Mesenchymal Stem Cells and Nanofibers as ... - Wiley Online Library

The Laryngoscope C 2014 The American Laryngological, V

Rhinological and Otological Society, Inc.

Mesenchymal Stem Cells and Nanofibers as Scaffolds for the Regeneration of Thyroid Cartilage Geraldo P. Jotz, MD, PhD; Paula R. da Luz Soster, PhD; Seno O. Kunrath, MD, PhD; Daniela Steffens, MSc; Daikelly I. Braghirolli, MSc; Claudio Galleano Zettler, MD, PhD; Carlos A. Beck, PhD; Marcelo Muccillo, MSc; Rui F. F. Lopes, PhD; Bernardo Mastella; Patricia Pranke, PhD Objectives/Hypothesis: The aim of this study has been to establish an alternative approach in the form of regeneration of the thyroid cartilage. Study Design: Four 1-month old pigs (Sus scrofa) were used (divided into 3 groups) and submitted to general anesthetic to perform cervictomy with exposure of the thyroid cartilage in a total of 12 (twelve) samples. Method: A resection of 4.0 cm2 of cartilage was carried out in the right upper region and in the left upper and lower left region of the cartilage, where a scaffold with or without stem cells was implanted. In the left lower region, no biomaterial was implanted and the defect was left open (lesion control [L]). Results: The average extension of the cartilaginous neoformation of L group was 136.3 lm (6 9.6) and 387.7 lm (6 43.2) in the scaffold (SCA) group, presenting a significant statistical difference (P < 0.01). The analysis carried out on the lesion site sections of the cartilage of the larynx of the animals from the SCA group 1 mesenchymal stem cells (SCA1MSC) showed an average of the extension of neocartilage of 825.4 lm (6 122.1), showing a more extensive area of neocartilage when compared to the other groups. These results demonstrated a high significantly statistical difference (P < 0.001) when compared with the L and SCA groups. Conclusion: In 100% of the cases for which SCA1MSCs were used, a significant success in the cartilage growth and closing of the lesion in the thyroid cartilage was obtained compared to the other two groups for which MSCs were not used. Key Words: Stem cells, thyroid cartilage, regeneration, scaffolds.. Level of Evidence: N/A. Laryngoscope, 124:E455–E460, 2014

INTRODUCTION Cancer of the larynx is diagnosed annually in approximately 10,000 men and women in the United States and is among the most common types of cancer of the upper aerodigestive tract.1 Reconstruction of the airways, mainly those with cartilaginous formation in their structure, continue to challenge medical science. A very large variety of techniques for cartilaginous reconstruction have been From the Department of Morphological Sciences (G.P.J., P.R.DLS., the Hematology and Stem Cells Laboratory, Pharmacy School (D.S., D.I.B., P.P.); the Post Graduate Program in Physiology (D.S., D.I.B, P.P.); the Medicine Veterinary School (C.A.B, M.M.); the Medicine School; Federal University of Grande do Sul (B.M.); the Federal University of Health Sciences of Porto Alegre (C.G.Z.); and the Stem Cell Research Institute (P.P.), Porto Alegre, RS, Brazil. Editor’s Note: This Manuscript was accepted for publication June 3, 2014. This work was supported by the National Council for Scientific Pesand Technological Development (CNPq), Fundac¸~ ao de Amparo a quisa do Rio Grande do Sul (FAPERGS), and the Stem Cell Research Institute. The authors have no other funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Geraldo Pereira Jotz, MD, PhD, Department of Morphological Sciences, Federal University of Rio Grande do Sul, Rua Dom Pedro II 891/Room 604. Porto Alegre, RS, 90550-142, Brazil. E-mail: [email protected] S.O.K., R.F.F.L.);

DOI: 10.1002/lary.24805

Laryngoscope 124: December 2014

described in laryngotracheal reconstruction surgeries— not always with very promising results in milder cases (level II of Cotton) and those of benign origin.2,3 These different therapeutic approaches have been developed around the world with the aim of offering an improvement in the life quality of this group of patients. The main types of tracheobronchial substitutes that have been used in airway transplantation are synthetic prostheses, bioprostheses, allografts, autografts, and bioengineered conduits. According to a recent review by Martinod et al.,4 despite the fact that research has been carried out in this area for more than 50 years, airway transplantation is still one of the biggest challenges for thoracic surgery and regenerative medicine.4 In the last decade, research using stem cells (SCs) has attracted a great deal of attention from the academic and scientific worlds because it shows enormous potential for modifying the concepts of traditional therapies, with a wide impact on genetic therapy, carcinogenesis, tissue damage, and regeneration, among others. The identification, isolation, and differentiation of embryonic stem cells have broadened the spectrum of potentials for cellular therapy. Recent findings5 have demonstrated the possibility of isolating skeletal muscular cells derived from SCs of embryoid bodies. The intramuscular and systemic transplantation of these cells in dystrophic Jotz et al.: The Regeneration of Thyroid Cartilage

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mice resulted in extensive engraftment of adult myofibers, with progressive contractile function and without the formation of teratomas. Significant advances with the use of SC of embryonic origin (ESC) have been described for the correction of spinal cord injury for which the application of ESC has demonstrated a beneficial effect for the restoration of locomotor ability in animals after spinal cord injury. This therapy should soon be tested on humans.6 The aim of this study has been to establish an alternative for the regeneration of thyroid cartilage in pigs, reconstructing the organ through the implant of human MSCs grown on nanofiber matrices, produced by biodegradable and biocompatible polymers using the electrospinning technique.

MATERIALS AND METHODS Animals Four 1-month-old pigs were used (Sus scrofa), maintained at the Hospital de Clinicas Veterin arias (Veterinary Hospital) of the Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil. The use of the animals followed the experiment protocol approved by the UFRGS ethics committee for the use of animals in research. Young animals were chosen with an average weight of 15 kilograms, which made surgery and anesthesia easier; however, the rapid growth and weight gain of these animals resulted in greater difficulties for the postoperative control.

Isolation and Cultivation of Stem Cells For the study, mesenchymal stem cells (MSCs) isolated from tissue pulp from deciduous teeth were used. The teeth were extracted from children in good general health enrolled in the Pediatric Dentistry Program of the Faculty of Dentistry at UFRGS, after informed written consent of the parents/guardians (no. 296/08). The collection, isolation, and characterization of the pulp tissue MSCs was carried out in accordance with the established protocol described by Luisi et al.7,8 After isolation, the adherent cells were cultivated in a Dulbecco’s Modified Eagle’s Medium culture medium (D5523, Sigma-Aldrich, St. Louis, MO), supplemented with 10% bovine fetal serum and 1% penicillin/ streptomycin (Gibco BRL, Grand Island, NY) at 37 C in a humid atmosphere of 5% de CO2. The culture medium was changed every 3 to 4 days. After reaching approximately 90% confluence, the primary cultures were washed with phosphate buffered saline 13 and trypsinized with a 0.25% solution of trypsin-EDTA (Sigma-Aldrich). The cells were resuspended in a culture medium and plated in new bottles of cellular culture for expansion. Subsequent passages were carried out in the same way. Cells between the fifth and 10th passage were used for the in vivo experiments.

the matrices were constructed with the following dimensions: 2 cm length, 2 cm width, and 2 mm density (2 cm 3 2 cm 3 2 mm). The process was carried out at the Hematology and Stem Cell Laboratory at the Faculty of Pharmacy of UFRGS. The nanofiber scaffolds were adhered onto plastic Petri dishes and were sterilized with ultraviolet light for 2 hours on a 24well plate in a laminar flow hood.

Seeding of the Mesenchymal Stem Cells For this purpose, 30,000 cells per cm2 were seeded onto the scaffolds. Two different cultures of mesenchymal stem cells from dental pulp were employed for use in the animals. The cells seeded onto the scaffolds were maintained in culture for 7 days before their implantation in the pigs. In this period, two changes of medium were performed.

Surgical Procedure The animals were preanesthetized with 10 mg/kg ketamine plus 0.3 mg/kg midazolam plus 2 mg/kg intramuscular meperidine. Immediately afterward, induction of the anesthetic was performed with 5 mg/kg propofol, and maintenance of the anesthetic with isoflurane and oxygen under the constant vigilance of a veterinary surgeon. The surgery was carried out with partial resection was performed of the thyroid cartilage in three distinctive locations and implantation of the matrices of the nanofibers cultivated/not cultivated with MSCs. Three distinctive experimental procedures were established in a group of four animals (12 samples). In all of the animals, the partial resection of the thyroid cartilage was performed with a resection of approximately 4.0 cm2 of the thyroid cartilage in the upper right region, 4.0 cm2 of the thyroid cartilage in the upper left region, and 4.0 cm2 of the thyroid cartilage in the lower left region. Figure 1 shows a representation of the surgical procedure. After the procedure, the treatments that differentiated the groups were the following: L group, which did not receive a matrix graft on the injury site; SCA group, which received a nanofiber matrix without stem cells; and SCA1MSCs group, which received a matrix graft with cultivated stem cells. One month after the procedure, euthanasia was performed on all animals in the experiment groups (Fig. 2). As a postoperative procedure, antimicrobian intramuscular therapy was carried out with penicillin (veterinary pentabiotic) 15,000 IU/kg, once a day for 5 to 7 days. The antiinflammatory intramuscular therapy was based on Flunixin meglumine (Banamine) 2 mg/kg, twice a day for 3 to 5 days. The antibiotic was administered in a prophylactic manner due to the presence of an implanted foreign object (i.e., the scaffold) in the animals’ organism. The animals were maintained at the veterinary facility of the Veterinary Hospital and Clinic of UFRGS. All the animals received clinical evaluation at least once a day during the postoperative period until they were submitted to euthanasia for the laryngectomy.

Scaffolds The polymer solution consisting of poly-DL-lactide (PDLLA) (molecular weight 75,000–120,000) (Sigma-Aldrich) was produced at a concentration of 7% (w/w) using 1,1,1,3,3,3Hexafluoro-2-propanol (Sigma-Aldrich). The construction of biomaterials was performed by the electrospinning method. The polymer solutions were placed between electrodes, which were connected to a high voltage. The voltage used for the solution of PDLLA was 20 kV, using an inner needle diameter of 0.45 mm and a flow rate of 1.88 mL/h. The distance between the needle and collector for the solution was 15 cm. For the present work,


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Process and Analysis of the Samples After euthanasia was performed following the aforementioned anesthetic procedure, the larynges of the animals were removed. The focus of interest was carefully removed, fixed in 10% buffered formalin and processed by the inclusion method in paraffin. Next, 7 lm sections were performed with a microtomus (Leica), deparaffined with xilol, and hydrated in decreasing alcohol series. The material was dyed with hematoxylin and eosin (HE). The laminas were examined with a Nikon Optiphot-

Jotz et al.: The Regeneration of Thyroid Cartilage

Fig. 1. Representation of surgical procedure. L group, SCA group, and SCA1MSC group. L 5 scaffold; MSC 5 mesenchymal stem cells; SCA 5 scaffold.

2 microscope (Tokyo, Japan) and the images captured with a Micrometrics CMOS 518 CU camera (Ottowa, Ontario, Canada) together with the microscope. The general composition of the lesion site was analyzed in the area of the cuts, with the aim of identifying the extremities of the thyroid cartilage maintained in a cartilaginous neoformation and the extension of this when present. The extension of the cartilaginous neoformation was measured in micrometers using the image analysis software Image Pro-Plus 6.0 (Media Cybernetics, Rockville, MD). The measurement was taken at the point where there was cartilage with basophilic matrix, suggesting an already established cartilage, and traced until the furthermost point from the cartilage in neoformation—tracing in a straight line all the area of the eosinophilic cartilage matrix (Fig. 3C and D). Ten measurements of each animal from each group were made in equidistant sections in the central area of the lesion. The first 0.5 cm of each lesion was discarded from the analysis from the initially selected cuts. The 0.5 cm of the free limit and inner part of the thyroid cartilage also were discarded (Fig. 4). For the section procedures, collected samples were analyzed from the centimeter median of the lesion. Figure 4 shows a detail of the pieces of tissue used for the histological analysis.

Under microscope examination, cartilaginous tissue growth was observed in just the SCA1MSCs group. On the laminas dyed with HE staining of the samples from the SCA and SCA1MSCs groups, the presence of granuloma (Fig. 3A) composed of macrophages, epithelial, and giant cells (Fig. 3B), as well as the presence of surgical thread was observed. In the L group, the extremities of the thyroid cartilage on the defect site presented an insignificant extension of neocartilage in two or three cuts, being the location of the reestablished defect by fibrous tissue (Fig. 3C). Observation of the histological cuts of the animals demonstrated that the lesion site remained present without complete closing and reconstruction of the cartilage. However, unlike the L group, the extension of regeneration was greater, presenting a far more significant area of neocartilage (Fig. 3D and 3E). In this experimental group, it was also observed that the point between the two extremities of cartilage where the defect had been created was filled with fibrous tissue,

Statistical Analysis The collected extension measurements of neoformed cartilage were compared between the different animal groups using one-way analysis of variance (ANOVA) followed by the post-hoc Tukey test (P < 0.05) with SPSS 11.5 software (SPSS Inc., Chicago, IL).

RESULTS During the 30-day study period, none of the animals showed abnormal clinical characteristics, and there was no observation of infection at the surgery site or respiratory difficulties. When euthanasia was performed, microscopic analysis showed that the lesion site, which was covered only by the scaffold or free of any type of covering, was easily identified. In the animals with induced defects covered with a scaffold containing mesenchymal stem cells, a fine layer of fibrous tissue was observed surrounded by cartilage, which made it difficult to see the lesions. Laryngoscope 124: December 2014

Fig. 2. Thyroid cartilage: right lateral view. (A) Used scaffold with stem cell to reconstruction. (B) Used-only scaffold to reconstruction.


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Fig. 3. Histological cross-sections of animal larynx cartilage. (A) SCA group, showing the region where granuloma was developed (scale bar 5 100 mm). (B) Black arrow shows the presence of giant cells in the formed granuloma (scale bar 5 50 mm). (C) Extremity of damaged cartilage from lesion group showing a small area of cartilaginous neoformation and presence of fibrosis. Fib was observed in the remains of the lesion (data not shown). (D) Extremity of damaged cartilage from SCA group showing that the extension of neocartilage was greater than in the lesion group. (E) Extremity of damaged cartilage from SCA1MSC group. The image shows the area of the cartilaginous neoformation. Between the lesion extremities, a bridge of cartilaginous matrix (*) was formed. (In C, D, and E, scale bar 5 100 mm). Fib 5 fibrosis; MSC 5 mesenchymal stem cells; SCA 5 scaffold.

and the said defect was not completely reestablished after 30 days of the scaffold grafting. The data described through microscopic analysis of the L and SCA groups was measured and analyzed statistically, and it was observed that the average extension of neocartilage formation of the L group was 136.3 lm (6 9.6 lm) and 387.7 lm (6 43.2 lm) of the SCA group, presenting a significant statistical difference (P < 0.01) (Fig. 5).

The analysis carried out on the sections from the lesion site of the cartilage from the larynges of the animals of the SCA1MSCs group showed a more extensive area of neocartilage compared to the SCA group. Furthermore, it was possible to observe in the fibrous extension formed between the extremities of the damaged cartilage, projections of cartilaginous matrix in a process of formation (Fig. 3D). The average of the neocartilage

Fig. 4. Histological analysis of biopsy (30 days after surgical procedure). The extension of neocartilage (red arrows) was measured by Image Pro-Plus software. Free limit and inner part of the thyroid cartilage were discarded.


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Fig. 5. Extension of cartilaginous neoformation after 30 days of lesion in the following groups: L, SCA, SCA1MSC. **P < 0.01. ***P < 0.001 using one-way ANOVA followed by Tukey post-hoc test. L 5 scaffold; MSC 5 mesenchymal stem cells; SCA 5 scaffold.

formation of the group that received a scaffold grafting cultivated with mesenchymal stem cells was 825.4 lm (6 122.1 lm), which was considered statistically significant (P < 0.001) when compared to the L and SCA groups (Fig. 5). The induced defect was not completely regenerated after a 30-day period. There was no formation of teratomas or tumors in consequence of the interposition of mesenchymal stem cells, and the scaffold was totally absorbed after 30 postoperative days.

DISCUSSION Engineered tissue could form the basis for novel therapies for millions of patients who suffer from the loss of tissue or its function, which includes patients with cancer of the larynx. In this present study, scaffolds with or without MSC were utilized as an alternative approach for the formation of the thyroid cartilage. It was observed that 30 days after the biomaterial had been implanted, a growth of cartilaginous tissue could be seen in the region in which the scaffold with the stem cells had been placed. Leukocyte infiltration in more than 50% of the histological cuts was observed, indicating an immunological response to this granulomatous reaction due to the prolonged presence of foreign material in the host tissue. However, no formation of tumors or teratomas was found under microscopic analysis. Furthermore, 1 month after the implant of the scaffolds with the stem cells, no evidence of material visible under a microscope was observed. After resection, it was also observed that the lesion of 4.0 cm2 in the cartilage had been partially repaired; this was observed in all animals that were analyzed. There was partial cartilaginous regeneration though the use of mesenchymal stem cells acquired from deciduous teeth pulp. Under microscopic observation, it was observed that there was not complete closing of the lesion area that was reconstructed with stem cells associated with the scaffold (group SCA1MSCs). Through palpation, a rigid frame of the closure of the lesion was observed. In the Laryngoscope 124: December 2014

area where only the scaffold was implanted (group SCA) with the aim of reconstructing the lesion, fibrous tissue was observed through palpation. In the area where no treatment was applied to reconstruct the lesion (group L), a fine layer of conjunctive tissue was observed. This grew, closing the cartilaginous orifice. In the group in which only a cartilaginous lesion was induced (group L) without any form of treatment, it was possible to microscopically observe a small formation of cartilaginous tissue. The small regenerated extension of cartilage observed in some sections can be explained by the differentiation of chondroblasts from the chondrogenic cells present in the perichondrium, which completely enwrapped the damaged extremities. The use of stem cells for tissue regeneration is an important chapter to be explored for daily clinical practice, mainly in relation to the reconstruction of structures that have been destroyed by tumorous diseases or trauma. Despite the observation that there was cartilaginous tissue growth in the group of stem cells cultivated on the scaffolds, it could not be confirmed that the stem cells were responsible for the growth of cartilage. Moreover, it can be confirmed that, according to the histopathological analysis, this cartilaginous tissue regeneration was observed with significant differences in relation to the other groups studied. There are numerous reports demonstrating that MSCs can repair tissue by directly differentiating into mesenchymal lineages. Recent studies have established that these cells can also enhance the differentiation of other progenitor cells into functional somatic cells. In addition, they may contribute to other aspects of local tissue repair via paracrine mechanisms.10,11 Thus, it is not clear which pathway was used by the MSCs to form the cartilage tissue in the animals. In this case, further analysis should be made; however, this was not the aim of this study. As with Lee et al.,12 the hypothesis of this present study is that techniques for the reconstruction of small and large cartilaginous defects must support for growth Jotz et al.: The Regeneration of Thyroid Cartilage

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and also for the mesenchymal stem cells. The present study corroborates this hypothesis; on a microscopic scale in the three studied groups, a small growth of cartilage was observed in the first group (L). However, in the second and third groups (SCA and SCA1MSCs, respectively), the presence of the scaffold was significant for the building of the cartilaginous structure, with a higher performance in the group in which there was a presence of stem cells. Observation of the histological cuts of the animals in the SCA group showed that the lesion site remained present without complete closing and reconstruction of the cartilage; however, it is believed that the presence of the scaffolds served as a support for the differentiation on a higher level of the chondrogenic cells from the perichondrium. Therefore, the scaffold here served as a bridge for the host cells and also as a support where MSC could attach to be transplanted in animal models and the cells could stay in the right place to be regenerated. Recently, Sharma et al.13 demonstrated significant results through the use of polymeric matrices as scaffolds for tissue engineering associated with mesenchymal stem cells. After preclinical testing in a caprine model, a pilot clinical study was initiated for which the biomaterials system was combined with standard microfracture surgery in 15 patients with focal cartilage defects on the medial femoral condyle. Their findings are corroborated with the results shown in the present study in which, within 1 month after the implant of the scaffolds, evidence of growth was shown in the cartilage in the in situ of surgically induced lesions in the thyroid cartilage. As in the study of Sharma et al.,13 the present study did not show side effects related to the use of mesenchymal stem cells in the cartilaginous reconstruction of the studied animals. Inanc¸ et al. 14 reported good results with the use of mesenchymal stem cells together with tissue engineering. The researchers analyzed the in vitro application of MSCs cultivated on biomaterials, with the aim of closing the defects of the conjunctive tissue and the upper airways, and there were no reports of the formation of tumors or teratomas. It was observed that tissue growth remained restricted to the limits defined by the scaffold when it was sutured in the cartilage, covering the lower, upper, and posterior areas of the lesion. The use of scaffolds with or without stem cells did not present difficulties or technical limitations for tissue regeneration. It was easy to implant within the lesion area; the stem cells only developed the area of tissue in which they were in contact. The limitations observed with this practice are still in reference to the time required to prepare the material for implantation at the receptor site, which is in the region of 7 days. In this study, in microscopic terms, the induced defect was not completely regenerated after a 30-day period, but the presence of mesenchymal stem cells on the scaffold accelerated reconstruction of the defective organ without any sign of teratomas. Therefore, it was shown that the use of MSCs presents itself as a differential in the formation of neighboring tissue, as in


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the case of thyroid cartilage. The scaffold does not only function as a “bridge,” but mainly as a suitable environment for the growth of cells and a link with the tissue that is to be regenerated. Although tissue regeneration has been small, there was a statistical difference among the studied groups. In future studies, the group intends to extend the analysis period of these surgical interventions, with the aim of evaluating macro and microscopic tissue repair over longer periods. Other strategies to be evaluated will be differentiation into chondroblasts and the use of scaffolds with encapsulated growth factors.

CONCLUSION The use of mesenchymal stem cells in association with nanofibers as scaffolds appears to be a promising strategy for the therapeutic regeneration of thyroid cartilage. To conclude, the use of the electrospinning technique for the production of nanofiber matrices associated with the cultivation of mesenchymal stem cells acquired from deciduous teeth presents an innovative alternative for tissue repair of cartilage from the larynx.

Acknowledgment The authors wish to thank Dr. Leder Leal Xavier for the support in the statistic analysis of this work.

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