Susanna Miettinen

Novel maxillary reconstruction with ectopic bone formation by GMP adipose stem cells

Microvascular reconstruction is the state-of-the-art in many fields of defect surgery today. Currently, reconstruction of large bony defects involves harvesting of autologous bone causing donor site morbidity and risk of infection. Specifically, utilizing autologous adipose stem cells (autoASCs), large quantities of cells can be retrieved for cell therapy applications and the risk of tissue rejection is diminished. The authors describe the first case report of a microvascular custom-made ectopic bone flap employing good manufacturing practice (GMP) level ASCs. The patient underwent a hemimaxillectomy due to a large keratocyst. After 36 months of follow-up, the defect was reconstructed with a microvascular flap using autoASCs, beta-tricalcium phosphate and bone morphogenetic protein-2. ASCs were isolated and expanded in clean room facilities according to GMP standards and were characterized in vitro. After 8 months of follow-up, the flap had developed mature bone structures and vasculature and was transplanted into the defect area. Postoperative healing has been uneventful, and further rehabilitation with dental implants has been started. The in vitro characterization demonstrated multipotentiality and mesenchymal stem cell characteristics in ASCs. This is the first clinical case where ectopic bone was produced using autoASCs in microvascular reconstruction surgery and it will pave way for new clinical trials in the field.

The Potential of Adipose Stem Cells in Regenerative Medicine

Adipose stem cells (ASCs) are an attractive and abundant stem cell source with therapeutic applicability in diverse fields for the repair and regeneration of acute and chronically damaged tissues. Importantly, unlike the human bone marrow stromal/stem stem cells (BMSCs) that are present at low frequency in the bone marrow, ASCs can be retrieved in high number from either liposuction aspirates or subcutaneous adipose tissue fragments and can easily be expanded in vitro. ASCs display properties similar to that observed in BMSCs and, upon induction, undergo at least osteogenic, chondrogenic, adipogenic and neurogenic, differentiation in vitro. Furthermore, ASCs have been shown to be immunoprivileged, prevent severe graft-versus-host disease in vitro and in vivo and to be genetically stable in long-term culture. They have also proven applicability in other functions, such as providing hematopoietic support and gene transfer. Due to these characteristics, ASCs have rapidly advanced into clinical trials for treatment of a broad range of conditions. As cell therapies are becoming more frequent, clinical laboratories following good manufacturing practices are needed. At the same time as laboratory processes become more extensive, the need for control in the processing laboratory grows consequently involving a greater risk of complications and possibly adverse events for the recipient. Therefore, the safety, reproducibility and quality of the stem cells must thoroughly be examined prior to extensive use in clinical applications. In this review, some of the aspects of examination on ASCs in vitro and the utilization of ASCs in clinical studies are discussed.

Characterisation of human dental stem cells and buccal mucosa fibroblasts

Human craniofacial stem cells are recently discovered sources of putative mesenchymal stem cells that hold great promise for autogenic or allogenic cell therapy and tissue engineering. Prior to employing these cells in clinical applications, they must be thoroughly investigated and characterized. In this study, the surface marker expression was investigated on dental pulp stem cells (DPSCs), dental follicle cells (DFCs), periodontal ligament stem cells (PDLSCs), and buccal mucosa fibroblasts (BMFs) utilising surface markers for flow cytometry. The osteogenic potential was also examined by bone-associated markers alkaline phosphatase, Runx2, collagen type I, osteocalcin, and osteopontin. The results from our study demonstrate that the dental cell sources exhibit comparable surface marker and bone-associated marker profiles parallel to those of other mesenchymal stem cell sources, yet distinct from the buccal mucosa fibroblasts. Our data support evidence towards clinical applicability of dental stem cells in hard tissue regeneration.

Serum-free, xeno-free culture media maintain the proliferation rate and multipotentiality of adipose stem cells in vitro

BACKGROUND AIMS Human adipose stem cells (ASC) are an abundant, readily available population of multipotent progenitor cells that reside in adipose tissue. ASC have been shown to have therapeutic applicability in pre-clinical studies, but a standardized expansion method for clinical cell therapy has yet to be established. Isolated ASC are typically expanded in medium containing fetal bovine serum (FBS); however, sera and other culturing reagents of animal origin in clinical therapy pose numerous safety issues, including possible infections and severe immune reactions. METHODS To identify optimal conditions for ex vivo expansion of ASC, the effects of seven serum-free (SF) and xeno-free (XF) media were investigated with both FBS and allogeneic human serum (alloHS; as a control media). Surface marker expression, proliferation, morphology and differentiation analyzes were utilized for investigating the effects of media on ASC. RESULTS The proliferation and morphology analysis demonstrated significant differences between ASC cultured in SF/XF culture media compared with serum-containing culture media, with medium prototype StemPro MSC SFM XenoFree providing significantly higher proliferation rates than ASC cultured in media containing serum, while still maintaining the differentiation potential and surface marker expression profile characteristic of ASC. CONCLUSIONS Looking forward, fully defined XF media formulations will provide the means for the development and approval of safer clinical cell therapy treatments. However, to fully recognize the capacity of these XF culture media, further pre-clinical safety and efficacy studies must be performed.

A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic, Induced Pluripotent and Adipose Stem Cells

Background The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable. Methodology/Principal Findings Here, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed. Conclusion/Significance Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.

Cranioplasty with adipose-derived stem cells and biomaterial: a novel method for cranial reconstruction.

BACKGROUND:There is no optimal method for reconstruction of large calvarial defects. Because of the limitations of autologous bone grafts and alloplastic materials, new methods for performing cranioplasties are needed. OBJECTIVE:To create autologous bone to repair cranial defects. METHODS:We performed a cranioplasty procedure with this new method in 4 patients who had large calvarial defects of different etiologies. We used autologous adipose-derived stem cells seeded in beta-tricalcium phosphate granules. For 2 patients, we used a bilaminate technique with resorbable mesh. RESULTS:During follow-up, there were no clinically relevant postoperative complications. The computed tomography scans revealed satisfactory outcome in ossification, and in the clinical examinations, the outcomes were good. The cranioplasty was measured in Hounsfield units from each computed tomography scan. The Hounsfield units increased gradually to equal the value of bone. CONCLUSION:The combination of scaffold material such as beta-tricalcium phosphate and autologous adipose-derived stem cells constitutes a promising model for reconstruction of human large cranial defects. The success of these clinical cases paves way for further studies and clinical applications to turn this method into a reliable treatment regimen.

Antiproliferative Action of Vitamin D

During the past few years, it has become apparent that vitamin D may play an important role in malignant transformation. Epidemiological studies suggest that low vitamin D serum concentration increases especially the risk of hormone-related cancers. Experimentally, vitamin D suppresses the proliferation of normal and malignant cells and induces differentiation and apoptosis. In the present review we discuss the mechanisms whereby vitamin D regulates cell proliferation and whether it could be used in prevention and treatment of hyperproliferative disorders like cancers.

Growth and Osteogenic Differentiation of Adipose Stem Cells on PLA/Bioactive Glass and PLA/β-TCP Scaffolds

The aim of this study was to compare the effects of novel three-dimensional composite scaffolds consisting of a bioactive phase (bioactive glass or beta-tricalcium phosphate [beta-TCP] 10 and 20 wt%) incorporated within a polylactic acid (PLA) matrix on viability, distribution, proliferation, and osteogenic differentiation of human adipose stem cells (ASCs). The viability and distribution of ASCs on the bioactive composite scaffolds was evaluated using Live/Dead fluorescence staining, environmental scanning electron microscopy, and scanning electron microscopy. There were no differences between the two concentrations of bioactive glass and beta-TCP in PLA scaffolds on proliferation and osteogenic differentiation of ASCs. After 2 weeks of culture, DNA content and alkaline phosphatase (ALP) activity of ASCs cultured on PLA/beta-TCP composite scaffolds were higher relative to other scaffold types. Interestingly, the cell number was significantly lower, but the relative ALP/DNA ratio of ASCs was significantly higher in PLA/bioactive glass scaffolds than in other three scaffold types. These results indicate that the PLA/beta-TCP composite scaffolds significantly enhance ASC proliferation and total ALP activity compared to other scaffold types. This supports the potential future use of PLA/beta-TCP composites as effective scaffolds for tissue engineering and as bone replacement materials.

Characterization of zinc-releasing three-dimensional bioactive glass scaffolds and their effect on human adipose stem cell proliferation and osteogenic differentiation

While the addition of zinc ions to bioactive ceramics has been shown to enhance the proliferation and osteogenic differentiation of osteoblast-like cells, contradictory results have been found. Therefore, the effect of zinc-releasing ceramics on cell proliferation and differentiation into osteogenic lineages requires further clarification. The aim of this study was to evaluate the effects of zinc addition on the degradation profile of three-dimensional bioactive glass scaffold, and on the proliferation and osteogenesis of human adipose stem cells (hASCs) in these scaffolds. Bioactive glass scaffolds containing Na(2)O, K(2)O, MgO, CaO, B(2)O(3), TiO(2), P(2)O(5) and SiO(2) were prepared. The degradation was evaluated by weight loss measurement, scanning electron microscopy and elemental analysis. The degradation profile of bioactive glass was shown to slow down with the addition of zinc. Qualitative live/dead staining showed that zinc addition to bioactive glass inhibits cell spreading and proliferation of hASCs. However, zinc addition had no significant effect on DNA content, alkaline phosphatase activity and osteopontin concentration of hASCs when measured quantitatively. Our results suggest that the possible stimulatory effect of addition of zinc on hASC proliferation and osteogenesis was not detected because addition of zinc slowed down the degradation rate of the studied bioactive glass scaffolds.

Adipose Stem Cells Used to Reconstruct 13 Cases With Cranio-Maxillofacial Hard-Tissue Defects

Although isolated reports of hard‐tissue reconstruction in the cranio‐maxillofacial skeleton exist, multipatient case series are lacking. This study aimed to review the experience with 13 consecutive cases of cranio‐maxillofacial hard‐tissue defects at four anatomically different sites, namely frontal sinus (3 cases), cranial bone (5 cases), mandible (3 cases), and nasal septum (2 cases). Autologous adipose tissue was harvested from the anterior abdominal wall, and adipose‐derived stem cells were cultured, expanded, and then seeded onto resorbable scaffold materials for subsequent reimplantation into hard‐tissue defects. The defects were reconstructed with either bioactive glass or β‐tricalcium phosphate scaffolds seeded with adipose‐derived stem cells (ASCs), and in some cases with the addition of recombinant human bone morphogenetic protein‐2. Production and use of ASCs were done according to good manufacturing practice guidelines. Follow‐up time ranged from 12 to 52 months. Successful integration of the construct to the surrounding skeleton was noted in 10 of the 13 cases. Two cranial defect cases in which nonrigid resorbable containment meshes were used sustained bone resorption to the point that they required the procedure to be redone. One septal perforation case failed outright at 1 year because of the postsurgical resumption of the patients uncontrolled nasal picking habit.