Characterization of human mesenchymal stem cells by six-color immunofluorescence

Oral Presentations/023. Tissue Engineering I

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References

[1] Guerrero CA, Bell WH, Meza LS. Intraoral distraction osteogenesis: maxillary and mandibular lengthening. Atlas Oral Maxillofac Surg Clin North Am 1999;7:111-51. [2] Liou EJW, Chen PKT, Huang C, Chen YR. Interdental distraction osteogenesis and rapid orthodontic tooth movement: a novel approach to approximate a wide alveolar cleft or bony defect. Plast Reconstr Surg 2000;4:1262-72.

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023. Tissue Engineering I

[-0--~--~ A LIVING TISSUE-ENGINEERED AUTOGENOUS BONE SU BSTITUTE

G.J. Meijer 1,2, R. Koole 1 , C.A. van Blitterswijk 2, J.D. de Bruijn 3. DENTAL IMPLANTS IN PATIENTS WITH OROFACIAL CLEFTS A F T E R BONE GRAFTING

A. Aref, EJ. Kramer, H. Schliephake. Department for Cranio-Maxillofacial

Surgery University of Goettingen 37099 Goettingen, Germany Propose of the study was to evaluate the success of implant treatment in patients with orofacial clefts and to ascertain relevant factors for their prognosis. This long term follow-up study evaluated prospectively 75 implants placed into 45 patients, which had received bone grafting at cleft sites before. The average period of observation was 5.5 years. All implants were examined clinically and radiographically. The results were compared to two control-groups: one control-group had received bone grafting and implants for other reasons and the other control-group had received implants into the non grafted maxilla. Ten implants were lost in eight patients, resulting a implant survival rate of 82.2%. The patient related parameters as age, gender and type of cleft had no significant influence. The length of an implant was significantly related to an improved implant survival rate (p < 0.01). The implant survival rate of the cleft patients was less when compared to the control-group without bone grafting, but improved when compared to the control-group with bone grafting for other reasons. The combination of implant insertion with bone grafting is a reliable alternative for patients with orofacial clefts.

[ - 0 - - ~ ' ~ PRIMARY AND SECONDARY TREATMENT IN CLEFT LIP AND PALATE, OUR EXPERIENCE F. Manna 1 , G. Clarich 1 , F. Radovich 1 , P. Di Emidio 1 , S. Pensiero 1 , C. Riberti 2 , G.F. Guarneri 2. 1Childen's Hospital Burlo Garofolo Surgery

Department, Trieste, Italy; 2plastic Department Udine, University Udine, Italy Aim of this study is to present a result in the treatment of cleft patients and emphasize the importance of surgical timing. Many protocols and technique are reported in the literature, but controversies are still present. Primary cleft nose repair, adequate rotation advancement technique, differential muscle repair, nasal floor closure and vermillion reconstruction- Millard or Tennison technique is used within 50/60 months of life. Monasterio technique is used for the soft palate within the 10 year of life. Perko-Widmaier technique for the hard palate within 30/40 year. Alveolar bone graft reconstruction about the 9°/110 year. Nasal correction after the 160 year. Good aesthetic result and a normal nasal breathing was achieved. Primary and velopharingeal competence and phonetic result was very satisfactory. A palatal fistula was present on 20% of patients. Midfacial growth is a complex process, even more if influenced by surgery. The severity of facial deformity and tissue volume of upper lips in infancy will be possible factors affecting maxillofacial growth of patients with unilateral cleft lip and palate. Already in the 1966 Ortiz-Monasterio concluded that in adult non operated cleft patients "growth defects of the middle third of the face are caused by early or repeated and aggressive surgery". We underline the need of a multidisciplinary management to minimize the psychological discomfort and the surgical stress. The integration of more techniques and the use of proper sequence in primary and secondary cleft patients are important to achieve satisfactory results. References

[1] Cleft Palate Craniofac J. 2004 Sep;41(5):550-8. Management of the impaired adult cleft patient: the last chance. Felemovicius J, Ortiz-Monasterio F.: Plast Reconstr Surg. 2005 Mar;115(3):687-695. [2] Maxillofacial Growth in Children with Unilateral Cleft Lip and Palate following Secondary Alveolar Bone Grafting: An Interim Evaluation. Chang HP, Chuang MC, Yang YH, Liu PH, Chang CH, Cheng CF, Lai JR: Plast Reconstr Surg. 2005 Feb;115(2):383-7. [3] Surgical technique for the correction of postpalatoplasty fistulae of the hard palate. Denny AD, Amm CA.

1Department of Oral Maxillofacial Surgery, University Medical Centre Utrecht, the Netherlands; 2Institute for Biomedical Technology,University of Twente, the Netherlands; 3Queen Mary University of London, United Kingdom The placement of dental implants requires the presence of adequate bone volume. In patients with inadequate bone volume, additional surgical techniques may be needed to achieve acceptable treatment results, such as onlay or inlay grafting procedures [1]. Autogenous bone is currently the standard and the most successful method for bone reconstruction. However, bone grafting procedures result in donor site morbidity and prolonged operation time. Moreover the available bone volume is limited. Therefore more than a century of research has been conducted with the goal of finding a suitable material to repair, replace or augment bony segments of the skull. The ideal bone graft material should be osteoinductive, i.e. capable of triggering osteogenesis. Only if growth factors, cellular elements or progenitor cells are present, as in autogenous bone grafts, osteogenesis will occur [9]. However, most bone substitutes of today are osteoconductive. As relatively inert filling materials, they only provide a scaffold to allow bone ingrowth [7]. The last decade, apart from the use of gene therapy and embryonic stem cells, two novel bone engineering technologies to enhance bone healing were introduced. The first approach is the use of growth factors such as bone morphogenetic proteins of the TGF-I-~ family combined with graft materials. A potential drawback of this approach, however, is that high, supra-physiologic concentrations are needed to obtain the desired osteoinductive effect with possible related side-effects and high costs. Furthermore, most if not all current techniques in which bone growth factors are used, result in a burst-release of the growth factor shortly after placement followed by a limited release over longer time periods, thus limiting the effectiveness of such an approach. The second and more biologically driven approach combines living osteogenic cells with biomaterial scaffolds ex vivo, to allow the development of a three dimensional tissue structure. Several investigators [2-8] have reported that bone marrow cells, when cultured under appropriate conditions, can maintain their viability and are able to differentiate into osteogenic cells. Although in most reported techniques, the cultured cells are inoculated for a few hours in the porous biomaterial scaffold prior to placement, an alternative technique can be followed that creates a truly living tissueengineered autogenous bone substitute. (Truncated)

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CHARACTERIZATION OF HUMAN MESENCHYMAL STEM CELLS BY SIX-COLOR IMMUNOFLUORESCENCE

C. Pautke 1 , E Haasters 2, A. Kolk 1 , H. GLilkan 2, W. Mutschler 2, S. Milz 3, M. Schieker 2 . 1Departmentof ©ral and Maxillofacial Surgery, Technical

University of Munich, Germany; 2Department of Sugery, LudwigMaximilians University of Munich, Germany; 3AO Research Institute, Davos, Switzerland Human mesenchymal stem cells (hMSC) are ideally suited for tissue engineering applications because of their ability to self-replicate and their differentiation capacity. However, they lack distinct phenotypic features. Although no single marker has been found to be specific for stem cells, certain surface proteins are used as putative stem cell markers, in particular CD44, CD105 and CD106. Because other cell types also express these markers, only their simultaneous detection at single cell level appears to be a suitable approach for the characterization of mesenchymal stem cells. Therefore, the aim of this study was to characterize human mesenchymal stem cells at the single cell level by six-color immunofluorescence of CD44, CD105, CD106, collagenIV, fibronectin, and actin. Indirect immunofluorescence was performed to detect antibody binding using secondary antibodies conjugated to different fluorochromes (AMCA, Texas Red, FITC, Alexa546, Cy2, and Alexa633, respectively). To discriminate between the different fluorescent spectra, spectral image acquisition was performed using a Sagnac type

Int. J. Oral Maxillofac. Surg. 2005; 34 (Supplement 1): $ 1 - $ 1 8 1

44 interferometer allowing subsequent linear unimixing and decomposition of each pixel in its spectral components. The hMSC turned out to be heterogeneous in morphology and immunocytochemistry. With the exception of collagen-IV, we found that the investigated antigens were expressed by most of the cells. However, individual antigen distribution was dependent on cell morphology. By six-color immunofluorescence hMSC could clearly distinguished from other cell types like fibroblasts or osteoblasts. The different distributions of the investigated antigens emphasize the heterogeneity of human mesenchymal stem cell populations. Thus, our methodological approach may provide useful information for further detailed stem cell characterization.

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PROLIFERATION AND DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS ON THREE DIMENSIONAL SCAFFOLDS

T. Materna 1, H.J. Rolf1, M. Gelinsky2, H. Schliephake 1. 1Klinik

f#r Mund-, Kiefer- u. Gesichtschirurgie, Georg-August-Universit#t GSttingen, Robert-Koch-Str. 40, 37075 GSttingen, Germany; 21nstitutffJr Werkstoffwissenschaft, Technische Universit#t Dresden, Haflwachstr. 6, 01069 Dresden, Maxillofacial Surgery, University of Goettingen, Germany To support in vitro osteogenesis in tissue engeneering, cells with bone building ability need a three dimensional scaffold on which to adhere, grow and proliferate. The present study examines the use of different scaffold materials for promoting proliferation and differentiation of bone marrow derived human mesenchymal stem cells (MSCs). The MSCs that were derived from the pelvic bone marrow were cultured in a standard medium to the second passage. A defined number of 1.25×104 cells/ml were added to each standardized scaffold of coralin calcium carbonate (CaCO3), bovine hydroxyapatite (HA), equine collagen and mineralized bovine collagen type I. After 4, 10, 18, 25, 32, 40, 46 and 52 days the cell population's density was measured by MTT. To determine the degree of cell-differentiation the relating amount of osteocalcin synthetised by the cells was measured in the culture medium. As osteocalcin correlates with the initial mineralisation of the extracellular matrix it is considered as a marker for the advanced stage of osteoblast differentiation. All tests were performed separatedly for each scaffold material as well as for the two dimensional controls. Within the 52-day-duration of the experiment the two-dimensional controls showed constantly a low proliferation-level. We found significant higher degrees of proliferation in all four three dimensional scaffolds. CaCO3 and HA showed the maximum proliferation on day 10, the equine collagen on day 4. The proliferation peak of cells seeded on mineralized bovine collagen type I was reached on day 52. The highest concentration of osteocalcin was observed on day 46 for the controls, on day 18 for CaCO3 and on day 32 for HA. Compared to the controls the equine collagen showed a delayed and similar low level proliferation. All three dimensional scaffold materials induced a comparably higher proliferation than the two dimensional controls. Under given culture conditions, without the addition of osteogenic supplements, the CaCO3- and HA-scaffolds showed earlier but less differentiation than the controls. The findings reported here suggest that during 52 days only the physical support of equine collagen promoted significant higher proliferation and osteoblast differentiation.

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INTRAINDIVIDUAL COMPARISON OF THE OSTEOGENIC DIFFERENTIATION POTENTIAL OF MESENCHYMAL STEM CELLS DERIVED FROM ADULT ADIPOSE TISSUE AND BONE MARROW

K. Weinzierl, D. Halama, A. Burkhardt, E Gaunitz, B. Frerich. Department of Oral and Maxillofacial Surgery, Facial Plastic Surgery, University of Leipzig, Germany Future cell based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. The methodes to isolate mesenchymal stem cells (MCS) from the bone marrow compartment (BMSC) has some potential and practical limitations. An alternative source for MCS is the human adipose tissue (ATSC), that is obtainable in large quantities with minimal discomfort for the patient. Human adipose tissue and bone marrow were obtained during routine operations in 4 patients. The isolated fibroblast-like cells from both sources were cultured and maintained in vitro for an extended period with stable population doubeling. Osteogenic differentiation was induced during 3rd passage by using dexamethason and beta-glycerophosphate (series 1) and by

administration of BMP-2 (series 2). After 5 weeks of osteogenic differentiation the expression of specific and typical markers of osteogenic differentiation (osteocalcin = OC; osteonectin = ON; osteopontin =OP; alkaline phosphatase=AP) was analysed by quantitative RT-PCR. The amount of mineralization was evaluated semiquantitatively by means of v. Kossa staining. The expression of stem cell associated markers markers was monitored by flow cytometry. The expression of OC, ON, OP, AP during the differentiation of BMSC increased more in series 1 than in series 2. In ATSC, expression of OC and AP increased markedly, whereas expression of ON increased not. The mRNA-level of OP even decreased in ATSC. Mineralisation was shown in series 1 both with BMSC and ATSC, but not by addition of BMP-2. Flow cytometry showed the decrease of stem cell associated surface markers (CD44, CD73=SH3, CD90, CD105=SH2), while Osteocalcin increased also in flow cytometry. An osteogenic differentiation is possible in BMSC and in ATSC by use of the dexamethasone/beta-glycerophosphate protocol as well as by administration of BMP-2. The osteogenic differentiation of ATSC showed a lower osteogenic potential than BMSC. The osteogenic differentiation potential of BMP-2 in our protocol is less effective than dexamethasone/beta-glycerophosphate. ~-'~

ADIPOSE TISSUE ENGINEERING IN SCID-MICE

B. Frerich, K. Weinzierl, A. B6ttner. Department of Oral and Maxillofacial

Surgery, Facial Plastic Surgery, University of Leipzig, Germany Adipose tissue can serve as a simple model for the engineering of a soft tissue requiring vascularisation. It has high significance since it provides volume and contour of many flaps used in maxillofacial reconstruction. Human adipose tissue stromal cells were amplified in culture and seeded onto collagen microparticles. Following adipogenic differentiation they were composed to "adipose tissue equivalents" together with endothelial cells. The expression of markers specific or typic for adipose tissue (Leptin, Adiponectin) was ascertained by RT-PCR. Furthermore, the tissue equivalents were implanted into immundeficient SCID-mice (n =20) and evaluated after 7 days, 3 and 6 weeks. Histologic examination of the in vitro aggregates revealed to more or less extent (bewteen 30% and 95%) mature adipose tissue with formation of univacuolar adipocytes, which stained positive with Sudan stain. The expression of leptin and adiponectin could be demonstrated on the mRNA-level after the differentiation process. A capillary-like network formation could be shwon in histological sections and in laser scanning microscopy. In vivo, engineered adipose tissue could be shown even after 6 weeks. Simultaneous implantation of endothelial cells improved survival fo the inner parts of the transplanted aggregates. The tissue engineering of fat is feasible in the SCID-mouse model. The in vivo results reflect the need for simultaneous vascular engineering. ~-'~

GENE EXPRESSION PROFILE OF HUMAN OSTEOBLASTS AND ENDOTHELIAL CELLS AS BASIS FOR TISSUE ENGINEERING

J. Kleinheinz 1, T. Fillies 1, B. Brandt2, U. Joos 1. 1Department of

Cranio-Maxillofacial Surgery, University Hospital, Muenster, Germany; 2Department of Clinical Chemistry and Laboratory Medicine, University Hospital, Muenster, Germany Aim of the study was to analyze the effect of human endothelial cells and human osteoblasts on pattern of gene expression of both cells in coculture. Preconfluent human umbilical vein endothelial cells (HUVEC) and human osteoblasts from mandibular periosteum were seeded separately in culture plates. The cells had no direct contact but the plates were put in a larger plate and cells were supported by the same medium. Monocultures of the cells with the same medium served as control groups. After 7 days cells were fixed and the RNA-expression profiles of monoculture cells and coculture cells were evaluated and compared using gene chip technology. Evaluation covered a three step statistical analysis to determine significantly increased or decreased expression rates. Validation of the results was carried out using selective RT-PCR of the significantly changed gene sections. Out of 31.000 genes in the beginning 12 could be selected in endothelial cells, and 15 in osteoblasts which demonstrated significantly differential gene expression in monoculture and coculture (fold change 2.5). Analysis of the encoded gene products revealed overexpression of vasculogenetic and angiogenetic factors in endothelial cells (e.g. ephrin B4, VE-Cadherine, integrins), demonstrating a higher grade of differentiation and structural