Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids

Three-dimensional skeletal muscle organ-like structures (organoids) formed in tissue culture by fusion of proliferating myoblasts into parallel networks of long, unbranched myofibers provide an in vivo-like model for examining the effects of growth factors, tension, and space flight on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commercial perfusion bioreactor system, we measured metabolism, protein turnover. and autocrine/paracrine growth factor release rates. Medium glucose was metabolized at a constant rate in both low-serum- and serum-free media for up to 30 d. Total organoid noncollagenous protein and DNA content decreased approximately 22-28% (P < 0.05) over a 13-d period. Total protein synthesis rates could be determined accurately in the bioreactors for up to 30 h and total protein degradation rates could be measured for up to 3 wk. Special fixation and storage conditions necessary for space flight studies were validated as part of the studies. For example, the anabolic autocrine/paracrine skeletal muscle growth factors prostaglandin F2alpha (PGF2alpha) and insulin-like growth factor-1 (IGF-1) could be measured accurately in collected media fractions, even after storage at 37 degrees C for up to 10 d. In contrast, creatine kinase activity (a marker of cell damage) in collected media fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.     

Anti-selectin therapy modifies skeletal muscle ischemia and reperfusion injury

Restoration of blood flow to ischemic skeletal muscle results in a reperfusion injury characterized by permeability edema in part mediated by neutrophils that adhere via the selectin family of adhesion molecules. Rats underwent 4 h of hindlimb tourniquet ischemia followed by 4 h reperfusion. The role of neutrophils was determined by rendering one group of animals neutropenic before ischemia. In additional experimental groups, selectins were blocked with either a soluble form of the selectin counter-receptor, sialyl-Lewis X (SLX) or a monoclonal antibody directed against P-selectin (PB1.3). Neutrophil depletion resulted in a 36.1% reduction in hindlimb permeability (p < .05). SLX reduced hindlimb permeability index (PI) 23.9% at 1 mg/kg and 36.1% at 10 mg/kg compared to a nonfucosylated oligosaccharide, sialyl-N-acetylactosamine (p < .05). SLX also reduced neutrophil sequestration by 48.6% (p < .05). PB1.3 reduced hindlimb injury by 26.5% (p < .05) but did not reduce leukosequestration. We interpret these data to indicate that ischemia and reperfusion lead to selectin-mediated neutrophil sequestration. The oligosaccharide SLX, while moderately effective in limiting neutrophil sequestration was as effective as neutrophil depletion in reducing hindlimb permeability. The lack of concordance between the ability of SLX and PB1.3 in limiting neutrophil sequestration and permeability indicate mechanisms of action of these two agents that are in addition to the blocking of adhesion.     

A DNA controlled-release coating for gene transfer: transfection in skeletal and cardiac muscle

In this paper we report a novel technique of DNA-polymer coating for gene transfer. A proprietary DNA polymer solution was used for thin-layer coating on a chromic gut suture as a model study. The coated sutures were characterized for physical properties such as coating thickness, mass of the DNA deposited on the suture, surface characteristics as determined by scanning electron microscopy, and in vitro DNA release characteristics under simulated physiologic conditions. The in vivo gene transfection using DNA-coated sutures was demonstrated in rat skeletal muscle and in canine atrial myocardium. A heat-stable human placental alkaline phosphatase (AP) plasmid was used as a marker gene. Incisions of 1 to 1.5 cm were made in the rat skeletal muscles or the canine atrial myocardium. The sites were closed with either the DNA-coated sutures or control sutures. Two weeks after the surgery, the tissue samples adjacent to the suture lines were retrieved and analyzed for AP activity. The DNA-coated sutures demonstrated a sustained release of the DNA under in vitro conditions, with an approximately 84% cumulative DNA release occurring in 26 days. An agarose gel electrophoresis of the DNA samples released from the suture demonstrated two bands, with the lower band corresponding to the input DNA (supercoiled). It seems that there was a partial transformation of the DNA from a supercoiled to an open circular form due to the polymer coating. The tissue sites, which received the DNA-coated sutures, demonstrated a significantly higher AP activity compared with the tissue sites that received control sutures. In the rat studies, the mean AP activity (square root of cpm/microgram protein) was 43.6 +/- 3.3 vs 20.6 +/- 2.1 (p = 0.001) at the control sites. Similarly, in the canine studies, the AP activity was 73.6 +/- 7.4 Vs 21.6 +/- 1.4 (p = 0.0009) at the control sites. Thus, our studies demonstrated a successful gene transfer using our DNA-polymer coating technique. This technique could be useful for coating sutures used in vascular and general surgery, and also for coating medical devices, such as stents, catheters, or orthopedic devices, to achieve a site-specific gene delivery.     

Progress, problems, and prospects for gene therapy in muscle

As the molecular defects that cause many muscle diseases have been identified, research has shifted to finding novel therapies. Gene therapy has been proposed both for correcting primary gene defects of muscle and as a way of using muscle for the production of proteins therapeutic in inflammatory or nonmuscle diseases. Several strategies have been developed to introduce foreign genes into diseased muscles, including myoblast transfer, direct injection of plasmids or DNA-liposome complexes, and infection with modified viruses. Related strategies, using antisense sequences or ribozymes, have been devised to modify gene expression in diseased cells. No method has yet proved itself in the clinic, although current work remains promising and some of the pitfalls that must be overcome have been identified.     

Dihydropyridine receptor gene expression in skeletal muscle from mdx and control mice

The expression of isoform-specific dihydropyrine receptor-calcium channel (DHPR) alpha 1-subunit genes was investigated in mdx and control mouse diaphragm (DIA) and tibialis anterior (TA). RNase protection assays were carried out with a rat DHPR cDNA probe specific for skeletal muscle and a mouse DHPR cDNA probe specific for cardiac muscle. The level of expression of the gene encoding the cardiac DHPR was very weak in TA muscle from both control and mdx mice. Compared to TA, DIA expressed mRNA for the cardiac isoform at significantly higher levels, but mdx and control mouse DIA levels were similar to one another. In contrast, mRNA expression levels for the DHPR skeletal muscle isoform were lower in control DIA than TA. However, there was a dramatic increase in the expression for the DHPR skeletal muscle isoform in mdx DIA compared with control DIA, reaching the TA expression level, whereas dystrophy did not affect TA expression. [3H]-PN200-110 binding was used to further assess DIA DHPR expression at the protein level. The density of binding sites for the probe was not significantly affected in DIA muscles of mdx vs. control mice, but it was reduced in older mdx and control mice. The increase in DHPR mRNA levels without a consequent increase in DHPR protein expression could be secondary to possible enhanced protein degradation which occurs in mdx DIA. The altered DHPR expression levels found here do not appear to be responsible for the severe deficits in contractile function of the mdx DIA.     

Myotonic dystrophy protein kinase gene expression in skeletal muscle from congenitally affected infants

Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida. Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem. The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida. Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp. The detection of Thiothrix spp. was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures. It was estimated through immunocytochemical procedures that Thiothrix spp. comprised 18% of the biofilm in the municipal water storage tanks. These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling.     

Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle

Nitric oxide (NO) is a pluripotent molecule that can be secreted by skeletal muscle through the activity of the neuronal constitutive isoform of NO synthase. To determine whether skeletal muscle and diaphragm might also express the macrophage-inducible form of NO synthase (iNOS) during provocative states, we examined tissue from mice at serial times after intravenous administration of Escherichia coli endotoxin. In these studies, iNOS mRNA was strongly expressed in the diaphragm and skeletal muscle of mice 4 h after intravenous endotoxin and was significantly diminished by 8 h after challenge. Induction of iNOS mRNA was followed by expression of iNOS immunoreactive protein on Western immunoblots. Increased iNOS activity was demonstrated by conversion of arginine to citrulline. Immunochemical analysis of diaphragmatic explants exposed to endotoxin in vitro revealed specific iNOS staining in myocytes, in addition to macrophages and endothelium. These results may be important in understanding the pathogenesis of respiratory pump failure during septic shock, as well as skeletal muscle injury during inflammation or metabolic stress.     

Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney

This experiment was an attempt to use the soybean residue derived from the production of "soy milk". The residue contains about 18% protein, 70% carbohydrates and 7.5% lipid as fish feed for rearing common carp, Cyprinus carpio. There were 4 types of diets: (1) soybean residue, (2) soybean residue digested with Papain, (3) soybean residue (64%) mixed with beef liver (34%) and (4) same mixture as (3) but digested with Papain. The results indicate that the percentage increase in weight and length of fish feeding with beef liver supplemented diets was higher than those feeding with soybean residue alone. This was possibly due to the fact that beef liver was able to supplement the nutrient deficiency in soybean. The two types of feeds (2 and 4) digested with Papain also yielded significantly better fish growth in terms of weight and length gains, than their counterparts without digestion. Furthermore, the water turbidity of the tanks added with digested feeds was significantly less, as Papain was able to hyrolyse the protein substrates suspended in the water, and thus lowered the turbidity.     

Alterations in skeletal muscle gene expression in the rat with chronic congestive heart failure

Congestive heart failure is often associated with skeletal muscle abnormalities that contribute to early fatigue and acidosis. Up to the present time, however, the mechanisms responsible for these changes are unclear. Myocardial infarctions were produced by coronary ligation in adult Sprague-Dawley rats. At 20 weeks, 10 control rats, and 15 animals with heart failure [defined by elevated LVEDP (26.1 +/- 3.1 v 2.5 +/- 0.5 mmHg) and RV hypertrophy (300 +/- 21 g v 158 +/- 9 mg)] underwent in vivo measurements of total body, and soleus total protein and myosin heavy chain (MHC) synthesis by [3H]leucine constant infusion. Soleus muscle was also analysed for protein content, and MHC isoenzyme content by SDS-PAGE. Northern blotting also was used to determine levels of the mRNA's encoding type I, IIa, IIb, and IIx MHC, alpha-skeletal actin, COX III, SDH and GAPDH. Soleus muscles in heart failure rats were smaller than controls (112 +/- 6 v 126 +/- 5 mg) and the degree of atrophy was significant when corrected for body mass (0.38 +/- 0.02 v 0.46 +/- 0.02 mg/g. P = 0.007). Although there was no significant difference in plasma leucine flux (an index of whole-body protein synthesis), soleus muscle total and MHC synthesis was reduced in heart failure animals. Whereas the Type I MHC isoenzyme (beta MHC) was the only MHC detected in the soleus of control animals, type II MHC isoenzyme comprised 11.8 +/- 3.1% of the MHC in the heart failure group. Furthermore, steady-state mRNA levels encoding beta MHC were significantly depressed in the heart failure rats, where those encoding Types IIb and IIx MHC were increased. Steady-state mRNA levels of alpha-skeletal actin, cytochrome C oxidase (COX III) and succinate dehydrogenase (SDH) were also significantly depressed. This animal model of chronic heart failure is associated with quantitative and qualitative alterations in skeletal muscle gene expression that are similar to those reported in skeletal muscle of patients with chronic heart failure. The altered phenotype and impaired metabolic capacity may contribute to exercise intolerance in CHF.     

Expression of deletion-containing dystrophins in mdx muscle: implications for gene therapy and dystrophin function

The expression of full-length dystrophin and various dystrophin deletion mutants was monitored in mdx mouse muscle after intramuscular injection of dystrophin-encoding plasmid DNAs. Recombinant dystrophin proteins, including those lacking either the amino terminus, carboxyl terminus, or most of the central rod domain, showed localization to the plasma membrane. This suggests that there are multiple attachment sites for dystrophin to the plasma membrane. Only those constructs containing the carboxyl terminus were able to stabilize dystrophin-associated proteins (DAP) at the membrane, consistent with other studies that suggest that this domain is critical to DAP binding. Colocalization with DAP was not necessary for membrane localization of the various dystrophin molecules. However, stabilization and co-localization of the DAP did seem to be a prerequisite for expression and/or stabilization of mutant dystrophins beyond 1 wk and these same criteria seemed important for mitigating the histopathological consequences of dystrophin deficiency.     

Capillary adrenoceptors in rat skeletal muscle

Coronary artery disease (CAD) is the most common cause of death in women in the United States. Dyslipidemia is a risk factor for CAD in both men and women. Low levels of high-density lipoprotein (HDL) cholesterol and hypertriglyceridemia, especially in association with a dense low-density lipoprotein (LDL) phenotype, may be of greater importance in women than in men. The relationship between CAD and dyslipidemia and the therapeutic approach to disorders of lipid metabolism in women have unique features because of the effects of exogenous and endogenous hormones on lipid pathways. Estrogen decreases LDL cholesterol and Lp(a) lipoprotein and increases triglyceride and HDL cholesterol levels. Progestogens decrease triglycerides, HDL cholesterol, and Lp(a), and they increase LDL cholesterol. Thus, oral contraceptives increase plasma triglycerides, whereas the effect of these agents on LDL cholesterol and HDL cholesterol levels is related to the androgenicity and dose of progestogen. Postmenopausal hormone replacement therapy increases triglycerides and decreases LDL cholesterol. The effect of hormone replacement therapy on HDL cholesterol is influenced by the addition of progestogen. Although no primary prevention studies have analyzed lipid lowering and CAD in women, secondary prevention studies have suggested that the response to drug treatment and the benefit of lipid lowering are similar in women and in men. Hormone replacement therapy should be considered in the treatment of hypercholesterolemia in postmenopausal women; however, individualization of treatment is important to avoid adverse effects.     

Coupling of skeletal muscle to a prosthesis for circulatory support

A durable bond between the end of skeletal muscles and prosthetic structures could, with appropriate linkage, allow circulatory support power by synchronous and/or sequential contraction of several in situ conditioned muscles. Potential advantages relative to a myoplasty wrap involve 1) less traumatic dissection, 2) efficient linear force development, 3) selectable contraction rate, 4) greater stroke work, 5) independent control of muscle pre-load and end diastolic pressure, and 6) independent control of duration of muscle tension and ejection time. However, no existing means of tissue-prosthetic bonding appears adequate. Practicality would demand that full tension bearing capacity by the bond take no longer than muscle conditioning. A prosthesis was developed to achieve those goals. As scaled for this study, it is made of 7,200-7,800 unspun, unplaited, 22 to 26 microns diameter polyester fibers swaged into four taper needles for weaving through distal muscle. The other end is formed into a polyurethane sheathed kernmantel cord for distal fixation. Devices were implanted in six 3 to 4 kg rabbits (unilateral posterior tibial tendon replacement, random side selection with contralateral dissection/closure controls), and their tensile strength was tested at 30 days. All healed well; leg movements were normal after 1 week. Limbs were frozen at -70 degrees C between death and testing. Control failure occurred at 243 +/- 94 N and experimental at 163 +/- 44 N (p = 0.065, t-test); highest estimated requirement was 17.2 N. Interface strength was adequate by 30 days. Continued investigations, addressing other questions, are warranted.     

A modified method for isolating alpha-amylase from rabbit skeletal muscle

A modified method for purifying alpha-amylase from rabbit skeletal muscle is described, which in comparison with the previously published method gives better results of isolation and is economical, less time-consuming, and require less material. The former method gave 1,340-fold purification of the enzyme with a yield of 0-6%, and the new method 2,014-fold purification and 7-6% yield. Electrophoresis in polyacrylamide gel showed absence of a strictly defined zone of enzyme activity and presence of inactive proteins.     

Quantitative analysis for histochemical grouping of muscle fibers after skeletal muscle transplantation by microneurovascular anastomoses

A poly-histochemical quantitative assay was carried out on fibers of grafted rectus femoris muscles of rabbit which had undergone simulated free muscle transplantation by microneurovascular anastomoses in speciments at 3, 6, 12 months postoperatively. At the same time, the same assay was done for two experimental control groups: either the motor nerve or the patella tendon was simply severed and immediately sutured. It was found that the contractile characteristics of whole muscle were depended on relative number of different muscle fibers. That meant it was depended on the relative number of different motor fibers which had run through the anastomosed site. The caliber change among different types of muscle fibers mainly represented there was cross reneurotization during nerve regeneration.     

Nerve stimulation and denervation induce differential patterns of immediate early gene mRNA expression in skeletal muscle

Many properties of skeletal muscle cells are closely regulated by motor nerves. Neuromuscular synaptic transmission (including the 'activity' it triggers) mediates many of these effects, while denervation results in a different spectrum of muscle cell changes. However, little is known about the early regulatory events that occur in mature muscle cells in response to muscle activity or denervation. We have examined the effects of motor nerve stimulation and denervation on the expression of 4 immediate early genes (IEGs)--c-jun, junB, zif268, and nur77--in mature mouse gastrocnemius muscle. Electrical stimulation of the sciatic nerve in a pattern of brisk intermittent exercise induced a marked rise in zif268 and c-jun mRNA levels within 45 min, a minimal rise in junB, and no change in nur77 mRNA levels. By contrast, surgical denervation resulted in a marked increase of c-jun, a slight rise in junB, and no change in nur77 or zif268 mRNA levels. These findings show that neural stimulation and denervation lead to differential patterns of IEG expression. The selectivity of these patterns suggests that differential IEG expression may play an important role in regulating the specific phenotypic changes in skeletal muscles that result from denervation, innervation, and various patterns of stimulation.     

Prophylactic gene therapy for cancer

BACKGROUND: Epithelial ovarian tumors of borderline malignancy are different from benign tumors and malignant neoplasms. They exist with relatively benign clinical course, younger age and better prognosis as compared with invasive malignant carcinomas. Most of them are discovered at early stage, for example, stage Ia. This retrospective review evaluates the clinical features, treatments and prognosis of 48 patients with borderline malignancy of ovarian tumors. METHODS: Forty-eight patients with ovarian tumors of borderline malignancy, aged from 14 to 69 years (mean: 39.2 years; median: 36 years), were retrospectively studied. The histopathologic diagnosis was based on the morphologic criteria published by Tazelaar et al. in 1985. All cases, including 16 cases diagnosed before 1985, were pathologically reviewed. All information of clinical stage, surgical intervention and prognosis was achieved by reviewing hospital record or contacting patients by telephone. Two patients were lost to follow up. One patient died of sepsis resulting from another operation for another gynecological cancer. Totally forty-five patients were included for evaluation. RESULTS: Thirty-nine of the 48 patients (81.3%) were at stage Ia, 6 cases (12.5%) were at stage Ib, 2 cases (4.1%) were at stage Ic, and the remaining one patient (2.1%) was at state IIIc. Thirty-four patients (71%) were with mucinous cystadenoma of borderline malignancy, 11 cases (23%) were of serous type, and 3 patients (6%) were of mixed serous and mucinous type. Twenty-two patients underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy (TAH and BSO), but one of them remained partial ovary due to young age (27 y/o). Twelve patients were treated with unilateral oophorectomy or unilateral salpingo-oophorectomy (USO). Twelve patients underwent USO and wedge resection of contralateral ovary. One case underwent debulking surgery. One patient underwent enucleation of ovarian tumor and biopsy of contralateral ovary. Eighteen patients were treated with chemotherapy after operation. One patient developed recurrence 4 months after the primary operation. Excluding two cases lost to follow up and one case with surgical mortality for another gynecological cancer, forty-five patients were alive and were followed from 9 months to 165 months. (median: 48 months; mean: 46 months) CONCLUSIONS: Most of the patients were at the early stage of disease when first diagnosed, 81.3% were at stage Ia and only one case was at stage IIIc. Sixty-three percent of our patients underwent surgical treatment alone while the rest of them (37%) had post-operative chemotherapy with either alkeran or PAC. The use of adjuvant chemotherapy seemed unwarranted as there was no difference in survival between those with and without it. (P > 0.05) The low recurrent rate of 2% in our patients again confirmed the 9 P relative benign clinical course of this disease.