From mesenchymal stem cells to myoblast cell sheets: A next-generation regenerative concept for fistulizing Crohn’s disease

Abstract

Crohn’s disease is a chronic relapsing inflammatory bowel disorder characterized by impaired healing and a high risk of fistula formation. Although biologic therapies effectively suppress inflammation, durable tissue repair remains difficult to achieve because of incomplete structural regeneration. Cell sheet technology, a scaffold-free cell delivery platform that preserves extracellular matrix integrity, offers improved cell retention, sustained paracrine activity, and enhanced tissue integration compared with conventional cell suspension. Preclinical studies across multiple organ systems have indicated that both mesenchymal stem cell and myoblast cell sheets promote angiogenesis and inflammation modulation. However, mesenchymal stem cell-based approaches predominantly induce regulatory repair with a limited capacity for structural reconstruction. In contrast, myoblast cell sheets possess intrinsic myogenic potential, enabling direct muscle layer regeneration and functional restoration of the skeletal muscle. This review discusses recent advances in cell sheet technology for Crohn’s disease and highlights myoblast cell sheets as a promising reconstructive strategy for fistulizing diseases.

Key Words: Cell sheet technology; Mesenchymal stem cells; Myoblasts; Regenerative medicine; Inflammatory bowel disease; Crohn’s disease

Core Tip: Crohn’s disease fistulas remain a clinical challenge because current therapies mainly suppress inflammation without restoring the damaged tissue’s architecture. This review highlights cell sheet technology as a scaffold-free regenerative platform with superior cell retention, extracellular matrix preservation, and sustained paracrine activity. While mesenchymal stem cell therapies mainly provide regulatory repair, myoblast cell sheets add intrinsic myogenic potential, enabling reconstruction of the disrupted muscle layer and functional tissue recovery. By shifting the therapeutic goal from inflammation control to structural regeneration, myoblast cell sheets may offer a next-generation strategy for the durable repair of complex Crohn’s disease fistulas.

INTRODUCTION

Crohn’s disease (CD) is a chronic and relapsing form of inflammatory bowel disease (IBD) with a steadily rising global prevalence, and has become an increasing public health and economic burden worldwide[1]. Among its complications, fistulizing disease is one of the most challenging to manage[2]. Fistulas commonly develop between segments of the intestine (enteroenteric), between the intestine and skin (enterocutaneous), or between the intestine and adjacent organs, such as the bladder or vagina, with the terminal ileum and rectum being the most frequently affected regions[3].

Fistula development is typically a consequence of persistent transmural inflammation. Deep mucosal ulceration may extend through the muscular layer and erode into the surrounding tissues, ultimately forming an abnormal epithelialized tract[4]. The presence of a fistula can lead to recurrent infection, abscess formation, tissue destruction, leakage of enteric contents, electrolyte imbalance, and malnutrition, all of which substantially impair the quality of life[5].

Current therapeutic approaches are inadequate. Biological agents, particularly tumor necrosis factor (TNF) inhibitors such as infliximab and adalimumab, can promote fistula closure in some patients; however, sustained remission is uncommon, and recurrence rates remain high[6]. When medical treatment fails, surgical intervention becomes necessary, including drainage, fistulotomy, or bowel resection[3]. However, surgery carries notable risks, and repeated procedures do not prevent recurrence and may eventually lead to short bowel syndrome and chronic nutritional deficiency[2].

Given these limitations, durable healing of Crohn’s fistulas remains difficult to achieve. There is a pressing need for therapeutic strategies that can not only control inflammation but also restore tissue architecture and function. With advances in regenerative medicine, cell-based therapies have emerged as a promising direction and may offer new possibilities for the treatment of complex Crohn’s fistulas[7-9].

Pathogenesis of CD: From chronic inflammation to fistula formation

CD is characterized by chronic immune dysregulation and transmural inflammation of the gastrointestinal tract, presenting in a stepwise pathological evolution[10]. Rather than resolving after the initial inflammatory insult, mucosal damage progresses toward pathological tissue remodeling, excessive extracellular matrix (ECM) deposition, and ultimately penetrating complications, including fistula formation[11,12]. CD is therefore increasingly recognized not merely as an inflammatory disorder but as a chronic, progressive, structure-altering disease driven by the interplay between immune imbalance, barrier dysfunction, disrupted ECM turnover, and epithelial plasticity[13-15]. Advances in transcriptomics and single-cell profiling have further refined the conceptual framework of CD from a purely inflammatory condition to a staged disease with distinct biological programs operating across its clinical continuum[16-19].

Chronic inflammation: In the early phase of the disease, persistent immune-mediated inflammation is the predominant pathological feature[10]. Genetic susceptibility plays a central role, with variants of NOD2, ATG16 L1, and IL23R contributing to altered responses to microbial and environmental stimuli[20,21]. Barrier disruption exposes underlying immune cells to luminal antigens, leading dendritic cells and macrophages to continuously recognize microbe-associated molecular patterns and activate the canonical nuclear factor kappa B, mitogen-activated protein kinase, and Janus kinase-signal transducer and activator of transcription signaling pathways[22,23]. This results in sustained secretion of proinflammatory cytokines, including TNF-α, interleukin (IL)-6, IL-17, and interferon (IFN)-γ, amplifying the inflammatory cascade[21,22]. The adaptive immune response, dominated by the T helper (Th)1/Th17 axis, further reinforces cytokine-driven inflammation, whereas reduced expression of tight junction proteins increases epithelial permeability and facilitates antigen translocation[24]. Dysbiosis - particularly expansion of adherent-invasive Escherichia coli and loss of commensal Bacteroides - creates a self-perpetuating loop of immune activation, promoting chronicity and refractoriness to spontaneous remission[25,26].

Fibrosis and structural remodeling: With persistent inflammation, the intestinal repair process gradually shifts from physiological regeneration to pathological ECM accumulation and structural remodeling[27,28]. Elevated profibrotic signaling, including that of tumor growth factor (TGF)-β, IL-13, and platelet-derived growth factor, drives the sustained activation of fibroblasts and myofibroblasts, resulting in the excessive production of type I and III collagen and fibronectin[29,30]. Dysregulation of ECM degradation, characterized by an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors, skews tissue metabolism toward deposition rather than breakdown[31]. Chronic inflammation also generates a hypoxic microenvironment, inducing hypoxia inducible factor (HIF)-1α expression and reinforcing the fibrotic phenotype[32]. Recent single-cell sequencing and transcriptomic studies have revealed the emergence of highly activated fibroblast subsets resembling cancer-associated fibroblasts, indicating that fibrosis is not a passive scarring phenomenon but an active, self-sustaining biological process[33]. Accumulation of ECM results in bowel wall thickening, rigidity, and ultimately luminal narrowing or obstruction[34].

Mechanisms underlying fistula formation: As fibrosis and inflammation coexist and progressively deepen within the bowel wall, tissue injury advances toward penetrating complications, ultimately leading to fistula formation. This process extends beyond passive tissue breakdown and reflects an active, biologically driven remodeling response shaped by sustained microenvironmental stress and cellular plasticity[35]. Under persistent inflammatory and hypoxic conditions, epithelial cells lining the fistula tract exhibit epithelial-to-mesenchymal transition-like or transitional phenotypic changes, characterized by loss of epithelial polarity and intercellular adhesion together with enhanced migratory and invasive properties[36]. These alterations are regulated, at least in part, by profibrotic and developmental signaling pathways, most notably TGF-β/Smad signaling, with the contributory involvement of Wnt/β-catenin pathways[37]. Concurrently, the upregulation of MMPs, including MMP-3 and particularly MMP-9, promotes ECM degradation and contributes to the persistence of the fistula tract[38]. Hypoxia-associated HIF-1α signaling is linked to aberrant angiogenesis and granulation tissue expansion, whereas colonization by luminal bacteria and microbial biofilm formation may sustain localized inflammation[39]. Collectively, these mechanisms provide a mechanistic basis for the chronicity, therapeutic refractoriness, and high recurrence rate of fistulizing CD.

MESENCHYMAL STEM CELLS

Mechanisms of action of mesenchymal stem cells

With an improved understanding of the pathological continuum of CD, therapeutic strategies have gradually shifted from suppressing inflammation to restoring tissue structure and rebalancing the intestinal microenvironment[40]. Mesenchymal stem cells (MSCs) have emerged as one of the most promising cellular therapies for refractory CD, particularly fistulizing phenotypes, owing to their low immunogenicity, robust immunomodulatory capacity, and regenerative potential[41]. Various MSC sources, including bone marrow, adipose tissue, and umbilical cord, have been explored clinically, with adipose-derived allogeneic MSCs being the most advanced in translational development[42]. The current research trajectory suggests that the therapeutic goal is transitioning from “controlling inflammation” to “restoring function and reversing damage”, positioning MSC therapy at the center of this paradigm shift[43].

The therapeutic efficacy of MSCs in CD arises from their multilayered regulatory functions, including immunomodulation, epithelial barrier restoration, and intervention in pathological tissue remodeling. Unlike single-target biologics, MSCs act through cell-cell contact, paracrine signaling, and extracellular vesicle-mediated communication, enabling the dynamic modulation of complex inflammatory and structural alterations characteristic of CD[41].

At the immunomodulatory level, multiple preclinical studies have demonstrated that MSCs attenuate Th1/Th17-driven inflammatory responses and reduce the secretion of key proinflammatory cytokines, including IFN-γ, IL-17, IL-6, and TNF-α, while enhancing anti-inflammatory mediators, such as IL-10 and TGF-β[44]. In vitro experiments on intestinal immune cells have shown that MSCs suppress dendritic cell maturation via indoleamine 2,3-dioxygenase- and prostaglandin E2-dependent signaling pathways, thereby limiting activation of naïve T cells[45]. Additionally, MSCs promote macrophage polarization from inflammatory M1 to reparative M2 phenotypes, a process considered pivotal for transitioning from chronic inflammation to mucosal healing, especially in fistulizing and fibrotic microenvironments[46].

With respect to epithelial barrier repair, MSCs and their extracellular vesicles promote intestinal stem cell proliferation and homeostasis by regulating the Wnt/β-catenin and Notch signaling pathways. In murine dextran sulfate sodium-induced colitis models, MSC administration has been shown to increase expression of E-cadherin, occludin, and zonula occludens-1, indicating the restoration of tight junction integrity and mucosal barrier function. Moreover, specific MSC-derived microRNAs (miRNAs), including miR-21, miR-146a, and miR-150, have been identified as regulators of inflammatory signaling and epithelial regeneration, suggesting a molecular basis for MSC-mediated barrier repair[47].

Regarding fibrosis and fistula-associated tissue remodeling, MSCs modulate the balance between MMP-2/MMP-9 and tissue inhibitor of metalloproteinases 1, thereby improving the degradation-deposition equilibrium of the ECM. Experimental evidence indicates that MSCs downregulate TGF-β/Smad signaling, suppress myofibroblast activation, and reduce collagen type I/III accumulation, effects that are particularly relevant in stenotic and chronic fibrotic diseases[35]. In addition, within the hypoxic and ischemic microenvironment characteristic of fistula tracts, MSCs influence HIF-1α signaling and stimulate angiogenesis, potentially improving tissue perfusion and creating favorable conditions for subsequent healing[39].

Clinical evidence of MSC therapy in CD

Multiple prospective and randomized clinical studies have evaluated the therapeutic potential of MSCs in refractory CD, particularly complex perianal fistulas (Table 1)[6,42,48-52]. The first randomized controlled evidence obtained in fistulizing CD using allogeneic MSCs was reported by Molendijk et al[48], with a 40% fistula closure rate in patients treated with bone marrow-derived MSCs; no closures were observed in the placebo group, indicating a clear treatment effect independent of the cell source.

Table 1 Summary of key clinical studies evaluating mesenchymal stem cell therapy in Crohn’s disease.

Ref.	Country	n (control vs MSC)	Autologous/allogeneic	Cell type/product	Follow-up	Main outcomes
Molendijk et al[48], 2015	Netherlands	6 vs 15	Allogeneic	BM-MSC	24 weeks	Fistula healing 40% vs 0%; good safety
Panés et al[42], 2016	Europe/Israel	107 vs 105	Allogeneic	AD-MSC (Cx601, darvadstrocel)	24 weeks	Combined remission 50% vs 34%; similar safety
Panés et al[6], 2018	Europe/Israel	103 vs 102	Allogeneic	AD-MSC (Cx601)	52 weeks	Sustained remission 56% vs 36%
Zhou et al[49], 2020	China	11 vs 11	Autologous	AD-MSC	52 weeks	Healing 63.6%; CDAI & PDAI improved; safe
Garcia-Olmo et al[50], 2022	Europe/Israel	15 vs 25	Allogeneic	AD-MSC (Cx601)	104 weeks	Sustained closure approximately 65%; no long-term safety issues
Lightner et al[51], 2023	United States	2-4 vs 4-15	Allogeneic	BM-MSC/Remestemcel-L	6 weeks to 3 months	Fistula healing; CDAI & CRP improvement; acceptable safety
Lindsay et al[52], 2017	United Kingdom	9 vs 13	Autologous	HSC	48 weeks	CDAI, SES-CD, IBDQ, MARIA, PRO2 improved; reversible AEs

MSC: Mesenchymal stem cells; BM-MSC: Bone marrow-derived mesenchymal stem cells; AD-MSC: Adipose-derived mesenchymal stem cells; CDAI: Crohn’s Disease Activity Index; PDAI: Perianal Disease Activity Index; CRP: C-reactive protein; HSC: Hematopoietic stem cell; SES-CD: Simple Endoscopic Score for Crohn’s Disease; IBDQ: Inflammatory Bowel Disease Questionnaire; AEs: Adverse events.

A major milestone was achieved in the phase III ADMIRE-CD trial led by Panés et al[6], which evaluated a standardized allogeneic adipose-derived MSC product (Cx601, later named darvadstrocel). In this multicenter study involving 212 patients from seven European countries and Israel, combined clinical and magnetic resonance imaging-confirmed remission at week 24 was achieved in 50% of the MSC-treated group compared with 34% in the control group. Subsequent analyses demonstrated sustained remission in 56% of treated patients at 52 weeks, and long-term follow-up further showed maintenance of fistula closure in approximately 60%-65% of cases at 104 weeks, without the emergence of new safety signals. These results led to the regulatory approval of darvadstrocel by the European Medicines Agency as the first MSC-based advanced therapy medicinal product for complex perianal CD.

Consistent efficacy has also been reported in independent cohorts from other regions of the world. In a randomized controlled study from China, Zhou et al[49] observed a fistula closure rate of 63.6% following treatment with autologous adipose-derived MSCs, accompanied by significant improvements in CD Activity Index, Perianal Disease Activity Index, and quality of life scores. In the United States, Lightner et al[51] reported rapid clinical responses in a phase I/II study of allogeneic bone marrow-derived MSCs, with a substantial proportion of patients achieving early fistula closure within weeks of treatment and an acceptable safety profile.

Collectively, these studies demonstrate that MSC therapy consistently induces clinically meaningful fistula closure, radiological and symptomatic improvement, and durable responses with favorable safety across different cell sources and geographic locations. The reproducibility of these outcomes across phase II-III trials and long-term follow-up cohorts provides strong clinical support for MSC-based regenerative therapy as a promising option for refractory perianal fistulizing CD.

Limitations of conventional MSC therapy

Retention limitations of MSCs and the constraints of the closure-based strategy: Despite the promising immunomodulatory and pro-regenerative potential of MSCs in the treatment of complex fistulizing CD, their therapeutic efficacy remains limited by poor cellular retention and a hostile local microenvironment[53-55]. When MSCs are directly injected into the fistula tract, they are rapidly lost due to ongoing inflammatory exudation, fluctuating intraluminal pressure, hypoxic conditions, and lack of ECM anchorage[56]. Multiple cell-tracking studies have demonstrated that a large proportion of transplanted MSCs progressively disappear over days to weeks post-injection, with only a small fraction transiently retained and very few integrating into host tissues[53-57]. Therefore, the principal effect of MSCs within the fistula is transient paracrine modulation rather than durable, structural reconstruction.

To improve cell retention and prolong local therapeutic activity, some researchers have proposed a “closure-based delivery strategy”, in which the internal and external openings of the fistula are closed immediately after MSC injection[42,50]. This approach aims to minimize cellular outflow and enhance contact between MSCs and the fistula wall. Representative findings of this strategy are summarized in Table 1. Overall, in chronic, fibrotic, and inflammation-quiescent mature fistulas without active drainage, this method achieves moderate and reproducible efficacy[42,50,51]. For instance, in the ADMIRE-CD phase III trial, allogeneic adipose-derived MSCs (darvadstrocel) were used to treat complex perianal fistulas, achieving a combined clinical and radiologic remission rate of approximately 50%-56% at 24-52 weeks[6,42]. Subsequent multinational studies have reported similar closure rates and favorable safety profiles[50,51]. These findings suggest that closure-based MSC therapy is effective and safe for well-drained, infection-free, and structurally stable chronic fistulas.

However, these “successful” outcomes are highly dependent on stringent patient-selection criteria. Nearly all MSC-related clinical trials explicitly excluded patients with active intestinal inflammation or high CD Activity Index, unresolved abscesses or infections, persistent perianal discharge without established drainage, acute perforation-like or non-epithelialized fistula tracts, severe proctitis or luminal strictures, and recently formed or immature sinus-like tracts[42,51,52]. In other words, the closure-based MSC approach is applicable only to chronic, well-drained, infection-free, and structurally mature fistulas and not to active, acute, leaking, or perforation-type lesions[42,55].

When fistula closure is attempted during active disease or in the presence of uncontrolled infection, it blocks the natural drainage pathway for intestinal pressure and inflammatory exudate, resulting in a closed, high-pressure cavity[55]. Under such conditions, MSCs not only fail to remain localized but are also prone to extrusion by fluid shear or rapid loss of viability under hypoxic/inflammatory stress[53,54]. Additionally, the accumulation of exudate within the sealed cavity may lead to abscess formation, re-perforation, or recurrent leakage[55]. Therefore, the strategy of “closing the fistula to improve retention” may paradoxically exacerbate biomechanical imbalance and infection risk in unsuitable cases. The fundamental issue is not the intrinsic inefficacy of MSCs but rather the mismatch between the closure-based MSC strategy and the pathophysiological characteristics of active Crohn’s fistulas.

Structural and myogenic limitations of MSCs: Although MSCs possess potent immunomodulatory and reparative capabilities, increasing evidence indicates that their therapeutic mechanism in CD-related fistulas primarily depends on paracrine modulation rather than direct structural or myogenic regeneration. MSCs exert their effects by secreting various cytokines, exosomes, and growth factors that suppress local inflammation, promote granulation tissue formation, and stimulate angiogenesis, thereby improving the local healing microenvironment[53,54]. However, their ability to survive long-term and structurally integrate within the fistulous tract remains limited, preventing durable anatomic closure or muscular reconstruction[56].

Clinical and imaging follow-up studies consistently suggest that even when early clinical closure is achieved, anatomical and functional reconstruction remains incomplete[42,55]. In the ADMIRE-CD trial and subsequent multicenter studies, approximately 50%-56% of patients achieved combined remission at 24-52 weeks, while nearly half failed to achieve complete healing or later relapse[6,42]. Magnetic resonance imaging evaluations further revealed that post-MSC treatment, most fistulous tracts exhibited reduced inflammation and increased fibrosis; however, muscle layer and sphincter continuity were not restored, suggesting that MSCs primarily induce regulatory repair rather than reconstructive repair[42,58].

This limitation of structural restoration is particularly evident in complex fistulas characterized by severe fibrosis, muscle disruption, or chronic epithelialization[59]. Animal models have shown that although MSCs significantly reduce inflammation and promote granulation tissue formation, they fail to restore muscular continuity or contractile function in fistulas with substantial myofiber damage. These findings reflect the biological nature of MSCs as regulatory stem cells, not constructive stem cells; their reparative role relies on microenvironmental modulation rather than tissue replacement[53,54].

Moreover, long-standing complex fistulas are often accompanied by ischemia, fibrosis, and chronic hypoxia, which further impair MSC proliferation and differentiation potential. Elevated levels of TNF-α and IFN-γ in the inflammatory microenvironment can activate the p53-p21 signaling pathway, inducing a senescence-like phenotype in MSCs that diminishes their secretion of reparative factors and reduces their paracrine activity. Consequently, even when inflammation is partially controlled, MSCs struggle to exert true regenerative effects on fibrotic or hypoxic fistula tissues[59,60].

Overall, MSC-based therapies may play a greater role in short-term immunomodulation and tissue repair enhancement than in long-term structural regeneration and functional recovery in CD fistulas. This “structural insufficiency” explains why many patients experience relapse or recurrent tract formation despite the initial closure. Future research must focus on enhancing MSC differentiation potential, improving their anchorage and integration within fistulous tissue, and developing hybrid or engineered cell-based systems that combine MSCs with ECM scaffolds or lineage-committed progenitors, ultimately advancing the field from regulatory repair toward true reconstructive regeneration.

MYOBLAST CELL SHEET

Cell sheet technology

Cell sheet technology, first developed by Shimizu et al[61] in Japan, is based on the use of temperature-responsive culture surfaces that allow cells to be detached as intact sheets simply by lowering the temperature, without enzymatic digestion. Unlike conventional cell-harvesting methods, this approach preserves the ECM components, intercellular junctions (including E-cadherin and connexin-mediated gap junctions), and integrin-mediated adhesion receptors, thereby maintaining the native architecture and adhesion capacity of the cells[62]. This property makes the cell sheet a scaffold-free engineered cell delivery platform, providing a biologically and structurally intact cellular construct that closely mimics native tissue organization.

Compared with conventional MSC suspensions, the cell sheets exhibited significantly enhanced cell retention and local reparative activity. Multiple studies have shown that transplanted MSC sheets can persist at the implantation site for prolonged periods, whereas injected MSCs undergo rapid loss within days[56,61]. In myocardial infarction models[62], skin wound repair[63], and bone defect repair[64], the ECM and integrin signals preserved in cell sheets were shown to form a “bio-adhesive interface”, which markedly improved cell attachment and tissue anchorage while minimizing early cellular washout.

In addition to improved retention, MSC sheets exhibit prolonged and enhanced paracrine activity. Owing to the preservation of cell-cell and cell-ECM interactions, MSCs within sheet constructs maintain a more physiological microenvironment, which supports sustained secretion of pro-angiogenic, anti-inflammatory, and anti-fibrotic factors, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), fibroblast growth factor-2, TGF-β, and IL-10[62,65]. In contrast, MSCs delivered as single-cell suspensions rapidly lose cell-cell contact, are exposed to anoikis and shear stress, and display attenuated survival and secretory function after transplantation.

Consistent with these mechanistic advantages, a growing body of preclinical studies has directly compared MSC sheets with conventional MSC suspensions across a range of disease models, demonstrating the consistently superior therapeutic efficacy of the sheet-based approach (Table 2)[62-67]. In large-animal and rodent models of myocardial infarction, MSC sheets achieved higher engraftment, enhanced neovascularization, and greater improvement in cardiac function than intramyocardially injected or intravenously infused MSC suspensions. Similar superiority was observed in renal disease models, in which MSC sheets more effectively attenuated inflammation and interstitial fibrosis and preserved microvascular integrity than systemically delivered MSCs. In musculoskeletal and cutaneous regeneration models, including critical-sized bone defects, cartilage injury, and full-thickness skin wounds, MSC sheets showed improved tissue integration, matrix deposition, and structural repair compared to cell suspensions.

Table 2 Comparative studies of mesenchymal stem cell sheets and cell suspensions across different disease models.

Ref.	Model	MSC source	Comparison	Outcome type	Main outcome
Gao et al[62], 2022	Porcine myocardial infarction	hUC-MSC	Cell sheet vs suspension	Functional recovery	Cell sheets improved cardiac function and reduced infarct size more effectively than MSC suspension
Takemura et al[66], 2020	Rat diabetic nephropathy	AD-MSC	Cell sheet vs IV suspension	Anti-inflammatory/anti-fibrotic	Cell sheets showed greater attenuation of renal inflammation and fibrosis than suspended MSCs
Imafuku et al[65], 2019	Rat renal ischemia-reperfusion injury	BM-MSC	Cell sheet vs IV suspension	Anti-fibrotic	Cell sheets more effectively reduced renal fibrosis and preserved microvasculature than MSC suspension
Chen et al[63], 2017	Mouse skin wound/subcutaneous implantation	AD-MSC	Cell sheet vs suspension	Retention/paracrine activity	Cell sheets exhibited higher cell retention and stronger paracrine factor secretion than suspended MSCs
Long et al[64], 2014	Mouse critical-sized femoral defect	BM-MSC	Cell sheet vs suspension	Structural regeneration	Cell sheets promoted superior bone regeneration and graft integration compared with MSC suspension
Qi et al[67], 2014	Rabbit cartilage/meniscal defect	BM-MSC	Cell sheet vs suspension	Cartilage repair	Cell sheets achieved better matrix deposition and defect repair than injected MSCs

Overall, mesenchymal stem cell sheet-based delivery showed superior therapeutic efficacy compared with mesenchymal stem cell suspension, as evidenced by improved cell retention, enhanced paracrine activity, and better tissue repair. MSC: Mesenchymal stem cell; hUC-MSC: Human umbilical cord-derived mesenchymal stem cell; AD-MSC: Adipose-derived mesenchymal stem cell; BM-MSC: Bone marrow-derived mesenchymal stem cell.

In IBD, particularly in CD-related models, rigorous head-to-head comparisons between MSC cell sheets and MSC suspensions are still lacking. Therefore, definitive conclusions regarding the superiority of cell sheet-based delivery over conventional suspension therapy cannot be drawn from direct comparative evidence. Nevertheless, recent studies have explored the endoscopic transplantation of MSC sheets directly onto inflamed or ulcerated colonic mucosa, demonstrating pronounced local anti-inflammatory and tissue repair effects, thereby highlighting the feasibility and therapeutic potential of cell sheet-based localized delivery in IBD[68]. In parallel, multiple systematic reviews and meta-analyses of MSC therapy in IBD and CD have consistently reported immunomodulatory activity, attenuation of intestinal inflammation, and promotion of mucosal healing, providing a strong theoretical and experimental foundation for further evaluation of cell sheet strategies through well-designed comparative studies in this field[43,69].

Myoblasts: A potential strategy for structural repair and functional reconstruction

Although cell sheet technology markedly improves the tissue retention and local persistence of MSCs, the underlying reparative mechanism of MSC sheets remains predominantly based on microenvironmental modulation of the transplanted cells. Their principal actions involve suppressing inflammation, promoting angiogenesis, and facilitating granulation tissue formation, rather than achieving true structural or functional reconstruction[64]. In various models, including the myocardium, bone, cartilage, and intestinal mucosa, MSC sheets can enhance tissue stability and healing; however, they generally lack the capacity to fully restore the muscle layer, connective tissue architecture, and contractile function[66]. This indicates that while cell sheet technology effectively overcomes the limitation of poor cell retention, its regenerative outcome is still constrained by the intrinsic biological properties of MSCs. Accordingly, MSC-based repair represents a form of “regulatory repair” rather than “reconstructive repair”.

Consequently, it has become increasingly recognized that genuine structural regeneration and functional recovery require cell types with intrinsic myogenic differentiation potentials. In this context, skeletal myoblasts derived from muscle satellite cells have emerged as a more advantageous cell source than MSCs for cell sheet-based regenerative therapy owing to their committed myogenic lineage identity. In addition to secreting reparative and immunomodulatory factors, such as VEGF, HGF, and stromal cell-derived factor 1(SDF-1), myoblasts can directly differentiate into mature myofibers, thereby reconstructing the muscle layer and restoring local contractile function[70,71]. When delivered in the form of cell sheets, myoblasts preserve intact cell-cell and cell-ECM interactions, exhibit high in vivo retention, and sustain paracrine signaling, while simultaneously enabling true structural regeneration of muscle tissue[70]. These combined properties confer myoblast sheets with the dual advantages of microenvironmental modulation and architectural reconstruction, making them particularly attractive for the treatment of inflammatory fistulas and tissue defects associated with muscular destruction, and highlighting their potential superiority over conventional MSC-based therapies.

Multiple animal and preclinical studies have demonstrated that skeletal myoblast sheets, as an adhesive cell delivery platform, provide more stable local cell retention and sustained paracrine support in ischemic and injured microenvironments, thereby promoting structural and functional tissue repair. Memon et al[70] reported that transplantation of myoblast sheets in myocardial injury models significantly improved cardiac function and reduced fibrosis and adverse remodeling, with the therapeutic benefit being associated with the sustained release of SDF-1 and other growth factors and possible recruitment of endogenous reparative cells. Hata et al[71] employed a pacing-induced canine heart failure and remodeling model to show that autologous skeletal myoblast sheets attenuated pathological ventricular remodeling and improved cardiac function. In addition, Wang et al[72] performed human myoblast transplantation into a porcine myocardial infarction model and found that the treatment downregulated expression of pro-inflammatory mediators, such as IL-1β and TNF-α, while upregulating IL-10, accompanied by reduced infarct size and fibrosis and improved cardiac performance, indicating a pronounced immunomodulatory and anti-fibrotic effect.

Comparative studies also support the potential advantages of myogenic cells over MSCs in terms of both structural and functional repair. In a rat model of ischemic cardiomyopathy, Shudo et al[73] found that skeletal myoblast cell sheets exerted stronger reparative effects than MSC-only sheets, with enhanced paracrine activity, increased HGF expression in the infarcted area, and activation of Akt-1/Bcl-2 survival signaling. Burdzinska et al[74] reported that autologous muscle-derived cells produced more durable functional recovery than bone marrow-derived MSCs in a urethral transplantation model using aged multiparous ewes, as evidenced by greater improvements in the maximum urethral closure pressure and functional area at 3 months post-treatment. Similarly, Guarita-Souza et al[75] showed that myoblast transplantation improved ejection fraction and promoted new myofiber formation more directly than MSC transplantation in rats with severe myocardial infarction. Yet, successful engraftment has not been observed in all settings. Kihara et al[76] reported that both myoblasts and adipose-derived MSCs failed to engraft effectively after in utero transplantation in a mouse model of Duchenne muscular dystrophy; nevertheless, under transplacental transplantation, the fetal survival was higher in the myoblast group than in the adipose-derived MSC group, suggesting better tolerability of myoblasts in this specific condition.

In the context of gastrointestinal tissue repair, skeletal myoblast sheets exhibit marked regenerative and anti-inflammatory properties. Yamaguchi et al[77] demonstrated in a rat acetic acid-induced gastric ulcer model that serosal application of myoblast sheets accelerated ulcer healing and was associated with the temporal upregulation of HIF-1α, SDF-1, and VEGF, along with enhanced angiogenesis. The Kanetaka’s group at Nagasaki University has conducted a series of studies on cell sheet-based regeneration in the gastrointestinal tract[78-81]. Matsumoto et al[78] reported in a porcine duodenal endoscopic submucosal dissection model that autologous myoblast sheets significantly reduced the incidence of delayed perforation and promoted connective tissue remodeling. Subsequently, Kanetaka et al[79] conducted the first-in-human clinical study in which autologous myoblast sheets were laparoscopically transplanted to duodenal post-endoscopic submucosal dissection defects, confirming the safety and feasibility of this approach without perforations or stricture formation. Furthermore, Tanaka et al[80] investigated a rat pancreatic fistula model and found that myoblast sheets reduced local inflammation and pancreatic juice-associated tissue injury, while Nakamura et al[81] investigated a high-risk colonic anastomosis model and found that multilayered human myoblast sheets increased anastomotic bursting pressure, enhanced early collagen deposition, and suppressed inflammatory cell infiltration, thereby promoting anastomotic healing and reducing postoperative complications.

Clinical translation and challenges of myoblast cell sheets

Although myoblast cell sheets have strong regenerative and reparative potential, their clinical translation still faces several challenges. Based on results from various other disease models, it may also be potentially useful for CD. Most current findings are derived from animal models of myocardial injury, gastric ulcers, duodenal post-endoscopic submucosal dissection defects, pancreatic fistula, or colonic anastomosis, rather than Crohn’s fistula models. This is partly because Crohn’s is a chronic immune-mediated inflammatory disease with a complex pathological process that is difficult to fully reproduce in animal models[82]. More disease-relevant models are needed to accurately assess their therapeutic value in this setting.

Before clinical application, more practical quality assessment criteria should be established, including myoblast purity, cell viability, microbial contamination, sheet thickness and integrity, and ECM preservation[83,84]. For CD-related fistulas, the delivery of the cell sheet and its stable retention at the target site remain important practical issues. Current cell sheet therapy usually requires exposure of the lesion by endoscopy, laparoscopy, or open surgery, followed by attachment of the cell sheet to the tissue defect[68]. However, Crohn’s fistulas are often not simple surface wounds, but instead they are deep, narrow, branching, or irregular tract-like structures frequently accompanied by chronic inflammation, fibrosis, infection, drainage, and fragile surrounding tissue[56]. These features make it difficult to accurately place the cell sheet and maintain its local retention for a time duration that is sufficient for reparative effects to be achieved. Therefore, future studies should focus on feasible delivery routes, fixation methods, and transplantation timing in fistula models that closely resemble clinical CD.

Given the complex inflammatory and structural features of CD-related fistulas, future studies should explore combination strategies that will enhance the therapeutic potential of myoblast cell sheets. For example, combining myoblast cell sheets with MSCs or other supportive cells may help promote angiogenesis, paracrine activity, tissue integration, and functional repair[84]. In addition, bioengineering strategies such as growth factor-releasing hydrogels, organoid therapy, intestinal tissue engineering, and 3D bioprinting may provide additional support for epithelial regeneration, local delivery of reparative factors, and structural reconstruction[85]. However, these strategies remain exploratory in clinical practice, and further studies are required to determine whether they can improve the therapeutic efficacy and stability of myoblast cell sheets.

Prospects of myoblast cell sheet therapy for CD

Compared with MSC-based therapies, which mainly act through immunomodulation and paracrine signaling, myoblast cell sheet therapy represents an alternative regenerative approach with intrinsic myogenic potential. MSCs primarily contribute to inflammation control, angiogenesis, and microenvironmental regulation; however, their ability to restore disrupted tissue architecture, particularly of the muscle layer, remains limited. In contrast, myoblast cell sheets may provide similar paracrine effects while offering the potential for structural support through muscle-like tissue formation. This distinction suggests that MSC-based therapies mainly promote regulatory repair, whereas myoblast-based approaches may contribute to aspects of structural repair, representing a potential extension of current regenerative strategies rather than direct replacement. The key differences between these approaches are summarized in Table 3.

Table 3 Comparison between mesenchymal stem cell therapy and myoblast cell sheet therapy.

Item	MSC therapy	Myoblast cell sheet therapy
Main effect	Immunomodulation and improvement of the local inflammatory environment	Local repair with potential muscle layer reconstruction
Delivery	Usually local injection; cells may be easily lost	Transplanted as a cell sheet, with better local retention
Repair pattern	Regulatory repair	Potential structural support and repair
Evidence in CD	Supported by several clinical studies	Direct evidence remains limited
Potential role	Promotes fistula closure and inflammation control	May support tissue reconstruction in complex fistulas

MSC: Mesenchymal stem cell; CD: Crohn’s disease.

From a clinical perspective, myoblast cell sheet therapy may help address an unmet aspect of Crohn’s fistula management: The incomplete restoration of tissue structure. Although current medical and surgical treatments can control inflammation and promote fistula closure in some patients, full reconstruction of the muscle layer and long-term functional recovery remain challenging. In this context, myoblast cell sheets could be considered a complementary option for supporting local tissue integrity. Importantly, this approach is unlikely to be effective as a standalone therapy. Given the systemic and immune-mediated nature of CD, the most likely potential role for myoblast cell sheet therapy would be in combination with established anti-inflammatory and surgical strategies. For example, MSC-based therapies may help regulate the inflammatory response and improve the local environment, while myoblast cell sheets can contribute to the structural support. In addition, emerging bioengineering approaches, such as growth factor delivery and tissue engineering platforms, may further expand the range of combinatorial strategies. Such integrated approaches may provide a more balanced way to address inflammation and tissue damage.

CONCLUSION

Myoblast cell sheet therapy, alongside MSC-based approaches, may represent a potential extension of current regenerative strategies for fistulizing CD, particularly with regard to structural tissue support. The integration of immunomodulatory treatments and other regenerative approaches may broaden the therapeutic options. Further studies will clarify its efficacy, safety, and clinical profiles for successful translation into clinical practice.