Muscle Regeneration of the Tongue: A Review of Current Clinical and Regenerative Research Strategies

Abstract

Various abnormalities of the tongue, including cancers, commonly require surgical removal to sequester growth and metastasis. However, even minor resections can affect functional outcomes such as speech and swallowing, thereby reducing quality of life. Surgical resections alone create volumetric muscle loss whereby muscle tissue cannot self-regenerate within the tongue. In these cases, the tongue is reconstructed typically in the form of autologous skin flaps. However, flap reconstruction has many limitations and unfortunately is the primary option for oral and reconstructive surgeons to treat tongue defects. The alternative, but yet undeveloped, strategy for tongue reconstruction is regenerative medicine, which widely focuses on building new organs with stem cells. Regenerative medicine has successfully treated many tissues, but research has inadequately addressed the tongue as a vital organ in need of tissue engineering. In this review, we address the current standard for tongue reconstruction, the cellular mechanisms of muscle cell development, and the stem cell studies that have attempted muscle engineering within the tongue. Until now, no review has focused on engineering the tongue with regenerative medicine, which could guide innovative strategies for tongue reconstruction.

Impact statement

Unlike other bodily organs, the current literature has inadequately addressed the tongue as a vital organ in need of tissue engineering. Therefore, this review seeks to highlight the clinical challenges of tongue reconstruction, alternative tissue engineering strategies, and to summarize the studies involving muscle regeneration within the tongue. This information will guide maxillofacial surgeons and tissue engineering scientists to pursue innovative strategies that alleviate volumetric muscle loss in the tongue.

Background

According to the American Cancer Society, the year 2020 expects more than 50,000 new cases of oral cancer, with one third of these cases occurring in the tongue.¹ The first treatment method for oral tongue cancer (OTC) is typically surgical resection, characterized as partial, hemi, subtotal, or total glossectomy.² As the tongue has complex physiological functions, radical resections can have lifelong consequences by impairing speech, food intake, mastication, and deglutition. Therefore, patients with severe tissue defects following resection of OTC are confined to extensive reconstruction.³

The primary aim of tongue tissue reconstruction is to restore a patient's ability to successfully speak and swallow post-resection. However, the standard care for functional repair of partial tongue defects, especially that of radical resection, lack consensus. As tumors invade muscle tissue, critical glossectomy defects stunt natural muscle regeneration, thereby promoting postoperative complication. Therefore, it is important to consider muscle tissue replacement to improve functional outcomes.

This review summarizes tongue anatomy, current clinical reconstructive methodologies, myogenesis, and muscle regenerative literature of the tongue. Our purpose is to aid maxillofacial surgeons and tissue engineering scientists in pursuing innovative strategies for alleviating volumetric muscle loss in the tongue.

Structure of the Tongue

The tongue is divided into two main sections: the anterior two third (the body) occupies the oral cavity and the posterior one third (the base) occupies the oropharynx, being proximal to the epiglottis. Average adult tongues are ∼3 inches long as measured from the epiglottis to the apex, doubling in size from the neonatal tongue.⁴

The tongue is predominantly a muscular organ that originates from occipital somites of the embryonic mesoderm germ layer.⁵ Interiorly, eight muscles entwine into a flexible matrix that allows its complex movements (Fig. 1). Four extrinsic muscles function to move the tongue around the mouth, whereas four intrinsic muscles change its shape and are the only muscles that function independent of the skeleton.

FIG. 1.

Muscle anatomy of the tongue. The tongue consists of four intrinsic muscles (superior longitudinal, inferior longitudinal, vertical, and transverse muscles) and four extrinsic muscles (genioglossus, hyoglossus, styloglossus, and palatoglossus muscles). The intrinsic muscles are the only muscles known to function without bone attachment. Color images are available online.

The muscles are both innervated by sensory (lingual, chorda tympani, and glossopharyngeal) and motor (hypoglossal and vagus) nerves. The hypoglossal nerve innervates all muscles of the tongue except for the palatoglossus muscle, which is controlled by the vagus nerve. During surgical procedures, attention is given to preserving innervation and sensation of the tongue. Exteriorly, the tongue's surface is coated with nonkeratinized stratified squamous epithelium tissue. Additionally, this surface has bumpy projections called papillae, which house thousands of taste buds.

Altogether, the dynamic structure of the tongue is critically important to speaking and swallowing. Therefore, any disease or trauma to its structure will require medical treatment. Being a vascularized organ rich in lymphatics, the tongue is highly predisposed to malignancy. In the United States, oral cancer is the eighth most common cancer among men, which frequently occurs in the tongue.¹ Oral cancers are usually associated with either the human papillomavirus or a history of alcohol and smoking.^6,7 More than 90% of oral cancers are squamous cell carcinomas, which typically begin on the tongue's lateral border.^8,9 The 5-year relative survival rate is 65% but increases to 84% when diagnosed locally.¹ Other rare conditions of the tongue include verrucous carcinoma, lymphoma, or benign tumors.^10–12

Tongue Reconstruction Methods

Despite various ailments of the tongue, a common treatment strategy is surgical resection, which effectively removes a tumor or precancerous lesion. However, the surgery itself creates a muscle defect, which predisposes functional debilitations. In other words, if the resection is too large, then muscle tissue will not self-renew, leaving patients with speech impediments, longer meal times, and trouble swallowing. For example, patients with complex tongue defects typically struggle to actively move their food bolus posteriorly within the oral cavity. Therefore, successful transfer of food bolus before the swallowing reflex requires a volumetric tissue presence.^13,14 In these cases, tongue reconstruction is necessary, which may occur simultaneously with tumor resection or in a follow-up surgery.

Smaller tongue defects may be closed primarily; however, complex defects often require local, regional, or microvascular flaps in an effort to reproduce the tongue's original biomechanics. In flap surgery, skin tissue with blood supply is extracted from a less needed site and implanted into the tongue. Flaps are named based on the original donor site, having various applications, advantages, and limitations, which are summarized in Table 1.^3,15–25 The two most common sites are the radial forearm free flap (RFFF) and anterolateral thigh flap (ALTF). The RFFF are advantageous due to its thin flexible tissue and its consistent anatomy that can achieve both neural and vascular anastomosis, from the lateral antebrachial cutaneous nerve and radial artery, respectively.³ In addition, the relative ease of extracting an RFFF allows simultaneous harvest of the tumor and the flap within one surgery.

Table 1.

Current Methods for Tongue Tissue Reconstruction

Reconstructive technique	Application/indication	Pros	Cons
Primary closure	Wedge-shaped resection Type 1	No flap required	Loss of bulk or volume to tongue
FRFF	Partial, hemi, total glossectomy, anterior tongue, floor of mouth¹⁵	Well established, common Does not necessitate a skin graft Does not involve the sacrifice of an artery or possible damage to the tendons of the hand Consistent flap vascular anatomy¹⁶ Acceptable donor site morbidity¹⁷ Thin tissue, easy to manipulate No sacrifice of muscle tissue¹⁵	Insufficient flap bulk for larger defects Risk of antebrachial lateral cutaneous nerve anastomosis¹⁸
ALTF	Total glossectomy, base of tongue, large defects	No noticeable donor site deformity No loss of a major artery Minor postoperative paresthesia¹⁹ Reduced incidence of infection compared with FRFF¹⁶	Bulky flap Difficult dissection Most common complication is marginal necrosis²⁰
PMMCF	Hemiglossectomy	Well established, reliable Require less operating time compared with FRFF²¹	Requires careful pedicle identification Slightly higher risk of necrosis compared with FRFF²¹
Infrahyoid flaps (sternothyroid, sternohyoid, thyrohyoid, and omohyoid)	Partial, hemi, total glossectomy	Do not require a second surgical team Viable alternative when free tissue transfer is contraindicated²⁰	Compromise the infrahyoid muscle group Rotational flap is limited by the length of the vascular pedicle²² Contraindicated by previous neck dissection or by the presence of metastatic nodal disease
RAFF	Hemiglossectomy, salvage surgery	Technical advantages including decreased subcutaneous fat with thinness and pliability²³ Covers large defects, bulky	Muscle loss Possible abdominal hernia³ Atrophic changes
LDF	Subtotal, total glossectomy	Bulky, covers large defects Ability to retain bulkiness if innervated³	Muscle loss Positioning is difficult, difficult to contour
FOSCFF	Mandible, floor of mouth	Easy to contour No psychiatric compromise²⁰	Skin paddle limited Potential for pathological fracture at donor site
Vertical lower trapezius island myocutaneous flap	Hemi, total glossectomy²⁴	Viable option for patients having poor health, radiation therapy, or neck dissections¹⁸ Donor site is closed primarily	Risk of shoulder weakness Prone positioning, often requiring two-stage procedure²⁵

ALTF, anterolateral thigh flap; FOSCFF, fibula osteocutaneous flap; FRFF, free radial forearm flap; LDP, latissimus dorsi flap; PMMCF, pectoralis major myocutaneous flap; RAFF, rectus abdominis free flap.

However, the RFFF does leave a noticeable scar and sometimes causes weakness and numbness of the hand.³ In contrast, the ALTF has a less noticeable site of scarring, no loss of a major artery, and no postoperative paresthesia.²⁰ However, the anatomy of the ALTF is less consistent than that of the RFFF.³ One study compared the functional outcomes of 30 OTC patients receiving an RFFF or ALTF for tongue reconstruction.²⁶ Although there were no significant differences in the success of oral feeding or intelligible speech, the ALTF were relatively bulkier, which may limit the tongue's mobility. Conversely, four patients receiving an RFFF developed infection due to inadequate flap volume. None of the patients had major complications postsurgery, but the original contours of the flaps did change due to gradual atrophy.

Other flaps are derived locally such as from the infrahyoid muscle group (isolated from the neck) or the lower trapezius muscle. Local flaps are advantageous for shorter operating times, but infrahyoid flaps are not ideal for patients with prior neck dissections, which are often necessary to prevent tumor metastasis.²⁰ In contrast, the extended vertical lower trapezius island myocutaneous flap has been recommended for patients with previous neck dissections.²⁰ Other nonlocal sites include the rectus abdominis musculoperitoneal flap, which is commonly used for vaginal defects, but can be used for tongue reconstruction due to its thinness and flexibility.²⁰

Structural Shortcomings of Current Reconstructive Techniques

Flap reconstruction is deemed necessary to restore tongue volume in patients who undergo resection of more than half their tongue.²⁷ Although these procedures offer an immediate improvement, they are not without complication or shortcomings. Flaps are the primary option for reconstructing the tongue postsurgery, leaving surgeons with limited treatment strategies. Surgeons must ensure enough tissue is extracted to construct the flap, but if the donor defect is too large, then this creates new complications. In layman's terms, flaps are a “robbing Peter to pay Paul” strategy. Additionally, some flaps undergo necrosis, which may be one reason why some patients do not have improved functional outcomes following tongue reconstruction.^28,29

Necrosis should not be confused with apoptosis, which is an organized cell death when cells have completed their function. Instead, necrosis is premature cell death caused by an insufficient blood supply that forces cells to swell and trigger an inflammatory response so that macrophages clean up the cell debris. Since flaps are derived from a second surgical site, this increases the risk of infection and necrosis. Radiation therapy following surgery is another concern, as radiation induces significant flap volume loss.³⁰ Overall, flap shrinkage makes it difficult to (1) approximate tissue amount and (2) confidence that the flap will permanently sustain.

Another complication of using flaps is hair growth on the tongue, which is likely due to the presence of hair follicles from the donor site. Intraoral hair growth in the tongue creates a tremendous amount of awkward discomfort and reduced quality of life. It could be treated with laser hair removal, but some patients have refused this option due to past surgical experiences and could result in further complications, such as cysts.³¹ Finally, flaps predominantly consist of skin and adipose tissue, thereby missing the overall goal of restoring functional muscle tissue. It is therefore our goal to encourage science to investigate alternative tongue reconstructive solutions by instead regenerating new tissue. Since tongue resections involve significant muscle deformation, we therefore will focus on muscle regeneration from stem cells and review the available muscle regenerative literature of the tongue.

Molecular Regulation of Muscle Regeneration

A potential alternative to flap reconstruction is engineering new muscle tissue with regenerative medicine. The core components of regenerative medicine are stem cells and biomaterials. In recent years, the cells of choice are the mesenchymal stem or mesenchymal stromal cells (MSCs). MSCs can be isolated from a variety of adult somatic tissue and hence alleviate the ethical concerns of using embryonic stem cells.³² According to the International Society for Cell and Gene Therapy (ISCT), MSCs are nonspecialized, adhere to plastic for proliferation, express specific cluster-of-differentiation markers, and have the ability to differentiate into a cell lineage with specific biological functions.³³ On the other side, biomaterials can (1) fill gaps for structural support and (2) attach, deliver, and cue stem cell differentiation into specialized cell lineages. Therefore, differentiating stem cells into functional muscle cells is a novel and feasible strategy. For this strategy to be efficacious, one must consider the genetic and cell signaling mechanisms involved in myogenesis.

When muscles are injured or damaged, native tissues cue satellite cells (SCs) to synthesize new muscle tissue. SCs are sandwiched between the basal lamina and sarcolemma of muscle fibers. Active SCs undergo myogenesis by distinct phases of proliferation, differentiation, fusion, and maturation (Fig. 2). SCs are unipotent stem cells but should not be confused with muscle-derived stem cells, which are multipotent and not restricted to the myogenic lineage.^34,35

FIG. 2.

The stages of muscle regeneration. Muscle regeneration occurs intrinsically from activated satellite cells, which rapidly proliferate before differentiating and fusing into myotube structures. Each stage of muscle regeneration is genetically regulated by Pax3/Pax7 (activation); MyoD, MyoG, Mrf5, Myf6 (differentiation); Mef2, Myomaker, MHC, desmin (fusion and maturation). MHC, myosin heavy chain. Color images are available online.

Rapidly proliferating SCs are referred to as adult myoblasts³⁶; when cued, myoblasts stop proliferating, exit the cell cycle, and begin differentiation. During differentiation, myoblasts morphologically transit from round to elongated myocytes. These myocytes fuse into extraordinarily long multinucleated myotubes, which either join native myofibers or collectively mature into new myofibers. A small population of myoblasts do not differentiate and instead reverse into quiescent SCs.³⁷

Pax3 and Pax7 are two genes that initiate myogenesis. However, Pax3 appears more important in embryonic myogenesis rather than muscle regeneration. It was found that Pax7 (but not Pax3) was upregulated in adult muscle regeneration, suggesting that Pax7 replaces the embryonic role of Pax3.³⁸ Pax7 is believed to play two roles during myogenesis by (1) stimulating myoblast proliferation and (2) inhibiting terminal differentiation by reversing some (but not all) myoblasts into quiescence as storage for the next future event.³⁶

Muscle differentiation is positively regulated by a family of transcription factors known as myogenic regulator factors (MRFs), which are: myoblast determination protein 1 (MyoD), also known as myogenic factor 3; myogenin (MyoG), also called Myf4; myogenic factor 5 (Mrf5); and myogenic regulatory factor 6 (Myf6), also known as Mrf4 or herculin. The MRFs are a family of basic helix-loop-helix transcription factors, which dimerize with E proteins and bind to genomic E-boxes, a motif found in the promoters of many muscle-specific genes.^39,40 Expression of any one of the four MRFs is known to establish myoblast identity, terminal differentiation, and stimulation of the entire skeletal myogenesis pathway.⁴¹ A co-regulator of the MRFs is myocyte enhancer binding factor 2 (MEF2), which binds promoter sites (MEF2 sites or TATA box) and thereby recruits MRFs for transcriptional activity.⁴¹

Although Pax7 switches on myogenesis, it also switches off myoblast proliferation and differentiation. At the start of muscle regeneration, Pax7 suppresses the MyoD activity so SCs have an opportunity to self-renew before differentiation.³⁶ When myoblasts stop proliferating, Pax7 expression declines while MyoD and MyoG expression increases.^36,42 It appears that MyoD functions downstream of Pax7 but upstream of MyoG; however, other reports characterize Mrf5 as the earliest marker of muscle differentiation.⁴³ When cells have differentiated, MyoD restimulates Pax7 expression as a negative feedback mechanism to suppress its function and terminal muscle differentiation.³⁶

A critical step of generating new muscle fibers is the fusion of myoblasts. Fusion is specific to skeletal muscle and is not involved in the formation of cardiac or smooth muscle. Myocyte fusion is considered an index of differentiation; many studies have found that C2C12 cells, murine myoblast cell lines, produce a fusion index (the percentage of nuclei incorporated into myotubes) of 30–50% within 7 days.^44–47 Therefore, C2C12 cells can be used as a positive control when investigating myogenic potential of other stem cell sources.

The exact mechanisms that regulate fusion are unclear, but studies suggest that myocyte enhancer factor 2 (Mef2) ^48,49 and myomaker (a muscle-specific membrane protein)^50–52 are essential to myoblast fusion and muscle formation. Other studies have demonstrated that in vitro and in vivo loss of protein phosphatase 2A-Bδ (PP2A-Bδ) impairs the fusion of multinucleated muscle fibers.⁵³ Myosin heavy chain (MHC) and desmin are also thought as late-stage markers of muscle formation.^54,55 In particular, MHC constitutes up to 50% of the total protein content of skeletal muscle tissue.⁵⁶

Cell Sources for Muscle Regeneration

Since natural muscle regeneration occurs from activated SCs (myoblasts), these cells would pose as candidates for engineering severe muscle defects, including the tongue. However, SCs are a rare population of muscle, which make up only 2–7% of adult muscle cells and hence would require a large amount of tissue for isolation.^57,58 Additionally, clinical injection of myoblasts has shown unpromising therapeutic applications. For example, about 99% of myoblasts die after injection due to inflammatory and cell-mediated immune responses, and the cells that do survive fail to migrate from the injection site.⁵⁹

The disappointing results of myogenic stem cells for in vivo muscle regeneration have turned attention to alternative stem cell resources, with particular focus on MSCs. MSCs are multipotent stem cells that can be derived from nearly any adult tissue in the body. Bone marrow is the major source of MSCs (BM-MSCs), which were identified in the 1970s by Alexander Friedenstein and his colleagues.⁶⁰ MSCs exhibit potential to undergo osteoblast, adipocyte, and chondrocyte differentiation and can differentiate into non-mesodermal cell types as well.^61,62 One of the most attractive features of MSCs is their immunosuppressive and antirejection properties, which is likely due to the absence of human leukocyte antigen class II, a main mediator of immune responses.⁶³ This is very important as MSCs eliminate the demand to extract cells from a patient in need and instead can be injected as an allograft procedure.

Most studies have shown that MSCs potentiate the skeletal myogenic process under co-culturing conditions with primary myoblasts.^64–69 Although these results are encouraging to muscle tissue engineering, co-culturing MSCs with other cell lineages adds complexity to determining mechanisms and in vivo translation. Dezawa et al. evaluated rat and human BM-MSCs and showed excellent induction of skeletal myogenesis independent of co-culturing methods.⁷⁰ Results showed the cells expressed MyoD, and a fusion index up to ∼40% after 2 weeks of induction. Myogenic induction of MSCs in vitro using xenogeneic-free growth supplements (i.e., human plasma, platelet lysate, and insulin-transferrin-selenite) is also being studied for clinical compatibility.^66,70,71 In vivo, transplantation of human BM-MSCs into rat pelvic muscles was found to survive and migrate toward muscle fibers without any adverse immunosuppressive side effects or signs of tumorigenesis, suggesting that MSCs can be safely transplanted into muscle tissue.⁶⁶

In addition to BM-MSCs, other banks of MSCs are adipose tissues, which are easily extracted from liposuction procedures, tissue that would normally be discarded as waste. Adipose-derived MSCs (AD-MSCs) were first isolated in 2001 and were originally termed processed lipoaspirate cells by Zuk et al.⁷² Like BM-MSCs, it was demonstrated that AD-MSCs can undergo chemically induced differentiation into adipogenic, chondrogenic, osteogenic, neurogenic, and myogenic cell lineages in vitro.⁷³ AD-MSCs exposed to myogenic media expressed multiple transcription factors including MyoD, Myf5, Myf6, MyoG, and myosin and desmin, the latter being major structural proteins.

A follow-up study showed that human AD-MSCs not only underwent differentiation but also showed a distinct multinucleated and striated appearance, whereas the control group was absent of this morphology.⁷⁴ A separate laboratory successfully replicated the same method for myogenic differentiation using MSCs that were isolated from human umbilical cord blood.⁷⁵ Their results showed significant mRNA expression of MyoD, MyoG, and MHC in comparison to cells cultured in a control medium. A summary of various MSC sources and their potential for muscle differentiation is described in Table 2. Overall, MSCs could be attractive candidates for regenerating new muscle tissue post-glossectomy procedures.

Table 2.

Summary of Mesenchymal Stem Cell Muscle Differentiation

MSC source	Major finding	References
BM-MSCs	Cells had a 40% fusion index after 2 weeks of chemical induction in vitro Cells migrated toward muscle fibers without any immunosuppressive side effects in vivo	Dezawa et al.⁷⁰ Gunetti et al.⁶⁶
AD-MSCs	Cells exposed to myogenic media up to 6 weeks expressed MyoD, Myf5, Myf6, MyoG, myosin, and desmin in vitro Cells demonstrated a distinct multinucleated and striated muscle morphology after 6 weeks in myogenic media in vitro	Zuk et al.⁷³ Mizuno et al.⁷⁴
UC-MSCs	Cells cultured in myogenic media up to 6 weeks expressed MyoD, MyoG, and MHC in vitro	Gang et al.⁷⁵
GMSCs	Differentiation and fusion into myotube structures occurred after 5–10 days in vitro	Zorin et al.⁷⁶

AD-MSCs, adipose-derived mesenchymal stem cells; BM-MSCs, bone marrow mesenchymal stem cells; GMSCs, gingiva-derived mesenchymal stem cells; MHC, myosin heavy chain; UC-MSCs, umbilical cord-mesenchymal stem cells.

Tongue Muscle Regeneration

The tongue is a complex multifunctional organ with vital roles in food intake, mastication, and deglutination. When cancer penetrates the tongue's surface, tumor resection usually includes muscular and neural tissues, thereby causing functional debilitation. Additionally, adjuvant therapies postsurgery such as chemotherapy and radiotherapy promote atrophy of remaining healthy tissue.^77,78 Several reports exist on taste and neural regeneration,^79–86 but there are a limited number of studies that specifically address tongue muscle regeneration, even though muscle is the predominant tissue of the tongue. These studies are summarized in Table 3.

Table 3.

Summary of Muscle Regeneration Studies of the Tongue

Study	Animal model	Defect	Cell line	Materials	Conclusions
Kim et al.⁸⁷	Male Lewis rats	Left-sided mucosa-sparing hemiglossectomy	Myoblasts derived from syngeneic rats	Isotonic NaCl solution; a collagen-rich hydrogel; or a collagen-rich hydrogel containing a suspension of neonatal myoblasts	Animals treated with a hydrogel–myoblast composite had increased tongue weight and demonstrated formation of muscle-like tissue relative to the nonoperated control side
Luxameechanporn et al.⁸⁸	Female Lewis rats	Left-sided mucosa-sparing hemiglossectomy	Myoblasts derived from Lewis rat fetuses	Collagen gel	After 6 weeks, transplanted myoblasts were shown to survive. Animals treated with a hydrogel–myoblast combination had increased desmin staining relative to the control
Charbonneau et al.⁹⁶	Axolotl (Salamander)	2.5 mm biopsy punch	N/A	N/A	Muscle regeneration begins with degradation up to 7 days before transitioning to a reconstructive phase
Xu et al.⁹¹	Female Sprague-Dawley rats; 6–7 weeks old	6 mm biopsy punch with a depth of 3 mm on the left-sided anterior dorsal tongue by way of a lateral approach	Human gingiva-derived MSCs	Decellularized porcine SIS-ECM	SIS-ECM and GMSC/SIS-ECM groups demonstrated less scar tissue than the control, but the GMSC/SIS-ECM group had the fastest wound healing rate. The SIS-ECM and GMSC/SIS-ECM groups expressed upregulation of myogenic markers
Kinoshita et al.⁴⁴	BALB/c-nu/nu male mice; 6 weeks old	2.5 mm ø biopsy punch in the center of tongue	C2C12 myoblasts	Hydrogel	Animals treated with a hydrogel and myoblasts had an increased number of newly regenerated myofibers

SIS-ECM, small intestinal submucosa extracellular matrix.

The first study to investigate muscle regeneration in the tongue was in 2003 by Kim et al.⁸⁷ In this study, a hemiglossectomy of the lateral left side and mucosa-sparing defect was created in male Lewis rats. Immediately following the defect, the animals received one of three intervention treatments: control group 1 received an isotonic NaCl solution; group 2 received a collagen-rich hydrogel; and group 3 received a hydrogel containing a suspension of neonatal myoblasts for 6 weeks. The animals that received the hydrogel–myoblast composite had increased tongue weight and demonstrated formation of muscle-like tissue in comparison to the nonoperated control side but were not compared quantitatively with the other groups. It was also unconfirmed if the muscle-like tissue was due to injected myoblasts, as the cells were not tracked. In contrast, the NaCl solution and hydrogel groups showed a loss of tongue weight, which was supported by histological evaluation.

A follow-up study using the same left-sided mucosa sparing hemiglossectomy defect in female Lewis rats tracked the myoblasts 6 weeks after transplantation.⁸⁸ Animals were divided into two groups receiving either a collagen gel (control group) or collagen gel with myoblasts (experimental group). The transplanted myoblasts survived 6 weeks after transplantation after being successfully tracked with 10 μM 5-chloromethylfluorescein diacetate. In addition, the group that received the collagen–myoblast composite had statistically higher desmin staining in the defect relative to the control group, suggesting cell differentiation and possibly myotube formation. There was no change in fibrosis or inflammation scores between the control and experimental groups. Although this study demonstrated successful myoblast transplantation in an animal model, the results of myoblast transplantation in human clinical trials have been disappointing.^89,90

No muscle regeneration studies of the tongue are reported between the years 2006 and 2015. With controversy surrounding the clinical relevance of myoblast transplantation in humans, a few studies turned attention to MSCs as a potential resource for tongue muscle regeneration. Xu et al. investigated human gingiva-derived mesenchymal stem cells (GMSCs) in combination with the Food and Drug Administration-approved decellularized porcine small intestinal submucosa extracellular matrix (SIS-ECM) as a novel tissue graft for tongue reconstruction.⁹¹ A 6-mm biopsy punch with a depth of 3 mm was created on the left-sided anterior dorsal tongue in female Sprague-Dawley rats.

Animals were divided into three groups: receiving no transplant, an SIS-ECM transplantation, or GMSC/SIS-ECM transplantation into the tongue defect (Fig. 3B) and were monitored up to 4 weeks. The GMSCs were tracked with PKH26 and seeded onto the SIS-ECM before being patched over the defect. Results showed that the GMSCs attached to the SIS-ECM construct and the animals tolerated the defect by maintaining body weights throughout the study (Fig. 3A, D). Two weeks postsurgery, both SIS-ECM and GMSC/SIS-ECM groups demonstrated less scar tissue than the control, but the GMSC/SIS-ECM group had a faster wound healing rate than both groups (Fig. 3C).

FIG. 3.

GMSC/SIS-ECM constructs promote tongue wound healing. (A) GMSCs were cultured on SIS-ECM constructs for 48 h and stained with PKH26 (red), anti-CD90 (green), and DAPI (blue). (B) A tongue defect model was treated with a control (defect only), SIS-ECM, or GMSC/SIS-ECM construct; images were taken at 0, 3, 5, and 7 days postsurgery. (C) The percent relative wound area of each group at 0, 3, 5, or 7 days postsurgery. (D) Body weight of each group at 1, 3, 6, 9, or 12 days postsurgery. (E) Images of tongue defects from each group at 2 weeks postsurgery. (F) H&E staining results from control, SIS-ECM, or GMSC/SIS-ECM 2 weeks postsurgery (10 × magnification). Images with a blue circle indicate the original defected area from surgery. Figure adapted from Xu et al.⁹¹ GMSC, gingiva-derived mesenchymal stem cells; H&E, hematoxylin and eosin; SIS-ECM, small intestinal submucosa extracellular matrix. Color images are available online.

Immunofluorescent studies showed that the transplanted GMSCs expressed Pax7, MyoD, and Myf5; however, overlapping this expression with all PKH26-tagged cells only accounted to about 10%, suggesting a small proportion of cell differentiation. Western blot analysis confirmed that Pax7 and MyoD were significantly upregulated in the SIS-ECM and GMSC/SIS-ECM groups but was more pronounced in the latter. In addition, histological analysis suggested muscle regeneration in the GMSC/SIS-ECM group relative to the defect control and SIS-ECM groups alone (Fig. 3F).

The most recent study to investigate muscle regeneration of the tongue was a study conducted by Kinoshita et al.⁴⁴ A 2.5 mm ø biopsy punch was created in a center area of BALB/c nude male mice. The defects were treated with primary closure (control), a hydrogel, or a hydrogel with C2C12 myoblasts. After 2 weeks of treatment, animals that received a hydrogel with myoblasts had a statistically increased number of newly regenerated myofibers. The control and hydrogel groups alone demonstrated 5% newly regenerated myofibers, whereas the hydrogel and myoblast composite group demonstrated 15% newly regenerated myofibers. The newly regenerated myofibers were identified by hematoxylin and eosin staining and quantified based on centrally located nuclei relative to the total number of nuclei (central + peripheral).

In vivo, the hydrogel fully degraded within 1 week, which may partially explain a lack of robust differentiation as cells could have dropped off during biodegradation. Additionally, myoblasts grown in a three-dimensional (3D) environment proliferate significantly slower than myoblasts grown in a two-dimensional environment.⁹² It should also be noted that some hydrogels need to be coated with a protein to facilitate cell attachment.^93,94 In this study, myoblasts attached when hydrogels were coated with fibronectin, a major cell adhesion protein of the ECM.

Although these methods were successful in accomplishing the purpose of these studies, immediately implanting a cell-based therapy following resection raises clinical concerns. As Vahabzadeh-Hagh et al. stated: “Very early wound manipulation (within 48 hours) can shift the growth factor and cell infiltration profile, and produce overly optimistic results that may not be relevant to most clinical scenarios.”⁹⁵ This statement is in part supported by a study that investigated wound healing after creating a 2.5 mm biopsy punch in an axolotl (regenerative amphibian) and found that muscle regeneration began to occur 7 days after the degradation phase.⁹⁶

Instead, postsurgery patients need time to recover, and second, many patients also undergo adjuvant chemotherapy and/or radiation therapy. Therefore, a cell-based therapy for tongue reconstruction should be implemented when the cancer is extinct. To overcome these issues, a group of researchers from the University of California, Los Angeles, David Geffen School of Medicine recently created a tongue defect model in athymic nude rats and waited 2 weeks before implementing a cell therapy intervention. The tongue defect was created by way of a unilateral partial glossectomy using a 4 mm dermal punch and the animals were placed on a soft food diet.⁹⁵

Human BM-MSCs were suspended in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and 1% penicillin/streptomycin and injected into the defect. The animals received this treatment for 3 weeks before being sacrificed. The results showed that a high concentration of BM-MSCs (250,000 cells in 300 μL media) reduced scar burden and pathological scores for inflammation and fibrosis in comparison to animals receiving a low concentration of BM-MSCs (70,000 cells in 300 μL). The animals demonstrated great tolerance of this defect as all animals had no weight loss and instead maintained a normal growth curve.

Similarly, a recent phase I clinical trial followed 10 patients who previously had oropharynx cancer and demonstrated swallowing impairment post-treatment.⁹⁷ All patients were treated with autologous muscle-derived cells (150 × 10⁶), which were injected into the tongue. Results found that there was significantly improved tongue strength at both 6 months (mean = 30.7 kPa) and 12 months (mean = 31.8 kPa) after cell therapy, in comparison to baseline levels (mean = 26.3 kPa). The authors concluded that injecting autologous muscle-derived cells into the tongue was safe and may demonstrate greater efficacy at larger doses, which is currently under investigation.

Challenges of Regenerative Medicine

In 1995, Langer et al. proposed the concept of tissue engineering, which applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function.⁹⁸ Thus, like any other tissue, a combination of viable cells and scaffolds could contribute toward the regeneration of muscle in the context of tongue repair. Typically, porous 3D scaffolds fabricated from natural or synthetic biomaterials provide the appropriate environment for the regeneration of tissues and organs. Scaffolds act as a template for tissue formation and can deliver cells or growth factors to the injury site for mechanical or chemical stimuli. Scaffolds alone have also been used for tissue regeneration. As a result, in addition to the donor cells delivered using a scaffold, the response of the host cells to the scaffolds cannot be overlooked. Hence, for a tissue engineering strategy to be successful, one has to control the variables related to both the scaffolds and cells.

The field of biomaterials and scaffolds is extensive and constitutes an important line of research and investigation.⁹⁹ The choice of biomaterials used in the fabrication of scaffolds is highly dependent on the in vivo applications. However, some of the key considerations important when designing or determining the suitability of a scaffold for use in tissue engineering include biocompatibility, biodegradability, mechanical, and physicochemical properties. Each one of these aspects is tissue specific and is being addressed by chemists and engineers in collaboration with basic molecular and cellular biologists. There is a multitude of literature related to each and every aspect of this field of research and hence is beyond the scope of this review article.

Treating tongue defects with tissue engineering strategies is a novel strategy and has the potential, but many challenges must be overcome before this is routinely employed in the clinic. With any cell-based therapy, there are many variables to consider such as age, sex, and medical history of the donor and the host cells. For these reasons, not every patient may respond to a similar case that receives stem cells from a different donor. In other cases, allogenic cells may not be available and could require the patient in need to undergo an additional procedure for cell isolation. There are also challenges in growing cells which is time consuming, has risks of contamination, and would require additional laboratory space in the clinic. Other considerations are personalizing treatment such as the proper stem cell dosage and whether or not a material or donor cells are needed to support a tongue defect. However, new technology such as 3D printing is revolutionizing personalized implants and may be useful in tongue tissue engineering.^100,101

As described above, every aspect of treatment strategy development is important, and arguably, the most challenging is the product testing and characterization that leads to safe and robust manufacturing processes. The next question that is raised is how best to regulate these novel therapies while getting them into the market as quickly as possible. Uncertainties over how cell therapies should be regulated, safety concerns over administration of live cells to a patient, and lack of sufficient funding to support the full development and commercialization of cell therapies all present additional and significant challenges to the development of cell therapies. As a result, cell therapies are still largely at the research and development phase, but there is an interest from academic and industrial arenas, and hence, this is starting to change. With investment growing every year, as well as the number of clinical trials testing cell therapies, interest in the cell therapy field will continue to grow in coming years.

Conclusions

Various metastatic cancers and benign tumors of the tongue typically require surgical resection. Although surgery is effective in removing these diseases, large tongue defects do not have the capacity to regenerate new muscle tissue and therefore requires reconstruction. The primary standard for tongue reconstruction is flap surgery, which has several limitations and inconsistent outcomes. Alternatively, a novel strategy is engineering new muscle that could replace the resected tongue tissue. Using regenerative medicine, stem cells can be signaled into specific cell lineages, which can treat a variety of defects. Although stem cell treatments have been successful for rebuilding several organs, this strategy is yet unknown for the tongue. Therefore, there are many challenges that need to be addressed before clinical translation. Overall, more regenerative studies are needed before new treatment strategies for tongue reconstruction can be introduced in the clinic.

Footnotes

Acknowledgment

The authors acknowledge the support of the Tissue Regeneration and Regenerative Medicine Program at the University of Tennessee College of Veterinary Medicine.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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