Sasa Janjanin

MOLD-SHAPED, NANOFIBER SCAFFOLD-BASED CARTILAGE ENGINEERING USING HUMAN MESENCHYMAL STEM CELLS AND BIOREACTOR

BACKGROUND Mesenchymal stem cell (MSC)-based tissue engineering is a promising future alternative to autologous cartilage grafting. This study evaluates the potential of using MSCs, seeded into electrospun, biodegradable polymeric nanofibrous scaffolds, to engineer cartilage with defined dimensions and shape, similar to grafts used for subcutaneous implantation in plastic and reconstructive surgery. MATERIALS AND METHODS Human bone marrow derived MSCs seeded onto nanofibrous scaffolds and placed in custom-designed molds were cultured for up to 42 days in bioreactors. Chondrogenesis was induced with either transforming growth factor-beta1 (TGF-beta1) alone or in combination with insulin-like growth factor-I (IGF-I). RESULTS Constructs exhibited hyaline cartilage histology with desired thickness and shape as well as favorable tissue integrity and shape retention, suggesting the presence of elastic tissue. Time-dependent increase in cartilage matrix gene expression was seen in both types of culture: at Day 42, TGF-beta1/IGF-I treated cultures showed higher collagen Type 2 and aggrecan expression. Both culture conditions showed significant time-dependent increase in sulfated glycosaminoglycan and hydroxyproline contents. TGF-beta1/IGF-I-treated samples were significantly stiffer; with equilibrium compressive Youngs modulus values reaching 17 kPa by Day 42. CONCLUSIONS The successful ex vivo development of geometrically defined cartilaginous construct using customized molding suggests the potential of cell-based cartilage tissue for reconstructive surgery.

Activin A Expression Regulates Multipotency of Mesenchymal Progenitor Cells

IntroductionBone marrow (BM) stroma currently represents the most common and investigated source of mesenchymal progenitor cells (MPCs); however, comparable adult progenitor or stem cells have also been isolated from a wide variety of tissues. This study aims to assess the functional similarities of MPCs from different tissues and to identify specific factor(s) related to their multipotency.MethodsFor this purpose, we directly compared MPCs isolated from different adult tissues, including bone marrow, tonsil, muscle, and dental pulp. We first examined and compared proliferation rates, immunomodulatory properties, and multidifferentiation potential of these MPCs in vitro. Next, we specifically evaluated activin A expression profile and activin A:follistatin ratio in MPCs from the four sources.ResultsThe multidifferentiation potential of the MPCs is correlated with activin A level and/or the activin A:follistatin ratio. Interestingly, by siRNA-mediated activin A knockdown, activin A was shown to be required for the chondrogenic and osteogenic differentiation of MPCs. These findings strongly suggest that activin A has a pivotal differentiation-related role in the early stages of chondrogenesis and osteogenesis while inhibiting adipogenesis of MPCs.ConclusionsThis comparative analysis of MPCs from different tissue sources also identifies bone marrow-derived MPCs as the most potent MPCs in terms of multilineage differentiation and immunosuppression, two key requirements in cell-based regenerative medicine. In addition, this study implicates the significance of activin A as a functional marker of MPC identity.

In Vitro Adipose Tissue Engineering Using an Electrospun Nanofibrous Scaffold

Electrospun 3-dimensional nanofibrous scaffolds share morphologic similarities to collagen fibrils, and promote favorable biologic responses of seeded cells. In this study, we have fabricated a 3-dimensional nanofibrous scaffold made of poly L-lactic acid, and examined its ability to support and maintain the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells in vitro. After a 21-day incubation, oil red O staining of constructs treated with adipogenic supplements revealed positive adipose-like staining, compared with lack of staining in untreated cultures. Semi-quantitative RT-PCR analysis of human bone marrow-derived mesenchymal stem cells cultured in adipogenic medium revealed highly elevated levels of adipogenesis-associated genes (1797-fold for lipoprotein lipase, and 5.6-fold for peroxisome proliferator-activated receptor γ). Immunofluorescence staining of cellular constructs in adipogenic culture media showed the presence of lipoprotein lipase vesicles, a characteristic feature of adipose tissue. These results suggest that the poly L-lactic acid-based nanofibrous scaffold is a promising candidate for adult stem cell-based engineering of adipose tissue.

Volumetric facelift: evaluation of rhytidectomy with alloplastic augmentation.

Objectives Facial aging occurs as a result of soft tissue atrophy and resorption of the bony skeleton, which results in a loss of soft tissue volume and laxity of the overlying skin. Volumetric augmentation is a key component of facial rejuvenation surgery, and should be considered of equal importance to soft tissue lifting. Augmentation can be accomplished with synthetic fillers, autologous grafts, soft tissue repositioning techniques, and/or alloplastic implants. Only alloplastic implants, however, provide truly long-term volumetric correction. To date, there have been no large series dealing with the complications and results of implantation performed concurrently with rhytidectomy, which we have termed “volumetric rhytidectomy.” We present our experience with 100 patients treated with a combination of malar and chin implants and rhytidectomy, compared to 200 patients who underwent rhytidectomy alone. Methods The authors performed a retrospective review of patients treated with a combination of silicone malar and chin augmentation with rhytidectomy versus patients treated with rhytidectomy alone. Both groups of patients underwent close postoperative evaluation at 3 days, 1 week, 2 weeks, and 1 month. All patients were surveyed at 6 months to assess aesthetic satisfaction. Complication rates were noted and tabulated. Statistical analysis was performed to evaluate for any differences in the two groups. Results Between 2002 and 2006, 100 patients underwent malar and chin implantation along with rhytidectomy; 200 patients underwent rhytidectomy alone. In the first group, there were a total of 6 cases in which implant removal was necessary, and 2 cases in which revision was required. There were no statistically significant differences (p < 0.05) observed between the two groups with respect to major or minor hematoma, seroma, infection, sensory nerve injury, facial nerve injury, hypertrophic scarring, dehiscence, skin sloughing, or revision. Conclusions Volumetric rhytidectomy reliably augments the malar and mental areas, allows for subtle skeletal contouring, and results in successful rejuvenation. Rhytidectomy is relatively safe to perform concurrently with silicone augmentation, and does not result in an increased complication rate as compared to rhytidectomy alone.

Does suture material and technique really matter? Lessons learned from 800 consecutive blepharoplasties.

Objectives: The purpose of this study was to evaluate established suture materials and techniques for blepharoplasty closure and evaluate for any differences in rates of complications between these groups.

Synchronous multicentric bilateral sinonasal inverted papilloma and frontal sinus osteoma.

Inverted papilloma is generally considered a benign unilateral sinonasal tumor. Its synchronous bilateral multicentric occurrence is extremely rare. A 22-year-old male patient presented with stage III inverted papilloma involving both ethmoid sinuses, both frontal sinuses, and cribriform area. The patient also had a large osteoma emanating from the basal part of the frontal sinus septum, which completely obstructed both nasofrontal recesses, leaving no communication between the sinuses and the nasal cavity. The frontal sinus septum was intact, so there was no communication between the 2 sides either. Following the era of aggressive surgical approaches dominated by lateral rhinotomy and medial maxillectomy, the advent of endoscopic techniques has dramatically improved visualization of sinus chambers and nasal cavity, resulting in lower morbidity and similar results to those achieved with open surgical procedures. In our patient, the concomitant presence of a huge frontal sinus osteoma posed an unacceptable risk for endoscopic resection due to the possible residual disease in the nasofrontal recess regions. Surgical resection remains the mainstay treatment and should be tailored in accordance with the localization and spread of disease. The surgeon should be ready to use different surgical approaches and, if intraoperatively needed, to modify them accordingly.

R410 – Immunosuppressive Properties of Tonsil-Derived MSCs

Problem We have previously shown that stroma of human palatine tonsils contains mesenchymal stem cells (MSCs) that can be isolated and expanded in culture. These tonsil-derived MSCs (T-MSCs) show multipotent differentiation properties, i.e. can differentiate along multiple mesenchymal lineages, including osteoblasts, chondrocytes, adipocytes, and myocytes. Recent findings also show that MSCs display immunoregulatory properties. Although the exact immunosuppressive mechanisms are unknown, the capacity of MSCs to suppress T-cell proliferation stimulated by allogeneic lymphocytes, dendritic cells, and phytohemaglutinin (PHA) is well documented. This study explores immunosuppressive characteristics of T-MSCs and compares them with characteristics of bone marrow-derived MSCs (BM-MSCs), a well-characterized cell population. Methods The mixed lymphocyte reaction (MLR) using human peripheral blood mononuclear cells (PBMC) from healthy donors and xenogeneic murine splenocytes was used to test the immunosuppressive properties of T-MSCs and BMMSCs. Indoleamine 2,3-dioxygenase (IDO) enzyme activity was measured spectrophotometrically based on tryptophan-to-kynurenine conversion in the supernatant. Interferon (IFN)-g in culture supernatants was quantified using a commercially available ELISA kit. Results Addition of BM-MSCs and T-MSCs both inhibited the PHA-induced proliferative response of PBMC and xenogeneic splenocytes. The difference in immunosuppressive activity correlates with the level of cell surface interferon (IFN)-g receptor as well as the differential ability of IFN-g to stimulate of IDO activity by T-MSCs compared to BM-MSCs. Conclusion T-MSCs share similar immunosuppressive characteristics as BM-MSCs in MLR. The immunosuppressive activity is significant and dose-dependent, although at a lower level than that of BM-MSCs. Significance Owing to their ease of isolation, rapid proliferation in the culture and self-renewal capacity, MSCs to date are considered an attractive candidate cell type for the development of novel cell-based therapies. They could be relevant in a wide range of clinical applications, including tissue and organ repair, drug or gene delivery to diseased tissues, improvement of allogenic hematopoietic cell transplantation, and the management of graft-versus-host disease. Support Supported by NIAMS Intramural Research Program (NIH ZO1 AR 41131). Sasa Janjanin is a recipient of the Fulbright Scholarship of the U.S. Department of State.

S168 – The Use of a Harmonic Scalpel in Head & Neck Surgery

Objectives The harmonic scalpel cuts and coagulates simultaneously using a mechanical vibration. We present our 1-year experience in the use Harmonic ACE(tm), Harmonic FOCUS(tm) and harmonic scalpel with 5 mm curved blade in head and neck surgery (Ethicon Endo-Surgery). Methods Over period of 1 year we performed 295 thyroidectomies, 23 parotidectomies, and 45 tonsillectomies using the harmonic scalpel. Control group consisted of 106 thyroidectomies, 9 parotidectomies, and 30 tonsillectomies that were performed over previous 5 months with the use of conventional hemostatic techniques (electrocautery and knot tying). Results The use of the harmonic scalpel shortened the duration of thyroid and parotid surgery by 20–30% as compared with the conventional techniques (p<0.05). The average skin incision length for those undergoing thyroidectomy with the harmonic scalpel was 1.8 cm shorter as compared with the conventional thyroidectomy group (p<0.05). Because of small skin incisions in thyroidectomy we preferred Harmonic ACE(tm) over Harmonic FOCUS(tm) due to longer shaft of scalpel, while Harmonic FOCUS(tm) was more suitable for parotidectomy. The use of Harmonic ACE(tm) shortened average duration of tonsillectomy by 5.5 minutes, while use of 5 mm curved blade (HF 105) did not affect significantly the average duration of tonsillectomy. Postoperative pain and complication rate were comparable for both groups (p>0.05). Conclusions The use of the harmonic scalpel reduces surgical time and intraoperative blood loss in head and neck procedures, and enables shorter incision length in thyroid surgery.