Sustained release of bone morphogenetic protein-4 in adult rabbit extraocular muscle results in decreased force and muscle size: potential for strabismus treatment.

Purpose. To assess the effect of a sustained-release preparation of bone morphogenetic protein-4 (BMP-4) on EOM force generation and muscle size. Methods. Sustained-release pellets, releasing 500 nanograms/day of BMP-4 for a maximum of 3 months, were implanted beneath the superior rectus muscle (SR) belly in anesthetized adult rabbits. The contralateral side received a placebo pellet as a control. After 1, 3, and 6 months, SRs were removed, and force generation at twitch and tetanic frequencies as well as fatigue resistance were determined in vitro. Myofiber size, myosin heavy chain isoform expression, and satellite cell density were assessed histologically. Results. SR force generation was significantly decreased by BMP-4 compared with the contralateral controls. Force generation was decreased by 25-30% by 1 month, 31-50% by 3 months, and at 6 months, after 3 BMP-4-free months, force was still decreased by 20-31%. No change in fatigue was seen. Significant decreases in muscle size were seen, greatest at 3 months. At all time points Pax7- and MyoD-positive satellite cell densities were significantly decreased. Conclusions. The decreased force generation and muscle size caused by sustained release of BMP-4 suggests that myogenic signaling factors may provide a more biological method of decreasing muscle strength in vivo than exogenously administered toxins. Treating antagonist-agonist pairs of EOM with titratable, naturally occurring myogenic signaling and growth factors may provide safe, efficacious, nonsurgical treatment options for patients with strabismus.

Modulating neuromuscular junction density changes in botulinum toxin-treated orbicularis oculi muscle.

PURPOSE: Botulinum toxin A is the most commonly used treatment for blepharospasm, hemifacial spasm, and other focal dystonias. Its main drawback is its relatively short duration of effect. The goal of this study was to examine the ability of corticotropin releasing factor (CRF) or antibody to insulin growth factor I-receptor (anti-IGFIR) to reduce the up-regulation of neuromuscular junctions that are associated with return of muscle function after botulinum toxin treatment. METHODS: Eyelids of adult rabbits were locally injected with either botulinum toxin alone or botulinum toxin treatment followed by injection of either CRF or anti-IGFIR. After one, two, or four weeks, the orbicularis oculi muscles within the treated eyelids were examined for density of neuromuscular junctions histologically. RESULTS: Injection of botulinum toxin into rabbit eyelids resulted in a significant increase in the density of neuromuscular junctions at one and two weeks, and an even greater increase in neuromuscular junction density by four weeks after treatment. Treatment with either CRF or anti-IGFIR completely prevented this increase in neuromuscular junction density. CONCLUSIONS: The return of function after botulinum toxin-induced muscle paralysis is due to terminal sprouting and formation of new neuromuscular junctions within the paralyzed muscles. Injection with CRF or anti-IGFIR after botulinum toxin treatment prevents this sprouting, which in turn should increase the duration of effectiveness of single botulinum toxin treatments. Future physiology studies will address this. Prolonging botulinum toxin's clinical efficacy should decrease the number of injections needed for patient muscle spasm relief, decreasing the risk of negative side effects and changes in drug effectiveness that often occurs over a lifetime of botulinum toxin exposure.

Botulinum toxin pretreatment augments the weakening effect of injection with ricin-mAb35 in rabbit extraocular muscle.

PURPOSE: To examine force generation and duration of effect of botulinum toxin pretreatment, followed by injection of ricin-mAb35 in extraocular muscle. METHODS: In normal adult rabbits, one superior rectus muscle was injected with either 5 units botulinum toxin or 1/50 maximally tolerated dose for rats (rMTD) of ricin-mAb35. Additional rabbits were first injected with 5 units botulinum toxin, and after 1, 2, or 4 weeks the same muscle was injected with either 1/10 or 1/50 rMTD ricin-mAb35. In each treatment group, the contralateral muscle was injected with an equal volume of saline. After 12 weeks (1/50 rMTD) or 6 months (1/10 rMTD), the rabbits were euthanized. Both SR muscles were removed and assayed physiologically, using an in vitro apparatus. RESULTS: Twelve weeks after treatment with either botulinum toxin or immunotoxin alone, only ricin-mAb35-treated muscles were weaker than control muscles at tetanic stimulation frequencies. Pretreatment with botulinum toxin prior to injection with immunotoxin, especially at shorter intervals between injections, resulted in significant decreases in twitch and tetanic force generation compared with controls and muscles treated with ricin-mAb35 only or botulinum toxin only. At 6 months, force generation was decreased from control only in muscles treated with the higher dose of ricin-mAb35. Botulinum toxin pretreatment did not augment this effect at 6 months. CONCLUSIONS: Upregulation of postsynaptic nicotinic acetylcholine receptors caused by botulinum toxin pretreatment amplifies the reduction of force generation in extraocular muscle following secondary injection of the immunotoxin ricin-mAb35 within 3 months of treatment.

Myogenic growth factors can decrease extraocular muscle force generation: a potential biological approach to the treatment of strabismus.

PURPOSE: Future pharmacologic treatment of strabismus may be optimized if drugs that are less potentially toxic to patients can be developed. Prior studies have shown that direct injection of extraocular muscles (EOMs) with insulin growth factor or fibroblast growth factor results in significant increases in the generation of EOM force. The purpose of this study was to examine the morphometric and physiological effects of direct EOM injection with the growth factors BMP4, TGFbeta1, Shh, and Wnt3A. METHODS: One superior rectus muscle of normal adult rabbits was injected with BMP4, TGFbeta1, Shh, or Wnt3A. The contralateral muscle was injected with an equal volume of saline to serve as a control. After 1 week, the animals were euthanatized, and both superior rectus muscles were removed and assayed physiologically. The muscles were stimulated at increasing frequencies to determine force generation. A separate group of treated and control superior rectus muscles were examined histologically for alterations in total muscle cross-sectional area and myosin heavy chain isoform (MyHC) composition. RESULTS: One week after a single injection of BMP4, TGFbeta1, Shh, or Wnt3A, all treated muscles showed significant decreases in generation of force compared with control muscles. BMP4, TGFbeta1, Shh, and Wnt3A significantly decreased the mean myofiber cross-sectional area of fast MyHC-positive myofibers. BMP4 resulted in a conversion of fast-to-slow myofibers and a significant decrease in the percentage of developmental and neonatal MyHC-positive myofibers. Alterations in mean cross-sectional area and proportion of MyHCs were seen after injection with TGFbeta1, Shh, and Wnt3A. TGFbeta1 and BMP4 injections resulted in increased Pax7-positive satellite cells, whereas BMP4, TGFbeta1, and Wnt3A resulted in a decrease in MyoD-positive satellite cells. CONCLUSIONS: These results suggest that, rather than using toxins or immunotoxins, a more biological approach to decrease muscle strength is possible and demonstrate the potential utility of myogenic signaling factors for decreasing EOM strength. Ongoing drug-delivery studies will elucidate means of extending treatment effect to make such agents clinically useful.

Myofiber length and three-dimensional localization of NMJs in normal and botulinum toxin treated adult extraocular muscles.

PURPOSE: The density and three-dimensional localization of neuromuscular junctions (NMJs) of normal and botulinum toxin-treated normal adult rabbit and monkey extraocular muscles (EOMs) were analyzed. To demonstrate average myofiber length, randomly selected individual myofibers were reconstructed and compared with total muscle length. METHODS: Normal adult rabbit and monkey EOM and normal adult rabbit tibialis anterior were dissected in their entirety, frozen, sectioned longitudinally, and immunostained for NMJ localization. In addition, adult rabbit EOMs were injected with 5 U botulinum toxin, and NMJ density was determined after 2 weeks. NMJ locations for the three groups of EOM were reconstructed, and density of NMJ was determined. Individual myofibers were reconstructed from the orbital and global layers to determine mean fiber length. RESULTS: NMJs were dispersed throughout the entire length of all EOMs examined from adult rabbits and monkeys and were visualized by alpha-bungarotoxin staining and three-dimensional reconstruction of serial sections. In leg muscle, two relatively tight bands of NMJs were seen. Botulinum toxin significantly increased total NMJ density. Mean fiber lengths were 1.9 and 4.83 mm in the orbital and global layers, respectively, approximately 10% and 24% of the total origin-to-insertion muscle lengths. In addition, individual myofibers continuously changed their intrafascicular relationships over their lengths. CONCLUSIONS: The density and distribution of NMJs in normal EOMs are more extensive than previously described. Individual myofibers are significantly shorter than the tendon-to-tendon muscle length in both muscle layers. Botulinum toxin results in a doubling of NMJ density. NMJ localization in normal EOMs has ramifications for understanding eye movement control, but it is also important when surgical or pharmacologic intervention is used for the treatment of strabismus, nystagmus, or other eye muscle disorders.

Increasing muscle strength as a treatment for strabismus: sustained release of insulin-like growth factor-1 in rabbit extraocular muscle.

PURPOSE: Currently, no drug treatment is available for strengthening underacting extraocular muscles (EOM) in strabismus. We showed previously that single injections of insulin-like growth factor (IGF-1) result in significant but short-term increases in muscle force generation. This study examined the effects of sustained release of IGF-1 on force generation in rabbit superior rectus muscles. METHODS: In adult rabbits, slow-release pellets containing IGF-1 were implanted on the global side of one superior rectus muscle. After 1 week, or 1, 2, 3, or 6 months, treated and control muscles were examined for force generation using an in vitro physiology apparatus. All muscles were prepared for histology and mean myofiber cross-sectional areas were determined. RESULTS: One and 3 months after pellet implantation, treated muscles generated significantly greater force than contralateral control muscles, whereas at 2 months, no significant difference was found. Force per cross-sectional area (mN/cm(2)) at 3 months also increased significantly in the treated muscles. Mean muscle cross-sectional area increased significantly after 1, 2, and 3 months of sustained exposure to IGF-1 compared with controls. After an additional 3 months without IGF-1 exposure, mean cross-sectional areas were significantly greater than controls but significantly reduced compared with areas at 1, 2, and 3 months. CONCLUSIONS: IGF-1 appears to be highly effective in increasing muscle force generation. Because slow release of IGF-1 results in sustained increases in EOM force generation, it may be a potentially useful alternative to surgical resection procedures because it avoids many of the potential long-term biomechanical hazards of resection surgery.

Increased extraocular muscle strength with direct injection of insulin-like growth factor-I.

PURPOSE: Previous work has demonstrated the effectiveness of insulin-like growth factor (IGF)-II in increasing force generation in extraocular muscle (EOM). Studies in the literature have suggested that IGF-I would be even more effective than IGF-II. This study was performed to assess the effects on muscle mass and force generation of IGF-I injection in adult rabbit superior rectus muscle. METHODS: Adult rabbits received a single injection of IGF-I at one of several doses into one superior rectus muscle. One week after treatment, the rabbits were euthanatized, and the superior rectus muscle from each orbit was removed. Force generation was measured using an in vitro apparatus, and injected muscles were compared with the contralateral control. A second group of animals were injected similarly, and the muscles were examined at 1 week for changes in cross-sectional area of individual myofibers. RESULTS: EOMs demonstrate significant numbers of cells expressing the IGF receptor. After the EOMs were injected with IGF-I, there were significant increases both in muscle force generation and cross-sectional area at all doses tested in this study. Doses of 10 and 25 microg IGF-I were most effective. CONCLUSIONS: Direct muscular injection of IGF-I effectively increases EOM force generation without the potential biomechanical hazards of surgery such as permanently altered muscle length or insertional position on the globe.

Gene delivery through cell culture substrate adsorbed DNA complexes.

Efficient gene delivery is a fundamental goal of biotechnology and has numerous applications in both basic and applied science. Substrate-mediated delivery and reverse transfection enhance gene transfer by increasing the concentration of DNA in the cellular microenvironment through immobilizing a plasmid to a cell culture substrate prior to cell seeding. In this report, we examine gene delivery of plasmids that were complexed with cationic polymers (polyplexes) or lipids (lipoplexes) and subsequently immobilized to cell culture or biomaterial substrates by adsorption. Polyplexes and lipoplexes were adsorbed to either tissue culture polystyrene or serum-adsorbed tissue culture polystyrene. The quantity of DNA immobilized increased with time of exposure, and the deposition rate and final amount deposited depended upon the properties of the substrate and complex. For polyplexes, serum modification enhanced reporter gene expression up to 1500-fold relative to unmodified substrates and yielded equivalent or greater expression compared to bolus delivery. For lipoplexes, serum modification significantly increased the number of transfected cells relative to unmodified substrates yet provided similar levels of expression. Immobilized complexes transfect primary cells with improved cellular viability relative to bolus delivery. Finally, this substrate-mediated delivery approach was extended to a widely used biomaterial, poly(lactide-co-glycolide). Immobilization of DNA complexes to tissue culture polystyrene substrates can be a useful tool for enhancing gene delivery for in vitro studies. Additionally, adapting this system to biomaterials may facilitate application to fields such as tissue engineering.

Substrate-mediated delivery from self-assembled monolayers: effect of surface ionization, hydrophilicity, and patterning.

Gene transfer has many potential applications in basic and applied sciences. In vitro, DNA delivery can be enhanced by increasing the concentration of DNA in the cellular microenvironment through immobilization of DNA to a substrate that supports cell adhesion. Substrate-mediated delivery describes the immobilization of DNA, complexed with cationic lipids or polymers, to a biomaterial or substrate. As surface properties are critical to the efficiency of the surface delivery approach, self-assembled monolayers (SAMs) of alkanethiols on gold were used to correlate surface chemistry of the substrate to binding, release, and transfection of non-specifically immobilized complexes. Surface hydrophobicity and ionization were found to mediate both DNA complex immobilization and transfection, but had no effect on complex release. Additionally, SAMs were used in conjunction with soft lithographic techniques to imprint substrates with specific patterns, resulting in patterned DNA complex deposition and transfection, with transfection efficiencies in the patterns nearing 40%. Controlling the interactions between complexes and substrates, with the potential for patterned delivery, can be used to locally enhance or regulate gene transfer, with applications to tissue engineering scaffolds and transfected cell arrays.

Crosslinked hyaluronic acid hydrogels: a strategy to functionalize and pattern.

The physiological activity of hyaluronic acid (HA) polymers and oligomers makes it a promising material for a variety of applications. The development of HA-hydrogel scaffolds with improved mechanical stability against degradation and biochemical functionality may enhance their application to tissue engineering. In this report, a crosslinking strategy targeting the alcohol groups via a poly(ethylene glycol) diepoxide crosslinker was investigated for the generation of degradable HA hydrogels. To provide support for cell adhesion in vitro, collagen was incorporated into the HA solution prior to the crosslinking process. The hydrogels have a continuous exterior and a porous interior, with pore diameters ranging from 6 to 9 microm. HA and HA-collagen hydrogels degrade in the presence of hyaluronidase and collagenase enzymes, indicating that the chemical modification does not prevent biodegradation. Complete degradation of the hydrogels occurred within 14 days in hyaluronidase (100 U/ml) and 3 days in collagenase (66 U/ml). Pattern transfer was employed to introduce a surface topography onto the hydrogel, which was able to orient cell growth. Furthermore, the hydrogels could be functionalized with the biomolecule neutravidin by incorporation of biotin along the HA backbone. This biotinylation approach may allow attachment of bioactive molecules that are conjugated to avidin.

Synthesis and characterization of injectable, water-soluble copolymers of tertiary amine methacrylates and poly(ethylene glycol) containing methacrylates.

Several homopolymers and copolymers of 2-(diethylamino)ethyl methacrylate (DEAEM) and poly(ethylene glycol) methyl ether methacrylate (PEGMEM) were synthesized using anionic polymerization initiated by potassium t-butoxide. The polymers were characterized by average molecular weight, polydispersity and monomeric unit composition. A very narrow molecular weight distribution was achieved with a well-controlled composition. The glass transition temperatures and compositions of the copolymers followed a Gordon-Taylor relationship. The water solubility and biocompatibility of the copolymers was compared to their parent homopolymers to determine if the addition of a poly(ethylene glycol) group was sufficient to solubilize the polymers in aqueous buffer solutions and to increase the biocompatibility of the polymers. These water-soluble, injectable cationic copolymers have potential applications in gene delivery as well as other biomaterial applications.

Al-Cu-Fe quasicrystal/ultra-high molecular weight polyethylene composites as biomaterials for acetabular cup prosthetics.

Polymer composites of Al-Cu-Fe quasicrystals and ultra-high molecular weight polyethylene (UHMWPE) were investigated for use in acetabular cup prosthetics. The wear properties of the Al-Cu-Fe/UHMWPE samples and a 440 steel ball counterface were measured. The mechanical strength of the Al-Cu-Fe/UHMWPE composites was compared to UHMWPE and alumina/UHMWPE. The biocompatibility of the composite material was tested using a direct contact cytotoxicity assay. Al-Cu-Fe/UHMWPE demonstrated lower volume loss after wear and higher mechanical strength than UHMWPE. This composite material also showed no increase in counterface wear or cytotoxicity relative to UHMWPE. These combined results demonstrate that Al-Cu-Fe/UHMWPE composites are promising candidate materials for acetabular cup prosthetics.

The effect of salts on the micellization temperature of aqueous poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) solutions and the dissolution rate and water diffusion coefficient in their corresponding gels.

Studies were performed to examine the effect of ionic salts on phase transitions, dissolution rates, and diffusion coefficients of water in gels of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) with polymer concentrations ranging from 22 to 32% w/w and salt concentrations ranging from 0 to 1.5% w/w. Salts tested include Na(3)PO(4), Na(2)SO(4), Na(2)HPO(4), NaH(2)PO(4), NaCH(3)CO(2), NaCl, and KI. Micellization transition temperatures were obtained using differential scanning calorimetry. The dissolution rates were obtained by measurement of the surface erosion rates, and diffusion coefficients were obtained by using a method to analyze the intrusion of water into the aqueous gels. It was found that salts had no effect on the dissolution rate of the polymer gels into deionized water. However, when the salt concentration in the aqueous dissolution media was adjusted to match the concentration in the gels, the dissolution rate of the polymer gel decreased with increasing salt concentration. The salts also had a profound effect on the critical micellization temperature (CMT) and the diffusion coefficient of water within the gel. The diffusion coefficient and CMT decreased in the presence of salts. The magnitude of these effects was comparable to their placement on the Hofmeister, or lyotropic series for salts. The effects of polymer and salt concentrations on the CMT were quantified, and a single correlation was proposed to predict the micellization temperatures for a wide range of salt and polymer concentrations.