Multipotent mesenchymal stromal cells in

Medical Hypotheses 82 (2014) 769–773

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Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy

Multipotent mesenchymal stromal cells in otorhinolaryngology Lukas Skoloudik a, Viktor Chrobok a, David Kalfert a,⇑, Zuzana Koci b, Stanislav Filip c a Department of Otorhinolaryngology and Head and Neck Surgery, University Hospital Hradec Kralove, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Czech Republic b Laboratory of Tissue Culture and Stem Cells, Institute of Experimental Medicine, Academy of Science of the Czech Republic, Czech Republic c Department of Oncology and Radiotherapy, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Czech Republic

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Article history: Received 12 November 2013 Accepted 19 March 2014

a b s t r a c t Multipotent mesenchymal stromal cells (MSCs) are primitive cells capable of restoring damaged mesenchyme and with the ability to differentiate into mature cells of bone, cartilage, muscle, fat, nerve or fibrous tissues. MSCs are therefore good candidates for applications in regenerative medicine and cell based therapy. They regenerate through self-renewal, differentiational capacity, immune modulation and secretion of bioactive molecules. Authors present a review of MSCs applications in otorhinolaryngology. The major interest is focused on phonosurgery, sensorineural deafness and reconstruction of large tissue defects with bone, cartilage or soft tissue replacement. Current evidence of MSCs treatment efficacy in otorhinolaryngology is based on animal models. The true impact on clinical treatment will not be known until clinical studies prove functional outcomes in human medicine. Ó 2014 Elsevier Ltd. All rights reserved.

Background MSCs are used in otorhinolaryngology due to their self-renewal capacity and multilineage differentiation ability to differentiate into cells of mesenchymal tissue (bone, cartilage, muscle, fat, nerve or fibrous tissues). MSCs produce bioactive molecules suppressing inflammatory response and support regeneration of injured tissues [1–4]. The immunomodulatory capacity of MSCs appears to facilitate their use for allogenic transplantation [5]. These qualities predetermine MSC as a great tool for regeneration of injured tissue or replacement of missing tissue after surgery, injury or chronic refractory wound [6]. Stem cells include embryonic stem cells derived from blastocysts which are pluripotent and adult or somatic stem cells including hematopoietic progenitor/stem cells, mesenchymal stem cells, and tissue specific progenitor cells, all having multipotent differentiation ability. Another type of stem cells present induced pluripotent stem cells which are reprogrammed differentiated cells, having differentiation potential of embryonic stem cells. MSCs have been firstly isolated from bone marrow in the 1960’s. Since then they have been isolated from numerous tissues, including adipose tissue, liver, gastric epithelium, synovial membrane, etc. MSCs from 0.001% to 0.01% of bone marrow aspirate, ⇑ Corresponding author. Address: Department of Otorhinolaryngology and Head and Neck Surgery, University Hospital Hradec Kralove, Sokolská 581, 500 05 Hradec Králové, Czech Republic. Tel.: +420 495832263; fax: +420 495832033. E-mail address: [email protected] (D. Kalfert). http://dx.doi.org/10.1016/j.mehy.2014.03.022 0306-9877/Ó 2014 Elsevier Ltd. All rights reserved.

which is 10 times less than hematopoietic progenitor/stem cells and 2% of adipose tissue aspirate [7]. Special attention is paid to their occurrence in various tissues of newborns. Postnatal tissues are useful and ethically uncontroversial alternative offering certain advantages for obtaining MSCs for therapeutic purposes. These tissues include MSCs from amniotic fluid, amniotic membrane, chorionic membranes, chorionic villi, placenta and umbilical cord blood [8]. International Society for Cellular Therapy defined MSCs on the basis of three criteria [9]: 1. MSCs under standard cultivation conditions adhere to plastic surface and form colonies; 2. MSCs carry surface markers CD105, CD73 and CD90 while lack markers characteristic for hematopoietic stem cells, CD34, CD45, CD11a, CD19 or CD79a, CD11b or CD14 or HLA-DR; 3. MSCs can be stimulated to differentiate into osteoblasts, adipocytes or chondrocytes. In addition to the necessity of the three surface molecules on MSCs, other criteria must be accompanied. For example the absence of many other specific markers, mainly expression markers of monocytes, endothelial cells and lymphocytes. This fact indicates that although MSCs have common surface molecules other different surface molecules can be crucial for further differentiation, and subsequently the functional manifestations of the damaged tissue. MSCs derived from umbilical cord blood (UCB MSCs) or adipose tissue (AT MSCs) have a significantly higher expression

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of CD44 in contrast to the bone marrow (BM MSCs) [10]. Currently, attention is given to the other markers that characterise certain subpopulations of MSCs. Some markers such as Stro-1 or adhesion molecule VCAM-1 meet these conditions. Nevertheless, expression of Stro-1 was also discovered in the small population of CD34+ hematopoietic stem cells and endothelial cells, as well as the expression of adhesion molecule VCAM-1 [11,12]. Nowadays, it is generally accepted that MSCs exhibit tissuespecific functional biodiversity, which is mediated by a direct ‘‘cell-to-cell’’ cellular communication associated with the activity of adhesion molecules, cytokines, growth factors and cell signaling pathways [13]. The ability of MSCs to differentiate into bone cells called osteoblasts led to their use in biologically based methods of bone repair. Cellular response realized by platelets, inflammatory cells and macrophages penetrating into the injured bone attract an invasion of mesenchymal stem cells to differentiate into osteoblasts and chondrocytes (Fig. 1). Preclinically, this was demonstrated on bone repair proportion of mesenchymal stem cells from periosteum, bone marrow, circulating blood and surrounding soft tissues. In the treatment of femoral defects in experimental animals it was shown that after 12 weeks there was significantly higher level of bone formation using porous biphasic hydroxyapatite/tricalcium phosphate serving as a carrier of cultured mesenchymal cells compared with scaffolds alone [14]. Published compositions were also subjected to another preclinical studies, all confirming better bone formation using scaffolds as carriers of mesenchymal stem cell differentiation into osteoblasts [15–17]. Bone replacement in otorhinolaryngology Bone replacement in otorhinolaryngology enables a skeletal reconstruction of injured bone, postoperative bone deficit or treatment of congenital abnormalities. MSCs transplantation is an alternative of extensive plastic surgery using autologous bone graft or vascularized flap reconstruction. MSCs have been explored to avoid complications associated with harvesting of the bone and decrease morbidity of both the donor and recipient reconstruction.

Bone regeneration is influenced by MSCs not only in the sense of tissue replacement. In addition MSCs have imunomodulatory and anti-inflammatory effect due to cell-to-cell contact or secretion of bioactive molecules. The osteogenic differentiation of MSCs was proven in several studies [3,4]. MSCs can be directly injected to the fracture sites to facilitate healing. In case of bone reconstruction the MSCs are compound with osteoconductive biomaterial. Ceramic based graft substitute materials include calcium sulphate based materials, such as hydroxyapatite, beta-tricalcium phosphate and bioactive glass. Cultured MSCs on scaffolds in osteogenic media induce bone development de novo. In otorhinolaryngology MSCs with osteogenic differentiation can be utilized for maxillofacial reconstruction, nasal defects, orbital wall fractures and frontal bone defects. Perhaps most promising is the MSCs augmentation of maxillary sinus for dental implants. The MSCs augmentation of sinus floor cause more new bone formation compared to augmentation with hydroxyapatite without MSCs [18–20]. MSCs have also been utilized for reconstruction of cleft palate. Demineralized bone matrix with calcium sulphate and MSCs were implanted into the bone defect, demonstrating new bone formation as the grafts were incorporated [21]. Temporal bone is another point of interest. MSCs facilitate reconstruction of postoperative defects in middle ear surgery [22]. Canal wall down technique cause patients discomforts with open mastoid cavity. Ear canal looses it’s self clearing ability and becomes a focus of chronic infection. Patient needs regular treatment with bathing restriction and hearing aids limitations. MSCs offer a new method for ear canal reconstruction. MSCs with osteogenic differentiation on ceramic scaffolds enable the new bone formation to close mastoid cavity and normalize the anatomy of the middle and external ear. Due to advances in the field of tissue engineering, biomaterials and cell biology, MSCs may play an important role in regeneration of the bone. However, there are a number of aspects in the application of mesenchymal stem cells requiring further research as there are currently few clinical trials reporting their application. The

Fig. 1. The mesengenic process. Adult mesenchymal stem cells (MSCs) isolated from bone marrow and mitotically expanded in culture are able to differentiate into a number of mesenchymal phenotypes, including those that form bone, cartilage, muscle, fat and other connective tissues.


main tasks in developing the repair and regeneration of bone strategies include optimizing the source of mesenchymal stem cells (bone marrow, fat), standardization of protocols to obtain maximum yield, three-dimensional culturing MSCs (ex vivo complex three-dimensional 3D hybrid tissue), standardization of growth factors used during cultivation, scaffold comparison to identify the most appropriate biomaterials to be used as cell carriers including biomechanical studies, and the use of nanomaterials. The prerequisite for addressing the above issues are prospective, randomized studies involving a larger number of patients with longer following periods and comparing the control group with the group undergoing treatment of bone defects. Cartilage replacement in otorhinolaryngology Cartilage is a favourite autograft in otorhinolaryngology. Auricle’s cartilage is obtainable without serious morbidity of donor site. Cartilage autograft is utilized particularly in middle ear surgery. However, the autograft is insufficient in case of large cartilage defect, such as functional trachea replacements. Large tracheal defects are consequences of extensive resection due to damage from intubation or malignancies. MSCs are utilized to seed decellularized donor trachea allografts [23], induce cartilage development de novo [24,25] or reduce the antigenicity of trachea allografts [26]. The target of tracheal reconstruction is development of biocompatible graft able to tolerate negative pressures during inspiration. Macciarini et al. were the first group to perform a transplantation of decellularized donor trachea seeded with MSCs. They used two cells lines. MSCs differentiated into chondrocytes were used on the external surface and epithelial cells in the lumen. Although animal studies confirmed the applicability of this approach and both cell lines work synergistically, the shortages in donor trachea limit its wide spread application. MSCs can significantly improve the long-term survival and efficiency of synthetic trachea replacement. MSCs are utilized on different scaffolds, such as collagen hydrogel [25] or collagen sponge with polypropylene [24]. Skin and soft tissue replacement in otorhinolaryngology MSCs are utilized for enhancing skin and mucosal wound healing. Application of MSCs enhances tissue reperfusion through their secretion of trophic factors, inhibit T-cell function and promote neovascularization [27]. MSCs stimulate extracellular matrix deposition, angiogenesis and native cell recruitment. MSCs can be used for skin replacement after extensive operation or serious injury, as well as enhancing healing of skin damage after heat or radiation burn [28]. Clinically the cell based therapy support a standard wound treatment, such as serial debridements, split thickness skin graft and local application of medicaments for enhancing wound healing and tissue repair. At present, MSCs are frequently used for soft tissue augmentation. In case of adipose tissue transfer the MSCs have antiinflammatory effects and improve long-term survival of the grafts, reduce postoperative atrophy and resorption of the fat tissue through enhanced angiogenesis and cell self-renewal [29–31]. Much of the interest in otorhinolaryngology focuses on phonosurgery. The applications of cell-scaffold engineering methods are used for treatment of injured vocal cords (VC) or augmentation of VC to compensate a glottic insufficiency. The application of cell-scaffold tissue adds a new quality for phonosurgery. Injection of MSCs into vocal cords supports their remodelation. The growth factors and extracellular matrix (ECM) proteins enhance long-term tissue regeneration and improve the pliability of vocal cords, particularly in their lamina propria. MSCs have been shown to provide

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antifibrotic benefits. MSCs derived from adipose tissue (AT MSCs) modulate scar formation by increasing expression of hyaluronic acid (HA) and hepatocyte growth factor (HGF) and decreasing collagen secretion and proliferation [32,33]. Bone marrow MSCs (BM MSCs) decrease the collagen type I [34]. In animal model the injection of BM MSCs produces optimal biomechanical properties of the superficial lamina propria [35]. By 3 months after MSCs injection the viscoelastic properties and thickenes of lamina propria of injured vocal cords were similar to unscarred cords [35]. The addition of MSCs provides their growth factors and ECM proteins. MSCs do not engraft into the VC tissue. No MSCs were found 3 months after injection [35]. Much of the interest focuses on biomechanical properties of the cell-scaffold composites, such as collagen, fibrin, HA or a combination [36–41]. Extensive research has been conducted on a HA hydrogel. In animal model the tissue engineered HA hydrogel produced optimal biomechanical properties for the superficial layer of the lamina propria, material is biocompatible and non-toxic [39]. The combination of HA hydrogels with BM MSCs enhances tissue regeneration compared to the biomaterial alone [42]. Addition of BM MSCs to the HA hydrogel facilitates ECM production, remodeling and regeneration of injured vocal cords [37,42]. The inclusion of MSCs into a HA hydrogel seems to be an optimal treatment for injured VC by accelerating the wound healing through the production of bioactive molecules and restoring volume to the superficial lamina propria. The effect of MSCs treatment for larger laryngeal lesions was tested in vitro. Long et al. presented replacing the entire vocal cord cover by two MSCs layers in a fibrin scaffold [41]. Functional studies showed that the AT MSCs fibrin construct produced similar elastic properties as the VC cover from a cadaveric larynx [43].

Neural tissue replacement in otorhinolaryngology MSCs are utilized for treatment of injured peripheral nerve. Facial nerve and laryngeal recurrent nerve are the most common iatrogenic peripheral nerve palsy in ENT. The efficacy of MSCs for neural regeneration after acute facial nerve injury was proven on an animal model [44]. MSCs promote facial nerve regeneration after axonotomy. The combined use of platelet-rich plasma and MSCs show a greater beneficial effect than use of either alone. The first case report of MSCs treatment for neural injury in human was published by Caylan et al. [45]. Facial nerve axonotomy in temporal part after mastoidectomy was treated with a nerve cable graft obtained from vicinity. One month after the surgery complete facial nerve paralysis persisted with EMG denervation. MSCs transplantation was performed 42 days after facial nerve trauma. The patient’s facial nerve paralysis has recovered from House–Brackmann grade VI to III in 5 months. Another common iatrogenic nerve trauma is laryngeal recurrent nerve (LRN) palsy after thyroidectomy. Although LRN palsy occurs in 0.5–3% of thyroidectomies current treatments for vocal cord paralysis are suboptimal and fail to restore dynamic function [46,47]. A promising potential therapy is the use of autologous muscle MSCs. Survival of autologous muscle MSCs and fusion of the MSCs with the denervated myofibers was demonstrated in animal model. The survival and fusion with myofibers are facilitated by combination MSCs with insulin-like growth factor 1 (IGF-1) or ciliary neurotrophic factor (CNTF). Extensive research has been focused on regeneration of inner ear. Treatment of sensorineural hearing loss (SNHL) is still unsatisfactory and results of current therapy are disappointing. A source of sensory cells and neurons for regeneration of inner ear cells would provide a valuable tool for clinical application as neurons

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and hair cells could eventually be employed in cell replacement therapy in hearing loss. One of the limits of MSCs replacement is the process of MSCs application into the inner ear. MSCs have to be injected directly to the lesion locus. In animal experiment MSCs survived in modiolus only after modiolar infection, not after scala tympani injection [9]. Another limit is the fact that stem cells do not spontaneously divide to replace damaged sensory cells. However, by a combination of growth factor stimulation and expression of the transcription factor, Math1, that is required for hair cell formation in the inner ear, BM MSCs can be induced to differentiate into hair cells [48]. The neurosensory progenitors obtained from bone marrow can be converted to sensory hair cells by co-culture with cells of the developing sensory epithelium.

Discussion – pros and cons MSCs are able to differentiate into bone, cartilage, muscle, marrow stroma, tendon–ligament, fat and other connective tissues. New insight and clinical experiences suggest that MSCs are released at sites of injury, where they secrete numerous bioactive factors that both possess regenerative and immunomodulatory effects on damaged tissue. These events report that MSCs are responsible for the normal turnover and maintenance of adult mesenchymal tissues [49]. Cells have half-lives and their natural expiration must be matched with their replacement. Therefore, we suggest that MSCs may be a source of new replacement cells. Analogy could be found in the known sequence of events involved in the turnover and maintenance of blood cells that are formed from haematopoietic stem cells (HSCs) [50,51]. Given the above, we have reconsidered our MSC logic for their therapeutic use. Surely, MSCs can still be of great value for use in tissue engineering therapies by virtue of their ability to differentiate into distinctive and specialized cells. The use of inductive or instructive delivery vehicles, or the jump-starting of MSCs down specific lineage pathways, would seem to be necessary. However, the MSCs are used as site-regulated multidrug dispensers in bronchial asthma, radiation exposure, neurological disorders, etc. [52–54]. The research of MSCs in otorhinolaryngology is still in its infancy. Most of the published evidences MSCs applications in otorhinolaryngology are based on animal model. We miss enough clinical data to compare the cell-scaffold engineering methods with alternative kinds of treatment. In laryngeal surgery much if the interest is focused on treatment of injured VC and VC augmentation. The studies [32–43] suggest efficacy of MSCs utilization in phonosurgery. However, the price of the MSCs treatment and leak long-term clinical data limit its wide spread application. In temporal bone surgery the research is focused on surgical trauma. The obliteration of mastoid cavity was studied on animal model [55]. Considering costs and benefits there are cheaper and surgically easer techniques for mastoid obliteration are available using autologous tissue (temporal muscle flap, musculocutaneous flap, etc.). However, compared to soft tissue obliteration the MSCs based surgery could facilitate accurate reconstruction of ear canal and middle ear cavity. More clinical studies are necessary to consider a true impact on clinical treatment and functional outcomes. Unsatisfactory results of current treatment of SNHL encourage MSCs based regeneration of inner ear. However, the fusion of MSCs with neural epithelium of inner ear has not been proved yet and paracrinal effect of MSCs bioactive molecules is not sufficient for inner ear regeneration [9,48]. The MSCs based treatment of injured peripheral nerve is studied on animal models. In human medicine only particular case

studies were published. The efficacy of MSCs for neural regeneration is limited by fusion of MSC and the neural cell. But the effect of MSCs bioactive molecules on facial nerve promotion was proven on an animal model [44]. MSCs tissue replacement enables reconstruction of bone, cartilage or skin after extensive operation or serious injury, as well as enhances healing of tissue damage after heat or radiation burn. MSCs transplantation is an alternative to an extensive plastic surgery using autologous grafts or vascularized flaps reconstruction. MSCs based treatment decreases complications associated with harvesting of the donor bone and morbidity of the donor and recipient reconstruction. In addition MSCs have imunomodulatory and anti-inflammatory effect due to cell-to-cell contact or secretion of bioactive molecules. Clinically the MSCs are used in combination with routine wound treatment, such as serial debridements, split skin grafts and local application of medicaments for enhancing wound healing and tissue repair. The main limits of wide spread utilization are disposal, price and time consumption of MSCs treatment. Stem cell-derived products constitute a promising therapeutic approach for the treatment of a wide range of disorders. Neurodegenerative diseases, like Parkinson’s disease or Huntington’s disease, cardiac failure and blood disorders and among others, may 1 day be treated using cellular therapies. In otorhinolaryngology the MSC treatment is still at the beginning. Considering current animal studies, the main impacts of MSCs treatment on clinical practice in otorhinolaryngology are following: 1. Phonosurgery – treatment of injured vocal cords and vocal cords augmentation. 2. Middle ear surgery – treatment of postoperative temporal bone defect. 3. Sensorineural hearing loss – regeneration of inner ear neural epithelium. 4. Neural regeneration – treatment of injured peripheral nerve. 5. Tissue replacement – tissue reconstruction after extensive operation or serious injury. Furthermore, owing to the potential positive compact on healthcare systems, transplantation of such stem cell technologies into the clinical practice will bring about social and economic advantages worldwide. Conclusion Tissue engineering development enables the use of MSCs across the entire medicine field. In otorhinolaryngology the research is targeted on repairing or replacing injured tissue of head and neck. The majority of the interest is focused on phonosurgery, sensorineural deafness and reconstruction of large tissue defects with bone, cartilage or soft tissue replacement. Most of the published evidence of MSCs utilization in otorhinolaryngology is based on animal model. The true impact on clinical treatment will not be known until clinical studies prove functional outcomes in human medicine. Conflict of interest The authors declare that there are no conflicts of interest regarding the preparation or content of this article. Acknowledgement The experiment was supported by MH CZ – DRO (UHHK, 00179906) and by the programme PRVOUK 37/06 (LF UK).


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