Skeletal muscle derived stem cells microintegrated into a biodegradable elastomer for reconstruction of the abdominal wall
Introduction
A variety of techniques have been applied to generate tissue engineered constructs, where cells are combined with degradable scaffolds followed by a period of in vitro culture or direct implantation [1], [2], [3]. For example, three dimensional (3D) printing where a cell-suspending bioink is printed onto a collector plate is attractive in that it does not require cell seeding and culture [4]. Furthermore, the fabrication can be altered by selecting cell type and bioink carrier. However, mechanical weakness is often an issue because the mechanical properties of the created construct is determined by the bioink utilized. This material has to both carry the cells and then be amenable to a process of stiffening to obtain the desired mechanical properties (often using crosslinking chemistries). On the other hand, polymer electrospinning has the advantage of being able to deposit polymers of relatively high mechanical strength using solvent processing, but with the disadvantage that subsequently achieving high cell seeding densities in the interfiber space can be problematic due to the high fiber densities normally generated. To overcome this limit, researchers have used methods to reduce the density of fibers during the deposition process, providing subsequently seeded cells larger pathways for migration inwards [5]. Scaffolds have also been processed after the fiber deposition process by laser ablation [6] or dissolution of secondary fiber populations [7] to provide for better cellular ingress. An alternative approach to this challenge is to utilize a process of concurrent polymer electrospinning and cell electrospraying, where fibers and cells are deposited together to result in a “microintegrated” cellularized construct [8], [9]. This technique has the advantage of occurring in one step such that subsequent cell seeding and cellular ingress into the scaffold are not required. With judicious selection of polymer and cell type, this concurrent electrodeposition technique offers the potential to rapidly generate conduit and sheet constructs for a variety of applications.
Our previous work has demonstrated the general feasibility of creating “microintegrated” tissue constructs by the concurrent electrodeposition technique [5], [8], [9]. This technique is unique in that cells are seeded three dimensionally while building the synthetic scaffold. The electrodeposited cells were well preserved in the processing (over 95% viability) and maintained proliferative ability while the scaffold achieved attractive tensile mechanical properties from the concurrently deposited fibers. However, the stability of the seeded cells and their potential contribution to the construct remodeling process has not been evaluated in vivo. To investigate the performance of one of these types of scaffolds, we have concurrently electrospun a biodegradable elastomeric polymer while electrospraying skeletal muscle-derived stem cells (MDSCs) in culture medium. The resulting micro-integrated tissue construct was evaluated in a rat abdominal wall muscle replacement model. The cell type selected, MDSCs, are considered a promising somatic stem cell for use in tissue engineering in that they may not be restricted to the myogenic lineage or mesenchymal tissues, and can differentiate into multiple lineages [10], [11]. These cells also are known to secrete many growth factors and cytokines to impact tissue regeneration [11], [12], [13], [14], [15], [16]. The polymer utilized, poly(ester urethane)urea (PEUU), has been previously characterized in a variety of soft tissue settings, including the abdominal wall [17], [18], [19], [20], and can be processed to possess mechanical properties that approximate certain soft tissue behavior. Since tracking of the MDSCs associated with the implanted construct was desired, green fluorescent protein positive (GFP+) transgenic rats were utilized as tissue donors to isolate GFP + MDSCs. The GFP + tissue constructs were implanted in GFP- strain-matched rats. Over an 8 wk implantation period, with construct explantation occurring at 4 and 8 wk, the presence of the loaded cells in the construct was determined. Also determined was the mechanical behavior and histological characteristics of the MDSC-loaded construct versus a control construct where culture medium without cells was electrosprayed. Biaxial, as opposed to uniaxial, tensile testing was performed since in situ the construct or tissue fibers would be constrained and forced to undergo more stretching than rotation. This would not be the case with uniaxial tension.
Section snippets
MDSC isolation
MDSCs were isolated from the skeletal muscle of 3-wk-old green fluorescent protein (GFP) transgenic SD rats hind limb, as previously described [21], by a modified preplating technique. The GFP transgenic rats were kind gifts from Dr. Kimimasa Tobita, University of Pittsburgh, originally provided by Prof. Masaru Okabe of Osaka University. The obtained MDSCs were expanded in proliferation media (Dulbecco's Modified Eagle's Medium, Invitrogen, Carlsbad, California), 10% fetal bovine serum
Construct characteristics pre-implantation
Cell integration into the scaffold after processing was confirmed with H&E staining (Fig. 1B) and SEM (Fig. 1C). MDSCs were seen to be deposited between the fibers and across the construct thickness. SEM images after 5 days of culture showed that the microintegrated cells had expanded and appeared to increase on the scaffold surface (Fig. 1D). The constructs were found to be mechanically robust with qualitatively good handling characteristics. Under equi-biaxial tension (Fig. 1E), constructs
Discussion
Volumetric muscle loss (VML) is a serious skeletal muscle defect due to traumatic or surgical damage or ventral hernia, where spontaneous muscle regeneration is not achievable and the result is a persistent functional deficit. Current approaches to this challenge are either biological or synthetic material implantation to the defect to obtain mechanical support, but accepting a lack of de novo functional muscle regeneration [26], [27]. Cell integrated scaffold implantation may provide a
Conclusions
In summary, a cellularized construct was created which consisted of MDSCs microintegrated among sub-micron scale PEUU fibers, fabricated by a concurrent electrodeposition method. In a rat abdominal wall defect model there was evidence of extended cell survival, together with cytokine expression by the implanted cells. Compared to acellular controls, the MDSC constructs showed positive effects for the generation of tissue in situ with higher vascularity, faster absorption of implanted material
Acknowledgements
This work was supported in part by the Armed Forces Institute for Regenerative Medicine (AFIRM, #W81XWH-08-2-0032, W81XWH-14-2-0004). We would also like to thank Prof. Michael S. Sacks for use of the biaxial mechanical testing device, Deanna Rhoads and Hongbin Jian for histological sectioning, and the Center for Biological Imaging at the University of Pittsburgh for assistance in imaging.
References (33)
- et al.
Material strategies for creating artificial cell-instructive niches
Curr. Opin. Biotechnol.
(2012) - et al.
Combination scaffolds of salmon fibrin, hyaluronic acid, and laminin for human neural stem cell and vascular tissue engineering
Acta Biomater.
(2016) - et al.
The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers
Biomaterials
(2008) - et al.
Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix
Biomaterials
(2006) - et al.
Fabrication of cell microintegrated blood vessel constructs through electrohydrodynamic atomization
Biomaterials
(2007) - et al.
Muscle-derived stem cells for tissue engineering and regenerative therapy
Biomaterials
(2007) - et al.
Effect of VEGF on the regenerative capacity of muscle stem cells in dystrophic skeletal muscle
Mol. Ther.
(2009) - et al.
Muscle derived stem cell contains the potential to enhance long term retention as well as an aesthetic outcome of autologous fat grafting
Med. Hypotheses
(2011) - et al.
A relationship between vascular endothelial growth factor, angiogenesis, and cardiac repair after muscle stem cell transplantation into ischemic hearts
J. Am. Coll. Cardiol.
(2007) - et al.
Morphological and mechanical characteristics of the reconstructed rat abdominal wall following use of a wet electrospun biodegradable polyurethane elastomer scaffold
Biomaterials
(2010)
Mechanical properties and in vivo behavior of a biodegradable synthetic polymer microfibereextracellular matrix hydrogel biohybrid scaffold
Biomaterials
Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes
Biomaterials
Surgical meshes coated with mesenchymal stem cells provide an anti-inflammatory environment by a M2 macrophage polarization
Acta Biomater.
Immunomodulation by mesenchymal stem cells combats the foreign body response to cell-laden synthetic hydrogels
Biomaterials
Enhanced osteogenic and vasculogenic differentiation potential of human adipose stem cells on biphasic calcium phosphate scaffolds in fibrin gels
Stem cells Int.
Bioprinting and differentiation of stem cells
Molecules
Cited by (31)
Preclinical research studies for treating severe muscular injuries: focus on tissue-engineered strategies
2023, Trends in BiotechnologyCitation Excerpt :Given their myogenic potential and preserved stemness [55], SatSCs have been used for skeletal muscle regeneration (Table 1) [24,25,29,35,37,42]. In general, myogenesis was improved when SatSCs were transplanted, independently of the biomaterial used [25,29,35,42], as evidenced by the expression of myogenic factors, such as dystrophin [29], Pax7 [25,42], MyoD [42], and myogenin [35]. Interestingly, myogenesis was achieved even when a very low number of cells (250 isolated cells) was used within the biomaterial [25].
Injectable laminin-biofunctionalized gellan gum hydrogels loaded with myoblasts for skeletal muscle regeneration
2022, Acta BiomaterialiaCitation Excerpt :Probably, additional probing is required at an earlier time-point. Although fast twitching/adult fibers (MHC) were found among all groups at early time-points, nonetheless, MHC expression decreased after eight weeks, as previously reported [49]. Although unexpected, it might be attributed to the myofiber type transformation under multiple factors including altered physical activity, to name a few [50].
A biomimetic triple-layered biocomposite with effective multifunction for dura repair
2021, Acta BiomaterialiaDecellularized biologic muscle-fascia abdominal wall scaffold graft
2021, Surgery (United States)