Osteointegration of hydroxyapatite-titanium implants coated with nonglycosylated recombinant human bone morphogenetic protein-2 (BMP-2) in aged sheep
Introduction
Optimization of bone/implant integration by administration of bone morphogenetic proteins (BMP) has been explored experimentally in various experimental setups [16]. Animal species used for testing include readily available species, from mice and rats to dogs and sheep. In the overwhelming majority of these studies, skeletally mature animals were chosen as targets. Occasionally, adolescent animals were selected. In general, the data obtained in those studies indicate above-average bone formation, irrespectively of the specific BMP subtype. However, at least to our knowledge, none of the studies focused on significantly aged animals as an experimental model. This situation is understandable considering the accumulated maintenance costs for old animals but it leaves a major question mark behind the extrapolation to the situation for human patients. Most problem patients who obtain hip, knee, or other replacements are of advanced age. Many, mostly female, patients display more or less advanced stages of osteoporosis or develop local osteoporosis at the site of implantation. More recently, due to the increased focus of research on osteoporosis, various animal models of osteopenia have been developed, including surgically or hormonally castrated animals, such as mice, rat, dogs, and sheep [8], [15]. Such animals develop a rapidly progressing osteoporosis based on a lack of control for metabolic calcium retention in the bone. In contrast, they of course do not display the biology of an aged animal, characterized by for example a significant loss of stem cells or precursor cells of skeletal tissues. The re-activation of such slumbering populations of regenerative cell types in part explains the usually significant response to experimental therapy of osteoporosis in those models.
In aged individuals, secondary osteoporosis or lack of implant healing is usually not a consequence of a hormonal deficit (even though it might have initially been so) but is based on insufficient bone healing as a consequence of age-related cellular deficits in the skeleton and the bone marrow. In other words, recruitment of cells involved in wound healing is restricted by the numbers of competent cells. Whether or not the residual cell population may be sufficient under appropriate stimuli has never been explored. Therefore, in a leap of rationalization, currently, methods are under development to enrich precursor cell populations from various autologous sources such as bone marrow, fat tissue, or blood. In general, those attempts report logarithmic depletions of competent precursor cells in the studied body compartments as a function of age, thus questioning the use of such procedures in the elderly. Most recently, a genetic analysis of Icelanders revealed a positive correlation between occurring osteoporosis and deficient expression of BMP-2, indicating a putative role of endogenous BMP-2 as a preventive or protective agent [20]. In a situation of thus conflicting backgrounds, we decided to exploit a particular occasion, having access to postreproductive ewes, age 8–13 years, and to practically unlimited quantities of BMP-2, the experimentally most widely applied BMP. Sheep are considered to be a good model for human osteoporosis, even with experimentally ovariectomized animals [15]. Due to tertiary structure and its surface hydrophobicity mature BMP-2 is relatively insoluble under physiological conditions. BMPs extracted from demineralized bone or overexpressed in mammalian cell culture are post-translationally modified through N-linked glycosylation at a conserved N-linked site. The lack of glycosylation in bacterially expressed BMP-2 leads to a further reduction of solubility that causes a delayed release from application sites and have promise for decreased BMP doses necessary for efficient induction of bone [17]. Using a model implant based on hydroxyapatite (HA)-coated titanium alloy impregnated either with or without nonglycosylated BMP-2, we demonstrated implant integration in aged sheep accompanied by bone remodeling, formation of new cancellous bone, and regeneration of bone marrow in close vicinity of the BMP-2-refined implant.
Section snippets
Expression and purification of BMP-2
Recombinant human BMP-2 was expressed in E. coli based on a maltose-binding protein (MBP)-fusion expression vector. The malE fusion vector was constructed from pMAL-c2X (New England Biolabs, Beverly, MA) by removing the factor Xa site with an AvaI–BamHI restriction digest and replacing them with an annealed pair of synthetic oligonucleotides (5′-GTC CCT CGG GCA TCA CCA TCA CCA TCA TTC TGG TCT GGT ACC ACG CGG ATC CGC GC-3′, 5′-GCG CGG ATC CGC GTG GTA CCA GAC CAG AAT GAT GGT GAT GGT GAT GCC CGA
HA surface structure
BMP-2 coating was performed based on the macroscopic dimensions of the implant cylinders. Since the HA has a certain microcrystallinity and an average particle size of 5 μm, the geometric surface of the titanium cylinder obtains a roughness that significantly expands its surface area. By using microscopic laser scanning, we estimated the surface expansion to be 5- to 6-fold at 3–5 μm resolution and almost 30-fold at 500 nm resolution (Table 1). Considering this, coating density changes from the
Discussion
To our knowledge, this is the first time that nonglycosylated BMP-2 has been tested in large mammals of advanced age. Working with older sheep caused some problems not existing with younger animals. Of 25 sheep, 18 were finally analyzed since 5 animals fractured at the implant site (possibly caused by advanced osteoporosis) within 2 weeks postoperatively and 2 animals died of unrelated causes (heart failure). Nonetheless, the model allowed to clearly differentiate effects caused by the surgical
Acknowledgments
This work was supported by a public grant from the Bundesministerium für Forschung und Technologie, Germany, AZ 031255. We appreciate the instrumental help of Cordula Mueller, Jana Schömburg, Dr. Silke Erdmann (Department of Orthopaedics) and Karin Blechschmidt (Department of Otolaryngology) in preparing the histological samples, of Andrea Galonska (Department of Orthopaedics) in preparing the manuscript (all affiliations University of Jena), and of Beat Gasser (RMS Foundation, Switzerland) for
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