Therapeutic Potential of Human Nasal Inferior Turbinate-Derived Stem Cells: Microarray Analysis of Multilineage Differentiation.

INTRODUCTION: Human nasal inferior turbinate-derived stem cells (hNTSCs) are attractive sources of adult stem cells for medical application because they can be easily obtained and cultivated with a highly proliferative capacity. The ability of hNTSCs to differentiate into chondrocytes, osteocytes, and neural cells makes them potential replacement therapeutic candidates in intractable disease. Nevertheless, detailed expression pattern of genes associated with trilineage differentiation (osteogenesis, chondrogenesis, and neurogenesis) in hNTSCs has not been revealed yet. METHODS: In this study, we aimed to evaluate gene expression patterns of various transcription factors and marker genes associated with a particular lineage (osteogenesis, chondrogenesis, and neurogenesis) of differentiation of hNTSCs by DNA microarrays. RESULTS: In microarrays, 36 transcripts such as E2F transcription factor 1, activating transcription factor 5, and AKR1B10 were upregulated and 36 transcripts such as CA9, PPFIA4, HAS2, and COL4A4 were downregulated in osteogenically differentiated hNTSCs. In chondrogenically differentiated hNTSCs, 3 transcripts (NUDT14, CPA4, and heparin-binding epidermal growth factor-like growth factor) were upregulated and 82 transcripts such as PTGS1, CLEC2D, and TET1 were downregulated. In neurally differentiated hNTSCs, 61 transcripts such as insulin-like growth factor-binding protein-1, nerve growth factor receptor, FGF1, OLFML1, and EPGN were upregulated and 98 transcripts such as ACAN, RUNX2, and C21orf96 were downregulated. In gene ontology (GO) analysis, cell signal-related GO terms were highly expressed. By contrast, catalysis GO terms and GO terms related to oxidoreductase were overrepresented in chondrogenically differentiated hNTSCs and osteogenically differentiated hNTSCs, respectively. CONCLUSION: Considering overall results, hNTSCs-specific genetic information may promote further studies on intracellular mechanisms defining key features of these cells.

Rapid Cartilage Regeneration of Spheroids Composed of Human Nasal Septum-Derived Chondrocyte in Rat Osteochondral Defect Model.

BACKGROUND: Cell-based therapies have been studied for articular cartilage regeneration. Articular cartilage defects have little treatments because articular cartilage was limited regenerative capacity. Damaged articular cartilage is difficult to obtain a successful therapeutic effect. In additionally these articular cartilage defects often cause osteoarthritis. Chondrocyte implantation is a widely available therapy used for regeneration of articular cartilage because this tissue has poor repair capacity after injury. Human nasal septum-drived chondrocytes (hNCs) from the septum show greater proliferation ability and chondrogenic capacity than human articular chondrocytes (hACs), even across different donors with different ages. Moreover, the chondrogenic properties of hNCs can be maintained after extensive culture expansion. METHODS: In this study, 2 dimensional (2D) monolayer cultured hNCs (hNCs-2D) and 3 dimensional (3D) spheroids cultured hNCs (hNCs-3D) were examined for chondrogenic capacity in vitro by PCR and immunofluorescence staining for chondrogenic marker, cell survival during cultured and for cartilage regeneration ability in vivo in a rat osteochondral defect model. RESULTS: hNCs-3D showed higher viability and more uniform morphology than 3D spheroids cultured hACs (hACs-3D) in culture. hNCs-3D also showed greater expression levels of the chondrocyte-specific marker Type II collagen (COL2A1) and sex-determining region Y (SRY)-box 9 (SOX9) than hNCs-2D. hNCs-3D also expressed chondrogenic markers in collagen. Specially, in the osteochondral defect model, implantation of hNCs-3D led to greater chondrogenic repair of focal cartilage defects in rats than implantation of hNCs-2D. CONCLUSION: These data suggest that hNCs-3D are valuable therapeutic agents for repair and regeneration of cartilage defects.

Accelerated Bone Regeneration via Three-Dimensional Cell-Printed Constructs Containing Human Nasal Turbinate-Derived Stem Cells as a Clinically Applicable Therapy.

Stem cell transplantation is a promising therapeutic strategy that includes both cell therapy and tissue engineering for the treatment of many regenerative diseases; however, the efficacy and safety of stem cell therapy depend on the cell type used in therapeutic and translational applications. In this study, we validated the hypothesis that human nasal turbinate-derived mesenchymal stem cells (hTMSCs) are a potential therapeutic source of adult stem cells for clinical use in bone tissue engineering using three-dimensional (3D) cell-printing technology. hTMSCs were cultured and evaluated for clinical use according to their cell growth, cell size, and preclinical safety and were then incorporated into a multicompositional 3D bioprinting system and investigated for bone tissue regeneration in vitro and in vivo. Finally, hTMSCs were compared with human bone marrow-derived MSCs (hBMSCs), which are the most common stem cell type used in regenerative medicine. hTMSCs from three different donors showed greater and faster cell growth than hBMSCs from two different donors when cultured. The hTMSCs were smaller in size than the hBMSCs. Furthermore, the hTMSCs did not exhibit safety issues in immunodeficient mice. hTMSCs in 3D-printed constructs (3D-hTMSC) showed much greater viability, growth, and osteogenic differentiation potential in vitro than hBMSCs in 3D-printed constructs (3D-hBMSC). Likewise, 3D-hTMSC showed better cell survival and alkaline phosphatase activity and greater osteogenic protein expression than 3D-hBMSC upon subcutaneous implantation into the dorsal region of nude mice. Notably, in an orthotopic model involving implantation into a tibial defect in rats, implantation of 3D-hTMSC led to greater bone matrix formation and enhanced bone healing to a greater degree than implantation of 3D-hBMSC. The clinically reliable evidence provided by these results is underlined by the potential for rapid tissue regeneration and ambulation in bone fracture patients implanted with 3D-hTMSC.

Characteristics of Nasal Septal Cartilage-Derived Progenitor Cells during Prolonged Cultivation.

Objective To produce alternate cell sources for tissue regeneration, human nasal septal cartilage-derived progenitor cells (NSPs) were tested to identify whether these cells meet the criteria of cartilage progenitor cells. We also evaluated the effects of prolonged cultivation on the characteristics of NSPs. Study Design In vitro study. Setting Academic research laboratory. Methods NSPs were isolated from discarded human nasal septal cartilage. NSPs were cultured for 10 passages. The expression of septal progenitor cell surface markers was assessed by fluorescence-activated cell sorting. Cell proliferation was measured with a cell-counting kit. Cytokine secretion was analyzed with multiplex immunoassays. Chondrogenic differentiation of NSPs without differentiation induction was analyzed with type II collagen immunohistochemistry. Cartilage-specific protein expression was evaluated by Western blotting. Under osteo- and adipodifferentiation media, 2 lineage differentiation potentials were evaluated by histology and gene expression analysis. Results Surface epitope analysis revealed that NSPs are positive for mesenchymal stem cells markers and negative for hematopoietic cell markers. Cultured NSPs showed sufficient cell expansion and chondrogenic potential, as demonstrated by immunostaining and expression of cartilage-specific protein. IL-6, IL-8, and transforming growth factor ß were secreted by over 200 pg/mL. The osteo- and adipodifferentiation potentials of NSPs were identified by histology and specific gene expression. The aforementioned characteristics were not influenced by prolonged cultivation. Conclusion NSPs represent an initial step toward creating a cell source from surgically discarded tissue that may prove useful in cartilage regeneration.

Evaluation of characteristic of human turbinate derived mesenchymal stem cells cultured in the serum free media.

We evaluated the effect of serum-free and xeno-cultivation (SFXFM) on the characterization, proliferation, and differentiation properties of human nasal stem cells (airway tissue; hTMSCs). hTMSCs were isolated from 10 patients, after which patient samples were separated into two groups, an SFXFM group and a control group. The control group was treated with bovine serum-containing medium. FACS analysis revealed that SFXFM-cultured hTMSCs maintained a characteristic mesenchymal stem cell phenotype. hTMSC proliferation was not influenced by SFXFM. In addition, upregulation of IL-8 and GM-CSF and downregulation of RANTES expression were shown in response to SFXFM. Moreover, two-lineage differentiation properties (osteocyte and adipocyte) of hTMSCs were enhanced under SFXFM. Finally, the genetic stability of SFXFM-cultured hTMSCs was demonstrated by normal karyotype results. SFXFM enables good expansion, multipotentiality, and normal genotype maintenance of MSCs. Moreover, this approach serves as a substitute to conventional media for the cultivation of capable MSCs for upcoming medical applications.

New application of three-dimensional printing biomaterial in nasal reconstruction.

OBJECTIVES: Polycaprolactone (PCL) is an U.S. Food and Drug Administration-approved synthetic biodegradable polymer and is easily fabricated into three-dimensional (3D) structures. In this study, the 3D-printed PCL implant for nasal augmentation was further evaluated for its suitability for nasal surgeries such as septoplasty and rhinoplasty. METHODS: Ten New Zealand White rabbits were included and divided into study and sham groups (7 and 3, respectively). A lateral incision was made on the nasal dorsum and a pocket formed in the subperichondrial plane between the upper lateral cartilage and nasal septum. Polycaprolactone was fabricated based on 3D printing technology into a 0.8 × 0.8-cm rectangular shape for use as a nasal implant. The material was inserted as a septal extension graft and sutured with alar cartilage for nasal reshaping. The implants were harvested 4, 8, and 12 weeks after implantation and evaluated by gross morphological assessment and histological examination. RESULTS: The initial shape of the implant was unchanged in all cases, and no definitive postoperative complications were seen over the 3-month period. Gross morphological evaluation confirmed that implants remained in their initial location without migration or extrusion. Histologic evaluations showed that the implant architectures were maintained with excellent fibrovascular ingrowth and minimal inflammatory reactions. CONCLUSION: Polycaprolactone can be used for nasal reconstruction such as nasal augmentation. Polycaprolactone is easy to work with and will avoid the increased operative time and morbidity associated with autograft harvesting. Therefore, PCL implants designed by 3D printing can serve as clinically biocompatible materials in craniofacial reconstruction in the future. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:1036-1043, 2017.

Characteristics of Human Turbinate-Derived Mesenchymal Stem Cells Are Not Affected by Allergic Condition of Donor.

The characteristics of mesenchymal stem cells (MSCs) derived from human turbinates (hTMSCs) have not been investigated in allergic rhinitis. We evaluated the influence of allergic state of the donor on the characteristics, proliferation, and differentiation potential of hTMSCs, compared with hTMSCs derived from non-allergic patients. hTMSCs were isolated from five non-allergic and five allergic patients. The expression of toll-like receptors (TLRs) in hTMSCs was measured by FACS, and cell proliferation was measured using a cell counting kit. Cytokine secretion was analyzed using multiplex immunoassays. The osteogenic, chondrogenic, and adipogenic differentiation potentials of hTMSCs were evaluated by histology and gene expression analysis. In allergic patients, FACS analysis showed that TLR3 and TLR4 were more highly expressed on the surface of hTMSCs than TLR2 and TLR5. The proliferation of hTMSCs was not influenced by the presence of TLR priming. The expression of IL-6, IL-8, IL-12, IP-10, and RANTES was upregulated after the TLR4 priming. The differentiation potential of hTMSCs was not influenced by TLR priming. These characteristics of hTMSCs were similar to those of hTMSCs from non-allergic patients. We conclude that the allergic condition of the donor does not influence TLR expression, proliferation, or immunomodulatory potential of hTMSCs.

Comparative effects of chlorhexidine and essential oils containing mouth rinse on stem cells cultured on a titanium surface.

Chlorhexidine (CHX) and Listerine (LIS), an essential oil compound, are the two commonly used adjunctive agents for mechanical debridement, for reducing the bacterial load in the treatment of peri-implant inflammation. However, antimicrobial agents have been reported to be cytotoxic to the alveolar bone cells and gingival epithelial cells. The present study was performed to examine the effects of antiseptics CHX and LIS, on the morphology and proliferation of stem cells. Stem cells derived from the buccal fat pad were grown on machined titanium discs. Each disc was immersed in CHX or LIS for 30 sec, 1.5 min or 4.5 min. Cell morphology was evaluated with a confocal laser microscope and the viability of the cells was quantitatively analyzed with the cell counting kit-8 (CCK-8). The untreated cells attached to the titanium discs demonstrated well-organized actin cytoskeletons. No marked alterations in the cytoskeletal organization were observed in any of the treated groups. The treatment with CHX and LIS of the titanium discs decreased the viability of the cells grown on the treated discs (P<0.05). The stem cells derived from the buccal fat pad were sensitive to CHX and LIS, and a reduction in cellular viability was observed when these agents were applied to the discs for 30 sec. Further studies are required to determine the optimal application time and concentration of this antimicrobial agent for maximizing the reduction of the bacterial load and minimizing the cytotoxicity to the surrounding cells.