Int Poster J Dent Oral Med 2007, Vol 9 No 03, Poster 373
Bone Engineering: Allogenic and Alloplastic Bone Transplants vitalized by Osteoblast-like Cells
Dr. Marc Hinze, Dr. Sebastian Sauerbier, Dr. Margit Wiedmann-Al-Ahmad, Ute Hübner, Prof. Dr. Dr. Rainer Schmelzeisen, Prof. Dr. Dr. Ralf Gutwald
all authors belong to:
Departement of Oral and Maxillofacial Surgery, Universitiy Hospital Freiburg
19th to 21th of Oktober 2006
AO-Biotechnology-Symposium, From basic research to clinical applications using biotechnology and bioengineering
The search for suitable techniques and materials for the reconstruction of
bone defects is a primary goal in many clinical disciplines. Implants made
of synthetic polymers, ceramics or metals as well as allogenic materials
like collagen or cartilage are used for bone grafting. Up to now no grafting
material exists with the quality of the original tissue. These artificial
materials show problems in anchoring and mechanical stability or induce
immunological reactions. A new approach in therapy is the application of
tissue engineered bone grafts. The possibility of cell culturing in vitro
and the exclusive use of endogenous cells opens the way for a "self cell
therapy" and thus avoids problems like limited resources. Additionally, the
risk of donor site morbidity is decreased because only small biopsies have
to be harvested. In this study, we focused on the search for a biomaterial
which represents a suitable matrix for three-dimensional growth of human
osteoblast-like cells in vitro and for the surgical management of intraoral
Material and Methods
Human osteoblast-like cells were cultured on two different biomaterials: a
human demineralised bone matrix (DBX® Mix, Musculoskeletal Transplant
Foundation, NJ, USA, distributed by symthes) and a non-sintered,
nanocrystalline, phase-pure hydroxylapatite (Ostim® Paste, Heraeus Kulzer,
Hanau, Germany). Cortico-lamellar bone was obtained during dental surgery.
Opti-minimal-essential-medium (Opti-MEM, Gibco Life Technologies, NY, USA)
was used for primary culture with 10% fetal calf serum (FCS, PAA
Laboratories, Linz, Austria), 2% HEPES (Gibco Life Technologies, NY, USA)
and the antibiotics penicillin (1%) and streptomycin (1%, PAA Laboratories,
Linz, Austria). The confluent primary osteoblasts were detached from the
culture flask by incubation with 0.5% trypsin (PAA Laboratories, Linz,
Austria) in phosphate buffered saline (PBS) for 5 min at 37°C. The cells
were filtered through a 100μm cell-strainer (Falcon, NJ, USA) in a 50ml
tube (Falcon, NJ, USA), centrifuged and resuspended in 1ml fresh medium RPMI
1640 (Gibco Life Technologies, NY, USA), supplemented with 10% FCS, 2%
HEPES, penicillin (1%) and streptomycin (1%). The osteoblasts were
transferred into a 75cm2 culture flask (Falcon, NJ, USA), filled up with
30ml culture medium. After 2-3 weeks, the cells were trypsinized again from
the culture flask, centrifuged and resuspended in 1ml medium. The cells from
the first passage were seeded on the two different biomaterials. An aliquot
of the same passage was seeded in cell culture plates and served as control
of the cell proliferation. Additionally, plates with 1x 105 cells/ml were
incubated for 1 week for the detection of alkaline phosphatase and collagen.
Cell cultures were kept in a humified atmosphere of 5% CO2 at 37°C. For the
staining of osteoblast-like cells an alkaline phosphatase assay kit (Sigma,
Deisenhofen, Germany) was used. The evaluation of collagen type-I was done
by light microscopy and the computer program Analysis 3.1 after
immuno-staining with anti-collagen I antibody (Sigma, Deisenhofen, Germany).
Osteocalcin was analysed using a competitive EIA kit (Osteomedical, Bünde,
Germany) and an ELISA-Reader (Anthos Labtech, Salzburg, Austria). For cell
proliferation analysis, the nonradioactive assay EZ4U (Biomedica, Wien,
Austria) was used. The cell vitality was evaluated by fluorescence
microscopy and a dichromogenic PI/FDA-staining. For the cell colonization
analysis the samples were examined by scanning electron microscopy at 15
|Fig. 1. Cell proliferation analysis of human osteoblast-like cells in cell culture (black) and seeded onto DBX® (gray) respectively onto Ostim® (white).
All cell culture supernatants of human osteoblast-like cells examined were
osteocalcin positive with approximately 10 ng/ml osteocalcin and the
alkaline staining of these cells typically resulted intensively positive
(about 36.9%). Immuncytochemistry of the fixed cells showed the presence of
collagen type-I in about 10.5% of the cells. Osteoblast-like cells seeded
onto the human demineralised bone matrix (DBX®) showed a ten times higher
rate of proliferation capacity than the cells cultivated on hydroxyapatite
Ostim® (Fig.1). After 3 weeks of cultivation the vital cells migrated over
the biomaterial and a beginning vitalization could be observed on DBX®
(Fig.2). The surface of Ostim® was sparsely covered by human osteoblast-like
cells after 3 weeks of cultivation indicating that there is no vitalization
in vitro (Fig.3). Thin sections of the demineralised bone matrix (DBX®)
showed a multilayered growth of human osteoblast-like cells already after 2
weeks of cultivation (Fig. 4). In comparison, Fig. 5 shows thin section of
osteoblasts after a period of two weeks grown on Ostim®. Scanning electron
microscopy after 3 weeks of cultivation on DBX® a dense network of
multilayered polygonal shaped cells could be observed (Fig. 6). Fig. 7 shows
an isolated and scattered growth of osteoblast-like cells upon Ostim®.
|Fig. 2. Fluorescence microscopy after PI/FDA-staining of human osteoblast-like cells after cultivation for three weeks on DBX® (magnification 25x).
||Fig. 4. Thin section of human osteoblast-like cells after a cultivation period of two weeks on DBX® (magnification 500x). (Toulidinblue)
||Fig. 6. Scanning electron microscopy of human osteoblast-like cells cultivated three weeks on DBX® (magnification 1000x).
|Fig. 3. Fluorescence microscopy after PI/FDA-staining of human osteoblast-like cells after cultivation for three weeks on Ostim® (magnification 25x).
||Fig. 5. Thin section of human osteoblast-like cells after a cultivation period of two weeks on Ostim® (magnification 500x).
||Fig. 7. Scanning electron microscopy of human osteoblast-like cells cultivated three weeks on Ostim® (magnification 1000x).
The topographic structure of the biomaterial surface could be a reason for
different proliferation rates. Anselme (2000) described the decisive role of
surface roughness, chemistry or surface energy regarding cell adhesion, cell
migration or cell proliferation upon biomaterials. The mitogene effect of
demineralised bone matrix can be attributed to the existence of various
growth factors in the bone matrix, such as BMP's (Urist 1965). Wozney et al.
(1992) showed that BMP's, belonging to the TGF-superfamily, are activated by
the process of demineralization. Furthermore, Zhang et al. (1997) described
that BMP's are directly bound to the bone mineral and the demineralization
process release them, indicating a proportional connection between the
demineralization level, the accessible BMP's and the osteoinductive effect.
Further in vivo studies are necessary to examine if the present in vitro
results correspond with the in vivo conditions. In future, it appears
conceivable to produce made-to-measure and biological integrative
biomaterials in combination with autologous cells. Pradel et al. (2006)
clinically applied demineralized bone matrix (Osteovit, Braun, Melsungen,
Gremany) cultured with osteoblasts in mandibular cysts. Nonetheless, further
research with regard to the clinical application of such biomaterial/cell
constructs are of essential importance for the further development of bone
- Anselme K. Osteoblast adhesion on biomaterials. Biomaterials 2000;21:667-681.
- Pradel W., Eckelt U., Lauer G. Bone regeneration after enucleation of mandibular cysts: Comparing autogeneous grafts from tissue-engineeered bone and iliac bone. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101(3):285-290.
- Urist MR. Bone formation by autoinduction. Science 1965;150:893-899.
- Wozney JM. The bone morphogenetic protein family and osteogenesis. Mol Reprod Dev 1992;32:160-167.
- Zhang M, Powers Jr. RM, Wolfinbarger Jr. L. Effect(s) of demineralization process on the osteoinductivity of demineralized bone matrix. J Periodontol 1997;68:1085-1092.
This Poster was submitted by Dr. Marc Hinze.
Dr. Marc Hinze
Departement of Oral and Maxillofacial Surgery
Universitiy Hospital Freiburg
Hugstetter Str. 55