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Extracellular matrix scaffolds derived from porcine small intestinal submucosa (SIS-ECM) have been shown to promote the formation of site-specific tissue in a number of preclinical animal studies. However, this constructive remodeling process requires that the scaffold be subjected to a site-specific mechanical environment. The specific quantitativ...
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... was synthesized from 1 mg total RNA using the Superscript Ô First-Strand Synthesis System for reverse tran- scription polymerase chain reaction (RT-PCR) (Invitrogen, Life Technologies) per the manufacturer's protocol. Semi- quantitative PCR was performed using the synthesized cDNA and gene primers specific for murine Col I, Col III, SMA, TN-C, MMP-2, MMP-9, TGF-b1, TGF-b3, and glyc- eraldehyde 3-phosphate dehydrogenase (GAPDH) ( Table 1) in EasyStart Ô Micro100 tubes (Molecular BioProducts, San Diego, CA). The PCR were performed in an Eppendorf Mastercycler with a typical reaction having the following parameters: an initial 5-minute denaturation at 958C, a gene- specific number of cycles (Table 1) consisting of 30-second denaturation at 958C, a primer-specific annealing tempera- ture (Table 1) for 30 seconds, and an elongation for 1 minute at 728C. ...
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... quantitative PCR was performed using the synthesized cDNA and gene primers specific for murine Col I, Col III, SMA, TN-C, MMP-2, MMP-9, TGF-b1, TGF-b3, and glyc- eraldehyde 3-phosphate dehydrogenase (GAPDH) ( Table 1) in EasyStart Ô Micro100 tubes (Molecular BioProducts, San Diego, CA). The PCR were performed in an Eppendorf Mastercycler with a typical reaction having the following parameters: an initial 5-minute denaturation at 958C, a gene- specific number of cycles (Table 1) consisting of 30-second denaturation at 958C, a primer-specific annealing tempera- ture (Table 1) for 30 seconds, and an elongation for 1 minute at 728C. The cycle numbers were chosen to be within the linear range in the relationship between the amount of mRNA in the original tissue sample and the PCR band in- tensities for each gene of interest. ...
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... quantitative PCR was performed using the synthesized cDNA and gene primers specific for murine Col I, Col III, SMA, TN-C, MMP-2, MMP-9, TGF-b1, TGF-b3, and glyc- eraldehyde 3-phosphate dehydrogenase (GAPDH) ( Table 1) in EasyStart Ô Micro100 tubes (Molecular BioProducts, San Diego, CA). The PCR were performed in an Eppendorf Mastercycler with a typical reaction having the following parameters: an initial 5-minute denaturation at 958C, a gene- specific number of cycles (Table 1) consisting of 30-second denaturation at 958C, a primer-specific annealing tempera- ture (Table 1) for 30 seconds, and an elongation for 1 minute at 728C. The cycle numbers were chosen to be within the linear range in the relationship between the amount of mRNA in the original tissue sample and the PCR band in- tensities for each gene of interest. ...
Citations
... 48,49 In addition, cyclic mechanical strain enhances the function and development of engineered tissues by improving the production of collagen and elastin. 50 Moreover, the mechanical properties of engineered tissues can also be improved by culturing under mechanical stimulation. 43 Therefore, cell sheets with enhanced thickness and mechanical properties obtained through appropriate cyclic mechanical stimulation possess great potential for bone tissue engineering applications. ...
Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades. However, efficient harvest and handling of cell sheets remain challenging, including insufficient extracellular matrix content and poor mechanical strength. Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types. However, currently, there are no effective ways to apply mechanical loading to cell sheets. In this study, we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide) (PNIPAAm) to poly(dimethylsiloxane) (PDMS) surfaces. The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting. Subsequently, MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates. Upon maturation, the cell sheets were harvested by lowering the temperature. We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning. Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated. After implantation into the critical-sized calvarial defects of mice, the mechanically conditioned cell sheets significantly promoted new bone formation. Findings from this study reveal that thermo-responsive elastomer, together with mechanical conditioning, can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.
... Prominent TE studies using this approach rely on materials such as porcine small intestinal submucosa (SIS) or decellularized tendon. In one such study, 3T3 mouse fibroblasts seeded in SIS scaffolds and subjected to dynamic loading exhibited upregulated tendon/ligament-specific gene expression, namely increased Col-I and decreased Col-III Fig. 4 With recent advances in bioreactor design, methods for applying mechanical stimulation to scaffold-based tendon and ligament constructs have shifted from stimulating explants and two-dimensional (2D) monolayer culture, to complex dynamic loading of three-dimensional (3D) cell-seeded scaffolds (adapted from [5], with permission) expression with increasing strain frequency [77]. Another group engineered tendon constructs by rolling tenocyte-seeded SIS scaffolds into tubular constructs that could be mounted into custom loading frames for static or dynamic loading within an incubator. ...
... (b) Computer-controlled application of a vacuum uniaxially strains the membranes, driving the loading pins apart to stretch the growth channel, and apply controlled dynamic strain to developing scaffold-free tendon fibers, emulating tensile strains seen in tendon development in vivo [90]. Static tension Yes " tendon-specific gene expression [86] " matrix deposition/remodeling [73,74,79,82] " construct alignment [73,74] " biomechanical properties [73,74,77] No / 2D culture Regulated intercellular communication [67,91] " tendon-specific gene expression [85,86] " matrix deposition [85] " biomechanics [86,87] Quasi-static (slow, increasing) Tension ...
... Yes " biomechanical properties [72] Dynamic/cyclic loading Equiaxial strain 2D culture " tendon-specific gene expression [92] Yes " tendon-specific gene expression [48,76,81,82] " cell alignment [48,76] " matrix synthesis [82] " biomechanical properties [76][77][78]81] Uni-axial tension ...
Tendon, ligament, and skeletal muscle are highly-organized tissues that largely rely on a hierarchical collagenous matrix to withstand high tensile loads experienced in activities of daily life. This critical biomechanical role predisposes these tissues to injury, and current treatments fail to recapitulate the biomechanical function of native tissue. This has prompted researchers to pursue engineering functional tissue replacements, or dysfunction/disease/development models, by emulating in vivo stimuli within in vitro tissue engineering platforms; specifically mechanical stimulation, as well as active contraction in skeletal muscle. Mechanical loading is critical for matrix production and organization in the development, maturation, and maintenance of native tendon, ligament, and skeletal muscle, as well as their interfaces. Tissue engineers seek to harness these mechanobiological benefits using bioreactors to apply both static and dynamic mechanical stimulation to tissue constructs, and induce active contraction in engineered skeletal muscle. The vast majority of engineering approaches in these tissues are scaffold-based, providing interim structure and support to engineered constructs, and sufficient integrity to withstand mechanical loading. Alternatively, some recent studies have employed developmentally-inspired scaffold-free techniques, relying on cellular self-assembly and matrix production to form tissue constructs. Whether utilizing a scaffold or not, incorporation of mechanobiological stimuli has been shown to improve the composition, structure, and biomechanical function of engineered tendon, ligament, and skeletal muscle. Together, these findings highlight the importance of mechanobiology and suggest how it can be leveraged to engineer these tissues and their interfaces, and to create functional multi-tissue constructs.
... In vitro mechanical stimulation of 3T3 fibroblasts in small intestinal submucosa extracellular matrix scaffold may increase the genetic expressions of collagen type I, aSMA, MMP-9, TGF-beta1 and TGF-beta3 42 . Another dynamic culturing of smooth muscle cells on small intestinal submucosa showed increases in collagen and elastin in the presence of VEGF or FGF-2, with the seeded small intestinal submucosa scaffold showing positive Verhoeff-van Gieson elastin fibrils staining 43 . ...
Autologous vascular grafts have the advantages of better biocompatibility and prognosis. However, previous studies that implanted bare polymer tubes in animals to grow autologous tubular tissues were limited by their poor yield rates and stability. To enhance the yield rate of the tubular tissue, we employed a design with the addition of overlaid autologous whole blood scaffold containing lipopolysaccharides (LPS). Furthermore, we applied in vivo dynamic mechanical stimuli through cyclically inflatable silicone tube to improve the mechanical properties of the harvested tissues. The effectiveness of the modification was examined by implanting the tubes in the peritoneal cavity of rats. A group without mechanical stimuli served as the controls. After 24 days of culture including 16 days of cyclic mechanical stimuli, we harvested the tubular tissue forming on the silicone tube for analysis or further autologous interposition vascular grafting. In comparison with those without cyclic dynamic stimuli, tubular tissues with this treatment during in vivo culture had stronger mechanical properties, better smooth muscle differentiation, and more collagen and elastin expression by the end of incubation period in the peritoneal cavity. The grafts remained patent after 4 months of implantation and showed the presence of endothelial and smooth muscle cells. This model shows a new prospect for vascular tissue engineering.
... In many of the other comparison substrates, these endpoints were not consistently observed, suggesting that the ECM plays an integral role in modulating the fate of hSMPCs. It appears that components of muscle ECM combined with the alginate and heparin niche facilitate activation of differentiation mechanisms by supplying physical and chemical cues via cell-cell and cell-matrix communication [45]. In these experiments, Alg-G-H-based substrates with VEGF could sequester the requisite proteins and chemical compounds and support sustained release for 2 weeks sufficient for myogenesis. ...
Statement of significance:
Alginate based biomaterials are commonly used in tissue engineering and regenerative medicine field, however, the inefficient sequestration of growth factors restricted its utilization. In this study, a novel alginate based substrates was produced covalently modified with gelatin and heparin, in order to capture more effective cytokines and proteins in the culture milieu, keep homeostasis for cell survival and tissue regeneration with growth factor sequestration and long-term release capacities. Combining with skeletal muscle derived ECM, the modified Alginate-Gelatin-Heparin gel could most effectively mimic the tissue specific microenvironment to support skeletal muscle progenitor cells proliferation, differentiation and myotube formation. Additionally, the economical and practical features will make it more promising in high-throughput application for regenerative medicine research.
... The application of a physiologic mechanical load (i.e., concomitant physical rehabilitation) during the entirety of the remodelling period following ECM implantation has been shown to promote favourable outcomes. [24][25][26][27] It has been suggested that ECM bioscaffolds contribute to force improvement by simple force transduction based on results of a rodent model in which postoperative physical therapy could not be controlled. 28 While the scar release of the procedure and the mechanical transduction effect of the ECM layer may both be contributing factors to the improved function, the histologic imaging and electrophysiologic evidence of vascularised, innervated skeletal muscle within the defect site in the present cohort of human patients suggests a positive and contributing role for new skeletal muscle in the functional outcomes. ...
Volumetric muscle loss (VML) is a severe and debilitating clinical problem. Current standard of care includes physical therapy or orthotics, which do not correct underlying strength deficits, and surgical tendon transfers or muscle transfers, which involve donor site morbidity and fall short of restoring function. The results of a 13-patient cohort study are described herein and involve a regenerative medicine approach for VML treatment. Acellular bioscaffolds composed of mammalian extracellular matrix (ECM) were implanted and combined with aggressive and early physical therapy following treatment. Immunolabeling of ultrasound-guided biopsies, and magnetic resonance imaging and computed tomography imaging were performed to analyse the presence of stem/progenitor cells and formation of new skeletal muscle. Force production, range-of-motion and functional task performance were analysed by physical therapists. Electrodiagnostic evaluation was used to analyse presence of innervated skeletal muscle. This study is registered with ClinicalTrials.gov, numbers NCT01292876. In vivo remodelling of ECM bioscaffolds was associated with mobilisation of perivascular stem cells; formation of new, vascularised, innervated islands of skeletal muscle within the implantation site; increased force production; and improved functional task performance when compared with pre-operative performance. Compared with pre-operative performance, by 6 months after ECM implantation, patients showed an average improvement of 37.3% (P<0.05) in strength and 27.1% improvement in range-of-motion tasks (P<0.05). Implantation of acellular bioscaffolds derived from ECM can improve strength and function, and promotes site-appropriate remodelling of VML defects. These findings provide early evidence of bioscaffolding as a viable treatment of VML.
... The positive outcomes in the aforementioned study are partially attributed to an aggressive, targeted physical therapy regimen prescribed to all patients, which was implemented within 24-48 h after ECM bioscaffold implantation. The application of physiologic mechanical load (i.e., concomitant physical rehabilitation) during the ECM-remodeling period has been shown to promote favorable pre-clinical and clinical outcomes including an increased cellular infiltrate, more rapid and extensive neovascularization, more organized and aligned connective tissue matrix, and influence upon gene expression and cellular behavior (47,48). VML patients treated with ECM bioscaffolds were subjected to exhaustive physical therapy prior to bioscaffold implantation during which time they achieved a plateau in their performance. ...
Tissue engineering and regenerative medicine-based strategies for the reconstruction of functional skeletal muscle tissue have included cellular and acellular approaches. The use of acellular biologic scaffold material as a treatment for volumetric muscle loss (VML) in five patients has recently been reported with a generally favorable outcome. Further studies are necessary for a better understanding of the mechanism(s) behind acellular bioscaffold-mediated skeletal muscle repair, and for combination cell-based/bioscaffold based approaches. The present overview highlights the current thinking on bioscaffold-based remodeling including the associated mechanisms and the future of scaffold-based skeletal muscle reconstruction.
... Although synthetic polymers such as polyglycolic and polylactic acids can be used as scaffolds for skin tissue engineering, 6 collagen-based matrices are the most common scaffolds used for fabricating bioartificial skin grafts. 1 Moreover, fibroblasts loaded on individual ECM components such as collagen 7 or glycosaminoglycans 8 are known to aid wound healing. There are also reports on the use of fibroblasts loaded on natural biomaterials like chitosan, 9 fibrin, 10 hyaluronic acid 11 and ECM-derived scaffolds 12 for accelerating wound healing. It has been shown that fibroblast loaded on to chitosan-alginate sponge modulates healing of full thickness excision wounds in rabbit model. ...
Graft-assisted healing is often proposed for clinical management of large-sized third-degree cutaneous burn wounds. Skin-graft substitutes prepared by loading appropriate cell types on suitable scaffolds have been found successful. We have previously shown that cholecyst-derived scaffold prepared by a non-detergent/enzymatic method can be used as skin-graft substitute for promoting healing of full thickness excision wounds in rabbit. This article examines the use of this scaffold for preparing bio-artificial grafts by loading homologous fibroblasts. The healing potential was evaluated in a rabbit model of full thickness skin-burn wound. The healing process was evaluated by gross morphology evaluation and histomorphology evaluation at 7, 14 and 28 days of healing. Ex vivo imaging of the wounded tissue was performed and it was found that the loaded fibroblasts remained viable at least for 14 days in the healing wound. By the first week, re-epithelialisation was evident in all animals treated with the cell-loaded graft. Histomorphological wound healing parameters such as the quickness of re-epithelialisation, the nature of collagen deposition and the extent of neo-vascularisation indicated that cell-loaded grafts promoted faster healing of the wounds. Results of immunohistochemistry indicated a parallel change in the number of proliferating cells and myofibroblast in the healing tissue. Although the pathophysiology of the healing reaction was not established, the observations suggested that homologus fibroblast-loaded cholecyst-derived scaffold promoted faster healing of third-degree wounds in rabbit model by modulating myofibroblast response. It was concluded that cholecyst-derived scaffold prepared by the non-detergent/enzymatic method is a potential scaffold for fabricating bioartificial skin grafts.
... Among the multiple components derived from the ECM hydrogels that benefits wound healing and tissue remodelling [17,36], we confirmed the partial preservation of growth factors in the ECM-SIS hydrogel after processing and enzyme digestion, i.e., FGF-2 by 50% and TGF-b1 by 90% of those found in the sheet form of ECM-SIS [28]. In addition, our results on the mRNA expressions of fibroblasts are comparable with those of a previous study with cells seeding on a sheet of SIS bioscaffold [37]. ...
Background/Objective
We have previously shown that an extracellular matrix (ECM) bioscaffold derived from porcine small intestine submucosa (SIS) enhanced the healing of a gap injury of the medial collateral ligament as well as the central third defect of the patellar tendon. With the addition of a hydrogel form of SIS, we found that a transected goat anterior cruciate ligament (ACL) could also be healed. The result begs the research question of whether SIS hydrogel has positive effects on ACL fibroblasts (ACLFs) and thus facilitates ACL healing.
Methods
In the study, ECM-SIS hydrogel was fabricated from the digestion of decellularised and sterilised sheets of SIS derived from αGal-deficient (GalSafe) pigs. As a comparison, a pure collagen hydrogel was also fabricated from commercial collagen type I solution. The morphometrics of hydrogels was assessed with scanning electron microscopy. The ECM-SIS and collagen hydrogels had similar fibre diameters (0.105 ± 0.010 μm vs. 0.114 ± 0.004 μm), fibre orientation (0.51 ± 0.02 vs. 0.52 ± 0.02), and pore size (0.092 ± 0.012 μm vs. 0.087 ± 0.008 μm). The preservation of bioactive properties of SIS hydrogel was assessed by detecting bioactive molecules sensitive to processing and enzyme digestion, such as growth factors fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta 1 (TGF-β1), with enzyme-linked immunosorbent assay. ACLFs were isolated and expanded in culture from explants of rat ACLs (n = 3). The cells were then seeded on the hydrogels and cultured with 0%, 1%, and 10% foetal bovine serum (FBS) for 3 days and 7 days. Cell attachment was observed using a light microscope and scanning electron microscopy, whereas cell proliferation and matrix production (collagen types I and III) were examined with bromodeoxyuridine assays and reverse transcription-polymerase chain reaction, respectively.
Results
The results showed that FGF-2 and TGF-β1 in the SIS hydrogel were preserved by 50% (65.9 ± 26.1 ng/g dry SIS) and 90% (4.4 ± 0.6 ng/g dry SIS) relative to their contents in ECM-SIS sheets, respectively. At Day 3 of culture, ACLFs on the SIS hydrogel were found to proliferate 39%, 31%, and 22% more than those on the pure collagen hydrogel at 0%, 1%, and 10% FBS, respectively (p < 0.05). Collagen type I mRNA expression was increased by 150%, 207%, and 100%, respectively, compared to collagen hydrogel (p < 0.05), whereas collagen type III mRNA expression was increased by 123% and 132% at 0% and 1% FBS, respectively (all p < 0.05) but not at 10% FBS. By Day 7, collagen type I mRNA expression was still elevated by 137% and 100% compared to collagen hydrogel at 1% and 10% FBS, respectively (p < 0.05). Yet, collagen type III mRNA levels were not significantly different between the two groups at any FBS concentrations.
Conclusion
Our data showed that the ECM-SIS hydrogel not only supported the growth of ACLFs, but also promoted their proliferation and matrix production relative to a pure collagen hydrogel. As such, ECM-SIS hydrogel has potential therapeutic value to facilitate ACL healing at the early stage after injury.
... 108,109 This latter is especially important, as a dermal construct should stimulate fibroblast proliferation and differentiation, but conversely, the balance between cell number and ECM (production) is crucial in skin regeneration as hypertrophic scarring involves too many myofibroblasts and too much ECM. Mechanical loading of wounds causes hypertrophic scarring in mice, 110 and both in vitro and in vivo studies have shown that static as well as cyclic strain increase myofibroblast formation, 111 proliferation, 37,112,113 and differentiation (the expression of several ECM genes including a smooth muscle actin [aSMA]). 37,[112][113][114] Strain was also found to reduce myofibroblast apoptosis, 110,115 whereas its release increased apoptosis. ...
... Mechanical loading of wounds causes hypertrophic scarring in mice, 110 and both in vitro and in vivo studies have shown that static as well as cyclic strain increase myofibroblast formation, 111 proliferation, 37,112,113 and differentiation (the expression of several ECM genes including a smooth muscle actin [aSMA]). 37,[112][113][114] Strain was also found to reduce myofibroblast apoptosis, 110,115 whereas its release increased apoptosis. 116 Others have shown that aSMA is down-regulated after cyclic equibiaxial strain in vitro, 117 and these differences may result from different strain profiles. ...
... 119,120 Finally, in lung fibroblasts differentiation is reduced after cyclic strain, 121 in contrast to dermal fibroblasts that generally showed increased differentiation. 37,112,113 In gingival fibroblasts, mechanical strain leads to proliferative and antiapoptotic stimuli, 122 whereas others confirmed the induction of pro-liferation but did not observe increased expression of myofibroblast markers. 123 Cell type is thus also important in responses to mechanical strain, and results should, therefore, specifically be defined for cells from the orofacial region. ...
Cleft lip and palate patients suffer from functional, aesthetical, and psychosocial problems due to suboptimal regeneration of skin, mucosa, and skeletal muscle after restorative cleft surgery. The field of tissue engineering and regenerative medicine (TE/RM) aims to restore the normal physiology of tissues and organs in conditions such as birth defects or after injury. A crucial factor in cell differentiation, tissue formation, and tissue function is mechanical strain. Regardless of this, mechanical cues are not yet widely employed in TE/RM. However, the effects of mechanical stimulation on cells are not straight-forward in vitro as cellular responses may differ with cell type and loading regime, complicating the translation to a therapeutic protocol. We here give an overview of the different types of mechanical strain that act on cells and tissues and discuss the effects on muscle, and skin and mucosa. We conclude that presently, sufficient knowledge is lacking to reproducibly implement external mechanical loading in TE/RM approaches. Mechanical cues can be applied in TE/RM by fine-tuning the stiffness and architecture of the constructs to guide the differentiation of the seeded cells or the invading surrounding cells. This may already improve the treatment of orofacial clefts and other disorders affecting soft tissues. This article is protected by copyright. All rights reserved.
© 2015 by the Wound Healing Society.
... In fact, the ECM can transmit forces and thereby place mechanical stress on the surrounding cells, thus altering intracellular signaling and cellular function. 65 We previously mentioned that TGF-b mediates scar formation. Wipff et al. showed that growth factors such as TGF-b, which form complexes with some components of the ECM, can be released and activated under stress conditions via conformational change of the ECM. ...
Significance: Delayed wound healing is one of the most challenging complications of several diseases, including diabetes. There is a vast interest in finding efficient treatments that promote scarless wound healing. The ability of the fetus to regenerate skin wounds after injury has generated much interest in the fetus as a model of regeneration. In this review, we evaluate the role and differential regulation of inflammation, extracellular matrix (ECM) composition, and mechanical stress in determining wound phenotype after injury. Recent Advances: Comparisons between postnatal and fetal wounds have revealed many differences in the healing process. Fetal skin wound healing is characterized by a reduced inflammatory response, an ECM rich in type III collagen and high-molecular-weight hyaluronic acid (HMW-HA), and minimal mechanical stress. In contrast, adult wounds have a sustained inflammatory response, an ECM with increased type I collagen, and low-molecular-weight (LMW-HA) and are subject to significant mechanical load. Critical Issues: The differential regulation of these processes in the fetus compared with the adult plays a critical role in promoting regeneration in the fetus while resulting in scar formation in the adult. Future Directions: Understanding the significance of inflammation and biomechanical forces in wound healing may help in designing therapeutic strategies for the management of chronic nonhealing wounds.
