed in culture. Viable fibers were liberated from muscle with collagenase and purified from non-muscle cells. The fibers were covered with a basal lamina and retained normal morphological characteristics. Each fiber contained two to three satellite cells per 100 myonuclei. Satellite cells showed little proliferative activity in medium with 10% serum but could be induced to enter the cell cycle by chick embryo extract or fibroblast growth factor. Other polypeptide mitogens such as epidermal growth factor, multiplication stimulating activity, and platelet-derived growth factor were ineffective. Mitogen-stimulated satellite cells fused to form new myotubes after 4-5 days in culture. These results imply that satellite cells are under positive growth control since they proliferate in contact with viable mature fibers when stimulated with mitogen. The mature fibers remained viable in culture but did not give rise to mononucleated cells. After several days, however, the fibers began to extend sarcoplasmic sprouts and underwent dedifferentiative changes that led to the formation of multinucleated cells resembling myotubes. These cells reexpressed embryonic isozymes of creatine kinase not made by the mature fibers.     

Expression of myostatin gene in regenerating skeletal muscle of the rat and its localization

The expression of myostatin mRNA was examined in regenerating skeletal muscle of the rat. Skeletal muscle regeneration was induced by injecting bupivacaine or hypertonic saline solution into the femoral muscle, and the tissues were collected 48 h after the treatment. In situ hybridization analysis revealed that the cells positive for myostatin message were localized in the regenerating area of the bupivacaine-treated tissues, where a numerous number of mononucleated cells were present. The myostatin-positive mononucleated cells contained both myogenic and nonmyogenic cells, as revealed by immunohistochemical staining for desmin and vimentin. Bupivacaine treatment to the testes resulted in no myostatin message expression in the testicular vimentin-positive cells, suggesting that the expression of myostatin message in vimentin-positive cells is a skeletal muscle-specific phenomenon. Furthermore, crushed muscle extract prepared from regenerating skeletal muscle had induced myostatin mRNA expression in skeletal muscle-derived fibroblasts in a dose-dependent manner. These results indicated that myostatin is expressed during skeletal muscle regeneration both in myogenic and nonmyogenic cells, and suggested that some factor(s) capable of inducing myostatin expression in fibroblasts are present in regenerating skeletal muscle.     

Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells.

The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that greater than 94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2'-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivacaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reptilian myotomal myogenesis-lessons from the sand lizard Lacerta agilis L. (Reptilia, Lacertidae)Update

Reptilian myotomal myogenesis is poorly understood. This paper reports on structural, ultrastructural and immunocytochemical studies of muscle differentiation in sand lizard (Lacerta agilis) embryos. During somitogenesis, the somites are composed of epithelial vesicles with a centrally located somitocoel. At later developmental stages the ventral portion of the somite cortex disaggregates into the sclerotome mesenchyme, while the dorsal wall of the somite differentiates into dermomyotome. At these developmental stages, mononucleated cells of the dermomyotome are Pax3-positive. The dermomyotome layer forms the dorsomedial and ventromedial lips. The myotome is first composed of mono- and then of multinucleated myotubes and small mononucleated cells that occur in the vicinity of the myotubes. These mononucleated cells exhibit low proliferative potential as revealed by the use of PCNA antibody. At subsequent stages of myogenesis the mononucleated cells express Pax7 protein, a marker of satellite cells, and assume ultrastructural features characteristic of satellite cells. Some of the mononucleated cells contribute to muscle growth, being involved in fusion with differentiating muscle fibers. This study revealed similarities of myotomal myogenesis in reptiles to that of other vertebrates.     

Appearance of adipose cells in connective tissue at the implantation site of bone matrix gelatin and bupivacaine injection.

Two types of adipose cells were found in the connective tissue on day 7 after bone matrix gelatin (BMG) implantation and an injection of bupivacaine: mature adipose cells with a large lipid droplet (2-140 microns) and immature adipose cells with many small lipid droplets (0.1-2 microns). On day 10 after BMG implantation, typical adipose tissue was observed near the implant. The immature adipose cells had small, spherical mitochondria, glycogen granules and cytoplasmic microvesicles, and they might differentiate from undifferentiated mesenchymal cells in the connective tissue or the peripheral cells around the vessels as a white adipose tissue. These findings suggest that the differentiation of adipose cells in the connective tissue near heterotopic bone formation might be induced not only by mechanical and/or bupivacaine injury, but also by some factor or factors of the BMG.     

Analysis of expression of heavy myosin chains during in vitro differentiation of satellite cells and myoblasts derived from rat skeletal muscles

Differentiation of cultured myogenic progenitor cells (satellite cells and mononucleated myoblasts) derived from hindlimb muscles of rat embryos and newborn animals was studied. Immunocytochemical methods and PCR analysis revealed expression of heavy myosin chains at the earliest stages of myogenesis (in mononucleated myoblasts). Expression of the gene encoding the embryonic form of myosin and a low level of expression of the gene encoding perinatal myosin in cultured progenitor cells derived from embryonic muscles was detected by PCR. Cells derived from muscles of newborn animals also expressed these two myosin forms, though at a lower level. The progenitor cells derived from muscles of rat embryos and newborn animals were found to express myosin 2a, which is characteristic of fast-twitch definitive muscle fibers.     

Phosphatase activity in the myenteric plexus

Intense and very intense reactions were obtained for acid phosphatase, calcium activated ATP-ase (pH 9.4), magnesium activated ATP-ase (pH 7.2) and glucose-6-phosphatase in the cytoplasms of the myenteric plexus nerve cells of the small intestine of Macacus rhesus and rabbit. Nucleotidase activity was moderate or slight and unspecific alkaline phosphatase activity absent. Both ATP-ases presented an intense activity in the myenteric plexus nerve cells of human fetuses 30, 33, and 34 weeks old; 5-nucleotidase activity, slight in the 30-week-old fetuses became more intense in the 33- and 34-week-old fetuses. The satellite neuroglial cells, nerve fibers and blood capillaries presented negative alkaline phosphatase reactions and intense or very intense activities of the other phosphatases.     

Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury

Adult bone marrow-derived cells (BMDC) are shown to contribute to muscle tissue in a step-wise biological progression. Following irradiation-induced damage, transplanted GFP-labeled BMDC become satellite cells: membrane-ensheathed mononucleate muscle stem cells. Following a subsequent exercise-induced damage, GFP-labeled multinucleate myofibers are detected. Isolated GFP-labeled satellite cells are heritably myogenic. They express three characteristic muscle markers, are karyotypically diploid, and form clones that can fuse into multinucleate cells in culture or into myofibers after injection into mouse muscles. These results suggest that two temporally distinct injury-related signals first induce BMDC to occupy the muscle stem cell niche and then to help regenerate mature muscle fibers. The stress-induced progression of BMDC to muscle satellite cell to muscle fiber results in a contribution to as many as 3.5% of muscle fibers and is due to developmental plasticity in response to environmental cues.     

Isolation and Biological Characteristics of Beijing Fatty Chicken Skeletal Muscle Satellite Cells

Abstract

Skeletal muscle satellite cells, a postulated multipotential stem cell population, play an essential role in the postnatal replenishment of skeletal muscles. In the present research, the skeletal muscle satellite cells were isolated from the pectorals of 15-day-old Beijing Fatty Chicken embryos using combined enzymatic digestion of 0.1% collagenase 1 and 0.25% trypsin. Myogenic markers such as MyoD, Pax7 and demin were detected, indicating their skeletal muscle satellite cell identity. Karyotype analysis showed that these in vitro cultured cells were genetically stable. Being exposed to bone morphogen and adipogenic factors, it was proved that they differentiated into osteocytes and adipocytes correspondingly.     

骨髓间充质干细胞成肌分化在骨骼肌再生中的应用

骨骼肌良好的再生能力是由于肌卫星细胞的存在,然而肌卫星细胞的数量仅占骨骼肌细胞数量的1%~ 5%,当肌肉损伤时,仅依靠这些卫星细胞还不足以促进骨骼肌修复与再生,并且这种再生能力会随着年龄的增大而衰减,并不能修复损伤严重的骨骼肌。骨髓间充质干细胞（BMSC）因其多向分化潜能,旁分泌潜能,免疫调节能力及容易获取等特点广泛用于损伤骨骼肌的修复与再生。但在某种程度上,仅仅采用BMSC治疗损伤的骨骼肌仍不能达到满意的效果。因此,大量研究采用药物、生物材料、细胞及细胞因子对BMSC进行预处理不仅可改善它的移植率,还可显著促进其向骨骼肌分化,从而最大限度的发掘骨骼肌间充质干细胞的成肌分化潜能以促进骨骼肌的修复。因此,本篇综述旨在概括BMSC成肌分化在骨骼肌再生中的应用。     

Estrogen influences satellite cell activation and proliferation following downhill running in rats.

To investigate the influence of estrogen on postexercise muscle repair processes, we examined the effects of estrogen supplementation (0.25-mg pellet) on numbers of myofibers positive for markers of total, activated, and proliferating satellite cells in rat skeletal muscles 72 h following downhill running. Ovariectomized female rats (n = 44) were divided into four groups (n = 11 per group): sham (no estrogen) controls (SC); sham, exercised (SE); estrogen-supplemented controls (EC); and estrogen-supplemented, exercised (EE). After 8 days of estrogen exposure, animals were exposed to 90 min of treadmill running at 17 m/min (-13.5 degrees ). Seventy-two hours later, soleus and white vastus muscles were removed and immunostained for total [paired box homeotic gene 7 (Pax7)], [activated myogenic differentiation factor D (MyoD)], and proliferating [5-bromo-2'-deoxyuridine (BrdU)] satellite cells. beta-Glucuronidase activity was increased (P < 0.05) in both muscles following exercise; however, the postexercise elevations in enzyme activity were attenuated in the EE group compared with the SE group in the soleus (P < 0.05). Immunohistochemical analysis revealed that exercised groups displayed increased numbers of myofibers containing total, activated, and proliferating satellite cells compared with control groups (P < 0.05). Furthermore, greater numbers of fibers positive for markers of total, activated, and proliferating satellite cells were observed postexercise in EE animals compared with SE animals for both muscles (P < 0.05). The results demonstrate that estrogen may potentially influence post-damage repair of skeletal muscle through activation of satellite cells.     

Effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone tissue

The effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone in guinea pig was studied. Heterotopic bone formation was induced by transplantation of allogenic urinary bladder epithelium. Starting from the day of transplantation the animals were treated subcutaneously with HEBP and Cl2MBP with a dose of 12.5 mg P/kg/day during 35 days. The control group was injected with 0.9% NaCl solution. The advantage of heterotopic bone induction as an experimental model is the fact that the applied drugs act on de novo bone formation. Collagen fibers were treated as markers of bone because their size and spatial arrangement reflect the structure and maturity of organic matrix of this tissue. Decalcified histological sections of induced bone, taken 35 days after implantation of inductor, were stained by the picrosirius method. This staining enhances the natural birefringency of collagen fibers and allows for better and specific visualization of collagen fibers bundles under polarizing microscope. In this way the amount of information in the analysed image is increased. Thirty five microphotographs were analysed from each of the investigated groups with the use of optical diffractometry. The radial distribution of light intensity in diffraction patterns was analysed what allowed to evaluate spatial frequencies connected with the width of collagen bundles in induced bone tissue. Since the spatial arrangement of collagen fibers in newly formed bone is random, analysis of angular distribution of light intensity in diffractograms was not performed. Using discriminant analysis the significant differences between all three studied groups of animals were found.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections.

We have developed a method for in situ hybridization of adult bone tissue utilizing undecalcified sections and have used it to histologically examine the mRNA expression of non-collagenous bone matrix proteins such as osteocalcin (bone Gla protein, BGP), matrix Gla protein (MGP), and osteopontin in adult rats. Expression was compared with that in bone tissues of newborn rats. In the adult bone tissue, osteocalcin mRNA was strongly expressed in periosteal and endosteal cuboidal osteoblasts but not in primary spongiosa near the growth plate. Osteopontin mRNA was strongly expressed in cells present on the bone resorption surface, osteocytes, and hypertrophic chondrocytes, but not in cuboidal osteoblasts on the formation surface. Osteopontin and osteocalcin mRNAs were expressed independently and the distribution of cells expressing osteopontin mRNA corresponded with acid phosphatase-positive mononuclear cells and osteoclasts. Expression of MGP mRNA was noted only in hypertrophic chondrocytes. In newborn rat bone tissues, expression of osteocalcin mRNA was much weaker than in adult rat bone tissues. These results clearly indicate the differential expression of mRNAs of non-collagenous bone matrix proteins in adult rat bone tissues.     

Cell-specific paracrine actions of IL-6 family cytokines from bone,marrow and muscle that control bone formation and resorption

Bone renews itself and changes shape throughout life to account for the changing needs of the body; this requires co-ordinated activities of bone resorbing cells (osteoclasts), bone forming cells (osteoblasts) and bone’s internal cellular network (osteocytes). This review focuses on paracrine signaling by the IL-6 family of cytokines between bone cells, bone marrow, and skeletal muscle in normal physiology and in pathological states where their levels may be locally or systemically elevated. These functions include the support of osteoclast formation by osteoblast lineage cells in response to interleukin 6 (IL-6), interleukin 11 (IL-11), oncostatin M (OSM) and cardiotrophin 1 (CT-1). In addition it will discuss how bone-resorbing osteoclasts promote osteoblast activity by secreting CT-1, which acts as a “coupling factor” on osteocytes, osteoblasts, and their precursors to promote bone formation. OSM, produced by osteoblast lineage cells and macrophages, stimulates bone formation via osteocytes. IL-6 family cytokines also mediate actions of other bone formation stimuli like parathyroid hormone (PTH) and mechanical loading. CT-1, OSM and LIF suppress marrow adipogenesis by shifting commitment of pluripotent precursors towards osteoblast differentiation. Ciliary neurotrophic factor (CNTF) is released as a myokine from skeletal muscle and suppresses osteoblast differentiation and bone formation on the periosteum (outer bone surface in apposition to muscle). Finally, IL-6 acts directly on marrow-derived osteoclasts to stimulate release of “osteotransmitters” that act through the cortical osteocyte network to stimulate bone formation on the periosteum. Each will be discussed as illustrations of how the extended family of IL-6 cytokines acts within the skeleton in physiology and may be altered in pathological conditions or by targeted therapies.     

The reaction of osteoclasts when releasing osteocytes from osteocytic lacunae in the bone during bone modeling

Osteocytes are released from the osteocytic lacunae when osteoclasts resorb the bone matrix during bone modeling and remodeling. It remains unknown how osteoclasts react when releasing osteocytes during bone modeling, and the fate of these released osteocytes is also unclear. Femoral mid-shafts of 2-day-old kittens were sectioned into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and examined by light microscopy (LM) and transmission electron microscopy (TEM). The sections showed many osteoclasts at the endosteum but there were no osteoblasts. There were many half-released, fully released, half-exposed, and fully exposed osteocytes on the bone surfaces. Many cell-like structures were seen in the cell bodies of osteoclasts by LM, and some semithin sections were re-sectioned into ultrathin sections for re-observation by TEM. By TEM, these were determinated to be mononuclear cells. The serial ultrathin sections showed that the mononuclear cells appeared to be engulfed in osteoclasts on one section but that the cell was connected with the bone surface of the osteocytic lacuna on another section. These results show that the mononuclear cells in the osteoclasts were osteocytes. The present study suggests that osteoclasts engulf some osteocytes but do not engulf others when releasing osteocytes during bone modeling.     

The phylogenetic origin and evolution of acellular bone in teleost fishes: insights into osteocyte function in bone metabolism

Vertebrate bone is composed of three main cell types: osteoblasts, osteoclasts and osteocytes, the latter being by far the most numerous. Osteocytes are thought to play a fundamental role in bone physiology and homeostasis, however they are entirely absent in most extant species of teleosts, a group that comprises the vast majority of bony ‘fishes’, and approximately half of vertebrates. Understanding how this acellular (anosteocytic) bone appeared and was maintained in such an important vertebrate group has important implications for our understanding of the function and evolution of osteocytes. Nevertheless, although it is clear that cellular bone is ancestral for teleosts, it has not been clear in which specific subgroup the osteocytes were lost. This review aims to clarify the phylogenetic distribution of cellular and acellular bone in teleosts, to identify its precise origin, reversals to cellularity, and their implications. We surveyed the bone type for more than 600 fossil and extant ray‐finned fish species and optimised the results on recent large‐scale molecular phylogenetic trees, estimating ancestral states. We find that acellular bone is a probable synapomorphy of Euteleostei, a group uniting approximately two‐thirds of teleost species. We also confirm homoplasy in these traits: acellular bone occurs in some non‐euteleosts (although rarely), and cellular bone was reacquired several times independently within euteleosts, in salmons and relatives, tunas and the opah (Lampris sp.). The occurrence of peculiar ecological (e.g. anadromous migration) and physiological (e.g. red‐muscle endothermy) strategies in these lineages might explain the reacquisition of osteocytes. Our review supports that the main contribution of osteocytes in teleost bone is to mineral homeostasis (via osteocytic osteolysis) and not to strain detection or bone remodelling, helping to clarify their role in bone physiology.