Effects of heat stimulation and l‐ascorbic acid 2‐phosphate supplementation on myogenic differentiation of artificial skeletal muscle tissue constructs

Although skeletal muscle tissue engineering has been extensively studied, the physical forces produced by tissue‐engineered skeletal muscles remain to be improved for potential clinical utility. In this study, we examined the effects of mild heat stimulation and supplementation of a l ‐ascorbic acid derivative, l ‐ascorbic acid 2‐phosphate (AscP), on myoblast differentiation and physical force generation of tissue‐engineered skeletal muscles. Compared with control cultures at 37°C, mouse C2C12 myoblast cells cultured at 39°C enhanced myotube diameter (skeletal muscle hypertrophy), whereas mild heat stimulation did not promote myotube formation (differentiation rate). Conversely, AscP supplementation resulted in an increased differentiation rate but did not induce skeletal muscle hypertrophy. Following combined treatment with mild heat stimulation and AscP supplementation, both skeletal muscle hypertrophy and differentiation rate were enhanced. Moreover, the active tension produced by the tissue‐engineered skeletal muscles was improved following combined treatment. These findings indicate that tissue culture using mild heat stimulation and AscP supplementation is a promising approach to enhance the function of tissue‐engineered skeletal muscles. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of implantation on engineered skeletal muscle constructs

The development of engineered skeletal muscle would provide a viable tissue for replacement and repair of muscle damaged by disease or injury. Our current tissue‐engineering methods result in three‐dimensional (3D) muscle constructs that generate tension but do not advance phenotypically beyond neonatal characteristics. To develop to an adult phenotype, innervation and vascularization of the construct must occur. In this study, 3D muscle constructs were implanted into the hindlimb of a rat, along the sciatic nerve, with the sural nerve isolated, transected and sutured to the construct to encourage innervation. Aortic ring anchors were sutured to the tendons of the biceps femoris muscle so that the construct would move dynamically with the endogenous muscle. After 1 week in vivo, the constructs were explanted, evaluated for force production and stained for muscle, nerve and collagen markers. Implanted muscle constructs showed a developing capillary system, an epimysium‐like outer layer of connective tissue and an increase in myofibre content. The beginning of α‐bungarotoxin clustering suggests that neuromuscular junctions (NMJs) could form on the implanted muscle, given more time in vivo. Additionally, the constructs increased maximum isometric force from 192 ± 41 μN to 549 ± 103 μN (245% increase) compared to in vitro controls, which increased from 276 ± 23 μN to 329 ± 27μN (25% increase). These findings suggest that engineered muscle tissue survives 1 week of implantation and begins to develop the necessary interfaces needed to advance the phenotype toward adult muscle. However, in terms of force production, the muscle constructs need longer implantation times to fully develop an adult phenotype. Copyright © 2012 John Wiley & Sons, Ltd.     

The MicroRNA-92a/Sp1/MyoD Axis Regulates Hypoxic Stimulation of Myogenic Lineage Differentiation in Mouse Embryonic Stem Cells

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Development of a novel smart scaffold for human skeletal muscle regeneration

Skeletal muscle defects are notoriously difficult to manage and the current methods used are associated with many limitations. Engineered skeletal muscle tissue has the potential to provide a solution that circumvents these disadvantages. Our previous work has identified a novel three‐dimensionally aligned degradable phosphate glass fibre scaffold that can support myoblast differentiation and maturation. This current study has further developed the scaffold by encasing the fibres within a collagen gel to produce a smart composite scaffold that provides key biomimetic cues and supports the formation of a tissue that may be implanted in vivo. The constructs formed were approximately 30 mm long and microscopic examination confirmed favourable unidirectional cell alignment. There was good cell survival, and gene expression studies demonstrated upregulation of the myogenic regulatory factors and developmental and adult myosin heavy chain isoforms indicating myofibre formation and maturation respectively. Compared with the three‐dimensional glass fibre scaffolds, the composite scaffolds had later gene upregulation, however, the use of collagen gels reinforced with degradable aligned glass fibres offers the opportunity to create a tissue analogue that can be easily manipulated. Furthermore, the glass fibre ends could support tendon/bone formation, and the channels formed as the fibres degrade could allow for vascular ingrowth. Copyright © 2013 John Wiley & Sons, Ltd.     

Electrical pulse stimulation of skeletal myoblasts cell cultures with simulated action potentials

Electrical pulse stimulation has an important effect on skeletal muscle development and maturation. However, the methodology for controlling these stimulation parameters to develop in vitro functional skeletal muscle tissues remains to be established. In this work, we have studied the effect of simulated action potentials on the growth and differentiation of skeletal myoblast cell cultures. A circuit simulating action potentials of 0.15 and 0.3 V/mm, at a frequency of 1 Hz and with a 4‐ms pulse width, is proposed. Results show an important improvement of the growth rate and differentiation of myoblasts at a voltage of 0.15 V/mm. Parameters such as electrodes geometry or type of signals must be considered in the development of in vitro skeletal muscle.     

痉挛型瘫痪大鼠骨骼肌肌球蛋白重链亚型的蛋白表达研究

目的:探讨痉挛型瘫痪大鼠骨骼肌肌球蛋白重链亚型的蛋白水平。方法:将50只5d龄新生Sprague-Dawley大鼠随机分成假手术组和模型组,每组25只。建模后大鼠20日龄时,HE染色观察锥体束病理损伤情况,Western blotting检测瘫痪侧比目鱼肌和腓肠肌中MHC-Ⅰ、MHC-Ⅱb蛋白的表达。结果:与假手术组相比,模型组大鼠患侧肢体屈曲痉挛、走路缓慢、跛行,HE染色显示仅锥体束处出现细胞坏死、核固缩;模型组比目鱼肌MHC-Ⅰ蛋白表达(3.78±0.32)显著高于假手术组(0.24±0.11),P0.001;模型组腓肠肌MHC-Ⅰ蛋白表达(3.68±0.28)显著高于假手术组(2.34±0.26),P0.001;模型组比目鱼肌MHC-Ⅱb蛋白表达(0.34±0.16)显著低于假手术组(0.77±0.21),P0.001;模型组腓肠肌MHC-Ⅱb蛋白表达(0.32±0.18)显著低于假手术组(0.78±0.22),P0.001。结论:痉挛状态下大鼠骨骼肌肌球蛋白重链亚型MHC-Ⅰ蛋白高表达、MHC-Ⅱb蛋白低表达。     

Synergy between myogenic and non-myogenic cells in a 3D tissue-engineered craniofacial skeletal muscle construct

In vitro skeletal muscle engineering involves the culture of isolated primary myogenic cells in an environment conducive to the formation of a three-dimensional (3D) tissue construct capable of generating force. Isolated human myogenic cells have been used to study cell-cell interactions, permitting identification of functions intrinsic to skeletal muscle in two dimensions (2D). However, the independent contribution of human myogenic and non-myogenic cell types that comprise skeletal muscle to myogenic cell differentiation, force generation and matrix remodelling has yet to be established in 3D. The objective of this study was to use isolated human myogenic and non-myogenic muscle-derived cells (MDC) seeded in 3D collagen constructs to engineer a biomimetic craniofacial skeletal construct. The aim was to purify the two subpopulations of myogenic and non-myogenic cells from human masseter muscle and quantitate myogenic cell differentiation, force generation and matrix remodelling of the 3D collagen construct. The results showed that both the heterogeneous mixture of cells and the purified myogenic cell population expressed myogenin, indicative of myogenic cell differentiation. Further, there was a synergistic effect as the heterogeneous co-culture of myogenic and non-myogenic cells generated the highest peak force and expressed the most MMP-2 mRNA compared to isolated individual cell populations.     

Promotion of skeletal muscle repair in a rat skeletal muscle injury model by local injection of human adipose tissue‐derived regenerative cells

Human adipose tissue‐derived regenerative cells (ADRCs) can be isolated easily and aseptically from unwanted subcutaneous fat without culturing. ADRCs have been used in clinical cosmetic therapy. In addition, they are expected to be an attractive and feasible source of cell‐based therapies in regenerative medicine. Therefore, this paper investigates whether transplantation of human adult ADRCs into skeletal muscle injury models promotes the repair of muscle tissues. This was done by locally injecting human ADRCs into an injured site after laceration of the nude‐rat tibialis anterior muscle. Phosphate‐buffered saline (PBS) and bone marrow mononuclear cells (MNCs) were injected as negative and positive controls, respectively. After injury, recovery of muscle strength was accelerated by transplantation of ADRCs compared to administration of PBS and MNCs. Moreover, transplantation of ADRCs also enhanced angiogenesis and myogenesis, but the number of vascular and muscular cells labeled with the human cell‐specific maker was limited at the injury site. Results showed that transplantation of ADRCs into a skeletal muscle injury model promoted repair of muscle tissues in a paracrine manner rather than differentiation of itself into blood vessels and myofibres. Thus, it is believed that ADRCs are a useful and feasible cell source not only for cosmetic therapy but also for regenerative therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

骨骼肌损伤与修复中内质网应激蛋白的表达变化

摘要 目的：通过观察骨骼肌损伤与修复过程中内质网应激（ERS）蛋白的表达变化,探讨内质网应激在骨骼肌损伤与修复过程中的可能作用。 方法：72只8周龄雄性SD大鼠随机分为对照组（C组, n=8）和运动组（E组, n=64）。采用一次性持续90min下坡跑（速度为16m/min,坡度为-16°）,分别在运动结束后的0h、6h、12h、1d、2d、3d、1周和2周处死,取大鼠右侧腓肠肌,用于组织学、SOD活性和MDA含量以及内质网应激蛋白GRP78和GADD153表达的测定。 结果：①运动后3d骨骼肌大量炎症细胞浸润,损伤程度最为严重,运动后2周肌纤维形态结构基本恢复正常;②SOD的活性运动后即刻上升,之后下降,在1d最低,3d出现第二次下降;MDA的含量运动后即刻上升,之后持续下降,2周时低于对照组水平。SOD/MDA表现为运动后下降,1d最低,之后持续上升。上述变化与对照组相比,均无显著性差异。③内质网应激蛋白GRP78、GADD153的表达以及GRP78/GADD153表现为运动后呈上升趋势,1d时出现第一次峰值,之后略有下降,1周达到最高,2周时出现下降趋势,但仍高于对照组。 结论：骨骼肌损伤修复过程中前期内质网应激蛋白的表达增加有利于发挥细胞保护的作用,后期的表达有利于促进骨骼肌损伤后的修复。     

超早期推拿对骨骼肌急性钝挫伤模型大鼠肌细胞膜修复相关蛋白dysferlin表达的影响

目的:观察超早期推拿对骨骼肌急性钝挫伤模型大鼠肌细胞膜修复相关蛋白dysferlin表达的影响,探寻dysferlin在骨骼肌钝挫伤肌细胞膜修复中的作用机制。方法:SD大鼠55只,随机分为正常对照组(n=5)、自然恢复组(n=25)、推拿组(n=25)。根据推拿治疗的天数计时,自然恢复组和推拿组分别在1d、2d、3d、5d、7d五个时间点处死,每亚组每个时间点5只,在腓肠肌标记的区域内取肌肉组织,HE观察组织的形态变化,运用Western blot检测蛋白dysferlin的表达情况,并进行统计学分析。结果:HE结果显示,与正常对照组相比,骨骼肌钝挫伤后,1d和2d肌纤维出现肿胀;3d出现炎性细胞浸润且自然恢复组比推拿组更加严重;5d炎性细胞浸润更加明显;7d推拿组炎性细胞较5d明显减少,结缔组织形成较少,7d自然恢复组有大量的结缔组织填充在肌纤维之间。WB结果显示:dysferlin在模型组中比在正常对照组中的表达明显增多;各个相应时间点推拿组较自然恢复组表达量明显增多(P0.05),且随着时间的推移dysferlin在推拿组各亚组、自然恢复组各亚组中的表达量呈逐渐上升趋势。结论:在骨骼肌急性钝挫伤模型大鼠腓肠肌修复过程中,超早期推拿可增加膜修复蛋白dysferlin的表达,促进破裂的肌细胞膜及时修复,可能有利于损伤肌细胞的修复,从而帮助受损的骨骼肌修复。     

Enteric neural differentiation in innervated,physiologically functional,smooth muscle constructs is modulated by bone morphogenic protein 2 secreted by sphincteric smooth muscle cells

The enteric nervous system (ENS) controls gastrointestinal (GI) functions, including motility and digestion, which are impaired in ENS disorders. Differentiation of enteric neurons is mediated by factors released by the gut mesenchyme, including smooth muscle cells (SMCs). SMC‐derived factors involved in adult enteric neural progenitor cells (NPCs) differentiation remain elusive. Furthermore, physiologically relevant in vitro models to investigate the innervations of various regions of the gut, such as the pylorus and lower oesophageal sphincter (LES), are not available. Here, neural differentiation in bioengineered innervated circular constructs composed of SMCs isolated from the internal anal sphincter (IAS), pylorus, LES and colon of rabbits was investigated. Additionally, SMC‐derived factors that induce neural differentiation were identified to optimize bioengineered construct innervations. Sphincteric and non‐sphincteric bioengineered constructs aligned circumferentially and SMCs maintained contractile phenotypes. Sphincteric constructs generated spontaneous basal tones. Higher levels of excitatory and inhibitory motor neuron differentiation and secretion of bone morphogenic protein 2 (BMP2) were observed in bioengineered, innervated, sphincteric constructs compared to non‐sphincteric constructs. The addition of BMP2 to non‐sphincteric colonic SMC constructs increased nitrergic innervations, and inhibition of BMP2 with noggin in sphincteric constructs decreased functional relaxation. These studies provide: (a) the first bioengineered innervated pylorus and LES constructs; (b) physiologically relevant models to investigate SMCs and adult NPCs interactions; and (c) evidence of the region‐specific effects of SMCs on neural differentiation mediated by BMP2. Furthermore, this study paves the way for the development of innervated bioengineered GI tissue constructs tailored to specific disorders and locations within the gut. Copyright © 2015 John Wiley & Sons, Ltd.     

电针对大鼠急性骨骼肌损伤修复中生长相关蛋白和聚集蛋白表达的影响

目的:通过观察电针治疗对急性骨骼肌损伤中生长相关蛋白(GAP-43)及聚集蛋白(agrin)表达的影响,探讨电针促进受损骨骼肌功能恢复的作用机制。方法:将32只SD大鼠随机分为正常组(A组,n=4)、模型组(B组,n=4)、自然恢复组(C组,n=12)、电针组(D组,n=12),A组为空白对照不做任何处理,其余各组使用自制重物打击器建立骨骼肌急性钝挫伤模型,C组不做电针处理自然恢复,D组于造模后48h开始电针干预,每日1次,每次15min。于造模后24h处死B组取材观察造模是否成功,C组及D组于建模后第7、14、21天三个时间点同时取材使用HE染色观察组织形态变化,Western-bolt检测GAP-43及agrin表达情况。结果:HE染色显示:与A组比较,各组肌细胞大量溶解、肌纤维排列紊乱及炎性细胞浸润。D组与C组比较,肌卫星细胞增殖较快,新生肌纤维明显增多,修复更为迅速。在正常组织中GAP-43表达量极少,骨骼肌损伤后各组中GAP-43表达量显著增高(P0.05);在第14天,GAP-43表达量达到高峰,D组继续表达呈上升趋势(P0.01),C组开始下降,但仍高于A组(P0.05)。与A组相比,骨骼肌受损后各时间点C、D两组agrin的表达明显增多(P0.05),其中D组差异最为显著(P0.01)。结论:电针有促进受损骨骼肌恢复的作用,可能与提高GAP-43和agrin的表达水平有关。     

Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude mice

Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue‐engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real‐time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round‐shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real‐time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright © 2009 John Wiley & Sons, Ltd.     

Biohybrids for spinal cord injury repair

Spinal cord injuries (SCIs) result in the loss of sensory and motor function with massive cell death and axon degeneration. We have previously shown that transplantation of spinal cord‐derived ependymal progenitor cells (epSPC) significantly improves functional recovery after acute and chronic SCI in experimental models, via neuronal differentiation and trophic glial cell support. Here, we propose an improved procedure based on transplantation of epSPC in a tubular conduit of hyaluronic acid containing poly (lactic acid) fibres creating a biohybrid scaffold. In vitro analysis showed that the poly (lactic acid) fibres included in the conduit induce a preferential neuronal fate of the epSPC rather than glial differentiation, favouring elongation of cellular processes. The safety and efficacy of the biohybrid implantation was evaluated in a complete SCI rat model. The conduits allowed efficient epSPC transfer into the spinal cord, improving the preservation of the neuronal tissue by increasing the presence of neuronal fibres at the injury site and by reducing cavities and cyst formation. The biohybrid‐implanted animals presented diminished astrocytic reactivity surrounding the scar area, an increased number of preserved neuronal fibres with a horizontal directional pattern, and enhanced coexpression of the growth cone marker GAP43. The biohybrids offer an improved method for cell transplantation with potential capabilities for neuronal tissue regeneration, opening a promising avenue for cell therapies and SCI treatment.     

Validity of real time ultrasound for measuring skeletal muscle size

Abstract

Objectives: To identify what is known about the validity of real time ultrasound (RTUS) measurements of skeletal muscle size and to summarise populations and conditions that have been assessed with RTUS.

Methods: Seven databases were searched for potential study inclusion. All identified studies were assessed with inclusion criteria and a quality assessment scale. Each study reported muscle size measurements with RTUS and a reference standard (either MRI or CT). Muscle sizes were measured as a muscle thickness (linear dimension), a muscle area (cross sectional area or CSA) or as a muscle volume (anatomical cross sectional area or ACSA).

Results: Thirteen studies were identified for inclusion in this systematic review. Measures of correlation coefficient, coefficient of determination and mean effect size changes were determined. All of the studies in the review came to the same conclusion – that RTUS is a valid alternative to objectively measure muscle size when compared to gold standards such as MRI or CT.

Discussion: All the studies in the review came to the conclusion that RTUS can provide valid measurements of skeletal muscle. The populations represented in this review were adequately varied to warrant generalisation of the results.     

Genipin‐crosslinked gelatin–silk fibroin hydrogels for modulating the behaviour of pluripotent cells

Different hydrogel materials have been prepared to investigate the effects of culture substrate on the behaviour of pluripotent cells. In particular, genipin‐crosslinked gelatin–silk fibroin hydrogels of different compositions have been prepared, physically characterized and used as substrates for the culture of pluripotent cells. Pluripotent cells cultured on hydrogels remained viable and proliferated. Gelatin and silk fibroin promoted the proliferation of cells in the short and long term, respectively. Moreover, cells cultured on genipin‐crosslinked gelatin–silk fibroin blended hydrogels were induced to an epithelial ectodermal differentiation fate, instead of the neural ectodermal fate obtained by culturing on tissue culture plates. This work confirms that specific culture substrates can be used to modulate the behaviour of pluripotent cells and that our genipin‐crosslinked gelatin–silk fibroin blended hydrogels can induce pluripotent cells differentiation to an epithelial ectodermal fate. Copyright © 2014 John Wiley & Sons, Ltd.     

Tracheal cartilage – evaluating the potential of a novel biomaterial for reconstructive cardiovascular procedures

Modern cardiovascular medicine aims for procedures that preferably involve biological materials and, ideally, living implants. Thereby, the regenerative capacity of the target organ may be preserved or even supported, with a potential implant growth capacity during the following time. In the current study we sought to evaluate the integrative capacity of vital and non‐vital tracheal cartilage rings (TCRs) of allogenic or xenogenic origin (allo‐/xeno‐vTCR; allo‐/xeno‐nvTCR) as biomaterials under the in vivo functional load of the circulatory system. Ovine and porcine vTCRs and nvTCRs were implanted in the mitral valve (MV) position for 3 and 9 months (n = 3 each), respectively, in lambs. MV function and TCR position were analysed by echocardiography. Tissue morphology (planimetry), vitality (live/dead‐assay) and implant endothelialization (scanning electron microscopy) were analysed. No functional impairment or significant MV insufficiency or stenosis was observed in any group. TCR shrinkage was observed in all xeno‐TCRs and allo‐nvTCRs at 3 months. Only TCRs of allogenic groups at 9 months and allo‐vTCRs at 3 months showed a ring area comparable to its size at implantation. Moreover, allogenic vital cartilage showed superior tissue integration, greater endothelialization, less inflammation and calcification. Interestingly, in this group viable cartilage cells were found up to 9 months after implantation. Allogenic viable cartilage may represent a well‐suited living material for reconstructive cardiovascular procedures, and further studies are warranted to elucidate the benefits of this novel material, particularly as a structurally supportive component in growing recipients. Copyright © 2012 John Wiley & Sons, Ltd.