A computerized mechanical cell stimulator for tissue culture: Effects on skeletal muscle organogenesis

Summary A tissue culture system has been developed which can mechanically stimulate cells growing on a highly elastic plastic substratum in a 24-well cell growth chamber. The collagen-coated substratum to which the cells attach and grow in the Mechanical Cell Stimulator (Model I) can be repetitively stretched and relaxed by stepper motor with linear accuracy of 30 μm. The activity controlling unit is an Apple IIe computer interfaced with the cell growth chamber via optical data links and is capable of simulating many of the mechanical activity patterns that cells are subjected to in vivo. Primary avian skeletal myoblasts proliferate and fuse into multinucleated myotubes in this set-up in a manner similar to normal tissue culture dishes. Under static culture conditions, the muscle cells differentiate into networks of myotubes which show little orientation. Growing the proliferating muscle cells on a unidirectional stretching substratum causes the developing myotubes to orient parallel to the direction of movement. In contrast, growing the cells on a substratum undergoing continuous stretch-relaxation cycling orients the developing myotubes perpendicular to the direction of movement. Neither type of mechanical activity significantly affects the rate of cell proliferation of the rate of myoblast fusion into myotubes. These results indicate that during in vivo skeletal muscle organogenesis, when substantial mechanical stresses are placed on skeletal muscle cells by both continuous bone elongation and by spontaneous contractions, only bone elongation plays a significant role in proper fiber orientation for subsequent functional work. Supported by grants NS16753, AR36266, and RR05818 from the National Institutes of Health, Bethesda, MD.     

Induced Formation and Maturation of Acetylcholine Receptor Clusters in a Defined 3D Bio-Artificial Muscle

Dysfunction of the neuromuscular junction is involved in a wide range of muscular diseases. The development of neuromuscular junction through which skeletal muscle is innervated requires the functional modulation of acetylcholine receptor (AchR) clustering on myofibers. However, studies on AchR clustering in vitro are mostly done on monolayer muscle cell culture, which lacks a three-dimensional (3D) structure, a prominent limitation of the two-dimensional (2D) system. To enable a better understanding on the structure–function correlation underlying skeletal muscle innervation, a muscle system with a well-defined geometry mimicking the in vivo muscular setting is needed. Here, we report a 3D bio-artificial muscle (BAM) bioengineered from green fluorescent protein-transduced C3H murine myoblasts as a novel in vitro tissue-based model for muscle innervation studies. Our cell biological and molecular analysis showed that this BAM is structurally similar to in vivo muscle tissue and can reach the perinatal differentiation stage, higher than does 2D culture. Effective clustering and morphological maturation of AchRs on BAMs induced by agrin and laminin indicate the functional activity and plasticity of this BAM system toward innervation. Taken together, our results show that the BAM provides a favorable 3D environment that at least partially recapitulates real physiological skeletal muscle with regard to innervation. With a convenience of fabrication and manipulation, this 3D in vitro system offers a novel model for studying mechanisms underlying skeletal muscle innervation and testing therapeutic strategies for relevant nervous and muscular diseases.     

Monocytes/macrophages express chemokine receptor CCR9 in rheumatoid arthritis and CCL25 stimulates their differentiation

Introduction  

Monocytes/macrophages accumulate in the rheumatoid (RA) synovium where they play a central role in inflammation and joint destruction. Identification of molecules involved in their accumulation and differentiation is important to inform therapeutic strategies. This study investigated the expression and function of chemokine receptor CCR9 in the peripheral blood (PB) and synovium of RA, non-RA patients and healthy volunteers.     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

Mechanical stimulation of skeletal muscle increases prostaglandin F2α production,cyclooxygenase activity,and cell growth by a pertussis toxin sensitive mechanism

Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increased the long-term production of prostaglandin F_2α, an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical stimulation, the enzymatic activity of cyclooxygenase (prostaglandin GH synthase [PGHS]), a regulatory enzyme in prostaglandin synthesis, was increased 82% (P <.005), and this increase was maintained for at least 24 h. Kinetic analysis of stretch-activated cyclooxygenase activity indicated a two to threefold decrease in the enzyme's K_m, with little change in its V_max. Immunocytochemical analysis of the cell cultures indicated the presence of high levels of the mitogen-inducible isoform of cyclooxygenase (PGHS-2) in the skeletal myofibers compared to the interstitial fibroblasts. While the stretch-induced increase in cyclooxygenase enzymatic activity was not inhibited by tetrodotoxin and therefore was independent of cellular electrical activity, the G protein inhibitor pertussis toxin prevented stretch-induced cyclooxygenase activation. Pertussis toxin also inhibited stretch-induced increases in PGF_2α production, phospholipase D activation, and cell growth. It is concluded that stretch of skeletal muscle increases muscle cell growth through a G protein-dependent process involving the activation of cyclooxygenase, an immediate early gene product. © 1995 Wiley-Liss, Inc.