Hannu Kuokkanen

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.

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.

Stem cell enrichment does not warrant a higher graft survival in lipofilling of the breast: a prospective comparative study.

BACKGROUND Stem cell enrichment is generally believed to be of crucial importance for success in lipofilling for cosmetic breast augmentation. No comparative clinical studies have been reported to support this. METHODS A total of 18 women underwent breast augmentation with water-assisted lipotransfer (WAL). In 10 of the cases, transferred lipoaspirate was enriched with stromal stem cells using the Celution(®) system (Cytori Therapeutics Inc., San Diego, Ca, USA). Magnetic resonance imaging (MRI)-based volumetric analysis was done preoperatively and 6 months after the procedure. To verify scientifically that stem cells were transplanted, samples of the transplanted tissues were processed in the laboratory to isolate the adipose stem cells (ASCs). RESULTS MRI volumetry revealed a volume survival of the whole (watery) graft of mean 54% (SD 7) in the WAL only and of 50% (SD 10) in the WAL with stem cell-enrichment patients. As centrifugation of the WAL grafts demonstrated an average adipose tissue of 68%, the average volume survival of adipose tissue itself was 79% (SD 13) in the WAL only and 74% (SD 14) in the WAL with stem cell-enrichment patients. This difference (4.5%) was not statistically significant (independent samples t test, p = 0.330, 95% confidence interval of difference, 4.8, 13.9%). CONCLUSIONS Breast augmentation by lipofilling using WAL alone is faster, cheaper, has a lower risk of contamination and offers at least an equal take rate. We do not see any advantage in stem cell enrichment by the Celution(®) system in cosmetic fat transplantation to the breast.

Postoperative wound complications after internal fixation of closed calcaneal fractures : A retrospective analysis of 126 consecutive patients with 148 fractures

Aim: The aim of the study was to analyse the number of soft-tissue complications after internal fixation of calcaneal fractures and to evaluate risk factors leading to these complications. Material: A retrospective analysis of 126 consecutive patients with 148 operatively treated calcaneal fractures was performed. Only primarily closed fractures were included in the study. Results: Wound healing was problematic in 35 cases (24 per cent). The wound was infected in 23 cases (16 per cent) and a wound edge necrosis was observed in 12 cases (8 per cent). The soft tissue complication needed operative treatment in 20 cases (14 per cent). The complications did not lead to amputations in any case. In the end of the follow up all wounds had healed. Conclusions: The statistical analysis identified a longer delay in surgery and longer operation time to be positive risk factors for wound complications.

The effects of vibration loading on adipose stem cell number, viability and differentiation towards bone-forming cells

Mechanical stimulation is an essential factor affecting the metabolism of bone cells and their precursors. We hypothesized that vibration loading would stimulate differentiation of human adipose stem cells (hASCs) towards bone-forming cells and simultaneously inhibit differentiation towards fat tissue. We developed a vibration-loading device that produces 3g peak acceleration at frequencies of 50 and 100 Hz to cells cultured on well plates. hASCs were cultured using either basal medium (BM), osteogenic medium (OM) or adipogenic medium (AM), and subjected to vibration loading for 3 h d–1 for 1, 7 and 14 day. Osteogenesis, i.e. differentiation of hASCs towards bone-forming cells, was analysed using markers such as alkaline phosphatase (ALP) activity, collagen production and mineralization. Both 50 and 100 Hz vibration frequencies induced significantly increased ALP activity and collagen production of hASCs compared with the static control at 14 day in OM. A similar trend was detected for mineralization, but the increase was not statistically significant. Furthermore, vibration loading inhibited adipocyte differentiation of hASCs. Vibration did not affect cell number or viability. These findings suggest that osteogenic culture conditions amplify the stimulatory effect of vibration loading on differentiation of hASCs towards bone-forming cells.

Late results of skin-sparing mastectomy followed by immediate breast reconstruction†

Skin‐sparing mastectomy (SSM) facilitates optimal immediate breast reconstruction (IBR) by preserving the inframammary fold and most of the breast skin. Concerns persist that SSM might increase the rate of local recurrence as the surgical approach is less extensive. Patients who had SSM and IBR over 10 years at a single institution were reviewed.

Intraoperative evaluation of blood flow in the internal mammary or thoracodorsal artery as a recipient vessel for a free TRAM flap

Although the free microvascular transverse rectus abdominis musculocutaneous (TRAM) flap is in routine use for breast reconstruction, little is known of its hemodynamics. The purpose of this study was to determine whether any differences exist when the free TRAM flap is anastomosed to the thoracodorsal or internal mammary vessels. The study comprised 25 patients receiving a free TRAM flap for breast reconstruction. The thoracodorsal vessels were used as recipients in 21 patients and the internal mammary vessels were used in 4 patients. Blood flow rate was measured directly in the donor and recipient arteries, and after anastomosis by a transit-time ultrasonic flowmeter (CardioMed). Two- and 3-mm probes were used. The blood flow rate in the donor artery (deep inferior epigastric) before flap dissection was 11 ± 6 ml per minute (mean ± standard deviation). The rate was significantly (p < 0.05) lower (5 ± 3 ml per minute) in the recipient thoracodorsal artery than in the donor, but after transplantation it increased to 14 ± 5 ml per minute (p < 0.05), attaining the same value as the donor artery. The blood flow rate in the intact internal mammary artery was significantly higher (25 ± 10 ml per minute) than in the donor and thoracodorsal arteries, but after anastomosis it dropped to the same value (12 ± 3 ml per minute;p < 0.05) as the donor artery. The intake of blood in TRAM flaps supplied by the internal mammary artery seems to be no greater than that in free flaps anastomosed to thoracodorsal vessels, although the flow in the internal mammary artery was much higher. The authors concluded that the blood supply in a free TRAM flap is independent of the flow in the recipient artery and that thoracodorsal vessels, although often in a scarred bed and radiated, are as suitable for anastomosing a free TRAM flap as are internal mammary vessels.

Human skin transcriptome during superficial cutaneous wound healing.

Healing of the epidermis is a crucial process for maintaining the skins defense integrity and its resistance to environmental threats. Compromised wound healing renders the individual readily vulnerable to infections and loss of body homeostasis. To clarify the human response of reepithelialization, we biopsied split‐thickness skin graft donor site wounds immediately before and after harvesting, as well as during the healing process 3 and 7 days thereafter. In all, 25 biopsies from eight patients qualified for the study. All samples were analyzed by genome‐wide microarrays. Here, we identified the genes associated with normal skin reepithelialization over time and organized them by similarities according to their induction or suppression patterns during wound healing. Our results provide the first elaborate insight into the transcriptome during normal human epidermal wound healing. The data not only reveal novel genes associated with epidermal wound healing but also provide a fundamental basis for the translational interpretation of data acquired from experimental models.

Treatment of subcapital fractures of the fifth metacarpal bone: a prospective randomised comparison between functional treatment and reposition and splinting.

We did a prospective study to compare the results of treatment of subcapital fractures of the fifth metacarpal bone by closed reduction and splinting or by functional treatment. Twenty-nine consecutive patients were randomly divided into the two treatment groups (functional n = 14, and reposition and splinting n = 15). The results of treatment were satisfactory in both groups. Functionally treated patients recovered their grip force and range of movement of the affected hand a little sooner. All fractures in both groups had united within three months. There were no complications. We conclude that subcapital fractures of the fifth metacarpal bone can successfully be treated without closed reduction and splinting.