Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier
Introduction
Currently, the gold standard for grafting of bone defects is the use of autogenous bone. In order to avoid morbidity at the donor site or if large amounts of autogenous bone are necessary, bone substitution materials can be used [1]. Bone substitution materials can be combined with vital cells such as mesenchymal stem cells (MSC) to increase bone formation [2], [3], [4], [5]. Both synthetic and allograft materials allow adhesion and growth of osteoblastic cells, or osteogenic differentiation of precursor cells in vitro [6], [7], [8]. Previously, our group had compared in vitro osteogenic induction of bone marrow derived MSC on small blocks of -tricalcium phosphate (-TCP), calcium deficient hydroxyapatite (CDHA) and demineralized bone matrix (DBM) [6]. These in vitro data indicated that the resorbable ceramic CDHA which characteristically has a high surface area per volume has properties comparable to -tricalcium phosphates with regard to the support of osteogenic induction of cells (alkaline phosphatase activity and osteocalcin content). However, due to its high specific surface area (approximately 48 m2/g) which approximates to that of bone (80 m2/g) [6], [9], cells can adhere more easily to CDHA than to -TCP (specific surface <0.5 m2/g) [6].
To further evaluate the novel high surface biomaterial CDHA in combination with MSC regarding its capacity to allow or induce bone formation in vivo, one can choose between an ectopic [10], [11], [12] or an orthotopic implantation [13], [14], [15], [16] model. We opted for an immunodeficiency mouse model with ectopic implantation because it is well established for the evaluation of bone substitution materials [10], [11], [12]. It allows the use of human MSC and biomaterials under non-loaded conditions [12], [17], [18], [19], [20] without the presence of growth factors at an orthotopic site whose influence may disguise differences between the performances of biomaterials. Furthermore, the mouse model allows a more cost effective and standardized comparison of different biomaterials and subgroups than a model with larger animals.
When bone substitution materials are combined with MSC to improve bone healing, one approach is to use undifferentiated MSC for bone formation in vivo [13], [14], [15], [16]. The other is to induce osteogenic induction of the MSC on the biomaterial prior to implantation in order to provide immediate new bone-forming capability after in vivo implantation [21], [22], [23]. Several authors found more extensive bone formation in vivo with MSC expanded in monolayer in the presence of dexamethasone and ascorbic acid or FGF-2 [11], [12]. However, to our knowledge there has been no direct comparison of a 3D osteogenic induction of MSC in a biocomposite with a biocomposite with freshly loaded cells supplemented by different biomaterials. If a direct application of cells without prior 3D cultivation proves to be equally effective, time and money could be saved in clinical applications. An important issue in clinical application is the use of xenogenic supplements such as fetal calf serum (FCS). Culturing MSC in FCS can cause immunologic reactions after re-implantation in vivo [24], [25] and FCS has been implicated as a potential vector for prion transmission [26]. Therefore, we eliminated all xenogenic supplements in our setting.
The aim of the current study was to examine ectopic bone formation in the new resorbable CDHA with a high specific surface area. We compared the novel ceramic with other biomaterials, for example -TCP, DBM and hydroxyapatite (HA) in vivo by subcutaneous implantation in severe combined immunodeficiency mice (SCID-mice). -TCP and HA were included as positive controls for ectopic bone formation in vivo because MSC/HA composites have been found to induce bone in previous studies [11], [12]. In contrast, DBM was unable to induce bone formation in a similar setting [12] and was used as a negative control. Histology, ALP enzyme activity and osteocalcin content were used to evaluate bone formation in empty and MSC loaded scaffolds 4 and 8 weeks after ectopic implantation in SCID mice. To assess the value of in vitro osteogenic pre-induction, one group of samples was kept under osteogenic conditions for 2 weeks prior to in vivo implantation.
Section snippets
Materials
-tricalcium phosphate (-TCP), HA and CDHA, Ca9(PO4)5(HPO4)OH, ceramic bodies (by Dr. h.c. Robert Mathys Foundation, Switzerland) were produced in an emulsion process as described earlier [9]. The macroporosity (pores ∅ 0.2–0.6 mm), microporosity (pores ∅ <5 μm) and specific surface area of the ceramics are shown in Table 1. Demineralized bone matrix (DBM, Grafton Flex® by Osteotech) was used as a control material. DBM is a soft, purely protein based matrix consisting mainly of collagen I and a
ALP activity
The mean values of specific alkaline phosphatase (ALP) activity were highest in -TCP, followed by CDHA and DBM () (Fig. 1, Table 2). Specific ALP activity was significantly higher in -TCP than in CDHA and higher () in all ceramics compared to DBM. There was no significant difference between -TCP and HA. Looking at all biomaterials, specific ALP values did not change significantly from 4 to 8 weeks. Specific ALP activity was significantly higher in the samples with MSC compared
Discussion
This in vivo study was performed to evaluate the novel ceramic CDHA. We demonstrated that there was no bone formation without MSC. Bone formation occurred at a similar frequency in CDHA (1/8) and -TCP carriers (2/8) with freshly loaded MSC. There appeared to be a trend towards improved in vivo bone formation after osteogenic induction of MSC for 2 weeks in CDHA (3/8) in comparison to -TCP (0/8). Bone was of human origin and no hematopoetic tissue or cartilage was found, although metachromasia
Acknowledgements
The authors are grateful to Beat Gasser and Marc Bohner, Dr. h.c. Robert Mathys Foundation, for help in designing the study. We thank K. Goetzke, R. Föhr and A. Krauthoff for technical assistance with histology and S. Schneider for statistical evaluation of the data. Furthermore, we would like to thank Irina Berger, Institute of Pathology, University of Heidelberg, for her help in evaluation of histological sections. This study was supported by the Dr. h.c. Robert Mathys Foundation and the
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